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Aquatic Biofilms

Ecology, Water Quality and Wastewater Treatment

Anna M. Romaní  Helena Guasch  M. Dolors Balaguer

Caister Academic Press

Aquatic Biofilms
Ecology, Water Quality and Wastewater Treatment

Edited by and

Anna M. Romaní M. Dolors Balaguer

Laboratory of Chemical and
Institute of Aquatic Ecology
Environmental Engineering (LEQUiA)
University of Girona
Institute of the Environment
University of Girona
Helena Guasch Spain

Institute of Aquatic Ecology

University of Girona

Caister Academic Press

Copyright © 2016

Caister Academic Press

Norfolk, UK

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ISBN: 978-1-910190-17-3 (hardback)

ISBN: 978-1-910190-18-0 (ebook)

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U.S. Government works.

Cover design adapted from scanning electron microscope images of aquatic biofilms. Images
were obtained by V. Díaz-Villanueva (front cover) and A. Freixa (back cover) with the
technical assistance of C. Carulla at the Technical Services of the University of Girona.

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Part I Biofilm Mode of Life 1

1 Limits of the Biofilm Concept and Types of Aquatic Biofilms 3
Juanita Mora-Gómez, Anna Freixa, Núria Perujo and Laura Barral-Fraga

2 Laser Microscopy for the Study of Biofilms: Issues and Options 29

Thomas R. Neu and John R. Lawrence

3 Interactions and Communication within Marine Biofilms 47

Priyanka Sathe and Sergey Dobretsov

4 Microbial Biodiversity in Natural Biofilms 63

Katharina Besemer

5 Aquatic Biofilms and Biogeochemical Processes 89

Laura Leff, Jonathon B. Van Gray, Eugènia Martí, Stephanie N. Merbt and
Anna M. Romaní

Part II Biofilms and Pollution 109

6 Benthic Diatom Monitoring and Assessment of Freshwater
Environments: Standard Methods and Future Challenges 111
Soizic Morin, Nora Gómez, Elisabet Tornés, Magdalena Licursi and
Juliette Rosebery

7 The Use of Biofilms to Assess the Effects of Chemicals on

Freshwater Ecosystems 125
Helena Guasch, Joan Artigas, Berta Bonet, Chloe Bonnineau,
Oriol Canals, Natàlia Corcoll, Arnaud Foulquier, Julio López-Doval,
Sandra Kim Tiam, Soizic Morin, Enrique Navarro, Stephane Pesce,
Lorenzo Proia, Humbert Salvadó and Alexandra Serra

8 Biofilm Development in Sewer Networks 145

Oriol Gutierrez, Guangming Jiang, Keshab Sharma and Zhiguo Yuan
iv  | Contents

Part III New Technologies Using Biofilms 165

9 Biofilm Biodegradation Potential 167
Freshta Akbari, Natasha Andrade, Merily Horwat and Birthe V. Kjellerup

10 Electroactive Biofilms in Water and Air Pollution Treatment 183

Anna Vilajeliu-Pons, Sebastià Puig, Alessandro Carmona-Martínez,
Nicolas Bernet, Marta Coma, Federico Aulenta, Jesús Colprim and
Maria Dolors Balaguer

11 Biofilms for One-stage Autotrophic Nitrogen Removal 205

José M. Carvajal-Arroyo, Tiago Rogeiro Vitor Akaboci, Maël Ruscalleda,
Jesús Colprim, Emilie Courtens and Siegfried E. Vlaeminck

Glossary 223
Index 227

Freshta Akbari Maria Dolors Balaguer

Department of Biological Sciences Laboratory of Chemical and Environmental
Goucher College Engineering (LEQUiA)
Baltimore, MD Institute of the Environment
USA University of Girona
Natasha Andrade
Department of Civil and Environmental Engineering
University of Maryland at College Park Laura Barral-Fraga
College Park, MD Institute of Aquatic Ecology
USA University of Girona
Joan Artigas
Laboratory Microorganisms: Genome and
Environment Nicolas Bernet
Clermont University INRA, UR0050
University Blaise Pascal Laboratoire de Biotechnologie de l’Environnement
Clermont-Ferrand Narbonne
France; and France
CNRS, UMR 6023
Laboratory Microorganisms: Genome and
Aubière Katharina Besemer
France School of Engineering
University of Glasgow Glasgow
United Kingdom; and
Federico Aulenta Department of Limnology and Bio-Oceanography
Water Research Institute (IRSA) University of Vienna
National Research Council (CNR) Vienna
Monterotondo Austria
vi  | Contributors

Berta Bonet Marta Coma

Institute of Aquatic Ecology Laboratory of Microbial Ecology and Technology
University of Girona (LabMET)
Girona Faculty of Bioscience Engineering
Spain Ghent University
Chloe Bonnineau
Institute of Life Sciences
Université Catholiqu de Louvain Natàlia Corcoll
Louvain-la-Neuve Department of Biological and Environmental Sciences
Belgium University of Gothenburg
Oriol Canals
Laboratory of Protistology
Department of Animal Biology Emilie Courtens
Faculty of Biology Laboratory of Microbial Ecology and Technology
University of Barcelona (LabMET)
Barcelona Faculty of Bioscience Engineering
Spain Ghent University
Alessandro Carmona-Martínez
INRA, UR0050
Laboratoire de Biotechnologie de l’Environnement Sergey Dobretsov
Narbonne Department of Marine Science and Fisheries
France College of Agricultural and Marine Sciences
Sultan Qaboos University
Jose M. Carvajal-Arroyo
Laboratory of Microbial Ecology and Technology
University of Ghent Arnaud Foulquier
Ghent Laboratoire d'Écologie Alpine (LECA)
Belgium UMR 5553 CNRS
Université Grenoble Alpes
Jesús Colprim
Laboratory of Chemical and Environmental
Engineering (LEQUiA)
Institute of the Environment Anna Freixa
University of Girona Institute of Aquatic Ecology
Girona University of Girona
Spain Girona
Contributors |  vii

Nora Gómez Birthe V. Kjellerup

Laboratory of Plankton and Biofilms Department of Civil and Environmental Engineering
Institute of Limnology “Dr. R.A. Ringuelet” University of Maryland at College Park
National Scientific and Technical Research Council College Park, MD
National Univeristy of La Plata (UNLP)
Buenos Aires
John R. Lawrence Environment Canada
National Hydrology Research Centre
Helena Guasch Saskatoon, SK
Institute of Aquatic Ecology Canada
University of Girona
Laura Leff Department of Biological Sciences
Kent State University
Oriol Gutierrez Kent, OH
Catalan Institute for Water Research (ICRA) USA
Spain; and
Advanced Water Management Centre
University of Queensland Magdalena Licursi
Brisbane Laboratory of Plankton and Biofilms
Australia Institute of Limnology “Dr. R.A. Ringuelet”
National Scientific and Technical Research Council; (CONICET)
National Univeristy of La Plata (UNLP)
Merily Horwat Buenos Aires
Department of Civil and Environmental Engineering Argentina
University of Maryland at College Park
College Park, MD
Julio López-Doval Department of Ecology
Laboratory of Limnology
Guangming Jiang University of São Paulo
Advanced Water Management Centre São Paulo
University of Queensland Brasil
Australia Eugènia Martí
Integrative Freshwater Ecology Group
Sandra Kim Tiam Center for Advanced Studies of Blanes (CEAB-CSIC)
Institut National de la Recherche Scientifique Girona
Centre Eau Terre Environment (INRS-ETE) Spain
viii  | Contributors

Stephanie N. Merbt Stephane Pesce

Integrative Freshwater Ecology Group Freshwater Systems, Ecology and Pollution Research
Center for Advanced Studies of Blanes (CEAB-CSIC) Unit (MALY)
Girona National Research Institute of Science and Technology
Spain for Environment and Agriculture (Irstea)
Juanita Mora-Gómez
Institute of Aquatic Ecology
University of Girona Lorenzo Proia
Girona Resources and Ecosystems Area
Spain Catalan Institute for Water Research
Soizic Morin
Aquatic Ecosystems and Global Change Research Unit Sebastià Puig
(EABX) Laboratory of Chemical and Environmental
National Research Institute of Science and Technology Engineering (LEQUiA)
for Environment and Agriculture (Irstea) Institute of the Environment
Cestas University of Girona
France Girona
Enrique Navarro
Department of Biodiversity and Restoration Anna M. Romaní
Pyrenean Institute of Ecology (CSIC) Institute of Aquatic Ecology
Zaragoza University of Girona
Spain Girona
Thomas R. Neu
Department of River Ecology Juliette Rosebery
Helmholtz Center for Environmental Research – UFZ Aquatic Ecosystems and Global Change Research Unit
Magdeburg (EABX)
Germany National Research Institute of Science and Technology
for Environment and Agriculture (Irstea)
Núria Perujo
Institute of Aquatic Ecology
University of Girona
Girona Maël Ruscalleda
Spain Laboratory of Chemical and Environmental
Engineering (LEQUiA)
Institute of the Environment
University of Girona
Contributors |  ix

Humbert Salvadó Anna Vilajeliu-Pons

Laboratory of Protistology Laboratory of Chemical and Environmental
Department of Animal Biology Engineering (LEQUiA)
Faculty of Biology Institute of the Environment
University of Barcelona University of Girona
Barcelona Girona
Spain Spain

Priyanka Sathe Tiago Rogeiro Vitor Akaboci

Department of Marine Science and Fisheries Laboratory of Chemical and Environmental
College of Agricultural and Marine Sciences Engineering (LEQUiA)
Sultan Qaboos University Institute of the Environment
Muscat University of Girona
Oman Girona
Alexandra Serra
Integrative Freshwater Ecology Group Siegfried E. Vlaeminck
Center for Advanced Studies of Blanes (CEAB-CSIC) Research Group of Sustainable Energy, Air and Water
Girona Technology
Spain Department of Bioscience Engineering
University of Antwerp
Antwerp; and
Laboratory of Microbial Ecology and Technology
Keshab Sharma (LabMET)
Advanced Water Management Centre Ghent University
University of Queensland Ghent
Brisbane Belgium

Elisabet Tornés Zhiguo Yuan

Institute of Aquatic Ecology; and Advanced Water Management Centre
Catalan Institute for Water Research (ICRA) University of Queensland
University of Girona Brisbane
Girona Australia

Jonathon B. Van Gray

Department of Biological Sciences
Kent State University
Kent, OH
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On any wet surface a biofilm is easily formed, man-made systems such as water engineering
whether it is on a building, a rock in a river, processes. Although in each specific environ-
marine sediments, a decaying leaf, a sewage ment a distinct biofilm may develop, the drivers
pipe, among others. The extensive appearance and gradients in biofilms show parallelisms. For
of the biofilm mode of life may be linked to instance, the oxygen gradient determining spe-
its properties such as nutrient entrapment and cific biogeochemical reactions is similar between
physical protection of cells from the surround- naturally occurring fluvial biofilms and those
ing environment. Also, this mode of life is old, developing on granules for water technology pur-
underlining its resistance. It is thought that poses. Other example is the knowledge gained
aggregated layer-structured biofilms similar to from anthropogenic disturbances effects on bio-
ancient stromatolites have been relevant for films, showing parallelisms to responses observed
the origin of first microbial cells on Earth. The from biofilms growing in extreme environments
unique and complex characteristics of biofilms and developing similar resistance strategies.
include mechanisms and processes occurring at The aim of this book was to compile in a
different scales addressed by different scientific single volume the latest, up-to-date theory, meth-
branches. Atomic forces and chemical bonds are odology, and applications of aquatic biofilm’s
keys for attachment processes, development of research. From the theory, a broad review of
the matrix, and chemical gradients. At the cell to biofilm history, architecture, cell communication,
organisms scale, life science analyzes cell-to-cell biodiversity and biogeochemistry is included,
communication, diversity of microbial metabo- updating both theory and methodology. Then,
lisms and food web interactions in biofilms. the study of biofilms developing in polluted
Aquatic biofilms are also a significant component systems as well as their use and relevance as
at the Earth sciences scale as shown for instance ecotoxicological sensors is reviewed. Finally,
by their relevance in biogeochemical cycles. application and profit of biofilms is shown in
From the first report of surface-associated three examples on new technologies using bio-
bacterial cells, aquatic biofilm research have been films. We believe the different points of view and
exponentially developed in the last decades, approaches presented in the book, from theory
covering the study of biofilms in marine and to application, from ecology to engineering, are
freshwater environments, including pristine but complementary and feed from each other con-
also those affected by pollution and anthropo- tributing to our understanding of biofilm mode
genic disturbances, and of those developing in of life.

Anna M. Romaní, Helena Guasch, Marilós Balaguer

Part I

Biofilm Mode of Life

Limits of the Biofilm Concept and
Types of Aquatic Biofilms
Juanita Mora-Gómez, Anna Freixa, Núria Perujo and
Laura Barral-Fraga

Abstract ecological health of freshwater ecosystems. Bio-

Nowadays, it is widely recognized that in natural films are crucial in ecosystem functioning and
aquatic settings bacterial cells are most often have an excellent ability to degrade and transform
found in close association with wet surfaces and pollutants, as they are involved in primary pro-
interfaces in the form of multicellular aggregates duction (photosynthetic activity) (Underwood
commonly referred to as biofilms, which also et al., 2005), carbon and nutrient cycling (Davey
involve algae, fungi and protozoa. However, since and O’Toole, 2000), retention of inorganic and
surface-associated bacteria were first reported, the organic nutrients (Pusch et al., 1998), and sup-
biofilm concept has been developed to include port of food webs (Anderson-Glena et al., 2008).
the great complexity of this microbial way of life. There are numerous benefits that a bacterial
Biofilms in natural and anthropogenic environ- community might obtain from the formation of
ments are modulated by the nature of the surface biofilms. For example, biofilms protect micro-
in which they grow and environmental factors, organisms from protozoa grazing, and host
which determine the biofilm composition and defences as in plant-associated biofilms (López
structure and, in consequence, its metabolism et al., 2010). Moreover, biofilms are also used to
and function. As a consequence there are many solve a huge problem in the treatment of waste-
types of biofilm and many terms have been used water, cleaning polluted water in water treatment
through years to try to describe particular biofilms plants by removing organic material (Lazarova
in relation to a type of surface or environment. In and Manem, 1995). In addition to the important
the present review, we summarize the knowledge practical use of biofilms in biological wastewater
on biofilms, starting from the origin and evolution treatment, they are also used in biodegradation
of the concept followed by a description of biofilm and bioremediation in bioreactors (De Beer and
types based on substratum characteristics. Finally, Stoodley, 2006).
we explain the general effects of environmental Biofilms also have negative effects in man-made
variables, contextualizing them in the wide range systems, including biofouling and microbial-
of natural aquatic ecosystems (including fresh induced corrosion, which affect industrial water
and marine water) and some man-made systems systems (Videla and Herrera, 2005) and drinking
(such as those associated with water distribution water distribution systems. Many problems are
systems and marine environments). due to biofilm growth (Berry et al., 2006) and the
biodeterioration processes from the interactions
between metal surfaces and biofilm. In marine
Introduction systems, man-made structures such as aquaculture
nets, oil and gas installations, and ship hulls are also
Aquatic biofilms: relevant role in the affected by biofilm attachment and growth, the
environment so-called microfoulers (Salta et al., 2013). Marine
Biofilm communities play a role in the environ- biofouling may cause some problems such as the
ment both in maintaining and improving the cost associated with increased fuel consumption
4  | Mora-Gómez et al.

by ships linked to increased frictional drag, as surfaces in lakes and rivers (Wetzel, 1983). By
reported in Schultz et al. (2011). The survival of this time scientists and engineers used developing
microorganisms on man-made surfaces is based technology to effectively study microbial com-
upon interactions of many variables, including munities, and biofilm research was progressively
temperature, pipe surface, nutrient levels and type established as a relevant scientific topic. Many
and concentration of disinfectants (Berry et al., definitions of aquatic biofilms appeared during
2006). It is also known that attached cells have the eighties referring to biofilm as an assemblage
certain ecological advantages over planktonic of autotrophic and heterotrophic microorganisms
cells such as having increased resistance to chlo- embedded in a polymeric matrix and developing
rine and other biocides (Emtiazi et al., 2004). The on wetted surfaces (Lock et al., 1984; Costerton
resistance of biofilm cells to disinfection consists et al., 1987; Characklis and Marshall, 1989).
of the formation of persistent cells and protection Later, the concept of biofilm evolved by including
linked to the increased production of extracellular new knowledge from research on biofilm forma-
polymeric substances. tion, organization, cell-to-cell communication,
interaction between microorganisms, and three-
Biofilm history and names dimensional structure. The great development of
Over the years, a considerable amount of research the research on biofilm formation in medicine
has dealt with the subject of biofilm. The first and animal health (i.e. cancer processes, trans-
official report of surface-associated microbial cells plants or prosthesis) has also helped to improve
was in 1684, when Van Leeuwenhoek informed the knowledge of aquatic biofilms by using rel-
the Royal Society of London that he had observed evant techniques such as confocal microscopy
an accumulation of microorganisms on tooth (see Chapter 2) and cell-to-cell signalling (see
surfaces using a simple microscope. But it was Chapter 3).
not until 1936 that Zobell and Anderson intro- The study of biofilm in different systems, where
duced the term periphytes, referring to bacteria distinct environmental variables and types of sub-
associated with surfaces in bottles used to store strata are found, led to the appearance of different
seawater (Zobell and Anderson, 1936). The terms when referring to biofilm such as periphyton
examination of biofilms was promoted just after and microphytobentos, or other less common ones
electron microscope development, which allowed like benthos, haptobenthos and herpobenthos. Thus,
high resolution at much higher magnifications there can be a dilemma when defining the termi-
than when observed under the light microscope nology used to describe a biofilm (Wetzel, 1983),
(Donlan, 2002). In the following decades many and sometimes it becomes a real problem when
microbiologists investigated aquatic bacteria, looking for the appropriate bibliography. In most
especially for the study and treatment of many scientific publications periphyton refers to fresh-
serious diseases. Some studies reported that bac- water ecosystems while microphytobenthos refers
teria attached to aquatic surfaces are often 1,000 to marine ecosystems. Periphyton is defined as an
or 10,000 times greater in number than planktonic assemblage of freshwater organisms mainly com-
(free-floating) bacteria (Costerton et al., 1978; posed of photoautotrophic algae, heterotrophic
Donland, 2002). and chemoautotrophic bacteria, fungi, protozo-
The first word proposed to describe micro- ans, metazoans and viruses which grow upon a
organisms attached to aquatic surfaces was benthic substrate (Wetzel, 1983; Larned, 2010).
‘aufwuchs’, a German word, meaning surface Microphytobenthos are defined as populations
growth (Ruttner, 1953). But it was not until 1975 of photoautotrophic microorganisms (diatoms,
that the term biofilm made its first appearance euglenids, crysophyceans, dinoflagelates) that
in the scientific literature (Mack et al., 1975). In colonize benthic substrata in marine systems,
1983, Wetzel described biofilms as assemblages especially in intertidal and lower supra-tidal sedi-
of bacteria, algae, fungi, and protozoa within a ments reached by light (MacIntyre et al., 1996;
protective matrix of extracellular polymeric sub- Jesus et al., 2009; Pan et al., 2013). The term bio-
stances and detritus, which colonize submerged film was initially used in engineering and referred
Limits of the Biofilm Concept |  5

to attached heterotrophic communities (Wetzel, drinking water distribution systems, MacLeod et

1983), but it has been spread to both natural and al., 1990). In these artificial substrates the concept
anthropogenic aquatic systems as a general term of biofouling (or microbial fouling) is widely used
referring to attached-microbial communities. to describe the accumulation process of micro-
Even though biofilm and periphyton are mostly organisms on wet surfaces, especially in shipping
used synonymously, Saikia (2001) reported slight equipment and water distribution systems (Melo
differences between the terms periphyton and bio- and Bott, 1997).
film, indicating that periphyton is linked to nutrient Similar to biofilms, a special and characteristic
dynamics in ecosystems and especially including form of attached microbial community is a micro-
photosynthetic organisms. Finally, benthos were bial mat. Microbial mats are organo-sedimentary
initially described as organisms associated to the structures that develop on solid surfaces, formed
bottom or solid–liquid interfaces in aquatic sys- by the trapping and binding of sediment and/
tems, haptobenthos as adhered organisms but not or the net carbonate-precipitating activities of
penetrating a solid surface, and herpobenthos as microorganisms, resulting in a layered structure.
organisms penetrating bottom sediments (Wetzel, They are mainly formed by cyanobacteria, col-
1983; Neuswanger et al., 1982). ourless sulfur bacteria, purple sulfur bacteria,
Additionally, since biofilms are formed in and sulfate-reducing bacteria, and can be found
sand, sediment, rocks and cobbles, wood and in lagoons, marine intertidal and subtidal zones,
leaves, and the surface of submerged plants, spe- hypersaline ponds, hot springs, fresh water rivers
cific names for each natural substrate have also and lakes (van Gemerden, 1993; Dupraz and Viss-
been used by adding the corresponding adjective che, 2005). Ancient microbial mats (stromatolites)
(such as epilithic, epixylic, or epipsammic biofilm; were abundant and diverse in shallow zones of the
see Table 1.1 and Fig. 1.1) (Lock, 1993; Romaní, oceans in the Proterozoic (Bottjer et al., 1996).
2010; Vadeboncoeur and Steinman, 2002). The Like the first photosynthetic communities, stro-
term biofilm is also commonly used in man-made matolites consumed the greenhouse gas CO2,
surfaces such as industrial equipment and in and produced free O2 and H2, playing a crucial
water management (water treatment plants and role for the early establishment of life (Dupraz

Table 1.1 Most common names, main factors from the substratum type (support media) affecting the
biofilm (intrinsic substratum factors), and dominant groups composing the biofilm for the different substrata
Dead plant Living Suspended Artificial
Rocks Sediment material plants aggregates (POM) (man-made)
Most Epilithic Epipsammic Epixylic biofilm Epiphytic Snow particles, Biofilm
common biofilm; biofilm; biofilm marine snow, lake (biofouling,
used names epilithon; epipsammon; snow, river snow; bioclogging)
periphyton epipelic biofilm; aggreggates,
epipelon; granules
hyporheic biofilm
Intrinsic Surface Grain size and Organic matter Biofilm– Particle density, Material
substratum roughness; roughness of characteristics plant size and shape; roughness;
factors stone size and grain; profile (chemical interaction particle settling type of
orientation zonation in depth composition) velocity; particle material
surface properties
Community Algae, Bacteria, Fungi, Algae, Diatoms, Bacteria,
composition cyanobacteria, cyanobacteria, bacteria, bacteria, filamentous fungi and
(dominant bacteria, fungi archaea, algae, archaea, fungi and cyanobacteria, protozoa
groups) and protozoa fungi and algae, and protozoa bacteria,
protozoa metazoans protozoans,

POM, particulate organic matter.

6  | Mora-Gómez et al.




Marine Snow Epiphytic

Algae Bacteria Fungi Cyanobacteria Protozoa

Zooplankton POM EPS Plant exudates WDS

Figure 1.1  Biofilm types with regard to differential substrata find in natural and man-made environments.
EPS, extracellular polymeric substances; POM, particular organic matter; WDS, water distribution system.
Modified from Romaní (2010).

and Vissche, 2005). However, in the latter part of However, several characteristics are shared by
the Proterozoic they declined most likely due to any type of biofilm. Nowadays it is widely rec-
metazoan diversification, increased grazing and ognized that in natural settings bacterial cells
sediment disturbance (Bottjer et al., 1996). are most often found in close association with
Within the limits of the biofilm concept, surfaces and interfaces, in the form of multicel-
aggregates or granules as suspended microbial lular aggregates commonly referred to as biofilms
flocs have many features in common with classi- which also involve other microorganisms such as
cal biofilms, such as the polymeric matrix and the algae, fungi, viruses and protozoa (Branda et al.,
association of diverse groups of microorganisms 2005). A general characteristic of biofilm struc-
such as bacteria, algae and protozoans, which ture is that these microorganisms, together with
interact structurally and functionally (De Beer extracellular enzymes and detritus, are enclosed
and Stoodley, 2006). Aggregates play a role in within a polymeric matrix (Golladay and Sin-
nutrient cycling and organic matter decomposi- sabaugh, 1991). These extracellular polymeric
tion in lakes, rivers and the sea (Azam and Cho, substances (EPS) mainly composed of polysac-
1987; Grossart and Simon, 1993). charides, provide mechanical stability to biofilms,
mediate their adhesion to surfaces, and form a
What is the definition of a biofilm? cohesive three-dimensional polymer network
The answer to this question is not always simple that interconnects and transiently immobilizes
due to the large variability in structure and biofilm cells (Wingender and Flemming, 2011;
composition of biofilm, and the different envi- Decho, 2000; Gerbersdorf et al., 2008). This
ronments in which biofilm develops. The high matrix provides protection from predation, toxic
complexity of aquatic biofilms is related to the substances and physical perturbations. Although
type of substratum and the environment in which the polymeric matrix is mostly constituted by
they are living (Karatan and Watnick, 2009). water (up to 97%, Zhang and Feng, 2001) and
Limits of the Biofilm Concept |  7

polysaccharides, other compounds are found, systems and man-made marine structures, bio-
such as extracellular DNA, proteins and lipids, film grows upon pipes and boats. The nature of
particulate material and detritus (Costerton et the substratum determines the composition and
al., 1995; Sutherland, 2001; Lawrence and Neu, structure of the biofilm and, in consequence,
2003). its metabolism (Romaní, 2004a). Furthermore,
As a consequence of this three-dimensional some studies have demonstrated that the nature
physical structure, tight interactions between of the substratum is the most important factor for
the organisms living within the biofilm occur, biofilm development, since substratum properties
building a ‘micro-ecosystem’ where feeding inter- may regulate patterns of cell accumulation and
actions, nutrient cycling, competition, synergism, distribution during the early stages of biofilm
and cell-to-cell signalling take place. In aquatic development (Terlizzi and Faimali, 2010).
biofilms, diverse trophic interactions have been
described, such as protozoa feeding on bacteria Rocks
and algae, rotifer feeding on protozoa, bacteria Biofilms attached to rock surfaces (also to gravel
and detritus, nematode feeding on algae, grazing and cobbles) are referred to as epilithic biofilms
by metazoans, etc., which might also cause struc- or epilithon in rivers (Guasch and Sabater, 1994;
tural changes to the biofilm (Augspurger et al., Sabater et al., 2006; Anderson-Glena et al., 2008),
2008; Früh et al., 2011; Majdi et al., 2011; Risse- marine environments (Thompson et al., 2004,
Buhl et al., 2012). Thus, biofilm can be seen as the 2005; Firstater et al., 2012; Smith et al., 2010),
well-defined microbial loop, for example, in the and lakes (Lowe, 1996; Vadeboncoeur and Stein-
pelagic ecosystem (Azam, 1998), but ‘squeezed’ man, 2002). Compared to other biofilms, such as
in the physical space, enhancing the relevance of those that grow on sand, epilithic biofilms have a
the spatial structure which facilitates cooperation more complex structure with a higher algal bio-
(Kreft, 2004). mass and they are more independent of seasonal
For the purposes of this review, we seek to give fluctuations (Romaní and Sabater, 2001; Graba
an overview of the limits of the biofilm concept et al., 2013). Data from river epilithic biofilms
by including microbial communities developing show a proportion of total carbon of 60–90% for
in natural aquatic environments and man-made algae, 10–40% for EPS, 1–5% for bacteria and
surfaces (associated with water treatment plants less than 1% for fungi (Romaní, 2010). However,
and drinking water distribution systems, as well in shaded environments (i.e. forested rivers and
as marine surfaces). In this sense, biofilm types streams), heterotrophic biomass (bacteria, fungi
are described based on the substratum where and protozoa) become more important (Romaní,
they develop, and the main environmental factors 2010).
influencing the different types of biofilm are also Stones provide a three-dimensional physical
reviewed. habitat for biofilm biomass (Bergey, 2008). The
developing biofilms are greatly influenced by rock
characteristics such as surface texture (Clifford
Types of aquatic biofilms et al., 1992; Sanson et al., 1995; Murdock and
defined by the substratum Dodds, 2007), stone size (Ledger and Hildrew,
where they develop 1998), and stone orientation (Murdock and
In aquatic environments, biofilms can be highly Dodds, 2007). On the one hand, rougher surface
diverse depending on the substratum where they can cause greater biofilm accumulation on rocks,
develop. In natural environments, biofilm grows since it increases sedimentation efficiency and
upon inert substrata such as sand, sediment, rocks cell adhesion, and protects the biofilm from dis-
and cobbles; non-living organic substrata such as turbances such as scouring and grazing (Murdock
wood, leaf litter or particular organic matter; and and Dodds, 2007). On the other hand, stone size
living plants such as aquatic macrophytes and and stability determine the algal resistance to
macroalgae (Table 1.1). In man-made substrata, scour (Peterson, 1996). Regarding stone orienta-
associated with drinking water distribution tion, biofilm accumulation is consistently greater
8  | Mora-Gómez et al.

on horizontal than on vertical surfaces (Baynes, diffusion into the biofilm (Larned et al., 2004;
1999; Kralj et al., 2006; Murdock and Dodds, Saikia, 2011).
Conversely, the effect of rock chemistry on Sediment
biofilms is unclear. It seems that the chemi- Microorganisms attached to the particles of sandy
cal composition of streambed stones is of little sediments (sand and gravel) are referred to as
importance to algal assemblages, which may epipsammic, while when developing on muddy
be caused by the low dissolution rate of stones sediments (clay or silt) they are known as epipelic
(Bergey, 2008). However, some evidence points (MacIntyre et al., 1996). Sediments are a hard,
out that rock chemistry may affect early stages of inert substratum, characterized by its smaller size
biofilm colonization (Blinn et al., 1980). A lack of than other substrates. In rivers, the biofilm devel-
chemical effect helps to explain the similarity of oping on sediment has been defined as playing
algal biomass and assemblages among such differ- a key role in organic matter decomposition, also
ent substrates as rock and wood (e.g. Sabater et al., being more heterotrophic (with higher contri-
1998; Townsend and Gell, 2005) and the success butions of bacteria and fungi) than the biofilm
of using artificial substrates, such as glass or clay developing on rocks (Pusch et al., 1998; Romaní
tiles, as substitutes for natural substrates in biofilm and Sabater, 2001).
studies (e.g. Tuchman and Stevenson, 1980; Lam- Colonization and microbial community
berti and Resh, 1985). development on sediments depends on the size,
Although autotrophic–heterotrophic rela- roughness and surface area of the grain. Specifi-
tionships may occur in most aquatic biofilms, cally, microbial colonization on sediment grains
algal–bacterial interactions have been mainly is proportional to the grain surface area while
described for epilithic biofilms. In biofilms surface area is inversely proportional to grain size.
developing on rocks, the structural stability and However, these assumptions could be applied
close spatial relationship between bacteria and only from coarse grains to fine silt (DeFlaun
algae favours the bacterial use of fresh labile and Mayer, 1983) because clay particles are very
organic compounds released by algae (Wetzel, rarely colonized since they are too small and
1993; Sobczak, 1996; Romaní and Sabater, 1999, smooth (Mayer and Rossi, 1982). For example, in
2000), affecting the whole biofilm metabolism. intertidal environments, non-cohesive sediments
In addition, when a thick biofilm is developed, (such as sand and gravel) have been described to
an anoxic layer may exist at the biofilm bottom exhibit greater diversity than cohesive sediments
and consequently the presence of anaerobic bac- (such as clay and silt). Jesus et al. (2009) showed
teria (Schramm et al., 1999). This micro-spatial that cohesive sediments were mainly colonized
variation contributes to the intra-site differences by diatoms, while sandy sediments support
observed in microbial communities (Anderson- three microphytobenthic groups (cyanobacteria,
Glena et al., 2008). euglenids and diatoms. Besides the size of parti-
Epilithic biofilms in aquatic environments cles, the degree of roughness plays an important
have been defined as playing a relevant role in role, increasing microbial colonization as parti-
nutrient uptake since they can use inorganic and cle roughness increases (Meyer-Reil, 1994). In
organic compounds from the flowing water by concordance, it has been reported that microbial
catalysing enzymes such as nitrogen reductase biomass is higher on grains with more surface
and alkaline phosphatase (Adey et al., 1993). irregularities than smooth grains of the same size
In addition, biofilm can trap particulate mate- (DeFlaun and Mayer, 1983).
rial from the water column and thus increase its In aquatic environments, sediments usually
concentration in contrast to the water column. show a profile zonation in depth if they are suf-
Nutrient uptake is potentially influenced by the ficiently thick, changing from an oxidized zone
degree and distribution of biofilm cover, ambient in the surface sediment to a reduced zone in
macronutrient concentration, grazing, sloughing, deeper layers creating anoxic zones. In this sense,
temperature, advective transport in the river, and sediment thickness creates marked physical and
Limits of the Biofilm Concept |  9

chemical gradients that determine changes in due to their ability to degrade lignocellulose
sediment biofilm community (Froelich et al., compounds, mainly constituent of plant tissues
1979). In this regard, surface sediments support (Nikolcheva and Bärlocher, 2004). However, bac-
heterotrophic communities that include more teria, archaea, algae and metazoans also form this
opportunistic species (Arnosti, 2011). Bacterial kind of biofilms (Manerkar et al., 2008; Gaudes et
density and activity is commonly higher in surface al., 2009; Duarte et al., 2010; Danger et al., 2013).
sediments and decrease with depth (Taylor et al., This particular type of attached microbial com-
2002). Hydrolysis rates of organic compounds munity is not commonly classified as a biofilm
have also been shown to decrease with depth by scientists working on decaying plant material,
likely due to the influx of fresh organic matter since colonizing microorganisms (mainly fungi)
that stimulates microbial activity at the sediment not only adhere just to the surface of dead plants
surfaces (Meyer-Reil, 1986; Poremba and Hoppe, but also enter and degrade the plant tissue. Fungi
1995; Boetius et al., 2000). In rivers, the sediment are able to penetrate leaves with their hyphae,
hyporheic zone (interface between the river chan- whereas bacteria, archaea and algae attach to the
nel and groundwater) offers protection to the external surfaces or colonize inside the leaves in
sediment biofilm communities against high dis- association with hyphae growth (Baschien et
charge, desiccations, and extreme temperatures al., 2009). However, epixylic biofilms share two
(Brunke and Gonser, 1997). At the hyporheic key characteristics with all aquatic biofilms: (1)
zone, grain size, sediment shape, and composition microorganisms in a sessile form are aggregated
determine the porosity and hydraulic conductiv- and interact among them, and (2) extracellular
ity of the sediment, and influence most physical material is produced for attachment between cells
and chemical processes (Boulton et al., 1998). and/or to the substratum.
Nutrients are adhered to sediment particles Microorganisms growing on dead plant
and are available for the community living in material are influenced by internal and external
them. In this sense, river sediment acts as a sink factors (Webster and Benfield, 1986). Internal
of nutrients in the water column (Wetzel, 1996) ones mainly refer to dead organic matter qual-
through nutrient uptake and deposition (Dodds, ity, such as cuticle toughness, nutrient content
2003). However, non-cohesive sediments (sand) (particularly nitrogen and phosphorus), and
have higher solute transport due to high resuspen- the amount of less palatable substances such as
sion and mixing (Ehrenhauss et al., 2004), as this lignin, or chemical inhibitors (Meentemeyer,
type of sediments have lower nutrient concen- 1978; Ostrofsky, 1997; Cornwell et al., 2008). The
trations than cohesive sediments (Underwood, quality of organic matter can vary significantly
2010). due to several reasons, such as the part of the
plant being decomposed, plant taxonomic dif-
Dead plant material ferences (Ostrofsky, 1997), intraspecies genetic
Biofilm developing on dead plant material such as variations (LeRoy et al., 2007), phenological
wood and leaves is named epixylic biofilm or epixy- status (Kochi and Yanai, 2006) or plant condition
lon (Vadeboncoeur and Steinman, 2002). In this (Lecerf and Chauvet, 2008). However, it is gener-
case, the substratum (i.e. dead organic matter sur- ally observed that slower decomposition occurs
faces) represents both a physical support medium in dead plant material with high lignin content
for microbial colonization and a source of organic levels, low nitrogen or phosphorus concentra-
matter for microbes (Golladay and Sinsabaugh, tion or greater toughness (e.g. Triska and Sedell,
1991). 1976; Gessner and Chauvet, 1994; Martínez et
The relevance of the different organisms form- al., 2013). External factors are environmental
ing epixylic biofilms is markedly different to other variables such as water temperature, nutrient con-
biofilm types. Taxa from several fungal phyla such centration or flow (Webster and Benfield, 1986;
as Chytridiomycota, Zygomycota, Ascomycota Tank et al., 2010). The rate of decomposition of
and Basidiomycota, are the principal microbial plant organic matter is determined by the effect of
groups growing on dead plant material, primarily interaction between the quality of plant material
10  | Mora-Gómez et al.

and the environmental variables (e.g. LeRoy and changes in bacterial densities and community
Marks, 2006). Additionally, decomposition veloc- composition (Hempel et al., 2008).
ity might be very important for the developing However, the interaction between the plant
biofilm since slower decomposition enables the and the epiphytic biofilm is not always positive.
organic substrate to remain longer in the system Negative affectation of biofilms on submerged
so consequently greater biofilm biomass develop- plants could arise from increased shading by thick
ment is achieved, as occurs in wood (Golladay biofilms and potentially also from pathogenic bac-
and Sinsabaugh, 1991). teria present in the biofilm (Underwood, 1991).
Moreover, it has been shown that, in some cases,
Living plants excessive biofilm growth decreases the exchange
Biofilm growing on living plants, such as that of nutrients, and reduces photosynthesis and
developing on macrophytes providing a relatively plant growth (Sand-Jensen and Søndergaard,
steady substratum, is named epiphytic biofilm 1981; Asaeda et al., 2004). Plants can also exert
(Vadeboncoeur and Steinman, 2002). Submersed negative effects on epiphytic bacteria due to the
plants and macro algae, living in wetlands and lit- release of secondary compounds such as poly-
toral zones, are a favourable habitat for microbial phenols, which may inhibit bacterial growth and
growth. Aquatic plants present vast potential for attachment (Van Donk and Van de Bund, 2002).
microbial colonization and are frequently covered
by a dense growth of algae, bacteria and other Suspended aggregates
organisms favouring biofilm formation (Carpen- Suspended aggregates made of microorganisms,
ter and Lodge, 1986). However, in deeper parts organic and inorganic particles, are highly fragile
of the plant, bacteria might be favoured due to structures suspended in fresh and sea water and
light reduction produced by the host plant, which are usually named lake snow, river snow or marine
affects algae development. Epiphytic bacteria snow. Aggregates typically occur during bloom
biofilms have been found to be dominated by the periods after an increased input of nutrients (De
bacterial group Cytophaga–Flavobacteria–Bac- Beer and Stoodley, 2006)
teroidetes and Alpha-proteobacteria in marine Marine snow consists of aggregates of diatoms,
(Burke et al., 2011) and freshwater environments filamentous cyanobacteria, bacteria, protozoans,
(Hempel et al., 2008). Moreover, high densities zooplankton carcasses, abandoned larvacean
of nitrifying and denitrifying bacteria have been houses, faecal pellets, macrophyte detritus, clay
reported on submersed macrophytes (Körner, and silt minerals, calcite and other particles from
1999). the surrounding water, glued together in a poly-
Plant and epiphytic biofilm interact in many meric matrix released from phytoplankton and
ways, and the interaction might be both syn- bacteria. Anoxic conditions may exist within the
ergic and antagonistic. Biofilm provides the snow particle so that diverse aerobic and anaero-
macrophytes with organic compounds and bic microbes colonize different niches (Alldredge,
carbon dioxide, and also mediates nutrient uptake 1998). Similar aggregates are formed in some
and enhances nutrient recycling (Wetzel, 1993; water treatment systems where they are called
Eriksson and Weisner, 1999). In addition, some granules (see Chapter 12).
bacterial species produce compounds against Many complex physical, chemical, biological
fouling organisms (Rao et al., 2006), and some and specific microbial processes are involved in
species enhance plant growth (Marshall et al., the formation of aggregates (Simon et al., 2002).
2006). In return, plants provide a substrate for Aggregation is a complex process and is con-
biofilm formation and exude organic compounds trolled mainly by particle density, size and shape,
and gases such as methane from the root zone, settling velocity and the surface properties of the
which are used by some biofilm bacteria (Heilman particle (Chen et al., 1994; Simon et al., 2002).
and Carlton, 2001). Consequently, different plant Furthermore, porosity is an important factor
species, plant part and environmental conditions controlling the sinking rate of aggregate, the flux
might influence epiphytic biofilms, determining of water through the aggregate moving relative to
Limits of the Biofilm Concept |  11

the surrounding water, and the flux of nutrients to develops (Watnick and Kolter, 2000). Generally,
and from the microorganisms colonizing the sur- biofouling can be prevented by a combination of
face of the aggregate (Alldredge and Silver, 1988; pre-treatment (by reducing microorganism and
Ploug, 2001). nutrient concentrations) and preventative chemi-
Snow particles or suspended aggregates are cal cleaning (Guo et al., 2012). In addition, there
ubiquitous in the water column, and sink rap- are various factors that affect biofouling formation
idly. For this reason, suspended aggregates play such as solid surface material, feed water char-
a key role in transporting organic matter from acteristics and operational conditions (mainly
surface water layers to the benthos. Due to their involving temperature, dissolved oxygen and flow
important role in the carbon cycle, aggregates velocity) (Le-Clech et al., 2006).
and their associated microbial communities are a The solid and inert man-made surface may
major focus of investigation (Simon et al., 2002). have several characteristics that are important in
Furthermore, aggregates are frequently identified the attachment process. Characklis and Marshall
as hotspots of carbon remineralization and micro- (1989) reported greater microbial colonization
bial activities, including enzyme activities (Smith with increased surface roughness in artificial sub-
et al., 1992; Grossart et al., 2007). Bacterial abun- strates. Different properties of the surface material
dance on suspended aggregates is around 108 may also exert a strong influence on the rate and
bacteria/ml, which is 100-fold higher than in bulk extent of attachment. Most investigators have
water (Grossart et al., 2007). Such aggregates are found that microorganisms attach more rapidly
amply described in marine environments (marine to hydrophobic, non-polar surfaces such as Teflon
snow), while few authors have studied them in and other plastics than to hydrophilic materials
river and lakes (river snow, lake snow). such as glass or copper pipes (Lehtola et al., 2005;
Fletcher and Loeb, 1979; Pringle and Fletcher,
Man-made surfaces 1983). In general, attachment will occur more
Biofilms may also form on a wide variety of arti- easily on surfaces that are rougher, more hydro-
ficial surfaces such as industrial or potable water phobic, and coated by surface ‘conditioning’ films
system piping, where biofouling may occur by (Cooksey and Wigglesworth-Cooksey, 1995).
deposition and the growth of bacterial cells or
flocs. In drinking water distribution systems
Gram-positive bacteria and Alpha- Beta- and Biofilm responses to
Gamma-proteobacteria are the dominating groups environmental conditions
(Tokajian et al., 2005). In marine environments, Biofilms are present in all aquatic environments
man-made structures such as oil and gas installa- including freshwater ecosystems (streams, rivers,
tions, aquaculture nets, as well as ship hulls, can lakes and reservoirs), marine environments (tidal,
also provide surfaces for attachment (Flemming, intertidal, and deep sea), wetlands (swamps,
2002). In this sense, the attachment of diatoms marshes, bogs and fens), and man-made struc-
along with bacteria on man-made structures con- tures (such as those associated with drinking
stitutes a major problem on artificial structures water distribution systems). However, depending
immersed in the marine environment (Salta et al., on the particular characteristics of each environ-
2013). ment, biofilms are determined by a specific set
The formation of attached microbial com- of environmental factors (Fig. 1.2). Some char-
munities to man-made surfaces follows similar acteristics of the water surrounding the biofilm
sequential steps to that observed for inert natural such as temperature, oxygen concentration, pH
substrates (such as rock and sediments). First, sur- and nutrient levels commonly affect all aquatic
face is conditioned with a film of polysaccharides environments. Other factors are only relevant in
and proteins, then pioneer planktonic cells attach some environments and, as an example, flow is
to the surface and progressively other microbes especially relevant in rivers and in drinking water
arrive and biological adhesion continues to pipes, while pressure and tides are especially rel-
form microcolonies until the mature biofilm evant in marine environments (Fig. 1.2).
12  | Mora-Gómez et al.

water distribution systems and surfaces in marine

environments such as boats are usually under dark
LAKE and oligotrophic conditions (Kelly et al., 2014).
As mentioned above, the environment plays a
significant role in determining the function and
Light Wave
structure of biofilms, all of them related with the
Tides colonizing substrata (Fig. 1.1) (Stoodley et al.,
Tº, 02, pH, Light 2002). Some environmental factors are physical
(flow, light, temperature, oxygen, salinity) and
Flow Salinity
others chemical (pH, nutrient availability, organic
matter composition), but biological aspects such
Light as grazing also determine biofilm development
(McIntyre et al., 1996; Sabater et al., 2002; Burns
and Ryder, 2001). General aspects of the envi-
ronmental factors affecting biofilms are reviewed
Figure 1.2  Principal factors affecting biofilm
development in natural and man-made
environments considered in this chapter. Light
Light is a fundamental variable affecting bio-
films since light availability is a primary factor in
Generally, rivers and streams are characterized determining the proportion of photosynthetic
by their unidirectional flow along a continuum, organisms in biofilms (Guasch and Sabater, 1995;
where factors such as sediment load, nutrient Hill, 1996; Romaní, 2010). However, benthic
status, temperature, pH and water velocity, as light environments are rather variable, and light
well as the input of autochthonous and alloch- intensity may regulate benthic algal biomass as a
thonous carbon sources, vary from upstream to function of depth, turbidity and canopy develop-
downstream (Koetsier et al., 1997; Battin et al., ment in riparian zones (Cahoon and Nearhoof,
2001; Winter et al., 2007). Lakes and reservoirs 1999; Hill, 1996). Some species are capable of
have predictable vertical gradients of temperature, tolerating conditions of low light and resist the
light, oxygen and pH, with cold, dark and anoxic stresses of light deprivation (Peterson, 1996),
conditions in the deepest zone (Lampert and while in some environments such as deep waters
Sommer, 2007). Wetlands are shallow environ- in marine environments and lakes, photosynthetic
ments (less than 5 m deep), which vary greatly in organisms are absent from biofilm communities
size and shape, and present standing water bodies (Lalli and Parsons, 1993).
with characteristic vegetation. These environ- Attenuation of light by the water column is
ments may contain water permanently, seasonally greater in lakes, sea and deep wetlands than in
or occasionally and water might range from being running waters, but turbidity from silt and other
fresh to salty (Hart et al., 1990). Benthic marine inorganic particles substantially reduces light
environments can be divided in three main zones penetration in many rivers, reservoirs, wave-swept
(intertidal, subtidal and deep-sea), each of them lakes, and estuaries. In lakes, phytoplankton abun-
with its own habitat characteristics, which in dance can control the quantity and quality of light
turn influence the benthic biofilm community that reaches benthic algae, resulting in an inverse
established (Chiu et al., 2006). Specifically, the relationship between phytoplankton biomass
intertidal zone is highly dependent on tidal effects and benthic algal biomass (Hill, 1996). In small
(Nybakken and Wallace, 1992); the subtidal zone streams, light availability is mainly determined by
is greatly influenced by wave action (Huzarska, riparian vegetation (Guasch and Sabater, 1995;
2013), and the deep-sea floor is characterized Roberts et al., 2004; Proia et al., 2012). In a marine
by having a disphotic zone with high pressures environment, light intensity changes in correla-
(Siebenaller and Somero, 1978). Finally, drinking tion to the different tidal levels.
Limits of the Biofilm Concept |  13

Biofilms exposed to high light conditions usu- light environments (such as open sites in clear
ally develop a significant biomass of phototrophic streams or tidal zones) have developed mecha-
organisms and thus also display photosynthetic nisms that reduce the potentially damaging effects
activity. They are thicker and more structured, of high irradiances. Therefore, algae exposed to
with a higher C/N ratio and a greater contribu- high irradiances accumulate carotenoids, which
tion of the EPS than those developing in the may diminish photoinhibition by absorbing
dark (Romaní et al., 2004b). In these conditions, photons in excess, and experience reduced quan-
microbes uptake organic compounds released by tum yields, or produce sheath pigments such as
algae (high-quality fresh molecules) as they are scytonemin (Hill, 1996). Similarly, light might
less dependent on the organic molecules avail- also have negative consequences on heterotrophic
able in the flowing water (Romaní et al., 2004b). organisms. It has been observed that UV radia-
Photosynthetic activity promoted by light may tion may limit microbial development in epixylic
also stimulate extracellular enzyme activity of biofilms, as fungi are more sensitive than bacteria
biofilms growing on both inert and organic (Denward et al., 2001). In addition, denitrifica-
substrates (Francoeur et al., 2006). In contrast, tion in epiphytic biofilms may be inhibited under
in low light conditions, a thinner biofilm is usu- light conditions (Eriksson, 2001). In marine
ally built, with lower algal biomass and, thus, ecosystems, it has been demonstrated that physi-
microbes mainly use molecules from the dis- ological responses to excessive insolation depress
solved organic matter (DOM) and particulate the growth of epilithic biofilms, leading to a
organic matter (POM) pool. These situations decline in productivity during summer (Lamon-
obviously also depend on other factors such as tagne et al., 1989; Thompson et al., 2004).
inorganic and organic matter available and flow
velocity (Romaní, 2010). Temperature
Complementarily, some aspects related to the Water temperature directly regulates metabolic
type of substrate also determine light intensity rates, microbial growth and activity, and conse-
reaching the biofilm. In rocks, substrata texture quently plays an essential role in biofilm function
changes light availability due to a greater area such as organic matter processing, primary
in rough surfaces, since the same amount of production and nutrient cycling (Brunke and
light energy is distributed across a larger area Gonser, 1997; Dang et al., 2009; Friberg et al.,
(Lambert’s law). Moreover, direct light intensity 2013; Ylla et al., 2014). Additionally, temperature
changes with whole-substratum orientation and variation may lead to alter community structure,
light direction due to microscale slope angle extracellular enzyme activity, biofilm metabolism,
(Murdock and Dodds, 2007). Thus, biofilms interspecific relationships and biodiversity in all
located on the rock sides receive significantly less kinds of biofilms (Romaní and Sabater, 2000;
direct light than cells on the top when the light Ferreira and Chauvet, 2011; Romaní et al., 2014).
source is directly overhead (Hill, 1996). Likewise, Optimal temperatures for most biofilm range
light decreases rapidly in depth within sediment. from 10 to 30°C, and higher temperatures induce
In natural sediments fine particles and the detritus heat stress and reduce growth (DeNicola and
stored inside the cavities between larger particles Hoagland, 1996). At the same time, species have a
reduces light penetration (Brunke and Gonser, temperature tolerance range and below and above
1997). In non-cohesive sediments (i.e. sand), this range their activity is reduced or suppressed,
light reaches a few centimetres in depth (Kühl making the response of the whole biofilm to
et al., 1994; Kühl and Jorgensen, 1995), while in temperature variation difficult to predict (Chau-
cohesive sediments (i.e. silt), light is restricted to vet and Suberkropp, 1998; Rajashekhar and
the top 2 mm (Kühl et al., 1994). Kaveriappa, 2000). A rise on temperature may
In contrast, high light may lead to photoinhibi- also increase bacterial attachment on man-made
tion, defined as a reduction in the photosynthetic surfaces (Donlan, 2002).
rate of the whole integrated biofilm (Underwood, Frequency and range of temperature oscilla-
2002). Phototrophic organisms inhabiting high tions can deeply affect the structure and function
14  | Mora-Gómez et al.

of biofilms, however, this effect varies depending In epilithic biofilms, autotrophs and hetero-
on biofilm type. For example, in epixylic biofilms trophs can respond differently to nutrient supply.
it was observed that diel temperature oscillations In biofilm heterotrophic functioning, nutrient
might accelerate microbial decomposer activity by availability can either directly (by influencing
up to 31%; however, under very low temperatures metabolic activities) or indirectly (by effect on
(~3°C) oscillations do not affect organic matter primary producers) affect biofilm communities,
decomposition (Dang et al., 2009). In contrast, and this response might change depending on
in sediments, the influence of diel and seasonal light availability (e.g. Romaní and Sabater, 2000;
temperature fluctuations are mainly observed in Rier and Stevenson, 2002). High availability of
surface biofilms while deep sediments are more nutrients can effectively enhance algal biomass
stable, with no sudden decrease/increase in tem- and productivity when light is present (e.g.
perature (Brunke and Gonser, 1997). Moreover, Guasch et al., 1995; Ylla et al., 2007).
in intertidal areas, adaptations to temperature are Sediments can play an important role as sinks
really important because these areas are regularly for inorganic nutrients by buffering their release
subjected to aerial temperature variation (Nybak- into the water column (Wetzel, 1996; Jarvie et al.,
ken and Wallace, 1992). 2005). As described by Dodds (2003), epipsam-
Studies evaluating the impact of expected tem- mic biofilms may remove phosphorous from the
perature increase in the next decades have shown water column, including uptake and sediment
that the interaction between temperature and the deposition. However, in eutrophic lakes, sedi-
quality of available organic matter may affect bio- ments can contribute to eutrophication through
film responses. For example, in epixylic biofilms, phosphorus release into the water column
microorganisms living on low degradable organic (Ribeiro et al., 2008; Martins et al., 2008).
matter are less affected by increasing temperature In attached communities growing on plant
than those feeding on labile substrates (Bärlocher detritus, high phosphorus and nitrogen con-
et al., 2013; Gonçalves et al., 2013). In contrast, centrations in water promote biomass accrual,
in epilithic biofilms some evidence points out that production and respiration of the microbial
degradation of more bioavailable organic carbon community, as well as its organic matter degra-
is not affected by temperature increase, while dation activity (Gulis et al., 2008; Suberkropp
degradation of recalcitrant substances might be et al., 2010). However, decomposer activity is
enhanced by warming (Ylla et al., 2012). only enhanced by nutrients up to a threshold,
Although less studied, a general effect of tem- beyond which it decreases again (Duarte et al.,
perature on attached microbial communities is 2009; Woodward et al., 2012). The effect of nutri-
that related to biogeographic variations. In this ent concentration on epixylic biofilm is further
sense, in epilithic biofilms it has been observed strongly influenced by the nutrient levels in the
that at small biogeographic scales, bacterial taxon main organic substratum where biofilm develops
richness is greater at latitudes closer to the equator and their balance with nutrients in water. For
and reduced at higher altitudes, both factors (lati- example, Royer and Minshall (2001) did not
tude and altitude) being inversely related with air find any effect of increasing nutrient on microbial
temperature (Lear et al., 2013). leaf decomposition, apparently due to the lack of
nutrient limitation of the studied stream. Ardón
Inorganic nutrients and Pringle (2007) observed that biofilm growing
Nutrients are essential resources, and their availa- on recalcitrant C sources did not show enhanced
bility could determine changes in the structure and respiration while biofilm growing on labile C
the function of microbial communities (Larned, sources was stimulated by nutrients.
2010). The trophic status of aquatic ecosystems However, epiphytic biofilm adapted to high
determines the microbial species dominance and nutrient concentrations may increase its deni-
microbial community composition of developing trifying capacity about a hundred times that of
biofilms (Coleman and Burkholder, 1994; Duarte surface-attached microbes adapted to lower nutri-
et al., 2009). ent levels (Eriksson and Weisner, 1996).
Limits of the Biofilm Concept |  15

Oxygen and pH utilization during photosynthesis in light biofilms

Oxygen gradients in aquatic ecosystems promote results in an increase in the internal pH and this
differential biochemical processes in biofilms, higher pH favours the removal of metals by pre-
from aerobic respiration at the biofilm surface to cipitation and possibly adsorption (Liehr et al.,
methane oxidation, anaerobic respiration and fer- 1994). In heterotrophic communities, low pH
mentation occurring in zones of oxygen depletion may also slow down organic matter decomposi-
(Brune et al., 2000), commonly found in fresh- tion mediated by microorganisms, which effect
water and marine sediments. Oxygen levels in is related to a reduced efficiency of pH-sensitive
water are strongly dependent on microbial activ- enzymes and changes in community composi-
ity and physical factors such as water re-aeration tions (Simon et al., 2009; Clivot et al., 2013a,b).
and depth. Additionally, human contamination
providing a large amount of organic and inorganic Flow
nutrients, as observed in sewage effluent, might In running water, flow velocity and water level
induce oxygen depletion and anaerobic condi- modulate biofilm growth and thickness (Aug-
tions. In these cases, biofilm activity may cause spurger and Kusel, 2010). Biofilm is disturbed
diurnal fluctuation in pH, oxygen and redox. by the mechanical effect of abrasion during high
Reduced redox conditions affect the mobility of flow periods. For example, disturbances in river
trace metals (i.e. Mn, Cu, Zn, Cd, Fe, Hg) in the systems (such as a flood) reset biofilm succession
interstices (Brunke and Gonser, 1997) and acidi- by removing biomass and clearing substrata for
fication is often accompanied by metal dissolution colonization (Webb et al., 2006). Biofilms have
and deposition (Sasaki et al., 2005). some mechanisms to avoid this disturbance as the
Biofilm biomass production and organic thickness of extracellular polymeric substances,
matter mineralization are often considered to be which contribute to biofilm stability versus flow
highly dependent on oxygen concentration, with (Costerton et al., 1995; Gerbersdorf, 2008).
lower rates under anoxic conditions. In sediments, Moreover, under flow disturbance, bacteria with
the oxygen content declines with increasing sedi- higher growth rates can survive on biofilm surfi-
ment depth affecting the microbial metabolism cial layers, while those slower growing bacteria are
and diversity (Brunke and Gonser, 1997). In the forced to remain at lower biofilm layers (Furumai
surface, oxic sediment biofilms are composed and Rittmann, 1994).
of a high diversity of aerobic bacteria while in Two types of flows are relevant to most natural
deeper sediment layers, with low or nil oxygen and man-made environments: laminar flow and
concentration, biofilms are mainly composed turbulent flow; both may affect biofilm microbial
of nitrifiers, sulfate-reducing and methanogenic composition and function. Biofilms under turbu-
bacteria (Brune et al., 2000). In contrast, the lent flow commonly form filamentous ‘streamers’
oxygen produced by photosynthetic algae also since they can oscillate rapidly in the flow (Stood-
has a negative effect on chemical processes such ley et al., 2002). In addition, when increasing
as denitrification, nitrification, metal oxidation the turbulence, the boundary layer between the
and sulfite oxidation in biofilms formed on rock, biofilm and the water column indirectly decreases,
sediments or living plants (Eriksson, 2001; Glud, and consequently, increases the nutrient supply to
2008). Additionally, a reduction of oxygen con- the biofilm (De Beer et al., 2006) but decreases
centration in water can affect fungal development biofilm microbial biomass and density (Biggs and
(i.e. slow growing and low sporulation rates), Hickey, 1994). In light-grown ephilitic biofilms
and decrease fungal diversity (Solé et al., 2008; flow velocity has been shown to stimulate photo-
Medeiros et al., 2009) reducing the microbial deg- synthesis and increase nutrient supply (Stevenson
radation velocity of dead plant material (Pascoal and Glover, 1993; Augspurger and Kusel, 2010;
et al., 2003). Romaní and Marxsen, 2002). Flow velocity can
Likewise, low pH may also affect biofilms in also affect the biofilm structure and algal com-
aquatic ecosystems. pH affects epilithic biofilm position and its response to higher shear stresses
functioning and composition. For example, CO2 (Graba, 2013). Flow velocity may be relevant
16  | Mora-Gómez et al.

during biofilm formation since it is reported that (Howell, 2009). This is mainly due to a reduction
bacterial abundances are higher in young biofilms of biofilm maturation and maintenance in bio-
grown under higher flow velocities than in bio- films in the early stages of development occurring
films grown under slower flow velocities (Hunt under high shear stress (Rochex et al., 2008).
and Parry, 1998). In marine systems, tides are relevant, especially
In sediments, flow velocity can influence in intertidal zones, because they determine the
bacterial activity and solute transport (Fischer community’s ability to tolerate immersion in air.
et al., 2003; Battin, 2000), but the effect of water Many intertidal species are well-adapted to tidal
movement decreases with sediment depth. High situations, so they are able to be quiescent when
flows affect biofilm biomass by increasing sedi- the tide is out and resume normal activity when
ment mobility, which leads to the abrasion of the the tide is in (Nybakken and Wallace, 1992).
biofilm by suspended sediment and substrate Tidal levels have been observed to cause seasonal
tumbling (Biggs et al., 1999). patterns in the abundance of cyanobacteria,
In plant detritus, slow flowing water current being maximal on the lower shore in summer
might reduce microbial respiration, fungal sporu- and on the upper shore during winter (Thomp-
lation and biomass, while high flow increases son et al., 2004). In addition, an abundance of
ATP, chlorophyll a concentration and the number photosynthetic microbiota can be greater on the
of aquatic hyphomycete species (Golladay and upper shore than on the lower shore (Aleem,
Sinsabaugh, 1991; Schlief and Mutz, 2009). 1950; Castenholz, 1963; Thompson et al., 2004,
However, flow changes do not always determine 2005). Additionally, microbial mats that develop
differential microbial decomposer activity (Fer- in intertidal environments are exposed to water
reira and Graça, 2006; Dewson et al., 2007). movement and wind as eroding forces. Mat
Similarly, increased photosynthesis and respira- formation increases the stability of sediments
tion are observed for biofilms growing on living cementing individual sand grains to each other
plants under high flow conditions, but denitri- and increasing the critical friction velocity (Van
fication capacity is favoured in stagnant waters Gemerden et al., 1989).
(Eriksson, 2001). However, waves and currents also exist in lakes
In general, in aquatic ecosystems, flow increases and produce sediment suspension and transport
biofilm colonization and metabolism up to a from shallower to deeper areas, a process called
certain level when flow destroys the biofilm, this sediment focusing. By this process, fine-grained
threshold depends on the specific environment. sediments tend to concentrate in deeper parts of
Similarly, in man-made systems, increased flow the lake. Wave action and currents may in part con-
velocity favours biofilm attachment, if flow dis- trol nutrient transport and availability (Mackay
turbance does not exceed critical levels (Donlan, et al., 2011). Additionally, waves and wind as
2002). physical factors may affect the formation of lake
snow aggregates which is mostly dependent on
Waves and tides wind-induced turbulences (Grossart and Simon,
Wave action is an important physical factor in 1993). In estuaries and tidal zones affected by
marine shallow water habitats and in some lakes. shallow seas, macroaggregates are usually smaller
In these habitats, turbulence produced by wave and more abundant than in lakes and in open sea
action keeps inshore water from becoming ther- due to high shear rates leading to disaggregation
mally stratified, and for this reason nutrients in and high resuspension rates (Chen et al., 1994;
marine shallow water habitats are rarely limited or Zimmermann, 1997).
locked up in a bottom reservoir (Nybakken and
Wallace, 1992). Waves and tides determine shear Pressure
stresses and influence biofilm diversity in lakes Pressure is a factor that greatly influences marine
and marine systems. Specifically, biofilms that systems where it ranges from 20 to more than
develop under high shear stresses are less diverse 1000 atm. Hydrostatic pressure is correlated to
than those developing under lower shear stresses an increase in depth (Carney, 2005). Pressure
Limits of the Biofilm Concept |  17

increases by 102 kPa every 10 metres in depth. diatom assemblages present in epipsamic biofilms
This means that pressure plays a major role in in estuaries and showed Navicula gregaria and N.
organisms’ adaptation to the deep-sea environ- phyllepta to be abundant at oligo- and mesohaline
ment (increased hydrostatic pressure but also low sites respectively, while Pleurosigma angulatum
temperature) (Siebenaller and Somero, 1978). and Plagiotropis vitrea were abundant at polyha-
Pressure affects the morphology of bacteria line sites.
(Zobell and Oppenheimer, 1950) and it may have Under natural conditions, some freshwater
mutagenetic effects (Zobell and Morita, 1957). ecosystems are also influenced by increasing salin-
Some deep-sea bacteria appear to be obligate ity concentrations, mainly because of evaporation
barophiles (adapted to high pressure). However, combined with intrusions of groundwater and
even under optimum conditions for reproduc- the mineral constitution of some catchments.
tion, barophilic bacteria in sediment found at The available data suggest that freshwater biota
great depths are highly specialized and are typi- will be adversely affected as salinity exceeds 1 g/l,
cally slower growing than shallow-water benthic however the sensitivity of prokaryote and eukary-
species and communities (Cowie, 2010). ote biofilm components seems to be different
(Nielsen et al., 2003). Species replacement in bac-
Salinity terial communities has been observed in salinized
Salinity variation is an important factor for freshwater systems, but the different communities
biofilms in marine environments and estuar- possess similar metabolic capabilities (Hempel
ies as well as in some characteristic continental et al., 2008; Hart et al., 1990). Conversely, the
aquatic ecosystems (salty wetlands, lagoons or majority of freshwater algal taxa do not appear to
rivers). Marine bacteria grow optimally at a salt tolerate increasing salinity (Nielsen et al., 2003).
concentration between 3.3% and 3.5% and do not In addition, the breakdown of organic matter is
develop or develop poorly when there is no NaCl seen to be reduced under high salinity concentra-
in the water. Variations in salinity influence bacte- tion, which suggests that fungi have low tolerance
rial growth, metabolism and cell attachment on of hyper saline environments (Schäfer et al.,
artificial substrates (Chiu et al., 2006). Gradients 2012).
in salinity are especially important in intertidal
biofilms and estuarine biofilms. Intertidal biofilms Grazing
may be exposed to low/high tide alternations that Biofilm is strongly regulated by herbivores in
influence salinity concentration due to lower/ aquatic ecosystems (Steinman, 1996; Thompson
higher dilution (Nybakken and Wallace, 1992). et al., 2004). According to Lowe (1996), biotic
Depending on the season, salinity has a stronger disturbance is usually associated to the activity of
effect on marine biofilms. Chiu et al. (2006) grazers in the biofilm community. Grazers impact
observed that in summer, community composi- biofilm communities both by direct consumption
tion on biofilms was affected by salinity, while in of algal cells and by dislodgment of cells from
winter it was more affected by temperature than the substratum (Lowe, 1996). Primary consum-
by salinity. ers (herbivores) in freshwater ecosystems span
Estuaries are dynamic ecosystems located at many taxonomic groups, but insects, molluscs
the interface between marine and terrestrial envi- (especially gastropods), crustaceans, and fish
ronments, and are exposed to salinity gradients appear to be particularly important (Lamberti,
of between 1.0% and 3.2%, although salinity at 1996; Burns and Ryder, 2001). Concretely, the
depth might be remarkably constant (Dunn et al., reduction of algal biomass due to herbivory has
2008). Salinity gradient in estuaries might regu- been demonstrated for a variety of grazer types
late nitrification since the denitrifier community (Steinman, 1996). Grazing maintains microbial
seems to be dominated by halotolerant bacteria biofilms at early successional stages and, when
(Magalhães et al., 2005). Similarly, algal commu- it is reduced, this invariably leads to an increase
nity composition is also influenced by the gradient in microalgal and then macroalgal biomass, sug-
of salinity. Underwood et al. (1998) analysed the gesting that top-down regulation is a key factor.
18  | Mora-Gómez et al.

This grazing effect has been observed in marine the foraging intensity of the principal molluscan
environments (Thompson et al., 2004; Firstater et grazers and limpets (Thompson et al., 2004).
al., 2012), estuaries (Sullivan and Currin, 2000), A specific example of excluding grazing influ-
rivers (Feminella and Hawkins, 1995; Lamberti, ence is that of microbial mats which are mainly
1996; Steinman, 1996), lakes (Lowe, 1996), and developed under conditions that exclude or limit
in man-made systems (Esselink et al., 2002). grazing pressure (Cohen, 1989). Since mat build-
Bacteria within biofilms may also be subjected ing depends on the suppression of invertebrate
to predation by free-living protozoa in freshwater grazers, extreme salinity, temperature and drought
systems (Hahn and Höfle, 2001). At the same conditions allow the development of microbial
time, because biofilms are a valuable food resource mats (Connor et al., 1982; Awramik, 1984).
in aquatic ecosystems, their abundance may
influence the physiological fitness of herbivores.
Moreover, herbivore growth rates are frequently Conclusions
controlled by biofilm availability (Lamberti, The main conclusions drawn from this review are
1996), summarized in the following points:
In plant detritus biofilms, invertebrates play a
dual role, some feeding on biofilm by grazing on • Through the bibliography, biofilm is also
plant surface, but others consuming dead plant referred to as: periphyton, aufwuchs, microphyto-
matter by shredding plant tissue. In a detrital bentos, benthos, haptobenthos and herpobenthos.
system, synergistic and antagonistic interactions Moreover, regarding to the type of substrate
have been observed between aquatic inverte- where the biofilm develops it is also known as
brates and microorganisms present on dead plant epilithic, epipsammic, epipelic, hiporheic, epix-
material (Canhoto and Graça, 2008). Microbes ylic, or epiphytic biofilm. The specific aquatic
are responsible for increasing plant tissue nutri- suspended aggregates behaving similarly to
ent content, favouring invertebrate consumption biofilm are called marine snow, lake snow, river
(Bärlocher and Kendrick, 1975; Foucreau et al., snow, aggregates and granules. In man-made sys-
2013), and some insect larvae can use microbial tems the most common term is biofilm and the
enzymes inside their guts (Canhoto and Graça, words biofouling or bioclogging are mainly used
2008). Meanwhile, invertebrate feeding can when biofilm growth has a ‘negative’ effect.
disrupt physical barriers of dead plants, enhance • Substrata in natural and man-made envi-
their surface-to-volume ratio, and increase basal ronments determine biofilm structure and
resources by egestion and excretion, favouring metabolism due to the particular character-
microbial performance (Sabetta et al., 2000; istics of each type of surface. Therefore the
Canhoto and Graça, 2008; Diaz Villanueva et roughness, size and chemical composition,
al., 2012). On the other hand, invertebrates and as well as the possible interaction between
microbes can compete for plant food resources attached community and substratum give the
(Bärlocher, 1980); and invertebrates also predate biofilm a particular configuration, favouring
bacterial cells, mycelia or fungal spores (Suberk- the development of either prokaryotes or
ropp et al., 1983; Suberkropp and Wallace, 1992; eukaryotes (see Fig. 1.2 for a summary of most
Hahn and Höfle, 2001). common biofilms).
However, grazing pressure on biofilms is • Biofilm characteristics are highly vari-
further modulated by specific environmental able depending on the chemical, physical
conditions favouring or limiting the development and biological conditions found in each
of grazers. Related to this, in estuarine and hyper- particular aquatic environment. For example,
saline environments (Wieland and Kühl, 2000), photosynthetic microorganisms are promoted
periodic drying (Lassen et al., 1992) or high in biofilms growing under high light conditions,
temperatures (Castenholz, 1984) can limit meio- and increased temperature or nutrient condi-
and macrofauna growth, thus reducing grazing tions enhance the rate of microbial processes.
activity. Moreover, sea/air temperature regulates The effect of most factors (light, temperature,
Limits of the Biofilm Concept |  19

nutrients, oxygen, pH, flow, pressure, salinity, Azam, F., and Cho, B.C. (1987). Bacterial utilization of
organic matter in the sea. In Ecology of Microbial
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range and sensitivity of species to those factors. Press, Cambridge, UK), pp. 261–281.
However, environmental variables also interact Azam, F. (1998). Microbial control of oceanic carbon flux:
among them and it is sometimes difficult to the plot thickens. Science 280, 696–696.
Bärlocher, F. (1980). Leaf-eating invertebrates as
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Bärlocher, F., Kebede, Y.K., Gonçalves, A.L., and
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groups (bacteria, archaea, viruses, algae, fungi, substrate quality modulate sporulation by aquatic
cyanobacteria, protozoa, small metazoa), (2) hyphomycetes. Microb. Ecol. 66, 30–39.
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Battin, T.J. (2000). Hydrodynamics is a major determinant
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Laser Microscopy for the Study of
Biofilms: Issues and Options
Thomas R. Neu and John R. Lawrence

Abstract cyanobacteria), fungi, bacteria, archaea and graz-

In this review, river biofilms and the various ers (protists and benthic macroinvertebrates).
structural aspects of environmental biofilms are This community is exposed to physicochemical
discussed. These include sample type and origin, parameters such as light, hydrodynamics, differ-
cellular and polymeric constituents as well as ential nutrient regimes and fluctuating conditions.
examination at the micro- and meso-scale. For this The main factors shaping aquatic biofilms are
purpose, selected studies are considered taking twofold: (1) a complex prokaryote/eukaryote
advantage of the confocal imaging approach for community including competitive and symbiontic
investigation of hydrated environmental bio- interactions as well as grazing, and (2) long-term
film samples. Emphasis is put on extracellular development and exposure to physicochemical
polymeric substances (EPS) as a multi-functional environmental effects. Consequently, biofilms in
component of microbial biofilms. The main tech- natural aquatic ecosystems are rather different if
nical focus is on laser scanning microscopy by compared to technical and particularly pure cul-
means of three-dimensional, multichannel imag- ture laboratory biofilms.
ing, specific staining techniques and digital image Aquatic biofilm systems are found in marine
analysis. The advantages and limitations of the and freshwater habitats. The latter can be sub-
approach are critically assessed. Finally, research divided into lake and river biofilm systems.
needs are listed in order to advance understanding The focus in this chapter will be on river/lotic
of complex, environmental, real world biofilms. biofilm systems. In this respect the following
philosophical statement might be important: ‘you
cannot step twice into the same river’ (Heraclitus
Introduction 535–475 bc). Thus heterogeneity, sampling,
By definition, biofilm systems are a collection of reproducibility and comparison represent critical
microorganisms and their extracellular polymeric issues in fluvial biofilm research. As a result, river
substances (EPS) associated with an interface. biofilms remained for a long time a neglected field
Aquatic biofilm systems comprise bio-films in microbiological research (Leff, 1994).
(stationary) and bio-aggregates (mobile), which This chapter is based on two previous reviews
both fulfil the definition of biofilm systems (see on river biofilms discussing general aspects (Neu
Chapter 1). EPS are organic polymers of micro- et al., 2003) and the use of model systems (Law-
biological origin which in biofilm systems form rence and Neu, 2003) for the study of biofilm
a matrix into which the microorganisms are composition and microstructure. Furthermore,
embedded. At the same time EPS are responsible some recent reviews analyse the use of laser scan-
for the interaction with interfaces as well as with ning microscopy for studying the general biofilm
dissolved and particulate compounds. Environ- structure (Neu and Lawrence, 2014b), the bio-
mental aquatic biofilm systems are heterogeneous film matrix (Neu and Lawrence, 2014a) and the
assemblages composed of phototrophs (algae and biofilm matrix functionality (Neu and Lawrence,
30  | Neu and Lawrence

Table 2.1  Review articles on laser microscopy applications in biofilm research

Focus of review Reference
First overview on CLSM applications Caldwell et al. (1992)
Comprehensive review of CLSM Lawrence et al. (1998b)
CLSM methodology Lawrence and Neu (1999)
Short CLSM overview Palmer, Jr. and Sternberg (1999)
Structured CLSM approach Neu and Lawrence (2002)
One-photon LSM versus two-photon LSM Neu and Lawrence (2005)
Spatio-temporal approaches Palmer, Jr. et al. (2006)
Environmental CLSM applications Lawrence et al. (2007a)
CLSM techniques and protocols Lawrence and Neu (2007a)
CLSM of aggregates Lawrence and Neu (2007b)
CLSM–MRI–STXM Neu et al. (2010)
CLSM of hydrocarbon biofilms Neu and Lawrence (2010)
In situ biofilm matrix analysis Neu and Lawrence (2014a)
CLSM applications Neu and Lawrence (2014b)

2009). Laser microscopy based methods for of the fragile 3-dimensional structure. In contrast,
the study of biofilms have also been extensively biofilms can be associated with solid environmen-
reviewed (Table 2.1). The main part of this chap- tal surfaces such as pebbles and rocks, sand and
ter has a focus on biofilm structural investigations sediments or plant surfaces and wood. A common
by means of confocal laser scanning microscopy strategy in studying river biofilms is the exposure
(CLSM). For this purpose the various interfaces of a defined substratum which is mounted on a
in river habitats are differentiated (solid–liquid, special device for holding it in a certain position.
liquid–liquid, liquid–gas). The challenge of In many cases the material used is readily available
analysing the biofilm matrix including its func- in the lab such as glass or plastic slides. Some-
tionality will also be dealt with. Before focusing times tailor-made materials have been employed
on the biofilm structure and matrix, some general as for example clay and ceramic tiles, wood, cut
issues with respect to current understanding of rocks and metals. Often the surface roughness
terms and models are discussed. Then practical and texture is manipulated in order to simulate
considerations regarding sample type and mount- environmental surfaces. A different approach
ing are touched upon. Finally research needs are takes advantage of micro- and mesocosms which
defined for future investigations of biofilm struc- are ideally set-up next to the river in order to
ture and function. assure continuous once-flow-through conditions.
Microcosms are usually set-up in the laboratory
(Singer et al., 2006; Zippel et al., 2007) or in a
Significance of sample type container next to the river (
and origin for laser microscopy magdeburg). Mesocosms consisting of large out-
studies door flow lanes are often built parallel to the river
With respect to sample types one has to distinguish to be as close as possible to the real environment
the two major forms of river biofilm systems, bio- (Battin et al., 2003; Besemer et al., 2009b).
aggregates and bio-films. Aggregates may come With respect to specimen preparation and
directly from the water column or be developed in mounting for CLSM another aspect comes from
microcosms such as plankton columns or rolling the type of microscope available, inverted or
flasks. This sample type has to be mounted in spe- upright. The upright version of any laser scanning
cial chambers to avoid deformation and squeezing microscope is often more flexible for analysing
Laser Microscopy of River Biofilm Systems |  31

a variety of environmental samples. Aggregates of protists. Most CLSMs are point scanners for
can be mounted and stained in coverwell cham- high resolution imaging and as a result motile or
bers with protective spacers of different heights. drifting objects may cause either irregular stripes
Defined surfaces such as glass slides can be placed or dotted tracks. However, there are instruments
in a Petri dish, stained and then examined with with different (fast) scanners available as well as
water immersible lenses. Similarly, pebbles, rocks, spinning disc CLSMs, which can handle motility
twigs or shells can be placed in a dish with higher issues. Finally, large differences in signal intensity
walls for observation of larger samples. It is also could be an issue as the signal to noise ratio can be
possible to form a moat on larger objects (i.e. with optimized for either the bright objects or for the
plasticine or silicone) to create a well for staining. very faint objects but not both.
For further details on laser microscopy of biofilm Biofilm analysis by means of CLSM usu-
samples the reader is referred to a number of ally starts with direct visual examination of the
reviews focusing on different technical and applied unstained sample in order to ‘see’ the original
aspects (see Table 2.1). More recently, optical structure and patterns as well as possible auto-
coherence tomography (OCT), a mesoscale fluorescence using the epifluorescence mode.
technique, was evaluated for studying microbial In a second step in the CLSM mode, the sample
biofilms. For OCT the water immersed biofilm is optically sectioned in order to record highly
sample can be placed directly under the imaging resolved images. In many cases one aim is to reveal
probe. First results of OCT imaging applied to bacterial cells and their distribution mostly by
biofilm visualization are supplied below. using a green emitting nucleic acid-specific fluo-
rochrome such as Syto9 or SybrGreen. In aquatic
biofilms this would mean using three laser lines
The laser microscopy approach for excitation of the nucleic acid stain, the auto-
for biofilm analysis fluorescence of the cyanobacteria (phycobilin and
Laser microscopy, mostly used as confocal laser chlorophyll a) and the autofluorescence of algae
scanning microscopy (CLSM), has evolved as (only chlorophyll a). In addition, the first line can
the method of choice for examination of biofilm be used for recording the reflection signal and the
structure from various habitats. Most importantly, second line could be used for adding, for example,
CLSM allows three-dimensional, multichan- a lectin in order to visualize glycoconjugates. As
nel microscopy of fully hydrated, living or fixed a consequence, emission signals are recorded in
samples. CLSM can be used for recording the four channels with the need for analysing co-local-
reflection (cellular or environmental) and auto- ization of the phycobilin and chlorophyll a signal
fluorescence signals (pigments of phototrophs) of in order to isolate the cyanobacteria and lectin
biofilms as well as the emission signals of added signal. Most laser microscopes offer the option of
fluorochromes specific for certain biochemi- non-confocal transmission imaging which can be
cal constituents. The fluorochromes may target a useful addition. The variety and complexity of
nucleic acids, proteins, glycoconjugates, lipids/ river biofilms examined by means of fluorescence
membranes, enzymes as well as the microhabitat techniques in combination with laser microscopy
(e.g. pH). The reader again is referred to a number is presented in Figs. 2.1 and 2.2.
of reviews covering staining procedures for micro-
bial biofilms (see Table 2.1).
Despite the many advantages of CLSM, the Laser microscopy of aquatic
technique has a few disadvantages to be aware of if biofilms
the biofilm structure has to be examined. One of CLSM has been employed for studying numer-
them is laser penetration into thick light scattering ous biofilm systems from very different habitats
and absorbing samples. Thus, for thick environ- for assessment of species specific features as well
mental biofilms (> 150 µm) or microbial mats, the as environmental and treatment effects (Neu and
sample still has to be embedded and sectioned. Lawrence, 2014b). In the following section several
Another critical point is motility for example selected studies with a focus on aquatic biofilms
32  | Neu and Lawrence

Figure 2.1  CLSM of river biofilms on pebble-sized stones illustrating heterogeneity of the biofilm landscape.
Data sets are shown as maximum intensity projections. Colour allocation: white, reflection; green, bacteria
(nucleic acid staining); red, glycoconjugates (lectin staining); turquoise, cyanobacteria (autofluorescence);
blue, algae (autofluorescence). Scale bar = 20 µm. (A) Location dominated by bacteria of different morphology
with only little glycoconjugate signal in between partly clustered. (B) Location with a bacterial base biofilm
covered with large cloud-like glycoconjugate clusters forming the interface to the bulk water phase. (C)
Location showing a cobblestone-like phototrophic biofilm of cyanobacteria (turquoise) and algae (blue) with
bacteria in between and some glycoconjugates. (D) Location with algal and bacterial filaments oriented
perpendicular to the rock surface with few glycoconjugates.

and using CLSM as a major tool will be discussed. annular reactors was investigated (Neu and Law-
One of the first studies demonstrating the suit- rence, 1997). The turbulent flow produced a
ability of CLSM for optical sectioning of biofilms biofilm architecture with ridges oriented to flow
showed the complex architecture in biofilms direction. These ridges increased the surface area
formed by different bacterial strains (Lawrence and created microhabitats. The bacteria were
et al., 1991). Depending on the species, cell accu- mainly colonizing the outer region of the ridges
mulations at the bottom or top of the biofilm were which may be composed of colloidal and par-
found. In between the cellular biofilm constitu- ticulate organic matter. Lectin staining showed
ents void spaces and channels could be identified. large areas/volumes with lectin-specific EPS
Similar findings were reported from experiments glycoconjugates. The CLSM technique employed
with contaminated groundwater in a fluidized was systematically tested and improved using the
bed reactor with biofilms developed on granular three-channel approach (Lawrence et al., 1998a).
activated carbon (Massol-Deya et al., 1995). In the meantime this approach was extended by
CLSM as a major tool for examining river adding another channel for reflection imaging as
biofilm systems was employed in a series of stud- well as cyanobacteria signals via co-localization
ies each with a different focus. In a first study the analysis of the red and far red channel as for exam-
structure of river biofilms developed in rotating ple shown in a study on rock biofilms (Horath
Laser Microscopy of River Biofilm Systems |  33

Figure 2.2  CLSM of a typical phototrophic river biofilm on a pebble stone demonstrating the 3-dimensional
arrangement of biofilm constituents. The data set is shown as XY maximum intensity projection from the
top (large image) as well as XZ and YZ projection from the side within the yellow cross in the XY image
(rectangular images). Colour allocation: white, reflection; green, bacteria (nucleic acid staining); red,
glycoconjugates (lectin staining); pink, cyanobacteria (autofluorescence); blue, algae (autofluorescence).
Note that the white signal in the two side view images is a result of triple signals from the red-green-blue
(RGB) overlay. The parallel lines in the side view images are caused by the user defined step size in axial
direction. Scale bar = 20 µm.

et al., 2006). Thereby five different channels can assessing the use of fluorescently labelled lectins
be used for imaging of river biofilm constituents. was carried out (Neu et al., 2001). For this pur-
In a subsequent study the river biofilms were pose the lectins were applied as single, or double
examined using rRNA targeted probes for Alpha-, and triple combinations to examine glycoconju-
Beta- and Gamma-proteobacteria as well as the gate distribution in hydrated river biofilms.
Cytophaga–Flavobacterium (Manz et al., 1999). One aspect of river biofilms is their role in
The river biofilms showed a distinct succession sorption and degradation of contaminants which
starting with Beta-proteobacteria, followed by a may affect biofilm structure and function. Thus,
shift towards Alpha-proteobacteria and bacteria by using biofilm reactors fed with river water,
of the Cytophaga–Flavobacterium group. In terms the fates of atrazine and diclofop methyl were
of morphology Beta-proteobacteria showed the followed (Lawrence et al., 2001). By using immu-
highest diversity. In order to run many replicate nostaining, the sorption of atrazine to a unique
reactors, a new simple and inexpensive rotating microcolony was demonstrated. Both herbicides
annular reactor design was developed (Lawrence became sorbed and degraded within the biofilm
et al., 2000). A comparison with the commercial which may act as a sink for both herbicides.
system showed similar results for biofilm struc- Another study focused on the degradation of
ture and composition. As the in situ analysis of the selected pharmaceuticals in river biofilms (Win-
biofilm matrix remains difficult, a detailed study kler et al., 2001). It could be shown that ibuprofen
34  | Neu and Lawrence

was readily degraded and two degradation prod- study the focus was on flow velocity in order to
ucts could be identified. In contrast, clofibric acid examine the effect of laminar, intermediate and
was not degraded in the reactor biofilms run with turbulent flow on biofilm architecture and com-
river water either from the South Saskatchewan or munity composition (Besemer et al., 2007). For
the Elbe River. With respect to elemental contam- this purpose, a laboratory flow lane microcosm
ination the effect of nickel, oxygen and nutrients with ceramic tiles was used. The flow velocity
on the river biofilm community was investigated showed a clear effect resulting in uniform bio-
(Lawrence et al., 2004). Nickel concentrations films under laminar flow to ridged biofilms with
at the industrial release rate had a dramatic effect streamers under turbulent flow. In addition,
on both cyanobacteria and the total phototrophic algae were identified as ecosystem engineers.
biomass. Nickel also reduced the carbohydrate Algae were initially part of garland-like streamers
utilization spectrum of the biofilm bacteria and composed of bacteria and EPS decorated with
had a negative impact on denitrification. diatoms. Later, filamentous algae dominated the
The response of biofilms towards nutrient biofilm which are, compared to bacteria, forming
additions was studied in another experimental larger building blocks thereby changing the fluid
series (Neu et al., 2005). The focus was put on dynamics and creating microenvironments. The
the presence of lectin-specific glycoconjugates effect of spatially structured landscapes on biofilm
produced during treatment with carbon, nitro- community composition was studied using large
gen, phosphorus and a combination thereof. streamside flumes (Besemer et al., 2009a,b). It
The individual treatments showed differences in was found that the community composition along
photosynthetic biomass, bacterial biomass and the spatially structured landscape showed a high
glycoconjugates when compared with control variation between base biofilm and streamers and
biofilms. It could also be shown that the reactors suggested species sorting within different micro-
with single nutrient additions had a similar glyco- habitats. This heterogeneity eventually led to
conjugate make-up. This was also the case when changes in resource use as studied by measuring
the carbon, nitrogen and phosphorus treatment carbon uptake in the form of glucose and dis-
was compared with the control biofilms. In a solved organic carbon (Singer et al., 2010).
detailed lectin study the microhabitat of river bio-
films and microcolonies was examined (Lawrence
et al., 2007b). Within the microcolonies, different Laser microscopy of aquatic
microdomains were identified by using a combi- aggregates
nation of lectins. It was found that some colonies Similar to biofilm studies, CLSM can be employed
had a specific glycoconjugate distribution located to examine bioaggregates, aquatic flocs or reactor
on the cell surface, in between the bacterial cells granules as indicated above. The first publication
and around the microcolony. Thereby bacteria in this field combined in situ photography, laser
have the potential to design and control their microscopy and chemical analysis for studying
immediate microenvironment. A further aspect in fixed marine aggregates (Cowen and Holloway,
this study was the combination of fluorescence in 1996). Later, the group summarized the approach
situ hybridization (FISH) and fluorescence lectin- in a methodological manuscript in which they
binding analysis (FLBA). This allowed allocation detailed the various fluorescence staining tech-
of both bacterial identity and glycoconjugate niques applied to marine snow (Holloway and
identity. Cowen, 1997). CLSM has also been applied for
The CLSM approach was also employed by examination of multiple parameters in non-fixed
Battin and co-workers in studies on river eco- river snow (Neu, 2000). The samples were imaged
system processes. They could show that biofilms in the reflection and fluorescence mode in order to
changed the physicochemical microhabitat and visualize mineral particles, autofluorescence and
thereby have a dramatic effect on transient stor- specific fluorescent probes added. With a focus on
age of organic compounds and the retention of glycoconjugates, the application of different fluo-
suspended particles (Battin et al., 2003). In a later rescently labelled lectins revealed the distribution
Laser Microscopy of River Biofilm Systems |  35

of various glycoconjugate clusters within individ- the oil–water interface. For example, Whyte et
ual river snow aggregates. In parallel, the identity of al. (1999) investigated the degradation of diesel
river snow bacteria can be assessed by FISH using at low temperature and showed the coloniza-
rRNA targeted probes (Böckelmann et al., 2000). tion of the droplets by Rhodococcus. Baldi et al.
In a follow up experiment the FISH technique was (2003) also studied the adhesion of bacteria to
combined with FLBA. By this means, the identity diesel droplets (Baldi et al., 1999) as well as the
of bacterial species or groups can be correlated growth of yeast on different hydrocarbons. The
with the production of extracellular glycoconju- interaction and degradation of PCB by bacteria
gates (Böckelmann et al., 2002). In a comparative was shown in a study with a mixed culture isolated
study of aggregates from the rivers Danube and from contaminated soil. In a simple aquarium
Elbe, methodological and seasonal aspects were experiment the bacteria from the sediment colo-
investigated (Luef et al., 2009b). Small aggregates nized PCB droplets attached to floating plastic
were examined directly whereas larger ones were slides (Macedo et al., 2005). A subsequent experi-
embedded, cryo-sectioned and post-stained. ment looked at the adaptation of the microbial
Samples were positively and negatively stained community to different PCB levels (Macedo et
with a focus on bacteria and glycoconjugate dis- al., 2007). Later, in a methodological publication
tribution. The CLSM approach was pushed to CLSM was again suggested as a tool for imaging
its limit in an investigation of viruses associated the colonization of aromatic hydrocarbons by
with river snow (Luef et al., 2009a). Although at Mycobacteria (Wouters et al., 2010). Nevertheless,
the edge of detection, viruses could be imaged imaging of oil–water mixtures by CLSM remains
on native hydrated aggregates. The report criti- difficult with respect to mounting the samples.
cally discussed technical challenges which can be Challenges arise due to the specific weight (float-
overcome by following certain protocols. The first ing of hydrocarbon droplets) and hydrophobicity
combination of a full FLBA with all commercially (sorption of hydrocarbon droplets).
available lectins combined with catalysed reporter An often overlooked interface in aquatic
deposition – fluorescence in situ hybridization habitats is represented by the water–air interface
(CARD-FISH) was applied to a >10 µm plankton (Norkrans, 1981). This extreme habitat, also
fraction collected at Helgoland surface waters called neuston, is characterized by an aquatic sur-
(Bennke et al., 2013). Focus was put on the face layer. The interface is enriched in biochemical
interaction of bacteroidetes with diatoms and the constituents forming a kind of gelatinous layer
role of glycoconjugates. It could be demonstrated (Wurl and Holmes, 2008; Cunliffe and Murrell,
that different bacteroidetes clades interacted with 2009). As a consequence, this nutrient-enriched
diatoms using different glycoconjugates suggest- layer attracts microorganisms (Franklin et al.,
ing their essential role in the formation of marine 2005). For studying sea surface microlayers, a
aggregates. range of different devices were employed in order
to collect material for analysis (Maki, 1993). The
formation and distribution of sea surface micro-
Laser microscopy of biofilms at layers was the subject of several reviews (Wurl et
liquid and gaseous interfaces al., 2011; Cunliffe et al., 2013). Somewhat similar
Interfaces in aquatic environments are not only are microbial surface layers forming in standing
represented by solid surfaces or associated with water samples (e.g. vases or puddles) or liquid
mobile aggregates. Biofilms may also be found microbial cultures (e.g. Bacillus). To the best of
at liquid–liquid interfaces such as water–oil our knowledge these layers were examined by
interfaces or at water–air interfaces. Hydrocarbon means of CLSM in only one survey (Krol et al.,
associated biofilms and their examination by 2011). The focus of this study was on plasmids
CLSM has already been reviewed and discussed transfer in E. coli biofilms. It turned out that,
in methodological detail elsewhere (e.g. Neu and compared to biofilms at the solid–liquid interface
Lawrence, 2010). Only a few groups have actually the plasmid transfer at the water–air interface was
used CLSM for looking at bacteria colonizing much higher. It was suggested that apart from
36  | Neu and Lawrence

population density, the oxygen concentration may against a wide range of feeding types (Weitere et
be the crucial factor for plasmid transfer. al., 2005). In experiments with Serratia the role
of quorum sensing in response to a suspension
feeder and a surface feeder was examined (Queck
Laser microscopy for studying et al., 2006). It was suggested that differentiation
biofilm grazing into cell chains and filamentous biofilms provides
In environmental habitats biofilms are exposed to protection against grazing. In a recent study with
grazing by protists and macroinvertebrates. This Vibrio cholerae WT and different mutants the
means there is a dynamic reciprocity between focus was on bacterial polysaccharide produc-
biofilm growth, biofilm detachment and slough- tion and quorum sensing (Sun et al., 2013). It
ing as well as biofilm grazing which eventually turned out that V. cholerae possesses various
will determine the overall biofilm architecture. defence mechanisms against grazing pressure. In
There are a number of reports on biofilm grazing a study with macroinvertebrates, CLSM was also
but only few examined biofilm structure by using employed as a tool to examine biofilm structure
CLSM. Initially, an attempt was made to visualize before and after grazing. For example the effect of
protists in pure culture as well as inside mature grazing was followed after exposing river biofilms
biofilms (Martin-Cereceda et al., 2001; Packroff to different benthic invertebrates (Lawrence et al.,
et al., 2002). It turned out that it is difficult to 2002). It could be demonstrated that grazing by
identify a staining procedure which separates the mayflies, ostracods and snails resulted in various
bacterial biofilm signal from the protist signal. biofilm patterns due to differential grazing strate-
As protists have different feeding modes it can gies of the individual species.
be expected that they affect the biofilm structure
in different ways. This was followed in a flow cell
study with stream biofilms (Böhme et al., 2009). Laser microscopy for exploring
As a function of the protists feeding strategy, graz- biofilm metabolism
ing led to rougher more porous biofilms and it It is also important, but difficult to determine,
may alter bacterial cell distribution, microcolony the metabolic status of microorganisms. CLSM
growth and settlement of drifting bacteria. Simi- however, offers a number of approaches based on
larly, the effect of a free-swimming filter feeder fluorescent reporters. In the meantime, many com-
and a surface-associated predator was investigated binations of fluorochromes have been suggested
on pure culture biofilms (Dopheide et al., 2011). for measuring the viability of microorganisms.
It was shown that protists have a preference for Relevant references were listed in a recent review
certain types of biofilms based on chemical cues. article (see tables 7 and 8 in Neu and Lawrence,
Furthermore, it turned out that the filter feeder 2014b). Live–dead staining, in particular, has
changed the biofilm structure more dramatically received a wide application and when used in a
resulting in holes, channels and isolated grazing- standardized protocol can provide a useful index
resistant microcolonies. In a series of reports of environmental impacts on biofilm communi-
on grazing by the group of Kjelleberg, several ties. Nevertheless, a number of issues exist with
different biofilms and protists were combined in in situ use of fluorescent reporters in ‘open’ and
order to detail their interactions. In a first study, complex environmental biofilms. In agreement
the effect of a surface feeding flagellate was tested with Caldwell and collaborators (Caldwell et al.,
against biofilms of various Pseudomonas mutants 1997) other authors have more recently found
(Matz et al., 2004). It was demonstrated that P. discrepancies between fluorescence staining and
aeruginosa formed microcolonies and produced culture results. These studies reinforce the dictum
toxins as a response to the grazer. A subsequent that all fluorescent staining should be subject to
survey used protists with different feeding strate- effective ‘ground truth’. Several reports which
gies. They could show that microcolony formation critically assessed the status of viability and the
is only useful in early stages of biofilm develop- application of fluorochrome combinations for
ment whereas inhibitor production was effective measuring viability are listed for further details
Laser Microscopy of River Biofilm Systems |  37

Table 2.2  Critical assessments of fluorochrome combinations for bacterial viability

Focus of review Reference
Vigour, vitality, viability Lloyd and Hayes (1995)
Viability assessment via fluorochromes Breeuwer and Abee (2000)
Molecular methods Keer and Birch (2003)
Application of the BacLight kit Stocks (2004)
BacLight kit in flow cytometry Berney et al. (2007)
Mechanisms of probes for viability Sträuber and Müller (2010)
Red but not dead? – Yeast study Davey and Hexley (2011)
Life, death and in between: … Davey (2011)
Confusion over live–dead staining Netuschil et al. (2014)

(Table 2.2). The timeliness of the topic ‘bacterial comparison of CLSM and OCT was reported by
viability’ culminated in an ongoing conference examination of biofilms developed in a flow lane
series with the title ‘How dead is dead?’ under laminar, transient and turbulent flow condi-
tions (Wagner et al., 2010). The biofilm showed
different porosities depending on flow conditions.
Large area imaging of biofilms It was also demonstrated that the mesoscale struc-
In order to image large biofilm areas there are ture was represented much better as compared to
two options if CLSM is employed. Firstly, a low- CLSM. Nevertheless, CLSM is needed in order
magnification lens with high numerical aperture to probe for different biochemical features of bio-
(NA) (e.g. 25x, NA 0.95) can be used. Secondly, films. The effect of biofilm grazing by eucaryotes
newer CLSM setups with a motorized microscope may be also investigated by OCT (Derlon et al.,
table have the option of running a so-called tile or 2012). This was shown on ultrafiltration mem-
mosaic scan. This allows recording of multiple branes driven by gravity and exposed to different
areas at high magnification and high resolution fluxes and grazing pressure. In a follow up manu-
which are finally stitched together into one large script, a more detailed study compared OCT with
dataset. Although an attractive option, issues CLSM, again in order to take advantage of both
arise in terms of time for recording data (several techniques (Derlon et al., 2013). Although OCT
hours), data size (several gigabytes) and flatness has certain advantages in terms of depth resolu-
of the substratum (imaging of black volumes if tion, similar to CLSM shading effects are an issue
substratum is tilted or structured in three dimen- with thick samples.
sions). The advantage of the CLSM however lies
in the multichannel mode which enables the col-
lection of multiple biofilm parameters. Another Biofilm matrix
option could be optical coherence tomography After many years of biofilm research, the bio-
(OCT) which originated in the medical field. film matrix still represents the ‘dark matter’ of
OCT is an interferometric technique and is based microbial biofilms. Although the extracellular
on reflection only. The advantages of OCT are its space and its constituents, often termed EPS, is
non-invasiveness, no need for adding contrast- accepted as an essential part of biofilm systems,
ing agents and a large scan area in the millimeter its characterization is still in its infancy. The
range. The first application of OCT for studying challenge is based on matrix complexity as well
biofilms was demonstrated in capillary flow cell as on the methods available for chemical and in
biofilms (Xi et al., 2006). Then the technique was situ analysis. Biochemically the EPS constituents
applied to follow biofilm development in a tube comprise polysaccharides, proteins, nucleic acids,
reactor in order to assess the effect of different amphiphilic/lipophilic compounds and bacterial
treatments (Haisch and Niessner, 2007). A direct refractory compounds. In terms of constituents
38  | Neu and Lawrence

they may be soluble (most), insoluble (cellulose, matrix. Clearly the ‘biofilm model’ remains a
mutan, amyloids) or structural (vesicles, nano- topic for debate and discussion.
tubes, pili). Consequently the biofilm matrix or,
in other words, the extracellular space defined
by the matrix constituents, initially meant to be Laser microscopy of the
only polysaccharides and/or proteins, becomes biofilm matrix
more and more differentiated. In this respect it This next section will elaborate on studies tar-
is important to look at the extracellular space geting the biofilm matrix. The studies will be
in general. It may comprise bacterial structures discussed according to their focus and approach
extruding from the cell surface into the extracellu- into: chemical analysis, in situ imaging, proteom-
lar space (tubes, pili), the components shed from ics and the functional role of EPS.
the cell surface (membrane vesicles, amyloids), To date the biofilm matrix has mainly been
the polymers actively released (polysaccharides, examined using two strategies, by traditional
proteins) but also low molecular metabolites pro- chemical techniques and by differential imaging.
duced and released by the bacteria. On top of that, Chemical analysis of the matrix is usually done via
compounds from the environment (e.g. humic extraction and subsequent biochemical analysis
compounds and colloids) will become associ- (Nielsen and Jahn, 1999). The critical issue is
ated and embedded into the matrix originally potential cell lyses and contamination of the EPS
produced by microorganisms. Thus, the question fraction by intracellular polymers. In any case, this
arises: what finally makes up the biofilm matrix? can be done easily in pure culture studies where
Maybe the acronym EPS standing for ‘extracel- usually only a few polymers are produced which
lular polymeric substances’ as a synonym for the can be purified and characterized. Whereas from
biofilm matrix has to be questioned and discussed environmental samples containing numerous
once again (Allison et al., 2003). polymer types (Staudt et al., 2003) only bulk
A further aspect to be discussed at this point chemical information in the form of per cent
is the common model with respect to biofilm polysaccharides, proteins, etc. becomes available.
structure and matrix which has been used in many References covering extraction with subsequent
publications. The so-called ‘mushroom model’ chemical analysis were compiled in a protocol
seems to be quite popular but in fact represents a on biofilm matrix assessment, see (Neu and Law-
flow-cell artefact. The model is of course correct rence, 2014a).
under laminar flow conditions in a small flow cell The second approach takes advantage of in situ
designed for microscopy of the developing pure imaging, e.g. by CLSM employing matrix-specific
or mixed culture biofilms (Crusz et al., 2012). probes. In comparison to electron microscopy
However, by looking at numerous biofilm samples where fixation and dehydration are necessary,
from the environment as well as technical habitats CLSM does not require these artefact prone
it was found that the structural variation is much procedures. Suitable fluorochromes and probes
larger and in most cases there are no mushroom for the biofilm matrix have been compiled in
structures. Similarly, the general drawing of bio- Table 2.3. Some of the fluorochromes, e.g. the cell
films as surface-associated bacteria surrounded impermeant ones may give false signals due to the
by a polymer matrix has to be questioned. Again staining of intracellular constituents in leaky cells.
this might be the case in many biofilms, however Other fluorochromes may give a double signal
from environmental and biofilm reactor samples from the biofilm matrix as well as from biofilm
often structural features with distinct filamen- cells as for example protein-specific dyes. Conse-
tous bacteria have been found. The filaments are quently differential imaging is required in order
often sticking vertically out of a basal biofilm to separate the individual signals. The most fre-
with no obvious polymer matrix surrounding quently used approach is now called fluorescence
them. Another observation is the coverage of lectin-binding analysis (FLBA). This comprises a
environmental biofilms by a network of very thin full scale screening with all the commercially avail-
filamentous bacteria also showing no polymer able lectins in order to identify a panel of useful
Laser Microscopy of River Biofilm Systems |  39

Table 2.3  Fluorochromes and probes suitable for biofilm matrix constituents
Fluorochrome/probe Target Reference
Fluorescently labelled lectins Glycoconjugates Neu and Lawrence (1999b), Neu and Lawrence
(general precedure) (1999a), Neu et al. (2001)
Lectin screening (using about 70 Selecting a panel of Staudt et al. (2003), Laue et al. (2006), Peltola et al.
lectins commercially available) useful lectins for a (2008), Luef et al. (2009b), Zippel and Neu (2011),
particular sample Bennke et al. (2013), Weissbrodt et al. (2013), Zhang
et al. (2014)
ELF 97 phosphate Phosphatase activity Paragas et al. (2002), Nedoma et al. (2003)
DDAO eDNA Allesen-Holm et al. (2006)
Cell impermeant dyes (TOTO-, eDNA Li et al. (1995), Roth et al. (1997), Lebaron et al.
TO-PRO-, SYTOX-series) (1998)
SYPRO series (e.g. SYPRO- Protein in general Zubkov et al. (1999)
FM lipophilic dyes (FM 1-43, Extracellular vesicles Sharp and Pogliano (1999)
FM 4-64) (potentially useful)

probes matching a particular biofilm system or matrix proteins which were found to be differ-
a specific structural cue. Screening with about ent and partly unknown ( Jiao et al., 2011). A
70 different lectins has been applied already to a combination of genomics (with focus on alginate
number of different biofilm systems such as reactor genes) and proteomics (with focus on amyloids)
biofilms inoculated with river water or activated was employed for the analysis of activated sludge
sludge (Staudt et al., 2003), Pseudomonas biofilms samples (Albertsen et al., 2013). Interestingly
(Laue et al., 2006), Deinococcus biofilms (Peltola most alginate related genes could be allocated to
et al., 2008), river aggregates (Luef et al., 2009b), bacteroidetes and not to Pseudomonas.
phototrophic biofilms (Zippel and Neu, 2011), EPS functionality represents a topic were two
marine aggregates (Bennke et al., 2013), aerobic clusters of publications can be discussed. One of
granules (Weissbrodt et al., 2013) and acidophilic them is represented by a series of review articles
archaeal biofilms (Zhang et al., 2014). Although compiling facts (polymer identities and proper-
potentially laborious, this approach gives rise to a ties) and references (of other reviews) finally
targeted panel of lectins with maximal effective- making suggestions of what could be a function.
ness for each system. In a first comprehensive book the many aspects of
More recently biofilms have been subjected to EPS identity, analysis and function were compiled
the -omic approaches including proteomics of the (Wingender et al., 1999). Several of these chapters
biofilm matrix. In a very limited number of papers together with other review articles were listed,
the extracellular proteins of pure culture biofilms assessed and critically discussed in a review on
were characterized with respect to their function. matrix functionality (Neu and Lawrence, 2009).
Examples are available for Haemophilus (Gallaher As a result and by focusing on the nature and
et al., 2006), Myxococcus (Curtis et al., 2007), role of the biofilm matrix a still evolving concept
Listeria (Dumas et al., 2008) and Shewanella (Cao of matrix functionality was suggested. The many
et al., 2011). In most of the studies proteins with functions maybe compiled as a list or allocated as
new or unknown functions were found. Even less a matrix showing multi-functionality (Table 2.4).
is known for complex environmental biofilms. In any case, the concept of matrix functionality
The few reports are from dental plaque where may help to streamline and focus the discussion
the effect of sucrose on matrix protein composi- as well as future research in this direction. The
tion was investigated (Paes Leme et al., 2008). other cluster is represented by pure culture studies
Another survey focused on biofilms in acid mine focusing mainly on a single polymer and its func-
drainage and compared cellular proteins with tion. Major studies were done with Escherichia,
40  | Neu and Lawrence

Table 2.4  Biofilm matrix characteristics and functionality arranged to indicate multi-functionality of matrix
constituents (Neu and Lawrence, 2009)
constructive sorptive adhesive anti-adhesive
polysaccharides polysaccharides polysaccharides amphiphiles
proteins proteins polysaccharides
cohesive active surface-active informative
proteins (amyloids) proteins amphiphiles nucleic acids
vesicles vesicles vesicles
proteins proteins (lectins)
nutritive locomotive conductive redox-active
polysaccharides amphiphiles proteins bacterial refractory
proteins polysaccharides nucleic acids compounds
nucleic acids proteins

Pseudomonas, Bacillus, Streptococcus, among data sets, online deconvolution on the fly, quanti-
others. However, the many articles published are fication and extraction of structural information.
beyond the scope of this chapter. Another area of progress will be in the field of
chemical imaging. The various tools (e.g. Raman
microscopy, nano-SIMS, synchrotron imaging)
Research needs have already been used in a limited number of stud-
CLSM is now an established technique for in situ ies (Neu et al., 2010). However, what is needed
assessment of microbial biofilm systems from should comprise traditional light/laser micros-
many different habitats. Nevertheless, in order copy and electron microscopy/tomography plus
to progress, new probes are needed especially for chemical imaging. This approach is already known
the complex constituents of the biofilm matrix. and partly established as correlative microscopy.
In terms of resolution, new techniques may help Finally, in the age of –omics, proteomics and
to elucidate new features of interfacial microbes. transcriptomics are required for addressing iden-
This includes, firstly, imaging at deep locations tity and function of matrix constituents. A totally
using multi-photon imaging (Neu and Lawrence, neglected -omics area is glycomics which still has
2005; Neu et al., 2004; Horton et al., 2013) or by to be applied in studies of microbial biofilms and
employing selective plane illumination micros- especially the biofilm matrix.
copy (SPIM) (Huisken and Stainier, 2009).
Secondly, the various nanoscopy techniques Acknowledgements
having a resolution across the diffraction barrier The expert support by Ute Kuhlicke and George
including structured illumination microscopy D.W. Swerhone over many years of laser micros-
– SIM (Schermelleh et al., 2008), stimulated copy on many types of biofilm systems is highly
emission depletion (STED) microscopy (Klar appreciated. We are very thankful to Norbert
et al., 2000) and blink microscopy – dSTORM/ Kamjunke for collecting rock samples in the Harz
GSDIM (van de Linde et al., 2008; Fölling et al., mountain creeks (Figs. 2.1 and 2.2).
2008) will allow further progress. These demand-
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Interactions and Communication within
Marine Biofilms
Priyanka Sathe and Sergey Dobretsov

Abstract Introduction
Biofilms are the prime mode of life for many Microbial communities in marine environment
microbial species and they are omnipresent in are abundant, diverse and complex. Metabolism
aquatic environment. Biofilms’ three-dimensional of aquatic ecosystem is mediated by microbial
architecture provides the concomitant microbial communities forming aquatic biofilms. Aquatic
populations with additional protection from microbial communities consist of two primary
predation and toxic substances. Marine biofilms forms: (1) planktonic (free-floating) and (2) ben-
(often termed as micro-fouling) are composed thic (attached to the substratum) organisms. The
of different species of bacteria, diatoms and pro- equilibrium between these two states depends on
tozoa surrounded by a matrix of extra polymeric environmental factors such as movement of water
substances (EPS). Formation of marine biofilms and substrate availability. Freshwater biofilms are
depends on the species present, their microbial composed of nutritionally diverse microorgan-
activity and environmental conditions. Marine isms, and many environmental factors, like light
biofilms attract or repel larvae of invertebrates and temperature, can affect the biofilm structure
and spores of macro-algae resulting in formation and community composition (Flemming et al.,
of macro-fouling communities ultimately lead- 2001). Freshwater biofilms are very well explained
ing to biofouling which is ever increasing threat in literature, unlike marine biofilms (Costerton et
for maritime industry. Function of any biofilm al., 1987; Characklis et al., 1990; Salta et al., 2013).
is dependent on symbiotic interactions between One litre of seawater in the surface of the
microbial communities. These interactions ocean will typically contain 109 bacterial cells
include competition, cooperation, and neutralism. and the total number of bacteria species, although
The best studied chemical interaction between currently unknown, has been estimated to be as
microbes is a cell-to-cell signalling mechanism many as 1 million (Curtis et al., 2002). In aqueous
between bacteria which is called quorum sensing systems, microbial cells are found as both ‘plank-
(QS). This process is based on production and tonic’ (floating) cells and ‘sessile’ (attached) cells
perception of simple chemical signals (inducers), on surfaces. In marine environment the majority
which at concentrations higher than thresholds of microorganisms are organized in aggregates or
ones triggered differential expression of target biofilms. In biofilms microbes are enclosed in the
genes and cascades of chemical reactions. In order matrix of self-produced extra polymeric substances
to prevent growth of competitors, some micro- (EPS) (Flemming et al., 2001). This extracel-
organism evolved ability to interfere with QS of lular matrix plays a major role in holding cells
bacteria. This chapter reviews current understand- together within the biofilm, while simultaneously
ing of the role of marine microbial communities, protecting them from external insults. Biofilms
interactions between different microorganisms developed on artificial and natural substrata in
within marine biofilms and novel anti-biofilm seawater contain different types of microorgan-
strategies. isms, e.g. bacteria, archaea, protozoa, fungi and
48  | Sathe and Dobretsov

algae (Dobretsov et al., 2010). In a biofilm, each This microbial biofilms attract or repel larvae of
group of microorganisms performs specialized invertebrates and spores of macro-algae result-
metabolic functions. Biofilms have three dimen- ing in formation of macro-fouling communities
sional structures (Costerton et al., 1999; see also (Fig.  3.1) (Yebra et al., 2004; Chambers et al.,
Chapter 2). Microbes form a biofilm in response 2006; Magin et al., 2010; Rosenhahn et al., 2010).
to many factors, which may include cellular recog- Over the last century, marine biofilm research
nition of specific or non-specific attachment sites has been primarily focused on its adverse role
on the surface, nutritional cues, or in some cases, in industrial biofouling. Biofouling is the term
by exposure to inhibitory concentrations of anti- used to describe the unwanted accumulation
biotics. This kind of survival strategy facilitates of microscopic (micro-fouling), large multicel-
microorganisms to respond and adapt to physi- lular (macro-fouling) organisms and deposits
ologically limited and versatile environment and of their waste products (chemical fouling) on a
protect them from harmful environmental condi- submerged surface (Wahl et al., 1989). The effects
tions ( Jefferson et al., 2004). When a cell switches of biofouling can be felt across numerous marine
to the biofilm mode of growth, it undergoes a phe- industries and is a particular costly problem for
notypic shift in behaviour, in which large groups desalination plants, nuclear plants, ships and
of genes are differentially regulated. Biofilms are other marine installations, and equipment work-
essentially multicellular organization of microbial ing in sea water.
communities, which involves physiological inter- Not only macro-fouling but also biofilms can
action, communication and signalling, mutual cause severe industrial problems by increasing
and antagonistic strategies similar to the higher drag force, promoting corrosion and reducing
forms of life (O’Toole et al., 2000). heat transfer efficiency (Yebra et al., 2004, 2006;
In aquatic milieu any natural and man-made Corteson et al., 1987). A biofilm 1 mm thick on
substrates are quickly colonized with different a ship hull leads to the penalty of hundreds of
species of micro- and macro-organisms (Railkin, dollars due to higher fuel consumption (Schultz
2004). Bacteria are considered to be the primary et al., 2011). Biofilms induce corrosion of metals
colonizers of substrata, forming ubiquitous bio- by a number of mechanisms including cathodic
films or bacterial aggregates attached to abiotic and anodic depolarization, hydrogen production,
and living surfaces within several hours (Zobell metal reduction, and production of corrosive
et al., 1943). Bacterial biofilms have a complex metabolites, such as organic acids and exopoly-
structure and are composed of multiple bacterial mers (Videla and Herrera, 2004; Landousli et al.,
species encased in EPS (Flemming et al., 2001). 2011).

Figure 3.1  Stages in the formation of a biofilm from planktonic form of cells to community mode, these
stages ultimately lead to biofouling.
Microbial Interactions Shaping Marine Biofilms |  49

Traditional way to control biofouling is based substratum surface free energy, substratum prop-
on using toxic biocides that kill fouling organ- erties and water turbulence (Zobell, 1943; Dexter
isms (Yebra et al., 2004). These biocides are et al., 1975; Characklis et al., 1990; Costeron et
highly toxic and kill other marine organisms as al., 1995). Most microorganisms attach strongly
well. Antifouling biocides can accumulate in to hydrophobic materials, such as Teflon™, than
sediments, fish and marine mammals (Thomas to hydrophilic materials, such as glass (Fletcher
and Brooks, 2010). Many publications have been et al., 1979; Railkin, 2004). Adhesion of bacte-
able to demonstrate that biocidal and fouling ria and microalgae to surfaces of living (tissue
release coatings are generally free from macro- of plants and animals) and non-living (metals,
fouling but not from microbial biofilms (Cassé plastics, and organic particles) materials are medi-
and Swain, 2006; Molino et al., 2009; Dobretsov ated by secretion of a slimy, glue-like substance
and Thomason, 2011; Zargiel et al., 2011; Briand of extracellular polymers (EPS), which contain
et al., 2012). Thus, eradication of biofilms and polysaccharides, lipopolysaccharides, proteins,
inhibition of their growth are major industrial nucleic and huminic acids (Chapter 2). Adhesion
concerns. of microorganisms is not always a passive process;
Despite the recent and significant increase microbes may respond to chemical substances
in the study of aquatic microbial communities, and swim away or towards to the substratum
little is known about the microbial interactions (Marshall et al., 1971; Vandevivere et al., 1993).
within complex ecosystems such as marine water. Additionally, attached microorganisms can
Current knowledge of these interactions within stimulate or suppress the co-adhesion of other
microbial communities in marine environment planktonic microorganisms causing formation
is scarce whereas these communities are hotspots of multispecies heterogeneous biofilms (McEl-
of exchanges between bacteria and macro-organ- downey et al., 1986).
isms. A number of investigations have shown that
The aims of this chapter are to discuss (1) various physical factors (for example, flow rate,
existing knowledge of formation and composi- hydrodynamic forces, substrate properties, and
tion of marine biofilms; (2) Shed light on various viscosity), chemical factors (for example, nutrient
microbial interactions responsible for mainte- availability and EPS production), and biological
nance of a biofilm in marine environments; (3) factors (for example, competition and predation)
Discuss novel anti-biofilm strategies that are affect biofilm architecture both separate and in
being investigated; (4) Highlight future directions combination (Qian et al., 2007). Colonization of
for advancing our understanding of dynamics of the surface of substratum by bacteria is affected
marine biofilms. by different temperatures, motility behaviour
(Kirboe and Jackson, 2001), surface chemistry
and energy (Ista et al., 2004). Microbial biofilms
Adhesion and communication in marine environment are very heterogenic and
during biofilm formation dynamic structures and these features make them
In order to generate a biofilm, cells switch from a difficult to model and investigate (Dobretsov,
planktonic to a sessile state by down-regulating the 2010). With improvement in imaging technol-
expression of flagellar genes concomitantly with ogy and the development of electron microscopy,
an up-regulation of genes involved in production scanning confocal laser microscopy (SCLM),
of the extracellular matrix (Fig. 3.1). During ini- real time image capture, atomic force microscopy
tial stages of marine biofilm production, proteins, (AFM), nuclear magnetic resonance imaging
glycoproteins, proteoglycans and polysaccharides (NMRI), various spectroscopic imaging tech-
present in the water adsorb to the substratum niques, like Raman imaging and fluorescent in situ
(Maki et al., 2002). A molecular fouling can either hybridization (FISH) methods, our understand-
inhibit microbial attachment or facilitate it and ing of biofilm’s structure and functions have been
furnish microorganisms with a carbon source for improved significantly (see Chapter 2 for more
their growth. Microbial adhesion depends on the details).
50  | Sathe and Dobretsov

Composition of marine biofilms in 1 year old biofilms developed on copper-based

Competition for space and nutrition among antifouling coatings (Muthukrishnan et al., 2014).
microorganisms determines the microbial com- Additionally, diatoms belonging to the genes
position of biofilms. Usually, marine biofilms Nitzschia, Navicula, Amphora and Licmophora
consist of different species of bacteria, Archaea and were reported in biofilms on antifouling coatings
unicellular organisms, such as diatoms and flagel- (Callow, 1986; Cassé and Swain, 2006; Briand et
lates (Dobretsov, 2010). The densities of fungi al., 2012).
and protista are generally low in marine biofilms In most of experiments, investigators have
(Railkin, 2004; Salta et al., 2013). Marine biofilms been studying mono-species microbial films in
are dominated by bacteria, which are the primary static conditions without the addition of nutri-
colonizer of any clean substratum (Armstrong et ents (Maki, 2002; Dobretsov et al., 2006). These
al., 2001). Viruses are possibly very abundant in studies give only limited information about the
marine biofilms but their presence has not been structure, function and the role of biofilms in
confirmed (Dobretsov, 2010). The second largest the field, as natural biofilms are formed under
group of microorganisms in marine biofilms is turbulent flow conditions and composed of
diatoms or Bacillariophyta. Diatoms belonging numerous species of microorganisms (Characklis
to genes Achnanthes, Amphora, Navicula and Lic- et al., 1990; Webster et al., 2004). Therefore, more
mophora dominate in marine biofilms (Cookesy et investigations should be done in order to investi-
al., 1995; Thornton et al., 2002). Protists (mainly gate marine biofilms in the field conditions.
flagellates) can be found in marine biofilms but in
low densities (Underwood et al., 2005; Maybruck
et al., 2004). Fungi, mainly Cladosporium species, Factors affecting biofilm
have been isolated from marine biofilms, while composition
their mycelia have been rarely found (Maybruck The species composition of early marine biofilms
et al., 2004). Marine fungi, protists and viruses in depends mainly on two factors: (1) presence of
marine biofilms are rarely studied, while, prob- colonizers; and (2) the physical and chemical
ably, they play an important role as decomposers, conditions of the substratum and environment.
grazers and parasites (Dobretsov, 2010). Marine biofilms are subjected to constant dynamic
Marine biofilms can develop even on the surface changes. Any modifications in abiotic conditions
of antifouling coatings that contain toxic biocides (e.g. climate, depth, light regime, season, water
(Callow, 1986; Dobretsov and Thomason, 2011; chemistry, nutrient supply and substratum charac-
Chen et al., 2013). However, there is only limited teristics) and biotic surroundings (e.g. availability
information about biofilms’ composition and and physiology of colonizing species, competi-
functions on antifouling coatings (Yebra et al., tion, predation, grazing and cooperation among
2004). Recent studies with culture independent species) change the composition of biofilms,
molecular-based techniques suggested that the density, productivity, architecture, succession
type of biocide and environmental conditions rate, and the production of chemical compounds
affected the composition of microbial biofilms by microorganisms (Salta et al., 2013). For exam-
(Briand et al., 2012; Chen et al., 2013). Bacteria ple, some microorganisms are found a whole year
belonging to the class Alpha-proteobacteria, around, while others are seen only during particu-
Gamma-proteobacteria, firmicutes and bacteroi- lar seasons (Moss et al., 2006; Forster et al., 2006)
detes have been found on the hulls of ships coated and even hours (Underwood et al., 2005). The
with cuprous oxide based coatings (McNamara et diatoms Gyrosigma balticum and Pleurosigma angu-
al., 2009). The presence of Delta-proteobacteria latum were found at the surface of Navicula and
was reported on copper-based biocidal coatings Nitzschia dominated biofilms only during midday
(Chen et al., 2013). The metagenomic study of (Underwood et al., 2005). This phenomenon can
marine biofilms developed on antifouling coat- be explained by rapid (within minutes) migra-
ings demonstrated high abundance of bacteria tions of diatoms that have tendency to adjust their
Acaryochloris marina and Maritimimonas rapanae position in a biofilm according to light intensity.
Microbial Interactions Shaping Marine Biofilms |  51

Additionally, biofilm sloughing and detachment siderophores (Davies et al., 1998; Dong et al.,
can open space for re-colonization by newcomers 2001; Dong and Zhang, 2005).
and existing microorganisms (Underwood et al.,
As an example, different biofilms have been Quorum sensing
developed on chemically inert black and white Quorum sensing (QS) is a form of bacterial
substrata exposed to fouling in tropical waters population density dependent cell-to-cell com-
(Dobretsov et al., 2013). Pyrosequencing via 454 munication and gene regulation that has been
of 16S rRNA genes of bacteria from white and shown to contribute to the formation and matu-
black substrata showed that while Alpha-proteo- ration of biofilms. QS allows them to coordinate
bacteria and firmicutes dominated on both type of their adhesion, biofilm maturation, swarming,
substrata, their quantities were different. In other luminescence and chemical compound produc-
words, biofilms reflected the key environmental tion (Waters and Bassler, 2005; Steinberg et al.,
factors of a substratum. This relationship has an 2011). Overall, QS helps to control the switch
important implication for the larval settlement of from the behaviours of single cell organisms
marine invertebrates (Hadfield et al., 2001; Salta to those appropriate to cells within a biofilm
et al., 2013). (Sauer et al., 2002). During this process, a class
of small molecules named ‘autoinducers’ is pro-
duced and released into the environment (Fig.
Communication strategies 3.2). When the concentration of these signals
within biofilms reaches a threshold level, these signals trigger
Marine biofilms are usually influenced by posi- an expression of target genes that consequently
tive, neutral or negative interactions within its change the behaviour of bacteria (Sauer et al.,
counterparts. Chemical substances secreted by 2002; Steinberg et al., 2011). Different species
one species of microorganisms greatly influence of Gram-positive and Gram-negative bacteria
the colonization of other species (Holmstro¨m use a different but potentially overlapping QS
et al., 2002). In biofilms, microbes grow in close signalling systems. There are three documented
proximity and compete or cooperate for space and QS systems: the N-acyl homoserine lactone
nutrients (Nadell et al., 2009). These interactions (AHL)-based signalling system of Gram-negative
between different microbial species are based on bacteria, an oligopeptide-based system in Gram-
the sophisticated cell-to-cell chemical communi- positive bacteria and a furanones-based system
cation mechanisms, such as QS, or the secretion that is shared between both groups (Roux et al.,
of secondary metabolites, like antimicrobials or 2009; Yin et al., 2012).

Figure 3.2  Generalized model of AHL-mediated quorum sensing in Gram-negative marine bacteria.
52  | Sathe and Dobretsov

Mechanism of AHL-based QS
signaling system
The best characterized QS signalling system is the
AHL-based one (Dobretsov et al., 2009). It has
gained a significant interest due to its frequent role
in microbial virulence mechanisms and wide dis- Figure 3.3  Generalized structure of acyl
tribution between marine environment (Huang et homoserine lactones (AHL) produced by bacteria.
All of them have common lactone ring with different
al., 2009; Taylor et al., 2004). This QS system is size (C4–C18) fatty acid acyl size chain R1. R2 = H,
able to regulate different physiological activities O or OH.
of Gram-negative bacteria, which include biolu-
minescence, exopolysaccharides, antibiotic and
enzyme production, virulence, conjugal plasmid marine snow particles (Gram, 2002), and sponges
transfer, biofilm formation, and growth (Irie and (Taylor et al., 2004; Mohamed et al., 2008) are
Parsek, 2008). producing different AHL signals. AHL producing
In AHL-based QS system, AHL signals, pro- bacteria isolated from sponges belonged to Roseo-
duced by ‘LuxI’ autoinducer synthase (Fig. 3.2), bacter sp., Marinobacter sp. and Vibrio sp. There
exit the cells after their synthesis (either by passive are few publications that investigated production
diffusion or active transport) and accumulate in of QS signals in biofilms and microbial mats.
the environment (Bassler et al., 2002). Different Subtidal biofilms produce different size chain
species of bacteria can produce different AHLs, AHLs during formation on artificial substrata
which differ from each other by the presence (Huang et al., 2007). AHL producing culturable
of functional groups, like OH, and the length of isolates from biofilms belonged either to Alpha-
acyl-side chain (Fig. 3.3). When the extracellular proteobacteria (all Rhodobacteriaceae isolates) or
concentration of AHLs reaches a certain thresh- to Gamma-proteobacteria (genera Pseudoaltero-
old, the cognate signals bind to the cytoplasmic monas, Vibrio and Thalassomonas) (Huang et al.,
LuxR-type receptor, and form active dimmers 2008). Production and degradation of AHLs by
(Williams et al., 2007). This complex binds to marine microbial mats (stromatolites) composed
specific regions (known as lux boxes) within mostly of cyanobacteria and sulfate-reducing bac-
promoter sequences and activates transcription teria was examined (Decho, 2009). Under natural
of QS genes that change bacterial behaviour (Fig. conditions microbial mats produced a wide range
3.2). In the absence of AHLs, LuxR proteins are of AHLs (C4 to C14 acyl side chain; Fig. 3.3)
degraded quickly; conversely, binding of LuxR and most of QS signals accumulated during the
to the cognate AHL signal stabilizes the protein night-time. In vivo production of QS signals and
(Cho, 2009). Some bacteria and marine organ- inhibitors in hot spring and desert crust micro-
isms, such as algae, are able to respond to the QS bial mats has been reported (Abed et al., 2011;
signals of other bacteria or to compromise those Dobretsov et al., 2011). These studies suggest
(Dobretsov et al., 2009). that QS signals are present in biofilms developed
on artificial and natural substrata and we lack of
QS in marine biofilms knowledge about production and turnover of
Information concerning the presence of AHLs and AHLs in the environment.
other QS molecules in the marine environment is
scarce (Dobretsov et al., 2007b). First evidence on Interactions within biofilms and with
the presence of QS signals in marine environment new colonizers
came from the study of the symbiotic bacterium In microbial biofilms different microor-
Vibrio fischeri, which colonizes the light organ of ganisms interact with each other and new
sepiolid squid Euprymna scolopes (Eberhard et prokaryotic and eukaryotic colonizers. These
al., 1981). Production of light by this bacterium intra- and inter-specific interactions between
is regulated by AHL signals (Fig. 3.3). Several biofilm inhabiting microorganisms include help-
studies have showed that bacteria associated with ful associations (i.e. mutualism), associations
Microbial Interactions Shaping Marine Biofilms |  53

in which one microorganism benefits but other antagonistically or synergistically with each other,
does not (commensalism), interactions benefi- the latter resulting in co-colonization of distinct
cial for one species and harmful for another one groups of bacteria having metabolic coopera-
(i.e. predation, grazing, parasitism, diseases) and tion (O’Toole, 2000; Armstrong et al., 2001;
various competitive interactions in which both Nadell et al., 2009). Microbial processes such as
species are adversely affected (Dobretsov, 2010). nitrification, anaerobic degradation of organic
In addition, chemical compounds produced by compounds, or bioremediation of xenobiotic
microorganisms can inhibit or induce settlement compounds, have been shown to require interac-
of invertebrate larvae and spores of macroalgae tions between different bacterial species within
(Dobretsov et al., 2006; Qian et al., 2007; Salta et the biofilm (Paerl and Pinckney, 1996). This
al., 2013; Marshall et al., 2006). These interactions metabolic cooperation is advantageous to the
between the biofilm and higher plants and animals microbial community. Nevertheless, cooperation
are of interest at several levels. Microbial biofilms among species is only expected under restricted
provide food and vitamins for grazing macro- conditions (Nadell et al., 2009).
organisms, while both seaweeds and invertebrates Under natural conditions, bacteria intra- or
supply organic nutrients to the heterotrophic inter-specifically compete with their neighbours
bacteria in the biofilm (Thompson et al., 2004). for space and resources. A surface (especially of
Micro-grazers, like heterotrophic flagellates, hosts) may itself also be a trophic source where
amoebas and ciliates, are among the major regula- attached microorganisms catabolize organic
tors of bacterial biomass and diversity. In marine or inorganic nutrients directly (Madigan and
snow biofilms the density of bacteria and their Martinko, 2006; Grossart, 2010). Therefore, the
behaviour was largely controlled by the grazing presence of other microorganisms on a surface
pressure from flagellates (Kiørboe et al., 2003). reduces the availability of food and space for
Presence of heterotrophic flagellates changed colonizing species (Salta et al., 2013). Under such
biofilm architecture and shifted the composition competitive selection it is not surprising that bac-
of bacterial communities towards less diverse with teria have developed special mechanisms in order
dominance of grazing resistant strains. to interfere with the capability of other antagonis-
Some of prokaryote–eukaryote interactions tic bacteria (Falagas et al., 2008). The mechanisms
have been widely investigated, others are remained of bacterial antagonism include depletion of some
undiscovered. One of interesting examples of this essential substances (e.g. substrate or a vitamin),
inter-kingdom communication is the green alga alteration in the microenvironment (e.g. changes
Ulva (Enteromorpha) sp. Zoospores of this alga in the gas concentration or pH), the presenta-
use bacterial QS signals in order to detect a suita- tion of an obstacle or barrier, and production of
ble surface for attachment ( Joint et al., 2007). The antagonistic substances (Reid et al., 2001).
agglutination and attachment of diatoms Navicula Clearing a space to colonize by eliminating
sp. enhanced in the presence of spent culture of prior residents can be accomplished by produc-
the bacterium Pseudoalteromonas sp. (Cooksey tion of antimicrobials, toxins, and molecules that
and Cooksey, 1995). Similarly, the bacterium facilitate the competitors’ dispersal without actu-
Pseudoalteromonas tunicata requires the presence ally killing those (Hibbing et al., 2010). It has been
of diverse species of bacteria in the inoculum for shown that bacterial species that belong to the
its effective colonization of the surface of the alga Streptomyces, Alteromonas, Pseudoalteromonas and
Ulva australis (Rao et al., 2007). Roseobacter genus produce a range of antibiotics
The complex architecture of a mature biofilm (Wiese et al., 2009). In the mixed species biofilms,
provides niches with distinct physico-chemical the antibiotic produced by the bacterium P. tuni-
conditions, differing, e.g. in oxygen availability, cata remove the competing bacterial strains unless
in concentration of diffusible substrates and its competitor is relatively insensitive to antibacte-
metabolic side products, in pH, and in the cell rial protein or produces strong inhibitory activity
density (Costerton et al., 1999). In such a mixed against the bacterium P. tunicate ( James et al.,
microbial community the strains may interact 1996). Antagonistic antimicrobial activity widely
54  | Sathe and Dobretsov

exists between common fouling bacteria and fungi (Teplitski et al., 2011). Two classes of bacterial
in marine biofilms (Qian et al., 2005). About 70% AHL degrading enzymes have been identified,
of tested fungal isolates (mostly Cladosporium) which are lactonases and acylases (Rajamani et
inhibited growth of marine bacterial isolates from al., 2011). For example, Bacillus spp. release an
biofilms and, in opposite, 17% of tested bacterial AHL-lactonase that cleaves the lactone ring of
strains (mostly Staphylococcus) inhibited growth AHL molecules and probably interferes with
of fungi in laboratory experiments. This suggests the AHL signalling of other species in the same
that the competitive interactions between differ- niche (Dong et al., 2002; Rajamani et al., 2011).
ent components of biofilms is quite common and Being Gram-positive bacteria, Bacillus species
cannot be neglected in future investigations. does not produce AHLs and communicate with
each other using peptide signals (Oh et al., 2012).
AHL-acylases cleave the acyl side chain from the
Anti-biofilm strategies lactone ring of AHL molecule (Dong et al., 2002).
Removal of detrimental biofilms from surfaces Recently, AHL-acylase, named ‘autoinducer
exposed in the marine environment remains a inhibitor from cyanobacteria’ (AiiC), was isolated
challenge. Biofilms can be controlled by mechani- from the marine cyanobacterium Anabaena sp.
cal, chemical and thermal treatments (Barraud PCC7120 (Romero et al., 2008).
et al., 2009). Conventional antifouling biocides Not only prokaryotes but also eukaryotes
have limited effectiveness against biofilm bacteria are able to control bacterial QS (Dobretsov
in industrial settings (Flemming and Ridgway, et al., 2009; Goecke et al., 2010). The red alga
2008). Along with the killing of bacteria, their Delisea pulchra produces halogenated furanones
removal from the surface is desirable (Pitts et al., structurally similar to AHLs that interfere with
2003). Controlling of bacterial biofilms is easier bacterial AHL-mediated QS signalling (Rasmus-
at the primary stages, when biofilm formation is sen et al., 2000). Furanones from D. pulchra and
initiated. their synthetic analogues are potent inhibitors
Antimicrobials, such as antibiotics and of QS-regulated behaviours in Gram-negative
halogen-based treatments, have been tradition- bacteria, including expression of virulence genes,
ally designed to inhibit bacterial growth in water. production of antibiotics, bacterial motility,
These treatments are not effective against bio- swarming, and biofilm formation (Steinberg and
films, as these methods require unacceptably high De Nys, 2002; Defoirdt et al., 2004). A unicellular
cost and energy inputs or lead to fatal outcomes chlorophyte, Chlamydomonas reinhardtii, secretes
in order to complete killing of microorganisms in a number of compounds that mimic the activity
the biofilms (Pitts et al., 2003). To address there is of AHLs and thus affect QS in bacteria (Teplitski
a need for novel and improved anti-biofilm treat- et al., 2004).
ments. Several novel antimicrobial treatments Many marine organisms are able to produce QS
against marine biofilms have been developed that inhibitors but in many cases the mode of action of
are the subject of this section. QS inhibitors cannot be determined (Dobretsov
et al., 2009; Goecke et al., 2010). The sponge Luf-
QS inhibition fariella variabilis produces manoalide, manoalide
Given that bacteria have evolved the ability to monoacetate and secomanoalide that inhibited
communicate via QS, it is reasonable that marine QS of LuxR-based reporters (Skindersoe et al.,
organisms have also evolved the ability to prevent 2008). Demethoxy encecalin and hymenialdisin
bacterial biofilms using QS inhibitors. There from sponges inhibited QS of the LasR-based
are examples of interference in which microbes and the LuxR-based reporters (Dobretsov et al.,
release enzymes that degrade autoinducers mol- 2011). The cyanobacterium Lyngbya majuscula
ecules (Dobretsov et al., 2009). AHL-degrading produced malyngamide C, 8-epimalyngamide C
microorganisms are taxonomically diverse and and malyngolide that inhibited QS of LasR-based
include true fungi (Ascomycetes and Basidiomy- reporters (Kwan et al., 2010; Dobretsov et al.,
cetes), Gram-positive and Gram-negative bacteria 2010)
Microbial Interactions Shaping Marine Biofilms |  55

Disruption of QS is considered to be an (NO2–), a process that involves NO (Schlag et al.,

important way to prevent microbial infections 2007). NO has been suggested to be an ancient
(Williams et al., 2007; Parsek et al., 2000), as well and highly conserved regulator of dispersal across
as to prevent biofouling (Dobretsov et al., 2009). eukaryotic organisms (Bishop and Brandhorst,
QS inhibitor – kojic acid – incorporated in a 2003). For example, dissolution and dispersal
non-toxic matrix was able to prevent biofouling of aggregated mycelial cells of fungi, Neurospora
in tropical waters (Dobretsov et al., 2011). Bac- crassa (Ninnemann and Maier, 1996) and Can-
teria producing AHL-degrading enzymes have dida tropicalis (Wilken and Huchzermeyer, 1999),
been identified as promising agents in reducing and the amoeba Dictyostelium discoideum (Tao et
biofouling (Oh et al., 2012). The introduction of al., 1997) were shown to rely on NO signalling.
micro-encapsulated transgenic E. coli that pro- Taken together, these observations strongly sug-
duces AHL-degrading enzymes strongly reduced gest that NO can helps in biofilm dispersal and
biofouling of membranes in wastewater treatment combined NO and antibiotic treatment can offer
facilities over an extended period of time (Oh et a novel strategy to control marine biofilms.
al., 2012).
Reactive oxygen species mediated
Nitric oxide mediated biofilm biofilm disruption
dispersal Photodynamic therapy (PDT), also known as
Many bacterial species, such as Shewanella woodyi photo radiation therapy or phototherapy, involves
(Liu et al., 2012), Shewanella oneidensis (Plate et the use of a photoactive dye (photo sensitizer)
al., 2012), and Legionella pneumophila (Carlson that is activated by exposure to light of a specific
et al., 2010) use nitric oxide (NO) in order to wavelength in the presence of oxygen (Hashimoto
regulate biofilm formation through a pathway et al., 2005). The transfer of energy from the acti-
involving cyclic-di-GMP metabolism. In these vated photo sensitizer to available oxygen results
bacteria, NO is sensed by H-NOX and ultimately in the formation of toxic reactive oxygen species
regulated the activities of a diguanylate cyclase (ROS), such as singlet oxygen and free radicals
and/or phosphodiesterase, either directly or (Hashimoto et al., 2005). These very reactive
indirectly through a histidine kinase, to control ROS can damage proteins, lipids, nucleic acids,
the intracellular concentration of cyclic-di-GMP and other cellular components (Li et al., 2008).
(Plate et al., 2012; Carlson et al., 2010). NO was It is suggested that the photocatalytic killing
identified as important factor mediating biofilm mechanism initially damages the surfaces weak
dispersal of the pathogen P. aeruginosa (Barraud points of the bacterial cells, before totally break-
et al., 2006). Low, non-toxic concentrations of age of the cell membranes (Li et al., 2008). The
NO induced a transition from the sessile, resistant internal bacterial components then leak from the
biofilm phenotypes of P. aeruginosa to planktonic cells through the damaged sites. Finally, the pho-
phenotypes (Darling et al., 2003; Van Alst et tocatalytic reaction oxidizes the cell debris.
al., 2007). Addition of antimicrobial treatment Photocatalysis of water create ROS using solar
to NO treated biofilms completely removed P. energy (Miller et al., 2010). Titanium dioxide
aeruginosa biofilms (Barraud et al., 2006). Cyclic- (TiO2) is one of such photo-catalyst that can gen-
di-GMP involved in NO mediated dispersal of P. erate ROS when illuminated with light and thus
aeruginosa is controlled by diguanylate cyclases eliminate microbes (Sapkota et al., 2011). How-
and phosphodiesterases (Ryan et al., 2006). ever, because of its large band p for excitation,
Genes encoding these enzymes and redox sensors only ultraviolet (UV) light irradiation can excite
are widely distributed in all bacteria (Delgado- TiO2, which limits its application in the living
Nixon et al., 2000; Romling et al., 2005). This environment. However, with impurity doping,
suggests that NO dispersal of biofilms may occur through metal coating and controlled calcinations
in different species of bacteria. Several studies TiO2 has been successfully modified to expand its
supported this hypothesis. Formation of Staphy- absorption wavelengths to the visible light region
lococcus biofilms is inhibited by exposure to nitrite (Liu et al., 2007). Previous studies have showed
56  | Sathe and Dobretsov

that the modified TiO2 significantly reduced the environment, their microbial activity and interac-
numbers of surviving bacterial cells of E. coli in tions with other species present in biofilms. QS is
response to visible light illumination (Liu et al., one of the types of chemical interactions between
2007). Several other metal oxide nanoparticles bacterial cells, as well as between prokaryotic
and nanocoatings are being investigated for their and eukaryotic organisms. Removal of biofilms
potent anti biofilm activity. It has been shown from surfaces exposed in the marine environment
that ZnO nano-coatings illuminated with light remains a challenge. Various novel techniques,
inhibit growth of bacterial pathogens, such as such as QS inhibitors, NO, and ROS can offer
Escherichia coli, Bacillus sp., Salmonella sp., Listeria a better solution to get rid of these biofilms in
monocytogenes and Staphylococcus aureus in fresh near future. Prevention of microbial attach-
water ( Jones et al., 2008; Liu et al., 2009; Jaisai et ment and biofilm dispersal rather than killing of
al., 2012). Recent study demonstrated that ZnO marine microorganisms should be future priority
nanorod coatings inhibited biofilm formation of directions. Close collaboration between microbi-
the marine bacterium Acinetobacter sp. and set- ologists, physicists, chemists and engineers will be
tlement of the bryozoan larvae of Bugula neritina necessary for the development of new non-toxic
(Al Fori et al., 2014). Vanadium pentoxide (V2O5 antifouling applications.
nanowires in sea water catalyse the oxidation
of bromide ions and produce singlet molecular References
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Microbial Biodiversity in Natural
Katharina Besemer

Abstract environment (Battin et al., 2008; Hall-Stoodley

Natural biofilms in aquatic ecosystems exist et al., 2004) and on a wide variety of organic and
as complex and dynamic communities, which inorganic substrates, which makes them promi-
harbour a considerable microbial biodiversity, nent players in organic matter remineralization
typically dominated by Alpha-, Beta- and Gamma- (Das et al., 2007; Golladay and Sinsabaugh, 1991)
proteobacteria, bacteroidetes and cyanobacteria. and in the stabilization of fine sediments against
A number of publications have highlighted the resuspension (Decho, 2000; Gobet et al., 2012),
importance of biofilm biodiversity for ecosystem for instance. In ecosystems with large surface-
processes in a range of environments. The aim to-volume-ratios, such as streams, benthic (the
of this chapter is to give an overview of different interface between stream water and sediment
aspects of microbial diversity in natural biofilms, exposed to light) and hyporheic (the streambed
including local diversity (alpha-diversity), among- sediment where stream water and groundwater
patch diversity (beta-diversity), taxonomic and mix) biofilms typically dominate microbial life
functional diversity. While its major focus lies (Battin, 2000; Battin et al., 2003ab; Boulton et
on biofilms in the benthic and hyporheic zone al., 1998; Marmonier et al., 2012), while biofilms
of streams and rivers, this chapter also includes on submerged plants or attached to suspended
examples from lake, tidal and marine sediments, aggregates (‘mobile biofilms’; Battin et al., 2008)
and biofilms associated with suspended aggre- are of major importance in larger rivers, estuaries
gates. The local and regional processes that have and lakes (Luef et al., 2007; Simon et al., 2002;
been proposed to sustain or constrain biofilm see Chapter 1). Furthermore, biofilms play a
biodiversity and community assembly, such as critical role in some marine ecosystems, such as
environmental heterogeneity, biotic interactions intertidal mats, which are important locations of
and dispersal dynamics are reviewed. Finally, primary productivity and the main food resource
this chapter considers the relationship between for many benthic grazers (Decho, 2000; Russell et
taxonomic diversity, functional diversity and eco- al., 2007).
system processes. The taxonomic and functional diversity har-
boured by natural biofilms in aquatic ecosystems is
huge. Extensive three-dimensional structures (see
Introduction Chapter 2), a complex network of interactions
Biofilms are an important form of microbial life and a surprising level of multi-cellular behaviour
in many aquatic ecosystems (Davey and O’Toole, (see Chapter 3) act in concert to provide diverse
2000; Hall-Stoodley et al., 2004; see also Chapter micro-habitats favouring taxonomic and func-
1), where they contribute to major ecosystem tional biodiversity (Flemming and Wingender,
processes and even to globally important bio- 2010; Stoodley et al., 2002; Watnick and Kolter,
geochemical fluxes (Battin et al., 2003a, 2008). 2000; Webb et al., 2003). Bacteria, archaea, algae,
Biofilms develop in virtually every interfacial fungi, protozoa and viruses contribute to the
64  | Besemer

biodiversity of these dynamic communities ( Jack- succession. Further, Hödl and colleagues (2011)
son and Jackson, 2008; Lock et al., 1984; Romaní, found suggestive evidence that biotic interactions,
2010; Stoodley et al., 2002). The rapid develop- such as cell-to-cell signalling or surface condition-
ment of novel technologies in the last decades has ing, play an important role already during primary
pushed the limits to which the diversity of micro- colonization and early biofilm formation. As
bial communities can currently be explored, and biofilm growth progresses, interactions between
unveiled the vast diversity of the microbial world. species and with the abiotic environment may
Still, a comprehensive appraisal of microbial bio- gain further importance, denoting a shift from
diversity is elusive for many ecosystems (Quince predominantly stochastic to deterministic pro-
et al., 2008; Zinger et al., 2012) and aquatic bio- cesses governing biofilm formation (Battin et al.,
films are no exception to this. Nevertheless, the 2007; Besemer et al., 2007).
unprecedented accuracy with which microbial Succession of biofilms is driven by differential
diversity can now be analysed has fostered new species performance in dispersal, survival and
insights in the distribution of biofilm biodiversity reproduction (Burns and Ryder, 2001). Based
and the underlying mechanisms (Besemer et al., on a study on freshwater biofilms, Jackson and
2013; Wang et al., 2012c, 2013). Novel statistical colleagues (2001) developed a conceptual model
methods (Quince et al., 2008) and meta-analyses of diversity dynamics during biofilm succession.
of published data enable comparisons between This model proposes that initial colonization
studies and help to set findings in a broader eco- leads to a rapid increase in species diversity, which
logical context (Lozupone and Knight, 2007). subsequently declines as the biofilm becomes
This chapter reviews findings on the microbial dominated by a limited number of strong com-
diversity contained in natural biofilms in a range of petitors. Finally, as biofilms mature, their complex
aquatic habitats, the mechanisms generating and three-dimensional structure may facilitate greater
maintaining this biodiversity, and the implications diversity through habitat diversification and
of biodiversity for ecosystem functioning. While broader resource diversity. Trophic interactions
the major focus of this chapter lies on the prokary- within the biofilm are likely to become more
otic diversity of benthic and hyporheic biofilms in important than competition during this late suc-
streams and rivers, examples from other environ- cessional state, thereby fostering a switch from
ments and from eukaryotic microbial groups are bottom-up to top-down control of biofilm diver-
included to provide a more comprehensive pic- sity ( Jackson, 2003).
ture of the diversity contained in natural biofilms. Several empirical studies support the predic-
tions of this conceptual model. For instance,
epilithic river biofilms showed a rapid increase,
Biofilm community assembly – followed by a decrease and, during late succes-
stochastic versus deterministic sion, again a slight increase in diversity during
processes a biomass accrual phase (Lyautey et al., 2005).
Biofilm formation is initiated when single cells Further, river biofilms developing on exposed
or microbial aggregates from the overlying water glass slides (Boulêtreau et al., 2014) and on
column attach to a submerged surface (Costerton decaying leaves (Fischer et al., 2009) showed a
et al., 1995; Hödl et al., 2011), where some will decrease in bacterial richness after a peak during
start to grow and eventually form a biofilm (Stood- early succession, potentially caused by competi-
ley et al., 2002). This initial colonization may be tive exclusion. In contrast, Lear and colleagues
largely stochastic, as it relies on dispersal from (2008) found that epilithic biofilm diversity was
the source community suspended in the water still increasing after 7–9 weeks of growth, indi-
(Battin et al., 2007; Jackson, 2003). However, the cating that biofilm succession in these streams
aptitude to attach to surfaces or to other microbes proceeded slowly. It is in fact plausible to assume
differs between species (Rickard et al., 2003; that differences in resource supply, physical
Yawata et al., 2014), which may select for certain habitat conditions or disturbance regime in the
species already during this early phase of biofilm various ecosystems modulate the successional
Microbial Biodiversity in Natural Biofilms |  65

patterns of aquatic biofilms. For instance, Fierer state (Burns and Ryder, 2001). Several studies
and colleagues (2010) predicted different suc- demonstrated the effects of disturbance frequency
cessional trajectories for microbial communities on biofilm diversity and succession. Cardinale
dominated by autotrophic organisms (e.g. algae- and colleagues (2006a) showed that algal species
dominated biofilms), heterotrophic microbial diversity in stream biofilms could be explained by
communities fuelled by external inputs of organic the joint effects of disturbance frequency, which
carbon (e.g. hyporheic biofilms), and communi- created new niche opportunities for species, and
ties whose major carbon sources are contained by the rate at which biomass accrual lead to suc-
within the substrate itself (e.g. biofilms growing cessional displacement of inferior by superior
on decaying leaves). Changes in the quality and competitors. In an experimental study, Milferstedt
quantity of resources as they are modified by the and colleagues (2013) found that biofilm diversity
microorganisms during biofilm growth are indeed was able to recover between weekly disturbances
likely to explain successional differences between indicating community resilience, while daily
epilithic (Lear et al., 2008) and leaf litter (Fischer disturbances led to a community dominated by
et al., 2009) biofilms, for instance. Interestingly, a single, disturbance-resistant organism. Further,
successional patterns in experimental biofilms bacterial richness in stream biofilms during suc-
were also shown to be altered by shear stress. cession increased faster in a Mediterranean stream
Biofilms grown at intermediate shear showed the subjected to frequent drying and flooding events
pattern predicted by Jackson’s model, whereas than in a more stable Central European stream,
biofilms grown at high shear rates showed no suggesting that the Mediterranean biofilm was
second increase of diversity, and biofilms grown dominated by taxa adapted to fast re-colonization
at low shear showed high diversity throughout the after disturbances, whereas diversity in the Central
experiment (Rochex et al., 2008). European stream was likely controlled by biotic
Disturbances change the microbial land- interactions (Artigas et al., 2012). Given the spa-
scape and set biofilm communities back to an tial and temporal heterogeneity of disturbances
earlier successional state by removing a part of and hydrodynamic conditions experienced by
the biodiversity, which then becomes available biota in aquatic environments (Lake, 2000) it
for colonization of new substrata (Battin et al., may, therefore, be assumed that natural biofilms
2007; Burns and Ryder, 2001). The temporal exist as assemblages of patches with differing suc-
and spatial scale of such disturbances may influ- cessional trajectories and at different successional
ence diversity by altering the relative importance states (Besemer et al., 2007, 2009a; Cardinale et
of biotic interactions and dispersal dynamics. al., 2002).
When disturbance frequency is low, the extended
time for species to recruit to habitat patches and
for local interactions to take place is expected to Metacommunity ecology –
favour competitively superior species, which are the roles of dispersal and
able to suppress or displace inferior competitors environmental conditions
under the given suite of environmental conditions At the regional scale, natural aquatic biofilms
(Tilman, 1987). At high disturbance frequency, exist as part of a larger microbial network in
conditions may favour dominance by species that which local communities are linked by dispersal
are either good dispersers and able to quickly of multiple interacting species to form a meta-
colonize empty habitat patches, or are resistant community (Battin et al., 2007; Holyoak et al.,
against disturbances (Cadotte, 2007; Cardinale et 2005). Numerous studies have assessed the rela-
al., 2006a). In streams with a dynamic hydrologic tive importance of regional (dispersal dynamics,
regime, frequent disturbances might therefore landscape patterns) and local processes (abiotic
preserve pioneering biofilm taxa by resetting habitat conditions, deterministic biotic interac-
the process of succession, while in more stable tions) for microbial community composition and
environments, like lakes for instance, biofilm diversity. In a conceptual model for freshwater
communities are more likely to reach a mature bacterioplankton, Logue and Lindström (2008)
66  | Besemer

proposed that in aquatic ecosystems with long 2013) were compared with random subsamples of
retention times such as lakes, community assem- the respective stream water communities, as they
bly is mainly driven by species sorting, where the might result from purely stochastic immigration
local environment and biotic interactions select to an empty habitat patch from a source commu-
from the metacommunity. Systems with short res- nity. It was found that such stochastic immigration
idence times such as streams, on the other hand, was unlikely to explain the observed community
are strongly affected by mass effects through the composition of these biofilms. Collectively, these
constant input of species, which maintain species findings suggest that the local environment and
in less favourable habitats and lessen the co-var- biotic interactions select microorganisms from
iance between environmental conditions and the stream water for biofilm formation (Fig. 4.1).
community composition (Logue and Lindström, In contrast, stream water communities can likely
2008). However, the attachment of microbial be explained by mass effects, determined by
cells to a stable surface increases their residence large cell influx rates and short residence times
time relative to those in the water column, which limiting the influence of environmental factors
potentially makes them more susceptible to local (Logue and Lindström, 2008). Streams collect
environmental conditions. microorganisms from various sources, such as the
Indeed, environmental conditions, rather surrounding terrestrial landscape, groundwater
than geographic distance, were repeatedly found and upstream tributaries, collectively forming
to explain biofilm community composition indi- the upstream metacommunity (Crump et al.,
cating that species sorting would be a candidate 2007, 2012; Wilhelm et al., 2013). The notion
mechanism for biofilm community assembly. For of stream water as an important integrator of
instance, Fierer and colleagues (2007) found that microbial diversity is indeed supported by the
pH could predict most of the variation between fact that significantly higher diversity was found
bacterial communities inhabiting fine benthic in the boreal and glacier-fed stream water com-
organic matter in streams, and, similarly, Horner- munities than in the biofilms (Besemer et al.,
Devine and colleagues (2004) found that bacterial 2012; Wilhelm et al., 2013). Similar results have
community composition in salt marsh sediments been reported for a marsh of Lake Hallwil in
was primarily driven by water chemistry and plant Switzerland, were the microbial diversity of the
cover, rather than geographic distance. However, lake water was slightly higher than that in sedi-
combined environmental and dispersal dynamics ments, and clearly higher than on plant litter and
were observed to explain community variation in epiphytic biofilms (Buesing et al., 2009). In
among marine sediment bacteria (Xiong et al., contrast, biofilm diversity was found to be higher
2014), and ammonia-oxidizing bacterial com- than planktonic bacterial diversity in Lake Baikal
munities in salt-marsh sediments, which showed (Parfenova et al., 2013) and in mountain lakes in
imprints of dispersal limitation at local, but not at the Pyrenees (Bartrons et al., 2012). According to
regional scale (Martiny et al., 2011). Furthermore, the conceptual model of Logue and Lindström
community composition and diversity of epilithic (2008), it can be expected that continuous inflow
stream biofilms across New Zealand were related of microbes could support the microbial diversity
to both geographical location and environmental in stream water and, to a lesser extent, in lakes
variables, though the relationship with the envi- with a short retention time (for instance, 3.8 years
ronmental properties was clearly stronger (Lear et in lake Hallwil; Buesing and Gessner, 2006),
al., 2013). Lear and colleagues suggested that the while species sorting and competitive interactions
structure of bacterial communities is largely deter- might constrain the diversity in a lake with such
mined by niche-based processes (that is, species a high retention time as lake Baikal (> 300 years;
sorting due to environmental factors) rather than Jasechko et al., 2013). However, the mountain
by dispersal limitations. To evaluate species sort- lakes studied by Bartrons and colleagues (2010,
ing as a possible mechanism of biofilm assembly, 2012) showed higher phylogenetic diversity in
the biofilm communities from boreal (Besemer et biofilms than in the plankton despite the short
al., 2012) and glacier-fed streams (Wilhelm et al., water residence times of these lakes.
Microbial Biodiversity in Natural Biofilms |  67

Surface-inflow from
terrestrial environment
Transport of cells with water flow

Source community suspended

in the stream water

Subsurface-inflow from
terrestrial environment


Species sorting


Figure 4.1 Theoretical scheme of stream biofilm assembly. The stream water collects and transports
microorganisms from the surrounding landscape into a source community, which eventually seeds the
benthic biofilm. The attachment to a stable surface and the encapsulation of microbial cells in the biofilm
matrix increase their residence time, making them more susceptible to the ambient environmental conditions.
Species sorting is therefore a candidate mechanism for the assembly of benthic biofilm communities. The
microbial diversity in the stream water, on the other hand, may be supported by mass effects through the
constant influx of species from the surrounding environment.

Landscape topography and water flow affect the streambed was flat. This result suggests that
dispersal in aquatic systems, but also gener- dispersal has a role in structuring biofilm compo-
ate microhabitats that differ in turbulence and sition at small scales, and agrees with the findings
shear (Battin et al., 2003b, 2007; Rickard et al., from Martiny and colleagues (2011) in salt marsh
2004). Experimenting with stream mesocosms sediments. However, this relatively simple model
containing streambed landscapes, epilithic bio- failed to capture the complexity of the heteroge-
film community composition was found to be neous flow landscapes. Furthermore, Woodcock
related to the spatial variation of hydrodynamic and colleagues (2013) found evidence that the
conditions (Besemer et al., 2009a). The resulting importance of immigration from the stream water
species turnover between habitat patches differing varied between microhabitats differing in water
in hydrodynamic conditions generated a gradient turbulence and flow velocity. These findings point
of beta-diversity that increased with habitat heter- to flow heterogeneity as a factor likely influencing
ogeneity at the landscape scale. It can be assumed biofilm biodiversity by altering both dispersal
that the flow landscape sorted species according to dynamics and physical habitat conditions.
their ability to cope with the local hydrodynamic The importance of flow-induced dispersal for
environment, which agrees with earlier findings biofilm diversity at the scale of an entire stream
showing that flow velocity and shear stress modify network was assessed by sampling more than 100
biofilm structure (Battin et al., 2003b). Using the streams in a pre-alpine catchment (Besemer et al.,
same experimental set-up, Woodcock and col- 2013). Confluences, as conspicuous nodes in the
leagues (2013) showed that the beta-diversity fluvial network, were expected to accumulate spe-
patterns among habitat patches could be suf- cies from multiple catchments and, thus, increase
ficiently reproduced by a neutral model of flow the size of the metacommunity from which the
induced dispersal when the hydrodynamic regime local biofilm communities assemble. If random
in the mesocosm was homogenous, that is, when invasion from the metacommunity has a role in
68  | Besemer

structuring a local community, the diversity of 2010). Matthiessen and colleagues (2010) found
this community will increase with the size of the that habitat heterogeneity in terms of varying
metacommunity from which it is drawn (Curtis light intensities among local patches increased
and Sloan, 2004). Alpha-diversity was therefore local and regional diversity, maintaining inferior
predicted to increase downstream of confluences, competitors in less favourable patches. However,
and, at the network scale, from headwaters to increasing dispersal rates among local com-
larger streams. However, biodiversity and even- munities homogenized the metacommunity by
ness of microbial biofilms were, on average, lower distributing the best competitor across the whole
downstream of confluences throughout the metacommunity, thereby decreasing diversity
fluvial network, ultimately causing biodiversity and evenness. Using a similar set-up to test for
to decrease downstream (Besemer et al., 2013). the combined effects of habitat connectivity
These results suggest that the local environment and heat stress, Eggers and colleagues (2012)
and biotic interactions, such as competition, may reported that high dispersal rates decreased local
modify the influence of metacommunity con- diversity and evenness, while, at the same time,
nectivity on local biofilm biodiversity throughout generating a rescue effect for extinction-prone
a stream network. The larger regional species rare species. Taken together, these studies indi-
pool downstream of confluences is more likely cate that the complex network of interactions,
to include superior competitors adapted to the which characterizes microbial biofilms (Stoodley
streambed environment. Cascading through the et al., 2002), is likely to play an important role
network, this accumulation of competitive taxa in the assembly and diversity of natural biofilm
could ultimately decrease mean alpha-diversity. communities.
Furthermore, beta-diversity among head-
waters was higher than between larger streams,
evoking dispersal limitation as a driver of beta- Spatial and temporal species
diversity among these systems (Besemer et al., turnover – the role of beta-
2013). Headwaters also encompass a larger diversity
geographical area compared with downstream
catchments, which results in a wider range of Beta-diversity across space
environmental conditions among these systems, Regional diversity depends on both local diver-
potentially enhancing beta-diversity (Clarke et al., sity and community variability between habitat
2008; Fierer et al., 2007). Controlling for a pos- patches. The biodiversity contained in a region
sible distance effect, it was found that the higher can therefore be large even when local diversity
beta-diversity among headwaters could not be of individual habitat patches is low, given that the
explained exclusively by the larger geographical species turnover between habitat patches is high
(and potentially environmental) distance among (Nemergut et al., 2013). Spatial beta-diversity was
them. This suggests that the dendritic nature of found to be an important component of biofilm
fluvial networks constrains microbial dispersal diversity at multiple spatial scales, comparing
and leads to elevated beta-diversity between head- streams from multiple catchments (Wilhelm et al.,
waters. Pronounced variability of alpha-diversity 2013), streams within a watershed (Besemer et al.,
among headwaters further indicated an imprint of 2013; Fierer et al., 2007), habitat patches within a
dispersal limitation, in combination with higher stream (Fischer et al., 2009; Wang et al., 2012a)
habitat and resource variation among headwaters and even architectural features within a biofilm
(Besemer et al., 2013; Brown et al., 2011). (Besemer et al., 2009b). Strikingly, beta-diversity
A negative effect of high metacommunity con- was higher among biofilms than among stream
nectivity, as indicated by this study, has indeed water samples in glacier-fed streams, contrasting
been predicted by a theoretical model (Mouquet the patterns observed for alpha-diversity (Wil-
and Loreau, 2003) and has also been shown for helm et al., 2013). This highlights the potential of
experimental metacommunities of marine algal biofilms to harbour significant biodiversity at the
biofilms (Eggers et al., 2012; Matthiessen et al., regional scale, even if their alpha-diversity is lower
Microbial Biodiversity in Natural Biofilms |  69

than that of the water column in some environ- and for the ecosystem functions and services they
ments. provide.
The high alpha-diversity found in some
habitats may actually reflect high beta-diversity Temporal beta-diversity
between micro-habitats pooled in a single Another aspect of beta-diversity is the temporal
sample (Nemergut et al., 2013). This might be turnover of communities, which can promote
especially true for biofilms developing on and in regional diversity through asynchronous environ-
sediments, were strong environmental gradients mental fluctuations in different habitat patches
and high structural complexity are assumed (Chesson, 2000). Microbial communities typi-
to enhance microbial diversity (Brunke and cally consist of a set of abundant taxa, which are
Gonser, 1997; Torsvik et al., 2002). Conducting assumed to perform most ecosystem functions,
a comprehensive meta-analysis of environmental and a huge diversity of rare taxa, potentially
samples, Lozupone and Knight (2007) found constituting a seed bank (Lennon and Jones,
that sediments harbour exceptionally high 2011; Pedrós-Alió, 2006). This seed bank com-
phylogenetic diversity, even higher than soils. prises populations that are potentially capable
Further, sediments were reported to feature high of being resuscitated following environmental
beta-diversity at the regional scale because of change (Lennon and Jones, 2011). Dormancy
their heterogeneity in terms of redox gradients is a common response to environmental stress
and nutrient availability (Nemergut et al., 2013). among microorganisms, which can prolong their
Similarly, in a study comparing bacterial commu- persistence and allows species to contend with
nities from different habitats, the highest spatial temporal variability of environmental conditions
beta-diversity was found in subsurface soils and ( Jones and Lennon, 2010). The accumulation of
sediments, followed by mountain stream bio- dormant organisms in the seed bank promotes
films, surface sediments and lake water (Wang co-existence of competing species by constituting
et al., 2013). Collectively, these studies suggest a temporal storage effect, which may be especially
aquatic sediments as one of the most important relevant in harsh and fluctuating environments
reservoirs for microbial diversity and underline (Chesson, 2000; Chesson and Huntly, 1997;
the necessity to consider both alpha- and beta- Lennon and Jones, 2011).
diversity in the study of sediment-attached Epilithic and epipsammic biofilms in glacier-
microbial communities. fed streams, as an example of an environment
Environmental heterogeneity is a key factor experiencing strong seasonal and diurnal fluc-
maintaining beta-diversity between habitat tuations of water chemistry and temperature,
patches. Human-induced loss of geomorphologi- were found to harbour a large number of rare taxa,
cal streambed heterogeneity was found to decrease which were more abundant in the active commu-
beta-diversity and gamma-diversity in benthic nity (based on rRNA analysis) than in the bulk
algal biofilms, while alpha-diversity remained high community (based on analysis of the rRNA gene;
in the altered streams (Passy and Blanchet, 2007) Wilhelm et al., 2014). This suggests that rare taxa
– similar to the patterns that were found for bacte- contribute disproportionately to microbial com-
rial beta-diversity in stream biofilms (Besemer et munity dynamics in glacier-fed streams. Transient
al., 2009a). On a larger spatial scale, headwaters, dormancy and high community turnover, where
as the smallest but most abundant components of taxa repeatedly enter and leave the seed bank,
a fluvial network, harbour the highest degree of could therefore be candidate mechanisms that
habitat heterogeneity (Brown et al., 2011) and the maintain high biofilm diversity in these systems.
highest level of microbial beta-diversity (Besemer This agrees with findings from hyporheic sedi-
et al., 2013). Habitat loss (e.g. by stream burial, ments from glacier-fed streams (Freimann et al.,
mountain-top mining) and homogenization (e.g. 2013a) where pronounced temporal turnover in
through stream regulation and streambed degra- community structure and function was observed,
dation) is, therefore, likely to have severe impacts compared to groundwater-fed streams. Fur-
for the microbial diversity in streams and rivers thermore, community turnover in glacier-fed
70  | Besemer

hyporheic communities was more closely related epilithic, epipsammic and hyporheic stream and
to changes in water chemistry than in the compar- river biofilms (Araya et al., 2003; Battin et al.,
atively stable groundwater-fed streams (Freimann 2001; Beier et al., 2008; Besemer et al., 2012;
et al., 2013b). Decreasing glacial runoff due to Bricheux et al., 2013; Gao et al., 2005; Marxsen
climate change could lead to reduced temporal et al., 2010; Olapade and Leff, 2005) and aggre-
beta-diversity and, thus, to a loss of diversity at the gate-associated microbial communities in lakes
landscape scale (Freimann et al., 2013b; Wilhelm (Schweitzer et al., 2001; Tang et al., 2009), while
et al., 2013). Alpha- and Gamma-proteobacteria are often
In a completely different environment, namely the most abundant bacterial groups in marine
epiphytic biofilms on brown macro-algae in the biofilms (Gobet et al., 2012; Lachnit et al., 2011;
Baltic Sea, Stratil and colleagues (2013) found McKew et al., 2011). However, a surprisingly
indications that members of the rare biosphere large number of studies found that Alpha-pro-
could become abundant and potentially contrib- teobacteria were also as abundant – or even more
ute to community functions when environmental abundant – than Beta-proteobacteria in freshwa-
conditions became appropriate, thus supporting ter biofilms ranging from epilithic and hyporheic
the seed bank hypothesis. In contrast, temporal stream biofilms (Anderson-Glenna et al., 2008;
variation in biofilm communities attached to sand Hall et al., 2012; Lear et al., 2009a; Romaní et al.,
grains of coastal sediments could be explained by 2014; Wang et al., 2012b) to biofilms on living or
constant input of transient taxa from other loca- decaying plants (Buesing et al., 2009; McNamara
tions by advection and physical mixing (Gobet et and Leff, 2004), and to diatom-aggregates in lakes
al., 2012). Though significant temporal commu- (Knoll et al., 2001). While some studies (Knoll
nity turnover was observed in this system, most et al., 2001; Schweitzer et al., 2001) observed a
was due to changes in the rare biosphere, and rare switch from an Alpha-proteobacteria dominated
taxa tended to stay rare, arguing against a dynamic to a Beta-proteobacteria dominated community
seed bank. Obviously, various mechanisms may during biofilm development, others (Manz et al.,
drive temporal – as spatial – beta-diversity pat- 1999) found the opposite pattern – decreasing
terns in various ecosystems. importance of Beta-proteobacteria, and increas-
ing dominance of Alpha-proteobacteria. Further,
a study on sediment bacteria along a river from
Taxonomic composition of freshwater to the sea showed that both Alpha- and
biofilms Beta-proteobacteria, together with Acidobacteria
Numerous studies have elucidated the taxonomic and Nitrospira, were overrepresented in fresh-
and phylogenetic structure of microbial biofilms water sediments, while Gamma-proteobacteria
across a broad range of habitats, of which only a were overrepresented in marine sediments and
few are mentioned here. The following paragraphs Epsilon-proteobacteria in intertidal sediments
give a short overview of the most abundant (Wang et al., 2012c). Deviating strategies to cope
microbial phyla within biofilms. The bacterial with grazing pressure or low substrate availability
diversity of biofilms is typically dominated by (Newton et al., 2011) might favour Alpha- or
Alpha-, Beta- and Gamma-proteobacteria, bacte- Beta-proteobacteria depending on habitat type
roidetes and cyanobacteria (Romaní et al., 2013). and environmental conditions (Romaní et al.,
Beta-proteobacteria is often the most abundant 2014).
bacterial phylum in freshwater habitats (Barberán Members of the bacteroidetes phylum (for-
and Casamayor, 2010; Glöckner et al., 1999; merly the Cytophaga/Flavobacterium/Bacteroides
Newton et al., 2011), while Alpha- and Gamma- group) often represent a substantial – or even
proteobacteria are commonly found to dominate the dominant – part of biofilm communities
marine systems (Glöckner et al., 1999; Rusch et (e.g. Anderson-Glenna et al., 2008; Bartrons et
al., 2007). Accordingly, Beta-proteobacteria has al., 2012; Gobet et al., 2012; Hall et al., 2012;
repeatedly been found to be the most important Parfenova et al., 2013) and especially aggregate-
bacterial group in freshwater biofilms, including associated communities (Knoll et al., 2001; Manz
Microbial Biodiversity in Natural Biofilms |  71

et al., 1999; Schweitzer et al., 2001). Gliding sediments (McKew et al., 2011). Buriánková
motility and the ability to use complex macro- and colleagues (2012) showed that methano-
molecules could qualify members of this group genic archaea can be an important component
to be important players in the degradation of sus- (>10%) of the microbial community in hyporheic
pended particles (Crump et al., 1999; Kirchman, sediments, indicating the presence of anoxic
2002; Newton et al., 2011). For instance, Crump micro-habitats within hyporheic biofilms. A
and colleagues (1999) reported that Cytophaga meta-analysis of archaeal sequences revealed that
(bacteroidetes) were the most abundant group on freshwater sediment communities contain lower
estuarine particles, where they might contribute archaeal biodiversity than planktonic freshwater
significantly to the estuarine food web. Further, communities (Auguet et al., 2010). Furthermore,
bacteroidetes have been found to be associated the lack of indicator species among freshwater
with cyanobacterial blooms (Newton et al., 2011) sediment archaea in the study by Auguet and col-
and can constitute the most abundant group on leagues might indicate late archaeal colonization
cyanobacterial aggregates (Tang et al., 2009). The of this environment.
relative abundance of bacteroidetes might increase Among the eukaryotic organisms, algae, most
with particle age, when labile organic compounds commonly Bacillariophyta and Chlorophyta, are
are depleted and the particle increasingly consists a major component of biofilms exposed to light
of refractory organic material (Knoll et al., 2001; (Arnon et al., 2007; Artigas et al., 2012; Battin et
Schweitzer et al., 2001). al., 2003b; Besemer et al., 2007; Wilhelm et al.,
Cyanobacteria are a common phototrophic 2013). Fungi, especially Ascomycota, are a promi-
compartment of microbial biofilms and are the nent structuring element of biofilms developing
dominant organisms in the complex laminated on submerged organic matter and are important
stromatolitic mats (Paerl et al., 2000). Cyano- players in the degradation of leaf litter and wood
bacteria were found to be the most abundant (Das et al., 2007; Gessner et al., 2007; Golladay
microbial group in lake biofilms (Parfenova et and Sinsabaugh, 1991). Protists, including flagel-
al., 2013), epipsammic (Hullar et al., 2006) and lates, ciliates, and amoebae can control biofilm
epilithic (Wang et al., 2012b) stream biofilms, growth and alter biofilm structure and function
and are often more abundant in biofilms than in (Böhme et al., 2009; Bott and Kaplan, 1989).
the water column (Parfenova et al., 2013; Ylla et Lastly, biofilms can also be an important reservoir
al., 2009). Acidobacteria can dominate the micro- of viruses in aquatic ecosystems, which may affect
bial community inhabiting fine benthic organic biofilms by structuring community composition
matter, especially at low pH (Fierer et al., 2007). and diversity ( Jackson and Jackson, 2008; Luef et
Further taxonomic groups typically present in al., 2009).
biofilms at lower abundances include Actinobac-
teria, firmicutes, Gemmatimonadetes, Delta- and
Epsilon-proteobacteria, Verrucomicrobia, Plancto- Biofilm diversity in different
mycetes, and Deinococcus-Thermus (e.g. Bartrons habitats
et al., 2012; Besemer et al., 2009b, 2012; Hullar et Biofilm community composition and diversity
al., 2006; Lachnit et al., 2011; Romaní et al., 2014; can differ significantly between different habitat
Wang et al., 2012b). types (e.g. benthic and hyporheic zone, living or
Archaea have generally been reported to con- decaying plant materials, suspended particles),
stitute minor, or even undetectable, proportions even within the same environment (Buesing et
of benthic biofilms in streams and rivers (Bricheux al., 2009; Wilhelm et al., 2014). The nature of the
et al., 2013; Lawrence et al., 2004; Manz et al., substratum (organic or inorganic, surface area,
1999; Marxsen et al., 2010; Romaní et al., 2014; stability), hydrologic conditions (shear stress,
Wilhelm et al., 2014); however, higher relative current velocity and turbulence influencing mass
abundances of archaea (in the range of 1–10%) transfer), temporal and spatial gradients (e.g.
have been found in streams (Battin et al., 2001, light, temperature, nutrients, oxygen) differenti-
2004; Olapade and Leff, 2005) and saltmarsh ate biofilm habitats and are all likely to influence
72  | Besemer

biofilm structure and function (Battin et al., microorganisms, as observed for bacteria in ben-
2003b; Brunke and Gonser, 1997; Romaní, 2010). thic biofilms (Romaní et al., 2004) and fungi in
Comparing biofilms from different streambed mixed-species biofilms on leaf litter (Danger et
substrates, Wilhelm and colleagues (2014) found al., 2013), potentially by increasing the availabil-
that epipsammic biofilms harboured a higher bac- ity and diversity of high-quality organic carbon
terial diversity than epilithic biofilms. The larger sources. In contrast, studies on experimental
specific surface area of sandy sediments may foster stream biofilms observed a slightly negative (Lear
the adhesion of microbial cells from the water et al., 2009b) or no consistent effect (Romaní et
flushing through it, thereby supporting microbial al., 2014) of light availability on microbial diver-
diversity. Further, biofilms attached to surface sity, though the bacterial community composition
sediment particles are more prone to translocation was significantly affected in both studies. Negative
than epilithic biofilms (Hullar et al., 2006), which effects of primary producers on the diversity of
might result in a patchy distribution of biofilm heterotrophic microorganisms could be caused by
biodiversity and high beta-diversity among micro- allelopathic compounds, which are produced by
habitats (Nemergut et al., 2013). Interestingly, the many algae and cyanobacteria (Leflaive and Ten-
diversity of bacterial morphotypes showed the Hage, 2007), or competition between algae and
opposite pattern with higher diversity in epilithic bacteria (Hulot et al., 2001). Indeed, a large field
than epipsammic biofilms, which might be related study in pre-alpine streams indicated a negative
to grazing pressure (Romaní and Sabater, 2001). relationship between the relative abundance of
Bacterial diversity was found to increase with cyanobacteria and the overall diversity and even-
sediment depth in coastal sands (Gobet et al., ness of epilithic biofilm communities (Besemer
2012), potentially reflecting increasing physico- et al., 2013). Furthermore, heterotrophic bacteria
chemical stability of the habitat. Decreasing shear attached to organic particles exhibited lower
stress with sediment depth could contribute to richness in lakes that experienced cyanobacterial
this pattern (Brunke and Gonser, 1997; Rickard blooms compared to lakes without such blooms
et al., 2004). Storage effects through dormancy (Tang et al., 2009).
mechanisms may also be prevalent in deep sedi- Suspended aggregates formed by microbes,
ments and in the hyporheic zone (Crump et al., their extracellular polymeric substances and
2012), which can constitute a refuge for microor- detrital particles are hotspots of microbial bio-
ganisms during unfavourable periods (Timoner et mass and activity that structure an otherwise
al., 2012). poorly structured water column (Battin et al.,
While benthic or epiphytic biofilms and 2008). Contradictory findings have been reported
suspended aggregates in the photic zone of regarding the relative importance of aggregate-
freshwater or marine systems contain substantial associated microbes to overall microbial diversity.
phototrophic components (Battin et al., 2001; Higher aggregate-associated microbial diversity
Besemer et al., 2007; Romaní, 2010), biofilms has been found in a wide range of environments,
within sediments or in the aphotic zone rely on including lakes (Bižić-Ionescu et al., in press), river
external carbon sources (Romaní et al., 2004) floodplains (Besemer et al., 2005), coastal marine
or, especially in deep-sea biofilms, on chemo- environments (Mohit et al., 2014; Zhang et al.,
autotrophic primary production (López-García et 2007), the open sea (Crespo et al., 2013), and the
al., 2003). The close spatial proximity of primary deep ocean (Eloe et al., 2011). In contrast, several
producers and heterotrophic microorganisms in studies indicated that the microbial diversity on
biofilms may promote internal cycling of organic suspended aggregates and particles was lower
carbon (Battin et al., 2003b; Singer et al., 2011), than in the free-living microbial community in
but may also have important implications for the lakes (Riemann and Winding, 2001), temperate
transformation and decomposition of alloch- marine (Acinas et al., 1999; Moeseneder et al.,
thonous organic matter through priming, for 2001) and Arctic marine environments (Kellogg
instance (Danger et al., 2013). The presence of and Deming, 2009), suggesting that particles
algae might enhance the diversity of heterotrophic recruit a fraction of the bacterial community
Microbial Biodiversity in Natural Biofilms |  73

present in the surrounding waters. However, might enable higher biofilm diversity (Bengtsson
those among the above cited studies using high- and Øvreås, 2010; Bengtsson et al., 2012). This
throughput sequencing methods, which can agrees with findings for the red macroalga Deli-
explore microbial diversity at a higher resolution sea pulchra, where thalli with bleaching disease
than classical molecular methods (Zinger et al., harboured a more diverse biofilm than healthy
2012), consistently reported higher diversity thalli, potentially due to decreased selective
in the particle-attached microbial community chemical defence of the macroalga (Fernandes
(Bižić-Ionescu et al., in press; Crespo et al., 2013; et al., 2012). Similarly, community composition
Mohit et al., 2014). Suspended aggregates in and diversity of biofilms growing on senescing
aquatic environments are highly diverse by means macrophytes differed according to plant species
of their origin, composition and age and likely and to the plant’s elemental composition (Mille-
constitute a heterogeneous micro-environment Lindblom et al., 2006). In contrast, experimental
harbouring a high number of potential niches for hyporheic biofilms fed with leaf leachates that
microbial growth (Bižić-Ionescu et al., in press). differed in chemical composition showed clear
Indeed, the diversity of aggregate-associated changes in bacterial community composition,
microbial communities was found to increase but no consistent change in diversity (Hall et al.,
with availability and diversity of suspended par- 2012).
ticles (Bižić-Ionescu et al., in press; Crespo et al., The simplest and the most complex biofilm
2013; Mohit et al., 2014). These studies suggest communities occur in extreme environments,
that aggregate-associated microbes, as ‘mobile including hot, nutrient-deprived, hypersaline,
biofilms’, contribute significantly to the overall calcareous, acidic and high-irradiance ecosystems
diversity of a range of aquatic habitats. In fact, the (Paerl et al., 2000). Biofilms forming streamers in
enormous microbial diversity of the oceans has acidic, metal-rich waters, for instance, were found
been proposed to result in part from a cob-web to harbour a limited bacterial diversity, dominated
like structure of polymers, colloids, gel particles by Beta-proteobacteria (Hallberg et al., 2006).
and large aggregates constituting micro-niches in Similarly, bacterial biofilms on substrates exposed
a seemingly homogeneous environment (Azam to metal-rich hydrothermal vent emissions at the
and Malfatti, 2007). Mid-Atlantic Ridge consisted almost exclusively
Biofilms associated with littoral plants or of Epsilon-proteobacteria, though considerable
macro-algae exhibit a wide array of interactions diversity was present within this group (López-
with their host, ranging from symbiotic to patho- García et al., 2003). However, high bacterial
logical, and can even be necessary to maintain diversity was found in adjacent sediments, indi-
normal macro-algal morphology (Goecke et al., cating that fast-developing Epsilon-proteobacteria
2010; Miranda et al., 2013; Morris and Monier, are the first colonizers where hydrogen sulfide is
2003). Epiphytic biofilm communities have been supplied and that, as communities evolve, they
found to differ depending on the macrophyte’s diversify and allow the creation of new niches
taxonomic affiliation, tissue type, age and state of (López-García et al., 2003). The most complex
disease. For a Planctomycetes-dominated biofilm biofilms are laminated cyanobacterial-bacterial
on kelp surface (Laminaria hyperborea), Bengts- mats, some of which facilitate the trapping and
son and Øvreås (2010) showed that overall binding and/or the precipitation of minerals
biofilm diversity increased, while the relative into organo-sedimentary structures to form stro-
abundance of Planctomycetes decreased with matolites (Paerl et al., 2000). In Antarctic desert
kelp tissue age. Ageing of the kelp tissue could streams, for instance, perennial, desiccation- and
be associated with reduced chemical defence freezing tolerant microbial mats are the most
against microbial colonization, resulting in a loss prominent form of life and hotspots of produc-
of the competitive advantage of Planctomycetes tivity in an otherwise inhospitable environment
towards other bacterial groups. Together with (Stanish et al., 2013). The diversity of diatoms in
the potential appearance of new niches involved these mats was found to exhibit an inverse rela-
in degradation of different kelp constituents, this tionship with both bacterial and cyanobacterial
74  | Besemer

diversity, and the diversity of these three groups ecosystems, whose body mass distribution is
showed different relationships to hydrology and skewed towards small organisms (Hillebrand,
water chemistry (Stanish et al., 2013). In a con- 2004).
trasting environment, stromatolitic mats from the Similar to latitude, species diversity may
Bahamas, increased salinity resulted in a decrease change with altitude, frequently showing either
in the relative abundance of cyanobacteria and an a monotonic decrease with altitude or a hump-
increase in the relative abundance of eukaryotic shaped pattern (Rahbek, 2005), or, as reported
chloroplasts, accompanied by a dramatic shift in for microorganisms living in soils and on plant-
the morphology of the mat (Ahrendt et al., 2014). surfaces, no patterns at all (Fierer et al., 2011). For
These findings indicate differential environmental stream biofilms, microbial diversity was found to
controls on the diversity of algae, cyanobacteria be reduced at higher elevations in streams across
and heterotrophic bacteria in these complex New Zealand (Lear et al., 2013), which agrees
microbial consortia. with findings from streams sampled above, at and
below the tree-line in the Austrian Alps (Wil-
helm et al., in press). A decrease of diversity with
Patterns of biofilm elevation was also observed for benthic biofilms
diversity along spatial and in alpine lakes, particularly related to the location
environmental gradients of the lake above or below the tree-line (Bartrons
et al., 2012). The higher complexity of the sur-
Spatial patterns of biofilm diversity rounding ecosystem and the increasing diversity
The large-scale distribution of biodiversity on of organic matter sources below the treeline are
earth varies along spatial gradients, such as lati- likely to contribute to this pattern (Bartrons et al.,
tude, elevation and depth (Gaston, 2000), likely 2012). Further, Wilhelm and colleagues (2013)
based on mechanisms related to spatial (e.g. the found that biofilm biodiversity decreased with
mid-domain effect, size of ecoclimatic zones) elevation among glacier-fed streams, though this
and climatic factors (e.g. light, temperature, pattern was weaker than for planktonic com-
water supply). For macro-organisms, it is widely munities of the same streams. These authors
accepted that species diversity within a specified suggested that, at lower altitudes, more sources
area increases with decreasing latitudes and peaks of microorganisms upstream in the catchment
close to the equator (Gaston, 2000; Hillebrand, (e.g. subglacial, englacial and supraglacial runoff,
2004), while it remains debated whether such a groundwater, adjacent soils) contribute to the
patterns exists for microorganisms. Bacterial rich- microbial community suspended in the stream
ness was found to increase towards lower latitudes water, which eventually seeds the biofilm. Spe-
in stream biofilms across New Zealand (Lear et cies sorting according to ambient environmental
al., 2013; Washington et al., 2013), which is in conditions might have attenuated this altitudinal
agreement with findings for marine (Fuhrman et pattern in stream biofilms (Besemer et al., 2012;
al., 2008; Pommier et al., 2007; Sul et al., 2013), Wilhelm et al., 2013).
and lake (Schiaffino et al., 2011) bacterioplank- Contrasting these findings, stream biofilm
ton, but contrasts findings from soils (Chu et al., richness and evenness increased with altitude in
2010; Fierer and Jackson, 2006). This finding that a Chinese stream spanning an altitudinal gradi-
stream biofilm microbes show a similar latitudinal ent of more than 2000 metres (Wang et al., 2011,
pattern as macro-organisms is notable because the 2012b). Furthermore, phylogenetic clustering of
strength of the latitudinal diversity gradient has biofilm bacteria increased with altitude, indicating
been shown to be weaker for small organisms, and that the high taxonomic diversity at high altitude
for freshwater compared to marine and terrestrial streams likely consists of many closely related spe-
environments (Hillebrand, 2004). High dispersal cies (Wang et al., 2012b). Large diel temperature
rates of microorganisms (e.g. Fenchel and Finlay, variations, increasing supply of dissolved organic
2004) may in fact be one of the reasons for the matter and frequent disturbances might have
weaker latitudinal diversity gradient in freshwater contributed to the high bacterial diversity at high
Microbial Biodiversity in Natural Biofilms |  75

altitudes (Wang et al., 2011, 2012b). Indeed, large and macro-invertebrates (Wang et al., 2012a) and
temporal variation in physical habitat conditions resulted in a significant distance-decay of similarity
was proposed to enhance diversity already in the between biofilm communities (Wang et al., 2013).
river continuum concept (Vannote et al., 1980), a Furthermore, biofilms beta-diversity among
mechanism, which was later conceptualized as the glacier-fed streams was found to decrease with
temporal storage effect (Chesson, 2000). Avail- increasing stream water temperature (Wilhelm
ability and diversity of dissolved organic matter et al., 2013). This study suggested that warming
have been proposed to be highest in the headwa- temperatures at high altitudes may contribute to
ter region of stream networks due to their intimate a homogenization of biofilm communities among
linkage to the terrestrial environment, which trans- glacier-fed streams – similar to patterns observed
lates into increased habitat diversity for bacteria for invertebrates ( Jacobsen et al., 2012). Col-
(McArthur et al., 1992). Further, disturbances lectively, these studies indicate that high altitude
might contribute to diversity because superior streams, in spite of apparent harsh environmental
competitors do not have the time to become dom- conditions, constitute unique habitats for stream
inant (Lake, 2000; Wang et al., 2012b). In contrast microbes with important implications for regional
to bacteria, Wang and colleagues (2011) found diversity.
that diatom richness decreased monotonically
with elevation in the same biofilms. This finding Biofilm diversity along
is surprising, given that both groups presumably environmental gradients
disperse readily through water flow (Crump et Microbial diversity and community composi-
al., 2007; Finlay, 2002), and indicates different tion vary along multiple environmental factors,
mechanisms governing their diversity. some of which are discussed in the following
In light of these contradictory observations, section. One of the driving forces behind lati-
some of the data from a study of biofilms in tudinal and elevational patterns of biodiversity
pre-alpine streams (Besemer et al., 2013) were is temperature, which influences the diversity
re-analysed with regard to elevation as a potential distribution of many organisms across a range of
force driving biofilm diversity. Elevation (ranging spatial scales (Gaston, 2000). Temperature was
from 500 to 1800 metres above sea level) showed identified as the most important factor govern-
no significant correlation with diversity when ing the diversity of microbial mats, sediments
tested alone, however, when catchment size and and soil in geothermal areas ranging from 7°C
elevation were included in multiple regression to 99°C (Sharp et al., 2014). Diversity along this
analysis, diversity (as richness, Shannon and large temperature gradient followed a unimodal
Simpson diversity) decreased significantly (P distribution with a peak at 24°C. Similarly, Stratil
< 0.05) with both increasing catchment size (that and colleagues (2013) found that the diversity
is, downstream) and with increasing elevation of epiphytic biofilms on Fucus vesiculosus was
(that is, upstream). Obviously, different mecha- highest at 15°C and decreased towards lower and
nisms are counteracting here to determine biofilm higher temperatures. At the temporal scale, the
diversity. The resulting elevational pattern of bio- influence of temperature on biodiversity receives
film biodiversity in any stream will likely depend increasing attention in the context of global
on the complex interplay of the spatial patterns climate change, often in combination with other
of environmental conditions, dispersal dynamics factors related to human-induced environmental
constrained by the fluvial landscape, and the size change. Díaz-Villanueva and colleagues (2011)
of the metacommunity seeding individual streams found that a temperature difference of 3°C had a
(Besemer et al., 2013; Wilhelm et al., 2013). significant effect on biofilm community composi-
Beyond the local scale, biofilm beta-diversity tion in young biofilms, while in mature biofilms
within and among streams has been shown to nutrients were more important; effects on biofilm
exhibit elevational patterns. Community turnover richness were not significant. This indicates that
of biofilm bacteria along an altitudinal gradient water temperature changes in the range that can
was in the same order of magnitude as for diatoms be expected for streams and rivers due to climate
76  | Besemer

change could have significant impacts on biofilm be more diverse in the coastal sea than in lakes
composition, while the taxonomic diversity might (Bižić-Ionescu et al., in press), and the microbial
be less affected. diversity of a freshwater-wetland receiving saltwa-
Another important environmental variable ter intrusions during storm events increased when
governing microbial diversity is pH, which has exposed to higher salinity ( Jackson and Vallaire,
been shown to explain much of the alpha- and 2009). The higher diversity found on marine
beta-diversity patterns of soil bacteria (Fierer aggregates could be related to the diversity of the
and Jackson, 2006). Epilithic biofilm diversity available particles (Bižić-Ionescu et al., in press),
decreased with increasing acidity in streams rang- while the observed pattern in the wetland sedi-
ing from relatively pristine to highly impacted by ments might be caused by lowered dominance of
acid mine drainage (Lear et al., 2009a), which strong competitors following the saltwater intru-
agrees with the findings from soil microbial sion ( Jackson and Vallaire, 2009).
communities (Fierer and Jackson, 2006). How- The hydrologic regime, including the fre-
ever, in another study by Lear and colleagues quency and intensity of hydrologic disturbances
(2013), biofilm diversity appeared unrelated to (Allan, 2004), influences the diversity, com-
the pH in stream water or upstream soils. In the munity structure and functioning of aquatic
context of global change, ocean acidification due biota (Marmonier et al., 2012; Romaní et al.,
to rising levels of atmospheric carbon dioxide 2013). Desiccation and flooding events promote
could impair microbial communities, especially physiological stress conditions for microbes, and
lithifying microbial mats. Intertidal benthic algal the way that biofilm microbes respond to these
biofilms exhibited decreasing algal and cyano- disturbances can modulate the ecology and bio-
bacterial richness when subjected to increasing geochemistry of the environment they inhabit
temperature and CO2 levels (Russell et al., 2007); (Romaní et al., 2013; Timoner et al., 2012).
however, exposing lithifying microbial mats to Biofilms in salt marsh sediments, as an example
elevated CO2 concentrations had no profound of an environment experiencing regular cycles of
effect on their diversity, community structure desiccation and rewetting, showed only a slight
or carbon precipitation, which might indicate reduction in biodiversity following desiccation,
that these mats are adapted to fluctuating CO2- but a more pronounced loss of diversity after
concentrations (Ahrendt et al., 2014). rewetting, indication intolerance of many dried
Salinity is a major determinant of microbial cells to sudden rewetting (McKew et al., 2011).
community composition – more important than The steady increase in salinity during desiccation
the distinction between aquatic and terrestrial favoured haloversatile taxa, which became domi-
environments – reflecting the importance of nant after rewetting, presumably because of the
osmotic adaption (Lozupone and Knight, 2007; founder effect coupled with their capacity to tol-
Tamames et al., 2010). Patterns of microbial diver- erate the sudden hypo-osmotic stress (McKew et
sity along salinity gradients, on the other hand, al., 2011). Community recovery after desiccation
are less clear. Bacterial richness and evenness in might depend on the presence of persistent humid
freshwater, intertidal and marine sediments were refuges, as was shown for a Central European and
shown to decrease with increasing salinity along a Mediterranean stream (Marxsen et al., 2010;
a river-estuary continuum (Wang et al., 2012c), Romaní et al., 2013). After rewetting of desiccated
which agrees with findings for bacterioplankton stream sediments, bacterial diversity was higher
from lakes and marine systems (Barberán and and microbial activity recovered faster in the Cen-
Casamayor, 2010). Furthermore, a meta-analysis tral European stream, suggesting better survival
of published studies found higher microbial diver- of the indigenous microbial stream community
sity in freshwater than in marine environments due to higher sediment humidity (Marxsen et al.,
(Tamames et al., 2010), potentially reflecting 2010). Timoner and colleagues (2012) showed
higher environmental heterogeneity among that autotrophic and heterotrophic microbes
freshwater systems. In contrast, suspended responded differently to desiccation and rewet-
aggregate-associated bacteria were reported to ting, indicating that the current increase in flow
Microbial Biodiversity in Natural Biofilms |  77

intermittency extent is likely to increase the might include reduced interspecific competition
relative importance of heterotrophic processes in at higher resource availability, which could sup-
stream ecosystems. port diversity (Burgos-Caraballo et al., 2014); on
The global changes in catchment land use from the other hand, some members of a community
pristine to human-dominated landscapes can might respond strongly to an increase in a limiting
affect and deteriorate the biodiversity of aquatic resource, thereby outcompeting other species and
systems through nutrient enrichment, contami- leading to a decline in species richness (Mittel-
nant pollution or hydrologic alteration (Allan, bach et al., 2001). The resulting effect of changing
2004). Numerous studies have assessed the nutrient conditions may depend on the number of
effects of human-induced alterations of aquatic limiting resources in the system, as proposed by
environments on the structure and diversity of the resource-competition theory (Interlandi and
microbial communities. In studies on epilithic Kilham, 2001).
stream biofilms in New Zealand, catchment land Runoff from urban areas and industrial release
use had a significant impact on the community increases the concentrations of heavy metals in
structure (Lear et al., 2013; Washington et al., aquatic systems, which get accumulated in bio-
2013), and Lear and colleagues (2013) found films and are then transferred to higher trophic
indications for a relationship between taxon levels (Ancion et al., 2010). Exposure to zinc,
richness and catchment properties, including copper and lead had lasting effects on commu-
land use. However, no significant effect of human nity structure and decreased diversity in biofilms
activities in the upstream landscape on biofilm grown from stream water (Ancion et al., 2010).
diversity was found in either of these studies. This Similarly, diversity of river biofilms decreased
agrees with findings from tropical streams, where when exposed to nickel, an effect, which was
prokaryotic and eukaryotic biofilm diversity alleviated by the concomitant supply of nutrients
showed no consistent patterns with respect to (organic carbon, nitrogen and phosphorus) at
land use (Burgos-Caraballo et al., 2014). In this high oxygen levels (Lawrence et al., 2004). Law-
study, nitrate concentration was the best single rence and colleagues (2004) further observed
predictor of bacterial diversity. a reduction of catabolic functions by nickel,
Increased inputs of nutrients accompanying reflecting a loss of a functional group or a loss of
intensified agricultural land use, riparian defor- diversity of functional groups.
estation and urbanization (Allan, 2004) can
have significant effects on biofilm community
composition; however, the responses of biofilm How many microbial taxa are
diversity vary markedly between studies. In there in biofilms?
the study by Burgos-Caraballo and colleagues Due to the enormous microbial diversity in
(2014), bacterial diversity increased with nitrate most environments, it is often impossible to get
concentrations. In contrast, epilithic stream an exhaustive census of the microbial diversity
biofilm diversity was lower in a highly impacted, contained in an environment (Caporaso et al.,
nutrient rich urban stream than in more pristine 2011; Logue et al., 2012; Sogin et al., 2006). To
streams (Lear et al., 2009c) and bacterial diversity get an idea of the true richness of a community it
in wetland sediments decreased when exposed to is therefore necessary to estimate the number of
increased nitrogen levels ( Jackson and Vallaire, unobserved – additionally to the observed – spe-
2009). Lyautey and colleagues (2003) found cies. A number of statistical techniques, including
similar bacterial richness in epilithic river biofilms parametric and nonparametric, frequentist and
upstream and downstream of a large urban centre, Bayesian methods have been developed and used
which increased the nitrogen and phosphorus load to assess the richness of microbial communities
of the river, while the highest diversity of marine (Bunge et al., 2014). However, it remains difficult
sediment bacteria was found at intermediate levels to compare the richness estimates from different
of nitrogen pollution (Xiong et al., 2014). The studies because of the different laboratory and
mechanisms behind these contradictory patterns statistical methods used. As one example, some
78  | Besemer

richness estimates for biofilms are compared here, Functional diversity in biofilms
which have been made using 454-pyrosequenc- The relationship between taxonomic diversity,
ing of the 16S rRNA and its gene. The selected functional diversity and ecosystem processes is
studies have the advantage of having used the a central issue of ecology (Gamfeldt et al., 2013;
same parametric method, the Diversity Estima- Loreau et al., 2005) and of uttermost importance
tion software of Quince and colleagues (2008) in the light of global biodiversity loss (Loreau et
based on a 97% sequence similarity cutoff to al., 2001; Sala and Knowlton, 2006; Worm et al.,
distinguish operational taxonomic units (OTUs). 2006). While it is widely accepted that micro-
The studies differ, however, in the primers used bial diversity exerts influence over ecosystem
for PCR [Hall and colleagues (2012) and Wil- functioning, the shape of this relationship and
helm and colleagues (2013, 2014) used the same the underlying causes remain debated (Mokany
primers, Besemer and colleagues (2012) and et al., 2008; Nielsen et al., 2011). Diversity can
Bengtsson and colleagues (2012) used different enhance functioning through niche differentia-
primers each], so the numbers presented here tion or facilitation, such that the performance of
can only give a first rough idea of the true rich- the community increases above the level expected
ness contained in microbial biofilms. Wilhelm by the performance of the individual contributing
and colleagues (2013, 2014) estimated that the species, often subsumed under the term comple-
microbial richness of epilithic and epipsammic mentarity effect; and through stochastic processes
biofilms in glacial streams ranged from 200 to involved in community assembly, such that more
2500 OTUs and from 500 to 2000, respectively. diverse communities have a higher probability
This is similar to the richness found in hyporheic of containing highly productive species, usually
biofilms in boreal streams (1000–1300 OTUs; referred to as sampling or selection effects (Car-
Besemer et al., 2012) and in experimental dinale et al., 2006b; Loreau et al., 2001). The close
hyporheic biofilms (600–2300 OTUs; Hall et proximity of microbial cells and the architectural
al., 2012). The estimated richness of marine and compositional differentiation of biofilms
epiphytic biofilms on kelp was lower, ranging may foster functional complementarity (Besemer
from <100 to 600 (Bengtsson et al., 2012), and et al., 2009b; Singer et al., 2010, 2011). Indeed,
Bengtsson and colleagues suggested that these indications for complementarity have been found
kelp biofilms can be regarded as low bacterial for epilithic biofilms using experimental stream
diversity habitats. However, Bengtsson and col- mesocosms differing in spatial flow heterogeneity
leagues fitted the OTU’s abundance distributions (Singer et al., 2010). Increasing flow heterogeneity
to the Inverse-Gaussian distribution, while the resulted in higher beta-diversity between hydro-
above-mentioned studies applied the Sichel dynamic microhabitats along the mesocosms and
distribution (Quince et al., 2008), which makes in a broader range of dissolved organic carbon
the results less comparable. Two of these studies compounds removed from the stream water.
also assessed the richness of the active biofilm These results suggested that biofilm communities
community, which was, on average, lower than differentiated into functionally non-redundant
that of the bulk community (700–900 OTUs in local communities in habitat patches character-
boreal hyporheic biofilms; Besemer et al., 2012; ized by different hydrology, whose diversified
< 100–2200 OTUs and 400–1400 in epilithic metabolic capabilities then induced complemen-
and epipsammic biofilms, respectively, in glacial tarity at the regional scale (Singer et al., 2010).
streams; Wilhelm et al., 2014). For comparison, Further, niche complementarity among species
the estimated true richness of the stream water was found to enhance biomass and nitrogen
microbial community was 6100–6500 and uptake of algal biofilms grown in heterogene-
400–6100 OTUs in boreal and glacial streams, ous flow environments (Cardinale, 2011). This
respectively, and lower (2800–3900 OTUs) in study provided evidence that communities with
the active than in the bulk community in boreal more species were able to take greater advantage
streams (Besemer et al., 2012; Wilhelm et al., of the niche opportunities provided by the envi-
2013). ronment, which allowed the system to capture
Microbial Biodiversity in Natural Biofilms |  79

a greater proportion of the available resources. number of similarly performing species (Nielsen
When, on the other hand, these niche opportu- et al., 2011), specific functions could be prone to
nities were experimentally removed by making be lost as result of decreasing microbial diversity.
the flow environment homogenous, increased Indeed, Peter and colleagues (2011) found that
biomass in more diverse communities resulted the likelihood of sustaining multi-functionality,
from a species-specific selection effect (Cardinale, measured as the activity of a range of extracellular
2011). This agrees with earlier findings showing enzymes, decreased with decreasing diversity in
that the relative importance of complementarity freshwater biofilms, indicating a limited level of
and selection effects can change with the degree of functional redundancy. This pattern was more
spatial and temporal environmental heterogeneity pronounced when biofilms were fed only recal-
(Cardinale et al., 2000). citrant organic matter than when a labile carbon
Changes of the underlying mechanisms with source was added (Peter et al., 2011).
environmental context suggest that there may be no Functional diversity of a community may
single, generalizable relationship between species often be a more suitable predictor of ecosystem
diversity and ecosystem functioning (Cardinale et processes than is taxonomic diversity (Lecerf
al., 2000). Strong selection effects could result in and Richardson, 2010; Tilman et al., 1997). For
an idiosyncratic relationship between community epiphytic biofilms, Burke and colleagues (2011)
diversity and function, where community compo- found that a core of functional genes associated
sition and functional identity are more important with a surface-attached mode of life were repre-
than species diversity per se; further, functional sented in a variety of microbial taxa, indicating the
redundancy within a community can lead to a presence of functional guilds formed by function-
diversity–function relationship which saturates ally redundant taxa. Their findings suggest that the
at low levels of diversity (Nielsen et al., 2011). specific species composition in a biofilm is driven
Especially for microbes, assumed high levels of by stochastic colonization of members of these
redundancy and metabolic plasticity have been guilds from the source community, which would
suggested to preclude a clear relationship between result in a weak coupling between taxonomy
microbial diversity and universal functions, such and function. Therefore, although taxonomic
as productivity or respiration (Langenheder et al., and functional diversity appear to be correlated
2000; Nielsen et al., 2011). For instance, Gobet at some point at least (Fierer et al., 2012), this
and colleagues (2012) found that taxonomic relationship can be modified by the degree of func-
diversity varied more than microbial functions tional redundancy in the community (Hooper et
in coastal sand biofilms, suggesting that a limited al., 2005; Nielsen et al., 2011).
number of continuously abundant, resident taxa Biofilm functional diversity, as taxonomic
performed most of the functions and/or a certain diversity, has been shown to respond to changing
level of functional redundancy among taxa. Still, environmental conditions, ultimately affecting
microbial abundance and extra-cellular enzymatic the biogeochemical cycling of organic matter and
activity were related to major changes in commu- nutrients in aquatic systems (Romaní et al., 2014;
nity composition in this study (Gobet et al., 2012). Ylla et al., 2014). Warming of stream water by 3°C
In contrast, a significant positive relationship increased functional diversity in stream biofilms,
between bacterial evenness and production was mainly due to a wider use of carbohydrates and
found in epiphytic kelp biofilms (Bengtsson et al., polymers (Ylla et al., 2014). In contrast, experi-
2012), which in this system indicates increasing mental stream biofilms showed a decrease in
productivity with decreasing dominance of a few functional diversity under elevated (2°C) water
Planctomycetes-associated OTUs. These results temperature towards a specialized use of a few
suggest that a few slow-growing Planctomycetes carbohydrates when grown under light, while
dominate on living kelp surfaces while a more dark-grown biofilms showed a slight increase
even bacterial community may be necessary for in functional diversity at higher temperature
the degradation of fragmented kelp. While uni- (Romaní et al., 2014). Furthermore, young bio-
versal functions may often be buffered by a high films appeared to be less specialized in the use
80  | Besemer

of organic carbon sources than mature biofilms, in stream water and biofilm from an urban river
determined by fluorescent in situ hybridization and
indicating that the capacity to use a wide range DGGE analysis. FEMS Microbiol. Ecol. 43, 111–119.
of organic compounds might be advantageous Arnon, S., Packman, A.I., Peterson, C.G., and Gray, K.A.
for pioneering species (Romaní et al., 2014). The (2007). Effects of overlying velocity on periphyton
structural and functional diversity of biofilms, structure and denitrification. J. Geophys. Res. 112,
reflecting the spatial and temporal heterogene- Artigas, J., Fund, K., Kirchen, S., Morin, S., Obst, U.,
ity of their environment, could thus have broad Romaní, A.M., Sabater, S., and Schwartz, T. (2012).
consequences for globally relevant carbon fluxes Patterns of biofilm formation in two streams from
(Singer et al., 2010). This might be especially true different bioclimatic regions: analysis of microbial
community structure and metabolism. Hydrobiologia
in systems in which biofilms dominate microbial 695, 83–96.
life, such as stream networks (Battin et al., 2008). Auguet, J.-C., Barberan, A., and Casamayor, E.O. (2010).
Global loss, deterioration and homogenization of Global ecological patterns in uncultured Archaea.
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Azam, F., and Malfatti, F. (2007). Microbial structuring of
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diversity and ecosystem processes of microbial Barberán, A., and Casamayor, E.O. (2010). Global
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Aquat. Microb. Ecol. 59, 1–10.
Acknowledgements Bartrons, M., Camarero, L., and Catalan, J. (2010).
I thank Tom J. Battin, Brigitte Rosenwirth, Jürgen Isotopic composition of dissolved inorganic nitrogen
Besemer and Michael Forisch for their helpful in high mountain lakes: variation with altitude in the
comments on this manuscript. Katharina Besemer Pyrenees. Biogeosciences 7, 1469–1479.
Bartrons, M., Catalan, J., and Casamayor, E.O. (2012). High
is supported by an Erwin Schrödinger fellowship bacterial diversity in epilithic biofilms of oligotrophic
(Austrian Science Fund: J3542–B22). mountain lakes. Microb. Ecol. 64, 860–869.
Battin, T.J. (2000). Hydrodynamics is a major determinant
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Aquatic Biofilms and Biogeochemical
Laura Leff, Jonathon B. Van Gray, Eugènia Martí, Stephanie N. Merbt
and Anna M. Romaní

Abstract conditions of sites were they develop, including

In this chapter, we discuss the biogeochemistry of the nature of the substrate to which the biofilm
biofilms with an emphasis on the unique features is attached. Ultimately, the characteristics of bio-
of biofilms that impact oxidation-reduction reac- films and their biogeochemical reactivity have
tions, such as retention of materials (nutrients, implications for ecosystem-scale processes, such
organic molecules, including enzymes), short- as nutrient uptake, in aquatic ecosystems.
ened uptake lengths, intercellular interactions As spatiotemporal refugia from the dynamic
due to proximity of the organisms, and redox flow of the bulk fluid, biofilms provide a unique
gradients over short spatial distances. Our focus immobilized microenvironment in which the
is on carbon, nitrogen and phosphorus with biological, chemical, and physical properties of
some inclusion of micronutrients. The role of the biofilm matrix facilitate abiotic–biotic interac-
biofilm structure and microbial community com- tions necessary to drive biogeochemical reactions.
position (including algal–bacterial relationships) This respite from the transience of the bulk fluid
in determining the spatiotemporal variations permits the aggregate microbial populations to
in biogeochemistry is discussed as well as the persist and diversify in close proximity (Hansen et
spatial scales of biogeochemical reactions within al., 2007; Poltak and Cooper, 2010), allowing for
biofilms. Methods for measuring biogeochemical greater exchange of genetic materials and metabo-
processes and environmental conditions in bio- lites within and between populations (Battin et al.,
films are reviewed, from molecular tools and fine 2003). These interactions result in a high uptake
scale measurements, to large scale biogeochemi- efficiency and increased retention time for biore-
cal measurements in flumes and mesocosms or active solutes (i.e. nutrients, organic compounds,
natural systems. The relevance of the spatial scale and extracellular enzymes) within the biofilm and
is highlighted as well as the challenge for scaling- provide a dynamic resource pool for internal use
up of methods. by those organisms within the sphere of the dif-
fusive capabilities of the associated matrix (Battin
et al., 2003; Flemming and Wingender, 2010;
Introduction Stewart, 2003).
Biofilms are ubiquitous when surfaces are exposed The extracellular polymeric substances (EPS)
to water and microorganisms, and have unique matrix serves to capture nutrients from water,
physical and chemical properties (e.g. reviewed retain extracellular enzymes, and provide protec-
by Costerton et al., 1987; see Chapter 1). The tion from disturbance and predation (Sutherland,
close physical spacing of cells and properties of 2001). Biofilms accumulate and concentrate
the extracellular matrix provide opportunities for nutrients (Lock et al., 1984) which are avail-
significant biogeochemical interactions allowing able for assimilatory or dissimilatory processes.
biofilms to serve as ‘hotspots’ of biogeochemical The three dimensional structure of the biofilm
cycling. These properties change over time as the includes passageways (channels and pores) by
biofilm develops and vary with environmental which nutrients can be transported (DeBeer and
90  | Leff et al.

Schramm, 1999). In addition, biofilms may alter In this chapter, we discuss the biogeochemis-
adsorption. Collectively, these features allow bio- try of aquatic biofilms with an emphasis on the
films to play a major role in ecosystem function. unique features of biofilms that impact oxidation-
The importance of this growth form has led to reduction reactions and retention of materials.
a large number of studies in freshwater ecosys- We focus on carbon, nitrogen and phosphorus
tems, such as those focused on production of with some inclusion of other elements (e.g. S,
extracellular enzymes and nitrogen cycling (e.g. Fe, etc.). The role of both extrinsic (environmen-
Romaní et al., 2013; von Shiller et al., 2009). The tal conditions, inorganic and organic nutrient
nature of the role of biofilms and the extent of availability), and intrinsic (biofilm structure)
our knowledge varies among ecosystems; argu- factors in determining the spatiotemporal vari-
ably there is more known about biofilm function ations in biogeochemistry and the spatial scales
in streams and rivers than in lakes or wetlands. of biogeochemical reactions within biofilms is
This is likely attributable to the more widely discussed. Methods for measuring biogeochemi-
acknowledged and studied role that benthos play cal processes and environmental conditions in
in streams. biofilms are introduced, such as use of molecular
One factor that contributes to differences in tools, assessment of microbial function linked to
biofilms among aquatic ecosystems is the nature biogeochemical processes (extracellular enzymes,
of the benthic substrate (see Chapter 1). Sub- respiration), fine scale measurements (micro-
strates for biofilm development can be generally electrodes, confocal microscopy) and the use of
considered organic (such as leaves or wood) or larger-scale methods (flumes, mesocosms, use of
inorganic (such as rocks). The surface properties stable isotopes).
of the substrate, such as charge, roughness, etc.,
influence the biofilm (Palmer et al., 2007) as
does leaching of materials from the underlying Biofilm biogeochemical
substrate. Research on biogeochemistry has been environment
received special attention for biofilms on rocks While the biofilm environment greatly enhances
and sand, as well as on leaves. the efficacy of the biogeochemical processes
Physical proximity of cells in biofilms provides occurring therein (Battin et al., 2003; Poltak and
the opportunity for inter-cellular communication, Cooper, 2011; Burmølle et al., 2014), capabilities
sharing of resources, modification of the physical of individual biofilms vary based on the specific
environment that enhances adhesion, and altera- extrinsic and intrinsic factors as they affect the
tion of redox conditions at a small spatial scale. biological, chemical, and physical components
For example, Reidel et al. (2001) discovered of the biofilm matrix. Extrinsic factors can be
that cross-species intercellular communication generalized as the external physico-chemical
occurred in mixed species biofilms. Studies such drivers affecting biofilm formation and stability
as this one, as well as those on co-aggregation, (Lyautey et al., 2005), while intrinsic properties
have often focused on interactions among organ- can be broadly characterized as factors associated
isms in laboratory communities and many of these with the internal heterogeneity of the microbial
phenomena have not been investigated in aquatic composition of the biofilm – each of which will be
ecosystems and are more widely known from oral explored in greater detail in the following sections
biofilms (Kolenbrander, 2000). However, these (Fig. 5.1).
types of interactions are potentially important in
biogeochemistry of aquatic biofilms. Unlike some Extrinsic biofilm properties affecting
other aspects of biofilms, this physical positioning biogeochemical processes
and structure is inherently connected to com- The ubiquity of biofilms within aquatic systems
munity composition, particularly in light of the suggests that biofilms form regardless of environ-
specific inter-species interactions that have been mental conditions as long as adequate resources
demonstrated in laboratory studies ( James et al., are provided (e.g. nutrients and space to colonize).
1995). Yet extrinsic physical and chemical drivers play a
Biofilms and Biogeochemistry |  91



•Nutrient availability:
organic/inorganic WATER
stoichiometry •Succession stage
quantity/quality •EPS matrix
•Temperature •Thickness
•Light •Community composition
BIOFILM •Interactions:
•Type: prokaryote/eukaryote
organic/inorganic SUBSTRATUM

Biofilm Biogeochemical Potential

C, N, P cycling

Figure 5.1 Diagram of the extrinsic and intrinsic factors affecting biofilm biogeochemical processes, and
finally determining the biofilm potential for nutrient cycling. Extrinsic factors mainly include those related to
physical and chemical drivers, while intrinsic ones are those derived from biofilm structure and composition.

significant role in affecting biofilm biogeochemical resource pool for microbial organisms in biofilms
processes, as the course of biofilm development (Whitehead and Verran, 2008). Comparisons
and the subsequent functional capabilities of the among substrata demonstrate that surface prop-
community are contingent upon these factors. For erties impact rates of biofilm formation, density,
our purposes here, we will focus on abiotic factors diversity, and resource retention (Golladay and
that affect biofilm biogeochemistry, including the Sinsabaugh, 1991; Sinsabaugh et al., 1991; Bar-
substrata type, hydrological variability, tempera- biero, 2000; Khatoon et al., 2007; Bergey et al.,
ture, and nutrient availability. 2010). Physical characteristics of the substrata are
The effects of substrata on biofilm biogeo- significant to biofilm biogeochemistry (White-
chemistry are closely linked to the nature of the head and Verran, 2008). Substratum stability and
substrata (e.g. organic versus inorganic) and topography have been shown to positively affect
the physical and chemical characteristics of benthic algae colonization, which act as a major
substrata surfaces. Organic (such as leaves and source of endogenous oxygen production and
woody debris) and inorganic (such as rocks or carbon fixation (see Burkholder, 1996). Similarly,
sand grains, clay tiles and glass slides or beads) in a field study comparing the effects of nutrient
substrata differ in their reactivity with the biofilm supply on biofilms forming on rock or sand sub-
(Whitehead and Verran, 2009). Organic surfaces strata, Romani et al. (2004a) identified substratum
provide a pool of supplemental resources (such as stability and roughness as key determinants of
reduced carbon, organic nitrogen, etc.) for hetero- biofilm structure and heterotrophic metabolism.
trophic use and may promote higher colonization In a larger scale, in a comparison of physical heter-
rates of aquatic fungi (Golladay and Sinsabaugh, ogeneity of stream substrate on biological activity,
1991), thereby increasing resource availability Cardinale et al. (2002) found significant increases
via substrate degradation. Inorganic substrata in algal productivity and biofilm respiration as a
tend to be less reactive or completely inert as a function of greater substrate heterogeneity.
92  | Leff et al.

By their very nature, aquatic biofilms are reduced as structural stability with the biofilm is
subjected to stresses associated with the flow of increased due to algal interactions.
the bulk fluid. Examination of physical response Temperature plays a significant role in mediat-
to flow velocities in a mixed bioreactor commu- ing biogeochemical activity in aquatic habitats due
nity (Beyenal and Lewandowski, 2002) found to the inherent ties with the metabolic activity,
that the internal structure of the biofilm matrix chemical kinetics, and compound stability. Due
is the result of an intricate balance between two to taxon-specific tolerances, temperature often
contrasting properties: the preservation of struc- dictates what organisms can persist in the environ-
tural integrity through increased density so as to ment and contribute to biofilm formation (Goller
withstand shear stress and increasing diffusivity and Romeo, 2008; Hostacka et al., 2010) and the
of nutrients deeper into the biofilm matrix (i.e. biogeochemical potential of the resulting commu-
reduced density). For instance, increased flow nity. Increased temperatures have been linked to
velocity has been implicated in reducing bio- increases in algal growth rates (Butterwick et al.,
film density and spatial heterogeneity (Battin 2005), bacterial productivity (Fischer et al., 2002;
et al., 2003b; Coundoul et al., 2014), while Adams et al., 2010), resource diffusivity (Bissett
simultaneously increasing the mass transfer of et al., 2008), enzymatic activity (Bouletreau et al.,
nutrients from the bulk fluid into the biofilm 2012; Ylla et al., 2014), as well as to modifications
matrix (Donlan, 2002; Taherzadeh et al., 2012). in community composition (Rubin and Leff,
In the same study, Battin et al. (2003b) found bio- 2007) and enzyme capabilities (Ylla et al., 2012).
films developed under slow velocity conditions to Additionally, changes in temperature can alter the
accrue more biomass, but were also more reliant overall metabolic state of the biofilm, as increased
on internal resource cycling than those developed temperatures have been shown to shift biofilms
under elevated velocities. While these results towards heterotrophy (Hancke and Glud, 2004;
provide great insight into the potential effects of Degerman et al., 2013).
flow on the biogeochemistry of biofilms, they are Aquatic resource dynamics have an immeas-
based on manipulative studies that compare con- urable effect on biofilm activity. In aquatic
trasting scenarios of low versus high flows rather systems the bulk fluid represents the largest pool
than the heterogeneity found in natural systems, of dissolved nutrients available to the biofilm
failing to capture details into the effects of flow community, especially to those developing on
heterogeneity on community development. In inorganic substrata. Consisting of a diverse mix-
a pair of studies, Besemer et al. examined the ture of allochthonous and autochthonous C, N,
effects of flow velocity and heterogeneity (lami- and P compounds, the bulk fluid resource pool
nar, transitional, or turbulent flows) on bacterial is highly heterogeneous, and as such, specific
community composition (Besemer et al., 2009) resource availability varies based on the concen-
and biofilm community succession (Besemer trations of resources in the bulk fluid and the
et al., 2007; see also Chapter 4). These studies ability of the biofilm community to satisfy their
found increasing bacterial diversity with flow het- stoichiometric requirements through resource
erogeneity (Besemer et al., 2009), as well as that assimilation. Thus, the bulk fluid resource pool
flow velocity and heterogeneity influenced early can effectively contribute to resource limitation
microbial successional trajectories. However, or saturation within the biofilm matrix, thereby
community composition converged upon biofilm altering the structural and functional dynamics
maturation as algal populations became domi- of the community (Tank and Dodds, 2003; Hoe-
nant and were believed to exert greater control llein et al., 2011). For instance, as described in a
over the internal biofilm environment (Besemer subsequent section, a number of studies found
et al., 2007). Taken together, these results sug- changes in the aqueous dissolved organic carbon
gest that during early succession, flow plays a (DOC) pool to significantly affect biofilm com-
significant role in determining the phylogenetic munity composition (Olapade and Leff, 2006), P
composition of the biofilm community and that, cycling (Ardón and Pringle, 2007) and N dynam-
as maturation continues, the effects of flow are ics ( Johnson et al., 2012). Studies looking solely
Biofilms and Biogeochemistry |  93

into the effects of nutrient (e.g. N or P) additions heterogeneous biofilm architecture that includes
saw similar results; increased fungal and bacterial numerous interstitial voids as well as particu-
activity with N and P additions (Gulis et al., 2008), lates captured from the bulk fluid. Successional
organic and inorganic N treatments resulted in stratification of microbial cells and populations
increased hyporheic biofilm nitrification (Find- results in a high degree of spatial heterogeneity at
lay and Sinsabaugh, 2003), N and P enrichment the micro-scale, with community depth varying
stimulated algal biomass accumulation (Artigas based on cell density, ranging from 1–2 microns
et al., 2013), and so forth. These results highlight in monolayered communities, to several millime-
not only the complexity of the effects of aquatic tres in low-disturbance, highly stratified biofilms
resources on biofilm biogeochemistry, but also (Wimpenny et al., 2000; Battin et al., 2003b).
the depth of potential alterations that can occur as Concurrently, as biofilm biomass and physical
the result of modifications to the aquatic resource complexity increase, the chemical concentrations
pool. of endogenous and exogenous solutes are affected
Obviously light availability determines as alterations in resource production/consump-
development of photoautotrophic community tion and diffusion rates lead to the development
and many adaptations to either high/low light of extensive physico-chemical micro-gradients
intensity have been described for algae and (Stewart, 2003; Stewart and Franklin, 2008;
cyanobacteria and thus influencing community Renslow et al., 2010). Stewart and Franklin (2008)
composition (e.g. Corcoll et al., 2012). For the identified three distinct gradient patterns that
purpose of this chapter, however, our discussion develop as a function of metabolite concentration:
of the effect of light is mainly focused on the decreasing metabolic substrate concentrations
consequence of photosynthetic organisms in the with biofilm depth, metabolic products increase
biofilm for biogeochemical processes as well as with biofilm depth, and metabolic intermediar-
their significance for autotrophic–heterotrophic ies originating within the biofilm will diffuse to
relationships, as described below. areas of lower concentrations, regardless of depth.
For instance, in many aquatic biofilms oxygen
Intrinsic biofilm properties affecting gradients are formed as a function of autotrophy
biogeochemical processes occurring within the surface layers of the biofilm.
The biogeochemical potential of the biofilm matrix Autotrophic oxygenation near the surface of the
is inextricably linked to its intrinsic properties. biofilm results in the formation of oxygen gradi-
Autogenic changes associated with community ents, which can extend several hundred microns
succession and biofilm maturation result in com- into the biofilm matrix, resulting in distinct verti-
positional shifts in microbial populations and with cal oxygen profiles in which O2 concentrations
them, the metabolic diversity and biogeochemical become increasingly depleted with depth as aero-
potential of the biofilm environment ( Jackson bic respiration gives way to anaerobic metabolic
et al., 2001; Lyautey et al., 2005; Burmølle et al., processes (Stewart, 2008). Similar gradients have
2014). During the early stages of succession, been identified in aquatic biofilms for a host of
the biogeochemical environment of the biofilm compounds including nitrite, nitrate, ammonium,
is constrained by a lack of physical complexity, sulfate, methane, and pH (Stewart, 2008).
as early colonizers in most aquatic systems are In addition to the structural and physical
likely dominated by metabolically generalist taxa and chemical changes, maturation affects the
reliant on resources present within the overlying functional capabilities of the biofilm as niche
bulk fluid (Sobczak, 1996; Romaní et al., 2014a). differentiation and the localized set of symbiotic
Biofilm maturation occurs as competitively adept interactions and communications necessary
populations expand and additional planktonic to stimulate increased biogeochemical activ-
taxa are integrated into the matrix via coadhe- ity develop (Hansen et al., 2007; Wintermute
sion/coaggregation mechanisms (Rickard et al., and Silver, 2010). Cross feeding and syntrophic
2003), forming a stratified biological architecture. interactions, the use of one organism’s meta-
EPS excretion by active cells creates a spatially bolic intermediates and by-products by another
94  | Leff et al.

organism as a metabolic substrate, serve to interactions of the autotrophic and heterotrophic

increase internal nutrient cycling, reduce energy populations within the biofilm may be highly
expenditures on resource acquisition from water influential on internal biogeochemical cycling,
column, and can create favourable environmental external modifications to the system can ulti-
conditions for novel niche development (Pfeiffer mately disrupt these processes.
and Bonheoffer, 2004; Okabe et al., 2005; Bull and
Harcombe, 2009; Wintermute and Silver, 2010;
Pande et al., 2013). Quorum sensing, a mecha- Biogeochemical cycling
nism for intercellular communication within and In this section, we highlight selected biogeo-
between prokaryotic and eukaryotic populations, chemical cycles (C, N and P cycling) in light of
can promote synergistic or antagonistic interac- the context of extrinsic and intrinsic properties
tions that affect the structural and functional that influence biofilm function discussed above.
attributes of the community (Valle et al., 2004; Thus, for each element discussed consideration
Irie and Parsek, 2008; Atkinson and Williams, is given to factors that influence biogeochemi-
2009; see Chapter 3). cal spatiotemporal changes. Although the three
Often-overlooked, the interactions between main elements are considered separately, there
the heterotrophic and photoautotrophic compo- are many connections among them since bio-
nents of the biofilm community are an integral geochemical processes. As shown in many of the
part of biofilm biogeochemistry. During early examples included below for C, N and P cycling,
stages of biofilm formation, the biofilm com- in many occasions their interactions and stoichi-
munity is predominantly heterotrophic in nature ometry matters and thus can be relevant for the
and reliant on the resources available in the bulk understanding of a single nutrient cycling. As an
fluid ( Jackson, 2003). As the biofilm matures and example, using a whole stream N and C addition,
autotrophic taxa are integrated, the heterotrophic Oviedo-Vargas et al. (2013) found that P cycling
community shifts to one dominated by autotro- appeared to be N limited and that the form of N
phy, with the heterotrophic community becoming (ammonia versus nitrate) was critical. Moreover
increasingly reliant on the labile carbon provided they conclude that C and P cycling are biologi-
by the photoautotrophic populations (Sobczak, cally coupled and that this coupling is mediated
1996; Jackson, 2003). This metabolic shift greatly by N form.
enhances the internalized cycling of resources
within the biofilm as the complementary aspects Carbon cycling
of heterotrophic and autotrophic metabolism Here we emphasize the forms and biogeochemi-
converge. For instance, Rier et al. (2007) found cal fate of organic matter (OM) with particular
evidence to suggest that photosynthesis-mediated emphasis on differences among broad categories
alterations to the physico-chemical state of the of OM: dissolved versus particulate, labile versus
biofilm may enhance heterotrophic extracellular recalcitrant, allochthonous versus autochthonous.
enzyme activity by altering biofilm pH and redox Because the OM pool is complex and undefined
gradients. Similarly, Romani and Sabater (2000) reliance on these coarse categories has been
saw increased enzyme activity in autotrophic common (Münster and Chróst, 1990), although
biofilms and attributed this to the algal exudate over time there have been advances in our ability
and metabolite availability to heterotrophic taxa. to chemically characterize the OM pool.
Conversely, heterotrophic taxa also affect the Coarse particulate organic matter (CPOM)
autotrophic populations. Under nutrient-poor plays host to a well-developed biofilm community
conditions, algal populations are often dependent and decomposition of plant litter has been widely
upon endogenous bacterial metabolites to sup- studied in aquatic ecosystems (e.g. Boyero et al.,
port productivity (Scott et al., 2008). However, 2011). However, only a portion of the studies
in the same study, Scott et al. (2008) found that on leaf litter decomposition include an explicitly
increased nutrient loading may de-couple bacte- stated focus on biofilms (e.g. Artigas et al., 2011).
rial–algal interactions, suggesting that while the Often in these studies, the biofilm per se is not the
Biofilms and Biogeochemistry |  95

focus, but rather the researchers measure some Jones and Lock, 1989; Tank and Webster, 1998;
aspect of microbial function which is presumed Rier et al., 2007). These factors contribute to the
to be attributed to biofilm dwellers. Among them, spatial and temporal variation of the extracellular
those that examine extracellular enzymes are enzyme activities, together with the seasonality of
the most common while relatively fewer studies litter inputs to the aquatic systems.
examined both microbial community structure Impacts of dissolved organic matter (DOM)
and function. Given that in this particular section on biofilms have been examined in the laboratory
our focus is on biogeochemical cycling, generali- and in the field; the latter has been accomplished
zations about function based on these studies are via whole stream additions and nutrient diffus-
presented here and microbial community com- ing artificial substrates among other approaches.
position is not extensively discussed. In addition, Collectively, this range of approaches provides
extracellular enzymes for N and P acquisition are insight into biofilm biogeochemistry with each
described subsequently but, of course, although approach having inherent pluses and minuses.
we discuss each element separately their bio- Regardless of the approach, there is an underly-
geochemistry is very much intertwined. In fact, ing pattern of spatiotemporal change linked to
Sinsabaugh et al. (2010) demonstrated that the variability in both extrinsic and intrinsic factors.
stoichiometric ratio of extracellular enzymes of For example, in Mediterranean streams, biofilms
biofilms tracked the C:N or C:P relative to plank- change in OM use as seasonally OM sources
tonic communities; and thus, they conclude that transition from allochthonous to autochthonous
biofilm and plankton communities have similar (Romaní et al., 2013). In these systems, seasonal
ecoenzymatic stoichiometric relationships with drying reduces bacterial production and chlo-
underlying microbiological community param- rophyll biomass, but the rapid recovery of both
eters. autotrophs and heterotrophs upon rewetting
Extracellular enzymes for degradation of plant indicates their responsiveness to such seasonal
polymers to monomers are among the most well changes. In the laboratory, biofilms on various
studied aspects of aquatic biofilm biogeochem- surfaces including glass (slides or beads), ceramic
istry regardless of habitat and are widely studied tiles, leaf disks, etc. have been exposed to various
in a range of ecosystems (Arnosti et al., 2014). DOM treatments. Response variables include
Leaves are the most commonly examined with those related to abundance or biomass, function
fewer studies on wood; and the most widely (such as enzyme activity or productivity), and
assayed enzymes include beta-glucosidase, community structure. For example, Ghosh and
N-acetylglucosaminidase, beta-xylosidase, cel- Leff (2013), by using glass beads as the substrate,
lobiohydrolase and phenol oxidase (e.g. Artigas found that OM treatments induced changes in
et al., 2011; Frossard et al., 2013). Enzymes are bacterial community composition and rates of N
produced to varying extents by both fungi and utilization. Ylla et al. (2009) used glass tiles and
bacteria; commonly, fungal biomass is thought to found that both glucose concentrations and light
peak after bacterial biomass on leaves decompos- levels impacted biofilms, including altering beta-
ing in streams. Also typically fungi are presumed glucosidase and peptidase activity. In contrast,
to contribute more to the degradation of lignocel- Freeman et al. (1990) found that high molecular
lulose and some bacteria in biofilms may be solely weight DOM had an inhibitory effect on extracel-
relying on monomers produced by the degrada- lular enzyme activity. Olapade and Leff (2005),
tion efforts of other microorganisms, and thus who used nutrient diffusing artificial substrates to
significant synergistic and antagonistic interac- look at DOM and nutrient impacts on biofilms,
tions between fungi and bacteria modulate plant found that the greatest impacts on biofilm were
litter decomposition (Romaní et al., 2006). Fac- attributable to labile, low-molecular-weight DOM
tors that appear to control extracellular enzyme amendments at times of year when chlorophyll a
in leaf biofilms include concentrations of metals concentrations were low. These four example stud-
(such as Al), nutrient availability, algal biomass, ies highlight the relevance of both DOM quantity
and physical conditions, such as temperature (e.g. and quality on determining changes in the biofilm
96  | Leff et al.

metabolism. In addition, it has been further shown transfer into biofilms (Pastor et al., 2013). The use
that responses to specific properties of DOM vary of 15N tracer additions in streams over the past
among bacterial taxa (McNamara and Leff, 2004). decade has allowed quantifying in situ rates of
At a larger scale, Singer et al. (2010), using stream assimilatory N uptake rates by biofilms developed
flumes, demonstrated that heterogeneity in water on either inorganic and organic substrata. Overall,
flow altered DOC uptake and that such responses results from these studies highlight the active role
were likely linked to community composition. of biofilms in N uptake and their contribution
In addition, significant DOC, especially biode- to in-stream N cycling (Mulholland et al., 2000;
gradable DOC, uptake have been measured for Tank et al., 2000; Dodds et al., 2000; Hamilton
light and dark grown biofilms in a fluvial system et al., 2001; Merriam et al., 2002; Ashkenas et al.,
(Romaní et al., 2004b). 2004; Simon et al., 2005; von Schiller et al., 2009;
Biofilms provide an opportunity for direct Sobota et al., 2012). In addition, the close interac-
interaction between bacteria and algae, which can tion among organisms and the biogeochemical
influence the dynamics of both organic and inor- gradients within biofilms facilitate the coupling
ganic carbon. These interactions are perhaps most between dissimilatory oxidation and reduction
pronounced in microbial mats, especially those reactions, such as nitrification and denitrification.
between heterotrophic bacteria and cyanobac- Lastly, depending on environmental conditions,
teria in which production of biological available nitrogen fixation may be also important (Grimm
DOM excreted by the photoautotrophs can be and Petrone, 1997).
used by heterotrophic bacteria (Marshall, 1989). Biofilms, especially epilithic ones, tend to rely
Among the approaches used to examine bacterial- on nitrogen from the water column. Nutrient
algal coupling in biofilms are those in which light diffusing artificial substrates have been used (as
and nutrients are manipulated experimentally. shown for some studies of DOM) to examine
Both light and nutrients have been found to be the effect of nutrient availability mostly for bio-
limiting for periphyton (Hepinstall and Fuller, film growth and nutrient uptake. For instance,
1994). It has been shown that photosynthesis Tank and Dodds (2003) found that responses
enhances peptidase activity and that this activity to nutrient amendment differed among biofilms
typically increases with higher chlorophyll con- dominated by heterotrophs and autotrophs,
tent within the biofilm, likely due to the use of and also differed between organic and inorganic
available labile peptides released by algae, but this substrates. Moreover, increased biofilm growth
tight link between algae and bacteria is weaken and nitrogen uptake after nitrogen enrichment
when glucose is added (Ylla et al., 2009). In addi- appears to be more relevant under light conditions
tion, photosynthesis can cause changes in pH that (von Schiller et al., 2007), suggesting a positive
impact biofilm function. For example, Espeland interaction between light and nutrients for bio-
and Wetzel (2001) found that such changes in pH film primary production (Ylla et al., 2007). Scott
reduced alpha and beta glucosidase activity and et al. (2009) also using nutrient diffusing artificial
increased alkaline phosphatase. substrates found measurable nitrogen fixation and
suggested that in the system studiednitrogen was
Nitrogen cycling limiting for primary producers. Likewise, Romaní
The increased amount of biological available et al. (2004) found that nutrient addition altered
nitrogen derived from human activities has led to extracellular enzyme activity and that the nature
many studies on nitrogen in biofilms. Biofilms are of the response varied among biofilms developed
highly metabolic active; and thus, assimilation of in different substrates (rock versus sand). Other
N from the water column is relevant to meet the studies have investigated the response of bio-
biotic demand of N (von Schiller et al., 2009). films in terms of N uptake to gradual increases
In fact, the relationship between the 15N natural in DIN concentration and found that while the
abundances of dissolved inorganic nitrogen and response follows a Michaelis–Menten model for
biofilms found across several stream locations ammonium increases, the response for nitrate is
within a catchment provides evidence for stream absent or even negative (Kemp and Dodds, 2002;
Biofilms and Biogeochemistry |  97

O’Brien and Dodds, 2007; Ribot et al., 2013). on assimilation of phosphate and production of
In the case of leaf decomposition, the effect of alkaline phosphatase as well as on internal loading
nitrate increases followed a Michaelis–Menten of phosphorus from sediments. Price and Carrick
model response to breakdown rate (Ferreira et (2014) found that there were rapid and high levels
al., 2006), and at the same time, leaf litter can be of phosphorus exchange between biofilms and the
relevant as nitrate retention (Duan et al., 2014). In overlying water and that seasonal changes strongly
addition, nutrients have been found to be limiting influenced phosphorus flux.
to biofilm communities on wood and leaves in Nutrient diffusing artificial substrates have also
studies involving whole stream nutrient additions been used to examine responses to phosphorus
(Stelzer et al., 2003). additions. For example, Rier et al. (2014) found
Various studies have examined dissimilatory that both light and phosphorus had a positive
processing of nitrogen in biofilms. Teissier et al. impact on biofilm algae and this, in turn, influ-
(2007) found that nitrifiers were present and enced extracellular enzyme activity, potentially
able to compete with algae in river biofilms. In as a result of priming by labile OM. Similarly, Ylla
fact, nitrifier bacteria and archaea are already et al. (2007) found inorganic phosphorus uptake
present at early stages of biofilm succession and by the epilithic biofilm was enhanced at high light
their abundance seems to be favoured by ammo- and high nutrient conditions. Other studies have
nium concentration in stream water (Merbt et also examined nitrogen and phosphorus together;
al., 2011). Nitrification activity of biofilms have for example, Sekar et al. (2002) found that light
been measured using recirculating chambers and levels impacted algal communities as well as the
results show that environmental conditions of N:P ratio of biofilms. As described for plank-
oxygen and ammonium concentrations favours tonic habitats, phosphatase activity in biofilms
nitrification rates (Kemp and Dodds, 2002). typically decreases when increasing P availability
Other studies show that denitrification in biofilms and increases when P is limiting, although this
can be relevant, thought its variability is related to depends on N:P balance (Sala et al., 2001). In
biomass and oxygen concentrations (Teissier et a long-term nutrient enrichment experiment,
al., 2007) and nitrate concentration in the water phosphatase activity decreased in river biofilms
column (Kemp and Doods, 2002). When the and, at the same time, biofilms were enriched in
nitrogen cycle is viewed as a whole, there is evi- P content after nine months of nutrient enrich-
dence that nitrogen biogeochemistry associated ment while biofilm nitrogen enrichment was only
with biofilms changes over succession. Teissier et significant after 2 years of experiment (Sabater et
al. (2007) indicated that at early stages, assimila- al., 2011).
tion by epilithic biofilms exceeds mineralization Phosphorus cycling is especially relevant
and storage, whereas in more mature biofilms in sediment biofilms. In aquatic sediment, dis-
nitrogen mineralization is greater than uptake. In solved inorganic phosphorus constitutes a small
addition, results from a translocation experiment percentage of the total phosphorus which is
using 15N natural abundances in mature biofilms, often limiting for microorganisms growth. Thus,
indicated that while biofilm N uptake rates seem hydrolysis of organic phosphorus compounds
to be controlled by environmental conditions by means of hydrolytic phosphatases becomes
(such as light and nutrient concentrations), N the main mechanism for microorganisms to
turnover rates are mostly determined by biofilm obtain phosphorus (e.g. Scholz and Marxsen,
intrinsic characteristics (Peipoch et al., 2014). 1996). Phosphatase activity measurement in
lake sediments indicate that phosphorus cycling
Phosphorus cycling is enhanced by higher organic matter availability
Because of its importance as a limiting nutrient, (Wang et al., 2012).
phosphorus has been extensively studied in fresh-
water ecosystems. Unlike nitrogen, the number of Cycling of sulfur and micronutrients
chemical forms available and transformations is Many fewer studies have examined sulfur,
restricted and thus much effort has been focused metals, and micronutrients in general, cycling in
98  | Leff et al.

biofilms relative to studies on carbon, nitrogen of these compartments at whole-ecosystem scale.

and phosphorus. Environments where these The influence of biofilm activity on water chem-
micronutrients have been studied, include those istry has been demonstrated in previous studies
impacted by acid mine drainage in which forms (Woodruff et al., 1999; Mulholland et al., 2000;
and concentrations of micronutrients such as Tank et al., 2000). However, how biofilm activity
sulfur, iron and aluminium are altered. In addition, contributes to whole-ecosystem processes is still
a number of studies have focused on wastewater an ongoing question in aquatic ecosystem ecol-
treatment facilities in which biofilms develop. ogy. Scaling-up activity of biofilms developed in
Generally, there is little information that is specific different habitats to whole-ecosystem scale pro-
to freshwater natural biofilms regarding sulfur and cesses is a first to empirically tackle this question,
micronutrients. However, presumably there are but for this it is necessary to have good characteri-
commonalities in the biotransformations of these zation of the biogeochemical activity of biofilms.
elements that transcend the conditions of the spe- In this section, we review some of the techniques
cific ecosystem. that are currently used to characterize the biogeo-
Sulfur is much more widely studied in marine chemical activity of biofilms at different scales of
ecosystems, including estuaries, than in fresh- organization, which could be merged to increase
water ecosystems (Odum, 1998). In freshwater our knowledge on the relevance of biofilms at
systems, sulfate-reducing bacteria which produce whole-ecosystem scale (Table 5.1).
hydrogen sulfide are present and play a potential
role in acid mine bioremediation. Molecular tools and functional
Oxidation coupled to reduction of manganese genomics
or iron occurs in sediments and these might be Biogeochemical processes in biofilms are mostly
linked to microbial processes since bacteria that driven by microorganisms; although we can
large use manganese or iron as the sole terminal learn a lot from community and ecosystem scale
electron acceptor have been cultivated (Tham- experiments, molecular phylogenetics and func-
drup, 2000). Iron is known to be taken up by tional genomics provide very powerful tools to
biofilms and microbes play an important role understand which are the key microbial players
in iron speciation through oxidation-reduction driving biogeochemical processes. Functional
reactions. Also organic compounds released genes encode for enzymes, which drive the
by bacteria influence iron dynamics in biofilms biogeochemical process. All microorganisms
(Duckworth et al., 2009). Julien et al. (2014) with the same functional gene are put together
experimentally demonstrated that iron is not in a functional group, which is linked to a bio-
homogenous in biofilms, rather it appears in ‘hot- geochemical process. However, the gene may be
spots’. Iron bacteria that oxidize ferrous salts are present in phylogenetic similar (monophyletic,
rather restricted in distribution based on specific e.g. ammonia-oxidizing bacteria) or in distinct
environmental conditions (Wetzel, 2001). Some microorganisms (polyphyletic, e.g. nitrogen-
of these species can also oxidize manganese salts. fixing bacteria or denitrifying bacteria), due to
evolutionary processes or horizontal gene trans-
fer. Therefore, members of the functional group
Measurements of may differ significantly in transformation rates and
biogeochemical processing in response to physical and chemical environmental
biofilms conditions. Consequently, characterization of
Biogeochemical cycling in aquatic biofilms the microbial community composition as well
typically plays a key role in organic and inorganic as abundance and activity of the particular func-
nutrient cycling in the aquatic habitat where tional group is important for prediction of activity
it develops. The contribution of biofilms to at the whole-biofilm scale (Fernàndez-Guerra and
emergent biogeochemical ecosystem functions Casamayor, 2012).
depends on the characteristics of the aquatic Functional genes can be tracked in the
ecosystem, as well as on the relative abundance environment. For instance, both the ammonia
Biofilms and Biogeochemistry |  99

Table 5.1 List of methods included in this chapter to study biofilm biogeochemical processes, including
their primary application and approximate spatial scale
Method Primary applications spatial scale

Examine biofilm spatial heterogeneity

Microsensors: optodes Obtain gradients in biofilm depth and spatial 10–6–10–3 m
distribution (e.g. for O2 and N inorganic forms)
Confocal microscopy combined with Localize specific metabolic activity within the biofilm 10–6–10–3 m
functional measurements structure (e.g. use of fluorescent substrata to detect
extracellular enzyme activity, or fluorescent in situ
hybridization (FISH) to identify microbial groups)

Consider the biofilm as a blackbox

Molecular tools (DNA, RNA) Examine diversity of specific functional genes; single- 10–6 m
cell analysis (e.g. FISH techniques)
Extracellular enzyme activities Capacity of biofilm to degrade specific organic 10–3–10–2 m
Electron transport system (ETS) activity, Obtain metabolic rates (respiration and gross primary 10–3–10–1 m
O2 and CO2 mass balances, use of production) of biofilms
hydrometabolic tracers (e.g. Resazurin)
Spikes of inorganic nutrients or Obtain uptake/release rates and uptake kinetics for 10–2–10–1 m
dissolved organic carbon (DOC) in inorganic nutrients and DOC
recirculating chambers or flumes.
Addition of stable isotopes of inorganic Follows fate of nutrients and organic carbon from the 10–1–102 m
nutrients and carbon water column into biofilms

oxidizing bacteria and archaea encode for the DNA subsequent of each elongation step and the
enzyme ammonia monooxygenase and its alpha emitted irradiance is measured. The quantification
subunit (amoA) can be traced in environmental takes place by comparing with a standard DNA
samples using specific primers [e.g. for amoA of from genome or clone DNA (Fierer et al., 2005).
bacteria (Rotthauwe et al., 1997) and archaea The activity of a functional group is measured
(Francis et al., 2005)] giving insights into compo- via quantification of the messenger RNA of the
sition, abundance, activity of functional genes and functional gene by reverse-transcriptase qPCR.
the organisms in which this gene occurs (Zak et Here, the messenger RNA is extracted from the
al., 2006). environmental sample, transcribed to copy DNA
The phylogenetic composition of the func- using the reverse transcriptase and subsequently
tional group can be characterized via molecular quantified via qPCR (e.g. Nicol et al., 2008).
fingerprinting methods. After the amplification of All these methods are useful as long as the
the functional gene, use of denaturating gradient functional group is familiar and a specific primer
gel electrophoresis (DGGE) or terminal restric- available. However, if this is not the case, stable
tion fragment length polymorphism (T-RFLP) isotope probing (SIP) is an approach that can
allows comparing the community composition of uncover active microorganisms in the biofilm
different samples among each other, but the meas- assemblage avoiding the need of knowledge about
urement of diversity is not possible. In contrast, the community composition. In this method, bio-
the sequencing of clone libraries allows determin- film is incubated with isotopic enriched substrate
ing diversity and evenness in a sample (Muyzer et (e.g. 13C or 15N). Active organisms in the assem-
al., 1993; Muyzer and Wawer., 1998). Abundance blage incorporate stable isotopes in their DNA.
of a functional group can be assessed using quan- Genomic DNA of the sample is extracted and
titative polymerase chain reaction (qPCR) where the DNA enriched in heavy isotopes is separated
a fluorescent dye attaches to the double bound from the not enriched via ultra centrifugation. The
100  | Leff et al.

part of the DNA representing the ‘active’ micro- Extracellular enzymes are those involved in the
organisms can be analysed via the amplification hydrolysis of common polysaccharides from
of the 16S ribosomal RNA and sequencing. This allochthonous/autochthonous origin (alpha- and
approach leads to sequences that are further com- beta-glucosidase, beta-xylosidase, cellobiohydro-
pared with existing data bases (e.g. GenBank) to lase), peptides (peptidase), lipids (lipases), but
unveil the active part of the community composi- also include those oxidative enzymes involved
tion of the sample. in the degradation of complex compounds such
These DNA based approaches are highly sen- as lignin (phenol oxidase, peroxidase). Biofilm
sitive, but also prone to overestimation because enzyme activities can be further involved in the
gene copy numbers are not always adaptable to degradation of chitin (from eukaryotes, for exam-
cell numbers. The target gene copy number per ple; chitinases), acquisition of inorganic nutrients
microbial genome is sometimes unknown and (phosphatase), and adhesion processes (certain
the number of genome copies per microbial cell proteases). Extracellular enzyme activity meas-
can vary in different growth phases (Ludwig ured in biofilms informs us about internal nutrient
and Schleifer, 2000). In contrast, cells of a func- and organic matter cycling and/or about use and
tional group can be visualized within a sample transformation of materials from the flowing
with fluorescent markers (fluorescent in situ water, this may depend on the biofilm thickness
hybridization, FISH) and further quantified by and the stages of biofilm formation (Romaní et al.,
fluorescence microscopy (Daims and Wagner, 2012).
2007). Although FISH is a time-consuming and However, classical enzyme activity measure-
less sensitive method than DNA approaches, it ments consider the biofilm as a black box while
allows direct visual assessment of the cells within enzymes and enzyme activity might be heteroge-
their structure and hence minimization of biases. neously distributed within the biofilm.
FISH is of high interest when investigating biofilm Investigations of biofilm structure with con-
architecture or interactions between functional focal laser scanning microscopy (CLSM), have
groups. In one analysis up to seven different shown the patchiness structure of aquatic biofilms
populations can be assessed. The FISH method in three dimensions as well as allow detecting
has been adapted to a great variety of questions to structural changes with nutrient enrichments,
phylogenetic assignment (Clone-FISH), activity toxic substances and light intensity (Neu et al.,
(FISH-MAR), quantification (SPIKE-FISH) and 2005; Lawrence et al., 2005; Zippel and Neu,
detection in complex samples when combined 2005; see Chapter 2). More recently, the use of
with confocal laser scanning microscopy (CLSM) ELF (enzyme labelled fluorescence)-substrata
(CARD-FISH, DOPE-FISH) (Wagner et al., together with CLSM has allowed measuring
2003). extracellular enzyme activity and locating where
it takes place within the biofilms. The measure-
Extracellular enzymes ment of cell-enzyme activities was first developed
Biogeochemical processes in biofilms have also for planktonic environments by the use of ELF-
been approached by the measurement of extra- substrata that crystallize at the site of enzyme
cellular enzyme activities. The microorganisms activity. This methodology has been mainly devel-
within biofilms have the capacity to decom- oped for the measurement of phosphatase activity
pose organic matter by producing extracellular of attached bacteria (Van Ommen Kloeke and
enzymes either located outside the cells or in the Geesey, 1999) and in planktonic algae (Nedoma et
periplasmatic space (in Gram-negative bacteria, al., 2003; Díaz-de- Quijano et al., 2014). The first
Chróst, 1990), or released into the biofilm EPS studies of cell-enzyme activity in attached commu-
matrix (Freeman and Lock, 1995; Thomson and nities (cyanobacterial mat, activated sludge) were
Sinsabaugh, 2000; Romaní et al., 2008). Thanks performed by disrupting the biofilm structure to
to the enzyme activity, microbes can uptake obtain a biofilm suspension (Sirová et al., 2006;
simple compounds from high molecular weight Schade and Lemmer, 2006). However, the use of
compounds usually found in the environment. the undisturbed biofilm is crucial to understand
Biofilms and Biogeochemistry |  101

its functioning (Barranguet et al., 2004). More of a biofilm were more affected by ZnO nanopar-
recently, intact biofilms have been used to localize ticles, as measured by a reduction of respiration
extracellular enzyme activity. Tielen et al. (2013) activity, than the deeper layers.
incubated Pseudomonas aeruginosa biofilms with In parallel, development of planar optodes pro-
ELF 97 palmitate and further CLSM visualization vides a tool for mapping the spatial distribution of
to localize lipase activity in the biofilm and showed oxygen concentration in two dimensions (Glud
its interaction with alginate matrix (Tielen et al., et al., 1996; Kühl and Polerecky, 2008; Staal et al.,
2013). In Nielsen et al. (2010), a combination 2011a). Planar optodes use luminescent oxygen
of methods to describe cell activity in biofilms indicators immobilized in a polymeric matrix,
is described, including extracellular enzyme which is permeable to oxygen and can be fixed
activity by a range of ELF-substrata and BODIPY- on foils or glass surfaces (Glud et al., 1996; Oguri
substrata, and fluorescence in situ hibridzation, et al., 2006). Kühl et al. (2007) also presented an
which has been mainly applied in activated sludge interesting combined method by applying confo-
communities (Kragelund et al., 2011; Xia et al., cal microscopy and imaging based on sensors
2007, 2008). Recently, a combination of these for microscopic oxygen measurements to show
techniques has been also used to further develop oxygen decay in a biofilm flow chamber mounted
a model to determine fluid dynamic processes on a microscope. Staal et al. (2011b) linked the
within the biofilm (Adav et al., 2010). However, biofilm biomass distribution to the oxygen spatial
most of these new techniques are mainly used in distribution and showed oxygen microenviron-
engineered systems (activated sludge, biofilms in ments linked to changes in flow conditions.
water treatment plants), and application to natural
aquatic biofilms is still under experimental phase. Influence of biofilm metabolism on
water column biogeochemistry
Biofilm metabolism at the fine scale In the paragraphs above, we described methods
Fine-scale measurements of biogeochemical pro- to examine the metabolism within the biofilms
cesses within biofilms can be inferred from vertical and the resulting gradients. To analyse how
profiles by using microsensors. For instance, verti- these internal processes impact water column
cal profiles of oxygen and nitrous oxide within biogeochemistry, biofilms can be examined as
biofilms have been used to measure denitrification a whole after being placed into either recirculat-
in biofilms (Revsbech et al., 1988; Nielsen et al., ing chambers or artificial flumes. This laboratory
1990). Since these early studies, microelectrodes experimental setting provides high reproducibility
have been used in many fields and are effective and and therefore is highly valuable for study biofilm
promising research tools that are able to capture in function in response to multiple treatments
situ bioreactivity of natural biofilms. For instance, (Singer et al., 2006). Overarching parameters to
the monitoring of biofilm development with estimate biofilm metabolic activity are biofilm
microsensors was able to show that nitrification respiration and primary production. Respiration
assemblages developed earlier than denitrification has been measured by incubating natural biofilms
assemblages in biofilms (Li and Bishop, 2004). in the experimental setting, recording oxygen con-
The use of microsensors has also been combined sumption and inferring it from an oxygen mass
with in situ hydridization to link the presence of balance (e.g. Guasch and Sabater, 1995). These
nitrifying bacteria to the nitrogen metabolism measurements have provided insights on the
detected by the microsensors (Okabe et al., 1999). effects of environmental factors on biofilm respi-
The results from microelectrodes have been also ration (e.g. temperature; Bouletreau et al., 2012).
used to model biofilm oxygen uptake kinetics, as Another method to measure respiration activity
described in Zhou et al. (2009). Microprofiles of of biofilms is the estimation of the electron trans-
oxygen concentration in biofilms have been used port system activity (ETS) by the reduction of the
to investigate the effect of different toxicants on electron transport acceptor INT (2–3 tetrazolium
biofilms (e.g. Zou et al., 2011). In this context, chloride) to INT-formazan (iodonitrotetrazo-
Hou et al. (2014), showed that the surface layers lium formazan) (Blenkinsopp and Lock, 1990).
102  | Leff et al.

More recently, respiration in biofilms has been nitrogen, cycling at whole-reach scale. Patterns of
estimated by using a phenoxazine compound, natural abundance between dissolved inorganic
resazurin (Raz) as ‘smart tracer’ (Haggerty et al., nitrogen in the water column and in biofilms
2008, 2009). In the presence of aerobic bacteria have allowed to examine not only the transfer of
Raz reduces irreversibly to resorufin (Rru) and, dissolved inorganic nitrogen into biofilms, but
at the same time, changes maximum excitation also rates at which nitrogen is incorporated into
wavelength from 602 to 570 nm, respectively. biofilms (Pastor et al., 2014; Peipoch et al., 2014).
The change from Raz to Rru has been shown to This technique is based on the fact that the lighter
be proportional to aerobic respiration and can be and heavier isotope vary in chemical and physical
measured using a spectrofluorometer (McNicholl behaviour and that they undergo fractionation
et al., 2007). due to a preferential use of lighter in front of heav-
Recirculating chamber and flumes also allow ier isotopes in several biogeochemical processes.
measurement of biofilm nutrient and organic The predominantly enzymatic driven fractiona-
matter demand from the adjacent water column tion leads to a distinct composition of isotopes
by estimating the uptake rates of these bioreactive in source and product and hence natural abun-
solutes. Basically, these measurements are based dance of stable isotopes can be used as tracers of
on spikes of target bioreactive solutes into the metabolic activities (Kendall and Caldwell, 1998;
mesocosms and the further trace of solute con- Sulzman, 2007). In addition, stable isotopes have
centrations over time in the water column (e.g. been used as tracers, which have allowed tracing
O`Brien and Dodds, 2008; Kemp and Dodds, the biogeochemical fate of bioreactive solutes
2002; Ribot et al., 2012). In these experiments, in the water column and how different biotic
changes over time in recirculating chambers or compartments use them at ambient conditions at
along space in artificial flumes are used to estimate whole-ecosystem scale (Mulholland et al., 2000).
processing metrics associated with biofilm activ- Addition of isotopically enriched nutrient tracers
ity based on standard approaches for nutrient can be used in mesocosm experiments focusing on
uptake in streams (Webster and Valett, 2006). For biofilms to identify rates of different processes or
instance, the decrease of added NH4+ over time in identifying the assimilatory responses of biofilm
recirculating chambers with biofilms is assumed uptake in front of changes in nutrient availability
to follow a first-order kinetic rate; and thus, the (Ribot et al., 2013).
slope provides an estimate of the rate constant of In addition, contribution of biofilms to whole-
a target process associated to biofilm activity. This reach rates of both metabolism and nutrient
approach is also useful to estimate kinetics of a processing can be estimated by scaling the data
biofilm in response to bioreactive solutes (Kemp derived from mesocosms studies if we assume
and Dodds, 2002; Ribot et al., 2012). that biofilms operate similarly under experimen-
The measurement of dissimilatory uptake pro- tal and in situ conditions (Kemp and Dodds,
cesses associated with the cycling of N by biofilms 2002). Alternatively, in the case of nitrogen
(e.g. nitrification or denitrification) can also be cycling, the use of stable isotopes can be done at
approached by the combined use of key substrate the whole-stream reach scale and thus particular
for the process and specific inhibitors of the par- contribution of biofilms to whole.-reach uptake
ticular process. For instant, nitrapyrine, acetylene rates can be evaluated. Here the water column
and dicyandiamid are inhibitors of nitrification of streams and rivers can be enriched by stable
which have been used in mesocosm studies to esti- isotopes via additions of isotopes without altering
mate the nitrification rates associated with biofilm the background concentrations as well as the habi-
activity by comparing rates between inhibited and tat conditions for particular biotic compartments.
control treatments (Powell et al., 1986; Teissier et This approach has been used to provide uptake
al., 2007; Smith and Schallenberg, 2013). data not only at the whole-reach scale (Peterson
In recent years, use of stable isotopes has et al., 2001; Johnson et al., 2012), but also to esti-
significantly increased our ability to assess the mate the relative contribution of different biotic
contribution of biofilms to nutrient, especially compartments occurring at whole-reach scale,
Biofilms and Biogeochemistry |  103

including biofilms (Mulholland et al., 2000). Sim- Battin, T.J., Sloan, W.T., Kjelleberg, S., Daims, H., Head,
I.M., Curtis, T.P., and Eberl, L. (2007). Microbial
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bloom-forming diatom Didymosphenia geminata.
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Part II

Biofilms and Pollution

Benthic Diatom Monitoring
and Assessment of Freshwater
Environments: Standard Methods and
Future Challenges
Soizic Morin, Nora Gómez, Elisabet Tornés, Magdalena Licursi and
Juliette Rosebery

Abstract faced by scientists to improve routine biomoni-

Since biofilms integrate the environmental effects toring.
of water chemistry, along with the physical and
geomorphological characteristics of rivers and
lakes, they have been widely applied in biomoni- Introduction
toring. In particular, diatoms are extensively used Various biological metrics have been developed to
as reliable environmental indicators. Diatoms assess the health of freshwater ecosystems. They
are microscopic, unicellular brown algae, which reflect integrative effects of present and past con-
often dominate the algal biomass of biofilms. The ditions, whereas traditional chemical and physical
shape and morphology of the siliceous skeleton, measurements only apply to the current situation.
the frustules, unique to each taxon are used for Several studies have shown that community-
taxonomical identification. The floras are diverse, based indicators are needed to evaluate water
in relation to their geographical location (climate, quality. Diatom biomonitoring has benefited,
geology, relief) and to the quality of the aquatic over other existing periphytic bioindicators, from
environments they inhabit. Indeed, species are a long research history. Indeed, typical water qual-
sensitive to the water physicochemical parameters ity assessment in flowing watercourses is mainly
and their presence/abundance is therefore cor- based on benthic diatoms, i.e. diatoms growing
related to water quality. on diverse natural (pebbles, macrophytes) or
Diatom sensitivity or tolerance towards dif- artificial (glass slides, ceramic tiles) substrates
ferent environmental parameters has long been immerged in the water. However, biofilms are
studied and used to implement bioassessment composed of diverse components other than dia-
methods. Such methods evolved from indices of toms. Although the planktonic microflora is used
saprobity designed first for European streams, to widely as biological quality element (especially in
developments of various diatom indicators world- lacustrine environments), benthic green algae and
wide, able to highlight different types of pollution cyanobacteria are poorly known and no consen-
(pH, salinity, nutrients, toxicants). sual bioindicator exists.
The objective of this chapter is to provide sci- Diatoms are a siliceous class of unicellular
entists and water managers with a broad overview algae known to be very diverse (Guiry, 2012)
of diatom tools helpful to monitor the ecological and sensitive to chemical conditions. They
status of freshwater environments. We describe are excellent bioindicators due to their short
the applicability range and the limitations of the generation time and their varying ecological
main existing methods, metrics (indices, traits) preferences. Moreover, the structural elements in
and types of surveys used, as well as the challenges their siliceous cell walls allow reliable taxonomic
112  | Morin et al.

determination at specific and subspecific levels. diatoms are relatively sedentary, and live their
They are distributed throughout the world in whole life cycle in the water implying that their
nearly all types of aquatic systems and usually community structure is tightly associated with
account for the highest number of species among local environmental conditions at the sample site.
the primary producers in aquatic systems. They Thus, diatoms are generally collected from hard
respond to environmental disturbances not surfaces immerged (natural or introduced, like
only at the community level through changes in glass slides or ceramic tiles; e.g. Sekar et al., 2004).
diversity, but also by shifts in dominant taxa. Over Moreover, their sampling requires minimal effort
the last decades, a great number of diatom-based (scraping or pipetting the surface, using corers),
methods (reviewed in Prygiel et al., 1999) have causes minimal disturbance to the sampling sites
been proposed to assess water quality. No one and it is possible at sites where other bioindica-
single metric is applied worldwide, but most of tors, e.g. benthic invertebrates and fish are absent
the diatom indices used are minor adjustments (Lear et al., 2012).
of a common approach based on the knowledge Besides being easy to sample, diatoms have
of the ecological spectrum of species, combining the great advantage that their frustules provide
sensitivity to pollution and indicator value. detailed features enabling reliable identification at
This chapter aims to review the main char- species and subspecies levels. These siliceous walls
acteristics of diatoms which have been used to are easily preserved and cleaned of organic mate-
develop bioassessment methods in freshwater rial (with hydrogen peroxide, hydrochloric acid;
environments. Various indicators are described, AFNOR, 2003), to be permanently mounted on
with respect to the types of pollution that can be microscope slides in refringent resin for identifica-
addressed, and to the kinds of approach imple- tion under light microscopy at high magnification
mented. The pros and cons of existing systems, (x1000), with oil immersion.
as well as future challenges in biomonitoring are On the other hand, microscopic determination
presented. of diatom taxa requires precise identification than
can only be reached by trained operators. Among
the main diatom features to be taken into account
Potential of diatoms for water when doing microscopic identification are: the
quality assessment general shape of the valve (the kind of symme-
Diatoms (Bacillariophyceae) are particularly try), cell dimensions, length–to-breadth ratio,
interesting as indicators of water quality (Table and ornamentations (presence or absence of one
6.1). These unicellular brown microalgae (size or two raphes, i.e. longitudinal slits on the valve;
ranging from < 10 to > 500 µm) occur in all orientation of the striae; presence of specific fea-
aquatic environments and are found at almost tures; stria density).
all levels of pollution. Diatoms can account for Diatom community structure reflects a gradi-
up to 80% of the taxa present in streams, rivers, ent of general pollution in water quality (although
lakes and wetlands (McIntire et al., 1996). They causes are not always identifiable) integrated over
are used worldwide as biological markers to assess time on a relatively short timescale, in relation to
water quality: in Europe to support the implemen- high sensitivity of individual species towards dif-
tation of the Water Framework Directive (Kelly et ferent levels of pollutions (Lowe and Pan, 1996;
al., 1998), but also in routine monitoring surveys McCormick and Cairns, 1994; Stevenson and
in Canada, USA, Japan, Australia, South America, Pan, 1999; Whitton and Rott, 1996). In order
etc. (e.g. Lobo et al., 2004; Gómez and Licursi, to determine species sensitivity, the methodol-
2001; UNESCO-WHO-UNEP, 1996). ogy most frequently used is a direct comparison
Diatoms occupy a diverse range of habitats: between the physicochemical water quality and
they can be planktonic (freely living in the water the species composition of the corresponding
column) or fixed on diverse substrata (periphytic records in relative abundances of the taxa using
on hard surfaces, epipsammic on sand, epiphytic appropriate multivariate analyses. Results are
on macrophytes, see Chapter 1). Periphytic generally presented as species optima towards
Diatom-based Monitoring and Assessment |  113

Table 6.1  Advantages and drawbacks of diatoms as bioindicators of water quality

Advantages Drawbacks

Biofilm presence generally visible to the naked eye on
the substrata
Quick and easy collection (scraping, pipetting, using
corers for soft sediments and sand)
Possible use of artificial substrates Risks of vandalism or loss (e.g. floods) of artificial

Taxon identification
Numerous identification resources (books, articles, Difficult and ever-changing systematics
Time-consuming counting/identification of samples
High quality microscope necessary

Bioassessment power
Widespread distribution, even in hostile environments Geographically specific distributions (endemism)
High species diversity (about 10 000 species known) Heterogeneous biomass distribution (e.g. light
Sensitive to numerous kinds of pollution Poorly sensitive to habitat disturbances
Conservation of the frustules (paleolimnological Integrative power lower than for higher organisms
applications) and integrative power variable depending
on the species

Index calculation
Dedicated software (e.g. Omnidia) Applicability of indices generated for other geographic

different environmental parameters (Charles et research and monitoring programmes, manage

al., 2006; Ponader et al., 2008), or species eco- data and calculate indices.
logical profiles expressed as species probability of
presence along ecological gradients (Potapova et
al., 2004). Towards a harmonized
Once the polluo-sensitivity or resistance of way of using diatoms for
species to defined classes of quality are hier- biomonitoring
archized (e.g. Van Dam et al., 1994), species can be The need to monitor water quality led to the
grouped and used as indicators for saprobic condi- development of standardized sampling proto-
tions (Sládeček, 1986), salinity (Ziemann, 1991), cols and assessment methods, through single,
acidification (Birks et al., 1990) and eutrophica- simplified indices. Such indices were created by
tion in lakes and rivers (Steinberg and Schiefele, adapting the formula of Zelinka and Marvan
1988; Hofmann, 1994). The relative abundances (1961), basically combining the abundances of
of the different species identified are reported and species and their individual ecological prefer-
used, e.g. for calculation of water quality indices. ences, into a single score of water quality. Among
Depending on the methods, a minimum of 400 them, DAipo (Diatom Assemblage Index to
individuals identified is required for reliable organic pollution; Watanabe et al., 1986), IBD
assessment (AFNOR 2000). Diatomists have cre- (Indice Biologique Diatomées, Coste et al.,
ated practical tools such as the software Ominida 2009), IDEC (Indice Diatomées de l’Est du
(Lecointe et al., 1993). This software allows to Canada; Lavoie et al., 2006), IPS (Indice de
efficiently compute diatom inventories from Polluosensibilité Spécifique; Coste in Cemagref,
114  | Morin et al.

1982), TDI (Trophic Diatom Index; Kelly and Implementation of diatom-

Whitton, 1995), and IDP (Pampean Diatom based assessment in rivers
Index; Gómez and Licursi, 2001) are successfully and streams: main approaches,
used in many monitoring programmes. advantages and limitations
Recently, limitations in these approaches Assessment of lotic systems has received more
have been identified, linked to the importance attention than lakes (King et al., 2005), although
of reliable autoecological taxon information and the dynamics of phytoplankton as well as paleo-
to the potential general application of indices ecological reconstructions have been extensively
developed in a particular geographical area investigated in these systems. However, lacustrine
(e.g. application of European indices to USA; biomonitoring of contemporary water quality is
Potapova and Charles, 2007). To overcome increasingly applied, and is based on diatoms sam-
critical limitations concerning pertinent bio- pled in the littoral zone and with methods similar
monitoring irrespective of regional differences to those used in rivers and streams.
in taxa, climate and other local constraints, Diatom monitoring is mainly used for regula-
approaches such as the use of a common metric tory purposes, with very large scale programmes
for the different European countries (intercali- (from basin to country) and allows mapping
bration exercises, Kelly et al., 2009; Almeida et water quality using simple biological indices. This
al., 2014) have been developed. approach gives information about the current state
Diatom assemblages respond rapidly (weeks) of rivers, and can also be used to monitor recovery
and sensitively to environmental change and processes following rehabilitation programmes.
provide highly informative assessment of the More specific approaches to determine the role
biotic integrity of aquatic ecosystems. To limit of certain pressures in modifying the community
the biases linked to regional distribution of spe- in situ can also be performed, such as before/after
cies, different characteristics can also be used in impact studies (that can be studied in space or
the monitoring such as taxa richness, diversity, time), or the use of translocation experiments.
biomass, autoecology of individual species, Applied examples of these three complementary
biotic indices, percentages of aberrant dia- approaches are provided below.
toms, percentages of motile diatoms, mortality,
among others descriptors (Stevenson and Bahls, Large scale monitoring programmes
1999). Other approaches are complementarily and data integration
employed, based on non-taxonomical indica- In the last decades, numerous countries from the
tors, assuming that a given pressure selects for northern hemisphere have included diatoms in
certain characteristics, whatever the location/ their biomonitoring programmes, while in the
site studied. The diagnosis therefore relies on the southern hemisphere their use is less frequent
proportion of ecological characteristics derived (Rimet, 2012).
from species distributions and on their clas- Communicating the conditions of biological
sification in terms of ecological preferences (as systems, and the impact of human activities on
described previously) or ‘traits’ (e.g. postures, aquatic ecosystems, is the ultimate purpose of
growth forms, motility, or the ecological guilds biological monitoring. As an example, the bio-
defined by Passy, 2007). monitoring programme of the Matanza-Riachuelo
Indices have been developed mainly to assess River, a highly polluted Argentinean hydrographic
organic pollution, eutrophication or pH. But they system (Fig. 6.1) is presented. This basin has a
fail when there is low nutrient enrichment or surface area of 2240 km2, and is inhabited by more
several superimposed anthropogenic influences. than five million people. The deterioration of the
Therefore, attempts are made to develop diatom water quality of the main watercourse and of most
descriptors more specific to toxic pollution (see of its tributaries highlights a strong polluting load
Chapter 8). They generally require a combination from household and industrial sewage waters.
of structural and morphological descriptors (e.g. The urban pollution widely exceeds the diluting
cell sizes, deformities). and self-depuration capacity of the river, as well
Diatom-based Monitoring and Assessment |  115

Figure 6.1  Water quality map corresponding to the 2010 monitoring results of Matanza-Riachuelo basin
employing the Pampean Diatom Index (IDP) and percentage of cytologic abnormalities.

as toxic contaminants, chromium, copper, lead organic matter and heavy metals can be diagnosed
and cadmium concentrations above the guideline through high values of IDP, but also high percent-
levels of water quality for protection of aquatic ages of cytological abnormalities.
life. In this case, a regional index, the Pampean
Diatom Index (IDP) (Gómez and Licursi, 2001), Before/after impact studies (in
is employed to describe the different environ- space or time)
mental conditions related to the eutrophication Diatoms have been used in experimental designs
and the enrichment with organic matter through for comparing time series or differences in a
five water qualities codified with different colours treated area (or impacted) and control area,
(Fig. 6.1). Biomonitoring results expressed on before or after the intervention or experimental
maps constitute a useful tool for stakeholders as treatment. The before/after impact assessment is
they provide quick visualization, through the use a very suitable methodology for assessing whether
of different graphic codes and colours, of the evo- or not a stress has changed the environment,
lution of the water quality in hydrographic basins to determine which components are adversely
through time and space. Likewise, the evaluation affected, and to estimate the magnitude of the
of cytological abnormalities (percentage of aber- effects. The simplest approach, referred to as the
rant frustules and cytoplasmic content impaired) before–after design (BA), considers the time scale
also contributes to the detection of changes of the and involves collection of data prior to the begin-
water quality mainly associated to toxic pollution ning of activity and compares it with data recorded
(Figs. 6.1 and 6.2). So, in the lower basin, strong after the start of the activity (Smith, 2002). On the
symptoms of eutrophication and high amounts of other hand the before-after-control-impact-paired
116  | Morin et al.

Figure 6.2  Normal cells (framed in green) and specimens with a deformed frustule or modified ornamentation
(Surirella angusta and Nitzschia palea) and impaired cytoplasmic content (Nitzschia sp.) (framed in red, the
arrows indicate the alterations) recorded in the Matanza-Riachuelo basin.

(BACIP) approach is a proper method, involv- bacillum) others showed minor changes. Fur-
ing as well space scale, to evaluate the effects of thermore nutrient enrichment favoured shifting
the stressor being assessed; in this approach the the proportion of stalked or filamentous to more
‘impact area’ is paired to another area referred to motile growth forms (Fig. 6.4). The fertilization
as ‘control area’ (Stewart-Oaten, 1996). Paired in La Choza caused a mild to moderate effect,
samples are collected a number of times, both not immediately felt, on the diatom assemblages.
before and after the perturbation, simultaneously Taking into account the increase in urbanization
(or nearly so) at both a ‘Control’ and ‘Impact’ and agricultural activity in the Pampean plain, it is
location. The standard analytical approach, using likely that biodiversity can be seriously impaired
the resulting BACIPS data, is to calculate the dif- if the entrance of nutrients to these ecosystems
ference between ‘Control’ and ‘Impact’ values on is not mitigated. A similar study carried out in an
each date (termed delta), and test whether the oligotrophic Mediterranean forest stream (Ver-
mean of these deltas changes from before to after aart et al., 2008) reported that long-term nutrient
the perturbation (Bence et al., 1996). This type addition has significant effects on the algal bio-
of approach allows the elucidation of potential mass and community composition, and this was
effects that may occur in aquatic environments as detectable despite the low light availability result-
a result of a stressor. ing from the dense tree canopy. Results obtained
A study conducted in a Pampean watercourse from field experiments are extremely valuable,
(La Choza stream) is an example of application illustrating the expected effects of different pollu-
of this methodology in assessing the impact tion scenarios.
of a stressor (Artigas et al., 2013). The diatom
assemblages inhabiting the epipelic biofilm of this Active biomonitoring: use of
stream were exposed to a continuous surplus of translocation experiments
inorganic nutrients; increasing concentrations of Over the last decades, artificial substrates (e.g.
nitrogen and phosphorus in water 3-fold the basal glass slides or plastic sheets for periphyton, plastic
concentration. Nutrient enrichment was achieved trays for epipelon) have been increasingly used
by the use of fertilizer bags distributed along the for collecting diatom communities; consequently
reach; the period of exposure was of 14 months. promoting the development of translocation
The changes in nutrient concentration were experiments (Ivorra et al., 1999; Morin et al.,
associated with a significant increase (BACIPS, 2010; Tolcach and Gómez, 2002; Sierra and
P < 0.001) in diatom density and a decrease in Gómez, 2010). Basically, artificial substrates are
species richness and diversity (Fig. 6.3). Changes immersed in a river site, and then transferred else-
in the relative proportions of the diatom taxa were where to build scenarios concerning the responses
also observed; while some taxa showed moderate of the community to changes in environmental
to high variations (Nitzschia palea, N. frustulum, conditions. Diatom communities from a refer-
Melosira varians, N. supralitorea and Caloneis ence (unpolluted) site can be transferred to an
Diatom-based Monitoring and Assessment |  117

Diatom composition differed significantly

between upstream, intermediate and downstream
sites. According to the gradient of eutrophication,
IPS values decreased going downstream. Com-
munities translocated from the two contaminated
sites recovered a taxonomic composition closer to
the reference (upstream) within 1 month, as well
as increasing IPS values.
The use of translocation to assess the recov-
ery potential of diatom communities has been
recently questioned due to the fact that it is
impossible to discriminate between the sole effect
of water quality improvement and recolonization
by immigrants from the upstream pool (Morin et
al., 2012a). However, translocation experiments
remain powerful monitoring tools to assess poten-
tial gains in ecological health after remediation,
especially in the case of connected hydrosystems.

Diatom-based assessment
shifts towards fundamental
The effects of anthropogenic modification of
abiotic factors on natural variations in water qual-
ity variables and biotic relationships should be
addressed. In contrast to studies on biofilm bacte-
rial communities that have focused on large-scale
biogeography (Martiny et al., 2006; Battin et al.,
2007), information on the relative importance of
natural and anthropogenically driven variations
in diatom communities is limited to recent ecore-
gional approaches (see below). Recent works
also aim at integrating the role of biotic factors,
i.e. competition or facilitation within the com-
Figure 6.3 (A) Richness, (B) diversity and (C)
densities in the control (green bars) and enriched
munity, in structuring the assemblages. Together,
(orange bars) reaches in La Choza stream. The these works provide information that may be
dotted line indicates the onset of the fertilization implemented in multimetric approaches. Finally,
period. molecular approaches have also been developed
to study the phylogeny of diatoms and more
precisely define the concept of species in diatoms,
impacted location, to explore the effects of water and shifting towards molecular biomonitoring.
quality degradation. Opposite translocations
(polluted to unpolluted site) can be performed Spatial distributions
to study simulated improvement of water qual- Many diatom species are known to have broad dis-
ity. Fig. 6.5 illustrates the recovery of community tributions; others seem limited to specific climatic
structure and water quality indices (using the IPS; zones or geographical regions, or are endemic
Coste in Cemagref, 1982) in diatom communities to a particular habitat. Despite the importance
sampled along a gradient of orthophosphates. of benthic diatoms as biomonitors, large and
118  | Morin et al.

Figure 6.4  Relative abundance of diatom growth forms in the Control (A) and Enriched (B) reaches in La
Choza stream. The dotted line indicates the onset of the fertilization period.

Figure 6.5 Translocation along a gradient of orthophosphates. (A) Location of the sample sites along
the River Morcille. Diatom communities are sampled after 2 months at the three sites (Up: upstream,
Int:  intermediate, Dw: downstream), or after 1 month in their original site followed by 1 month upstream
(Int → Up and Dw → Up). Average orthophosphate concentrations are shown in mg/l. (B) Principal component
analysis based on relative abundances of the 40 dominant diatom taxa showing the discrimination between
sampled communities, and corresponding average IPS values.

regional scale knowledge of their structure and broader-scale factors determines the composition
function is still scarce. Their distributional pat- of diatom communities (Leira and Sabater, 2005).
terns respond to a multitude of different factors, In addition, the relative effects of anthropogenic
from the biogeochemical characteristics of water variation over natural variation in determining
and its nutrient content, to geomorphological and the distribution of a given community should also
physiographical features, but also to biotic inter- be considered. Nutrient enrichment and human
actions, that operate over a wide range of spatial disturbances act to change local and large-scale
and temporal scales (Menge and Olson, 1990). factors reducing the regional differences (Tornés
Water chemistry, light availability, variations in et al., 2007). An obvious consequence of the over-
temperature, water velocity, substrata type and riding effect of human activities is that differences
grazing are among the factors that potentially in relative abundance and composition of diatom
affect benthic diatoms (Stevenson et al., 1996). communities are clearer among relatively undis-
The respective relevance of water quality varia- turbed sites than among sites severely affected
tion and physiographical processes in a particular by nutrient enrichment. It has been proved then
geographical area are expressed in a complex gra- that this knowledge is crucial in the assessment of
dient, in which the interaction between local and biological quality, which is based on the degree of
Diatom-based Monitoring and Assessment |  119

deviation between expected and observed condi- coexistence and facilitation. But how to disentan-
tions (Tornés et al., 2012). gle the relative importance of this biotic process in
Recently, an increasing body of research comparison to the others? In reality two types of
supports the idea that microorganisms exhibit approaches exist to unravel the causes: mathemat-
biogeographical patterns with no strict evidence ically by comparing real communities to virtual
of ubiquitous and global distribution (Hillebrand ones with no biotic interactions, and practically
et al., 2001; Heino and Soininen, 2005). At the by conducting laboratory experiments.
landscape scale, local species belong to broader As diatoms are part of a complex three-dimen-
metacommunities, shaped by dispersion, con- sional matrix, we can assume that competition
nectivity, biotic interactions and habitat area plays a significant role in community structure.
(Altermatt et al., 2013; Chapter 4). In particular, However, to our knowledge, there is no strict
historical processes (i.e. colonization, extinction, evidence of competitive exclusion between
dispersion, migration) may determine global diatom species from natural stream ecosystems.
diversity patterns of diatoms (Vyverman et al., To date, only Heino and Soininen (2005) have
2007). Potapova and Charles (2002) demon- tried to highlight such patterns for stream diatom
strated that large-scale spatial patterns of species communities, by a mathematical approach. From
dispersal, independent of local environmental Finish data they calculated an index of species
characteristics, cannot be neglected in broad- pairs that do not co-occur, i.e. chequerboard pairs
scale studies. In their study almost one-third (C-score, Stone and Roberts, 1990), which is a
of the explainable variation in diatom species measure of the exclusion rate between species.
composition at the USA national scale was attrib- They compared the results to those obtained from
uted to spatial factors. Similarly, another recent random communities, with no biotic interactions
spatial concept applied to diatoms is nestedness (Gotelli, 2000). Despite the higher number of
(Soininen, 2008; Tornés and Ruhí, 2013). Nest- chequerboard pairs in the real dataset, the particu-
edness is a metacommunity-based concept which lar structure of the environmental dataset used did
quantifies the overlap in species composition not allow interpretation of the results as evidence
between high and low diversity sites (Atmar and of competitive exclusion.
Patterson, 1993). Nested structures occur when Concerning positive biotic relationships,
assemblages of species-poor sites are subsets of such as niche complementarity, Burkholder et
the assemblages of species-rich sites. The extent al. (1990) by laboratory autoradiographic tech-
to which environmental variables determine nest- niques, reported a direct comparison of phosphate
edness is still poorly understood. For example, uptake by adnate and by loosely attached diatoms
Tornés and Ruhí (2013) identified hydrological in an intact biofilm matrix. They highlighted
stability as the main driver of nestedness of diatom the fact that loosely attached cells took up sig-
communities in Mediterranean rivers. nificantly more radiolabel than did the underlying
adnate cells, which were more isolated from the
Integrating the role of biotic water column nutrient source. The results gave
interactions evidence of a physiological assimilation gradient
Assembly rules, through general principles, try to among diatoms, where loosely attached cells can
explain the different processes leading to the pres- form a significant barrier to nutrient entry. It can
ence of a particular species at a given site (Weiher then be concluded from those seminal works
and Keddy, 1999). The species sensitivity towards that such species could facilitate the persistence
the environmental conditions and their ability of underlying sensitive species. Moreover, in an
to participate in spatial dispersion represent two experimental investigation of periphytic succes-
important processes (see above). A third major sion in recirculating laboratory streams, Passy
process concerns biotic interactions, which have and Larson (2011) examined the density and the
been very poorly studied to date. This consists relative abundance of diatoms across gradients of
in studying species co-occurrence in biofilms low to high nutrient supply and low to interme-
through the concepts of competition, passive diate current velocity. They concluded that the
120  | Morin et al.

mechanism of species succession, especially at a riparian disturbances (percentage of the species

functional level, was a neutral coexistence where A. minutissimum for example) or metal pollution
sensitive species were neither facilitated nor out- (number of diatom cells). More recently Delgado
competed by tolerant species but controlled by et al. (2010) developed a diatom multimetric
the environment. index (MDIAT) as a combination of metric values
(existing European diatom-based indices and
Multimetric developments to better percentage of local reference taxa) responding to
diagnose the type of stressors organic and nutrient stressors.
Analysis of diatom assemblages can be based on
taxonomic (e.g. composition and abundance, Progress in taxonomic identification
richness, diversity, species sensitivity, growth using molecular tools
form, motility) or non-taxonomic measures (e.g. Molecular biology is increasingly used for diatom
enzyme activity, chlorophyll content). The most phylogenetic analyses (Bruder and Medlin, 2007;
common taxonomic-based techniques used for Medlin et al., 2008; Medlin, 2010; Medlin and
monitoring rivers worldwide belong to three basic Kaczmarska, 2004), but classifications based on
approaches: indicator species or traits towards molecular studies and current systematics based
different environmental parameters, indices of on morphological features rarely cross (Cox,
community structure (e.g. diversity, richness, 2009). Resulting identifications are difficult to
evenness), or biotic indices. Multimetric indices compare, with in some cases generic boundaries
can combine these three types of approaches incorrectly drawn (Medlin et al., 2008). Chal-
into a unitless measure, to be able to respond lenge in correct identification concerns species
both to specific sources of stress and to general with very subtle morphological differences
perturbations (Karr and Chu, 1997). Ideally, the (Behnke et al., 2004) or original species descrip-
series of indicators should represent key informa- tions covering more than one genotype (Evans et
tion about structure, function and composition al., 2008; Mann et al., 2008; Sarno et al., 2005)
(Dale and Beyeler, 2001). Then, such indices and vice-versa with species of large phenotypic
integrate metrics that must show clear relation- plasticity, e.g. leading to identification of the
ships with different types of stressors, and should extremes as different taxa (Mann et al., 2010).
reduce uncertainty and increase robustness of Primers specific to diatoms have been proposed
assessment in comparison to single metrics by by Valiente Moro et al. (2009) but they are not
combining different types of metrics indica- specific enough to characterize the real diversity
tive of different environmental conditions and of samples (Morin et al., 2012b). Next-generation
community features. Several studies in Europe sequencing was recently attempted to inventory
(Hering et al., 2006) and in USA (Barbour et taxonomic diversity in diatom communities
al., 1999; Karr and Chu, 1999) have proved the (Kermarrec et al., 2013a). Although taxonomic
multimetric index to be a valuable approach for assignation was not always stringent, barcoding
assessing the ecological status of water bodies. The approaches offer promising perspectives for high
first attempt to settle a diatom-based multimetric throughput screening of diatom diversity, and
index was that of Hill et al. (2000) who proposed a may represent a powerful tool for biomonitoring
Periphytic Index of Biotic Integrity (PIBI) for the in the future.
Mid-Appalachian region (USA). PIBI includes Today, molecular tools are rather used to
diatom and non-diatom criteria, linked to the support taxonomy, particularly in the case of
relative abundance of some genera, the percent- cryptic species where they are needed to refine
age of acidophilic or eutraphentic diatoms, the identifications (e.g. Evans et al., 2008; Mann et al.,
percentage of motile diatoms. These metrics were 2008). The combination of molecular techniques
well correlated with both water quality and stream with microscopic observations allows progress
depth and width. Then Griffith et al. (2002) in classification, especially when dealing with
proposed for the Southern Rockies ecoregion of complicated species complexes (Kermarrec et al.,
Colorado (USA) diatom metrics rather linked to 2013b).
Diatom-based Monitoring and Assessment |  121

Conclusions streams from different biomes. Environ. Res. Lett. 8,

Diatom-based biomonitoring is widely used for Atmar, W., and Patterson, B. (1993). The measure of
regulatory purposes, at large scales or for many order and disorder in the distribution of species in
local applications. Here we reviewed methods fragmented habitat. Oecologia. 96, 373–382.
differing in their objectives (evaluation of the Battin, T.J., Sloan, W.T., Kjelleberg, S., Daims, H., Head,
I.M., Curtis, T.P., and Eberl, L. (2007). Microbial
general water quality, diagnostic of local impacts) landscapes: new paths to biofilm research. Nat. Rev.
and highlighted the recent inflexions in diatom Microbiol. 5, 76–81.
research, towards increasing consideration of fun- Barbour, M.T., Gerritsen, J., Snyder, B.D., and Stribling,
damental ecology and multi-proxy approaches. J.B. (1999). Rapid bioassessment protocols for use
in streams and wadeable rivers: periphyton, benthic
Molecular tools are also considered to potentially macroinvertebrates and fish, second edition. EPA-
contribute to a better assessment of water quality, 841-B-99-002. (US Environmental Protection Agency,
as they may improve our knowledge of diatom Office of Water, Washington, DC, USA).
taxonomy and help to refine taxa ecological Behnke, A., Friedl, T., Chepurnov, V.A., and Mann, D.G.
(2004). Reproductive compatibility and rDNA
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control-impact paired series design: the predictive
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The Use of Biofilms to Assess the
Effects of Chemicals on Freshwater
Helena Guasch, Joan Artigas, Berta Bonet, Chloe Bonnineau,
Oriol Canals, Natàlia Corcoll, Arnaud Foulquier, Julio López-Doval,
Sandra Kim Tiam, Soizic Morin, Enrique Navarro, Stephane Pesce,
Lorenzo Proia, Humbert Salvadó and Alexandra Serra

Abstract Introduction
Nowadays, biofilms are one of the principal The increasing worldwide contamination of
targets of community ecotoxicology in aquatic freshwater systems with thousands of industrial
ecosystems with a high potential for future use and natural chemical compounds is one of the
in ecotoxicology. A large set of methods derived key environmental problems facing humanity.
from biofilm ecology has successfully been Developing and refining tools to assess the impact
applied in ecotoxicology providing a diverse and of these pollutants on aquatic life is still a chal-
comprehensive toolbox. Our ability to quantify lenging issue (Hering et al., 2010). In spite of
the effects of pollution on different biofilm com- the inherent complexity of natural systems, the
ponents, allows the direct effects of pollutants on basis for using natural biofilms to assess acute and
the most sensitive community and their indirect chronic effects of pollution is rather simple. It is
effects on the rest of biofilm components to be expected that the effects of toxicity will first trig-
evaluated. Biofilms are also a site for biotrans- ger a biochemical response, e.g. by the activation
fomation and/or transfer of chemicals to other of detoxification mechanisms, causing thereafter
aquatic organisms, supporting a more general- physiological alterations, such as a reduction in
ized use of biofilms in environmental chemistry. photosynthetic activity and respiration, and lead-
Investigations aiming to describe processes at ing finally to a reduction in the growth of the most
biofilm scale, like nutrient dynamics and those sensitive species and the selection of the most
including simple food chains, have recently been tolerant species causing a shift in the structure (i.e.
applied, providing the opportunity of upscaling species composition) of the biofilm community.
the effects of pollutants on biofilms to food webs Together with the analysis of water chemistry,
and ecosystems. Finally, biofilm ecotoxicology and the prevailing environmental conditions, a
should now focus on providing the theoretical set of biofilm parameters (i.e. endpoints) may be
background for understanding the complex set used to assess the effects of pollutions under real-
of responses of natural communities to pollution. exposure scenarios (Fig. 7.1).
This knowledge should also be the basis for guid- It has been shown that the use of biofilms in
ing the selection of the most appropriate tools ecotoxicology is rather common, either in field or
and the development of new approaches for a laboratory investigations. The majority of studies
better detection of the impact of pollution on deal with metals and pesticides, but several inves-
aquatic life. tigations have recently been focused on emerging
compounds (Guasch et al., 2012). Here we aim to
update previous reviews and to provide a critical
overview of the most common endpoints used to
126  | Guasch et al.

assess the biological and ecological effects of pol- radiation (Navarro et al., 2008). The analysis of
lution, the results obtained in different exposure accessory pigments (e.g. β-carotene, diatoxanthin,
scenarios, and future trends in the use of biofilms diadinoxanthin, pheophytin, etc.) has also been
in ecotoxicology. shown to suitably detect early toxicity of com-
pounds targeting directly and/or indirectly the
photosynthetic apparatus (Laviale et al., 2010;
Biofilm ecotoxicology – a multi- Corcoll et al., 2012b,c; Bonnineau et al., 2013).
component approach Chronic exposure to contaminants exerts a
The biological composition of biofilms is very selection pressure on the community that may be
broad, including several types of communities: reflected by physiological changes at species level
algal, bacterial, fungal, protozoan and microin- (modifying its tolerance against contaminants) or
vertebrate communities, each of them including by changes in the abundance and composition of
a large list of species involved in many ecological algal communities (i.e. biomass, species composi-
processes. tion). PAM fluorescence, or high-performance
Owing to the prominent role that algae play liquid chromatography (HPLC), is used to
in biofilms growing in illuminated surfaces, the quantify the relative distribution of algal groups
majority of ecotoxicological investigations have (green, blue and brown algae) within a biofilm
focused on the algal component of biofilms based on photosynthetic pigments (e.g. Corcoll et
(reviewed in Corcoll et al., 2012a), while fewer al., 2012a,b). In biofilms, taxonomic community
studies have focused on the bacterial component identification is generally performed for diatoms
(reviewed in Proia et al., 2012a). Studies dealing (Chapter 6), a highly diverse, cosmopolitan class
with other biofilm components, such as fungi or of brown algae. Shifts in structure have led to
protozoa, in spite of their important role, have classifications based on species sensitivity/toler-
received little attention. ance to contaminants (Morin et al., 2009, 2012,
2014; Ricart et al., 2009). Specific morphological
Effects of pollutants on the endpoints, e.g. teratologies (Falasco et al., 2009)
autotrophic component of biofilms or cell sizes (Luís et al., 2011), have also proved
If light is available, algae and other phototrophic to detect metal pollution successfully. Algal
organisms become the main component of bio- taxonomy has been largely used to study toxicant-
films. Effects of pollutants have been investigated induced selection in biofilm communities, due
on both the function and structure of the auto- to its tradition but also its high sensitivity. More
trophic component of biofilms (Table 7.1). recently, molecular tools using DNA sequences
Among the functional descriptors used, photo- have been described as promising tools to assess
synthesis-related parameters are some of the most the prevalence of specific gene sequences in toler-
relevant endpoints for assessing toxicity towards ant communities and their taxonomic affinities in
algae. Pulse amplitude modulated (PAM) fluo- natural biofilms (Eriksson et al., 2009).
rescence techniques were developed to measure Quantitative real-time polymerase chain reac-
among other parameters, photosynthetic capacity tion (qPCR) techniques have been used with
and efficiency, and non-photochemical photosyn- success in the field of ecotoxicology in order to
thetic processes. These functional endpoints are assess the effects of various contaminants on dif-
largely applied to evaluate the effects of chemicals ferent diatom species (planktonic and benthic).
on biofilms for their sensitivity to a large panel of For instance, after the exposure of Thalassiosira
chemicals, especially those targeting photosystem pseudonana to PAHs, Bopp and Lettleri (2007)
II, like herbicides or certain metals (e.g. copper) observed strong up-regulation of lacsA, which is
(Serra et al., 2009; Ricart et al., 2009; Laviale et involved in the fatty acid metabolism and repres-
al., 2011). Fluorescence techniques are easy to sion of sil3, contributing to the formation of the
apply (for more details, see review by Corcoll et silica shell; highlighting then a possible impact of
al., 2012a) and are even useful for assessing the such compounds on these functions. In a different
impact of physical stressors, such as ultraviolet study, Guo et al. (2013) reported up-regulation of
Biofilms in Ecotoxicology |  127

Table 7.1 Summary of biofilm endpoints (in bold) and methods (in italics) in ecotoxicology
Functional responses at molecular, cell, Effects on the structure and
community or ecosystem level Changes in biomass architecture of the community

Autotrophic organisms
Photosynthesis: PAM, 14C-HCO3 uptake Chlorophyll concentration: Algal groups: microscope, HPLC
Tolerance induction: toxicity assays, DNA Algal density: microscope, Species composition: microscope
sequences flow cytometry
Diatom cell size, teratoforms:
microscope, flow cytometry
Genetic diversity: fingerprinting

C uptake: 3H-thymidne incorporation Bacterial density: Genetic diversity: fingerprint, FISH,
microscope, flow cytometry CARD-FISH, NGS
Respiration: substrate-induced respiration
Physiological profile: MicroRespTM
Antibiotic resistance genes

Respiration: substrate- induced respiration Fungal density: microscope Species composition: microscope
(mycelium growth)
Reproduction: sporulation Biomass: ergosterol Genetic diversity: fingerprint, NGS
Extracellular degradation of organic
matter: EEA by enzymatic assays or qPCR

Duplication rate: dynamics of cell density Cell density: microscope Cell damage: lysosomal membrane
stability, cytoplasmatic vacuolization,
Grazing activity and endocytotic rate: Species composition: microscope
clearance assays, intake of particles
Genetic diversity: fingerprint

Whole biofilm
CR, GPP and NPP: O2 change, MicroResp AFDM: weight of organic 3D structure: confocal microscopy
material after burning biomass
PO4 and NH4 uptake: nutrient addition DW: weight of the whole Accumulation and bio accumulation:
biofilm after drying biomass intracellular/total metal concentration,
total concentration of chemicals
Antioxidant response: antioxidant enzyme Contaminant transfer: food web
activities (AEA) experiments
Extracellular degradation of organic
matter: EEA by enzymatic assays
Leaf litter breakdown: biomass changes

PAM, pulse amplitude modulated fluorescence; HPLC, high-performance liquid chromatography; fingerprint, DGGE
(denaturing gradient gel electorphoresis); T-RFLP, terminal restriction fragment length polymorphism; FISH, fluorescence
in situ hybridization; NGS, next-generation sequencing; MicroResp, basal/substrate induced respiration; EEA,
extracellular enzyme activity; AEA, antioxidant enzyme activity; qPCR, quantitative polymerization chain reaction; AFDM,
ash-free dry mass; CR, community respiration; GPP, gross primary production; NPP, net primary production; DW, dry
The endpoints and methods used to assess the effects of chemicals can be specific to the different biofilm communities:
phototrophic organisms; bacteria; fungi and protozoa, or affect the whole biofilm. These methods provide information
about functional attributes (from molecular and physiological responses to biofilm-mediated ecosystem functions),
changes in biomass, effects on the community structure (e.g. community composition) and architecture (3D structure) of
biofilms or accumulation and trophic transfer of chemicals.
128  | Guasch et al.

heat shock protein 70/90 (HSP70 and HSP90) communities to degrade or mineralize organic
on the diatom Ditylum brightwellii after copper compounds (e.g. pesticides, pharmaceuticals or
and nickel exposure but not after exposure to endocrine disruptors) can also be viewed as a
endocrine-disrupting chemicals (BPA, PCB, and promising ecotoxicological tool (Paje et al., 2002;
endosulfan), revealing that these genes are differ- Pesce et al., 2009; Writer et al., 2011a, 2011b). In
entially involved in the defence response against addition to their functional impact, toxicants may
various environmental stressors. Moreover, gene affect the structure and diversity of biofilm bacte-
expression is an early and sensitive biomarker of ria. Those effects can be assessed quantitatively, by
toxicant exposure. Actually, qPCR tools are able determining bacterial cell densities using micros-
to reveal toxic effects, whereas other endpoints copy (Proia et al., 2011, 2012b) or flow cytometry
like growth inhibition are not (Bopp and Lettleri, (Villeneuve et al., 2011), and semi-quantitatively,
2007; Kim Tiam et al., 2012). They were also by using fluorescence in situ hybridization (FISH)
shown to respond at environmental concentra- and catalysed reported deposition-fluorescence
tions. Indeed Kim Tiam et al. (2012) observed in situ hybridization (CARD-FISH) to detect the
early differential expression of genes involved in impact of toxicants on community composition
regulation of mitochondrial metabolism (cox1, at a broad phylogenetic level (Brummer et al.,
nad5, 12S) and photosynthesis (psaA, d1) on the 2000; Lawrence et al., 2007; Proia et al., 2013a).
diatom Eolimna minima after exposure to cad- Toxicant effects on the bacterial community com-
mium concentrations of 10μg/l. position can also be evaluated by using molecular
fingerprint techniques (Dorigo et al., 2010; Tlili
Effects of pollutants on bacteria et al., 2010). New perspectives are now given by
Given the generally close link between bacterial next-generation sequencing (NGS) that provide
and algal production in stream biofilms (Scott et a more detailed characterization of community
al., 2008), effects of toxicants on biofilm bacterial composition and allow taxonomic identification
communities can be either direct, or indirect by of bacterial community members, as shown by
following changes in the autotrophic compo- recent studies aimed at assessing bacterial diver-
nent (Ricart et al., 2009; Proia et al., 2011). The sity on river biofilms using NGS-based approaches
functional response of biofilm bacteria to environ- (Besemer et al., 2012; Hall et al., 2012; Bricheux et
mental stressors can be evaluated using a large set al., 2013) (Table 7.1).
of global descriptors, including bacterial growth In the last decade, ecotoxicology has also been
(Lawrence et al., 2007), bacterial production, by focused on investigating the fate and effects of
measuring incorporation of radiolabelled thymi- antibiotics in nature. As an example, the preva-
dine (Paulson et al., 2000; Blanck et al., 2003), and lence of antibiotic resistance genes in bacteria of
bacterial survival rates (Ricart et al., 2010) (Table stream biofilms has recently been demonstrated
7.1). Toxicants can also affect biogeochemical pro- (e.g. Dutour et al., 2002; Fox et al., 2008; Marti
cesses associated with bacterial metabolism, such et al., 2013), as well as the effects of real mixtures
as organic matter decomposition and nutrient of antibiotics detected in the bacterial compart-
cycling. Such effects on biofilm bacterial commu- ment of highly impacted river biofilm (Proia et al.,
nities can be assessed through the measurement 2013a).
of extracellular enzyme activities (EEA) involved
in carbon, nitrogen or phosphorus acquisition Effects of pollutants on fungi
(Ricart et al., 2009; Tlili et al., 2010; Fechner et Evaluation of chemical stress in aquatic fungal
al., 2012), or through the measurement of gas communities has been mostly performed in leaf
production to evaluate basal or substrate-induced biofilms, because of the great fungal biomass
respiration (Tlili et al., 2011a,b), denitrifica- accrual (ca. 98% of total microbial biomass) and
tion (Chénier et al., 2006; Wang et al., 2014) or strong toxicant adsorption potential in this sub-
community-level physiological profile (Lawrence stratum. Responses of leaf fungal communities to
et al., 2004, 2007; Boivin et al., 2006; Tlili et toxicants are mostly evaluated through the litter
al., 2011b). The potential of biofilm bacterial breakdown (Moreirinha et al., 2011; Artigas et al.,
Biofilms in Ecotoxicology |  129

2012; Flores et al., 2014), a key ecosystem process to aquatic pollution. Compared to other aquatic
used as an indicator of functional stream integrity consumers, protozoa communities have a faster
(Gessner and Chauvet, 2002). Metals (e.g. copper physiological response and succession process
and zinc) and organic pesticides (e.g. azole fungi- (i.e. the replacement of species over time) due
cides) can depress litter decomposition (Duarte to their higher growth rate (Salvadó et al., 1995;
et al., 2008; Artigas et al., 2012) above a certain Nicolau et al., 2001; Zhou et al., 2008; Madoni,
threshold concentration. Toxicant effects may 2011). Indeed, protozoa are also affected by pol-
be based on the respiration (substrate-induced lutants. Heavy metals (Niederlehner and Cairns,
respiration) and reproduction (sporulation) activ- 1992; Madoni, 2000; Holtze et al., 2003; Díaz et
ities of the fungal community (Tlili et al., 2010; al., 2006; Martín-González et al., 2005; Rico et
Moreirinha et al., 2011). Functional descriptors, al., 2009; Ancion et al., 2013), ammonia (Nied-
such as cellulolytic (cellobiohydrolase), hemicel- erlehner and Cairns, 1990), pesticides (Shi et al.,
lulolytic (β-xylosidase) and ligninolytic (phenol 2013), polycyclic aromatic hydrocarbons, PAHs
oxidase) extracellular enzyme activities, have (Lara et al., 2007) and nanoparticles (Mortimer
been used to determine toxicant impairment et al., 2010) among other pollutants (Bringmann
on fungal capacities to degrade organic matter and Kühn, 1980; Nalecz-Jawecki et al., 1993; Seli-
and alter carbon cycling in rivers (Artigas et al., vanovskaya et al., 1997) have been demonstrated
2012). Methodological approaches based on gene to affect protozoa. The effects of each pollutant
regulation encoding for extracellular enzymes vary depending on its concentration and its expo-
(e.g. quantitative real-time PCR, Solé et al., 2012) sure time (Cairns and Pratt, 1993) and by the
have become promising tools to advance in the specific capability of each species to acclimatize,
understanding of molecular mechanisms control- to recover its population and to bioaccumulate
ling microbial activities involved in carbon cycling the pollutant (Martín-González et al., 2006). In
and mitigation of environmental pollution (e.g. that sense, the study of structural and functional
pesticide degradation). From a structural point attributes of the protozoa community provides
of view, the density and taxonomic composition several useful endpoints for assessing pollution
of aquatic hyphomycete communities (dominant in aquatic ecosystems. Effects of pollutants have
in submerged leaves) are shown to be sensitive been observed on protozoa richness (Gracia et
to heavy metals (Duarte et al., 2008) and organic al., 1994; Fernandez-Leborans and Novillo, 1995;
pesticides (Bundschuh et al., 2011). Genetic Nicolau et al., 2005) or species composition
approaches (including fingerprint, and NGS- (Fernandez-Leborans and Novillo, 1995; Canals
techniques) are considered as useful tools to et al., 2013), e.g. the stalked ciliate Opercularia
identify toxicant effects in aquatic hyphomycete spp is normally associated to stressed or polluted
communities (Moreirinha et al., 2011; Artigas ecosystems. In addition to classical endpoints,
et al., 2012; Tolkkinen et al., 2013; Flores et al., such as mortality (Bergquist and Bovee, 1976;
2014), but in situ approaches are lacking regarding Salvadó et al., 1997) or duplication rate (Salvadó
the literature. In parallel, the use of stable isotope et al., 1997; Gomiero et al., 2012), effects of pol-
probing techniques (optimized for soil microbial lutants on cell viability (e.g. Nalecz-Jawecki et al.,
communities, Park et al., 2006) are promising 1993; Salvadó et al., 1997; Mortimer et al., 2010),
tools to identify populations capable of degrading grazing activity or endocytotic rate (Ke) (Nico-
pollutants and, therefore, of comprehending the lau et al., 2001; Gomiero et al., 2012) have also
adaptation potential of fungal communities in been measured. Finally, effects of toxicity at cel-
contaminated ecosystems including their use in lular level, such as lysosomal membrane stability
bioremediation (Table 7.1). (Gomiero et al., 2012), cytoplasmatic vacuoliza-
tion and mitocondrial degeneration have also
Effects of pollutants on protozoa been observed (Martín-Gonzalez et al., 2006).
As unicellular organisms associated to biofilms, In addition to classical methods based on micro-
protozoa are closely in contact with the sur- scopic analyses, fingerprinting techniques, such as
rounding environment and show high sensitivity denaturing gradient gel electrophoresis (DGGE)
130  | Guasch et al.

and terminal restriction fragment length polymor- al., 1998; Sabater et al., 2002; Meylan et al., 2004;
phism (T-RFLP), are gaining greater prominence, Lundqvist et al., 2012), but also by biological
as these approaches are increasing our knowledge proprieties of the biofilm, such as its age, thick-
of the complexity of biofilm protozoa communi- ness or EPS composition (Headley et al., 1998;
ties (Dopheide et al., 2008, 2009). Nevertheless, Lawrence et al., 2001). Bioaccumulation kinetics
combining microscopic and molecular analyses is of chemicals are rather complex and depend on
recommended to obtain further information. the substance’s chemical properties, as well as
on uptake mechanisms that may be passive and/
or active. Metal bioaccumulation in biofilms has
Ecotoxicological responses of been studied extensively, and is described as a
the whole biofilm two-step process. Metals are first adsorbed extra-
Biofilms are not only an assemblage of aquatic cellularly (in the EPS or onto cell surfaces), before
organisms but ubiquitous complex structures being absorbed into cells by uptake mechanisms
with a large proportion of non-living organic and (Holding et al., 2003). Intracellular and total
inorganic matter with a high adsorption capac- metal content in biofilms can be measured easily,
ity. While many ecotoxicological investigations to improve the description of exposure (Meylan
focus on the effects of chemicals on specific et al., 2003; Morin et al., 2008a; Serra et al., 2009).
compartments of the biofilm, endpoints provid- In spite of the expected variability in bioaccu-
ing information about the effects of chemical mulation capacity of biofilms, a large number of
exposure on the whole biofilm, such as bioaccu- studies reported a strong relation between metal
mulation, oxidative stress or nanoparticle toxicity, bioaccumulation and changes in the structure,
are also interesting (Table 7.1). Investigations composition and function of algal and bacterial
describing processes at biofilm scale, like primary communities living in biofilms (Duong et al., 2008;
production and nutrient dynamics, provide the Morin et al., 2008b; Ancion et al., 2010; Bonet et
opportunity of upscaling the effects of pollutants al., 2012; Corcoll et al., 2012c). Studies reporting
on biofilms to ecosystem functioning. herbicide bioaccumulation in biofilms are rather
numerous (Headley et al., 1998; Lawrence et al.,
Accumulation of pollutants in 2001). However, the investigations of the link to
natural biofilms toxicity are scarce, probably because of the highly
Total concentrations of chemicals in water complex and diverse toxicokinetics of these com-
fluctuate in time, and do not always reflect the pounds and the impossibility to separate between
integrated exposure to water chemicals of organ- intracellular and extracellular accumulation. More
isms living in that environment, thus complicating recently, several authors have reported the bioac-
the establishment of direct relationships to toxic- cumulation of pharmaceuticals and endocrine
ity. Monitoring chemical bioaccumulation may disruptors in biofilms (Writer et al., 2011a, 2013;
overcome this problem because it can represent Wunder et al., 2011). However, their link to tox-
real bioavailability and exposure. Thus, the icity on biofilm is still not confirmed. As many
accumulation of pollutants in biofilms can be con- compounds susceptible to provoking deleterious
sidered the first step in the exposure of microbial impacts on the biota are likely to be accumulated
organisms living in the biofilm matrix and of those in biofilms (e.g. Lawrence et al., 2001; Sabater,
placed at higher trophic levels. In addition, it can 2003), measuring toxicant concentrations in this
also be considered as a detoxification pathway ‘natural passive sampler’ – the biofilm – may be a
(see Chapter 10). valuable alternative to traditional chemical moni-
Bioaccumulation of chemicals in biofilms is toring. This measure would provide ecologically
influenced by several interacting physical and relevant information about the potential risk of
chemical parameters of the environment like contaminants for the aquatic ecosystem and may
current velocity, temperature, pH, nutrients and be especially useful and reliable for those com-
organic matter concentration in water or the pounds not undergoing metabolization into the
hydrophobicity of each compound (Headley et biofilm (e.g. metals).
Biofilms in Ecotoxicology |  131

Detecting biofilm under oxidative inhibition) observed in the majority of individuals

stress and species within the community (Bonnineau et
Chemical contamination in biofilm is likely al., 2012).
to induce direct or indirect oxidative stress by Biofilm AEAs have been used at different
enhancing reactive oxygen species (ROS) produc- scales in both laboratory and field studies, mainly
tion or impairing cellular antioxidant responses. to determine the antioxidant response of the
The resulting excess in ROS can provoke lipid community to a specific chemical. For instance,
peroxidation, membrane disruption, alteration in several studies, AEAs have been found to be
in cell structures and mutagenesis (Scandalios, more sensitive to contaminant than traditional
1993; Mittler, 2002; Edreva, 2005; Wolfe-Simon biomarkers such as photosynthetic parameters
et al., 2005; Lesser, 2006). Though oxidative stress (Dewez et al., 2005; Guasch et al., 2010b; Bonet
can be specifically induced by some toxicant (e.g. et al., 2013, 2014). Measuring AEA response
copper), it can also result from general metabo- throughout a gradient of oxidative stress can also
lism alteration and thus indicates a low ‘health’ provide information on the antioxidant capacity
status of biofilm. Therefore, the detection of of the community. Indeed, AEAs are expected
oxidative stress damage and response within the to increase with increasing oxidative stress until
whole biofilm community is expected to provide ROS overcomes the cell defence system and AEAs
information on biofilm stress status and its ability eventually decrease due to cellular damage. From
to cope with further oxidative stress (Bonnineau this unimodal (bell shape) pattern of response,
et al., 2013) a range of oxidative stress levels by which AEAs
Lipid peroxide quantification is a common increased can be defined; within this range the
measure of cellular oxidative damage that can be community is expected to be able to alleviate
estimated at community level. For instance, Vera oxidative stress. This range defines the antioxidant
et al. (2012) used the thiobarbituric acid-reactive capacity of a community and is influenced by vari-
substances (TBARS) assay to show how exposure ous parameters (e.g. biofilm age, pre-exposure to
to an environmentally relevant concentration contamination). For instance, chronic exposure
of a glyphosate formulation provoked oxidative of biofilm to the herbicide oxyfluorfen led to an
damage in the biofilm community. increase in biofilm CAT capacity. Biofilms chroni-
Nevertheless, most of the recent work has cally exposed to oxyfluorfen were able to respond
been focused on biofilm antioxidant capacity, to higher concentrations of oxyfluorfen by an
rather than on oxidative damage. In fact, to keep increase in CAT activity while in non-adapted
the oxidative balance under control, organ- biofilms (those not previously exposed), CAT
isms have non-enzymatic mechanisms (e.g. activity decreased in response to acute exposure
glutathione, carotenoids and phenolics; Okamoto to high levels of oxyfluorfen, probably because of
et al., 2001) as well as enzymatic mechanisms oxidative damage (Bonnineau et al., 2013).
(e.g. glutathione-S-transferase: GST, catalase: Since oxidative stress can be greatly influenced
CAT, ascorbate peroxidase: APX, glutathione by environmental parameters, such as light or tem-
reductase: GR and superoxide dismutase: SOD perature (Butow et al., 1997; Aguilera et al., 2002;
activities). In particular, several authors have pro- Li et al., 2010), both laboratory and field studies
posed using antioxidant enzyme activities (AEAs) are needed to better understand AEAs responses
as biomarkers of pollution due to their capacity to and interpret their variations (Bonnineau et al.,
respond to both organic and inorganic pollutants 2013). For instance, Bonet et al. (2012) showed
(Valavanidis et al., 2006; Guasch et al., 2010a,b; that, under controlled conditions (microcosm
Maharana et al., 2010; Bonnineau et al., 2011; study), APX clearly decreased due to Zn expo-
Bonet et al., 2012, 2013, 2014). In biofilms, AEAs sure while, in the field, the inhibition of GST was
are defined as a global indicator of the ‘health’ shown to be a biomarker of Zn exposure (Bonet et
status of the whole biofilm, then considered as a al., 2013, 2014). These differences were attributed
black box. AEA measurement at community level to variations in environmental parameters and a
is expected to reflect the tendency (activation or specific effort has been made to better understand
132  | Guasch et al.

field variability. In an annual monitoring, Bonet present in the environment. Among other effects
et al. (2013) observed that AEA followed the in rivers, ENPs may impact on photosynthetic
seasonality of the system, changing as a response organisms. The ENPs have direct and indirect tox-
to light and water temperature fluctuations. How- icological effects on different organisms present
ever, seasonality was not observed in the polluted in biofilms (Navarro et al., 2008). The physical
site, where Zn masked this pattern of variation. characteristics of ENPs facilitate their transport
Detecting oxidative stress in biofilms provides in suspension. In addition, their large density, as
information on oxidative damage (e.g. Lipid per- that of metallic nanomaterials and their surface
oxidation), antioxidant responses and antioxidant properties, which may enhance agglomeration
capacity of the community. These markers of processes, may provoke sedimentation under
oxidative stress can be used to detect alteration reduced hydrodynamics. This process will deposit
within biofilm community due to pollutant expo- ENPs in biofilms where biouptake may take place,
sure but also to environmental variations (e.g. thus leading to toxic effects. In addition, since bio-
climate change) (Bonet et al., 2013). film ecotoxicological testing can be done under
the controlled conditions of micro or mesocosms
Differential biofilm gene expression (artificial channels), certain methodological prob-
In aquatic ecosystems, biofilm ecotoxicology lems associated to ENP experimentation, mostly
has been used to investigate contaminant effects related to the lack of control and characterization
at different levels of biological organization, of ENPs and the environmental conditions pre-
from species composition to biogeochemical vailing during the exposure of the organisms, will
processes. New approaches suggest going even be avoided (Handy et al., 2012).
deeper within biofilm and investigating structure
and function at molecular level. Differential gene
expression has been studied until now on single Biofilm ecotoxicology –
species (i.e. diatom cultures). Nevertheless, the link between pollution and
tools developed for diatoms are extremely prom- ecosystem health
ising and in the future could be expanded to the Biofilms are considered biological entities which
whole biofilm. The qPCR tools have been tested play a key role in ecosystem functioning, and are
with less success at the community level (i.e. a in turn very sensitive to chemical exposure. Inves-
biofilm composed of different diatom species tigations aiming to describe processes at biofilm
and other organisms), using these specific gene scale like nutrient dynamics and those including
sequences principally because of the lack of avail- simple food chains, are common in ecological
able nucleotide sequences of such organisms in research but less used in ecotoxicology. These
genomic databases (Tiam, unpublished data). approaches have recently been applied, providing
the opportunity of upscaling the effects of pollut-
Biofilms in nanoparticle ants on biofilms to food webs and ecosystems.
Experiments with biofilms are an optimal target Upscaling biofilm responses to
for assessing the environmental risks related to ecosystem processes
new emerging toxicants such as nanoparticles. Biofilm communities are composed of many
As biofilms grow on submerged surfaces, they microbial species with a key role in ecosystem
are especially exposed to engineered nanopar- functioning offering important insights regarding
ticles (ENPs). Nanotechnology development mechanisms occurring from the single cell level
is leading to a proliferation of products that are to biogeochemical processes at a larger scale by
likely to become a source of many different engi- mediating processes, such as oxygen production,
neered nanoparticles (ENP) in the environment, nutrient uptake and organic matter transforma-
where their fate, behaviour and effects are mostly tion (Battin et al., 2003; see Chapter 5). In fluvial
unknown. Their nano-size allows these materi- systems, for example, combining community-
als to interact at molecular scale with organisms scale (such as mesocosm experiments) and
Biofilms in Ecotoxicology |  133

2 3



5 100 %

50 %

0 %
0 10
1 102 103 104

Figure 7.1 Conceptual framework for the use of freshwater biofilms in aquatic ecotoxicology. Water
samples (1) from a river affected by chemical pollution are analysed (2). The chemicals present in the water
(3) are classified according to their mode of action (different shaded colours), and a reduced number of
chemicals representing the different modes of action (represented by different colours) are selected (4).
In parallel, artificial substrata (5) are placed at the sampling points, and during a few weeks or months
(depending on the type of community and local conditions), the environmental variables are monitored (6).
This information can be used later during the experimental design, to study their role as modulating factors
of the measured ecotoxicity. After the colonization time, the substrata containing the freshwater biofilms
are sampled (7), some are preserved for analytical purposes (8), and others are used for dose–response
experiments (9). Results are plotted and modelled (10) and the relevant concentrations measured (as the
Effective Concentration reducing by 50% the biological parameter measured: EC50). In addition, biofilms can
be processed to extract (11) and analyse (2) the chemicals adsorbed and/or bioaccumulated. The list of the
detected chemicals (12) can complement the information obtained from water samples (3).

whole-reach scale (field or ecosystem) measure- industrial discharge on biofilm functioning.

ments together with the characterization of the These authors found a rapid shift (one week)
structure and function of biofilms contribute to from autotrophic- to heterotrophic-dominated
a better understanding of the effects of human metabolism when the communities from the
activities on ecosystem processes. non-impacted site were translocated to impacted
Rosi-Marshall et al. (2013) in a field study, sites. In contrast, when communities were trans-
using Nutrient Diffusing Substrata (NDS) found ferred back to the reference site, the recovery to
that a mix of pharmaceuticals produced changes autotrophy took up to four weeks (Sierra and
in biofilm metabolism parameters, consisting Gómez, 2010).
of a decrease in community respiration (CR) Hill et al. (1997) used chambers to measure
and gross primary production (GPP). Another benthic metabolism in a rocky mountain stream
study used field translocation between impacted which had elevated metal pollution and found
and non-impacted sites to assess the effects of that GPP and net primary productivity (NPP)
134  | Guasch et al.

decreased with increasing metal concentrations These studies exemplify how classical biofilm
by one order of magnitude from the reference site processes, such as nutrient uptake and community
to the most impacted site. The effects of different metabolism commonly investigated in ecosys-
pharmaceuticals on biofilm metabolism and nutri- tem ecology, are sensitive tools for assessing the
ent uptake were assessed in vitro and in situ (using ecotoxicological effects of pollutants on fresh-
NDS in the field) in a central Indiana river (USA). water communities and ecosystems. In addition,
The in vitro experiments showed that ammonium addressing these endpoints allows the ecological
uptake was reduced after exposure to nicotine relevance of the observed effects at different levels,
and caffeine, and nitrate uptake was increased by from community to whole ecosystem scales, to be
nicotine exposure, while no effects were observed increased.
on microbial metabolism. On the other hand, an
in situ experiment showed that nicotine increased Biofilms in ecotoxicological food
microbial respiration (Bunch and Bernot, 2011). web studies
Nutrient uptake was also used to assess the effects Studies based on food-web relationships between
of metals on fluvial biofilms. Serra et al. (2009) biofilms and their grazers provide a high degree
found a slight decrease in phosphate uptake after of environmental realism. Due to the increased
chronic copper exposure of the biofilm to 26 µg/l complexity, these studies allow us to assess the
in artificial channels. In another mesocosm study, responses of communities and within communi-
Proia et al. (2011) showed that triclosan (60 µg/l) ties and the evaluation of the direct and indirect
inhibited biofilm phosphate uptake up to 71% effects of pollutants at different trophic levels
and uptake rates did not recover until two weeks (Culp et al., 2000; Geiszinger et al., 2009).
after the end of exposure. The negative effect of These investigations are mainly performed
triclosan on biofilm capacity to uptake phosphate in experimental conditions in order to ensure
was confirmed in other investigations using control of environmental variables (Fig. 7.2), but
microcosms and revealed the persistence of this some field studies also exist. Literature reviews
effect over time (Proia et al., 2013b; Guasch et al., provide interesting experimental models (Culp
in press). et al., 1996; Ledger et al., 2009). In most cases,

Figure 7.2  Experimental settings of biofilm ecotoxicology. Biofilm communities growing on artificial substrata
(e.g. sandblasted glass substrata) are exposed to chemicals under controlled conditions. Exposure can be
done in (A) crystallizing dishes (1.5 l volume) with recirculating water; (B) recirculating indoor channels (1 m
long); (C) one flow through indoor channels (2 m long). (D) detail of a snail (grazer) placed on top of biofilms
in a grazing experiment. (E) one flow through outdoor channels (5 m long).
Biofilms in Ecotoxicology |  135

food-web experiments involve biofilms and a bioavailability and bioaccumulation of insecti-

grazer in order to study biomagnification and cides.
transfer of the test substance from primary pro- The presence or absence of grazers can inter-
ducer to consumer. Other experiments address fere in the effects of toxicants on functional or
the possible additional effects of grazing pressure structural characteristics of biofilms. Muñoz et al.
on a chemically stressed biofilm or the possible (2001) studied the effects of atrazine in a single
indirect effects of pollutants on grazers or biofilms food web and described reduction of carbon
due to toxicant-induced alterations of ecologi- incorporation and algal diversity in biofilm due
cal relevance. Generally speaking, insects and to the interaction of grazers (Physa acuta) with
molluscs have been used as grazers. Food-web the herbicide. Evans-White and Lamberti (2009)
experiments with biofilms have been applied observed that toxicants in combination with graz-
in order to study the ecotoxicological effect of ers increased chlorophyll concentration and algal
pesticides (Muñoz et al., 2001; Real et al., 2003; diversity. On the contrary, similar experiments
López-Doval et al., 2010; Lundqvist et al., 2012), with food webs did not find interactive effects of
metals (Irwing et al., 2003; Conley et al., 2011; grazing and toxicants on biofilm (Real et al., 2003;
Xie and Buchwalter, 2011; Kim et al., 2012; Li et López-Doval et al., 2010). Indirect effects on
al., 2012), nanoparticles (Kulacki et al., 2012) and the structure and function of biofilms have been
emerging pollutants (Evans-White and Lamberti, observed as a consequence of the changes in the
2009), among other compounds. physiology and behaviour of P. acuta induced by
Several authors demonstrated the importance the toxicant (Evans-White and Lamberti, 2009).
of biofilms in the introduction of toxicants in the Overall, it is reasonable to expect that grazing
food web by means of food-web experiments. may influence the response of biofilms to toxic
In the case of zinc, bioaccumulation in biofilm, exposure. Communities suffering both grazing
metal transfer and bioaccumulation in the grazer pressure and the effects of toxic substances will
Centroptilum triangulifer were shown (Kim et al., have less ability to overcome grazing effects than
2012). Irwing et al. (2003) demonstrated that non-exposed communities, because toxicity will
mayflies grazing on biofilms contaminated with limit algae regrowth and facilitate the extinction
cadmium showed significant inhibition in growth of the less abundant species after grazing. This
and feeding in comparison to those exposed to interaction may have remarkable ecological impli-
contaminated water. Xie and Buchwalter (2011), cations since grazing pressure will magnify the
using biochemical responses in the mayfly C. tri- negative effects that toxicants exert on ecosystem
angulifer, confirmed that cadmium is more toxic by processes, such as primary production and nutri-
ingestion of contaminated biofilm than by direct ent cycling (Fig. 7.3).
exposure to contaminated water. Experiments
with food webs demonstrated that high nutri- Environmental factors modulating
tional quality and quantity of available biofilm biofilm response to pollutants
diminish the toxicological response of mayflies to In the field, environmental conditions are highly
selenium (Conley et al., 2011). Bioavailability of variable and organisms are rarely under optimal
pollutants is modulated by the influence of envi- conditions. There is growing awareness that these
ronmental factors on biofilm, as demonstrated abiotic parameters can strongly constrain ecosys-
with food-web experiments. Increasing levels of tem responses to anthropogenic contamination
phosphate enhanced bioaccumulation of copper (Fisher et al., 2013). Nevertheless, their influ-
in biofilms and dietary toxicity to the amphipod ence is rarely taken into account in single-species
Hyalella azteca (Li et al., 2012). In an experiment ecotoxicological tests. Indeed, single species have
with freshwater snails and biofilms, Lundqvist et a limited range of acclimation to environmental
al. (2012) reported that dissolved organic matter parameters and studies performed at community
in water interferes in the sorption of pesticides level appear to be better suited for investigating
(carbofuran, lindane and chlorpyrifos) to bio- the influence of environmental factors on con-
films and is, therefore, a factor that can modulate tamination effects (Clements et al., 2009). In
136  | Guasch et al.

species-function 1
species-function 2
species-function 3 grazing toxicity
species-function 4
↓ photosynthesis
↓↓ Biomass ↓ sensitive spp.
grazing +
↑ photosynthesis


Recovery Lower biomass Selection of

Lower amount of tolerant species
species and loosing ecosystem
ecosystem functions functions

Figure 7.3  General model of the individual and combined effects of grazing and toxicity on biofilms. Based
on a simple four-species biofilm model, it is expected that toxicity will constrain the ability of the community
to recover from grazing pressure. In addition to the selection pressure exerted by toxicity, causing a
reduction in activity (i.e. photosynthesis) and an increase in the relative abundance of the most tolerant
species (sp1 and sp3), the reduction in population size caused by the non-selective effect of grazing on
biofilms, will increase the risk of extinction for the less abundant species. Overall, grazing and toxicity will
have cumulative negative effects on biofilms causing a reduction in the number of species, the biomass and
ecosystem functions.

aquatic ecosystems, environmental parameters, While flow regime can affect chemical bioavail-
such as light intensity, flow regime or temperature, ability (Osorio et al., 2014), this highly variable
strongly influence biofilm structure and function abiotic factor can also modulate biofilm structure
(Chapter 1) and these factors can have a critical and function (Graba et al., 2013). Therefore, the
effect on biofilm community response to contami- flow regime under which biofilm is grown is also
nation (Fig. 7.4). susceptible to alter the capacity of biofilm to cope
Light intensity and regime is highly variable in with chemical toxicity. For instance, a simulated
the field due to seasonal variations and/or changes drought event in artificial streams reduced biofilm
in riparian vegetation. Nevertheless, light is the first capacity to recover from a subsequent 48 hour
energy source for the autotrophic component of exposure to a bactericide (87 µg/l of triclosan)
biofilm and therefore modulates not only biofilm at both structural (high bacterial mortality) and
structure and function but also biofilm response functional level (reduced phosphate uptake)
to herbicides and metals, as shown by several (Proia et al., 2013b). Villeneuve et al. (2011) also
authors at both laboratory and field scale (Guasch showed that biofilms grown under a turbulent
et al., 2003; Laviale et al., 2010; Bonnineau et al., flow regime have a higher sensitivity to pesticides
2012; Bonet et al., 2013). Not only was biofilm than biofilms grown under a laminar flow regime.
grown under high light intensity more sensitive to The influence of other factors like sediment
the herbicide atrazine (field study, Guasch et al., deposition (Magbanua et al., 2013), temperature
2003), but it was also more tolerant to glyphosate (Larras et al., 2013), nutrient concentration (Tlili
(laboratory study, Bonnineau et al., 2012). et al., 2010) or salinization (Rotter et al., 2013)
Biofilms in Ecotoxicology |  137

Figure 7.4  Interactions between environmental factors and contamination in river biofilms. The main abiotic
factors modulating biofilm structure and function are indicated in a schematic view of a biofilm (adapted
from Romani, 2010). Environmental parameters and toxicants are likely to affect similar biofilm parameters,
as indicated in the table; the expected negative impact of a toxicant is indicated by an arrow.

on biofilm response to pollutants has also been On the one hand, our ability to quantify the
investigated (Fig 7.4). effects of pollution on different biofilm com-
These previous studies have shown how ponents, allows us to evaluate the direct effects
environmental parameters can constrain com- of pollutants on the most sensitive community
munity capacity to respond to a pollutant but (e.g. algae in the case of herbicides or bacteria
also to recover from contamination exposure. for antibiotics) and also their indirect effects on
To better understand ecosystem responses to the rest of biofilm components and on higher
contamination, it is essential to take into account trophic levels because all of them are closely
these parameters in toxicity assessment. Since the related through biological interactions. For exam-
influence of abiotic factors on biofilm structure ple, the model presented for biofilms exposed to
and function has been intensively investigated toxicants under grazing pressure exemplifies the
in ecology, the use of biofilm in ecotoxicology advantage of using complex biological models
appears then as a realistic approach, in which like biofilms and their grazers to improve our
environmental parameters can be integrated into ability to predict the effects of pollution in
toxicity assessment. multiple-stress scenarios (Fig. 7.3). On the
other hand, enormous progress has been made
regarding sensitivity. The application of early
Conclusions and future warning systems, for example the study of AEAs
recommendations in whole biofilms, may allow us to detect early
Biofilms are nowadays one of the principal tar- responses of the community by the activation
gets of community ecotoxicology with a high of mechanisms of defence towards toxicity. In
potential for future uses in ecotoxicology. A large terms of analytical chemistry, different methods
set of methods derived from biofilm ecology have been refined to quantify low concentrations
have successfully been applied in ecotoxicology of a large panel of chemicals in biota, including
providing a diverse and comprehensive toolbox. biofilm samples. In addition to metals, recent
138  | Guasch et al.

investigations have shown that many organic progress of genetics. As an example, metagen-
pollutants have a tendency to adsorb and/or be omics is envisaged as a promising approach for
uptaken in biofilms, acting as ‘natural passive targeting the effect of contaminants on specific
samplers’. Biofilms are also a site for biotrans- biofilm functions, and the microorganism respon-
fomation and/or transfer of chemicals to other sible behind them.
aquatic organisms, supporting a more general- While biofilm ecotoxicology studies have
ized use of biofilm samples in environmental inherited the methods and basics of biofilm
chemistry. This methodological progress is also ecology and community ecotoxicology, a general
visible in terms of new applications like the use framework to formulate a hypothesis about the
of biofilms to investigate nanoparticle toxicity. response of this model of an aquatic community
The set of biofilm endpoints described (Table to human perturbations is still lacking. As shown
7.1) provides a powerful toolbox covering the in this review, a large set of methods has been
expected responses of biofilms to pollution at refined and validated. Bearing this in mind, bio-
different temporal scales: from early responses film ecotoxicology should now focus on providing
to acute exposure (e.g. by the activation of the theoretical background for understanding the
mechanisms of detoxification, the inhibition complex set of responses of natural communities
of photosynthesis or respiration), to long-term to pollution. This knowledge should also be the
effects after chronic exposure (e.g. extinction basis to guide the selection of the most appropri-
of the most sensitive species and changes in the ate tools and the development of new approaches
whole community structure). It is important to for a better detection of the impact of pollution on
highlight the potential that different molecular aquatic life.
approaches may have on our ability to detect the
effects of pollution on the diversity of species of Acknowledgements
the different biofilm components (Table 7.1). This study has been carried out with financial sup-
In contrast to the study of some of the biofilm port from the French National Research Agency
components, such as algae, with a long tradition (ANR) in the framework of the Investments
in taxonomy (i.e. the use of diatom species com- for the Future Programme, within the Cluster
position as biological indicators; see Chapter 6), of Excellence COTE (ANR-10-LABX-45) and
assessing the effects of toxicity on the species ANR CESA PoToMAC (ANR-11-CESA-0022).
composition of other biofilm components is Financial support was also provided by the Span-
less common (i.e. bacteria). In this regard, the ish Science and Education Ministry (project
application of molecular tools may contribute to CTM2009-14111-CO2–01) and the Spanish
overcome this limitation, understood, however, Economy and Competitiveness Ministry (project
as a complement of rather than a substitute for CGL2013–43822-R).
microscope observation classical taxonomy.
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Biofilm Development in Sewer
Oriol Gutierrez, Guangming Jiang, Keshab Sharma and Zhiguo Yuan

Abstract wastewater from its source to the point where it

Wastewater collection systems, or sewers, are is discharged. The discharge point is usually a
crucial sections of the urban water cycle where Wastewater Treatment Plant (WWTP) but may
complex microbial, chemical and physicochemi- also be natural environments (Fig. 8.1). Sewer
cal processes take place. This chapter aims to give systems are crucial in protecting public health as
an overview of the diversity and importance of these prevent the spread of diseases by avoiding
biofilms and bioreactions occurring in sewers, population exposure to the contaminated waste-
paying special attention to its detrimental effects. water. By definition, wastewater is water that has
Sewer biofilms can be divided in two main classes: been adversely affected in quality by anthropo-
genic influence, either from domestic households
1 Submerged biofilms: including activities of (such as showers, toilets and washing machines)
sulfate-reducing bacteria (SRB) responsible or from industrial processes (Metcalf and Eddy,
for the formation of sulfide (H2S, an odorous, 2003). Thus composition of wastewater, although
toxic and corrosion-inducer compound), contains more than 99% of water, varies widely
Methanogenic Archaea (MA) responsible for depending upon the source. Sewage can contain
the formation of methane (CH4, an explosive suspended solids (that can create sludge deposits
and potent greenhouse gas) and the Fer- and anaerobic conditions in sewers), biodegrada-
mentation processes that increase the two ble organics (proteins, carbohydrates and fats that
previous biofilms metabolism. can lead to the depletion of oxygen in water and
2 Unsubmerged biofilms: activities of biofilms develop septic conditions), pathogens, nutrients
growing on the gas phase of sewers that (nitrogen and phosphorus), priority pollutants
causes loss of concrete mass, cracking of (organic and inorganic compounds with acute
the sewer pipes and ultimately, structural toxicity, and heavy metals from industrial pro-
collapse. This process is known as microbially cesses) and dissolved inorganics (such as calcium,
induced concrete corrosion (MICC). sodium and sulfate).
Sewers are very important assets of the urban
The structure of sewer-biofilms and mecha- water systems. For instance, in Spain only, the
nisms for the control of its harmful effects are sewer networks have an extension of around
described. 89,900 kilometres (equivalent to twice the equa-
tor distance) that collect the wastewater generated
from 8110 municipalities, covering 86% of the
Introduction total population. Similarly, the length of sewer
Wastewater collection systems, or sewers, consist pipes are 1,200,000 and 117,000 km in the USA
of an underground network of physical struc- and Australia, respectively (AWA, 2011; US EPA,
tures-installations composed of pipelines, pump 1991). The total asset value of these networks is
stations, manholes and channels that convey the estimated to be about one trillion dollars in the
146  | Gutierrez et al.

Figure 8.1  (A) Scheme of the urban sewer system; (B) picture of sewer trunk main in Paris (France), courtesy
of; and (C) distribution of a sewer network in the domestic suburb of Burleigh Heads,
courtesy of Gold Coast City Council (Australia).

USA and $100 billion in Australia (Brongers, gravity is not possible, pump stations need to be
2001). employed. Pump stations are normally installed at
Depending on the topography, sewers are low elevation points of the sewer network in order
classified to include two types of pipes: gravity to pump the sewage up through a pressure main
sections and pressure mains (Fig. 8.2). Gravity to another gravity pipe, to convey wastewater over
sewers are used to collect wastewater from multi- a hill, and/or in case of nearly flat terrains up to a
ple sources and convey the wastewater by gravity treatment facility. Depending on the topography
to a central location. Gravity pipes have sufficient of each catchment, gravity pipes or pressure mains
slopes to keep the wastewater flowing naturally may be predominant in a sewer network.
through the system without having excessive Sewers have been traditionally considered
solid deposition. Whenever wastewater has to be only as a system for hydraulic transport of sewage.
transported to a higher location and flow under However processes occurring in sewer systems are

Pressure sewer

station Gravity sewer

Figure 8.2  Sewer network types of pipe depending on topography.

Biofilm Development in Sewer Networks |  147

much broader than solely the hydraulic processes. Biofilms and biological reactions have
Sewers are ‘reactors’ where complex microbial, important impacts on sewers functioning.
chemical and physicochemical processes take Microorganisms are widely present in the waste-
place. The complexity of those reactions depends water and are exposed to a range of substrates
very much on the inherent sewer features such as: that contain fractions both of organic matter and
inorganic compounds (Metcalf and Eddy, 2003).
1 Wastewater matrix, which includes a diver- Sewer biofilms grow attached to sewer surfaces
sity of microorganisms and pollutants (such including walls, sediments and other physical sup-
as organic matter, nutrients and particles) ports (Hvitved-Jacobsen, 2002). They take the
varying with the location and time within the form of a concentrated layer of microorganisms
network. with self-imbedded matrix of extracellular poly-
2 Presence of different sewer phases such meric substances that hold together in a shape of
as suspended wastewater phase, biofilms, slime. According to Hvitved-Jacobsen (2002),
sediments and surface of the sewer in contact sewer biofilms typically have a water content of
with the sewer atmosphere (Fig. 8.3). 70–90%, 50–90% of organic matter and a rela-
3 Microbial processes that occur under tively high content of carbohydrates and proteins.
changing environmental conditions such Different types of microorganisms will prevail
as aerobic (presence of dissolved oxygen in particular sections of sewers depending on
in wastewater), anoxic (presence of nitrate hydraulic features and wastewater composition.
or nitrite), anaerobic (absence of oxidant Hydraulic features are related to the reareation
compounds in the wastewater) and at zones of the pipes and include turbulence flow of
different level of redox potentials. sewage, ventilation of the systems and depth of
the wastewater column. Wastewater composition
Fig. 8.3 presents the most important processes characteristics are related to substrate availability,
occurring in sewer systems. It can be seen that dissolved organic matter, temperature, pH and
some processes are biologically mediated by redox of sewage.
biofilms. The diversity and importance of bioreac- This chapter aims to give an overview of the
tions in sewers is going to be described in sections most common biofilms found in sewers, paying
below. special attention to its detrimental effects.

Urban atmosphere


Wastewater Sludge
Sewer Atmosphere
Wastewater from Treated
Mass transfer
households & industry water

Water Phase Receiving

Mass environment
Runoff water from transfer Overflow
urban surfaces
Sediments Biofilm

Infiltration Groundwater
and soils

Figure 8.3  Wastewater flows and processes occurring in sewers.

148  | Gutierrez et al.

Submerged sewer biofilms thermodynamically efficient electron acceptor.

dynamics The anaerobic sulfate respiration is carried out
As widely explained in this book, biofilms need by a functional group of bacteria commonly
the presence of water, therefore sewer biofilms called sulfate-reducing bacteria (SRB) under
develop mainly in submerged conditions. Anaero- anaerobic conditions as a dissimilatory reduc-
bic sewer bioprocesses, related to both the sulfur tion reaction (equations 8.1–8.5). SRB obtain
and the carbon cycles, are very important in energy by oxidizing organic compounds and/or
sewers. The interaction of those processes and molecular hydrogen (H2) while reducing sulfate
the aerobic transformation of wastewater are (SO42–) to hydrogen sulfide (H2S-HS–) during its
crucial for the performance of urban wastewater metabolism. Desulfovibrio and Desulfotomaculum
systems. To alleviate and control the sewer cor- are the dominant genera of SRB (Muyzer and
rosion and odour problems, various liquid-phase Stams, 2008).
technologies have been used to reduce the forma- SRB grow inside the biofilm in sewer walls
tion/emission of H2S or CH4 (WERF, 2007). The and take the sulfate and organic matter present
most commonly used chemicals for liquid-phase in sewage for their metabolism (Fig. 8.4). Sulfate
technologies include oxygen, nitrate, magnesium concentrations between 15–30  mg S-SO42–/l
hydroxide, iron salts, and caustic shocking to deac- are typical in domestic sewage but those can go
tivate sewer biofilms (Gutierrez et al., 2008, 2009, higher depending on the source of drinking water
2014; Jiang and Yuan, 2013a; Jiang et al., 2011a; or if the water comes from mineral sources with
Zhang et al., 2009). The dynamics of submerged strong presences of salts like sulfate (Pikaar et al.,
sewer biofilms under different environmental 2014).
conditions are presented in this section. Within sewer networks, sulfide is formed
mainly in completely filled rising main sections,
Anaerobic sewer biofilms more prone to septicity than partially filled grav-
Anaerobic conditions develop under the absence ity sections where reareation from the gas phase
of dissolved oxygen leading to sewage septicity. can occur (Hvitved-Jacobsen, 2002). Sulfide
Anaerobic conditions are very common in sewers concentration in rising mains depends upon the
and promote the development of biofilms which sewer features such as the hydraulic retention time
are the cause of the majority of process-related (HRT, time that sewage remains in a certain pipe)
problems. The main anaerobic bioprocesses in and the area/volume ratio (A/V ratio, the inner
sewers are: surface area to volume ratio, i.e. 2/r, where r is the
radius of the pipe, relative surface of sewer inner-
• anaerobic sulfate respiration walls to the volume of sewage that contains).
• methane generation The production and accumulation of hydrogen
• fermentation. sulfide is a major concern for wastewater utilities
and health of residents nearby the sewers. The
Anaerobic sulfate respiration problems related with sulfide depend upon the
In situations where there is lack of oxygen extent by which H2S escapes from the liquid
and nitrate, sulfate (SO42–) becomes the most phase of sewers (where is formed) to the sewer

Sulfate-reducing reactions ∆Go' (kJ/reaction)* Equation no.

4H2 + SO4 2– + H+ → HS– + 4H2O –151.9 8.1
Acetate– + SO42– → 2HCO3– + HS– –47.6 8.2
Propionate– + 0.75SO42– → Acetate– + HCO3– + 0.75HS– + 0.25H+ –37.7 8.3
Butyrate– + 0.5SO42– → 2 Acetate– + 0.5HS– + 0.5H+ –27.8 8.4
Lactate– + 0.5SO42– → Acetate– + HCO3– + 0.5HS– –80.2 8.5

*Thauer et al. (1977).

Biofilm Development in Sewer Networks |  149

Figure 8.4  Conceptual representation of the anaerobic sulfate respiration of sewer biofilms. sCOD: Organic
matter dissolved in wastewater expressed as soluble chemical oxygen demand.

atmosphere, the rate of H2S stripping or transfer irritation, a sore throat and cough, nausea, short-
to the sewer atmosphere that takes place under ness of breath, and fluid in the lungs. These effects
turbulent conditions (drop structures, manholes, are believed to be due to the fact that hydrogen
wetwells) and in gravity sewers. The extent of sulfide combines with alkali present in moist
transfer depends mainly on the pH of sewage. H2S surface tissues to form sodium sulfide. Exposure
is a weak diabasic acid which dissociates As Per to concentrations higher than 300 ppmv in air
the equation 8.6. can cause death by pulmonar oedema and over
1000 ppmv cause immediate collapse with loss of
H2S ↔ H+ + HS– breathing, even after inhalation of a single breath.
pKa = –logKa = 7.1 (8.6) A third major effect of biogenic H2S consists
of the induced corrosion of sewer walls and
As long as H2S remains dissolved in waste- infrastructures. Biogenic sulfide corrosion is a
water, it does not present any harm and will be bacterially mediated process in which hydrogen
eventually oxidized chemical or biochemically. sulfide gas is subsequently converted to sulfuric
The ionic form (HS–) is soluble in water and acid that attacks concrete and steel within waste-
remains dissolved. On the other hand the molecu- water environments. The hydrogen sulfide gas is
lar form (H2S) is more volatile, and can be easily biochemically oxidized in the presence of mois-
released from the sewer liquid phase. When H2S ture to form sulfuric acid. This process causes
is released to the sewer headspace and build-up critical problems to wastewater managers in terms
there, it causes major detrimental effects including of repairing and rehabilitation expenses. Sewer
odour nuisance and toxicity. H2S is a flammable assets are under serious threat with an estimated
and poisonous gas with a characteristic odour of annual asset loss of around $14 billion in the USA
rotten eggs. Its odour concentration threshold is alone (Brongers et al., 2002). Sulfide induced con-
very low, 0.0047 ppmv in gas, and is potentially crete corrosion is recognized as a main cause in
dangerous because its smell is quickly lost as most cases (US EPA, 1991). For instance, in Aus-
the concentration increases. Exposure to lower tralia alone the costs of infrastructure depreciation
concentrations (10–100 ppmv) can result in eye due to sulfide-induced corrosion are estimated to
150  | Gutierrez et al.

be in the order of 100 million dollars per year. Full Methane production and emission from
details of this process are presented below. sewers induce serious concerns as it is a potent
greenhouse gas with a global warming potential
Methane generation 21 times that of carbon dioxide over a 100 year
Hydrogen sulfide is not the only detrimental horizon. To date, methane production from
compound produced in anaerobic sewer systems. sewer systems has been largely overlooked in
Recent field studies showed that a significant the GHG inventories. The latest report from the
amount of methane (CH4) is formed in sewers, Intergovernmental Panel on Climate Change
particularly in pressure pipes (Foley et al., 2009). (IPCC) concerning greenhouse emissions did
Methanogenesis is carried out by Methanogenic not consider methane production from closed or
Archaea (MA), a domain distinct from bacteria. underground sewer systems (IPCC et al., 2013),
Bacteria and archaea differ in cell wall charac- despite some previous indications that domestic
teristics, membrane lipid composition, in RNA sewage could be one of the anthropogenic meth-
polymerase structure and, therefore, protein ane sources (Minami and Takata, 1997). Methane
synthesis (Gantner et al., 2011). Biogenic forma- is explosive at low concentrations, thus represents
tion of methane is a form of anaerobic respiration a safety risk in confined spaces like sewer man-
in which the terminal electron acceptor is not holes because of its low explosion limit (lower
oxygen but carbon compounds of low molecular explosive level is approximately 5% mix in air)
weight. In contrast to sulfate reducers, methano- (Spencer et al., 2006). Further, methane produc-
gens use a limited number of substrates for growth tion in sewers inevitably consumes the soluble
and energy production. Quantitatively, hydrogen, organic compounds (sCOD) including volatile
carbon dioxide and acetate are the most impor- fatty acids (VFAs), which may be required in the
tant and best-known substrates for methanogens receiving wastewater treatment plant for biologi-
(Muyzer and Stams, 2008). Equations 8.7–8.9 cal nutrient removal.
represent the process of methane production. In anaerobic environments with low redox
Ongoing studies in Australia, USA and Spain potentials (<  −200 mV), SRB compete with
are addressing this lack of knowledge on the CH4 other anaerobes, including fermentative bacteria,
formation from sewers. Methane formation in proton-reducing acetogenic bacteria, homoaceto-
sewers has recently been reported. For instance, gens and methanogens, for the available common
Guisasola and co-workers (2008) measured two substrates. In the presence of sulfate in excess,
rising mains in Gold Coast (Australia) where dis- sulfate reducers compete with methanogens for
solved methane concentrations ranging between the common substrates hydrogen and acetate and
5 and 30 mg/l, equivalent to 20–120 mg COD/l, with syntrophic methanogenic communities (Dar
were measured. Gutierrez and co-workers et al., 2008). However Guisasola et al. (2009)
detected CH4 production in pressure sewers of showed that methane and sulfide are simultane-
la Costa Brava (Spain) (Gutierrez et al., 2012). ously produced in sewer systems, which implies
Measurements at multiple locations of a rising the coexistence of MA and SRB in sewers biofilms
main have shown methane at concentrations and that these bacteria function simultaneously.
between 1.5 and 9 mg COD/l. Similarly to H2S, The simultaneous functioning of the SRB and
methane concentration in sewers has shown to MA is related to the spatial arrangement of these
be dependent on the HRT of wastewater and the bacteria in sewer biofilms. Sewer biofilms are
A/V ratio of the pipe. relatively thick (several hundred micrometres;

Methanogenic reactions ∆Go' (kJ/reaction) Equation no.

4H2 + HCO3  + H → CH4 + 3H2O
– +
–135.6 8.7
CO2 + 4H2 → CH4 + 2H2O– –130.7 8.8
Acetate– + H2O → CH4 + HCO3–
–31.0 8.9
Biofilm Development in Sewer Networks |  151

Mohanakrishnan et al., 2007) and the Sulfate/ this reason, the lower affinity of MA for these
Organic Matter ratio (S/COD) shows spatial vari- precursors is not a handicap to the growth of
ation inside the biofilm, being relatively high near methanogens deeper within the biofilm. With
the surface in contact with the bulk liquid and sulfate most likely only partially penetrating the
close to zero in the inner zone adjacent to the pipe biofilm, conceptually two different zones may
surface (Sun et al., 2014). Fig. 8.5 depicts a sche- appear in the biofilm: a sulfate-reducing anaerobic
matic view of this hypothesis, which is supported zone (nearer the surface, dominated by SRB) and
by the sulfide profile measured in sewer biofilms a deeper anaerobic zone dominated by MA Thus,
by Mohanakrishnan et al. (2007). In contrast, the extent of methanogenesis in a sewer system is
the supply of methanogenesis precursors (VFA) inversely proportional to the sulfate penetration
is unlikely to be limiting within the biofilm. For length into the biofilm.

0 (%) (%)

Depth into the Biofilm (µm)


(%) (%)



0 20 40 60 80 100
Relative abundance (%)

Figure 8.5 (A) Conceptual stratified biofilm model under anaerobic conditions including SRB (sulfate-
reducing bacteria) and MA (methanogenic archaea); (B) The SRB and MA proportions of total microorganisms
(bacteria and archaea) detected by FISH within the sewer biofilms; (C) Heatmap displaying the abundance
and distribution of the predominant SRB genera in different sewer biofilm layers from the biofilm surface to
the bottom (Layer 1 to Layer 5). (D) Heatmap displaying the abundance and distribution of the predominant
MA genera in different sewer biofilm layers from the biofilm surface to the bottom (layer 1 to layer 5) (Sun et
al., 2014).
152  | Gutierrez et al.

Fermentation/acetogenic reactions ∆Go' (kJ/reaction) Equation no.

Propionate– + 3H2O → Acetate– + HCO3– + H+ + 3H2 +76.1 8.10
Butyrate– + 2H2O → 2 Acetate– + H+ + 2H2 +48.3 8.11
Lactate– + 2H2O → Acetate– + HCO3– + H+ + 2H2 –4.2 8.12

In fact, the competition between SRB and strong interaction between the carbon and sulfur
MAis not unique to sewer systems. Environmen- cycles in anaerobic sewers that promote the effects
tal microbiologists have devoted much attention described above.
to such competition in natural anaerobic environ-
ments, for example aquatic sediments or paddy Anoxic sewer microbial processes
rice soils (Abram and Nedwell, 1978; Sørensen Nitrate salts (sodium and calcium nitrate) are
et al., 1981; Lovley et al., 1982; Oremland and sometimes artificially added in to pressure sewers
Polcin, 1982; van Bodegom and Stams, 1999; to avoid anaerobic conditions and prevent sulfide
Abram and Nedwell, 1978; Bodegom and Stams, formation ( Jiang et al., 2009; Mohanakrishnan et
1999; Lovley et al., 1982; Oremland and Polcin, al., 2009; Okabe et al., 2007; Zhang et al., 2008).
1982; Sørensen et al., 1981) and anaerobic digest- In the last 70 years, nitrate dosing has been used
ers (Bhattacharya et al., 1996; Gupta, 1994; by water industry to control hydrogen sulfide
Kalyuzhnyi and Fedorovich, 1998). production in sewers (Ganigue et al., 2011).
The addition of a thermodynamically favourable
Fermentation electron acceptor to anaerobic sewers aims to shift
Fermentation consists of the partial breakdown redox conditions and prevent the sulfate respira-
of dissolved organic matter that yields organic tion. Anoxic sulfide oxidation by sewer biofilms
by-products of low molecular weight. Under using nitrate can be described as a two-step pro-
anaerobic conditions, degradation of easily cess, namely the oxidation of sulfide to elemental
biodegradable organic matter is the dominating sulfur, and oxidation of elemental sulfur to sulfate
process. Although fermentation processes do not (equations 8.15 and 8.16, respectively) ( Jiang et
present direct harmful effects in sewers, its by- al., 2009).
products are essential to SRB and MA activities Saracevic et al. (2006) applied 40 mgN-NO3–
since anaerobic fermentation is the source of H2 /l nitrate to a 5.0 km long rising main sewer, and
and VFAs. Fermenters create a syntrophic relation discovered that after a lag time of 3–4 days, the
with SRB and MA populations in which H2 and nitrate application reduced sulfide concentrations
acetate (preferred electron donors) are rapidly from 10–20 mg S-S2–/l to below 2–3 mg S-S2–/l.
scavenged while being produced. Fermentation Nitrate concentration of 5 mg N-NO3–/l in waste-
takes place in the suspended water phase, biofilms water were reported to be sufficient to inhibit
and sewer sediments. The number of fermentation sulfide production in a 61-km-long gravity sewer
reactions occurring in sewers is quite extensive. (Rodríguez-Gómez et al., 2005).
The more relevant ones are presented in equations Nitrate does not have an immediate or long-
8.10–8.14. lasting inhibitory/toxic effect on sulfate reduction
In addition, fermentation also plays a role on sewer biofilms. Biofilms in a nitrate-receiving
in the formation of odorous compounds under laboratory sewer system were found to fully main-
strictly anaerobic conditions. Therefore there is a tain their sulfidogenic activity (sulfide production

Homoacetogenic reactions ∆Go' (kJ/reaction) Equation no.

4H2 + 2HCO3– + H+ → Acetate– + 4H2O –104.6 8.13
Lactate →
– 1.5 Acetate– + 0.5H+ –56.5 8.14
Biofilm Development in Sewer Networks |  153

Nitrate denitrification reactions ∆Go' (kJ/reaction) Equation no.

5S2– + 2NO3– + 12H+ → 5S0 + N2 + 6H2O –955 8.15
5SO + 6NO3– + 2H2O → 5SO42– + 3N2 + 4H+ –2738 8.16

rate in the absence of nitrate) during several major role and H2S and CH4 control are related
months of nitrate addition (Auguet et al., 2014; to penetration of nitrate into the biofilm. Sulfate-
Mohanakrishnan et al., 2009). Two main mecha- reducing activity and methanogenesis would
nisms have been suggested to control sulfide persist respectively in the deeper parts of the
production by nitrate addition in sewers: anoxic biofilm where soluble chemical oxygen demand
sulfide oxidation and competitive exclusion of would still be able to penetrate but not nitrate.
SRB. The first involves the growth of a chemo- These sulfide-oxidizing bacteria (SOB),
lithotrophic sulfide-oxidizing nitrate-reducing e.g. Thiobacillus denitrificans, are mostly che-
community, able to oxidize sulfide to elemental moautotrophic and have been extensively studied
sulfur as a major intermediate coupled to nitrate (Sublette and Sylvester, 1987; Cadenhead and
reduction. The latter triggers the development of Sublette, 1990; Chazal and Lens, 2000; Yang et al.,
a heterotrophic, nitrate reducing bacteria (hNRB) 2005).
community, competing with SRB for organic More recently, nitrite (NO2–) has also been
electron donors. Jiang et al. (2013) proposed a used for sulfide and methane control in sewer
conceptual biofilm model with competitive and pipes ( Jiang et al., 2010; Mohanakrishnan et al.,
synergistic interactions among hNRB, sulfide- 2008; see equations 8.17 and 8.18 as reaction
oxidizing nitrate-reducing bacteria (soNRB), SRB examples). In addition to the oxidizing capacity as
and MA occurring in anoxic sewer biofilms (Fig. NO3–, NO2– has a toxic effect on microorganisms
8.6). Similarly as presented in sections before, thanks to its metabolic inhibitor properties. Nitrite
microbial stratification within the biofilm plays a blocks sulfate reduction in SRB by inhibiting the

Figure 8.6 Conceptual stratified biofilm model under anoxic conditions: Competitive and synergistic
interactions among heterotrophic nitrate reducing bacteria (hNRB), sulfide-oxidizing nitrate-reducing
bacteria (soNRB), sulfate-reducing bacteria (SRB), and methanogenic archaea (MA). Blue arrows indicate
the sulfur cycling between soNRB and SRB.
154  | Gutierrez et al.

Nitrite denitrification reactions ∆Go' (kJ/reaction) Equation no.

3HS− + 8NO2− + 5H+ → 3SO42  − + 4N2 + 4H2O −2944 8.17
3HS− + 2NO2− + 5H+ → 3S0 + N2 + 4H2O −917 8.18

dissimilatory sulfite reductase gene that catalyses Sewer biofilms in lab reactor exposed to FNA
the conversion of sulfite to sulfide (Hubert et al., concentrations above 0.2 mgN-NO2–/l decreased
2005; Nemati et al., 2001). Some SRB possess a its viable fraction by 80% only with 6 hours of
nitrite reductase gene (which prevents the inhibi- contact time. The recovery of methane produc-
tion of SRB by nitrite), but the function of the tion was about seven times slower compared to
enzyme is purely for detoxification, and energy the recovery of sulfide production. The biocidal
is not generated by the reduction (Greene et al., effect was found to be strongly dependent on the
2003). Denitrifying bacterial species like Thioba- FNA concentration rather than the nitrite con-
cillus denitrificans can oxidize sulfide to elemental centration or the pH level separately (for the pH
sulfur simultaneously reducing nitrogenous spe- range of 6.0–7.6). Based on the biocidal effects of
cies to dinitrogen (Mahmood et al., 2007). Nitrite FNA, intermittent dosing of nitrite with acid (to
toxicity produces a long-lasting inhibitory effect form FNA) is potentially a cost-effective strategy
for H2S and CH4 even after the termination of the to control sulfide and methane production in
nitrite addition Conversely, other oxidants (such sewers. Several studies have been confirmed this
as nitrate or oxygen) would need to be present in hypothesis ( Jiang et al., 2011a, 2013a).
the bulk at all times and in all sections of the sewer
in order to effectively prevent sulfide accumula- Aerobic sewer microbial processes
tion. Aerobic microorganisms are commonly found in
Jiang and coauthors (2011) revealed that the sewer systems especially in the gravity sections
protonated form of nitrite, HNO2, free nitrous where turbulence and reareation promote condi-
acid (FNA) is responsible of its biocidal effect. tions optimal to their growth. These organisms

Figure 8.7 Conceptual stratified biofilm model under aerobic conditions: competitive and synergistic
interactions among heterotrophic bacteria (HB), sulfide-oxidizing bacteria (SOB), sulfate-reducing bacteria
(SRB), and methanogenic archaea (MA). Blue arrows indicate the sulfur cycling between SOB and SRB.
Biofilm Development in Sewer Networks |  155

use oxygen as a terminal electron acceptor and concentration in the bulk (Ganigué and Yuan,
consume dissolved organic matter in the sewage 2014; Gutierrez et al., 2008). This is in agreement
in their respiration process (Metcalf and Eddy, with the stratification theory proposed by Jiang
2003). Aerobic processes consist of a major sink and co-authors for oxidants addition to sewers
of sulfide in sewer systems (Fig. 8.7). The shift ( Jiang et al., 2013a). Both studies reported that
between aerobic and anaerobic conditions in sulfide and methane accumulation resumed on
wastewater of a sewer system can be very dynamic. complete depletion of oxygen at a certain depth
Solubility of oxygen depends on the temperature of biofilm. Oxygen did not exhibit any toxic effect
(8.3 mg O2/l at 25°C, and this increases with the on sulfate-reducing bacteria (SRB) in the biofilm.
decrease in the temperature) and the respiration With regards to the SRB, it further stimulated its
rate depends on the availability of degradable growth and increased its activity in biofilms in
organic matter and temperature and may reach downstream sewer sections due to increased avail-
values as high as 20–40 mg O2/l·h (Hvitved- ability of sulfate at these locations as the result
Jacobsen, 2002). Thus the reareation rate in of oxic conditions upstream. Furthermore the
gravity sewers could be insufficient to maintain oxygen uptake rate of the system increased with
the oxic conditions. Thus, the gravity sewers repeated exposure to oxygen, with concomitant
with low slopes and in warm climate are likely to increase in the consumption of organic carbon in
develop anaerobic conditions. the wastewater due to the development of an het-
Artificial injection of oxygen to sewers is erotrophic community within the sewer (Ganigué
widely used by wastewater authorities to mitigate and Yuan, 2014; Gutierrez et al., 2008).
the sulfide problems (Boon et al., 1998; Ganigue
et al., 2011; Hvitved-Jacobsen, 2002; Zhang et al.,
2008). A laboratory study carried out in lab-sewer Unsubmerged sewer biofilms
simulating system showed that oxygen is an effec-
tive chemical and biological oxidant of sulfide Microbially induced concrete sewer
but it did not prevent the sulfide and methane corrosion
production in the biofilm, which continued in the Concrete corrosion in sewers is primarily a result
deeper layers of biofilm irrespective of the oxygen of biological processes occurring in the biofilms


1998 2002

Figure 8.8 Rapid corrosion of a large gravity sewer pipe: 10 km length, 2 m width and 3 m height. The
rehabilitation cost of the pipe was 100 Million Australian $. Courtesy of Sydney Water Corporation.
156  | Gutierrez et al.

growing on the sewer pipe surface exposed to the leads to the formation of two important corrosion
gas phase. Due to the significant role played by products: gypsum and ettringite, according to the
microorganisms in sewer corrosion, it is termed as reactions below (equations 8.19–8.22):
microbially induced concrete corrosion (MICC).
Corrosion causes loss of concrete mass, crack- CaO∙SiO2∙2H2O + H2SO4 →
ing of the sewer pipes and ultimately, structural CaSO4 + Si(OH)4 + H2O(8.19)
collapse (Fig. 8.8). The rehabilitation and replace-
ment of corrosion damaged sewers involves very H2SO4 + CaCO3 → CaSO4 + H2CO3(8.20)
high costs. It is estimated that the annual cost of
concrete corrosion within the water and waste- H2SO4 + Ca(OH)2 → CaSO4∙2H2O (gypsum)
water infrastructure is about US$36 billion in (8.21)
USA (Koch et al., 2002). This cost is expected to
increase as the ageing infrastructure continues to 3CaSO4∙2H2O + 4CaO∙Al2O3∙13H2O
fail (Sydney et al., 1996; US EPA, 1991). + 14H2O → (CaO)3∙Al2O3∙(CaSO4)3∙32H2O
As shown in Fig. 8.9, sulfide production by (ettringite)(8.22)
sulfate-reducing bacteria and the subsequent
emission to the sewer headspace are the primary Both gypsum and ettringite have significantly
causes for concrete sewer corrosion. The biologi- higher volumes than intact cement, estimated to
cal production of sulfuric acid from oxidation of range from 124% to 700% (Monteny et al., 2000;
hydrogen sulfide with oxygen ( Joseph et al., Parande et al., 2006). The expansion is believed to
2012a; Parker, 1945) causes mass loss of concrete cause internal cracking and pitting, which in turn,
(Islander et al., 1991; Ismail et al., 1993). The exposes more surface area for acid attack. How-
corrosion-causing biofilms grow on the surface of ever, recent findings revealed that micro-cracking
corroding concrete (Okabe et al., 2007). at the corrosion front is more likely caused by the
A range of corrosion products is formed during iron rust deposition ( Jiang et al., 2014).
various stages of the corrosion process. The
components of uncorroded cement are mainly Microbial structure and populations
hydrated calcium silicate (CaO∙SiO2∙2H2O) and of corrosion biofilms
portlandite (Ca(OH)2). Abiotic processes that Fresh concrete in sewer pipes immediately after
include carbonation and H2S acidification, result construction is usually immune to biological
in CaCO3, and Ca(HS)2 and S0 as the main prod- attack because of its high alkalinity (pH around
ucts (Wei et al., 2014). During active concrete 12), a result of the formation of calcium hydrox-
corrosion, biologically produced sulfuric acid ide (Ca(OH)2) as the byproduct of cement

Figure 8.9  Processes involved in the microbially induced concrete corrosion in sewers.
Biofilm Development in Sewer Networks |  157

hydration. Anaerobic conditions in wastewater Ca(OH)2 + 2H2S → Ca(HS)2 + 2H2O(8.26)

favour the production of hydrogen sulfide and
carbon dioxide, which builds up in the sewer air Ca(HS)2 + O2 → 2S + Ca(OH)2(8.27)
(Guisasola et al., 2009; Lahav et al., 2004; Nielsen
et al., 2005). Over time, the pH of the alkaline When the surface pH reaches 9, various
concrete surface is gradually reduced by the microorganisms can colonize on the concrete
carbonation (equations 8.23–8.24) and neutrali- surface with the availability of moisture and
zation of hydrogen sulfide (equations 8.25–8.27) nutrients (i.e. stage 2 of corrosion development
(Bagreev and Bandosz, 2004, 2005; Joseph et al., shown in Fig. 8.10). Due to the abundance of
2012b). It was also found that the attachment and hydrogen sulfide and oxygen, some neutrophilic
colonization of some pioneer microorganisms sulfide-oxidizing bacteria (SOB) start to colonize
(mainly heterotrophic, halotolerant, and neutro- and grow on the concrete (Mori et al., 1992).
philc bacteria) on the concrete surface could have The dominant SOB species of the second stage
a great impact on the initial pH decrease (Okabe of corrosion include Thiothrix sp., Thiobacillus
et al., 2007). The initial acidification processes can plumbophilus, Thiomonas sp., and Halothiobacillus
reduce the pH of concrete sewer from around 12 neapolitanus (Okabe et al., 2007). These SOB spe-
to 9, this can establish suitable growth conditions cies were probably responsible for the production
for the subsequent emergence and propagation of of sulfuric acid from the gaseous hydrogen sulfide
sulfur and sulfide oxidizing microorganisms on (Vollertsen et al., 2008; Zhang et al., 2008; Zivica
the concrete surface ( Joseph et al., 2012a). and Bajza, 2001). The sulfide-oxidizing bacteria,
along with some fungi, algae, and lichens form
Ca(OH)2 + CO2 → CaCO3 + H2O (8.23) biofilms (Fig. 8.10), which further reduce down
the surface pH of concrete (Domingo et al., 2011;
CaO·SiO2·2H2O + CO2 → CaCO3 + Silica Nica et al., 2000).
+ H2O(8.24) In the third stage, at which the pH remains
around 2, the acidophilic SOB Acidithiobacil-
CaCO3 + H2S → Ca(HCO3)2 + Ca(HS)2 lus thiooxidans appeared and became the most
(8.25) dominant microbial species (Okabe et al., 2007;

Figure 8.10  The succession of different sulfide-oxidizing bacteria with the changes of surface pH due to
the development of concrete corrosion in sewers. Adapted from Islander et al. (1991). Neutrophilic sulfide-
oxidizing microorganisms (NSOM); acidophilic sulfide-oxidizing microorganism (ASOM).
158  | Gutierrez et al.

Table 8.1  Sulfide-oxidizing bacteria (Thiobacillus) species involved in the microbially induced concrete
corrosion in sewers, and their characteristics (Okabe et al., 2007; Roberts et al., 2002)
Species Growth pH Temperature (°C) Lifestyle Sulfur substrates
Thiobacillus thioparus 4.5–10 15–42 Autotrophic Thiosulfate, sulfide,
aerobe thiocyanate
Starkeya novella (Thiobacillus novellus) 5–9.2 10–37 Mixotroph Thiosulfate
Halothiobacillus neapolitanus 4.5–8.5 8–39 Autotroph Sulfur, sulfide, thiosulfate
Thiomonas intermedia 5–7.5 15–37 Mixotroph Sulfur, thiosulfate
Thiobacillus plumbophilus 4–6.5 9–41 Autotroph Sulfide
Thiothrix spp. Neutral pH 10–30 Mixotroph Sulfide, thiosulfate
Thiobacillus intermedius (Thiomonas 1.7–9 15–37 Mixotroph Thiosulfate
Acidithiobacillus thiooxidans 0.5–5.5 10–37 Autotroph Thiosulfate, sulfur
Acidiphilum acidophilum 1.5–6.0 10–35 Heterotroph Sulfur, thiosulfate

Wei et al., 2014), although there are some con-

flicting findings about the dominant species in
different environments (Cayford et al., 2012;
Parker, 1951; Sand and Bock, 1984). The high
amount of sulfuric acid produced by corrosion
biofilm leads to significant loss of concrete mass
at this stage.
The important species like Thiobacillus sp. (syn.
Acidithiobacillus sp.) and other SOB species found
in corrosion biofilms are listed in Table 8.1. The
first seven species are neutrophilic sulfide-oxidiz-
ing microorganisms (NSOM), while the last three
Figure 8.11 Concrete surface (scanning electron
are acidophilic sulfide-oxidizing microorganism
microscopy image, 4000×) covered with a layer
(ASOM). The NSOM, and probably some fungi, of biofilms containing many 1–3 mm-sized rods of
further reduce the concrete pH to 4–5. At the end thiobacilli (Monteny et al., 2000).
of stage 2, ASOM starts to grow in the sewer cor-
rosion biofilm due to both the low pH and also
the sulfur products produced by NSOM. Sulfur are heterotrophic bacteria (Pseudomonas,
generated by NSOM is an essential substrate for Streptomyces, Arthrobacter, Bacillus, Flavobacter,
ASOM, which produce significant amounts of Micromonas, etc.) and fungi which may grow as
sulfuric acid ( Jensen et al., 2008; Kelly and Wood, co-cultures with SOB (Coleman and Gaudet,
2000). The optimum growth pH, trophic prop- 1993; Nica et al., 2000). It was suggested that
erty (e.g. autotrophic or mixotrophic), and ability fungus, e.g. Fusarium sp., act synergistically with
to utilize different sulfur compounds (e.g. H2S, thiobacilli by either oxidizing sulfide to thiosulfate
S0, and S2O32–) by SOB determine the order of which can then be used by thiobacilli, or excret-
appearance and the dominance of different SOB ing extracellular polymeric substances (EPS) that
species on corroding concrete surfaces in sewer facilitates thiobacilli to attach to elemental sulfur
systems (Okabe et al., 2007). (Cho and Mori, 1995; Gu et al., 1998) (Fig. 8.11).
In addition to the important SOB species It was found that over 50% of the corrosion biofilm
in the corrosion biofilm, there are many other community was heterotrophic bacteria other than
microorganisms being identified that are able SOB (Okabe et al., 2007). The variety of micro-
to oxidize sulfur compounds, most of which organisms, both heterotrophic and autotrophic
Biofilm Development in Sewer Networks |  159

bacteria and fungi, isolated from corroding sewers was found that humidity is important for the cor-
shows the complexity of the microbiological rosion biofilms which are far from the water level
population in the corrosion biofilms. in sewer pipes ( Jiang et al., 2014). This is because
high humidity leads to high moisture content in
Factors impacting the corrosion concrete that facilitates increased biological H2S
biofilm activities oxidation and sulfate production.
The H2S concentration in real sewers varies greatly Short-term changes of temperature are typical
due to the different hydraulic retention time, flow for sewer systems and the interactions between
velocity and wastewater characteristics. In addi- sewer systems and sewage (Vollertsen et al.,
tion to high relative humidity (see below), a H2S 1999). One important process for sewer concrete
level >2 ppm was suggested to be required for the corrosion is the air-water transfer of hydrogen
sulfide oxidation to proceed on concrete sewers sulfide, which was found to increase with increas-
(O’Dea, 2007). It is traditionally assumed that ing temperature at a constant turbulence level
corrosion biofilm activity (i.e. biological sulfide (Yongsiri et al., 2004). It was widely accepted
oxidation) is directly proportional to H2S emis- that sulfide oxidation rate, both chemically and
sion rate (De Belie et al., 2004). The well-known biologically, increases with temperature, which
Pomeroy model can be used to calculate the dete- can be described with an Arrhenius relationship
rioration rate of concrete sewer pipes (Pomeroy, (Nielsen et al., 2004, 2006). The sulfide oxidation
1990). rate was reported to be doubled for a temperature
increase of 7–9°C. In addition, sewer systems
Cr =
(8.28) located in different climates would have been
alk acclimated to different temperatures. The sulfide
where Cr = corrosion rate (mm/year); k = factor oxidation rates, and accordingly corrosion rates,
related to the acid formation, based on climate would thus be very different for different climatic
conditions, 0.8 in moderate climates; fsw= sulfide regions. However, no clear effects of temperature
flux at the air–wall interface [g H2S/(m2 ·h)]; and (5–17°C) was found for the hydrogen sulfide
alk = alkalinity of the pipe material (g CaCO3/g oxidation kinetics (Nielsen et al., 2012). This was
concrete). attributed to the population dynamics of SOB in
A recent report established a relationship the corrosion layers. Another study also reported
between corrosion rate and controlling factors non-significant effects of temperature between
including H2S gaseous concentration, relative 18–35°C on the sulfide oxidation and corrosion
humidity and temperature for concrete sewers rates ( Jiang et al., 2014). Further studies are
either exposed to air or near the wastewater needed to clarify the effects of temperature on the
surface ( Jiang et al., 2014). The corrosion rate actual corrosion rates in sewers.
is proportional to the biofilm activity that is
generally formulated as a power function of H2S Control of concrete sewer corrosion
concentration (Nielsen et al., 2005). To alleviate and control the concrete sewer cor-
In sewers, water and nutrients provided by rosion problems, various gas-phase technologies
sewage are found to promote the microbial corro- have been used to reduce or remove H2S from
sion, especially for the area close to the wastewater sewer air. The sewer air treatment technologies
level in a sewer pipe (Mori et al., 1992). For the include activated carbon adsorption, chemical
pipe surface further away from wastewater level, scrubbing, and biotrickling filters for the biologi-
the relative humidity of the sewer air and the cal oxidation of H2S (Sivret and Stuetz, 2010). In
condensation process on the concrete surface addition to removing H2S from sewer air, the ven-
would generate a water film for microbial growth. tilation also reduce the humidity in the sewer gas
It was reported that humidity plays a role in phase, which can significantly reduce the sewer
surface neutralization at the early stage of sewer concrete corrosion ( Jiang et al., 2014).
concrete corrosion ( Joseph et al., 2012a). During However, it is difficult to control sulfide in the
a long-term investigation of sewer corrosion, it entire sewer network using chemical dosing or
160  | Gutierrez et al.

sewer air treatment due to costs and site restric- Conclusions and perspectives
tions. It is thus relevant and essential to make the Biofilms are naturally present in urban sewer
sewer itself resistant to corrosion by concrete- systems. Sewer biofilms can grow under different
based technologies that construct new sewers environments (anaerobic/aerobic) leading to the
with corrosion-resistant concrete (proactive growth of different types of microorganisms. The
prevention) or repair, coat and line the corroded release of detrimental compounds from sewer
concrete surfaces (passive rehabilitation) (Haile biofilms, such as H2S or CH4, has been thoroughly
et al., 2010; Hewayde et al., 2007). This approach described in this chapter. A significant amount of
includes applications of admixtures, protective knowledge has been generated in recent years but
coatings, and acid-resistant cement to prevent further investigations are still required to fully
chemical attack by sulfuric acid. Antimicrobial understand sewer biofilms and control its impacts
coatings and admixtures, such as silver/copper on urban water systems.
zeolites (Zeomic®) and water-stabilized silicone It is important to have a good understanding
quaternary ammonium salt (Conshield®) among of the biofilm-functioning under the specific
a few other commercialized products, were also sewer conditions in order to develop appropriate
applied to reduce or eliminate the microbial activ- control strategies. In this regard, new methods
ity (De Muynck et al., 2009; Rivera-Garza et al., for the control of the production of detrimental
2000; Yamanaka et al., 2002). compounds in sewer biofilms are currently being
Recently, it was reported that Escherichia coli developed and tested. For instance, free nitrous
DH5α biofilm showed great potential to control acid, an emerging chemical, has been shown to be
and minimize microbially induced concrete cost-effective and environment-friendly in both
corrosion by Thiobacillus neapolitanus and Thio- lab studies and full scale scenarios ( Jiang and
bacillus thiooxidans (Soleimani et al., 2013a). A Yuan, 2013a,b; Jiang et al., 2011b, 2013b).
protective biofilm layer with a depth of 20–40 Integration of sewers with different subsys-
mm was successfully grown on the surface of tems of the urban water cycle is also receiving a
cement mortars (Soleimani et al., 2013b). How- most deserved attention. Recent studies have
ever, due to the highly oligotrophic condition on demonstrated that using sulfate-based coagulants
the sewer surface exposed to air, it is unlikely this in drinking water can have a major impact in
technology can be implemented in real gravity sulfate content of sewer downstream, enhancing
sewer. the development of sewer biofilms (Pikaar et al.,
For new sewers, it is preferable to be con- 2014).
structed with corrosion-resistant concrete because Moreover further research is required to
the rehabilitation after corrosion damage is usu- understand how the sewer biofilms contribute to
ally difficult and expensive. This can be achieved the in-sewer biotransformation of various chemi-
by using cementitious materials containing the cals of different interests. Very little is known
corrosion inhibitors as an admixture (Saraswathy about the role of biofilms in the transformation of
and Song, 2007). Different cements or cements many micro pollutants detected in sewers, includ-
with admixtures have been trialed with limited ing pharmaceuticals and personal care products.
success. Current admixtures mainly focus on A limited number of recent studies have shown
changing the physiochemical properties of con- sewer biofilms to play an important role in the
crete, such as reducing permeability or increasing degradation of illicit drugs and residues, pharma-
buffering capacity. Concrete with a lower content ceuticals and health-related compounds ( Jelic et
of tricalcium aluminate (C3A) was shown to have al., 2014; Thai et al., 2013, 2014).
better resistance to the sulfate attack (Monteny With regards to the microbially induced corro-
et al., 2000). Polymer additions are also used to sion problem, microbial communities in corrosion
modify concrete, with its corrosion resistance to biofilms and their biological processes are still not
acid attack being improved or worsen for different fully delineated. A better understanding of the
polymers (Vincke et al., 2001). microorganisms involved and their metabolisms
Biofilm Development in Sewer Networks |  161

will lead to more efficient control strategies and Cayford, B.I., Dennis, P.G., Keller, J., Tyson, G.W., and
Bond, P.L. (2012). High-throughput amplicon
optimal sewer management. sequencing reveals distinct communities within a
corroding concrete sewer system. Appl. Environ.
Acknowledgements Microbiol. 78, 7160–7162.
Oriol Gutierrez would like to acknowledge Cho, K.-S., and Mori, T. (1995). A newly isolated fungus
participates in the corrosion of concrete sewer pipes.
the project CTM 2011-27163 funded by the Water Sci. Technol. 31, 263–271.
Spanish Government Ministerio de Economía Coleman, R.N., and Gaudet, I.D. (1993). Thiobacillus
y Competitividad, the European Commission neopolitanus implicated in the degradation of concrete
project 2010-RG277050 and the support from tanks used for potable water storage. Water Res. 27,
the Economy and Knowledge Department of the Dar, S.A., Kleerebezem, R., Stams, A.J.M., Kuenen,
Catalan Government through a Consolidated J.G., and Muyzer, G. (2008). Competition and
Research Group (2014 SGR 291)-Catalan Insti- coexistence of sulfate-reducing bacteria, acetogens
tute for Water Research. Dr Guangming Jiang is and methanogens in a lab-scale anaerobic bioreactor
as affected by changing substrate to sulfate ratio. Appl.
the recipient of a Queensland State Government’s Microbiol. Biotechnol. 78, 1045–1055.
Early Career Accelerate Fellowship (Australia). Domingo, J.W.S., Revetta, R.P., Iker, B., Gomez-Alvarez,
V., Garcia, J., Sullivan, J., and Weast, J. (2011).
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