Seviour Bible 2010

Microbial Ecology of Activated Sludge
Edited by
Robert Seviour and Per Halkjær Nielsen
Published by IWA Publishing
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ISBN 10: 1843390329

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Table of contents
Preface ..................................................................................................................................... vii

Contributors ............................................................................................................................. xi
1 AN OVERVIEW OF THE MICROBES IN ACTIVATED SLUDGE ................................ 1

2 THE ACTIVATED SLUDGE PROCESS ......................................................................... 57
3 MICROBIAL COMMUNITIES IN ACTIVATED SLUDGE PLANTS .............................. 95
4 PROTOZOA IN ACTIVATED SLUDGE PROCESSES ................................................. 127
5 FACTORS AFFECTING THE BULKING AND FOAMING FILAMENTOUS
BACTERIA IN ACTIVATED SLUDGE ........................................................................... 139
6 THE CURRENT TAXONOMIC STATUS OF THE FILAMENTOUS BACTERIA
FOUND IN ACTIVATED SLUDGE PLANTS ................................................................. 169
7 MICROBIOLOGY OF BULKING ..................................................................................... 191
8 FOAMING ......................................................................................................................... 215
9 THE MICROBIOLOGY OF NITROGEN REMOVAL ...................................................... 259
10 THE MICROBIOLOGY OF PHOSPHORUS REMOVAL ............................................... 281
11 METHODS FOR THE EXAMINATION AND CHARACTERIZATION OF THE
ACTIVATED SLUDGE COMMUNITY ............................................................................. 321
11.1 Microscopy and microscopic examination of activated sludge .................. 322
11.2 Preparation of specimens for microscopy ..................................................... 329
11.3 Stains used for examination of activated sludge samples .......................... 331
11.4 Isolation of filamentous bacteria from activated sludge .............................. 341
11.5 Collection of data from microscopic analysis and use of worksheets ...... 342
11.6 DNA and RNA extraction .................................................................................. 343
11.7 Polymerase chain reaction (PCR) technology ............................................... 350
11.8 Clone library generation ................................................................................... 360
11.9 FISH probes and their design .......................................................................... 370
11.10 Microautoradiography (MAR) ........................................................................... 393
11.11 Microarrays for studying composition and function of communities ........ 397
vi Microbial Ecology of Activated Sludge
11.12 Terminal restriction fragment length polymorphism (T-RFLP) ................... 412

11.13 PCR-DGGE .......................................................................................................... 421
11.14 Stable isotope probing ...................................................................................... 427
11.15 Flow cytometry ................................................................................................... 439
11.16 Microsensors ...................................................................................................... 448
12 DESCRIPTIONS OF ACTIVATED SLUDGE ORGANISMS ......................................... 453
13 COLOUR IMAGE SECTION ........................................................................................... 489
14 REFERENCE LIST .......................................................................................................... 537
Index ....................................................................................................................................... 643
Preface
‘‘Science is an endless search for truth. Any representation of reality we develop can be only partial.
There is no finality, sometimes no single best representation. There is only deeper understanding, more
revealing and enveloping representations. Scientific advance, then, is a succession of newer representa-
tions superceding older ones, either because an older one has run its course and is no longer a reliable
guide for a field or because the newer one is more powerful, encompassing and productive than its
predecessor(s)’’. [C.R. Woese (2004)]
It is now a decade since the book ‘The microbiology of activated sludge’ was published (Seviour and
Blackall, 1999), but so much new, exciting and useful information has been generated in the short period
since then that its use-by-date has well and truly passed. Therefore this is an opportune time to produce a
new book which can now develop some of the ideas expressed there, and critically review this material.
In the intervening years, several other publications concerned with various aspects of activated sludge
microbiology have appeared (Bitton, 2002, 2005; Eikelboom, 2006; Jenkins et al., 2004; Mara and Horan,
2003; Tandoi et al., 2006; Wiesmann et al., 2006), but we believe a book like this one, covering all
aspects in a holistic and coherent way at an advanced level, still has value. We would like to think that it
represents much more than simply a few cosmetic changes to what was written earlier, but instead through
a complete rewrite, takes a fresh and challenging look at the complex microbial ecology of activated
sludge. It also provides an opportunity to correct some of the embarrassing mistakes we made in the first
book, some of which have been drawn to our attention by colleagues. Instead of writing most of this in
house, as happened with the first edition, we decided it was now necessary to invite acknowledged experts
from around the world to contribute their special skills and expertise to make it stronger and hopefully
more useful to the intended audience.
In the first book we tried to foreshadow the impacts the application of applying molecular methods to
studying the microbial communities in activated sludge systems were likely to have on our understanding.
These have probably exceeded our most optimistic expectations, and many of the questions we thought
then needed urgent attention have been resolved, at least partly. For example, we now have a much better
knowledge than we did of the phylogenetic diversity of the filamentous bacteria causing bulking and
foaming, and 16S rRNA targeted FISH probes are available for the in situ identification of several,
suggesting that the conventional microscopic methods, long recognized as limited in their value, may
soon be complemented and even eventually replaced by molecular methods. Commercial kits based on
FISH probes are already available for a few of the particularly problematic filaments, and this number is
likely to increase as the advantages in using such systems become more apparent to the industrial end
users. DNA microarrays, allowing simultaneous in situ identification of multiple populations have
already been designed and applied to activated sludge systems for populations considered functionally
important there (Adamczyk et al., 2003; Kelly et al., 2005; Loy et al., 2005). FISH probes (Nielsen et al.,
2009) in combination with microautoradiography (MAR) have allowed us to begin to elucidate their
in situ physiology too, and already several interesting strategies for controlling bulking caused by some
filaments have been proposed based on these studies (Andreasen and Nielsen, 2000; Kragelund et al.,
2005, 2006, 2007a,b).
# 2010 IWA Publishing. Microbial Ecology of Activated Sludge. Edited by Robert Seviour and Per Halkjær Nielsen.
ISBN: 9781843390329. Published by IWA Publishing, London, UK.
viii Microbial Ecology of Activated Sludge
Our understanding of which bacteria are important in nitrification has changed completely as a result of
molecular data from their community analyses (Burrell et al., 1998; Juretschko et al., 1998; Maixner et al.,
2006; Park et al., 2006), although which are the important denitrifying bacteria in activated sludge
systems is not as clear (Ginige et al., 2004, 2005; Nielsen et al., 2009; Thomsen et al., 2007).
Nevertheless, such dramatic recent shifts in some of our previously held views, based as they were on old
information, raises the important question of whether the plants we currently operate for nitrogen removal,
are designed optimally for the organisms we now realize are actually performing the important tasks.
The change in our understanding of the microbiology of phosphorus removal from molecular studies is
equally profound (Oehmen et al., 2007; Seviour and McIlroy, 2008; Seviour et al., 2003), and again what
we once thought were major players in this game appear now to be no more than ‘laboratory weeds’
(Amann et al., 1995a). However, this story still has some way to go, and it seems likely that the
phylogenetic diversity among the polyphosphate accumulating organisms (PAO) and their possible
competitors, the glycogen accumulating organisms (GAO), will turn out to be larger than now recognized.
Again FISH/MAR and stable isotope probing (SIP) is helping address some of these issues by providing
information on the ecophysiology of these activated sludge populations (Ahn et al., 2007; Burow et al.,
2007; Kong et al., 2002, 2004, 2005, 2006, 2007; Meyer et al., 2006; Schroeder et al., 2008).
We have always suspected that activated sludge represented a considerable microbial resource in terms
of the biodiversity of the organisms there, but it took these molecular methods to reveal just how diverse
the community really was. An important question now is whether all this diversity reflects a similar
level in ecophysiological niche diversity (Jaspers and Overmann, 2004; Maixner et al., 2006). Already we
know that some populations we would probably never have suspected of finding in activated sludge in
fact appear to flourish there in large numbers, although what role/s these might play in contributing to the
chemical transformations is in most cases still a mystery (Blackall and Yeates, 2004; Kong et al., 2007).
So while we might have described our understanding of activated sludge microbiology in 1998/9 as
representing the ‘start of the beginning’, now it can perhaps be more adequately described as the ‘end of
the beginning’. Much remains to be discovered, and as the technology improves at great pace, more
informative but probably expensive methods will be directed to its study (Blackall and Yeates, 2004).
More rapid DNA sequencing methods including pyrosequencing (Hall, 2007; Sanapareddy et al., 2009)
and metagenomics (Bertin et al., 2008; Handelsman, 2004; Kunin et al., 2008a; Steele and Streit, 2005;
Streit and Schmitz, 2004; Ward, 2006) will become increasingly used. The whole genome sequence of
Candidatus ‘Accumulibacter phosphatis’ the bacterium thought to be an important PAO (Martin et al.,
2006), is now available. Having this information available has already helped us understand better the
physiology and biochemistry of these PAO (e.g. Burow et al., 2008a,b). Metaproteomics (Wilmes et al.,
2008a,b) has also been used in attempts to recognize which functional proteins are expressed in situ by
these Accumulibacter.
Undoubtedly, the big challenge now, as it was when the first edition of this book was written, is to try
to communicate with engineers to convince them that all this elegant science can be applied productively
to improve how they design and operate their activated sludge systems. This is no easy task, but a start has
been made to try to bring the engineering and microbiology together (Curtis et al., 2003; Daims et al.,
2006; Grady and Filipe, 2000; Iranpour et al., 2003; Keller et al., 2002; Rittmann, 2006a). Unless practical
value emerges from this microbiological research, then its benefit to the community in contributing to
water reuse will be unrealized.
We have decided to keep the structure of the book very similar to how it was in the first book. The
feedback received from this earlier version was that, in general colleagues found the ‘methodology’
section useful, bringing together in convenient ways information needed to study this microbial
community. So it has been extended to incorporate some of the newer techniques now applied to activated
Preface ix
sludge microbiology, allowing that several excellent methodological manuals are already available. As
then, we have also deliberately used recent authoritative reviews where appropriate for dealing with some
of the more general topics discussed in this book, hoping to save the reader time searching for papers.
Finally we would like to thank colleagues around the world with whom we have had discussions on
activated sludge, our postgraduate students who have stimulated interest in this project and all the
contributing authors, who have written such insightful chapters. Their patience in hanging on until this
book was finally finished deserves special mention. We must thank those colleagues (mainly again our
postgraduate students) who supplied many of the micrographs and other photos included here, and these
are acknowledged individually in the text. Special thanks go to Phil Hugenholtz for his phylogenetic tree,
Jiri Wanner for assisting with the account of the history of wastewater treatment, Mike Goodfellow for his
systematics advice and Rick Webb for providing many of the beautiful electron micrographs. Hilde
Lemmer and Valter Tandoi and their colleagues are also thanked for micrographs of the ‘new’ filaments
they have described. We must also thank Michael Dunn from IWA publishers who first encouraged a 2nd
edition, and to Ian Borthwick and David Burns and their predecessors at IWA Publishers who threatened,
encouraged and cajoled us into finishing this project. Finally, thanks to Anne Archer and Simon McIlroy
who checked and corrected all the chapters.
Let’s hope this book is a suitable reward for all their efforts. It was written to serve the professional
interests of an increasing number of microbiologists attracted to the challenge of delving into the
complexities of activated sludge. We also hope that it is as enjoyable as it is informative, and that it
reflects some of the sentiments expressed so elegantly by Woese (2004) above.
Bob Seviour
Per Nielsen
January 2009
Contributors
Jeremy J. Barr
Australian Wastewater Management Centre,
University of Queensland,
St Lucia, Queensland 4072, Australia
Philip L. Bond
Andrew E. Cook
Colin R. Curds
Research Associate,
Department of Zoology,
The Natural History Museum,
Cromwell Road,
London SW7 5DB, UK
Holger Daims
Lehrstuhl für Mikrobielle Ökologie,
Institut für Ökologie und Naturschutz (IECB),
Universität Wien, Althanstr.
14, A-1090 Wien, Austria
Toshikazu Fukushima
Department of Environmental Engineering,
National Cheng Kung University,
Taiwan 70101
Maneesha P. Ginige
CSIRO Division of Land and Water,
Perth, W.A., Australia
Shaomei He
Department of Civil and Environmental Engineering,
University of Wisconsin-Madison, Madison,
Wisconsin, 53706, USA
xii Microbial Ecology of Activated Sludge
Dae Wook Kang

Caroline Kragelund
Department of Biotechnology,
Chemistry and Environmental Engineering,
Aalborg University,
Sohngaardsholmsvej 49
DK-9000 Aalborg, Denmark
Jeremy Lenaerts
DakoCytomation Ltd.,
Denmark House, Angel Drove, Ely,
Cambridgeshire CB7 4ET, UK
Kenneth C. Lindrea
Biotechnology Research Centre,
La Trobe University,
Bendigo, Vic 3552, Australia
Wen-Tso Liu
University of Illinois,
Urbana, IL61801-2352, USA
Alexander Loy
Simon McIlroy
La Trobe University,
Bendigo, Vic 3552, Australia
Katherine McMahon
3204 Engineering Hall,
1415, Engineering Drive,
University of Wisconsin,
Madison, WI 53706, USA
Contributors xiii
Rikke L. Meyer
Department of Microbial Ecology,
Aarhus University,
Building 540,
Ny Munkegade,
8000 Aarhus C, Denmark
Gerard Muyzer
Environmental Biotechnology,
Delft University of Technology,
Julianalaan 67, NL-2628 BC Delft,
The Netherlands
Per H. Nielsen
Aalborg University,
Jeppe L. Nielsen
Aalborg University,
Daniel R. Noguera
Adrian Oehmen
Chemistry Department,
Faculty of Science and Technology,
New University of Lisbon,
2829-516 Caparica, Portugal
Kate Porter
Biota Holdings Ltd.,
585 Blackburn Road, Notting Hill,
Victoria, 3168, Australia
xiv Microbial Ecology of Activated Sludge
Jonathon Porter
National Laboratory Service,
Starcross Laboratory,
Staplake Mount,
Exeter EX4 4PS, UK
David McLean Roberts

Head of Protista & Mathematics Division,
The Natural History Museum,
Cromwell Road,
London SW7 5DB, UK
Humberto Salvadó
Animal Biology Unit,
Faculty of Biology,
University of Barcelona,
Av. Diagonal 645,
Barcelona-08028, Spain
Robert J. Seviour
La Trobe University, PO Box 199,
Bendigo, VIC 3552, Australia
Elizabeth M. Seviour
La Trobe University, PO Box 199,
Bendigo, VIC 3552, Australia
Michael W. Taylor
School of Biological Sciences,
University of Auckland,
Auckland, New Zealand
Michael Wagner
Contributors xv
Jiri Wanner
ICT Prague,
Department of Water Technology and Environmental Engineering,
Prague 6 16628,
Czech Republic
Alan Warren
Natural History Museum,
Cromwell Road,
London SW7 5BD, UK
E. Yildirim
Environmental Biotechnology Group,
Delft University of Technology,
Delft, Netherlands
L. Safak Yilmaz
1
An overview of the microbes in
activated sludge
Robert J. Seviour
INTRODUCTION
The need for wastewater treatment
Domestic sewage contains undigested food remnants still rich in an array of organic compounds, including a
diverse range of lipids, proteins and carbohydrates, and bacterial cells (Gray, 2005). So although possibly
carbon limited, sufficient N and P are present to be confident that biologically based treatment systems for
domestic sewage should be successful. The same will not always apply to industrial wastes (see below). Its
composition will of course vary with community water usage rates, dietary habits, culture and general life
style (Gray, 2005), and so any generalizations made here about what is present in a typical sewage are, in our
view, of limited value. In healthy people, the majority of bacteria present in faeces are probably harmless
commensals, but they are still excreted in very large numbers (more than 109 cells per gram of faeces).
However, people with gastrointestinal upsets or who are carriers of certain pathogenic organisms
(a surprisingly large proportion of our society) will also continually excrete disease causing organisms.
Wastewater treatment systems ultimately need to remove or reduce the numbers of many different
pathogenic enteroviruses known to be excreted in faeces, some of which are present in very high numbers
there (Wyn-Jones and Sellwood, 2001; Oragui, 2003; Percival et al., 2004). These include the viruses
causing hepatitis (de Serres et al., 1999), as well as rotaviruses, and enteric adenoviruses.
Pathogenic bacteria found in faeces, especially enterohaemorrhagic and other enteropathogenic
Escherichia coli strains, Salmonella and Shigella spp. (Percival et al., 2004), Campylobacter jejuni
2 Microbial Ecology of Activated Sludge
(Jones, 2001) and Vibrio cholerae, represent potentially fatal threats to our health (Danielson and Cooper,
2007). Cyst producing protozoa like Entamoeba histolytica (Theron and Cloete, 2002), Cryptosporidium
spp. (Rose et al., 2002; Carey et al., 2004; Fayer and Xiao, 2007) and Giardia lamblia (Gerardi and
Zimmerman, 2005) have become increasingly of concern, while intestinal helminth parasites including
tapeworms, flatworms and roundworms may also be present (Stott, 2003). Pathogenic fungi including
Aspergillus fumigatus (Brakhage and Langfelder, 2002), and Candida spp. have also been detected.
Because of how we use our water, the health problems resulting from a single incident involving any
water borne pathogen inevitably have catastrophic outcomes, simultaneously affecting often thousands of
people. Our increasing reliance in some countries on treated wastewater as a source of potable water
makes their prior eradication even more essential (Curtis, 2003; Percival et al., 2004; Gerardi and
Zimmerman, 2005). Fortunately for many pathogens including some viruses, we now have rapid, sensitive
and specific molecular based methods for their detection, and in some cases quantification, in treated
water (Leclerc et al., 2001; Lemarchand et al., 2004; Percival et al., 2004).
Antibiotic residues (Yang and Carlson, 2004) are readily detected in wastewater (Watkinson et al.,
2007) and antibiotic resistant bacterial strains (Dröge et al., 2000; Furuya and Lowy, 2006; Auerbach
et al., 2007) are frequently isolated from treatment plants and effluents in countries where they have been
sought (e.g. Börjesson et al., 2009), further emphasizing the potential risk these systems represent to our
health (Reinthaler et al., 2003; Novais et al., 2005; Ferreira de Silva et al., 2006; Martins da Costa et al.,
2006; Schlüter et al., 2007).
Aerosol production from the forced aeration of activated sludge is also a public health concern (Brandi
et al., 2000; Carducci et al., 2000; Bauer et al., 2002; Fracchia et al., 2006; Pillai and Ricke, 2002), since
these aerosols may contain viruses, and pathogenic bacteria and fungi (Orsini et al., 2002). Surface
mechanical aerators appear to represent a higher risk than submerged diffusers (Brandi et al., 2000). Those
who work in treatment plants should be made aware of these hazards (Thorn and Beijer, 2004), and in
some countries like the U.K., there are now occupational health and safety guidelines explaining the risks
in working with sewage (‘‘sewage workers’ syndrome’’ including gastroenteritis, fever and general
malaise). Requirements for all who work with it are to carry O.H. and S. information cards, and be
vaccinated against viral infections like hepatitis A and B (Gray, 1999). The debilitating Whipple’s disease
caused by the actinobacterial Trophyrema whipplei is also common among those who work with sewage
(Schöniger-Hekele et al., 2007).
The concentration of faeces in sewage, and hence its microbial content is not a constant, but changes
continuously with time, on a daily, weekly and seasonal basis, reflecting both weather conditions and
patterns of human activity. This material has to be efficiently disposed of in such a way that the spread of
all these pathogenic microbes, which are mainly water borne, is limited, and epidemics of the diseases
they might cause thus avoided (Theron and Cloete, 2002; Gerardi, 2005). However, probably no
wastewater treatment plant has ever been constructed where this has been the deliberate primary design
aim, and so the plant effluent is eventually disinfected usually with chlorine, U.V. irradiation or ozone to
help ensure its microbiological safety. This requirement can never be guaranteed, with cyst-producing
pathogenic protozoa like Cryptosporidium surviving both the treatment process and subsequent effluent
disinfection (Chauret et al., 1999; Robertson et al., 2000, 2006).
Equally importantly, sewage needs to be treated so that the levels of both the organic and inorganic
nutrients it contains are substantially reduced. Only then can it be safely diluted by unloading into another
receiving body of water like a river or the sea without destroying the so-called self purification properties
of that water body. Untreated sewage will induce serious and often irreversible changes in its ecology, and
this practice is now strictly forbidden in developed urbanized countries. Organic compounds will serve as
substrates for the growth of the bacteria present, leading to oxygen utilisation and eventual depletion, and
An overview of the microbes in activated sludge 3
subsequent death of the animal and plant life. So water is routinely analysed for its levels of organic
matter. Enrichment with nitrogen and particularly phosphorus stimulate the growth of green algae and
cyanobacteria, often accompanied by toxin production (Burkholder, 2002; Falconer, 2002; Sangolkar et al.,
2007), and when these die, eventual stagnation of the water. This process of eutrophication (Philips, 2002)
is now a serious problem in many countries around the world, and microbiological methods for its
prevention figure prominently in this book (chapters 2, 9, 10).
A brief history of wastewater treatment

Only very recently has our pollution of the environment in this way occurred on a scale sufficient to cause
such concern. However, wherever human beings have lived together in large numbers, they have
intuitively understood the need to dispose of their wastes. Sewage collection systems are not new.
Historical records show that they have existed for thousands of years, with the Babylonians and Assyrians,
and later the Romans all constructing quite advanced processes (Hughes, 1977; Hughes and Stafford,
1976), as anyone who has walked among the ruins of Rome and the Roman forts in Hadrian’s Wall in the
north of England can testify. Unfortunately, medieval communities were not so environmentally aware or
health conscious, and in most European communities living conditions and health standards became
appalling, with many people dying prematurely of infectious diseases. Sewers (derived from the word
‘seaward’), consisting of nothing more than open ditches that had been constructed by the middle ages
in many cities including London, took wastes from houses towards the River Thames and eventually
out to sea.
Unfortunately, when these inevitably became blocked, their contents spilled into the streets and houses.
Since it was not accompanied by the provision of any adequate disposal facilities for the labour force,
rapid urbanisation which occurred in England during the 19th century as a result of the Industrial
Revolution exacerbated this situation and eventually political intervention and action was necessary.
Overbearing odour problems from the cess pits constructed under houses made living intolerable (e.g. the
big stink in London in 1858), and in attempts to protect themselves from the odiferous ‘night air’, many
people died in their beds, probably from H2S poisoning. Rivers, which received most of the untreated
sewage, became deadly dispersal systems for faecal pathogenic organisms. No one was safe, and
thousands of people died of diseases like typhoid (Salmonella), and cholera (Vibrio) in the 1840/50s from
drinking faecally contaminated water, eventually forcing the authorities to take action.
In 1848 a national Public Health act was passed in England, requiring that each house installed some
means of treating its sewage, and in 1855 the London Metropolitan Board of Works was formed, with
the responsibility for rectifying these problems. After several other legislative attempts had been made
to improve the safety of river drinking water, the Rivers Pollution Prevention Act was passed into law
in 1876. Unfortunately, it had little effect in preventing further river pollution, but led to a later Royal
Commission on Sewage Disposal in 1898, whose purpose was to coordinate evaluation of new
treatment processes. Its major and long lasting contribution was the stipulation of standards for treated
water, and in 1908, it recommended that all treated sewage should meet a 30:20 standard (for BOD5
and suspended solids (SS) respectively (see chapter 2 for definitions). This standard was adopted in
1912 in the UK, together with the recommendation that plants should achieve ‘full nitrification’, and is
still widely used to indicate satisfactory plant performance. It also undoubtedly challenged engineers to
design and build treatment systems capable of delivering this effluent quality.
A range of simple small scale biological treatment systems including cess pits, septic tanks, land
effluent percolation (sewage farms) and trickling filters all operating on a small scale, had been
introduced by then. All these early systems, constructed to cope with small populations, and dealing only
with domestic wastes were very slow and inefficient. They were clearly not suitable for treating the huge
volumes of wastes efficiently and rapidly in a fairly small space, created by modern societies from both
domestic and industrial sources. The treatment systems were based on the by then familiar observation
that polluted water left long enough in contact with the air would eventually clarify (Hughes, 1977).
Although empirically designed and constructed, they, and in fact all modern systems, operate on the same
underlying principle, that the microbes present will metabolise the organic and inorganic compounds in
the waste water as nutrients to support their growth. These substrates are converted to more microbial
cells, which can then be collected and removed from the liquid phase of the sewage. Carbon dioxide
(CO2) the final product of aerobic biological oxidation is also produced, which disperses to the
atmosphere.
THE ACTIVATED SLUDGE PROCESS

The activated sludge process was conceived and developed in 1914. It grew from a visit in 1912 by
Dr Gilbert Fowler, employed at the University of Manchester and representing Manchester Corporation to
the Lawrence Experimental Station in Massachusetts, to assess novel sewage treatment processes,
including those involving aeration. Back home he passed on his experiences to Edward Ardern and
William Lockett, both at the Manchester-Davey Hulme treatment plant (Lindrea and Seviour, 2002).
They quickly understood the importance of the American research, and were inspired to perform small
scale trials during 1913 and 1914, which led to the development of the activated sludge process. Their
original brilliant but simple contribution was to retain the sedimenting biomass generated during the
treatment process and reuse it to inoculate further fresh untreated waste. Standard practice until then was
to discard this ‘sludge’ and start again. By adopting such a strategy they demonstrated that the time
required to produce a 30:20 quality effluent and achieve ‘full nitrification’ was markedly less than before
(Lindrea and Seviour, 2002). They referred to the harvested biomass as ‘activated sludge’, and published
their findings in a series of papers (Ardern and Lockett, 1914a,b), which must be rated among the most
important ever in environmental sanitary engineering. A pilot plant was established in 1914 to test this
process under both continuous flow and fill-and draw configurations (chapter 2), and the outcomes were
so promising that a full scale continuous flow plant, the first activated sludge plant in the world, was
constructed in the city of Worcester in the English Midlands. It was fitted with a mechanical aeration
system, since trials suggested submerged aerators were unsuitable because of their propensity to clog
rapidly. The same aerator has survived and still operates at the plant in Stockport, England. Very quickly
this activated sludge process with so many advantages over all its competitors was adopted throughout
Europe and the British Commonwealth, and there are probably now well in excess of 70,000 plants in
operation around the world.
Since its development almost 100 years ago, the activated sludge process has undergone and continues
to experience many changes in its operational features and design to improve both its efficiency and
flexibility. Some of these will be discussed in this book. Conventional systems were originally designed
to remove carbonaceous organic compounds and ammonia which is toxic to fish, but in some parts
of the world, most plants are now constructed which attempt to remove other nitrogen and
phosphorus containing compounds microbiologically, and these are discussed in detail later in this book
(chapters 2, 9, 10).
Operationally, efficient treatment of nutrient rich domestic sewage by conventional systems is
relatively simple, and a flow diagram showing the general features of an activated sludge plant is given in
Figure 1.1. These are engineered ecosystems, where a biomass, consisting of microbes (mainly bacteria
and protozoa) and other solids, and organised into discrete 3-D entities called flocs (chapter 3) is
maintained in a constant state of suspension in an aeration basin, either by some agitation system, often
surface aerators, or through the mixing action of bubbles rising from diffusers on the basin floor
(Grady et al., 1999). This ensures the bacteria are held in intimate contact with nutrients in the bulk liquid.
These nutrients are rapidly oxidised or respired in the presence of oxygen (O2), which is continuously
replenished by the aeration system. Cells therefore grow and increase in numbers, while organic substrates
are degraded to CO2.
Influent Effluent
2° CLARIFIER
AER
Waste
sludge
's' - recycle
Fully aerobic (conventional) process
Figure 1.1. Flow diagram for conventional activated sludge plant. AER ¼ aerated zone; ‘s-recycle’ ¼ return
activated sludge.
Much of the material entering the basin is in the form of insoluble particles, which become
incorporated into the floc, where they are then degraded by exocellular enzymes, albeit at a reduced rate,
during this aeration period (Wanner, 1994a,c). It is generally assumed that most of the organic
compounds, particularly naturally occurring ones, will be readily metabolised by the microbes present,
although as mentioned above, some xenobiotics are recalcitrant and may not be degraded rapidly enough
in these conventional systems (Byrns, 2001). The technology of aerobic granulation (chapter 2)
providing very high reactor biomass concentrations to deal with such situations still needs to show it can
handle adequately industrial wastes at full scale, but such systems are operating or under construction
in Europe.
Presented with new compounds, most microbes require a period of acclimatisation or adaptation
before they can be degraded. Therefore, whether or not a compound is oxidised in the aerobic basin
depends on whether microbes capable of its degradation are present, and, very importantly, maintained
within the system (Hamer, 1987, 1997). Survival will rely upon several competitive attributes of an
organism, including its growth rate and probably more importantly, its ability to participate in floc
formation and cope with the inevitable changes in nutrient availability, as discussed later in this book
(chapter 3). Eventually some cells will die and lyse (probably mediated often by bacteriophage attack),
releasing their cell contents, some of which will also be metabolised by the biomass, a phenomenon
referred to as cryptic growth (Hamer, 1984). Therefore sufficient time must be allowed for the biomass to
metabolise the majority of the biodegradable compounds present, especially the particulate material in the
flocs (a process called sludge reactivation), which in turn will depend upon how much solid material is
entering the plant (its sludge loading) and the capacity of the aeration basin to cope with the load (Gray,
1989, 1990; Grady et al.,1999).
Finally, gravity clarification, where the sludge separates from the liquid phase, is allowed to occur,
either in the aeration basin itself, as in a fill and draw or sequencing batch reactor system (chapter 2), or
more commonly, in a separate clarifier for continuous flow systems. Problems associated with effective
solids separation occupy considerable space in this book because many have a microbiological basis, and
the conception and design of membrane bioreactors (chapter 2) has been in large part an attempt to
overcome them. The settled biomass, now enriched with those microbes, selected as being the best able
to degrade the nutrients entering the plant, is then recycled as return activated sludge (RAS) and used to
reinoculate the incoming raw sewage. Some of this biomass or sludge is also wasted at regular intervals,
the frequency of which can be manipulated to control the so called sludge age (defined in chapter 2) of
the system. This is the term used to describe the average length of time the biomass resides within the
aeration basin (or its mean cell residence time, MCRT).
In most conventional activated sludge systems, other important communities of microbes, capable of
oxidising inorganic nitrogenous compounds, may be present, as long as there is sufficient dissolved
oxygen to support their growth, and the MCRT is long enough to enable these slow growing bacteria to
survive. Such populations oxidise reduced inorganic compounds like ammonia to nitrite (NO2 ) and
nitrate (NO3 ) and the diversity and physiology of these nitrifying bacteria is discussed in more detail in
chapter 9. In the absence of O2, other microbes, the so called denitrifying bacteria (chapter 9) then
reduce this NO3 hopefully eventually to environmentally harmless dinitrogen (N2) gas, which disperses
into the atmosphere.
In activated sludge systems especially designed for removal of nitrogen containing compounds, the
systems are deliberately engineered so these nitrifying and denitrifying bacteria can grow and work in
unison. The proliferation of bacteria capable of accumulating and storing phosphorus intracellularly as
polyphosphate (poly P) is also deliberately promoted in phosphorus removing plants, designed to provide
them with a clear competitive advantage and become dominant there (Blackall et al., 2002; Seviour and
McIlroy, 2008; Seviour et al., 2003; Oehmen et al., 2007). Biological nutrient removal systems and their
microbiology are described in detail in chapters 2, 9 and 10.
GENERAL FEATURES AND CHARACTERISTICS OF MICROBES

RELEVANT TO ACTIVATED SLUDGE SYSTEMS
Before the operation of activated sludge systems is discussed in more depth later (chapter 2), some
general principles of microbiology need to be outlined, since it is the microbes which bring about
the chemical changes occurring in these plants, and whose role is so important to plant operation.
This book, like the first edition, was written to be read by both microbiologists and engineers,
professionals whose spheres of interest and activity frequently and inevitably overlap (Wilderer et al.,
2002; Keller et al., 2002; Rittmann, 2006a; Verstraete, 2007). Yet rarely do they communicate
effectively with each other, and even more rarely are they educated in ways to encourage them in this
vital activity (Daims et al., 2006c; McMahon et al., 2007a). Part of the problem is an inability to
understand the jargon each uses, or for microbiologists to think in mathematical/engineering terms.
So familiarization with just a few basic microbiological principles may help both. In the next
few pages, attempts will be made to introduce some of the basic concepts of biochemistry, and
microbiology, especially as they pertain to the rest of our discussions on activated sludge systems, and
hopefully presented in a manner understandable to non-microbiologists. Of course it is even more
difficult a task to cover the material adequately in such a small space than it was for the earlier version
of this book, as the pace of acquiring new information accelerates. So there is always the risk
of trivializing. We hope we avoid that trap, but list some of the better modern general textbooks on
microbiology at the end of this chapter, each of which in our view will fill in the inevitable gaps left
by our superficial coverage.
WHAT IS MICROBIOLOGY?
Trying to define what microbiology is seems a good place to start. Microbiology is the study of organisms
called microbes, which are so small (50.1 mm diameter) that they can not be seen with the naked eye
(although some microbes produce as part of their life cycles macroscopic structures, like several algae and
the mushrooms and toadstools formed by some fungi). Therefore we need to use a microscope, and until
microscopes were constructed in the mid-17th century, we had no idea that these organisms existed,
although they had long been harming us and crudely exploited by us for our benefit. Microbes are like the
curate’s egg. One very popular misconception is that all are dangerous and to be avoided if at all possible.
Some certainly are, even some of those we once thought were harmless. Yet the vast majority are of such
benefit that we depend on them for our survival. After all, we are made up substantially of bacterial cells.
Our genomes contain genes of bacterial origin, we carry large numbers on our skin and mucous
membranes, where they serve to protect us from attack by more harmful microbes, and each of our guts
contains massive numbers (about 500g worth), performing vital functions including for example,
supplying us with essential vitamins and amino acids we can not synthesise ourselves. They form
beneficial interactions with many other organisms, which in some cases help us grow our crops and the
animals we eat, and produce metabolites like antibiotics, which make all of our lives safer, longer and
more enjoyable.
Most we know little or nothing about, and it seems that more microbes live below ground in oceans and
subterranean habitats than on or above it (Whitman et al., 1998). It is generally agreed that we have
probably successfully grown in the laboratory 51% of all the microbes existing on earth (Amann, 2000;
Amann et al., 1995; Green and Keller, 2006; Hugenholtz et al., 1998a,b; Rappé and Giovannoni, 2003;
Schleifer, 2004; Tyson and Banfield, 2005). Those we do have information on reveal an incredible level of
biodiversity and phylogeny. Microbes are found everywhere on this planet where water is available
(Schlegel and Jannasch, 2006), and their abilities to live and flourish in extreme habitats deadly to all
other cellular organisms, reflect remarkable molecular and physiological adaptations to the prevailing
conditions (Vorgias and Antraikian, 2004). They also carry out most of the chemical transformations
taking place on this planet (Bull, 2004), and so keep the carbon, nitrogen, phosphorus, sulphur and other
elemental cycles working. Thus, although not visible to us, we ignore them at our peril. A very readable
account of just how important these organisms are is provided by Postgate (2002) in his excellent book
written primarily for the non-microbiologist.
HOW DO WE STUDY MICROBES?

Microbiologists routinely use several techniques to help them understand what microbes look like and
what they do. Microscopes visualise them. The light microscope provides only limited information, but
most of the studies, for example with the filamentous bacteria occurring in activated sludge, routinely use
this instrument for their initial characterization. It helps us determine their cell shape, arrangement and
size, none of which show much variation among bacteria, and so are of little assistance in helping us
identify them. Instead, it is their physiological and phylogenetic diversity which never cease to amaze.
With protozoa, algae and fungi, light microscopy is much more useful, since their morphological
characters are far more diverse and taxonomically relevant. Selected staining reactions, to detect cell
associated structures can in some bacteria provide additional valuable information on their likely identity.
Some of these microscopic methods, because they are so widely applied to activated sludge samples, are
described later (chapter 11).
It took the advent of the electron microscope before detailed information of the ultrastructure of cells
became available. The electron microscope has much greater resolving power than the light microscope,
and so fine details of cell anatomy and morphology were revealed for the first time. The transmission
electron microscope (TEM) views very thin sections and provides intracellular detail, while the scanning
electron microscope (SEM) is appropriate for examination of surfaces. These techniques are also
discussed later (chapter 11). Unquestionably, as the techniques of microscopy have continued to develop,
so have our abilities to learn more about the activated sludge community. In particular, fluorescence
microscopy coupled with confocal laser scanning microscopy and image analysis (e.g. Lawrence et al.,
2002; Lawrence and Neu, 2007; Daims et al., 2006a) provides now an opportunity for us to acquire
quantitative information on individual microbial populations not readily obtainable otherwise.
Because microbes are usually so small, it is very difficult for microbiologists to learn much from
studying individual cells (¼ individual organisms for most microbes), although techniques to assist in this
are becoming available (Brehm-Stecher and Johnson, 2004; Nielsen and Nielsen, 2005). Generally, they
study populations, consisting of millions of genetically identical individual cells. These populations, or
clones, have traditionally always been studied as pure or axenic cultures (a basic requirement stressed in
the education and training of microbiologists). So they have to be isolated first on to a medium which
supports their growth, and then cultured and maintained unchanged in pure culture (Overmann, 2006).
These steps are followed regardless of the kind of microbe under study (Fry, 2004).
Yet microbes rarely if ever grow in nature as pure cultures, and so this philosophy is not appropriate to
studying natural communities like activated sludge. With the advent of molecular methods for identifying
microbes, and with no a priori need to grow them first (Amann et al., 1995; Smalla, 2004), many
microbiologists thought culturing these organisms from nature was no longer relevant, and that these
molecular tools alone could provide all the information needed to understand microbial ecology. This is
no longer such a widely held view. There are still important scientific reasons for their culture (Janssen,
2006; Leadbetter, 2003; Palleroni, 1997; Zengler, 2008) if we wish to more fully understand microbial
diversity from all perspectives. This point is clearly demonstrated in the present context by how much
better we understand the relationships among the bulking and foaming filamentous bacterial morphotypes
in activated sludge as a result of their successful culture (Kämpfer and Wagner, 2002).
Nevertheless, we should not understate the contribution the ‘culture independent’ approach has made to
understanding activated sludge microbiology, which as this book will make very clear, is immense
and unequivocal. Obtaining more natural isolates from habitats like activated sludge represents real
challenges to microbial ecologists, and novel isolation and culturing methods will be needed. For
example, the method described by Zengler et al. (2002) for recovering individual cells encapsulated in gel
microdroplets, may be applicable to these communities. However, the solutions to acquiring cultured
isolates may be simpler. Notable success has been achieved by Janssen and colleagues by mimicking the
conditions encountered in the natural habitat. Using a combination of very dilute artificial media, longer
incubation times and slowly metabolized substrates, his group have successfully grown isolates from
soil of the Verrucomicrobia and Acidobacteria phyla, organisms considered by many as unculturable
(Janssen, 2006; Janssen et al., 2002; Sait et al., 2002), and some halophilic Archaea (Burns et al., 2004).
A similar approach was employed by Zhang et al. (2003b) to obtain pure cultures of Gemmatimonas
aurantiaca, the first cultured representative of the Gemmatimonadetes phylum. Ghosh et al. (2006), in
their attempts to isolate the polyphosphate accumulating organism or PAO Candidatus ‘Accumuli-
bacter phosphatis in enrichment cultures where the selective pressures thought to favour organisms
phylogenetically related to these were imposed, ended up growing another bacterium Stenotrophomonas,
also exhibiting the behaviour of PAO (see chapter 10). Evidence from molecular analyses in fact
may assist in developing better isolation strategies for bacteria (Leadbetter, 2003), although as this book
shows, the outcomes from applying this principle to activated sludge communities have not always been
encouraging.
WHAT ARE THESE MICROBES?

Some cell biology
The term, microbe, has no strict taxonomic meaning, because it is used to describe a large and diverse
collection of phylogenetically different organisms. Although we know that some bacteria are extremely
large (Schulz and Jørgensen, 2001), most are microscopic, and commonly exist as unicellular organisms.
Most biologists now accept that three levels of organisation exist in the biological world, based upon the
structures of their cells, the fundamental units of living organisms, and these will be briefly described now
after briefly considering the subcellular viruses.
Subcellular organisation: the viruses

Viruses are submicroscopic and non-cellular particles, being visible only with the electron microscope.
They consist of a protein coat called a capsid together with other chemicals and are surrounded by a
capsule in some viruses. The capsid is designed to protect their genetic material, the genome which in
viruses is either Deoxyribonucleic acid (DNA) or Ribonucleic acid (RNA), but never both, as is seen
in the truly cellular organisms described next. These viruses alternate between metabolically inert
infectious particles called virions, and entities inside a suitable host cell where they replicate. In the
replication process, the virus often completely takes over the host cell’s metabolism, which becomes
directed towards producing multiple copies of the virus, and so eventually the host cell is killed and the
viral progeny released to infect further susceptible cells. Viruses have been described which infect
bacteria, fungi, protozoa, algae and higher plants and animals. Some cause diseases in humans. These
can be quite trivial while others are life threatening. Viral infections are generally unreceptive to most
chemotherapy and unaffected by antimicrobial antibiotics, because they lack the cellular structures and
processes these target.
Viruses can also act as agents of genetic change, which in some cases results from the viral genome
upon infection becoming integrated into the host cell genome and remaining dormant there. This is seen
with many double stranded DNA bacterial viruses (bacteriophages or phages), the so called temperate
phages. Some animal viruses, like the retroviruses can behave in a similar way and these may eventually
induce tumor formation frequently enough to make us think now that about 20% of human cancers are
caused by them. Phages can also act as gene vectors, carrying genes from one cell to another by the
process of transduction (see below). Viruses come in a diverse range of shapes and sizes (Fauquet et al.,
2005), and many (e.g. Figure 1.2) can be detected in activated sludge (chapter 3). Phages probably play
an important role in controlling bacterial population densities there, and in our view should be examined
further for possible exploitation in controlling some of the microbiological problems these systems suffer
from, as discussed later in this book.
Cellular organisms
Cellular microbes are characterised by the possession of both DNA and RNA, are not usually dependent
upon another cell for their replication (although some like the Rickettsias and Chlamydiae are mostly
obligate parasites inside animal cells), and are surrounded by a functionally active (usually) double
layered cytoplasmic membrane.
Biologists had long struggled to place these cellular microbes in the biological kingdom (Rosella-
Mora and Amann, 2001), because of difficulties associated with the characters available to them to
distinguish between a shared evolutionary ancestry and recognizing where convergent evolution may have
been responsible for present apparent similarities in these attributes. Chatton (1938) first used the terms
Figure 1.2. T-even bacteriophages isolated from activated sludge (negatively stained TEMicrographs)
showing the icosahedral head (H), tail (T) and tail fibres (F) (J. Thomas). Bar mark ¼ 100 nm.
‘eukaryote’ and ‘prokaryote’ for distinguishing between cells possessing a nucleus and those with
none, and a fascinating account of the history of the ‘prokaryotic’/eukaryotic dichotomy is given by
Sapp (2005). The assumption was that these cell types each represented a group of organisms with a
single phylogenetic lineage, an interpretation consolidated by the publication of Stanier and van Niel
(1962) describing their ‘concept of a bacterium’. Woese (2004) strongly criticises this paper, stating that
they had never properly tested the ‘prokaryote’/eukaryote dichotomy, and that the term ‘prokaryote’ was
never meant to be used in the way it was by them (Sapp, 2005). He argues that microbiologists were
distracted into thinking that was the end of the story, and a belief that nothing more was to be learnt about
phylogenetic relationships between bacteria. Woese suggests that the term ‘prokaryote’ should have
no phylogenetic relevance, but be used exclusively to describe a cellular organization which is
non-eukaryotic (Woese, 2004; 2006). We agree. For the next decade and beyond, the most profound
evolutionary gap in the biological world was viewed by biologists as that existing between ‘prokaryotic’
and eukaryotic cells. Woese’s contributions to our current understanding of microbial phylogeny are
remarkable and revolutionary, and can not be overstated. Despite some stern opposition still from
eminent microbiologists (Whitman, 2009), his views have generally, if not universally, prevailed, and he
has completely changed how we believe these organisms relate to each other. He used ribosomal
RNA (rRNA) sequence comparisons as molecular clocks to explore non-eukaryotic cell phylogeny,
showed that all ‘prokaryotes’ were not the same, and had more than a single evolutionary past. His work
extended earlier observations that the chemical composition and organization of the cell walls and
cytoplasmic membranes among bacteria differed so markedly, as detailed below, that they probably were
phylogenetically diverse. The ‘bench mark’ molecular clock currently adopted is 16S rRNA, and analyses
of its sequence and the 18S rRNA eukaryotic equivalent, has enabled us to construct a tree of life
(Yarza et al., 2008). One example for non eukaryotes is presented in Figure 1.3, see colour image section
(in chapter 13). It has also provided us with the tools to explore the true level of diversity of microbes in
natural communities like activated sludge, with equally original and exciting outcomes, as hopefully this
book will demonstrate.
This 16S rRNA molecule has a number of attractive features as an indicator of evolutionary ancestry
for bacteria (Rossella-Mora and Amann, 2001; Ludwig and Klenk, 2005; Yarza et al., 2008). These
include:
. Its presence in all non-eukaryotic organisms.

. It is selectively neutral and has a conserved function in cells, being required for formation of
ribosomes, structures essential for protein synthesis.
. It possesses a large information content of about 1500 nucleotides, containing both conserved and
variable regions.
. This property allows phylogenies covering a broad range of relationships from individual species
to Domain level to be elucidate.
. The gene encoding it is easily sequenced.
. Large and growing publicly available data bases of 16S rRNA gene sequences (4400,000) are
freely available (Schloss and Handelsman, 2004; DeSantis et al., 2006), although these contain
many chimeric sequence data and so are not without their problems (Ashelford et al., 2005, 2006;
Yarza et al., 2008).
However, this gene is not beyond criticism as a molecular clock, and so several other genes and
proteins molecules have also been used in attempts to understand evolutionary relationships among
‘prokaryotic’ cells, as discussed in chapter 6. These criticisms include (Yarza et al., 2008):
. An individual cell may contain more than one copy of the 16S rRNA gene (and most do), each
possibly with small differences in their sequences.
. Only a single gene and not the whole genome of the organism is compared (we will discuss this
point later in chapter 6).
. There is now evidence that this gene in some organisms can move from one population to another
of a different taxon by the process of lateral or horizontal gene transfer (see later), so unrelated
cells may acquire it.
. This molecule is highly conserved, and so it is difficult to interpret the taxonomic significance
of two organisms having high (498%) similarity between their respective 16S rRNA gene
sequences, whether they are members of the same or different species, especially in organisms
with fast rates of evolutionary change.
. It is not possible to correlate the changes in its sequence to an evolutionary time scale.
Woese (reviewed in 1987) proposed from the 16S rRNA sequence data that ‘prokaryotic’ organisms
should be placed into two distinct separate Domains (Figure 1.3, see colour image section chapter 13),
now known as the Bacteria and Archaea [probably etymologically incorrect, and should be replaced
(Trüper, 2005b) by Archaeobacteria], with the third being the Eukarya. He argued that the Bacteria and
Archaea differ from each other in fundamental ways as much as either does from eukaryotic cells.
Furthermore, as we shall see in this chapter, where we explore the major features of these organisms, each
of the Bacteria and the Archaea are in some ways more similar to the Eukarya than they are to each other,
which raises several interesting evolutionary questions. There are still many eminent cell biologists who
believe in the ‘prokaryote’/eukaryote dichotomy. They argue that there is no justification for dividing
organisms with a ‘prokaryote’ cell organization into the two Domains of Bacteria and Archaea because
they differ from each other no more than animal and plant cells differ (e.g. Mayr, 1998). This is an
argument Woese (1998) and others (e.g. Pace, 2009a,b) dismiss by suggesting that we need to look at
genes not phenotypic characters before we make these judgements. Not all cell biologists agree, and
severely criticise his disregard for comparative cell biology (Margulis et al., 2000; Cavalier-Smith,
2002c). We have no space here to discuss this issue in more detail, because we now need to see what it is
that separates organisms in these three domains from each other.
CELLS WITH A NON-EUKARYOTIC ORGANIZATION

‘Prokaryotic’ cells are unified principally by structures they lack, and embrace members of the Domains
Bacteria and Archaea. Lacking a true nucleus and nuclear membrane, and other separate intracellular
membrane bound structures or organelles distinguish their ultra structural organization from eukaryotic
cells (described next). The location of their DNA or genomic material is a region called the nucleoid
(Figure 1.4). Of course there are always exceptions, and the Planctomycetes, unusual members of the
Bacteria, have their genome enclosed within a membrane (Fuerst, 2005). Otherwise the only biological
membrane ‘prokaryotes’ possess is their cytoplasmic membrane. All the intracellular membranes we see
in electron micrographs of some Bacteria like the Cyanobacteria, purple and green bacteria, methane
oxidizing bacteria and nitrifying bacteria (see later) represent foldings, extensions and invaginations
of this cytoplasmic membrane (Figure 1.5), increasing membrane surface area usually for the purpose
of maximizing energy (ATP) generation (see later). However, appearances may be deceptive, because
despite their apparent structural simplicity, ‘prokaryotic’ cells are highly organized, efficient and
successful, and able to adapt rapidly to changing environmental conditions.
Their morphological diversity is fairly minimal, being restricted to a very small range of cell shapes
like rods, cocci, spirals, budding and appendaged cells and filaments, and even squares (Walsby, 2005),
and many can change shape or adopt several. It is one reason why we struggled in the past to resolve their
natural interrelationships, where in the absence of other useful attributes, the assumption was made that
cells sharing the same morphology (microscopic look-alikes) were probably phylogenetically related
(e.g. cocci). We now know that bacterial morphology like physiology may reflect their phylogeny (Siefert
and Fox, 1998) but not always. This is an issue which still impacts on activated sludge microbiologists,
who struggle to ‘identify’ the filamentous bacteria causing bulking and foaming on their morphological
features (chapter 6).
Figure 1.4a. TEMicrograph of Bacillus sp., showing lack of nuclear membrane and DNA nucleoid (R.Webb).
Bar mark ¼ 250 nm.
Figure 1.4b. TEMicrograph of cell envelope of the Gram negative bacterium E. coli. (R. Webb). Bar
mark ¼ 50 nm.
Figure 1.4c. TEMicrograph of the cell wall of the Gram positive bacterium Bacillus sp. (R. Webb). Bar
mark ¼ 100nm.
The remarkable phenotypic diversity among ‘prokaryotes’ is expressed primarily in terms of their
physiology and biochemistry, and the unusual mechanisms they have for obtaining energy for growth.
These abilities only emphasise their importance in the utilization and degradation of chemically diverse
waste materials in processes like activated sludge, and their abilities to cope with changing conditions.
The bacteria show relatively little evidence of any morphogenetic differentiation leading to formation
of specialized cellular structures with specialized function, so prevalent among eukaryotic organisms.
Exceptions include endospores (Figure 1.6), remarkable survival structures synthesized by a few Bacteria,
providing them with the capacity to withstand extreme environmental conditions especially temperature,
at limits fatal to all other cellular organisms. Some Cyanobacteria form structures called heterocysts, sites
for diazotrophy where atmospheric nitrogen gas is fixed into organic nitrogenous compounds, and many
Actinobacteria differentiate to produce aerial spores or conidia, structurally quite unlike the endospores, to
aid in their dispersal through space.
So we need now to see how the non-eukaryotic cells of the Bacteria and Archaea are assembled and
how different they are to each other. These differences are summarized in Table 1.1.
Figure 1.5. TEMicrograph of a nitrifying bacterium showing intracellular membrane stacks (M), and surface
associated S-layer (S). (R. Webb). Bar mark ¼ 200 nm.
STRUCTURAL FEATURES OF ‘PROKARYOTIC’ CELLS

We believe from the 16S rRNA gene sequence data now available that nearly 100 different ancestral
lineages or phyla of the Bacteria exist (DeSantis et al., 2006), which is more than three times the number
we believed existed about a decade or so ago, and it is likely to increase (Figure 1.3). For more than half of
these we have yet to culture a single representative and most of those we have grown represent no more
than 4–5 phyla. However, the number of cultured and validly described bacterial species has almost
doubled during the past decade. If we believe that there may be more than 1 million species existing on
this planet, we still have a long way to go before we even start to understand how bacteria in all these
various forms might function, and what their roles in nature might be. How many of these phyla are
represented in activated sludge is equally uncertain, although as we shall see later, its community contains
some surprises.
HOW DO WE DIVIDE THESE GROUPS UP?

In the Bacteria, all the evidence supports the view that differentiation into two groups the Gram negative
and Gram positive Bacteria is vindicated since these clearly represent two natural groupings, reflecting
Figure 1.6. TEMicrographs of endospores of Bacillus sp (subterminal, ellipsoidal) and Clostridium (terminal,
spherical), showing complex multilayered nature of the spore coat (R. Webb). Bar mark ¼ 200 nm.
S ¼ endospore inside vejetative cell.
fundamental differences in their cell wall structure and organizations (see below), with the Gram negative
bacteria staining RED and the Gram positive bacteria PURPLE after the Gram stain (chapter 11).
The Gram positive bacteria appear to be phylogenetically more coherent than their Gram negative
counterparts, representing only two phyla, the low mol% GþC Mollicutes and Firmicutes and high mol%
GþC Actinobacteria. Although the Proteobacteria are the largest known group of Gram negative
Bacteria, many more distinct ‘Gram negative’ phyla exist (Figure 1.3).
The Archaea (Garrett and Klenk, 2007) contain the phyla (Figure 1.3);
(i) the Euryarcheota, including the obligately anaerobic methanogenic bacteria used for anaerobic
digestion of activated sludge to generate methane gas, and the aerobic halophilic bacteria, only
able to grow at extremely high NaCl concentrations
(ii) the Crenarcheota, once thought to contain only hyperthermophiles, organisms preferring to live
under extremely hot conditions, but now shown to be the most widely distributed members
of the Archaea, being detected in moderate or even very cold habitats (Schleper et al., 2005;
Robertson et al., 2005; Cavicchioli, 2006). These may be separated from each other in future
classifications.
Table 1.1. Distinguishing Between Cell organization in the Bacteria, Archaea and Eukarya (adapted from Krieg, 2005).
Feature Bacteria Archaea Eukarya
Genome surrounded by nuclear ve, except some ve þve
membrane Planctomycetales
Mitochondria ve ve usually þve
Chloroplasts in phototrophs ve ve usually þve
Golgi bodies present ve ve þve
Endoplasmic reticulum (ER) present ve ve þve
Lysosomes present ve ve þve in some
Vacuoles surrounded ve ve þve
by unit membranes
Gas vacuoles þve in some þve in some ve
Cytoskeleton present probably þve probably þve þve
Endocytosis and exocytosis ve ve þve
Cytoplasmic streaming ve ve þve
Peptidoglycan in cell wall þve in majority ve ve
Muramic acid and diaminopimelic þve in majority ve ve
acid synthesised
Sterols in cytoplasmic membranes ve in most ve in most þve
Use poly-b-hydroxybutyrate as þve for some ve ve
intracellular storage compound
Can fix atmospheric N2 þve for some þve for some ve
An overview of the microbes in activated sludge
Chemolithoautotrophy þve in some þve in some ve

Glycerol diethers ve þve ve
and tetraethers in membranes
Membrane linkage bond ester ether ester
Ribosome size 70S (16S, 23S, 5S) variable, often 470S 80S (18S, 28S, 5.85S, 5S)
except in organelles (70S)
Ribosome location dispersed in cytoplasm dispersed in cytoplasm attached to ER
(continued )
17
Table 1.1. Continued
18
Feature Bacteria Archaea Eukarya

Flagella submicroscopic, consisting submicroscopic microscopic and 9 £ 2þ2
of helically arranged subunits microtubule arrangement
of flagellin
Chromosome arrangement usually circular usually circular usually linear
Mitosis and Meiosis ve ve usually þve
First amino acid incorporated usually N-formylmethionine methionine methionine
during translation
m-RNA binding site at þve þve ve
AUCACCUCC
at 30 end of rRNA
t-RNA genes contain introns ve þve þve
Ribothymidine present in common þve ve ve
arm of t-RNA
Translation inhibited ve þve þve
by diphtheria toxin
DNA polymerases inhibited by þve ve ve
rifampicin
Protein synthesis inhibited by þve ve ve
kanamycin
and chloramphenicol
It now seems likely that this scheme does not reflect the total phylogenetic diversity existing among
the Archaea, since evidence is accumulating that some are so different to those above that they should
be placed in the Korarchaeota, for which no cultured members yet exist (Auchtung et al., 2006;
Dawson et al., 2006), and the Nanoarchaeta, currently with a single uncultured representative,
Nanoarchaeum equitans (Waters et al., 2003).
Bacteria and Archaea both have cells containing many unique chemical components, and their
distribution and occurrence often supports the 16S rRNA based phylogenetic evidence (Ludwig and
Klenk, 2005), but the processes these use to carry out most of the biochemical reactions required to
metabolize and grow are shared by all cells, and all use essentially the same genetic code for storing
information in their DNA (see later).
Outer cell layers

Some Bacteria possess an outer capsule or slime layer, mainly polysaccharide in nature, and it probably
protects cells against environmental harm or assists in their attachment to other cells as part of their
pathogenic lifestyles, as well as preventing our immune systems from dealing with them. Capsules are
not always present, but in some of the filamentous bacteria found in activated sludge including
Sphaerotilus natans, Eikelboom types 1701, 1851 and 0041/0675, this material is organized as a sheath
(chapters 11, 12), within which individual cells are located. In many Bacteria and Archaea an outer
crystalline protein or S-layer is present (Figure 1.5), although its function is not always clear.
The cell walls of Bacteria and Archaea

While a few lack a cell wall (the Mollicutes), most Bacteria have a rigid wall providing the cell with its
shape, and protection (Dmitriev et al., 2005). With the exception again of the Planctomycetes where the
wall is made of protein, this wall consists of many parallel polysaccharide chains made of two alternating
sugar derivatives, N-acetyl glucosamine and N-acetyl muramic acid (Figure 1.7), a sugar never detected
anywhere else in nature. These are joined together probably as a vertical scaffold-like layer, in a number
of different arrangements, by short (usually 4 in the tetrapeptide) chains of sometimes very unusual amino
acids attached to the N-acetyl muramic acid residues (Figure 1.7) to give a 3D structure called a
PEPTIDOGLYCAN or MUREIN layer. In some Bacteria, these sugars are not N-substituted, or have
N-glycolyl substitutions (Vollmer et al., 2008).
In Gram positive Bacteria the cell wall is mainly peptidoglycan in nature (Figure 1.4c), containing
unusual sugar phosphate polyols known as teichoic acids. A few also have complex fatty acids known as
mycolic acids which render their cells very hydrophobic, and we believe contribute towards their
involvement in the production and stabilization of foams in activated sludge plants, as discussed later
(chapter 8).
While the peptidoglycan occurs also in Gram negative Bacteria, it is a minor component, and no
teichoic acids and mycolic acids are detectable. Instead, their walls possess a chemically unique outer
membrane (Figure 1.4b) containing a lipopolysaccharide, which in turn contains a unique lipid called
LIPID A, and long polysaccharide chains, the core-polysaccharide and the more chemically diverse
somatic (O) or side-chain polysaccharides attached to it (Figure 1.8). In some cases these incorporate
sugars not seen anywhere else in nature. This Lipid A can behave as an endotoxin against mammalian
cells, including our own. Proteins called porins form channels in this membrane allowing direct passage
of small molecules through it. The space between the outer membrane and cytoplasmic membrane
1-4 linkage attacked by N-Acetylmuramic acid

lysozyme
H2C OH H2C OH
O O
β ,1-4
β,1-4
O O O
OH
NH NH
C O O C O
CH3 H3C
CH CH3
N-Acetylglucosamine (G) C O
NH
O
L-alanine
H3C CH C
NH
O CH2 HC O D-glutamic acid
The tetrapeptide chain

C CH2 C
NH2 HN OH
O meso-diaminopimelic
C HC CH2 CH O
acid (DAP)
HO CH2 CH2 C
HN
O D-alanine
H3C CH C
OH
Figure 1.7. The chemical structure of the peptidoglycan glycan tetrapeptide repeating unit in Bacteria (E.Quill).
Adapted from Seviour & Blackall (1999).
(see below) in Gram negative Bacteria is known as the periplasmic space, and is a region containing
many functionally important periplasmic proteins.
Although some lack cell walls, most Archaea have them, where they fulfil the same functions as those
in the Bacteria. None however, have an outer membrane or any peptidoglycan or murein material.
Their walls are more chemically diverse (Garrett and Klenk, 2007). A few of the methanogens have a
peptidoglycan-like cell wall, a pseudomurein, with a similar organization but radically different chemical
structure to the peptidoglycan. Others have walls made of polysaccharides or glycoproteins, and S-layer
proteins are found in all the main archaeal groups. Absence of any peptidoglycan means that the Archaea
are resistant to antibiotics affecting its biosynthesis, like the penicillins and cephalosporins.
External environment
O-polysaccharide
Protein Porin Lipid A
Lipopolysaccharide
Outer membrane
Phospholipid
Lipoprotein
Periplasm
Peptidoglycan
Protein
Phospholipid
Inner membrane
Internal environment
Figure 1.8. The chemical organization of the lipopolysaccharide outer membrane and peptidoglycan of Gram
negative bacterial cells (S. McIlroy).
The cytoplasmic membranes of Bacteria and Archaea

Immediately underlying the cell wall is the cytoplasmic membrane, which largely determines what
material enters and leaves the cell, acting as a selective permeability barrier. The processes whereby cells
transport materials into their cells are well understood and vary, but will not be dealt with here. This
membrane is made up of hydrophilic moieties chemically joined to a hydrophilic backbone, which is
glycerol. Its composition differs markedly between the Bacteria and the Archaea. In the Bacteria (and
Eukarya), the membrane is highly fluid and dynamic, and consists of a phospholipid bilayer, (seen as two
parallel electron dense lines with transmission electron microscopy), where the hydrophobic fatty acids
are attached to the glycerol by ester (C ¼ O-R-) linkages. Proteins involved with substrate transport or
energy generation are associated with this membrane and in many Bacteria, membrane stabilizing
compounds called hopanoids are also present.
In the Archaea, the cytoplasmic membranes are uniquely different in their chemical organization, since
the hydrophilic and hydrophobic components are linked by the much stronger ether (-C-O-C-) linkage,
probably necessary to survive under the harsh conditions where many live. The hydrophobic components
are chemically complex branched isoprenoid or phytanyl units, and while in some these are organized into
double layered arrangements called glycerol diethers (generating parallel electron dense images in the
TEM), others form diglycerol tetraethers producing a tougher single layered membrane. No hopanoids
are found in archaeal membranes.
Organization of DNA in Bacteria and Archaea

We know neither possesses a membrane bound nucleus. Instead the DNA or genomic material exists as a
single chromosome arranged in a circular closed double stranded form in most ‘prokaryotic’ cells called
a nucleoid (Figure 1.4). It consists of sequences of four chemicals called nucleotides or bases. The two strands
are held together by Adenine (A) in one strand pairing with Thymine (T) in the other, by two hydrogen bonds.
Similarly Guanine (G) pairs with Cytosine (C), but now by three hydrogen bonds, making G more difficult to
separate from C than A is from T. The sequences of these bases in the DNA represent the genes encoding for
all the organisms’ properties. The genome in Bacteria and Archaea can vary in size (e.g. ranging from 0.58
megabases (mb) in Mycoplasma genitalium, and even smaller in the archaeal Nanoarchaeum equitans with
0.49 mb, to 8.66 mb in Streptomyces coelicolor), but are generally smaller than those seen in eukaryotic cells.
The sequence information in these genes is first transcribed into a messenger called messenger RNA by
transcription, whose sequence is then translated into the corresponding amino acid sequence of a functional or
structural protein, where three bases (a codon) code for each amino acid. Translation requires structures called
ribosomes, which are found in the cytoplasm of all ‘prokaryotic’ cells. Ribosomes have a distinctive shape and
size, properties expressed as Svedburg (S) units. In Bacteria and Archaea, the ribosomes are 70S and made of
two subunits, a 50S and 30S subunit, each containing several proteins and ribosomal RNA or rRNA. This
rRNA is found in three different sizes, 5S, 16S and 23S rRNA consisting of about 125, 1500 and 3000
nucleotides respectively. In eukaryotic cells, ribosomes are larger, 80S, and consist of 60S and 40S subunits.
We will hear about these rRNA molecules repeatedly throughout this book, because their sequences are used
in classifying and identifying bacteria, including of course, those in activated sludge. Any event (e.g. mutation)
which changes the sequence of the bases in the DNA of the gene will frequently eventually alter the amino acid
sequence of the protein for which it encodes, often leading to synthesis of a non functional product.
DNA replication and cell division in the Bacteria or Archaea involves neither of the ordered processes
of mitosis and meiosis seen in Eukarya. The proteins involved in DNA replication in Archaea are much
more similar to those in Eukarya than they are to the corresponding proteins in the Bacteria (Grabowski
and Kelman, 2003), a feature which in the past has raised interesting evolutionary possibilities about the
origin of the eukaryotic nucleus (e.g. Margulis et al., 2000; Martin, 2005a).
Most ‘prokaryotic’ cells also contain small fragments of non-chromosomal double stranded DNA, which
is usually circular but can be linear. Some of these are called plasmids, which can replicate autonomously, but
appear not to be essential to the cell harbouring them. Plasmids usually only carry small numbers of genes,
but are important because some encode for enzymes imparting antibiotic resistance, or permit these plasmids
to move from one cell to another, as discussed later. Other non-chromosomal DNA fragments exist in
‘prokaryotic’ cells including the mobile transposable elements, including fragments known as insertion
sequence elements and transposons, able to move from one place on the chromosome to another, and in
the case of transposons, carrying genes with them, where they may then replicate in their new home.
Genetic recombination and horizontal gene transfer in ‘prokaryotes’

Although ‘prokaryotes’ are asexual organisms, three processes for exchanging genetic information
between them have been described, and constitute methods of horizontal or lateral gene transfer.
Because sexual differences are not apparent in the participating cells, they are referred to as DONOR and
RECIPIENT. These processes are;
TRANSFORMATION: where small fragments of cell-free or naked DNA from one bacterial cell are
assimilated and incorporated into the genome of a so-called ‘competent’ recipient cell. Although this
process is known to occur in many unrelated Bacteria and Archaea, not all cells are naturally competent
and thus transformable (about 1%). It is probably a frequent event in natural communities like activated
sludge, and some of the DNA detected in activated sludge flocs (see chapter 3) may be transformable.
Transformation initially involves the Donor DNA binding (eventually irreversibly) to the surface of the
Recipient cell by DNA binding proteins, and only competent cells can bind DNA. Then either before or
during the time the DNA enters the Recipient cell it becomes single stranded (S/S) and bound to other
proteins called competence specific proteins, protecting it from being degraded during its transfer to the
Recipient chromosome, where with the assistance of another protein RecA, the S/S Donor DNA is
integrated into the genome, replacing some of the Recipient S/S DNA. If the Donor DNA derives from a
virus, the process is called transfection.
TRANSDUCTION: where Donor DNA is transferred to the Recipient using a bacteriophage as the carrier
or vector, either by a process called Generalized Transduction, where any Donor genes can be transferred, or
by Specialized Transduction, where only certain Donor genes are transferred. Again we see transduction in
both Bacteria and Archaea. Donor genes replace essential genes needed by the bacteriophage to infect its
bacterial host and replicate, and so the phage is said to be defective. Like Transformation, not all
‘prokaryotes’ are transducible and not all bacteriophages can carry Donor genes to another cell.
CONJUGATION: a plasmid-encoded process of gene transfer requiring close physical contact between
the Donor and Recipient cells, and the method most closely resembling sexual reproduction in many
eukaryotes. Whether a cell can behave as a Donor is determined by whether it contains a particular plasmid,
which carries the genes facilitating its transfer from Donor to Recipient cell. These plasmids are called
conjugative plasmids, and some can be transferred to a diverse range of different Recipient cells including
plants and fungi, while others have a much narrower range. The plasmid (in E. coli, the Fertility or F plasmid)
provides the Donor (F1) cell with an ability to synthesize a structure called a sex pilus, which can recognize
receptors on the surface of Recipient (F-) cells, and by retraction back into the Donor, bring the Donor and
Recipient cells together. Then the plasmid replicates as it transfers from Donor to Recipient, ensuring that
both now contain a copy of that plasmid. Thus the Donor (Fþ) cell remains a Donor and the Recipient (F-) cell
becomes a Donor (Fþ) cell, also losing the surface receptors recognized by the sex pilus. Thus, the plasmid
and the genes it carries will spread between and throughout populations.
In some E. coli strains, the F plasmid is integrated into its chromosome at several different sites. These
strains are known as Hfr strains, and they too can act as Donor cells. However, now the chromosome
and its genes are replicated, and one copy transferred to a Recipient (F-) cell during conjugation in a
predictable linear sequence, where these genes may be integrated into the Recipient genome and
expressed. The Hfr strain remains an Hfr strain because it still has a copy of the transferred genes, and the
Recipient (F-) cell usually remains an (F-) cell because only rarely does it acquire a copy of the F plasmid.
Horizontal or lateral gene transfer

It is now clear from the available whole genome sequence data that most ‘prokaryotic’ genomes contain
genes which originated in other organisms, and that the extent of their occurrence there and their origins
often reflects the organism’s way of life (Koonin et al., 2001; Thomas and Nielsen, 2005). These were
acquired by the processes just described. How we detect their presence depends on being able to
recognize any genes with odd or unexpected features for that organism. For example, genes coding for
proteins previously detected only in very distantly related organisms would raise eyebrows, as would
those where the GC content of the gene and the particular codon used to code for each individual amino
acid in the expressed protein are markedly different to those in the majority of the genes. Some Bacteria
possess genes of eukaryotic and archaeal origin, and vice versa, and in some cases a considerable part
of an organism’s genome seems to have arrived there by horizontal gene transfer. The impact of
horizontal gene transfer confuses our attempts to resolve detailed evolutionary relationships among
cellular organisms (Katz, 2002; Lawrence and Hendrickson, 2003; Ochman et al., 2005; Philipe and
Douady, 2003).
Intracellular inclusion bodies in ‘prokaryotes’

Many ‘prokaryotes’, especially those commonly seen in activated sludge systems, contain intracellular
membrane enclosed granules. These are usually carbon and/or energy storage compounds, which can be
metabolized by the cell when other sources are not available (Dawes, 1992). The ability to store substrates
in this way represents an adaptation of cells to competitive environments like activated sludge, which
are subject to repeated ‘feast’/‘famine’ conditions, and in the systems designed to remove phosphate
microbiologically, selective pressures are deliberately imposed to favour such populations, which
eventually dominate their communities and make them work (see chapter 10). Most of these inclusion
bodies can be visualised by simple staining techniques, some of which are described later in this book
(chapter 11). Their presence is considered a helpful character for the microscopic identification of some
of the filamentous bacteria causing foaming and bulking in activated sludge, and other populations, as
detailed in chapter 12. These inclusion bodies include:
(i) poly b hydroxybutyrate (PHB), a polymer of b-hydroxybutyric acid monomers. The PHB chains
aggregate into granules which can be readily stained (chapter 11), although these stains are not
always specific, and so the term poly b hydroxyalkanoate is used unless the precise chemical
properties of the stained storage material is known. Many filamentous and other bacteria in
activated sludge plants contain PHB, for reasons discussed in chapters 3,5,7.
(ii) polyphosphate (polyP), or volutin or metachromatic granules are long chain polymers of
orthophosphate, and are probably present in most microbes, but particularly the polyphosphate
accumulating organisms (PAO) like Candidatus ‘Accumulibacter phosphatis’ seen in biological
phosphorus removal (EBPR) activated sludge plants and discussed in chapters 3,10.
(iii) glycogen which is a polymer of glucose, is also thought to be used for storage in activated sludge
microbes, especially the so-called glycogen accumulating organisms (GAO), seen in EBPR
communities, and considered possible competitors of the PAO, which also store glycogen
(chapters 3,10). Unfortunately glycogen is not easily visualized by staining in ‘prokaryote’ cells,
and so the GAO are not identifiable directly by microscopy as the cells accumulating polyP or
PHB are (Serafim et al., 2002a).
In some ‘prokaryotes’ the inclusion bodies are inorganic, and include:
(i) sulphur granules of elemental sulphur (S– ) are found inside or external to some ‘prokaryotic’
cells which oxidise reduced sulphur compounds like H2S as their source of energy and reducing
power. Sometimes the S– granules disappear as they are further oxidised to sulphate, often with a
corresponding decrease in pH. These granules are visible under phase contrast light microscopy,
and can be seen spectacularly in some of the bulking filamentous bacteria like Thiothrix spp.
discussed in chapter 12.
(ii) magnetite particles of Fe3O4, making up the magnetosomes seen in the magnetotactic bacteria,
enabling them to recognise and respond to the earth’s magnetic field, a mechanism shared by all
organisms with this ability (Frankel and Bazylinski, 2006).
Vacuoles in ‘prokaryotic’ cells

Some Bacteria and Archaea may contain vacuoles, but unlike eukaryotic vacuoles discussed below, these
are never surrounded by a bilayered lipid biological membrane. Of these, the gas vacuoles are especially
elegantly designed to reflect their function. They allow aquatic photosynthetic Cyanobacteria to float and
consequently increase their efficiency of light energy capture. These vacuoles consisting of gas vesicles
are surrounded by a protein membrane, constructed to be strong enough to avoid bursting, and permeable
only to gases, and are the reason why polluted lakes and streams are covered with a surface layer of
Cyanobacteria. Marine Beggiatoa contain intracellular vacuoles which contain high nitrate concentra-
tions. These are not seen in fresh water populations (Mussmann et al., 2003)
Motility in organisms with ‘prokaryotic’ cells

Many ‘prokaryotes’ move because they possess structures called flagella made of special proteins, on
their cell surfaces. These are sub-microscopic structures and so require special staining techniques to
visualize for light microscopy. They operate like nanoelectric motors, moving cells by flagellar spinning,
establishing a cone of revolution, similar to a propeller, allowing them to move towards and away from
chemical and physical environmental signals they can sense by cell surface receptors (Harshey, 2003).
This information is transmitted to the flagellar motor allowing it to direct cells accordingly by
changing its direction of revolution. Both flagellate Bacteria and Archaea demonstrate these chemotactic
responses using very similar signal transduction systems, although considerable diversity exists within
each (Szurmant and Ordal, 2004).
Some phylogenetically unrelated organisms share an ability to move in a different and distinctive way,
by gliding across solid surfaces. Gliding is seen with some filamentous bacteria described in this book.
There appear to be several mechanisms by which this is achieved, some of which involve structures like
pili, while others seem to involve special cytoplasmic proteins. How either works is not well understood
(Harshey, 2003; McBride, 2001). Some Gram negative cells also possess numerous and short structures on
their surfaces called fimbriae, which may be associated with motility, but they also provide pathogenic
bacteria with a mechanism for attachment to surfaces of other host cells, and cells to each other.
Do ‘prokaryotic’ cells possess a cytoskeleton?

One of the major differences recognized early by cell biologists distinguishing ‘prokaryotic’ organization
from eukaryotes was the lack of a cytoskeleton in the former, and used to explain the absence of functional
attributes like cytoplasmic streaming and mitotic spindle formation (see below) in ‘prokaryotes’.
However, it is now clear that bacterial cells also possess cytoskeletal proteinaceous elements homologous
with those seen in eukaryotes, actin and tubulin, which also participate in DNA separation during their
cell division (Shih and Rothfield, 2006). Their presence indicates these elements probably evolved in
‘prokaryotes’ first and are not eukaryotic inventions, or the genes encoding them were acquired from
eukaryotes by horizontal gene transfer.
CELLS WITH A EUKARYOTIC ORGANIZATION

In contrast to the ‘prokaryotes’ (Table 1.1), eukaryotic cells under the TEM possess a true nucleus
surrounded by a double nuclear membrane, and contain membrane bound organelles like mitochondria
and chloroplasts (in photosynthetic members) and endoplasmic reticulum (Figure 1.9), and in some
protozoa and fungi, organelles called lysosomes and hydrogenosomes (Martin, 2005b). Cells perform
specialised functions within these, as described later. Furthermore, eukaryotic organisms, even the
microbial members, demonstrate a considerably greater morphological diversity than seen with
‘prokaryotes’, and a frequent formation of specialised structures with specific functions.
rough ER
ER
Figure 1.9. TEMicrograph of an animal cell showing endoplasmic reticulum (ER) covered with ribosomes
(rough ER). (R. Webb). Bar mark ¼ 1 mm.
Eukaryotic microbes include the Fungi, Algae and Protozoa, each of which represents a group of
diverse organisms, but all with essentially the same cell ultrastructure and organisation. Again we can
now see, as we noted with ‘prokaryotes’, how the application of molecular methods has revolutionized
our understanding of their phylogenetic relationships to each other, and raises interesting questions
concerning their evolutionary links. These will be discussed briefly later. Furthermore, ecological
application of molecular approaches has already revealed unexpected levels of eukaryotic microbial
biodiversity in nature (e.g. Moreira and Lopez-Garcia, 2002; Baker et al., 2004).
Some of the main features of eukaryotic cells can be described very briefly here, and their similarities
and differences to the corresponding structures in ‘prokaryotic’ cells emphasized.
The cell walls of eukaryotic microbes

Both fungi and algae possess cell walls, at least during parts of their life cycles, but a peptidoglycan-like
material has never been detected in their walls. Instead, fungal walls are made up mainly of chitin (a polymer
of N-acetyl glucosamine). In the so-called ‘Water Fungi’ or Stramenopiles, once considered strange fungi,
but now recognized as algal-like, cellulose (a polymer of glucose) is the structural polysaccharide, together
with proteins and other polysaccharides like glucans, or mannans in yeasts. Most algae have a cellulosic
cell wall too, but in some like the diatoms, it is silica based. The protozoa lack a true wall, but many possess
a pellicle layer under the cytoplasmic membrane, imparting stable cell shape.
The membranes of eukaryotic microbes

In all eukaryotic cells including the microbial members, the cytoplasmic membrane is very similar in its
chemical composition to that found in the Bacteria, with an ester linkage joining the hydrophilic and
hydrophobic components. Sterols are used to stabilize these membranes in place of the hopanoids of
some ‘prokaryotic’ organisms. Similarly, fungi, algae and protozoa all contain a porous double nuclear
membrane enclosing the DNA in the nucleus, together with an extensive communication network of
membranes called the endoplasmic reticulum, which is often organised into structures called Golgi
bodies, involved in secretion of materials from their cells (Figure 1.10).
Organelles in eukaryotic microbes

In aerobic eukaryotes, mitochondria and chloroplasts (found in the algae), are sites for the synthesis of
energy in the form of the chemical adenosine triphosphate (ATP), needed by the cells to grow.
Chloroplasts (Figure 1.10) with the aid of light capturing pigments called chlorophylls convert light
energy to ATP by a process called photophosphorylation during aerobic photosynthesis. In mitochondria
(Figure 1.10), the process of respiration produces ATP from electron transport phosphoryllation. These
are described briefly later in this chapter.
It is worth repeating again here that no similar organelles are seen in ‘prokaryotes’, where the same
energy generating processes are performed on their cytoplasmic membrane or invaginations of it. Both
mitochondria and chloroplasts have similar ultrastructures, with an outer and an extensively folded inner
membrane, together with their own circular and D/S DNA, RNA and ribosomes, which are 70S, just like
those in ‘prokaryotic’ cells. This feature, together with other evidence, including their 16S rRNA gene
sequences, confirm that both mitochondria and chloroplasts were once free living ‘prokaryotic’ organisms,
which became ingested by another cell type. The endosymbiotic theory for the origin of these organelles
suggests that both then evolved together to give rise to our present day eukaryotic cell. It now seems likely
that chloroplasts were once Cyanobacteria, aerobic photosynthetic Bacteria, while mitochondria are most
similar to Alphaproteobacteria like the chlamydias, present day obligately intracellular bacterial parasites.
Which was the original host cell is not so clear. These organelles do not have complete autonomy within
their host and it is clear that considerable horizontal gene transfer has taken place, where many organellar
(‘prokaryotic’) genes have become integrated into the host cell’s genome.
A few unicellular anaerobic eukaryotes, including both fungi and protozoa lack mitochondria
(Bullerwell and Lang, 2005). These include the protozoan Giardia, a water borne parasite responsible for
serious outbreaks of gastroenteritis in humans from drinking faecally polluted water. The answer to the
G
C
Figure 1.10. TEMicrograph of an algal cell showing intracellular structural features of eukaryotic cells.
Nucleus (N), mitochondrion (M), Golgi apparatus (G), chloroplast (C) with thylakoid membranes, and
vacuoles (V). (G. McFadden).
question of whether these anaerobic amitochondriate eukaryotes evolved differently to the mitochondriate
versions appears to be no. Molecular evidence supports the view that organisms like Giardia once possessed
mitochondria, but then lost them later, presumably because they were not required for ATP production in
eukaryotes living anaerobically. Instead these protozoa possess organelles for ATP generation called
hydrogenosomes, which as their name suggests, generate H2. It is often claimed that these hydrogenosomes
share the same ancestor as the mitochondria, and so should be seen as their ‘anaerobic’ counterparts
(Voncken et al., 2002; Bullerwell and Lang 2005), yet some claim that hydrogenosomes evolved quite
independently and on several separate occasions (Tjaden et al., 2004; Martin, 2005b).
Vacuoles are also commonly seen in eukaryotic microbes, especially protozoa, where they play a
vital role in removing excess water from the cells (the contractile vacuole) or in the capture and bulk
transport of solid food particles, which often include bacterial cells, by a process called endocytosis or
phagotrophy. Unlike vacuoles in ‘prokaryotic’ cellular organisms, these are surrounded by a bi-layered
biological membrane. The food vacuoles containing the particulate matter fuse with other structures called
lysosomes, which are membrane-packaged cocktails of hydrolytic enzymes. These enzymes break the
food down to soluble compounds which then pass into the cell’s cytoplasm. This is the feeding mechanism
used by most of the protozoa in activated sludge, and the different organelles involved in it are collectively
referred to as the vacuome. As we shall see (chapter 4), protozoa are important organisms there, and
possess beautiful and highly specialised structures for food capture in this way.
The nuclear arrangement in eukaryotic microbes

In eukaryotes, the DNA within the nucleus is segmented into fragments known as chromosomes, which
are associated with special proteins called histones. Also present in the nucleus is a structure called the
nucleolus, where the rRNA in the ribosomes is synthesised. As in the ‘prokaryotes’, the sequence of bases
or nucleotides in the eukaryotic cell’s DNA determines its ultimate properties, by again determining the
sequence of amino acids in the proteins after transcription and translation (again involving mRNA), which
often occur in strikingly similar ways to how Archaea process their genetic information. Protein synthesis
requires ribosomes. However, as mentioned earlier, these are larger than those in ‘prokaryotes’ being 80S
(with 60S and 40S subunits) compared to 70S in ‘prokaryotic’ organisms.
Cytoskeletal elements in eukaryotic microbes

One other striking feature of eukaryote cells is the ubiquitous presence of a cytoskeleton made of
structures called microtubules and microfilaments. Microtubules occur in the spindle body involved in
the process called mitosis, where the chromosomes separate during nuclear and cell division. They are
also found in the microscopic cilia and flagella, the organelles of motility in eukaryotes. These organelles
both have the same ultrastructure, but are described as cilia if present in very large numbers on each cell,
and are relatively short, while the term flagellum is used for structures which are much longer and present
in few copies on each cell. Both are surrounded by an outer membrane and have the distinctive [932]12
arrangements of the microtubules, which is quite different to the structure of ‘prokaryotic’ flagella. They
propel cells by setting up a wavelike or beating motion, and their arrangements are particularly important
in the identification of the protozoa in activated sludge (chapter 4).
Microfilaments are made from a protein called actin, which, after complexing with another protein
myosin, acquires the ability to contract and expand. These microfilaments are believed to mediate the
process of amoeboid movement, demonstrated by the amoeboid protozoa seen in activated sludge plants
(chapter 4). Structural homologues of actin and tubulin are now known to occur in ‘prokaryotic’ cells, as
mentioned above, where they too may be involved in DNA separation during cell division.
Conclusions
Many questions remain unanswered about the origin of distinctive eukaryotic cell structures like the
nucleus and endoplasmic reticulum, and 80S ribosomes, and speculation supported by molecular evidence
continues. Cavalier-Smith (e.g. 2002a,b) has made many provocative and original contributions to aspects
of the evolutionary origin of eukaryotic cells and their structures, and the interested reader is advised to
read his work.
A BRIEF DESCRIPTION OF THE FUNGI

Fungi are non-photosynthetic eukaryotic microbes using preformed organic compounds as sources of
cell carbon and energy, which they absorb from their environment. Some like the mushrooms
and toadstools (Basidiomycota) often produce macroscopic multicellular structures during their life
cycles, and although traditionally thought of as primitive plants for study by botanists, it is clear
from analyses of rRNA genes and other genes encoding proteins that fungi are much more closely
related phylogenetically to animals. So the long held belief that Fungi evolved from the Algae is clearly
wrong. Their vegetative structures are either unicells, as seen with the yeasts, or much more commonly
branching filaments or hyphae, organised into a loose aggregate called a mycelium. Asexual and sexual
reproduction may be carried out, both processes which involve the formation of spores of different
types. Both have a survival function in Fungi, and many sexual spores are highly resistant and
often dormant structures, allowing the Fungi producing them to disperse in both space and time. Fungi
were once thought sufficiently different to be recognised as a completely separate group of eukaryotic
organisms, but now this view is changing.
Molecular techniques often using 18S rRNA and 26S rRNA gene sequences (the eukaryotic equivalents
of the ‘prokaryotic’ 16S rRNA and 23S rRNA genes) have already markedly changed our views on how
fungi should be classified and their phylogenetic relationships. There is no space here to deal with these
issues in detail, and the interested reader is directed to the fascinating reviews of Guarro et al. (1999),
Redecker et al. (2002), Lutzoni et al. (2004) and Bullerwell and Lang (2005) to see how these approaches
have made us change our opinions. Several groups of organisms traditionally considered fungal-like,
including the slime molds and the ‘water-fungi’, the Oomycota, are now placed elsewhere with this
molecular evidence, in the former case with the protozoa and in the latter with the brown algae and diatoms.
They represent examples of convergent evolution, very common among the Fungi, where phylogenetically
quite different organisms living in very similar ways end up looking superficially alike. Consequently, all
the early classifications based on fungal morphology inevitably have been exposed as flawed when
molecular characters are used instead. On the other hand, some rapidly evolving unicells previously
thought to be unrelated to the Fungi from 18S rRNA sequence comparisons, like the Microsporidia
(mammalian parasites lacking mitochondria, and thus once considered as primitive eukaryotic organisms)
emerge as members of the Fungi (Bullerwell and Lang, 2005; Cavalier-Smith, 2001).
Consequently fungal classification has become unrecognizable from that included in the earlier version
of this book. Then and until quite recently, most mycologists thought (with reservations) that the true
Fungi or Eumycota could be grouped into five Sub-Divisions, some of which were thought by then not to
represent phylogenetically based groupings, since they were generated almost totally from morphological
characters. Examples of reproductive structures produced by each are given in Figure 1.11.
Mastigomycotina – all producing motile cells (monoflagellate or biflagellate) during their sexual and/
or asexual life cycles, consisting of two Classes, the Chytridiomycetes and Oomycetes.
Zygomycotina – carrying out asexual reproduction by non-motile sporangiospores in a sac-like
structure called a sporangium, and sexual reproduction involving gametangial conjugation and formation
of a resting spore called a zygospore.
Ascomycotina – carrying out sexual reproduction leading to formation of sexual spores called
ascospores produced within a sac-like ascus, which in many is organized on a fertile layer (Hymenium) in
a fruiting body called an ascocarp.
Basidiomycotina – carrying out sexual reproduction leading to formation of sexual spores called
basidiospores produced on a special cell called a basidium, which is usually organized on a hymenial
layer in a fruiting body called a basidiocarp.
Deuteromycotina – a dumping place for all fungi not identifiable as either Ascomycotina or
Basidiomycotina because essential structures involved in sexual reproduction were not produced to allow
such a placement.
OOMYCOTA
sporangium
sporangia
4µm 20µm
zoospores zoospores
4µm 4µm
Saprolegnia ferax Phytophthora cinnamomi

Asexual reproduction involves sporangia and motile
biflagellate sporangiospores (zoospores)
O
F
20µm
Thraustothecium clavatus.
OOMYCOTA- sexual reproduction by Oogamy- oospheres
(O) fertilized by male gametes delivered by fertilization tube
(F) and resistant oospore is formed
Figure 1.11a. SEMicrographs of structures associated with asexual and sexual reproduction in members of
the non-fungal Oomycota. Bar markers are given on Figures.
Allomyces macrogynus
Resting sporangium
Maturing pore through

which zoospores will emerge
10µm 10µm
CHYTRIDIOMYCOTA – asexual reproduction involving sporangia (S) and motile uniflagellate zoospores
Allomyces arbusculus
20µm 4µm
CHYTRIDIOMYCOTA- motile uniflagellate motile gametes
Figure 1.11b. SEMicrographs of structures associated with asexual and sexual reproduction in the
Chytridiomycota. Bar markers given on Figures.
Now our views have changed markedly from the increasing amount of molecular (e.g. 18S rRNA
sequence data) acquired, and so many believe at the moment that Fungi as a whole should be classified
into the following Divisions.
Chytridiomycota – all aerobic fungi producing zoospores with single posterior flagellum (Figure 1.11a),
but now containing no oomycetous fungi or Oomycota with biflagellate zoospores (Figure 1.11b).
Evidence suggests that these are not monophyletic.
Zygomycota – including most the members of the Zygomycotina and possibly also not a monophyletic
group, although most closely related to the Chytridiomycota (Figure 1.11c).
Glomeromycota – thought by some to include probably the most ancient monophyletic group of fungi:
proposed to house some members known to participate in symbiotic relationships (mycorrhizas) with
S
C
40µm 20µm
Rhizopus sp
20µm 40µm
Gilbertella persicaria
ZYGOMYCOTA – asexual reproduction involves Sporangia (S)

and non-motile sporangiospores. Columella(C) often present
Z
Z
100µm 100µm
Zygorrhyncus moelleri Phycomyces blakesleeanus
Z
Z
40µm 100µm
Gilbertella persicaria Absidias pinosa

ZYGOMYCOTA-sexual reproduction by formation of zygospore (Z)
Figure 1.11c. SEMicrographs of structures associated with asexual and sexual reproduction in the
Zygomycota. Bar markers given on Figures.
ASCOMYCOTA -Asexual Reproduction involving conidia
conidium
= arthrospore
2µm 20µm
Oidium candidum Aspergillus niger
4µm
Penicillium digitatum Scopulariopsis brevicaulis

Figure 1.11d. SEMicrographs of structures associated with asexual reproduction in the Ascomycota.
Bar markers given on Figures.
4µm 20µm 100µm
Schizosaccharomyces octosporus– Peziza-Apothecium Sordaria fimicola - Perithecium

naked ascus
200µm 4µm
Sordaria fimicola-asci Emericella stellatus–

cleistothecium and- ascospores
ASCOMYCOTA-produce ascospores (usually 8?) in ascus usually in ascocarp
Figure 1.11e. SEMicrographs of structures associated with sexual reproduction in the Ascomycota.
Bar markers given on Figures.
BASIDIOMYCOTA
2µm 2µm
Phragmobasidium Holobasidium – 4 basidiospores/basidium
2µm
2µm
2 basidiospores/basidium 2 basiodiospores on sterigmata from the yeast Sporobolomyces

Figure 1.11f. SEMicrographs of structures associated with sexual reproduction in the Basidiomycota. Bar
markers given on Figures.
plant roots, and previously placed in the Zygomycotina. They are probably most closely related to the next
two fungal Divisions.
Ascomycota – to include many of the Deuteromycotina, now confirmed by molecular methods to
belong to this monophyletic group, which is probably the largest and most diverse (Figure 1.11d,e).
Basidiomycota – also probably monophyletic and also expanded to include some Deuteromycotina, a
decision based on molecular evidence. Closely related to the Ascomycota (Figure 1.11f).
However, fungal systematics is still in a state of great flux, as more molecular data accumulate, and
Hibbett et al. (2007) for example, have suggested a quite different classification, where some groups
traditionally placed in the Chytridiomycota and Zygomycota have been relocated into other phyla. The
Ascomycota and Basidiomycota are also separated into the sub-kingdom Dikarya in their scheme, as these
alone form dikaryotic cells during sexual reproduction.
Currently about 80,000 species of fungi have been described, but this probably seriously
underestimates their likely number on earth, which some believe is well in excess of 1.5million!
(Hyde 2001). As with ‘prokaryotes’, culture independent techniques are revealing more about their
biodiversity in natural communities. Ecologically they are vital to life on earth, especially in nutrient
(C,N,P) cycling, and form symbiotic relationships such as lichens and with most plant roots
(mycorrhizae), where they assist in their nutrition and growth (Anderson et al., 2007). They produce
some of the foods we enjoy, like alcoholic beverages, breads and cheeses, and are exploited by us to
provide a number of enzymes and complex metabolites we depend on to save our lives (Adrio and
Demain, 2003; Papagianni, 2004), including the penicillin antibiotics and the cyclosporin immunosur-
pressants, which in the latter case extend survival times of transplant patients by reducing organ rejection.
A BRIEF DESCRIPTION OF THE ALGAE AND PROTOZOA

It probably now makes more sense to discuss these organisms together. Algae have been described
traditionally as walled, aerobic photosynthetic eukaryotes, while the Protozoa have included the non-
photosynthetic eukaryotic microbes lacking cell walls. However, these definitions are simplistic. For
example, the euglenoids (e.g. Euglena) are phylogenetically photosynthetic protozoa, which can readily
lose their plastids when no longer of value i.e. in the absence of light. Thus, it is becoming increasingly
clear from molecular data gathered in the past decade that the phylogenetic boundaries between the algae
and protozoa (and indeed the fungi) are not at all sharply defined. The ability of chloroplasts or plastids to
be lost from and gained by other cells by endosymbiosis, as discussed below, has emphasized that the early
classifications separating photosynthetic algal and non-photosynthetic protozoal members were unlikely to
have a sound phylogenetic basis. Recent work has in fact encouraged the need for a more integrated
approach to the taxonomy of all eukaryotic microbes, as described below (Taylor, 2003).
ALGAE
With the photosynthetic algae, their plastids originated from a primary endosymbiotic capture of
cyanobacterial cells as mentioned above, an event occurring maybe 1–2 million years ago or even
earlier (Line, 2002). Electron microscopy shows that plastids in some algal lineages are enveloped
within three or more membranes, suggesting that these organisms (sometimes called secondary algae)
have probably arisen from one alga capturing another, by a process of secondary symbiosis. Phenotypic
characters traditionally used for algal classification include chemistry of their photosynthetic pigments,
their cell wall composition, cell morphology and intracellular storage compounds. However, molecular
approaches based mainly on 18S rRNA and 26S rRNA and plastid encoded gene sequences suggest
that algal phylogeny differs profoundly from this more traditional view. Yet many important phylo-
genetic questions remain unanswered, since it is also clear that horizontal gene transfer has played
an important role in the evolution of algae, with many plastid genes moving into the host cell genome.
Consequently, elucidating their phylogeny is difficult, and although several molecular phylogenetic
markers have been assessed, many believe that mitochondrial morphology and cell ultrastructure will
continue to play an important role in providing answers.
For our purposes, we can recognize the following algal groups, which it must be stressed are not
phylogenetically based:
Green Algae, showing great diversity in their size and morphology, and including the Chlorophytes
and Chlorarachniophytes, containing chlorophyll a and b in a double membrane plastid with starch
stores.
Red Algae with the Rhodophytes and Glaucophytes, containing chlorophyll a and phycobilin
pigments.
Yellow-brown Algae or Chromophytes, containing chlorophyll a and c but no chlorophyll b,

and carotenoids – a polythetic group divided into the dinoflagellates, haptophytes, cryophytes and
heterokonts (now all grouped in the Chromalveolates), with the former having plastids surrounded by
three membranes and the latter three having plastids with four membranes, suggesting a secondary
symbiotic origin for all of them. As plastids in the cryptophytes contain phycobilins, it is likely that these
arose by secondary symbiotic ingestion of a red alga. The heterokonts are a very diverse collection of
‘algal’ groups, and now include the diatoms and oomycetous ‘fungi’.
This is not the place to discuss algal phylogeny in detail and the often controversial nature of the
relationships between them, but the interested reader is directed towards the reviews of Andersen (2004),
Keeling (2004a,b), Lewis and McCourt (2004), Palmer et al. (2004) and Saunders and Hommersand
(2004) for more recent thoughts.
THE PROTOZOA
Again, a detailed discussion of protozoal systematics is not appropriate in a book of this kind, but
these organisms impact greatly on the activated sludge process. They lack a true cell wall, and are typically
motile, able to actively pursue food and ingest solid particles like bacterial cells by the process of endocytosis
or phagotrophy. As we shall see (chapter 4), they represent arguably the most spectacular examples of
morphological specialization shown in any of the eukaryotic microbes, in terms of their prey-capturing
mechanisms, which are crucially important in the efficient functioning of activated sludge plants. Traditionally
they have been classified on their means of motility. As with the other eukaryotic microbes discussed above,
molecular methods have rendered such a classification of little more than historic value, although it is useful
when considering in general terms how they behave in activated sludge, and their identification there.
So for the purposes of this book, we will again classify the protozoa into several groups which we know
are not phylogenetically justified, as follows:
Flagellate Protozoa (Mastigophora) – possess one or several flagella for motility, and contain a single
nucleus. Found in activated sludge, and some cause serious diseases in humans. Include the plastid
containing euglenoids (algae?).
Amoeboid Protozoa (Sarcodina) – move by creeping amoeboid movement, involving cytoplasmic
streaming into pseudopodia. Often produce protective shells (tests) and only possess a single nucleus.
Again some are parasitic.
Ciliate Protozoa (Ciliophora) – move by cilia or often sessile and stalked, with sophisticated feeding
mechanisms involving a mouth and cilia, and the only group to possess two nuclei, the macronucleus and
micronucleus. In some, the cilia are modified into organelles (cirrhi) to assist in crawling, as described in
chapter 4.
Sporozoa – contain obligately parasitic protozoa, responsible for potentially fatal diseases like malaria
(Plasmodium spp.). These have complex life cycles where sporozoites are produced to move between
different animal hosts, and unlike other groups which capture their prey by endocytosis. Sporozoa absorb
nutrients through their cell membranes. They contain a degenerative pigment-less plastid, the apicoplast,
derived possibly from an ingested red alga, but with an important biosynthetic role.
A COMPREHENSIVE SINGLE CLASSIFICATION OF ALL THE EUKARYA

Because of these phylogenetic overlaps, several attempts have been made to collate all this phylogenetic
data from eukaryotic microbes into a comprehensive single classification (Taylor, 2003), which could be
adopted by mycologists, phycologists and protozoologists alike. One example (see Adl et al., 2005 for
details) no longer groups them separately into fungi, algae or protozoa but takes a polyphasic approach
(see chapter 6), where mitochondrial ultrastructure is an important character, and argues convincingly
that all eukaryotic microbes can be placed into six monophyletic groups. Its practical value is not
yet clear.
HOW DO THESE MICROBES GROW?

After this very rapid guided tour through some of the organizational differences between ‘prokaryotes’
and eukaryotes, the underlying theory of their growth must now be mentioned briefly, because these are
essential pieces of information for understanding some of the principles applied in the operation of
activated sludge plants. Again inevitably the discussion will be superficial.
Like all cellular organisms, microbes need energy to grow. This energy is usually stored or conserved
in the form of the chemical adenosine triphosphate (ATP), which can be generated by cells in several
ways to be described next, and then used by them to synthesize the chemical compounds they need to
grow, and eventually replicate. It is convenient to view ATP as an energy carrier between energy
generating reactions (CATABOLISM) and energy consuming reactions (ANABOLISM), inside cells.
How these metabolic events interconnect can be neatly summarised diagrammatically as shown (Stanier
et al., 1987) in Figure 1.12.
Inorganic Reducing
organic ATP power
compounds + N, S, P ATP ATP
Catabolic Precursor Building
or fueling metabolites Macromolecules Cell structures
blocks
reactions + Proteins Walls, nucleoid
Light ATP Amino acids, Reducing Reducing
Reducing trace sugars, power DNA power ribosomes
power metals nucleotides RNA membranes
growth fatty acids lipds etc.
factors PHB
Glycogen
Figure 1.12. Metabolic changes leading to eventual growth of cells.
This flow diagram also emphasizes that as well as needing energy (ATP), the cell has other growth
requirements. Thus many of the anabolic or biosynthetic reactions need a source of reducing power, or
electrons. These electrons are obtained from the oxidation of energy rich substrates or in photosynthetic
organisms from exposure of their photosynthetic pigments to light energy. The cell uses chemicals called
electron carriers, which pick up these electrons and then transfer them to where they are needed.
Two important electron carriers used (and there are several with different abilities to acquire and donate
their electrons, determined by their redox potentials) are nicotinamide adenine dinucleotide (NAD1) and
nicotinamide adenine dinucleotide phosphate (NADP1) which then become reduced to NADH 1 H1
(NADH2) and NADPH 1 H1 (NADPH2) respectively. Their importance in cell growth will be
mentioned later.
MECHANISMS CELLS USE FOR GENERATING ENERGY

Microbes, especially ‘prokaryotes’ can obtain energy from a staggeringly diverse number of sources,
which emphasises their importance in activated sludge systems, and in a remarkable diversity of ways.
There is insufficient space here to discuss all these different energy generating processes, but the
interested reader should refer to the general microbiology textbooks mentioned at the end of this
chapter, where this material is dealt with very adequately.Two basic mechanisms exist in cells where
the energy released in catabolic reactions is coupled to the synthesis of ATP. The first is by chemical
coupling, involving a process called substrate level phosphorylation. This leads to ATP formation by
the transfer of a high energy phosphate bond to adenosine diphosphate (ADP) from a chemical
compound undergoing oxidation during catabolism. The other method is electrical coupling where
ATP is produced by the release of energy from the transfer of electrons along a chain of electron
carrier molecules like the cytochromes, which are capable of reversible oxidation and reduction.
These carriers are precisely located in membranes of cells, and some carry both electrons and protons
(Hþ), while others transport electrons only. The protons are pushed outside the membrane and a
proton gradient or proton motive force is then established across it. In some ‘prokaryotes’ (like the
GAO? – see chapter 10) a sodium gradient is established across the membrane, serving the same
purpose. The potential energy of this gradient is used by cells to generate ATP by a process of
electron transport phosphorylation. This process is also how cells reoxidise their NADHþHþ, which
they need to recycle for further involvement in oxidation reactions, in a process called respiration,
which is discussed later.
It is conventional and very useful to categorise all organisms into groups depending upon the sources
they obtain their energy, carbon and reducing power needed for growth. The names given to these
groups will be used continuously throughout this book, so it is important to define what each means
Table 1.2.
Table 1.2. Sources of Carbon, Energy and Reducing Power for Cell Growth.
Carbon source
Carbon dioxide or carbonate AUTOTROPHS
Reduced organic compounds HETEROTROPHS
Energy source
Light PHOTOTROPHS
Organic or inorganic chemicals CHEMOTROPHS
Reducing power
(Electron Source)
Oxidisable reduced inorganic compounds LITHOTROPHS
Oxidisable organic compounds ORGANOTROPHS
All organisms, ‘prokaryotes’ or eukaryotes, can be described as being either HETEROTROPHS,

meaning they obtain all their cell carbon from pre-existing organic compounds, or AUTOTROPHS who
obtain all their cell carbon from CO2. Each of these groups can then be further subdivided on the basis of
how they obtain their energy and reducing power needed for cell growth (Table 1.3). The terminology
conventionally used by microbiologists for these metabolic features differs from that recommended by the
IAWQ/IWA for engineers (Grady et al., 1996), a situation not conducive to effective communication
between these two groups. In particular the description of light and inorganic compounds as substrates and
not as energy sources is very confusing to microbiologists.
Each of these categories will now be considered briefly.
Table 1.3. Nutritional Categories of Organisms.

Source of energy, reducing Examples of organisms
power and carbon with these features
Chemoorganoheterotrophs Oxidisable organic Most Bacteria and Archaea, Fungi, Protozoa,
compounds Higher Animals
Chemolithoautotrophs Oxidisable inorganic Hydrogen Bacteria, Sulphur oxidising
compounds, CO2 Bacteria, Nitrifying Bacteria, Iron oxidising
Bacteria, some Archaea, Anammox Bacteria
NO EUKARYA
Photoorganoheterotrophs Light, Oxidisable organic Purple non sulphur Bacteria
compounds Green non sulphur Bacteria
Photolithoautotrophs Light, H2O, H2S, reduced Purple and Green sulphur Bacteria,
S compounds CO2 Cyanobacteria, Eukaryotic Algae,
Higher Plants
Chemoorganoheterotrophs
These are organisms which obtain all their cell carbon and energy from the oxidation of organic
compounds. We humans fall into this group, as do most of the bacteria and the protozoa important in
activated sludge plants. Two basic methods are used for their energy generation.
In fermentation, oxidation of substrates occurs in the absence of an external, usually inorganic,
acceptor for the electrons generated from these oxidations. Therefore, in fermentation organic
compounds act as both electron donors (i.e. substrates being oxidised) and electron acceptors, to
regenerate the electron acceptor NADH þ Hþ which becomes reoxidised to NADþ. ATP production
mainly occurs by substrate level phosphorylation, although in some cases so little energy is released
that this method is not possible. Instead, cells can establish gradients of protons or sodium across their
membranes for their ATP production. Since substrates are partially oxidised, only a small proportion of
the total chemical energy they contain is released and conserved as ATP. As a consequence of
fermentation, a range of partially oxidised fermentation end products, particularly organic acids and
gases like hydrogen (H2) and CO2 accumulate. ‘Prokaryotes’ produce a wide range of different
fermentation end products, which are often valuable in their identification and classification, and some
formed in large amounts we exploit industrially. Their production by fermentative organisms in the
anaerobic zones of biological nutrient removal plants is also crucial to high P removal capacity, as we
will see in chapter 10. In eukaryotes only two are produced, lactic acid and ethanol. Fermentation
occurs in organisms referred to as anaerobic (i.e. those which can only grow in the absence of O2) or
facultatively anaerobic (these can grow in the presence or absence of O2). These organisms can
ferment only a limited range of organic compounds, which is restricted by their ability to balance their
internal oxidation reduction (redox) potentials.
Respiration describes the process where substrates are oxidised, and the electrons released are
eventually transferred to an external, usually inorganic electron acceptor, to reoxidise NADHþHþ.
Respired substrates may be completely oxidised via the tricarboxylic acid cycle (TCA), to CO2 with more
energy released, and ATP is produced by both substrate level and electron transport phosphorylation,
where the electrons are passed along an electron transport chain containing electron carriers like
cytochromes differing in their redox potentials, accepting and passing on the electrons to the final electron
acceptor. A wider range of substrates or organic compounds can be respired than fermented, and it is
assumed that most organic compounds will be degraded eventually by respiring bacteria and fungi. If cells
use O2, they are called aerobic and the process referred to as aerobic respiration. This is the process
probably used by most of the aerobic chemoorganoheterotrophs in conventional activated sludge tanks.
Other organisms can respire using electron acceptors other than O2, a process known as anaerobic
respiration, occurring under anoxic (oxygen free) conditions. These include:
(a) Sulphate (SO2

4 ), used by some obligately anaerobically respiring sulphate reducing bacteria like
Desulfovibrio, which are found in sewerage systems running under anaerobic conditions. They
contain a unique cytochrome, c3, located in their periplasm, and sequentially reduce SO2 4 to
hydrogen sulphide (H2S), which is toxic and has a strong distinctive odour, as follows:
ATP → PP
2e– 6e–
SO42– → APS → SO32– → H2S
sulphate ATP adenosine AMP sulphite hydrogen
sulphurylase 50phosphosulphate sulphide
Oxidation of hydrogen is responsible for establishing the proton motive force in Desulphovibrio
from which ATP is generated.
(b) Nitrate (NO3 ), accepting electrons from a cytochrome and used by the so called denitrifying
bacteria. They are facultative aerobes, meaning they only use NO3 if O2 is unavailable as terminal
electron acceptor in respiration. Denitrifying bacteria are taxonomically diverse, belonging mainly
to the Proteobacteria, although Gram positive denitrifiers are known (chapters 3,9). Some are
metabolically flexible and will use other electron acceptors for respiration and even obtain energy
by fermentation. They are probably always present in activated sludge systems, but their identity is
less clear. The NO3 is sequentially reduced to more reduced forms, although not all bacteria form
N2 gas, the desired chemically inert and most reduced final product.
e– e– e– e–
NO3 → NO2 → NO → N2O → N2
nitrate nitrate nitrite nitrite nitric nitric nitrous nitrous dinitrogen

reductase reductase oxide oxide oxide oxide
reductase reductase
Nitric oxide (NO), nitrous oxide (N2O) and N2 are all gases, and the enzymes (reductases)
involved in their production are synthesised only when NO3 is available. Less energy is obtained
than with O2 as electron acceptor. Many bacteria only carry out the reduction of NO3 to NO2 , a
process referred to as dissimilatory nitrate reduction (Robertson and Kuenen, 1992). There is also
evidence that some organisms denitrify even when O2 is present (McDevitt et al., 2000), although
how important they are in activated sludge systems is not known. Denitrifying bacteria flourish in
activated sludge plants incorporating anoxic zones (chapter 2), from where N2 bubbles can be
seen emerging. However partial denitrification to NO or N2O does happen and is undesirable, since
both are serious atmospheric pollutants, causing long term ozone depletion. These processes are
discussed in more detail later (chapter 9).
(c) Carbonate can be used by two groups of obligately anaerobic organisms, the homoacetogenic
Bacteria and methanogenic Archaea. Both use H2 and a range of organic compounds as sources
of energy and electrons to reduce carbonate (CO3 ) to acetate or methane gas (CH4). Both grow
either as chemoorganoheterotrophs or as chemolithoautotrophs (see below), in both cases using
the non-cyclic acetyl-CoA pathway. Establishment of either a proton or sodium gradient is used
for ATP production.
4H2 þ Hþ þ 2HCO3 ! CH4 þ 3H2 O for the methanogens
4H2 þ Hþ þ 2HCO3 ! CH3 COO þ 4H2 O for acetogens
Whether the obligately anaerobic acetogens are present in activated sludge is not clear, although
methanogens have been detected (chapter 3). Both are important members of the microbial
communities responsible for CH4 generation in anaerobic sludge digesters (Hedderich and
Whitman, 2006).
(d) Other Inorganic Electron Acceptors. Ferric iron acts as electron acceptor for anaerobic respiration
via a ferric iron reductase to produce ferrous iron in some chemoorganoheterotrophic bacteria
using a wide range of organic, often aromatic compounds as electron donors. These iron reducing
bacteria are found in activated sludge (Nielsen, 1996; Nielsen et al., 1997; Nielsen et al., 2002a).
Manganese (Mn4þ) can also serve a similar function in bacteria, including some filamentous
bacteria, with a variety of organic electron donors. Selenium and arsenic compounds may also
participate in anaerobic respiration, with arsenate reduction to the highly toxic arsenite held
responsible for many deaths in people in underdeveloped countries from drinking contaminated
water (Silver and Phung, 2005; Oremland and Stoltz, 2005; Stolz et al., 2006). The identity of some
arsenate reducing organisms is now known.
(e) Organic Electron Acceptors function in some cells carrying out anaerobic respiration. These
acceptors include fumarate which becomes reduced to succinate, dimethyl sulphoxide being
reduced to dimethyl sulphide, and trimethylamine oxide to trimethylamine. In each case cells are
able to couple these reductions to ATP generation by electron transport phosphorylation. Reductive
dechlorination is a term used to describe reactions where chlorinated organic compounds act as
electron acceptors in some anaerobically respiring bacteria, processes often important in their
environmental detoxification.
This brief account illustrates how remarkably adept ‘prokaryotes’ are in how they can carry out
respiration and how they can adjust to prevailing conditions in the most efficient manner.
Chemolithoautotrophs
Organisms in this category are restricted exclusively to a few aerobic and anaerobic ‘prokaryotes’ able
to obtain energy from oxidation of a range of inorganic compounds. All their cell carbon comes from
CO2 (Table 1.4), which needs to be reduced with expenditure of considerable amounts of ATP,
to organic compounds. Thus, it is not surprising that some will use organic carbon sources if
available, saving this energy. These organisms are called mixotrophs. Some chemolithoautotrophs are
important in wastewater treatment plants, as we shall see later. Several groups of bacteria can live this
way. They are:
(a) Hydrogen oxidising organisms include many Bacteria and Archaea, which use H2 as an energy
source. Since organic compounds if present can serve as energy sources they are referred to as
facultative chemolithoautotrophs. Some use O2 as their electron acceptor (aerobic), but for others
NO3 or SO24 .
hydrogenase
1 O →
H2 + —
2 2 H2O
(b) Iron oxidising bacteria also include both Bacteria (e.g. Acidithiobacillus ferrooxidans) and
Archaea (e.g. Ferroplasma), which use ferrous ions (Fe2þ) as their energy source, forming ferric
ions (Fe3þ) in an oxidation which releases only small amounts of energy. Thus, these bacteria
grow very slowly. They also include Sphaerotilus natans, and several other iron oxidising bacteria
are present in activated sludge (chapter 3).
1
2Fe2þ þ 2Hþ þ O2 ! 2Fe3þ þ H2 O
2
Some electron acceptors involved in ATP production in these organisms are very unusual. Ferric
ions precipitate as insoluble ferric hydroxide (Fe (OH)3), and as Fe2þ is insoluble at neutral pH,
these iron oxidising bacteria are mainly found in highly acid pH environments (5pH 1).
Table 1.4. Major differences between the mechanism of photosynthesis in Bacteria and Eukarya.
‘Prokaryotes’ Eukarya
Algae and
Proteobacteria Green Bacteria Cyanobacteria higher plants
Major photosynthetic Bacteriochlorophyll Bacteriochlorophyll Chlorophyll a,b? Chlorophyll a,b
pigments a or b c,d,e
Phycobiliproteins absent absent present absent except
in Red Algae
Site for membraneous chlorosomes thylakoids chloroplasts
photo-synthesis lamellae
Photosystem I cyclic present present present present
photophosphoryllation
Photosystem II non absent absent present present
cyclic photophos-
phoryllation
Source of reducing H2S, S2 O2
3 H2S, S2 O2
3 H2O H2O
power So granules So granules – –
sometimes seen sometimes seen
Generation of oxygen none none occurs occurs
Examples Purple sulphur Green sulphur – –
and nonsulphur bacteria and
bacteria ‘Chloroflexi ’
(c) Sulphur oxidising bacteria are sometimes referred to as the colorless sulphur bacteria and obtain
their energy from the oxidation of reduced inorganic sulphur compounds like H2S, S2 O2 3 or
elemental sulphur (So) as follows:
So
or APS reductase
S2O3 – → S2O4 –
2 2
H2S → + 2H+
or
S2O32 – sulphite oxidase
These bacteria often accumulate So granules inside the cells, where they can be seen with phase
contrast microscopy. Some filamentous bacteria like Thiothrix spp, Beggiatoa and several other
filaments including type 021N (chapter 12) probably carry out these oxidation steps for energy
production. Often this So can then be further oxidised to SO2
4 and so it disappears from the cells.
The end result is the formation of H2SO4 which leads to a drop in pH. Members of the genus
Thiobacillus obtain energy in this way. Their role if any, and that of other similar bacteria in
activated sludge plants is not understood, but they can cause serious corrosion in the sewer pipes.
(d) Ammonia (NH3) and Nitrite (NO2 ) oxidising bacteria are aerobes which obtain their energy
from the oxidation of either NH3 or NO2 . No ‘prokaryote’ is known which will oxidize NH3 all
the way to NO3 (Costa et al., 2006). The following reactions are carried out in these bacteria
which are of great importance in activated sludge systems. Nitrifying bacteria have extensive
membrane systems inside their cells (Figure 1.5), which differ in their arrangements among
different genera, for ATP production.
(i) Those oxidising NH3 to NO2 the nitroso-Fying bacteria or ammonia oxidising bacteria
(AOB): a 2-stage process
e.g. Nitrosomonas
ammonia
monooxygenase
NH3 + O2 + H+ → NH2OH + H2O

hydroxylamine
where O2 is molecular oxygen, and as the reaction is energetically unfavourable, no energy

from this step is made available to the cells.
hydroxylamine
oxidoreductase
NH2OH + O2 → –
NO2 + 2H2O + H+
nitrite
where the energy now released can be utilised by the cell. Medium pH drops during
nitrification from the production of Hþ.
In summary:
NH3 þ 2O2 þ Hþ ! NO2 þ 2H2 O
(ii) Those oxidising NO2 to NO3 – the NITRO-FYING bacteria or nitrite oxidising bacteria
(NOB),
e.g. Nitrobacter and Nitrospira:
nitrite oxidase
– 1 O
NO2 + — → NO3
–
2 2
where the O2 used in this reaction comes from water.

The energy yield from these oxidations is very low, and so these organisms grow very
slowly.
Archaeal nitrifying organisms are now known to exist (Francis et al., 2007; Prosser and
Nichol, 2008), but whether they are widely found in activated sludge is uncertain. These
will be discussed in more detail in chapter 9. It is now clear that some of the nitrite
oxidizing bacteria can behave as mixotrophs using organic substrates when available and
not CO2 as their carbon sources to save energy.
(iii) Some Bacteria can oxidize NH3 under anaerobic conditions in a process referred to as
ANAMMOX, an acronym for anaerobic ammonia oxidation. These use NHþ 4 as their energy
source and NO2 as electron acceptor, and the same enzyme we saw in the aerobic nitroso
bacteria above, ammonia monooxygenase, as follows:
NHþ
4 þNO2 ! N2 þ ATP
They produce hydrazine as an intermediate in this process, the only recorded biological
system known to generate it. This highly toxic, high energy compound is contained within an
organelle called an anammoxosome surrounded by an impermeable membrane of unique
chemical composition (Fuerst, 2005; Strous and Jetten, 2004). ANAMMOX is an attractive
option for removing N from wastes high in NO2 since it requires no expensive aeration
(chapter 2), and all the biochemical changes proceed in one population. Organisms carrying
out this oxidation have never been grown but are chemolithoautotrophic Planctomycetales,
all sharing essentially the same physiology. Several are known, including Candidatus
‘Brocadia spp.’ Candidatus ‘Kuenenia stuttgartiensis’, Candidatus ‘Scalindua sorokinii’ and
Candidatus ‘Anammoxoglobus propionicus’. More will inevitably be described. These
anammox bacteria now seem widespread in nature (Penton et al., 2006), and may play a
major environmental role in oxidising ammonia in anaerobic marine ecosystems (Strous and
Jetten, 2004; van der Vossenberg et al., 2008). All cell carbon is acquired from CO2, and is
fixed by the same metabolic pathway as in the aerobic nitrifiers.
(e) arsenite oxidizing members of the Bacteria and Archaea are known, and some behave as
chemolithoautotrophs, obtaining their energy from arsenite oxidation (Silver and Phung, 2005).
Photolithoautotrophs
These are organisms which use light as an energy source and CO2 as their sole source of carbon. Both
‘prokaryotic’ and eukaryotic photolithoautotrophs are known. They all obtain their energy in the form of
ATP by processes of electron transport phosphoryllation, but substantial differences exist in the methods
used, and the compounds generating reducing power for CO2 reduction. This is not the place to discuss
in detail what these systems are, because photolithoautotrophs are probably not found in large numbers
in activated sludge systems. However, they all need certain pigments for the capture of the light energy,
a source of reducing power for the subsequent reduction of CO2 into organic compounds and electron
transport systems to generate their ATP. Their main features, summarised in Table 1.4, suggest they
belong to two fundamentally different groups.
(a) The anoxygenic photosynthetic ‘proteobacterial’ purple sulphur and non-sulphur bacteria, and
the green sulphur (phylum ‘Chlorobi’) and green non-sulphur (phylum ‘Chloroflexi’) bacteria
(not all of whom are photosynthetic). All possess only one light system, and obtain their
reducing power from H2S, So or H2, and in the former case, sometimes generating So granules
stored inside or dumped outside the cells. Many demonstrate a remarkable ability to obtain
energy by methods other than phototrophy when light is not available. Their photosynthetic
pigments are bacteriochlorophylls, chemically distinct from the pigments in the oxygenic
photosynthetic organisms described next. Autotrophic CO2 reduction in the ‘Chloroflexi’ occurs
by the so-called hydroxypropionate pathway, found in no other phototroph, but seen in some
Archaea. In the ‘Chlorobi’ it is achieved exclusively by the so-called reverse citric acid cycle,
but this pathway has also been detected in several non-phototrophic Archaea, possibly as a
result of horizontal gene transfer.
(b) The oxygenic photosynthetic Cyanobacteria and photolithoautotrophic eukaryotes, both posses-
sing two light systems, and both use H2O as a source of reducing power to generate O2. They
share some of the same chlorophylls, and as we discussed earlier, considerable evidence supports
the view that eukaryotes inherited their plastids and thus photosynthetic mechanisms from
Cyanobacteria by endosymbiosis.
THE GROWTH AND NUTRITION OF MICROBES

To grow, all cells, including the bacteria and protozoa in activated sludge systems must be provided with
several essential nutrients to form more cell material, and eventually to divide. Unfortunately, what these
requirements are for many organisms including most of the bacteria in activated sludge systems is not
clearly understood. Thus, it is not easy to isolate and grow many readily in axenic culture, a problem
which has impacted on our understanding of its community composition (chapter 3).
Most microbes (the protozoa being notable exceptions) absorb their nutrients from their environment
through their cytoplasmic membranes. As most live in conditions where the substrates they need are at
much lower levels than those already existing within their cells, they require mechanisms for transporting
these against a concentration gradient. Several mechanisms exist, and cells often need to expend energy to
achieve this.
Briefly, the nutritional requirements cells need for growth include:

An energy source to provide the organism with the ATP needed for biosynthetic and other reactions.
These have already been discussed briefly above.
A carbon source needed for the production of new cell compounds. Heterotrophs use organic compounds.
‘Prokaryotes’ and fungi are able to metabolize a vast array of diverse organic compounds including both
naturally occurring and xenobiotic compounds. Autotrophs use CO2 as their sole carbon source, but as
explained above, this has first to be "fixed" or reduced into organic compounds, a step requiring
considerable energy, and explaining why some prefer to grow as mixotrophs or heterotrophs where
possible.
A nitrogen source, for the synthesis of proteins and nucleic acids is also required, and may be obtained
from several sources. Some aerobic and anaerobic ‘prokaryotes’ can fix atmospheric dinitrogen gas into
amino acids (diazotrophy), but most ‘prokaryotes’ and all known eukaryotes can not. They need either an
inorganic source like NH3 or NO3, which is then incorporated into amino acids, or preformed amino acids,
acquired directly or from protein degradation.
A source of sulphur is needed for the synthesis of amino acids like cysteine or methionine, and vitamins
like biotin. Most cells prefer SO2
4 or H2S, which are again assimilated directly into amino acids, or they
can obtain sulphur from utilizing the sulphur containing amino acids directly.
Macronutrients like phosphorus (P), potassium, magnesium and calcium, are required either for the
synthesis of key cell components (as with P), or to stabilise cell structures like ribosomes or cells walls
(calcium and magnesium) or to allow key enzymes to function (magnesium and potassium). Iron is also
required by cells for the synthesis of proteins like cytochromes.
This book will deal at length with the organisms able to accumulate P at levels far greater than those
needed for cell growth, and who remove P from wastewater during its treatment, a process known to be
influenced by the availability of Mg2þ (Schönborn et al., 2001; Wu et al., 2006).
Growth factors are needed by some cells which have lost the ability to synthesise these essential
compounds themselves. They include amino acids or vitamins, and must be present in the environment for
the organism to grow. Often the precise requirements for culturing particular organisms is poorly
understood, and so these factors are added to laboratory media in the form of crude animal or other cell
digests, like peptone or yeast extract (Atlas, 2005; Overmann, 2006).
In addition, if the organism is to grow, the abiotic culture conditions like the prevailing pH and
temperature, its water activity (aw) and the gaseous environment (e.g. pO2 or pCO2) must be appropriate
(Schlegel and Jannasch, 2006). Microbes have tolerance ranges for all of these and if they are exceeded,
then no growth will occur. Certain organisms can tolerate levels close to the limits capable of supporting
life, and are called extremophiles. Hyperthermophiles for example, have special biochemical and
molecular attributes which protect heat sensitive sites within their cells allowing them to grow at
temperatures 4 100– C, and survival features are shown in cells thriving for example, at low and high pH
(Sorokin, 2005) and low aw. Generally, ‘prokaryotes’ seem able to tolerate many environmental extremes
better than eukaryotes. This is especially true of temperature, probably because eukaryotic cells possess
more intracellular membrane systems, which may be their Achillean heels, and phototrophic ‘prokaryotes’
are more temperature sensitive than non-phototrophic members, for the same reason.
THE GROWTH KINETICS OF MICROBES

The end result of these catabolic and anabolic reactions is usually an increase in cell size, followed then by
cell division. Filamentous fungi and possibly some of the filamentous bacteria like the Mycolata found in
foams on activated sludge plants (chapter 8) grow by apical extension of their filaments and their growth
kinetics will not be discussed further here. The reader is referred to Prosser and Tough (1991) for a
review. With many filamentous bacteria including some discussed in this book, extension at the apex of
the filaments does not occur (Seviour et al., 1994; Rossetti et al., 2005). Instead in most bacteria cell
division occurs by a process called binary fission, where the parent cell divides equally (usually) into two
daughter cells, a process whose frequency is affected by a wide range of factors. The time taken for a cell
to divide is referred to as its mean generation or doubling time (td) and is usually more rapid for most
bacteria than for eukaryotic microbes like fungi under most environmental conditions.
Cells can be grown in the laboratory using two types of culture systems (Overmann, 2006). The first
is Closed or Batch Culture, characterised by no input or output of materials once the medium has been
inoculated with cells. In batch culture, the environment and therefore the organism’s physiology is
continuously changing, and as neither can be controlled adequately, so-called steady state conditions are
not achievable.
On the other hand Open or Continuous Culture, is achieved by a balanced input of fresh growth
medium and removal of spent medium and cells from the growth vessel. It is possible to maintain
exponential growth (see later) of a population indefinitely and to achieve steady state conditions
(Monod, 1949; Herbert, 1961). These systems may be run as turbidostats where a constant desired
population size is maintained by measuring culture turbidity and using this parameter to control the flow
of fresh medium into the reactor. More commonly, continuous culture systems are run as chemostats,
where the specific growth rate (m) of the organism is controlled by controlling the rate at which the growth
limiting substrate (for example, the carbon or nitrogen source) is provided to the culture. Therefore in a
chemostat it is possible to grow our population at any desired rate, which provides a very powerful
experimental system. Activated sludge systems are considered to operate in a similar manner to
chemostats, except that most of the cells which grow are added back into the system (chapter 2).
Batch culture systems

In batch culture, microbial population changes follow a characteristic growth curve which consists of
several phases (Figure 1.13).
Stationary
In X
De
l
tia
ath
nen
po
Ex
Lag
Time
Figure 1.13. A typical batch culture growth curve, showing the recognisable phases of growth.
First is the Lag phase (where m ¼ 0) which does not always occur, and its length can vary with
organism and environmental conditions. A lag phase is seen when old cells in stationary or death phase
(see later) are used to inoculate fresh medium of the same or different composition, or when cells at any
stage of growth are inoculated into a medium of different chemical composition. It is not found when cells
in exponential growth phase (see next) are transferred to fresh medium of the same chemical composition.
The lag phase is believed to reflect a period of either recuperation or resuscitation, where cell components
required for exponential growth have first to be assembled, or adaptation, where cells from a different
medium have to synthesise other enzymes not already present to metabolise the newly available
substrates.
The Exponential phase is considered to have started when m is a constant, and all cells are viable and
actively dividing. The value of m is specific for each organism grown under specified conditions. It is
affected by the growth limiting substrate and factors like culture pH, temperature and dissolved O2 (DO)
concentration, and most of these may change continuously in batch culture.
Stationary phase is where m again becomes 0 after a deceleration phase, and growth ceases. This
happens because cells either run out of nutrients or are adversely affected by changes in medium pH or
pO2 (in aerobic organisms), brought about by their own metabolic activity.
Finally a Death phase can be detected where m is negative and the number of viable cells declines at an
exponential rate.
KINETIC ANALYSIS OF BATCH CULTURES

Under favourable growth conditions, and with plenty of nutrients available,
1 cell → 2 cells → 4 cells → etc.

division division division
or
2o cells → 21 cells → 22 cells → etc. → 2n cells
Thus the growth of the population is logarithmic or exponential. However, organisms will grow
arithmetically if conditions for exponential growth are not present, where the population size increases as
a direct function of time (Figure 1.14).
exponential
No. of cells
arithmetic
Time
Figure 1.14. Differences in the pattern of increase in cell numbers during exponential and arithmetic
population growth.
In exponential growth, if X is the final cell density after time t, Xo the initial cell density and
n ¼ number of generations,
X ¼ X o 2n or X ¼ Xo t=td ; where td ¼ double time

Taking natural logarithms
ln X ¼ ln Xo þ t=td
and this equation allows us to predict what X or td is after a known time.

We more usually express exponential growth as:
dX
¼ mx ð1Þ
dt
where m ¼ specific growth rate constant (h 1)

Integrating gives the expression:
ln X ¼ ln Xo þ mt
which is a straight line relationship, and means that a plot of lnX against t gives a straight line whose
slope ¼ m, can then be calculated.
slope ¼ m ¼ 0:693=td
Exponential growth of a population in batch culture does not continue for long, and is self limiting. The
growth limiting substrate concentration will therefore affect m according to the following expression of
Monod (1949).
S
m ¼ mmax ð2Þ
S þ Ks
Plotting m against the concentration of growth limiting substrate gives the relationship in Figure 1.15.
Here mmax is the maximum growth rate of the population, which occurs when S (substrate
concentration) is very large, or ks (substrate saturation co-efficient) is very small. All are characteristic for
each organism, and the value for ks is specific for each substrate.
When m ¼ 12 mmax, then the numerical value of ks ¼ S. The ks is a numerical expression of the affinity
of an organism for the growth limiting substrate. A low value of ks means an organism has a high affinity
for that substrate and vice versa.
How can we determine values for Ks and mmax?

If equation (2) is rearranged, it gives the following.
1 1 Ks 1
¼ þ ð3Þ
m S mmax mmax
which is a straight line relationship.

µmax
0.8
µ(h–1) 0.6
0.4 0.5µmax
0.2
0 20 40 60 80
S (mg/l)
Figure 1.15. Relationship between growth rate m and S, the concentration of growth limiting substrate.
By plotting
1 1
·v·
S m
Ks and mmax can be calculated as shown in Figure 1.16.
UTILISATION OF SUBSTRATES IN BATCH CULTURE

Although cells utilise substrates mainly for production of more cells, some substrate is used for cell
maintenance, and for synthesising metabolic products. A balance equation can therefore be written to
allow for these demands.
Substrate Accumulation ¼ Substrate used for cell growth Substrate used for maintenance
Substrate used for product formation
dS mX qp X
¼ mx ð4Þ
dt Yx=s YP=s
Yx/s ¼ cell yield coefficient (g cells dry wt/g substrate utilised).

YP/s ¼ product yield coefficient (g product/g substrate utilised).
1/µ(h)
1 1/µmax
–0.1 0 0.1 0.2 0.3 0.4
1/S (l/mg)
1/KS
Figure 1.16. Determination of Ks and mmax from a plot of 1/m .v. 1/S.
qp ¼ product formation rate (h 1).

m ¼ maintenance coefficient (g substrate g 1 biomass l 1).
In most batch cultures, m initially is insignificant and if no metabolic products are formed, then the
equation becomes simplified to
dS mx
¼ ð5Þ
dt Yx=s
It is then possible to calculate qs (the specific rate of substrate utilisation) from Equation (4) as
1 dS m qp
qs ¼ ¼ m ð6Þ
x dt Yx=s YP=s
Cell yield (Yx/s) and product yield (Yp/s) coefficients are measures of the efficiency whereby cells
convert substrate into biomass and product respectively, and can be expressed mathematically as follows.
dx dS dP dS
¼ Yxs and ¼ YP=s
dt dt dt dt
m m
or as Yx=s ¼ and YP=S ¼
qs qp
If there is a maintenance requirement, then some of the substrate utilised will be used for this purpose and
so the measured
DX
Yx=sm ¼ þ DSm
DSt
where St ¼ true substrate used.

Sm ¼ substrate used for maintenance.
DX
The true Yx=St ¼
DSt
And the rate of substrate utilisation

dS dS dS
¼ þ
dt dt maintenance dt true

dS dS mx
where ¼ mx and ¼
dt maintenance dt true Yx=St

dS mx
\ ¼ mx þ ð7Þ
dt Yx=St
Multiplying by 1/X
ds 1 m
¼mþ
dt X Yx=st
1 m 1
or ¼ þ ð8Þ
Yx=s m Yx=st
which is a straight line relationship.
1 1
Thus, if is plotted against , the straight line will have a slope of the maintenance coefficient, m.
YðX=sÞ m
This determination is usually carried out in chemostat culture, where m can be varied readily (see later).
KINETIC ANALYSIS OF CHEMOSTAT CULTURES

Simple balance equations can be written which attempt to explain what might happen in a chemostat,
where fresh medium is added continually to a vessel of constant volume, and spent medium and cells
removed.
For biomass production to illustrate the net change in cell density (X),
the increase in X ¼ growth of cells – cells lost by dilution – cell death.
or
dX Flow Rate ðml· h1 Þ f

¼ mx Dx aX where D ¼ dilution rate ðh1 Þ ¼ or
dt Volume ðmlÞ V
(a ¼ death rate constant)

dx
¼ xðm DÞ aX
dt
In continuous culture, a is probably insignificant
dX
¼ xðm DÞ ð9Þ
dt
When m4D, then dX/dt is positive, and so the population increases.

When m5D, then dX/dt is negative, and cells are washed out, until eventually X ¼ 0.
The value of D at which this occurs is called the Critical Dilution Rate or Dcrit, and above this value,
steady state conditions cannot be achieved.
When m ¼ D, then dX/dt ¼ 0, and so called steady state conditions are achieved.
This expression shows that the value of X or biomass concentration is self regulating, and that, by
setting a value for D (below Dcrit), steady state will eventually be reached. Knowing D, then m is known.
This means that m can be set and maintained at any predetermined value by setting the value of D.
Chemostats work best at low values of D.
Substituting (9) into (2),

dX S
¼ X mmax D ð10Þ
dt S þ ks
UTILISATION OF SUBSTRATES IN CONTINUOUS CULTURE

A similar balance equation to that for batch culture can be written for chemostats, where fresh nutrients
are added and spent nutrients removed.
Substrate Accumulation ¼ Substrate entering vessel Substrate leaving vessel Substrate used for
cell production Substrate used for maintenance Substrate used for
product formation
ds mx qp X
¼ DSR DS mx
dt Y Yp=s
SR ¼ concentration of growth limiting substrates entering vessel.

S ¼ concentration of growth limiting substrate in vessel.
In most chemostat experiments, except those run at low D with carbon limitation, m is insignificantly
small, and product formation negligible.
ds mx
Thus ¼ DSR DS ð11Þ
dt Yx=s
Under conditions of steady state,
dS
¼0
dt
mx
¼ DðSR SÞ ð12Þ
Yx=s
Also at steady state, D ¼ m.
X ¼ Yx=s ðSR SÞ ð13Þ
where X ¼ biomass concentration at steady state and S ¼ substrate concentration at steady state.
At steady state where D ¼ m, (2) becomes
S
D ¼ mmax
S þ Ks
Ks D
and S¼
mmax D
Substituting from (13),

Ks D
x ¼ Yx=s SR
mmax D
These equations show that S only varies as a function of D, while X varies as a function of both SR and D.
This introduction to the growth kinetics of cells considers the simplest ideal systems only, and
chemostats often show departures from these models. Also no attempt has been made here to model
continuous culture systems with biomass recycle, which activated sludge systems incorporate, or other
flow configurations like fed batch systems. The reader is referred to Pirt (1975), Scragg (1991) and
Panikov (1995) for further information on the kinetic analysis of microbial growth.
THE CONTROL AND PREVENTION OF MICROBIAL GROWTH

The growth and viability of cells may be affected by a range of chemical and physical factors. Many
chemicals kill cells, but often these chemicals are non-selective. In other words, they kill mammalian cells
as well as bacterial cells. These are the disinfectants and antiseptics and consequently are more
commonly used to treat inanimate objects. Examples of these include heavy metals like silver nitrate
(AgNO3) or copper sulphate (CuSO4), halogens like iodine or chlorine (Cl2), and ozone, phenolic
compounds, and cationic detergents like quaternary ammonium compounds. Most have little value in the
control of microbial infections of man, but some, like chlorine and ozone are applied to activated sludge
systems to try to eliminate some of the filamentous bacteria causing bulking and foaming. Often they are
not always successful and run the risk of harming the rest of the biomass, although ozone has been
successful in eliminating foaming (chapter 8).
Some chemicals are selectively toxic and because of their modes of action, often kill ‘prokaryotic’ cells
but leave eukaryotic cells unharmed, and vice versa. These are the selectively toxic antibiotics,
compounds usually produced by other biological systems and effective at very low concentrations.
Antibiotics are often synthesized by slower growing organisms, and possibly provide them with the ability
to compete with those growing faster in natural communities. They specifically target certain cellular
metabolic activities (Franklin and Snow, 2005). Examples include the penicillins, cephalosporins and
vancomycin which inhibit peptidoglycan synthesis in the Bacteria only, the ionophores like valinomycin
which act on cell membranes, those antibiotics affecting the synthesis of DNA and RNA like rifamycin
and nalidixic acid, and the large number which disrupt protein synthesis by targeted action against 70S
ribosomes, like streptomycin, the tetracyclines, erythromycin, and 80S ribosomes like cycloheximide.
They are selective, because they attack sites and processes differing between and within ‘prokaryotes’ and
eukaryotes, as already mentioned. Strains resistant to all the antibiotics used clinically to treat disease
causing organisms have now emerged, and many can be readily isolated from activated sludge
communities (see earlier). So the search for novel antibiotics continues. Some believe that other
approaches are now needed, and interest in using bacteriophages to deal with human pathogens is
increasing. As discussed later in the book, a similar approach to controlling activated sludge bulking and
foaming is also attractive.
CONCLUSIONS
This very brief outline of the microbial world and how these organisms behave and grow should clarify
some of the discussion to follow on the microbiology of activated sludge systems. The following listed
books on general introductory microbiology are valuable additions to any activated sludge library.
Both have strengths and weaknesses but contain up to date information, are superbly presented and very
readable.
Madigan, M.T., Martinko, J.M., Dunlap, P.V. & Clark, D.P. (2008). Brock Biology of Microorganisms (12th ed.),
Pearson Benjamin Cummings, San Francisco.
Willey, J.M., Sherwood, L.M. & Woolverton, C.J. (2008). Prescott, L.M., Harley, J.P. & Klein, D.A. Microbiology
(7th ed.), McGraw Hill, New York, USA.
2
The activated sludge process
Robert J. Seviour, Kenneth C. Lindrea, and Adrian Oehmen
INTRODUCTION
Treatment of wastes with activated sludge systems represents a component of probably the largest
biotechnology industry in the world. Yet these processes differ from most conventional biotechnological
processes, which require pure cultures and controlled aerobic fermentations for large scale production
of economically important metabolites or biomass from microbes (Gray, 1990; Matsui et al., 1991;
Grau, 1992). Instead their communities are mixed populations, having to deal with a diversity of inorganic
and organic compounds entering the system irregularly and differing in their chemical compositions and
molecular/particle sizes. Until quite recently, the products of activated sludge systems (the biosolids or
sludge and treated water) were considered valueless, to be disposed of as cheaply as possible. Times have
changed.
What do these systems need to deal with?

Activated sludge systems need to be robust. As well as containing faecal material, domestic and industrial
wastes carry a range of naturally occurring and xenobiotic organic compounds, including bioactive
pharmaceuticals (Lindqvist et al., 2005; Ternes 1998; Vieno et al., 2007), and surfactants, which appear
able to affect community composition by reducing population diversity (Kraigher et al., 2008). Some
represent possible sources of endocrine disrupting compounds (see below) (Lozada et al., 2004) and
plasticiser phthalate esters (Marttinen et al., 2003). Many of these will be adsorbed and/or degraded by the
bacterial populations enriched in the activated sludge communities exposed to them (Lozada et al., 2004;
Roslev et al., 2007), but some are toxic, and will not be degraded (Byrns, 2001; Lindquist et al., 2005).
Understanding their fate clearly depends on having sensitive chemical or biological assays for their
detection in the influent raw sewage and liquid effluent, and harvested sludges (Matamoros et al., 2008;
Ren and Frymier, 2002; Ricco et al., 2004; Sotero-Santos et al., 2005).
The view has been expressed (Bott and Love, 2004) that toxic chemicals probably seriously affect
performances of many plants in the US and there is no reason to believe that plants in other countries are
any different. It is easy to demonstrate that some bacterial populations in plants show protective stress
responses to such chemicals. Thus, communities exposed to shock loads of electrophilic chemicals leak
Kþ from their cells, probably from induction of a glutathione-gated Kþ efflux stress response, leading to a
deflocculation (Bott and Love, 2002, 2004; Henriques and Love, 2007). Monitoring for the presence of
stress proteins in activated sludge systems may help us prevent their possible failure from this cause.
Work to develop suitable biosensors is required. Detecting increased levels of precursor 16S rRNA
from cells exposed to primary effluents from activated sludge is again consistent with harm caused by
chemicals entering the plants (Stroot and Oerther, 2003), or possibly from products of the metabolism of
less hazardous substrates. Several rapid and sensitive commercial kits (e.g. MicrotoxT with Vibrio
fischeri) based on viability of bioluminescent reporter bacteria, or suitable genetically engineered
bacterial strains, have been assessed for their ability to predict effects of influent toxic chemicals on
plant communities treating industrial wastes (Bitton, 2002a; Gutiérrez et al., 2002; Kelly et al., 1999;
Kungolos, 2005; Lajoie et al., 2002; Ricco et al., 2004; Wiles et al., 2003). In general these systems
show promise, but how popular they currently are for routinely monitoring plant influents or effluents
is unclear.
The endocrine disrupters pose particular potential environmental problems, not to the operation of the
activated sludge process itself, but subsequently. As these include the degradation products of the
alkylphenol polyethoxylate non-ionic surfactants widely used in industry (Ahel and Terzic, 2003),
and natural and synthetic oestrogens, both excreted by many women, wastewater is probably their major
environmental point source. Endocrine disrupters including estrone, 17a-ethinylestradiol, 17b-estradiol
and nonylphenol (Birkett and Lester, 2002) are biologically active at very low concentrations and
immensely harmful environmentally. They lead to a feminization of males and altered reproductive
activity and pattern in many animals, and an increase in the rates of several cancers including breast,
testicular and prostate cancers. Clearly, understanding their fate in wastewater treatment plants and their
influence on the composition of the microbial communities there (Kraigher et al., 2008) is of concern.
Several Bacteria including rhodococci capable of degrading oestradiol have been isolated from
activated sludge (e.g. Fahrbach et al., 2006; Fujii et al., 2002, 2003; Yoshimoto et al., 2004), and their
activities there demonstrated (Yu et al., 2005). Zang et al. (2008) provide in situ physiological evidence
to suggest that novel Betaproteobacteria and Gammaproteobacteria are both important in estrone
degradation.
However, most published data would seem to suggest that activated sludge plants are only variably
effective at removing such compounds (Carballa et al., 2004; Hashimoto et al., 2007; Johnson et al., 2005;
Svenson et al., 2003), an ability possibly determined by their hydraulic retention times (Johnson et al.,
2005), although they seem more effective than trickle filter systems (Kasprzyk-Hordern et al., 2009).
Thus, low but biologically active levels often end up in the treated effluents, meaning additional treatment
processes are required to improve removal efficiency of these and other bioactive pharmaceuticals
(Ternes et al., 2003; Zhang and Zhou, 2005). Some have been assessed, and both nanofiltration and
reverse osmosis reportedly achieve high removal (Kim et al., 2007).
Heavy metals will be present in both industrial and domestic sewage too, although their discharge
levels into sewerage systems are closely regulated in most countries (Binkley and Simpson, 2003).
An accumulation of some to high concentration in the sludge, probably involving their chemical
The activated sludge process 59
adsorption, complexing with bacterial extracellular polymeric substances there (Nielsen et al., 2004b), and
intracellular metal accumulation (Binkley and Simpson, 2003) may largely dictate how the sludge can be
finally disposed (Arthurson, 2008).
Several studies have followed the fate of highly toxic metals like chromium, cadmium, mercury and
nickel, all used in large amounts by industry, in activated sludge systems, especially factors affecting their
adsorption (Battistoni et al., 1993; Imai and Gloyna, 1990; Kasan and Baecker, 1989), and how they affect
plant performance (Dilek et al., 1998; Vankova et al., 1999; Yetis et al., 1999). Because of their biocidal
properties (directly related to their solubility), it is not surprising that activated sludge respiration and
nitrification rates (Madoni et al., 1999), and general plant performance fall in their presence. Solids
settlability may also deteriorate from induced deflocculation events (e.g. Stasinakis et al., 2003),
increasing effluent turbidity.
Changes in microbial community composition in response to their presence have been reported. For
example, Shuttleworth and Unz (1991) showed that certain filamentous bacteria were harmed by them and
Abraham et al. (1997), Fernandes-Leborans and Herrero (1999), Hoffman and Atlas (1987) and Madoni
et al. (1996) have all demonstrated harmful effects of heavy metals on the protozoan populations in
activated sludge. Clear differences in sensitivities of different protozoan species have also been reported.
However, some evidence suggests that the community may adapt to Ni presence (Yetis and Gokcay, 1989)
and Cr (VI) resistant bacterial strains have been recovered from chromium contaminated activated sludge
(Francisco et al., 2002). Technology is now available to recover these often valuable heavy metals from
sludges with both chemical and biological leaching processes (Rulkens, 2004).
The activated sludge system needs to disperse all the incoming substrates with a suitable mixing
system, so that the microbes come into intimate contact with them. Crucially in this process, these
microbes must be encouraged to grow not as freely dispersed cells but as aggregated communities called
flocs (see chapter 3), possessing settling properties allowing an efficient separation from the liquid
supernatant in the clarifiers (see chapters 7 and 8). Sufficient O2 has to be provided at considerable
expense, to help mix the reactor biomass and satisfy the demands of the aerobically respiring organisms
responsible for substrate biodegradation. The process must also cope with massive fluctuations in flow
rates, and consequently large periodic changes in the strength and composition of the wastes. These will
occur on an hourly, daily and seasonal basis (Gray, 1989, 1990, 2005). Fluctuations in mixed liquor
temperatures and pH, which affect differentially microbial metabolic activities and their growth rates
(and hence plant performance), will also be encountered. Both for example, are known to alter the
competitive balance between the GAO and PAO in EBPR processes (chapter 10) (Oehmen et al., 2005a,
2007; Panswad et al., 2003; Whang and Park, 2006; Zhang et al., 2005b). With all these uncontrollable
variables, it might be considered remarkable that activated sludge plants work as well as they do. There
seems to be general agreement that, because of their reliability, versatility and adaptability, they will
probably provide the foundation of systems for aerobic treatment of wastes well into the future (Downing,
1992; Grady et al., 1999; Gray, 1989, 1990, 2005; Qasim, 1994; Tchobanoglous et al., 2003). However, as
this chapter will demonstrate, fundamental changes to the biotechnology of wastewater treatment are
occurring at such a rate to suggest that many of the operational features of traditional activated sludge
systems are under threat and likely to change as we seek to overcome their shortcomings, and respond to
changes in how we view their primary purposes.
WASTE DISPOSAL SYSTEMS OR WATER RESOURCES FOR RECYCLING?

No longer do we, or can we, view these simply as systems for waste disposal, but instead they have
emerged as processes we need now to exploit, to develop them as environmentally friendly and safe
methods for regenerating water and other materials for recycling purposes. With widespread community
concerns for global warming and its effect on our climate and environment, politicians in many developed
countries (where generally water in the past has been viewed as a cheap and endlessly renewable resource)
have realized, usually somewhat belatedly, that we can no longer continue to use it profligately. Increases
in how much we are charged for our water should rapidly reinforce that message.
Furthermore, no longer can we afford to dispose of our treated wastewater by simply dumping it into
oceans or another receiving body of water to be forgotten. The technology already exists, and in several
countries is in use, to safely and reliably recycle this end product for eventual re-entry into the drinking
water supply, and to replace pristine potable water with grey water for domestic functions like toilet
flushing and garden watering (Bolwidt, 2005). Consumer prejudice based largely on ignorance is probably
the main reason why this is not a more common practice. Inevitably, many people will eventually have no
realistic choice. Many industrial water needs could be, and in some countries are being met with recycled
water. It will become a much more valuable and prized commodity, and wastewater treatment systems
will need to keep pace with community views on how we should more sensibly use it. The better the
quality of the treated water produced then the more economically attractive options we have for its
reuse. So without doubt we are in the midst of a major shift in our philosophy on wastewater treatment
(Sala and Serra, 2004).
WHAT ABOUT DISPOSAL OF THE SLUDGE?

A similar change in our attitude is now noticeable in how we view the other end product of the process,
the microbial sludge or biosolids, which is a largely unexploited resource in most countries. Traditionally
it has been considered a liability to be disposed of, or wasted, often at great expense and inconvenience
(Grady et al., 1999). Sludge represents the final waste product of the activated sludge process, but is being
viewed now increasingly as a potentially attractive source of chemicals. For example, considerable
interest exists in recovering phosphorus (P) as struvite especially from sludges from plants removing P
microbiologically, for use as a fertilizer (Balmer, 2004; de-Bashan and Bashan, 2004). As other natural
reservoirs of P from countries like Nauru become exhausted, and the economics become more attractive,
this interest is likely to accelerate. Recovery of polyhydroyxyalkanoates (PHA) for biodegradable plastics
production from activated sludge, which often contain these at high levels, has long been an attractive idea
(Dai et al., 2007a; Reis et al., 2003; Salehizadeh and van Loosdrecht, 2004; Satoh et al., 1998; Serafim
et al., 2002b, 2006; Verlinden et al., 2007; Yan et al., 2006), and more is now known on how PHA yields
in sludge can be increased (Chua et al., 2003; Lemos et al., 1998; Ma et al., 2000; Majone et al., 2006;
Serafim et al., 2006), and the molecular biology of their production (Ciesielski et al., 2006).
Many other uses have been suggested for sludge (Øegaard, 2004; Tay et al., 2004), ranging from
construction materials like bricks to providing carbon substrates for producing biopesticides from Bacillus
thuringiensis (Brar et al., 2006; Vidyarthi et al., 2002; Yezza et al., 2005). These are all driven by the
challenge to find legal and profitable means for disposal of the huge amounts of this waste product, being
generated at an estimated 20–40 kg/population equivalent/year (Kroiss, 2004).
Land application with or without dewatering is probably still economically the most attractive option in
countries with sufficient space, and with laws to allow it. However, risk management considerations like
sludge toxic chemical and pathogen levels then become important, especially if that land is used
subsequently for crop production (Arthurson, 2008). Although sludges contain high levels of pathogens
(Sahlström et al., 2004; Westrell et al., 2004), evidence suggests that any risk to human health from this
practice is probably acceptably low (Gale, 2005), especially if first treated by high rate composting
processes like aerobic thermophilic autothermal digestion (ATAD) systems. Here sludge pasteurization
and pathogen inactivation is thought to occur at the elevated temperatures (460– C) employed. Methane
production from anaerobic sludge digestion, also a stabilizing technology, has long been employed in
many activated sludge plants to provide cheap energy (Sanders et al., 2002) and the microbial community
in these reactors is now well understood (Narihiro and Sekiguchi, 2007). In countries where land is
relatively scarce (e.g. Japan and parts of Europe), incineration is often the method of sludge disposal, or as
an energy source for generation of electrical power.
Other approaches to tackle problems of sludge disposal have been directed at improving its quality and
safety, by preventing toxic chemicals from entering the treatment system; or by reducing its production
(Pérez-Elvira et al., 2006) during the activated sludge process by biological degradation (Wei et al.,
2003), expensive ozone disintegration (Böhler and Sigrist, 2004; Saktaywin et al., 2005), or by
manipulating plant configuration and operational conditions (Wei et al., 2003). Many experimental plant
designs have been developed with the primary aim of reducing or in some cases totally eliminating sludge
production (Pérez- Elvira et al., 2006), but most are not implemented at full-scale plant level, and so their
ultimate value and impact on the industry is uncertain.
THE CURRENT STATE OF THE ACTIVATED SLUDGE PROCESS

As Downing pointed out back in 1992, apart from the incorporation of more sophisticated instrumentation
for in situ plant monitoring, and computerised control systems, the basic design for conventional plants
had changed little over the preceding 80 or so years. Much of this still applies. In most, the aerobic reactor
still consists of a rectangular basin with either submerged diffusers or mechanical surface agitators
supplying the mixing and aeration (Gray, 1990; Grady et al., 1999; Tchobanoglous et al., 2003). The
effluent mixed liquor is still run into a clarifier, where sludge separates from the liquid supernatant
(often inadequately), and the majority of this sludge is recycled to inoculate the incoming raw wastes
(Tchobanoglous et al., 2003).
Downing (1992) also discussed what he considered were the main stimuli for innovation in plant
design since its introduction. Originally, these systems were conceived primarily to remove carbonaceous
material from domestic sources containing naturally occurring organic compounds, and to produce treated
effluent with low enough levels of BOD5 and suspended solids (SS) to allow safe discharge into another
body of water. The 30:20 standard for BOD5 and SS respectively (chapter 1) is still used in many
countries as the acceptable level for effluents from conventional plants (Gray, 1990). Later, these had to
satisfy the requirements for lowered NH3 content, because NH3 was more toxic to fish than NO3 , and so
plant design was modified to encourage the process of nitrification. In the past three decades, with
increased community concerns for the long term environmental hazards posed by eutrophication, plants
have been designed and built which remove both nitrogen (N) and phosphorus (P) microbiologically.
These nutrient removal systems have become more common as governments enforce stricter standards on
effluent quality. Addition of extra reactor tanks providing different environmental conditions, especially in
terms of O2 availability, encouraging growth of particular physiological groups, has expanded the performance
capabilities of conventional activated sludge plants to satisfy these increased demands. Thus, incorporation of
anoxic zones with low dissolved oxygen levels but plentiful NO3 permits denitrification and N removal
(chapter 9), and anaerobic zones lacking both NO3 and O2 are thought to provide the polyphosphate
accumulating organisms (PAO) with the selective advantage needed for P removal (chapter 10). Some plant
configurations and the factors affecting their performance are briefly considered here (Tchobanoglous et al.,
2003). Attempts will also be made to suggest what wastewater treatment processes might look like in future as
their roles change. Possibly many will feed microbial fuel cells (Rabaey et al., 2007; Rozendal et al., 2008),
where their microbial communities generate electrical power from the electron donors present in the waste.
DESIGN CONFIGURATIONS FOR FULL-SCALE ACTIVATED SLUDGE

SYSTEMS: THE ENGINEER’S PERSPECTIVE
One of the main engineering appeals of activated sludge systems is their operational flexibility. Engineers
have always had prime responsibility for designing and constructing wastewater treatment processes.
Only rarely, if at all, were biologists involved or consulted. Inevitably most engineers will have a different
perspective of what the activated sludge process essentially is and how it should work. The term ‘Black
Box’ is often used to describe the engineer’s view of activated sludge. As long as what comes out of the
system is satisfactory, the how and the why of what takes place in the reactor/s was considered by most to
be of little interest or importance. Encountered operational problems were considered to have an
engineering basis and therefore should be addressed with engineering solutions. Some current engineering
and operational challenges associated with improving the performances of nutrient removal plants are
discussed by Barnard and Steichen (2006), where this philosophy is clear.
As one of its aims, this book would like to persuade engineers that there is considerable advantage to
them in understanding the basic microbiology of these processes (Wilderer et al., 2002), as seen in the
development of plants like the SHARON, ANAMMOX and CANON processes, each conceived to remove N
in non-traditional ways (Schmidt et al., 2003). In these, the engineering outcomes have come primarily from
basic microbiological studies, and not vice versa, which has more often been the case with wastewater
treatment processes like EBPR. No equivalent innovative process development has occurred for EBPR
plants, maybe because we lack the same level of microbiological understanding we have for N removal
(see chapter 9). Furthermore, high level of P removal now demanded by regulatory bodies in most countries
is best achieved not by improvements to existing plant design but with final chemical P precipitation.
There is no doubt that early activated sludge designs have evolved into forms, which were intended by
their designers to treat incoming waste cheaper and more efficiently in a smaller space, to improve
treatment of particular types of wastes, or to meet increasingly stringent discharge criteria. By and large
these aims have been achieved, but much remains to be done, and so evolutionary forces to improve
activated sludge processes will continue to operate.
The following discussion groups these systems on the basis of their purpose in terms of effluent quality.
CONVENTIONAL ACTIVATED SLUDGE PLANTS REMOVING ORGANIC

CARBON
Many of the early plants including the original design were fill and draw, semicontinuous systems (Ardern
and Lockett, 1914a,b) which, are now becoming popular again as sequencing batch reactor SBR systems
(discussed later) because of their versatility and flexibility (Irvine et al., 1997; Irvine and Wilderer, 2002).
However, most conventional systems currently run continuously. These can be differentiated on the basis of
their mixing regimes (Grady et al., 1999; Gray, 1989, 1990; Tchobanoglous et al., 2003), being referred to
as either plug flow or completely mixed systems. As Gray (1989) explains, these categories are imprecise,
since the necessary aeration of plug flow systems will inevitably produce some mixing, and total complete
mixing in the so-called completely mixed systems at full scale is unlikely to be achieved in practice.
PLUG FLOW SYSTEMS

The features of plug flow systems are shown in Figure 2.1. These are thought to encourage less
filamentous bacterial growth (chapter 5; Wanner, 1994 b,d) and hence produce better settling sludge than
completely mixed systems (Poole, 1984).
Influent Effluent
Plug flow conditions
Aeration distributed uniformly 2° Clarifier
Waste
Air sludge
's'-recycle
Fully aerobic plug flow (conventional) process
Figure 2.1. General features of a plug flow activated sludge system. ‘s’ recycle for return activated sludge
(RAS).
Such plants often operate inefficiently from uneven load distribution along the reactors. Thus O2
demand at the inlet is high, reducing the dissolved oxygen (DO) level to near zero. A long hydraulic
retention time guarantees complete nitrification and high reactivation of the sludge. To overcome
imbalances in O2 demand in plug flow systems, tapered aeration systems (Figure 2.2), which attempted to
distribute the O2 according to biomass requirements have been installed. However, in regions where air
input velocity is low, possibility of biomass settlement increased. Step aeration was thus introduced,
(Figure 2.3), achieved by dividing air supply into two portions and supplying the larger to the inlet half of
the plant.
Influent Effluent
Aeration supply tapered 2° Clarifier
along plant length
Waste
Air sludge
's'-recycle
Fully aerobic plug flow (Tapered aeration) process
Figure 2.2. A plug flow activated sludge system with tapered aeration.
Influent Effluent
Aeration distributed at approx 2° Clarifier
2/3:1/3 along plant
Waste
Air sludge
's'-recycle
Fully aerobic plug flow (Tapered aeration) process
Figure 2.3. A plug flow activated sludge system with step aeration.
The same effect of tapered aeration is achievable in plants with surface agitators (Gray, 1990), by
varying their speed and immersed depth, or in others by introducing the raw feed at several points along
the length of a plug flow system, for incremental or step feed (Figure 2.4). This design may also provide
increased operational flexibility (Grady et al., 1999; Gray, 1990). Return activated sludge (RAS) can be
fed back into the aeration tank at several points, to achieve the same effect.
Influent (feed) distributed at intervals along plant length

Effluent

2° Clarifier
Aeration distributed uniformly
Waste
Air sludge
's'-recycle
Fully aerobic plug flow (Step feed) process
Figure 2.4. A plug flow activated sludge system with a step feed.
COMPLETELY MIXED SYSTEMS

Modifications overcame the perceived aeration inefficiency inherent with plug flow systems, and led to the
development of completely mixed systems (Figure 2.5). However, these are considered more susceptible
to bulking by filamentous bacteria, for reasons discussed in chapters 5, 7.
Influent Effluent
2° Clarifier
AER
Waste
Air
sludge
's'-recycle
Fully aerobic (complete mix) process
Figure 2.5. A diagrammatic flow diagram of a completely mixed activated sludge process.
Such systems consist of square or circular reactor tanks, in which the RAS and incoming raw sewage are
mixed rapidly with the biomass, reducing any risk of toxic shock possible in plug flow systems, although
slightly increasing chances of short circuiting of bulk liquid. However, organic overloading and under-
aeration will restrict nitrification (Downing, 1992; Gray, 1989, 1990), and so these designs often
incorporate several tanks in series, to produce what is really a ‘pseudo plug flow’ generating a better settling
sludge, and avoiding any possibility of eventual denitrification and N2 gas evolution in the clarifiers.
CONTACT STABILIZATION
This (Figure 2.6) aims to achieve a more rapid adsorption of solids onto the flocs, by allowing contact
between the incoming waste and RAS in a small aerobic tank for up to one hour (Gujer and Jenkins,
1975a,b). Improved removal of particulate and readily biodegradable organic substrates is claimed, but
not removal of the slowly degraded particulate matter (terms defined later) (Gray, 1989; Tchobanoglous
et al., 2002). Mixed liquor from the contact tank is clarified and the sludge returned to a larger aeration
basin where it is conditioned separately for 5–6 hours to allow for oxidation of adsorbed material. Among
advantages claimed are an extended sludge age in the same size plant, lowered sludge production and
ability to cope with any toxic or shock loads (Lesouef et al., 1992). However, it does not produce a high
quality effluent, nitrifies poorly (if at all) and is best suited to treatment of wastes containing large amounts
of particulates (Gray, 1989, 1990).
Influent
Effluent
2°
Clarifier
AER AER
Waste
sludge
's'-recycle
Contact process
Figure 2.6. The contact stabilization process.
EXTENDED AERATION SYSTEMS

These operate with low loadings and high SS levels, and long aeration periods and sludge ages
(Eckenfelder and Grau, 1992; Gray, 1989, 1990; Tchobanoglous et al., 2002; Wanner, 1994a,c), allowing
complete oxidation and improved stabilisation of the sludge. They were first introduced by Pasveer (1959)
for low cost treatment in small rural communities in the Netherlands, and consisted of a shallow earthen
oval ditch with a horizontal brush aerator providing both aeration and circular mixing. The main principle
of design was to achieve high D.O. conditions close to the aerator to encourage nitrification while
anoxic conditions away from it permitted subsequent denitrification. These are completely mixed plants
with settle/decant phases, allowing periodic discharge of the treated effluent. Although generating
reduced quantities of sludge because of extended periods of endogenous respiration, sludge wasting is
still required to maintain a constant mixed liquor suspended solids level, the parameter often used to
control these plants. Such systems need little ongoing routine maintenance, although adequate mixing can
be a problem.
Subsequent scaling up and inclusion of separate clarification produced plants capable of handling larger
loads. One such design was the Carrousel process (Figure 2.7) with an increased liquid depth (requiring less
space) and larger low energy surface aerators (Downing, 1992; Gray, 1990; Tchobanoglous et al., 2002), and
expandable to include multiple channels (Gray, 1990) with increased numbers of aerators.
Waste
sludge
‘s’-recycle
AER ANX
Influent Clarifier
Centre wall Effluent
ANX AER
Carrousel (extended) aeration process
Figure 2.7. The Carrousel oxidation ditch.

PACKAGED PLANTS
Commonly found serving small, often isolated communities are self contained prefabricated steel or concrete
package activated sludge systems. Their essential operational features are discussed by Gray (1990). They
are usually designed to contain both sludge separation and recirculation systems, meaning they can operate at
long hydraulic retention times in the absence of any primary solids separation. Sludge production is
generally low, and many different designs are still in use around the world (Gray, 1990).
HIGH RATE TREATMENT SYSTEMS

These systems are commonly used to treat strong industrial wastes like food and dairy effluent, or when
nitrification is deliberately being avoided. They operate with very high sludge loading rates, which require
maintaining MLSS concentrations much greater than in conventional plants, efficient aeration systems and
very short sludge retention times. Sludge production is high and its settling properties often poor, although
coagulants can be used to assist in sludge dewatering (Gray, 1990). Full nitrification is seldom achieved,
and these high rate processes operate with little regard for final effluent quality (Gray, 1990). They include
the A-B process developed and widely used in Germany, consisting of a high rate stage followed then by a
second low loaded stage.
Now systems serving basically the same purpose and based on ‘aerobic granule’ technology (see below) are
under development, and these are discussed later.
MULTISTAGE TREATMENT SYSTEMS

These depend on the ability of the biomass to adapt to toxic or inhibitory influent materials, which may
affect nitrification (chapter 9). If a well nitrified effluent is required, removal of toxic components may be
accomplished by an adapted sludge in the first stage, which partially treats the influent, followed by a
second system which completes the treatment and nitrifies the effluent from the first stage (Figure 2.8).
One disadvantage is the expense of installing and maintaining two treatment systems.
Influent Effluent
Clarifier Clarifier
AER AER
Waste Waste
sludge sludge
's'-recycle 's'-recycle
2-stage Conventional process
Figure 2.8. A two stage-two sludge activated sludge process.
ACTIVATED SLUDGE PROCESS DESIGNED FOR NITROGEN (N) REMOVAL

Partial biological N removal in the activated sludge process will always occur through sludge production
(growth) and wasting, but in municipal systems this accounts for only a small part of the total N input
(chapter 9). Plants which incorporate an anoxic zone (see below) and aerobic zone may combine
denitrification and nitrification, and so achieve N removal (Bryan, 1993; Grady et al., 1999; Robertson
and Kuenen, 1992; Tchobanoglous et al., 2003). However, as we shall see below, the microbiological
conversion of ammonia to dinitrogen gas can be achieved in other ways (Schmidt et al., 2003).
Traditionally the main requirements for denitrification have been thought to be;
(i) Generation of NO3 , which requires an aerobic zone for the aerobic process of nitrification carried
out by the chemolithoautotrophic nitrifying bacteria, since NO3 levels in municipal waste waters
are usually low. These organisms can use CO2 as their carbon source since they are autotrophs.
(ii) Absence of O2 in the anoxic zone, which instead contains the NO3 generated aerobically as
the electron acceptor, encouraging anaerobic respiration by the chemoorganoheterotrophic
denitrifying organisms there.
(iii) A community which can denitrify, and presence of a suitable organic electron donor or energy
source to allow denitrification or anaerobic respiration. Methanol was an early choice, but became
prohibitively expensive, and so later processes were based on other cheaper substrates or those
already present in the biomass (Shin et al., 1992; Bryan, 1993). Some of these designs are
discussed next.
The ability to denitrify is widespread among bacteria (chapter 9). Denitrification has always been
considered to occur only under anoxic conditions where NO3 and not O2 acts as the terminal electron
acceptor, since it was always thought that O2 is preferred by these facultatively anaerobic cells, if
available to them. However, it is clear that many bacteria can denitrify aerobically too (Robertson and
Kuenen, 1992), but its importance in activated sludge systems has never been demonstrated.
PROCESS CONFIGURATIONS IN SYSTEMS REMOVING NITROGEN

Most conventional nitrogen removing systems are single sludge processes (Barnard, 1973), where
a single microbial community is employed for both the nitrification and denitrification stages
(Tchobanoglous et al., 2003; Toerien et al., 1990).
Two anoxic reactor types may be distinguished conveniently by the energy sources used by the
denitrifying bacteria. Thus, Wuhrmann (1957) proposed a design where the aerobic zone preceded the
anoxic zone (Figure 2.9).
Influent Waste Effluent
sec 2° Clarifier
AER ANX
's'-recycle Waste
sludge
Wuhrmann process (N/D)
Waste
Influent sludge Effluent
2° Clarifier
AER ANX
's'-recycle
Wuhrmann process (N/D) adapted to improve
denitrification by carbon substrate addition
Figure 2.9. The Wuhrmann activated sludge configuration for nitrogen removal, showing carbon (as
methanol) supplementation.
Consequently, organic substrates required for denitrification were probably supplied mainly from
endogenous death and lysis of active biomass, since most had been utilised already in the aerobic reactor.
As the rate of substrate release is low, so is the denitrification rate, although it can be boosted by methanol
or acetate addition (Figure 2.9). Such anoxic zones are usually referred to as secondary anoxic zones.
Ludzack and Ettinger (1962) changed this configuration by placing the anoxic reactor in front of the
aerobic tank to receive the incoming raw sewage, and in partial contact with it (Figure 2.10). These anoxic
zones are called primary anoxic zones.
Influent Effluent
ANX AER 2° Clarifier
's'-recycle Waste
sludge
Ludzack-Ettinger process (N/D)
Figure 2.10. Ludzack-Ettinger plant configuration.
Much of the organic material was removed in this anoxic zone, reducing the active heterotrophic
community in the aerobic zone and the competition for O2, and allowing increased nitrification to
occur (Tchobanoglous et al., 2003; Toerien et al., 1990). Crude mixing of contents of the anoxic and
aerobic zones was achieved by the action of aerators and allowed denitrification to occur, but lack of
control of this mixing gave variable plant performances (Bryan, 1993). Separation of the anoxic and
aerobic zones and recycles between the two was considered necessary by Barnard (1975a,b, 1976) to
improve plant performance, and the outcome is known as the modified Ludzack-Ettinger process
(Figure 2.11). It will not eliminate NO3 completely, because that formed in the aerobic zone is
recycled in both the ‘a’ recycle and effluent flows (Bryan, 1993; Randall, 1992).
Waste
ANX AER 2°Clarifier
‘a’-recycle
‘s’-recycle
Modified Ludzack-Ettinger process
Methanol Waste
2°Clarifier
ANX AER
‘a’-recycle
‘s’-recycle
Modified Ludzack-Ettinger process adapted to improve
Figure 2.11. The Modified Ludzack-Ettinger activated sludge process showing carbon (as methanol)
supplementation to enhance denitrification. ‘a’ recycle for return of mixed liquor from the aerobic to anoxic
reactor.
Then Barnard developed the Bardenpho process (Figure 2.12) by adding another anoxic reactor after
the aerobic reactor, giving a four stage system, where both primary and secondary anoxic zones were
incorporated into the one design (Tchobanoglous et al., 2003; Toerien et al., 1990). This had the dual
effect of increasing denitrification rates and producing a nearly NO3 free effluent. A re-aeration reactor
was also incorporated after the secondary anoxic reactor, where air stripping of N2 gas from the sludge
was achieved, preventing it rising in the clarifier subsequently, and permitting nitrification of any NH3
produced in the secondary anoxic reactor (Toerien et al., 1990). This design configuration, which often
removed additional phosphate, is particularly important since it can be considered the precursor of most of
the plants which have since followed.
Waste
re
sec A
ANX AER ANX E 2° Clarifier
R
‘a’-recycle
‘s’-recycle
4 stage Bardenpho process (N/D)
Waste Methanol
re
A 2° Clarifier
sec E
ANX AER ANX R
‘a’-recycle
‘s’-recycle
4 stage Bardenpho process (N/D) adapted to improve
Figure 2.12. Configuration of the Bardenpho process showing carbon (as methanol) supplementation to
encourage denitrification.
The R-D-N and Biodenitro processes

In the Czech Republic and other European countries, the R-D-N (Regeneration-Denitrification-
Nitrification) process (Kos et al., 1992) is popular for N removal (Bejvl et al., 2002). This process,
designed to overcome shortcomings of other N removal processes, has the configuration shown on
Figure 2.13. By incorporating a contact stabilization (see Figure 2.6) RAS reaeration (regeneration) step into
the process, it effectively increases the aerobic sludge age, reducing the nitrification reactor volume. Sludge
with good settlability is reportedly generated (Kos et al., 1992). It has since been modified to remove P as
well, with the R-AN-D-N process (see Figure 2.20).
The Biodenitro process consists of at least two oxidation ditches in series and so is complex to operate
and expensive to construct but is used around the world because of its high N removal capacity
(Tchobanoglous et al., 2003). Its operating principle is different to the plants discussed so far, as it runs
as a continuous batch process consisting of several operational phases (4) over a 4 hour cycle. Influent
alternatively enters zones configured to operate either as anoxic or aerobic reactors permitting anoxic
denitrification (90 min phase duration) and aerobic nitrification (30 min duration) to occur across the
cycle. The volume of influent feed is small compared to the total reactor volume, and mixing ensures the
biomass is kept in suspension during the anoxic phases. The process was outlined in the first edition of this
book (Seviour and Blackall, 1999).
Influent Waste
sludge Effluent
R D N 2° Clarifier
Anoxic IR
selectors
RAS
R-D-N process
Figure 2.13. The R-D-N process configuration.
NOVEL N REMOVAL PROCESSES

As described later, some of these configurations designed for N removal were the immediate precursors
of system removing both N and P, which generally were obtained after only fairly minor modifications
to existing designs. However, before P removal processes are described, we need to mention the several
novel processes for N removal that have been developed. Some are operating at full-scale (Schmidt
et al., 2003; van der Star et al., 2007). These arose from increases in our understanding of basic and
novel aspects of bacterial N metabolism. They demonstrate precisely the advantages arising
from engineers and microbiologists working together, and point the way to how other plants might
be conceived and designed in the future. Some will be discussed here, although the reader may need to
go to chapter 9 where the microbiology of N removal is discussed to understand more fully how
they function.
The SHARON (single reactor system for high rate ammonium removal over nitrite) operates as
a chemostat with no biomass retention, and runs usually at quite high temperatures under conditions
generally where the selected D is 4mmax for the nitrite oxidising bacteria (NOB) but not the ammonia
oxidising bacteria (AOB). Consequently only partial nitrification occurs and nitrite accumulates as the
end-product. This partial oxidation requires less aeration, and if used with conventional denitrification,
less organic substrate is required (commonly methanol) since only nitrite has to be reduced to dinitrogen
gas (Schmidt et al., 2003).
However, SHARON is more often used in combination with the ANAMMOX process where the nitrite
is reduced to dinitrogen gas, with ammonia as the electron donor (chapters 1, 9), in the absence of any
aeration. In this combination, no methanol is added to SHARON to permit chemoorganoheterotrophic
denitrification. After 50% of the ammonia has been oxidized by the Anammox bacteria, the pH drops and
so further oxidation ceases (Schmidt et al., 2003). This process, which is quite sensitive, is now operating
at full-scale in Amsterdam (van der Star et al., 2007), with all the design and economic advantages
discussed by Schmidt et al. (2003). However, because the Anammox organisms grow so slowly, start-up
times are long, and overloading can affect performance, by shifting the very delicate balance between the
different populations.
The CANON (completely autotrophic nitrogen removal over nitrite) process is an extension of the
SHARON/ANAMMOX process, but carried out in a single aerated reactor (Schmidt et al., 2003), and
possibly involves previously undescribed Anammox organisms (Third et al., 2005a,b). Here the aerobic
AOB oxidise ammonia to nitrite, consume all the oxygen and thus allow the anaerobic Anammox bacteria
to oxidise ammonia and reduce the nitrite to dinitrogen gas.
ACTIVATED SLUDGE SYSTEMS REMOVING NITROGEN AND

PHOSPHORUS
Further design advances have led to plants removing not only carbonaceous compounds and nitrogen, but
also phosphate (Grady et al., 1999; Henze et al., 1995c; Meganck and Faup, 1988; Tchobanoglous et al.,
2003; Toerien et al., 1990). These can be divided into mainstream and sidestream P removal processes,
but only the former are described here. Briefly, the process of biological P removal in excess of their
normal metabolic or growth requirements is achieved by the accumulation of polyphosphate (polyP)
granules in bacteria, a process which occurs under particular operating conditions (Blackall et al., 2002;
Oehmen et al., 2007; Seviour and McIlroy, 2008; Seviour et al., 2003). This phenomenon is sometimes
called ‘enhanced biological phosphorus removal’ (EBPR). The immobilized intracellular P is then
removed by normal sludge wasting. A detailed microbiological understanding of this process is only now
becoming clearer (chapter 10) although the conditions required for successful P removal in such plants
are extensively documented. They operate on the principle of establishing alternating feast/famine
regimes where the anaerobic zone is the feast and the aerobic zone the famine stage. The PAO are
considered to be selectively favoured under these cyclic conditions, as explained later, and so eventually
dominate the EBPR community.
The requirements include (Barnard and Abraham, 2006; Oehmen et al., 2007; Meganck and Faup,
1988; Mino et al., 1998; Toerien et al., 1990; Seviour et al., 2003; Tchobanoglous et al., 2003; van
Loosdrecht et al., 1997a)
(i) The raw influent sewage to enter an anaerobic zone (feed stage), selectively favouring the PAO by
providing them with the readily metabolizable organic carbon and energy sources present in the
influent sewage.
(ii) The necessity to limit the levels of NO3 entering this anaerobic zone, since denitrifying bacteria
are thought to utilise these organic substrates by respiration, and so reduce the level available to
the PAO. Thus, plants removing P also remove N (Barker and Dold, 1996b; Oehmen et al., 2007;
Seviour et al., 2003; Tchobanoglous et al., 2003).
(iii) Strict maintenance of anaerobic conditions in the anaerobic zone, by limiting the aeration
caused by any turbulence, will also restrict any aerobic respiration of the organic substrates
present.
(iv) Alternating anaerobic/aerobic regimes for cycling the biomass, where the aerobic zone represents
the famine stage, and where P assimilation and polyP synthesis by the biomass occur.
Most of these requirements were discovered empirically, but they are the bases for all the full-scale
EBPR plant configurations now operating (Grady et al., 1999; Mino et al., 1998; Tchobanoglous et al.,
2003), and are discussed briefly next. However, it is clear that EBPR can occur under conditions markedly
different to these, again illustrating the flexibility and adaptability of the process and the organisms
involved. Thus, a famine anoxic zone can substitute for a famine aerobic zone in EBPR (Oehmen et al.,
2007; Seviour et al., 2003), and a continuously aerated EBPR process has been described where selective
pressures on the microbial community similar to those brought about by the anaerobic: aerobic feed/
famine cycling can be imposed in a fundamentally different manner (Ahn et al., 2007).
The pioneer work of Barnard was crucial in helping to resolve the process requirements for
EBPR (Barnard, 1994, 1998; Wentzel et al., 1991a,b), by modifying the Bardenpho or Phoredox
system (Figure 2.12). This gave the five-stage Phoredox process (Figure 2.14) where adding an anaerobic
zone to the head of the plant led to reliable phosphate removal.
Waste
re
A
sen E 2° Clarifier
ANA ANX AER ANX R
‘a’-recycle
‘s’-recycle
5 Stage Phoredox process
Figure 2.14. The five stage Bardenpho process.
Barnard (1982) also agreed that NO3 recycle in the RAS must be minimised to avoid possible
denitrification in the anaerobic zone. Here volatile fatty acids (acetate), either present in the incoming
waste, or formed in situ by the microbial community as fermentation end products are utilised by the PAO
biomass (chapter 10) and stored as PHB or poly b-hydroxyalkanoates (PHA). Energy required for
PHB/PHA synthesis is derived from the anaerobic degradation of polyP, resulting in the release
of orthophosphate into the bulk liquid, and/or from glycogen metabolism, as described in detail later
(chapter 10). Aerobically, the reverse process is thought to occur in the same populations, with the
catabolism of stored PHA providing energy for P uptake and the synthesis of polyP, and glycogen
replenishment (Blackall et al., 2002; Mino et al., 1998; Oehmen et al., 2007; Seviour et al., 2003; van
Loosdrecht et al., 1997a).
Subsequent simplification of the Phoredox process by increasing the size of the primary anoxic reactor
with increasing denitrification efficiency, and negating the need for any secondary anoxic and reaeration
reactor (Bryan, 1993; Toerien et al., 1990) gave rise to the three-stage Phoredox process. This design led
to less NO3 being returned to the head of the plant, and so increased EBPR capacity resulted.
Unfortunately, commercial interests leading to process patents have often had undesirable outcomes in
restricting and delaying the introduction of some EBPR processes in certain parts of the world, and
limiting opportunities to test them under different conditions. Examples are the patents successfully taken
out by Air Products in the US on the two-stage high rate Phoredox and three-stage Bardenpho systems,
processes, which they called A/O and A2O respectively (Barnard, 1994; Oehmen et al., 2007), and
shown in Figure 2.15 and Figure 2.16. The Bardenpho configured plants generally tend to be long
sludge age plants while A/O systems operate at shorter sludge age, and are popular in many countries
including Japan.
Waste
2° Clarifier
ANA AER
's'-recycle
2 Stage Phoredox or A/O process
Figure 2.15. The A/O activated sludge system configurations.

Waste
ANA ANX AER 2° Clarifier
'a'-recycle
's'-recycle
3 Stage Phoredox or (A2/O) process
Figure 2.16. The AO2 activated sludge system configurations.
Plants treating influents with high levels of readily biodegradable COD (RBCOD- see later)
appeared to be less sensitive to NO3 poisoning (Barker and Dold, 1996a; Bryan, 1993; Ekama et al.,
1983; Pitman, 1991; Wentzel et al., 1992; WRC, 1984) because of their capacity to rapidly denitrify
(Toerien et al., 1990). Some plants now incorporate pre-fermenters at their heads to increase the
levels of volatile fatty acids in their feed to increase and make EBPR more reliable (Barnard and
Abraham, 2006).
The Johannesburg process was developed from the Phoredox system, with the aim also to overcome
detrimental effects of NO3 on EBPR (Barker and Dold, 1996a), by introducing an endogenous sludge
denitrification vessel in the RAS line (Barnard, 1994), as shown in Figure 2.17. High biomass concen-
trations in this vessel were intended to ensure that complete denitrification would occur (Pitman et al.,
1991). However, this configuration increases the anoxic biomass fraction to a level which restricts the size
of the primary anoxic or anaerobic zones. Yet such plants have been built and operate, apparently
successfully, in several countries (Barnard, 1994).
Influent Waste
sludge Effluent
sec 2° Clarifier
ANX ANA ANX AER
'a'-recycle
's'-recycle
Johannesburg process
Influent Waste
sludge Effluent
2° Clarifier
ANX ANA ANX AER
'a'-recycle
's'-recycle
Johannesburg process (modified)
Figure 2.17. The Johannesburg activated sludge configuration.
A further modification to the basic Phoredox process with the same aim of avoiding problems of NO3
inhibiting P removal was that from the University of Cape Town (UCT) (Ekama et al., 1984), and is
called the UCT process (Argaman, 1991; Bryan, 1993; Toerien et al., 1990; Wentzel et al., 1991b, 1992).
Here the RAS (s recycle) passes first into the anoxic rather than the anaerobic zone (Figure 2.18) and a
mixed liquor recycle runs from the anoxic reactor to the anaerobic reactor (r recycle). This configuration
was designed to ensure that any NO3 in the RAS is fully denitrified in the anoxic reactor before reaching
the anaerobic zone (Oehmen et al., 2007; Toerien et al., 1990).
Waste
‘r’-recycle sludge Effluent
Influent
2° Clarifier
ANA ANX AER
‘a’-recycle
‘s’-recycle
University of Cape Town (UCT) (VIP) process
Figure 2.18. The UCT activated sludge configuration.
The so called modified UCT process (Figure 2.19) divided the single anoxic zone into two compartments,
allowing for improved individual control of the mixed liquor and RAS streams (Oehmen et al., 2007;
Tchobanoglous et al., 2003; Toerien et al., 1990), and such plants operate successfully globally.
Waste
‘r’-rec sludge Effluent
Influent
A
N 2° Clarifier
ANA X ANX AER
‘a’-recycle
‘s’-recycle
Modified University of Cape Town (MUCT) process
Figure 2.19. The modified UCT activated sludge system.
Like most South African systems, this configuration uses long (420 day) sludge ages, with
accompanying problems from bulking filamentous bacteria (chapter 7), but a shorter sludge age variant
called the Virginia Initiative Project or VIP process (Daigger et al., 1988) has been derived from it.
The Biodenipho configuration evolved in a similar way from the Biodenitro configuration described
earlier, by incorporating an anaerobic zone at the head of that plant configuration to encourage high EBPR
across the cycles (Seviour and Blackall, 1999c).
OTHER EBPR SYSTEMS

Alternatively, our understanding now that some PAO assimilate P and store it as polyP under anoxic
conditions where NO3 not oxygen, acts as the electron acceptor (Seviour et al., 2003: chapter 10) has
been exploited. One attraction is a simultaneous removal of N and P. This principle forms the basis for the
Dephanox process (Bortone et al., 1996, 1999), which in laboratory trials performed favourably when
compared to the Johannesburg process, consistently generating a treated effluent with 51 mg/l P. It also
deals well with influents with low COD/TKN ratios, (i.e. those not suitable for most other EBPR systems),
and with considerable savings in aeration and sludge disposal costs. However, it is still only experimental,
and requires two clarifiers.
The N removing R-D-N system mentioned earlier has also evolved into the R-AN-D-N [Regeneration-
Anaerobic-Denitrification (anoxic)-Nitrification (aerobic)] process for N and P removal. Like its
ancestor it has been adopted for treating industrial effluents at full scale in the Czech Republic, but is
not used much elsewhere despite its claimed advantages (Bejvl et al., 2002; Wanner et al., 2000). It too
incorporates a regeneration zone to increase sludge age and an anaerobic zone to encourage P removal
(Figure 2.20). Occasional foaming and bulking problems have been reported, with Candidatus ‘Microthrix
parvicella’ appearing in the winter (Bejvl et al., 2004).
Waste
R AN D N 2° Clarifier
Anoxic IR
selectors
RAS
R-AN-D-N Process
Figure 2.20. The R-AN-D-N configuration.
SEQUENCING BATCH REACTORS (SBR)

Sequencing batch reactors (SBRs) have been defined as ‘time orientated periodic unsteady state cyclical
processes’ (Irvine and Ketchum, 1989; Irvine and Wilderer, 2002; Irvine et al., 1997; Ketchum, 1997).
Maybe they should more appropriately be referred to as ‘sequentially fed reactors’. SBR systems consist
of a series of one, or sometimes more than one reactor, each of which operates under batch mode. They
run on a ‘fill and draw’ principle, similar to how many of the earliest activated sludge systems
operated (Irvine and Ketchum, 1989; Ketchum, 1997). They represent the ideal plug flow system, and
differ fundamentally from the continuous flow systems discussed above, in a number of essential features
(Irvine and Wilderer, 2002);
(i) Separation of the biomass is achieved in the reactor itself and not in a separate clarifier system.
(ii) SBRs are ‘time orientated’ and not ‘space orientated’ systems like the continuous flow processes,
in that all the unit processes occur in a single reactor, where conditions can vary over a repeated
time cycle. Each cycle can consist of a number of separate operational phases of fill, react, settle,
decant and idle, the duration and conditions of each set to a predetermined value.
(iii) SBR influent and effluent streams are uncoupled in time.
Because the length and imposed operational parameters for each individual phase can be manipulated,
SBRs are versatile and flexible, and make very attractive research systems. With repeated cycles, the
bacterial populations in a sense become ‘synchronized’, allowing relationships between SBR population
dynamics and chemical transformational changes easier to interpret than in completely mixed systems.
Much of what we know about the microbiology of N and P removal, for example, has come from studies
with laboratory scale SBR systems (Keller, 2005; Oehmen et al., 2007; Seviour et al., 2003; chapters
9, 10), where the feast/famine conditions required for EBPR can be imposed in strictly targeted ways.
They also generally generate sludge with excellent settling properties, possibly as a consequence of the
feast/famine conditions established there (Irvine and Wilderer, 2002; chapters 5, 7).
SBRs are increasingly being installed as full scale systems around the world for a range of applications.
Plants vary from the single tank Unifed process to complex multitank (24 SBR reactors) plants like that
operating at Ringsend in Dublin. They are used for N and P removal from both domestic and industrial
wastewaters (Keller, 2005; Keller et al., 2001; Peters et al., 2004), and for treating high strength
wastes, often modified to incorporate aerobic granule technology (Keller, 2005) discussed below. Their
applications are likely to expand as current SBR laboratory scale processes achieving desirable outcomes
like simultaneous nitrification and denitrification (chapter 9) are eventually scaled up.
FULL SCALE SBR DESIGN AND OPERATION

Careful thought is needed to operate SBRs effectively at full scale. Operational aspects to consider are
their hydraulic capacity, sludge settlement rates and whether N and/or P removal is sought. With a single
reactor trying to achieve the three outcomes of waste treatment, clarification and flow equalization, all
intimately interlinked, some compromise in efficiency of operation of each is inevitable. Such operational
interdependence probably represents the largest weakness in designing and operating SBRs.
As SBR operation is time-sequenced, it is imperative that the storage basins are available at all times
to store incoming flows. So a minimum cycle time has to be set, which first requires characterizing
the plant in terms of its maximum flow, duration of maximum flow incidents and average flow rates.
Under all these conditions sufficient time must be allowed for the sludge to settle and effluent to be
decanted. The depth the sludge is required to settle will also dictate the settle period, illustrating how
design parameters interact in SBR design.
Operation will depend on the desired outcomes. P removal is readily achieved in SBRs because of their
abilities to impose the selective pressures necessary (Keller, 2005). N removal is less easily achievable.
The denitrifiers and RBCOD need to be brought together in an anoxic environment, but in a conventional
SBR cycle, anoxic conditions are observed only during settle and decant, by which time no RBCOD is
available. So to achieve denitrification by the chemoorganoheterotrophic bacteria, basins can be fed
during both settle and decant phases. However, this strategy may have implications for final effluent
quality from short-circuiting, and so reactor baffling may be required. It is possible to have a mixed/
nonaerated fill period during the react phase, but this will reduce the effective sludge retention time or
sludge age (see later), thus increasing the cycle time and subsequently increasing basin size. Some
systems seem to achieve denitrification by ‘preserving’ RBCOD stored intracellularly as PHA by the
biomass, a process encouraged by installing an anoxic/anaerobic selector prior to the SBR allowing
P release and PHA production. By carefully controlling plant aeration and DO levels, these stores of PHA
may be maintained and used for denitrification, either during settle and decant phases, or during the
fill/aerate phase through simultaneous nitrification and denitrification (SND). For efficient nitrification,
the slow growing nitrifying bacteria require adequate time, and to achieve a sufficiently long sludge age,
a larger SBR reactor may be required.
MEMBRANE BIOREACTORS (MBR)

Membrane reactors are systems where separation of the solid/liquid phases is achieved not by gravity
settling in a clarifier as it is in a conventional activated sludge system, but with a membrane solid/liquid
separation system (Judd, 2008; Rittman, 2006b; Stephenson et al., 2000, 2001). Consequently the final
effluent has lower suspended solids and hence lower turbidity than that from clarifiers. Biomass
concentrations in the reactor are maintained because it is completely retained there, a feature allowing
maintenance of long sludge retention times in small volume reactors (Ben Aim and Semmens, 2002;
Daigger and Crawford, 2005; Visvanathan et al., 2000). Other advantages over conventional systems
using clarifiers include;
(a) formation of flocs with good settling properties is no longer essential and the problems of bulking
(chapters 7, 8) are eliminated, although foaming can occur
(b) all solids have the same sludge residence times determined by the discharge flow rate, and so
knowing hydraulic residence times is not relevant in MBRs
(c) sludge production rates are claimed to be lower, but the wasted sludge is less easily dewatered
before disposal
(d) important slow growing organisms like the nitrifying bacteria are always retained within the
reactor.
In some systems the membrane is external to the actual reactor, but in others, membranes are
immersed. Several different membrane module designs are used in MBRs depending on manufacturer.
Flat sheet membranes and vertical and horizontal hollow fibres have all been used, but the general view is
that designs are likely to improve as our understanding of the main problem associated with MBRs, that of
membrane fouling (Ben Aims and Semmens, 2002; Liao et al., 2004a,b; Trussell et al., 2006) and
subsequent decreases in transmembrane liquid flux, increases. The technology is also likely to become
cheaper as the market expands. This fouling seems to be not from the solids but from the colloidal fraction
in the waste (Ben Aims and Semmens, 2002; Geng and Hall, 2006; Rosenberger et al., 2006), and possibly
biofilm formation on the membrane involving specialized microbial populations different to those in the
reactor community (Zhang et al., 2005a). Defouling is achieved by backwashing or cleaning with
chemicals like chlorine or acids and bases. Full-scale MBRs need to be designed to accommodate this
requirement (Chang et al., 2002), and the possibility of membrane damage directly resulting from
chemical cleaning recognized (Wintgens et al., 2003).
Although considered more suitable for small scale treatment plants because of membrane costs and
performance requirements, some large-scale MBRs (e.g. Swanage, England) operate successfully for
applications including N and P removal (Daigger and Crawford, 2006). Both capital and operating costs
are higher than with clarifier-based systems, but again these are likely to fall. Microbiological studies with
MBR communities are well underway. Although generally similar to those seen in conventional systems
carrying out the same processes like EBPR (Miura et al., 2007a,b), they appear to be dominated
often by very slow growing novel strains favoured under the stringent conditions of nutrient limitation
imposed by the prolonged biomass retention times characterizing MBRs (e.g. Chen and Lapara, 2006;
Lapara et al., 2002). Claims have been made that MBRs are more efficient in removal of pharmaceuticals
(Radjenovic et al., 2007), some viruses, Giardia and Cryptosporium cysts and bacterial faecal indicators
than unit processes like tertiary filtration and conventional activated sludge systems (Ottoson et al., 2006;
Zhang and Farahbakhsh, 2007).
AEROBIC GRANULE TECHNOLOGY

SBR reactors designed to operate with the biomass organized as aerobic granules not as flocs, are
theoretically attractive options to conventional activated sludge systems (Figure 2.21). Their attractions
include; a small reactor volume can be used because granules provide a very high biomass
concentration; a very high settling rate (de Kreuk and de Bruin, 2004). Aerobic granule formation from
pre-existing flocs seems to be encouraged with very short SBR settling times (Ivanov et al., 2006),
ensuring loosely aggregated biomass is washed out of the reactor, and increasing shear which might
stimulate EPS production (McSwain et al., 2004) improving granule density and strength, and increase
their diameter (Liu and Tay, 2004). High sludge loading rates and low HRT seem to have the same
outcome, and temperature may also play a role in stabilizing aerobic granule organization (de Kreuk
et al., 2005).
50.00 µm
Figure 2.21. SEM of an aerobic granule generated in an EBPR SBR removing P (J. Ahn).
However, the precise mechanism for their formation (Li et al., 2006), as with flocs (chapter 3) is still
unclear, although slow growth rates and cell starvation appear to help. It seems likely that several different
mechanisms may exist, and it might not be possible to come up with a single mechanistic explanation for
granulation. Each application may require a separate explanation.
However, this technology possibly provides a means for more carefully controlling and manipulating
how the microbial community is organized than is achievable with flocs, with all the associated operating
advantages. There is still much to be learned about the influence of parameters like granule diameter on
their performance (Chiu et al., 2007). They have been shown to remove N and P successfully from a range
of wastes (de Kreuk et al., 2005; Li et al., 2005; Lemaire et al., 2008) and to treat industrial wastewaters
(Schwartzenbeck et al., 2005; Nancharaiah et al., 2006), but in most cases have been tested with a
synthetic sewage and simple carbon substrates. However, their operational flexibility and ruggedness is
shown by their ability to remove COD, N and P from complex wastes (Cassidy and Belia, 2005). What we
know about their microbiology is best left to chapter 3.
THE FUTURE FOR ACTIVATED SLUDGE PLANT DESIGN?

It is likely that activated sludge plants will continue to evolve, and new designs continue to appear, in
response to many of the same stimuli mentioned earlier. We have just looked at examples where plant
design was less empirically based. Pressure will also be brought to bear on existing plants to perform
better (Barnard and Abraham, 2006; Barnard and Steichen, 2006), encouraged by increasingly stringent
regulatory requirements for effluent quality, especially as water recycling demands become more
imperative. As the microbiology and chemistry of activated sludge becomes better understood, monitoring
these systems will be more sophisticated, automatic and holistic. We suggested in the earlier version of
this book that dedicated single unit processes, containing a specialised microbial community capable
of carrying out one particular function or process will become commonplace (Seviour et al., 1999c). Such
extensions are much cheaper than constructing completely new plants and provide the necessary
flexibility to cope with changing demands, especially if they operate as SBRs. These add-on units
are already in use to treat some industrial wastes containing toxic xenobiotics, but their application is
likely to extend to systems for the removal of endocrine disrupter substances and other environmentally
harmful compounds, as our understanding of the systematics, ecology and physiology of the microbial
communities involved increases.
Membrane bioreactors will become more attractive as membrane technology improves and they
become cheaper to manufacture. It is hard to suggest how soon solids separation problems like bulking
will be rare events, but at the present rate of acceptance of membrane based clarification and advances in
technology it may not be too long before clarifiers are no more than archaeological curiosities.
However, probably the most profound shift in how this industry operates will be the one we discussed
earlier, where these systems will no longer be viewed as disposal systems for polluted water, but valuable
sources of recyclable and recoverable water, energy and other materials. It is likely that this will be the
major impetus for change well into this century (Tchobanoglous et al., 2003; Wilderer, 2004), as the
consequences of climate change begin to impact.
MONITORING THE PROCESS – THE ENGINEERS’ REQUIREMENTS

In any design and control exercise, the engineer would probably characterize the plant in a way quite
different to a chemist or microbiologist, although attempts to model the design concept (see later) requires
information on the chemical nature of the raw sewage and treated effluent. Microbiological information of
the same detail would not normally be sought, although perhaps that will change with time. The widely
used IWA ASM models (Henze et al., 2008) with their increasing reference to physiological bacterial
types might help persuade the industry of its value and encourage widespread application of monitoring
technology like microarray screening to their plants (e.g. Loy et al., 2005). This issue will be raised later.
Some of the parameters process design engineers would monitor (Pipes and Zmuda, 2007) are
discussed next.
Influent flow data and hydraulic load

Hydraulic retention time (HRT) measures the time wastewater spends in the reactor, and needs to be
long enough for floc formation and desired metabolic processes to occur. Hydraulic load will vary,
depending for example, on weather conditions, and accurate flow data are crucial in assisting plant
design and operation, since knowing flow will help determine parameters like F/M ratios, MLSS and
MLVSS (see below), and in deciding plant aeration requirements (Grady et al., 1999; Gray, 1989;
Tchobanoglous et al., 2003; Wanner, 1994a,c).
Influent alkalinity and pH

The optimal pH range for activated sludge is assumed to be between 6.5 and 8.5 (Gray, 1989). So influent
pH is important, and buffering ability of the mixed liquor (its alkalinity) in full scale plants should
normally be sufficient to cope with small pH changes. However, in laboratory scale systems, this may not
always happen. Microbial activity will moderate any effects of high pH by CO2 evolution producing
bicarbonate (HCO3 ) during respiration. Consequences of low influent pH are probably more important,
and influent pre-treatment may need to raise pH and alkalinity, since nitrification is particularly
susceptible to low pH (chapter 9). It may also contribute to it through release of Hþ ions by the AOB, and
pH may fall to levels where it no longer occurs (Painter, 1986; Winkler, 1984). However, denitrification
increases pH to counter this. Low pH influents can encourage bulking by fungi (Jenkins et al., 2004b) and
affect EBPR processes by changing the balance between the PAO and GAO (Chen and Gu, 2006; Oehmen
et al., 2007), as detailed in chapter 10.
Dissolved oxygen and redox levels

Most plants install dissolved oxygen (DO) probes in their aerobic reactors, but redox probes are less
commonly used. Both provide valuable information about the biological activity of the biomass, and
the data are often used to control aeration rates, cutting operating costs. Biomass specific oxygen
uptake rates (SOUR) can also be determined in situ. Rugged probes are available for measuring both
parameters, but their membranes can become fouled by cell growth, and they should be regularly (at least
daily) calibrated.
Mixed liquor suspended solids (MLSS) and mixed liquor volatile

suspended solids (MLVSS)
These provide crude estimates of biomass concentrations. Both are used with influent BOD5 values
to determine F/M ratios (see next). If influent BOD5 is not measured regularly, as is often the case,
then MLSS or MLVSS (mixed liquor volatile suspended solids), becomes the major control parameters.
The MLVSS represents the organic proportion of the biomass, but not necessarily its active
portion (Gray, 1989). Both are functions of influent load, process volume and particularly sludge age
(Wanner, 1994a,c).
Sludge loading or food/microorganisms ratio (F/M ratio)

The sludge loading rate, organic loading rate or the F/M ratio is adopted in many plants as the key
operational parameter (Gray, 1989; Hawkes, 1983; Tchobanoglous et al., 2003).
F/M ¼ BOD5 · Dilution Rate D/
MLVSS concentration of biomass · volume of reactor
However, since the BOD test is slow, and MLVSS measures neither the active biomass, nor is
necessarily proportional to it, this parameter needs to be viewed critically. F/M ratio is controlled by
sludge wasting rates, where high rates decrease sludge age and increase the F/M ratio. It may be useful in
monitoring conventional plants which usually operate with F/M ratios of 0.1–0.4, since if M is large and
F is small, less substrate is available for biomass production. So low F/M processes produce less biomass
per unit of applied organic load and generate a high quality effluent with good floc formation, as seen with
MBRs. However, O2 requirements are greater, and the principle behind the design of the so-called
extended aeration or low rate systems operating at about 0.05 kg/kg MLVSS/day is to minimize this.
Conversely, high F/M systems use less O2 per unit load, but sludge production is higher and effluent
quality poorer, as with high rate systems.
Chudoba et al. (1991) and Grady et al. (1996, 1999) and others have supported replacing F/M with the
S0/X0 ratio, where S0 is the initial substrate concentration and X0 the initial biomass concentration. This is
not the same as the F/M ratio, but it has been adopted by many modellers to measure biomass growth
kinetics, although its biological significance is uncertain.
Sludge age or sludge residence time (SRT) or mean cell residence

time (MCRT)
Although sludge age control is important in modern plant operation (Henze et al., 1995c; Tchobanoglous
et al., 2003; Wanner, 1994a,c), adequate methods for controlling it are not always used. Sludge age is
considered the fundamental parameter in any design exercise (Orhon and Artan, 1994) and all other
operational parameters are then self-regulating (Beccari et al., 1993; Ekama and Marais, 1977, 1979a,b;
Ekama et al., 1983, 1984; Osborn et al., 1986). It can be determined by dividing the total amount of sludge
solids in the system by the rate of loss of sludge from it, although it is not usually feasible to calculate
either precisely (Gray, 1990). Sludge age is controlled by regulating the rate at which sludge is wasted
from the system before clarification (Gray, 1989, 1990; Henze et al., 1995c; Tchobanoglous et al., 2003).
MONITORING THE PROCESS – THE CHEMIST’S REQUIREMENTS

The chemist would probably assess the process differently to an engineer, by seeing it in terms of
comparing the chemical nature of the influent and the effluent, and trying to quantify the chemical
transformations occurring in the reactors. Asked to diagrammatically represent this process the chemist
might produce a flow diagram like that shown in Figure 2.22, quite unlike the engineers’ interpretation.
However, there is general agreement that chemists can provide engineers with invaluable information
since many parameters indicate plant operational performance.
Organic Degradation or catabolism

substrates
{C,H,O,N,P,S} Mineralized
products
Oxygen Reducing
power
CO2+H2O + SO42–
Energy
Biomass (ATP) PO43–+ NO–3
Precursors
Waste
Outline of the
activated sludge
process
Biosynthesis or anabolism
Biomass recycle
Figure 2.22. The Chemist’s view of an activated sludge process.
Although the process is a biological one, on-site monitoring still predominantly assays chemical and
physical rather than microbiological parameters (Henze et al., 1995a,b,c; Pipes and Zmuda, 2007), and
relatively fewer small plants employ qualified microbiologists. Chemical monitoring is preferred because
the tests are generally more sensitive and more rapidly performed, especially for modelling purposes
(Henze, 1995). These methods are not discussed here because they are described fully elsewhere
(APHA, 2005). The value of each chemical parameter measured will depend on the plant configuration
and the model adopted (Gray, 1990; Henze et al., 1995a,b, 2008), but those frequently analysed for are
considered next.
MONITORING THE CHEMICAL PROPERTIES OF THE INFLUENT

AND EFFLUENT
An understanding of influent and effluent characteristics is important for modelling and routine plant
operation (Henze et al., 1995a,b, 2008; Tchobanoglous et al., 2003). Regulatory authorities issue dis-
charge licenses based largely on the chemical quality of the final treated effluent, and effluent toxicity
(Bitton, 2002b), although toxicity assays are not routine and are often performed only following an
incident. In many plants, samples are taken at regular intervals by mechanical sampling systems, meaning
only their average concentrations are determined. Some chemical changes may occur to the sewage prior
to analyses while passing along the sewer (Nielsen et al., 1992), or held in the sampler. Therefore
estimates of total plant loads can be in error, since most systems do not receive wastes at a steady flow and
load but in peaks. Daily maximum variation in influent flow and concentration can be one order of
magnitude or more, and each may occur at different times. Consequently, weighted mean concentrations
should be used. Many chemical parameters discussed here should also be determined for the final treated
effluent. Suitable methods are available (e.g. APHA, 2005) but only for some. Methodologies for many
of the parameters required for modelling purposes are not considered adequate (Gujer, 2006), and
standardized protocols for most are still to be agreed (Henze et al., 1995b,c).
The amount of organic material entering or leaving the plant

(organic loading)
The BOD5 test
Several parameters determine organic loading. While BOD5 measurements are still widely used, they have
limited operational value (Tchobanoglous et al., 2003; Vanrolleghem and Lee, 2003; Wentzel et al.,
2003). BOD5 is based on the amount of O2 required to microbiologically degrade a sample under arbitrary
standard conditions of a five day incubation at 20– C. These conditions were originally imposed from
conclusions reached in the UK that receiving river water was never likely to exceed 20– C, and no river
took longer than 5days to deposit its contents into the sea. Clearly they are met in few other countries.
BOD5 represents the total amount of O2 consumed from several physiological processes including
utilization of organic compounds during chemoorganoheterotrophic bacterial respiration, respiration by
any predatory protozoa present, and the subsequent death and lysis of the biomass and the respiration of
the products (Gray, 1989; APHA, 2005). For influent domestic wastewater the BOD:COD (see next) ratio
is approx. 0.5. New and supposedly improved methods for its determination have been described
(see Vanrolleghem and Lee, 2003). All are sensitive to toxicity and inhibition effects, and the data are not
available for at least five days after sampling. However, the industry is innately conservative, and there is
considerable resistance among some to changing to another indicator.
The COD test

The Chemical Oxygen Demand (COD) test has many advantages over the BOD5 test, including its relative
speed and reliability (Henze et al., 1995c; Marais and Ekama, 1976). Methodology has evolved to
automate the assay, but hazardous chemicals are used (Vanrolleghem and Lee, 2003). This test measures
the total amount of biodegradable and non-biodegradable oxidisable material present in a sample, and
COD values are invaluable in constructing carbon mass balances for modelling, as discussed later
(Dold et al., 1980, 1986; Henze et al., 1987, 1995a,b, 2008; Wentzel et al., 1992).
Under batch conditions, oxygen uptake rates (OUR) vary during extended aeration of sewage (Figure 2.23),
suggesting that different organic fractions vary in their susceptibility to microbial degradation and respiration
(Dold and Marais, 1986; Dold et al., 1980).
High respiration rate

(utilization of sample
DO Decay rate (mg O2/L/hr)
really degradable substrates)
Slow respiration rate

(utilization of biodegradable
particulate substrates)
Endogenous respiration rate
Time (hours)
Figure 2.23. Pattern of change in oxygen uptake rates of an initially aerated sample of activated sludge
biomass, illustrating the pattern of levels of microbial activity.
Consequently, Dold et al. (1980) suggested that the COD should be subdivided into the so-called
biodegradable and unbiodegradable components, the BISUBSTRATE hypothesis (Ekama et al., 1986a;
Henze et al., 1987; Lessard and Beck, 1993) (Figure 2.24). Each of these fractions has been further
subdivided into soluble readily biodegradable (Sbsi), particulate slowly biodegradable (Sbpi), soluble
nonbiodegradable (Susi), and particulate nonbiodegradable (Supi) (Figure 2.25). As Wanner (1994a,b)
correctly points out, these terms are all relative, and what may not be biodegradable under some
conditions may be under others. Further division of these has also been suggested (Henze, 1995a,b,c;
Tchobanoglous et al., 2003) for modelling purposes, but their ease of determination and routine value to
plant operators is debatable. Also, models incorporating so many parameters may become too complex
and user unfriendly, defeating their purpose. Their significance to modelling this process is discussed later
in this chapter.
Influent total COD
Influent Influent
unbiodegradable biodegradable
COD COD
Influent Influent Influent Influent

soluble particulate particulate soluble
unbiodegradable unbiodegradable biodegradable biodegradable
COD COD COD COD
Figure 2.24. Fractionation of Influent COD suggested by Marais and Ekama (1976) and Dold et al. (1980).
Influent total COD
Influent Influent Influent Influent

soluble particulate particulate soluble
unbiogradable unbiogradable biogradable biogradable
COD COD COD COD
"RBCOD"
Effluent Inert Enmeshed Soluble Soluble

soluble mixed liquor biodegradable biodegradable biodegradable
unbio- volatile COD COD COD
degradable suspended (convertible) short chain
COD solids RBCOD fatty acids
(no change) (VSS) (SCFA)
Figure 2.25. Fractionation of Influent COD suggested by Marais and Ekama (1976) and Dold et al. (1980).
The amount of nitrogenous material in the influent

Knowing the influent Total Kjeldahl Nitrogen (TKN) is essential to understanding operation and
modelling of denitrifying plants. It chemically measures non-oxidised nitrogen compounds, including the
fraction untreated by the process, which is usually low (APHA, 2005).
TKN/COD ratio
This is popular with engineers as a ‘rule of thumb’ in assessing the ‘treatability’ of waste water by a
specified process (Ekama et al., 1983; van Haandel et al., 1982). For example, it is believed that high
ratios encourage nitrification and correspondingly low denitrification, and vice versa (Marais et al., 1983;
Pitman, 1991). However, common sense would suggest that the relevance of its determination, like the
F/M ratio is confounded by many factors, not the least of which is the inherent variability in influent
characteristics, and thus values for both TKN and COD.
MONITORING THE PROCESS – THE MICROBIOLOGIST’S

REQUIREMENTS
Activated sludge is a microbiological process and relies on naturally selected microbial populations to
succeed. The microbiologist would like to monitor the community by identifying which populations
carry out the transformations measurable by the chemist and then monitor their functional roles there
(Figure 2.26).
Following changes in their population dynamics in response to operational conditions and plant
performance is the most meaningful, if not the simplest way, to monitor activated sludge processes. Much
of what follows in this book is concerned with how far we have come in achieving this, and so will
be dealt with later. There is now a sense of optimism that the tools needed to accomplish this are
within reach. We have made considerable progress since the earlier version of this book was published.
FISH based DNA microarray fingerprinting of targeted populations (for example, bulking or foaming
filaments, nitrifiers, denitrifiers or PAO) in activated sludge communities seems to be one method to
provide sensitive early warning systems, allowing detectable population changes and activities over time to
be related to plant operation and performance, but this technology is yet to impact on the industry.
O2 CO2 O2 CO2
New
bacterial New Effluent
Wastewater
Influent growth cells
Organics Bacteria Protozoa
Clarifier
Biological
Recycled cellular organics floc
released by death and lysis
Waste organics are incorporated into the floc

(biomass) by synthesis and protozoan predation
Waste
Mixed aeration tank
sludge
Return sludge recycle
Generalized biological processes in aerobic activated sludge

Figure 2.26 The microbiologist’s view of the activated sludge process.
Automated confocal laser scanning microscopic methods and image analysis (Daims et al., 2001c) with
user friendly software (e.g. Daims et al., 2006a; Zhou et al., 2007b), combined with FISH and/or other
fluorescent staining methods (chapters 3, 11) have already been applied successfully to characterizing
floc arrangement and its organization (Schmid et al., 2003a; Thill et al., 1999), and for identifying and
quantifying a range of bacterial populations in laboratory scale and full scale systems. FISH based kits
for in situ identification of nitrifiers and some filamentous bacteria are now available (e.g. Vermicon) and
used routinely for monitoring plant communities in some countries. They require access to adequate
fluorescence microscopy, a need already recognized and accommodated by the availability of a relatively
cheap system by Vermicon to accompany their FISH kits.
Real time qPCR based assays (Smith, 2005) to detect the expressed activities of genes encoding for key
enzymes involved in processes like nitrification (amoA gene for ammonia monooxygenase activity),
denitrification (nirS and nirK genes for nitrite reductase) and P removal (ppk for polyphosphate kinase)
are available to allow key biological activities of the important populations to be routinely monitored
(chapter 3). Of course these expensive facilities would not be supportable at smaller individual plants, but
centralized laboratory facilities or out-sourcing of analyses could make all such assays economically
attractive. The main hurdle probably is in having the information they provide recognized by the industry
as sufficiently valuable for it to invest in this technology. That may take time, unless regulatory authorities
intervene to enforce it.
AUTOMATIC ON-LINE MONITORING OF ACTIVATED SLUDGE SYSTEMS

There is a greater need for continuous plant on-line monitoring. When the earlier version of this book was
published, such systems were already in place in many large-scale plants. As sensors and automatic
systems like flow injection analysers capable of continuously measuring important chemical parameter
and computer control algorithms have improved, so has the view that such systems can provide vitally
important data. Their installation is recognized now as a cost effective way of improving plant
performance, in preference often to structural plant modification. Many of these automated methods have
been critically reviewed by Vanrolleghem and Lee (2003), and so will not be dealt with in detail here.
Ion selective electrodes will become more popular to measure for example in situ nitrification and
denitrification rates, and biosensors (e.g. the NOx biosensor to monitor generation of highly undesirable
gaseous products like N2O produced during denitrification) will continue to be developed and applied.
Robust fluorescence based sensors to measure intracellular NADH levels reflecting the physiological
status of the biomass (Wos and Pollard, 2006) also lend themselves to automation and real time
measurement (Farabegoli et al., 2003). As with the microbiological methods mentioned above, these
would probably not be installed at smaller plants, although again, a single centralized data logger system
could store and handle such information from widely scattered systems, as already happens commonly
with other plant operational data.
MODELLING THE ACTIVATED SLUDGE PROCESS

The biochemical activity and/or performance of activated sludge processes are often described with
mathematical models, to aid in process optimisation, design and control. Their application enables
the user to evaluate multiple operational scenarios that can lead to improved performance of a new or
existing wastewater treatment plant. Successful process operation depends on the metabolic activity of
the populations present and these are subject to large variations depending on associated operational
(e.g. process configuration, hydraulics, aeration intensity, etc.) and environmental conditions (e.g.
wastewater composition, temperature, pH, etc.). Strategies are available to describe the process as a series
of mathematical expressions, where the most frequently implemented are mechanistic models, which
involve using material balance equations requiring an understanding of the reaction stoichiometry and
kinetics involved in the process. Mechanistic models will be the main focus of this section.
Activated sludge model no. 1 (ASM1)

The most popular models are those (see Henze et al., 2000) developed by the International Water
Association (IWA). Many of the concepts incorporated into ASM1 (Henze et al., 1987) were based on
work by the University of Cape Town (Marais and Ekama, 1976; Ekama and Marais, 1979; Dold et al.,
1980; van Haandel et al., 1981). ASM1 (as is its predecessors and successors) is a mechanistic model that
consists of stoichiometric and kinetic expressions describing the bulk biochemical transformations
of soluble and particulate compounds in the sludge. With stoichiometric expressions, the components
(e.g. COD, ammonia, oxygen) may be utilised or produced in a given biochemical process (e.g. biomass
growth, biomass decay, hydrolysis) according to a set of yield coefficients. The rate of each biochemical
process is expressed in a series of Monod-type kinetic reactions. Such an expression is shown below to
describe the rate of aerobic biomass growth (rx):

SS SO
rx ¼ m X ð1Þ
KS þ SS KO þ SO
where m ¼ maximum specific growth rate.

SS,O ¼ concentrations of soluble substrate and dissolved oxygen, respectively.
KS,O ¼ affinity coefficients for soluble substrate and dissolved oxygen, respectively.
X ¼ biomass concentration.
With Monod-type ‘‘switching functions’’ as shown above, it is possible to separate biochemical

transformations that occur only under certain conditions. Thus, when soluble substrate or DO concen-
trations approach zero, biomass growth is predicted not to occur. When growth is not limited by these
(i.e. when their concentrations are high relative to their affinity coefficients), biomass growth occur at its
maximum specific rate.
Initially, both ASM1 (Henze et al., 1987) and South African models were developed to describe
removal of organic matter (i.e. COD) and N removal by nitrification and denitrification. To adequately
describe removal of organic matter in the activated sludge process, the influent COD was partitioned
into separate fractions, based on their ease of biodegradability (see earlier and Figure 2.25). The
non-biodegradable COD fraction consists of soluble and particulate components that pass through the
activated sludge plant unchanged in form (i.e. ‘‘inert’’ fractions). The biodegradable COD fraction was
further separated into readily biodegradable COD (RBCOD, or SS) and slowly biodegradable COD
(SBCOD, or XS). RBCOD consists of simple, soluble substrates directly metabolisable by heterotrophic
biomass for growth. SBCOD consists of more complex compounds, often insoluble particulates, which
are enmeshed into the biomass and require hydrolysis to RBCOD before being metabolised for
growth. Conversion of SBCOD to RBCOD is a slow process compared to RBCOD utilisation, and this
parameter is required for the model to describe adequately the kinetics of COD utilisation and growth
(Dold et al., 1980).
In ASM1 (Henze et al., 1987), nitrogen-containing compounds were also divided into fractions.
The non-biodegradable (inert) nitrogenous fraction exists in particulate form, associated with non-
biodegradable particulate COD. The biodegradable fraction is characterised in terms of ammonia,
together with soluble and particulate organic nitrogen. Particulate organic nitrogen is hydrolysed to
soluble organic nitrogen, which in turn, is converted to ammonia by heterotrophic organisms.
By analogy to the conversion of SBCOD to RBCOD, these reactions assist in describing the kinetics of
metabolism of nitrogenous matter. Ammonia is utilised by both heterotrophic and autotrophic biomass for
growth, where a fraction of the ammonia is assimilated into both population groups, while the remainder
is nitrified to nitrate to supply energy for autotrophic growth. It was assumed in ASM1 that ammonia
was completely converted to nitrate during nitrification, with no accumulation of nitrite. While this
assumption is appropriate for most wastewater treatment systems, later modelling efforts incorporated
nitrite production where its accumulation is substantial (see Sin et al., 2008). Under anoxic conditions,
heterotrophic growth involves denitrification. In the kinetic expression for this a parameter was inserted to
account for the slower growth rate observed anoxically compared to aerobically, which also reflects that
not all heterotrophs can denitrify (Henze et al., 1987).
The transformations described by ASM1 thus include:
. Aerobic growth of heterotrophs and autotrophs.

. Anoxic growth of heterotrophs.
. Decay processes for heterotrophs and autotrophs.
. Hydrolysis of SBCOD and organic nitrogen compounds.
Biomass decay occurs from processes resulting in cell lysis or predation by protozoa. Decay is modelled
according to the so-called death-regeneration hypothesis (Dold et al., 1980), where it results in a release
of SBCOD and small amounts of inert particulate material. Since SBCOD is converted to RBCOD, which
is then used for growth, decay results in regeneration of new biomass. The amount formed however, is
always less than the amount lost to decay, in accordance with the stoichiometric yields of the model.
Since the creation of ASM1, many additions and adjustments have been made to the model to improve
upon problems that have arisen in its application, and to expand its scope (Gernaey et al., 2004). However,
ASM1 should be considered as the reference model, encouraging the general acceptance of activated
sludge modelling in the industry, and it is still that chosen for numerous applications.
Incorporating enhanced biological phosphorus removal (EBPR)

processes into ASM models
The Activated Sludge Model No. 2 (Henze et al., 1995b; Gujer et al., 1995) is an extension of ASM1,
which simulates the behaviour of systems incorporating COD removal, nitrification, denitrification, and
biological and chemical phosphorus removal. It must be said that our understanding of mechanisms for
EBPR was weaker than that for nitrification and denitrification when ASM2 was developed. Thus the
model contains numerous simplifications. During its development, the authors always intended that later
model development would provide an improved description for process design purposes. Subsequent work
has partially achieved this objective, as detailed later.
In incorporating EBPR into model development, the stoichiometric behaviour was based largely
on earlier EBPR biochemical models (Comeau et al., 1986; Wentzel et al., 1986; Mino et al., 1987;
Wentzel et al., 1991b: see chapter 10), while Wentzel et al. (1992) made substantial contributions to
the kinetic model development. Major additions to ASM2 include recognising a portion of heterotrophs
as PAO, which take up fermented carbon sources like volatile fatty acids (VFAs: considered to consist
only of actetate in the model: other VFAs e.g. propionate may also be taken up by PAO) and store them as
PHA (considered to be only poly-b-hydroxybutyrate (PHB) in the model, though other PHA fractions may
be produced). During anaerobic VFA uptake and PHA synthesis, PAO hydrolyse their stored polyP and
release phosphate into the bulk liquid. Aerobically, PAO oxidise stored PHA for growth, and phosphate
uptake and storage as polyP.
In ASM2, the sole carbon-based storage polymer was assumed to be PHB. However, the anaerobic-
aerobic cycling of glycogen by PAO has since been widely accepted (Blackall et al., 2002; Mino et al.,
1998; Oehmen et al., 2007; Seviour et al., 2003: see chapter 10). Incorporating glycogen metabolism into
ASM2 was proposed in modified versions (Manga et al., 2001; Mino et al., 1995).
To distinguish between the carbon sources taken up by PAO (like acetate) from the remainder of the
RBCOD in ASM2, RBCOD was split into two fractions: fermentation products (SA: since it is considered
to be acetate only), and fermentable soluble COD (SF). PAO were modelled to take up only SA, where
it is usually fully consumed under anaerobic conditions. However, acetate present under anoxic or
aerobic conditions is also consumed by PAO and stored as PHB (Kuba et al., 1994; Ahn et al., 2002a,b;
Pijuan et al., 2005), which is predicted by the model. Other heterotrophs were modelled to directly take up
either SA or SF, and use them for growth under aerobic or anoxic conditions.
ASM2 did not incorporate initially simultaneous phosphorus removal and denitrification by PAO.
This process and anoxic growth of PAO was incorporated into a later version, known as ASM2d
(Henze et al., 1999). ASM2d has since become the more widely applied model incorporating EBPR, and
further developments have often arisen from its adaptation.
Activated sludge model no. 3 (ASM3)

ASM3 (Gujer et al., 1999) describes COD removal, nitrification and denitrification, as does ASM1. ASM3
was proposed as the standard model to improve upon some of the difficulties encountered in ASM1. One
was that heterotrophic biomass was modelled assuming that all RBCOD is taken up and stored
intracellularly (modelled to be PHB) prior to biomass growth. This presented a useful (yet simplified)
approach to model COD storage. Another difference between ASM1 and ASM3 is that the death-
regeneration concept for biomass decay was replaced with endogenous respiration, where biomass is
converted largely into inert particulate matter. This facilitates calculating rates of endogenous processes
(e.g. decay), performed by respirometry.
The aim of ASM3 is that its calibration is easier than with ASM1 (model calibration is described in
more detail later). Parameters difficult to determine were eliminated or simplified. In ASM1, all variables
are influenced directly by a change in a parameter value, whereas this direct influence is reduced in ASM3
(Gernaey et al., 2004). ASM3 is also superior to ASM1 in describing cases where storage of RBCOD is
substantial, for example in industrial wastewater treatment (Gernaey et al., 2004; Ky et al., 2001). This
ASM3 model has since been extended to describe EBPR, in ASM3-BioP, also known as the EAWAG
model (Rieger et al., 2001).
Combining ASM models with structured metabolic models

An alternative to modelling EBPR is with metabolic models (Kuba et al., 1996; Murnleitner et al., 1997;
Smolders et al., 1994a, 1994b, 1995), where PAO metabolic processes are described in more detail.
In metabolic modelling, overall reactions describing anaerobic, anoxic and aerobic biochemical
transformations are calculated through mass, energy and reduction-oxidation (redox) balances from
proposed metabolic pathways (usually those of the Accumulibacter PAO, see chapter 10). Yield
coefficients and kinetic parameters are typically validated experimentally by measuring production/
consumption rates of key substrates and storage products in enriched PAO cultures (e.g. Smolders et al.,
1995). Typical anaerobic and aerobic/anoxic processes taken into account in such models of PAO are
shown in Table 2.1. Subsequent experimental studies agree well with the bulk biochemical
transformations and model predictions, supporting the validity of the proposed metabolism
(Accumulibacter has not yet been obtained in pure culture: chapter 10). The metabolism of GAO has
also been described by similar metabolic models (Filipe et al., 2001a; Zeng et al., 2003c), and also
correlate well with experimental data from enriched GAO cultures.
Table 2.1. Anaerobic and aerobic/anoxic processes accounted for in metabolic models of PAO. Metabolic
models of GAOs incorporate the same processes, but excluding phosphate release and uptake.
Anaerobic Aerobic/Anoxic
VFA uptake (e.g. acetate) PHA degradation
Polyphosphate degradation and phosphate release Phosphate uptake and polyP storage
Glycogen consumption Glycogen production
PHA production Biomass growth
Cell maintenance Oxygen/Nitrate consumption
Cell maintenance
Metabolic models of PAO have been incorporated into ASM models to model P and N removal
in full-scale plants. The combined metabolic/ASM2d model developed at the Technical University of
Delft (known as the TUDP model) has been applied successfully to domestic activated sludge systems
operating with different configurations (Brdjanovic et al., 2000; Hao et al., 2001; Meijer et al., 2001;
van Veldhuizen et al., 1999). A combined TUDP/ASM3 model called A3DX was developed by Ky et al.
(2001) and described well a full-scale SBR plant treating industrial wastewater. The calibration of
the TUDP and A3DX models requires adjustment of only 3 or 4 parameters. Therefore, compared to
conventional ASM models, combining metabolic and ASM models improves prediction of process
performance, with the advantage of requiring fewer parameter calibrations (see discussion below on
model calibration).
Model calibration and wastewater characterisation

Influent wastewater characteristics and activated sludge properties tend to vary widely on a site-by-site
basis. Consequently, before these models are applied, it is common practice to adjust some parameters
within the modelled expressions and ensure the influent wastewater is properly characterised (see earlier).
Model calibration and wastewater characterisation are critical and challenging issues in modelling, if better
prediction of the process performance of a given process is sought (Sin et al., 2005). Factors known to
affect model parameters include temperature, pH, presence of inhibitory compounds, and the floc structure
of the biomass. Although ASM models are mainly used to describe plants treating domestic wastewater,
they can be adapted to industrial wastewater plants with adequate calibration (Gernaey et al., 2004).
Starting point for model calibration and wastewater characterisation is usually the default parameter set
provided with the ASM models. Generally model parameters are optimised by ad-hoc tuning of selected
parameters; while other approaches use mathematical algorithms exclusively for parameter optimisation
(Gernaey et al., 2004). Both approaches are subject to large biases, and can lead to multiple sets of
unrealistic estimations that happen to describe a given set of data.
So it is preferable to constrain model parameters within realistic boundaries. This is often performed
through a series of measurements of relevant influent/effluent variables at the plant (and in the various
unit processes), in combination with lab-scale batch tests using activated sludge obtained from it
(Hulsbeek et al., 2002; Melcer et al., 2003; Petersen et al., 2002; Sin et al., 2005). Any cyclic variations in
wastewater composition need to be taken into account during the calibration procedure, to ensure the
measurements are representative (Koch et al., 2000; Rieger et al., 2001). Sensitivity analyses of model
outputs to changes in parameter values allow identification of the most important model parameters for
calibration as well as their interdependencies (Koch et al., 2000; Brun et al., 2002; Petersen et al., 2002).
It is common practice that calibration is only performed for key process parameters, while default values
are used for parameters for which the model outputs display a low sensitivity (Hulsbeek et al., 2002;
Melcer et al., 2003).
To minimise the uncertainty and errors involved, efforts have been made to standardize the calibration
procedure (Sin et al., 2005), which is becoming common practice. Although several protocols exist, each
agrees on key procedures (Sin et al., 2005). The first step involves project definition. Models can be used
for optimisation, design, upgrade or control of plants, and the calibration procedure is tailored to suit the
intended purpose (e.g. plant design tends to involve a simpler procedure, considering the generally
high amount of uncertainty in influent wastewater and process operation – see Melcer et al., 2003).
Protocols also agree that data collection, verification and reconciliation are necessary (Sin et al., 2005).
Since calibration depends on the accuracy of experimental data, errors can be identified through a series
of mass balance calculations, typically using phosphate, COD, nitrogen and oxygen flows (Meijer et al.,
2002). This improves the accuracy of the experimental data, and prevents unjustifiable changes to
model parameters during calibration (Meijer et al., 2002). Finally, each modelling protocol agrees that
calibrated models should be validated against a set of different data from the plant (Sin et al., 2005),
generally where one or more key conditions (e.g. sludge age, temperature, etc.) have been changed.
Validation increases further confidence in the model accuracy, and if the validation step fails, the model
must be re-calibrated.
The differences among calibration protocols are generally the experimental methods for wastewater
characterisation and parameter estimation, the sampling frequency during the measurement campaign and
the selection of parameters to calibrate (Sin et al., 2005). Currently, an IWA task group is focusing on
good modelling practice in the industry, where the aim is to develop a unified protocol that provides an
internationally standardised model calibration procedure (http://www.modeleau.org/GMP_TG/).
ADVANCES AND FUTURE DIRECTIONS IN ASM MODELLING

Microbial population dynamics
An increasing number of modelling studies are now being devoted to describing microbial population
dynamics in lab scale and full scale activated sludge systems (Gujer, 2006). These include denitrification
with external methanol addition (Purtschert and Gujer, 1999), where two groups of denitrifiers displaying
different growth kinetics and yield coefficients were analysed to promote operational conditions that
selected the most efficient denitrifier populations. Hug et al. (2006) have developed a model describing the
proliferation of Candidatus ‘Microthrix parvicella’ (a bulking filament – see chapter 7) in a full scale
plant. Its main focus was to describe its growth dynamics in response to changes in temperature from
seasonal variations, to identify conditions encouraging bulking.
Furthermore, bases for competition between PAO and GAO in EBPR systems (chapters 3, 10) have
used metabolic modelling techniques (Lopez-Vazquez et al., 2009; Yagci et al., 2003; Zeng et al., 2003d)
together with ASM-based models (Manga et al., 2001; Mino et al., 1995; Yagci et al., 2006). Yagci et al.
(2006) showed an adapted ASM2d model accounting for GAO activity could more meaningfully describe
a lab scale SBR performance than the ASM2d could, over a wide range of influent COD/P ratios. While
most modeling studies assume that acetate is the sole carbon source used by PAO and GAO, adapted ASM
and metabolic models can describe PAO and GAO metabolism with propionate, incorporating production
of other PHA fractions like PHV and PH2MV (Oehmen et al., 2005c; Oehmen et al., 2006; Pijuan et al.,
2004). Propionate may be abundant in some systems, especially those employing pre-fermentation, and
impacts on community selection of PAO and GAO (Oehmen et al., 2005b). As well as carbon source, pH
and temperature also affect competition between PAO and GAO (Oehmen et al., 2007: chapter 10).
Lopez-Vazquez et al. (2009) developed a metabolic model describing the combined effects of carbon
source, pH and temperature on Accumulibacter (PAO), Competibacter (GAO), and Defluviicoccus
(GAO). It was used to assess the population dynamics of these PAO and GAO, and predict conditions
favoring minimisation of GAO and corresponding optimisation of EBPR capacity.
It seems likely that future advances will be made in predicting population dynamics, which may
prove invaluable in optimising and controlling full scale treatment plants. Clearly, positive collaborations
between microbiologists and engineers will be essential for this level of description in such complex
microbial communities to be achieved.
Linking activated sludge models with the entire WWTP

Recently the concept of wastewater treatment processes control has changed, where an integrated control
approach to entire plant operation is more highly favoured (Olsson, 2006). Thus, the focus is on modelling
the activated sludge system in combination with the other unit processes present (see Copp et al., 2008;
Jeppsson et al., 2006, 2007). An IWA task group is focusing on benchmarking control strategies for
WWTP (www.benchmarkwwtp.org).
This commenced with the Benchmark Simulation Model 1 (BSM1 – see Copp, 2002; Jeppsson and
Pons, 2004; Spanjers et al., 1998). BSM1 combined the ASM1 model with a detailed model of the settling
dynamics in the secondary clarifier (Takacs et al., 1991), the assumption being that no biological reactions
took place in the settler. This model was expanded into BSM2, where other key processes of the entire
treatment plant were included (Copp et al., 2008; Jeppsson et al., 2006, 2007). These were the primary
clarifier (Otterpohl and Freund, 1992) and sludge treatment processes including sludge thickeners,
anaerobic digestion processes (i.e. ADM1, Batstone et al., 2002; several studies have focussed on
modelling anaerobic digestion processes as reviewed by Batstone et al., 2006), and sludge dewatering.
During development of BSM2, model interfaces were developed between ASM1 and ADM1, since
components of these two models are different, whereby ASM1 variables are converted into ADM1
variables (while maintaining COD and N balances), and vice versa (Jeppsson et al., 2006).
Influent wastewater dynamics (accounting for rainfall, seasonal changes and dynamics in household
and industry water usage) have been modelled to evaluate their possible effects of disturbances on
treatment plants (Gernaey, 2006). Storage tanks were included in the model for equalization of wastewater
flows. Further, since treatment plant control usually relies on information from on-line sensors, the noise,
response and drift of these sensors may introduce error (Jeppsson et al., 2006). Thus, BSM2 also
incorporates the modelling of realistic sensor behaviour, based on developments by Reiger et al. (2003).
The purpose of benchmark modelling is to link the entire wastewater treatment together to establish
integrated process control schemes. Incorporation of each unit process facilitates evaluation of the effect
of each sub-process on the global plant operation. The goal is to account for as many factors as possible
(including disturbances). This is a more sophisticated and complex model that allows the study of unit
process interaction and is likely to become increasingly applied. Given the onset of climate change, which
can lead to more frequent disturbances to treatment plants and an increased need for process control such
an approach is likely to become more attractive.
Biofilm, granular and membrane bioreactor (MBR) processes

Conventional activated sludge processes generally occupy large areas of potentially valuable land, mainly
due to the large secondary clarifiers. Strategies exist to reduce their size or eliminate the need for them,
thus improving the cost-effectiveness of the treatment process. Describing such processes through
modelling approaches has become increasingly used.
Treatment processes have been developed based on microbial attachment to surfaces or biofilms,
including the integrated fixed-film activated sludge (IFAS) process and the moving bed biofilm reactor
(MBBR). Biofilm models exist varying in their complexity reflecting their intended application;
i.e. simpler models are often used in practice, while more complex models have been developed mainly as
research tools to improve our understanding of biofilms (Boltz et al., 2008). Models generally describe
biofilm structure (i.e. its development and accumulation of biomass, biomass detachment from it, its
thickness, cell density, etc.) and substrate (i.e. COD, oxygen, nutrients) flux through it (Wanner et al.,
2006a,b). Mass transfer limitations of the substrate to and within the biofilm are usually considered. One
current challenge is identifying the high number of model parameters, given often limited experimental
data consisting often only of substrate concentrations in the bulk phase (Brockmann et al., 2008). Some
models describe the activity of multiple microbial groups differing in their biokinetic parameters for
growth, respiration, and production of extracellular polymeric substances (EPS) and internal storage
products (Picioreanu et al., 2004; Xavier et al., 2005). These models thus incorporate concepts of
microbial competition. In this work, biofilm images obtained with confocal laser scanning microscopy
(CLSM) agreed well with model-predicted biofilm structures, and CLSM was suggested as a useful tool in
model validation (Xavier et al., 2005).
Aerobic granular sludge processes are now available (see earlier) and utilised in practice
(de Kreuk et al., 2007b). Several models are available, and usually apply the principles of biofilm
modelling (de Kreuk et al., 2007a; Xavier et al., 2007). They have been used to analyse influence of
substrate concentration, temperature, granule size, on nutrient removal performance (de Kreuk et al.,
2007a). Models have also been developed to describe competition inside granules, especially between
heterotrophs, nitrifiers and PAO (Xavier et al., 2007), where granule structure is also described in detail.
Membrane bioreactors (MBRs) have also become popular to treat both domestic and industrial
wastewater (LeClech et al., 2006). Secondary clarifiers are not required since biomass is removed from
effluent by membrane filtration (see earlier). Operational challenges relate largely to prevent membrane
fouling, whereby effluent flux is reduced from clogging or blockage of membrane pores, usually caused by
EPS, or more specifically, colloidal material referred to as soluble microbial products (SMP) (Jiang, 2007;
LeClech et al., 2006). These are characterised as biomass associated products from biomass decay (BAP),
and utilisation associated products (UAP) from biomass growth (Jiang, 2007). MBR processes have been
modelled (Jiang, 2007; Ng and Kim, 2007), through describing effects of SMP on membrane fouling.
Jiang et al. (2007) linked a modified ASM1 model for biological characterisation (ASM1 was maintained
intact except for a few altered parameters) with a hydrodynamic model describing flux through the
membrane and resistance to transport. Further development and application of MBR models, especially
with full-scale systems, is necessary.
The presence of micropollutants

The occurrence and fate of micropollutants in activated sludge systems, including heavy metals,
herbicides, pesticides, hormones, antibiotics and pharmaceuticals and personal care products, has attracted
increased research interest (see earlier) because of their potentially harmful effects even at very low
concentrations (often present in the ng/L to mg/L range). Efforts have been made to model micropollutant
biodegradation (Joss et al., 2006; Schönerklee et al., 2008), and their adsorption to sludge. Nevertheless,
development of appropriate models remains a challenge, arising from the wide range of compounds to
assess, difficulties in quantifying accurately levels of each at these low concentrations (requiring time-
consuming and expensive analytical procedures) and their less predictable influent dynamics compared to
COD, N and P. Future modeling will undoubtedly focus on these important compounds, which are of a
particular concern in wastewater recycling projects, where they pose a high risk to humans.
Empirical and hybrid models

An alternative approach to modelling activated sludge is with empirical (or ‘‘black-box’’) models. Here
statistical methods are used to link the input and output data from a treatment plant, to determine
appropriate process control actions based on repeated variations in the data. Empirical models do not rely
on describing the biological, chemical or physical processes occurring, and are most widely used
in situations where mechanistic models do not provide adequate description of a process (Gernaey et al.,
2004). The main challenge with them is to interpret and link results of the model with processes
occurring within the plant, and to use that information effectively for process control and optimisation
(Gernaey et al., 2004).
Consequently empirical models have been used in combination with mechanistic models
(e.g. Rodriguez-Roda et al., 2002). These hybrid models generally consist of a simplified mechanistic
model incorporating information about process reactions, while the empirical model is usually an artificial
neural network (Zhao et al., 1997; Thibault et al., 2000). They rely on a preliminary prediction of desired
outputs (e.g. effluent COD, ammonia, phosphate concentrations) by the simplified process model, where
any errors are corrected by a ‘trained’ neural network, which can adjust specific inputs (e.g. aeration
intensity, recycle flow rates) to generate a specific target output (Baeza et al., 2002). If the neural
network improves prediction of some outputs, this indicates that further improvement is needed in the
mechanistic model (Gernaey et al., 2004).
Hybrid models have potential to become more widely implemented, and can be attractive options for
process control purposes, especially given the increasing availability of reliable on-line measurement
tools. Comas et al. (2008) implemented a hybrid model of BSM1 combined with a statistical set of input/
output correlations to evaluate several short-term and long-term control strategies. They showed that
control strategies that led to minimised operating costs and best effluent quality were also likely to lead to
settling problems in the secondary clarifier. These results are consistent with the concept of sludge
population optimisation (Yuan and Blackall, 2002; Yuan et al., 2008), whereby short-term improvements
from process-control strategies can result in negative effects on the microbial community and sludge
composition, leading to long-term performance deficiencies. Thus, it is desirable to incorporate a ‘‘global’’
approach to process control of wastewater treatment systems (Olsson, 2006), whereby the effects
on microbial community structure and interactions between unit process operations are all taken into
consideration (Comas et al., 2008; Yuan et al., 2008).
3
Microbial communities in activated
sludge plants
Robert J. Seviour and Per H. Nielsen
INTRODUCTION
Despite activated sludge systems being in operation for nearly 100 years, we have understood little about
their microbiology until quite recently. Which organisms were there, or more importantly, what roles
they played in the process were largely a mystery. When early microbial ecologists, especially those
predisposed to working in the laboratory with pure cultures, tried to address these questions, many soon
gave up. They recognized that the methodology available, based predominantly as it was on isolating and
attempting to culture and identify individual populations, was inadequate for elucidating biodiversity in
such complex systems. Inevitably they became cataloguers of descriptive information, which suffered
from the biases inherent in these approaches (chapter 1). While early microscopic descriptions of the
morphological diversity among activated sludge metazoa and protozoa and filamentous bacteria can still
be applied (with some limitations) for monitoring plants the same is not true for most other members of
the activated sludge community. Thus, it is clear that bacterial populations we once grew and considered
important in processes like N and P removal are marginally relevant at best, and in retrospect, much of our
time and energy has been wasted chasing what have been aptly called ‘laboratory weeds’ (Amann et al.,
1995). Consequently, much of the early published culture dependent information on activated sludge
microbiology, with a few notable exceptions, is of little more than historical interest, and should be
viewed as such.
Everything changed with the advent of molecular methods (Amann and Kuhl, 1998; Daims, 2005;
Loy et al., 2002a; Wagner and Loy, 2002; Wagner et al., 2002, 2006; Wilderer et al., 2002), and continues to
as new techniques for resolving microbial community structure and function continue to emerge (Akkermans
et al., 1995; Daims et al., 2006a; Gilbride et al., 2006; Kowalchuk et al., 2004; Leadbetter, 2005; Osborn and
Smith, 2005; Rochelle, 2001). This approach is sometimes referred to as the ‘full RNA cycle’ approach
(Figure 3.1). There is still much to do, especially in applying these methods to full-scale plants, although this
now happens (Beer et al., 2006; Juretschko et al., 2002; Kong et al., 2004, 2005, 2007; Wagner and Loy,
2002; Wong et al., 2005). This chapter will show hopefully how remarkable our progress has been since the
early version of this book was published, in our attempts to understand activated sludge communities.
Environmental Sample
Ribosome
small
subunit
(ssu) FISH DNA Genomic
extraction DNA
large
subunit
ssu
rRNA
full rDNA
rRNA amplification
DNA cycle
probe
rDNA
genes
rRNA
Clone
Cloning,
Probe Library
Sequencing
Design
Sequence
Comparison
1 500 1000 1500
Figure 3.1. The full rRNA-cycle for analysis of microbial communities containing organisms, shown with
rectangles, circles, and triangles. DNA is extracted from all the organisms in the amplified, cloned, and
sequenced. Sequence information is then used in FISH probe design. In this example, a probe is designed to
target organisms represented as rectangles. Optimization of the FISH protocol is required to find which
conditions maximize target organism specificity (L.S. Yilmaz, D.W. Kang and D.R. Noguera).
MICROBIAL POPULATIONS IN ACTIVATED SLUDGE – THEIR LOCATION

In any discussion on activated sludge microbiology, we need to be reminded that much of the biomass is
not freely suspended in the bulk liquid, but is organised into flocs (Nielsen, 2002). The organism’s spatial
location within the system (inside the floc or on its surface or freely suspended in the bulk liquid) is
crucial, since ultimately it will determine whether a population is retained within the reactor or leaves in
the clarified liquid phase, and hence its contribution towards final effluent quality. This location may also
influence its access to nutrients including O2, and its sensitivity to toxic compounds (Henriques and Love,
2007; Henriques et al., 2005). The floc organization will probably determine also how successfully
cells can be recovered from mixed liquor samples in attempts to isolate them into pure culture (Banks
and Walker, 1976), as discussed below. Activated sludge populations have been categorized into
Microbial communities in activated sludge plants 97
‘floc-forming’ and ‘non-floc-forming’ populations for modelling, especially the population dynamics of
the filamentous bacteria (Kappeler and Gujer, 1994a,b), and the validity of such a distinction is discussed
in chapter 5. Measurements with selective microsensors (chapter 11) show that many different
microenvironments can exist within a single floc, and confocal laser scanning microscopy (CLSM) has
revealed, not unexpectedly that individual bacterial populations may occupy different locations there
(Daims et al., 2001; Thill et al., 1999). Considerable substrate gradients occur within individual flocs,
although their possible influence on floc community composition and function/activity is generally still
unclear (Schramm et al., 1999a,b).
The floc
We should view the floc as a complex heterogenous structure (Droppo et al., 2005), composed of aggregates
of bacteria and other organisms embedded in a polymeric matrix or gel, which is often referred to as
extracellular polymeric substances or EPS (Fleming and Wingender, 2002; Jorand et al., 1995; Keiding
et al., 2001; Li and Ganczarczyk, 1986, 1989, 1990, 1991; Nielsen, 2002). Attached particulate organic
matter and inorganic particles may also be integrated into its matrix (Figure 3.2(a)). Most, but probably not
all the bacteria there are thought to be metabolically active (Lopez et al., 2005; Nielsen and Nielsen,
2002a,b), but overall floc structure, its community composition and activity may change (Forster et al.,
2002) with both process type and operation (Liao et al., 2006). Most bacteria exist as microcolonies or
microflocs bound into the floc structure (Figure 3.2(b), see colour image section (chapter 13)), although
some are always present as single cells or as filamentous bacteria (Jenkins et al., 2004b; Jorand et al., 1995;
Zartarian et al., 1994). Evidence suggests that microcolonies of certain populations e.g. the PAO and
nitrifiers are more resistant to imposed shear than others (Larsen et al., 2006, 2008b; Wilén et al., 2008),
Thus, in an EBPR community, the Alphaproteobacteria and Firmicutes were shown to produce weaker
microcolonies than those consisting of Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria
and Actinobacteria. Flocs with microcolonies of the PAO Candidatus ‘Accumulibacter phosphatis’ were
especially resistant to shear. Interestingly, only a few individual populations from each of these
phylogenetic lineages appeared to be present in the floc (Klausen et al., 2004).
Figure 3.2. (a) A diagrammatic representation of the organization and structure of an activated sludge floc.
The chemical composition of the floc polymeric matrix is highly variable and still incompletely
characterized. Increasingly sophisticated techniques continue to be applied to it in the hope that a clearer
picture of its chemistry and physical properties will emerge (Garnier et al., 2005; McSwain et al., 2005).
The major component is proteinaceous material, but high levels of polysaccharide, humic substances
and nucleic acids are also detected (Comte et al., 2006; Frølund and Keiding, 1994; Frølund et al., 1996;
Horan and Eccles, 1986; Kerley and Forster, 1995; Morgan et al., 1990; McSwain et al., 2005;
Nielsen and Jahn, 1999; Wilén et al., 2003a,c). Their source is probably microbial, although some of the
protein and DNA in the floc EPS may originate from the organic food we eat and from other sources in the
incoming sewage. Amyloid adhesion proteins from floc bacterial surfaces seem to be major components,
and may be important in encouraging bacterial adherence and aggregation (Larsen et al., 2008a).
The humic components also originate in the wastewater, and these may cause problems when PCR based
methods are used for community composition analyses (chapter 11).
Generally, the bacterial cell fraction contributes only 5–20% of the organic matter, while the EPS
matrix is responsible for more than 50%, and thus particularly important in determining floc properties
(Frølund et al., 1996, Wilén et al., 2003a,b,c). The precise chemical composition of the macromolecules,
especially polysaccharides probably reflect which bacterial populations are present, and their growth
conditions (Branda et al., 2005), and so relating floc properties to the presence of each macromolecule
group is not possible. However, some evidence suggests that the EPS protein composition may change in
poorer settling flocs, where lower molecular weight proteins are more dominant (Garnier et al., 2005).
The EPS matrix can be divided into two major pools; a relatively loosely attached ‘‘cloud’’ that is easy
to extract, and a more firmly bound and difficult to remove fraction (Keiding and Nielsen, 1997;
Wilén et al., 2003b; Zhang and Fang, 2001). The first is thought to enable formation of the entire floc by
embedding each of the components in a common gel matrix, while the latter may determine the properties
of individual microcolonies. Evidence suggests that higher levels of the loosely attached EPS affect floc
structure as a consequence of poor flocculation (Li and Yang, 2007). The EPS-matrix ensures all the
above-mentioned constituents are held together by inter-particle forces like the DLVO-forces (Van der
Waal’s forces and electrostatic interactions), cross-linkage of charged polymers by cations, hydrophobic
interactions, steric interactions and polymer entanglements (Hermansson, 1999). In particular, surface
charge and mainly negative charges of the floc polymers and presence of multivalent cations including
Mg2þ, Ca2þ and Fe3þ are considered important in determining their essential hydrogel-like properties
(Keiding and Rasmussen, 2003; Keiding et al., 2001; Liao et al., 2001).
However, flocs can disintegrate, releasing EPS and colloidal material, as for example when Fe3þ is
reduced to Fe2þ either from the activity of chemoorganoheterotrophic iron reducing bacteria (Caccavo et al.,
1996) or in the presence of sulphide (Nielsen and Keiding, 1998). Wilén et al. (2004) showed deflocculation
may also occur under anaerobic operating conditions, with subsequent reflocculation, resulting probably from
biological not chemical activities, when conditions again become aerobic. Changes in cation concentrations
in the influent may also cause floc disintegration. Watanabe et al. (1999) interpreted deflocculation in
their system fed phenol as a shift in community composition to domination by non-flocculating organisms
under toxic conditions. Of course effluent quality will then deteriorate in each of these cases.
The floc matrix also contains high hydrolytic enzymatic activity (Geesey and Kloeke, 2005; Yu et al.,
2007). As much of the organic matter in most wastewaters is particulate or colloidal in nature
(Levine et al., 1985) it must first be degraded by exocellular enzymes before assimilation by the bacterial
cells. Enzymatic activity of the matrix can be measured directly by degradation of protein, carbohydrate
and lipid (Dueholm et al., 2001; Geesy and Kloeke, 2005; Morgenroth et al., 2002), formation of
ammonium by deammonification of organic matter (Henze, 1991) or indirectly using a range of model
substrates (e.g. Frølund et al., 1995; Xia et al., 2007, 2008b). All exoenzyme activity is bound in the EPS
matrix and not detectable in the bulk liquid (Frølund et al., 1995), and so is most probably associated with
the microbial cell surface (Cadoret et al., 2002; Gessesse et al., 2003; Kloeke et al., 1999; Nielsen, 2002).
Esterase is the dominant exoenzyme activity detected in flocs, but protease, lipase, phosphatase and other
enzymatic activities have been reported there (Geesy and Kloeke, 2005). Different bacterial populations
seem to express exoenzyme activities like lipases, proteases or phosphatases, suggesting that degradation
of macromolecules may involve particular populations of bacteria (e.g. Xia et al., 2007, 2008a,b).
The mechanisms involved in floc formation have been the subject of considerable controversy and debate.
Some of the physiological and environmental factors aiding and encouraging cell aggregation into flocs, and the
possible mechanisms involved, were discussed by Bossier and Verstraete (1996). However, since most bacteria
grow as microcolonies within the floc, direct aggregation of single cells may be of relatively minor importance.
Instead, it is widely agreed that some of the filamentous bacteria discussed later in this book are important in
small numbers in maintaining floc structural integrity, by providing a skeletal matrix or microstructure for all
the other components (Eriksson and Alm, 1991; Jenkins et al., 2004b), as shown in Figure 3.2.
Methods for preparing flocs for detailed microscopic examination have been described (Droppo et al.,
1996), but not widely applied to studies into the dynamics of floc organization. It was once thought that
members of the bacterial genus Zoogloea played an important role in floc formation (Pipes, 1967). Now
we know that these are just one of many microcolony formers, although they may dominate flocs in some
treatment plants (Juretschko et al., 2002). Any possible involvement of bacterial intercellular signalling or
quorum sensing in floc formation is unclear, although N-acyl-l-homoserine lactones, the chemicals
thought responsible, are synthesised by some activated sludge bacteria (Morgan-Sagatume et al., 2005),
and may affect both community composition and in situ cell function (Valle et al., 2004).
Suspended bacteria and other particles entering the reactors adsorb very rapidly to existing flocs
(Olofson et al., 1998; Pujol and Canter, 1992; Wanner, 1994a,b,c; Zita and Hermansson, 1997), by a
process of adhesion, encouraged by their high cell surface hydrophobicity (Olofsson et al., 1998). Less
hydrophobic cells may remain in suspension, leading to their high levels (107 ml 1) in the final effluent
(Morgan-Sagastume et al., 2007; Wilén et al., 2000). So flocs clearly play an important role in removal of
bacterial cells and other particulate matter from the mixed liquor.
Aerobic granules
As mentioned in chapter 2, activated sludge flocs can be encouraged (at least in SBR systems), to
transform into spatially heterogeneous large granules called aerobic granules. This induced
transformation may result from increased EPS production (Liu et al., 2005a,b), and selectively favours
bacterial populations with high co-aggregation propensity (Jiang et al., 2006). It leads to marked changes
in floc chemical properties and organization. Thus, McSwain et al. (2005) showed that aerobic granules
had much higher protein and polysaccharide contents than the flocs from which they derived, and a less
diverse microbial community. Their spatial organization was different too, and the cells were embedded
in EPS, but located on their outer edge only. However, Lemaire et al. (2008) showed in larger granules
carrying out simultaneous nitrification and dentrification and P removal that while the PAO
Accumulibacter dominated the outer region of the granules, the Competibacter GAO were located
centrally, and this GAO population was responsible for denitrification. Yi et al. (2002) have demonstrated
large shifts in granule microbial community composition during their development, and suggested that
particular populations, unfortunately not identified or reported in their study, were especially important in
their maturation. Novel bacteria can be isolated readily from aerobic granules (e.g. Maszenan et al., 2007),
and some evidence suggests that certain bacterial populations may be able to coexist only within them
(Jiang et al., 2007). Frequent excessive growth of filamentous bacteria can occur in these aerobic granules,
for reasons not clearly resolved (Liu and Liu, 2006), which markedly affects their settlability properties, in
a similar way to bulking of flocs (chapter 7).
METHODS FOR STUDYING MICROBIAL POPULATIONS

IN ACTIVATED SLUDGE
The culture dependent methods we were forced to use until quite recently are heavily biased, and so the
organisms successfully isolated with them reflect more the methods used than the existing community
biodiversity. Although studies with culture independent methodology make it clear that the community of
activated sludge systems is phylogenetically more diverse than these early approaches led us to believe
(Amann et al., 1996, 1998; Blackall and Nielsen, 2000; Daims, 2005; Loy et al., 2002a; Snaidr et al.,
1997; Stahl et al., 2006; Wagner and Loy, 2002; Wagner et al., 2002), it seems that certain groups of
organisms appear consistently more frequently than others. Why this happens is not clear (e.g. Lozada
et al., 2006; Prosser et al., 2007; Sloan et al., 2006). One major question we still need to answer is whether
this high level of biodiversity reflects a similar level of functional and niche diversity (He et al., 2007a;
Maixner et al., 2006), or whether functional redundancy exists in these communities (Franklin and Mills,
2006; Siripong and Rittmann, 2007).
We now know from a range of molecular fingerprinting techniques like 16S rRNA based clone library
generation, PCR-DGGE or T-RFLP (chapter 11) that the community will vary continuously in response
to changes in influent composition and plant operating characteristics, and it probably never attains a true
steady state. Furthermore, in plants designed for example, for N and P removal, the selective pressures
deliberately imposed encourage the growth of organisms which are not as commonly seen in conventional
plants (chapters 9, 10).
What can be forgotten is that PCR based ‘culture independent’ methods based usually on 16S/23S
rRNA sequence analysis also suffer from biases (Acinas et al., 2005; Daims, 2005a; Loy et al., 2002a;
Osborn and Smith, 2005; Osborne et al., 2005; Sipos et al., 2007; von Wintzingerode et al., 1997). How
these impact on our understanding of activated sludge microbiology is discussed frequently throughout the
book. They affect how we should handle samples and extract their DNA/RNA (Dionisi et al., 2003; Daims,
2005; Juretschko et al., 2002; Keith et al., 2005), interpretation of outcomes from the PCR protocols we
use, clone library construction and analysis (Figuerola and Erijman, 2007; Röling and Head, 2005; Sipos
et al., 2007), and consequently the reliability of the 16S rRNA databases we depend on (Ashelford et al.,
2005, 2006) to analyse the data generated from techniques like DGGE and FISH community analyses.
Sampling populations
Despite all the attractions of using culture independent methods of analyses, isolating populations of
interest into pure culture is still desirable and should be encouraged. Who would dispute that the possession
of pure cultures of putative PAO populations like the Candidatus ‘Accumulibacter phosphatis’ and GAO
like Candidatus ‘Competibacter phosphatis’ or Defluviicoccus would benefit our attempts to understand
EBPR processes better? As much of the biomass is associated with flocs, floc-associated organisms require
prior resuspension, and the methods described for this purpose, including floc homogenisation and
sonication have been assessed (Banks and Walker, 1976). Both carry the risk of physical damage to the
cells, affecting their subsequent recovery. Other more directed sampling approaches have been applied.
Micromanipulation
Micromanipulation has been employed to isolate populations of interest directly, especially the
filamentous bacteria (Blackall et al., 1994b; Hornsby and Horan, 1994; Levantesi et al., 2004, 2006a,b;
Seviour et al., 1994, 1997). Many of these can be first recognised microscopically with confidence, or even
more confidently after FISH probing (Thomsen et al., 2004), although an initial FISH probing is not
necessary for all morphotypes. Most of the axenic cultures of the filamentous bacteria we have acquired
were obtained in this way, and with practice and skill it is still a method of choice for their attempted
isolation (Blackall et al., 1991a; Blackall et al., 2000; Bradford et al., 1996; Levantesi et al., 2004,
2006a,b; Liu et al., 2000, 2001a; Seviour et al., 1994; Thomsen et al., 2006a). Kragelund et al. (2008),
Levantesi et al. (2004, 2006a) and Thomsen et al. (2004) have employed it successfully to enrich for
single unculturable filament morphotypes before using their biomass to generate 16S rRNA sequence data
by RT-PCR (reverse transcription PCR). This permits construction of a clone library and subsequent
design of targeted FISH probes for their in situ identification. Micromanipulation-RT-PCR seems an
attractive method for identifying other non-culturable filaments, especially where large amounts of
filament biomass can be ‘harvested’ in this way. It should be encouraged, especially for plant samples
where a single morphotype dominates.
Micromanipulation has also been successful in isolating into culture phylogenetically diverse TFO,
including some putative GAO (Seviour et al., 2000), and clustered cells of Acinetobacter from EBPR
plants were obtained by this method (Beacham et al., 1990; Duncan et al., 1988). Lemos et al. (2008) have
also employed it to isolate PHA accumulating populations. Although it requires considerable practice,
inexpensive micromanipulation equipment suited for this purpose (Skerman, 1968) is available, and
described in more detail in chapter 11.
Differential centrifugation
Another approach, applied mainly to enrich populations of putative PAO for subsequent molecular based
identification rather than their culture, relies on the assumption that because they contain high levels of
polyP, glycogen or PHA (chapter 10), their cells will be heavier than those lacking these polymers. Hence
centrifugation in a support like Percoll should allow their separation and recovery (e.g. Schuler et al.,
2002; Streichan et al., 1990), and eventual identity. However, the outcomes have not always agreed with
data obtained from clone libraries generated from the same samples, possibly because of difficulties in
complete separation of the populations sought from other floc material.
Flow cytometry
Flow cytometry (Porter, 1999; Steen 2000; Vives-Rego et al., 2000) is a versatile technique which has
been applied to studying activated sludge communities (chapter 11) for several purposes (Forster et al.,
2003). It has been used successfully to enrich and recover FISH targeted activated sludge populations,
again mainly for their subsequent identification, (McIlroy et al., 2008a; Miyauchi et al., 2007; Snaidr et al.,
1999; Wagner et al., 1993; Wallner et al., 1995) or those immunofluorescently tagged (Porter et al., 1995).
Staining with fluorescent stains for polyP (e.g. DAPI) may allow enriched recovery of PAO (Hung et al.,
2002; 2004; Kawaharasaki et al., 1999, 2002; Zilles et al., 2002a,b). However, activated sludge sample
handling can be problematic (Perez-Feito et al., 2006). Biomass flocculation may prevent or limit
cell separation and recovery, and samples may need some prior homogenization to freely suspend the
individual cells of interest. Fluorescence-activated cell sorting (Park et al., 2005) with flow cytometry
and a combination of fluorescent dyes targeting different cell sites has been applied to activated sludge
and claims made that levels of biodiversity greater than those achievable with many other culture
independent methods can be detected.
Culturing activated sludge organisms

All the culture media chosen to grow activated sludge organisms are selective to differing extents, and
collectively allow us to recover only a small percentage of the populations present there (Loy et al.,
2002a; Wagner and Loy, 2002; Wagner et al., 2002; Wilderer et al., 2002). However, this does not mean
necessarily that cells are a priori unculturable. Clearly we need to understand better the nutritional
requirements of the organisms we seek, and then design media which we are more confident will succeed
in their culture. Without any prior information on what these requirements are this is no easy task.
Unfortunately, it is the situation we face with the Accumulibacter PAO, Competibacter GAO and many
filamentous bacteria causing bulking and foaming. Knowing an organism’s closest cultured phylogenetic
relative generally has not always provided helpful clues about how we might grow it, since phylotype and
phenotype infrequently match (e.g. Beer et al., 2002). Nor have substrate assimilation data from in situ
physiological microautoradiography (MAR) based experiments helped much. For example, data
from the filamentous bacterium type 0041, a member of the candidate TM7 phylum with no cultured
representatives, would suggest that this organism has very mundane and easily satisfied nutritional
requirements (Thomsen et al., 2002).
Yet it should not be forgotten that many activated sludge bacteria have been cultured successfully
in the past, including several filaments (Blackall et al., 1994b, 2000; Kampfer and Wagner, 2002;
Levantesi et al., 2004, 2006a,b; Liu et al., 2001a; Seviour et al., 1997; Snaidr et al., 2002) and represen-
tatives of other morphotypes like the TFO (Seviour et al., 2000, 2003). Preliminary screening of cultured
bacteria with targeted FISH probes to identify possibly novel populations before detailed characteriza-
tion has yielded several quite interesting strains (Kampfer et al., 2005; Spring et al., 2004, 2005). Of
the many media described for recovering activated sludge organisms (Seviour, 1999a; Atlas, 2005),
most success has been achieved with dilute complex media (chapter 5), which reflects the conditions
experienced by these organisms in situ, where nutrient limitation is the usual state of affairs.
While not wishing to discuss at length here the validity of the concepts of ‘viable but nonculturable’
cells (see Kell, 2004; Kell et al., 1998) and their impact on the culture of activated sludge microbes, it may
be that the filamentous bacteria and TFO we have successfully grown so far represent the ‘easy to obtain’
ones. Future efforts may be less productive, even if different strategies are adopted. Experience with
some filaments, where they grow well on initial isolation but subsequently die (Levantesi et al., 2006a;
Seviour et al., 1994), suggests they may lack the ability to synthesize an essential nutrient not supplied,
but one present in adequate amounts from sample carryover. Alternatively these filaments may produce
toxic metabolites from the substrates provided in the medium, which they initially metabolize, but
eventually ‘commit’ ecological suicide (Seviour et al., 1994). The same rationale may apply to most
activated sludge populations, or they may require other populations to supply them with (unknown)
essential nutrients, as seems to be the case with some filamentous bacteria like Candidatus ‘Alysiosphaera
spp.’ requiring co-culture with a yeast to grow (Snaidr et al., 2002).
Identifying and quantifying populations in activated sludge

Our ability to identify in situ bacterial populations and consequently quantify them and study their
ecophysiology has also been revolutionized by molecular methods (Daims et al., 2006c; Rowan et al.,
2005; Wagner et al., 2006; Wilderer et al., 2002). However, for some important populations
‘identifications’ are entirely on their distinctive microscopic morphological features, as with many
unidentified filamentous bacteria and the protozoa. The characters used may be based on descriptions
and keys generated in countries other than where the identification is attempted. The many problems
associated with microscopic ‘identification’ of the filamentous bacteria in activated sludge will be
addressed in chapter 6. Image analysis based systems appear particularly attractive for identifying
the protozoa and metazoa (e.g. Ginoris et al., 2007; chapter 4), because of the easily determined
morphologically diverse features they possess.
Chemotaxonomic markers like fatty acid profiles and quinones have been used to identify putative
PAO populations in activated sludge (Seviour et al., 2003), but probably lack sufficient specificity. Both
markers may also change with the organism’s physiological status. Fluorescent antibodies have been
used for populations like Candidatus ‘Microthrix parvicella’ (Connery et al., 2002) and the ammonia
monooxygenases (amo) of the Nitroso-bacteria (Fiencke and Bock, 2004), although antibody specificity
again is a potential problem, as is the high background autofluorescence from non-specific antibody
reactions (Loy et al., 2002a).
FISH
We now base culture independent identifications of bacterial populations on characters seemingly without
these drawbacks. Although several different genes or their products have been used, the 16S rRNA gene
has been exploited most, because its product (16S rRNA) is in ribosomes, which occur in multiple copies
in metabolically active cells in all ‘prokaryotes’ (chapter 1). Knowing this sequence, for example from
clone library data, means we can design fluorescently tagged probe/s targeting an organism’s 16S rRNA,
with software (e.g.ARB) designed for this purpose. FISH and fluorescence microscopy (chapter 11) then
permit its in situ identification (Amann and Fuchs, 2008; Amann and Ludwig, 2000; Fuchs et al., 2007).
FISH probes can be designed theoretically to target populations ranging across the whole taxonomic
hierarchy, from individual species (not strains) to Domains (Amann and Fuchs, 2008; Amann and Ludwig,
2000; Amann and Schleifer, 2005).
The reader will find in this book frequent mention of FISH and the many probes now available to detect
individual phylotypes among the filamentous bacteria (Table 6.4) (Beer et al., 2002; Davenport et al., 2000;
de los Reyes et al., 1998a,b; Kragelund et al., 2005, 2006, 2007a; Levantesi et al., 2004, 2006a,b; Liu and
Seviour, 2001a; Oerther et al., 2001; Wagner et al., 2003; Xia et al., 2007, 2008b), nitrifying bacteria
(Daims et al., 2001a,b,c; Gieseke et al., 2001; chapter 9), the PAO (Crocetti et al., 2000; Hesselmann et al.,
1999; Kong et al., 2004, 2005; Zilles et al., 2002b) and the GAO (Crocetti et al., 2002; Kong et al., 2002a,b:
2006; Meyer et al., 2006; Wong and Liu, 2007; Wong et al., 2004). They are also available for other important
physiological groups of bacteria in activated sludge (Nielsen et al., 2002a, 2009; Schramm et al., 1999a;
Thomsen et al., 2006b, 2007). A guided tour through the FISH protocol (Daims et al., 2005b) is given in
chapter 11. It is an elegant technique, which has been responsible (in some cases alone) for changing
fundamentally our views about activated sludge microbiology, but is not without problems, which have been
reviewed critically (chapter 11; Amann and Fuchs, 2008; Amann and Ludwig, 2000; Nielsen et al., 2009).
The main weakness with FISH lies with the databases we have available for probe design. Although
they now contain 4400,000 16S rRNA sequences (De Santis et al., 2006; Schloss and Handelsman,
2004), this number still represents just a small percentage of all the ‘prokaryotes’ thought to exist on this
planet. A surprising number of these sequences (5%) are thought to be chimaeric artefacts generated
during sequencing (Ashelford et al., 2005). Consequently, FISH probes designed with these data sets may
inevitably target organisms whose sequences are not yet included, or miss some members of a taxon
against which it was originally designed, giving false positives and negatives respectively. An example of
the latter is the original EUB338 probe designed to target all members of the domain Bacteria sequenced
at that time. We know this did not detect the Verrucomicrobia or Planctomycetales for which sequence
information was then unavailable, and so needed redesigning to accommodate these (Daims et al., 1999).
The failure of some Aquificales and ‘Chloroflexi’ to respond to the EUB338 mix probes (Amann and
Fuchs, 2008; Bjornsson et al., 2002; Kragelund et al., 2007a; Speirs et al., 2009) suggests further
modification to their design is needed.
Thus, probe design is an evolving activity, and we should expect to see similar changes with others
we now use with confidence. An additional problem was mentioned in chapter 1. 16S rRNA genes are
highly conserved, and so it is not always possible to design a probe to distinguish between closely
related organisms. Often competitor probes are required to achieve this resolution (Erhardt et al., 1997).
Or the 23S rRNA is targeted by FISH instead, as happens with members of the closely related
Betaproteobacteria and Gammaproteobacteria (Manz et al., 1992). However, the problems of coverage
apply even more to these because of the smaller sequence databases available, and often require their
redesign too, as with FISH probes targeting the Gammaproteobacteria (Siyambalapitiya and Blackall,
2005; Yeates et al., 2003).
Although it seems that bacteria like the nitrifying bacteria always retain high ribosome numbers
independent of their physiological state, metabolically inactive cells would generally contain fewer
ribosomes, and hence be more difficult to detect by FISH. Accessing inadequately its target site can also
lower FISH probe signal strength (Behrens et al., 2003a,b, 2004; Fuchs et al., 2000, 2001; Yilmaz et al.,
2006). Several methods can increase probe signal strength, including using non-fluorescent helper probes
(Behrens et al., 2003a; Fuchs et al., 2000, 2001; Meyer et al., 2006), which bind to the 16S rRNA, and
alter its conformation, allowing readier probe access. Changing the chemistry of the probes themselves
may also increase the fluorescence signal (Amann and Fuchs, 2008), as achieved with the LNA/DNA
probes described by Kubota et al., (2006). CARD-FISH (see p. 373) (Pernthaler et al., 2002) achieves the
same result (chapter 11), as do peptide nucleic acid probes (Wilks and Keevil, 2006). Probe permeability
(Kragelund et al., 2007b) may also be a problem, as with some foaming mycolic acid bacteria (Mycolata)
(chapter 8), especially when FISH is used as a quantitative tool (Davenport et al., 2000), and if the cells
generating low or no FISH signals still stabilize foams (Carr et al., 2005; Eales et al., 2005, 2006).
Other genes of functional importance in processes like nitrification, denitrification and P removal are
not present in cells in such large copy numbers, and so identifying cells expressing them is problematic,
even if their gene sequences are known. FISH techniques detecting specific transcribed mRNA have been
described for such purposes (Coleman et al., 2007; Pernthaler and Amann, 2004), although not applied yet
to activated sludge communities.
qPCR based methods

Having sequence information for a targeted gene generally allows its detection by PCR based techniques,
and from which populations this gene might originate can be deduced by analysing its diversity within the
community. Although we know conventional PCR data can not be used quantitatively (von Wintzingerode
et al., 1997), real-time PCR (qPCR) (Smith, 2005; Smith et al., 2006) and competitive PCR (cPCR)
systems (Dionisi et al., 2002b,c) allow us to quantify individual bacterial populations and copy numbers
of functional genes of interest (chapter 11). qPCR has been used to quantify many activated sludge
populations, including some bulking and foaming filamentous bacteria (Vervaeren et al., 2005) like
Candidatus ‘M. parvicella’ (Kaetzke et al., 2005), Several nitrifying and denitrifying bacterial populations
(Geets et al., 2007; Limpiyakorn et al., 2006, 2007), and the levels of their functional Amo genes
(e.g. Dionisi et al., 2003; Sekido et al., 2007) and Accumulibacter PAO (Fukushima et al., 2007;
He et al., 2007a) can be quantified by the same approach. Gene expression in situ can also be quantified
using extracted mRNA and reverse transcriptase qPCR, and better estimates of the actively involved
populations reached. Real-time t-RFLP combining qPCR and t-RFLP (chapter 11) has been used to
quantify diverse oestrogen degrading populations in activated sludge (Yu et al., 2005), and this method
seems applicable for similar studies with other clearly defined physiological groups.
Other quantification methods

For reasons which should be clear by now, no longer do we depend on viable counts on artificial lab media
to estimate population numbers in activated sludge, although many media have been designed for this
purpose, and may still be valuable for non-quantitative studies (Pike et al., 1975). In addition, most
probable number (MPN) techniques, although not used as frequently now, are still helpful in quantifying
populations with known physiological properties, like nitrifying bacteria and sulphate reducing bacteria
(Ingvorsen et al., 2003). These can be used in combination with FISH to increase their detection
sensitivity. Enumeration of functional groups capable of assimilating particular substrates in situ under
specified operating conditions is achieved with MAR (Nielsen and Nielsen, 2002a,b, 2005). DAPI or more
sensitive fluorescent DNA targeted stains (e.g. Sybr green, Syto9 etc.) are used frequently for determining
total cell numbers in activated sludge, and often provides the basis for estimating the relative numerical
importance of other groups (e.g. Beer et al., 2006; Snaidr et al., 1997) after more targeted techniques
like FISH.
Because it does not suffer from the biases inherent in conventional PCR based methods, and is
relatively simple, FISH has been exploited not only for identification purposes, but for quantification of
individual populations (Amann and Ludwig, 2000), either in combination with image analysis software
(Daims et al., 2001c), which may be automated for such purposes (Daims et al., 2006a; Zhou et al.,
2007b), or flow cytometry (Forster et al., 2003). So far these applications have been directed mainly at
quantifying populations of particular importance like the bulking and foaming filaments (Hug et al.,
2005), PAO populations (Beer et al., 2006; Crocetti et al., 2000; Kong et al., 2005; Wong et al., 2005),
and denitrifying (Thomsen et al., 2007) and nitrifying bacteria (Daims et al., 2001a,b), but they can be
used equally for any group of organisms that can be appropriately tagged.
Total microbial biomass levels, important for modelling purposes (chapter 2) have been quantified by
measuring total DNA levels, determined chemically or after staining with fluorescent stains like DAPI, but
only with varying success when correlated against other indicators on the same samples. This discrepancy
may reflect difficulties in estimating DNA content on a single cell basis, possible contamination with large
amounts of extracellular DNA (eDNA, Böckelmann et al., 2006; Palmgen and Nielsen, 1996) or problems
in translating values for cell numbers (or biovolume) into biomass measurements.
Detecting viable cells in activated sludge

Even if we can identify and quantify individual populations reliably, such data often tell us little or
nothing about their activity or function within activated sludge plants (Amann and Kühl, 1998;
Wagner et al., 2006). The once widely held view was that many organisms, especially those associated
with flocs are either moribund or dead, since total microscopic counts and viable counts carried out on the
same samples of mixed liquor always compared badly (Pike, 1975). Although we explain such
discrepancies in other ways now, this view may still be valid in some cases (Lopez et al., 2005). However,
application of rRNA targeted probes to activated sludge, relying as they do on high ribosome levels in
physiologically active cells to generate bright signals, suggests that most cells there (usually about 80% –
see later) are metabolically active (Amann and Ludwig, 2000; Amann et al., 1995; Loy et al., 2002a).
As mentioned above, the problems of distinguishing methodologically between what we mean by
cell ‘viability’ and cell ‘activity’, and how we apply these terms to microbes, have been discussed by
Kell (2004) and Kell et al. (1998). Several methods for determining both parameters have been applied to
activated sludge communities, but measure different cell properties. So which one is the most suitable, and
how the data are interpreted, both need careful consideration (Nebe-von-Caron et al., 2000).
Knowing what proportion of the community is viable and/or metabolically active is useful information
for example before attempting to micromanipulate a filamentous bacterium into pure culture, or if there is
some concern that the community might have been exposed to toxic chemicals, or after an episode of
chlorination to control a bulking incident. Some viability methods rely on the inability of fluorescent
nucleic acid stains like SYTOX green to enter viable cells with intact membranes (Roth et al., 1997).
Others (e.g. the LIVE/DEAD1 method) use double staining (chapter 11), based on differentiating
between cells with (fluoresce green ¼ alive) and without membrane integrity (these fluoresce
red ¼ dead). This technique did show that many mycolata (Figure 3.3, see colour image section
(chapter 13)) in stable activated sludge foams are dead (Carr et al., 2005). CLSM coupled with image
analysis has used the same method to map quantitatively the distribution of living and dead cells in flocs
formed under different conditions (Lopez et al., 2005), and together with flow cytometry (Ziglio et al.,
2002) to estimate overall sludge biomass viability. However, interpreting LIVE/DEAD1 staining may be
problematic with complex samples like activated sludge (Biggerstaff et al., 2006).
Detecting and identifying active cells in activated sludge and measuring

their activities
Several methods can assess total biomass activities, and identify and quantify active cells and measure
their rates of activity. Respirometry when carried out under recommended strictly standardized conditions
(Copp et al., 2002; Spanjers et al., 1998) allows estimating the rates of bulk activities of important
metabolic processes (for example respiration, nitrification and denitrification), and provides reliable and
essential data for modelling purposes (chapter 2), but not at the level of single cells or individual
populations. Biomass ATP content as an indicator of activity (Jorgensen et al., 1992a) suffers from the
same problem, as do many of the enzyme assays used to assess biomass activities (Nybroe et al., 1992),
but which may not arise from viable cells.
Enzyme labeled fluorescence (ELF) techniques (chapter 11) allow localization of activity of
ectoenzymes to individual cells, and have been applied successfully (chapters 5, 7) with filamentous
bacteria to suggest which substrates they might degrade in situ (Eales et al., 2006; Kragelund et al., 2005,
2006, 2007a,b). ELF again is not necessarily a feature of viable cells only, and is limited by lack of
commercial substrate availability (Eales et al., 2006; Kloeke and Gessey, 1999; Kragelund et al., 2005, 2006,
2007a; Schade and Lemmer, 2005). Detection of esterase and dehydrogenase activity in single cells with flow
cytometry has been claimed to quantify the numbers of actively respiring cells (Veal et al., 2000).
With dehydrogenases the electrons generated are transferred to a redox dye (the electron acceptor),
which on becoming reduced is converted to a detectable product. Early studies used colorless tetrazolium
salts like 2-p (iodo-phenyl0-3(nitro-phenyl)-5(phenyl)tetrazolium chloride (INT), which is reduced to a
red formazan precipitate, retained within the cells. INT was used to determine the activity of filamentous
bacteria (Awong et al., 1985; Caravelli et al., 2004) and to follow heavy metal inhibition of activated
sludge biomass (Anderson et al., 1988). However, its suitability for such purposes has been questioned
(Maurines-Caroneill et al., 1998), and it has been replaced largely now by CTC (5-cyano-2,3-ditolyl
tetrazolium chloride) (Rodriguez et al., 1992), which is reduced at a different site to INT during electron
transport (Smith and McFeters, 1997). However, whether CTC measures cell viability or activity is
controversial (Créach et al., 2003; Nielsen et al., 2003a). Reduction of CTC generates a fluorescent
formazan precipitate, which may also be formed abiotically (Smith and McFeters, 1997), or by floc EPS
material (Griebe et al., 1997; Wuertz et al., 1998), and some evidence suggests that at the concentrations
used in experiments with activated sludge, it may inhibit bacterial metabolism (Créach et al., 2003).
In comparison to MAR (see below), CTC staining gave much lower estimates for numbers of active/viable
cells in activated sludge (Nielsen et al., 2003a).
Both reduced forms of INT and CTC are retained within the cells, and so quantitative analyses of their
generation rates require prior extraction. Tetrazolium salts are available which are reduced to water
soluble formazan products (Bensaid et al., 2000; McCluskey et al., 2005). These have been assessed
against activated sludge bacterial cultures and appear suited for routine monitoring of microbial
respiratory activity, in for example assessing possible toxic responses of the community. Resazurin, a
cheaper alternative, has also been used for the same purpose to estimate and monitor activities of the total
biomass in plants, again with encouraging results (McNicholl et al., 2007).
Fingerprinting of carbon utilization patterns with systems like BIOLOG can assess overall metabolic/
degradative potentials of activated sludge communities (Fulthorpe and Allen, 1994; Paixâo et al., 2007;
Victorio et al., 1996). It has the attraction of allowing custom-made substrate screening, to detect
whether a particular compound of interest is being degraded. However, it too is not without its problems
(Preston-Mafham et al., 2002), and is seldom used. Instead, MAR (Andreasen and Nielsen, 1997;
Lee et al., 1999; Nielsen and Nielsen, 2005; Ouverney and Furhman, 1999; Wagner et al., 2006), usually
in combination with FISH has replaced much of what it aimed to achieve.
Together these now provide the means to determine microbial activity in situ (Lee et al., 1999; Nielsen
and Nielsen, 2005; Wagner et al., 2006) by detecting assimilation of substrates of interest by a single defined
population at an individual cell level (see Figure 3.4). Their elegance and ability to answer important
questions about which populations may be responsible for certain biochemical processes in activated sludge
means they have been applied increasingly to resolving the ecophysiology of bulking and foaming
filamentous bacteria (chapters 5, 7, 8) and the PAO and GAO (chapter 10). FISH-MAR has been used
quantitatively to enumerate populations assimilating acetate under different operational conditions, and
several other functional organism groups there (Nielsen and Nielsen, 2002a). The protocol has been
modified (Het CO2–MAR) to discriminate between growing and non-growing cells of the filamentous
bacterium Candidatus ‘Microthix parvicella’ and to indicate conditions under which in situ growth might
occur (Hesselsoe et al., 2005). This is based on MAR determined heterotrophic 14CO2 assimilation. Isotope
arrays using labelled RNA in combination with FISH-MAR have also been designed and tested against the
chemolithoautotrophic nitroso-bacteria (Adamczyk et al., 2003), allowing simultaneous identification of
multiple populations assimilating a particular substrate. MAR can be quantified (FISH-QMAR), and
substrate (acetate) assimilation rates and Ks values in FISH-identified filamentous bacteria and their
individual cells have been measured in this way (Nielsen et al., 2003b), as discussed in chapter 5.
FISH-MAR in combination with ELF has also provided valuable information on the in situ physiology of
several bulking and foaming filamentous bacteria (Kragelund et al., 2005, 2006, 2007a,b).
MAR can be criticized in that with suitable controls it clearly demonstrates assimilation of a substrate,
but not necessarily its subsequent metabolic fate (Wagner et al., 2006). There is the further possibility that
the substrate provided (especially under anaerobic conditions) is first metabolized by other populations in
activated sludge, and it is the fate of the product of these events that is actually visualised (Kong et al.,
2005). Despite such concerns, it seems on balance the best indicator of in situ cell activity/viability. When
MAR, FISH and CTC reduction in the filament Thiothrix were compared as indicators of cell activity/
viability (Nielsen et al., 2003a), FISH and MAR outcomes agreed well, while cells responding positively
to them were often CTC negative. However, in G. amarae, some FISH positive cells in each filament in
foams were sometimes MAR negative, and vice versa (Carr et al., 2006). FISH negative but MAR positive
cells have also been noticed with aquatic Actinobacteria (Nielsen et al., 2006), emphasizing the problems
of defining which are the truely active populations in natural communities. Further discussions on
FISH-MAR are given in Nielsen and Nielsen (2005) and Wagner et al. (2006), and in chapter 11.
Figure 3.4. MAR showing acetate uptake by an individual filament of Skermania piniformis (K. Eales). Bar
marker ¼ 10 mm.
Stable-isotope probing (SIP), like FISH-MAR, allows us to relate physiological/biochemical activity

to a particular population/s in complex communities (Dumont and Murrell, 2005; Friedrich, 2006;
Neufeld et al., 2007; Wagner et al., 2006) including activated sludge (chapter 11). In this method, 13C
enriched substrates of interest are supplied to the community, and if assimilated are incorporated into the
DNA of populations metabolizing them (Neufeld et al., 2007). This ‘heavy’ DNA can be recovered from
‘normal’ 12C-DNA and the phylogenetic diversity of 16S rRNA genes of the populations from which it
derived then determined (Neufeld et al., 2007). Because specific mRNA transcribed from the functional
genes in active populations will also be 13C enriched, RNA based recovery protocols and phylogenetic
analysis are more likely to reveal the active functional populations, although RNA extraction methods are
more troublesome than those for DNA (Milling et al., 2005; Nogales, 2005; chapter 11).
Following changes in activated sludge community composition

Some molecular techniques allow us to follow changes in activated sludge community composition, either
in response to changes in operational conditions and/or over time. Clone libraries are too slow to allow
analyses of sufficient samples for these purposes, but PCR-profiling methods based on 16S rRNA can
provide such qualitative information. Selecting other primers targeting functional genes or gene fragments
allows them and their populations to be profiled and monitored too (Hallin et al., 2006; Lee et al., 2005).
Thus, DGGE (Boon et al., 2002; Chen and Lapara, 2006; Gilbride et al., 2006; Lapara et al., 2000,
2002; Stamper et al., 2003; Whitely and Bailey, 2000), TGGE (Eichner et al., 1999; Watanabe et al.,
1999) as well as T-RFLP (Hoshino et al., 2005; Liu et al., 1997; Saikaly et al., 2005) and SSCP
(Dabert et al., 2001, 2005), ribosomal intergenic spacer length (RIS-rRNA) polymorphism fingerprinting
(Smith et al., 2004), LP-RAPD (Yan et al., 2007) and cloning-T-RFLP (Hoshino et al., 2006) have all
been applied to activated sludge communities for such purposes. Each allows simultaneous analyses of
large numbers of samples, and population changes in them to be detected. Their relative advantages and
disadvantages have been discussed by Smalla et al. (2007) for soil analysis, and in chapter 11, and as
these are all PCR-based methods, quantitative profile comparisons are not realistically possible. FISH or
qPCR for targeted populations like nitrifiers (e.g. Layton et al., 2005; Limpiyakorn et al., 2005, 2006;
Maixener et al., 2006; Sekido et al., 2007) or Accumulibacter PAO (He et al., 2007a) may be preferred
instead. Problems associated with comparative analyses of the large numbers of profile patterns generated
with these methods and their ecological significance have been addressed (e.g. Abdo et al., 2006;
Blackwood et al., 2007; Osborne et al., 2006). Other marker characters have been used to achieve the
same purpose, but suffer from limitations. Thus, Werker and Hall (1998) and Werker et al. (2003) showed
community shifts in activated sludge from fatty acid isopropyl ester patterns, but not identification of
individual populations.
Resolving population structure/function relationships in activated sludge

communities
With techniques like FISH-MAR and SIP and their ability to identify in situ which populations or
phylotypes are responsible for performing key biochemical changes in activated sludge at a single cell
level, it is possible to relate these functional attributes to targeted organisms (Gray and Head, 2001;
Nielsen and Nielsen, 2006; Wagner et al., 2006). For example, when used with sensitive histochemical
staining techniques (Serafim et al., 2002a) for storage polymers polyP and polyhydroxyalkanoates,
FISH-MAR has changed profoundly our understanding of the ecophysiology of filamentous bacteria
and PAO in EBPR processes (chapters 5, 7, 10). Tagging cells with the green fluorescent protein marker
(Eberl et al., 1997; Larrainzar et al., 2005), also allows us to follow the fate of populations and assess their
possible role in degrading compounds of interest.
Applying PCR based techniques for detecting individual functional genes, and subsequent
phylogenetic analyses where the populations possessing these genes can be identified was mentioned
earlier. This was the approach taken to identify populations degrading 2,4-D in activated sludge (Lee et al.,
2005). A broader strategy involves extracting, separating and identifying the proteins, hoping that some
responsible for carrying out the process of interest will be revealed. This megaproteomics approach was
that adopted by Wilmes and Bond (2004; 2006; 2008a,b), with 2-D protein electrophoresis and rapid
amino acid sequence analyses, to help identify collectively the functional proteins involved in EBPR
(chapter 10).
THE ACTIVATED SLUDGE MICROBIAL COMMUNITY COMPOSITION

Viruses and bacteriophages
Humans excrete viruses in their faeces, and so not surprisingly raw sewage (Lodder and de Roda Husman,
2005) and activated sludge communities contain large numbers of these enteric viruses. They include the
polioviruses, enteroviruses, hepatitis A virus, noroviruses, caliciviruses, the adenoviruses and rotaviruses.
Many are serious pathogens of humans, causing gastroenteritis and other more serious diseases. Thus, they
pose as serious potential health hazards if allowed to contaminate potable water supplies and food, and
many believe both should be assayed routinely for their presence (Fong and Lipp, 2005). Some of
these viruses are robust and will survive in the environment (Rzežutka and Cook, 2004). As methodology
for their detection and quantification improves (Fong and Lipp, 2005) so will our understanding
of their ecology and survival capabilities in activated sludge increase (Haramato et al., 2005; Sedmak
et al., 2005).
As we also excrete large numbers of bacteria, the general view is that phages will be present wherever
their bacterial hosts are (Chibani-Chennoufi et al., 2004). Our understanding of bacteriophage ecology in
activated sludge systems is still relatively poor (Otawa et al., 2007; Wagner, 2005; Weinbauer, 2004), as a
consequence of methodological problems, in not being able to culture most of their bacterial hosts for
phage detection and quantification. Consequently we lack satisfactory answers to several important
questions. We still do not know how many phages are present nor how diverse they are, especially in
terms of their host ranges. It is likely that their numbers are considerable (Otawa et al., 2007) if the phage:
bacteria ratios are similar to those (i.e. 410:1 would give up to 1012 ml 1) estimated for other aquatic
habitats (Weinbauer, 2004). Even these are likely to be underestimates since they would not accommodate
any prophages, which almost certainly will exist.
It is easy to isolate phages which infect enteric bacteria like E. coli and related populations. These
appear to decline sharply in numbers during the activated sludge process, possibly by adsorption to flocs
(Tanji et al., 2002), and are sensitive to final chlorination (Bitton, 1987; Tanji et al., 2003). The suggestion
has been made that coliphages are suitable indicators of general viral removal from treatment systems
(Arraj et al., 2005; Gino et al., 2007). Some temperate coliphages may carry the stx2 gene encoding the
Shiga toxin, which has health implications for personnel at treatment plants, especially those exposed to
aerosols (Muniesa et al., 2004).
Hantula et al. (1991) published data which imply many activated sludge phages have broad host
ranges, although they were not thought to play important roles in determining community function.
However, their data are open to criticism since only phages whose bacterial hosts are culturable were
examined, and only superficial characterization was performed with them. The results of Khan et al.
(2002a,b) who targeted phages infecting organisms involved in EBPR, suffer from the same limitations.
They suggested their preparations had broad host ranges too, and some were infective against both
Gram-negative and Gram-positive bacteria. This might result from them being mixtures of several
different phages, although Jensen et al. (1998) showed the vast majority of those they examined in sewage
were also polyvalent. Fluorescently tagged phages provide a simple and specific method to rival FISH for
identifying and quantifying their host cells (Khan et al., 2002a,b; Lee et al., 2006), and in assisting in
revealing phage ecology.
Clearly more work is required to extend our understanding of activated sludge phage diversity before
the roles they play in determining community population dynamics become clearer. Lee et al. (2006)
successfully isolated and characterized phages they claimed were specific against the putative
PAO Microlunatus phosphovorus, and suggested that these and other phages might affect the chemical
transformations they carry out, but with no strong supportive evidence. It is generally believed that
virulent phages are important in controlling bacterial numbers by cell lysis, but whether the consequences
of such activity are beneficial in operational terms remains speculative.
Equally unclear is the role of these phages as agents of transduction (chapter 1) and thus genetic
change (Gino et al., 2007; Jensen et al., 1998; Sander and Schmieger et al., 2001). With the interest in
phage therapy to treat infectious diseases no longer susceptible to antibiotic chemotherapy (Chanishvili
et al., 2001; McNerney and Traoré, 2005), the application of similar technology to tackling operational
problems in activated sludge systems has been considered (Withey et al., 2005). This approach seems
particularly attractive in attempts to control or eliminate bulking and foaming filaments. It is most likely
that phages capable of lysing the causative bacteria are present, and Thomas et al. (2002) readily isolated
several actinophages, some with broad host ranges, capable of lysing members of the foaming Mycolata
(Figure 1.2). This technology is theoretically safe, highly specific and environmentally friendly, although
some potential problems with it, including possible bacterial host resistance (Withey et al., 2005) still
need resolution.
Bacteria and Archaea

The (largely untested) assumption is that all activated sludge plants of similar configurations and treating
similar wastes will have roughly similar community compositions, regardless of their global location.
The bacterial communities of particular interest, including those responsible for P and N removal, and
bulking and foaming will be dealt with only briefly here, and so this section will aim to give a more
general overview of activated sludge bacteriology. Many earlier studies, especially those directed at
understanding which populations are involved in P and N removal used lab scale systems (often SBRs) fed
a standardized synthetic sewage, where some process control could be exerted (Seviour et al., 2003), and
less attention was paid to the more complex and less controllable full-scale plants. However, this
imbalance has been addressed (Beer et al., 2006; Kong et al., 2004, 2005, 2007; Tykesson et al., 2006;
Wong et al., 2005; Xia et al., 2007; 2008a,b), and there are now sufficient data to permit a few
generalizations about activated sludge bacteriology. Examples where FISH has been used to quantify their
populations are given in Table 3.1.
We have known for many years that these communities, not unexpectedly (chapters 1, 2) inevitably
contain a diverse group of faecal commensals and pathogenic organisms in high numbers, and although
their elimination is one aim of the treatment process, many survive it to threaten us. PCR based assays
have expanded the list of bacterial pathogens known to occur in sewage and plant effluent, increasing this
concern (chapter 1).
The number of bacteria in activated sludge has been estimated to be in the range of 1–10 £ 1012/g VSS
(Nielsen and Nielsen, 2002b), based on DAPI staining that imparts fluorescence to DNA of most bacteria
regardless of whether they are ‘alive’ (the total count). The percentage of alive or metabolically active
cells from independent studies is typically around 80% of this total count. This fraction can be assessed by
FISH which detects cells with high ribosome contents and thereby is believed to indicate present or very
recent cell activity. Alternatively it can be quantified as those cells taking up substrates after MAR
(Nielsen and Nielsen, 2002b), and a similar value of around 80% has been obtained using this measure.
The extent of the biodiversity of activated sludge bacterial communities?

What has emerged from the accumulated molecular data are the high numbers of unidentified taxa never
detected by culture dependent methods in activated sludge (Figure 1.3) (Blackall and Yeates, 2004;
Seviour and Maszenan, 2002). Some of these must play roles in the chemical transformations occurring
there, but in the majority of cases we are yet to find out what these are, since they represent cloned rRNA
sequences only and their phenotypes are unknown.
How the levels of biodiversity of these communities may be determined has been assessed in several
ways (Daims et al., 2006c; Juretschko et al., 2002; Loy et al., 2002a). FISH has frequently confirmed results
from 16S rRNA clone library analyses, but they do not always produce the same outcomes from the same
community (Ahn et al., 2007; Juretschko et al., 2002; Loy et al., 2002a; Meyer et al., 2006; Wagner and
Cloete, 2002). This presumably reflects the biases which are inherent in all PCR based methods, and the
relative insensitivity of FISH microscopic methods when analysing small sample sizes (Daims, 2005b).
The number and range of new bacterial genera and species which have been and continue to
be cultured from activated sludge in the past decade seems endless. Studies with cultured isolates have
been invaluable in identifying populations capable of performing metabolic reactions of importance
Table 3.1. Composition of full scale activated sludge plant communities as determined by semi-quantitative FISH analyses. ND ¼ not
112
detected.
Population composition (biovolume % against Eubmix)
Nitropira
(LGCmix)
(HoAc1402)
(Eub338II)
Firmicutes
(HGC69a)
Proteobacteria
(CFX1223)
‘ Chloroflexi’
Acidobacteria
(CF319a+b)
Actinobacteria
Bacteroidetes
Planctomycetales
Plant
(Ntspa1026,662,712)
Plant name configuration Alpha Beta Gamma Reference

Dietersheim Unspecified 16 ^ 2 51 ^ 7 11 ^ 4 22 ^ 6 13 ^ 5 6^1 ND 51 ND ND Schmid et al.
(2003a)
GroBlappen Unspecified 13 ^ 3 24 ^ 3 4^1 21 ^ 16 7^4 6^2 ND 4^1 ND ND Schmid et al.
(2003a)
Poing Unspecified 9^2 18 ^ 3 7^1 36 ^ 9 25 ^ 5 1^0 ND 2^1 ND ND Schmid et al.
(2003a)
Ariake_A2O A2O 9 20 9 10 51 51 ND ND ND ND Wong et al.
(2005)
Nakano AO 12 30 6 19 51 51 ND ND ND ND Wong et al.
(2005)
Kraftisried Industrial 16 47 2 ND ND ND 5 6 1 12 Juretschko
N-removal et al. (2002)
A Modified UCT 10 ^ 4 17 ^ 7 13 ^ 4 8^3 56 56 25 ^ 7 ND ND ND Beer et al.
(2006)
B 3 stage bardenpho 27 ^ 9 6^4 57 23 ^ 11 57 57 51 ND ND ND Beer et al.
(2006)
C Carousel/UCT 35 ^ 15 56 16 ^ 7 10 ^ 9 51 13 ^ 6 56 ND ND ND Beer et al.
(2006)
D 3 stage bardenpho 19 ^ 10 16 ^ 11 7^5 42 ^ 14 51 56 10 ^ 5 ND ND ND Beer et al.
(2006)
E Modified UCT 66 ^ 15 51 6^5 22 ^ 8 51 13 ^ 9 29 ^ 14 ND ND ND Beer et al.
(2006)
F Carrousel/UCT 37 ^ 18 6^5 8^4 16 ^ 6 ND 11 ^ 8 24 ^ 11 ND ND ND Beer et al.
(2006)
G Modified UCT 32 ^ 12 9^4 5^2 12 ^ 8 55 55 55 ND ND ND Beer et al.
(2006)
H Modified UCT 30 ^ 9 16 ^ 11 7^4 13 ^ 8 51 13 ^ 3 55 ND ND ND Beer et al.
(2006)
J Modified UCT 33 ^ 10 13 ^ 9 17 ^ 8 7^6 55 56 15 ^ 7 ND ND ND Beer et al.
(2006)
(Yoshimoto et al., 2004), but such information does not by itself allow their role in situ to be predicted.
Applying innovative selective enrichment techniques with activated sludge brings its rewards. For
example, they have generated the first cultured isolates of some bacterial phyla (e.g. Zhang et al., 2003b).
The rapidly expanding list of activated sludge organisms emphasizes how largely unexploited this habitat
still is as a source of novel organisms.
More 16S rRNA clone library data are available from lab-scale systems usually fed synthetic wastewater
containing a single carbon source (see Loy et al., 2002a), but because the selective pressures are likely to
differ markedly from those in full scale plants, any conclusions do not necessarily translate readily to them.
Surprisingly few extensive quantitative studies have been performed on detailing the complete community
structure of full-scale plants. Thus, only one industrial treatment plant community performing nitrification
and denitrification (Juretschko et al., 2002) and a plant receiving wastewater from a petroleum refinery
(Figuerola and Erijman, 2007) have been characterized by 16S rRNA gene clone library analysis and
subsequent Q-FISH with probes designed to target sequences recovered in the clone library. Data from the
one full-scale EBPR community comprehensively characterized by Kong et al. (2007) seemed to suggest
that we now know the identity of all the main populations in these systems, and no surprises are left.
Whether this applies to all such plants seems unlikely. It is more probable that we still lack an encompassing
overview of biodiversity in full scale systems, and that activated sludge bacteriology will continue to hold
many surprises, as illustrated below. Many rRNA sequences may arise from populations entering the plants
in the raw effluent, providing a high diversity ‘background’, but these may be unimportant in terms of how
these processes function, and most may even fail to survive long there.
ACTIVATED SLUDGE PLANT COMMUNITY STRUCTURE

The Archaea
It now seems that methanogenic Archaea occur in activated sludge (Gray et al., 2002), albeit in small
numbers (51% of total cells; Nielsen and Nielsen, 2002b), Whether these actively grow there or are
seeded from the sewers is not clear. Since so few studies have explored archaeal populations in this system
it is premature to comment further on their possible importance, but PCR analyses targeting the amoA
genes of the Crenarchaeota suggest that archaeal ammonia oxidizing organisms (Francis et al., 2007)
may be commonly occurring nitrifying members (Park et al., 2006). As mentioned earlier (chapter 1),
members of this domain are ecologically much more widely distributed than once considered likely
(e.g. Cavicchioli, 2006), so the increasingly large number of FISH probes now available targeting them
(Crocetti et al., 2005) may provide further insight into their role.
Chemoorganoheterotrophic bacteria
Data would suggest that these are the major populations in activated sludge. While culture dependent
methods using nutrient rich media favour the growth and hence detection of Gammaproteobacteria
(Wagner et al., 1993; Kämpfer et al., 1996), with conventional plants, both FISH and clone library data
using 16S rRNA sequence analyses suggest that the beta one subgroup of the Betaproteobacteria, and
especially members of the Comomonadaceae (Khan et al., 2002c; Spring et al., 2004) including
Acidovorax spp (Schulze et al., 1999), are among the dominant bacteria. The Alphaproteobacteria include
the commonly detected sphingomonads (Neef et al., 1999). Caulobacter can be readily isolated from
activated sludge too (Holm et al., 1996; Winkler and Cox, 1980), and Hyphomicrobium species, possible
denitrifying members, have been detected in activated sludge communities, sometimes on a seasonal
basis (Gliesche and Fesefeldt, 1998; Layton et al., 2000). Gammaproteobacteria, and Actinobacteria
are also generally well represented in communities treating both domestic and industrial wastes.
The Actinobacteria often occur in high numbers, especially in some foaming plants and EBPR processes
(Beer et al., 2006; Kong et al., 2005; Seviour et al., 2008; Xia et al., 2008a). Deltaproteobacteria have
been detected in many communities, represented mainly by the sulphate reducing bacteria (SRB)
Desulfovibrionaceae and Desulfobacteriaceae (Manz et al., 1998).
Less expected has been the detection of large numbers of generally previously undescribed and
phylogenetically diverse members of the ‘Chloroflexi’ (Beer et al., 2006; Bjornnson et al., 2002;
Kragelund et al., 2007a), Bacteroidetes (Hong et al., 2008; Kragelund et al., 2008; Xia et al., 2008b)
and unusual Planctomycetales (Bond et al., 1995, 1998; Chouari et al., 2003; Neef et al., 1998) in
conventional and EBPR plants. ‘Chloroflexi’ include representatives of the bulking filamentous bacterial
morphotypes Eikelboom type 1851 (Beer et al., 2002) and type 0092 (Spiers et al., 2009), and ‘Nostocoida
limicola’ III is a planctomycete (Liu et al., 2001a). Their in situ function is still largely a mystery. Also
frequently detected in clone libraries are representatives of phyla with few or no cultured representatives
including the Verrucomicrobia and Acidobacteria (Loy et al., 2002a), and the OM11 and TM7 group, the
latter including some members of the Eikelboom filament morphotype type 0041 (Hugenholtz et al., 2001;
Thomsen et al., 2002). Firmicutes appear to be common but not generally dominating populations (Meier et al.,
1999), and they, together with some Actinobacteria, may be major fermentative populations (Kong et al.,
2008). The insect pathogen Bacillus thuringiensis is frequently recovered into culture (Mizuki et al., 2001).
As discussed later (chapters 5, 7), many more filamentous bacterial morphotypes than once thought
existed have been described in plants treating industrial wastes (Eikelboom, 2006; Eikelboom and Geurkink,
2002; van der Waarde et al., 2002) (chapter 7), and other populations also occur more commonly there
e.g. Arcobacter sp. (Snaidr et al., 1997), and possibly Paenobacillus (Simpson et al., 2006).
The phylogenetic diversity now apparent within a single filament morphotype and among the TFO
morphotypes (Figure 3.5, see colour image section (chapter 13)) is also striking, emphasizing the risks in
relying solely on morphology for their identification (chapters 5, 7, 10, 12). The latter include both cultured
(after micromanipulation) and uncultured (after FISH) members of Alphaproteobacteria (Maszenan et al.,
1997, 2005a; Meyer et al., 2006; Wong and Liu, 2007; Wong et al., 2004) and Betaproteobacteria
(Maszenan et al., 2002), as well as the Actinobacteria (Maszenan et al., 1999b, 2000; 2005b; Seviour et al.,
2000). Some of these TFO, more of which await identification, appear especially frequently in EBPR plants
(chapter 10), and in those treating wastes containing high carbohydrate levels (Tsai and Liu, 2002). Novel
foaming Actinobacteria continue to be grown in pure culture (Nam et al., 2003, 2004; Soddell et al.,
2006a,b), supporting the notion that our understanding of the microbial ecology of these communities is
poor (Soddell et al., 1998; Stainsby et al., 2002) (chapter 8). Activated sludge communities also seem to
represent a rich reservoir of new species of well established genera, as seen for example, with Acinetobacter
(Carr et al., 2003), Acidovorax (Schulze et al., 1999) and Aeromonas (Nsabimana et al., 2000).
Function of these populations?

As mentioned previously, knowing only which organisms are present will not reveal all the secrets of
activated sludge systems. Recognizing and attributing functions to individual populations or phylotypes
(most of which are uncultured) in the main physiological groups is essential, and in some cases applying
the techniques mentioned in this chapter is assisting this, as a few examples will demonstrate.
Denitrifying bacteria
Denitrifying bacteria are present in all plants capable of complete N-removal but may be relatively scant in
plants carrying out only nitrification (Nielsen and Nielsen, 2002a). These can be recognized tentatively either
by their abilities to assimilate and grow on respirable substrates like acetate or methanol in the presence of NO2
or NO3, as determined with FISH-MAR (Nielsen and Nielsen, 2002a), by stable isotope probing (Ginige et al.,
2004) or by applying PCR based methods to recognize populations possessing the marker norB (Braker and
Tiedje, 2003), nirS, nirK and nosZ denitrifying genes (Goregues et al., 2005; Hallin et al., 2006; Throbäck et al.,
2004). Which are the dominant populations of denitrifiers in activated sludge is unclear, and because this ability
is so widespread, populations may vary between individual plants, and with changes in influent and operational
conditions (Hallin et al., 2006) (chapter 9). Ginige et al. (2007) and Morgan-Sagastume et al. (2008) confirmed
they are adaptable organisms, and showed by FISH that different survival strategies are used by them in
response to changes in their substrate/nitrite to biomass ratios. Many potential denitrifier candidates have been
isolated and described in pure culture, but subsequent FISH-MAR analyses have yet to confirm the importance
of most of these in situ. Those detected in plants designed to remove N include Aquaspirillum, Azoarcus and
Thaurea species (Morgan-Sagastume et al., 2008; Thomsen et al., 2004, 2007), while in industrial plants
Azoarcus, Thauera and Zoogloea spp have been detected, and proposed as the dominant denitrifiers. How many
bulking and foaming filamentous bacteria can denitrify is unclear (chapter 5), although some by MAR can
reduce nitrate. When attempts were made to optimize conditions for culturing denitrifiers from activated sludge
(Heylen et al., 2006), the isolates obtained were predominantly Alphaproteobacteria and Betaproteobacteria,
with few Gram-positive bacteria, but individually the in situ importance of all of these was unclear. In EBPR
plants (chapter 10) there is now strong evidence that Accumulibacter PAO denitrify (Carvalho et al., 2007;
Kong et al., 2004; Morgan-Sagastume et al., 2008; Seviour et al., 2003; Thomsen et al., 2007).
Polymer degrading bacteria

As many of the substrates entering plants are polymeric in nature, organisms responsible for their enzymatic
degradation are functionally important, in providing low molecular weight RBCOD to other members of the
community. In situ methods employing BODIPYL fluorescein labeled proteins coupled with FISH could
identify some of the protein hydrolyzing populations (Xia et al., 2007, 2008b), revealing among them novel
epiphytic members of the Saprospiraceae in the Bacteroidetes. These were named Candidatus ‘Epiflobacter
spp.’ (Xia et al., 2008b). Other protein hydrolyzing populations included filamentous members of the TM7
and ‘Chloroflexi’ phyla as well as Betaproteobacteria, and members of this physiological group are likely
to be more phylogenetically diverse than the data so far suggest. Which proteins are degraded by which
organisms is not clear (Morales-Belpaire and Gerin, 2007). A similar approach was used to identify
the a-amylase producing starch degrading bacteria in EBPR plants (Xia et al., 2008a), using BODIPYL-
fluorescein tagged starch. These were all Tetrasphaera related Actinobacteria and were present in numbers
large enough to suggest they are important community members.
Iron bacteria
Fe3þ reduction by dissimilatory iron-reducing bacteria (DIRB) is a well-documented process in activated
sludge (Nielsen et al., 1997; Rasmussen et al., 1996), but with the exception of a single isolate, the
obligate iron reducing Geobacter sulfurreducens, these DIRB remain largely unidentified (Nielsen et al.,
1997, 2002a). As mentioned earlier iron is important for strong floc structure and for biological and
chemical P removal (Caccavo et al., 1996; Nielsen, 1996). Oxidation of Fe (II) may take place chemically
with oxygen or biologically with oxygen or nitrate as terminal electron acceptors. Some Bacteria have
been described in activated sludge that oxidize Fe(II) with nitrate as electron acceptor, thereby carrying
out an iron dependent denitrification (Nielsen and Nielsen, 1998). However, the process is not considered
as important as when oxygen is used. The identity of the bacteria responsible is unknown, but organisms
possessing this capability have been isolated (Straub et al., 1996).
Sulfate reducing bacteria

As mentioned above, sulfate reducing bacteria (SRB) are present in many activated sludge systems
(Lens et al., 1995), and are physiologically active during extended anaerobic periods e.g. in sludge storage
tanks (Rasmussen et al., 1994). As iron is usually present too, the generated sulfide precipitates as black
iron sulfide. About 0.5–8% of the total bacteria have been identified as sulfate reducers from FISH-MAR
or MPN enumeration, and sulfate reduction rates (Nielsen and Nielsen, 2002b; Manz et al., 1998; Vester
and Ingvorsen, 1998). Dominant SRB belonged to the Desulfovibrionaceae and Desulfobacteriaceae
(Manz et al., 1998). They actively grow in sewer lines and are transported to the plant where they may
replicate under anaerobic conditions, as for example in the anaerobic reactors of EBPR plants (Kjeldsen
et al., 2004, 2005). Evidence suggests that prolonged aeration failed to lower their activity, and oxygen
tolerant sulphate reducing Desulfovibrio strains have been described (Mogensen et al., 2005).
PHA accumulating bacteria

Many phylogenetically diverse activated sludge bacteria including the PAO and GAO (chapter 10), other
TFO (Seviour et al., 2000) and some filamentous bacteria (e.g. Blackall et al., 2000; Kragelund et al.,
2005, 2006, 2007a,b) (chapters 5, 7) accumulate large amounts of intracellular PHA in situ, facilitating
their survival there (see later). With the increased interest in exploiting sludge (chapter 2) as a cheap
source of these biopolymers (Salehizadeh and van Loosdrecht, 2004), some effort has been directed at
identifying the major PHA accumulating populations, either with PCR based methods (Majone et al.,
2006) and FISH (Serafim et al., 2006), in combination with nile blue A staining (Serafim et al., 2002a;
chapter 11), or alternatively from PCR-clone library-based analyses of the phaC gene encoding for two
classes of PHA synthases (Michinaka et al., 2007), or after their micromanipulation (Lemos et al., 2008).
This ability is common among members of the activated sludge community.
The glycogen accumulating organisms (GAO)

The GAO are probably ubiquitous in EBPR plants. They are ‘defined’ (Hesselmann et al., 1999) as
populations having a phenotype distinguished by its ability to assimilate substrates anaerobically for PHA
synthesis, but incapable of accumulating P aerobically. Other populations probably possess this anaerobic
GAO phenotype without accumulating glycogen aerobically. Unfortunately obtaining direct evidence
for an in situ ability to accumulate glycogen is difficult, because it is not detected readily by staining
(Serafim et al., 2002a). Several Actinobacteria (some of which are TFO) grown from laboratory scale and
full-scale plants, accumulate glycogen (Seviour et al., 2000, 2003). However, their importance in situ is
not clear, and FISH probes targeting these have generally failed to detect them in high numbers in full
scale plants (Beer et al., 2006; Wong et al., 2004).
Recognizing which populations might have the GAO phenotype is now easier with FISH-MAR, but is
still based largely on indirect evidence. This suggests they are phylogenetic diverse. The putative EBPR
GAO described so far (chapter 10), include Competibacter members of the Gammaproteobacteria
(Crocetti et al., 2002; Kong et al., 2002b, 2006; Nielsen et al., 1999a) and Alphaproteobacteria. The latter
are closely related to members of the genus Defluviicoccus (Burow et al., 2007; Meyer et al., 2006; Wong
and Liu, 2007; Wong et al., 2004), but phylogenetically diverse and distinct from its only cultured
member, D. vanus (Maszenan et al., 2005a). It seems that Candidatus ‘Monilibacter batavus’ a filamentous
alphaproteobacterial Nostocoida limicola morphotype (Levantesi et al., 2004) is also a member of
this group (Nittami et al., 2009), and cluster II Defluviicoccus have been seen dominating the community
of plants treating paper and pulp mill wastes (McIlroy et al., unpublished).
The PAO
Our views of which organisms are important in P removal have changed dramatically in the past decade
(chapter 10), and Acinetobacter, the favorite PAO candidate when the first edition of this book was
written, has since been discarded. Instead, evidence from EBPR plants around the world support the
importance of the betaproteobacterial Rhodocyclus related Candidatus ‘Accumulibacter phosphatis’
(Blackall et al., 2002; He et al., 2007a; Oehmen et al., 2007; Seviour and McIlroy, 2008; Seviour et al.,
2003), and a DNA microarray targeting these populations has been designed and applied to activated
sludge samples (Loy et al., 2005). From in situ DAPI staining many different activated sludge bacteria
including several bulking and foaming filamentous bacteria like Candidatus ‘Microthrix parvicella’
(chapters 5, 6) accumulate intracellular polyP granules. However, whether these and other cultured
organisms accumulating polyP (Ghosh et al., 2005; Hanada et al., 2002; Nakamura et al., 1995; Maszenan
et al., 2000; Spring et al., 2005; Zhang et al., 2003b) behave according to the models of how PAO are
expected to, is uncertain (chapter 10). Yet their combined contribution to final P removal may be
important. While their participation in EBPR in full scale plants is not clear, populations related to
members of the genus Tetrasphaera (Maszenan et al., 2000) and possibly Stenotrophomonas (Ghosh et al.,
2005) may eventually turn out to be important PAO.
Thus, observations (Kong et al., 2005) that Tetrasphaera can assimilate substrates other than acetate
anaerobically (as generally deemed necessary in defining the PAO phenotype), but not to support PHA
synthesis, might suggest a more flexible working definition of a PAO is required. This is especially since
subsequent aerobic P assimilation and polyP synthesis occurred in that same population. Also important
to remember is that the numerical contribution of the currently identified putative Accumulibacter
PAO to the total EBPR community composition in full scale plants is often small (Figure 3.6).
Understanding better the role of polyphosphate kinases and the phylogenetic diversity of the ppk gene
in EBPR plants operating under different conditions may provide a sensitive means for monitoring
EBPR capacity, and PCR based methods for its assay are available (He et al., 2007a; McMahon
et al., 2007b).
Chemolithoautotrophic bacteria
The nitrifying organisms
Nitrification performed by the chemolithoautotrophic nitrifying bacteria is a key process in N removal
(chapter 9). As with P removal, molecular methods have changed our views totally on which nitrifying
populations are important in activated sludge. Not only have they revealed a previously unsuspected level
of phylogenetic diversity among both the AOB and NOB (Daims et al., 2001a; Paungfoo et al., 2006;
Spieck et al., 2006), but 16S rRNA clone library data and subsequent FISH analyses have shown that
nitrite oxidation is carried out, not by Nitrobacter spp, from which most of our understanding of this
process was originally derived, but by uncultured Nitrospira spp. (Burrell et al., 1998; Daims et al., 2000;
Juretschko et al., 2002; Maixner et al., 2006) (chapter 9).
Nitrifiers appear to differ substantially in their ecology, with Nitrobacter considered a probable
r-strategist and Nitrospira a slower growing K-strategist, better able to cope with the low nitrite (energy
souce) levels found in activated sludge, and explaining its presence there (Nogueira and Melo, 2006;
Maixner et al., 2006; Schramm et al., 1999b). However, Nitrobacter and Nitrospira may co-exist in
nitrifying plants (Siripong and Rittman, 2007). Differences in competitive abilities based on oxygen and
ammonia affinities, both potentially important selective factors, have also been recorded among
Nitrosomonas lineages (Geets et al., 2006; Lydmark et al., 2007; Park and Nogueira, 2006), and may
determine which phylotype succeeds in a particular plant. Yet FISH reveals that nitrifying bacterial
UC TM7
(2±1%, TM7905)
UC Bacteroidetes
Bacteroidetes Alphaproteobacteria
(1%, Bac993*)
17% (7±2%, ALF968*)
UC Betaproteobacteria
UC bacteria in Saprospiraceae (2±1%, Bet135*)
(16±2%, Bac111*) UC Betaproteobacteria
(3±1%, Bet65*)
UC Rhodocyclus-related PAOs
Actinobacteria
UC Actinobacterial PAOs (9±2%, PAOmix*)
Betaproteobacteria
(5±1%, Actino-658*) Azoarcus-related bacteria
13%
(4±2%, Azo644*)
38%
UC Actinobacterial PAOs
Thauera-related bacteria
(8±2%, Actino-221*)
(1%, Thau-646*)
UC nitrite-oxidizing bacteria in
Nitrospira (subdivision 1) Aquaspirillum-related
(4±1%, Ntspa1431*) bacteria (16±3%, Aqs997*)
Chloroflexi
UC Nitrosomonas-related ammonia-
(8±2%, GNSB-941 & CFX1223)
oxydizing bacteria
Deltaproteobacteria (3±1%, Cluster6a-192*)
(2±1%, SRB385)
UC GAOs in GB group UC Gammaproteobacteria
(5±1%, GBmix*) (3±1%, Gam829*)
UC Gammaproteobacteria
(1%, Gam455*)
Gammaproteobacteria
9%
Figure 3.6. Composition of the community of a full-scale EBPR plant in Skagen, Denmark using FISH
probes. Percentages represent the biovolumes of each probed population as percentages of the total
biovolume estimated with the EUBmix probes. For further details, see Kong et al. (2007). UC ¼ uncultured.
communities change little with time or operational conditions, except possibly in response to seasonal
changes (Siripong and Rittman 2007).
It also shows their cells are not randomly dispersed throughout the flocs, but instead are organized into
large microcolonies (Daims et al., 2006b; Mobarry et al., 1996), with the potential survival advantages
such clustered arrangements bring (see later). Even more intriguing is the close physical proximity of
clusters of the AOB to those of the NOB, revealing syntrophic interaction between them. Thus, we know
that the NOB depend on the AOB to provide them with nitrite (their energy source), while in turn
removing this highly toxic product (Mobarry et al., 1996). Fine differences in their ecology seem to exist
which relate to their phylogeny. Thus, Daims et al. (2006b) showed that members of the two recognized
Nitrospira phylotype clusters differ in their tolerances to nitrite (chapter 9), a factor likely to affect their
distribution in plant biomass samples.
Because of their functional importance many PCR-based methods have been described for their
detection and quantification (chapter 9). DNA microarrays have also been designed for them and applied
to activated sludge communities (Kelly et al., 2005; Stahl et al., 2006), and a FISH based kit (Vermicon)
is commercially available for their in situ detection and enumeration, but not yet their nitrifying activity.
The whole genome sequences of Nitrosomonas europea and N. eutropha are also available, showing
evidence of niche specialization (Chain et al., 2003; Stein et al., 2007). Our current views on nitrification
may need further reappraisal with the possibility that some Archaea in activated sludge may nitrify
(Francis et al., 2007; Park et al., 2006).
The sulphur oxidizing bacteria

Some filamentous bacteria including Eikelboom type 021N/Thiothrix spp, Beggiatoa and Eikelboom
type 0914 (chapters 5, 11) contain distinctive intracellular So granules (see Figure 12.7, 12.11 and 12.30,
(see colour image section chapter 13)). These probably derive from their chemolithoautotrophic
metabolism, obtaining ATP from oxidizing reduced S compounds. It is also possible that they might grow
mixotrophically (Nelson, 1989). FISH-MAR in situ data from Thiothrix suggest both are likely and in fact
evidence for heterotrophic, mixotrophic and autotrophic growth was obtained, depending on
environmental conditions (Nielsen et al., 2000). The So granules resulted from aerobic autotrophic
growth on thiosulphate as energy source, and disappeared under anaerobic conditions after switching to
heterotrophic growth (Nielsen et al., 2000). Rossetti et al. (2003) also showed with a Thiothrix isolated
from activated sludge that it grew either heterotrophically on acetate or autotrophically with CO2. Such
metabolic flexibility would increase its competitive abilities in activated sludge.
Activated sludge plants may contain phototrophic bacteria that use H2S as their source of reducing
power. Some can denitrify, presumably when photosynthesis is not possible, suggesting they are also
metabolically versatile, and hence able to adjust to changing environmental conditions (Bond et al., 1995).
Several have been obtained in pure culture (Zhang et al., 2003a), but little else is known about them or
their possible in situ impact.
Cyanobacteria
These are seen commonly as slime masses attached to the walls of the reactors, yet few studies have targeted
their identification and possible function in activated sludge. Microscopic data was consistent with them
occurring in large numbers in plants treating paper and pulp mill wastes (Kirkwood et al., 2001), where they
appeared to have both beneficial and detrimental effects on plant operation (Kirkwood et al., 2005).
Molecular methods have not been applied yet to detect whether any are toxigenic.
Protozoa
The importance of protozoa in activated sludge is well documented. However, much still needs to be
learned about the dynamics of their feeding, which in most occurs by active predation involving
endocytosis and bulk transport of suspended particulate material like bacterial cells, and especially their
feeding selectivity, and how it may influence community composition (Hahn et al., 1999; Jürgens et al.,
1999; Petropoulos and Gilbride, 2005). It is now clear that bacteria have mechanisms to protect
themselves from protozoal grazing (Matz and Kjelleberg, 2005), although whether these operate in
activated sludge has not been explored. The protozoa seen there are beautifully adapted in their
morphology and physiology as predators. All reports show they can occur in large numbers in activated
sludge, and their role in the process (Curds, 1975, 1982) is probably better understood than for most other
groups of organisms discussed above. Representatives of all the major taxa have been reported in plant
samples analysed in Britain (Curds, 1969a) and Europe (Augustin and Foissner, 1992a; Esteban et al.,
1990, 1991; Foissner, 1988; Madoni, 2002; Madoni et al., 1993; Martin-Cereceda et al., 1996;
Salvado et al., 1995) from where most of the taxonomic descriptions used globally come (chapter 4).
Members include Cryptosporidium parvum, recognised as a serious intestinal parasite, infecting humans
drinking faecally contaminated water, because their oocysts may survive the activated sludge process and
any subsequent effluent chlorination (Fayer and Xiao, 2007; Robertson et al., 2006). Its increasing
importance has encouraged the search for suitably sensitive molecular methods to detect it at low
concentrations in water (Guy et al., 2003). Cysts of the flagellate amitochondriate pathogenic protozoan
Giardia lamblia (Adam, 2001; Rochelle, 2002) and other species can also now be detected and identified by
molecular methods at very high dilutions (e.g. Miller and Sterling, 2007), but these organisms are apparently
more readily removed by the activated sludge process and chlorination. Pathogenic amoeboid protozoa have
also been reported in activated sludge (Curds et al., 1991; chapter 4).
The ecology of the ciliate protozoa has attracted most attention, because they represent the numerically
dominant group in activated sludge, but are particularly sensitive to processes like chlorination
(Salvadó et al., 2000). Compilations of descriptions of these are given in the popular manual of Curds
(1969b), in British samples, and Augustin and Foissner (1992a) and Foissner (1988) in Europe, and a free
interactive software package facilitating their identification is also available from the Natural History
Museum, London (chapter 4) (www.nhm.ac.uk/zoology/ciliate/). How closely their descriptions accord
with the ciliate protozoa seen in samples from other countries is not clear (Duchene, 1991; Horan, 1992),
but applying techniques like FISH (e.g. Fried et al., 2002) will help address uncertainty surrounding their
biogeography. Far less effort has been directed towards understanding the influence of either the flagellate
or amoeboid forms on activated sludge population dynamics, and needs review. The importance of protozoa
in the activated sludge process warrants detailed separate discussion, and chapter 4 is devoted to them.
Fungi
Fungi can be isolated and cultured from activated sludge systems (Cooke and Pipes, 1970; Tomlinson and
Williams, 1975). However, the popular view is that they are not important members of the community.
This is mainly because generally they have lower mmax values than bacteria. preventing them from
competing successfully, except under some circumstances. Thus, in plants with a low (54.5) pH, where
the normal bacterial microflora are out-competed (Jenkins et al., 2004b), they may cause bulking. A check
of the published lists of isolated cultured fungi suggests that the majority are members of the Ascomycota
and Basidiomycota, and most probably originate from the normal air microflora, where they may
enter reactors after their spores settle out by sedimentation, or from being washed out by aerosols, or
from soil erosion into the collecting sewers. Predaceous fungi consuming the rotifiers there have been
identified in plants as Zoophagus insidians (Cooke and Ludzack, 1958), and Gray (1984) has detected
microscopically both nematode capturing and epiparasitic fungi in activated sludge systems in Ireland.
They probably occur more widely than this, but have not been reported elsewhere. Ergosterol has been
used for estimating total fungal biomass levels in some activated sludge plants (Gardner et al., 1993).
We know of no detailed published study where activated sludge fungal communities have been
analysed with culture independent techniques (Grant et al., 2006). Experiences from bacterial studies
suggest that such work is likely to be revealing, especially with plants treating industrial wastes, where the
well known abilities of fungi to degrade complex substrates may provide them with some advantage.
Molecular techniques now available borrow heavily from those employed for bacteria, and their impact
on fungal systematics has been discussed in chapter 1. Fungal community DNA extraction methods
applicable to conidia as well as vegetative cells have been assessed for several habitats, but not
yet activated sludge (Haugland et al., 1999; Karakousis et al., 2006). PCR based protocols are well
established now using either the 18S rRNA gene (May et al., 2001; O’Brien et al., 2005; Wu et al., 2003)
and/or the internal transcribed rRNA (ITS) genes as phylogenetic markers (Anderson and Cairney, 2007;
Bougoure and Cairney, 2005). Primers have been designed for qPCR of targeted fungal groups
(Haugland et al., 2004; Vesper et al., 2005) and many are listed at (http://www.epa.gov/microbes/
moldtech.htm). Both DGGE and T-RFLP fingerprinting methods have also been adapted for soil fungal
community analyses (Bougoure and Cairney, 2005; Nikolcheva et al., 2003), and targeted FISH probes
have been designed and applied to fungal communties (Baker et al., 2004), although hyphal permeability
problems with these may limit their application. As might be predicted, data from studies where such
methods have been used reveal a level of fungal biodiversity far greater than suggested from culture
dependent approaches. Whether activated sludge communities show similar trends requires attention.
Algae
Like the fungi, activated sludge algal communities have not been exposed to molecular methods of
analyses, and so our understanding of their biodiversity is based almost entirely on early microscopic
data (Benson-Evans and Williams, 1975). Currently the general conclusion is that they are unlikely to be
important members of this community.
Metazoa
Little further information on the occurrence and function of these has become available since the earlier version
of this book. Most activated sludge biomass samples contain larger metazoan organisms like Nematodes,
Rotifers and Oligochaete worms. These probably play important roles as predators, consuming bacterial cells
(Poole, 1984; Ratsak et al., 1993), but their importance is not as well understood as with the protozoa (Woombs
and Laybourn-Parry, 1987). How selectively they prey on bacterial cells, and their consequent influence on the
composition of the activated sludge community are where further data would be helpful. Some of the more
frequently seen metazoa in plant samples are shown in Figure 3.7, and the factors which seem to determine their
frequencies in activated sludge communities have been discussed (Ratsak et al., 1996; Salvadó, 1994). Interest
has been shown in using the oligochaetes to reduce sludge production (Ratsak and Verkuijlen, 2006).
FACTORS AFFECTING SURVIVAL OF AN ORGANISM IN ACTIVATED

SLUDGE SYSTEMS
The activated sludge system is a highly competitive habitat and considerable selective pressures are
brought to bear on the organisms there. It behaves essentially as a continuous culture system with biomass
recycling (Hamer, 1984, 1987; Hamer et al., 1985). So theoretically populations not well suited for the
prevailing conditions will be uncompetitive, and be washed out in the clarified liquid effluent. Some of the
factors believed to affect an organism’s ability to remain within this system are discussed here, although
often the experimental data supporting these is scant and unconvincing.
An organism’s specific growth rate (m)

Once we can identify and reliably quantify our population of interest by methods like FISH, it is
theoretically possible to measure how its numbers change with time, and thus determine its growth rate
(Rossetti et al., 2007). This value may decide its ability to compete with other populations. Simple
chemostat theory shows that the availability of growth limiting substrates and any other factor which
might affect m will determine an organism’s fate. At high D or low MCRT, slower growing organisms
should be lost from the system, as now D4m, reflecting their inability to compete for the growth limiting
substrate. This model may apply to freely suspended cells but not necessarily for floc forming organisms
(Wanner, 1993). Nor has it always applied to mixed bacterial populations in laboratory chemostat
studies, whose communities may achieve long term stability, probably reflecting complex positive and
negative interdependent relationships between member populations. However, wash out based on this idea
is a popular control measure in some countries (Jenkins et al., 2004b) for some of the slower growing
filamentous bacteria causing foaming (chapter 8).
Figure 3.7. Examples of common activated sludge metazoa. (a) nematodes with long thin bodies feeding on
bacteria. (b) rotifers with highly specialized ciliate mouthparts and often a branched’tail’. (c) oligochaete, the
most common being the ‘bristle worm (Aeolosoma spp.), with tufts of bristles on each body segment.
(d) tardigrade, soft-skinned, vaguely segmented organism, with sucking proboscis as its mouth and four pairs
of short, clawed, unjointed legs. Bar graphs for all ¼ 100 mm.
Most populations in activated sludge are chemoorganoheterotrophs, obtaining cell carbon and energy
from oxidising organic compounds entering the plant. The nitrifying chemolithoautotrophs use inorganic
chemical compounds like NHþ
4 and NO2 for energy production, and CO2 as a carbon source (chapter 9),
although some (Nitrospira) may behave as mixotrophs (Daims et al., 2001a). The diversity of substrates
the chemoorganoheterotrophs have to deal with is likely to be considerable, and not all organic
compounds in the influent (e.g. EDCs) will necessarily be degraded completely to CO2, especially in
plants operating with short sludge ages. Biomass is metabolically very active, as judged from the range of
enzyme activities detected there, which include phosphatases, peptidases, glucosidases, lipases and
proteases and esterases (Boczar et al., 2001; Frølund et al., 1995; Gessesse et al., 2003; Lemmer et al.,
1994). In some cases their substrate specificities in situ have been determined (Dueholm et al., 2001), and
for some the populations synthesizing them (Xia et al., 2007, 2008a,b).
The basic nutritional requirements for microbial growth were described in chapter 1, and domestic
sewage would probably satisfy most of them. Essential sources of N, S and P will be present in ample
supply but this may not always hold true for industrial wastewater like those from paper mills, where N
and P supplementation is required. Cations like Mg2þ and Ca2þare needed for growth too, and limiting
concentrations of Mg2þ may affect detrimentally the performance of EBPR plants (Lindrea et al., 1998;
Schönborn et al., 2001; Wu et al., 2006).
Developing sound mathematical models depends on being able to determine key kinetic parameters
like m for populations of interest, preferably in situ. However, understanding the possible interactions,
both beneficial and antagonistic, which might occur between individual populations affecting their
growth rates is a more daunting task. Any kinetic data are scant, because of problems with methodology.
Pollard and Greenfield (1997) have used rates of incorporation of [methyl – 3H] – thymidine into
DNA (the so called thymidine growth assay) to obtain overall bacterial community m values for EBPR
communities. They have tested and recommended a protocol suitable for activated sludge and with it
could show differences in m for communities in various zones of the plant. This approach was then refined
to determine the m of individual populations of Zoogloea ramigera in combination with targeted DNA
probe hybridization (Pollard, 1998). It has since been modified further for in situ m determinations for
nitrifiers (Pollard, 2006). Q-MAR (Nielsen et al., 2003b) has also been used to determine apparent Ks
and m values at a single cell level in FISH targeted populations.
Image analysis based on changes in filament length has helped determine the growth kinetics of the
filamentous bacteria Thiothrix, type 1863 (Acinetobacter johnsonii) and Candidatus ‘M. parvicella’ in
both axenic culture and in situ. For the latter, the influence of temperature and pH on its m was also
estimated (Rosetti et al., 2005, 2007; Tomei et al., 1999).
Tolerance to abiotic factors and toxic chemicals

Equally important will be an organism’s ability to tolerate fluctuations in temperature, pH, and redox
potential, all of which may affect its physiology, growth rate and viability (Schlegel and Jannasch, 2006).
We do not understand the variations in these parameters existing in the microenvironments within and on
individual flocs, which probably provide environmental conditions quite different to those measurable
in the plant as a whole (Li and Ganczarczyk, 1990; Schramm et al., 1999a; Zartarian et al., 1994).
Microsensors (chapter 11) will reveal how heterogenous these may be, but such data are relatively scarce.
Organisms intimately associated with flocs may be protected by the EPS component (Henriques and Love,
2007; Wanner, 1994b) from toxic effects of compounds like heavy metals. Our lack of in situ data makes
it difficult to assess how important these abiotic parameters and toxic compounds are in determining
community composition and its performance (chapter 2). However, the nitrifiers (Principi et al., 2006)
and protozoa seem especially sensitive. Techniques like DGGE, TGGE and T-RFLP may allow us to
follow such population changes, and some data have already been reported (Nadarajah et al., 2007).
It seems (Rossetti et al., 2005) that temperature in particular can influence the composition of
foaming filamentous bacterial communities (chapters 5, 8), and both pH and temperature markedly
influence competition between the PAO and GAO populations (chapter 10), and hence EBPR capacity
(Oehmen et al., 2005a; 2007).
An ability to contribute to floc formation

Many freely suspended organisms in the bulk liquid, regardless of their in situ growth rates will leave the
system with the clarified effluent after biomass separation. As long as the flocs have good settling
properties, those populations associated with them will be reinoculated with the return activated
sludge (RAS) into the reactor (Jenkins et al., 2004b). The ability of the slow growing sessile stalked
protozoa and filamentous bacteria to integrate into and onto floc surfaces explains, partially at least, how
they manage to remain in activated sludge. Equally impressive is the ability of populations like
the nitrifying bacteria (Daims et al., 2006b; Larsen et al., 2008b; Mobarry et al., 1996), the PAO
(Crocetti et al., 2000) and GAO (Ahn et al., 2007; Crocetti et al., 2002; Meyer et al., 2006) to grow within
flocs as large clusters often of heavily capsulated cells (see Figure 11.11). This arrangement may also
protect them from predatory grazing by the floc associated protozoa (Matz and Kjelleberg, 2005), but
unfortunately render them less susceptible to DNA extraction protocols (chapter 11).
An ability to withstand starvation conditions

Equally, little will be gained by an organism having a high growth rate (i.e. adopting an r-strategy) if it is
unable to maintain this under the dynamic conditions prevailing in activated sludge, including inevitable
fluctuations in substrate availability and periods of possible starvation. These unbalanced growth
conditions impact markedly on the organisms’ physiology (Majone et al., 1999). Such unpredictable feast
and famine conditions mean that survival is best achieved by populations having mechanisms for rapidly
assimilating substrates when they become available, but instead of using them to support an increased m
(‘growth’ response), converting most to intracellular carbon and/or energy stores like PHA, glycogen etc
(‘storage’ response) (Majone and Tandoi, 2002; van Loosdrecht et al., 1997a,b). These storage polymers
are then available to allow growth when exogenous substrates are no longer present (Kadouri et al., 2005).
Thus, PHA accumulation will provide that population with an advantageous ability to respond better to
sudden increases in substrate availability (Frigon et al., 2006b).
It is not surprising then that many activated sludge bacteria contain high levels of PHA and glycogen,
and polyP (Majone and Tandoi, 2002), although the role of the latter is less clear (Seviour et al., 2003),
and possibly other storage compounds as yet uncharacterized (Kong et al., 2005; Nielsen et al., 2002).
In EBPR processes we deliberately impose selective pressures on the community to favour PAO
populations (chapter 10) able to assimilate substrates anaerobically for synthesis of PHA, which can then
be used aerobically to provide energy for phosphate uptake and polyP formation (Majone and Tandoi
2002; Mino et al., 1998; Oehmen et al., 2007; Seviour et al., 2003). The GAO, are also selected under
the same operational conditions for similar reasons. In some activated sludge systems, deliberate
attempts have been used to selectively disadvantage the bulking filamentous bacteria by manipulating the
feeding regimes and organisms’ subsequent ‘growth’ and ‘storage’ responses (Martins et al., 2004a,b)
(chapters 5, 7), but are not successful with all filaments.
Some may have alternative strategies to cope with starvation. For example, Candidatus ‘M. parvicella’
can maintain substrate assimilation functions even after prolonged starvation, an attribute which may also
explain its frequent appearance in EBPR plants where such conditions are deliberately established
(Andreasen and Nielsen, 2000; Nielsen et al., 2002; Rossetti et al., 2005). The nitrifying bacteria appear to
tolerate prolonged starvation (Bollmann et al., 2005) by always maintaining high ribosome numbers,
which allow cells to become metabolically active immediately growth is possible. Similar abilities to
tolerate starvation may be shared by other populations that store PHA or glycogen, and novel bacterial
populations have been isolated into pure culture following selective enrichment by prolonged starvation
(Kataoka et al., 1996; Zhang et al., 2003b).
HORIZONTAL GENE TRANSFER IN ACTIVATED SLUDGE COMMUNITIES

Horizontal transfer of genes between populations in natural microbial communities by transformation,
transduction and conjugation (described briefly in chapter 1) has played an important role in bacterial
evolution (Davison, 1999). Transfer of plasmid borne antibiotic resistance genes in nature has been
documented extensively. Movement of other genes has not received so much attention, but ideal
conditions for such activity probably exist in activated sludge (Schlüter et al., 2007a,b). Although bacteria
like Acinetobacter baylyi are naturally transformable (Vaneechoutte et al., 2006), and transduction
involving phages has been reported, little information is available on their role if any in horizontal gene
transfer in activated sludge plants (van Elsas and Bailey, 2002). On the other hand there is evidence
that conjugation does occur (Geisenberger et al., 1999; Marcinek et al., 1998). Conjugative antibiotic
resistance plasmids sometimes carrying multiple resistance genes have been detected and isolated from
activated sludge (Dröge et al., 2000; Schlüter et al., 2007a,b; Szczepanowski et al., 2005), and some
carrying these and other genes involved in xenobiotic catabolism appear able to infect a wide range
of recipient cells (de Gelder et al., 2005; Goris et al., 2003). Integrons, which are small mobile genetic
elements moving genes between cells contain gene cassettes, and are active participants in horizontal gene
transfer (Hall et al., 1999; Holmes et al., 2003). These can be detected readily in activated sludge biomass,
and are remarkably diverse in the gene elements they contain (Beer and Seviour, 2006), further implying
that such communities are sites for intense levels of bacterial evolutionary activity.
MANIPULATING THE MICROBIAL COMMUNITY IN ACTIVATED SLUDGE

PLANTS
Microbiologists have been attracted for a long time to the concept of adding microbes with particular
metabolic attributes to activated sludge plants (Bioaugmentation) to improve their capability. The general
explanation given for why the results obtained have not been encouraging (e.g. Wilderer et al., 1991), is
that so few unoccupied niches remain for any introduced populations to fill. Most of the commercially
available microbial cultures with claims to improve plant performance assessed by Stephenson and
Stephenson (1992) proved completely ineffective, illustrating this point. However, many of these are still
promoted within the industry. With molecular methods, the population dynamics of communities can be
monitored following any attempt at bioaugmentation (Boon et al., 2003; Dabert et al., 2005), and the little
published data suggest that newly introduced populations do indeed disappear, largely from protozoal
predation (Bouchez et al., 2000). More studies are required to see if this is a general mechanism.
Two rational bioaugmentation strategies seem to have been adopted. Selected whole cells whose
genes of importance are chromosomally borne, and whose subsequent survival may be enhanced by
preadaptation treatment like starvation (Watanabe et al., 2000) are inoculated. (e.g. Boon et al., 2003;
Dabert et al., 2005; Quan et al., 2004; Watanabe et al., 1998). Alternatively, suitable host cells carrying
desired conjugative plasmid borne genes are used. Most studies have been carried out so far in lab scale
reactors, but with some success. Thus, even if the host cells eventually disappear, the plasmids may still
survive, transform other competent host cells and then in turn be transferred to other bacteria.
The view held by many microbiologists is that one outcome from a better understanding of
activated sludge microbiology, especially from being able to identify the populations thriving under the
operating conditions desired, is that it may allow more rational bioaugmentation strategies. Earlier failures
probably resulted from trying to introduce populations unsuited for that particular plant configuration
(Loy et al., 2002a).
4
Protozoa in activated sludge processes
Alan Warren, Humberto Salvadó, Colin R. Curds, and
David McL. Roberts
INTRODUCTION
The taxonomy of the eukaryotes has been in a state of flux for many years (chapter 1), but six major
assemblages are now recognised (Adl et al., 2005; Simpson and Roger, 2002). Older classifications were
based on easily determined physiological characters, for example, photosynthetic pigments (algae, plants)
or their absence (protozoa, fungi, animals). The predominantly unicellular forms are traditionally grouped
as the protists, i.e. the unicellular algae and protozoa, but not the fungi. Photosynthetic organisms are rare
in activated sludge, and this chapter will discuss those protists traditionally called protozoa. They are
enormously diverse, and range in size from a few microns to a couple of millimetres; some are colonial
and some colonies are readily visible to the naked eye.
OCCURRENCE IN PLANTS
Activated sludge plants are almost invariably colonized by protozoa, and their presence was noted virtually
as soon as the process was developed (Johnson, 1914). They can occur in very large numbers
and commonly reach concentrations of 105–106 cells ml 1 in the mixed liquor, constituting about 5% of
the dry weight of the suspended solids in aeration tanks (Curds, 1968). However, there has been
some debate concerning methods for their enumeration, particularly with respect to the number and
volume of samples that need to be examined in order to obtain accurate estimations, and their numbers
vary e.g. 5 £ 10 ml (Augustin and Foissner, 1992b; Augustin et al., 1989), 2 £ 25 ml (Madoni, 1994b),
3 £ 25 ml (Martin-Cereceda et al., 1996), 10 £ 100 ml (Ettl, 2000). The main trade-offs are that large
volume samples are likely to contain a more representative fauna, especially with respect to the large
colonial forms, but they take longer to examine and the smaller organisms are more likely to be
overlooked.
Representatives of all the major functional groups of free-living protozoa have been reported in plant
samples analysed in Britain (Curds and Cockburn, 1970a; Poole, 1984) and elsewhere in Europe
(Augustin and Foissner, 1992a; Ettl, 2000; Esteban et al., 1990, 1991; Foissner, 1988; Madoni et al., 1993;
Martin-Cereceda et al., 1996; Salvado et al., 1995) from where most of the descriptions used around the
world come. The composition of the protozoan community is known to vary with most of the plant
operational conditions examined, as well as a host of environmental factors including temperature,
pH, ammonia, salinity and heavy metal concentrations (Abraham et al., 1997; Al-Shahwani and Horan,
1991; Curds, 1975; Esteban et al., 1991; Hoffman and Atlas, 1987; Lishman et al., 2000; Madoni, 1994a;
Madoni et al., 1993, 1996; Martin-Cereceda et al., 1996; Nicolau et al., 2005, 2007; Poole, 1984;
Puigagut et al., 2005; Salvado, 1994; Salvado and Gracia, 1993; Salvado et al., 1995, 2001).
Ciliates
The ciliate protozoa usually dominate the protozoan community and consequently have attracted the most
attention. They have been divided into a number of useful ecological groups on the basis of their feeding
habits, mode of locomotion, and physical location within the biomass (Table 4.1) (Curds, 1975, 1982,
1992). According to Curds (1968), Madoni (1991) and Madoni et al. (1994b) these groupings are:
(a) free-swimming ciliates (Figure 4.1a,b, see colour image section (chapter 13));
(b) crawling ciliates, which crawl over and through the flocs (Figure 4.1c,d);
(c) attached ciliates, which are fixed to flocs through holdfasts, and extend into the bulk liquid,
ensuring these sediment with the flocs in the clarifiers, and are recycled with the settled activated
sludge (Figure 4.2, see colour image section (chapter 13)).
(d) carnivorous ciliates, feeding on other protozoa (Figure 4.2f).
Table 4.1. Behavioural and feeding patterns of common ciliated protozoa in activated sludge plants.
Species Habit Food
Aspidisca cicada C B
Carchesium polypinum A B
Euplotes moebiusi C B
Operculara coarctata A B
Paramecium aurelia F B
Uronema nigricans F B
Vorticella alba A B
Vorticella convallaria A B
Vorticella microstoma A B
Acineria uncinata C Carn
Acineta tuberosa A Carn
Holophrya discolor F Carn
A ¼ sessile, attached to flocs.

B ¼ bacterivore, feeding on bacteria.
C ¼ crawling in and over flocs.
Carn ¼ carnivore, feeding on other protozoa.
F ¼ free-swimming.
Protozoa in activated sludge processes 129
Most of those belonging to (A), (B), and (C) are bacterivorous while (D) includes examples that
are free-swimming, crawling and attached. The mathematical modelling work of Curds (1971)
showed that the habit of protozoa (and indeed other microbes) affects their chances of survival, growth
rates and quality of the effluent emerging from activated sludge plants. This will be discussed more
fully later.
Detailed compilations of descriptions of most of the more important ciliate protozoa found in mixed
liquors are given in Curds (1969), Curds et al. (2008), Fiałkowska et al. (2005), Foissner et al. (1991,
1992, 1994, 1995) and Serrano et al. (2008). Most of the organisms within these guides were isolated from
sites in Europe. How closely these resemble the ciliate protozoa in biomass samples from plants in other
parts of the world is not really known (Duchene, 1991; Horan, 1992) although where studies have
been carried out the evidence suggests that activated sludge ciliate communities are similar worldwide
(Banina et al., 1983; Chen et al., 2003; Curds, 1975; Lee et al., 2004; Liu et al., 2008; Morishita, 1970;
Pillai and Subrahmanyan, 1942, 1944; Zhou et al., 2006).
More than 200 ciliate species have been reported in activated sludge processes, 175 of which were
accepted by Curds et al. (2008) as ‘true’ sewage ciliates. However, only a few of these are commonly seen
in individual plants, making identification less of a deterrent (Esteban et al., 1991; Madoni et al., 1993).
Ciliate identification is traditionally based on morphology and involves microscopical examination of
cells either in vivo or following fixation and staining. The most commonly used staining protocols employ
silver but other stains such as the fluorescent taxoid FLUTAX have also been utilised for activated
sludge ciliates (Arregui et al., 2003). Identification of genera is generally straightforward but species
identification is often problematic, especially for non-specialists, even using the best identification
guides. Advances in modern technology have allowed this situation to be addressed by, for example the
development of semi-automated identification systems using image analysis (Ginoris et al., 2007; da
Motta et al., 2001) and of interactive guides that use a variety of data including digitized video sequences
(Curds et al., 2008; Fiałkowska et al., 2005). Molecular methods will undoubtedly become increasingly
important as tools for ciliate identification when they become more refined for the study of protozoa
in the wastewater environment. Such methods include total community DNA analysis by techniques such
as DGGE, T-RFLP and comparative sequences analysis of rDNA clones (Marsh et al., 1998), and FISH
(Fried et al., 2002; Weber et al., 2007).
Some of the more commonly observed morphologically distinctive forms reported in samples of mixed
liquors are shown in Figures 4.1 and 4.2 (chapter 13). Many of these have highly specialised feeding
mechanisms involving cilia which set up vortices sweeping suspended material, particularly bacterial
cells, into their mouth parts. Here they enter the organism by the process of endocytosis or phagocytosis in
food vacuoles, and are then degraded by protozoan enzymes. Other ciliates include carnivores that feed on
other protozoa, and specialist feeders that have the ability to engulf filamentous bacteria and algae.
Several flagellate protozoa and amoeboid forms are also seen in activated sludge samples (Curds, 1975,
1992; Madoni, 1991) and a selection of these is shown in Figure 4.3 (see colour image section (chapter 13)).
Flagellates
The traditional collective term ‘flagellate’ refers to organisms that possess one or more flagella (long,
tapering, hair-like appendages ultrastructurally identical to cilia), which act as organelles of locomotion
and feeding. They are not a taxonomic grouping. The flagella can
(a) beat with an undulating or whiplash motion that propels the organism through the water; create
feeding currents to draw food particles toward the organism
Arcella vulgaris Euglypha tuberculata

(c) (d)
Figure 4.3. Examples of amoebae commonly seen in activated sludge plants. (c) Arcella vulgaris; this is a
testate amoeba with a proteinaceous test (shell) that is circular in outline and has a centrally located, circular
aperture through which the pseudopodia project. (d) Euglypha tuberculata; in this testate amoeba the test is
made of overlapping silica scales. Figures (c) and (d) from the Colin Ogden SEM archive at the Natural
History Museum, London.
(b) trap food directly on their surface or

(c) act as the locus of gliding motion when pressed against a solid surface.
Commonly, where two flagella are present, one may project forward while the other trails behind.
Often the organism‘s flagella are longer than its body.
Some protozoan flagellates, especially certain members of the euglenoid and dinoflagellate groups,
possess plastids containing chlorophyll and are wholly or partially photolithoautotrophic. Such flagellates
form a substantial part of the protozoan communities in other biological wastewater-treatment processes
such as oxidation ponds and in the upper layers of trickling filters, but are usually not well represented in
activated sludge, probably due to a lack of light penetration of the turbid mixed liquor. Other flagellates
are colourless and heterotrophic, obtaining their food either as particulate or in a dissolved form. Perhaps
the most common types found in activated sludge are the bodonids (Figure 4.3a, see colour image section
(chapter 13)).
The true importance of flagellate protozoa in activated sludge systems has probably been
underestimated, since most feed actively on bacteria. A few including Peranema can be carnivorous,
and some are able to utilize soluble organic compounds in competition with bacteria. Furthermore, the
last detailed review of flagellates in activated sludge was that of Hänel (1979). Clearly more work is
needed with them.
Amoebae
The primary characteristic of the amoebae is their possession of pseudopodia, temporary processes that
serve as organelles of locomotion and feeding. They feed primarily by phagocytosis of particulate
food and often contain food vacuoles that are visible under the light microscope. There is a considerable
diversity of structure in the amoebae, particularly in the character of any shell or skeletal material that may be
present, and in the type of pseudopodium (for example, broadly lobed, needle-like, or reticulate), and
their identification is based primarily on such characters. The best contemporary guides to the amoebae
are those of Clarke (2003), Ogden and Hedley (1980), Page (1988) and Smirnov and Brown (2004).
Amoebae found in activated sludge plants belong to one of three morphological groups:
(a) naked amoebae (without a shell);

(b) testate amoebae (with a shell) and
(c) Heliozoa (planktonic forms with stiff, radiating actinopodia), although the latter is relatively
uncommon.
Figure 4.3b (see colour image section (chapter 13)) shows an example of a naked amoeba. The cell matrix
(the cytoplasm) is divided into a clear outer layer (the ectoplasm) that surrounds a granular inner area or
endoplasm. Within the endoplasm are the nucleus, contractile vacuole, food vacuoles, storage granules, and
other inclusions. Figure 4.3c,d shows examples of testate amoebae. The shell, or test, may be proteinaceous,
agglutinate, siliceous, or calcareous in composition. In the activated sludge process, naked and testate
amoebae are about equally well represented although little work has been carried out on the identification of
either (Ramirez et al., 1993). They are normally present in low numbers but can, on occasion, become
dominant. Although no more common than naked amoebae, testate amoebae are perhaps more easily noticed
because of their distinctive test which can remain intact long after the amoeba has died.
Pathogenic and parasitic protozoa

Obligate and facultative pathogenic or parasitic protozoa also occur in activated sludge plants. Obligate
parasites such as Giardia and Cryptosporidium are present in the form of resistant cysts and oocysts
respectively, whereas facultative pathogens, such as the amoebae Naegleria and Acanthamoeba, may also
be present as active trophozoites (Ramirez et al., 1993). Other parasitic protists found in activated sludge
include Cyclospora, Entamoeba, Isospora and microsporidia (now considered fungi – chapter 1),
although information on these with respect to wastewater treatment is still scarce (Robertson et al., 1999).
In recent years Cryptosporidium has emerged as an important agent of diarrhoeal disease, waterborne
outbreaks of which are increasingly associated with wastewater-derived oocysts (Fayer and Xiao, 2008;
Patel et al., 1998). Giardia is a flagellated protozoan which also causes severe diarrhoeal disease and
whose cysts are often plentiful in sewage. The removal of Cryptosporidium and Giardia from wastewaters
is therefore becoming of increasing importance for public health (chapter 3).
Traditional methods for the detection and enumeration of Cryptosporidium and Giardia in
environmental samples are often unsuitable for wastewater because of its high turbidity. Therefore
alternative methods have been developed which greatly improve the capture efficiency of cysts and
oocysts and their selective separation from particulate debris. One of the most commonly used of these is
immunomagnetic separation followed by immunofluorescence assay (Iacovaski et al., 2004). However,
despite the improvements that this method brings, it has been criticised for being time-consuming
and insufficiently specific for the target species (Chesnot and Schwartzbrod, 2004). Density-based
purification and molecular detection methods are now developed to address these problems with most
attention focused on Percoll-sucrose flotation followed by real-time PCR detection (Bertrand et al., 2004;
Lebuhn et al., 2004). Other advantages offered by molecular detection methods include the
differentiation of human pathogenic species and strains from those that originate from non-human
sources (Xiao et al., 2001).
The efficacy of the activated sludge process for the removal of parasitic and pathogenic protozoa from
wastewaters was reviewed by Robertson et al. (1999). The ranges of reported removal efficiencies were
97 >99% for Giardia, 79–91% for Cryptosporidium and 83% for Entamoeba. Subsequent studies have
reported removal efficiencies of 87.0–98.4% (Caccio et al., 2003) and 66–94% (Robertson et al., 2000) for
Giardia, and of 28–98% (Robertson et al., 2000) and 99% (Suwa and Suzuki, 2003) for Cryptosporidium.
However, as Robertson et al. (1999) noted, only generalizations can be made when comparing data such
as these because recovery efficiencies differ markedly according to the sampling protocols employed, the
techniques used for purification and identification of cysts/oocysts, and the turbidities of the samples.
Although the removal mechanisms involved in the activated sludge process have not been systematically
investigated, it is known that Giardia cysts are largely concentrated in the mixed liquor solids and the sludges
(Casson et al., 1990). Nevertheless, it has been reported that over 20% of Giardia cysts and 33–100% of
Cryptosporidium oocysts in treated effluent are viable (Smith et al., 1994; Parker et al., 1993). In order to
increase the efficiency of protozoan parasite inactivation and removal from the liquid phase in activated
sludge plants, additional treatments have been suggested, but with varying degrees of success. These include:
UV and gamma irradiation (Liberti et al., 2003; Neto et al., 2006; Thompson and Blatchley, 2000;); ozone
(Liberti et al., 2000); electrochemically generated mixed oxidants (Casteel et al., 2000); addition of
coagulants (Suwa and Suzuki, 2003); tertiary treatment in algal ponds (Araki et al., 2001).
A range of assays has been used in order to assess the viability of cysts and oocysts isolated from
wastewaters. These include: differential interference contrast microscopy; vital dye exclusion (e.g. DAPI
and propidium iodide); cell culture assay; infectivity to laboratory mammals (Casteel et al., 2000;
Garcia et al., 2002; Thiriat et al., 1998). Using such techniques the effect of the activated sludge process
on cyst and oocyst viability has been investigated. Robertson et al. (2000), for example, found that the
viability of Cryptosporidium oocysts was unaffected by all stages of the activated sludge process apart
from the sludge holding tank. By contrast, Garcia et al. (2002) found that none of the Giardia cysts
isolated from tertiary-treated effluents were infective in laboratory gerbils and concluded that the health
risks associated with these effluents are probably over-estimated.
It has long been known that the commonly used indicators of faecal pollution such as coliform bacteria
and somatic coliphages do not take account of Giardia and Cryptosporidium, largely because of the highly
resistant nature of the cysts and oocysts of the latter. Alternative indicators such as the spore-forming
anaerobic bacterium Clostridium perfringens and aerobes such as pseudomonads have therefore been
suggested. Indeed the European Drinking Water Directive 98/83/CE states that Cryptosporidium has to be
determined in water intended for human consumption if C. perfringens is detected. However, work, has
suggested that a wide range of factors including environmental conditions, background flora and abiotic
substances may affect the relative survival and fate of protozoan parasites differently to that of the target
organisms, thus limiting the value of the latter as indicators (Brianesco and Bonadonna, 2005;
Chauret et al., 1999). Similar studies have been carried out for activated sludge but to date there is little
consensus among the conclusions. Chauret et al. (1999), for example, found no correlation between
protozoan parasites and any of the microbial indicators tested, with the exception of that between
Cryptosporidium and faecal enterococci. By contrast, Brianesco and Bonadonna (2005) found a
relationship between Giardia and C. perfringens but none for Cryptosporidium, while Bonadonna et al.
(2002) concluded that none of the microbial indicators tested were reliable surrogates for protozoan
parasites. Clearly more work is needed in this area and no reliance should be placed on the currently used
microbial indicators for assessing protozoan parasites in activated sludge.
THE ROLE OF PROTOZOA IN ACTIVATED SLUDGE PLANTS

Most of the literature supports the view that the ciliate protozoa (and probably the other protozoan groups
like the flagellates) play a vital direct role in reducing the numbers of freely suspended and loosely
attached bacterial cells in the bulk liquid by the process of grazing, and thus improve considerably the
clarity of the final effluent leaving the plant (Curds, 1973a; Curds et al., 1968; Holubar et al., 2000;
Madoni, 1994a). Indeed following a series of studies of predator-prey relationships among bacteria and
protozoa, Curds (1992) concluded that protozoa could, by predation alone, easily account for all the
observed bacterial removal in the activated sludge process, although in reality a number of other removal
processes are probably involved. The importance of protozoan predation was further highlighted by
Eberl et al. (1997) who, following the introduction of fluorescently labelled bacterial cells into an
activated sludge system, demonstrated that predation by protozoa was the main mechanism for their
removal. Some of the factors which affect this grazing process and the possible roles of protozoa
in substrate degradation have been reviewed by Ratsak et al. (1996). Their role in influencing the
community composition in simulated activated sludge systems has been studied by Pernthaler et al. (1997)
and Simek et al. (1997).
Work carried out by Curds and co-workers (reviewed by Curds, 1982, 1992) has shown convincingly that
effluent quality with domestic wastewater is much poorer by several criteria when ciliate protozoa are not
present, as shown in Tables 4.2 and 4.3. Overgrazing of nitrifying bacteria, however, can lead to increased N
and P concentrations in the effluent (Ghyoot and Verstraete, 1999). What is still not well understood is how
rapidly bacterial cells are assimilated by these protozoa in activated sludge plants, or how selective they are
in their feeding patterns, although there is much evidence to suggest that feeding by grazing is not a random
process (Simek et al., 1997). For example, Fenchel (1980, 1986) suggested that each ciliate species selects
particles as food on the basis of their size, which reflects the morphological features of each protozoan
species’ mouthparts. Gurijala and Alexander (1990) also demonstrated a selectivity of feeding, and showed
that one feature protecting bacterial cells against protozoan predation was their hydrophobicity. Cech et al.
(1994) proposed that bacteria which accumulate glycogen instead of polyP (the so-called ‘‘G-bacteria’’ or
GAO – chapters 3, 10) survive in activated sludge plants because of their ability to form capsules. These
become integrated into the flocs and so avoid predation, a view favoured for the survival of other bacteria
(Ratsak et al., 1996). Likewise Luxmy et al. (2000) found that the size distributions both of suspended
bacteria and flocs are markedly influenced by ciliate grazing, with lower percentages of small (ca.1 mm)
bacteria and small (510 mm) flocs occurring in the presence of high numbers of bacterivorous ciliates.
Evidence for selective predation by the flagellate Adriamonas peritocrescens on the nitrifying bacteria was
presented by Verhagen and Laanbroek (1992). Common sense suggests that filamentous bacteria, because
of their morphology, would be less readily consumed than unicells, and there is some experimental data
supporting this view (Güde, 1979). However, both Inamori et al. (1991) and Terashi and Hamada (1991)
have described certain ciliate protozoa that readily prey on some filamentous bacteria, and suggest these
may even represent a means for controlling these filaments in plants, an idea which has not been pursued.
Table 4.2. Relationship between the presence of certain protozoa and the biochemical oxygen demand of
the final effluent in activated sludge plants (after Curds and Cockburn, 1970b). Values are the percentage
frequency for each organism seen in mixed liquors.
Frequency of occurrence (%)
BOD range (mg l 1) 0–10 11–20 21–30 430
Vorticella convallaria 63 73 37 22
Vorticella fromenteli 38 33 12 0
Carchesium polypinum 19 47 12 0
Aspidisca cicada 75 80 50 56
Euplotes patella 38 25 24 0
Flagellates 0 0 37 45

Euplotes patella is rarely seen in some mixed liquors, in Spain for example, whereas E. aedaculatus is often very common.

At least some flagellates are probably present in most mixed liquors although numbers are so small that they may not be
detected.
Table 4.3. The effect of the presence of ciliate protozoa on the quality of final effluent from laboratory-scale
activated sludge plants (after Curds et al., 1968).
Ciliates
Effluent parameter Present Absent
BOD (mg l 1) 7–24 53–70
COD (mg l 1) 24–142 198–250
Organic nitrogen (mg l 1) 7–10 14–21
Suspended solids (mg l 1) 11–34 86–118
Turbidity (A620) 0.05–0.29 0.95–1.42
Viable count of bacteria (£106ml 1) 1–9 106–160
Most studies on the role of protozoa in the activated sludge process have been carried out for domestic
wastewaters. EBPR plants and those treating industrial or agricultural effluents, for example, remain
comparatively poorly studied, although results so far indicate that protozoa play a similarly important role
in these too (Aescht and Foissner, 1992; Bécares et al., 1998; Çech et al., 1994; Holubar et al., 2000;
Hughes and Stafford, 1976; Liu et al., 2008; Ovez and Orhon, 2005; Salvado et al., 2003).
In addition to predation on suspended bacteria, a number of other roles of protozoa in the activated sludge
process have been suggested. These include inducing and aiding floc formation by one of three processes:
(1) secretion of soluble polysaccharide into the medium which changes the surface charge of the
colloidal particles;
(2) ingested particles becoming glued together by a mucin during cytosis;
(3) removal of dispersed bacteria by predation inducing the growth of floc-forming and filamentous
bacteria.
The role of ciliates in the development of activated sludge flocs has also been investigated (Arregui
et al., 2007; Weber et al., 2007) although Curds (1992) concluded that protozoan-induced flocculation is
unlikely to be important in this process. It is also known that protozoa can utilise soluble substrates but to
date no work has been carried out on the quantitative aspects of substrate utilisation when they also have a
plentiful supply of bacteria present as a food source. It is unlikely that protozoa can compete successfully
with bacteria for such substrates, so mineralization of dissolved organic matter is probably not an
important function either. The effects of particulate vs. soluble organic compounds on the microfaunal
community and treatment efficiency were investigated by Puigagut et al. (2007).
A third role may be the ingestion of flocculated bacteria and mineralization of sludge. Published and
unpublished data suggest that these processes may reduce sludge production by more than 25% (Ghyoot
and Verstraete, 1999; Salvado et al., in preparation) while other authors have also reported substantial
reductions in sludge production as a result of protozoan predation (Lee and Oleszkiewicz, 2003; Lee and
Welander, 1996). Finally, it has been suggested that bacterial activity, and therefore purification processes
in activated sludge, are stimulated by the predatory activities of protozoa (reviewed in Curds, 1977).
There is little doubt, however, that the primary role of protozoa in the activated sludge process is the
removal of dispersed bacteria by predation.
PROTOZOA AS INDICATORS OF PLANT PERFORMANCE

The relationship between the clarity and quality of the liquid effluent from clarifiers and the occurrence of
protozoa has led to the suggestion that the presence of certain protozoan assemblages or of particular
ciliate species may be used to indicate overall plant performance (see Table 4.4) (Curds, 1982; Curds and
Cockburn, 1970b; Lee et al., 2004; Madoni, 1994a; Madoni et al., 1993; Martin-Cereceda et al., 1996;
Ovez and Orhon, 2005; Poole, 1984; Zhou et al., 2006). Thus, frequent microscopic examination of the
biomass may provide a rapid, simple and convenient method for indicating sudden changes in
performance, as long as the protozoa can be accurately identified by the laboratory staff at their particular
plant, and this should happen with practice. There are published statements that the composition of the
ciliate protozoan community can be used confidently to suggest whether the plant is operating well,
i.e. neither under- or over-loaded, adequately aerated, suitable sludge-retention time, etc. Some of
these suggestions are summarised in Table 4.5 (Madoni, 1994a; Madoni et al., 1993; Salvado, 1994;
Salvado et al., 1995). The desirable protozoa have been suggested to be the crawling and (most) attached
ciliates and the testate amoebae, while certain attached ciliates such as Opercularia spp. and Vorticella
microstoma, swimming bacterivorous ciliates, and small flagellates are said to indicate poor effluent
quality (Madoni, 1994a; Madoni et al., 1993). Other studies have suggested different relationships
(Esteban et al., 1991; Martin-Cereceda et al., 1996; Poole, 1984).
Table 4.4. Effect of sludge loadings on the presence of a few common genera of ciliate protozoa in activated
sludge plants (after Madoni, 1991).
Sludge loading
Protozoan Low Medium High
Aspidisca cicada þ þ þ
Vorticella picta þ
Vorticella convallaria þ þ þ
Vorticella microstoma þ
Operculara microdiscum þ
Operculara coarctata þ
Acineria uncinata þ
Table 4.5. Possible relationships between presence of particular dominant groups of protozoa, plant
performance and operational characteristics.
Dominant Protozoa Performance Possible causes
Small flagellates poor poorly aerated sludge,
(except choanoflagellates) overloaded plant
Small free-swimming ciliates mediocre poorly aerated sludge,
low sludge age
Large free-swimming ciliates mediocre overloaded plant
Certain attached ciliates failing performance transient perturbations
(i.e. Opercularia spp. and
Vorticella microstoma)
Crawling ciliates good –
Attached and crawling ciliates good –
Small flagellates and small poor high load
naked amoebae
Testate amoebae unknown underloaded
Salvado et al. (1995) considered that the less commonly seen protozoa like Acineta tuberosa,
Euplotes sp. and Zoothamnium sp. all indicated a high quality effluent in the three plants they monitored.
They suggested the most frequently observed protozoa including Vorticella microstoma and Opercularia
coarctata were associated with low quality effluent, agreeing with the views expressed by Madoni
(1994a). These protozoa have often been associated with plants with high loadings (Table 4.4).
Esteban et al. (1991) closely examined the effects of a wide range of factors, both biological and
process-related, on the protozoan populations in a plant in Spain and reaffirmed the importance of some of
these ciliates in determining effluent quality. By following individual ciliate species they were able to
recognise which factors appeared to favour, or otherwise, their presence in mixed liquor. Using principal
components analysis, their data suggest that the quality of the raw wastewater entering the plant and the
organic load were both particularly important in determining both ciliate numbers and their diversity
(see also Esteban and Tellez, 1992). This infers that each plant may be expected to develop its own
distinctive protozoan community. These views support the results of Al-Shahwani and Horan (1991) who
also proposed that by carefully monitoring the ciliate populations and operating conditions of each
individual plant, it may be possible for plant operators to predict plant performance based on the presence
of particular species. Although no clear correlation was seen with any change in any single plant variable
and any individual species of protozoa, they suggested that as few as 20 ciliate species would be sufficient
to achieve this aim. However, a prolonged period of data collection and establishment of a sound data
base would seem to be required on each individual plant before such a scheme could be confidently
implemented. There is no published evidence that this follow-up has occurred widely. Some of their
identifications of protozoa were later queried by Esteban (1992). The user-friendly key to identification of
ciliated protozoa being completed by Curds et al. (2008) aims to improve this situation. This CD will not
only enable the non-specialist user to identify the species present but will also aid in predicting effluent
quality. It aims to provide global starting parameters concerning the indicator value of each species whose
accuracy can then be improved automatically from results obtained locally. This will also enable
comparisons between plants, works and countries.
It has been proposed that certain protozoan species and assemblages are associated with nitrifying
plants (Chen et al., 2003; Cybis and Horan, 1997; Poole, 1984). In a study of 13 activated sludge plants
with a range of configurations and operating conditions, Poole (1984) reported that the mixed liquor
fauna of nitrifying plants are characterized by holotrichous free-swimming ciliates such as Prorodon
and Blepharisma and by testate amoebae such as Euglypha. Chen et al. (2004) also reported an
association between nitrification and Euglypha as well as with the peritrich Epistylis lacustris, while
others have suggested indicators of nitrification include the testate amoeba Centropyxis and the loricate
peritrich ciliate Thuricola kellicottiana. By contrast, with sequencing batch reactors, Cybis and Horan
(1997) reported that the onset of nitrification was marked by a change in the protozoan community from
one dominated by crawling and attached ciliates to one where free-swimming flagellates dominated,
although this may have been a response to the sudden drop in pH (from 7.0 to 5.8) that accompanied
the change. As denitrification became established, and the pH increased, attached ciliates became
dominant again.
All the general observations that the relative dominance of different protozoan groups changes with
changes in plant performance, and that the numbers of morphological species drop as plant performance
deteriorates were used by Madoni (1994a) to develop the concept of a sludge biotic index (SBI).
He proposed that this index would allow the biological quality of the sludge to be expressed objectively
in numerical terms, and he categorised mixed liquors into four classes based on their SBI value. Madoni
suggested that this scheme has more general value than those of Esteban et al. (1991) and Al-Shahwani
and Horan (1991) for assessing plant performance, since he believes there is less risk of subjective
influences on data collection and interpretation. Madoni (1994a) also details a protocol for carrying out
these analyses for assessing the mixed liquor microfauna. Nevertheless, the reliability of the SBI values
obtained depends largely on the ability of the operator to identify accurately the protozoa present, and
in many plants that may not be achieved because of the staff and resources available. As with the other
schemes discussed, the general applicability of the SBI to plants of different configurations in different
parts of the world is still to be tested. Only then will its value be apparent.
A summary of the literature would appear to suggest that an efficiently operating plant would have the
following protozoan community characteristics (Madoni et al., 1993), although the relationships between
causes and effects are often not well understood for these empirical observations.
(a) A high population density of ciliated protozoa of more than 103 per ml;
(b) A community consisting mainly of crawling and attached ciliates, and few or no flagellates;
(c) The ciliates would be taxonomically diverse, and no one species would dominate the rest by a
factor of more than 10.
The guidelines shown in Table 4.5 may help the operator diagnose what might be wrong with the plant
and what remedial action is needed.
It is usually recommended that simple microscopic analysis of plant samples should be carried out
frequently and, if possible, daily (Madoni, 1994a). There also seems to be a need to set up courses for
laboratory staff in each country to train them to identify the protozoa they commonly encounter, so that
some of the ideas discussed above may be more widely tested.
How generally applicable globally these findings are awaits further work and there appears to be little
evidence in the literature that these ideas have been examined fully or pursued in countries other than
those in western Europe where the initial work was done (although for examples see Banina et al., 1983;
Chen et al., 2005; Lee et al., 2004; Morishita, 1970, 1976; Ovez and Orhon, 2005). Little systematic work
has been carried out comparing protozoa from different parts of the world (Liu et al., 2008), a situation
similar to that with the filamentous bacteria, and co-operative studies between different groups seem to be
required. As mentioned previously the technology is now available to develop gene probes for some of
these protozoa (Marsh et al., 1998; Fried et al., 2002), so in theory they may be identified unequivocally in
plants around the world, and their population dynamics more exactly determined.
POPULATION DYNAMICS OF ACTIVATED SLUDGE PROTOZOA

Although the ecology of the activated sludge system is still quite poorly understood (chapter 3), attempts
have been made to model the process to see how densities of the different populations of protozoa vary
with changes in certain operating conditions. These models are based on several assumptions about the
feeding patterns of different groups of organisms and allow for whether they are freely in suspension or
associated with flocs, which are thus recycled (Curds, 1992; Fried and Lemmer, 2003). One possible food
chain providing the basis for such a model is given in Figure 4.4. If Monod kinetics (chapter 1) are
adopted, computer simulations have shown that after oscillations in population densities of these protozoa,
and their corresponding bacterial prey, they eventually become stable and in balance by negative feedback
control. These models clearly show that free-swimming ciliates feeding upon suspended bacteria, at
steady state, have a much higher growth rate (equivalent to 1/sewage retention time) than those ciliates
which settle with the flocs and thus are recycled (equivalent to 1/sludge age). Monod kinetics infer that
organisms with a high growth rate require the availability of greater concentrations of substrate (in this
case suspended bacteria) to be present. Applying this to an activated sludge plant means that if the
predominant ciliate population is free-swimming then the concentration of bacteria in the effluent will be
greater than when the ciliates are predominantly attached forms. There will be larger numbers of slower
growing attached ciliates than free-swimming ciliates which will be fewer in number but growing at a
faster rate. The reader is referred to the original papers by Curds (1971; 1973b,c; 1974) and to Curds
(1982, 1992) for a more detailed summary of this work and its application, although our understanding of
predator-prey relationships upon which this is based is still uncertain (e.g. Williams, 1980).
Substrate Sludge
Nutrients Bacteria
Activated
sludge
Flagellate Floc
Protozoa
Attached Attached
Sewage & carnivorous
Bacteria crawling ciliate
Ciliate Protozoa
Protozoa
Free-swimming
Free-swimming carnivorous
ciliate Protozoa ciliate protozoa
Figure 4.4. The food chain of activated sludge biomass, showing probable interactions between ecological
groups of the protozoa.
ACKNOWLEDGEMENT
We would like to thank Dr Janusz Fyda, Jagiellonian University, Krakow, Poland, for supplying several of
the photomicrographs.
5
Factors affecting the bulking and foaming
filamentous bacteria in activated sludge
Robert J. Seviour
INTRODUCTION
Filamentous bacteria should be considered as normal components of the microbial community of
activated sludge plants, although where they originate and how they arrive there is not clear (Wanner,
1993; 1994a,b), since many e.g. Candidatus ‘Microthrix parvicella’ have been detected nowhere else.
They seem to play an important role in providing a matrix for the formation of flocs (chapter 3) with
structures appropriate for rapid settling (Jenkins et al., 2004b). However, major problems arise when these
filamentous bacteria proliferate, leading to the problems of bulking and foaming. Surveys carried out
around the world (see Tandoi et al., 2006 for summaries) suggest that most activated sludge plants will
suffer from one or both of these operational disorders, but little proactive attempts are made in the industry
to prevent either. Certainly from the author’s experiences in Australia, few operators are experienced in
filament identification or regularly examine their plant biomass microscopically. This is despite the
availability of courses designed to help identify these bacteria. Equally, very few water authorities employ
professional microbiologists to carry out these routine analyses.
With practice microscopic analysis of activated sludge biomass is simple, cheap and rapid (chapter 11).
It can provide information on floc structure and mixed liquor filament composition, and in detecting sudden
or gradual changes in both, which may pre-warn of changes to plant performance (Jenkins et al., 2004b;
Tandoi et al., 2006). Microscopy also presents visually the organisms which constitute the activated sludge
community, bringing operations and management staff closer to the process. An increasing availability of
FISH probes and FISH-based commercial kits makes identification and even quantification of many
filaments in activated sludge more feasible on a routine basis (see later), and these are applied routinely in
larger treatment plants in several countries.
REASONS FOR INADEQUATE SOLIDS SEPARATIONS IN CLARIFIERS

Activated sludge processes depend ultimately for their successful operation on a rapid and complete
separation of the biomass from the liquid supernatant in the clarifiers, and production of non-turbid final
effluents. This requirement is not always met, which is why membrane bioreactors and aerobic granules
are seen as attractive alternative technologies (chapter 2). Wanner (2002) and Jenkins et al. (2004b) have
listed some reasons for poor solids separation, and these are summarised in Table 5.1. Not all separation
problems are caused by an excessive growth of filamentous bacteria, although this section will deal with
those that are, namely BULKING and FOAMING (Dionisi et al., 2006).
Table 5.1. Problems associated with separation of biomass in activated sludge secondary clarifiers
(Jenkins et al., 2004b).
Problem Possible cause Operational consequence
Dispersed growth No true flocs formed but microbial Effluent is turbid and solids do
cells freely suspended in liquid not settle
Slime or non-filamentous Large production of exopolysaccharide of Poor solids settlability and
bulking microbial origin. Often seen in plants solids carry over from clarifier
treating industrial effluents which are
deficient in either nitrogen or phosphorus.
Incorrectly called ‘zoogloeal’ bulking
Pinpoint floc Small weak flocs which are easily broken up. Turbid effluent
Smaller fragments fail to settle.
Found in plants operating Low SVI
at low F/M ratios
Filamentous bulking Filamentous bacteria extend from flocs and High SVI and, if severe, solids
interfloc bridging occurs, or development of overflow from clarifiers
open diffuse flocs
Blanket rising As consequence of denitrification in the Surface of clarifiers covered
clarifiers, the N2 gas generated gives with buoyant layer of biomass
buoyancy to the flocs, which float to the
surface. Exacerbated by long retention time
of solids in the clarifiers
Foaming or Scumming Probably caused mainly by hydrophobic Very stable foams bringing
bacteria like Mycolata, biomass to surface of clarifiers.
Candidatus ‘Microthrix parvicella’, Can cause carryover of
Candidatus ‘Microthrix calida’? solids from clarifiers.
Type 0041/0675, Candidatus Potential health risk from
‘Nostocoida limicola’ (Tetrasphaera) foam associated opportunistic
and Acinetobacter spp. Mycolata pathogens
Bulking sludge and its properties

Martins et al. (2004b) and Wanner (2006a,b) recognize a bulking sludge as one which settles slowly and
compacts poorly. This serious problem, discussed more fully in chapter 7, results from an excessive
Factors affecting the bulking and foaming filamentous bacteria in activated sludge 141
growth of filamentous bacteria away from the floc surface, even though these may still represent a minor
component of the total microbial community. Not all filaments will have the same effects because they
differ in their abilities to grow out from the flocs to form inter-bridges between them (Jenkins et al.,
2004b; Wanner, 2002). Floc morphology can be characterised in several ways which reflect differences
in their shape, firmness, compactness and size (Jenkins et al., 2004b), and several microscopic/image
analysis methods for automatically determining floc morphology have been published (e.g. Banadda et al.,
2005; Jenné et al., 2006; Liwarski-Bizukojc, 2005). One would expect that floc macrostructure
would reflect the amount and arrangement of the filamentous bacteria, and Martins et al. (2004b), and
Jenkins et al. (2004b) have both recognised what they describe as:
(a) the ideal floc, where the filaments are restricted largely to the floc interior, and only limited
extension into the bulk liquid is seen;
(b) pinpoint flocs with little or no filamentous matrix, but possessing floc microstructure. These flocs
are small and fragile, and very easily disrupted by low shear and any turbulence. The small floc
fragments settle poorly, producing a turbid effluent;
(c) filamentous bulking sludge, with extensive growth of filamentous bacteria, both inside and
crucially, extending from the floc. These filaments make the flocs diffuse and/or provide interfloc
bridging, which both lead to poor settlability and compaction. However, the supernatant is usually
very clear, as suspended particles are readily filtered out by this matrix.
Examples of the diversity in floc morphology and structure in plants as revealed by microscopy are shown
and described in Figure 5.1, see colour image section (chapter 13). The image analysis methods mentioned
above reveal more subtle variations than light microscopy can provide, but are still mainly research tools.
HOW DO WE PREDICT WHETHER PLANTS WILL SUFFER FROM

FOAMING AND BULKING?
Foaming
Production of stable foam is thought to result from a flotation process, where hydrophobic particles
(bacterial cells) attach to air bubbles, which are then stabilized by the presence of surfactant material
(Soddell and Seviour, 1990). Established foams are easily recognised by their visible presence on the
surface of the reactors, but often overflow onto the surrounding walkways and lawns etc (Figure 5.2, see
colour image section (chapter 13)). However, not all foams are microbiologically induced. Some may be
caused by detergents, especially in newly commissioned plants, and these often readily disperse with no
intervention (Jenkins et al., 2004b). Sometimes a stable foam can appear very rapidly (overnight), as a
result of dumping of detergent cleaning material into the sewers. Methods have been sought to monitor
plant biomass to provide early warning systems for foaming incidents (chapter 8). Apart from regular
quick qualitative microscopic examinations of mixed liquor samples, looking particularly for, and
identifying foam causing bacterial morphotypes like the distinctive Gordonia amarae-like organisms
(GALO), and Candidatus ‘M. parvicella’ (chapter 8), more semi-quantitative methods have been
reported. Pitt and Jenkins (1990) and Ho and Jenkins (1991) both described filament counting methods for
routinely estimating GALO levels, and Cha et al. (1992) tried to correlate these with foam production.
However, such methods should be used cautiously, since some foaming Mycolata filaments may fragment
as part of their life cycles, yet still stabilize foams (Davenport et al., 2000; Soddell and Seviour 1990).
The extent of this fragmentation probably varies between Mycolata organisms and within the same
filament under different conditions (Soddell, 1999; Soddell and Seviour, 1990, 1993).
Hernandez et al. (1994) were among the first to seek more targeted identification methods, applying
fluorescently tagged antibodies against ‘Nocardia’ (probably G. amarae). These suffer inherently from poor
specificity, and can only be generated from organisms previously grown in pure culture. FISH probes
targeted at the foaming Mycolata and other filament phylotypes (Davenport et al., 2000; de los Reyes et al.,
1997, 1998; Eales et al., 2006; Erhart et al., 1997; Kämpfer and Wagner, 2002; Liu and Seviour, 2001) in
combination with either epifluorescent (Hug et al., 2005) or confocal laser scanning microscopy (CLSM)
and image analysis (Daims et al., 2001c, 2006a; Lopez et al., 2005; Zhou et al., 2007b) are generally more
suitable for filament identification and subsequent quantification. They do not depend on organism
morphology, and so any unicellular Mycolata populations for example, will still be detected by FISH. With
this approach Davenport et al. (2000) with 16S rRNA targeted probes designed for most of the Mycolata,
suggested that a threshold of about 2 £ 106 cells ml 1 existed, above which foams were produced. They
later proposed a universal threshold value for foaming (Davenport et al., 2008). de los Reyes and Raskin
(2002) refined this to differentiate between the levels of G. amarae (in terms of the total filament lengths, not
cell numbers) required for initiating or stabilizing foam, and suggested that about 5£ more was needed for
foam stabilization. Unicellular forms would not have been included in their estimations.
The FISH approach is not above criticism. For example, there is the danger of reaching unwarranted
generalizations from such limited data, since similar estimations are not available for other foaming
bacterial populations, and the number of plants examined in this way is small. It is best tested on an
individual basis against each plant biomass community, whose filament populations are well documented.
Furthermore, we still do not understand the full extent of the phylogenetic diversity among foam forming
Mycolata (Goodfellow et al., 1998; Soddell et al., 1998; Stainsby et al., 2002), but are certain that the
FISH probes currently available do not cover all of them (Kragelund et al., 2007b). FISH probe detection
relies on cell ribosome content. There is evidence that G. amarae, and probably other foaming bacteria
like Candidatus ‘M. parvicella’ (de los Reyes et al., 2002) are either dead (Carr et al., 2005), or have very
low ribosome (rRNA) levels (Oerther et al., 1999a; 2001) in situ, yet may still be able to generate and
stabilize foams. Hence FISH based methods may underestimate the filament levels required to produce a
stable foam. For quantification it was suggested that FISH should used be in combination with antibody
staining (Oerther et al., 2001), which is insensitive to differences in cell activities, and should incorporate
quantification of population specific rRNA levels for validation. Such complex analyses will probably
ensure routine on-site monitoring will never use them.
Early warning recognition of foaming incidents has also been attempted by monitoring biomass
cell surface hydrophobicity (CSH) levels with the MATH assay (Rosenberg 2006), and whether these
change before or during foam development (Khan et al., 1991; Stratton et al., 2001, 2003). However,
results with it have been equivocal, and when Eikelboom (1991) critically examined the available
methods for estimating CSH, he found none were suitably reliable for this purpose. Therefore,
whether CSH shifts relate to changes in biomass foaming potential is unclear. Certainly when CSH
values were compared to the foam forming abilities and mycolic acid contents of pure cultures of
Rhodococcus isolates from foam, little correlation between them was seen (Stratton et al., 2002,
2003). In situ CSH methods using fluorescent microspheres (Zita and Hermansson, 1997) have also
been used with some success in combination with FISH in detecting relative differences in the CSH
of some filamentous bacteria at an individual cell level in foams (Nielsen et al., 2001; Eales et al.,
2005, 2006; Kragelund et al., 2007a,b). However, similar difficulties in interpreting these data are
encountered. Blackall and Marshall (1989) and Stratton (1997) both described standard laboratory
scale foaming tests and simple foam rating systems to measure their stability, and this is probably the
most direct and valuable way to monitor plants for potential foaming incidents. The microbiology of
foaming is discussed in detail in chapter 8.
Bulking
Bulking sludge is less obviously detectable visually in its early stages than is a foaming episode (Cha et al.,
1992). However, since sludge only bulks when the filaments extend from the surface of the flocs
(Liao et al., 2004a; Lou and de los Reyes, 2005a,b; Martins et al., 2004b; Vervaeren et al., 2005),
microscopic monitoring is helpful. Several other empirical methods are used to monitor plants routinely
(Jenkins et al., 2004b; Wanner, 2002, 2006a), and these include:
(i) Settlability Tests – which are simple to perform and should indicate the settling characteristics of
the sludge actually taking place in the secondary clarifiers. Several tests are available, which are
of different value to the plant operator. Briefly, these are:
– 30 Minute Settling Test (V30) ¼ the volume of biomass which settles out from a known
volume of mixed liquor in 30 minutes.
– Sludge Volume Index (SVI) – unlike the V30 test, this makes some allowance for differences in
solids concentrations in the mixed liquor, by basing the measurement on the level of MLSS
(chapter 2). A SVI of 150–200 ml g1 is often used to define a bulking sludge, but this value
has been questioned (Schuler and Jassby, 2007). However, SVI is popular with plant
operators, as is the slightly modified version for obtaining the Stirred Sludge Volume Index
(SSVI) (Jenkins et al., 2004b; Wanner, 2002). Both methods have been criticised as unreliably
indicating bulking, and as not representing what actually happens in a secondary clarifier.
Instead the Diluted Sludge Volume Index (DSVI), which does, has gradually become the
method of choice in many countries.
Filament quantification methods in bulking sludge

The early microscopic techniques (Sezgin et al., 1978) for filament quantification, while relatively simple
and quick, do not provide reliable identifications of the problem causing bacteria (see chapter 7). Data
from these assays are usually expressed as total extended filament length (TEFL) values, expressing the
extent of filament growth out from the floc surface. Attempts to correlate TEFL with parameters like DSVI
are discussed in Jenkins et al. (2004b). While Lee et al. (1983) showed that at 120 ml g 1 DSVI (where
their sludge started to bulk), the TEFL was 30 km g 1 MLSS, Gabb et al. (1988) suggested that 10 km g 1
MLSS TEFL represents a more precise boundary between non-bulking and bulking sludges. More people
probably use the simpler subjective filament abundance scale suggested by Eikelboom and van Buijsen
(1983) and Jenkins et al. (2004b), which is listed in Table 5.2. The filament abundance determinations
given there were carried out in the current author’s laboratory, and the data obtained (not presented)
suggest that a score of very common, abundant or excessive corresponds to a DSVI greater than that
considered necessary for bulking. Similar studies with similar outcomes have been carried out in other
countries (Cingolani et al., 1994; Kristensen et al., 1994; Rossetti et al., 1994). Alternatively, rapidly
monitoring changes in the thixotropic properties of activated sludge, a non-Newtonian fluid when
filaments are abundant, indicated it was possible to detect a bulking episode by following the reduced
hysteresis area (rHa), where the rHa/TSS ratios increased with increasing SVI (Tixier et al., 2003).
FISH based quantification methods for monitoring bulking

As with foaming, most recent work has been directed at developing more targeted FISH/image analysis
based methods for estimating both total and extended filament length, and the extent of bulking from
individual filamentous populations (e.g. van der Waarde et al., 2002), However, many of the same criticisms
Table 5.2. Subjective scheme of Jenkins et al. (2004) for determination of filament abundance in mixed
liquor.
Score value Assessment of filament abundance Comments
0 None seen
1 Few present Only seen in an occasional floc
2 Some present Commonly seen but not in all flocs
3 Common Seen in all flocs but only at low frequencies
(1–5 filaments per floc)
4 Very common Seen in all flocs at frequencies of 5–20 per floc
5 Abundant presence Seen in all flocs, at frequencies of $20 per floc
6 Excessive numbers Seen in all flocs. Biomass mostly filaments
which are plentiful in the bulk liquid
levelled earlier against suitability of FISH analyses in foaming studies apply equally here. Problems can
arise from background autofluorescence which complicates image analysis, and the sampling plans used
need to be statistically soundly based (Liao et al., 2004a). Attempts to calibrate bulking more precisely with
FISH by estimating the TEFL required for its onset (Liao et al., 2004a) have suggested that such a value may
vary with each filament type. Vervaeren et al. (2005) applied quantitative FISH and qPCR to bulking
biomass containing type 021N, and showed good correlation between SVI and levels of its 16S rRNA genes.
They also suggested that determining the specific 16S rRNA: DNA ratios for each dominant filament may
provide a sensitive early warning method for episodes of bulking. It is unclear how generally applicable
this approach is, especially for unidentified bulking filaments for which qPCR methods are not available,
and whether it works adequately for full-scale bulking plants. Furthermore, as with some other methods
mentioned here, it may be too complex for routine on-site use. However, Hug et al. (2005) claim their non-
automated FISH-epifluorescence methods for filament quantification using an abundance rating system is
sufficiently quick and reliable for routine monitoring of both bulking and foaming. Commercially available
FISH based methods for quantifying a few filaments at treatment plants and cheap dedicated epifluorescence
microscopes are now available from Vermicon, who clearly see a market for such technology.
WHAT SELECTIVE PRESSURES ARE THOUGHT TO FAVOR THE

FILAMENTOUS BACTERIA IN ACTIVATED SLUDGE?
Considerable effort has been directed towards understanding how and why some filamentous bacteria
proliferate sufficiently to cause bulking and foaming problems. As Martins et al. (2004b) point out, this
work has been carried out by both engineers and microbiologists working largely independently of each
other. Each has generally asked different research questions in attempts to reach an all-embracing single
solution to the problems. Martins et al. (2004b) believe, as we do, that both groups working together
might achieve better outcomes, and only when we understand more fully the identity and ecology of the
bacteria responsible can we formulate and implement practical strategies to counter them together.
The microbiological approach

Surveying filament populations in plants
Traditionally the efforts of microbiologists have been directed at trying to understand which factors might
determine the filamentous bacterial communities in plants, primarily by identifying them in systems
running with different operational parameters. These studies have been based largely on surveying
biomass samples for morphotypes identifiable by microscopy, often from a single grab sample taken at
one or a few arbitrary times and generally from a relatively small number of bulking and/or foaming
plants. The plants sampled for each survey are often geographically scattered, and treat waste from
populations whose cultural and dietary habits may be diverse. This general approach, which has been
adopted in many countries (see summary in Tandoi et al., 2006), will tell us which morphotypes are
present at that sampling time, but not always how or why they occur, as the plant operational data
accompanying the microbiological survey data are in many cases scant and unreliable.
Even so, the data available (Tandoi et al., 2006) seem to indicate that the nature of the influent (where
detailed) and prevailing plant configuration and operating conditions (where provided) do contribute
towards determining which dominant filamentous bacteria occur (see chapter 7). Eikelboom and van
Buijsen (1983) and Jenkins (1992) with their survey data based on microscopic examination of samples
from plants in The Netherlands and U.S.A. respectively, tried to correlate particular filament morphotypes
with plant operational parameters. Not surprisingly, given the number of interacting variables likely to be
involved in filament selection, their data do not always agree (Wanner, 1994d), but both support the view
that certain morphotypes are favoured selectively under particular plant operating conditions (Table 5.3).
However, often only a single operational factor was recognized as favouring each one, and the reality
that plants usually contain several different dominant filament morphotypes was not fully addressed.
Nevertheless, such information is used on an empirical basis by operational staff around the world as the
initial step in trying to control bulking by modifying operating conditions.
Table 5.3. Suggested grouping of filamentous bacteria based on plant operational conditions which appear
to favour their growth (Eikelboom and van Buijsen, 1983; Jenkins et al., 2004).
Filaments Favoured operational parameter?
Sphaerotilus natans Low levels of dissolved oxygen, although exact
Haliscomenobacter hydrossis values for these levels are not known.
Type 1701
Candidatus ‘‘Microthrix parvicella’’ Low F/M. Longer sludge ages or higher mean
Haliscomenobacter hydrossis cell residence times.
Nocardia spp.
Types 021N, 0041/0675,
0092, 0581, 0961, 0803, 0914
Type 1863 High F/M, short sludge age (55 days)
Thiothrix spp. Nitrogen or phosphorus deficiency in mixed liquors.
Sphaerotilus natans
Haliscomenobacter hydrossis
Type 021N, 0041/0675
Sphaerotilus natans Readily biodegradable soluble substrates.
Thiothrix spp.
‘‘Nostocoida limicola’’
Type 1851
Candidatus ‘‘Microthrix parvicella’’ Slowly biodegradable or particulate substrates.
Type 0041/0675
Type 0092
Fungi Low pH (56)
Thiothrix spp. Presence of H2S and septic wastewater
Beggiatoa provides energy source for mixotrophic growth.
Type 021N
Of course these rules of thumb will be compromised if their original filament identifications were not
precise, which, as we shall see, is often the case with microscopic based methods, failing generally to
reveal any information about distributions of individual filament strains or species. They will too if these
‘relationships’ do not hold true globally for each particular filament morphotype, as a consequence of
phylogenetic differences or physiological/ecological variations among its different members. We know
that both these situations may occur (chapter 6).
Ideally relating cause and effect to control bulking should take into account every factor which might
determine the competitive ability of each filamentous bacterium in each individual plant, and we are a
long way from understanding what these are. Resolving their ecophysiology by techniques like FISH-
MAR may help in this, as shown impressively for Candidatus ‘M. parvicella’ (Rossetti et al., 2005).
FISH-based surveys can also be criticised, since probes for many filaments are not yet available. Those
that are do not target individual species, because of inadequate understanding of filament taxonomy
(chapter 6). Instead, many target phylotypes, each of which may contain several different ‘species’ or
strains. Despite this, FISH surveys mapping the distribution of filament phylotypes have been carried out
(e.g. Kim et al., 2002; Lemmer et al., 2005), and provided information not possible from traditional
microscopy. For example, FISH has confirmed what was long suspected, that the phylogenetic diversity of
bulking filaments, especially in industrial plant biomass samples (Eikelboom and Geurkink, 2002;
Kragelund et al., 2005, 2006; Levantesi et al., 2004, 2006a; van der Waarde et al., 2002) is much greater
than surveys based on traditional microscopy had suggested. How exclusive these industrial filaments are
to industrial plants is unclear from a survey restricted to a few countries, since FISH has demonstrated that
Candidatus ‘Alysiosphaera europea’ (Levantesi et al., 2004; Snaidr et al., 2002;) for example, is widely
distributed in plants in Japan and Australia, and can occur in amounts high enough to cause bulking in
plants treating only domestic sewage (E. Seviour and A. Chua, unpublished).
So are survey data helpful?

Even allowing for these criticisms, some further comment on the published survey data is warranted. They
show that even though their phylogeny is relatively unexplored, the diversity of filament morphotypes
dominating foams in plants globally is relatively small. Thus, the GALO, unicellular Actinobacteria and
Candidatus ‘M. parvicella’ are almost always found (Seviour et al., 2008), and often appear together
(chapter 8). However, population shifts may occur. Hence, Candidatus ‘M. parvicella’, while dominating
during the winter, is frequently replaced in foaming plants in the summer by GALO (Lacko et al., 1999;
Seviour et al., 1990b; Tandoi et al., 2006; Wanner et al., 1998b). An unidentified N. limicola morphotype
exhibits a similar pattern (Andreasen et al., 2006). Survey data (Seviour et al., 1990) clearly showed the
morphologically distinctive Skermania piniformis is a major foaming organism in Australia. It was
thought to be rare elsewhere, but surveys now suggest it is probably globally widespread in foams (de los
Reyes et al., 2002; Eales et al., 2005; Kragelund et al., 2007b; Soddell and Seviour 1998). The global
distribution of Candidatus ‘Microthrix calida’ (Levantesi et al., 2006b) is not known.
Bulking filaments vary more in their relative importance between plants and countries (Tandoi et al.,
2006), but again for reasons not always apparent (see chapter 7). Thus, Candidatus ‘M. parvicella’ is more
frequently reported in surveys of bulking (and foaming) plants in countries in the Northern hemisphere
(Eikelboom et al., 1998) i.e. those in colder climates (see later). On the other hand, while type 0092 was the
dominant bulking filament in surveys from France (Gravelau et al., 2005) and South Africa (Blackbeard et al.,
1986a, 1988), type 021N is more commonly seen in Japan, Italy and in non-EBPR plants in Denmark. In the
Czech Republic, N. limicola (probably N. limicola II) appears more frequently than it once did, and more so
there than in other European countries surveyed. Yet there is no persuasive explanation for all these trends.
Clearly some survey data (Tandoi et al., 2006) add support to the earlier generalizations listed in
Table 5.3, that plants with particular operating configurations favour certain filament morphotypes.
Thus, most from several countries show that types 0092, 0914 and 0041/0675 and Candidatus
‘M. parvicella’ are especially common in low F/M EBPR plants, while types 021N and 0961 are rarely
seen there (chapter 8). On the other hand, high F/M plants also seem generally to encourage types
1863 and 0803. Filamentous bacteria like H. hydrossis occur in most activated plant communities
regardless of their design, operating conditions, or location, but usually as minor populations, and so
may probably never cause operational problems on their own. Some Eikelboom morphotypes like types
1852, 1702 and 0211 are rarely if ever reported in surveys, which may reflect in part the comparative
difficulty in recognizing them microscopically, and also lack of information on their taxonomy and
FISH probes to detect them.
The engineering approach

Engineers have tackled the operational problems of bulking and foaming differently, by trying to come up
with unifying theories to cover all filaments, without worrying too much about their precise identity in
plants, or direct experimental evidence to support their theories. They ignore the likelihood that each
individual filament morphotype will differ in its in situ behaviour from the others. However, their ideas
continue to be popular within the industry in attempting to reach engineering solutions to what are
essentially microbiological problems. Some of their theories are discussed briefly here, and are reviewed
more fully by Wanner (1994a,c; 2002) and Martins et al. (2004b).
The kinetic and metabolic selection theories

Chudoba et al. (1973a,b; 1985) proposed that all the bacteria in activated sludge systems could be clumped
neatly into two inclusively delineated groups, with no apparent overlap between them. The so-called kinetic
selection theory is based on recognizing these two generic groups with hypothesized basic differences in their
kinetic properties. They are;
(i) floc-formers, and

(ii) filamentous bacteria
Making this distinction is very popular among people attempting to model the growth of bulking and
foaming filaments, and is based on assumed differences in their respective competitive abilities in
differently operated activated sludge systems (Chudoba, 1985; Kappeler and Guyer, 1994a,b). These form
the basis for the following theories.
The kinetic selection theory

The assumption here is that all ‘floc formers’ have higher mmax and Ks values for the growth limiting
substrate than all the (slower growing) filamentous bacteria. Consequently, because of their much lower
Ks values, the filaments will out-compete the floc formers best at low substrate concentrations (nutrient
deficient or nutrient limiting conditions) where S<<KS (Wanner, 1993, 1994a,b). Thus, communities
dominated by filaments would be more commonly associated with completely mixed systems, but plug-
flow systems and SBR systems would rarely bulk (see chapter 7).
The storage selection theory

The metabolic/storage selection theory is an extension and refinement of this (Martins et al., 2004b;
Wanner 2002, 2006a), but still relies on the same generalized fundamental differences existing between
the so-called ‘floc formers’ and filamentous bacteria (Martins et al., 2004b). In this theory the ‘floc-
formers’ are hypothesized as being able to store substrates supplied at high levels into the storage
polymers PHA, lipids and glycogen, while the filamentous bacteria can not. Consequently the filaments
would be out-competed in plants where high substrate concentrations are found.
Organic material in mixed liquor is categorized by modellers into two main types (see chapter 2);
. that present in a soluble, readily biodegradable form (RBCOD),

. or the majority occurring as colloidal/particulate material (which can be assimilated by cells only
slowly after its exocellular enzymatic degradation (SBCOD).
Consequently, Kappeler and Gujer, (1994a) thought that filamentous bacteria, because of their physical
extension from the flocs into the bulk liquid (the distinguishing feature of bulking sludge) would have
immediate access to the soluble RBCOD, and hence a competitive advantage over the ‘floc formers’, since
this fraction must first diffuse into the floc. On the other hand, the particulate material, being intimately
associated with the floc and not available to the extended filaments, is assumed to be enzymatically
hydrolysed only within the floc by the ‘floc formers’, which then preferentially metabolize the hydrolysis
products (e.g. Kappeler and Gujer, 1994a; Wanner, 1994a,c). It is accepted, almost as dogma, that all the
filaments must have low apparent Ks values for the readily biodegradable substrates, but high Ks values for
the particulate ones, and that all floc formers show the opposite behaviour (e.g. Ramadori and Tandoi, 1993).
The popular engineering approach to controlling bulking and foaming with selectors (chapters 7, 8) is
largely based on these hypotheses. Such units are designed to provide conditions so that the filaments can
no longer compete with the ‘floc-formers’. Now disadvantaged, they should disappear from the system
(Chudoba and Pujol, 1994; Martins et al., 2004a,b; Wanner, 1994a). However, selectors do not work for
all filaments (Martins et al., 2004a,b), reflecting our ignorance either of their growth kinetics and/or
weaknesses in the theories on which they are based.
The metabolic selection theory

In EBPR activated sludge systems the decisive fraction of RBCOD is utilized not under aerobic, but under
anaerobic and/or anoxic conditions. Thus, populations able to metabolize substrates under these
conditions will be selectively favoured. This is the principle of metabolic selection.
Alternation of anaerobic and aerobic conditions is a prerequisite for EBPR (chapter 10). As filamentous
organisms including S. natans, type 021N and others are considered unable to utilize substrates under such
conditions at a rate comparable to that of ‘floc-formers’, their suppression in EBPR systems is based on
metabolic principles. While S. natans and type 021N are commonly seen in conventional plants, surveys of
EBPR systems in Europe, South Africa, U.S.A. and Australia indicate that these filaments are not favoured
in such plants (Tandoi et al., 2006). Their low competitive abilities are thought to be based on differences in
denitrification rates between them and the floc-formers (Wanner, 1994a).
The NOx inhibition selection theory for low F/M filaments

Casey et al. (1992; 1994; 1999) attempted to explain why certain filaments cause bulking in configurations
with alternating anoxic and oxic conditions operating at low F/M ratios (chapter 2). They worked with
mixed communities where the dominant filament was Candidatus ‘M. parvicella’, but suggested that their
conclusions might apply to all low F/M filaments (Table 5.3). To explain this domination they
hypothesized that the ‘floc-formers’ denitrified in anoxic reactors of low F/M systems to reduce NO3 to
the toxic intermediate NO (they measured NO2 levels and assumed NO was formed from it), which then
inhibited them. On the other hand, because the filaments could only reduce NO3 to NO2 and no further,
they were not similarly affected and so out-competed the floc-formers by using the SBCOD aerobically
i.e. under conditions where little or no RBCOD would be present. Although pure culture studies suggest
that NO2 reduction in Candidatus ‘M. parvicella’ does not occur (Rosetti et al., 2005; Tandoi et al.,
1998b), some in situ FISH-MAR data (Andreasen and Nielsen, 2000) show that NO2 may act as electron
acceptor in this filament.
This hypothesis has since been modified and extended (Tsai et al., 2003) to propose that Candidatus
‘M. parvicella’ control can be achieved by allowing the nitrifying bacteria to remove the NH3 it requires
to grow in the aerobic zone. Again the evidence presented does not take into account information now
available on the in situ physiology of this filament (chapter 7), which suggests other aspects of its
physiology are likely to be more influential in its dominance in low F/M EBPR plants (Andreasen and
Nielsen, 2000; Rossetti et al., 2005).
WHAT EVIDENCE IS THERE TO SUPPORT THESE SELECTION THEORIES?

The obvious question then is which of these theories is correct? Or are several factors responsible for
encouraging the proliferation of filamentous bacteria? The theories assume that all filamentous bacteria
are fundamentally different in their growth kinetics to all non-filamentous ‘floc-formers’. Each hypothesis
is distinguished mainly by the lack of convincing experimental evidence to support the underlying
assumptions. So, several very important questions arise (Martins et al., 2004b; Wanner, 1994a,c).
Firstly, why should organisms growing as filaments always differ so much in their basic kinetic
properties from non-filamentous bacteria (Martins et al., 2004b)? Pipes (1987) thought that bulking
filaments proliferate because their morphology would ensure a higher surface area/volume ratio than for
individual cells, allowing better nutrient uptake especially at low substrate concentrations. However, this
explanation fails to take into account that many bulking bacteria are in fact septate filaments, made up of
chains of individual cells (see chapter 12), where such an advantage should not occur.
Martins et al. (2004b) suggested that filaments are selectively advantaged under substrate limiting
conditions by being able to grow out from flocs into the bulk liquid, a feature of bulking sludge, and thus
access nutrients there. They (2003, 2004b) believe that bulking is induced by substrate gradients
established in the flocs, and filaments gain a competitive advantage at low substrate levels at the floc
surfaces. At high substrate concentrations they can compete without extending from the floc surface, and
hence bulking does not occur. There is some qualified experimental data to support this so-called
‘diffusion limitation theory’ (e.g. Liao et al., 2004a; Lou and de los Reyes 2005a,b, 2008), but no
identification of which substrate/s, presumed to be different for each filament by Martins et al. (2003), are
responsible. Whether floc organization in bulking plants allows such gradients to be established is not
known, but some of the in situ methods we now have available (FISH-MAR etc) may permit these ideas to
be tested experimentally.
Secondly, and relating directly to what has just been discussed, how neatly do these activated sludge
bacteria fit into these location-based categories, and, importantly, do all the filamentous bacteria in the
biomass exist in such different physical and chemical habitats to the floc formers at all times and in all
plants? Microscopic evidence, admittedly based on an incomplete understanding of floc organization,
suggests caution is needed here, and many filaments like Candidatus ‘M. parvicella’ and types 0041/0675
(Wanner, 1993, 1994a) are seen as both freely suspended filaments extending from the flocs and as
integral components of them. This is the case even in sludge considered to be bulking, which based as it is
on settlability tests, is often an arbitrary decision. MAR experiments might reveal whether their capacity
for substrate assimilation depends on their location within the floc. Substrate uptake capacity is thought to
vary between filaments of the same phylotype, but not along each individual septate filament
(Nielsen et al., 2003a), although this statement may not apply to all filaments (Seviour et al., 2006).
Similarly, some ‘floc formers’ must always be close enough to the floc surface to have ready access to
soluble substrates in the bulk liquid, even if present at low concentrations.
So it seems that their chemical and physical microenvironments overlap to a considerable extent. Gujer
and Kappeler (1992) and Kappeler and Guyer (1994b) proposed that some of the foam-forming Mycolata
existing inside and outside flocs should be assigned arbitrarily intermediate Ks values for both particulate
and soluble substrate categories for modelling purposes (see above). Clearly the situation is much more
complex than this, and substantial gradients of a range of environmental factors both physical and
chemical, which may not be measurable by us, may exist among most organisms associated with flocs.
Furthermore, floc structure is probably much more open than was once thought likely (chapter 3),
meaning organisms inside the floc may still have ready access to substrates in the bulk liquid (Li and
Ganczarczyk, 1990), a hypothesis in need of further testing.
Thirdly, how much kinetic data are available for these filamentous (and floc forming) bacteria, and
how do their mmax and Ks values compare? In other words, can differences between their kinetic constants,
if indeed they exist, be used to explain their presence in plants and support these hypotheses? Most kinetic
data come from pure culture studies, and even then only a small number of strains grown on a limited
range of usually simple substrates have been characterised kinetically. Furthermore, the designated ‘floc
formers’ examined in the few experiments reported seem to have been chosen fairly arbitrarily on
morphological criteria alone (i.e. being non-filamentous). So these may not necessarily be those
competing with the filaments in situ, but represent further examples of readily cultured laboratory weeds
(chapters 1, 3). True ‘floc forming’ competitors may be as difficult to isolate into pure culture as many
filamentous bacteria are (chapter 6).
Pure culture kinetic data for some cultured filaments were given in the earlier version of this book
(Seviour and Blackall, 1999), but may not be helpful in addressing this question. It seems improbable that
the mmax and Ks values given for pure cultures would resemble those for the same organisms growing
in situ where multiple substrates are available, and both biotic and abiotic interactions impact upon them.
However, Rosseti et al. (2002) showed little difference between pure culture and in situ kinetic behaviour
of Candidatus ‘M. parvicella’ under their experimental conditions. FISH based methods like those
described by Rosetti et al. (2007) for in situ m determinations with type 1863 should be more generally
applied to other filaments.
What the sparse available pure culture data do suggest is that there is insufficient evidence to support
confidently many of the kinetic arguments put forward above. Thus, differences between filaments and
floc-formers in their values for mmax and Ks, if available, are often small enough to suggest that the
concept of competition between them, based on kinetic selection theory, is difficult to sustain (Wanner,
1994a,c).
Quantitative MAR experiments (Nielsen et al., 2003b) have generated in situ Ks values but with only a
few filaments and substrates. Similar low Ks values for acetate were obtained for both Thiothrix sp and
Meganema perideroedes, data which could be interpreted to support the kinetic selection theory (Nielsen
et al., 2003b). TEFL image analysis was used to generate in situ mmax and Ks data for Candidatus
‘M. parvicella’ identified by FISH and growing on mixed substrates (Rosetti et al., 2002, 2005), and
confirmed that this filament grows slowly in activated sludge. Pure culture studies also obtained a low Ks
value for uptake of combined substrates, as they did for Candidatus ‘Microthrix calida’ (Levantesi et al.,
2006b), again consistent with the kinetic selection theory. However, it is not possible to extrapolate from
data with a few filaments to all, and much more work is needed before any generalizations about their
in situ growth kinetics can be made. Other published data suggest they may not be feasible anyway, since
chemostat experiments with pure cultures of S. natans (Contreras et al., 2000) have yielded kinetic data
inconsistent with kinetic selection theory. In situ kinetic data for these and other selected filament are
presented in Table 5.4.
Fourthly, little convincing experimental data exist to justify claims that filamentous bacteria play little
or no role in the degradation of particulate SBCOD (Salcher et al., 1982). Although Jenkins (1992)
pointed out that the few filaments grown in pure culture seem able to utilise low molecular weight
compounds like sugars and amino acids, they may still possess the necessary metabolic machinery in situ
to utilise complex macromolecules too, and be more nutritionally versatile in situ than this hypothesis
demands. Proteolytic activity is commonplace, especially among epiphytic Saprospiraceae attached to the
sheath of some filamentous bacteria (Xia et al., 2007, 2008b), and may provide their host filaments with
their substrates. Starch degradation seems to be restricted mainly to Actinobacteria (Xia et al., 2008a).
Surface associated in situ lipase activity in Candidatus ‘M. parvicella’ has also been documented (Nielsen
et al., 2002b; Schade and Lemmer 2005), consistent with its preference for long chain fatty acids. Pure
cultures of Candidatus ‘Microthrix calida’ (Levantesi et al., 2006b) also possess lipase and other
enzymatic activity, suggesting it might be more nutritionally versatile than Candidatus ‘M. parvicella’. As
discussed in chapter 3, activated sludge flocs possess high soluble enzymatic activities, including some
capable of degrading larger molecules, and so their products will be available for all cells. More relevant,
in situ enzymatic activity assays with ELF (see chapter 11) suggest that not all filamentous bacteria
possess surface associated enzymes capable of degrading simple soluble molecules. For example, no
ectoenzyme activities were detectable with healthy M. perideroedes filaments (Kragelund et al., 2005) and
only lipase activity was seen with several other alphaproteobacterial filaments (Kragelund et al., 2006).
These results may reflect the limited range of ELF substrates available commercially for in situ work of
this kind. However, existing evidence is probably insufficient to state confidently that filamentous bacteria
are unable to utilize SRBCOD, and more in situ ELF studies are required with other filaments. In any case,
the protozoa with their ability to capture solid particles of food by endocytosis must play an important
role in the utilisation of particulate substrates, and their possible influence on filament behaviour has not
been considered.
Fifthly, one of the central dogmas of the metabolic selection theory is that filamentous bacteria in
activated sludge lack the ability to synthesize and store intracellular carbon energy reserves like PHA
(Chiesa and Irvine, 1985), and hence do not survive starvation conditions as well as the ‘floc-formers’. The
many advantages to bacteria being frugal in times of plenty by storing carbon and energy reserves were
pointed out earlier (chapter 3), but what evidence is there that filaments lack this feature? Many like
Candidatus ‘M. parvicella’ and Candidatus ‘M. calida’ (Levantesi et al., 2006b) under the microscope
contain material with the staining properties of lipophilic material, produced under a wide range
of environmental conditions (Rossetti et al., 2005). PolyP granules have been seen in Candidatus
‘M. parvicella’ (Erhart et al., 1997) and many GALO (Beer et al., 2006; Jenkins et al., 2004b; Soddell 1999).
In M. perideroedes and other alphaproteobacterial filaments, the PHA storage capacity is considerable and
its synthesis is supported by a wide range of substrates (Kragelund et al., 2005, 2006) (Table 5.5). Although
PHA can be detected in pure cultures of Candidatus ‘Nostocoida limicola’, these filaments do not always
contain it in situ (Dionisi et al., 2002a; Liu et al., 2001a; McKenzie et al., 2006; Seviour et al., unpublished),
and some filaments like type 0092 never stain positively for it (Speirs et al., 2009).
Table 5.4. Physiological features of selected filamentous bacteria cultured from activated sludge. Only those organisms which have been
152
‘identified’ reliably are included. Often only single strains of each have been characterized. nr ¼ not recorded; ve ¼ not found
Substrates
Nutritional De- supporting pH Temp DO Ks Storage
Filament Phylogeny category nitrification growth range range– C requirements mmax mgl 1 polymers References
Meganema Alpha- Chemoorgano- ve Vary with strain nr 15–35 Aerobic nr nr PHA, (Levantesi
perideroedes proteobacteria heterotroph but diverse. PolyP et al., 2004;
Utilize sugars, Thomsen
fatty acids amino et al.,
acids and some 2006a)
polymers, and
inorganic
N sources.
Type 1851 ‘Chloroflexi ’ Chemoorgano- ve, Vary with 7–7.5 15–35 Aerobic and Slow nr PHA, (Beer et al.,
Kouleothrix heterotroph, but nitrate isolate but anaerobic growing 2002;
aurantiaca growing reduced diverse range with 0.48–0.93 Kohno,
mainly on to nitrite of sugars fermentative day 1 on 2002;
sugars and in some metabolism GPY Kragelund
able to strains medium et al.,
ferment 2007a)
glucose. Grow
very slowly
anaerobically
‘Nostocoida ‘Chloroflexi ’ Chemoorgano- nr Complex, 6–7.8 nr Aerobic nr nr Variable (Schade
limicola’ heterotroph requirements polyP? et al., 2002)
and growth not
supported by
simple amino
acids, sugars
or organic acids
Candidatus Actinobacteria Chemoorgano- ve but Wide range 6.7–8.0 Wide Microaeroph Depends Very Unknown (Tandoi
‘Microthrix heterotroph nitrate of organic temp ilic on strain low for lipid et al.,
parvicella’ reduced compounds, range and culture COD and material, 1998b;
to nitrite but long chain 7–25 conditions 8 mgl 1 polyP, Rosetti
fatty acids optimum but slow for oleic PHA et al., 2002,
preferred. varies growing, acid 2005)
Inorganic N with strain 0.36–
sources used 0.66 d 1
on mixed
substrates,
in batch
culture, but
1.44 d 1
under
chemostat
conditions
(continued )
Substrates
Filament Phylogeny category nitrification growth range range– C requirements mmax mgl ÿ1 polymers References
Candidatus Actinobacteria Chemoorgano- ÿve, but Specialized Alkaline 15–36.5 Aerobic but Slow Indirect Unknown (Levantesi
‘Microthrix heterotroph nitrate long chain fatty (‡7.8) anaerobic growing, evidence lipid et al.,
calida’ reduced acids preferred, substrate 0.36 dÿ1 suggests material 2006b)
to nitrite but other assimilation a very
substrates low Ks
possibly used
Sphaerotilus Beta- Chemoorgano- ÿve, but Varies with 6.5–7.5 10–40, Aerobic Relatively 4.6 mg PHB (Williams
natans proteobacteria heterotroph nitrate isolate. Diverse opt. rapidly COD l ÿ1 and Unz,
reduced range of 20–30 growing For citrate 1985b;
to nitrite polymers and 0.3 h ÿ1 Contreras
monomers on citrate et al., 2000;
Sugars and Kämpfer
organic acids and Spring,
used, and grow 2005)
best on organic
N sources.
Require
Vitamin B12
Candidatus Actinobacteria Chemoorgano- ÿve, but Grows on few nr 15–35 Aerobic nr nr PolyP? (Blackall
‘Nostocoida heterotroph nitrate sugars and Lipophilic et al., 1999;
limicola’ reduced acetate and granules McKenzie
(Tetrasphaera to nitrite peptone, but et al., 2006)
spp.) not on glucose,
oleate or oleic
acid. Inorganic
N sources
support growth
Haliscomeno- ‘Bacteroidetes’ Chemoorgano- ÿve, but Glucose and 7.0–8.0 8–30 Aerobic 1.2–2.2 d ÿ1 5 mgl ÿ1 Poly- (Mulder
bacter heterotroph nitrate sucrose support opt. for glucose for sacchar- and
hydrossis reduced its growth as do 26 glucose ide Deinema,
to nitrite both inorganic 2006)
and organic
N sources like
glutamate and
aspartate and
casamino
Factors affecting the bulking and foaming filamentous bacteria in activated sludge
acids. Thiamine
and vitamin B12
required
(continued )
153
154
Substrates
Filament Phylogeny category nitrification growth range range– C requirements mmax mgl ÿ1 polymers References
Trichococcus Firmicutes Chemoorgano- ÿve, Range of 5.8–9.0 25–30 anaerobic, nr nr nr (Liu et al.,
flocculiformis heterotroph although substrates opt. mixed acid 2002;
some strains fermented with pH 8 fermentative Scheff et al.,
could reduce acid and gas metabolism 1984)
nitrate production
Mycolata Actinobacteria Chemoorgano- ÿve Varies with Wide Wide Aerobic 2.5 d ÿ1 0.5 mgl ÿ1 PHA, (Goodfel-
G. amarae, heterotrophs isolate. Diverse range range for for polyP low and
S. piniformis range of G. amarae G. amarae Maldonado,
Rhodococcus substrates, but on acetate 2006;
spp. some Blackall
Tsukamurella evidence that et al.,
spp. more hydropho- 1991d)
bic substrates
are preferentially
metabolized in
some strains
Thiothrix sp. Gamma- Metabolically ÿve, but Some strains Wide Wide Aerobic or 2.5 day ÿ1 nr PolyP, (Aruga
strain C3 proteobacteria flexible, with some can use narrow range range microaero- chemo- PHB and et al., 2002;
(Type O21N) some strains reduce range of organic philic organo- sulphur Rossetti
showing nitrate to substrates hetero- granules et al., 2003;
Chemoorgano- nitrite chemoorgano- trophically Unz and
heterotrophic heterotrophic- and Head,
Chemolitho ally, and obtain mixotro- 2005;
autotrophic energy from phically Howarth
and reduced sulphur with acetate et al., 1999;
Mixotrophic compounds 1.8 day ÿ1 Tandoi
metabolism chemolithoauto- Chemo- et al., 1994)
trophically. lithoauto-
Where mixotrophically

trophic with thio-
growth occurs sulphate
cells use reduced (strain C3)
sulphur
compounds as
energy sources,
and a narrow
range of organic
carbon sources
Table 5.5. In Situ Ecophysiology of FISH defined filamentous bacteria in activated sludge communities as determined by MAR, FISH/MAR
and ELF analyses. nr ¼ not recorded
In situ
Ks mgl 1 enzyme Storage Ecological
Filament Substrates Assimilated mmax production polymers strategy Comments References
Aerobic Anoxic NO2 Anoxic NO3 Anaerobic
Meganema In one Only acetate Only acetate, No evidence 1.8 mM No High capacity This filament Relatively (Kragelund
perideroedes European and glucose propionate, for anaerobic for ectoenzyme for PHB seems to lack hydrophobic. et al., 2005;
industrial taken up, glucose and substrate acetate activity production the ability to Filaments Kragelund
plant, wide suggesting galactose assimilation detected, under aerobic utilize polymeric were FISH et al., 2006;
Group 2 range of an ability assimilated, and anoxic substrates, but defined but Nielsen
Alpha- sugars and to denitrify and weaker conditions, has a versatile only from a et al.,
Proteobacterial amino acids than and synthesis capacity to small 2003b)
filaments assimilated. aerobically, was supported assimilate low number
No evidence but by a range molecular weight of plants
for chemolitho- suggestive of substrates substrates. It in Denmark.
autotrophic of denitrifying may denitrify,
growth potential which is an
unusual feature
of filamentous
bacteria in
activated sludge
‘Candidatus’ Restricted No evidence Glucose and No anaerobic nr No All substrates (Kragelund
Alysiosphaera in one for substrate possibly substrate ectoenzyme assimilated et al., 2006)
europea European assimilation acetate assimilation activities aerobically
industrial under these assimilated in Europe, detected supported
plant to conditions in Europe but possibly PHB
Group 2 acetate, but not aspartate production
Alpha- pyruvate, in Australia in Australia. aerobically
Proteobacterial glucose, or Japan and glucose
filaments mannose and anoxically
leucine, but with nitrate.
not propionate No PHB
or oleic acid. seen with
In one nitrite
Japanese
plant, glucose
was also
assimilated
aerobically,
and in one
Australian
plant, glycerol,
aspartate and
glutamate
155
(continued )
156
In situ
Ks mgl ÿ1 enzyme Storage Ecological
Candidatus Restricted Pyruvate Pyruvate No anaerobic nr Only lipase Aerobic PHB Possibly capable (Kragelund
‘Monilibacter in one and acetate and acetate substrate activity production of denitrification et al., 2006)
batavus’ European (weakly) (weakly) assimilation detected, with acetate,
industrial assimilated, assimilated, although propionate
Group 1 plant to but few but few oleic acid and pyruvate,
Alpha- acetate, substrates substrates not but anoxically
Proteobacterial propionate examined examined assimilated only with
filaments and acetate and
pyruvate pyruvate
Candidatus In one Only Pyruvate No anaerobic nr No PHB Possibly capable Inter-plant (Kragelund
‘Alysiomicrobium European pyruvate and acetate substrate ectoenzyme production of denitrification differences et al., 2006)
bavaricum’ industrial and acetate (weakly) assimilation activities supported by in MAR data
plant diverse (weakly) assimilated, detected diverse range
substrates assimilated but relatively of substrates
Group 1 assimilated, few (organic acids
Alpha- including substrates sugars and
Proteobacterial acetate and examined amino acids)
filaments propionate under aerobic
(but not oleic and organic
acid), glucose, acids anoxically
mannose and
leucine. Some
differences
between
phylotypes
Candidatus Restricted to Pyruvate Pyruvate No anaerobic nr No PHB synthesis Possibly capable Inter-plant (Kragelund
‘Sphaeronema acetate, and and acetate and acetate substrate ectoenzyme supported by of denitrification differences et al., 2006)
italicum’ pyruvate. (weakly) (weakly) assimilation activities limited range in MAR data
Oleic acid assimilated, assimilated, detected of organic
assimilated but few but few acids
Group 1 by one substrates substrates aerobically
Alpha- phylotype, examined examined and anoxically
Proteobacterial while the
filaments other took
up propionate
and butyrate
in two
European
industrial plant
(continued )
In situ
Thiothrix sp. In one nr Acetate only Acetate only 2.4 mM nr nr MAR data show Data agree (Nielsen
European taken up in taken up in for it can behave as generally et al., 2000;
industrial plant, presence of presence of acetate a mixotroph with those Nielsen
acetate but not thiosulphate thiosulphate as chemoorgano- from studies et al.,
glucose uptake. or So or So chemo- heterotroph and with pure 2003b)
Increased granules granules, organo- chemolitho- cultures of
acetate and but no hetero- autotroph, under Thiothrix
bicarbonate bicarbonate troph aerobic (see Table
uptake with assimilation. conditions, 7.4), except
thiosulphate; Unclear and grow for
So granules whether anoxically, suggestions
produced with anaerobic demonstrating here of
thiosulphate. growth considerable anaerobic
occurs metabolic metabolic
flexibility, function
enabling it to
grow under a
wide range of
conditions in
activated sludge
Type O21N/ Varied nr nr nr nr nr nr nr identification’ (Nielsen

Thiothrix spp. between of type et al., 1998)
plants, but O21N/
acetate was Thiothrix
taken up used FISH
by all, while and micro-
glucose, scopy with
ethanol, samples from
glycine leucine different
and oleic acid plants, so
were each possible
assimilated filaments
by some from different
taxa were
examined
(continued )
157
158
In situ
‘Chloroflexi ’ Diverse range None None None nr Wide range nr May grow on Only active (Kragelund
of substrates detected detected detected detected in dead or moribund under et al.,
(mainly different bacterial cells, aerobic 2007a)
carbohydrates) phylotypes, since some conditions,
assimilated including can assimilate although
in several esterase, N-acetyl often seen
European glucuroni- glucosamine, in EBPR
plants dase, or polymeric plants.
chitinase, colloidal Filaments are
galactosidase carbohydrates hydrophilic.
activities entrapped in Some
the floc EPS. differences
in MAR data
between pure
cultures and
their in situ
behaviour
which was
less diverse
Candidatus A specialized Oleic acid Oleic acid Evidence 0.66 d ÿ1 Lipase Lipid material Slow growing, In situ (Andreasen
‘M. parvicella’ feeder uptake seen uptake seen that oleic at 20– C activity whose but high substrate nutritional and
restricted acid uptake by TEFL detected chemistry is storage capacity requirements Nielsen,
to long is highest method by ELF unresolved, under a wide seem to 2000;
chain fatty under but filaments range of differ from Rosetti
acids like anaerobic are strongly conditions, those et al., 2005)
oleic acid. conditions. hydrophobic especially reported in
In 4 Probably after MAC anaerobically pure cultures,
European does not assays allows this where a

plants support filament to greater
growth, survive periods diversity of
but is of prolonged substrates
stored in starvation as appear to
a form still occur in EBPR support
unclear. plants, and growth
thus thrive there.
Also grows at low
temperatures and
thus is selectively
favored in cold
climates and
winter over
other filaments
(continued )
In situ
Gordonia Diverse range Glycerol, Glycerol Glycerol, and nr Esterase, polyP and Metabolically Many (Carr et al.,
amarae of both and palmitic and glycine palmitic acid phosphatase PHB diverse, unlike appeared 2006)
hydrophilic acid and assimilated and glycine and production Candidatus to be
and glycine assimilated glucuroni- ‘M. parvicella’ nonviable in
hydrophobic (both weakly) dase, but suggesting their the plants
substrates assimilated no lipase control not examined.
assimilated activity readily Behave
in one plant achievable differently
in Australia in situ to
their axenic
properties,
which
suggest a
strictly
aerobic
metabolism,
and different
substrate
utilization
patterns
Gordonia Acetate, None Acetate, None nr Lipase, PHB Filaments are An improved (Kragelund
amarae propionate detected propionate detected esterase synthesised hydrophobic, permeabili- et al.,
and glucose and glucose and but varies among zation 2007b)
assimilated assimilated, phosphatase filaments by MAC method for
in three but weaker activities assay. Diverse FISH, but
plants in than substrate uptake still many
Europe aerobically pattern, making GALO did
control not respond
problematic to FISH
probes for
G. amarae,
but again
many GALO
did not
respond to
the FISH
probes
(continued )
159
160
In situ
Skermania In two None Same None nr Esterase PHB Hydrophobic Australian (Eales et al.,
piniformis Australian detected substrates detected glucuroni- synthesised filaments but FISH/MAR 2005, 2006;
plants, both in either as dase and variation among data differ Kragelund
hydrophilic Australian assimilated phosphatase individual from that et al.,
and or aerobically, activities, filaments from Europe. 2007b)
hydrophobic European but weaker but no In situ data
substrates studies lipase activity differ from
assimilated, in Australian those
including studies. reported for
glycerol, In Europe pure cultures
glycine, oleic lipase,
acid and phosphatase
palmitic acid, and esterase
but neither activities were
glucose or detected
acetate. In
three plants
in Europe,
only acetate
and propionate,
were
assimilated
Type 0041 Can not take nr Glucose Glucose nr nr nr Not clear In situ (Thomsen
up acetate and assimilated, substrate et al., 2002)
but assimilates galactose but not by assimilation
range of taken up all filaments patterns are
sugars, and consistent
glycine. with this

Variability in phylotype
uptake capacity having simple
for glycine, nutritional
leucine, oleic requirements,
acid and yet it has
galactose, and never been
between 2 isolated in
European axenic culture
plants
(continued )
In situ
Candidatus Neither Uptake Similar to Uptake of nr no exolipase PHA in pure Nutritionally Differences (Seviour
‘N. limicola’ glucose, patterns results for palmitate activity cultures but versatile using between et al., 2006;
glutamate nor vary but NO2 and detected not shown both pure Seviour
Tetrasphaera acetate were weak uptake possibly in situ convincingly hydrophilic cultures et al.,
spp. utilized in two of glycerol, oleate in in situ and and in situ unpub-
Australian aspartate same plant hydrophobic uptake of lished)
plants and palmitate samples substrates with acetate. Blackall
although in biomass different electron et al. (2000)
glycerol and from plants acceptor
aspartate in Japan
were, and
and data for Australia
oleic acid and
palmitate
varied between
plants. In two
Japanese
plants, glucose,
glycerol, oleic
acid, glutamate
and aspartate
were
assimilated
in both but
acetate was not
161
What conditions influence synthesis of PHB or polyP in other filaments in mixed liquors of activated
sludge systems, are not well understood. So their presence or absence by staining should be interpreted
with caution, especially for the purpose of their identification (chapter 6).
THE ECOLOGY OF FILAMENTS IN ACTIVATED SLUDGE; WHAT OTHER

FACTORS MIGHT ALSO DETERMINE THEIR INFLUENCE ON PLANT
PERFORMANCE?
Attempts to construct a single theory to embrace all the filaments to explain why bulking and foaming
occur, are unlikely to succeed. With foaming there is considerable in situ evidence (chapter 8) that the
cells of all the causative organisms are comparatively hydrophobic. So they will preferentially attach to
the air bubbles, be carried to the surface and there stabilize the foam. Considerable debate still exists as to
what cell components are responsible for this CSH. While Mycolata cell walls possess mycolic acids, their
foaming abilities in pure culture do not always correspond to either their mycolic acid composition or
CSH (Stratton et al., 2002, 2003). What determines hydrophobicity in organisms like Candidatus
‘M. parvicella’ is less clear and more generally what operational conditions might affect the CSH of these
foaming bacteria is equally uncertain. It seems highly probable that a filamentous growth form is not
essential for participation in foam formation and stabilization, and many foams contain predominantly
unicellular Actinobacteria (Davenport et al., 2000; Soddell, 1999) and even unicellular Proteobacteria
(de los Reyes et al., 2002; Lemmer et al., 2005). In situ MAC studies suggest that other Gram negative
filaments, not normally considered as foaming bacteria, have a relatively hydrophobic cell surface
(Kragelund et al., 2006), although again the chemical basis for this property is not clear.
Both bulking and foaming result from an excessive proliferation of some bacteria, and so
understanding what affects their growth in situ might provide us with clues on how we might limit it
and hence control these events. Like all organisms, filaments will only grow if the prevailing abiotic and
biotic conditions are appropriate (chapter 1), but much of the relevant data come from pure culture
studies of phylogenetically characterized isolates, and so is restricted inevitably to those we can grow
under laboratory conditions. Some information has derived from following the fate of individual filament
phylotypes in situ, taking advantage of semi-quantitative FISH/image analysis and FISH-MAR. With so
many interacting variables, it is difficult usually to relate directly a single factor to the fate of an individual
filament, and so caution is necessary in interpretation of the discussion that follows.
Tolerance to abiotic factors

Growth rates of all bacteria will be affected by abiotic factors like pH, redox potential, temperature and
pO2 (Schlegel and Jannasch, 2006). However, our information on how most filamentous bacteria respond
to these parameters either in pure culture or in activated sludge systems is scant. Yet such information
may provide valuable clues as to how their growth might be controlled.
Temperature
All cells have a temperature range over which they can grow, and an optimal temperature at which they
grow best (Schegel and Jannasch, 2006). Activated sludge plants inevitably will show marked seasonal
variations in temperature, and mixed liquor temperatures will depend on plant location, being consistently
higher in northern Australia say than in northern Scandinavia. Often filamentous bacteria respond in
a fairly predictable manner to changes. For example, at low temperatures (520– C down to 7– C)
Candidatus ‘M. parvicella’ often predominates, because it grows best at lower temperatures (Rossetti
et al., 2005). On the other hand, Mycolata like G. amarae and S. piniformis appear to be more favoured by
warmer temperatures (Frigon et al., 2006a; Tandoi et al., 2006) than Candidatus ‘M. parvicella’
(Andreasen and Nielsen, 2000; Nielsen et al., 2002b). Reports of the M. parvicella morphotype in plants
operating 420– C (Soddell and Seviour, 1990) may be a case of mistaken identity, and the presence
instead of its look-alike Candidatus ‘M. calida’, able to grow in pure culture at least up to 30– C (Levantesi
et al., 2006). This possibility of mistaken identity is now readily solved by FISH probing. Such seasonal
changes might also be explained in terms of changes in amounts and kinds of fats in the diet (Frigon et al.,
2006a), which can reach high levels (40% of COD) in wastewater (Chipasa and Medrzycka, 2006).
pH
Mixed liquor pH is not always monitored routinely in plants, although it is generally believed that if low
enough (5pH 5), fungal growth is favoured over bacterial (Jenkins et al., 2004b). Little is known about its
selective role in determining filamentous bacterial community composition, or how, for example, pH affects
filament growth rates even in pure culture. Exceptions are with some Mycolata, which grow over fairly
narrow pH ranges axenically, and in one strain of Candidatus ‘M. parvicella’ (Rossetti et al., 2005), which
shows a much broader tolerance, growing equally well between pH 6.7 and 8.0, and possibly even higher.
pO2
Most plants continuously monitor and attempt to control the pO2 level, as O2 is an essential substrate for
both the aerobic chemoorganoheterotrophs and chemolithoautotrophic nitrifying bacteria (chapter 1). It is
also expensive to supply. Some survey data (Table 5.3) suggest that DO levels can influence growth of
some filaments, and anecdotally S. natans, H. hydrossis and Type 1701 appear to be favoured at lower
pO2. In fact FISH based evidence suggests that repetitive stress induced by oxygen deficiency, either alone
or in combination with plant overload can encourage sudden explosions in populations of filaments like H.
hydrossis, Thiothrix and type 021N (Gaval and Pernelle, 2003; Pernelle et al., 2000). Martins et al. (2003)
showed too that type 021N and Thiothrix spp and type 1851 were favoured in SBR systems run at low
pO2. There is convincing evidence that Candidatus ‘M. parvicella’ is a microaerophilic organism (Rossetti
et al., 2005), growing best at low pO2 levels, where it probably has considerable selective advantage.
Thus, it possesses a high capacity for substrate storage under anaerobic and anoxic conditions, which, in
the absence of very much evidence, is considered unusual for filamentous bacteria, where storage seems to
occur mainly under aerobic conditions (Rossetti et al., 2005).
Redox potential
Very few plant operators in our experience monitor the redox potential of their mixed liquors, although its
influence on the metabolism and growth of the bacterial populations is likely to be substantial. Yet how
filamentous bacteria respond to changes in mixed liquor redox levels has never been documented in the
literature. As with pH and pO2, microelectrode techniques to determine redox gradients within the flocs
(see chapter 11) may show they differ to those measurable in bulk plant samples.
Metabolic attributes of filaments

Chemical composition of the influent wastewater and whether it is of domestic or industrial origin may be
important in determining filament community composition (chapter 7). Jenkins et al. (1992) and Wanner
(1994a,c) have suggested how filaments might be ‘classified’ on their nutritional attributes (Tables 5.6 and
5.7), but these schemes were constructed largely on descriptive and anecdotal data using the flawed
Table 5.6. The ‘classification’ of filamentous bacteria (Wanner and Grau, 1989) on the basis of their
metabolic properties.
Group Properties
Group S: Sphaerotilus-like oxic zone growers Sheathed organisms, with strictly aerobic
e.g. Sphaerolitus natans respiratory mode of metabolism. May store
Type 1701 polyphosphate and PHB as cell inclusions, but
Type 0041/0675 only incidentally. Found in wastewaters rich
in organic compounds operating at low
dissolved oxygen levels and short retention times.
Group C: Cyanobacteria-like oxic Non-photosynthetic but also aerobic
zone growers organisms with a strictly respiratory mode of metabolism,
e.g. Thiothrix spp. chemolithoautotrophs with the ability to obtain energy
Type 021N (?) from oxidation of inorganic sulphur compounds.
Group A: All zone growers Facultatively aerobic chemoorganoheterotrophs able
e.g. Candidatus ‘Microthrix parvicella’ to carry out aerobic and anaerobic respiration,
Type 0092 and synthesize cell inclusion bodies as energy stores
‘Nostocoida limicola’
Type 0803
Group F: Foam forming organisms Hydrophobic cells, so they form and stabilize foams.
e.g. Mycolata like May also synthesize surfactants
Gordonia spp.
Tsukamurella spp.
Skermania piniformis
‘‘Nostocoida limicola’’?
Type 0092?
Type 0041/0675?
Acinetobacter spp.
approach of microscopic morphotype identifications. Consequently, these classifications probably will

never predict successfully under which sets of conditions a particular filament will always be found.
Mention was made earlier of the filamentous bacteria identified in plants treating industrial wastes
(Eikelboom, 2006; Eikelboom and Geurkink, 2002), but what determines their distribution there is equally
unclear. Large numbers of interacting influent variables are likely to be involved in deciding the fate of
any filament, which should be borne in mind especially when control strategies are implemented based on
manipulating a single operating parameter only.
Their growth rates will be determined by how well they can generate energy from the substrates
available to them, and how versatile their energy generating systems are in being able to accommodate
changes in plant operating conditions. Thiothrix was selected earlier as an example of a filament with a
highly versatile metabolism, suited to such variations (Nielsen et al., 2000). Most filaments are probably
aerobic chemoorganotrophs, although Trichococcus flocculiformis (Liu et al., 2002) has a heterolactic
fermentative metabolism, and in situ FISH-MAR data for Candidatus ‘N.limicola’ suggested some ability
for both anoxic and anaerobic substrate assimilation (Seviour et al., unpublished). Claims for pure cultures
of (supposedly) a type 0961 isolate (Horan et al., 1988) and the ‘Chloroflexi’ type 1851 (Kohno et al.,
2002) having a fermentative metabolism need confirming in situ with MAR.
Where evidence for autotrophy among the filaments has been sought, results generally have been
negative (Kragelund et al., 2005, 2006; Nielsen et al., 2000). One notable exception is Thiothrix,
(Majone et al., 2007; Nielsen et al., 2000; Tandoi et al., 1994,). We know less about the detailed
ecophysiology of most other filaments, but FISH-MAR has revealed which substrates can be assimilated
Table 5.7. Categorisation of floc forming and filamentous bacteria into groups according to their known and
sometimes hypothesised kinetic and physiological properties (adopted from Jenkins, 1992).
Floc formers Filamentous bacteria
Feature Group A Group B Group A Group B
Floc formers Floc formers Mycolata; Sphaerotilus Candidatus
in stirred plants in plants with natans; ‘Microthrix
with anaerobic aerobic and Haliscomenobacter parvicella’;
selectors anoxic selectors hydrossis; ‘Nostocoida type 0092
limicola’; Thiothrix
spp.; type 1701
mmax medium high medium low
soluble carbon transport rate
aerobic medium high medium low?
anaerobic high zero zero possibly
Ks for:
soluble carbon medium high low low
oxygen medium medium low low
Carbon substrate uptake rate medium high low low
þ
Max. NH4 uptake rate low low high ?
under N limitation
Max P uptake rate under low low high ?
P limitation
Denitrification rate medium high low/zero ?
extent complete complete incomplete incomplete?
Storage capacity
Soluble carbon medium high low high
Anaerobic P high low zero high?
Able to oxidize sulphides medium zero high zero
Growth rate at pH 6 low low high ?
by some under different plant conditions (Table 5.5). Their uptake patterns often vary between and within
each phylotype, making practical exploitation of such data problematic until the extent of this variation
and the reasons for it are better understood. As suggested earlier, this variation may reflect partly the
specificity of some FISH probes used in these studies (Kragelund et al., 2006; 2007b).
More MAR experiments with more filaments are required, but preferably involving an agreed set of
substrates and standardized experimental protocols for all. Only then can sensible comparisons be drawn
between data from different laboratories. Even so the data already generated (Table 5.5) encourage hopes
of developing targeted control strategies for filaments like Candidatus ‘M. parvicella’. This organism
seems to be a ‘specialized’ feeder, in the sense of utilizing only long chain fatty acids like oleic acid for
growth, albeit under a wide range of operational conditions (Rossetti et al., 2005). In this regard it differs
from its closest relative Candidatus ‘M. calida’ and its fellow foaming bacteria G. amarae and
S. piniformis, with both having more catholic tastes, assimilating hydrophilic substrates in situ too
(Carr et al., 2006; Eales et al., 2006; Kragelund et al., 2007b). Others like type 0041 (Thomsen et al.,
2002) and M. perideroedes (Kragelund et al., 2005) also seem to assimilate a diverse range of substrates,
implying that controlling them by manipulating their nutritional requirements is less simple than with
specialist feeders. M. perideroedes (Kragelund et al., 2005; Thomsen et al., 2006a) and several other
alphaproteobacterial filaments (Kragelund et al., 2006) may be unusual among filaments in being able to
denitrify using organic substrates as electron donors (Kragelund et al., 2005). However, it is too early yet
to know if this is really the case, and more strains need examining (chapter 7).
Data obtained with pure cultures of some of the Gram negative bulking filaments (e.g. Kragelund et al.,
2005; Thomsen et al., 2006a,b) and with Candidatus ‘M. parvicella’ (Rossetti et al., 2005) often differ
to those generated from in situ MAR experiments. So, extrapolating from pure culture data to explain
behaviour in activated sludge should be undertaken cautiously. This view is reinforced when G. amarae
and S. piniformis ecophysiology is assessed. Their in situ FISH-MAR data (Carr et al., 2006; Eales et al.,
2006) raise doubts about either being obligate aerobes or preferring hydrophobic substrates, as pure
culture data suggest (Soddell, 1999). In situ substrate assimilation patterns reveal both take up a range of
hydrophilic and hydrophobic substrates under aerobic and anoxic (and possibly) anaerobic conditions.
Whether they can grow on these substrates under all these conditions is unclear.
Implicit selective factors

Most microbial communities are characterised by a level of stability or homeostasis, where sizes of
individual populations remain at reasonably stable levels (Bull 2004), and only when this homeostasis
breaks down will one group of organisms assume dominance. With so many variables, activated sludge
communities probably never achieve true ‘steady state’ or homeostasis, but sudden onset of incidents
of foaming and possibly bulking reflects some loss of it, initiated by factors we still know little about
(e.g. Frigon et al., 2006a).Community homeostasis is generally believed to operate through feedback
control (Bull and Slater, 1982; Little et al., 2008) when populations respond to any changes to dampen
and eventually eliminate them, by each closely interacting with others within the community. Our
understanding of whether such interactions occur in activated sludge systems is poor, but they must exist,
and probably play a crucial role in determining community composition. Few appropriate examples are
available for filamentous bacteria, but several exist for other populations. Such interactions can be either
beneficial or antagonistic (Little et al., 2008).
Beneficial – where both interacting populations benefit from the interaction. These include Mutualism,
which can take many forms, as seen with the syntrophy between the Nitroso-AOB and Nitro-NOB
(chapters 3 and 9). Similar interactions may explain why it is often difficult to culture filamentous
bacteria, and why others only survive briefly after isolation (Levantesi et al., 2004, 2006a; Seviour et al.,
1994; Snaidr et al., 2002).
Antagonistic – where one or often both of the interacting populations suffers. Examples include
competition for any limiting resource (a growth limiting nutrient or energy source), as illustrated above
with the kinetic selection theory. The dominance of Nitrospira and not Nitrobacter cells in many
nitrifying plants is explained (Daims et al., 2001a) in terms of the ability of the former to out-compete the
latter for nitrite as their shared energy source at the low nitrite levels found there (chapter 9). Such
populations whose competitive strategies rely on more than just a fast growth rate (the r-strategists) are
called K-strategists. So organisms able to achieve some competitive advantage over others by avoiding
starvation by storing substrates as PHA etc., or becoming an integral part of the activated sludge floc, both
of which filamentous bacteria do, and forming hydrophobic clusters which are not susceptible to grazing
protozoa, would fall into this category.
If one population inhibits another by producing inhibitory chemicals like acids or antibiotics or by
changing the pH, pO2, etc. to a level outside the tolerance range of its competitors (amensalism), its
chances of survival may increase (Bull, 2004). This ability seems commoner among slower growing
organisms that use it to remove faster growing likely competitors. No filamentous bacterium from
this habitat is yet known which synthesises antimicrobial compounds, with the possible exception of
T. flocculiformis producing lactic acid (Liu et al., 2002). Type 021N has been reported to undergo lysis
from activity of other bacteria (Yaguchi et al., 1991), although why or how this occurs is not understood,
and infective phages could be involved instead (chapter 3). Nitric oxide (NO) is a toxic metabolite
produced by denitrifying bacteria and failure to generate it has been used to explain (Casey et al., 1992,
1994) why filaments like Candidatus ‘M. parvicella’ proliferate, as discussed earlier. Some sublineage
phylotypes of Nitrospira seem to be selectively inhibited at higher NO2 levels than others, ensuring that
each occupies a distinct niche in the nitrifying activated sludge community (Maixner et al., 2006).
There is little doubt that parasitism and predation occur. Thus, bacteriophages capable of lysing
some foaming filamentous bacteria (Thomas et al., 2002), and Bdellovibro strains can both be isolated
readily from activated sludge samples, Protozoa are important bacterial predators (chapter 4), and some
apparently preferentially feed on filamentous bacteria (Inamori et al., 1991; Terashi and Hamada, 1991),
although how commonly is unclear. Whether they and others can affect community composition by
selective grazing seems probable (Cech et al., 1994; Gude, 1979; chapters 3, 4).
6
The current taxonomic status of the
filamentous bacteria found in activated
sludge plants
Robert J. Seviour
INTRODUCTION
To understand better some of the problems we face in our attempts to identify bacteria in activated sludge,
it is helpful first to discuss a few general principles of taxonomy and systematics (Goodfellow, 2000). Do
not be discouraged at this point. We need to know with certainty which organisms are present there before
we can understand this process. Furthermore systematics unquestionably represents an exciting and
rapidly progressing area of microbiology (Stackebrandt, 2006a), as hopefully the material in this and other
chapters demonstrates. This discussion will be brief, and the more interested reader is directed towards the
review articles cited throughout.
Taxonomy consists of three separate sub-disciplines, sometimes called the trinity of taxonomy. They
are all closely interrelated, but intellectually distinct. These are:
1. CLASSIFICATION
Classification describes the exercise of arranging organisms into groups called taxa, on the basis of their
similarities and differences (Stackebrandt, 2006b). Classifications evolve as our ability to obtain additional
information about the objects we wish to arrange (for example the filamentous bacteria) improves. Those
we have for ‘prokaryotic’ and eukaryotic microbes have changed profoundly in the last couple of decades
from the application of molecular characteristics like rRNA and other gene sequences (see chapter 1), and
will continue to. It is important to remember that there is no official classification of bacteria or any other
microbe. All are constructed by microbiologists, and hence their adoption by the scientific community will
depend ultimately on their value and usefulness to those they serve.
The ‘prokaryotic’ species – an artificial concept

In biological classifications, the taxa are organised hierarchically, with the species representing the basic
group or unit (Brenner et al., 2005). How we define what we mean by a species for both eukaryotic and
‘prokaryotic’ organisms has always troubled biologists (Roselló-Mora and Amann, 2001). In particular the
species concept for ‘prokaryotes’, which do not reproduce sexually still remains the subject of
considerable controversy, generating passionate debate (Achtman and Wagner, 2008; Fisher et al., 2006;
Roselló-Mora and Amann, 2001; Roselló-Mora and Kampfer, 2004). The discussion if anything has
increased in its intensity with the availability of molecular information, and evidence for extensive
horizontal gene transfer between populations. Any consensual outcome to its resolution seems unlikely in
the immediate future (Roselló-Mora, 2003). Formal bacterial species descriptions based on only a single
isolate have been criticised (Roselló-Mora, 2006), but delaying publication until several isolates of what
might be a rare strain become available will prevent effective communication among taxonomists. As a
compromise, Felis and Dellaglio (2007) have proposed that all such species should be given the status of
species propenda. However, basing species descriptions on a single isolate, as seen with some of the
Mycolata from activated sludge is helpful as it indicates the extent of the diversity embraced by individual
genera. A standardized format for formally publishing bacterial species descriptions has been suggested to
assist in information transmission (Kämpfer et al., 2003). The reader is directed to the following reviews
for detailed discussions of why defining a bacterial species is so problematic (Cohan, 2002; Fisher et al.,
2006; Gevers et al., 2005; Lan and Reeves, 2000; Roselló-Mora, 2003; Roselló-Mora and Amann, 2001;
Roselló-Mora and López-López, 2008). The impact of promiscuous horizontal gene transfer among
‘prokaryotes’ (Achtman and Wagner, 2008) compounds the difficulty.
Current working definitions acknowledge these difficulties and reflect a certain pragmatism in
their wording. For example, what has now become almost an ‘official’ definition suggested by
Stackebrandt et al. (2002) delineates each ‘prokaryotic’ species as ‘a group of strains characterized by a
certain degree of phenotypic consistency, a significant degree (50–70%) of DNA hybridization and more
than 97% of 16S ribosomal RNA (rRNA) gene sequence identity’. In discussing the suitability of this
definition, they urged the pursuit of reaching a more satisfactory version, since the molecular characters
chosen and the values given for each for delineating individual ‘species’ are somewhat controversial
(Ludwig and Klenk, 2005). Relying entirely on a single and highly conserved 16S rRNA gene (Coenye et al.,
2005; Gevers et al., 2005; Roselló-Mora and Amann, 2001) can present problems (chapter 1). Thus, some
bacteria, including the filament Trichococcus flocculiformis (Liu et al., 2002) share very high (499%) 16S
rRNA similarities with other isolates, but DNA: DNA hybridization values below the stated 70% value.
In fact basing genomic speciation on a 70% hybridization value is seen increasingly as too rigid and
unsustainable (Roselló-Mora, 2006). Many feel it should be more flexible, and whole genome analyses
have revealed large sequence diversity among strains of a single species constructed on this basis
(Coenye et al., 2005; Goris et al., 2007; Roselló-Mora and Amann, 2001), reflecting substantial horizontal
gene transfer activity. Historically this value has also been applied inconsistently, for example for
delineating certain clinically important bacterial species like Shigella spp. from non-pathogenic close
relatives like Escherichia spp. even though the criteria of Stackebrandt et al. (2002) mean they should
The current taxonomic status of the filamentous bacteria found in activated sludge plants 171
belong to the same species (Konstantinidis and Tiedje, 2005a,b). Individually determined hybridization
values are often laboratory specific, as the methods available are both difficult and slow, and they can be
used only with cultured strains. Critically, unlike 16S rRNA sequences, no accumulative DNA:DNA
hybridization data bases exist against which other individual strains may be compared (Gevers et al.,
2005; Goris et al., 2007).
The belief is that a more suitable species definition (see Achtman and Wagner, 2008; Coenye et al.,
2005) will eventually emerge from comparing whole genome sequences, which are now being generated
at an accelerating rate with improvements in DNA sequencing technology (Hall, 2007). These data are not
suitable yet for routine taxonomic purposes, and so other characters more easily acquired experimentally
than DNA:DNA hybridization values and which reflect whole genome sequences, are being sought.
Common sense would suggest that, if only for practical reasons, whatever replaces DNA:DNA
hybridization as the benchmark should agree substantially with the current classifications based largely on
it (Roselló-Mora, 2006). Multilocus sequence analysis/typing (MLSA/T) using core or ‘housekeeping’
genes, has many supporters (Coenye et al., 2005; Cohan, 2002; Gevers et al., 2005; Maiden, 2006),
especially since relationships emerging with it usually do reflect those from whole genome sequence
comparisons. Importantly they generally reveal relationships similar to those achieved with DNA:DNA
hybridization. These core genes encode for proteins not involved in strain adaptation to specific selective
pressures (Cohan, 2002), and criteria for their selection have been recommended (Coenye et al., 2005;
Maiden, 2006). They seem able to recognize and delineate ecotypes, or ecologically distinct clusters of
organisms, and it has been proposed that the best definition ultimately of a bacterial species would
incorporate the concept of ecotypes (Cohan, 2002), an idea receiving increasing support.
Principles of classification
Classifications should satisfy several aims, including summarising and cataloguing information, and
providing a sound basis for identification of new or previously undescribed isolates (Brenner et al., 2005;
Krieg, 2005a,b). To achieve these aims, a classification should;
. have a high information content

. be stable, in being able to accommodate new information without any major restructuring, which
requires that the organisms included should always be as comprehensively characterised as
possible
. be constructed empirically
Unfortunately, most early classifications for bacteria did not satisfy these criteria, and different
characters were often given different importance by individual taxonomists (Roselló-Mora and Amann,
2001), leading to confusion, uncertainty and instability.
Classifications can be of several types (Brenner et al., 2005; Logan, 1994; Sneath, 2005a):
(a) Artificial (monothetic) – are usually designed for a special purpose, including only those
organisms of particular interest (e.g. pathogenic strains). They use key characters which are given
extra weight or taxonomic importance, are not stable and change as more characterization data
become available for the organisms involved. Unfortunately, they are the only ‘classifications’
available for many of the filamentous bacteria we see in activated sludge plants, as discussed later.
(b) Natural or Phenetic – are general purpose classifications, attempting to include all organisms and
all aspects of their physiology, biochemistry, genetics, etc. These are based on the characteristics
an organism currently possesses, and ignore its possible evolutionary past (Sneath, 2005a). Since
as many characters as possible are used, the derived taxa are referred to as polythetic. Variation in
any single character among groups of organisms can be readily accommodated. These polythetic
classifications have high information content, are stable, and consequently have predictive value.
Computers can be used to handle and analyse the large amount of data involved, and this so-called
computer assisted or numerical classification (Sneath, 2005a) has had a considerable impact on
bacterial classification. It has become less popular now, replaced by phylogenetic approaches
(see next).
(c) Natural or Phylogenetic – are based on the evolutionary relationships between organisms, to
reflect their lines of ancestry (Brenner et al., 2005). They have long been constructed for higher
plants and animals, but were once considered unachievable for bacteria (see chapter 1). These
organisms leave few fossil remains, are genetically highly variable, and have a simple
morphology. This makes it almost impossible to use such characters to distinguish between those
showing true evolutionary homology or likeness, and those arising from processes of convergent
evolution. 16S rRNA gene sequences have provided us with the framework for a phylogenetic
classification of ‘prokaryotes’ (Ludwig and Klenk, 2005) (chapter 1). Because of its highly
conserved nature, many now argue that 16S rRNA gene sequences should be replaced by genes
evolving more rapidly and other genes, including those encoding aminoacyl-tRNA synthetases,
rpoC1, RNA polymerases, recA and gyrB proteins and several others, have been assessed as
alternative phylogenetic markers (Woese et al., 2000). Generally they produce outcomes very
similar to those using 16S rRNA, but not always, probably as a consequence of horizontal gene
transfer (chapter 1) between populations (Ludwig and Klenk., 2005; Purkall, 2006).
2. NOMENCLATURE
There is only ONE official system for bacterial nomenclature, regulated by an internationally agreed set of
strict rules, or a code of nomenclature (Sneath, 2005b; Trüper, 2005a,b). Proper names (Trüper, 2005b)
are essential to permit effective communication between bacteriologists in different laboratories. Because
of early confusion in naming bacteria, a new starting date of January 1st, 1980 was chosen for the future
recognition and authorisation of valid bacterial names (Skerman et al., 1989; Sneath, 2005b). Latinised
systems following code rules must be used, with distinctive names given for each taxon within the
hierarchy. These names must also be stable, unambiguous, and easy to pronounce (Tindall et al., 2006;
Trüper, 2005a,b). The reader is referred to Trüper (2005a,b) for further information on how the code
operates, and how names should be constructed. New names, which should describe some distinctive
feature of the organism (Trüper, 2005a,b), and ideally reflect its phylogeny (Dellaglio et al., 2004) are
only considered valid when its description has been published (Tindall et al., 2006), or special reference to
it made in the International Journal of Systematic and Evolutionary Microbiology (IJSEM). For an
uncultured organism, where a 16S rRNA sequence but little else might be available, or an isolate for
which insufficient characterization is possible for valid speciation according to the code rules, the prefix
‘Candidatus’ is used (e.g. Candidatus ‘Microthrix parvicella’). This denotes its provisional taxonomic
status, without placing it definitively into any recognized taxon (Murray and Stackebrandt, 1995). Several
filamentous bacteria have this status, as detailed elsewhere in the chapter.
3. IDENTIFICATION
This procedure is different intellectually to that of classification, although they are often confused, since
a sound system of identification requires a sound classification, where the individual taxa are
comprehensively described. Identification is an exercise in comparison, where an unknown is compared to
known isolates, and so identification schemes can only be constructed after such isolates have been
properly classified (Krieg, 2005a), a requirement which in turn demands that the unknown is appropriately
characterized (Trüper and Schleifer, 2006). Several simple rules exist for the process of identification of
an unknown, and these have been outlined by Kreig (2005a):
(a) A pure culture of the unknown is required (but not for Candidatus status).
(b) The characters selected should be few in number and easily determined, a rule not necessarily
applying to characters used for classification. Many of importance are not always obtainable from
the filamentous and other bacteria in activated sludge, since they usually grow (if at all) poorly in
pure culture (see later).
(c) Standardised and if possible, cheap and reliable test methods should always be employed, so that
inter-laboratory comparisons are possible, and outcomes should be rapidly achieved.
(d) Comparison with a type or reference strain of the suspected known organism is needed for reliable
identification. If available, these are held by culture collections around the world. Reference to
classifications like those in Bergey’s Manual of Systematic Bacteriology or The ‘Prokaryotes’
(Dworkin, 2006) also helps in identifying isolates.
APPROACHES TO IDENTIFICATION
A number of approaches to bacterial identification can be taken, some of which do not fit the rules given
above (Krieg, 2005a). These include:
1. The blunderbuss approach, where as many characters as possible are determined, which can be
time consuming and expensive, but generates enough data to give a high chance of correct
identification using, say, Bergey’s Manual. This approach is not suited to a busy diagnostic
laboratory or one with few resources, or where extensive characterisation is not feasible for other
reasons, as with many activated sludge filamentous bacteria.
2. A step wise or deductive/intuitive approach, with diagnostic schemes, where the microbiologist,
usually experienced, thinks he/she knows the answer, and then chooses the appropriate tests
accordingly to pursue it.
3. Dichotomous keys, where one proceeds along progressively branched routes, selected on whether
the test result for the previous character is positive or negative. A single wrong decision leads
down the wrong branch, to the inevitable wrong identification. These have been devised for the
filamentous bacteria in activated sludge, but all suffer from this inherent weakness (Jenkins et al.,
2004b).
4. A simultaneous approach, which uses computer derived identification systems. These overcome
one of the major weaknesses of dichotomous keys, because non-uniformity of test results with
collections of strains can be readily accommodated. An identification matrix is generated where
each test or character is expressed as an estimate of the probability of a positive response for each
organism. These systems have been commercialised into rapid kits, which allow for the
identification of many bacteria, but are only as good as the data bases from which the probabilities
are calculated. They include the API, APIzyme and Biolog systems, all of which have been used,
if not always successfully, to identify foaming filamentous bacteria (Mycolata) and other activated
sludge populations (Soddell et al., 1992). They do not permit the identification of most other
filamentous bacteria, but have contributed towards phenoypic characterization for resolving their
taxonomic status (Liu et al., 2000; Soddell and Seviour, 1994).
CHARACTERS USED IN THE CLASSIFICATION AND IDENTIFICATION

OF BACTERIA
It is generally accepted that the best approach to classification is to use phenotypic and genotypic
(phylogenetic) characters together, to reflect as much as possible of the organism’s biological properties.
This philosophy is called Polyphasic Taxonomy (Gillis et al., 2005; Roselló-Mora and Amann, 2001;
Vandamme et al., 1996). Depending on the purpose, choice of characters is still important, since not all
will reveal phylogenetic relationships or reflect similarities in whole genome sequences between strains.
Furthermore, some are more suitable for identification than classification, which also requires more
extensive strain characterization. In higher plants and animals, morphological characters allowed the early
construction of sound phylogenetic classifications, although many of these are changing too with
molecular data. Unfortunately, as mentioned previously, morphology with ‘prokaryotes’ is not useful
because it varies so little among them, and is a notoriously unreliable indicator of their phylogenetic
relatedness. In other words, because organisms look alike under the microscope does not necessarily mean
that they are closely related phylogenetically, a comment which applies to many of the filamentous
bacteria discussed in this book.
We would expect closely related organisms to share a high level of similarity in their whole genome
sequences (Coenye et al., 2005). No such sequence data are available yet for any activated sludge
filamentous bacterium, but the whole genomes of the PAO Candidatus ‘Accumulibacter phosphatis’
(chapter 10) and Nitrosomonas europea and Nitrospira defluvii (chapter 9) have been sequenced.
A more rapid but indirect indication of DNA relatedness between strains can be obtained from a range
of DNA typing/fingerprinting methods (Krieg, 2005a; Purkall, 2006; Vandamme et al., 1996), which are
briefly outlined below.
GENOTYPIC CHARACTERS
Whole genome sequence data have already impacted on ‘prokaryotic’ systematics (Konstantinidis and
Tiedje, 2005b), and their importance will continue to increase as sequencing and sequence analyses
become quicker and cheaper (Coenye et al., 2005). This approach is not (yet) suitable for routine strain
characterization and identification. However, the value of 16S rRNA gene sequence data for the design
and application of targeted fluorescently tagged oligonucleotides and their in situ identification by FISH
(Amann and Ludwig, 2000; Amann and Schleifer, 2005) is clear (chapter 3), and their impact on our
understanding of the systematics of the activated sludge filamentous bacteria will be described later.
In vitro DNA/DNA hybridization is an indirect way of comparing genomic sequences between strains,
and its limitations and value in defining genomic species (Roselló-Mora, 2006) were discussed above.
Several other methods, including many based on PCR amplification of a designated region of the genome
(e.g. the intergenic spacer region, ISR in the rrn operon) are available for fingerprinting ‘prokaryotes’ for
their classification and identification. These approaches, including ribotyping, REP-PCR and RAPD-PCR
are critically discussed by Gürtler and Mayall (2001) and Purkall (2006). Any differences in the patterns
obtained usually after the amplified DNA fragment is digested with suitable restriction enzymes into
smaller fragments before separation by gel electrophoresis, will generally reflect differences in their
genomic DNA sequences. These approaches are generally more useful for resolving interspecies
relationships.
Indirect comparisons between genomes can also be made on the basis of their DNA base ratios
(expressed as the Guanine þ Cytosine [G þ C] mol% value), which vary between about 25–80 mol%
among ‘prokaryotes’. It will tell us if two organisms are different (45% difference between two strains
means they probably belong to different species), but not necessarily whether they are closely related, and
is not based directly on DNA sequence comparisons. However, this character is a required piece of
information in any description of a new bacterial genus. The reader is referred to Goodfellow and
O’Donnell (1993, 1994), Roselló-Mora and Amann (2001), Stackebrandt and Goodfellow (1991) and
Vandamme et al. (1996) for further details and taxonomic applications of these different techniques.
PHENOTYPIC CHARACTERS
These were the only ones available before the advent of genomically based information, and all required
pure cultures of the strains of interest (Roselló-Mora and Amann, 2001). The so-called ‘classical’
phenotypic characters do not reveal phylogenetic relationships between strains, a problem which
exasperated early taxonomists. Some biochemical properties may be plasmid determined and therefore
readily lost or gained from the host cell, making them taxonomically unreliable (Harwood, 1993).
However, phenotypic attributes are still important contributors to bacterial strain identification and in
reaching descriptions of novel organisms in polyphasic studies (Roselló-Mora and Amann, 2001;
Vandamme et al., 1996). They include morphological, biochemical and physiological attributes. Most
require strictly standardized methodologies for their determination, yet inter laboratory comparisons have
often revealed considerable differences in the test results from the same bacteria (Sackin and Jones, 1993;
Vandamme et al., 1996). However, each only reflects a partial expression of the total genomic information
of an organism, requiring that large numbers of them are used.
With advances in biochemical analytical methodology, similarities or differences in the chemical
composition of certain cell components have been used successfully for both classifying and
identifying bacteria (Goodfellow and O’Donnell, 1993, 1994). Employing such characters, which may
depend on the conditions under which strains are grown, is called ‘chemotaxonomy’, and has had a
considerable impact on our understanding of relationships for example, among the Actinobacteria
(Roselló-Mora and Amann, 2001). In particular, comparisons of cell wall and lipid (menaquinone
structure) composition of the mycolic acid containing Mycolata, found in large numbers in activated
sludge foams, has changed radically our understanding of their taxonomy (Goodfellow and Maldonado,
2006), as discussed in chapter 8. Electrophoretic patterns of whole cell or cell envelope proteins, the
products of transcription and translation, were once popular characters for examining relationships
between closely related strains (Kersters et al., 1994), but have largely been replaced by the genotypic
approaches mentioned above.
SO HOW DO WE CLASSIFY AND IDENTIFY THE FILAMENTOUS

BACTERIA?
We might begin now to see how difficult it is to properly classify and identify the activated sludge
filamentous bacteria, when many of the methods used in their study conform to so few of the taxonomic
principles discussed above. Some of these difficulties need to be considered more thoroughly.
LACK OF AVAILABILITY OF PURE CULTURES

Relatively few of the filamentous bacteria in activated sludge have been grown axenically, and then only from
a small number of treatment plants around the world. Examples of some whose true identity we now know are
listed in Tables 6.1 and 7.1. Our limited success in increasing their number since the earlier version of this
book was published might raise doubts if we will ever manage to grow all of them. It is frustrating and slow
work of the kind universities were once willing to support, but in the current climate such long term
investigations with uncertain outcomes are less likely to attract research funding. Results from the EC funded
MACOBS and Dynafilm project directed at culturing and identifying industrial bulking filaments illustrate
how hard-won success is (Levantesi et al., 2004, 2006 a,b). Of course, in the absence of information on what
their detailed nutritional requirements are (Liu et al., 2001a), this culturing problem is not easily overcome.
Organisms closely related phylogenetically do not always share similar phenotypes (Achenbach and Coates,
2000), as seen for example, with the bulking filamentous bacterium type 1851 (Beer et al., 2002). Its closest
phylogenetic relative (but only 84%) is Roseiflexus castenholtzii a thermotolerant, carotenoid producing
gliding bacterium, a phenotype not shared with type 1851. Hopes that in situ MAR data (chapters 3, 7, 11)
may provide valuable clues as to which substrate/s to incorporate into culture media may disappoint if the
experiences with type 0041 are any indication. As mentioned in chapter 7, this morphotype appears to have
very simple nutritional requirements for members of a phylum (the TM7 phylum), from which not a single
cultured representative exists (Thomsen et al., 2002).
Some media successfully used in one attempt to grow a filament morphotype often fail in another. For
example, the chemically defined medium Slijkhuis (1983) designed for Candidatus ‘Microthrix
parvicella’ has never led since to its subsequent successful isolation and growth (Rossetti et al., 2005),
which might reflect substantial physiological variations exist among individual strains. Pure cultures of
the GALO Mycolata show large physiological differences, despite them all having a similar
morphological appearance in mixed liquor (Goodfellow and Maldonado, 2006; Soddell et al., 1992,
1999). With Skermania piniformis, the opposite seems to be the case (Eales et al., 2006; Soddell and
Seviour, 1994, 1998). Pure cultures of organisms, all identified on morphological features as Zoogloea
ramigera are clearly phylogenetically diverse (Shin et al., 1993), as are those of type 021N (Aruga et al.,
2002; Howarth et al., 1999; Kanagawa et al., 2000), type 1863 (Seviour et al., 1997) and H. hydrossis
(Kragelund et al., 2008). Isolates of the Nostocoida limicola II morphotype belong to several quite
different phylogenetic lineages (Blackall et al., 2000; Levantesi et al., 2004; McKenzie et al., 2006;
Schade et al., 2002). Not only do these observations reinforce the earlier stated view that morphology in
bacteria is an unreliable indicator of their true relatedness (Woese, 1987), but also raise important
questions about the microscopic methods used to identify them (see later).
It is unproductive to systematically empirically screen media (Kämpfer et al., 1995) for those capable of
supporting the growth of filamentous bacteria. Ways of increasing our capacity to improve their recovery rate
from activated sludge samples are not self evident, and novel methods will probably be required. Already
these have had some success. Thus, Liu et al. (2001a) successfully grew several isolates of novel filamentous
Planctomycetes (Nostocoida limicola III?) on medium made with clarified final plant supernatant, and
D-arabinose as carbon source, selected after screening an enriched harvest of micromanipulated filaments
with BiologT. Growing pure cultures of sheathed filaments like types 1701/ S. natans or 0041/0675 is further
complicated by attached cells of phylogenetically diverse organisms. These may include Caulobacter
(MacRae and Smit, 1991; Winkler and Cox, 1980) and Paenibacillus (Takeda et al., 2002), but many are
probably novel members of the Saprospiraceae in the Bacteroidetes (Kragelund et al., 2008; Thomsen et al.,
2002; Xia et al., 2007, 2008b). However, micromanipulation of filament regions free of these led to successful
isolation of type 1851 (Beer et al., 2002). Their presence may be fitness- or age-dependent, meaning their
value as stable identificatory characters is questionable (Wanner, 1994d).
Table 6.1. Examples of filamentous bacteria isolated into pure culture from activated sludge including those whose 16S rRNA genes have
been sequenced, and the extent of their characterisation [Mycolata not included].
Organism Isolation medium Isolation method Extent of characterisation Comments References
Meganema GS or MSV Micromanipulation 16S rRNA sequence data Nine isolates from different Levantesi et al. (2004,
perideroides mineral medium and extensive phenotypic plants all sharing high 16S 2006a) (Europe)
plus acetate plus characterization rRNA sequence similarities
Alpha- vitamins but differing markedly in
proteobacteria their phenotypic properties. Thomsen et al. (2006a)
Validly named (Europe)
Sphaerotilus CGY Streak plating Biochemical and 6 isolates Richard et al. (1982)
natans physiological all similar (US)
characterisation used in
Beta- S, SS, GS Streak plating all these studies, but tests 4 isolates Williams and Unz
Proteobacteria differed. Organisms all similar (1985b) (US)
considered easy to culture.
I, GS, ACS Streak plating Validly named. 16S rRNA 46 isolates but did not Ziegler et al. (1990)
sequence data and FISH distinguish from Type 1701 (Germany)
probe now described
Type 1701 CGY Streak plating Physiology and 10 isolates Richard et al. (1982)
biochemistry all similar (US)
Beta- Physiology and
proteobacteria SS, CGY Streak plating biochemistry 6 isolates Williams and Unz
Nutritional studies all similar (1985b) (US)
RS ? 24 strains Kämpfer et al. (1995)
(Germany)
Type 0803 R2A Micromanipulation 16S rRNA sequence and Single isolate, but different Bradford et al. (1996)
some phenotypic data phenotype to above (Australia)
Beta-
proteobacteria
Type 1863 R2A Micromanipulation 16S rRNA sequence data Single isolate identified as Rossetti et al. (1997b)
Acintobacter johnsonii (Italy)
5 isolates
Gamma R2A Micromanipulation 16S rRNA sequence data 2 were Cytophaga spp. Seviour et al. (1997)
proteobacteria 2 were Acinetobacter (Australia)
johnsonii
1 was Moraxella sp.
The current taxonomic status of the filamentous bacteria found in activated sludge plants
(continued )
177
178

Thiothrix spp. GS Streak plating Biochemistry and physiology 3 isolates, variable Richard et al. (1982)
phenotype (US)
Gamma- L, GS, LT, MSV Streak plating Biochemistry and physiology 4 isolates, variable Williams and Unz
proteobacteria phenotype (1985a; 1989) (US)
BTA Micromanipulation Biochemistry and physiology strain CT3, possibly a new Tandoi et al. (1994)
Thiothrix species? Rosetti et al. (2003)
(Italy)
16S rRNA sequence data
Type 021N GS Streak plating ? 10 isolates variable Richard et al. (1982)

phenotype (US)
Members of the GS, ASC, LT Streak plating Biochemical and 13 isolates variable Williams and Unz
genus Thiothrix, physiological tests used phenotype (1985a) (US)
now known to I, GS, ACS, LT Streak plating for this filament in these 29 isolates, variable Ziegler et al. (1990)
represent several different studies phenotype (Germany)
different species I,GS,ACS,LT Streak plating Nutritional studies 15 isolates Kampfer et al. (1995)
I, GS, EGG Micromanipulation 16S rRNA sequence data 6 isolates of Thiothrix and Hudson et al. (1994)
8 isolates of type 021N, (Australia, Japan);
and description of 4 new Howarth et al. (1998,
Thiothrix spp. 1999) (Australia, US,
Japan)
MSV ? 16S rRNA sequence data 4 isolates Wagner et al. (1994c)
(Germany)
EGGC, EGGCH Streak plating 16S rRNA sequence data 15 isolates of type 021N, and Kanagawa et al. (2000)
two new species recognized Aruga et al. (2002)
(Japan)
‘Nostocoida HA Streak plating 16S rRNA sequence data Novel organism; ‘N. limicola’ II Schade et al. (2002)
limicola’ and phenotypic morphotype; FISH probe (Europe)

characterization available
‘Chloroflexi’
Type 1851 R2A Micromanipulation 16S rRNA sequence data Single isolate from plant Beer et al. (2002)
(Kouleothrix and limited phenotypic treating domestic wastewater (Australia)
aurantiaca) characterization
‘Chloroflexi’ IA plus activated Micromanipulation 16S rRNA sequence data Five isolates from industrial Kohno et al. (2002)
sludge extract plus phenotypic wastewater (Japan)
characterization
(continued )
MSV plus acetate Micromanipulation 16S rRNA sequence data Three isolates from industrial Kragelund et al. (2007a)
plus vitamins or wastewater treatment plants (Europe)
R2A
Trichococcus UA Streak plating Chemotaxonomy Several isolates from 5 plants Scheff et al. (1984)
flocculiformis biochemistry and in Germany. Not reported (Germany)
(Nostocoida physiology elsewhere
limicola I?)
Firmicutes R2A Micromanipulation 16S rRNA sequence data Variable morphology, often Liu et al. (2000, 2002)
and extensive genotypic and growing as unicells or swollen (Australia)
phenotypic data. Valid name cells in pairs
Candidatus SM Streak Plating Extensive physiology and Isolation medium never used Slijkhuis (1983)
‘Microthrix biochemistry successfully elsewhere (Holland)
parvicella’
Actinobacteria R2A, NTM Micromanipulation 16S rRNA sequence data Single isolate Blackall et al. (1994b)
obtained (Australia)
R2A Micromanipulation 16S rRNA sequence data Single isolate, but the only Rosetti et al. (1997,
very similar to the Australian one well characterized 2005) (Italy)
isolate kinetically
Candidatus Strain dependent Micromanipulation 16S rRNA sequence Several isolates from industrial Levantesi et al. (2006b)
‘Microthrix calida’ but MSV mineral data and phenotypic plants. Media supporting (Europe)
base plus acetate, characterization growth different to that
Actinobacteria thiosulphate and/ successful with Candidatus
or bicarbonate ‘Microthrix parvicella’
and /or high pH (8)
Candidatus R2A Micromanipulation 16S rRNA sequence Now shown to represent Blackall et al. (2000)
‘Nostocoida data and phenotypic several species in the genus (Australia and Italy)
limicola’ data obtained for Tetrasphaera, and so in future
several strains should be referred to by its
Actinobacteria valid names. FISH probes McKenzie et al. (2006)
available (Australia)
(continued )
179
180

Isosphaera spp R2A Micromanipulation 16S rRNA sequence data These probably represent Liu et al. (2001a)
(Nostocoida after screening from five isolates all similar, different and novel species (Australia)
limicola III????) with Biolog, and but little phenotypic data of Isosphaera, whose
then growth with phenotype they do not share
Planctomy- D-arabinose as
cetales sole carbon source
Abbreviations:
I, SCY and D media described by van Veen (1973).
GS, ACS, SS and S media described by Richard et al. (1982).
L, LT and MSV media described by Williams and Unz (1985a).
CGY medium described by Dondero et al. (1961).
UA medium described by Scheff et al. (1984).
SM medium described by Slijkhuis (1983).
TSGA medium described by Nowak and Brown (1990).
R2A medium described by Reasoner and Geldreich (1982).
RS medium described by Wagner et al. (1994c).
BTA medium described by Tandoi et al. (1994).
ECG medium described by Hudson et al. (1994).
EGGC, EGGCH media described by Kanagawa et al. (2000) and Aruga et al. (2002).
Although some filamentous bacteria reportedly have been obtained simply by streaking biomass samples
onto solid medium (Kämpfer et al., 1995; Kämpfer and Wagner, 2002), their subsequent morphotype
‘identification’ should be viewed as equivocal, especially in the case of those never grown since with the
same media. Plating out cells from homogenised samples often makes it impossible to relate the filaments
seen in the sample under the microscope to those eventually growing in pure culture. Micromanipulation
(chapter 3) is the preferred method (Blackall et al., 1991a; Levantesi et al., 2004; Liu et al., 2001a; Soddell
and Seviour, 1990) since particular filaments can be recognised on the basis of their morphology by direct
microscopic examination prior to isolation. This technique is described in chapter 11.
CHARACTERIZATION OF FILAMENTS
With the non-culturable filaments we were once restricted by necessity to characters only obtainable from
microscopic examination of biomass samples. This has changed. Construction of rRNA based clone
libraries and subsequent design of 16S rRNA or 23S rRNA targeted FISH probes (see chapters 3, 7, 11,
12) has allowed resolution of their phylogeny and subsequent in situ identification. However, many people
in the industry still rely on microscopy for their routine identification, since FISH probes are still not
available for all the filament morphotypes (Eikelboom, 2006; Jenkins et al., 2004b). The microscopic
characters used in ‘identification’ (Eikelboom, 2000; Jenkins et al., 2004b) are listed in Tables 6.2 and 6.3.
As already mentioned, some attributes like PHB and polyP inclusion bodies and attached particles may
only be seen in cells growing under certain culture conditions (Wanner, 1994d), and there is often no easy
way of knowing what these conditions are or how they might change within or between different treatment
plants. The popular filament identification manuals often list different attributes for the same filament
morphotype (Eikelboom, 2006; Jenkins et al., 2004b)
Table 6.2. Microscopic and morphological characters used in identifying filamentous bacteria in activated
sludge.
Cells
Dimensions length and diameter
Shape rods, cocci, discs
Septa presence or absence
Inclusions presence of polyphosphate, poly-b-hydroxyalkanoates or sulphur granules
Motility by gliding or possession of flagella
Trichomes
Dimensions length and diameter
Shape straight, bent or coiled
Location within floc or free in bulk liquid
Attached growth presence or absence of attached bacterial cells
Branching true or false branching
Sheath presence or absence
Staining Reactions
Gram Stain positive (purple) or negative (red)
Neisser Stain positive (mauve) or negative (brown)
Even with the filaments successfully cultured, rarely has sufficient characterization data been obtained
to allow their valid naming following the code rules. Exceptions are listed in Tables 6.1 and 7.1. Certainly
their slow growth rates on laboratory media present practical difficulties, and cultured filaments often
Table 6.3. Summary of typical morphological and staining characteristics of examples of the filamentous organisms commonly observed in activated
sludge (Modified extensively from Jenkins et al. 2004b).
Bright field Phase contrast
Neisser Inclusions Filament Septa Cell shape
Gram stain Sulphur Diam. Length Loca- Indenta- Attached and size
Filament stain A B A B Other mm mm Shape tion Seen tions Sheath growth (mm) Features

Meganema V ve ve ve ve PHB, 1.5–2.2 5500 C, F þve þve ve ve Irregular, disc Similar
perideroides B shaped cells microscopically
to ‘N. limicola’ II

Candidatus V ve þve ve ve PHB, 1.5–2.0 200 C, F þve þve ve ve Irregular, disc Similar
‘Alysiosphaera polyP B shaped cells microscopically
europea to ‘N.limicola’ II

Candidatus ve ve þve ve ve PHB, 0.8–1.4 200 C F þve þve ve ve Irregular, disc Similar
‘Sphaeronema polyP shaped cells microscopically
italicum’ to ‘N.limicola’ II

Candididatus ve ve ve ve ve PHB c.a 1.5 200 C F þve þve ve ve Irregular, disc Similar
‘Alysio- shaped cells microscopically
microbium to ‘N.limicola’ II
bavaricum’

Candidatus V ve þve ve ve PHB, 1.3–2.0 5200 C F þve þve ve ve Irregular, disc Similar
‘Monilibacter polyP shaped cells microscopically
batavus’ to ‘N.limicola’ II

S. natans ve ve ve ve ve PHB 1.0–1.4 5500 ST E þve þve þve ve Round-ended False
cells branching,
1.4 £ 2.0 Sheath,
Gonidia
Type 1701 ve ve ve ve ve PHB 0.6–0.8 20–80 ST, I, E þve þve þve þþ Round-ended Similar to
B cells S. natans Cell
0.8 £ 1.2 septa harder
to discern
Type 0803 ve ve ve ve ve ve 0.8 50–150 ST E, F þve ve ve ve Rectangular
cells
0.8 £ 1.5

Type 1851 weakly ve ve ve ve PHB 0.5–1.0 100–300 ST, E þ/ve ve þve /þve Rectangular Trichomes
þve, V SC cells in bundles
0.5 £ 1.0

Type O21N ve ve / ve þve PHB 1.0–2.0 50–1000 ST, E þve þve ve ve Barrels, Rosettes, gonidia
þve SC rectangles,
discoid cells
1.0–2.0 £
1.5–2.0
(continued )
Thiothrix I / ve / þ/ þve PHB 1.4–2.5 100 ST, E þve ve þve ve Rectangular Rosettes, gonidia
þve þve ve 4500 SC cells
2.0–3-5.0
Thiothrix II ve ve / þ/ þve PHB 0.8–1.4 50–200 ST, E þve ve þve ve Rectangular So granules
þve ve SC cells
1.0 £ 1.5
Beggiatoa / ve / þ/ þve PHB 1.2–3.0 100- ST F /þve ve ve ve Rectangular Gliding motility
spp. þve þve ve 4500 cells and flexing
2.0–6.0

Trichococcus þve ve ve ve ve Not 1.0–1.5 V, ST, E, F þve þve ve ve 1.0–1.5 £ Coccoid cells
flocculiformis reported can be SC 1.0–1.5 in long chains,
[‘N. limicola’ I ?] 5100 sometimes
forming knots

Candidatus þve ve þve ve ve Lipid, 0.3–0.85 50–200 C I ve ve ve ve Individual Unbranched,
‘M. parvicella’ polyP cells not spaghetti-like
readily seen tangled filaments,
often uneven
in appearance
from ‘spherical
bodies’

Candidatus þve ve ve Lipid 0.3–0.7 nr C I ve ve ve ve Individual Like M. parvicella,
‘M. calida’ cells not but possibly
readily seen thinner and
‘spherical bodies’
common.

‘N. limicola’ þ/ve, / V ve / PHB? 1.0–1.4 4100– C, B I, E þve þve ve ve Discoid, Gram and
Known to V þve, þve 200 flattened cells Neisser variable
describe several V and coiled-bent
different filament
phylotypes, appearance
which
vary micros-
copically

G. amarae þve ve þve ve ve PHB 1.0 5–30 I I, F þve ve ve ve Variable Right-angled
Other GALO PolyP branched
will vary filaments, but may
fragment
(continued )

S. piniformis þve ve þve ve ve PHB, V V I I, F þve ve ve ve Variable Acute angled
PolyP branching of
filaments, although
branching patterns
vary considerably
in situ

Type 0041 þve, V ve / ve ve ve 1.4–1.6 100–500 ST, I, E þve ve þve þþ/ Squares Neisser þve
þve SC ve reaction can occur
Type 0675 þve, V ve / ve ve ve 0.8–1.0 50–150 ST, I, E þve ve þve þþ/ Squares Neisser þve
þve SC ve reaction can occur

Isosphaera V þve ve ve ve PHB? 2.0 200–300 C I, E þve þve ve ve Discoid or Cells in ‘string
spp. flattened of pearls’
[‘N.limicola’ cells ‘arrangement
III?]

H.hydrossis ve ve ve ve ve ve 0.5 10–100 ST, B E, F ve ve þve / ? ‘needle’ straight
þve

Type 1863 ve ve / ve ve PHB 0.8 20–50 B, I E, F þve þve ve ve Oval rods ‘strings of
Acinetobacter þve 0.8 £ 1.0–1.5 sausages’
spp.
Type 0411 ve ve ve ve ve ve 0.8 50–150 B, I E þve þve ve ve Elongated Cells in chains
rods
0.8 £ 2–4.0
Type 0092 ve þve ve ve ve ve 0.8–1.0 20–60 ST, B I þ/ve ve ve ve Rectangular þve uniform
0.8 £ 1.5 Neisser stain
Type 0961 ve ve ve ve ve ve 0.8–1.2 40–80 ST E þve ve ve ve Rectangular ‘ghost-like’
0.8–1.4 £ transparent cells
2.0–4.0
Type 0581 ve ve ve ve ve ve 0.5–0.8 100–200 C I ve ve ve ve Unknown
Notation:
Gram stain: þ, positive; , negative; V, variable; þ,, Variable, most commonly positive; ,þ Variable, most commonly negative.
Neisser stain: A, Complete filament staining; B, Granules of Neisser positive regions.
Sulphur inclusions: A, Observed in situ; B, Present after sulphur test.
Other inclusions: PHB, Poly-b-hydroxybutyrate, PolyP, polyphosphate.
Filament shape: St, Straight; B, Bent; SC, Smoothly curved; C, Coiled; I, Irregularly shaped.
Filament locations: E, Extends from floc surface; I, Found mostly within the floc; F, Free in liquid between the flocs.

FISH PROBES DESCRIBED FOR THE FILAMENT MAKING ITS MICROSCOPICALLY BASED IDENTIFICATION LESS AMBIGUOUS.
seem to have low levels of metabolic activity, producing negative results for many of the common
standard phenotypic biochemical tests applied.
It is probable that the culture conditions we have chosen for this purpose are not optimal, although there is
evidence to suggest that some may grow equally slowly in activated sludge (Rossetti et al., 2005).
Consequently, their 16S rRNA sequence, which reveals their phylogeny is often the only character obtainable.
We have detailed and extensive characterisations of many but not yet all of the different foaming
Mycolata (see chapter 8), yet this information is not applied in the industry for their routine identification.
All are assumed to have similar phenotypes. They are still referred to collectively (even in some of the
literature) as GALO or incorrectly as Nocardia spp. even though members of this genus are relatively
rarely seen in activated sludge, or described equally imprecisely as ‘nocardioforms’ (Jenkins et al., 2004b;
Soddell and Seviour, 1990).
HOW DO WE IDENTIFY THESE FILAMENTS?

It was mentioned earlier in this chapter that identification is an exercise of comparison, and thus requires a
comprehensive set of reliable data for the strains against which comparisons are to be made. In most cases
they are not available for many of the Eikelboom filament morphotypes, and very few reference or type
strains are held in culture collections. Most are not mentioned or described in Bergey’s Manual of
Systematic Bacteriology or The ‘Prokaryotes’ (Dworkin et al., 2006), both widely used references for
bacterial identification. Among the few exceptions are Sphaerotilus natans, Beggiatoa sp., Haliscome-
nobacter hydrossis, Thiothrix nivea, Candidatus ‘‘Nostocoida limicola’, now included in the genus
Tetrasphaera (McKenzie et al., 2006), Gordonia spp., Skermania piniformis and other Mycolata genera.
In the past few years, additional filaments like Meganema perideroides (Thomsen et al., 2006a) (see
chapter 12) have been formally described and validly named in IJSEM, and type strains properly
deposited in culture collections. So progress is being made, albeit slowly. FISH probes are now available
for the filaments listed in Table 6.4. However, because of our poor understanding of their taxonomy, the
probes we possess more often target phylotypes, and not individual filament ‘species’.
NAMING THESE FILAMENTS?

Most of the Eikelboom filament morphotypes were not given proper valid names that follow the
International Code of Nomenclature in his original study. Even those that have (Table 6.1 and 7.1) are still
often referred to in the literature and more widely in the industry by their original numerical type names
(i.e. type 1701, 1851, 021N, etc.) from the system of Eikelboom (1975). This does not permit effective
communication, and can lead to confusion and argument as to which bacterium is under discussion. The
system has been very valuable in the past, but now with our increasing knowledge of the taxonomic status
of many of these filaments, it is time to encourage change. So where filaments have been validly named,
scientific journals should insist that these names always be given priority.
CURRENT CLASSIFICATIONS OF THE FILAMENTOUS BACTERIA

As mentioned above, the majority of the filamentous bacteria in activated sludge are not included in the
most widely used manuals for classifying and identifying bacteria because of inadequate characterization
data. The classifications available which have been published for them are mainly artificial or ‘‘special
purpose’’, and suffer from their disadvantages.
186
Table 6.4. FISH probe sequences of filamentous bacteria in activated sludge.

Organism Taxonomic status Probe Probe sequence (50 to 30 ) References
Meganema perideroedes Alphaproteobacteria Meg983 CGGGATGTCAAAAGGTGG Thomsen et al. (2006a)
Meg1028 CTGTCACCGAGTCCCTTGC
Ca. ‘Monilibacter Alphaproteobacteria MC2-649 CTCTCCCGGACTCGAGCC Levantesi et al. (2004)
batavus’
Ca. ‘Sphaeronema Alphaproteobacteria Sita-649 CCWCTCCCGGACCCCAG Levantesi et al. (2004)
italicum’ Nost993 CAGCCGAAACGGGCATGT Kragelund et al. (2006)
Ca. ‘Alysiomicrobium Alphaproteobacteria PPx3-1428 TGGCCCACCGGCTTCGGG Levantesi et al. (2004)
bavaricum’ PPx1002 GGTCTCCCCGGGCCGCGG Kragelund et al. (2006)
Alysiosphaera europaea Alphaproteobacteria Noli-644 TCCGGTCTCCAGCCACA Levantesi et al. (2004)
Ca. ‘Combothrix italica’ Alphaproteobacteria Combo-1031 CACCTGCAGTGGCCTCCCGA Levantesi et al. (2004)
Sphaerotilus natans Betaproteobacteria SNA CATCCCCCTCTACCGTAC Wagner et al. (1994a)
Aquaspirillum (identified Betaproteobacteria Aqs977 CTCTGGTAACTTCCGTAC Thomsen et al.
as Eikelboom Type 1701, (2004; 2006b)
Type 0041/0675 and
possibly Type 1851)
Acinetobacter (including Gammaproteobacteria ACA652 ATCCTCTCCCATACTCTA Wagner et al. (1994c)
Eikelboom Type 1863) (ACA23a)
Thiothrix and Eikelboom Gammaproteobacteria G123T CCTTCCGATCTCTATGCA Kanagawa et al. (2000)
Type 021N
Eikelboom G1B TGT GTT CGA GTT CCT TGC Kanagawa et al. (2000)
Type 021N group I
Eikelboom G2M GCA CCA CCG ACC CCT TAG Kanagawa et al. (2000)
Type 021N group II
Eikelboom G3M CTC AGG GAT TCC TGC CAT Kanagawa et al. (2000)
Type 021N group III
Thiothrix nivea Gammaproteobacteria TNI CTCCTCTCCCACATTCTA Wagner et al. (1994a)
Kouleothrix aurantiaca Phylum Chloroflexi EU25-1238 CTGCGCATTGCCACCGACAT Kragelund et al. (2007a)
Eikelboom Type 1851 Phylum Chloroflexi CHL 1851 AATTCCACGAACCTCTGCCA Beer et al. (2002)
(continued)
Organism Taxonomic status Probe Probe sequence (50 to 30 ) References

Nostocoida limicola-like Phylum ‘Chloroflexi ’ AHW183 CCGACACTACCCACTCGT Schade et al. (2002)
organisms
Unidentified filaments Phylum ‘Chloroflexi ’ CFX1223 CCATTGTAGGGTGTGTGTMG Björnsson et al. (2002)
CFX109 CACGTGTTCCTCAGCCGT
CFX784 ACCGGGGTCTCTAATCCC Gich et al. (2001)
GNSB-941 AAACCACACGCTCCGCT
Nostocoida limicola I Firmicutes NLIM191 CGCCACTATCTTCTCAGT Liu et al. (2001a)
(Trichococcus flocculiformis)
Bacillus spp. Firmicutes LGC353b GCGGAAGATTCCCTACTG Felske et al. (1998)
Ca. ‘Microthrix calida’ Actinobacteria Mpa-T1-1260 TTCGCATGACCTCACGGTTT Levantesi et al. (2006b)
Ca. ‘Microthrix Actinobacteria MPA CCGGACTCTAGTCAGAGC Erhart et al. (1997)
parvicella’ MPA60 GGATGGCCGCGTTCGACT
MPA223 GCCGCGAGACCCTCCTAG
MPA645 CCGGACTCTAGTCAGAGC
MPA650 CCCTACCGGACTCTAGTC
TM7 phylum (including Phylum TM7 TM7305 GTCCCAGTCTGGCTGATC Hugenholtz et al. (2001)
Eikelboom Type 0041) (subdiv1)
TM7905 CCGTCAATTCCTTTATGTTTTA
‘Nostocoida limicola’ III Planctomycetales NLIMIII 301 CCCAGTGTGCCGGGCCAC Liu et al. (2001a)
‘N. limicola’ III NLIMIII 729 AGCATCCAGAACCTCGCT Liu et al. (2001a)
(strain Ben220)
‘N. limicola’ III NLIMIII 830 CCATCGGCGAGCCCCCTA Liu et al. (2001a)
(except Ben 220)
Haliscomenobacter Bacteroidetes HHY GCCTACCTCAACCTGATT Wagner et al. (1994a)
hydrossis (Cytophaga-Flexibacter
group)
Haliscomenobacter spp. Bacteroidetes HHY-654 GCCTACCTCAACYTGATT Kragelund et al. (2008)
(Cytophaga-Flexibacter
group)
Ca. ‘Magnospira bakii’ Bacteroidetes Bak655 CGCCAACCCTAACCCATTT Snaidr et al. (1999)

(Cytophaga-Flexibacter Bak1408 TCCAAACTCCCATGGCTT
group)
187
Although unable to culture most of the 29 different morphological types he recognised, Eikelboom
(1975) published one of the earliest, which predates the advances made in filament taxonomy (Kämpfer
and Wagner, 2002; Martins et al., 2004b; Mckenzie et al., 2006; Thomsen et al., 2006a), by dividing them
into several groups, solely on their microscopic properties and staining reactions. These are listed here;
1. Sheath Forming Gram Negative Filaments

Sphaerotilus natans, Haliscomenobacter hydrossis, types 1701, 1702, 0321
2. Sheath Forming Gram Positive Filaments
types 0041, 0675, 1851
3. Sheathless Curled Multicelled Filaments
Thiothrix spp., Nostocoida limicola, Cyanobacteria, type 021N
4. Slender, Coiled Filaments
Microthrix parvicella, types 0581, 0192
5. Straight Short Multicelled Filaments
types 0803, 1091, 0092, 0961
6. Gliding Motile Filaments
Beggiatoa spp., types 0914, 1111, 1501
7. All others that did not fit into these groups
Nocardia spp., Fungi, types 1863, 0411
Of course this ‘classification’ was constructed before 16S rRNA sequence analysis was available, and it
made the most of the sparse morphological data obtainable from these organisms. It undoubtedly provided
a valuable framework for future work with these organisms but its flaws are obvious. It uses mainly
invalid names for the bacteria, and the groupings are arbitrary and rely on unjustified selected weighted
morphological characters. More industry orientated ‘‘classifications’’ for these bacteria were generated by
Jenkins (1992) and Wanner (1993), who both grouped filaments based on their presence in plants with
particular operating parameters. These schemes discussed earlier (Tables 5.7 and 5.8) are open to the same
criticisms mentioned there. All were designed primarily to assist plant operational staff control the
problems of bulking and foaming, and not to increase our understanding of the basic systematics of these
filamentous bacteria. They extrapolate from a small knowledge base, and like all artificial classifications,
will become increasingly less useful as our understanding of these bacteria continues to increase.
CURRENT STATUS OF FILAMENT IDENTIFICATION PROCEDURES

Attempts to identify these filaments, especially those where no FISH probes are available, invariably rely on
the reference manuals of Eikelboom (2000, 2006) and Jenkins et al. (2004b). Their approach is based largely
on the morphological characters and staining responses (Table 6.2), given in Table 6.3, and recognizing
morphotypes. Although these manuals are widely used, they should be used with some caution. They often
show little regard for the taxonomic conventions and rules discussed earlier. For example, deciding
unilaterally and with no experimental justification, that the morphotype name N. limicola II no longer exists
(Eikelboom, 2000), but should be considered a synonym of N. limicola III (both incidentally are invalid
names, and probably different bacteria) illustrates the problems that can arise when general taxonomic
conventions are ignored. The two reference manuals do not always agree, and sometimes present
confusingly different descriptions for the ‘‘same’’ morphotype. Such disagreements include variations in
filament dimensions, and outcomes of certain staining reactions, which may arise because the organisms
described are different. Both manuals also fail to describe many of the more recently characterized filaments.
The point has been made often that morphological criteria are unreliable indicators of relatedness
among these filaments, a problem exacerbated by some able to revert to unicellular forms as a regular
feature of their life cycles (Ramothokang et al., 2006; Seviour et al., 1997; Wagner et al., 1994a,b,c).
Thus, organisms which look the same, even to a trained and experienced eye, may not necessarily be so.
Filaments from different plants, particularly in different countries, can readily fit the morphology–based
descriptions given in the manuals, and yet strains within each morphological type might vary considerably
in their phylogeny and ecophysiology (for example the GALO and ‘N.limicola’).
WHICH IDENTIFICATION METHOD IS BEST?

Listing the advantages and disadvantages of both microscopic and FISH based identification methods for
these filamentous bacteria may help in summarizing some of the points raised, which suggests that neither
is perfect (M}uller et al., 2007).
MICROSCOPIC IDENTIFICATION METHODS

Advantages
. Relatively inexpensive since it only requires a good quality basic microscope fitted with bright
field and phase contrast optics (chapter 11).
. Rapid with regular practice and so regular examination of each individual plant biomass should
allow the filaments there to be ‘identified’ with confidence, and any changes in their community
composition readily recognized.
. Courses now run in many countries to help teach these ‘identification’ methods.
Disadvantages
. Relies on filament morphology, so only recognizes morphotypes.
. A single filament morphotype may contain several phylogenetically unrelated filamentous bacteria
indistinguishable under the microscope, and as this method does not reveal these differences,
potential filament diversity is inevitably underestimated.
. Morphology of a filament may change (eg. to single cells), and if so would not be ‘identified’ as
that filament by microscopy.
rRNA TARGETED FISH-BASED IDENTIFICATION METHOD

Advantages
. Can design probes covering the whole taxonomic spectrum from species to Domain level.
. Is independent of an organism’s morphology, so will recognize filaments and unicells equally well.
. Will reveal whether each filament morphotype consists of one, or more than one bacterial
phylotype, which all look indistinguishable under the microscope.
Disadvantages
. Probes not available for all filaments.
. May not access its target sites (ribosomes), either because of problems with cell permeability (as with
some foaming Mycolata) or from probe/target secondary structure constraints, giving false negatives.
. Need metabolically active cells containing plenty of targets (ribosomes) for positive response, so
false negatives are possible.
. Data bases of rRNA gene sequences available for probe design are incomplete, and so the risk of
false positives from populations whose 16S rRNA sequences have not yet been deposited (495%
of those which exist?) is possible.
. Each FISH analysis will only detect one or a few filament phylotypes in the biomass, and so rRNA
based DNA microarrays (chapter 11) are needed for comprehensive community fingerprinting.
. Requires more expensive equipment and skilled personnel.
THE IMPACT OF MOLECULAR APPROACHES ON OUR UNDERSTANDING

OF THE SYSTEMATICS OF FILAMENTOUS BACTERIA
Frequent mention has been made in this book of how acquisition of rRNA gene sequence data and
application of FISH probes designed from them has allowed us to address many questions relating to
in situ properties of activated sludge filament populations (Nielsen et al., 2009). It seems appropriate to
summarize here their impact on our understanding of their systematics (chapter 7).
(a) This rRNA approach has revealed that the phylogenetic diversity among these filaments is
considerable, and novel phylotypes continue to be recognized (e.g. Ajithkumar et al., 2002;
Eikelboom and Geurink, 2002; Levantesi et al., 2004, 2006a; Speirs et al., 2009).
(b) It has helped to show that some filaments are often phylogenetically very unusual bacteria. These
include Candidatus ‘M. parvicella’ (Blackall et al., 1994b, 1996) and Candidatus ‘M. calida’
(Levantesi et al., 2006b), type 1851 (Kouleothrix aurantiaca?) (Beer et al., 2002), type 0041
(Hugenholtz et al., 2001), N. limicola III? (Isosphaera sp) (Liu et al., 2001a), Meganema
perideroides (Levantesi et al., 2004; Thomsen et al., 2006a), Candidatus ‘Combothrix italica’,
Candidatus ‘Alysiosphaera europea’, Candidatus ‘Sphaeronema italicum’, Candidatus
‘Monilibacter batavus’ (Nittami et al., 2009) and Candidatus ‘Combothrix italica’ (Levantesi
et al., 2004, 2006a), and type 0092 (Speirs et al., 2009).
(c) It has also confirmed what we have long suspected, that a single filament morphotype often
contains more than one phylogenetically unrelated organism, as for example with type 021N
(Aruga et al., 2002; Howarth et al., 1999; Kanagawa et al., 2000), type 1863 (Seviour et al.,
1997), Nostocoida limicola II (Blackall et al., 2000; Levantesi et al., 2004; Schade et al., 2002)
and G. amarae – like organisms, GALO (Soddell 1999; Soddell et al., 2006b).
(d) It has also helped to show that some filaments are in fact members of previously described
bacterial genera, some of which were not known previously to grow as filaments (chapter 12).
These include type 1863 [Acinetobacter spp.] (Rossetti et al., 1997b; Seviour et al., 1997; Wagner
et al., 1993, 1994c), Candidatus ‘N. limicola’ [Tetrasphaera spp.] (Mckenzie et al., 2006),
Candidatus ‘M. batavus’ [Defluviicoccus] (Nittami et al., 2009) and again type 021N [Thiothrix
spp.] (Aruga et al., 2002; Howarth et al., 1999; Kanagawa et al., 2000).
(e) In combination with MAR, FISH probes, where available, have now provided us with the means
of elucidating the in situ ecophysiology of individual filament phylotypes, which may provide
clues as to how these might be controlled (Nielsen and Nielsen, 2005; chapters 3, 7, 11).
We might now see how having these tools have increased our understanding of their roles in bulking
and foaming in chapters 7 and 8, which follow.
7
Microbiology of bulking
Jiri Wanner, Caroline Kragelund, and Per H. Nielsen
INTRODUCTION
Excessive growth of filamentous bacteria has caused serious operational problems in activated sludge
wastewater treatment plants for decades. This filamentous overgrowth is known as bulking, and prevents a
proper flocculation and settling of the biomass in the clarifier (chapter 4). Some filamentous bacteria also
form foam on surfaces of reactor tanks and clarifiers, which may be a thin cover, although occasionally up
to half a meter or more of foam can be observed (chapter 8). In the past 30 years numerous studies have
tried to develop ways to control these filamentous organisms, either by adopting general universal
methods, or by more targeted approaches based on knowledge of their identity, physiology and ecology.
As discussed in chapter 5, more than 30 different morphotypes (‘identified’ by microscopy) have been
observed in systems treating primarily municipal wastewater (Eikelboom, 1975, 2000; Jenkins et al.,
1993, 2004b), and many more are encountered in industrial treatment plants (Eikelboom et al., 2006;
Eikelboom and Geurkink, 2002).
Based only on morphological identifications and extensive pilot-scale and full-scale experience, control
measures have been developed against many of them. However, we now know that only few individual
filament species can be identified reliably based on their morphology (chapter 6), and so the development
of culture-independent molecular methods (chapter 3) allows a more reliable identification. Furthermore,
detailed studies on their ecophysiology have revealed large differences among them, which suggest that
individual control measures might be needed for each. Such knowledge is already being used for bulking
control and management purposes as detailed below. The ecophysiological data can also help explain
why some filaments in activated sludge are so difficult to control. However, again as described below,
a thorough knowledge of treatment plant design, operation and wastewater characteristics is crucially
important in selecting an appropriate control measure, and so a detailed audit of treatment plants with
bulking problem is recommended.
THE ABUNDANCE AND OCCURRENCE OF BULKING IN ACTIVATED

SLUDGE SYSTEMS
A number of surveys have been published (chapter 5) to establish the relative abundance of the
filamentous bacteria in bulking activated sludge plants in different countries. Most of these have described
the various morphotypes present in plants obtained with the identification manuals of Eikelboom
(Eikelboom, 2000; Eikelboom and van Buijsen, 1983) or Jenkins et al. (1993; 2004b). Only quite recently
have surveys been carried out using FISH probes that generally allow a better and more reliable
identification. As mentioned in chapter 3, much of the early published information on activated sludge
microbiology is of little more than historical interest and should be viewed as such, because of problems
resolving the true level of population biodiversity in these processes, difficulties applying equally to most
of the filamentous bacteria there. So attempts to relate full-scale or pilot-scale observations of ‘filamentous
bacterial morphotypes’ to their real identity remained problematic until very recently and even now many
studies are carried out without their proper identification. As detailed in chapter 6, we now know that
only a few of the filaments can be identified reliably with the manuals based only on their morphology and
microscopy (see Table 7.1). These are restricted to Candidatus ‘M. parvicella’, Thiothrix, perhaps some
Mycolata and S. natans, but few others. Furthermore, as shown in Table 7.1 a single filamentous
morphotype often embraces several different organisms (chapter 6).
This can be exemplified by filaments included in the morphotype Nostocoida limicola, which affiliate to
at least 5 different bacterial phyla: Proteobacteria (class Alphaproteobacteria) (Levantesi et al., 2004;
Snaidr et al., 2002; Thomsen et al., 2006a; van der Waarde et al., 2002), Firmicutes (Liu et al., 2000),
Actinobacteria (Liu et al., 2001a) Planctomycetales (Liu et al., 2001a) and Chloroflexi (Schade et al.,
2002). Also, the thin needle-like filaments morphologically identified frequently as Haliscomenobacter
hydrossis in the Bacteroidetes may in fact be short, thin ‘Chloroflexi ’ filaments that can be detected
readily with subdivision FISH probes (Bjornsson et al., 2002). Furthermore, many H. hydrossis-like
species do not hybridize with the widely used probe (HHY), but only with broad group-specific probes,
indicating the presence of a large yet still unidentifiable population of thin needle-like filaments in
activated sludge (Kindaichi et al., 2004; Kragelund et al., 2008; Schauer and Hahn, 2005). The different
filament morphotypes with attached growth (types 0041/0675, 1701 and 1851) are also difficult or
impossible to identify precisely without FISH analysis. For these reasons, it is highly recommended that
FISH or other molecular methods be applied in all future surveys, together with the traditional microscopic
examination methods. Some morphotypes which may occur in large amounts in activated sludge
(e.g. 0961, 0914 and 0803) can not be identified presently by FISH since suitable probes are not available,
but new probes are continuously being developed, for example for type 0092 (Spiers et al., 2009).
Surveys based on morphological identification

In addition to the 30 or so different filamentous morphotypes ‘identified’ in municipal activated sludge
plants (Eikelboom, 2000; Jenkins et al., 2004b), an additional 40 morphotypes have since been described
in industrial plants, although many of these are rarely seen (Eikelboom and Geurkink, 2002). Most may
cause bulking or foaming, but it is important to note that about ten of all these morphotypes account for at
least 90% of the bulking incidences in both municipal and industrial WWTP (Eikelboom and Geurkink,
2000; Eikelboom, 2000; Jenkins et al., 2004b). Some are capable of forming thick foam layers on process
Microbiology of bulking 193
Table 7.1. Overview of the most common morphotypes and their possible phylogenetic identity.
Morphotype Possible phylogenetic identity Reference
Haliscomenobacter Haliscomenobacter hydrossis (Wagner et al., 1994a;
hydrossis (Bacteroidetes) Kindaichi et al., 2004; Okabe
Other species in the phylum et al., 2005; Weller et al., 2000)
Bacteroidetes (Bjornsson et al., 2002;
Species within the phylum ‘Chloroflexi ’ Kragelund et al., 2007a)
Microthrix parvicella Candidatus ‘Microthrix parvicella’ (Erhart et al., 1997)
(Actinobacteria)
Candidatus ‘Microthrix calida’ (Levantesi et al., 2005b)
(Actinobacteria)
Nostocoida limicola Candidatus ‘Nostocoida limicola’ (Blackall et al., 2000)
morphotypes (Actinobacteria)
Several species of Alphaproteobacteria (Kragelund et al., 2005,2006);
e.g. Meganema perideroedes Levantesi et al., 2004)
Species within the phylum Firmicutes (Liu et al., 2000)
Species within the phylum (Liu et al., 2001)
Plantomycetes
Species within the phylum Chloroflexi (Schade et al., 2002)
Thiothrix species (Kragelund et al., 2006;
(Gammaproteobacteria) Levantesi et al., 2004)
Nocardioforms/ Gordonia species and G. amarae (Soddell, 1999)
Mycolata (Actinobacteria)
Skermania species and (Soddell and Seviour, 1998)
S. piniformis (Actinobacteria)
Unknown species (Kragelund et al., 2007b)
Thiothrix Thiothrix species (Kanagawa et al., 2000)
(Gammaproteobacteria)
Species within the phylum (Kragelund et al., 2007a)
‘Chloroflexi ’ without
attached growth
Type 0041/0675 Species within the phylum (Kragelund et al., 2007a)
‘Chloroflexi ’
Curvibacter-related (Thomsen et al., 2006b)
(Betaproteobacteria)
Candidate division TM7 (Hugenholtz et al., 2001;
Thomsen et al., 2002)
Type 0092 Species in the phylum Bacteroidetes (Bradford et al., 1996)
‘Chloroflexi’ Speirs et al. (2009)
Type 021N Thiothrix species (Kanagawa et al., 2000)
(Gammaproteobacteria)
Meganema perideroedes (Levantesi et al., 2004;
Alphaproteobacteria Thomsen et al., 2006b)
Species within the phylum Firmicutes (Liu et al., 2000)
Species within the phylum ‘Chloroflexi ’ (Blackall et al., 2000;
Schade et al., 2002)
(continued)
Morphotype Possible phylogenetic identity Reference

Type 0803 Species in the phylum (Bradford et al., 1996)
Betaproteobacteria
Unknown
Type 1701 Curvibacter-related (Betaproteobacteria) (Thomsen et al., 2006b;
Candidatus Division TM7 (TM7) Thomsen et al., 2002)
Unknown
Type 1851 Kouleothrix aurantiaca (‘Chloroflexi ’) (Beer et al., 2002;
Kohno et al., 2002;
Kragelund et al., 2007a)
Species within the phylum ‘Chloroflexi ’ (Bjornsson et al., 2002)
Curvibacter-related (Betaproteobacteria) (Thomsen et al., 2006b)
tanks without being present in high numbers in the activated sludge mixed liquor (Seviour et al., 1994).
Foaming is described in detail in chapter 8. The relative abundances and distribution of the dominant
individual filament morphotypes was believed to vary with geographical location (Chua and Le, 1994).
The most abundant filamentous species identified in municipal activated sludge treatment plants are
Candidatus ‘M. parvicella’, type 0041 and the Mycolata (Table 7.2). Candidatus ‘M. parvicella’ is one of
the dominant filaments in Europe, South Africa and Australia (Blackbeard et al., 1986a; Eikelboom et al.,
1998; Seviour et al., 1994) but is ranked much lower in plants surveyed in Thailand and the US (Mino,
1995; Richard et al., 1982). In general, this filament ranking order is more or less the same for plants
in Europe, South Africa, and Argentina, but shows some differences to those in Australia and the US.
For example, in the reported surveys, type 1701 and the Mycolata, both very common in US plants are not
seen as frequently in those in Europe. However, several more recent small surveys indicate that these
types may in fact be more universally distributed than the earlier data indicate (Eales et al., 2005;
Kragelund et al., 2008; Thomsen et al., 2006b).
These discrepancies in their relative abundances may be explained partly by differences in the identity
of some morphotypes, but most probably is due to differences in the selective pressure imposed on their
populations in different parts of the world from variations in plant design and operating conditions, and in
influent characteristics like wastewater composition and strength (Jenkins et al., 1993; Wanner, 1987;
Wanner et al., 1987a,b). In particular, filament types and their abundance in treatment plants only
removing organic carbon and performing only nitrification are markedly different to those seen in plants
with full N-removal (nitrification-denitrification). Thus, it seems likely that their distribution and
abundance reflect more their nutritional requirements than geographical location, and that their ranking
order may change in different parts of the world according to changes in treatment plant design and
operation, as seen in plants with carbon removal versus nutrient removal.
Surveys based on FISH probing

Only relatively few surveys have been performed based on traditional microscopic ‘identification’ and
FISH analysis (Levantesi et al., 2004; van der Waarde et al., 1998, 2002). These have all been conducted on
industrial samples, and the main findings are presented in Table 7.3. In the report by van der Waarde et al.
(2002), 17 different published species-targeted and group-targeted FISH probes were used and 81 samples
Table 7.2. Ranking of dominant filamentous organisms in bulking and foaming activated sludge based on morphological observations.
Europe South Africa Australia Asia America
Czech France KwaZulu- South Africa USA
The The Denmark Italy Republic (Pujol Natal (Blackbeard Australia Japan (Richard
Netherlands Netherlands Germany (Kristensen (Rossetti (Wanner and (Lacko et al., 1986a; (Seviour Thailand (Martins et al., 1982; Argentina
(Eikelboom (Eikelboom, (Wagner, et al., et al., et al., Canler, et al., Blackbeard et al., (Mino, et al., Strom and (Di Marzio,
et al., 1998) 1977)a 1982)a 1994) 1994) 1998b) 1994) 1999) et al., 1986b) 1994) 1995) 2004a) Jenkins, 1984) 2002)
Filamentous 93 200 315 38 40 86 12 6 111 65 6 ? 270 10
organism WWTP WWTP WWTP WWTP WWTP WWTP WWTP WWTP WWTP WWTP WWTP WWTP WWTP WWTP
(c)
Candidatus 1 1 2 1 1 1 X 2 1 7 2
‘M. parvicella’
Type 2 6 3 2 3 5 X 1 6 2 X X 4 2
0041/0675
Type 1851 3 12 – 6 – – 2 3 8 13
Type 021N 4 2 1 3 6 7 2 – 1 X X 3 1
S. natans 5 7 4 – – – – 14 X 6 1
Type 0092 6 4 – 3 4 3 X 1 3 X 8 3
Nocardioform/ – 2 6 1 6 7 X X 1 3
Mycolata
N. limicola I, 11 7 – 5 2 X 3 8 5 12
II, and III
H. hydrossis 3 6 – – 11 9 5 9
Thiothrix spp. 9 – – 7 8 4 9 13 X 5 2
Type 1701 5 8 – 9 13 5 8 12 X 2 2
Type 0803 7 10 – – 4 7 9 11
Type 0914 – – 5 10 10 4 5 7 18
Type 0961 10 9 – 8 9 – 10 14
Beggiatoa spp. 8 – – – – 5 – 15 16
a
Nocardioforms/Mycolata not included, cited in Martins et al. (2004); X ¼ unranked.
Table 7.3. Filamentous bacteria identified by specific gene probes in bulking and foaming activated sludge from Danish, German, Dutch
196
and Italian industrial and municipal wastewater treatment plants. Percentage of the plants investigated with the probe-defined populations
is shown.
70 WWTP 86 126 126 114
(van der WWTP WWTP WWTP WWTP
Waarde (Levantesi (Kragelund (Kragelund (Levantesi
Oligonucleotide Species, genus, et al., 2002) et al., 2004) et al., 2007a) et al., 2008) et al., 2005b)
Phylum probe class or phylum % % % % %
Proteobacteria ALF968# Class Alphaproteobacteria 58 65

EU12-645+ Meganema perideroedes 17 3
EU26-653
PPx3-1428 Candidatus ‘Alysiomicrobium 21 16
bavaricum’
MC2-649 Candidatus ‘Monilibacter 11 10
batavus’
Noli-644 Candidatus ‘Alysiosphaera 33 24
europaea’
Sita-649 Candidatus ‘Sphaeronema 10
italicum’
Combo1031 Candidatus ‘Combothrix 6
italica’
LDI Leptothrix sp. 2
SNA Sphaerotilus natans 25
TNI Thiothrix sp. 38
21N Thiothrix eikelboomii 4

ACA652/23a Acinetobacter 16
LMU Leucothrix 4
Bacteroidetes No phylum probe 62
(sum of probes below) 24¤
CFB719 Class Bacteroidetes 20
and Class Sphingobacteria
CFB563 Class Flavobacteria 10
(continued )
70 WWTP 86 126 126 114

(van der WWTP WWTP WWTP WWTP
Waarde (Levantesi (Kragelund (Kragelund (Levantesi
Oligonucleotide Species, genus, et al., 2002) et al., 2004) et al., 2007a) et al., 2008) et al., 2005b)
Phylum probe class or phylum % % % % %
CFB286 Class Bacteroidetes 0

SAP309 Family Saprospiraceae 42
HHY-654 Haliscomenobacter 42
hydrossis and Iso10B
HHY Haliscomenobacter 58 32
hydrossis
HHY-T5 Clone T5 10
‘Chloroflexi’ CFX1223 Phylum ‘Chloroflexi ’ 50
and GNSB941 26
CFX1223 Unknown Chloroflexi species 25
CFX784 Subdivision 1 of Chloroflexi 51
CFX109 Subdivision 3 of Chloroflexi 3
Chl1851 Eikelboom morphotype 1851 51

EU25-1238 Isolate EU25 1

Actinobacteria HGC-69a# Actinobacteria 16
NLI265 Nostocoida limicola 6
Gor596 Gordonia amarae 7
MPA645 Candidatus ‘Microthrix 13
parvicella’
Mpa-all-1410 Candidatus ‘Microthrix parvicella’ 21
and Candidatus ‘M. calida’
Mpa-T1-1260 Candidatus ‘M. calida’ 5
#
Broad group specific gene probes
¤
Filament index exceeding 1.5 for Bacteroidetes

Filament index exceeding 1.5 for Chloroflexi

Most likely unknown filamentous bacteria. Candidatus ‘Microthrix spp.’ are not targeted by this probe.
197
examined from 70 different European plants. With FISH it was possible to identify the dominant
filamentous populations in only 45% of the samples, showing that many were still undescribed.
Large filamentous populations (filament number 3–5 on a scale from 0–5 (none to very many filaments –
chapter 5) were identified in 59% of the samples. Filamentous Alphaproteobacteria and H. hydrossis–like
filaments were detected in 58% of all samples while S. natans and Thiothrix spp. occurred in 25% and 38%
of plants respectively. However, abundances of the different filaments in the industrial samples were not
listed, and so the dominant filaments potentially responsible for bulking remain unclear.
Another interesting observation was the frequent presence of several co-dominating filaments. This
was more frequently observed in industrial (between 2–4 dominating species) than in municipal (between
1–2 species) samples (Eikelboom and Geurkink, 2002). Gram-positive filaments were generally poorly
represented in industrial plants in contrast to their frequent dominance in many municipal processes, based
only on morphological identification, although Candidatus ‘M. parvicella’ was detected in some industrial
plants. This difference in the filamentous communities between municipal and industrial WWTP may be
explained in terms of different wastewater composition and process design. However, it could also arise
from insufficient permeabilization procedures conducted prior to FISH. Both Candidatus ‘M. parvicella’
and members of the Mycolata can only be detected by FISH after they have been subjected to some
pretreatment (enzymatic, acid, or other) (Carr et al., 2005; Erhart et al., 1997; Kragelund et al., 2007b).
In several studies, novel species-targeted or group-targeted FISH probes have been developed and
applied to small numbers of treatment plants in order to gain an impression of the abundance of that
particular filament. The most detailed work of this kind was that carried out with the filamentous
Alphaproteobacteria as they appeared to be important in plants treating industrial wastes (Table 7.3).
Several probes targeting Alphaproteobacteria with a Nostocoida limicola-like or type 021N-like
morphology were designed and applied to biomass samples (Levantesi et al., 2004). These two
morphotypes appeared to be those most commonly involved in bulking incidents in 86 of the plants.
Filamentous Alphaproteobacteria were detected in 65% of all biomass samples and were thought to be
responsible for bulking incidents in 26% of these. Four FISH-probe defined species were identified among
them (Table 7.3). The study showed that most of the filaments ‘identified’ in industrial plants as
N. limicola in earlier studies were different species of filamentous Alphaproteobacteria. This proposal is
also supported by investigations into bulking in the pulp and paper industry, where filamentous
Alphaproteobacteria often cause severe bulking problems (Kragelund et al., 2006). The study also showed
that some filamentous Alphaproteobacteria have a morphotype indistinguishable from that of type 021N
(except for the presence of sulfur granules), and may have been misidentified in many earlier studies using
microscopic identification methods as type 021N (Howarth et al., 1999).
Among the Betaproteobacteria, some filamentous bacteria hybridizing with a probe designed to target
Aquaspirillum (re-classified as Curvibacter, Ding and Yokata., 2006) have a very similar morphology to
type 1701 (Thomsen et al., 2006b). This filament with or without attached growth, was abundant in
several Danish nutrient removal plants but was often hidden inside flocs, and therefore easy to overlook
without FISH analyses (Thomsen et al., 2006b). Filamentous bacteria affiliated to phylum ‘Chloroflexi ’
have been observed in about half of all the industrial plants screened with a selection of ‘Chloroflexi ’
targeted probes, and in 26% they contributed substantially to the overall filament index (Kragelund et al.,
2007a). Filamentous members of the Bacteroidetes have also been pursued in plants, and it seems they are
commonly present in both industrial and municipal activated sludge samples (Kragelund et al., 2008).
They were identified in 62% of those examined and held responsible for bulking in 24%. These filaments
were often situated inside sludge flocs or in bundles, and without epiphytic growth, making morphology
based ‘identification’ difficult. Candidatus ‘M. parvicella’ and Candidatus ‘M. calida’ were estimated to
occur in about 21% and 5% respectively of these industrial plants (Levantesi et al., 2005). FISH probes
targeting filamentous bacteria belonging to the candidate group TM7 hybridize with many type 0041
filaments and in some treatment plants they constituted up to 15% of the 0041/0675 morphotypes
(Hugenholtz et al., 2001; Thomsen et al., 2002). They seemed especially common in EBPR plants.
Employing FISH in such surveys is not without difficulties. If we wish to continue to learn more about
the identity and phylogenetic diversity of these filaments, it is important to ensure that appropriate and
updated probes are applied. For example, the early and widely used probes for Thiothrix and type 021N
(TN1 and 21N, Wagner et al., (1994a)) are now known to target only a fraction of the entire Thiothrix-
type 021N group (Kanagawa et al., 2000). Thus, many of these filament morphotypes may be overlooked
if only these probes are used. Instead the newer designed probes covering a broader phylogenetic range of
these filaments should be applied (Kanagawa et al., 2000). Therefore, it is important to test coverage of
the probes in the databases (probeBase, Ribosomal Database Project or others) before use, and it is
recommended to apply simultaneously a general broad range probe together with a more specifically
targeted one. Application of FISH with a large battery of probes is also much more labour intensive than
normal microscopic examination based on morphology (chapter 6). However, molecular methods are
necessary if reliable identification of filaments is sought, and hopefully the development of faster methods
like qPCR or high-throughput methods such as DNA-arrays (chapters 3, 11) will become available in the
near future.
Evaluation of the dominant filamentous microorganisms has been conducted traditionally by estimating
the relative contribution different filament types make to the filament index (Eikelboom, 2000). FISH
combined with digital image analysis now allows quantification of individual filaments beyond the
filament index, so it is possible to monitor the length of a particular probe-defined filament and compare
it to the total filament length per volume or per g. VSS. Or it can be related to all the bacteria in the
sample detected by FISH or DAPI. In one such study Liao et al. (2004b) developed a statistical tool
for monitoring S. natans and type 1851 populations in lab-scale and full-scale plants. Candidatus
‘M. parvicella’ and the Mycolata were also quantified by FISH and image analysis and new filament
abundance categories suggested from these data (Hug et al., 2005). Real-Time qPCR protocols have been
used to quantify individual filamentous bacterial phylotypes from their 16S rRNA gene copy numbers as
compared to the total bacterial 16S rRNA copies present in activated sludge samples (Kaetzke et al., 2005;
Vervaeren et al., 2005). Detection of foam forming Gordonia using PCR and DGGE directly in foam has
been successful (Shen et al., 2007). In pulp and paper samples containing Thiothrix species-degenerate
primers for amplification of the chaperonin subunit (cpn60) were applied. These revealed molecular
phylogenetic information and are more generally suitable for characterization of complex microbial
communities (Dumonceaux et al., 2006; Hill et al., 2004).
The most abundant filamentous species in municipal

and industrial WWTP
The most abundant filamentous species or types found in different types of treatment plants are
summarized in Table 7.4. Rankings are based on all available FISH surveys and an evaluation by the
authors of this chapter of the ‘‘true identity’’ of each population detected in the morphology-based surveys
(Table 7.2 and 7.3). The most abundant filaments in EBPR plants treating mainly municipal wastewater
are Candidatus ‘M. parvicella’, some filaments belonging to Candidate division TM7 (type 0041
and others), some ‘Chloroflexi ’ species (type 1851 and others), some Curvibacter-related filaments
(type 1701 and others), some Bacteriodetes (H. hydrossis-like), some Mycolata (Gordonia and Skermania
morphotypes), type 0092, and type 0803. Of these, only Candidatus ‘M. parvicella’ causes serious
operational problems, while the others may cause sporadic episodes of bulking or foaming. In plants
treating municipal wastewater with carbon removal but no N-removal, it is mainly the Alphaproteo-
bacteria (N. limicola morphotypes), Thiothrix spp. (different Thiothrix types and Type 021N), Mycolata
(Gordonia) and possibly type 1701 (identity not yet revealed by FISH). All can cause serious bulking
or foaming problems. In plants treating industrial wastewater, bulking problems are often caused by
Alphaproteobacteria (N. limicola morphotypes), Thiothrix spp. (different Thiothrix morphotypes and
type 021N), Mycolata (Gordonia and Skermania morphotypes) and possibly some ‘Chloroflexi ’ species
(type 1851 and others). These filaments occur in plants treating many different types of industrial
wastewaters and with different process designs (Eikelboom and Geurkink, 2002). Furthermore, occasional
severe bulking incidents are associated by the presence of rare and so far unidentified filaments, possibly
favored by very specific growth conditions with certain industries (Pellegrin et al., 1999).
Table 7.4. Ranking of most abundant filamentous bacteria causing bulking in activated sludge systems
primarily treating municipal or industrial wastewater.
Filamentous species Municipal plants Industrial plants
Alphaproteobacteria (‘N. limicola’-like) 1
Betaproteobacteria 7
(Type 0803-like)
Thiothrix eikelboomii 2
(Type 021N)
Thiothrix types I,II 2
Bacteroidetes 6
(Type 0092-like)
Haliscomenobacter hydrossis 4
‘Chloroflexi ’ 2 4
(Type 1851, Type 0041/0675 and Type 1701)
TM7-related (Type 0041/0675)/ 3
Curvibacter-related bacteria (Type 1701)
Candidatus ‘Microthrix parvicella’ 1
Mycolata 5 3
(Nocardioforms)
BULKING CONTROL METHODS

Filamentous bulking is a problem of poor activated sludge compaction. In systems with sludge separation
by gravity in secondary clarifiers bulking results in:
. low return and wasted activated sludge solids

. difficulties in maintaining the required activated sludge concentration in reaction basins (problems
with sludge age control)
. poor sludge dewaterability
. hydraulic overloading of sludge handling facilities
Filamentous bacteria interfere with the sedimentation and compaction of the flocs in two ways:
1. Some grow preferentially inside flocs, and produce flocs with a diffuse open structure (chapter 3).
These open flocs provide space for water retention, so although aggregation of individual flocs is
not mechanically hindered by filaments extending from them, excessive water remains ‘‘captured’’
in the settled sludge.
2. Alternatively and more commonly, filaments detrimentally affect sedimentation and compaction
of flocs when most extend from compact and firm flocs into the bulk liquid. These filaments,
which in low numbers form the backbone of firm flocs, are able in large numbers to prevent
mechanically the compaction of individual flocs. This problem is called ‘‘bridging’’ (chapter 3).
However, final floc settling properties depend not only on the quantity and type of filamentous
bacterial populations. Their settling properties result from the balance between the detrimental effects of
excessive filamentous bacterial growth and the weighting effect of various floc-forming bacteria. It has
been observed empirically in EBPR systems that presence of large clusters (microcolonies) of the PAO
and nitrifiers contribute to better sedimentation of flocs (Larsen et al., 2006; 2008b) with FISH. These
floc-forming populations do not form a uniform group either. As shown in chapter 5 and earlier here,
techniques for identification of the filamentous bacteria in activated sludge are now more precise and
accurate. However, determining possible relationships between filamentous bacterial populations and
bulking is still based largely on their ‘identification’ to morphotypes. In addition, the classification of
these filaments into groups according to their properties available in the literature works well in most
cases with these morphotypes. On the other hand, there is enough literature, including this book
(e.g. Table 7.1), which provide data on the correlation between identification into morphotypes and
phylotypes (identification based on FISH).
The identification and classification of floc-forming bacteria is even less well understood, and it is
generally accepted that the so-called floc-formers are not a homogeneous group of organisms. Jenkins
et al. (1993; 2004b) and Wanner (1994a,c) distinguished at least two different groups:
(a) Floc-formers growing in oxic completely mixed reactors or in activated sludge systems with
anaerobic zones
(b) Floc-formers growing in systems with substrate concentration gradients, both oxic and anoxic.
Our current knowledge of both filamentous and floc-forming microbes, although still incomplete,
enables engineers to apply bulking control measures based on sound scientific principles. In chapter 5 the
so-called microbiological and engineering approaches to bulking control were described. Let us try to
combine the current microbiological knowledge of bulking causes with the practically verified methods
of bulking control. In this way these methods can be called ‘‘bioengineering’’ or ‘‘biotechnological’’
methods for bulking control. They specifically target at the biological level the reasons for bulking
problems (i.e. balance of competition between floc-forming vs. filamentous microbes). However, it is
important to realize that these methods have been developed over many years and often the causative
filaments were not identified reliably using FISH. So, unsuccessful treatments may have resulted from an
inappropriate choice of methods arising from filament misidentification. There is also another approach,
which can be described as non-specific bulking control methods. These are not aimed at curing the
reasons of bulking but at compensating for the negative effects of excessive presence of filamentous
bacteria in activated sludge.
SPECIFIC, BIOENGINEERING METHODS FOR BULKING CONTROL

Outside factors affecting composition of activated sludge
Wastewater composition
Wastewater is a complex mixture of substrates, nutrients and micronutrients which support the community
of activated sludge microbes. In addition, wastewater continuously inoculates this consortium with the
bacteria from sewers. These can be described (chapter 2) as:
Readily biodegradable substrates. Low molecular weight organic compounds, i.e., simple molecules,
which can be utilized directly by cells. These (e.g. monosaccharides, alcohols, volatile fatty acids and
amino acids) represent ca. 10–20% of COD of most municipal wastewaters, and are thought to favor the
growth of filamentous bacteria including type 021N, Sphaerotilus natans, type ‘Nostocoida limicola’ and
perhaps H. hydrossis. Special attention has to be paid to the design of activated sludge systems for treating
waste containing a high fraction for example, from the food processing industry.
However, the danger of filamentous bulking from these readily biodegradable substrates in modern
activated sludge systems is limited. Systems now are designed and operated in most countries as low-
loaded (long-sludge-age) systems carrying out nitrification and denitrification. The denitrification zone is
placed before that for nitrification (w.r.t. place in continuous systems and time in SBRs), so that biomass
is contacted with readily biodegradable substrates in the absence of oxygen (chapter 2). Thus RBCOD
is preferably used by denitrifying bacteria (or some DPAOs) and not by the filamentous bacteria (see
metabolic selection below).
Reduced sulphur compounds can also be readily metabolized as energy sources (chapter 1) and
support the growth of filaments like type 021N/Thiothrix or Beggiatoa. However, the fraction of readily
available reduced sulphur compounds in municipal wastewater is not sufficiently high to lead to serious
bulking problems. These can be more pronounced in towns with extended sewer systems in flat areas,
where collected municipal wastewater becomes septic because of long residence time and limited aeration
in them. On the other hand, many equipped with pressure sewer systems with long pipes are reporting the
problems with hydrogen sulphide production and subsequent bulking problems caused by type 021N/
Thiothrix. Elevated sulphide levels may occur in some industrial wastewaters, for instance, from coal and
crude oil processing, or in plants with poorly working pre-settling or equalization tanks.
Particulate, slowly biodegradable substrates. Most organic compounds in municipal wastewaters are
present as particulate substrates (chapter 2), consisting of high molecular weight organic compounds
present either as colloids or as true suspended solids. In both cases they have to be degraded by extracellular
enzymes before they become available to microbial cells, by a mechanism of hydrolysis, and the hydrolysis
products are similar chemically to the readily biodegradable substrates in wastewaters (chapter 2).
However, hydrolysis is a surface phenomenon and is connected with populations in flocs (chapter 3).
The impact of particulate substrate hydrolysis on filamentous bulking will depend on many factors,
including:
(i) the ratio between the levels of ‘‘entrapped’’ particulate substrate to those of bacterial biomass in
the flocs:
(ii) this ratio governs the rate of consumption of hydrolysis products inside the flocs, and the higher
the ratio, the more hydrolysis products released by diffusion out from the flocs.
(iii) The rate of diffusion of hydrolysis products is determined by the concentration gradient between
the floc interior and bulk liquid.
While the concentration inside the flocs is given by factor i), the concentration in the bulk liquid is
affected mainly by factor iii), and depends on the hydraulic regime of the aeration basin.
In completely mixed aeration basins the hydrolysis products will be diluted immediately in the whole
volume of the tank, resulting in low concentrations. Because of the high affinity of filamentous bacteria for
substrates at low concentrations (lower KS value than for floc-forming microbes), this may lead to their
proliferation (but see chapter 5 for a critical examination of this assumption).
The effects of the above factors will depend also on the types of filamentous bacteria present. Some
grow preferably either outside or inside the flocs. The different impact of particulate substrate hydrolysis
on their growth is explained by Wanner (1994a) using the hydrolysis hypothesis of Ekama and Marais
(1986) and Gujer and Kappeler (1992).
The recommended control strategy for filament problems caused by elevated concentrations of
particulate substrates is as follows:
(a) To maintain a low ratio between particulate substrate and biomass levels in activated sludge flocs;
which can be achieved by operating the process at a higher sludge age, resulting in higher biomass
concentrations. Thus, nutrient removal systems operated at higher sludge ages to support
nitrification should resist this kind of bulking.
(b) To reduce the danger of excessive growth of filaments on hydrolysis products released into
the bulk liquid in the main aeration basin, it is recommended to compartmentalize even the
nitrification stage.
Some individual particulate substrates seem to favor the growth of selected filamentous bacteria,
as seen with fats and grease, which are selectively concentrated in foams and support the growth of
some Mycolata (chapter 8). Long-chain fatty acids preferentially support the growth of Candidatus
‘M. parvicella’ (Andreasen and Nielsen, 1997, 2000; Nielsen et al., 2002b), as discussed later.
Inoculation of activated sludge systems from wastewaters. The sewerage network and wastewater
treatment plants form one unit system, and processes in sewers may markedly affect the composition of
the activated sludge community. For example, readily biodegradable substrates can be taken up by
bacteria growing in inner slimes especially in extended sewer systems. These biofilms are continuously
sloughed off and inoculate the treatment plant. Filaments like S. natans may be found there, but whether
they are the source of any other filaments is not clear.
Temperature, pH, toxic compounds

The physical properties and chemical composition of the influent wastewater are certainly among the
more important parameters determining microbial community composition of activated sludge biomass.
Besides variations in its ionic strength and alkalinity, other parameters reflecting the chemical
transformations of the bacteria present may differ from plant to plant and affect ultimately their
importance. However, in most plants return activated sludge (RAS) is not mixed directly with the raw
influent sewage, which is first subjected to mechanical pretreatment (screening, grit and oil and grease
removal). In Europe, where primary sedimentation and anaerobic digestion is part of state-of-the-art
treatment technology, wastewater composition can change markedly by processes occurring in this
mechanical pretreatment, and include:
1. stripping in step screens and aerated grit chambers

2. pH changes from fermentation in primary clarifiers
3. neutralization and precipitation reactions

4. coagulation of larger molecules in primary clarifiers
For instance, concentrations of inorganic toxic compounds like heavy metals or sulphide can be
reduced by precipitation, while those of strongly reactive compounds like chlorine can be changed by
reactions with organic compounds in wastewater. Chemical reactions in primary treatment may also
change wastewater alkalinity which can result in pH changes. Thus, in expressing the effects of the above
factors on filamentous bulking it is always better to take into account their actual concentrations in the
aeration basin of the plant.
Intrinsic factors
Biomass retention time YX
Biomass retention time YX (‘‘sludge age’’) impacts on the distribution of individual populations in the
activated sludge community, according to their growth and decay rates. Low retention times YX may
result in wash-out of slower growing populations whose net growth rate is slower than the dilution rate
D ¼ 1/YX. On the other hand, high YX values favour slower-growing populations. Unfortunately, the
‘‘safe’’ YX values, at which individual filamentous bacteria are washed out to the extent necessary for
bulking control, are too low to be applied as a common bulking control measure. The practical problem is
that the limiting value of YX for the growth of most filaments seems to be close to that for nitrifying
bacteria. Thus a ‘‘wash-out’’ strategy for filaments usually results in washing out the nitrifying organisms,
unless nitrification is stabilized by some ‘‘side-stream’’ process like bioaugmentation of nitrification in a
regeneration zone (Krhutkova et al., 2006; Parker and Wanner, 2007). Such bioaugmentation methods
help retain more nitrifying bacteria than corresponds to a particular sludge age.
Actual substrate concentration in reactor

Communities in activated sludge cultured in reactors with substrate concentration gradients acquire
certain features, which is often described as the ‘‘selector’’ effect. They are:
(1) high rates of substrate consumption

(2) high oxygen (or more generally external electron acceptor) uptake rate
(3) enhanced growth of floc-forming bacteria.
This substrate concentration gradient produces a combined effect of both kinetic and metabolic
selection together with storage selection (see chapter 5).
Most current specific bioengineering methods for bulking control exploit the existence of substrate
concentration gradients in reactors, regardless of other growth conditions dominating in them (oxic,
anoxic and anaerobic reactors).
A substrate concentration gradient is achieved by proper compartmentalization of the whole volume of a
reactor, or at least of its head-end. This compartmentalized region is also called a selector. The effect of
metabolic selection is generated by changing aerated and non-aerated compartments. Storage selection
needs the concentration gradient for promoting unbalanced growth, and time for the metabolism of stored
substrates (regeneration of storage capacity) tEM (Figures 7.1, 7.2 and 7.3). Full-scale plant experience
suggests that it is more economic to provide this regeneration time not in the main aeration basin (Figure. 7.4)
but in a special regeneration zone inserted into the return sludge stream (Figure 7.5). As is clear from
equations 7.1 and 7.2, the regeneration zone provides conditions for endogenous metabolism with higher
concentrations XR, so that the volume necessary for achieving the same tEM and also aerobic sludge age is
lower.
The rules for practical design of selectors, even in this era of mathematical modeling, are based mostly
on practical knowledge verified by long-term operational experience. Some of the basic rules can be found
in the literature (Jenkins et al., 1993; Wanner, 1994a,b,d; Martins et al., 2004a,b).
Storage
products
Substrate AC
Metabolisms
(Regeneration)
Cell membrane
AC = Accumulation
Figure 7.1. Schematic diagram of substrate accumulation/storage and metabolism in cells with unbalanced
growth.
tS tS
S
tR tR
tEM tEM
TIME
Figure 7.2. Time for endogenous metabolism tEM (regeneration) in SBR activated sludge systems.
S ¼ Substrate concentration
tS ¼ time of sedimentation period
tR ¼ time of reaction period
tEM
TIME
Figure 7.3. Time for endogenous metabolism tEM (regeneration) in plug-flow activated sludge system.
VN :XN þ VR :XR
YX ;AER ¼ YX :
VN :XN þ VR :XR þ VD :XD
Equation 7.1. Aerobic sludge age YX,AER in activated sludge with nitrification (N), denitrification (D) and
return sludge regeneration (R) zones (Kos et al., 1992).
V – volume of the zone; X – concentration of activated sludge biomass (as SS)
Q ·S
BX ¼
V ·X
Equation 7.2. Sludge loading BX

Q – activated sludge inflow
S – substrate concentration in the inflow
V – volume of activated sludge reactor
X – activated sludge biomass concentration.
Contact
I zone MAB SC
RAS
WAS
Figure 7.4. Activated sludge system with compartmentalized contact zone.

I – influent, E – effluent, WAS – waste activated sludge, RAS – return activated sludge
Contact zone: compartmentalized zone with substrate concentration gradient (accumulation and storage of
organic substrate), MAB – main aeration basin: a part of activated sludge system, where organic carbon
removal is finished and where the endogenous metabolism of accumulated/stored substrate takes place
(regeneration), SC – secondary clarifier.
Contact
zone MAB SC
I
E
RAS
Regeneration
WAS
Figure 7.5. Activated sludge system with compartmentalized contact zone.

I – influent, E – effluent, WAS – waste activated sludge, RAS – return activated sludge
Contact zone: compartmentalized zone with substrate concentration gradient (accumulation and storage of
organic substrate), MAB – main aeration basin: a part of activated sludge system, where organic carbon
removal is finished, Regeneration: a part of the activated sludge system, where the endogenous metabolism
of accumulated/stored substrate takes place (regeneration), SC – secondary clarifier.
Dissolved oxygen, organic substrate, nutrients, pH and temperature in aeration basins

Dissolved oxygen and organic substrate. Some filamentous bacteria (e.g., S. natans/type 1701 which were
not identified reliably by FISH, and H. hydrossis) exhibit a high affinity for dissolved oxygen (DO)
because of low values of their saturation constant KO. The boundary between ‘‘bulking’’ and ‘‘non-
bulking’’ DO concentrations is not fixed because this value depends on the actual value of activated sludge
loading (BX – equation 7.2) in the reactor. The sludge loading rate increases the rate of substrate
consumption, which then increases the rate of oxygen consumption in the flocs. Thus, even relatively high
DO concentrations in the bulk liquid do not automatically prevent the growth of the so-called ‘low DO
filaments’ (chapter 6). Nevertheless, DO concentrations less than 0.5 mg/l are dangerous for low DO
bulking, especially when soluble substrates still remain in the mixed liquor (Jenkins et al., 1993; Wanner
2004). Some filamentous organisms may also have a very high affinity for organic substrates, probably
higher than most floc-formers have (summarized by Jenkins, 1992), as for example Thiothrix spp. and
Meganema perideroedes (Kragelund et al., 2005; Nielsen et al., 2003b).
Nutrients. As with DO, some filaments exhibit a higher affinity than others for certain nutrients
(i.e. nitrogen, phosphorus and micronutrients), again as a result of adopting a KS-strategy under conditions
of balanced growth. Therefore, addition of nutrients to control bulking is sometimes necessary. However,
as with DO bulking, the extent of problems caused by ‘low nutrient filaments’ depends on the organic
substrate flux into the flocs. A lack of N can be also masked by its chemical form, as exemplified in
processes for biological pretreatment of dairy wastewater. The dairy wastewater was concentrated (BOD5
around 1, 500 mg/l), and its N content corresponding to a BOD5 to N ratio suitable for biological
treatment. However, this N, analytically revealed as organic N, was only slowly degradable and thus
unavailable for growth of floc-forming organisms. The fact that this system was paradoxically N limited
was indicated by almost zero concentrations of ammonia in the effluent and excessive amounts of type
021N. Once ammonium sulphate was added at a level to keep effluent NHþ 4 concentrations between
2–4 mgN/l (the plant discharged the effluent to a public sewer network), settling properties visibly
changed within a month and the sludge started to settle in secondary clarifier – (Figures 7.6, 7.7, see
colour image section (chapter 13)). Further operation of the plant proved that this dosage had to be
maintained to keep SVI values acceptable. Similar experiences can be found in biological treatment of
pulp and paper wastewaters, which are often short of both N and P.
As guidelines for nutrient dosing, the following empirical concentrations in the final effluent might
be used: ammonia nitrogen at least 1 mg/l, soluble orthophosphate phosphorus more than 0.2 mg/l
(Wanner, 1994a).
pH. As far as is known, none of the common filaments exhibits a special preference for extreme pH
values. Some fungi prefer low pH values, although such a decrease in the pH in municipal wastewater
treatment plants (e.g. by nitrification) supporting excessive fungal growth is improbable. Lime dosing
equipment should be considered when an acid stream might be discharged into the plant.
Temperature. Temperature affects rates of all biochemical processes as well as O2 solubility in mixed
liquor. Thus, elevated temperatures may support growth of filamentous bacteria associated with such low
DO concentrations, and the oxygenation capacity of the aeration systems should be calculated for the
highest mixed liquor temperatures expected during the year, to guarantee the recommended DO
concentration can be achieved in warm weather.
There are reports of striking seasonal shifts in the dominance of individual filament types. Typically,
Candidatus ‘M. parvicella’, which dominates in winters, is replaced by Mycolata, type 0041 or Nostocoida
limicola in warmer months (chapter 5). Thus, according to more than ten years of microscopic
examination of activated sludge from the Central WWTP of Prague, more severe Candidatus
‘M. parvicella’ bulking problems are to be expected in winters than in summers when other types
dominate (e.g. Mycolata). This shift is almost certainly connected with temperature changes and one
possible persuasive explanation for it is given by Andreasen and Nielsen (2000).
Control measures based on ecophysiological features of bulking filamentous bacteria

Several studies on the ecophysiology of filamentous bacteria using FISH-MAR and associated techniques
(chapters 3, 5) have been conducted, providing some understanding of their in situ physiology that may
explain why they are present in particular treatment plants and therefore indicate how they may be
controlled. Detailed description of individual filaments are presented in other chapters (chapter 12), so
here we have summarized their key features. Table 7.5 shows which filaments can use electron acceptors
other than O2, and their primary sources of nutrients, and whether these are soluble or particulate, based
on such ecophysiological studies. Several filamentous bacteria appear well adapted to grow on a wide
range of soluble substrates by having a high substrate uptake rate, or a large storage capacity, a relatively
high growth rate and lack of any exoenzymatic activity for degradation of macromolecules. These occur
mainly in systems with high levels of soluble substrates, such as industrial plants, and include some of the
Alphaproteobacteria (e.g. those with a Nostocoida limicola morphotype) and the gammaproteobacterial
Thiothrix and type 021N.
Another group seems well adapted to grow in systems, typically low loaded municipal plants, which
require degradation of particulate substrates. These are characterized by being able to assimilate only a few
substrates at low uptake rates, with low or no storage capacity, and production of many exoenzymes.
Furthermore, many of these bacteria are typically present inside the flocs where they often form the
important structural backbone (chapter 3). This group includes the filamentous bacteria with attached
growth (‘Chloroflexi ’, Curvibacter-related species and TM7-related species, which include most of
morphotypes types 0041, 1701 and 1851), H. hydrossis and related species, and Candidatus ‘M. parvicella’.
Table 7.5. Overview of the ecophysiology exhibited by common gene-probe defined filamentous bacteria present in activated sludge and
possible control measures.
Growth on
Primary Soluble(S)/
Carbon/energy Particulate(P) Storage Possible control
e-acceptors sources substrates capacity measure
Alphaproteobacteria
(Candidatus O2, (NO3, NO2)# SCFA and other S Large Selectors (anoxic),
‘Monilibacter soluble substrates substrate gradients
batavus’ , Candidatus
‘Sphaeronema italicum’,
Candidatus
‘Alysiomicrobium
bavaricum’, Candidatus
‘Alysiosphaera
europaea’,
and M. perideroedes)
Betaproteobacteria
Curvibacter-related O2, (NO3, NO2, Proteins P Small Longer sludge age
(some morphotype anaerobic)# Compartmentation
1701 and others) of aerobic tanks
Gammaproteobacteria
Thiothrix O2, (NO3, NO2, SCFA, reduced S Average Selectors (anoxic),
anaerobic)# sulfur compounds substrate gradients
Removal of sulfide
Type 021N O2, (NO3,)# SCFA, glucose, S Nd Selectors (anoxic),
(Thiothrix eikelboomii ) (reduced sulfur substrate gradients
compounds) Removal of sulfide
Bacteroidetes
Haliscomenobacter O2 Polysaccharides, S+P Small Longer sludge age
hydrossis glucose and proteins Compartmentation
of aerobic tanks
‘Chloroflexi ’
Eikelboom O2 Polysaccharides P Small Longer sludge age
Type 1851 and and proteins Compartmentation
related filaments of aerobic tanks
Thin ‘Chloroflexi’ O2 Sugars P Small Longer sludge age
Compartmentation
of aerobic tanks
209
(continued)
210
Growth on
Primary Soluble(S)/
Carbon/energy Particulate(P) Storage Possible control
e-acceptors sources substrates capacity measure
Candidate division TM7

Some Type O2, ( NO3, NO2, Polysaccharides and P Nd Longer sludge age
1851 and 0041 anaerobic)# proteins Compartmentation
of aerobic tanks
Actinobacteria
Candidatus O2, NO3, (NO2, LCFA P Large Removal of lipids,
‘Microthrix parvicella’ anaerobic)# high oxygen and low
ammonium in
nitrification tanks
PAC (polyaluminium
chloride)
Gordonia amarae O2, (NO3, NO2, SCFA; LCFA, other S+P Large Dosage with chemicals
anaerobic)#
Skermania piniformis O2, (NO3, NO2, LCFA and SCFA S+P Large Dosage with chemicals
anaerobic)#
Candidatus O2, (NO3, NO2, LCFA, other P Nd ?
‘Nostocoida limicola’ anaerobic)#
Nd: not determined

#
Substrate uptake less than under aerobic conditions, possibly only for storage
+
not all species, for details consult Aruga et al., 2002
SCFA ¼ short chain fatty acids

LCFA ¼ long chain fatty acids
A third group consists of the foam forming Mycolata (primarily Gordonia and Skermania spp.). They
also grow relatively slowly on soluble substrates but seem primarily associated with plants treating
wastewater containing high levels of lipids (particulates), but not always. They usually have very
hydrophobic cells and stabilize foams, but occasionally large amounts of these filaments can be present in
flocs without excessive foam formation (chapter 8). Gordonia and Skermania can be controlled by
chemical addition too (Kragelund et al., 2007c).
If we look at the electron acceptors used by these filamentous bacteria, they all use primarily O2. Many can
also use nitrate and nitrite, but generally with a large reduction in growth rate. Instead several take up
substrate in the presence of nitrate or nitrite, probably for storage, so these substrates can then be used
subsequently in periods of famine. It is still unclear to what extent they can carry out complete denitrification.
These include the filamentous Alphaproteobacteria (Kragelund et al., 2006). On the other hand, Candidatus
‘M. parvicella’ assimilates substantial amounts of long chain fatty acids under anaerobic conditions, but
growth is only apparently in the presence of nitrate or O2 (Hesselsoe et al., 2005; Rossetti et al., 2005).
This ecophysiological information helps explain why different filamentous bacteria may grow in
different types of treatment plants and importantly, how they might be controlled. For example, those able
to assimilate a wide range of soluble substrates could be controlled in plants by introducing substrate
gradients (e.g. selectors), and preferably also by including anoxic or anaerobic stages. For Thiothrix and
type 021N, any sulfide should also be removed. Most filaments able to degrade particulate substrates
are especially present in plants treating municipal wastewater, operating with relatively long sludge
ages, and with nutrient removal. They rarely cause severe bulking problems there (except Candidatus
‘M. parvicella’) and could not be controlled by selectors, although longer sludge ages and
compartmentation of aerobic tanks may help. Candidatus ‘M. parvicella’ can be controlled quite
efficiently by certain polyaluminium chloride compounds (PACs) (Roels et al., 2002; Nielsen et al., 2005).
Non-specific, abiotic methods of bulking control

All the above methods are targeted at controlling excessive growth of filamentous bacteria. However, in
wastewater treatment practice other methods of bulking control are popular. These are not aimed at
controlling filament growth but at suppressing the problems resulting from their presence, or at their total
elimination.
Elimination of the filamentous population

This method is applicable for eliminating the problem of interfloc bridging. It exploits the fact that the
relatively thin filaments are exposed directly and immediately to concentrations of chemicals in the bulk
liquid, while most of the floc-forming organisms are exposed to lower concentrations because of diffusional
resistance in the outer layer of the flocs. Therefore, any filaments protruding from the flocs will be
selectively killed by adding toxic compounds to the RAS stream, while most of the floc-forming organisms
will survive inside the flocs. For the full-scale control of filamentous bulking, chlorine is used most often
(Jenkins et al., 2004b), but not in all countries. However, chlorination of activated sludge is a remedial
method, and should not be chosen as a permanent solution to bulking problems (Wanner, 1994a,d).
One promising method, which is now being studied in both pilot-scale units and full-scale plants, is the
selective destruction of filaments by the effect of cavitation, which can be started by ultrasound (Show
et al., 2007), although cavitation generated by hydraulic phenomena is more popular. For instance,
Schmid (2005) explained this cavitation whereby imploding bubbles generated by it create a highly
turbulent flow regime which stresses filamentous bacteria by inducing an intra cellular protection strategy
against the imposed mechanical stress. This reduces their selective growth advantage and thus reduces
the risk of bulking sludge development. The cavitation bubbles are generated inside a nozzle fed by a
submersible pump, where activated sludge is accelerated until evaporation pressure is reached and
cavitation starts at the smallest diameter. Specific energy input is small and lies significantly under the
critical value for microbial disintegration. Continuous exposition of the sludge into such conditions
reduces not only the length of the filaments but their total numbers. The flocculation properties of the
biosolids sludge are not harmed by this technique. This non-specific process of bulking control requires no
chemicals for operation. The technology is marketed under various trade names like ‘‘cavitation selector’’
(EMU GmbH, Germany) or ‘‘sludge squeezer’’ (Hans Huber AG, Germany). Figure 7.8 shows the pilot-
plant cavitation nozzle developed and tested in the Czech Republic and Figure 7.9 the settling test data
during the trial operation.
Figure 7.8. Cavitation nozzle of pilot plant installation for bulking control (courtesy of SIGMAINVEST,
Engineering Division, Olomouc, Czech Republic).
Increase of sedimentation velocities

As described earlier, excessive presence of filamentous bacterial populations reduces the sludge settling
velocities by generating floc bridging and an ‘‘open’’ floc structure. One way to compensate for this is to
increase the specific weight of the individual activated sludge flocs, as follows.
. addition of mineral suspensions to the mixed liquor (e.g. soil suspension, primary effluent particles
by reducing the capacity of primary settling tanks)
. addition of digested sludge to the mixed liquor (this must be performed very carefully to avoid any
DO deficit in the aeration basins)
. formation of heavy ‘‘cores’’ in the flocs by precipitation of mineral salts, the most common
method of bulking control with this approach is adding ferric salts to the mixed liquor (Wanner
et al., 2000)
Figure 7.9. Setting tests on sampler of mixed liquor taken during the operation of the cavitation pilot plant
unit.
Figure 7.10. Classical construction of the flocculation zone in a secondary clarifier.

The experience from Central WWTP of Prague shows that the current operation with rather limited
capacity of secondary clarifiers would not be possible without continuous dosing of the mineral salts
(ferric and aluminum salts).
Compensation of slow settling velocities

One possible solution is to construct new secondary clarifiers which are less susceptible to poor settling
properties of the activated sludge than the older constructions. Secondary clarifiers more resistant to
bulking sludge should exhibit the following features (ATV Design Standard A131, 2000; Ekama et al.,
1996; Wanner, 2006b):
. a specially designed zone for efficient flocculation of activated sludge (Figure 7.10)
. deeper settling tanks providing enough ‘‘buffering’’ capacity
. reduction of any hydraulic or mechanical disturbances in the layer of settled sludge to avoid
sludge re-suspension.
The final solution to bulking problems in the future will probably be the application of membrane
separation instead of secondary clarification (see chapter 3).
8
Foaming
Francis L. de los Reyes III
INTRODUCTION
Foaming, the ‘‘formation of a stable, viscous, chocolate-colored scum layer on the surface of aeration
basins and secondary clarifiers’’ (Jenkins et al., 2004b) is a widespread solids separation problem in
activated sludge (Figure 5.2). While other types of foaming may occur, such as the white foam arising
from poorly biodegradable surfactants (Jenkins et al., 2004b), the foaming problem discussed here is the
highly stable brown foam or scum caused by certain bacteria. The first published report appearing in 1969
(Anon, 1969), described the presence of thick masses of microbial filaments, which under the microscope
revealed the dominant bacteria were Gram positive, right angled branching Actinomycetes. These were
later identified as members of the genus Nocardia (Lechevalier and Lechevalier, 1974). It seems that
foaming problems were becoming more common around that time, and reports of scum formation began
to appear in the literature (e.g. Wells and Garrett, 1971).
Consequently, these foams were called Nocardia foams, but this term is incorrect for every episode, for
several reasons. Firstly, other organisms have been shown, or are thought to cause foaming, as described
later. Secondly, continuing work on the taxonomy of the Actinobacteria Mycolata has led to major
changes to their classification including the transfer of Nocardia rhodochrous to the genus Rhodococcus.
The best known foam former, Nocardia amarae, has been reclassified as Gordona amarae (Goodfellow
et al., 1994; Klatte et al., 1994b; Ruimy et al., 1994), since changed to the etymologically more correct
name Gordonia (Stackebrandt et al., 1997), which will be used throughout this chapter. It is thus prudent
to specify which is the causative organism, if known, for an individual foam (e.g. Gordonia amarae
foam), or to use a more general term ‘Mycolata foaming’ to describe foaming caused by mycolic acid-
containing Actinobacteria, whose precise identification may not be known.
OCCURRENCE AND INITIAL STUDIES

The extent of this foaming problem is best discussed in terms of survey data from large numbers of
treatment plants. In the U.S., early surveys were undertaken originally to delineate bulking problems
in activated sludge. In 1982 (Richard, 1989) showed 31% of 270 activated sludge (AS) plants from 35
states contained ‘Nocardia’, which were reported as the second most abundant filament. A 1984 survey
(Strom and Jenkins, 1984) of 78 laboratory, pilot, and prototype plants from 20 states also showed
‘Nocardia’ was the second most frequently occurring filament. Those specifically designed to delineate
foaming problems were also conducted. Thus, Pitt and Jenkins (1990) in the U.S., found that of 114
responses, 75 plants (66%) had experienced some foaming. In Illinois, 30 of 31plants (97%) had foamed
at one time or another (de los Reyes et al., 2002), while in North Carolina, 88% of 47 plants responding
had foaming problems (Keith, 2002).
Foaming occurs worldwide. Of 129 plants sampled in 1988 in Queensland, New South Wales, and
Victoria (Australia), 66 foamed during the sampling period (Seviour et al., 1990b). Later, of 65 Australian
plants, Seviour et al. (1994) reported that 53 experienced bulking, while 44 produced foams. Blackall et al.
(1991c) showed that in 46 plants in Queensland, only 8% had never experienced foaming. A survey of
39 Italian plants (Rossetti et al., 1994) also revealed a predominance of both bulking and foaming
filamentous bacteria, while a later study showed that 84 of 167 plants had foaming problems
(Madoni et al., 2000). A comprehensive survey of 6013 plants in France (Pujol et al., 1991a) representing
75% of all French plants operating at that time, revealed that 1192 plants were affected by foaming, while
in 111 South African plants, (Blackbeard et al., 1986a) foaming was reported in 68% of them. The larger
plants (flows 440 m3/d) described the problem as difficult to manage (‘impossible’?) while foaming was
more manageable in the smaller plants. In the Netherlands, of 70 Carrousel (oxidation ditch) plants
48% and 17% of those examined foamed during spring and autumn respectively (Eikelboom, 1994a),
while 50% of EBPR plants in Denmark had foaming problems (Wanner, 1994d). Similar work in the
Czech Republic (Wanner et al., 1998b) suggested that foaming incidents were increasing. Foaming has
also been reported in activated sludge plants in Japan (Hiraoka and Tsumura, 1984a; Mori et al., 1992),
Sweden (Holmstrom et al., 1996), Austria (Franz and Matsche, 1994), Germany and Switzerland
(Lemmer and Kroppenstedt, 1984), Saudi Arabia (Goddard and Forster, 1987b), Hong Kong (Chua and
Le, 1994), and the UK (Goddard and Forster, 1986).
PROBLEMS ASSOCIATED WITH FOAMING

The global occurrence of filamentous foaming has attracted considerable interest over the past two
decades. A foam layer can be more than a meter thick (Goddard and Forster, 1987a,b), and accumulate
to such an extent that it overflows the basin freeboard and covers walkways, handrails, and surrounding
areas, creating hazardous, slippery conditions, and preventing access to equipment (Jenkins et al., 1993)
(Figure 5.2). The following have been cited as operating problems associated with foaming (Blackall
et al., 1985): a) extra housekeeping on the part of the operator; b) blockage of foam removal systems;
c) reduction of oxygen transfer at the surface of mechanically aerated basins; d) carriage and possible
dispersal of pathogens in wind blown foam; e) drying of foam with resultant cleaning and odour
problems; and f) reduction of effluent quality through an increase in effluent suspended solids and BOD,
if foam reaches the final effluent. Furthermore, foams have been known in covered aeration basins to
accumulate to such an extent that the increased water head in the basin prevents influent wastewater
from entering the basin (Jenkins et al., 1993). Process control becomes extremely difficult in situations
where high solids inventory is trapped in the foam, and Hao et al. (1988a) estimate that up to 20% of
Foaming 217
the total sludge inventory can be in the foam. In sealed pure oxygen activated sludge systems, foam
may enter the oxygen compressors, creating a fire hazard (Hanson et al., 1992).
Problems with foaming in anaerobic digesters are frequent and linked to occurrence of filamentous
foaming in the aerobic reactors. A survey by the American Society of Civil Engineers revealed anaerobic
digester foaming is a persistent problem, with over half of the installations surveyed having experienced
foaming problems at one time or another (Filbert, 1985). Similarly, van Niekerk et al. (1987b) showed
that 54% of anaerobic digesters surveyed had foaming problems that could be attributed to ‘Nocardia’
in the waste AS feeds. Such foams have caused blockage of gas mixing devices, inversion of solids
profiles, entrapment in and fouling of gas collection pipework, gas binding of sludge recirculation plants
leading to inability to heat the digesters, foam penetration between floating covers and digester walls,
and tipping of floating covers by rapid expansion and then collapse of the sludge volume when gas mixers
are operated intermittently (Jenkins et al., 1993; van Niekerk et al., 1987b). Pagilla et al. (1996a) reported
that foam in the digested sludge covered the surface of sludge storage basins at the Sacramento Regional
Wastewater Treatment Plant (Sacramento, CA), thereby preventing sunlight from reaching photosynthetic
algae, and creating odor problems. Foams may also occupy large volumes in anaerobic digesters, and
Hernandez (1994) showed that at the Southeast Water Pollution Control Plant (San Francisco, CA), foam
occupied up to one-half their volume.
Perhaps the greatest impetus for foaming research lies in the possible public health hazard of pathogens
spreading through creation of aerosols. Several members of the genus Nocardia are pathogenic. However,
Nocardia are comparatively rarely seen in foams (see later) and so far there has been no reported case of
nocardiosis or other disease attributed to foams or aerosols from foams among wastewater treatment
plant personnel. Nevertheless, the public health implications of foaming should be of grave concern, and
underscore the importance of distinguishing pathogenic from non-pathogenic isolates of the causative
organisms.
WHICH MICROBES ARE IN FOAM?

Identification of the causative microorganisms in foam is important in attempts to understand their growth
and ecophysiology. In turn, such understanding will allow the identification of operational causes, and
guide rational control methods. The three approaches used to identify microbes in foam are: microscopy,
isolation and identification in pure culture, and molecular methods.
Microscopic identification and relative incidence of foam microbes

Early studies of microbial communities in activated sludge relied mainly on microscopic examination.
One of the first attempts to develop an identification key based on morphological and physiological
tests was reported by Farquhar and Boyle (1971a). A more practical and useful method for
filament identification was developed by Eikelboom (1975, 1977). His identification key (chapter 6)
used morphological characteristics and simple staining reactions, and with it he was able to ‘classify’
filamentous organisms into 26 ‘morphotypes’. Because this method was not based on phylogeny or
conventional taxonomy, over half of the filaments he described were given ‘‘type’’ numbers and not valid
names (see discussion in chapters 5,6) Subsequent modifications to Eikelboom’s key took these
limitations into account (at least partially). They were adopted to ‘de-emphasize the need for the
observation of cell septa, which can depend on quality and adjustment of the microscope used; and to
include some filamentous organisms in the key twice where an important characteristic is variable,
e.g. Gram stain reaction and the observation of intracellular sulfur granules. . .’ (Jenkins et al., 1993).
Early microscopic examination of foam (pre-1980s) showed a dominance of Actinobacteria with

characteristic Gram positive, right-angled branching filamentous cells. Since then, other organisms have
been identified as dominant in the foam layer. Table 8.1 summarizes those identified in plant surveys from
around the world.
Table 8.1. Bacteria identified in activated sludge foams by microscopy.

NALO/GALO Most Gram positive, right angled branching bacteria are
reported as ‘Nocardia’ or NALO (Nocardia amarae-like
Gordonia spp. Nocardia spp.
organism). Some describe these morphotypes as GALO
(Gordonia amarae-like organism), after the transfer of N.
amarae to the genus Gordonia. The designations NALO
and GALO are used since rhodococci, mycobacteria and
other Mycolata may share a similar morphology under
certain conditions. A better term is to refer to these as
Mycolata, which includes all members of the mycolic
acid-containing Actinobacteria classified in the suborder
Corynebacterineae
Candidatus ‘Microthrix parvicella’ Major cause of foaming in Europe, Australia and South
Africa. Becoming more common in the US as a cause of
both foaming and bulking
Skermania piniformis A mycolic acid-containing actinomycete with a charac-
teristic acute-angled branching. Originally termed PTLO
(Pine tree-like organism). Has been observed in
Australia and Europe, and occasionally in the US
‘Actinomycetes’ Seviour et al. (1994) define these as Gram positive
branched rods without the distinctive branching patterns
of NALO/GALO and S. piniformis
Eikelboom Type 0092 Dominant organism found in South Africa. Other work
raises doubts as to whether it is a true foam former
Eikelboom Type 0041 Type 0675 was observed in French and German
surveys. Some studies do not distinguish between
Eikelboom Type 0675 0041/0675 morphotypes (chapter 12)
‘Nostocoida limicola’ II More commonly observed in Czech surveys
Eikelboom Types 0803, 0581, 1851, 0413, Occasionally reported as dominant in foam, but their
021N, 0914, 1701, Haliscomenobacter incidence is low
hydrossis, Sphaerotilus sp., Acinetobacter sp.,
and Cyanobacteria.
Based on data in (Blackall et al., 1989; Blackbeard et al., 1986b; Chun et al., 1997; Duchene, 1994b; Eikelboom, 1975;
Eikelboom et al., 1997; Goddard and Forster, 1987; Lechevalier and Lechevalier, 1974; Lemmer et al., 2005; Madoni et al.,
2000; Pujol et al., 1991a; Richard, 1989; Seviour et al., 1994; Seviour et al., 1990; Sezgin, 1988; Wanner and Grau, 1989;
Wanner et al., 1998b).
The first detailed report of a microscopic examination of foam described the presence there of thick
masses of filaments (Anon, 1969), which were ‘identified’ as members of the genus Nocardia. There was
no mention of cause and effect (the relationship of these filaments to the foaming problem) (Anon, 1969).
Many early surveys show that Nocardia was dominant in bulking and foaming plants (Jenkins et al.,
2004b), although data from around the world revealed the dominance of different organisms (Table 8.2).
Table 8.2. Ranking of dominance of filamentous bacteria reported in activated sludge foams.
Czech
Organism USA France Australia Netherlands South Africa Italy Germany Republic
NALO/GALO 1 3 2 2 2 2 3 2
Candidatus 6 1 1 1 1 1 1 1
‘M. parvicella’
PTLO/Skermania – – 4 – – – – –
piniformis
‘Actinomycetes’ – – 7 – – – 2 –
or Gram positives
Type 0092 7 4 6 – 1 – 4 –
Type 0041/0675 4 2 3 – 3 3 6 –
‘Nostocoida 9 – 5 3 – – 5 3
limicola’
Foaming
Eikelboom Types varies minor – – varies – minor –

0803, 0581, 1851,
0413, 021N, 0914,
1701,
Haliscomenobacter
hydrossis,
Sphaerotilus sp.,
Acinetobacter sp.,
and Cyanobacteria
References Jenkins Pujol et al. Seviour Eikelboom Blackbeard Madoni Lemmer Wanner
et al. (2004b); (1991a) et al. et al. (1997) et al. (1986a) et al. et al. et al.
Richard (1989); (1994b) (2000) (2005) (1998b)
Strom and
Jenkins (1984)
– not reported or rated as very low.

219
Several observations can be drawn from these data in Table 8.2.
(i) It is evident that Candidatus ‘Microthrix parvicella’, a Gram-positive unbranched filament previously
associated with bulking (Eikelboom, 1975), is the commonest dominant organism in most foam
surveys, especially in Europe, Australia, and South America, but less so in the U.S. A more
comprehensive survey of treatment plants in the U.S. is necessary to resolve the question of which
organism/s is really predominant in foam. It should be noted that these US survey data combine
results from 1982 (Richard, 1989) and 1984 (Strom and Jenkins, 1984). High levels of Candidatus
‘M. parvicella’ in biological nutrient removal plants were noted in South Africa (Blackbeard et al.,
1986a), Denmark (Kristensen et al., 1994), Germany (Kunst and Reins, 1994), the Netherlands
(Eikelboom, 1975), and in the European Union survey (Germany, Greece, Denmark, the Netherlands)
(Eikelboom et al., 1997). This may reflect the changing redox conditions in EBNR systems (anoxic-
aerobic for N removal; anaerobic-aerobic for P removal) and the capability of some filaments to
utilize substrates under both anoxic and anaerobic conditions (Nielsen et al., 2000). Temperature also
seems influential with Candidatus ‘M. parvicella’ whose levels in plants are higher at lower (420– C)
operating temperatures (Eikelboom, 1994b). It occurs more commonly than Mycolata in plants in
colder areas in Australia (Seviour et al., 1990a) and the U.S. (Richard, 1989) (chapters 5, 7).
(ii) While earlier surveys in the US and Australia showed the Mycolata were prevalent in foam, these
now seem the second most frequently reported foamers worldwide. In Australia, Blackall et al.
(1991c) reported that 27 of 46 plants had foams dominated by actinomycetes, while pine tree
like organisms (PTLOs) were ranked third. These were later identified as Nocardia pinensis
(Blackall et al., 1989), now Skermania piniformis (Chun et al., 1997). Soddell and Seviour (1990)
suggested that S. piniformis is not restricted to Australia, since several other studies
showed photomicrographs of organisms with the same characteristic pine tree branching. This
has been confirmed by reports of PTLO (presumably S. piniformis) in foams in North America
(de los Reyes et al., 2002) and Denmark (Eales et al., 2005). Other unidentified Mycolata with the
PTLO morphotype certainly exist in foams (Kragelund et al., 2007b).
(iii) Organisms other than unicellular ‘actinomycetes’ and Candidatus ‘M. parvicella’ have also been
reported in foam. Both the N. limicola II morphotype and Type 0041 were held responsible for the
first serious case of foaming in the Czech Republic (Wanner and Grau, 1989), while Duchene
(1994a) reported that Eikelboom Types 0675 and 0092 were associated with 20% and 5%,
respectively of 1192 foaming cases in France. Type 0803 was also present in 1% of these,
especially in very old foams. Blackbeard et al. (1986a) also found that Type 0092 was enriched in
foams compared to the corresponding mixed liquor, although only by 1.35 fold compared to 2.3
and 2.1 fold for Candidatus ‘M. parvicella’ and ‘Nocardia’ spp.
(iv) The following organisms have also been reported as major members of some foam communities:
Type 0914 (Seviour et al., 1990b; Wanner and Grau, 1989), Type 0581 (Seviour et al., 1990b),
Nostocoida limicola (Goddard and Forster, 1987), Type 0041, Type 0803, Type 0675, Type 1841,
and Haliscomenobacter hydrossis (Blackbeard et al., 1986a). Goddard and Forster (1987)
also described Type 0041 in foams in laboratory-scale reactors. Soddell and Seviour (1990)
have reported the frequent presence of unidentified Gram-positive cocci in Australian foams,
and hypothesized that they may be Rhodococcus spp. in their coccal life cycle stage. Gram
positive unicells were also observed as the third most important organisms in foams in Germany
(Lemmer et al., 2005), while Gram positive rods were the second most common in Czech
Republic foams (Ruzickova et al., 2006).
Foaming 221
Isolation and identification in pure culture

The contention that Nocardia filaments were major causative foaming organisms probably arose because
Lechevalier and Lechevalier (1974) isolated and described a new filamentous organism from activated
sludge foam, which they named Nocardia amarae, from the Greek amara meaning sewage duct. It is
interesting that the authors did not insist that this organism was causing the foam, only that it was
commonly seen in foams in the plants they examined (10 from 5 states in the U.S.). Furthermore, they
isolated other species including Nocardia rhodochrous (now Rhodococcus rhodochrous) and another
strain related to N. rhodochrous. Sezgin et al. (1988) later report that N. asteroides, N. caviae,
Streptomyces spp., Micromonospora spp., and Actinomadura sp. were all present in the same samples.
Nevertheless, most cases of foaming until quite recently have been given the generic name ‘‘Nocardia
foams’’. The choice of this term was probably vindicated by repeated isolations of N. amarae from foams
(Blackall et al., 1988; Dhaliwal, 1979; Hiraoka and Tsumura, 1984b). Soddell et al. (1992) questioned the
adequacy of this term in light of the isolation of several taxonomically diverse branching actinomycetes
from foams. They asserted that the term NALO, or ‘nocardioforms’, was a better descriptive term for
filaments ‘identified’ on the basis of their morphology alone, for measuring their diversity in foam.
High levels of biodiversity among actinomycetes in foams have emerged slowly after numerous isolation
and culturing studies. For example, Sezgin et al. (1988) obtained 105 isolates from two wastewater treatment
plants. Of these 51 were ‘N. amarae’, and 24 were N. asteroides. The others were identified as Rhodococcus
coprophilus, R. equi, R. erythropolis, R. maris, Mycobacterium fortuitum, and strains belonging to the
genera Oerskovia, Amycolaptosis, and Micromonospora. Cluster analysis was performed by Lemmer and
Kroppenstedt (1984) on strains from plants in Germany and Switzerland. They isolated 11 strains of
Rhodococcus, two of Gordonia aurianticus (now Tsukamurella spp.), and a single strain of ‘N. amarae’.
However, a more realistic estimate of the extent of actinomycete diversity in foam was revealed
by Goodfellow et al. (1996, 1997) after selective isolation, chemotaxonomy, and pyrolysis mass spectro-
metry (PyMS). Actinomycetes presumptively identified as Gordonia, Rhodococcus, and Tsukamurella were
obtained, and most fell into groups corresponding to several Gordonia spp. (G. amarae, G. hydrophobica,
G. aichiensis, G. bronchialis, G. obuensis, G. rubropertincta, G. sputi, G. terrae).
While culturing techniques are useful in revealing such population diversity, methods like dilution
plating can be criticized (chapters 1, 3) for their biases and limitations, and thus their failure to reveal
most organisms present in a sample, their true biodiversity, and their activity in natural environments
(Amann et al., 1995; Brock, 1987). The majority of cells that do not grow on laboratory media are:
(i) known species for which the applied cultivation conditions are unsuitable, or which have entered a
non-viable state, and (ii) unknown species that have never been cultured for lack of suitable methods
(Amann et al., 1995). Additionally, isolation of a strain from a particular habitat provides no evidence of
its potential activity (function) in that environment (Cross, 1981). These considerations are especially
important for activated sludge filamentous bacteria, which are often very fastidious and grow slowly
(Slijkhuis, 1983; Soddell and Seviour, 1990; Tandoi et al., 1998b).
Soddell and Seviour (1990) recommended micromanipulation (Skerman, 1968) as the technique which
allows the ‘‘isolation, in pure culture, of individual filaments directly observed under the microscope’’,
and proposed that all future studies should utilize this simple and inexpensive technique. It involves
‘identifying’ individual filament morphotypes under the microscope, separating these from the rest of the
biomass with a glass ‘hook’, then growing the separated cells on a suitable medium. Micromanipulation
was used to isolate N. pinensis (Skermania piniformis) from foams in Australia (Blackall et al., 1989),
and subsequent studies have used this technique successfully to obtain Candidatus ‘M. parvicella’
(Blackall et al., 1994b, 1996; Blackall et al., 1991a) and other filaments (Hornsby and Horan, 1994).
Soddell et al. (1992) successfully used it for isolating filaments with the characteristic branching of
G. amarae or S. piniformis, and obtained 26 strains subsequently grouped into 8 taxonomic groupings
based on cluster analysis of 91 morphological and biochemical characteristics (Goodfellow et al., 1982b).
These groupings revealed novel rhodococci and mycobacteria, as well as Nocardia species, G. amarae,
and S. piniformis. Differences in morphologies in pure culture and in situ arising from growth rate
changes, and branching and fragmentation patterns revealed the complexity of foaming actinomycete
ecology. Thus, it became clear from this study that a typical ‘Nocardia’ foam under the microscope was
composed of a very diverse community of organisms. A total of 54 isolates of S. piniformis were obtained
(Soddell and Seviour, 1994). While large variations in morphology (branching angle, branch lengths, and
interbranch distances) were observed, these cultures were consistent in their physiological properties. This
led to the hypothesis that the variation in filament morphology in situ reflects differences in environmental
conditions. Interestingly, a shift in morphology from filamentous to single-cell forms was also observed
by Ramothokang et al. (2006), which they attributed to a change in nutrient levels, pH, and other factors
during culturing.
Table 8.3 shows which bacteria have been isolated into pure culture from foams. While many Mycolata
have been obtained, growing Candidatus ‘M. parvicella’, the most common foam-forming organism,
in pure culture has been problematic. The original isolates of Slijkhuis (1983) are no longer viable. The
review of Rossetti et al. (2005) summarizes the saga of the efforts made to culture this filament . Currently
only three recognized strains are held as pure cultures and data from them have advanced considerably our
knowledge of its physiology and phylogeny (Rossetti et al., 2005). Six out of 12 putative Candidatus
‘M. parvicella’ morphotypes isolated from industrial activated sludge plants were shown to be members
of another ‘Microthrix’ species. These were named Candidatus ‘Microthrix calida’, and some of their
physiological characteristics were distinctively different (Levantesi et al., 2006b). FISH probing data
suggest the possibility of further biodiversity within the Microthrix morphotype.
Table 8.3. Dominant microbes isolated in pure culture from activated sludge foam.
Current name of foam isolates Reference(s)

Mycolic acid-producing actinomycetes
Dietzia Sezgin et al. (1988)
Gordonia amarae Lechevalier and Lechevalier (1974)
Lechevalier et al. (1976)
Dhaliwal (1979)
Sakai et al. (1983)
Lemmer and Kroppenstedt (1984)
Hiraoka and Tsumura (1984)
Blackall (1987)
Sezgin et al. (1988)
Soddell et al. (1992)
Gordonia spp. Goodfellow et al. (1996)
Soddell and Seviour (1997)
Soddell et al. (2006b)
Millisia brevis Soddell et al. (2006a)
Mycobacterium fortuitum Sezgin et al. (1988)
Mycobacterium spp. Lechevalier et al. (1976)
Foaming 223
Table 8.3. Continued.
Current name of foam isolates Reference(s)

Nocardia asteroides Lechevalier et al. (1976)
Nocardia farcinica Stratton et al. (1996a)
Nocardia otitidiscaviarum Lechevalier et al. (1976)
Nocardia takedensis Yamamura et al. (2005)
Nocardia spp. Sezgin et al. (1988)
Rhodococcus coprophilus Lemmer and Kroppenstedt (1984)
Rhodococcus equi Lemmer and Kroppenstedt (1984)
Rhodococcus erythropolis Sezgin et al. (1988)
Rhodococcus globerulus Sezgin et al. (1988)
Rhodococcus rhodochrous Lechevalier et al. (1976)
Sakai et al. (1983)
Lemmer and Kroppenstedt (1984)
Rhodococcus ruber Lemmer and Kroppenstedt (1984)
Rhodococcus rubra Lemmer and Kroppenstedt (1984)
Rhodococcus spp. Lemmer and Kroppenstedt (1984)
Soddell and Seviour (1997)
Skermania piniformis Blackall et al. (1989)
Soddell and Seviour (1994, 1998)
Tsukamurella paurometabola Lemmer and Kroppenstedt (1984)
Tsukamurella pseudospumae Nam et al. (2004a)
Tsukamurella spumae Goodfellow et al. (1997)
Nam et al. (2003)
Other actinomycetes
Candidatus ‘Microthrix parvicella’ van Veen (1973)
Eikelboom (1975)
Slijkhuis (1983a)
Blackall et al. (1994a)
Tandoi et al. (1998b)
Amycolatopsis sp. Sezgin et al. (1988)
Micromonospora sp. Lechevalier et al. (1976)
Actinomadura sp. Lechevalier et al. (1976)
Streptomyces sp. Lechevalier et al. (1976)
Oerskovia sp. Sezgin et al. (1988)
The importance of bacterial identification

From this discussion, we see why it is imperative that identification techniques not relying solely on
microscopic attributes are adopted in foaming research. Pure culture isolation and characterization, as well
as nucleic acid based techniques, particularly rRNA sequence analysis, are needed for more precise and
reliable identification (chapter 6). While these are technically more difficult and time-consuming than
microscopy, there are important advantages in being able to differentiate among the various Mycolata.
1. Although the foaming Mycolata are all closely related taxonomically, they may differ substantially
in their ecophysiology and growth kinetics. For example, many rhodococci grow fast, producing
colonies on agar in 2–3 days, while G. amarae may take a week, and S. piniformis up to three weeks.
These growth rate differences may explain the conflicting reports of success in controlling foaming
using MCRT reduction. Reducing MCRT may not always result in successful washout of the
particular Mycolata species if the organism involved is a fast grower. Richard (1989) early on
warned that the MCRT needed to control Mycolata foaming may depend on which populations are
involved, and this feature may be plant-specific. Similarly, conflicting reports on the effect of
temperature on foaming can be found in the literature, with some reports that ‘Nocardia’ foaming
occurs at temperatures less than 13– C (Dhaliwal, 1991b). It is more likely that these are
‘rhodococci’ able to grow at lower temperatures than G. amarae. The ‘causes’ supposedly favoring
filamentous growth, according to surveys of operators (de los Reyes et al., 2002; Jenkins et al.,
2004b), include: long MCRT (low F/M), high MLSS, high oil/grease, presence of industrial waste,
short MCRT (high F/M), low MLSS, nutrient deficiency, high temperature, low DO, and low flow.
One likely explanation for such conflicting reasons is that the foaming events observed by operators
are each caused by different organisms with different ecophysiologies.
2. It is important to know if pathogenic organisms are present in foams, because of their potential to
spread through creation of aerosols. Some Mycolata are known to be pathogenic, and can cause
diseases in humans including (a) pulmonary, neural, and/or systemic nocardiosis; (b) actinomycetic
mycetomas, which are tumor-like growths of the organisms within the tissues; and (c) localized
cutaneous or subcutaneous infections (Lechevalier, 1989). Nocardia asteroides, N. brasiliensis,
and N. otititidiscaviarum may act as primary pathogens in cases of actinomycetoma, or as causative
agents of secondary opportunistic infections (i.e. nocardiosis) in immunocompromised individuals
(Harris, 1980; Jimenez et al., 1990). Several species of Gordonia are opportunistic pathogens and
have been isolated from the sputum of humans (G. bronchialis, G. sputi, G. aichiensis) suffering
from pulmonary lesions (Bendinger et al., 1995). The isolation of Nocardia farcinica from foam has
served to highlight these concerns (Stratton et al., 1996). N. farcinica causes human infections
including pneumonia, cutaneous infections, and brain abscesses in traumatized or immunocom-
promised patients (Wallace and Steele, 1988). The risks may be greater than currently thought, since
this organism is resistant to many antibiotics (Wallace et al., 1991).
Thus, it is clear that more thorough analysis of isolates and more rapid and precise identification of
foaming organisms is needed, which explains the attraction of using molecular techniques such as the ones
described below.
MOLECULAR APPROACHES
Molecular techniques, particularly those based on the 16S rRNA gene, have made possible the study of
foam communities without any need for phenotypic identification or culturing. These techniques have
Foaming 225
expanded our knowledge of the occurrence, prevalence, and ecophysiology of organisms in foam. The
techniques used in the study of foam communities have been discussed in chapter 3, and include: PCR-
cloning-sequencing (with universal 16S rRNA gene primers), targeting specific populations with either
FISH probes or labeled oligonucleotide probes hybridized to extracted and immobilized rRNA, or PCR
approaches based on more specifically targeted primers. PCR-cloning-sequencing (chapter 11) is used to
answer the question of ‘who is present’ in the sample.
Schüppler et al. (1995b) used DNA extraction, PCR with universal 16S rRNA gene primers, cloning,
and sequencing to identify the ‘nocardioforms’ in activated sludge. Random sequencing of clones
revealed a high diversity. Colony hybridization of clones with an oligonucleotide probe targeting most
members of the Mycolata showed that only 27 of 3000 clones were ‘nocardioforms’. Comparative 16S
rRNA analysis further showed that all these sequences differed from those then deposited in the EMBL
and Genbank databases. Phenotypic identification of isolates cultured from the same samples revealed a
large discrepancy from those identified with 16S rRNA gene sequence data, where only one 16S rRNA
gene sequence of a cultured isolate was represented in the clone library.
A similar PCR-cloning-sequencing approach was performed on foam samples from a plant in South
Africa (Wagner and Cloete, 2002). The 25 sequences obtained clustered with Dietzia maris and other
members of the Mycolata, ‘Nostocoida limicola’ II, the Bacteroidetes group, bacterial clones from oral
cavities, Type 1701 and Sphaerotilus natans, Frankia, and other uncultured bacteria. Interestingly, the
dominant filament observed microscopically at the plant, Candidatus ‘M. parvicella’, was not
represented in their small clone library. This highlights the potential biases inherent in all PCR-cloning
methods (chapter 11), particularly when basing diversity estimates on small clone libraries. Similar
results were obtained when PCR-DGGE (chapter 11) was used to characterize populations in foam and
the mixed liquor. While Candidatus ‘M. parvicella’ by microscopy and FISH appeared to be dominant,
the DGGE bands representing Candidatus ‘M. parvicella’ had lower signal intensities than expected
after gel staining, (Eales, 2006). Again, this reflects problems in applying PCR based methods
quantitatively (chapter 11).
FISH has been used frequently to determine the in situ presence of foam-causing organisms, and
several FISH probes have been designed to target these (Table 8.4). Their application to cells in
foams has yielded important data. Several have revealed that FISH probes targeting all Mycolata
members detect filaments lacking the typical right-angled branching morphology of the GALO. FISH
probes have also revealed the presence of non-filamentous Gordonia (de los Reyes et al., 1997c; de
los Reyes and Raskin, 2002), ‘rhodococci’ (Davenport et al., 1998), Dietzia (Schuppler et al., 1995a),
and other Mycolata (Davenport et al., 2000). In fact quantitative FISH data have suggested that non-
filamentous (coccoid, rod-shaped) Mycolata in some foams may be more numerous and thus, more
important than those in the filamentous form (Davenport and Curtis, 2002; Davenport et al., 2000).
Considering the mechanisms proposed for foam formation, there is no fundamental reason why a
filamentous morphology is necessary for its formation and stabilization. The Mycolata filamentous
forms may have received most attention in the past, since they are more readily recognizable
microscopically, but that morphology is not necessarily why foams form (Davenport and Curtis,
2002). A study following changes in the morphology of Gordonia spp. as foaming progressed in US
(de los Reyes and Raskin, 2002) revealed that short, single rods were observed initially in March–
April, followed by brightly fluorescent clusters of rods in May, giving rise to the typical right-angled
branching filaments in June. These results raise interesting questions on how Mycolata morphology
may affect foaming potential, since foaming at this plant was visually observed only beginning
in May. A similar change in morphology has been recorded in pure culture chemostat studies
(see below).
Table 8.4. FISH oligonucleotide probes targeting the 16S rRNA of organisms in foam.
226
Target site site

(rRNA positions, % formamide
Probe name Target group E. coli numbering) Probe Sequence (5’ to 3’) in FISH Reference
S--Myb-0736-a-A-22 Mycolata 0736–0757 CAGCGTCAGTTACTACCCAGAG 30 de los Reyes

et al. (1997)
S--Myb-0736-b-A-22 Mycolata 0736–0757 CAGCGTCAGTTACTxCCCAGAGb 30 de los Reyes
et al. (1997)
S-G-Gor-0596-a-A-22 Gordonia 0596–0617 TGCAGAATTTCACAGACGACGC 20 de los Reyes
et al. (1997)
S-S-Gam-0192-a-A-18 G. amarae 0192–0209 CACCCACCCCCATGCAGG 30 de los Reyes
et al. (1997)
S-S-G.am-0205-a-A-19 G. amarae 0205–0223 CATCCCTGACCGCAAAAGC 30 de los Reyes
et al. (1998c)
MNP1 Mycolata 0152–0172 AACCCATGCAGGCCGTAGTCC 50 Schuppler
et al. (1998)
DLP Dietzia, 0182–202 CCACCATGCGGCAGGAGCTCA 40 Schuppler
Rhodococcus et al. (1998)
GLP2 G. amarae 0178–0197 AAGGGCAGGTCATATCCGGT 45 Schuppler
et al. (1998)
Rco1 Rhodococcus 0181–0193 ACCATGCAACCGGAGGTCAT 40 Davenport
et al. (1998)
Rco2 Rhodococcus 0611–0631 AGCCCGCAGTTGAGCTCCGGG 40 Davenport
et al. (1998)
Myc657 Mycolata 0657–0672 AGTCTCCCCTGYAGTA 30 at 37– C Davenport
et al. (2000)
Spin 1449 Skermania 1449–1466 CCGCTCCCTCCCACAAAG 35 Eales

piniformis et al. (2006)
MPA60 Candidatus 0060–0077 GGATGGCCGCGTTCGACT 20 Erhart
‘Microthrix et al. (1997)
parvicella’
MPA223 Ca. ‘M. parvicella’ 0223–0240 GCCGCGAGACCCTCCTAG 20 Erhart
et al. (1997)
MPA 645 Ca. ‘M. parvicella’ 0645–0661 CCGGACTCTAGTCAGAGC 20 Erhart
et al. (1997)
MPA650 Ca. ‘M. parvicella’ 0650–666 CCCTACCGGACTCTAGTC 20 Erhart
et al. (1997)
Foaming 227
FISH studies have also shown that individual cells within a filament may fluoresce at different intensities,
while some within the same filament show no FISH signal (Carr et al., 2005; de los Reyes et al., 1998; de los
Reyes and Raskin, 2002; Eales et al., 2006b; Kragelund et al., 2007b; Schuppler et al., 1998). It may be that
these cells are dead or moribund, or reflect differences in permeability to the FISH probes. Several studies
have shown that a high portion of cells in foam may have very low metabolic activity. Viability stains
(e.g. LIVE/DEAD stains – chapter 11) show that not all cells in a single filament had functionally intact
membranes (Carr et al., 2005). Similar observations have been made for Candidatus ‘M. parvicella’
(Eales, 2006). In addition, membrane hybridization studies showed that both Gordonia and Candidatus
‘M. parvicella’ have low intracellular rRNA contents, and importantly, that metabolic activity is not
essential to cause and stabilize foam (de los Reyes et al., 1998a,b,c; de los Reyes and Raskin, 2002;
de los Reyes et al., 2002; Oerther, 1999a,b). Thus, the important conclusion is how to prevent growth of
Mycolata in the first place, since even dead cells can be problematic (de los Reyes and Raskin, 2000).
An important issue in applying FISH probes to foam organisms is to ensure they can enter the
Mycolata cells, which are notoriously impermeable because their cell walls contain hydrophobic mycolic
acids which complex with arabinogalactans to produce a waxy outer layer (Macnaughton et al., 1994).
The standard parformaldehyde fixation procedure suitable for most Gram negative cells is not appropriate
with these Mycolata. Several FISH permeabilization procedures for FISH have been described
(Eales et al., 2006; Kragelund et al., 2007b), involving organic solvents (Schuppler et al., 1998), acid
hydrolysis (Macnaughton et al., 1994), and treatment with enzymes (Carr et al., 2005; Davenport et al.,
2000; Schuppler et al., 1998). These are summarized in Table 8.5.
Table 8.5. Permeabilization methods used for Mycolata and Candidatus ‘M. parvicella’.
Treatment Chemicals used Reference Success
Acid hydrolysis Hydrochloric acid Macnaughton et al. Worked with most
(1994) Mycolata tested, but
not all
Short-term fixation Paraformaldehyde de los Reyes et al. Varied results,
(1997) generally less
successful than
other treatments
Enzyme treatment Mutanolysin and Erhart et al., 1997; Worked with most
lysozyme Schuppler et al. Mycolata tested, but
(1998) not all; worked for
Candidatus
‘M. parvicella’
Organic solvents Diethyl ether, Xylene Davenport et al. Worked for very few
(2000) Mycolata
Enzyme treatment Lipase, proteinase K Davenport et al. Worked with some but
(2000) not all Mycolata tested
Combination Acid/Mutanolysin/lysozyme Carr et al. (2005) Appears to be effective
Acid/lipase/proteinase K for many Mycolata tested
Combination Lysozyme/achromopeptidase/HCl Kragelund et al. Appears to be the
(2007b) most effective yet for
Mycolata and Candidatus
‘M. parvicella’
Another method providing useful insights into foaming populations is hybridization after RNA
extraction, or membrane hybridization, typically using radiolabeled or enzyme-labeled oligonucleotide
probes. Quantification requires at least two probes: a universal or domain-targeted probe, and a population
targeted probe. The ratio of the signals from these is then used to express the percentage of hybridizable
rRNA attributable to the target population. The first Mycolata and Gordonia probes were in fact designed
for this protocol. They revealed that up to 18% of the rRNA in foam can be attributed to Mycolata,
with most from Gordonia (de los Reyes et al., 1997). Subsequent studies using group-, genus-, species-
and strain-targeted probes showed more Mycolata rRNA in foam than in the corresponding mixed liquor
and in anaerobic digesters (de los Reyes, 1998c), and a dominance of G. amarae during foaming (de los
Reyes and Raskin, 2002). Oerther et al. (2001) used quantitative membrane hybridizations with RNA
extracted from samples from a full-scale plant experiencing seasonal foaming, and probes targeting
Gordonia. A five-fold increase in Gordonia rRNA (0.25% to 1.25% of total rRNA) corresponded to the
appearance of foam, and quantitative FISH showed an increase in Gordonia biomass from 4% to 20% of
the MLVSS concentration. When foam disappeared, the levels of Gordonia rRNA and biomass decreased
correspondingly.
The power of membrane hybridization in tracking changes in community structure over the course of a
foaming episode has been demonstrated (de los Reyes et al., 2002). It could show that Gordonia was
present in mixed liquor, foam, RAS and anaerobic digester sludges in seven full-scale wastewater
treatment plants in California, Wisconsin, and Illinois, USA. Values ranged from 1 to 8% of VSS as
determined by FISH, while rRNA percentages ranged from 0.5 to 8% as determined by quantitative
membrane hybridizations. Interestingly, hybridizations to samples from other plants revealed that in
several, members of the Gammaproteobacteria were also enriched 25- to 100-fold in the foam, suggesting
a role for members of this class in foaming.
qPCR primers targeting the 16S rRNA gene of Candidatus ‘M. parvicella’ were applied to
samples from German plants (Kaetzke et al., 2005), and its 16S rRNA gene copies in those foaming
ranged from 0–8% of the total, while non-foaming plants had levels below 3%. Primers targeting the 16S
rRNA gene of members of the genus Gordonia have also been designed (Shen and Young, 2005), and
could be used potentially in a similar way.
DNA microarrays analyze complex environments and construct microbial community profiles. They
consist of oligonucleotides spatially arrayed on the surface of a small solid matrix (chapter 11), to which
fluorescently labeled nucleic acid samples are hybridized, scanned, and analyzed (Zhou and Thompson,
2002). Kim et al. (2004) used a microarray with random unsequenced DNA fragments from pure cultures
of G. amarae and two other pure cultures for hybridization with activated sludge samples. A follow-up
study included random sequences from thirteen strains including G. amarae and S. piniformis (Kim et al.,
2005). Both showed in general, that the probes were specific, with minimal cross-hybridization, as
assessed against pure and mixed culture hybridizations.
Other approaches have also been used to detect Mycolata in foams. An immunofluorescent (antibody)
stain targeting Gordonia (de los Reyes et al., 1998a; Hernandez et al., 1994) was able to detect and quantify
Gordonia in foam and mixed liquor and anaerobic digester samples. An enzyme-linked immunosorbent
assay (ELISA) based on an anti-G. amarae polyclonal antibody has also been reported (Iwahori et al.,
2000). Subsequent work with an anti-G. amarae mycolic acid polyclonal antibody produced an assay for
detecting other mycolata, including Nocardia, Rhodococcus, Dietzia, Mycobacterium, and Tsukamurella
spp., but not Corynebacterium (Iwahori et al., 2001a). Production of antibodies requires pure cultures,
and since isolation of many filaments in pure culture is problematic, immunodetection is limited in
its application. In addition, antibody penetration into flocs can be prevented by the extracellular
polymeric substances (EPS – chapter 3), and by unspecific binding to organic particles and fungal spores
Foaming 229
(Kampfer, 1997). Nevertheless, advances in antibody probing technology have been made. For example, to
isolate targeted Mycolata, bound antigens were separated with paramagnetic beads (Morisada et al., 2002).
This technique had recoveries of 80 to 100% at 103 to 107 CFU/ml and 107 beads/ml. Another method has
exploited the hydrophobic nature of Mycolata cell walls and their ability to utilize paraffin as sole carbon
source. Paraffin-coated slides and miniaturized devices were developed to recover and subsequently
quantify Mycolata from environmental samples (Polaczyk et al., 2006). Fatty acid methyl ester (FAME)
analysis of membrane lipids has also been used to detect G. amarae in activated sludge (Cha et al., 1999).
The GC peak areas correlated well with ‘Nocardia’ filament counts (expressed as number of intersections/g
VSS – chapter 11).
DETERMINING CAUSATIVE ORGANISMS

How does one decide if an organism is a foam-former? Clearly, a population found in large quantities in
foam is a presumptive candidate. Blackbeard et al. (1986a) suggested that the mere presence of certain
bacteria in large quantities, or their dominance, in foam does not necessarily mean they are causative, and
point out that these selectively accumulate in the foam, and are not simply carried there. By comparing
their frequency in mixed liquor and the foam layer, they concluded that Type 0092, Candidatus
‘M. parvicella’, and Nocardia spp. were all foam-causing filaments. However, it is possible that these are
entrapped in the foam from the mixed liquor, and do not actually ‘cause’ its formation or stabilization.
This was apparently the case for Type 0092, which was later shown to be equally dominant in foam and
mixed liquor in EBPR plants (Blackbeard et al., 1988). The question of causation is one of considerable
interest, given the preponderance of molecular methods to detect and quantify organisms, and the
complexity of ecological phenomena.
The ‘gold standard’ for assigning microbial causality is based on Koch’s postulates which can be
summarized as follows (Fredricks and Relman, 1996):
(i) The parasite occurs in every case of the disease, under circumstances that can account for the
pathological changes and clinical course of the disease.
(ii) The parasite occurs in no other disease as a fortuitous and nonpathogenic parasite.
(iii) After being fully isolated from the body and repeatedly grown in pure culture, the parasite can
induce the disease anew.
Later reviewers added a fourth postulate: a requirement to reisolate the microbe from the
experimentally inoculated host.
Postulate 1 is a criterion for necessity, while Postulates 2, 3 and 4 are criteria for sufficiency. Can we
use these postulates for determining causality in environmental microbiology (e.g. that a specific
organism is causing foaming)? Unfortunately, these postulates (as Koch himself realized) have severe
limitations, the greatest being the requirement for a pure culture of the organism in question. If not
satisfied, postulates 3 and 4 can not be met, and in environmental samples it is difficult, if not impossible
to eliminate possible effects of co-agents or other factors that accompany the organism or the effect. A
microbe that fulfils Koch’s postulates is most likely the causative organism. However one that fails to
fulfill Koch’s postulates may still represent the causative organism. A new framework for assigning
causality is needed, one that is reflective of the technological and intellectual tools available today, and
one based on a revised set of criteria is suggested here. These are shown in Table 8.6.
Applying the above criteria to the Mycolata shows that they, and especially G. amarae, are foam
causing organisms. For example, high levels of Mycolata correlate strongly with foam occurrence
Table 8.6. Hill’s epidemiologic criteria for causal association1, modified to apply to environmental
microbiology.
Causal criterion Causal association
1. Strength of association What is the statistical correlation?
2. Consistency of association Agreement among repeated observations in different places,
at different times, using different methodology, by different
researchers, under different circumstances?
3. Specificity of association Is the outcome unique to the organism?
4. Temporality Does exposure precede the outcome variable?
5. Biological gradient Evidence for a dose-response relationship?
6. Plausibility Does the causal relationship make biological sense?
Is there a mechanism?
7. Coherence Is causal relationship compatible with present knowledge
of the phenomenon?
8. Experimentation Does controlled manipulation change the outcome?
9. Analogy Does causal relationship conform to a previously described relationship?
1
Modified from Fredricks and Relman (1996).
(Criterion 1), in studies from all over the world by different researchers (Criterion 2). Occurrence of
foam is preceded by an increase in Gordonia levels (de los Reyes and Raskin, 2002; Oerther et al., 2001)
(Criterion 4) and a dose-response relationship in terms of threshold levels has been demonstrated (de los
Reyes and Raskin, 2002). The causal relationship is plausible and compatible with current knowledge
(hydrophobicity of cell wall and biosurfactants aid in foam formation, ability to grow on hydrophobic
substrates) (Criteria 6 and 7). Finally, controlled manipulations (e.g. washout) result in the decrease
of foam (de los Reyes and Raskin, 2002) (Criterion 8). While Criterion 3 is not met, this only means
that other organisms may also be causative. Note that we need to distinguish between necessity and
sufficiency in applying these criteria. Similarly, a case can be made for Candidatus ‘M. parvicella’ and
S. piniformis, although some of the studies available (e.g. dose-response and experimentation) are less
rigorous than is the case with Gordonia.
However, applying the same criteria to other organisms raises questions about their role as foam-
formers. For example, a case for the morphotype ‘Nostocoida limicola’ II is weaker from a lack of
mechanism, consistency and strength of association, gradient, and experimentation. The same can be
said for Eikelboom types 0092, 0581, 1851, 0803, 0041/0675, and other ‘non famous scum bacteria’
(Lemmer et al., 2005). It is possible that individual plants may have features that allow some of these to
become foam formers. For example, some Gammaproteobacteria (de los Reyes et al., 2002) and Gram
positive coccoidal cells (Lemmer et al., 2005) (possibly a stage in the life cycle of some Mycolata) may
be true foam formers in some plants.
It is also evident that determining the causative organisms necessitates their unequivocal identification,
better detection of less conspicuous morphologies, and more quantitative procedures. These make
selecting molecular methods more important in future work. Isolating organisms into pure culture will
also allow direct testing of ‘dose-response’ relationships. For example, adding varying amounts of cells to
non-foaming sludge should result in corresponding changes in foam indices, as occurred with Gordonia
(de los Reyes and Raskin, 2002). However, care should be taken with this approach, since changes in
Foaming 231
environmental conditions may affect the organisms’ physiology (Carr et al., 2006; Eales et al., 2005), or
mechanisms of causation may not be amenable to such ‘dose-response’ interpretations.
QUANTIFYING FILAMENTS AND MYCOLATA IN FOAMS

Filaments in activated sludge
Quantification techniques are traditionally based on microscopy, and thus suffer from subjectivity. For
bulking sludge, where total filament length was used, subjective scales are common, such as the ‘þ to
þþþ scale’ of Rensink (1974), the ‘slight, moderate, or excessive scale’ of Farquhar and Boyle (1971b),
and the ‘arbitrary 10-point scale’ of Forster and Dallas-Newton (1980). Eikelboom (1982) proposed a five
point scale based on a rough ‘eye estimation’, which was ‘calibrated’ with reference images. Observers
are required to give category numbers based on a photograph that best resembled the microscopic view.
Jenkins et al. (2004b) presented another subjective scale of filament abundance ranging from 0 to 6, with 0
being ‘‘none’’, and 6 ‘‘excessive’’. Representative ‘reference’ microphotographs were again provided as
aids. Another older technique involved estimating the relative proportion of the microscope field occupied
by filamentous and ‘zoogleal’ bacteria (Chudoba et al., 1973a,b).
Pipes (1979) attempted a true numerical estimation by counting the total number of actual bulking
filaments, but did not determine filament lengths, while Finstein and Heukelekian (1967) measured total
lengths of filaments protruding through the floc. Sezgin et al. (1978) proposed using a technique which
involved a microscopic estimation of total extended filament length (TEFL) expressed on a per mass or
per volume basis (of MLSS). Another method described by Walker (1982), used a Lund nanoplankton
counting chamber, where the numbers of filaments inside squares running diagonally across a microscope
graticule were counted, and expressed as filament length per mass of MLSS using appropriate formulae.
Matsui and Yamamoto (1984) displayed their data on a color TV technique to fit filament length
distribution to a Gaussian distribution.
Quantifying Mycolata using microscopy and colony counts

None of these above methods quantify filaments in foam, but instead were applied originally to assess
bulking (chapter 7). A rapid technique for Nocardia (Gordonia?) was developed by Vega-Rodriguez
(1983) and modified by Pitt and Jenkins (1990). They both consist of microscopically counting the
number of branched Gram-positive filaments 41 mm long in a diluted Gram-stained sample of mixed
liquor (Jenkins et al., 2004b). Their number is determined by counting how many intersections they make
with three equally spaced lines drawn across a stained microscope slide. Knowing VSS concentrations,
counts are expressed as ‘‘number of intersections per gram of VSS’’. This method has been applied
to both bench-scale and full-scale plants (Cha et al., 1992; Pitt and Jenkins, 1990), and is routinely
used in the Sacramento, California treatment plants (Hernandez et al., 1994). Cha et al. (1992) reported
that replicate counts were achievable within ^20% of each other, and that foam occurred at around
1 £ 106 intersections/g VSS, (1 £ 104 intersections/g VSS was their detection limit). Some Mycolata
filaments fragment during their life cycles and its extent may vary between organisms and within the
same organism under different conditions. Therefore, this scoring system is likely to reflect these
morphological shifts.
Relationships between actinomycete numbers (by plate counts) and onstart of foaming have been
assessed, and counts of 104 to 106 colonies per mg MLSS were linked to foam formation (Hiraoka and
Tsumura, 1984b; Pipes, 1978). Colony counts with n-octadecane as sole carbon source showed levels of
9 £ 105 colony forming units (CFU)/mg SS (4.08 £ 105 CFU/ml) of ‘Nocardia’ (G. amarae?) in mixed
liquor and 8.67 £ 106 CFU/ml in the scum (Fujita et al., 1994). These values represent a 20 fold con-
centration, and show that about 3% of the total viable cells in the scum were ‘Nocardia’. Goodfellow et al.
(1996) used DAPI and epifluorescence microscopy to determine total viable counts. They ranged from
0.13 £ 108 CFU/ml for a sample with no stable surface scum to 1.31 £ 108 for an extensive stable foam
(Goodfellow et al., 1996). The mean percentages of presumptive Mycolata (gordoniae, rhodococci, and
tsukamurellae) were 5% and 54% respectively.
Antibody stains and molecular methods

One early attempt to identify and quantify Gordonia filaments without relying on morphology or staining
was that of Hernandez et al. (1994), with polyclonal antibodies against Gordonia. A secondary antibody
conjugated to fluorescein isothiocyanate (FITC) allowed its in situ detection by epifluorescent microscopy.
They determined the relationship between total filament length of pure cultures of G. amarae and biomass
levels (as VSS), and so could estimate the mass (VSS) of filaments detected by the antibody probe. Its mass
fraction in the San Francisco Southeast Plant by this method was much higher (10–28% of VSS) than the
mass fraction measured by Gram staining (2–10%).
Subsequently, a semi-quantitative FISH technique based on the relationship between VSS and filament
length was developed (de los Reyes et al., 1998c), and used with antibody staining. In general, antibody
stains gave higher biomass percentages than did FISH probes, although results were comparable. Filament
levels measured in foam and RAS samples ranged from 2 to 18% of the VSS. Some branched filaments
(presumably Gordonia) were stained clearly by the antibodies, but gave low FISH signals, which varied
considerably from sample to sample. This was thought to reflect combined effects of low cellular rRNA
content, very stringent hybridization conditions, and uneven cell permeability to the FISH probe.
One advantage of these molecular approaches is their ability to quantify individual populations
associated with foaming. An important criterion any causative organisms must meet is that the level of
their cells corresponds to the extent of foaming. This has been shown for the Mycolata in several culture
dependent and microscopy-based studies (Cha et al., 1992; Fujita et al., 1994; Goodfellow et al., 1996).
Quantitative rRNA hybridization probing also revealed that a five-fold increase in Gordonia rRNA
corresponded to the appearance of foaming (Oerther et al., 2001), while with semi-quantitative FISH
(de los Reyes et al., 1998c) an increase in Gordonia biomass from 4% to 32% of the mixed liquor volatile
suspended solids (MLVSS) concentration (Oerther et al., 2001) was detected when foam appeared. When
foam disappeared, the levels of Gordonia rRNA and biomass decreased.
Thresholds for foaming

If Mycolata cells cause foaming, then it is reasonable to assume that foaming will begin once these reach a
threshold level in the mixed liquor. The concept of threshold concentrations for foaming was explored by
de los Reyes (2000) in a three-tiered series of experiments: (1) batch tests involving adding Gordonia cells
to mixed liquor, (2) analysis of a full-scale plant experiencing seasonal foaming, and (3) a study with lab-
scale reactors augmented with foam-forming organisms. The results obtained were consistent among all
three, and showed the existence of two foaming thresholds (de los Reyes and Raskin, 2002). The first was
termed the ‘‘formation threshold’’, which reflects a sudden increase in foaming potential. The second was
termed a ‘‘stability threshold’’ which corresponds to a situation in which a stable foam is maintained as a
thick viscous layer on the surface of the aeration basin. The former (filament length) was determined to be
2 £ 108 mm/ml, while the latter was 1 £ 109 mm/ml (de los Reyes and Raskin, 2002). It was hypothesized
that plants with Gordonia levels above the first threshold would experience episodic (e.g. seasonal)
Foaming 233
foaming, while in plants with year-round foaming, Gordonia levels would probably be above the second
threshold, and that the values for these are most likely to be plant-specific. Davenport et al. (2000)
had suggested a value of 2 £ 106 cells/ml or about 4 £ 1012 cells/m2 as a foaming threshold for a baffled
reactor, and stated that for an unbaffled reactor it should be greater than this. Jolis and Marneri (2006)
confirmed the existence of formation and stability thresholds in a California, USA plant, and generated
values similar to those of de los Reyes and Raskin (2002) for an Illinois, USA plant. Based on FISH data
from 14 treatment plants, a universal threshold value for foaming of 2 £ 106 mycolata cells ml 1 was
suggested by Davenport et al. (2008). However, this fails to consider that dead or metabolically inactive
FISH negative filaments, may still be hydrophobic and participate in foam stabilization (Carr et al., 2005).
TAXONOMY OF FOAM-FORMING ORGANISMS

The systematics of foam-forming microorganisms has undergone massive changes in the last two decades
as a result of improvements in chemotaxonomic, numerical phenetic, and molecular systematic proce-
dures (Goodfellow, 1992). Only the most current phylogeny of the foam-forming organisms is reviewed
in this section.
Mycolic acid-containing Actinobacteria – the Mycolata

Historically, classification of actinomycetes was based primarily on colonial and microscopic morphology,
but several warnings had been made about the risks in over-relying on these (Cross and Alderson, 1988;
Minnikin, 1980; Williams, 1980). The taxonomic value of cell wall and lipid composition, numerical
taxonomy, and nucleic acid sequence analysis proved to be greater. The concept of cell wall types as
taxonomic characters was introduced by Becker (1965) and Lechevalier and Lechevalier (1970). Based on
characteristic components of their peptidoglycan, actinomycetes can be placed in one of nine cell wall type
groups (Table 8.7).
Table 8.7. Characteristic components of major actinomycete cell wall types.

Cell wall type Major constituents Representative genera
I L-DAP, glycine Streptomyces
II Meso-DAP, glycine Micromonospora
III meso-DAP Actinomadura,
Nocardiopsis, Saccharothrix
IV Meso-DAP, arabinose, galactose Nocardia, Rhodococcus,
Gordonia, Tsukamurella,
Skermania, Mycobacterium,
Dietzia, Amycolata, Amycolaptosis
V Lysine, ornithine Actinomyces israeli
VI Lysine (galactose, aspartic acid) Oerskovia, Actinomyces, Microbacterium
VII DAB, glycine (lysine) Agromyces
VIII ornithine Bifidobacterium, Cellulomonas
Data are based on Lechevalier and Lechevalier (1980). Abbreviations: DAB, diaminobutyric acid; DAP, diaminopimelic
acid. Parentheses indicate variable appearance.
Cell wall types were once important characters in actinomycete taxonomy (Lechevalier, 1989), since
their peptidoglycans contain major amounts of alanine, glucosamine, glutamic acid, and muramic acid
(Soddell and Seviour, 1990). Analysis of whole cell hydrolysates by paper chromatography allows
identification of wall types (Types I to IV), and for a long time were used to differentiate between
actinomycete genera. Thus, Mycolata have cell wall Type IV, containing meso-diaminopimelic acid
(DAP), arabinose, and galactose (Lechevalier, 1989). These can be divided into two groups based on
whether they possess a-branched, b-hydroxylated fatty acids, mycolic acids. Most foam-forming
Actinobacteria isolated so far contain mycolic acids, which are thought to be responsible for their cell
hydrophobicity. They are high molecular weight 3-hydroxy 2-alkyl branched chain fatty acids containing
between 30 and 90 carbon atoms (Macnaughton et al., 1994; Minnikin et al., 1984). The different structural
types include so-called a-mycolates and acids having oxygen functions (4C ¼ O, 4CHOCH3,
COOH), in addition to the 3-hydroxy acid unit (Minnikin et al., 1984).
At the subgeneric level, numerical taxonomy was the most effective early method for establishing
taxonomic relationships (Goodfellow and Cross, 1984). However, it did not adequately resolve some
persistent taxonomic problems, as highlighted by the incorrect early classification of N. amarae and N.
pinensis. Both were placed originally in the genus Nocardia, although enough evidence was available to
suggest that they differed markedly from other members of this genus (Blackall et al., 1994a; Blackall
et al., 1989). Phylogenetic analysis has now clarified the taxonomic relationships among members of the
Mycolata. However, a polyphasic approach, in which both phylogenetic as well as chemotaxonomic
criteria (particularly mycolic acid and menaquinone structure) are considered, should be used (chapter 6).
Table 8.8 shows the chemotaxonomic properties of the currently accepted genera of the mycolic acid
containing Actinobacteria.
Current classification
The Mycolata form a distinct phyletic line within the suborder Corynebacterineae (Stackebrandt et al.,
1997), which encompasses the genera Corynebacterium, Dietzia, Gordonia, Millisia, Mycobacterium,
Nocardia, Rhodococcus, Segniliparus, Skermania, Tsukamurella, and Williamsia, based primarily on
chemical, molecular, and morphological markers (Butler et al., 2005; Chun et al., 1997; Soddell et al.,
2006a). The most notable foam forming member has been Nocardia amarae, now reclassified as
Gordonia amarae (Goodfellow et al., 1994; Klatte et al., 1994b; Ruimy et al., 1994). Note that the
previous genus name, Gordona, should be replaced by Gordonia, following proper etymology
(Goodfellow et al., 1998; Stackebrandt et al., 1997). Nocardia pinensis has also been reclassified into
a new genus Skermania as Skermania piniformis (Chun et al., 1997) (Table 8.9).
Candidatus ‘M. parvicella’

Candidatus ‘‘M. parvicella’’ (Bradford et al., 1998) is an unsheathed Gram-positive, unbranched filament
associated with both bulking and foaming incidents around the world. First descriptions were made by
Pasveer (1969), who thought it was a filamentous form of Escherichia coli. First isolations were reported
by van Veen (1973), Eikelboom (1975), and Slijkhuis (1983). A feature of all these isolates was the
difficulty in storing them and their slow growth rates in pure culture (Tandoi et al., 1998b). Some reports
of its isolation and characterization (Chacin et al., 1994; Kerley et al., 1994; Kocianova et al., 1994) need
to be treated with caution, as the organism described differs markedly to pure cultures of Candidatus
‘M. parvicella’ described in the literature. These include morphology, variable Gram staining, motility,
Table 8.8. Chemotaxonomic characteristics differentiating different mycolic acid-containing Mycolata (Based on data by Chun et al., 1997).
Mycolic acids Fatty acid esters
No. of No. of released on Predominant Phosphatidyl- Mol % GþC
Species Morphology carbons double bonds pyrolysis menaquinone ethanolamine in DNA
Corynebacterium Straight to slightly 22–38 0–2 8–18 MK-8(H2) – 51–67
curved rods, which MK-9(H2)
reproduce by snapping
division; club-shaped
elements may also be formed
Rhodococcus Rods to extensively branched 34–52 0–4 12–16 MK-8(H2) þ 63–73
substrate mycelium that
fragments into irregular rods
and cocci
Dietzia Short rods and cocci. 34–38 ND ND MK-8(H2) þ 73
Nocardia Substrate mycelium that 46–60 0–3 12–18 (w-cyclo)- þ 64–72
fragments into rods and MK-8(H4)
coccoid elements
Gordonia Rods, cocci and filaments 46–66 1–4 16–18 MK-9(H2) þ 63–69
Foaming
Skermania Filaments with acute 58–64 2–6 16–20 (w-cyclo)- þ 67.5

branching angles and MK-8(H4)
tree-like appearance
early in growth
Tsukamurella Straight to slightly 64–78 1–6 20–22 MK-9 þ 67–68
curved rods occur singly,
in pairs or in masses
Mycobacterium Slightly curved or 60–90 1–3 22–26 MK-9(H2) þ 61–71
straight rods, sometimes
branching filaments that
fragment into rods and
coccoid elements
Millisia Characteristic rudimentary 44–52 ND 16–18 MK-8(H2) þ 64.7
right-angled branching
Williamsia Irregular rods or cocci 50–56 MK-9(H2) þ 64–65
Segniliparus Rods ND ND 10–24 ND ND 68–72
235
ND ¼ no data.
Table 8.9. Taxonomic status (March 2007) of Mycolata containing mycolic acids in the cell wall, excluding Corynebacterium and
236
Mycobacterium.
Current classification References/Taxonomic comments
Dietzia spp.
Dietzia maris Rhodococcus maris first described by Nesterenko et al. (1982). Reclassified as new genus by Rainey et al.
(1995b) on phylogenetic grounds
Dietzia psychralcaliphila Yumoto et al. (2002)
Dietzia kunjamensis Mayilraj et al. (2006)
Gordonia spp.
Gordonia aichiensis Rhodococcus aichiensis first described by Tsukamura (1982). Reclassified as Gordonia
by Klatte et al. (1994b) on phylogenetic grounds
Gordonia alkanivorans First described by Kummer et al. (1999). G. nitida (Yoon et al., 2000b) was later determined
by Arenskotter et al. (2005) to be the same species as G. alkanivorans
Gordonia amarae Nocardia amarae first described by Lechevalier and Lechevalier (1974). Further characterized by
Goodfellow et al. (1982b). Classified as Gordonia by Blackall et al. (2004), Klatte et al. (1994b),
Goodfellow et al. (1994) and Ruimy et al. (1994) on phylogenetic grounds
Gordonia amicalis Kim et al. (2003)
Gordonia bronchialis Originally described by Tsukamura (1971) as Gordona bronchialis but Goodfellow and Alderson (1977)
reclassified it as Rhodococcus bronchialis using numerical taxonomy. Stackebrandt et al. (1988)
reinstated it to Gordonia on phylogenetic grounds
Gordonia defluvii Soddell et al. (2006b)
Gordonia desulfuricans Kim et al. (1999)
Gordonia hirsuta Klatte et al. (1996)
Gordonia hydrophobica Bendinger et al. (1995)
Gordonia jacobaea de Miguel et al. (2000)
Gordonia namibiensis Brandao et al. (2001)
Gordonia paraffinivorans Xue et al. (2003)
Gordonia polyisoprenivorans Linos et al. (1999)
Gordonia rhizosphera Takeuchi and Hatano (1998)

Gordonia rubropertincta Goodfellow and Alderson (1977) classified it as Rhodococcus rubropertinctus using numerical
taxonomy, with Rhodococcus corallinus as a distinct cluster. Mordarski et al. (1980) found that
R. corallinus belonged to the same DNA group and classified it as Gordonia on phylogenetic grounds
Gordonia shandongensis Luo et al. (2007)
Gordonia sihwensis Kim et al. (2003)
Gordonia sinesedis Maldonado et al. (2003)
Gordonia soli Shen et al. (2006)
Gordonia sputi Rhodococcus sputi first described by Tsukamura (1978). Name revived by Tsukamura
and Yano (1985). Transferred to Gordona by Stackebrandt et al. (1988) on phylogenetic grounds.
R. chubuensis first described by Tsukamura (1982). Rainey et al. (1995a) classified it as a Gordona but
suggested its transfer to G. sputi. Rhodococcus obuensis described by Tsukamura (1982), but DNA
homology indicates it is a subjective synonym of G. sputi (Zakrzewskaczerwinska-Czerwinska, 1988)
(continued)
Gordonia terrae Originally described as Gordonia terrae by Tsukamura (1971) but reclassified as Rhodococcus terrae
by Goodfellow and Alderson (1977) using numerical taxonomy. Reinstated as Gordonia by
Stackebrandt et al. (1988) on phylogenetic grounds
Gordonia westfalica Linos et al. (2002)
Nocardia spp.
Nocardia abscessus Yassin et al. (2000b)
Nocardia africana Hamid et al. (2001)
Nocardia alba Li et al. (2004b)
Nocardia anaemiae Kageyama et al. (2004h)
Nocardia aobensis Kageyama et al. (2004c)
Nocardia araoensis Kageyama et al. (2004f)
Nocardia arthritidis Kageyama et al. (2004d)
Nocardia asiatica Kageyama et al. (2004a)
Nocardia asteroides Described by Goodfellow and Lechevalier (1989). The Nocardia asteroides complex has not yet been fully
clarified. Nocardia farcinica and Nocardia nova are probably separate species (Steingrube, 1995; Laurent,
1996), but Nocardia asteroides sensu stricto may still be heterogeneous (Steingrube, 1995; Laurent, 1996)
Nocardia beijingensis Described by Wang et al. (2001)
Nocardia brasiliensis Described by Goodfellow (1989)
Foaming
Nocardia brevicatena Originally described as Micropolyspora brevicatena. Goodfellow (1982b) transferred it to Nocardia
Nocardia carnea Described by Goodfellow (1989) and Gordon et al. (1978)
Nocardia caishijiensis Described by Zhang et al. (2003c)
Nocardia cerradoensis Described by Albuquerque de Barros et al. (2003)
Nocardia crassostreae Described by Friedman et al. (1998)
Nocardia cummidelens Described by Maldonado et al. (2000) within the N. salmonicida clade
Nocardia cyriacigeorgica Described by Yassin et al. (2001a). Formerly N. cyriacigeorgici
Nocardia farcinica Although considered to be part of the N. asteroides complex it has been separated from N. asteroides
sensu stricto using RFLP (Steingrube, 1995), ribotyping (Laurent, 1996) and 16S rRNA sequencing (Chun
and Goodfellow, 1995; Rainey, 1995a). Rainey (1995a) linked it to Nocardia otitidiscaviarum but Chun and
Goodfellow (1995) could not confirm this
Nocardia flavorosea Chun et al. (1998)
Nocardia fluminea Described by Maldonado et al. (2000) as within the N. salmonicida clade
Nocardia higoensis Kageyama et al. (2004e)
Nocardia ignorata Described by Yassin et al. (2001b)
Nocardia inohanensis Kageyama et al. (2004i)
Nocardia kruczakiae Conville et al. (2004)
Nocardia lijiangensis Xu et al. (2006)
Nocardia mexicana Rodrı́guez-Nava et al. (2004)
Nocardia neocaledoniensis Saintpierre-Bonaccio et al. (2004)
237
(continued)
238

Nocardia niigatensis Kageyama et al. (2004i)
Nocardia nova First described by Tsukamura (1982) but classified as species incertae sedis by Goodfellow (1989).
Further characterized by Yano (1990) using numerical taxonomy. Separated from N. asteroides sensu
stricto by RFLP (Steingrube, 1995) and ribotyping (Laurent, 1996)
Nocardia otitidiscaviarum Described by Goodfellow (1989). Originally called Nocardia caviae
Nocardia paucivorans Yassin et al. (2000a)
Nocardia pigrifrangens Wang et al. (2004)
Nocardia pseudobrasiliensis First described by Ruimy et al. (1996) based on taxon described by Wallace (1995)
Nocardia pseudovacinii Formerly N. vacinii (Goodfellow, 1989; Gordon et al., 1978)
Nocardia pneumoniae Kageyama et al. (2004f)
Nocardia puris Yassin et al. (2003)
Nocardia salmonicida Maldonado et al. (2000). Formerly Streptomyces salmonica
Nocardia seriola Described by Kudo et al. (1988). Formerly N. seriolae
Nocardia shimofusensis Kageyama et al. (2004e)
Nocardia sienata Kageyama et al. (2004g). Formerly Nocardia senatus
Nocardia soli Maldonado et al. (2000)
Nocardia takedensis Yamamura et al. (2005)
Nocardia tenerifensis Kämpfer et al. (2004)
Nocardia testaceae Kageyama et al. (2004g). Formerly Nocardia testaceus
Nocardia thailandica Kageyama et al. (2004b)
Nocardia transvalensis Described by Goodfellow (1989) and Gordon et al. (1978)
Nocardia uniformis Isik et al. (1999)
Nocardia vermiculata Kageyama et al. (2004b)
Nocardia veterana Gürtler et al. (2001)
Nocardia vinacea Kinoshita et al. (2001)
Nocardia xishanensis Zhang et al. (2004)
Nocardia yamanashiensis Kageyama et al. (2004i)
Rhodococcus spp.
Rhodococcus australis First described by Ferreira (1984). Although name is not valid, this ‘species’ appears in commercial
identification kits like Biolog. Reclassified by Yassin et al. (2007) as Gordonia malaquae, isolated from foam
Rhodococcus coprophilus Rowbotham and Cross (1977) and Goodfellow and Alderson (1977)
Rhodococcus equi Corynebacterium equi reclassified as Rhodococcus equi by Goodfellow and Alderson (1977) and
Goodfellow et al. (1982a) after numerical taxonomy Nocardia restricta renamed as R. equi by
Goodfellow and Alderson (1977) using numerical taxonomy. This was confirmed by Rainey (1995a)
on phylogenetic grounds
Rhodococcus erythreus Rainey (1995a)
(continued)
Rhodococcus erythropolis Nocardia erythropolis classified as R. erythropolis by Goodfellow and Alderson (1977) using numerical
taxonomy. Nocardia calccarea reclassified as R. erythropolis by Goodfellow et al. (1982c) using
numerical taxonomy, confirmed by Rainey (1995a) on phylogenetic grounds. Arthrobacter
picolinophilus transferred to R. erythropolis by Koch et al. (1995)
Rhodococcus fascians Reclassified by Goodfellow (1984) as Rhodococcus from the genus Corynebacterium on chemical,
genetic, and phenetic data
Rhodococcus globerulus Strains classified as Nocardia globerula, Nocardia corynebacteroides and Mycobaterium
globerlum were reclassified as R. globerlus by Goodfellow et al. (1982c) after numerical taxonomy
Rhodococcus Strains classified as Nocardia globerula, Nocardia corynebacteroides and Mycobacterium
corynebacteroides globerulum were reclassified as R. globerulus by Goodfellow (1982c) using numerical taxonomy
Rhodococcus marinonascens Nocardia corynebacteroides reclassified as Rhodococcus by Rainey (1995a) on phylogenetic grounds
Rhodococcus opacus Klatte (1994a)
Rhodococcus percolatus First described by Briglia et al. (1996) based on chemotaxonomic and phylogenetic data
Rhodococcus pyridinivorans Yoon et al. (2000a)
Rhodococcus rhodnii Nocardia rhodnii reclassified by Goodfellow and Alderson (1977) using numerical taxonomy
Rhodococcus rhodochrous Mycobacterium rhodocochrous and Nocardia rhodocochrous reclassified as Rhodococcus
by Goodfellow and Alderson (1977) using numerical taxonomy. Rhodococcus roseus reclassified as
R. rhodochrous by Rainey (1995a) on phylogenetic grounds
Rhodococcus ruber Nocardia rubra and Nocardia pellegrino reclassified as R. ruber by Goodfellow and Alderson (1977)
Foaming
using numerical taxonomy

Rhodococcus zopfii Stoecker et al. (1994)
Skermania sp.
Skermania piniformis Originally called ‘Pine Tree-like Organism’ (PTLO) because of its morphology. First described by
Blackall et al. (1989) as Nocardia pinensis. Later work (Blackall et al., 1994a) led to change to
Skermania piniformis by Chun et al. (1997)
Tsukamurella spp.
Tsukamurella inchonensis Yassin et al. (1995)
Tsukamurella paurometabola First described as Gordona aurantiaca by Tsukamura (1971), further described by Goodfellow et al. (1978)
Classified as R. aurantiacus by Tsukamura and Yano (1985), Corynebacterium paurometabolum first
described by Steinhaus (1941). Then R. aurantiacus and C.paurometabolum combined as new
genus Tsukamurella by Collins (1988) on phylogenetic grounds
Tsukamurella pseudospumae Nam et al. (2004b)
Tsukamurella pulmonis Yassin et al. (1996)
Tsukamurella spumae Nam et al. (2003)
Tsukamurella tyrosinosolvens Yassin et al. (1997)
Williamsia spp.
Williamsia maris Stach et al. (2004)
Williamsia muralis Descibed by Kampfer et al. (1999) as the first member of the genus Williamsia
239
abilities to undergo rod-filament transition and formation of spores. It has been suggested that this isolate
may be a filamentous Bacillus sp. (Blackall et al., 1995).
Several early workers (Eikelboom, 1994b; Foot et al., 1992; Wanner and Grau, 1989) had speculated
that Candidatus ‘M. parvicella’ was an actinomycete. With micromanipulation and 16S rRNA
sequencing, Blackall et al. (1994) showed it to be a deep branching member of the Actinobacteria
closely related to an unnamed iron-oxidizing strain, now classified as Acidimicrobium ferroxidans
(Clark and Norris, 1996). An Italian isolate of Candidatus ‘M. parvicella’ strain RN1, had the same 16S
rRNA gene sequence as the Australian isolate DAN1-3 (Rossetti et al., 1997a). This is important, in that
independently obtained strains emerged as members of the same species, as evidenced by near identical
16S rRNA gene sequences. An extensive and critical review of the microbiology and physiology of
M. parvicella has been published (Rossetti et al., 2005).
HOW IS FOAM FORMED?

Several mechanisms for filamentous foaming have been proposed. The branched morphology of
‘nocardioforms’ was visualized to form a net between the sludge flocs, trapping oil and grease droplets
and gas bubbles, causing the sludge to float to the surface (Pipes, 1978). Another hypothesis suggests that
the high lipid content of cells of Candidatus ‘M. parvicella’ and the Mycolata and foam (8–23%)
(Hao et al., 1988b; Soddell and Seviour, 1990), leads to a lower density, causing the fats and sludge to
float. Lemmer (1986) calculated that with an average density of 0.9–0.95 g/cm3 for oil and grease, and an
average of 1.8 g/cm3 for dry sludge, roughly 85% of the dry weight of sludge as grease and oil, would
achieve buoyancy, making this hypothesis unlikely. The currently favored theory involve a selective
enrichment of mixed liquor organisms by a flotation process (Blackall and Marshall, 1989).
Jenkins et al. (1993) and Soddell and Seviour (1990) also favoured this flotation mechanism. The
filaments, having hydrophobic cell walls, attach to surfaces of solid particles rendering them hydrophobic,
so they then attach to gas bubbles, making a three-phase foam. These particles must be small (less than
300 mm) in order to be lifted by buoyancy of the bubbles (Soddell and Seviour, 1990), and must also
bridge the water film between two air bubbles to create a dam that prevents liquid drainage and film
thinning. Thus, the foam is rendered stable. An analogy of filamentous foaming with mineral separation by
flotation has been discussed (Jenkins et al., 1993). In mineral flotation, a chemical called a collector is
added to the mineral-impurities-water mixture. The collector specifically adsorbs to the mineral particles
and makes them hydrophobic. Surfactant is then added, generating a stable foam. The mineral is then
floated and skimmed off. In activated sludge, the hydrophobic filaments can be considered as collectors
that cause flocs to float and form a stable foam in the presence of surfactant. Thus, for stable foam
formation, the following must be present: gas bubbles, hydrophobic particles, and surfactants. These are
discussed next.
Gas bubbles
When the structure of foam was examined by TEM and SEM (Foot et al., 1993), it consisted of a dense
matrix of filamentous organisms interspersed with air bubbles, which were surrounded by a membrane
composed of cellular and exocellular material. Since all activated sludge systems are aerated, it is
not feasible to eliminate the gas phase from the aeration basin. However, the size of bubbles may
be controllable. Thus, van Niekerk et al. (1987b) found that foam levels produced in laboratory-scale
anaerobic digester reactors depended on the mixing method. Fine bubble gas mixing produced more foam
than coarse bubble mixing. Mechanical mixing, which minimizes introduction of gas bubbles into the
Foaming 241
digester, supported the least foam formation. Increasing bubble size decreased the ability of an organism
to be enriched at the liquid-gas interface (Wozniak et al., 1976). However, effects of bubble size on
foaming in an aeration basin have yet to be investigated thoroughly. Lemmer and Baumann (1988)
suggested it was the aeration system that was responsible for flotation and not denitrification or H2S
production by cells.
Hydrophobicity
A common feature of the Mycolata is their hydrophobic cell surface. Cell surface hydrophobicity (CSH) is
assumed to be imparted by large amounts of mycolic acids covalently bound to sugars in the cell wall,
creating a waxy cell surface (Stratton et al., 1997). However, as discussed below, this assumption needs
closer examination. The various Mycolata wall constituents have different physical functions (Minnikin
and O’Donnell, 1984). The closest layer to the peptidoglycan contains the a-branch of mycolic acids
bound to it, and this is believed to be a structural permeability layer. The next layer has double bonds and
oxygen atoms associated with the mycolic acid alkyl chains, and consequently is less dense. This layer is
thought to serve as a parallel binding region for adhesion of loosely associated or ‘‘free’’ complex lipids.
The third layer, defined by the terminal ends of the covalently bound mycolic acids and ‘‘free’’ associated
lipids has been established as a ‘‘hydrophobic interaction region’’. Thus the Mycolata appear to possess an
‘outer membrane’ analogous in function possibly to that seen in the Gram negative bacteria (chapter 1).
The initial linking of hydrophobicity to foaming was by Khan et al. (1991), who showed that sludges
with foaming problems had higher hydrophobicity than those that did not. This relationship encouraged
them to propose a categorization of sludge samples based on solids hydrophobicity. Interestingly, no
hydrophobicity-foaming relationship was observed for their Nostocoida limicola-dominated sludges.
Hydrophobicity was measured by them with the MATH assay (Rosenberg et al., 1980). Kocianova et al.
(1992) showed a linear relationship between solids hydophobicity and the percentage of aeration tank
foam coverage. Similar results were obtained by Stratton et al. (1995), who monitored four activated
sludge systems over four months, and showed that onset of foaming often correlated with an increase in
MLSS hydrophobicity, the types and relative numbers of filamentous bacteria present, and other
operational parameters.
Relationships between mycolic acid content and CSH (Bendinger et al., 1993) showed that the Mycolata
were more hydrophobic than other bacteria. Furthermore, increasing mycolic acid chain length paralleled
increased CSH, although the opposing influence of other cell surface compounds (e.g. glycopeptidolipids,
trehalose-containing lipo-oligosaccharides, weakly hydrophobic exoploysaccharides, proteins, and
peptidolipids) reduced this overall effect. There is evidence that inactive G. amarae cells have high
CSH, since when mixed liquor with high levels of this organism was chlorinated, higher foaming potential
and foam stability were still observed compared to non-chlorinated samples (Pagilla et al., 1998).
Kerley and Forster (1995), on the other hand, detected no marked differences in the hydrophobicity of
foaming and non-foaming sludges. However, when Candidatus ‘M. parvicella’-dominated foams were
analyzed, they suggested that increasing uronic acid levels in the EPS were related to increasing
hydrophobicities, by the uronic acid negative charge neutralizing the positive charge of a polyvalent metal
ion, leaving a hydrophobic surface. However, they also suggested that shifts in EPS chemistry from
branched to unbranched chains are analogous to changes from smooth to rough bacterial colony forms,
and these could account for foam formation.
Studies focusing on individual cell surface characteristics and their relation to foaming have also been
reported. Thus, Sunairi et al. (1997) studied four strains of Rhodococcus rhodochrous with different
colony morphologies (two rough strains, one circular smooth, and one circular mucoidal). They showed
that mycolic acid levels were not responsible for differences in their CSH and that exocellular
polysaccharides (EPS) produced by R. rhodochrous decreased CSH, by acting as hydrophilins. In
addition, they reported that the rough mutants were hydrophobic, and smooth strains and mucoidal
mutants hydrophilic. Such data suggest that CSH changes in R. rhodochrous modifying their culture
characteristics and scum forming ability. Changes in CSH with culture age and C:N ratio were shown for
some ‘nocardioforms’ (Stratton et al., 1993), but no consistent pattern was found. When Stratton et al.
(1997) monitored CSH of R. rhodochrous and changes in their mycolic acid content and foaming ability,
they showed it changed with culture age (older cells were more hydrophobic), and growth temperature and
carbon source. Interestingly, mycolic acid composition did not appear to influence either their CSH or
foaming ability. These results contradict earlier studies relating mycolic acid chain length to CSH, and
suggest that mycolic acid composition cannot be held solely responsible for CSH. In addition, they
postulated that CSH may not be the only factor in foaming. Later, Stratton et al. (2002) showed there was
no clear relationship in three Rhodococcus spp. isolated from foam between their CSH determined by the
MATH assay, and their ability to produce a stable foam. No correlation was found between mycolic acid
composition, in terms of their chain lengths or degree of unsaturation, and either CSH or foaming ability.
The manipulation of Mycolata cell surface characteristics has been attempted. The basic premise
has been to try to change a hydrophobic particle to one that is more hydrophilic, thereby preventing
its attachment to bubbles. Stratton et al. (1997, 2002) used calcium chloride, dextrin, zinc sulfate,
carboxymethylcellulose, talc, bentonite, muloorina, and zeolite in attempts to alter Mycolata cell surface
chemistry, and found that only bentonite, talc, and zeolite were effective in lowering foaming potential.
However, none of these additives markedly changed CSH, again raising doubts about the role of CSH, as
measured by the MATH assay, on foaming. Instead they suggested that interactions between cell surfaces
and the bulk liquid (e.g. zeta potential) may play an important role in foaming.
Surfactants (surface active agents)

A surfactant is a substance that when present at low concentration, adsorbs onto the surfaces or interfaces
of a system altering the surface or interfacial free energies of those surfaces (Rosen, 1978). Surface-active
agents have a characteristic amphipathic structure, consisting of a lyophobic structural group with
little attraction for the solvent, and a lyophilic group with a strong attraction (Lang and Wagner, 1987).
When dissolved in a solvent, the lyophobic group in the solvent causes a distortion of the solvent liquid
structure, increasing the free energy of the system (Rosen, 1978), meaning that less energy is needed to
bring a surfactant molecule than a solvent molecule to the surface. Thus, the surfactant concentrates on
the surface. The presence of the surfactant also decreases the work needed to create a unit area of surface
(the surface free energy or surface tension) (Rosen, 1978). The lyophilic group prevents the surfactant
from being expelled completely from the solvent, and so the molecule has a lyophilic group in the
aqueous phase and a lyophobic group oriented away from it.
Microbial surfactants have been used in biotechnology for many years (see Kosaric et al., 1996). With the
Mycolata, the same mycolic acids that confer hydrophobicity also confer surface activity. Lechevalier
(1975) was first to show that N. amarae (G. amarae) grown in glucose medium reduced its surface tension,
and Cairns et al. (1982) reported that it produces a cell-bound surfactant when grown with hydrocarbon
substrates like hexadecane. Similar results were obtained with other Mycolata, where higher surfactant
production was recorded in media containing hydrocarbons (Soddell and Seviour, 1990). With acetate
and hexadecane present, biosurfactant production by G. amarae increased compared with hexadecane
alone (Pagilla et al., 2002). In addition, this biosurfactant was utilized as sole carbon source by G. amarae
(Pagilla et al., 2002). Participation by both cells and extracellular biosurfactants in foam formation and
Foaming 243
stabilization was shown for G. amarae SC1 culture (Iwahori et al., 2001b). Other foaming organisms like
Rhodococcus rubra, also produce surfactant (Khan and Forster, 1991).
Measurements of surface tensions of mixed liquor samples show correlation between these values
and foaming. Thus, when foam and foam-causing organisms were present, there was a marked decrease
in surface tension of the mixed liquors (Goddard and Forster, 1986), Yet because surface tension did
not relate to levels of extractable lipids, the surfactants involved were considered not to be fatty acid
homologues. Subsequently Goddard and Forster (1987) pointed out that higher concentrations of lipids,
proteins, and carbohydrates were present in foaming than in non-foaming plants, which they attributed
to presence of biosurfactants whose composition depended on which substrate was being used. They
also hypothesized that their putative foaming organism (N. limicola?) was producing biosurfactants.
However, Blackall et al. (1991b) could not correlate foam quantity and its stability with changes in
surface tension values in their work. In a related study, Khan and Forster (1990) showed that biosurfactants
from R. rubra generated a foam whose stability was similar to that produced with p-octylbenzene
sulphonate, a chemical surfactant. However, this stability was not comparable with that in treatment
plants where Rhodococcus sp. was dominant. They hypothesized that changes in plant feed substrate
composition may be an additional factor in foam formation (Khan and Forster, 1990).
Jenkins (1992) postulated that for surfactants to exert foam-producing and stabilizing effects, they must
be poorly or slowly biodegradable in the aeration basin. This was demonstrated by Ho and Jenkins (1991),
with the nonionic surfactant alkyl phenol ethoxylate. They also showed that surfactants alone could not
generate a stable foam if the sludge did not contain ‘Nocardia’ cells, and foaming of the ‘Nocardia’ –
containing sludge reduced the ‘Nocardia’ count by almost three-fold.
FACTORS AFFECTING GROWTH OF FOAM FORMERS

Nutrient requirements
Foaming Mycolata can use a range of substrates, including readily degradable sugars and short chain fatty
acids, as well as slowly degradable substances like high molecular weight polysaccharides, proteins and
aromatic compounds including pesticides (Goodfellow, 1992; Lemmer, 1986; Lemmer and Kroppenstedt,
1984). Organic and inorganic nitrogen, sulfur and phosphorus sources are also used by most strains (Lemmer,
1985), while some Nocardia spp. are reported to grow saprobically on dead cell material (Jenkins et al.,
1993). Other work suggests that Nocardia and related organisms also metabolize hydrocarbons, complex
lipids, steroids, phenols, aromatic carboxylic acids, chlorinated aromatics, dinitiles, triphenylmethane
dyes, and other xenobiotics (Bell et al., 1998; Bengis-Garber and Gutman, 1989; Boyle, 1989; Finnerty,
1992; Golovlev et al., 1978; Haggblom et al., 1989; Klatte et al., 1994a; Leahy and Colwell, 1990;
Pelczynska-Czoch and Mordarski, 1983; Rast et al., 1980; Sorkhoh et al., 1990; Tarnok, 1976; Uotila et al.,
1992; Warhurst and Fewson, 1994; Whalen et al., 1993; Williams et al., 1989; Yatome et al., 1993).
It should be noted that the name ‘Nocardia’ used in many of these studies covers a range of
phylogenetically diverse Mycolata, including species from Rhodococcus, Millisia, Nocardia, Gordonia,
Dietzia, and Tsukamurella. Nevertheless, it is clear that the Mycolata, particularly the rhodococci, are
extremely versatile metabolically, and so should compete well with other bacteria in activated sludge,
even though they may grow slower on simple readily degradable substrates.
Studies using microautoradiography (MAR) show a more complicated picture of which substrates
are taken up by Mycolata in situ. Thus, GALO Mycolata ‘identified’ solely by their characteristic
right angled morphology assimilated acetate, propionate, oleic acid, palmitic acid, trioleic acid, glucose,
leucine, ethanol, thymidine and an amino acid mixture in both foam and mixed liquor but only under
aerobic conditions (Kragelund et al., 2007b). However, Mycolata identified more precisely with FISH
probes targeting Gordonia and S. piniformis assimilated only acetate and propionate, and only the
Gordonia probe-identified filaments (Kragelund et al., 2007b) took up glucose. In another MAR study
(Carr et al., 2006), no glucose or oleic acid uptake was detected by Gordonia, but palmitic acid
assimilation did occur, while S. piniformis (Eales et al., 2006a) showed uptake of only palmitic and oleic
acid, and not glucose.
How is it then that pure culture work (Blackall et al., 1991a; Soddell and Seviour, 1996; Soddell
and Seviour, 1990) suggests that S. piniformis and G. amarae can both grow on glucose and acetate?
Furthermore, why do the MAR studies show such inconsistencies in uptake patterns? The first question
highlights the probable differences between organisms grown as pure cultures and their in situ behaviour.
It is not feasible to deduce in situ behavior from pure culture data. The second question may be answered
by considering the possibility of environmental stresses, such as UV, dessication, etc., and foam age,
which were hypothesized (Kragelund et al., 2007b) might lead to restricted substrate uptake ability.
However, there is no direct evidence for this, and more MAR studies are urgently needed to clarify this
issue. It is also important to note that uptake of a particular substrate shown by MAR does not necessarily
mean that the organism can grow on that substrate.
Candidatus ‘M. parvicella’ appears to have a more limited range of organic substrates for growth than
the Mycolata, and is a ‘specialist feeder’. However, controversy surrounds the behaviour of different
isolates. The original Slijkhuis isolate (Slijkhuis, 1983) grew on oleic acid, and its polyoxyethylenesorbitan
ester (Tween 80), as well as the polyoxyethylenesorbitan esters of stearic acid (Tween 60), palmitic acid
(Tween 40), and lauric acid (Tween 20), but not free long-chain fatty acids and volatile fatty acids. On the
other hand, the only other isolate characterized extensively in pure culture RN1 (Tandoi et al., 1998b),
could grow on a wide range of carbon sources including organic acids (acetic and pyruvic acid), yeast
extract, casamino acids, and sodium oleate, but now not oleic acid or the sorbitan esters. Australian isolates
did not grow on oleic acid or its sorbitan esters either (Blackall et al., 1994b). In contrast, in situ MAR
studies (Andreasen and Nielsen, 1997, 1998) showed that Candidatus ‘M. parvicella’ did not take up
acetate, ethanol, glucose, and other simple carbon sources, but only the long chain fatty acids oleic acid and
palmitic acid. Again, differences in environments have been cited as a possible explanation: competition for
easily biodegradable substances is more intense in activated sludge, and thus Candidatus ‘M. parvicella’
may have acquired a possible competitive advantage by preferentially using more complex compounds
in situ (Rossetti et al., 2005).
Oxygen
The belief has been that Mycolata required oxygen to grow (Goodfellow et al., 1982a,b,c; Soddell and
Seviour, 1998). Pure culture studies have shown, for example, that G. amarae could not grow on acetate
either anoxically or anaerobically (Blackall et al., 1991d). However, in situ MAR data have shown
substrate uptake under both conditions (Carr et al., 2006; Eales et al., 2005; Eales et al., 2006a). Here
again, several discrepancies exist that need further examination. Carr et al. (2006) reported uptake of
acetate, glycine, and glycerol under aerobic, anaerobic, nitrite-reducing, and nitrate-reducing conditions
for FISH probe-identified G. amarae. However, Kragelund et al. (2007b) observed little uptake of acetate,
propionate, and glucose for genus probe-identified Gordonia organisms under nitrate-reducing conditions,
less uptake of propionate under nitrite-reducing conditions, and no uptake under anaerobic conditions. For
PTLO, uptake of oleic acid under aerobic, anaerobic, nitrite-reducing, and nitrate-reducing conditions
occurred in Danish plants (Eales et al., 2005), but no uptake of oleic acid under anaerobic conditions was
detected in Australian plants for probe defined S. piniformis (Eales et al., 2006a). In the latter study,
Foaming 245
S. piniformis also assimilated glycerol, glycine, oleic acid, and palmitic acid under nitrate-reducing
conditions, but no substrate uptake was observed under nitrite-reducing conditions.
It was suggested that either the PTLO in the Danish study were not S. piniformis, or physiological
differences exist between strains from the different countries. The situation with substrate uptake by the
Mycolata with different electron acceptors is confused by different pre-incubation protocols in these MAR
studies. Thus, Kragelund et al. (2007b) used a 2 hour preincubation step with unlabeled substrates in the
absence of oxygen, while in the other studies (Carr et al., 2006; Eales et al., 2005; Eales et al., 2006a) no
or 0.5 to 1 hour pre-incubation was used. It is reported that many filamentous bacteria are MAR-negative
if this pre-incubation step is included, and MAR-positive without it (Andreasen and Nielsen, 1997), which
in the former case is thought to reflect that these substrates are not used by cells for growth but for storage.
However, the physiological explanation for this is not always clear. Future MAR studies should be
performed under standardized conditions to take into account the importance of this pre-incubation and
that at least 2 h pre-incubation with unlabeled substrate should be performed to differentiate between
substrate assimilation for storage and for growth.
The redox conditions supporting growth of Candidatus ‘M. parvicella’ have now become clearer. The
consensus is that it can grow over a wide range of oxygen partial pressures. It has a high affinity for
oxygen at low concentrations, which probably gives it a competitive advantage under low dissolved
oxygen conditions (Slijkhuis and Deinema, 1982; Tandoi et al., 1998b). Thus, Candidatus ‘M. parvicella’
can be considered a microaerophilic organism, and high DO concentrations (46 mg/l) may be toxic to it
(Rossetti et al., 2005; Slijkhuis and Deinema, 1982). While an Italian isolate showed no growth under
anaerobic conditions in pure cultures, it could partially reduce nitrate to nitrite (Tandoi et al., 1998b).
On the other hand, in situ MAR studies (Andreasen and Nielsen, 2000; Hesselsøe et al., 2005) showed
that oleic acid was taken up under anaerobic, anoxic, and aerobic conditions. Certainly lab-scale and full-
scale plant data show it proliferates in their anoxic and anaerobic zones (particularly in EBPR plants)
(Mamais et al., 1998; Blackbeard et al., 1986a; Eikelboom et al., 1994).
Several explanations attempt to account for the ability of Candidatus ‘M. parvicella’ to survive and
take up substrate under low oxygen conditions. Firstly, it was suggested it requires reduced nitrogen
compounds and reduced sulfur compounds for growth (Slijkhuis, 1983), and high oxygen levels probably
reduce their concentrations (Slijkhuis and Deinema, 1988). Secondly, MAR data under anaerobic-aerobic
dynamic phases show that it behaves like a phosphorus accumulating organism (PAO), in that takes up
and stores long chain fatty acids (LCFA) under anaerobic conditions (but not short chain fatty acids or
glucose), and then subsequently uses these when oxygen or nitrate are present (Nielsen et al., 2002b).
Because Candidatus ‘M. parvicella’ does not show an active uptake-release of orthophosphate, it has been
posited that intracellular glycogen or lipids may be the storage material used as energy source for
anaerobic LCFA uptake (Nielsen et al., 2002b).
Temperature
The effect of temperature on Mycolata growth has been investigated in pure culture and in treatment plant
experiments. Based on anecdotal information, Jenkins et al. (1993) suggested that the presence of
‘‘Nocardia’’ is associated with higher rather than lower bulk liquid temperatures. A correlation between
foaming problems and temperatures above 14– C was reported by Lechevalier (1975), and characterization
of ‘‘N. amarae’’ (G.amarae) showed that it grew between 23– C and 37– C, although a few strains also
grew at 10– C (Lechevalier and Lechevalier, 1974). Supporting information was presented by Pipes (1978)
who reported Mycolata foaming only at temperatures above 18– C, and by Pitt and Jenkins (1990) who
showed their ‘‘Nocardia’’ grew in bench-scale experiments at 18– C, 20– C, and 25– C, but not at 13– C. In a
rejoinder, Dhaliwal (1991a) disputed these findings, stating he observed ‘Nocardia’ foaming at treatment
plants in St. Paul, Minnesota and Detroit, Michigan, at 7–10– C, possibly because the two causative
populations strains were different. Soddell and Seviour (1995) reported that Lemmer and Popp described
year-round foaming in Germany at 8–10– C (winter) and 16– C (summer), but the causative organism was
not ‘‘Nocardia’’ but R. rhodochrous.
Soddell and Seviour (1995) showed that controversy about ‘Nocardia’ foaming and temperature
is essentially irrelevant, since Mycolata from activated sludge grew over a wide range of temperatures
(5–50– C) and that some, particularly rhodococci, could grow as low as 5– C. Thus, reports of ‘‘Nocardia’’
foaming at low temperatures were more likely due to Rhodococcus spp. whose cells are microscopically
indistinguishable from ‘‘Nocardia’’ spp. (Soddell and Seviour, 1995), emphasizing that the precise
identification of the causative organisms is crucial in such studies. Their maximum growth temperatures
may be relevant, given that foam is heated by direct sunlight, and ambient temperatures can reach 30– C or
higher, and Sakai et al. (1983) reported isolation of a Rhodococcus from mixed liquor at 40– C.
Slijkhuis (1983) suggested an optimum growth temperature for Candidatus ‘M. parvicella’ of 25– C,
although growth could occur at 8– C and no growth was detected at 35– C. However, Knoop et al. (1998)
determined its optimum growth to be at #12– C, and no growth at $20– C. An ability of Candidatus
‘M. parvicella’ to grow as low as 7– C provides it with a competitive advantage during winter. Seasonal
patterns, where it is more dominant during the winter, and increases as the temperature decreases, have
been observed in full-scale plants in the Czech Republic, Italy, South Africa, and the Netherlands
(Rossetti et al., 2005).
pH
The influence of pH on ‘Nocardia’ populations was determined by Cha et al. (1992), where at MCRTs of
3 and 8 days, its optimum pH was 6.5. Jenkins et al. (1993) theorized that the lower pH (6.5) in oxygen-
activated sludge plants would contribute to ‘Nocardia’ foaming, although foam trapping would have a
greater influence than pH. Furthermore, they postulated that plants carrying out nitrification are more
prone to foaming because of depressed pH values from denitrification. Sakai et al. (1983) on the other
hand, found the optimum pH for G. amarae was much higher, at 7.8. Again, these contradictions may
reflect different strains (or even genera) of ‘Nocardia’ examined in the two studies. Most strains currently
recognized as Nocardia spp. grow between pH 6 and 9, but not at 5, and many also grow at pH 10
(Goodfellow, 1971). A few Mycolata (Rhodococcus erythropolis and Gordonia terrae) can grow at
pH values as low as 4, and most rhodococci, gordoniae and tsukamurellae will grow up to pH 9
(Goodfellow et al., 1990; Goodfellow et al., 1991).
With pure cultures of Candidatus ‘M. parvicella’, growth occurs at pH 47.0, but with the optimal
around pH 8.0 (Slijkhuis and Deinema, 1982). This suggests that treatment plants (pH 6–7.5) represent
suboptimal conditions for this filament (Soddell and Seviour, 1990). However, for strain RN1, less
sensitivity to pH was observed, with no marked difference in its maximum growth rate between pH 6.7–8.0
(Tandoi et al., 1998b).
MCRT
The general consensus is that ‘Nocardia’ is favored at longer, rather than shorter mean cell residence
times (MCRTs) (Jenkins, 1992). This is consistent with pure culture data where S. piniformis may take
10–21 days to produce colonies, while G. amarae requires 4–7 days, and rhodococci take only 3–4 days
(Soddell and Seviour, 1990). In the original case history of foaming (Anon, 1969), the problem was solved
Foaming 247
by reducing sludge age. Pipes (1978) reported that foaming only occurred at MCRT 49 days, and this has
become a benchmark value in countries like USA for its control. Pitt and Jenkins (1990) described
increases in ‘‘Nocardia’’ counts with increasing MCRT. Washout MCRTs (MCRTs used for foam control)
of between 1.6–2.7 days appeared to be effective in several California plants (Cha et al., 1992). Despite
this, many reports describe foaming episodes at lower MCRTs (Sezgin and Karr, 1984; Soddell and
Seviour, 1990), and Dhaliwal (1979) concluded that foam was not related to sludge age or MCRT. Soddell
and Seviour (1990) suggest one explanation might rest with problems calculating sludge age and applying
different formulae. For example, solids levels in the secondary settler are not always included, and the
manner of wasting sludge differs within the same plant and from plant to plant. Another problem is
calculating a MCRT for foam, because floating solids are not usually considered (Blackall et al., 1985).
Still another issue is the question of organism identity, since most of these studies relied solely on
microscopic identification. Consequently, these contradictions may be explained readily by the presence
of quite different causative organisms in each study.
HOW DO FOAM FORMERS COMPETE IN ACTIVATED SLUDGE?

Given that most known foam forming organisms grow slowly in pure culture (compared to many other
activated sludge bacteria), how is it possible that such high masses of filaments are routinely observed in
foams? Since it is likely that some episodes are caused by non-filamentous cells, which cannot be
identified by staining and microscopy, relying on morphology for identification probably underestimates
the amount of Mycolata in activated sludge. So the question of how Mycolata compete in activated sludge
needs to be addressed.
r-K strategy?
Lemmer (1986) proposed a kinetic-based hypothesis to explain actinomycete growth and foaming, which
was based partially on their ecological niches in soil and activated sludge. According to this hypothesis,
Mycolata grow slowly using mainly slowly degradable complex organic substrates, thus occupying a
niche not available to many other populations. This occurs under normal activated sludge plant conditions,
and if the MCRT is sufficiently long, a stable population of these can build up without interfering with
plant operation (Soddell and Seviour, 1990). An ability to grow competitively at low substrate concen-
trations, characterized by high substrate uptake efficiency and relatively low cell yield, is a K-strategy.
However, when high concentrations of readily biodegradable substrates are available, these populations
switch to a mmax strategy (also called r-strategy, involving high growth rates at high substrate concen-
trations). In other words, Lemmer postulates that the Mycolata switch between K and r strategies. Data
supporting this hypothesis came from the work of Koronelli and Nesterova (1990), who showed that
Dietzia maris switches from a K-strategy with glucose to an r-strategy when grown on n-hexadecane.
Chua et al. (1996) suggested that G. amarae is also a K strategist that grows slowly, having a strong
affinity towards, and able to survive on, low concentrations of stearic acid. It would be interesting to see
similar kinetic studies extended to other Mycolata, although the capability of G. amarae to outcompete
floc-formers at both low and high substrate concentrations has been questioned by Blackall et al. (1991d).
In chemostat studies with acetate, they showed that G. amarae strains ASF3 and ASAC1 had lower
growth rates at both low and high acetate concentrations compared to Zooglea ramigera FFG3 and
filament type 021N. It should be noted that this study was performed only with acetate, so the data may not
apply to all substrates. In addition, the r-K strategy for Mycolata was proposed based on early literature
data and before differentiation between individual species was common practice (Eales, 2006). It is almost
certain that different species and strains of Mycolata will vary in their kinetic substrate uptake
characteristics.
Growth at the air-water interface

Another possible competitive strategy for Mycolata in foaming activated sludge involves the selective
enrichment of substrates and nutrients at the water-air interfaces (between bulk of water and aeration basin
surface, or surface of aeration basin bubbles). Lemmer (1986) hypothesized that the Mycolata, because of
their high CSH and branching hyphae, have selective access to substrates such as fats, oils, and greases
that float. Supporting this are the many anecdotal reports (Eikelboom, 1975; Forster, 1992; Franz and
Matsche, 1994; Pipes, 1978) associating ‘Nocardia’ foaming with fatty material. However, some reports
disagree and show no such relationship (Jenkins, 1992; Lemmer and Popp, 1982; Soddell and Seviour,
1990), or report no foaming in the presence of high fat loadings (Hrudy, 1981; Young, 1979). It is
pertinent here that a mathematical model showed the positive effect of substrate enrichment at the air-
water interface on Mycolata growth (Fougias and Forster, 1997).
Soddell et al. (1997) tested several hydrophobic substrates, including olive oil, safflower oil, coconut
oil, glycerol trioleate, paraffin oil, hexadecane, and kerosene, and found a wide range of foam isolates
grew on vegetable oil (an important component of domestic wastewater) and other oils. Interestingly,
with all insoluble hydrophobic substrates, these isolates clearly attached to droplets of substrate rather
than growing in the aqueous phase. Earlier (Soddell and Seviour, 1996) showed these Mycolata grew
on the oil droplets and did not appear in the aqueous phase until most of the oil had been degraded.
Cairns et al. (1982) found that G. amarae could de-emulgate oil in water emulsions, and Lemmer (1986)
postulated that oil droplets then served as a selective food supply, and conferred further competitive
advantage to it. At the air-water interface, Mycolata will be exposed to higher levels of hydro-
phobic substrates and nutrients, meaning higher effective growth rates compared to other populations
exposed to lower substrate concentrations in the mixed liquor. Strictly speaking, this is not a switch to
r-strategy, but reflects access to higher substrate levels, resulting in a higher m, independent of ecological
strategy.
Do foam formers have a preference for hydrophobic substrates? Some data suggest that Mycolata grow
better on hydrophobic substrates than on sugars. R. erythropolis and S. piniformis produced more cell
biomass with olive oil as a substrate, than with sugars like glucose or fructose (Kurane et al., 1986;
Soddell and Seviour, 1996). Baryshnikova (1985) reported that rhodococci grow considerably slower on
glucose than on aliphatic compounds like fatty acids, alcohols, and n-alkanes. Neu and Poralla (1988)
described a Rhodococcus species producing larger quantities of hydrophobic polysaccharide capsular
material in medium containing hexadecane than in one with glucose.
However, some data are inconsistent with this general view. Thus, foam isolates of G. amarae seemed
to produce more biomass with glucose, acetate, and trehalose than with hexadecane and cooking oil
(Blackalland Marshall, 1991), although difficulties were experienced with dry weight determinations
with the hydrophobic substrates (Blackall, 1987). R. rhodochrous also grew better on glucose than
on dodecane (Sorkhoh et al., 1990). However, Mycolata grew better on longer (C16–C18) carbon chain
alkanes (Fujita et al., 1991; Iwahori et al., 1995) than on shorter chain alkanes.
In an important statistical analysis of 8 years of data from a plant in Illinois experiencing seasonal
foaming, several hypotheses were tested to explain these episodes. The causal variables included: system
configuration, influent composition and concentration, MLSS concentration, effect of winter temperature
on minimum growth, oxygen transfer limitation, and the rate of hydrolysis of slowly degradable substrates
(Frigon et al., 2006a). Statistical analyses included path analysis, multivariate regression, and principal
Foaming 249
component analysis. Taken together, results showed that high lipid loading rate related to the primary
clarifier bypass at the plant, was the most likely cause. However, the rate of lipid hydrolysis was also
affected by temperature, increasing with increasing temperature, and thus an interactive mechanism
of temperature effects and lipid loading rates was proposed to explain the seasonal foaming episodes
(Frigon et al., 2006a). The importance of the lipid bypass stream and tripalmitin was experimentally
verified by an increase in the rRNA percentage of Mycolata in lab-scale reactors.
Lemmer (1986) also postulated that Mycolata have advantages at surfaces of secondary clarifiers,
which include adaptation to dryness (being former soil inhabitants), protection from solar radiation
by pigments, survival by saprobic growth on sludge particle during nutrient scarcity, and an ability to
synthesize polyP and PHB as reserve material to cope with starvation. All these factors would provide
advantages in foam.
Foam trapping
Blackall et al. (1991d) showed with chemostats that subsurface withdrawal of cells tended to favor
filamentous growth with G. amarae, while surface overflow culture removal favored cell clumping or
aggregation, forms selectively retained in the reactor. On the basis of these data, Jenkins (1998)
hypothesized that foam trapping in treatment plants increases ‘‘Nocardia’’ filament numbers, leading to a
greater propensity for them to foam. In other words, filamentous growth may be a survival mechanism to
prevent washout, and work by Narayanan (2003), in lab-scale experiments, has verified the effect of foam
trapping on encouraging dispersed growth forms of Mycolata.
Enrichment in the foam layer

It is possible that filaments have an advantage in foam, and that foam formation is an ecological strategy
for these organisms (see above). Alternatively, foaming may merely be the product of physical-chemical
factors (i.e. cell hydrophobicity and surface tension). Thus, they are there simply because they are
enriched in, and cause and stabilize foams.
If we assume that foaming is primarily a physical-chemical phenomenon arising from the
hydrophobicity of filament cell surfaces, then these filaments need not necessarily be metabolically
active. Studies with INT-(2-{p-iodo-phenyl}-3-tetrazolium chloride) to detect metabolically active cells
showed similar levels of activities in both foams and mixed liquors (Awong et al., 1985; Hernandez et al.,
1994). Awong et al. (1985) hypothesized that continuous, extensive movement of Mycolata from the
mixed liquor to the foam must be ocurring to explain their metabolic activity in foam. Note that the use
of tetrazolium salts to measure respiratory activity (chapter 11) suffers from limitations imposed by
extracellular redox activity (Wuertz et al., 1997). In addition, INT is considered less suitable for activated
sludge studies because of residual activity of the electron transport system (Maurines-Carboneill et al.,
1998). Thus, these salts may overestimate metabolic activity.
As stated earlier, FISH studies with foam have shown that filaments with the typical morphology of
GALO and PTLO do not always show FISH signals (de los Reyes et al., 1997; Eales, 2006) and that
empty regions within filaments are common (Schuppler et al., 1998; Carr et al., 2006). These observations
point to a low level of metabolic activity of cells in foam. Measuring cell activities in foam samples from
full-scale plants by quantitative membrane hybridizations and FISH (de los Reyes et al., 1998; Oerther
et al., 2001) revealed marked differences between the Gordonia rRNA fractions and Gordonia VSS
percentages in the same samples. These reflected the contributions of Gordonia to growth activity (rRNA
fractions) and biomass levels (VSS percentages) respectively.
Antibody probing also showed marked differences in the Gordonia VSS when measured by
quantitative FISH and fluorescent antibody staining. In general, higher Gordonia biomass levels were
obtained with antibody stains than with FISH (de los Reyes et al., 1998c). This was expected, because
antibody stains do not depend on cell ribosome levels (and therefore activity). These different outcomes
from the two methods indicate that a large number of G. amarae were not actively growing. Alternatively,
it is possible that Gordonia cells have a low intrinsic rRNA content (de los Reyes and Raskin, 2002). FISH
analyses targeting Gordonia in samples before and during foaming have revealed marked differences in
fluorescence signal intensity (de los Reyes and Raskin, 2002). Cells hybridized during foam initiation
gave much brighter signals than those examined during the foaming period, suggesting that growth related
activity was higher then. The presence of Gordonia showing the lower FISH signals suggests a retention
of non-growing filaments in foam. These data imply that methods to control the growth of filaments after
foam initiation may be ineffective, as inactive cells can still cause and stabilize foam. They point to the
need for more careful studies immediately before and during the early phases of foaming and stress the
importance of the foam initiation phase in any control strategies.
MEASUREMENT OF FOAMING POTENTIAL AND STABILITY

General methods
The measurement of foaming potential and stability has suffered from the arbitrary nature of most of
the methods used (chapter 5). Ideally, a ‘‘foaming test’’ should describe a parameter independent of
the apparatus and procedure used, and only reflect the nature of the solution tested (Bikerman, 1973).
Unfortunately, no such standard method exists. Nevertheless, several methods are available. Wilson
(1996) divided these into three groups: a) methods for characterizing foam structure; b) methods for
assessing foamability and foam stability; and c) methods for measuring foam rheology. Of these, methods
for assessing foamability and foam stability are most relevant to studies of activated sludge foaming.
Foamability (foam potential) is a measure of how easily a liquid foams. It is the foam-generating power
of a liquid, the suspensions contained within it, and the factors that help to attain immediate foam
stabilization (Wilson, 1996). It directly relates to physical factors such as viscosity, surface tension, and
other properties. Foam stability, on the other hand, involves factors that lead to its breakdown and
collapse, and is a measure of a foam’s persistence. Methods for measuring stability of foams include
(Wilson, 1996): a) the sparge tube technique; b) conductimetric method; c) pressure methods; d) light
scattering and reflection methods; and e) forced destruction/disruption methods. These are discussed in
more detail by Wilson (1996).
Methods applied to activated sludge

Several foaming measures have been described. Pretorius and Laubscher (1987) suggested the use of a
Scum Index (SI), calculated as follows:
biomass levels in foam
SI ð%Þ ¼ · 100
total biomass
The portion of biomass in foam is determined by fractionary flotation performed at a standard aeration
rate of 10 m3/m3-h in a batch flotation cell, 80 mm in diameter and 500 mm in height. Flotation is
repeated until all foam-forming organisms are transferred into the scum layer. This method has been
described as ‘‘probably the most exact method to predict the amount of foam formed in aeration basins’’
(Wanner, 1994d). A similar test was proposed by Ng et al. (2000). They called what was measured the
Foaming 251
‘‘foam index’’, which was related to the filament count. The extent of problems arising from foaming
according to different SI value is shown in Table 8.10.
Table 8.10. Scum Index values and expected operational problems

(Pretorius and Laubscher, 1987).
SI (%) Extent of problems
0–0.5 Insignificant
0.5–6 Low
6–10 Moderate
10–15 Serious
415 Disastrous
Foaming tests based on subjective foaming scales have also been used. For example, Blackall et al.
(1991b) developed a foaming test and a corresponding scale that combined foamability and stability. The
test is based on a foaming apparatus consisting of a glass cylinder (40 mm diameter, 500 mm height) fitted
with a sintered glass disc (max. pore size 40–90 mm) through which 200 ml/min of air is supplied. Foam
generation and stability of a 50 ml sample are recorded in terms of foam volume, bubble size, speed of
formation, and time of foam to collapse. Their classification to assess foams is shown in Table 8.11.
Table 8.11. Classification of foams (Blackall et al., 1991b).

Foam rating Description
0 Reaction to aeration as for pure water. Bubbles break at surface but are unable to foam
or have no stability
1 1.0–3.0 cm of foam with fragile, ill-formed bubbles. Insufficient stability to form films.
Immediate collapse when aeration is halted
2 Intermittent films sufficiently stable for at least 45–10 s. Usually generated from
a fragile foam structure of limited height. Films unstable when aeration is halted
3 Foam of some substance (i.e. bubbles about 1 cm diameter) to 3–8 cm height.
Infrequent to regular film formation, with both film and foam semi-stable when
aeration is halted. Films have 10–30 s of stability
4 Initially 8–15 cm of foam (about 1 cm diameter bubbles) with stable films being formed
at regular intervals. Body of the foam and films stable for 3–5 min once aeration ceases
5 Condition of stable foam 5–10 cm in height in 2 min, after which collapse to 3–5 cm
in height, which is stable when aeration is halted. No films
6 Stable foam 15–30 cm in height with no films. Bubble size about 0.5 cm during
production and only increases to 2–3 cm diameter in 3–5 min from time that aeration
is halted
7 Dense, stable foam higher than 30 cm after 2 min aeration. Bubble size about 0.3 cm
during production of foam, and max. 1 cm diameter 3–5 min after aeration is halted.
Foam is sufficiently stable to show no change in height 10–15 min after aeration is halted
Foam heights from foaming tests have also been used to quantify foaming. Ho and Jenkins (1991) used
a test requiring a 1-liter graduated cylinder aerated at 4 ft3/h through a sintered silica sand diffuser. In this
test, 400 ml of a mixed liquor of 3000 mg/l SS was used. Instantaneous foam heights were estimated after
every 10 s for 20 min, and the average foam height was measured at 5 min. Ho and Jenkins (1991) also
used an ‘Alka-Seltzer’ foaming test to estimate the foam potential of surfactant solutions alone.
Khan and Forster (1990) measured both foam height and stability with a pneumatic system similar to
those described above. In their dynamic method, foam heights (taken after 1 min) were measured while
air was being passed through the samples. For ‘‘foam-positive’’ samples, a low aeration rate, static
method was used (aerated for 30 min, foam height read 1 or 5 min after aeration was stopped). They also
expressed their results in terms of a foam stability index, defined by the time it takes for a foam to
collapse to 50% of its initial value (T50). This method has been used by other workers (Kerley and
Forster, 1995; Kocianova et al., 1992), while Kocianova et al. (1992) reported a method involving
estimating the percentage of foam cover on reactor surfaces in full-scale plants. Their classification is
given in Table 8.12.
Table 8.12. Classification of foam cover in full-scale activated sludge plants (Kocianova et al., 1992).
Cover (%) Feature
0 No foam or light froth in corners
10 Foam in corners extending to one quarter the length of the pocket
20 Foam extending from corners meeting in center of side walls
35 Foam forming circle around aerator drawing away from side wall
50 Foam extending towards aerator by one third the radius of the tank
70 Foam extending towards aerator by one half the radius of the tank
Kocianova et al. (1992) also measured foam accumulation levels in the secondary settling tank with a
translucent cylinder with holes (9 holes, 1.5 mm dia.) drilled into the bottom. The cylinder was drawn up
through the foam, allowing the mixed liquor to pass through the holes and the foam to form a compact
plug at the bottom. The volume of foam was then measured and expressed as rate of foam accumulation
in ml/h. Their data showed that foam accumulation and percent cover were linearly related within the
95% confidence limits. Furthermore percent foam cover was correlated with biomass hydrophobicity,
and they suggested this could act as a simple operational technique for assessing foam problems.
A comparison of the SI (Pretorius and Laubscher, 1987), foaming rate (Blackall et al., 1991b),
hydrophobicity (Rosenberg et al., 1980), aeration basin coverage (Kocianova et al., 1992), and oil and
grease and DSVI methods was performed by Torregrossa and Viviani (1997). Their results showed no
clear correlation between these parameters, although SI, hydrophobicity, and foaming rate all increased as
aeration tank percent coverage increased. No correlation was established between SI and mixed liquor
biomass hydrophobicity. The authors proposed that SI and foam hydrophobicity are more reliable
estimates, although they agreed that no single parameter should be taken as a working standard. A similar
lack of correlation between SI, hydrophobicity, aeration tank coverage, and Candidatus ‘M. parvicella’
population was found by Hladikova et al. (2002). Later Torregrossa et al. (2005) showed that both SI and
foaming rates correlated with hydrophobicity of the mixed liquor and foam, but not with aeration basin
coverage, because of effects of surface hydraulic arrangements.
Another foam potential test (Oerther et al., 2001) modified the Alka-Seltzer foaming test of Ho
and Jenkins (1991). It involved placing 250 ml mixed liquor samples into 500 ml graduated cylinders
and adding 2 tablets of commercial Alka-Seltzer antacid tablet. The frothy effervescence generated
caused foamy material to rise. The maximum height reached was expressed as maximum foam height.
To standardize this test, it was conducted on samples from a full-scale plant and different solids
Foaming 253
concentrations. Its optimization involved developing a quick method for determining solids levels by
microwave drying (de los Reyes, 1997) which allowed an a priori knowledge of their levels. This test
has been applied to a full-scale plant before, during, and after a foaming episode (Oerther et al., 2001)
and in determining foam formation and stability thresholds (de los Reyes and Raskin, 2002). Maximum
foam heights generated during foaming were markedly higher than those before or after foaming at all
solids levels, outcomes suggesting that this foam potential test can be used as an index for quantifying
foaming.
FOAM CONTROL METHODS

Surveys of foaming wastewater treatment plants have shown that the most common methods employed are
water sprays, contact zones, chlorination of return activated sludge, and skimming of aeration tanks,
aerated channels, and final clarifiers (Wanner, 1994d; WEF, 1992a). Their application and success rates
are shown in Table 8.13.
Table 8.13. Strategies used to control biological foaming in the USA, Australia, and France (Blackall et al.,
1991c; Pitt and Jenkins, 1990; Pujol et al., 1991b).
Number of US Success Success Success
plants using Rate, % Rate, % Rate, %
Foam control strategy strategy US Australia France
Reduction in sludge age 44 73 57 n/a
Chlorination (or oxidant addition) 48 58 20 66
of mixed liquor and/or return AS
Use of water sprays 58 88 28 n/a
Initial contact zones n/a n/a n/a 73
Use of antifoaming agents 35 20 n/a 57
Coagulant addition n/a n/a n/a 46
Reduced aeration 5 60 33 n/a
These data illustrate that adoption and success rates of control methods differ widely, and that there is
no one universal ‘‘magic’’ solution. In fact, the complexity of the problem has generated considerable
scepticism for foam control methods. Pujol et al. (1991b) found that technical measures taken against
foaming had been used in only 16% of plants with foaming problems, and the average success rate did not
exceed 30%. Wanner (1994d) sensibly suggested that when discussing foam control, methods that target
causative filaments should be distinguished from the non-specific (e.g. removal/destruction of foams)
methods, to avoid possible misinterpretation.
Manipulation of biomass retention time

The first reported case of foaming occurred suddenly, following a decrease in organic loading in the
aeration basin of the East Plant of the Jones Island municipal wastewater treatment plant operated by
the Sewerage Commission of the City of Milwaukee (Anon, 1969). The other aeration basin running in
parallel was not subjected to the same conditions, and did not experience foaming. Consequently, when
the organic loading of the foaming basin was increased by lowering the MLSS (and therefore, increasing
the F/M ratio), the strategy proved successful, and has since been used commonly to control foam
(Goddard and Forster, 1987b; Pipes, 1978). Reducing MLSS also reduces MCRT, assuming the same
sludge wasting rate. Its success is based on kinetic washout. Slow-growing organisms (such as Mycolata)
are washed out at lower MCRTs. Thus, a variety of workers have proposed ‘benchmark’ values for
MCRT, F/M, and MLSS.
Pipes (1978) reported that at MCRTs 49 days, ‘nocardioforms’ (¼ Mycolata) were present in large
numbers above 18– C. However, others have reported foaming at shorter MCRTs (Soddell and Seviour,
1990 and references therein). Dhaliwal (1979) questioned the effectiveness of using MCRT and MLSS
reduction, and stated that foaming arises because of the presence of ‘N. amarae’ (G. amarae) and is not
related to sludge age, F/M ratio, or MLSS concentration. Goddard and Foster (1987a) also suggested that
foaming is independent of F/M ratios and HRT. Cha et al. (1992) showed that ‘Nocardia’ levels generally
increased as MCRT increased over 1.5–20 days. At 16– C, Nocardia washout occurred at an MCRT of 2.2
days, while at 24– C, it occurred at an MCRT of 1.5 days. Their data underscore the importance of
temperature effects on MCRT. In general, the higher the temperature, the lower the sludge age needs to be
to accomplish washout. However, caution should be exercised when using these values as guides, since
different Mycolata will behave differently at different MCRT-temperature combinations (see earlier).
Contradictions with these ‘benchmark’ values may reflect differences in calculating MCRT (Soddell
and Seviour, 1990), and with calculating sludge ages for floating solids. Once a stable foam layer is
formed, traditional calculations may not be valid (Soddell and Seviour, 1990). Gujer and Kappler (1992)
suggested a correction for floating biomass in the MCRT calculation:
yx; foam ¼ 1=ðDx kF Þ
where Dx ¼ 1/yx (at steady-state) and kF is the flotation rate constant, which varies from zero (with free
water surface), or less than zero (floating cells are washed out from the system at a rate higher than the
mixed liquor wastage rate), to Dx in systems with high foam accumulation.
Use of chemicals and antifoam agents

A popular non-specific control method for controlling foaming is applying chemicals. Chlorination of
RAS or the mixed liquor is widely used, although not as effectively as in bulking control (Wanner, 1994d).
Because foam-forming filaments are selectively retained in the foam, and thus not concentrated in the
RAS lines, chlorination may not be adequate for filament death (Wanner, 1994d). For example, Sezgin
and Karr (1986) reported chlorinating RAS in a foaming plant led to deteriorating effluent quality and
floc dispersion without eliminating the Mycolata. In addition, Candidatus ‘M. parvicella’ may require a
10–100 times greater dosage of chlorine than other activated sludge filaments (Neethling et al., 1985).
The basic idea is to apply sufficient chemical to the foam layer so as not kill the floc-forming microbes.
Wanner (1994d) suggested spraying chlorine solution, or sprinkling powdered calcium hypochlorite
directly onto the foam. This technique was employed by Albertson and Hendricks (1992) in a Phoenix,
Arizona plant, where spraying of 2000–3000 mg/l Cl2 for two days was adequate. Chlorination can also be
applied to disinfect wasted foam and solids returned to the primary tank inlet to prevent reinfection of the
aeration basin (Tipping, 1995). The use of ozone for controlling filamentous growth has also been used
(Goi et al., 1994), and it and peroxide (van Leeuwen, 1992) have both been reported to suppress foaming,
and to control successfully G. amarae foams.
Coagulants such as FeCl3 are popular. Thus, Duchene (1994a) added 4 g FeCl3/kg MLSS/d,
which reduced foaming markedly within two weeks. In Germany, Lemmer and Kroppenstedt (1984)
added 4 mg/l of Fe3þ to a full-scale plant, although in the laboratory, 10 mg/l of Fe did not control several
Foaming 255
Gordonia or Rhodococcus strains. G. amarae growth was inhibited. Reduction in phosphorus availability
by precipitation with pickle liquor (Fe3þ) was suggested as another mechanism for foam control (Soddell
and Seviour, 1990).
Antifoaming agents are generally not effective against this thick, stable viscous filamentous scum.
Duchene (1994a) concluded that commercial enzymatic or microbial products also do not work when used
at realistic dosage rates, agreeing with Blackall et al. (1991c) who showed two products marketed for
scum control had no effect on foam in full-scale plants in Australia. In anaerobic sludge digesters, no
difference was seen between control reactors and reactors receiving 500 and 1000 ml/l of defoamant (Ross
and Ellis, 1992). ‘‘Bacteria-enzyme’’ additives were investigated by Franz and Matsche (1994), who
reported no difference between the foaming of a control basin and a parallel basin to which additives were
applied. Interestingly, they did observe a shift in filamentous population from ‘Nocardia’ to Candidatus
‘M. parvicella’ without any effect on the extent of the foaming. These examples highlight the general
experience that all antifoams and commercial ‘‘bacteria-enzyme’’ additives are not effective. Addition of
particles which modify the cell surface and cell surface hydrophobicity is an area of some interest
(Stratton et al., 1997). Initial results are still mixed; only bentonite, talc, and zeolite were effective in
decreasing foaming potential (see previous discussion). Anti-mycolate compounds inhibiting cell wall
(mycolic acid) synthesis, and thus growth of Mycolata, have been tested (Davenport et al., 2005), and of
seven compounds tested, one (3-hydroxyhexanoic acid) showed some potential as a foam control agent,
although the economics of its application were not discussed.
Using polymers to control ‘Nocardia’ foaming was reported in the 1300 l/s (30-mgd) Terminal Island
Treatment Plant in Los Angeles, California (Shao et al., 1997). A dose of 0.5 mg/l of polyacrylamide
cationic polymer (Clarifloc LA-2691) into the mixed liquor channel reduced aeration basin foam coverage
and ‘Nocardia’ counts. No attempts were made to investigate the mechanisms for its action, but two
hypotheses were presented. The cationic polymer may have neutralized (precipitated) anionic surfactants,
thus eliminating their foam-stabilizing effects. Alternatively, it may coagulate dispersed ‘Nocardia’
filaments into the flocs, enabling them to be wasted along with the rest of the mixed liquor (Shao et al.,
1997). Later work (Narayanan, 2003) showed the latter mechanism to be more likely.
Addition of polyaluminum chloride (PAC) is now an accepted control strategy for Candidatus
‘M. parvicella’ in Europe (Eikelboom, 1997; Roels et al., 2002). Jacobsen et al. (1997) reported
that polyaluminum chloride (PAX 14) doses of up to 15 g Al/kg MLSS effectively inhibited M. parvicella.
At higher doses, inhibition of nitrification was also observed. Nielsen et al. (2005) used higher doses
(10–15 g Al/kg SS), than Badoer et al. (2004) (1.5 g Al3þ/kg MLSS/day). The recommended dose of
PAX-14 is 2–3 g Al/kg MLSS/day in the RAS for at least three weeks (Rossetti et al., 2005), and
Roels et al. (2002) suggested an empirical formula: 60/SRT ¼ # g of Al3þ/kg MLSS. Eales (2006) tested
Alchlor-AC, another commercial aluminum chloride to control Candidatus ‘M. parvicella’, and showed
no effect of dosing on reactor pH and MLSS.
What is the mechanism of action of polyaluminum chloride on Candidatus ‘M. parvicella’? Eikelboom
(1997) suggested that PAC altered its cell surface characteristics, changing its physiology or substrates
used, while both Roels et al. (2002) and Rossetti et al. (2005) proposed that its effect is on the
hydrophobicity of the cell wall, reducing its competitive advantage in mixed liquor, or preventing it from
metabolizing hydrophobic substrates. Badoer et al. (2004) suggested that the rapid decrease in foam
coverage following PAC addition may reflect some denaturation of its cell wall, but Nielsen et al. (2005)
showed marked decreases in lipase activity after PAC addition, and no effect on CSH. Eales (2006) also
showed no decrease in CSH after addition of Alchlor-AC, although she could not detect any decrease in
in situ lipase production. It should be noted that the dosages used in the Nielsen et al. (2005) study were
much higher than those used by Eales (2006). PAC has also been tested on some Mycolata. Batch tests
(Eales, 2006) showed that Alchlor-AC addition as low as 0.001% (v/v) prevented the growth of G. amarae,
but that higher concentrations (10%, v/v) were needed to lyse cells that were already growing. Thus the
effect appears to be on cell viability, not on enzyme activity or CSH. On the other hand, Roels et al.
(2002) found no effect of PAX-14 on ‘Nocardia’ in situ. Clearly, more controlled full-scale studies with
more Mycolata are needed to resolve these questions, as any insensitive Mycolata may not have been
G. amarae. Successful control of filaments was also reported by Bidault et al. (1997), and Eikelboom and
Grovenstein (1997) where a mixture of talc and chlorite (PE 8418) controlled bulking. Neither study
mentioned which filaments were affected, and so this talc mixture should be investigated specifically for
defined (e.g. Candidatus ‘M. parvicella’) control.
Physical methods
An obvious method to reduce problems caused by foam is to skim the foam layer off from secondary
clarifiers and even aeration tanks. The design recommendation of the Water Environment Federation
manuals is to equip both rectangular and circular secondary settlers with skimmers which can act over
the whole surface (WEF, 1992b). Baffles that form foam traps should be provided with a foam removal
device. Pumps and pipes designed for foam removal must take into account the higher viscosity of the
foam compared to the mixed liquor (Wanner, 1994d). Because non-viable filaments may also cause
foaming in anaerobic digesters (van Niekerk et al., 1987b), any recycling of foam through the plant should
be avoided at all costs. Fahmy and Hao (1990) suggested the hydrolysis of foam using low pH and heat
treatment as an attractive alternative to foam disposal to eliminate transferring any foam-formers to the
anaerobic digester. Thermal hydrolysis of the separated foam at 9 bars and 170– C appears to be a
treatment option for separated foam, as thermal hydrolysis completely destroys Gordonia cells (Jolis and
Marneri, 2006).
The process of selective flotation has been used for the enrichment of valuable minerals from bulk ores
(Leja, 1982). The same concept has also been applied to foaming. Pretorius and Laubscher (1987) showed
that more than 95% of the foam-forming organisms were removed within a few hours. The concept of
flotation and selective foam wasting was extended successfully to full-scale plants by Richards et al.
(1990) in Atlanta. Classifying selector (a term coined by Brown and Caldwell (Parker et al., 2003)) refers
to the mechanism by which foam is separated from mixed liquor by virtue of its propensity to float.
Pagilla et al. (1996b) investigated both lab- and full-scale classifying selectors. The former consisted of a
foam window, a ‘foam through’, and a foam collector. Selective classification was practiced once a day
for 10 min by stopping both influent flow and RAS pumps, increasing aeration rate, then manually
scraping surface foam into the ‘foam through’ and the foam collector. In the full-scale system, the
classifying selector was placed in one of the RAS channels and used a surface foam wasting system
(Pagilla et al., 1996b). Results showed that classifying selectors reduced the ‘Nocardia’ levels to less than
those in a non-foaming reactor, and addition of a nonionic biodegradable surfactant (NEODOL 25–7)
enhanced their removal. Parker et al. (2003) reported that several full-scale plants have been in operation
in the U.S. since the 1990’s, which employ retrofit devices that waste the foam continuously, which they
claim are effective in reducing foam and filament counts. In addition to classifying selectors (for retrofit
applications), proper design (e.g. eliminating foam trapping features such as baffles) is now considered
vital to prevent foaming from becoming a serious operational problem, and Jolis et al. (2006) have used a
combination of selective wasting, SRT control, and polymer addition to control Mycolata counts in a full-
scale plant.
In general, spraying with water or mixed liquor may be useful against light foams, but is ineffective
against heavy, stable foams (Jenkins et al., 1993; Soddell and Seviour, 1990). Water sprays may be
Foaming 257
viewed as emergency control measures to prevent massive loss of solids in the secondary clarifier
(Wanner, 1994d). High volumes of water may dilute the foams instead of mechanically breaking them,
and thus secondary clarifiers need to be equipped with adequate scum traps to handle the increased scum
(Soddell and Seviour, 1990). Other mechanical foam breaking systems have had some success, although
they do not remove foam completely (Soddell and Seviour, 1990).
Addition of supernatant from anaerobic digesters

Soddell and Seviour (1990) cite the early experiments of Lechevalier showing that addition of anaerobic
digester supernatant back into the primary clarifier reduced foaming in the aeration basin. However, the
finding that anaerobic digester foaming can also occur discredits these findings (Hao et al., 1988a), and
competition for nutrients by organisms in the supernatant was used to explain the ‘‘toxicity’’ effects.
Kinetic and metabolic selection

Originally proposed in the 1970s (Chudoba and Pujol, 1994), application of selectors to control
filamentous growth has increased over the past few years. Their effectiveness depends on kinetic and/or
metabolic advantages conferred to floc-formers over filaments (chapters 5, 7). Kinetic selection depends
on the fact that floc formers exhibit higher soluble substrate uptake rates compared to filaments at high
substrate concentrations. Metabolic selection is based on a postulated inability (or decreased ability) of
many filaments to take up substrate under anoxic or anaerobic conditions (Jenkins et al., 1993).
Since Mycolata have traditionally been considered as aerobes, aerobic selectors rely on kinetic
selection. For example, in chemostat studies under aerobic conditions, ‘Nocardia’ (Gordonia) amarae
demonstrated lower acetate uptake rates than Zooglea ramigera at high dilution rates (i.e. low MCRTs)
(Blackall et al., 1991d). It was suggested that aerobic selectors may not be as effective in controlling
G. amarae growth at higher MCRTs. This was supported by data from Cha et al. (1992) who reported that
Nocardia (Gordonia?) levels decreased with an aerobic selector at a 5-day, but not a 10-day MCRT.
A ‘feast-famine’ approach that alternately raised and lowered the F/M ratio was investigated in Hong
Kong (Chua et al., 2000). By increasing it to 0.8 mg BOD/mg MLSS-day in the ‘‘feast unit’’ and lowering
it to 0.2 in the ‘‘famine unit’’, the average F/M was kept below 0.5 mg BOD/mg MLSS-day. It also
appeared to decrease foaming at lab-scale.
Control of Gordonia using anoxic selectors was also successful in stirred tank reactors with 12 day
MCRT (Cha et al., 1992). After 5 MCRTs, the control reactor had Gordonia counts 4 times higher than
those in the selector system. When their contents were interchanged, the control CSTR Gordonia count
decreased rapidly under anoxic selector conditions (Cha et al., 1992). Gasser (1987) reported that periodic
anoxia in full-scale plants controlled foaming caused by Gordonia. The same result was obtained by
Gerardi (1986), who advocated shutting off the aeration for two hours, settling, and transferring the
supernatant to an on-line aeration basin. Addition of 50 to 150 mM of calcium nitrate per m3 of sludge,
together with a reaction zone with an HRT of 1 hour, was also claimed to be effective in controlling
‘Nocardia’ growth (Yu, 2007).
As G. amarae could not grow nor take up acetate under completely anaerobic conditions (Blackall
et al., 1991d), it was hypothesized that anaerobic selectors should be effective in controlling this
organism. However, only small and inconsistent reductions in G. amarae levels were obtained with
anaerobic selectors in laboratory- and full-scale experiments (Jenkins et al., 1993; Pitt and Jenkins, 1990).
Presence of Gordonia filaments in anaerobic digester foam and sludge is also problematic and may
reflect the effects of anaerobic conditions. van Niekerk et al. (1987b) reported that Gordonia filaments
concentrated in the foam in anaerobic digester sludge, and while Gordonia may not be expected to survive
there, filament fragments still retained their ability to produce foam. In fact Hernandez and Jenkins (1994)
showed that Gordonia filaments are not completely degraded during anaerobic digestion (approx. 37%
filament reduction at MCRT of 14 days). Furthermore, a large fraction (approx. 60%, as judged by INT
reduction) of Gordonia filaments were capable of respiration after 14 days. These results point out
the likelihood of Gordonia survival in anaerobic digestion, and the risk of their consequent recycling
to the treatment mainstream. Nevertheless, there are some reports of success with anaerobic selectors
(Fainsod et al., 1999; Jolis et al., 2007; Mori et al., 1992), which once again might be explained by the
presence of different Mycolata in each study.
Candidatus ‘M. parvicella’seems to compete well in anaerobic selector systems. It has a high substrate
storage and uptake capacity (Jenkins et al., 1993), and an ability to take up substrates under anaerobic
conditions (Rossetti et al., 2005). It seems that M. parvicella can compete particularly well in anaerobic-
aerobic systems like EBPR systems, since surveys show that these are particularly prone to bulking, and
that Candidatus ‘M. parvicella’ often dominates there.
CONCLUSIONS
Before understanding comes confusion (Grady, 2003). This certainly has been the case with foaming in
activated sludge. From attributing it to one species of filamentous bacteria to realizing the high level of
diversity among the foam-forming filamentous and non-filamentous organisms; from anecdotal evidence
for causes to quantitative assessment of mechanisms and organisms; and from trial-and-error ‘solutions’
to more rational control methods, much progress has been made in the last four decades. However, there
is still more to be learned on the nature of the organisms involved, the specific mechanisms leading
to foaming, and the most effective prevention and control methods. The tools that have been used
traditionally by microbiologists and engineers, like microscopy and pure culture studies, are still valuable,
and should be employed alongside molecular methods. Modern approaches for studying foaming should
be more quantitative (both in measuring parameters, and their statistical analysis), and should more
accurately mimic in situ conditions. For example, more detailed studies with qPCR, DNA microarrays,
stable isotope probing, proteomics techniques, advanced modeling approaches, and other methodological
innovations (chapter 11) will surely generate more meaningful data. Even then, some confusion will still
precede understanding, and only careful analysis of the data will allow us to understand the microbiology,
causes, and control of activated sludge foaming.
9
The microbiology of nitrogen removal
Holger Daims and Michael Wagner
INTRODUCTION
Most of the nitrogen (N) in municipal and many industrial wastewaters occurs in the form of ammonium
(NHþ 4 ), which is in pH-dependent equilibrium with ammonia (NH3), or is bound in urea or other organic
molecules, for example in the amino groups of proteins. From there it is set free, also as ammonia/
ammonium, during the biodegradation of organic matter which is carried out by heterotrophic microbes in
the activated sludge system. The microbially catalyzed release of ammonia/ammonium from organic
matter is known as ammonification (Figure 9.1). In two-sludge treatment plants ammonification proceeds,
along with BOD reduction, in the aerobic high-load first stage. As the heterotrophic microbes need N for
their cellular processes and growth, a fraction of the ammonium is incorporated into activated sludge
biomass. However, this assimilation of N (Figure 9.1) usually is not sufficient for its removal from
wastewater. The average C:N ratio of heterotrophic biomass is 12:1, whereas the C:N ratio of domestic
wastewater is shifted towards a higher N content (Bever et al., 1995). The excess N remains in the effluent
and without further processing would be discharged into receiving waters.
This has to be prevented, because ammonia and its oxidation product nitrite, are highly toxic to aquatic
life, and N pollution leads to the eutrophication of natural waters, causing long-term damage to invaluable
ecosystems (Henze et al., 1997). Therefore, its elimination from sewage is among the most important
steps of modern biological wastewater treatment and the permitted N concentrations in effluents of
treatment plants are strictly limited. For example: while the total N load of typical municipal wastewaters
is between 20 and 80 mg N · l 1 (Henze et al., 1997), the effluent of a large municipal treatment plant with
more than 100,000 population equivalents must not contain more than 10 mg N · l 1 (of total N) according
to regulations in the European Union. However, several EU countries have stricter limits, where the
NO–2
Nitrification
Ammonification
NO–3 Biomass NH3 NH+4

Assimilation Assimilation +
NO–2
Anammox
NO–2 N2
Denitrification
NO
N2O
Figure 9.1. Schematic illustration of the biogeochemical nitrogen cycle. Processes that are relevant to
nitrogen removal in wastewater treatment plants are marked by bold arrows. See text for details.
maximum permitted N level is 8mg N · l 1. Efficient N removal often requires the operation of separate
reactors, because the growth requirements of the involved microbes differ substantially from the optimal
conditions for heterotrophic carbon oxidation (chapter 2).
Conventional N removal comprises two completely different microbial processes: nitrification and
denitrification (Figure 9.1). During nitrification, ammonia is oxidized to nitrite that is subsequently
oxidized to nitrate. The microbes contributing to nitrification (nitrifiers) form two distinct functional
groups which are specialized in either ammonia or nitrite oxidation. Interestingly, no known single
organism is able to oxidize ammonia completely to nitrate although it has been speculated that such
organisms might exist (Costa et al., 2006). Both functional groups of nitrifiers are aerobic and
chemolithoautotrophic, i.e. they fix CO2 and gain energy for growth by oxidizing inorganic substances –
in this case ammonia or nitrite, respectively (but some nitrifiers can also denitrify under anaerobic
conditions; Bock et al., 1995; Ehrich et al., 1995; Freitag et al., 1987; Stüven et al., 1992). Although the
nitrifiers are aerobic microbes and nitrification requires oxygen, this process can often not be combined
efficiently with heterotrophic BOD-reduction in the same activated sludge tank. The nitrifiers would be
inhibited by the high concentrations of organic carbon and thus not able to compete with the faster
growing heterotrophs for O2. Instead, in many treatment plants nitrification takes place in a second tank
receiving the effluent of the first stage with a lower C:N ratio that favours growth and activity of the
nitrifying organisms.
The oxidation of ammonia and nitrite yields only low amounts of free energy (Bock and Wagner, 2001;
Costa et al., 2006), and nitrifiers must maintain an energy-demanding reverse electron flow to supply their
anabolic metabolism with the required reducing equivalents (Bock and Wagner, 2001). Consequently,
all nitrifiers are slow-growing microbes and the sludge age in nitrifying tanks must be higher than in
systems for carbon oxidation. This causes practical problems. Firstly, a long retention time favours the
proliferation of slow-growing filamentous organisms that may cause activated sludge bulking and foaming,
for example Candidatus ‘Microthrix parvicella’ (chapters 7, 8). Secondly, nitrifying populations recover
only slowly after any breakdown of nitrification. Breakdowns happen occasionally and are caused for
example, by changes in temperature, pH, or wastewater composition. However, in many situations a clear
correlation between nitrification performance and environmental conditions is not obvious. In general,
The microbiology of nitrogen removal 261
such problems are more common in industrial treatment plants where the wastewater composition is more
variable and concentrations of inhibitory substances are often higher than in municipal systems. As
nitrifiers grow slowly, nitrifying reactors have often been implemented as biofilters, such as trickling
filters and rotating disc filters. Here microbes form sessile biofilms, so that high amounts of slow-growing
biomass accumulate and can be maintained. In such systems, carbon oxidation occurs near the inlet of the
reactor where the organic matter concentration is higher and heterotrophs out-compete the nitrifiers for
O2. Nitrification takes place farther away from the inlet, in those regions of the tank where the carbon
concentration is lower and sufficient O2 is available to the nitrifying bacteria (Bever et al., 1995; Henze
et al., 1997).
The second step of biological N removal, denitrification, is an anaerobic process catalyzed mainly by
facultatively anaerobic heterotrophic microbes. These bacteria often use organic compounds as electron
donors for reducing nitrate (NO3 ) and nitrite (NO2 ) to the gaseous N compounds NO, N2O and eventually
N2 (Zumft, 1997). Their demand for organic carbon is often not satisfied during periods of high N load and
relatively low carbon content of the wastewater, or if the sewage contains mainly complex, recalcitrant
carbon compounds. It is common practice to increase the denitrification rates under such conditions by
adding readily biodegradable organic compounds like methanol and acetate to the denitrification tanks
(chapter 2) (Henze et al., 1997). Methane in biogas has also been suggested as a cheap carbon source
suitable for denitrification (Werner and Kayser, 1991). Alternatively, the first stage of a treatment plant is
by-passed by channelling a fraction of the carbon-rich raw wastewater directly into the denitrification
stage to supply the denitrifying bacteria with sufficient organic carbon (Wandl et al., 2002).
Some designs place an anoxic denitrification tank between the high-load first stage and the aerobic
nitrification stage (chapter 2). In this case, the denitrification tank directly receives the effluent from the
first stage, which can still contain relatively high amounts of (chemically complex and slowly degradable)
carbon sources. Again, denitrification can be enhanced by addition of less complex organic compounds
or raw wastewater. With this setup, N removal is achieved by circulating wastewater between the
nitrification and denitrification tanks. Other implementations combine nitrification and denitrification in
partly or intermittently aerated activated sludge tanks, or sequencing batch reactors (chapter 2).
The classical N removal pathway via nitrification and denitrification is costly, because the nitrification
stage must be aerated and, if needed, organic carbon sources must be added to maintain denitrification.
Therefore, current interest focuses on pathways to N elimination that require less aeration and external
carbon supply. In this context, the anaerobic ammonium oxidizers (‘‘Anammox’’ organisms; Strous et al.,
1999) are highly relevant. These autotrophic bacteria can transform ammonium and nitrite directly to N2
with nitrate as a by-product of the reaction (Strous et al., 1998, 1999). Thus, this anaerobic process
constitutes a ‘shortcut’ in the biogeochemical N cycle (Figure 9.1). Future full-scale implementations
of the Anammox process could markedly reduce the space requirements and costs of N removal from
wastewater.
This chapter addresses the microbiology of microbes that are relevant to N elimination in activated
sludge treatment plants. As this broad topic embraces bacteriology, biochemistry and genomics as well as
physics of mass transfer and aspects of process engineering, an encompassing discussion is not possible
in the scope of a single chapter. Therefore, we have focussed on the identity, diversity and physiology of
these bacteria and other selected aspects of their biology, which are interesting for novel process designs.
In particular, we emphasize recent findings regarding the ecological niche differentiation of closely related
organisms or members of the same functional groups. The efficiency of wastewater treatment is ultimately
determined by the composition and functioning of microbial communities, but which selective forces
shape these communities? Niche differentiation is the response of microbes (and, in principle, all other
living organisms) to competition for resources in spatially and temporarily fluctuating environments.
Thus, more insight into this phenomenon could reveal factors that influence the abundance, activity, and
diversity of process-relevant microorganisms in treatment plants, and it is more than likely that this
strongly influences the resilience of the N-removal processes against perturbations (Daims et al., 2006b).
CHEMOLITHOAUTOTROPHIC AMMONIA-OXIDIZING BACTERIA

AND ARCHAEA
Phylogeny and habitats of the ammonia oxidizers
Ammonia-oxidizing bacteria (AOB) are responsible for the first and rate-limiting step of nitrification, the
conversion of ammonia to nitrite. All known chemolithoautotrophic AOB are Proteobacteria, with the
majority belonging to the Betaproteobacteria and two species, Nitrosococcus halophilus and N. oceani,
being members of the Gammaproteobacteria (Figure 9.2). The existence of these two distinct lineages of
AOB within the Proteobacteria was first derived from analyses of 16S rRNA oligonucleotide catalogues
(Woese et al., 1985; Woese et al., 1984b) and later confirmed by comparative phylogenetic analyses of
16S rRNA gene sequences (Head et al., 1993; Purkhold et al., 2000; Purkhold et al., 2003; Teske et al.,
1994; Utåker et al., 1995). Based on ultrastructural criteria, the betaproteobacterial AOB have been
subdivided into the four genera Nitrosomonas, Nitrosospira, Nitrosovibrio, and Nitrosolobus. The latter
three are closely related to each other, and despite morphological differences among them, 16S rRNA-
based phylogeny does not convincingly support their separation at the genus level. Therefore, Head et al.
(1993) suggested combining these three in a single genus Nitrosospira, which forms a monophyletic clade
closely related to the genus Nitrosomonas (Figure 9.2). Later analyses confirmed that splitting this
Nitrosospira lineage into subgroups is not supported consistently by phylogenetic treeing methods
(Purkhold et al., 2000). In contrast, the genus Nitrosomonas can be subdivided into several sublineages
which form stable branches within the betaproteobacterial AOB (Figure 9.2). The 16S rRNA-based
phylogeny of AOB has been complemented by comparative sequence analyses of the amoA gene, which
encodes the alpha subunit of ammonia monooxygenase (Amo), an enzyme specific for ammonia oxidizers.
This gene is not only suitable as a phylogenetic marker, but is also a functional marker and allows specific
detection and identification of ammonia oxidizers in the environment (Holmes et al., 1995; Purkhold et al.,
2000; Rotthauwe et al., 1997). Generally, phylogenies based on 16S rRNA and amoA gene sequences
correlate very well, indicating that lateral gene transfer of amoA was uncommon or did not take place at
all during evolution of AOB.
Betaproteobacterial AOB are widely distributed in natural and man-made ecosystems. The genus
Nitrosomonas occurs in aquatic and terrestrial habitats. Results of culture-dependent and -independent
surveys strongly suggest that the known phylogenetic sublineages of the nitrosomonads (Figure 9.2)
represent organisms with different ecophysiological traits and environmental distribution patterns
(Koops and Pommerening-Roser, 2001; Koops et al., 2003; Kowalchuk and Stephen, 2001). Most notably,
all known members of the N. europaea/N. mobilis lineage are halotolerant or moderately halophilic and
seem to occur mainly in eutrophic habitats. Their halotolerance and preference for higher substrate
concentrations may explain why these organisms constitute a large fraction of the ammonia-oxidizing
populations in many treatment plants (Wagner and Loy, 2002) and are often isolated from activated
sludge in culture media with high ammonia content.
Most isolates related to the Nitrosospira lineage have been obtained from soil, and Nitrosospira-like
bacteria are thought to be the dominant AOB in terrestrial habitats (Kowalchuk et al., 1997; Laverman et al.,
2001; Mendum et al., 1999; Stephen et al., 1998). In contrast, they have only been detected occasionally
Ammonia-oxidizing Crenarchaeota, for example

Nitrosopumilus maritimus, Cenarchaeum symbiosum
Archaea
Gamma-
N. communis proteobacteria
lineage
Nitrosococcus oceani
(AY690336)
Nitrosococcus
halophilus
Nitrosomonas (AF287298)
Nitrosomonas
nitrosa
communis Nitrosomonas sp. Nm58
(AF272425)
N. europaea / (AF272417) (AY123799)
Nc. mobilis Nitrosospira

lineage Nitrosomonas
lineage sp. Nm148
(AY123792)
Nitrosomonas sp. NM 104 (AF272415)
Nitrosococcus mobilis (AF037105)
Nitrosomonas europaea (AF037106) other Betaproteobacteria
Nitrosomonas
Nitrosomonas eutropha (AY123795)
marina
Nitrosomonas halophila (AF272413) (AF272418)
Nitrosomonas cryotolerans (AF272423) Nitrosomonas aestuarii
Nitrosomonas sp. Nm143 (AY123794) (AF272420)
Nitrosomonas ureae
environmental clone (AJ003775) (AF272414)
environmental clone (U62885) Nitrosomonassp. Nm47
environmental clone (AY123810)
(AF216675) Nitrosomonas oligotropha
(AF272422)
Beta-
proteobacteria
N. oligotropha lineage
0.10
Figure 9.2. 16S rRNA sequence-based phylogenetic tree showing the affiliation of the currently known
ammonia-oxidizing microorganisms. Phylogenetic lineages, which have unequivocally been detected by
cultivation-dependent or by molecular methods in wastewater treatment plants, are indicated by names and
arrows. The tree topology was determined by Maximum Likelihood analysis of representative sequences from
each lineage. The scale bar indicates 0.1 estimated change per nucleotide. For more details on the
phylogeny of ammonia oxidizers please refer to Koops et al. (2003).
in nitrifying reactors (e.g. Schramm et al., 1998). It is still unclear to which extent members of the
Nitrosospira lineage differ with respect to their ecophysiological features (Koops et al., 2003).
The gammaproteobacterial AOB are halophilic organisms found in brackish water and marine
ecosystems (Koops et al., 1990; Watson, 1965). These organisms have been detected by cultivation and by
sequence analyses of environmental 16S rRNA and amoA genes, in seawater sampled at geographically
distant locations and thus may be globally distributed in the world’s oceans (Ward and O’Mullan, 2002).
However, they have not been shown yet to play any important role in nitrification in wastewater treatment.
Only recently, the long-lasting assumption that chemolithoautotrophic nitrification is restricted to
members of the domain Bacteria has been disproved by the discovery of ammonia-oxidizing Archaea
(AOA). After environmental genomics had provided strong clues that AOA may occur in soils and oceans
(Schleper et al., 2005; Venter et al., 2004), the existence and nitrifying activity of these organisms was
eventually confirmed by isolation of an ammonia-oxidizing archaeon from gravel of a saltwater aquarium
(Könneke et al., 2005). Molecular studies have demonstrated that AOA are more abundant than AOB in
different soils (Leininger et al., 2006), suggesting that AOA have high impact on N cycling not only in
marine habitats (Francis et al., 2005; Wuchter et al., 2006), but also in terrestrial ecosystems. Thermophilic
AOA have now been identified in hot springs where they drive ammonia oxidation in these extreme
environments (de la Torre et al., 2008; Hatzenpichler et al., 2008). All known AOA are Crenarchaeota and
carry amoA genes that are distantly related to the amoA genes of AOB (Schleper et al., 2005). Although
amoA genes of AOA have been detected by PCR in treatment plants (Park et al., 2006), it remains unclear
whether they play an important role in nitrification in wastewater treatment. The reported detection
of crenarchaeal amoA genes in activated sludge may also be explained by the presence of naked DNA or of
AOA cells originating from soil that show no or little nitrifying activity and would not proliferate in
these systems. Clearly, further research is required to assess their importance in N removal from sewage.
Physiology of ammonia oxidation

The chemolithoautotrophic oxidation of ammonia to nitrite is a two-step process catalyzed by two
enzymes, the membrane-bound ammonia monooxygenase (Amo) (e.g. Hollocher et al., 1981; Hooper
et al., 1997; Hyman and Wood, 1985; McTavish et al., 1993) and the periplasmic hydroxylamine
oxidoreductase (HAO) (e.g. Bergmann et al., 1994; Olson and Hooper, 1983). The first intermediate of
the reaction is hydroxylamine that Amo produces from ammonia and oxygen (equation 1):
Amo
NH3 þ O2 þ 2Hþ þ 2e ! NH2 OH ð1Þ
As the first step is endergonic, the actual energy source for AOB must be the intermediate
hydroxylamine, which is the substrate of the second and exergonic step catalyzed by HAO (equation 2):
HAO
NH2 OH þ H2 O ! HNO2 þ 4Hþ þ 4e ð2Þ
Indeed, addition of hydroxylamine to mineral media also containing ammonia shortens the lag phase of
cultured AOB, probably because oxidation of hydroxylamine by HAO delivers reductants for the first step
catalyzed by Amo (Bock and Wagner, 2001). However, AOB can not grow in media which contain
hydroxylamine, but no ammonia, unless Amo is simultaneously inhibited chemically. It might be that in
the absence of ammonia, a functionally active Amo forms toxic O2 radicals if reductants are available
(Bock and Wagner, 2001). This could also cause nitrification failure in treatment plants if large amounts
of organic electron donors are present and ammonia concentrations are very low. The complex
biochemistry of Amo and HAO and the coupling between the steps catalyzed by these two enzymes are
not yet understood fully.
The preferred way for AOB to gain energy and carbon is via the oxidation of ammonia to nitrite
and the autotrophic fixation of CO2, respectively. However, their metabolism is far more versatile.
N. europaea is not restricted to CO2 as carbon source, but also can obtain carbon from several organic
substrates including pyruvate (Clark and Schmidt, 1966), amino acids (Clark and Schmidt, 1967), and
fructose (Hommes et al., 2003). Furthermore, N. europaea and N. eutropha can nitrify and denitrify
simultaneously under mixotrophic conditions (when ammonia and suitable organic compounds are
present), and O2 limitation by using nitrite in addition to O2 as electron acceptor (Bock et al., 1995).
Under such conditions, considerable amounts of gaseous N2O and dinitrogen are produced. In the absence
of O2, N. eutropha and N. europaea have been observed to denitrify with H2 or simple organic compounds
serving as electron donors and nitrite as electron acceptor (Bock et al., 1995; Stüven et al., 1992).
Experiments performed with pure cultures of AOB suggested that nitrogen dioxide (NO2) or rather its
dimeric form N2O4, and not O2, is the agent for ammonia oxidation by Amo (Schmidt and Bock, 1998;
Schmidt et al., 2001b, 2001c; Zart et al., 2000). In a hypothetical model of a ‘NOx cycle’, O2 oxidizes NO
to NO2. Two molecules of NO2 form N2O4, which serves as oxidant in the Amo reaction. As the Amo
reaction yields not only hydroxylamine but also NO as a by-product (not shown in equation 1), the cycle is
closed and NO can again be oxidized to NO2 (Schmidt et al., 2001a). The initial amounts of NO required
to initiate this NOx cycle may be provided by aerobic denitrification with nitrite as electron acceptor
(Schmidt et al., 2004). Consistent with this hypothesis, ammonia oxidation can be inhibited by removal of
NO and NO2 from the growth medium, and is enhanced by an external supply of these compounds
(Schmidt et al., 2001c; Zart and Bock, 1998; Zart et al., 2000). This model also provides an explanation
for the phenomenon that N. eutropha oxidizes ammonia under anoxic conditions if the atmosphere is
supplemented with NO2 (Schmidt and Bock, 1997). It should be noted that this O2-independent ammonia
oxidation by AOB is fundamentally different from anaerobic ammonium oxidation (‘‘Anammox’’), which
uses ammonium and nitrite as substrates and is catalyzed by unusual planctomycetes (see below).
Ammonia-oxidizing bacteria and wastewater treatment

Culture-based surveys of AOB from nitrifying reactors result mainly in enrichment and isolation of
organisms closely related to Nitrosomonas europaea and N. eutropha, which grow well in the synthetic
mineral media used. Members of the genus Nitrosomonas are also frequently detected in treatment plants by
16S rRNA and amoA sequence analyses and by FISH with rRNA-targeted oligonucleotide probes, and
therefore are considered to be the most important AOB in wastewater treatment (Purkhold et al., 2000).
However, the culture-independent methods have revealed that no less than three different phylogenetic
sublineages of the genus Nitrosomonas in addition to N. europaea and N. eutropha (Figure 9.2) occur
in municipal and industrial activated sludge basins and biofilm reactors (e.g. Gieseke et al., 2003; Layton et al.,
2005; Purkhold et al., 2000). The detection of AOB related to N. marina and to ‘‘Cluster 5’’ members
(Purkhold et al., 2000), which as yet contains no cultured representative, in activated sludge awaits future
verification with more detailed phylogenetic analyses and in situ detection methods (Rowan et al., 2003).
Interestingly, the distribution patterns of different Nitrosomonas-like AOB seem to depend on the type
of treatment plant and its operational conditions. While some reactors are dominated by only one species
(Juretschko et al., 1998), other systems can contain as many as five different populations (Daims et al.,
2001b; Gieseke et al., 2003). Quantitative FISH revealed that in an industrial treatment plant, both
Nitrosomonas europaea and Nitrosococcus mobilis occurred, but N. mobilis was much more abundant
than N. europaea (Juretschko et al., 1998, 2002). As a brackish water organism, N. mobilis is moderately
halophilic (Koops et al., 1976) and seems to be predominant in reactors receiving high loads of ammonia
and salts (Daims et al., 2001b; Juretschko et al., 1998; Mota et al., 2005). Bollmann et al. (2002) carried
out competition experiments with N. europaea and a different Nitrosomonas-like ammonia oxidizer
affiliated to N. oligotropha. Between these two AOB, which belong to lineages found frequently in
treatment plants, marked differences in substrate affinity and starvation behaviour were observed.
Thus, the phylogenetic diversity of AOB in sewage treatment plants reflects adaptations to different
environmental conditions, and this niche differentiation may be relevant to reactor operation, performance
and stability. For example, bioaugmentation with nitrifying activated sludge from other treatment
plants or special cultures of nitrifiers has been suggested in attempts to counteract weak performance of
nitrifying reactors. Unfortunately, such measures are often not as successful as expected. One biological
explanation for such failures is a lack of adaptation among the organisms in the source activated sludge to
the conditions in the target reactor. These organisms may not proliferate in their new environment
and/or may be exposed to predators and bacteriophage attack. In such cases, bioaugmentation is
unlikely to improve reactor performance and may even lead to a complete breakdown of the system
(Bouchez et al., 2000).
In contrast to the nitrosomonads, members of the Nitrosospira lineage have only occasionally
been detected in full-scale activated sludge or biofilm reactors. However, in accordance with their
wide distribution in soils, Nitrosospira species might be functionally important in rhizoremediation

treatment plants (Haleem et al., 2000). Based on microsensor measurements of substrate conversion,
Schramm et al. (1999a) proposed that Nitrosospira cells have a lower Km value for nitrite and a lower
growth rate than those for Nitrosomonas species, suggesting that Nitrosospira may be better adapted to
low substrate concentrations. This could explain why Nitrosospira are found more frequently than
Nitrosomonas in soils where ammonia concentrations are less than those in sewage, and why they are
outcompeted by Nitrosomonas in most treatment plants.
It should be noted that 16S rRNA- or amoA-based phylogenies may capture only a small fraction of the
(functional) diversity of AOB in treatment plants. Marine bacterial populations with highly similar or even
identical 16S rRNA sequences can differ substantially in their gene content and genomic organization
(Coleman et al., 2006; Thompson et al., 2005) and may have different ecophysiological properties.
The degree of genomic and functional diversity of very closely related AOB in treatment plants is as
yet unknown, but this kind of variability may have a profound impact on the nitrification process.
Whole-genome analyses of cultured AOB (Chain et al., 2003; Klotz et al., 2006) provide an excellent
knowledge base for future research in this direction, and should in future be complemented by
environmental genomics approaches, which allow one to compare efficiently large numbers of (partial)
genomes without the restrictions imposed by cultivation.
As mentioned above, some AOB simultaneously nitrify and denitrify under oxygen limitation and in
the presence of simple organic compounds. At first glance, it seems that this feature could be exploited
for single-reactor N elimination where ammonia is oxidized only to nitrite, which is then reduced to
gaseous nitrogen by the same organisms. This apparently straightforward approach could reduce the
oxygen demand for nitrification by as much as 50% and would require less organic substrate for
denitrification, because the reduction of nitrate to nitrite is skipped. Unfortunately, N elimination under
these conditions would be hampered because the ammonia oxidation rates of AOB decrease markedly
when O2 becomes a limiting factor. However, Zart and Bock (1998) made the interesting observation
that rates of ammonia oxidation and aerobic denitrification by Nitrosomonas eutropha increased in the
presence of NO or NO2. This finding has lead to the ‘‘NOx process’’, where the aerobic denitrification
activity of AOB is stimulated by supplying the reactor with a mixture of air and NO2 (Schmidt et al.,
2003). A small amount of nitrite, which is not denitrified by the AOB, must subsequently be removed
in a small anoxic denitrification tank (Schmidt et al., 2003). The NOx process reduces the overall costs
for nitrification/denitrification at the expense of the added NO2. Other innovative solutions to reduce
the costs for N removal are discussed later in sections on denitrification and anaerobic ammonium
oxidation.
CHEMOLITHOAUTOTROPHIC NITRITE-OXIDIZING BACTERIA

Phylogeny and habitats of the nitrite oxidizers
Nitrite-oxidizing bacteria (NOB) catalyze the second step in nitrification, the oxidation of nitrite to nitrate.
This functional group is phylogenetically more heterogeneous than the AOB (Figure 9.3). All known
nitrite oxidizers belong to one of the genera Nitrobacter, Nitrospira, Nitrococcus, and Nitrospina.
The physiologically best characterized NOB are members of the genus Nitrobacter (Winogradsky, 1892)
in the class Alphaproteobacteria (Stackebrandt et al., 1988; Woese et al., 1984a), which contains the
four described species N. winogradskyi (Watson, 1971; Winslow et al., 1917), N. hamburgensis
(Bock et al., 1983), N. vulgaris (Bock et al., 1990), and N. alkalicus (Sorokin et al., 1998). Nitrobacter
Gamma-
proteobacteria
Thiobacillus Beta-
Escherichia coli ferrooxidans proteobacteria
Nitrococcus
mobilis Gallionella ferruginea
Delta-
Nitrospina
proteobacteria
gracilis
genus Nitrotoga
Bdellovibrio
bacteriovorus
Rhodobacter
capsulatus
Paracoccus
denitrificans
Sublineage I other Bacteria Bradyrhizobium
N. defluvii japonicum
genus Nitrobacter
genus Nitrospira
Sublineage II
Alpha-
N. moscoviensis
proteobacteria
Sublineage III
(Nullarbor caves) Thermodesulfovibrio
Sublineage IV islandicus
N. marina Leptospirillum Magnetobacterium
ferrooxidans bavaricum
Phylum 0.10
Nitrospirae
Figure 9.3. 16S rRNA sequence-based phylogenetic tree showing the affiliation of the currently known
nitrite-oxidizing bacteria. Phylogenetic lineages, which have been detected by cultivation-dependent or
by molecular methods in wastewater treatment plants, are marked with arrows. The tree topology was
determined by Maximum Likelihood analysis of representative sequences from each lineage. The scale bar
indicates 0.1 estimated change per nucleotide.
species are found in a large range of terrestrial and aquatic habitats, including unusual environments
such as building stones (Mansch and Bock, 1998) and sediments of highly alkaline soda lakes
(Sorokin et al., 1998).
Each of the genera Nitrococcus and Nitrospina harbours only one known species (Watson and
Waterbury, 1971). Nitrococcus mobilis is a member of the Gammaproteobacteria, whereas 16S rRNA-
based phylogeny suggests that Nitrospina gracilis belongs to the Deltaproteobacteria (Teske et al., 1994)
despite phenotypic differences between this species and other proteobacterial NOB (Lipski et al., 2001).
Both were isolated from seawater and have never been detected outside marine environments.
The genus Nitrospira constitutes the most diverse group of known NOB. It contains only two described
species, N. marina (Watson et al., 1986) and N. moscoviensis (Ehrich et al., 1995), but phylogenetically
diverse 16S rRNA sequences of uncultured Nitrospira-like bacteria have been found in many different
soil, sediment, freshwater, and marine samples (e.g. Daims et al., 2001a). This genus is unrelated to
the other NOB and belongs to the distinct bacterial phylum Nitrospirae, which contains also the
iron-oxidizing genus Leptospirillum, the thermophilic, sulphate-reducing genus Thermodesulfovibrio, and
the peculiar magnetotactic Magnetobacterium bavaricum (Ehrich et al., 1995). The genus Nitrospira has
been subdivided into phylogenetic sublineages I-IV (Daims et al., 2001a) (Figure 9.3).
Physiology of nitrite oxidation

In Nitrobacter, the key step of nitrite oxidation is catalyzed by nitrite oxidoreductase (Nxr), a membrane-
bound enzyme that oxidizes nitrite to nitrate using water as the source of oxygen:
NO2 þ H2 O $ NO3 þ 2Hþ þ 2e ð3Þ
The electrons released in this reaction are transferred to oxygen via a respiratory chain with a
cytochrome c oxidase of the aa3-type as terminal oxidase:
1
2Hþ þ 2e þ O2 ! H2 O ð4Þ
2
The molecular properties of Nxr and other components of the nitrite oxidation system of Nitrobacter
have been described in detail by Bock and Wagner (2001) and references cited therein. The genes of two
subunits, norA and norB, of Nxr from Nitrobacter have been sequenced (Kirstein and Bock, 1993). They
cluster together with an additional open reading frame, nxrX, in the order nxrA-nxrX-nxrB. Consistent
with biochemical data, the alpha- and beta-subunits of Nxr have high sequence similarities to the alpha-
and beta-subunits of dissimilatory nitrate reductases found in several chemoorganoheterotrophic
bacteria (Kirstein and Bock, 1993). Much less is known about the nitrite-oxidizing enzyme apparatus
of NOB other than Nitrobacter. Experiments performed with pure cultures of Nitrospira marina and
N. moscoviensis showed that the biochemistry of nitrite oxidation in Nitrospira differs from Nitrobacter
(Ehrich et al., 1995; Watson et al., 1986). However, antibodies directed against the Nxr of Nitrobacter
cross-react with the nitrite-oxidizing systems of the other nitrite oxidizers (Bartosch et al., 1999;
Spieck et al., 1998), and have been used to detect Nitrobacter and Nitrospira in enrichments of NOB
from both activated sludge and soil samples (Bartosch et al., 2002; Bartosch et al., 1999).
Not all NOB are obligate chemolithoautotrophs, and Nitrobacter and Nitrospira at least exhibit a
surprisingly high degree of physiological flexibility. Thus, in the absence of nitrite, Nitrobacter can grow
on simple organic compounds like acetate and pyruvate (Bock, 1976; Smith and Hoare, 1968), and
Nitrospira marina grows better in media containing both nitrite and pyruvate than in media with nitrite
alone (Watson et al., 1986). We showed by FISH-MAR (chapter 11) that uncultured Nitrospira-like
bacteria used pyruvate, but not acetate, under aerobic conditions in a nitrifying biofilm (Daims et al.,
2001a). In the absence of O2 Nitrobacter and some Nitrospira at least could reduce nitrate with electrons
derived from H2 (N. moscoviensis; Ehrich et al., 1995) or from organic compounds (Nitrobacter;
Bock et al., 1988; Freitag et al., 1987).
Nitrite oxidizers and wastewater treatment

Nitrobacter can be enriched and isolated from virtually every nitrifying activated sludge or biofilm sample
by incubation in mineral nitrite media. Therefore, Nitrobacter-like bacteria were long considered to be
the key nitrite oxidizers in biological wastewater treatment (e.g. Henze et al., 1997). This view was
challenged for the first time when Nitrobacter cells were not detected by FISH with rRNA-targeted
oligonucleotide probes in nitrifying activated sludge samples from several treatment plants (Wagner et al.,
1996). It should be noted that the sensitivity of FISH and other microscopy-based techniques (103–104
cells per ml) is lower than that of culture-based methods. The latter can detect a single microbial cell that
proliferates in the used medium. However, if one ml of activated sludge contains 108–109 microbial cells,
104 cells correspond to not more than 0.01% of the total microbial community. An organism carrying out
such a key process in a treatment plant must be more abundant than this, and so should be detectable by
FISH. Subsequently, application of the full-cycle rRNA approach (Amann et al., 1995) to a full-scale
industrial treatment plant revealed that novel NOB (and not Nitrobacter) closely related to the
genus Nitrospira were predominant nitrite oxidizers in that system (Juretschko et al., 1998). While all
attempts to enrich these Nitrospira-like bacteria from the sludge were unsuccessful, a Nitrobacter strain
was readily isolated from the same sample. This demonstrates that the once assumed importance of
Nitrobacter in wastewater treatment was merely an artifact caused by cultivation biases. The wide
distribution of Nitrospira in full-scale activated sludge tanks and biofilm reactors has been confirmed in
follow-up studies (e.g. Daims et al., 2001a), and their nitrite-oxidizing activity quantified by microsensor
measurements in lab-scale reactors (Gieseke et al., 2003; Okabe et al., 1999; Schramm et al., 1998).
Although Nitrobacter and Nitrospira share a common physiological feature – both can grow as
chemolithoautotrophs with nitrite as their source of energy – they belong to completely different
phylogenetic lineages (Figure 9.3). This large phylogenetic distance almost certainly reflects (as yet
unknown) ecophysiological differences, which could have important practical implications. While
cultured Nitrobacter species have been investigated in detail, our knowledge about the physiology of the
as yet uncultured Nitrospira-like bacteria in treatment plants is still scarce.
A major advance was made by the successful enrichment of a sublineage I Nitrospira strain from
nitrifying activated sludge (Spieck et al., 2006). This strain was described provisionally as Candidatus
‘Nitrospira defluvii’ (Figure 9.3) and used for sequencing the first Nitrospira genome by an environmental
genomics approach. Ongoing genome analysis has led to the discovery of an unexpected enzyme,
chlorite dismutase (CLD) in N. defluvii (Maixner et al., 2008). This enzyme detoxifies chlorite (ClO2 ) by
conversion to chloride and O2, a reaction reported previously only in heterotrophic (per)chlorate-reducing
bacteria. Heterologous expression of the Nitrospira cld gene and enzymatic characterization of the CLD
gene product showed it to have a high chlorite-degrading activity. Hence, environmental genomics have
revealed that Nitrospira may be involved in (per)chlorate and chlorite degradation, and might provide a
link between the N cycle and important bioremediation processes at industrially contaminated sites or in
sewage. Genome analysis is expected to yield further novel insights into the biology of Nitrospira.
However, one aspect highly relevant to practice in wastewater treatment – substrate turnover kinetics –
has already been studied in Nitrobacter and also in Nitrospira. By a combination of FISH and microsensor
measurements, Schramm et al. (1999a) monitored quantitatively the nitrite-oxidizing activity of
Nitrospira in a nitrifying biofilm. They then estimated cell-specific nitrite oxidation rates and found these
were much lower than the known rates from Nitrobacter pure cultures. In addition, the estimated Km value
of Nitrospira for nitrite was markedly below the known Km values from different Nitrobacter species.
Hence, Nitrospira and Nitrobacter seem to adopt different survival strategies (Schramm et al., 1999a).
Nitrospira appear to be K-strategists, able to exploit efficiently low levels of nitrite, and reach relatively
high population densities under nitrite-limited conditions. On the other hand Nitrobacter are r-strategists,
being outcompeted by Nitrospira as long as nitrite concentrations are very low, but able to grow faster
than Nitrospira as soon as more nitrite becomes available.
This ‘‘K/r’’ hypothesis for NOB may explain the wide distribution of Nitrospira in many natural
habitats and their predominance in treatment plants. As nitrite is an intermediate of nitrification, it does
not usually accumulate but is rapidly consumed by NOB soon after its production by AOB. Thus, under
steady-state conditions nitrite is a growth-limiting factor and low concentrations (‘famine’ conditions)
favour Nitrospira over Nitrobacter. Accordingly, proliferation of Nitrobacter to higher cell densities
probably depends on a temporarily elevated nitrogen input (‘feast’ conditions). In natural habitats, this
may occur during the degradation of dead biomass or in faeces rich in nitrogen. In those nitrifying
treatment plants, where nitrite peaks occur frequently as a consequence of influent composition and
operational conditions, periodic changes in its concentration theoretically should create ecological niches
for NOB populations adapted to different nitrite levels. Consistent with this hypothesis, stable coexistence
of Nitrospira and Nitrobacter has been observed in a high-load nitrifying sequencing batch biofilm
reactor where pronounced nitrite peaks occurred during each operational cycle (Daims et al., 2001a;
Gieseke et al., 2003). However, other factors such as O2 availability may also exert selective effects on
different nitrite-oxidizing bacterial populations (Schramm et al., 1999a).
The ‘‘K/r’’ hypothesis could explain why Nitrospira are more abundant in treatment plants than
Nitrobacter, but this does not imply that all Nitrospira-like bacteria have identical ecophysiological
properties. As shown in Figure 9.3, the genus Nitrospira is phylogenetically complex and consists of at
least four different sublineages. To date, members of sublineages I, II and IV have been detected in
wastewater treatment plants (Daims et al., 2001a). Interestingly, FISH with highly specific rRNA-targeted
probes has revealed that representatives of sublineages I and II can co-exist in the same nitrifying reactors.
Such co-existence without competitive exclusion requires niche differentiation, where a difference in at
least one important feature enables the co-existing organisms to inhabit (slightly) different ecological
niches in the same ecosystem. In the case of Nitrospira sublineage I and II, niche differentiation is realized
by preference for higher (sublineage I) or lower (sublineage II) nitrite concentrations (Maixner et al.,
2006). In addition, these organisms exhibit a particular spatial localization pattern in biofilms, where they
are localized at different distances from cell clusters of AOB (sublineage I Nitrospira were shown to be
localized closer to the AOB than sublineage II) (Figure 9.4, see colour image section (chapter 13)).
Models of nitrite fluxes predicted that in a biofilm, pronounced nitrite concentration gradients can form
within only 20 mm because of nitrite production by AOB and the almost immediate nitrite consumption by
juxtaposed NOB. Thus, nitrite concentrations are highest at the boundaries of AOB cell aggregates and
decrease sharply within a few mm distance from the AOB. These modelled gradients and the preference
for different nitrite concentrations can explain the aforementioned localization pattern of the different
Nitrospira, providing an example for co-existence enabled by a combination of physiological traits and
spatial heterogeneity within the habitat (Maixner et al., 2006).
Like other bacteria, nitrifiers are involved in biological interactions with other microbes. AOB and
NOB are mutualistic symbionts, because AOB produce the nitrite required by NOB, which in turn prevent
accumulation of nitrite to toxic concentrations (Stein and Arp, 1998). On the other hand, both groups
compete for O2 the terminal electron acceptor (Sliekers et al., 2005). It is tempting to speculate that the
interactions between AOB and NOB could be more complex and might involve the exchange of vitamins
and other important growth factors. Furthermore, the autotrophic nitrifiers are primary producers that
release organic compounds used by heterotrophic bacteria (Kindaichi et al., 2004; Okabe et al., 2005a;
Rittmann et al., 1994). The current knowledge of mutualistic or antagonistic interactions between nitrifiers
and heterotrophs is sparse, although such interactions might be important in determining the activity and
environmental distribution of AOB and NOB.
The examples described above show that the biology of nitrification in treatment plants is much
more complex than assumed earlier. Of course, one might ask whether this micro-scale complexity
could be of any relevance to practical applications in wastewater treatment. The physiological
differences between functionally similar organisms may indeed be important, because they are the
basis for a pronounced diversity of ecotypes that carry out the same key reactions but are not
identical with respect to additional capabilities. Theoretically, a high diversity of such organisms in
the same reactor should result in better process stability, because the likelihood is high that at least
one of these populations resists a particular disturbance (e.g. shifts in nutrient load or concentrations
of toxic compounds or phage attacks) and continues to catalyze the process. In contrast, a
monoculture of only one strain with limited physiological flexibility would be more prone to process
failure from altered environmental conditions. This effect is illustrated by comparing a natural pristine
forest to an agricultural monoculture: While the (highly diverse) forest constitutes a stable ecosystem
that can exist for centuries despite temporarily harsh conditions, the monoculture would not survive
without constant and expensive protection from biotic and abiotic disturbance, e.g. by irrigation and
application of pesticides. One imaginable strategy to support a high microbial diversity in a treatment
plant community is to confront the system intentionally with controlled shifts of environmental
conditions, which create new niches for additional populations (Daims et al., 2001b). Such shifts may
comprise temporal changes of nutrient load, aeration, pH, temperature, amount and kind of added
carbon sources, and other operational parameters. To a certain extent this strategy resembles the
concept of SBRs (chapter 2), but it would be much more flexible and could be applied to a wide
range of reactor designs including conventional activated sludge tanks.
DENITRIFICATION AND DENITRIFYING BACTERIA

Diversity and physiology of the denitrifiers
Denitrification has been defined as ‘[. . .] the dissimilatory transformation of nitrate or nitrite to a gas
species concomitant with energy conservation [. . .]’ (Zumft, 1997). Thus, denitrifying microbes can use
the N compounds NO3 and NO2 (but also the gases NO and N2O) as terminal electron acceptors in their
respiratory metabolism. Denitrification is generally considered to be an anaerobic process although
it may occur under aerobic and microaerophilic conditions (Robertson and Kuenen, 1984). In the
absence of O2, denitrifiers have substantial selective advantages as they can shuttle electrons through a
respiratory chain, a process which yields more energy than fermentative metabolism. In addition,
denitrification allows these organisms to use substrates that cannot be metabolized by fermentation
pathways (see chapter 1). Denitrification is a widespread attribute among ‘prokaryotes’, and denitrifiers
are a phylogenetically extremely heterogeneous group. Furthermore, denitrifying ‘prokaryotes’ are
highly diverse with regard to their modes of growth and major physiological features. Denitrifiers are
found, for example, among the organotrophs, lithotrophs (using inorganic electron donors such as H2),
phototrophs, N2-fixing organisms (diazotrophs), and extremophiles like the halophilic and thermophilic
bacteria and Archaea (Zumft, 1997). As mentioned above, nitrifiers (AOB and NOB) can also denitrify
under O2-limited conditions. In agreement with their physiological diversity, denitrifiers have been
detected in many different soil, sediment and aquatic habitats. Most known denitrifiers are not obligate
anaerobes but can respire with O2 as electron acceptor, so that denitrification is their ‘alternative way of
life’ which substitutes aerobic respiration when O2 is unavailable. As aerobic respiration yields more
energy, many of these organisms switch to denitrification only when the pO2 falls below a threshold.
Such physiological flexibility demands strict genetic and biochemical regulation based on the amounts
of available electron donors, O2, and respirable nitrogen oxides. Indeed, in many denitrifiers the nitrogen
oxide reductases, which are needed for denitrification (see below), are repressed under aerobic
conditions. However, the amount of O2 required for repression differs over a wide range among the
denitrifying microbes (Knowles, 1982). Thus, denitrifiers in total encompass a wide range of adaptations
to different environmental conditions, with pure anaerobiosis and aerobic denitrification as two extremes
on either end of the scale.
Denitrification is a four-step process by which four metalloenzymes sequentially reduce nitrate to
nitrite, nitrite to nitric oxide (NO), NO to nitrous oxide (N2O) and N2O to N2:
Nar Nir Nor Nos

NO3 ! NO2 ! NO ! N2 O ! N2 ð5Þ
This process does not always proceed to complete reduction to N2. Depending on their community
composition and prevailing environmental conditions, denitrifiers may release considerable amounts of
the gaseous intermediates NO and N2O. Both compounds pose a threat to the stratospheric ozone layer,
and N2O is a more potent greenhouse gas than CO2. Thus, on a global scale the activity of denitrifying
organisms not only plays a major role for biogeochemical N-cycling but also affects atmospheric
chemistry and probably contributes to global warming.
The respiratory nitrate reductases (Nar) of E. coli and Paracoccus spp. are membrane-bound
complexes of three protein subunits. The large (a) subunit contains a molybdenum cofactor and is the
active site of Nar; the small (b) subunit binds several iron-sulphur clusters, and the third (g) subunit is a
b-type cytochrome, which also anchors the ab complex in the cytoplasmic membrane. The electron flow
within the Nar complex is thought to begin at the g subunit and continue via the b to the a subunit
(Zumft, 1997). Another class of dissimilatory nitrate reductases, the periplasmic nitrate reductases (Nap)
differ in both their structure and location. At least in some organisms, their expression is not repressed by
O2 (Philippot, 2002), suggesting that Nap is involved in aerobic denitrification.
Two different nitrite reductases (NirS and NirK) occur in denitrifiers, but are never found in the same
organism. In Gram negative bacteria both are periplasmic enzymes, which differ in their structure and
metal cofactor. The crystal structure of NirS (cytochrome cd1) of Paracoccus pantotrophus revealed a
dimeric protein with one heme C and one heme D1 – binding domain per monomer (Fülöp et al., 1995).
Thus, the metal cofactor of NirS is iron. In contrast, NirK species are trimeric enzymes and contain Cu as
prosthetic metal (e.g. Godden et al., 1991). Concerning the distribution of these nitrite reductases among
the denitrifiers, it is thought that NirS is the more frequent type but NirK occurs in a greater variety of
phylogenetically unrelated taxa. However, a survey of nir genes in cultured denitrifiers has revealed that
nirK was the prevalent form in 4100 analyzed isolates from different phyla of Gram negative and Gram
positive bacteria (Heylen et al., 2006). The selective pressures that favour the evolution of two widely
distributed nitrite reductases remain unknown. Clearly, the metal content of all denitrification enzymes
causes an increased demand for these metals. Hence, the use of iron in NirS and copper in NirK might
confer a selective advantage on different denitrifiers in situations when one of these metals is depleted
(Zumft, 1997).
Nitric oxide reductases (Nor) of bacterial denitrifiers are membrane-bound complexes of at least two
subunits, with the larger subunit being a hydrophobic b-type cytochrome (e.g. Heiss et al., 1989). Nor
plays a critical role in denitrification as it prevents the accumulation of NO, which is toxic to bacteria
including denitrifiers. Accordingly, nitrite reductase and nitric oxide reductase are genetically linked
in some denitrifiers (Zumft, 1997). The reduction of N2O to N2 is catalyzed by periplasmic Nos, a
dimer of two identical subunits that contain several Cu atoms and in some cases also Zn, Ni and Fe
(Zumft, 1997). The reduction of N2O constitutes a separate respiratory process, which occurs also in
some non-denitrifying bacteria. The diversity in the prosthetic metal content of N2O reductases
may reflect functional fine-tuning of this enzyme family in organisms with different habitats and
ecophysiological traits.
Denitrifying bacteria and wastewater treatment

Activated sludge has often been used as inoculum for enrichment and isolation of denitrifying bacteria.
Based on this approach many isolates have been obtained, which belong to the genera Alcaligenes, Bacillus,
Hyphomicrobium, Methylobacterium, Paracoccus, Pseudomonas and others (reviewed by Knowles, 1982).
However, in view of their biases (Wagner et al., 1993), conventional culture-dependent methods are
unlikely to yield a realistic picture of the complex denitrifying communities in treatment plants.
Furthermore, an organism that denitrifies in pure culture does not necessarily exhibit the same phenotype
in situ in a full-scale plant. Unfortunately, the application of culture-independent methods to investigate
denitrifiers in environmental samples is also limited. The ability to denitrify is not restricted to a few
phylogenetically coherent groups (as is the case with nitrification), but instead is distributed over
many ‘prokaryotic’ lineages, which contain also non-denitrifying microbes. This lack of a reliable link
between the denitrifying activity and the phylogenetic affiliation of an organism hampers identification of
uncultured denitrifiers by rRNA-based phylogeny, and their quantification by related methods like FISH.
To overcome this problem, the genes encoding the nitrite reductases, nirS and nirK, have been used
as functional markers to detect denitrifiers in the environment (e.g. Braker et al., 1998; Taroncher-
Oldenburg et al., 2003). For example, fingerprinting techniques like DGGE (chapter 11) applied to nir
genes can help assess the overall diversity of denitrifying microbes in a sample. However, the nirS and
nirK genes should not be used as phylogenetic markers to identify the denitrifying populations in
environmental samples, although this has been attempted. Phylogenetic trees calculated with NirS and
NirK sequences differ from trees based on 16S rRNA sequences in their branching order and apparent
affiliations of the organisms (Casciotti and Ward, 2001; Philippot, 2002). Incongruence of Nir- and 16S
rRNA-based phylogenies has been observed in an encompassing phylogenetic analysis of nirS and nirK
(Heylen et al., 2006). This study revealed also (i) that denitrifiers, which are not directly related
(according to 16S rRNA-based phylogeny), can possess sequence-identical nirK genes; and (ii) that
similarities between the nirK genes of different denitrifiers correlated with the common habitat of these
organisms, and thus with environmental selective pressures, and not with phylogenetic affiliation.
Moreover, Etchebehere and Tiedje (2005) found that a Thauera-related denitrifying bacterium possessed
and expressed two nirS genes, which fell at different positions in NirS-based phylogenetic trees.
Altogether, these results strongly suggest that lateral transfer of nir genes have occurred even between
distantly related denitrifiers. Comparisons of protein sequence- and 16S rRNA-based phylogenies showed
also that Nor (norB, norZ), Nos (nosZ), and Nap (napA) have been transferred horizontally between
different denitrifiers, and thus are unsuitable as phylogenetic markers (Philippot, 2002). In contrast,
phylogenetic trees calculated with a limited set of respiratory nitrate reductase (narG)-sequences
showed good congruence with 16S rRNA-based trees of the same organisms (Philippot, 2002). However,
narG occurs also in non-denitrifying bacteria like E. coli, which can reduce nitrate but lacks the other
denitrification enzymes that generate the gaseous nitrogen compounds. Thus, a suitable specific
phylogenetic and functional marker for denitrifiers is lacking.
Sometimes the in situ quantification of candidate denitrifiers with culture-independent techniques can
provide hints regarding their possible functional importance in a denitrifying community. For example, based
on quantitative FISH with specific rRNA-targeted probes, Neef et al. (1996) found that bacteria related to
Paracoccus and Hyphomicrobium were more abundant in a denitrifying than in a non-denitrifying sand filter.
However, an unequivocal proof of function is achieved only by combining culture-independent identification
with in situ physiological characterization. This is offered by FISH-MAR (chapter 11) (Lee et al., 1999),
which is the method that has revealed most insight into the composition of denitrifying microbial communities
in treatment plants (Table 9.1). For example, bacteria related to members of the genera Azoarcus and Thauera
(Rhodocyclaceae, Betaproteobacteria) were shown to be abundant in a full-scale industrial nitrifying/
denitrifying activated sludge tank (Juretschko et al., 2002). Their denitrifying activity was demonstrated in situ
by FISH-MAR (Juretschko, 2000; Wagner and Loy, 2002). Subsequently, Thauera-like bacteria were
detected in other full-scale plants. Again, FISH-MAR (Ginige et al., 2005) and/or stable isotope probing (SIP)
(Ginige et al., 2005; Osaka et al., 2006) confirmed their denitrifying activity, suggesting that the Azoarcus/
Thauera-complex may be a widely distributed group of key denitrifiers in wastewater treatment. This was
confirmed by Thomsen et al. (2007), who analysed denitrifying communities in two municipal wastewater
Table 9.1. Summary of studies using cultivation-independent methods to identify and physiologically characterize denitrifying bacteria in
274
treatment plants.
Type of sample Denitrifiers identified Selected key findings Methods applied Reference
AS1 from an Bacteria related to High abundance and 16S rRNA analysis, Juretschko (2000),
industrial plant the genera Azoarcus denitrifying activity FISH-MAR3 Juretschko et al. (2002),
and Thauera Wagner and Loy (2002)
3 nitrifying-denitrifying No specific organism Large fractions (47–93%) MAR Nielsen and Nielsen
AS samples was studied of all bacteria in the (2002a)
samples consumed
acetate under denitrifying
conditions
AS from 9 Bacteria related to High abundance and Micro-manipulation, Thomsen et al. (2004)
municipal or Curvibacter potential denitrifying 16S rRNA analysis,
industrial plants delicatus and C. activity in municipal FISH-MAR
lanceolatus WWTPs
(formerly
Aquaspirillum
delicatum and
Pseudomonas
lanceolata,
respectively)
AS from a Methylophilales-related Key denitrifiers were SIP4, 16S rRNA Ginige et al. (2004)
methanol-fed, bacteria; Methylophilales; no analysis, FISH-MAR
lab-scale SBR2 Hyphomicrobium, comparable functional
Paracoccus importance of the other
organisms in this system
AS from 2 Rhodocyclus-related Denitrifying activity FISH-MAR Kong et al. (2004)
municipal and 1 polyphosphate- with NO3 and NO2 ,

industrial EBPR accumulating acetate as organic
plant organisms substrate
(continued )
Return AS from Betaproteobacteria Abundance of these SIP, 16S rRNA Ginige et al. (2005)
a full-scale plant related to the genera organisms in the analysis, FISH-MAR
and sludge from Acidovorax, full-scale system,
a lab-scale SBR Dechloromonas and enrichment
Thauera in the SBR, and
denitrification with
acetate as organic
substrate
AS from an Meganema Denitrification with NO3 FISH-MAR Kragelund et al. (2005)
industrial plant perideroedes and NO2 but reduced
organic substrate
spectrum compared to
aerobic conditions
AS from 21 Phylogenetically Occurred in most FISH-MAR Thomsen et al. (2006b)
plants heterogeneous WWTPs; some
filaments showed denitrifying activity
AS from 7 Alphaproteobacterial All 5 filaments showed FISH-MAR Kragelund et al. (2006)
industrial plants filaments: Ca. denitrifying activity
‘Monilibacter with different organic
batavus’; Ca. substrate uptake
‘Sphaeronema patterns; different substrate
italicum’; Ca. uptake compared to
‘Alysiomicrobium aerobic conditions
bavaricum’; Ca.
The microbiology of nitrogen removal
‘Alysiosphaera
europaea’;
Meganema
perideroedes
Biofilm grown Bacteria related to High in situ abundance FISH and Satoh et al. (2006)
on carriers Hyphomicrobium and denitrifying activity micro-electrodes
inserted into an with CH3OH as organic
AS tank of an substrate
industrial plant
(continued )
275
276
AS from a Bacteria related to Supply with either acetate SIP, 16S rRNA and Osaka et al. (2006)
municipal plant the genera Azoarcus, or methanol as organic nirS/nirK sequence
Thauera, substrate favoured analysis
Comamonas, different groups of
Acidovorax, denitrifying bacteria
Paracoccus,
Rhodobacter,
Methylophilus,
Methylobacillus,
Hyphomicrobium
and others
As from two Bacteria related to Different substrate FISH-MAR Thomsen et al. (2007)
municipal plants Curvibacter, spectra of predominant
Azoarcus and denitrifying groups
Thauera
AS from three Bacteria related to Highly abundant FISH-MAR Hagman et al. (2008)
plants without Azoarcus, Zoogloea Azoarcus- related
external carbon and Alpha- and denitrifiers used both
supply or with Gamma- acetate and methanol
addition of proteobacteria as carbon sources
ethanol or
methanol
respectively
AS from two Bacteria related to Relatively high abundance FISH-MAR Morgan-Sagastume
domestic plants Curvibacter, of non-betaproteobacterial et al. (2008)
Azoarcus, denitrifiers
Thauera,
Accumulibacter,
Alpha-proteobacteria,
Gamma-proteobacteria
and Actinobacteria
1
AS ¼ Activated sludge
2
SBR ¼ Sequencing batch reactor
3
MAR ¼ Microautoradiography
4
SIP ¼ Stable isotope probing.
treatment plants and their carbon assimilation profiles with FISH-MAR. The predominant denitrifiers were
closely related to Curvibacter (Betaproteobacteria; formerly Aquaspirillum), Azoarcus and Thauera.
Interestingly Curvibacter used only amino acids under aerobic or denitrifying conditions. Azoarcus was
more versatile and assimilated amino acids but also acetate and pyruvate, while Thauera took up acetate,
propionate, pyruvate, ethanol and amino acids. The data demonstrate niche partitioning among the key
nitrifiers, with Curvibacter being the most specialist and Thauera the most versatile (generalist) of these
populations (Thomsen et al., 2007). Culturable denitrifiers of the genus Thauera have been isolated from
activated sludge (Etchebehere et al., 2001), soil and groundwater samples. Many of these strains grow on
aromatic and halogenated hydrocarbons (Mechichi et al., 2002; Rabus et al., 1999), indicating that Thauera
may contribute to removal of recalcitrant and toxic compounds from wastewater.
In EBPR plants, where P and N removal are combined, some PAO also act as denitrifiers, as reported
for Candidatus ‘Accumulibacter phosphatis’ (chapter 10).
Feeding activated sludge with acetate or methanol, two carbon sources often used to enhance
denitrification in treatment plants, seems to select for different groups of denitrifiers. While acetate
apparently supports members of the Comamonadaceae and Rhodocyclaceae such as Comamonas,
Acidovorax and Thauera (all Betaproteobacteria) (Ginige et al., 2005; Osaka et al., 2006), methanol
favours methylotrophic denitrifiers like Methylophilus, Methylobacillus (both Betaproteobacteria) and
Hyphomicrobium (Alphaproteobacteria) (Ginige et al., 2004; Osaka et al., 2006). However, Hagman et al.
(2008) detected a high abundance of Azoarcus (30% of the total bacterial community) in a denitrifying
sludge fed with methanol, and FISH-MAR revealed that it assimilated both acetate and methanol,
suggesting that the supplied carbon source may not always select for different denitrifying populations.
Thus these findings and those of other studies (Table 9.1) show that the abundant denitrifiers are members
of the genera Curvibacter, Azoarcus, Thauera, Zoogloea and Accumulibacter (all Betaproteobacteria).
However, considering the vast diversity of the Bacteria (Rappé and Giovannoni, 2003), the currently known
complexity of denitrifying microbial communities appears to represent just the tip of the iceberg.
Morgan-Sagastume et al. (2008) described a heterogenous denitrifying community, which in addition
to the Betaproteobacteria mentioned above, included Alphaproetobacteria, Gammaproteobacteria
and Actinobacteria in two municipal full scale treatment plants. Clearly we are still some way from
understanding possible interrelationships between denitrifying community composition, reactor type and
operation, wastewater chemistry, and process performance and stability.
One innovative way to lower operational costs of N removal is the SHARON (single-reactor high-
activity ammonium removal over nitrite) process (Hellinga et al., 1998), which involves ammonia-
oxidizing and denitrifying bacteria (chapter 2). SHARON operates at high temperatures of 30–40– C and
pH 7–8. Under these conditions, AOB grow faster than NOB, which are selectively washed out of the
system if an appropriate flow rate is maintained without biomass retention (Hellinga et al., 1998). Hence,
the effluent of the SHARON reactor contains N mainly in the form of nitrite, which is then removed in a
subsequent non-aerated denitrification stage. As the oxidation of ammonia to nitrite instead of nitrate
requires less oxygen and the denitrification of nitrite requires less carbon source than that of nitrate, the
total operational costs are markedly reduced.
ANAEROBIC AMMONIUM OXIDATION (ANAMMOX)

Phylogeny and physiology of anaerobic ammonium oxidizers
Almost three decades ago, and based on thermodynamic calculations, Engelbert Broda (1977) predicted
that lithotrophic microbes could theoretically grow by oxidizing NHþ
4 to N2 with NO3 or O2 as electron
acceptors. However, evidence for the existence of such a lithotroph did not exist until Mulder et al. (1995)
observed loss of ammonium from an anoxic fluidized bed reactor. Indeed, the decrease in ammonium
was accompanied by consumption of nitrate, indicating that NO3 was used as electron acceptor for the
anaerobic oxidation of NHþ 4 . Additional experiments confirmed the biological nature of this process,
but showed also that nitrite and not nitrate, was the electron acceptor for anaerobic ammonium oxidation
(‘‘Anammox’’) (van de Graaf et al., 1995). Based on these findings, biomass showing Anammox
activity was enriched in a lab-scale sequencing batch reactor (Strous et al., 1998). However, all attempts
to isolate the peculiar Anammox organisms from this enrichment were unsuccessful. Eventually, culture-
independent molecular methods revealed that these novel microbes were members of a deep-branching
lineage in the bacterial phylum Planctomycetes (Strous et al., 1999).
Identification of the first Anammox bacterium, which was named Candidatus ‘Brocadia anammoxidans’,
was followed by discoveries of additional closely related anaerobic ammonium oxidizers in treatment
plants [Candidatus ‘Kuenenia stuttgartiensis’ (Schmid et al., 2000); Candidatus ‘Scalindua brodae’ and
Candidatus ‘Scalindua wagneri’ (Schmid et al., 2003b); Candidatus ‘Anammoxoglobus propionicus’
(Kartal et al., 2007)] and in anaerobic marine waters [Candidatus ‘Scalindua sorokinii’ (Kuypers et al.,
2003)]. We now know that Anammox organisms are widespread in wastewater treatment systems, sea- and
freshwater habitats and sediments (Egli et al., 2001; Kuypers et al., 2005; Penton et al., 2006; Schubert et al.,
2006).
Anammox bacteria propagate by budding instead of binary fission and possess an intracellular
compartment (Lindsay et al., 2001) called an ‘‘anammoxosome’’. In addition, they have an unusual cell
envelope structure and contain ladderanes (Damsté et al., 2002). These unique membrane lipids with
concatenated cyclobutane rings have already been used as biomarkers to detect Anammox organisms in
environmental samples. Anammox bacteria are chemolithoautotrophic organisms. They catalyze the
following reaction:
NHþ
4 þ NO2 ! N2 þ 2H2 O ð6Þ
Insight into the intricate Anammox pathway has been obtained by sequencing and analyzing the
genome of the uncultured Anammox bacterium Candidatus ‘K. stuttgartiensis’ (Strous et al., 2006). Based
on these data, experimental results and thermodynamic calculations, the following mechanism has been
proposed:
Nitrite is first reduced to NO by a nitrite reductase (NirS). Subsequently, a novel enzyme (hydrazine
hydrolase) forms hydrazine (N2H4) from nitric oxide and ammonium by reducing NO and oxidizing NHþ 4.
Finally, hydrazine is oxidized and converted to N2 by an enzyme with some similarity to HAO of aerobic
AOB. In this step, four high-energy electrons per oxidized N2H4 are transferred to an electron transport
chain and from there to nitrite and NO (see above). Released energy is conserved in a membrane potential.
The carbon requirements for growth are satisfied by CO2 fixation via the acetyl-CoA pathway (Strous et al.,
2006). The high-energy electrons, needed for carbon fixation, are transferred by another novel enzyme
(hydrazine dehydrogenase) from hydrazine via ferredoxin to the enzymes of the acetyl-CoA pathway
(chapter 1). As this reaction also utilizes N2H4 and converts it to N2, the hydrazine pool becomes diluted.
Its replenishment is achieved by oxidation of nitrite to nitrate and a reverse transport of these electrons to
nitrite and NO, which leads to the formation of new hydrazine (see above). This explains why the
Anammox reaction yields some nitrate in addition to N2.
Anammox bacteria are extremely slow-growing microbes with doubling times of 10–20 days and a low
biomass yield. Interestingly, they are not limited to energy conservation via the Anammox pathway.
Candidatus ‘K. stuttgartiensis’ can respire with iron and manganese oxides as electron acceptors and
formate as electron donor, and oxidizes iron with nitrate as electron acceptor (Strous et al., 2006).
Furthermore, Güven et al. (2005) demonstrated that at least some Anammox organisms can oxidize
propionate to CO2 with nitrate as electron acceptor. This reaction is independent from the oxidation of
ammonium and takes place in parallel to the Anammox process. The same study revealed that methanol
and ethanol inhibit the metabolism of Anammox bacteria.
Anammox organisms and wastewater treatment

The Anammox process is suitable for N removal from wastewaters with a high ammonium content and
a low C:N ratio, such as sludge digester effluents (Jetten et al., 2001), and has attractive advantages
over the traditional nitrification-denitrification pathway. The autotrophy of Anammox bacteria means no
requirement for organic carbon sources, which are often needed to enhance denitrification. Aeration of
Anammox reactors is unnecessary because Anammox organisms are obligate anaerobes. However, there
still is a requirement for nitrite as substrate, which can be supplied by aerobic AOB (see below). Thus, the
Anammox process has the potential to save a high portion of costs for N removal (Jetten et al., 2001;
Schmidt et al., 2003).
As with denitrification (see above), the Anammox process can be combined with SHARON to achieve
nitrogen removal with reduced operational costs (chapter 2). In the combined SHARON-Anammox
process (van Dongen et al., 2001), AOB convert approximately 50% of the ammonium in the influent to
nitrite. Thus, the effluent of the aerated stage contains a mixture of ammonium and nitrite in a ratio
optimal for Anammox, which then takes place in a second anaerobic reactor.
In contrast, the CANON (completely autotrophic nitrogen-removal over nitrite) system (Sliekers et al.,
2002) relies on a stable interaction between aerobic AOB and Anammox to develop in a single reactor
(chapter 2). The required inhibition of NOB is achieved by maintaining ammonium saturation and
concomitant O2 limitation in the CANON reactor. Under these conditions, NOB must compete with
highly active AOB for O2 and with Anammox bacteria for nitrite. In contrast, O2 is the only limiting factor
for AOB while the Anammox organisms are only nitrite-limited. As NOB experience double limitation,
they are outcompeted by AOB and Anammox (Third et al., 2001). However, this system is highly
sensitive to a decrease in the ammonium concentration in the influent. Ammonium limitation causes a
lower activity of AOB, which leads to less oxygen consumption and an increase in the dissolved O2
concentration. The unavoidable inhibition of Anammox from presence of O2 causes an increase in
nitrite, which eventually favours the growth of aerobic NOB (Third et al., 2001). Therefore, full-scale
implementations of CANON would require precise control of aeration and ammonium supply. Similar to
CANON, the OLAND (O2-limited autotrophic nitrification-denitrification) system consists of one reactor
that is operated under O2 limitation (Kuai and Verstraete, 1998). Molecular analyses confirmed that the
microbial community in lab-scale rotating biological contactors operated under OLAND conditions
contained aerobic AOB and Anammox bacteria (Pynaert et al., 2003).
A successful up-scaling of the Anammox process and prolonged operation of full-scale Anammox
reactors has been reported (Kartal et al., 2004; van der Star et al., 2007; Wett, 2006). Despite these
promising developments of novel N-removal processes that integrate nitritation and Anammox, some
problems remain to be solved before the Anammox process can widely be applied in full-scale treatment
plants. As already mentioned, Anammox bacteria grow extremely slowly. Indeed, the time needed to
start (or re-start) an Anammox reactor from scratch has been estimated to be as long as 100–200 days
(Güven et al., 2005). Thus, any event that causes inhibition of the Anammox process in a running
bioreactor, as for example an increase of toxic compounds in the influent, could have long-term negative
effects on the efficiency of the affected process. Consequently, molecular tools to detect early changes in
the activity of Anammox bacteria before such changes become manifest in a decreased reactor
performance could be highly beneficial to large-scale applications of Anammox. Such a tool is FISH with
oligonucleotide probes that target the intergenic spacer regions (ISRs) between the 16S rRNA and 23S
rRNA genes. These spacer-targeted probes bind only to metabolically active Anammox cells and when
applied in combination with other suitable rRNA-targeted probes, are excellent markers to distinguish
active from inactive Anammox bacteria in biofilms and activated sludge samples (Schmid et al., 2001).
Because of the extraordinarily slow growth of Anammox bacteria, highly efficient biomass retention is
required. Therefore, Anammox is particularly sensitive to the consequences of poor sludge settling,
problems discussed in detail by Dapena-Mora et al. (2004). Theoretically, the effects of Anammox reactor
breakdowns could be alleviated by re-inoculating the reactor with previously stored Anammox biomass.
However, long-term storage may not be practicable everywhere, and as mentioned above, bioaugmenta-
tion of a reactor with bacteria from another source is prone to failure (Bouchez et al., 2000). From a
microbiological perspective, it could make more sense to find operating conditions which favour the
growth of Anammox organisms that are more resistant to perturbation. This may not be a single
population but a more diverse community of Anammox strains with different physiological traits. Future
research on the physiological versatility and niche differentiation of Anammox bacteria may reveal
strategies to increase the diversity of Anammox organisms in engineered systems.
10
The microbiology of phosphorus removal
Katherine D. McMahon, Shaomei He, and Adrian Oehmen
INTRODUCTION
Phosphorus (P) is an essential element for the growth of all organisms and thus plays a critical role in
stimulating the excessive growth of phytoplankton in many surface waters, leading to eutrophication.
The resulting primary productivity can fuel hypoxia, increase water turbidity, lead to production of toxins
that are harmful to fish and animals, and cause a general deterioration in water quality. Two major inputs
of P into water bodies are agricultural runoff and point wastewater discharges. Conventional aerated
activated sludge processes (chapter 2) were designed initially to remove organic carbonaceous material,
represented as COD or BOD, but P levels are not reduced substantially by them. For example, the typical
P content is 1.5 to 2.0 percent of the dry weight of biosolids, and by wasting the activated sludge, the
amount of P removed is about 10–30% (Stensel, 1991).
Phosphorus can also be reduced to low levels by chemical precipitation with aluminum chloride
(or sulfate), ferric chloride and calcium hydroxide (Jenkins and Hermanowicz, 1991). However, chemical
costs can be very high, and this strategy generates large quantities of chemical sludge that is difficult to
dewater and dispose of.
Enhanced biological P removal (EBPR) is an alternative approach. Some microbes in activated sludge
can take up larger amounts of P than required for their growth and store it intracellularly in the form of
polyphosphate (polyP) under cyclic anaerobic and aerobic conditions (Mino et al., 1998). These
organisms are referred to as polyphosphate accumulating organisms (PAO). Their intracellular P content
is higher than conventional activated sludge, ranging from 3 to 6% or more of their biomass dry weight
(Stensel, 1991). This immobilized P is subsequently removed from the bulk water through sludge wastage.
Many activated sludge processes have been modified to optimize conditions favorable to EBPR and these
produce treated effluents with total-P levels as low as 0.1–0.2 mgl 1 (Blackall et al., 2002). Compared to
chemical P removal, EBPR requires no precipitant addition and generates a well-settling sludge with a
high nutrient content that can be applied potentially as a fertilizer substitute in agriculture. Thus, the
EBPR process should be viewed as cost effective and environmentally friendly.
THE DISCOVERY AND APPLICATION OF EBPR

The EBPR phenomenon was discovered accidentally. In the late 1950s and early 1960s, it was noticed that
activated sludge could take up P in excess of that required for microbial growth in plug flow activated
sludge plants (Alarcon, 1961; Greenburg et al., 1955; Srinath, 1959). This phenomenon was called luxury
uptake of P (Levin and Shapiro, 1965), and enables higher or enhanced removal efficiency of P than
through its stoichiometric coupling to growth. A similar enhanced P removal was reported subsequently in
more full-scale activated sludge plants, all with a common configuration of a long, narrow plug flow tank
with high dissolved oxygen (DO) levels near the end of the tank. P release occurred near the front of the
tank, close to the inlet of the return activated sludge and primary effluent, which was essentially anaerobic.
In the latter tank region, P uptake was observed, associated with these elevated DO levels.
These findings generated considerable debate about the possible mechanism responsible for this
enhanced P removal, and whether it was from chemical precipitation or by biological removal.
Consistently observed pH increases with higher aeration rates towards the end of the aeration basin,
causing a stripping of CO2, suggested that inorganic phosphate (Pi) might be precipitating through
formation of calcium phosphate (Menar and Jenkins, 1969). However, when Levin and Shapiro (1965)
added the respiratory inhibitor 2,4 di-nitrophenol to a reactor performing EBPR, P uptake was inhibited,
confirming it was a biological process.
Yet, the fundamental mechanisms involved remained a mystery. Based on many consistent
observations, empirical process guidelines were developed (Milbury, 1971; Vacker, 1967). The critical
design factor appeared to be the alternating anaerobic and aerobic zones, and the return of sludge with the
addition of substrate in the influent to the anaerobic zone. In the 1970s, the first biological P removal
facilities with anaerobic/aerobic cycling were established in South Africa and United States (Barnard,
1976), and subsequently many more EBPR facilities were constructed, with modified configurations
(chapter 2), all achieving P removal but without understanding how or why.
Despite its many advantages EBPR processes can suffer from performance instability. With appropriate
wastewater composition and well managed operation, EBPR can generate treated effluents with soluble P
levels less than 0.1 mgl 1. However, many struggle to achieve reliable effluent P levels below 1.0 mgl 1
(Neethling et al., 2005; Stephens et al., 2004). A sudden loss of EBPR performance in many plants can
occur with no apparent explanation, and attempts to recover high EBPR capacity are not always effective.
Thus, expensive chemical treatment processes are often installed as back up systems during these plant
failures (Jenkins and Hermanowicz 1991; Neethling et al., 2005). Clearly the lower the effluent P levels
from the biological process, the less chemical precipitation is required, and this in itself is justification for
better understanding the mechanisms involved in the microbial ‘black box’ that drives EBPR.
BIOCHEMICAL TRANSFORMATIONS AND METABOLIC MODELS

Biochemical transformations
Because no pure culture of a confirmed PAO is available (discussed later), the existing metabolic models
have been developed based on measuring chemical transformations occurring in the bulk liquid and
The microbiology of phosphorus removal 283
sludge biomass in complex communities, whose members are assumed to be a single PAO population
with homogeneous metabolic behavior. These biochemical transformations involve (Oehmen et al., 2007;
Mino et al., 1998; Seviour et al., 2003):
During the anaerobic (feed) phase:
1. Short chain volatile fatty acids (VFAs) are rapidly assimilated and stored as poly-b-hydroxy-
alkanoate (PHA), whose chemical composition depends on the available carbon source. With
acetate as carbon source, poly-b-hydroxybutyrate (PHB) is synthesized, and with propionate, poly-
b-hydroxyvalerate (PHV) and poly-b-hydroxy-2-methylvalerate (PH2MV) are produced.
2. Intracellular polyP levels decrease, with a corresponding increase of Pi levels in the bulk liquid.
3. Other intracellular storage carbohydrates, especially glycogen are degraded.
During the subsequent aerobic (famine) phase:
1. PHA levels in the biomass drop, correlating with an increase in biomass growth rate.
2. Pi levels in the bulk liquid decrease while intracellular poly P levels increase.
3. Biomass storage carbohydrates, especially glycogen, are replenished.
Based these biochemical transformations, several models of EBPR metabolism have been proposed
and reviewed (Oehmen et al., 2007). These models are generally based on acetate as the model substrate,
although PAO can assimilate other substrates including other VFAs, amino acids and low-molecular
weight substrates (see later). The Comeau-Wenzel model and the Mino model are both widely accepted.
Comeau-Wentzel model
Comeau and Wentzel were the first to describe a mechanistic model attempting to explain these EBPR
chemical transformations (Comeau et al., 1986; Wentzel et al., 1986). The model is thus referred to as the
Comeau-Wentzel model, and is presented in Figure 10.1, see colour image section (chapter 13).
Essentially this model suggests that under anaerobic feed conditions, stored polyP is degraded to produce
ATP, which is thought to provide the energy required to synthesise the energized form of acetate
acetyl-CoA (AcCoA) and re-establish the proton motive force (PMF) consumed by substrate transport.
ATP degradation leads to Pi release. Some of the AcCoA undergoes oxidation via the tricarboxylic
acid (TCA) cycle, which generates NADH,. This provides the reducing power for converting AcCoA to
PHB. Metallic cations like Ca2þ and Mg2þ (Mþ) are co-transported with Pi. Under subsequent aerobic
famine conditions, the intracellularly stored PHB is oxidized via the TCA cycle, which generates the PMF
used for ATP production, which PAO cells use for growth and replenishment of intracellular polyP stores.
One major problem with this model is the accumulation of FADH2 (reduced flavin adenine
dinucleotide) in the TCA cycle operating under anaerobic conditions from the oxidation of succinate to
fumarate. Only NAD(P)H can be used for PHB formation in the PAO. Without an external electron
acceptor (like O2), FADH2 accumulation will shut down the TCA cycle. To overcome this problem, a
split TCA cycle was proposed in which the left arm of the TCA cycle operates in reverse (Figure 10.8,
see colour image section (chapter 13)), so that a fraction of the fumarate is reduced to succinate and then
succinyl-CoA, consuming FADH2. Succinyl-CoA is then converted to methylmalonyl-CoA, propionyl-
CoA, and eventually to PHV (Hesselmann et al., 2000; Pramanik et al., 1999), explaining the small
amount of PHV formed even with an acetate feed.
An alternative proposal for how the TCA cycle might operate anaerobically was through a glyoxylate
shunt, but this does not solve the FADH2 accumulation problem (Louie et al., 2000). Metagenomic
analysis of a PAO-enriched sludge suggested another mechanism allowing for anaerobic oxidation of
FADH2 involving quinones as electron acceptors (Garcia Martin et al., 2006), and a novel cytochrome
b/b6 functioning in reverse to transfer electrons from these reduced quinones to NAD(P)þ, at the expense
of an established PMF (see Figure 10.8). However, the hypothesized role of this enzyme needs further
examination (see later).
Mino model
One issue fiercely debated among EBPR metabolic modellers is the source of reducing power required for
PHA formation in the anaerobic stage, especially since PHA is more highly reduced than acetate. In the
Comeau-Wentzel model, NADH is generated by oxidation of acetate through the TCA cycle (Figure 10.1).
Mino et al. (1987) proposed that NADH is also generated from anaerobic degradation of intracellularly
stored glycogen to AcCoA in addition to its partial oxidation via the TCA cycle to CO2. This model
is now referred to as the Mino model (Figure 10.2, see colour image section (chapter 13)). Arun et al.
(1988) showed that intracellular carbohydrates were consumed during anaerobic acetate uptake and
PHB production and that these were degraded using the Embden-Meyerhoff-Parnas (EMP) pathway
(chapter 1), and converted to AcCoA, a series of reactions which provided NADH for PHB synthesis.
However, other experimental evidence has suggested that the glycogen is degraded using the
Entner-Doudoroff (ED) pathway (Hesselmann et al., 2000; Maurer et al., 1997), which is not consistent
with data from the genomic analysis of model PAO enriched with acetate (Garcia Martin et al., 2006),
which indicates that the ED pathway may not be present in these populations (see later).
Another difference between the two models is the energy source thought to be used for anaerobic
metabolism. In the Comeau-Wentzel model, degradation of polyP is considered to be the sole energy source
used by the PAO for substrate assimilation, PHA synthesis, and cell maintenance. However, in the Mino
model, glycogen catabolism is also thought to provide ATP for these functions (Figure 10.2). Evidence to
support glycogen degradation as the primary source of reducing power has accumulated and the Mino
model is thought more likely to be reflect the biochemical changes (Mino et al., 1998; Seviour et al., 2003).
For example, stoichiometry from the Mino model explains well the experimentally observed anaerobic
transformations of acetate, PHB, glycogen and CO2 by PAO-enriched sludges (Satoh et al., 1992;
Smolders et al., 1994b). Sludge fed with radiolabeled acetate showed that acetate was not oxidized to
CO2 through the full TCA cycle (Bordacs and Chiesa, 1989). Then with 13C nuclear magnetic resonance
spectroscopy (NMR), Pereira et al. (1996) could demonstrate that the PHA synthesized anaerobically was
converted to glycogen in the subsequent aerobic phase using full-scale EBPR sludge, and importantly
Kong et al. (2004) confirmed with metabolic inhibitors and a model PAO that glycolysis, and not the TCA
cycle, was providing the reducing power for PHA synthesis during anaerobic substrate assimilation.
Although the Mino model is generally favored, the possibility that some NADH production is generated
by the TCA cycle cannot be totally excluded, since some data indicate that glycogen degradation
alone is insufficient to provide the required reducing power for PHA synthesis (Hesselmann et al., 2000;
Pereira et al., 1996). It seems likely that both glycogen and the TCA cycle are used, as subsequently
confirmed on a full-scale EBPR sludge (Pijuan et al., 2008). The contribution of each source may depend on
environmental conditions and the physiological status of PAO cells.
It must be emphasized that all these experiments were performed on EBPR sludges with mixed
bacterial communities. Glycogen accumulating non-polyP organisms (GAO) (discussed later), which
exhibit carbon transformation under alternating anaerobic and aerobic conditions similar to the PAO but
without any polyP accumulation, were probably also present in these communities (Blackall et al., 2002).
It is difficult to distinguish relative contributions of each to these biochemical transformations, but GAO
presence may explain some of the discrepancies observed in the different studies and question the value of
both these metabolic models. Thus, many aspects of PAO metabolism remain unresolved.
Denitrifying PAO (DPAO)

The metabolic models described above were established based on O2 serving as the electron acceptor for
aerobic P uptake. However, in treatment plants like the MUCT process (chapter 2) designed to remove
both P and nitrogen (N), some P uptake can occur in the anoxic zone, indicating that NO3 is being used as
terminal electron acceptor for respiratory energy generation and P uptake (referred to here as anoxic P
uptake to distinguish it from aerobic P uptake). The PAO able to use both O2 and NO3 for P uptake are
referred to as denitrifying PAO (DPAO). Coupling P uptake with denitrification provides advantages
including lowered energy costs for aeration, decreased organic carbon demand for denitrification and
decreased biomass generation and sludge disposal problems, since denitrification is metabolically less
efficient in generating energy for cell growth than aerobic respiration. Some studies suggested that DPAO
and regular PAO are different populations (Meinhold et al., 1999), while others have argued that they are
the same, switching their metabolism between using NO3 and O2 (Zeng et al., 2003a,b). This issue is
discussed in more detail later.
STORAGE POLYMERS IN EBPR

From the proposed EBPR metabolic models with acetate as substrate, cyclical transformation of three
polymers involving energy, carbon, and electron flows provides PAO with a competitive advantage
allowing them to sequester carbon anaerobically. Polymers are generally synthesized by bacterial cells in
response to stress or nutrient limitation (chapter 3), where growth is no longer possible, but also
transiently under nutrient-rich conditions. The three polymers important in EBPR, and their biochemical
transformations are discussed next.
Polyphosphate and its metabolism

Polyphosphate
Inorganic polyphosphate (polyP) is a linear polymer of many tens to hundreds, or even thousands, of
orthophosphate residues linked by high-energy phosphoanhydride bonds (Figure 10.3). As a molecular
fossil, polyP is ubiquitous in nature and occurs in every organism so far examined: Bacteria, Archaea,
fungi, protozoa, plants, and animals (Kornberg, 1995). PolyP can be visualized inside cells by staining
with basic dyes, including toluidine blue or methylene blue, followed by light microscopy (chapter 11).
More sensitively, polyP can also be stained with DAPI (chapter 11), which under fluorescence
microscopy, fluoresces yellow/white.
O– O– O– O–
–O P O P O P O P O–
O O O n O
Figure 10.3. Inorganic polyP. The value of n in the chains varies from many tens to hundreds.
Functions of polyP
As a ‘‘molecule for many reasons’’ (Kornberg, 1995), polyP has important and variable functions in
cells, depending on the species, its cellular location and physiological status and environmental
conditions. Many attempts have been made to understand the physiological roles of polyP. Those
proposed include: substituting for ATP and energy source by converting polyP to ATP (Kornberg,
1999); a reservoir of Pi, generated by converting polyP to free Pi, whose stable level is essential
for metabolism and growth (Kornberg, 1999); a strong chelator of ions (e.g. Mn2þ, Mg2þ, Ca2þ) with
the Pi polyanion (Dunn, 1994); a buffer against alkali conditions by neutralising protons released from
enzymatic hydrolysis of polyP (Pick et al., 1990); a channel for DNA entry by formation of a polyP-
DNA Ca2þ complex in the cell membrane, rendering cells competent for transformation (chapter 1)
(Reusch and Sadoff, 1988); regulation of gene expression in response to stress, and promotion of
survival in stationary phase cells (Rao and Kornberg, 1996, 1999); a component of the cell capsule in
some pathogenic Neisseria spp. (Tinsley et al., 1993); an essential component of the Lon protease
complex involved in ribosomal protein degradation (Kuroda et al., 2001); and an important regulator of
expression of virulence factors in the stationary phase of some pathogens (Rashid and Kornberg, 2000a;
Rashid et al., 2000b).
It is not yet clear whether some of these functions apply to the PAO in EBPR systems. EBPR metabolic
models indicate a central role for polyP as an energy source and in re-establishing the PMF in the
anaerobic phase. There has been considerable debate on the mechanism for acetate transport across
the cell membrane. The Comeau-Wentzel model assumes a passive diffusion process (Figure 10.1), while
the stoichiometric models usually assume active transport is involved, which requires ATP generation
from polyP degradation (Smolders et al., 1994b). The former consumes PMF, and was demonstrated
experimentally as the acetate transport mechanism for sludge enriched with Candidatus ‘Accumulibacter
phosphatis’ a putative PAO not yet cultured, (as discussed later) by Saunders et al. (2006) and Burow et al.
(2008a). Both also proposed that anaerobic PMF generation was achieved by the efflux of protons in
symport with Pi resulting from polyP degradation via an inorganic phosphate transport (Pit) system.
In either case, polyP plays an important role in acetate uptake.
Once inside the cell, acetate is activated to AcCoA through a high-affinity AcCoA synthase (acs) or a
low-affinity acetate kinase (ack) and phosphotransacetylase (pta) system. There is also debate on which
pathway is used for PAO acetate activation, but the agreement is that both pathways consume energy in
the form of ATP, which is provided in some way by polyP degradation. Thus, polyP seems important for
acetate uptake and activation under anaerobic conditions, conferring a selective advantage for PAO to
allow them to compete effectively for carbon sources. PolyP by its degradation may also regulate the
intracellular pH of PAO under alkaline conditions (Bond et al., 1999b).
PolyP metabolism and polyphosphate kinase

Because of its multiple and important biological functions, the biochemistry of polyP is relatively well
studied in model organisms. Its metabolism is of particular interest in EBPR systems since its synthesis is
the direct mechanism by which P is removed from wastewater.
Polyphosphate kinase 1 (PPK1) is the enzyme primarily responsible for polyP synthesis (Ahn and
Kornberg, 1990). It catalyzes the reversible conversion of the g phosphate of ATP to polyP, as indicated in
equation 1. Most studies conducted with PPK1 have been performed on model bacteria, like Escherichia
coli (Ahn and Kornberg, 1990; Akiyama et al., 1992; Keasling et al., 1998; Rao and Kornberg, 1996;
Tzeng and Kornberg, 2000), Pseudomonas aeruginosa (Ishige et al., 1998; Rashid et al., 2000; Zago et al.,
1999), Neisseria meningitidis (Tinsley and Gotschlich 1995; Tinsley et al., 1993), and Acinetobacter spp.
(Gavigan et al., 1999; Geissdoerfer et al., 1998; Trelstad et al., 1999; van Niel et al., 1999). It should be
noted that the ‘‘1’’ in the naming of PPK1 was introduced upon discovery of PPK2 (discussed later), and
PPK1 (or PPK2) here should be distinguished from the PPK allele 1 (or 2).
polyPn þ ATP ! polyPnþ1 þ ADP (1)
PPK1 in model bacteria

PPK1 has been purified from several bacteria, including E. coli, Acinetobacter sp. strain ADP1,
Pseudomonas aeruginosa, Neisseria meningitidis and Klebsiella aerogenes. The genes encoding PPK1
(ppk1) have been cloned and sequenced from several pure cultures, and show PPK1 is highly conserved in
many bacterial species (Tzeng and Kornberg, 1998).
The E. coli PPK1 was the first PPK partly purified and characterized in 1956 by Kornberg and
coworkers (Kornberg et al., 1956). They found a strict dependence on Mg2þ and an adherence to
Michaelis-Menten kinetics with respect to its substrate ATP. In 1990, the E. coli PPK1 was purified
to homogeneity by Ahn and Kornberg, (1990) and characterized. They revealed that this PPK1 is a
tetramer of 69 KDa subunits and its characterization suggested that it was autophosphorylated during
polyP synthesis, as evidenced by an N-linked phosphoenzyme reaction intermediate. They also found
PPK1 occurred in lysates attached to a particulate cell fraction and either sonication or strong salt was
required for its solubilization. The E. coli ppk1 gene has also been cloned, sequenced, and over-expressed
(about 100 fold) (Akiyama et al., 1992). It possesses an open reading frame (ORF) coding for 687 amino
acids (80 KDa). Akiyama et al. (1993) also reported that PPK1 binds peripherally to the outer membrane
of cells. After being purified from overproducing cells and released from attachment to the cell membrane,
the soluble PPK1 could reassociate with the cell membrane fraction. The same group showed a gene,
ppx, encoding a novel exopolyphosphatase of 513 amino acids (58,133 Da) was located downstream of
ppk1, and its transcription depended on the ppk1 promoter, indicating a polyP metabolism operon
comprised of ppk1 and ppx (Akiyama et al., 1993).
Expression of ppk1 was shown to be under the control of the Pho regulon in E. coli (Wanner, 1996)
and Klebsiella aerogenes (Kato et al., 1993), but this does not appear to regulate ppk1 expression
in all organisms. In Pseudomonas aeruginosa, ppk1 expression was not affected by extracellular
Pi concentration, but rather was under the control of a stress response sigma factor (Zago et al., 1999). In
an Acinetobacter sp., low Pi concentrations induced expression of ppk1 (Geissdoerfer et al., 1998), but the
highest PPK1 activity was at high Pi concentrations following a period of Pi starvation that may induce
enzyme synthesis (Trelstad et al., 1999). Such conditions of Pi starvation and availability are similar to
those found in EBPR processes, and so a similar regulatory system may operate there.
PPK2. Kornberg and colleagues also discovered a previously uncharacterized PPK activity from a null
mutant of Pseudomonas aeruginosa strain PAO1 lacking PPK1 (Ishige et al., 2002; Zhang et al., 2002).
They named the novel PPK as PPK2, which differs from PPK1 by the following: 1) PPK2 can synthesize
polyP from GTP or ATP, and GTP is preferred, 2) Mn2þ is preferred over Mg2þ for polyP synthesising
activity, 3) The reverse reaction, the synthesis of GTP from GDP by degradation of polyP was 75 fold
greater than the forward reaction, and was stimulated by polyP.
PPK1 in activated sludge. The first study on PPK1 in EBPR activated sludge was that of McMahon et al.
(2002a,b). Exploiting the high level of conservation of the PPK1 amino acid sequence among bacteria,
they designed degenerate primers to retrieve fragments of putative ppk1 genes from activated sludge
samples from acetate-fed laboratory-scale EBPR SBRs, where Candidatus ‘Accumulibacter phosphatis’
(an uncultured putative PAO, discussed in detail later) comprised approximately 80% of the total bacteria
in the sludge (McMahon et al., 2002a,b). Two genotypes of ppk1 were retrieved from Accumulibacter,
designated Type I and Type II. The Type I ppk1 was shown to be expressed during EBPR by mRNA dot
blot analysis. A full-length Type I ppk1 sequence was obtained by inverse PCR, it was cloned and
overexpressed in E. coli and the protein then purified to near homogeneity. This purified recombinant
Type I PPK1, appearing to be a 75 kDa monomer, was capable of synthesizing polyP from ATP, had a
specific activity comparable to the purified PPK1 from Acinetobacter sp. and E. coli, and a requirement
for Mg2þ. In activated sludge samples, PPK1 activity was located largely in the insoluble cell debris
fraction of the lysed sludge. In the same study, a larger genetic fragment containing ppk1 was also
retrieved. Interestingly, this putative ORF was flanked by a ppx homolog upstream and a relA homolog
downstream, in both Type I and II ppk (Figure 10.4). In other model organisms, exopolyphosphatase
(PPX) (see below) catalyzes hydrolysis of polyP to release Pi, and relA encodes for the synthase of
guanosine tetraphosphate (ppGpp) and pentaphosphate (pppGpp), whose elevated levels were shown to
inhibit PPX activity and thus favouring polyP formation in E. coli (Rao et al., 1998). Subsequently,
McMahon et al. (2007b) retrieved ppk1 fragments from several full-scale EBPR activated sludges.
The ppk1 fragments from the full-scale sludge were nearly identical to the Type II ppk1 obtained earlier
from the lab-scale reactors, and this sequence type was not observed in conventional non-EBPR aerobic
activated sludge systems.
ppx ppx1 relA
Type I 214 bp 67 bp
Type II 211 bp 52 bp
Figure 10.4. Organization of the gene clusters retrieved from large fragments of genomic DNA from
Accumulibacter PAO, using inverse PCR (McMahon et al., 2002b). Intergenic spacer region lengths are
noted for the two Types. Not to scale.
Ppk1 expression was examined on Accumulibacter-enriched sludge using reverse transcription

quantitative PCR (RT-qPCR) (Saunders, 2005), who found that its expression during an anaerobic/aerobic
EBPR cycle varied only minimally, with slight evidence of induction at the end of the cycle. Furthermore,
ppk1 expression was not repressed during the anaerobic phase or by carbon limitation during the later part of
the aerobic phase. Compared with low Pi concentrations (510 uM), its expression was repressed by about
3-fold and 13-fold at Pi concentrations of 3 mM and 12 mM respectively. Such a repression by high external
Pi levels suggests a Pho regulon may be functioning in Accumulibacter. However, ppk1 transcript
quantification was performed against 16S rRNA as a baseline for normalization of expression levels. Since
16S rRNA abundance on an individual cell basis may vary during the course of an EBPR cycle (He and
McMahon, unpublished data), their accuracy of quantification may be compromised.
Other enzymes involved in polyP metabolisms

Exopolyphosphatase
Exopolyphosphatase (PPX) is a highly processive enzyme catalyzing hydrolysis of terminal residues of long
chain polyP to Pi. In E. coli, PPX is a homodimer of 58 kDa subunits, mainly located at the membrane
(Kornberg et al., 1999), and the ppx gene is located 7bp downstream of ppk1 in the same operon.
Phosphotransferase
The polyP:AMP phosphotransferase enzyme (PAP) catalyzes phosphorylation of AMP to ADP at the
expense of polyP, while adenylate kinase (ADK) catalyzes the reversible conversion of 2 ADP to ATP and
AMP. The PAP:ADK system has been suggested to be involved in polyP transformation in EBPR
communities. However, studies were conducted primarily on isolates including Acinetobacter spp. from
EBPR sludges, populations subsequently shown to be of minor importance in these systems. However, the
metagenomic analysis of Accumulibacter-enriched EBPR sludge has indicated a presence of genes encoding
both PAP and ADK (Garcia Martin et al., 2006). Therefore, their possible roles in EBPR need further study.
Polyhydroxyalkanoates
Polyhydroxyalkanoates (PHAs) are linear polyesters produced by bacteria under conditions where carbon
sources are readily available, but other essential substrates are not. These water-insoluble polymers are
deposited as intracellular inclusions or granules, which can be stained by Nile Blue or Sudan Black
(chapter 11). In EBPR, the PHAs synthesized vary in their chemistry and reflect the nature of VFAs
present in wastewater, as mentioned above. The biosynthetic pathways for PHB have been extensively
documented in model organisms like Ralstonia eutropha (Kessler and Witholt, 2001) (Figure 10.5).
TCA cycle
Glucose
Acetyl-CoA Citrate synthase
β-Ketothiolase HSCoA
Acetoacetyl-CoA
NADP+H+
Acetoacetyl-
CoA-reductase
NADP+
(R)-3-OH-Butyryl-CoA
PHB synthase
HSCoA
PHB
Figure 10.5. PHB synthesis pathway in Ralstonia eutropha. The three-step pathway involves b-ketothiolase
(PhaA), acetoacetyl-CoA-reductase (PhaB), and PHB synthase (PhaC). The b-ketothiolase is negatively
regulated by coenzyme A (HSCoA), which is produced when acetyl-CoA enters the TCA cycle under non-
limited conditions. High concentrations of NADPH and NADH inhibit the citrate synthase of the TCA cycle,
which modulates the flux of carbon providing enough acetyl-CoA for the b-ketothiolase. Dotted lines indicate
negative regulatory effects. Modified from Kessler and Witholt (2001).
In most bacteria, a b-ketothiolase (PhaA), an NADPH-dependent acetoacetyl-CoA reductase (PhaB)

and a PHB synthase (PhaC) comprise a three-step PHB-biosynthetic pathway. PHB synthesis occurs under
unbalanced growth conditions where excess carbon is provided, while O2, N, Pi, Mg2þ, or some other
nutrient is in growth limiting amounts (Kessler and Witholt, 2001). Regulation of PHB metabolism occurs
at both transcriptional and/or enzymatic levels. In Acinetobacter spp. transcription of PhaABC genes
is induced by phosphate starvation through a promoter seemingly under the control of the pho regulon
(Schembri et al., 1995). Regulatory proteins associated with transcription of PHA metabolic genes
have also been identified, including PhaR (Matsusaki et al., 1998) and PhaF (Prieto et al., 1999) in
Pseudomonas spp. At the level of regulating enzymatic activity, the redox status of cells and their
concentrations of CoA play important regulatory roles in PHB biosynthesis (Stubbe et al., 2005). Activity
of thiolase is inhibited by free CoA, but as increased levels of NAD(P)H would decrease CoA, this
inhibition is alleviated, thus favoring PHB synthesis. High levels of NAD(P)H inhibit citrate synthase,
causing blockage of the TCA cycle. Therefore a high NAD(P)H/NADþ ratio favors carbon flux into PHB
production. Under anaerobic EBPR conditions, and lack of any external electron acceptor, the cell is
under relatively reduced redox conditions. Whether PHB production in PAOs is controlled by redox
conditions is worth investigating.
Glycogen
Glycogen is a major carbohydrate storage compound in bacterial cells, and is accumulated by
many species under nutrient-limited conditions. There has been considerable debate about how
anaerobic glycogen degradation occurs during EBPR. When it was first proposed as a major source of
NAD(P)H for PHA production, the prevailing view was that glycogen was degraded by the EMP pathway
(Arun et al., 1988). However, 13C labeled NMR experiments suggested that the ED pathway was operative
(Maurer et al., 1997). These pathways differ in the amount of ATP they generate. Thus, the overall
energy balance computed in metabolic models depends heavily on which pathway is incorporated into the
model. Indeed, some models have been used to provide support for operation of the ED pathway
(Smolders et al., 1994b; Filipe et al., 2001b). However, as described later, genomic analyses suggest that
Accumulibacter PAO lack genes encoding key enzymes in this pathway (Garcia Martin et al., 2006).
Thus, much remains to be resolved regarding glycogen metabolism in PAO too.
THE ROLE AND NECESSITY OF THE ANAEROBIC ZONE

For high P removal, physical separation of electron donor and acceptor by creating a strict anaerobic zone
free from oxygen and nitrate is generally thought to be required. Our current understanding of EBPR
assumes that the anaerobic phase acts as a selection zone for PAO in the following way. Fermentative
bacteria degrade complex carbon polymers into smaller RBCOD favored by PAO. In the absence of any
external electron acceptor, oxidative phosphorylation cannot occur, preventing respiring heterotrophs
from assimilating these substrates. However, PAO can sequester during the anaerobic feed phase by using
energy derived from their stored polyP and glycogen. Furthermore, decoupling of electron donor and
acceptor probably encourages the biotransformation of the three key polymers (see above), which
provides PAO with a selective advantage, allowing them to thrive under alternating anaerobic and aerobic
conditions.
However, several studies have suggested that EBPR activities can still be maintained if acetate was
provided under aerobic conditions (Guisasola et al., 2004; Pijuan et al., 2005, 2006). Substantial aerobic
P-release has been observed, coupled with acetate-uptake, as occurs under anaerobic conditions in EBPR
communities. P uptake occurred immediately after complete consumption of acetate. Furthermore,
reactors operated under continuous aeration were maintained for several months with stable P removal
(Ahn et al., 2007) by feeding acetate and P separately. This raises the question of whether separation of
electron donor and acceptor is necessary for the PAO to accumulate polyP, or if it only prevents other
heterotrophs from accessing available organic substrates aerobically.
It has been suggested that when influent COD is relatively high (e.g. 460–120 mg/L) and DO in the
bulk liquid insufficient (e.g. 54 mg/L), the aerobic region of flocs is restricted to their surface layers
because of O2 utilization by other microbes (Li and Bishop, 2004). This may explain the presence of PAO
in granular aerobic sludges (chapters 2, 3) (de Kreuk and van Loosdrecht, 2004, 2005; Lemaire et al.,
2008; Lin et al., 2003), as described in a hybrid biofilm-mixed reactor kinetic model which quantified
effects of diffusion gradients on EBPR activity within granules (de Kreuk et al., 2007a).
In the continuously aerated EBPR studies of Guisasola et al. (2004) and Pijuan et al. (2005, 2006) DO
levels in the bulk liquid were maintained only above 2 mg/L, and with Ahn et al. (2007), DO levels
rapidly fell to ,10% of saturation (51 mg/L) in the feed phase, corresponding to P release. It is important
to clarify whether cells in the center of the flocs experience similar aerobic conditions to those located in
the outer floc layers.
Presence of NO3 in the anaerobic zone decreases Pi release rate and negatively impacts on EBPR
capacity (chapter 2). This may be explained by i) competition for organic carbon from other denitrifying
heterotrophic microbes using NO3 as electron acceptor (Stensel, 1991); ii) inhibitory effect of NO3 or
denitrification intermediates like NO, on PAO metabolism (van Niel et al., 1998); iii) PAO shifting their
metabolism to use NO3 as electron acceptor for acetate oxidation, leading to simultaneous P release and
uptake by PAO, ultimately resulting in a decrease in net P release (Kuba et al., 1994). Which of these is
responsible still awaits resolution.
THE MICROBES RESPONSIBLE FOR EBPR – CULTURE-DEPENDENT

APPROACHES
Being able to identify which microbes are primarily responsible for EBPR would facilitate our
understanding of the biochemical mechanisms, functional regulation and population dynamics of PAO,
therefore allowing process and operational optimization to maximize P removal and achieve system
stability by rationally and intentionally exploiting the natural capabilities of activated sludge microbes.
Most of the early literature suggests Acinetobacter spp. (a member of the Gammaproteobacteria) as the
primary PAO, a conclusion based on their ready isolation from sludges accumulating polyP around the
world. The first report was in 1975, when Fuhs and Chen cultured an organism from an EBPR plant and
identified it as a strain of Acinetobacter lwoffii. They described batch experiments in which carbon-starved
A. lwoffii cultures released Pi upon carbon addition. Intracellular polyP and PHB granules were also
observed. Subsequently many other groups reported isolating Acinetobacter spp. from EBPR systems
of various configurations (Jenkins and Tandoi, 1991; Mino et al., 1998b; Seviour et al., 2003). All these
studies relied on culture-dependent methods (chapter 3), employing artificial culture media and growth
conditions (using agar plates or batch incubations of sludge dilutions), a strategy heavily biased
for characterization of microbial community structure (Amann et al., 1995b, 1998; Hugenholtz et al.,
1998a,b).
Despite their initial popularity, many studies have since demonstrated that Acinetobacter spp. are
NOT primarily responsible for EBPR (Oehmen et al., 2007; Mino et al., 1998; Seviour et al., 2003),
a conclusion supported by the following experimental evidence:
1. When polyamine diaminopropane (DAP) was used as a biomarker for Acinetobacter in full scale
sludges carrying out EBPR with both high and low organic loading rates (Auling et al., 1991), it
was detected in plants with low loading rates, whereas those with high loading rates and efficient
P removal had very low DAP content.
2. The respiratory quinone profiles of PAO-enriched sludges did not match those of Acinetobacter,
suggesting that Acinetobacter were minor populations there (Hiraishi et al., 1989).
3. Analyses of community structure with methods based on 16S rRNA gene sequences have
repeatedly shown that Acinetobacter are not present in large numbers in EBPR sludges (e.g. Bond
et al., 1995; Kampfer et al., 1996; Wagner et al., 1994c).
Members of the Actinobacteria were also isolated frequently from EBPR systems. For example,
Nakamura et al. (1995) isolated Microlunatus phosphovorus, from activated sludge (Nakamura et al.,
1995). Kinetics studies combined with 31P and 13C NMR have revealed that this organism can
anaerobically release and aerobically assimilate Pi as polyP at rates comparable to those seen in activated
sludge. However, P and carbon metabolism are not tightly coupled, and glucose, its favored carbon
source, is fermented to acetate during the anaerobic phase without it being channeled to PHA or other
carbon reserves. Thus, M. phosphovorus does not exhibit the predicted PAO phenotype (Santos et al.,
1999). Another Gram-positive coccus, Tetrasphaera, which is a tetrad forming organism (TFO) and
accumulates polyP, was isolated from activated sludge biomass by micromanipulation (Maszenan et al.,
2000). Later the rod shaped Tetrasphaera elongata, another species was also isolated from an activated
sludge reactor. Other Actinobacteria have also been shown to accumulate polyP (Hanada et al., 2002), but
in no case has it been demonstrated that these isolates have the required PAO phenotype (see earlier).
It is common knowledge now that the majority (estimated 499%) of bacteria in natural environments
are currently ‘unculturable’ (Amann et al., 1995; Hugenholtz et al., 1998a,b). Thus, culture-based
approaches inevitably underestimate the true level of biodiversity, and provide a skewed picture
of microbial community structure in the environment. Therefore, culture-independent methods, which
themselves are also biased (chapter 3) have been used increasingly to resolve EBPR community
composition.
MOLECULAR BIOLOGY TECHNIQUES USED IN STUDYING EBPR

MICROBIAL ECOLOGY
Applying DNA and RNA sequences as biomarkers has revolutionized our view of the biodiversity of
EBPR communities, and identification of major PAO populations (chapter 3). PCR-based techniques
have been used to retrieve 16S rRNA genes from lab-scale SBRs (Bond et al., 1995; Hesselmann et al.,
1999) and full-scale systems (Zilles et al., 2002b), and FISH (Table 10.1) then used to confirm the
abundance of phylogenetically-defined bacterial groups e.g. (Crocetti et al., 2000; Liu et al., 2001b;
Zilles et al., 2002b). Besides 16S/23S rRNA genes, other structural or functional genes have also
been used including the 16S–23S internal transcribed spacer (ITS) region (He et al., 2006), genes
encoding PPK1 (McMahon et al., 2002a), nitrite reductases (Miyauchi et al., 2007), and PHA synthases
(Wang et al., 2008a). These genes provide a higher resolution of population phylogeny since they evolve
faster than the 16S rRNA genes and thus exhibit higher degrees of sequence variation among lineages.
Further advancement in DNA sequencing technology and computation means it is now possible to
obtain whole genome sequences of populations at a community level from environmental samples.
Metagenomics, the study of all the genetic material present in an environmental sample, consisting of the
genomes of many individual organisms (metagenome) (Handelsman, 2004), has been applied successfully
to EBPR communities and the outcomes will increase our understanding of this technologically important
yet sophisticated microbial process (Seviour and McIlroy, 2008).
Table 10.1. Summary of 16S rRNA-targeted FISH probes for putative PAO and GAO in EBPR systems (from Oehmen et al., 2007).
Probe Sequence 50 –30 Specificity Reference
Probes designed for (putative) PAO:
PAO462 CCGTCATCTACWCAGGGTATTAAC Most Accumulibacter Crocetti et al. (2000)
PAO651 CCCTCTGCCAAACTCCAG Most Accumulibacter Crocetti et al. (2000)
PAO846 GTTAGCTACGGCACTAAAAGG Most Accumulibacter Crocetti et al. (2000)
RHC439 CNATTTCTTCCCCGCCGA Rhodocyclus/Accumulibacter Hesselmann et al. (1999b)
RHC175 TGCTCACAGAATATGCGG Most Rhodocyclaceae Hesselmann et al. (1999b)
PAO462b CCGTCATCTRCWCAGGGTATTAAC Most Accumulibacter Zilles et al. (2002b)
PAO846b GTTAGCTACGGYACTAAAAGG Most Accumulibacter Zilles et al. (2002b)
Actino-1011 TTGCGGGGCACCCATCTCT Tetrasphaera-related Actinobacteria Liu et al. (2001b)
Actino-221 CGCAGGTCCATCCCAGAC Tetrasphaera-related Actinobacteria Kong et al. (2005)
Actino-658 TCCGGTCTCCCCTACCAT Tetrasphaera-related Actinobacteria Kong et al. (2005)
BET135 ACGTTATCCCCCACTCAATGG Dechloromonas-related organisms Kong et al. (2007)
AMAR 839 CTGCGACACCGAACGGCAAGCC Amaricoccus spp. Maszenan et al. (2000)
MIC184 CATTCCTCAAGTCTGCC Micropruina glycogenica Kong et al. (2001)
Probes designed for (putative) GAO:
Gam1019 GGTTCCTTGCGGCACCTC Some Gammaproteobacteria Nielsen et al. (1999a)
Gam1278 ACGAGCGGCTTTTTGGGA Some Gammaproteobacteria Nielsen et al. (1999a)
The microbiology of phosphorus removal
GAOQ431 TCCCCGCCTAAAGGGCTT Some Competibacter Crocetti et al. (2002)

GAOQ989 TTCCCCGGATGTCAAGGC Some Competibacter Crocetti et al. (2002)
GB CGATCCTCTAGCCCACT Competibacter (GB group) Kong et al. (2002b)
GB_G1 (GAOQ989) TTCCCCGGATGTCAAGGC Some Competibacter Kong et al. (2002b)
(continued )
293
294
Probe Sequence 50 –30 Specificity Reference

GB_G2 TTCCCCAGATGTCAAGGC Some Competibacter Kong et al. (2002b)
GB_1 and 2 GGCTGACTGACCCATCC Some Competibacter Kong et al. (2002b)
GB_2 GGCATCGCTGCCCTCGTT Some Competibacter Kong et al. (2002b)
GB_3 CCACTCAAGTCCAGCCGT Some Competibacter Kong et al. (2002b)
GB_4 GGCTCCTTGCGGCACCGT Some Competibacter Kong et al. (2002b)
GB_5 CTAGGCGCCGAAGCGCCC Some Competibacter Kong et al. (2002b)
GB_6 (Gam1019) GGTTCCTTGCGGCACCTC Some Competibacter Kong et al. (2002b)
GB_7 CATCTCTGGACATTCCCC Some Competibacter Kong et al. (2002b)
TFO_DF218 GAAGCCTTTGCCCCTCAG Defluviicoccus-related organisms Wong et al. (2004)
(cluster 1)
TFO_DF618 GCCTCACTTGTCTAACCG Defluviicoccus-related organisms Wong et al. (2004)
(cluster 1)
TFO_DF862 AGCTAAGCTCCCCGACAT Defluviicoccus vanus Wong et al. (2004)
DF988 GATACGACGCCCATGTCAAGGG Defluviicoccus-related organisms Meyer et al. (2006)
(cluster 2)
DF1020 CCGGCCGAACCGACTCCC Defluviicoccus-related organisms Meyer et al. (2006)
(cluster 2)
TFO_DF629 AGGACTTTCACGCCTCAC Defluviicoccus-related organisms Wong and Liu (2007)
(cluster 2)
TFO_DF776 GCTATAGCGTCAGTTACGG Defluviicoccus-related organisms Wong and Liu (2007)

(cluster 1)
BET65 CAGTTGCCCCGCGTACCG Some Betaproteobacteria Kong et al. (2007)
GAM455 CTGACGTATTCGGCCAGTGC Some Gammaproteobacteria Kong et al. (2007)

Requires competitor or helper probes.
CANDIDATUS ‘ACCUMULIBACTER PHOSPHATIS’

Based primarily on culture–independent approaches, it is now widely accepted that one of the most
important organisms responsible for EBPR in acetate-fed sequencing batch reactors and many full-scale
municipal plants is a relative of the Rhodocyclus group in the Beta subclass of the Proteobacteria.
Hesselmann et al. (1999) first proposed a genus and species name for this still uncultured group of
organisms enriched in their EBPR bioreactor, Candidatus ‘Accumulibacter phosphatis’. In the literature,
this group has been called ‘Rhodocyclus-related PAO’ or ‘Accumulibacter’ or ‘CAP’. Here we use the
name Accumulibacter to circumscribe a coherent lineage comprised of multiple potential species.
A constructed phylogeny of this lineage is presented in Figure 10.6.
Identification and confirmation of Accumulibacter as a bona fide PAO

Accumulibacter-related organisms were identified by Bond et al. (1995) from the first application of 16S
rRNA gene-targeted PCR to EBPR communities. They compared the community structures of two
sludges, one removing Pi, and one removing minimal Pi. Both sludges were cultivated in anaerobic-
aerobic SBRs with settled full-scale sewage serving as the feed. However, the organic content of sewage
was too low to stimulate significant Pi-removal capacity. Thus, one SBR was supplemented with acetate to
encourage PAO growth. Community 16 rRNA genes were amplified from the two sludges using Bacteria-
specific primers and approximately 90 clones from each library were randomly chosen for partial
sequencing. Both libraries contained a large number of sequences affiliated with the Betaproteobacteria,
but the library from acetate-supplemented Pi-removing sludge contained substantially more betaproteo-
bacterial sequences. These were mainly affiliated with the Rhodocyclus group. While acknowledging the
quantitative limitations of PCR-based techniques (chapter 3), the authors hypothesized that members of
the Rhodocyclus group play an important role in P removal, since so many more clones of this group were
found in the acetate-supplemented sludge.
Rhodocyclus-like sequences were also obtained from similar laboratory scale SBRs by Hesselmann et al.
(1999). Reactors were operated with a 7 d MCRT (chapter 2) and fed mineral medium with acetate as carbon
source. Based on results of Bond et al. (1995), they designed PCR primers targeting Rhodocyclus-like
organisms, and used these to construct two PCR-based clone libraries. The primers retrieved two distinct
types of sequences, arbitrarily named R1 and R6. Five fold more R1 clones were obtained in the library than
R6. However, it was determined subsequently by FISH that the true abundance of R1 sequences was heavily
over-represented in these clone libraries, and that the R6 type actually dominated in the sludge. The R6
sequence was 99% identical to the Rhodocyclus-like clone sequence (SBRA220) obtained by Bond et al.
(1995). Hesselmann et al. (1999b) proposed a species name Candidatus ‘Accumulibacter phosphatis’ for this
Rhodocyclus-related PAO.
At about the same time that Hesselmann and colleagues were publishing their data, 16S rRNA clone
libraries were being constructed from acetate-fed EBPR sludges maintained in laboratory-scale SBRs
by three different groups working on three different continents: Australia (Crocetti et al., 2000), Asia
(Liu et al., 2001b), and North America (McMahon, 2002b). As with Bond et al. (1995), Bacteria- targeted
primers were used to amplify community 16S rRNA genes. In each case, a large number of library clones
were affiliated with the Rhodocyclus group, and their sequences were more than 97% similar to that of
clone SBRA220 (Bond et al., 1995) and clone R6 (Hesselmann et al., 1999b). FISH probes designed
against these (Table 10.1) were used to confirm the abundance of Accumulibacter-related cells in each
community, some of which were enriched in Accumulibacter up to 80% of all FISH probed Bacteria.
Crocetti et al. (2000) designed a set of Accumulibacter-targeted FISH probes (PAO462, PAO 651 and
PAO 846), which are referred to as the ‘PAOMIX’ probes (Table 10.1), and are still widely used. When
1000 bootstrap
Dechloromonas and Rhodocyclus as outgroups VIR D2 (EF565150)
79
DUR D8 (EF565149)
80 NAN E6 (EF565157)
VIR AI (EF565152) IIB
76 UCT NS 16(AY064186)
74 Orbal D51 (AF450474)
75 Orbal D32 (AF450466)
Orbal D42 (AF450469)
73
68 LVI A12 (EF565160)
70 LV2 H10 (EF565161)
71 69 LV1 A10 (EF565158) IIC
LV2 B12 (EF565159) (and IID)
72
UCT 032 (AY062125)
VIR D5 (EF565151)
67 UCB HP1 A49 (AF502229)
US_Metagenome (2000209150)
VIR H3 (EF565153)
49 VIR A12 (EF565155)
NAN A4 (EF565156)
UCB HP1 B75 (AF502227) IIA
UCB HP1 A26 (AF502231)
51
UWMH 4 (EF56147)
66 Oz_Metagenome (2000470820)
SBR B34 (AF204247)
UCB HP1 A31 (AF502230)
UWMH 9 (EF565148)
SBR A220 (AF204244)
UCB HP1 B23 (AF502225)
54 UCB HP1 A13 (AF502224)
65 Ebpr 15 (AF255641) I
UCT N173 (AY064179)
60 UCT N161 (AY064178)
Cand. Accumulibacter phosphatis (R6) (AJ224937)
UCT NS01 (AY064180)
Orbal D5 (AF450459)
64 Orbal D5 (AF450471)
VIR B4 (EF565154)
62 Orbal D41 (AF450468)
Orbal D54 (AF450476)
Rhodocyclus tenuis (D16209)
Dechloromonas agitatus (AF047462)
0.05
Figure 10.6. Unrooted maximum likelihood tree for Accumulibacter – related 16S rRNA sequences
constructed using GARLI (Genetic Algorithm for Rapid Likelihood Inference) Version 0.96. A total of 1022 bp
(E. coli 16S rRNA position 439 to 1459) were included for phylogenetic analysis. The bootstrap values
generated from 1000 bootstrap resamplings are shown next to the nodes. The original type strain (clone R6)
identified by Hesselmann et al. (1999) is included with the full proposed genus and species name. Sequence
names starting with LV, UCT, DUR, NAN, and VIR were recovered from full-scale plants as described in the
original publication (He et al., 2007a). Sequence names starting with ‘‘UCT’’ were recovered from plant NS
and those starting with ‘‘Orbal’’ were recovered from an Orbal plant (Zilles et al., 2002a). Sequence names
starting with UCB and UWMH were recovered from lab-scale SBRs in Berkeley, CA, USA, (McMahon et al.,
2002a) or in Madison, WI, USA (He et al., 2007a), respectively. Clade affiliations indicated to the right of the
tree were inferred upon comparison of the 16S rRNA phylogeny to that constructed using ppk1 (Figure 10.7),
which are congruent (He et al., 2007a). Scale bar indicates number of changes per site.
they applied this probe set they showed a clear linear relationship between Accumulibacter abundance and
sludge non-soluble P content. Co-localization of polyP granules and FISH-positive cells confirmed their
identity as PAO. Liu et al. (2001b) could also co-localize stained PHB with FISH positives cells. These
results reinforced evidence for the role of Accumulibacter in laboratory scale EBPR reactors, and provided
additional Accumulibacter 16S rRNA sequences for use in phylogenetic analyses and FISH-probe design.
Distribution patterns, population dynamics, and ecology

of Accumulibacter
Since their initial identification, many studies have explored the distribution and ecology of Accumulibacter-
related organisms in activated sludge, which also emerged as the dominant PAO in lab-scale acetate-fed
SBRs in other studies using several techniques (Dabert et al., 2001; Levantesi et al., 2002; McMahon et al.,
2002b; Onuki 2002; Zeng et al., 2003b). Thus, when Onuki et al. (2001) analyzed the community of a
lab-scale EBPR sludge using DGGE (chapter 11) over a two-month start-up period, the dominant band on
the DGGE gels at high EBPR capacity was closely related to members of the Rhodocyclus group after its
excision and sequencing. The relative abundance of Accumulibacter has also been assessed using the
PAOMIX probes and targeted FISH, in acetate-fed lab-scale SBRs effectively removing P, where these
usually dominate such communities, often ranging from 40 to 80% of total bacteria (Carvalho et al., 2007; He
et al., 2006; McMahon et al., 2002a; Oehmen et al., 2004; Zeng et al., 2003b). In fact its dominance has been
greater than 90% when the reactor was fed with alternating acetate and propionate supplies (Lu et al., 2006)
Accumulibacter also dominated an EBPR reactor fed solely with propionate, contributing as much as
55% (Pijuan et al., 2004) or 89% of total Bacteria (Carvalho et al., 2007). This lineage was also abundant
and important in anaerobic/anoxic lab-scale EBPR systems (Zeng et al., 2003a,b), aerobic granular sludge
that simultaneously removed N and P (Lemaire et al., 2008), and continuously aerated EBPR systems
(Ahn et al., 2007).
Although its role in P removal was now well established in lab-scale sludge fed with acetate or
propionate, abundance of Accumulibacter and its role in full-scale EBPR systems needed further study
to determine whether this group is responsible primarily for P removal in practice. Furthermore, there
may be regional difference in wastewater composition, climate, and preferred plant configuration and
operational conditions. Therefore, it was necessary to survey a range of full-scale EBPR systems and
map the distribution of Accumulibacter. In the USA, Zilles et al. (2002a,b) estimated these populations
and perhaps even more importantly their participation in P-removal in two full-scale EBPR WWTPs by
combining FISH, polyP staining and buoyant density centrifugation and cell recovery. In a UCT process
(chapter 2), Accumulibacter comprised 18% of the total bacterial biovolume, after FISH. With the
method developed in this study, their cells accumulating polyP (‘Rhodocyclus-related PAO’) were
estimated to be 20% of the total bacterial community, representing 73% of total PAO. In the other plant
operated as an aerobic-anoxic Orbal process, Accumulibacter accounted for only 13% of the biovolume.
However, the fraction participating in polyP accumulation was only 6% of total cells, representing 26%
of total PAOs. Zilles et al. (2002a,b) suggested that the fraction involved in P removal depended on the
type of treatment process applied. Another survey of five full-scale treatment plants in the USA using
the same approach applied by Zilles et al. (2002a) suggested that Accumulibacter were present in full-
scale EBPR facilities at levels ranging from 9 to 24% of total cells, and more than 80% had a high
polyP content, confirming their involvement in EBPR in these facilities (He et al., 2008). Elevated
abundance strongly correlated with systems without prefermentation, but with a high anaerobic volume
percentage, suggesting that Accumulibacter might have a higher affinity for VFAs, compared to other
possible PAO.
In Australia, Saunders et al. (2003) estimated that Accumulibacter were present in four full-scale plants
at levels ranging from 7–12% of total Bacteria. In contrast, another survey on nine full-scale EBPR plants
in the eastern states of Australia showed that very few Accumulibacter could be detected by FISH in most
of the plants examined, and even where present, not all these cells accumulated polyP (Beer et al., 2006).
Compared to other surveys where detailed plant operational information was provided, their SRT was
relatively much longer in most of the plants.
In Denmark, ten full-scale EBPR plants were studied by Kong et al. (2005) who found that they were
more abundant in domestic plants, ranging in relative abundance from 9 to 17%. They were present
at much lower levels (53%) in most industrial plants where the wastewater had a higher protein content,
suggesting its composition may be an important factor in determining PAO population composition
(Kong et al., 2005). In the Netherlands, a survey of seven full-scale EBPR plants indicated that
Accumulibacter abundances ranged from 5.7 to 16.4%, with an average of 9.2% of the total bacteria
(López-Vázquez et al., 2008) and its abundance was positively correlated with pH of the anaerobic reactor.
A survey of three Japanese full-scale EBPR treatment plants mainly treating municipal wastewater in
Tokyo showed that they accounted for 8–17% of total bacteria and accumulated polyP as indicated by
co-localization with FISH and polyP staining (Chua et al., 2006). However, another survey including nine
full-scale facilities (eight in Tokyo and one in Kawasaki), most treating mainly domestic wastewater,
showed that although Accumulibacter was more than 10% of the total Bacteria in most samples, a large
fraction contained little or no polyP (Wong et al., 2005). Taken together, these studies clearly demonstrate
the importance of Accumulibacter in full-scale EBPR processes. Differences in their abundance and
involvement in P removal suggest that further full-scale surveys are needed to explain their distribution in
plants with different configurations and operational conditions.
Ecophysiology of Accumulibacter
To study the in situ physiology of bacteria in EBPR communities, Liu et al. (2001b) applied 16S rRNA-
targeted FISH probes in combination with polyP and PHA staining on their acetate-fed EBPR sludge,
and observed that Accumulibacter accumulated both polyP and PHA, confirming their physiological traits
agreed with those proposed in the EBPR metabolic models. However, not all cells accumulate large amounts
of polyP at all times, especially in full-scale EBPR plant communities (Beer et al., 2006; Kong et al., 2004;
Wong et al., 2005; Zilles et al., 2002b), indicating a physiological heterogeneity among its members.
FISH-MAR (chapters 3, 11) is a powerful tool for linking phylogenetic identity with metabolic
functions in situ. With this approach, Kong et al. (2004) characterized the ecophysiology of
Accumulibacter in sludges collected from three full-scale EBPR plants in Denmark, and also showed
the metabolic behavior of this group was consistent with the biochemical models proposed for PAO.
Accumulibacter assimilated a range of organic substrates under anaerobic conditions including acetate,
propionate, pyruvate and glutamic acid, but not formate, ethanol, butyrate, glucose and several amino
acids tested, except for glutamic acid. These data suggest Accumulibacter assimilate a narrow range of
low molecular weight substrates, and other fermentation products provide potential ecological niches
for other non-Accumulibacter PAO to strive. Furthermore, Accumulibacter could use O2, NO3, and NO2
as electron acceptors for P uptake, confirming that these were the DPAO contributing to NO3 removal.
Population ecology of Accumulibacter

Most studies into the occurrence and phylogeny of Accumulibacter have relied on 16S rRNA as the
phylogenetic marker. A single bacterial species, defined as detailed in chapter 6, generally contains
several strains often with substantial phenotypic and genetic differences. With Accumulibacter, some of
their 16S rRNA sequences recovered from geographically and temporally distinct EBPR systems are
nearly identical (Garcia Martin et al., 2006). Whether these sequences represent essentially identical
Accumulibacter populations or different ecotypes possessing genotypic and/or phenotypic differences
among them and/or across EBPR systems is not easily resolved. The single-copy gene in Accumulibacter
(Garcia Martin et al., 2006) ppk1, encoding polyP kinase 1 appears to have evolved faster than 16S rRNA
genes (Kunin et al., 2008b), and thus provides greater phylogenetic resolution allowing finer-scale
differences within this lineage to be recognised.
Thus, McMahon et al. (2007b) designed and validated a ppk1 primer set based on their retrieved ppk1
fragments from an Accumulibacter-enriched lab-scale EBPR reactor (McMahon et al., 2002a) and several
full-scale plants together with degenerate PCR primers targeting ppk1 of most bacteria (McMahon et al.,
2007b). He et al. (2007a) also retrieved fragments of Accumulibacter 16S rRNA and ppk1 genes from a
lab-scale and several full-scale EBPR systems. Phylogenies reconstructed with 16S rRNA genes and ppk1
were largely congruent (Figures 10.6 and 10.7), with ppk1 providing higher phylogenetic resolution and
clearer tree topology. At least five ppk1 sequence clades were recovered within the lineage, and specific
ppk1 primer sets were then designed and qPCR used to to determine the relative distribution of these five
clades in lab-scale and full-scale EBPR communities. They found Accumulibacter subpopulation structure
varied among different EBPR systems and also temporally within a single system. They further suggested
that the Accumulibacter lineage is more diverse than previously realized, and postulated that different
clades within the lineage are ecologically distinct.
Peterson et al. (2008) have since shown that Accumulibacter also inhabit environments other than
activated sludge EBPR systems, including freshwater and estuarine sediments. They recovered several
new clades of Accumulibacter not previously detected in activated sludge, to give a total of 12 clades,
which clearly form two major groups, Type I and Type II (Figure 10.7). They also found habitat
characteristics were key factors in determining Accumulibacter distribution, and enrichment of particular
clades in EBPR communities seemed to impact on population distribution in the local environment,
providing evidence of ecological differences among the different subpopulations.
Besides ppk1, the 16S-23S internal transcribed spacer (ITS) region (He, 2006), and genes encoding
PHA synthase (phaC) (Wang et al., 2008a) have also been used as phylogenetic markers to study fine
differences among the Accumulibacter lineage. So far ppk1 would appear to provide the most suitable.
Species delineation with Accumulibacter

The problems of defining a bacterial species were discussed at length in chapter 6, and problems associated
with using 16S rRNA sequence similarity values to delineate individual species emphasized (Stackebrandt
and Ebers, 2006). Also discussed there was the ‘ecotype’ species concept of Cohan (2002) as an alternative
where each currently recognized species would contain many ecotypes, with each comprising a set of strains
occupying the same ecological niche. Cohan (2002) claims that recognizing ecotypes is more biologically
meaningful in studies in microbial ecology. With Accumulibacter, two Types, each comprising several clades
can be defined with ppk1 as phylogenetic marker (Figure 10.7), as discussed above. From the definition and
properties of an ecotype of Cohan (2002), each is expected to be identifiable as a sequence cluster, where
average sequence divergence between clusters is greater than the average sequence divergence within a cluster
(Cohan, 2002). These Accumulibacter clades meet this criterion and thus may represent putative individual
ecotypes. Possible differences in their ecophysiological traits have being tested (Carvalho et al., 2007;
Flowers et al., submitted), although whether these clades are truly ecologically distinct is still unclear.
Currently, basing Accumulibacter nomenclature on Type and clade using ppk1 sequence data is the best option
until a better classification is feasible from ecologically more meaningful evidence.
100 IIA
61
100
IIB
100
IID
100 Type II
IIG
100
IIE
100
93 IIF
100
IIC
100
93 IA
82
99 IB
100 Type I
64 IC
85 ID
100
100
IE
Rhodocyclus tenuis
0.10 changes per site
Figure 10.7. Maximum likelihood tree of the Accumulibacter lineage based on comparative analysis of
aligned ppk1 gene nucleotide sequences. The tree was constructed using an alignment with positions of less
than 50% maximum frequency masked, using GARLI with a general time reversible model and Gamma-
distributed among-site rate heterogeneity (Zwickl, 2006) to find the best tree and RAxML for 100 bootstrap
resamplings (Stamatakis et al., 2008). Two primary lines of descent in Accumulibacter, Types I and II, are
indicated by brackets. Monophyletic clades within these Types are shown as compressed wedges. Fully
expanded trees for each of these clades can be found elsewhere (Peterson et al., 2008). Notably, the sludges
from which the metagenome sequences were derived were enriched in clade IIA. Other lab-scale sludges
were enriched in clade IA (McMahon et al., 2002a) or IID (Wilmes et al., 2008b), but the population structure
within a bioreactor is also dynamic over time (He and McMahon, unpublished data). Surveys of EBPR
sludges (both lab scale and full scale) (He et al., 2007a; McMahon et al., 2007; Peterson et al., 2008) have
recovered members of all clades except ID, IE, and IIG.
The nitrate reduction conundrum

Accumulibacter is reported to use nitrate as electron acceptor and to achieve anoxic P uptake (Dabert et al.,
2001; Kong et al., 2004; Zeng et al., 2003b). However, the metagenomic analysis of Accumulibacter clade
IIA-enriched EBPR sludges indicated an absence of genes encoding a respiratory nitrate reductase (Garcia
Martin et al., 2006). With full-scale EBPR sludges when NO3 was provided as sole electron acceptor,
some but not all Accumulibacter could take up P, as determined by FISH-MAR (Kong et al., 2004). This
difference in NO3 reduction capability was also seen more clearly in two lab-scale reactors fed with
acetate and propionate respectively and dominated by two Accumulibacter populations with different
morphologies (Carvalho et al., 2007). The coccus morphotype predominated in the acetate SBR and
appeared unable to use NO3 as electron acceptor, while the rod morphotype was highly enriched in the
propionate SBR and could use NO3, NO2 and O2 for P uptake. However, this study did not distinguish
phylogenetically between these two subpopulations. When the capacity of Accumulibacter to use NO3 as
electron acceptor was investigated further using two lab-scale acetate-fed EBPR sludges each dominated
by a different clade (Flowers et al., submitted), the clade IA members could couple NO3 reduction with
P uptake, while those in clade IIA could not, suggesting marked physiological difference between
members of these different clades.
The Accumulibacter genome

Identification of PAO enables their populations to be tracked, but does not provide a direct link to their
polyP-accumulating function and P removal performance, Thus their functional attributes need to be
known to understand how the biochemistry of EBPR is regulated. Because key metabolic features are
likely to be shared by potentially phylogenetically diverse PAO groups, it has been suggested that
common key genes involved in EBPR metabolism may be found among them. Therefore it is important to
identify which key genes these are and to study their genetic and enzymatic regulation (Mino, 2000).
However, little has been known until recently about the enzymes and functional genes involved in EBPR
from a lack of PAO pure cultures.
A revolutionary advance in understanding EBPR microbiology has been achieved through
metagenomic sequencing (Mino and Satoh, 2006). High-throughput shotgun sequencing technique and
powerful bioinformatics tools have made genome sequencing of members of mixed natural microbial
communities possible. The US Department of Energy’s Joint Genome Institute (DEO-JGI) carried
out metagenome sequencing of two Accumulibacter-dominated lab-scale EBPR sludges, one from
Wisconsin, USA (US) and the other from Queensland, Australia (OZ) (Garcia Martin et al., 2006). At the
time of sampling, the SBRs cultivating the sludges were enriched with ,80% (US) and ,60% (OZ)
Accumulibacter species as determined by FISH. Approximately 98 and 78 Mbp of shotgun sequence data
were obtained from the US and OZ sludges respectively, constituting nearly complete coverage of the
Accumulibacter strains present in the two communities. These genomes had an average content of
63 mol% GþC and an average read depth of 8X and 5X respectively for the US and OZ assemblies.
Metagenomic analyses often include ‘binning’ sequence reads or assembled contigs into phylogenetic
groups. The largest contigs and scaffolds could be reliably binned as Accumulibacter clade IIA members,
suggesting that this clade was dominant in both reactors at the time samples were collected. However,
representatives of clade IA were also present at low abundance (based on genome sequence coverage)
with genomes up to 15% divergent at the nucleotide level compared to the clade IIA genome. It was not
possible to reliably bin genomic fragments from clade IA or other Accumulibacter strains present because
the low sequence coverage did not allow for appreciable assembly of the shotgun reads. A total of thirteen
16S rRNA phylotypes were identified in the assembled reads, with only Accumulibacter overlapping
between the two sludges. Thus, the flanking community constituted a distinct and diverse assemblage,
comprised of members affiliated with the Xanthomonadales, Flavobacteriales, and Rhizobiales. The
significance of the flanking community composition to Accumulibacter ecology or EBPR performance is
unknown but should be a fruitful topic of future research.
One especially surprising result of the metagenomic analysis was the finding that clade IIA genomes
from the US and OZ reactors were more than 95% identical to each other at the nucleotide level over
79% of the reconstructed US genome. This level of sequence conservation was unexpected because
of the geographic distance between the two source reactors (Garcia Martin et al., 2006; Kunin et al.,
2008b). To explain this, the authors suggested that Accumulibacter is relatively rapidly dispersed around
the world, with full scale EBPR wastewater treatment plants being sparse high-density point sources
linked by dispersal through widespread diffuse reservoirs like aquatic environments (Kunin et al., 2008b;
Peterson et al., 2008).
In 2008, the DEO-JGI finished the genome of the US Accumulibacter strain with additional
sequencing, assembly, and manually curated annotation (McMahon, unpublished data). The strain was
named Candidatus ‘Accumulibacter phosphatis’ clade IIA str. UW-1 and its finished genome sequence
was deposited in publicly available databases including GenBank, the Genomes Online Database
(GOLD), and JGI’s Integrated Microbial Genomes database (IMG) (see Figures 10.8 and 10.9, see
colour image section (chapter 13)). The final genome sequence includes a circular 5.06 Mbp
chromosome and three plasmids (168 kbp, 42 kbp, and 38 kbp). This genome sequence contains 4,620
predicted genes, among which, 3,337 of the proteins have known functions, while for the remainder,
their functions are unknown. Curiously, one plasmid encodes 156 predicted features, none of which
have a predicted function. It is hoped to improve the genome annotation using post-genomic analyses of
gene expression.
Metabolic reconstruction
The original unfinished assembly of the US and OZ Accumulibacter genomes allowed for a near complete
metabolic reconstruction of the biochemical pathways involved in EBPR (Garcia Martin et al., 2006).
This analysis seemed to resolve several long-standing conflicts regarding EBPR metabolism, at least for
clade IIA strain UW-1. The extent to which other Accumulibacter clades/strains/populations exhibit
genome differences that map to physiological differences relevant to EBPR process design and
performance is currently a mystery.
A gene-centric analysis was first conducted in comparison with other metagenomic datasets available
at the time to identify over-represented gene families. Many gene families predicted to be important in
EBPR were over-represented in the sequence datasets (Figures 10.8 and 10.9). These included genes
encoding for proteins involved in phosphate transport (components of both low and high affinity
transporters), VFA handling (symporters and PHA synthetases), anaerobic operation of the TCA cycle,
and cobalt uptake. Some over-represented gene families have no obvious fit in the current metabolic
models. These include 11 families annotated as Ca2þ-binding proteins related to RTX toxins (repeat
toxins). The authors speculated that they are part of the extracellular capsular polymeric substance (EPS)
(chapter 3), given the importance of divalent cations to EPS and flocculation. Other interesting
gene families include iron transporters and those involved in DNA exchange. About 15% of these
overrepresented families have no predicted function, suggesting that many functionally important genes in
EBPR remain to be characterized.
The reconstruction yielded a new map of EBPR metabolism that includes many of the elements
proposed in the Comeau-Wentzel and Mino models described earlier (Figures 10.8 and 10.9). Genes
encoding enzymes required for phosphate transport, synthesis and degradation of the three key
biopolymers (polyP, PHA, and glycogen), central metabolism (e.g. the TCA cycle), energy generation,
exocellular polymeric substance (EPS) synthesis, and basic cell maintenance and replication could be
identified. A detailed description of the most salient genes follows.
The metagenomic analysis also provided clues about the possible life style of Accumulibacter outside
EBPR systems. The surprising findings include a full complement of genes encoding for components
involved in nitrogen fixation and many of the key genes for CO2 fixation, both of which are energetically
expensive and unlikely to be expressed in EBPR systems where both C and N are plentiful. Genes
presumably involved in flagellar biosynthesis were also identified, though no flagella have been observed
for this organism in EBPR. Perhaps most importantly, the recovery of an Accumulibacter genome makes
possible functional studies at the levels of gene transcription [(meta)transcriptomics] and translation
[(meta)proteomics], at a genome or even at a community scale, both of which are certain to revolutionize
our understanding of EBPR microbiology.
Carbon metabolism in Accumulibacter

Genes were identified encoding for enzymes probably involved in anaerobic acetate uptake and activation
(e.g. acetate permease (ActP), acetyl-CoA synthetase (Acs), acetate kinase (AckA), and phosphotransa-
cetylase (Pta)). Propionate is likely to be assimilated through the same transporter and activated via a
propinyl-CoA synthetase (PrpE). In E. coli, acetate is transported by the ActP permease encoded by gene
yjcG, which is co-transcribed with acs in the same sequence cluster (Gimenez et al., 2003). This process is
driven by a transmembrane electrochemical potential, generated by Hþ or Naþ. Acetate can be activated
to form acetyl-CoA irreversibly through the high-affinity Acs (Kumari et al., 1995), or reversibly with
the low-affinity acetate kinase/phosphotransacetylase (AckA/Pta) pathway (Kakuda et al., 1994).
In Accumulibacter, a homolog of yjcG is located with acs. Experiments with the protonophore carbonyl
cyanide m-chlorophenylhydrazone (CCCP), a PMF uncoupler, suggest that acetate transport was occurring
through a permease mediated process energized by the PMF (Saunders et al., 2007) generated by phosphate
efflux in symport with Hþ through the Pit transporter (Burow et al., 2008a).
As described earlier, the routes used for glycogen degradation in PAO have been topics of heated
debate, mainly because conflicting results have been obtained experimentally and from mathematical
modelling. The controversy is important because it impacts on the cellular energy budget, with the EMP
pathway yielding more ATP (Hesselmann et al., 2000). All genes encoding enzymes for the EMP pathway
were recognized in the Accumulibacter clade IIA genome (Garcia Martin et al., 2006). In contrast, several
key genes encoding enzymes unique to the ED pathway were absent, including those encoding
6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase. Other enzymes
typically feeding into the ED pathway were also not seen in Accumulibacter clade IIA (e.g. gluco-
6-phosphate dehydrogenase). A more careful analysis of the finished genome confirmed that these genes
were absent (McMahon, unpublished data).
Also as described earlier, the source of reducing power for PHA synthesis in the anaerobic phase has
been controversial. The amount of glycogen degraded is generally insufficient to explain the observed
level of PHA produced in acetate-fed systems (Schuler and Jenkins, 2003a,b). The original Comeau-
Wentzel model included anaerobic operation of the TCA cycle for NAD(P)H generation, but no
explanation was given for how reduced quinones produced by succinate dehydrogenase were recycled in
the absence of external electron acceptors. A novel cytochrome b/b6 was identified during metagenomic
analysis, and seemed to be a fusion of a cytochrome b/b6 with five transmembrane helices and a soluble
NAD(P)- and flavin-binding domain. Garcia Martin et al. (2006) suggested that this fusion protein
functions in reverse as a quinol-NAD(P) reductase, fueled by the PMF. However, they also note
that operation of a split TCA cycle is plausible since a fumarate reductase gene was identified in the
metagenome.
Phosphate metabolism
Genes predicted to be involved in polyP manipulation and storage were identified in the Accumulibacter
clade IIA genome, including those encoding polyphosphate kinase 1 (ppk1), polyphosphate kinase 2
(ppk2), exopolyphosphatase (ppx), polyphosphate AMP phosphotransferase (pap), adenylate kinase (adk),
a membrane bound proton-pumping pyrophosphatase, high affinity phosphate specific transporter (Pst),
and a low affinity phosphate transporter (Pit) (Figures 10.8. and 10.9).
Many model organisms including E. coli and Pseudomonas aeruginosa use polyphosphate kinase 1,
polyphosphate AMP phosphotransferase, and adenylate kinase to synthesize and degrade polyP
(Kornberg, 1999). Consequently it is not surprising to find these in the Accumulibacter genome.
Polyphosphate kinase 1 can be used to synthesize or degrade polyP while consuming or generating
respectively ATP directly. It is interesting that in Accumulibacter clade IIA the genes for adenylate kinase
and membrane bound pyrophosphatase genes occur next to each other on the genome, suggesting that
if polyP degradation occurs via pap and adk, ATP production is linked to maintenance of the PMF
(Garcia Martin et al., 2006). Exopolyphosphatase can also degrade polyP in a non-reversible reaction that
does not generate ATP directly, and polyphosphate kinase 2 degrades polyP producing GTP from GDP.
As observed in an Accumulibacter-enriched sludge cultivated in California (McMahon, unpublished data)

(see also Figure 10.4), the ppk1 and ppx genes were located in a cluster along with relA. While ppx and
ppk1 are often found next to each other in bacterial genomes, the cluster of ppx-ppk-relA has been
reported so far in only one other sequenced genome (Azoarcus sp. EbN1).
As described earlier, the synthase encoded by relA produces the intracellular ‘‘alarmone’’ guanosine
30 ,50 -bisdiphosphate (ppGpp) which initiates global changes in RNA expression via the stringent
response (Cashel et al., 1996). Interestingly, E. coli mutants lacking relA do accumulate polyP under
certain conditions (Ault-Riche et al., 1998), and E. coli engineered to produce large quantities of
(p)ppGpp produced massive amounts of polyP (Kuroda et al., 1997). It is thought that (p)ppGpp inhibits
polyphosphatase by binding to the enzyme, thus preventing polyP breakdown. Additional links between
polyP metabolism, amino acid starvation, and ribosomal protein degradation have also been reported
(Kuroda et al., 2001). The proximity of relA to ppk and ppx in the Accumulibacter genome suggests a
similar role for ‘‘magic spot’’ in EBPR metabolism, although the exact mechanism probably involves
a complex regulatory network also involving RpoS (Kornberg, 1999).
In model organisms like E coli, two major phosphate transporters exist, the high affinity phosphate
specific (Pst) and the low affinity inorganic phosphate (Pit) systems (van Veen, 1997). The Pst system
belongs to the ATP binding cassette (ABC) transporter superfamily. It is composed of two integral
membrane proteins (PstA and PstC) that form a transport channel, a membrane-associated protein dimer
(PstB) that has an ATP binding motif and acts as ATPase to provide energy for active transport and a
periplasmic substrate binding protein (PstS) that binds to phosphate in the periplasm and shepherds it to
the transporters in the cytoplasmic membrane. The Pit system consists of a single transmembrane protein,
and functions as a symporter of Hþ and phosphate, and usually transports metal-neutralized phosphate
rather than ionic species of HPO24 .
In Accumulibacter, two phosphate permeases in the Pit system are located in the same gene cluster
(Garcia Martin et al., 2006). Such a tandem set of Pit genes is very uncommon and reflect an adaptation
of Accumulibacter to the EBPR environment, and also provides it with the ability to transport phosphate
effectively. Accumulibacter also has three highly conserved clusters of Pst genes (Garcia Martin et al.,
2006). It was postulated from the genome sequence that the Pit system is ready to use under high Pi
conditions, while the Pst system is suppressed most of the time during EBPR operation.
The imprint of phage

Some of the largest sequence-level differences between the US and OZ Accumulibacter genomes were
associated with phage-related genomic inserts, particularly CRISPR elements (Kunin et al., 2008b).
CRISPRs are hypothesized to be linked to phage insertions that confer resistance to phage based
on foreign DNA ‘‘spacers’’ within the clustered interspaced short palindromic repeats (CRISPRs).
These rapidly evolving segments of the genome may confer resistance to local phages within a genomic
background that seems remarkably homogeneous across spatially separate EBPR systems. The
implications of phage pressure on EBPR communities are substantial. Kunin et al. (2008b) hypothesize
that phage predation creates a ‘‘kill-the-winner’’ situation in which dominant Accumulibacter strains are
rapidly wiped out and replaced by previously rare strains. Occasional community collapse could result if
a large diversity of flanking Accumulibacter strains were not present. This proposal is in conflict with
common engineering objectives of selecting for ‘‘the best PAO’’ since a low biodiversity in this situation
would lead to lack of system robustness (McMahon et al., 2007a). There is clearly an urgent need to
better understand the fundamental principles governing activated sludge community assembly, resistance,
and resilience.
Post-genomic insights
Although the metagenomic sequences provide a considerable amount of information about the metabolic
capabilities of Accumulibacter (Figures 10.8 and 10.9), post-genomic studies looking at gene expression
may confirm which of the predicted pathways are those actually involved in EBPR. Wilmes et al.
(2008a,b) have conducted shotgun metaproteomics analyses on Accumulibacter-enriched EBPR sludge,
using 2D-PAGE combined with mass spectrometry (MS) analysis, and liquid chromatography and
tandem mass spectrometry (LC-MS/MS). These two studies have identified many of the in situ expressed
proteins including enzymes involved in key metabolic pathways, including PHB synthesis, glycolysis,
gluconeogenesis, and glycogen synthesis. Furthermore, the authors suggested a contribution from fatty
acid metabolism and the glyoxylate bypass in EBPR. Comparisons of the protein profiles of EBPR
communities under anaerobic and aerobic conditions, revealed a strong similarity between them.
Contrary to the metagenome analysis (Garcia Martin et al., 2006), Wilmes et al. (2008a) suggest
that Accumulibacter is capable of denitrification, with the expression of a periplasmic nitrate reductase
(NAP) subunit encoded by a napC/nirT homolog (IMG/M OID 2000207840). The highest BLAST match
has an 80% amino acid similarity to the predicted napC/nirT [YP_285780] of the chlorite dismutase
operon in the Dechloromonas aromatica RCB genome on a contig reliably binned as Accumulibacter.
However, the gene product is more likely to be a component of nitrite reductase because an un-annotated
open reading frame in the same Accumulibacter operon (IMG/M OID 2000207830) shares 64%
similarity with nirB, a gene encoding a biochemically verified diheme cytochrome c551 (Genbank
Accession number P83170) in Rhodocyclus tenuis (which does not denitrify) (Devreese et al., 2000).
NAPs generally do not couple nitrate reduction to proton motive force generation, although they do
employ nitrate as an electron sink for growth on reduced substrates (Gonzalez et al., 2006). In any case,
it is not possible to confirm whether the expressed napC/nirT homolog observed by Wilmes et al.
(2008a,b) was involved in nitrate or nitrite reduction.
Overall, analysis of the data from their study proved to be difficult because the Accumulibacter culture
available was affiliated with a clade other than IIA. Instead genotyping based on ppk1 suggested members
of clade IID and an unidentified Type I clade were present. Although we do not know yet the extent of
genomic sequence variation among clade members, it is possible that many of their proteins had masses
different to those predicted from their the gene sequences, making protein identification difficult. This
might explain also the difficulties encountered by Wilmes et al. (2008a,b) in assigning their detected
proteins to a phylogenetic bin.
Gene expression can also be detected by targeting gene transcripts (mRNA) as used by Burow et al.
(2008b), where expression of several genes involved in anaerobic carbon metabolism in EBPR was
investigated using reverse-transcriptase quantitative RT-qPCR. They confirmed the expression of
isocitrate lyase (ICL) in the glyoxylate shunt, aconitase (acn) in the TCA cycle, succinate dehydrogenase
(SDH) and the proposed cytochrome b/b6 fusion protein (see also Figure 10.8). A functional glyoxylate
shunt was further confirmed by inhibitors of aconitase (fluoroacetate) and ICL (3-nitropropionate and
itaconate), which caused a marked decrease in acetate uptake and PHA production. Interestingly,
abundances of ICL and acn transcripts increased under aerobic conditions. Burow et al. (2008a,b)
suggested on this evidence that the TCA cycle probably operates together with the glyoxylate cycle
aerobically (bypassing the decarboxylation steps), thereby carefully regulating the balance between
carbon used for energy and growth and glycogen replenishment.
Despite their preliminary nature, these data provide direct evidence that links the metabolism of a defined
population to the chemical transformations they carry out. Therefore, integrating cultivation-independent
metagenomics with metaproteomics (and metatranscriptomics) is very powerful in functional analysis
of these biotechnologically important but complex ecosystems involving uncultured bacterial groups.
OTHER PAO
Despite being the most commonly reported PAO, Accumulibacter cell numbers do not account for
all the PAO observed in lab-scale and especially full-scale EBPR systems. For example, when Lee et al.
(2002a) applied FISH-MAR to a pilot-scale EBPR community, they found considerable 33Pi uptake
by the Proteobacteria (some related to Rhodocyclus, but the identity of the rest unknown) and the
Actinobacteria. Not all the Accumulibacter showed 33Pi uptake. No statistically significant correlation
was observed between Accumulibacter relative abundances, or any other investigated bacterial groups,
and the P content of the activated sludge. Thus, P removal in these systems was believed to be
performed by a mixed population consisting of Accumulibacter and other unidentified bacteria. Futher-
more even in surveyed treatment plants containing abundant Accumulibacter, often this group only
accounted for a small fraction of the total PAO (He et al., 2008; Kong et al., 2004; Zilles et al., 2002b),
and some Accumulibacter appeared incapable of accumulating polyP (Kong et al., 2004; Wong et al.,
2005). Clearly, searching for other putative PAO populations was required.
Actinobacterial PAO
Several putative PAO belonging to the phylum Actinobacteria have been cultured, but there is no strong
evidence suggesting their physiology is that required by the EBPR metabolic models. Nevertheless,
Actinobacteria have been suggested as putative PAO from data obtained from culture-independent
approaches. Community structure analyses in a full-scale EBPR plant using 23S rRNA-targeted FISH
have revealed that high mol% GþC Gram-positive Actinobacteria targeted by probe HGC69a can
dominate EBPR systems, and the presence of polyP in these bacteria indicate that they might play a role in
P removal (Wagner et al., 1994c; Kampfer et al., 1996). Similar studies with an acetate-fed, continuous
lab-scale EBPR sludge revealed the predominating clones (31% of the sequenced clones) in the 16S rRNA
gene clone library were also Actinobacteria (Christensson et al., 1998). However, relative abundances
derived from PCR-based techniques, like clone library analysis, should be viewed cautiously (chapters 3,
11) because of potential biases associated with DNA extraction and PCR amplification (Reysenbach et al.,
1992; von Wintzingerode et al., 1997).
Nevertheless, when comparing the community compositions of ‘efficient’ and ‘non-efficient’ EBPR
sludge by FISH, Bond et al. (1999a) found that Actinobacteria were the second largest group, behind the
subclass-2 Betaproteobacteria in the former community. At the same time, these bacteria were reported
as the dominant populations with high polyP content after combined FISH and polyP staining with DAPI
(Kawaharasaki et al., 1999). In a detailed characterisation of a lab-scale EBPR sludge community,
Liu et al. (2001b) constructed a 16S rRNA clone library, and showed that as well as Accumulibacter, a
large number of sequences related to the Actinobacteria were also present. Among these, two affiliated
with members of the genus Tetrasphaera, an organism earlier isolated from EBPR sludge and suggested
to be a PAO (Maszenan et al., 2000). They designed a 16S rRNA based probe Actino_1011 to target these
Tetrasphaera-related Actinobacteria and with FISH analysis combined with polyP and PHA staining,
showed that these were the second most abundant group. Furthermore these accumulated polyP but not
PHA aerobically (Liu et al., 2001b). Applying the same probe Actino_1011, revealed that these
Tetrasphaera-related Actinobacteria also dominated the PAO community in other pilot-scale EBPR
systems (Eschenhagen et al., 2003).
Applying FISH-MAR with 33Pi to ten full-scale EBPR plants in Denmark, Kong et al. (2005) found
that in addition to Accumulibacter two morphologically different cells (cocci in clusters of tetrads (TFO)
and short rods in clumps) hybridized with the probe Actino_1011 targeting Tetrasphaera-related
Actinobacteria were also actively involved in P uptake (Figure 10.10). After constructing 16S rRNA clone
libraries, they designed two probes, Actino-658 and Actino-221 to target the short-rod and coccus
Tetrasphaera-related morphotypes respectively, and showed that both were common in all surveyed
plants. Further FISH-MAR analysis revealed that both morphotypes assimilated organic substrate
anaerobically and Pi aerobically, confirming their PAO phenotype. However, they only utilized amino
acids, and not short-chain fatty acids like acetate, or glucose or ethanol, thus exhibiting a narrower
substrate utilization pattern than Accumulibacter. Furthermore, these actinobacterial PAO did not
accumulate PHA anaerobically, and the glycolytic pathway seemed important in anaerobic substrate
uptake, suggesting that unidentified storage compound(s) other than PHA and glycogen play a role in
their carbon and energy transformations. Evidence was provided to show that actinobacterial PAO use
both O2 and NO3, but not NO2 as electron acceptors for growth and P uptake.
99
55 Uncultured actinobacteria (8 clones), (AY710272)
60 Lochheadia duodecas, (AB072496)
Uncultured actinobacterium (AF387312)
58 Tetrasphaera elongata, (AB030911)
99 Tetrasphaera sp. Ellin115, (AF408957)
65 Tetrasphaera sp. MSFC 3-M5-R-7, (EF028236)
93 Tetrasphaera japonica, (AF125092)
Uncultured actinobacteria (5 clones), (AY710276)
50
99 Tetrasphaera australiensis, (AF125091)
69 Tetrasphaera australiensis, (AF125090)
Tetrasphaera nostocoidensis, (DQ007320)
96 Candidatus Nostocoida limicola, (X85212)
99 Candidatus Nostocoida limicola, (X85211)
81 Tetrasphaera jenkinsii (DQ007321)
58 Candidatus Nostocoida limicola, (Y14597)
Tetrasphaera jenkinsii (DQ007319)
Candidatus Nostocoida limicola, (Y14596)
0.02
Figure 10.10. Distance matrix tree of the genus Tetrasphaera and related bacteria in the Actinobacteria
where putative PAOs may be present. The tree is based on comparative analysis of aligned 16S rRNA gene
nucleotide sequences, and was calculated using the neighbour-joining method with a Poisson correction
model and a 100 replicate bootstrap analysis. A bootstrap consensus tree was calculated and average
bootstrap values are included on the trees. The two clades representing uncultured Actinobacteria may be
PAOs (Kong et al., 2005). Most sequences from uncultured Actinobacteria are from Kong et al. (2005). Tree
constructed by H. Nguyen and P. H. Nielsen.
These physiological differences between Actinobacteria- and Accumulibacter-related PAO would

allow them to occupy different ecological niches, to exhibit different distributions and play roles of
different importance in EBPR systems. Indeed, among the ten activated sludge plants surveyed in
Denmark (Kong et al., 2005), the Actinobacteria-related PAOs accounted for 3–16% of the total Bacteria
in those treating domestic wastes, and 17–35% in industrial plants, where the influent wastewater had
higher protein levels. In these industrial plants, their abundance was much higher than that of
Accumulibacter, consistent with the observation that actinobacterial PAO assimilate amino acids. Thus
organic carbon substrate composition might be a key factor in selecting which PAO populations dominate.
The presence and importance of actinobacterial PAO was also demonstrated in nine full-scale EBPR
facilities surveyed in Australia (Beer et al., 2006). Actinobacteria hybridizing with probe HGC69a
accounted for 7–35% of total Bacteria in these systems, and more than 50% of these Bacteria contained
polyP and accounted for most of polyP positive cells (PAO) seen. As observed by Liu et al. (2001b) and
Kong et al. (2005), these Actinobacteria did not accumulate PHA. Most did not respond to probes
Actino-1101, Actino-221 or Actino-658, designed against the Actinobacteria-related PAO, suggesting
further actinobacterial PAO members probably exist.
Dechloromonas-related PAO
In the same survey of full-scale EBPR plants in Australia (Beer et al., 2006), some Betaproteobacteria
also contained polyP after DAPI staining, while most cells targeted by probes for Accumulibacter did not.
Therefore, Betaproteobacteria not affiliated with Accumulibacter behave as PAO in these communities.
In a full-scale EBPR treatment plant operated as an aerated-anoxic Orbal process, Zilles et al. (2002b)
found that while Accumulibacter accounted for 6% of the total Bacteria, they were only 26% of the
total PAO community. Clone libraries generated from this plant contained Accumulibacter-related 16S
rRNA genes, but also sequences closely related to Dechloromonas agitatus, a distantly related organism
that is still clustered within the larger ‘‘Rhodocyclus group’’. The authors used their phylogenetic tree
to define a ‘‘Rhodocyclus tenuis subgroup’’ (Accumulibacter) and a ‘Dechloromonas agitatus subgroup’
to distinguish between these two sequence types (see also Figure 10.6). To further identify which
non-Accumulibacter PAOs played more important roles in P removal in such a system, a lab-scale EBPR
reactor was operated at a low dissolved oxygen concentration in the anaerobic phase to simulate the Orbal
process (Goel et al., 2005). The reactor achieved good P removal under such conditions. By applying
full-cycle rRNA analysis, they showed that instead of the Accumulibacter group, Dechloromonas sp. was
enriched under such operational conditions. In Denmark, Kong et al. (2007) conducted a detailed study of
PAO community structure and function in a full-scale EBPR plant sample, by combining full-cycle
rRNA approach, chemical staining and microautoradiography. They found that bacteria affiliated with
Dechloromonas could take up short chain fatty acids like acetate anaerobically and accumulate PHA and
polyP, thus exhibiting PAO phenotype.
However, the community for a lab-scale SBR operated under continuously aerobic conditions
contained Dechloromonas-related bacteria which assimilated acetate and accumulated intracellular PHA
in the feed stage, but did not accumulate polyP in the famine stage (Ahn et al., 2007). It seems possible
that Dechloromonas spp. are capable of behaving as PAO under some conditions. They are closely related
phylogenetically to Accumulibacter, and may share physiological traits in common. Just as some
Accumulibacter do not accumulate polyP under some conditions, some Dechloromonas-related PAO may
not always demonstrate the PAO phenotype. Nevertheless, their importance and role generally in EBPR
systems needs further investigation.
In the past decade, with molecular tools available to investigate the microbial community structures,
the high microbial diversity of EBPR communities has been demonstrated repeatedly (Seviour et al.,
2003). It seems more likely that PAO may consist of several phylogenetically and taxonomically diverse
bacterial groups, as Mino (1998) had suggested, and PAO community members may vary among different
treatment systems.
OTHER EBPR COMMUNITY MEMBERS

It is also important to identify the other (flanking) community members in EBPR microbial communities,
since they may also perform important roles in P removal.
In lab-scale reactors fed synthetic wastewater containing usually acetate or propionate, 16S rRNA gene
clone libraries and/or FISH frequently detect other major flanking members of the Alphaproteobacteria,
Gammaproteobacteria, other Betaproteobacteria, planctomycete group and Bacteroidetes (CFB) group
(Bond et al., 1995; Crocetti et al., 2000; Kawaharasaki et al., 1999; Liu et al., 2001b). Among these, the
Bacteroidetes comprised half of all the clones in one library, although FISH data suggested that this was a
PCR-generated artefact (Liu et al., 2001b). It should be mentioned that the Gammaproteobacteria
detected by Liu et al. (2001b) accumulated both polyP and PHA, as did the Alphaproteobacteria detected
by Kawaharasaki et al. (2001). Unfortunately these features were not pursued.
In both pilot-scale (Eschenhagen et al., 2003) and full-scale EBPR communities (Beer et al., 2006;
Kong et al., 2007), commonly FISH-detected flanking members have again included members of
the Alphaproteobacteria, Gammaproteobacteria, other Betaproteobacteria and Bacteroidetes (CFB)
group. In particular, Bacteroidetes comprised 10–22% of the total bacteria in 11 EBPR plants in Denmark
(Kong et al., 2007). As major populations in these communities, it was postulated that they had a important
function in EBPR, being responsible for protein hydrolysis (Xia et al., 2007).
Not all these flanking members contribute positively to EBPR. Some compete with PAO for organic
carbon substrates in the feed stage, but do not contribute to P removal in the famine stage, thus negatively
affecting EBPR capacity. In particular, some Alphaproteobacteria and Gammaproteobacteria frequently
detected in EBPR systems at high numbers may function as glycogen accumulating non-polyP organisms
(GAO) discussed below and be responsible for EBPR failure.
GLYCOGEN ACCUMULATING NON-POLYP ORGANISMS (GAO)

Glycogen accumulating non-polyP organisms (GAO) are a group of functionally and numerically
important bacteria found in EBPR communities. Like PAO, GAO take up organic substrates anaerobically
and store these as PHA, which are subsequently used under aerobic or anoxic conditions for
energy generation and growth. However, in contrast to PAO, glycogen degradation is the only source of
energy for anaerobic organic substrate uptake by GAO. Hence, GAO do not degrade polyP anaerobically,
nor do they accumulate it under aerobic or anoxic conditions. Thus, GAO compete with the PAO for
organic carbon substrates since they grow under identical operational conditions (i.e. anaerobic followed
by aerobic or anoxic conditions), without contributing to P removal. Consequently, GAO are widely
considered to be a bacterial group detrimental to EBPR systems.
Historically, GAO were originally detected as a morphologically distinct group of bacteria in
EBPR systems with deteriorated performance and were referred to as ‘‘G-bacteria’’. It was reported that
some EBPR failures occurred even under well controlled operational conditions in the laboratory (Fukase
et al., 1985; Cech and Hartman, 1990), where anaerobic organic substrate uptake was not coupled to
P release. A group of bacteria with coccoid cells in the form of tetrads (tetrad forming organisms, TFO)
were enriched in a reactor fed with a mixture of acetate and glucose (Cech and Hartman, 1990; 1993).
This enriched culture could take up substrates anaerobically and proliferate under alternating anaerobic
and aerobic conditions without any polyP accumulation. This group of TFO was tentatively named
‘G-bacteria’ (Cech and Hartman, 1993).
When glycolysis was inhibited by iodoacetate in a ‘G-bacteria’ dominated sludge, a decrease in acetate
uptake was observed. Hence Liu et al. (1994) proposed that glycogen accumulation and its degradation
through glycolysis was providing the energy and reducing power for these ‘G-bacteria’ to assimilate
organic substrates anaerobically. Therefore, the ‘G-bacteria’ were henceforth referred to as glycogen
accumulating organisms (GAO) (Mino et al., 1995). The definition of ‘G-bacteria’ is based on
morphology as distinct TFO, while the definition for GAO is phenotype-based. Morphological
identification can be problematic and sometimes misleading, since bacteria exhibiting the GAO
phenotype without the typical tetrad morphologies have been detected, and not all TFO are necessarily
capable of competing with PAO for anaerobic substrate uptake. Therefore, GAO defined on their
physiological traits of glycogen accumulation without polyP accumulation is more appropriate in

discussions of EBPR processes, and this term has since been more widely adopted.
GAO phylogeny (Figure 10.11)

Culture-Dependent Approaches
As with the PAO, culture-dependent methods have also been employed in attempts to identify these
GAO, where the morphologically distinct TFO have been primary targets with this approach. Isolates of
the same ‘G-bacteria’ observed by Cech and Hartman (1993) were identified as Amaricoccus spp.
(Maszenan et al., 1997), although in subsequent study, these were unable to assimilate either glucose or
acetate under anaerobic conditions (Falvo et al., 2001), and have not been observed by FISH analysis
in other poorly performing EBPR systems (Seviour et al., 2003). Most other isolated TFO have also not
been detected in lab-scale or full-scale EBPR processes, with the exception of Micropruina glycogenica
(Shintani et al., 2000), and Actinobacteria, which Kong et al. (2001) have observed in a lab-scale
reactor. Further, M. glycogenica can assimilate glucose and acetate anaerobically, and store glycogen
(Shintani et al., 2000; Kong et al., 2001). Unfortunately, further reports implicating this organism as a
GAO are unavailable (see e.g. Beer et al., 2006), and so the relevance of M. glycogenica in EBPR
systems, and its role as a competitor of PAO, remains in doubt. Several attempts at isolating another group
of GAO from the Gammaproteobacteria have been unsuccessful (Crocetti et al., 2002). However,
Defluviicoccus vanus isolates have been obtained (Maszenan et al., 2005a) and have been shown to take
up glucose anaerobically, with simultaneous glycogen degradation and PHA production without cycling
polyP (Wong and Liu, 2007). D. vanus isolates have thus been confirmed to display the GAO phenotype,
although it should be noted that Wong and Liu (2007) observed phylogenetic and phenotypic diversity
between D. vanus NCIMB 13612 (the isolated strain of Maszenan et al., 2005a) and the D. vanus-related
GAO enriched in their lab-scale reactor (see discussion below). Further studies investigating GAO
phylogeny and ecophysiology have generally employed culture-independent methods, as is the case with
PAO. The current view is presented in Figure 10.11.
Candidatus ‘Competibacter phosphatis’

A group of organisms belonging to the Gammproteobacteria, Candidatus ‘Competibacter phosphatis’
(also known as the GB lineage) have been shown in situ to display the GAO phenotype using culture-
independent techniques (Crocetti et al., 2002). These organisms do not exhibit a TFO morphology.
Nielsen et al. (1999a) first identified a novel group of Gammaproteobacteria by analyzing the dominant
DGGE bands of PCR-amplified 16S rRNA genes. FISH probes were then designed to target this
group of organisms, which accounted for 35% of the total bacteria in the community of a poorly
performing EBPR sludge. Crocetti et al. (2002) generated a 16S rRNA gene clone library from their
deteriorated EBPR reactor, and with these sequences developed two FISH probes (GAOQ431 and
GAOQ989) (Table 10.1) that target a group of Gammaproteobacteria in the same cluster as Nielsen et al.
(1999a) (Figure 10.11), with a high specificity. Crocetti et al. (2002) proposed the name Candidatus
‘Competibacter phosphatis’ (commonly known as Competibacter) for this group of organisms and used
FISH and post-FISH chemical staining to show that they performed anaerobic-aerobic cycling of PHA but
not polyP, in lab-scale and full-scale sludges, thus confirming their GAO phenotype. Further phylogenetic
analysis of the Competibacter group was performed by Kong et al. (2002b), who analyzed sequences
from three additional 16S rRNA gene clone libraries extracted from three lab-scale EBPR systems
displaying different P removal performances (Figure 10.11). From these analysis, ten FISH probes
were developed targeting this group of organisms at different hierarchical levels, where the GB probe
Actinobacteria
Kineosphaera limosa, AF109792
Micropruina glycogenica, AB012607
Amaricoccus kaplicensis, U88041
Tetracoccus cechii, Y09609
Uncultured activated sludge clone TFOa44, AY351640
'Defluviicoccus related'
Uncultured activated sludge clone pGAO17, DQ146466
1
Uncultured activated sludge clone TFOa43, AY351639
Alpha–
Defluviicoccus vanus, AF179678
Uncultured activated sludge clone 43, DQ413102 2
Uncultured activated sludge clone LPB46, AF527585
Uncultured activated sludge clone C17, AB445109
Uncultured bioreactor clone mlel-13, AF280850 3
Uncultured activated sludge clone B29, AB445108
Beta–
Uncultured activated sludge clone Skagenf97, DQ640667
Leptothrix ginsengisoli, AB271046
Quatrionicoccus australiensis, AY007722
Proteobacteria
Thialkalivibrio thiocyanodenitrificans, AY360060
Nitrosococcus halophilus, AF287298
Uncultured activated sludge clone SBRT185, AF361095
Uncultured activated sludge clone SBRH10, AF361096 1
Uncultured activated sludge clone SBRL1_8, AY098896
Uncultured activated sludge clone G115, FJ356057 2
Uncultured activated sludge clone H3, EU529718
Uncultured activated sludge clone SBRQ157, AF361092
Gamma–
'Competibacter' (GB)
Uncultured activated sludge clone SBRQ185, AF361090 3
Uncultured activated sludge clone SBRQ191, AF361091
Uncultured activated sludge clone SBRC_60, AY098902 4
Uncultured activated sludge clone C13, FJ356025
Uncultured activated sludge clone H68, EU529733
Uncultured activated sludge clone SBRL2_40, AY098900 5
Uncultured activated sludge clone SBRC_20, AY098905
0.10
Figure 10.11. Phylogenetic tree based on 16S rRNA sequences showing the relationships between the
putative glycogen accumulating organisms (GAO) in activated sludge, using the neighbour joining algorithm
(S. Mcllroy).
was the most general, targeting the entire group (Table 10.1). The probes targeting the ‘GB’ group
have been subdivided into GB_G1 (identical to those targeted with probes GAOQ989) and GB_G2
(which incorporates a one-base mismatch from GAOQ989), and probes GB_1 to GB_7 targeting the 7
subgroups. Thus, the known diversity of Competibacter can be covered with probes GAOQ989 and
GB_G2, or the GB probe (Oehmen et al., 2007). Competibacter have been repeatedly observed by FISH
in abundance in numerous lab-scale and full-scale EBPR sludges (Crocetti et al., 2002; Gu et al., 2008;
Kong et al., 2002b, 2006; Lopez-Vazquez et al., 2008a,b; Oehmen et al., 2005c; Saunders et al., 2003;
Wong et al., 2005), where their ability to compete with PAO for anaerobic uptake of organic substrates
has often been displayed.
Defluviicoccus vanus – related GAO

Another group of GAO have also been observed in numerous lab-scale reactors and some full-scale EBPR
plants, and these are related to Defluviicoccus vanus in the Alphaproteobacteria. Wong et al. (2004) found
that these organisms, which displayed a TFO morphotype, became highly enriched in a membrane
bioreactor with deteriorated P removal performance. They prepared a 16S rRNA gene clone library
selective for Alphaproteobacteria and designed two FISH probes (TFO_DF218 and TFO_DF618) for this
group of organisms that was closely related to D. vanus (Table 10.1). These probes targeted most of the
Alphaproteobacteria in their sludge, although a third probe developed to target D. vanus (TFO_DF862)
did not bind to any cells in the biomass, suggesting that the dominant organisms were phylogenetically
distinct from D. vanus (Figure 10.11).
Wong et al. (2004) also performed post-FISH chemical staining to confirm that their D. vanus-related
organisms performed anaerobic-aerobic PHA cycling, thus displaying the GAO phenotype. However, they
did not detect these organisms in a survey of full-scale EBPR plants.
Meyer et al. (2006) then identified a separate group of D. vanus-related organisms from a lab-scale
reactor using rRNA-based stable isotope probing prior to 16S rRNA clone library construction.
Phylogenetic analysis revealed a second cluster of organisms (Figure 10.11) related to D. vanus, and
FISH probes (DF988 and DF1020, plus helper probes) were designed to target these Cluster 2
D. vanus-related organisms (Table 10.1). Neither probes DF988 nor DF1020 hybridize with the Cluster
1 D. vanus-related organisms identified by Wong et al. (2004). These Cluster 2 D. vanus-related
organisms were detected in abundance in three lab-scale reactors displaying poor P-removal
performance, and were also found in small numbers in two full-scale EBPR plants (Meyer et al.,
2006). Post-FISH/chemical staining showed that these organisms performed anaerobic-aerobic PHA
cycling, and thus displayed the GAO phenotype.
Since then other studies have contributed to an understanding of the phylogenetic diversity of
the D. vanus-related GAO. Wong and Liu (2007) constructed additional 16S rRNA gene clone libraries,
and additional sequences were added to their phylogenetic tree (Figure 10.11). They proposed a third
Cluster, which consisted of a single sequence obtained from a non-EBPR system treating pharmaceutical
wastewater, and designed additional FISH probes to target members of Clusters 1 and 2. These were
used to investigate substrate assimilation patterns with FISH-MAR of their mixed-culture enrichment,
consisting mainly of Cluster 1 D. vanus-related GAO. They showed Cluster 1 organisms assimilated
acetate and propionate anaerobically, but not glucose, in contrast to results with a D. vanus-related GAO
isolate (also hybridizing with FISH probes for Cluster 1) that could assimilate glucose. Thus, it seems that
differences may exist in the ecophysiology of the D. vanus-related GAO, a view substantiated by
Schroeder et al. (2008).
McIlroy et al. (2008a) also investigated the phylogeny of D. vanus-related GAO detected in a
continuously aerated EBPR system (Ahn et al., 2007). A 16S rRNA gene clone library was performed after
flow cytometry was used to sort cells hybridizing with the ALF968 FISH probe for Alphaproteobacteria.
They revealed sequences similar to the aforementioned studies, despite the difference in the operational
mode of the bioreactor. Our understanding of the phylogenetic diversity and coverage of members
of this GAO group is incomplete. Thus, it is clear that the bulking filamentous organism Candidatus
‘Monilibacter batavus’ (Levantesi et al., 2004) is a member of the Cluster 3 D. vanus-related GAO
(Nittami et al., 2009), and McIlroy et al. (unpublished data) have also found 16S rRNA sequences suggesting
existence of members of a Cluster 4 D. vanus-related organisms, whose importance in EBPR systems is
unknown.
While the abundance of D. vanus-related GAO has been demonstrated clearly in lab-scale EBPR
systems, relatively few reports are available where their abundance has been studied in full-scale plants.
Thus, Burow et al. (2007) surveyed 10 full-scale EBPR plants in Denmark, and found that these were
present in small numbers 41% of all Bacteria in only 2 plants, although they were more abundant in one
plant, comprising 9% of all Bacteria. Interestingly, D. vanus-related GAO hybridized only with the
Cluster 2 FISH probes in both plants. Burow et al. (2007) confirmed with FISH-MAR that these organisms
took up acetate and propionate anaerobically, illustrating that they have the capacity to compete with
PAO for the anaerobic uptake of organic carbon. A survey of EBPR plants in the Netherlands revealed
that these D. vanus-related GAO were not abundant (51% of Bacteria) there (López-Vázquez et al.,
2008). Further attempts assessing the abundance of these organisms in full-scale EBPR plants would
be valuable.
Other GAO
Some studies have suggested that other populations with a GAO phenotype may exist in EBPR
communities. However, a re-evaluation of the Sphingomonas related alphaproteobacterial GAO
(Beer et al., 2006) suggests it is a member of the Cluster 1 D. vanus-related GAO (McIlroy et al.,
unpublished data). Oehmen et al. (2006) observed a group of Alphaproteobacteria GAO that displayed
anaerobic-aerobic PHA cycling where relatively few cells hybridized with the FISH probes designed for
D. vanus Cluster 1 or Cluster 2 members suggesting the presence of other currently unidentified GAO.
Two other groups of organisms found in Danish EBPR plants, belonging to the Betaproteobacteria and
Gammaproteobacteria respectively, took up acetate under anaerobic conditions (after FISH-MAR)
and stored PHA, but not polyP (Kong et al., 2007), thus displaying the GAO phenotype. FISH probes were
designed (BET65 and GAM455) for these organisms (Table 10.1), which Kong et al. (2007) observed
in varying abundances (1–6%) in a survey of several Danish plants. Further studies investigating the
abundance of these and other GAO, are now needed to more fully understand the biodiversity of this
important group and their roles in EBPR processes.
Physiological differentiation between GAO and PAO

A number of important metabolic differences exist between PAO and GAO, most notably concerning
their primary energy source for anaerobic organic carbon uptake. While PAO cycle polyP, the GAO use
glycogen as their sole energy source, and thus consume much higher amounts of glycogen anaerobically
compared with PAO to produce sufficient ATP for organic carbon uptake (Oehmen et al., 2007). During
this process, reducing equivalents (i.e. NADH) are produced in excess of metabolic requirements, and are
thought to be consumed through conversion of a portion of the glycogen via pyruvate, the reductive
branch of the TCA cycle and the succinate-propionate pathway to propionyl-CoA (Liu et al., 1994;
Satoh et al., 1994) (Figure 10.8). This is used by the GAO to regulate the redox balance within their cells,
and consequently increased levels of propionyl-CoA are produced compared to PAO. Thus GAO tend to
produce higher levels of PHV from acetate uptake (Mino et al., 1998; Oehmen et al., 2007).
Similar differences in the overall biochemical activity between PAO and GAO can be elucidated by
comparing the metabolic models proposed for each group. These models were developed to describe
the mass, energy and redox transformations associated with PAO and GAO metabolism, and show
high correlation with experimentally determined stoichiometry associated with acetate and propionate
uptake by enriched cultures of PAO (Smolders et al., 1994a,b; Smolders et al., 1995; Filipe et al., 2001b;
Oehmen et al., 2005b) and GAO (Filipe et al., 2001a; Zeng et al., 2003c; Oehmen et al., 2006).
Comparing biochemical activities of mixed cultures of PAO and GAO with the theoretical stoichiometry
has been performed in efforts to characterize the relative activities attributed to each group (Schuler and
Jenkins, 2003a,b; Yagci et al., 2003; Zeng et al., 2003d).
Glycogen degradation pathway

The original metabolic pathway hypothesized for glycogen degradation by GAO was the EMP pathway
(Satoh et al., 1994), which was supported by the observation that glucose-6-phosphate dehydrogenase,
an enzyme distinctive of the ED pathway was not active in an enriched culture of unidentified GAO
(Filipe et al., 2001a). However, Lemos et al. (2007) showed that an enriched culture of GAO degraded
glycogen through the ED pathway using in vivo NMR experiments with 13-C labeled acetate. They also
found a portion of the labeled acetate was metabolized through the oxidative branch of the TCA cycle.
Their enriched culture contained both Competibacter (46%) and D. vanus-related GAO (36%, Cluster 1
and 6% Cluster 2) with 51% Accumulibacter. It has not been determined if different GAO populations
possess different glycogen degradative pathways.
Mechanisms for organic carbon uptake

Mechanisms of acetate uptake have been studied for Accumulibacter, Competibacter and D. vanus-related
GAO (Cluster 1) using highly enriched cultures of each population (Saunders et al., 2007; Burow et al.,
2008a), and the outcomes summarized in Figure 10.12 (a–c). In each case, the energy required for acetate
uptake is driven by a proton motive force (PMF), as determined through experiments with metabolic
inhibitors. While Accumulibacter is suggested to generate its PMF by phosphate efflux through the Pit
transporter (see earlier), Competibacter is thought to generate its PMF by proton efflux across the cell
membrane through both the F1F0-ATPase (at the expense of ATP) and fumarate reductase (in the
reductive branch of the TCA cycle, consuming NADH) (Saunders et al., 2007). However, generation of
the PMF in Defluviicoccus, is not considered to occur through the F1F0-ATPase, since DCCD (N,N,Ç-
dicyclohexylcarbodiimide) did not inhibit its acetate uptake (Burow et al., 2008a), unlike Competibacter
(Saunders et al., 2007).
Instead, Burow et al. (2008a) proposed that Defluviicoccus generated its PMF through proton
efflux across the cell membrane from fumarate reductase (similar to Competibacter), or by the efflux of
sodium ions via methylmalonyl-CoA decarboxylation (in the succinate-propionate pathway). These
findings correlate well with the hypotheses put forward in metabolic modelling studies. Thus, Zeng et al.
(2003a,b) proposed that ATP consumption was necessary for acetate uptake in their community
(where Competibacter were the dominant organism), while Oehmen et al. (2006) suggested that a
negligible level of ATP consumption was necessary for propionate uptake (where Alphaproteobacteria
GAO including some Defluviicoccus dominated their sludge). These observations are consistent with
differences in the operational importance of the F1F0-ATPase in Competibacter and Defluviicoccus
(Saunders et al., 2007; Burow et al., 2008a).
Nitrate and nitrite utilization by GAO

As with the PAO, GAO are also capable of denitrification using NO3 as terminal electron acceptor.
However, as with the different Accumulibacter clades (see earlier), Competibacter and D. vanus-
related GAO differ in their denitrification abilities (Zeng et al., 2003e; Wang et al., 2008b). Thus,
Zeng et al. (2003e) found an enriched culture of GAO (consisting mainly of Competibacter: see
Wang et al., 2008b) could denitrify all the way from NO3 to dinitrogen gas (using their stored PHA
as energy source), although they noted that N2O was the main denitrification product from nitrite
accumulation in the bulk phase. On the other hand, Wang et al. (2008b) found an enriched culture
of D. vanus-related GAO (Cluster 1) denitrified from NO3 to NO2 (also using their stored PHA), but
were unable to reduce nitrite. Thus, they proposed that D. vanus-related GAO (Cluster 1) are
incomplete denitrifiers.
(a) H+ MeHPO4
Pit transporter
+
+
+ – ii
+ –
Acetate –
–
permease
(x>1)H+
Acetate– i
(b) (x>1)H+/Na+ Acetate–
Acetate permease
+ +
+
+ – – ii
–
Fumarate –
Propionyl CoA
reductase Fumarate
NADH
H+ 2Na+
i NAD+
Succinate Methylmalonyl
Methylmalonyl CoA
CoA
(c) (x>1)H+ Acetate–
Acetate permease
+ +
+
+ – – ii
–
Fumarate –
ADP +
reductase Fumarate Pi
NADH
H+ 3H+
i NAD+ iii
Succinate ATPase
ATP
Figure 10.12. Proposed mechanisms of PMF generation for acetate uptake by PAO and GAO, adapted from
Burow et al. (2008a) and Saunders et al. (2007). (a) In Accumulibacter, (i) Acetate permease-mediated
symport of acetate is energized primarily by the electrical membrane potential. (ii) Pit efflux of Hþ in symport
with inorganic Pi generates the electrochemical gradient and is used for acetate uptake. (b) In Defluviicoccus
(i) The fumarate reductase system generates an electrochemical potential. (ii) Permease-mediated symport
of acetate is energized by the membrane potential and likely the sodium potential. (iii) Methylmalonyl-CoA
decarboxylation may generate PMF through a sodium potential. (c) In Competibacter (i) The fumarate
reductase system generates an electrochemical potential. (ii) Acetate permease-mediated symport is
energized by the membrane potential generated through the fumarate reductase system as well as the
ATPase. (iii) Export of protons through the ATPase generates PMF, at the expense of ATP.
In FISH-MAR experiments, Kong et al. (2006) showed that different subgroups of the GB lineage
(i.e. Competibacter) possessed different denitrification abilities when acetate was supplied as the carbon
source. Thus, while subgroup 6 showed full denitrification capability (i.e. acetate uptake was observed in
the presence of nitrate and nitrite), subgroups 1, 4 and 5 took up acetate only in the presence of nitrate.
In similar experiments with Cluster 2 D. vanus-related GAO, acetate uptake was not observed in the
presence of either NO3 or NO2 (Burow et al., 2007), although Kong et al. (2007) showed that their
GAM455 and BET65 GAOs were capable of acetate uptake when NO3 was present. These studies clearly
illustrate that the denitrification potential of GAO depends on their phylogeny.
Competition between GAO and PAO

Since they are competitors in EBPR systems, it is important to study the physiological differences between
these two functionally-defined groups in order to identify operational conditions that might favor the
growth of the PAO over GAO, thus enhancing the reliability of EBPR. Most studies have focused on
maximizing Accumulibacter PAO population densities, and minimizing those of the Competibacter or
D. vanus-related GAO (also assessed as the more general Alphaproteobacteria GAO). Much remains to be
learned about factors affecting the abundance of other PAO like the Actinobacteria PAO, and GAO, The
main factors that are currently thought to affect the balance between the PAO and GAO are discussed
below (Oehmen et al., 2007).
COD/P ratio
One of the first factors shown to impact on the balance between PAO and GAO is the concentration ratio
of influent organic carbon to orthophosphate (COD/P ratio) (Liu et al., 1994; Mino et al., 1998). A high
COD/P ratio may reduce the growth of PAO from polyP limitation, leading to proliferation of the GAO.
Such a strategy has been exploited to obtain enriched cultures of GAO (not always identified) in lab-scale
reactors (Bond et al., 1999a; Kong et al., 2002a; Liu et al., 1994; Oehmen et al., 2006; Zeng et al., 2003b).
Thus, a low COD/P ratio (e.g. 10–20 mgCOD/mgP) favors the growth of PAO, although an adequate
amount of COD must still be provided to achieve high P removal performance in EBPR systems (Oehmen
et al., 2007).
However, these PAO are very adaptable organisms, with the potential to survive P-limited conditions
(i.e. high COD/P ratios) by modifying their metabolism. Thus, Zhou et al. (2008) showed a highly
enriched Accumulibacter culture could switch from a PAO to a GAO-like metabolism upon depletion of
their polyP reserves, where now their primary energy source for acetate uptake was glycogen. They also
showed the rate of acetate uptake by this Accumulibacter culture was much lower with glycogen than
with polyP, and concluded that GAO would probably out-compete PAO under P-limited conditions.
Nevertheless, such as the capability of PAO to adjust their metabolism in this way in response to changes
in their environment would provide a means for their survival under variable plant P loadings (likely in
activated sludge plants), impacting on the PAO versus GAO struggle.
Carbon Source
The carbon source metabolized by PAO and GAO is an important factor in selection. Accumulibacter,
Competibacter and D. vanus-related GAO tend to compete anaerobically for uptake of short-chain fatty
acids, where acetate and propionate are present in the highest abundance, and so these have attracted the
most attention (Oehmen et al., 2007). Furthermore, each enriched community may display different
preferences for acetate and propionate uptake (Figure 10.13). Thus, enriched cultures of Accumulibacter
assimilate acetate and propionate at similar rates (Pijuan et al., 2004; Oehmen et al., 2005c), while
enriched Competibacter cultures take up propionate very slowly (Oehmen et al., 2005c). Kong et al.
(2006) have observed with FISH-MAR that Competibacter are capable of propionate uptake, although
their data suggest that these GAO populations are likely to be less competitive for propionate
than acetate (Oehmen et al., 2007). In contrast, D. vanus-related GAO (Burow et al., 2007; Meyer et al.,
2006; Schroeder et al., 2008; Wong and Liu, 2007; Wong et al., 2004) and other Alphaproteobacteria
GAO (Oehmen et al., 2005c) seem capable of competing with PAO for both acetate and propionate.
Nevertheless, some data suggest that these GAO are more efficient in propionate than acetate uptake
(Dai et al., 2007b; Oehmen et al., 2005c). In fact Dai et al. (2007b) showed an enriched culture of
D. vanus-related GAO (Cluster 1) took up propionate anaerobically at a much faster rate than acetate
and preferentially assimilated propionate when both carbon sources were present simultaneously. They
explained that the D. vanus-related GAO probably took up propionate preferentially because they require
lower amounts of energy (i.e. from glycogen) for carbon uptake according to the GAO metabolic
models (Filipe et al., 2001a; Zeng et al., 2003c; Oehmen et al., 2006). Thus D. vanus-related GAO may
maximize their anaerobic PHA storage and subsequent aerobic growth through propionate metabolism
(Dai et al., 2007b).
Accumulibacter
PAO
Acetate
Competibacter
GAO
Fermentation
Propionate
Defluviicoccus
GAO
Glucose
Other organic
carbon sources
Actinobacteria
PAO
Figure 10.13. Schematic representation of the abilities of PAO and GAO to assimilate anaerobically different
carbon substrates.
Based on these different carbon source preferences (Figure 10.13), one strategy to eliminate GAO in
lab-scale reactors involved a regular switching of the carbon source from acetate to propionate (Lu et al.,
2006). This proved successful in eliminating the GAO and producing a high enrichment of Accumulibacter.
Lopez-Vazquez et al. (2009) with an integrated metabolic model also suggested that such a combined
acetate/propionate feed should be less favourable theoretically to both Competibacter and D. vanus-
related GAO compared to Accumulibacter, even at high temperatures (see below).
This received some support from analysis of a full-scale plant in northern Australia, where
improved EBPR was observed after increasing the propionate fraction of the feed by molasses dosing to
a prefermenter (Thomas, 2008; Thomas et al., 2003). This strategy replaced direct acetate addition, and
appeared to lead to a decrease in the unidentified GAO population and an improved P removal. Improved
long-term P removal performance was also observed in lab-scale bioreactors fed with real wastewater
after the propionate feed fraction was increased (Chen et al., 2004, 2005). Propionate may provide
selective advantages to the PAO because Competibacter (but not the Defluviicoccus related GAO) is less
competitive for this substrate, while Accumulibacter need to compete potentially with a wider range of
organisms for acetate (Oehmen et al., 2007).
Other organic carbon sources including other short chain fatty acids, sugars and amino acids can also
be taken up differentially by PAO and GAO, thus influencing the competition between them. However, in
mixed cultures some of these substrates may be fermented to acetate and propionate prior to uptake by
PAO or GAO (Wentzel et al., 1991a,b; Oehmen et al., 2007). This has been supported by FISH-MAR
experiments, showing that Accumulibacter and Competibacter are unable to consume glucose directly,
but consume the products of fermentation by other populations (Kong et al., 2004, 2006). On the other
hand however, the D. vanus-related GAO appear able to take up glucose directly (Burow et al., 2007), and
D. vanus also assimilates glucose in pure culture (Maszenan et al., 2005a; Wong and Liu, 2007). Thus,
D. vanus-related GAO seem able to assimilate a wider range of substrates than either Accumulibacter
or Competibacter, suggesting that they may be more competitive than either under certain conditions.
One such situation could be in industrial wastewater plants, where Burow et al. (2007) detected
these in high abundance. Industrial wastewaters generally contain more complex carbon sources than
domestic wastewaters. Kong et al. (2005) also observed higher abundances of Actinobacteria PAO than
Accumulibacter in plants receiving industrial discharges, which may also reflect the carbon sources
available in the influent. Actinobacteria PAOs assimilate mixtures of amino acids using FISH-MAR,
rather than short chain fatty acids or glucose (Kong et al., 2005).
Thus, considerable experimental evidence clearly shows that the carbon sources available in the
influent and metabolized by each PAO and GAO group can play a substantial role in the selection of
the community members in EBPR plants, and thus determine the efficiency of EBPR performance.
pH
The ambient pH of the wastewater may also affect competition between PAO and GAO where a high pH
(e.g. 7.25–8.0) has been reported to advantage the PAO and a low pH (57.0) the GAO (Oehmen et al.,
2007). Anaerobically, the PAO (not identified) consume a larger amount of polyP at high pH to meet the
increased energy demand for organic carbon assimilation (Bond et al., 1999b; Filipe et al., 2001c;
Liu et al., 1996; Smolders et al., 1994b), while their rates of acetate uptake, glycogen degradation and
PHA accumulation remain relatively constant, at least over a pH range of 6.5–8.0 (Filipe et al., 2001c).
On the other hand, GAO (also not identified) exhibited a higher glycogen consumption rate at a high
pH to compensate for this increased energy demand for carbon uptake. However, their acetate uptake
kinetics were slower (Filipe et al., 2001c). Thus, it seems likely that the advantage of PAO over GAO in
organic carbon uptake at high pH stems from their extra energy source (i.e. polyP), which means they
are more able to cope with increased energy demand (Oehmen et al., 2007). Furthermore, Filipe et al.
(2001c) observed that aerobic rates of P uptake, biomass growth and PHA consumption in PAO were
all markedly reduced at a pH of 6.5, compared to rates at pH 7.0 or 7.5, while aerobic GAO kinetics
remained unchanged over the same pH range. Thus, it seems that the GAO will possess an advantage at
such low pH.
These studies have been useful in suggesting a potential control mechanism for EBPR plant operation,
where high operational pH reduces the level of GAO competition with PAO. This strategy has been
validated experimentally, where FISH determined increases in the relative ratios of Accumulibacter/
Competibacter and Accumulibacter/Alphaproteobacteria GAO were observed after increasing operational

reactor pH (Oehmen et al., 2005a).
Temperature
Another key factor influencing the competition between PAO-GAO is temperature. It has been widely
reported that EBPR performance deteriorates at elevated temperature (420– C), hypothesized to
result from a shift in the community from PAO to GAO (Erdal et al., 2003; Panswad et al., 2003; Whang
and Park, 2006). Panswad et al. (2003) estimated the PAO and GAO mass fractions of their sludges
non-microbiologically from assessing changes in the P release to acetate uptake ratios, used frequently
as an indicator of PAO-GAO activity in other studies (Saunders et al., 2003; Schuler and Jenkins,
2003a,b). Furthermore, when Lopez-Vazquez et al. (2007) compared acetate uptake kinetics of an
enriched Accumulibacter sludge and an enriched Competibacter sludge at a range of temperatures in
batch tests, they found acetate uptake rate in the two sludges were similar between 5–20– C. However,
between 20–35– C, acetate uptake rates of the Competibacter sludge increased relative to those of
the Accumulibacter sludge. Thus, Competibacter displayed a clear advantage in acetate uptake at
temperatures 420– C. At 520– C, Lopez-Vazquez et al. (2007) suggested that Accumulibacter would
hold the advantage because of the lower cell maintenance energy requirements they observed over this
temperature range. Later focussing on the aerobic metabolism of Competibacter, Lopez-Vazquez et al.
(2008b) showed that Accumulibacter and Competibacter share similar aerobic temperature dependencies,
and concluded that any competitive advantages are established in the anaerobic EBPR stage. The situation
with the D. vanus-related GAO is less clear.
Other Factors
A range of other factors may potentially influence competition between the PAO-GAO. One is the
accumulation of NO2 in the form of free nitrous acid, which has been shown to inhibit PAO (Saito et al.,
2004; Zhou et al., 2007a) and thus potentially favour the growth of GAO. Other factors hypothesized
to affect competition between them include sludge retention time (SRT), the DO concentration in the
aerobic phase, and the presence of chemical precipitants. It is currently believed that a low SRT, a lower
DO concentration and absence of chemical precipitants favor the PAO (Oehmen et al., 2007), although
the evidence available is often scant. Feed strategy may also affect EBPR stability. Thus, Burow et al.
(2008a) detailed data suggesting that Accumulibacter PAO assimilate acetate more efficiently at lower
concentrations than Defluviicoccus- related GAO, and suggested that a trickle feed system, where such
conditions are established and maintained, might improve P removal.
11
Methods for the examination and
characterization of the activated sludge
community
Robert J. Seviour and Per H. Nielsen (editors)
INTRODUCTION
In this chapter, descriptions of methods useful for studying activated sludge communities are given. We
have expanded this section from the earlier version to cover the increasing range of techniques now
available, and many of these have been written by acknowledged experts.
Included are protocols for microscopic examination of samples, and associated staining techniques.
Also described are molecular methods used routinely now for analysing community composition and
elucidating the functional roles of populations there. This is not meant as a comprehensive guide to
practical microbiology, and laboratories should be encouraged to purchase one or more of the excellent
methodological books available providing details not found here. In particular, every laboratory should
have a copy of workshop manuals describing the molecular methods referred to often in this book. The
following are recommended.
(i) Akkermans, A.D.L., van Elsas, J.D. and de Bruijn, F.J. (Eds) (1995). Molecular Microbial
Ecology Manual. Dordrecht; Kluwer Academic Publishers.
(ii) Edwards, C. (Ed) (1999). Environmental Monitoring of Bacteria. Totowa, N.J.; Humana Press.
(iii) Kowalchuk, G.A., de Bruijn, F.J., Head, I.M., Akkermans, A.D.L. and van Elsas, J.D. (Eds)
(2004). Molecular Microbial Ecology Manual (2nd edition) Volumes 1 and 2. Dordrecht; Kluwer
Academic Publishers.
(iv) Leadbetter, J.R. (Ed) (2005). Environmental Microbiology. Methods in Enzymology, Volume
397. New York; Academic Press.
(v) Osborn, A.M. and Smith, C.J. (Eds) (2005). Molecular Microbial Ecology. New York; Taylor and
Francis.
(vi) Reddy, C.A., Beveridge, T.J., Breznak, J.A., Marzluf, G., Schmidt, T.M., and Snyder, L.R. (Eds.)
(2007). Methods for General and Molecular Microbiology (3rd Edition). Washington DC; ASM
Press.
(vii) Nielsen, P.H., Daims, H. and Lemmer, H. (2009). FISH Handbook for Biological Wastewater
Treatment. London; IWA Publishing.
11.1 MICROSCOPY AND MICROSCOPIC EXAMINATION OF

ACTIVATED SLUDGE
Elizabeth M. Seviour, Kenneth C. Lindrea, and Robert J. Seviour
THE LIGHT MICROSCOPE

The light microscope still plays an indispensable role in examining activated sludge biomass. Purchase
of a good quality (binocular) instrument and its subsequent maintenance are essential. Most reputable
manufacturers produce microscopes which are more than adequate, but as microscopy expands and new
applications are described access to more sophisticated systems is desirable. To obtain the best outcomes,
some knowledge of basic microscopy principles is useful.
PRINCIPLES OF THE COMPOUND LIGHT MICROSCOPY

The microscope magnifies objects making them visible, but it must also possess resolution or resolving
power to allow separation of closely aligned objects. These must have sufficient contrast against their
background to be seen. Light microscopes are compound microscopes consisting of three different lens
systems. When correctly positioned, the condenser situated between the light source and the specimen
focuses parallel rays of light onto it. Its position must be established before use, but once set, no further
adjustment is necessary. It should supply a cone of light capable of filling the objective lens aperture (see
below) required for optimal resolution. The objective and ocular or eyepiece lenses magnify the image
passing to the eye. The iris diaphragm controls the amount of light passing through the condenser onto the
specimen, and therefore affects brightness and more importantly, contrast of the image. Its optimal setting
has to be established for each objective lens.
Magnification and resolution

Magnification is the product of the separate magnifications of the objective and ocular lenses. However,
the resolving power of the microscope is more important i.e. its ability to reveal closely adjacent details as
separate and clear.
Methods for the examination and characterization of the activated sludge community 323
The distance between two points which are distinguishable is called the resolving limit (d).
0:5 l
d¼ ð1Þ
N sin a
where l is the wavelength light

a is the ½ angle of the objective
N is the refractive index of the objective medium below lens.
N sin a is referred to as the numerical aperture (NA) of a lens. Increasing NA increases its resolving
power, but in air, NA can not exceed 0.65. If placed in suitable oil, then NA can be increased theoretically
to 1.4, although on most microscopes, the oil immersion lens has a numerical aperture of 1.25. Murray and
Robinow (2007) provide a more detailed explanation.
Setting up the light microscope

The microscope must be set up properly to provide high quality images. Remember, the data acquired
reflects microscope performance. The following routine is suggested. Initially the reader should follow
this carefully, but with practice it may be shortened.
Lower power (L.P.) Objective (10£)

(i) Place the microscope in a convenient position with its base close to the edge of the bench.
(ii) Select the eyepiece lens of 10£, and the objective of 10£. Adjust both eyepiece lenses for personal
comfort.
(iii) Place a prepared slide on the microscope stage so that the specimen is central to the stage opening.
(iv) Rack the condenser up to its maximum setting and fully open the iris diaphragm.
(v) Lower the lens until it is about 5 mm from the slide, and looking down the eyepiece, carefully turn
the coarse adjustment until the image comes into view. Focus sharply with the fine adjustment.
(vi) Adjust the iris diaphragm to a point where the image is sharp. As a check, remove the eyepiece
and look down the tube. About two-thirds to three quarters of the diameter of the back lens should
be light-filled, so adjust if necessary.
(vii) Still looking down the tube, adjust the condenser position until the iris diaphragm is sharply
focussed. (It may also be necessary to readjust the iris diaphragm so that it again just encroaches
on the circle of light). Best resolution is when the condenser is raised to near its maximum height,
but where any dust on the lens is out of focus.
(viii) Replace the eyepiece and examine the preparation using fine focus adjustment and adjustable
mechanical stage.
High Power (H.P.) objective (£40)

(i) Rotate the nose piece so the high power objective clicks into place. As objectives are para-focal
(i.e. each close to being in focus at the same setting) fine control adjustment only focus specimen.
(ii) When the object is in focus, adjust the iris diaphragm to the point of optimal contrast (as in (vii).
(iii) Re-examine the sample.
Oil Immersion (O.I.) objective

(i) Adjust the stage so that the specimen is in the centre of the field of view.
(ii) Raise all objectives clear of the stage.
(iii) Place a single drop of immersion oil on slide in the centre of the specimen (1 cm diam.).
(iv) Rotate the nosepiece so that the oil immersion lens clicks into place. This is the only lens designed
for oil, so if oil contaminates other lenses, clean them immediately with lens tissue.
(v) With the eye at the level of the stage and coarse adjustment, lower the lens until in contact with
the oil. Then very carefully lower the lens further towards the slide without actually touching it, so
it is as close as possible to the specimen without damaging either.
(vi) Looking down the eyepiece lens, slowly and carefully focus up with the fine adjustment until the
image comes into focus. If this fails, repeat the whole procedure. NEVER FOCUS BY TURNING
THE OBJECTIVE DOWN TOWARDS YOUR SPECIMEN.
(vii) Adjust the iris diaphragm until a sharp image is obtained.
(viii) Re-examine the specimen.
Important notes
1. The condenser once focused should require no further adjustment.
2. The iris diaphragm is used to alter the cone of light to suit the N.A. of the objective; thus, it
requires adjustment whenever a change is made from one objective to another.
3. Keep both eyes open when looking through the eyepiece (this might require practice) - it should
be possible to look down the microscope with one eye and focus on the worksheets with the other.
COMMON DIFFICULTIES IN MICROSCOPY

Difficulties may be encountered, and the following hints may be useful.
Inability to obtain a sharp image with the oil immersion object

(i) Check there is no dirt or dried oil on the front lens of the objective. If so, clean with lens tissue.
(ii) Check that the microscope slide carrying the specimen is not upside down.
(iii) Check the immersion oil is not so viscous that the slide adheres to the objective lens and travels
upwards during focus adjustment.
(iv) Check whether the specimen slide and cover slip has a film of dried immersion oil and dirt left on
it by a previous viewer.
(v) Some high-power objectives have such a short working distance that a thick cover slip or a thick
layer of Canada balsam beneath the cover slip may prevent bringing the lens close enough to bring
the object into focus.
(vi) Streaming and marked distortion can follow when different makes of immersion oil are mixed, if
their refractive indices are different.
A dark shadow passes into the field with the loss of definition
of the image
Is usually caused by movement of an air bubble in the oil, and may be overcome by raising the objective
lens so that contact between the oil and lens is broken, and then refocussing. If this fails, clean objective
and cover slip with suitable cleaning agent and begin again.
Poor illumination or the field of view in semi-darkness

(i) Check that condenser has been racked up to its full height. Occasionally it slips downwards in its
mounting ring.
(ii) Check that the sub-stage diaphragm is open.
CARE OF THE MICROSCOPE

Microscopes are precision instruments, expensive to repair or replace, and should be carefully maintained.
The following tips are useful.
(i) Store in a cool dark place, and protect from dust by keeping plastic cover on when not in use.
(ii) Check that all parts of the microscope are clean before and after use.
(iii) The oil immersion lens (see later) must be cleaned after use by wiping it with lens tissue. If oil is
left on the lens, it may dry and need to be removed with a proper cleaning solution. DO NOT use
solvents (e.g. alcohol) which might damage it.
(iv) Never rub the lens surface with a dry cloth or coarse tissue, since it may become scratched.
Always use lens tissue.
OPTICAL SYSTEMS FOR LIGHT MICROSCOPY

Most microscopes are now fitted with a range of options allowing systems for bright field, phase contrast,
Nomarski or fluorescence microscopy. The following may help how to set them up to expand their
application to activated sludge characterization.
Bright field microscopy

First adjust the phase setting on the condenser to 0, then use as for an ordinary microscope.
Phase contrast microscopy

In bright field microscopy transparent objects like cells lack contrast, which can be overcome by staining.
Some used for examining activated sludge are detailed later. For unstained preparations, contrast can be
increased by closing the iris diaphragm. However, the objective NA and hence its resolving power is reduced.
Phase contrast microscopy provides an alternative for live preparations. Thus, intracellular S– granules
in filamentous bacteria like Thiothrix can be seen clearly under phase contrast (Figures 12.10a; 12.11b).
It depends on altering the wavelength phase of light rays passing through translucent objects. These are
invisible to the human eye which can only detect differences in wavelength (colour) or amplitude
(intensity).
Phase microscopy converts changes in phase into changes in amplitude and so renders translucent
objects visible (Figure 11.1, see colour image section (chapter 13)). Light refracted by the specimen is
retarded by a phase plate in the objective lens causing differences in intensity with no reduction in its NA
or resolving power. An annular diaphragm is mounted in the back focal plane of the condenser so the
specimen is illuminated by a hollow cone of light. A phase-retarding annulus or phase plate is positioned
in the back focal plane of the objective lens exactly corresponding to the annulus in the condenser. Light
refracted by the specimen does not pass through this annulus, and so is retarded 1/4 of a wavelength
compared with the directly transmitted beam.
Phase microscopy must use designated objectives supplied with the phase microscope. Those supplied
with ordinary light microscopes are NOT suitable.
Protocol
Select an objective and set microscope up for bright field microscopy, align the phase rings and adjust the
phase ring to the number corresponding to the power of the objective lens. Please consult the
manufacturer’s Instruction Manual for detailed procedures if problems arise. The specimen will now be
contrasted against the background.
Nomarski interference microscopy

In Nomarski interference microscopy, the light beam is sheared by polarising prisms beneath the
condenser, and this light, consisting of two bundles of rays differing by 90– in their phases of polarisation
is used to illuminate the specimen. The amount of shear is kept less than the resolving limit of the
objective lens. Both beams pass through the specimen and another set of polarising prisms, re-combining
the beam. The two sheared images produced are not seen as such, since the shear is too small to be
resolved. This technique produces clearly defined 3-D relief-like images with high contrast in a range of
interference colours, and is recommended for activated sludge examination. A comparison of the images
produced by various optical systems is given in Figure 11.1 showing improvement in the 3-D detail
provided by Nomarski optics.
Nomarski interference optics requires appropriate condenser and polarising prisms. Setting up the
microscope requires practice, but the results are worth the effort involved. Manufacturers’ Instruction
Manuals will explain how to use this system for each brand of microscope.
Fluorescence microscopy
In fluorescence microscopy, the specimen is excited with short ultraviolet wavelength light, and emits
light of a longer visible wavelength, where it is said to fluoresce. Special lenses able to transmit U.V.
light, especially if the excitation light is directed at the specimen from above (epifluorescence) need to be
purchased, together with an appropriate source of excitation light (Murray and Robinow, 2007). These
can be expensive, but provide the user with a powerful facility for certain staining procedures for
detecting populations storing PHA and polyP and in combination with FISH probes for in situ
identification of filamentous bacteria and organisms like PAO (Figures 11.2, 11.3, see colour image
section (chapter 13)).
IMAGE ANALYSIS
Most manufacturers of epifluorescent microscopes or CLSM systems (see below) sell their own
instrument-compatible digital image analysis software, although these are not the only commercial
sources (Daims et al., 2001c, 2006a). In our experience most systems are powerful and user-friendly, and
almost essential for FISH based quantification and detailed studies on floc structure and shape (Jenné et al.,
2006; Lopez et al., 2005; Schmid et al., 2003a). In fact they have become routine tools for activated
sludge microbiologists. In our view, a publicly accessible and versatile system suitable for such work is
daime or digital image analysis in microbial ecology (Daims et al., 2006a). Designed for use with the
CLSM, this software not only quantifies microbial populations of interest and evaluates/validates new
FISH probes in situ, but can be used to quantify patterns of spatial organization between populations with
a technique called ‘3-D FISH’. It can be downloaded from (http://www.microbial-ecology.net/daime), and

a user manual is also available. See Nielsen et al. (2009) for further details.
THE ELECTRON MICROSCOPE

This microscope uses beams of electrons with the properties of electro-magnetic waves which are
accelerated through an electrical field. The wavelength is inversely proportional to the square root of the
accelerating voltage applied. Because this is much shorter than visible light, the resolving limit of the
electron microscope is much lower. The electron beam is focussed onto the specimen using magnetic lenses,
and an electronic image on a phosphorescent screen is produced. Biological specimens have to be fixed and
dehydrated before viewing, which requires skill. The review by Beveridge et al. (2007b) is recommended.
In transmission electron microscopy (TEM), adequate specimen fixation is critical for success. Thin
sections are used (550 nm), and the image is generated by differences in contrast, resulting from
differential scattering of the electrons as they pass through. Contrast is achieved or enhanced by staining
with heavy metals like lead. At such high resolution, important ultrastructural information is revealed, as
seen in Figure 11.4, showing the unusual intracellular membranes of the planctomycete Isosphaera sp
(‘Nostocoida limicola’ III?).
Figure 11.4. TEM of Isosphaera (‘N. limicola’ III?), showing characteristic planctomycete intracellular
membranes (J.R. Liu).
In scanning electron microscopy (SEM), surfaces of specimens are bombarded by electrons that
generate secondary electrons. These are collected with a scanning coil which is positively charged, and
eventually an image is generated. SEM provides high resolution, three dimensional images, and
invaluable information on morphology for example of filamentous bacteria, as shown in Figure 11.5 with
Trichococcus flocculiformis.
Figure 11.5. SEM of the filamentous bacterium Trichococcus flocculiformis showing its distinctive coccoid
cells in chains (J.R. Liu). Bar mark ¼ 4mm.
THE CONFOCAL LASER SCANNING MICROSCOPE (CLSM)

Confocal microscopy has provided a powerful tool with which to study microbial community structure. It
is a technique where the light microscope has been transformed from an instrument able to view samples
in two dimensions to one which can explore structures in three dimensions (Lawrence et al., 2002).
Imaging with the CLSM is achieved by focussing a point of light or a band of light from a laser onto
the sample with the objective lens. As with epifluorescent microscopy, quality of the objective lens
determines overall image quality. The diameter of the focus light probe depends on the numerical aperture
of the lens. If a sample is stained with fluorescent dye (e.g. a fluorescently labelled oligonucleotide for
FISH), light emitted from this point is imaged by the same objective and passed through an optical
transfer path to reach a light sensitive detector like a photomultiplier tube. The confocal effect, or
elimination of the ‘‘out of focus’’ information, is achieved by having an aperture (the confocal aperture or
pinhole) placed in front of the detector, at a point equivalent to the back focal point of the objective lens.
At this point, a spot is formed which contains all the information from the focal point within the sample,
and the aperture can be set to a size that allows only this spot to pass through the aperture onto the
detector. The detector therefore, only ‘‘sees’’ light from the focal plane of the objective. This forms the
optical section. Useful imaging information about the whole sample is gained by having this spot scanned
across the whole specimen. Scanning is achieved simply by moving the laser beam. Lasers are used to
produce a spot with sufficient illumination to generate an adequate fluorescence signal from the sample.
Confocal laser scanning microscopy is now employed widely in studies with activated sludge,
especially to visualize and quantify FISH probed populations (chapters 3, 5) (Daims et al., 2006a; Snaidr
et al., 1997; Wagner et al., 1994b), especially those probed simultaneously with multiple probes, each
tagged with a different fluorochrome to confirm their identity (Nielsen et al., 2009). CSLM can display
color complementarity where cells targeted with probes tagged for example with green (FLUOS) and red
(CY-3) fluorochromes fluoresce yellow. Those hybridizing with probes with green (FLUOS), red (CY-3)
and blue (CY-5) fluorochromes when applied together appear white (Figure 11.6, see colour image section
(chapter 13)).
11.2 PREPARATION OF SPECIMENS FOR MICROSCOPY

Elizabeth M. Seviour and Kenneth C. Lindrea
Equipment and materials

Displacement tube, (a P.V.C. or glass tube of approx 150 cm length and 2 cm in diameter used for taking
dip samples), sample bottles (100–200 ml and 25 ml). Clean microscope slides, cover slips, wash bottles
for distilled water and methylated spirits (label carefully), Pasteur pipettes or glass droppers, forceps,
staining solutions in dropper bottles and a deep tray with a rack for slides.
Sampling of activated sludge, mixed liquors and foams

Unrepresentative samples provide data of limited value. A larger sample than is required should be taken
(4100 ml). Samples of mixed liquor should be drawn from beneath the surface with a suitable
displacement tube, while samples of foam must be carefully skimmed from the surface of the mixed liquor
to avoid dilution and contamination, making sure that ‘fresh’ foam is collected. In plants with several
reactors, samples should be taken from each.
Sample storage
Samples should be examined as soon as possible after collection. No detailed studies have been conducted
on effects of prolonged storage on protozoan populations, but these organisms seem to survive at ambient
temperature for a few days after collection. Longer storage times should be avoided. Refrigeration does
not appear to be essential, but is recommended. For transport, a small bottle (425 ml or less) with an air
space about equal to the sample volume is adequate. Such samples are sent to this laboratory from
throughout South Eastern Australia without apparent problems, given reasonable delivery times (1–2 days)
and handling conditions (i.e. maintenance of moderate temperatures).
Slide handling
In preparing slides for microscopy, cleanliness is essential. If slides are handled, their surface may become
greasy, and so they should be picked up by their edges only. Similarly, cover slips must not be
contaminated with fingerprints. Forceps may be used to handle both during staining, and unused slides
stored in methylated spirits.
Preparation of samples for microscopy

The reader is referred to the review by Beveridge et al. (2007a) for details.
Air-dried smears
This method precedes staining. The main consideration is not to put too much sample on the slide.
(i) A drop of well-mixed sample taken with a Pasteur pipette is placed on the centre of the slide and
spread to cover about half its area by gently smearing it with the tip of the pipette.
(ii) It is then allowed to dry at ambient temperature or by holding high above a Bunsen flame to
speed up the process. In some protocols, a short exposure to heating in a low flame (gas or spirit
lamp) can ‘fix’ the specimen while others specify that air-drying only be used. Heat fixed slides
are not used in this laboratory as it is not necessary, and excessive heating may alter the
specimen.
(iii) Staining must be carried out in a tray and gloves must be worn. Stains will permanently mark
benches, floors and clothing. Any deep kitchen tray is suitable, the slides being supported by a
wire cake rack or similar item. During staining slides are laid horizontally, stain applied by a
dropper and the rack or slide tipped to allow the stain to flow off the slide.
(iv) Rinsing may be carried out over the tray with a wash bottle with a large jet opening or over a sink
under a gentle flow of tap water. Alcohol rinses may also be performed over the tray. When
staining is complete, rinsings etc. are disposed of following laboratory rules.
(v) Slides are finally allowed to air-dry prior to examination. They may be stored without substantial
deterioration unless immersion oil has been used. The oil will collect dust and lint if left exposed,
rendering the slide unusable unless kept in a closed slide box.
Wet mounts
A wet mount is prepared by placing a drop of well mixed sample on a clean slide and carefully placing a
cover slip over it to exclude air bubbles. Care must be taken to use an appropriate sample volume, since if
too small, air will be entrained under the cover slip and evaporation will occur. Too much sample may
allow the cover slip to float off, and the sample to spill onto the microscope stage. A tissue can be
employed to soak up the excess, or preferably prepare a new slide. The wet mount must be examined
immediately before it dries out.
Preparation of sludge and foam samples for SEM

Although not suggested as a routine exercise, examination of samples by SEM can be rewarding,
particularly in revealing morphological features of protozoa and filamentous bacteria not visible by light
microscopy, and in looking at floc structure and organisation. The critical steps are to prepare the samples
adequately, with a method suitable for the wide diversity of organisms present. The literature contains
descriptions of many methods, but the one described here has been applied successfully to mixed liquor
and foam samples. It is a slightly modified version of the method of Seviour et al. (1984).
Procedure
(i) Samples are fixed in small sealed glass vials in equal volumes of 2% (w/v) osmium tetroxide and
6% (w/v) glutaraldehyde for at least 24 h at 4–5– C.
(ii) They are then dehydrated through a graded isopropanol series of 0, 25, 50, 75 and 100% (v/v)
isopropanol for 15 min in each, before being stored in 100% isopropanol. All stages are carried
out in glass vials with samples contained in small reservoirs of glass tubing (1 cm internal diam)
cut into 1 cm sections, with polycarbonate membranes (0.45 mm pore diameter) attached firmly to
their base with silicon glue.
(iii) Solutions are replaced by putting these reservoirs onto absorbent toilet tissue and allowing the
solvent to drain out, leaving the sample still moist on the membrane.
(iv) Specimens are critical point dried still in their reservoirs, membranes gently removed, and they or
the samples transferred to aluminium stubs coated with double sided tape.
(v) After gold coating specimens are examined under the microscope. Some of the micrographs of
filamentous bacteria obtained with this method are shown in this book (e.g. Figure 11.5).
11.3 STAINS USED FOR EXAMINATION OF ACTIVATED SLUDGE

SAMPLES
Elizabeth M. Seviour and Kenneth C. Lindrea
Staining may be considered a means of colour coding organisms to provide characteristics as aids to cell
differentiation and identification. Many stains are available, but for activated sludge only a few are used
routinely (Jenkins et al., 2004b). More details are provided by Beveridge et al. (2007a).
Gram stain (differentiation on the basis of cell wall structure)

The Gram stain, which differentiates between two natural groups of Bacteria based on their cell wall
chemistry (chapter 1) is probably the most frequently used. A stain, crystal violet enters the cell and
complexes with Gram’s iodine. Subsequent rinsing with alcohol washes this complex from Gram negative
cells, leaving these colourless. In Gram positive cells the complex is retained and a darkly stained
specimen results. Gram negative bacteria are usually counterstained with an aqueous solution of Safranin
(red), providing a colour contrast. Many protocols have been published but some work better than others,
and equivocal results can be produced. Cells staining Gram positive when ‘young’ often become Gram
variable or negative as they age. The method described below works well with activated sludge samples.
Preparation of reagents
Solution 1
Prepare the following separately and combine prior to use.
Part A
Crystal Violet 2.0 g
Ethanol 95% v/v 20 ml
Part B
Ammonium Oxalate 0.8 g
Distilled water 80 ml
Solution 2
Iodine 1.0 g
Potassium Iodide 2.0 g
Solution 3
Safranin (2.5% w/v in 95% ethanol 10 ml)
Procedure
(i) Prepare air dried smears and stain for 1 minute with solution 1, pour off stain and rinse with water.
(ii) Shake off excess and stain with solution 2 for 1 min. Pour off and rinse as before.
(iii) Holding the slide at an angle, decolourize smear by gently rinsing with alcohol (methylated
spirits) from a wash bottle until rinsings have no visible colour (5–60 sec). Shake dry.
(iv) Counterstain for 10 sec with solution 3, and gently wash with tap water. Then examine under oil
immersion. Gram positive cells are purple. Gram negative cells are red. Filamentous bacteria
and TFO/GAO contain members of each.
Comments
(i) Most solutions may be purchased from chemical suppliers.
(ii) Many recipe variations are used to prepare the stain.
(iii) No special storage conditions are required, but it is wise to replace these every six months.
Alternative Gram staining methods

Several other Gram staining protocols have been applied to activated sludge communities, and may
be adapted to provide additional information. For example, Forster et al. (2002) showed that the
fluorescent dye hexidium iodide, as well as selectively staining only Gram positive bacteria (Mason
et al., 1998), can be used simultaneously with 5(6)-carboxyfluorescein diacetate (CFDA) and flow
cytometry (see section 11.15) to quantify their active populations. A kit for detecting both viability and
Gram stain reaction with hexidium iodide is available from Molecular Probes (LIVE BacLight Gram
Stain1 kit).
Detecting acid fast bacteria

Although not commonly used in activated sludge studies, this stain can detect the often high numbers of
acid fast Mycolata bacteria in foams (chapter 8). Acid fastness reflects chemical differences in cell walls
of a few Actinobacteria, especially Mycobacterium spp. Wall polymers like arabinogalactans and
complex branched fatty acids, mycolic acids prevent these being decolorised by acid/alcohol after staining
with carbolfuschin, and render cells hydrophobic. Several methods are available for this staining
procedure, but the Ziehl-Nielsen method works well with foam samples.
Solution 1
Carbol fuschin stain.
Basic Fuschin 0.3 g
95% Ethanol 10 ml
Phenol, heat melted crystals 5 ml
(i) Dissolve the basic fuschin in the ethanol before adding the phenol dissolved in the water. Mix,
leave for several days and filter before use.
Solution 2
Decolorising solvent.
95% Ethanol 97 ml
HCl (conc) 3 ml
Solution 3
Counterstain.
Methylene blue chloride 0.3 g
Procedure
(i) Carefully place a small piece of filter paper over a dried, fixed smear and flood with carbol
fuschin.
(ii) Gently heat the slide with a torch of cotton wool soaked with ethanol held under the slide until the
stain just steams, then leave for 5 min with no further heating.
(iii) Remove the filter paper and wash with tap water before decolorising the smear with the acidic
ethanol solution, until no further colour is removed. This may take a few minutes. Wash gently
with tap water before counterstaining with methylene blue for about 30 sec.
(iv) Finally wash with tap water and allow to air dry before examining under oil immersion. The
typical appearance of acid fast bacteria in a foam sample is shown in Figure 11.7, see colour
image section (chapter 13).
Comments
(i) This stain can be used to indicate the presence of bacteria other than Nocardia spp.
i.e. Mycobacterium spp. in foams.
(ii) The heating step for carbol fuschin can be eliminated by extending staining period to 20 min.
Neisser stain (detection of polyP)

Identification of polyphosphate ‘volutins’ or beads may be valuable in bacterial identification, but are not
visible without staining. The ‘active’ ingredient in the Neisser stain is methylene blue which is cationic, and
links to anionic sites on the polymeric polyP chains, colouring them a lilac colour, the Neisser positive reaction.
Solution 1
Prepare and separately store the following:
Part A Methylene Blue 0.1 g
Ethanol 95% 5 ml
Acetic Acid, glacial 5 ml
Part B Crystal Violet (10% w/v in 95% ethanol) 3.3 ml
Ethanol 95% 6.7 ml
Mix together, 2 parts by volume of A and 1 part by volume of B. Prepare fresh daily.
Solution 2
Bismark Brown (1% w/v aqueous) 33.3 ml
or Chrysoidin Y (1% w/v aqueous)
Distilled water 66.7 ml
Procedure
(i) Using solution 1, stain air dried smear for 30 sec. and follow with a 1 second rinse with water.
(ii) Shake off excess water and stain with solution 2 for 1 min, rinse well with water and allow to dry
prior to examination.
Comments
(i) Virtually no limitations on storage of solutions A, B and 2. These may be purchased ready made.
(ii) With filamentous organisms, if granules are present they almost always indicate the presence of
Candidatus ‘M. parvicella’ or some of the GALO. As far as we know, Candidatus ‘N. limicola’
(Tetrasphaera spp.) and Type 0092 are the only filamentous bacteria staining entirely lilac
color. Consequently, Neisser positive filaments are relatively easily ‘identified’, although some
alphaproteobacterial N.limicola morphotypes can stain Neisser negative (chapter 12) (Figure 11.8,
see colour image section (chapter 13)).
(iii) Some unicellular bacteria especially Candidatus ‘Accumulibacter phosphatis’ store polypho-
sphate (polyP) intracellularly. Dark clusters of these bacteria may be observed in the sludge floc
after Neisser staining.
DAPI staining (detection of polyP) and in combination with FISH

Intracellular polyP granules can also be detected in PAO cells with the fluorescent stain 40 ,6-diamidino-2-
phenylindole (DAPI) (Liu et al., 2001b), and imparts to them a white/yellow fluorescence This is
distinguished by epifluorescence microscopy from the blue fluorescence with DNA after careful choice of
filters. It can also be combined with FISH (section 11.9) to identify in situ the Accumulibacter populations
accumulating polyP (Serafim et al., 2002a), as shown in Figure 11.3.
Procedure
(i) An aliquot (10 ml) of a DAPI solution (50 ppm) is added to each well of slides containing samples
prepared for subsequent FISH (section 11.9), and left for 2 min.
(ii) Slides are quickly rinsed with milliQ water, dried and 2 ml of a suitable antifade mountant
(e.g. Vectashield1) added.
(iii) Samples are viewed by epifluorescence microscopy with a suitable UV filter. DAPI positive cells
fluoresce white/yellow.
Comments
(i) DAPI also imparts fluorescence to DNA, but it fluoresces blue not white/yellow.
Sudan black stain (detection of lipophilic material including poly-b-hydroxy

alkanoates (PHA))
The ability of the lipophilic sudan black stain to dissolve in fatty material allows it to detect poly-b-
hydroxy butyrate (PHB) granules, which would otherwise not be visible under light microscopy. These
occur inside cells of some bacteria (e.g. the PAO and some filaments). However sudan black is not specific
for PHB, and stains most lipophilic material.
Solution 1:
Sudan Black B (IV) 0.3 g
Ethanol 60% 100 ml
Solution 2:
Safranin 0 0.5 g
Procedure
(i) Using solution 1, stain air dried smears for 10 min. adding extra stain if slide begins to dry, and
follow with a 1 second rinse with water.
(ii) Shake off excess water and stain with solution 2 for 10 sec, rinse well with water and allow to dry
prior to examination.
(iii) Examine the prepared specimen through a bright-field objective lens (400–1000 £ magnification).
Blue/black granules in a clear or lightly coloured background indicates the presence of
lipophilic material usually poly b hydroxy butyrate (PHB) (Figure 11.9, see colour image
section (chapter 13)).
Comments
(i) Virtually no limitations on storage of solutions 1 and 2.
Nile blue A stain for detection of PHA

Nile Blue A is considered a more specific stain for PHB than sudan black, and is now commonly used
in studies of activated sludge. The original method of Ostle and Holt (1982) has been modified
(e.g. Kitamura and Doi, 1996) to avoid requirements for slide incubations at 55– C.
Solution 1
1% (w/v) aqueous solution of nile blue A (Sigma Chemicals).
Solution 2
8% (w/v) acetic acid.
Procedure
(i) Incubate heat fixed smears with solution 1 at 55– C for 10 min, wash with water to remove excess
stain and add solution 2 for 1 min.
(ii) Wash with water and blot dry, before remoistening and covering smear with a cover slip. Do not
place immersion oil directly onto the smear as it will extract the fluorescent dye, but place on
top of the cover slip.
(iii) View by epifluorescence microscopy with an appropriate filter providing an excitation wavelength
of 360–400nm. PHB granules fluoresce strongly red/bright orange (Figure 11.2).
Comments
(i) This stain can also be used in combination with FISH to identify in situ PHA accumulating
organisms, like filamentous bacteria and PAO/GAO (chapter 12). After nile blue A staining,
slides are immersed in absolute ethanol for 30 min, and then FISH is carried out as detailed in
section 11.9.
Crystal violet stain (for detection of sheaths)

This stain can be used to detect sheaths on filamentous bacteria. However, results are often inconclusive
and it is often easier to see sheath material with phase contrast or Nomarski optics.
Crystal Violet 0.1% w/v aqueous solution.
Procedure
(i) Add 1 drop of the stain to 1 drop of sample on a clean slide.
(ii) With a cover slip in place, examine with oil immersion, where sheaths have the appearance of
those shown in Figure 11.10, see colour image section (chapter 13).
Comments
(i) Other staining protocols for sheath or capsular materials include the negative staining technique
with India ink, where with low (£400) magnification and bright field or phase contrast optics,
sheaths can be seen as a halo of non staining material around cells or flocs (Figure 11.11, see
colour image section (chapter 13)). These are more apparent in smears obtained by ‘dragging’ the
cover slip across the slide leaving a thin film of sample as it moves.
Alcian blue (1% solution in 95% ethanol) has also been used detect
polysaccharide sheaths
Procedure
(i) A 1:9 dilution in water of this solution is added to smears for 1 min.
(ii) The smear is washed in tap water, dried, counterstained very briefly (410 sec) in carbolfuschin,
and washed immediately in tap water, before being air dried and examined under oil immersion.
(iii) Sheath appears blue and cells red.
However in this laboratory this method gives no better results than the crystal violet stain.
Viability/activity stains
How viable the bacteria in the community are can be valuable information, for example, if the intention is
to monitor the fate of filamentous bacteria after say chemical treatment (Maurines-Carboneill et al., 1998),
or exposure to heavy metals (Yin et al., 2005), or to isolate and culture them. Several microscopic
methods have been described for assessing cell viability. Most measure cell respiratory activity, and
depend metabolically active cells being able to reduce tetrazolium redox salts to insoluble products
detectable under the microscope. Several salts have been used with activated sludge, and include
2 (p-iodophenyl)-3-p(nitrophenyl)-5-phenyl tetrazolium chloride) or INT, which is reduced to red INT-
formazan crystals (Maurines-Carboneill et al., 1998) visible under light microscopy.
INT activity staining

Solution 1
INT – 0.02% aqueous solution, membrane sterilised if necessary, and stable at 5– for 1 month. INT can be
purchased from Kodak Chemicals.
Solution 2
37% solution of formaldehyde (or replaced by 6% glutaraldehyde), both of which are toxic and need to be
handled carefully.
Solution 3
Malachite green 0.05% aqueous solution.
Procedure
1. Add 1 ml of the INT solution to 10 ml mixed liquor, and incubate at room temperature for 30 min
in the dark.
2. After fixation with formaldehyde, spread 2 drops onto a clean slide and allow the smears to air
dry; gently heat fix.
3. Flood with malachite green stain for 1 min, and drain off (do not wash) before examining under
oil immersion.
CTC activity staining

More widely used is the more sensitive 5-cyano-2,3-tolyl-tetrazolium chloride (CTC), which is reduced to
an insoluble intracellular discrete fluorescent CTC-formazan precipitate, visible by epifluorescence
microscopy (Rodriguez et al., 1992). Other tetrazolium salts producing water soluble formazan products
which can be more readily quantified are also available, and have been applied to activated sludge
communities (McCluskey et al., 2005). Critical assessments of the value of CTC data (chapter 3) are
published (Créach et al., 2003; Gruden et al., 2003a,b; Nielsen et al., 2003a).
Procedure
Several recipes for CTC have been used with activated sludge, but a concentration between 2–6 mM
gives optimal formazan production. Higher levels may inhibit electron transport and hence reduce
product formation (Rodriguez et al., 1992). The best advice is to assess each sample with a range of
concentrations and select the best one. We use an equal volume of sample (mixed liquor or foam) and
5 mM CTC, and incubate samples at room temperature in the dark for 2h. Controls of formaldehyde
exposed cells should always be run in parallel to accommodate abiotic CTC reduction. Smears are
viewed by epifluorecence microscopy and a suitable mountant (e.g. Vectashield1, Citifluor1) with a
suitable excitation (420 nm) and a 590 nm emission cut-off filter. Reduced formazan precipitate appears
as brightly fluorescing red intracellular particles, which are not always visible separately microscopically
(Figure 11.12, see colour image section (chapter 13)). CTC can be purchased from Polysciences Inc. and
Sigma Chemicals.
LIVE/DEAD staining
Several kits are commercially available for assessing in situ cell viability. Included is the LIVE/DEAD1
BacLight1 method (Molecular Probes), based on dual staining of samples with the green and red DNA/RNA
stains SYTO1 (although others like SYBR1 gold can be substituted) and propidium iodide respectively.
These differ in terms of cell permeability properties. While SYTO1 penetrates all cells regardless of their
membrane integrity, propidium red only enters cells whose membranes are no longer functionally intact
(Williams et al., 1998). Thus, after staining, cells with intact membranes (i.e. live cells) fluoresce green,
while those with damaged membranes (i.e. dead cells) fluoresce red, where propidium iodide quenches the
green fluorescence. Suitable choice of excitation and emission filters allows ready distinction between these
by epifluorescence microscopy. However, results are not always clear cut, and ‘intermediate’ reactions often
occur, especially with Gram negative bacteria with their outer membrane (Berney et al., 2007).
Procedure
Optimise the protocol for your own samples, by adjusting relative amounts of the two stains to provide the
best color balance in viewed specimens. We do not use the kit but purchase separately the two stains,
SYBR1 gold and propidium iodide (both from Molecular Probes), which work well.
(i) A 40 ml aliquot of a 1:100 dilution of SYBR1 gold commercial stock in dimethly sulphoxide and
3 ml of propidium iodide (Molecular Probes) solution (1 mgl 1) is added to 1ml of either foam or
mixed liquor, and incubated at room temperature in the dark for 15–30 min.
(ii) An aliquot is then placed onto a clean microscope slide and viewed by epifluorescence
microscopy with suitable excitation and emission filters. Green cells are viable and red cells are
assumed to be dead (Figure 3.3).
This stain has been used with foaming Mycolata (Carr et al., 2005; Eales et al., 2006) and to monitor
effects of chlorination on filamentous bacteria causing bulking (Seka et al., 2003). Propidium monoazide
can be used instead of propidium iodide (Nocker et al., 2007).
In situ display of exoenzyme activity (ELF assays)

This method is used for in situ physiology studies of activated sludge organisms at a single cell level. It
provides information on which substrates organisms are metabolizing there, and several studies with
filamentous bacteria have employed it (chapters 5, 6, 7). ELF is based on enzyme linked fluorescent
(ELF1 97, Molecular Probes) probes, where the non-fluorescent substrate is converted enzymatically to
a fluorescent product, which precipitates on the surface of the active cell. CLSM or epifluorescence
microscopy allows that cell to be recognized. The range of substrates available is limited, so only a
few enzymes can be detected by ELF. Several methods have been published (Kragelund et al., 2005;
Nielsen et al., 2002b), but the one detailed here usually works well. Suitable controls (formaldehyde
treated samples) should always be included.
(i) A 20–100 ml aliquot of mixed liquor or foam is incubated with a 0.2 mM solution of the ELF
substrate for up to 3 h in the dark at room temperature.
(ii) Samples are placed on clean slides and viewed with an epifluorescence microscope at excitation
and emission wavelengths of 320–380 nm and 420 nm respectively (Kragelund et al., 2005).
Enzymatic activity is indicated by localized regions of the filaments fluorescing green
(Figure 11.13, see colour image section (chapter 13)).
ELF substrates are available from Molecular Probes for detecting esterase, lipase, chitinase/N-
acetylglucosaminidase, phosphatase, b-D-galactosidase and b-D-glucuronidase activity.
In situ detection of organisms with proteolytic activity

It is also possible to identify in situ cells showing exocellular proteolytic activities with 4-bora-3a, 4a-diaza-
s-indacene (BODIPY) derivatives (Xia et al., 2007, 2008b), covalently bound to proteins. BODIPY FL
bovine serum albumen and BODIPY FL casein are available from Molecular Probes. Samples are incubated
with these following the method of Xia et al. (2007), and spread onto gelatin coated welled slides in the dark.
Slides are mounted in Citifluor1 or Vectashield1 and examined under the epifluorescent microscope using
the 450–490 nm excitation filter and the 515 nm emission filter (Figure 11.14).
This can be combined with FISH to identify the populations involved, after fixing samples on slides
(following removal of the mounting fluid with 70% ethanol in a 1 min washing step), with either 4%
paraformaldehyde or 50% ethanol for 2 h for Gram negative and Gram positive bacteria respectively
(Xia et al., 2007, 2008b). FISH is carried out as detailed later (section 11.9). Suitable heated controls are
incorporated (Xia et al., 2007).
(a) (b)
Figure 11.14. In situ detection of protein hydrolyzing organisms (a) bright field image of filament with
attached cells and (b) after incubation with protease BODIPY substrate showing these attached cells
fluorescing, indicating their protein hydrolyzing properties (Xia et al. 2008b).
In situ detection of cell hydrophobicity (microsphere adhesion to cells,

MAC, assay)
Availability of fluorescent polystyrene microspheres (diam 0.02 mm) with different hydrophobicities allows
for in situ estimates of cell surface hydrophobicity (CSH). It is especially valuable for foaming filamentous
bacteria (Eales et al., 2006; Nielsen et al., 2001), whose morphology lends itself well to techniques of this
kind. Both hydrophilic and hydrophobic microspheres are available (Fluospheres1, Molecular Probes),
meaning cell surface types can be distinguished by the fluorescence from their binding preferences. The
method is that of Nielsen et al. (2001), and is the same for hydrophilic and hydrophobic microspheres.
(i) Microspheres are suspended in water to give a 0.2% (w/v) suspension, and dispersed by sonication
for about 90 min at 60 W output.
(ii) An equal volume of this suspension and mixed liquor/foam sample (10–20 ml of each) in 0.1 ml
water are vortex mixed for about 2 min, and then viewed by epifluorescence microscopy at
excitation/emission wavelenghts of 505 nm and 515 nm respectively.
Figure 11.15 shows the appearance of Gordonia amarae in foam after exposure to hydrophobic
microspheres, demonstrating its relatively high cell surface hydrophobicity.
It can be used in combination with FISH (Nielsen et al., 2001), where samples are immobilized on
gelatin coated slides before being treated, as described in section 11.9.
Figure 11.15. Fluorescence micrograph showing a GALO filament in activated sludge foam, with high in situ
cell surface hydrophobicity from its strong fluorescence. The MAC assay was carried out with hydrophobic
microbeads (K. Eales).
11.4 ISOLATION OF FILAMENTOUS BACTERIA FROM ACTIVATED

SLUDGE
Elizabeth M. Seviour
More basic microbiology needs to be carried out with the bulking and foaming filamentous bacteria if we
hope to understand them. More morphotypes need to be cultured from more plants, and
Micromanipulation is the method of choice, since these can be ‘identified’ under the microscope, and
physically isolated (Blackall et al., 1991a, 1994b; Levantesi et al., 2004, 2006b; Seviour et al., 1997).
Conventional isolation methods do not allow this, and so it is not possible to relate what might be growing
on the medium to what was present in the specimen. It has been used with success for isolating clustered
cells of Acinetobacter and phylogenetically diverse TFO from biomass of EBPR plants (Beacham et al.,
1990; Duncan et al., 1988: Maszenan et al., 1997, 2000), and denitrifying bacteria (Thomsen et al., 2004).
Micromanipulation has been used very successfully to harvest large amounts of a single filament
morphotype for subsequent molecular characterization and identification (Levantesi et al., 2004; Nittami
et al., 2009).
Micromanipulator
A Skerman (Skerman, 1968) micromanipulator attached to a 32£ long working distance objective lens
(Wild Leitz) or a 10£ objective lens is used (Seviour and Blackall, 1999c).
Procedure
(i) A drop of mixed liquor or foam diluted with water is placed on the surface of a plate of the chosen
solid medium.
(ii) A hook, formed by the method described by Skerman (1968) is then used to drag filaments
first recognized under the microscope away from surrounding biomass to the edge of the plates.
A ‘spoon’ (Skerman, 1968) is used for TFO micromanipulation.
(iii) Up to 10 filament morphotypes can be isolated in this way on each plate, which is then incubated
to allow these isolated colonies to grow.
Regular checking is essential to monitor their growth and possible overgrowth from contaminants.
If necessary further micromanipulation can then be used to physically move these isolates from
contaminants.
Comments
In our experience the technique requires practice to master, and even then the success rate for filament
isolation is still very low (51%). This probably reflects the isolation medium used, but the organisms may
be moribund. It is worth checking biomass first with the CTC viability stain detailed above. In this
laboratory the R2A medium of Reasoner and Geldreich, (1985) seems to perform better for filaments than
all the others tested (Beer et al., 2001; Blackall et al., 1994, 2000; Liu et al., 2000, 2001a). MSV has been
more successful in other laboratories (Levantesi et al., 2004, 2006b). However, being adventurous and
innovative (e.g. Liu et al., 2001a), may bring rewards (chapter 5).
11.5 COLLECTION OF DATA FROM MICROSCOPIC ANALYSIS AND USE

OF WORKSHEETS
Kenneth C. Lindrea and Elizabeth M. Seviour
With activated sludge samples, one must be aware that the sample volume viewed is small, and may
not necessarily be a true reflection of the original specimen. Both wet mounts and dried smears are
two-dimensional views of what is a three-dimensional environment. Methods for preparation of specimens
may affect the quality of the data collected. Consequently, conclusions drawn from any single sample
should be interpreted conservatively. Even within a single drop of sample, objects are not normally
distributed, i.e. one portion of the slide may show a tangled clump of filaments or a colony of higher
organisms like the protozoan Epistylis, which may not be seen elsewhere in the specimen. The flocs on
one quadrant may have extended filaments which are either absent elsewhere, or present in lower amounts
in the other areas of view. Examination of the specimen along the slide is recommended.
In ‘normal’ sludges, information sought concentrates on floc size and structure, presence or otherwise
of higher organisms, and filamentous bacteria and free cells and inorganic particles in the bulk liquid. Any
uncertainty of operations staff in interpreting their observations is understandable, since the activated
sludge community is complex. A daily examination may reveal changes in the nature of the biomass.
However, more commonly such changes take place slowly and imperceptibly, making their detection
difficult unless some objective record of the state of the sample is made, e.g. a carefully filled out work-
sheet or photomicrographic records.
Experience and practice in the microscopic examination of activated sludge is essential (Wilson and
Wharfe, 1989). There is no substitute for this, irrespective of the previous training of the observer
(Seviour et al., 1990). If more than one person is involved, then collaboration is essential to reach
agreement. Since evaluations are subjective, it is imperative that patience be employed, and careful
recording of the results of the observations must be made. The only reliable way is to use a work-sheet,
which is filled out as observations are made, and then kept as part of the plant’s records. Reference
photographs are available in a number of publications (Jenkins et al., 2004b; Eikelboom, 2006),
including this book (chapter 13). Alternatively, personnel can prepare a set of digitally acquired images
of their own. Colour posters from several sources and online images of high quality are available to
help. A poster produced by us and available from our laboratory (Seviour, 2004) showing the diagnostic
features of most commonly seen filamentous bacteria and information on their FISH probing is
recommended for laboratory staff with little prior experience in filament identification, but who are keen
to learn.
Laboratory worksheets for data collection

The worksheet used in this laboratory is included in the earlier version of our book, so is not shown here.
It was adapted from the two formulated by Jenkins et al. (2004b), allowing an assessment of.
[1a] Filament abundance,

[1b] The effect of filaments on floc structure,
[1c] Floc morphology,
[1d] Floc diameter and,
[1e] The presence or absence of free cells in suspension, Zoogloeal organisms and inorganic and
organic particles.
Alternative worksheets
As tools for in situ identification of activated sludge populations become less dependent on recognizing
morphotypes and more molecular, this worksheet will not summarize the information from such
approaches. Consequently, if methods like FISH are used, the one suggested by Wilderer et al. (2002)
with appropriate modification, might be worth considering as an initial guide, and provide the basis for a
personalized version.
11.6 DNA AND RNA EXTRACTION

Simon McIlroy and Kate Porter
INTRODUCTION
Critical to the success of culture independent analyses of activated sludge communities is the isolation of
high quality nucleic acids. However, the extraction method used may result in considerable bias (see von
Wintzingerode et al., 1997). So selecting a suitable protocol is essential, or populations resistant to the
lysis method chosen will be either under-represented in subsequent analyses or completely excluded from
them, producing a skewed starting point for all further explorations (von Wintzingerode et al., 1997).
A limited number of methods have been described for isolating nucleic acids from activated sludge
samples (Bourrain et al., 1999; Gabor et al., 2003; Goubin et al., 2008; Lemarchand et al., 2005; McIlroy
et al., 2008b; Roh et al., 2006; Orsini and Romano-Spica, 2001; Purohit et al., 2003; Smalla 1995; Yu and
Mohn, 1999). Most studies have utilised either commercially available kits, or adapted methods designed
for soil populations, without exploring whether these protocols are suitable.
While many problems encountered in bacterial nucleic acid extraction are common to all
environmental samples, each presents its own set of challenges, and the techniques used need to be
adapted accordingly (Purdy, 2005). This chapter discusses important principles in dealing with activated
sludge samples and details two validated methods for simultaneous extraction of DNA and RNA.
SAMPLING
To prevent nucleic acid degradation (in particular RNA), activated sludge samples should be processed
immediately on acquisition. Since this is not always possible, especially when working in the field or with
large numbers of samples, an alternative is to use solutions such as RNA later (sold by Ambion and
QIAGEN), designed to prevent degradation of RNA between sampling and processing. Samples can be
frozen immediately and processed at a later date (Juretschko et al., 2002; Nogales et al., 2002;
Rochelle et al., 1994), although some RNA degradation may still occur. They may be fixed with ethanol
or formaldehyde, although those fixed in ethanol should be processed within 24 h of sampling so as to
avoid lysis of Gram negative cells (Juretschko et al., 2002). In addition, fixing may affect adversely the
ability to obtain PCR amplifiable DNA (Wallner et al., 1997).
Pre-treatment of sample
Activated sludge samples frequently contain polysaccharides, proteins and humic acids which can co-
purify with the nucleic acids and affect downstream applications. To help reduce their impact, biomass
can be washed before the lysis stage (Fortin et al., 2004; Orsini and Romano-Spica 2001; Purohit et al.,
2003), or the sample can be centrifuged and resuspended in an appropriate buffer, like Tris-
ethylenediaminetetraacetic acid (EDTA). Given the short half-life of bacterial mRNAs (Kaberdin
and Bläsi, 2006), if RNA is a desired target then any pre-treatment steps should be limited and performed
at 4– C.
CELL LYSIS
Adequate lysis of all the populations present in a sludge sample is the most important step in
the extraction process. Approaches to enhance cell lysis for environmental samples have included
chemical (Zhou et al., 1996), enzymic (Porteous et al., 1994), mechanical (Ogram et al., 1987), heat
(Picard et al., 1992), and freeze-thawing (Tsai and Olson, 1991) cell disruption. These are often used in
combination to increase lytic efficiency. Applying multiple extraction methods to the same sample
(Juretschko et al., 2002; von Wintzingerode et al., 1997) increases the probability of obtaining represen-
tative nucleic acids from all the populations present. This is supported by community profile studies where
data suggest that they vary in their susceptibility to different lysis methods (Frostegård et al., 1999; Krsek
and Wellington, 1999). It is important to note that the ‘fragile’ strains (i.e. those easily lysed by most
methods) can be frequently over-represented, while ‘tough’ cells (i.e. those resistant to most lysis
techniques) may not be represented at all in the final nucleic acid pool.
Mechanical lysis
Activated sludge biomass contains cellular aggregates or flocs that vary in size and can be robust (Yu and
Mohn, 1999; McIlroy et al., 2008b). These flocs and the individual microcolonies they contain are often
heavily encapsulated, which presents an additional barrier to achieving complete cell lysis (Ahn et al.,
2007). Mechanical lysis may be essential in breaking apart aggregates and allowing individual cells within
the flocs to become accessible to subsequent lysis methods (Bourrain et al., 1999; McIlroy et al., 2008b;
Watanabe et al., 1998; Yu and Mohn, 1999). Bead beating is the most popular mechanical method and
involves vigorously shaking the sample in the presence of small glass, zirconia or ceramic beads that tear
the flocs and cells apart. Unfortunately, bead beating also shears high molecular weight nucleic acid and
reduces its quality for some applications. The smaller DNA fragments increase the probability of chimeric
sequences forming during PCR (Liesack et al., 1991). An alternative to bead beating is grinding samples
in liquid nitrogen. This technique provides more intact nucleic acids (Zhou et al., 1996), although it is
time consuming and hence inappropriate for high throughput analysis. Sonication is another approach to
mechanical lysis, which uses ultrasound to physically break apart cells. However, it requires careful
monitoring to avoid shredding of the nucleic acids (Krsek and Wellington, 1999; Picard et al., 1992).
Chemical lysis
A range of chemicals are included in nucleic acid extraction buffers to aid in cell lysis. Surfactants
like sodium dodecyl sulphate (SDS), sarkosyl or cetyl trimethylammonium bromide (CTAB) break
membrane lipids, while phenol and/or chaotropic agents (sodium perchlorate, guanidinium thiocyanate
or sodium trichloroacetate) denature proteins and separate them from other cellular components (Purdy,
2005; Summerton et al., 1983). Chemical lysis is also frequently combined with heat to increase lysis
efficiency (Roose-Amsaleg et al., 2001), although this can also accelerate degradation of nucleic acids,
particularly RNA.
Enzymatic lysis
Enzymatic digestions of cell walls can facilitate release of nucleic acids and have been incorporated into
extraction protocols. Enzymes utilised include lysozyme, mutanolysin, achromopeptidase and pronase
(Roose-Amsaleg et al., 2001). Although these can assist in achieving representative cell lysis, enzymatic
digestion steps should be avoided if RNA is required, as these are also ideal for RNase activity.
Furthermore, as incubation periods are long, care must be taken to ensure that the digestion step does not
encourage a shift in the community composition.
Freeze-thaw lysis
Freeze-thaw involves successive rapid freezing and thawing of cells, creating instability in the cell
membranes and making them more susceptible to subsequent chemical and enzymic lysis (Moore et al.,
2004). Freeze-thaw is rarely used alone, but is typically applied in combination with other methods.
ASSESSING BIASES IN DNA EXTRACTION

Proving that unbiased cell lysis has occurred is difficult with environmental samples. Several methods have
been used to gauge lysis efficiency. A relatively simple approach is to quantify nucleic acids yields and
compare them to theoretical yields (Zhou et al., 1996), although nucleic acid losses during extraction make
this of little value except in the most extreme cases of poor lysis. Alternatively the cell material remaining after
the lysis stage can be examined - the presence of large amounts of intact cellular material would suggest lysis
is inadequate. Nucleic acid dyes like 40 ,6-diamidino-2-phenylindole (DAPI) (Miller et al., 1999), acrinidine
orange (Tsai and Olson, 1991), or 5-(4,6-dichlorotriazin-2-yl)amino fluorescein (DTAF) (Zhou et al., 1996)
should be used in any microscopic assessment, as cells can appear intact even when their nucleic acid has been
extracted (‘ghost cells’). Both approaches provide only a rough guide to lysis efficiency and should not be
used alone.
Lysis efficiency can be assessed by spiking a sample with (or applying the method on) an organism
known to resist lysis (Moré et al., 1994; Picard et al., 1992). Lysis of this ‘tough’ strain may then be
assessed either microscopically, or with targeted primers and PCR. Addition of either naked DNA, or
readily lysed cells, reveals whether the early nucleic acid released from fragile target cells is lost from the
final DNA pool (Frostegård, et al., 1999). Neither provides a true indication of unbiased lysis as lysis of
the ‘spiked’ organisms does not guarantee necessarily that those naturally present in the sample have also
lysed (Zhou et al., 1996), especially as they would not be associated with flocs.
An alternative is to use molecular methods to identify target groups known to be present in the sample
(Kuske et al., 1998; Picard et al., 1992). Ideally these should be diverse populations, one very sensitive to
lysis and another resistant (for example see Figure 11.16b). This approach requires a prior knowledge of
the community composition, which can be obtained with methods like FISH, or community profiling
techniques such as DGGE. Such profiles can be used after each extraction step to assess changes in levels
of diversity (Krsek and Wellington, 1999). However, it is difficult to determine from DGGE profiles
whether changes in diversity reflect the extraction protocol, or result from other downstream biases like
PCR artefact formation.
It seems that no single definitive way exists to assure unbiased isolation of nucleic acid from activated
sludge samples, and so multiple parameters should be applied to validate the method chosen.
(New Eng. Biolabs)

2-log DNA ladder
1kb DNA ladder
NaTCA Method
NaTCA Method
NaTCA Method
Phenol method
Phenol method
(Promega)
MobioKit
MobioKit
(a) (b)
10,000 bp
DNA
Defluviicoccus
Dechloromonas
23S
1,500 bp
16S 500 bp
200 bp
250 bp
5S
Figure 11.16. (a) Total nucleic acid extract from the biomass in an EBPR SBR run on a 1% agarose gel.
(b) PCR targeting groups within the SBR community with DNA extracts from different extraction methods run
on a 1.5% agarose gel. The phenol method was adapted from McVeigh et al. (1996).
PURITY
Polysaccharides, protein and humic acids in activated sludge will co-purify with nucleic acids, which can
affect downstream applications. Some of these contaminants may be removed by biomass washing, but
additional treatments may be necessary. The treatment required to remove these contaminants varies as
their types and levels will differ between samples depending on the composition of the waste being
treated, and plant configuration. Commercially available columns such as the Elutip-d1 column
(Whatman) can be used to further increase nucleic acid purity (Picard et al., 1992). Nucleic acids may be
extracted from agarose gels after electrophoresis, a technique that also removes any small fragments of
DNA (Porteous et al., 1994). It is important to bear in mind that each additional step sacrifices yield, time
and money, so the extent of purification will depend on the quality of nucleic acids required for each
purpose.
Protein contaminants
Nucleases may degrade nucleic acids or interfere with later downstream techniques. They may be
removed with phenol-chloroform extraction, caesium chloride gradients, proteinase K digestion,
hydroxyapatite chromatography (Purdy, 2005), or sodium trichloroacetate (Summerton et al., 1983).
In addition, nucleases may be inactivated by the inclusion of diethylpyrocarbonate (DEPC) (Yu and
Mohn, 1999), reducing agents such as dithiothreitol (DTT) or b-mercaptoethanol, or divalent cation-
chelating compounds including EDTA (Wilkins and Lawrence, 1996).
Humic acid contaminants

Humic acids are present in most activated sludge samples, although often at lower concentrations than
in soil. These can interfere with PCR, restriction enzyme digests and hybridization based methods
(Milling et al., 2005). Addition of compounds like CTAB, polyvinylpyrrolidone (PVP) and polyvinyl-
polypyrrolidone (PVPP) to the extraction buffer may help in their removal (Wilkins and Lawrence, 1996;
Zhou et al., 1996).
Polysaccharide contaminants
Polysaccharides can make up a large proportion of activated sludge suspended solids (see chapter 3),
and interfere with resuspension of nucleic acids and their electrophoresis (Wilkins and Lawrence,
1996). Addition of CTAB will precipitate acidic polysaccharides (Moore et al., 2004). Alternatively,
potassium ethyl xanthogenate may be used to render the polysaccharides more soluble (Tillett and
Neilan, 2000).
WORKING WITH RNA

RNA is particularly susceptible to degradation and so special precautions need to be taken. It is important
to remember that autoclaving equipment and solutions will not always deactivate RNases (Sambrook and
Russell, 2001). The following precautions are recommended.
. All glassware/spatulas should be baked at 300– C for 4 h.

. Treat plasticware with DEPC or soak in 1% SDS for 2 h.
. Always use certified nuclease free chemicals and disposable plasticware.
. All solutions must be treated by exposure to 0.1% DEPC and incubated at 37– C for at least 1 h or
overnight at room temperature. Solutions that can not be treated (such as Tris buffers) should be
made with DEPC treated water.
. Use fresh solutions or fresh aliquots of solutions.
. If possible, dedicate equipment and space to RNA work only.
. Work quickly and analyse extracted RNA as soon as possible.
. Avoid breathing on samples and if possible use filter tips.
METHOD FOR THE SIMULTANEOUS EXTRACTION OF DNA AND RNA

FROM ACTIVATED SLUDGE
A range of methods have been described for extracting nucleic acids from environmental samples. We
have investigated many of these and detail here one protocol we found was successful with activated
sludge samples (McIlroy et al., 2008b). It allows the simultaneous extraction of DNA and RNA and
provides efficient biomass lysis (Figure 11.16a). The nucleic acids obtained are of sufficient quality for
downstream applications, including PCR, reverse transcription PCR, real time PCR, and restriction
enzyme digestion. The method also eliminates the use of phenol and chloroform, both of which
are toxic.
NaTCA METHOD
Solutions/reagents
. 4.5 M sodium trichloroacetate (NaTCA)1
. 6 £ Lysis Buffer2
300 mM Tris-HCl, pH 8.5
90 mM EDTA, pH 8.5
6% (w/v) N-lauroylsarcosine
6% (w/v) PVP
60 mM DTT
. 50% (v/v) Dow Corning 1520-US antifoam (Dow Corning, Midland, USA)3
. Tris-HCl EDTA (TE) buffer
10 mM Tris-HCl, pH 8.0
1 mM EDTA, pH 8.0
. 70% (v/v) ethanol
. 2-propanol
. 2 M NaOH
. 10 M NaOH
. DEPC
4.5 M NaTCA stock preparation

(i) Add trichloroacetic acid (TCA) in a sterile beaker on ice on a magnetic stirrer to a small volume of
sterile distilled water4 and continuously stir.
(ii) Slowly add 10 M NaOH with a dropping funnel, never allowing the solution to exceed 50– C, until
the TCA is almost neutralized5.
(iii) Adjust the pH of the solution to between 6.7 and 7.3 using 2 M NaOH solution and adjust the final
volume to give a final concentration of 4.5 M NaTCA.
(iv) Store the solution at 20– C6.
Equipment
. 0.1 mm diameter glass beads (Biospec, OK, USA)
. 1 mm diameter glass beads (Biospec, OK, USA) (optional)7
. 1.5 ml microcentrifuge tubes
1
See below for preparation instructions.
2
Store at 20– C.
3
This antifoam is quite viscous so dilution to 50% makes it easier to pipette. This antifoam may be
autoclaved.
4
Use only enough water to allow the magnetic stirred to mix the TCA. The TCA will dissolve as
NaOH is added. Too much water will ‘overshoot’ the final desired concentration.
5
The volume required should be pre-calculated.
6
This solution will degrade at 4– C. If degradation has occurred a brown pellet sometimes forms during
2-propanol precipitation.
7
A small number of larger sized beads can help in the disruption of larger bacterial aggregates.
. 2.0 ml microcentrifuge tubes

. 2.0 ml screw-cap microcentrifuge tubes (bead beater compatible)
. Mini bead beater (Biospec, OK, USA)
. Refrigerated microcentrifuge
. Heating block or waterbath
. Magnetic stirrer
. Dropping funnel
. Thermometer
Protocol
Before beginning:
. Set the heat block to 37– C

. Prepare NaTCA lysis buffer by mixing (per sample þ 1):
W 1000 ml 4.5 M NaTCA
W 250 ml 6 £ lysis buffer
W 50 ml 50% (v/v) antifoam
W 200 ml DEPC treated distilled water8
(i) Add 10–15 mg (wet weight) of biomass to a 1.5 ml microcentrifuge tube and centrifuge at 7,000 g,
5 min, 4– C.
(ii) Decant the supernatant and resuspend the biomass pellet in 1,500 ml NaTCA lysis buffer and 0.6 g
of 0.1 mm diameter glass beads. Homogenize the cells in a mini- bead beater at maximum speed
for 3 min9.
(iii) Pellet cell debris by centrifugation (20,000 g, 5 min, 4– C).
(iv) Transfer the supernatant to a fresh 1.5 ml microcentrifuge tube containing 0.6 vol 2-propanol. Mix
well.
(v) Precipitate the nucleic acids on ice for 15 min before collecting them by centrifugation (20,000 g,
5 min, 4– C).
(vi) Carefully remove the supernatant and discard.
(vii) Wash the nucleic acid pellet twice with 1 ml 70% (w/v) ethanol.
(viii) Remove as much of the ethanol as possible and air dry the pellet for 15 min.
(ix) Resuspend the pellet in 50–100 ml TE buffer and incubate at 37– C for 15 min.
(x) Mix the pellet by pipetting up and down and centrifuge the tube at 20,000 g, 30 sec, at room
temperature.
(xi) Transfer the supernatant to a fresh tube and store at 70– C until required10.
8
If the sample is being added directly to the buffer then adjust the volume of water added accordingly.
9
It is essential that beating time is optimized as samples will require different beating times and
intensities to lyse cells. Inadequate times will result in non-representative extraction and excessive beating
time will unnecessarily damage the nucleic acids.
10
If required, DNA or RNA can be removed by DNase or RNase digestions respectively.
11.7 POLYMERASE CHAIN REACTION (PCR) TECHNOLOGY

Toshikazu Fukushima and Philip L. Bond
INTRODUCTION
Polymerase chain reaction (PCR) is used routinely for culture-independent analyses of activated sludge
communities and their functions. The ribosomal RNA (rRNA) gene has been targeted to determine overall
population biodiversity while genes, for example, like polyphosphate kinase (ppk; McMahon et al., 2002a, b)
nitrite reductase (nir; Braker et al., 1995) and ammonia monooxygenase (amo; Rotthauwe et al., 1997)
targets those of functional relevance like the PAO, denitrifying organisms and the AOB nitrifying bacteria.
However, as detailed in chapter 3, bias and artefact formation can occur in multi-template PCR
for several reasons, and thus provide misleading information on natural community composition
(Acinas et al., 2005; Chandler et al., 1997; Kanagawa et al., 2003; Kurata et al., 2004). Consequently,
PCR based methods do not provide quantitatively valid information.
PCR techniques have thus been developed to overcome some of these problems by increasing amplification
specificity and amplicon yield. Real-time quantitative PCR (qPCR) methods were also developed to allow
targeted gene copy numbers to be quantified. Additionally reverse transcription PCR (RT-PCR) allows RNA
such as rRNA and messenger RNA (mRNA) to be amplified, which allows detection of active populations
and because of easier extraction of RNA instead of DNA, are more sensitive. This technique has been
used successfully to identify small numbers of micromanipulated filamentous bacteria in bulking activated
sludge (e.g. Levantesi et al., 2004). The basic steps involved in PCR are represented below (Figure 11.17).
This section discusses in detail aspects of PCR particularly relevant to activated sludge samples.
PCR PRIMERS USED FOR AMPLIFICATION FROM ACTIVATED SLUDGE DNA

Many different targeted primer sets have been described and applied to activated sludge communities
process. These target rRNA genes and appropriate functional genes.
PCR primers for the 16S rRNA gene

An average bacterial 16S rRNA molecule is about 1,500 nucleotides in length. When fully or almost
fully sequenced (at least 41,000 nucleotides), it contains sufficient conserved and variable nucleotide
regions for reliable phylogenetic analyses (Amann et al., 1995). Therefore, 16S rRNA targeted primers
have been used widely to obtain PCR amplicons directly from DNA extracted from activated sludge.
Universal and domain-targeted primers for 16S rRNA genes were described by Lane (1991), and are still
widely used (Table 11.1) although as databases (e.g. Ribosomal Database Project (http://rdp.cme.msu.edu/
index.jsp) (Cole et al., 2009) increase in size, these primers often need to be modified and updated
(chapter 3). Often, primers that target a particular phylogenetic group are required. It can be a challenge
to find sequence regions with the appropriate consensus that capture 100% of the group sought, and
which at the same time exclude all other organisms. As new sequences are added continually to the
database, group specific primers may become obsolete, as they have mismatched sequences for intended
species. The repeated discovery of new higher taxa after 16S rRNA gene sequence analyses indicates
that ‘prokaryotic’ biodiversity is still poorly represented in these data bases (Baker et al., 2003;
Guermazi et al., 2008), and so design of domain and group specific primers requires further development
(Forney et al., 2004).
1st cycle
dsDNA fragment to be amplified
Denature
Annealing
Primer
Extension
2nd cycle
20-30
cycles
Figure 11.17. A schematic of PCR amplification procedure. Initial heating causes the dsDNA duplex (top) to
separate into two single strands (denature). Then targeted primers hybridize to the appropriate sites on each of
the denatured single strands (annealing). Finally complementary DNA strands are synthesised with DNA
polymerase and dNTP (extension) using the single strands as templates. At the end of the 1st cycle, two DNA
duplexes are presents. The same three- step process of (1) denature, (2) annealing, and (3) extension is
repeated in each PCR cycle. After 20–30 cycles, targeted DNA sequences are amplified more than 106-fold.
PCR primers for 23S rRNA genes

The 23S rRNA gene shares suitable features with the 16S rRNA gene (e.g. universal distribution,
conserved function and both conserved and variable regions), yet it includes additional diagnostic
nucleotide sequence stretches because of its increased length (approx. 3,000 nucleotides), characteristic
insertions and/or deletions, and possibly better phylogenetic resolution resulting from its greater sequence
variation (Ludwig and Schleifer, 1994).
Domain targeted primers targeting the 23S rRNA gene have also been also described by Lane (1991)
(Table 11.2). However, application of of the 23S rRNA gene for bacterial community analysis is limited
by lack of established broad-range bacterial PCR amplification and sequencing primers (Hunt et al.,
2006). Hence more 23S rRNA gene sequences are required so that the universal primers currently
available can be re-evaluated.
Table 11.1. Universal or domain-targeted primers targeting the 16S rRNA gene (Lane, 1991).
Position according
Primer to E. coli numbering Target Sequence (50 -30 )
27f 8–27 Most Bacteria AGAGTTTGATCMTGGCTCAG
357f 339–357 Most Bacteria CTCCTACGGGAGGCAGCAG
530f 515–530 Most Bacteria, Archaea GTGCCAGCMGCCGCGG
926f 907–926 Most Bacteria, Archaea AAACTYAAAKGAATTGACGG
1114f 1099–1104 Most Bacteria GCAACGAGCGCAACCC
1406f 1391–1406 Most Bacteria, Archaea TGYACACACCTCCCGT
342r 357–342 Most Bacteria CTGCTGCSYCCCGTAG
519r 536–519 Most Bacteria, Archaea GWATTACCGCGGCKGCTG
907r 926–907 Most Bacteria, Archaea CCGTCAATTCMTTTRAGTTT
1100r 1114–1100 Most Bacteria GGGTTGCGCTCGTTG
1392r 1406–1392 Most Bacteria, Archaea ACGGGCGGTGTGTRC
1492r 1513–1492 Most Bacteria, Archaea TACGGYTACCTTGTTACGACTT
1525r 1542–1525 Most Bacteria, Archaea AAGGAGGTGWTCCARCC
Table 11.2. Universal bacterial primers for 23S rRNA gene (Lane, 1991).
Primer Sequence (50 -30 )
115r GGGTTBCCCCATTCGG
130f CCGAATGGGGVAAGGG
242r KTTCGCTCGCCRCTAC
256f AGTAGYGGCGAGCGAA
559r CATTMTACAAAAGGYACGC
577f GCGTRCCTTTTGTAKAATG
1091r RGTGAGCTRTTACGC
1104f WGCGTAAYAGCTCAC
1608r CYACCTGTGWCGGTTT
1623f AAACCGWCACAGGTRG
1930r CGACAAGGAATTTCGCTAC
1948f GTAGCGAAATTCCTTGTCG
2053r CAYGGGGTCTTTRCGTC
2069f GACGYAAAGACCCCRTG
2241r ACCGCCCCAGTHAAACT
2253f GGYACAAADTGACCCC
2498r GAGYCGACATCGAGG
2512f CCTCGATGTCGRCTC
2654r CCGGTCCTCTCGTACT
2669f AGTACGAGAGGACCGG
2747r GYTTAGATGCYTTC
2758f YTGAARGCATCTAA

Probable typographic error in the published primer sequence. (Hunt et al., 2006).
PCR primers for functional genes

Functional genes that code for proteins with important roles in activated sludge communities can be the
target of PCR amplification. They may provide additional phylogenetic information on population
biodiversity of the functional populations there. Several primers for functional genes have been designed
for activated sludge community analyses, including those targeting genes thought to be important for
substrate or nutrient removal (i.e. carbon, nitrogen, phosphorus cycles) (Table 11.3).
Table 11.3. Examples of PCR primers for detection of functional genes in activated sludge.
Primer Target gene Sequence (50 -30 ) Reference
nirK1F nitrite reductase (nirK) GGMATGGTKCCSTGGCA Braker et al. (1995)
nirK4R nitrite reductase (nirK) GGRATRARCCAGGTTTCC Braker et al. (1995)
nirS1F nitrite reductase (nirS) CCTAYTGGCCGCCRCART Braker et al. (1995)
nirS5R nitrite reductase (nirS) CTTGTTGWACTCGSSCTG- Braker et al. (1995)
CAC
amoA-1F ammonia monooxygenase GGGGTTTCTACTGGTGGT Rotthauwe et al. (1997)
(amoA)
amoA-2R ammonia monooxygenase CCCCTCKGSAAAGCCTTC- Rotthauwe et al. (1997)
(amoA) TTC
NLDE-0199F polyphosphate kinase (ppk) CGTATGAATTTTCTTGGTA- McMahon et al. (2002a)
(NLDE-F) TTTATTGTACTAATCT-
ngaygarttyta
TGNY-1435R polyphosphate kinase (ppk) GTCGAGCAGTTTTTGCAT- McMahon et al. (2002a)
(TGNY-R) GAwarttnccngta
phaCF1 PHAb synthase (phaC) ATCAACAARTWCTACRT- Sheu et al. (2000)
CYTSGACCT
phaCR4 PHAb synthase (phaC) AGGTAGTTGTYGACSMMR- Sheu et al. (2000)
TAGKTCCA
(Forward primer)c Aconitase B (acnB)d GATGCTCGGTGGCTA- Burow et al. (2008b)
CAACG
(Reverse primer)c Aconitase B (acnB)d CTTGACATCGTGGAA- Burow et al. (2008b)
GAAGTCG
(Forward primer)c Isocitrate lyase (ICL)d AAGGTCGTCGAGCGCATC Burow et al. (2008b)
(Reverse primer)c Isocitrate lyase (ICL)d CATCAGTTCGAAGGCGTT- Burow et al. (2008b)
GAG
(Forward primer)c Succinate dehydrogenase ATCCGGACAAGTTCGTCGG Burow et al. (2008b)
(sdhB)d
(Reverse primer)c Succinate dehydrogenase GGACAGACATCGACG- Burow et al. (2008b)
(sdhB)d CAATTC
(Forward primer)c Cytochrome b/b6 (cyb/b6)d ATGCTGCTGCACCTCGTTC Burow et al. (2008b)
(Reverse primer)c Cytochrome b/b6 (cyb/b6)d CAGTTGGTCCCAGAC- Burow et al. (2008b)
GATCC
a
The consensus clamp is shown in capital letters, while the degenerate core is shown in lowercase letters.
b
polyhydroxyalkanoate
c
not named.
d
Only Candidatus ‘Accumulibacter phosphatis’ metabolic genes are targeted.
Primer design
The specificity of a primer is a crucial factor in successful PCR amplification. To amplify only the
intended fragment, primers should have 100% complementarity to the target sequence only. Computer
programs like Primer3 (Rozen and Skaletsky, 2000), and PRIMROSE (Ashelford et al., 2002) or the
PROBE-DESIGN tool of the software package ARB (Ludwig et al., 2004) are helpful in finding suitable
primer sequence regions.
Melting temperature (Tm), primer length and GC content (GþC mol%) are also key parameters in
primer design. The Tm is the temperature at which half the d/s DNA strands are single-stranded. The Tm
can estimated by the nearest-neighbour method (Breslauer, et al., 1986) or other generic formula
(e.g. Tm ¼ 4(G þ C) þ 2(A þ T)– C) (Roux, 1995). Thus, both primer length and GþC mol% contribute
to the primer Tm. Recommended ranges of Tm, primer length and GþC mol% are 56–62– C, 18–24 bases,
and 40–60%, respectively (Dieffenbach, et al., 1995b; Hyndman and Mitsuhashi, 2003) It is also
recommended that the Tm for each primer of a pair is within 2 to 5– C of the other (Grunenwald, 2003).
A primer sequence that contains self complementarity may lead to formation of hairpins and primer-
dimers, and should be avoided (Hyndman and Mitsuhashi, 2003). A hairpin is a structure formed by a
single DNA molecule in which a portion of one part of the DNA hybridizes to a complementary region
within the same DNA strand. The hairpin adversely affects the primer’s ability to bind to the target site.
Primer-dimer formation occurs by hybridization of the primer to itself or to the other primer in the pair.
Again this will affect the primer’s ability to anneal to the target site. Thus, to prevent primer-dimer
formation, complementary sequences within a primer and between the primer pair should be avoided.
PRINCIPLES AND FACTORS AFFECTING PCR AMPLIFICATION

PCR is a fundamental technique and is extremely successful in generating amplified regions (amplicons)
of DNA. However, challenges exist in obtaining enough PCR product and achieving specificity. These are
often sample specific, as particular DNA templates are poorly or not amplifiable under standard PCR
reaction conditions. Performance of PCR is susceptible to substances that interfere with the DNA
polymerase reaction and intrinsic DNA properties, as detailed below. Therefore, optimizations of PCR
reagents, amplification conditions (annealing temperature and number of PCR cycles) and purification of
template DNA are imperative.
Magnesium concentration
Magnesium (Mg2þ) concentration is important for successful amplification. In the absence of adequate
free Mg2þ, the DNA polymerase is inactive, while overly high Mg2þ concentrations may lead to spurious
priming (Eckert and Kunkel, 1990). The concentration of template DNA and presence of proteins may
affect the amount of free Mg2þ. Therefore, optimizing its concentration is required for individual nucleic
acid extracts with the aim of achieving the highest amplification efficiency and specificity. Although its
optimal concentration range can vary with the DNA polymerase used, a typical two-step optimization
series might first include adding Mg2þ at 0.5 mM increments from 0.5 to 5.0 mM and, after the range is
narrowed, at several 0.2 or 0.3 mM increments (Roux, 1995).
Co-solvents and other additives

To increase PCR efficiency and specificity, PCR co-solvents have been popular. Betaine improves DNA
amplification by reducing formation of any secondary structure caused by GC-rich regions, and therefore,
may be applicable generally to ameliorate amplification of GC-rich DNA sequences (Henke et al., 1997).
Dimethyl sulfoxide (DMSO) and formamide are also useful with GC-rich DNA templates by interfering
with the formation of hydrogen bonds between the two DNA strands (Geiduschek and Herskovits, 1961;
Kolmodin and Birch, 2002).
Adding other purified proteins to the PCR reaction mixture may improve amplification, serving to
‘mop-up’ any components that may interfere with the activity of the DNA polymerase. The presence of
400 ng/mL of bovine serum albumen (BSA) or 150 ng/mL gp32 increases the PCR tolerance by 10 to 1000-
fold towards inhibitors like FeCl3, fulvic acids and tannic acids (Kreader, 1996). Although these co-
solvents and additives help some PCR reactions, their use should be tested empirically for each template/
primer combination (Kolmodin and Birch, 2002).
Primer annealing temperature

The annealing temperature of primer to template DNA is a critical factor in PCR and needs to be
optimized for each primer pair. In addition, annealing of primer to template DNA is a function of the
thermodynamics of the hydrogen bonding between the base pairs. Thus, the annealing temperature is
sequence specific, and is related to the Tm, whose estimation is useful as a guide for its empirical
optimization. Generally, as annealing temperature is increased, binding stringency increases, which
reduces non-specific primer annealing and thus decreases the amount of undesired products generated.
Further increases in temperature may decrease the annealing reaction and lower amplification product
yields. Typically the objective of any PCR optimisation is to use a low annealing temperature where the
reaction is still specific and non-specific products are not generated (Sipos et al., 2007).
Cycle number
The number of PCR amplification cycles also requires optimization. Increased amounts of PCR product
are obtained by increasing this number. However, this will eventually decrease the activity of the DNA
polymerase, and so other reagents in the PCR reaction mix will become limiting. Generally composition
of the PCR reaction mix is designed for cycle numbers of between 20 to 35.
An important consideration when PCR is performed on DNA samples that are mixtures of homologous
genes is the formation of heteroduplexes and amplification of chimera sequences. A heterodupulex can
form by cross-hybridization of two different sequences that may share high sequence homology. If such a
recombination occurs during the PCR denaturation-annealing step, then amplification will lead to
chimeric products composed of parts of two different sequences. Chimera formation is thought to occur at
higher rates over the last few cycles of a PCR reaction, and thus incorrect information on abundance of
individual genes and diversity of gene sequences in any community will result (Kanagawa, 2003). This
problem is very common in analyses of complex microbial communities and is not restricted to 16S
rRNA gene amplification (von Wintzigerode and Goebel, 1997). Such problems can be avoided partially
by reducing the number of PCR cycles. In addition, chimeras of 16S rRNA gene sequences can be
identified with the several chimera check programs now available (e.g. that in the Ribosomal Database
Project – Maidak et al., 1999).
DNA template
Successful PCR amplification depends on the quality of the DNA template used. Factors that may inhibit
amplification of DNA by PCR arise from several sources, and many substances in activated sludge inhibit
enzyme activity, including proteins, humic acids, polysaccharides and metal ions (Straub et al., 1994).
Prior to embarking on any study, especially one encompassing many samples, it is critically important to
determine that the DNA extraction method selected is suitable in providing a template amplifiable by
PCR (section 11.6). Performance of PCR will be affected too by the propensity of DNA to form
secondary structures and its GC mol% content, which affects denaturation (Ralser et al., 2006).
Furthermore, some chemicals and reagents used for nucleic acid extraction may inhibit PCR, and these
include residual sodium dodecyl sulfate (SDS) and phenol, inhibitors of Taq DNA polymerase (Griffin
and Griffin, 1994) and Tth DNA polymerase (Katcher and Schwartz, 1994) respectively. Thus,
appropriate purification methods during DNA extraction from activated sludge samples are essential (see
section 11.6).
AN EXAMPLE PCR METHOD USING DOMAIN TARGETED BACTERIAL

PRIMERS
Here, a typical protocol adapted from Ahn, et al. (2007) is described for PCR amplificaton of bacterial
domain sequences. As stressed above, individual PCR protocols should be optimized for cycle conditions
and primer, reagent and DNA template concentrations (Table 11.4).
Table 11.4. Components of a PCR reaction mix of 50 mL.

Component Volume Final Conc.
10£ AmpliTaq Gold reaction buffer 5 mL 1£
2 mM dNTP 5 mL 200 mM
10 mM Forward primer (27f) 0.125 mL 25 pM
10 mM Reverse primer (1525r) 0.125 mL 25 pM
10% (vol/vol) dimethyl sulfoxide 7.5 mL 1.5%
50U AmpliTaq Gold Taq polymerase 2.5 mL 2.5U
25 mM MgCl2 solution 5 mL 2.5 mM
Template DNA prepared from a dilution of the extracted DNA X mL 51 mg/reaction
Distilled Water (DNase/RNase Free) Y mL
Solutions/reagents
. AmpliTaq Gold reaction buffer (Applied Biosystems)
. Deoxynucleoside triphosphates (dNTP; Promega)
. 10 mM Forward Primer (27f)
. 10 mM Reverse Primer (1525r)
. Dimethyl sulfoxide (Sigma)
. AmpliTaq Gold Taq polymerase (Applied Biosystems)
. MgCl2 solution (Applied Biosystems)
. DNase/RNase free distilled water (ultra PURE; invitrogen)
Equipment
. 0.2mL thin-walled PCR tubes
. Thermal cycler (e.g. iCycler; Bio-Rad)
. Clean laboratory bench
. Ice or aluminum tube rack
Protocol
(i) Thaw the AmpliTaq Gold reaction buffer, dNTP, Primers and MgCl2 solution.
(ii) Prepare the PCR reaction mix (Table 11.4) in 0.2 mL thin-walled PCR tube on ice or cooled tube
rack. Any combination of template DNA (X mL) and distilled water (Y mL) can be used as long as
the total volume of the PCR reaction mix sample equals 50 mL.
(iii) Place the reactions in a thermal cycler.
(iv) Perform the following incubations.

96– C for 10 min, followed by 30 cycles of denaturation at 96– C for 1 min, annealing at 52– C for
30 sec, and extension at 72– C for 2 min.
A 7-min extension at 72– C is performed at the end of the final cycle.
(v) Store the reaction at 4– C (short term storage) or 80– C (long term storage).
(vi) Apply an aliquot of the reaction to agarose gel electrophoresis to estimate size and concentration
of the PCR product.
ADVANCED PCR TECHNIQUES

Here we describe more specialized PCR techniques.
Hot start PCR

Hot start PCR (D’Aquila et al., 1991) is popular to maximize sensitivity and specificity. The aim is to
withhold at least one of the critical components from participating in the PCR reaction until the
temperature in the first cycle rises above the Tm of the reactants (Roux, 1995). Typically the hot start
temperature may be 94–96– C, but this will vary depending on the PCR enzyme. Some PCR enzyme kits
are in fact inactive until they reach a certain temperature (e.g. AmpliTaq Gold; 95– C for 5 min).
Touchdown PCR
The occurrence of spurious primer annealing during gene amplification resulting in non-specific
PCR products is frequently encountered. As mentioned above, utilizing co-solvents, optimization of
PCR condition and Mg2þconcentration may overcome this problem. However, optimizing these
conditions and factors can be time consuming. Touchdown PCR (Don et al., 1991; Hecker and Roux,
1996) can be applied rapidly instead.
For touchdown PCR, the initial annealing temperature is set to 10– C above the expected denaturation
temperature or previously determined annealing temperature. During subsequent PCR cycles the
annealing temperature is progressively decreased, for example by 1– C every second cycle, until a touch
down to the expected annealing temperature is reached. Then the remaining additional cycling reactions
are performed. In the earlier PCR cycles, spurious primer annealing is avoided from the high annealing
temperature. Even though this decreases in later cycles (to the spurious annealing temperature), the
targeted sequence region has been preferentially amplified, and thus is in a position to outcompete any
non-specific PCR amplicon generation during the remaining cycles (Roux, 1995).
Nested PCR
Nested PCR can also be used to decrease any undesired primer annealing. This approach is often quite
successful in reducing or eliminating unwanted PCR product formation while at the same time markedly
increasing sensitivity (Roux, 1995).
Nested PCR is achieved by using two pairs of PCR primers in two subsequent PCR amplifications.
DNA templates are amplified first with an initial primer pair of a targeted gene. Then, an aliquot of the
PCR product is subjected to further PCR amplification with a second primer pair with sequences
complementary to those internal to the initial primer pair. The strategy of nested PCR is that if the wrong
region was amplified by spurious priming, the probability is very low that it would also be amplified
during a second round of PCR.
REVERSE TRANSCRIPTION PCR FROM AN RNA TEMPLATE

Reverse transcription RT-PCR can be used to detect rRNA (Boon et al., 2003) and mRNA (Qin et al.,
2008) from samples of activated sludge. Amplification of rRNA has been used to obtain more information
on the distribution of microbial activities within such communities (Felske et al., 1996). In analyses of
community rRNA, RT-PCR products are often compared to DNA amplified products to compare the
presence with activity of individual populations (Anderson et al., 2008). The mRNA can be used to
indicate potential metabolic activities because its turnover is rapid in living bacterial cells, and amounts of
mRNA expressed reflect their functional activity at any particular time (Alifano et al., 1994).
Since RNA cannot serve as a template for PCR, a reaction is required to synthesize complementary
DNA (cDNA) from RNA extracts with the enzyme reverse transcriptase and appropriate primers.
Synthesized cDNA is then used as template for the normal PCR reaction.
RNA extracts will contain contaminating DNA, which may interfere with the downstream detection of
RNA. Thus, extracts must be treated with DNase, and then be checked by PCR amplification to confirm its
removal. Since RNA is susceptible to degradation, it is important to use RNase free laboratory equipment
and chemicals (see section 11.6).
Often the RT step is used to generate the DNA encoding important functional genes, using appropriate
primers (Bustin, 2000). Other applications of RT-PCR include using random hexamers (six oligonucleotides
of random sequence (d(N)6) that anneal randomly). This random hexamer approach is used to synthesize
cDNA from as much of the RNA in an extract as possible. It is used typically to analyse overall expression
of mRNA from bacteria (Dieffenbach and Dveksler, 1995a). RT-PCR can also be used to analyse
mRNA expression from eukaryotes, with oligo-dT primers (homo-oligomeric deoxyribonucleotide) for
reverse transcription, as poly (A) tails occur in the vast majority of eukaryotic mRNAs (Frohman et al., 1988).
REAL-TIME QUANTITATIVE PCR

Real-time quantitative PCR (qPCR) enables quantification of the number of targeted gene copies. A range of
different platforms for qPCR have been developed. These include SYBR1 Green I (Wittwer et al., 1997),
TaqMan probe (Heid et al., 1996), Molecular beacons (Tyagi and Kramer, 1996), and Quenching primer/
probe (Kurata et al., 2001). The principal of qPCR using SYBR1 Green I is that fluorescence intensity
increases in proportion to the amount of amplified products formed in a given time (Figure 11.18), which
is proportional to the concentration of the specific template DNA in the original sample. Real-time qPCR
has been applied to activated sludge systems to quantify targeted functional genes including amoA and
ppk1 (Harms et al., 2003; He et al., 2007) and the 16S rRNA gene (Boon et al., 2003; Vervaeren et al., 2005).
As an example of its application, qPCR has been used to quantify specifically 16S rRNA gene
copies of the PAO Candidatus ‘Accumulibacter phosphatis’ (chapter 10). Several PCR conditions for
amplifying Accumulibacter 16S rRNA genes have been described (Fukushima et al., 2007; He et al.,
2007; Okunuki et al., 2007). Fukushima, et al. (2007) have estimated Accumulibacter numbers in
laboratory-scale and full-scale activated sludge samples and compared the quantitative results obtained
with both qPCR and FISH. These agreed well, with an R2 value of 0.6871 showing a statistically
significant correlation (p50.001) (Figure 11.19). This real-time qPCR method is suitable to quantify
target organisms in activated sludge even at abundances of less than 1% of the total bacterial population.
qPCR targeting the rRNA gene can be used determine relative abundances of targeted bacteria with
high sensitivity, reproducibility and throughput. For example, with 30 extracted samples, results can be
obtained within a day (DNA extraction 3 h; sample preparation 2 h; qPCR amplification 2h). So this
technique is a fast and efficient tool to quantify targeted population abundances in activated sludge and is
more sensitive than FISH.
®
SYBR Green I
Primer
Denature
Annealing
Extension
Figure 11.18. Principle of real-time qPCR with SYBRÒ Green I. Initially free SYBRÒ Green I dye has very low
fluorescence and will not bind to denatured DNA. During the annealing step, a double stranded DNA
structure is formed, to which SYBRÒ Green I dye now binds leading to a dramatic increase in fluorescence
signal intensity. Then in the extension step, this fluorescent signal strength increases proportionally to the
number of SYBRÒ Green I dye molecules bound per double stranded DNA molecules. Fluorescence
intensities are detected at the end of the extension period of each PCR cycle.
40
y = 1E-05x + 3.4221
% of PAOMIX positive cells
R2 = 0.6871
30
(FISH)
20
10
0
0.0E+00 5.0E+05 1.0E+06 1.5E+06 2.0E+06 2.5E+06
Copies per reaction (Quantitative PCR)
Figure 11.19. Comparison of results of laboratory-scale (•) and full-scale (o) activated sludge by the real-
time quantitative PCR method and by FISH for Accumulibacter (Fukushima, et al., 2007).
11.8 CLONE LIBRARY GENERATION

Jeremy J. Barr, Andrew E. Cook, Toshikazu Fukushima, and Philip L. Bond
INTRODUCTION
A clone library is a collection of DNA sequences, usually derived from PCR amplification, inserted into a
plasmid and cloned into a bacterial host cell (usually E. coli). Further amplification of the plasmid occurs
within each host cell. The 16S rRNA gene is a popular gene for investigating phylogenetic diversity in
activated sludge. However, several functional genes have also been used, and two examples are the ppk
(He et al., 2007a) (chapter 10) and nir (Osaka et al., 2006) genes.
A number of methods are available to screen clone libraries, which include individual plasmid
extractions followed by their subsequent restriction endonuclease digestion, or colony PCR with plasmid
targeted primers flanking the cloned DNA insert. These are suitable for screening libraries containing
relatively low numbers of clones. However, if larger clone libraries require screening, particularly in
quantitative ecological investigations, high throughput methods are preferable (e.g: other PCR based
methods, DGGE or non-PCR based methods like FISH).
Importantly clone library analysis provides raw sequence data of genes or gene fragments not so
readily obtained by other means. These sequences are analyzed to examine population diversity (Du et al.,
2008) and phylogeny (Blackall et al., 1998) within samples, using dedicated computer programs. Clone
library analysis can clarify the level of biodiversity of members of a community, and assist in putative
identification of unculturable bacteria, and elucidate their possible gene functions. Furthermore, the DNA
sequences obtained can assist in probe/primer design for FISH or PCR (see Figure 3.1).
This chapter discusses (i) clone library generation (Figure 11.20) for analysis of activated sludge
communities, (ii) screening of these clone libraries and (iii) clone analyses by sequencing and phylogenetics.
Figure 11.20. Basic schematic outlining general procedure for clone library generation and analysis. Steps
include; bulk DNA extraction, ligation of DNA into a cloning vector, transformation of cloning vector into host
cell, generation of clone library, screening of clones followed by further downstream analysis. (F. Slater).
CLONE LIBRARY GENERATION

Generation of DNA inserts
Most cloning kits exploit plasmids called T-vectors, which, when linearised, exhibit a single deoxythymidine
residue (T) at their 30 end (Marchuk et al., 1991). The use of T-vectors coupled with non-proof reading DNA
polymerases (e.g. AmpliTaq Gold Taq polymerase), which exhibit terminal deoxynucleotide transferase
activity and add a deoxyadenosine residue (A) to the 30 ends of PCR products (Clark, 1988), greatly enhances
the efficiency of sticky-end ligation. Conversely, blunt end ligation (Weisburg et al., 1991) products are
generated with Taq with 30 to 50 proofreading function. These do not add a deoxyadenosine residue (A) to the
30 end of PCR products. For this reason, blunt end ligation procedures are less efficient than sticky-end
ligations, and so it is recommended that sticky-end ligations be used (Rheims et al., 1996).
If a proofreading DNA polymerase (e.g. pfu DNA polymerase) is used to generate PCR fragments for
T-vector cloning, a 30 deoxyadenosine residue (A) will need to be attached to the 30 end of the fragment, an
additional step known as A-tailing. Using this T-A cloning strategy eliminates the need to create sticky
end ligations (including restriction sites on the PCR primers which require further processing steps
between the PCR and ligation procedures).
METHODS FOR A-TAILING

Solutions/reagents
. AmpliTaq Gold DNA Taq polymerase (Applied Biosystems)
. AmpliTaq Gold DNA Taq polymerase reaction 10£ buffer (Applied Biosystems)
. Deoxyadenosine triphosphate (dATP; Promega)
. MgCl2 solution (Applied Biosystems)
. Fresh PCR product
Equipment
. 0.5 mL Eppendorf tube
. Water bath or fridge
Protocol
(i) Thaw DNA Taq polymerase reaction 10£ buffer, dATP and MgCl2 solution.
(ii) To the 0.5 mL Eppendorf tube add 1–2 ml of fresh PCR product, 1 ml of DNA Taq polymerase
reaction 10£ buffer, 1 ml of 25 mM MgCl2, 2 ml of 2 mM dATPs, 5 units of DNA Taq polymerase
and deionised water to a final reaction volume of 10 ml.
(iii) Incubate at 70– C for 15 to 30 min or at 4– C overnight.
(iv) Use 1–2 ml in a ligation reaction with suitable vector.
LIGATION OF INSERTS INTO CLONING VECTOR

Following amplification of DNA inserts, the next step towards clone library generation is ligation of PCR
fragments into a cloning vector. As an example we describe here the pGEM-T Easy Vector. However,
many similar commercial T-A vectors are available that use convenient and low cost systems for cloning.
pGEM vectors are already prepared for use, having been linearised with EcoRV and having a 30 terminal
thymidine added to both ends to promote sticky end cloning. Hence recircularisation of the vector has been
prevented (Mezei and Storts, 1994; Robles and Doers, 1994). pGEM-T Easy Vectors also contain the T7
and SP6 RNA polymerase promoters flanking a multiple cloning region and an ampicillin resistance gene.
METHODS FOR pGEM-T EASY VECTOR LIGATION

This section should be read in conjunction with the pGEM-T Easy Vector, Technical Manual provided by
Promega.
Solutions/reagents
. 2£ Rapid Ligation Buffer (Promega)
. pGEM-T Easy Vector (50 ng; Promega)
. PCR product
. Control Insert DNA (Promega)
. T4 DNA Ligase (3 Weiss units/ml; Promega)
Equipment
. 0.5 mL Eppendorf tube
. Vortex
. Centrifuge
. Fridge
Protocol
(i) Briefly centrifuge pGEM-T Easy Vector, Control Insert DNA and PCR product tubes to collect
contents at the bottom of the tubes.
(ii) Set up ligation reactions as described in Table 11.5 below;
Table 11.5. Components of a Ligation reaction mix of 10 mL.

Component Standard reaction Positive control Background control
2X Rapid Ligation Buffer 5 ml 5 ml 5 ml
pGEM-T Easy Vector (50 ng) 1 ml 1 ml 1 ml
PCR product (ng) X ml – –
Control Insert DNA – 2 ml –
T4 DNA Ligase (3 Weiss units/ml) 1 ml 1 ml 1 ml
Distilled water to a final volume of 10 ml 10 m l 10 m l 10 m l

PCR product concentration (ng) depends on insert size (kb) and insert: vector ratio, refer to Technical Manual, Promega.
(iii) Mix reactions by pipetting.

(iv) Incubate at 1 h room temperature, or for maximum transformants, at 4– C overnight.
Positive controls are included to determine both ligation and transformation efficiency of the competent
host cells. Background controls will determine the number of colonies resulting from non-T-tailed or
undigested pGEM-T Easy Vector with no insert. Background colonies are differentiated from those with
inserts by the blue/white screening method described below. If ligation and transformation procedures
have already been optimized, positive and background controls may be omitted from future cloning.
A feature of the pGEM-T Easy Vector is its ability to allow recombinant clones containing inserts to be
identified directly by blue/white colour screening on indicator plates, a feature known as insertional
inactivation. It is performed by ligation of the PCR fragment into the multiple cloning site of the vector,
which is positioned within the same gene coding the a subunit of a b-galactosidase (lacZ). The E. coli host
contains an inactive version of lacZ. When transformed cells containing a vector with no insert are plated
onto a medium containing an inducer for the lacZ gene (IPTG) and the chromogenic substrate (X-Gal),
colonies turn blue because of b-galactosidase expression. However, when transformed cells containing a
vector with an insert, which interrupts b-galactosidase expression, are plated onto the same medium, cells
can not produce a functional b-galactosidase. Hence colonies remain white (Promega. 2007). With this
method known as blue/white screening, it is possible to generate large numbers of clone libraries.
TRANSFORMATION OF COMPETENT CELLS

Transformation describes where competent E. coli cells are transformed with recombinant vector DNA
(vector and inserted PCR product). Competent cells are adapted to take up extracellular DNA readily
(chapter 1). If the vector is successfully transformed into a host cell, genes encoded on the vector may be
expressed there and potentially can confer a new function. After transformation, cells are plated onto a
suitable medium, selecting for a trait conferred by the vector as evidence of successful transformation,
usually in the form of an antibiotic resistance gene.
Methods for one Shot TOP10 Competent cells (Invitrogen 2004)

Competent cells are highly sensitive to mechanical shearing and changes in temperature, so they should to
be handled with care. Thaw competent cells on ice and do not pipette them. One Shot TOP10 Competent
cells do not require the substrate IPTG to induce expression from the lac promoter. The substrate X-Gal is
spread onto the agar medium with desired antibiotic to induce blue/white screening.
Solutions/reagents
. 50 ml vial of One Shot cells (Invitrogen)
. 250 ml S.O.C medium (Invitrogen)
. Ligation reaction
Equipment
. 37– C shaking and non-shaking incubator
. 10 cm diameter LB agar plates with appropriate antibiotic (100 mg/ml) and 40 ml of 40 mg/ml
X-Gal spread on top of plate
. Ice bucket with ice
. 42– C water bath
Protocol
(i) Centrifuge tubes containing ligation reactions briefly and place on ice.
(ii) Thaw, on ice, one 50 ml vial of One Shot cells for each transformation.
(iii) Pipette 1 to 5 ml of ligation reaction directly into the vial of competent cells and mix by tapping
gently; do not pipette.
(iv) Incubate the vial on ice for 30 min.

(v) Incubate for exactly 30 sec in the 42– C water bath; do not mix or shake.
(vi) Remove vial from water bath and place on ice.
(vii) Add 250 ml of room temperature S.O.C medium to vial, using sterile technique to prevent
contamination.
(viii) Shake vial at 37– C for exactly 1h at 225 rpm in a shaking incubator.
(ix) Spread 20 ml to 200 ml from vial onto LB agar plates; remaining transformation may be stored at
4– C overnight if desired.
(x) Invert plates and incubate at 37– C overnight.
Protocol for DH5a competent cells (Chung et al., 1989)

Non-commercial DH5a cells will require IPTG to induce expression from the lac promoter, which requires
preparing the agar media with X-Gal plus IPTG and the desired antibiotic to induce blue/white screening.
Solutions/reagents
. E. coli strain DH5a
. PYE broth
. TSB (LB broth pH 6.5 supplemented with 10% w/v PEG4000; 5% v/v DMSO; 10 mM MgCl2;
10 mM MgSO4)
. Ligation reaction
Equipment
. 1.5 ml Eppendorf tube
. Ice bucket with ice
. 42– C water bath
. 37– C shaking and non-shaking incubator
. Centrifuge
. 10 cm diameter LB agar plates with appropriate antibiotic (100 mg/ml) and 40 ml of 40 mg/ml
X-Gal spread on top of plate
Procedure
(i) Take a small loopful of an overnight culture of the E. coli strain DH5a and inoculate into 5 ml
PYE broth.
(ii) Incubate at 37– C shaking and grow to OD600 0.3–04 (growth for approximately 60 to 90 min).
(iii) Add 1 ml of incubated cells to a 1.5 ml Eppendorf and pellet cells by centrifugation at 13,200 rpm
for 5 min.
(iv) Discard the supernatant and gently resuspend cells in 200 ml of cold TSB and incubate on ice for
10 min.
(v) Add 5–10 ml of ligation reactor directly to the competent cells and incubate on ice for a further
30 min.
(vi) Place tube in 42– C water bath for 30 sec, and incubate at 37– C for 5 min.
(vii) Add 1 ml PYE broth to the tube and incubate at 37– C for a further 60 min.
(viii) Spread plate 100, 200 and 400 ml samples of transformants onto selective media and incubate at
37– C overnight.
After transformation and growth on the selective media, blue colonies containing vectors without
inserts, and white colonies with vectors with inserts, should be visible. The ratio of blue to white colonies
reflects the transformation efficiency of the competent cells, where a typical efficiency should be
,1 £ 109 transformants/mg vector DNA. However, this can vary depending on the insert: vector ratio and
so optimization of this ratio is required. A low ratio may yield a poor transformation efficiency and a high
ratio can create concatomers. Several false positives may occur among the white colonies (clones which
appear white but do not contain inserts, or have inserts of the incorrect size). Thus more precise clone
library screening should be performed.
CLONE LIBRARY SCREENING

Initial screening of clone libraries is performed to reduce the numbers of clones to sequence. By screening
it is possible to remove duplicate clones, those with incorrectly sized inserts and without inserts.
The majority of this work is completed after plasmid extraction from the clones (Anderson and McKay,
1983), which is not covered in this section.
Vector PCR
After plasmid extraction, PCR is performed with each plasmid to amplify inserts for further analysis.
By using the cloning vector’s internal primer sites, which flank the inserted DNA, it is possible to
amplify the sequence from the extracted plasmid. The T7 (50 -TAATACGACTCACTATAGGG-30 ) and
SP6 (50 -TATTTAGGTGACACTATAG-30 ) primers are designed for amplifying and sequencing inserts
cloned into pGEM Vectors. PCR may also be performed directly from host cells without plasmid
extraction. Instead of adding purified template DNA to the PCR reaction, a sterile pipette tip is used to
touch the surface of a freshly grown clone colony. The tip is immersed into a PCR reaction mixture
containing the primers mentioned above, and PCR then proceeds as normal.
Agarose gel electrophoresis of PCR products is used to confirm presence or absence of cloned inserts.
A suitable DNA ladder allows for determination of insert sizes. Incorrectly sized cloned inserts can be
discarded. Amplified inserts are used for a number of downstream applications including restriction
fragment digests and sequencing, and can be stored at 4– C for short-term storage.
Restriction enzyme analysis (REA) of cloned inserts

DNA Restriction Enzyme analysis (REA) is a simple method to detect sequence variations within cloned
genes by digestion with appropriate restriction endonucleases. It is popular for screening from its ability to
analyze large numbers of clones at high-speed at a low cost in a simple and reproducible manner.
Selection of restriction enzymes depends solely on the cloned insert being analyzed, as many different
enzymes are available, each suited to a range of applications. For this purpose, four base cutters
(those recognizing 4 base pair sequences) are used. These endonucleases exhibit a high cutting frequency
and will cut the 16S rRNA gene into fragments of ,250 bp. Two commonly used enzymes in activated
sludge clone library screening are Alu1 and Msp1 (Gich et al., 2000; McMahon et al., 2007b), chosen on
the basis of their high average number of restriction sites per taxon (Moyer et al., 1996), and form the
basis of this discussion.
PROTOCOL FOR REA USING ALU1 AND MSP1 (Di Cello et al., 1997)
Solutions/reagents
. 10£ NE Buffer2 (New England BioLabs)
. Alu1 restriction enzyme (New England BioLabs)
. Msp1 restriction enzyme (New England BioLabs)
Equipment
. 1.5 ml Eppendorf tube
. 37– C incubator
. 65– C incubator
Protocol
(i) To a 1.5 ml Eppendorf tube add approximately 1.5 mg of DNA.
(ii) Add 3 units of Alu1 and Msp1 restriction enzyme.
(iii) Add 3 ml of 10£ NE Buffer 2.
(iv) Add distilled water to a final volume of 30 ml and mix thoroughly by pipetting.
(v) Briefly centrifuge and incubate at 37– C for 3 h.
(vi) Inactive enzymes at 65– C for 10 min.
Amplified ribosomal DNA restriction analysis (ARDRA) method is where REA is used directly on
mixed culture DNA, and has been applied directly to activated sludge samples (Barberio and Fani, 1998;
Gich et al., 2000). It provides little information about identity of the microbes present, but can provide
a quick assessment of genotypic changes in the community over time, or for comparing different
communities. However, other techniques e.g. T-RFLP (Osborn et al., 2000; Eschenhagen et al., 2003) and
DGGE (Beer et al., 2004; Liu et al., 2007) have also been applied to activated sludge samples
(sections 11.11 and 11.12).
It is also important to estimate the species richness of the sampled community from rRNA gene clone
library analyses. Since sequences with high similarity are assigned typically to the same species, such
sequences can be grouped together. Each group is called an operational taxonomic unit (OTU). OTUs are
generally defined based on the sequence differences between groups. The recommended 16S rRNA gene
sequence threshold for sequence differences within each OTU should not exceed 3% (i.e. 497%
similarity). However, OTU determination has not been based on standardized thresholds, and those used
vary from 1 to 5%, which makes statistical comparisons between clone libraries difficult (Martin, 2002).
Analysis of REA products is performed usually with a 3% agarose gel electrophoresis because of the
relatively small bands expected (Figure 11.21). Clones are grouped into individual OTUs based on
differences in banding patterns. These can be visualized by eye or with software (e.g. PDQuert Quantity
One). Such groupings can reduce considerably the number of clones to be sequenced, depending on
sample diversity.
CLONE LIBRARY SEQUENCE ANALYSES

Classification systems have been revolutionized with the advent of biological molecular analysis, which
has contributed considerably to our understanding of evolutionary relationships between bacteria
1 2 3 4 5 6 7 8 9 10
Figure 11.21. Agarose gel of REA showing molecular weight markers in lanes 1 and 10, one OTU in lanes 2,
4, 5, 7 and 8, another OTU in lane 3 and a final OTU in lanes 6 and 9.
(chapters 1, 6). Cloning and sequencing of marker genes has provided an insight into phylogenetic
relationships existing among ‘prokaryotes’ at the genus, species and subspecies level (Anderson et al.,
2001). The 5S, 16S and 23S rRNA genes and ribosomal protein sequences are highly conserved within
bacteria and their sequences provide insights into diversity from domain to species level. The 16S rRNA
gene in particular has become a reliable tool in resolving phylogenetic relationships among bacteria from
many environments including activated sludge (Liao and Dennis, 1994; Amann et al., 1995).
Although 16S rRNA gene sequencing may not always allow a strain to be placed into a species it is a
powerful tool in determining to which species a strain most likely belongs (i.e. it identifies its closest
relatives) (Fox et al., 1992). It is generally believed that 500 nucleotides of an unknown sequence (such as
from clone libraries) are sufficient for phylogenetic placement, but only if compared to a longer known
sequence in the data base against which it is more than 90% similar (Hugenholtz et al., 1998a).
Sequencing
Determination of nucleotide sequences was revolutionized by the introduction of the chain-termination
DNA sequencing method (Sanger et al., 1977) known as Sanger sequencing. This method provided an
opportunity to many laboratories for small scale, rapid turn around time and affordable DNA sequencing
of approximately 1200 bp reads. Now most sequencing is more economically out-sourced and many
facilities offer sequencing as a service. Sample submission guidelines differ between these and should be
checked before following prescribed guidelines.
The two most important factors in fluorescent Sanger DNA sequencing are the quality and quantity
of the DNA template. DNA quality should always be checked by absorbance measurements on a
spectrophotometer and should lie between OD260/OD280 ¼ 1.8 to 2.0. DNA quantity will vary depending
on size of the PCR product or plasmid being sequenced (for 16S rRNA gene PCR fragments, 20–80 ng of
DNA can be sufficient although this should be checked against the sequencing facility guidelines).
Following REA, clones exhibiting similar restriction patterns are placed into a single OTU. Thus,
different OTUs will exhibit different restriction patterns. Several clone representatives from each OTU can
be selected and sequenced, using either 16S rRNA primers or internal vector primers.
Sequence analysis
Information regarding the sequence identity of each clone can be determined by comparing each sequence to
one of the online databases, such as GenBank at the National Centre for Biotechnology Information (NCBI)
(Benson et al., 2009) or European Molecular Biology Laboratory (EMBL) nucleotide sequence database
(Kulikova et al., 2004). To obtain initial sequence identity values, FASTA (Pearson and Lipman, 1988) or basic
local alignment search tool (BLAST) (Altschul et al., 1990) analyses are performed. The BLAST algorithm
enables a user to recognize high identity between a query sequence and those already on the database.
GenBank in combination with BLAST (see http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) is widely used
for initial sequence identification. GenBank is part of the International Nucleotide Sequence Database
Collaboration, which also comprises the DNA DataBank of Japan (DDBJ) and EMBL. Thus, it is
comprehensive containing publicly available nucleotide sequences.
For sequence analysis of rRNA genes, the Ribosomal Database Project (Cole et al., 2009) and
Greengenes (DeSantis et al., 2006) may be used. RDP-II provides quality-controlled bacterial and
archaeal small subunit rRNA alignments and analysis tools. A sequence match program enables the
researcher to find sequences similar to the query sequences using a word matching strategy not requiring
prior alignment. This program is more accurate than BLAST at finding closely related rRNA sequences
(Cole et al., 2005). In addition, chimera sequences composed of two different gene sequences (see
chapter 3) can be identified with the chimera check program (Maidak et al., 1999). Greengenes is a 16S
rRNA gene database, which addresses the limitations of public repositories by providing chimera
screening, standard alignment, and phylogenetic classifications using multiple published taxonomies.
Once the OTUs are allocated, it may be useful to determine the level of diversity covered in the sample
by the clone library, which can be achieved through construction of an accumulation curve. Figure 11.22
shows an ideal accumulation curve, where the number of clones sequenced from a library is plotted
against the number of unique sequences detected there. If the accumulation curve is near stage ‘a’ of the
curve, the majority of the biodiversity, or richness, has not been recovered and further sequencing of
clones is recommended. However, if the curve is near stage ‘b’ the analysis has probably exhausted
biodiversity and so further sequencing is unlikely to produce more unique sequences, and represents a
waste of time and money.
PHYLOGENETIC SEQUENCE ANALYSIS

DNA sequences can be compared for phylogenetic analysis to estimate evolutionary relationships between
them. The first step in constructing a phylogenetic tree is selecting sequences to be used after which their
alignment can be performed. This is the most critical step in tree construction as it is where comparison of
homologous characters between sequences takes place. Several online bioinformatics software programs
are available to perform such alignments (e.g. ClustalX). Ideally same length query sequences should be
used in the alignment, but this is rarely possible as sequence lengths obtained from each clone will vary.
Alignment between conserved regions of 16S rRNA genes is straightforward, but becomes more
problematic between regions of greater variability for 16S rRNA molecules, such as secondary structure
interactions. These problems can be resolved by comparing sequences to secondary structure models.
Regions of sequences that cannot be aligned unambiguously should not be used in subsequent
phylogenetic analyses.
Several online software programs are available to construct phylogenetic trees from alignment data.
Two are briefly discussed here.
1. ARB: a software environment for sequence data (Ludwig et al., 2004).
Accumulation curve analysis
sequences detected
Number of unique
Number of clones sequenced

Figure 11.22. Accumulation curve analysis of a clone library plotting number of clones sequenced against
number of unique sequences obtained. Point a indicates too few clones sequenced to cover majority of
biodiversity richness in clone library, point b indicates majority of richness has been sequenced and further
clone library analysis is not required.
The word derives from the Latin word, arbor meaning tree. The ARB program originated 15 years ago
and comprises several software tools for sequence analysis. It can be used for any nucleic or amino acid
sequence. The central database contains all processed (aligned) primary structures to which new sequences
may be added. Additionally ARB contains software tools for data import and export, sequence alignment,
primary and secondary structure editing, phylogenetic analyses and other components used for data analysis.
2. Molecular Evolutionary Genetics Analysis (MEGA) software (Kumar et al., 2001).
MEGA software functionality has been evolving for many years. The most recent release, the fourth
version MEGA4, (Tamura et al., 2007) includes sequence data alignment and assembly features,
estimation of sequence divergence and reconstruction and visualization of phylogenetic trees. MEGA can
be used as a workbench to explore sequence data from a microbial ecological perspective. Additionally
MEGA comes with online help, explaining the different aspects of its user-interface.
Phylogenetic trees may be unrooted (no common ancestor used) or rooted, by adding sequence
information of taxa known to lie outside the presumed group. These are called outgroup taxa and do not
necessarily have any evolutionary significance to the other sequences. Inference of the phylogenetic
significance of a particular 16S rRNA gene sequence is best performed using several algorithms in
combination with a measurement of confidence placed on the phylogenetic groups identified (e.g.
bootstrap analysis). Three algorithms used to infer phylogenetic relationships between different 16S rRNA
sequences are the Neighbour-Joining, Maximum Likelihood and Maximum Parsimony. Rooting a
phylogenetic tree and inference can be automatically selected for when using the MEGA software.
Applying several phylogeny methods instils more confidence in tree topology. Analysis with distance
method algorithms like Neighbour Joining and Maximum Likelihood provides distance in formats in the
tree. For example, the horizontal distances on branches between nodes is proportional to differences
between the sequences (Figure 11.23).
A. coerulea IFO 14679T (U49002)

51 A. luteofluorescens IFO 13057T (U49008)
94 A. verrucosospora IFO 14100T (U49011)
98 A. citrea IFO 14678T (U49001)
0.01 A. macra IFO 14102T (U49009)
A. formosensis DSM 43997T (AJ420140)
A. pelletieri JCM 3388T (AF163119)
A. meyerii A288T (AY273787)
A. madurae DSM 43067T (X97889)
A. latina DSM 43382T (AY035998)
78 Unknown clone strain
A. rugatobispora IFO 14382T (U49010)
A. fulvescens IFO 14347T (U49005)
A. atramentaria IFO 14695T (U49000)
A. oligospora ATCC 43269T (AF163118)
A. viridilutea IFO 14480T (D86943)
A. hibisca JCM 9627T (AF163115)
Actinocorallia herbida IFO 15485T (D85473)
A. kijaniata DSM 43764T (X97890)
99 A. namibiensis DSM 44197T (AJ420134)
Spirillospora albida IFO 12248T (D85498)
A. fibrosa ATCC 49459T (AF163114)
58 A. nitritigenes DSM 44137T (AY035999)
97 A. vinacea JCM 3325T (AF134070)
A. viridis DSM 43175T (AJ420142)
A. yumaensis JCM 3369T (AF163122)
69 68 A. catellatospora AS4.1522T (AF154127)
99 A. livida JCM 3387T (AF163116)
A. umbrina JCM 6837T (AF163121)
A. echinospora IFO 14042T (U49004)
A. spadix JCM 3146T (AF163120)
Nocardia farcinica DSM 43665T (X80610)
Figure 11.23. 16S rRNA gene sequence neighbour-joining phylogenetic tree showing the relative positions
of an unknown clone strain and the type strains of Actinomadura species. The sequence of Nocardia
farcinica served as the out group. The scale bar represents 0.01 nucleotide substitutions per position.
Accession numbers are shown in brackets.
11.9 FISH PROBES AND THEIR DESIGN

L. Safak Yilmaz, Dae Wook Kang, and Daniel R. Noguera
INTRODUCTION
Fluorescence in situ hybridization (FISH) relies on DNA/RNA hybridizations taking place within whole
microbial cells. While in situ hybridizations with DNA oligonucleotides were conducted initially with
radiolabeled probes (Giovannoni et al., 1988), DeLong et al. (1989) pioneered the transition to
contemporary FISH methods by introducing the more convenient fluorophore-labeled probes. Later,
Amann and coworkers optimized FISH protocols (Amann et al., 1990a,b; Manz et al., 1992; Roller et al.,
1994; Stahl and Amann, 1991), and FISH became an integral part of the so called rRNA cycle (Figure 3.1)
for identifying and quantifying microbes in activated sludge or other environments. Now FISH is
considered a diagnostic method of choice with widespread environmental and medical applications, and
has been reviewed often (Amann and Fuchs, 2008; Amann et al., 2001; Nielsen et al., 2009; Moter and
Gobel, 2000; Wagner et al., 2003). Many FISH probes have been described in the literature, most
of which are listed in probeBase (Loy et al., 2003) (http://www.microbial-ecology.de/probebase/).
The reader is directed towards the excellent general accounts of Daims et al. (2005b), Amann and Fuchs
(2008) and Nielsen et al. (2009).
In FISH, abundant rRNA molecules are probed with fluorescently labeled DNA oligonucleotides that
target specific sites on the rRNA of organisms of interest. The DNA probes hybridize to their target sites
forming DNA/RNA duplexes under favorable, but stringent conditions, permitting detection of target cells
by fluorescence after excess probe is removed. Detection is by microscopy or flow cytometry. Used with
other methods described in this book, FISH corresponds to the last step in the rRNA cycle (Figure 3.1).
Here the general protocol for FISH is described, together with a mechanistic approach to designing FISH
probes using rRNA gene sequence information.
SENSITIVITY AND SPECIFICITY: CORNER STONES OF PROBE DESIGN

The ideal probe for FISH should offer both high sensitivity and high specificity. Sensitivity refers to the
level of fluorescent signal obtained from hybridized target cells as compared to the background
fluorescence of non-targeted cells. That is, high fluorescent signal is a requirement for the visualization
of targeted cells within the mixed community that characterizes activated sludge. Specificity is the
ability of the FISH probe to allow differentiation between targeted organisms and non-targeted but
closely-related organisms that could affect the interpretation of results. Thus, the probe design and
optimization of the FISH protocol should ensure as far as possible that any false positive hybridizations
are eliminated.
Sensitivity
The first requirement for successful FISH is a detectable signal from target cells. Low signal is associated
with four factors:
(i) impermeability of cell walls/envelopes to the probe.

(ii) low ribosome (target site) content of cells.
(iii) poor hybridization efficiency of probe to the target site.
(iv) quenching of the fluorophore once the probe is hybridized.
All these factors will be discussed below.
Specificity
The second requirement for successful FISH is to determine stringent hybridization conditions under
which the probe fails to bind to non-target organisms, but still hybridizes with target cells providing
a strong fluorescent signal. Stringency is adjusted usually with formamide, a mild denaturant that tends to
dissociate the probe-target duplex while not affecting cell morphology. Incremental addition of formamide
to the hybridization buffer results in a sigmoidal decrease in average cell fluorescence signal intensity
(Figure 11.24). Dissociation is more rapid for duplexes with base mismatches, and therefore, an optimal
range of formamide concentrations that makes any mismatch discrimination possible is generally
attainable. In the given example (Figure 11.24), 45–55% formamide is the optimum, since only organism
with no mismatches between target site and probe will yield detectable fluorescent signals in that range.
250 Perfect
1 Mismatch
200 2 Mismatches
Fluorescence intensity (a.u.)
3 Mismatches
150
100
50
0
0 10 20 30 40 50 60 70 80
Formamide (%)
Figure 11.24. Typical formamide denaturation profiles in FISH. Formamide concentration is expressed as
percentage (v/v) in the hybridization buffer.
KEY STEPS IN A FISH EXPERIMENT

FISH generally consist of six consecutive steps: probe synthesis, cell fixation, hybridization, wash, pre-
analysis treatment, and sample analysis. Procedures involved in moving from one step to the next one will
depend on whether flow cytometry (section 11.15) or microscopy is used to analyse hybridized samples.
In flow cytometry protocols, cells are pelleted by centrifugation, old hybridization buffer is decanted, and
cells re-suspended in new buffer by vortexing. For microscopy, cells remain attached to glass slides after
fixation, and buffer changes between steps is achieved by rinsing slides and applying the next buffer. All
six steps of typical FISH protocols are briefly described next.
Probe synthesis
Chemical synthesis of FISH probes, generally a 15–30 oligonucleotide with a fluorophore attached at the 50
end, is carried out with automated methods that involve extending the probe attached to a solid support, one
nucleotide at a time. The technology for achieving this is cost-effective and performed in many commercial
laboratories. Rigorous purification of the synthesized probe is not generally required for FISH applications.
The choice of fluorophore will depend on the capabilities of the instrument used for analysis, since
appropriate filter sets are required for adequate visualization of the fluorescent signal. Common fluorophores
for FISH emit light at wavelengths spanning a large spectrum, and include fluorescein (518 nm), CY-3
(565 nm), and CY-5 (670 nm). A high background fluorescence at particular wavelengths or cost of the
fluorophore may also influence selection. Furthermore, although not yet well understood some fluorophores
are quenched by nucleotides after hybridization, with the level depending on which nucleotides are present
in the vicinity of the fluorophore. Thus, in developing a new FISH protocol, it is not uncommon to evaluate
probes with different fluorophores to select experimentally the one performing the best.
As well as usual single-fluorophore probe labeling, other labeling techniques which enhance signal
intensity when the number of target molecules is small have been described, as in the case when ribosome
content is low or when mRNA molecules are targeted instead of rRNA. Thus signal intensity can be
enhanced by labeling probes with enzymes like horseradish peroxidase, in a procedure called catalyzed
reported deposition (CARD)-FISH (Amann et al., 1992; Pernthaler and Amann, 2004; Pernthaler et al.,
2002; Schonhuber et al., 1997). After hybridization, the enzyme linked to the hybridized probes catalyzes
the deposition of a supplied detectable substrate, thus amplifying signal yield from a single hybridization
event (van Gijlswijk et al., 1997) and markedly enhancing sensitivity. Hoshino et al. (2008) determined
that while detection of target microbes in activated sludge by conventional FISH required cells to contain
at least 1,400 rRNA copies per cell, those with as few as 36 rRNA copies per cell were detected with
CARD-FISH. However, the large size of the enzymes attached to the oligonucleotide probes means
modified cell fixation and hybridization protocols are required.
Cell fixation
Cell fixation aims to inactivate microbial cells, preserve their morphology, permeabilize them for probe
penetration, and inhibit any enzyme activity liable to lead to cell lysis and degradation of target molecules.
The most commonly used fixative in FISH is formaldehyde, applied by mixing samples with solutions
prepared from paraformaldehyde (polymerized formaldehyde) to reach a final concentration of 3% (w/v) in
the mixture (Amann, 1996; Amann et al., 1995). Although a short exposure to formaldehyde (10–30 min)
is sufficient for most samples, cell walls of some Gram-positive bacteria are not adequately permeabilized
with this fixative (Braun-Howland et al., 1992; de los Reyes et al., 1997; Macnaughton et al., 1994).
Alternative procedures include heat and ethanol fixation, combined use of several fixatives, and additional
treatment with lytic enzymes like lysozyme (Hahn et al., 1992; Jurtshuk et al., 1992; Macnaughton et al.,
1994; Roller et al., 1994).
After fixation and fixative removal (if necessary), cells can be stored at 20– C either in a 1:1
(v/v) mixture of ethanol and phosphate buffered saline (PBS) (Amann et al., 1990a) or in PBS at 4– C
(Amann et al., 1990b) for weeks to months, without detectable loss of FISH sensitivity.
Does cell fixation affect FISH outcomes by altering structure of the ribosome, and thereby, target site
accessibility? Although changes involved in fixation are not well understood, formaldehyde is thought to
cross-link biopolymers and modify nucleic acid bases (Park et al., 1996; Masuda et al., 1999), while
ethanol precipitates proteins and nucleic acids, and heat denatures biopolymers. Thus, the ribosome, a
complex of RNA (ca. 2/3 by mass) and proteins (ca. 1/3 by mass) (Ramakrishnan and Moore, 2001) might
become distorted during fixation. In the only study published where this has been considered, with probes
targeting the 16S rRNA of Escherichia coli, Behrens et al. (2003a) concluded that target accessibility did
not change from formaldehyde fixation to ethanol fixation.
Hybridization
Hybridization involves incubating cells in a hybridization buffer under conditions favorable for duplex
formation, but stringent enough to minimize formation of duplexes where mismatches exist between
probe and target. Factors affecting RNA/DNA hybridization need to be considered and adjusted
accordingly, and include temperature and NaCl concentrations. Compositions of hybridization buffers and
hybridization conditions are given in Table 11.6.
Table 11.6. Composition of typical hybridization and wash buffers.

Hybridizationa Washa
M FC M FC
b b
Probe 1.5–5 ng/ml 1.5–5 ng/ml – –
Na1 0.9 M 0.9 M varied 0.9 M
SDS c 0.01% 0.10% 0.01% 0.10%
Formamide varied varied – varied
pH 7 7 8 7
Temperature,– C 46 46 48 46
a
M, microscopy protocols; FC, flow cytometry protocols.
b
This range corresponds to roughly 225–755 nM for an average-sized probe labeled with CY-3.
c
SDS, sodium dodecyl sulfate.
Probe concentration in the buffer is generally 1.5 to 5 ng/ml, as lower amounts may cause inefficient
hybridization, while higher concentrations may result in non-specific binding (Fuchs et al., 1998).
Including 0.9 M sodium chloride provides the counterion (Naþ) that neutralize the negative charge on the
nucleic acid backbone, thus facilitating formation of the DNA/RNA duplex (Bloomfield et al., 2000).
Sodium dodecyl sulphate (SDS) helps to disrupt cell membranes and walls, improving probe penetration
(Burggraf et al., 1994) as well as target site accessibility, by denaturing or removing proteins
(Behrens et al., 2003a; Rajagopal et al., 2002). One possible explanation for the need for higher SDS
levels in flow cytometry (Table 11.6) is that SDS may reduce cell clumping. The role of formamide is to
adjust RNA/DNA hybridization stringency, and so the optimum formamide concentration for each probe
needs to be determined first (usually 0–60% v/v). The pH of hybridization is at neutral. Although nucleic
acid interactions will not be affected by pH changes within pH 5 to 9 (Bloomfield et al., 2000), some
fluorophores are pH sensitive. Since formamide levels are adjusted in FISH to obtain the appropriate
stringency, incubation temperature is typically kept constant at 46– C, a value traceable to early studies
into optimizing hybridization sensitivity and specificity by adjusting temperature (Amann et al., 1990a).
An important variable is the hybridization period, which usually ranges from 1.5 hours to overnight
(Amann et al., 1990a; Davenport et al., 2000; DeLong et al., 1989; Moller et al., 1996). In theory,
increasing incubation time increases hybridization efficiency by overcoming potential kinetic barriers of
target site accessibility and/or probe diffusion across cell boundaries. Figure 11.25 illustrates the effect of
hybridization period on the fluorescence intensity of probes targeting different regions on the 16S rRNA of
E. coli. It demonstrates that differential probe accessibility to target regions affects hybridization rate
(Yilmaz et al., 2006). On the other hand, with formamide to optimize probe specificity, some kinetic
barriers are removed as it denatures the higher order structure of the ribosome (Gamper et al., 1987;
Yilmaz and Noguera, 2004). Therefore, the incubation period is a variable that should be optimized
during the development of a FISH protocol, with overnight hybridizations expected to approach
equilibrium-like conditions in most cases.
Wash
The washing step aims to remove excess unhybridized probe from samples, without compromising its
sensitivity and specificity, by creating a probe sink outside cells, while mimicking the stringency conditions
of the hybridization step. In flow cytometry-FISH, this is readily fulfilled by transferring cells to
hybridization buffer minus probe (Fuchs et al., 1998). However, in microscopy FISH, glass slides are
immersed in wash buffer, and since this step requires relatively large buffer volumes, formamide is preferably
500
Fluorescence intensity (a.u.)

400
300
200
100
0
0 25 50 75 100 125 150 175
Hybridization period (hrs)
E146–165 E962–980 E1153–1172 E1235–1255 non-EUB
Figure 11.25. Time series of fluorescence intensity in FISH obtained by flow cytometry using four selected
probes targeting the 16S rRNA of E. coli K12 (Yilmaz, 2006). Target site positioning is indicated in probe
name. Vertical lines show 3 h and one day hybridizations. Fluorescence of the non-EUB probe indicates
background signal intensity. Note how the signal of E1235–1255 begins relatively low but ends up becoming
the brightest as hybridization period is extended. The probes with the greatest gains of signal intensity with
increased time of hybridization (E1153–1172 and E1235–1255) target regions packed with protein-rRNA
interactions (Yilmaz, 2006).
not used to avoid generating large amounts of toxic waste (Moter and Gobel, 2000). Without formamide,
stringency of the buffer is adjusted by changing the Naþ concentration. In theory, lowering [Naþ] by a known
amount can make wash stringency equivalent to that achieved with formamide during hybridization. The
required [Naþ] in wash buffer corresponding to an equivalent formamide level in hybridization buffer ([FA])
is based on Equation 1 (Lathe, 1985; Manz et al., 1992), where; Mh and Mw denote [Naþ] in hybridization and
wash buffers respectively, and FA stands for the equivalent formamide percentage in the hybridization
buffer. The constant a is the rate of decrease in dissociation temperature (Td) with increase in formamide
levels, and ranges between 0.5 to 0.7– C/% (a value of 0.6 is commonly used).
Equation 1 can be derived from Equation 2 by making the Td values for hybridization and wash
equal11. In Equation 2, M is the molar concentration of monovalent cations, GC% is the percentage of G
and C nucleotides in the oligonucleotide sequence, and L is the number of nucleic acid bases (i.e. probe
length). This equation stems from empirical formulations for DNA/DNA hybridizations (Lathe, 1985), but
is commonly applied to FISH experiments.
a½FA
Mw ¼ Mh 10 16:6 ð1Þ
Td ¼ 81:5 þ 16:6 log M þ 0:41GC% 820=L þ a½FA ð2Þ
Other differences in the wash conditions from hybridization conditions in microscopy FISH include an
increase of pH to 8 and an incubation temperature of 48– C (Table 11.6), although the reasons for these
shifts are not clear. The wash period is typically 10–30 min (Amann et al., 1990b; Davenport et al., 2000;
de los Reyes et al., 1997; Schramm et al., 1998).
11
Note that [FA] is zero in the wash buffer and all terms other than 16.6 logM cancel out in the
equality, leading to Equation 1.
Pre-analysis treatment
As well as preparing samples for analysis by flow cytometry or microscopy, any pre-analysis treatments
must avoid dissociation of hybrids and bleaching of fluorescent dyes to maintain probe sensitivity between
wash and analysis steps. In flow cytometry FISH, samples from wash are transferred to a cold storage
buffer amended with sufficient salt (e.g. 1£ PBS at 4– C) to prevent duplex dissociation (Amann et al.,
1990b; Fuchs et al., 1998; Wallner et al., 1993). Its pH is important if pH-sensitive fluorophores like
fluorescein derivatives, are used. Samples need to be analyzed on the same day, which is possible because
of the high throughput nature of flow cytometry analyses (section 11.15). In microscopy FISH, after the
wash buffer is rinsed off and slides are dried, antifading mounting solutions like 1,4-diazabicyclo(2.2.2)
octane (DABCO), Citifluor, or Vectashield are applied to wells and slides are covered with cover slips,
which can be sealed to block any oxidation of the antifade agent. Since these procedures increase shelf-
life of the slides, they can be stored for several weeks in the dark.
Sample analysis
FISH samples are analyzed on the fluorescent signal from cells containing the probe/target hybrids. To
generate this the fluorophore is excited with light at an appropriate wavelength, depending on the
fluorophore. Generally, arc lamps (mostly mercury and xenon burners) and lasers are used for excitation.
The response comes in the form of emitted light with a wavelength spectrum depending on the
fluorophore. Flow cytometry FISH yields data in the form of thousands of collected events with assigned
fluorescence intensities (section 11.15). Ideally, bright events represent hybridized (target) cells, while
dim ones arise from background noise or non-hybridized (non-target) cells. Typical output is a histogram
of cell events distributed over fluorescence intensity (Figure 11.26, see colour image section (chapter 13)).
Thus, given that the collection of 10,000 events takes only seconds, flow cytometry provides high
throughput and statistically reliable quantification of populations of interest (Amann et al., 1990b; Perez
Feito et al., 2006; Wallner et al., 1993, 1995). However, its main disadvantage is an inability to
discriminate between individual cells and cell aggregates, affecting the validity of cell quantification
(Biegala et al., 2003; Perez Feito et al., 2006; Wallner et al., 1995).
On the other hand microscopy FISH can visualize spatial arrangement of FISH hybridized cells
e.g. within flocs (Figure 11.26), although quantification is tedious and subjective if epifluorescence
microscopy and manual counting are used (Pernthaler et al., 2002; Wagner et al., 2003). Semi-automated
(Daims et al., 2001c) or fully automated (Kuehn et al., 1998) quantification using confocal microscopes
equipped with appropriate image analysis software is now routine. Confocal laser scanning microscopy
captures the third dimension of densely aggregated cells (Wagner et al., 1998), thereby allowing
quantification of probe-labeled cells as biovolume ratios or percentages. Absolute cell numbers can be
determined if a known amount of tagged reference organisms is spiked into samples as internal standard
(Daims et al., 2001c). The intensity of probe-conferred fluorescence can also be quantified by microscopy,
based on pixel intensities of hybridized cells (e.g. de los Reyes et al., 1997).
In both flow cytometry and microscopy FISH, it is useful to counter-stain all organisms (target and non-
target) so cells can be discriminated from debris, and the relative abundances of target cells determined. This
is achieved with applying domain-level probes together with more specific probes in the hybridization step
(Amann et al., 1990b; Zheng et al., 1996) (see Figure 11.26c, see colour image section (chapter 13)) or by
applying stains like DAPI (e.g. Biesterfeld et al., 2001; Glockner et al., 1996) or Hoeschst (e.g. Wallner et al.
(1995)) that bind to DNA, during pre-analysis treatment. FISH targeted populations can then be expressed
as percentages of these.
EXAMPLE OF FISH PROTOCOL

A guided tour is provided on how FISH can be applied to activated sludge samples, where visualization of
hybridized cells is by microscopy.
Required equipment
. Magnetic hot plate stirrer
. 10-well, Black, 8mm diagnostic slide
. No. 1 cover slips (Size: 24 £ 50 mm, thickness: 0.12 to 0.16 mm)
. Incubator for 46– C and 48– C
. Epifluorescence or confocal microscope
. Thermometer
. Desiccator
CELL FIXATION
Solutions/reagents
. FOR FIXATIVE (4% PARAFORMALDEHYDE):
W Paraformaldehyde
W 10 M NaOH
W 1 M HCl
W 3£ PBS (390 mM NaCl, 30 mM Na2HPO4, pH 7.2)
. FOR GELATIN COATED SLIDES:
W Ethanolic KOH solution: 10% (w/v) KOH in ethanol
W Gelatin solution: 0.1% (w/v) gelatin plus 0.01% (w/v) chromium potassium sulfate in distilled
water
Preparation of paraformaldehyde fixative solution (4% w/v)

(i) Add 20 ml 10 M NaOH and 2 g paraformaldehyde to 33 ml distilled water.
(ii) Working in a fume hood, dissolve by heating to 65– C and gently mixing for 10–20 min.
(iii) Add 17 ml of 3£ PBS, mix and cool to room temperature.
(iv) Adjust pH to 7.2 with 1 M HCl (,10–20 ml needed).
(v) Filter the pH-adjusted solution through a 0.2 mm pore-size cellulose filter.
(vi) Store filtrate at 4– C in the dark; use within 48 h.
Preparation of gelatin coated slides

(i) Soak untreated 10-well slides in ethanolic KOH solution for 1 h.
(ii) Rinse slides with distilled water; let slides air dry for about 20 min.
(iii) Prepare gelatin solution by dissolving gelatin and chromium potassium sulfate in distilled water at
60– C.
(iv) Allow gelatin solution to cool to 40– C.
(v) Dip slides in the 40– C gelatin solution for 15 min; allow to air dry.
(vi) Store gelatin coated slides at 4– C.
Fixation and immobilization of activated sludge sample on

gelatin-coated slides
(i) Optional: To reduce sizes of activated sludge flocs, use a syringe to pass each biomass sample
through a 26G needle at least 30 times.
(ii) Aliquot 0.3 ml of sample into a 1.5 ml microcentrifuge tube.
(iii) Add 0.9 ml of paraformaldehyde fixative solution and incubate for 30 min at room temperature.
This gives a 3% paraformaldehyde solution.
(iv) Then aliquot 100 ml of sludge/fixative suspension into 10 ml distilled water. The volume can be
adjusted depending on cell concentration in the activated sludge sample.
(v) Filter through a black polycarbonate filter (pore size: 0.2 mm; diameter: 25 mm) to collect cells on
the filter.
(vi) Filter an additional volume of distilled water (30 ml) to rinse out any remaining fixative from the filter.
(vii) To transfer cells from the filter onto gelatin-coated slides, position the filter on the slide so that it
covers 4 different wells, and gently press the filter against the gelatin with the thumb.
(viii) Dry slides in a desiccator for 60–90 min.
(ix) Carefully peel off the filter. The cells should be retained by the gelatin in the slide.
(x) Dehydrate the slide by sequentially immersing it in an ethanol series (50, 80, and 95% solutions
by volume), for 3 min in each solution.
(xi) Allow slides to air dry for 15–30 min, and store at 4– C if needed.
HYBRIDIZATION
Solutions/reagents
. For hybridization buffer:
W 1.8 M NaCl
W 1 M Tris-HCl (pH 7.2)
W 10% Sodium dodecyl sulfate (SDS)
W Formamide
Preparation of hybridization buffer

(i) Determine the volume of formamide to be added to the hybridization buffer to achieve the
required formamide concentration. If preparing 10 ml of hybridization buffer with 20%
formamide, then 2 ml of formamide will be used in the following step.
(ii) To prepare 10 ml of hybridization buffer, mix 5 ml 1.8 M NaCl, 0.2 ml 1 M Tris-HCl (pH 7.2),
10 ml 10% SDS, formamide, and distilled water. Water volume will depend on the volume of
formamide added. The final concentrations in the hybridization buffer are 0.9 M NaCl, 20 mM
Tris-HCl (pH 7.2), and 0.01% SDS.
(iii) Store hybridization buffer at room temperature.
Preparation of hybridization chamber

A hybridization chamber can be as simple as a 50 ml plastic tube wide enough to fit a microscope slide.
(i) To maintain moisture in the chamber during hybridization, soak a piece of tissue or absorbent
paper in 0.9 M NaCl and placed it in the chamber.
(ii) Close the chamber and preheat it in a 46– C incubator.
HYBRIDIZATION
Probes can be stored at 80– C in roughly 10 to 40 mM aliquots depending on the targeted final
concentration (see Table 11.6 for range of concentrations). A dilution of the probe received from the
manufacturer is usually required.
(i) Probe solutions should be thawed on ice in the dark before use.
(ii) Mix 0.5 ml of probe solution with 15.5 ml of hybridization buffer.
(iii) Add hybridization buffer to each well of the gelatin-coated slide that contains cells for FISH
(add 16 ml per well).
(iv) Carefully place the slide in the preheated hybridization chamber so that hybridization buffer
covers the well at all times.
(v) Incubate the hybridization chamber at 46– C for 1.5 h or another pre-determined incubation period.
WASH
Solutions/reagents
. For wash buffer:
W 1.8 M NaCl
W 10% SDS
Preparation of wash buffer

(i) Use Equation 1 to calculate the molar concentration of Naþ equivalent to the amount of
formamide used in the hybridization buffer. For instance, if the hybridization used 30%
formamide and 0.9 M NaCl, then the equivalent Naþ concentration according to Equation 1 is
Mw ¼ 0.07 M.
(ii) Calculate the volume of NaCl stock solution needed to reach the required Naþ concentra-
tion in 40 ml of buffer. For instance, if 30% formamide was used in the hybridization
buffer, then the volume of stock solution needed to prepare 40 ml of wash buffer is
v ¼ (0.07 M/1.8 M)(40 ml) ¼ 1.6 ml. The volume prepared should be sufficient to completely
immerse the slide in the buffer during the wash step.
(iii) Prepare 40 ml of wash buffer by adding the calculated volume of 1.8 M NaCl stock solution,
0.8 ml of 1 M Tris-HCl (pH 8.0), 40 ml 10% SDS, and make up to 40 ml with distilled water. For
30% formamide, the volumes of NaCl solution and distilled water would be 1.6 ml and 37.6 ml,
respectively.
(iv) The wash buffer can be pre-heated to 46– C in the oven during the hybridization step.
Wash
(i) Gently rinse slides 2–3 times with 1 ml preheated wash buffer to remove most of the hybridization
buffer.
(ii) Incubate rinsed slide in the remaining preheated wash buffer for 20 min at 48– C. We typically use
40 ml wash buffer in a 50 ml tube, and place the slide inside the tube.
(iii) After incubation, gently rinse slides 2–3 times with 1 ml distilled water.
(iv) Let slides air dry for 30 min.
PRE-ANALYSIS TREATMENT
Solutions/reagents
. For DAPI solution:
W DAPI (40 ,6-diamidino-2-phenylindole)
. For DABCO antifading solution:
W DABCO (1,4-diazabicyclo[2.2.2] octane)
W glycerol solution (86% v/v)
W filter sterilized water (0.2 mm)
. For Citifluor/Vectashield antifading solution:
W Citifluor (Citifluor Ltd., London, UK)
W Vectashield (Vector Laboratories, Burlingame, CA, USA)
Preparation of DAPI solution

(i) Dissolve 40 mg DAPI in 40 ml distilled water, giving a 1 mg DAPI/ml solution.
(ii) Store at 4– C.
Preparation of DABCO antifading solution

(i) Combine 0.466 g DABCO, 400 ml 1 M Tris-HCl (pH 8.0), 1.6 ml sterile water, and 18 ml
glycerol solution.
(ii) Dissolve by warming to 70– C.
(iii) Vortex mixture.
(iv) Aliquot and store at 20– C. This should not be stored for more than one year.
(v) Once thawed the solution should only be used once.
Preparation of Citifluor/Vectashield antifading solution

(i) Prepare antifading solution by mixing 4 parts of Citifluor and 1 part of Vectashield (4:1 volume
ratio).
DAPI staining and pre-analysis treatment

(i) Apply 40 ml DAPI solution to each well and incubate in the dark for 5 min.
(ii) Gently rinse 2–3 times with 1 ml distilled water; and air dry or blot dry.
(iii) Apply 4 ml of antifading solution per well. Our experience is that the DABCO antifading solution
works well when DAPI is used as a counter stain and when CY-3 is the fluorophore. With
fluorescein the Citifluor/Vectashield solution is preferred. However, quality of DAPI visualization
is less with Citifluor/Vectashield than with DABCO.
(iv) Place cover slip over the wells and seal with fingernail polish. Cover slip thickness may be
important depending on the microscope. We use 24 £ 50 mm No. 1 cover slips, (0.12–0.16 mm
thick).
(v) Allow time for the fingernail polish to dry before viewing.
(vi) Sealed slides can be stored in the dark at 4– C prior to examination by microscopy.
DESIGN OF FISH PROBES

Design of oligonucleotide probes for FISH has been largely an empirical trial-and-error approach
(Hugenholtz et al., 2002). First, a potential target site specific for the organisms of interest is selected, and
probe length is determined, using empirical dissociation temperature formulations (e.g. Equation 2) so that
the dissociation temperature is above the hybridization temperature (Daims et al., 2005b, 2006b). The
probe is then synthesized and experimentally tested. If pure cultures of the target organisms and potential
false-positive organisms are available, formamide denaturation curves (for example Figure 11.24) are
obtained and an optimal formamide concentration is selected such that false-positives are eliminated while
maintaining fluorescent signal from targeted organisms. If pure cultures are not available, formamide
optimization is performed by the risky approach of directly testing new probes on activated sludge
samples. Signal intensities and morphologies of hybridized cells are observed, and the optimal formamide
concentration is selected largely based on the morphology of observed hybridized cells being uniform.
With this empirical approach, it is common to reach the experimental validation step only to find out
that the probe fails to produce high signal intensities, in which case a new potential target site is selected,
and testing begins again. Although this trial-and-error procedure is highly inefficient, its persistent use
by those who pioneered FISH methods has generated many good probes targeting a large array of
phylogenetically diverse microorganisms (see probeBase).
To improve FISH probe development, probe design needs to be rationalized with mechanistic
approaches. A conceptual model of probe-conferred fluorescence is shown in Figure 11.27. Factors
considered in this model arise from the nucleic acid research literature, systematic experimental
evaluations of hundreds of FISH probes targeting model organisms (Behrens et al., 2003b; Fuchs et al.,
1998, 2001; Inácio et al., 2003), and mathematical models of FISH that bridge the gap between the
literature and FISH applications (Yilmaz and Noguera, 2004; Yilmaz et al., 2006).
Brightness
Hybridization efficiency
Affinity Accessibility
• Tertiary
• DNA/rRNA rRNA/rRNA
interactions • Secondary interactions
(∆G°1) rRNA/rRNA
interactions
• DNA/rRNA (∆G°3) • Protein/rRNA
interactions interactions
(∆G°2)
• Probe size
and structure
• Fluorophore quenching
Figure 11.27. Venn diagram representation of factors affecting probe brightness. Shaded set includes the
free energy terms used in thermodynamic models of FISH. Subscripts in free energy terms correspond to
reactions in Figure 11.28.
As shown in Figure 11.27, probe brightness is a function of two factors, namely hybridization
efficiency and fluorophore quenching. Hybridization efficiency can be defined as the ratio of target
molecules bound to the probe to the total number of target molecules present in the cell. Fluorophore
quenching occurs when the energy absorbed from light is transferred undesirably to a nucleotide or amino
acid within the vicinity of the fluorophore, thereby reducing its quantum yield and hence its fluorescence
intensity (Crockett and Wittwer, 2001; Marras et al., 2002; Nazarenko et al., 2002; Seidel et al., 1996;
Torimura et al., 2001). Currently only a limited ability exists for predicting the extent of quenching given
the target site of a probe, although fluorescein derivatives are more prone to quenching than other
fluorophores, and the CY-3 fluorophore appears not to be affected by quenching. Thus, potential
quenching issues can be addressed at the stage of fluorophore selection, while design of probes can be
based primarily on hybridization efficiency concepts.
Hybridization efficiency depends largely on stability of structural interactions within the ribosome
during hybridization, although probe size and shape also seem to influence hybridization by mechanisms
not yet understood (Yilmaz, 2006; Yilmaz and Noguera, 2004). Factors associated with structural
interactions are considered in two groups; namely, those associated with probe affinity and those dictating
target site accessibility, although the two are not mutually exclusive (Yilmaz and Noguera, 2004). The
affinity set shown in Figure 11.27 includes critical secondary12 nucleic acid interactions taking place
during FISH, which are relatively stable and expected to drive FISH thermodynamics. These interactions,
represented in Figure 11.28, include;
(i) formation of the duplex between the DNA probe and the rRNA target molecule,
(ii) DNA/DNA interactions originating from the self-folding of the probe, and
(iii) RNA/RNA interactions within the secondary structure of the rRNA molecule.
DNA Probef rRNATargetf
Reaction 2 Reaction 3
Reaction 1 DNA/rRNA
DNA Probeu + rRNATargetu Probe/Target
Duplex
Figure 11.28. Hybridization scheme used in mathematical models of FISH (Yilmaz and Noguera, 2004).
Subscripts ‘‘f’’ and ‘‘u’’ indicate folded and unfolded structural states, respectively.
Stability of these structures is predictable, based on free energy rules deduced from extensive
experimental and theoretical work (reviewed in Turner (2000)), although predictive ability is lower for
more complex structures (Mathews et al., 1999). Mathematical models of FISH use these free energy
values to calculate probe affinity to the target site, defined as an overall free energy term (DG–overall) for the
three reactions represented in Figure 11.28 (Yilmaz and Noguera, 2004) and calculated with Equation 3,
where R is the ideal gas constant (1.99 £ 10 3 kcal · mol 1 · K 1), T is the hybridization temperature
(typically 319.15 K), and the DG–i terms correspond to the free energy changes for reactions in
Figure 11.28. This thermodynamically defined affinity can be converted to hybridization efficiency using
equilibrium chemistry, assuming the remaining elements of the hybridization efficiency set in Figure
11.27 do not affect markedly stability of the probe/target hybrid at equilibrium. With this concept, Yilmaz
and Noguera (2004) demonstrated that a probe with DG–overall greater than 10 kcal/mole will have poor
12
The term ‘‘secondary’’ refers to two dimensional interactions, or structures, such as in helix
formation.
hybridization efficiency resulting in low detectable fluorescence signal, while those with DG–overall lower
than 13 kcal/mole have 499% hybridization efficiencies, and accordingly, high fluorescent signals.
Note that accuracy of these affinity-based hybridization efficiency estimations depends on accuracy of free
energy predictions13.
2 3
DG 8
6 1 7
6 e RT 7
DG8overall 6
¼ RT ln6 7 ð3Þ
DG 8 DG 8 7
4 2 3 5
1 þ e RT 1 þ e RT
Hybridization efficiency will be limited if the probe cannot reach the targeted site on the rRNA molecule,
a concept known as target site accessibility. As illustrated in Figure 11.27, accessibility is affected by
strong secondary RNA/RNA interactions at the target site, interactions between rRNA and proteins, and
also tertiary rRNA interactions. In theory, tertiary rRNA/rRNA and protein/rRNA interactions are less
stable generally than any secondary interactions defined in the affinity set. They may pose kinetic
limitations on FISH by slowing required conformational changes to the ribosome complex that eventually
allow the probe to bind to its target site. Therefore, as a first approximation, probe hybridization efficiency
can be predicted from probe affinity provided that hybridization is brought to equilibrium.
This was demonstrated by Yilmaz et al. (2006) for the 16S rRNA of E. coli. By designing probes with
high thermodynamic affinity and eliminating kinetic barriers by extending hybridization times, they
showed all regions of the 16S RNA of E. coli could be made accessible to FISH probes.
A final step in rationalization of probe design is the in silico prediction of formamide dissociation
profiles (Yilmaz and Noguera, 2007). This was accomplished with a model defining each free energy term
related to affinity (Figure 11.27) as a linear function of formamide, thus allowing the calculation of
DG–overall (Equation 3) for each formamide concentration.
THERMODYNAMIC APPROACH FOR THE DESIGN OF FISH PROBES

To describe the different steps involved in design of a FISH probe, and to illustrate how the mechanistic
model of FISH can be used, a step-by-step design of a probe targeting a specific sub-cluster of organisms
within the genus Nitrospira is presented.
Background and design objective

Nitrospira is one of the two recognized genera of nitrite oxidizing bacteria (NOB) important in activated
sludge (chapter 9). Daims et al. (2001a) described Nitrospira diversity (based on 16S rRNA sequence
analyses) consisting of four distinct lineages, with only members of Lineages I and II detected so far in
activated sludge (Daims et al., 2001a; Maixner et al., 2006). The characterized strains of N. moscoviensis
and N. marina belong to Lineages II and IV, respectively. A representative of Lineage I, Candidatus
‘Nitrospira defluvii’, has also been described although not yet purified (Spieck et al., 2006), and Candidatus
‘Nitrospira bockiana’, not related to any of the four lineages has been isolated (Lebedeva et al., 2008).
We have detected Nitrospira in drinking water chloraminated distribution systems belonging to
Lineages I and II (Noguera et al., 2009; Regan et al., 2002, 2003), with most Lineage II-related sequences
13
The concern with predictive power is especially about DG–3 due to the many conformational states
that the large target molecule can assume, and thus, different approaches have been proposed for DG–3
calculation (Yilmaz and Noguera, 2004).
forming a phylogenetically distinct cluster, identified as DW (Drinking Water) (Figure 11.29). Thus, the
design objective in this example is to develop a FISH probe and protocol for the detection of this NOB
cluster in environmental samples. Since none of the sequences within this DW cluster are from activated
sludge, it is not known whether these organisms are present there.
DW NsrC7-6; AY048956
PL1169; AWWARF #3088
DW NsrC7-2; AY048952
FP0956; AWWARF #3088
C203616; AWWARF #3088
C109523; AWWARF #3088
TH_c156; EU273233
DW Ntspa445
unc. Nitrospira sp Nsp13; AY876625
Nitrospira sp.; AF035813
Nsr R2-8; AF454659
Nsr R2-6; AF454658
Nsr R2-4; AF454656 Ntspa1151
Nsr R2-2; AF454654
DW NsrE7-10; AY048960
unc. Nitrospira sp.;4; DQ414433
unc. Nitrospira sp.;3; DQ414437
Ntspa662
Nitrospira sp.; AJ224039
Nitrospira sp.; Y14644
unc. bacterium A14; EF069263 Lin-II
Nsr LWCE-2; AF454664

Nitrospira cf. moscoviensis; AF155152
DW NsrB8-6-6; AY048965
Nsr R1-1; Af454652
unc. bacterium A-11; AF033558
unc. Nitrospira sp.; 1; DQ414434
unc. Nitrospira sp.; 3; DQ414436
Nitrospira sp.; Y14640 Lin-I Ntspa1431
Candidatus nitrospira defluvii; DQ059545
Candidatus Nitrospira bockiana; EU084879
unc. bacterium Cc033; AY942757
Thermodesulfovibrio islandicus (T)
0.1
Figure 11.29. Phylogenetic tree showing Lineages I and II in the genus Nitrospira, which include NOB
relevant in activated sludge. Sequences retrieved from drinking water distribution systems define a separate
cluster (DW) related to Lineage II. The example in this section describes the design of a FISH probe targeting
the DW cluster (probe Ntspa445). Coverage of other Nitrospira-related probes (Maixner et al., 2006) is
indicated. Bold font shows sequences from authors’ clone library; other sequences were retrieved from
GenBank. Underlined sequences have single mismatches to the Ntspa445 probe (see Table 11.7).
Step-by-step probe design

Figure 11.30 illustrates the general steps involved in FISH probe design. The procedure has several
decision making steps, and where it may be necessary to start the process again, by re- selecting the probe
target site.
Clone
Selection Sequence library
of alignment
target site Public
database
Public
databases:
Too many
Specificity check RDPGenBank
Non-targets
SILVA
…
None or few
non-targets
Probe affinity ∆Goov=f(∆Go1, ∆Go2, ∆Go3)

I∆GoovI< 13 kcal/mol
I ∆GoovI > 13 kcal/mol
Too many
Mismatch stability ∆∆Go1MM=f(MMtype, NNMM)
Stable MMs
None or few
stable MMs
FAm ≥ 60% (v/v) Formamide

dissociation LFEM
profiles
FAm< 60% (v/v)
Experimental optimization
Figure 11.30. Thermodynamics-based probe design. Side nodes (rectangular) provide descriptions for the
main steps (diamond). Abbreviations: MM, mismatch; NNMM, nearest neighbors of mismatch; LFEM, linear
free energy model.
SELECTION OF TARGET SITE: The first step is the search for a site on the target molecule specific
to populations of interest (the 16S rRNA molecule?). A comparative analysis of aligned sequences
representing targeted and closely related organisms that need differentiating from the targets is performed.
An appropriate target site would (i) be 15–30 nucleotides long, (ii) maximize the coverage of target
organisms, and (iii) eliminate or minimize the number of non-targets sharing this target sequence.
Table 11.7 shows examples of target and non-target sequences used in this example14. The probe is 21
nucleotides long, a perfect complement to targeted organisms, and has two or more mismatches to all
other organisms in the set of sequences used. Thus, the selected target site fits these three criteria. The
exact target site length was determined iteratively using the specificity check and probe affinity steps
described below. We have included only the final sequence in Table 11.7, although shorter and longer
segments were also evaluated.
14
Only a subset of sequences used in comparative analyses is shown in Table 11.6. For manual
analyses as in the given example, the size of the total set of target and non-target sequences used for
comparison can be in the order of 50 sequences.
386
Table 11.7. Probe design example: Target site specificity of probe Ntspa445.
Probe Sequence and Target Sitec
Organism Stepa Source Treeb 30 -CCCTTCTACCTCACCCATTGG-50 MM typed DDG–1MMe
DS Clone FP0956 Selection Clone Lib. DW 50 -GGGAAGATGGAGTGGGTAACC-30 na na
AF035813 Nitrospira sp. Selection Public DW ............................................................. na na
DQ414434 uncultured Nitrospira sp 1 Selection Public Lin-I ..............................................C.A.C..T. na nd
DQ059545 Candidatus ‘Nitrospira defluvii’ Selection Public Lin-I ..............................................C.A.C..T. na nd
DQ414437 uncultured Nitrospira sp 3 Selection Public Lin-II ..........................................AC.............. na nd
X82558 Nitrospira moscoviensis (T) Selection Public Lin-II ..........................................AC.............. na nd
EU084879 Candidatus ‘Nitrospira bockiana’ Selection Public out .................................C..CCCAC.AC.A. na nd
AY876625 uncultured Nitrospira sp Nsp13 Check Public DW ........................................G.................. dCTC/rGGG 1.3
EU273233 uncultured bacterium; TH_c156 Check Public DW .........................................................G. dTGG/rAGC 3.5
AY048960 uncultured bacterium; DW_NsrE7-10 Check Public DW .............................................C............. dCAC/rGCG 3.1
AY048952 uncultured bacterium; DW_NsrC7-2 Check Public DW .................................................T......... dTCA/rATT 3.6
AY942757 uncultured bacterium; Cc033 Check Public out ..A........................................................ dCCT/rGAA 5.8
AJ224041 Nitrospira sp. Check Public LinI ..................................................C........ dTAC/rACG 3.3
EF069263 uncultured bacterium; A14 Check Public LinII .................................................A......... dTCA/rAAT 3.6
a
Indicates whether organism was considered in target site selection or specificity check steps of probe design.
b
Phylogenetic group according to Figure 11.29. out, outside of the three lineages of interest.
c
Selected target site for detection of Nitrospira within the DW cluster (see Figure 11.29). Top row represents probe sequence from 30 to 50 end. The first target
sequence is also explicitly indicated, while others are represented by letters at mismatches to the probe and dots in other positions.
d
Trinucleotide group with single mismatch and its nearest neighbors. Orientation is: probe sequence (30 –50 )/target sequence (50 –30 ). Letters d and r stand for
DNA and RNA, respectively.
e
Free energy indicating mismatch stability, in kcal/mol. Values were calculated only for single mismatches using DNA/RNA nearest neighbor parameters and
averages of DNA/DNA and RNA/RNA type mismatch parameters, according to Yilmaz et al. (2008). na, not available; nd, not determined.
SPECIFICITY CHECK: Although the selected target site may look promising in the alignment with
the small sequence sets used, its actual specificity is uncertain without searching comprehensive sequence
databases. Such a search provides a reliable assessment of any risk of a false identification. With 16S
rRNA as the target, the Ribosomal Database Project (RDP) seems to be a useful resource, (http://
rdp.cme.msu.edu/) (Cole et al., 2005). The latest version (release 10) contains more than 700,000
sequences, with about 600,000 designated as ‘Good Quality’ sequences. With the Probe Match tool,
sequences that perfectly match the probe and those with up to three mismatches can be readily found.
For our example (Table 11.7), the RDP Probe Match tool identified 36 perfect matches and 25 single
mismatches to the proposed probe sequence15, with all but one sequence within the genus Nitrospira.
Eighteen of the perfect oligonucleotide matches were sufficiently long for comparative analyses, and all
belonged to the DW cluster16. These findings suggest the selected target site is relatively specific17. Single
mismatches comprise seven different sequence types, all of which are represented in Table 11.7.
Careful re-evaluation reveals that four mismatch types belong to organisms in the targeted DW lineage
(Figure 11.29). These could be included in the probe coverage by introducing appropriate degeneracy into
its sequence (e.g. a G or a C in the second position from the 50 end), which allows the mismatching
organism TH_c156 to be targeted [Table 11.7]). However, for simplicity, in this example no degeneracy is
introduced. The remaining three mismatch types belong to organisms falling outside this subcluster, and
therefore, probe and protocol designs should aim at ensuring that they do not bind to the probe.
If the database search yields too many sequences with perfect and single mismatches outside the target
lineage (not the case in this example, but often encountered in initial stages of probe design), a different
target site should be evaluated (Figure 11.30).
Other useful rRNA databases include SILVA (http://www.arb-silva.de/) (Pruesse et al., 2007), the
Greengenes project (http://greengenes.lbl.gov/) (DeSantis et al., 2006), and the European rRNA database
(http://www.psb.ugent.be/rRNA/) (Wuyts et al., 2004). probeCheck (http://www.microbial-ecology.net/
probecheck) (Loy et al., 2008) provides a probe specificity checking tool that enables the directed use of
RDP, Greengenes, and SILVA databases from one site. Among these the broadest content belongs to
SILVA where the type of rRNA, and number of sequences are considered21. GenBank (Benson et al.,
2006) (http://www.ncbi.nlm.nih.gov/Genbank/index.html) can also be used, being the largest source of
publically available sequences regardless of target molecule. However, its raw rRNA sequences mean
much more work (i.e. alignment, taxonomic classification, etc.) than the rRNA-specific databases.
It is important to mention also ARB software (Ludwig et al., 2004), a phylogenetic analysis tool widely
used. ARB offers a probe design routine equipped with an algorithm that finds optimum target sites with
respect to specificity. Thus, with a large enough database, this tool automates the target site selection and
specificity check steps given in Figure 11.3018. SILVA provides large ARB-compatible databases
convenient for ARB users (Pruesse et al., 2007).
PROBE AFFINITY: While comparative sequence analyses yield a potential target region, the exact
target site (and the probe length) is decided based on probe affinity. We favor using the thermodynamic
15
Only good quality sequences in RDP database were used.
16
RDP server has a built-in tree construction tool that helps in rapid identification of organisms based
on phylogenetic clusters.
17
Sometimes it is not possible to eliminate perfect matches to non-targets outside the taxonomic group
of interest (species, genus, phylum, etc.).
18
However, the up-to-date thermodynamic parameters and variables used in the mechanistic approach
described in this chapter are not yet included in ARB.
affinity concept discussed above (i.e. DG–overall), and recommend that overall free energy change of
hybridization be less than 14 kcal/mol for efficient hybridization.19 However, values too negative20 may
compromise specificity, as stability of probe/non-target hybrids also increases with increased affinity. Thus,
the initial selected target site is extended or shortened until a reasonable DG–overall value is achieved.
Specificity of the target site may be compromised during this and adjustments require iterations with
previous steps (Figure 11.30). Occasionally, when the target region is associated with a highly stable rRNA
secondary structure (large negative DG–3 values), it may not be possible to achieve the desired DG–overall
values without extending the probe beyond 30 nucleotides in length. Since probe length can offset the
potential gains in hybridization efficiency (Figure 11.27), a different target site should then be considered.
Probe folding free energy (DG–2) should also be separately evaluated, as highly negative values
(DG–2 52 kcal/mol) may create stable probe structures, causing potentially lower hybridization
efficiencies than predicted from DG–overall (Yilmaz, 2006). In the example here, thermodynamic affinity of
the Ntspa445 probe was adjusted to a nearly ideal value of 16.4 kcal/mol, with a positive DG–2
(Table 11.8), which indicates that it has no substantial self-complementarity. Both values were calculated
for typical FISH conditions (T ¼ 46– C, [Naþ] ¼ 0.9M) by methods described in Yilmaz and Noguera
(2004). An online resource for calculating these free energies can be found at http://homepages.cae.wisc.edu/
,noguera/fish.html.
MISMATCH STABILITY: In this step, risk of false positives is evaluated thermodynamically for the
potential target site. Since probe specificity depends on destabilizing effects of mismatches in probe/rRNA
hybrids, more stable mismatches pose a higher risk of false identification. Mismatch destabilization can be
quantified as an increase in the free energy change of the formation of probe/rRNA duplex (DG–1) from
insertion of a mismatch, a variable defined as DDG–1 (Yilmaz et al., 2008). This variable is a function of
mismatch type21 and nearest neighbors in the perfect match duplex that become eliminated upon
mismatch insertion. Since effects of mismatch type have not been quantified for all DNA/RNA duplex
combinations (Sugimoto et al., 2000), the average of thermodynamic parameters defined for DNA/DNA
and RNA/RNA mismatches has been proposed for FISH, with reasonable predictions (Yilmaz et al.,
2008). Based on single mismatch stability tests with E. coli 16S rRNA, a threshold DDG–1 value of 2.0
kcal/mol is the set value used for deciding whether a mismatch is unstable (Yilmaz et al., 2008). In other
words, mismatch discrimination in FISH is predicted to be difficult when the DDG–1 is 155 2.0 kcal/mol.
The experimental solution is to use competitor probes, unlabelled oligonucleotides with perfectly
complementarity to non-target organisms with the target sequence mismatch (Manz et al., 1992). Since
competitor probes also tend to decrease probe sensitivity because of competition for the target site, using
too many competitors in a FISH experiment cannot be recommended. So target sites with too many stable
mismatches should be avoided, bringing the designer back to the target site selection step (Figure 11.30).
For the given example, Table 11.7 lists the DDG–1 calculated for the seven different mismatch types
identified. Based on this parameter, only one type of stable mismatch is below the threshold of 2 kcal/mol,
but it belongs to an organism from the targeted lineage. Therefore, there is no need for competitor probes.
The position of a mismatch affects mismatch stability, although this information derives from
experimental observations other than FISH (Urakawa et al., 2002; Pozhitkov et al., 2006). Generally,
19
The more negative DG–overall the more stable the probe/target hybrid, as follows from thermodynamic
principles.
20
See the formamide dissociation profile step for the criterion of ‘‘too negative’’.
21
In this context, mismatch type includes the mismatch pair and the nearest neighbors of this pair on
the duplex.
Table 11.8. Characteristics of FISH probes used in the design example.
Target siteb Thermodynamic parametersd
a c
Probe Sequence 50 30 Target organism DG–1 DG–2 DG–3 DG–overall Reference
Ntspa445 GGTTACCCACTCCATCTTCCC 445 465 FP0956 27.8 0.7 11.2 16.4 This section
Ntspa1431 TTGGCTTGGGCGACTTCA 1431 1448 DQ059545 20.6 0.4 2.9 17.4 (Maixner et al., 2006)
Candidatus
‘Nitrospira defluvii’
Ntspa1151 TTCTCCTGGGCAGTCTCTCC 1151 1170 AF035813; 26.3 1.0 4.6 21.5 (Maixner et al., 2006)
Nitrospira sp.
Ntspa662 GGAATTCCGCGCTCCTCT 662 679 FP0956 22.6 1.0 8.5 14.1 (Maixner et al., 2006)
a
From 50 to 30 end.
b
Position of target site according to E. coli numbering.
c
Target organism used to calculate DG–3 value.
d
Values, in kcal/mol, calculated according to Yilmaz and Noguera (2004). See http://homepages.cae.wisc.edu/,noguera/fish.html for an online DG–overall
calculator.
Methods for the examination and characterization of the activated sludge community
389
mismatches close to the probe end are more stable than centrally located ones. In the absence of an
empirical positional weight for FISH, judgment of the designer should decide if a competitor is needed
when DDG–1 4 2.0 kcal/mol and the mismatch is close to the probe sequence end. ARB software provides
default parameters for a weighted mismatch score (WM) to evaluate mismatch stability, which takes
positional effects into account. However, ARB does not provide documentation on how these default
parameters were derived. Modifications to default parameters for specific FISH applications have been
recommended based on tests with E. coli 16S rRNA (Yilmaz et al., 2008). Thus, ARB users can benefit
from the WM scores for evaluating mismatch stability, instead of from the thermodynamic DDG–1 concept,
although with additional care. Non-users of ARB can use probeCheck (Loy et al., 2008), which employs
the ARB probe match tool, and provides default WM scores online22.
THEORETICAL FORMAMIDE DISSOCIATION PROFILES: The purpose of this step is two fold;
The first is to design a probe that melts at a moderate concentration of formamide during optimization of
probe specificity. For this, mathematical models predicting formamide dissociation can be used (Yilmaz and
Noguera, 2007; Yilmaz et al., 2008)23. If the theoretical melting point is too high (i.e. $ 60% formamide),
then mismatch discrimination ability may be compromised. This is where the designer can decide that
the DG–overall is too negative (see above), as the melting point strongly depends on this value, and bring the
melting point down by adjusting (generally shortening) the target site (Figure 11.30). This also lowers the
magnitude of DG–overall. Thus, designing a probe with a moderately negative DG–overall value in the affinity step
helps reduce the number of iterations. In the Ntspa445 probe example, the estimated melting point is around
50% formamide (see Figure 11.31 for the predicted dissociation profile), dictated partially by a relatively low
(negative) DG–overall value of 16.4 kcal/mol. More negative values (observed with longer probes) were
avoided, since the corresponding melting points were estimated as greater than 60% formamide.
The second objective is to evaluate the risk of false positives. In this the mismatch discrimination
ability is based on comparing formamide denaturation profiles for target and mismatched organisms
(Figure 11.31), and not the mismatch stability (DDG–1) alone. As well as the DDG–1 value determined in the
previous step, the difference between melting points of perfect and mismatched duplexes (DFAm, see
Figure 11.31) is a function of many variables including probe length and DG–3 values (Yilmaz and
Noguera, 2007; Yilmaz et al., 2008). Bearing in mind the relatively large uncertainty in DG–3 values of
different organisms (Yilmaz and Noguera, 2004), and that the theoretical predictions have not been tested
against multiple-organism systems, these curves should be currently handled with care. Competitor probe
use is recommended when DFAm 5 20% (Yilmaz et al., 2008).
Experimental optimization
While the thermodynamics-based design provides an up-to-date in silico assessment of probe
performance, accuracy of the predictions is not perfect because of the complex nature of in situ
hybridizations. Therefore, final optimization of FISH probe sensitivity and specificity can be performed
only experimentally. Design-level evaluations should be viewed as a means of maximizing the probability
of developing a workable probe with minimal effort in its optimization.
Optimization of sensitivity starts at the level of probe synthesis, when type and position of the
fluorophore are decided. For example, the most economical approach is to attach a fluorescein tag to the
50 end, but this fluorophore is generally associated with lower sensitivity, probably from nucleobase-
22
DNA/DNA associations which may not be relevant to FISH (Yilmaz et al., 2008).
23
Formamide dissociation profiles depend on probe concentration. For all calculations in this chapter,
a probe concentration of 250 nM was assumed.
1 Perfect match
Theoretical hybridization efficiency

dCAC/rGCG
dTCA/rAAT
0.75
dCCT/rGAA
∆[FA]m
0.5
0.25
0
0 20 40 60
Formamide (%) (v/v)
Figure 11.31. Theoretical formamide dissociation profiles for probe Ntspa445. Profiles were derived with the
SmM model in Yilmaz and Noguera (2007) using sequences of clone FP0956 (perfect match), Nitrospira sp.
(dCAC/rGCG-type mismatch), uncultured bacterium A14 (dTCA/rAAT), and uncultured bacterium Cc033
(dCCT/rGAA). See Table 11.6 for details of sequences. The difference in formamide melting points of perfect
match and dCCT/rGAA-type mismatch (D[FA]m) is illustrated.
specific quenching, discussed above. From published reports on fluorophore quenching (e.g. Marras et al.
(2002)) CY-3 seems to be one attractive option, but is relatively expensive. Perhaps the most aggressive
approach is enzyme labeling for subsequent signal amplification (i.e. CARD-FISH), which is not subject
to quenching. However, this option is also expensive, and is rarely necessary in activated sludge studies,
as sensitivity is not limited by ribosome content (but see chapter 8).
Another variable suitable for optimization for improving sensitivity is the hybridization time, as
some target sites are kinetically less accessible. However, extended hybridization times may increase
background fluorescence in some samples. While no theoretical models exist to predict which probes
may benefit, empirical accessibility maps of 16S (Behrens et al., 2003b; Fuchs et al., 1998) and 23S
(Fuchs et al., 2001) rRNA have been developed. Yilmaz et al. (2006) showed that the seemingly
inaccessible sites on these maps can be made more accessible by increasing probe affinity or hybridization
period. Yilmaz et al. (2006) also created a map of kinetic accessibility for E. coli 16S rRNA, to be used as
a guide as to whether incubation time may be optimized.
Optimization requires an experimental derivation of formamide curves (Figure 11.24) with cultured
target and non-target organisms, but these are not always available. Clone-FISH (Schramm et al., 2002),
circumvents this by making in situ copies of the cloned rRNA molecule in a host cell, but is limited by
clone availability, This is especially problematic when some of the most stable mismatches are found in
sequences deposited in public databases, and not in those in one’s own clone library, as with the probe
design example here (Table 11.7). Furthermore, both pure culture and clone-FISH may be tedious when
potential false positives are numerous. Then optimizations can be based on the most stable mismatches
and not on the whole set of mismatched organisms, following theoretical guidelines in the design process
(e.g. using the DDG–1 parameter; Table 11.7).
Clearly possible false identifications in the complex activated sludge community cannot be ruled out
after limited numbers of tests. Therefore the philosophy of optimization should be centered around the
concept of confidence in the results, rather than in a solid proof for the presence of target organisms. An
effective way to achieve this is to use multiple probes for targeting organisms of interest (Amann, 1996;
Amann and Ludwig, 2000; Poppert et al., 2002; Fieseler et al., 2004). The rationale for this approach is
clear. If false positives exist for one probe, it is unlikely that the same organisms would act as false positives
for other probes to which they have different mismatches. The multiple probe approach is most effective
when two probes with different labels are used simultaneously, such that the target organisms would be
those generating a signal at both wavelengths. For this to work, probes should either be designed to have
similar formamide melting points, or should be hybridized successively at the optimum formamide
concentration for each probe, in increasing order of formamide concentrations (Wagner et al., 1994b).
EPILOGUE
This example targeting the Nitrospira DW cluster illustrates the in silico steps for the design of probe
Ntspa445. To complete the design it is necessary to carry out the experimental optimization steps
described above. First, because of potential problems of fluorophore quenching and the likelihood that
dual labeling with other Nitrospira probes is desirable (see Figure 11.29), the probe should be tagged
with different fluorophores. Optimum formamide concentration for probe Ntspa445 was predicted at
about 40–50%, and the predicted DFAm of two single-mismatch types was close to the 20% threshold
(Figure 11.31). Since no pure culture representatives of this cluster are available, the only possible way to
confirm the formamide dissociation predictions would be using clone-FISH with both a target clone and
the closest non-targets in Figure 11.31 (also see Table 11.7). The outcome of this work may or may not
point to the need for unlabeled competitors to minimize false positive hybridizations. At the predicted
formamide levels, any kinetic barriers should be removed at this moderately inaccessible (Yilmaz et al.,
2006) target site. Consequently, conventional or overnight hybridizations should be sufficient for optimal
FISH sensitivity.
Finally, to maximize confidence in hybridization results with Ntspa445, dual fluorophore labeling with
other probes targeting Nitrospira should be evaluated experimentally (see Figure 11.29). For instance,
dual labeling with the Ntspa1151 probe would allow quantification of the relative abundance of the
DW cluster compared to that of Lineage II members. Similar experiments with Ntspa662 and Ntspa1431
probes would allow relative abundances of DW, Lineage I, Lineage II, and the Nitrospira outside
these lineages to be determined. In an experiment with activated sludge collected at the Nine Springs
Wastewater Treatment Plant (Madison, WI), with overnight hybridizations and 35% formamide in the
hybridization buffer, we determined that Lineage I members dominated the Nitrospira population in this
sludge (Figures 11.26 c,d), while no positive hybridizations were observed with Ntspa1151 and Ntspa445
probes at 35% and 40% formamide repectively24. Lack of hybridization with the Ntspa1151 probe and
double positive hybridizations with Ntspa1431 and Ntspa662 probes for most cells provide confidence
that the DW Nitrospira are not abundant in this activated sludge sample. Of course, without full
experimental validation of probe design, and lacking a positive control for hybridizations to Ntspa445
(which should be carried out with clone-FISH since pure cultures are not available), possible fluorophore
quenching or sub-optimal formamide concentrations cannot be ruled out completely.
ACKNOWLEDGMENTS
Funding from the National Science Foundation (grants CBET-9875642 and CBET-0302618), the Water
Research Foundation (Project AwwaRF 04165), and the Water Environment Research Foundation (Paul
L. Busch award to D.R.N) is gratefully acknowledged.
24
Maixner et al. (2006) reported optimal formamide concentrations of 35% for Ntspa662 and
Ntspa1431, and 35–40% for Ntspa1151, based on clone-FISH tests.
11.10 MICROAUTORADIOGRAPHY (MAR)

Per H. Nielsen and Jeppe L. Nielsen
Microautoradiography (MAR) is a powerful tool used first in microbial ecology in the 1960s (Brock and
Brock, 1966, 1968). It has since been applied to a range of activated sludge studies (chapter 3) including
detection of cell viability, enumeration of bacteria capable of consuming specific organic substrates,
studies of autotrophic activity, uptake of orthophosphate and potential use of various electron acceptors
by PAO, GAO and several filamentous bacteria (e.g. Andreasen and Nielsen, 1997, 2000; Daims et al.,
2001a; Eales et al., 2006; Kong et al., 2004, 2005; Kragelund et al., 2005, 2006, 2007a,b; Lee et al.,
1999; Nielsen and Nielsen, 2002a; Nielsen et al., 2003a). The method is based on the observation that
radiolabelled substrate taken up by individual cells can be visualized with a radiation-sensitive silver
halide emulsion, which is placed over the radiolabelled bacteria and subsequently processed by standard
photographic procedures.
The radiotracers used are typically the soft beta-emitters like 3H, 14C and 33P. Common for all is that
the silver grains on top and near the labelled bacteria can be visualized by bright field or phase contrast
microscopy. Low energy emitters give the highest resolution. Tritium, for example, has a resolution of
approximately 0.5 mm while that for 14C and 33P is 2–3 mm.
A detailed description of general procedures for MAR can be found in several publications (Carman,
1993; Meyer-Reil, 1978; Rogers, 1979; Tabor and Neihof, 1982) and as applied to activated sludge
(e.g. Andreasen and Nielsen, 1997, 2000; Nielsen and Nielsen, 2002a,b, 2005).
MAR has been used in combination with staining techniques (Andreasen and Nielsen, 1997, 2000;
Meyer-Reil, 1978; Nielsen et al., 2003b; Tabor and Neihof, 1982). However, the great advantage is being
able to combine MAR with FISH. Together, they provide the means to link identity of individual bacterial
populations to their in situ physiology. Several modifications of the FISH-MAR protocols have been
described (Gray et al., 2000; Lee et al., 1999). They include STAR-FISH (substrate tracking
autoradiography-FISH: Ouverney and Fuhrman (1999) and MICRO-FISH (microautoradiography-FISH:
Cottrell and Kirchman (2000). The protocol presented here for application to activated sludge is based on
those described by Lee et al., (1999) and Nielsen and Nielsen (2005). The value of the information
obtained by combining FISH and MAR is reviewed by Wagner et al. (2006).
The FISH-MAR procedure is outlined in Figure 11.32 (a,b). For figure 11.32b, see colour image
section (chapter 13).
(i) The sample is incubated under defined conditions for sufficient time to allow enough of the
radiotracer to be taken up by active cells. Several factors need to be considered in choosing
incubation conditions: type and specific activity of the radiotracer, concentration of unlabelled
substrate, concentrations of electron acceptors (oxygen, nitrate or others), inhibitors, biomass
concentration and temperature and incubation time. It is advisable to first measure substrate
consumption and incorporation into the biomass to determine optimal incubation conditions.
(ii) After incubation, samples are fixed in 4% paraformaldeyde (PFA) or ethanol, and washed to remove
surplus radiotracer. Subsequent sample homogenization, cryosectioning or dilution may be
necessary for optimal visualization of the FISH-MAR signal. For FISH, standard protocols can be
followed (section 11.9) with FLUOS-, and CY-3-labelled oligonucleotide rRNA targeted probes.
(iii) The stained MAR-positive bacteria are examined by combining bright field or phase-contrast with
epifluorescence microscopy, or if available, CLSM.
Incubation
of sample with radioactive labelled e-donor
under defined e-acceptor conditions
Fixation
Washing step
Sample preparation
e.g.dilution, homogenisation or cryosectioning
Immobilisation on slides
FISH
Film emulsion
Exposure
Film development
Microscopic examination
Figure 11.32. (a) A schematic diagram showing the major steps in the FISH-MAR protocol.
PROTOCOL
The protocol presented here is based on those of Lee et al. (1999) and Nielsen and Nielsen (2005). It is
difficult to describe one suitable for all samples, as many factors are experimental aim-specific. Therefore,
this protocol is general and may need modification for individual applications.
Safety
It is important to conduct all experiments in rooms dedicated to isotope work and to follow all local safety
instructions. Obtain all necessary approval from the appropriate radiation safety officer. All involved
personnel should be properly trained and advised in handling radioisotopes. If all safety instructions are
followed, MAR experiments carry a very low risk.
Notes
(i) Samples must be obtained preferably on the same day as experiments are conducted, although
storage for 1–2 days at 4– C can be used in most cases. Before incubation, it is wise to keep
the sludge aerated for 1–2 h to remove the RBCOD. If anaerobic experiments are to be performed,
the sludge can be kept anoxic for 1–2 h to remove residual nitrate.
(ii) The optimal biomass concentration to use is typically around 1 gSS/l, and any sample dilution
should use supernatant from the activated sludge sample. This level ensures that the substrate
added (typically 0.5–1 mM) is not depleted during a 1–2 h incubation period. However, a lower
biomass concentration or a higher substrate concentration may be required, which should be
determined empirically.
(iii) The radiolabelled tracer must be diluted properly with the same unlabelled substrate to assure that
only metabolically active cells take up sufficient substrate to be visualized eventually as MAR-
positive cells. The amount of tracer required to detect one active cell is approx. 10 15 Ci or
3 · 10 5 Bq, corresponding to a final specific activity of the substrate of approx. 10 6 mCi/mmol.
Typically, 10 mCi tracer is added per ml of diluted activated sludge with a concentration of non-
labelled substrate of 0.5–1 mM. The length of incubation is adjusted to ensure a measurable
uptake, which is often 1–3 h for organic substrates and 3–5 h for uptake of radioactive
bicarbonate.
(iv) A suitable incubation volume is 2–3 ml and 10 ml glass vials closed with butyl rubber stoppers
are recommended. For anoxic and anaerobic incubations, any residual oxygen should be removed
by flushing/evacuation several times with oxygen-free nitrogen and an evacuation pump. Trace
amounts of oxygen can also be removed by introducing a pre-incubation step prior to addition of
radiolabelled tracer. Substrates and tracers are added to the biomass samples for anoxic or
anaerobic experiments using strict anaerobic techniques.
(v) A negative control allowing for chemography must be included in all experiments. A pasteurized
sample heated to 80– C for 10 min is suitable, but must be made just prior to the experiment to
avoid possible germination of bacterial spores.
Termination of MAR incubation and washing and staining

(i) Incubations are terminated by adding freshly made paraformaldehyde (PFA) directly to the vials
(final concentration 4%). For fixing Gram positive bacteria, a final concentration of 50% EtOH/
50% PBS is used. Samples are fixed for 1–3 hours at 4– C.
(ii) They are then pelleted by centrifugation and pellets washed 3 times with filtered (pore size
0.45 mm) tap water. Washed samples are immobilised on hydrophilic (gelatine coated) glass
slides or cover slips (see below) and air-dried at room temperature on the bench. Remaining
sample can be stored at –20– C in 50% EtOH/50% PBS.
Notes
(i) Before immobilization samples may be treated further. If floc-associated bacteria are to be
investigated, samples must be homogenised or sectioned into thin slices by cryosectioning to
distinguish MAR-signals from individual cells. Gentle use of a glass tissue grinder can often be
adequate for disrupting the floc without destroying individual microcolonies and without lysing
cells. Filamentous bacteria external to the flocs can be investigated without any further treatment.
(ii) Special staining procedures like FISH, DAPI, Gram and Neisser staining (section 11.3) can be
made at this point. The FISH-MAR combination is best performed by adding the rRNA targeted
probes directly onto the sample at the center of the cover glass. FISH is then applied as detailed in
section 11.9.
(iii) After the desired staining, slides or coverslips are allowed to air-dry.
Autoradiographic procedure
The following steps are performed in a darkroom with a safety light suitable for black and white film
development procedures.
(i) The film emulsion (LM-11, Amersham Biotech) is placed at 43– C in a water bath and shaken
gently every 2 min for 10 min to remove any air bubbles, before being carefully poured into a
dipping vessel to a height of c.a 3 cm, again making sure to avoid air bubble formation.
(ii) Glass slides/cover slips are carefully dipped into the emulsion for 5 sec, removed and placed
vertically on a folded paper tissue. The sides without any sample are then cleaned with paper tissue.
(iii) These are placed horizontally and dried on a plastic tray for 2 h in the dark, before being placed
in a slide box containing water free silica gel. The box is covered with aluminum foil and exposed
at 4– C. The optimal exposure time is normally 2–10 days but will depend on the sample,
radioactivity of substrate, incubation time etc, and needs to be determined empirically.
(iv) For emulsion development, slides/cover slips are placed in developer (Kodak1 D19, 40 g/l) for
3 min, and carefully drip-dried before being placed in distilled water for 1 min, and then into the
fixer (Na-thiosulphate, 300 g/l) for 4 min. Finally to remove fixer, slides are washed carefully
under running tap water for 10 min followed by 2 £ 2 min washing in distilled water.
Notes
(i) The slides/cover slips must air dry for at least 4 h before microscopic investigation.
(ii) Storage at 4– C preserves slides for at least 1 year.
Microscopic evaluation
(i) Slides are covered by a small drop of antifading agent (e.g. Citifluor1 or Vectashield1) and a
cover slip before examination, either through the supporting cover slip or from above through the
autoradiographic film. Microscopy can be performed by either bright field microscopy or CLSM
with transmitted light from the laser in combination with fluorescence microscopy (epifluores-
cence or LSM).
(ii) Fluorescence of either (5(6)-Carboxyfluorescein-N-hydroxysuccinimide ester (FLUOS) or the
sulfoindocyanine dye CY-3-labelled oligonucleotide FISH probes usually provides sufficient
contrast to the silver grains. CY-5-labelled probes are not recommended since this fluorochrome
results in reflections from silver grains, which may be misinterpreted as FISH–positive signals.
Cover slips have the advantage that the fluorescent signal from cell staining is visible through
them, and thus is not shielded by silver grains from the MAR-signal.
Notes
(i) To find optimal exposure times, additional glass slides should be prepared to allow daily
inspection. If so, it is convenient to combine DAPI staining with examining the MAR signal, by
switching between bright field and fluorescence microscopy.
(ii) Quantification of the MAR signal (QMAR, Nielsen et al., 2003b) can give valuable information
about substrate uptake by the individual cells. It is possible to count the number of silver grains on
top of the bacteria (Nielsen et al., 2000, 2003b). A proper quantification requires procedures that
ensure prevention of substrate leakage from the cells, and use of internal standards, as detailed by
Nielsen et al. (2003b).
PROBLEMS WITH FISH-MAR

(i) MAR and especially FISH-MAR have limitations. Extensive experience with both techniques, and
preferably access to and experience with CLSM are required. It is time consuming, and relatively
expensive from the cost of radiolabelled substrates. In some countries safety regulations require
dedicated laboratories and equipment for isotope work.
(ii) Commercial availability of suitable radiolabelled substrates may be a problem. Often, suppliers
offer a limited selection of either 3H or 14C-labelled organic compounds, while more esoteric
compounds can be produced only on request at high cost. However, shop around as most common
substrates can be purchased at a reasonable price.
(iii) Some of these problems may be solved with another approach, HetCO2-MAR (Hesselsoe et al.,
2005), where labeled bicarbonate is taken up when heterotrophic organisms assimilate and
transform unlabelled organic matter, a process known as heterotrophic CO2 assimilation. This
method only works if the organic substrate is incorporated into biomass so that CO2 is
incorporated concomitantly, which is not always the case. However, for screening potential
organic substrates, it can be useful.
(iv) Interpretation of a positive or negative MAR-signal must be carried out carefully. Controls and
duplicates should always be included. If unexpected results are obtained, these must always be
checked against possible errors in the protocol, particularly in relation to incubation conditions.
This is critical for incubations under anoxic and anaerobic conditions (Lee et al., 1999).
11.11 MICROARRAYS FOR STUDYING COMPOSITION AND FUNCTION

OF COMMUNITIES
Michael Taylor, Alexander Loy, and Michael Wagner
INTRODUCTION
Microarrays were developed for genome-wide analysis of gene expression (Schena et al., 1995, 1996),
and with the continued expansion of genomics it is for this application they are best known. However,
their potential as tools for studying community composition was recognised in 1997 (Guschin et al.,
1997), which has led to an increase in the number of studies using arrays for this purpose (reviewed by
Bodrossy and Sessitsch, 2004; Wagner et al., 2007; Zhou, 2003; Zhou and Thompson, 2002). Current
interest in microarrays reflects their advantages over other molecular techniques, especially their potential
for high-throughput sample analysis once full automation is achieved. This feature alone will be of
considerable benefit in monitoring engineered systems like activated sludge plants.
Despite this, few examples of the full-scale application of microarrays for analysis of complex
communities exist, attributed to complexities associated with method optimization and standardization
(Bodrossy and Sessitsch, 2004). Whether to use DNA or RNA as targets for analyses to achieve desired
results must be considered carefully. Other factors are important, including, but not limited to
microarray platform, probe type and length, amplification and detection strategies, and choice of
functional or rRNA marker genes. The approach chosen depends on the biological question being asked
and the environment under study. One system for which microarrays should prove particularly useful is
activated sludge.
These systems contain diverse communities of microbes whose composition has been described with a
range of molecular techniques (chapter 3). However, all suffer from limitations which can be overcome,
at least in part, with microarrays. Their beauty lies in the highly parallel nature of organism detection,
i.e. hundreds to tens of thousands of probes can be applied simultaneously to a single sample, compared
with only a few as with formats like FISH (Wagner et al., 2003). This advantage is especially important
when dealing with phylogenetically diverse groups of organisms, where no single specific probe or primer
might target all members. In addition, advances in microarray technology hold promise for the analysis of
community-level gene expression (Dennis et al., 2003; McGrath et al., 2008), providing the opportunity to
link organism function and phylogeny in a high-throughput manner (Adamczyk et al., 2003; Neufeld et al.,
2007).
An overview of microarrays for community analysis is presented here, outlining the applications for
which they have been used, and highlighting the benefits and pitfalls of the different approaches. Key
factors leading to successful usage are introduced, and their potential for revealing community structure
and function in activated sludge is discussed.
MICROARRAY FUNDAMENTALS
The term microarray refers to a small solid surface, (e.g. a glass microscope slide), to which is anchored a
set of oligonucleotide or PCR product-based probes. The essential steps of an environmental microarray
analysis are outlined in Figure 11.33, see colour image section (chapter 13). Under appropriate
hybridization conditions, the microarray is covered with a solution including labelled target nucleic acids.
For microbes, these are often (but not exclusively) fluorescently-labelled rRNA gene fragments or the
rRNA itself. Labelled targets then form duplexes with complementary nucleotide sequences present on
relevant probes. Probe sequences may be highly conserved, in that they target most or all microbes, or
targeted to a lower phylogenetic level like genus or species. A washing step then takes place, whereby
weak duplexes (indicating a poor complementarity of probe and target) are denatured, and these targets
removed. Remaining probe-target duplexes can then be identified and in some cases quantified by image
analysis. Identity of organisms present in the sample is inferred from the pattern of probe hybridization.
METHODOLOGICAL CONSIDERATIONS FOR MICROARRAY ANALYSES

Successful applications of microarrays for community analysis (e.g. Brodie et al., 2007; Loy et al., 2002b,
2004; Neufeld et al., 2006; Stralis-Pavese et al., 2004; Wilson et al., 2002a) should be viewed as
methodological triumphs, given the many complex, interrelated factors involved. These should be
considered before beginning any microarray study and are discussed next.
Surface type
Three main solid supports are currently used for environmental microarrays: Affymetrix arrays, planar
glass arrays and three-dimensional array formats (Bodrossy and Sessitsch, 2004; Loy and Bodrossy,
2006).
Affymetrix supply microarrays for whole-genome expression studies, and apply the same technology
(photolithography) to produce high-density microarrays for community analysis. Oligonucleotides are
synthesized directly onto the glass surface by a photolithographic masking method. One such array
comprising 31,179 16S rRNA gene-targeted probes was described and successfully tested on air
(Wilson et al., 2002a), clinical (Flanagan et al., 2007) and contaminated aquifer samples (Brodie et al.,
2006). One advantage of this array type is the high number of probes included, allowing for parallel
detection of many target organisms. However, its high cost has limited its application (but see Wilson et al.,
2002a,b). Furthermore, photolithography is only appropriate for the manufacture of short (530
nucleotides) probes (Hashsham et al., 2004; Schena, 2003) and oligonucleotide quality is not easily
assessed.
Planar glass microarrays are the most widely used array format, reflecting their relatively low cost and
high flexibility. Here, slide surfaces are treated with reactive chemical groups (aldehydes, amino or poly-
lysine groups) to facilitate probe attachment. Probes (oligonucleotides, PCR products or whole genomes)
are generated separately, quality-tested (at least for oligonucleotides), then deposited by a spotting device
onto the surface. For oligonucleotides, this is achieved usually by covalent binding of probes to the
reactive groups on the slide via a terminal amino-modification. For longer probes (approximately 450
nucleotides), no such modification is required. After spotting, reactive groups not bound to probes are
chemically inactivated. Immobilized probes are then available for subsequent hybridization with labelled
target molecules. Many commercially produced microarray spotters are available, and any choice should
take into account both price and particular features required. It may also be worth considering different
surface chemistries since both signal intensity and background levels can vary (Denef et al., 2003;
Taylor et al., 2003).
A third array type involves 3-D structures, comprised of a matrix like polyacrylamide gel built onto the
slide surface. Probes are spotted onto individual gel pads and hybridization takes place inside this 3-D
matrix. This requires highly specialized equipment and is limited to a small number of users. Its benefits
include a higher probe load applied to the gel pads compared with planar glass arrays, and better probe
accessibility due to the 3-D matrix. Gel pad-based microarrays have been combined with special detection
devices to enable simultaneous monitoring of non-equilibrium dissociation curves (melting curves) for all
probes on an array (Guschin et al., 1997, Urakawa et al., 2002, 2003). This approach holds much promise
to improve discrimination between perfect-match and mismatch targets, although debate about certain
aspects of it continues (Pozhitkov and Noble, 2007a,b; Siripong et al., 2006).
Probe type: Oligonucleotides or PCR products?

Microarray probes come in three main types; short (530 bases in length) and long (50–100 bases)
chemically synthesized oligonucleotides, and longer probes (4100 bases) derived from PCR products.
Deciding which type is best comes down to two competing priorities: sensitivity or specificity. As a
general rule, longer probes exhibit greater sensitivity, but lower specificity (Hughes et al., 2001;
Relogio et al., 2002). In environmental studies, where target organisms may be present at very low
abundances within the community, sensitivity is likely to be critical. In such cases, PCR product-based
probes (several hundred bases in length) stand the best chance of detecting them. Such probes led to a
reported detection limit of approximately 1 ng of pure genomic DNA (Wu et al., 2001), compared with
5–10 ng for 50mer oligonucleotides (Rhee et al., 2004; Tiquia et al., 2004). When probe length effects
were systematically assessed, amplicon probes of 500 bp or more produced much stronger hybridization
signals than were obtained with those up to 100 nucleotides long (Letowski et al., 2004). Similar increases
in signal intensity were generated with increasing probe length (PCR product-based) up to at least 700
bases (Franssen-van Hal et al., 2002; Stillman and Tonkinson, 2001). Conversely, Kane et al. (2000)
found 50mer oligos to be as sensitive as PCR product-based probes for detecting 3-fold changes in mRNA
levels, although only probes 5400 bp were used.
Sensitivity is clearly important, but in many contexts specificity may be equally so. Indeed, an ability to
detect routinely single-base mismatches is widely acknowledged as one of the most important goals in
environmental microarray studies (Peplies et al., 2003; Li et al., 2004a; Urakawa et al., 2003). Thus, the
high specificity of microarrays with short (15–25 bases) oligonucleotides has made them popular
(Bodrossy et al., 2003; Brodie et al., 2006, 2007; Chandler et al., 2003; El Fantroussi et al., 2003; Franke-
Whittle et al., 2005; Guschin et al., 1997; Koizumi et al., 2002; Loy et al., 2002b, 2004, 2005;
Peplies et al., 2003, 2004; Sanguin et al., 2006; Small et al., 2001; Wilson et al., 2002a). Their ability to
discriminate between organisms with a single base mismatch at the target site has been confirmed (e.g.
Urakawa et al., 2002, 2003), whereas probes based on long oligonucleotides or PCR products reportedly
discriminate only among sequences with less than about 80–85% similarity (e.g. Taroncher-Oldenburg
et al., 2003; Wu et al., 2001). Even for short oligonucleotides, the likelihood of detecting single base
differences depends on the type and position of the mismatch within the probe-target duplex. Terminal
mismatches, for example, are particularly problematic (Bodrossy et al., 2003; Urakawa et al., 2002, 2003),
although some enzyme-mediated detection methods (e.g. ligase detection reaction; Busti et al. (2002);
Castiglioni et al. (2004)) actually work on this basis. In the ligase reaction, probe-target hybridization
occurs in solution; probes contain a ‘Zip code’ extension, which then binds with complementary
sequences on the microarray (Busti et al., 2002).
Although detection sensitivities of short oligonucleotide probes do not match those of longer probes, it
is possible to use them for direct detection of rRNA in environmental samples i.e. without PCR
amplification and its associated biases (Adamczyk et al., 2003; El Fantroussi et al., 2003; Peplies et al.,
2004; Small et al., 2001). Often this was achievable only with a two-probe detection system, in which
‘capture probes’ are immobilized on the slide surface and, as their name implies, bind to the unlabelled
target nucleic acid in solution and thus ‘capture’ it to the slide (Chandler et al., 2003; Small et al., 2001).
A labelled chaperone or detector probe, designed to bind near to the capture probe, is added free in
solution and facilitates the opening of secondary structures of the target molecule, enabling improved
target-capture probe hybridization. Although this approach is elegant in principle, requirement for a second
probe of reasonable specificity to target the rRNA close to the capture probe may often negate its use.
Finding a sufficient number of specific probe binding sites in a conserved molecule like 16S rRNA can be
difficult (Fuchs et al., 2000): so probe design is not a trivial exercise! Furthermore, using similar but
unlabelled, ‘helper oligonucleotides’ reportedly resulted in a reduction in specificity (Peplies et al., 2003).
Oligonucleotide probe design

Microarray probe design is deceptively simple: probes should detect only those organisms that we wish to
target, and exclude all others. This seemingly straightforward objective is often difficult to achieve, and a
myriad of factors must be considered (Hashsham et al., 2004; Wagner et al., 2007). Fortunately, many
programs are available to assist. One of the most popular is the ARB package (Ludwig et al., 2004),
whose foundation is a sequence database of the target gene. For 16S rRNA a database containing more
than 50,000 aligned sequences is available, while the ARB-compatible SILVA database contains
4500,000 aligned 16S rRNA sequences (Pruesse et al., 2007). Other design programs are also freely
available, including PRIMROSE (Ashelford et al., 2002), PRIMEGENS (Xu et al., 2002), Osprey
(Gordon and Sensen, 2004) and OligoArray (Rouillard et al., 2003). Furthermore, the Ribosomal Database
Project (Cole et al., 2007) offers a Probe Match option, where probe specificity can be checked against a
database of 4700,000 16S rRNA sequences. probeCheck (Loy et al., 2008) is another centralized option
for evaluating probe specificity and coverage (section 11.9).
An important, yet often ignored aspect of probe design is the need for a sound phylogenetic basis. If
sequences used for probe design are incorrect or, alternatively, correct sequences are poorly aligned, both
may generate faulty probes and ultimately a skewed view of community diversity. Previously published
probes should be checked, as some were designed based on much smaller sequence datasets than are
available now. A solid approach to probe design is exemplified by the ‘multiple probe concept’ (Behr
et al., 2000; Loy and Bodrossy, 2006), where probes are designed to target organisms at different levels of
phylogenetic specificity. Some will target the same organisms by complementing several unique regions
of the applicable gene, while others exhibit hierarchical (nested) specificity. Thus, general probes, which
target higher phylogenetic levels, can confirm or otherwise the information obtained with more specific
probes (Behr et al., 2000). In a 16S rRNA gene-based microarray for detecting all known lineages of
sulphate-reducing bacteria (Loy et al., 2002b), each organism of interest was targeted by a minimum of
three probes. Such probe redundancy should increase confidence in the results obtained, since all probes
targeting a given taxon must exhibit signals before a positive identification is made. However, this
approach requires that all probes are initially tested with suitable reference nucleic acids, and only probes
which give a signal with these are retained on the final microarray (Loy et al., 2002b, 2005). Figure 11.34,
see colour image section (chapter 13) illustrates the hierarchical and parallel nature of probes based on
their specificities.
The underlying phylogeny remains one of many considerations in probe design. It is also vital to
consider the hybridization characteristics of any probe set, especially how differences in probe
composition might affect formation of perfectly matched and mismatched probe-target duplexes.
Immobilization of probes onto supports like glass slides makes any prediction of hybridization behavior
difficult. Steric effects may reduce hybridization efficiency from (i) overcrowding of immobilized probes
on a surface, or (ii) reduced access of the target molecule to the probe (Shchepinov et al., 1997;
Southern et al., 1999). The latter can be ameliorated with addition of spacers (in our lab spacers comprise
15–20 ‘T’ nucleotides at the 50 end of the probe), which displace the probe away from the slide surface
(Guo et al., 1994; Shchepinov et al., 1997), or by fragmentation of target molecules. This reduces any
effects of secondary structure in the target, and shorter molecules are more compatible for hybridization
with short immobilized probes. Signal strength is often enhanced compared with non-fragmented targets
too (Bodrossy et al., 2003; Chandler et al., 2003; Small et al., 2001). Probe overcrowding can be
minimized by empirically determining the optimal probe concentration (Peterson et al., 2001;
Relogio et al., 2002).
A major aim with microarrays in environmental work is to discriminate between perfectly-matched
and mismatched targets. Mismatch characteristics (position and type) are important in this. Central
mismatches are easier to discriminate against than those in terminal positions with short oligonucleotide
probes (Urakawa et al., 2002, 2003), and where possible, probes should be designed with this in mind.
However, distributing mismatches evenly over the entire length of the probe increases specificity for long
oligonucleotides (Letowski et al., 2004). The type of mismatch is another factor (Bodrossy et al., 2003),
although this may be less critical than mismatch position (Urakawa et al., 2002).
Parallel hybridization requires that all probes on a microarray display similar thermodynamic
properties, i.e. melting behaviour. In some labs, oligonucleotide probes are designed of varying length so
they all have similar predicted melting temperatures (e.g. Bodrossy et al., 2003), while others use probes
of the same length, but compensate for differences in melting temperature by addition of tetramethyl
ammonium chloride (TMACl) or other tertiary amine salts (e.g. Loy et al., 2002b, 2005). Such compounds
reduce differences in melting behavior among different oligos by equalising stabilities of AT- and GC-rich
duplexes (Jacobs et al., 1988; Maskos and Southern, 1992). Other points to consider are base-stacking
interactions (i.e. influence of adjacent nucleotides) and putative secondary structures of both probe and
target molecules. All influence probe-target hybridization, and consequently the likelihood of achieving
desired results. Parameters which integrate many of these characteristics, like binding free energy (DG) or
melting temperature of probe-target hybrids, may therefore prove useful in predicting their hybridization
behavior and thus facilitate design of specific and thermodynamically homogeneous probe sets (Loy et al.,
2005; Stralis-Pavese et al., 2004; Taroncher-Oldenburg et al., 2003) (section 11.9).
The importance of probe design cannot be overstated, particularly with short oligonucleotide probes
where minor sequence changes may compromise specificity. Subsequent success or otherwise with
complex communities ultimately rests upon the quality of the designed probe set. Specificity, sensitivity
and thermodynamic homogeneity are all important considerations. Moreover, in silico probe design
should be followed by a rigorous evaluation process, whereby the array is tested against a wide range
of reference organisms (pure cultures and/or environmental clones). Such testing reveals essential
information about the specificity and hybridization behavior of individual probes, which may not be
predicted from the in silico design process. Non-specific probes or probes giving no or poor signal can
then be eliminated from the array prior to application.
Amplification vs direct detection of nucleic acids?

A key issue with microarrays in microbial ecology is detection sensitivity. Often target genes or organisms
are present at very low abundance against a background of high diversity. In such cases, the standard
approach has been to amplify extracted nucleic acids using PCR, which can generate millions of gene copies
from a low initial number, and greatly increase the likelihood of detecting rare organisms. PCR products that
can be labelled during or after amplification (see below), are then available for hybridization. However, a
drawback of all PCR-based approaches is the bias associated with amplification of mixed templates (see
chapter 3) (Polz and Cavanaugh, 1998; Suzuki and Giovannoni, 1996; von Wintzingerode et al., 1997). It is
now accepted that PCR-based methods may not necessarily provide an accurate picture of microbial
community structure. The question is whether one has a choice i.e. is it possible to detect the target
organisms in an environment of interest without amplification? Here we examine the available evidence.
Several studies have successfully employed PCR amplification of target genes prior to microarray
hybridization (Bodrossy et al., 2003; Brodie et al., 2007; Loy et al., 2002b, 2004, 2005; Siripong et al.,
2006; Taroncher-Oldenburg et al., 2003; Wilson et al., 2002a). The 16S rRNA gene has been utilized,
with partial (Loy et al., 2005; Wilson et al., 2002a) or almost-entire (Loy et al., 2002b, 2004, 2005) gene
fragments being amplified. The general approach is to use conserved or universal primers; detection
specificity can then be achieved with group- or species-specific probes on the microarray. However,
greater sensitivity may be achieved with direct use of group-selective PCR primers. Detection of members
of the betaproteobacterial Rhodocyclales in activated sludge was enhanced with PCR primers targeting
these (Loy et al., 2005). Indeed, Rhodocyclales populations comprising less than 1% of all Bacteria
(determined by FISH) could be detected. However, this approach may prove less feasible for
phylogenetically diverse target groups, since many separate PCR reactions would be needed. Multiplex
PCR, where several different primer pairs are combined in one reaction, may offer a partial solution
(Panicker et al., 2004; Vora et al., 2004). PCR amplification of the functional marker gene particulate
methane monooxygenase (pmoA) allowed Bodrossy et al. (2003) to detect targeted methanotrophs down
to 5% of the total methanotroph community. Similarly, a PCR protocol targeting nitrifying bacterial
populations in wastewater improved their detection sensitivity (Siripong et al., 2006).
The best approach ultimately involves direct application of extracted RNA (i.e. without prior
amplification) onto the microarray (Adamczyk et al., 2003; El Fantroussi et al., 2003; Peplies et al., 2004;
Small et al., 2001). By removing the PCR step and its biases, the potential for quantification can be increased
(Bavykin et al., 2001). However, inferring cell numbers from measuring rRNA contents is not possible,
because rRNA concentration of a population depends on its growth rate too. Sensitivity is the key issue here,
as the target RNA may be present only at low abundances in a diverse community. However, this
methodology has been applied successfully to activated sludge (Adamczyk et al., 2003), soil (Chandler et al.,
2003; Small et al., 2001), marine sediments (El Fantroussi et al., 2003) and seawater samples (Peplies et al.,
2004). All these studies utilized naturally amplified rRNAs, which can be present in thousands of copies per
cell. Direct detection of DNA or RNA is also more straightforward if one exploits the higher sensitivity of
longer oligonucleotide probes (e.g. Taroncher-Oldenburg et al., 2003) or PCR product-based probes
(e.g. Wu et al., 2001). Their advantages and disadvantages were discussed earlier.
Although difficult practically, microarray work with messenger RNA (mRNA) offers the exciting
possibility of monitoring community-level gene expression (Dennis et al., 2003). Using a similar protocol
to that for pure-culture gene expression experiments, mRNA can be reverse-transcribed (RT-PCR) to form
cDNA, which is subsequently labelled and hybridized with immobilized probes. However, sensitivity is
again the major issue, since mRNA comprises only a minor fraction of total RNA transcripts in each cell.
As it will contain transcripts from many different genes, detecting expression of a specific gene in an
environmental sample is challenging, but some progress has been made (also see below), with measuring
induction of resin acid-degradative genes in an activated sludge sample (Dennis et al., 2003).
Somewhat unexpectedly, reported detection limits for microarrays with prior PCR amplification differ
little to those involving direct detection of nucleic acids. In the latter, bacteria with relative abundances of
c.a. 5% have been detected (Cho and Tiedje, 2002; Peplies et al., 2004), similar to results using PCR
amplification (Bodrossy et al., 2003; Loy et al., 2005). However, such comparisons based solely on the
literature should be treated with caution, since probes with differing lengths and specificities were used.
Moreover, even the method for determining the proportion of the community detected is not constant.
Thus, what is required is a systematic comparison of detection limits for amplified and directly-detected
nucleic acids, under standardized conditions and with identical probes. To our knowledge, no such study
has been reported.
Labelling strategies
Many options for labelling nucleic acid targets prior to hybridization exist; here we focus on the most
common approaches, using fluorescent dyes. Most utilize the cyanine dyes CY-3 and CY-5 or the related
Alexa dyes (Alexa Fluor 555 and Alexa Fluor 647, respectively), since their discrete spectral qualities
enable their respective signals to be discriminated when both are present on the same slide. We use
random prime labelling (Loy et al., 2002b), in which short (10mer) oligonucleotides of random sequence
are mixed with denatured target DNA (i.e. PCR products) in the presence of CY-labelled dCTP
nucleotides. These are then incorporated into newly synthesised strands (synthesis is catalysed by the
Klenow fragment included in the reaction mixture). Random prime labelling has been used in other
environmental studies (Cho and Tiedje, 2002; Rhee et al., 2004; Wu et al., 2001).
Direct incorporation of labelled nucleotides during PCR amplification is also an option (e.g. Behr et al.,
2000), as is using fluorescently-labelled PCR primers, except that only the ends of the target fragments
(i.e. where primers have bound) contain labels. This may result in a weaker signal than when multiple
labelled nucleotides are used. However, such modified nucleotides can also create problems, as PCR
efficiency is reduced from their bulky size. When DNA templates are converted into RNA, labelling
during in vitro transcription allows efficient dye incorporation (Bodrossy et al., 2003).
Post-labelling offers yet another alternative where a chemically reactive amino-allyl nucleotide analog
is enzymically incorporated into the target DNA during PCR, followed by post-hybridization coupling
with an antibody specific for the label and bound with horse radish peroxidase. Fluorescent tyramide
molecules (e.g. CY-3-tyramide) are then enzymatically deposited onto the microarray surface at sites of
duplex formation, amplifyin the signal by up to 1000 fold (Schena, 2003). Denef et al. (2003) showed that
TSA labeling compared favorably with amino-allyl and direct nucleotide incorporation methods, where
70mer oligonucleotide probes plus TSA allowed detection of 1% of the total community without PCR
amplification. Amino-allyl groups are smaller than the fluorescent bases, so nucleotide analogs
incorporate into DNA or RNA efficiently, resulting in more uniform labeling. The additional advantage
is that CY-3 and CY-5 labelling efficiencies are equal (valuable if performing competitive hybridizations
with two samples on the same microarray). Dennis et al. (2003) found at least a two-fold increase in signal
intensity with amino-allyl labelling of mRNA compared to when CY-labelled dNTPs were used directly in
reverse transcriptase reactions.
Tyramide signal amplification (TSA) labelling is another promising technique. It involves enzymatic
incorporation of labelled nucleotides into target nucleic acids, followed by post-hybridization coupling
with an antibody specific for the label and bound with horseradish peroxidase. Fluorescent tyramide
molecules (e.g. CY-3-tyramide) are then enzymatically deposited onto the microarray surface at sites of
duplex formation, amplifying the signal up to 100-fold under optimal conditions (Schena, 2003). TSA
labelling compared favourably with amino-allyl and direct nucleotide incorporation methods (Denef et al.,
2003). Using 70mer oligonucleotide probes together with TSA, 1% of the total community could be
detected without PCR amplification. This should encourage further exploration of this approach.
Biotinylation offers an alternative to fluorescence labelling, and has been used in several microarray
studies (Chandler et al., 2003; Vora et al., 2004; Wilson et al., 2002a).
POTENTIAL FOR QUANTIFICATION

The value of microarrays will be enhanced once quantification is achievable. Unfortunately, many
potential biases are associated with microarray manufacturing, sample preparation (e.g. DNA/RNA
extraction, PCR amplification, labelling) and hybridization (e.g. steric effects, secondary structures of
probes and targets), all of which make quantification difficult (Table 11.9).
One of the first attempts to quantify genes in mixed communities was that of Cho and Tiedje (2002).
An identical reference gene fragment (consisting of l DNA) was added to each probe solution prior to
spotting on the array. An environmental sample labelled with CY-3 was mixed with CY-5-labelled l
DNA then hybridized with the array. Quantification was based on the resulting ratio of CY-3 (¼ sample)
to CY-5 (¼ reference) signals (Cho and Tiedje 2002). In other studies, strong linear relationships between
DNA concentration and signal intensity have been observed with mixed samples, using both PCR product-
based probes (Wu et al., 2001) and 50mer oligonucleotides (Tiquia et al., 2004), suggesting that some
level of quantification should be achievable even in complex microbial systems. However, comparison
among different probes on a microarray is difficult because the slopes of their linear correlations vary
(Tiquia et al., 2004; Wu et al., 2001).
Efforts have been undertaken using shorter (15–26mer) probes (Bodrossy et al., 2003), where a reference
mixture of known composition was labelled with CY-5 and hybridized on the microarray with CY-3-labelled
target molecules. By comparing signal ratios (CY-3/CY-5) a semi-quantitative description of methanotroph
community composition was achieved, where variations in spot morphology and hybridization efficiency
among probes was accounted for (Bodrossy et al., 2003). Competitive hybridizations of two samples, one
CY-3- and one CY-5-labelled, were used to compare relative abundances in, say, control and treatment, or
the same environment sampled at two points in time. This eliminates slide-to-slide and spot-to-spot
variation, but is still prone to biases arising from differential incorporation of the two dyes (amino-allyl
labelling or dye-switch experiments could help circumvent this problem; see above).
Table 11.9. Potential barriers to quantification of environmental microarray data, and how to address the
problems.
Potential bias Possible improvements
Inconsistent spot size and shape • Check spots using nucleic acid stain (only for selected
slides in a batch, as staining can prevent subsequent
hybridisation) (but see Battaglia et al., 2000)
• Include lDNA during probe spotting. CY-5-lDNA is added
with CY-3-target during hybridisation; normalise according
to CY-3/CY-5 ratios (Cho and Tiedje, 2002)
• Include CY-3-labelled nucleotide during probe spotting;
after spots checked, CY-3 washed off prior to hybridisation
(Shearstone et al., 2002)
DNA/RNA extraction biases • Assess results using different extraction methods
(e.g. some cells may be lysed less • Include an internal standard during extraction-
efficiently than others) see section 11.6
PCR amplification biases • Ideally, detect extracted RNA directly (e.g. Adamczyk et al.,
2003; El Fantroussi et al., 2003)
• If amplification necessary, minimise PCR bias
(e.g. increase template concentration, reduce cycle
number) (Polz and Cavanaugh, 1998)
Labelling biases • For two-colour analyses, normalise for incorporation
efficiency of CY-3 vs CY-5 through addition of internal
standard (e.g. Cho and Tiedje, 2002)
• Difficult to control for bias occurring among different
samples labelled with same dye
Steric effects • Employ spacer molecules to hold probe away from slide
– target access to probes may be surface
limited for larger fragments • Shear target DNA/RNA
– probe overcrowding • Optimise amount of probe spotted ! select concentration
with highest hybridisation signal
Differences in hybridisation behaviour • During probe design, try to make melting temperatures as
among probes (even seemingly similar similar as possible (e.g. Bodrossy et al., 2003) or minimise
probes can yield markedly different differences between A:T and G:C pairs using TMACl (e.g.
hybridisation signals with their respective Loy et al., 2002b).
perfect match targets) • Probe (and target) secondary structure are also potential
factors. ! predict probe-target hybridisation properties
using DG calculations (e.g. Loy et al., 2005).
• Test specificity and sensitivity of each probe on the
microarray empirically
Marcelino et al. (2006) described an algorithm which could separate true probe-target signal from
additional signal from cross hybridization and noise. They claimed a capacity for specific and accurate
quantification of rRNA sequences in complex environmental samples and identified Vibrio spp.,
comprising50.05% of the total bacteria in a marine sample. Others have reported considerable problems
in predicting microarray hybridization behaviour (Pozhitkov et al., 2007a, 2008), suggesting that at the
very least, quantification potential should be analysed rigorously for each array format.
rRNA VS FUNCTIONAL GENE MICROARRAYS

Microarrays for microbial ecology can be grouped into three categories: community genome arrays,
functional gene arrays (PCR product- or oligonucleotide-based) and rRNA-targeted oligonucleotide arrays
(PhyloChips) (Zhou 2003). The first of these relies on initial culturing of organisms for probe construction
and is considered only briefly. Most studies of complex communities involve either rRNA or functional
gene arrays, and these will be discussed in detail.
PhyloChips invariably target the 16S or 23S rRNA (genes) (Table 11.10), which share several useful
features: they occur in all ‘prokaryotes’, they contain both conserved and variable regions (allowing
targeting at various phylogenetic levels), and a wealth of sequence information is publicly available
(Ludwig and Schleifer, 1999). This is especially true for 16S rRNA genes (Ludwig et al., 2004); currently,
more than 700,000 16S rRNA sequences are available through the Ribosomal Database Project (http://
rdp.cme.msu.edu)), greatly facilitating probe design. The main limitation of the 16S rRNA approach is the
high overall sequence conservation of this gene (chapter 1). Strain- and even species-level differentiation
is often impossible, thus often limiting the level of phylogenetic resolution. An ability to discriminate
between single mismatches therefore assumes even greater importance.
Table 11.10. Key differences between PhyloChips and functional gene arrays.
PhyloChip Functional gene arrays
Targetable genes 16S and 23S ribosomal RNA Any gene encoding for a specific
relevant for activated genes (present in all ‘prokaryotes’) cellular function
sludge microorganisms e.g. ammonia monooxygenase amoA,
nitrite reductases nirS/nirK, nitrogenase
nifH, dissimilatory (bi)sulphite reductase
dsrAB
Use of nested probes Yes Limited due to variable ‘wobble’ position
which render design of probes for
broader target groups difficult
Number of available 16S rRNA: 4114,000 for amoA: 1158
sequences1 Bacteria (Cole et al., 2005) nirS: 411
23S rRNA: ,1100 dsrAB: 924
(Wuyts et al., 2004) nifH: 1784
(from Schadt et al., 2005)
Specificity Single base difference Short oligonucleotides: single base
(these arrays invariably use difference
short oligonucleotides) Long oligonucleotides: 588% sequence
similarity (Rhee et al., 2004)
PCR products: 585% sequence similar-
ity (Wu et al., 2001)
Taxonomic resolution Species/strain level Short oligonucleotides: strain level
(depending on taxa) Long oligonucleotides: species level
PCR products: species level in best-case
scenario
Possibility of examining Yes, by combining with Isotope Yes, by extracting mRNA and measuring
activity? Array (radioactivity readily detected gene expression
in rRNA)
1
For some genes (e.g. 16S rRNA gene), extensive databases of aligned sequences are freely available
(e.g. http://arb-db-central.swiki.net/1). Others must be aligned after extracting relevant sequences from public databases.
In our laboratory we have used the PhyloChip approach (Loy et al., 2002b, 2004, 2005) (Figure 11.35,
see colour image section (chapter 13)), based on an encompassing phylogenetic analysis of a particular
functional or phylogenetic group of organisms. This is not to be confused with the functional gene
approach discussed below. Rather, it reflects the fact that some functionally related taxa are related at the
16S rRNA level. For example, the ammonia-oxidizing bacteria share a common function and are almost
exclusively contained within a monophyletic subset of the Betaproteobacteria (Koops et al., 2003;
Purkhold et al., 2000, 2003) (chapter 9). In others like the sulphate-reducing ‘prokaryotes’ (SRPs), these
functionally related organisms are spread among distinct phylogenetic groups (Loy et al., 2002b). Since
no single specific primer pair or probe can detect all SRPs, the high parallelism of microarrays offers the
best option for their study. Irrespective of the organisms targeted, a sound phylogeny offers the
opportunity for rational probe design. Wherever possible, PhyloChips contain hierarchical, nested probes,
adhering to the multiple probe concept introduced earlier (Figure 11.34). Redundancy of probes gives
added confidence, since the presence of an organism is only confirmed when all relevant probes return
positive signals. Moreover, the multiple probe concept can lead to detection of novel organisms closely
related to those directly targeted by the array. If probes targeting higher phylogenetic levels (i.e. lower
probe specificity) are positive, but highly specific strain- or species-level probes are negative, then this
could imply presence of hitherto undiscovered populations.
The key to the PhyloChip approach is the subsequent validation of results by independent methods
(Figure 11.35), allowing confirmation of microarray results and potentially, the design of new probes
(e.g. if a clone library yields novel sequences not targeted by the initial array). Relevant organisms can be
quantified by FISH or membrane hybridization methods. Thus, applying the microarray to an
environmental sample is part of an integrated approach to explore community structure. The flexibility
provided by planar glass microarrays enables new probes to be added at will; in effect, any given
PhyloChip is in a constant state of evolution.
Probe redundancy is also readily achievable with high density Affymetrix arrays (Wilson et al.,
2002a,b), although the on-chip oligonucleotide manufacturing process allows less flexibility
(Hashsham et al., 2004). The strategy of Wilson et al. (2002a) involved amplification of an c.a 83-bp
region of the 16S rRNA gene, followed by design of at least 24 probes to target each sequence type. A
taxon was only considered present if 22 of the 24 probes were positive. One drawback is that resolution is
only possible at higher phylogenetic levels.
Functional gene arrays (FGAs) target genes responsible for a specific metabolic process, e.g. ammonia
monooxygenase (for ammonia oxidation), nitrate reductase (for denitrification), dissimilatory (bi)sulphite
reductase (for sulphate reduction) (Table 11.9; Schadt et al., 2005). As mentioned above, these so-called
‘functional guilds’ of organisms can comprise phylogenetically coherent groups, while others are spread
among divergent taxa. In either case, FGAs provide information which directly links function and identity.
One well-evaluated FGA targets the particulate methane monooxygenase (pmoA) gene of all known
methanotrophs (Bodrossy et al., 2003; Stralis-Pavese et al., 2004). It comprises 68 short probes (17–27
bases in length), and also includes probes for the evolutionarily related ammonia monooxygenase (amoA)
gene (Stralis-Pavese et al., 2004). Hierarchical probes are also included, highlighting that even some
functional genes can be targeted in a nested manner. However, a constraint on developing nested probes
with such protein-coding genes is the existence of ‘wobble’ positions, i.e. the third nucleotide in a given
codon is often degenerate or highly variable. The extent of this and the practicalities of designing probes
targeting broader groups of organisms, depends on the level of sequence conservation for any given gene
fragment.
Most functional gene arrays developed so far for environmental applications contain either long
oligonucleotide probes (440 bp) (Denef et al., 2003; He et al., 2007b; Rhee et al., 2004; Taroncher-
Oldenburg et al., 2003; Tiquia et al., 2004) or probes based on PCR products (up to ,1200 bp)
(Dennis et al., 2003; Wu et al., 2001). As mentioned already, increased sensitivity can be achieved by
increasing probe length, but the price paid is a reduction in specificity. However, achieved specificity may
still be adequate, depending on the biological question posed. For example, the reported thresholds for
differentiating among sequences with these longer probes ranges from 75–88% (Kane et al., 2000;
Rhee et al., 2004; Taroncher-Oldenburg et al., 2003; Tiquia et al., 2004; Wu et al., 2001). Since many
ammonia oxidizer species differ by more than 20% in their amoA gene sequences (Koops et al., 2003), it
may be feasible to discriminate among some using the relevant functional gene and stringent hybridization
conditions. Of course, this ‘‘species threshold’’ may differ with other functional genes (depending on
probe length and target site), and must be checked. In principle this highlights one advantage of the FGA
over PhyloChip arrays, as a long oligonucleotide probe targeting the conserved 16S rRNA gene would
hybridize with undesired organisms. Thus, FGAs are more flexible in their achievable sensitivity/
resolution. It has been argued that lower specificity may be advantageous if the objective is to target a
family of closely related genes (Lemarchand et al., 2004). FGAs so far have targeted mainly those genes
responsible for nitrogen cycling (Jenkins et al., 2004a; Taroncher-Oldenburg et al., 2003; Steward et al.,
2004) or biodegradation (Dennis et al., 2003; Rhee et al., 2004). However, the GeoChip, which contains
24,243 50mer probes targets 4150 functional groups involved in N, C, S and P cycling, and populations
involved in bioremediation of organic contaminants and metals (He et al., 2007b).
Community genome arrays (Wu et al., 2004) offer a further option. This approach is conceptually
similar to that of reverse sample genome probing (RSGP) (Greene and Voordouw, 2003; Voordouw et al.,
1991, 1993) and involves spotting genomic DNA of cultured organisms onto a microarray slide. Its
application can indicate whether the cultured organisms (or close relatives), are present. As with RSGP,
the drawback is a requirement for culturing strains, and so the composition of any community is expressed
in terms of its cultivable component. One extension overcoming the need for cultivation is the spotting of
bacterial artificial chromosome (BAC) clones (representing uncultured organisms) from metagenome
libraries onto the array.
MICRO- AND MACROARRAYS APPLIED TO ACTIVATED SLUDGE

Considering the complexities associated with microarray development and application, it is reasonable to
ask why they should be used at all for analyzing activated sludge communities when so many other
techniques are available (chapter 3). Fortunately, the answer is as simple as the question. No other
method can describe, in a high-throughput manner, community structure and function at such a high
resolution and specificity. They offer the possibility of screening large numbers of samples from different
plants, providing information which might then be correlated with indices of plant efficiency.
Alternatively, the same plant could be monitored through time, allowing detection of subtle changes in
the resident community which may signal problems ahead. Such early detection may be crucial in
preventing subsequent process failures.
To date, only a handful of studies have used microarrays with activated sludge samples. However,
several diagnostic arrays are suitable. These include arrays for organisms involved in nitrification
(Adamczyk et al., 2003; Taylor, Loy, Adamczyk and Wagner; unpublished data), denitrification
(Bulow et al., 2008; Loy et al., 2005; Taroncher-Oldenburg et al., 2003), nitrogen fixation (Moisander
et al., 2007; Steward et al., 2004) and phosphorus removal (Loy et al., 2005). A microarray has been
described targeting members of the Rhodocyclales, a physiologically diverse group of bacteria important
in sewage treatment (Blackall et al., 2002:, Hesselmann et al., 1999; Juretschko et al., 2002). The
uncultured Candidatus ‘Accumulibacter phosphatis’ is responsible for enhanced biological phosphorus
removal (EBPR) (chapter 10), while other representatives of this order may contribute to denitrification
(Juretschko et al., 2002). Loy et al. (2005) designed and successfully applied a 16S rRNA gene-targeted
microarray, the RHC-PhyloChip to identify all known members (cultured and uncultured) of the
Rhodocyclales. The array employed a nested probe set consisting of 79 probes, and was rigorously
evaluated with 29 reference organisms. With Rhodocyclales-targeted PCR primers, uncultured
Sterolibacterium-, Ferribacterium/Dechloromonas- and Zoogloea-related bacteria were detected in
industrial activated sludge (e.g. Figure 11.36, see colour image section (chapter 13)). The sensitivity
provided with specific primers allowed their detection despite comprising 51% of all bacteria in the
sludge (Loy et al., 2005). Importantly, the results obtained with the RHC-PhyloChip were confirmed by
subsequent 16S rRNA gene sequence analysis (Figure 11.36). The proven involvement of Candidatus
‘Accumulibacter phosphatis’ in EBPR suggests that this microarray will be valuable in the study of both
EBPR and denitrification processes.
Denitrification is a key step in treating nitrogen-rich wastewaters, and other published arrays may be of
interest here. Denitrifying organisms are spread across many bacterial phyla (and include Archaea).
Microarrays offer the possibility of detecting these organisms by targeting the nitrite reductase (nirS/nirK)
genes in a functional gene approach (chapter 9). Several microarray studies have included nirS probes of
varying lengths and specificities (Bulow et al., 2008; He et al., 2007b; Taroncher-Oldenburg et al., 2003;
Tiquia et al., 2004; Wu et al., 2001), but, to our knowledge, these are yet to be used with wastewater
samples.
Microarrays targeting nitrifying bacteria also exist, ranging from small prototype arrays used for
method optimization (Adamczyk et al., 2003; Guschin et al., 1997) to an encompassing array targeting all
known chemoautotrophic nitrifiers (Taylor, Loy, Adamczyk, Wagner; unpublished data). These arrays
should provide important and useful data. The Nitrifier-PhyloChip, comprises some 200 probes targeting
ammonia-oxidising, nitrite-oxidising and anaerobic ammonia-oxidising (Anammox) organisms at various
specificities. Collectively, these are spread across six bacterial classes, rendering simultaneous FISH
analysis impractical from the high number of probes required.
A radically different prototype microarray for ammonia-oxidising bacteria (AOB) represents one
of the few published applications with activated sludge. With it Adamczyk et al. (2003) showed
ammonia-oxidisers were present in industrial and municipal treatment plants. Their study employed prior
incubation of samples with 14C-labelled substrate in order to link bacterial community structure with
function, making it radically different to other microarray work. The ‘Isotope Array’ (Adamczyk et al.,
2003) is dealt with below.
Nitrogen fixation is responsible for nutrient removal in some pulp mill effluents (Gauthier et al.,
2000; Slade et al., 2003, 2004), and a DNA macroarray offers new insights into the diversity of the
functional gene dinitrogenase reductase (nifH) (Moisander et al., 2007). The array, comprising 706 nifH
targeted probes spotted onto nylon membranes, was applied to aquatic nitrogen fixers but could prove
equally useful in studying nitrogen-fixing sludges. Array technology also offers promise for the
detection of pathogens and indicator organisms in wastewater and drinking water (Lemarchand et al.,
2004; Straub and Chandler, 2003). Behr et al. (2000) constructed an rRNA gene-based array in a
microwell plate format to detect and identify enterococci, common indicators of faecal contamination.
Screening of different treatment plants resulted in positive signals from several subgroup- and two
species-specific probes. The enterococcal probe set for hybridization on microarrays has been evaluated
(Lehner et al., 2005).
Sulphate-reducing bacteria occur in activated sludge (Lens et al., 1995; Manz et al., 1998;
Schramm et al., 1999b) where they may influence floc stability (chapter 3) and compete with PAO in the
anaerobic basins of EBPR plants for electron donors (Nielsen, 1996; Nielsen and Keiding, 1998). A 16S
rRNA gene-based microarray which targets all known lineages of sulphate-reducing ‘prokaryotes’
(Loy et al., 2002b) should provide valuable insights into their diversity there. It might be argued that the
sulphate reducers are a perfect target group. Phylogenetically they are highly diverse and are thus
impossible to target employing a small number of primer pairs or probes. The high parallelism of
microarrays offers the ideal solution.
Another option is to construct a ‘habitat array’ to monitor all organisms in a specified environment
(activated sludge plants). This requires prior construction of a clone library to identify key members,
followed by design of oligonucleotide probes to target them, or alternatively, the direct use of PCR
products from the clone library as probes. Furthermore, large probe collections targeting organisms of
relevance in treatment plants are available (Loy et al., 2004). Or a ‘habitat’ array can be constructed to
monitor all populations in for example, a wastewater treatment plant. This requires the prior construction
of a clone library, followed by design of oligonucleotide probes to target the members, or alternatively
direct use of PCR products from the library as probes. Large collections of probes relevant to treatment
plants are already available (Loy et al., 2003, 2007).
LINKING FUNCTION WITH PHYLOGENY

Microarrays for monitoring community-wide gene expression
Microarrays offer the exciting prospect of being able to monitor gene expression of an entire community,
based on extraction of mRNA, conversion of this into cDNA by reverse transcription, then detecting
labelled cDNA on the microarray. As mentioned earlier, the challenge with this relates to mRNA
instability. Its susceptibility to enzymatic degradation means that extra care is required (section 11.6).
However, it is this instability that makes working with mRNA worthwhile: its short half-life coupled with
its role as an intermediate in gene expression ensures it is an excellent indicator of gene activity at a given
point in time.
Dennis et al. (2003) developed a microarray to measure expression of bacterial metabolic genes in
sludge-fed pulp mill effluent. Induction of genes responsible for resin acid degradation was detected
following addition of resin acid to pulp mill bioreactor cultures. Considerable optimisation is required to
enhance sensitivity and specificity of detection, but this study is a promising start. In theory, any
functional gene could be utilized. However, with a few exceptions (Garcia-Martin et al., 2006) little is
known about the metagenome of activated sludge communities. Therefore, for most ‘‘functions’’ no
encompassing gene sequence database exists, and thus no adequate microarray probes can be designed.
The ‘isotope array’

Unquestionably sequence analysis of rRNA genes has revolutionised our understanding of microbial
diversity (Pace, 1997; Hugenholtz et al., 1998a; DeLong and Pace, 2001). However, 16S rRNA sequence
identity usually provides little information about an organism’s physiology, and attempting to correlate
bacterial function with identity has become a major focus in contemporary microbial ecology (Gray and
Head, 2001; Wagner, 2004; Wagner et al., 2006). One method with great promise is the ‘isotope array’
developed by Adamczyk et al. (2003) (Figure 11.37, see colour image section (chapter 13)). These
authors were able to demonstrate incorporation of isotopically-labelled substrates into the rRNA of
targeted AOB. Two nitrifying activated sludge samples were incubated with 14C-bicarbonate for 412 h
(sufficient time for maximum 14C incorporation; Adamczyk et al., 2003), after which total RNA was
extracted from a subsample. Fluorescent labelling of this RNA was achieved followed by hybridization of
the RNA on the microarray. Fluorescence and radioactivity were then quantified for each microarray spot
using a microarray scanner and beta-imager, respectively (NB: variable mode imagers, such as the
Typhoon 9410 from Amersham Biosciences, are capable of measuring both signals simultaneously). Since
radioactivity is only detected in spots representing populations metabolizing the labelled substrate, this
method provides a direct link between their function and phylogeny. For sludge taken from a municipal
plant, members of the Nitrosomonas oligotropha lineage were dominant among the AOB community,
whereas the N. europaea/N. eutropha lineage dominated in an industrial plant (Adamczyk et al., 2003).
Correspondence between fluorescent and radioactive signals implied that at least some of the detected
AOB were metabolically active during the experiment. Importantly, results obtained using the isotope
array were consistent with those obtained with two independent methods, FISH-MAR and amoA clone
library analysis.
This isotope array poses several unique technical challenges. Sensitivity is of most concern, since
substrate utilization by any organism can only be detected if sufficient isotope is incorporated into its
RNA. Adamczyk et al. (2003) detected 14C incorporation into 16S rRNA of bacteria comprising about 5–
10% of the total community. Given the large amounts of RNA which should be present in activated
sludge, these values can probably be considered the best-case scenario with the current technology. Cross-
feeding of radioactively-labelled substrate, particularly when longer incubation times are used is another
consideration (see section 11.14), when trying to differentiate between primary substrate consumers and
populations metabolizing their waste products (Adamczyk et al., 2003). Yet cross-feeding could be
exploited to trace the flux of labelled carbon through the community. Work is underway to apply the
isotope concept to a full scale array (Hesselsoe, Loy and Wagner, unpublished), when it should prove very
useful in linking function and identity.
FUTURE PROSPECTS
Microarrays show great promise for the study of activated sludge populations. Although they are not yet
widely applied to such processes, the microarrays already available could reveal useful information about
their communities. The potential for high throughput monitoring, with a high phylogenetic resolution of
multiple taxa, sets microarrays apart from other methods. Indeed, the high diversity of organisms present
in activated sludge can only be effectively monitored with such techniques. Advances in our
understanding should resolve many of the current limitations associated with their use. In particular,
enhanced labelling and detection procedures should overcome the problem of sensitivity. Similarly, the
factors which influence hybridization are becoming clearer, and routine detection of single base
mismatches must surely be our ultimate goal. Quantification, too, is a crucial aim. In addition, an area
which should become a key focus is the further development of methods linking structure with function
(e.g. isotopic labelling methods, community-level gene expression).
It is likely that activated sludge will continue to be a fertile testing ground for the next generation of
microarrays. In many ways it is an ideal model system for testing such tools. High numbers of organisms
representing diverse taxa are typically nutrient-replete and thus contain high levels of ribosomal RNA,
facilitating their direct detection. Finally, data obtained developing such methods can be valuable for both
applied and basic research. Process failures in activated sludge systems can be expensive and time-
consuming to correct, and by close monitoring of their communities we may detect changes before
problems arise. Microarrays offer perhaps our best tool yet.
11.12 TERMINAL RESTRICTION FRAGMENT LENGTH POLYMORPHISM

(T-RFLP)
Wen-Tso Liu
INTRODUCTION
Terminal restriction fragment length polymorphism (T-RFLP) of PCR-amplified DNA is a sensitive and
reproducible method for providing semi-quantitative analysis of the diversity and dynamics in microbial
ecosystems (Hartmann and Widmer, 2008; Marsh and Nakatsu, 2007; Marzorati et al., 2008). The basic
protocol for this method as illustrated in Figure 11.38, see colour image section (chapter 13) includes.
(i) DNA extraction,

(ii) PCR amplification,
(iii) restriction enzyme digestion,
(iv) terminal fragment separation and detection,
(v) data analysis, and
(vi) phylogenetic identification of major terminal restriction fragments (T-RFs).
Notes
(i) In the DNA extraction step, total nucleic acids are extracted with a suitable protocol (Hurt et al.,
2001; Liu et al., 1997; Zhou et al., 1996). In general, all include cell lysis followed by phenol/
chloroform purification and ethanol precipitation to ensure high recovery of good quality and
representative DNA (section 11.6).
(ii) In PCR amplification, targeted markers are amplified with a primer set that contains a fluorescent
label(s) such as 6-carboxy-fluorescein (FAM) and HEX at the 50 end of the forward primer, the
reverse primer, or both. As Table 11.11 indicates, the most popular marker is the SSU ribosomal
RNA gene for the domains Bacteria (Liu et al., 1997), Eukarya (Marsh et al., 1998), and Archaea
(Lueders and Friedrich, 2000; Moeseneder et al., 2001; van der Maarel et al., 1998). Primer sets
targeting each of these are available at different levels of specificity (e.g. domain, class and
family), and should be systematically evaluated in silico before being validated in in situ studies.
For Bacteria, most use the primer sets, 8F/926R and 8F/1492R, because the T-RFs generated in
this targeted 16S rRNA gene region are more diverse (Liu et al., 1997).
(iii) Other functional genetic markers including the mercury resistance (mer), nitrogen fixation (nifH
and anf), ammonia monooxygenase (amoA), nosZ, PAB pufM, and methyl-coenzyme reductase
(mcrA) genes have been used. The primer sets for these are also listed in Table 11.11.
(iv) After PCR amplification, DNA products are purified, concentrated and completely digested with
one or more endonucleases (usually tetrameric restriction enzymes) separately. Digested products
are subjected to size separation with gel or capillary electrophoresis and automatic DNA
sequencers. Gel electrophoresis was the method used to generate T-RFLP profiles in early studies
using this technique, but capillary electrophoresis, because of its simplicity and accuracy in
fragment sizing, has become more popular, even though it requires a desalting step (Grüntzig
et al., 2002).
(v) T-RFLP resolution can be maximized further by determining the proper combination of primer
and restriction enzyme with the computer simulation programs, Patscan (http://www.mcs.anl.gov/
home/overbeek/PatScan/HTML/patscan.html) (Dsouza et al., 1997) or TAP T-RFLP (http://
www.cme.msu.edu/RDP/html/analyses.html) (Marsh et al., 2000). Restriction enzymes can be
assessed as to which one yields the highest number and most even distribution of terminal
restriction fragments. Liu et al. (1997) showed that 686 amplifiable sequences (8–927, E. coli
numbering) from 1102 complete SSU rRNA sequences obtained in the Ribosomal Database
Project (RDP) could be classified into 233 different terminal restriction fragments (i.e. ribotypes).
An hierarchical approach employing phylum- and group-level (or below) primers can further
increase the sensitivity of analysis. For example, the sulfate reducing bacteria (Wieland et al.,
2003), the nitrifying bacteria (Regan et al., 2002), the Acidobacterium (Kuske et al., 2002), the
Actinobacteria (Conn and Franco, 2004; Liu et al., 1998), and Planctomycetes (Derakshani et al.,
2001) have been all selectively analyzed in this manner (Table 11.11).
(vi) T-RFLP provides a sensitive, reproducible and robust methodology for the comparative analysis of
microbial diversity in environmental samples. Each electropherogram represents a fingerprint of the
community. Profiles with different size fragments reveal the level of diversity and the relative
abundances of each population. On average, 30–50 predominant terminal restricted fragments (i.e.
different OTUs or ribotypes) can be detected, with the least dominant accounting for less than 0.2%
of the total amplified community rDNA (Clement et al., 1998; Kerkhof et al., 2000; Liu et al., 1997).
(vii) Therefore, it is possible to compare samples according to the presence/absence of peaks, as well as
their relative intensity through statistical analysis (Abdo et al., 2006). Two most commonly used
approaches are cluster analysis (Liu et al., 1997) and principal component analysis (Clement et al.,
1998) and simple statistical software like SSCP or image analysis software (e.g. GelCompar II by
Applied Math Inc.), although others claim to be more discriminating (e.g. Abdo et al., 2006;
Culman et al., 2008; Nakaqno et al., 2008; Osborne et al., 2006).
(viii) Several software packages are available to infer the possible phylogenetic affiliation of predom-
inant T-RFs within a sample. These include TAP T-RFLP (http://35.8.164.52/html/TAP-trflp.html
#introduction), the T-RFLP Phylogenetic Assignment Tool (PAT) (http://trflp.limnology.
wisc.edu/index.jsp), the Microbial Community Analysis (MiCA) (http://mica.ibest.uidaho.edu/),
and tRFLP fragment sorter version 4.0 (http://www.oardc.ohio-state.edu/trflpfragsort/default.htm).
(ix) It is important to note that data from a T-RFLP characterization may not reflect fully the native
population structure. Biases associated with DNA extraction and PCR amplification steps have
been emphasized throughout this book. Osborn et al. (2000) demonstrated that some biases could
be minimized if key experimental parameters (e.g. sample replication and handling, PCR DNA
template concentration, PCR cycle time, Taq polymerase types, and the minimum restriction
enzyme digestion time) were systematically standardized. Highly degenerate primers can lead to a
biased representation of abundance of individual targets (Lueders and Friedrich, 2003). Likewise,
possible pseudo T-RFs may be produced from single-stranded amplicons in the T-RFLP, but these
can be eliminated by digestion with single-strand-specific mung bean nuclease prior to T-RFLP
analysis (Egert and Friedrich, 2003).
(x) In addition to DNA and PCR related biases, a single terminal fragment may actually include one
or more than one, different or closely related sequence arising from a high conservation of the
restriction site positions in rRNA sequences. Consequently T-RFLP profiles may underestimate
levels of microbial diversity by a factor of three to four, and population phylogeny resolved only
Table 11.11. Primers used in T-RFLP population analysis.
414
Forward primer and Sequence Reverse primer and Sequence

Specificityc (50 to 30 )ab (50 to 30 )ab c Labeld Reference
16S rRNA gene
Bacteria 8F 926R HEX, TET, FAM, Egert and Friedrich (2003);
(AGAGTTTGATCMTGGCTCAG) (CCGTCAATTCMTTTRAGTTT) NED, CY-5 Gong et al. (2002);
Leser et al. (2000);
Liu et al. (1997); Onstott et al.
(2003); Ovreas et al. (2003);
Schmitt-Wagner et al. (2003);
Sessitsch et al. (2002);
Urakawa et al. (2000);
Wieland et al. (2003)
Bacteria 8F 1392R HEX, FAM Avrahami et al. (2002);
(ACGGGCGGTGTGTACA) Blackwood et al. (2003);
Braker et al. (2001);
Dollhopf et al. (2002);
Marsh et al. (2000);
Petrie et al. (2003)
Bacteria 8F 1492R HEX, TET, FAM, Barkovskii and Fukui (2004);
(GGTTACCTTGTTACGACTT) JOE, NED Dunbar et al. (2000); Gong
et al. (2002); Kent et al.
(2003); Kibe et al. (2004);
Konstantinidis et al. (2003);
Kuske et al. (2002); Luna et al.
(2004);
Matz and Jurgens (2003);
Moeseneder et al. (1999);
Richardson et al. (2002);

Trotha et al. (2002);
Wilson (1997)
Bacteria 47F 927R CY-3 Chen et al. (2004)
(CYTAACACATGCAAGTCG) (ACCGCTTGTGCGGGCCC)
Bacteria 63F 1389R HEX, FAM Osborn et al. (2000)
(CAGGCCTAACACATGCAAGTC) (ACGGGCGGTGTGTACAAG)
Bacteria 516f 1492R HEX Nagashima et al. (2003)
(TGCCAGCAGCCGCGGTA)
(continued)

Archaea Ar21F Ar915R HEX, TET Takai et al. (2003);
(TTCCGGTTGATCCYGCCGGA) (GTGCTCCCCCGCCAATTCCT) Takai et al. (2001)
Archaea Ar21F Ar958R HEX, FAM, JOE Dollhopf et al. (2002); Lee et al.
(TTCCGGTTGATCCYGCCGGA) (YCCGGCGTTGAMTCCAATT) (2003); Moeseneder et al.
(2001); Ovreas et al. (2003);
Winter et al. (2004)
Archaea Ar109F Ar912R FAM Egert and Friedrich (2003);
(ACKGCTCAGTAACACGT) (CTCCCCCGCCAATTCCTTTA) Friedrich et al. (2001); Lue-
ders and Friedrich (2000)
Archaea Ar109F Ar934b FAM Chin et al. (1999a,b); Fey et al.
(GTGCTCCCCCGCCAATTCCT) (2001); Fey and Conrad
(2000); Kemnitz et al. (2004);
Lueders et al. (2001);
Nusslein et al. (2001)
Eukaryotes Primer A Primer B TET Marsh et al. (1998)
(CCGAATTCGTCGACAA- (CCCGGGATCCAAGCTT-
CCTGGTTGATCCTGCCAGT) GATCCTTCTGCAGGTTCACC-
TAC)
SRB 8F BSR385R HEX, TET
(CGGCGTCGCTGCGTCAGG)
ß-subclass AOB EUB338F Nso1225 FAM Regan et al. (2002)
(ACTCCTACGGGAGGCAGC) (CGCCATTGTATTACGTGTGA)
NOB NIT3 Ntspa0685 M FAM Regan et al. (2002)
(CCTGTGCTCCATGCTCCG) (CGGGAATTCCGCGCTC)
Actinobacteria 8F HGC678R Liu et al. (1998)
(GGTGTTCCTCCTGATATCTG)
Actinobacterium 243F 1492R TET Conn and Franco (2004)
(GGATGAGCCCGCCGCCTA)
Acidobacterium 31F 1492R FAM Dunbar et al. (2000);
Methods for the examination and characterization of the activated sludge community
(GATCCTGGCTCAGAATC) Kuske et al. (2002)
(continued )
415
416

Planctomycetes PLA-40F 1492R FAM Derakshani et al. (2001)
(CGGCRTGGATTAGGCATG)
Functional gene
amoA amoA-1F amoA-2R FAM Horz et al. (2001); Mintie et al.
(GGGGGTTTCTACTGGTGGT) (CCCCTCKGSAAAGCCTTCTTC) (2003); Regan et al. (2002)
amoA, pmoA A189 A682 FAM, IRD700 Horz et al. (2001); Pester et al.
(GGNGACTGGGACTTCTGG) (GAASGCNGAGAAGAASGC) (2004); Sakano et al. (2002)
nirS nirS1F nirS6R FAM Braker et al. (2001)
(CCTAYTGGCCGCCRCART) (CGTTGAACTTRCCGGT)
nirK nirK1F nirK5R FAM Avrahami et al. (2002)
(GGMATGGTKCCSTGGCA) (GCCTCGATCAGRTTRTGG)
nifH Zehr-nifHf Zehr-nifHr CY-5 Tan et al. (2003)
(TGYGAYCCNAARGCNGA) (ADNGCCATCATYTCNCC)
nosZ Nos661F Nos1773R Sakano et al. (2002)
(CGGCTGGGGGCTGACCAA) (ATRTCGATCARCTGBTCGTT)
PAB PufM PB557F PB750R HEX, TET Wieland et al. (2003)
(CGCACCTGGACTGGAC) (CCCATGGTCCAGCGCCAGAA)
mcrA MCRf MCRr FAM Lueders et al. (2001);
(TAYGAYCARATHTGGYT) (ACRTTCATNGCRTARTT) Lueders and Friedrich (2003)
a
Primers: 8F and 27F (positions 8–27 of Escherichia coli 16S rRNA), 907R and 926R (positions 907–926 of Escherichia coli 16S rRNA), 27F and 907R have
been changed to 8F and 926R, respectively (for standardization purpose).
b
Y ¼ (CT), W ¼ (AT), S ¼ (GC), K ¼ (GT), R ¼ (AG), M ¼ (AC), H ¼ (ACT), D ¼ (AGT), B ¼ (GCT), N ¼ (AGCT).
c
SRB: sulfate-reducing bacteria; AOB: ammonia-oxidizing bacteria; NOB: nitrite-oxidizing bacteria; PAB: purple anoxygenic bacteria.
d
HEX: 5-hexachlorofluorescein; TET: 5-tetrachlorofluorescein; FAM: 5-carboxyfluorescein; JOE: 6-carboxy-40 ,50 -dichloro-20 ,70 -dimethoxyfluorescein.
at the level of higher-order groups. Without prior knowledge of community composition, the
phylogenetic position based on the terminal restriction fragment lengths may not be reflected
correctly (Liu et al., 1997; Marsh et al., 2000). To further minimize the effects of such biases, it is
recommended to apply more than one molecular technique to resolve community structure and
follow changes within it (Chen et al., 2004; Richardson et al., 2002).
In summary, T-RFLP has been applied extensively to resolve community composition and monitor
how it changes temporally and spatially in samples for example from activated sludge processes
(Chen et al., 2004; Liu et al., 1997; Regan et al., 2002), soil (Chin et al., 1999a; Dunbar et al., 2000;
Kemnitz et al., 2004), marine environments (Ovreas et al., 2003), termite and human gut environments
(Kibe et al., 2004; Leser et al., 2000).
PROTOCOL
Reagents and equipment required
PCR amplification
Box with ice
Pipettes and tips
Sterile 1.5 ml tubes (for preparation of the master mix)
Sterile 0.2 ml PCR tubes
PCR machine
dNTP solution (2 mM)
Primers (10 mM)
Taq polymerase (5 U/ml)
PCR buffer (10£)
Sterile milliQ water
Concentration and purification of PCR products

Pipettes and tips
Sterile 1.5 ml tubes
1£ TAE buffer
Agarose
Microwave oven
Ethidium bromide solution (10 mg/ml)
Gel electrophoresis system
Loading buffer for agarose gel
Parafilm
QIAquick PCR purification kit (Qiagen, cat. 28106)
UV-table
Centrifuge
Restriction digestion of the PCR products

Sterile 0.2 ml PCR tubes
Restriction enzymes
Restriction enzyme buffers (10£)
Incubator
Detection of labeled fragments

PCR machine or incubator (suitable for 95– C denaturation)
Gel-based or capillary electrophoresis-based DNA auto-sequencer
EXPERIMENTAL PROCEDURES
DNA extraction – see section 11.6
PCR amplification
(i) Add the following reagents to a sterile 0.1 ml tube:
Forward primer (10 mM) 2 ml
Reverse primer (10 mM) 2 ml
PCR Buffer from kit (10£) 10 ml
MgCl2 (50 mM) 5 ml
dNTP (2 mM) 10 ml
H2O 70.5 ml
Taq polymerase (5 U/ml) 0.5 ml
DNA template 1 ml
(ii) Use the following PCR thermal program:
Hot start 94– C 1 min
30 cycles of the following
Denaturation 94– C 30 sec
Annealing varying 30 sec
Extension 72– C 30 sec
Final extension 72– C 5 min
. Note 1: Duplicate or triplicate PCR reactions should be made for each sample. The
combination of multiple PCRs can reduce the bias of individual amplification reactions.
. Note 2: Annealing temperature and the time applied in each step vary with different primer sets.
(iii) Analyze the amplified PCR product on a 1% agarose gel containing 10 ug/ml of ethidium bromide
solution in 1£ TAE buffer (Sambrook et al., 2001).
(iv) Purify the PCR product of the correct size using a QIAquick PCR Purification Kit according to the
manufacturers’ instructions (Qiagen).
Digestion of the purified PCR product with one to three different tetrameric
endonucleases
These tetrameric endonucleases include for example MspI (CCGG), RsaI (GTAC), HhaI/CfoI (GCGC),
HaeIII (GGCC), AluI (AGCT), MvnI/FnuDII (CGCG), TaqI (TCGA).
(i) Add the following reagents to a sterile 0.1 ml tube

Restriction endonuclease (10–20U) 1 ml
Buffer (10£) 1 ml
Purified PCR sample 8 ml
Total volume 10 ml
(ii) Incubate the tube in the PCR machine or water bath at the suggested optimal temperature for the
restriction enzyme for at least 3 h.
(iii) Inactivate the restriction enzymes by heating at 65–80– C for 5–20 min.
(iv) (optional step) The restricted DNA is precipitated with 0.1 volume of sodium acetate (3 M, pH 5.2)
and 2 volumes of 96% chilled ethanol. After centrifugation at 13,000 rpm, carefully wash the
DNA precipitate twice with 70% ice-cold ethanol, dry the pellet, and dissolve the DNA in 5 ml of
sterile milliQ water.
Analyzsis of the T-RFLP pattern of the digested PCR product using a DNA
auto-sequencer (Figure 11.39)
Add the following reagents into a 0.1 ml tube.
Loading buffer 0.5 ml
Deionized formamide 2 ml
Digested PCR products 2–2.5 ml
Standard size marker 0.5 ml
(i) Denature the DNA mixture at 94– C for 2–5 min, and transfer the tube immediately on ice.
(ii) Analyze the T-RFLP patterns of the denatured and digested PCR products with a gel-based or a
capillary-based DNA auto-sequencer. Table 11.12 shows the operational conditions and para-
meters used for different DNA auto-sequencers.
(iii) After electrophoresis, determine the lengths of fluorescently labeled T-RFs with internal size
standard using the fragment sizing software provided for individual DNA auto-sequencers.
30000
136.34
25000
20000
400.30
188.83 328.53
15000
103.31
10000
187.72 326.92
323.99
106.26
105.15 628.55
5000 192.95
102.11
184.82
100.63 183.67
0
0 50 100 150 200 250 300 350 400 450 500 550 600 650 700
Size (nt)
Figure 11.39. A typical electropherogram of T-RFLP pattern from CEQ 8000 genetic analysis system
(Beckman coulter). The sizes of individual T-RFs are determined and labeled as shown.
Note 1: The standard size marker depending on the DNA autosequencer used can be GeneScan-500, 1000,
2500-labeled with ROX or TAMARA (PE Applied Biosystems), or a mixture of Low Range Standard and
100 bp Molecular Ruler (both Texas red labeled; Bio-Rad), or CEQ DNA size standard kit-600 (Beckman
Coulter).
Note 2: It is recommended to desalt the digested PCR product (Grüntzig et al., 2002). In capillary
electrophoresis, the injection of DNA samples is usually achieved by two methods: hydrodynamic
Table 11.12. Conditions used for analyzing T-RFLP patterns on (A) a gel-based or (B) a capillary-based DNA
auto-sequencer.
(A)
System Gel Electrophoresis Software
ABI Prism 373, 12, 24, 36 cm 4–6% 2,500 V, 40 mA, 27 W for GeneScan
377 series denaturing polyacrylamide 6–20 hours
automated gel (7–8.3 M urea)
sequencer(ABI)
Model 4200 5.5–6% 1,200 V, 25 mA, 30 W, Gelscan Professional
(LI-COR) at 50– C for 10 hours
(B)
System Capillary Injection Electrophoresis Software
ABI Prism 310 POP-4 polymer, 15 kV for 15 kV at 60– C GeneScan
capillary 47 cm by 50 mm 5–20 sec for 30–45 min
sequencer (ABI)
ABI Prism 3100 Performance-optimized 30 V/cm for 244 V/cm for GeneScan
capillary polymer type 6; 20 sec 20 sec
sequencer (ABI) or POP-4 polymer
Biofocus 3000 Polyacryloylaminoet 10 kV for 3 kV for 75 min
capillary electrophoresis hoxyethanol-coated, 20 sec
apparatus (Bio-Rad) 44 cm by 75 mm
CEQ 8000 genetic CEQ separation 2.1 kV for 4.8 kV at 55– C CEQ 8000-fragment
analysis system capillary array 30 sec for 1–2 hr analysis program
(Beckman coulter) 33–75B, and CEQ
separation gel-LPA I
injection by different pressure and electrokinetic injection, with a combination of electrophoresis and
electroendosmosis to inject the sample. The ABI Prism 310 and 3100 genetic analyzers (PE, ABI) and
CEQ 8000 analysis system (Beckman coulter) use electrical injection, where the presence of ions
(from salt) can interfere with the uptake of DNA. An insufficient fluorescent signal will produce an
electropherogram with small number of peaks all of low intensity. Therefore, it is essential to desalt the
inactivated restriction digest with Microcon columns (Amicon), Qiaquick Nucleotide Removal Kit
(Qiagen) or ethanol precipitation. Furthermore, injection times and voltages can be adjusted to increase
uptake of DNA from samples to generate a higher fluorescent signal and more peaks.
Data analysis
Several software packages are available commercially to perform cluster analysis and principle
component analysis. A few examples are provided below.
a. GelCompar II by Applied Math Inc. (http://www.appliedmaths.com/gelcompar/gelcompar.htm)

b. SPSS (http://www.spss.com)
c. SAS (http://www.sas.com)
PHYLOGENETIC IDENTIFICATION OF MAJOR T-RF

In silico analysis for phylogenetic identification of major T-RFs can be performed with the following programs.
(i) TAP T-RFLP (http://35.8.164.52/html/TAP-trflp.html#introduction),

(ii) T-RFLP Phylogenetic Assignment Tool (PAT) (http://trflp.limnology.wisc.edu/index.jsp),
(iii) the Microbial Community Analysis (MiCA) (http://mica.ibest.uidaho.edu/), and
(iv) tRFLP fragment sorter version 4.0 (http://www.oardc.ohio-state.edu/trflpfragsort/default.htm).
Acknowledgement
Many thanks for assistance from Chialung Chen.
11.13 PCR-DGGE
Gerard Muyzer and Esengül Yilirim
INTRODUCTION
Cloning (section 11.7) is laborious, time-consuming, and expensive, especially when a large number of
samples are analysed. An alternative is to use genetic fingerprinting techniques, such as denaturing
gradient gel electrophoresis (DGGE; Muyzer, 1999) or terminal restriction fragment length polymorphism
(T-RFLP; section 11.12) Marsh, 1999: Marsh and Nakatsu, 2007; Marzorati et al., 2008).
Here we describe the procedure for DGGE analysis of 16S rRNA gene fragments obtained after PCR
amplification of genomic DNA or cDNA from bacteria.
(i) Genomic DNA is isolated from samples containing mixed microbial populations (section 11.6).
A fragment of the SSU rRNA encoding gene is amplified in the polymerase chain reaction (PCR)
using domain specific primers (Tables 11.13 and 11.14).
(ii) The mixture of fragments obtained from all the different bacteria in the sample is then analysed
by DGGE, which separates DNA fragments by difference in their melting behaviour. DNA
molecules of equal size but different nucleotide sequences have different melting behaviour
and can therefore be separated on a polyacrylamide gel containing a linear gradient of the
DNA denaturants urea and formamide.
(iii) The electrophoresis pattern gives an indication of the complexity of the sample’s microbial community
(see Figure 11.40 for an example). The number of bands is a rough estimation of the number of
different predominant bacterial populations in the sample, while the intensity of individual bands
can be a measure for the proportional abundance of the particular population (Nübel et al., 1999).
(iv) PCR-DGGE allows analysis of many samples simultaneously, which facilitates monitoring changes
in the composition of communities over time after natural or induced perturbations. The approach
was first used by Muyzer et al. (1993), and it is a now a well-established and widely used method
in microbial ecology.
(v) DGGE has been used to study the complexity of microbial populations of Bacteria (e.g. Schäfer
et al., 2001), Archaea (e.g. Coolen et al., 2004; Vetriani et al., 1999), and Eukarya (Diéz et al.,
2001), and to study virus diversity (Scanlan and Wilson, 1999; Short and Suttle, 1999).
(vi) PCR-DGGE analysis is a robust method; it is reproducible and rapid, and allows identification of
individual populations by sequencing of excised bands or hybridisation analysis with taxon
specific probes. It is not restricted to the analysis of SSU ribosomal RNA genes, and has been used
to analyse PCR products from other genes, including the [NiFe]-hydrogenase gene (Wawer and
Muyzer, 1995), which makes it suitable to determine differential gene expression in environ-
mental samples, e.g. by genetically distinct but closely related populations (Wawer et al., 1997).
(vii) Apart from suffering from the biases and limitations inherent in PCR protocols (see von
Wintzingerode et al. (1997)) DGGE analysis is limited to DNA fragments up to ca. 500 bp.
Furthermore, a bacterial species might harbour more than one rRNA operon with sequence
variation, which may generate more than one band from it (Nübel et al., 1996). DNA fragments
with different sequences may also fail to separate on the denaturant gradient gel if they retain the
same melting behaviour, resulting in mixed sequences upon their reamplification and sequencing.
(viii) However, despite this, PCR-DGGE analysis of naturally occurring communities is a valuable and
popular technique (see Muyzer and Smalla (1998); Schäfer and Muyzer (2001) for reviews), and
has been used successfully to study those in activated sludge (e.g. Ahn et al., 2007; Gray et al.,
2002; Limpiyakorn et al., 2004; Tanaka et al., 2003).
Table 11.13. Primers used for DGGE analysis of SSU rRNA fragments of Bacteria, Archaea, and Eukarya.
Primera,b Target sitec Sequence (50 -30 ) Specificity Reference
341F-GCe 341–357c CCT ACG GGA GGC AGC AG Bacteria Muyzer et al. (1993)
518R 518–534c ATT ACC GCG GCT GCT GG Universal Muyzer et al. (1993)
907R 907–926c CCG TCA ATT CMT TTG AGT TT Bacteria Muyzer et al. (1998)
Parch519F 519–533c CAG CCG CCG CGG TAA Archaea Coolen et al. (2004)
ARC915R-GCe 915–934c GTG CTC CCC CGC CAA TTC CT Archaea Stahl and Amann (1991)
Euk1AF 4–20d CTG GTT GAT CCT GCC AG Eukarya Diéz et al. (2001)
Euk516R-GCd 563–548d ACC AGA CTT GCC TCC Eukarya Diéz et al. (2001)
a
F (forward) and R (reverse) indicate the orientation of the primers in relation to the rRNA sequence.
b
The following primers have been used together: 341/518, 341/907, Parch519F/ARC915R-GC, Euk1AF/Euk516R-GC.
c
E. coli numbering according to Brosius et al. (1981).
d
Saccharomyces cerevisiae positions.
e
GC denotes that a GC-rich sequence (50 -CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC G-30 ) is
attached to the 50 -end of the primer.
Table 11.14. PCR conditions for SSU rRNA gene amplification.

Primer
combination Specificity PCR program Reference
341F-GC/518 Bacteria 5 min at 94– C, followed by 20 cycles of 1 min at Muyzer et al. (1993, 1998)
341F-GC/907R 94– C, 1 min at 65 . . . 55– C (touchdown -0.5– C
per cycle), 3 min at 72– C, followed by 15 cycles
of 1 min at 94– C, 1 min at 55– C, and 3 min at
72– C, followed by 7 min final extension at 72– C
Parch519F/ Archaea 4 min at 94– C, followed by 35 cycles of 30 sec Coolen et al. (2004)
ARC915R-GC at 94– C, 40 sec at 57– C, 40 sec at 72– C,
followed by 10 min final extension at 72– C
Euk1AF/ Eukarya 130 sec at 94– C, followed by 35 cycles of Diéz et al. (2001)
Euk516R-GC 30 sec at 94– C, 45 sec at 56– C, 130 sec at 72– C
0.10 Uncultured bacterium (AY212641)

EY46
Thauera aromatica (AJ315681)
A B C D Thauera terpenica (AJ005817)
Uncultured sludge bacterium (AF234693)
EY41
Uncultured sludge bacterium (AF234684)
Zoogloea ramigera (X74913)
Uncultured bacterium (AF204250)
EY8
β Uncultured bacterium (AF280861)
Nitrosomonas europaea (AF037106)
Uncultured bacterium (AB166775)
EY44
γ Xanthomonas sp (AF139997)
Frateuria aurantia (AJ010481)
Xanthomonas campestris (X99299)
Uncultured bacterium (AJ311962)
EY39
Sphingomonas natatoriaa (AB02488)
Sphingomonas Trueperi (X97776)
α Mesorhizobium loti (U50166)
Brevundimonas diminuta
Flavobacterium sp. (AM177620)
EY24
EY36
Flavobacterium omnivorum (AF433174)
Uncultured bacterium (AY212665)
EY37
Flavobacterium columnare (AB015481)
Empedobacter brevis (M59052)
B Cytophaga baltica (AJ005972)
EY35
C Chlorobium limicola (M31769)
Chlorobium phaeobacteroides (Y10651)
Candidatus nitrospira defluvii (DQ059545)
EY33
EY9
Nitrospira sp. (Y14639)
Nitrospira marina (X82559)
Leptospirillum ferrooxidans (AJ 295685)
N Magnetobacterium bavaricum (X71838)
Thermodesuifovibrio islandicus (X96726)
Figure 11.40. Community analysis of activated sludge and aerobic granular sludge. DGGE of 16S rRNA
gene fragments derived from bacterial genomic DNA of samples taken from an activated sludge reactor and
different aerobic granular sludge reactors for municipal sewage treatment, i.e. full-scale activated sludge
(EBPR) reactor (lane A), pilot-scale aerobic granular sludge reactor fed with raw wastewater (lane B), pilot-
scale aerobic granular sludge reactor fed with presettled wastewater (lane C), and a lab-scale aerobic
granular sludge reactor fed with synthetic wastewater (lane D). The sequences obtained from the excised
fragments were used to infer the phylogenetic affiliation of the community members. Names in bold were
obtained from the DGGE profiles. The letters on the branches indicate the group name: a, Alphaproteo-
bacteria; b, Betaproteobacteria; g, Gammaproteobacteria; B, Bacteroidetes; C, Chlorobi; N, Nitrospirae. The
bar indicates 10% sequence variation.
PROTOCOL
Extraction of nucleic acids – see section 11.6
Purification of RNA
(i) Add the following reagents to a microcentrifuge tube:
. _ 2 ml water
. _ 2 ml 10£ DNase buffer
. _ 15 ml nucleic acid extract
. _ 1ml DNase enzyme
(ii) Incubate for 30 min at 37– C in a water bath or thermocycler. Add 280 ml of water and 30 ml of
sodium acetate, and vortex briefly.
(iii) Add 300 ml of PCI and invert the tube end-over-end. Centrifuge briefly to separate the aqueous
and organic phase. Transfer the aqueous phase to a clean tube.
(iv) Add 2.5 vol 100% (v/v) ethanol and incubate for 2 h at –20– C. Centrifuge for 1 h at 4– C to pellet
the RNA. Remove the supernatant by gentle aspiration and rinse the pellet with ice-cold 70% (v/v)
ethanol.
(v) Dry the pellet under vacuum at room temperature. Re-dissolve it in 15 ml water and use directly
for cDNA preparation, or store at –80– C for later use.
Reagents
. _ DNase I, RNase free: 10 units per ml (Amersham Pharmacia Biotech, Uppsala, Sweden, cat. no.
27-0514-01)
. _ DNase buffer (10£ concentrated): 400 mM Tris, 60 mM MgCl2, pH 7.5
. _ Sodium acetate (3 M, pH 5.2)
. _ Phenol:chloroform:isoamylalcohol (PCI, 25:24:1, Sigma, Missouri, USA, cat. no. P3803)
. _ Water: molecular biology grade water (Sigma, Missouri, USA, cat. no. W4502)
Preparation of cDNA
(i) Add 1 ml of hexanucleotides (ca. 40 ng/ml) to 10 ml of RNA solution in a microcentrifuge tube.
(ii) Incubate for 10 min at 70– C in a water bath or thermocycler to denature the RNA. Cool on ice.
(iii) Centrifuge briefly to collect the liquid in the bottom of the tube, and add 4 ml of 5£ RT buffer, and
4 ml of dNTP solution. Incubate for 2 min at 37– C.
(iv) Add 1 ml (200 units) of MMLV reverse transcriptase, and incubate for 1 h at 37– C. Incubate at
95– C for 5 min to stop the reaction. Cool on ice.
(v) Use 1–5 ml of the solution as template for PCR, or dilute if necessary. Store the remainder of the
prepared cDNA at –80– C.
Reagents
. _ dNTP solution: 2.5 mM of each dNTP (Amersham Pharmacia Biotech, Uppsala, Sweden, cat.
nr. 27-2035-01) in water (Sigma, Missouri, USA, cat. no. W4502)
. _ Reverse transcriptase (RT) buffer (5£ concentrated): 250 mM Tris-HCl, pH 8.3, 375 mM KCl,
15 mM MgCl2, 50 mM dithiothreitol (Promega, Madison, USA, cat. no. M1701)
. _ MMLV-reverse transcriptase: 200 units/ml (Promega, Madison, USA, cat. no. M1701).
. _ Hexanucleotides: Dilute the hexanucleotides (BoehringerMannheim GmbH, Mannheim,
Germany, cat. no. 1277081) 1:50 [v/v] in molecular biology grade water (Sigma, Missouri,
USA, cat. no. W4502)
Polymerase chain reaction (PCR)

(i) Prepare a master mix for 100 ml PCR reactions by adding the following reagents per reaction:
. _ 10 ml 10£ PCR buffer
. _ 10 ml dNTPs
. _ 1 ml forward primer
. _ 1 ml water
. _ 0.2 ml Taq polymerase
(ii) Vortex and spin briefly to collect reagents in the bottom of the tube. Dispense 99 ml of the master
mix to each of the PCR reaction tubes. Add 1 ml of template DNA (or cDNA) solution to each
tube. Add two drops of mineral oil (e.g. Sigma, Missouri, USA, cat. nr. M8862) if the
thermocycler is not equipped with a heated lid.
(iii) Insert the tubes in the thermocycler, and start the appropriate PCR program (see Table 11.14).
Inspect 5 ml of the PCR product by electrophoresis on 1.5% (w/v) agarose gels together with the
Precision Molecular Mass Standard.
Reagents
. _ PCR buffer: 100 mM Tris-HCl, pH 9, 15 mM MgCl2, 500 mM KCl (Amersham Pharmacia
Biotech, Uppsala, Sweden)
. _ Taq DNA polymerase: 5 units/ml (Amersham Pharmacia Biotech, Uppsala, Sweden, cat. no. 27-
0799-02)
. _ Primers: 50 mM (See Table 11.13)
. _ Water: molecular biology grade water (Sigma, Missouri, USA, cat. no. W4502)
. _ Precision Molecular Mass Standard: Bio-Rad, California, USA, cat. no. 170-8207
DENATURING GRADIENT GEL ELECTROPHORESIS

Casting of denaturing gradient gels
(i) Clean the glass plates, spacers and combs with water and soap. Rinse them thoroughly with
de-mineralised water. Wipe with 70% (v/v) ethanol and a dust-free cloth.
(ii) Assemble the glass plates with 1 mm spacers and 1 mm comb according to instructions. Set up the
gradient former.
(iii) Connect the tubing of the peristaltic pump to the gradient former. Attach a needle to the end of the
tubing and place the needle in the middle of the gel sandwich.
(iv) Add 8 ml TEMED and 60 ml APS to the 6% acrylamide/ 0% UF and to the 6% acrylamide/ 80%
UF solutions.
(v) Pour the solutions into the gradient former, switch on the magnetic stirrer, start the pump and
slowly open the valve between the two reservoirs. Cast a gel of 6% acrylamide without urea and
formamide on top of the gradient gel to obtain good slots. Allow the gel to polymerise for at
least 2 h.
Running of denaturing gradient gels

(i) Remove the combs and rinse the slots with 1£ TAE buffer. Put the gel sandwich into the
electrophoresis tank, and fill with 1£ TAE buffer. Set the temperature to 60– C, and switch on the
heater and pump.
(ii) Prepare the samples; add 1/10 of volume of gel loading solution to each sample. Load samples
onto the gel with a Hamilton syringe. Rinse the syringe thoroughly with 1£ TAE between each
sample.
(iii) Connect the electrophoresis unit to the power supply, and run the gel at a constant voltage of 100 V
for 18 h for primer pair 341F-GC/907RM or the optimised running conditions for the particular
fragment.
(iv) After electrophoresis, stain gels for 30 min in an ethidium bromide solution, destain for 30 min in
Milli-Q water, and photograph with UV illumination and digital camera. Take several photos with
different exposure times for the detection of faint bands, and to resolve details in intensely stained
band (see Note 2).
Reagents
. _ Acrylamide/bisacrylamide: Ready-to-use stock solution of 37.5:1, 40% acrylamide/bisacryla-
mide (Bio-Rad, California, USA, cat. no. 161-0149)
. _ Formamide (de-ionised): Add 10 g of mixed bed resin (Sigma, Missouri, USA, cat. no. M8032)
to 100 ml of formamide and stir for 60 min. Filter formamide over a paper filter and store in 32 ml
volumes at –20– C
. _ Urea: Bio-Rad, California, USA, cat. no. 161-0731
. _ Tris Acetate EDTA (TAE) buffer (50£ concentrated): 2 M Tris, 1 M acetic acid, 50 mM EDTA,
pH 8.3)
. _ 6% acrylamide, 0% urea/formamide (UF) solution: 15 ml acrylamide/bisacrylamide stock
solution, 2 ml 50£ TAE, adjust to 100 ml with Milli-Q water
. _ 6% acrylamide, 80% urea/formamide (UF) solution: 15 ml acrylamide/bisacrylamide stock
solution, 2 ml 50£ TAE, 33.6 g urea, 32 ml formamide, adjust to 100 ml with Milli-Q water
. _ Ammonium persulphate solution (APS, 10% [w/v]): Dissolve 1 g of ammonium persulphate in
10 ml water. Aliquot in single use portions and store at –20– C
. _ TEMED: Ready-to-use solution from Bio-Rad, California, USA, cat. no. 161-0801
. _ Gel loading solution (10£ concentrated): 5 ml glycerol, 25 mg bromophenolblue, 25 mg
xylenecyanole, and 5 ml water
ANALYSIS OF DGGE PATTERNS

DGGE patterns from mixed microbial communities may be complex. Different kinds of information can
be extracted from them i.e. the number, position, and relative intensity of the bands (Marzorati et al.,
2008). Furthermore, the nucleotide sequence of individual bands can be determined to identify the
microbial population (see below).
Unweighted Pair-wise Grouping with Mathemical Averages (UPGMA) or MultiDimensional Scaling
(MDS) are powerful statistical methods to facilitate interpretation of large sets of complex DGGE
patterns. The first step is the creation of a binary matrix representative of the bands in a set of DGGE
patterns.
Presence or absence of DGGE bands in one sample is scored, as present (1) or absent (0), relative to
bands detectable in all samples of a set of DGGE patterns. The binary matrix is translated into a distance
matrix with a similarity coefficient, like the Jaccard coefficient. This is then used for UPGMA or MDS.
Software programs, such as Phoretix (Shimadzu Biotech, Japan) and Bionumerics (Applied Maths,
Belgium) are available to perform these analyses. For more information, see Schäfer and Muyzer (2001)
and Fromin et al. (2002).
DGGE-derived values of genetic richness and abundance (defined as relative fluorescence of bands)
can be used to calculate diversity indices (e.g. Gafan et al., 2005; Nübel et al., 1999). Nübel et al. (1999)
compared the diversity of oxygenic phototrophic organisms in microbial mat samples from different sites.
Using targeted PCR (Nübel et al., 1997), they amplified their 16S rRNA gene fragments and separated
them by DGGE. Samples were compared according to the number of bands (i.e. the genetic richness) and
their relative staining intensity (i.e, evenness, although fluorescence intensity does not necessarily relate
to relative fragment abundance in original sample because of PCR biases- section 11.7). With these values
they could calculate a Shannow-Weaver index.
DGGE-defined diversity indices showed high congruency with those calculated by two other culture-
independent approaches. Gafan et al. (2005) determined diversity indices of microbial biota in plaque
from children with and without gingivitis, and found a reduced diversity in those with.
EXCISION AND RE-AMPLIFICATION OF BANDS

(i) Transfer the gel to a UV-table. Photograph the gel, and mark the bands to be excised. Cut these
out with a clean scalpel blade (see note 3). Transfer each to a labelled tube containing 200 ml
water.
(ii) Incubate for 2 h at room temperature. Remove the water by gentle aspiration (use a clean tip for
each band). Add 25–50 ml water and incubate overnight at 4– C. Use the water as template
for re-amplification with the same primers as used for the original PCR, and store the remainder
at 20– C.
(iii) Check this PCR product alongside the original DGGE pattern to make sure that it corresponds
to the desired fragment, and is a single band. Purify the DNA fragment using the Qiaquick
Purification Kit (Qiagen, Germany), determine its concentration, and send it out for sequencing.
Notes
1. For all solutions molecular biology grade water (Sigma, Missouri, USA, cat. no. W4502) must be
used, unless other water, such as Milli-Q water, is explicitly indicated.
2. Denaturing gradient gels can also be stained with SYBR Green (Molecular Probes Inc., Eugene,
USA, cat. no. S-7567), GelStar (BioWhittacker Molecular Applications, Rockland, USA, cat. no.
50535), or silver (e.g. Bio-Rad Laboratories, USA, cat. no. 161- 0449). Furthermore, gels can be
blotted onto nylon membranes for hybridisation analysis with radioactive or digoxigenine-labelled
probes (see Muyzer et al., 1998).
3. UV-light will damage the DNA that is to be re-amplified. Therefore excision should proceed as
quickly as possible, and UV exposure has to be kept as short as possible.
11.14 STABLE ISOTOPE PROBING

Maneesha Ginige
Stable isotopes are those that do not undergo radioactive decay. Hence, their nuclei are stable and their
masses remain constant. Some of these, the building blocks of most organic compounds, are detailed in
Table 11.15.
Table 11.15. Selected isotopes and their natural abundance

(Radajewski et al., 2003).
Stable isotope Natural abundance (%)
1
H 99.99
2
H 0.01
12
C 98.93
13
C 1.07
14
N 99.63
15
N 0.37
16
O 99.76
17
O 0.04
18
O 0.20
31
P 100
13
C, 15N, 2H are in low abundance in living organisms, and useful as tools in culture independent
methods to identify populations involved in ecological processes. Linking phylogeny with community
function is a two step process.
Firstly, function is established with these isotopes. Some introduce isotopes externally (stable isotope
probing (SIP)) while others exploit naturally abundant isotopes. The methods adopted in SIP are DNA-,
RNA- and PLFA-SIP, while isotope ratio mass spectrometry (IRMS) and secondary ion mass
spectrometry (SIMS) establish function through natural abundance of stable isotopes.
Secondly, the functionally linked communities are identified using rRNA analysis, PLFA analysis,
fluorescence in situ hybridisation (FISH) and rRNA capture approaches. Figure 11.41 summarizes these
methods.
Mixed microbial
community
Externally introduce
Establish natural
and track migration
abundance of stable
of naturally rare stable
isotopes (e.g. δ13C)
isotopes into biomarkers
using IRMS and SIMS
(DNA-SIP / RNA-SIP
for signatures
/ PLFA-SIP)
Microorganisms linked
to ecological process
rDNA analysis / FISH / PLFAs /

PLFA analysis rRNA capture
approaches
Microorganisms linked
with thier taxonomic
identity
Figure 11.41. Schematic diagram illustrating how stable isotopes can be used to link ecological processes
and microbial population identity.
STABLE ISOTOPE PROBING (SIP)

SIP uses stable isotopes to track migration of selected substrates into cellular components
(biomarkers), providing phylogenetic information. Three biomarkers, namely DNA, RNA and polar
lipid derived fatty acids (PLFAs) are used (Boschker et al., 1998; Manefield et al., 2002b; Radajewski
et al., 2000). Very few studies with SIP so far have been directed towards understanding functionality
in activated sludge, and so examples discussed here come mainly from work with communities from
other habitats.
DNA BASED STABLE ISOTOPE PROBING (DNA-SIP) – THE PRINCIPLES

DNA-SIP was first introduced by Radajewski et al. (2000). However, its rationale was justified by
Meselson et al. (1958), who endeavored to show how the DNA structure proposed by Watson and Crick
engaged in a replication process. By tracking migration of 15N labelled ammonia into DNA by density
gradient centrifugation, they showed that one subunit of daughter DNA is derived from the parental DNA
while the other is newly synthesized, incorporating 15N labelled nitrogen. Radajewski et al. (2000) used
DNA-SIP to target active methylotrophic populations in soil by exposing cells to 13CH3OH. These would
then incorporate 13C atoms into DNA while growing on 13CH3OH. Both studies revealed that tracking
migration of an isotopically labelled substrate into DNA was feasible, by resolving isotopically labelled
DNA from the non-labelled form with density gradient centrifugation. Additionally, Radajewski et al.
(2000) recognized the elegance of being able to alienate specifically DNA of an active population utilizing
a chosen substrate, thereby facilitating an approach to link phylogeny with community function.
DNA separation and recovery

For successful separation of isotopically labelled DNA from non-labelled DNA on a density gradient, its
buoyant density should be relatively high. It varies naturally according to its GC content, and DNA with a
GþC content of 30–70 mol% ranges in density from about 1.69–1.73 g cm 3 (Rolfe and Meselson,
1959). Lueders et al. (2004a) confirmed that members from all three domains of life contain non-
isotopically labelled DNA which falls into this range. They also highlighted the importance of achieving a
buoyant density greater than 1.73 gcm 3 with DNA-SIP to resolve isotopically labelled DNA from
unlabelled high GC content DNA.
Radajewski et al. (2003) examined the possibility of using 13C, 15N and 2H to isotopically label DNA.
Table 11.16 details the elements that constitute nucleic acids and their abundance, to appreciate which
isotopes are best suited for DNA-SIP.
Table 11.16. Number of atoms per nucleotide unit in DNA (Data taken from Radajewski et al. (2003)).
Nucleotide unit H C N O P
b-D-2-Deoxyribose backbone 7 5 0 5 1
(A) Deoxyadenylate 11 10 5 5 1
(G) Deoxyguanylate 11 10 5 6 1
(T) Deoxythymidylate 12 10 2 7 1
(C) Deoxycytidylate 11 9 3 6 1
Thus, the two most abundant elements are C and H. However, incorporation of 2H and 13C may not
necessary yield DNA with sufficiently high buoyant densities if the 2H or 13C DNA content is not 100
atom%. Radajewski et al. (2003) reported that 13C-DNA with a GþC content of 35–70% theoretically
results in a buoyant density of 1.75–1.79 g cm 3 if its 13C content is 100 atom%. They speculated that
the 13C content of isotopically labelled DNA has to exceed 50 atom% to avoid isolation of high GC
content 12C-DNA in the 13C-DNA fraction. Accordingly, DNA-SIP requires a uniform labelling of DNA.
With 13C isotopes, one-carbon substrates are attractive since the single carbon atom becomes the building
block for all macromolecules (with the assumption that the one-carbon substrate is the only carbon source
available). Manefield et al. (2002a) showed with 13C6-phenol that uniform labelling of RNA could be
achieved by strategically positioning the 13C atom in the phenol ring. However, it is unlikely the same
would apply to more complex substrates.
13
C isotopes have been widely used in SIP. Cadisch et al. (2005) demonstrated that migration of 15N
labelled isotopes could also be tracked into DNA. As Table 11.16 shows, since the number of N atoms per
nucleotide unit is approximately half that of carbon, a successful separation of 15N-DNA from 14N-DNA
is only achieved when the 15N enrichment is 4 50 atom%. Depending on AT to GC ratio, N concentration
can vary theoretically between 13.9 and 15.8% (Cadisch et al., 2005). Bearing this in mind and the
hypothetical calculations of Radajewski et al. (2003), in mixed cultures that the 15N enrichment required
may need to be markedly higher than 50 atom%, to eliminate confidently contamination by high GC
14
N-DNA. Using 2H in SIP has also been assessed (Radajewski et al., 2000). Even though the number of
H atoms per nucleotide unit is greater than C (Table 11.16), Radajewski et al. (2000) showed that a
2
H-DNA separation with CD3OD as substrate was ,50% less compared to a 13C-DNA separation, with
13
CH3OH.
The basic DNA-SIP protocol described by Radajewski et al. (2000) to identify methylotropic microbes
from soil exposed to 13CH3OH, has undergone only minor modifications since. After incubation, they
extracted soil DNA and subjected it to CsCl density centrifugation, following the protocol of
Sambrook et al. (2001). The 13C-DNA (‘heavy’) separated from the 12C-DNA (‘light’), forming a second
band in the gradient, and both bands were visible with ethidium bromide under UV (Figure 11.42).
According to Cadisch et al. (2005) and Lueders et al. (2004a) a minimum of 13–15 mg of DNA is required
to see clear bands with ethidium bromide in a CsCl gradient. Smaller quantities (2 mg) give only faintly
visible bands making their extraction difficult. Rickwood (1992) suggested using a UV light having a
wavelength of 300 nm to minimize any DNA damage.
(A) (B)
12CH OH, 13CH OH, 13CH OH,

3 3 3
pure culture pure culture mixed culture
control control soil
Figure 11.42. Localization of 13C isotopically labelled DNA from non-labelled DNA on a density gradient. (A)
DNA banding positions of pure cultures exposed to 12CH3OH and 13CH3OH. (B) DNA banding positions of a
mixed microbial community exposed to 13CH3OH.
Separation of isotopically labelled DNA on an ethidium bromide-containing CsCl gradient depends on

three additional factors: the average density of the CsCl gradient, the centrifugal force applied, and
duration of centrifugation. Lueders et al. (2004a), recommended an average CsCl gradient density of
1.725 g ml 1 (prepared by mixing 4.9 ml of a 1.9 g ml 1 of CsCl (Sigma) stock solution, 0.9 ml of
gradient buffer (GB; 0.1 M Tris-HCl, pH 8; 0.1 M KCl; 1 mM EDTA) and DNA in a total volume of 6 ml),
and performed ultracentrifugation at 20– C for 36 h at 177,000 g. Similar protocols have been used by others
(e.g Cadisch et al., 2005, Ginige et al., 2004, Hutchens et al., 2004, Radajewski et al., 2000, 2002).
However, others have been used for band separation (0.5 mm – 2 cm distance apart). Radajewski et al.
(2002) and Morris et al. (2002) reported successful separation at 20– C with 265,000 g for 12–16 h, while
Hutchens et al. (2004) and Cadisch et al. (2005) applied 140,000 g for 69 h at 20– C. The latter two
reported that lowering the speed makes the density gradient less steep, increasing separation of the
isotopically labelled DNA.
Cadisch et al. (2005) further emphasized the benefits of low speed extended centrifugation for 15N-
DNA-SIP even at relatively low 15N atom% enrichments. The influence of fixed-angle rotors compared to
swing-out rotors for isopycnic centrifugation was demonstrated by Rickwood (1992). Longer path lengths
of swing-out rotors make them vulnerable to spin speeds, resulting potentially in crystallization of the
gradient medium at the tube bottoms, leading in extreme cases to rotor failure.
Under UV illumination, a red RNA pellet is sometimes seen at the bottom of the tube while proteins
band on the top (Rickwood, 1992). Extraction of the two separated DNA fractions is achieved either
by needle and syringe or by fractionation. Radajewski et al. (2000) performed a second round of
centrifugation with the 13C-DNA fraction to obtain ultra-pure 13C-DNA, although the need for large
amounts of DNA to visualize a clear band with UV may prevent this. Purification of DNA from ethidium
bromide can be achieved with isopropanol, while dialysis will remove CsCl (Hutchens et al., 2004,
Radajewski et al., 2000, 2002; Rickwood, 1992). Ginige et al. (2004) used Microcon YM-10 (Millipore,
Australia Pty. Ltd.) filter devices successfully to remove ethidium bromide and CsCl without losing
much DNA.
The 13C-DNA fraction recovered by Radajewski et al. (2000) harbored combined genomes of
organisms growing on 13CH3OH. The application of ‘universal’ PCR primers to amplify the SSU rRNA
genes of the three domains present in an isotopically labelled DNA fraction facilitates linking phylogeny
with function. If PCR primers are available for functional genes of the metabolic pathway involved, a
functional gene amplification and analysis may also be carried out.
Ginige et al. (2004) with FISH-MAR proved that the FISH probes designed against the 16S rRNA
sequences of the 13C-DNA fraction successfully targeted methanol utilizing denitrifiers in an activated
sludge community in a laboratory scale bioreactor. A similar approach (Ginige et al., 2005) allowed the
acetate utilizing organisms there to be identified. Design of FISH probes in both studies was only achieved
after establishing the metabolic pathways of the target organisms, and allowed a better understanding of the
impact of external carbon usage on performance of full-scale activated sludge systems (Ginige et al., 2007).
Limitations of DNA-SIP
The most serious limitation is a need for long incubation periods to permit incorporation of isotopically
labelled atoms into target DNA. Since incorporation occurs only during DNA replication, incubation
times depend on target population generation times. Further, to generate a sufficiently high isotopically
labelled atoms% DNA, several generations are required if satisfactory separation of labeled DNA is to be
achieved. Thus, incubation periods 440 days were applied by Radajewski et al. (2000) and Morris et al.
(2002), while shorter incubations were carried out by Whitby et al. (2001) (5 days) and Ginige et al.
(2004) (24 h).
Any incubation may lead to production of labelled by-products, which in turn become substrates for
non-target microbes (cross-feeding), syntrophic and non-syntrophic to the target organisms. Predation of
labelled target cells by non-target cells might also impact on the application value of DNA-SIP. However,
Morris et al. (2002) speculated that observing discrete 13C and 12C DNA fractions would indicate that
little or no cross feeding had occurred, possibly because 12C-labelled exogenous compounds and other
trophic interactions result in a marked dilution effect. Thus, smeared 13C DNA fractions would indicate
cross feeding. Radajewski et al. (2002) proposed that DNA-SIP time-course experiments would reveal
any such events, and Manefield et al. (2002a) using RNA-SIP highlighted the sensitivity of such
experiments by successfully demonstrating cross feeding of phenol-13C6 atoms to the RNA of
P. chlororaphis, a non-phenol degrading organism, after 72 h of incubation. Gallagher et al. (2005) as an
enhancement of the DNA-SIP approach, showed that DNA-SIP could be successfully applied with
minimal DNA labelling (i.e. with very short incubation periods) using carrier DNA to assist visualization
of the heavy DNA fraction.
Thus, DNA-SIP should be applied cautiously, taking note of the above. Ginige et al. (2004) adopted an
approach where DNA-SIP was used to bias the clone library towards methanol -using denitrifiers in
activated sludge, but adopted several other methods to confirm that populations identified in the 13C-DNA
fraction were those sought. Meyer et al. (2006) used a similar approach to enrich for Defluviicoccus GAO
populations using 13C-propionate as feed.
RNA BASED STABLE ISOTOPE PROBING (RNA-SIP) – PRINCIPLES

Manefield et al. (2002a) used a novel RNA-based stable isotope probing approach (RNA-SIP) to link
population function with taxonomy. They anticipated RNA-SIP would eliminate some drawbacks of
DNA-SIP since RNA synthesis in metabolically active cells occurs at high rates, and isotope incorporation
into RNA could then take place in target cells without cell replication. RNA-SIP is probably a more
sensitive approach, requiring reduced amounts and/or concentrations of isotopically labelled substrates
and incubation periods. However, labelling of non-target microbial RNA from cross feeding is still a
possibility.
Unlike DNA-SIP, RNA-SIP relies on the ‘transcriptome’ of the active target population(s), so has the
advantage of tapping into the naturally amplified phylogenetic signature molecule (rRNA) in active cells.
Manefield et al. (2002a) successfully demonstrated migration of 13C atoms into RNA with both pure and
mixed bacterial cultures. They used phenol-13C6 while others have since used a range of substrates like
13
C CO2, methanol, benzene, toluene and pentachlorophenol to bio-mark RNA (Lu and Conrad, 2005,
Lueders et al., 2004a, Mahmood et al., 2005). With pure cultures, Manefield et al. (2002b) showed that a
minimum of 20 13C atom% RNA enrichment is required to separate labelled from non-labelled RNA on
an isopycnic gradient. Griffiths et al. (2004) also stressed the importance of substantial isotopic RNA
enrichment to achieve adequate separation. With mixed cultures however, Manefield et al. (2002a)
showed evidence of separation with only a 4 13C atom% RNA enrichment.
RNA-SIP has so far focused on 13C labelled substrates, and 15N labelled substrates have yet to be used,
but these may provide opportunities to trace active populations capable of utilizing nitrogenous substrates.
If so limitations on the use of 15N labelled substrates (Cadisch et al., 2005) then need proper consideration.
RNA separation and recovery

As in DNA-SIP, isotopically labelled ‘heavy’ RNA can be tracked and isolated with density gradient
centrifugation. However, according to Rickwood (1992), higher buoyant densities of native RNA do not
facilitate its separation on a CsCl gradient at 20– C, and CsCl/Cs2SO4/H2O (2 : 2:1) gradients. KI
gradients and NaI gradients may improve separation. Manefield et al. (2002b) found that caesium
trifluoroacetate (CsTFA)/formamide gradients (Carter et al., 1983; Zarlenga and Gamble, 1987)
(1.86 ml of a 1.99 g ml 1 CsTFA solution (Amersham Pharmacia Biotech), 375 ml of H2O and 75 ml of
deionised formamide) effectively separated labelled and non-labelled RNA. Formamide disrupts
hydrogen bonding between amino groups of RNA bases, thus resolving RNA secondary structure and
preventing aggregation (Rickwood, 1992). The former ensures that density differences result from
differences in RNA isotopic enrichment. Since the destructive effects of formamide on some
polycarbonate plastics are known, the centrifuge tubes must be chosen carefully to prevent any rotor
damage and injury. Thus, Manefield et al. (2002a) used polyallomer bell-top Quick-Seal centrifuge
tubes (11 £ 32 mm) on a TLA120.2 rotor.
Manefield et al. (2002b) detail the problems associated with CsTFA/formamide gradients. CsTFA is
comparatively expensive, it hinders migration of nucleic acids in agarose gels, absorbs UV light and
inhibits PCR at 450 mg ml 1. Thus, direct observation of RNA on such a gradient with UV illumination
is problematic. Hence the experimental approach taken for RNA-SIP studies is different to DNA-SIP, as
summarized in Figure 11.43.
Cultivation of microbes on stable

isotope labelled substrates
RNA extraction
Isopycnic IRMS
centrifugation analysis
Gradient
fractionation
Reverse transcription
&PCR
Denaturing gradient
gel electrophoresis Cloning & sequencing
Figure 11.43. Flow diagram illustrating the steps involved in the RNA-based stable isotope probing
procedure (adapted from Manefield et al. (2002b)).
The success of RNA-SIP largely depends on a successful extraction of RNA from samples, and
McDonald et al. (2005) and Manefield et al. (2002a) both point to this as a major limitation. However,
methods for obtaining high quality RNA from soil and activated sludge are published (Griffiths et al.,
2000; Lu and Conrad, 2005; Mahmood et al., 2005; Meyer et al., 2006). Most RNA-SIP studies have
chosen the protocol of Griffiths et al. (2000), with minor modifications (Manefield et al., 2002a). While
Manefield et al. (2002b) found an RNeasy Mini kit from Qiagen suitable for RNA extraction from
laboratory-grown cultures, they used this protocol with environmental samples, and then further purified
the extracted RNA with the RNeasy Mini kit. Griffiths et al. (2000) stressed the importance of working in
an RNase free environment, treating all glassware with diethyl pyrocarbonate (Blumberg (1987), and
using plasticware certified RNase- and DNase-free (section 11.6).
Verification of quality and quantity of extracted RNA is important. Quality has been estimated by gel
electrophoresis or with ultramicro volume cuvettes in a GeneQuant pro RNA/DNA calculator (Amersham
Pharmacia Biotech) (Griffiths et al., 2004; Manefield et al., 2002a,b). Nucleic acids are quantified utilizing
this calculator, or PicoGreen and RiboGreen nucleic acid quantification dyes (Griffiths et al., 2004;
Lueders et al., 2004a; Manefield et al., 2002a,b). Both Lueders et al. (2004a) and Manefield et al. (2002a)
stress the importance of loading low quantities of total RNA onto CsTFA/formamide gradients (,500 ng
per gradient) to ensure successful isopycnic separation, since Lueders et al. (2004a) showed that RNA
quantities 41 mg cause aggregation and incomplete separation.
Prior to isopycnic centrifugation, most RNA-SIP studies validate extracted RNA with continuous
flow isotope ratio mass spectrometry (IRMS) (Lueders et al., 2004a; Manefield et al., 2002a,b; Meyer
et al., 2006), which reports isotopic ratios (e.g. 13C/12C ratio (%)) of a sample against a global
standard. However, it requires a large sample size (e.g. 410 mg-C for a 13C/12C ratio measurement)
(Meyer et al., 2006). Manefield et al. (2002b) detail a different protocol when extracting sufficient
RNA for IRMS analysis and isopycnic centrifugation is not feasible. They used 0.36 mg-C of RNA
(,1 mg of total RNA) for IRMS analysis. Since the minimum C content required is 25 mg-C
(Manefield et al., 2002b), they supplemented the balance by mixing the RNA aliquot with 24.64 mg-C
of glucose (,61.6 mg of glucose). The following Equation (1) was then used to express 13C atom% in
the composite sample.
" #
13 Rsample
C atoms% ¼ · 100 ð1Þ
Rsample þ 1
where Rsample is the m/z 45/44 (13C/12C) ratio determined by IRMS on analyte CO2 gas released on
combustion of the sample.
Manefield et al. (2002b) subsequently used a mass balance calculation (Equation. (2)), to determine the
actual 13C RNA atom% in the sample. Eqn. (2) subtracts the mass of 13C attributed to glucose from the
total mass of 13C in the measured sample to reveal the mass of 13C attributed to RNA.

ðS% · SmÞ ðG% · GmÞ
RNA13 C atom% ¼ ð2Þ
Sm Gm
where S% ¼ sample 13C atom%, Sm ¼ sample C mass, G% ¼ glucose 13C atom% and Gm ¼ glucose C
mass. A similar approach could be used to determine isotopic ratios of 15N and 2H labelled nucleic acids.
RNA-SIP studies so far each use different conditions for isopycnic centrifugation. Manefield et al.
(2002a,b) applied an average centrifugal force of 145,424 g at 20– C for 36 h while Meyer et al. (2006)
and Lueders et al. (2004a) operated at 130,000 g at 20– C for an extended period of 60 h. Certainly
Cadisch et al. (2005) did show that isotopically labelled DNA is separated effectively by applying a
reduced centrifugal force over an extended period of time, but whether a similar approach allows adequate
separation of isotopically labelled RNA is unknown.
The BDs for 12C RNA range from 1.755 to 1.83 g ml 1 and such differences could not be attributed to
variability in its GC-content (Lueders et al., 2004a). Lueders et al. (2004a) found that 12C-RNA of
members of all three domains often band between 1.776 and 1.79 g ml 1 in CsTFA gradients. Yet they
found that fully labelled 13C-RNA from M. extorquens banded at ,1.815 g ml 1. However, according to
Manefield et al. (2002a), RNA tends to span throughout the entire length of the CsTFA gradient on
isopycnic centrifugation, and RT-PCR of fractions along the entire CsTFA gradient indicated RNA
present in all. This highlights the importance of systematically fractioning the gradient, IRMS analysing
each fraction (to detect isotopically labelled RNA) and profiling them to reveal the populations utilizing
the stable isotopic substrate.
One efficient way of fractionating an isopycnic gradient is by displacing it with a solution whose
density is comparatively low or high (Rickwood, 1992). Manefield (2002a) recommended water with a
low density, and with it displaced the gradient from the top while collecting fractions from the tube
bottom with a syringe pump at a flow rate of 3.3 ml s 1. Lueders et al. (2004a) obtained 14 equal ,400 ml
fractions while Meyer et al. (2006) obtained 10 equal ,400 ml fractions. Each contained a minimal
amount of RNA, and not sufficient for both IRMS analysis and RT-PCR amplification. Thus,
Manefield et al. (2002a) produced six replicates of an isopycnic centrifugation and identically fractionated
all of them. By pooling fractions according to their respective densities, they obtained more RNA for each.
Density of a fraction is measured with a refractometer or by weighing a known volume of the fraction on a
four-figure milligram balance (Lueders et al., 2004a; Manefield et al., 2002a). The isolation of RNA from
gradient fractions can also be achieved by precipitation with isopropanol (Sambrook et al., 2001).
Comparing community profiles along fractions of a single isopycnic centrifugation, with those from
similar fractions along a time-course experiment, allows recognition of populations actively utilizing the
isotopic substrate. To confirm its active incorporation, Manefield et al. (2002b) stressed the need for an
increase in abundance of a relevant RNA species in the high-density fractions over time, with no
concomitant increase in its abundance in lower density fractions. Such an experiment also detects possible
cross feeding effects, and excludes erroneous inclusion of high mol% GC RNA as isotopically labelled
RNA simply because it occurs in high-density fractions (Manefield et al., 2002a).
Community profiling has been based on 16S rRNA. The amplification of its genes by RT-PCR of RNA
in gradient fractions with universal primers has generated community profiles of each fraction over time.
Techniques like DGGE (section 11.13) and T-RFLP (section 11.12) have been used to generate such
profiles, which revealed subtle changes in population densities across fractions (Griffiths et al., 2004; Lu
and Conrad, 2005; Lueders et al., 2004b; Manefield et al., 2002a).
Meyer et al. (2006) used RNA-SIP in attempts to identify the visually abundant TFO in activated
sludge in a deteriorated bioreactor operated to achieve EBPR. Abundance of Alphaproteobacterial TFO by
FISH was more than 50%, but clone libraries failed to reveal their identity or functionality. It was through
RNA-SIP that Meyer et al. (2006) succeeded in obtaining 16S rRNA sequence data identifying them as
Defluviicoccus spp. allowing the design of targeted FISH probes, and resolution of their ecophysiology as
putative GAO (chapter 10).
Limitations of RNA-SIP
DNA-SIP protocols are simpler and more robust than RNA-SIP protocols. In a comparative study with
pure cultures Lueders et al. (2004a) found that CsCl gradients resolved isotopically labelled DNA more
effectively than CsTFA gradients did with RNA. However, RNA-SIP is favored because of its continuous
turnover making it a more responsive biomarker than DNA. High turnover rates also ensure high
enrichment of this biomarker, thus eliminating a need for prolonged incubation periods.
However, Griffiths et al. (2004) have shown that isotopic RNA enrichment with some ecosystems does
not allow an unambiguous identification of active organisms. When polar lipid derived fatty acid (PLFA)
biomarkers were used (see below), a high isotopic enrichment was achieved, facilitating identification
(Treonis et al., 2004). This serves to highlight that despite low enrichments of some biomarkers, others
may become substantially enriched. Griffiths et al. (2004) suggest several reasons for poor isotopic RNA
enrichment. The limited supply and dilution effect of the isotopic substrate and/or possible presence of
only a small percentage of active microbes utilizing it were proposed.
Lueders et al. (2004b) demonstrated the power of combining RNA- and DNA-SIP to identify
methylotrophs in soil using 13C-methanol. RNA-SIP identified some members of the Methylobacteriaceae
(Alphaproteobacteria) and a novel clade within the family Methylophilaceae in the Betaproteobacteria.
Low levels of 13C-methanol and short incubation periods were successful because of the superior
sensitivity of RNA-SIP. However, DNA-SIP not only gave insight into primary methanol oxidising
populations, but also identified fungi and other microbes either involved in a predatory or a synergistic
relationship with them. Such experiments may yield valuable information about microbial interactions and
pave the way to a better understanding of food webs in nature.
POLAR LIPID DERIVED FATTY ACID-BASED STABLE ISOTOPE

PROBING (PLFA-SIP) – PRINCIPLES
DNA-SIP and RNA-SIP are laborious, expensive and time consuming, and hence not appropriate to
monitor community changes routinely. PLFA profiles are. However, they do not always provide species
resolution, but a broader diversity measurement at a phenotypic level. As with DNA- and RNA-SIP,
PLFA profiles may elucidate the expressed population diversity, but not the metabolic transformations
carried out by that community.
According to Lechevalier (1989), under normal conditions, PLFAs contribute a constant proportion of
the biomass and are not found in storage products or dead cells. PLFAs have been used to derive
phylogenetic information and monitor abundance of populations in complex ecosystems (Ringelberg et al.,
1997; Zelles, 1999), where profiles of mixed communities were compared to those from commercial
databases and pure cultures. The former rely on whole-cell saponification fatty acid profiles, and provide
taxonomic description of unidentified microbes. However, profiles in databases are restricted to organisms
available as pure cultures. Hence, PLFAs are of limited value.
Based on existing PLFA profiles of sulphate-reducing bacteria, Boschker et al. (1998) successfully
linked their identity to their capability to utilise acetate. They exposed sediment communities to 13C-
labelled acetate and analysed 13C enriched PLFAs. Profiles indicated that Desulfotomaculum acetoxidans-
like organisms were responsible for acetate-coupled sulphate reduction. These had not been reported
associated with marine sediments previously.
Boschker et al. (1998) clearly demonstrate the effectiveness of PLFA-SIP and were the first to use SIP
to directly link microbial activity with identity. They extended PLFA analysis by applying stable isotopes
and speculated that further extension was possible to estimate growth rates and yields of functional
populations as polar lipid synthesis is closely linked to bacterial growth.
Besides providing taxonomic information, PLFA profiles can provide information on overall structure
of active communities and biomass levels of selected groups there (Spring et al., 2000). They have been
used as rapid and inexpensive tools to monitor community changes in complex communities. Thus,
Boon et al. (1996); Cavigelli et al. (1995) and Elferink et al. (1998) used complete PLFA patterns as
fingerprints to monitor changes in several microbial communities while Findlay et al. (1990) followed
changes in a single community under different environmental conditions. This ability may provide vital
clues to process engineers to forecast behavior in processes like activated sludge reactors. PLFA-SIP
facilitates the measurement of functional changes in ecosystems in relation to community changes.
Additionally, since PLFA profiles indicate the physiological state of populations (Spring et al., 2000),
PLFA-SIP could be an alternative for ecophysiological investigations, when identification of a wide
diversity of microbes is not the focus.
LIMITATIONS OF STABLE ISOTOPE PROBING

SIP is a powerful technique, which can provide valuable insight into the diversity and activity of
communities present in complex ecosystems. Dumont and Murrell (2005) predict that in future SIP may
also prove useful with communities colonizing humans, as stable isotope labelled substrates have
advantages over currently used radioisotopes. According to Radajewski et al. (2000) the single most
important factor to consider in SIP investigations is dilution of the labelled substrate before its
assimilation and incorporation. With 13C and 15N labelled substrates, indigenous 12C and 14N substrates
cause dilution, reducing the chances of labelled atoms from becoming incorporated into DNA, RNA and
PLFAs. The solution requires using long incubation periods and excess amounts of labelled substrates.
Both strategies are likely to bias results. High substrate concentrations may promote the growth of
copiotrophic populations, which typically may not be those active under normal conditions (Gray and
Head, 2001). According to Gray and Head (2001), traditional pure-culture based methods impose similar
biases and inevitably fail to reveal the functionally active populations. Extended incubation periods are
also problematic as non-primary organisms tend to be exposed to stable isotopes by consuming metabolic
products and intermediates, which are also isotopically labelled (Radajewski et al., 2000). However,
where complete degradation of a labelled compound involves a group of organisms, it may not always be
possible to dissect the role of each without using labelled intermediates formed in the degradation
pathway. Time-course experiments and community profiling can demonstrate stable isotope flux through
different communities.
Most SIP investigations have assumed that stable isotopes have no preference or effect towards natural
populations. According to Matwiyoff et al. (1973) this is the case against many labeled substrates.
However, Zengler et al. (1999) and Uphaus et al. (1967) and others have shown that some cells possess a
capability to discriminate between isotopically labelled substrates and their non-labelled counterparts. In
particular, Zengler et al. (1999) found that uniformly labelled 13C-hexadecane retarded growth of an
enrichment culture more than a mixture of 13C- and 12C-hexadecane did, and Uphaus et al. (1967) showed
that an alga alters the quantity and distribution of its nucleic acids when 1H, 12C, 16O and 14N sources are
replaced with 2H, 13C, 18O and 15N.
Therefore, it is clear that SIP has limitations. However, each of these methods (DNA-, RNA- and
PLFA-SIP) compensates the other by addressing some of these. Additionally, SIP based techniques must
be combined with molecular and classical microbiological methods to minimize their limitations and
obtain a clearer view into the metabolic functions of naturally occurring populations (Radajewski et al.,
2003).
THE USE OF NATURALLY ABUNDANT STABLE ISOTOPES TO LINK

MICROBIAL PHYLOGENY WITH COMMUNITY FUNCTION
The application of stable isotopes may not always reveal the true identity of a functionally active natural
population as biases similar to those seen with culture dependent methods (chapters 1, 3) may influence
outcomes (Gray and Head, 2001). Natural abundances of stable isotopes in cells have been used as
biomarkers to link phylogeny with community function (MacGregor et al., 2002; Orphan et al., 2001,
2002). This approach is still in its infancy but holds considerable potential to investigate community
function, eliminating some of the limitations inherent in SIP. The following are two examples.
Fluorescence in situ hybridisation and secondary ion mass spectrometry

(FISH-SIMS)
Secondary ion mass spectrometry is used widely in earth and planetary sciences, but only in 2001 was it
applied to active cells in marine sediments (Orphan et al., 2001, 2002) showing with FISH and SIMS that
Archaea belonging to Methanosarcinales could oxidise methane anaerobically.
Stable isotope characterisation of small-subunit rRNA using IRMS

(SSU-IRMS)
The limitations of FISH and PLFAs encouraged MacGregor et al. (2002) to use SSU rRNA to identify
bacteria. It relies on using naturally abundant stable isotopic signature molecules to establish community
function. MacGregor et al. (2002) highlighted several features of SSU rRNA as a biomarker. Among these
was its occurrence in all living organisms; an ability to design probes against conserved and the highly
variable regions to reflect phylogeny; its transient existence in cells ensuring that its detection there
reflects populations that have been active recently. They also speculated that C and N cycles could be
studied based on differences in isotopic fractionation in SSU rRNA since both are elements of rRNA.
MacGregor et al. (2002) used a novel method to isolate the SSU rRNA of particular phylogenetic
groups from mixtures of total RNA, employing biotin-labelled oligonucleotide probes and streptavidin-
coated paramagnetic beads. They then determined the isotope levels of isolated SSU rRNA by IRMS, and
demonstrated its potential with pure cultures, showing that at natural abundance levels, d13C values of
SSU rRNA in each phylogenetic group closely reflected those of the carbon source used.
Its major limitation is the need to isolate large amounts (10 – 100 mg) of rRNA. This is not practical
with natural samples, where RNA concentrations are low, but commercial kits (bead beaters and freezer
mills) handling large sample volumes (10 – 50 g) may facilitate its wider application. Improving capture
efficiency is one way to reduce required quantities. MacGregor et al. (2002) anticipated a several-fold
improvement in capture efficiency with unlabelled helper FISH probes (Fuchs et al., 2000), with
sequences complementary to those on either side of the capture probe target site. Radajewski et al. (2003)
also suggested using rRNA-based capture of entire cells (Stoffels et al., 1999) to facilitate subsequent
IRMS analysis not only of rRNA but also other biomarkers.
PROS and CONS of using naturally abundant stable isotopes

A large heterogeneity in natural stable isotopic values in cell populations, PLFAs and SSU rRNAs is often
observed even in Archaea producing methane, a strong natural isotopic signature molecule (MacGregor
et al., 2002; Orphan et al., 2001; Thiel et al., 2001). Orphan et al. (2001) suggest that large d13C variations
reflect relative abundances of different phylogenetic groups. Abraham et al. (1998) revealed that a strong
isotopic fractionation is associated with lipid biosynthesis and so total cell d13C values could markedly
differ to those of the PLFAs. Similarly (Radajewski et al., 2003), biosynthetic and scavenging pathways
result in higher isotopic fractionation than nucleic acids biosynthetic pathways. Therefore, establishing
community function based on natural isotopic abundances must be carried out with caution.
Both FISH-SIMS and SSU-IRMS can be performed on samples systematically exposed to isotopic
signature molecules. Their enrichment in target communities will increase the sensitivity of both methods
but may contain biases, as discussed above. However, the sensitivity of SIMS and IRMS means that
the isotopic enrichment required to resolve community function reliably is lower in FISH-SIMS and
SSU-IRMS than in either DNA-SIP or RNA-SIP.
OUTLOOK
Stable isotope methods linking phylogeny with function are powerful and already have provided valuable
insight into the diversity and activity of many ecosystems including activated sludge. The five
experimental approaches described above each possess limitations, and careful consideration is needed to
decide which approach best suits each application. They fall into two distinct groups. Both DNA-SIP
and RNA-SIP involve phylogenetic analysis of stable isotope-labelled biomarker material, while
PLFA-SIP, FISH-SIMS and SSU-IRMS involve stable isotope analysis of phylogenetic groups (Manefield
et al., 2004).
As a guideline Manefield et al. (2004) suggest that PLFA, DNA and RNA-SIP are most appropriate if
the aim is to investigate ‘‘which organism/s assimilates a particular substrate?’’ and FISH-SIMS and SSU-
IRMS when the aim is to investigate ‘‘Does an identifiable organism assimilate a particular substrate?’’.
Stable isotope based methods in environmental microbiology appear to offer a clear view into the
metabolic functions of microbial populations by facilitating an in situ characterization of their
biochemical pathways, genes and proteins. Such knowledge must enhance our ability to manipulate
wastewater treatment systems more effectively to achieve industrial and environmental benefits.
11.15 FLOW CYTOMETRY

Jonathon Porter and Jeremy Lenaerts
INTRODUCTION
Flow cytometry (FCM) has much to offer those interested in activated sludge microbiology as it is one of
the few techniques that can make measurements on large numbers of individual cells, with techniques like
flow cytometry-cell sorting (FCM-CS).
Direct examination of samples by microscopy has limitations. FCM can be considered an alternative
and complementary technique, although it extends the value of microscopy, with quantitative analysis of
thousands of cells, one at a time, every second. Many instruments also offer subsequent cell sorting, which
physically recovers targeted cells. A detailed discussion of the physical basis of FCM-CS can be found
elsewhere (Porter, 1999; Robinson et al., 2004; Shapiro, 2003). In addition, several excellent websites
dedicated to FCM-CS exist (e.g. www.cyto.purdue.edu).
Many different makes and models of flow cytometers exist, but most follow the same basic principle. In
its most simple configuration, a cytometer is an automated microscope. Microscopy involves examining
slide-mounted specimens. If individual cells were fed sequentially into a fluid stream flowing along a
defined path on a slide, each cell could be analysed in turn. Provided that each cell arrived singly and
separately, and remained in the field of view sufficiently long, all the desired information could be
recorded. This is essentially how FCM works. Cells are brought into a sensing region, passed through a
light beam of uniform wavelength and intensity (so each cell receives uniform illumination), and both
scattered light and fluorescence (should a cell be fluorescently labelled) is collected. The light signals are
converted to an electrical signal which is stored and analysed. Plots can then be made of scattered light
against dye-specific fluorescence for every cell analysed.
Cell sorting takes the process a stage further. The most common set up is the jet-in-air sorter. Particles are
analysed in the fluid stream, as above. Data signals are processed, and a decision is made on each particle
whether to sort it or allow it to go to waste. These decisions are directed by the operator, based on criteria such
as whether it is fluorescent or not, or whether it can be sorted cleanly (i.e. whether non-target particles would be
co-sorted). The operator has full control over these criteria, although some decisions may be forced – e.g. when
sorting very rare target cells, sort purity may be sacrificed to ensure enough target cells are recovered. These
issues also relate to subsequent processing of sorted samples – e.g. if PCR amplification of target cell genes is
desired, sort purity is not necessarily a problem if the selected PCR primers are sufficiently specific.
To sort, the instrument is set up accordingly. Part of this is to cause deformation of the fluid stream
surrounding the cells, which does not affect cell analysis. However, after a cell passes through the light
beam, the fluid stream becomes unstable, and breaks up into droplets (which contain the cells). If the
decision is made to sort, the instrument waits until the target cell is about to reach this unstable point,
when a charge is placed onto the whole of the sheath fluid. This charge is maintained until the target cell is
contained in a droplet and that droplet has lost contact with the fluid stream. The charge is then turned off.
The target cell falls, surrounded by charged fluid, between two charged plates, and the droplet is deflected
(by repulsion/attraction of the charged plates) from the main stream of droplets. It can then be recovered
by placing an appropriate vessel at the side of the main stream. Moving the vessel at appropriate intervals
allows deposition of defined numbers of target cells in the same place.
Several areas are now highlighted which influence the successful application of FCM to activated
sludge. These are sample preparation, instrument set-up, sample labeling and data analysis.
Sample preparation
FCM-CS was developed for blood analysis, and lends itself especially well to samples where cells are in
suspension. In activated sludge, cells are both suspended and in flocs. Suspended cells can be analysed by
allowing flocs to settle prior to analysis. Sample preparation is likely to require disrupting the flocs to
some extent. Disruption will be determined by the question being asked. The size of flocs may cause
blocking or clogging in many instrument fluid systems if no disruption is involved. However, instruments
designed to analyse and sort objects up to 1.5 mm diam. are available (see below).
Floc dispersal has been achieved with physical (e.g. blending or sonication) and chemical (e.g.
detergent) treatments (chapter 3). For reproducibility, sonication treatments should be screened
individually by each laboratory, with volume of sample, probe diameter, time and amplitude (energy)
recorded. Bottle shape may affect sonication, with conical profiles ‘‘focusing’’ the waves. Even the
material used to make the bottle may have an effect, as more elastic polymers absorb energy. Forster et al.
(2002) suggest that 15 ml of mixed liquor in a 30 ml bottle (conical end), sonicated with a probe of 4 mm
diameter at 5 mm amplitude is the starting point for flow analysis. Under these conditions, cell lysis was
insignificant when monitored by microscopy and by cfu counts. Samples sonicated in this way allow
analysis and sorting (jet-in-air) of bacteria with a 70 mm nozzle (Forster et al., 2002).
Analysing filamentous bacteria and other bacteria (e.g. nitrifying bacteria, PAO) that exist in
microcolonies is important. Applying FCM to them is possible, but needs thought. The process of
injecting sample into the main sheath flow for FCM (hydrodynamic focusing) is presumed to align ‘linear’
forms with the direction of fluid flow. This does not imply that filaments are necessarily straightened,
rather that the longer axis will tend towards parallel with the direction of sheath flow, and is another
reason for selecting larger calibration beads for instrument set up, to ensure light is collected from a
broader sensing region. Filaments are large enough for one section to pass through the excitation light
before the remainder – thus, gathering an integrated light signal, or the pulse width of the signal, should be
considered (contrast with analysis of smaller bacteria, which can fit entirely within the light beam).
Analyzing microcolonies requires the same consideration. Sorting larger particles is readily achieved with
correct instrumentation (see below); thus whole flocs containing cells of interest may be separated for
subsequent analysis, with no prior disruption. Whether flocs are sorted without damage is not clear,
although instruments can be set up to handle fragile cells (e.g. apoptotic cells, dendritic cells, some
activated sludge cells) by keeping sheath pressures to 15–25 psi. In practice, this will only limit high-
speed sorting. Wallner et al. (1995) noted by microscopy after FISH hybridization, that flocs were
generally smaller, and cells were released from them; thus some impact seems unavoidable. For
investigations into three dimensional structures of flocs (e.g. spatial arrangement of cells), FCM will not
necessarily be the preferred instrument.
Instrument set-up
Instrument set up will be guided by the instrument and the manufacturer’s operating instructions. In
general, FCM bacterial analysis will require the logarithmic signal amplification of narrow (forward) and
wide (side) angle light scatter, and the removal of neutral density filters. It may be beneficial to replace the
traditional photodiode for narrow angle light scatter detection with the more sensitive photomultiplier
tube. Bacterial analysis may also require fewer milliwatts (e.g. 7 to 15 mW) of laser power output than
protocols analysing eukaryote cells. Some general points are discussed here.
General FCM
Mixed liquor contains many particles, some of which are bacterial cells; others are particles the size of
bacteria. It is still important to ensure that noise is minimised (i.e. any objects that could be bacteria of
interest have come only from the sample).
(i) All buffers and sheath fluid must be filtered meticulously before use. Some stock dyes may come
in solution, or only a few mg. only may be purchased to prepare stock. As often less than 1 ml may
be added finally to label cells, it is generally unnecessary to filter such stocks (although they
should be prepared in filtered buffer). If they are thought to contain particles, centrifuge them and
use the dye in the supernatant. Cheaper dyes, working stocks or larger volumes should be filtered
where possible. Pour some filtered solution from the first filtration run into the final container, cap,
shake and decant the solution to be filtered again. Repeat at least once more. In this way, solutions
will be filtered three times, and the containers are rinsed in particle free solution twice. Keep
dedicated containers for filtered buffers and sheath fluid.
(ii) If possible, use the same buffer for sheath fluid and for preparing/suspending samples.
Commercial sheath fluids may contain antimicrobial agents, so if the assay requires viable cells,
prepare sheath fluid (e.g. distilled water or phosphate-buffered saline; PBS) in-house. Routinely
filter all sheath fluid through 0.1 mm pore size filters, prior to autoclaving.
(iii) Keep stocks sterile; any sheath fluid left over should be discarded. The instrument will have an in-
line sheath fluid filter prior to the sensing region. If altering this (e.g. reducing pore size), make
sure the effective filtration area is large enough to maintain sheath flow rates and pressure.
(iv) If the sheath fluid is used for sorting, it will need dissolved salts for the droplet-charging process.
Salts and buffers may adversely affect fluorescent labelling (e.g. with DNA stains). However, the
concentration of salt required may be less than that in commercial sheath fluids, or PBS. Provided
that stable side streams (droplet deflection) can be achieved, there is no need for more salt. Salt
deposits from dried sheath fluid will have to be cleaned eventually.
Cleaning
Cleaning and disinfecting the instrument is important to prevent microbial growth when not in use. Some
operators also place sample tubes containing cleaning solution in place overnight.
(i) Wash and flush the instrument fluid systems thoroughly. Rinse thoroughly after bleach or detergent
cleaning. Unless the instrument has a dedicated operator, have a standard cleaning and shut down
procedure that all users must follow, and monitor users to identify those that do not leave it clean.
(ii) Waste from the instrument may contain live microbes – it may be possible to add concentrated
disinfectant to treat biological and chemical hazards in the waste collection tank.
Calibration and focusing

(i) The instrument will require daily calibration before, during and at the end of the experimental
work. This is generally achieved using particles of known size and shape. Keep a record of the
calibration data to monitor performance over time.
(ii) Uniform fluorescent microspheres of bacterial size are available in a range of sizes and
fluorescence intensities (numbers of fluorescein equivalents per microsphere). Such beads will
help instrument set up. Instruments that utilise high numerical aperture microscope objective
lenses to collect light signals may require precise focusing to allow precise measurements.
(iii) Small beads (0.5 mm diameter) can be used. However, for heterogeneous activated sludge
samples, which contain a wide range of particle sizes, and for jet-in-air systems, larger (1–2 mm
diam.) beads may allow for cells following alternative trajectories to be in focus.
(iv) It is important that all cells are exposed to the same amount of excitation light. Instrument settings
will generally require logarithmic gains on the photo-multiplier tubes, with voltages of 500–750 V,
or even greater. Detecting fluorescently labelled bacterial cells may require more sensitive settings
than those for detecting beads.
Successful detection of bacteria will require fluorescent tagging to identify them from background
events (particulates, noise or non-target cells. Dyes or probes of relevance allow total cell enumeration
(DNA staining), specific cell enumeration (surface molecules or oligonucleotide labelling), activity
assessment and combinations, such as dual or multi-colour analysis. Such a wide range of applications
precludes presenting a defined protocol for each here. However, the methods described in the references
have proven to be robust, and should provide a good starting point for each laboratory, instrument and
activated sludge sample. All protocols should be tested to ensure they are optimized. Additionally, the
range of fluorescent probes continues to expand (Molecular Probes; www.probes.invitrogen.com). It is
worthwhile spending time designing the experimental procedure carefully.
EXPERIMENTAL DESIGN AND DATA ACQUISITION

The experimental design will be influenced by the instrument, the experimental question and downstream
processing of the sample. Many instruments use single laser light sources.
(i) A fluorescent label is generally required for activated sludge samples. Microbiological
applications generally have a limited number (two) of fluorochromes assessed from a single
excitation wavelength. This could be said to limit the number of useful parameters assessed for
each cell, but probing is also limited in the choice of target molecules available for a satisfactory
signal. In practice however, two fluorescent labels generally allow useful measurements.
(ii) The instrument is set to trigger (record an event) based on a light signal, which may be light
scatter (an inherent property of all particles), with subsequent analysis of how many appropriate
particles possess a fluorescent signal, or fluorescence.
(iii) It is sometimes useful to examine data from activated sludge samples when the instrument is set
to trigger on light scatter, as problems of identifying target signals against the heterogeneous
background become very clear.
(iv) The major criticism of triggering on light scatter alone is doubt about whether an event was a
bacterial cell or a particle; the major criticism of triggering on fluorescence is that it may limit
options in subsequent fluorescence measurements.
(v) An instrument with two or three lasers allows more flexibility (exemplified for FCM-CS analysis of cell
phenotypes in human blood, utilising 17 colours – so-called polychromatic flow cytometry; Perfetto
et al., 2004). However, as stated above, this number of possible targets does not exist for bacteria.
(vi) A fluorescent label with e.g. high specificity for DNA will allow more effective discrimination
between particles that contain DNA and particles that do not. Two (or more) light sources expands
the available options, as the instrument can trigger on DNA fluorescence (often u.v. laser), leaving
green and red fluorescence available for other measurements (e.g. oligonucleotide probing).
However, success may be achieved with a single laser instrument used correctly, some experience
of the system being analysed and appropriate data processing.
(vii) Data shown below (Figure 11.44a) demonstrate activated sludge analysed by forward angle light
scatter. This parameter is probably the least diagnostic to trigger on, and both cells and non-
cellular particles are recorded. However, detection of relatively weak fluorescence (oligonucleo-
tide probing) of rare target cells (approximately 0.001% of the total events; 1 £ 104 cells in a
background of 109 cells per ml.) is possible.
(A) 1000 (B) 1000
800 800
Green fluorescence
Green fluorescence
600 600
400 400
200 200
0 0
0 200 400 600 800 1000 0 200 400 600 800 1000
Forward scatter Forward scatter
(C) 1000 (D) 1000
800 800
Green fluorescence
Green fluorescence
600 600
400 400
200 200
0 0
0 200 400 600 800 1000 0 200 400 600 800 1000
Forward scatter Forward scatter
Figure 11.44. Probing AS samples for pseudomonads using FISH. AS samples were sonicated and
analysed, with the instrument set to trigger on narrow (forward angle) light scatter (A, B), to detect all particles
with size and scatter properties indicative of bacteria. Using data processing to reduce the number of non-
target events (C, D) the proportion of target events detected in total increased. Use of a molecular beacon
probe (C) in place of a standard, linear probe (D) improved the signal:noise ratio by controlling noise. Using
the most effective discrimination of target events (achieved under the settings for panel C), cell sorting was
also the most effective, and yielded cell preparations most suitable for PCR template, as judged by specific
PCR product yield from 500 events.
(viii) This shows that finding rare target events is much improved by instrument set up and data
processing, to reduce the number of non-target events (Figure 11.44c,d) and by maximising
the signal:noise ratio (in this case by controlling noise, rather than increasing signal strength;
Figure 11.44c). Triggering on a fluorescence parameter initially eliminates a substantial
proportion of non-target particles for which data are collected (Wallner et al., 1995). A further
excellent example of this is given in Perfetto et al. (2004).
DETECTION AND ENUMERATION OF BACTERIA IN

ACTIVATED SLUDGE
Clearly, bacteria in flocs are of great interest, and floc dispersal is probably the major limiting factor in
finding target cells by FCM. Several options may help in the search for rare events, including using
different sort modes, multiple rounds of sorting, magnetic bead-based sorting, and PCR to amplify
sequences from target cells.
(i) Accurate enumeration requires limiting the number of washing steps. Repeated pelleting/
resuspension of cells to remove unbound dyes etc. affects their numbers, light scatter charac-
teristics and cell-cell associations.
(ii) This can be demonstrated with a laboratory culture centrifuged for different times and speeds. As
both are increased, events per ml fall, the proportion of single cells decreases and mean side scatter
(arbitrary units) increases.
(iii) Thus, adding concentrated buffers, and using lower concentrations of dyes is the favored option.
Empirical testing of labeling protocols is straightforward and worthwhile. It is also preferable to
keep analysis rates low enough to reduce coincidence events (two or more cells passing through
the sensing region together).
Most DNA stains are inherently fluorescent, but become more fluorescent when bound to the
molecule,giving a high signal to noise ratio, and effective detection of bacteria. Thus, the most effective
approach is to trigger the instrument on DNA fluorescence, which ignores all non-cellular particles.
Enumeration can be achieved by measuring flow rates and time, and/or by adding a known amount of
calibration beads to the sample, and counting these beads (representing a distinct population).
Specific cell enumeration

Detection of specific cells requires them possessing a unique feature. Light scatter characteristics are
unpredictable, although a relationship exists between increased scatter characteristics and cell size. Most
approaches use some fluorescent tag.
(i) Antisera have been used for direct detection and for immuno-magnetic separation of bacteria from
environmental samples.
(ii) Perhaps the technique of most interest is FISH. The reader is referred to section 11.9 for further
detail on FISH protocols. For FCM, cells are generally probed in suspension (McIlroy et al.,
2008a), although cells have been probed on slides and then washed off for subsequent FCM-CS
(Sekar et al., 2004).
(iii) Two points are emphasised here. Sample fixation using 3 volumes of freshly prepared 4%
paraformaldehyde has consistently proven the best approach for probing single cells
(section 11.9).
(iv) The Mycolata require additional consideration. Hydrochloric acid improves cell permeabilisation,
but Davenport et al. (2000); Carr et al. (2005) and Kragelund et al. (2007b) have each suggested
improved protocols, with that of Kragelund et al. (2007b) currently the most effective.
(v) The second point regards probe concentration, hybridization times and subsequent washing, to
remove any unbound and non-specifically-bound probe. Wallner et al. (1993) demonstrated that
low probe concentrations combined with long hybridization times reduce the need for washing,
thus improving the ability to count.
(vi) Molecular beacon probes can likewise reduce noise by being closed (dark) when not hybridized
(Lenaerts et al., 2007; Xi et al., 2003). Signal strength has also been maximised by probe-linked
reporter enzyme activity (Sekar et al., 2004).
(vii) Using FISH to detect and enumerate target groups allows culture-independent monitoring of cell
numbers, and thus levels of indicator groups readily monitored (e.g. all Mycolata morphotypes;
Davenport et al., 2000).
ACTIVITY ASSESSMENT
The question of proving whether an organism is alive or not remains difficult to answer (chapter 3).
However, demonstrating some form of cellular activity is more straightforward. Whether the measured
activity directly assesses a particular cellular function may require validation.
As discussed, detection of cells requires a satisfactory signal: noise ratio. So, a general
recommendation for activated sludge samples is to use substrates which are non-fluorescent until
modified by the cell, rather than those which are inherently fluorescent, and require cellular uptake and
concentration. Probes of the latter type (e.g. those often used for membrane potential) may still be of
value for cultured species. Three main groups of fluorescent dyes are discussed here, namely those
measuring enzyme activity (with associated membrane integrity), dye exclusion and respiratory chain
activity (section 11.3).
Intracellular enzyme activity and membrane integrity

Applying dyes like 5(6) carboxyfluorescein diacetate (CFDA) provide very robust procedures with a
widely available substrate. Published protocols are simple and readily optimised empirically for each
sample source.
(i) Samples are diluted or concentrated to c.a 1 £ 106 cells/ml, and between 1 to 10 ml of stock
(1 mM in acetone) per ml of sample is added. Stocks are stable at 20– C for at least 3 months.
(ii) Some form of cell permeabilization should be used, such as exposure to Tween-20 (0.1% final
concentration). Incubations are carried out at 25–37– C. It is worth checking the pH of the sample,
and incorporating a buffer if necessary to keep the pH above neutral.
(iii) Samples can be gently heated to ensure dye access into the cell, in which case subsequent chilling
on ice may help ensure dye retention. The fact that the dye is non-fluorescent until hydrolysed
ensures a low background signal. Labelling efficiency may vary according to the growth phase of
cells.
(iv) Analysis of 16S rRNA gene PCR products, by DGGE profiling (section 11.13) of bacteria sorted
after labelling with either of the two (5 and 6) isomers of CFDA showed no differences,
suggesting no selectivity in dye uptake (Forster and Porter, unpublished data). Cocktails of
fluorogenic ester dyes did not increase active bacterial counts, also suggesting that different dyes
label the same bacterial cells in heterogeneous samples (Porter et al., 1995, 1997).
(v) Extended incubation times do not generally improve the viable cell count, and may cause non-
specific hydrolysis of the substrate. Controls prepared from fixed samples may be necessary.
Membrane exclusion assays

A popular assay is the BacLight Live/Dead kit from Molecular Probes (see section 11.3). This is based on
the ability of a green fluorescent DNA dye (SYTO 9) to cross all membranes, and stain all cells effectively
enough for detection, coupled with a red fluorescent dye (propidium iodide) which only enters cells with a
compromised membrane, and counter-stains sufficiently well for red fluorescence to dominate (chapter 3;
section 11.3).
(i) A single laser FCM (as opposed to microscopy) will quickly demonstrate that the green dye is
present in all exposed cells. As the bright green fluorescence spills over into the red fluorescence
detection channel, appropriate filters (i.e. longer wavelength than standard) for the red
fluorescence detector may help.
(ii) Other workers have suggested substituting alternative green dyes (such as SYBR Green;
Molecular Probes), which passes fluorescence energy onto the propidium dye when both dyes are
in cells, thus causing quenching of the green fluorescence and enhanced discrimination of sub-
populations (Gregori et al., 2001).
(iii) Propidium iodide alone allows estimation of proportions of cells with a compromised membrane.
Other dyes with multiple positive charges, such as ethidium, have also been used. Efflux pumps
exist in some species for some dyes.
(iv) It is often possible to scale down manufacturer’s protocols to allow more assays from an
expensive kit.
Respiratory chain activity

As a fluorescent label is the best approach for activated sludge bacteria, choice of dye is limited to 5-cyano
2,3 di-tolyltetrazolium chloride (CTC) (chapter 3; section 11.3). Again, empirical optimisation of
incubation conditions is straightforward.
(i) A good starting point is to incubate samples, after dilution to 106 to 107 cells per ml., with
3.5 mM CTC (molecular weight 312) for 1–4 h at 25– C. CTC stock solutions are stable in the
dark at 4– C for at least 6 weeks. However, some cells in biomass samples will label regardless.
(ii) Controls may be prepared from fixed (formalin or heat) samples if necessary. Inhibition by
traditional specific inhibitors such as cyanide or azide may not be effective for different formazan
structures (Smith and McFeters, 1997).
SORTING
Routine application of cell-sorting has not been widely adopted in microbial ecology, but the approach
has a great deal to offer. Physical separation of target cells allows subsequent identification by DNA
sequencing (e.g. McIlroy et al., 2008a), and possible culture.
(i) The instrument system requires sterilisation prior to sorting, with e.g. bleach; any sterilising agents
then need to be flushed away. A 0.22 mm pore size in-line filter in the sheath fluid placed after a
three-way luer lock valve may achieve this, since bleach can be passed into the sheath fluid lines via
the valve, through the in-line filter until it flows from the nozzle tip, and left to soak. It can then be
flushed out with standard sheath fluid, avoiding the need to introduce fluids upstream of the filter.
(ii) Controls for sorting are readily achieved, by e.g. sorting non-target events and by analysing the
sheath fluid for contamination.
(iii) Safety considerations should also be mentioned, especially with respect to sorting, which may
well deliberately generate (contained) aerosols. Thus, work with ‘live’ activated sludge samples
should be risk assessed, and the instrument containment features should be used.
(iv) Placing appropriate collection vessels (plates or tubes of broth) around the sort chamber will
confirm that any aerosols are contained. Virus containment can be confirmed by passing phage
suspensions through the instrument and placing host lawns around the chamber.
(v) These precautions should also be considered when using cells labeled with radioisotopes to help
elucidate function.
(vi) The number of events/cells sorted is recorded by the instrument; sorts can thus be large scale
(e.g. 50–60,000 events; Sekar et al., 2004) or much smaller scale, for a defined number of cells.
(vii) To generate a representative clone library of unknown populations (section 11.8), large starting
numbers may be appropriate, although Sekar et al., (2004) used 10,000 cells from marine water
samples.
(viii) To confirm target cell identification, far fewer cells could be considered to enhance starting
template purity and detection of a single sequence. PCR amplification of 16S rRNA genes of
laboratory cultures has been reported from 1000 (Ferrari et al., 2004) and 500 (Wallner et al.,
1995) E. coli cells, and from 1000 Bacillus subtilis cells (Wallner et al., 1995). The rRNA gene
copy number is 7 for E. coli and 8–10 for B. subtilis (Klappenbach et al., 2001).
(ix) Nested PCR amplification was reproducibly successful for 100 Erwinia cells (Ferrari et al., 2004;
rRNA gene copy number 2).
(x) With a jet-in-air sorter, nested PCR products have been obtained routinely for 50 E. coli cells, and
from 10% of sorts using 10 cells (Porter, unpublished). This study used live cells, which would not
have been suitable for FISH tagging.
(xi) For activated sludge samples, 500 fixed, FISH-probed gammaproteobacterial pseudomonad cells
reproducibly yielded PCR products (Lenaerts et al., 2007; rRNA gene copy number of 4–7;
Klappenbach et al., 2001).
(xii) Successful identification of unknown bacteria from this number or even fewer cells, following RT-
PCR, using the higher copy ribosomal RNA as template, has been reported by Thomsen et al.
(2004), who demonstrates that fixation conditions affected subsequent polymerase enzyme activity.
FUTURE DEVELOPMENTS
FCM-CS has great potential for studies of bacteria in activated sludge communities. Its main strengths are
the individual examination and possible separation of many thousands of bacteria quickly and objectively.
Identification of target cells within the heterogeneous community depends on the ability to obtain an
effective signal: noise ratio from the fluorescent tag. Its main weakness is the need to disaggregate flocs
prior to analysis (although labelling target cells may require floc disruption). However, this limitation may
be overcome, with advances in flow technology like the COPAS system (www.unionbio.com). Unlike
traditional jet-in-air sorters, which rely on vibrating the fluid stream to form droplets, this instrument
literally blows air at the stream to divert a section of fluid into a collecting device. Although not yet applied
to flocs, this avoids problems associated with jet-in-air sorting of large particles – the energy required to
form large droplets, and their reliable deflection. Further advances to help study activated sludge bacteria
with less floc disruption include imaging flow cytometry (www.amnis.com). This essentially photographs
events as they pass through the sensing region. Thus, each particle is represented by a bright field image
and up to four fluorescence images, and should offer the advantages of FCM, but with greater within-floc
spatial resolution. However, at present, the analysis rate of such instruments is much lower.
The level and sophistication of technology applied to answering questions about activated sludge
microbiology will undoubtedly vary between research laboratory and instrumentation for routine
monitoring. However, technological advances and improvements from other FCM applications are driving
data acquisition and analysis developments at a considerable rate (Perfetto et al., 2004). Plankton analysers
may be towed behind research vessels (Herman et al., 2004) or used while moored or submerged
(Dubelaar et al., 1999, 2004). These approaches have applied traditional flow cytometry instrumentation to
permit continual measurements, and developed scanning flow cytometry in conjunction with light scatter
and autofluorescence measurements to determine particle sizes, distributions and identification. Several
operational limitations may apply with activated sludge samples (e.g. high cell/particle densities requiring
effective data acquisition and processing, to determine particle identity and to cope with coincidence events)
but there is a real possibility of on-line analysis instruments with FCM principles soon.
Acknowledgements
Financial support for the development and validation of flow cytometric protocols was provided by the
Natural Environment Research Council, and by the Biotechnology and Biological Science Research
Council, Swindon, UK.
11.16 MICROSENSORS
Rikke Meyer
INTRODUCTION
Microsensors were employed in microbial ecology as early as 1969 (Bungay et al., 1969) to measure
oxygen profiles in wastewater biofilms, but widespread application of the technology had to wait for the
methodological advances made by Revsbech (1989) following seminal work in the 1980’s with studies of
photosynthesis in sediments and microbial mats (Revsbech et al., 1980; Revsbech and Jorgensen, 1983).
A wide range of sensors now contribute to studies of processes with microscale spatial resolution,
as outlined in this section.
Broadly two detection principles are employed in microsensors: electrochemical and optical.
Electrochemical microsensors are classed as either potentiometric or amperometric. Potentiometric
sensors are also known as ion-selective electrodes because their measuring principle is based on allowing
certain ions to enter the sensor tip through an ion-selective barrier. This results in build-up of an electric
potential which is measured against a reference electrode.
The most successful potentiometric microsensor is an adaptation of the common glass pH electrode
(Thomas, 1978) with a lifetime of about 1 year, and suffers least interference from other chemical
compounds. Others use a liquid ion exchange (LIX) membrane as the barrier, but it is not 100% selective
and interference from other ions may occur. LIX microsensors benefit from a high resolution facilitated by
their small tip size, but have a lifetime of only hours to weeks. The only microsensors available for NHþ 4
are LIX (de Beer and Vandenheuvel, 1988), and other examples are those used for NO3 (de Beer and
Sweerts, 1989), NO2 (de Beer et al., 1997), and Ca2þ (Ammann et al., 1987).
Amperometric detection involves oxidation or reduction of the measured compound on a cathode or
anode, which produces a current proportional to the amount of analyte consumed. In the commonly used
Clark-type sensors, the cathode is contained in a casing and separated from the surrounding environment
by a gas-permeable membrane. Figure 11.45 shows a diagram of a Clark-type oxygen microsensor
(redrawn from Revsbech and Jørgensen 1986) with an oxygen profile measured in a photosynthetically
active algae mat (unpublished data). H2S (Jeroschewski et al., 1996), H2 (Witty, 1991), and N2O
(Andersen et al., 2001) sensors are also Clark-type sensors. As the ‘‘raw’’ N2O sensor is sensitive to O2 it
has an extra casing around the tip containing an O2 scavenging solution. These gas-sensing microsensors
form the basis of diffusivity or flow sensors (Revsbech et al., 1998), where they detect outward diffusion
of a tracer gas from an internal reservoir to the surrounding environment. All have a lifetime of months to
years, and few problems with chemical interference, except from H2S.
Oxygen (µmol l–1)

–400 –200 0 200 400
-0.1
Diffusive boundary
0.0
Glass
Electrolyt 0.1
Depth (µm)
Glass
0.2
Platinum
0.3 O2 production
Gold O2 concentration (model)
O2 concentration (measured)
Silicone
0.4
–1.0 –0.5 0.0 0.5 1.0
10 µm Oxygen production (nmol cm–3 s–1)
Figure 11.45. A schematic diagram of a Clark-type oxygen microsensor (redrawn from Revsbech and
Jørgensen, 1986) and an oxygen profile measured in a photosynthetically active algae mat. The area graph
shows the oxygen production rate calculated from the concentration profile.
Optical microsensors collect light at the sensor tip and direct it through an optical fibre to the
measuring system. These either detect light (irradiance or scalar irradiance) directly in the sample
(Kühl et al., 1994; Lassen et al., 1992), or optical changes in an indicator compound immobilised on the
sensor tip. The latter type is referred to as ‘‘optode’’. Application of such compounds has led to optical
sensors for O2 (Klimant et al., 1995), temperature (Holst et al., 1997), pH (Kohls et al., 1997b) and CO2
(Kohls et al., 1997a). The O2 optode has excellent long-term stability and is free from interference.
Some sensors incorporate a biological element by immobilising enzymes or cells at the tip. These
microscale biosensors exploit the specificity of biological molecules to transform otherwise ‘‘immeasur-
able’’ compounds into a product that is now detectable. The best known is the glucose sensor, developed
in a microscale version by Cronenberg et al. (1991). It contains glucose oxidase immobilised on the tip of
a platinum anode, where it oxidises glucose aerobically with concurrent production of H2O2, which is then
reduced on the platinum surface, generating a detectable current.
A simple design for an organic carbon microsensor was that of Neudörfer and Meyer-Reil (1997), with
a pure culture of yeast immobilised on the tip of an O2 microsensor. As carbon is oxidised by the cells, O2
is consumed, and the signal of the sensor decreases. A similar principle is used in the CH4 biosensor,
containing a pure culture of methanotrophic bacteria oxidising CH4 aerobically. It measures consumption
of O2 from an internal reservoir and thus can be applied in anoxic environments.
Sensors for NOx (Larsen et al., 1997) and NO2 (Nielsen et al., 2004a) contain pure cultures of
denitrifying bacteria immobilised in front of an N2O sensor. They are provided with an electron donor
from an internal medium reservoir but lack any electron acceptor. As NO3 or NO2 diffuse into the sensor
tip, both are reduced to N2O and measured by the electrochemical sensor. A biosensor for volatile fatty
acids (Meyer et al., 2002) is based on same principle, where bacteria are provided with NO3 from the
internal medium reservoir, but do not reduce NO3 to N2O unless provided with an electron donor (volatile
fatty acids) present in the sample being analysed.
With a tip diameter 420 mm, biosensors are comparatively larger, which affects spatial resolution of
the measurements. They have a relatively short life, ranging from a few days to weeks, which restricts
their exploitation. Manufacturers include Unisense A/S (www.unisense.com), AMT Analysenmesstech-
nich GmbH (www.amt-gmbh.com), and Diamond General (www.diamondgeneral.com), each offering a
range of sensors. For a more extensive discussion, the reader is referred to Kühl and Revsbech (2001).
Substrate transport by diffusion

The unique properties of microsensors relate to their small size and speed of molecular diffusion at this
scale. Molecular diffusion is very fast over small distances (tens of mm) but much slower over large
distances, as the time taken to diffuse correlates with that distance squared (Revsbech and Jorgensen,
1983). Therefore microsensors usually have rapid response times. Furthermore, samples require no
stirring due to the diffusive flux of analyte to the sensor’s tip, which is beneficial if measuring gradients in
stagnant media like sediments or biofilms. Microsensors can resolve chemical gradients occurring over
fractions of a mm. Activity of organisms in an unstirred matrix drives a diffusive flux of substrate into the
matrix, which generates a concentration gradient. If activity is high enough to deplete the substrate, its
overall uptake rate is limited by the diffusive flux and not organism activity, and mass transfer limitation
occurs.
The diffusive flux (J) of molecules across a known area can be described by Fick’s first law of diffusion
(Crank, 1983):
qC
J ¼ Ds ·
qx
where qC/qx is the slope of the concentration gradient, and Ds is the effective matrix diffusion coefficient.
Thus, flux is calculated from the first derivative (the slope) of the concentration profile. Metabolic activity
at different matrix depths causes a variation in the flux of substrate with depth, and the net consumption of
substrate at different positions through the matrix is calculated as the change in flux over distance, i.e. the
second derivative of the concentration profile. This relationship can be described by a mathematical
equation (Revsbech et al., 1981):
q2 CðxÞ
Ds ðxÞ · ¼ RðxÞ PðxÞ
qx2
where R(x) is the rate of consumption at a certain position (x), P(x) is the production, C(x) is the
concentration, and Ds is the effective diffusion coefficient. A linear concentration gradient (constant flux
over distance) thus represents zero activity, and a concave profile represents consumption whereas a
convex profile represents production (Figure 11.45).
MICROSENSOR MEASUREMENTS AND INTERPRETATION OF DATA

Microsensor profiles are measured by fixing the sensor to a micromanipulator and lowering it step-wise
into the sample, collecting a data point at each step. The spatial resolution depends on sensor tip diameter
and a step size at least twice the tip diameter is recommended. Measurements are most easily and
accurately performed with a motorized, computer-controlled micromanipulator, so that no physical
disturbance is imposed. The software (e.g. Profix, Unisense) controlling the micromanipulator can be used
to log sensor output and display the concentration profile as it is measured.
Concentration profiles measured during steady state, i.e. when no change in the profile occurs over
time, can be used to calculate a profile of the depth-specific production or consumption rates as described
above. Berg et al. (1998) published a numerical procedure that models activity profiles, and found a
statistically supported best fit to the measured data. It offers an objective interpretation of concentration
profiles, aiming to obtain the simplest possible interpretation of the measured data. The oxygen production
profile shown in Figure 11.45 was calculated with Berg et al.’s procedure. Circles (•) represent measured
data points, and the line the concentration profile modeled from the production profile.
Such calculations require an accurate value for the diffusion coefficient of the analyte within the
matrix. It also assumes that mass transport processes in the matrix other than diffusion are either absent or
can be quantified. If these parameters are not accurately known, substantial errors in the rate calculations
can arise, so it is prudent to determine them empirically using a diffusivity sensor.
One of the practical challenges in measuring microsensor profiles is to create a hydrodynamic regime
surrounding the sample in the experimental set-up, which accurately replicates in situ conditions. All
surfaces are surrounded by a diffusive boundary layer (DBL), through which molecular transport occurs
by diffusion (Figure 11.45). DBL thickness depends on the topography of the sample surface and direction
and velocity of the flow above it (Roy et al., 2002). Because chemical gradients in biofilms and bacterial
aggregates are often steep and a particular substrate may only penetrate a few hundred mm into the
biomass, the DBL makes up a substantial part of the total distance a molecule has to diffuse. A change in
its thickness can have a dramatic effect on the overall substrate uptake rate of the biomass, since the
diffusive flux is proportional to the distance (dx, Equation 1) a molecule has to diffuse.
Inaccurate determination of the sample surface can have a similar effect on calculated rates. Its
determination can be done visually by observing the sample through a stereomicroscope as the sensor
approaches the sample. In samples with a relatively plane surface, it can be detected by parallel
measurement with a field irradiance sensor, measuring backscattering of near-infrared light directed from
the optical fiber onto the sample surface (Klimant et al., 1997).
In conclusion, microsensor measurements provide detailed information about the position and relative
activity of different processes in gradient environments and such analysis can explain the processes
underlying the overall transformation of compounds in greater detail than that which is obtained from
bulk liquid measurements alone. Commercial availability has made the technology more accessible to the
non-specialist research group, and further developments continue to extend the range of compounds
and environments that can be studied. Examples of their applications to activated sludge to measure
NOx, oxygen gradients in flocs, biofilms and aerobic granules can be found in Kishida et al. (2005),
Lemaire et al. (2008); Meyer et al. (2005); and Schramm et al. (1997).
12
Descriptions of activated sludge organisms
Elizabeth M. Seviour, Simon McIlroy and Robert J. Seviour
Since the earlier version of this book appeared, the phylogeny of what we think are some of the important
activated sludge microbial populations has become clearer, and we are now able to place many of them
taxonomically into recognized bacterial phyla (Nielsen et al., 2009). However, their functional importance
in many cases is still largely unknown. With some filaments, we know no more about them now than we
did when Eikelboom (1975) first described them from their microscopic features. Some are rarely seen in
plants around the world, and are often difficult to recognize on morphology alone. As we have already
discussed, for others rRNA sequence data reveal that a single filament morphotype may contain several
quite different phylotypes. We frequently see filaments which do not share the properties for any of those
given in the manuals of Eikelboom (2000) or Jenkins et al. (2004b), and so their ‘identification’ becomes
impossible, even though we often try to adjust to fit them into these schemes. This chapter and chapter 13
includes micrographs showing (where relevant) the diagnostic features of some of these filaments,
together with short summaries of their known features. We have imposed some order on how we deal
with these organisms, and taxonomically related members are now discussed together. Their previous
arrangement into similar morphotypes no longer seems appropriate, except by default for populations
where no phylogenetic data are available.
We have divided our organisms into FILAMENTOUS BACTERIA and NON-FILAMENTOUS
BACTERIA, mainly for reader convenience, and hope that this information, together with that presented
in other sections of this book, will be useful in assisting in their ‘recognition’. The nitrifying bacteria and
the PAO are not included here as these are discussed in detail in chapters 9 and 10. However, we have
included brief descriptions of some of the TFO which embrace putative members of some GAO and
possibly PAO.
DESCRIPTIONS OF THE FILAMENTOUS BACTERIA IN ACTIVATED

SLUDGE
Gram negative filaments
Alphaproteobacteria
(a) Candidatus ‘Alysiomicrobium bavaricum’
Organism Description (Figure 12.1 (a,b), see colour image section (chapter 13)).
Cell properties
Size ca. 1.5 m m
Shape irregular, discoid cells
Septation 1ve
Inclusions PHB
Motility ve
Trichome properties
Length 200 m m
Shape curled/coiled
Location free in mixed liquor
Attached growth absent
Branching absent
Sheath absent
Staining reactions
Gram stain ve
Neisser stain ve
Diagnostic features
Microscopic appearance of the ‘Nostocoida limicola’ morphotype.
COMMENTS
(i) Ecological distribution: detected by FISH in plants in Europe treating a range of industrial wastes;
its occurrence in plants treating domestic wastes not reported, although it is probably there.
(ii) Physiology: chemoorganoheterotroph, able to assimilate in situ a diversity of substrates
aerobically, including sugars and organic acids like acetate but not oleic acid, and a few organic
acids anoxically in the presence of both nitrate and nitrite, but none anaerobically; assimilation
patterns differ with phylotype, but only a few plants examined; no exoenzymatic activity,
suggesting preference for soluble substrates.
(iii) Systematics: a new genus in the Alphaproteobacteria.
(iv) FISH probes: available (see Table 6.4).
(v) Other: not yet grown in pure culture.
(vi) REFS: Kragelund et al. (2006); Levantesi et al. (2004; 2006a).
(b) Candidatus ‘Alysiosphaera europea’

Organism Description (Figure 12.1 (e,f), see colour image section (chapter 13)).
Descriptions of activated sludge organisms 455
Cell properties
Size 1.1–1.6 m m
Shape discoid/rounded
Septation 1ve
Inclusions PHB
Motility ve
Trichome properties
Length 200 m m
Shape curled
Branching absent
Sheath absent
Staining reactions
Gram stain variable
Neisser stain 1ve
Diagnostic features
Microscopic appearance of ‘Nostocoida limicola’ morphotype.
COMMENTS
(i) Ecological distribution: detected by FISH in plants treating a wide range of both industrial and
domestic wastes.
(ii) Physiology: chemoorganoheterotroph, assimilating a range of substrates aerobically including
sugars and acetate, but not oleic acid, and a few anoxically with nitrate, but none anoxically with
nitrite or anaerobically; only a few plants examined in this way; no exoenzymatic activity.
(iii) Systematics: a novel genus in the Alphaproteobacteria.
(v) Other: grown in pure culture and may be a common filament in activated sludge systems.
(vi) REFS: Kragelund et al. (2006); Levantesi et al. (2004, 2006a).
(c) Candidatus ‘Combothrix italica’

Organism Description (Figure 12.1 (k,l), see colour image section (chapter 13)).
Diagnostic features Microscopic appearance of ‘Nostocoida limicola’ morphotype, and sharing similar
morphological features to the other alphaproteobacterial N.limicola morphotypes described here.
(i) FISH probe sequence: available.

(ii) REFS: Levantesi et al. (2004).
(d) Candidatus ‘Monilibacter batavus’

Organism Description (Figure 12.1 (c,d), see colour image section (chapter 13)).
Cell properties
Size 1.3–2.0 m m
Shape discoid
Septation 1ve
Inclusions PHB, PolyP
Motility ve
Trichome properties
Length <200 m m
Shape curled
Branching absent
Sheath absent
Staining reactions
Gram stain variable
Neisser stain 1ve
Diagnostic features
COMMENTS
(i) Ecological distribution: detected by FISH in plants treating a wide range of industrial wastes and
are often dominant in EBPR plants in Australia.
(ii) Physiology: chemoorganoheterotroph, but only assimilating organic acids including acetate and
propionate (but not oleic acid) aerobically, and acetate weakly anoxically with both nitrate and
nitrite, but none anaerobically; only a few plants examined in this way; no exoenzymatic activity.
(iii) Systematics: a member of cluster 3 Defluviicoccus.
(v) Other: not yet grown in axenic culture.
(vi) REFS: Kragelund et al. (2006); Levantesi et al. (2004; 2006a; Nittami et al., 2009).
(e) Candidatus ‘Sphaeronema italicum’

Organism Description (Figure 12.1 (g,h), see colour image section (chapter 13)).
Cell properties
Size 0.8–1.4 m m
Shape discoid/rods
Septation 1ve
Motility ve
Trichome properties
Length 200 m m
Shape curled

Branching absent
Sheath absent
Staining reactions
Gram stain ve
Neisser stain 1ve
Diagnostic features
COMMENTS
(i) Ecological distribution: detected by FISH in plants treating wide range of industrial wastes, but
not reported yet in those treating domestic wastes.
(ii) Physiology: chemoorganoheterotroph, but assimilating aerobically in situ a limited range of organic
acids including acetate, and in one strain only oleic acid, and acetate weakly anoxically with both
nitrate and nitrite; none assimilated anaerobically; patterns differ between phylotypes; only a few
plants examined; no exoenzymatic activity.
(iii) Systematics: a novel genus in the Alphaproteobacteria.
(iv) FISH probe: available (see Table 6.4).
(v) Other: not grown in axenic culture.
(vi) REFS: Kragelund et al. (2006); Levantesi et al. (2004; 2006a).
(f) Meganema perideroedes

Organism Description (Figure 12.1 (m,n), see colour image section (chapter 13)).
Cell properties
Size 1.5–2.2 m m
Shape discoid
Septation 1ve
Motility ve
Trichome properties
Length <500 m m
Shape curled
Branching absent
Sheath absent
Staining reactions
Gram stain variable
Neisser stain 1ve
Diagnostic features Features of type 021N morphotype and presence of gonidia and rosettes in some,
but no So granules.
COMMENTS
(i) Ecological distribution: detected by FISH in plants treating industrial wastes, but its occurrence
in those treating domestic wastes is unknown.
(ii) Physiology: chemoorganoheterotroph, assimilating in situ a wide range of substrates aerobically
and acetate, propionate and glucose anoxically with both nitrate and nitrite; lipase activity
detected.
(iii) Systematics: a validly named organism in the Methylobacterium/Xanthobacter group of the
Alphaproteobacteria, and cultures deposited in culture collections. Described earlier by
Howarth et al. (1999) as type 021N, strain Ben 47.
(iv) FISH probes: two available that should be used together (see Table 6.4).
(v) Other: several strains now grown in axenic culture, and extensively characterized; two
morphotypes recognized which differ in their filament diameters and Gram staining reactions.
(vi) REFS: Kragelund et al. (2006); Levantesi et al. (2004; 2006a); Thomsen et al. (2006a).
The phylogenetic relationships between these alphaproteobacterial filaments are shown in

Figure 12.2.
Thiothrix fructosivorans, L79962

Thiothrix sp. str. CT3, AF148516
'Thiothrix ramosa', U32940
Thiothrix unzii, L79961
Thiothrix nivea, L40993
Thiothrix defluvii, AF127020
Thiothrix flexilis, AB042545
Gamma-
Thiothrix disciformis, AB042532
Thiothrix eikelboomii, L79965
Acinetobacter calcoaceticus, M34139
Eikelboom type 1863 str. Ben59, X95305
Acinetobacter johnsonii, X81663
Moraxella osloensis, AF005190
Beggiatoa alba, AF110274
Uncultured filamentous activated sludge clone, AY322151
Curvibacter gracilis, AB109889
Eikelboom type 0803 str. Ben04B, X86071
Beta-
Acidovorax delafieldii, AF078764

Eikelboom type 1701 str. RC2, L79964
Sphaerotilus natans, Z18534
Zoogloea ramigera, X74913
Defluviicoccus vanus, AF179678
Candidatus 'Monilibacter batavus', AY590701
Candidatus 'Sphaeronema italicum', AY428765
Azospirillum brasilense, X79739
Alpha-
Candidatus 'Alysiosphaera europaea', AY428766

Candidatus 'Alysiomicrobium bavaricum', AY428762
Candidatus 'Combothrix italica', AY590698
Methylosinus sporium, AJ458489
Meganema perideroedes, AY170119
Meganema perideroedes, AF180468
0.10
Figure 12.2. Maximum likelihood phylogenetic tree of the 16S rRNA gene sequences of the filamentous
Proteobacteria in activated sludge and related sequences. All sequences were at least 1000 b.p. long. The
scale bar corresponds to 0.1 substitutions per nucleotide position.
Betaproteobacteria
(a) Sphaerotilus natans
Organism Description (Figure 12.3, see colour image section (chapter 13)).
Cell properties
Size 1.2–2.5 3 2–10 m m
Shape rod-rectangular
Septation 1ve, and indentations obvious
Inclusions PHB
Motility ve, but motile gonidia are formed
Trichome properties
Length 200–1000 m m or more
Shape straight or slightly bent
Location radiate from within flocs
Attached growth rare but some dispute in the literature
Branching distinctive false branching in nutritionally poor medium
Sheath present, but mutants known which produce no sheath
Staining reactions
Gram stain ve
Neisser stain ve
Diagnostic features
False branching and gonidia, and sheath clearly visible in portions of trichomes devoid of cells.
COMMENTS
(i) Ecological distribution: once thought to be the main causative organism of bulking, but
microscopy- based surveys often suggest it is infrequently present and if so, associated with
nutrient deficiency in plants.
(ii) Physiology: chemoorganoheterotrophic aerobic organism, but can grow at very low levels
of oxygen, which encourage PHB synthesis; no in situ ecophysiological data available, but
data from axenic cultures suggest it can use a wide range of substrates to support growth.
(iii) Systematics: only one species currently recognized (S. natans), and 16S rRNA sequences of
readily cultured isolates from many sources all very similar. Most closely related phylogenetically
to type 1701.
(iv) FISH probes: available (see Table 6.4) and target type 1701 too.
(v) Other: easily identified in biomass.
(vi) REFS; Howarth et al. (1998); Kämpfer and Spring (2005); Wagner et al. (1994a).
(b) Type 1701

Organism Description (Figure 12.4, see colour image section (Chapter 13)).
Cell properties
Size 0.6–1 3 1.2–6 m m
Shape rods with rounded ends
Septation 1ve and indentations clearly visible

Inclusions PHB
Motility ve
Trichome properties
Length 20–200 m m
Shape straight, sometimes curved or bent
Location within flocs or extending into mixed liquor
Attached growth extensive
Branching false branching rarely seen
Sheath close fitting sheath visible
Staining reactions
Gram stain ve
Neisser stain ve
Diagnostic features
Sheath and perpendicularly attached cells.
COMMENTS
(i) Ecological distribution; reported as a major filament in some but not all countries in early
microscopy based surveys, and possibly in plants operating at low dissolved oxygen levels.
(ii) Physiology: chemoorganoheterotroph, but no in situ ecophysiological data available; in pure
culture, grows slowly.
(iii) Systematics; unknown, although 16S rRNA sequence and FISH data suggest strain RC2 is closely
related to S. natans, but whether all are is not clear; availability of pure cultures, which vary
markedly in their phenotype, should allow this to be addressed.
(iv) FISH probes: responds to that for S. natans (see Table 6.4), and some are targeted with probes
designed for Aquaspirillum/Curvibacter.
(v) Other: readily recognized in biomass samples.
(vi) REFS; Howarth et al. (1998); Kämpfer and Spring (2005); Thomsen et al. (2006b); Wagner et al.
(1994a).
(c) Type 0803

Cell properties
Size 0.8–1.5 m m
Shape square/rectangular
Septation 1ve but indentations not readily seen
Inclusions incidental polyP granules in pure cultures
Motility ve
Trichome properties
Length 50–300 m m
Shape straight
Location free in mixed liquor, but can extend from floc
Branching absent
Sheath absent
Staining reactions
Gram stain ve
Neisser stain usually ve
Diagnostic features
Few useful identificatory features, but can form thick bundles.
COMMENTS
(i) Ecological distribution: found widely in microscopy-based surveys in the bulk liquid and foams
as one of the dominating filaments in plants around the world.
(ii) Physiology: largely unknown.
(iii) Systematics: the 16S rRNA sequence of a single isolate grown in axenic culture in Australia
places it in the Betaproteobacteria, but existence of other phylotypes is likely since this
morphotype in European plants does not respond to a FISH probe designed against this sequence.
More effort should be directed at resolving its phylogeny.
(iv) FISH probes: available against the cultured isolate (Table 6.4).
(v) Other: difficult to ‘identify’ microscopically since it has few distinctive features; can be confused
with type 0914, although its filaments are thinner and those in bundles tend to align in parallel
while those in type 0914 often traverse each other.
(vi) REFS: Bradford et al. (1996); Jenkins et al. (2004b).
(d) Zoogloea spp.

Cell properties
Size 1.0–1.3 3 2.1–3.6 m m
Shape straight to slightly curved rods
Septation not applicable (na)
Inclusions PHB
Motility 1ve with single polar flagellum
Trichome properties na
Staining reactions
Gram stain ve
Neisser stain unknown
Diagnostic features
Formation of a zoogloeal matrix, often with distinctive finger-like projections.
COMMENTS
(i) Ecological distribution: commonly seen by microscopy in plants and may play an important role
in floc generation, providing the EPS matrix around which the floc forms; excessive growth can
lead to viscous bulking?
(ii) Physiology: aerobic chemoorganoheterotrophs with a respiratory mode of metabolism; able to

utilize a wide range of substrates.
(iii) Systematics: 16S rRNA sequence analyses have resolved the taxonomy of this organism; clearly a
member of the Betaproteobacteria, although speciation is still controversial, with two, Z. ramigera
and Z. resiniphila currently recognized.
(v) Other: can exist with either an amorphous or finger like morphology.
(vi) REFS: Roselló–Mora et al. (1995); Shin et al. (1993); Unz (2005).
The phylogenetic relationships between these betaproteobacterial filaments are shown in

Figure 12.2.
Gammaproteobacteria
(a) Beggiatoa
Cell properties
Size highly variable, 1–50 3 2–10 m m
Shape discoid/cylindrical
Septation visible
Inclusions sulphur granules, and PHB and PolyP reported
Motility rapid gliding
Trichome properties
Length often reaching 1 cm and containing several thousand individual cells
Shape curved but flexes during gliding movement
Location moves between flocs
Attached growth rarely seen
Branching absent
Sheath absent
Staining reactions
Gram stain ve
Neisser stain ve
Diagnostic features
Gliding motility and refractile sulphur granules seen by phase contrast microscopy.
COMMENTS
(i) Ecological distribution: not commonly seen in plants and always present in small numbers;
associated with septicity in plants and often increases in numbers in stored biomass samples.
(ii) Physiology: chemoorganoheterotroph with strictly respiratory metabolism; aerobic/microaer-
ophilic but no in situ physiological studies reported; can possibly behave mixotrophically and
chemolithoautotrophically depending on conditions.
(iii) Systematics: one recognized species B. alba, but the taxonomic diversity of activated sludge
isolates is likely to be higher, and several species may exist.
(iv) FISH probe sequence: not available.
(v) Other: readily cultured, and large size makes it easy to recognize.
(vi) REFS: Strohl (2005).
b) Type 1863
Cell properties
Size 0.8 3 1.5 m m
Shape ovoid or coccoid in plants
Septation clearly visible
Inclusions absent under the microscope
Motility ve
Trichome properties
Length <150 m m
Shape coiled or tangled
Location free in bulk liquid between flocs
Branching absent
Sheath absent
Staining reactions
Gram stain ve
Neisser stain ve
Diagnostic features
Individual ‘sausage’ shaped cells are easily seen in filaments.
COMMENTS
(i) Ecological distribution: in FISH based studies, not often a dominating filament in biomass
samples; favored by low F/M short sludge age (55 days) plants?; found in foams too.
(ii) Physiology: chemoorganoheterotroph, but no in situ physiological data available.
(iii) Systematics; taxonomically diverse, but many are Acinetobacter spp (A. johnsonii); other isolates
appear to be members of the closely related genus Moraxella, or the ‘Bacteroidetes’.
(iv) FISH probes: some respond to that designed against Acinetobacter spp.
(v) Other: may also exist in activated sludge as cells in pairs or short chains.
(vi) REFS: Rossetti et al. (1997b); Seviour et al. (1997); Wagner et al. (1994c).
(c) Type 021N

Cell properties
Size variable 0.6–2.2 3 4–7 m m
Shape variable, but can be discoid, barrel-shaped, cuboidal, bead like or swollen in appearance
Inclusions sulphur granules often present, but PHB and polyP may also be seen
Motility ve, although gonidia which glide may be formed in some
Trichome properties
Length variable, 100–200 m m, but up to 500 m m, depending on culture conditions
Shape variable
Location extends from floc
Branching absent
Sheath has been reported in some
Staining reactions
Gram stain variable, mostly gram negative but with gram positive regions in the trichomes
associated with location of sulphur granules
Neisser stain variable
Diagnostic features
Gonidia, motile by gliding, rosettes and knots also seen in some species.
COMMENTS
(i) Ecological distribution: its importance in activated sludge plants from microscopy and FISH
surveys varies between countries, and is more commonly seen in say US and Japan than in
Australia, although the reasons why are not clear; associated with low F/M plants experiencing
septicity and nutrient deficiencies; has been seen in foams.
(ii) Physiology: chemoorganoheterotrophs, but chemolithoautotrophic and mixotrophic behaviour has
been found in both axenic cultures and in situ with some strains (e.g. C3).
(iii) Systematics: 16S rRNA sequence data show that most type 021N isolates are members of several
new species in the genus Thiothrix, including T. eikelboomii, T. unzii, T. fructivorans, T. defluvii
(Thiothrix 1?) , T. disciformis and T. flexilis; strain Ben 47 is related (99% similar) to a member of
the Alphaproteobacteria, closely related to Meganema perideroedes.
(iv) FISH probes: several have been designed for different taxonomic groups (see Table 6.4).
(v) Other; unclear how Eikelboom Thiothrix type II is related to type 021N.
(vi) REFS: Aruga et al. (2002); Howarth et al. (1999); Kanagawa et al. (2000); Rossetti et al. (2003);
Unz and Head (2005).
(d) Thiothrix 1
Cell properties
Size 1.4–2.2 3 3–5 m m
Inclusions sulphur and polyP granules sometimes seen
Motility apical gonidia with flagella may be released
Trichome properties
Length 100–500 m m
Shape straight/smoothly curved
Location extend from floc surface
Attached growth usually absent
Branching absent
Sheath present
Staining reactions
Gram stain ve, but may appear 1ve with large amounts of sulphur granules inside trichomes
Neisser stain ve
Diagnostic features
Sulphur granules and distinctive rosettes, but neither always present.
COMMENTS
(i) Ecological distribution: reported in microscopy-based surveys of plants in most countries, and favoured
by influents rich in reduced sulphur compounds or having nutrient deficiency or in low F/M plants.
(ii) Physiology: probably metabolically flexible, growing heterotrophically and autotrophically, and
mixotrophically, depending on conditions.
(iii) Systematics: uncertain where it lies within the genus Thiothrix, but 16S rRNA gene sequence data
of one cultured isolate places it into a new species, T. defluvii.
(iv) FISH probes: available for Thiothrix, but not deliberately targeting this morphotype? (Table 6.4).
(v) Other: similar to Thiothrix II, but trichome is thicker, and microscopically distinguishable from
type 021N.
(vi) REFS: Howarth et al. (1999); Unz and Head (2005).
(e) Thiothrix II
Cell properties
Size 0.8–1.4 3 1–2 m m
Shape rectangular
Septation clearly visible, no indentations
Inclusions sulphur granules extensive and polyP?
Motility motile gonidia may be released
Trichome properties
Length 50–100 m m
Shape straight/smoothly curved
Location extending from floc surface
Branching absent
Sheath present but difficult to see
Staining reactions
Gram stain ve
Neisser stain ve
Diagnostic features
Rosettes more commonly seen with Thiothrix II than with Thiothrix I.
COMMENTS
(i) Ecological distribution: similar to Thiothrix I, although some microscopy based survey data do
not distinguish between Thiothrix I and II.
(ii) Physiology: deposition of intracellular sulphur granules suggest autotrophy and mixotrophy,
depending on the conditions.
(iii) Systematics: its relationship to other Thiothrix species and type 021N is unclear, as this
morphotype has never (knowingly) been grown in pure culture and its 16S rRNA sequenced.
(iv) FISH probes: available for Thiothrix spp. but not known which if any of these target this
morphotype; responds to the probe for Gammaproteobacteria.
(v) Other: its phylogenetic relationship to the type 021N morphotype is unclear.
(vi) REFS: Howarth et al. (1999); Unz and Head (2005).
The phylogenetic relationships between these gammaproteobacterial filaments are shown in Figure 12.2.
‘CHLOROFLEXI ’
(a) Type 1851/ ‘Kouleothrix aurantiaca’
Cell properties
Size 0.5–1.0 m m
Shape rectangular
Septation sometimes visible
Inclusions PHB?
Motility ve (although slow gliding has been reported)
Trichome properties
Shape straight or curved
Location extending from flocs or in bulk liquid
Attached growth present (but not always) and perpendicular to surface
Branching absent
Sheath present but not readily seen
Staining reactions
Gram stain variable, but often weakly positive
Neisser stain ve
Diagnostic features
Bleached appearance of cells, and thin filaments often in thick bundles.
COMMENTS
(i) Ecological distribution: found incidentally in bulking and foaming plants (including EBPR
plants) treating domestic and industrial wastes in many countries.
(ii) Physiology: chemoorganoheterotroph, and growing mainly on sugars in pure culture; in situ
physiological data suggest obligately aerobic organisms assimilating sugars and bacterial cell wall
debris, and diverse ectoenzyme production suggests polymeric substrates utilized too.
(iii) Systematics: current isolates are all members of the ‘Chloroflexi’, but phylogenetically different;
for type 1851, closest relative (84% similarity) is the phenotypically different Roseiflexus
castenholtzii, a thermotolerant pigmented gliding organism; for K. aurantiaca, strains are only
about 93–95% similar to type 1851.
(iv) FISH probes: have been designed for type 1851 and ‘K. aurantiaca’ (Table 6.4).
(v) Other: other ‘Chloroflexi’ filaments with morphologies different to type 1851 are seen in
activated sludge plants, and these may be important in protein degradation.
(vi) REFS: Beer et al. (2002); Kohno et al. (2002); Kragelund et al. (2007a); Xia et al. (2007).
(b) ‘Nostocoida limicola’ morphotype

Cell properties (from pure culture of single isolate)
Size 1.0–1.2 m m
Shape ovoid/discoid
Septation easily visible
Inclusions PolyP?
Motility ve
Trichome properties
Length >100 m m
Shape coiled
Location extending from floc into bulk liquid
Branching absent
Sheath absent
Staining Reactions
Gram stain usually Gram 1ve, but can be Gram variable
Neisser stain ve but with 1ve granules
Diagnostic features
Possess the ‘Nostocoida limicola’ II morphotype.
COMMENTS
(i) Ecological distribution: only found by FISH in large numbers in plants treating domestic wastes
in Germany; not found in plants treating paper mill or petrochemical wastes; its global importance
not known.
(ii) Physiology: chemoorganoheterotroph, but different to the other ‘N. limicola’ phylotypes, in
having complex growth requirements? No in situ data.
(iii) Systematics: novel member of the ‘Chloroflexi’.

(v) Other: Not able to grow at low nutrient concentrations?
(vi) REFS: Schade et al. (2002).
(c) Type 0092

Cell properties
Size 0.5–0.8 3 1–1.5 m m
Shape rectangular
Septation not readily seen
Inclusions no sulphur or PHB granules
Motility ve
Trichome properties
Length 10–60 m m
Shape straight/irregularly curved
Location within and extending from flocs
Branching absent
Sheath absent
Staining Reactions
Gram stain ve
Neisser stain þve filament
Diagnostic features
Neisser stain where entire trichome stains distinctively lilac, and often the only means to detect its
presence.
COMMENTS
(i) Ecological distribution: from microscopy based survey data, important bulking filament in many
countries; often dominates in EBPR plants with low F/M; seen in foams but role in their formation
and stabilization is unclear.
(ii) Physiology; unknown.
(iii) Systematics: claims that it has been grown in pure culture must be treated cautiously since its
identity was not confirmed, and the 16S rRNA sequence data suggesting it belongs to the
‘Bacteroidetes’ may need reviewing, as FISH shows it belongs to the ‘Chloroflexi’ (Speirs et al.,
2009).
(iv) FISH probes: responds to the CFX1223 and GNSB941 probes (Bjornsson et al., 2002) in
Australian plants, and specific probes available for two type 0092 morphological variants (Speirs
et al., 2009).
(v) Other: another common filament whose identity is you known.
(vi) REFS: Bradford et al. (1996); Speirs et al. (2009); Ramothokang et al. (2003).
The phylogenetic relationships between these ‘Chloroflexi’ filaments are shown in Figure 12.15.
Eikelboom type 1851 str. Ben52, AY063760

'Kouleothrix aurantiaca', AB079638
Roseiflexus castenholzii, AB041226
Chloroflexus aurantiacus, D38365
Chloroflexus aggregans, D32255
Chloroflexi
Filamentous activated sludge isolate str. Ben15, X86447
Herpetosiphon aurantiacus, M34117
Uncultured activated sludge clone A31, AF234694
Anaerolinea thermophila, AB046413
Uncultured activated sludge clone H1, AF234710
Uncultured Eikelboom type 0092 clone A26, AB445103
Uncultured activated sludge clone SBR2037, X84576
Dehalococcoides ethenogenes, AF004928
Bacterioidetes
Chryseobacterium balustinum, M58771
Saprospira grandis, AB088636
Candidatus ‘Magnospira bakii’, AF087057
Filamentous activated sludge isolate str. Iso10B, DQ232755
Haliscomenobacter hydrossis, M58790
Uncultured activated sludge clone TNO5, DQ232756
Planctomycetales
Singulisphaera acidiphila, AM850678
'Nostocoida limicola' III str. Ben225, AF244752
Isosphaera pallida, X64372
0.10
Figure 12.15. Phylogenetic tree of the 16S rRNA gene sequences of the ‘Chloroflexi ’, TM7, Planctomycetes
and Bacteroidetes filamentous bacteria characterized in activated sludge, constructed with the maximum
liklihood algorithm. All sequences were at least 1100 bp long. The scale bar corresponds to 0.1 substitutions
per nucleotide position.
GRAM POSITIVE FILAMENTS

Firmicutes (Low mol% GþC)
(a) Trichococcus flocculiformis
Cell properties
Size highly variable in pure culture but in situ 1–1.5–1–2.5 m m
Shape highly variable in pure culture, but in situ as coccoid cells in chains
Septation clearly visible and indentations readily seen
Inclusions refractile in older cultures?
Motility ve
Trichome properties
Length variable, but can reach >100 m m
Shape curved or straight
Location extending into bulk liquid
Branching absent
Sheath absent
Staining Reactions
Gram stain 1ve
Neisser stain not reported
Diagnostic features
Trichome appearance and occasional presence of knots.
COMMENTS
(i) Ecological distribution: microscopy based surveys report its presence in bulking plants in Europe
and Australia, so probably widely distributed.
(ii) Physiology: anaerobic chemoorganoheterotroph with heterolactic acid fermentative metabolism.
(iii) Systematics: Member of low GC mol% Firmicutes, and phylogenetically closely related to
Lactosphaera, Ruminococcus and Carnococcus spp, which are reclassified as other Trichococcus
spp.
(v) Other: has been suggested to be the same organism as the N. limicola I morphotype (sensu
Eikelboom and van Buijsen 1983), which may be polythetic; not included in identification manuals.
(vi) REFS: Liu et al. (2000, 2002); Scheff et al. (1984).
(b) Bacillus spp. (including Bacillus cereus, Bacillus mycoides, Bacillus funiculus)
Cell properties
Size variable
Shape rods with rounded ends sometimes containing endospores
Septation visible
Inclusions endospores and PHB?
Motility flagella
Trichome properties
Length variable but often very long
Shape straight/curved
Location in bulk liquid
Branching absent
Sheath absent
Staining Reactions
Gram stain 1ve
Neisser stain ve
Diagnostic features
Endospore formation in rod shaped cells in long chains visible under phase contrast.
COMMENTS
(i) Ecological distribution: probably very common but not often recorded in microscopy based surveys.
(ii) Physiology: aerobic chemoorganoheterotrophs, with capacity to denitrify; resting structures
(endospores) provide survival capacity.
(iii) Systematics: in the Firmicutes but taxonomic diversity of Bacillus spp. in activated sludge not
resolved.
(iv) FISH probes: available but not applied to activated sludge samples.
(v) Other: easy to identify and culture.
(vi) REFS: Ajithkumar et al. (2002); Seviour et al. (1999a).
The phylogenetic relationships between these Firmicutes filaments are shown in Figure 12.17.
Streptococcus suis, AF009497
Firmicutes
'Nostocoida limicola' I str. Ben204, AF244377
Trichococcus flocculiformis, AJ306611
Bacillus funiculus, AB049195
Candidatus 'Microthrix parvicella', X89774
Candidatus 'Microthrix calida', DQ147277
Tetrasphaera elongata, AB030911
Tetrasphaera australiensis, AF125091
Tetrasphaera nostocoidensis, DQ007320
Tetrasphaera japonica, AF125092
Tetrasphaera jenkinsii, DQ007321
Tetrasphaera veronensis, Y14595
Dietzia maris, X79290
Tsukamurella sunchonensis, AF150494
Tsukamurella pseudospumae, AY238513
Tsukamurella spumae, Z37150
Tsukamurella paurometabola, X80628
Millisia brevis, AY534742
Gordonia malaquae, AM406674
Actinobacteria
'Gordonia spumae', AJ251724
Gordonia amarae, X80635
Gordonia defluvii, AY650265
Skermania piniformis, Z35435
Nocardia takedensis, AB158277
Nocardia asteroides, X80606
Nocardia otitidiscaviarum, X80611
Nocardia farcinica, X91041
Rhodococcus equi, X80614
Rhodococcus zopfii, X81934
Rhodococcus rhodochrous, X79288
Rhodococcus ruber, X80625
Rhodococcus coprophilus, X80626
Rhodococcus erythropolis, X81929
Rhodococcus globerulus, X80619
Mycobacterium phlei, AF480603
Mycobacterium fortuitum, AY457066
0.10
Figure 12.17. Phylogenetic tree of the 16S rRNA gene sequences of the Firmicutes and Actinobacteria
Gram positive filaments characterized in activated sludge, using the maximum likelihood algorithm. All
sequences were at least 1300 bp long. The scale bar corresponds to 0.1 substitutions per nucleotide position.
Actinobacteria (High mol% GþC)

(a) Candidatus ‘Microthrix calida’
Cell properties
Size 0.3–0.7 m m
Shape not readily seen, but trichomes contain swollen spherical cells
Septation not readily seen in situ
Inclusions lipophilic granules in swollen cells and rest of trichome
Motility ve
Trichome properties
Length >100 m m
Shape ‘spaghetti-like’ tangled
Location extending from flocs
Branching absent
Sheath absent
Staining reactions
Gram stain 1ve
Neisser stain not reported
Diagnostic features
Can be mistaken for Candidatus ‘M. parvicella’, but filaments thinner, so requires FISH analyses for
precise identification.
COMMENTS
(i) Ecological distribution: only detected so far by FISH in plants in Europe treating diverse
industrial wastes.
(ii) Physiology: slow growing and in some ways very similar to Candidatus ‘M. parvicella’ in
preferentially utilizing long chain fatty acids under aerobic anoxic (with nitrate) and anaerobic
conditions, and storing these as lipid granules; however grows at higher temperatures; can
assimilate nutrients at very low concentrations (K-strategy?).
(iii) Systematics: several isolates grown in pure culture, and all phylogenetically most closely related
to Candidatus ‘M. parvicella’ in the Actinobacteria.
(iv) FISH probes: available to distinguish it from Candidatus ‘M. parvicella’ (see Table 6.4).
(v) Other; probably mistaken for Candidatus ‘M. parvicella’ in plants operating at higher
temperatures, but filaments noticeably thinner.
(vi) REFS: Levantesi et al. (2006b).
(b) Candidatus ‘Microthrix parvicella’

Cell properties
Size 0.4–0.85 m m
Shape not readily apparent but trichomes contain swollen cells
Septation not readily seen, but are present
Inclusions polyP granules give trichomes a beaded appearance
Motility ve
Trichome properties
Length >200m mm
Shape ‘spaghetti-like’ tangles
Location extending from flocs
Branching absent
Sheath absent
Staining reactions
Gram stain 1ve
Neisser stain 1ve granules in trichomes
Diagnostic features
Can be mistaken for Candidatus ‘M. calida’, but filaments thicker; otherwise morphotype easily recognised
microscopically.
COMMENTS
(i) Ecological distribution: causes episodes of both foaming and bulking in plants around the world;
seems to be more prevalent in those in colder climates (bulk liquid temperatures 520– C), and
EBPR systems with anaerobic zones.
(ii) Physiology: a specialized ‘feeder’ and in situ MAR data show it grows selectively on long chain
fatty acids (but not always in pure culture); can survive starvation conditions under anaerobic and
anoxic, but not aerobic conditions, for long periods because of high storage (lipid) capacity;
microaerophilic, and high DO levels possibly toxic; chemoorganoheterotroph.
(iii) Systematics: an unusual member of the Actinobacteria.
(iv) FISH probes: available (see Table 6.4), and can distinguish between this filament and
Candidatus ‘M. calida’.
(v) Other: very slow growing organism in activated sludge, stores polyP, but phenotype not that of a
PAO according to the accepted definition (see chapter 11); inter strain variations in physiology
not clear.
(vi) REFS: Blackall et al. (1996); Rossetti et al. (2005).
(c) Candidatus ‘Nostocoida limicola’

Organism Description (Figure 12.20, see colour image section (Chapter 13) and Figure 12.21).
Cell properties
Size 1.0–1.4 m m
Shape spherical/ovoid/discoid in situ, but highly variable in pure culture
Septation present and usually visible
Inclusions PolyP and PHB in pure cultures but not always present in situ
Motility ve
Trichome properties
Length <200 m m, and often much shorter
Shape coiled and bent
Location extending from flocs into bulk liquid
Figure 12.21. Candidatus ‘Nostocoida limicola’ SEMs showing morphological variations exhibited by
this filament in pure culture, ranging from swollen irregular coccoid cells to the typical filament morphology
seen in activated sludge samples depending on the culture conditions used (McKenzie et al., 2006).
Bar mark ¼ 2 mm.
Branching incidental branching has been reported but not in FISH defined trichomes
Sheath: absent
Staining reactions
Gram stain variable, with Gram negative regions in trichomes
Neisser stain variable but whole filament stains
Diagnostic features
Coiled and bent filaments, with often irregular discoid cells, and often Neisser positive.
COMMENTS
(i) Ecological distribution: recorded globally in microscopy based surveys of plants treating
domestic and industrial wastes, and possibly increasing in occurrence; causes bulking and
foaming?
(ii) Physiology: in pure culture can grow on a range of substrates aerobically, but no evidence that
they can denitrify; in situ FISH-MAR data differ especially in their ability to use acetate, and in
assimilating some substrates anaerobically and anoxically as well as aerobically.
(iii) Systematics: cultured isolates belong to several species in the genus Tetrasphaera in the
Actinobacteria.
(iv) FISH probes: available (see Table 6.4), but not all target individual species.
(v) Other: corresponds to the ‘Nostocoida limicola’ II morphotype of Eikelboom and van Buijsen
(1983).
(vi) REFS: Blackall et al. (2000); Liu and Seviour (2001); McKenzie et al. (2006).
(d) Gordonia amarae–like organisms

These include Gordonia, Nocardia, Rhodococcus, Dietzia, Tsukamurella, Mycobacterium and
Millisia spp.
Cell properties
Size variable
Shape irregular
Septation readily seen
Inclusions PolyP and PHB granules
Motility ve
Trichome properties
Length variable
Shape distinctive right angled branching of trichomes
Location in flocs and free in bulk liquid
Branching absent
Sheath absent
Staining Reactions
Gram stain 1ve, but often uneven
Neisser stain 1ve granules may be present but trichome stains negatively
Diagnostic features
Trichomes show right angled branching, although they may also fragment to single coccoid cells.
COMMENTS
(i) Ecological distribution: major morphotype seen in mixed liquor and especially foams in plants
around the world, and higher temperatures may favour their presence over other foaming filaments
like Candidatus ‘M. parvicella’; hydrophobic cells from synthesis of mycolic acids, and may be
how these cells form and stabilize foams; grow faster than many other filaments in pure culture.
(ii) Physiology: chemoorganoheterotrophs, able to utilize wide range of substrates; in situ studies
with G. amarae showed no obvious preference for hydrophobic substrates.
(iii) Systematics: ‘GALO’ describes a taxonomically diverse group of organisms likely to differ
markedly in their phenotypic properties; belong to different genera in the Corynebacterinae in the
Actinobacteria.
(iv) FISH probes: available for some but not all foaming GALO, and cells often require
permeabilization before FISH.
(v) Other: it is clear that many more GALO in activated sludge are yet to be isolated and
characterized.
(vi) REFS: Carr et al. (2005); Goodfellow and Maldonano (2006); Kragelund et al. (2007b);
Stainsby et al. (2002)
(e) Skermania piniformis or ‘pine tree’ like organisms (PTLO)

Cell properties
Size 1.0 3 1.0–2.0 m m
Shape irregular
Septation present but not always visible
Inclusions PolyP and PHB often detected
Motility ve
Trichome properties
Length variable
Shape ‘tree-like’ pattern of acutely angled branches
Location present in flocs and free in bulk liquid
Branching distinctive patterns, present as acutely angled branches, but branching angles vary
Sheath ve
Staining Reactions
Gram stain 1ve
Neisser stain 1ve granules sometimes seen in –ve trichomes
Diagnostic features
Branching pattern of acutely angled branches, although these angles and branching frequency vary in situ.
COMMENTS
(i) Ecological distribution: Once thought to be more common in foams in Australia; now recorded
in several other countries, so it probably is widely distributed.
(ii) Physiology: slow growing chemoorganoheterotroph; like G. amarae, in situ studies suggest it has
no preference for hydrophobic over hydrophilic substrates, and can assimilate substrates
anoxically in the presence of nitrate but not nitrite; substrate assimilation patterns differ to those
obtained with pure cultures, and may vary among trichomes within and between plants.
(iii) Systematics: originally placed in the genus Nocardia as N. pinensis, but now reclassified as new
genus in the family Gordoniaceae; all isolates fall into the one species despite differences in
branching patterns.
(iv) FISH probes: available, but permeabilization required (see Table 6.4).
(v) Other: easily ‘identified’ in activated sludge, but suggestions other Mycolata may share this
morphotype.
(vi) REFS: Eales et al. (2005, 2006); Goodfellow and Maldonado (2006); Soddell and Seviour (1994,
1998).
(f) Millisia brevis
Cell properties
Size variable
Shape filaments fragment to coccoid elements as part of life cycle
Septation present but not visible
Inclusions polyP granules
Motility ve
Trichome properties
Length variable
Shape branched filaments which fragment into cocci
Location unclear
Branching rudimentary right-angled branching
Sheath ve
Staining reactions
Gram stain 1ve/variable
Neisser stain ve
Diagnostic features: short filaments with short branches, fragmenting to coccoid elements in stationary
phase.
COMMENTS
(i) Ecological distribution: original isolates were obtained from activated sludge foam in Australia,
but an organism with an identical 16S rRNA sequence has been isolated from a marine
invertebrate in Puerto Rica.
(ii) Physiology: chemoorganoheterotroph growing slowly in pure culture on a wide range of
substrates, with no obvious preference for hydrophobic compounds.
(iii) Systematics: a member of the mycolic acid producing bacteria in the family Gordoniaceae.
(iv) FISH probe sequence: not reported.
(v) Other: its frequency of occurrence in foams is not known as it has only been reported in
Australia, but FISH surveys would clarify this.
(vi) REFS: Soddell et al. (2006a).
The phylogenetic relationships between these actinobacterial filaments are shown in Figure 12.17.
Candidatus Division TM7

Type 0041
Cell properties
Size 1.0–1.9 3 1.0–2.3 m m
Septation visible in regions where attached growth is absent, otherwise masked
Inclusions polyP?
Motility ve
Trichome properties
Length 100–300 m m or longer
Shape smoothly curved
Location in flocs and forming inter-floc bridges
Attached growth abundant
Branching rare
Sheath present but not always easily seen under light microscope without staining
Staining reactions
Gram stain 1ve
Neisser stain intracellular 1ve granules sometimes seen
Diagnostic features
Cell shape and attached growth
COMMENTS
(i) Ecological distribution: from microscopy based survey data is often the dominant filament in
plants from around the world; seen in both foams and mixed liquor.
(ii) Physiology: chemoorganoheterotroph with simple nutritional requirements; in situ FISH/MAR
data suggest it is unable to utilize acetate aerobically, but assimilates several simple sugars and
amino acids; weak anaerobic uptake of glucose and galactose recorded; ecophysiology varied
between plants.
(iii) Systematics: the type 0041 morphotype is probably phylogenetically heterogenous; some fail to
respond to FISH probes targeting members of the TM7 division; others respond to FISH probes
targeting the ‘Chloroflexi’; the TM7 type 0041 have a typical Gram positive cell wall; the
taxonomic relationships between type 0041 and type 0675, difficult often to separate
microscopically, is unknown; whether type 0065 responds to the FISH probes designed against
TM7 members has not been reported; the only Gram positive sheathed filament?
(iv) FISH probes: available against members of the TM7 division (see Table 6.4).
(v) Other: attached bacteria have different ecophysiology to the type 0041 trichomes in assimilating
acetate, and represent largely unidentified bacteria.
(vi) REFS: Hugenholtz et al. (2001); Kragelund et al. (2007a); M}uller et al. (2007); Thomsen et al.
(2002); Xia et al. (2007; 2008b).
Planctomycetales
(a) Isosphaera spp. (‘Nostocoida limicola’ III?)
Cell properties
Size 1.6–2.0 m m
Shape spherical/discoid
Inclusions PHB?
Motility ve
Trichome properties
Length <200 m m
Shape coiled, curved or bent
Location often inside floc but can extend into bulk liquid
Branching absent
Sheath absent
Staining reactions
Gram stain variable and uneven
Neisser stain positive sometimes
Diagnostic features
Appearance of ‘poppet beads’.
COMMENTS
(i) Ecological distribution: seen incidentally by microscopy in many plants, but all isolates grown in
pure culture have come from those treating dairy wastes in Australia, where it dominated.
(ii) Physiology: little known, but probably an aerobic chemoorganoheterotroph.
(iii) Systematics: shown to be in the Planctomycetales, probably as new Isosphaera species; possess
many features considered unique to members of this Division.
(v) Other: those grown in pure culture considered by some to be the same organism as the
Nostocoida limicola III morphotype of Eikelboom and van Buijsen (1983).
(vi) REFS: Liu and Seviour (2001); Liu et al. (2001a).
The phylogenetic relationships of Isosphaera spp. are shown in Figure 12.15.
‘Bacteroidetes’
(a) Haliscomenobacter hydrossis
Cell properties
Size 0.35–0.45 3 3–5 m m
Shape rectangular, but not readily seen microscopically
Septation not obvious

Inclusions polysaccharide globules
Motility ve
Trichome properties
Length <100 m m
Shape straight or bent
Location always extends from floc
Attached growth often present
Branching absent
Sheath present but not easily seen
Staining Reactions
Gram stain ve
Neisser stain ve
Diagnostic features
Very thin needle like trichomes extending often in large numbers from flocs, giving them a ‘pin cushion’
appearance.
COMMENTS
(i) Ecological distribution: found from microscopy based surveys in most plants as an incidental
filament; rarely dominant; factors determining their presence there are largely unknown, but low
DO may be one.
(ii) Physiology: chemoorganoheterotrophs in pure culture; no in situ physiological data reported;
grow well on simple substrates but require vitamin B12 and thiamine; ecophysiology suggest these
use mainly sugars.
(iii) Systematics: once thought all isolates belong to the one species validly named as H. hydrossis in
the Bacteroidetes; strains are deposited in culture collections. However, probably phylogenetically
diverse.
(iv) FISH probe sequences: available (see Table 6.4).
(v) Other: often difficult to see except after staining because of its small diameter.
(vi) REFS: Kragelund et al. (2008); Mulder and Deinema (2006); van Veen et al. (1973).
(b) Candidatus ‘Magnospira bakii’

Cell properties
Size 0.8–2 m m
Shape bent rods
Septation not recorded
Inclusions not recorded
Motility 1ve by rotational gliding
Trichome properties
Length 10–30 m m
Shape corkscrew
Location in bulk liquid
Attached growth ve

Branching ve
Sheath ve but possess ‘slime belt’
Staining reactions
Gram stain not recorded
Neisser stain not recorded
Diagnostic features
Corkscrew shape and movement.
COMMENTS
(i) Ecological distribution: unknown but present probably in small numbers in many plants; FISH
probe availability should assist in determining its global distribution.
(ii) Physiology: unknown
(iii) Systematics: member of the ‘Bacteroidetes’ phylum, most closely related to Saprospira.
(iv) FISH probe sequence: available (see Table 6.4).
(v) Other: not been grown; is this organism the same as that ‘identified’ as Spirillum in the popular
identification manuals?
(vi) REFS: Snaidr et al. (1999).
The phylogenetic relationships between these Bacteroidetes filaments are shown in Figure 12.15.
FILAMENTOUS BACTERIA OF UNKNOWN TAXONOMIC AFFILIATION

(a) Type 0675
Cell properties
Size 0.5–1.0 3 0.7–2.5 m m
Septation visible in regions lacking attached growth
Inclusions PHB?
Motility ve
Trichome properties
Length 50–100 m m
Shape straight/curved
Location free and also extending from flocs
Attached growth abundant, although not completely covering trichome surface
Branching absent
Sheath present but not always readily seen
Staining reactions
Gram stain 1ve/variable
Neisser stain ve
Diagnostic features
Attached particles and cell shape.
COMMENTS
(i) Ecological distribution: similar to type 0041; they usually appear together in both foams and
mixed liquor.
(ii) Physiology: unknown.
(iii) Systematics: its taxonomic status and relationship to type 0041 are unknown.
(iv) FISH probes: not available.
(v) Other: the current distinction between type 0675 and type 0041, based on trichome dimensions is
not convincing, as this may vary with culture conditions; we prefer to group these together until
their taxonomy is resolved, but others separate them.
(vi) REFS: Jenkins et al. (2004b).
(b) Type 0411

Cell properties
Size 0.5–0.8 3 2–4 m m
Shape elongated rods
Septation clearly visible and indentations obvious
Inclusions absent
Motility ve
Trichome properties
Length 50–200 m m
Shape irregular/bent
Location extending from floc edge
Branching absent
Sheath not visible
Staining reactions
Gram stain ve
Neisser stain ve
Diagnostic features
Few, except appearance of cells in regular chains.
COMMENTS
(i) Ecological distribution; infrequently seen in microscopy based surveys of plants around the
world; not clear what might determine its presence.
(ii) Physiology: unclear, and no in situ data available.
(iii) Systematics: 16S rRNA sequence of a single isolate grown in Australia suggests it is a member of
the ‘Bacteroidetes’; this has not been confirmed elsewhere.

(v) Other: more effort should be directed at resolving its phylogeny.
(vi) REFS: Bradford et al. (1996).
(c) Type 0961

Cell properties
Size 0.8–1.5 3 2–4 m m
Shape rectangular
Septation visible
Inclusions absent
Motility ve
Trichome properties
Length 20–150 m m
Shape straight/bent
Location extending from flocs, but also seen free in bulk liquid
Branching absent
Sheath absent, but sheath present?
Staining Reactions
Gram stain ve
Neisser stain ve
Diagnostic features
Transparent cells with an empty appearance, sometimes in bundles
COMMENTS
(i) Ecological distribution: commonly seen in microscopy based surveys in plants around the world;
sometimes dominating; favoured in plants (EBPR?) with low F/M.
(iii) Systematics: claims for its isolation in pure culture, but not by micromanipulation, so uncertain
identification; no 16S rRNA sequence data.
(v) Other: clone library construction or RT-PCR from biomass samples where this filament is
dominant might assist in its identification.
(vi) REFS: Buali and Horan (1989); Jenkins et al. (2004b); Levantesi et al. (2004).
(d) Type 0914

Cell properties
Size 0.7–1.0 3 1.0 m m
Septation present but no indentations visible
Inclusions sulphur and PHB granules usually present and often polyP granules
Motility ve
Trichome properties
Length 50–200 m m
Shape straight or smoothly curved
Location often form interfloc bridges as bundles
Attached growth rarely seen
Branching absent
Sheath absent
Staining reactions
Gram stain ve, but 1ve if abundant sulphur granules are present
Neisser stain ve, but 1ve granules sometimes seen
Diagnostic features
Trichomes form long parallel bundles or cables, and PHB granules can obscure septa.
COMMENTS
(i) Ecological distribution: microscopy suggests this filament occurs in bulking plants and foams,
and especially in EBPR plants, but rarely as dominant filament.
(iii) Systematics: a member of the ‘Chloroflexi’ (McIlroy et al., unpublished).
(iv) FISH probe: unavailable, but fluoresces with GNSB914 and CFX1223 probes for the
‘Chloroflexi’.
(v) Other: this filament has never been grown in pure culture; efforts should be directed at resolving
its phylogeny with culture independent methods.
(e) Type 0581

Cell properties
Size 0.4–0.7 m m
Shape individual cells not discernable by microscopy
Septation not visible
Inclusions absent
Motility ve
Trichome properties
Shape variable
Location within floc and free in bulk liquid
Branching absent
Sheath absent
Staining Reactions
Gram stain ve
Neisser stain ve
Diagnostic features
Few, making its identification difficult.
COMMENTS
(i) Ecological distribution: uncommon, but may be missed because of difficulties in its
identification; suggestions from microscopy studies that it is more prevalent in low F/M plants.
(iii) Systematics: unknown.
(iv) FISH probes: unavailable.
(v) Other: looks superficially like Candidatus ‘M. parvicella’ under light microscopy, but is clearly
Gram and Neisser negative.
PAO, TFO AND PUTATIVE GAO IN EBPR SYSTEMS

(a) Candidatus ‘Accumulibacter phosphatis’
Organism Description (Figure.12.32, see colour image section (chapter 13)).
COMMENTS
(i) Ecological distribution: has been detected in both lab-scale and full-scale EBPR processes
around the world, so is certainly a major contributor to EBPR. Has the in situ phenotype expected
of a PAO, in being able to assimilate substrates (acetate and probably others) anaerobically to
synthesize PHA. This PHA is then metabolized aerobically for energy production, some of which
is used to assimilate phosphate and synthesize polyP. Little known about occurrence in other
habitats, but have been detected in freshwater and estuarine sediments.
(ii) Physiology: favored by anaerobic:aerobic cycling but evidence suggests it can also act as PAO
under both and anoxic:aerobic conditions, consistent with an ability to denitrify. Physiological
diversity may exist among strains of this organism.
(iii) Systematics: a member of the Betaproteobacteria, and increasing evidence that it may contain
several species.
(iv) FISH probes: available (Table 10.1), but these probably do not target individual species.
(v) REFS: Crocetti et al. (2000); Kong et al. (2004); Oehman et al. (2007); Peterson et al. (2008)
Seviour et al. (2003); chapter 10.
(b) Amaricoccus spp

COMMENTS
(i) Ecological distribution: TFO probably widely found in activated sludge systems, and has
never been detected elsewhere, but initial claims (Cech and Hartman 1993) that this organism is
a GAO responsible for EBPR failure in systems fed glucose seems very unlikely; does not
possess the GAO phenotype, since no ability to assimilate substrates and synthesize PHB
anaerobically.
(ii) Physiology: aerobic, synthesise large amounts of PHB from wide range of simple substrates, and
do not store polyP.
(iii) Systematics: in the Alphaproteobacteria, and four species currently recognized, all isolated from
activated sludge, A. kaplicensis (synonym Tetracoccus cechii), A. macauensis, A. tamworthensis
and A.veronensis.
(iv) FISH probe: available (Table 10.1) but not for individual species.
(v) Other: was Amaricoccus in fact the TFO dominating the community in the reactor fed glucose in
the study by Cech and Hartman (1993)?
(vi) REFS: Blackall et al. (1997); Cech and Hartman (1993); Maszenan et al. (1997, 2000, 2005a).
(c) Defluviicoccus spp.

COMMENTS
(i) Ecological distribution; TFO, originally isolated from a plant in the Czech republic treating
brewery wastes, but since seen dominating the communities of lab-scale anaerobic:aerobic SBR
and membrane bioreactors, and also detected in small numbers in full scale plants in Australia and
Denmark, but not Japan; their importance in deteriorating EBPR systems is uncertain.
(ii) Physiology: increasing evidence from in situ FISH-MAR data that these have the phenotype of a
GAO, in being able to assimilate acetate and propionate anaerobically to synthesize PHB, and not
assimilating phosphate aerobically into polyP; can also assimilate substrates aerobically, but the
assimilation patterns may vary with some (eg glucose).
(iii) Systematics: in subgroup 1 of the Alphaproteobacteria; one isolate, D. vanus has been obtained
in pure culture, but is different phylogenetically to Defluviicoccus clones obtained from lab scale
reactors; 16S rRNA sequence data divide these up into two clusters Cluster 1 and Cluster 2, with
those in Cluster 2 more commonly detected in full scale plants; also seen in a lab-scale
continuously aerated SBR EBPR process; Candidatus ‘Monilibacter spp’ (Figure 12.1) are
members of Cluster 3 Defluviicoccus.
(iv) FISH probes: several available to detect members of the two Clusters (Table 10.1).
(v) Other: how do these compete with the PAO, with whom they are always found, for substrates in
EBPR systems?
(vi) REFS: Burow et al. (2007); Maszenan et al. (2005a); Meyer et al. (2006); Nittami et al. (2009);
Schroeder et al., 2008; Wong and Liu, (2007); Wong et al. (2004);
(d) Candidatus ‘Competibacter phosphatis’

COMMENTS
(i) Ecological distribution: large cocci/coccobacilli frequently found in anaerobic:aerobic EBPR
lab-scale and full-scale plants with poor phosphorus removal capacity; never been cultured.
(ii) Physiology: FISH-MAR data from in situ studies demonstrate the phenotype of a GAO,
assimilating substrates like acetate and amino acids but not glucose, anaerobically for PHB
synthesis and showing no aerobic P uptake.
(iii) Systematics; members of the Gammaproteobacteria, but phylogenetically very diverse; seven
sub-groups recognized but probably more; named Candidatus ‘Competibacter phosphatis’.
(iv) FISH probes: several available for members of sub-groups (Table 10.1).
(v) Other: share similar ecophysiologies with the Rhodocyclus related PAO, so competition between
them would be expected, although always found together in these communities; PAO may be
favoured over the GAO at higher (47.5) pH.
(vi) REFS: Crocetti et al. (2002); Kong et al. (2002b, 2006); Nielsen et al. (1999a); Oehman et al. (2005).
(d) Tetrasphaera spp.

Organism Description (see Figure 12.36, colour image section (chapter 13))
COMMENTS
(i) Ecological distribution: TFO (but highly variable morphology) mostly isolated from activated
sludge plants; closely related populations are PAO in some EBPR systems.
(ii) Physiology: aerobic, but in situ data suggest some can assimilate some substrates (but not acetate)
anaerobically and store these, but not as PHB; these may then synthesize polyP aerobically.
(iii) Systematics: Actinobacteria and the genus now contains isolates of the filamentous bulking
filament Candidatus ‘Nostocoida limicola’; seven species currently recognized.
(iv) FISH probes: available (Table 10.1).
(v) Other: may be important PAO in some systems.
(vi) REFS: Kong et al. (2005); Maszenan et al. (2000); McKenzie et al. (2006).
13
Colour image section
Figures from Chapter 1
WCHD3 Halobacteria
Thermoplasmata Methanomicrobia
Methanobacteria
Thermococci
OP11
WSA2
M
SA eth
GM an ZB2
pMC2A209 EG oco Elev_16S_1754
ABY1_OD1
cc
i
SM2F11
Cenarchaea
SR1 GN02 OP10
Chloroflexi
Sd WS6
TM7
pUWA2
SHA Thermi
SC3
C2 pSL4 BRC1
AD3
ria
Thermoprotei
cte
Gemmatimonadetes
iba
YNPFFA4
Por
s
Korarchaeota GN04 ere
WS3 rix act bia
ob cro
pMC2A15 dithTM6 Fibr u s imi
l El Planctomycetes
Ca AC1
pMCA25
nella
theo
Entoacterium ia
o su lfidobesulfobacter
0.1 od
ThermThermCCM11b
SPAM
Nitrospirae
NKB19
OP8
NC10
Proteobacteria Acidobacteria
Th
erm WS1 OP9_JS1
Di od
esu
cty lfo
og biu
Chlorobi lo m
Bacteroidetes ZB3 m
i
WPS
Marine_group_A GN01 Thermotogae Actinobacteria
Deferribacteres
Aq
OP5 OP1
Ch
uif
Synerg
Cyanobacteria Coprothermobacteria
ica
lam
e
yd
OP3
iae
Len
istetes
tisp
hae
WWE1
rae
Verrucomicrobia
Spirochaetes
Firmicutes Fusobacteria
Figure 1.3. Phylogenetic tree based on 236,469 rRNA sequences to illustrate the known phyla of the
Archaea and Bacteria and the relationships among these. Image was created using the NAST alignment of
16S rRNA gene sequences from greengenes. Tree was built with the software, fasttree by Morgan Price then
edited in ARB by Phil Hugenholtz. Color coding: red 410% of sequences in the phylum come from activated
sludge; orange 1–10%; blue 51% (P. Hugenholtz).
Colour image section 491
Figure 3.2. (b) A floc after staining with Live Dead1stain, showing presence of living (green) and dead cells
(red).
Figure 3.3. Live: Dead staining of Gordonia amarae like organisms (GALO) in an activated sludge foam,
showing both living and dead regions within an individual filament (E. Harvey). Bar marker ¼ 10 mm.
Figure 3.5. CSLM of an activated sludge granule containing GAO/TFO after FISH with FLUOS (green)
labeled EUBmix probes for all Bacteria, CY-3 (red) labelled GB probe for Competibacter spp. and PAOmix
probes labelled with CY-5 (blue). Some TFO only fluoresced with the EUBmix probe (green cells) while other
fluoresced with both the EUBmix and GB probes (yellow cells). The PAO fluoresced with both the EUBmix
and PAOmix probes (lightblue) (S. McIlroy). Bar marker ¼ 10 mm.
C. colpoda Spirostomum sp
(a) (b)
50 µm
Aspidisca lyncheus Euplotes affinis
(c) (d)
50 µm 20 µm
Figure 4.1. Examples of free-swimming and crawling ciliated protozoa in activated sludge plants.
(a) Colpidium colpoda, dorsal view; the body is usually about 100 £ 50 mm, covered with cilia and with a
distinct oblique depression on the right side which marks the opening of the mouth. (b) Spirostomum sp.; this
is the largest ciliate commonly seen in activated sludge. It is covered in cilia and has a long adoral zone of
membranelles in the anterior region of the body. (c) Aspidicsa lynceus, lateral view; the body is rigid, about
50 £ 30 mm, dorsal side domed and ventral side flat. It typically crawls over flocs using compound ciliary
structures called cirri. (d) Euplotes affinis, ventral view; the body is 50 £ 30 mm, dorso-ventrally flattened,
with longitudinal ridges on both the ventral and dorsal surfaces. The cirri are arranged in two main groups
and, like Aspidisca, it crawls over flocs. Figures (a) – (c) courtesy of Dr Janusz Fyda, Jagiellonian University,
Krakow, Poland.
Vorticella convalaria Epistylis plicatilis

(a) (b)
50 µm 100 µm
Epistylis coronata Opercularia microdiscum

(c) (d)
50 µm
Thuricolla folliculata Sphaerophya magna

(e) (f)
100 µm 50 µm
Figure 4.2. Examples of attached ciliates from activated sludge. (a) Vorticella convallaria; the zooid is borne
upon an unbranched, contractile stalk that contains a spiral, thread-like organelle called the spasmoneme
(or myoneme). The macronucleus is J-shaped and there is a single anterior contractile vacuole. (b) Epistylis
plicatilis. A colonial form with elongate-conical zooids borne upon a branched, non-contractile stalk (i.e. lacks a
spasmoneme). Colonies are often large, sometimes reaching 4 mm in size. (c) Epistylis coronata; the zooids
are broader than those of E. plicatilis and distinctly constricted beneath the peristomial lip. Note the food
vacuoles and anterior contractile vacuole. (d) Opercularia microdiscum. A colonial form with a non-contractile
stalk. Note the narrow persitomial disc born upon a stalk-like projection that extends beyond the peristomial
rim. (e) Thuricola folliculata. Loricate ciliate, each lorica typically contains two zooids, one long and one short,
that are attached to the base of the lorica without a stalk. When the zooids contract a valve closes the lorica
aperture. (f) Hypophrya sp. A suctorian ciliate that attaches to flocs via a non-contractile stalk. Note the
characteristic tentacles arranged in bunches (or fascicles) on the body. Figures (a) – (c), (e) and (f) courtesy
of Dr Janusz Fyda, Jagiellonian University, Krakow, Poland.
Bodonid flagellated Amoeba thecamoeba

(a) (b)
Figure 4.3. Examples of flagellates and amoebae commonly seen in activated sludge plants. (a) Bodonid
flagellate; note the two flagella, one short anterior flagellum that bears minute hairs (mastigonemes) which
are only visible with the electron microscope, and a long, naked retrograde flagellum that typically exceeds
the body length. (b) Amoeba thecamoeba sp. The body is typically flattened with longitudinal surface wrinkles
or folds and covered with a thick, dense layer of glycocalyx.
(a) (b) (c)
10 µm
(d) (e) (f)
10 µm 10 µm
Figure 5.1. Light micrographs of activated sludge floc structures (a) pin point flocs, (b) weak, open
and diffuse flocs, (c,d,e) well formed compact and irregular flocs and (f) bulking sludge showing inter-floc
bridging. Bar markers ¼ 10 mm.
(a) (b)
Figure 5.2. An incident of foaming at an activated sludge plant (a) affecting liquid pumping and (b) reducing
clarifier performance (Courtesy W. Murdoch).
Figure 7.6. Bulking activated sludge (nitrogen deficient) in dairy wastewater treatment plant in secondary
clarifies.
Figure 7.7. The change in the settling characteristics of this mixed liquor after 4 weeks of ammonium
sulphate dosing.
Figure 9.4. Microcolonies of co-existing ammonia- and nitrite-oxidizing bacteria, labelled by fluorescence
in situ hybridization with specific rRNA-targeted oligonucleotide probes, in a nitrifying biofilm. AOB are shown
in blue, nitrite-oxidizing Nitrospira of sublineage I are coloured red and Nitrospira of sublineage II are shown in
green. The spatial arrangement of the two nitrite oxidizers reflects their niche differentiation with respect to
nitrite. Sublineage I Nitrospira, which prefer higher nitrite concentrations, closely co-aggregate with the AOB.
In contrast, sublinage II Nitrospira prefer lower nitrite concentrations and are often found at larger distances
away from the AOB. They are also surrounded by sublineage I Nitrospira, which probably consume most of
the nitrite produced by the AOB. The image was produced by direct volume rendering of a confocal image
stack. Bar ¼ 10 mm. (Image courtesy of Frank Maixner)
H+ Acetate
H+
H+
H+ H+ New biomass
energy
(via ATP) HAc
H+ polyP
ATPase
Ac-CoA Ac-CoA
H+ polyP ATP
TCA H+ H+ TCA
H+ + + H+ ADP Cycle
H+ H+ Cycle
H H
NADH2
Pi NAD CO2 Pi NAD CO2
NADH2
M+ M+ H+
PHB PHB
Pi M + Pi M+ Electron
transport
O2 chain
H+
H2O
(pmf)
Figure 10.1. The anaerobic and aerobic EBPR metabolism described in the Comeau-Wentzel model
(redrawn from Comeau et al., 1986).
H+ Acetate
H+
H+
H+ H+
New biomass
HAc
ATP ATP Glycogen
polyP
ATPase
Ac-CoA
+
ATP
polyP Glycogen H+ H
NADH2 TCA
H+ +H+ H+ ADP Ac-CoA
H Cycle
NAD+ Pi
Pi
NAD+ CO2
M+ M+ H+ NADH2
PHB
PHB
Pi M+ Pi M+ Electron
transport
O2 chain
H+
H2O (pmf)
Figure 10.2. Modified Mino model of anaerobic and aerobic EBPR metabolism (as described in Arun et al.,
1988). The ATP requirement for acetate transport is controversial, thus it is shown as a dashed arrow.
Pit
H g2
+
M
(low affinity)
+
H g2
+
M
+
Acetate
H g2
Glycogen
+
M
V-ATPase EMP
+
H + (high aff.) (low aff.)
,N H+
a + M ackA
g 2+ acs
pta
PDC Symporter
H+ PHB
Proton transport ,N tate
H+
a+ PhaA Ace inate
pyrophosphatase MDH PhaB Pr+op +
(OA dec.)
PhaC PHV PH2MV H ,N
H+
PhaA
PhaB PhaA
MalDH PhaC PhaB
Pi acn PhaC
ppx AMP
MS ICL
IDH
pap adk ATP
Polyphosphate OGDC
G-STK
(degradation) A-STK complex
ADP QH2 SDH Propionate
ppK2
Q
MCM
GTP
H
+
,N
GDP
a
+
OH
H
O
H
+
,N
a
+
mmdA,B,C
Quinone
dehydrogenase
NADH NAD
PPi Pi
(pyrophosphate)(ortophosphate)
CoASH
(unacetylated
coenzyme A) Membrane bound
ATP AMP
ADP
Figure 10.8. EBPR-relevant metabolism inferred from the Accumulibacter composite genome. In the
anaerobic phase acetate and propionate are stored as four types of PHA; polyhydroxybutyrate (PHB, from
acetate only), polyhydroxyvalerate (PHV, from acetate and propionate), polyhydroxy-2-methylbutyrate
(PH2MB, from acetate and propionate) and polyhydroxy-2-methylvalerate (PH2MV, from propionate only).
PHA production requires energy (ATP) and reducing power (NAD(P)H). ATP (in red) is supplied by polyP
degradation and, to a lesser degree, glycogen degradation. NAD(P)H (in blue) is provided by glycogen
degradation and the TCA cycle (enabled by a novel quinol reductase). A possible alternative use of the TCA
cycle splits it in two branches through the use of fumarate reductase (dashed line). The glyoxylate shunt could
also be active (dashed line). Reproduced from Garcia Martin et al. (2006).
F-ATPases Flipase
H
+
Polymerase
H
V-ATPase
+
H
+
,N
a EPS
+
H
+
H
+
Pst
(high affinity) H
+
,N
a EMP
+
PhaB, croR
FDox FDred ccR, pccAB
PhaZ ibd2, meaC
PHB meaA
+
POR pyruvate
H synthesis
Mg 2 MDH PhaZ PHV PH2MV
Pst H+ (OA dec.)
Mg 2 CS
(low affinity) Pi PhaZ
+ + acn
H H AMP
2
Mg2 Mg (synthesis)
Polyphosphate pap adk ATP IDH

OGDC
ADP G-STK complex
A-STK
ppk1 QH2 SDH
mcmAB, epm
Q meaB, meaD
GTP PccAB
NADH NAD
GDP
PPi Pi
(pyrophosphate)(ortophosphate) E.T
.C.
CoASH +
H
O2
(unaacetylated
coenzyme A) Membrane bound HO
2 NO3
NO2
ATP AMP
ADP
Figure 10.9. In the aerobic EBPR phase, PHA is broken down to fuel the TCA cycle, ATP generation, and
growth. PolyP and glycogen stores are regenerated. The dashed line represents an alternative pathway for
PHB degradation for which not all genes have been characterized. The dotted lines leading from the high
affinity phosphate transporters (Pst) indicate that these transporters are unlikely to be active for most of the
aerobic phase. Reproduced from Garcia Martin et al. (2006).
(a) (b) (c)
Figure 11.1. Comparison between images of an activated sludge floc when examined by bright field (a),
phase contrast (b) and Nomarski interference microscopy (c). Bar mark ¼ 20 mm.
(a) (b)
Figure 11.2. Fluorescence micrograph of the same image of a cluster of Accumulibacter cells identified
by FISH probing with CY-3 tagged PAO mix probes, after staining with nile blue A for PHA: (a) FISH image;
(b) PHA positive cells (S. Schroeder). Bar mark ¼ 10 mm.
(a) (b)
Figure 11.3. Fluorescence micrograph of the same image of a cluster of Accumulibacter cells identified
by FISH probing with CY-3 tagged PAO mix probes, after staining with DAPI for polyP: (a) FISH: (b) polyP
fluorescing cells (yellow) (S. Schroeder). Bar mark ¼ 10 mm.
Figure 11.6. CLSM image of an aerobic granule after FISH probing with FLUOS tagged EUBmix probe
(green cells), the CY-3 tagged DF988 probe (yellow cells from complementarity between green and red)
and the CY-5 tagged probe DF1020 for the GAO Defluviicoccus, where its cells appear white from
complementarities between green, yellow and blue fluorochromes (S. McIlroy). Bar mark ¼ 10 mm.
Figure 11.7. Acid fast stain of a sample from a foaming activated sludge plant, showing acid fast bacteria
(staining with crystal violet) associated with non acid fast GALO. Bar mark ¼ 10 mm.
Figure 11.8. Neisser stain showing Neisser positive (lilac) type 0092 filaments and Neisser negative (beige)
‘N. limicola’ filaments. Bar mark ¼ 10 mm.
Figure 11.9. A cluster of PAO (Accumulibacter?) taken from the anaerobic zone of an EBPR plant after
staining with Sudan Black stain, showing presence of intracellular PHA. Bar mark ¼ 10 mm.
Figure 11.10. Crystal violet stain revealing the presence of a sheath on the filamentous bacterium type 0041.
Bar mark ¼ 10 mm.
Figure 11.11. Negative staining with India ink, revealing the presence of a capsular material surrounding the
cluster of TFO in an activated sludge floc (S. Schroeder).
Figure 11.12. Fluorescence micrograph of an activated sludge floc and GALO filaments after staining for cell
viability with CTC. Viable cells fluoresce red (E. Carr). Bar mark ¼ 10 mm.
(a) (c)
(b)
Figure 11.13. Combination of ELF (Enzyme-labelled fluorescence) and FISH. (a) shows a FISH positive
aggregate of Skermania piniformis filaments in a foam detected by the Spin1449 probe (CY-3 ¼ red), and
(b) the same aggregate showing glucuronidase exoenzyme activity after ELF assay (green precipitate).
(c) combines FISH positive S. piniformis in foam and associated glucuronidase activity (C. Kragelund and J.L.
Nielsen).
(a) (b)
Non-target
Non-target cells
cells
CAP
CAP
(c) (d)
Figure 11.26. (a) Flow cytometry histogram plot showing an activated sludge sample hybridized with three
FLUOS-labelled probes (PAO462b, PAO651, and PAO846b; Zilles et al. (2002)) targeting ‘Candidatus
Accumulibacter phosphatis’ (CAP). Horizontal axis shows signal intensity (FL1). (b) Flow cytometry dot plot of
the sample shown in (a). Vertical axis indicates forward scatter (FSC). (c) showing activated sludge
hybridized with FLUOS-labelled bacterial probe EUB338 (Amann et al., 1990b) and CY-3-labelled Ntspa662
targeting Nitrospira, which is the genus used in the probe design example described in chapter 11
(see Table 11.7 and Figure 11.29). Since FLUOS is indicated with green and CY-3 with red, yellow cells are
positive for both probes (i.e., Nitrospira in the domain Bacteria), while green cells correspond to Bacteria other
than Nitrospira. (d) showing activated sludge floc hybridized with fluorescein-labelled (green) Ntspa1431
targeting Nitrospira Lineage I (Table 11.7 and Figure 11.29) and CY-3-labelled (red) Ntspa662 targeting all
Nitrospira. Yellow cells hybridized with both probes, indicating that Lineage I dominates the Nitrospira
population in this sample.
Microautoradiography (MAR)
1. Incubation 3. FISH (with fluorescent probe)

Radio-labelled
substrate
4. Photographic emulsion
& developing
2. Immobilization on cover slip
5. Microscopy
Figure 11.32. (b) Diagrammatic representation of the major steps in the FISH-MAR protocol (S. Schroeder).
Environmental sample
DNA/RNA isolation
PCR amplification
(if applicable)
Labelling of target
e.g. fluorescence
Hybridisation
Washing step
Image acquisition
Image analysis
Normalisation
Signal-to-noise ratios
Presence-absence data
Inventory list of organisms

present in the sample
Figure 11.33. Schematic representation of a typical environmental microarray analysis.

Figure 11.34. The nested Multiple Probe Concept. All organisms represented in the phylogenetic tree at left
are targeted by at least four probes of hierarchical (nested) and/or parallel specificity. Putative identities of
organisms can then be inferred from the pattern of probe hybridisation on the microarray.
PhyloChip
analysis
1 Phylogenetic
fingerprint
Quantification
Quantitative
hybridisation Environmental
e.g. FISH 3 sample
Detailed
phylogenetic
analysis 2
rRNA Selective PCR

New probes
sequence cloning
(if applicable)
analysis sequencing
Figure 11.35. The PhyloChip approach. Application of the PhyloChip (1) provides a preliminary description
(fingerprint) of a microbial community. These results are subsequently validated (2) in a more detailed
phylogenetic analysis, and quantitative information can also be obtained (3). rRNA sequence analysis offers
the additional possibility of designing new probes to target those organisms which were detected in the rRNA
clone library (but not targeted on the initial array).
Activated sludge sample from year 2002
General
Bacteria Rhodocyclales- Genus Zoogloea Most Zoogloea species Yet uncultivated
UNIV1390a specific probes ZOGLO828 but not Z. resiniphilia Zoogloea species
Not shown due
2 EUB338
to complexity
ZOGLO1416 ZOGLO647 ZOGLO455
Increasing specificity of hierarchical probes
Kraftisried wwtp clone KRZ64

Kraftisried wwtp clone KRZ70
ZOGLO455
Kraftisried wwtp clone H10
Kraftisried wwtp clone H40
Kraftisried wwtp clone A16
3 EBPR reactor clone PHOS-HC20 ZOGLO647
Kraftisried wwtp clone KRZ65 ZOGLO828
Zoogloea ramigera ZOGLO1416
EBPR reactor clone HP1 A03
TCB-transforming microbial consortium clone SJA-21 EUB338
Zoogloea resiniphila
UNIV1390a
FISH probe OTU1-1415 targeting the genus Zoogloea
1996: 17% 2002: <1%
Figure 11.36. RHC-PhyloChip-based diversity analysis of activated sludge from the Kraftisried (Germany)
industrial wastewater treatment plant: example using the genus Zoogloea (modified from Loy et al., 2005).
Probes exhibiting positive microarray hybridisation signals as indicated by boxes (1) were compiled according
to their phylogenetic specificities (2), and identities of the organisms present inferred based on the probe
hybridisation pattern (3). Subsequent quantification of members of the genus Zoogloea by FISH revealed much
lower abundances compared with an earlier study of the same treatment plant (Juretschko et al., 2002) (4).
Incubation of sample
with radioactive substrate
RNA extraction
Fluorescence labelling
PhyloChip
hybridisation
Fluorescence scan (diversity)
Radioactivity scan (diversity)
Figure 11.37. Schematic representation of the steps involved in an Isotope Array analysis.
(2) PCR with a

C (1) DNA extraction fluorescently labeled
forward primer
B Mixture of microbial DNA

50 30
Environmental sample
30 50
50 30
30 50
Image analysis
Cluster analysis 50
30
Principal-component analysis 30 50
Web-based database analysis
Fluorescent PCR products of a
a. TAP T-RFLP
b. T-RFLP Phylogenetic Assignment Tool (PAT) Outline of T-RFLP method specific gene
c. the Microbial Community Analysis (MiCA)
d. tRFLP fragment sorter version 4.0
(3) Restriction
enzyme digestion
(6) Data analysis
B 50 1200 bp 300 bp 30
Fluorescent intensity
1200 bp
1100 1100 bp (4) Separation fragments 30 50
800 bp 50 1100 bp 400 bp 30
C A by electrophoresis
(RFU)
600 bp 30 50
600 1200
400 bp 50 600 bp 800 bp 30
300 bp (5) Detection of
30 50
100 bp fluorescent
fragments 100 bp
Cut at specific site
Typical electropherograms of Fragments
T-RFLP fingerprint on gel
Figure 11.38. The basis and procedure of terminal restriction fragment length polymorphism (T-RFLP)
method. (i) DNA extraction, (ii) PCR amplification, (iii) restriction enzyme digestion, (iv) terminal fragment
separation and detection, (vi) data analysis, and (vii) phylogenetic identification of major terminal restriction
fragments (T-RFs).
Figure 12.1. FISH of alphaproteobacterial filamentous bacteria, showing corresponding bright field and
fluorescence micrographs. (a, b) Candidatus ‘Alysiomicrobium bavaricum’ with PPx3-1428 probe; (c, d)
Candidatus ‘Monilibacter batavus’ with MC2-649 probe; (e, f) Candidatus ‘Alysiosphaera europea with
NOLI-644 probe; (g, h) Candidatus ‘Sphaeronema italicum’ with Sita-649 probe; (k, l) Candidatus
‘Combothrix italica’ with Combo-1031 probe; (m, n) Meganema perideroedes; (o, p) Meganema
perideroedes (i, j) Candidatus ‘Combothrix italica’ with combo-1031 probe. All FISH probes were tagged
with CY-3. (Reprinted from C. Levantesi et al. (2004) Syst Appl Microbiol, 27, 716–727, and reproduced with
permission of Elsevier). Bar mark ¼ 10 mm.
Figure 12.3. Sphaerotilus natans (a) false branching (phase), (b) Gram stain showing Gram –ve sheathed
filaments and (c) FISH with CY-3 tagged SNA655 probe where a sheath is clearly shown enclosing
rectangular cells (K. Eales). Bar mark ¼ 10 mm.
Figure 12.4. Type 1701, closely related phylogenetically to S. natans, (a) sheathed filaments with attached
particles (b) double FISH probed sample with FLUOS tagged EUB338mix showing green attached epi-parasitic
cells and CY-3 tagged BET42a probe for Betaproteobacteria, where the type1701 filament appears yellow from
responding to both probes colour complementarity (D. Wong Man Tak). Bar mark ¼ 10 mm.
Figure 12.5. Type 0803, Gram stain showing Gram negative filaments arranged in typical bundles. Bar
mark ¼ 10 mm.
Figure 12.6. Zoogloea ramigera, (a) showing amorphous cell clusters (phase) and (b) Gram negative cells
organized into finger-like projections. Bar mark ¼ 10 mm.
Figure 12.7. Beggiatoa (a) dark trichome with barely visible S0 granules (phase) and (b) abundant refractile
S0 granules now seen in trichome (phase). Bar mark ¼ 10 mm.
Figure 12.8. Type 1863 (Acinetobacter johnsonii) (a) distinctive chain of ‘sausage’ shaped cells (phase),
(b) tangled chain of Gram –ve cells, (c) interfloc bridging with these Gram negative cells in chains. Bar
mark ¼ 10 mm and (d) SEM showing the often pleiomorphic appearance of some cells appearing as irregular
rods. Bar mark ¼ 2 mm.
Figure 12.9. Type 021N (a) filament of irregularly shaped, often discoid cells and clear indentations
separating them (phase) and (b, c) Phase and corresponding FISH with CY-3 tagged 21N probe showing
characteristic appearance of type 021N in activated sludge (K. Eales). Bar mark ¼ 10 mm.
Figure 12.10. ‘Thiothrix’ I (a) rosette production, (b) rosette with S0 granules visible inside filaments (phase)
and (c) S0 granules more apparent after methylene blue staining. Bar mark ¼ 10 mm.
Figure 12.11. ‘Thiothrix’ II (a) large rosette of filaments with intracellular S0 granules and (b) FISH with CY-3
tagged GAM42a probe showing rosette. Bar mark ¼ 10 mm.
Figure 12.12. Type 1851 (Kouleothrix) (a) individual sheathed filament with rod shaped bacterial cells
attached (phase), (b) Gram stain showing weakly staining Gram –ve filaments in characteristic bundles and
(c) FISH probed filaments with EUBmix (Fluos-tagged) probe, CY-3 tagged CHL1851 probe and PAO mix
(CY-5 tagged) probe showing interfloc filament bundles (yellow). Bar mark ¼ 10 mm.
Figure 12.13. ‘Chloroflexi ’ (Nostocoida limicola ) morphotype with typical coiled and bent appearance
(a) bright field and (b) FISH with CY-3 tagged AHW 183 probe (M. Schade and H. Lemmer). Bar mark ¼ 10 mm.
Figure 12.14. Type 0092, (a) diagnostic Neisser positive (lilac) short blunt filaments extending from floc and
(b) CLSM image of FISH probed biomass: magenta cells ¼ those responding to both GNSB941/CFX1223
mix (CY-5) for ‘Chloroflexi ’ and CFX223 (CY-3) FISH probes for ‘Chloroflexi’ type 0092. Note localised
fluorescence signal; blue cells ¼ those responding to the GNSB941/CFX1223 mix probes alone, lacking
EUBmix FISH target sites; green cells ¼ those responding only to the EUBmix (FLUOS) probes; light blue
cells ¼ those responding to EUBmix þ GNSB941/CFX1223 mix probes together. (L. Speirs & S. Schroeder)
Figure 12.16. Trichococcus flocculiformis (a) Gram stain showing interfloc bridging with these filaments.
Bar mark ¼ 10 mm, (b) SEM of filament morphology with regular coccoid cells dividing by binary fission. Bar
mark ¼ 3 mm, (c) change in filament morphology to highly irregular swollen and smaller cells in chains.
Bar mark ¼ 3 mm and (d) FISH probing with Fluos tagged EUBmix and CY-3 tagged NLIM191 probes.
Bar mark ¼ 5 mm (Jian Rong Liu).
Figure 12.18. Candidatus ‘Microthrix calida’ (a) unbranched tangled filaments of strain TNO-1 (phase)
Bar mark ¼ 10 mm, (b) Gram stained filaments in pure culture, (c) colony appearance on agar medium,
(d) swollen spherical cells containing lipid material (phase), (e) fluorescent lipophilic material after nile blue
A staining, (f–g) FISH with CY-3 tagged Mpa-T1-1260 probe and (h) in situ Gram staining. (Reprinted from
Levantesi et al. (2006) Environ. Microbiol. 8, 1552–1563, and reproduced with permission from Blackwell
Publishing). Bar mark ¼ 5 mm, except (c) ¼ 20 mm.
Figure 12.19. Candidatus ‘Microthrix parvicella’ (a) irregular ‘spaghetti-like’ tangled unbranched filaments
with no septa visible, (b) Gram stain showing Gram positive filaments, (c) SEM showing irregular appearance of
the unbranched filaments and (d) FISH with CY-3 labelled Mpamix probes (H. Lemmer). Bar mark ¼ 10 mm.
Figure 12.20. Candidatus ‘Nostocoida limicola’ (a) coiled and angled appearance of filaments containing
flattened discoid cells (Nomarski), (b) Gram variable appearance of filaments, (c) both Neisser negative and
Neisser positive filaments of this morphotype exist, probably from phylogenetically different organisms
and (d) FISH probed filament of typical appearance with CY-3 labelled NLIM175 probe (Jian Rong Liu). Bar
mark ¼ 10 mm.
Figure 12.22. Gordonia amarae like organisms (GALO) (a) Gram stained short fragment showing typical
right angled branching, (b) foam sample containing high numbers of this Gram positive morphotype, (c) DAPI
staining reveals GALO can store polyphosphate granules (yellow fluorescence) and (d) FISH probed
G. amarae filament with CY-3 tagged GAM 205 probe (E. Carr). Bar mark ¼ 10 mm.
Figure 12.23. Skermania piniformis (a) distinctive appearance of filament with acute angle branching,
(b, c) Gram stained filaments showing variations in branching frequency and branch length and (d) FISH
probed filaments with SPIN1449 probe (K. Eales). Bar mark ¼ 10 mm.
Figure 12.24. Type 0041 (a) sheathed filament with attached bacterial cells obscuring septa (Nomarski),
(b) Gram þve filament with Gram –ve attached bacteria Bar mark ¼ 10 mm, (c) TEM showing typical Gram
positive septal wall and sheath. Bar mark ¼ 200 nm and (d) clear demonstration of sheathed nature of this
filament. Bar mark ¼ 200 nm (R. Webb).
Figure 12.25. ‘Isosphaera’ spp. (a) typical beaded appearance of filaments with often flattened, discoid cells,
(b, c) SEMS showing morphological variation in cells of this morphotype, ranging from discoid to spherical in
shape (2 mm, 5 mm), (d) biomass sample probed with CY-3 tagged NLIM301 probe (10 mm), (e) negatively
stained TEM preparation showing crateriform cell surface typical of Planctomycetes (100 nm) and (f) TEM
showing regular array of tubular intracellular membranes of this filament (200 nm) (Jian Rong Liu and R.Webb).
Figure 12.26. Haliscomenobacter hydrossis (a) needle like filament with no visible septa emerging
from floc (Nomarski), (b) these occasionally occur in bundles and (c) FISH probed filament with CY-3 tagged
HHY655 probe where many filaments are bent (K. Eales). Bar mark ¼ 10 mm.
Figure 12.27. Type 0675 (a) sheathed filament with heavy attached growth (phase), (b) shows regions of
this filament with no attached cells and (c) Gram variable filament with Gram –ve attached bacterial cells. Bar
mark ¼ 10 mm.
Figure 12.28. Type 0411 (a) typical filament appearance with elongated rod-shaped cells and constrictions
visible at septa (phase) and (b) Gram stain showing the Gram negative individual cells enclosed in sheath.
Bar mark ¼ 10 mm.
Figure 12.29. Type 0961 (a) filaments consisting of irregular rectangular cells with distinctive ‘empty’
transparent appearance (phase) and (b) Gram stain showing Gram –ve filaments in bundles. Bar
mark ¼ 10 mm.
Figure 12.30. Type 0914 (a) bundle of Gram –ve filaments showing interfloc bridging, (b) sudan black
staining reveals lipophilic granules. Bar mark ¼ 10 mm and (c) FISH with CY-3 tagged GNSB941 probe for
‘Chloroflexi’ showing characteristic bundled filaments fluorescing.
Figure 12.31. Type 0581 (a) Gram negative tangled filaments superficially very similar in appearance to
Candidatus ‘M. parvicella’ and (b) Neisser negative filaments (V. Tandoi). Bar mark ¼ 20 mm.
Figure 12.32. Candidatus ‘Accumulibacter phosphatis’ (a) typical cell cluster after staining with
methylene blue showing metachromatic appearance of cells (pink) consistent with polyP presence and
(b) CLSM of Accumulibacter after double FISH probing with the EUBmix (Fluos ¼ green) probes for all
Bacteria and the PAO mix (CY-5 ¼ blue) probes for Accumulibacter PAO (S. Schroeder). Bar mark ¼ 10 mm.
Figure 12.33. Amaricoccus (a) Gram negative tetrads held together by dried capsular material and (b) after
Neisser staining, showing no intracellular polyP, but walls are Neisser þve (A.M. Maszenan). Bar
mark ¼ 10 mm.
Figure 12.34. Defluviicoccus activated sludge floc after FISH with EUBmix (fluos) and CY-3 tagged DF 988
probe showing clusters of Defluviicoccus (yellow). Blue cells are Accumulibacter PAO with CY-5 labelled
PAO mix probes. Bar mark ¼ 10 mm.
Figure 12.35. Candidatus ‘Competibacter phosphatis’ showing (a) large ovoid cells (yellow) in clusters
after FISH probing with EUBmix (green) and GB-mix (CY-3 tagged) probes and PAOmix (CY-5 tagged) and
(b) phase contrast micrograph showing their coccobacillus shape. Bar mark ¼ 10 mm.
14
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Index
Note: Numbers in italics relate to figures.
A polyphosphate-accumulating organisms (PAO) 117,

A-B process 66 295–297, 301, 502, 506
A-tailing 361 population dynamics 91, 297–298
A/O system 72–73 proton motive force (PMF) 315
abiotic factor tolerance 123, 162–163 quantitative PCR (qPCR) 359
abiotic methods 211–214 sampling 100
Absidia spinosa 33 species delineation 299–300
AcCoA see acetyl-CoA unrooted maximum likelihood tree 296, 300
Accumulibacter 8, 297–298 acetate kinase (ack) 286
acetate uptake 302, 314, 315, 316 acetate uptake 257, 284, 286, 290, 305
bacteriophages 304 Accumulibacter 302, 314, 315, 316
carbon 302–303, 317 glycogen-accumulating organisms (GAO) 309, 313,
culture 102 315–319
denitrification 115, 276–277 Skermania piniformis 108
distribution 297–298 acetogens 42
ecology 297–299 acetyl-CoA (AcCoA) 283
ecophysiology 298 synthase (acs) 286, 302–303, 500
enhanced biological phosphorus removal (EBPR) 302, acetyl-CoA pathway 42
500, 506 acid fast bacteria 332–333, 504
FISH 104, 293, 359 Acinetobacter 190, 196, 218–219, 286–292, 341, 463
genes 288, 305 Acinetobacter johnsonii 123, 177, 458, 521
genome 301–305 aconitase 305
glycogen 290 B 352–353
identification of 295–297 actin 25, 29
metabolic pathways 89 Actinobacteria 14, 16, 112–116, 118, 471–477
nitrate reduction 300 bulking 197
phage imprint 304 carbon 317
phosphate metabolism 303–304 ecophysiology 152–154, 162, 210
phosphotransferase 289 enhanced biological phosphorus removal (EBPR)
polyphosphate 24, 286, 288 306–308
flocs 97 glycogen-accumulating organisms (GAO) 503

foaming 215, 218, 233–234, 240 technology 66–70, 76–79, 78
nitrogen removal 276–277 aerobic organisms
phosphorus removal 292–293, 306–308, 307, bacteria 16, 27, 45
310–311, 316–318 eukaryotes 27, 36
phylogeny 193, 471, 490 fungi 32
polyphosphate-accumulating organisms (PAO) prokaryotes 42, 47
306–308 aerobic processes 5, 5
staining 332 fermentation 57
T-RFLP 413, 415 respiration 41, 71, 164, 209–211
taxonomy 179, 187, 233 aerobic reactors 61, 63–64, 80, 85, 86
Actinomadura 221, 223, 233, 370 aerobic thermophilic autothermal digestion (ATAD) 60
actinomycetes 220–223, 233 air-dried smears 330
activated sludge air-water interface 248–249
bulking 140–141, 143 alanine 20, 234
see also bulking Alcian blue 336
characterization 321–452 algae 26–27, 28, 36–37
filamentous bacteria 169–190 biodiversity 121
flocs 97–99, 97 cellulosic 27
foaming 141–142, 496 green see green algae
see also foaming microbial communities 121
granules 99 photosynthesis 43, 449
metazoa 122 red see red algae
microbial communities 95–126 secondary 36
organisms 453–536 yellow-brown see yellow-brown algae
protozoa 127–138, 138 algorithms 369
taxonomy 169–190 Alka-Seltzer test 252–253
viruses 10 alkalinity 80
activated sludge plants 96–127 Allomyces arbusculus 32
conventional 5 Alphaproteobacteria 113–115, 177, 186, 454–458
activated sludge processes 4–6, 57–94 bulking 192, 200, 208
biomass 83, 83, 138 characterization 435
chemists 81 culture 275, 277
microbiologists 85 denaturing gradient gel electrophoresis (DGGE) 423
models 86–89 ecophysiology 209
multistage treatment systems 66, 66 enhanced biological phosphorus removal (EBPR)
oxygen uptake rates (OUR) 83, 83 308–309, 316–317
active cells 106–108 FISH 517
activity assessment 445–446 flocs 97, 151–152
adenine 22 glycogen-accumulating organisms (GAO) 116, 118, 312
adenosine diphosphate (ADP) 39 phylogeny 193, 196, 198, 266, 267, 423
adenosine triphosphate (ATP) 27, 38 see also Betaproteobacteria; Gammaproteobacteria,
biomass 106 Proteobacteria
adenylate kinase (ADK) 289 Amaricoccus 485–486, 534
Aeolosoma 122 FISH 293
aeration systems 65 phylogeny 310, 311
aerobic conditions amensalism 166–167
enhanced biological phosphorus removal (EBPR) 71 ammonia monooxygenase 45
88–89, 501 PCR 352–353
famine 71, 283 ammonia-oxidizing Archaea (AOA) 263–264
oxidation 4, 152–161 ammonia-oxidizing bacteria (AOB) 44, 70
phosphorus uptake 117, 285 ammonia oxidation 264–265
sludge age 205–206 anaerobic ammonium oxidation (ANAMMOX)
aerobic granulation 5, 92, 99 277–280
enhanced biological phosphorus removal (EBPR) 78 characterization 350, 409–411, 415–416
Index 645
habitats 262–264 ANAMMOX see anaerobic ammonia oxidation

microcolonies 117, 118, 498 anammoxosome 45, 278
mutualism 166 annealing temperatures 355
nitrogen removal 262–266, 270, 277–278 anoxia
pH 80 anoxygenic bacteria 46
phylogeny 262–264, 263 phosphorus uptake 285
wastewater treatment 265–266 zones 41
ammonification 259 antibiotics 56
ammonium 259, 261 antiseptics 55
anaerobic oxidation see ANAMMOX A2O activated sludge system 72, 72–73
control measures 210 AOB see ammonia-oxidizing bacteria
detergents 56 apicoplast 37
enzymic activity 98 apothecium 34
removal 70, 277–278 arabinogalactans 227, 332
ammonium chloride, tetramethyl (TMACl) 401, 405 Archaea 12
ammonium sulphate 207, 497 arsenite 45
Amoeba thecamoeba 495 cell organization 17–25, 29
amoebae 495 characterization 353, 368, 409, 412, 415, 421–422,
amoeboid movement 29 437–438
amoeboid protozoa 37 cytoplasmic membranes 21–22
amperometric sensors 449 microbial communities 111–113, 118
amplification nutrition 40–46
microarrays 402–403 phylogeny 11, 262, 263, 490
PCR 351, 354–355 polyphosphate 285
PCR-DGGE 422 transduction 23
amplified ribosomal DNA restriction analysis (ARDRA) ARDRA see amplified ribosomal DNA restriction
366 analysis
anabolism 38, 38 arsenic 42
anaerobic ammonium oxidation (ANAMMOX) 45, 70 arsenite 42
ammonia-oxidizing bacteria (AOB) 277–280 Archaea 45
nitrogen removal 277–280 bacteria 45
anaerobic conditions 163, 203, 244–245 artificial (monothetic) classification 171
anaerobic organisms 16 ascocarp 30, 34
Archaea 437 Ascomycota 34, 35
bacteria 132, 152–161, 165, 209–211 Ascomycotina 30
eukaryotes 27–28 ascospores 34
glycogen accumulating organisms (GAO) 116, ascus 30, 34
309–319 Aspergillus 2, 34
phosphorus-accumulating organisms (PAO) 308 Aspidisca lynceus 493
prokaryotes 40, 47 ATAD see autothermal digestion
sulphate-reducing bacteria 116 ATP see adenosine triphosphate
sulphur-oxidizing bacteria 119 automatic monitoring 85–86
anaerobic processes autoradiography, microautoradiography (MAR) 393–397
fermentation 40 autothermal digestion (ATAD) 60
methane production 61 autotrophs 39–40, 46, 67, 87
respiration 41–42, 67, 164
anaerobic reactors 71–76 B
enhanced biological phosphorus removal (EBPR) BAC see bacterial artificial chromosome
88–89, 297 Bacillus
feed conditions 283 cell organization 13–14, 16
foaming 217, 240, 255–258 FISH 187
nitrogen removal 260–261, 266 phylogeny 470–471, 471
phosphorus removal 281–286, 290–291 wastewater 272
r recycle 74 Bacillus subtilis 447
R-AN-D-N configuration 75, 75 Bacillus thuringiensis 60, 114
bacteria 12 sulphur 44, 119, 145, 202

abiotic factor tolerance 162–163 taxonomy 185, 188
arsenite 45 Betaproteobacteria 459–462, 485
cell organization 17–22, 20–21 bulking 200
flow cytometry (FCM) 444–445 characterization 402, 407, 435
foam 224 denaturing gradient gel electrophoresis (DGGE) 423
genotypes 174–175 ecophysiology 153, 209
Gram-negative 15–16 enhanced biological phosphorus removal (EBPR)
Gram-positive 15–16 308–309
growth rates 162–163 estrone 58
identification of 173–174, 224 FISH 104, 186, 294
magnetotactic 25, 267 flocs 97
microbial communities 111–113 habitats 262
pH 163 microbial communities 113–117, 118
phenotypes 175 nitrogen removal 272, 275–277
photosynthesis 43 phosphorus removal 295, 305, 308–309, 313
phylogeny 11, 490 phylogeny 193–194, 198, 263, 267, 423, 519
pO2 163 sequencing 177
redox potential 163 binary fission 48
temperature 162–163 bioaugmentation 125
bacterial artificial chromosome (BAC) 408 biochemical oxygen demand (BOD)
bacteriochlorophylls 43, 46 BOD5day 3, 61, 80, 82–83, 207
bacteriophages 9, 10 foaming 216, 257
Accumulibacter 304 nitrogen removal 259–260
coliphage 132 phosphorus removal 281–283
defective 23 protozoa 133–134
imprint 304 biodegradable fractions 83, 87
microbial communities 109–111 Biodenitro process 69, 74
sorting 447 biodiversity 100–101, 111–113
Bacteroidetes 479–481 algae 121
denaturing gradient gel electrophoresis (DGGE) 423 characterization 350, 352, 360, 368–369
enhanced biological phosphorus removal (EBPR) 118, EBPR 292
308–309 fungi 121, 221–222
FISH 112, 114 bioengineering 202–214
phylogeny 118, 423, 469 biogeochemical nitrogen cycle 260, 261, 272
polymers 115 biomass
baffles 256 associated products (BAP) 93
BAP see biomass associated products ATP content 106
Bardenpho process 69, 69, 71, 72 bulking 204
three-stage (A2O) 72, 112 Candidatus ‘Microthrix calida’ 140, 146, 151, 153
see also Phoredox process Candidatus ‘Nostocoida limicola’ 140, 151, 153
basidiocarp 30 foaming 253–254
Basidiomycota 30, 35, 35, 120 nucleic acids 346
Basidiomycotina 30 oxygen uptake rates (OUR) 83, 83
basidiospores 30, 35 retention time 204, 253–254
basidium 30, 35 bisubstrate hypothesis 83
batch culture BOD see biochemical oxygen demand
closed 48 bodonids 130, 495
growth 48–49, 51 bright field microscopy 182–184, 189, 325–326, 502,
kinetics 49–50, 49, 52 517, 524
substrates 51–53, 51–52 bulking 12
Beggiatoa 25, 195, 462–463 abiotic methods 211–214
granules 44, 520 abundance 192–200
phylogeny 458 Actinobacteria 197
staining 183 activated sludge plants 140–141
Index 647
Alphaproteobacteria 192, 200, 208 phylogeny 193, 222

Betaproteobacteria 200 Candidatus ‘Microthrix parvicella’ 472–473, 527
bioengineering 202–214 permeabilization 227
biomass 204 taxonomy 234
cavitation nozzle 212 Candidatus ‘Monilibacter batavus’ 116, 182, 190, 196,
Chloroflexi 192, 196–200, 208 312, 456, 517
clarifiers 200–208, 213, 214 phylogeny 458
dissolved oxygen (DO) 207–208, 212 Candidatus ‘Nostocoida limicola’ 179, 210, 487, 528
ecophysiology 208–211 biomass 140, 151, 153
filamentous bacteria 139–168, 200, 211–212 morphology 473–475, 474
Firmicutes 192 phylogeny 193
FISH 143–144, 147, 194–199 Candidatus ‘Sphaeronema italicum’ 182, 190, 196, 517
inter-floc bridging 496 phylogeny 456–457, 458
intrinsic factors 204–211 CANON (completely autotrophic nitrogen removal over
microbiology 191–214 nitrite) process 62, 70, 279
monitoring 143–144 see also nitrogen removal
morphology 192–194 CAP see Candidatus ‘Accumulibacter phosphatis’
nutrients 207–208 capsid 9
pH 203–204, 208 capsules 19, 508
plants 143 carbol fuschin stain 332–333
sedimentation 212–214 carbon
substrates 204–207, 205–206 Accumulibacter 302–303, 317
temperature 203–204, 208 Actinobacteria 317
toxic compounds 203–204 assimilation 317
wastewater 202–203, 497 C:N ratio 259–260
Competibacter 314, 316–317, 317
C Defluviicoccus 314, 317
Candidatus ‘Accumulibacter phosphatis’ 24, 100, 174, glycogen-accumulating organisms (GAO) 314,
485 316–318, 317
enhanced biological phosphorus removal (EBPR) growth 39
295–305, 409 metabolism 302–303
flocs 97, 534 polyphosphate-accumulating organisms (PAO)
flow cytometry (FCM) 510 316–318, 317
nitrogen removal 277 removal 62
PCR 353 sources 39–40, 46–47, 316–318
phosphorus removal 286–287, 295 uptake 314
staining 334 carbon dioxide 42
Candidatus ‘Alysiomicrobium bavaricum’ 181, 196, carbonate 41
209, 275, 454 carboxyfluorescein diacetate (CFDA) 332, 445–446
FISH 156, 186, 517 carboxyfluorescein-N-hydroxysuccinimide ester
phylogeny 458 (FLUOS) 510
Candidatus ‘Alysiosphaera europea’ 146, 190, 454–455 CARD see catalyzed reported deposition
FISH 517 carotenoids 37
Candidatus ‘Combothrix italica’ 190, 196, 455 Carrousel process 65, 65
FISH 517 catabolism 38, 38
phylogeny 458 catalyzed reported deposition (CARD)–FISH 104, 373,
Candidatus ‘Competibacter phosphatis’ 100, 487 391
flocs 535 cavitation 211–212, 212–213
phylogeny 310–311 cell organization 9–12
see also Competibacter actinomycetes 233
Candidatus Division TM7 194, 478 Archaea 17–25, 29
Candidatus ‘Magnospira bakii’ 469, 480–481 Bacillus 13–14, 16
Candidatus ‘Microthrix calida’ 471–472, 526 bacteria 17–22, 20–21
biomass 140, 146, 151, 153 envelopes 13
characterization 179 Escherichia coli 13
Eukarya 12, 17–18, 22 chlorophylls 27, 36–37, 43, 46, 130

eukaryotes 27 bacteriochlorophylls 43, 46
layers 19 chloroplasts 26–28, 28, 36
membranes 27 chromosomes 18, 22–23, 29, 301–302, 408
walls 14, 19–20, 27, 233 Chytridiomycetes 30
cells Chytridiomycota 32, 32, 35
fixation 373, 377–378 cilia 29, 37, 129, 493
growth 48–49 ciliated protozoa (Ciliophora) 37, 128–129, 133–137,
hydrophobicity 340 493–494
lysis 344–345 clarifiers 5, 5, 61, 63–70, 64, 72–75, 77, 85, 91–93
cellulose 27 bulking 200–208, 213, 214
cellulosic algae 27 foaming 215, 249, 253, 256–257, 496
cephalosporins 56 solids separation 140–141, 143
CFDA see carboxyfluorescein diacetate Clark-type oxygen microsensor 449, 449
chemical oxygen demand (COD) 73–74, 82–83, 83–84, classification
86–88, 91–93, 152–153, 316 artificial (monothetic) 171
readily biodegradable (RBCOD) 73, 76, 84, 87–89, filamentous bacteria 164–165, 169–172, 174
115, 148–149, 202, 290, 394 floc forming bacteria 165
slowly biodegradable (SBCOD) 87, 149, 151 kinetic properties 165
see also oxygen metabolic properties 164
chemicals natural (phenetic) 171–172
cell lysis 344 natural (phylogenetic) 172
coupling 39 physiological properties 165
tolerance to 123 principles of 171–172
chemists 81–84, 81 prokaryotes 170–171
chemolithoautotrophs 17, 40, 42–45, 462, 464 CLD see chlorite dismutase
ecophysiology 154–155, 157, 163–164 cleistothecium 34
microbial communities 117–119, 122 clone libraries
nitrogen removal 67, 107, 262–271 A-tailing 361
chemoorganoheterotrophs 40, 41–42, 67, 454, accumulation curves 369
477–480 competent cells 363–365
ecophysiology 152–154, 157, 163–164 DNA inserts 361
microbial communities 113–114, 122 ligation 361–363
nitrogen removal 67, 70, 268 pGEM-T vectors 362–363
see also organotrophs phylogenetic sequence analysis 368–370
chemostats 48 plasmids 360–361, 365, 367
chemotrophs 39 restriction enzyme analysis (REA) 365–366
chimeras 355, 368 schematics 360
chitin 27 screening 365–366
chlorine 55–56, 77, 204, 211, 254 sequence analysis 366–368
chlorite dismutase (CLD) 269 transformation 363–365
Chlorobi 46 vector PCR 365
denaturing gradient gel electrophoresis closed culture see batch culture, closed
(DGGE) 423 Clostridium 16
phylogeny 423, 490 Clostridium perfringens 132
Chloroflexi 466–469, 478, 484, 524–525, 533 CLSM see confocal laser scanning microscopy
bulking 192, 196–200, 208 coccoid cells 183, 225, 230, 235, 309, 328, 463, 469,
characterization 178 474, 475, 477, 525
ecophysiology 152, 158, 209 coliphage 132
FISH 103, 112, 186–187 Colpidium colpoda 493
foaming 196–197 Comeau-Wentzel model 283–284, 302–303, 499
microbial communities 114–115, 118 compartmentalized contact zones 204, 206–207
morphotype 524 competence specific proteins 23
photosynthesis 43, 46 competent cells 23, 362–365
phylogeny 193–194, 469, 490 Competibacter 91, 99–100, 112, 116
Index 649
carbon 314, 316–317, 317 decant phase 65, 75–76

FISH 293–294 Dechloromonas 275, 296, 305, 308, 346, 409
phosphorus removal 314–319, 315 Dechloromonas-related bacteria 293, 308
phylogeny 311 polyphosphate-accumulating organisms (PAO) 308
proton motive force (PMF) 315 Defluviicoccus 91, 100, 190, 346, 456, 486, 492, 535
see also Candidatus ‘Competibacter phosphatis’ carbon 314, 317
completely mixed systems 62, 64, 64, 65, 147, 201 glycogen-accumulating organisms (GAO) 116, 432,
confocal laser scanning microscopy (CLSM) 84, 92, 503
328–329 granules 435, 492, 503
conidia 14, 34, 120 proton motive force (PMF) 315
conjugation 23, 30, 124–125 Defluviicoccus vanus 294, 310, 311, 312–313, 458
contact stabilization process 64–65, 65, 69 Defluviicoccus-related organisms 294, 318–319
continuous culture dehydrogenases 106, 278, 303, 305, 314, 353, 500
open 48 denaturing gradient gel electrophoresis (DGGE)
substrates 54–55 421–427
contractile vacuoles 28 Alphaproteobacteria 423
control methods Bacteroidetes 423
bulking 200–214 Betaproteobacteria 423
foaming 253–258 Chlorobi 423
convergent evolution 172 Gammaproteobacteria 423
crateriform cell surfaces 531 microbial communities 108–109
Crenarchaeota 16 Nitrospirae 423
Critical Dilution Rate 54 PCR-DGGE 425–426
cryophytes 37 populations 108
cryptic growth 5 denitrification 64–76, 80, 84–88, 91, 152–156, 260–266,
crystal violet stain 336 271–277
CSLM see confocal scanning light microscopy Accumulibacter 115, 276–277
culture R-AN-D-N configuration 69, 75
Accumulibacter 102 R-D-N process 6, 69, 70, 75
Alphaproteobacteria 275, 277 denitrifying bacteria 6, 41
5-cyano-2,3-ditolyltetrazolium chloride (CTC) diversity 271–272
activity 337–338 ecophysiology 271–272
populations 106–107 microbial communities 114–115
cyanobacteria 3, 12, 14, 25, 27, 40, 43, 46 wastewater 272–277
foaming 218–219 see also nitrifying bacteria
microbial communities 119, 164, 188 denitrifying polyphosphate-accumulating organisms
phylogeny 490 (DPAO) 202, 285, 298
cytochromes 39–41, 47 density gradients 429–430, 430, 432
b/b6 272, 284, 303, 305, 352–353 deoxyribonucleic acid see DNA
c oxidase 268, 272 DEPC see diethylpyrocarbonate
cytoplasmic membranes 9 Dephanox process 74–75
Archaea 21–22 design
bacteria 21–22 FISH probes 381–390
prokaryotes 12, 15 sludge systems 61–62
cytosine 22, 175 detergents 56
cytoskeleton 25 Deuteromycotina 30, 35
eukaryotes 29 DGGE see denaturing gradient gel electrophoresis
prokaryotes 25 4, 6-diamidino-2-phenylindole (DAPI) 334–335, 380
diatoms 27, 30, 37
D 1, 4-diazabicyclo(2.2.2)octane (DABCO) 376, 380
DAP see meso-diaminopimelic acid diazotrophy 14, 47
data diethylpyrocarbonate (DEPC) 346–349
analysis 420, 451–452 Dietzia 222, 225–226, 228, 233–236, 243, 247, 471,
collection 342–343, 442–444 475–476
DBL see diffuse boundary layer differential centrifugation 101
diffuse boundary layer (DBL) 451 foaming 217

diffusion limitation theory 149 glycogen-accumulating organisms (GAO) 314–316
diglycerol tetraethers 22 ED see Entner-Doudoroff
Diluted Sludge Volume Index (DSVI) 143, 252 EDC see ethylene dichloride
dimethyl sulphide 42 EDTA see ethylenediaminetetraacetic acid
dimethyl sulphoxide 42, 354, 356 effluent, final 133–134
dinoflagellates 37, 130 electrical coupling 39
direct detection microarrays 402–403 electrochemical microsensors 449
disinfectants 55 electron microscope 327–328
dissimilatory iron-reducing bacteria (DIRB) 115 electrons 38
dissimilatory nitrate reduction 41, 268, 271–272 acceptors 42
dissimilatory sulphate reductase 406–407 carriers 38
dissolved oxygen (DO) 80, 83, 152–154 transport 27, 39–40
bulking 207–208, 212 electropherograms 419
foaming 224, 245 ELF see enzyme-labelled fluorescence
growth kinetics 49 ELISA see enzyme-linked immunosorbent assay
phosphorus removal 291, 319 Embden-Meyerhoff-Parnas (EMP) pathway 284, 290,
DNA 9 303, 314, 500–501
auto-sequencer 419–420 Emericella stellatus 34
binding proteins 23 empirical models 93
DNA-based stable isotope probing (DNA-SIP) endocytosis 17, 28, 37, 119, 129, 151
429–432 see also phagotrophy
extraction 343–349, 418 endoplasmic reticulum (ER) 17, 26–27, 26
humic acid 347 endospores 14, 16, 470
inserts, clone libraries 361 endosymbiosis 27, 36, 46
microarray fingerprinting 84 energy
microbial communities 343–349 growth 39
NaTCA 348–349 mechanisms 38–46
organization 22 sources 39–40, 46
PCR 355 engineering 79–81, 147–149
polysaccharide contamination 347 enhanced biological phosphorus removal (EBPR) 71
protein contamination 346–347 Accumulibacter 302, 500, 506
purity 346–347 Actinobacteria 306–308
T-RFLP 418–420 activated sludge system model ASM2 88
DO see dissolved oxygen (DO) aerobic granulation 78
DPAO see denitrifying PAO aerobic phase 501
DSVI see Diluted Sludge Volume Index Alphaproteobacteria 308–309, 316–317
anaerobic reactors 88–89, 297
E Bacteroidetes 118, 308–309
EBPR see enhanced biological phosphorus removal Betaproteobacteria 308–309
ecology, Accumulibacter 297–298 biodiversity 292
ecophysiology 102, 155–161 Candidatus ‘Accumulibacter phosphatis’ 295–305,
Accumulibacter 298 409
Actinobacteria 152–154, 162, 210 Comeau-Wentzel model 499
Alphaproteobacteria 209 competitors, glycogen-accumulating organisms
anaerobic ammonium oxidizers 277–279 (GAO) 316–319
Betaproteobacteria 153, 209 Dechloromonas-related bacteria 308
bulking 208–211 extracellular polymeric substances (EPS) 501
chemolithoautotrophs 154–155, 157, 163–164 FISH probes 293–294
chemoorganoheterotrophs 152–154, 157, 163–164 Gammaproteobacteria 308–309
Chloroflexi 152, 158, 209 glycogen-accumulating organisms (GAO) 292–294,
denitrifying bacteria 271–272 309–313, 316–319, 485–487
extracellular polymeric substances (EPS) 158 modified Mino model 499
filamentous bacteria 152–162, 209–210 molecular biology techniques 292–294
Firmicutes 154 planctomycetes 308–309
Index 651
polyphosphate-accumulating organisms (PAO) structure 26–29

292–308, 485, 506 thylakoid membranes 28
storage polymers 285–290 vacuoles 28, 28, 29
tetrad-forming organisms (TFO) 485–487 Eumycota 30
Entner-Doudoroff (ED) pathway 284 Euplotes affinis 493
envelopes 13 Euryarchaeota 16
environmental microarray analysis 398, 512 exopolyphosphatase (PPX) 288
enzyme-labelled fluorescence (ELF) 106–107, 338–339 exponential growth 48
filamentous bacteria 155–162 extracellular polymeric substances (EPS) 106, 461
Skermania piniformis 509 ecophysiology 158
enzyme-linked immunosorbent assay (ELISA) 228 enhanced biological phosphorus removal (EBPR)
enzymes 5 501
activity 98 flocs 92–93, 97–99, 123
exoenzymes 338–339 foaming 228, 241–242
flow cytometry (FCM) 445–446 granules 78, 99
lysis 345 membrane bioreactors 92–93
see also ‘individual enzymes’ phosphorus removal 302–303
Epistylis coronata 494 Zoogloea 461
Epistylis plicatilis 494 extremophiles 47, 271
EPS see exocellular polymeric substances; extracellular
polymeric substances F
ER see endoplasmic reticulum F-plasmid 23
erythromycin 56 facultative aerobes 41, 164
Escherichia coli 1 facultative anaerobes 40, 67, 261
cell envelope 13 facultative chemolithoautotrophs 42, 164
cell organization 13 FADH2 (reduced flavin adeninedinucleotide) 283–284
clone libraries 360, 363–364 FAM (6-carboxy-fluorescein) 412, 414–416
conjugation 23 famine, aerobic conditions 71, 283
FISH 375, 383 famine regime see feast/famine regimes
phosphate metabolism 303–304 fatty acid methyl ester (FAME) 229
phylogeny 267, 296 fatty acids
PPK1 287–288 complex 19, 21, 332
esterases 99, 106, 122, 158–160, 399 long chain 151, 153–154, 158, 243–245, 472–473
estrone, Betaproteobacteria 58 polar lipid derived (PLFA) 428
ethylene dichloride (EDC) 122 short chain 308, 316, 318
ethylenediaminetetraacetic acid (EDTA) 344, 347–348, volatile (VFA) 72–73, 88, 202, 244, 450
426, 430 FCM see flow cytometry
euglenoids 36–37 feast/famine regimes 71
Eukarya feed stage 71, 291, 308–309
cell membranes 27 feed strategy 319
cell organization 12, 17–18, 22 feeding 128, 133
cell organization 27 anaerobic reactors 283
cell walls 27 fermentation 40–41, 317
chloroplasts 27–28, 28 aerobic processes 57
classification 37–38 anaerobic processes 40
cytoskeleton 29 ferric ions 43
endoplasmic reticulum (ER) 26 ferric iron 42
eukaryotes 10–12 reductase 42
Golgi apparatus 27, 28 ferrous ions 43
mitochondria 27–28, 28 ferrous iron 42
nucleus 26, 28, 29 filamentous bacteria 12, 454
organelles 27–28 apical extension 47
PCR-DGGE 421–422 bulking 139–168, 200, 211–212
photosynthesis 43 characterization of 181–185
ribosomes 26 classification 164–165, 169–172, 174, 185–188
cultures 176–181 Candidatus ‘Alysiosphaera europea’ 517

ecology 162–167 Candidatus ‘Combothrix italica’ 517
ecophysiology 152–162, 209–210 Candidatus ‘Monilibacter batavus’ 517
elimination of 211–212 Candidatus ‘Sphaeronema italicum’ 517
engineering solutions 147–149 cell fixation 373, 377–378
enrichment 249–250 Chloroflexi 103, 112, 186–187
enzyme-labelled fluorescence (ELF) 155–162 Competibacter 293–294
FISH 186–187 DAPI staining 334–335, 380
FISH/MAR 155–162 Defluviicoccus 492
foaming 139–168, 219, 231, 249–250 ecophysiology 155–162
gene probes 196–197, 209–210 enhanced biological phosphorus removal (EBPR)
gene sequencing 177–180 293–294
Gram-negative filaments 454–469 Escherichia coli 375, 383
Gram-positive filaments 469–481 Firmicutes 187
growth conditions 145 FISH-MAR see FISH-MAR
identification 173–175, 185, 188–190 fluorescence intensity 375
isolation of 341 foam formers 226
kinetics 147 formamide 372
metabolic selection theory 147–148 glycogen accumulating organisms (GAO) 492
metabolism 163–166 hybridization 373–374, 378–379, 382
microautoradiography (MAR) 155–162 Meganema perideroedes 190, 517
molecular approaches 190 microbial communities 112
morphology 182–184, 195 Nitrospira 383, 384, 386–387, 389, 391–392, 498
nomenclature 172, 185 polyphosphate accumulating organisms (PAO) 492
NOx inhibition 148–149 pre-analysis treatment 376, 380
performance indicators 162–167, 265 probes see FISH probes
quantification 143, 231 protocol 377–380
selection theories 148–149, 162 pseudomonads 443
selective pressures 144–149 quantitative PCR 359
staining 182–184 rRNA 189–190
surveying 144–147 sample analysis 376
systematics 190 secondary ion mass spectrometry (FISH–SIMS) 437
taxonomy 169–190, 481–485 Skermania piniformis 509
wastewater treatment 199–200 tetrad-forming organisms (TFO) 294, 492
see also non-filamentous bacteria washing steps 374–375, 379
fill period 76 FISH probes 370–371
fimbriae 25 Amaricoccus 293
Firmicutes 16 brightness 381
bulking 192 characteristics 390
characterization 179 design 381–390
FISH 187 dissociation profiles 391
flocs 97 Ntspa445 design 386, 389
microbial communities 112, 114 optimization 390–391, 390–392
phylogeny 193, 469–471, 471, 490 sensitivity 371
physiology 154 sequences 186–187
FISH 84–85 specificity 371–372
Accumulibacter 104, 293, 359 synthesis 372–373
Alphaproteobacteria 517 thermodynamics 383–390, 385
Amaricoccus 293 FISH-MAR
Bacillus 187 detection 107
Bacteroidetes 112, 114 filamentous bacteria 155–162
Betaproteobacteria 104, 186, 294 limitations of 397
bulking 143–144, 147, 194–199 microbial communities 108–109
Candidatus ‘Alysiomicrobium bavaricum’ 156, 186, populations 107–109
517 protocol 394, 511
Index 653
five-stage Bardenpho (Phoredox) process 71 causative organisms 229–231

flagella 18, 25, 29–30, 181 Chloroflexi 196–197
flagellates 31–32, 37, 129–130, 133, 135–136, 495 classification of 251–252
flocculation 98, 191, 212–214, 213 cultures 221–224
deflocculation 59, 98 cyanobacteria 218–219
flocs 4–5, 59, 97–99, 97 dominant 222–223
Actinobacteria 97 filament enrichment 249–250
Alphaproteobacteria 97, 151–152 filament quantification 231
amorphous 520 filamentous bacteria 139–168
Betaproteobacteria 97 FISH probes 226
bright field microscopy 502 identification 217–220, 224
Candidatus ‘Accumulibacter phosphatis’ 97, 534 kinetic selection 257–258
Candidatus ‘Competibacter phosphatis’ 535 mean cell residence time (MCRT) 246–247
capsular material 508 metabolic selection 257–258
Defluviicoccus 535 Mycolata 215, 231–232
diffuse 140, 496 Nocardia 215–224, 229–232
extracellular polymeric substances (EPS) 92–93, non-acid fast Gordonia amarae-like organisms
97–99, 123 (GALO) 504
Firmicutes 97 nutrients 243–244
floc-forming organisms 97, 123–124, 147, 165 oxygen 244–245
Gordonia amarae-like organisms (GALO) filaments pH 246
509 staining 232
ideal 141 taxonomy 233–240
inter-floc bridging 496 temperature 245–246
Live Dead stain 491 foaming 12, 137–138, 141–142, 496
Normarski interference microscopy 502 Actinobacteria 215, 218, 233–234, 240
phase contrast microscopy 502 air-water interface 248–249
pinpoint 141 Alka-Seltzer test 252–253
structures 496 anaerobic digester supernatant 257
tetrad-forming organisms (TFO) 508 anaerobic reactors 217, 240, 255–258
weak 496 antifoam agents 254–256
flow cytometry (FCM) 101 baffles 256
activity assessment 445–446 biochemical oxygen demand (BOD) 216, 257
bacteria 444–445 biomass retention time 253–254
calibration 442 clarifiers 215, 249, 253, 256–257, 496
Candidatus ‘Accumulibacter phosphatis’ 510 control methods 253–258
cleaning 441 dissolved oxygen (DO) 224, 245
data acquisition 442–444 ecophysiology 217
enzymes 445–446 extracellular polymeric substances (EPS) 228,
instrument set up 441 241–242
membranes 445–446 measurement of 250–253
respiratory chain activity 446 molecular methods 224–229, 232
sample preparation 440 occurrence 216
sorting 447 physical control methods 256–257
fluorescence problems 216–217
intensity 375 r-K strategy 247–248
in situ hybridization see FISH Scum Index (SI) 250–252
fluorescence microscopy 8, 85, 325–326, 396 selective flotation 256
epifluorescence 232, 334, 336–338, 340, 393 thermal hydrolysis 256
FLUOS see carboxyfluorescein-N-hydroxysuccinimide thresholds 232–233
ester (FLUOS) trapping 249
foam formers 496 water sprays 256–257
acid fast bacteria 504 food/microorganisms (F/M) ratio 80–81
Actinobacteria 215, 218, 233–234, 240 formamide 372
bacteria 224 freeze–thaw lysis 345
fumarate 42, 283 acetate uptake 309, 313, 315–319

reductase 283, 314, 315, 500 aerobic granulation 503
functional genes 104, 108–109 Alphaproteobacteria 116, 118, 312
enhanced biological phosphorus removal (EBPR) 292, carbon 314, 316–318, 317
301 chemical oxygen demand (COD)/P ratio 316
microarrays 406–410 Defluviicoccus 116, 432, 503
PCR 352–353, 431 ecophysiology 314–316
T-RFLP 412, 416 enhanced biological phosphorus removal (EBPR)
fungi 26–27, 29 292–294, 309–313, 316–319, 485–487
biodiversity 121, 221–222 feed strategy 319
classification 29–30 FISH 492
microbial communities 120–121 glycogen degradation pathway 314
reproduction 30–35, 31–35 granules 492
true 30 metabolic models 89
microbial communities 116–117
G nitrate 314–316
GALO see Gordonia amarae-like organisms nitrite 314–316
gametangial conjugation 30 nitrous acid 319
Gammaproteobacteria 58, 113, 462–466 pH 318–319
characterization 178, 276 phosphorus removal 309–319
denaturing gradient gel electrophoresis (DGGE) 423 phylogeny 310–313
ecophysiology 154, 209 and polyphosphate-accumulating organisms (PAO)
enhanced biological phosphorus removal (EBPR) 313–319
308–309 proton motive force (PMF) 315
FISH 186, 293–294 sludge retention time (SRT) 319
flocs 97 temperature 319
glycogen accumulating organisms (GAO) 116, 118, GOLD see Genomes Online Database
310 Golgi bodies 27, 28
phylogeny 193, 262, 263, 267, 267, 423 Gordonia 164, 185, 193, 199–200, 218, 222, 224–236,
gas bubbles 240–241 475–476
gel electrophoresis 421–427 Gordonia amarae-like organisms (GALO) 141, 218,
generalized transduction 23 222, 340, 475–476
genes ecophysiology 159, 210
Accumulibacter 288, 305 flocs 509
clusters 288 foaming 215
expression 305 Live Dead stain 491
probes 196–197, 209–210 morphotype 529
recombination 22–23 probes 197
sequencing 177–180 taxonomy 236, 529
transfer 11 Gram stain 331–333
genomes 12, 22 Gram-negative bacteria 13, 15–16, 21, 332, 454–469
Accumulibacter 301–305 Gram-positive bacteria 15–16, 332, 469–481
Genomes Online Database (GOLD) 301 granules 24–25, 92, 99
genotypes 174–175 Beggiatoa 44, 520
Gilbertella persicaria 33 Defluviicoccus 435, 492, 503
gliding 25, 130, 181, 183 extracellular polymeric substances (EPS) 78, 99
Gliding Motile Filaments 188 glycogen-accumulating organisms (GAO) 492
Glomeromycota 32–35 sulphur 24–25
glucans 27 technology 77–78
D-glutamic acid 20 tetrad-forming organisms (TFO) 492
glycerol diethers 17, 22 Thiothrix 523
glycogen 24, 38, 88–89, 124, 290 green algae 36
Accumulibacter 290 growth 38, 38, 46–47
glycogen-accumulating organisms (GAO) abiotic factor tolerance 162–163
acetate 309, 313, 315–319 bacteria 162–163
Index 655
batch culture 48–49, 51 hyperthermophiles 16, 47

carbon 39 hyphae 30, 248
conditions, filamentous bacteria 145 Hypophyra 494
dissolved oxygen (DO) 49
energy 39 I
kinetics 47–51, 47–56 ideal flocs 141
pH 163 IFAS see integrated fixed-film activated sludge
pOU2u 163 imaging
redox potential 163 analysis 326–328
reducing power 39 problems 324–325
temperature 162–163 IMG see Integrated Microbial Genomes
guanine 22 inclusion bodies 24–25, 164, 181
guanosine tetraphosphate (ppGpp) 288, 304 influent 3–4, 5, 58
alkalinity 80
H flow data 79
habitats monitoring 81–90
ammonia-oxidizing Archaea (AOA) 263–264 pH 80
ammonia-oxidizing bacteria (AOB) 262–264 insertion sequence 22
Betaproteobacteria 262 INT see 2-p-iodo-phenyl0-3(nitro-phenyl)-5(phenyl)-
nitrite-oxidizing bacteria (NOB) 266–267 tetrazolium chloride (INT)
Haliscomenobacter hydrossis 145, 479–480, 531 integrated fixed-film activated sludge (IFAS) process 92
bulking 192–193, 200 Integrated Microbial Genomes database (IMG) 301, 305
ecophysiology 153, 165, 209 inter-floc bridging 140–141, 496, 521, 525, 533
FISH 187 intergenic spacer regions (ISRs) 174, 280
foams 218–220 internal transcribed spacer (ITS) 120, 184, 292, 299
gene probes 197 2-p-iodo-phenyl0-3(nitro-phenyl)-5(phenyl)tetrazolium
phylogeny 469 chloride (INT) 106–107, 337
haptophytes 37 ionophores 56
heterokonts 37 IRMS see isotope ratio mass spectrometry (IRMS)
heterotrophs 39–40 iron-oxidising bacteria 40, 43
Hfr strains 23 iron-reducing bacteria 42, 98, 115
high rate treatment systems 66 isocitrate lyase 305, 352–353
Hill’s epidemiological criteria 230 isoprenoid unit 22
histones 29 Isosphaera 180, 184, 190, 327, 327, 469, 479–480, 531
Holobasidium 35 isotope ratio mass spectrometry (IRMS) 428, 433,
homoacetogenic bacteria 41–42 434–435, 438–439
hopanoids 21 small-subunit rRNA (SSU-IRMS) 438
horizontal gene transfer 11 isotopes 427–439, 428, 430
algae 36 arrays 410–411, 515
Archaea 46 ISRs see intergenic spacer regions (ISRs)
microbial communities 124–125 ITS see internal transcribed spacer
prokaryotes 22–24, 22–25, 27, 170, 172
hot start PCR 357 J
humic acid 343, 346–347, 355 Johannesburg process 73, 73
hybrid models 93–94
hybridization 373–374, 378–379, 382 K
hydraulic retention time (HRT) 58, 63, 66, 79 K-strategists 166
hydrazine 45, 278 kinetics
hydrogen-oxidising organisms 42 batch culture 49–50, 49, 52
hydrogenosomes 26, 28 growth 47–51, 47–56
hydrophobicity Michaelis–Menten 287
detection of 340, 340 Monod 137
foam 241–242, 249, 252 selection 147, 165–166, 257–258
hydroxypropionate pathway 46 Korarchaeota 19, 490
hymenium 30 Ks values 50–51
L characterisation 177, 182, 185–186, 207

ladderanes 278 ecophysiology 275
lateral gene transfer FISH 190, 517
bacteria 262 phylogeny 193, 196
prokaryotes 22–24, 23–24 membranes
ligation 361–363 bioreactors (MBR) 6, 76–77, 92–93
light microscope cell organization 27
bright field 325 cytoplasmic 9, 11
care of 325 exclusion assays 446
compound 322–324 flow cytometry (FCM) 445–446
fluorescence microscopy 326 Gram-negative bacteria 21
image analysis 326–327 integrity 445–446
image problems 324–325 Isosphaera 327
magnification 322–323 nuclear 12, 27
microscopy 322–327 pores 93
Nomarski interference microscopy 326 stacks 15
objectives 323–324 meso-diaminopimelic acid (DAP) 20, 233
phase contrast microscopy 325–326 messenger RNA see mRNA
resolution 322–323 metabolic models 89
set up 323 glycogen-accumulating organisms (GAO) 89
lipid A 19, 21 metabolic pathways, Accumulibacter 89
lipophilic granules 151, 153, 335–336, 472, 526, 533 metabolic selection
lipopolysaccharide 19, 21 foaming 257–258
lithotrophs 39, 271, 277–278 theory 147–148
LIVE/DEAD stains 338, 491 metabolism 302–303
long primer random amplified polymorphic DNA metazoa 121, 122
(LP-RAPD) 109 methane 61
Lutzack-Ettinger process 68, 68 methanogenic Archaea 41–42
lysosomes 17, 26, 29 MiCA see Microbial Community Analysis
Michaelis-Menten kinetics 287
M microarrays
macroarrays 408–410 amplification 402–403
macromolecules 38, 98–99 communities 397–411
macronutrients 47 direct detection 402–403
magnetite 25 functional gene 406–408
Magnetobacterium 267, 423 isotope arrays 410–411
magnetotactic bacteria 25, 267 labelling strategies 403–404
magnification 322–323 microbial communities 397–411
manganese 42, 278 nucleic acid detection 402–403
mannans 27 PhyloChip 406–408
manual monitoring 79–85 phylogeny 410–411
MAR see microautoradiography probe design 400–402
Mastigomycotina 30, 31–32 probe type 399–400
mastigonemes 495 quantification 404–405
Mastigophora 37 RHC-PhyloChip-based diversity analysis 514
maximum likelihood algorithm 369 rRNA 406–408
ppk1 gene sequences 300 schematics 512
rRNA gene sequences 458 screening potential 408–410
maximum parsimony algorithm 369 surface type 398–399
MBR see membrane bioreactors microautoradiography (MAR)
MCRT see mean cell residence time autoradiography 396
MDS see MultiDimensional Scaling filamentous bacteria 155–162
mean cell residence time (MCRT) 81 FISH-MAR 394, 511
foam formers 246–247 incubation 395
Meganema perideroedes 150, 152, 155, 457–458 microscopy 396
Index 657
populations 105, 107 rRNA analysis cycle 96

protocol 394–396 selective factors 166–167
washing 395 SIP 108–109
microbial communities 6–12, 38–46 species richness 366
abiotic factor tolerance 123 specific growth rate (mu) 121–123
activated sludge plants 95–126 starvation response 124
Aeolosoma 122 structure 113–121
algae 121 study methods 100–109
Archaea 111–113, 118 sulphate-reducing bacteria 116
bacteria 111–113 sulphur-oxidizing bacteria 119
bacteriophages 109–111 survival factors 121–124
batch culture 49–50, 49, 51–53, 51–52 T-RFLP 108–109
Betaproteobacteria 113–117, 118 tardigrades 122
biodiversity 111–113 TGGE 108–109
cellular organisation 9–12 viruses 109–111
chemical tolerance 123 Microbial Community Analysis (MiCA) 413, 421, 516
chemolithoautotrophs 117–119, 122 microbiologists 84–85, 85
chemoorganoheterotrophs 113–114, 122 microcolonies
chemostatic culture 53–54 ammonia-oxidizing bacteria (AOB) 117, 118, 498
Chloroflexi 114–115, 118 nitrite-oxidizing bacteria (NOB) 498
composition 109–113 microfilaments 29
continuous culture 54–55 micromanipulation 100–101
cyanobacteria 119, 164, 188 micromanipulators 341
denaturing gradient gel electrophoresis (DGGE) micropollutants 93
108–109 microscopy 322–329
denitrifying bacteria 114–115 air-dried smears 330
enhanced biological phosphorus removal (EBPR) 118, confocal laser scanning microscopy (CLSM)
291–292 328–329
Firmicutes 112, 114 data collection 342–343
FISH 112 electron microscopy 327–328
FISH-MAR 108–109 identification 189, 217–220
floc forming 123–124 light microscopy 322–327
foaming 217–224 microautoradiography (MAR) 396
fungi 120–121 quantification 231–232
glycogen-accumulating organisms (GAO) 116–117 sample preparation 329–331
growth 38, 38, 46–51, 55–56 sample storage 329
horizontal gene transfer 124–125 sampling 329
iron bacteria 115 slide handling 329
isotopes 428 wet mounts 330
location 96–99 microsensors 448–452
manipulation 125 data interpretation 451–452
metazoa 121 substrate transport 450–451
microarrays 397–411, 410–411 microtubules 29
nematodes 122 Millisia 234–235, 243, 475–476
nitrifying bacteria 117–118 Millisia brevis 222, 471, 477–478
nutrition 46–47 Mino model 284–285
oligochaetes 122 mitochondria 26–28, 28
PCR 108–109 mixed liquor 59, 74
PHA-accumulating bacteria 116 ammonium sulphate dosing 497
phylogeny 423 cavitation pilot plant 213
polymer-degrading bacteria 115 suspended solids (MLSS) 66, 80, 143
population dynamics 91 volatile suspended solids (MLVSS) 79–81, 84
protozoa 119–120 mixotrophs 42, 45, 47, 145, 154, 157
RIS-RNA fingerprinting 108–109 MMBR see moving bed biofilm reactor
rotifers 122 models 86–94
modified Mino model 499 nematodes 120–122, 122

molecular biology 190, 224–229, 232, 292–294 nested PCR 357
Monod kinetics 137 nicotinamide adenine dinucleotide (NADSþs) 38
monitoring 79–85, 143–144 phosphate (NADPSþs) 38
bulking 143–144 Nile blue A stain 335–336
influent 81–90 NirS see nitrite reductase
monothetic classification see artificial (monothetic) nitrate 41
classification Accumulibacter 300
morphology glycogen-accumulating organisms (GAO) 314–316
bulking 192–194 reduction 300
Candidatus ‘Nostocoida limicola’ 474 utilization 314–316
filamentous bacteria 182–184, 195 nitric oxide 41
morphotype 193–194 nitrification
Chloroflexi 524 nitrogen removal 260–261, 260–262
Gordonia amarae-like organisms (GALO) 529 R-AN-D-N configuration 75
Nostocoida limicola 524 R-D-N process 70
most probable number (MPN) techniques 105 nitrifying bacteria 6, 40, 44–45
motility 25, 29, 37, 181 membrane stacks 15
moving bed biofilm reactors (MBBR) 92 microbial communities 117–118
MPN see most probable number (MPN) techniques see also denitrifying bacteria
mRNA 22, 29 nitrite 314–316
MultiDimensional Scaling (MDS) 426 oxidation 268
Multiple Probe Concept 513 nitrite oxidoreductase (Nxr) 268
multistage treatment systems 66, 66 nitrite reductase (NirS) 41, 272–273, 278
municipal wastewater treatment plants 196–200 gene arrays 406, 409
mureins 19–20 primers 350, 352–353
pseudomurein 20 nitrite-oxidizing bacteria (NOB) 44–45, 70
mutualism 166 habitats 266–267
ammonia-oxidizing bacteria (AOB) 166 microcolonies 498
mycelium 30, 235 nitrite oxidation 268
mycobacteria 218, 222 nitrogen removal 266–271
Mycobacterium 221–222, 228, 233–236, 239, 471, phylogeny 266–267, 267
475–476 wastewater treatment 268–271
Mycolata 47, 140–142, 150, 154, 165 Nitrobacter 45, 117, 266, 267, 268–270
bulking 193–195, 198–200 nitrogen 71–75
characterization 225–231, 235 Actinobacteria 276–277
foam formers 215, 231–232 ammonia 259
foaming 162–164, 215, 218, 220, 222, 240–258 ammonia-oxidizing Archaea (AOA) 263–264
quantification 231–233 ammonia-oxidizing bacteria (AOB) 262–266, 270,
taxonomy 224, 233–240, 236–239 277–278
mycolic acids 19, 227, 233–236, 241–242, 332 ammonium 259
anaerobic ammonium oxidation (ANAMMOX)
N 277–280
N-acetyl glucosamine 19, 20, 27, 158 anaerobic reactors 260–261, 266
N-acetyl muramic acid 17, 19, 20, 234 Betaproteobacteria 272, 275–277
nalidixic acid 56 biochemical oxygen demand (BOD) 259–260
Nanoarchaeota 19 C:N ratio 259–260
Nanoarchaeum equitans 19, 22 Candidatus ‘Accumulibacter phosphatis’ 277
NaTCA method 348–349 chemolithoautotrophs 67, 107, 262–271
natural (phenetic), classification 171–172 chemoorganoheterotrophs 67, 70, 268
natural (phenetic) classification 171–172 cycle 260
natural (phylogenetic) classification 172 denitrification 260–262, 271–277
naturally abundant isotopes 437–438 dioxide 264
Neighbour-Joining algorithm 307, 311, 369, 370 gas 41
Neisser stain 181–182, 184, 333–334, 505 nitrification 260–262
Index 659
nitrite-oxidizing bacteria (NOB) 266–271 organotrophs 39–40

oxidation 260 see also chemoorganoheterotrophs
plant configurations 67–69 oxidation 4, 38–40, 43–45
sources 47 aerobic conditions 152–161
see also CANON process oxygen 2, 5–6
Nitrospira 45, 117–118, 118, 166–167, 266–270, 267 foam formers 244–245
denaturing gradient gel electrophoresis (DGGE) 423 photosynthesis 46, 449
FISH 383, 384, 386–387, 389, 391–392, 498 uptake rates (OUR) 83, 83
FLUOS-labelled probes 510 see also chemical oxygen demand (COD); dissolved
phylogeny 384, 423, 490 oxygen
nitrous acid 319
nitrous oxide 41, 271 P
Nocardia 142, 145, 475–476 PAC see polyaluminum chloride
foam 215–224, 229–232 PAO see polyphosphate-accumulating organisms
growth 243–257 PAP see polyphosphate (polyP):AMP phosphotrans-
phylogeny 370, 471 ferase enzyme
taxonomy 233–239 paraformaldehyde (PFA) 227, 239, 273
nomenclature 172, 185 PCR 108–109
non-filamentous bacteria 140, 149–150 16S rRNA 350, 352
see also ‘individual bacteria’ 23S rRNA 351–352
Normarski interference microscopy 325–326, 502 aconitase B 352–353
Nostocoida limicola ammonia monooxygenase 352–353
I 187, 471 amplification 351, 354–355
II 190, 225, 230 annealing temperature 355
III 114, 180, 187, 469, 479–480 Candidatus ‘Accumulibacter phosphatis’ 353
see also Candidatus ‘Nostocoida limicola’ clone libraries 365
Ntspa445 386, 389 cycle number 355
nuclear membrane 12, 13, 27 cytochrome b/bU6u 352–353
nucleic acid 346, 402–403 DNA template 355
nucleoid 12, 13, 22, 38 functional genes 352–353
nucleolus 29 hot start 357
nucleus 10, 12, 37, 494 isocitrate lyase 352–353
eukaryotes 26, 28, 29 method 356–357
prokaryotes 22 nested 357
nutrients nitrite reductase 352–353
bulking 207–208 PCR-DGGE 421–427
foam formers 243–244 polyhydroxyalkanoate (PHA) synthase 352–353
nutrition 36, 40, 46–47 polyphosphate kinase 352–353
Archaea 40–46 primers 350–354
product digestion 418–420
O profiling 108–109
obligately anaerobic organisms 41 real-time quantitative (qPCR) 358–359
Oidium candidum 34 reverse transcription (RT-PCR) 358
OLAND (O2-limited autotrophic succinate dehydrogenase 352–353
nitrification-denitrification) system 279 T-RFLP 418–420
oligochaetes 121, 122 technology 350–359
Oomycota 30, 31, 32 touchdown 357
oospores 31 vector PCR 365
open culture see continuous culture penicillins 20, 36, 56
open reading frame (ORF) 287–288 Penicillium digitatum 34
operational taxonomic unit (OTU) 366–367 pentaphosphate (pppGpp) 288
Opercularia 128, 135, 494 peptidoglycans 17, 19–20, 20–21, 27, 233–234
optical microsensors 449–450 performance indicators 3, 58–59, 125
optode 449–450 filaments 162–167, 265
organelles 27–28 protozoa 134–137
periplasmic space 20 glycogen-accumulating organisms (GAO) 309–319

perithecium 34 Mino model 284–285
permeabilization phosphotransferase 289
Candidatus ‘Microthrix parvicella’ 227 polyhydroxyalkanoates 289–290
probes 104, 159, 198, 227, 337, 446 polyphosphate 285–286, 288–289
staining 338 polyphosphate kinase 286–288
Peziza 34 uptake 117, 285
PFA see paraformaldehyde phosphorylation 27
pGEM-T vectors 361–363, 365 phosphotransferase 289
pH 80 photolithoautotrophs 40, 40, 45–46
ammonia-oxidizing bacteria (AOB) 80 photolithotrophs 40
bacteria 163 photoorganoheterotrophs 40, 40
bulking 203–204, 208 photophosphorylation 27
foam formers 246 photosynthesis
glycogen-accumulating organisms (GAO) 318–319 algae 43, 449
growth 163 bacteria 43
polyphosphate-accumulating organisms (PAO) Chloroflexi 43, 46
318–319 Eukarya 43
PH2MV see polyhydroxy-2-methylvalerate prokaryotes 43
PHA see polyhydroxyalkanoate phototrophs 39
phage see bacteriophage Phragmobasidium 35
phagotrophy 28 PHV see polyhydroxyvalerate
see also endocytosis phycobilins 36–37
phase contrast microscopy 325–326, 502 Phycomyces blakesleeanus 33
PHB see polyhydroxybutyrate PhyloChip 406–408, 513
phenetic classification see natural (phenetic) phylogenetic classification see natural (phylogenetic)
classification classification
phenotypes 175 phylogenetic sequence analysis 368–370
Phoredox process 71–72 phylogeny
Five-stage 71–72, 72 Actinobacteria 193, 471
Three-stage 72 Actinomadura 221, 223, 233, 370
Two-stage (A/O) 72, 72–73 Alphaproteobacteria 193, 196, 198, 266, 267, 423
see also Bardenpho process Amaricoccus 310, 311
phosphate 303–304 ammonia-oxidizing bacteria (AOB) 262–264, 263
phospholipid 21 anaerobic ammonium oxidizers 277–279
phosphorus 71–75 Archaea 11, 262, 263, 490
Actinobacteria 292–293, 306–308, 307, 310–311, Bacillus 470–471, 471
316–318 Bacteria 490
anaerobic reactors 281–286, 290–291 bacteria 11, 490
anaerobic zone 290–291 Bacteroidetes 118, 423, 469
anoxia 285 Beggiatoa 458
Betaproteobacteria 295, 305, 308–309, 313 Betaproteobacteria 193–194, 198, 263, 267, 423,
biochemical oxygen demand (BOD) 281–283 519
Candidatus ‘Accumulibacter phosphatis’ 286–287, Candidatus ‘Competibacter phosphatis’ 310–311
295 Candidatus ‘Microthrix calida’ 193, 222
Comeau–Wentzel model 283–284 Chlorobi 423, 490
Competibacter 314–319, 315 Chloroflexi 193–194, 469, 490
denitrifying polyphosphate-accumulating organisms community analysis 423
(DPAO) 285 Competibacter 311
dissolved oxygen (DO) 291, 319 cyanobacteria 490
enhanced biological phosphorus removal (EBPR) 282, Defluviicoccus vanus 294, 310, 311, 312–313, 458
285–290 Defluviicoccus vanus-related organisms 312–313
exopolyphosphatase 288 Escherichia coli 267, 296
extracellular polymeric substances (EPS) 302–303 Firmicutes 193, 471, 490
glycogen 290 Gammaproteobacteria 193, 262, 263, 267, 267, 423
Index 661
glycogen-accumulating organisms (GAO) 310–313 polyhydroxybutyrate (PHB) 24, 88, 182–184, 288,
Haliscomenobacter hydrossis 469 335–336
Meganema perideroedes 193, 196 FISH 151, 154–156, 159–160
microarrays 410–411 polyhydroxyvalerate (PHV) 283, 501
morphotypes 193–194 polymer degrading bacteria 115
nitrite-oxidizing bacteria (NOB) 266–267, 267 polymerase chain reaction see PCR
Nitrospira 384, 423, 490 polymers 115
planctomycetes 469 polyP see polyphosphate
Proteobacteria 458 polyphasic taxonomy 174
rRNA 263, 267, 296, 370, 423, 458, 469, 471, 490 polyphosphate 24
rRNA gene sequences 263, 267, 296, 370, 423, 458, Accumulibacter 24, 286, 288
469, 471, 490 AMP phosphotransferase enzyme (PAP) 289
sequence analysis, clone libraries 368–370 Archaea 285
T-RFLP 421 function, phosphorus removal 286
tetrad-forming organisms (TFO) 311, 312 inorganic 285
physical control methods 256–257 phosphorus removal 285, 288–289
physiology see ecophysiology stains 333–334
phytanyl unit 22 polyphosphate kinase (PPK)
Phytophthora cinnamomi 31 1 (PPK1) 287–288, 300
phytoplankton 281 2 (PPK2) 287
pili 23, 25 phosphorus removal 286–287
pin point flocs 496 primers 352–353
pine tree-like organisms (PTLO) 218–220, 239, polyphosphate-accumulating organisms (PAO) 281, 288
244–245, 249, 476–477 Accumulibacter 117, 295–297, 301, 502, 506
pinpoint flocs 141 Actinobacteria 306–308
Planctomycetales 479 carbon 316–318, 317
planctomycetes 12, 19, 114, 531 COD/P ratio 316
enhanced biological phosphorus removal (EBPR) Dechloromonas-related bacteria 308
308–309 denitrifying (DPAO) 285
membranes 327, 327 enhanced biological phosphorus removal (EBPR)
phylogeny 416, 469, 490 292–308, 485, 506
plant configurations, nitrogen 67–69 feed strategy 319
plasmids 22–23, 124–125 FISH 492
clone libraries 360–361, 365, 367 and glycogen-accumulating organisms (GAO) 313–319
plastids 36–37, 46, 130 metabolic models 89
PLFA see polar lipid derived fatty acid nitrous acid 319
plug flow systems 62–64 pH 318–319
return activated sludge (RAS) 63 probes 502
step aeration 63 proton motive force (PMF) 315
step feed 64 sludge retention time (SRT) 319
substrates 206 temperature 319
tapered aeration 63 polysaccharides 19, 27, 347
PMF see proton motive force exocellular 242
pO2 163 lipopolysaccharide 19, 21
bacteria 163 polythetic taxa 172
growth 163 polyvinylpolypyrrolidone (PVPP) 347
polar lipid derived fatty acid (PLFA) 428, 428, 435 polyvinylpyrrolidone (PVP) 347–348
stable isotope probing (PLFA-SIP) 436–439 population dynamics 91, 297–298
polyaluminum chloride (PAC) 210–211, 255 population ecology 298–299
polyhydroxy-2-methylvalerate (PH2MV) 283, 500 populations
polyhydroxyalkanoate (PHA) 60, 72, 88–89, 289–290, 16S rRNA profiling 108
335 Accumulibacter 297–299
accumulating bacteria 116 active cell detection 106–108
Mino model 284 biomass ATP content 106
synthase 116, 299, 352–353 CTC detection 106–107
dehydrogenase detection 106 genetic recombination 22–23

denaturing gradient gel electrophoresis (DGGE) 108 groups 15–25
differential centrifugation 101 horizontal gene transfer 22–24, 22–25, 27, 170, 172
dynamics 137–138, 297–298 intracellular inclusion bodies 24–25
ecology 298–299 lateral gene transfer 22–24, 23–24
enzyme labelled fluorescence (ELF) 106 motility 25
esterase detection 106 nucleoid 12, 13
fingerprinting 107 photosynthesis 43
FISH 101–105 structure 15
FISH-MAR 107–109 vacuoles 25
flow cytometry (FCM) 101 proteins
identification 102–103 competence specific 23
INT detection 106–107 contamination 346–347
LP-RAPD 109 DNA binding 23
microautoradiography (MAR) 105, 107 hydrolyzing organisms 339
micromanipulation 100–101 Proteobacteria 16, 41, 43, 112, 196, 262, 311, 458, 490
most probable number (MPN) techniques 105 see also Alphaproteobacteria; Betaproteobacteria;
protozoa 137–138 Gammaproteobacteria
qPCR 104, 109 proteolytic organisms 339
quantification 102–103 proton gradient see proton motive force
respirometry 106 proton motive force (PMF) 39, 283, 314, 315
ribosomal intergenic spacer length (RIS-rRNA) protozoa 26, 36–37
108–109 activated sludge process 57–94
sampling 100–101, 100–102 Aspidisca lynceus 493
SSCP analysis 108 behaviour patterns 128
stable isotope probing (SIP) 107–109 biochemical oxygen demand (BOD) 133–134
T-RFLP 108–109 ciliated 493
temperature gradient gel electrophoresis (TGGE) 108 feeding patterns 128
viable cell detection 105–106 final effluent quality 134
pores, membranes 93 food chain, activated sludge biomass 138
porins 19, 21 microbial communities 119–120
potentiometric sensors 449 performance indicators 134–137
PPK see polyphosphate kinase plant performance effects 135
PPX see exopolyphosphatase sludge loading effects 135
primers pseudomonads 132, 443
design 353–354 pseudomurein 20
nitrite reductase (NirS) 350, 352–353 PTLO see pine tree-like organisms
PCR 350–354 pure cultures 221–224
PCR-DGGE 422 PVP see polyvinylpyrrolidone
T-RFLP 414–416 PVPP see polyvinylpolypyrrolidone
Probe Match tool 387 pyrolysis mass spectrometry (PyMS) 221
probes
brightness 381 Q
design 400–402 qPCR see quantitative PCR
dissociation profiles 391 quantification
FISH 372–373, 381, 391 microarrays 404–405
microarrays 399–402 populations 102–103
permeabilization 104, 159, 198, 227, 337, 446 quantitative PCR (qPCR) 104, 109
polyphosphate-accumulating organisms (PAO) 502 Accumulibacter 359
synthesis 372–373 and FISH 359
prokaryotes 10–25
classification 170–171 R
cytoplasmic membranes 12, 15 R-AN-D-N [Regeneration–Anaerobic–Denitrification
cytoskeleton 25 (anoxic)–Nitrification (aerobic)] process 69,
endospores 14, 16 75, 75
Index 663
R-D-N (Regeneration–Denitrification–Nitrification) respiratory chain 268, 271, 445–446

process 69, 70 respirometry 106
r-K strategy 247–248 restriction enzyme analysis (REA) 365–366
r-strategists 166 restriction fragment length polymorphism (RFLP),
Ralstonia eutropha 289, 289 terminal (T-RFLP) 412–421
random amplified polymorphic DNA (RAPD) retention time 204, 253–254
long primer (LP-RAPD) 109 return activated sludge (RAS) 63, 74
RAPD-PCR 174 reverse citric acid cycle 46
RAS see return activated sludge reverse sample genome probing (RSGP) 408
RBCOD see readily biodegradable COD reverse transcription PCR (RT-PCR) 358
react phase 76 rHa see reduced hysteresis area
readily biodegradable chemical oxygen demand RHC-PhyloChip-based diversity analysis 514
(RBCOD) 73, 87 Zoogloea 514
real-time quantitative PCR (qPCR) 85, 358–359, 359 Rhizopus 33
RecA 23, 172 Rhodococcus 154, 218, 221–226, 233–239, 471, 475–476
red algae 36 ribonucleic acid see RNA
redox potential 40, 80, 163 ribosomal DNA restriction analysis (ARDRA),
reduced hysteresis area (rHa) 143 amplified 366
reducing power 38–40, 39 ribosomal intergenic spacer length (RIS-rRNA) 108–109
growth 39 ribosomal RNA see rRNA
reductive dechlorination 42 ribosomes 22, 26
regeneration 69–70, 70, 75, 75 70S 29
reproduction 80S 29
Absidias pinosa 33 rifamycin 56
Allomyces arbusculus 32 RNA 9
Ascomycota 34 cell lysis 344–345
asexual 30 communities 343–349
Aspergillus niger 34 degradation of 347
Basidiomycota 35 NaTCA 348–349
Chytridiomycota 32 ribosomal see rRNA
Emericella stellatus 34 sampling 343–344
fungi 30–35, 31–35 stable isotope probing (RNA-SIP) 432–436, 433
Gilbertella persicaria 33 rods
Holobasidium 35 Acinetobacter johnsonii 521
Oidium candidum 34 Type1863 521
Oomycota 31 rotifers 122
Penicillium digitatum 34 rRNA 29
Peziza 34 16S 27
Phragmobasidium 35 18S 30
Phycomyces blakesleeanus 33 26S 30
Phytophthora cinnamomi 31 analysis 96
Rhizopus 33 FISH 189–190
Saprolegnia ferax 31 microarrays 406–408
Schizosaccharomyces octosporus 34 phylogeny 263, 267, 296, 370, 423, 458, 469, 471, 490
Scopulariopsis brevicaulis 34 RSGP see reverse sample genome probing
sexual 30 RT-PCR see reverse transcription PCR
Sordaria fimicola 34
Sporobolomyces 35 S
Thraustothecium clavatus 31 S-layer 19
Zygomycota 33 samples
Zygorhynchus moelleri 33 FISH 376
resolution 322–323 flow cytometry (FCM) 440
respiration 27, 39–40 microscopy 329–331
aerobic processes 41, 71, 164, 209–211 RNA extraction 343–344
anaerobic processes 41–42, 67, 164 stains 331–340
storage 329 sludge

sampling 100–102 age 6, 76, 81, 205–206
Accumulibacter 100 disposal 60–61
Sanger sequencing 367 DNA extraction 343–349
Saprolegnia ferax 31 glycogen-accumulating organisms (GAO) 319
Sarcodina 37 loading 80–81, 135
SBCOD see slowly biodegradable chemical oxygen polyphosphate-accumulating organisms (PAO) 319
demand protozoa 135
SBRs see sequencing batch reactors retention time (SRT) 81, 319
scanning electron microscopy (SEM) 8, 327–328 RNA extraction 343–349
sample preparation 330–331 sludge plants
Schizosaccharomyces octosporus 34 configurations 67–69
Scopulariopsis brevicaulis 34 performance 135, 162–167
screening Sludge Volume Index (SVI)
clone libraries 365–366 small-subunit rRNA (SSU), IRMS (SSU-IRMS) 438
macroarrays 408–410 SND see simultaneous nitrification and denitrification
microarrays 408–410 sodium dodecyl sulfate (SDS) 344, 347, 355, 374,
scum see foam 378–379
Scum Index (SI) 250–252 sodium trichloroacetate (NaTCA) 348–349
SDS see sodium dodecyl sulfate solid separation 140–141, 143
secondary clarifiers 91–93, 140, 206–208, 213 soluble microbial products (SMP) 93
secondary ion mass spectrometry (SIMS) 437 soluble nonbiodegradable (Susi) fraction 83
sedimentation 212–214 soluble readily biodegradable (Sbsi) fraction 83
selection 149–162 Sordaria fimicola 34
factors 166–167 sorting 447
foaming 256 specialized transduction 23
pressures 144–149 species
selective flotation 256 delineation 299–300
selenium 42 richness 366
SEM see scanning electron microscopy specific growth rate (m) 48, 121–123
sequence analysis 366–368 specific oxygen uptake rates (SOUR) 80
sequencing 177 Sphaerotilus natans 19, 43, 145, 458, 459, 518
batch reactors (SBRs) 5, 62, 75–78, 78, 205 characterization 177, 186, 196, 219
settleability 59, 69, 99, 140–141 ecophysiology 153, 165
tests 143 spindle body 29
settle phase 76 Spirostomum 493
sex pilus 23 sporangia 30, 31–33
SHARON (single reactor system for high rate sporangiospores 30, 31
ammonium removal over nitrite) 70 see also zoospores
sheaths spores 30
bacteria 19, 188 coat 16
stains 336 Sporobolomyces 35
type0041 507 sporozoa 37
SI see Scum Index sporozoites 37
SIMS see secondary ion mass spectrometry SRB see sulphate-reducing bacteria
simultaneous nitrification and denitrification (SND) 76 SS see suspended solids
SIP see stable isotope probing SSCP 108, 413
Skermania piniformis 476–477, 530 SSVI see Stirred Sludge Volume Index
acetate uptake 108 stable isotope probing (SIP) 107–109
enzyme-labelled fluorescence (ELF) 509 DNA-based (DNA-SIP) 429–432
slender coiled filaments 188 isotopes 427–439
slides 329 naturally abundant isotopes 437–438
slow settling velocities 214 polar lipid derived fatty acid-based (PLFA-SIP)
slowly biodegradable chemical oxygen demand 436–437
(SBCOD) 87 RNA-based (RNA-SIP) 432–436, 433
Index 665
staining sulphur 47
Actinobacteria 332 Beggiatoa 44, 119, 145, 202
Beggiatoa 183 granules 24–25
Candidatus ‘Accumulibacter phosphatis’ 334 sulphur-oxidizing bacteria 43–44, 119
foam formers 232 surfactants 242–243
permeabilization 338 survival factors 121–124
stains Susi see soluble nonbiodegradable (Susi) fraction
acid fast bacteria 332–333 suspended solids (SS) 61, 65
Actinobacteria 332 mixed liquor (MLSS) 66, 80, 143
Alcian blue 336 SVI see Sludge Volume Index
carbol fuschin 332–333 symbiosis 36
crystal violet 336
5-cyano-2, 3-ditolyltetrazolium chloride (CTC) T
337–338 T-even bacteriophages 10
DAPI 334 T-RFLP 108–109, 516
filamentous bacteria 182–184 Actinobacteria 413, 415
foaming 232 data analysis 420
Gram 331–333 DNA auto-sequencer 419–420
INT activity 337 DNA extraction 418
lipophilic material 335–336 electropherograms 419
LIVE/DEAD 338 PCR 418–420
Neisser 333–334 phylogeny 421
Nile blue A 335–336 primers 414–416
polyhydroxyalkanoates (PHA) 335 tapered aeration systems 63, 63
polyhydroxybutyrate (PHB) 335–336 tardigrades 122
polyphosphate detection 333–334 taxonomy
samples 331–340 Actinobacteria 179, 187, 233
sheath detection 336 Beggiatoa 185, 188
Sudan black 335 Candidatus ‘Microthrix parvicella’ 234
viability 337–338 filamentous bacteria 169–190
starch 36, 115, 151 foam formers 233–240
starvation 124 Gordonia amarae-like organisms (GALO) 236, 529
step feed 63, 63–64 Mycolata 224, 233–240, 236–239
sterols 17, 27, 120 TCA see tricarboxylic acid cycle
Stirred Sludge Volume Index (SSVI) 143 TEFL see total extended filament length
storage selection theory 148 teichoic acids 19
Straight Short Multicelled Filaments 188 TEM see transmission electron microscopy
Streptococcus 471 temperature
Streptomyces 22, 221, 223, 233, 238 bacteria 162–163
streptomycin 56 bulking 203–204, 208
substrates foam formers 245–246
batch culture 51–53, 51–52 glycogen-accumulating organisms (GAO) 319
bulking 204–207, 205–206, 207 gradient gel electrophoresis (TGGE) 108–109
continuous culture 54–55 growth 162–163
microsensors 450–451 polyphosphate-accumulating organisms (PAO) 319
phosphorylation 39–40 terminal restriction fragment length polymorphism see
plug flow systems 206 T-RFLP
SBR systems 205 tetracyclines 56
transport 450–451 tetrad-forming organisms (TFO) 101–102, 114, 534
unbalanced growth 205 enhanced biological phosphorus removal (EBPR)
succinate 42 485–487
dehydrogenase 352–353 FISH 294, 492
Sudan black stain 335 flocs 508
sulphate 41 granules 492
sulphate-reducing bacteria (SRB) 41, 114, 116, 415–416 phylogeny 311, 312
tetramethyl ammonium chloride (TMACl) 401, 405 U

Tetrasphaera 115, 140, 153, 161, 179, 292–293, 307, UAP see utilisation associated products
471, 487 UCT see University of Cape Town
thermal hydrolysis 256 unicells 30
foaming 256 unified process 76
thermodynamics 383–390, 385 University of Cape Town (UCT) 73
Thiothrix 465–466, 523 sludge configuration 74, 74
Thraustothecium clavatus 31 unrooted maximum likelihood tree 296, 300
Thuricola folliculata 494 Unweighted Pair-wise Grouping with Mathematical
thylakoid membranes 28, 43 Averages (UPGMA) 426
thymine 22 utilisation associated products (UAP) 93
TMAC1 see tetramethyl ammonium chloride
total extended filament length (TEFL) 143–144, 150, V
158, 231 vacuoles 25
Total Kjeldahl Nitrogen (TKN) 84 contractile 28
TKN/COD ratio 84 eukaryotes 28, 28, 29
total suspended solids (TSS) 143 prokaryotes 25
touchdown PCR 357 vacuome 29
toxic compounds 203–204 valinomycin 56
bulking 203–204 vancomycin 56
transcription 22 vector PCR 365
transduction 9, 23 VFAs see volatile fatty acids
transfection 23 viability 105–106, 337–338
transformation 23 Virginia Initiative Project (VIP) process 74
clone libraries 363–365 virions 9
translation 22 viruses 9, 10, 109–111
transmission electron microscopy (TEM) 8, 327 volatile fatty acids (VFAs) 88–89, 283, 289, 297, 302
transposons 22 volatile suspended solids (MLVSS), mixed liquor
trapping 249 79–81, 84
tricarboxylic acid (TCA) cycle 40, 283–284, 289–290, volatile suspended solids (VSS) 84, 111, 199, 228,
302–303, 499–501 231–232, 249–250
Trichococcus flocculiformis 183, 328, 469–470, Vorticella 128, 133, 135, 494
471, 525
trimethylamine 42 W
TSS see total suspended solids walls 14, 19–20, 27, 233
Tsukamurella 154, 164, 221, 223, 233–234, 239, washing
475–476 FISH 374–375, 379
tubulin 25 microautoradiography (MAR) 395
turbidostats 48 wastewater 1–4, 199–200
two stage–two sludge activated sludge 66, 66 ammonia-oxidizing bacteria (AOB) 265–266
Type021N 463–464, 522 anaerobic ammonium oxidizers 279–280
Type0041 507, 530 Bacillus 272
Type0092 468–469, 525 bulking 202–203, 497
Type0411 482–483, 532 characterisation 90
Type0581 484–485, 534 composition 202–203
Type0675 481–482, 532 denitrifying bacteria 272–277
Type0803 460–461, 519 dynamics 91–92
Type0914 483–484, 533 nitrite-oxidizing bacteria (NOB) 268–271
Type0961 483, 533 water sprays 256–257
Type1701 519 wet mounts 330
Type1851/‘Kouleothrix’ 466–467, 524 worksheets 342–343
Type1863 463, 521 Wuhrmann activated sludge configuration 67, 67
Index 667
Y Zoogloea ramigera 123, 176, 423, 458, 520

yeasts 30 zoospores 30, 32
yellow-brown algae 37 see also sporangiospores
Zygomycota 32, 33, 35
Z Zygomycotina 30, 32, 35
Zoogloea 99, 140, 276–277, 461–462 Zygorhynchus moelleri 33
extracellular polymeric substances (EPS) 461 zygospores 30, 33
RHC-PhyloChip-based diversity analysis 514

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Microbial Ecology of Activated Sludge

Microbial Ecology of Activated Sludge

First published 2010

Cover design by www.designforpublishing.co.uk

British Library Cataloguing in Publication Data

Library of Congress Cataloging-in-Publication Data

ISBN 10: 1843390329

Preface ..................................................................................................................................... vii

1 AN OVERVIEW OF THE MICROBES IN ACTIVATED SLUDGE ................................ 1

11.12 Terminal restriction fragment length polymorphism (T-RFLP) ................... 412

Dae Wook Kang

David McLean Roberts

A brief history of wastewater treatment

THE ACTIVATED SLUDGE PROCESS

Fully aerobic (conventional) process

GENERAL FEATURES AND CHARACTERISTICS OF MICROBES

HOW DO WE STUDY MICROBES?

WHAT ARE THESE MICROBES?

Subcellular organisation: the viruses

. Its presence in all non-eukaryotic organisms.

CELLS WITH A NON-EUKARYOTIC ORGANIZATION

STRUCTURAL FEATURES OF ‘PROKARYOTIC’ CELLS

HOW DO WE DIVIDE THESE GROUPS UP?

Chemolithoautotrophy þve in some þve in some ve

Feature Bacteria Archaea Eukarya

Outer cell layers

The cell walls of Bacteria and Archaea

1-4 linkage attacked by N-Acetylmuramic acid

The tetrapeptide chain

Protein Porin Lipid A

The cytoplasmic membranes of Bacteria and Archaea

Organization of DNA in Bacteria and Archaea

Genetic recombination and horizontal gene transfer in ‘prokaryotes’

Horizontal or lateral gene transfer

Intracellular inclusion bodies in ‘prokaryotes’

In some ‘prokaryotes’ the inclusion bodies are inorganic, and include:

Vacuoles in ‘prokaryotic’ cells

Motility in organisms with ‘prokaryotic’ cells

Do ‘prokaryotic’ cells possess a cytoskeleton?

CELLS WITH A EUKARYOTIC ORGANIZATION

The cell walls of eukaryotic microbes

The membranes of eukaryotic microbes

Organelles in eukaryotic microbes

The nuclear arrangement in eukaryotic microbes

Cytoskeletal elements in eukaryotic microbes

A BRIEF DESCRIPTION OF THE FUNGI

Saprolegnia ferax Phytophthora cinnamomi

Maturing pore through

CHYTRIDIOMYCOTA- motile uniflagellate motile gametes

ZYGOMYCOTA – asexual reproduction involves Sporangia (S)

Zygorrhyncus moelleri Phycomyces blakesleeanus

Gilbertella persicaria Absidias pinosa

ASCOMYCOTA -Asexual Reproduction involving conidia

Oidium candidum Aspergillus niger

Penicillium digitatum Scopulariopsis brevicaulis

4µm 20µm 100µm

Schizosaccharomyces octosporus– Peziza-Apothecium Sordaria fimicola - Perithecium

Sordaria fimicola-asci Emericella stellatus–

Phragmobasidium Holobasidium – 4 basidiospores/basidium

2 basidiospores/basidium 2 basiodiospores on sterigmata from the yeast Sporobolomyces

A BRIEF DESCRIPTION OF THE ALGAE AND PROTOZOA

Yellow-brown Algae or Chromophytes, containing chlorophyll a and c but no chlorophyll b,

A COMPREHENSIVE SINGLE CLASSIFICATION OF ALL THE EUKARYA