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ENVIRONMENTAL SCIENCE, ENGINEERING AND TECHNOLOGY
WASTEWATER TREATMENT
PROCESSES, MANAGEMENT STRATEGIES
AND ENVIRONMENTAL/HEALTH IMPACTS
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Wastewater Treatment : Processes, Management Strategies and Environmental/Health Impacts, edited by Lydia M. Barrett, Nova Science
ENVIRONMENTAL SCIENCE, ENGINEERING AND TECHNOLOGY
WASTEWATER TREATMENT
PROCESSES, MANAGEMENT STRATEGIES
AND ENVIRONMENTAL/HEALTH IMPACTS
LYDIA M. BARRETT
EDITOR
New York
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Library of Congress Cataloging-in-Publication Data

Wastewater treatment (Hauppauge, N.Y.)
Wastewater treatment : processes, management strategies and environmental/health impacts / editors, Lydia
M. Barrett.
pages cm. -- (Environmental science, engineering and technology)
Includes bibliographical references and index.
ISBN H%RRN
1. Sewage--Purification. 2. Sewage--Purification--Environmental aspects. 3. Sewage--Purification--Health
aspects. I. Barrett, Lydia M. II. Title.
TD430.W36275 2014
628.3--dc23
2015008478
Published by Nova Science Publishers, Inc. † New York

CONTENTS
Preface vii
Chapter 1 Removal of Pharmaceuticals in Conventional Wastewater
Treatment Plants 1
A. Dordio and A. J. P. Carvalho
Chapter 2 Molecular Characterization of Microbial Communities
in Constructed Wetlands: The Effect of Plant Species, Organic
Matter and Hydraulic Design 45
Ricardo Sidrach-Cardona, Olga Sánchez, Laura Garrido,
Jordi Mas and Eloy Bécares
Chapter 3 Assessment of the Presence of Triclosan in Wastewater Treatment
Plants in the Czech Republic and Alternative Fungal Treatment 69
Zdena Křesinová, Radim Žebrák, Jana Langová
and Tomáš Cajthaml
Chapter 4 Removal of Pharmaceuticals and Personal Care Products

from Wastewater 81
Ma Ying, Jian Wu, Ken D. Oakes and Anming Hu
Chapter 5 Application and Environmental Compliance of Anaerobic
Sequencing Batch Reactors Applied to Hydrogen /Methane
Bioenergy Production 111
Giovanna Lovato, Roberta Albanez,
Daniel Moureira Fontes Lima,
Irina Soraya Moncayo Bravo,
Weriskiney Araújo Almeida,
Suzana Maria Ratuznei
and José Alberto Domingues Rodrigues
Chapter 6 Microbial Exopolysaccharides As Efficient Bioflocculants
in Wastewater Treatment 163
Habib Chouchane, Mohamed Neifar, Atef Jaouani,
Mouna Mahjoubi, Ahmed Slaheddine Masmoudi
and Ameur Cherif
vi Contents
Chapter 7 Extremozymes As Efficient Green Biocatalysts in Bioremediation

of Industrial Wastewaters 191
Mohamed Neifar, Habib Chouchane, Atef Jaouani,
Ahmed Slaheddine Masmoudi and Ameur Cherif
Index 215
PREFACE
This book discusses processes involved with wastewater treatment as well as

management strategies and their impact on the environment.
Chapter 1 – Over the latest years, the occurrence of pharmaceutical residues in the
environment has attracted great interest, in particular in regard to the possible harmful effects
of many of these pollutants to living organisms. One of the main sources of pharmaceuticals
in the environment is the discharge of effluents from wastewater treatment plants (WWTPs),
where their removal is often incomplete. The widespread use of pharmaceuticals and their
generally inefficient removal by most WWTPs are the main reasons for their frequent
detection in many water quality monitoring studies. In fact, most WWTPs are inefficient for
the removal of micropollutants in general (especially hardly biodegradable organic
xenobiotics) as these conventional systems were mainly designed for removing bulk
pollutants. It would therefore be valuable to determine if WWTPs could be cost-effectively
modified/expanded to reduce pharmaceutical discharges.
Recent research has been dedicated to evaluate the application of several advanced
treatment technologies, such as advanced oxidation processes, adsorption processes and

membrane processes, either for the removal of organic micropollutants in general or
specifically for the removal of pharmaceutical residues. However, despite the sometimes high
removal efficiencies attained, these technologies are in most cases too expensive to be
considered as viable solutions on a large scale. Moreover, some of these treatments may
originate some transformation products that are potentially more persistent or toxic than the
parent compounds. Therefore, this field of research remains very active as the search for cost-
effective treatment processes continues to be pursued.
In this work the authors intend to present a review on the fate and the removal
efficiencies of pharmaceuticals in conventional WWTPs, describe the main mechanisms
involved in pharmaceuticals removal in WWTPs processes, as well as a discussion of the
major factors influencing that removal. In addition, the authors present some of the research
work that has been carried out over the latest years in attempting to improve the removals of
these pollutants in WWTPs. Thereafter, they describe some of the main alternative processes
that are suggested by that research that can be used to complement conventional WWTPs and
attain more efficient treatment of wastewaters in regard to contamination with
pharmaceuticals.
Chapter 2 – Constructed wetlands are becoming an interesting alternative for wastewater
reuse since high concentrations of contaminants and pathogenic microorganisms can be
viii Lydia M. Barrett
removed with these natural treatment systems. In this study, experimental constructed
wetlands treating diluted wastewater were used to study the effect of plant species (Typha
angustifolia or Phragmites australis), hydraulic design (free-water flow or sub-subsuperficial
flow) and organic loading (3 or 9 gBOD5/m2/d) on the microbial composition of the
rhizoplane, gravel biofilm and interstitial water. The analysis of DGGE band patterns showed
statistically significant differences in community assemblages between plant species.
Hydraulic configuration, and plant presence in a lesser extend, were more important than
organic load in shaping microbial communities in the studied wetlands. Distinctive
communities were found for roots, gravel biofilm and interstitial water inside the same
mesocosm, being differences among these communities higher for Phragmites than Typha
planted tanks. Plants had an effect on all the microbial communities studied into the
mesocosms, proving that their influence affect interstitial water and gravel-associated bacteria
far beyond their roots. Environmental conditions, mainly redox, are suggested as main driving
forces in organizing microbial assemblages in the studied wetlands.
Chapter 3 – Wastewater treatment plants (WWTPs) are considered to be a major source
of various organopollutants in the aquatic environments as a consequence of insufficient
treatment processes. This study was performed to investigate the occurrence of triclosan
(TCS), an antimicrobial personal care product, in inlets and outlets of several WWTPs in
order to provide information about the presence of the compound in the Czech Republic.
Samples were collected at nine WWTPs located in Bohemia, downstream from variously
populated cities in the summer/autumn of 2011 and spring of 2012. At two WWTPs, TCS
concentrations were found to be under the limit of detection of the method at both inlets and
outlets. At the other WWTPs, the concentrations of TCS at the inlets ranged from 0 to 1830
ng l-1 (median 597 ng l-1, mean 528 ng l-1). At the outlets, the concentrations varied from 59 to
1051 ng l-1 (median 205 ng l-1, mean 335 ng l-1). The removal of TCS from wastewater is a
process consisting of several mechanisms, such as adsorption and microbial and as well as
abiotic degradation. Part of TCS is usually converted into methyl-triclosan (Me-TCS) by
microbes in activated sludge under aerobic conditions and this metabolite is very often used
as a pollution marker. In this study, no Me-TCS was detected at the outlets, suggesting that
the major removal mechanism was adsorption or some other unknown mechanisms.
Fluctuations in TCS concentrations over 24 hours were monitored at both the inlet and the
outlet of one selected WWTP. The highest outlet concentration was observed at 8 PM (1267
ng l-1), while the lowest outlet concentration was at 5 AM (152 ng l-1) and the concentration
of a pooled 24h sample was 556 ng l-1. TCS concentrations in the inlet samples peaked at 8
AM (2845 ng l-1) and decreased at about 10 AM to 923 ng l-1. Because the results document
the inadequacy of the WWTP treatment processes, the authors also tested an alternative TCS
degradation method using a white rot fungus in a bioreactor. Pleurotus ostreatus was tested
for the treatment at one of the WWTP outlets in a continuous trickle bed fungal bioreactor,
which was filled with lignocellulosic support inoculated with the fungus and placed directly
in the WWTP object. The results for non-sterile conditions showed more than 80% removal
of TCS following the fungal treatment. The application of Pleurotus ostreatus in the
bioreactor has been shown to be an efficient tool for removal of TCS from real wastewaters.
Chapter 4 – Pharmaceuticals and personal care products (PPCPs) have attracted much
recent attention as widespread emerging environmental contaminants, both due to their near
ubiquitous detection in surface waters adjacent urban areas, but also their potential to generate
endocrine modulating responses at low concentrations in exposed organisms. Although
Preface ix
usually detected in environmental matrices only at low ng/L or µg/L ranges, adverse effects in
exposed aquatic organisms, especially with respect to estrogenic compounds, have been
widely reported and prompted concerns over the potential for human health effects.
Consequently, there has been increasing research attention paid to cost-effectively removing
PPCPs from wastewater prior to its‘ discharge to the environment. Numerous studies have
examined the occurrence and fate of PPCPs in wastewater and adjacent receiving
environments, focusing on their removal by conventional and advanced treatment processes at
varying scales ranging from lab to bench experiments to full treatment plant manipulations.
This review will discuss various removal mechanisms such as surface absorption, membrane
filtration, advanced oxidization treatments (including photocatalytic degradation and
electrolysis) with advantages and limitations of each treatment approach (or combinations
thereof) in removing PPCPs explored. Elucidating the fate and effects of PPCPs in the
environment, and the potential of recent technologies to limit their environmental
contamination, are key elements in protecting future ecosystem and human health.
Chapter 5 – Energy production from renewable resources and from processes with low
energy demand has an important role in the creation of a more sustainable world. The
development of anaerobic processes for wastewater treatment focused on biogas production
with the potential for energy production has shown great progress in the last few years due to
new projects involving reactors that are more efficient and more stable and that have lower
costs of maintenance and operation. Considering this background, the aim of this review was
to describe and discuss the factors that influence biomethane and biohydrogen production
from several wastewaters in ASBR and AnSBBR reactors. The review was focused on the
following variables: inoculum type and pre-treatment, biomass arrangement (free or
immobilized cells), organic load, operation mode, cycle time, feeding time, temperature, pH,
substrates (wastewaters) and kinetic studies.
Chapter 6 – Flocculants are chemicals that stimulate flocculation by aggregation of
colloids and other suspended particles. They are extensively applied in domestic and
industrial wastewaters treatment. Synthetic organic flocculants as well as inorganic

flocculants, including polyaluminium chloride salts and aluminium sulphate, were shown to
be associated to various human health problems such as neurotoxicity, cancer and
Alzheimer‘s disease. They are also known to be non biodegradable and not environment-
friendly. Because of their adverse nature, more attention has been given to the use of
microbial flocculants. A number of microbes i.e. bacteria, yeast, and fungi, are described as
bioflocculants producers with interesting bioremediation potential.
Current applications are mainly in industrial sectors associated with textiles, detergents,
adhesives, microbial enhanced oil recovery, and wastewater treatment. These biomolecules
have particular advantages such as their safety for ecosystems as they are biodegradables and
harmlessness to humans and animals and their high flocculating effects. Microbial
bioflocculants are mostly composed of polysaccharides, proteins, glycoproteins, nucleic acids
and lipids. For examples, flocculant produced by Rhodoocus erythropolis is predominantly
protein in nature, those of Bacillus mucilaginosus (Lian et al., 2008) and of Proteus mirabilis
were shown to be glycoproteins, whereas, Paenibacillus elgii, Serratia ficaria and Klebsiella
mobilis bioflucculants are mainly polysaccharides. Exopolysaccharides bioflocculants have
gained increasing attention since they contain ionizable functional groups such as carboxyl,
amine, sulphate, acetate, and hydroxyl groups which enable these biopolymers, to be effective
in removing suspended solids, heavy metals and in reducing the turbidity of different types of
x Lydia M. Barrett
industrial wastewater effluents. They promote flocculating by bridges formation between

particles resulting in their aggregation and precipitation.
This chapter discusses various studies conducted so far about microbial flocculants. An
attempt was taken to recapitulate the main types of flocculants, flocculation mechanisms, the
most important bioflocculant microorganisms, physicochemical factors influencing the
flocculating activity with a special focus on applications of exopolysaccharide based
flocculant in traitment of wastewater.
Chapter 7 – Recalcitrant pollutants are present in the wastewaters of a great number of
industries, including coal conversion, petroleum refining, resins and plastics, textiles, oil
milling, tanning, mining, pulp and paper. The removal of significant concentrations of
pollutants from industrial wastewater is required to achieve the environmental standards. The
current methods using physical and chemical techniques suffer from factors such as high cost,
hazardous by-products formation, and poor removal efficiency. Therefore, research studies
are being conducted to develop alternative approaches to the pollutants removal from
wastewaters using enzyme-based technology. Varous enzymes offer a number of advantages
over conventional chemical catalysts including a high degree of specificity, operation and
stability under harsh conditions of industrial wastewaters (acidic or alkaline pH, low or
elevated temperatures, high salt concentrations, or high metals, etc.), and a high reaction
velocity which reduces processing costs. Selectivity and versatility are also important
properties responsible for wide and diversified range of applications of extremozymes in the
wastewater treatment.
Several scientific reports and patents described the use of hydrolytic extremozymes such
as lipases and proteases for the biological treatment of effluents of many industries like
dairies and tanneries, which are rich in oil and proteins, respectively. The lignin-degrading
enzymes (lignin peroxidase, manganese peroxidise, versatile peroxidise and laccase), known
so far, are extracellular and nonspecific enzymes, participating in different oxidative reactions
where the aromatic structure of lignin and bonds between the basic units are broken. These
biocatalysts have been extensively used to oxidatively detoxify various aromatic xenobiotic
pollutants found in industrial wastewaters. The interest in laccases was strongly increased
upon discovering that they can oxidize nonphenolic aromatic compounds in the presence of
low-molecular-mass redox mediators. Furthermore, laccases have an advantage compared to
ligninolytic peroxidases, as they use oxygen, a non-limited electron acceptor, for their
catalytic activity, which makes them more suitable for industrial wastewater treatment. The
present chapter reviews the occurrence, modes of action and biochemical properties of
oxidative and hydrolytic extremozymes, as well as their applications in industrial wastewater
treatment. The main advantages as well as the main drawbacks that are still present in the
extensive application of enzyme-based technology in wastewater treatment will be also
mentioned.
In: Wastewater Treatment ISBN: 978-1-63482-467-5
Editor: Lydia M. Barrett © 2015 Nova Science Publishers, Inc.
Chapter 1
REMOVAL OF PHARMACEUTICALS
IN CONVENTIONAL WASTEWATER
TREATMENT PLANTS
A. Dordio1,2 and A. J. P. Carvalho1,3

1
Chemistry Department, School of Sciences and Technology,
University of Évora, Évora, Portugal
2
MARE – Marine and Environmental Research Centre,
University of Évora, Évora, Portugal
3
CQE – Évora Chemistry Centre, University of Évora, Évora, Portugal
ABSTRACT
Over the latest years, the occurrence of pharmaceutical residues in the environment
has attracted great interest, in particular in regard to the possible harmful effects of many
of these pollutants to living organisms. One of the main sources of pharmaceuticals in the
environment is the discharge of effluents from wastewater treatment plants (WWTPs),
where their removal is often incomplete. The widespread use of pharmaceuticals and
their generally inefficient removal by most WWTPs are the main reasons for their
frequent detection in many water quality monitoring studies. In fact, most WWTPs are
inefficient for the removal of micropollutants in general (especially hardly biodegradable
organic xenobiotics) as these conventional systems were mainly designed for removing
bulk pollutants. It would therefore be valuable to determine if WWTPs could be cost-
effectively modified/expanded to reduce pharmaceutical discharges.
Recent research has been dedicated to evaluate the application of several advanced
treatment technologies, such as advanced oxidation processes, adsorption processes and
membrane processes, either for the removal of organic micropollutants in general or
specifically for the removal of pharmaceutical residues. However, despite the sometimes
high removal efficiencies attained, these technologies are in most cases too expensive to
be considered as viable solutions on a large scale. Moreover, some of these treatments
may originate some transformation products that are potentially more persistent or toxic

Corresponding author: Tel: +351 - 266 745343; E-mail address: avbd@uevora.pt.
2 A. Dordio and A. J. P. Carvalho
than the parent compounds. Therefore, this field of research remains very active as the
search for cost-effective treatment processes continues to be pursued.
In this work we intend to present a review on the fate and the removal efficiencies of
pharmaceuticals in conventional WWTPs, describe the main mechanisms involved in
pharmaceuticals removal in WWTPs processes, as well as a discussion of the major
factors influencing that removal. In addition, we present some of the research work that
has been carried out over the latest years in attempting to improve the removals of these
pollutants in WWTPs. Thereafter, we describe some of the main alternative processes
that are suggested by that research that can be used to complement conventional WWTPs
and attain more efficient treatment of wastewaters in regard to contamination with
pharmaceuticals.
1. INTRODUCTION
Pharmaceuticals are compounds that are developed and used to provide a specific
biochemical activity in the diagnosis, treatment, or prevention of diseases. Increasing amounts
of pharmacologically active substances are consumed yearly in human and veterinary
medicine.
The following characteristics of pharmaceuticals differentiate them from conventional
industrial chemical contaminants:
 they can be formed by large and chemically complex molecules that vary widely in
molecular weight (ranging typically from 200 to 1000 Dalton), structure,
functionality, and shape; due to the variety of functions that these substances must
perform, pharmaceuticals present very different physicochemical properties and
biological functionalities;
 in general they are polar molecules with more than one ionizable group, and the
degree of ionization and its properties depend on the pH of the medium; most notably
they can be characterized by, at least, some moderate solubility in water as they
commonly must take effect in aqueous media, but some of them also present some
lipophilicity;
 some pharmaceuticals (e.g. erythromycin, cyclophosphamide, naproxen, and
sulfamethoxazole) can persist in the environment for more than a year, and others
(e.g., clofibric acid) can persist for various years and become biologically active
through accumulation;
 after their administration, the molecules are absorbed, distributed, and subjected to
metabolic reactions that can modify their chemical structure, frequently rendering
them in a less biodegradable form.
In summary, pharmaceuticals are usually resistant to degradation, highly persistent in

aqueous medium, and potentially able to produce adverse events in water organisms and also
have a negative impact on human health.
It is important to note that the classification of pharmaceuticals by their active substances,
within subgroups of pharmaceuticals, does not imply that they follow a certain chemical
behavior. Small changes in chemical structure may have significant effects on solubility,
Removal of Pharmaceuticals in Conventional Wastewater Treatment Plants 3
polarity and other properties, which will in turn will determine a difficult to predict
environmental distribution in air, sediments and soils, water and biota (Kummerer, 2009)
Pharmaceuticals have become one of the most important emerging classes of pollutants
that have been detected in raw and treated wastewater, biosolids and sediments, receiving
waters, groundwaters and even drinking water worldwide (Jones et al., 2005; Fent et al.,
2006; Nikolaou et al., 2007; Picó and Andreu, 2007; Aga, 2008; Miège et al., 2009;
Mompelat et al., 2009; Calisto and Esteves, 2009; Bolong et al., 2009; Monteiro and Boxall,
2010; Kummerer, 2010; Fatta-Kassinos et al., 2011a; Verlicchi et al., 2012; Lapworth et al.,
2012; Kumar et al., 2012; Michael et al., 2013; Rivera-Utrilla et al., 2013; Milic et al., 2013;
Daghrir and Drogui, 2013; Li, 2014; Luo et al., 2014; Manzetti and Ghisi, 2014). Despite the
only recent public awareness, pharmaceuticals, their metabolites and transformation products
have been entering the environment for many years. However, it was the recent advances in
chemical analysis methodologies and instrumentation (significantly lowering the detection
and quantification limits for analyses of organic compounds in complex environmental
matrices) that have allowed in-depth environmental monitoring studies, thus highlighting the
extent of this problem and consequently initiating some public awareness to this emergent
environmental issue.
Through excretion or improper disposal of unused or expired products, pharmaceuticals
and their metabolites are continuously introduced into the sewage system. Many of these
compounds do not receive efficient treatment in wastewater treatment plants (WWTPs),
because municipal WWTPs were originally just designed to deal with bulk pollutants and are
generally inefficient in removing micropollutants (especially the chemically varied class of
pharmaceutical compounds) from domestic wastewaters. Therefore, pharmaceutical residues
remaining untreated or only partially treated in the WWTPs effluents eventually end up being
released in the receiving water bodies (or in treated wastewater reused in irrigation, and in
biosolids reused in agriculture as soil amendment or disposed to landfill). There are several
possible sources and routes for pharmaceuticals to reach the environment, but WWTPs have
been identified as the main point of their collection and subsequent introduction in the
environment via both effluent wastewater and sludge (Jones et al., 2005; Fent et al., 2006;
Nikolaou et al., 2007; Aga, 2008; Bolong et al., 2009; Kummerer, 2010; Verlicchi et al.,
2012; Michael et al., 2013; Luo et al., 2014). Once released into the receiving water bodies
and soils, pharmaceuticals are subjected to the same potential types of transport, transfer and
transformation/degradation processes as other organic contaminants. Thus, the interaction of
pharmaceuticals with soil, surface and ground water and aquatic sediments is similarly
complex.
Over the last years, the detection of pharmaceutical residues in environmental samples
(mostly in aquatic sources) has been a hot topic and numerous monitoring studies have been
carried out. In such studies, the most frequently detected pharmaceutical residues belong to
the pharmaco-therapeutic classes of lipid regulating drugs, analgesics and anti-inflammatory
drugs, antibiotics, hormones, antidiabetics, neuroactive drugs and beta-blockers (Jones et al.,
2005; Fent et al., 2006; Nikolaou et al., 2007; Picó and Andreu, 2007; Aga, 2008; Miège et
al., 2009; Mompelat et al., 2009; Calisto and Esteves, 2009; Bolong et al., 2009; Monteiro
and Boxall, 2010; Kummerer, 2010; Fatta-Kassinos et al., 2011a; Verlicchi et al., 2012;
Lapworth et al., 2012; Kumar et al., 2012; Michael et al., 2013; Rivera-Utrilla et al., 2013;
Milic et al., 2013; Daghrir and Drogui, 2013; Li, 2014; Luo et al., 2014; Manzetti and Ghisi,
2014).
The occurrence of organic micropollutants in the aquatic environment have been

frequently associated with a number of negative effects, including short-term and long-term
toxicity and endocrine disrupting effects (Scrimshaw and Lester, 1996; Stangroom et al.,
1998; Fent et al., 2006; Pal et al., 2010; Ikehata et al., 2010; Narvaez and Jimenez, 2012; da
Silva et al., 2013; Pal et al., 2014). However, despite available research documenting
pharmaceuticals presence in the environment, relevant information concerning their fate and
long-term effects is still lacking. Therefore, risks of long-term toxicity are not well-known
neither the influences on the evolution of aquatic and terrestrial ecosystems and their fauna
and flora. Ecotoxicity studies have demonstrated, however, that pharmaceutical pollutants
could affect the growth, reproduction and behaviour of animals (e.g. birds, fishes,
invertebrates), plants and bacteria, even at levels as low as a few ng L-1 (Cleuvers, 2003;
Boxall, 2004; Cleuvers, 2004; Cleuvers, 2005; Farré et al., 2008; Calisto and Esteves, 2009;
Kümmerer, 2009; Santos et al., 2010; Cuong et al., 2011; Escher et al., 2011; Kumar et al.,
2012; Michael et al., 2013; Arnold et al., 2014; Gabet-Giraud et al., 2014). Threats to human
health are posed by the presence of trace concentrations in soils, which directly affect the
safety of food and drinking water. Even if absorbed quantities from water or food are below
therapeutic concentrations and no acute toxicity is observable, long-term effects and the
potential for cumulative or synergistic effects between several drugs are still unpredictable
and undocumented. In particular, the extensive use of antibiotics and consequent continual
exposure of bacteria to antibiotic residues in the aquatic environment has contributed to the
development of antibiotic resistance genes and bacteria, reducing the therapeutic potential
against human and animal pathogens (Brown et al., 2006; Kemper, 2008; Kümmerer, 2009;
Zhang et al., 2009; Kumar et al., 2012; Michael et al., 2013; Rizzo et al., 2013; Milic et al.,
2013; Daghrir and Drogui, 2013; Borghi and Palma, 2014; Amador et al., 2015).
The foreseeable environmental consequences of high environmental loads of
pharmaceuticals points out to the urgent need of finding ways to retain and remove these
pollutants before they reach the receiving water bodies. Optimization of the WWTP processes
has been tried by increasing hydraulic and solid retention times, for example. In addition,
some advanced technologies have been evaluated to decrease their discharge into water
bodies. However, despite the sometimes high removal efficiencies attained, these processes
are generally not cost-effective on a large scale (Fent et al., 2006; Kummerer, 2009; Michael
et al., 2013; Luo et al., 2014; Ganzenko et al., 2014; Plumlee et al., 2014). In fact, it remains a
crucial necessity to find applicable technologies for removing pharmaceuticals from
wastewater with higher efficiencies at reasonable cost of operation and maintenance.
In this work, a review is presented of the current wastewater treatment technology, the
fate of pharmaceuticals in conventional wastewater treatment plants as well as an
identification of the main factors affecting their fate, and an overview of recent reports of the
pharmaceutical inputs to WWTPs and their efficiency in removing them. The main advanced
treatment technologies that have been more frequently considered and studied are presented
along with a discussion of their main advantages and disadvantages. Hopefully, an up-to-date
overview is herein given of the present situation regarding how successfully pharmaceutical
contaminants are being handled by current wastewater treatment technology and the options
for complementary/alternative treatment, which may enlighten anyone committed to address
this problem.
2. PHARMACEUTICALS FATE IN CONVENTIONAL WWTPS

In recent years, the effects of many pharmaceuticals on the environment have become
more clearly understood. Ongoing research nowadays aims not only at determining the
environmental effects of potentially toxic and biologically active substances found in
wastewaters, but also increasingly to develop strategies for removing these substances in both
conventional and advanced wastewater treatment processes. As a result wastewater treatment
requirements are becoming more stringent in the establishment of both the limit
concentrations of many of these substances in the WWTPs effluents and of whole effluent
toxicity limits. To meet these new requirements, many of the existing conventional WWTPs
may have to be adapted or reformed. Hence, more comprehensive studies are required to
thoroughly understand the behavior of pharmaceuticals under both conventional and
advanced wastewater treatment processes and to gain more knowledge on the removal
processes within the WWTPs. The evaluation of the fate of pharmaceutical residues in
WWTPs and the assessment of the parameters affecting the efficiency of their removal is
imperative for the optimization of treatment processes.
Municipal WWTPs normally receive wastewater that contains a lot of different trace
pollutant compounds (both of synthetic and natural origins), which conventional treatment
technologies have not been specifically designed for. The degree to which such pollutants are
removed varies from near completion to almost none. In fact, the treatment processes in
municipal WWTPs are designed to remove bulk constituents of wastewater, such as
suspended solids, biodegradable organic matter, pathogens and nutrients, by physical,
chemical and biological processes available along the consecutive stages of a conventional
treatment. These conventional WWTPs typically comprise three or four stages of treatment,
with different biological and physicochemical processes available for each treatment stage
(Figure 1):

 preliminary treatment (pre-treatment): removal of gross solids and grit that may
damage some equipment (e.g. bar screening and grit removal);
 primary treatment: removal of suspended solids, oils and fats and some organic
matter (e.g. fine screening and sedimentation);
 secondary treatment (biological treatment and secondary sedimentation): removal of
biodegradable organics, nutrients and suspended solids. Several biological treatments
are being used in modern municipal WWTPs (e.g. activated sludge, trickling filters,
rotating biological contactors, aerated lagoons), but the most common method is the
conventional activated sludge process, either with or without a
nitrification/denitrification unit to enhance nitrogen removal. Disinfection (typically
by chlorination or UV irradiation) is also sometimes included in the definition of
conventional secondary treatment;
 tertiary and/or advanced treatment: removal of dissolved and suspended materials
remaining after conventional secondary treatment (e.g. slow sand filtration,
membrane processes, disinfection, oxidation processes, activated carbon adsorption).
This stage is always associated with higher treatment costs but their use is justified
with the necessity to meet more stringent discharge and water reuse requirements,
generally based on public and environmental health objectives.
After wastewater treatment, both treated effluent and solid waste streams are produced.
The effluent is usually discharged into surface waters and the solid waste, also known as
sewage sludge, is disposed after treatment into a landfill or can also be recycled and used as
fertilizer on agricultural fields. The treatment of the sludge has the aim of increasing and
stabilizing the solid organic content and reducing pathogens. Treatment processes include
thickening (in order to increase the solid content of sludge), stabilization (e.g. anaerobic or
aerobic digestion), conditioning (e.g. chemical or other), dewatering, drying and thermal
reduction (e.g. incineration). As result of treatment, the water and organic content should be
considerably reduced, and the processed solids should be suitable for reuse or final disposal.
Figure 1. Diagram of a conventional wastewater treatment plant (adapted from Tchobanoglous et al.
(2003)).
Due to the fact that the term pharmaceuticals refers collectively to compounds with the
single common characteristic of being used in the prevention and treatment of diseases, but
are otherwise formed by many chemically unrelated families, drugs span a wide variety of
physical and chemical properties. Hence, the efficiencies by which pharmaceuticals are
removed in WWTPs may vary quite substantially and unpredictably. However, some broad
features of pharmaceuticals‘ fate in WWTPs can be outlined.
In general it is observed that there is very little elimination of most organic
micropollutants in the preliminary and primary treatments of wastewaters and, as such, it is
also unlikely that many pharmaceutical compounds will be removed during screening or
primary sedimentation (Carballa et al., 2004; Ternes et al., 2004b; Jones et al., 2005; Zorita et
al., 2009; Monteiro and Boxall, 2010; Zhang et al., 2011b; Verlicchi et al., 2012; Hamid and
Eskicioglu, 2012; Luo et al., 2014). In some cases, compounds may even be released during
these stages, caused by the simultaneous presence of deconjugable substances (i.e. human
metabolites) of these compounds in the raw influent (Carballa et al., 2004).
Given the low biological activity at these initial stages, any pollutant removal here will
rely on both the tendency of the individual drugs to sorb to solids (primary sludge) and the
degree of suspended solids removal in the primary sedimentation tank (Zhang et al., 2008;
Monteiro and Boxall, 2010; Hamid and Eskicioglu, 2012; Luo et al., 2014). Normally, there
is little change in dissolved polar organics at this point and, as well, little to no loss may be
expected specifically for polar drugs. In general, elimination of any compound by sorption to
sludge is considered relevant only when the log Kd for that compound is higher than ~ 2.5 –
2.7 (i.e. Kd > 300 – 500 L kg-1) (Ternes et al., 2004c; Joss et al., 2005). The removal of
organic compounds may also be affected by factors such as pH, retention time, temperature,
and amount and type of solids present in the wastewater (Ternes et al., 2004c; Joss et al.,
2005; Carballa et al., 2008; Hamid and Eskicioglu, 2012; Luo et al., 2014).
Removal of drugs in secondary (biological) treatment by activated sludge may occur by
the same mechanisms as other organic micropollutants, which include sorption to secondary
sludge, chemical degradation or transformation (such as hydrolysis or photolysis) and
biotransformation/biodegradation (aerobic, anoxic and anaerobic) (Monteiro and Boxall,
2010; Verlicchi et al., 2012; Hamid and Eskicioglu, 2012; Luo et al., 2014). Little loss by
volatilization during aeration (stripping) is expected due to the typically low volatility of most
pharmaceutical compounds (Larsen et al., 2004; Miège et al., 2009; Caliman and Gavrilescu,
2009; Verlicchi et al., 2012; Luo et al., 2014); in fact, it is found that Henry coefficients of
over ~ 10−3 are required for significant stripping in a bioreactor with fine bubble aeration
(Larsen et al., 2004). Pharmaceuticals removal at this stage is also affected by environmental
and operation conditions (Joss et al., 2005; Clara et al., 2005; Vieno et al., 2005; Vieno et al.,
2007; Aga, 2008; Onesios et al., 2009; Verlicchi et al., 2012; Luo et al., 2014).
Drugs remaining in wastewaters after primary and secondary treatment may be
eliminated by tertiary or advanced treatments. However, in most countries only a small
number of WWTPs have these adaptations. Advanced treatment techniques such as advanced
oxidation processes, membrane processes and adsorption processes applied in water treatment
have been shown to remove pharmaceuticals to levels below detection limits (Fent et al.,
2006; Esplugas et al., 2007; Snyder et al., 2007; Simazaki et al., 2008; Klavarioti et al., 2009;
Rosal et al., 2010; Kit Chan et al., 2012; Dolar et al., 2012; Feng et al., 2013; Ek et al., 2014;
Yu et al., 2014; Rodriguez-Mozaz et al., 2015; Rizzo et al., 2015) but how effectively they do
so varies with the treatment conditions employed. Notwithstanding, despite the sometimes
high removal efficiencies attained, most of these technologies are too expensive to be
considered as viable solutions on a large scale in wastewater treatment (Fent et al., 2006;
Klavarioti et al., 2009; Tahar et al., 2013). Moreover, some of these treatments may originate
some transformation products which, in some cases, may be more persistent or toxic than the
parent compounds (Farré et al., 2008; Fatta-Kassinos et al., 2011b; Postigo and Richardson,
2014; Wang and Lin, 2014; Evgenidou et al., 2015).
In summary, pharmaceuticals are subjected in WWTPs to a variety of phenomena
(dispersion, dilution, partition, sorption, biodegradation and abiotic transformation)
contributing to their elimination or transfer to solid phase, thereby lowering their
concentration in the aqueous medium. The total removal during treatment generally refers to
the losses of a parent compound contributed by all of those different processes.
3. OCCURRENCE AND REMOVAL OF PHARMACEUTICALS

IN CONVENTIONAL WWTPS
Recent reviews provide a general overview on the occurrence and fate of pharmaceutical
residues in WWTPs (Fent et al., 2006; Miège et al., 2009; Pal et al., 2010; Deblonde et al.,
2011; Cuong et al., 2011; Verlicchi et al., 2012; Ratola et al., 2012; Hamid and Eskicioglu,
2012; Michael et al., 2013; Evgenidou et al., 2015) and, ultimately, in the environment (Fent
et al., 2006; Mompelat et al., 2009; Caliman and Gavrilescu, 2009; Calisto and Esteves, 2009;
Pal et al., 2010; Fatta-Kassinos et al., 2011a; Li, 2014; Arnold et al., 2014; Postigo and
Richardson, 2014; Meffe and de Bustamante, 2014; Van Doorslaer et al., 2014; Taylor and
Senac, 2014; Manzetti and Ghisi, 2014).
According to the literature, many of the pharmaceuticals that have become ubiquitous in
the environment and in conventional WWTPs are mostly from the therapeutic classes of
analgesic/anti-inflammatory, antibiotic, beta-blocker, lipid regulator, and psycho/neuroactive
drugs (Fent et al., 2006; Nikolaou et al., 2007; Aga, 2008; Miège et al., 2009; Caliman and
Gavrilescu, 2009; Calisto and Esteves, 2009; Kümmerer, 2009; Verlicchi et al., 2012; Luo et
al., 2014).
a) Analgesics and Anti-Inflammatory Drugs
Analgesics are pain-relief drugs that include narcotic analgesics, non-narcotic analgesics,
and non-steroidal anti-inflammatory drugs (NSAID). They act in various ways on the
peripheral and central nervous systems and are widely used to alleviate the pain present in
almost all diseases (Bueno et al., 2012; Li, 2014).
NSAIDs are the most used class of drugs for the treatment of acute pain and
inflammation. They are administered both orally and topically and are available as
prescription and over-the-counter (non-prescription) drugs. As result of the very large
consumptions of this type of drugs and their mode of administration, NSAIDs are, among all
pharmaceuticals, one of the top classes usually present in domestic wastewaters and in
environmental samples (Fent et al., 2006; Miège et al., 2009; Caliman and Gavrilescu, 2009;
Monteiro and Boxall, 2010; Verlicchi et al., 2012; Li, 2014; Luo et al., 2014). Top-selling
NSAIDs worldwide include ibuprofen, diclofenac, naproxen, ketoprofen, fenoprofen,
indomethacin and mefenamic acid. Common non-narcotic analgesics include acetaminophen
(paracetamol) and acetylsalicylic acid. Narcotic analgesics include codeine, methadone,
morphine and oxycodone (Bateman, 2012; Li, 2014). All these are also the compounds of this
therapeutical classes more frequently detected in the environment.
Diclofenac, ibuprofen and naproxen are some of the most studied NSAIDs in regard to
their ecotoxicity (Ferrari et al., 2003; Cleuvers, 2003; Cleuvers, 2004; Parolini et al., 2009;
Santos et al., 2010; van den Brandhof and Montforts, 2010; Escher et al., 2011; Narvaez and
Jimenez, 2012).
b) Antibiotics
Antibiotics are among the most important groups of pharmaceuticals used. Their usage
was introduced in the late 1930s and has kept increasing ever since. These drugs can be used
for either prevention (although lately this type of use is being disapproved) or treatment of
disease caused by microorganisms such as bacteria and fungi. In a broad sense the generic
term ―antibiotic‖ refers to any class of organic molecules that inhibit or stop the growth of
microorganisms by specific interactions with the targets (Kümmerer, 2009; Davies and
Davies, 2010; Michael et al., 2013). They are used extensively in human (domestic and
hospital use) and veterinary medicine as well as in aquaculture for the purpose of preventing
or treating microbial infections, while in livestock farming they are also used to promote the
growth of animals. Some antibiotics have additional use in growing fruit and in bee keeping.
Antibiotics can be grouped and classified based on chemical structure, mechanism of
inhibition of microorganisms, or spectrum of activity. The variety of chemicals comprising
the antibiotics class can be divided into subgroups such as -lactams, quinolones,
tetracyclines, macrolides, sulfonamides and others.
The active compounds of antibiotics are often complex molecules which may possess
different functionalities, even within the same molecule. Under diverse environmental
conditions these molecules can be neutral, cationic, anionic, or zwitterionic. Because of the
different functionalities within a single molecule, their physico-chemical and biological
properties, such as log Kow, sorption behavior, photo reactivity and antibiotic activity and
toxicity, may change with pH (Cunningham, 2008; Kümmerer, 2009; Michael et al., 2013).
These pollutants are considered as ―pseudo-persistent‖, meaning that they enter into the
environment continuously, which leads to their permanent presence at more or less stable
concentrations. Water contamination with antibiotics assume special significance among the
wide variety of pharmaceutical residues detected in the environment and in WWTPs due to
their extensive use and the high frequency of their detection in environmentally relevant
concentration levels. Such conditions have been associated to chronic toxicity and the
prevalence of resistance to antibiotics in bacterial species (Andersen, 1993; Hirsch et al.,
1999; Brown et al., 2006; Kemper, 2008; Kümmerer, 2009; Dìaz-Cruz et al., 2009; Zhang et
al., 2009; Davies and Davies, 2010; Mokracka et al., 2012; Kumar et al., 2012; Michael et al.,
2013; Johnning et al., 2013; Rizzo et al., 2013; Milic et al., 2013; Daghrir and Drogui, 2013;
Borghi and Palma, 2014; Amador et al., 2015). In fact, the extensive use of antibiotics and
consequent continual exposure of bacteria to antibiotic residues in the aquatic environment
has contributed to the development of antibiotic resistance genes and bacteria, reducing the
therapeutic potential against human and animal pathogens (Brown et al., 2006; Kemper,
2008; Kümmerer, 2009; Zhang et al., 2009; Kumar et al., 2012; Michael et al., 2013; Rizzo et
al., 2013; Milic et al., 2013; Daghrir and Drogui, 2013; Borghi and Palma, 2014; Amador et
al., 2015). Furthermore, the biological treatment in WWTPs creates an environment
potentially suitable for resistance development and spreading. Concomitantly, the number of
studies focusing on the chronic toxicological assessment of antibiotics in the environment is
constantly increasing. Several works have been published presenting comprehensive reviews
on the ecotoxicity of antibiotics (Boxall, 2004; Sarmah et al., 2006; Kemper, 2008;
Kümmerer, 2009; Zhang et al., 2009; Garcia-Galan et al., 2009; Du and Liu, 2012; Kumar et
al., 2012; Boxall et al., 2012; Narvaez and Jimenez, 2012; Kar and Roy, 2012; Michael et al.,
2013; Rizzo et al., 2013; Milic et al., 2013; Daghrir and Drogui, 2013).
The antibiotic therapeutic classes more frequently detected in conventional WWTPs
include sulfonamides, macrolides, tetracyclines, and fluoroquinolones (see next section).
Although members of the -lactams class are sold in the highest amounts, the concentrations
found in environmental samples are low compared to expected from their extensive use
(Hirsch et al., 1999; Michael et al., 2013). The reason may be the chemical instability of the
-lactam ring of the penicillins, which is rapidly hydrolyzed and degraded (Hirsch et al.,
1999).
Another issue, that is related with the use of reclaimed wastewater for irrigation or reuse
of sludge as soil fertilizer, is the plant uptake of antibiotics (Eggen et al., 2011; Du and Liu,
2012; Kang et al., 2013; Bassil et al., 2013; Mathews and Reinhold, 2013; Datta et al., 2013).
Antibiotics accumulation may represent an important exposure pathway of these compounds
to humans and other biota (European Medicines Agency-EMEA). There are also new
concerns that antibiotics decrease the biodegradation of leaf and other plant materials, which
serves as the primary food source for aquatic life in rivers and streams (Richardson and
Ternes, 2011).
c) Blood Lipid Regulators
Lipid regulators are substances used to regulate the metabolism of lipids and to lower
levels of triglycerides and low-density lipoproteins (LDL) and increase levels of high-density
lipoproteins (HDL) in the blood (Monteiro and Boxall, 2010). These pharmaceuticals are used
in the treatment and prevention of cardiovascular disease.
There are three kinds of lipid regulators, fibric acid derivatives (or fibrates, e.g.
clofibrate, bezafibrate, gemfibrozil and fenofibrate), statins (e.g. atorvastatin, simvastatin and
lovastatin) and niacin (or nicotinic acid).
Fibrates are more commonly studied and detected in the environment than statins.
However, in the last decade in many countries, statins became the drug of choice instead of
fibrates to lower cholesterol levels and their usage is increasing. Notwithstanding, clofibric
acid, which is a metabolite of clofibrate and other fibrates, is currently one of the most
frequently detected drugs in the environment and one of the most persistent, with an
estimated persistence in the environment of 21 years (Khetan and Collins, 2007). In addition,
many analogues of clofibrate, such as gemfibrozil, bezafibrate and fenofibrate and, among
statins, the drugs atorvastatin, mevastatin and prevastatin were detected in various
environmental matrices and raw and treated wastewater as well as surface water (Metcalfe et
al., 2003; Miao and Metcalfe, 2003; Radjenovic et al., 2009a; Gros et al., 2010; Jelic et al.,
2011).
d) Beta-Blockers
β-Blockers (or β-adrenergic receptor antagonists) are anti-hypertensives, i.e. they act on
blood vessels, to prevent vasodilatation and reduce the speed and force of heart contractions.
This is another very important class of prescription drugs, very commonly used in the
treatment of cardiovascular diseases. The most common pharmaceuticals of this class are
atenolol, metoprolol, and propranolol which have all been frequently identified in
wastewaters and surface waters (Fent et al., 2006; Hernando et al., 2007; Maurer et al., 2007;
Miège et al., 2009; Gabet-Giraud et al., 2010; Deblonde et al., 2011; Verlicchi et al., 2012;
Luo et al., 2014). Like the non-steroidal anti-inflammatory drugs, beta-blockers are not highly
persistent, but they are present in the environment due to their high consumptions.
The ecotoxicity of several beta-blockers has been studied in several papers (Cleuvers,
2003; Fraysse and Garric, 2005; Cleuvers, 2005; Santos et al., 2010; Alder et al., 2010;
Escher et al., 2011; Claessens et al., 2013; Maszkowska et al., 2014). One of the problems
that has been identified for these compounds is that mixtures of beta-blockers show
concentration addition, due to the fact that beta-blockers work through essentially the same
mode of action (Cleuvers, 2005; Maszkowska et al., 2014).
e) Psycho/Neuroactive Drugs
The group of psycho-active drugs that act on the central nervous system providing
anxiolytic, sedative and hypnotic effects, comprise, essentially, benzodiazepines, buspirone
(an azapirone), zoplicone (a cyclopyrrolone), zolpidem (an imidazopyridine) and barbiturates
(Chouinard et al., 1999; Calisto and Esteves, 2009). Among these, the benzodiazepines is one
of the most prescribed groups of pharmaceuticals throughout the world (van der Ven et al.,
2004). Ecotoxicological studies on this class of substances have been often performed but, in
spite of its high consumption, benzodiazepines are among those pharmaceuticals less
commonly addressed in environmental research (Ternes et al., 2001; Kosjek et al., 2012;
Mendoza et al., 2014). Nowadays, there are several dozens of benzodiazepines under
international control for therapeutic use, among which alprazolam, diazepam, lorazepam and
oxazepam are the most important ones, with special relevance to diazepam as the top selling
substance of this family worlwide (Calisto and Esteves, 2009). Diazepam is thought to have
the potential for a long lasting action in the environment because its active metabolites have
long half-lives ((Calisto and Esteves, 2009; Kosjek et al., 2012).
Benzodiazepines are the drugs of choice in the pharmacotherapy of anxiety and related
emotional disorders, sleep disorders, status epileptics, and other convulsive states. They are
also used as centrally acting muscle relaxants, for premedication and as inducing agents in
anaesthesiology (Calisto and Esteves, 2009). However, these compounds are not exclusively
used in human medicine. Prescription of benzodiazepines is also common for veterinary use
(Courtheyn et al., 2002; Gaskins et al., 2008) for their anxiolytic and appetite stimulant
effects in domestic and wild animals (Gaskins et al., 2008). Their potential shortcomings in
human usage include tolerance, withdrawal symptoms, and their abuse potential (Riss et al.,
2008; Kosjek et al., 2012).
Antidepressants are also widely prescribed pharmaceuticals (Calisto and Esteves, 2009).
Among the most common antidepressants are the class of selective serotonin re-uptake
inhibitors, comprising five drugs available in the market, namely fluoxetine, fluvoxamine,
paroxetine, sertraline and citalopram (Johnson et al., 2007). These drugs act by modulating
the levels of the serotonin neurotransmitter (Brooks et al., 2005), and are largely used for the
treatment of depression, compulsive–obsessive disorder, panic disorder, as well as to other
cases in which selective inhibition of serotonin re-uptake is desirable (Brooks et al., 2003;
Calisto and Esteves, 2009). Veterinary uses of antidepressants comprise the treatment of
pathologies such as separation anxiety, obsessive–compulsive and fearful behavior among
other behavioral problems (Mills, 2003).
A large number of antidepressants have already been identified in water, wastewater,
sludge and biological tissues of aquatic organisms (Metcalfe et al., 2003; Brooks et al., 2005;
Schultzt and Furlong, 2008; Santos et al., 2010; Metcalfe et al., 2010; Schultz et al., 2010;
Verlicchi et al., 2012; Lajeunesse et al., 2012; Best et al., 2014; Sheng et al., 2014), fluoxetine
and its metabolite norfluoxetine being the most commonly studied antidepressants.
One other very notorious case of a psycho-active drug that, despite not being one of the
top-selling drugs, it is nevertheless among the most frequently detected and prevalent
pharmaceuticals in the environment due to its recalcitrant behavior in WWTPs, is the anti-
epileptic drug carbamazepine (Fent et al., 2006; Aga, 2008; Miège et al., 2009; Verlicchi et
al., 2012; Luo et al., 2014).
Pharmaceuticals acting on the nervous system have a great relevance on the regulation of
behavior, with a major aptitude to directly affect the central nervous system and disrupt
neuro-endocrine signaling. Possible adverse effects of this group of pharmaceuticals in
organisms can be illustrated by, for example, the observed modification of reproductive
patterns in non-target aquatic organisms (van der Ven et al., 2004; van der Ven et al., 2006;
Calisto and Esteves, 2009; Santos et al., 2010; Schultz et al., 2010). Several studies have
demonstrated that these compounds can affect physiological systems at very low
concentrations (van der Ven et al., 2004; van der Ven et al., 2006; Calisto and Esteves, 2009;
Santos et al., 2010; Schultz et al., 2010). A number of other studies on fluoxetine, diazepam,
sertraline, paroxetine, and others, clearly showed significant adverse effects of
antidepressants and anxiolytics in aquatic organisms (Brooks et al., 2005; van der Ven et al.,
2006; Johnson et al., 2007; Gaworecki and Klaine, 2008; Fong and Molnar, 2008; Calisto and
Esteves, 2009; Gust et al., 2009; Santos et al., 2010; Verlicchi et al., 2012; Kosjek et al.,
2012; Mendoza et al., 2014; Bean et al., 2014). Furthermore, the increasing number of studies
about chronic toxicity on non-target aquatic organisms pointed out that no extrapolations
between acute and chronic toxicity should be done, underlining the need of developing a
distinct approach to better clarify this issue (Cunningham et al., 2006). In fact, seeing that
aquatic organisms are extensively exposed to pharmaceuticals, it would be more important to
understand life cycle toxicity rather than perform acute toxicity tests (Calisto and Esteves,
2009).
The fate of incoming pharmaceutical residues in the WWTP is not straightforward to
predict. Some substances seem to be almost completely eliminated, while others remain
unaffected as they pass the different treatment stages in the WWTP. Pharmaceutical residues
that are excreted in conjugated form as result of human metabolism may undergo de-
conjugation during WWTP treatment, sometimes contributing to an increase on the levels of

the free pharmaceutical leaving the WWTP in comparison with the influent. Some highly
consumed pharmaceuticals have not been detected in WWTP influents, indicating that
complete metabolism and degradation must occur already in the human body or during the
transport of wastewater from the household to the WWTP. Other less commonly prescribed
pharmaceuticals are frequently detected even in the WWTP effluents due to their recalcitrant
behavior.
3.1. Occurrence in WWTP Influents and Effluents and Removal Efficiency

The characterization of pharmaceuticals occurrence in wastewaters as well as their
removal by WWTPs has been under focus of several recent reviews (either restricted to
specific pharmaceutical classes or covering wider ranges of compounds) which is a clear
demonstration of the importance that has been given to this subject lately (Fent et al., 2006;
Nikolaou et al., 2007; Aga, 2008; Miège et al., 2009; Calisto and Esteves, 2009; Kümmerer,
2009; Onesios et al., 2009; Deblonde et al., 2011; Cuong et al., 2011; Fatta-Kassinos et al.,
2011a; Verlicchi et al., 2012; Michael et al., 2013; Tijani et al., 2013; Luo et al., 2014; Van
Doorslaer et al., 2014; Evgenidou et al., 2015). A summary of the data collected in those
reviews on the occurrence of the most relevant and ubiquitous pharmaceuticals, grouped in
alphabetic order of their therapeutic classes and names, is presented in Table 1. The data
reports typical WWTP influent and effluent concentration ranges of each pharmaceutical and
the assessed efficiency of pharmaceutical removal in WWTPs.
A brief overview of Table 1 reveals that, typically, pharmaceuticals are detected in
WWTPs influents or effluents at concentration levels in the range of ng L-1 to µg L-1. On the
other hand, significant variations in reported concentration levels is a feature that also stands
out from the analysis of that data. This variability results from the fact that pharmaceutical
concentrations are typically low and strongly affected by many factors having some spatial
and temporal variability. This includes variations in time and world location of
production/sales/consumption levels of the pharmaceuticals, thus affecting WWTPs inputs
(rates of production, sales volume and market strategies, local prescription and usage
practices, spatial and seasonal distribution of diseases prevalence), as well as some aspects
that relate with WWTP design and operation that affect the characteristics of the final WWTP
effluent (water consumption per person and per day, environmental conditions, WWTP size,
plant configuration especially the type of bioreactor, hydraulic retention time, solids retention
time).
Table 1. WWTP influent and effluent concentrations and respective removal efficiencies
for selected pharmaceuticals in conventional WWTPs (adapted from Luo et al. (2014))
Therapeutic class Selected Compounds Influent Effluent Removal

(g L-1) (g L-1) (%)
Analgesic/
anti-inflammatory
Acetaminophen/ Paracetamol 1.57-292 ND-0.03 98.7-100
Diclofenac <0.001-94.2 <0.001- 2.5 <0-81.4
Ibuprofen <0.0004-603 ND-69 72-100
Ketoprofen <0.004-8.56 <0.003-3.92 10.8-100
Mefenamic acid <0.017-3.20 <0.005-2.40 0-70.2
Naproxen <0.002-611 <0.002-33.9 43.3-98.6

Salicylic acid 0.58-63.7 ND-0.50 89.6-100
Antibiotic
Amoxicillin 0.018-6.94 ND-0.27 99-100
Norfloxacin ND-0.46 0.0139-0.320 76-93
Sulfamethoxazole <0.003-1.25 <0.003-1.15 4-88.9
Erythromycin 0.07-10 0.02-2.84 0-82.5
Oxytetracycline 0.007-0.35 0.005-0.070 29->88
Tetracycline 0.029-1.300 0.016-0.85 12-100
Trimethoprim 0.06-6.80 <0.01-3.05 0-81.6
-Blocker
Atenolol 0.1-33.1 0.13-7.60 0-85.1
Metoprolol 0.002-1.52 0.003-0.25 3-56.4
Propranolol 0.05-0.12 0.01-0.615
Lipid regulator
Bezafibrate 0.05-7.6 0.02-4.30 9.10-70.5
Clofibric acid 0-0.74 0.042-0.33 0-93.6
Gemfibrozil 0.10-17.1 <0.025-5.24 0-92.3
Psycho/
Neuroactive drugs
Carbamazepine <0.04-3.78 <0.05-4.60 0-62.3
Pharmaceuticals that are more commonly detected in WWTPs belong mostly to the
therapeutic classes of the anti-inflammatories and analgesics, antibiotics, blood lipid
regulators, beta-blockers and psycho/neuroactive drugs (Fent et al., 2006; Nikolaou et al.,
2007; Aga, 2008; Miège et al., 2009; Calisto and Esteves, 2009; Verlicchi et al., 2012;
Michael et al., 2013; Luo et al., 2014; Vieno and Sillanpaa, 2014). In fact non-steroidal anti-
inflammatory drugs (NSAIDs) are very commonly reported as the pharmaceuticals with the
most abundant loads in WWTPs, which may be attributed to their high consumption because
these are over-the counter drugs. Among NSAIDs ibuprofen, naproxen, diclofenac and
ketoprofen are mentioned as being detected most frequently and at the highest concentrations
in WWTP influents. Even though the concentrations of these compounds are markedly
lowered at the effluent, because of the high inputs, they leave the WWTPs at concentrations
that are frequently quite far from negligible (Aga, 2008; Caliman and Gavrilescu, 2009;
Calisto and Esteves, 2009; Kümmerer, 2009; Kummerer, 2009; Cuong et al., 2011; Kumar et
al., 2012; da Silva et al., 2013; Li, 2014; Frade et al., 2014).
Several studies also evidence the ubiquity of several antibiotics (e.g. ofloxacin,
roxithromycin, ciprofloxacin and sulfamethoxazole) in effluents of many WWTPs which
reaffirms the concern with these pharmaceuticals, in particular the recurrent worry with
bacteria resistance threat.
Among blood lipid regulators, the fact that fibrates are highly prescribed drugs might be
seen as supporting the prediction that they would also be detected frequently in wastewaters.
In fact, they are not present in their native form but in the form of their bioactive metabolite
clofibric acid. This compound has been one of the earliest detected and most notorious water
contaminants of pharmaceutical origin.
Many studies also report the detection in WWTPs influents and effluents of a number of
beta-blocker drugs, namely atenolol, metoprolol, propanolol and sotalol. Among these,
however, atenolol seems to be the beta-blocker most frequently found worldwide.
Carbamazepine, fluoxetine, and diazepam are the compounds that are more often
detected among neuroactive pharmaceuticals. The anti-epileptic carbamazepine is a

prominent case among pharmaceutical pollutants as it is an especially common contaminant
of the aquatic environment, a fact that is due to its long history of clinical usage and to its
notoriously recalcitrant behavior. Common WWTP influent concentrations are in the order of
magnitude of several hundreds ng L–1 (Table 1).
Among highly consumed drugs, those harder to biodegrade were observed in most cases
to be also more frequently present in the treated WWTPs effluents. Nevertheless, even some
easily biodegradable pharmaceuticals for which conventional wastewater treatment processes
may achieve high removal efficiencies may also still occur in non-negligible amounts in the
treated effluents, because they arrive at WWTPs in heavy influent loads. Consequently, these
compounds may end up being discharged into the receiving water bodies even though the
WWTP efficiency may be considered reasonably high in such cases.
In addition to the parent pharmaceutical compounds, a series of ensuing metabolites and
transformation products are also of great environmental importance and concern as, in some
cases, their ecotoxicity may even exceed that of the original drugs. However, up to now the
study of pharmaceutical metabolites and transformation products and their occurrence in
WWTPs effluents and the aquatic environmental is still very scarce. Notable exceptions from
some specific compounds are clofibric acid, 10,11-dihydro-10,11-epoxycarbamazepine, 3 –
hydroxycarbamazepine, salicylic acid, hydroxyl-ibuprofen and carboxy-ibuprofen (Farré et

al., 2008; Miège et al., 2009; Leclercq et al., 2009).
3.2. Occurrence of Pharmaceuticals in Sewage Sludge

Sludge, originating from the wastewater treatment processes, is the semisolid residue
generated during the primary (physical and/or chemical), the secondary (biological), and the
tertiary (often nutrient removal) treatment.
Due to the physical-chemical processes involved in the treatment, the sludge tends to
concentrate heavy metals and poorly biodegradable trace organic compounds as well as
potentially pathogenic organisms (viruses, bacteria, etc.) present in wastewaters. Sludge is,
however, rich in nutrients such as nitrogen and phosphorous and contains valuable organic
matter that is useful when soils are depleted or subject to erosion.
Agricultural application of sewage sludge has become a widespread method for its
disposal, since it is the most economical outlet for sludge (compared to incineration) and, at
the same time, offers the opportunity to recycle plant nutrients and organic matter to soil for
crop production stimulating biological activity. In general, it is considered that the re-use of
sludge should be encouraged since it represents a long-term solution, provided that the re-
used sludge quality is compatible with public health and environmental protection
requirements. However, in regard to limits of pharmaceutical levels in sludges for soil
application, current legislation (both in the U.S. and in the E.U.) is lacking in prescribing any
such control. Considering the hydrophobic/lipophilic nature and interactions with sludge
particles (e.g., cation bridging, hydrogen bonding), it is believed that pharmaceuticals, can
sorb onto sludge during primary and secondary sedimentation. Moreover, for some
pharmaceuticals, such as antibiotic fluoroquinolones, sorption to sewage sludge represents the
main removal route during wastewater treatment (Lillenberg et al., 2010; Giger et al., 2003;
Jones-Lepp and Stevens, 2007; Picó and Andreu, 2007; Kümmerer, 2009; Michael et al.,
2013; Zhou et al., 2013; Frade et al., 2014). In general, data on the occurrence of
pharmaceuticals in sludge are sparse. Most of the studies on the fate of pharmaceuticals in
WWTPs are focused only on the aqueous phase, due to the considerable complexity of the
sludge matrix and, as consequence, to the difficulties of performing chemical analyses on that
medium.
Where available, studies on pharmaceuticals contamination in sludge have more
frequently targeted the group of antibiotics (which is reported in several studies to be
typically the most abundant pharmaceuticals class found in sludge samples), in particular
fluoroquinolones, tetracyclines and sulfonamides (Lillenberg et al., 2010; Giger et al., 2003;
Kim et al., 2005; Jones-Lepp and Stevens, 2007; Picó and Andreu, 2007; Kümmerer, 2009;
Tang et al., 2009; Prado et al., 2009; Yang et al., 2010; Clarke and Smith, 2011; Hu et al.,
2012; Yang et al., 2012; Michael et al., 2013; Pamreddy et al., 2013; Dorival-Garcia et al.,
2013; Zhou et al., 2013; Frade et al., 2014). Only few studies reported data on the occurrence
of anti-inflammatories and some other therapeutic classes (psychoactive drugs, lipid-lowering
drugs, etc.) (Wu et al., 2008; Spongberg and Witter, 2008; Okuda et al., 2009; Nieto et al.,
2010; Clarke and Smith, 2011; Jelic et al., 2011; Yu et al., 2011; Verlicchi et al., 2012; Jelic
et al., 2012; Vieno and Sillanpaa, 2014).
4. MAIN REMOVAL PROCESSES FOR PHARMACEUTICALS IN WWTPS

In the preliminary, primary and secondary stages of treatment in conventional WWTPs
the primary removal mechanisms for pharmaceutical residues are one or more of: sorption
onto suspended solids (with the subsequent elimination in sludge separation), biodegradation
(aerobic, anoxic and anaerobic) and chemical transformation (via processes such photolysis or
hydrolysis). Further elimination may be achieved in tertiary treatment (through advanced
treatment processes) where the previous mechanisms may also be used in especially
optimized conditions, but other mechanisms may also be involved such as catalytic oxidation,
membrane filtration; or in alternative to such advanced treatments, through the complex
action of natural systems (as in the case of phytoremediation treatment) where a variety of
physical, chemical and biological processes interoperate cooperatively as has been described
in detail elsewhere (Deegan et al., 2011; Jones et al., 2005; Aga, 2008; Matamoros et al.,
2008; Onesios et al., 2009; Monteiro and Boxall, 2010; Verlicchi et al., 2012; Feng et al.,
2013; Tijani et al., 2013; Tahar et al., 2013; Luo et al., 2014; Verlicchi and Zambello, 2014;
Ganzenko et al., 2014; Gude et al., 2014; Li et al., 2014b; Rodriguez-Mozaz et al., 2015;
Cincinelli et al., 2015).
a) Sorption on Sludge
The main removal mechanism of most recalcitrant pharmaceuticals in conventional

WWTPs is sorption to sludge, rather than biodegradation. In fact, sorption can be an
important removal mechanism especially when a pharmaceutical is hardly biodegradable and
has a high sorption potential.
The extent of sorption of a compound to sludge is described by the sorption constant (Kd).
Sorption to sludge can be considered as a relevant elimination process for compounds with Kd
values ~ 2.5 – 2.7 (i.e. Kd > 300 – 500 L kg-1) (Ternes et al., 2004c; Joss et al., 2005; Luo et
al., 2014).
Two different kinds of sorption mechanisms can take place (Verlicchi et al., 2012):
i) absorption, which is due to hydrophobic interactions of the aliphatic and aromatic

groups of a compound with the lipophilic cell membrane of the microorganisms or
the lipid fractions of the suspended solids (Ternes et al., 2004c). The ability of a
substance to be absorbed by sludge is defined by its octanol-water partition
coefficient (Kow), which is usually used as a measure of the hydrophobicity of a
compound. Charged compounds and uncharged ones with log Kow < 2.5 are assumed
to be poorly absorbed in sewage sludge (Rogers et al., 1986).
ii) adsorption, which is due to electrostatic interactions of positively charged groups of
chemicals with the negatively charged surfaces of the microorganisms or microbial
flocs in sludge (Ternes et al., 2004c; Carballa et al., 2008).
It consequently follows that lipophilicity and the electrostatic state are two important
properties with a determining influence on the amount of pharmaceutical that is sorbed to the
sludge. Due to the ionic nature of many pharmaceuticals, adsorption is the most plausible
mechanism for sorption of cationic compounds in WWTPs. For non ionic, weekly polar
pharmaceuticals, the ability of a compound to be retained in the sludge through absorption
will in general be related to its Kow. However, mechanisms other than hydrophobic
partitioning and electrostatic-driven physical adsorption, namely chemical interactions
through hydrogen bonding, ionic interactions, and surface complexation, may play a
significant role in the sorption of pharmaceuticals to sludge. Therefore, also compounds with
low log Kow and log Kd values may in some cases extensively sorb to sludge. For example,
despite their negative log Kow, fluoroquinolones have a high tendency for sorption and were
reported to be present in sewage sludge samples from various WWTPs (Golet et al., 2002;
Golet et al., 2003; Vieno et al., 2007; Picó and Andreu, 2007; Radjenovic et al., 2009b; Yang
et al., 2010; Van Doorslaer et al., 2014). Also tetracycline and sulfonamides exhibit strong
sorption onto sludge particles, higher than expected based on their hydrophobicity
(Kümmerer, 2009; Verlicchi et al., 2012).
Sorption on the sludge is a mechanism depending on many factors, including
temperature, pH, wastewater composition, and chemical characteristics of the compounds (in
addition to the already mentioned Kow, other properties include the ionization constant Ka, the
water solubility, etc.) (Ternes et al., 2004a; Ternes et al., 2004c; Joss et al., 2005; Carballa et
al., 2008; Kümmerer, 2009; Verlicchi et al., 2012; Michael et al., 2013; Luo et al., 2014; Van
Doorslaer et al., 2014).
Observed differences in Kd values of some pharmaceuticals on primary and on secondary
sludge seem to indicate that sludge composition and pH are crucial, at least for specific
sorption interactions.
In the secondary sludge, microorganisms represent the greater proportion of the
suspended solids, whereas the primary sludge contains fewer microorganisms and is primarily
characterized by a large lipid fraction (Carballa et al., 2008).
b) Biodegradation
During biological degradation in WWTPs, pharmaceutical contaminants can undergo: (1)

mineralization; (2) transformation to more hydrophobic compounds, which partition onto the
solid portion of the activated sludge; and (3) transformation to more hydrophilic compounds,
which remain in the liquid phase and are eventually discharged into surface waters.
Furthermore, the biological transformation/degradation of pharmaceuticals in WWTPs can
occur by direct metabolization or by co-metabolization (Aga, 2008; Onesios et al., 2009; Luo
et al., 2014). In the former, bacteria use the compound as their primary carbon source,
whereas in the latter, bacteria break down or partially convert the compound but do not use it
as the primary carbon source.
Despite the wide consortium of microorganisms present in biological treatments, it is
unlikely that pharmaceuticals present as microcontaminants in wastewater can be fully
removed by biodegradation alone. Firstly, due to the relatively low concentrations of
pharmaceuticals relative to other pollutants in the wastewater, it is unlikely that
pharmaceuticals will be favorable energy or carbon sources for microorganisms compared to
other organic constituents in the wastewater, and co-metabolization has been considered as
the more plausible mechanism of biodegradation.
In addition, such low concentrations may be insufficient to induce a significant activity

increase on enzymes that are capable of degrading pharmaceuticals. Secondly, many of these
compounds are bioactive, which can inhibit growth or metabolism of microorganisms. In
addition, for those pharmaceuticals that are excreted mostly in unmetabolized form, the fact
that they leave the body without modification may be indicative that those compounds are
more resistant to biological transformations and, therefore, will in general be recalcitrant to
biodegradation in WWTPs too. Moreover, even for biodegradable compounds, the rates of
these processes are too low for the complete biodegradation of the compounds during typical
hydraulic retention times in WWTPs. As such, their elimination has been found to be
incomplete (Vieno et al., 2007; Verlicchi et al., 2012).
Biodegradation processes are strongly influenced by the characteristics of the biomass,
the compound‘s physical and chemical properties (in particular, the complexity of the
chemical structure and the presence of some structural features and functional groups, e.g.
aromatic rings, halogens, etc.), the plant configuration (e.g. activated sludge, trickling filters,
rotating biological contactors, aerated lagoons), environmental conditions (e.g. temperature,
redox conditions) and operation parameters (e.g. hydraulic retention time, solid retention
time, agitation and aeration conditions) (Joss et al., 2005; Clara et al., 2005; Vieno et al.,
2005; Vieno et al., 2007; Aga, 2008; Miège et al., 2009; Onesios et al., 2009; Verlicchi et al.,
2012; Luo et al., 2014).
c) Abiotic Transformation
Abiotic transformations of pharmaceuticals in WWTPs may occur via hydrolysis and

photolysis. However, pharmaceuticals are typically administered in aqueous solution and,
thus, are generally resistant to hydrolysis. Therefore, photodegradation is probably the
dominant process for their abiotic transformation (Andreozzi et al., 2003; Monteiro and
Boxall, 2010).
c1) Photodegradation
Photodegradation can be an important mechanism for removal of many pharmaceuticals
from aqueous media. Several pharmaceutical classes, including analgesics, anti-
inflammatories, antibiotics and -blockers, have shown some susceptibility to be
photodegraded in laboratory experiments and field observations (Andreozzi et al., 2003;
Packer et al., 2003; Boreen et al., 2003; Chiron et al., 2006; Liu and Williams, 2007; Piram et
al., 2008; Radjenovic et al., 2009b; Bonvin et al., 2013; Kawabata et al., 2013; Wang and Lin,
2014; Gotostos et al., 2014; Ma et al., 2014; Batchu et al., 2014; Challis et al., 2014; Lin et
al., 2014; Loaiza-Ambuludi et al., 2014)
Photolysis can occur directly by absorption of solar light or indirectly involving the
action of natural photosensitizers. These include humic acids and other photolytically excited
natural organic matter, nitrates, carbonates, or iron present in the aqueous media (Andreozzi
et al., 2003; Boreen et al., 2003; Jones et al., 2005; Monteiro and Boxall, 2010; Zhang et al.,
2013; Wang and Lin, 2014; Lee et al., 2014; Ma et al., 2014; Batchu et al., 2014) that can
generate strong oxidant species such as hydroxyl radicals and singlet oxygen under solar
irradiation (Packer et al., 2003; Liu and Williams, 2007; Piram et al., 2008; Radjenovic et al.,
2009b; Fatta-Kassinos et al., 2011b; Wang and Lin, 2014; Ma et al., 2014; De Laurentiis et
al., 2014).
Direct photolysis depends essentially on two major factors, the rate of light absorption (as
determined by the pharmaceutical‘s molar absorptivity and by the light intensity in the UV-B
and UV-A wavelength ranges) and the quantum yield (the measure of how efficiently the
substance reacts after the absorption of a photon; it is characteristic of each compound over a
given wavelength range). The extent of direct photolysis can be determined experimentally
through common procedures for obtaining a direct photolysis rate constant under a given
irradiation source (sunlight or artificial light).
Indirect photolysis is often responsible for a major contribution to the overall photolytic
fate of pharmaceuticals, in particular when the absorption of light above 290 nm by the
compound is not very significant. Indirect mechanisms can be very complex and difficult to
predict, as pharmaceuticals can react via multiple pathways through interaction with naturally
occurring photo-generated transient species.
The extent of photolysis of a given compound is highly dependent on its chemical
structure. The presence of aromatic rings and conjugated- systems, as well as various
functional groups and heteroatoms, favor the direct absorption of solar radiation. These
structures typically enhance the strong absorption of radiation in the UV-C wavelength range,
with tailing absorption into the UV-B and even, in some cases, also the UV-A ranges. The
potential spectral overlap of the absorptivities of many pharmaceuticals with natural sunlight
wavelength range ( > 290 nm) suggests that, in a considerable number of cases, there are
favorable conditions for them to be degraded at least partially by direct photolysis.
In addition to the intrinsic compound's characteristics, the photodegradation of
pharmaceuticals also depends on conditions such as temperature, light intensity, composition
of the wastewater (e.g. dissolved organic matter, nitrate concentration, pH) and operation
parameters in WWTPs (Andreozzi et al., 2003; Boreen et al., 2003; Ternes et al., 2004b; Kim
et al., 2005; Mai et al., 2010; Majewsky et al., 2011; Kawabata et al., 2013; Niu et al., 2013;
Wang and Lin, 2014; Batchu et al., 2014; Challis et al., 2014; Young et al., 2014).
One major issue with photodegradation processes is the potential toxicity of degradation
products, in particular the possibility that in some cases they exhibit higher toxicity than the
original compounds (Trovo et al., 2009; Kawabata et al., 2012; Wang and Lin, 2014; Ma et
al., 2014; Bergheim et al., 2014).
In the case of pharmaceuticals, all removal processes in WWTPs (mainly sorption,
biodegradation, and photodegradation) are in some way limited: i) the extent of sorption is
dependent on the characteristics of sludge; ii) pharmaceuticals are designed to be biologically
stable; and iii) even though many pharmaceuticals are photoactive, because they frequently
possess aromatic rings, heteroatoms, and other functional groups that could be susceptible to
photodegradation, they may also yield products that present some environmental concern.
5. FACTORS AFFECTING THE REMOVAL OF PHARMACEUTICALS

IN WWTPS
Several factors have been mentioned already that have some minor or major influence
over the efficiency of pharmaceuticals removal from wastewater by WWTPs processes.
Several studies are available in the literature where the effects of some of these factors are
addressed (Carballa et al., 2004; Clara et al., 2005; Vieno et al., 2005; Vieno et al., 2007;
Cirja et al., 2008; Carballa et al., 2008; Aga, 2008; Miège et al., 2009; Onesios et al., 2009;
Verlicchi et al., 2012; Michael et al., 2013; Vieno and Sillanpaa, 2014).
Following, a more comprehensive account of most of all such factors is presented, which
regard to the physico-chemical properties of the pharmaceuticals, environmental conditions
and factors which are specific to WWTPs, such as wastewater characteristics and operation
parameters.
a) Physico-Chemical Properties of Pharmaceuticals
Some of the most important properties of pharmaceuticals that affect their fate and
removal in WWTPs are:
 Molecular Structure: pharmaceutical structural factors of major importance are

molecular size, hydrophobicity, molecular charge, ability for hydrogen bonding,
arrangement and interactions of molecular fragments and its coordination (Dragun,
1998). In general, pharmaceuticals are large and complex molecules with several
functional groups. In this respect they form a very heterogeneous group varying
widely in their molecular structures, functionalities, molecular weights and other
properties (Cunningham, 2008).
 Polarity: due to their typically varied heteroatom content and multifunctional
composition many pharmaceuticals are polar or ionizable, and this fact plays a major
role in their solubility and mobility in WWTPs (Cunningham, 2008). A polar
pharmaceutical will be very water soluble and tend to not be adsorbed onto organic
matter. On the other hand, a non-polar compound will have a higher tendency to
leave water and be adsorbed to sludge (Cunningham, 2008). However, a fact that can
not be ignored is that the ionization of some pharmaceuticals may lead to more
complex ionic, ion pairing or complexation mechanisms of sorption which are not
accounted for in the conventional simple mechanism of non-polar partitioning to
organic matter in sludge (Cunningham, 2008).
 Ionization constant (pKa): whether an acid or basic pharmaceutical exists as the
neutral molecule or as its conjugate ionic species will have a profound influence on
its behavior, bioavailability and, ultimately, its WWTPs fate (Cunningham, 2008).
Firstly, the ionic species will be much more water soluble than its neutral
counterpart. Consequently, if the acid or basic pharmaceutical is easily ionized, it
will not distribute as readily into a hydrophobic (lipid-like) compartment, neither will
it have a tendency to volatilize. Secondly, the pharmaceutical‘s charge can directly
affect its affinity with the solid phase and determine the type of sorption mechanism
(hydrophobic or ionic). It is not uncommon for pharmaceuticals to have more than
one ionizable functional group which results, in such cases, in several degrees of
ionization for the molecules, controlled by the pH of the medium (Cunningham,
2008).
 Water solubility (Sw): the tendency of a compound to be in the aqueous medium is

an important factor in determining its mobility and distribution. Solubility in water is
mainly determined by the compound‘s polarity or tendency to ionize (as indicated by
its pKa). However, other factors may strongly influence a compound‘s solubility such
as temperature, pH and ionic strength of the solution, and dissolved organic matter
content (Tinsley, 2004).
 Octanol-water partition coefficient (Kow): is a measure of the hydrophobicity of an
organic compound. An inverse relation between the Kow and aqueous solubility is
intuitive since compounds that are soluble in water are usually less soluble in non-
polar organic solvents like octanol (playing the role of a surrogate for lipids) and vice
versa. Therefore, the more hydrophobic a compound is, the less water soluble it
becomes and the more likely it will adsorb to inorganic as well as organic solid
particles (Cunningham, 2008).
 Sorption or solid-water distribution coefficient (Kd): is commonly used as a
quantitative measure of the sorption of compounds from the aqueous compartment
onto sludge. The Kd value is simply a ratio of the sorbed phase concentration to the
solution phase concentration at equilibrium (Cunningham, 2008). The Kd value is
often estimated from the log Kow of the contaminant and the organic matter content of
the sludge. However, this approach is based on the assumption that the partitioning
will be essentially into the organic fraction of the solid which is valid only for neutral
and hydrophobic compounds. In fact, many pharmaceuticals are multifunctional
ionizable compounds with a much more complex partitioning behavior which may
include ionic, ion pairing and/or complexation interactions with solid particles. In
this case, Kd should be experimentally determined at different pH values and solution
ionic strengths in order to assess the role of ionic interactions.
 Volatility: this depends on the Henry‘s law constant (KH) of the compound.
Alternatively, for practical applications, it is most convenient to use a

―dimensionless‖ air-water partition constant or Henry‘s coefficient (Kaw) which is
defined as the ratio between concentrations in the vapor and in the water phase
(Schwarzenbach et al., 2003). Pharmaceuticals generally have low volatilities (Kaw <
10-5 (POSEIDON, 2006)), since these compounds are mostly meant to take effect in
an aqueous environment and, therefore, are rather hydrophilic. The volatility of a
compound depends on temperature and may also be diminished if the compound is
not available in water, e.g. when it is sorbed.
 Persistence: the ability of a pharmaceutical to remain active in the environment is
measured by its half-life. Half-life is not an absolute characteristic of a substance but
depends on environmental conditions, which helps explain why values of this
property reported in the literature for the same pharmaceutical sometimes span very
broad ranges of values. Persistence is a function of the chemical and biological
degradation processes which break down the pharmaceuticals into simpler
compounds.
b) Environmental Conditions
The main environmental conditions that affect the removal of pharmaceuticals in

WWTPs include:
 Temperature: some physical properties such as vapor pressure, water solubility and,
thereby, also the Henry's law constant are strongly temperature dependent.
Temperature variations can also affect biodegradation and partition (sorption and
volatilization) of pharmaceuticals (Vieno et al., 2005; van Nuijs et al., 2009;
Daneshvar et al., 2010; Verlicchi et al., 2012; Golovko et al., 2014). In fact, in the
case of biodegradable pharmaceuticals, environment temperature (the variations of
which have a seasonal character) is one of the factors to have a major influence in the
removal efficiency in WWTPs (Vieno et al., 2005; van Nuijs et al., 2009; Daneshvar
et al., 2010; Verlicchi et al., 2012; Golovko et al., 2014). On the other hand, poorly
biodegradable pharmaceuticals, which may be removed primarily by sorption
processes, are affected by temperature in a less significant extent.
 Rainfall: rainfall events lead to a dilution of the wastewater but also significantly
increase the flow rate in the WWTP that consequently reduces the hydraulic retention
times (Musolff et al., 2009; Phillips et al., 2012; Baker and Kasprzyk-Hordern,
2013). Rainfall events also disturb the microbial activity of the WWTP enormously,
with potential washout of important microorganism species (Ternes, 1998). These
events lead to a (sometimes significant) decrease in the elimination rates of
pharmaceuticals.
 Sunlight: rates of photodegradation processes, which are responsible for some
transformation of less photostable compounds, are directly correlated with sunlight
intensity, especially in the elimination of photodegradable pharmaceuticals with
absorbances in the range of the visible electromagnetic radiation (290-800 nm)

(Andreozzi et al., 2003; Boreen et al., 2003; Bergheim et al., 2014).
c) Wastewater Characteristics
Some characteristics of the wastewater also have a significant impact on the effectiveness
of the processes responsible for pharmaceuticals removal in WWTPs. These include:
 pH: the acidity or alkalinity of the aqueous environment can as well affect the fate
and removal of pharmaceuticals by influencing some of their properties such as the
degree of ionization, solubility and, in some cases, even the chemical stability of the
compound. Under different pH conditions, ionizable molecules can be neutral,
cationic, anionic or zwitterionic and, consequently, their physical, chemical and
biological properties (e.g. sorption, photo-reactivity, bio-activity and toxicity) will
shift according to the changes in the degree of ionization and charge state (Cirja et
al., 2008; Kummerer, 2009; Verlicchi et al., 2012). Additionally, the physiology of
microorganisms and the activity of extra-cellular enzymes is strongly influenced by
the medium pH, as biological processes are usually only effective within a narrow
range of pH values.
 Organic matter: the content of organic matter in a given medium influences the
sorption affinity of the more hydrophobic compounds for that medium, as well as
influencing the level of biological activity. Organic matter also serves as an energy
source for microorganisms that are essential for the breakdown of some
biodegradable pharmaceuticals.
 Redox conditions: the biodiversity of the microbial flora present in the wastewater or
sludge as well as the type of chemical and biological processes that occur during
treatment are influenced by the redox conditions of the environment (Gobel et al.,
2007), which, in turn, may have an impact on the efficiency of biodegradation. In
general, it is observed that pharmaceuticals are more efficiently degraded in aerobic
conditions than in anoxic or anaerobic conditions (Jones et al., 2005).
d) Operation Parameters
Some of the main WWTP operation parameters that also have an important influence in
the efficiency of the treatment are:
 Hydraulic retention time (HRT): the residence time of the wastewater in the
bioreactor affects the elimination of the biodegradable pharmaceuticals (Kim et al.,
2005; Gros et al., 2010; Verlicchi et al., 2012; Vieno and Sillanpaa, 2014). The
extent of biodegradation and, consequently, more efficient elimination of the
pharmaceuticals results from increased contact time between the microorganisms and
the wastewater to be treated (Kim et al., 2005; Gros et al., 2010; Verlicchi et al.,
2012; Vieno and Sillanpaa, 2014).

 Solids retention time (SRT) or sludge age: the residence time of activated sludge
solids in the bioreactor seems to have some correlation with treatment efficiency. In
fact, biological wastewater treatments employing higher SRTs (>10 days) are
reported to yield higher removal efficiencies for pharmaceutical compounds
compared with cases where sludge is removed more frequently (Kreuzinger et al.,
2004; Clara et al., 2005; Kim et al., 2005; Maurer et al., 2007; Gobel et al., 2007;
Oppenheimer et al., 2007; Onesios et al., 2009; Majewsky et al., 2011; Luo et al.,
2014). This observation implies that there is an enhanced biodegradation ability or
different sorption capacity for pharmaceuticals in sludge with a higher SRT. Several
researchers have, in fact, observed increased biomass hydrophobicity at higher SRTs.
Increasing sludge age enables populations of slower growing bacteria to develop and
also serves to increase the potential for the acclimation of the population to the
compounds encountered. Among such slower growing bacteria, the nitrifying
bacteria have been found to have positive effects on the removal of a range of
micropollutants such as pharmaceuticals because, under nitrifying conditions, co-
metabolism using ammonium monooxygenase enzyme is a possible degradation
pathway for many such micropollutants (Suarez et al., 2010; Fernandez-Fontaina et
al., 2012). This change in the bacterial population with time means any chemicals in
the wastewater are exposed to a greater array of bacteria and bacterial enzymes,
increasing the likelihood that they will be degraded to less harmful compounds;
however, recalcitrant polar organics may still pass through (Jones et al., 2005).
 Dissolved oxygen level: the oxygen concentration in the wastewater will determine
the redox conditions and, consequently, the extent and type of redox phenomena
present, both in chemical and in biological processes (aerobic, anoxic or anaerobic).
6. ALTERNATIVE/COMPLEMENTARY REMOVAL TECHNOLOGIES

IN WWTPS
Although commonly adopted as the best cost-effective wastewater treatment technology

available to date, conventional treatment typically attains only partial removal of most
pharmaceutical residues, especially polar ones. Thus, it has become clear that more advanced
technologies must complement (or, in some specific cases, substitute) conventional treatment
in order to fulfill more stringent WWTP effluent quality goals or to comply with requirements
for municipal wastewater reuse.
The high cost of implementation and/or operation is the major drawback of most
advanced treatment technologies currently available. As a more economic alternative, the use
of aquatic plants-based technologies such as constructed wetlands (CWs) for the removal of
pharmaceuticals residues has been increasingly explored in recent years. In fact, these
systems are becoming an option for secondary wastewater treatment systems or as treatment
units for polishing secondary effluent from WWTPs. In addition to low cost, simple operation
and maintenance (thereby not requiring highly skilled labour), and environmental friendliness
are some of their most attractive characteristics.
6.1. Advanced Treatment Technologies
In recent years, a broad number of advanced treatment technologies for pharmaceutical

compounds have been researched. Among them activated carbon adsorption, membrane
treatment using both biological (membrane bioreactors) and non-biological processes
(reversed osmosis, ultrafiltration, nanofiltration), and advanced oxidation processes (AOP)
are the most frequently studied at lab experiments and pilot scale for removal of emerging
polar contaminants, such as pharmaceutical compounds, at trace concentrations (Fent et al.,
2006; Ikehata et al., 2006; Esplugas et al., 2007; Snyder et al., 2007; Cirja et al., 2008;
Klavarioti et al., 2009; Melo et al., 2009; Fatta-Kassinos et al., 2011b; Verlicchi et al., 2012;
Kit Chan et al., 2012; Dolar et al., 2012; Siegrist and Joss, 2012; Acero et al., 2012; Nguyen
et al., 2012; Michael et al., 2013; Feng et al., 2013; Tijani et al., 2013; Tahar et al., 2013; Luo
et al., 2014; Ek et al., 2014; Ganzenko et al., 2014; Secondes et al., 2014; Evgenidou et al.,
2015; Rodriguez-Mozaz et al., 2015; Cincinelli et al., 2015).
a) Activated Carbon Adsorption

Activated carbon is one of the oldest, most popular and widely used adsorbents
(Ramakrishna and Viraraghavan, 1997; Rodriguez-Reinoso, 1997; Derbyshire et al., 2001;
Babel and Kurniawan, 2003; Yu et al., 2009; Tahar et al., 2013; Margot et al., 2013; Ruhl et
al., 2014). It has long been used in water and wastewater treatment, being a recognised
technology for the removal of both natural and synthetic organic contaminants. Hence,
researchers have explored the possibility of using this process for removing emerging
pollutants including pharmaceuticals (Snyder et al., 2007; Bolong et al., 2009; Rivera-Utrilla
et al., 2009; Rossner et al., 2009; Yu et al., 2009; Gabet-Giraud et al., 2010; Michael et al.,
2013; Rivera-Utrilla et al., 2013; Tahar et al., 2013; Margot et al., 2013; Luo et al., 2014; Yu
et al., 2014; Ruhl et al., 2014; Rakic et al., 2015; Meinel et al., 2015).
Its capacity and versatility is mainly due to its porous texture that provides it with a large
surface area, its controllable pore structure and its thermo-stability (Chen et al., 2011). Its
chemical nature can also be easily modified by chemical treatment in order to enhance its
properties.
There are two most common physical forms in which activated carbons are used, namely
as a powdered feed or as granular form in packed bed filters. There are other forms that have
recently been attracting increasing attention, among them fibers mainly obtained from
isotropic coal and petroleum pitch, cloths, felts and other fibrous natural wastes (Mestre et al.,
2011; Nabais et al., 2012; Reza et al., 2014).
Almost any carbonaceous material may be used as a precursor for the preparation of
activated carbons, the main requirement being that it possesses a high carbon content.
Granular activated carbon can be used as a replacement for anthracite media in
conventional filters, providing both adsorption and filtration. It can be applied following
conventional activated sludge treatment, as an adsorption bed. However, carbon regeneration
and disposal require environmental considerations (Snyder et al., 2007). In fact, the biggest
barrier to the general use of activated carbon is its cost and the difficulties associated with
regeneration (Ramakrishna and Viraraghavan, 1997; Derbyshire et al., 2001; Babel and
Kurniawan, 2003; Chen et al., 2011; Tahar et al., 2013). Activated carbon is quite expensive
and the higher the quality the greater the cost. The use of carbons based on relatively
expensive starting materials is unjustified for most pollution control applications (Streat et al.,
1995). Also, both chemical and thermal regeneration of spent carbon is expensive, impractical
on a large scale and produces additional effluent and results in considerable loss of the
adsorbent.
b) Membrane Processes
A diversity of membrane system types (including microfiltration, ultrafiltration,
nanofiltration, reverse osmosis, electrodialysis reversal, membrane bioreactors and
combinations of membranes in series) have already been tested for the removal of
pharmaceuticals from contaminated water either at pilot or full scale (Deegan et al., 2011;
Snyder et al., 2007; Cirja et al., 2008; Bolong et al., 2009; Dolar et al., 2012; Siegrist and
Joss, 2012; Nguyen et al., 2012; Luo et al., 2014; Secondes et al., 2014; Coday et al., 2014;
Liu et al., 2014; Li et al., 2014a; Rodriguez-Mozaz et al., 2015; Cincinelli et al., 2015).
Microfiltration and ultrafiltration have been regarded as being mostly ineffective in removing
organic contaminants as pore sizes vary from 100-1000 times larger than such micro
pollutants, thus allowing them to slip through the membranes (Deegan et al., 2011). On the
other hand, reverse osmosis and nanofiltration were assessed as promising alternatives for
eliminating micropollutants including pharmaceuticals and these membrane technologies
have been evaluated and employed in tertiary wastewater treatment for wastewater
reclamation and reuse (Park et al., 2004; Nghiem et al., 2005; Yoon et al., 2006; Snyder et al.,
2007; Nghiem and Hawkes, 2007; Yoon et al., 2007; Acero et al., 2010; Benitez et al., 2011;
Siegrist and Joss, 2012; Acero et al., 2012; Plumlee et al., 2014) but studies concerning
pharmaceutical removal is still somewhat limited.
Comparatively, the nanofiltration membrane is ―looser‖ than reverse osmosis. The latter
will give, in many cases, almost complete removal, but the higher energy consumption is a
less attractive feature of this option (Vedavyasan, 2000; Plumlee et al., 2014).
b1) Membrane Bioreactor (MBR) for Wastewater Treatment

Membrane bioreactor (MBR) is the combination of a membrane process like
microfiltration or ultrafiltration with a suspended growth bioreactor (Melin et al., 2006; Judd,
2008; Li et al., 2014a). Thus MBRs combine biodegradation in a conventional activated
sludge process with a separation step to retain sludge in the system for higher removal of
micropollutants such as pharmaceuticals (Melin et al., 2006; Judd, 2008; Onesios et al., 2009;
Santos et al., 2011; Chon et al., 2011; Jia et al., 2011; Dolar et al., 2012; Siegrist and Joss,
2012; Nguyen et al., 2012; Chon et al., 2012; Galan et al., 2012; Kovalova et al., 2012;
Schroder et al., 2012; Trinh et al., 2012; Melo-Guimaraes et al., 2013; Nguyen et al., 2013a;
Nguyen et al., 2013b; Dutta et al., 2014; Vieno and Sillanpaa, 2014; Kim et al., 2014; Li et
al., 2014a).
These reactors can be composed of two units: a bioreactor tank and a membrane module,
but generally, these two units are combined in only one, where the membrane unit is
submerged inside the bioreactor. The micro or ultrafiltration membranes in an MBR are
available in different configurations, namely as flat-sheets (plate and frame), hollow fibers
(outside-in) and (multi)tubular (inside-out) and can be made from different materials
(metallic, ceramic or polymeric) (Melin et al., 2006; Li et al., 2014a).
MBRs make hydraulic retention time (HRT) independent of sludge retention time (SRT),
which facilitates a more flexible control of operation parameters. The possibility of
uncoupling HRT and SRT in tangential filtration is a clear advantage with respect to
traditional gravity settling, as it allows MBRs to achieve a high SRT within compact reactor
volumes, which is a great improvement in comparison to conventional activated sludge
systems. Because of this ability to reach higher contact times and then to maintain in reaction
a slow-growing biomass, microorganism species are more diversified with higher
physiological capacity (Auriol et al., 2006) and are more adapted to resistant compounds.
Moreover, the higher the biomass concentration, the more effective the process for treating
wastewater.
The separation of membrane and biology compartments allows different aeration systems
and oxygen concentrations in both compartments. In the biology part, fine bubble aeration
supplies oxygen to the bacteria, necessary for the oxidation of substrates, while in the
membrane compartment coarse bubble aeration is used to generate enough turbulence to
decrease, as much as possible, membrane fouling and clogging, which are the major
drawbacks of such processes. The membranes are in contact with the sludge but reject the
sludge solids while the water permeates through the membranes. Additionally, anoxic or
anaerobic biology tanks can be added for nitrogen and biological phosphorous removal.
The mechanisms involved in MBR for pharmaceuticals removal may include physical
retention by membrane, biotransformation, air stripping, sorption, and photo-transformation
(Larsen et al., 2004; Zhang et al., 2008; Judd, 2008; Suárez et al., 2008; Sipma et al., 2010;
Verlicchi et al., 2012; Pomiès et al., 2013; Li et al., 2014a). Since the molecular size of most
pharmaceuticals (molecular weight between 100 and 1,000 g mol-1) is at least 100 times
smaller than the pore size of membranes used for MBR process, no direct physical retention
of the compounds by MBR membranes can be expected (Larsen et al., 2004). Therefore, the
main mechanism for removal of most pharmaceutical micropollutants in MBR is via
biodegradation.
c) Advanced Oxidation Processes (AOP)

Conventional chemical oxidation is an important treatment process for both water and
wastewater that is commonly used for providing disinfection, but these reactions also have the
potential to attain some removal of emerging pollutants. Thus, substantial research effort has
been put into optimizing and enhancing their effects using advanced oxidation processes
(AOPs), which has recently emerged as an important class of technologies for the oxidation
and destruction of a wide range of organic pollutants in water and wastewater including
pharmaceuticals (Andreozzi et al., 2003; Ikehata et al., 2006; Klavarioti et al., 2009; Naddeo
et al., 2009; Rosario-Ortiz et al., 2010; Lester et al., 2011; de Witte et al., 2011; Scheers et al.,
2012; De la Cruz et al., 2012; Michael et al., 2013; Ibanez et al., 2013; Schulze-Hennings and
Pinnekamp, 2013; Klamerth et al., 2013; Luo et al., 2014; James et al., 2014). AOPs involve
the generation and use of free oxygen radicals and other active oxygen species (e.g.
superoxide radical anions, O2• -, hydroperoxyl radicals, HO2•, triplet oxygen, 3O2, and organic
peroxyl radicals, R-O-O•), the primary species of which being the hydroxyl radical, HO•. This
is a powerful oxidizing agent leading to oxidation and mineralization of organic matter with
elevated reaction rate constants, although also being characterized by a lack of selectivity of
attack (Ikehata et al., 2006; Esplugas et al., 2007; Michael et al., 2013; Rivera-Utrilla et al.,
2013).
These processes are characterized and differentiated by the methods of hydroxyl radicals
production, which involve combinations of chemical agents (e.g., ozone (O3), hydrogen
peroxide (H2O2), transition metals, and metal oxides) and auxiliary energy sources (e.g.,
ultraviolet-visible (UV-Vis) radiation, electronic current, -radiation, and ultrasound).
Examples of AOPs that have been used and evaluated (mainly at a bench scale, but many of
the processes are being developed at a pilot-scale as well) include O3/H2O2, O3/UV,
O3/H2O2/UV, H2O2/UV, Fenton (Fe2+/H2O2), photo- and electro-Fenton, chelating agent
assisted Fenton/photo-Fenton, heterogeneous photo-oxidation using titanium dioxide
(TiO2/h), -radiolysis, and sonolysis (Ikehata et al., 2006; Esplugas et al., 2007; Belgiorno et
al., 2007; Melo et al., 2009; Fatta-Kassinos et al., 2011b; Sires and Brillas, 2012; Michael et
al., 2013; Rivera-Utrilla et al., 2013; Feng et al., 2013; Ganzenko et al., 2014; Brillas, 2014).
This diversity of ways of producing HO• is responsible for the versatility of AOPs (Ikehata et
al., 2006; Esplugas et al., 2007; Michael et al., 2013; Rivera-Utrilla et al., 2013; Feng et al.,
2013) that facilitates the compliance with specific treatment requirements.
Some of the advantages of AOPs include the possibility of complete mineralization of
organic contaminants, or production of less harmful and more biodegradable by-products, and
ability to handle fluctuating wastewater flow rates and compositions (Zhou and Smith, 2001).
However, most studies do not include information on the by-products formed during the
application AOPs or any information related to the activity of the by-products. Therefore,
these processes should be carefully monitored and ecotoxicological investigations should be
conducted to investigate the formation of potentially toxic transformation products (Hollender

et al., 2009; Rizzo, 2011; Fatta-Kassinos et al., 2011b; Michael et al., 2013; Ganzenko et al.,
2014).
The effectiveness of oxidative processes for degrading pharmaceuticals will be largely
determined by the specific wastewater matrix. However, the effects of the matrix
characteristics on pharmaceuticals removal are more poorly understood than for other
technologies. For example, dissolved organic matter can result in the formation of oxidation
by-products that may cause wastewater quality to deteriorate beyond its initial state.
Similarly, the presence of nitrates, carbonates and dissolved organic matter can interfere with
the destruction of the target pollutants and ultimately reduce the effectiveness of the selected
AOP. The performance of AOPs is also affected by the presence of other water and
wastewater constituents such as suspended solids, as well as by pH and temperature (Ikehata
et al., 2006). For example, suspended solids and color can hinder photochemical reactions by
light scattering and absorption, and may impair the performance of photochemical AOPs,
such as O3/UV, H2O2/UV, photo-Fenton, and TiO2/h processes. Carbonate, bicarbonate, and
chloride ions, as well as some natural organic compounds, are known to act as radical
scavengers. These compounds compete with target pollutants for hydroxyl radicals. Therefore
their presence increases oxidant demands and lowers the treatment efficiency. In addition,
materials and equipment costs as well as energy requirements and efficiency must be taken
into account when assessing the overall performance of AOPs (Ikehata et al., 2006).
Reviews on the advantages and disadvantages of the different technologies are available
(Ternes et al., 2004b; Jones et al., 2005; Jones et al., 2007; Dalrymple et al., 2007; Wenzel et
al., 2008; Klavarioti et al., 2009; Melo et al., 2009; Bolong et al., 2009; Kummerer, 2009;
Kummerer, 2010; Fatta-Kassinos et al., 2011b; Rivera-Utrilla et al., 2013; Ganzenko et al.,
2014).
Much higher efficiencies are generally attainable by advanced treatment technologies, but
all of these technologies have more or less specific shortcomings:
 Many of these technologies have high costs of implementation and/or operation and
maintenance, and also require more expensive skilled labor;
 Efficiency may depend strongly on the type of compound;
 None of the technologies can remove all of the compounds (Ravina et al., 2002;
Schroder, 2002; Wenzel et al., 2008; Melo et al., 2009; Rivera-Utrilla et al., 2013;
Ganzenko et al., 2014);
 Will they work for new compounds in the future? (Kummerer, 2009);
 Mutagenic and toxic properties have been found for the reaction products of (photo)
oxidation processes (Rizzo, 2011; Fatta-Kassinos et al., 2011b; Michael et al., 2013;
Ganzenko et al., 2014);
 Resistance in biomembrane reactors: would the enrichment of antibiotics and
resistant bacteria cause increasing resistance? No information is available on this
topic (Kummerer, 2009);
 Resistance material will not fully be retained by membranes (Kummerer, 2009).
6.2. Constructed Wetlands
Constructed wetlands systems (CWS) can be defined as designed, man-made structures

composed of water saturated beds, containing (in addition to the water column) soil or other
selected solid support matrix, emergent and/or submergent wetland vegetation and microbial
populations as the main components. These systems emulate natural wetlands for human use
and benefits (Hammer and Bastian, 1989; USEPA, 1993; Cooper et al., 1996; Vymazal et al.,
1998; Kadlec and Wallace, 2009). In fact, for a long time, natural wetlands have been
credited with a capacity to depurate the waters that inundated such areas. CWS take
advantage of many of the same processes that occur in natural wetlands, but do so within a
more controlled environment. CWS are optimized to attain greater efficiencies by enhancing
the concerted action of all the components (support matrix, vegetation and microbial
population) through a variety of interdependent chemical, physical and biological processes
(sorption, microbial degradation, plant uptake, etc.).
In the past, CWS have been used mainly as wastewater treatment alternatives or
complementary to the conventional treatment for domestic wastewaters of small
communities. Thus, CWS have been mostly applied in the removal of bulk wastewater
pollutants such as suspended solids, organic matter, excess of nutrients and pathogens.
More recently, targeting use of CWS for dealing with more specific pollutants, such as
pharmaceuticals among other types of organic pollutants, especially those which are more
recalcitrant to conventional WWTPs‘ treatment, have been the subject of an increasing
number of studies both at pilot and at full scale (Matamoros and Bayona, 2006; Matamoros et
al., 2007; Matamoros et al., 2009; Dordio et al., 2009b; Dordio et al., 2010; Avila et al., 2010;
Hijosa-Valsero et al., 2010; Zhang et al., 2011a; Hijosa-Valsero et al., 2011a; Matamoros et
al., 2012; Breitholtz et al., 2012; Dan et al., 2013; Verlicchi et al., 2013; Verlicchi and
Zambello, 2014; Berglund et al., 2014; Zhu and Chen, 2014; Li et al., 2014b). In many of
such studies, CWS have been proving to be efficient and cost-effective solutions for the
removal of some pharmaceuticals (Matamoros et al., 2008; Hijosa-Valsero et al., 2010; Zhang
et al., 2011a; Breitholtz et al., 2012; Verlicchi et al., 2013; Verlicchi and Zambello, 2014;
Berglund et al., 2014; Zhang et al., 2014). In fact, CWS are increasingly being used to
provide a form of secondary or tertiary treatment for wastewaters.
It is not easy to accomplish a comprehensive description of organic xenobiotics removal
processes in CWS and these wastewater treatment systems have been (and to some extent still
are) frequently operated as ―black boxes‖. In the past, much of the design of CWS has been
done with little knowledge of, or consideration for, the roles played by each component and
how their effects could be enhanced and optimized. However, over the most recent years
some accumulated knowledge has been increasingly applied in the construction and operation
of new systems, which results in a much greater variety of plant species, support matrix
materials and constructed wetland designs being seen, studied and introduced in newly
constructed CWS (Brix, 1997; Sundaravadivel and Vigneswaran, 2001; Stottmeister et al.,
2003; Akratos and Tsihrintzis, 2007; Brisson and Chazarenc, 2009; Truu et al., 2009;
Calheiros et al., 2009; Dordio et al., 2009a; Dordio et al., 2010; Zhang et al., 2010; Li et al.,
2010; Albuquerque et al., 2010; Dordio et al., 2011; Vymazal, 2011; Dordio and Carvalho,
2013; Verlicchi and Zambello, 2014; Carvalho et al., 2014; Zhang et al., 2014; Cardinal et al.,
2014).
Major design parameters, removal mechanisms and treatment performances have been
studied and reviewed by several researchers (Vymazal et al., 1998; Sundaravadivel and
Vigneswaran, 2001; García et al., 2004; Garcia et al., 2005; Kadlec and Wallace, 2009;
García et al., 2010; Vymazal, 2011; Hijosa-Valsero et al., 2011b; Reyes-Contreras et al.,
2012; Zhang et al., 2012a; Zhang et al., 2012b; Ranieri et al., 2013; Avila et al., 2013;
Verlicchi and Zambello, 2014; Avila et al., 2014; Li et al., 2014b).
CONCLUSION
Typical conventional WWTPs consist of the common stages of preliminary, primary and
secondary treatments (the latter employing most commonly the technology of activated
sludge systems). The final liquid effluent is eventually discharged in a receiving water body
or reused for irrigation purposes whereas the treated sludge is disposed in a landfill or reused
as soil fertilizer. In any case, it is necessary to assure high standards of effluent quality and
careful monitoring of the most problematic (prioritary) pollutants, especially if treated
wastewater and/or sludge reuse is intended. Concomitantly, knowledge about
ecotoxicological effects of this class of pollutants (not limited to the parent pharmaceutical
substances but also their metabolites and transformation products, which in many cases are
not well identified, much less their potential toxicity) has to be vastly improved. Such
knowledge is crucial for assessing the risk to the environment (and possibly to public health)
that can be caused by this type of substances as well as to establish the need to address their
removal in WWTPs (and which concentration limits must be attained).
Conventional WWTPs were never designed to deal with pharmaceutical compounds and
other such organic micropollutants. Efficiencies of pharmaceuticals removal in WWTPs vary
substantially for different compounds (and even for the same substance), as was shown
above, in result of the wide range of physicochemical properties that characterize this ample
group of chemical substances, in addition to the variability of environmental and operational
conditions. Because in many cases WWTPs efficiencies for removal of pharmaceuticals can
be considered insufficient, optimization of wastewater treatment processes remains a task
with top priority. In this regard, one of the main weaknesses to be overcome is the efficiency
of biological treatment, which is commonly low for polar persistent micropollutants.
Improvements have been attempted under more favorable conditions, e.g. increasing contact
times (SRT and HRT), optimizing temperature, and fine tuning redox conditions. In order to
achieve more effective optimizations, it is necessary to have a comprehensive knowledge of
the fate of pharmaceuticals in WWTPs and a deeper understanding of the factors that affect
such fate.
The application of advanced treatment processes downstream of conventional biological
process can significantly enhance the removal of more recalcitrant pollutants before effluent
disposal. In fact, advanced technologies such as adsorption processes, advanced oxidation
processes and membrane processes, seem to be promising alternatives for pharmaceuticals
removal, considering the high efficiencies that have been attained in many cases. However,
two major issues need to be address that may hinder the full-scale application of some
advanced treatment processes: high costs of implementation and operation which compromise
their economic viability, and the formation of by-products that need to be identified and
whose toxicity needs to be well characterized. In regard to the latter, not all advanced
treatment processes involve the occurrence of chemical reactions with formation of by-
products (e.g. adsorption processes); for those that do, further studies need to indicate
conditions to favor mechanisms that avoid formation of such by-products or achieve their
complete decomposition. In regard to economical considerations, recent research has been
increasingly turning to the search and study of low cost reagents and materials (e.g. easily and
widely available natural materials or agricultural wastes) and the study and optimization of
cheaper alternatives such as constructed wetlands systems.
As public awareness to the issue of emergent pollutants is increased, in particular in
regard to water contamination with pharmaceuticals which have recently been gaining
attention of the press, it is to be expected that sooner or later that concern will have its effect
in more strict regulations on water and wastewater quality. At some point the efficiencies of
WWTPs in dealing with pharmaceutical pollutants will need to be addressed and the
conventional plants will need to be reformed or adapted. Cost-effective options need to be
evaluated, optimized and made ready for that foreseeable future.
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Chapter 2
MOLECULAR CHARACTERIZATION OF MICROBIAL

COMMUNITIES IN CONSTRUCTED WETLANDS:
THE EFFECT OF PLANT SPECIES, ORGANIC MATTER
AND HYDRAULIC DESIGN
Ricardo Sidrach-Cardona1, Olga Sánchez2*†,

Laura Garrido2, Jordi Mas2 and Eloy Bécares1
1
Department of Ecology, Faculty of Biology, University of León, León, Spain
2
Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona,
Bellaterra, Spain
ABSTRACT
Constructed wetlands are becoming an interesting alternative for wastewater reuse

since high concentrations of contaminants and pathogenic microorganisms can be
removed with these natural treatment systems. In this study, experimental constructed
wetlands treating diluted wastewater were used to study the effect of plant species (Typha
angustifolia or Phragmites australis), hydraulic design (free-water flow or sub-
subsuperficial flow) and organic loading (3 or 9 gBOD 5/m2/d) on the microbial
composition of the rhizoplane, gravel biofilm and interstitial water. The analysis of
DGGE band patterns showed statistically significant differences in community
assemblages between plant species. Hydraulic configuration, and plant presence in a
lesser extend, were more important than organic load in shaping microbial communities
in the studied wetlands. Distinctive communities were found for roots, gravel biofilm and
interstitial water inside the same mesocosm, being differences among these communities
higher for Phragmites than Typha planted tanks. Plants had an effect on all the microbial
communities studied into the mesocosms, proving that their influence affect interstitial
water and gravel-associated bacteria far beyond their roots. Environmental conditions,

These authors contributed equally to this work.
†
Corresponding author: Email: olga.sanchez@uab.es.
46 Ricardo Sidrach-Cardona, Olga Sánchez, Laura Garrido et al.
mainly redox, are suggested as main driving forces in organizing microbial assemblages
in the studied wetlands.
INTRODUCTION
Natural wastewater treatment systems such as constructed wetlands constitute a relevant
option to conventional methods due to their efficiency, low stablisment costs and reduced
operation and management requirement. In these systems, microbial processes are crucial
since many reactions are microbiologically mediated (Stottmeister et al., 2003; Kadlec &
Wallace, 2009). Thus, the wetland rhizosphere is characterized by the presence of oxic-anoxic
interfaces in which obligatory aerobic to strictly anaerobic microbes operate in close
proximity, thereby facilitating elemental cycling (Schlesinger, 2004). Despite the impact of
microbes in wetland ecosystems, little is still known about the species present and their
ecology and functioning in situ, especially in the rhizosphere (Bodelier et al., 2006).
However, former limitations of cultivation dependent techniques on the study of wetland
microbiology have been overcome by the development of molecular techniques like FISH
(Criado & Bécares, 2005) and specially fingerprinting methods (Gutknecht et al., 2006; Truu
et al., 2009; Adrados et al., 2014, Morató et al., 2014). Thus, it has been proved that wetland
plants enhance microbial density and activity in their rhizosphere (Gagnon et al., 2007;
Nikolausz et al., 2008), mainly due to the fact that plants provide carbon compounds through
root exudates and a microaerobic environment via root oxygen release (Armstrong &
Armstrong, 2001; Lu et al., 2006). Microbial densities and activities can also differ depending
on the presence of plants or on the species present in the wetland (Angeloni et al., 2006;
Gagnon et al., 2007; Li et al., 2008; Ruiz- Rueda et al., 2008; Wang et al., 2008). A few
studies found higher functional microbial diversity or higher microbial activity in rhizosphere
sediments compared to non-rhizosphere (bulk) sediments (Tam et al., 2001; Vacca et al.,
2005) indicating some kind of connection between plants and their adjacent microbial
communities. On the other hand, other studies (Ahn et al., 2007; Baptista et al., 2008) pointed
to an absence of a vegetation effect on microbial community. The influence of plants on their
surrounding environment, clearly evidenced in terrestrial plants (see e.g. Hawkes et al., 2007)
seems to be much less clear in aquatic ecosystems, probably due to a potentially more
homogeneous environmental conditions in wetlands in comparison with soil.
Polluted areas and constructed wetlands have been the focus of the majority of
microbially-based research on plants-bacteria coupling (Gutknecht et al., 2006). Constructed
wetlands have been extensively developed in the last decades for the treatment of point and
diffuse pollution and their widespread use and research has also improved the knowledge on
the functioning of, not only constructed, but also natural wetlands (Reddy and DeLaune,
2008). An important part of the treatment in these systems is attributable to the presence and
activity of plants and to the interactions between plant and bacteria. Nevertheless, until
recently the microbial ecology of constructed wetlands has remained relatively
uncharacterized (Truu et al., 2009) and there is still little understanding of microbial
community structure in constructed wetlands and how it is influenced by plants or other
potential factors like hydraulic fluxes or organic matter. In this study an experimental
constructed wetland was built with the objective to study, a) the effect of plants on their
surrounding rhizosphere by comparing planted and unplanted tanks b) the differences
Molecular Characterization of Microbial Communities ... 47
between Typha angustifolia and Phragmites australis concerning their microbial root-
associated communities and, c) the effect of hydraulic design (superficial vs. sub-superficial)
and organic loading on the main microbial habitats of planted tanks.
MATERIALS AND METHODS
Experimental Pilot Plant
The constructed wetland pilot plant was located in León (north-west of Spain) and
received primary settled wastewater from the urban wastewater treatment plant as influent
(mean influent BOD5 of 105 mg O2/L). Further details on the pilot plant and wastewater
characteristics are described elsewhere (Hijosa-Valsero et al., 2012; Pedescoll et al., 2013).
Briefly, eight mesocosm tanks 1 m2 surface 0.5 depth were used for the experiment and
divided in three sets (Figure 1).
Typha-FM-SF Typha-FW-SF Typha-FW-SSF Unplanted-FW-SSF

(CW1) (CW2) (CW3) (CW4)
Phragmites-FM-SF Phragmites-SSF Phragmites-SSF Unplanted-SSF

(CW5) (CW6) (CW7) (CW8)
Figure 1. Schematic design characteristics of the CWs. FM: floating macrophytes, FW: free-water
layer, SF: surface flow; SSF: sub-surface flow. Systems CW1 and CW5 had a water layer of 0.25 m
and plant growth was supported by 25 cm length and 15 cm diameter garden-net cylinders (2 cm pore
size). Systems CW2, CW3 and CW4 had a 0.25 m of free-water (FW) layer over a 0.25 m layer of
silicious gravel. Systems CW6, CW7 and CW8 consisted of a 0.5 m silicious gravel layer with sub-
surface flow (SSF).
A set of three tanks (tanks numbers 2, 3 and 4) were free-water flow systems (FW), half-
filled with gravel (0.7-15 mm diameter) up to 0.25 m and water up to 0.5 m. Two of the tanks
(tanks 2 and 3) were planted with Typha angustifolia and the other (tank 4) was unplanted and
considered as control for plant effect comparisons. Influent and effluent were superficial in
tank 2 (i.e. effluent from the surface at the end of the tank), whereas effluent was sub-
superficial, (i.e. effluent from the bottom at the end of the tank) in tanks 3 and 4. Comparison
of tanks 2 and 3 was therefore used to test flow effect (superficial vs. sub-superficial) and
comparison of tanks 3 and 4 to test differences between Typha planted and unplanted tanks.
Another set of three tanks (tanks numbers 6 to 8) were conventional sub-surface flow systems
(SSF). Tanks were filled up (0.5 m depth) with siliceous gravel, water level being 4 cms
below gravel surface. Two tanks (tanks 6 and 7) were planted with Phragmites australis and
the other tank (tank 8) was left unplanted and considered as control. Tank 6 was loaded with 9
gBOD5/m2/d whereas the rest of the tanks used in the experiment were loaded with 3
gBOD5/m2/d. Comparison of tanks 6 and 7 allowed to check for loading effects while
comparison of tank 7 and 8 for Phragmites effect. The last set of tanks (tanks 1 and 5) were
filled up with 0.25 m water without any gravel inside (hydroponic tanks). Tank 1 was planted
with Typha and plant 5 with Phragmites to compare bacterial community differences among
plant species. Plant support in the hydroponic tanks followed previous experiences (Soto et
al., 1999), using rolled strips (15 cm diameter, 25 cm length) of garden plastic nets 2 cms
pore size to allow both, root support and development. Rolled strips were tided-up to PVC-
tube frames to give more stability to the structure when plants reached full development. All
tanks received the same wastewater as influent with a load of 3 g BOD5/m2/d, with exception
of the aforementioned tank 6 which received three times more. Detailed information on
wastewater characteristics and efficiencies are presented elsewhere (Hijosa-Valsero et al.,
2012).
Collection of Samples
Samples were taken in late summer, when plants were in their maximum development
(100% coverage). Biofilm samples from the gravel and plant roots, and from the interstitial
liquid of each of the tanks were simultaneously taken in two zones in the last third of the
tanks, close to the final effluent. Bacteria attached to the gravel were dislodged using
protocols described by Pierzo et al. (1994). About two hundred grams of gravel was firstly
washed with sterile tap water to remove all particles loosely attached to the gravel and later
with buffered miliQ water. Sample was divided in tubes with 100 g gravel each and 100 ml
buffered miliQ. Tubes were shaken in an orbital shaker for 15 min at 1000 rpm and sonicated
later at 3 min interval 1 min rest for four times following previous experiences. Supernatant
was centrifuged in 45 ml tubes at 14.000 g for 30 min in a refrigerated (-4ºC) centrifuge.
Pellet was preserved at -20ºC until DNA extraction.
Bacterial biofilm from plant roots was extracted using protocols described in Lu et al.
(2006), Yang & Crowley (2000) and Ibekwe et al. (2003). About 25g fresh weight of small
active roots were taken form the plants rhizomes. Roots were first washed with sterile tap
water and buffered miliQ water and later homogenized with an Ultra-turrax. Homogenized
samples were kept in 30 ml Falcon tubes at -20ºC until DNA extraction. Interstitial water
from the gravel bed was sampled with a 100 mL syringe at 15 cm. depth from the gravel
surface. Samples were centrifuged at 14.000 g for 30 min in a refrigerated (-4ºC) centrifuge
and kept at -20ºC until extraction.
Several replicates from each tank and environment were taken but due to DGGE
limitations not all replicates were included in the same gel.
DNA Extraction
Different kits were utilized for DNA extraction. The DNA Power Soil kit from MOBIO
(12888-50) was used for DNA extraction of interstitial liquid and gravel biofilm, while the
PowerMax soil kit from MOBIO (12988-10) was utilized for roots. DNA from the influent
was extracted with the UltraClean water DNA kit (MOBIO, 14880-25). DNA integrity was
checked by agarose gel electrophoresis, and quantified using a low DNA mass ladder as a
standard (Invitrogen).
PCR-DGGE Fingerprinting
Fragments of the 16S rRNA gene suitable for DGGE analysis were obtained by using the
bacterial specific primer set 358f-907rM (Sánchez et al., 2007). Polimerase chain reaction
(PCR) was carried out with a Biometra thermal cycler using the following program: initial
denaturation at 94ºC for 5 min; 10 touchdown cycles of denaturation (at 94ºC for 1 min),
annealing (at 63.5-53.5ºC for 1 min, decreasing 1ºC each cycle), and extension (at 72ºC for 3
min); 20 standard cycles (annealing at 53.5ºC, 1 min) and a final extension at 72ºC for 5 min.
PCR mixtures contained the template DNA, each deoxynucleoside triphosphate at a
concentration of 200 µM, 1.5 mM MgCl2, each primer at a concentration of 0.3 µM, 2.5 U
Taq DNA polymerase (Invitrogen) and PCR buffer supplied by the manufacturer. BSA
(Bovine Serum Albumin) at a final concentration of 600 µg·ml-1 was added to minimize the
inhibitory effect of humic substances (Kreader, 1996). The volume of reactions was 50 µl.
PCR products were verified and quantified by agarose gel electrophoresis with a low DNA
mass ladder standard (Invitrogen). The DGGE was run in a DCode system (Bio-Rad) as
described by Muyzer et al. (1998). A 6% polyacrylamide gel with a gradient of 30-70%
DNA-denaturant agent was cast by mixing solutions of 0% and 80% denaturant agent (100%
denaturant agent is 7 M urea and 40% deionized formamide). Seven hundred ng of PCR
product were loaded for each sample and the gel was run at 100 V for 18 h at 60°C in 1xTAE
buffer (40 mM Tris [pH 7.4], 20 mM sodium acetate, 1 mM EDTA). The gel was stained with
SybrGold (Molecular Probes) for 45 min, rinsed with 1xTAE buffer, removed from the glass
plate to a UV-transparent gel scoop, and visualized with UV in a Gel Doc EQ (Bio-Rad).
Prominent bands were excised from the gels, resuspended in milli-q water overnight and
reamplified for its sequencing.
rRNA Sequencing
Purification of PCR products from DGGE bands and sequencing reactions were
performed by Macrogen (South Korea) with primer 907rM. They utilized the Big Dye
Terminator version 3.1 sequencing kit and reactions were run in an automatic ABI 3730XL
Analyzer-96 capillary type. Sequences were subjected to a BLAST search (Altschul et al.,
1997) to obtain an indication of the phylogenetic affiliation, and to the Bellerophon program
(Huber et al., 2004) to determine potential chimeric artefacts.
Accession Numbers
Fifty-eight 16S rRNA gene sequences were sent to the EMBL database (http://www.
Ebi.ac.uk/embl) and received the following accession numbers: from FM991973 to
FM992030.
Quantitative Analyses
Digitalized DGGE images were analyzed with Quantity One software (Bio-Rad). Bands
occupying the same position in the different lanes of the gels were identified. A matrix was
constructed for all lanes, taking into account the presence or absence of the individual bands.
This matrix was used to calculate similarity index for presence-absence data using the Past
program (Hammer et al., 2001). Raup-Crick index was utilized as this method uses a
randomization (Monte Carlo) procedure, comparing the observed number of species
occurring in both associations with the distribution of co-occurrences from 200 random
replicates. Dendrograms using unweighted-pair group average (UPGMA) given by other
three different algorithms (Jaccard, Dice and Simpson) were also compared in order to assess
the robustness of the groupings. Statistical differences between groups were checked with
non-parametrics Man-Withney and Kolmogorov-Smirnov test, and with the ANOSIM
permutation/randomization test using PRIMER v5 (Clarke & Gorley, 2001). The R statistic
produced by this test fluctuates from 0 (no differences) to 1 (perfect separation) with R>0.75
indicating well separated groups, R>0.5 overlapping but clearly different, or barely separated
at all when R<0.25. Non-metric multidimensional scaling MDS was also used to check for
2D grouping of sampling points. Raup Crick similarity matrixes from the PAST program
(Hammer et al., 2001) were also used as data input for both, ANOSIM and MDS tests. As
groups from MDS were mostly the same than those from UPGMA dendrograms only the
MDS graphs will be presented.
RESULTS
Bacterial Community Composition
Analysis of the bacterial community composition by PCR-DGGE was performed on

influent, gravel biofilm, interstitial liquid and root samples collected in summer 2007 for the
different experimental wetlands. DGGE was performed independently for each of the
environments (roots, gravel and interstitial samples), and also independently for the different
hydraulic designs studied: hydroponic, subsurface flow (SSF) and free-water flow (FW).
Banding patterns for the 16S rRNA DGGE-PCR amplicons are presented in Figure 2. The
number of bands per lane varied from 10 to 31 (mean values of 20.2, 16.7 and 19.8 for gravel
biofilm, roots and interstitial liquid respectively). Some differences could be observed in band
position, intensity, and number of bands present in the different samples for each
environment, demonstrating that different bacterial communities developed.
Figure 2. DGGE fingerprints from samples of influent, gravel biofilm, interstitial liquid and roots
obtained from the different CW tanks. Numbers on the top of the lanes refer to the experimental tank;
letters A, B and C refer to replicates inside the same tank (when available); CT: central sampling, S:
surface sampling, I: lower part sampling, unpl: unplanted, SSF: sub-surface flow, FW: free water flow,
INF: influent.
Dendrograms and multidimensional scaling (MDS) were applied to discriminate

microbial assemblages between the different microenvironments. The dendrograms based on
the DGGE banding pattern from roots, gravel biofilm and interstitial water (Figure 3)
separated the samples according to the different wetland design and plant species.
Dendrograms were very consistent independently of the algorithm used, being groups fully
coincident with those observed in the MDS (Figure 4).
Samples from roots split in three main clusters or MDS associations according to the
different types of constructed wetlands, and on the base of plant type (Figures 3 and 4). MDS
clearly differentiated 3 groups: Samples from Typha planted tanks (samples 1A, 1B, 1C, and
root samples from tanks 2 and 3), samples from the hydroponic Phragmites tank (samples
5A, 5B and 5C), and samples from the gravel (SSF) Phragmites planted tanks (samples 6A,
6B, 7A and 7B). DGGE gel patterns from Typha and Phragmites roots proved to be
statistically different when using one-way ANOSIM test (R = 0.582, P = 0.001). Two-way
ANOSIM test also showed differences for plant type (R= 0.864, P=0.007) and also for the
type of tank, being the hydroponic tank planted with Phragmites (tank 5) statistically different
from the gravel planted ones (tanks 6 and 7) (R= 0.663, P=0.009).
Figure 3. Dendrograms of the bands using similarity Raup-Crick index. Robustness of the associations
were confirmed by using three different algorythms (Jaccard, Dice and Simpson), obtaining the same
groups. Dendrograms were carried out individually for root associated bacteria, gravel biofilm and
interstitial water, as well as for Phragmites and Typha tanks. Letters A, B and C refer to replicates
inside the same tank (when available); CT: central sampling, S: surface sampling, I: lower part
sampling, unpl: unplanted, SSF: sub-surface flow, FW: free water flow.
Figure 4. Multidimensional scaling (MDS) analyses generated from the DGGE profiles of the different
samples analyzed for each environment (gravel biofilm, interstitial liquid and roots), as well as for each
planted tanks (Phragmites and Typha planted tanks). Type of flow, sub-surface (SSF) and free-water
(FW) of planted or unplanted tanks is indicated. Sample number refers to tank type. Letters A, B and C
refer to replicates inside the same tank (when available); CT: central sampling, S: surface sampling, I:
lower part sampling, INF: influent to the tanks. Circles refers to main groups in the dendrograms
(Figure 3).
Bacterial assemblages growing on the roots of Typha are therefore statistically different
from those growing on Phragmites roots. With regard to the hydraulic design of the tank,
Typha roots harboured the same community composition independently on the type of tank as
no differences were found between hydroponic (tank 1) and FW tanks (tanks 2 and 3) (Figure
4). On the other hand, bacterial community from Phragmites roots growing on the hydroponic
tank was statistically different from that growing on SSF gravel tanks, being no statistical
differences between high organically loaded (tank 6) and low loaded (tank 7) conditions (K-S
test, p=0.73). Microbial community composition on Phragmites roots was therefore
statistically different depending on the support media (hydroponic or gravel-based) whereas
no differences were found for Typha.
Within the gravel biofilm (Figure 4), samples split into two main groups depending on
the type of constructed wetland (SSF or FW) being these two groups statistically different
using ANOSIM (R= 0.519, p=0.029). Statistical differences between plant types (Typha vs.
Phragmites) (R= 0.64, p=0.067), planted vs. un-planted (control) tanks (R=0.143, p= 0.4), or
high vs. low loaded tanks (R=0.25, p=0.66) did not show significant differences using
ANOSIM. The type of flow (superficial in tank 2 vs. sub-superficial in tank 3) did not
influence bacterial communities present on the gravel of FW tanks. Presence (FW) or absence
(SSF) of free water surface is therefore statistically more important than other experimental
conditions when comparing bacterial communities growing on gravel biofilm.
The community composition of interstitial liquid was strongly separated (MDS stress:
0,01) according to wetland design (SSF or FW) or plant presence (Figure 4). MDS clearly
separated the unplanted SSF tank (tank 8) from the Phragmites SSF planted tanks (tanks 6
and 7) and from the FW tanks (tanks 2, 3 and 4) being differences statistically significant
(R=1, p= 0.017). Pairwise test showed significant differences between plant species (Typha
vs. Phragmites tanks, R= 1, p=0.01), and between planted vs. control SSF tanks (R=1,
P=0.033). These results suggest that interstitial communities were significantly influenced by
the type of hydraulic design (SSF or FW), and by the presence of plants in the case of sub-
superficial (SSF) systems. Following the pattern previously observed in the gravel biofilm, no
differences were found between loading conditions (tank 6 vs. 7), neither between Typha
planted and unplanted tank (tanks 2 or 3 vs. tank 4) nor between surface (tank 2) vs. sub-
surface flow (tank 3) Typha planted tanks with regards to bacterial communities in the
interstitial water.
DGGE gels with samples belonging either to the Typha planted tanks (tanks 1, 3 and its
control tank 4) or to the Phragmites planted tanks (tanks 5, 7, and its control tank 8),
combining the three different environments, and the influent, in the same gel were also run
(Figure 2). Both, the dendrograms and MDS obtained from these fingerprints showed that
samples separated on the base of the microenvironment (gravel biofilm, interstitial water and
roots) (Figure 4). One-way ANOSIM for Phragmites planted tanks including unplanted tank
8 showed significant statistical differences among the three environments (gravel, roots and
interstitial) (R= 0.954, P=0,001). Pairwise tests showed significant differences for all the
three combinations, gravel vs. interstitial (R= 0.821, p=0.05), gravel vs. roots (R=1, p=0.008),
and interstitial vs. roots (R=1, p= 0.04). No differences were found between planted and
unplanted gravel tanks (samples 7 vs. 8) neither between roots from the hydroponic and
gravel planted tanks (samples 5 vs. 7). Concerning FW tanks (gravel tanks 2 and 3 planted
with Typha including control tank 4) (Figure 4), differences among the three environments
were significant for the global ANOSIM test (R=0.85, p=0.005), being pairwise test almost
significant for gravel (samples 4 and 3) vs. roots (samples 1 and 3) (R=1, p=0.06), interstitial
(samples 3 and 4) vs. roots (R=0.857, p=0.06) and clearly not significant for gravel vs.
interstitial (R=0.25, p=0.33). In general, bacterial communities from the three environments
(roots, interstitial and gravel biofilms) were statistically different inside the same tank, being
differences more evident for Phragmites than Typha planted tanks.
Analysis of Predominant Bacterial Species by PCR-DGGE
A total of 58 band positions were excised and sequenced in order to determine their
phylogenetic affiliation. The closest matches (and percentages of similarity) for the sequences
retrieved were determined by a BLAST search (Table 1). The number of bases used to
calculate each similarity value is also shown in Table 1 as an indication of the quality of the
sequence. Five bands could be retrieved from the DGGE corresponding to the influent. They
belonged preferentially to different Betaproteobacteria, while one band (band 5I) affiliated
with a high similarity (>97%) to Acinetobacter johnsonii, a Gammaproteobacterium. Bands
affiliated to Betaproteobacteria constituted a constant in the different environments studied in
this work. Thus, some DGGE bands corresponding to different gravel biofilm samples
showed similarities higher than 94.4% to uncultured members of this phylogenetic group,
while some DGGE bands of samples corresponding to interstitial liquid, significantly
affiliated to the freshwater widespread Polynucleobacter sp., as well as to uncultured
Betaproteobacteria. One DGGE band obtained from root samples (band 3R) was affiliated to
Comamonas compostus, although at a low similarity value, and the rest of Betaproteobacteria
bands from this environment matched well with non-cultured members of this group.
Members of uncultured Bacteroidetes were present in all samples of constructed wetlands.
Besides, sequences of Chryseobacterium joostei were found in gravel biofilm samples (bands
1L and 14L), whereas sequences of Flavobacterium sp. were present in interstitial liquid and
roots of Phragmites (band 13R).
Firmicutes (Gram+ bacteria with low G+C content) could also be observed in the three
environments studied from artificial wetlands, although closest matches corresponded to
uncultured members of this group. On the other hand, sequences from Deltaproteobacteria
were only present in samples from gravel biofilm and roots, and some taxonomic groups were
found exclusively in one of the environments. Thus, uncultured Actinobacteria predominated
in some samples of interstitial liquid, while groups such as Acidobacteria, Chlorobi or
Alphaproteobacteria were specific of roots samples. Sequence identity of most of the bands in
Table 1 showed that they were closely associated with different species or bacteria from
sludge or wastewater environments.
Table 1. Phylogenetic affiliation of sequences obtained from DGGE bands, with closest
uncultured and cultured matches. Number of bases used to calculate the sequence
similarity is shown in parentheses in the third column
Band Closest match Similarity Taxonomic group Acc nº (Gen Cultured closest
(%) Bank) match
(nº bases) (% similarity)
INFLUENT
WETLE-1I Acinetobacter 97.6 (532) -proteobacteria DQ870719
johnsonii
WETLE-2I Diaphorobacter 98.9 (527) ß-proteobacteria EU342381
oryzae
WETLE-3I Acidovorax defluvii 99.1 (528) ß-proteobacteria AM943035
WETLE-4I Uncultured ß- 98.9 (537) ß-proteobacteria AM940952 Acidovorax
proteobacterium temperans (98.7)
WETLE-5I Brachymonas 99.3 (540) ß-proteobacteria EU434449
denitrificans
Table 1. (Continued)
Band Closest match Similarity (%) Taxonomic group Acc nº (Gen Cultured closest
(nº bases) Bank) match
(% similarity)
GRAVEL BIOFILM
WETLE-1B Uncultured diatom 99.4 (521) Bacillariophyta AY168726 Phaeodactylum
chloroplast tricurnutum (97.9)
WETLE-2B Uncultured bacterium 97.6 (523) Bacteroidetes AB237701 Flexibacter canadensis
(89.9)
WETLE-3B Uncultured ß- 95.6 (517) ß-proteobacteria AY947965 Azovibrio sp. (93.3)
proteobacterium
WETLE-4B Uncultured bacterium 96.2 (525) -proteobacteria EU443000 Desulfobacula
toluolica (94.3)
WETLE-5B Uncultured bacterium 97.6 (401) Bacteroidetes AB237701 Flexibacter canadensis
(87.8)
WETLE-6B Chryseobacterium 99.4 (522) Bacteroidetes EF204455
joostei
WETLE-7B Chryseobacterium 99.4 (522) Bacteroidetes EF204455
joostei
WETLE-8B Uncultured 99.6 (534) Bacteroidetes EF111172 Alistipes onderdonkii
Bacteroidetes (85.3)
WETLE-9B Uncultured bacterium 98.9 (534) Firmicutes AY532555 Erysipelothrix
rhusiopathiae (92.4)
WETLE-10B Uncultured bacterium 94.2 (506) Bacteroidetes AM086159 Prolixibacter
bellariovorans (90.1)
WETLE- Uncultured 99.3 (542) -proteobacteria EU234252 Desulfobacter
11B bacterium postgateii (98.9)
WETLE- Uncultured 99.3 (541) ß-proteobacteria AJ318917 Dechloromonas
12B bacterium denitrificans (96.9)
WETLE- Uncultured 94.4 (388) ß-proteobacteria EU529730 Rhodocyclus tenuis
13B bacterium (93.4)
WETLE- Uncultured 97.1 (529) ß-proteobacteria AM909879 Siderooxidans
14B bacterium paludicola (94.5)
WETLE- Uncultured 97.2 (529) ß-proteobacteria AB252929 Denitratisoma

15B Gallionella oestradiolicum (94.5)
WETLE- Uncultured 98.9 (539) ß-proteobacteria AB355063 Denitratisoma
16B bacterium oestradiolicum (95.6)
INTERSTITIAL LIQUID
WETLE-1L Flavobacterium sp. 99.8 (533) Bacteroidetes AB426577 Flavobacterium
succinicans (98.7)
WETLE-2L Uncultured 98.9 (539) ß-proteobacteria EU234274 Azovibrio restrictus
bacterium (94.9)
WETLE-3L Polynucleo-bacter 99.6 (533) ß-proteobacteria AB426572 Polynucleobacter
sp. necessarius (98.7)
WETLE-4L Uncultured 100 (526) Actinobacteria AY948008 Salinibacterium
actinobacterium aquaticus (98.1)
WETLE-5L Uncultured 98.1 (517) Actinobacteria AJ863316 Leifsonia xyli (97.9)
bacterium
WETLE-6L Uncultured 98.5 (517) Actinobacteria AJ863316 Leifsonia xyli (98.3)
bacterium
WETLE-7L Polynucleo-bacter 99.4 (541) ß-proteobacteria AB426572 Polynucleobacter
WETLE-8L Polynucleo-bacter 100 (542) ß-proteobacteria AB426572 Polynucleobacter
WETLE-9L Uncultured 98.8 (513) Firmicutes AY754834 Clostridium botulinum
bacterium (90.6)
Band Closest match Similarity (%) Taxonomic group Acc nº (Gen Cultured closest
(nº bases) Bank) match
(% similarity)
WETLE-10L Haslea salstonica 93.0 (494) Bacillariophyta AF514854

chloroplast
WETLE-11L Uncultured 89.7 (490) ß-proteobacteria EU133809 Rubrivirax
bacterium gelatinosus (88.8)
ROOTS
WETLE-1R Uncultured 98.9 (538) Acidobacteria EU499471 Geobacter
bacterium pickeringii (83.2)
WETLE-2R Lolium perenne 99.8 (522) Spermatophyta AM777385
chloroplast
WETLE-3R Comamonas 90.1 (491) ß-proteobacteria EF015884
compostus
WETLE-4R Uncultured 96.9 (402) Acidobacteria EU499471 Desulfomonile
bacterium limimaris (84.9)
WETLE-5R Lolium perenne 100 (519) Spermatophyta AM777385
chloroplast
WETLE-6R Uncultured 93.5 (507) ß-proteobacteria FJ037637 Candidatus
bacterium Nitrotoga arctica
(91.3)
WETLE-7R ß-proteo- 91.9 (487) ß-proteobacteria AY297807 Rhodocyclus sp.
bacterium (91.1)
WETLE-8R Uncultured 95.2 (511) Bacteroidetes DQ093919 Bacteroides sp.
bacterium (91.8)
WETLE-9R Uncultured 98.7 (512) Firmicutes AY754834 Clostridium
bacterium botulinum (90.0)
WETLE-10R Uncultured 89.3 (469) ß-proteobacteria AY785239 Rhodocyclus tenuis
bacterium (86.9)
WETLE-11R Uncultured 95.1 (504) ß-proteobacteria EU266786 Dechloromonas
Rhodocyclaceae hortensis (94.3)
WETLE-12R Uncultured 99.4 (540) ß-proteobacteria EF667706 Dechloromonas
bacterium hortensis (98.7)
WETLE-13R Flavo- 99.8 (533) Bacteroidetes AB426577 Flavobacterium
bacterium sp. succinicans (98.7)

WETLE-14R Uncultured 98.3 (509) -proteobacteria EU284319 Rhodobacter
bacterium blasticus (97.5)
WETLE-15R Uncultured 99.4 (515) -proteobacteria EU284319 Rhodobacter
bacterium blasticus (97.5)
WETLE-16R Uncutlured 98.2 (534) Acidobacteria DQ676412 Holophaga fetida
Acidobacteria (83.1)
WETLE-17R Uncultured 98.2 (532) Acidobacteria EU499471 Desulfofrigus sp.
bacterium (82.9)
WETLE-18R Uncultured 98.5 (533) Acidobacteria EU499471 Desulfofrigus sp.
bacterium (82.7)
WETLE-19R Uncultured 95.2 (499) -proteobacteria AF502221 Rhizobium giardinii
bacterium (90.8)
WETLE-20R Uncultured soil 95.2 (519) ß-proteobacteria DQ297980 Methylibium sp.
bacterium (94.3)
WETLE-21R Lolium perenne 100 (519) Spermatophyta AM777385
chloroplast
WETLE-22R Lolium perenne 99.2 (516) Spermatophyta AM777385
chloroplast
WETLE-23R Uncultured 99.3 (542) -proteobacteria AM159357 Pelobacter
Geobacter sp. propionicus (98.7)
WETLE-24R Uncultured ß- 99.1 (540) ß-proteobacteria AB265946 Denitratisoma
proteobacterium oestradiolicum
(95.4)
WETLE-25R Chlorobium sp. 98.3 (524) Chlorobi AB210277
DISCUSSION
Bacterial Assemblage Diversity as Revealed by DGGE
Excision of prominent DGGE bands and subsequent sequencing allowed characterization

of predominant microorganisms from the influent as well as from gravel biofim, interstitial
water and roots. Most of these sequences belonged to uncultured microorganisms, while
others matched well with cultured bacteria. In general, they were closely associated with
different species or bacteria from sludge or wastewater environments. Thus, within the Beta
and Gammaproteobacteria predominating in the influent, the genus Acinetobacter (a
Gammaproteobacterium) and the facultative anaerobe Brachymonas denitrificans (a
Betaproteobacterium) occur in a wide variety of activated sludges in which enhanced
biological phosphate removal is observed (Korstee et al., 1994; Ivanov et al., 2005). On the
other hand, Acidovorax defluvii (a Betaproteobacterium) was isolated from activated sludge
samples from a municipal wastewater treatment plant (Schulze et al., 1999). Furthermore,
Diaphorobacter sp. (Beta) is able to perform partial nitrification followed by further aerobic
removal of nitrite in high-nitrogen-containing wastewaters. Overall, ammonia-oxidizing
bacteria seem to play an important role in the nitrogen cycle in natural and constructed
wetlands.
Also, sequences of Betaproteobacteria, Bacteroidetes and Firmicutes could be retrieved
from the three environments studied in the different wetlands. Within the Betaproteobacteria,
most sequences had uncultured closest matches; in these cases, cultured closest matches
corresponded mostly to microorganisms related to the nitrogen cycle. For example, several
sequences from gravel biofilm and roots were associated to Denitratisoma oestradiolicum, a
denitrifying bacterium isolated from activated sludge of a wastewater treatment plant
(Fahrbach et al., 2006), or to Dechloromonas denitrificans, a N2O-producing facultative
aerobe (Horn et al., 2005). On the other hand, samples of interstitial liquid abounded in
sequences closely related to Polynucleobacter sp., a typical freshwater microorganism
detected in acidic, neutral and alkaline habitats located in different climatic zones (Hahn,
2006). A number of sequences belonged to the phylum Bacteroidetes. For instance,
Flavobacterium sp. is also a typical genus which can be found in activated sludge (Park et al.,
2007; Shokrollahzadeh et al., 2008; Yu et al., 2007); it was retrieved in samples of interstitial
water and roots.
Other sequences, however, belonged to microorganisms present only in specific
environments. Thus, sequences affiliated to uncultured Deltaproteo-bacteria could be detected
in gravel biofilm and roots, but were not retrieved in prominent bands of interstitial liquid.
Some of these sequences were associated to sulphate-reducing bacteria, a ubiquitous group of
microorganisms that can be found in many natural and engineered environments where
sulphate is present, such as anaerobic wastewater treatment plants (Ben-Dov et al., 2007; Dar
et al., 2005).
Other groups were just detected in one particular environment. Thus, sequences affiliated
to Actinobacteria could only be retrieved in interstitial water samples, while samples
belonging to Acidobacteria, Alphaproteo-bacteria and Chlorobi were found in roots.
Actinobacteria include some of the most common soil life microorganisms, performing an
important role in decomposition of organic materials, and thereby playing a vital part in
organic matter turnover and carbon cycle. Some studies have reported their ability to degrade
environmental pollutants in activated sludge from wastewater treatment (Kim et al., 2007).
Acidobacteria was another taxonomic group only observed in root samples. Sequences
belonging to this widespread distributed phylum have been found traditionally in soil, aquatic
environments and wastewater treatment plants (Ludwig et al., 1997; Juretschko et al., 2002).
On the other hand, Alphaproteobacteria related to the phoheterotrophic purple nonsulfur
bacterium Rhodobacter blasticus have also been detected in roots; this microoganism has
been isolated mainly from wastewater and polluted environments such as photosynthetic
sludge processes (Hiraishi et al., 1995; Okubo et al., 2005). In general, the genus Rhodobacter
includes species with an extensive range of metabolic capabilities which allow them to
survive in a number of varied habitats, thus appearing in all types of aquatic environments. In
fact, the group Alphaproteobacteria has also been found as attached bacteria from different
species of plants in aquatic environments (Crump & Koch, 2008). Sequences belonging to
Chlorobium sp., a green sulphur bacterium, were also detected only in roots. The ability of
this microorganism to use sulphide has been exploited in the past for the treatment of
sulphide-containing wastewaters and gas streams (Cork et al., 1983; Kobayashi et al., 1983).
Thus, many of the species recovered from DGGE bands in this work have the potential to act
as degraders of environmental pollutants in natural and constructed wetlands.
Differences between Plant Species Microbial Communities
Graphical and statistical analysis showed significant differences between Typha and
Phragmites root-associated microorganisms. Our protocol did not differentiate between
endophytic and rhizoplane bacteria (McClung et al., 1983; Chelius & Tripplet, 2001),
therefore both root habitats were pooled in the analysis. Plants species in soils clearly
influence the microbial communities of their roots, each plant species harbouring a distinctive
microbial assemblage (see e.g. Hawkes et al., 2007; Ehrenfeld et al., 2005, for revision).
Nevertheless, a small body of research has demonstrated this aspect in the roots of wetland
plants as mostly are focused on the rhizosphere and not on the proper roots or their
rhizoplanes. Abundances of Fe-oxidizing bacteria were different among five wetland species
studied by Emerson et al. (1999) which isolated different strains in the rhizoplane of different
plant species. Bergholz et al. (2001) compared the physiological diversity of root-asociated
bacteria of Spartina patens with results from a previous work studying S. alterniflora and
Juncus roemerianus rhizoplains (Bagwell et al., 1998). Substantial differences among roots
communities were explained due to bacterial oxygen preference and therefore to differences
in oxygen production by the plant species. The same reasoning was applied by Briones et al.
(2003) to explain differences in band patters among different rice cultivars. A more evident
work on species differences was carried out by Chelius & Lepo (1999), who made RFLP
analysis from the rhizosphere of Spartina alterniflora and Sesbania macrocarpa, revealing
differences in the community structure of N2-fixing bacteria. Concerning constructed
wetlands, Ruiz-Rueda et al. (2008) also found different nitrogen-related microbial
communities when comparing Typha and Phragmites species. In our work, Typha
angustifolia roots harboured a significantly distinctive community than Phragmites australis,
with substantial variations between hydroponically and gravel grown plants in this last
species.
Differences in bacterial assemblages between hydroponically and gravel-filled

Phragmites tanks could be due to the environmental differences in these tanks. Wirsel et al.
(2001) found that endophytic fungi biodiversity in Phragmites roots changed depending on
the type of soil media. In our case, the study of main environmental conditions (redox, pH, or
dissolved oxygen) showed that pH was the only variable with statistical differences
(ANOVA, F= 116, p<0,001) between hydroponic tank 5 (pH=7) and gravel planted tanks 6
and 7 (both with a pH= 6.6). Organic loading, another important factor potentially
influencing root development and oxygen release due to the toxic effect of low redox
conditions (see e.g. Armstrong & Armstrong, 2005) did not affect differences among redox
conditions in the experimental tanks probably due to the low loadings applied (redox values
of 162mV and 166 mV for high and low loaded tanks, respectively). Growth substrata (i.e.
hydroponic or gravel) seemed therefore to have more influence on bacterial assemblages of
Phragmites roots than the organic load assayed.
The Effect of Plants and Hydraulic Design on Microbial Communities
Hydrology has consistently been proved to be an important control variable in wetland

communities (Gutknecht et al., 2006). In our work the influence of hydraulic design on
bacterial communities was significant in Phragmites roots, interstitial water and gravel
biofilm associated communities when considered separately (Figures 3 and 4), as bands from
SSF and FW systems (or hydroponic for Phragmites roots) kept significantly different in all
environments. These results agree with Vacca et al. (2005), who found that hydraulic design
(horizontal or vertical) produced differences in band patterns in bulk soil of constructed
wetlands. Hydraulic patterns were also driving forces shaping bacterial communities in a 0.1
ha constructed wetland (Popko et al., 2006). Hydraulic design mainly affects redox conditions
in water due to a better exchange of oxygen in FW systems than SSF systems, FW having
therefore higher redox than SSF systems (Lin et al., 2008; Kadlec & Knight, 1996). The
presence of a water layer in FW wetlands also allows the development of a planktonic and
periphytic community in these wetlands which could also influence the microbial community
of their sediments and their interstitial water (Reddy & DeLaune, 2008). Studies have
revealed that the redox state of the rhizosphere has a significant effect on the intensity of
oxygen release through the roots of helophytes (Wiessner et al., 2002; Armstrong &
Armstrong, 2001). In our experiment mean redox conditions were clearly different between
FW and SSF tanks (mean values of 234mV and 137 mV, respectively), with significantly
lower values for the unplanted gravel tank 8 (80 mV) than the Phragmites planted (tanks 6
and 7, 164 mV mean) (ANOVA, F=23.5, p<0,001). In accordance with the previously
mentioned results on bacterial communities in the roots, higher organic loading in SSF tank 6
(9 g BOD5/m2/d) did not make effect on microbial communities in comparison with low-
loaded tank 7 (3 g BOD5/m2/d). This could be due to the still low loading assayed as 9 g
BOD5/m2/d is below the recommended design value in some wetland manuals (e.g. Reed,
1990).
Also, no difference was found between gravel and interstitial communities of tank 2 (FW
superficial flow) and tank 3 (FW sub-superficial flow). This is in accordance with the absence
of differences in redox conditions between tank 2 (120 ±75 mV) and tank 3 (180±71mV). The
effect of redox conditions in shaping the rhizosphere microbial communities has been
observed in both, natural and constructed wetlands (Dong et al., 2006; Nikolausz et al., 2008;
Wang et al., 2008; Ahn et al., 2009). Typha and Phragmites are clearly different in root
development and oxygen production (Brix, 1990; Reddy et al., 1990; Armstrong et al., 1990),
being plausible to think, as it has already shown with other wetland species (Lu et al., 2004;
2006), that these differences could affect the microbial composition of their rhizospheres.
Separate analysis of DGGE band patterns for SSF and FW tanks significantly differenced
three community groups: roots, interstitial and gravel communities (Figure 4, Phragmites and
Typha tanks). Differences were, in accordance with previously mentioned results, more
significant for SSF tanks than for FW tanks. Microbial communities are therefore different
for the different habitats under the same hydraulic design, having plant species a secondary
influence on these differences. Differences between planted and non-planted control tanks
were only evident for Phragmites interstitial water communities but such clear results were
not found for Typha. These results agree with Vacca et al. (2005) who found differences in
band patterns between planted and non planted Phragmites tanks and also between the type of
soil used for growing. Lower differences among studied habitats for Typha tanks could be
explained as consequence of a higher influence of this species on their surrounding
rhizosphere and to the ―planktonic‖ effect of the free-water environment developed on top of
the tanks. In accordance to this hypothesis, redox, dissolved oxygen and pH were
significantly higher for Typha than Phragmites planted tanks (mean redox values of 208±42
mV and 163±49 mV, 1.3±0.1 and 0.46±0.2 mgO2/L of dissolved oxygen, 7±0.1 and 6.6±0.2
pH units, for Typha and Phragmites tanks, respectively).
Comparison of our results with previous works on the same topic is problematic because
experiments in constructed wetlands treating wastewater are highly variable in operational
conditions (e.g. organic loading, redox values, wastewater characteristics, plant species, plant
density). Moreover, constructed wetlands experiments are usually designed to test their
efficiency for wastewater treatment, and not specifically to test the effect of plants on
wastewater treatment. A detailed look on these papers suggest that either high organic
loading, low plant density or inadequate redox conditions for root development could have
influenced the absence of plant effects in constructed wetlands (Baptista et al., 2008; Ahn et
al., 2007; DeJournett et al., 2007; Gorra et al., 2007), whereas in other works opposed
conditions could suggest plant effects on their microbial communities (Wang et al., 2008; Li
et al., 2008; Ruiz-Rueda et al., 2008; Ibekwe et al., 2007; Vacca et al., 2005; see also
Stottmeister et al., 2003). This also fits with Ravit et al. (2006) suggestions that plant effects
on associated microbial communities are less evident in human-disturbed (organically loaded)
natural wetlands. Plant effects on their associated bacterial communities could be therefore
more evident as physico-chemical conditions in constructed wetlands approach those in
natural wetlands. In our work, plants fully covered all tanks and ground biomass reached high
values (mean values of 10 and 4 kg DW/m2 for Typha and Phragmites planted tanks,
respectively) which maximize oxygen release into the rhizosphere (Wiessner et al., 2002).
Moreover, organic load was kept much lower than recommended in constructed wetlands
manuals (EPA, 2000). Under these circumstances, we were able to find significant differences
between plant species with regard to the microbial community structure of their roots.
CONCLUSION
Constructed wetlands harbour microbial communities which play an important role in
processes such as degradation of contaminants or nutrient cycling, therefore controlling
wastewater treatment efficiency. These populations could be influenced by aspects like the
type of plant species, organic matter load or hydraulic design. In this chapter, experimental
constructed wetlands were used to study the effect of those factors in microbial communities
of rhizoplane, gravel and interstitial water. Hydraulic design of the wetland (superficial or
sub-superficial flow) seemed to be more important than plant presence in shaping microbial
assemblages of their rhizosphere for Typha but not for Phragmites planted tanks. Microbial
communities from roots, gravel biofilm and interstitial water were clearly different inside the
same planted tank being these differences more important for Phragmites than Typha planted
mesocosms. This paper gives therefore evidences to support the influence of wetland plant
species on their root-attached microbial communities and their effect on their surrounding
habitats, and also how this last influence is also driven by the hydraulic patterns of the
wetland.
ACKNOWLEDGMENTS
The authors would like to thank Juan Carlos Sánchez, María Hijosa-Valsero, Mercedes
López-Vázquez and Héctor Astiarraga for their valuable help in field and laboratory work.
This work was funded by the projects MICROWET (CTM2005-06457-C05-03/TECNO),
CONSOLIDER-TRAGUA (CSD2006-00044) and PET2008-0165-02.
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closely associated with common reed. New Phytologist, 149, 589-598.
Yang, C. H. & Crowley, D. E. (2000). Rhizosphere microbial community structure in relation
to root location and plant iron nutritional status. Appl. Environ. Microbiol., 66(1), 345-
351.
Yu, C. P., Roh, H. & Chu, K. H. (2007). 17beta-estradiol-degrading bacteria isolated from
activated sludge. Environ. Sci. Technol., 41, 486-492.
Chapter 3
ASSESSMENT OF THE PRESENCE OF TRICLOSAN IN

WASTEWATER TREATMENT PLANTS IN THE CZECH
REPUBLIC AND ALTERNATIVE FUNGAL TREATMENT
Zdena Křesinová1, Radim Žebrák2, Jana Langová3

and Tomáš Cajthaml1,3,*
1
Institute of Microbiology, Academy of Sciences of the Czech Republic,
Prague, Czech Republic
2
DEKONTA a.s, , Prague, Czech Republic
3
Institute for Environmental Studies, Faculty of Science,
Charles University in Prague, Prague, Czech Republic
ABSTRACT
Wastewater treatment plants (WWTPs) are considered to be a major source of

various organopollutants in the aquatic environments as a consequence of insufficient
treatment processes. This study was performed to investigate the occurrence of triclosan
(TCS), an antimicrobial personal care product, in inlets and outlets of several WWTPs in
order to provide information about the presence of the compound in the Czech Republic.
Samples were collected at nine WWTPs located in Bohemia, downstream from variously
populated cities in the summer/autumn of 2011 and spring of 2012. At two WWTPs, TCS
concentrations were found to be under the limit of detection of the method at both inlets
and outlets. At the other WWTPs, the concentrations of TCS at the inlets ranged from 0
to 1830 ng l-1 (median 597 ng l-1, mean 528 ng l-1). At the outlets, the concentrations
varied from 59 to 1051 ng l-1 (median 205 ng l-1, mean 335 ng l-1). The removal of TCS
from wastewater is a process consisting of several mechanisms, such as adsorption and
microbial and as well as abiotic degradation. Part of TCS is usually converted into
methyl-triclosan (Me-TCS) by microbes in activated sludge under aerobic conditions and
this metabolite is very often used as a pollution marker. In this study, no Me-TCS was
detected at the outlets, suggesting that the major removal mechanism was adsorption or
some other unknown mechanisms. Fluctuations in TCS concentrations over 24 hours
were monitored at both the inlet and the outlet of one selected WWTP. The highest outlet
*
Corresponding author: cajthaml@biomed.cas.cz.
70 Zdena Křesinová, Radim Ţebrák, Jana Langová et al.
concentration was observed at 8 PM (1267 ng l-1), while the lowest outlet concentration
was at 5 AM (152 ng l-1) and the concentration of a pooled 24h sample was 556 ng l -1.
TCS concentrations in the inlet samples peaked at 8 AM (2845 ng l -1) and decreased at
about 10 AM to 923 ng l-1. Because the results document the inadequacy of the WWTP
treatment processes, we also tested an alternative TCS degradation method using a white
rot fungus in a bioreactor. Pleurotus ostreatus was tested for the treatment at one of the
WWTP outlets in a continuous trickle bed fungal bioreactor, which was filled with
lignocellulosic support inoculated with the fungus and placed directly in the WWTP
object. The results for non-sterile conditions showed more than 80% removal of TCS
following the fungal treatment. The application of Pleurotus ostreatus in the bioreactor
has been shown to be an efficient tool for removal of TCS from real wastewaters.
Keywords: Triclosan, wastewater treatment plants, monitoring, biodegradation, Pleurotus

ostreatus
INTRODUCTION
Triclosan (5-chloro-2-[2,4-dichloro-phenoxy]-phenol; TCS) is an organic compound used
in household and personal care products because of its antimicrobial properties. TCS is
widely present and detected in environmental samples and several studies have documented
its endocrine disrupting activity [1, 2]. TCS belongs among the most frequently detected
compounds in receiving water bodies downstream of intense urbanization, because of its
insufficient removal during wastewater treatment plant (WWTP) processes [3, 4]. Therefore,
particular attention has been paid to the fate of TCS during the treatment and several
monitoring studies have been performed in western European countries and North America.
The reported TCS environmental concentrations differ from study to study but also often
in individual sampling campaigns, as reflected in many works carried out in Europe, North
Africa, Australia and Asia. The detected amount of TCS usually ranged from <1 to 100 ng l-1
[3, 4, 5] in surface waters. Several times higher concentrations have been detected at WWTPs
(101 ng l-1 to 102 µg l-1) [4, 6, 7]. In his study, Kasprzyk-Horden [3] focused on 5-month
monitoring at two WWTPs and their receiving rivers located in the UK. TCS concentrations
detected in the influents were about 102-103 ppt, and one order of magnitude lower values
(units to hundreds of ppt) were found in the effluents and rivers. A high TCS concentration
level (µg l-1) was detected in the Ontario River (USA)[8] and in the respective receiving
WWTP effluents. In contrast, very low TCS concentrations were detected in Spain (Ebro
River)[9] and also in the Moravia region (Czech Republic)[10].
The TCS removal efficiency of the WWTPs varied according to the type of cleaning
steps involved and 32% removal has been reported for only primary treatment [11]. When
secondary treatment was used, the efficiency of the WWTPs ranged from 60 to 90%, [5, 12]
where 90% of the degraded TCS is assumed to be removed by activated sludge [13]. It is
worth noting that the removal process involves two major mechanisms: sorption and
degradation [14]. Methyltriclosan (5-chloro-2-(2,4-dichlorophenoxy)anisole) (Me-TCS) is an
environmental bacterial transformation product that is formed under aerobic conditions [12]
and is thought to be even more persistent than TCS. Me-TCS appears to be a suitable marker
for monitoring lipophilic contaminants released into the aquatic environment from WWTPs.
Assessment of the Presence of Triclosan in Wastewater Treatment Plants … 71
Because of the only partial TRC elimination in WWTPs, many studies focused on other
mechanisms and potential methods for removing TRC from the environment. Several studies
have been published recently focusing on the use of bioreactors for effective removal of TCS
and other endocrine disrupters [13, 15, 16, 17]. White rot fungi (WRF), an important
widespread group of microorganisms responsible for biodegradation of lignin, have
frequently been documented to decompose a wide range of organopollutants resistant to
bacterial degradation, including TRC [18, 19, 20].
This work was performed to monitor TRC at several WWTPs in the Czech Republic in
order to obtain new and original data on the actual state of this chemical in the Czech
Republic. This study also provides a reference status, which could be used as a basis for
future monitoring and for establishing sustainable policies. Finally, removal of TCS using an
alternative bioremediation strategy was also investigated using a trickle bed bioreactor. The
fungal strain Pleurotus ostreatus was selected on the basis of our previous biodegradation
study,[21] where P. ostreatus represents a very efficient and promising microorganism for
TCS removal.
MATERIAL AND METHODS

Chemicals. TCS (97%) was used as an analytical standard and as a substrate for
degradation experiments. The compounds were purchased from Aldrich, Germany. Other
chemicals (pure or of higher purity) used as standards were supplied by Fluka, Aldrich or
Merck (Germany). All the solvents of trace analysis quality or gradient grade were purchased
from Chromservis (Czech Republic).
Organisms and cultivation. The ligninolytic fungal strain Pleurotus ostreatus HK-35
was obtained from the Culture Collection of Basidiomycetes of the Academy of Sciences,
Prague. Inocula were prepared in 250 mL Erlenmeyer flasks containing 20 mL of malt-extract

medium starting from 2 mycelial plugs (0.7 mm Ø). Cultures grown for 7 days at 28 °C were
homogenized by Ultraturrax-T25 (IKA-Labortechnik, Germany) and 5% aliquots of the
mycelial suspension were used to inoculate sterile straw pellets (25 g/flasks). The static
cultures were incubated in 500 mL Erlenmeyer flasks at 28 °C.
Bioreactor operating protocol. A pilot scale polycarbonate cylindrical reactor with a
total volume of 47 L (working volume 30 L) was washed with 20 L of tap water and placed in
an iron frame holder in the area of a WWTP (A) in Central Bohemia. A fixed bed reactor with
continuous flow in a trickle bed arrangement was filled with spent fungal substrate (Farma
Volek, Vrbno nad Lesy, Czech Republic) and with previously sterilized straw pellets (2/1,
v/v). An aeration pump supplied air to the bioreactor through a diffuser placed at the bottom
of the reactor. The fungal substrate was aerated for 3 days before the start of the continuous
flow and then the reactor was purged with 3 L of dechlorinated tap water (10 cycles) after 3
days in order to flush the color pigments released from the straw substrate. Then experiments
were carried out with the wastewater effluent and also with the fortified wastewater effluent
(concentration level increased to 1.0 µg L-1). The flow rate was set at 2 L h-1. The hydraulic
retention time was approx 5 minutes. In the case the real wastewater, the influent and effluent
were sampled every 60 minutes and the collected sampled were pooled together create a
representative 24 hours sample.
Monitoring sample collection. Nine selected WWTPs (A, D-K) downstream from
variously populated cities in the summer/autumn of 2011 and spring of 2012 were tested for
the presence of TCS in their influents and effluents. The samples were collected directly after
the primary treatments (influents) and before entrance into the receiving water (effluents).
Fluctuations of TCS concentrations during 24 hours were monitored at both the inlet and the
outlet of one selected WWTP (A). These composite samples were collected every 30 min
(approx. 50 mL for each sampling) into glass bottles and thereafter stored in the dark at 4 °C.
Chemical analysis. Extraction of TCS and Me-TCS from waste water was carried out
using SPE columns (C18 Chromabond, Chromservis). After conditioning the SPE columns
(ethyl acetate, methanol, water), 1000 mL of filtered wastewater (pH 2.5, adjusted HCl) was
applied. The samples were eluted by 10 mL of ethyl acetate. The extracts were dried with
sodium sulfate, trimethylsililated (using BSTFA, 60 °C, 2 hours), and measured with GC/MS.
GC/MS analyses were performed with a SCION SQ (Bruker, USA) instrument equipped
with a CP-800 injector (Bruker, USA) and MS Workstation 8.0 software (Bruker, USA). 1 μL
of the sample solution was injected into the GC/MS system. The analyses were performed
with a 30 m long × 0.25 mm I.D., 0.25 mm film thickness DB-5MS column (Agilent, USA).
The GC oven temperature program started from 60°C (hold 1 min), then heated up to 120 °C
at 25°C min-1, and finally to 240°C at 2.5°C min-1 and was held isothermally for 28 min.
Helium (99.999%) was used as the carrier gas, with a constant flow rate of 1 mL min-1. The
injector was operated in the split/splitless mode, with a splitless time of 1 min. The injector
temperature was 240°C. The source, ion trap and transfer line temperatures were 250, 220 and
280 °C, respectively. The mass spectra were recorded at 3 scans s-1 under electron impact (EI)
at 70 eV and mass range 50-500 amu.
Enzyme activity, PLFA determination. Laccase activity was determined by oxidation
of 5 mM 2,2-azinobis-3-ethylbenzo-thiazoline-6-sulfonic acid [22]. One unit of enzyme
produced 1 μmol of the reaction product per min under the assay conditions at room
temperature.
The straw-substrate samples for PLFA analyses were extracted, fractionated and analyzed
with GC/MS. Methylated fatty acids were identified according to their mass spectra using a
mixture of chemical standards obtained from Sigma (Germany). The biomass of Gram-
positive (G+) bacteria was quantified as the sum of i14:0, i15:0, a15:0, i16:0, i17:0 and a17:0.
Gram-negative (G-) bacteria were determined on the basis of 16:1ω7, 18:1ω7, cy17:0, cy19:0,
16:1ω5. Fungi were quantified using 18:2ω6,9 [33].
RESULTS AND DISCUSSION

Concentrations of TCS and in WWTPs (Pre-Screening)
TCS was detected at the inlets and outlets of all the 9 selected WWTPs except two inlets,
where the TCS concentration was under the limit of detection of the method (‹1 ng L-1). The
concentrations of TCS in wastewaters are illustrated in Table 1 and an overview of the
samples from all the WWTPs compared with results of 24 hour-monitoring of WWTP A are
shown in Figure 1. According to the results, the mean concentrations of TCS in the influents
of all the WWTPs ranged between ‹LOQ and 1830 ng L-1 and between 59 and 1051 ng L-1 in
the effluents, while Me-TCS was not detected in any of the tested WWTP samples. The high
variance in the TCS concentrations is in accordance with the patterns reported in the
literature. To date, several authors have published various concentration levels of TCS in the
influents and effluents of WWTPs. Gonzales-Marino et al. [23] detected TCS concentrations
ranging from ‹LOQ to 936 ng L-1 in raw wastewater in Spain. Similarly, Stakinakis et al. [24]
found TCS in effluents from Greek sewage plants in the range from 0.13 to 6.88 µg L-1 and
0.17 to 23.9 µg L-1.
Table 1. TCS concentrations detected at the selected 9 WWTPs in the Czech Republic.
ND – not detected
Influent Effluent
WWTPs
TCS (ng l-1) STD TCS (ng l-1) STD
A 1830.0 183.0 1051.9 199.7
D ND 104.8 0.8
E 598.3 8.0 89.2 15.7
F ND 58.8 0.8
G 596.7 15.7 475.8 15.1
H 122.0 6.5 152.0 28.2
I 819.3 81.9 258.8 46.6
L 679.4 6.8 621.8 87.1
K 112.0 13.4 69.5 4.9
3000
2000
1000
WWTPs in WWTPs out A 24h in A 24h out
Figure 1. Box plots of TCS concentrations in individual representative WWTP inlet (WWTPs in) and
outlet (WWTPs out) samples and 24-hour monitoring of WWTP A (A 24 h in; A 24 h out). Each box
displays the median, upper and lower quartiles of the respective distribution. For WWTP sample plots,
n=9; for 24-hour plots, n=16. Box whiskers represent the maximum and minimum range excluding any
extreme outliers (shown as a dagger).
3
3,0x10 Influent
Effluent
3
2,5x10
TCS concentration (ng l )

-1
3
2,0x10
3
1,5x10
3
1,0x10
2
5,0x10
0,0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (hours)
Figure 2. TCS concentrations in the effluents and influents measured over 24 hours at the WWTPs A.
The results are expressed as the mean concentrations (ng L-1) ± the standard deviations (n=3).
The results of the screening analyses shown in Table 1 suggest that the TCS
concentrations detected in our study were generally higher than the concentrations detected at
another site in the Czech Republic by Grabic et al. [10]. In their work, a passive sampling
method was used for monitoring EDCs in the Svratka and Svitava rivers (Moravia) and the
related receiving WWTP effluents in 2006 and 2007. The detected TCS and Me-TCS
concentrations in the WWTP effluent ranged from 9.5 to 16 ng L-1 and 23 to 38 ng L-1,
respectively. The influent concentrations were 33 – 84 ng L-1 and 0.15 – 0.29 ng L-1,
respectively. Very similar concentrations of TCS and Me-TCS were detected by the same
authors in 2007 and 2008 using the same method at the same site [25]. To the best of our
knowledge, no data has been reported on expanded screening of the presence of TCS in
WWTP influents and effluents in the Czech Republic. The results in Figure 1 document that
TCS was not totally removed in the present study by the employed wastewater treatment
processes and part of the initial concentrations was also detected in the effluents.
TCS Concentration Course in 24 Hours Experiment
According to the pre-screening results, WWTP A was also selected for 24-hour TCS
monitoring because it yielded the highest TCS concentration. Samples were taken 16 times
during 24 hours and analyzed using GC/MS. TCS varied greatly in the individual inlet
samples (see Figure 2). The lowest concentrations were detected in the morning, except for
the 6 to 9 AM rush-hour period (see Figure 2). For the inlet, two obvious TCS peaks
culminated at 8 AM and 6 PM. A similar situation was found for the output samples, with
slight shifts to 6 AM and 10 PM. The composite pooled sample values from WWTP A
exhibited approximately the same concentration level (influent: 1375 ng L-1; effluent: 556 ng
l-1) as the calculated means of the 24-hour monitoring (1584 ng L-1 for the influent and 479 ng
L-1 for the effluent).
TCS Removal in Bioreactor
A pilot scale bioreactor was constructed based on previous optimization on a laboratory

scale batch and continuous flow reactor experiments carried out in our laboratory (data not
shown). The pilot reactor was installed directly at the site of the WWTP A effluent. Very high
elimination of 87% TCS was achieved with real wastewater effluents and about 75% with the
fortified wastewater. All the experiments were performed under non-sterile conditions.
Figure 3 shows the degradation of TCS in the real wastewater and in the fortified
wastewater. Initially, the fortified wastewater was used to quantify the degradation potential
of P. ostreatus under the conditions and technical arrangements employed. The results are
displayed in Figure 3A, where the average concentration of TCS is expresses as a percentage
of the initial concentration (1 μg L-1). TCS degradation during 5 sampling campaigns (C_1-
C_5) of real wastewater (Figure 3B) ranged from 57% up to 100%. In the fortified
wastewater, TCS was substantially degraded from the first 0.5 hours after its addition and the
degradation rate culminated within 3 hours, probably reaching maximal degradation capacity
of the reactor under the conditions employed. A possible explanation for the lag phase could
be the adaptation of the fungal organism in terms of induction of enzymes together with
production of the necessary redox mediators [26]. It should be borne in mind that, in addition
to the degradation, sorption could also contribute to the TCS removal within first 0.5 h.
However, biosorption can also play a positive role through facilitation of further degradation
[27]. Based on its physical and chemical properties, it is not anticipated that triclosan could be
removed by volatilization; therefore biodegradation and (bio)sorption are the two major
mechanisms of TCS removal. As already described above, sorption could take place in the
first phase of TCS removal, but after a 24 hours cycle the results indicate that biodegradation
is the main removal mechanism.
According to our PLFA analysis results (Figure 4) from the bioreactor with the fortified
wastewater, the P. ostreatus biomass had increasing tendency during the whole course of the
test. In contrast, bacterial biomass decreased slightly in the initial period; however, after the
application of the fortified wastewater, the bacterial biomass gradually increased in the
bioreactor. However, comparing these two parameters, the results showed that
fungal/bacterial ratio decreased during the experiment. Especially Gram negative bacteria
were responsible for the bacterial biomass increase data visible form the Figure 4. Despite the
fact that bacterial degradation is probably ineffective, as was demonstrated by previously
published bioreactor studies, and fungal biodegradation is the major TCS elimination
mechanism in this study, the PLFA results indicate that the fungus was not negatively
influenced by the autochthonous bacterial microflora. The results of our study regarding the
major role of fungal TCS degradation are in good agreement with our previous study with
pure fungal culture [20] and also with recent studies published in the literature [15, 28]. The
laccase activity was detected in the reactor effluent and the activity did not significantly differ
from the initial values (ANOVA, P < 0.05) during the whole bioreactor process. These results
also indicate that the bacterial cultures did not negatively influence the P. ostreatus
physiology during the test, as has been reported in some studies [29, 30, 44].
Fortified wastewater Wastewater Bioreactor influent

Bioreactor effluent
A TRC
B
100
% of TCS influent concentration

100
% of TCS influent concentration 50
50
0
C_1 C_2 C_3 C_4 C_5
Sampling campaigns
0 30 min 1 hour 3 hours 5 hour 1 day 2 days 4 days 7 days

Time
Figure 3. TCS degradation in the pilot scale bioreactor with the wastewater (B) and the fortified
wastewater (A). Degradation is expressed as a percentage of the TCS influent concentrations
(fortification level 1.0 µg L-1).
Bacteria G+ G-
Fungi/Bacteria Fungi
140
40
120
Bacterial PLFA markers (g g )
Fungal PLFA marker (g g )

-1
-1
30 100
80
20
60
40
10
20
0 0
-3 0 3 6 9 12
Time (days)
Figure 4. The results of PLFA analysis during the experiment with the fortified water detected in the
bioreactor solid support (straw substrate). The error bar represents the standard deviation (n=3).
CONCLUSION
The analytical data on the presence of TCS in the effluents and influents of nine WWTPs
in the Czech Republic suggest that WWTPs are not completely effective in TCS removal and
therefore represent an important source of environmental contamination. Elevated TCS
concentrations in effluents flowing into receiving streams may potentially increase toxicity in
aquatic environments and emphasize the need for further research and monitoring. Given the
potential adverse effects of TCS on aquatic organisms and human health, it is also necessary
to develop and to improve methods and processes for TCS removal. The results of this study
document that the ligninolytic strain P. ostreatus is effective in TCS removal even under non-
sterile conditions and that it can be employed in continuous flow bioreactor arrangements.
ACKNOWLEDGMENT
This work was funded by the grants Competence Center TE01020218 of the Technology
Agency of the Czech Republic and by the Grant No. 15-02328S of the Czech Science
Foundation.
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pharmaceuticals and endocrine disruptor compounds. Sci Total Environ. 2014, 493,
365-76.
[29] Gao, D; Zeng, Y; Wen, X; Qian, Y. Competition strategies for the incubation of white
rot fungi under non-sterile conditions. Process Biochemistry. 2008, 43(9), 937-44.
[30] Libra, JA; Borchert, M; Banit, S. Competition strategies for the decolorization of a
textile-reactive dye with the white-rot fungi trametes versicolor under non-sterile
conditions. Biotechnol Bioeng. 2003, 82(6), 736-44.
Chapter 4
REMOVAL OF PHARMACEUTICALS AND PERSONAL

CARE PRODUCTS FROM WASTEWATER
Ma Ying1, Jian Wu1, Ken D. Oakes3 and Anming Hu1,3*

1
Institute of Laser Engineering, Beijing University of Technology, Beijing, P. R. China
2
Verschuren Centre, Department of Biology, Cape Breton University, Sydney, Canada
3
Department of Mechanical, Aerospace and Biomedical Engineering,
University of Tennessee, Knoxville, TN, US
ABSTRACT
Pharmaceuticals and personal care products (PPCPs) have attracted much recent
attention as widespread emerging environmental contaminants, both due to their near
ubiquitous detection in surface waters adjacent urban areas, but also their potential to
generate endocrine modulating responses at low concentrations in exposed organisms.
Although usually detected in environmental matrices only at low ng/L or µg/L ranges,
adverse effects in exposed aquatic organisms, especially with respect to estrogenic
compounds, have been widely reported and prompted concerns over the potential for
human health effects. Consequently, there has been increasing research attention paid to
cost-effectively removing PPCPs from wastewater prior to its‘ discharge to the
environment. Numerous studies have examined the occurrence and fate of PPCPs in
wastewater and adjacent receiving environments, focusing on their removal by
conventional and advanced treatment processes at varying scales ranging from lab to
bench experiments to full treatment plant manipulations. This review will discuss various
removal mechanisms such as surface absorption, membrane filtration, advanced
oxidization treatments (including photocatalytic degradation and electrolysis) with
advantages and limitations of each treatment approach (or combinations thereof) in
removing PPCPs explored. Elucidating the fate and effects of PPCPs in the environment,
and the potential of recent technologies to limit their environmental contamination, are
key elements in protecting future ecosystem and human health.
*
Corresponding author: anminghu@bjut.edu.cn.
82 Ma Ying, Jian Wu, Ken D. Oakes et al.
1. INTRODUCTION
In the past few decades, aging demographics and consumer demand has lead to sharp
increases in the production and consumption of pharmaceuticals and personal care products
(PPCPs), along with the detection of parent compounds, metabolites, and degradation
products in soil, sediment, and water matrices. It was not until Daughton and Ternes [1]
published a review on environmental pharmaceuticals and personal care products (PPCPs) in
1999 that widespread public and researcher attention was focused on the distribution,
particularly in surface waters, of these chemicals. The United States Environmental
Protection Agency (USEPA) formally defines PPCPs as including not only prescription drugs
and biologics, but also diagnostic agents, ―nutraceuticals,‖ fragrances, sun-screen agents, and
numerous other consumer ingested or applied compounds which ultimately pass through
municipal waste water treatments plants (MWWTPs) where they are detected, almost
invariably only in trace concentrations, in the environment. In recent years, PPCPs in aquatic
environments have been recognized as emerging contaminants due to their high polarity, low
volatility, and near continuous infusion within MWWTP effluents. [2] With the advent of
new analytical techniques, these compounds can be detected at extremely low concentrations
in very complex matrixes. [3,4] As pharmaceuticals are designed to exert specific
physiological effects on humans and animals at trace concentrations, their inadvertent release
to the environment, and subsequent uptake by exposed aquatic organisms or human
populations via drinking water intakes downstream of MWWTPs may produce unintended
consequences in biota or medically compromised patients. As some PPCPs resist microbial
degradation in MWWTPs, and in light of their consequent continuous input to the
environment, PPCPs were considered to be a potential threat to human and animal health,
depending on the properties of each compound, in both high and low concentrations. [5,6]
Consequently, many questions regarding the environmental fate of PPCPs, their
ecotoxicological and human health risks, and the ability of current water and wastewater
treatment infrastructure to effectively remove these compounds remain unanswered. [4]
Within this context, this chapter will summarize the current knowledge and uncertainties
regarding PPCPs in aquatic environments, as well as, the effectiveness of varying water
treatment processes (from conventional water treatment to advanced oxidation processes,
with special attention to emerging nanotechnologies4) to remove PPCPs from water.
2. OCCURRENCE OF PPCPS IN AQUATIC ENVIRONMENTS

2.1. PPCPs in Water Supply Sources
The occurrence of PPCPs in aquatic environments has been investigated and described in
many countries globally; [7] although animal feeding operations [8] can be the dominant
source of PPCPs in rural areas, in more urban areas, most PPCPs in surface waters originate
from MWWTP effluents, although other sources and pathways are possible (Fig 1). These
PPCPs are introduced to MWWTPs mainly through ingested medicines excreted as un-
metabolized parent compounds (or reactive metabolites) via urine, effluents from
pharmaceutical production facilities, hospitals and direct disposal of medication in toilets,
Removal of Pharmaceuticals and Personal Care Products from Wastewater 83
where many will eventually be introduced to surface water. MWWTP sludges and manure
applied to land can also be significant non-point sources of PPCPs, especially during severe
rain-events, where residues can leach into surface water and groundwater. Further, PPCPs
may also be introduced to groundwater via aquaculture facilities and septic systems. [9] As
worldwide environmental contaminants, the occurrence and concentration of PPCPs in water
sources can vary significantly, ranging from a few ng/L to high μg/L values (Table 1), and
can differ significantly even in the same river, typically increasing with additional
downstream MWWTP effluent additions. [10]
Medicinal products Veterinary products
Pharmaceutical
hospitals Waste disposal manure
production facilities
Wastewater treatment soil

WWTP sludge
plants(WWTPs)
Surface water Ground water Septic systems

Aquaculture failities
Drinking water
Figure 1. Scheme showing possible sources and pathways for the occurrence of pharmaceutical residues
in the aquatic environment. Adapted from Ref. [3].
Table 1. PPCP concentration ranges in source water
PPCP compound Concentrations in source water (μg/L) References

Clofibric acid 3.2–26.7 [11]
Phenazon 0.06-0.155 [12]
Salicylic acid 1.601-89.133 [12,13]
Ibuprofen 0.002–34.0 [14-16]
Paracetamol 0.069–26.09 [17-18]
Acetaminophen 0.027–65.2 [11,19]
Caffeine 0–38.0 [11-12,20-21]
Sulfamethoxazole 0.0017-2.17 [12,16,22]
Trimethoprim 0.002-0.18 [12,19,21]
Naproxen 0–135.2 [11,23-25]
PPCP compound Concentrations in source water (μg/L) References

Ciprofloxacin 0.017-0.036 [29-30]
Bisphenol A 0–147.2 [11,26-27 ]
Diclofenac 0.003–5 [12,15.28]
Carbamazepine 0.014–0.5 [12,18,29-31]
Gemfibrozil 0.3-1.652 [14,20]
Estrone 0.0006–4.7 [11,16,32]
17β-estradiol 0.0004–4.5 [11,16.33]
Triclosan 0.39–37.8 [12,31,34-35]
Nonylphenol 0.006–37 [26-27,36-37]
Octylphenol 0.0008–0.7 [26-27,36-37]
2.2. PPCPs in Drinking Water
Drinking water treatment plants (DWTPs) remove microorganisms and organic

contaminants from raw water sources to meet drinking water standards, with the treatment
process employed mainly a function of the best technology affordable that is adequate for the
local raw water characteristics. Conventional treatment processes usually include coagulation
(flocculation and sedimentation), filtration and disinfection steps, occasionally also including
ion exchange (or water softening) processes. [38] Despite these treatment processes, studies
demonstrate some recalcitrant PPCPs are not completely removed, [39,40] although relative
to MWWTPs, there is less known of the behavior of PPCPs in drinking water. One reason
may be the lack of systematic monitoring programs at many municipalities, or insufficient
analytical sensitivity to detect PPCPs in drinking water, which are usually present at
concentrations of sub-ng/L levels. [41] However, at least 25 PPCPs are reported more than
once in drinking water in the reviewed literature, with the β-blocker atenolol being the most
frequently detected compound in drinking water with concentrations above the limit of
quantification (LOQ), followed by salicylic acid and carbamazepine, one of which were
characterized at detectable levels in more than 30% of the drinking water samples. Table 2
reports the maximum measured concentrations and frequency of PPCP detection in drinking
water samples from France [15], Spain [42], China [43] and the United States [44] in recent
years.
Table 2. Maximal measured concentrations and frequency of PPCPs detection in

drinking water samples from France, Spain, China, and the United States
PPCP compounds Maximum Concentration (ng/L) Frequency

Atenolol 23.00 25
Atorvastatin 18.00 8
Bisphenol A 6.00 2
Butylparaben 28.00 5
Carbamazepine 32.00 15
Carbazochrome 0.89 2
Chlofibric acid 19.00 5
Chloramphenicol 2.00 1
PPCP compounds Maximum Concentration (ng/L) Frequency

Diclofenac 18.00 4
Fenofibric acid 16.00 3
Fluoxetine 0.82 2
Gemfibrozil 2.10 10
Hydrochlorthiazide 7.00 10
Ibuprofen 39.00 8
Ketoprofen 7.00 3
Meprobamate 42.00 14
Metoprolol 1.00 2
Methylparaben 12.00 6
Naproxen 11.00 10
Oxazepam 2.50 2
Paracetamol 45.00 4
Phenytoin 19.00 15
Pravastatin 0.20 1
Propylparaben 9.00 5
Salicylic acid 31.00 21
Sotalol 3.00 4
Sulfamethoxazole 3.00 6
Sulpiride 0.17 3
Thiamphenicol 7.00 6
Triclocarban 13.00 2
Triclosan 1.20 1
Trimethoprim 1.00 1
3. REMOVAL OF PPCPS DURING TREATMENT PROCESSES

Both MWWTPs and DWTPs are designed to remove organic and inorganic suspended
materials, flocculated matter, and pathogens from wastewater or drinking water sources;
however, neither drinking nor wastewater treatment processes are specifically designed to
remove PPCPs from water (although both do to some degree, varying with the
physicochemical properties of individual PPCPs). Due to their high chemical stability and
low biodegradability, most PPCPs are unlikely to be completely removed by conventional
treatment processes (Figure 2). In recent decades, several technologies were developed to
meet more stringent water treatment demands, including advanced oxidation processes
(AOPs) and membrane technologies. [45]
3.1. Advances Oxidation Processes (AOPs)
Advanced oxidation processes can be defined as ―near ambient temperature and pressure
water treatment processes utilizing the generation of hydroxyl radicals in sufficient quantity
to effect water purification‖ [46], and are generally used to remove organic and sometimes
inorganic materials via oxidation by hydroxyl radicals (OH•), which are usually produced
with the help of oxidants (e.g. O3, H2O2) and/or energy sources (e.g. UV light) or catalysts
(e.g. TiO2, LaFeO3). AOPs can reduce contaminant concentrations from several hundred ppm
to less than 5 ppb, earning the moniker ―the water treatment processes of the 21st century‖
[46].
“Filters”
Drinking water
Wastewater
treatment
Surface water
Bank filtration
Drinking water
treatment
Figure 2. ―Filters‖ able to remove pharmaceutical compounds on the path from wastewater to drinking
water. The different filters mainly reflect engineered solutions, but also pathways and environmental
compartments where mechanisms of biodegradation, sorption, photolysis, and oxidation can occur.
Adapted from Ref. [40].
3.1.1. Photocatalysis
Photocatalysis is a change in the rate of a chemical reaction when the photocatalyst
absorbs ultraviolet, visible, or infrared radiation. Titanium dioxide (TiO2) is the most widely
utilized semiconductor photocatalyst in water treatment applications due to its low cost and
commercial availability in various crystalline forms, particle characteristics, non-toxic nature,
and high photochemical stability. [47] From a mechanistic viewpoint, photocatalysis occurs
through the absorption of photo energy greater than the TiO2 band gap, to produce valence
band holes and conduction band electrons. The holes and electrons can either: (1) recombine,
with the resultant absorbed energy dissipated as heat, or (2) make their separate ways to the
TiO2 surface where they can react with and degrade waterborne contaminant species adsorbed
on the catalyst surface. [48]
Valence band holes can also react with water and produce hydroxyl radicals, which are
among the most powerful reactive oxygen species, capable of oxidizing a wide range of
substrates. In addition to TiO2, other semiconductor catalysts such as ZnO and LaFeO3 have
also been employed in water treatment applications. [49, 50]
3.1.1.1. TiO2 Photocatalysts under UV Irradiation

Typically, UV illumination is the primary energy source driving photocatalytic activity,
with pure TiO2 in the anatase phase possessing a band gap energy of 3.2eV, and numerous
studies have investigated the potential of using TiO2 as a photocatalyst for removal of
waterborne PPCPs.
In most of these studies, the focus has been on determining optimal operating conditions,
such as photocatalyst loading, initial concentration of the PPCP under investigation, and
solution pH and light intensity to maximize degradation and mineralization. [51]
Hu et al. [52,53] synthesized highly entangled TiO2 nanowires (10-20 nm diameters, 100
μm lengths) and their photocatalytic degradation kinetics towards PPCPs under UV
irradiation, demonstrating both that selected PPCPs can be degraded in a process described by
pseudo-first order kinetics, and that TiO2 nanowire membranes have potential for water
treatment applications. As with TiO2 nanowires, TiO2 anatase phase nanobelts (30-100 nm
widths and 10 μm lengths) exposed to 365 nm UV illumination can also photocatalyze the
oxidation of PPCPs. [54] These TiO2 nanobelts under high reaction temperatures, alkaline
conditions, and in a concentration-dependent manner, facilitated faster photodegradation of
theophylline, while amoxicillin conversely had mineralization retarded as solution pH was
increased from 5 to 7.5. [55]
CB O₂
e¯
O₂¯ H₂O₂
e¯ e¯
hν₃
hν₁ OH¯
hν₂ h⁺
h⁺ OH¯
h⁺
VB OH*
Figure 3. Photocatalystic mechanisms: hυ1 pure TiO2, hυ2 metallic-doped TiO2, hυ3 non-metallic-doped
TiO2. Valance Band (VB) and Conduction Band (CB) illustrated by horizontal lines.
The anti-inflammatory Naproxen is one of the most detected pharmaceuticals in water,

and studies on photocatalytic and photolytic degradation of naproxen demonstrated that under
Xe-lamp (290-400 nm), naproxen degradation by photolysis was twice as efficient as by
photocatalysis. [56]
Low adsorption of naproxen on the catalytic surface, as well as possible recombination
and deactivation of the OH• radical may have have contributed to the lower photocatalytic
efficiency. In contrast with naproxen, diclofenac showed excellent removal by UV/TiO2
treatment. [57] The highest photocatalytic efficiency (residual diclofenac concentration =
0.4%) was obtained with a TiO2 loading/initial concentration of 624 mgL-1/8.17 mg L-1 with
complete mineralization achieved within 2 h of irradiation.
Degradation efficiencies of other PPCPs such as iopromide, acetaminophen,
sulfamethoxazole, and carbamazepine under UV/TiO2 treatment were also investigated, [58]
with more efficient degradation observed when the reactor influent was in buffered electrolyte
than when in wastewater effluent (Figure 4) demonstrating the influence of wastewater
effluent matrix in inhibiting photocatalytic deactivation.
Figure 4. Degradation efficiencies in buffered electrolyte and wastewater effluent. Adapted from Ref.
[57].
3.1.1.2. Modified TiO2 Photocatalysts under Visible Irradiation

As UV accounts for only 5% of visible spectrum radiation, incident solar energy is not
efficient for the photoexcitation of unmodified TiO2; consequently, there has been a great deal
of interest in preparing a TiO2 photocatalyst capable of efficiently utilizing sunlight. One
means of reducing the wide band gap of TiO2, which requires an energy threshold only
associated with the UV portion of the visible spectrum, is by modifying or ―doping‖ TiO2
with other constituents. Both metallic and non-metallic doping can reduce the energetic band
gap by either raising the position of the valence band, or by lowering the position of the
conduction band. As shown in Figure 3, electrons are excited from the defect state, imparted
by metal doping, to the conduction band (CB) with excitation by a photon of energy hv2.
Transition metal doping can also improve the trapping of electrons and prevent electron–hole
recombination during irradiation, resulting in enhanced photoactivity. With non-metallic
doping, electrons are excited in the valance band (VB) by impurity energy level hv3 (Fig 3)
under UV irradiation, experiencing excitation from impurity energy levels under visible light
irradiation. [59]
Platinum ion-doped TiO2 (Ption-TiO2), one of several metallic-doping methods,
demonstrated good photocatalytic activities with respect to the degradation of dichloroacetate
under visible light irradiation. [60] The visible activity of Ption-TiO2 can be ascribed to the
role of doped Pt ions as a charge generating center, producing free and trapped charges (Pt3+).
( )
( )
In addition to Ption-TiO2, other metallic-doped TiO2 compounds, such as Zr-doped TiO2,

have also had their photocatalytic performance evaluated using the degradation of ibuprofen
under UV-Vis light as a model. [61] Relative to P25, Zr-TiO2 demonstrated improved
performance due to its increased specific surface area and improved adsorption properties.
Iodine doping is a commonly employed non-metallic doping method using iodic acid as a
dopant [62,63] Studies suggest rutile iodine-doped TiO2 nanowires produce the highest
methylene blue degradation relative to other synthesized nanomaterials. Their enhanced
photocatalytic activity was attributed to existing oxygen vacancies, iodine multi-valences in I-
O-Ti bonds, and 3d state Ti3+ sites in the TiO2 lattice.
Another further means of achieving visible light-driven photocatalytic activity is coupling
TiO2 with narrow band gap semiconductors, which should have more negative and less
positive valence band potentials. [64] As shown in Fig. 5, the mechanism underlying this
technique enhances photocatalytic efficiency by promoting the separation of photo-induced
electron–hole pairs through changing the carrier transfer pathway. For example titanium
dioxide, when coupled with tungsten trioxide, as TiO2–WO3 composite nanotubes,
completely removed a mixture of PPCPs (caffeine, metoprolol and ibuprofen) through
photocatalytic ozonation using simulated solar light and photocatalytic ozonation in less than
40 min (with up to 64% TOC removal after 2 h). [65] However, the obvious drawbacks of
semiconductor coupling are the reduction in capacity of conduction band electrons of the
coupled semiconductor, while the high oxidation capacity of valence band holes of TiO2
cannot be utilized, according to the Z-Scheme electron transfer model. [66]
3.1.1.3. LaFeO3 Perovskite Photocatalyst under Visible Irradiation

Perovskite-type oxides of the general formula ABO3 are exciting materials for water
treatment owing to their stability and high photocatalytic activity in water. LaFeO3, a typical
ABO3-type perovskite oxide, has attracted considerable attention among researchers due to its
broad potential for diverse applications such as catalytic oxidation, surface electronic states,
and gas-sensitive characters. Additionally, LaFeO3 has been found to be photocatalytically
active in the presence of visible light due to its unique optoelectronic properties and narrow
band gap. [67] To date, there is a paucity of data on PPCP removal by LaFeO 3, despite many
studies focussing on the visible light photocatalytic activity of LaFeO3. [68,69] Hu et al., [49]
synthesized LaFeO3 and La2FeTiO6 by glucose sol–gel methods and evaluated their potential
as photocatalysts by assessing p-chlorophenol degradation under visible light. La2FeTiO6

demonstrated 62.1% degradation of p-chlorophenol with 56.6% chemical oxygen demand
(COD) removal after 5 h of visible-light irradiation, while LaFeO3 degraded 49.0% of p-
chlorophenol with 45.1% COD removal under identical conditions. The bigger surface area
and smaller grain diameter contribute to the excellent photocatalytic activities of La2FeTiO6.
Supported perovskite oxide, LaFeO3/SBA-15, was also investigated for photocatalytic
efficacy in the oxidation of organic dyes using hydrogen peroxide as the oxidant. [70]
Comparative tests on various organic dyes demonstrated after 60 min of reaction time that
LaFeO3/SBA-15 is most efficient in degrading Rhodamine B (80% removal) relative to
Methylene Blue (66% removal), Reactive Brilliant Red X-3B (42% removal),, and Direct
Scarlet 4BS (42% removal).
3.1.2. Photolysis Processes

Photolysis is the interaction of light (natural or artificial) with a molecule to induce
photochemical reactions leading to the molecules degradation to intermediate products, and
ultimately, to complete mineralization. [71] Physical disinfection of drinking water is largely
dependent on ultraviolet radiation-driven photolysis, which has the advantage of minimizing
the formation of disinfection by-products relative to chlorination. [72] However, a typical
dose of 40 mJ/cm2 applied for drinking water treatment, while sufficient for microbial
disinfection, is largely ineffective for removal of most targeted organic compounds. For
example, 17α-ethinylestradiol (EE2), diclofenac, sulfamethoxazole, and iopromide
experienced only modest removals (0.4–27%) in dilute solutions of buffered water
undergoing photolysis at pH 7.0. [73] Some macrolide antibiotics (i.e. such as clarithromycin,
erythromycin and azithromycin) exposed to UV alone exhibited very low (4-7%) removal
efficiencies, even with the considerable UV dose of 2768 mJ/cm2 for 15 min, which is a much
higher dose than the 40-140 mJ/cm2 required for typical disinfection. [74]
O₂¯
CB e¯
e¯ CB
O₂
VB OH¯
h⁺
OH˙
Coupled
VB semiconductor
TiO₂
Figure 5. Schematic illustrating the visible light photocatalytic mechanisms of TiO2 coupled with a
semiconductor.
The efficiency of UV photolysis can be enhanced when irradiation is combined with

hydrogen peroxide (H2O2), to form high reactive hydroxyl radicals (OH•), thus facilitating
non-specific degradation of a broad suite of organic compounds.
The efficiency of UV/H2O2 co-treatment depends upon the absorbance spectrum of the
PPCP or other organic compounds, along with the quantum yield of photolysis, the
concentration of hydrogen peroxide employed, and the other dissolved and suspended
constituents comprising the water matrix. Relative to UV treatment alone, the combination of
UV/H2O2 yielded over a 90% removal efficiency for 39 of 41 evaluated pharmaceuticals at a
UV dose of 923 mJ/cm2, the exceptions being norfloxacin and caffeine (69% and 67%
removal efficiencies, respectively). As illustrated in Fig. 6, the addition of H2O2 substantially
enhanced removal of pharmaceuticals during UV irradiation processes, to the extent that it
may be possible to reduce UV energy inputs required for effective water treatment. [74]
However, the presence of natural organic matter (NOM) in water will quench OH• radicals
and decrease degradation efficiency [75]; however, Doll and Frimmel [76] suggested that
while NOM can act as a radical scavenger, it can also serve as a precursor of reactive species,
which can enhance degradation of recalcitrant PPCPs such as carbamazepine by the
production of photochemically-induced reactive species.
Figure 6. Removal efficiency of 41 pharmaceuticals during UV and UV/H2O2 water treatment

processes. Reproduce permission from Ref. [74].
3.1.3. Ozonation Processes

Ozone is a strong oxidant able to decompose into hydroxyl radicals, which are stronger
oxidizing agents than ozone itself. Therefore ozonation incorporates the so-called indirect
oxidation afforded by hydroxyl radicals, and their attack on certain functional groups of
organic molecules through an electrophilic mechanism. [77] Unlike ozone, hydroxyl radicals
are non-specific oxidants able to degrade a broader range of PPCPs and other organic
micropollutants via radical addition, hydrogen abstraction or electron transfer mechanisms.
[78] Hydroxyl radical production by ozone can be described as:
Ozonation is commonly employed in drinking water treatment systems as an alternative

disinfection to free chlorine, mitigating odor and taste aesthetic complaints. Considerable
research to date demonstrates the interest of researchers and water managers alike in the
application of O3, O3/H2O2, and O3/UV for removal of PPCPs from water. Snyder et al. [79]
conducted bench-scale investigations of PPCP ozonation in surface water and wastewater
matrices demonstrating that the majority of investigated PPCPs were more than 90% removed
at O3 exposures commonly used for disinfection. Notably, almost complete (>99%) removal
of some fairly ubiquitous and recalcitrant PPCPs including carbamazepine, diclofenac,
naproxen, sulfamethoxazole and trimethoprim demonstrated the effectiveness of ozone as
shown in Fig. 7. However, other PPCPs and common micropollutants were relatively resistant
to ozonation, with iopromide, meprobamate, and tris-chloroethylphosphate (TCEP)

exhibiting, less than 60% removal at the highest ozone concentrations investigated (2.5
mg/L). The addition of H2O2 to O3 to achieve advanced oxidation was of little benefit for
those PPCPs resistant to ozonation, relative to O3 alone. O3/H2O2 marginally enhanced
removal of dilantin, diazepam, DEET, iopromide and meprobamate, while advanced
oxidation actually decreased removal efficacies of pentoxifylline, caffeine, and
androstenedione relative to ozonation alone. [79]
100
80
Precent Removal (%)
60
40
20
H2O2 Dose 0mg L-1

H2O2 Dose 0.5mg L-1
0
az ne
py e
m ste e
Su Pro en en
nz tr ne
D ac
O apr te
ne en
xa e
Te T ole
e
M pr en
ho e
ba e
up in
Tr ost EP
ba af e
ze T
im
T
ic am
a) in
lfa ge zon
ho n
et on
in
r o id
ar C ren
D DEE
D
Ib nt
N ma
n
pr
xy ox
o
te h
et ro
m fei
Io rof
o( az
z
ep
ep om
st C
D
D p
fe
ila
os op
di
im er
lo
dr in
ia
b
An tam
e
Ac
Be
Figure 7. PPCPs removal efficiencies using O3 and O3/H2O2 processes. Adapted from Ref. [79].
In O3/H2O2 system, the removal efficiency of COD and dissolved organic carbon (DOC)
depends on the degree of OH• radical generation, which can be described as the following
reaction: [80]
Degradation efficiency differs among different H2O2 to O3 molar ratios, with O3/H2O2
applied at a 0.5 molar ratio demonstrating the highest efficiency for removal of organics from
wastewater effluent (Fig. 8), which suggests that the ratio of H2O2 injected with respect to
ozone dosage is critical. [81]
Figure 8. Effect of O3 and O3/H2O2 treatment on COD and DOC removal from wastewater effluent.
Adapted from Ref. [81].
UV-irradiation of O3 promotes the formation of OH• radicals via the rapid decomposition
of O3. The observed reaction pathways for the UV/O3 process were similar to those of the
ozonation process, but with faster kinetics, presumably induced by non-selective OH• radical
reactions. [81]
Table 3. Electrical Energy and Operating Cost Required for O3/UV Processes.
Adapted from Ref. [82]
Applied process O3 alone O3/UV 21.5W O3/UV 65W

O3 dose (mg L-1) 2 4 6 2 4 6 2 4 6
Total electrical
0.03 0.06 0.09 0.37 0.4 0.43 1.06 1.09 1.12
energy(kWh m−3)
No. of PPCPs
removed
24/37 32/37 35/37 15/35 23/35 31/35 24/38 34/38 35/38
≥90%/No. of
detected PPCPs
Operating cost
0.5 0.9 1.4 5.6 6.0 6.5 15.9 16.3 16.8
(Yen m−3)
Advanced oxidation (UV/O3) removal of PPCPs is superior to O3 alone due the additional
degradation afforded by direct UV photodegradation, but most importantly, OH• radical
oxidation. While advanced oxidation is more effective, when considering cost-effectiveness,
ozonation alone removed more PPCPs per unit cost (Table 3). [82]
3.1.4. Electrocatalysis
Electrocatalysis is catalytic action generated on an electrode surface achieved through
changing the rate and selectivity of electrochemical reactions, and is closely connected with
the adsorption of reacting, intermediate and end products. [83] The main difference between
electrocatalytic and heterogeneous catalytic reactions is the direct anodic oxidation of
electrocatalysis where the pollutants are adsorbed on the anode surface and destroyed by the
anodic electron transfer reaction.
3.1.4.1. Photoelectrocatalytic (PEC) with TiO2 Nanofilm Modified Electrode

To prevent the fast recombination of the photo-generated electrons/holes (e−/h+) and
improve the photocatalytic efficiency of TiO2 films, the photoelectrocatalytic (PEC)
technique was devised by applying an external bias potential onto TiO2 film-coated
electrodes, combining both electrochemical and photocatalytic technologies. [84] The
potential of a PEC oxidation process for the treatment of carbamazepine was evaluated using
TiO2 photo-anodes prepared by pulsed laser deposition (PLD). [85] The PLD TiO2 coatings
were found to be of anatase structure consisting of nanocrystallites of approximately 15 nm
diameter. The contribution of treatment time and pollutant concentration accounts for 70.6%
and 23.3% of carbamazepine PEC degradation, respectively, whereas the contributions of
cathode material and current intensity were 2.4% and 0.75%, respectively. The PEC process
applied under optimal conditions is able to oxidize 73.5% ± 2.8% of carbamazepine and
ensure partial mineralization of 21.2% ± 7.7%. The TiO2 thin film can also be deposited onto
surfaces via the liquid phase deposition (LPD) process. [86] In evaluating degradation of the
model compound 4-aminoantipyrine, PEC processes exhibited a much higher efficiency of
62.1% after 120 min treatment, while electrolysis, direct photolysis, photocatalysis were 3%,
38% and 50.8% efficient, respectively. Recently, magnetic loading has been demonstrated as
a novel means of preparing TiO2 electrodes, using the magnetic force between an external
magnet and TiO2/SiO2/Fe3O4 (TSF) nanoparticles to facilitate the desired adherence of
catalyst to the electrode, which has been successfully applied in the degradation of diclofenac.
[87]
3.1.4.2. PEC with TiO2 Nanotube Arrays (TNAs) Electrode

In a typical TiO2-based PEC process, there are two key factors affecting overall treatment
efficiency; the TiO2 photocatalyst and the reactor configuration. Recently, highly ordered
titania nanotube arrays (TNAs) grown by the electrochemical anodisation of titanium foils
have attracted considerable attention due to their unique microstructures. [88,89] Bai et al.,
[90] developed a novel thin-layer PEC reactor with a double-faced TNA electrode and
successfully applied it for the degradation of tetracycline. The titania nanotubes are highly
ordered and well aligned (Figure 9), with diameters of ∼100 nm, wall thicknesses of ∼10 nm
and lengths of ∼420 nm. The efficiency of organic compound degradation by the new PEC
with TNA reactor was proven much higher than conventional PEC processes under similar
treatment conditions. With UV illumination of 5 mW cm-2, the PEC with TNA tetracycline
removal rate reached 54.8-96.4% when using 20–120 mg L−1 tetracycline in wastewater,
higher than that achieved by conventional PEC reactors (about 14.6-80.4%) within a 1 h
timeframe. Similar to pristine crystalline TNAs, cathodised dark TNAs also have proven
excellent photocatalytic performance. [91] The cathodised dark TNAs were obtained by rapid
cathodisation for 30 s, which resulted in significantly enhanced optical absorbance, covering

the full spectrum of visible light. The degradation of four PPCPs (phenol, ibuprofen,
carbamazepine and caffeine) showed that the dark crystalline TNAs have a 107-131%
enhancement in degradation kinetics for the targeted organic contaminants relative to the
pristine crystalline TNAs (annealed in air).
3.2. Filtration Processes
Filtration in water treatment usually describes some mechanical or biological processes

through which undesirable constituents are removed by physical hinderance or absorption
into a biological film grown on (or in) the filter medium.
3.2.1. Sand Filtration

Sand filtration is a typical treatment of secondary waste water effluent used to remove
suspended solids and persistent turbidity after clarification. It is possible to remove a portion
of dissolved PPCPs associated with the retained solids, but this contribution is relatively
small. Considering the removal efficiencies of 24 different PPCPs during sand filtration, the
hydrophobicity of these organic compounds is likely to be a dominant factor governing their
adherence to colloidal particles, and hence filterability. [92] This is consistent with the survey
conducted by Oulton et al., [93] where the highest removal efficiencies were obtained for
hydrophobic compounds which are hydrophobic enough to partition from water to sludge
phases, making these higher log Kow compounds more bioavailable to sludge
microorganisms, and hence more susceptible to degradation during conventional activated
sludge treatments with adequate hydraulic and solids retention times.
3.2.2. Membrane Filtration

Membrane filtration has been used for removal of pathogens, micropollutants and salts in
drinking water treatment over the past several decades, but in recent years, novel membrane
technologies have also attracted interest for removing PPCPs from wastewater. Membranes
can be divided into four classes according to their molecular weight cut-offs (MWCO):
microfiltration (MF), ultrafiltration (UF), nanofiltration (NF) and reverse osmosis (RO).
Micro- and ultrafiltration are usually installed in low hydraulic pressure contexts as pre-
treatment devices, while nanofiltration and reverse osmosis are often used with high pressures
to facilitate micropollutant removal. Based on physical dimensions of most PPCPs, only NF
and RO membranes would be considered suitable for PPCP removal based purely on size
exclusion mechanisms, and negating other factors.
Yoon et al., [94] investigated removal efficiencies for 52 PPCPs varying in physico-
chemical properties such as size, hydrophobicity, and polarity using NF and UF membranes
in a dead-end stirred-cell filtration system. Experiments were performed at environmentally
relevant initial PPCP concentrations typically ranging from 2 to <250 ng L-1. The results
demonstrated that the NF membrane retained many PPCPs due to both hydrophobic
adsorption and size exclusion, while the UF membrane typically retained only hydrophobic
PPCPs attributable to hydrophobic adsorption. But NF and RO were capable of significant
rejection of nearly all target compounds in both pilot- and full-scale investigation, although
some compounds were still detectable at trace levels in permeates. [95] Fig. 10 shows NF
devices employed in wastewater treatment processes within a chemical company in China.
The schematic mechanism of RO exclusion is shown in Fig. 11.
Figure 9. FE-SEM top-view and cross-section (inset) of titania nanotube arrays on front (a) and back of
electrode (b), XRD patterns of the TNAs (c). Reproduced with permission from Ref. [90].
Table 4. Physical properties of different filter membranes
Pore size Molecular Process Filtration Removal of

mass
>0.1 μm >5000 KDa Microfiltration <2 bar larger bacteria, particles
2-100 nm 5-5000 kDa Ultrafiltration 1-10 bar bacteria, proteins, larger
viruses
1-2 nm 0.1-5 kDa Nanofiltration 3-20 bar viruses, 2-valent ions
<1 nm <100 Da Reverse 10-80 bar salts, small organic
osmosis molecules
Figure 10. Nanofiltration devices in operation treating chemical plant wastewater.
Heavy
Suspended Organic
metal virus
solid bacteria compound
Untreated
Retentates
water
RO membrane
Pure
water
Fiure 11. Schematic mechanism of reverse osmosis filtration.
However, a byproduct of RO filtration is retentate which can contain high concentrations

of contaminants, which may require further treatment prior to environmental disposal, with 18
of 27 pharmaceuticals identified in some retentates at μg L-1 concentrations. [96] Hydroxyl
radical removal of PPCPs from RO retentate approached 80 to 100% as measured by
excitation-emission matrix (EEM) fluorescence spectroscopy, while there was a 40 to 50%
reduction of UV and visible humic-like peaks associated with OH• oxidation of the effluent
organic matter (EfOM) as monitored by decreases in retentate fluorescence. Removal and
disposal of concentrated brine produced during RO processes is also problematic, as is the
associated energy consumption. Economic analysis suggests the use of membrane filtration
technologies for water treatment may be ultimately both economically and ecologically
undesirable due to these high energy footprints. [97]
3.3. Adsorption Processes
Adsorption is a surface-based process in which atoms, ions or molecules from a gas,

liquid or dissolved solid adhere to a surface. In water treatment, activated carbon is the most
widely used adsorbant of organic substances and non-polar adsorbates due to its large
micropore volume and the resulting high surface area.
3.3.1. Activated Carbon Adsorption

Activated carbon can be made from carbonaceous source materials such as coal, peat,
wood or nutshells that are pyrolyzed at temperatures ranging from 600-900℃ in absence of
oxygen. Powder activated carbon (PAC) and granular activated carbon (GAC) are the most
widely used adsorbents for removing organic micropollutants in drinking water, and growing
evidence supports their use for removing PPCPs from water. [98] In activated carbon
adsorption processes, the octanol-water partitioning coefficient (log Kow values) are strongly
correlated with PPCP removal, [99] but characteristics of activated carbon and solute,
including pore volume distribution, surface area and slurry pH, are also believed to influence
sorption. As demonstrated in Table 6, the addition of 5 mg/L of PAC with a 4 h contact time
resulted in high removal rates of between 75% to >98% for PPCPs with log Kow values higher
than 3. However, PPCP compounds with low log Kow or deprotonated acidic functional
groups exhibited lower rates of removal with PAC under identical conditions. For those
higher log Kow compounds such as fluoxetine which were efficiently (>90%) removed at low
PAC dosage, adding more PAC did not significantly enhance removal, but for other
compounds like diclofenac, over the range of PAC dosages commonly used in water
treatment plants (WTPs), increasing PAC dosage may help improve their removals (Table 5).
Table 5. Percentage PPCP and waterborne contaminant removal as a function of PAC

dose. Adapted from Ref. [99]
PPCP compounds 1 mg/L 5 mg/L 20 mg/L

Diclofenac 0 44±11 92±1
Ibuprofen 2±3 35±15 80±1
Meprobamate 8±4 44±4 94±1
DEET 21±6 73±4 98±1
Oxybenzone 75±1 92±1 95±1
Fluoxetine 91±3 96±1 96±1
Similar to PAC, GAC are also proved efficacious in removing most PPCP compounds
evaluated by greater than 90%; [100] however, as with PAC, water soluble contaminants can
still pass through GAC much more readily than strongly hydrophobic contaminants. Further,
full-scale GAC applications, in drinking water facilities with or without regular regeneration,
and varying in total organic carbon (TOC) resulted in vastly different outcomes. A facility
with on-site and regular regeneration and low total organic carbon (TOC) demonstrated good
removal of trace organics from source water while a second facility with relatively high TOC
reported very little removal (Table 6). Therefore for water treatment, pretreatment of higher
TOC source waters and the rate of activate carbon regeneration (or replacement) are key
design criteria.
Table 6. Results of full-scale GAC testing at different facilities.

Adapted from Ref. [100]
Facility 1 with regular Facility 2 without regular

regeneration regeneration
Waterborne
GAC INF GAC EFF GAC INF GAC EFF
Contaminant
Caffeine 17 <10.0 42,650 35,700
Carbamazepine 2.2 <1.0 199 168
DEET 1.8 <1.0 701 256
Dilantin 1.8 <1.0 110 85
Erythromycin 1.8 <1.0 139 128
Gemfibrozil 1.2 <1.0 3710 3405
Ibuprofen 1.1 <1.0 8760 7325
Iopromide 3.3 <1.0 5 1.4
Meprobamate 1.2 <1.0 226 196
Sulfamethoxazole 6 <1.0 426 69
3.4. Biological Processes
As MWWTPs were (and are) not designed for removal of PPCPs, the incomplete removal
of many of these organic compounds results in their continuous infusion to receiving
environments, with MWWTPs considered to be the major point sources by which PPCPs
enter surface waters. [101] Nevertheless, biological treatment processes within MWWTPs
make a substantial contribution towards PPCPs removal.
3.4.1. Conventional Activated Sludge (CAS)

Conventional activated sludge is the most commonly employed biological wastewater

treatment system, with PPCP removal during CAS treatment processes attributable both to
biodegradation and adsorption to the sludge. For acidic PPCPs, biodegradation is the
mechanism chiefly responsible for removal, while fragrances and higher log Kow
pharmaceuticals are predominantly removed from the overlying aqueous phase by
partitioning into sludge. [102] Zhou et al., [103] identified bacteria with bioaugmentation
characteristics to enhance PPCP removal, with eight of eleven bacteria isolated capable of
removing triclosan, bisphenol A, ibuprofen, or 17 β-estradiol to below 10 ng/L, indicating
these isolated bacteria may enhance PPCP removal from MWWTPs if present at sufficient
concentrations. However, carbamazepine, diclofenac, iopromide, metoprolol, sotalol and
other PPCPs are recalcitrant to CAS, passing largely unabated through this treatment with no
more than 30% removal via solids and CAS from conventional treatment facilities. [104]
3.4.2. Constructed Wetlands (CWs)

With considerable advantages of low-cost and simple maintenance, environmentally
friendly constructed wetlands employing aquatic plants have long been used for treatment of
all kinds of wastewater. [105] Recently, the potential of CWs for PPCP removal or final
polishing has been increasingly explored. [106] Although CWs appear to have a promising
future as an alternative for secondary wastewater treatment systems or as supplementary
treatment units for polishing secondary effluent from WWTPs, the mechanisms involved are
largely unknown. Specifically, only influent and effluent concentrations can be routinely
measured to evaluate wetland performance, precluding in many cases detailed studies on the
actual fate of the compounds or their removal pathways. A general pharmaceutical removal
mechanism for CWs is illustrated in Fig. 12, including sorption and sedimentation,
photodegradation, plant uptake and accumulation, as well as microbial degradation. Hijosa-
Valsero et al., [107] assessed the PPCP removal abilities of three different full-scale hybrid
pond-constructed wetlands and a conventional WWTP. The PPCPs monitored as they passed
through the wetlands and the WWTP represented a cross-section of commonly detected
environmental contaminants including analgesic–anti-inflammatory drugs (i.e. ketoprofen,
naproxen, ibuprofen, diclofenac and salicylic acid), anti-epileptic drugs (carbamazepine),
lipid regulators (clofibric acid), diuretic drugs (furosemide), stimulant drugs (caffeine) and
fragrances (methyl dihydrojasmonate). Results demonstrated that the hybrid systems were at
least as efficient in PPCP removal as the WWTP, achieving removal efficiencies exceeding
70%. Other investigations using seven mesocosm-scale CWs of different configurations
proved that soil-less CWs consistently demonstrated the highest removal efficiencies for
ketoprofen, ibuprofen and carbamazepine, while free-water CWs with effluent draining
through the bottom of the tank performed well for the degradation of ketoprofen, salicylic
acid, galaxolide and tonalide. [108] Finally, subsurface horizontal flow CWs behaved
efficiently for the removal of caffeine.
Figure 12. Pharmaceutical removal mechanisms in constructed wetlands. Adapted from Ref. [108].
3.4.3. Membrane Bioreactors (MBRs)

Relative to CAS, membrane bioreactors enjoy several advantages in removal of PPCP
compounds. Similar to CAS, biodegradation is the dominant removal mechanism of MBRs,
but MBRs operate with higher sludge concentrations, leading to greater biological activities
compared to CAS. Relative evaluations of PPCP removal by MBR relative to CAS using
municipal wastewaters often reported enhanced removal with MBRs [109,110] while other
studies found removal rates comparable to CAS. [111,112] A recent survey conducted by Sui
et al., [113] comparing removal of 12 PPCPs by CAS and MBR highlighted the notable
difference that for the easily biodegradable PPCPs, MBR performance was more stable than
CAS, especially during winter months. During colder periods, diclofenac, trimethoprim,
metoprolol, and gemfibrozil were removed with moderate effectiveness by MBR, while their
removal by CAS was much lower, or even negligible. However, recently Fernandez-Fontaina
et al., [114] noted the jury is still out on the relative performance advantages of MBR, as both
biodegradation and sorption coefficients for MBR were in the same range as those in CAS
systems, suggesting that biological degradation and/or sorption of PPCPs were roughly
comparable with MBR and CAS reactors.
CONCLUSION AND OUTLOOK

The occurrence and fate of pharmaceuticals and personal care products in aquatic
environments have generated widespread public concern during the past few decades.
Pharmaceuticals are designed to exert specific physiological effects on human and animals at
relatively low therapeutic doses, are usually resistant to biological degradation, and as
receptors are often conserved across phyla, these environmental drug residues can lead to
adverse changes in biota inhabiting MWWTP receiving environments and concern among
human populations drawing their drinking water from these surface waters. Although usually
detected only at vanishingly small (ng/L to μg/L) concentrations in surface waters near large
urban areas or intensive agricultural operations, these waterborne drugs, and some personal
care products, are nonetheless quite bioactive and can be readily detected by modern
analytical techniques. Despite the public and research attention, studies demonstrating
toxicological effects of PPCPs at environmentally relevant concentrations, let alone those of
their metabolites and degradation products, remain limited. Current experimental data
demonstrates PPCP removal, while limited with conventional treatment processes, can be
enhanced with recent treatment processes such as advanced oxidation or the use of membrane
technologies such as RO and NF. While effective at removing PPCPs, such advanced
treatment processes may not be economically feasible due to high treatment cost per unit
volume, and further studies on inexpensive methods to remove PPCPs compounds are still
warranted.
ACKNOWLEDGMENT
The authors are grateful for financial support from a Strategic Research Grant
(KZ40005001) from the Education Commission, Beijing Municipal Government; the Oversea
Talent Project of Beijing, and a special grant for the Advanced Laser Processing and
Development Project of Beijing. We appreciate the research initiative funding provided by
the University of Tennessee to A.H. as a component of a new hire package.
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Chapter 5
APPLICATION AND ENVIRONMENTAL COMPLIANCE

OF ANAEROBIC SEQUENCING BATCH REACTORS
APPLIED TO HYDROGEN /METHANE BIOENERGY
PRODUCTION
Giovanna Lovato1, Roberta Albanez1,

Daniel Moureira Fontes Lima1,
Irina Soraya Moncayo Bravo1,
Weriskiney Araújo Almeida1, Suzana Maria Ratuznei2
and José Alberto Domingues Rodrigues2*
1
Departamento de Hidráulica e Saneamento, Escola de Engenharia de São Carlos,
Universidade de São Paulo (USP), São Carlos-SP, Brasil
2
Departamento de Engenharia Química e de Alimentos, Escola de Engenharia Mauá,
Instituto Mauá de Tecnologia (IMT),
São Caetano do Sul-SP, Brasil
DRAFT ABSTRACT
Energy production from renewable resources and from processes with low energy
demand has an important role in the creation of a more sustainable world. The
development of anaerobic processes for wastewater treatment focused on biogas
production with the potential for energy production has shown great progress in the last
few years due to new projects involving reactors that are more efficient and more stable
and that have lower costs of maintenance and operation.
One of these reactors is the anaerobic sequencing batch and fed-batch reactor whose
typical cycle comprises four steps: (i) feeding in which fill time may vary, defining the
feed strategy as batch and/or fed-batch; (ii) the treatment itself, by biotransformation of
the wastewater constituents by microorganisms; (iii) sedimentation, when the biomass is
in granular form (ASBR) because when the biomass is immobilized on an inert support
*
To whom all correspondence should be addressed: E-mail rodrigues@maua.br.
112 G. Lovato, R. Albanez, D. M. Fontes Lima et al.
(AnSBBR) this step is not required; and (iv) discharge, i.e., removal of treated and
clarified liquid. The major advantages of this type of operation are its simplicity, its good
effluent quality control and the possibility of applying this type of treatment to a wide
range of wastewaters.
It can be verified in the literature that there has been an increase in the potential
application of these reactors to bioenergy generation. This application occurs in a wide
range of wastewaters that have been defined as a raw material and not as the residue of a
process. This retains the aim of viably producing energy from hydrogen and methane
while assuring the proper environmental disposal of all residues. Although such studies
have many objectives, they have one in common, which is the production of biomethane
and biohydrogen to generate energy.
New advancements in understanding the anaerobic sequencing batch and/or fed-
batch reactor may develop even more options for its use and identify limits and
drawbacks. Within this context, the aim of this review was to describe and discuss the
factors influencing biomethane and biohydrogen production from several wastewaters in
ASBR and AnSBBR reactors. The review was focused on the following variables:
1. Inoculum type and pre-treatment;

2. Biomass arrangement (free or immobilized cells);
3. Organic load;
4. Operation mode;
5. Cycle time;
6. Feeding time;
7. Temperature;
8. pH;
9. Substrates (wastewaters); and
10. Kinetic studies.
A review of the processes of the biological production of biohydrogen and

biomethane will also be presented in this chapter, along with the theoretical foundations
of performance indices applied to bioenergy production which appropriately allow the
assessment of the efficiency, production, productivity and yield of the process.
ABSTRACT
Energy production from renewable resources and from processes with low energy
demand has an important role in the creation of a more sustainable world. The
development of anaerobic processes for wastewater treatment focused on biogas
production with the potential for energy production has shown great progress in the last
few years due to new projects involving reactors that are more efficient and more stable
and that have lower costs of maintenance and operation. Considering this background, the
aim of this review was to describe and discuss the factors that influence biomethane and
biohydrogen production from several wastewaters in ASBR and AnSBBR reactors. The
review was focused on the following variables: inoculum type and pre-treatment, biomass
arrangement (free or immobilized cells), organic load, operation mode, cycle time,
feeding time, temperature, pH, substrates (wastewaters) and kinetic studies.
Keywords: ASBR, AnSBBR, biomethane, biohydrogen, bioenergy, environmental

compliance
Application and Environmental Compliance ... 113
1. INTRODUCTION
In recent decades, two research fields in wastewater treatment have converged:
purification of wastewaters and their reuse as a renewable source of energy and products.
Both fields lead to the common objective of economic benefit by means of a rational water
use and production of products and energy from a clean and renewable source (Leite et al.,
2008).
The development of anaerobic processes for wastewater treatment has shown great
progress in recent years due to new projects involving reactors that are more efficient and
more stable and that have fewer maintenance and operation costs. One of these reactors is the
anaerobic reactor operated in batch or fed-batch modes, whose typical cycle comprises four
steps: (i) feeding with different feeding times, which defines the feeding strategy as batch or
fed-batch; (ii) the treatment itself or reaction time, in which the wastewater is converted to
biogas by microorganisms; (iii) settling if the biomass is in granules (ASBR), but when the
biomass is immobilized in inert support (AnSBBR), this step is not necessary; and (iv)
discharge of the treated and clarified effluent (Dague et al., 1992; Fernandes et al., 1993;
Zaiat et al., 2001). The major advantages of this type of operation are its simplicity, the
efficient effluent quality control and the possibility of application to a large variety of
wastewaters.
Currently, reactors using biomass in granules (ASBR) have been applied to the treatment
of several types of wastewaters, such as effluents from the food industry, landfill leachate,
and piggery and chemical wastewaters (Selma et al., 2010; Silva et al., 2012; Carvalhinha et
al., 2010; Mockaitis et al., 2006; Chebel et al., 2006 and Bezerra et al., 2005). Cases in which
a reactor with immobilized biomass (AnSBBR) is used include the treatment of wastewaters
from synthetic fiber production, cheese whey, the textile industries and the automotive and
biodiesel industries, (Oliveira et al., 2009; Bezerra et al., 2011; Lovato et al., 2012; Lovato et
al., 2013; Rodrigues et al., 2011; Bezerra et al., 2009; Bezerra et al., 2007; Oliveira et al.,
2008; Lullio et al., 2014 and Albanez et al., 2014).
It can be verified in the literature that there has been an increase in the potential
application of these reactors to bioenergy generation. This application occurs in a wide range
of wastewaters that have been defined as a raw material and not as the residue of a process.
This retains the aim of viably producing energy from hydrogen and methane while assuring
the proper environmental disposal of all residues. Although such studies have many
objectives, they have one in common, which is the production of biomethane and
biohydrogen to generate energy. Within this context, the aim of this review was to describe
and discuss the factors influencing biomethane and biohydrogen production from several
wastewaters in ASBR and AnSBBR reactors, which are inoculum type and pre-treatment,
biomass arrangement, organic load, operation mode, cycle time, feeding time, temperature,
pH, substrates (wastewaters) and co-substrates and kinetic studies.
1.1. Biological Production of Methane
Methane production by anaerobic digestion and from a large variety of biologic residues
is growing globally, and it is considered ideal in many cases because of its environmental and
economic benefits. Moreover, the crucial benefits achieved by changing the use of natural gas
to biogas are as follows: (i) biogas is produced from renewable sources; (ii) biogas is locally
produced and does not depend on imported oil or natural gas stocks; (iii) it reduces the
amount of pollution by organic residues, which are responsible for the greatest part of the
pollution of natural waters; and (iv) it helps to retard the problems associated with the
management of residues (Chandra et al., 2012). Moreover, the amount of energy in
methane is 55.6 kJ.g-1, which is 17% greater than the amount of energy in gasoline
(47.5 kJ.g-1).
The methanogenic process of wastewater treatment based on carbon includes the
following microbiologic activities: hydrolysis, acidogenesis, acetogenesis, methanogenesis
and cell lysis. The first and last of these activities are considered non-growing processes,
while the others are considered growing processes. These processes can be divided into sub-
processes according to the system and components of interest.
As microorganisms are not capable of assimilating particles of organic matter, the first
step of anaerobic digestion is the hydrolysis of these complex particles into simpler dissolved
materials, which can cross the cell wall of fermentative bacteria. This conversion of the
particles of organic matter to dissolved material is achieved by the action of exoenzymes
excreted by hydrolytic bacteria. Under anaerobic conditions, it is normal for the hydrolysis of
polymers to occur slowly. Different factors can affect the level and speed of substrate
hydrolysis: temperature, residence time, substrate composition, size of the particles, medium
pH and hydrolysis product concentration (volatile acids, etc.).
In acidogenesis, many groups of microorganisms are used to convert sugars into acids
with or without release of hydrogen and production of lactic, butyric, propionic and acetic
acids. In acetogenesis, a group of acetogenic microorganisms which can produce hydrogen
convert volatile acids (butyric and propionic acids) into acetate, hydrogen and carbon dioxide.
This is a thermodynamically unfavorable process unless the partial pressure of hydrogen is
maintained at less than 10-3 atm. This pressure is normally conserved by the hydrogen
consumer microorganisms as it is in the case of hydrogenotrophic methanogenic Archaea and

of homoacetogenic Archaea. In methanogenesis, acetoclastic and hydrogenotrophic
methanogenic microorganisms are responsible for the conversion of acids to carbon dioxide
and methane by reducing carbon dioxide with hydrogen to form methane.
Methanogenic microorganisms are divided into two main groups according to their
substrate affinity and their magnitude of methane production. One group produces methane
from acetic acid or methanol; these are the acetoclastic methanogenic. The other group
produces methane from hydrogen and carbon dioxide, i.e., the hydrogenotrophic methanogens
(Rittman and McCarty, 2001; Kumar, 2008; Chernicharo, 1997).
Equation (1) presents the reaction of the oxidative process of the process of acetotrophic
methanogenesis, which is performed by acetoclastic methanogens. Equation (2) presents the
reaction of the hydrogenotrophic methanogenic organisms.
CH 3 COOH 
 CH 4  CO 2 (1)
4 H 2  CO 2 
 CH 4  2 H 2 O (2)
The acetoclastic methanogens and the hydrogenotrophic organisms have great

importance in the maintenance of anaerobic digestion because they are responsible for the
essential function of consuming the hydrogen produced in the latter steps. This provides a
reduction in the partial pressure of hydrogen.
The last step in the process is cell lysis, a process by which live cells are converted into
non-living particles and soluble material (Rodrigues et al., 2004).
Figure 1 illustrates the process of conversion of COD and their respective percentages of
conversion in each step of the volatile part of biodegradable residues degradation.
Figure 1.Conversion of the COD available in biodegradable residues (Adapted from Chandra et al.,
2012).
1.2. Biological Production of Hydrogen
Nearly 80% of the global energy demand is from fossil fuels (oil, natural gas and coal).
The oxides produced, such as COx, NOx and SOx, are precursors of the greenhouse effect,
damage the ozone layer, and produce acid rain and pollution. Recently, struggles have been
focused on developing clean energy alternatives. Among all of the potential candidates,
hydrogen (H2) is being highlighted as a promising energy source for the future because of its
high amount of energy per mass (141.9 kJ.g-1) and, more importantly, because only water is
produced when hydrogen reacts with oxygen in fuel cells. Following this line of reasoning,
changing the use of fossil fuels by using H2 seems to be quite a good option to sustain the
growing global economy and, at the same time, mitigate global climate problems (Leite et al.,
2008 and Wu et al., 2013).
Biological hydrogen production is an interesting way to produce this gas because this
process is lower in cost compared to other technologies and requires less energy for its
generation. It can occur by either the photosynthetic or fermentative process. Fermentation is
technically simpler, and the hydrogen can be produced from the organic load in wastewaters.
Many studies have demonstrated that the use of biologically produced H2 for energy
generation is viable (Wu et al., 2009; Alzate-Gaviria et al., 2007; Leite et al., 2008).
According to Tanisho (2001) and Lee et al. (2011) molecular hydrogen can be formed by
two reaction pathways, i.e., formic acid decomposition (Equation 3) or the reoxidation of
nicotinamide adenine dinucleotide (NADH to NAD+) (Equations 5 and 6).
(3)
This first pathway is closely related to the fermentation via acetate because pyruvate is
transformed into acetate and formic acid (Equation 4).
(4)
Pyruvate Acetic Acid Formic Acid
In the second case, hydrogen is formed by reoxidation of NADH, which, according to

Tanisho (2001), occurs during fermentation in both the acetone-butanol pathway and the
butyrate pathway.
(5)
→ (6)
The hydrogen concentration produced depends directly on the metabolic pathway

followed by the fermentative bacteria (Equations 7 and 8) (Das and Veziroglu, 2001; Lee et
al., 2011).
Figure 2. Metabolic pathway during the fermentation of several substrates (Adapted from Sá et al.,
2013 and Silva et al., 2009). [Notation: 1: Sucrose 6-P hydrolase; 2: Hexokinase; 3: phosphoglucose
isomerase; 4: Phosphofructokinase; 5: Aldolase; 6: Glyceraldehyde-3-P-dehydrogenase; 7:
Phosphoglycerokinase; 8: Enolase; 9: Pyruvate kinase; 10: Pyruvate-ferredoxin oxidoreductase;
11: Hydrogenase; 12: NAD(P)H-ferredoxin reductase; 13: Ferredoxin-NAP(P)+ reductase; 14:
Phosphotransacetylase kinase; 15: Acetate kinase; 16: Phosphotransbutylase kinase; 17: Butyrate
kinase; 18: Glycerol dehydrogenase; 19: DNA kinase; 20: Glycerol dehydratase; 21: 1,3-
propanediol dehydrogenase; 22: Glycerol kinase; 23: Glycerol-3-P-deydrogenase; 24: Xylose
isomerase; 25: Xylulokinase]
(7)
Glucose Acetic Acid
(8)
Glucose Butyric Acid
The ideal situation would be that the process always follows the acetate pathway,
guaranteeing maximum production. However this is not possible due to the equilibrium
between the concentrations of NADH and NAD+ that must be maintained. One mole of
acetate is produced by the consumption of two moles of NAD+. One mole of butyrate does
not consume or produce NAD+. One mole of ethanol or one mole of propionate produces two
moles of NAD+ (Ren et al., 2006).
To maintain the dynamic equilibrium between oxidation and reduction of NAD+/NADH,
NADH must be consumed, producing propionate, lactate, ethanol, and butanol. This
consequently reduces the yield of H2 production (Lee et al., 2011; Ren et al., 2006). Thus, the
only way to influence the metabolism in order to favor the desired pathway is by the
operational strategy for the reactor.
Sá et al. (2013) studied the metabolic pathways of microorganisms of the genus
Clostridium in the conversion of glycerol, glucose, fructose and xylose for hydrogen
production. Figure 2 illustrates the processes. It is interesting to note that in the metabolic
pathway followed from sucrose to glyceraldehyde-3-phosphate, some reactions only release
energy, while in the path from glycerol to the same compound, the reaction of NADH to
NAD+ occurs (which consumes energy). Therefore, it is a thermodynamically unfavorable
process and is more difficult to perform.
1.3. ASBR and AnSBBR Reactors

Figures 3 and 4 show typical ASBR and AnSBBR reactors setups (Manssouri et al.,
2013; Lovato et al., 2012). To improve the biological treatment system, there are many
operational control strategies in the reactors. The following sections are dedicated to
describing the variables that play important roles in controlling the studied reactors.
2. THEORETICAL FOUNDATIONS OF THE PERFORMANCE INDICES

APPLIED TO BIOENERGY PRODUCTION
An important factor regarding the production of bioenergy from organic biodegradable
residues is how to appropriately assess the efficiency, production, productivity and yield of
each process.
First, it is necessary to define AVOL, ASOL, RVOL and RSOL, which are terms related
to the organic load and largely studied in systems for the anaerobic biological production of
biogas and wastewater treatment.
Figure 3. ASBR / AnSBBR with mechanical stirring by an impeller (MS). (Adapted from Manssouri et
al., 2013) [Notation: 1 – Reactor; 2 – Basket containing inert support to biomass (only in case of an
AnSBBR; for an ASBR, this basket is not used); 3 – Affluent; 4 – Feeding pump; 5 – Discharge
pump; 6 – Effluent; 7 – Biogas outlet; 8 – Stirring system; 9 – Temperature control (water bath
thermostatically controlled); 10 – Automation system].
Figure 4. ASBR / AnSBBR with liquid phase recirculation mixture (LPR). (Adapted from Lovato et al.,
2012) [Notation: 1 – Reactor containing immobilized or granular biomass; 2 – Side reservoir; 3 –
Flow meter; 4 – Recycle pump; 5 –Discharge pumps; 6 – Feeding pumps; 7 – Affluent; 8 –
Automation system; 9 – Effluent outlet; 10 – Biogas outlet].
.
The applied volumetric organic load (AVOL) is defined as the amount of organic matter
applied to the reactor per unit time and volume of medium (in gCOD.L-1.d-1). For reactors
operated in batch mode, it is calculated by Equation (9), in which VA is the volume of
wastewater fed to reactor per cycle, N is the number of cycles per day, CSAFL is the organic
matter concentration in the effluent in non-filtered samples and VR is the total volume of
wastewater in the reactor per cycle.
( )
(9)
The specific applied organic load (ASOL) is defined as the amount of organic matter
applied to the reactor per unit time and mass of total volatile solids (MTVS) in the reactor
(gCOD.gTVS-1.d-1). For reactors operated in batch mode, it is calculated by Equation (10).
( )
(10)
The removed volumetric organic load (RVOL) for filtered samples is defined as the
amount of organic matter removed by the reactor per time unit and volume of medium in the
reactor (gCOD.L-1d-1). For reactors operated in batch mode, it is calculated by Equation (11),
where CSF is the organic matter concentration in the filtered effluent.
( )( )
(11)
The specific removed organic load (RSOL) for filtered samples is defined as the amount
of organic matter removed per unit time and mass of total volatile solids in the reactor
(gCOD.gTVS-1.d-1). For reactors operated in batch mode, it is calculated by Equation (12).
( )( )
(12)
The efficiencies of organic matter removal in the form of COD for filtered samples (εSF)
and non-filtered samples (εST) can be calculated by Equations (13) and (14), where CST is the
organic matter concentration in non-filtered samples of effluent.
ε ( ) (13)
ε ( ) (14)
The efficiency of organic matter removal in filtered samples from the liquid medium in
the reactor (εS) for cycle-length profiles can be calculated by Equations (15) and (16), the
former being used for batch reactors and the latter for fed-batch reactors, where CS0 is the
organic matter concentration in filtered samples from the liquid medium in the reactor at time
zero, VRes is the volume of the liquid medium in the reactor at time zero or residual volume,
FAFL is the feeding flow of affluent in the fed-batch period, ti is the feeding time (fed-batch
period), CSF is the organic matter concentration in form of COD in filtered samples from the
liquid medium in the reactor on time ti, and tBA is the time of fed-batch in the operating
conditions.
( ) ( ( ))
ε ( ) ( )
(15)
( ) ( ( ))
ε ( ) ( )
(16)
The molar volumetric productivity (MPr) is defined by the amount of biogas (H2 or CH4)
produced per time unit and usable reactor volume (molH2.m-3.d-1 or molCH4.m-3.d-1), which is
calculated by Equation (17), where n is the molar amount of biogas produced daily.
(17)
The specific molar productivity (SMPr) is defined as the amount of biogas (H2 or CH4)
produced per unit time and the mass of total volatile solids in the reactor (molH2.kgTVS-1.d-1
or molCH4.kgTVS-1.d-1), which is calculated by Equation (18). Productivity is a particularly
interesting parameter in dimensioning treatment units, permitting the assessment of the
capacity of production of a reactor from the volume (or mass of microorganisms) or, in
contrast, estimating the volume (or mass of microorganisms) necessary to reach a desired
production.
(18)
The molar yield of biogas produced per applied load (MYAL) is defined as the amount of
biogas (H2 or CH4) produced (in moles) by the amount of organic matter applied (in mass)
(molH2.kgCOD-1 or molCH4.kgCOD-1), which is calculated by Equation (19).
(19)
The molar yield of biogas produced per removed load (MYRL) is defined as the amount
of biogas (H2 or CH4) produced (in moles) by the amount of organic matter removed (in
mass) (molH2.kgCOD-1 or molCH4.kgCOD-1), which is calculated by Equation (20).
( )
(20)
The molar yield of biogas produced per substrate removed (YS) is defined as the moles of
biogas (H2 or CH4) produced per mole of substrate removed (molH2.molsubstrate-1 or mol
CH4.molsubstrate-1), which is calculated by Equation (21), where CSubsAFL is the molar
amount of substrate in the effluent and CSubsF is the molar amount of substrate in the filtered
effluent.
(21)
( )
Additionally, the volumetric yield of biogas (H2 or CH4) produced (VB) and organic
matter removed (NmL H2.gCOD-1 or NmL CH4.gCOD-1) is shown in Equation (22):
( )
(22)
3. FACTORS INFLUENCING THE PRODUCTION OF BIOHYDROGEN

AND BIOMETHANE
3.1. Influence of the Type and Pretreatment of the Inoculum
Anaerobic digestion is a complex process that involves many types of microorganism and
intermediate phases, as mentioned above.
The methanogenic microorganisms, which were previously classified as bacteria, are
currently classified as archaea. Archaea is a unique group of microorganisms that differ from
'true' bacteria by the presence of lipidic membranes, the absence of basic cell characteristics
(peptidoglycan, for example) and the presence of distinct ribosomal RNA. Methanogenic
archaea are obligatorily anaerobic and are considered the limiting species of the anaerobic
wastewater treatment process. Large numbers of methanogenic microorganisms are found in
the rich organic matter in anaerobic environments such as marshes, swamps, ponds, lakes and
marine sediments, as well as the bovine rumen (Boone et al., 1993), and do not need any type
of pretreatment.
Methane production from the anaerobic digestion of effluents and waste (including
sewage sludge, manure and the organic fraction of municipal waste) is a process that is
already widely applied. In this case, as previously mentioned, hydrogen is an intermediate
product that does not remain available due to its rapid uptake and conversion to methane by
methanogenic microorganisms. In biological processes for hydrogen production, the
production and consumption of H2 are disconnected, so the biohydrogen is available as a final
product. These processes are still being researched and developed. Thus several investigations
are still needed before the use of this technology on a commercial scale, as was the case with
the production of methane (Khanal, 2008).
Several microorganisms can produce hydrogen, provided that they possess the enzyme
hydrogenase or nitrogenase, highlighting the Clostridium genus; Escherichia coli;
Termonaerobacter and Enterobacter. According to Maintinguer (2008), the main
fermentative bacteria known for the production of hydrogen include species of Enterobacter,
Bacillus and Clostridium. There are also molds which also possess hydrogenase enzymes and
are thus able to produce hydrogen, but they are rarely used for this purpose (Boxma et al.
2005).
Microorganisms belonging to the genus Clostridium are the most important due to their
high potential for hydrogen production by its rapid metabolism and its ability to form spores.
According to Hawkes et al. (2002), the yield of hydrogen from species of Clostridium is
generally greater than that obtained by facultative aerobic bacteria such as Enterobacter sp. In
their studies, Lay (2001) determined that the genus Clostridium is the predominant producer
of hydrogen. In anaerobic processes, hydrogen is produced during the exponential phase of
growth of Clostridium, with production of solvents in the stationary phase. This happens
when the pH drops below 4.5. Apparently, the accumulation of volatile fatty acids and
hydrogen in the exponential growth phase causes this change of route. The genus Clostridium
can produce hydrogen from the degradation of proteins, amino acids or carbohydrates.
Clostridium acetobutyricum has attracted great interest because of its ability to ferment
various substrates into valuable end-products such as acetone, butanol and ethanol (Mathews
and Wang, 2009).
The fermentation processes utilizing microbial consortia have advantages over those
operating with pure cultures because the first operation is simpler, the process control is
easier and a great variety of substrates can be used, unlike the pure cultures, which have a
narrow range of substrate choices. However, in a fermentation process with microbial
consortium, the hydrogen produced by microorganisms can also be consumed by micro-
organic consumers of hydrogen, which can reduce the rate at which hydrogen can be obtained
(Hung et al., 2011).
Some techniques or treatments can be applied before or during the process of producing
hydrogen to select a particular bacterial population. These include heat shock, acids, bases,
chemicals and successive aeration (Gioannis et al., 2013.). Several studies highlight the need
for the inoculum to be pretreated to eliminate methanogens and thus optimize the hydrogen
production by the elimination of consumer species, but one should be careful with the
economic aspect of the treatment used.
Papers that reported investigations of the influence of the pretreatment of the inoculum
for biohydrogen production are focused mostly on works that used serum bottles in simple
batch mode. Sá et al. (2013) conducted a study on the influence of the treatment of the
inoculum from a plant treating domestic wastewater to produce hydrogen. The pre-treatments
studied were heat, acid and alkaline shock; the best treatment considering the yield between
the hydrogen produced and the sucrose consumed was the heat shock (100 °C for 60
minutes).
Danko et al. (2008) studied two inocula (a suspended sludge and a granular sludge) and
two types of pretreatment (2-bromoethanesulfonic acid and autoclave) and observed that
pretreatment with 2-bromoethanesulfonic acid showed the best results for the hydrogen
production, reaching in some cases three times the volumetric production observed with the
thermal pretreatment process.
Wan and Wang (2008) evaluated the inoculum pretreatment for five methods (acid, base,
heat shock, aeration and chloroform), with tests conducted on batches in glass vials and fed
with glucose. The thermal shock pretreatment achieved the best results for hydrogen
production.
Some recent methods have been gaining ground, as in the work of Yin et al. (2014a) in
which the authors tested different types of pre-treatment, including ionizing radiation, heat
shock, and treatment with acid and base, comparing them based on their applicability to the
enhancement of a microbial community that produces hydrogen using glucose as substrate.
The experimental results showed that the inoculum pretreated with ionizing radiation reached
the optimum production of hydrogen among the various pretreatment methods tested. The
same authors (Yin et al. 2014b) used gamma radiation as a pretreatment method for enriching
the bacterial community that produces hydrogen in batch flasks fed with glucose. They
obtained results superior to those obtained using traditional methods of pretreatment, such as
heat shock, acid, base, aeration and chloroform. Using apple pomace as a substrate, Wang et
al. (2010) conducted a series of experiments in batch flasks to investigate the effect of
pretreatment by UV radiation and ultrasound in the production of bio-hydrogen, achieving the
best results when using ultraviolet radiation for 15 minutes.
Only a few studies have investigated the effect of the pretreatment of the sludge using a
reactor configuration. By operating a CSTR fed with a synthetic medium based on glucose,
Lee et al. (2009) tested three strong acids (HCl, H2SO4 and HNO3) as pretreatment for the
inoculum for hydrogen production, achieving the best results using hydrochloric acid.
Penteado et al. (2013) investigated the influence of different inocula (anaerobic sludge
originating from a UASB used for treating effluent from a poultry slaughterhouse, anaerobic
sludge originating from a UASB used in the treatment of swine effluent and self-fermented
synthetic medium) and different methods of pretreating them (thermal shock and acid
treatment) to produce hydrogen in an upflow anaerobic fixed bed reactor fed with sucrose; the
first treatment achieved the highest hydrogen yield, while the second achieved a more stable
production of H2. Lin et al. (2013) applied digesters on a small scale fed with waste paper and
food residues to evaluate the influence of acid pretreatment (H2SO4) and base (NaOH) in the
two-stage production of hydrogen followed by methane. The results showed that both
treatments showed a 50% increase in H2 production compared with the process conducted
without pretreatment. Methane production, however, did not increase.
No study regarding the influence of the inoculum and its pretreatment was accomplished
in sequencing batch reactors (ASBR or AnSBBR). Only the paper of Venkata Mohan et al.
(2007a) studied the benefits of the pretreatment of the inoculum for hydrogen production in
an AnSBBR, but without comparing different types of pretreatment. The authors applied heat
shock in combination with acid treatment to eliminate non-spore forming microorganisms and
to inhibit the growth of methanogens in a process they called "bioaugmentation" with the
specific hydrogen production after applying this method.
It is possible to observe that there is no type of definitive pretreatment for biohydrogen

production. The various types of substrates used and the various configurations of reactors
operated show that each case requires a directed study.
3.2. Biomass Arrangement
The first configuration of the anaerobic sequencing batch reactor (ASBR) had its biomass
in granular form, i.e., suspended in the reaction medium (Dague et al. 1992). The main factors
affecting the overall performance of this reactor were agitation, the ratio substrate/biomass
(S/X), the geometric configuration of the reactor and the feeding strategy (Zaiat et al., 2001).
Agitation can be performed by recirculation of the liquid or gas phase of the reactor and
by using mechanical stirrers. Currently, the method less commonly employed is that of gas
agitation, which has some problems, such as the release of volatile compounds in water and
insufficient agitation when using low loads with low biogas production (Brito et al., 1997;..
Zaiat et al., 2001). A positive operation for the reactor performance is intermittent agitation
because it improves the sedimentation by increasing the efficiency of gas-liquid separation
(Zaiat et al. 2001). This is important because the removal of organic matter occurs mainly in
sedimentation, as observed in the work of Hawkins et al. (2001). The ratio of
substrate/biomass (S/X) is a very important factor in discontinuous systems with self-

immobilized biomass because it affects the granulation of the biomass. Low S/X ratios can
result in better retention of biomass, with superior granulation and good grain sedimentation,
with rapid and stable reactor start-ups. Moreover, high S/X ratios can reduce the removal
efficiency of organic matter and cause inhibition of microbial activity (Zaiat et al. 2001).
Experiments showed that the geometric configuration of the reactor ASBR significantly
influences the characteristics of biomass (flocculent or granular) and favors the selection of
microorganisms (Sung and Dague, 1995). The feeding strategy, in contrast, affects the
performance of the reactor. It relates the ratio S/X and the solids retention (Zaiat et al. 2001).
The ASBR has advantages over the UASB reactor, which also operates with suspended
biomass. Veronez et al. (2005) evaluated the characteristics of anaerobic granules treating
synthetic wastewater at a low load with mechanically agitated ASBR and UASB operated at
low and high surface speeds. The ASBR maintained the characteristics of the inoculated
sludge and the granules had similar characteristics throughout the reactor. The UASB
granules, however, varied from the top (flakes) to the bottom of the reactor (granules),
leading to the need for speed reduction so that flushing would not occur. Recirculation
improved microbiological characteristics in UASB, but the ASBR with an agitation rate of 50
rpm was better.
The search for improving ASBR processes led to the development of a new configuration
in which support material has been introduced for immobilization of the biomass. The reactor
was called an anaerobic sequencing batch reactor operated with mechanical agitation and
immobilized biomass (AnSBBR) (Ratusznei et al., 2000).
Garcia et al. (2008) studied some types of support for AnSBBR reactors and found that,
comparatively, the polyurethane foam was the best support material for the reactor
performance, kinetic analysis and adherence of methanogenic bacteria.
Pinho et al. (2005) investigated the influence of the size of the inert support on the
kinetics of degradation, which is equivalent to analyzing different conditions for mass transfer
in the solid phase, and concluded that the particle size significantly influenced both the
kinetic parameters of degradation and the reactor performance.
Comparing the ASBR with the AnSBBR, the immobilization of the biomass improves
solids retention and does not require the formation of granules or the sedimentation step,
which leads to a shorter operation (Ratusznei et al. 2000). Furthermore, the self-immobilized
biomass can float due to the presence of polymers, such as those reported by Mockaitis et al.
(2006) and Fuzzato et al. (2009). A disadvantage of the immobilized biomass systems is the
difficulty of mass transfer, but by using recirculation, for example, it is possible to fix this
problem (Ramos et al. 2003).
Sarti et al. (2006) observed significant similarity in the performances of ASBR and
ASBBR in domestic sewage treatment regarding organic matter removal, but the reactor
containing granular biomass produces biogas at an earlier stage compared to the reactor
containing immobilized biomass. The microbial structure in the two reactors was also
assessed, and it was found that the structures from the bacteria dominium from day 35 and
day 70 were different; thus, it can be inferred that biomass immobilization may have caused
selection of the bacterial populations. Microbial structure in the Archaea domain showed
greater similarity in the reactors, probably because these populations use specific substrates
formed at the end of the anaerobic process. Therefore, the choice of reactor must take into
account each particular process.
The methodology proposed by Zaiat et al. (1994) has been widely used for
immobilization of the anaerobic sludge. According to this methodology, the inert support
particles are placed in a container and the sludge is added until the entire support is in contact
with it. The contact should last 2 hours. After this period, the adhered cell matrices are placed
in the influent (the same influent that will be used in the experiment) to wash out the weakly
adhered solids. The influent will be drained and the inoculum is ready to be placed in the
reactor. Figure 5 shows the granulated biomass, and Figure 6 shows its immobilization on
polyurethane and polyethylene foam.
Figure 5. Photograph of granulated biomass.

(a)
(b)
Figure 6. Inert support particles of polyurethane (a) and low density polyethylene (b) before and after
immobilization (left and right, respectively).
3.3. Organic Load
The availability of a substrate for biomass should be evaluated by volumetric or specific

organic load applied. This factor was the direct object of investigations focusing on the
influence of organic load in the production of bioenergy in sequencing batch reactors.
However, some authors studied the production of hydrogen due to the variation of parameters
inherent to the organic load, as the effect of initial concentrations (Buitron and Carvajal,
2010), the feeding volumes (Badiei et al., 2011) or the retention times (Cheong et al., 2007;.
Arooj et al., 2008;. Chen et al., 2009). These parameters were studied separately or in a
combined manner but were not related to organic loads.
Several scientific studies address the influence of the applied organic load on the stability
of the biological process and the production of hydrogen and methane in sequencing batch
reactors (ASBR and AnSBBR).
In the study of Mockaitis et al. (2006), the increase in the applied volumetric organic load
was from 0.6 to 4.8 gDQO.L-1.d-1; a respective increase in the volumetric quantity of methane
has been reported. The profiles of the gas production over a period of one cycle showed a
high production rate followed by a period during which the rate was reduced. These authors
concluded that the period of reduced rate decreased with the increase in the influent organic
load, indicating a possible need to increase the cycle time. Moreover, the authors reported the
appearance of substances similar to polymers, whose origin was likely attributable to the
microorganisms. These substances caused flotation of the biomass, which was granular,
especially for higher loads. Fuzzato et al. (2009) have investigated the increase of the organic
load from 1.1 to 4.5, then to 8.0 and finally to 12 gDQO.L-1.d-1 upon treating dairy
wastewater in AnSBBR. The reactor was stable and efficient in removing the organic load,
but the appearance of polymer secretions has been reported, which did not affect the process
but, according to the authors, must be better studied.
The work of Bezerra et al. (2007) consisted of the application of organic loads of 3, 6 and
12 gDQO.L-1.d-1 in an AnSBBR with recirculation. The findings showed that the increased
load results in increased total production of methane. The authors explain that the increase in
organic load means a higher amount of substrate available to the acidogenic bacteria, which
in turn, produce more acid. The increased availability of acetate for the methanogens
therefore leads to an increased formation of methane.
Cheong and Hansen (2008) investigated the effect of the feeding strategy and the increase
of the organic load on the processing capacity and stability of an ASBR. Various organic
loads were applied (1.5 to 12 gDQO.L-1.d-1) according to the feeding strategy. The highest
methane productivity occurred with fed-batch mode and an organic load of 12 gDQO.L-1.d-1,
with a value of 3170 mL-CNTPCH4.L-1.d-1.
Oliveira et al. (2008) studied the application of an AnSBBR to the wastewater treatment
from automobile industry and showed that the increased load did not favor the process. The
authors concluded that the system was stable with the increase in the organic load, although
by the analysis of the kinetic parameters, an inhibition of anaerobic metabolism may have
occurred due to the constituents of the wastewater used when the organic load increased from
2.61 to 6.19 gDQO.L-1.d-1.
Carvalhinha et al. (2010) studied the metallurgical industry wastewater treatment using
an AnSBBR with increasing load. Organic loads from 1.5 to 6.16 gDQO.L-1.d-1 were applied,
and the authors concluded that the increased load led to an increased concentration of
methane in the biogas and its ratio to CO2. Selma et al. (2010) used a reactor with granular
biomass. The influent concentration was increased from 500 to 3000 mgDQO.L-1, when the
system lost much of its efficiency and stability. Unlike the previous work, the production of
methane decreased with the increasing load, and the greatest ratio between the concentration
of methane produced and organic matter consumed was 11.8 mmolCH4.gCOD-1, when the
lowest COD concentration was applied.
Almeida et al. (2014) studied the influence of increasing the organic load and the feed
strategy on the stability, treatment efficiency and methane production in an AnSBBR with
mechanical agitation treating vinasse. The organic loads were 1, 2 and 3 gDQO.L-1.d-1. The
results showed that the reactor remained stable under all experimental conditions studied and
the productivity and yield of methane consumed by organic load consumed increased with the
increase in organic load.
The papers that reported the production of methane showed some tendency toward an
increased organic load and a concomitant increase in stability and methane production. The
effect of the organic load on hydrogen production, in turn, showed a variable effect, although
the influence of this parameter has been noted.
Venkata Mohan et al. (2007b) studied the treatment of dairy-industry wastewater for
hydrogen production using an ASBR. Organic loads of 2.4, 3.5 and 4.7 gDQO.L-1.d-1 were
applied. According to the authors, the data showed that hydrogen production was higher and
faster (12 h) during the application of the two highest loads. When using the lowest organic
load, they concluded that overall adaptation of the biomass to the wastewater may not have
occurred, or perhaps the reduced availability of substrate led to lower gas production.
Goud and Mohan (2012) showed the importance of the effect of shock load in an ASBR
for hydrogen production. The authors found that the shock load modifies the process to favor
acidogenesis, inhibiting methanogenic bacteria (although not eliminating them) and enriching
the microbial composition with hydrogen-producing bacteria. The maximum value of the
ratio between produced hydrogen and organic matter removed was 16.6 molH2.kgDQO-1
using an organic load of 2.14 kgDQO.m-1.d-1 and a shock load of 35 kgDQO.m-3.d-1. With the
same organic load, but without the shock load treatment, a rate of 3-4 molH2.kgDQO-1 was
achieved. This was a much lower value than the one under the first conditions.
Manssouri et al. (2013) developed a study on hydrogen production by an AnSBBR
treating sucrose-based wastewater with organic loads of 9, 12, 13.5, 18, and 27 kgDQO.m-3.d-
1
. The study showed that the increase in the load by enhancing the influent concentration
resulted in an increased volumetric production of hydrogen. The opposite effect was noted
when increasing the load by reducing the cycle time. Thus, a trend was not observed in these
parameters with the variation of the load, in the same manner as the indicators of hydrogen
production.
In the work of Santos et al. (2014), an AnSBBR with recirculation of the liquid phase for
the production of biohydrogen was used; the organic load was modified by the influent
concentration and the cycle time. The variation in the organic load by means of the influent
concentration led to the generation of more methane than did varying the cycle time. Another
important conclusion of this work is that the reduction in cycle time with a proportional
reduction in the influent concentration (to maintain the same organic load) did not favor the
production of biogas. The authors found that in the condition with the longest cycle time (4
h), the maximum ratio of hydrogen generated by organic load removed occurred in the
application of lower concentrations of organic matter. For the other conditions (2 h and 3 h),
the maximum occurred at the highest concentration. The same happened to the productivity of
hydrogen. The experimental conditions with a higher influent concentration and
intermediate/lower cycle time and the condition with a lower influent concentration and
longer cycle showed the best results in terms of the productivity of hydrogen.
3.4. Continuous/Discontinuous Operation Mode
The choice between a reactor operating in continuous or discontinuous mode can be

crucial to the efficiency and effectiveness of the process. Continuous reactors such as UASB
and RALF are characterized by higher sludge retention time, high efficiency and volumetric
capacity process treatment but have long periods of start-up and are unsuitable for
wastewaters with low organic load. However, reactors operated in batch mode are more
flexible, have lower installation costs, are safer and can achieve more control over the quality
of the effluent.
There is also a third mode of operation called fed-batch, which is very common in the
fine chemicals industry and biotechnological processes. In this type of operation, a feed
stream is slowly added to the reactor. This mode is usually used to increase the selectivity or
the safety of the process. Table 1 shows a comparison between the three types of operation.
Table 1. Comparison between modes of reactor operation
Operation mode Advantages Disadvantages

Time to load and unload the
Simple equipment, flexible operation reactor, the reaction conditions
Batch mode, low installation cost, ease to shut change over time,
down and perform cleaning. inappropriate for large flows
of effluent.
Requires flow control and
stability of microorganisms,
Great treatment capacity, good for
unexpected breakdowns can
Continuous kinetic studies, uniformity of the
be extremely costly and it is
treated effluent.
inappropriate for low organic
loads.
Maintains low concentrations of the
compounds in the reaction medium and
thus inhibits the effects of substrate
degradation; it is effective to treat high
Fed-batch organic loads, absorbing peaks of Requires feeding strategy.
volatile acids that could destabilize the
process. It is more flexible for the
selection of optimum operation
conditions.
The sequencing batch reactors (ASBR), as mentioned above, have characteristics that
ensure advantages over other anaerobic digestion processes, such as a greater retention of
biomass inside the reactor compared to a continuous reactor, even at low food/microorganism
ratios, resulting in effluents with a smaller suspended solids concentration and greater quality
control because the cycle time can be controlled so that the desired efficiency is reached
(Sung and Dague, 1995;. Zaiat et al., 2001). Thus, the anaerobic sequencing batch reactor is
an alternative for specific cases, such as for treating wastewater that is generated
intermittently, with poorly degradable compounds or where release patterns are very
restrictive. The desired removal can be ensured by conditioning certain reactor operating
parameters, such as the cycle time and the initial load. In addition, this type of reactor has
other advantages, such as a high rate of organic matter removal, stable operation and
simplicity of its system (Zaiat et al. 2001).
Among the continuous reactors, the UASB - upflow anaerobic sludge blanket – reactor is
one of the most substantial and best known for sewage treatment; over 1,000 UASB reactors
have been installed worldwide (Chong et. Al., 2012). One of the advantages of the UASB is
self-immobilization of the biomass in a granule form, which ensures a longer retention time
of the sludge in the reactor. Another advantage is the presence of a gas/liquid/solid separator
on top of the reactor, which prevents biomass loss and maintains the high efficiency of the
process treatment.
Despite the success in treating various types of wastewater, the UASB has some
limitations, mainly due to the granulation process. These include the need for a long period of
start-up, a relatively high operating temperature and unsuitability for low organic load
residual. Furthermore, the granulation technology is not suitable for the removal of nutrients
(N, P) (Liu and Tay, 2004). Rizvi et al. (2014) also mentioned that the start-up of an UASB is
a limiting parameter for its application because a large number of factors affect the growth of
the sludge bed, including wastewater characteristics, acclimatization, pH, nutrients, presence
of toxic compounds, loading rate, flow rate, hydraulic retention time, liquid mixing and
reactor design.
3.5. Influence of the Hydraulic Retention Time and the Cycle Time
According to Kumar (2008), the production of hydrogen and methane is affected by the
hydraulic retention time (HRT) basically due to two reasons: (i) a dilution effect of the
substrate and product, and consequently the organic load, occurs, and (ii) it allows the
selection of the microorganisms according to their growth rate.
The second reason is closely related to the control of the hydrogen and methane
production process. Thus, the maximum growth rate of methanogenic microorganisms (μmax =
0.017 h-1) (Lettinga and Haandel, 1993) compared to the maximum growth rate of the
hydrogen-producing bacteria (μmax = 0.14 to 0.33 h-1) (Khanal, 2009) is noticeably smaller.
Therefore, the higher the hydraulic retention time the higher the probability of growth of
slow-growing methanogenic microorganisms and the greater likelihood of producing
methane.
When the goal is to obtain hydrogen, the establishment of a specific HRT must be made
taking into account the growth rate of the producer and consumer microorganisms and the
removal efficiency of organic matter. To excessively decrease the HRT to avoid the presence
of methanogens could result in low efficiencies of substrate consumption because variations
in the HRT have a direct effect on the applied volumetric organic load (AVOL) and the
separation of these two factors is unlikely (Kumar , 2008).
It is worth noting that work with a short HRT, and working with pH values lower than
6.5, does not guarantee the absence of methanogens in the medium. Fang and Liu (2002)
reported production of methane on a CSTR reactor operated with a pH of 6 and a HRT of
only 6 hours.
For anaerobic reactors operated in batch and sequencing batch reactors, the concept of
reaction time that best fits is called cycle time, corresponding to the residence time of
wastewater in the reactor, recorded from the load until the discharge.
Recent studies developed for hydrogen production have conducted tests with various
cycle times in order to evaluate its effect. Most studies use cycle times of 2, 3 and 4 hours. It
is important to remember that the applied volumetric organic load is directly proportional to
the number of cycles per day in the operation of batch reactors and the number of cycles
depends on the cycle time. The shorter the cycle time is, the larger the number of cycles per
day, and therefore, the greater is the number of times that the pump system feeds the reactor
with fresh substrate. In some cases, the continuous replenishment of nutrients is beneficial for
the hydrogen production, but in other cases and under some conditions, the result is harmful
because it accelerates the metabolic pathway contributing to the production of methane, as
noted by Moncayo Bravo et al. (2014). Lovato et al. (2014) and Moncayo Bravo et al. (2014)
tested the use of glycerol as the carbon source using an AnSBBR reactor. The difference
between the studies was the type of the feeding. Lovato et al. (2014) worked with fed batch,
while Moncayo Bravo et al. (2014) used fast feed batch. Due to this difference, Lovato et al.
(2014) achieved the best results with a cycle time of 3 hours, while Moncayo Bravo et al.
(2014) achieved the best results in 4 hours. The higher replacement of nutrients fed to the
reactor when it was operated in fed batch was positive, but this did not happen to the fast feed
batch. The explanation may be linked to the better utilization of the substrate by the
microorganisms in the first case.
Inoue et al. (2014), Manssouri et al. (2013) and Santos et al. (2014) operated AnSBBR
reactors with mechanical agitation and recirculation of the liquid phase. The carbon source
used was sucrose. For the three cases, the highest hydrogen molar yields were achieved by
operating the reactors with two hours of cycle time, compared to three and four hours of cycle
time tested.
Then, it is observed that the production of hydrogen, beyond the cycle time, is connected
to other variables such as the type of substrate and the type of feeding of the reactor.
In relation to methane production, the most commonly used cycle times have been 4, 6, 8,
12 and 24 hours. The best results have been achieved with a cycle time of 8 hours in most
studies, as seen in Table 2. Lullio et al. (2014) operated an AnSBBR reactor with 8 and 12
hours of cycle time and fed with sucrose. Contrary to expectations, the highest methane molar
yield was achieved when the reactor was operated with 8 hours of cycle time. The same
authors fed the reactor with effluent from an acidogenic reactor fed with sucrose, with cycle
times of 6 and 8 hours. The best results were also achieved in 8 hours. Thus, excessively long
times can have a negative effect on methane production.
Oliveira et al. (2009) tested cycle times of 8, 12 and 24 hours, feeding an ASBR reactor
with the effluent from the personal care products industry. Again, the best results were
achieved using a time cycle of 8 hours. Silva et al., 2013 operated an ASBR reactor fed with
glycerin from the biodiesel industry with cycle times of 4 and 8 hours. These authors
observed the highest levels of methane production when a cycle time of 8 hours was used.
The research of Selma et al. (2010), Bezerra et al. (2011), Lovato et al. (2012) and Lovato et
al. (2013) were also characterized by the operation of the reactor with a cycle time of 8 hours,
using a synthetic wastewater similar to that used by Silva et al. (2013).
Thus, it can be concluded that the production of both hydrogen and methane maintains a
relationship with the cycle time or hydraulic retention time, together with other parameters of
influence such as volumetric applied organic load, the type of reactor, the type of feed and
other factors.
3.6. Feeding Time
One of the most important variables of batch reactors is the feeding strategy. It is possible
to provide better efficiency and stability using the most appropriate filling time. While the
batch mode achieves higher concentrations of biogas, the fed batch mode allows the
maintenance of low concentrations of the compounds in the reaction medium and thus
decreases the effects of inhibition of substrate degradation. Furthermore, it is possible to treat
effluents containing high organic loads, absorbing high peaks of volatile acids that normally
destabilize the process (Rodrigues et al., 2011).
The application of fed batch operation of any wastewater treatment system should be
made after a thorough study of the process conditions. Shizas and Bagley (2002) concluded
that long feeding times have a positive influence on the performance of an ASBR system
treating glucose-based wastewater. However, Ratusznei et al. (2003) observed that removal
efficiency may decrease and the stability cannot be achieved when long filling times are
applied to AnSBBR and polyurethane foam is used as the inert support. Negative results were
credited to "stress" of biomass and food shortages, as part of the bed was without substrate
during the filling period, a problem that was not observed by Rodrigues et al. (2003) who
used an ASBR with granular biomass, concluding that the fed batch mode is suitable.
Bezerra et al. (2009) demonstrated that the influence of the filling time is greater when
the organic load applied to the system increases, and, contrary to expectations, the use of
higher fill times did not result in the minimization of negative effects when a shock load is
applied to the system. Selma et al. (2010) have also observed this effect of improvement of
the organic matter removal when the organic load was increased to larger fill times. Oliveira
et al. (2008) attested that the kinetic constants of substrate consumption and the formation of
volatile fatty acids are higher when fed batch mode is used. Damasceno et al. (2007) found
that higher filling times result in lower maximum concentrations of volatile fatty acids and the
peaks of these acids tend to happen at the end of the cycle.
The use of batch or fed batch as a feed strategy is important not only in the removal of
organic matter, but in the removal of compounds such as sulfur and nitrogen. Mockaitis et al.
(2010) showed that the fed-batch mode in an ASBR increased the sulfate removal efficiency
compared to the batch strategy, but there is a limit to the sulfate load that can be applied
according to the first mode; Friedl et al. (2009) found that an increased filling time favors
sulfur-reducing bacteria in the competition for organic matter and disfavors other anaerobic
microorganisms in the assimilation substrate.
The trend is that the utilization of the fed-batch mode increases the efficiency of the
reactor in relation to the removal of organic matter and nutrients, especially due to the ratio
between the filling time and the cycle time (tF/tC) ≤ 0.5, but there are exceptions, such as in
the work of Carvalhinha et al. (2010). In this case, the decrease in the efficiency of removal of
organic matter and the decrease in the concentration of methane in the biogas in the fed batch
mode are credited to the difficulty of degradation of the substrate and not to the inhibitory
effect of the effluent from the metallurgical industry. Then, the reaction rate was lower in the
fed batch mode (due to lower substrate concentrations), although there was nothing that
would inhibit the reaction in batch mode. The ratios tF/tC > 0.5, despite the stability, tend to
lose efficiency of removal of organic matter and form a viscous material that may be harmful
to the operation as shown by Borges et al. (2004)
The use and influence of the filling time related to the production of biomethane can be
ascertained in various works in the literature. Selma et al. (2010), Silva et al. (2013),
Rodrigues et al. (2011) and Oliveira et al. (2008) found biogas with very similar
concentrations of biomethane in batch and fed-batch mode, and the results of the latter are
therefore generally slightly lower. However, comparing the work of Bezerra et al. (2011) with
batch mode and of Lovato et al. (2012) with fed-batch mode, who worked with AnSBBR
treating synthetic effluent from the biodiesel production process, it is possible to notice a
significant improvement with the use of fed-batch mode. Lovato et al. (2012) reached
concentrations between 9.2 and 12.2 mmol.L-1 of methane in the biogas generated by the
process against 1.4 to 2.4 mmol.L-1 found by Bezerra et al. (2011).
Cheong and Hansen (2008) also found a significant improvement in the molar yield of
methane using the fed-batch mode because while the maximum productivity reached in the
batch mode was 32 mol CH4.m-3.d-1, in the fed batch mode this value reached 141 molCH4.m-
3 -1
.d . This demonstrates the necessity of a lab-scale experiment to determine the best mode of
operation of the bioreactor. Table 2 shows the results of some works that studied the influence
of filling time on the production of biomethane, where VCH4 is the volumetric production of
methane and CCH4 is the concentration of methane in the biogas produced.
The use and influence of the fed-batch mode for hydrogen production reactors
ASBR/AnSBBR is still scarce in the literature. Inoue et al. (2014) achieved better results than
Manssouri et al. (2013) by using the fed-batch mode (tF/tC = 0.5) in the treatment of synthetic
effluent based on sucrose in AnSBBR with mechanical stirring. The use of fed batch
interfered in the rates of substrate consumption (Inoue et al., 2014, reached sucrose removal
of approximately 98-99% and Manssouri et al., 2013, achieved sucrose removal of
approximately 97-99%) and in the formation of biohydrogen (the maximum productivity
achieved by Inoue et al., 2014, was 81.2 molH2.m-3.d-1 against 38.9 molH2.m-3.d-1 achieved by
Manssouri et al., 2013), minimizing the inhibition caused by the excess of substrate. Lovato
et al. (2014) and Moncayo Bravo et al. (2014) also conducted studies with similar conditions
in batch and fed-batch modes, respectively.
The objective of these two works was the production of biohydrogen in an AnSBBR with
recirculation of the liquid phase and treating effluent from the biodiesel production process.
Lovato et al. (2014), in fed-batch mode with tF/tC = 0.5, reached 100.9 molH2.m-3.d-1 against
67.5 molH2.m-3.d-1 in the work of Moncayo Bravo et al. (2014) treating pure commercial
glycerin; when the two studies used the industrial effluent, the result was that the fed-batch
mode reached 9.7 molH2.m-3.d-1 and the batch mode reached 8.3 molH2.m-3.d-1.
Table 2. Comparison between papers that aimed for methane production that studied the influence of filling time
AVOL (gDQO.L-1.d-1)
(molCH4.kgDQO-1)
CCH4 (mmol.L-1)
References Reactor Mode (B/FB) Effluent
VCH4 (NmL.
(L.cyicle-1)
MYRLC,m
(molCH4.
cycle-1)
tC(min)
m-3.d-1)
% CH4
VR (L)
T(°C)
MPr
tF/tC
VA
B 0.02 480 2 5 30 0.60 2.4 4.3 90.7 5.51 71
B 0.02 480 2 5 30 1.29 4.1 3.7 153.3 8.95 74
B Biodiesel 0.02 480 2 5 30 2.44 6.2 3.5 231.9 11.8 69
Selma et al., ASBR/ production
FB 0.25 480 2 5 30 1.27 - - - 8.21 70
2010 MS process
FB (synthetic) 0.50 480 2 5 30 1.33 - - - 8.04 71
FB 0.25 480 2 5 30 2.44 - - - 11.4 69
FB 0.50 480 2 5 30 2.36 - - - 11.3 70
B 0.02 480 1.5 3 30 1.5 1.8 1.3 40.0 0.6 72
Biodiesel
B 0.02 480 1.5 3 30 3.0 4.4 1.8 99.0 1.4 64
Bezerra et al., AnSBBR/ production

2011 RLF B process 0.02 480 1.5 3 30 4.5 7.6 2.5 171 2.4 57
(synthetic)
B 0.02 480 1.5 3 30 6.0 4.8 1.6 108 1.6 51
FB 0.25 480 1.5 3 30 4.5 - - - 13.6 75
FB 0.50 480 1.5 3 30 4.5 9 - 652* 9.20 71
FB Biodiesel 0.75 480 1.5 3 30 4.5 - - - 11.4 72
Lovato et al., AnSBBR/ FB production 0.25 480 1.5 3 30 6.0 - - - 11.2 67
2012 RLF process
FB (synthetic) 0.50 480 1.5 3 30 6.0 12 - 839* 11.9 73
FB 0.75 480 1.5 3 30 6.0 - - - 12.2 74
FB 0.5 480 1.5 3 30 7.5 14 - 962* 14.3 65
Wastewater Treatment : Processes, Management Strategies and Environmental/Health Impacts, edited by Lydia M. Barrett, Nova Science Publishers, Incorporated, 2015. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/unam/detail.action?docID=2130698.
AVOL (gDQO.L-1.d-1)
(molCH4.kgDQO-1)
CCH4 (mmol.L-1)
References Reactor Mode (B/FB) Effluent
VCH4 (NmL.
(L.cyicle-1)
MYRLC,m
(molCH4.
cycle-1)
tC(min)
m-3.d-1)
% CH4
VR (L)
T(°C)
MPr
tF/tC
VA
B 0.02 480 2 5 30 2.4 8.5 4.2 318.9 13.9 68
B 0.04 240 1 5 30 1.2 8.9 2.2 166.0 8.92 65
B Biodiesel 0.04 240 1 5 30 1.8 9.9 1.9 184.4 7.33 64
Silva et al., ASBR/ production
FB 0.50 480 2 5 30 2.4 - - 12.8 72
2013 MS process
FB (industrial) 0.50 480 2 5 30 3.6 - - 12.9 64
FB 0.50 240 1 5 30 1.2 - - 8.50 68
FB 0.5 240 1 5 30 3.6 - - 10.1 64
B 0,02 480 2 5 30 3.12 - - - 1.65 61
Metallurgical
Carvalhinha ASBR/
B industry 0,02 480 2 5 30 6.08 - - - 5.20 78
et al., 2010 MS
(synthetic)
FB 0,50 480 2 5 30 6.16 - - - 4.10 77
3.7. Temperature
Anaerobic processes, as well as other biological systems, are strongly influenced by

temperature. In nature, methane is formed over a wide temperature range from 0 °C to 97 °C
by complex communities consisting of hydrolytic, syntrophic, methanogenic and
homoacetogenic microorganisms that degrade organic matter producing CO2 and methane
under anaerobic conditions (Zeeman et al. 1988). In the anaerobic systems, there are three
optimal temperature ranges for methanogenesis: psychrophilic, mesophilic and thermophilic.
Thus, methanogenesis can be classified as psychrophilic, mesophilic and thermophilic,
respectively. Anaerobic conversion rates generally increase with increasing temperature up to
60 °C (Pohland, 1992). Anaerobic degradation (and consequently the biological methane
production) reaches its maximum efficiency between 5 and 15 °C for psychrophilic processes,
between 35 and 40 °C for mesophilic and at approximately 55 °C for thermophilic processes,
with a decrease in the rates between these optimal values. Significant temperature changes
should be made in the treatment of wastewater, and the project must provide sufficient
flexibility to ensure the stability of the process (Lettinga and Haandel, 1993).
Despite the consensus of the optimal temperature range for methane production, there is
considerable disagreement about the ideal temperature or even an ideal range for biological
hydrogen production temperature. According to the Zhang and Shen (2006), the best process
efficiencies occur in the range between 25 °C and 40 °C, particularly under conditions near
the ideal conditions (35 °C) for the function of the biochemical catalysts. Several studies have
been conducted and have shown that there is a certain temperature range between 25 °C and
60 °C in which the high capacity of hydrogen-producing bacteria to produce this gas through
fermentation can be noted. However, a decrease in production can occur when the
temperatures are high (Wan and Wang, 2009). Several studies converge to temperatures
between 30 °C and 36.8 °C, such as setting optimum temperature for anaerobic fermentation
systems producing hydrogen (Fang et al., 2002;. Lin and Chang, 2004). Lee et al. (2006)
investigated the effect of temperature on hydrogen production using sucrose as substrate and
concluded that, in general, the production rate of hydrogen and the yield of hydrogen tended
to increase with increasing temperature from 30 °C to 40 °C, while it tended to decrease with
further temperature increases from 40 °C to 45 °C. The optimum temperature reported by
Valdez-Vazquez et al. (2005), in contrast, was 55 °C. Gavala et al. (2006) studied two
continuous reactors using complete mix, one at 35 °C and another at 55 °C, and observed that
the hydrogen yield and volumetric hydrogen production were higher under thermophilic
conditions than under mesophilic conditions. Several reasons can be noted as the cause of so
many differences to define a temperature range or ideal temperature for hydrogen production:
reactor configurations used, types of substrates and concentrations applied, temperature
ranges studied and microorganisms involved.
In addition to the influence on the microorganisms, temperature has a direct influence on
the solubility of biogas originated from fermentation. This gas is produced in liquid medium
and needs to come off as a gaseous medium because only then will it become available for
use. Hydrogen is a gas with a low solubility in an aqueous environment. According to Guwy
et al. (1997), the solubility of hydrogen is 0.017 mLH2.ml-1 in water at 37 °C and 1 bar.
Taking into account Henry's Law, where the solubility is proportional to the partial pressure
of the gas, the higher the temperature, the lower the solubility of hydrogen. As a consequence,
the interaction of the microorganisms present in the reactor with the biogas is lower, putting
at a disadvantage the routes of the consumers of the hydrogen produced, as homoacetogenesis

and hydrogenotrophic methanogenesis (Hawkes et al., 2002).
Despite the importance of temperature, few studies address the influence of these
operating parameters in the production of biohydrogen or methane in sequencing batch
reactors. Some studies using this reactor configuration are focused only on the removal of
organic matter in the form of COD without quantifying the production of methane from this
process of environmental adaptation. Following this reasoning, Agibert et al. (2007) tested
five different temperatures (15, 20, 25, 30 and 35 °C) using an AnSBBR that recirculated the
liquid phase in the treatment of wastewater, obtaining the best efficiency for the removal of
organic matter in the form of COD (approximately 80%) at the higher temperatures (25, 30
and 35 °C), with a significant drop in the efficiency of the process (i.e., removal of organic
matter approximately 60%) for the lower temperatures (15 and 20 °C). Bergamo et al. (2009)
studied four different temperatures (15, 20, 25 and 30 °C) using a fluidized bed ASBR,
achieving greater efficiency of removal of organic matter in the form of COD at 20 °C (80%)
and low efficiency at 15 °C (79%). The authors concluded that the fluidized bed ASBR with a
granular biomass was more sensitive to the temperature than the ASBR with immobilized
biomass used by Agibert et al. (2007). In view of the difference in organic matter removal
obtained with the different temperatures tested, the work in these cited papers probably
experienced some influence of the same temperatures in the production of biogas.
In their work, Ndon and Dague (1997) studied the influence of temperature (35, 25, 20
and 15 °C) in an anaerobic reactor operated in sequencing batch (ASBR) treating a synthetic
wastewater based on dried skim milk. The amounts of methane obtained were practically the
same when the ASBR was operated at 35, 25 and 20 °C. A decrease in the amount of methane
was observed at 15 °C due to the decrease in the COD removal.
Operating an ASBR fed with brewery effluent, Agler et al. (2010) started its operation at
a reactor temperature of 37 °C. After reaching a stable level of methane production, the
temperature of operation was decreased to 22 °C, causing a decline in production of CH4
from 1.10 to 0.64 LCH4.L-1.d-1. After that there was an increase in temperature from 37 °C to
41 °C, with an increase in CH4 production from 1.10 to 1.70 LCH4.L-1.d-1.
In their study, Kayranli and Ugurlu (2011) showed that at low temperatures, it is possible
to anaerobically treat effluents with low organic load as domestic sewage. Three different
temperatures (10, 15 and 20 °C) were tested with the removal of organic matter and methane
production decreasing at the lower temperature. The authors concluded that longer cycles
times are needed at lower temperatures (15 °C) for the process to achieve stability.
Related to the biological production of hydrogen, Shi et al. (2013) evaluated the effect of
temperature (35, 50, and 65 °C) in an ASBR fed with Laminaria japonica without
pretreatment. A stable biohydrogen production was reached. The maximum hydrogen yield
achieved in mesophilic (35 °C), thermophilic (50 °C) and hyperthermophilic (65 °C)
conditions were 61.3, 49.7 and 48.1 mLH2.g-1TS, respectively.
3.8. pH
The hydrogen ion concentration as expressed by pH is a very important factor influencing

the formation of biohydrogen and biomethane in biological systems, considering that the pH
can affect both the activity of hydrogenase and the metabolic pathways, as well as the
substrate hydrolysis. The concentration of H+ ion in the system is also critical for maintaining
appropriate levels of ATP because in the presence of excess H+, ATP is used to ensure the
neutrality of the cell instead of producing hydrogen (Gioannis et al., 2013).
The influence of this parameter in the fermentation of hydrogen, however, is quite
controversial in the literature. Studies have shown that an appropriate range of pH for the
production of hydrogen is between 4.0 and 7.0. When the pH increases from 2.0 to
approximately 7.0, an increase in the capacity of hydrogen-producing bacteria can be noted.
However, high pH values between 10 and 14 may decrease the H2 production (Wan and
Wang, 2009). It was found that a lower initial pH offers a greater potential for hydrogen
production, but a lower production of this gas. Investigations were carried out in batches
varying the initial pH and measuring the hydrogen production, with the maximum yield being
obtained at a pH of 4.5 treating synthetic effluent based on sucrose (Khanal et al. 2003), and
6.0 treating cheese whey (Ferchichi et al., 2005). Some studies show that the optimum pH for
hydrogen production is in the range between 5.0 and 6.5 (Fang et al., 2002; Lin and Chang,
2004), while others point to a single value of 5.5 as the best (Fang and Liu, 2002; Mu et al.,
2006; Shin, 2007). Such disagreements can be attributed to differences between studies in
terms of substrates, inoculum and initial pH ranges. A pH value of approximately 5.0 seems
to be the most suitable.
Despite the disagreements about optimal values, Lay et al. (1999) emphasize that a pH
much below 4.5 is unfavorable for the production of hydrogen, having in mind that under
these conditions the inhibition of the hydrogenase activity and of other enzymes with a
consequent alteration of the metabolic pathways involved in the process may occur.
While the first stages of anaerobic digestion can occur in a wider range of pH values,
methanogenesis is developed only at neutral pH (Haandel and Lettinga, 1993). For pH values
outside the range 6.5 to 7.5, the rate of methane production is low. Given that the
methanogenesis is considered as the limiting step of the process, it is necessary for the pH of
the reactor to be maintained in the neutral range, if the objective is the production of CH4.
The number of studies about the influence of pH on biohydrogen production in

sequencing batch reactors is low. Cheong et al. (2007) operating a mesophilic ASBR fed with
synthetic glucose-based effluent tested three pH values (5.2, 5.7 and 6.2), finding no
statistically significant effect on the parameters examined. Wu et al. (2010) tested the
influence of five pH values (4.4, 4.7, 5.0, 5.3 and 5.6) for hydrogen production in an ASBR
with an HRT of 16 hours using pig manure supplemented with glucose, obtaining an optimum
yield of 1.50 molH2.mol-1glucose and a maximum production rate of 2.25 LH2.d-1L-1 at a pH
of 5.0. Chen et al. (2009) investigated the effect of pH (4.9, 5.5, 6.1 and 6.7) on the
production of biohydrogen in an anaerobic sequencing batch reactor (ASBR) fed with
carbohydrate-rich organic waste. The results showed that a pH value of 4.9 was most
favorable for the fermentative production of hydrogen in terms of H2 yield and conversion
efficiency, reaching a maximum yield 2.53 mole H2 per mole of sucrose consumed. However,
this pH gave a low concentration of volatile suspended solids, suggesting that low pH can
inhibit the growth of mixed cultures. Even so, the operation of the ASBR at a pH of 4.9 may,
according to the authors, have caused a greater activity of the hydrogen-producing bacteria
while the development of non-hydrogen producing bacteria may have decreased.
In their studies, Won and Lau (2011) studied the effect of pH in an ASBR fed with
sucrose as substrate and inoculated with sludge from anaerobic digestion without treatment.
The maximum volumetric hydrogen production (3.04 LH2.L-1.d-1) and the maximum
hydrogen yield (2.16 molH2.mol-1hexose) were achieved at a pH of 4.5. The same authors
(Won et al. 2013) evaluated the effect of pH (4.5, 5.0 and 5.5) again in the same reactor
configuration, this time using a wastewater from the refining of sugar as a substrate, obtaining
an optimal value of 5.5 for the parameter in question.
3.9. Substrate
Given that the processes of obtaining biohydrogen have living organisms as protagonists,
nutrients in the environment are crucial to their existence. The nutrients considered most
important by the percentage of the physical constitution of microorganisms are carbon,
nitrogen and phosphorus. Various C/N ratios have been studied to establish the optimum
value. Lin and Lay (2004), for example, tested C/N ratios of 130, 98, 47 and 40 for the
production of hydrogen. The maximum production was reported with a C/N ratio of 47 using
hexose as a carbon source. Morimoto et al. (2004) reported a value of 10 as their best value
using glucose as the carbon source and yeast extract as a nitrogen source.
As for the C/P ratio, some studies have established an optimum range of 120 to 130
(Hawkes et al., 2002. Lay and Lin, 2004). Hawkes et al. (2007) and Ren et al. (1995) have
reported hydrogen production using sugar or starch as the sole carbon source by adding
nitrogen and phosphorus salts; however, in most studies a more complex mixture of salts is
used. Hawkes et al. (2007) mention that the presence of sulfur and iron has an importance
equal to that of nitrogen and phosphorus due to the constitution of the hydrogenase enzyme.
According to Lee et al. (2001), a limited iron medium has low hydrogen production and a
change in the metabolic pathway for the production of acids (hydrogen generators) to
generate solvents (hydrogen consumers).
Table 3, obtained from Hawkes et al. (2007), presents a list of macro and trace elements
considered important for the production of hydrogen and the COD:N and COD:P ratios used
by the authors mentioned. It is important to mention that they all used hexoses as a carbon
source, which allows an easier comparison between them.
As seen in Table 3, no specific nutritional relationships may be established as absolute
for hydrogen production. As well as the factors presented above, the production of hydrogen
depends on the action between the nutritional ratio and other factors as well.
A variety of substrates has been used for the biohydrogen production by dark
fermentation. Glucose (Fang and Lui, 2002; Zhang et al., 2004;. Zoetemeyer et al., 1982;. Lin
and Chang, 1999;. Mizuno et al., 2000) and sucrose (Kyazze et al., 2006;. Lin and John, 2003;
Wu et al., 2006;. Chang et al., 2004;. Hussy et al., 2005;. Chen and Lin, 2003) are the most
widely used substrates because the greatest theoretical hydrogen production values are
achieved with carbohydrates (Reith et al. 2003).
Manssouri et al. (2013) and Santos et al. (2014) used sucrose as a carbon source and
obtained molar yields of 38.9 and 27.9 molH2.m-3.d-1, respectively. Souza et al. (2014)
achieved a molar productivity of 60.9 molH2.m-3.d-1 using glucose. Among these two pure
substrates, glucose was the substrate with the highest hydrogen production.
Although these substrates are commonly used in the literature, their high costs make the
process unsustainable from the economic point of view (Hawkes et al., 2002). For the process
of hydrogen production to be sustainable and competitive compared to other alternative
energy sources, the substrates must have a low cost and high availability. That is how
discharges of various origins have value when used as food for fermenting micro-organic
generators of biohydrogen. Examples of several wastewaters and solid waste include (Lay et
al., 1999; Noike and Mizuno, 2000;. Wang et al., 2003a and Wang et al., 2003b) industrial
wastewaters (Ueno et al., 1996;), food waste (Han e Shin, 2004), industrial food sewage (Han
e Shin, 2004; Youn e Shin, 2005), among others.
Table 3. Elements (mg L-1) and COD:N and COD:P used in some studies for hydrogen
production (Hawkes et al., 2007)
Fang Mu and
Lin and Chang Mizuno et al. Zoetemeyer et al. Hussy et al.
Elements and Liu Yu
(1999) (2000) (1982) (2003)
(2002) (2006)
N 131 929 680 351 680 72.9
K 112 56 113 97 183.7 53
S 42 5.5 0.6 12.8 9.9 -
Mg 32 13 32 3.6 37.8 12.0
Ni 12 - 0.13 - 12.05 1.23
Ca 14 - - 0.20 18.0 18.02
B 0.4 - 0.07 - 1.36 0.69
Mo 0.01 - 0.20 - 7.61 8.66
Zn 11 - 0.24 0.40 11.04 2.4
Co 6.2 0.04 0.74 - 3.72 1.24
Cu 3.7 1.3 - - 3.73 1.42
Mn 8.3 - 0.69 - 6.95 1.39
I - - 1.91 - - -
Fe 17.2 5.03 1.0 2.9 17.3 11.0
P 102.3 22.0 44.5 76.2 101.5 21.03
DQO:N 57:1 23:1 16:1 30:1 11.4:1 73:1
DQO:P 73:1 970:1 240:1 140:1 77:1 253:1
Industrial wastewater from food, beer and agricultural industries as well as from animal
confinement have great potential for use because they contain a large amount of
biodegradable material (Angenent et al., 2004). Arooj et al. (2008) and Lima et al. (2014)
obtained the mean and maximum molar productivities of 183.3 e 31.1 molH2.m-3.d-1 by using
cornstarch and cheese whey, respectively.
Even though these wastewaters are considered to only be slowly degradable, because of
the low presence of organic matter of fast degradation, they have also shown an interesting
potential. Brazil is a biodiesel producer country, which is a fast-growing industry there, and
with this rise, a large amount of glycerin is co-produced in a proportion of 10 kg for each 100
kg of biodiesel (Chatzifragkou and Papanikolaou, 2012). Moncayo Bravo et al. (2014) and
Lovato et al. (2014) used glycerol as a carbon source under the same environmental and
nutritional conditions, varying only the type of feeding. The former used a batch feed strategy
in the AnSBBR reactor, and the latter used fed-batch mode. Moncayo Bravo et al. (2014)
obtained a molar productivity of 67.5 molH2.m-3.d-1, while Lovato et. al. (2014) reached a
molar productivity of 100.9 molH2.m-3.d-1. These results indicated that a slow supply of a less
degradable substrate, in comparison with a fast supply, would contribute to degradation and
would optimize conversion.
As observed from the results presented and detailed in Tables 4 and 5, glycerol permitted
the achievement of a greater molar production of hydrogen than carbohydrates. Hydrogen
production, besides the substrate, is related to other operational parameters, which optimize
the results.
Methanogenic archaea organisms use as substrates the simple molecules resulting from
the hydrolysis and fermentation of complex compounds. These substrates are formate,
methanol, methylamines, acetate and the inorganic molecules, hydrogen and carbon dioxide.
Therefore, methane is formed by both inorganic and organic molecules, as shown in
Equations (23) and (24). In Equation (23), methane is produced from acetate, while in
Equations (24) and (25), it can be observed that methane is formed by the interaction of
hydrogen and carbon dioxide with bicarbonate HCO3- or carbonate CO32- (Gerardi, 2003).
(23)
(24)
(25)
Several substrates are used to produce methane. Domestic wastewater is among the
substrates that are easily degradable. Chebel et al. (2006), Bezerra et al. (2005) obtained
volumetric molar productions of 12 e 7 mmol.L-1, respectively. The difference between these
studies was the applied volumetric organic load. While Chebel et al. (2006) used 2.36
gDQO.L-1.d-1, Bezerra et al. (2005) used 1.5 gDQO.L-1.d-1.
Another substrate widely used and easy to degrade because of its richness in organic
matter is cheese whey. Bezerra et al. (2007) and Mockaitis et al. (2006) obtained maximum
molar productivities of 16.3 and 60.8 molCH4.m-3.d-1, respectively. The differences between
these studies are the reactor setup and the volumetric organic load used, the details of which
are presented in Tables 4 and 5.

There are also several studies focused on industrial wastewater treatment, for instance,
the studies of Selma et al. (2010), Bezerra et al. (2011), Silva et al. (2013), Lovato et al.
(2012) and Lovato et al. (2013). All of these studies were conducted with glycerin, a co-
product of biodiesel production. The higher volumetric molar productions of 12.9
mmolCH4.L-1 e 14.3 mmolCH4.L-1 were achieved by Silva et al. (2013) and Lovato et al.
(2012), respectively. The former conducted experiments with an ASBR reactor and a
volumetric organic load of 3.6 gDQO.L-1.d-1, and the latter with an AnSBBR with
recirculation of the liquid phase and a volumetric organic load of 7.5 gDQO.L-1.d-1. As a
result, it can be observed that the reactor set up and the applied organic load influence the
process.
Researchers have been studying other types of synthetic industrial wastewaters, such as
those from the personal care industry (Rodrigues et al., 2011; Oliveira et al., 2009) and from
automotive industries (Oliveira et al., 2008).
As previously stated, methanogenic microorganisms use the products of acidogenic
fermentation to produce methane. With this background, the effluents of acidogenic reactors
are also continuously being used to generate methane. Lullio et al. (2014) performed three
experiments that independently assessed the use of an acidogenic reactor effluent fed with
sucrose and another fed with glucose. The results were close. The molar production of
methane from the sucrose effluent was 25.9 mmolCH4.L-1 and from the glucose effluent was
25.2 mmolCH4.L-1. When the experiment was repeated feeding the methanogenic reactor
directly with sucrose, the volumetric molar production was only 15.9 mmolCH4.L-1. These
results demonstrate that methane production is more efficient when the reactor is fed with a
substrate previously processed by acidogenic microorganisms.
From the results of different studies, it can be concluded that there are various substrates
that can be used to obtain methane and hydrogen. However, the results depend on a mixture
of conditions, such as the volumetric organic load, temperature and pH, the variations of
which permit optimization of the results.
3.10. Kinetic Modelling of the Biomethane Production Process
Kinetic modelling is a useful tool to the process of analysis, design and control of
biological systems and should be performed by a precise determination of kinetic coefficients.
There have been several models developed to describe microbiological growth in reactors.
The first order model is the simplest model that can be used with the first order degradation
hypothesis. It is frequently used to describe the digestion limited by hydrolysis with respect to
a particular substrate (Gavala et al., 2003). The Monod model is the most widely applied
model that was developed by empirical analysis (Monod, 1949). Grau et al. (1975) and Chen
and Hashimoto (1978) improved the Monod model by perceiving that the substrate
concentration in the effluent is proportional to the substrate concentration in the affluent. Ma
et al. (2013) applied these four models to an ASBR reactor used to methane production and
treating animal sewage. They concluded that the Chen and Hashimoto (1978) model is the
best model.
Another kinetic model of organic matter degradation was developed by Rodrigues et al.
(2004), from the Bagley e Brodkorb (1999) model. The latter is an adaptation of another
model, proposed by the International Water Association (IWA) and is applied to activated
sludge treatment systems.
In the adopted kinetic model, which was developed for sequencing batch anaerobic
reactors, the process of organic matter degradation is simplified and divided in two
consecutive steps. In the former, the substrate is converted into total volatile acids, and in the
latter, these acids are converted into methane. Moreover, in these two steps, the conversion
reactions were considered as being of the first order. The model admits the existence of a
residual concentration of substrate and of total volatile acids, in which the respective reaction
velocities tend to zero.
The organic matter degradation pathway is shown hereafter, in which a, b and c are the
stoichiometric coefficients, and k1 and k2, the apparent kinetic parameters of the adopted
model.
a  S 
k1
b  AVT 
k2
cM (26)
The equations of substrate consumption velocities (rS), total volatile acids formation
(rAVT) and methane formation (rM) are shown hereafter as k1S, k1AVT, k2AVT and k2M, which are
the same apparent kinetic parameters, but are related to the substrate consumption, to the total
volatile acids formation and to methane formation, respectively. CS and CAVT are the
concentrations of the substrate and total volatile acids, and CSR and CAVTR, are their residual
values.
rS  k1S  (CS  CSR ) (27)
rAVT  k1AVT  (CS  CSR )  k 2AVT  (C AVT  C AVTR ) (28)
rM  k 2M  (C AVT  C AVTR ) (29)
The material balance of the reactor is shown below, and two distinct operational
conditions must be considered. The former is the batch mode, in which the feeding time is
much smaller than the total cycle time (i.e., F = 0), and the latter is the fed-batch mode, in
which the feeding time is significant in relation to the total cycle length (i.e., F ≠ 0), in which
CSo, CAVTo, CMo and Vo correspond to initial values of CS, CAVT, CM and V, respectively.
dV
F V  Vo (30)
dt
dCS F
  (CSo  CS )  rS CS  CSo (31)
dt V
dC AVT F
  (C AVTo  C AVT )  rAVT C AVT  C AVTo (32)
dt V
dC M F
   C M  rM C M  C Mo (33)
dt V
Because they are differential equations, the Runge-Kutta numerical integration method of
the 4th order and the constant integration step can be used by implementing it in an Excel®
worksheet to determine these parameters by Newton's algorithm, using the Solver tool from
Excel.
The kinetic parameters k1S, k1AVT, k2AVT and k2M are apparent because they comprise the
mass transport effects. On the other hand, the parameters CSR and CAVTR correspond only to
an estimative of the residual value of substrate and volatile acids contained in the reactor at
the end of a cycle of operation. Moreover, this kinetic model is a simplification of the
complex process of anaerobic degradation.
3.11. Kinetic Modelling of the Hydrogen Production Process (Wu et al., 2013)
Advanced investigations regarding any biological process always encompass kinetic

studies because the kinetic constants obtained can be used for the design, operation and
increase of the scale of other reactors with the same biological process. There have been
many acidogenic kinetic models developed and applied with success for these biological
processes, including the Monod model, modified Monod model and Cantois model. Although
the Monod model is preferred by many studies, the application of this model to complex
acidogenics with mixed cultures proved to be relatively problematic. Therefore, the effort to
find other models that can be used to describe the fermentative production of hydrogen is
valid.
One of the models that is receiving growing attention from researchers is the Gompertz
model, which was developed in the early 1990s to describe the growth of pure cultures and is
increasingly being used to describe the fermentative H2 production process. The substrate
consumption model using the Gompertz modified model can be visualized in Equation (34),
in which S0 is the initial substrate concentration (g.L-1), S is the substrate concentration (g.L-
1
), ΔSmax is the maximum concentration of substrate consumption (g.L-1), Rmax,S is the
maximum speed of substrate consumption (g.L-1.h-1), and λS is the time lag of substrate
consumption (h).
{ [ ( ) ]} (34)
Calculating the derivative of Equation (34) results in the speed of substrate consumption
which is described by Equation (35) in which rS is the speed of substrate consumption (g.L-
1 -1
.h ):
{ [ ( ) ] [ ( ) ] } (35)
Production of H2 would also be described by the Gompertz modified model, expressed in

Equation (36), in which parameters can be explained by Figure.7 and H is the accumulated
hydrogen (L), Hmax is the maximum accumulated hydrogen (L), Rmax,H2 is the maximum
speed of hydrogen production (L.h-1), and λH is the time lag of hydrogen production (h).
{ [ ( ) ]} (36)
8
Accumulated hydrogen
6 Hmax
production, H2(L)
max
4
ax
2
x
Rmax,H2
0 max,H2
0 20 40 60 80 100 120
ax,H2
Time (h)
 x,H2
Figure 7. Typical curve of hydrogen production fitted the Gompertz modified model.
Calculating the derivative of Equation (32) results in the speed of hydrogen production,
which is expressed by Equation (37), where rH2 is the speed of hydrogen production
(L.L-1.h-1).
{ [ ( ) ] [ ( ) ] }
(37)
The modified Gompertz model was used with success by several studies until the present
(Selembo et al., 2009; Akutsu et al., 2009; Gioannis et al., 2013).
4. FINAL COMPARISONS AND FUTURE APPROACH

The literature analysis of biomethane and biohydrogen production in ASBR/AnSBBR
reactors showed that many variables in the process can affect productivity and stability of
bioenergy generation. Parameters that influenced production, as indicated in this review, were
pH, temperature, inoculum, substrates (wastewaters), operation mode, cycle and feeding
times, organic load and biomass immobilization. It cannot be forgotten that these factors
interact mutually, and this can lead to important variations in the magnitude of the process
performance depending on the specific combination of the adopted operational variables.
Tables 4 and 5 show, respectively, comparisons between the studies that aimed at
biomethane and biohydrogen production using the reactors presented in this review.
Methane production viability can be verified in numerous studies, resulting in
conclusions about what the optimal operational conditions would be.
An inoculum for methanogenic reactors is easily found in anaerobic environments full of
organic load, such as wetlands, swamps, lagoons, lakes, marine sediment, bovine rumen and
slaughterhouses. In most cases, there is no need for inoculum pretreatment.
Many substrates are being used with good results for methanogenesis. Among them are
cheese whey and glucose as well as the effluents of biodiesel production, automotive and
personal care industries and ethanol and sugar.
With regard to the volumetric/specific applied organic load of these substrates, there is a
clear trend toward more stability and methane production when organic loading is rising until
a maximum. Greater values cause the system to fail.
With regard to the cycle time of methanogenic reactors, a time of 8 hours is frequently
used with good results. When fed-batch modes are chosen, it is also possible to see some
tendency in the use of the ration between feeding time and cycle time equals to 0.5.
When the temperature and pH of biomethane production processes are assessed, the
optimal temperature ranges are 5-15°C for psychrophilic processes, 35-40°C for mesophilic
process and approximately 55°C for thermophilic. The pH must be neutral, between 6.5 and
7.5.
Numerous kinetic models can be used to fit methanogenic experimental data. The first
order model can be highlighted from these because of its simplicity of use.
In general, if the main purpose of the process is the organic matter removal, with energy
production only a secondary objective and advantage, a methanogenic treatment is an
excellent option.
On the other hand, studies focused in biohydrogen production have many divergent
conclusions about optimal operational conditions.
Table 4. Comparison between studies aimed at biomethane production in ASBR/AnSBBR
VF AVOL MPr MYRL VCH4

Feed strategy tC VR T CM
Reference Reactor Effluent tF/tC (L. (gCOD. (molCH4. (molCH4. (NmL.cycle- %CH4
(B/FB) (min) -1 (L) (°C) -1 -1 (mmol.L-1)
cycle ) L .d ) m-3.d-1) kgCOD-1) 1
)
B 0.02 480 2 5 30 2.44 6.2 3.5 231.9 11.8 69
Biodiesel
Selma et al.,
ASBR/ MS FB production process 0.25 480 2 5 30 2.44 - - - 11.4 69
2010
(synthetic)
FB 0.50 480 2 5 30 2.36 - - - 11.3 70
B 0.02 480 1.5 3 30 1.5 1.8 1.3 40.0 0.6 72
Biodiesel
Bezerra et
AnSBBR/ LPR B production process 0.02 480 1.5 3 30 4.5 7.6 2.5 171 2.4 57
al., 2011
(synthetic)
B 0.02 480 1.5 3 30 6.0 4.8 1.6 108 1.6 51
B 0.02 480 2 5 30 2.4 8.5 4.2 318.9 13.9 68
B Biodiesel 0.04 240 1 5 30 1.8 9.9 1.9 184.4 7.33 64
Silva et al.,
ASBR/ MS production process
2013 FB 0.50 480 2 5 30 2.4 - - 12.8 72
(industrial)
FB 0.50 480 2 5 30 3.6 - - 12.9 64
Effluent of
B acidogenic reactor 0.02 480 1.5 3.0 30 8.22 128 16 7567 25.6 85
treating sucrose
Lullio et al., Effluent of

AnSBBR/ MS
2014. B acidogenic reactor 0.02 480 1.5 3.0 30 7.21 123 16 8112 25.2 87
treating glucose
B sucrose 0.02 480 1.5 3.0 30 7.04 41 7 3765 15.9 50
FB 0.5 480 1.5 3 30 4.5 9* - 652* 9.20 71
Biodiesel
Lovato et al.,
AnSBBR/ LPR FB production process 0.5 480 1.5 3 30 6.0 12* - 839* 11.9 73
2012
(synthetic)
FB 0.5 480 1.5 3 30 7.5 14* - 962* 14.3 65
FB Biodiesel 0.50 480 1.5 3 30 6.0 - - - - -
Lovato et al.,
AnSBBR/ LPR production process
2013 FB 0.50 480 1.5 3 30 7.5 8* - 170* 3.62 62
(industrial)
VF AVOL MPr MYRL

Feed strategy tC T VCH4
Reference Reactor Effluent tF/tC (L. VR (L) (gCOD. (molCH4. (molCH4. CM (mmol.L-1) %CH4
(B/FB) (min) -1 (°C) (NmL.cycle-1)
cycle ) L-1.d-1) m-3.d-1) kgCOD-1)
Mockaitis et B 0.02 480 2.0 2.0 30 2.50 26.6 - 397.9 - 60
ASBR/ MS Cheese whey
al., 2006 B 0.02 480 2.0 2.0 30 4.79 60.8 - 908.3 - 60
Bezerra et B 0.02 480 2.5 2.5 30 6.0 10.1 - 189 - 70

AnSBBR/ LPR Cheese whey
al., 2007. FB/B 0.50 480 2.5 2.5 30 12.0 16.3 - 304 - 65
Albanez et B 0.02 480 1.0 3.0 30 4.41 37 10.7 2508 21.8 76

AnSBBR/ MS Vinasse
al., 2014 B 0.02 480 1.0 3.0 30 5.54 43 9.8 2916 23.1 77
Table 5. Comparison between studies aimed at biomethane production in ASBR/AnSBBR
VF AVOL MPr MYRL VCH4 CM

Feed strategy tC VR T
Reference Reactor Effluent tF/tC (L. (gCOD. (molCH4. (molCH4. (NmL.cycle- (mmol. %CH4
(B/FB) (min) (L) (°C)
cycle-1) L-1.d-1) m-3.d-1) kgCOD-1) 1) L-1)
Rodrigues et Personal
AnSBBR/ LPR B 0.02 480 2.5 2.5 30 1.5 - - - 0.25 -
al., 2011 care industry
FB/B 0.50 480 0.88 2.5 30 1.5 - - - 1.60 -
FB/B 0.50 480 0.88 2.5 30 0.9 - - - 1.00 -
Bezerra et Cheese
AnSBBR/ LPR FB 0.75 480 1.5 2.5 30 6.0 - - - 17.68 -
al., 2009 whey
FB 0.25 480 1.5 2.5 30 12.0 - - - 23.29 -
FB 0.50 480 1.5 2.5 30 12.0 - - - 20.19 -
B 0.02 480 2.0 2.0 30 2.86 - - - 3.0 -
Oliveira et Automotive
AnSBBR/ MS B 0.02 480 2.0 2.0 30 2.61 - - - 4.0 -
al., 2008 industry
B/FB 0.02/0.50 480 1.0/1.0 2.0 30 2.57 - - - 3.1 -
B Low- 0.02 480 2.5 2.5 30 1.5 - - - - 68
Borges et FB strength 0.25 480 2.5 2.5 30 1.5 - - - 2.2 59
AnSBR/ MS
al., 2004 wastewater
FB 0.50 480 2.5 2.5 30 1.5 - - - 1.0 43
(synthetic)
B Domestic 0.01 720 2.0 5.0 30 1.55 - - - 12.0 -
Chebel et
ASBR/ MS wastewater
al., 2006 B 0.02 480 2.0 5.0 30 2.36 - - - 12.0 -
(synthetic)
B Domestic 0.02 480 1.87 2.50 30 1.5 - - - 7.0 64
Bezerra et
ASBR/ MS wastewater
al., 2005 B 0.02 480 1.25 2.50 30 1.5 - - - 3.0 56
(synthetic)
Cheong and B 0.02 720 - 2.6 35 12.0 32 929
Hansen, ASBR Glucose
FB 0.83 720 - 2.6 35 12.0 141 4121
2008
HDT =
B - 6.2 - 2.5 39 20.4 187.5 - - - -
Kwon and
Canned fruit days
Nakasaki, ASBR
industry HDT =
2013
B - 3.1 - 2.5 39 20.4 44.6 - - - -
days
Table 6. Comparison between studies aimed at biohydrogen production in ASBR/AnSBBR
tC VF VR T AVOL MPr SMPr MYAL MYRL YS

Reference Reactor set up Substrate (L. (gCOD. (molH2.m .-3
(molH2. (molH2. (molH2. (molH2.
(h) (L) (°C)
cycle-1) L-1.d-1) d-1) kgTVS-1.d-1) kgCOD-1) kgCOD-1) molsubs-1)
AnSBBR/ LPR in Pure glycerine 3.0 1.5 3.5 30 17.8 100.9 33.6 5.7 20.0 1.19
Lovato et al. (2014) fed-batch (tF/tC = Industrial
0.5) 3.0 1.5 3.5 30 19.0 9.7 0.6 0.5 1.3 0.10
glycerine
Pure glycerine 4.0 1.5 3.5 30 12.9 67.5 19.8 5.2 21.1 -
Moncayo Bravo et
AnSBBR/ LPR Industrial
al. (2014) 4.0 1.5 3.5 30 12.8 8.3 1.0 0.7 1.5 -
glycerine
AnSBBR/ MS in Sucrose based 4.0 1.5 3.5 30 13.5 54.9 7.45 4.03 22.1 1.48
Inoue et al. (2014) fed-batch (tF/tC = synthetic
0.5) effluent 2.0 1.5 3.5 30 27.0 81.2 8.11 3.11 25.3 1.40
Sucrose based 4.0 1.5 3.6 30 13.4 33.3 5.1 2.51 14.1 1.04
Manssouri et al.
AnSBBR/ MS synthetic
(2013) 2.0 1.5 3.6 30 18.1 38.9 5.5 2.13 12.4 0.87
effluent
Sucrose based 4.0 1.9 4.5 30 17.1 43.4 8.5 2.43 11.5 1.4
Santos et al. (2014) AnSBBR/ LPR synthetic
effluent 2.0 1.9 4.5 30 9.3 35.2 6.9 3.66 19.6 1.0
Glucose based 4.0 1.9 4.5 30 10.5 51.2 4.9 4.9 20.6 0.94
Souza et al. (2014) AnSBBR/ LPR synthetic

effluent 3.0 1.9 4.5 30 20.0 60.9 5.8 3.1 20.2 0.58
Sucrose 4.0 1.5 3.5 30 10.9 31.1 - 3.32 16.4 4.45
Lima et al. (2014) AnSBBR/ MS Lactose 4.0 1.5 3.5 30 10.9 10.5 2.08 1.19 6.92 1.41
Cheese whey 3.0 1.5 3.5 30 14.6 18.1 4.32 1.37 4.50 1.74
Corn-starch
based synthetic 6.0 1.5 3.0 35 40.0 114.7 12.9 2.7 - 0.51*
Arooj et al. (2008) ASBR/ MS effluent
3.0 1.5 3.0 35 80.0 183.3 21.4 2.3 - 0.44*
Liquid manure
Wu et al. (2009) ASBR/ LPR added with 4.0 1.0 4.0 37 15.2 107.1 - - - 1.63
glucose
Table 7. Comparison between studies aimed at biohydrogen production in ASBR/AnSBBR
tC VF VR T AVOL MPr SMPr MYAL MYRL YS

Reference Reactor set up Substrate (L. (gCOD. (molH2.m-3. (molH2. (molH2. (molH2. (molH2.
(h) (L) (°C)
cycle-1) L-1.d-1) d-1) kgTVS-1.d-1) kgCOD-1) kgCOD-1) molsubs-1)
Searmsirimongkol et Alcohol distillery

ASBR/ MS 4.0 1.0 4.0 37 60.0 147.7 12.0 2.4 7.7 -
al. (2011) effluent
Cheong and Hansen Glucose based 2.0 1.3 2.6 34.5 150 0.6 6.7 - 0.8 -
ASBR/ MS
(2006) synthetic effluent 3.0 1.3 2.6 34.5 50 0.4 16.8 - 4.7 -
Venkata Mohan et al. Distillery
AnSBBR/ LPR 24 0.8 0.8 28 9.6 32.5 - - 6.98 -
(2008)(a) wastewater
Venkata Mohan et al. Glucose based
AnSBBR/ LPR 24 1.4 1.4 28 4.75 4.7 - - 9.3 2.1
(2008)(b) synthetic effluent
Effluent of palm
Badiei et al. (2011) ASBR/ LPR 24 1.0 3.0 37 6.6 260 - 13.4 36.9 -
oil mill
Saraphirom and
Sweet sorghum
Reungsang et al. ASBR/ MS 12 0.7 1.3 30 17.5 147.5 - 8.41 0.68
syrup
(2011)
Cassava alcohol
Intanoo et al. (2012) ASBR/ MS 4 0.75 4.0 55 68 78.0 20.8 1.11 4.8 -
distillery
Solid waste of
Mohanakrishna et
AnSBBR / LPR diluted vegetable 24 1.4 1.4 28 44.3 14.1 - 0.3 2.22 -
al., 2011
market
Buitrón and Carvajal,
ASBR / MS Tequila vinasse 6 3.0 6.0 35 6.0 48 - 8 - -
2010
A large variety of microorganisms produce hydrogen, with the genus Clostridium as one
of the most important species. The inoculum used requires a pretreatment to eliminate biogas
consumer species (methanogenic microorganisms) to optimize the process. Among
pretreatments cited in this review, the most important are thermal shock, acid and radiation
pretreatments.
A great number of substrates is being used in biohydrogen production by dark
fermentation. Glucose and sucrose are the most often used substrates because the greatest
theoretical values of hydrogen production are reached by carbohydrate degradation. To make
the process of hydrogen production sustainable and competitive compared to other alternative
energy sources, substrates must have both low cost and high availability. Urban and industrial
wastewaters and urban solid waste fit well in this description. The influence of
volumetric/specific organic load of these substrates applied to acidogenic reactors was
detected, although it is varied.
Concerning the acidogenic reactor cycle time, these are usually shorter than the
methanogenic reactor cycle time because the maximum speed of methanogenic
microorganisms growth is lesser than that of hydrogen producer bacteria. Cycle times
between two or four hours seem to be indicated. For the feeding time, the ratio feeding
time/cycle time of 0.5 is proved to enhance quality and production of biohydrogen.
From the results, an optimal temperature or even an optimal range of temperatures was
not observed for the biological production of hydrogen. A great number of studies converge
to temperature values varying between 30.0°C and 36.8°C, but a meticulous analysis of each
process is necessary, in particular to define the best values for this parameter.
Once again, for pH, there is a disagreement about the optimal value for hydrogen
production. It seems that a pH close to 5.0 is the most indicated by the published literature.
Many acidogenic kinetic models are being developed and applied with success for these
biological process. The Gompertz model was developed in the early 1990s and is receiving
increasingly attention from researchers. This model describes the growth of pure cultures and
is being continuously used to analyze the fermentative process of H2 production. The literature
lacks mathematical models that consider the metabolic pathways of the microorganisms
involved in the process of biohydrogen production.
At this moment, given the uncertainties regarding the individual or combined influence of
the operational factors, the most secure alternative for the implementation of any process at
the industrial scale is the systematic study by laboratory scale studies regarding the process
itself and not only the use of existent studies. The biological processes of hydrogen
production are currently under study and development. They represent a key component of
the generation of clean energy and of the construction of a more sustainable world.
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Chapter 6
MICROBIAL EXOPOLYSACCHARIDES AS EFFICIENT

BIOFLOCCULANTS IN WASTEWATER TREATMENT
Habib Chouchane1,, Mohamed Neifar1,2, Atef Jaouani2,

Mouna Mahjoubi1, Ahmed Slaheddine Masmoudi1
and Ameur Cherif1
1
Laboratory of Biotechnology and Bio-Geo Resources
Valorization, Higher Institute for Biotechnology, Biotechpole
Sidi Thabet, University of Manouba, Ariana, Tunisia
2
Laboratory of Microorganisms and Active Biomolecules,
Faculty of Sciences of Tunis, University of
Tunis El Manar, Tunis, Tunisia
ABSTRACT
Flocculants are chemicals that stimulate flocculation by aggregation of colloids and
other suspended particles. They are extensively applied in domestic and industrial
wastewaters treatment. Synthetic organic flocculants as well as inorganic flocculants,
including polyaluminium chloride salts and aluminium sulphate, were shown to be
associated to various human health problems such as neurotoxicity, cancer and
Alzheimer‘s disease. They are also known to be non biodegradable and not environment-
friendly. Because of their adverse nature, more attention has been given to the use of
microbial flocculants. A number of microbes i.e. bacteria, yeast, and fungi, are described
as bioflocculants producers with interesting bioremediation potential.
Current applications are mainly in industrial sectors associated with textiles,
detergents, adhesives, microbial enhanced oil recovery, and wastewater treatment. These
biomolecules have particular advantages such as their safety for ecosystems as they are
biodegradables and harmlessness to humans and animals and their high flocculating
effects. Microbial bioflocculants are mostly composed of polysaccharides, proteins,
glycoproteins, nucleic acids and lipids. For examples, flocculant produced by Rhodoocus

Corresponding authors: Habib Chouchane (chouchane_habib@voila.fr); Mohamed Neifar (mohamed.naifar@
gmail.com).
164 Habib Chouchane, Mohamed Neifar, Atef Jouani et al.
erythropolis (Guo et al., 2015) is predominantly protein in nature, those of Bacillus

mucilaginosus (Lian et al., 2008) and of Proteus mirabilis (Zhang et al., 2009) were
shown to be glycoproteins, whereas, Paenibacillus elgii (Li et al., 2013), Serratia ficaria
(Gong et al., 2008) and Klebsiella mobilis (Wang et al., 2007) bioflucculants are mainly
polysaccharides. Exopolysaccharides bioflocculants have gained increasing attention
since they contain ionizable functional groups such as carboxyl, amine, sulphate, acetate,
and hydroxyl groups which enable these biopolymers, to be effective in removing
suspended solids, heavy metals and in reducing the turbidity of different types of
industrial wastewater effluents. They promote flocculating by bridges formation between
particles resulting in their aggregation and precipitation.
This chapter discusses various studies conducted so far about microbial flocculants.
An attempt was taken to recapitulate the main types of flocculants, flocculation
mechanisms, the most important bioflocculant microorganisms, physicochemical factors
influencing the flocculating activity with a special focus on applications of
exopolysaccharide based flocculant in traitment of wastewater.
Keywords: Microbial exopolysaccharides, bioflocculant, flocculant properties,

bioremediation, wastewater treatment
INTRODUCTION
Industrial and municipal wastewaters and effluents usually contain fine suspended solids,
dissolved solids, inorganic and organic particles, metals and other impurities and pollutants.
Various traditional and advanced technologies have been used to remove the colloidal
particles from wastewater, such as ion exchange, membrane filtration, precipitation, flotation,
solvent extraction, adsorption, coagulation, flocculation, biological and electrolytic methods
(Sharma et al., 2006; Mishra et al., 2011; Lee et al., 2014). Among these methods,
flocculation is one of the most widely used solid-liquid separation process for the removal of
suspended and dissolved solids, colloids and organic matter present in wastewater (Renault et
al., 2009; Rojas et al., 2010). Flocculation is a simple and efficient method extensively used
in the cases palm oil mill effluents, textile wastewater, pulp mill wastewater, oily wastewater,
and others effluent treatments (More et al., 2014; Wei et al., 2008). The flocculants are
classified into three groups i) inorganic flocculants ii) organic flocculants and iii) natural or
bioflocculants (Li et al., 2008; Bezawada et al., 2013). Inorganic and organic flocculants have
been implicated in human health and environmental. As an example, polyacrylamide contains
acrylamide monomers verified to be neurotoxic and carcinogenic to humans (Sharm et al.,
2006; Beland, 2010; Lee et al., 2014). In addition a clear link was demontrated between
aluminium in drinking water and human neurological disorders (Banks et al., 2006,
Tomljenovic, 2011). These alarming concerns argued the need for safe and environment-
freindly alternatives. Microbial flocculants are considered a potential promising alternative to
conventional chemical polymers because of their biodegradability, non-toxicity, non-
secondary pollution and efficiency. To date, several species of bioflocculant-producing
microorganisms have been recently reported and their bioflocculant were characterized (Table
1). They are mainly composed by exopolysaccharides (EPS) and/or proteins (Table 2). Their
production depends on the type of microbial strain used, physical conditions maintained
during fermentation and on kind of media components (Donot et al., 2012).
Microbial Exopolysaccharides As Efficient Bioflocculants … 165
This production is generally favoured by high carbon and low nitrogen substrate ratio
(Lim et al., 2004; Luo et al., 2009). Recent reports in literature indicate a regain of interest on
the optimization of culture conditions for bioflocculant-producing bacteria (Liu et al., 2014)
and on the characteristics of microbial EPS flocculants (Liu et al., 2009). In need, EPS have
been widely investigated in the treatment of several industrial effluents such as brewery
wastewater (Lian et al., 2008; Gong et al., 2008; Ugbenyen and Okoh, 2014), dying
wastewater (Li et al., 2013), swine wastewater (Guo et al., 2015), pharmaceutical wastewater
(Lian et al., 2008) textile effluents (Simphiwe et al., 2012) pulp and paper mill effluent (Gong
et al., 2008), and dairy woolen wastewater (Patil et al., 2011) (Table 2). This chapter provides
current state of the art knowledge with regard to microbial EPS flocculants and their
avantageous. Special emphasis has been laid on mechanisms of flocculation, physico-
chemical factors influencing the flocculating activity, and potential application of EPS in
wastewater treatment. Throughout this chapter the terms microbial EPS, microbial polymer
and bioflocculants have been used synonymously to adress exopolysaccharide.
I. TYPES OF FLOCCULANTS
Flocculants are materials used in fast solid-liquid separations, they act on the molecular
level on surface of the particles to reduce repulsive forces and mounting attractive forces
(Wei et al., 2008; Yang et al., 2010; Kalia and Avérous, 2011). Their addition enables
dispersed particles to aggregate together to form flocs of a size thus allowing them to settle
speedily and clear the system. In wastewater treatment they improve the stability of flocs
(Wong et al., 2006; Zhang and Li, 2010; More et al., 2014). The present chapter classifies the
flocculants that have been applied in wastewater treatment into three categories including
chemical flocculants, bioflocculants and grafted flocculants as shown in Figure 1. i) Chemical
flocculants derived from chemically/petroleum based materials, ii) Bioflocculants are mainly
explored on the past few years and sourced from natural materials, and iii) Grafted flocculants
are explored recently and synthesised by combining the properties of chemical and
bioflocculants. In this chapter more attention was given to microbial bioflocculants.
1. Chemical Flocculants
Chamical flocculants that are applied in wastewater traitement can be devided into groups
inorganic mineral salts and organic polymeric materials.
Inorganic Flocculants
Inorganic flocculants fall into inorganic small molecular flocculants and inorganic high
molecular flocculants by different molecular weight. Inorganic high molecular flocculants are
used more and more due to its excellence of high molecular weight, strong ability of
adheresion, flocculation and charge neutralization, immediate sedimentation and short
consumption compared with inorganic small molecular flocculants (Zhao-kun et al., 2000;
Lee et al., 2014).
Table 1. Important bioflocculant producing microorganisms
Bioflocculant Flocculant
Microorganisms Carbon source Nitrogen source References
yield (g/L) activity
Bacteria
Paenibacillus elgii sucrose peptone 25,63 87% Li et al. (2013)
Staphylococcus sp. yeast extract, urea,
brewery wastewater 15 96,8% Zhang et al. (2007)
Pseudomonas sp. (NH4)2SO4
yeast extract, urea,
Agrobacterium sp. sucrose 14,9 97,3% Li et al. (2010)
(NH4)2SO4
Bacillus mojavensis L-glutamic acid NH4Cl 5,2 96,12% Elkady et al. (2011)
Halomonas sp. glucose NH4Cl 4,52 95-97% He et al. (2009)
Ochrobacterium ciceri corn stover hydrolysates yeast extract, urea 3,8 94% Wang et al. (2007)
Rhizobium,radiobacter
rice straw fermentation + glucose yeast extract, urea 2,79 92,45% Zhao et al. (2012)
Bacillus sphaericus
Pseudomonas alcaligenes glucose nd 2,89 96,8% Wang et al. (2009)
yeast extract, urea,
Klebsiella pneumoniae glucose 2,84 97,5% Zhao et al. (2013)
(NH4)2SO4
Klebsiella mobilis dairy wastewater + ethanol 2% nd 2,58 95,4% Wang et al. (2007)
Sirrata ficaria lactose beef extract, urea 2.41 97,15% Gong et al. (2008)
Waste fermenting liquor
Bacillus subtilis, Bacillus
(ethanol, acetate, propionate, urea, yeast extract 2,12 >99% You et al. (2008)
fusiformis, Bacillus flexus
butyrate, valerate) + glucose
Sirratia sp. wastewater sludge peptone, caséine 2.45 70.4% Bezawada et al. (2013)
Cobetia spp. glucose casein 2.1 92,2% Ugbenyen et al. (2012)
Yeasts and fungi
Penicillium purpurogenum glucose yeast extract 6.4 > 96% Liu and Cheng (2010)
Streptomyces sp. glucose (NH4)2SO4 3.37 89,26% Nwodo et al. (2012)
Aspergillus niger palm oil mill effluent glutamic acid 2.6 79% Aljuboori et al., (2014a)
Penicillum sp. glucose or starch or molasses yeast extract nd 93% Liu and Cheng (2010)
Table 2. Application of microbial flocculants in wastewater treatment
Composition and nature of the Molucular Main results

Microorganisms FA (%) Type of wastewater References
bioflocculant weight (Da) %COD %TSS %TU %DY
Bacteria
% carbohydrate: 97.5
Domestic wastewater 74.6 93.3 nd nd
% protein: 2.5 Lian et al.
nd Bacillus mucilaginosus 89% Brewage wastewater 70.5 93.6 nd nd
Exopolysaccharide protein based (2008)
Pharmaceutical wastewater 66.2 88.4 nd nd
bioflocculant
Brewery wastewater 80.7 91.8 nd nd
% carbohydrate:100
5 Meat processing wastewater 76.3 93.7 nd nd Gong et al.
% protein: 0 3.13x10 Serratia ficaria 95.4%
Sauce brewing wastewater 64.1 93.7 nd nd (2008)
Exopolysaccharide based bioflocculant
Pulp and paper mill effluent 72.1 nd nd 99.9
%carbohydrate:100
Li et al.
% protein: 0 3.5x 106 Paenibacillus elgii 87% Dying wastewater 68 83 nd 88
(2013)
% carbohydrate:100
Wang et al.
% of protein: 0 nd Klebsiella mobilis 95.4% Dyes in aqueous solutions nd nd nd 91
(2007)
% protein: 30.9 Dyes in aqueous solutions Zhang et
1.2x105 Proteus mirabilis nd nd nd nd 2.005g/g
Exopolysaccharide protein-based (basic blue 54 (BB54) al.(2009)
bioflocculant
% polysaccharide: 3.3
Rhodococcus Guo et al.,
% protein: 95.6 3.93x105 93.8% Swine wastewater 48.3 nd 75.8 nd
erythropolis 2015
Protein based bioflocculant
% carbohydrate: nd
Patil et al.
% protein: nd 2x106 Azotobacter indicus 72% Dairy woolen wastewater 79 nd 68 nd
(2011)
Exopolysaccharide based bioflocculant*
A consortium of bacteria
% carbohydrate: nd
% protein: nd Staphylococcus sp. Indigotin printing ˃79.2 nd nd nd Zhang et al.,
nd nd
Exopolysacharide protein based Pseudomonas sp. Dyeing wastewater nd nd nd ˃86.5 (2007)
bioflocculant
% carbohydrate: nd
Two strains of Rhizopus Pu et al.,
% protein: nd nd 95.46% Potato starch wastewater 54.09 nd 92.11 nd
sp (2014)
Exopolysacharide based bioflocculant
Composition and nature of the Molucular Main results

Microorganisms FA (%) Type of wastewater References
bioflocculant weight (Da) %COD %TSS %TU %DY
A consortium of bacteria
% carbohydrate 66.7
% protein 31.5 Brewery wastewater 95.4 nd 94.8 nd
Cobetia sp. Ugbenyen et
nd 90% Dairy wastewater 99.1 nd 81.2 nd
Bacillus sp. Okoh, (2014)
Exopolysacharide protein based River water 73.9 nd 98.9 nd
bioflocculant
% carbohydrate: nd Brewery wastewater 99.7 nd 93.9 nd
Halobacillus sp. Cosa et Okoh,
% protein: nd nd 72% Dairy wastewater 99.9 nd 88.3 nd
Oceanobacillus sp. (2013)
Exopolysaccharide based bioflocculant River water 70.8 nd 98.6 nd
Bacillus subtilis,
Exiguobacterium
acetylicum, Klebsiella
% carbohydrate: nd Simphiwe et
nd terrigena, nd Textile industrial effluent nd nd nd 97.04
% protein :nd al., (2012)
Staphylococcus aureus,
Pseudomonas
pseudoalcaligenes,
fungi
Deng et al.,
% protein: 21.6 3,2x105 Aspergillus parasiticus 98.1% Dyeing wastewater nd nd nd 92.9
2005
Exopolysaccharide protein based flocculant
% carbohydrate:69.7
Aljuboori et al.,
% protein: 28.5 2.574x104 Aspergillus flavus >90% nd nd nd nd nd
(2014b)
Aljuboori et al.,
% protein: 31.4 nd Aspergillus niger 79% River water nd nd 63 nd
(2014a)
FA: flocculation activity in kaolin suspensions, COD: the maximal removal ration of chemical oxygen demand, TSS: the total removal of solid suspension,
TU: turbidity, nd: not determined.
Microbial Exopolysaccharides As Efficient Bioflocculants ... 169
Flocculants
Chemical Grafted Bio-

flocculants flocculants flocculants
Organic Inorganic Microbial Plant
flocculants flocculants flocculants flocculants
Poly- St-g-PAM
Polyacryla Polysaccharides
aluminium CMG-g-PAM
mide chloride;
polyamine, ferric
etc. Protein
chloride,
etc.
Glycoprotein
Lipid
Figure 1. Categories of flocculants.
Among the inorganic high molecular flocculants, the salts of multivalent metals like
aluminium polyaluminium chloride, ferric chloride, ferrous sulphate, calcium chloride and
magnesium chloride (Joo et al., 2007). According to many studies (Brown and Emelko, 2009;
Rojas-Reyna et al., 2010) polyaluminium chloride (PAC) has large market demand as the
wastewater treatment flocculant in the world.
Organic Flocculants
During last decade, many synthetic polymers have been used as the main organic
flocculants. As reported by Suopajärvi et al., (2013) these flocculants are mostly linear water
soluble polymers which are based on repeating units of various monomers such as acrylamide
and acrylic acid. The most used organic flocculants involve polyacrylamide, polyamine,
polyacrylic acid and poly diallyl dimethyl ammonium chloride. Based on the charge type,
these polymers are classified into four forms: cationic (positive charge), anionic (negative
charge), amphoteric (contains both positive and negative charge) and non ionic. The nature of
the charges is the principal parameter that influences the efficiency of flocculation process
followed by molecular weight and the charge density (Sharma et al., 2006; Ahmad et al.,
2008).
Chemical flocculants have many advantages of being effective in terms of flocculating
efficiency and availability. However, inorganic flocculants have been implicated in various
human health problems, they have been reported to be neurotoxic and carcinogenic (Banks et
al., 2006, Beland, 2010; Lee et al., 2014; Tomljenovic, 2011). These are also known to be non
biodegradable and not environmentally friendly (Matthys et al., 2005).
Sharma et al., (2006) added that they are highly sensitive to pH, inefficient towards very
fine particles and in cold water. They are also applicable only to a few disperse systems. The
use of organic flocculants is also debatable because of environmental consequence and health
hazards. Bolto and Gregory (2007) reported that cationic polyelectrolytes are very toxic,
especially to aquatic organisms. Moreover, the majority of synthetic polymer showed an
extremely slow biodegradation and their degraded products are considered hazardous due to
the release of monomers that coold enter in the food chain and cause carcinogenic effect
(Sharm et al., 2006; Brostow et al., 2009). For these reasons and because of the adverse
nature of chemical synthetic flocculants, there is an increasing demand for environment-
freindly and effective flocculants. In this respect, more attention has been given to the use of
flocculants from natural sources with the potential to substitute the chemical flocculants (Li et
al., 2009).
2. Bioflocculants
During recent years, micobial flocculants have emerged to be promising environment-

freindly alternative materials to replace chemical flocculants. They are kinds of metabolites
produced by all types of microbes i.e. bacteria, fungi during their growth. They are mainly
composed of high biopolymers such as extracellular polysaccharide, glycoprotein, protein and
nucleic acid (Gong et al., 2008; Labille et al., 2010).
Many studies reported that these biopolymers are harmless, biodegradable, and do not
lead to secondary pollution. As a result, bioflocculants gained much wider attention and are
being considered as a good replacement for chemical flocculants used in wastewater
treatment and other industrial processes (He et al., 2004; Xia et al., 2008; Salehizadeh et
Shojaosadati 2011; Wang et al., 2011; Feng et al., 2013).
Biopolymers such as microbial exopolysaccharides have been attracting many
researchers. As showen in table 2 sevral microbial flocculants were investigated in

wastewater treatment to remove inorganic solid suspensions (Lian et al., 2008; Gong et al.,
2008; Guo et al., 2015; Patil et al., 2011; Ugbenyen et Okoh, 2014), dye solutions (Deng et
al., 2005; Li et al., 2013; Wang et al., 2007; Zhang et al., 2009, Simphiwe et al., 2012), and
heavy metals (Salehizadeh et al., 2003; Gong et al., 2008).
3. Grafted Flocculants
It is evident that all polymers, whether natural or synthetic, have one or another
disadvantage. Microbial exopolysaccharides are fairly shear stable, in contrast with longchain
synthetic polymers, and are biodegradable.
However, they have lower efficiencies and thus higher concentrations are needed. Many
attempts have been made to obtain modified flocculants by combining the best properties of
biopolymers and synthetic polymers. Many grafted flocculants have been synthesised by
grafting synthetic polymers onto the backbone of natural polymers, hydroxypropyl methyl
cellulose grafted with polyacrylamide (HPMC-g-PAM) (Das et al., 2013); polyacrylamide
grafted starch (St-g-PAM) (Mishra et al., 2011); polyacrylamide grafted carboxymethyl guar
gum (CMG-g-PAM) (Pal et al., 2011).
They were also tested in synthetic wastewater using kaolin suspension. The positive
findings suggested the possible application of modified flocculants in wastewater treatement.
II. FLOCCULATION MECHANISMS

As reported by Lee et al. (2014) development of flocs formation involves several steps
occurring sequentially: i) Dispersion of the flocculant in the solution ii) Diffusion of the
flocculant towards the solid-liquid inter-face. iii) Adsorption of the flocculant onto the surface
of the particles iv) Collision of particles carrying an adsorbed flocculant with other particles
v) Adsorption of the flocculant onto other particles in orderto form microflocs vi) Growth of
the microflocs to larger and stronger flocs by successive collision and adsorption.
1. Mechanism for Chemical Flocculants
According to several studies (Salehizadeh and Shojaosadati, 2002; Bolto and Gregory,
2007; Sharma et al., 2006; Renault et al., 2009; Eric et al., 2014) many mechanisms of
flocculation have been proposed to explain the destabilisation of colloids and the mechanism
of flocs formation such as polymer bridging, polymer adsorption and charge neutralisation,
depletion flocculation, etc.
In many studies, it has been found that the main mechanisms of flocculation involved in
the removal of dissolved and particulate contaminants are charge neutralisation, bridge
formation and electrostatic patch. Conceptual illustrations of these mecanisms are shown in
figure 2, 3 and 4 (Bohuslav Dobias, 2005; Sharma et al., 2006; Lee et al., 2014).
Bohuslav Dobias, 2005.
Figure 2. Schematic of a charge neutralisation flocculation mechanism.
In the bridging mechanism (Figure 3a), polymer segments are adsorbed on multiple
adjacent particles, bridging them together to form an extended network of connections.
Because of the electrostatic repulsions, anionic polymers can attach to negatively charged
surfaces through ion binding if there is a sufficient concentration of metal ions. As an
example, calcium ions (Ca2+) can promote adsorption by linking carboxyl (-COOH) groups
with anionic sites on the particle surface.
The length of the polymer chains should be sufficient to extend from one particle surface
to another (figure 3b). A polymer with longer chains (high molecular weight) should be more
effective than with shorter chains (low molecular weight) (Lee et al., 2012; Razali et al.,
2011). For more effective bridging, Sher et al., (2013) explain that there should be sufficient
unoccupied surface on particle for attachment of segments of polymer chains absobed on
other particles. Hence the amount of polymer should not be excessive, otherwise the particle
surfaces will be overly coated with polymer such that no sites are free to bridge with other
particles. In this case particles are said to be restabilised (figure 3b). The mechanism is much
preferred for high molecular weight polyelectrolytes of low large density.
Charge neutralisation is postulated as the major mechanism for cases where the flocculant
and the absorption site are of opposite charge. Generally, particles in wastewater are
negatively charged and thus inorganic flocculants (metal salts) and cationic poly-electrolytes
are preferable. It follows that flocculation occur as a result of the reduced surface charge of
the particles (reduction of zeta potential) and hence a decreased electrical repulsion force
between colloidal particles, which allows the formation of van der Waals force of attraction to
encourage initial aggregation of colloidal and fine suspended materials to form microflocs
(figure 4). For effective flocculation, many studies reported that optimum flocculation occurs
at polyelectrolytes dosages around that needed to give a zeta potentiel close to zero (just
neutralize the particle charge).
In such a case, particles agglomerate under the influence of the van der Waal‘s forces.
These particles can disperse again if too much polymer is used because of the charge reversal.
The contribution of van der Waals forces renders negligible as the particle size increases (Eric
et al., 2014).
Bridging capability is reduced when high charge density polyelectrolytes with low
molecular weight adsorb on negative surfaces with a fairly low density of charged sites.
Sharma et al., 2006; Lee et al., 2014.
Figure 3. (a) Schematic of polymer flocculation by bridging mechanism, (b) Restabilisation of particles.
Arrows in (c) show attraction of opposite charges. (Sharma et al., 2006; Lee et al., 2014).
Figure 4. (a) negatively charged particles, (b) cationic flocculant, (c) charge neutralisation flocculation
by electrostatic patch mechanism.
Under these conditions, another mechanism of flocculation arise, which is known as

electrostatic patch mechanism (Lee et al., 2014). As shown in figure 4.c when a highly
charged cationic polymer adsorbs on a weakly charged negative surface, to induce overall
neutrality, it is not physically possible for each surface charged site to be neutralised by a
cationic polymer segment. As reported by Bolto and Gregory (2007), in these conditions there
is formation of cationic patches between surfac of uncoated negatively charged surfaces. An
important result of these cationic is that, as particles approach closely, there is an electrostatic
attraction between positive patches and negative areas, which can give particle attachment
and hence flocculation (figure 4.c).
2. Mechanism for Bioflocculants

The flocculation mechanism of microbial flocculent has been widely investigated in

many laboratories. Studies have shown that the chemical and physical properties of
bioflocculants determined the bioflocculation mechanism and performance.
The properties of cationic microbial exopolysaccharides, including their cationic
behaviour (reactive amino and hydroxyl groups) and their high molecular weight, are used for
flocculation by charge neutralisation and by bridging mechanism.
Guibal and Roussy, (2007) investigating flocculation of dye-containing solutions using
the chitosan, reported that the anionic dye was electrostatically attracted by protonated amine
groups from chitosan leading to neutralisation of the anionic charges of dyes and then the
flocculation was further enhanced by the bridging mechanism which bind the agglomerates
together and settle. Proteus mirabilis TJ-1 (Xia et al., 2008) and Bacillus megaterium TF10
(Yuan et al., 2011) both have been found to produce polysaccharides with a number of active
sorption sites and bridging has been proposed as the primary mechanism for flocculation for
these bioflocculants. Many findings (Ebeling et al., 2005; Yuan et al., 2011) indicating that
the bridging mechanism is common for bioflocculants given the chemical composition,
molecular weight, and molecular structure of many bioflocculants, all properties which
influence the effectiveness of the bridging mechanism.
In a recent study (Yin et al., 2014) high molecular weight polysaccharides were identified
as the main constitutes of a bioflocculant produced by Klebsiella sp. The polysaccharides
have long backbones (composed of rhamnose, mannose, glucose, and galactose) with a large
number of functional groups (-OH, -NH and -COOH). High molecular weight and active sites
are likely to adsorb particles and forming large flocs. Bridging is suggested as the main
mechanism for flocculation with kaolin.
Anionic exopolysaccharides are unable to flocculate anionic contaminants from the
wastewater without the assistance from a cationic flocculant. As demonstrated by several
studies (Roussy et al., 2005; Wu et al., 2012) addition of inorganic metal salts (e.g. ferric and
aluminium salts) or cationic polymer (e.g. chitosan) before addition of anionic
exopolysaccharides is necessary for charge neutralisation of negatively charged impurities.
After charge neutralisation, anionic exopolysaccharides with negatively charged
backbone (hydroxyl and carboxyl groups) allowed the polymer molecules to be extended into
solution and promote bridging of flocs. However, Satish et al., (2010) reported that an
exopolysaccharide produced by Azobacter indicus, identified as aliginate like polymer,
showed cation-dependent flocculating activity. Bridging, charge neutralization and a
combination of the two mechanisms have been identified as dominant mechanisms for
microbial bioflocculants.
III. EXOPOLYSACCHARIDE BASED BIOFLOCCULANTS

III.1. Exopolysaccharide Based Biofloculant Producing Microrganisms
Because of the adverse nature of chemical synthetic flocculants, more attention has been
given to microorganisms producing flocculants. According to the findings of Xia et al.,
(2008) screening for microorganisms with high bioflocculant-producing capability and high
flocculating efficiency is vital for success in this field. As presented in table 1 and table 2
many bacteria and fungi have been reported to produce extracellular biopolymers such as EPS
with considerable flocculating activity. These bioflocculant-producing microorganisms
include Klebsiella mobilis (Wang et al., 2007), Bacillus subtilis, Bacillus fusiformis, Bacillus
flexus (You et al., 2008), Bacillus sp. (Zheng et al., 2008), Serratia ficaria (Gong et al.,
2008), Pseudomonas alcaligenes (Wang et al., 2009), Halomonas sp. (He et al., 2010),
Bacillus moavensis (Elkady et al., 2011), Paenibacillus elgii (Li et al., 2013), Rhizobium
radiobacter, Bacillus sphaericus (Zhao et al., 2012), Agrobacterium sp. (Li et al., 2010),
Staphylococcus sp. Pseudomonas sp. (Zhang et al., 2007), Penicillium purpurogenum (Liu
and cheng, 2010), Ochrobacterium ciceri (Wang et al., 2007), Klebsiella pneumoniae (Zhao
et al., 2013), Streptomyces sp. (Nwodo et al., 2012), Aspergills niger (Aljuboori et al., 2014a),
Bacillus licheniformis (Li et al., 2009), Rhodococcus erythropolis (Guo et al., 2015),
Halobacillus sp, oceanobacillus sp. (Cosa and Okoh, 2013), Bacillus subtilis,
Exiguobacterium acetylicum, Klebsiella terrigena, Staphylococcus aureus (Simphiwe et al.,
2012), Rhizobium radiobacter, Bacillus sphaeicus (Wang et al., 2011), Cobetia sp. Bacillus
sp. (Ugbenyen et al., 2014).
As stated in table 2 the majority of bioflocculants produced by different microorganisms
are usually high molecular weight polymers such as EPS, proteins, and glycoproteins.
Although a number of microorganisms have been screened for their bioflocculant-

producing capabilities, to-date, very little has been accomplished on a commercial scale.
Industrial utilisation of exopolysaccharide based bioflocculants is hindered by their costly
productions and poor yields.
To overcome these limitations, studies have focused on the identification of bioflocculant
producer microorganisms, improvement of strains and bioflocculating efficiency and search
for low cost feedstocks. The use of two or more microbes in consortium was to improve the
yield of EPS and their flocculating activity.
Zhang et al. (2007) found that the flocculing activities of the bioflocculant produced by
combined cultivation of Staphylococcus sp. and Pseudomonas sp. was better than that of the
bioflocculant produced by either of the strains under the same condition. In addition, Zhao et
al., (2012) demonstrated that a compound bioflocculant produced by a mixed culture of
strains Rhizobium radiobacter and Bacillus sphaericus showed higher flocculating activity
when compared to individual strains.
Similarly, the bioflocculant produced by a consortium of Cobetia sp. and Bacillus sp.
showed 99%, 90.2 and 78.8% flocculating activities for riverwater, brewery and dairy
wastewaters, indicating that bioflocculant promises high potential in wastewater treatment
(Ugbenyen and Okoh, 2014).
As a possible cost-cutting measure several studies investigated the use of low-cost
substrates in growth media for bioflocculant production. Some low cost substrates such as
wastewater (Zhang et al., 2007; Liu and Cheng., 2010; Bezawada et al., 2013), corn stover
hydrolysates (Wang et al., 2007), agro-industrial waste (Sam et al., 2011; Pu et al., 2014),
palm oil mill effluent (Aljuboori et al., 2014a), rice straw fermentaion (Zhao et al., 2014),
soybean residues (Zulkeflee and Sanchez, 2014) have been documented as alternative carbon
source components in production media (table 1).
Zhang et al., (2007) obtained an exopolysaccharide based flocculant from multiple
microorganisme using brewey wastewater as carbon and energy source. This bioflocculant
was applied to treat indigotin printing and dyeing wastewater and two processes of removals
of COD and color were developed.
Palm oil mill effluent was used as carbon source to produce an exopolysaccharide based
bioflocculant from Aspergillus niger with high potential to treat river water colloids (63% of
turbidity removal with the present of Ca2+ ion) (Aljuboori et al., 2014a).
III.2. Physico-Chemical Factors Influencing the Flocculating Activity
As presented in table 1 several studies have reported the flocculation efficiency of the
exopolysaccharide based flocculants synthesized by different microorganisms. Most of these
studies used kaolin suspensions as a standard to evaluate the flocculating activity of the EPS.
In bioflocculants practical application, flocculating activity was also evaluated.
Many findings indicate that EPS flocculation efficiency is influenced by factors that
govern bacterial metabolism (Yuan et al., 2011; Li et al., 2013; Zhao et al., 2013; Aljuboori et
al., 2014; Yin et al., 2014; Guo et al., 2015).
The main factors include flocculant dosage, pH of suspension, presence of cations,
molecular weight of the bioflocculant, temperature, and forms of EPS bioflocculant.
1. Effects of the Dose of EPS-Bioflocculant

Several studies showed that there is an optimal bioflocculant dose (Salehizadeh and
Shojaosadati, 2002; Yuan et al., 2011, Aljuboori et al., 2014b). According to these studies
lower doses do not offer sufficient amounts of the bioflocculant to adsorb the suspended
kaolin clay particles. On the contrary, higher doses inhibited flocs from forming due to
stronger repulsive forces between them. As demonstrated by Aljuboori et al., (2014b) the
flocculating rate increased dramatically as the bioflocculant (IH-7) concentration increased
from 0.06 to 0.25 mg/L. The highest flocculating rate of 97.4% was recorded with 1mg/L of
IH- 7, above which, the flocculating rate gradually decreased.
2. Effects of the pH of the Suspension

The pH of the suspension is another important factor influencing the flocculating activity.
Exopolysaccharide based bioflocculant have a high flocculating efficiency across a wide pH
range and suggests that it is tolerant to acidic and basic conditions. However, efficiency
decreased at higher pH values. Yin et al., (2014) repoted that the flocculation activity of ZZ-3
produced by Klebsiella sp was stable a cross a pH rang of 3-7 (90.4- 94.5). Similarly the
bioflocculant produced by Enterobacter cloacae WD7 has been found to be tolerant of the
same pH range (3–9) (Prasertsan et al., 2006). Moreover, the bioflocculant produced by
Rhodococcus erythropolis (Guo et al., 2015) exhibited a strong flocculating capability in
alkaline conditions (7.0 - 9.0) and its flocculating activity reached 93.8% at pH 8.0. Yin et al.,
(2014) concluded that the deterioration of flocculation activity above pH 8 is probably due to
the degradation and rearrangement of the molecular structure of polysaccharides. These result
was slightly different from other similar studies, which claimed that bioflocculant had a
strong flocculating capability in alkaline conditions (>8.0) (Li et al., 2014, Zhang et al.,
2002). Ji et al., (2010) reported that the EPS produced by Bacillus licheniformus showed
strong flocculating activity at a wide range of pH values from 2 to 12. It appears that the
variations in the optimum pH of the suspensions for EPS depend on EPS composition, nature
of the suspensions, type (mono or multivalent) and doses of additive cations used. More et al.,
(2014) suggested that finding out the optimum pH range is a basic step during the flocculation
via EPS.
3. Effects of Presence of Cations

Several studies reported that flocculating activity is directly influenced by adding cations.
These cations acts as binding agent by neutralizing negatively charged particules and stabilize
the negative surface charged of bioflocculant. However results of these studies are conflicting
and divergent. More et al., (2012) rapported that the role of cations is to increase the initial
adsorption of the bioflocculant and suspended particules by decreasing the negative charge on
both the bioflocculant and thus improve flocculation. The flocculating activity of
Enterobacter sp. increases in the presence of Al3+‫ ‏‬, Fe3+‫‏‬, Fe2+‫ ‏‬, and Ca2‫‏‬+ (Yokoi et al., 1997).
Similarly the presence of CaCl2 significantly increased the flocculating activity of the
EPS produced by several species such as Bacillus, Pseudomonas, and Serratia (More et al.,
2012; Subramanian et al., 2010). Yin et al., (2014) demonstrated that the flocculation activity
of ZZ-3 produced by Klebisiella sp. was not impacted by Na+, K+, Ca2+, and Mg2+; slightly
impacted by Al3+; and dramatically impacted by Fe3+. The addition of Fe3+ into solution was
found to reduce flocculation for both bioflocculants from Klebisella (Yin et al., 2014) and
from Bacillus sp (Zheng et al., 2008).
Very few studies reported that bioflocculant have high flocculation activity without a
cation aid (Satish et al., 2010; Zhao et al., 2013; Yin et al., 2014).
Interactions of the exopolysaccharide based bioflocculant with different cations are very
crucial for the potential applications of EPS as a bioflocculant material. Optimization studies
must be conducted to understand these interactions and to figure out the best combination of
EPS and cations.
4. Effects of the Molecular Weight of EPS-Bioflocculant

The molecular weights of the EPS reported in different studies have been presented in
table 2. The molecular weight range of EPS bioflocculant varies from 2.574x104 Da
(Aljubouri et al., 2014b) to 3.5 x 106 Da (Li et al., 2013). The efficiency of the bridging
mechanism in flocculation is related to the size of the EPS (Yuan et al., 2011; Yin et al.,
2014). According to Ni and Yu, (2011) EPS of high molecular weight can form stronger
flocs. Consequently, high molecular weight EPS involves more adsorption points, stronger
bridging, and higher flocculating activity than does flocculation with a low-molecular-weight
EPS. The presence of large number of bonds among aggregated components increases the
floc compaction.
5. Effects of Temperature
The thermal stability of the biological products depends on their activity ingredients, as
known to all, The EPS bioflocculant with protein or peptide backbone in the structure showed
generally a poor heat stability, but those made of carbohydrates are heat-stable (Guo et al.,
2015). The EPS derived from Klebsiella pneumoniae (Zhao et al., 2013)), Halomonas sp. (He
et al., 2010), Serratia ficaria (Gong et al., 2008), Bacillus mucilaginosus. (Lian et al., 2008),
Aspergillus flavus (Aljuboori et al., 2013, 2014b), Klebisella sp (Yin et al. 2014) are heat-
stable and retain more than 50% of the initial flocculating activity after 15 min of boiling in
water. While the bioflocculant produced by Rhodococcus erythropolis (Guo et al., 2015) was
thermolabile. The poor heat stability confirmed that the main backbone of this bioflocculant
was a protein rather than a carbohydrate (see table 2).
On the other hand, according to Lu et al., (2005) the flocculating activity of the EPS from
Enterobacter aerogenes had an optimal temperature range of 35-65 °C and the highest
activity at 45°C. EPS from Bacillus licheniformis (Li et al., 2009) have also showed strong
flocculating activity over a broad range of temperature (4-95°C), and it maintained excellent
flocculating activity at lower temperatures, exhibiting its great application potential in treating
low temperature (below room temperature) drinking wastewater compared with commonly
used chemically flocculants, polyaluminium chloride (PAC) and polyacrylamide (PAM).
6. Effects of Forms of EPS-Bioflocculant

The difference in physicochemical nature of different types of EPS (tightly bound or
capsular EPS, loosely bound or slime EPS, broth EPS, etc.) is another factor which influence
the flocculation activity.
As reported by More et al. (2012) the slime, capsular and broth EPS produced by
different bacterial strains revealed distinct flocculation. Subramanian et al. (2010) reported
that the slime EPS produced in the synthetic mineral medium had better flocculation than that
of broth and capsular EPS respectively.
In addition, Bezawada et al., (2013) demonstrated that tightly bound EPS was found to be
the most effective and showed the highest flocculation activity compared to loosly bound EPS
and broth EPS. The variation in the flocculation was attributed to physiological and
biochemical differences of slime, capsular and broth EPS.
IV. APPLICATIONS OF EXOPOLYSACCHARIDES

IN WASTEWATER TREATMENTS
1. Wastewater Flocculation
The application of microbial flocculent on treatment of different kinds of wastewater has

been widely studied during the past decades, including the treatment of wastewater with high
concentration of organic compounds, the removal of turbidity, the decoloration, and the
dewatering of sludge. Instead of the single species of flocculent producing microorganisms,
the compound microbial flocculent has been used to treat the wastewater (Gong et al., 2008;
Li et al., 2013; Liu et al., 2009; Zhang et al., 2012; Yang et Zhao, 2013).
Many studies have proved that use of bioflocculant showed better removal in the
reduction of environmental concerned parameters (>90% generally) such as chemical oxygen
demand (COD), total suspended solids (TSS), turbidity and colour. As an exemple, the
bioflocculant from Bacillus mucilaginosus was used by Lian et al. (2008) to treat various
wastewaters (domestic, brewage, and pharmaceutical wastewater).
In the optimal conditions, the bioflocculant has a desired effective flocculation ability
with a significant removal ration of COD (˃70%), and TSS (˃88%) for wastewaters (Table
2). Authors postulate that polyvalent ions from wastewaters can complex with functional
groups of bioflocculant and cause an effective bridge and alteration of the polymer charge.
Similarly, bioflocculant produced by Paenibacillus elgii was purified, characterized, and

identified as an exopolysaccharide.
Its purification properties of real wastewater were investigated using a typical sample
combined with urban, agricultural, and industry wastewater (Li et al. 2013). The pollutant
removal efficiencies for COD, TSS and color were 68%, 83% and 88%, respectively. These
results are similar to those of commercial synthetic polymers.
According to Gong et al., (2008) a bioflocculant from Serratia ficaria was used to
flocculate a variety of real wastewater including river water brewery wastewater, meat
processing wastewater and soy sauce brewing wastewater.
It was also used to treat pulp effluent. For river water with low turbidity, the removal
efficiency of COD and turbidity obtained were 87.1% and 84.2%, respectively. The results of
COD removal in treating brewery wastewater, meat processing wastewater, and soy sauce
brewing wastewater were 80.7%, 76.3% and 64.1%, respectively. When treating pulp effluent
COD and the removal rate of color were up to 72,1% and 99,9%.
According to authors the removal efficiencies of real wastewater was similar and even
better than using traditional chemical flocculants (table 2).
2. Heavy Metal Removal from Wastewater
Most wastewaters contain various types of chemicals and different kinds of heavy metals.
Methods of treating heavy metals consist of chemical precipitation, solvent extraction,
dialysis, electrolytic extraction, reverse osmosis, evaporative methods, treatment with ion
exchange resins, etc. In recent years, there has been a significant effort to investigate new
methods of metallic element removal from contaminated sites. Biosorption of heavy metal
can be considered as an ecofreindly alternative technology able to compete with the
conventional technologies.
Many studies demonstrated that EPS can be an effective tool for the removal of toxic
heavy metals from industrial effluents (Comte et al., 2006; Salehizadeh and Shojaosadati,
2003; Gomma, 2012; Wang et al., 2013, Chien et al., 2013; Ozturk et al., 2014). The removal
capacity of exopolysaccharides is attributed to the polysaccharides, the proteins and lipid
contents. The EPS contains a large number of functional groups (such as -NH, -COOH, -OH,
-PO43-, etc.), these active groups induce strong attractive forces and serve as binding sites for
divalent cations. The number of binding sites and complexion capacities of EPS are correlated
with proteins, polysaccharides and humic substances content because of the presence of
functional groups (Lesmana et al., 2009; Ozturk et al., 2014; Solís et al., 2012). The
effectivness of metal uptake depends on initial metal concentration, pH, tempertaure, EPS
concentration, charge density, type of conformation of polymer with adsorbed ions effective
contact area between EPS and adsorbate and the type of microorganism (Comte et al., 2006;
Gomma, 2012; Lin and Harichund, 2011; Solís et al., 2012). It involves several mechanisms,
including physical adsorption, ion exchange, complexation, and precipitation.
According to Comt et al., (2006) functional groups with negative charges allow the EPS
components to be powerfull trappers of cations. The presence of uronic acids and sulfates
confers an overall negative charge and acidic property to the exopolymer and sulfated EPS
are of biotechnological importance. Exopolysaccharides produced by Bacillus firmus have
been reported could remove 98.3% Pb2+, 74.9% Cu2+, and 61.8% Zn2+ at optimum pH and
ambient temperature (Salehizadeh and Shojaosadati, 2003).
According to Rawat and Rai, (2012) Paenibacillus validus MP5 EPS shows maximum
adsorption values of 27% for Zn2+, followed by 16% for Ni2+ and Cd2+, 15% for Cr2+, 9% for
Co, and 7.5% for Pb2+. Li et al., (2013) found that EPS of Paenibacillus elgii had the highest
adsorption activity with Al3+, followed by Pb2+, Cu2+ and Co2+. The maximum metal
adsorption capacities for an exopolysaccharide based bioflocculant produced by
Pseudomonas aeruginosa was recorded as Cu2+ (87.39%) and Hg+ (89.09%) at a
concentration of 20 mg/L; Cd2+ (79.93%) and Pb2+ (79.7%) at a concentration of 40 mg/L;
and arsenate (72.96%) and Zn2+ (80.5%) at 60 mg/L (Gomma, 2012). Lin and Harichund
(2011) demonstrated the ability of EPS produced by several bacterial species belonging to
Pseudomonas sp., Herbaspirillium sp., and Paenibacillus sp to remove heavy metals from
industrial wastewater effluents. The bioflocculant produced by Pseudomonas sp adsorbed
more than 90% of Pb2+ and 78% of Hg+, respectively. Up to 66% of Cd2+ was removed by
Herbaspirillium sp bioflocculant.
The researchers showed that most bioflocculants display the higher heavy metal-tolerant
capacity at low concentrations. The EPS producing Pseudomonas sp. (strain EJ01) was also
observed to tolerate cadmium ions (2 mM and above) present in the growth medium and it
was directly related to the increase (50%) in the EPS concentration (Chien et al., 2013).
3. Dyes Removal from Wastewater
The use of synthetic chemical dyes in various industrial processes, including paper and
pulp manufacturing, plastics, dyeing of cloth, leather treatment and printing has increased
considerably over the last few years. Several studies reported that release of untreated dye-
containing wastewater into the natural water bodies has harmful effects on the aquatic life
(Zhang et al., 2009; Lee et al., 2011; Solís et al., 2012).
In wastewater treatment, microbial flocculant have been used to treat dye solutions (Deng
et al., 2005; Gao et al., 2009; Liu et al., 2009; Zhang et al., 2009, Simphiwe et al., 2012).
Several EPS producing microrganisms (such as Paenibacillus, Klebsiella, Proteus,
Pseudomonas, Staphylococcus, Serratia, Aspergillus, etc.) have been reported (table 2). The
results of several studies indicate that parameters like pH, temperture, bioflocculant
concentration and its functional groups play an important role in the decolorization activities.
Deng et al., (2005) found that EPS-based bioflocculant from Aspergillus parasiticus was
effective in flocclating some soluble anionic dyes in aqueous solution, in particular reactive
blue 4 and acid yellow 25 with a decolorization efficiency of 92.4 and 92.9%, respectively
with flocculant concentration of 50 ml/L at pH 6. While the same decolorization for acid
orange and reactive orange were achived with a flocculant dosage of 100-150ml/L. When the
decolorization ability of an exopolysaccharide based bioflocculant from Paenibacillus elgii
was investigated by Li et al., (2013), its decolorization efficiency for methylene blue and red
X-GRL was 65% and 72%, respectively with flocculant concentration of 30 ml/L. However,
authors indicated that lower rates were observed when used with anionic and neutral dye
(<50%). Similaly, The EPS produced in dairy wastewater by Klebsiella mobilis was efficient
in flocculating some disperse dyes in aqueous solutions with a decolorization efficiency of
91% for disperse violet HFRL at pH 7 and 25°C (Wang et al., 2007). In addition, Zhang et al.,
(2009) reported that an exopololysaccharide synthesized by Proteus mirabilis was effective in
hazardous dye (basic blue 54) removal from aqueous solution in batch systems. Authors
indicated that the maximum dye (BB 54) uptake rate of 2.005 g/g of EPS was observed. Gong
et al. (2008) demonstrated that exopolysaccharides produced by Serratia ficaria was able to
remove the colour of pulp and paper mill effluent more effectively than traditional chemical
flocculants. The removal rate of color was 99.9% with concentration of 0.4mg/L (table 2).
The optimal pH for the decolorization efficiency was in the rang of 6-8.
Solis et al., (2012) found that bacteria in the consortium possess higher colour removal
efficiency as compared to the single bacterium. Zhang et al. (2007) observed that the EPS
synthesized by a consortium of Staphylococcus sp. and Pseudomonas sp. had capability of
treating printing and dye wastewater (table 2). At pH 9 and flocculant concentration of
17ml/L, the maximum removal efficiencies of chroma from indigotin printing and dyeing
wastewater was up to 86.5%. Recently a bioflocculant produced by a consortium of Bacillus
subtilis, Exiguobacterium acetylicum, Klebsiella terrigena, Staphylococcus aureus,
Pseudomonas pseudoalcaligenes, and Pseudomonas plecoglossicida isolated from activated
sludge, was very effective for de colourizing different dyes, with a removal rate of up to
97.04%. Authors of the study (Simphiwe et al., 2012) reported that a pH of 7 was found to be
optimum for the removal of both whale and mediblue dyes, but the optimum pH for fawn and
mixed dye removal ranged from 9 to 10. Optimum temperature for fawn and mixed dye
varied between 40-45°C and 35-40°c, respectively. For whale and mediblue the optimum
temperature was 35°C.
The effectiveness of the decolourization efficiency depends on EPS concentration, pH,

temperature, ionic strength, and time of contact, dye concentration, dye structure and
microorganism. Results discussed in this paragraph suggest that microbial polysaccharide
based bioflocculants may provide a promising economical and cleaner alternative to replace
or supplement current treatment processes for the removal of very high concentrations of dyes
in industrial wastewater effluents.
4. Toxic Organic Compounds Removal from Wastewater
Recent findings indicate that exopolysaccharide based flocculants have ability to remove
polycyclic aromatic hydrocarbons (PAHs) from different wastewater and soils (More et al.,
2014; Jia et al., 2011; Zhang et al., 2011).
EPS produced by Zoogloea sp. and Aspergillus niger were employed in studies of
degradation of pyrene in contaminated soils (Jia et al., 2011). Authors demonstrated that
pyrene could be degraded in the presence of any one EPS. These bacteria had degraded more
than 80% of pyrene in 35 days, the degradation was increases with increase of EPS
concentration for both of these bacteria. In order to elucidate the role of EPS in removal of
PAHs Zhang et al., (2011) invistigated the biodegradation of phenantherene (PHE) in two-
liquid-phase system (TLPs). The demonstration was conducted in typical TLPs-silicone oil-
water PHE degrading bacteria capable of producing EPS (Sphingobium sp and Micrococcus
sp). Authors observed that the presence of both strains enhanced mass transfer of PHE from
silicone oil to water and that biodegradation of PHE mainly occured at the interfaces.
Exopolysaccharides faverd bacterial uptake of PHE and facilate the degradation of PAHs.
These results indicate that organic compounds removal from wastewater and soils could
be enhanced by increasing the specific surface area of the EPS in contact with PAHs.
CONCLUSION
The literature discussed above illustrate that the potential application of microbial
exopolysaccharide based bioflocculants in wastewater treatment has been verified and
investigated. Many studies have successfully demonstrated the treatment of wastewater
employing EPS produced by individual or a consortium of various microorganisms.
They have shown remarkable results in reduction or removal of environmental concerned
parameters such as TSS, turbidity, COD, dyes, heavy metals, and organic compounds with
more than 90% removal was achieved in treatment of various types of wastewaters. High
production cost is the main factor that has hampered the use of bioflocculants in industrial
operation. Application of exopolysaccharide in wastewater treatment mainly depends on
further decreasing the bioflocculant production cost using cheap substrates and novel
fermentation/recovery strategies. A cost benefit analysis of using EPS flocculants for
wastewater treatment and optimisation study to develop a standard protocol of production and
improve the flocculating efficiency needs to be carried out to determine the economic
feasibility of its practical use. Moreover, mostly work on EPS bioflocculant was conducted at
the lab scale (test tube) experiments.
These test tube studies results do not simulate actual potential of EPS as compared to the
conventional chemical polymers at field operating conditions. The complexity of the
flocculation systems justifies that an exopolysaccharide based bioflocculant cannot be
selected for a given application without industrial trials or practices for confirming the
flocculant selection and for determining its amount and its molecular weight. The yield of the
EPS and the effect of the different extraction methods need to be taken into consideration. It
is also not very well clear if purification is required for application of EPS as flocculants in
various wastewater treatement.
The literature discussed in this chapter showed lack of information on this aspect. Very
limited information exists on the flocculation efficiency of different types of EPS (crude,
purified, tightly bound, loosely, etc.) for real practical application.
Most of the studies have been conducted using kaolin as the medium to flocculate. This
warrants investigating which form of EPS produced by different microbial strains on different
media (semi-synthetic or complex or waste material) will be efficient for flocculation of
targeted system (wastewater flocculation, dyes removal, metal removal, toxic compounds
removal, etc.). It‘s also important to conduct more future studies for best flocculants with the
ability to produce very promising results in pollutants removal even at wider variations of pH,
temperature, salinity and other contaminants of the wastewater. Future research needs to look
into how molecular weight and charge density distribution affect the flocculation performance
to produce a better choice of flocculants for specific industrial applications. Optimisation of
these factors could significantly increase the treatment efficiency.
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Chapter 7
EXTREMOZYMES AS EFFICIENT GREEN

BIOCATALYSTS IN BIOREMEDIATION
OF INDUSTRIAL WASTEWATERS
Mohamed Neifar1,2, Habib Chouchane1,, Atef Jaouani2,

Ahmed Slaheddine Masmoudi1 and Ameur Cherif1
1
Laboratory of Biotechnology and Bio-Geo Resources
Valorization, Higher Institute for Biotechnology, Biotechpole
Sidi Thabet, University of Manouba, Ariana, Tunisia
2
Laboratory of Microorganisms and Active Biomolecules, Faculty
of Sciences of Tunis, University of Tunis El Manar, Tunis, Tunisia
ABSTRACT
Recalcitrant pollutants are present in the wastewaters of a great number of industries,
including coal conversion, petroleum refining, resins and plastics, textiles, oil milling,
tanning, mining, pulp and paper. The removal of significant concentrations of pollutants
from industrial wastewater is required to achieve the environmental standards. The
current methods using physical and chemical techniques suffer from factors such as high
cost, hazardous by-products formation, and poor removal efficiency.
Therefore, research studies are being conducted to develop alternative approaches to
the pollutants removal from wastewaters using enzyme-based technology.
Varous enzymes offer a number of advantages over conventional chemical catalysts
including a high degree of specificity, operation and stability under harsh conditions of
industrial wastewaters (acidic or alkaline pH, low or elevated temperatures, high salt
concentrations, or high metals, etc.), and a high reaction velocity which reduces
processing costs. Selectivity and versatility are also important properties responsible for
wide and diversified range of applications of extremozymes in the wastewater treatment.
Several scientific reports and patents described the use of hydrolytic extremozymes
such as lipases and proteases for the biological treatment of effluents of many industries

Corresponding authors: Mohamed Neifar (mohamed.naifar@gmail.com), Habib Chouchane (chouchane_habib@
voila.fr).
192 Mohamed Neifar, Habib Chouchane, Atef Jaouani et al.
like dairies and tanneries, which are rich in oil and proteins, respectively. The lignin-
degrading enzymes (lignin peroxidase, manganese peroxidise, versatile peroxidise and
laccase), known so far, are extracellular and nonspecific enzymes, participating in
different oxidative reactions where the aromatic structure of lignin and bonds between the
basic units are broken. These biocatalysts have been extensively used to oxidatively
detoxify various aromatic xenobiotic pollutants found in industrial wastewaters. The
interest in laccases was strongly increased upon discovering that they can oxidize
nonphenolic aromatic compounds in the presence of low-molecular-mass redox
mediators. Furthermore, laccases have an advantage compared to ligninolytic
peroxidases, as they use oxygen, a non-limited electron acceptor, for their catalytic
activity, which makes them more suitable for industrial wastewater treatment. The
present chapter reviews the occurrence, modes of action and biochemical properties of
oxidative and hydrolytic extremozymes, as well as their applications in industrial
wastewater treatment. The main advantages as well as the main drawbacks that are still
present in the extensive application of enzyme-based technology in wastewater treatment
will be also mentioned.
Keywords: Industrial wastewater, biodegradation, detoxification, green catalysis, hydrolytic

and oxidative extremozymes
INTRODUCTION
The increasingly stringent environmental regulation on hazardous, harmful wastes has
encouraged the search for innovative solutions for reducing and even eliminating pollution in
contaminated wastewaters. In this field, research efforts have been devoted to develop new,
low-cost, low-technology, eco-friendly treatments to restore polluted wastewaters.
Enzymatic treatment is suitable for wastewaters that contain relatively high amounts of
the hazardous target pollutants in comparison to others. They are catalysts with broad
specificity or incredibly high chemo-selectivity, region-selectivity and stereoselectivity, and

then they can be applied to a large range of different wastewater pollutants (Rao et al., 2010;
Mugdha and Usha, 2012). Moreover, they may produce modifications of structural and
toxicological properties of recalcitrant compounds, and even their complete conversion into
innocuous end products. In addition, they may perform processes for which no efficient
chemical transformations have been devised (Rao et al., 2010). The potential advantages of
the enzymatic treatment, as compared with conventional physico-chemical and biological
wastewater treatments are summarized in Table 1. The most representative enzymatic classes
used in the treatment of municipal and industrial wastewaters are hydrolases (e.g. lipases,
proteases, cellulases, xylanases, amylases and pectinases) and oxidoreductases (e.g. laccases,
lignin, manganese and versatile peroxidases) (Table 2). Their main producers are bacteria and
fungi. Enzymes were often preferred over intact producing microbes, because the isolated
enzymes appeared to have greater specificity, better standardization and easy handling and
storage (Husain and Husain 2008). These enzymes could be used as crude extracts or in pure
forms. Crude extracts are preferred for use in wastewater treatment over pure enzymes,
mainly because they are relatively inexpensive (Rao et al., 2010). The functionality of the
enzymes depends largely on their conformation which can be altered by the harsh conditions
prevailing in wastewaters like high or low temperature, very high or low pH, high ionic
strength, high concentrations of reactants, and presence of inhibitors.
Extremozymes As Efficient Green Biocatalysts in Bioremediation … 193
To be useful, the added enzymes must be stable under these extreme conditions. The
biocatalysts, called extremozymes, produced by microbial extremophiles offer new
opportunities for biotransformation of hazardous compounds in industrial wastewaters taking
into account their extreme stability at harsh conditions (Van den Burg, 2003; Gomes and
Steiner, 2004). However, due to the difficulties associated with large-scale culture of
extremophiles and downstream processing of extremozymes, corresponding genes from
extremophiles have been and are being expressed successfully in mesophilic hosts (Schiraldi
and De Rosa 2002; Gomes and Steiner, 2004). Most of the extremozymes that have found
their way into industrial applications are produced using E. coli expression systems
(Demirjian et al., 2001; Eichler, 2001; Gomes and Steiner, 2004).
To improve the performance of enzymes in wastewater treatment, enzymes are mostly
immobilized on natural and synthetic supports of different nature and through different
immobilization mechanisms. Immobilized enzymes have usually a long-term operational
stability. Furthermore, they may be reused and recovered at the end of the process (Nisha et
al., 2012; Mugdha and Usha, 2012; Jenitta et al., 2013). This chapter represents a short review
of oxidative and hydrolytic extremozymes including laccases, peroxidases, lipases, proteases,
cellulases, xylanases, amylases, and pectinases with particular attention to their use in
wastewater treatment as an alternative to conventional treatment methods.
USE OF HYDROLYTIC EXTREMOZYMES FOR THE TREATMENT

OF SLAUGHTERHOUSES AND DAIRY WASTEWATERS
Wastewaters from slaughterhouses and dairies usually contain high levels of fats, proteins
and carbohydrates. The pretreatment of wastewaters with hydrolytic enzymes should allow
reducing the concentrations of substances with negative effect or completely eliminating
them, and therefore could improve the efficiency of biological treatment of wastewaters
(Matsui et al., 2005; Nakhla et al., 2006; Efremenko et al., 2008).
Lipolytic Enzymes
Lipases (triacylglycerol ester hydrolases, E.C. 3.1.1.3), or more generally carboxylic ester
hydrolases, are among the most thoroughly studied and industrially important enzymes
(Sharma et al., 2001; Hasan et al., 2006; Liszka et al., 2012). Lipases catalyze the hydrolysis
of carboxyl ester bonds present in acylglycerol with the consequent release of organic acids
and glycerol (Mobarak-Qamsari et al., 2012). Fungal and bacterial lipases are largely
diversified in their biochemical properties and substrate specificity and selectivity, which
make them potential source for industrial applications (Gurung et al., 2013; Gopinath et al.,
2013). They are of great interest for treatment of wastewaters containing high oil and grease
contents (Massé et al., 2001; Massé et al., 2003; Sharma et al., 2001 Cammarota and Freire,
2006; Mobarak-Qamsari et al., 2012). Lipases produced by Bacillus subtilis, Bacillus
licheniformis, Bacillus amyloliquefaciens, Serratia marcescens, Pseudomonas aeruginosa,
and Staphylococcus aureus were reported to degrade palm oil mill, diary, slaughter house,
soap industry, and domestic wastewaters (Prasad and Manjunath, 2011). Application of a
pretreatment process to hydrolyze and dissolve fats may improve the biological degradation
of fatty wastewaters, accelerating the process and reducing time of anaerobic digestion
(Mobarak-Qamsari et al., 2012). Cammarota et al. (2001) evaluated the efficacy of an
enzymatic hydrolysis stage in dairy industry wastewater prior to the anaerobic biological
treatment. The authors employed an enzymatic preparation with high lipase activity with a
relatively low production cost. The average COD and fat removal efficiencies were 92% and
89%, respectively. Effluent turbidity and volatile suspended solids also exhibited notable
improvement, being reduced by 75% and 90%, respectively, in relation to the raw
wastewater. Leal et al. (2006) used an enzymatic extract with a high level of lipase activity
for the treatment of dairy wastewaters with high fat contents. The COD removal efficiencies
for hydrolyzed effluents (80-95%) were higher than those obtained for raw effluents (without
enzymatic pre-hydrolysis; 19-55%) at different oils and greases concentrations. Recently,
Stathopoulou et al. (2013) analysed Meat industry wastewater (oil and grease concentrations
from 250 to 3,000 mg·L-1) treated in batch anaerobic reactors with and without a commercial
lipase (Lipolase 100T, Novozymes) and a lipase obtained by solid state fermentation (Lipase
SEP). The benefits of the enzymatic prehydrolysis became evident when the wastewater was
treated with 0.1% (w/v) of lipases. When the wastewater containing 1,200 mg·L-1 of oil and
grease was pretreated with the Lipase SEP, removal of COD increased 22%, compared to a
control reactor fed with wastewater without any treatment (Stathopoulou et al., 2013).
Several microbial extremophile groups (thermophiles and hyperthermophiles,
psychrophiles, halophiles, alkalophiles/acidophiles, and solvent-resistant microorganisms),
able to produce lipases have been isolated and the corresponding enzymes have been purified
either from the wild-type culture supernatants or following cloning and expression in
mesophilic hosts (Buchon et al., 2000; Stathopoulou et al., 2013; Levisson et al., 2009;
Polizelli et al., 2013; Liszka et al., 2012; Saranya et al. 2014). The thermo-tolerant and
extreme acidophilic microorganism Bacillus pumilus was isolated from the soil collected
from a commercial edible-oil extraction industry. Its acidic thermostable lipase established
hydrolysis efficiency of 96 % for palm oil in wastewater at 50 °C (Saranya et al. 2014). Tsuji
et al. (2013) examined 305 isolates of fungi including eight Ascomycetous and six
Basidiomycetous species collected from Antarctica and found that Mrakia is a major
mycoflora highly adapted to the Antarctic environment.
The authors screened Mrakia isolates for their ability to decompose milk fat under low
temperature conditions and evaluated their potential for application to an active sludge system
in a region with a cold climate. Their results showed that 56 Mrakia spp. exhibited fat
decomposition ability. Lipase of the Antarctic yeast Mrakia blollopis SK-4 showed high
stablity in wide ranges of temperature and pH conditions and was not affected by the
existence of EDTA, various metals ions, or organic solvents. Extremophilic SK-4 lipase may
be considered as a promising bio-remediation agent for cleaning up unwanted milk fat curdles
from dairy milk wastewater under low temperature conditions.
Proteolytic Enzymes
Proteolytic enzymes or proteases are complex group of degradative enzymes collectively

known as peptidyl-peptide hydrolases and are responsible for hydrolysis of peptide bonds in
proteins (Gupta et al., 2002). Proteases are still the most dominant enzyme type in the market
due to their extensive use in detergent and dairies (Alves et al., 2014). Microbial proteases are
used in waste treatment from various food-processing industries and household activities to
solubilize proteinaceous waste and reduce the biological oxygen demand of aquatic systems
(Gupta et al., 2002). Fungal and bacterial proteases are of three types: acidic, neutral and
alkaline. Alkaline proteases are efficient under alkaline pH conditions and consist of a serine
residue at their active site. Several researchers reported the novel biochemical characteristics
of alkaline proteases identified from different microorganismes (Mital et al., 2006; Nowlan et
al., 2006; Patel et al., 2006a; Patel et al., 2006b; Thumar and Singh, 2007; Dodia et al., 2008;
Indhuja et al., 2012). They have the largest applications in bio-industry, since these possess
high activity and stability in abnormal conditions of extreme physiological parameters (work
under high pH, salt and in presence of different inhibitory compounds) (Johnvesly and Naik,
2001; Hadj-Ali et al., 2006; Vijayalakshmi et al., 2011). Bacterial extremophilic proteases
have received attention as a viable alternative for bioremediation of protein rich tannery
wastewater, which is characterized by high organic load, high suspended solids (sand, lime,
hair, flesh, dung, etc.) and high salinity (Sivaprakasam et al., 2011; Ahmad and Ansari, 2013).
A novel salt tolerant alkaline protease obtained from P. aeruginosa (isolated from tannery
saline wastewater) was used for enzymatic degradation studies. Tannery saline wastewater
treated with identified salt tolerant protease showed 75 % protein removal at 6 h duration and
2 % (v/v) protease addition was found to be the optimum dosage value (Sivaprakasam et al.,
2011). Using alkaline salt tolerant proteases as a primary agent for wastewater treatment
allowed overcoming some of the problems associated with traditional chemical and biological
treatments (Sivaprakasam et al., 2011). The production of extremophilic proteases using
wastewater sludge as raw materials would make it possible to market them at low production
cost (Haki and Rakshit, 2003; Verma and Baiswar, 2013). Further studies will be focused on
using immobilized enzyme reactors for treatment of protein rich wastewaters in order to
reduce the amount of enzyme dosage as well as cost involved in its operation (Srinivasan et
al., 2009; Joshi, 2010; Sivaprakasam et al., 2011).
Glycosyl Hydrolases
Various carbohydrases, primarily cellulases, amylases and polygalacturonases, represent

the second largest group in the market (Alves et al., 2014). Cellulolytic enzymes are a group
of hydrolytic enzymes distinguished by their ability to hydrolyze β- 1.4 glycosidic linkages in
cellulose to smaller oligosaccharides and eventually to glucose. The cellulose enzyme system
has 3 major enzymes i.e. endoglucanase (EC 3.2.1.4), exoglucanaes (EC 3.2.1.91) and
cellobiase (EC 3.2.1.21) whose synergistic effect leads to the hydrolysis of cellulose to
glucose (Zhang and Zhang, 2013). Some wastewaters generated from agricultural fields and
agroindustries contain a large amount of unutilized cellulose, thereby causing environmental
pollution. Lee et al. (2008) reported the transformation of particulate and recalcitrant organic
compounds in the swine wastewater into soluble and relatively easily biodegradable organic
products by the cellulase enzymatic hydrolysis. Pretreated swine wastewater by cellulase
enzymatic process showed 10.7% higher anaerobic digestibility at the end of 20 days
incubation and 29% higher initial methane production rate compared to untreated swine
wastewater.
Table 1. Advantages and disadvantages of the current methods used in wastewater treatment plants
Wastewater treatment Methods Advantages Disadvantages

Simple, economic and effective adsorption,
Regeneration of support is difficult, larger
Adsorption good removal efficiency for a wide variety of
contact times, huge volume required
Physical wastewater pollutants
Effective removal of a wide range of colorants Required high dissolved oxygen, ineffective for
Irradiation
at low volumes light resistant pollutant compounds
Coagulation and Short detention time, low capital costs, good High cost of chemicals for pH adjustment,
precipitation removal efficiencies problems of dewatering, sludge handling
Electrochemical No additional chemicals required, products are
High cost of electricity
oxidation nonhazardous
Chemical Unsuitable for some wastewater pollutants (e.g.
Ozonation Effective for various pollutant removal
disperse dyes)
Photochemical No sludge formation Formation of by-products
Decolorization of wide range of wastewaters, no
Fenton‘s reagent Highly expensive
alternation in volume
Longer detention times, less resistant to
Aerobic process Pollutant removal along with COD removal
recalcitrants
Resistant to a wide variety of complex

Biological Anaerobic process Longer acclimatization phase
pollutants, produced biogas
Single cell (fungal, algal, Good COD removal efficiency for low volumes, Expensive culture maintenance, applicable only
bacterial) very effective for specific pollutant for low effluent volume
Limited stability under industrial operational
Hydrolytic and oxidatives Great specificity, high COD removal efficiency, conditions for free mesophilic enzymes,
Enzymatic*
enzymes good standardization, easy handling and storage difficulties in the recovery and recycling of
biocatalysts
*
fall between chemical and biological processes, since they involve chemical reactions based on the action of biological catalysts.
Robinson et al., 2001; Karigar and Rao, 2011; Jegannathan and Nielsen, 2013.
Table 2. An overview of hydrolytic and oxidative enzymes potentially useful in wastewater treatment
Classes Enzymes Catalytic mechanism Main bioremediation functions WWT applications References
One electron oxidation of phenolics, Removal of polyphenols,
aromatic amines and other aromatic degradation of pesticides, textile
Laccase (EC 1.10.3.2)
hydrogen donors with simultaneous dyes and Polycyclic aromatic
reduction of O2 to H2O hydrocarbons, removal of lignin
Petroleum refinery
Oxidation of lignin side chains by one Durán and Esposito, 2000
wastewater
electron abstraction in the presence of Degradation of lignin (non-phenolic Ruggaber et al., 2006
Lignin peroxidase Textile dyeing
Oxidoreductases H2O2, generating reactive radicals. lignin units), mineralization of Latif et al., 2010
(EC 1.11.1.14) wastewater
(Lignin-modifying Cleavage of C-C and ether bonds in recalcitrant aromatic compounds Neifar et al., 2012
Paper mill wastewaters
enzymes) lignin Jenitta et al., 2013
Olive mill wastewaters
Oxidation of Mn2+ into Mn3+ Mn3+ Pakshirajan and Radhika,
Manganese peroxidase Oxidation and degradation of lignin, Leather effluents
acts as a diffusible oxidizer on 2013
(EC 1.11.1.13) phenols, dyes, and other xenobiotics Pharmaceutical effluents
phenolic or non-phenolic lignin units
Oxidation of Mn2+ to Mn3+ and also Oxidation of phenols, dyes,
Versatile peroxidase
phenolic and nonphenolic aromatic hydroquinones, aromatic alcohols
(EC 1.11.1.16)
compounds and xenobiotics
Split up animal and vegetable fats
Hydrolysis of ester bonds in water-
Lipases (EC 3.1.1.3) and oils into their component part
insoluble, lipid substrates
glycerol and fatty acids
hydrolysis of peptide bonds in Masse et al., 2001
Solubilization of proteins to enhance

Proteases proteins to produce peptide chains and Mendes et al., 2005
the lysis efficiency of total sludge
amino acids Dairy wastewaters Cammarota and Freire
Hydrolysis of cellulose to simple Hydrolyze of cellulose in sludge floc Swine wastewaters 2006
Cellulases
carbohydrates treatment Slaughterhouses Leal et al., 2006
Hydrolases Destroying coloured substances wastewaters Lee et al., 2008
Xylanases Degradation of xylan in wood
(enzymatic bleaching) Poultry wastewaters Sivaprakasam et al., 2011
Hydrolysis of starch by randomly Tannery saline Habib et al., 2012
cleaving the glucosidic bonds, thus Starch liquefaction and wastewaters Mobarak-Qamsari et al.,
Amylases
giving rise to smaller, more soluble sachcarification 2012
compounds Polizelli et al., 2013
Hydrolyse pectin (cleavage of α-1,4-
Pectinases
glycosidic bonds) into galacturonic Bioscouring and bleaching
(Polygalacturonases)
acid and small sugars
Table 3. Some oxidative and hydrolytic extremozymes from extremophilic bacteria with potential applications in wastewater treatment
Potential application in wastewater

Strain sources Extremophilic strain Extremophilic enzyme Enzyme characteristics References
treatment
Performance in saline and acidic
Hypersaline man-made Obligate halophilic wastewaters from phosphate-containing
Polyextremophilic amylase Best activity at 5 pH, 60°C, and 30% NaCl Ali et al., 2014
saltern from Thailand Aspergillus gracilis fertilizer industry, fish sauce production
units, and phenol production industries
Salt-tolerant alkaline Activity at 4-7% NaCl; optimum NaCl, 2% Tannery saline wastewater (75 % protein Sivaprakasam et
Tannery saline wastewater P. aeruginosa
protease Activity at 5-11 pH range; optimum pH, 9 removal after 6 h of treatment) al., 2011
extreme acidophilic and Palm oil wastewater (hydrolysis
Soil from a oil extraction Extremely acidophilic Extremely acidic thermostable lipase: pH, Saranya et al.,
thermo-tolerant Bacillus efficiency of 96 % for palm oil in
industry thermotolerant lipase 1; temperature, 50 °C 2014
pumilus wastewater at 50 °C. 3 h; pH, 1)
Sediments of Naga-ike, a Optimum temperature 60−65°C; optimum
Promising biological agents for parlor
lake in Skarvsnes, located Antarctic yeast strain pH 8.0-9.0; stable over a wide pH range
Polyextremophilic lipase wastewater treatment even in low Tsuji et al., 2013
near Syowa station, East Mrakia blollopis SK-4 (4.0−10.0) and up to 65°C; resistance to
temperature regions of the world
Antarctica various metal ions and organic solvents
Laccase resistant to inactivation caused by Potential in textile and pulp wastewaters
Bacillus licheniformis Alkali-resistant and high temperature and alkaline conditions. (final decolorization> 91% for reactive
Forest soil samples Lu et al., 2012
LS04 salt/solvent-tolerant laccase Tolerance towards NaCl and organic black 5, reactive blue 19 and indigo
solvents carmine after 2 h of treatment)
Potential in textile and kraft wastewaters
Stability at high pH and temperature, (complete decolorization and Molina-Guijarro et
Streptomyces ipomoea pH-versatile, salt-resistant

Not mentioned resistance to high concentrations of NaCl detoxification of the azo-type dye al., 2009 Eugenio
CECT 3341 laccase
and typical inhibitors Orange II in the presence of the natural et al., 2011
mediator, acetosyringone)
Thermophilic Alkali-resistant and Textile wastewater (ability to decolorize
Resistance to alkaline conditions and
Composting environments actinomycete moderately thermostable indigo carmine and diamond black with Lu et al., 2013
relatively high temperatures
Streptomyces sp. C1 laccase syringaldehyde as mediator)
Food dumps, food waste
waters, spoiled fruits, Alkaline pectinolytic Production at pH of 10.5 and temperature Ample application in food waste water Ramakanth et al.,
Alkaline polygalacturonase
vegetables and alkaline Bacillus sp of 60°C treatment 2014
soils
Alkaline-tolerant Active and stable at temperatures of 50-80 Removal of the lignin-associated
Soil of gold teak forest, Techapun et al.,
Streptomyces sp. Ab106 thermostable Cellulase-free °C, active at alkaline pH (pH 7-9), half-life hemicellulosic fraction of wood pulp
Thailand 2002
xylanase at 70 °C, pH 9.0 of 5 h (bio-bleaching)
Cellulases have been produced by a range of microorganisms including fungi and

bacteria (Peérez et al., 2002). Cellulases of the mesophilic fungi Trichoderma reesei and
Phanerochaete chrysosporium are the most thoroughly studied (Kirk and Cullen, 1998;
Peérez et al., 2002).
Several thermophilic fungi can degrade cellulose faster than T. reesei. The cellulases of
thermophilic fungi are thermostable and show optimal activity between 55 and 80° C
(Maheshwari et al., 2000). Among cellulolytic bacteria and archea, species from the genera
Cellulomonas, Pseudomonas, Streptomyces, and Pyrococcus are the best studied (Perez et al.,
2002; Haki and Rakshit, 2003). Thermostable cellulases of Pyrococcus furiousus and
Pyrococcus horikoshi, showing optimal activity at 102- 105°C (Haki and Rakshit, 2003). The
capacity of these microbial extremophiles inhabiting wide variety of environmental niches
such as high temperature and alkaline pH, help these strains to produce novel cellulotyc
extremozymes which are stable under harsh conditions leading to increase rates of cellulose
hydrolysis in industrial wastewaters (Acharya and Chaudhary, 2012).
Xylanases (EC 3.2.1.8) catalyze the hydrolysis of xylan, the major constituent of
hemicelluloses found in plant cell wall. The xylanolytic enzyme system is usually composed
of a repertoire of hydrolytic enzymes: β-1,4-endoxylanase, β-xylosidase, α-L-
arabinofuranosidase, α-glucuronidase, acetyl xylan esterase, and phenolic acid (ferulic and p-
coumaric acid esterase. All these enzymes act cooperatively to convert xylan into its
constituent sugars (Thomas et al., 2013). In the past two decades, the use of extremophilic
hemicellulases, has revolutionized the pulp and paper industry and provided global
environmental benefits since they can reduce the industrial pollution and effluent‘s toxicity
(Dhiman et al., 2014). The wood used for the production of the pulp is treated at high
temperature and basic pH, which implies that the enzymatic procedures require proteins
exhibiting a high thermostability and activity in a broad pH range (Haki and Rakshit, 2003).
Treatment with alkali-thermostable xylanase disrupts the cell wall structure facilitating lignin
removal in the various stages of bleaching. The optimum temperature for the activity of most
microbial xylanases is reported to be 50-60 °C with a half-life of about 1 h at 55 °C (Haki and

Rakshit, 2003; Polizeli et al., 2005; Ramakrishnan and Narayanan, 2013).
However, some xylanases have been reported to exhibit higher thermal stability and
optimal activity ranging from 80 to 100 °C (Haki and Rakshit, 2003; Ramakrishnan and
Narayanan, 2013; Yang et al., 2013). Alkaliphilic and thermostable cellulase-free xylanases
from Thermoactinomyces thalophilus and Clostridium abusonum CFR-702 (Haki and
Rakshit, 2003) were also reported. These enzymes have offered a major step in the reduction
of chlorine consumption in the bleaching process of kraft pulp, thus, lowering environmental
pollution by organic halogens in wastewaters.
However, scaling up of the enzyme production from the respective microorganisms to the
level required by the industry remains to be developed (Garg et al., 2011; Dhiman et al.,
2014). Amylolytic enzymes act on starch and related oligo- and polysaccharides to generate
progressively smaller polymers composed of glucose units. They include α-amylases,
glucoamylases or β-amylases and isoamylases or pullulanases.
The enzymes are classified into endo-acting and exo-acting enzymes. α-amylase is an
endo-acting enzyme and hydrolyses linkages in a random fashion and leads to the formation
of linear and branched oligosaccharides, while the rest are exo-acting enzymes and attack the
substrate from the non-reducing end, producing oligo and/or monosaccharides (Haki and
Rakshit, 2003; Sivaramakrishnan et al., 2006; Nigam, 2013).
A number of attempts were made to isolate and characterize extremophilic amylases from
diversified microbial sources to meet the requirements apply to industrial wastewater
treatment. The extremophilic amylases from microbial sources have often been reported to
work better at more than one extremity of available conditions, thus showing the
polyextremophilic behavior (Kiran and Chandra, 2008; Asad et al., 2011; Moshfegh et al.,
2013). For example, the polyextremophilic (acidophilic, thermophilic, and halophilic)
behavior of 𝛼-amylase obtained from obligate halophilic Aspergillus gracilis TISTR 3638
provides the options of using it in industrial operations occurring in mild acidic and
temperature conditions and at saline and low water activity. The comparison of 𝛼-amylase
from this study with the commercial grade amylase for wastewater remediation has shown
that the amylase from A. gracilis TISTR 3638 can be efficiently applied for saline wastewater
remediation (Ali et al., 2014).
Petinolytic enzymes or pectinases hydrolyze pectic substances. Protopectinases,
polygalacturonases (PGases), lyases and pectin esterases are among the extensively studied
pectinolytic enzymes (Jayani et al., 2005; Ramakanth et al., 2014). PGases catalyze the
hydrolytic cleavage of the polygalacturonic acid chain with the introduction of water across
the oxygen bridge. They are the most extensively studied among the family of pectinolytic
enzymes. Most of the microbial PGases work in acidic pH and a temperature range of 30-40
°C, but a few PGases, which can catalyze the hydrolysis of pectic substances at higher
temperatures or pH have been reported (Hoondal et al., 2000; Jayani et al., 2005; Ramakanth
et al., 2014). Pretreatment of wastewaters from the citrus-processing industry with alkaline
pectinolytic enzymes facilitates removal of pectinaceous material and renders it suitable for
decomposition by activated sludge treatment (Hoondal et al., 2000; Jayani et al., 2005).
Recently, Ramakanth et al. (2014) reported that alkaline PGases from thermotolerant
pectinolytic bacteria from diverse sources could have ample application in food wastewater
treatment.
USE OF OXIDATIVE EXTREMOZYMES FOR THE TREATMENT

OF EFFLUENTS WITH HIGH AROMATIC POLLUTANT CONTENTS
Aromatic compounds, including phenols, biphenols, anilines, benzidines, dyes and

polycyclic aromatic hydrocarbons, constitute the major xenobiotics and recalcitrant pollutants
found in wide variety of industrial wastewaters. Most aromatic compounds are toxic and must
be removed from these poor biodegradable wastewaters before they are discharged into the
environment. Several methods of enzymatic treatment have been proposed as potential
alternatives to conventional methods (Otlesa and Seleka, 2012). Oxidative enzymes in the
lignolytic systems of white-rot fungi (WRF) are the most important class of enzymes suited
for enzymatic industrial pollutant‘s degradation (Whiteley and Lee 2006). WRF which are
mostly basidiomycetes can secrete extracellular and non-specific lignin modifying enzymes
(LMEs) that play an important role in the degradation of lignin and xenobiotics. The LMEs of
WRF mainly comprise of lignin peroxidase (LiP), manganese dependent peroxidase (MnP),
and laccase (Asgher et al. 2008; Baldrian 2006; Yang et al. 2013). However, new reports
demonstrate thermal robustness and more alkaline and saline activities profiles of bacterial
laccases compared to fungal lignolytic enzymes (Molina-Guijarro et al., 2009).
Ligninolytic Peroxidases
Manganese peroxidases (MnP) are glycoproteins with an iron protoporphyrin IX (heme)

prosthetic group. They are usually secreted in multiple isoforms in carbon and nitrogen
limited media by several fungi. MnP catalyzes the peroxide dependent oxidation of Mn2+ to
Mn3+ which is stabilized by chelators such as oxalic acid. Chelated Mn3+ acts as a highly
reactive low molecular weight diffusible redox-mediator. Thus, MnP are able to oxidase and
depolymerise their natural substrate, i.e., lignin as well as recalcitrant xenobiotics, such as
nitroaminotoluene and textile dyes (Wesenberg et al., 2002). In vitro depolymerization can be
enhanced in the presence of co-oxidants such as thiols or unsaturated fatty acids (Hofrichter,
2002). Lignin peroxidases are N-glycosylated enzymes, which catalyzes the oxidation of non-
phenolic aromatic lignin moieties and similar compounds by one-electron abstraction to form
reactive radicals (Conesa et al. 2002, Martínez 2002). They contain heme in the active site
and show a classical peroxidase mechanism. The role of LiP in ligninolysis could be the
further transformations of lignin fragments which are initially released by MnP. LiP are not
essential for the attack on lignin: several highly active WRF and litter-decaying fungi do not
produce this enzyme. LiP have been used to mineralize a variety of recalcitrant aromatic
compounds, such as PAHs, polychlorinated biphenyls, and also dyes (Novotny et al., 2004).
Usually, the natural fungal secondary metabolite veratryl alcohol (VA) acts as redox mediator
to stimulate the LiP catalyzed oxidation of a wide range of recalcitrant substrates (Christian et
al., 2005). Versatile peroxidases (VP) are heme containing enzymes, structural hybrid
between MnPs and LiPs, since they can oxidize not only Mn2+ but also phenolic and
nonphenolic aromatic compounds including dyes, in manganese-independent reactions
(Shrivastava et al., 2005). VPs have been recently described in Pleurotus and Bjerkandera
(Honda et al., 2006; Rodakiewicz-Nowak et al., 2006). Decolourisation of some textile dyes
by VPs from B. adusta has been reported only in the presence of oxidizing mediators (Tinoco
et al., 2007). WRF peroxidases may find considerable use as a green treatment for COD
abatement and color removal in molasses wastewater (Vahabzadeh et al., 2004), Olive mill
wastewater (Jaouani et al., 2003; Jaouani et al., 2006; Neifar et al., 2012) and textile dyeing
wastewaters (Pakshirajan and Radhika, 2013). However, factors limiting the bioremediation
use of these peroxidases included their low production level and rather low stability (Conesa
et al. 2002). In addition, the peroxidases need the addition of H2O2 for their catalytic cycle.
Immobilization onto inert carriers may be the most effective means for improving stability of
WRF peroxidases. Immobilized forms may be incorporated into continuous process streams
and can be continuously recycled when used with batch systems. Heterologous expression of
ligninolytic peroxidases has been attempted, using several recombinant systems with various
degrees of success (Conesa et al. 2002). The challenge is now to move enzyme production
from laboratory scale to industrial scale.
Laccases
Laccases (benzenediol: oxygen oxidoreductase; EC 1.10.3.2), a family of multi-copper

polyphenol oxidases, catalyze the oxidation of a certain range of inorganic and aromatic
substances (particularly phenols), with the concomitant reduction of O2 to water (Baldrian,
2006; Giardina et al., 2010; Fang et al., 2014).
In the presence of small molecules capable to act as electron transfer mediators, the
substrate range of laccases can be further extended (Figure 1). Laccase are unique,
environmentally friendly and efficient biocatalysts that can degrade recalcitrant pollutants
(Zhang et al., 2015). They have been extensively investigated for new and challenging
decontamination programs because they affect the oxidation of many aromatic compounds
towards more benign and less toxic products. Indeed, laccases can be involved in the
detoxification of phenols, chlorinated phenolics, organophosphorus pesticides, synthetic dyes
and polycyclic aromatic hydrocarbons which are known for their carcinogenic as well as
mutagenic effect and are persistent in the environment (Durán et al., 2000; Bollag et al., 2003;
Torres et al., 2003; Pozdnyakova et al., 2004; Alcalde et al., 2006; Ruggaber et al., 2006).
Because of their wide substrate range, requirement of no cofactors, and use of readily
available oxygen as an electron acceptor, laccases have been successfully applied for the
bioremediation of effluents from textile, pulp and paper, petrol refinery and olive oil
industries (Vargas and Ramírez, 2002; Jaouani et al., 2005; Sigoillot et al., 2004; Olivieri et
al., 2006; Neifar et al., 2010; Neifar et al., 2011; Neifar et al., 2012; Otlesa and Seleka, 2012;
Kaushik and Thakur, 2013; Viswanath et al., 2015). The application of laccase in municipal
wastewater has also been largely explored (Ba et al., 2013). In nature, white rot fungi are the
most efficient laccase producers, and many fungal laccases have been isolated and
characterized (Baldrian, 2006). Many of fungal laccases exhibit high redox potentials and
therefore possess high activities towards their substrates. However, their use is restricted to
acidic reaction conditions and mesophilic temperatures.
4e -
Laccase mediators Subtrates oxidized by laccase mediator system
Synthetic mediators Polycyclic aromatic hydrocarbons
 ABTS [2,2′-azino-bis-(3-  Acenaphthene
ethylbenzothiazoline-6-sulfonic acid)]  Acenaphthylene
 1-hydroxybenzotriazole  Fluorine
 violuric acid  anthracene
 N-hydroxyacetanilide Recalcitrant synthetic dyes
 N-hydroxyfthalimide  Reactive Black 5 (diazo dye)
Natural mediators  Azure B (heterocyclic dye)
 Phenol  Acid Blue 74 (indigoid dye)
 Aniline  Reactive Blue 19 (anthraquinoid dye)
 4-hydroxybenzoic acid  Aniline Blue (triarylmethane-type dye)
 4-hydroxybenzyl alcohol  Solophenyl Red 3BL (tetraazo dye)
 Acetosyringone Polychlorinated biphenyls
 syringaldehyde pentachlorophenol
 Lignin-derived compounds Herbicides and pesticides
Camarero et al., 2005; Canas and Camero, 2010; Neifar et al., 2011.
Figure 1. Oxidation of non phenolic recalcitrant substrates with laccase mediator system.
Moreover, fungal laccases are highly glycosylated enzymes and cannot be produced with
bacterial expression systems (Gunne and Urlacher 2012). In the last two decades, laccases
have been found to be widespread in bacteria and archae (Claus, H. 2003; Sharma et al.,
2006; Sharma et al., 2007; Uthandi S et al., 2012; Maupin-Furlow et al., 2012).
These laccases have been found to possess distinctive properties, including excellent
activity and stability under extreme thermal, alkaline, and saline conditions (Sharma et al.,
2007: Fang et al., 2011; Lu et al., 2011). For examples, laccase from Bacillus tequilensis
SN4, isolated from the activated sludge of paper mill effluent treatment plant, was found to be
highly thermostable (optima at 80-90°C, retained more than 80% activity at 70°C for 24 h)
and alkali-stable (optimum pH of 8 for 2, 6-dimethoxyphenol and guaiacol, retained more
than 75% activity at pH 9.0 for 24 h) which makes this laccase a potential candidate for
bioremediation applications (Sondhi et al., 2014a, b). Similarly, laccase from Bacillus
licheniformis LS04, isolated from forest soil, demonstrated a broad pH range for catalyzing
substrates. It was quite stable at high temperature (up to 80°C) and alkaline pH (up to 9).
The unusual biochemical properties and dye decolourization abilities for this novel
laccase indicated its high potential in textile wastewater treatment (Lu et al., 2012). Recently,
researchers at the University of Florida have discovered a laccase in Haloferax volcanii that
lives in extreme saline environments, such as the Dead Sea. This new Archaea-based laccase
maintained activity at high temperature and extreme pH, as well as high salt conditions,
making it a great candidate for various industrial applications. The simple extraction and
purification offers cheaper and more effective yields for potential manufacturers, making the
technology competitive in the market (Uthandi S et al., 2010; Uthandi S et al., 2012; Maupin-
Furlow et al., 2012). Actinomycetes laccases with unusual properties have also been
discovered (Endo et al., 2003; Suzuki et al., 2003; Machczynski et al., 2004; Niladevi et al.,
2008; Molina-Guijarro et al., 2009; Gunne and Urlacher, 2012; Lu et al., 2013). As examples,
an unusual halotolerant-alkaline laccase from Streptomyces psammoticus has been described
by Niladevi et al. (2008). The relatively high activity at alkaline pH (up to 9.5) and high salt
concentrations (up to 1.2 M) makes this enzyme different from other reported laccases and
increase its potential for use in diverse industrial applications. A novel pH-versatile,
thermostable and salt-resistant laccase from Streptomyces ipomoea has been reported by
Molina-Guijarro et al. (2009). Microtox analysis confirmed that laccase-natural mediator
(acetosyringone) system was able not only to degrade azo-type textile dyes but also to
detoxify them completely at alkaline pH. Specific features of bacterial laccases have been
exhaustively adapted in order to reach the industrial demands for high catalytic activity and
stability with a low production cost. Review of Mate and alcade (2015) examined the most
significant advances in this exciting research area in which rational, semi-rational and
directed evolution approaches have been employed to ultimately convert laccases into high
value-added biocatalysts. The remediation of contaminated wastewater could be achieved by
using a combination of enzyme technology and nanotechnology known as the Single Enzyme
Nanoparticle (SEN). The use of nanoparticles in reactive remediation technology is of great
interest to wastewater treatment, since it involves the complete degradation of contaminants
to harmless products such as carbon dioxide and water. SENs are able to withstand more
drastic conditions of temperature, pH, salinity and pollutant concentration compared to free
enzymes. Novel nanoparticles such as nanosponges could be synthesized using laccases and
could find application in remediation of wastewaters from dyeing and textile processing
industries (Mugdha and Usha, 2012).
CONCLUSION
A large number of ligninolytic, lipolytic, proteolytic, cellulolytic, hemicellulolytic,
amylolytic and pectinolytic enzymes from different microbial sources have been reported to
play an important role in a wide array of wastewater treatment applications.
Enzyme-based technologies in this field can be considered as eco-friendly energy- and
material-saving processes. Enzymes can act on specific recalcitrant pollutants, to remove
them by precipitation or transformation to other products. They can also modify the properties
of a given wastewater to render it more acceptable for further treatment or help in
transforming waste material to value-added products. This increasing interest in enzymes in
wastewater treatment is triggered by the pressing need to use greener molecules rather than
environmental harmful compounds in treatment processes and by the necessity to effectively
eliminate a wide variety of emerging pollutants that are unsatisfactorily removed by existing
effluent treatment technologies.
Despite their advantages, the use of enzymes for the treatment of industrial/municipal
wastewater applications has been limited by several factors and specific requirements need to
be considered in the application of enzymes to wastewater treatment processes. In fact, most
of research studies focused on demonstrating the enzymatic degradation of various pollutants
in wastewaters but did not take into account economic and engineering aspects mandatory to
assess the process feasibility. Many enzymes that have been studied are still costly due to the
considerable costs o isolation, purification and production. Another limitation of using
enzymes is their inherent fragility in environmental conditions. All these constraints could be
solved by the following strategies:
 Improvements in growth techniques as well as advances in the development of

molecular strategies and tools for working with currently non-culturable
extremophilic strains. The identification of novel extremophiles promises the

discovery of new extremozymes;
 Enhancement in the number of completed extremophiles genomes as well as
improvements in annotation techniques that will help to reveal novel extremozymes
potentially useful in wastewater treatment;
 Use of site specific mutagenesis to improve existing biodegradation pathways. For
the degradation of recalcitrant compounds where no natural pathways are known,
novel pathways can be generated by combining pathway‗cassettes‘ from various
genetic sources;
 Reducing the cost of enzyme production through the use of zero or negative cost
substrates, such as wastewaters. Efforts on the design of special reactors and
reduction of mass transfer limitations are needed. Recombinant expression in suitable
hosts could also represent a promising tool to obtain higher production yields at
affordable price;
 For commercial availability, it is often necessary to immobilize the enzyme in order
to achieve improved stability and facilitate recovery and reuse. If properly designed,
an immobilized biocatalyst greatly increases the stability of enzymes, and eases the
burden of enzyme cost, and thus, is widely pursued for efficient, selective, and
environmentally friendly catalysis.
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INDEX
aesthetic, 91
# age, 23, 158
aggregation, ix, x, 163, 164, 172
21st century, 86
Agricultural Research Service, 183
agriculture, 3
A Agrobacterium, 166, 174, 184
alcohols, 197
abatement, 201 algorithm, 51, 142
Abraham, 207 alkalinity, 22, 151, 156
abstraction, 197, 201 alternative energy, 138, 150
abuse, 11 aluminium, ix, 163, 164, 169, 174
acclimatization, 129, 196 aluminium sulphate, ix, 163
acetaminophen, 8, 87 amine(s), ix, 164, 173, 197
acetic acid, 114 amine group, 173
acetone, 116, 122 amino, 122, 173, 197
acid, 2, 8, 10, 13, 14, 20, 72, 78, 83, 84, 85, 88, 100, amino acid(s), 122, 197
105, 107, 115, 122, 123, 126, 150, 153, 155, 169, ammonia, 58, 64
180, 184, 197, 199, 200, 201, 202 ammonium, 23, 169
acidic, x, 58, 98, 99, 102, 103, 104, 109, 176, 179, amylase, 198, 199, 200, 205, 208
191, 194, 195, 198, 200, 202 anaerobe, 58
acidity, 22 anaerobic digesters, 154
acrylic acid, 169 anaerobic digestion, 78, 113, 114, 121, 128, 137,
activated carbon, 5, 24, 25, 98, 108, 155 153, 194, 209
active compound, 9, 103 anaerobic processes, ix, 111, 112, 113, 122
active oxygen, 27 anaerobic sludge, 123, 125, 129, 152, 153
active site, 174, 195, 201 analgesic, 8, 100
adaptation(s), 7, 75, 127, 136, 141 anatase, 86, 87, 94
adenine, 115 androgen, 79
adhesives, ix, 163 animal feeding operations, 82
adjustment, 196 annealing, 49
adolescents, 185 annotation, 204
adsorption, vii, viii, 1, 5, 7, 16, 24, 25, 30, 69, 87, 88, anodization, 107
94, 95, 98, 99, 164, 171, 172, 176, 177, 179, 183, ANOVA, 60, 75
196 AnSBBR, ix, 112, 113, 117, 118, 123, 124, 126, 127,
advancements, 112 130, 131, 132, 133, 136, 139, 140, 144, 145, 146,
adverse effects, ix, 12, 77, 81 147, 148, 149, 151, 152, 153, 154, 155, 156, 157,
adverse event, 2 158, 159
aerobe, 58 antibiotic, 4, 8, 9, 15, 105
aerobic bacteria, 121 antibiotic resistance, 4, 9
216 Index
antidepressants, 11, 12 bicarbonate, 28, 140

anti-inflammatories, 14, 15, 18 bioavailability, 20
anti-inflammatory drugs, 3, 100 biocatalysts, x, 192, 193, 196, 202, 203, 211
anxiety, 11 biodegradability, 85, 107, 164
appetite, 11 biodegradables, ix, 163
aptitude, 12 biodegradation, 7, 10, 16, 17, 18, 19, 22, 23, 26, 27,
aquaculture, 8, 83 70, 71, 75, 78, 86, 99, 101, 170, 181, 188, 192,
aquatic life, 10, 180 204, 205, 208, 209, 210
aquatic organisms, ix, 11, 12, 77, 81, 82, 170 biodiesel, 113, 130, 132, 139, 140, 144, 151, 152,
aquatic systems, 195 156, 159
aqueous solutions, 167, 180, 211 biodiversity, 23, 60
arginine, 212 bioenergy, 112, 113, 117, 126, 144, 151
aromatic compounds, x, 192, 197, 200, 201, 202, 207 biofuel, 152, 158
aromatic hydrocarbons, 197, 202 biogas, ix, 111, 112, 113, 114, 117, 120, 121, 123,
aromatic rings, 18, 19 124, 127, 131, 132, 135, 136, 150, 156, 196
aryl hydrocarbon receptor, 79 biohydrogen, ix, 112, 113, 121, 122, 123, 127, 132,
ASBR, ix, 111, 112, 113, 117, 118, 123, 124, 126, 136, 137, 138, 139, 144, 148, 149, 150, 151, 153,
127, 128, 130, 131, 132, 133, 134, 136, 137, 140, 154, 155, 156, 158, 159, 160
141, 144, 145, 146, 147, 148, 149, 151, 155, 156, biological activities, 101
158, 159, 160 biological activity, 6, 15, 23
Asia, 70 biological processes, 5, 16, 23, 24, 29, 95, 121, 143,
assessment, 5, 9, 42, 62, 112, 120, 185, 207 150, 153, 196
assimilation, 131 biological systems, 135, 136, 141
atmosphere, 63 biomass, ix, 18, 23, 26, 61, 67, 72, 75, 111, 112, 113,
atoms, 98 118, 123, 124, 125, 126, 127, 128, 129, 131, 136,
ATP, 137 144, 152, 154, 157, 158, 184
attachment, 172, 173 biomethane, ix, 112, 113, 132, 136, 144, 145, 147
awareness, 3 biomolecules, ix, 163, 211
biopolymer(s), ix, 164, 170, 174, 184, 185, 186, 187
bioreactor, viii, 7, 13, 23, 26, 70, 71, 75, 76, 77, 78,
B 79, 109, 132, 151, 155, 189
bioremediation, ix, 71, 78, 163, 164, 195, 197, 201,

Bacillus mucilaginosus, ix, 164, 167, 177, 178, 185
202, 203, 208, 210, 213
Bacillus subtilis, 166, 168, 174, 180, 193, 209
birds, 4
bacteria, viii, ix, 4, 8, 9, 14, 15, 17, 23, 26, 28, 45,
bisphenol, 99, 103
46, 52, 55, 58, 59, 63, 64, 65, 66, 67, 72, 75, 78,
bleaching, 197, 198, 199, 211
96, 99, 114, 116, 121, 124, 126, 127, 129, 131,
blood, 10, 14, 182
135, 137, 150, 158, 160, 161, 163, 165, 167, 168,
blood vessels, 10
170, 174, 180, 181, 185, 188, 192, 198, 199, 200,
blood-brain barrier, 182
203, 205, 207, 209, 211
bonding, 15, 17, 20
bacterial strains, 177, 185, 187
bonds, x, 89, 177, 192, 194, 197
bacterium, 56, 57, 58, 59, 180, 183, 184, 185, 206,
Brazil, 139
210
breakdown, 23
band gap, 86, 88, 89
bulk pollutants, vii, 1, 3
barbiturates, 11
by-products, x, 27, 28, 30, 77, 89, 191, 196
barriers, 63
base, 51, 54, 122, 123
beef, 166 C
beer, 139
behavioral problems, 11 Ca2+, 172, 175, 176
Beijing, 81, 101 cadmium, 179
benefits, 29, 114, 123, 194, 199 caffeine, 89, 90, 92, 95, 100, 102, 103
benign, 202 calcium, 169, 172
bias, 94 campaigns, 70, 75
Index 217
cancer, ix, 163 coal, x, 25, 98, 115, 191

candidates, 115 coatings, 94
capillary, 49 collenchyma, 212
carbamazepine, 12, 14, 84, 87, 91, 94, 95, 99, 100, color, 28, 71, 175, 178, 180, 201
102, 103, 105, 107 commercial, 86, 121, 132, 175, 178, 186, 194, 200,
carbohydrate(s), 122, 137, 138, 140, 150, 152, 158, 204
167, 168, 177, 186, 193, 197 communities, viii, 45, 47, 50, 54, 59, 60, 61, 62, 63,
carbon, 17, 24, 25, 46, 59, 63, 65, 92, 98, 114, 130, 64, 66, 135
138, 139, 140, 159, 165, 175, 182, 188, 201, 203 community, viii, 45, 48, 50, 53, 54, 59, 60, 61, 62,
carbon dioxide, 114, 140, 203 65, 67, 122, 160
carboxyl, ix, 164, 172, 174, 193 compaction, 177
carcinogenicity, 182 competition, 131
cardiovascular disease, 10 complement, vii, 2, 24
casein, 166 complexity, 15, 18, 182
catalysis, 192, 204 compliance, 27, 112, 151
catalyst, 86, 94 composition, viii, 17, 19, 20, 45, 50, 53, 54, 61, 62,
catalytic activity, x, 192, 203 65, 66, 67, 78, 114, 127, 173, 176, 186
cation, 15, 174, 177 compost, 208
C-C, 197 compounds, vii, ix, 2, 3, 5, 6, 7, 8, 10, 11, 12, 14, 16,
cellulose, 195, 197, 199, 210, 212 17, 18, 19, 21, 22, 23, 24, 26, 27, 28, 30, 46, 70,
central nervous system, 8, 11, 12 71, 78, 79, 81, 82, 84, 85, 86, 88, 95, 96, 98, 100,
ceramic, 26 101, 104, 106, 108, 123, 128, 129, 131, 140, 182,
CFR, 199 192, 193, 195, 196, 197, 200, 201, 202, 204, 205,
charge density, 169, 172, 179, 182 206
cheese, 113, 137, 139, 140, 144, 153, 155 conception, 161
chemical catalysts, x, 191 conditioning, 6, 72, 129
chemical characteristics, 17 conduction, 86, 88, 89
chemical degradation, 7 configuration, viii, 13, 18, 45, 94, 123, 124, 136, 138
chemical interaction, 17 confinement, 139
chemical properties, 6, 18, 20, 75, 95 conjugation, 12
chemical reactions, 31, 196 consensus, 135
chemical stability, 22, 85 constituents, 5, 17, 28, 88, 90, 95, 111, 126, 188
China, 39, 64, 81, 84, 96, 103, 105, 107, 189 constructed wetlands, viii, 24, 31, 33, 42, 45, 46, 51,
chitosan, 173, 174, 184, 186 55, 58, 59, 60, 61, 62, 64, 65, 66, 67, 99, 100, 109
Chitosan, 183, 186 construction, 29, 150
chlorination, 5, 89 consumers, 122, 136, 138
chlorine, 91, 199 consumption, 11, 13, 14, 82, 107, 117, 121, 129,
chloroform, 122, 123 131, 132, 141, 143, 165, 199
chloroplast, 56, 57 contact time, 23, 26, 30, 98, 196
cholesterol, 10 contaminant, 14, 21, 77, 85, 86, 98
chromatography, 77, 103, 104 contaminants, vii, 2, 3, 4, 14, 17, 24, 25, 27, 45, 62,
circulation, 150, 151, 157, 158 70, 78, 82, 84, 95, 97, 98, 104, 106, 171, 174,
citalopram, 11 182, 203
cities, viii, 69, 72 contaminated sites, 179
classes, 3, 8, 9, 12, 14, 15, 18, 95, 192 contaminated soil(s), 181
classification, 2 contaminated water, 25
clean energy, 115, 150 contamination, vii, 2, 9, 15, 31, 102
cleaning, 70, 128, 194 controversial, 137
cleavage, 197, 200 conversion rate, 135
climate, 115, 194 conversion reaction, 141
cloning, 194 COOH, 115, 117, 140, 172, 174, 179
clusters, 51 coordination, 20
CO2, 63, 127, 135 copper, 201
218 Index
corn stover, 166, 175, 187 digestibility, 195

correlation(s), 23, 103 digestion, 6, 121, 141, 151, 161
cost, vii, ix, x, 1, 2, 4, 24, 25, 29, 31, 81, 86, 93, 99, discharges, vii, 1, 139
101, 115, 128, 138, 150, 175, 181, 191, 192, 194, diseases, 2, 6, 8, 13
195, 196, 203, 204 disinfection, 5, 27, 84, 89, 91
cost benefit analysis, 181 disorder, 11
covering, 12, 95 dispersion, 7, 209
crop, 15, 152 dissolved oxygen, 60, 61, 196
crop production, 15 distribution, 3, 13, 21, 50, 73, 82, 98, 109, 182
crystalline, 86, 94 diuretic, 100
CSF, 119, 120 diversity, 25, 27, 46, 59, 63, 64, 66, 67, 153, 185
CST, 119 DNA, 48, 49, 116
CT, 40, 51, 52, 53 DNA polymerase, 49
cultivars, 59, 63 DOC, 92, 93
cultivation, 46, 71, 175, 185 DOI, 211, 213
culture, 75, 78, 153, 155, 159, 165, 175, 183, 185, donors, 197
187, 193, 194, 196 doping, 88
culture conditions, 165, 185 dosage, 92, 98, 175, 180, 195
cycles, 49, 71, 119, 130, 136 drawing, 101
cycling, 46, 62 drinking water, 3, 4, 40, 82, 84, 85, 86, 89, 91, 95,
cyclophosphamide, 2 98, 101, 102, 103, 104, 105, 106, 108, 164, 185
cylindrical reactor, 71 drugs, 3, 4, 6, 7, 8, 10, 11, 13, 14, 15, 77, 100, 101,
Czech Republic, v, viii, 69, 70, 71, 73, 74, 77, 78 103
drying, 6
dyeing, 175, 180, 197, 201, 203, 208
D dyes, 89, 173, 180, 181, 182, 187, 196, 197, 200,
201, 202, 203, 205, 208, 212
dairies, x, 192, 193, 195
dairy industry, 194
database, 50, 62, 103 E
deacetylation, 184
decomposition, 31, 58, 93, 115, 194, 200 ecology, 46, 64, 65
decontamination, 202 ecosystem, ix, 81

degradation process, 3, 21 ecotoxicological, 27, 30, 82
degradation rate, 75, 210 electricity, 196
denaturation, 49 electrocatalysis, 94
denitrification, 5 electrochemical behavior, 154
denitrifying, 58, 64, 65, 66 electrode surface, 94, 107
depolymerization, 201 electrodes, 94, 107
deposition, 94, 107 electrolysis, ix, 81, 94
depression, 11 electrolyte, 87, 88
depth, 3, 47, 48 electromagnetic, 22
derivatives, 10, 187 electron(s), x, 72, 86, 88, 89, 91, 94, 192, 197, 201,
destruction, 27, 28 202
detectable, 84, 96 electrophoresis, 49, 64, 65, 66
detection, vii, viii, 1, 3, 7, 9, 14, 69, 72, 81, 82, 84, emission, 97
104 emotional disorder, 11
detention, 196 endocrine, viii, 4, 12, 70, 71, 77, 78, 79, 81, 102,
detergents, ix, 163 103, 104, 107, 108
detoxification, 192, 198, 202, 212 endocrine-disrupting chemicals, 108
DGGE band patterns, viii, 45, 61 endothelial cells, 102
dialysis, 179 energy, ix, 17, 23, 26, 27, 28, 85, 86, 88, 90, 93, 97,
differential equations, 142 111, 112, 113, 114, 115, 117, 144, 175, 204
diffusion, 107 energy consumption, 26, 97
Index 219
energy input, 90 fermentation, 115, 116, 122, 135, 137, 138, 140, 150,
energy production, ix, 111, 112, 144 152, 153, 154, 155, 157, 158, 159, 160, 164, 166,
engineering, 42, 107, 156, 204, 209 181, 188, 189, 194
environment(s), vii, ix, 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, ferredoxin, 116
12, 14, 21, 22, 23, 29, 30, 33, 46, 48, 50, 54, 53, fibers, 25, 26
55, 58, 59, 61, 66, 69, 70, 71, 77, 78, 81, 82, 83, film thickness, 72
99, 101, 102, 103, 121, 135, 138, 144, 163, 164, films, 94
170, 194, 198, 200, 202, 203, 211 filters, 5, 18, 25, 86
environmental conditions, 9, 13, 18, 20, 21, 22, 46, filtration, ix, 5, 16, 25, 26, 81, 84, 95, 97, 108, 164,
60, 204 183
environmental contaminants, viii, 81, 83, 100 financial, 101
environmental contamination, ix, 77, 81 financial support, 101
environmental effects, 5 fine tuning, 30
environmental management, 153 fingerprints, 51, 54
Environmental Protection Agency, 15, 82 fish, 198
environmental standards, x, 191 flexibility, 135
enzyme(s), x, 18, 22, 23, 24, 72, 75, 78, 121, 137, flocculants, ix, x, 163, 164, 165, 167, 169, 170, 171,
138, 191, 192, 193, 194, 195, 196, 197, 198, 199, 172, 174, 175, 177, 178, 180, 181, 182, 183, 184,
200, 201, 203, 204, 205, 206, 207, 208, 210, 211, 186, 189
212 flocculation, ix, x, 84, 163, 164, 165, 168, 169, 171,
EPA, 42, 61, 64 172, 173, 174, 175, 176, 177, 178, 182, 183, 184,
EPS, 164, 165, 174, 175, 176, 177, 178, 179, 180, 185, 186, 187, 188
181, 182, 183, 184, 187, 188 flora, 4, 23
equilibrium, 21, 117 flotation, 126, 164
equipment, 5, 28, 128 fluid, 152
erosion, 15 fluidized bed, 136, 154
ester, 193, 197, 208 fluorescence, 97
ester bonds, 193, 197 fluoroquinolones, 9, 15, 17
ethanol, 117, 122, 144, 166 fluoxetine, 11, 12, 14, 98
ethyl acetate, 72 fluvoxamine, 11
EU, 57 foils, 94
Europe, 43, 70 food, 4, 10, 113, 123, 128, 131, 139, 153, 154, 161,
evidence, 14, 98 170, 195, 198, 200
evolution, 4, 203, 209 food chain, 170
excitation, 88, 97 food industry, 113
exclusion, 95 force, 10, 94, 172
excretion, 3, 183 formamide, 49
exopolysaccharides, 164, 170, 173, 174, 179, 180, formation, x, 28, 30, 89, 93, 124, 126, 131, 132, 136,
183, 185, 186 141, 164, 171, 172, 173, 183, 191, 196, 199
experimental condition, 54, 127, 128 formula, 89
exposure, 4, 9, 10, 77 fouling, 26
extraction, 48, 49, 77, 103, 104, 164, 179, 182, 183, foundations, 112
194, 198, 203 fragility, 204
extracts, 72, 192 fragments, 20, 64, 201
France, 84
freshwater, 55, 58, 62, 64
F fructose, 117
fruits, 198
families, 6
fuel cell, 115
fat, 194, 209
funding, 102
fatty acids, 66, 72, 122, 131, 197, 201
fungi, ix, 8, 60, 67, 71, 78, 79, 163, 166, 168, 170,
fauna, 4
174, 192, 194, 199, 200, 201, 202, 205, 207, 208,
feedstocks, 175
210
220 Index
fungus, viii, 70, 75, 207, 213 human, ix, 2, 4, 6, 8, 9, 11, 12, 29, 61, 77, 81, 82,
101, 102, 103, 104, 163, 164, 169
human body, 12
G human health, ix, 2, 4, 77, 81, 82, 104, 163, 164, 169
hybrid, 100, 184, 201
gamma radiation, 122
hydraulic design, viii, 45, 47, 50, 53, 54, 60, 61, 62
gel, 48, 49, 52, 54, 64, 65, 66, 89
hydrogen, 15, 17, 20, 27, 65, 89, 90, 91, 105, 112,
gene expression, 77
113, 114, 115, 116, 117, 121, 122, 123, 126, 127,
genes, 4, 9, 63, 193
129, 130, 131, 132, 135, 136, 137, 138, 139, 140,
genetics, 183, 208
141, 143, 144, 150, 151, 152, 153, 154, 155, 156,
genome, 206
157, 158, 159, 160, 161, 197
genus, 58, 59, 117, 121, 150
hydrogen abstraction, 91
Germany, 33, 71, 72
hydrogen peroxide, 27, 89, 90, 105
global economy, 115
hydrogen sulfide, 65
glucose, 89, 117, 122, 123, 131, 137, 138, 140, 144,
hydrogenase, 121, 136, 137, 138
145, 148, 153, 155, 156, 157, 159, 160, 161, 166,
hydrolysis, 7, 16, 18, 114, 137, 140, 141, 193, 194,
174, 195, 199
195, 197, 198, 199, 200, 208, 209
glutamic acid, 166
hydrophobicity, 16, 17, 20, 21, 23, 95
glycerin, 130, 132, 139, 140, 156, 158
hydrothermal process, 106
glycerol, 117, 130, 139, 140, 152, 159, 193, 197
hydrothermal synthesis, 106
glycoproteins, ix, 163, 174, 201
hydroxide, 64
gracilis, 198, 200, 205
hydroxyl, ix, 15, 18, 27, 28, 85, 86, 90, 91, 107, 164,
grants, 77
173, 174
granules, 113, 124, 157
hydroxyl groups, ix, 164, 173
grasses, 63
hypothesis, 61, 141
gravity, 26
Greece, 102
greenhouse, 115 I
groundwater, 65, 83
grouping, 50 ibuprofen, 8, 14, 15, 88, 89, 95, 99, 100, 106
growth, 4, 8, 18, 26, 63, 105, 122, 123, 129, 137, ID, 213
141, 143, 150, 154, 157, 161, 170, 175, 179, 204 ideal, 113, 117, 135
growth rate, 129 identification, 4, 65, 175, 187, 204

identity, 55
illumination, 86, 87, 94
H images, 50
immobilization, 124, 125, 129, 144, 193
habitat(s), 47, 58, 59, 61, 62, 209
improvements, 204
hair, 195
impurities, 152, 164, 174
half-life, 21, 198, 199
in vitro, 102
halogens, 18, 199
in vivo, 77
harmful effects, vii, 1, 180
India, 209, 210
hazardous materials, 107
induction, 75
hazards, 170
industrial sectors, ix, 163
health, ix, 5, 81, 82, 170
industries, x, 113, 139, 140, 144, 191, 195, 198, 202,
heavy metals, ix, 15, 164, 170, 179, 181, 183, 184,
203
186
industry, 126, 127, 128, 130, 132, 134, 139, 140,
heme, 201
147, 157, 158, 178, 189, 193, 194, 195, 198, 199,
hemicellulose, 210
200, 208, 211
high fat, 194, 209
INF, 51, 53, 99
history, 14
inflammation, 8
hormone(s), 3, 77, 103, 104
infrastructure, 82
host, 63
ingredients, 177
inhibition, 9, 11, 65, 106, 124, 126, 131, 132, 137
Index 221
initial state, 28 liquid phase, 17, 94, 107, 118, 127, 130, 132, 136,
inoculum, ix, 112, 113, 122, 123, 125, 137, 144, 150 140
integration, 142, 156, 158 livestock, 9
integrity, 49 localization, 65
interaction effect, 153 Louisiana, 102, 103
interphase, 63 lovastatin, 10
invertebrates, 4 low density polyethylene, 125
iodine, 89, 106 low temperatures, 136
ionization, 2, 17, 20, 22 low-density lipoprotein, 10
ionizing radiation, 122 Luo, 3, 4, 6, 7, 8, 10, 12, 13, 14, 16, 17, 18, 23, 24,
ions, 28, 88, 96, 98, 172, 178, 179, 194 25, 27, 38, 165, 185
Iran, 66 lysis, 114, 115, 197
iron, 18, 65, 67, 71, 138, 155, 161, 201
irradiation, 18, 19, 87, 88, 89, 90, 102, 161
irrigation, 3, 9, 30 M
isolation, 187, 204
macrolide antibiotics, 90
magnesium, 169
J magnet, 94
magnitude, 14, 70, 114, 144
Japan, 107 major issues, 30
majority, 46, 91, 170, 174
man, 29, 198
K management, vii, 46, 108, 114
manganese, x, 192, 200, 201, 207, 212
K+, 176
manufacturing, 180
kinetic constants, 109, 131, 142
manure, 83, 121, 137, 148, 156, 160, 161
kinetic model, 141, 142, 144, 150
marsh, 63, 66
kinetic parameters, 124, 126, 141, 142
mass, x, 49, 72, 77, 78, 79, 96, 103, 104, 115, 119,
kinetic studies, ix, 112, 113, 128, 142
120, 124, 142, 181, 188, 192, 204
kinetics, 87, 93, 95, 105, 106, 107, 124, 151
mass spectrometry, 78, 79, 103, 104
Klebsiella mobilis, ix, 164, 166, 167, 174, 180, 187
materials, 5, 10, 25, 26, 28, 29, 31, 58, 67, 85, 89,
Korea, 103
98, 105, 114, 153, 165, 170, 172, 184
matrix, 15, 28, 29, 50, 87, 90, 97, 185
L matrixes, 50, 82
matter, 20, 23, 28, 59, 85, 114, 119, 124, 131, 132,
laccase, x, 75, 78, 192, 198, 200, 202, 203, 205, 206, 136
207, 208, 209, 210, 211, 212 measurements, 78
lactose, 166 meat, 178
lakes, 121, 144 media, 2, 18, 25, 54, 60, 164, 175, 182, 201
LDL, 10 median, viii, 69, 73
lead, 20, 22, 82, 101, 113, 144, 170 medication, 82
legislation, 15 medicine, 2, 8, 11
life cycle, 12 Mediterranean, 66, 104
light, 18, 19, 28, 82, 86, 88, 89, 90, 95, 105, 106, Mediterranean climate, 66
107, 196 medium composition, 185
light scattering, 28 membranes, 25, 26, 27, 28, 87, 95, 96, 105, 108, 121
lignin, x, 71, 192, 197, 198, 199, 200, 201, 205, 207, Metabolic, 116, 156
210, 212 metabolic pathways, 117, 136, 137, 150
lipases, x, 191, 192, 193, 194, 207, 209, 211 metabolism, 10, 12, 18, 23, 117, 121, 126, 175
lipids, ix, 10, 21, 163, 209 metabolites, 3, 6, 11, 14, 30, 82, 101, 170
lipoproteins, 10 metabolized, 82
liquid chromatography, 78, 79, 103, 104 metal ion(s), 172, 186, 198
metal oxides, 27
222 Index
metal salts, 172, 174 mutagenesis, 204

metals, x, 164, 169, 179, 191, 194 mutant, 184
meter, 118
methadone, 8
methanol, 72, 114, 140 N
methodology, 103, 125, 209
Na+, 176
methyl cellulose, 170, 183
NaCl, 198
methylene blue, 89, 180
NAD, 115, 116, 117
methyl-triclosan, viii, 69
NADH, 115, 116, 117
Mg2+, 176
nanobelts, 87
mice, 182
nanomaterials, 89
microbes, viii, ix, 46, 63, 69, 163, 170, 175, 192
nanoparticles, 94, 107, 203
microbial communities, viii, 45, 46, 59, 60, 61, 62,
nanotechnology, 203
63, 64, 65, 155
nanotube, 94, 96, 107, 108
microbial community, 46, 60, 61, 62, 65, 66, 67, 78,
nanowires, 87, 89, 105
122, 154
NAP, 116
microcrystalline, 155
narcotic, 8
microcrystalline cellulose, 155
narcotic analgesics, 8
microenvironments, 51
natural gas, 114, 115
microorganism(s), vii, x, 8, 9, 16, 17, 18, 22, 23, 26,
natural polymers, 170
45, 58, 59, 67, 71, 84, 95, 111, 113, 114, 117,
natural resource management, 63
120, 121, 122, 123, 124, 126, 128, 129, 130, 131,
negative effects, 4, 131
135, 138, 140, 150, 154, 164, 166, 174, 175, 178,
nervous system, 12
179, 181, 188, 194, 199
Netherlands, 43
micropollutants, vii, 1, 3, 4, 6, 7, 23, 25, 26, 27, 30,
neurotoxicity, ix, 163
91, 95, 98, 106, 109
neurotransmitter, 11
microstructures, 94
neutral, 9, 20, 21, 22, 58, 104, 137, 144, 180, 195
mineralization, 17, 27, 86, 87, 89, 94, 105, 197
niacin, 10
Mississippi River, 102
nicotinamide, 115
mixing, 49, 129
nicotinic acid, 10
modelling, 63, 141
nitrates, 18, 28
models, 141, 143, 150

nitrification, 5, 58
modifications, 192
nitrifying bacteria, 23, 66
molar ratios, 92
nitrite, 58
molar volume, 120
nitrogen, 5, 15, 26, 58, 59, 62, 131, 138, 155, 156,
molasses, 166, 201, 212
165, 188, 201
molds, 121
nitrogen gas, 156
mole, 117, 120, 137
nitrogenase, 121
molecular structure, 20, 173, 176
non-polar, 20, 21, 98
molecular weight, 2, 20, 27, 95, 165, 169, 172, 173,
non-steroidal anti-inflammatory drugs, 8, 10, 14, 102
174, 175, 177, 182, 184, 186, 201
North Africa, 70
molecules, 2, 8, 9, 20, 22, 89, 91, 96, 98, 140, 202,
North America, 70
204
NSAIDs, 8, 14
monomers, 164, 169, 170
nucleic acid, ix, 163, 170
monosaccharide, 186
nucleotide sequence, 212
Montana, 102
nutrient(s), 5, 15, 29, 62, 67, 129, 130, 131, 138
Montenegro, 41
nutritional status, 67
Moon, 152
morphine, 8
mosquitoes, 66 O
multidimensional, 50, 51
municipal solid waste, 151, 155 ofloxacin, 14
muscle relaxant, 11 OH, 64, 85, 87, 90, 92, 93, 97, 174, 179, 184
Index 223
oil, ix, x, 114, 115, 163, 175, 181, 191, 192, 193, peroxidise, x, 192
194, 198, 205, 209, 211 personal care products, viii, 40, 70, 78, 81, 82, 101,
olive oil, 202, 207 102, 103, 104, 105, 108, 109, 130
operations, 101, 200 pesticide, 78
opportunities, 193 petroleum, x, 25, 165, 191, 197
optimization, 5, 30, 31, 75, 141, 154, 165, 189 pharmaceutical residues, vii, 1, 3, 5, 7, 9, 12, 16, 24,
optoelectronic properties, 89 83, 102
organic compounds, 3, 7, 15, 28, 90, 95, 99, 178, pharmacotherapy, 11
181, 195 phenol, 70, 95, 107, 108, 198
organic matter, 5, 15, 18, 19, 20, 21, 23, 27, 28, 29, phenolic compounds, 103
46, 59, 62, 65, 90, 97, 107, 114, 119, 120, 121, phosphate, 58, 117, 198
123, 124, 127, 129, 131, 135, 136, 139, 140, 141, phosphorous, 15, 26, 154
144, 153, 164 phosphorus, 62, 65, 138
organic solvents, 21, 194, 198, 208, 211 photocatalysis, 86, 87, 94, 105
organism, 66, 75 photocatalysts, 89, 105, 106
organopollutants, viii, 69, 71 photodegradation, 18, 19, 22, 87, 93, 100, 107
osmosis, 24, 25, 95, 96 photolysis, 7, 16, 18, 19, 86, 87, 89, 90, 94, 106
overlap, 19 photosensitizers, 18
oxidation, vii, 1, 5, 7, 16, 24, 26, 27, 28, 30, 72, 78, photo-transformation, 26
82, 85, 86, 87, 89, 91, 92, 93, 94, 97, 101, 105, Phragmites, viii, 45, 47, 48, 51, 52, 53, 54, 55, 59,
106, 107, 108, 117, 196, 197, 201, 202, 212 60, 61, 62, 63
oxidation products, 78 Phragmites australis, viii, 45, 47, 48, 59, 63
oxidative reaction, x, 192 phylum, 58, 59, 65
oxygen, x, 18, 24, 26, 27, 46, 59, 60, 61, 63, 67, 89, physical properties, 22, 173
98, 106, 115, 168, 178, 192, 195, 200, 201, 202 physicochemical characteristics, 157
ozonation, 89, 91, 93, 106, 107, 108 physicochemical properties, 2, 30, 85
ozone, 27, 91, 92, 105, 115 Physiological, 63, 65
physiology, 22, 75, 208
phytoremediation, 16
P pitch, 25
plant growth, 47
Paenibacillus elgii, ix, 164, 166, 167, 174, 178, 179,
plant type, 51, 54

180, 184
plants, vii, viii, 1, 3, 4, 24, 31, 46, 48, 54, 58, 59, 61,
pain, 8
63, 64, 66, 67, 69, 70, 73, 78, 79, 82, 84, 98, 99,
pairing, 20, 21
103, 104, 109, 196
Pakistan, 205, 208
plaque, 64
palm oil, 149, 151, 164, 166, 175, 182, 193, 194,
plastics, x, 180, 191
198, 211
platinum, 88, 106
panic disorder, 11
playing, 21, 58
paroxetine, 11, 12
Pleurotus ostreatus, viii, 70, 71, 207, 210
partition, 7, 16, 17, 21, 22, 95
PM, viii, 70, 74
patents, x, 191
polar, 2, 6, 17, 20, 24, 30
pathogens, 4, 5, 6, 9, 29, 85, 95
polarity, 3, 21, 82, 95
pathways, 19, 82, 83, 86, 93, 100, 105, 107, 115, 204
pollutants, vii, x, 1, 2, 3, 4, 5, 9, 14, 17, 25, 27, 28,
PCR, 49, 50, 55, 63, 64, 65
29, 30, 31, 59, 78, 94, 164, 182, 191, 192, 196,
Pearl River Delta, 103
200, 202, 204, 205, 207, 208, 212
peat, 98
pollution, viii, 25, 46, 63, 69, 114, 115, 164, 170,
peptide, 177, 194, 197
192, 195, 199
peptide chain, 197
polyacrylamide, 49, 164, 169, 170, 177, 183, 185,
permeability, 63
186
permission, 91, 96
polyaluminium chloride salts, ix, 163
permit, 141
polyamine, 169
perovskite oxide, 89
polycarbonate, 71
peroxide, 201
224 Index
polychlorinated biphenyl, 102, 201

polychlorinated biphenyls (PCBs), 102
Q
polycyclic aromatic hydrocarbon, 102, 181, 188,
quality control, 112, 113, 129
200, 202
quantification, 3, 64, 84, 102
polymer, 126, 165, 170, 171, 172, 173, 174, 178,
179, 184, 186, 187
polymer chain(s), 172 R
polymer molecule, 174
polymerase, 63 radiation, 19, 22, 27, 86, 88, 89, 105, 106, 122, 150
polymerase chain reaction, 63 radical reactions, 93
polymeric materials, 165 radicals, 18, 27, 28, 85, 86, 90, 91, 93, 197, 201
polymers, 114, 124, 126, 164, 169, 170, 172, 174, rainfall, 22
182, 183, 199 raw materials, 195
polymorphism, 63 REA, 184
polyphenols, 197 reactants, 192
polysaccharide(s), ix, 163, 167, 170, 173, 174, 176, reaction medium, 123, 128, 131
179, 181, 184, 185, 186, 199 reaction rate, 27, 132
polyurethane, 124, 125, 131 reaction rate constants, 27
polyurethane foam, 124, 131 reaction temperature, 87
ponds, 121 reaction time, 89, 113, 130
population, 23, 29, 122 reactions, 2, 27, 28, 46, 49, 89, 94, 117, 201
Portugal, 1 reactive oxygen, 86
potassium, 158 reactivity, 9, 22
potato, 186 reagents, 31
poultry, 123 reasoning, 59, 115, 136
PPCPs, viii, 81, 82, 84, 85, 86, 87, 89, 91, 92, 93, 95, receptors, 101
97, 98, 99, 100, 101, 102, 103, 107, 108, 109 recombination, 87, 88, 94
precipitation, x, 164, 179, 196, 204 recovery, ix, 163, 181, 196, 204
preparation, 25, 194 recycling, 196
prescription drugs, 10, 82 regeneration, 25, 98, 99
prevention, 2, 6, 8, 10 regions of the world, 198
probability, 129 regulations, 31

process control, 122 rejection, 96
producers, ix, 163, 192, 202 relevance, 11, 12
programming, 189 relief, 8
project, 135 remediation, 194, 200, 203, 205, 211, 213
proliferation, 102 reproduction, 4
propranolol, 10 repulsion, 172
proteins, ix, x, 96, 122, 163, 164, 174, 179, 192, 193, requirements, 5, 15, 24, 27, 28, 200, 204
194, 197, 199 researchers, 23, 25, 30, 89, 91, 143, 150, 170, 179,
Proteus mirabilis, ix, 164, 167, 173, 180, 188 195, 203
Pseudomonas aeruginosa, 179, 183, 193, 211 residues, vii, 1, 3, 4, 5, 7, 9, 12, 16, 24, 83, 101, 102,
psychoactive drug, 15 112, 113, 115, 117, 123, 175, 189
public awareness, 3, 31 resins, x, 179, 191
public concern, 101 resistance, 9, 14, 28, 183, 198
public health, 15, 30 resources, ix, 111, 112
pulp, x, 164, 165, 178, 180, 182, 186, 191, 198, 199, response, 209
202, 206, 208, 211 reverse osmosis, 25, 26, 95, 97, 108, 179
pumps, 118 rhizoplane, viii, 45, 59, 62, 63
purification, 113, 178, 182, 203, 204, 205, 211, 212 Rhizopus, 167, 186
purity, 71 Rhodoocus erythropolis, ix, 164
PVC, 48 ribosomal RNA, 121
risk assessment, 103
Index 225
risk(s), 4, 30, 33, 82, 103 software, 50, 72

rodents, 182 solid phase, 7, 20, 77, 104, 124
room temperature, 72, 177 solid state, 194
root(s), viii, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, solid waste, 6, 139, 150, 205
58, 59, 60, 61, 62, 63, 64, 65, 67 solubility, 2, 17, 20, 21, 22, 135, 206
routes, 3, 136 solution, 15, 18, 21, 72, 86, 87, 171, 174, 176, 180,
Royal Society, 31, 32 186
rural areas, 82 solvents, 71, 122, 138
rutile, 89 sorption, 6, 7, 9, 15, 16, 17, 19, 20, 21, 22, 23, 26,
29, 70, 75, 86, 98, 100, 101, 109, 173
sorption process, 22
S South Africa, 187
South Korea, 49, 103, 104
safety, ix, 4, 128, 163
SP, 111
salinity, 182, 195, 203
Spain, 41, 45, 47, 70, 73, 84
salt concentration, x, 191, 203
species, viii, 9, 18, 19, 20, 22, 26, 27, 29, 45, 46, 48,
salt production, 183
50, 51, 54, 55, 58, 59, 61, 62, 63, 64, 66, 67, 86,
salts, ix, 95, 96, 138, 163, 165, 169, 174
91, 121, 122, 150, 164, 176, 178, 179, 183, 187,
scaling, 50, 51, 53, 199
194, 199, 210
scavengers, 28
specific surface, 88, 181
science, 103, 106
spectroscopy, 97
second generation, 152
spore, 123
secrete, 200
Spring, 66
sedative, 11
stability, x, 25, 48, 64, 86, 89, 126, 127, 128, 131,
sedimentation, 5, 6, 15, 84, 100, 111, 123, 124, 165
132, 135, 136, 144, 153, 155, 165, 177, 191, 193,
sediment(s), 3, 46, 60, 62, 64, 77, 78, 82, 104, 121,
195, 196, 201, 203, 204, 206, 209
144
stabilization, 6
seed, 157
standard deviation, 74, 76
selectivity, 27, 94, 128, 192, 193
standardization, 192, 196
semiconductor(s), 86, 89, 90
starch, 138, 148, 151, 154, 166, 167, 170, 185, 186,
sensitivity, 84
187, 197, 199
sequencing, 49, 58, 111, 112, 123, 124, 126, 128,
state(s), 11, 16, 22, 60, 71, 88, 89, 165, 189
130, 136, 137, 141, 150, 151, 152, 153, 154, 155,
sterile, viii, 48, 70, 71, 75, 77, 79
156, 157, 158, 159, 160, 161
steroids, 77
serine, 195, 206
stimulant, 11, 100
serotonin, 11
storage, 192, 196
Serratia ficaria, ix, 164, 167, 174, 177, 178, 180,
stress, 54, 131
183
structure, x, 2, 9, 18, 19, 25, 46, 48, 59, 61, 62, 66,
sertraline, 11, 12
67, 94, 124, 154, 160, 177, 181, 184, 188, 192,
serum, 65, 122
199, 207
serum albumin, 65
subgroups, 2, 9
sewage, 3, 6, 15, 16, 17, 40, 63, 65, 73, 78, 79, 104,
subsurface flow, 50, 66, 109
107, 108, 109, 121, 124, 129, 136, 139, 141, 153,
sucrose, 117, 122, 123, 127, 130, 132, 135, 137, 138,
158
140, 145, 150, 152, 154, 155, 156, 158, 166
shape, 2
sulfate, 63, 64, 72, 131, 153, 156, 158
shear, 170
sulfonamides, 9, 15, 17, 109
shock, 122, 123, 127, 131, 150, 151, 152, 154
sulfur, 63, 131, 138
showing, 83, 199, 200
sulphur, 59
side chain, 197
Sun, 36, 183, 188
SiO2, 94, 107
supplementation, 151
SLAC, 209
suppression, 78
sleep disorders, 11
surface area, 25, 89, 98
small communities, 29, 109
sodium, 49, 72, 188
226 Index
surface waters, viii, 6, 10, 17, 70, 78, 79, 81, 82, 99, transformation product, vii, 1, 3, 7, 14, 28, 30, 70,
101, 103, 104 104
surfactant, 208 transition metal, 27
susceptibility, 18 translocation, 212
suspensions, 105, 168, 170, 175, 176 transport, 3, 12, 66, 142
Sustainable Development, 34 treatment methods, 193
synergistic effect, 4, 195 triclosan, viii, 69, 75, 77, 78, 79, 99, 104
synthesis, 106, 183, 185 triglycerides, 10
synthetic fiber, 113 tryptophan, 212
synthetic polymers, 169, 170, 178 tungsten, 89
turbidity, ix, 95, 164, 168, 175, 178, 181, 194
turbulence, 26
T turnover, 59
Typha, viii, 45, 47, 51, 52, 53, 54, 59, 61, 62
Taiwan, 103
Typha angustifolia, viii, 45, 47, 59
tanks, viii, 26, 45, 46, 47, 48, 51, 52, 53, 54, 60, 61,
62
target, 12, 28, 96, 192 U
TBG, 207
TCS, viii, 69, 70, 71, 72, 73, 74, 75, 76, 77 UK, 33, 63, 66, 70
techniques, x, 7, 46, 82, 101, 122, 191, 204, 208 ultrasound, 27, 123
technologies, vii, ix, 1, 4, 5, 7, 24, 25, 27, 28, 30, 40, unit cost, 93
81, 85, 95, 97, 101, 115, 164, 204, 208, 211 United States, 82, 84, 182, 183
technology, x, 4, 24, 25, 30, 42, 84, 103, 106, 121, urban, viii, 47, 77, 81, 82, 101, 102, 103, 104, 150,
129, 156, 161, 179, 186, 191, 192, 203, 213 178
temperature, ix, 7, 17, 18, 19, 21, 22, 28, 30, 72, 85, urban areas, viii, 81, 82, 101
112, 113, 114, 129, 135, 136, 141, 144, 150, 151, urbanization, 70
152, 153, 154, 156, 157, 158, 159, 161, 175, 177, urea, 49, 166
179, 180, 181, 182, 184, 185, 192, 194, 198, 199, urine, 82
200, 203 USA, 34, 36, 37, 41, 42, 65, 70, 72, 102
terrestrial ecosystems, 4 UV, 5, 19, 27, 28, 49, 85, 86, 87, 88, 90, 91, 93, 94,
testing, 99 97, 105, 106, 107, 123
tetracyclines, 9, 15 UV irradiation, 5, 87, 88, 90

textiles, ix, x, 163, 191 UV light, 85
texture, 25 UV radiation, 123
Thailand, 198 UV-irradiation, 93
therapeutic use, 11
thermal stability, 177, 199
thermostability, 199, 206 V
thyroid, 77
vacancies, 89, 106
time periods, 152
valence, 86, 88, 89
titania, 94, 96, 106, 108
validation, 151
titanium, 27, 89, 94, 106, 107
vapor, 21, 22
total product, 126
variables, ix, 112, 117, 130, 131, 144
toxic, vii, 1, 5, 7, 28, 60, 86, 129, 170, 179, 182, 200,
variations, 13, 22, 59, 108, 129, 141, 144, 176, 182
202
vegetables, 198
toxic effect, 60
vegetation, 29, 46, 65, 66
toxic products, 202
velocity, x, 191
toxicity, 4, 5, 9, 12, 19, 22, 30, 31, 77, 105, 164, 199
versatility, x, 25, 27, 191
toxicology, 103
vinasse, 127, 149, 151
trace elements, 138
viruses, 15, 96
transformation(s), vii, 1, 3, 7, 14, 16, 17, 18, 22, 28,
volatility, 7, 21, 82
30, 65, 70, 104, 108, 154, 192, 195, 201, 204, 205
volatilization, 7, 22, 75
Index 227
WWTPs, vii, viii, 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 13, 14,

W 15, 16, 17, 18, 19, 20, 22, 24, 29, 30, 31, 69, 70,
71, 72, 73, 74, 77, 82, 100
Washington, 42
waste, 6, 65, 72, 79, 82, 95, 103, 104, 121, 123, 137,
139, 149, 152, 154, 159, 160, 161, 175, 182, 186, X
188, 195, 198, 204, 207, 208
waste treatment, 65, 195 xenobiotic pollutants, x, 192
waste water, 72, 82, 95, 103, 198 xenobiotics, vii, 1, 29, 197, 200, 201
wastewater reuse, vii, 3, 24, 45 XRD, 96
water purification, 85
water quality, vii, 1, 65, 103
water resources, 77 Y
wetlands, vii, 29, 36, 37, 43, 45, 46, 50, 55, 58, 60,
61, 62, 63, 64, 65, 66, 100, 109, 144 yeast, ix, 138, 163, 166, 194, 198
WHO, 182 Yeasts, 166
wide band gap, 88 yield, 19, 23, 90, 112, 117, 120, 121, 122, 123, 127,
wild animals, 11 130, 132, 135, 136, 137, 138, 155, 159, 166, 175,
withdrawal, 11 182
withdrawal symptoms, 11
wood, 98, 197, 198, 199, 208 Z
work study, 59
worldwide, 3, 8, 14, 83, 129 ZnO, 86
worry, 14

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Copyright © 2015. Nova Science Publishers, Incorporated. All rights reserved.

Additional books in this series can be found on Nova‘s website

Additional e-books in this series can be found on Nova‘s website

NOTICE TO THE READER

competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED

Library of Congress Cataloging-in-Publication Data

Published by Nova Science Publishers, Inc. † New York

Chapter 4 Removal of Pharmaceuticals and Personal Care Products

Chapter 7 Extremozymes As Efficient Green Biocatalysts in Bioremediation

This book discusses processes involved with wastewater treatment as well as

treatment technologies, such as advanced oxidation processes, adsorption processes and

industrial wastewaters treatment. Synthetic organic flocculants as well as inorganic

industrial wastewater effluents. They promote flocculating by bridges formation between

A. Dordio1,2 and A. J. P. Carvalho1,3

In summary, pharmaceuticals are usually resistant to degradation, highly persistent in

The occurrence of organic micropollutants in the aquatic environment have been

2. PHARMACEUTICALS FATE IN CONVENTIONAL WWTPS

3. OCCURRENCE AND REMOVAL OF PHARMACEUTICALS

a) Analgesics and Anti-Inflammatory Drugs

c) Blood Lipid Regulators

conjugation during WWTP treatment, sometimes contributing to an increase on the levels of

3.1. Occurrence in WWTP Influents and Effluents and Removal Efficiency

Therapeutic class Selected Compounds Influent Effluent Removal

Naproxen <0.002-611 <0.002-33.9 43.3-98.6

detected among neuroactive pharmaceuticals. The anti-epileptic carbamazepine is a

hydroxycarbamazepine, salicylic acid, hydroxyl-ibuprofen and carboxy-ibuprofen (Farré et

3.2. Occurrence of Pharmaceuticals in Sewage Sludge

4. MAIN REMOVAL PROCESSES FOR PHARMACEUTICALS IN WWTPS

The main removal mechanism of most recalcitrant pharmaceuticals in conventional

i) absorption, which is due to hydrophobic interactions of the aliphatic and aromatic

During biological degradation in WWTPs, pharmaceutical contaminants can undergo: (1)

In addition, such low concentrations may be insufficient to induce a significant activity

Abiotic transformations of pharmaceuticals in WWTPs may occur via hydrolysis and

5. FACTORS AFFECTING THE REMOVAL OF PHARMACEUTICALS

a) Physico-Chemical Properties of Pharmaceuticals

 Molecular Structure: pharmaceutical structural factors of major importance are

 Water solubility (Sw): the tendency of a compound to be in the aqueous medium is

Alternatively, for practical applications, it is most convenient to use a

The main environmental conditions that affect the removal of pharmaceuticals in

absorbances in the range of the visible electromagnetic radiation (290-800 nm)

2012; Vieno and Sillanpaa, 2014).

6. ALTERNATIVE/COMPLEMENTARY REMOVAL TECHNOLOGIES

Although commonly adopted as the best cost-effective wastewater treatment technology

6.1. Advanced Treatment Technologies

In recent years, a broad number of advanced treatment technologies for pharmaceutical

a) Activated Carbon Adsorption

b1) Membrane Bioreactor (MBR) for Wastewater Treatment

c) Advanced Oxidation Processes (AOP)

conducted to investigate the formation of potentially toxic transformation products (Hollender

6.2. Constructed Wetlands

Constructed wetlands systems (CWS) can be defined as designed, man-made structures

Bassil, R. J.; Bashour, I. I.; Sleiman, F. T.; Abou-Jawdeh, Y. A. Journal of Environmental

Clarke, B. O.; Smith, S. R. Environment International 2011, 37, 226-247.

Dalrymple, O. K.; Yeh, D. H.; Trotz, M. A. Journal of Chemical Technology and

A. P. Bioresour. Technol. 2011, 102, 7827-7834.

2013, 461, 480-498.

water reuse (EVK1-CT-2000-00047). Final report; 2006. Available online at:

Zhang, Y.; Geißen, S. U.; Gal, C. Chemosphere 2008, 73, 1151-1161.

MOLECULAR CHARACTERIZATION OF MICROBIAL

Ricardo Sidrach-Cardona1, Olga Sánchez2*†,

Constructed wetlands are becoming an interesting alternative for wastewater reuse

MATERIALS AND METHODS

Experimental Pilot Plant

Typha-FM-SF Typha-FW-SF Typha-FW-SSF Unplanted-FW-SSF