Anaerobic

Wastewater
Treatment
Other Titles by the Author

Biology of Wastewater Treatment

Second Edition
ISBN: 978-1-86094-328-7
ISBN: 978-1-86094-332-4 (pbk)

Fixed-Film Reactors in Wastewater Treatment

ISBN: 978-1-78634-924-8

Natural Wastewater Treatment Systems and Sustainability

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Activated Sludge: Developments and Sustainable Solutions

ISBN: 978-1-80061-387-4
 Anaerobic
Wastewater
Treatment

N I C H O L A S F G R AY
Trinity College, University of Dublin, Ireland

World Scientific
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ANAEROBIC WASTEWATER TREATMENT

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 Dedicated to Lucy, Cat and Becky
‘To you it’s just crap, to me it’s bread and butter.’
Spike Milligan
Recollections of the Latrine Orderly
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 Preface

Achieving sustainable wastewater treatment is one of the most

important engineering challenges facing society, as the need to mitigate
the dual problems of population growth and global warming becomes
critical. The traditional roles of protecting surface and ground waters,
preserving biodiversity, and preventing pathogen transfer have all
become more challenging as wastewaters become more complex with
new chemicals that are increasingly inhibitory or toxic to treatment.
Today, in fulfilling the needs of sustainability, circularity has become a
defining layer on top of our existing environmental and health
priorities with carbon harvesting, recovery of metals, and other useful
products, and, of course, reuse of water — all achievable expectations.
Traditionally, wastewater treatment has relied on separation and high-
energy aerobic processes, with a large portion of the carbon in the
wastewater lost as carbon dioxide (CO2) through aerobic metabolism,
with anaerobic treatment occasionally used for sludge stabilization.
To achieve sustainability, the emphasis of treatment has to change to
primarily anaerobic treatment, with aerobic treatment restricted to
effluent polishing where membrane filtration technologies are unable
to achieve the final effluent quality, although many natural treatment
options are available with low carbon footprints.
Traditionally, anaerobic digestion was a slow process, operating at
low microbial concentrations requiring large reactors and subject to
many operational problems. But today’s modern designs, coupled
with a greater understanding of the microbial basis of anaerobic
treatment, especially of the methanogenic archaea, have seen a rapid

vii
viii Anaerobic Wastewater Treatment

development in compact, rapid, and highly efficient processes —

many utilizing granular sludges.
Modern wastewater treatment has embraced the circular economy
ideology, becoming increasingly sustainable. Among the huge range
of valuable byproducts that can be generated by fermentation are
biomethane, bioplastics, biodiesel, and volatile fatty acids (VFAs).
VFAs are a valuable raw material used in the food, chemical, and
pharmaceutical sectors, as well as a carbon source for a wide range of
bio-based compounds. All these byproducts are harvesting the carbon
in the waste for reuse, preventing direct CO2 release into the
atmosphere. The disadvantage of using methane as a fuel is that CO2
is produced when it is utilized — this can be overcome by producing
hydrogen instead, which, when used, produces only water.
Biohydrogen (Bio-H2) is a carbon-free renewable fuel that is mainly
generated using hydrocarbons or gas. Today, there are increasing
opportunities to use anaerobic fermentation to produce Bio-H2,
making it a truly renewable fuel. Sulfate reducing bacteria (SRB) can
be used to recover metals from wastewaters, safeguarding our future
supplies of important metals as well as protecting the environment
from heavy metal pollution. The key advantage to using anaerobic
treatment in the recovery of metals is that even at very low
concentrations, they can be effectively recovered, producing highly
insoluble salts at low capital and operating costs. Metal sulfide
nanoparticles are an exciting byproduct that has a wide range of uses,
from the manufacturing of batteries, LEDs, and solar cells to
electroplating and use in environmental remediation, biomedicine,
and agriculture.
We tend to think of nutrient removal as requiring an aerobic step,
but today, both nitrogen and phosphorous can be effectively treated
using anaerobic technologies, including anaerobic ammonium
oxidation (anammox) and sulfur-based autotrophic denitrification
(SAD). Membrane technology can improve the efficiency of anaerobic
wastewater treatment by retaining solids and biomass within the
reactor while also ensuring a high-quality final effluent, which is
particle-free with extremely low pathogen and antibiotic gene
concentrations.
 Preface ix

The major advance in anaerobic treatment is due to our rapidly

growing knowledge of the key bacteria and archaea involved in this
syntrophic process. Research is also exposing the critical role of
quorum sensing and signal molecules, which is allowing researchers
to develop new and more efficient fermenter designs.
It is estimated that between 3 and 4% of global electricity supplies
are consumed by wastewater treatment plants (WWTPs) being
primarily required for aeration systems, pumping, and sludge
dewatering. The objective of wastewater sustainability is to obtain net
zero in terms of energy consumption and, if possible, even become
net positive. However, as effluent standards increase and loadings to
WWTPs increase, more energy is required. Carbon neutrality is only
possible using natural treatment systems or anaerobic processes, with
the latter being the only option for centralized wastewater treatment
systems. The primary objective of sustainable wastewater treatment is
to ensure that greenhouse gases are not released directly from unit
processes. When anaerobic systems are used instead of traditional
aerobic-based treatment processes, the advantages can be directly
related to significant reductions in carbon footprint and take us ever
closer to real carbon neutrality.
Concerning the quote in the frontispiece, which has been used in
all the editions and reprints of Biology of Wastewater Treatment, which
this series replaces. I was working late one evening, hand-drawing the
illustrations for the first edition as there were no computer graphics
available at that time. While listening to the radio, I heard Spike
Milligan utter that phrase, which seemed to apply so aptly not only to
myself but to all wastewater engineers and scientists. When I wrote to
him to ask for permission to use it, he replied that he was delighted
that he belonged to “a greater brotherhood” and so allowed the
phrase to be freely reproduced. This series of books is dedicated to all
those very special men and women throughout the world who are
part of that very special family of wastewater operators, managers,
designers, manufacturers, and researchers.

Nick Gray
November 2024
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 Acknowledgments

I am very grateful to the many people who have so very kindly

provided me with information, especially manufacturers. I would
especially like to thank all the publishers, organizations, and compa-
nies who have given me permission to reproduce or modify copyright
material in the text, in particular, the Chartered Institution of
Water and Environmental Management. This book is all about
those amazing researchers who continue to develop and refine the
technologies and those companies who make those dreams a reality.
I would like to particularly thank Dr. Amelia Rotaru of the University
of Syddanski, Denmark for allowing me to reproduce the images of
her anaerobic work bench, and Dr.-Ing Sören Weinrich for allowing
me to reproduce his equations of hydrolysis. My special thanks go to
Amanda Yun, Senior Editor at World Scientific Publishing, for all her
help and patience and especially to Gregory Lee, who has been such
an enormous help and support in getting this book, and indeed this
series, into print.

xi
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 Contents

Prefacevii
Acknowledgmentsxi

A Basis of Anaerobic Treatment

1 Introduction to Anaerobic Treatment 3
1.1 Scope of Anaerobic Treatment 3
1.2 The Anaerobic Microbial Process 5
1.3 Anaerobic Unit Processes  10
1.4 The Role of Anaerobic Wastewater Treatment 16
References  19

2 Anaerobic Metabolism and Microorganisms 21

2.1 Introduction 21
2.2 Anaerobic Degradation of Organic Matter 24
2.2.1 Hydrolysis 25
2.2.2 Acidogenesis or Fermentation  29
2.2.3 Acetogenesis  32
2.2.4 Methanogenesis  33
2.2.5 Importance of Redox Potential  36
References39

3 Methane and Other Volatile Products of Digestion 43

3.1 Introduction  43

xiii
xiv Anaerobic Wastewater Treatment

3.2 Methane  44
3.2.1 Formation  44
3.2.2 Health and Environmental Effects  54
3.3 Hydrogen Sulfide  56
3.3.1 Formation  56
3.3.2 Health and Environmental Effects  58
3.3.3 Corrosion  59
3.4 Ammonia  63
3.5 Volatile Organic Compounds and Terpenes  65
3.5.1 Volatile Organic Compounds  65
3.5.2 Terpenes  70
3.6 Siloxanes  72
References73

B Flow-Through (Conventional) Anaerobic Treatment

Processes 
4 Combined Anaerobic Wastewater Treatment Systems  81
4.1 Introduction  81
4.2 Septic Tanks  85
4.2.1 Cesspools  85
4.2.2 Septic Tanks  86
4.2.3 Septic Tank Modifications  100
4.2.4 Microbial Community Structure of Anaerobic
Sludge and Pathogen Removal  104
4.3 Imhoff Tanks 106
4.4 Anaerobic Lagoons  111
4.4.1 The Process  111
4.4.2 Microbiology  114
4.4.3 Application  118
4.4.4 Design of Anaerobic Lagoons 121
4.4.5 Gas Emissions from Lagoons  124
4.4.6 Odors  127
4.4.7 Denitrification  132
4.4.8 Covered Lagoons for Biogas Collection  133
References136
 Contents xv

5 Sludge Digesters 143

5.1 Introduction  143
5.2 Design  148
5.3 Hydrolysis  154
5.3.1 Enzymatic or Biological Hydrolysis  155
5.3.2 Pasteurization  157
5.3.3 Thermal Hydrolysis  159
5.4 Digester Operational Management  159
5.4.1 Composition of Raw Sludge 160
5.4.2 Method of Sludge Addition to Digester
and Commissioning  175
5.4.3 Internal Mixing and Circulation  178
5.4.4 Solids Retention Time  181
5.4.5 Temperature  182
5.4.6 pH and Alkalinity  186
5.4.7 Foaming  190
References200

C Contact (High-rate) Anaerobic Wastewater

Treatment Systems
6 Biofilm (Attached Growth) Systems 213
6.1 Introduction to Contact Systems  213
6.2 Anaerobic Biofilters or Static (Fixed) Bed Reactors 215
6.3 Rotating Biological Contactors  222
6.4 Anaerobic Moving Bed Biofilm Reactors 228
6.5 Expanded and Fluidized Bed Reactors  240
6.5.1 Anaerobic Expanded Bed Reactors 242
6.5.2 Anaerobic Fluidized Bed Reactors  244
6.5.3 Anammox Process  249
References251

7 Floc and Granular-based Systems (Dispersed Growth) 259

7.1 Up-flow Anaerobic Sludge Blanket Reactors 260
7.2 Expanded Granular Sludge Bed Reactors 264
7.3 Internal Circulation Systems 266
7.4 Anaerobic Continuously Stirred Tank Reactors 271
xvi Anaerobic Wastewater Treatment

7.5 Anaerobic Sequencing Batch Reactor  272

7.6 Baffled Reactors  277
7.7 Anaerobic Migrating Blanket Reactor 282
7.8 Hybrid Reactors  283
References286

D Biogas Production Systems

8 Biogas Production and Cleaning  297
8.1 Methane Production and Collection 297
8.2 Hydrogen Sulfide Control and Removal  301
8.2.1 Adsorption  305
8.2.2 Absorption  312
8.2.3 Catalytic Scrubbing 314
8.2.4 Biological Oxidation  314
8.2.5 Metal Sulfide Precipitation 319
8.2.6 In-situ Sulfide Control 320
8.3 Carbon Dioxide Removal Technologies  322
8.4 Removal of Water  326
8.5 Removal of Other Impurities  326
References327

E The Future
9 Sustainability and Future of Anaerobic Wastewater
Treatment333
9.1 Sustainability and the Circular Economy  333
9.1.1 Biohydrogen and Dark Fermentation  334
9.1.2 VFA Production  339
9.2 Biotechnology and the Future of Anaerobic
Wastewater Treatment  344
9.2.1 Granulation  344
9.2.2 Role of Metals  355
9.2.3 Membrane Technology  368
9.2.4 The Future  375
References389

Index401
 Section A

Basis of Anaerobic Treatment

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 1
Introduction to Anaerobic Treatment

1.1 Scope of Anaerobic Treatment

Anaerobic processes are used to treat strong organic wastewaters
(BOD >500 mg L–1) and for the treatment of organic wastes, includ-
ing primary and secondary sludges from domestic wastewater treat-
ment, industry, and agriculture. Strong organic wastes generated by
the agricultural and food industry sectors, often in large quantities,
provide particularly difficult treatment challenges. When treating
wastewaters containing large quantities of biodegradable organic mat-
ter, conventional aerobic treatment is beset with numerous opera-
tional difficulties. For example, the difficulty of maintaining aerobic
conditions, especially if the wastewater has a high concentration of
suspended solids, sludge bulking, inability to take high BOD (bio-
chemical oxygen demand) or COD (chemical oxygen demand) load-
ings, high operational and energy costs, and a high production of
biomass as wasted sludge that requires subsequent treatment and
disposal. In contrast, anaerobic treatment, although slower, offers a
number of attractive advantages in the treatment of strong organic
wastes, such as a high degree of purification, the ability to treat
high organic loads, production of a small quantity of excess sludge,
which is normally very stable, and the production of a wide range of
potential byproducts, such as methane, hydrogen, volatile fatty acids
(VFAs), and much more. Our current need to reduce carbon emis-
sions has resulted in a reappraisal of the carbon and nitrogen cycles in
wastewater treatment. During aerobic treatment, high energy usage is

3
4 Anaerobic Wastewater Treatment

required primarily from aeration, and where this comes from fossil
fuels, then this results in a high carbon footprint. Also, as the majority
of the carbon comprising the COD or BOD loading is oxidized to
produce carbon dioxide (CO2) and water, this increases the carbon
footprint per m3 of wastewater treated. In contrast, anaerobic treat-
ment requires far less energy, as no aeration is required, and all the
carbon in the wastewater and sludge is retained for recovery and
reuse, thereby significantly reducing carbon emissions. It is only
through anaerobic treatment that net zero for centralized wastewater
treatment plants (WWTPs) can be achieved. Unlike aerobic systems,
complete removal of organic matter is not always possible anaerobi-
cally, and subsequent aerobic treatment of anaerobic effluents is nor-
mally necessary, which indicates the potential of combined anaerobic
and aerobic units in series, although the use of natural treatment
systems or membrane filtration are low-carbon alternatives (Gray,
2022). The most widely used aerobic treatment process, activated
sludge, has an energy requirement of 0.7–1.2 kWh kg–1 COD
removed at a loading rate of between 0.5 and 2.0 kg COD m–3 d–1,
compared to loading rates for anaerobic treatment, which are as high
as 25 kg m–3 d–1, making anaerobic processes far more compact and
easier to maintain (Table 1.1). Energy usage for the anaerobic process
is generally ten times less than activated sludge, with most of that sup-
plied by combined heat and power (CHP) units operated by the

Table 1.1. Typical ranges for operational parameters for the main anaerobic biore-
actor designs (Zieliński et al., 2023). Reproduced under the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Normal Load Hydraulic Retention Solid Retention
Bioreactor (kg COD/m3/d) Time (day) Time (day)
CSTR 0.25–4.0 10–60 10–60
Anaerobic filter 1–40 0.5–12 20
Fluidized bed reactor 1–100 0.2–5 30
UASB 10–30 0.5–7 20
Hybrid reactor 1–100 0.2–5 20
AnMBRs 0.5–12.5 3–30 20–40
 Introduction to Anaerobic Treatment 5

Table 1.2. The advantages and disadvantages of anaerobic treatment compared to

aerobic treatment.
Advantages Disadvantages
Low operational costs High capital costs
Generally require heating
Low sludge production Long retention times required (>24 h)
Reactors sealed giving no odor or Corrosive and malodorous compounds
aerosols produced during anaerobiosis
Sludge is highly stabilized Not always as effective as aerobic
stabilization for pathogen destruction
Methane gas produced as end product Hydrogen sulfide also produced
Low nutrient requirement due to Reactor may require additional alkalinity
lower growth rate of anaerobes
Can be operated seasonally Slow growth rate of anaerobes can result in
long initial startup of reactors and
recovery periods.
Rapid start-up possible after Only used as pretreatment for liquid wastes
acclimation

methane produced during the process so that most anaerobic pro-

cesses achieve net zero or better in terms of carbon emissions. The
advantages and disadvantages of anaerobic treatment are summarized
in Table 1.2, while the typical carbon balances of both aerobic and
anaerobic systems are compared in Fig. 1.1.

1.2 The Anaerobic Microbial Process

Anaerobic heterotrophic bacteria are either obligate (i.e., unable to
grow in the presence of oxygen) or facultative (i.e., can adapt to envi-
ronments, either with or without oxygen). The latter form a bridge
between obligate aerobes and obligate anaerobic species. Growth and
metabolism of obligate anaerobes are inhibited by oxygen, with oxy-
gen toxicity depending on redox potential (Eh), partial pressure, com-
position of the substrate, growth rate, and cell density.
The traditional role of anaerobic heterotrophs is in sludge diges-
tion, converting unstable sewage sludge (biosolids) into a more stabi-
lized form. Digestion normally takes place in specially constructed
6 Anaerobic Wastewater Treatment

Figure 1.1. Comparison of the mass balance of carbon utilization in aerobic and
anaerobic processes.

reactors, although anaerobic digestion also occurs in the sludge blan-

ket of waste stabilization ponds. The presence of anaerobic activity in
aerobic treatment processes is generally undesirable, with the com-
monest effects of anaerobiosis being the production of foul odors and
interference of floc settling due to denitrification occurring in the
sedimentation tank after the aeration basin in the activated sludge
process.
There is a complex consortium of microorganisms involved in
the anaerobic degradation of high molecular weight organic com-
pounds to methane. Predominately bacterial and archaea, although
protozoa and fungi have been reported (Finlay and Fenchel, 1991),
anaerobic breakdown of organic matter is complex, involving synergis-
tic reactions between different groups. There are four groups involved
in this synergistic relationship: (1) hydrolytic bacteria, (2) fermentative
 Introduction to Anaerobic Treatment 7

Figure 1.2. Major steps in anaerobic decomposition.

acidogenic bacteria, (3) acetogenic bacteria, and (4) methanogens

(Fig. 1.2; Table 1.3). These are reviewed in Chap. 2.
Anaerobic degradation occurs in the absence of oxygen, with
methanogens adversely affected by even trace amounts of oxygen.
Anaerobic biomass can form a granular sludge structure under certain
circumstances (e.g., up-flow anaerobic sludge blanket (UASB) reac-
tor), and where this occurs, the methanogens are protected from the
effect of inhibitory compounds within the granules (Kato et al.,
1993) (Chap. 5). The basic difference between aerobic and anaerobic
oxidation is that in the aerobic system, oxygen is the ultimate hydro-
gen acceptor with a large release of energy, but in anaerobic systems,
the ultimate hydrogen acceptor is nitrate, sulfate, or an organic com-
pound with a much lower release of energy. Briefly, the process of
anaerobic decomposition involves four discrete stages (Fig. 1.3). The
first stage is the hydrolysis of high molecular weight carbohydrates,
fats, and proteins that are often insoluble by enzymatic action, into
8 Anaerobic Wastewater Treatment

Figure 1.3. Key microbial groups involved in biochemical steps in anaerobic

digestion.

Table 1.3. Main conversion steps and key microorganisms in anaerobic digestion
(Zieliński et al., 2023). Reproduced under the Creative Commons Attribution (CC
BY) license (https://creativecommons.org/licenses/by/4.0/).
Step Substrates Products Species
Hydrolysis Protein, fats, Amino acids, fatty
carbohydrates acids, polyols,
simple sugars
Acidogenesis Hydrolysis Fatty acids: acetic Aerobacter, Alcaligenes,
products acid, propionic Clostridium,
acid, butyric acid, Flavobacterium,
isobutyric acid,
 Introduction to Anaerobic Treatment 9

Table 1.3. (Continued)

Step Substrates Products Species

   valeric acid,    LactobacilIus,
isovaleric acid, Micrococcus,
caproic acid, Streptococcus,
lactic acid Pseudomonas,
Alcohols: ethanol, Lactobacterium
methanol,
propanol; H2, CO2
Acetogenesis Earlier products Acetics, H2, CO2 Syntrophobacter
wolinii,
Sytrophomonos
wolfei, Clostridium
spp., Peptococcus
anaerobius,
Lactobacillus, and
Actinomyces
Methanogenesis Acetogenesis Biogas (CH4, CO2) Methanobacterium,
products Methanobacillus,
Methanococcus,
Methanosarcina.
Methanosaeta

soluble polymers. The second stage involves the acid-forming bacte-

ria, which convert the soluble polymers into a range of organic acids
(acetic, butyric, and propionic acids), alcohols, hydrogen, and carbon
dioxide. Acetic acid, hydrogen, and carbon dioxide are the only end-
products of the acid production that can be converted directly into
methane by methanogenic archaea. A third stage is where organic
acids and alcohols are converted to acetic acid by acetogenic bacteria.
It is in the fourth and final stage, which is perhaps the most sensitive
to inhibition, where methanogenic archaea convert the acetic acid to
methane. Although methane is also produced from hydrogen and
carbon dioxide, about 70% of the methane is produced solely from
acetic acid. Obviously, the methanogenic stage is totally dependent on
the production of acetic acid, and so it is the third stage, the ace-
togenic phase, which is normally the rate-limiting step in biogas pro-
duction. The biochemistry of anaerobic decomposition is fully
explored in Sec. 2.1.
10 Anaerobic Wastewater Treatment

1.3 Anaerobic Unit Processes

There is a large range of diverse anaerobic processes available for
treating wastewater and solids, including anaerobic lagoons, digesters,
and filters. Present anaerobic technology can be divided into two
broad categories:
Flow-through systems for the digestion of concentrated wastes,
such as animal manures or sewage sludges that have a solids concen-
tration in the range of 2–10% — these include conventional com-
pletely mixed reactors (Fig. 1.4a), which are used primarily for sewage

(a) (b)

(c) (d)

(e) (f)

Figure 1.4. Schematic representation of digester types. Flow-through digesters

((a)–(b)) and contact systems ((c)–(f)) (adapted from (Casey, 1981)).
 Introduction to Anaerobic Treatment 11

sludges, and plug flow reactors, which are used to a limited extent for
the digestion of animal manures (Fig. 1.4b).
Wastewaters with a lower solids concentration are treated
anaerobically by contact systems, in which the wastewater is brought
into contact with an active microbial biomass that is retained within
the system. Contact systems are primarily used for treating industrial
effluents and are widespread in France, the Netherlands, and Germany.
Of the 400 European anaerobic systems treating industrial wastewa-
ters, 278 are employed in treating food, drink, and fermentation
wastes, and 48 in treating paper wastes. In flow-through systems, the
residence time of the waste (i.e., the hydraulic retention time (HRT))
and microbial biomass (i.e., sludge or solids residence time (SRT)) in
the unit are the same, whereas in a contact system, the SRT is far
greater than the HRT of the wastewater. The biomass is retained
within the reactor in a number of ways, e.g., by allowing the anaero-
bic microorganisms to develop as an attached film on a static medium
or to develop as flocs maintained in suspension, either by mechanical
mixing or by the upward flow of effluent through the reactor. By
using a system similar to the activated sludge process (contact (con-
tinuously) stirred tank reactors (CSTRs)), the anaerobic biomass is
present as suspended flocs, which are recovered in a separate settle-
ment chamber and recycled back to the main reactor (Fig 1.4d). This
process can also take place within a single reactor, where the depth of
the sludge blanket is controlled by the up-flow rate of the influent
wastewater (Fig. 1.4e). Attached anaerobic films can be on a static
filter medium of natural stone or plastic, similar to a percolating
filter (Fig. 1.4c), whereas fluidized or expanded beds incorporate a
fine grained medium such as sand (Fig. 1.4f). Media reactors are com-
pletely flooded to ensure anaerobic conditions and, like the sludge
blanket process, are operated in the up-flow mode. All these processes
are able to capture carbon by methanogenic conversion to methane.
Anaerobic treatment is an effective method for the complete
treatment of many organic wastes, especially animal wastes and
organic effluents from the food processing industries. The organic
substrate is degraded in the absence of oxygen to carbon dioxide and
methane, with only a small amount of bacterial growth. Approximately
90% of the available chemical energy, in the form of organic material,
12 Anaerobic Wastewater Treatment

is retained as methane production. Examples of methane yield from

dairy wastewaters are given by Zieliński et al. (2023) (Table 1.4).
Apart from the economic value of the methane gas produced,
anaerobic treatment has many advantages over aerobic treatment pro-
cesses, such as less biomass produced per unit of substrate utilized
(the lower biomass production means a lower requirement for nitro-
gen, phosphorus, and other nutrient and growth factors), and higher
organic loadings are possible as anaerobic processes are not limited by
oxygen transfer rates and the lower constructional and operational
costs compared with aerobic processes. However, the major disadvan-
tage in temperate and colder climates is the elevated temperatures
required to maintain microbial activity at a reasonable level.
In 1994, there were over 2,570 anaerobic digesters in Europe,
with 60% of the sewage sludge produced by member states before
final disposal. There were a further 400 digesters treating industrial
effluents, 400 treating farm slurry, and 200 treating landfill leachate
(Nyns, 1994). So, anaerobic digestion is processing almost one
billion tonnes of European waste each year. By 2020, this had risen
to 19,000 digesters, producing 167 TWh of biogas. There are also
a further 727 plants producing 26 TWh of biomethane operating
over 18 European countries, with 232 operating in Germany, 131 in
France, and 10 in the United Kingdom (UK). Both biogas and biom-
ethane are produced by anaerobic digestion; the difference is that
biogas comprises 60% methane and 40% carbon dioxide and is used
in CHP systems while carbon dioxide, hydrogen sulfide, and water
are removed from biomethane so that it has the same properties as
natural gas and can be loaded directly into the grid. Biogas and biom-
ethane production is expected to rise to 467 TWh by 2030 and dou-
ble by 2050. Ireland, for example, aims to replace 20% of its natural
gas with biomethane by 2030, primarily from the wastewater and
agricultural sectors (European Gas Association, 2023; Energy Ireland,
2024).
Because so many different groups of microorganisms are involved,
the digestion process is not easily modeled, especially as the methano-
genic archaea have a much lower growth rate than the acid-producing
bacteria. However, as the conversion of volatile acids to biogas is
Table 1.4. Examples of different anaerobic treatment processes treating dairy wastewaters showing the HRT, organic loading rate
(OLR) and performance (Zieliński et al., 2023). Reproduced under the Creative Commons Attribution (CC BY) license (https://
creativecommons.org/licenses/by/4.0/).
OLR Initial COD COD CH4 Yield
Reactor Type Wastewater HRT (d) (kgCOD/m3d) (mg O2/dm3) Removal (%) (m3CH4/kg COD)
Anaerobic filter Whey 1–5 1–4 5–20 72–90.2 0.089–0.28
Whey 10–15 2.2–3.3 33 90 NA
Synthetic whey 1 1–10.2 1–10.2 85–93.8 0.158–0.35
Biofilm-supported Synthetic dairy 10 NA NA 46–59 NA
CSTR
Fluidized bed reactor Dairy 7.5–11.3 10 3.2 85–98 0.37
Simulated milk 0.75–5 2.22–31 10–77.5 78 0.27

Introduction to Anaerobic Treatment 13

Ice-cream 1 1–12 5 70–90 0.157–0.193
wastewater
Hybrid UASB Dairy 0.25 8–20 5 65–93 0.25–0.31
Dairy 1.9 0.97–2.82 1.9–5.34 91–97 0.27–0.359
Hybrid reactor Dairy 0.5–1 1–2 1–2 64–76 0.03–0.24
Anaerobic moving Milk permeate 9.7–116.5 0.5–6.5 50–75 57.9–77 0.341
biofilm reactor Whey 0.6–17 1–21 6–17 70–97 0.33
Anaerobic sequencing Whey 0.33 2–12 1–6 76–95 NA
biofilm batch reactor
(ASBBR)
NA: not available.
14 Anaerobic Wastewater Treatment

generally considered to be the rate-limiting step of the overall reac-

tion, the methanogenic phase is usually used for modeling purposes.
The majority of models are applicable to the continuously stirred
tank reactor (CSTR), as they are the most widely used and operate
closest to the steady-state, which is easier to model. However, the
development of dynamic models has demonstrated the need for more
accurate ways of examining the operational problems of digesters.
Steady-state models are usually derived from one or two differential
equations that treat the digester contents as a single substrate and the
bacteria as a single population. Among the best-known models of this
type are those using Monod kinetics. Of particular interest are models
based on Contois kinetics developed by Chen and Hashimoto (1978,
1980; Vavilin et al., 2001). The dynamic models available all use
Monod kinetics and normally consider the interactions between sev-
eral substrates and bacterial populations. This results in a system of
differential equations that can only be solved by computer. Andrews
and Graef (1971) considered that relating substrate concentration to
the specific growth rate (the Monod function) was invalid for anaero-
bic reactors, as the volatile acids not only acted as substrate for metha-
nogenic bacteria but were also inhibitory at higher concentrations.
They replaced the Monod function with an inhibition function
(Haldane, 1930) so that:

μ = μm/[1 + Ks/s + s/Ki]

where Ki is the inhibition function. Whereas this early dynamic

model only included a single substrate and organism, more recent
models of this type consider more than one substrate or more than
one bacterial population (Hill and Barth, 1977; Hill and Nordstedt,
1980; Batstone et al., 2002a). Anaerobic digestion model No. 1
(ADM1) includes multiple steps that describe both biochemical pro-
cesses (e.g., disintegration from homogeneous particles to carbohy-
drates, proteins, and lipids; extracellular hydrolysis of these particulate
substrates to sugars, amino acids, and long chain fatty acids (LCFA),
respectively; acidogenesis from sugars and amino acids to VFAs and
hydrogen; acetogenesis of LCFA and VFAs to acetate; and separate
methanogenesis steps from acetate and hydrogen/carbon dioxide), as
 Introduction to Anaerobic Treatment 15

Figure 1.5. The IWA anaerobic digestion Model No 1 (ADM1) (Batstone et al.,
2002b). (1) Acidogenesis from monosaccharides (MSs), (2) acidogenesis from amino
acids (AAs), (3) acetogenesis from long chain fatty acids (LCFAs), (4) acetogenesis
from propionate, (5) acetogenesis from butyrate (HBu) and valerate (HVa), (6) ace-
toclastic methanogenesis, and (7) hydrogenotrophic methanogenesis. Reproduced
with permission of IWA Publishing.

well as physicochemical processes (e.g., ion association and dissocia-

tion and gas-liquid transfer) (Fig. 1.5). The model comprises a set of
differential and algebraic equations using 26 dynamic state concentra-
tion variables and 8 implicit algebraic variables per reactor vessel or
element. It can also be implemented using differential equations only
when 32 dynamic state concentration variables are used (Batstone
et al., 2002b). The ADM1 model has been extensively modified by
researchers over the past decade to reflect changes in substrate
complexity and process upgrading, especially in relation to fermenta-
tion and biogas production, which has been excellently reviewed by
16 Anaerobic Wastewater Treatment

Figure 1.6. The ratio of plug flow reactor volume to completely mixed flow reactor
volume for a first-order reactor process.

Mo et al. (2023). The use of models in anaerobic digestion has also

been reviewed by Emelu et al. (2022), who explore the development
of single-stage to multistage modeling of anaerobic digestion.
In plug-flow systems, there is a substrate concentration gradient
between the inflow and outflow ends of the reactor. As the rate of
reaction is dependent on the substrate, the average rate of reaction in
a plug-flow system is theoretically higher than for a continuous stirred
tank reactor (CSTR). Casey (1981) illustrates the significance of this
difference in terms of the reactor volume required for a first-order
reaction process (Fig. 1.6). This shows that the advantage of the plug-
flow reactor over the CSTR increases with the required degree of
reaction completion. Although a number of contact systems can be
considered as plug-flow processes, those treating domestic sewage
sludge are largely of the completely mixed type. Modeling of indi-
vidual processes is further explored when looking at specific process
designs.

1.4 The Role of Anaerobic Wastewater Treatment

When wastewater treatment was pioneered in the late 1890s, it was
to bring to an end the misery of waterborne diseases such as cholera
and typhoid that were killing millions of people globally, especially in
 Introduction to Anaerobic Treatment 17

urban areas, as well as changing how society could operate. The rapid
expansion of sewerage schemes in Victorian times, followed by the
rapid development of wastewater treatment between 1910 and 1960,
coupled with secure water supplies and water treatment, especially
chlorination, achieved not only the elimination of almost all water-
borne diseases in the developed world but also a massive improve-
ment in surface water quality in terms of organic and ammonia
pollution. During the latter part of the 20th century, priorities in
terms of wastewater treatment included the recovery of useful
byproducts and the removal of nitrogen and later phosphorous to
control eutrophication. The EU directive of wastewater treatment set
new treatment objectives in the 1990s, including the phasing out of
sewage sludge dumping at sea and the increasing controls of its use
on agricultural land, requiring sludges to receive advanced treatment,
which included thermal hydrolysis, which made anaerobic digestion
more effective and stable as a process. The directive also expanded the
use of anoxic and anaerobic zones in what had been strictly aerobic-
activated sludge processes for biological nutrient removal bringing
anaerobic treatment into the mainstream of wastewater treatment.
With the final acceptance of global warming and the need to reduce
carbon emissions, combined with the problem of failing sewer
infrastructure and aging treatment plants, and exacerbated by ever-
increasing loading due to population expansion, the need to improve
wastewater treatment capacity as well as efficiency in terms of sustain-
ability became urgent. So, for the first time in 150 years, wastewater
engineers are beginning to look at new concepts in treatment, driven
by the need to improve efficiency and reliability, reduce carbon diox-
ide emissions and energy consumption, expand the scope of material
recycling, and the production of renewable energy and other low
carbon byproducts. The circular economy concept has been an
important tool in achieving increasingly sustainable wastewater treat-
ment, and anaerobic processes are at the heart of this new revolution
(Zieliński et al., 2023).
The following chapters give the reader a broad overview of mod-
ern anaerobic wastewater treatment, starting with the key metabolic
pathways (Chap. 2). Gaseous products of digestion include methane,
hydrogen sulfide, ammonia, volatile organic compounds (VOCs),
18 Anaerobic Wastewater Treatment

and siloxanes, all of which are explored in terms of their formation,

impacts on health, and environmental effects (Chap. 3). Treatment
processes are broken down into flow-through (conventional) systems
and contact (high-rate) systems. Flow-through systems include more
traditional designs such as septic and Imhoff tanks used primarily for
individual houses or very small community systems and anaerobic
lagoons, which are widely used by the food processing industries as
well as wastewaters in warmer countries (Chap. 4). Sludge digestion
is now the norm for sewage sludge treatment and stabilization, with
many WWTPs using regional sludge digestion plants that cater for a
number of small to medium-sized WWTPs producing small quanti-
ties of waste sludge. Considered a difficult and often unreliable pro-
cess with the gas often flared off up to the 1970s, sludge digestion
has now evolved into a reliable process, from which many other
anaerobic processes have developed with many other sectors, espe-
cially agriculture, taking advantage of waste solids, including food
wastes, to generate biogas (Chap. 5). Modern anaerobic wastewater
treatment has been made possible through the development of con-
tact systems, from biofilm processes using carriers to dispersed
growth systems, such as the well-established UASB and the evolution
of granular-based reactors (Chaps. 6 and 7). As society makes the
slow transition from fossil fuels, anaerobic wastewater and sludge
treatment technologies are both contributing to the production of
renewable fuels, primarily through methane production for use at
WWTPs or by cleaning to remove hydrogen sulfide, carbon dioxide,
excess water, and other impurities to produce biomethane that can
then be fed directly into the national gas networks (Chap. 8). So,
although anaerobic processes are already making a huge contribution
toward a cleaner and more sustainable environment, future prospects
are extremely exciting as our understanding of how quorum sensing
can be used to enhance gas yields, the production of biohydrogen as
a new highly efficient fuel, or the production of new materials from
volatile acid production, and the recovery of other materials such as
metals (Chap. 9). But perhaps it is our improved understanding of
the microorganisms that carry out this complex synergistic set of
reactions that comprise anaerobic digestion and our ability to now
rapidly identify them and the community structures that provide us
 Introduction to Anaerobic Treatment 19

with the unique communities that are able to deliver such a wide
range of services transforming WWTPs from pollution control or
waste disposal centers to resource recovery hubs. The future of
anaerobic wastewater treatment is very exciting and will change
wastewater treatment for the better.

References
Andrews, J.F. and Graef, S.P. (1971) Dynamic modelling and simulation of
the anaerobic digestion process. In Anaerobic Biological Treatment (ed.
R.F. Gould). American Chemical Society, pp. 126–162.
Batstone, D.J., Keller, J., Angelidaki, I., Kalyuzhnyi, S.V., Pavlostathis, S.G.,
Rozzi, A., Sanders, W.T.M., Siegrist, H. and Vavillin, V.A. (2002a) The
IWA Anaerobic Digestion Model No. 1 (ADM1). IWA Publishing,
London, UK.
Batstone, D.J., Keller, J., Angelidaki, I., Kalyuzhnyi, S.V., Pavlostathis, S.G.,
Rozzi, A., Sanders, W.T.M., Siegrist, H. and Vavillin, V.A. (2002b) The
IWA anaerobic digestion model No. 1 (ADM1). Water Science and
Technology, 45, (10), 65–73.
Casey, T.J. (1981) Developments in anaerobic digestion. Transactions of the
Institute of Engineers in Ireland, 105, 25–32.
Chen, Y.R. and Hashimoto, A.G. (1978) Kinetics of methane fermentation.
Biotechnology and Bioengineering Symposium, 8, 269–282.
Chen, Y.R. and Hashimoto, A.G. (1980) Substrate utilization kinetic model
for biological treatment processes. Biotechology and Bioengineering, 22,
2081–2095.
Emebu, S., Pecha, J. and Janáćová, D. (2022) Review on anaerobic digestion
models: Model classification and elaboration of process phenomena.
Renewable and Sustainable Energy Reviews, 160, 112288. https://doi.
org/10.1016/j.rser.2022.112288
Energy Ireland (2024) https://www.energyireland.ie/the-future-of-anaero-
bic-digestion-in-ireland/
European Gas Association (2023) The ‘European Biomethane Map 2020’
shows a 51% increase of biomethane plants in Europe in two years.
https://www.europeanbiogas.eu/the-european-biomethane-map-
2020-shows-a-51-increase-of-biomethane-plants-in-europe-in-two-
years/#:~:text=European%20Biomethane%20Map’.-,The%20
analysis%20of%20the%20data%20collected%20shows%20that%20the%20
number,countries%20producing%20biomethane%20in%20Europe.
20 Anaerobic Wastewater Treatment

Finlay, B.J. and Fenchel, T. (1991) An anaerobic protozoan, with symbiotic

methanogens, living in municipal landfill material. FEMS Microbial
Ecology, 85, 169–180.
Gray, N.F. (2022) Natural Wastewater Treatment Systems and Sustainability.
World Scientific Publishing, London.
Haldane, J.B.S. (1930) Enzymes. Longman, London.
Hill, D.T. and Barth, C.L. (1977) A dynamic model for simulation of animal
waste digestion. Journal of the Water Pollution Control Federation, 49,
2129–2143.
Hill, D.T. and Nordstedt, R.A. (1980) Modelling techniques and computer
simulation of agricultural waste treatment processes. Agricultural
Wastes, 2, 135–156.
Kato, M., Field, J.A. and Lettinga, G. (1993) The high tolerance of metha-
nogens in granular sludge to oxygen. Biotechnology and Bioengineering,
42, 1360–1366.
Mo, R., Guo, W., Batstone, D., Makinia, J. and Li, Y. (2023) Modifications
to the anaerobic model no. 1 (AMD1) for enhanced understanding and
application of the anaerobic treatment process — A comprehensive
review. Water Research, 244, 120504. https://doi.org/10.1016/j.
watres.2023.120504
Nyns, E.J. (1994) A Guide to Successful Industrial Implementation of
Biomethanisation Technologies. The Institute Wallon, Namur, Belguim.
Vavilin, V.A., Rytov, S.V., Lokshina, L.Y., Rintala, J.A. and Lyberatos, G.
(2001) Simplified hydrolysis models for the optimal design of two-stage
anaerobic digestion. Water Research, 35, (4), 4247–4251.
Zieliński, M., Kazimierowicz, J. and Dębowski, M. (2023) Advantages and
limitations of anaerobic wastewater treatment — Technological basics,
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83. https://doi.org/10.3390/en16010083
 2
Anaerobic Metabolism and
Microorganisms

2.1 Introduction
A wide range of anaerobic microorganisms are found in the environ-
ment, with obligate species associated with anaerobic conditions,
including deep soil sediments, waterlogged soils, deep lakes, and
marine environments. Wastewater also contains anaerobes from the
human gut, especially Bacteroidetes spp.
Anaerobic bacteria are classified as facultative, obligate, and aero-
tolerant anaerobes.

Facultative anaerobes can grow in both aerobic and anaerobic envi-

ronments, being able to metabolize energy in the form of adenosine
triphosphate (ATP), either by respiration (aerobic) or by fermentation
(anaerobic). ATP production is much lower by fermentation than by
aerobic respiration, but sufficient is produced by fermentation to pro-
vide anaerobes with sufficient cellular energy. They form a bridge
between obligate aerobes and obligate anaerobic species. Growth and
metabolism of obligate anaerobes are inhibited by oxygen, with oxy-
gen toxicity depending on the redox potential (Eh), partial pressure,
composition of the substrate, growth rate, and cell density.

Obligate anaerobes only grow in the absence of oxygen and are

generally inhibited or even killed when oxygen is present. ATP is

21
22 Anaerobic Wastewater Treatment

generated by fermentation or anaerobic respiration. Many obligate

anaerobic bacteria produce hydrogen sulfide.

Aerotolerant anaerobes are not inhibited by the presence of oxygen

but can only generate ATP by fermentation (Pavlostathis and Giraldo-
Gomez, 1991). Aerobic respiration constantly produces reactive oxy-
gen species (ROS) as a byproduct, which are highly damaging to
cellular structures but which are broken down cellularly by the pro-
duction of enzymes, such as superoxide dismutase, peroxidase, and
catalase. Obligate anaerobes are generally unable to produce these
enzymes, which is the cause of their destruction when exposed to
aerobic environments where ROS are present. In contrast, aerotoler-
ant anaerobes are able to produce superoxide dismutase and
NADH oxidase/NADH peroxidase but not catalase, which give them
the ability to tolerate oxygen (Hansson and Häggström, 1984;
Brioukhanov and Netrusov, 2004; 2007). The most common ROS
are superoxide (O2−), peroxides (H2O2), and hydroxyl radical (OH•).
ROS formation starts with molecular oxygen being reduced to
superoxide:

O2 + e− → O2−(2.1)

The superoxide is disproportionated (i.e., dismutation) to hydro-

gen peroxide:

2H+ + O2− + O2− → H2O2 + O2 (2.2)

Partial or full reduction of hydrogen peroxide then follows:

H2O2 + e− → HO− + OH• (2.3)

2H+ + 2e− + H2O2 → 2H2O(2.4)

Aerotolerant anaerobes produce superoxide dismutase (SOD)

that breaks down superoxide anions to hydrogen peroxide:

2O2− + 2H+ → H2O2 + O2 (2.5)

 Anaerobic Metabolism and Microorganisms 23

Singlet oxygen (1O2) and hypochlorite ion (OCl−) are also types
of ROS.
The American Type Culture Collection (ATCC) not only holds
one of the largest reference collections of microorganisms (i.e.,
viruses, bacteriophages, bacterial, archaea, fungi, yeasts, cyano­
bacteria, and protists), it also provides a wide variety of aerobic and
anaerobic microbial species for bioremediation, biocontrol, and bio-
fuel production. They also produce a wide range of standardized
protocols and methods for handling and growing anaerobes. Similar
collections are found worldwide; for example, the Leibniz Institute
(DSMZ) in Germany has over 3,500 holdings of anaerobes, including
over 2,000 type strains.
Anaerobes require very specific growth conditions, which are
devoid of all oxygen in the case of obligate anaerobes. Anaerobic jars
or chambers that contain chemical packs to remove any oxygen and
release carbon dioxide are most widely used. Anaerobic chambers
usually have sealed gloves that allow technicians to work within the
chamber from the outside, preventing any ingress of air (Fig. 2.1).
The handling, growth, and identification of anaerobes is explained in
a report prepared by the Leibniz Institute (DSMZ, 2023), with
growth media specific to particular genera or species.
A recent development in taxonomic identification for anaerobic
bacteria and archaea is the Microbial Database for Activated Sludge
(MIDAS) developed by the Center for Microbial Communities at
Aalborg University in Denmark (https://www.midasfieldguide.org/
guide) (Dueholm et al., 2022). It is a remarkable resource that
includes details of both the physiology and ecology of the key species
and taxonomic details. It allows close examination of how environ-
mental and biogeographical factors affect anaerobic community struc-
ture in digesters. The database includes the results of full-length 16S
rRNA gene sequencing of bacteria and archaea collected from 285
full-scale anaerobic digesters from around the world. It also allows
sequences to be classified directly online. The most recent version of
the database, MIDAS-5, can identify 692 genera and 1013 species
(Dueholm et al., 2024).
24 Anaerobic Wastewater Treatment

(b)

(a)

Figure 2.1. Anaerobic chambers are used to ensure oxygen-free environments

when culturing and transferring anaerobic microorganisms. (a) Front view and (b)
side view showing the inside chamber. Reproduced with the kind permission of
Professor Amelia-Elena Rotaru, University of Sydney.

2.2 Anaerobic Degradation of Organic Matter

Anaerobic digestion can be carried out in a wide range of reactor
types, treating both wastewaters and sludges. However, it is perhaps
easiest to understand the process of anaerobic degradation by consid-
ering its use in domestic wastewater sludge treatment using digestion.
The settled sludge, from both primary and secondary settlement
tanks, is converted from its highly degradable odorous state to a sta-
ble and disposable product that neither smells nor undergoes further
decomposition on storage. The main constituents of raw sludge are
protein, fats, and polysaccharides, both soluble and insoluble, often in
particulate form. Typical chemical analysis of sewage sludge is protein
(25%), cellulose and lignin (25%), and fats (20%) (Gray, 2017). If
anaerobic breakdown is complete, then the end-products will be
methane, carbon dioxide, hydrogen sulfide, ammonia, water, and new
bacterial cells, although volatile organic compounds and siloxanes
may also be produced (Chap. 3). However, incomplete breakdown
due to environmental conditions or inhibitors can result in highly
 Anaerobic Metabolism and Microorganisms 25

polluting intermediate products being formed, such as volatile fatty

acids (VFAs) and alcohols, as well as ammonia.
The process of anaerobic breakdown of organic matter comprises
four discrete stages: hydrolysis, acid formation, and acetogenesis (i.e.,
the non-methanogenic phases), followed by methane formation
(methanogenic phase) (Fig. 1.3). This requires a complex consortium
of microorganisms, which can be classified as fermentative bacteria,
hydrogen-producing acetogenic bacteria, hydrogen-consuming ace-
togenic bacteria, carbon-dioxide-reducing methanogens, and aceti-
clastic methanogens.
It is convenient to think of these stages as different trophic levels,
and although all four stages normally occur simultaneously within a
digester, the microorganisms involved at each stage are metabolically
dependent on each other. For example, the methanogenic archaea
require the catabolized end-products of the acid-forming bacteria.
However, the latter species would eventually become inhibited by their
own end-products if these were not degraded in turn by the methano-
gens. Although bacteria are the major group of microorganisms
involved in anaerobic breakdown, archaea, fermentative ciliate and
flagellate protozoa, and some anaerobic fungi also occur. The process
does not readily occur in the presence of electron acceptors such as
oxygen, sulfate, or nitrate. Energy transformation is by the ATP sys-
tem, with energy being stored by the reaction of adenosine diphos-
phate (ADP) and inorganic phosphate (Pi) to form ATP. The energy
conserved in the pyrophosphate bond is used by splitting ATP into
either ADP + Pi or adenosine-5-monophosphate (AMP) and pyroph-
osphate (PPi).

2.2.1 Hydrolysis
The particulate solids present in the waste stream cannot be assimi-
lated by anaerobic microorganisms directly but need to have the large
organic polymers, which comprise these solids, broken down into
simpler soluble compounds that can be readily absorbed by the fer-
mentative bacteria. Nearly all organic wastes are amenable to being
anaerobically digested, although solid and semi-solid organic wastes,
26 Anaerobic Wastewater Treatment

as well as those comprising large particle sizes, are pretreated using

physicochemical methods to ensure maximum hydrolysis (Sec. 5.3).
Domestic wastewaters and sludges contain high percentages of biode-
gradable solids comprising proteins, carbohydrates (including cellu-
loses and lignins), and lipids. So, in the first stage of anaerobic
digestion, the major substrates in the sludge or wastewater are hydro-
lyzed, with the proteins converted to peptides and amino acids, fats
to glycerol and long-chain fatty acids (LCFAs), and polysaccharides
to mono- or disaccharides (Fig. 1.3), which are subsequently used by
the acidogenic bacteria. Wastewaters and sludges, including excess
biomass from bioreactors, are rich in bacteria, including pathogens.
Hydrolysis results in the death of a large proportion of any aerobic
cells present, including pathogens, followed by cell lysis that releases
the cellular contents back into solution.
As the process of anaerobic digestion involves four discrete steps,
each mediated by a consortium of specialist bacteria, archaea, and
other microorganisms, it is not surprising that the rate-limiting step
for the overall process is hydrolysis, on which the reactor design is
largely based. The rate of hydrolysis depends primarily on tempera-
ture and pH, with the hydrolytic fermentative bacteria extremely
temperature sensitive (Veeken and Hamelers, 1999). Noike et al.
(1985) compared the rates of substrate utilization for a range of sub-
strates and found that the rate of hydrolysis of cellulose was the rate-
limiting step overall in digestion.
Reactor design has to consider the minimum reactor temperature
and pH but also substrate composition, substrate residence time, the
size of particulate matter resent, and the concentration of end-prod-
ucts (e.g., VFAs and ammonia), which can inhibit hydrolysis if
allowed to build up. The rate of hydrolysis is largely dependent on the
nature of the organic matter being used as the main substrate. In turn,
this will determine the relative composition of the breakdown prod-
ucts. Hydrolysis is modeled as a first-order process with coefficient
rates of between 0.1 and 0.3 d−1 (Batstone et al., 2002).
The extracellular enzymes responsible for hydrolysis (i.e., hydro-
lases) are produced by hydrolytic fermentative bacteria, with the
products of hydrolysis used as their substrate.
 Anaerobic Metabolism and Microorganisms 27

Proteins are complex macromolecules comprising 20 different

amino acids, the sequence of which determines both the structure of
the protein and its properties. Proteins are more difficult to hydrolyze
than either carbohydrates or lipids. They are broken down into
smaller building blocks, such as polypeptides, oligopeptides, or even
individual amino acids, by proteolytic extracellular enzymes called
proteases, which are produced by only a small proportion of the bac-
teria present during anaerobic degradation. The majority of bacteria
are able to utilize these smaller soluble peptides or the amino acids,
which are absorbed directly across the cell wall and are broken down
intracellularly. Protease production is far in excess of that required,
even though protease-producing bacteria represent such a small per-
centage of the total bacteria present overall. Estimates indicate that
this over-production could be in the order of 50 times more than is
required (Hattingh et al. 1967), with the most active proteolytic bac-
teria, the spore-forming Clostridium spp. In anaerobic sludge, pro-
teolytic bacteria can reach concentrations of 6.5 × 107 mL−l, of which
65% of the isolates examined by Toerien (1970) were spore formers,
21% cocci, and the remainder non-sporing rods and bifid-like bacte-
ria. The rate of breakdown is dependent primarily on the solubility
and overall structure of the proteins, although temperature and pH
are also important. Duong et al. (2022) observed that increasing VFA
concentration reduced protein hydrolysis rates while carbohydrate
concentration did not. An example of a simple oligopeptide compris-
ing just two amino acids (i.e., a dipeptide) being hydrolyzed by pro-
tease into its component amino acids is shown below:

Fats and oils (i.e., lipids) are common in wastewaters and are esters
of fatty acids, the vast majority of which are triacylglycerol (commonly
28 Anaerobic Wastewater Treatment

known as triglycerides), which are triple esters of glycerol. Hydrolysis

of lipids is mediated by enzymes known as lipases or esterases, produced
by lipolytic bacteria, splitting triacylglycerol (C6H8O6) into glycerol and
three long chain fatty acids (LCHAs), as shown below:

Hydrolysis of fats is usually completely achieved during digestion,

although more slowly compared to other substrates. Relatively little is
known about the lipolytic bacteria, even though they have been shown
to be highly effective in anaerobic digesters. They are present in densi-
ties of up to 7 × 104 mL−1, and the addition of used vegetable oil to
digesters to enhance gas production is practiced in some countries.
Carbohydrates are found both as simple sugars or monosaccha-
rides that can be directly absorbed by bacteria or common disaccha-
rides (e.g., sucrose and maltose), and complex oligosaccharides and
polysaccharides, which need to be broken down into their component
monosaccharides by hydrolysis by glucosidases such as cellulase pro-
duced by cellulolytic bacteria before being absorbed by bacteria.
Wastewaters are particularly rich in long chain polysaccharides,
including pectin, starch, and celluloses. The hydrolysis of the disac-
charide cellobiose ((C6H7(OH)4O)2O) is shown below:
 Anaerobic Metabolism and Microorganisms 29

Disaccharides are hydrolyzed very rapidly; however, the hydrolysis

of polysaccharides takes much longer. Agricultural wastewaters and
wastes are rich in lignocellulosic compounds, which can only be par-
tially hydrolyzed, as lignin is not broken down under anaerobic con-
ditions (Brune and Sahm, 1981).
Anaerobic cellulolytic bacteria are present in anaerobic sludge at
concentrations of between 104 and 105 mL−1 and are predominantly
Gram-negative coccobacilli, with Bacteroides ruminicola being a par-
ticularly common species. Other species, including a Gram-positive
species forming curved rods, which formed short chains, were origi-
nally isolated by Hobson and Shaw (1971). In sewage sludge, the
ability to hydrolyze starch is the most common activity of these bac-
teria (Tables 1.3 and 3.2).

2.2.2 Acidogenesis or Fermentation

The heterogeneous groups of facultative and anaerobic bacteria, which
are responsible for hydrolysis, are also responsible for acid formation.
In this second stage, the hydrolyzed substrate is converted to organic
acids and alcohols, with new cells also being produced. Various bio-
chemical pathways are utilized, including fermentation and b-oxida-
tion. There is very little stabilization of the substrate in terms of BOD
or COD removal, with the products of acid fermentation being large
organic molecules. Obligate anaerobic bacteria occur in much larger
numbers than facultative bacteria, with ratios of 1:100 and numbers
in the order of 107–108 mL−l not uncommon (Kirsch, 1968). Major
acid-forming organisms include Bacillus sp., Micrococcus sp., and
Pseudomonas sp. (Table 1.3). Mono- and disaccharides, LCFA, glyc-
erol, amino acids, and short-chain peptides provide the main carbon
source for growth, with saturated fatty acids, carbon dioxide, and
ammonia being the main end-products. In practice, it is the monosac-
charides and amino acids that are the most abundant substrates.
Alcohols, aldehydes, and ketones are also produced but only in minute
quantities (Fig. 1.3). The concept of pyruvate as the pivotal com-
pound in metabolism was discussed earlier. When no external electron
acceptor is present, as is the case in acid fermentation, pyruvate
can undergo several alternative reactions that regenerate NAD from
30 Anaerobic Wastewater Treatment

Figure 2.2. Pyruvic acid is a pivotal compound in metabolism from which many
other compounds can be metabolized, depending on the organism and environmen-
tal conditions.

NADH (Fig. 2.2). Acetic, propionic, butyric, and lactic acids are the
most frequently produced end-products during fermentation.
Propionic and LCFAs are degraded by an intermediate microbial
group called the obligate hydrogen-producing acetogenic bacteria,
whereas other acid-producing bacteria, referred to as the homace-
togenic bacteria, produce acetic and sometimes, other acids (McInerney
et al., 1980).

Fermentation of monosaccharides
Monosaccharide fermentation is by two pathways, the Meyerhof-
Parnas (EMP) or Entner Doudoroff (ED) pathways. The final
products via acetyl-CoA are C3 (lactate or propionate), C2 (acetate),
C4 (butyrate), or, occasionally, C6 (caproate) products. The most
common products formed during fermentation are acetate, butyrate,
and ethanol. The main degradation pathways of glucose during aci-
dogenesis are:
 Anaerobic Metabolism and Microorganisms 31

C6H12O6 + 2H2O → 2CH3COOH + 2CO2 + 4H2(2.6)

      Glucose Acetic acid

C6H12O6 → CH3 [CH2]2COOH + 2CO2 + 2H2(2.7)

      Glucose Butyric acid

C6H12O6 → 2CH3CH(OH)COOH(2.8)
          Glucose Lactic acid

C6H12O6 + 2H2 → 2CH3CH2COOH + 2H2O(2.9)

       Glucose Propionic acid

C6H12O6 → CH3CH2COOH + CH3COOH + 2CO2 + 2H2(2.10)

Glucose      Propionic acid Acetic acid

C6H12O6 → 2CH3CH2OH + 2CO2(2.11)

           Glucose Ethanol

C6H12O6 + H2O → CH3CH2OH + CH3COOH + 2CO2 + 2H2 (2.12)

Glucose Ethanol Acetic acid

C6H12O6 + H2O → CH3CH2OH + CH3CH(OH)COOH + 2CO2

Glucose Ethanol Latic acid(2.13)

In these reactions, glucose is used as an electron acceptor to pro-

duce oxidized (e.g., acetate) or reduced products (e.g., propionate,
ethanol) (Angelidaki et al., 2011). The EMP and ED pathways can
coexist, as shown by Jojima et al. (2021), who studied the ethanol-
producing bacteria Corynebacterium glutamicum.

Fermentation of amino acids

Two different amino acids generally degrade together in a coupled
oxidation-reduction reaction known as the Stickland fermentation,
with one amino acid oxidized while the other is reduced. Uncoupled
amino acid degradation is also possible, e.g., glutamate fermentation.
Winter et al. (1987) give the example of coupled fermentation of
alanine with glycine by the Stickland reaction, with alanine oxidized
and glycine reduced:
32 Anaerobic Wastewater Treatment

CH3CHNH2COO− + 2H2O → CH3COO− + CO2 + NH3 + 4H+ + 4e−

Alanine Acetate(2.14)

  2CH2NH2COO− + 4H+ + 4e− → 2CH3COO− + 2NH3– (2.15)

   Glycine Acetate

2.2.3 Acetogenesis
Acetate is synthesized during acetogenesis either by the reduction of
carbon dioxide or from organic acids. Hydrogen-producing aceto-
gens oxidize ethanol and organic acids to acetate, hydrogen, and
carbon dioxide.

CH3CH2OH + H2O → CH3COO− + H+ + 2H2(2.16)

     Ethanol Acetate

  CH3[CH2]2COO− + 2H2O → CH3COO− + H+ + 2H2(2.17)

   Butyrate Acetate

CH3CH[OH]COO− + 2H2O → CH3COO− + HCO3− + H+ + 2H2

Lactate Acetate(2.18)

CH3CH2COO− + 3H2O→ CH3COO− + HCO3− + H+ + 3H2(2.19)

Propionate Acetate

CH3[CH2]3COO− + 2H2O → CH3COO− + → CH3CH2COO−

Valeriate Acetate + H+ + 2H2(2.20)

These reactions are generally endergonic under normal

operating conditions, and to make them exergonic, the resulting
hydrogen must be continuously removed. This is done by hydrogen-
utilizing archaeal methanogens (i.e., hydrogenotrophic methanogen-
esis), which require a close relationship between the bacteria and
archaea-creating interspecies hydrogen transfer requiring a close
physical proximity between the two (i.e., syntropy). The closer the
inter-microbial distance, the greater the specific growth rates.

Hydrogenotrophic methanogenesis 4H2 +2CO2 → CH4 + 2H2O(2.21)

 Anaerobic Metabolism and Microorganisms 33

This relationship also allows the degradation of fatty acids

that cannot be broken down by acetogens or methanogens alone
(Angelidaki et al., 2011). An example of syntrophic acetogenesis and
hydrogenotrophic methanogenesis is shown below:

At low temperatures or acidic conditions, hydrogen-utilizing ace-

togens (homoacetogens) can compete with the hydrogen-utilizing
archaea for the available hydrogen where they reduce CO2 to acetate:

Homoacetogenesis 4H2 + 2CO2 → CH3COO− + H+ + 2H2O(2.22)

Hydrogen can also be removed by sulfate-reducing bacteria,

although this can impact methane production by reducing the avail-
able substrate for hydrogenotrophic methanogenesis.

Sulfate reduction 4H2 + SO42− + H+ → H+ + HS− + 4H2O(2.23)

2.2.4 Methanogenesis
There are three principle anaerobic pathways in methane formation:
(i) Aceticlastic methanogenesis uses acetate to form methane, (ii)
hydrogenotrophic methanogenesis uses hydrogen and carbon diox-
ide, while (iii) methylotrophic methanogenesis utilizes methylated C1
compounds, such as methanol (CH3OH) or methylamines (CH3NH2).
These pathways are represented below:

       (i) CH3COO− + H+ → CH4 + CO2 (2.24)

       (ii) 4H2 + CO2 → CH4 + 2H2O(2.25)

      (iii) 4CH3OH → 3CH4 + CO2 + 2H2O(2.26)

34 Anaerobic Wastewater Treatment

In practice, it is aceticlastic and hydrogenotrophic methanogene-

sis that are the principle methane formation pathways in the digestion
of wastewater sludges, representing 70 and 30% of the overall meth-
ane generation respectively. Many hydrogenotrophic methanogens
can also use formate. Further details of methane formation and
microbial kinetics of methanogens are given in Sec. 3.2.1.
During methanogenesis, complete stabilization of the substrate
occurs, which is more efficient than complete aerobic stabilization,
with only gaseous end-products formed. Methane is an ideal end-
product as it is non-toxic, easily separated from the site of production,
is not very soluble, inert under anaerobic conditions, and can be read-
ily collected for use as an energy source. In the overall anaerobic fer-
mentation of carbohydrates to carbon dioxide and methane, equal
volumes of each gas (a) are produced.

  (C6H12O6)x × aH2O → aC6H12O6 → 3aCH4 + 3aCO2 (2.27)

The carbon dioxide produced only partially escapes as gas because,

unlike methane, it is relatively soluble in water. It also reacts with any
hydroxide ions (OH−) in the system to produce bicarbonate ions
(HCO3−). The evolution of carbon dioxide gas is, therefore, a func-
tion of factors such as pH, bicarbonate concentration, temperature,
and substrate composition. The biodegradable protein is deaminated
to produce ammonia, which reacts with water:

NH3 + HOH ↔ NH4+ + OH−(2.28)

This is the major source of hydroxide ions, which react with the
carbon dioxide evolved during methanogenesis to form bicarbonate
ions:

CO2 + HOH ↔ H2CO3 ↔ H+ + HCO3−(2.29)

H2CO3 + OH− ↔ HCO3− + HOH (2.30)

Therefore, the protein content of the wastewater substrate will

significantly affect the quantity of carbon dioxide actually released
from solution as well as the buffering capacity of the system in terms
of bicarbonate. The portion of carbon dioxide incorporated in the
 Anaerobic Metabolism and Microorganisms 35

bicarbonate ion is eventually removed from the reactor in the liquid

rather than in the gas phase.
The current taxonomy classifies methanogens, which are from the
domain Archaea and not bacteria, into six well-established orders:
Methanobacteriales, Methanocellales, Methanococcales, Methano­
microbiales, Methanosarcinales, and Methanopyrales, excluding the
order Methanomassiliicoccales, which is methylotrophic (Buan,
2018) (Sec. 3.2.1). The order Methanopyrales is hyperthermophilic
and closely related to both Methanobacteriales and Methanococcales
(Tables 2.1 and 3.1). There is currently only one known species,
Methanopyrus kandleri (Liu, 2010). Methanogens are unusual in that
they are composed of many species with very different cell morphol-
ogy. For example, long filaments (Methanosarcinales), short rods
(Methanobacteriales and Methanopyrales), and coccoidal cells
(Methanococcales and Methanomicrobiales).
Acetoclastic methanogenesis that uses acetate to form methane is the
most important pathway for methane production (Sec. 3.2.1) and is
carried out predominantly by the Methanosarcinales. Methylotrophic
methanogenesis is mainly carried out by archaea from the Metha­no­
sarcinales, although Methanosphaera spp. (Methano­bacteriales) can also
use methylated C1 compounds. Hydrogenotrophic methanogenesis
uses hydrogen and carbon dioxide and is carried out by members of the
Methanobacteriales, Methanocellales, Methanococcales, Metha­
nomicrobiales, and Methanopyrales (Tables 2.1 and 3.1) (Angelidaki
et al., 2011). Digesters support a wide range of methanogens, with the

Table 2.1. Anaerobic pathway, temperature, and pH ranges for the main six orders
of methanogens. Adapted from (Angeliidki et al., 2011).
Temperature pH
Order Predominant pathway range (°C) range
Methanobacteriales Hydrogenotrophic 15–60 6.1–8.0
Methanocellales Hydrogenotrophic 25–40 6.5–7.8
Methanococcales Hydrogenotrophic 20–88 4.5–9.8
Methanomicrobiales Hydrogenotrophic 15–60 6.1–8.0
Methanosarcinales Aceticlastic, hydrogenotrophic 10–70 4.0–10.0
Methanopyrales Hydrogenotrophic 84–110 5.5–7.0
36 Anaerobic Wastewater Treatment

community structure influenced by a wide range of operating param-

eters and designs. For example, acetoclastic methanogen Methanosarcina
thermophila and Methanosaeta are particularly common at higher
(thermophilic) temperatures. A decrease in pH, caused by increasing
the loading rate, which raises the VFA concentration, favors the acid
tolerant methanogens, such as Methanobrevibacter acididurans.

2.2.5 Importance of Redox Potential

Microaerobic or anaerobic conditions are required for fermentation
and methanogenic reactions to occur, not only in the treatment of
wastewaters and waste sludges but in an enormous array of other fer-
mentation processes, such as ethanol production. This is measured
and controlled by the use of redox potential, also known as oxidation-
reduction potential (ORP). ORP reactions in biological reactions are
normally defined in terms of the loss and gain of hydrogen or elec-
trons. Each oxidation is accompanied by a reduction and can be sum-
marized as:

where AH2 is the hydrogen donor, and B is the hydrogen acceptor.

Each couple (AH2/A or B/BH2) has a tendency to either donate
reducing equivalents and be oxidized (AH2 → A) or accept them and
be reduced (B → BH2). When both couples combine in a complete
redox reaction, the net flow of the reaction can be determined by the
relative tendency of each couple to donate or accept reducing equiva-
lents, which is the redox potential. This can be measured using a
galvanic cell consisting of two electrodes connected by a conducting
solution: oxidation occurs at the negative electrode (anode), and elec-
trons are produced, whereas electrons are consumed and reduction
takes place at the positive electrode (cathode). The redox potential is
quantified by comparison with a standard redox couple. By conven-
tion, the standard redox couple is present at a hydrogen electrode
 Anaerobic Metabolism and Microorganisms 37

consisting of a platinum electrode, with hydrogen ions in solution. In

the presence of platinum as the catalyst, the reaction is:

H2 ↔ 2H+ + 2e−(2.31)

and the tendency to donate reducing equivalents, as electrons in this

case, is measured as the voltage (potential) of the electrical current
generated when the electrode is coupled in series with another redox
couple electrode. Under standard conditions at 25°C, 1 atm of hydro-
gen, and at pH 0, the redox potential of the hydrogen electrode is
zero. At pH 7, the potential of the redox couple H2/2H+ + 2e− is –420
mV. The symbol Eh is used for the redox potential under standard con-
ditions. So fascinatingly, the ORP provides information on the activity
of electrons within the liquid medium as well as at the cellular level.
A couple of lower redox potential will always donate reducing
equivalents to a couple of higher potential and, during the oxidation
of a substrate, reducing equivalents are transferred in the direction of
increasing potential. This transfer is accompanied by the release of
free energy, the magnitude of which is given by the standard free
energy change,
∆Go′ = –nF∆Eh (2.32)

where n is the number of electrons transferred in the reaction, F is the

charge on one mole of electrons, which is Faraday’s constant (96.649
kJV−1 mol−l ), and ∆Eh is the standard electrode potential. Biological
systems have evolved to conserve this energy and convert it into bio-
logically useful forms (i.e., oxidative phosphorylation).
In practical terms, ORP can be used to indicate which oxidative-
reduction reactions will occur within a wastewater system and is par-
ticularly useful in the management of anaerobic systems (Fig. 2.3).
The ORP gives a measure of the general condition of the liquid.
Anaerobic processes similar to those found in sludge lagoons and
digesters will have low values of Eh (< –200 mV), whereas aerobic
processes will have higher values (> +50 mV). More precisely, values
of Eh –150 mV to –420 mV are found in anaerobic environments,
whereas aerobic environments vary between –200 mV and +420 mV.
38 Anaerobic Wastewater Treatment

Figure 2.3. The redox potential indicates which inorganic electron acceptor is
being used by the microbial community.

Facultative environments change from aerobic to anaerobic systems at

about +100 mV. ORP is a more reliable measure of aerobic condi-
tions than a dissolved oxygen concentration measurement, as it is
often used in the control of environmental conditions in activated
sludge tanks. Because odors are produced from anaerobic rather than
aerobic processes, ORP can be used as a convenient measure to deter-
mine the onset of odor production during the treatment of high-
strength wastes, such as animal wastes. The VFA concentration in the
liquid phase can be controlled by using ORP and pH, as can the ratio
of particular VFAs produced. This is widely used for the industrial
production of acetic, propionic, and butyric acids by anaerobic fer-
mentation using hydrolytic, acidogenic, and acetogenic bacteria
(Chen et al., 2022). So, the control of ORP is critical in all fermenta-
tion reactions, including wastewater anaerobic treatment and sludge
digestion. At the intracellular level, when electrons are transferred,
oxygen will be the prior acceptor, with the next acceptor utilized in
 Anaerobic Metabolism and Microorganisms 39

Table 2.2 Examples of the ORP of redox pairs

in intracellular metabolism (Liu et al., 2013).
Reproduced with permission of Elsevier Publishing.
Redox pairs ORP Eh (mV)
H+/H −420
NAD+/NADH −320
NADP+/NADPH −315
Lipoate; ox/red −290
Acetoacetate/3-hydroxybutyrate −270
GSSG/2GSH −240
Pyruvate/lactate −190
Oxaloacetate/malate −170
Fumarate/succinate +30
Cytochrome b; Fe3+/Fe2+ +80
Ubiquinone; ox/red +100
Cytochrome c1; Fe3+/Fe2+ +220
Cytochrome a; Fe3+/Fe2+ +290
Oxygen/water +820

its absence, according to Table 2.2. In anaerobic fermentation, the

key cofactors NAD+ (oxidized nicotinamide adenine dinucleotide and
NADP+ (nicotinamide adenine dinucleotide phosphate) are able to
accept electrons to support the metabolism of a large range of intra-
cellular reactions. They are reduced to NADH and NADPH, respec-
tively, so that the ratio of NADH/NAD+, in particular, determines
the intracellular ORP (Liu et al., 2013).

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 3
Methane and Other Volatile
Products of Digestion

3.1 Introduction
Biogas produced from organic waste streams by anaerobic digestion
is primarily a mixture of methane (CH4) and carbon dioxide (CO2)
with hydrogen (H2), nitrogen (N2), and water vapor (H2O) also pre-
sent. However, a wide range of other compounds can also be present
in biogas, several of which are potentially damaging to the environ-
ment and human health or cause damage over time to equipment,
either by corrosion or abrasion. These include hydrogen sulfide
(H2S), ammonia (NH2), siloxanes, and a wide range of volatile
organic carbon compounds (VOCs), including terpenes and halogen-
ated hydrocarbons (AOXs). Sludge digestion often produces high
hydrogen sulfide concentrations, while wastewater anaerobic treat-
ment is more associated with AOXs and siloxanes (Rasi et al., 2007;
Calbry-Muzyka et al., 2022).
Traditionally, biogas has been used at wastewater treatment plants
(WWTPs) to operate combined heat and power (CHP) engines,
which provide electricity to supplement plant operation as well as heat
for digestion and space heating. However, to be able to convert
biogas into a more valuable and versatile energy source, it must be
converted into either compressed natural gas (Bio-CNG) or liquefied
natural gas (Bio-LNG). In order to be able to inject biogas directly
into the gas grid, the purity of the methane must be 99%, with only

43
44 Anaerobic Wastewater Treatment

trace contaminants left. This purified biogas is known as biomethane.

The purification process comprises a number of steps, which may
include activated carbon, bio-scrubbers, and membrane filtration and
are discussed in detail in Chap. 8. Biomethane can also be used to
produce methanol and hydrogen.

3.2 Methane
3.2.1 Formation
Methane is a naturally formed alkane discovered by Alessandro Volta
in 1776. It is colorless, odorless, lighter than air, slightly soluble, and
highly flammable. It is produced in anoxic and anaerobic habitats
such as the sediments of lakes, rivers, and estuaries, as well as swamps,
marshes, and peat bogs from where it diffuses into the atmosphere,
although 60–70% is currently emitted from human activities that
include livestock rearing, landfill, waste, oil and gas extraction, waste-
water treatment, and leakage from gas networks. It is the third-largest
energy resource after oil and coal. Methane is produced by anaerobic
archaea by fermentation (digestion). As described in Chap. 2, anaero-
bic digestion comprises four discrete stages: hydrolysis, acid forma-
tion, and acetogenesis (the non-methanogenic phases), followed by
methane formation (methanogenic phase) (Fig. 1.2), with the process
of methanogenesis already explained in Sec. 2.2.4.
Methanogenic archaea conserve energy for adenosine triphos-
phate (ATP) synthesis through the production of methane gas via the
Wolfe Cycle (Thauer, 2012). Until the late 1970s, methanogens were
thought to be bacteria until Dr. Carl Woese, using genetic analysis,
discovered they were in a completely new domain, which he named
the Archaea. This means that the domain prokaryotes, which was
originally composed solely of the bacteria, has been replaced by two
new domains, the Bacteria and Archaea, alongside eukaryotes. What
is exciting about archaea is that they are extremophiles, with metha-
nogens so far isolated between pH 3.0 and 10.2, with temperatures
from –2 to 122oC, and are from a wide range of salinities. They are
strict anaerobes and obligate methane producers.
 Methane and Other Volatile Products of Digestion 45

Table 3.1. There are actually seven methanogen orders, which are listed below,
along with common species and methanogenesis pathway (Buan, 2018). Reproduced
under the Creative Commons Attribution license CC BY-NC-ND 4.0 DEED.
Order Representative organism Methanogenesis pathways
Methanopyrales Methanopyrus kandleri Hydrogenotrophic
Methanococcales Methanococcus maripaludis Hydrogenotrophic
Methanobacteriales Methanobacterium Hydrogenotrophic
thermoautotrophicum
Methanosarcinales Methanosarcina mazei Hydrogenotrophic,
methylotrophic,
carboxydotrophic,
acetoclastic
Methanomicrobiales Methanospirillum hungatei Hydrogenotrophic
Methanocellales Methanocella paludicola Hydrogenotrophic
Methanomassiliicoccales Methanomassiliicoccus Methylotrophic
luminyensis

The archaea responsible for methanogenesis in sludge digesters

and anaerobic wastewater treatment processes are similar to those
found in other anaerobic environments, including the digestive tract
of animals. Most methanogens belong to the genera Methanobacterium,
Methanosarcina, Methanospirillum, and Methanococcus (Table 3.1).
The methanogenic genera isolated so far are limited to the catabolism
of either one carbon (e.g., H2/CO2, CH3OH, CO, HCOOH, CH3NH2)
or two carbon compounds (e.g., CH3COOH) (Sec. 2.2.4). Methanogens
are unusual in that they are composed of many species with very dif-
ferent cell morphologies. They require a strict anaerobic environment
for growth with a redox potential (ORP) below –300 mV and have
simple nutritional requirements, primarily carbon dioxide, ammonia,
and sulfide. Ammonia is the essential nitrogen source for growth, as
no methanogenic species are known to utilize amino acids or pep-
tides. While sulfide is the most common sulfur source, although some
species can use cysteine instead. Methanogens contain a number of
unique co-enzymes, for example, co-enzyme 420, which is involved
in electron transfer instead of the usual ferredoxin, co-enzyme M,
which is used in methyl transfer reactions, and factor B, which is
46 Anaerobic Wastewater Treatment

required for the enzyme formation of methane from methyl co-

enzyme M. The synthesis of ATP appears to be via electron transport
linked to phosphorylation. The group is defined by not having
muramic acid in their cell walls, unique co-enzymes, and unique
oligo-nucleotide sequences of the 16S ribosomal RNA molecule
(Buan, 2018). Research on methanogens has been slow compared to
other fields of microbiology due to the culturing requiring oxygen-
free culture media as well as a reducing environment with an ORP
<50 mV.
There is a close relationship between gas production and the
number of methanogens present in a digester, generally being present
in numbers between 106 and 108 mL–1 (Table 3.2). It is from acetic
and, to a lesser extent, propionic acid that the greatest percentage of
methane is derived during methane fermentation, whereas formic acid
fermentation and methane fermentation associated with b-oxidation
of long chain fatty acids (LCFAs) probably account for most of the
small percentage of methane not derived from acetic and propionic
acids (Fig. 3.1).
Methane formation from acetic acid is a single-step process car-
ried out by one group of methanogenic archaea:

CH3COOH → CH4 + CO2(3.1)

             (acetic acid) (methane)

Table 3.2. Enumeration of anaerobic microbial

populations in sewage sludge digesters (adapted
from (Zeikus, 1980)).
Group Numbers per mL–1
Hydrolytic species
Total 108–109
Proteolytic 107
Cellulolytic 105
Acetogenic species 106
Homoacetogenic species 105–106
Methanogenic species 106–108
Sulfate reducers 104
 Methane and Other Volatile Products of Digestion 47

Figure 3.1. Pathways in methane formation of complex waste. Percentages repre-

sent the conversion of waste COD by various routes.

Methane fermentation of propionic acid is a two-step process

involving two groups of methanogens, with acetic acid as the inter-
mediate step:

Step 1: 
CH3CH2COOH + 0.5H2O → CH3COOH + 0.25CO2 + 0.75CH4 (3.2)
(propionic acid)            (acetic acid)           (methane)

Step 2: CH3COOH → CO2 + CH4(3.3)

       (acetic acid) (methane)

Overall: CH3CH2COOH + 0.5H2O → 1.25CO2 + 1.75CH4(3.4)

       (propionic acid)            (methane)
48 Anaerobic Wastewater Treatment

The acid-fermenting archaea are tolerant to changes in pH and

temperature and have a much higher rate of growth than the meth-
ane-fermenting archaea. This difference in growth rate, linked with a
greater sensitivity to environmental factors, results in the methano-
genic archaea becoming the major factor controlling the overall rate
of anaerobic digestion. However, in the digestion of other wastes, the
first stage of anaerobic digestion, the enzymic hydrolysis of polymers
to monomers and cellulose hydrolysis, in particular, has been reported
as the rate-limiting step in the conversion of cellulose to methane and
in the digestion of household waste. The effect of environmental fac-
tors and the inhibition of toxic compounds in the anaerobic process
are considered in Chap. 5.
The utilization of the organic fraction of wastewater is directly
related to methane production and vice versa. Where the exact chemical
nature of the sludge is known, the quantity of methane produced can
be estimated by the following equation (Buswell and Mueller, 1952):

  CnHaOb + (n-a/4-b/2)H2O → (n/2-a/8+b/4)CO2

+ (n/2+ a/8-b/4)CH4  (3.5)

However, McCarty (1964) estimated the theoretical methane

production from the complete stabilization of 1 kg of COD (chemical
oxygen demand) as 0.348 m3 at standard temperature and pressure.
It is useful to be able to calculate the proportion of substrate uti-
lized either as energy or synthesis. Both the amount of electron accep-
tor required and the end-products produced can be calculated by
knowing what proportion of the substrate is synthesized into new
cellular material. As part of the substrate goes into energy formation
and the remainder goes to cell synthesis, all the reacting material can
be expressed as:
fe + fs = 1 (3.6)

where fe is the fraction of electron donor used for energy and fs is the
fraction of electron donor used for cell synthesis. McCarty (1975)
calculates the amount of substrate metabolized by bacteria to form
energy and new cells by using balanced half equations. The overall
reaction is constructed from three half-reactions (Table 3.3): one for
 Table 3.3. Oxidation half-reactions. Reactants and products are given at unit activity except for (H+) = 10–7.
∆G°(W) * kcal per
Equation Half-reaction no. electron equivalent
Reactions for bacterial cell synthesis (Rc)
Ammonia as nitrogen source:
1 1/5CO2 + I/20HCO3– + 1/20NH4+ + H+ + e– = 1/20C5H7O2N + 9/20H2O
Nitrate as nitrogen source:
2 1/28NO3– + 5/28CO2 + 29/28H+ + e– = 1/28C5H7O2N + 11/28H2O

Methane and Other Volatile Products of Digestion 49

Reactions for electron acceptors (Ra)
Oxygen
3 l/402 + H+ + e– = 1/2H2O –18.675
Nitrate:
4 1/5NO3– + 6/5H+ + e– = 1/10N2 + 3/5H2O –17.128
Reactions for bacterial cell synthesis (Rc)
Sulphate:
5 1/8SO42– + 19/16H+ + e– = 1/16H2S + 1/16HS– + 1/2H2O 5.085
Carbon dioxide (methane fermentation):
6 1/8CO2 + H+ + e– = 1/8CH4 + 1/4H2O 5.763
(Continued)
 Table 3.3. (Continued)

50
∆G°(W) * kcal per

Anaerobic Wastewater Treatment

Equation Half-reaction no. electron equivalent
Reactions for electron donors (Rd)
Organic donors (heterotrophic reactions)
Domestic wastewater:
7 9/50CO2 + 1/50NH4+ + 1/5OHCO3– + H+ + c– = 1/5OC10H19O3N + 9/25H2O 7.6
Protein (amino acids, proteins, nitrogenous organics):
8 8/33CO2 + 2/33NH4+ + 31/33H+ + e– = 1/66C16H24C5N4 + 27/66H2O 7.7
Carbohydrates (cellulose, starch, sugars):
9 1/4CO2 + H+ + e– = 1/4CH2O + 1/4H2O 10.0
Grease (fats and oils):
10 4/23CO2 + H+ + e– = 1/46C8H16O + 15/46H2O 6.6

Acetate:
11 1/8CO2 + I/8HCO3– + H+ + e– = I/8CH3COO– + 3/8H2O 6.609
Propionate:
12 1/7CO2 + 1/14HCO3– + H+ + e– = 1/14CH3CH2COO– + 5/14H2O 6.664
Benzoate:
13 1/5CO2 + 1/30HCO3– + H+ + e– = 1/30C6H5COO– + 13/20H2O 6.892
 Ethanol:
14 1/6CO2 + H+ + e– = l/2CH3CH2OH + 1/4H2O 7.592
Lactate:
15 1/6CO2 + 1/12HCO3– + H+ + e– = 1/12CH3CHOHCOO– + 1/3H2O 7.873
Pyruvate:
16 1/5CO2 + 1/10HCO3– + H+ + e– = 1/10CH3COCCO– + 2/5H2O 8.545
Methanol:
17 1/6CO2 + H+ + e– 8.965

Methane and Other Volatile Products of Digestion 51

Inorganic donors (autotrophic reactions) = 1/6CH3OH + 1/6H2O
Fe + e
3+ –
= Fe2+ –17.780
19 1/2NO + H + e
–
3
+ –
= 1/2NO + 1/2H2O
–
2 –9.430
20 1/8NO + 5/4H + e
–
3
+ –
= 1/8NH + 3/8H2O
+
4 –8.245
21 1/6NO + 4/3H + e
–
2
+ –
= 1/6NH + 1/3H2O
+
4 –7.852
22 1/6SO42– + 4/3H+ + e– = 1/6S + 2/3H2O 4.657
23 1/8SO + 11/16H + e
2–
4
+ –
= 1/16H2S + 1/16HS + 1/2H2O
–
5.085
24 1/4SO + 5/4H + e
2–
4
+ –
= 1/8S2O + 5/8H2O
2–
3 5.091
25 H +e
+ –
= 1/2H2 9.670
26 1/2SO42– + H+ + e– = 1/2SO32– + 1/2H2O 10.595
52 Anaerobic Wastewater Treatment

the synthesis of new bacterial cells, which are assumed to be C5H7O2N

(Rc), one for the electron acceptor (Re), and one for the electron
donor (Rd), which combine to give the relationship where R is the
overall reaction:
R = fsRc + feRe − Rd  (3.7)

The volume of bacteria biomass and methane produced in an

anaerobic sewage sludge digester can be calculated using stoichiom-
etry. Using the tables devised by McCarty (Tables 3.3 and 3.4), the

Table 3.4. Maximum cell yield (fs)max (i.e., the maximum

fraction of the electron donor used for synthesis) for vari-
ous electron donors and acceptors.
Electron donor Electron acceptor ( fs) max
Heterotrophic reactions
Carbohydrate O2 0.72
Carbohydrate NO3 0.60
Carbohydrate SO4 0.30
Carbohydrate CO2 0.28
Protein O2 0.64
Protein CO2 0.08
Fatty acid O2 0.59
Fatty acid SO4 0.06
Fatty acid CO2 0.05
Glucose O2 0.79
Lactose O2 0.74
Sucrose O2 0.75
Glycine O2 0.52
Alanine O2 0.52
Propionate O2 0.58
Acetate O2 0.58
Methanol NO3 0.36
Methanol CO2 0.15
 Methane and Other Volatile Products of Digestion 53

Table 3.4. (Continued)

Electron donor Electron acceptor ( fs) max

Propionate CO2 0.07
Acetate CO2 0.06
Glucose CO2 0.27
Sewage sludge CO2 0.11
Autotrophic reactions
S O2 0.22
S2O3 O2 0.11
S2O3 NO3 0.20
NH4 O2 0.10
H2 O2 0.24
H2 CO2 0.04
Fe O2 0.07

appropriate half-reactions can be selected with carbon dioxide as the

electron acceptor:

Rc: 1/5CO2 + 1/20HCO3– + 1/20NH4+ + H+ + e– = 1/20C5H7O2N

+ 9/20H2O(3.8)
Re: 1/8CH4 +1/4H2O = 1/8CO2 + H+ + e–(3.9)
Rd: 1/50C10H19O3N + 9/25H2O = 9/50CO2 + 1/50NH4+
+ 1/50HCO3– + H+ + e–(3.10)

Table 3.4 gives a ( fs)max value of 0.11 for sewage sludge, and as the
digester has a mean cell residence time of 20 days, fs is calculated as:

fs = ( fs)max 1 − [(0.8 b ϕc)/(1+ b ϕc)](3.11)

where ϕc is the sludge age in days and the coefficient b represents the
rate of cell death and decay, which is assumed to be 0.03.

fs = 0.11 (1 − [(0.8(0.03)(20)/1 + 0.03(20)]) = 0.077

   As fs = 1 − fe,
    fe = 0.923
54 Anaerobic Wastewater Treatment

Therefore, the revised reactions using the relationship R = fs Rc +

fe Re − Rd are

Rc × fs:
0.0154CO2 + 0.0039HCO3– + 0.0039NH4+ + 0.007H+ + 0.007e–
= 0.0039C5H7O2N + 0.035H2O  (3.12)

Re × fe:
0.115CO2 + 0.923H+ + 0.923e– = 0.115CH4 + 0.231H2O(3.13)

Rd:
0.02C10H19O3N + 0.36H2O = 0.18CO2 + 0.02NH4+
+ 0.02HCO3– + H+ + e–(3.14)

When added and normalized by dividing by 0.02, the overall

reaction R is:

C10H19O3N + 4.7H2O = 0.195C5H7O2N + 5.75CH4 + 2.48CO2

+ 0.81NH4+ + 0.81HCO3–(3.15)

Therefore, for each mole of sewage sludge, 0.195 mole of new

cells are produced and 5.75 moles of methane is released.
A more precise value for ( fs)max can be calculated from basic
constituents using Table 3.3. For example, if domestic wastewater
contains 50% protein, 40% carbohydrate (CHO), and 10% fat, then
( fs)max wastewater of domestic wastewater for anaerobic digestion, where
carbon dioxide is the electron acceptor, is:

( fs)max wastewater = (% protein × ( fs)max protein) + (% CHO × ( fs)max CHO)

+ (% fat × ( fs)max fat)(3.16)
( fs)max wastewater = (0.50 × 0.08) + (0.40 × 0.28) + (0.10 × 0.05 )
( fs)max wastewater = 0.157

3.2.2 Health and Environmental Effects

Methane (CAS: 74-82-8) is an extremely flammable gas that can
displace oxygen, causing the risk of suffocation and explosion in
 Methane and Other Volatile Products of Digestion 55

low-lying areas and especially inside confined spaces. Explosions due

to methane have occurred at atmospheric concentrations of between
5 and 15%. It is an odorless gas, so is not detected by smell, making
it potentially very dangerous in the workplace, especially where anaer-
obic treatment processes or sludge digestion are employed at WWTPs.
People are frequently subjected to low levels of methane exposure
due to the use of natural gas appliances or when walking in areas
where it is naturally produced, such as wetlands. There are no
associated health risks with low-level exposure. However, as the con-
centration in air increases, it reduces the amount of oxygen breathed
in, causing a range of effects — initially rapid breathing, increased
heart rate, facial flushing, balance problems, numbness leading to
mood changes, slurred speech, vision problems, memory loss, and
eventually nausea, vomiting, and then collapse and unconsciousness.
Exposure has also been linked to pulmonary toxicity (Jo et al., 2013).
Excessive and prolonged exposure can be fatal (PHE, 2019).
Being lighter than air, methane displaces the oxygen present, which is
extremely serious in confirmed spaces as it will displace the oxygen
from the ceiling downward. So, all confined spaces or low-lying areas,
such as inside sedimentation or aeration tanks emptied for mainte-
nance, especially when covered, should be checked before entry using
a gas monitor to detect the presence of methane and sufficient oxygen
(i.e., >18% oxygen), especially if there is residual wastewater or sludge
present (CCOHS, 2023).
Global carbon dioxide emissions in 2022 from fuel combustion
and industrial processes was 36.8 Gt (giga tonnes), while 589 Mt
(million tonnes) of methane were released — 60% from manmade and
40% from natural sources (IEA, 2023). Although methane is far less
abundant in the atmosphere than carbon dioxide at 1.91 compared to
417 ppm, respectively, in 2022, it has a significantly higher global
warming potential (GWP). Current GWPs for methane vary over time
at 84–87 over 20 years, falling to 27–36 over 100 years (UNEP,
2021). Due to the higher emission levels, nearly all climate action has
focused on reducing carbon dioxide emissions, although there is
growing concern over the role of methane in global warming and a
general belief that more attention should be paid to reducing meth-
ane emissions overall. It is now thought that about half of the 1.1oC
56 Anaerobic Wastewater Treatment

rise above pre-industrial temperatures is due to methane. Another

factor is that methane has a relatively short half-life of 12 years com-
pared to carbon dioxide at 300–1,000 years, so action on methane,
with its high GWP and short half-life, could slow down global warm-
ing much faster in the short term than trying to focus solely on car-
bon dioxide reduction. Ocko et al. (2021) have predicted that
removing 300 Mt of methane, which is equivalent to sequestering
25.8 billion tonnes of carbon dioxide over 20 years, could reduce
global warming to 0.22°C by 2050, which would dampen the effects
of global-warming-induced climate change.
Methane-removal technologies can be divided into those based
on chemical oxidation or biological assimilation and have been
reviewed by Wang and He (2023). However, in wastewater treatment
and other waste processes, anaerobic digestion is used to capture car-
bon from the substrate in the form of methane, which is then ther-
mally oxidized as a fuel releasing carbon dioxide. The amount of
methane in the atmosphere has doubled since pre-industrial times,
with the rate of release steadily increasing since 1980. So, while it is a
key driver of global-warming-induced climate change, it is important
to exploit methane’s enormous potential as a valuable and sustainable
resource (UNEP, 2021). Using methane as an energy source reduces
overall greenhouse gas impact and is accepted as a carbon-capture
method.

3.3 Hydrogen Sulfide

3.3.1 Formation
The production of hydrogen sulfide is the most common and
well-known manifestation of anaerobiosis. Sulfide can be produced
by anaerobic microorganisms in two ways: (1) Protein is broken
down to amino acids, and those that contain sulfur, such as cysteine,
cystine, and methionine, are degraded further, with sulfide being
produced. Most anaerobic bacteria can produce sulfide from protein,
including Proteus, Bacteroides spp., and some Clostridium spp.
Although all can grow anaerobically, only Bacteroides spp., which can
 Methane and Other Volatile Products of Digestion 57

be present in feces at concentrations of up to 1010 g–1, are obligate

species. (2) However, in wastewater treatment systems, most sulfide
is produced from sulfate reduction by the anaerobic sulfate-splitting
bacterium Desulfovibrio desulfuricans, although species of the genus
Desulfotomaculum are also routinely isolated from digesters. The
former species is present in low numbers in sewage (60–600 mL–1)
but rapidly increases on storage up to concentrations in excess of
100,000 mL–1 after 14 days.
Sulfate reducing bacteria (SRB) only utilize a restricted range of
carbon compounds, such as lactate and malate, and rely on the meta-
bolic products of other anaerobic bacteria that can utilize more com-
plex organic compounds (Fig. 3.2). SRB are found in a wide range of
anaerobic environments, where there is a supply of sulfate, which they
utilize instead of oxygen for respiration, and organic matter to pro-
duce compounds such as lactate. Sulfate is a major ion in seawater,
and bacterial sulfate reduction is an important reaction in anoxic
estuarine and marine environments. When heavy metals are present in
the sediment and where sulfate-reducing bacteria are active, then the
sulfide produced reacts to form insoluble metal salts; the black discol-
oration so often associated with anaerobic sediments in rivers is due
largely to the formation of ferrous sulfides (Sec. 8.2.1). If no metals

(a) (b)

Figure 3.2. (a) Sulfide formation during anaerobic digestion and (b) the dissimila-
tory sulfate reduction pathway, including the main enzymes responsible (Jung et al.,
2022). Reproduced with permission of Elsevier Publishing.
58 Anaerobic Wastewater Treatment

are present, the sulfide escapes into the water column or the atmos-
phere as hydrogen sulfide gas (H2Sg).
The sulfide can be used as a source of energy by SRB or as an
electron donor by some of the photosynthetic bacteria. The nature of
the sulfides produced during anaerobic digestion is dependent primar-
ily on the temperature, pH, and sulfate loading, with dissolved sulfides
S2–, HS–, and H2Saq produced along with gaseous hydrogen sulfide gas.
The interaction between these forms of sulfide is shown below:

H2Saq ↔ HS– + H+(3.17)

HS– + H+ ↔ S2– + 2H+(3.18)
H2Saq + H2Sg (3.19)

The production of hydrogen sulfide is limited by the sulfate con-

centration in the wastewater or waste, which can range between
20 and 200 mg L–1 in domestic wastewater and 0.3 to 2.3% (wt%) in
sewage sludge (Dewil et al., 2008; Jung et al., 2022). The higher the
sulfur content of the waste stream, which comes mainly from proteins
(Tian et al., 2020), the greater the concentration of sulfide potentially
produced. Expected hydrogen sulfide gas in biogas is normally 0.1 to
2% (v/v). Sulfides can be toxic to a number of anaerobes, especially
methanogens, which can affect process operation, including the com-
plete inhibition of the methanogenic step. Depending on actual oper-
ating conditions, sulfide inhibition of methanogenic activity has been
reported at concentrations of between 100 and 800 mg L–1 as total
dissolved sulfides (S2– + HS– + H2Saq ) and between 50 and 400 mg L–1
of hydrogen sulfide gas. This is exacerbated by SRB competing directly
with methanogens for key substrates such as acetate and hydrogen
(Muyzer and Stams, 2008). The metabolism of the SRB has been
extensively studied and reviewed (Li et al., 2017; Zhang et al., 2022).

3.3.2 Health and Environmental Effects

Hydrogen sulfide (CAS No. 7783-06-4) is a colorless gas with a smell
of rotten eggs at low concentrations, which becomes less obvious
as the concentration increases, as it paralyzes the olfactory senses.
 Methane and Other Volatile Products of Digestion 59

The ability to detect the gas is lost at between 70 and 140 mg m–3
(50–100 ppm), so odor is not a safe indicator of overexposure. The
mean odor threshold for the general population is just 0.0063 mg m–3
(0.0045 ppm) with safe concentrations <0.18 mg m–3 (<0.13 ppm),
although lower values are recommended for continuous exposure.
WWTPs and the sewerage system are potentially high-risk areas for
hydrogen sulfide exposure, with concentrations in excess of 700 mg m–3
(≡502 ppm) reported (Locey, 2005). At low concentrations, it can
cause eye irritation and tissue damage, dizziness, headaches, nausea,
and respiratory problems. However, at high concentrations, it causes
almost instant unconsciousness and rapid asphyxiation. The gas is
heavier than air and tends to accumulate in underground tanks and
manholes, where it displaces oxygen, leading to a highly toxic and
potentially explosive environment, with concentrations in air above
4.3% being potentially explosive. Multiple deaths have occurred in
underground pumping stations, sludge wells, sewers, and even septic
tanks, so gas alert monitors should always be worn when working in
confined spaces at the treatment plant, where the possibility of expo-
sure to hydrogen sulfide, methane, carbon dioxide, carbon monoxide,
or ammonia is possible. All potential risk areas should be well-vented,
and warning signs posted with access restricted to those with gas alert
monitors and, if necessary, a positive-pressure, self-contained breath-
ing apparatus (Centre for Disease Control, 1977).
During the combustion of biogas, any hydrogen sulfide present is
burnt to form sulfur dioxide:

H2S + 1.5 O2 → SO2 + H2O(3.20)

In the atmosphere, sulfur dioxide is oxidized to sulfur trioxide,

which causes sulfuric acid to form when absorbed into water, causing
acid rain.

3.3.3 Corrosion
Concrete corrosion can occur at dissolved sulfide concentrations as
low as 0.1 mg S L–1, becoming severe at 2.0 mg S L–1 (Hvitved-
Jacobsen et al., 2002). The release of hydrogen sulfide gas depends
60 Anaerobic Wastewater Treatment

on the pressure, temperature, and pH. When produced in sewers by

SRB, the gas can cause severe corrosion of the concrete pipework.
The slightly acidic gas is absorbed into condensation water, which
collects at the top or crown of the sewer or on its side walls. Here, the
acidophilic sulfur oxidizing bacteria (SOB) in the surface biofilm,
which are able to tolerate pH levels of <1.0, oxidize the hydrogen
sulfide to sulfuric acid using atmospheric oxygen (Figs. 3.3 and 3.4):

H2S + O2 Acidithiobacillus thiooxidans → H2SO4 + energy (3.21)

The sulfuric acid reacts with the alkalinity in the concrete to

form a range of calcium salts, predominantly gypsum (CaSO4·2H2O)
(Eqs. (3.22), (3.23), and (3.24)) and ettringite, chemically known as

Figure 3.3. Crown erosion in concrete sewers due to microbial sulfide production,
where SRB are sulfate reducing bacteria, SOB are sulfide oxidizing bacteria, and MA
are methanogenic archaea (Zhang et al., 2023). Reproduced with permission of
Elsevier Publishing.
 Methane and Other Volatile Products of Digestion 61
(a) (b)

Figure 3.4. An example of crown erosion in concrete sewers in Sydney, Australia. (A) Internal upstream view of a sewer showing
erosion of the concrete ceiling. Sewer dimensions are 2 m (height) × 3 m (width) with a flow depth of 0.8–1 m. (B) Close-up of
the corroded ceiling surface. (C) Scrapings of the corrosion layer. (D) Scanning electron microscopy image of the corrosion scrap-
ings showing calcium salt crystals and acidophilic sulfur-oxidizing bacteria (Cayford et al., 2012). Reproduced with permission of
the Australian Centre for Water and Environmental Biotechnology. https://acweb.uq.edu.au/
62 Anaerobic Wastewater Treatment

calcium sulfoaluminate (3CaO·Al2O3·3CaSO4·32H2O) (Eq. (3.25)).

This leads to the breakdown of the concrete structure, making
it weaker, leading to structural destabilization and cracking. Over
time, the structural stability of the concrete pipe can become so weak-
ened that it eventually collapses (Li et al., 2017; Zhang et al., 2023;
Hvitved-Jacobsen et al., 2013).

H2SO4 + CaCO3 → CaSO4 + H2CO3(3.22)

H2SO4 + Ca(OH)2 → CaSO4 + 2H2O(3.23)
  H2SO4 + CaO⋅SiO2⋅2H2O → CaSO4 + Si(OH)4 + H2O(3.24)
3CaSO4 + 3CaO⋅Al2O3⋅6H2O + 26H2O → 3CaO⋅Al2O3⋅
3CaSO4⋅32H2O(3.25)

Crown erosion is particularly a problem in sewers receiving heated

effluents, with wastewaters containing high sulfur content or suffer-
ing from saline intrusion and sewers that are inadequately vented. The
problem is extremely critical for sustaining sewer infrastructure, with
annual damage estimated at US$100 million in Australia and a stag-
gering US$14 billion in the United States (Brongers et al., 2002;
Gutierrez et al., 2016).
Corrosion-resistant pipe materials, such as vitrified clay or plas-
tics, prevent corrosion in medium size sewers. In larger diameter
sewers, where concrete is the only possible material, corrosion is
reduced by ventilation, which expels the hydrogen sulfide and
reduces condensation. Control methods include chemical addition
to reduce sulfide formation or the removal of H2Saq after its forma-
tion. Inhibition of SRB is achieved using biocides, or the sulfides can
be oxidized by oxygen injection or the addition of nitrate salts.
Precipitation using iron salts converts the sulfides present into metal
sulfides, which are insoluble, while increasing the pH by the addi-
tion of an alkali such as NaOH or Mg(OH)2, reduces the H2Saq in
the waste stream wastewater that can be converted to H2Sg. The
addition of chemicals can, however, result in problems with the bio-
logical oxidation of wastewater at the WWTP, especially where acti-
vated sludge is used. Chemical addition is a continuous process,
 Methane and Other Volatile Products of Digestion 63

making it extremely expensive in the medium to long term. Another

alternative is coating concrete sewers with a protective layer, which
is expensive and technically difficult, or replacing sewers with more
corrosion-resistant concrete. Control strategies have been compared
by Zhang et al. (2023), who also explore future options. Hydrogen
sulfide is also highly corrosive to metal parts involved in handling
and processing biogas.
There are numerous post-treatment processes for the removal of
hydrogen sulfide gas in biogas, and these are explored in Sec. 8.2,
with in-situ sulfide control particularly of interest as it is cheaper
than adding a post-digestion process, although the in-situ technolo-
gies are at an early stage of development (Jung et al., 2022). The
easiest method of reducing sulfur in sewage sludge is to ensure the
sludge is dewatered before it becomes anoxic or anaerobic, thus
ensuring soluble sulfate is discharged with the effluent stream
(Dewil et al., 2008).

3.4 Ammonia
Nitrogen is normally absent from biogas and, if detected, is indicative
of denitrification or an air leak. Ammonia can be produced during
anaerobic digestion by the microbial reduction of proteins, and if in
excess to nutritional requirements of the anaerobic microbiota, will be
present in the biogas. Its presence can cause a number of operational
problems, including process inhibition, reduced biogas production,
reduced COD removal, malodor, and a reduction in the calorific
value of biogas and so its energy density. It also results in corrosion
to gas-processing equipment, including pipes and valves, due to react-
ing with H2O to form a strong base, reducing efficiency and the life
expectancy of equipment. During combustion, the ammonia in
biogas is converted into polluting NOx, which are also greenhouse
gases, and released into the environment (Strik et al., 2006).
Ammonia is essential to anaerobic microorganisms for the synthe-
sis of amino acids, proteins, and nucleic acids for bacterial growth,
and so should be in excess of 30–70 mg N L–1 but <200 mg N L–1 to
64 Anaerobic Wastewater Treatment

maintain optimal activity, although, at higher concentrations, it

becomes increasingly inhibitory. Ammonia also helps to neutralize
organic acids produced during fermentation, helping to maintain a
more neutral pH. Ammonia specifically inhibits the methanogenic
phase, with reported critical threshold values between 1,500 and
7,000 mg L–1 (Rajagopal et al., 2013). Inhibition is thought to be
linked to the concentration of free ammonia nitrogen (FAN) rather
than total ammonia nitrogen (TAN) (Jiang et al., 2019). Where there
is a high concentration of ammonia in the feedstock to the reactor,
then it begins to appear in the biogas, with concentrations of up to
445 ppm recorded (Strik et al., 2006).
In the liquid phase, ammonia is present either as ionized ammonia
(NH4+) or free ammonia (NH3) (Eq. (3.26)).

NH3 + H+ ↔ NH4+(3.26)

The relative fraction of each is dependent on both pH and tem-

perature (Fig. 3.5):
−1
 
 10 − pH  (3.27)
FAN = TAN × 1 +
 
−  0.09018+
2729.92  
 T (K )  
10 
Where FAN and TAN are the free and total ammonia nitrogen
concentrations (mg L–1) in the digestate, respectively, and T (K) is the
temperature in oK. The more protein that is available, then the greater
the pH leading to an increase in FAN, with temperature a key reason
why ammonia inhibition is more often a problem in thermophilic
than mesophilic digesters. The inhibitory process and adaptation
of the microbial population to high ammonia concentrations are
explored in depth by Jiang et al. (2019), including examples of avail-
able mitigation strategies, including adjusting the C:N ratio of the
feed, air stripping ammonia gas from the digestate, and chemical
precipitation of ammonia with magnesium and phosphorus to form
struvite (MgNH PO4∙6H2O).
The role of ammonia in anaerobic digestion, including the effects
on methanogens, is explored in detail in Sec. 5.4.1.
 Methane and Other Volatile Products of Digestion 65

Figure 3.5. The general variation between the proportion of unionized to free
ammonia at varying pH and temperatures.

3.5 Volatile Organic Compounds and Terpenes

3.5.1 Volatile Organic Compounds
Volatile organic compounds (VOCs) are a wide group of organic
compounds with a high vapor pressure, meaning that at ambient tem-
perature and pressures, they become gases. The main sources of
VOCs are vehicle emissions, industrial processes using solvents, petro-
leum industries, chlorination, food extraction, paints, laundry print-
ing, pharmaceutical industries, pesticide manufacture, and, increasingly,
wildfires. The situation at WWTPs is more complex and depends on
the nature and source of the wastewater streams being treated, while
VOCs can be removed or generated during the treatment process,
especially during anaerobic steps.
During anaerobic digestion, non-methane VOCs are produced in
large quantities. These include terpenes, sulfur-containing compounds
66 Anaerobic Wastewater Treatment

(e.g., dimethyl sulfide, carbon disulfide), oxygenated compounds

(e.g., ethanol, acetone), halogenated hydrocarbons (e.g., ethyl chlo-
ride, chloroethylene, trichloroethylene), and aromatic hydrocarbons
(e.g., benzene, toluene, xylene, and ethyl benzene). A breakdown of
VOCs found at an anaerobic digestion plant is given in Fig. 3.6.
VOCs are a concern, as many of them are known carcinogens,
with the remainder generally associated with other health-related
problems. Environmental problems include odor generation during
digestion, mainly due to sulfur-containing compounds and terpenes;
ozone formation in the troposphere, resulting in photochemical
smog; and the acceleration of ozone depletion in the stratosphere.
VOCs can also absorb surface infrared radiation, enhancing global
warming, although they have a short atmospheric life (Song et al.,
2019; David and Niculescu, 2021). The measurement of VOCs, espe-
cially in terms of air pollution monitoring, is based on four common
and easily measured VOCs: benzene, toluene, ethylbenzene, and
xylene, collectively known as the BTEX suite. BTEX monitors are
now widely available and can be located around the WWTP or within
buildings where digestion or sludge handling occurs.
While odors emitted during anaerobic digestion are primarily
associated with volatile sulfur compounds, especially hydrogen sulfide,
other non-sulfur VOCs also contribute to odor impact. These include
amines, ketones, and a range of aromatic compounds, including
indole, skatole, ρ-cresol ethylbenzene, and toluene (Chen et al.,
2004). Comparing VOC emissions from six different WWTPs
in Australia, Fisher et al. (2017) found different sludge processing
configurations resulted in distinct types and concentrations of VOCs,
with the widest range and highest concentrations emitted from dewa-
tered and stored biosolids. The most important odors detected were
ρ-cresol and butanoic acid, with trimethylamine, indole, and phenol
emitted from the dewatered and stored biosolids, and volatile fatty
acids associated with the digester were also problematic. The authors
concluded that effective odor management requires the monitoring
of both volatile sulfur compounds and selected non-sulfur VOCs.
Zheng et al. (2020) examined the release of VOCs at an anaero-
bic digestion plant processing 300 tons of food waste per day over a
period of 12 months and found that VOCs were released from all
 Methane and Other Volatile Products of Digestion 67

(a) (b)

(c) (d)

(e)

Figure 3.6. Seasonal concentration of VOCs from various process steps at a food
waste anaerobic digestion system (i.e., SR (sorting crushing room), HH (hydrother-
mal hydrolysis unit), BP (biodiesel production unit), AD (anaerobic digestion unit))
showing (a) terpenes, (b) sulfur-containing compounds, (c) oxygenated compounds,
(d) halogenated compounds, and (e) aromatic hydrocarbons (Zheng et al., 2020).
Reproduced with permission of Elsevier Publishing.
68 Anaerobic Wastewater Treatment

Figure 3.7. Process flow chart of a food waste anaerobic processing plant in
Suzhou, China. Asterisks indicate VOC sampling points used in Fig. 3.4 (Zheng
et al., 2020). Reproduced with permission of Elsevier Publishing.

areas of the process (Fig. 3.7). Emissions varied seasonally, being

highest in spring and lowest in winter, with concentrations of 3.49 to
104 mg m–3 recorded in the hydrothermal hydrolysis unit, 8.97 to
103 mg m–3 in the sorting/crushing room, 6.21 to 102 mg m–3 from
the anaerobic digestion unit, and 2.01 to 102 mg m–3 in the biogas
production unit (Fig. 3.6). Oxygenated compounds and terpenes
accounted for >98% of total VOC emissions. They concluded that
while emissions were below those associated with non-carcinogenic
risk, workers should minimize the time spent in the hydrothermal
hydrolysis unit. Some 380 different VOCs were recorded in a study
comparing sewage sludge, thickened sludge, digested sludge, and
dewatered sludge at a WWTP in France, of which 81 compounds
were found in all sludge types (Haider et al., 2022). Potential emis-
sions of VOCs increased as the organic matter content rose, although
dewatering digested sludge to a dry solids (DS) content of >60% did
reduce emissions of sulfur compounds.
The use of VOCs produced by microorganisms (mVOCs) during
anaerobic digestion as an early warning system of potential system
failure has been proposed by Nie et al. (2023). Currently, physico-
chemical parameters are used as indicators, such as pH, ORP, ammo-
nia, alkalinity, and volatile fatty acids, but are not fully reliable. Using
actual microbial metabolites as indicators could be far more precise,
especially when used in conjunction with existing early warning
parameters. They identified a number of potential pathways during
digestion where mVOCs are produced and a number of potential
candidate compounds (Fig. 3.8)
 Methane and Other Volatile Products of Digestion 69
Figure 3.8. Potential pathways for the production of microbial VOCs during anaerobic digestion, which could be used as indica-
tors of performance and operational stability (Nie et al., 2023). SAO: synoptic acetate oxidation; TCA cycle: the tricarboxylic acid
cycle. Reproduced with permission of Elsevier Publishing.
70 Anaerobic Wastewater Treatment

3.5.2 Terpenes
Terpenes are odorous compounds associated with specific plants such
as citrus and coniferous species that find their way into the sludge feed.
In biogas, two terpenes are of importance, ρ-cymène and
D-limonène, which are found in hundreds of different foods. For
example, ρ-cymène (C10H14) is found in many widely consumed prod-
ucts, such as dairy produce, carrots, orange juice, raspberries, lemon
oil, and many spices, while D-limonène (C10H16) is found in many
plants, such as fennel, dill, celery, and lemons. It is also widely used
as a food additive and in body-care products, and more recently, as a
substitute for petroleum-based solvents in detergents and paints
(Arrhenius et al., 2017).
The increasing use of food waste co-digested with wastewater
sludges to increase biogas yields has led to an increase in terpene levels
in the gas. Terpenes are only a problem in biogas when it is converted
to biomethane and must be removed before being pumped into the
gas network. Terpenes can corrode the polyethylene pipework as well
as rubber and plastic gaskets (Arrhenius et al., 2017), result in con-
densation within the pipework, and also mask the sulfurous odor
tetrahydrothiophene (THT), which is added to natural gas for leak
detection. They can also cause workplace problems, both in terms of
odors and potential health risks. Terpenes are also flammable, so if
contaminated biogas is used in normal on-site CHP systems for heat
recovery and electricity generation, they are destroyed as well as add-
ing to the overall energy generated.
Five terpenes are regularly monitored in biomethane, α-pinène,
β-pinène ρ-cymène, D-limonène, and 3-carene. They are analyzed
using either a gas chromatography-thermal conductivity detector
(GC-TCD) or, more recently, a gas chromatography-flame ionization
detector (GC-FID) (Budiman et al., 2015). Sampling and analysis of
trace compounds in biogas and biomethane, including terpenes, are
reviewed by Rasi et al. (2006; 2007) and Lecharlier et al. (2022).
When food waste is the main source of digester feed, D-limonène is
the main terpene present (>90%), with ρ-cymène found in small con-
centrations (1%). Both the concentration of specific substrates, espe-
cially citrus fruits, the consumption of which varies seasonally and
 Methane and Other Volatile Products of Digestion 71

Figure 3.9. The concentration of main terpenes (mg m–3) produced at eight differ-
ent treatment plants using different proportions of food waste (%) in the feedstock
using either mesophilic (Mes.) or thermophilic (Term.) digestion. There is a linear
correlation between the proportion of food waste in the feedstock and the concentra-
tion of terpenes in the biogas of up to 70%, after which the concentration of terpenes
levels out (Arrhenius et al., 2017). Reproduced with permission of Energiforsk AB,
Sweden. www.energiforsk.se

digester temperature determine the dominant terpene as well as the

concentration. During digestion, D-limonène is largely converted to
ρ-cymène, with the latter making up (>90%) of terpenes in the diges-
tate. In thermophilic digestion, comparatively more D-limonène is
normally found in biogas compared to mesophilic digestion, where
the conversion of D-limonène to ρ-cymène should be more complete,
resulting in higher concentrations of ρ-cymène evaporating into the
biogas (Fig. 3.9).
Arrhenius et al. (2017) reported terpene concentrations in biogas
generated from different percentages of food waste in the feedstock
varied between 360 and 1,650 mg m–3. After treatment, this was
reduced to 20 to 240 mg m–3 after upgrading the biogas to biometh-
ane using a variety of removal systems. Removal efficiency varied
with pressure swing adsorption (PSA) (100%) and water scrubbers
(83–96%), achieving higher removal efficiencies for terpenes than
amine scrubbers (61–85%) (Chap. 8).
72 Anaerobic Wastewater Treatment

3.6 Siloxanes
Organosilicons (siloxanes) are a large group of water-soluble man-
made synthetic polymers utilizing the linkage between silicon atoms
and an organic group, usually a methyl group. They are widely used
throughout society in cosmetics and toiletries, detergents, paper coat-
ings, anti-foaming agents, textile manufacture, sealants, and cooking
oils, and as food additives generally ending up concentrated in waste-
water sludge (McBean, 2008; Paglini et al., 2022).
Biogas can become contaminated by volatile organic silicon com-
pounds (VOSiCs), and when these are oxidized during biogas com-
bustion, microcrystalline silicon dioxide is formed, which has similar
properties to glass. This can severely damage engines and turbines,
increasing wear and blocking valves and pistons. Volatile methyl silox-
anes (VMSs) are the most common VOSiCs found in digester gas,
and these are shown in Table 3.5. The combustion of one of the com-
monest occurring VOSiCs in biogas, octamethylcyclotetrasiloxane
([(CH3)2SiO]4), is shown in Eq. (3.28) with silicon dioxide formed.

[(CH3)2SiO]4(g) + 16O2 → 4SiO2(s) + 8CO2(g) + 12H2O(g)(3.28)

Table 3.5. Chemical composition and key physicochemical properties of the most
frequently occurring volatile methyl siloxanes in biogas (de Arespacochaga et al.,
2015). Reproduced with permission of Elsevier Publishing.
Water
Molecular Boling solubility
Chemical weight point (mg/L at
Abbreviation formula (g/mol) (°C) 25 °C)

Hexamethyldisiloxane L2 C6H18OSi2 162 107 0.93

Octamethyltrisiloxane L3 C8H24O2Si3 237 153 0.034
Decamethyltetrasiloxane L4 C10H30O3Si4 311 194 0.00674
Dodecamethylpentasiloxane L5 C12H36O4Si5 385 232 0.000309
Hexamethylcyclotrisiloxane D3 C6H18O3Si3 223 135 1.56
Octamethylcyclotetrasiloxane D4 C8H24O4Si4 297 176 0.056
Decamethylcyclopentasiloxane D5 C10H30O5Si5 371 211 0.017
Dodecamethylcyclohexasiloxane D6 C12H36O6Si6 444 245 0.005
Trimethylsilanol TMOH C3H9SiOH 90 99 42,600
 Methane and Other Volatile Products of Digestion 73

The highest concentrations of siloxanes are found in biogas

derived from domestic and municipal wastewaters, with concentra-
tions as high as 41 ppm. Lower values are recorded from landfill gas
(4–9 ppm), with the least amount recorded in farm gas (Arnold and
Kajolinna, 2010; Wasajja et al., 2020). Removal of siloxane in biogas
is discussed in Sec. 8.5.

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 Section B

Flow-Through (Conventional)
Anaerobic Treatment Processes
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 4
Combined Anaerobic Wastewater
Treatment Systems

4.1 Introduction
Anaerobic treatment is a biological process in which the organic frac-
tion of wastewater sludge, comprising proteins, carbohydrates, and
lipids, is degraded in the absence of oxygen to methane and carbon
dioxide by bacteria and archaea. The process occurs quite widely in
natural environments, such as lake sediments, marshes, peat bogs, and
even in the rumen of certain herbivorous animals.
The first direct application of anaerobic treatment of wastewater
is attributed to Louis H. Mouras of Vesoul in France, who developed
a flow-through cesspool (c. 1860) in which he claimed, “sewage sol-
ids are liquified by fermentation.” He applied for a patent on 22
September 1881, with the design submitted very similar to the basic
septic tank of today (Fig. 4.1). By 1895, digester gas, also known as
biogas or methane, was being collected and used as fuel. Donald
Cameron constructed a large septic tank in Exeter (United Kingdom
(UK)) and was able to use the biogas for lighting the area around
the treatment plant (Fig. 4.2). By the turn of the century, it had
become common practice to incorporate a digestion chamber within
sedimentation tanks, with the Travis hydraulic tank and the Imhoff
tank the most widely adopted. The Imhoff tank, in particular, was
installed at most small to moderate-sized treatment plants and is still
to be found today in older wastewater treatment plants (WWTPs) in

81
82 Anaerobic Wastewater Treatment

Figure 4.1. The actual design used in the patent application (1881) for the first
anaerobic wastewater treatment system designed by Louis Mouras in France. It was
described as a flow-through cesspool, where A is the fermentation tank, B is the inlet
pipe, and C is the outlet pipe to the sewer.

Figure 4.2. It was the English engineer Donald Cameron who coined the term
“septic tank,” with his patented system used to collect biogas. In 1895, he was able
to produce enough biogas from his community septic tank to power the gas street-
lights in Exeter.
 Combined Anaerobic Wastewater Treatment Systems 83

Ireland. While few have been constructed in-situ since 1950, they are
still commercially available as packaged units. The development of
specific digesters for the anaerobic breakdown of sludges began in the
early 1920s, with their use restricted to large cities. During World War
II, there was renewed interest in biogas production from wastewater
sludges due to fuel shortages for transport, but very little develop-
ment occurred after the war. However, there has been a resurgence of
interest in anaerobic digestion in the past 20–25 years, especially in
France and China, with much fundamental research and development
work being done, leading to the creation of new, more robust and
reliable, digester, and anaerobic reactor designs. The basic flow-
through system can be separated into two categories: systems that
combine settlement with digestion and separate systems built for
digestion only (Fig. 4.3).
Combined systems of settlement and digestion are restricted to
situations where there is only a small volume of wastewater to be
treated. The combined system provides primary settlement and then
sludge stabilization by digestion, all within a single unit. By reducing

Figure 4.3. The principle conventional and high-rate anaerobic wastewater treat-
ment systems currently in use.
84 Anaerobic Wastewater Treatment

the volume of sludge produced and storing it in such a way so as

not to impede the settlement process, such systems are ideal for single
houses, hotels, restaurants, or small communities. Anaerobic lagoons
can also be categorized as combined systems, although their use
in Europe is largely restricted to strong organic wastes from the food
processing and agricultural industries. The selection, installation,
and maintenance of small wastewater treatment systems, including
combined anaerobic processes, are covered by a series of standards (EN
12566) by the Comité Européen de Normalisation (CEN) (Table 4.1).

Table 4.1. The European Committee for Standardization (CEN) Standards for
septic tanks and small treatment systems. CEN is the provider of standards for all 30
Member States of the EU, including EN 12566, which is a series of standards on
small wastewater treatment systems. https://standards.cen.eu/
Standard Title Function Reference
EN 12566-1 Prefabricated septic Design, installation and CEN 2000;
tanks testing of septic tanks 2003
EN 12566-2 Soil infiltration systems Design, installation of CEN 2005
percolation areas
EN 12566-3 Packaged and/or site Requirements and test CEN 2009
assembled domestic methods for packaged
wastewater secondary treatment units
treatment plants
EN 12566-4 Septic tanks assembled Assembly, installation and CEN 2007
in situ from pre- testing of site-assembled
fabricated kits septic tanks
EN 12566-5 Pre-treated effluent Design, construction, CEN 2008
filtration systems installation and testing
of secondary treatment
filtration units for
septic tanks
EN 12566-6 Pre-fabricated treatment Test methods and evaluation CEN 2013a
units for septic tank for conformity for
effluent secondary treatment units
used with septic tanks
EN 12566-7 Pre-fabricated tertiary Treatment requirements and CEN 2013b
treatment units test methods for packaged
and site assembled
tertiary treatment units
 Combined Anaerobic Wastewater Treatment Systems 85

4.2 Septic Tanks

Septic tanks are often confused with cesspools or cesspits. A cesspool
is an underground chamber constructed solely for the reception and
storage of wastewater, with no treatment taking place. A cesspool is a
storage tank that requires periodic emptying and is not intended as
a septic tank in which decomposition of the settled material occurs.
It is important to differentiate between the two, as these terms are
used interchangeably in many areas. A cesspit is the predecessor of the
cesspool, which allowed the liquid fraction to soakaway into the soil
layer. Made of brick or stone with gaps to allow liquid to percolate
into the surrounding soil, they were generally circular in design and
approximately 2 m in depth. They were common in rural areas but
were frequently associated with contamination of nearby wells and
boreholes. They were made illegal in the UK by the Public Health Act
of 1936. Where a septic tank or cesspool is not watertight, it acts as a
cesspit and is thus illegal.

4.2.1 Cesspools
Cesspools can be constructed out of concrete, plastic, or fiberglass,
and according to the British Code of Practice (BSI, 1972; 1983),
must be: (a) impervious (i.e., watertight), (b) not be able to overflow,
(c) have a minimum capacity of 18 m3 or 45 days retention for two
people assuming a per capita water usage of 180 L d–l, (d) be con-
structed so that it can be completely emptied, (e) adequately venti-
lated, and, most importantly, (f) adequately covered to ensure safety.
Clearly, cesspools are only used where no other form of treatment is
possible, and the need to have them regularly emptied means that
they are the most expensive form of treatment for domestic dwellings
in terms of both capital and operational costs. The only improvement
in this system can be achieved by adopting water-saving improve-
ments that reduce the volume of wastewater discharged. There are
advantages, however, such as no power requirement, no quality con-
trol required, no mechanism that can go wrong, the process is not
injured by intermittent use, and as there is no effluent discharge,
there is no immediate environmental impact. The major limitation on
86 Anaerobic Wastewater Treatment

the use of cesspools is the cost of emptying, although the construc-

tion of large underground storage tanks can be both difficult and
expensive. Sealed prefabricated units are available up to 54 m3 of
capacity and are cylindrical in design (12,000 mm × 2,740 mm) and
must be embedded in a minimum of 150 mm of concrete. Several
units can be used in series to increase capacity. The advantages and
disadvantages of cesspools have been examined by Mann (1979).
One area where storage tanks are still widely used is on farms.
Storage of farm effluents is expensive, and ideally, raw effluent should
be spread immediately onto the land. However, this is impossible
because of seasonal crop cycles and, at certain times of the year, the
risk of surface runoff causing pollution. The use of slurry tanks, both
of underground and above-ground construction, is widespread, and
the design of such systems is discussed in (DAFM, 2022).

4.2.2 Septic Tanks

Septic tanks are essentially a chamber in which settleable solids settle
out of suspension to form a sludge, which undergoes anaerobic break-
down. The process provides partial treatment only and cannot pro-
duce an effluent equivalent to a normal WWTP but is similar in
composition to settled sewage (Table 4.2). Therefore, further treat-
ment is required either by a secondary treatment process or by the
provision of a percolation (i.e., irrigation) area in which the sewage
percolates into the soil via a system of underground distribution pipes

Table 4.2. Variation in the strength of the effluent from 28 Irish septic tanks
(n = 84)
Mean Standard deviation Minimum Maximum

pH 7.0 0.24 6.3 7.5

BOD (mg L–1) 264 94.1 110 510
COD (mg L–1) 500 175.7 225 900
Suspended solids (mg L–1) 124 53.4 40 260
NH4–N (mg L–1) 55 21.4 16 106
Total phosphorus (mg L–1) 16.2 6.4 2.0 29.0
COD:BOD 1.9 0.21 1.3 2.4
BOD: biochemical oxygen demand; COD: chemical oxygen demand.
 Combined Anaerobic Wastewater Treatment Systems 87

and undergoes treatment by the formation of a biomat (Gray, 2022).

If the effluent from a septic tank is discharged directly to surface
waters, then a minimum dilution factor of 300–400 is required.
Although most commonly used for individual houses, they can be
used for small communities of up to 500 and are commonly used in
combination with a percolating filter, rotating biological contactor,
reed bed, or a submerged aerated filter system for small rural villages.
Septic tanks themselves require no power, and where subsurface
drainage is used, quality control can be confined to suspended solids
removal only to prevent blockage of the percolation pipework. They
are not adversely affected by intermittent use, have a very small head
loss, and can achieve 40–50% BOD (biochemical oxygen demand)
and 80% suspended solids removal.
With 50% of the global population living in rural areas, a large
portion of these households employ septic tanks or small-packaged
systems incorporating a septic tank section for the treatment of their
wastewater (Singh et al., 2019). In its simplest form, the septic tank
consists of a single chamber (Fig. 4.4a) with a single input and
output. They can be of any shape, although they are traditionally
rectangular and made from concrete comprising two or, frequently
in Scandinavia and China, three chambers (Fig. 4.4b). From 2015
to 2020, 10 million three-chambered septic tanks were installed in
China alone, with the final effluent used for irrigation (Tan et al.,
2021). New prefabricated units in plastic or fiberglass come in a vari-
ety of shapes, designs, and even colors. The tank consists of three
separate zones, a scum layer made up largely of emulsified fats that
floats on top of the clarified liquid, with a sludge layer in the base.
Wastewater enters and leaves the tank via T-shaped pipes that prevent
disturbing the scum layer or allowing solids or floatable material to be
carried out of the tank. As the wastewater moves through the cham-
ber, settleable solids gradually settle forming a sludge, and any fats or
buoyant material floats to the surface where they are retained by the
baffle edge of the T-pipe and form the scum layer. The scum layer,
although not vital to the successful operation of the tank, helps its
operation in three ways: it prevents oxygen transfer through the air-
water interface, insulates the anaerobic chamber by preventing heat
88 Anaerobic Wastewater Treatment

(a)

(b)

(c)

Figure 4.4. Examples of manufactured septic tanks: (a) Original single-chamber

system, (b) more common two-chamber system, and (c) GRP septic tank.
 Combined Anaerobic Wastewater Treatment Systems 89

loss, and by attracting and retaining fats, floatable material, including

raised solids from the sludge layer. It is in the sludge layer where
anaerobic decomposition takes place, with the organic fraction slowly
being degraded. Often, decomposition is incomplete, and the settled
material is only hydrolyzed and not broken down to methane. This
results in intermediate products of anaerobic digestion, such as short
chain fatty acids, being produced and slowly diffused back into the
clarified liquid to be discharged from the tank. This incomplete
anaerobic activity results in unpleasant odors, which the scum layer
helps to retain within the tank.
Methane production is inhibited by a number of factors in septic
tanks, most notably, low temperatures and insufficient sludge storage
capacity. Methane production in septic tanks is low, even in the sum-
mer, whereas in winter, it may cease completely, resulting in an overall
increase in sludge accumulation rate. However, gas formation occurs
even in the absence of methane formation, which can carry solids
back into the liquid phase and cause an increase in the rate of scum
accumulation. Most wastewaters contain a significant proportion of
non-degradable solids, and even if anaerobic digestion is highly effi-
cient, there will be a gradual increase in solids in the tank. As these
build up, the volume of the liquid zone in the chamber is reduced,
thus reducing the retention time of the wastewater and the degree of
settlement that is possible. If the discharge of solids to the next treat-
ment phase is to be minimized, then the septic tank must be regularly
desludged. Ideally, the floor of the chamber is sloped toward the inlet
end (Fig 4.4b), above which a manhole is situated into which a suc-
tion pipe can be lowered for sludge removal. Normally, they have a
flat bottom so that sludge accumulation tends to be deeper at the
outlet end of the tank (Fig. 4.4a). It is recommended septic tanks
should be desludged every 12 months, leaving a small quantity of the
sludge to re-seed the tank, ensuring rapid recommencement of anaer-
obic activity. In practice, it is easier for operators to completely empty
the tank, and it is felt that enough sludge will be left behind in the
corners of the chamber and adhering to the walls to ensure seeding.
If possible, the tank, which is designed to operate full, should be
90 Anaerobic Wastewater Treatment

refilled with water as soon as possible after desludging. This also pre-
vents the tank from rising up out of the ground if the soil is saturated
with water. In practice, the scum collapses as the sludge is pumped
out below. If this occurs, then the scum must also be pumped out to
prevent the overflow pipe becoming blocked as the tank refills. Also,
an excessively thick scum layer should be removed to prevent the inlet
and outlet pipes from becoming blocked. Where excessive scum for-
mation occurs, a grease trap should be installed.
When commissioning septic tanks, they should be filled with
water before use and tested for leaks by checking the water level over
a period of a few days. This is a common problem with prefabricated
concrete tanks, which come in two sections — a top and bottom,
often with a rubber seal that is placed between the two sections as
they are assembled. Although not strictly necessary, it is advantageous
if tanks can be seeded with a few liters of sludge when first commis-
sioned or a kilogram of cow or sheep dung added to help fermenta-
tion to begin. Commercial seeds are now available, which are easier
to handle and more convenient. There are a number of chemicals and
biological bacterial seeds advertised for septic tank problems. There is
no shortcut to effective tank care and the addition of yeast, enzymes,
and bacteria are not necessary for digestion within the tank. It is a
common fallacy that odors produced by septic tanks can be reduced
by cleaning the chamber using a proprietary cleaner. Under no cir-
cumstances should a tank be disinfected, as this will only result in even
worse odors being produced on recovery and inhibiting the degrada-
tion processes already taking place in the chamber. If odors are a
problem, then the tank should be desludged.

Septic tank design

The design and construction details of septic tanks are given by the
European standard EN12566 (Table 4.1), British Standard Institution
BS 6297 (BSI, 1983; 2007), and the Irish Environmental Protection
Agency (2009; 2021), although most countries have their own stand-
ards. The two most important design criteria for septic tanks are the
suitability of the land and the capacity of the tank. Many by-laws insist
 Combined Anaerobic Wastewater Treatment Systems 91

on minimum distances for the siting of tanks and percolation

areas from houses, wells, streams, and boundaries. These vary, but in
Ireland, no part of the percolation area should be closer than 10 m
from the nearest habitable building (or 4 m from the nearest road)
and 10 m from a ditch or stream (or 3 m from the boundary of the
adjoining site). Distances from wells vary from 30 to 60 m, depend-
ing on percolation characteristics, the minimum depth of subsoil
above rock, and the minimum depth of the water table (EPA, 2009).
The capacity and hence the retention time for settlement and sludge
digestion is calculated according to the number of people discharg-
ing. The minimum capacity of a septic tank must not be less than
2720 L, with the actual capacity calculated as:

C = (180P + 2000) liters (4.1)

where C is the capacity in liters, and P is the number of people dis-

charging into the system. It is quite common for people to purchase a
country cottage in which an elderly couple had lived for many years,
only to find that once modernized, with mains water, a new bathroom,
and a kitchen full of labor-saving devices, that the septic tank system
can no longer cope. For example, a disposal unit for household kitchen
waste (garbage grinder) results in a 30% increase in the BOD and a
60% increase in suspended solids discharged to the tank. Where these
are installed, the capacity of the septic tank must be calculated as:

C = (250P + 2000) liters  (4.2)

Therefore, in the design of a septic tank, the future size of the

family, number of bathrooms, visitors, and other potential develop-
ments must be included in the design. It is unacceptable to use
half values for children in such calculations as their water demands are
just as great as adults. In Ireland, for example, the capacity of the
septic tank is based on the number of people served, with two sizes
recommended. For 2–5 people, a tank of nominal capacity of 3 m3 is
required, or 4 m3 for 6–10 people. However, in practice, larger tanks
may be needed if water consumption is high or if the household
works from home. Some wastewaters are not suitable for discharge to
92 Anaerobic Wastewater Treatment

septic tanks, for example, inert, dense, or highly compactable solids.

Septic tanks are generally unsuitable for industrial wastewaters or agri-
cultural use.
While the majority of septic tanks are made from precast concrete
or are cast in situ, new prefabricated units in plastic or fiberglass
are now widely available and come in a variety of shapes, designs,
and even colors. The Klargester septic tank has a characteristic onion
shape (Fig. 4.4c). It is a three-chamber design that must be posi-
tioned vertically in the ground. To prevent misalignment, it must be
set in concrete, and the immediate site protected from traffic. The
wastewater enters at the base (zone 3), where settlement and sludge
storage occur. Settled wastewater is displaced upward into the second
chamber (zone 2), where the inclined base encourages further settle-
ment, with solids falling into the lower chamber through the periph-
eral slots. The wastewater passes into the final chamber (zone 1), from
where it is discharged via the outlet to the percolation area. These
plastic or fiberglass tanks are ideal where the location of the site does
not permit access by heavy-lifting equipment, such as a JCB machine,
required for moving concrete tanks. A standard plastic or fiberglass
septic tank can be easily carried and installed by two people.
Septic tanks are easy to uprate by adding extra chambers in series.
This reduces the effects of surge flows and excessive sludge accumula-
tion in the first chamber. Existing multi-chambered tanks can be
uprated by adding new septic tank units in parallel. The commonest
faults associated with septic tanks include: (i) Leaking joints when
tanks have been constructed from concrete sections or concrete rings.
It is important that septic tanks should be watertight to prevent con-
tamination of the groundwater and should be constructed, if possible,
without joints. (ii) Non-desludging is the commonest fault, resulting
in a reduced retention time and a stronger effluent due to less settle-
ment. The loss of solids under these circumstances can block pipes,
percolation areas, or increase loadings to secondary treatment units.
(iii) Blocked outlet pipes are frequently a problem because of the
scum layer becoming too thick or sludge physically blocking the out-
let pipe, which causes the tank to overflow. Tampons tend to become
suspended in the liquid phase of the tank and can easily be transferred
 Combined Anaerobic Wastewater Treatment Systems 93

into the final chamber and eventually into the percolation area and so,
if possible, should not be flushed along with nappies, sanitary and
incontinent pads, wipes, and cotton wool balls or pads. The insertion
of a circular mesh sieve into the final outlet T-piece can reduce
the loss of solids from the septic tank but must be regularly cleaned
to prevent blockage and overflow through the access manhole. In
Ireland, many systems have inadequate percolation areas or suitable
access for the sludge tanker, with desludging pipes occasionally having
to pass through the owners’ houses to reach the tank itself. The
design of percolation (sub-surface irrigation) areas and problems
associated with soil percolation have been reviewed by Gray (2022).

Operation
Little information is available on the operation of septic tank systems,
but on the whole, they are very robust. However, care should be
taken when using the following: Disinfectants should be used moder-
ately, as their bactericidal properties inhibit or kill the anaerobic bac-
teria, which can result in unpleasant odors being produced during
recovery. Phenol-based disinfectants can cause significant odors. It is
best to use disinfectants having free chlorine, as it reacts with the
organic matter in the sewage, rendering it harmless by the time it
reaches the tank. Caustic soda, which is often used to remove grease
from drains, can cause the sludge to flocculate and rise. Excessive use
can result in sludge passing out of the tank and blocking the percola-
tion area. Small amounts of acidic or alkaline cleaners do no lasting
harm and should be used in preference. Some strong cleaners can
upset the pH of the tank, which should be as near to neutral as pos-
sible. Under good operational practice, the tank will buffer itself. High
sodium concentrations in the water do not affect the septic tank sys-
tem directly but can impair the drainage properties of the soil. Those
who have water softeners and use soil percolation as a secondary treat-
ment process of septic tank effluents should take advice. Detergents,
especially alkyl benzene sulfonate, are known to inhibit the digestion
process, although, providing the tank capacity is sufficient, if normal
concentrations of detergents are used, the performance of the system
94 Anaerobic Wastewater Treatment

will not be impaired. Enzyme-based washing powders have no effect

on septic tank systems. Large flushes of water to the tank should be
avoided if possible to prevent scouring of the sludge unless the tank is
large enough to withstand them or a three-chambered system has
been installed. Wherever legally permissible, bathwater should be
diverted to a soakaway, as should rainwater and melted snow from
roofs and paved areas. Solid materials such as disposable nappies, cof-
fee grinds, bones, cigarette ends, and cat litter will not degrade in the
tank and should not be discharged to the septic tank as they will
reduce the volume of the settlement chamber very quickly and can be
difficult to remove due to compaction. Excessive amounts of fats, oils,
and greases should also be disposed of separately whenever possible,
or a grease trap installed before the septic tank.
If laundry wastewater exceeds 10% of the total flow to the septic
tank, then this should be treated separately using a treatment system
with a separate percolation area. Alternatively, it can be stored and
sprayed onto lawns during dry periods. Catering facilities such as res-
taurants produce a large quantity of grease that cannot be dealt with
by the system, so fats must be kept to a minimum, especially cooking
oils. A grease trap should be installed on the kitchen waste line as
close to the source as possible and before any foul water enters. Any
settleable solids will also be retained in the trap, which reduces its
efficiency and rapidly blocks it. So, where necessary, a dedicated dis-
posal line should be installed for grease and oils, or solids should not
be allowed to enter the drain. It is important that oils must be cooled
before entering the trap. High-protein wastewaters can also lead to
excessive hydrogen sulfide formation (Sec. 3.3).
Petrol and oil from car parking areas, garages, and petrol station
forecourts must be removed prior to septic tank or secondary treat-
ment. This is best done by the use of oil interceptors, which are
installed before the foul water from paved areas enters the system.
They are designed to ensure maximum separation with retention
times for adequate separation ranging from 6 to 20 minutes, depend-
ing on the density of the oil or petrol. Units are normally prefabri-
cated in plastic or glass fiber and vary in size from 2,000 to 54,000 L.
The design of the unit depends on the area served and the maximum
 Combined Anaerobic Wastewater Treatment Systems 95

expected rainfall per hour. They are also useful for containing spillages
of light-density liquids.

Maintenance
Maintenance of the septic tank, like all sewage treatment units, is
important in maximizing treatment efficiency and preventing pollu-
tion. The scum and sludge accumulation should be inspected twice a
year in systems serving more than one household, depending on its
volume. In many countries, it is a requirement to keep an up-to-date
record book noting inspection dates, observations, and details of
desludging. In the United States (US), the scum and sludge depth
should be measured twice a year. The depth of the sludge and the
thickness of the scum can be measured in the vicinity of the outlet
pipe in a single-chambered tank, which can help to predict when
desludging is necessary. The tank should be cleaned whenever the
bottom of the scum layer is within 7.5 cm of the bottom of the outlet
pipe or the sludge level is within 25–30 cm of the bottom of the out-
let pipe in a single-chambered tank. In practice, there should be as
little accumulated sludge as possible in the final chamber of a two or
three-chambered septic tank to ensure minimal solids loss from the
tank. Scum thickness is measured using a hinged flap device, which is
a weighted flap attached to a long rod (Fig. 4.5a). Any device can be
used, which allows the bottom of the scum mat to be felt. The meas-
uring device is pushed through the scum layer until the hinged flap
falls into the horizontal position. It is then gently pulled upward until
the flap engages against the bottom of the scum layer. The handle is
marked to correspond to a reference point on top of the tank. The
same procedure is used to locate the lower end of the outlet pipe.
The difference in height on the handle corresponds to the distance
the scum is from the outlet. Sludge level detectors, using a light-
sensitive cell or ultrasonic immersion sensors, are now widely used by
professionals to monitor sludge levels (Fig 4.5b). Ultrasound is not
only used to measure the blanket surface but to actually measure the
TSS (total suspended solids), thereby giving a profile of sludge den-
sity with depth. A transducer sends an ultra-sonic pulse, which is
96 Anaerobic Wastewater Treatment

(a)

(b)

Liquid
 Combined Anaerobic Wastewater Treatment Systems 97

reflected back to a second transducer. However, if not available, the

depth of sludge can be measured by wrapping a long stick in rough
light-colored toweling, which is tied securely. The stick is slowly low-
ered into the tank through the vertical piece of the outlet pipe to the
bottom of the tank to avoid the scum. It is left for a few minutes and
then slowly removed. The depth of the sludge can be distinguished
on the toweling by black particles clinging to it (Fig. 4.5a). If the
depth of the sludge is more than one-third of the total liquid and
sludge depth at this point, desludging should be arranged. In prac-
tice, measuring sludge depth in septic tanks can be quite difficult and
messy, requiring experimentation to get it right, making annual
desludging a simpler option.
In theory, the rate of sludge accumulation determines the rate of
desludging required, although in the UK and Ireland, desludging
every 12–18 months is recommended. Twenty-eight septic tank
systems of various sizes and configurations were studied in Ireland,
with sludge accumulation rates observed to decline steadily over time,
with the rate significantly less after 12 months (Gray, 1995). Specific
sludge accumulation per capita per day fell from 0.254 L ca–1d–1
(equivalent to 92.7 L ca–1yr–1) over the first 6 months of operation to
0.178 L ca–1d–1 (64.9 L ca–1yr–1) after 60 months. The mean sludge
accumulation rate, irrespective of sludge age, was 0.234 L ca–1d–1
(85.3 L ca–1yr–1). Increasing the desludging interval reduces the vol-
ume of sludge produced, and with the average cost of desludging in
Ireland between €300 and €400 (2025), this could amount to a
significant saving over time. There is also an increasing problem in
finding WWTPs willing to take septic tank sludge due to reported

Figure 4.5. (Continued on facing page) Sludge level detection. (a) Simple equip-
ment and procedures for measuring the accumulation of sludge and scum in a septic
tank. (b) The Sludge Gun™ portable sludge blanket level detector. A pulse of infrared
light is emitted and received by a phototransistor. In clear water, nothing happens,
but as the solids content of the water increases, a sound is emitted, which increases
in pitch and volume until the thickest sludge is located. This allows the exact location
of the sludge blanket and the nature of the overlaying liquid by reading off the depth
marked on the cable. Reproduced with permission of Markland Speciality Engineering
Ltd, Georgetown, Canada. https://sludgecontrols.com/
98 Anaerobic Wastewater Treatment

problems of septicity causing bulking problems when treated by the

activated sludge process (Gray, 2023). In theory, the longer the
sludge age, the more stabilized the sludge, which in warmer areas
would mean that sludges would not necessarily have to be disposed
to a sewage treatment works. Therefore, a standard 3,000 L septic
tank serving a population of 4 should require desludging every 3–5
years if operating and loaded correctly while retaining a minimum
hydraulic retention time of 2.0 days and a liquid settlement volume
of 48% overall. A disadvantage is that there is a discernible increase in
the carryover of solids from the primary chamber of some septic tanks
with sludge age. This suggests a potential for better-designed systems
to take advantage of longer sludge ages, with mutual benefits for
owners and the environment. An earlier study conducted by Philip
et al. (1993) reached similar conclusions, suggesting that desludging
intervals of up to 5 years were possible. They found that over the first
two years of operation, hydrolysis led to a steady build-up of volatile
fatty acids, with methane activity only recorded after 2 years and opti-
mal sludge reduction seen after 2.5 years, with a n average sludge
accumulation rate of 82 L ca–1yr–1 after 3 years falling to 60 L ca–1yr–1
after 3.5 years (Fig. 4.6). Elmitwalli (2013) recorded similar sludge

Figure 4.6. The average sludge accumulation rate over time in septic tanks in
France expressed as liters per capita per day (Philip et al., 1993). Reproduced with
permission of IWA Publishing.
 Combined Anaerobic Wastewater Treatment Systems 99

accumulation rates of between 0.19 and 0.22 L ca–1d–1 for domestic

wastewater and 0.13–0.15 L ca–1d–1 for black water. He concluded
that conversion rates of organic matter to methane were 50% and 60%
for the different wastewaters, respectively, but that to obtain a high
and stable conversion rate, desludging intervals had to be greater than
one year.
A detailed study of 27 two-chambered septic tanks by MacMahon
et al. (2022) recorded significantly higher accumulation rates of 250
L ca–1yr–1 over the first 12 months, falling to 150 L ca–1yr–1. This rep-
resents a much higher rate of accumulation than previously recorded
but could reflect a change in lifestyle and disposal practices over the
past decade or the effects of working from home, which became
prevalent due to COVID-19. Figure 4.7 uniquely shows the sludge
depth in both the primary and secondary chambers from the study by
MacMahon et al. (2022). This is interesting as it illustrates that it can-
not be assumed that septic tanks are all behaving in a predictive man-
ner, as would be expected in a normal WWTP primary settlement
tank, so each septic tank must be considered unique due largely to
behavior differences between households, water consumption, and
wastewater characteristics, as well as tank design and installation. So,
as a general rule of thumb, if the solids accumulation in the primary
chamber is >40% or >25% in the secondary tank, then desludging
should be considered. If these increase to >50 and >35%, respectively,
then the tank should be desludged immediately for the tank to oper-
ate effectively. Measuring sludge depth is very difficult in the primary
chamber, but measuring the sludge level in the secondary chamber is
much simpler. So, routine monitoring could be based on sludge
carryover into the secondary tank, as the primary function is to pro-
tect the percolation area. MacMahon et al. (2022) recommend that
desludging intervals could be extended in a temperate region, such as
Ireland and the UK, to at least 3–5 years, so long as the accumulated
sludge volume does not exceed 50%. They also agree with Gray’s
(1995) conclusions that the volume of the primary chamber could be
enlarged to facilitate longer desludging intervals and that this should
be considered as a possible change to current guidelines. What is clear
from their study is that more information is needed in relation to
sludge production in septic tanks, especially as work practices change.
100 Anaerobic Wastewater Treatment

Figure 4.7. Fractional sludge depth in the first and second chambers of two-
chambered septic tanks sampled in Ireland for different sludge ages. Of the 27 tanks
sampled, only 12 appear to be operating within guidelines, with 7 with high sludge
accumulations (>50% sludge). A further 8 septic tank systems are showing operational
problems. Those marked (A) show equal sludge accumulation in both chambers and
appear to be operating as a single chamber unit, and those marked (B) indicate sig-
nificant carryover of sludge to the secondary chamber, which could result in solids
loss. Both of these scenarios are indicative of design, installation, or operational
problems. Tanks with ≥ 50% or ≥ 35% in the first and second chambers, respectively,
require immediate desludging. Data from MacMahon et al. (2022). Reproduced
under the Creative Commons Attribution (CC-BY) license 4.0.

However, in practice, if the sludge levels cannot be checked, then

all septic tanks should be desludged at least every two years, if not
annually.

4.2.3 Septic Tank Modifications

With septic tanks, the world’s most common treatment system,
research into making them more effective and sustainable is an excit-
ing research area that encompasses all aspects of anaerobic treatment
 Combined Anaerobic Wastewater Treatment Systems 101

technologies. Conventional septic tanks offer limited treatment to

wastewaters, being only able to remove settleable solids and their
associated BOD. This means that a secondary treatment process is
required, and when this is subsurface irrigation, this often results in
ground or surface water pollution. A number of modifications to
septic tanks have been made, including the inclusion of media for
biofilm development or passing the wastewater through the sludge to
enhance anaerobic microbial activity (Fig. 4.8). Early modifications
were in the design of septic tanks, with longer narrower tanks improv-
ing effluent quality by improving laminar flow, reducing turbulence
and mixing, and ensuring all the tank volume was used. Small orifices
between chambers also caused short-circuiting of the wastewater and
turbulence, both affecting effluent quality, especially TSS.
Anaerobic-baffled septic tanks use a series of vertical plates within
the tank to ensure that the wastewater flows over and under each
plate as it flows from the inlet to the outlet. Within each zone settle-
ment, anaerobic bacteria and archaea develop, forcing the wastewater
to pass through the sludge as it passes under the vertical plates or
baffles, which increases the wastewater microbial biomass contact
time, whereas, in a standard septic tank, the wastewater passes over
the sludge biomass with little or no contact. These systems have
higher BOD and COD removal rates and enhanced specific methane
production rates (Chen et al., 2014) and are considered in more
detail in Sec. 7.6, where they can be operated using granular sludge
or by the inclusion of a carrier medium.
In up-flow septic tanks (USTs), also known as UASB-septic tanks,
the flow direction is changed from being horizontal to vertical, with
the influent wastewater passing upward through the sludge blanket,
with solids separation occurring as the wastewater rises to the over-
flow at the top of the tank. This increases the contact between the
solids and the microbial community within the sludge blanket,
improving removal efficiency, and also improves solids removal par-
tially by enmeshment within the sludge blanket but also due to the
flocculation of smaller solids as the wastewater moves upward, with
the solids slowly settling within the vertical flow tank (Coelho et al.,
2003; Kujawa-Roeleveld et al., 2005; Luostarinen et al., 2007).
102 Anaerobic Wastewater Treatment

(a)

(b)

(c)

Figure 4.8. Schematic septic tank modifications: (a) baffled septic tank, (b) up-flow
septic tank, and (c) integrated up-flow baffled system.
 Combined Anaerobic Wastewater Treatment Systems 103

Moussavi et al. (2010), using a pilot UST treating sewage, observed

that treatment efficiency was related to hydraulic retention time
(HRT), with 85, 77, and 86% BOD, COD, and TSS removal achieved
at an HRT of 24 hours. At 12 hours of removal, this fell to 71, 67,
and 67%, and at an HRT of 6 hours, this fell further to 33, 31, and
33% respectively. The system had a high specific methanogenic activ-
ity at 15.2 mL CH4 g–1 VSS d–1, with a significant stabilization of
sludge with a VSS/TSS ratio of 0.57.
Filter media has been used in anaerobic reactors to support an
active biofilm to enhance carbonaceous removal. A full-scale anaero-
bic packaged plant was developed by Sharma and Kazmi (2015) in
India, where they used the system to treat the wastewater from a toilet
block at a residential school. The single low-density polyethylene tank
comprised a septic tank (950 L), followed by an up-flow anaerobic
filter (380 L) containing plastic pall rings with a specific surface area
of 100 m2m–3 (Fig. 4.9). Enhanced removal efficiency was achieved
with average percentage removals in the final effluent of 73% for total

Figure 4.9. Combined septic tank and anaerobic biofilter (Sharma and Kazmi,
2015). Reproduced with permission of Elsevier Publishing.
104 Anaerobic Wastewater Treatment

COD, 78% BOD, and 83% TSS, with a 2.84 Log reduction in ther-
motolerant coliforms.
Except for the use of media for anaerobic biofilm development
housed in the final chamber of a two or three-chamber septic tank,
these adaptations have only been tested at the pilot level. The small
size of conventional septic tanks would encounter problems if retrofit-
ted, both due to the nature of the raw sludge and its accumulation,
causing reduced flow rates into the tank, resulting in the backup of
wastewater in the sewer pipe. Therefore, while these systems do work,
they have to be designed as conventional anaerobic biofilm or floc-
based reactors for individual houses or small communities, which
would be more complex, and larger units than septic tanks and are
explored in Chaps. 6 and 7, respectively. Even with modification,
the final effluent will still need subsequent treatment using subsurface
irrigation, a biological process, or a membrane system, before the
effluent can be safely discharged. The majority of packaged plants for
population equivalents of <20 employ an integrated septic tank (set-
tlement and fermentation), with a secondary treatment option (Gray,
2017). It is likely that in warmer climates, anaerobic package plants
for small communities may become the norm, coupled with treatment
wetlands or ponds. Sabry (2010) describes an integrated UST and
anaerobic baffle reactor in Eygpt that achieved an average COD,
BOD, and TSS removal rate of 84, 88, and 89%, respectively. Once
established, the baffled reactor acted as a polishing step for the UST.
While TSS removal was unaffected by temperature, the difference
between mean summer (35°C) and winter temperatures (22°C), both
COD and BOD removal rates fell by 9%. In temperate and cold cli-
mates, septic tanks will remain primarily a settlement step prior to
secondary treatment due to temperature. This has led to creating
tanks with insulated walls and, even in some cases, heated ones.

4.2.4 M
 icrobial Community Structure of Anaerobic
Sludge and Pathogen Removal
There are remarkably few specific studies on the microbial community
structure of septic tank sludges. Sharma and Kazmi (2015) produced
 Combined Anaerobic Wastewater Treatment Systems 105

(a) (b) (c)

(d) (e) (f)

Figure 4.10. SEM images of different types of septic tank sludge bacteria identi-
fied by Sharma and Kazmi (2015): (a) inert material with aggregation of
Methanococcus, (b) Methanobrevibacter-like species, (c) coccoid-shaped Archaeal spe-
cies, (d) Methanosarcina-like species, (e) filamentous group Methanothrix-like spe-
cies and (f) rod-shaped Methanosaeta-like species. Reproduced with permission of
Elsevier Publishing.

some excellent SEM images of the surface sludge in their experimen-

tal full-scale septic tank in India, where the temperature varied from
22 to 30°C (Fig. 4.10). They used morphological characteristics to
identify the genera and found methanogens very common, including
Methanosaeta, Methanothrix, Methanococcus, Methanospirillum,
Methanobrevibacter, and Methanosarcina. It is unclear whether this
would be the case in temperate climates, where little methanogenic
activity is observed. They also observed a strong correlation between
BOD and TSS removal and the reduction in total and fecal coliforms
and believed that TSS, in particular, could be used as an indicator of
coliform removal.
In a comparative study of the influents and effluents from
seven rural household three-chambered septic tanks in Tianjin, China,
between 73.5 and 98.7% of microbes isolated were bacteria, with
Archaea and viruses accounting for 0.6 and 1.5%, respectively (Tan
et al., 2021). All the bacteria were associated with human feces and
106 Anaerobic Wastewater Treatment

sewage in general, specifically the phyla Firmicutes (28.9% ± 14.0%),

Proteobacteria (25.2% ± 22,1%), and Bacteroidetes (16.4% ± 11.5%).
This represented 110 microbial classes, with 59 shared by all seven
households, with Gammaproteobacteria, Clostridia, and Bacteroidia
as the most dominant, representing between 47 and 95% of the
microbial community (Fig. 4.11). What was disturbing was that 104
pathogens and 83 virulence-related genes were also recorded from
just the seven households, with each system having between 47 and
82 different pathogenic species. Thirty-two species were found in all
samples, while 70 species were recorded in 50% of the samples tested.
There was no significant difference in either diversity or abundance of
pathogenic species between the influent and effluent, indicating that
septic tanks are poor at removing pathogens, highlighting the high
risk of contamination to ground and surface waters, especially when
used for irrigation. The study highlights how vulnerable the environ-
ment is to contamination from septic tanks by a wide range of patho-
gens, which ultimately reflects the general health of the people served.
In this study, Enterobacteriaceae (relative abundance of Klebsiella
pneumoniae (1.3 ± 3.7) × 10–3 % and Escherichia coli 6.4 (± 7.9) × 10–4 %)
were dominant with surprisingly Chlamydia trachomatis, an STI,
1.7 (+ 2.2) × 10–3 %.The genus of Pseudomonas (e.g., Pseudomonas
syringae, P. aeruginosa, P. tolaasii, P. fuscovaginae, P. cichorii, and
P. savastanoi) was also common (Fig. 4.12). The study also showed
that antibiotic-resistant genes (ARGs) proliferated in septic tanks,
with a total of 441 ARGs conferring resistance to 26 antibiotic classes,
allowing them to be disseminated into the environment and posing a
significant risk to human and animal health when used for irrigation,
which is common in China (Tan et al., 2021).

4.3 Imhoff Tanks

These combined settlement and digestion units are similar to septic
tanks, except they are specifically designed for sewage treatment
plants rather than individual houses. They fill the design gap between
septic tanks and small sedimentation tanks being used for small
WWTPs. They were widely adopted throughout the world during the
 Combined Anaerobic Wastewater Treatment Systems 107

Figure 4.11. Community microbial structure at class level and clustering using the
Euclidean distance of the influent and effluent from seven different septic tanks (SP1
to SP7) (Tan et al., 2021). Reproduced with permission of Elsevier Publishing.
108 Anaerobic Wastewater Treatment

Figure 4.12. Heatmap and clustering using the Euclidean distance showing the
abundance of pathogens recovered from the influent and effluent from seven differ-
ent septic tanks (SP1 to SP7) (Tan et al., 2021). Reproduced with permission of
Elsevier Publishing.

1930s through to the 1960s for both domestic and industrial waste-
waters but have been superseded by separate sedimentation and diges-
tion units. Although simple in design, they are not easy to construct
on-site in concrete, and soil stability is especially important, as Imhoff
tanks are fairly deep. The cost of construction is the main reason
for their selection as a primary step at small WWTPs. There has been
a resurgence of interest in recent years due to new prefabricated
designs using thinner reinforced concrete walls and stainless steel or
aluminum internal settlement chambers allowing rapid installation,
either as single units or several operated in parallel.
Imhoff tanks are rectangular in plan and comprise a settlement
zone with a digestion chamber below (Fig. 4.13a). Single or double
 Combined Anaerobic Wastewater Treatment Systems 109

(a)

(b)

Figure 4.13. Imhoff tanks. (a) Design of a single tank and (b) double Imhoff tanks
at Las Vegas, Honduras (Mikelonis et al., 2010). Reproduced under the Creative
Commons Attribution (CC-BY) license 4.0.
110 Anaerobic Wastewater Treatment

units were made in a variety of sizes, serving populations ranging

from 300 to 10,000 (Fig. 4.13b). The advantage over conventional
sedimentation tanks is that Imhoff tanks do not need to be desludged
regularly (i.e., once or twice daily) to prevent anoxic conditions from
developing, as the settlement chamber above is completely separated
from the fermenting sludge below. Basic design and operational data
are summarized in Table 4.6 (see page 131).
Screened sewage enters the V-shaped settlement chamber with a
retention time of 2 to 4 hours, and as the solids settle, they fall down
the inclined sides of the settlement unit and pass through the wide
longitudinal slot that runs along the base of the settlement chamber
and collects as a sludge in the digestion compartment below. The
overlapping lip of the base of the settlement chamber prevents solids
buoyed up by gas or gas bubbles themselves from entering the settle-
ment chamber and interfering with the sedimentation process. These
escape via two longitudinal vents that run alongside the outer wall of
the settlement tank, where a surface scum slowly forms. The digestion
compartment generally has a large storage capacity and sludge only
needs to be removed at long intervals. The long sludge retention time
allows digestion to take place, with any gas produced vented to the
atmosphere (Fig. 4.13). In some warmer climates, digestion proceeds
at a more rapid rate, allowing the gas to be collected. Imhoff tanks are
more efficient than septic tanks, with enhanced settlement due to the
better longitudinal flow. They are operated in the same way as a pri-
mary sedimentation tank, with a 50–70% reduction in suspended
solids and a 30–50% reduction in BOD being possible. They are com-
pact treatment units that require little maintenance, making them
ideal for small communities. However, as digestion is psychrophilic,
there is an incomplete breakdown of the sludge during winter, which
produces odors and reduces gas production. In the summer, gas pro-
duction can become excessive and interfere with settlement, causing
foaming and bubbling.
The main problems associated with their operation in Ireland are:
infrequent desludging, so that sludge is retained within the settlement
chamber, reducing the time for settlement; hydraulic overloading
again, reducing the retention time; the longitudinal slot at the base of
 Combined Anaerobic Wastewater Treatment Systems 111

the settlement chamber becoming blocked with large solids and

grease; and while in the summer, gas accumulates under the hardened
crust of scum, creating a potential safety hazard. Apart from proper
operational management, the more interesting attempts to uprate
Imhoff tanks have included converting them into rotating biological
contactor (RBC) units (Gray, 2021). Modern prefabricated units are
now available and are being used for small WWTPs and specific indus-
trial applications (Fig. 4.14a and 4.14b)

4.4 Anaerobic Lagoons

4.4.1 The Process
Treatment in anaerobic lagoons relies on the development of a bio-
logically active sludge layer. The sludge takes several months to build
up before maximum biological activity is reached. Oxygen transfer
through the air–water interface is undesirable and is minimized by
reducing the surface area to volume ratio by increasing the depth of
lagoons to between 2 and 5 m, effectively minimizing both oxygen
transfer and heat loss. Construction of lagoons of this depth requires
specialist engineering training and must be lined first with sand, and
then several layers of welded butyl rubber or PVC sheeting to prevent
any seepage to the unsaturated soil below that may ultimately result
in groundwater pollution. Anaerobic lagoons need to be desludged
occasionally, which may require the use of heavy machinery working
from the edge of the lagoon, so simple earthen banks may not always
be suitable, although smaller lagoons can be desludged using pumps.
Sludge accumulation rates, sludge characteristics, and disposal are
reviewed by Owusu-Twum and Sharara (2020).
As in septic tanks, three identifiable zones are observed in lagoons:
the scum, the supernatant, which contains about 0.1% volatile solids,
and the sludge, with 3–4% volatile solids (Fig. 14.15). The scum or
grease layer, which forms on the surface of the lagoon, can become
40–60 mm thick and has a number of important functions. It insu-
lates the lagoon, thus preventing heat loss and suppressing odors, but
most importantly, maintains anaerobic conditions in the supernatant
112 Anaerobic Wastewater Treatment

(a)

(b)

Figure 4.14. Modern packaged Imhoff tanks. (a) View of the settlement area
showing slits in the base for settleable solids to enter into the digestion chamber
below and ventilation pipes for digester gas. (b) Prefabricated unit being lowered
onto the concrete platform. Reproduced with permission of GAZEBO S.p.A.,
Gatteo, Italy. https://www.gazebo.it/
 Combined Anaerobic Wastewater Treatment Systems 113

Figure 4.15. Basic design of an anaerobic lagoon used for treating strong organic
wastewaters.

by eliminating oxygen transfer between the air–water interface. If the

grease content of the wastewater is low (e.g., vegetable-processing
wastewaters), then the lagoon surface can be covered using 50–100 mm-
diameter HDPE plastic balls. To be as effective as a grease layer, at
least 90% of the surface must be covered. Some animal wastewaters
can also be problematic; for example, while cattle slurry rapidly forms
a crust due to the high fiber content of their feces, pig slurry does not.
The scum layer can become a breeding ground for a variety of flies,
and in warmer climates, this can become a serious nuisance. Crust
formation is quicker in summer, and in a well-constructed anaerobic
lagoon, the BOD and suspended solids of the slurry will be reduced
by 80–90%. During the summer, methane production can be high,
and large pockets of gas will be seen breaking through the crust.
Desludging of farm lagoons should be done every 3–5 years, depend-
ing on depth, total capacity, and degree of anaerobic degradation
achieved, with the sludge spread directly onto the land without any of
the associated pathogen problems. As most of these lagoons are
between 2 and 3 m deep, they are potentially dangerous, and the
crust rapidly becomes covered with grass, weeds, and even small
bushes, giving it a false appearance of stability. Although in certain
circumstances, they may be strong enough for an animal or even a
child to walk on, they cannot support the weight of a small vehicle or
possibly an adult. So, for safety, they should be securely fenced. The
114 Anaerobic Wastewater Treatment

operation of anaerobic lagoons in series is not recommended as it is

difficult to maintain an adequate scum cover on the second lagoon.
The reduced loading results in the surface layer of the lagoon becom-
ing aerobic; thus, the system becomes facultative rather than strictly
anaerobic (Gray, 2022). Under-loaded lagoons will also tend to
become facultative ponds.
Although the inlet pipe can be at the surface, anaerobic lagoons
function most efficiently if the influent wastewater enters near the
bottom of the basin. This prevents the short-circuiting of the liquid
and ensures that it mixes with the microbial solids in the active anaer-
obic sludge layer. The flow pattern of the wastewater and the constant
release of methane and carbon dioxide resulting from the degradation
of organic matter ensure that some sludge particles remain in suspen-
sion within the supernatant. In this way, decomposition of organic
matter continues, both in the liquid phase and within the sludge
blanket; thus, a high mixing efficiency results in a high rate of BOD
removal (Parker et al., 1950). The discharge pipe is located just below
the scum layer at the opposite end of the basin to ensure an upward
flow pattern. This encourages the bacterial flocs to settle out of sus-
pension, ensuring a clarified effluent and also eliminating the need for
sludge return by retaining the microbial biomass in the lagoon. Full
design details are given by the USEPA (1983; 2011).

4.4.2 Microbiology
Anaerobic lagoons have a widely diverse population of bacteria and
archaea. In a study of four anaerobic lagoons treating swine wastewa-
ter, Ducey and Hunt (2013) identified 15,682 phylotypes that could
be classified into 839 individual genera, with 223 genera common to
all four lagoons. These belonged primarily to four major phyla: the
Firmicutes (54.1%), Proteobacteria (15.8%), Actinobacteria (7.2%),
and the Bacteroidetes (4.1%) (Fig. 14.16). These anaerobes are respo­
nsible for a wide range of functions, including fermentation, sulfate
reduction, odor production, and both nitrogen and sulfur cycling.
Twenty-five genera accounted for 20% of the operational taxonomic
units (OTUs) measured in the four lagoons (Table 4.3). Each lagoon
had a unique community structure controlled by key environmental
 Combined Anaerobic Wastewater Treatment Systems 115

(a)

(b)

(c)

Figure 4.16. Relative abundance of bacterial 16S rDNA genes from swine waste-
water anaerobic lagoons expressed as operational taxonomic units (OTUs) at (a) the
phylum level, (b) breakdown of proteobacteria, and (c) Firmicutes (Ducey and Hunt,
2013). Reproduced with permission of Elsevier Publishing.
Table 4.3. The relative abundance (Ra) (%) and the number of operational taxonomic units (#OTUs) of the commonest 25 genera

116 Anaerobic Wastewater Treatment

isolated from four anaerobic lagoons treating piggery wastewater (Ducey and Hunt, 2013). Reproduced with permission of Elsevier
Publishing.
Lagoon #1 Lagoon #2 Lagoon #3 Lagoon #4

Phylum Genus Ra #OTUs Ra #OTUs Ra #OTUs Ra # OTU

Firmicutes Anaerovorax 2.58% 96 2.45% 126 1.94% 73 2.78% 85
Proteobacteria Thiolamprovum 1.59% 59 3.81% 196 1.38% 52 1.14% 35
Firmicutes Clostridium sp. cluster XI 3.14% 117 0.70% 36 0.40% 15 2.16% 66
TM7 TM7 0.89% 33 1.24% 64 1.04% 39 1.34% 41
Firmicutes Clostridium (sensu stricto) 1.53% 57 0.64% 33 0.48% 18 1.63% 50
Firmicutes Oscillibacter 0.43% 16 1.13% 58 0.85% 32 0.98% 30
Actinobacteria Leucobacter 1.67% 62 0.45% 23 0.37% 14 0.78% 24
Proteobacteria Methylocystis 0.11% 4 0.10% 5 2.77% 104 0.00% 0
Firmicutes Tissierella 0.67% 25 0.86% 44 0.05% 2 1.18% 36
Firmicutes Acetanaerobacterium 1.05% 39 0.56% 29 0.16% 6 1.05% 32
Firmicutes Turicibacter 1.42% 53 0.40% 19 0.13% 5 0.85% 26
Firmicutes Saccharofermentans 0.67% 25 0.70% 36 0.67% 25 0.33% 10
Firmicutes Lachnospiracea 0.38% 14 0.80% 41 0.56% 21 0.62% 19
Actinobacteria Corynebacterium 0.73% 27 0.52% 27 0.59% 22 0.56% 17
Firmicutes Sporobacter 0.54% 20 0.76% 39 0.05% 2 0.75% 23
Synergistetes Aminobacterium 0.73% 27 0.51% 26 0.24% 9 0.26% 8
Firmicutes Lactobacillus 0.38% 14 0.45% 23 0.64% 24 0.29% 9
Proteobacteria Desulfomonile 0.38% 14 0.06% 3 1.23% 46 0.20% 6
Proteobacteria Acinetobacter 0.40% 15 0.45% 23 0.19% 7 0.59% 18
Actinobacteria Mycobacterium 0.13% 5 0.08% 4 1.25% 47 0.03% 1
Cyanobacteria Group IIa 0.00% 0 0.00% 0 1.52% 57 0.00% 0
Actinobacteria Actinomyces 0.70% 26 0.25% 13 0.11% 4 0.39% 12

Combined Anaerobic Wastewater Treatment Systems 117

Proteobacteria Thioflavicoccus 0.00% 0 0.00% 0 1.46% 55 0.00% 0
Bacteroidetes Prevotella 0.16% 6 0.27% 14 0.53% 20 0.42% 13
Actinobacteria Klugiella 0.02% 1 0.00% 0 1.09% 41 0.07% 2
Total 20.30% 755 17.19% 882 19.70% 740 18.40% 563
118 Anaerobic Wastewater Treatment

parameters, such as Kjeldhal nitrogen, COD, orthophosphate, total

suspended solids, and dissolved oxygen (DO) (Ducey and Hunt,
2013). The microbial community structure changes seasonally, which
may also affect odor production (Cook et al., 2010).
In anaerobic lagoons, the organic substrate is degraded in the
absence of oxygen to carbon dioxide and methane, with only a small
amount of bacterial growth. Approximately 90% of the available
chemical energy, in the form of organic material, is retained as meth-
ane production (McInerney et al., 1980). Apart from the economic
value of the methane gas produced (Sec. 4.4.8), anaerobic treatment
has many advantages over aerobic treatment processes, such as less
biomass produced per unit of substrate utilized (the lower biomass
production means a lower requirement for nitrogen, phosphorus, and
other nutrient and growth factors), higher organic loadings, as anaer-
obic processes are not limited by oxygen transfer rates, and lower
constructional and operational costs. However, the major disadvan-
tage in temperate and colder climates is the elevated temperatures
required to maintain microbial activity at a reasonable level.
When operating under ideal conditions, the rate of volatile acid
production equals the rate of conversion to methane. However,
methanogenic activity is adversely affected by fluctuations in organic
loading, temperature, and pH. So, if the methanogens are inhibited,
then there is an immediate reduction in the conversion of volatile
organic acids, leading to an increase in their concentration and a rapid
reduction in pH, causing further inhibition of methanogenic activity,
leading to process failure. Optimum (and maximum) operating con-
ditions for methane formation are temperature 30–35°C (25–40°C),
pH 6.8–7.4 (6.2–7.8), redox (or ORP) –520 to –530 mV (–490
to –550 mV), volatile organic acids 50–500 mg acetic acid L–1
(50–2,000 mg L–1), and alkalinity 2,000–3,000 mg CaCO3 L–1
(1,000–5,000 mg L–1) (USEPA, 2011).

4.4.3 Application
Anaerobic lagoons are generally used as a preliminary treatment for
strong organic wastes, with an 80% reduction in BOD and a 90%
 Combined Anaerobic Wastewater Treatment Systems 119

reduction in suspended solids possible. This means that the lagoon

only partially stabilizes the wastewater, and secondary treatment is
required before the effluent can be discharged to natural waters —
usually, this is achieved using an aerobic process either in the form of
facultative ponds in series or activated sludge, although recent develop-
ments in granular anaerobic processes, in particular, those employing
membranes, means that a full anaerobic treatment process could be
feasible, enhancing carbon recovery through extra methane produc-
tion and reducing energy use. However, anaerobic treatment can con-
siderably reduce the organic loading to secondary treatment units, thus
reducing the secondary treatment capacity required. If the subsequent
aerobic treatment process is a facultative pond, then the sludge will be
less likely to rise to the surface in warm weather as the sludge has
already undergone stabilization by anaerobic breakdown. Most indus-
trial ponds operate as anaerobic lagoons, and the process is widely used
for the treatment of slaughterhouse waste, animal production, sugar
beet, paper pulp, potato, and other food processing wastewaters
(Porges and Brackney, 1962; Barnes et al., 1984; Vuillot and Boutin,
1987). Wastewaters most amenable to stabilization by anaerobic break-
down in lagoons have a high organic strength, are rich in fats and
protein, have a relatively high temperature, are free from toxic materi-
als, especially heavy metals, and contain sufficient biological nutrients.
Slaughterhouse and meat-processing wastewaters are particularly
suitable for treatment by anaerobic lagoons as they have a high
BOD (~1,400 mg L–1), grease content (~500 mg L–1), temperature
(~28°C), and a neutral pH. Minimum pre-treatment is required prior
to discharge to an anaerobic lagoon, which should normally be limited
to blood recovery, screening to remove coarse solids, and dissolved air
flotation or skimming to remove excessive grease. If pre-treatment is
too extensive, then insufficient grease may remain in the wastewater
to form a scum when discharged to the lagoon. Domestic sewage is
normally not suitable for treatment by this method due to a relatively
low BOD concentration and grease content, although facultative
ponds work well in warmer climates. When sewage has a high indus-
trial content, anaerobic treatment often leads to serious odor prob-
lems due to the inhibition of certain microbial groups.
120 Anaerobic Wastewater Treatment

Anaerobic lagoons used for vegetable-processing wastewaters

tend to be shallower in design and are widely used to treat sugar beet
and potato wastewaters. The organic strength of sugar beet process-
ing wastewaters can be very high, and although a 60–70% reduction
of BOD and 80–90% reduction in suspended solids is possible, the
final effluent is still far too strong (BOD >500 mg L–1) for direct dis-
charge to surface waters, requiring further aerobic biological treat-
ment. These wastewaters are difficult to treat, as they are seasonal,
with <100 days wastewater production per year as is the case with
sugar beet processing. This can leave insufficient time for adequate
active anaerobic biomass to accumulate, resulting in partial anaerobic
breakdown and the release of odors. The shallow lagoons can also
result in partial reaeration, while low temperatures and a high C:N
ratio, common with vegetable wastewaters, all increase the inhibition
of anaerobic activity.
In arid and semi-arid areas, anaerobic-lagoon-treated effluent may
be used for irrigation, although it is more commonly further treated.
In the US, new regulations governing wastewater irrigation have been
introduced, which include the use of nutrient (N and P) management
plans, which are also commonly used in Europe (Sharpley et al., 2011).
Microbial standards for wastewater reuse on agricultural land are not
included, but the World Health Organization (WHO) has set stand-
ards based on fecal coliform counts of ≤1 × 105 100 mL–1 (WHO,
2006). However, little attention has been paid to the transfer of fecal
bacteria and zoonotic pathogens from anaerobic lagoons treating
animal rearing and slaughterhouse wastewaters. In a study of water
quality from an anaerobic lagoon treating piggery wastewater,
McLaughlin et al. (2012) studied the seasonal variations in both
nutrients and pathogens, recording higher nitrogen and carbon losses
at temperatures above 19°C, with the N:P ratio falling from 9.7 to
2.8 during the warmer months when irrigation was used. Enterococci
and zoonotic pathogens were present throughout the year but decre­
ased at higher summer temperatures, while water-soluble metals,
including Cu and Zn, increased. Temperature is a key factor in patho-
gen inactivation and removal, with aerobic systems more effective
than anaerobic lagoons (Pandey et al., 2016). A key nutrient in
 Combined Anaerobic Wastewater Treatment Systems 121

eutrophication is phosphorus, and the effluent from a piggery waste

anaerobic lagoon is rich in this nutrient with N:P ratios normally
between 2:1 and 4:1. In order to comply with crop requirements of
a ratio of 4:1 to 9:1, either additional N must be added or the P con-
centration reduced. As both NH4+ and Mg2+ are common in the
lagoon effluent, Nelson et al. (2003) have proposed P removal by
struvite (magnesium ammonium phosphate hexahydrate MgNH4PO4·
6H2O) precipitation, which can reduce effluent PO4-P concentration
to 2 mg L–1 as well also reducing the ammonium concentration and
creating a useful fertilizer.

4.4.4 Design of Anaerobic Lagoons

The design loadings for stabilization ponds are normally measured in
grams of BOD per unit area (g BOD m−2d–1), but as light is not an
important factor in anaerobic lagoons, loading is expressed as BOD
per unit volume (g BOD m−3d–1) or as the HRT (m−3h–1). The rate of
treatment is temperature dependent (Table 4.4), so in temperate or
cold climates, lagoons may have very reduced anaerobic activity in the
winter, acting primarily as settlement ponds. BOD reduction is a
function of both hydraulic retention time and temperature. The
WHO (1987) has given different BOD reduction values at tempera-
tures >20°C based on HRT as 1 day for 50%, 2.5 days for 60%, and
5 days for 70% reductions. Hammer (1977) gives a mean organic
loading value of 320 g BOD m−3d−1 at a minimum temperature of
25°C to achieve 75% BOD removal at a minimum retention time of
4 days. Similar figures are given by Gloyna (1971), who cites exam-
ples where longer retention times of up to 19 days are needed for
strong organic industrial wastewaters. The volume of the lagoon can
be calculated as:

λv = LiQ/Va g m−3d−1(4.3)

where λv is the organic loading rate (g BOD m−3d–1), Li is the influent

wastewater BOD (mg L–1 or g m−3), Q is the influent flow rate (m3d−1),
and Va is the volume of the lagoon (m3) (Mara and Pearson, 1998).
122 Anaerobic Wastewater Treatment

Table 4.4. Design values calculated at various

temperatures for the organic loading rate and per-
centage BOD removal in anaerobic lagoons.
Temperature Organic loading rate % BOD
(°C) (g BOD m–3 day–1 removal
≤10 100 40
11 120 42
12 140 44
13 160 46
14 180 48
15 200 50
16 220 52
17 240 54
18 260 56
19 280 58
20 300 60
21 300 62
22 300 64
23 300 66
24 300 68
≥25 300 70

The value λv increases with temperature but lies between 100 and 350
g m−3d−1, the former to maintain anaerobic conditions and the latter
to avoid odor release (Table 4.4). The mean temperature of the
lagoon is used to select λv from Table 4.4, from which the lagoon
volume (Va) can be calculated using Eq. (4.3). The mean hydraulic
retention time (θa) is calculated as:

θa = Va/Q days (4.4)

The major advantages of anaerobic systems are that less sludge is

produced compared with an aerobic system, and no aeration equip-
ment or power supply is required. The main operating limitations of
the process are the temperature and strength of the wastewater. The
temperature of the lagoon controls the BOD removal, and below
 Combined Anaerobic Wastewater Treatment Systems 123

15°C, purification will be due to physical settlement only, with limited

anaerobic breakdown of organic matter occurring. At higher tempera-
tures, anaerobic digestion proceeds rapidly, with biogas (70% CH4,
30% CO2) bubbling to the surface. The same bacterial groups are
involved as with other anaerobic systems. Thus, the process is inhib-
ited by the same conditions (e.g., pH <6.2). In temperate climates,
anaerobic lagoons act as settling tanks during the winter months, and
the accumulated sludge is subsequently degraded during the warmer
summer months. This was originally demonstrated by Parker et al.
(1950), who recorded a 65–80% BOD removal in lagoons during the
summer, with a retention period of 1.2 days, which fell to 45–65%
removal in the winter, even after 5 days retention. Therefore, special
attention should be paid to those factors affecting the temperature of
the lagoon, such as ambient and wastewater temperature and insuffi-
cient scum development for insulation. A well-designed anaerobic
lagoon will achieve, on average, 40% BOD removal at 10°C, rising to
60% at 20°C. The required hydraulic retention time is a function of
temperature and wastewater strength. Low BOD and grease contents
of wastewater will also reduce scum development; where no scum
formation occurs, then a thin surface film of the algae Chlamydomonas
often develops. Protection from the wind is important in preventing
mixing and the resultant break-up of surface scum. Increasing con-
cern over methane released into the atmosphere, which is a green-
house gas, from lagoons will eventually lead to all lagoons being
covered to capture the biogas (Sec. 4.4.8).
The large depth of anaerobic lagoons used to conserve heat and
maintain anaerobicity can lead to the stratification of key nutrients,
trace elements, and the microbial community (Lovanh et al., 2009;
Cook et al., 2010). These effects have been shown to be both sea-
sonal as well as temperature-dependent. Lagoons have been recorded
as being up to 9 m in depth, resulting in some becoming vertically
thermally stratified, which can result in the sludge layer remaining
colder or warmer than the upper water phase, depending on the sea-
son. When located in exposed areas, lagoons are also affected by wind
action, which causes mixing deep within the lagoon, resulting in the
settled solids being mixed with the water phase. The temperature vari-
ation within lagoons is illustrated in Fig. 4.17.
124 Anaerobic Wastewater Treatment

Figure 4.17. The temperature profile of an anaerobic lagoon (0.4 ha) treating pig-
gery wastewater (approximately 2,000 sows) (Lovanh et al., 2009). Reproduced with
permission of Elsevier Publishing.

4.4.5 Gas Emissions from Lagoons

The final phase in anaerobic digestion is methane fermentation,
where the end products of acid fermentation are converted to gases,
mainly methane and carbon dioxide, but also ammonia and hydrogen
sulfide, by several different species of obligate anaerobic archaea, see
Eq. (4.5). At this stage, complete stabilization of the substrate occurs,
and as the end products are only gases, it is more efficient than com-
plete aerobic stabilization.

Organic matter → CH4 + CO2 + H2 + NH3 + H2S(4.5)

In anaerobic lagoons, the biogas production rate is a function of

organic acid concentration and the lagoon temperature. Safley and
Westerman (1988) studied a number of covered lagoons (Sec. 4.4.8)
and found a minimum organic loading rate of 0·06 kg VS m−3 was
required for consistent biogas production, with rates varying between
 Combined Anaerobic Wastewater Treatment Systems 125

0·02 and 0·50 m3m−2d−1, which is equivalent to 0·03 to 0·23 m3m−3d−1,

although the carbon dioxide content was generally less than 35%.
These researchers later determined a relationship between methane
production and lagoon temperature (range: 6.7 to 27.2°C) after a
four-year study on an anaerobic lagoon treating dairy manure:

CH4 = 89.514e–0.00381T (4.6 )

where CH4 is the percentage methane concentration and T is the

lagoon temperature (°C) (Safley and Westerman, 1992).
Ammonia can be found at high concentrations in anaerobic
lagoons and can, therefore, be an important local source of atmos-
pheric ammonia. Aneja et al. (2001) studied a number of different
anaerobic lagoons (i.e., primary, secondary, and tertiary) treating
swine wastewater, with ammonia concentrations ranging from 37 to
909 mg N L–1. The rate of ammonia volatilization was correlated with
surface water temperature and the NHx (i.e., NH3 + NH4+) concen-
tration in the lagoon, with the highest mean emission rate of 120.3
µg N m–2 min–1 (range 50–374 µg N m–2 min–1) recorded in a primary
lagoon and falling to the lowest rate 40.7 µg N m–2 min–1 (range
22–84 µg N m–2 min–1) in a tertiary anaerobic lagoon over a tempera-
ture range of 10.3–23.3°C. They were able to model the emission
rate from their anaerobic lagoons as:

In(NH3-N flux) = 1.0788 + 0.0406 TL + 0.0015 (NHx)(4.7)

where the NH3-N flux is the loss of ammonia from the lagoon surface
(µg N m–2 min–1), TL is the surface water temperature of the lagoon
(°C), and NHx is the total ammonia-nitrogen concentration (mg N L–1).
A similar study by Grant et al. (2016) expressed the annual mean
ammonia emissions rate in terms of sow live weight, which ranged
from 0.124 to 0.240 g N d–1 kg–1 for breeding and weaning units and
0.072 to 0.208 g N d–1 kg–1 for finishing units (i.e., growing on to
market). They also produce several models: (i) incorporating wind
and temperature, which accounted for 74% of emissions variability,
and (ii) incorporating suspended organic concentration, pH, and
nitrogen excretion rates, which accounted for 66% of the emissions
126 Anaerobic Wastewater Treatment

Figure 4.18. Influence of air temperature on emissions from an anaerobic lagoon.

The unit AU represents 500 kg live pig weight (Grant et al., 2016). Reproduced with
permission of Elsevier Publishing.

variability. The solubility of NH3 and dissociation of NH4+ to NH3 in

the lagoon liquid is temperature-dependent, resulting in more NH3
volatilizing from the surface at higher temperatures, creating a sea-
sonal variation in emissions (Fig. 14.18). There is also a significant
diurnal variation in the release of ammonia into the atmosphere from
lagoons, reaching a maximum during the warmest time of day (Aneja
et al., 2001). The temperature in the lagoon is directly related to air
temperature, although the scum layer or cover, where used, does
insulate the wastewater to a certain extent (Fig. 4.19). Grant et al.
(2016) calculated that for each 1°C rise in the mean daily tempera-
ture, there is a corresponding 9.5% increase in maximum ammonia
emissions and a 6.9% increase in annual emissions. Ammonia emis-
sions from a dairy waste anaerobic lagoon were 55 kg NH3 per cow
per annum, which is 24% of the annual 180 kg N excreted per cow
per annum (Rumburg et al., 2008). An intetgrated physical/chemical
model for anaerobic lagoons has also been produced by Wu and
Chen (2011).
 Combined Anaerobic Wastewater Treatment Systems 127

Figure 4.19. Correlation between the daily mean air temperature measured 2 m
above the lagoon surface and the daily mean wastewater temperature measured 0.3
m below the lagoon surface (R2 0.87) (Grant et al., 2016). Reproduced with permis-
sion of Elsevier Publishing.

4.4.6 Odors
Many of the breakdown products of anaerobic fermentation in anaer-
obic lagoons are odorous. Odors are caused by the release of volatile
or gaseous compounds into the atmosphere and can have very low
odor thresholds, which is generally defined as the average concentra-
tion of the compound at which an odor is detected. The commonest
odor encountered is the smell of rotten eggs caused by hydrogen
sulfide, which is produced by sulfate reducing bacteria. The key
organic acids produced during anaerobic breakdown are also odor-
ous, and so when the final methanogenic phase is inhibited, these can
cause odor problems.
128 Anaerobic Wastewater Treatment

Some food-processing wastewaters produce extremely strong

odors, especially during treatment and storage (Gerick, 1984; Gray,
1988). For example, sugar beet wastewater undergoes partial anaero-
bic breakdown within the process and subsequently upon storage in
lagoons, with the production of a variety of odors. This is due to the
processing season taking place from October to January, with the cold
temperatures inhibiting the full completion of the fermentation pro-
cess. The major odors come from the volatile acids that comprise
most of the organic fraction of the effluent. The odor threshold con-
centrations for the volatile organic acids produced during the treat-
ment of sugar beet wastewater and waste are 24.3 ppm for acetic acid,
20.0 ppm for propionic acid, 0.05 ppm for iso-butyric acid, 0.24 ppm
for butyric acid, 0.7 ppm for iso-valeric acid, and 3.0 ppm for valeric
acid. Therefore, by measuring the volatile acid concentration and
dividing it by the appropriate odor threshold concentration, a meas-
ure of the odor production or concentration, known as the odor
number, can be calculated:

Compound concentration
in wastewater (4.8)
Odor number of wastewater =
Odor threshold concentration
for compound

Other odors associated with sugar beet processing include tri-

methylamine, which has a fishy odor, and organic sulfides, which
produce a strong odor of rotting cabbage, as do the thiol compounds
methyl mercaptan (CH3SH) and ethyl mercaptan (C2H5SH), both of
which have very low odor thresholds of 0.0011 ppm and 0.00019
ppm, respectively (Shore et al., 1979). A detailed list of odor thresh-
old values has been compiled by Fazzalari (1978), while a list of com-
mon odor compounds associated with anaerobic fermentation is
given in Table 4.5.
Under normal operating conditions, no odors are released due to
complete anaerobiosis and adequate scum cover. However, problems of
odor because of sulfide formation at low temperatures or high sulfate or
protein concentrations in the wastewater can occur. Sulfate can also be
a problem in some paper mill wastes that use alum as a sizing agent.
 Combined Anaerobic Wastewater Treatment Systems 129

Table 4.5. Some characteristic odors produced by compounds present in

wastewaters. These degradation products can be categorized into two main groups,
either degradation products of nitrogenous or sulfurous compounds. There are other
odorous compounds, such as those associated with chlorine and phenolic wastes.
Compounds General formulae Odor produced
Nitrogenous
Amines CH3NH2, (CH3)3N Fishy
Ammonia NH3 Ammoniacal, pungent
Diamines NH2(CH2)4NH2, NH2(CH2)5NH2 Rotten flesh
Skatole C8H5NHCH3 Faecal, repulsive

Sulfurous
Hydrogen sulfide H2S Rotten eggs
Mercaptans CH3SH,CH3(CH2)3SH Strong decayed cabbage
Organic sulfides (CH3)2S,CH3SSCH3 Rotten cabbage
Sulfur dioxide SO2 Pungent, acidic

Other
Chlorine Cl2 Chlorine
Chlorophenol Cl.C6H4OH Medicinal, phenolic

Hydrogen sulfide is produced by the reduction of sulfate by a wide

range of sulfate reducing bacteria that use the breakdown products of
organic matter, in particular, acetate, butyrate, ethanol, lactate, and pro-
pionate, while using sulfate as a terminal electron acceptor. There are
over 30 known genera of sulfate reducing bacteria in anaerobic lagoons,
with Desulfobacterium, Desulfobulbus, and Desulfovibrio all commonly
recorded (Zeikus, 1980; Rabus et al., 2006; Cook et al., 2008).
Although present in relatively small numbers in wastewater, sulfate
reducing bacteria rapidly grow under the ideal conditions found in
anaerobic lagoons. For example, Desulfovibrio desulfuricans is present in
low numbers in sewage (60–600 mL–1) but rapidly increases on storage
up to concentrations in excess of 100,000 mL–1 over 14 days. Ducey
and Hunt (2013) recorded 26 genera of sulfate reducing bacteria in
the four lagoons they studied. The genera Desulfatirhabdium,
Desulfobulbus, Desulfocurvus, Desulfomonile, Desulfovibrio, Desulfovirga,
130 Anaerobic Wastewater Treatment

and Thermodesulfobium were recorded in all four separate lagoons;

Desulfoglaeba, Desulfonispora, Desul­ fosporosinus, Desulfatiferula, and
Desulforegula in three lagoons; Desulfobacca, Desulfofaba, Desulfofustis,
Desulfococcus, and Desul­fomicrobium in two lagoons; while Desulfarculus,
Desulfonema, Desul­forhabdus, Desulforhopalus, Desulfospira, Desulfovermiculus,
Desul­furispora, Syntrophobacter, and Thermodesulforhabdus were recorded
in just one lagoon. The redox is fairly critical for sulfate reduction, with
ORPs of <–50 mV normally required for hydrogen sulfide production,
which normally occurs within the sludge layer. The mean concentration
of hydrogen sulfide released with biogas (CH4 80.4%; CO2 14.7%) dur-
ing a four-year study on an anaerobic lagoon treating dairy manure was
272 ppm over a lagoon temperature range of 6.7 to 27.2°C (Safley and
Westerman, 1992).
With better design, it is now not always critical to have a surface
scum as long as sufficiently high organic loading can be maintained to
ensure anaerobic conditions. Hydrogen sulfide is present in aqueous
solution as hydrogen sulfide gas, the bisulfide ion (HS−), or the sulfide
ion (S2−). Thus, for any given total sulfide concentration, the amount
present as odorous hydrogen sulfide is pH dependent (Fig. 4.20).
Mara and Pearson (1998) explain that designers have been reluctant

Figure 4.20. The effect of pH on the equilibrium of hydrogen sulfide–bisulfide–

sulfide.
 Combined Anaerobic Wastewater Treatment Systems 131

Table 4.6. Design and operational data for Imhoff tanks (Mikelonis et al., 2010).
Reproduced under the Creative Commons Attribution (CC-BY) license 4.0.
Value

Design parameter Range Typical

Settling compartment
Overflow rate, peak hour m3/m2/h 1–1.75 1.36
Detention time, h 2–4 3
Length to width ratio 2:1–5:1 3:1
Slope of settling compartment ratio 1.25:1–1.75:1 1.5:1
Slope opening, cm 15–30 25
Slot overhang, cm 15–30 25
Scum baffle
Below surface, cm 25–41 30
Above surface, cm 31 30
Freeboard, cm 45–60 60
Gas vent area
Surface area, % total surface area 15–30 20
Width of opening, cm1 45–75 60
Digestion section
Volume (unheated), storage capacity, 6 months sludge
m3/capita
Volume, m3/6 month storage2 0.06–0.10 0.07
Sludge withdrawal pipe, cm 20–30 25
Depth below slot to top of sludge, m 0.30–0.90 0.60
Tank depth
Water surface to tank bottom, m 7–10 9
1
Minimum width of opening must be 45 cm to allow a person to enter for cleaning
2
Based on a 6-month digestion period

to use anaerobic lagoons at treatment plants in case of odor forma-

tion. They stress that odor can be controlled by not exceeding the
design loadings (Table 4.4) and ensuring that influent wastewater
sulfate concentrations are <500 mg SO4 L−1. Sulfide reacts with heavy
metals to form insoluble metal sulfides, but if concentrations exceed
132 Anaerobic Wastewater Treatment

<50 mg L−1, there is a progressive inhibition of methanogenesis

(Pfeffer, 1970). No odor should arise from a lagoon if the liquid tem-
perature is above 20°C.

4.4.7 Denitrification
Jones et al. (2000) observed nitrogen gas emissions from anaerobic
lagoons treating piggery wastewater. Under normal circumstances,
denitrification requires the pre-step of nitrification, which is impossi-
ble under anaerobic conditions but may be possible under micro­
aerobic conditions found at the air–water interface of open lagoons.
A wide range of bacteria and archaea are able to denitrify under anoxic
conditions, using NO3– and NO2– as terminal electron acceptors.
Reduction of nitrate follows a sequence of steps, each mediated by a
specific enzyme.

  NO3– (aq) → NO2– (aq) → NO (g) →N2O (g) → N2 (g) (4.9)

It is during incomplete denitrification that both NO and N2O can

be released into the atmosphere, causing damaging effects to the
environment, both in terms of toxicity and global warming.

Denitrification
NO3– + 1.25CH2O → 0.5N2 + OH– + 0.75H2 + 1.25CO2(4.10)

As can be seen from the equation above, denitrification releases

OH– ions into the effluent, increasing the alkalinity. However, under
the strict anaerobic environment of the lagoons, nitrification would
be very limited or, at best, incomplete. Jones et al. suggested alterna-
tive microbial-mediated denitrification processes, such as anaerobic
ammonium oxidation, better known as anammox. Ammonium is
used as an energy source by the anammox bacteria and carbon dioxide
as a carbon source, with NO2– and NO3–, if present, used as electron
acceptors (Gray, 2021). Kartel et al. (2011) have described the pro-
cess in detail, where the enzyme nitrite oxidoreductase reduces nitrite
to nitrogen oxide, which then reacts with ammonium to form hydra-
zine (N2H2), enabled by the enzyme hydrazine synthase, which is
 Combined Anaerobic Wastewater Treatment Systems 133

oxidized by hydrazine dehydrogenase to nitrogen. The bacteria are

extremely slow-growing and strict anaerobes, unable to function if
the dissolved oxygen concentration rises above 0.5 mg L–1.

         Anammox
NH4+ + NO2– → N2 + 2H2O(4.11)

The autotrophic bacteria responsible for the oxidation of

ammonia and the simultaneous reduction of nitrite belong to the
widely distributed phylum Planctomycetes. Not all the bacteria in this
phylum are able to oxidize ammonium, this being restricted to the
Anammox-Planctomycetes, which have a characteristic membrane-
bound cell compartment called the anammoxosome (Fuerst and
Sagulenko, 2011) (Sec. 6.5.3).

4.4.8 Covered Lagoons for Biogas Collection

In the early 1990s, with the increasing concern over the release of
methane into the atmosphere, which has a high global warming
potential (GWP), operators began to cover anaerobic lagoons (Green
et al., 1995; Hodgson and Paspaliaris, 1996). This was initially done
using a sheet of geotextile membrane floated over the surface of the
lagoon, with the biogas passively recovered and flared off (Fig. 4.21).
Covering also means that scum formation or the use of plastic balls
to act as a surface barrier is no longer required, and that odor
control can be managed, opening up the process to a wider range of
wastewaters that can be treated and also increasing its sustainability.

Figure 4.21. Covered anaerobic lagoons can be constructed of any size and are
comparatively cheap to recover biogas from small to large-sized agricultural, indus-
trial, and wastewater operations.
134 Anaerobic Wastewater Treatment

Figure 4.22. Floating scum can become compacted under covers of anaerobic
lagoons due to methane gas pressure, occasionally causing covers to become dam-
aged (Bobage et al., 2022). Reproduced with permission of Elsevier Publishing.

Scum in anaerobic lagoons is primarily caused by methane bubbles

bringing undigested material to the surface. The buoyancy of these
solids causes surface compaction of the scum and an increase in its
strength, which forces the scum against the covers (Bobage et al.,
2022). This can be a major problem by reducing hydrodynamic
retention time, interfering with methane capture and removal, and
damaging covers (Fig. 4.22).
Although mesophilic digestion using standard digesters has been
used for agriculture, the cost and complexity of operation have seen
its limited application. In the past decade, the use of farm-based
covered anaerobic lagoons has spread throughout Europe, the US,
Australia, New Zealand, and elsewhere for the treatment of animal
wastewaters, primarily cattle and pig manures. This is hugely advanta-
geous for both farmers and the environment. For the farmers, it pro-
vides safe storage, commercial biogas production, and also the onsite
generation of electricity as well as heat, allowing farmers to diversify
into new production areas, such as high-profit greenhouse fruit,
herbs, and vegetables. Farm-based lagoons only need to be desludged
 Combined Anaerobic Wastewater Treatment Systems 135

every 3–5 years, depending on depth, total capacity in relation to

loading, and degree of anaerobic breakdown achieved. A highly sta-
bilized and largely odorless sludge is produced, which is generally
used for land disposal (Owusu-Twum and Sharara, 2020). The efflu-
ent is rich in nutrients and can be used for surface irrigation of grass-
land for silage or hay.
Anaerobic lagoons are widely used in New Zealand for the treat-
ment of predominantly dairy and also other animal wastewaters.
Although originally open, these lagoons are increasingly being cov-
ered to collect the biogas to reduce greenhouse gas emissions as well
as generate energy. Such ponds are operated below the optimum
mesophilic temperature and so have a reduced volatile solids (VS)
loading of 0.02–0.46 kg VS m–3d–1. Park and Craggs (2007) com-
pared two anaerobic lagoons: one treating piggery wastewater (influ-
ent BOD 11,500 mg L–1; BOD loading rate 0.30 kg BOD m–3d–1; VS
5,450 mg L–1) and the other dairy wastewater (influent BOD 1,147
mg L–1; BOD loading rate 0.01 kg BOD m–3d–1; VS 1,980 mg L–1).
Mean BOD and VS removal efficiency were 60.5 and 73.8% for
the piggery and 81.1 and 78.5% for the dairy wastewater anaerobic
lagoons, respectively. Mean biogas production 0.78 and 0.03 m3m–2
d–1, containing 72.0 and 80.3% methane from piggery and dairy
wastewater, respectively, which is equivalent to 0.81 and 0.54 m3CH4
kg–1 VS.
Biogas production is closely linked to temperature, as is COD
reduction and process stability. Optimal biogas production occurs in
mesophilic systems at 38°C, which is rarely achieved under field
conditions without the use of heat exchangers. In a study of covered
anaerobic lagoons treating abattoir wastewater in Queensland,
Australia, Schmidt et al. (2019) found that even the highest natural
lagoon temperature of 31°C represented a reduction of potential
biogas production of 30%. This increased to an 80% reduction at the
lower winter temperature of 25.5°C. They concluded that the natural
ambient temperatures found in covered anaerobic lagoons cannot
ensure sustainable or profitable production of biogas and recom-
mended the use of heat exchangers or waste heat, where available, to
maximize biogas production. Some farms have incorporated passive
136 Anaerobic Wastewater Treatment

solar heating to optimize the temperature. The move from low-rate

anaerobic lagoons operated at ambient temperatures to high-rate
lagoons maintained at 38°C maximizes the returns on investment
(ROI) and overall confidence in the technology, as operating at higher
temperatures also increases operational stability, improves effluent
quality, and improves sludge quality.
Anaerobic lagoons are often used in series with a facultative pond,
followed by at least one maturation pond. The loading to the faculta-
tive pond after the anaerobic stage is calculated using Table 4.4,
which gives the expected percentage BOD reduction at the medium
operating temperature.

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 5
Sludge Digesters

5.1 Introduction
Modern wastewater treatment practice separates primary settlement
from digestion, with septic tanks and Imhoff tanks now superceeded
with larger and more efficient primary settlement tanks, with the set-
tled sludge then being mixed with the settled waste biomass from the
secondary biological unit process, which is then thickened before
being anaerobically digested in a stand-alone reactor. Digestion is car-
ried out either in large open tanks or lagoons (Sec. 4.4) at ambient
temperature (psychrophilic digestion) or more rapidly in covered tanks
heated to between 30 and 35°C (mesophilic digestion) or at 50–70°C
(thermophilic digestion). Psychrophilic digesters are usually unmixed,
and, as they are often uncovered or vented, any gas produced is dis-
persed to the atmosphere as it is uneconomic to collect it. In winter,
the rate of digestion will be extremely low or even zero, whereas in the
summer, digestion may become rapid, inducing mixing as the gas rises.
Therefore, long retention times are required of between 6 and
12 months to stabilize sludges under these conditions and to balance
between sludge accumulation and sludge degradation. Psychrophilic
digesters are restricted to smaller works, where the output of sludge is
low, and land for sludge disposal is readily available. Both septic tanks
and Imhoff tanks fall within the psychrophilic range, which operates
from 5 to 25°C.
Heated digesters are much more cost-effective at larger treat­ment
plants, where there is sufficient sludge available to ensure a continuous

143
144 Anaerobic Wastewater Treatment

Figure 5.1. Single-stage anaerobic sludge digester.

operation, with gas collected and used to either directly or indirectly

heat the digesters. In their simplest form, digesters are intermittently
fed with no recycling. They can be a single-stage reactor (Fig. 5.1),
but in the United Kingdom (UK) and Ireland, conventional digestion
is usually a two-stage process, with the primary digester heated to the
desired temperature to allow for optimum anaerobic activity, with acid
formation and biogas production occurring simultaneously (Fig. 5.2).
The primary reactor is continuously stirred, unlike the earlier stratified
digesters (Fig. 5.3), which reduces the retention time from 30 to 60 d
to <15 d. The secondary digester is unheated and can be used for two
functions. Either to continue digestion under psychrophilic condi-
tions, in which case it will be stirred to ensure complete mixing as in
the primary digester, or it can be used for sludge separation. However,
as the evolution of gas interferes with settlement, the second reactor
cannot be used for both functions. When used for sludge separation,
the secondary digester is not stirred and is generally uncovered
(Fig. 5.4). Under quiescent conditions, the solids separate, leaving a
strong supernatant liquor on top of the stabilized sludge. The liquor
 Sludge Digesters 145

Figure 5.2. Two-stage anaerobic digester.

Figure 5.3. Comparison of conventional and high-rate digestion of sludge.

has a high soluble organic content with a high biochemical oxygen

demand (BOD), which needs to be recirculated back to the inlet of
the treatment plant. The settled solids are removed, dewatered, and
disposed. When the secondary digester is used for digestion, a third
146 Anaerobic Wastewater Treatment

Figure 5.4. Conventional sludge digestion plant.

digester may be employed if thickening is required. This type of fre-

quently fed reactor with no recycling of solids is used for the treatment
of sludges with high solids content, such as animal slurries and sewage
sludge. For example, the waste from a 300-pig unit was sucessfully
treated in such a reactor with a hydraulic retention time (HRT) of 10
days, with a gas yield of 0.3 m3 kg total solids d–1 (Hobson, 1984).
Sludges and liquors with a lower solids concentration can only be
digested in a conventional reactor where there is some form of solids
recycle to prevent wash-out of bacteria. Generally, such systems have
a short HRT of 0.5–5.0 days, and an efficient secondary settlement
system is vital to ensure an adequate SRT in the primary reactor. The
other type of flow-through reactor is the plug-flow type (Fig. 5.5),
which is used mainly for animal manure. However, adequate sludge
recycle is required for seeding purposes, while mixing can be achieved
by gas recycling. The major advantage of this system is that it allows
simple tank configurations to be used, resulting in significant savings
on capital cost (Casey, 1981).
Digestion is used to stabilize both primary and secondary
sludges having a solids content of between 20,000 and 60,000 mg
L–1 (2–6%). About 70% of the sludge is degradable, and up to 80%
of this will be digested, reducing the solids content by about 50%.
The remaining solids form a relatively stable sludge, which has a
characteristic tarry odor and can be difficult to dewater. The mixture
 Sludge Digesters 147

Figure 5.5. Schematic representation of a flow-through plug-flow type anaerobic

reactor.

of primary and secondary domestic wastewater sludges fed into a

digester is rich in carbohydrates, lipids, and proteins, which are ideal
substrates for microbial degradation, and although the sludge is
already rich in a variety of anaerobic bacteria, in order to obtain the
correct balance of hydrolytic acid producers and methanogens, the
raw sludge needs to be initially seeded. This is particularly important
as the main bacterial and archaea groups responsible for digestion
are dependent on the end-products of the other (Sec. 2.2; Fig. 1.3).
Hydolysis and fermentation are the first two stages where particulate
and high molecular weight organic matter is broken down to lower
fatty acids, hydrogen, and carbon dioxide. The end-products are
converted to acetic acid by acetogenic bacteria (third stage), which
is subsequently converted in the final and fourth stage by methano-
genic archaea to methane and carbon dioxide. Obviously, anaerobic
digestion will proceed most efficiently when the rates of reaction at
each stage are equal. However, if either stage one or two is limited,
then the nutrient supply to the other stages will be reduced with the
overall effect of suppressing the rate of digestion and biogas forma-
tion but not inhibiting it (Fig. 1.3). If either of the subsequent
stages is restricted, then the first-stage products will accumulate,
causing a gradual rise in the carbon dioxide fraction in the biogas
(>30%) and a gradual fall in the pH as volatile fatty acids (VFAs)
accumulate until the pH falls below 7.0 and the whole process
becomes stressed. All stages proceed simultaneously within the same
reactor, with the microbial groups in close proximity to each other.
However, as the anaerobic chain of reaction proceeds, the microbial
groups become progressively more sensitive to their environmental
148 Anaerobic Wastewater Treatment

conditions. Hydrolysis and acid formation are carried out by a

diverse and large group of facultative bacteria that can tolerate a
wide variation in temperature, pH, and a range of inhibitory sub-
stances (Sec. 5.4.1). In contrast, the acetogenic bacteria and metha-
nogenic archaea are far more sensitive, being highly specialized and
severely inhibited by even minor changes in operating conditions.
Thus, it is more common to have operational problems involving the
third and fourth stages of digestion (Secs. 2.1.3 and 2.1.4).

5.2 Design
Primary digesters are normally covered with a fixed or floating top for
gas collection. Floating covers rise and fall according to the volume of
gas and sludge, as they are in the same design as conventional gas
holders, with the weight of the floating cover creating the gas pres-
sure (Fig. 5.6). Methane is biochemically inert and can be stored

Figure 5.6. Primary digestion tank with a screw mixing pump and external heater
(Institute of Water Pollution Control, 1979). Reproduced with permission of the
Chartered Institution of Water and Environmental Management.
 Sludge Digesters 149

above the sludge within the reactor without affecting or inhibiting the
process. This allows flexible covers to be used to collect and store gas
above large reactors such as anaerobic lagoons (Fig. 4.21). Modern
digesters generally use fixed covers, which require a separate gas
holder that allows the gas to move freely in both directions (Figs. 5.7
and 5.8). This is particularly important if a vacuum is not to be pro-
duced when sludge is removed from the digester, which would not
only affect operation but could affect the structural stability of a reac-
tor with a fixed cover.
Digesters are usually circular in plan, with maximum diameters of
approximately 25 m, and to ensure the minimum depth, the ratios of
the reactor’s height to diameter is normally between 1:3 and 1:2,
although smaller digesters tend toward a ratio of 1:1. Conventional

Figure 5.7. Two-stage anaerobic digestion with a heated mesophilic digester on

the right and an unheated settlement stage on the left. Excess supernatant (sludge
liquor) is stored in the smaller tank on the extreme left prior to being returned to the
inlet for treatment.
150 Anaerobic Wastewater Treatment

Figure 5.8. Gas storage bag with gas flare in the background where excess gas is
burnt to prevent methane, which is a greenhouse gas, from escaping to the
atmosphere.

tank designs are susceptible to solids settlement and scum formation,

which is exacerbated when the width to height ratio is increased. The
floor of the digester is sloped between 12 and 30°, with facilities
to withdraw heavier solids and grit from the base. Large tanks are
built above ground, and smaller units can be constructed below
ground level or surrounded by earth embankments to provide addi-
tional insulation. Modern digesters are based on an egg-shaped
design, which prevents any settlement or scum formation, ensuring
the entire tank is always fully mixed and available for treatment
(Ditchtl, 1997) (Fig. 5.9).
The entire contents of the tank need to be turned over once every
3–4 hours, with the sludge warmed by heat exchangers using hot
water from gas boilers or the cooling systems of gas engines. Heating
and mixing of digester contents can be achieved by a number of tech-
niques, the most common being a central screw mixing pump with an
 Sludge Digesters 151

Figure 5.9. Design of a modern egg-shaped anaerobic digester that prevents both
settlement of solids and scum formation.

external heat exchanger, or a circulating pump and heat exchanger

housed in a projecting chamber, or internal gas-lift pumps. All these
methods are fully reviewed by the Institute of Water Pollution
Control (1979). The system shown in Fig. 5.6 has a screw pump
mounted on the top of a vertical uptake tube. Known as the “Simplex
System,” it is designed and manufactured by Ames Crosta Ltd. The
pump circulates the contents of the tank by drawing sludge from
the bottom of the reactor and then spraying it out over the surface of
the main body of sludge, providing surface scum control as well as
efficient mixing. At regular intervals, the pump is reversed for a short
152 Anaerobic Wastewater Treatment

period to prevent blockages and improve mixing. The heat exchanger

is external to the digester, the sludge being drawn off from near the
base, heated, and then returned toward the surface so that it is not
immediately drawn up the central updraught tube, thus ensuring
maximum utilization of the heat within the reactor. A simpler impel-
lor system is used in the design (Fig 5.9), which pulls or drives the
sludge up and down a draft tube.
The amount of heat required to raise the temperature of the
incoming sludge to the optimum temperature and maintain it is cal-
culated as:

H = WC ∆T + UA ∆T(5.1)

where H is the heat required to heat the incoming sludge and com-
pensate for heat losses (kg cal h–1), W is the mass rate of influent
sludge (kg h–1), ∆T is the difference between the digester temperature
and influent sludge temperature, U is the heat transfer through the
digester walls (kg cal m–2h–1 °C), A is the surface area of digester los-
ing heat, and C is the mean specific heat of the feed sludge (1 kg cal
kg–1 °C). The basic design criterion is the provision of sufficient
capacity to ensure an adequate sludge retention time. It is common
practice to use 25–30 days as the design retention period to allow for
variation in daily sludge loading. However, as the theoretical reten-
tion time can be as low as 7–10 days, many feel that with the improve-
ments in mixing and heating digesters now available, 15 days allows
an adequate safety margin to cover most operational difficulties.
Digester capacity can also be calculated on a population equivalent
(PE) basis. Assuming a per capita sludge production of 1.8 L of raw
sludge containing 4.5% dry solids, the capacity of a digester with a
25-day retention time is:

1.8 × 2.5 × PE m3
 (5.2)
1000

The volume of a batch digester can be more accurately calculated

using the equation:

Vs = [V1 – 0.66 (V1 – V2)]t (5.3)

 Sludge Digesters 153

where Vs is the digester volume (m3), V1 is the initial sludge volume

(m3), V2 is the final sludge volume (m3), and t is the retention time
(days).
For example, the digestion tank volume required to treat waste-
activated sludge from a treatment plant treating a population equiva-
lent of 35,000 is calculated as follows where the sludge production
rate is 0.1 kg dry solids (DS) ca–1 d–1, the solids content is 3.5% with
a volatile content of 78% and a specific gravity of wet sludge of 1.017.
After digestion, the sludge is 6% DS with a specific gravity of 1.025
and with 65% of the volatile solids destroyed.

Influent sludge production = 35000 × 0.1 = 3500 kg d–1

Volatile suspended solids (VSS) = 3500 × 0.78 = 2730 kg d–1
Fixed suspended solids (FSS) = 3500 × 0.22 = 770 kg d–1
VSS destroyed = 2730 × 0.65 = 1774.5 kg d–1

Remaining VSS in sludge after digestion = 2730 – 1774.5 = 955.5 kg d–1

Remaining FSS in sludge after digestion = 770 kg d–1
Total solids in sludge after digestion = 955.5 + 770 = 1725.5 kg d–1

Influent sludge volume = 3500 × (100/3.5) × (1/1.017) = 98.3 m3d–1

Digested sludge volume = 1725.5 × (100/6) × (1/1.025) = 28.1 m3d–1

Using Eq. (5.3) above, the volume of digested sludge (Vs) is cal-
culated using a sludge residence time of 25 days:

Vs = [98.3 – 0.66 (98.3 – 28.1)]25 = 1299.2 m3

As the sludge in the digester only occupies two-thirds of the tank,

the total tank volume required is:

1299.2 × 1.5 = 1948.8 m3

Although often quoted, organic loadings expressed as kg organic

or volatile matter m–3 are not appropriate parameters for digester
design. With the standard 25-day retention time, the typical sludge
feed of 4.5% total solids with an 85% organic (volatile) matter content
results in an organic loading equivalent to 1.5 kg m–3d–1. However, in
the UK, organic loading ranges from 0.3 to 2.8 kg m–3d–1, indicating
154 Anaerobic Wastewater Treatment

the varying water contents of the sludges being digested. So, in order
to achieve the optimum organic loading of 1.5 kg m–3d–1 with a thin
sludge, a much shorter retention time will result in incomplete diges-
tion. For this reason, sludges should be characterized and loadings
specified as a concentration of total (dry) solids (%).
Secondary digestion tanks are generally used for storage and sepa-
ration, although they can be used for further digestion and gas collec-
tion. Sludge is passed as frequently as possible from the primary
digester into the uncovered tank, where it cools and allows the liquor
to separate from the solids so that each can be withdrawn separately.
Temperature differences between the cool and warm sludge can cause
convection currents within the digester that will hinder settlement, as
will gas production so that it becomes difficult to obtain a solids-free
liquor. To overcome this, secondary digesters are relatively shallow,
with a maximum depth of 3.5 m. The capacity of older tanks was
between 50 and 70% of the primary digester, providing 15–20 days of
retention. Newer tanks are approximately the same size as the primary
tanks and provide similar retention times for the sludge. Tank volume
can be estimated on a population equivalent (PE) basis using the
equation:

Secondary digester volume = 0.035 × PE m3(5.4)

In order to ensure adequate operating conditions for digestion,

the pH, concentration of carbon dioxide in the biogas, and volatile
acid concentrations should all be continuously monitored. The nor-
mal operating conditions are a pH of 7.0–7.2, alkalinity (as CaCO3)
of 4,000–5,000 mg L–1, and a concentration of volatile acids (as acetic
acid) of <1,800 mg L–1. The carbon dioxide content of the biogas
should not exceed 30%, and once any of these values are exceeded,
then immediate remedial action is required.

5.3 Hydrolysis
In order to increase digestibility of sludges, they are often hydrolyzed
prior to entering the digester. This breaks down long-chain organic
molecules into water-soluble and readily degradable short-chain
 Sludge Digesters 155

molecules. The cells rupture, releasing enzymes and increasing the

removal of volatile solids, thus reducing the final volume of sludge
requiring disposal (>20%) and increasing biogas yields by >30% com-
pared to normal digestion. Hydrolysis reduces the HRT in the
digester and allows for more compact and efficient designs (Fig. 5.9),
with the sludge more viscous and easier to pump. Hydrolysis has also
fully replaced thermophilic digestion due to significant cost savings in
heating the digester. There are three types of hydrolysis widely
employed: (i) enzymatic or biological hydrolysis (42–55°C), (ii) pas-
teurization (55–70°C), and (iii) thermal hydrolysis (combined high
pressure and temperature), although chemical pretreatment is also
possible. All these processes ensure complete pathogen destruction as
well as making the sludge far more digestible. The presence of extra-
cellular polymeric substances (EPS) that bind activated sludge flocs
together makes the penetration of extracellular enzymes produced by
hydrolytic bacteria much more difficult, slowing natural hydrolysis
and the rate of digestion overall. The EPS is made up of protein, car-
bohydrates, lipids, and humic acids, which are all potentially degrada-
ble (Sun et al., 2016).

5.3.1 Enzymatic or Biological Hydrolysis

A wide range of enzymes are able to breakdown complex organic
macromolecules, including protease, amylase, lipase, cellulase, urease,
and xylanases, while there are many specific enzymes for treating con-
taminants in wastewaters and sludges as well as specific industrial
waste streams. These include oxygenases to treat fossil fuel derivatives,
peroxidases for treating aromatic compounds, dyes, and organic per-
oxides, laccases to treat phenols and dyes, and tyrosinases for treating
phenolic compounds. While all these enzymes are commercially avail-
able, their use in the pre-treatment or conditioning of biosolids is
largely limited to their use in septic tanks or industrial processes due
to cost.
Biological enzyme treatment is also being considered as a condi-
tioner for waste-activated sludge to increase dewaterability by acting
on the EPS in the sludge flocs. The enzymes destroy the colloidal floc
156 Anaerobic Wastewater Treatment

structure, releasing water and breaking down the larger organic mol-
ecules. Usually, protease and lipase are used, although α-amylase,
alkaline protease, neutral protease, and cellulase are also widely used.
Kang et al. (2023) used two enzymes to determine the optimal condi-
tions for the use of enzymes for waste activated sludge (WAS) condi-
tioning. They observed that for α-amylase, it was 3 hours at pH 6 and
45°C using a dosage rate of 30 mg g–1 TSS (total suspended solids),
and 2 hours for neutral protease at pH 6.5 and 40°C, also at a dosage
rate of 30 mg g–1 TSS. The sludge concentration of proteins and poly-
saccharides both increased in the supernatant, as did the soluble
chemical oxygen demand (sCOD) fraction. This suggests that the
problem of EPS degradation during anaerobic digestion can be solved
by pre-treating the WAS stream separately from the primary sludge
before digestion, ensuring hydrolysis of the EPS. Quintero-García
et al. (2023) have reviewed the use of enzymes in biosolids treatment
and indicate that their future may be in the production of value-added
products from biosolids. However, natural enzymes can be released
from the actual biosolids themselves by fragmenting the organic
material, including the bacteria releasing the enzymes into solution.
This can be done mechanically, by microwaves, or most successfully
using ultrasonication (Chu et al., 2002; Aldin et al., 2009). The
advantage is that not only are the enzymes released immediately, but
the solids are reduced in size, allowing better and more rapid degra-
dation overall. The various sludge pretreatment options are compared
by Uthirakrishnan et al. (2022).
Biological pre-hydrolysis (BPH) separates both hydrolysis and
acidogenesis from methane generation by having two or more anaero-
bic reactors in series, which significantly reduces HRT and also
increases solids loading to between 4 and 6 kg VS m–3d–1. When diges-
tion is completed in a single reactor, any of the non-methanogenic
stages can become the rate-limiting step, with methanogenesis inhib-
ited by a lack of substrate or resulting operational factors such as pH
or alkalinity. Hydrolysis, acidogenesis, and acetogenesis operate more
efficiently under acidic conditions, while methanogenesis operates
better at neutral pH. BPH is achieved by raising the temperature of
stored thickened sludge to encourage hydrolytic and acidogenic bac-
teria to break down large molecules of proteins, lipids, and
 Sludge Digesters 157

polysaccharides, including extra cellular polymers (ECPs), into smaller

soluble molecules. Ding et al. (2017) found that using a temperature
of 42°C for 6 days increased the sCOD from 175 to 3,315 mg L–1,
with the dominant VFA produced being acetic acid, with the concen-
tration increasing from 41.5 to 786 mg L–1, resulting in a significant
increase in methane yield. Wessex Water has installed a successful
BPH system at Avonmouth WWTP (wastewater treatment plant)
using six completely stirred reactors in series before the sludge finally
enters the methane phase digester. Under the trade name of Monsal
Enzymic Hydrolysis™, the plug flow nature of the design ensures no
short-circuiting and allows hydrolysis, acid production, and ace-
togenic phases to occur in sequence. The six tanks together give an
HRT of 2–3 days and are operated at 33°C, although optimum
enzyme production has been recorded at 42°C, with the final meth-
ane digesters operated at 35°C. The hydraulic loading is 1,400 m3d–1
at 5.5% DS, which is equivalent to a loading rate of 78 t DS d–1. The
system has an average solids reduction of 52%, with a biogas output
of between 390 and 430 m3 t–1 DS (Bungay and Abdelwahab, 2008).

5.3.2 Pasteurization
Pasteurization requires the sludge to be preheated to 70°C for a mini-
mum of 30 minutes. There are a number of systems available, for
example, Veolia’s BioPasteur™ system, where biosolids are passed
through batch pasteurization tanks and maintained at 75°C for 30
minutes. Heat is recovered using heat exchangers, and the condi-
tioned biosolids are fed continuously into the digester operated either
at mesophilic or thermophilic ranges. Anglian Water has used pas-
teurization in their HpH technology, which is an acronym for heat
recovery, pasteurization, and biological hydrolysis, which integrates
these three steps. Hot water and steam from existing combined heat
and power (CHP) engines, which convert biogas into electricity, are
used to heat the thickened biosolids to 40–45°C. The sludge is
pumped into the first pasteurization tank and heated to 55°C by recir-
culating it through a steam injector. Once at temperature, it is stored
in a second pasteurization tank for five hours where pasteurization is
completed. The final stage is for the pasteurized sludge to be cooled
158 Anaerobic Wastewater Treatment

to between 38 and 42°C in a separate tank, where it is held for 36

hours for biological hydrolysis to occur. It is then fed into the digest-
ers as required. The process allows much greater organic loading rates
to the digesters with enhanced methane yields 30% greater than con-
ventional digesters, producing on average >1 MWh of renewable
electricity per ton of dry solids via CHP, which is equivalent to 2.5
MWh of biogas energy. With 25% more volatile suspended solids
(VSS) destruction, fewer biosolids are produced, saving costs on
dewatering and producing excellent and pathogen-free Class A bio-
solids, which can be used for a wide range of uses. Both capital and
operational costs are lower than other hydrolysis systems, with all
the required heat coming from the CHP engines helping WWTPs
to become net zero. HpH technology is marketed by Royal
HaskoningDHV under the trade name of Helea™ (Fig. 5.10).

Figure 5.10. The Helea process comprising, from left to right, a heating tank, fol-
lowed by the two pasteurization tanks, and finally, the larger hydrolysis tank. Hot
water and steam are pumped around the system in highly insulated pipework. The
heat exchanger can be seen in the foreground in front of the heating tank. The pho-
tograph is of Basildon WWTP, where 10,300 tons of dry solids are processed annu-
ally. Reproduced with permission of Royal HaskoningDHV. https://www.
royalhaskoningdhv.com/en/services/helea
 Sludge Digesters 159

5.3.3 Thermal Hydrolysis

Thermal hydrolysis uses a feed sludge with 18% DS, which is diluted
with hot water to 12–14% DS. This is then heated to 100°C using
process steam and then fed into a batch reactor where it is heated to
165°C at 6 bar pressure for 90–110 minutes (Fig. 5.11). The heat
disintegrates and liquefies the sludge, which makes it far more degra-
dable. It is then fed into the digester as required (Kepp et al., 2000;
Morgan-Sagastume et al., 2011). The process is energy-intensive, and
it is not always possible to supply all the required power via energy
generated on the plant.

Figure 5.11. Example of a thermal hydrolysis unit used in Ireland. In this case, the
sludge is heated to 180°C at 12 bar of pressure for 40 minutes prior to digestion.

5.4 Digester Operational Management

Successful operational management of anaerobic digesters depends
on six major factors: composition of the raw sludge, method of
160 Anaerobic Wastewater Treatment

addition of raw sludge to the digester, internal mixing and circulation,

temperature, pH and alkalinity, and solids retention time.

5.4.1 Composition of Raw Sludge

Anaerobic microorganisms require certain substrates, growth factors,
trace elements, and nutrients for successful development. Mosey
(1983) compared raw and digested sewage sludge and showed quite
clearly that major substrates such as lipids, cellulose, and some pro-
teins are present as solids in suspension and that a substantial fraction
of the organic matter is either converted to microbial biomass or not
metabolized at all, as is the case with lignin (Table 5.1). Nitrogen and
phosphorus are both vital for bacterial growth and are required at

Table 5.1. Typical composition of sewage sludges before

and after digestion (g per 100 g total solids).
Constituent or test Raw sludge Digested sludge
Suspended solids 95 97
COD 140 100
Organic carbon 40 31
Organic matter 60–80 45–60
Greases and fats 7–35 3.5–17
Cellulose 4 0.6
Hemicelluloses 3 1.6
Lignin 6 8
Protein 22–28 16–21
Anionic detergents 0.5–1.5 0.7–2.2
Zinc 0.09 0.14
Copper 0.035 0.055
Lead 0.016 0.026
Chromium 0.01 0.016
Nickel 0.0092 0.015
Cadmium 0.0022 0.0035
 Sludge Digesters 161

minimum concentrations of 2.5% and 0.5% of the dry organic matter

content of the sludge, respectively. This is equivalent to a C:N ratio
of between 10 and 16:1 and an N:P ratio of 7:1. Huss (1977) found
the optimum BOD:N:P ratio to be 100:0.5:0.1, whereas the
COD:N:P ratio was found to range between 42:0.7:0.1 and
150:0.7:0.1 by Henze and Harremoes (1983). In practice, sewage
sludges have excess nitrogen and phosphorus, ensuring adequate
digestion, although certain trade wastes, including sugar beet and
other food-processing wastes, may be deficient in these nutrients,
making them less amenable to the digestion process (Lettinga, 1995).
Sewage sludges are mixtures of complex organic materials and vary
widely both in composition and strength from place to place. The
average solids content in the UK for sewage sludge is often quoted as
4.5%, although this will vary even from the same source, depending
on the operation of the settlement tank from which the sludge is
removed.
Secondary sludges from activated sludge or percolating filter units
are less amenable to digestion than primary sludges, as they contain a
lower proportion of digestible matter per unit mass of solids as well
as generally contain much more water than primary sludge. Only
about 30% of the available organic and volatile matter is utilized in
activated sludge due to the presence of EPS (Sec. 5.3), compared
with 50% in most primary sludges.
A wide variety of compounds normally present in sewage can
inhibit the digestion process when present in excessive concentra-
tions, most notably detergents, chlorinated hydrocarbons, heavy met-
als, and ammonia. The problem is that many toxic substances are
separated at the primary settlement stage, with organic compounds
such as chloroform being heavier than water and thus collecting at the
outlet hopper in the primary settlement tank and many heavy metal
salts being precipitated at this stage as hydroxides. All inhibitory sub-
stances will affect gas production, as the methanogenic archaea are
particularly prone to inhibition, unlike the diverse hydrolytic and
acid-forming bacteria. Another important factor is that the hydrolytic
and acid-forming bacteria are present in the raw sludge and are con-
stantly replaced; in contrast, the methanogenic bacterial population is
162 Anaerobic Wastewater Treatment

self-sustaining, and once the population has been reduced, it will take
time for the population density to be restored, and, therefore, re-
seeding may be necessary.

Detergents/surfactants
Detergents contain surfactants, which can be classified as non-ionic,
cationic, anionic, or amphoteric (Table 5.2). Non-ionic, cationic, and
amphoteric detergents have little effect on anaerobic digestion, being
largely biodegradable, even at high concentrations (Bruce et al.
1966), whereas anionic detergents are much less or non-biodegrada-
ble anaerobically, often inhibiting the process (Merrettig-Bruns and
Jelen, 2009).

Table 5.2. Common examples of surfactants used in household detergent

formulations.
Surfactants

Anionic Non-ionic Cationic Amphoteric

Soaps Alcohol ethoxylates Mono- or di-alkyl Alkyl betaine
(AE) quaternary
compounds
(DTDMAC)
Alkylbenzene Alkylphenol Esterified mono- Alkylamidopropyl
sulfonates ethoxylates or di-alkyl betaine
(ABS) (APE) quaternary
compounds
(esterquats)
Alkyl sulfates Fatty acid Imidazoline Betaines derived
(AS) alkanolamides derivatives from
(FAA) imidazolines
Alkyl ether Alkylamine oxides Alkylamphoacetates
sulfates (AO)
(AES)
Secondary alkane N-Methylglucamides
sulfonates (NMG)
(SAS)
Alkylpolyglycosides
(APG)
 Sludge Digesters 163

Soaps, which are salts of natural fatty acids, are readily degraded
anaerobically up to concentrations of 1,000 mg L–1. Most household
detergents comprised a significant proportion of anionic surfactants,
primarily alkylbenzene sulphonates (ABS) and, in particular, linear
alkylbenzene sulphonates (LAS). LAS are non-degradable anaerobi-
cally, and as they are strongly adsorbed onto organic solids, they are
invariably present in sewage sludge. Anionic detergents at concentra-
tions >1.5% of the dry raw sludge solids (expressed as Manoxol OT)
inhibit anaerobic digestion, even in heated digesters with relatively
long retention times of up to 40 days, with gas production being
particularly sensitive. Concentrations <1.5% will reduce the tolerance
of the process to other operational variables, such as temporary over-
loading. The effects of ABS on the microbial community are numer-
ous and include direct toxicity. Although degraded aerobically, it is
clear that these detergents are not utilized in the anaerobic process
because the enzymes responsible for their degradation, such as mono-
oxygenase (Cain, 1981), are not able to function due to the lack of
oxygen. These concentrations are quite common in domestic sewage
sludges, and although bacterial populations can become acclimatized,
serious inhibition will occur if the concentration is allowed to rise to
2.0% of the dry sludge solids. As digestion proceeds, the concentra-
tion of anionic detergents will increase from 1.5% in the raw sludge
to 2.5% DS in the digested sludge. This is not a problem when the
sludge is finally disposed of to agricultural land, as it will rapidly
degrade the aerobic soil environment. If inhibition occurs during
digestion, then the anionic detergents can be rapidly neutralized by
the addition of long-chain fatty amines, such as stearine amine, to the
sludge prior to digestion (Swanwick and Shurben, 1969). Some ani-
onic surfactants are biodegradable anaerobically, and these include
linear alkyl sulfosuccinates and alkyl sulfates (EU, 2016).
The biodegradability of detergents is summarized in the European
Union’s Detergents Ingredients Database (DID), which gives both
the acute and chronic toxicity levels for all detergents used in the
European Union, including details of whether they are degradable
under aerobic and anaerobic conditions (EU, 2016). The anaerobic
breakdown of surfactants has been reviewed by Merrettig-Bruns and
164 Anaerobic Wastewater Treatment

Jelen (2009), who give more precise biodegradability data than the
DID. The current problem with the use of detergents is that they are
only required under European Commission (EC) legislation to be
biodegradable under aerobic conditions, but with the increased use of
anaerobic wastewater treatment, there is a need for them to also be
biodegradable anaerobically. However, as can be seen in the EU DID,
only a fraction of the permitted surfactants used in detergent formula-
tions have been tested for their biodegradability under anaerobic
conditions (EU, 2016).

Chlorinated hydrocarbons
Although high concentrations of these solvents are unusual in sewage
sludges, they can cause a problem in certain industrial wastes or where
domestic sewage contains industrial discharges. The most frequent
source of this group of inhibitors is from dry-cleaning operations that
discharge chlorinated hydrocarbons into the sewer. Although their
effect on the process depends on several factors, it is the concentra-
tion in the sludge that is most critical. It appears that many chlorin-
ated hydrocarbons act selectively on the methanogen organisms,
which is why they are so toxic to anaerobic digestion compared to
aerobic systems (Blum and Speece, 1992). The concentration at
which 20% inhibition occurs is summarized in Table 5.3 for the most
widely used chlorinated hydrocarbons, although their inhibitory
effect will be increased if other inhibitory substances are also
present.
The most common chloro-aliphatic compound, chloroform
(CHCl3), is toxic to methanogens, and while most of the compound
will be lost by volatilization in the sewers or during aerobic treatment,
it can still cause detectable inhibition to methane production at con-
centrations as low as 10 mg kg–1 DS. Hickey et al. (1987) reported
complete inhibition of methane production at concentrations >1 mg
L–1 due to the compound binding to intercellular proteins, thereby
blocking essential binding sites in the methanogenic pathway (Jha and
Schmidt, 2017). Where chloroform is a problem, it should be
 Sludge Digesters 165

Table 5.3. Concentration of chlorinated hydrocarbons

in digesting sludge at which 20% inhibition occurs
(Institute of Water Pollution Control, 1979). Reproduced
with permission of the Chartered Institution of Water
and Environmental Management, London.
Concentration
Chemical (mg kg–1 dry solids)
Chloroform 15
Trichlorethane 20
1,1,2-Trichlorotrifluoroethane 200
Carbon tetrachloride 200
Trichloroethylene 1800
Tetrachloroethylene 1800

removed from the effluent by air-stripping prior to discharge into the

sewer, although some acclimation to the compound is possible (Lumb
et al., 1977). Benzene ring compounds such as benzene, toluene,
phenol, and chlorophenols all inhibit methanogens (Patel et al.,
1991; Wang et al., 1991). Even when protected by the granulated
sludge in floc-based reactors (Chap. 7), organics such as aromatic
compounds are still strong inhibitors of methanogens (Donlon et al.,
1995; Fang et al., 1997). Chlorophenols are all inhibitory to metha-
nogenesis, the most toxic being pentachlorophenol (PHP), with a
50% inhibition of biogas production at 8 mg L–1 reported. This has
been related to their hydrophobicity, which allows the compound to
adhere to bacteria membranes, affecting their integrity and cellular
metabolism (Sikkema et al., 1995). There is some evidence that
anaerobic microorganisms can become acclimatized to chlorinated
hydrocarbons. For example, Lian et al. (2020) demonstrated how
hexachlorocyclohexanes can be used as a food substrate during anaer-
obic digestion, with the genus Methanosaeta developing a high toler-
ance. The effects of aromatic compounds and halogenated aliphatics
on anaerobic digestion have been reviewed by Jha and Schmidt
(2017) and Nielsen et al. (2020).
166 Anaerobic Wastewater Treatment

Heavy metals
The most quoted cause of inhibition of sludge digesters is by heavy
metals, in particular, chromium, copper, nickel, cadmium, and zinc,
although many metals are essential for the function or activation of
critical enzymes and coenzymes during digestion (Takashima and
Speece, 1989). Metals are non-degradable and so can accumulate
within the digester, and can also be released into solution during fer-
mentation, but in practice, high concentrations of metals are required
for a significant inhibitory effect (Table 5.4). It is thought that the
heavy metals inactivate enzymes and co-enzymes, interfering with
their function and thereby inhibiting microbial activity and growth
(Oleszkiewicz and Sharma, 1990).
Toxicity of heavy metals to anaerobic digestion increases as the
metal affinity for sludges decreases. Nickel has the lowest affinity for
sludge, and so is the most toxic, and vice versa for lead (i.e., toxicity
to digestion is Ni>Cu>Cd>Cr>Pb). Lin (1992) gives IC50 values for
Cd2+, Cu2+, Zn2+, and Pb of 7.7, 12.5, 16, and 67 mg L–1, respectively.
While using pure cultures of Methanobacterium thermoautotrophicum
and Methanobacterium formicicum, Jarrell et al. (1987) found signifi-
cant differences in the IC50 values for the two archaea, respectively, for
Cu2+ at 4.8 and 3.5 mg L–1, Zn2+ 3.0 and 8.2 mg L–1, and for Ni2+ 1.2
and >15 mg L–1.
Synergistic effects have been noted with heavy metals and a num-
ber of other inhibitory substances, and under certain conditions, even

Table 5.4. Concentrations of heavy metals in digesting sludge that cause a 20%
reduction in gas production in laboratory experiments (Institute of Water Pollution
Control, 1979). Reproduced with permission of the Chartered Institution of Water
and Environmental Management, London.
Batch digesters: concentration Typical concentration in digested
Metal (mg kg–1 dry solids) sludges (mg kg–1 dry solids)
Nickel 2,000 30–140
Cadmium 2,200 7–50
Copper 2,700 200–800
Zinc 3,400 500–3,000
 Sludge Digesters 167

low concentrations may cause problems. Apart from the concentra-

tion of heavy metals in the raw sludge, other factors such as solubility,
pH, and the concentration of sulfide present will all affect their avail-
ability in the digester. Anaerobic microorganisms can withstand quite
high concentrations of total metals, as a considerable percentage of
the metal ions can be precipitated out of solution as either sulfides or
carbonates. The concentrations of metals that can be present in waste-
water without adversely affecting the anaerobic digestion of the
resulting sludge have been summarized in Table 5.5. There is always
more than one heavy metal present in domestic wastewater sludges,
and the inhibitory effects are additive on an equivalent weight basis
(Mosey, 1976). Where the milligram equivalent weight (meq) per kg
dry sludge solids (K) exceeds 400 meq kg–1, there is a 50% chance of
digester failure, which rises to 90% when K exceeds 800 meq kg–1. To
ensure a 90% probability that digestion will not be affected, the value
of K should be <170 meq kg–1.
K is measured using the concentrations of the most abundant
heavy metals (mg L–1) in sewage sludge, excluding chromium, using
the equation:

K =
(Zn/32.7 ) + (Ni/29.4) + (Pb/103.6) + (Cd/56.2) + (Cu/47.4) meq kg −1
Sludge solids concentration (kg L−1 )
(5.5)

Table 5.5. Highest metal concentrations in sewage that will allow

satisfactory digestion of sewage sludge.
Concentration in influent
sewage (mg L–1)

Primary sludge Combined sludge

Metal digestion digestion
Chromium (hexavalent) >50 >50a
Copper 10 10
Nickel >40 >10a
Zinc 10 10
a
Higher dose not studied.
168 Anaerobic Wastewater Treatment

The effects of heavy metals on anaerobic digestion are reviewed

by Jha and Schmidt (2017).
Some metals at trace concentrations (e.g., nickel, cobalt, man-
ganese, iron, and molybdenum) stimulate methanogens and increase
biogas production (Murray and van der Berg, 1981; Basiliko and
Yavitt, 2001). Other metals (e.g., Cu2+, Cd2+, and Cr4+) have also
been shown to have stimulatory effects at very low concentrations
in experimental studies on the anaerobic degradation of cattail
waste using rumen cultures (i.e., 2.4, 1.6, and 4.0 mg L–1, respec-
tively), rapidly accelerating inhibition as the concentration increased,
resulting in complete inhibition of biogas production at 30 mg Cu2+
L–1, 20 mg Cd2+ L–1, and 60 mg Cr4+ L–1 (Yue et al., 2007). The role
of metal in anaerobic wastewater treatment is explored further in
Sec. 9.2.2.

Ammonia
Ammonia or ammonium ions are essential nitrogen sources for anaer-
obic digestion but can be inhibitory when present at high concentra-
tions. The optimum C:N:P:S ratio for methanogenesis is 600:15:5:3,
so not all waste streams will have significant excess nitrogen. A mini-
mum concentration of 200 mg L–1 is required to prevent nitrogen
deficiency. Ammonia also plays an important role in buffering the pH
during digestion, requiring a balance between free ammonia nitrogen
(NH3) (FAN) and ammonium ions (NH4+), with the total ammonia
nitrogen (TAN) the total concentration of both. The relative propor-
tions of the NH3 and NH4+ in the digester are largely dependent on
the pH and temperature (Eqs. (3.26) and (3.27)) (Sec. 3.5).
However, it is FAN which methanogens are particularly sensitive to,
showing the importance of maintaining as near a neutral pH as pos-
sible during methanogenesis. Methanogenic activity in anaerobic
digestion has been reported as being 50–100% inhibited at FAN con-
centrations of >600 mg L–1 or at TAN concentrations >4,000 mg L–1,
although the exact level of inhibition varies depending on digester
temperature and pH, the microbial community, and reactor
configuration.
 Sludge Digesters 169

Inhibition of methanogenic metabolism by ammonia is probably

caused by a number of mechanisms, such as FAN limiting the expres-
sion of key enzymes and/or proton imbalance and deficiency in
potassium (Duc et al., 2022; Yu et al., 2021; Li et al., 2023). It is
generally accepted that acetoclastic methanogens are more sensitive to
ammonia than hydrogenotrophic archaea, although there are a num-
ber of examples showing the opposite being the case. In a pilot study,
Yan et al. (2019) studied the acclimatization of methanogens over a
period of 127 days, where the ammonia concentration was increased
in seven steps up to 8.5 g NH4+ L–1. They clearly demonstrated that
archaea had different tolerances to ammonia inhibition. There was a
discernible shift from the original community structure dominated by
both hydrogenotrophic and acetoclastic methanogens to more
ammonia-tolerant and metabolically versatile acetoclastic methano-
gens, with Methanosaeta concilii (specialist species only able to use
acetate) and Methanosarcina soligelidi (general species able to use
H2/CO2, acetate and methanol), the dominant archaea at low (1.1 g)
and high (8.5 g) ammonia concentrations, respectively. What is inter-
esting is the authors were able to show how M. soligelidi changed its
metabolic pathway from acetoclastic to hydrogenotrophic methano-
genesis as acclimatization progressed. Ammonia toxicity also targets
the pathways from propionate to acetate (propionate-oxidizing bacte-
ria) and acetate to methane (archaea). Under normal operating condi-
tions, it is expected that hydrogenotrophic methanogens will
outcompete acetoclastic methanogens due to higher growth rates and
greater ammonia tolerance (Guo et al., 2023). However, this is not
always the case. Guo et al. (2023) compared two pilot anaerobic
digesters operated constantly at 600 mg NH4+-N L–1 (control, R-C)
and at 2,000, 3,000, 4,000, and 5,000 mg NH4+-N L–1 each for a
period of 30 days, starting at the control concentration of 600 mg
NH4+-N L–1 through urea regulation (R-A). They found that lignin-
and cellulose-degrading bacteria dominated both reactors R-C and
R-A, with increasing ammonia not affecting the species richness of
hydrolysis bacteria. The total abundance of methanogens in R-C
slowly increased from 1.6% to 3.8% over the test period of 150. In
R-C, five species of methanogens were recorded: the acetoclastic
170 Anaerobic Wastewater Treatment

methanogens Methanosaeta harundinacea, Methanosaeta concilii, and

Methanosaeta thermophile, and the hydrogenotrophic methanogens
Methanolinea tarda and Methanolinea sp. Ammonia reduced the
diversity of methanogens as the concentration increased, with
Methanosaeta concilii and Methanosarcina barkeri recorded. Both are
acetoclastic archaea, with the former dominant up to 2,000 mg
NH4+-N L–1 and the latter becoming increasingly dominant up to
4,000 mg NH4+-N L–1. At 5,000 mg NH4+-N L–1, both species were
inhibited (Fig. 5.12). Like Methanosarcina soligelidi, Methanosarcina
barkeri is also able to utilize several metabolic pathways, including
acetate, H2/CO2, methanol, and pyruvate.
Methanogenesis is more sensitive to ammonia inhibition than
acidogenesis, so as the former is inhibited, the latter continues to
produce VFAs, which build up as the methanogens are unable to uti-
lize them at the same rate. The VFAs, especially propionate acid, cause
an increase in acidification of the digestate, which initially inhibits
methanogenesis, even further creating a feedback loop that increas-
ingly reduces methane production (Yuan et al., 2016). However, as
Mosey (1983) explains, the system is largely self-regulating, with the
drop in pH converting FAN to the less toxic ammonium ionic form
(NH4+), thus alleviating inhibition and allowing some degree of
microbial acclimatization. These high TAN and FAN concentrations
are mainly found in very thick sewage sludges and undiluted farm
slurries. Ammonia N is also generated within the digester by the deg-
radation of proteins, amino acids, urea, and nucleic acids.
Ammonia inhibition remains a common operation problem, with a
number of control options available to operators. Where process opera-
tion allows, then ammonia concentrations in the feed can be reduced
by extraction processes such as air-stripping, blending sludges to
reduce overall TAN concentrations, the use of trace element supple-
ments (Cai et al., 2023), or altering operational parameters such as pH,
temperature, or C:N ratio (Cai et al., 2021; Chen et al., 2021).
Ammonia-stripping (e.g., pre-treatment, in-situ, side stream, and post-
treatment) is an effective method for ammonia removal to prevent
ammonia inhibition during digestion (Fig. 5.13), producing a range of
useful byproducts, and has been reviewed by Yellezuome et al. (2022).
 Sludge Digesters 171
Figure 5.12. Shift in microbial community structure, with ammonia concentration using a pilot-plant anaerobic digester over a
period of 150 days. Ammonia concentrations are at 600 mg NH4+-N L–1 for the control digester (RC) and for the experimental
digester (RA) at 600 (-0d, -30d), 2,000 (-39d, -60d), 3,000 (-69d, -90d), 4,000 (-99d, -120d), and 5,000 (-129d, -150d) mg
NH4+-N L–1, each for a period of 30 days. The values in parentheses are the dates of sampling in days from the start of the experi-
ment (Guo et al., 2023). Reproduced with permission of Elsevier Publishing.
 172 Anaerobic Wastewater Treatment
(a) (b)

(c) (d)

Figure 5.13. Various configurations of ammonia stripping used in anaerobic digestion: (a) Pre-treatment, (b) in situ, (c) side-
stream, and (d) post-treatment (Yellezuome et al., 2022). Reproduced with permission of Elsevier Publishing.
 Sludge Digesters 173

The addition of carbon- or iron-based materials, including biochar and

hydrochar, has also been successful (Shi et al., 2021; Cai et al., 2022;
Wang et al., 2024). Usman et al. (2021) found that the addition of
hydrochar to the feed not only increased methane yields up to an
ammonia concentration of 6,000 mg L–1 but also promoted methano-
genic activity and diversity. They found that under ammonia inhibition
conditions, the hydrochar regulated the metabolites in the methane
metabolic pathway from hydrogenotrophic methanogens, using hydro-
gen and carbon dioxide as electron acceptors, to multiple pathways
(i.e., acetoclastic, using acetate and/or methylotrophic, using metha-
nol or methylamines) for methanogenesis, thereby maintaining meth-
ane yields. Other control methods include microbial acclimatization or
bioaugmentation (Shi et al., 2021; Li et al., 2023). Acclimatization to
high ammonia concentrations is probably due to changes in the meta-
bolic pathways (Sec. 2.2), although whether this is due to particular
species changing pathways or changes in diversity to more tolerant
species with different metabolic capabilities is unclear (Niu et al., 2015;
Esquivel-Elizondo et al., 2016).

Other ions
Methane formation does not occur readily in the presence of electron
acceptors, such as sulfate and nitrate (Jha and Schmidt, 2017). Sulfate
can be a particular problem in the digestion process if present in suf-
ficient quantities. The sulfate is reduced to sulfide by bacterial action,
with hydrogen sulfide eventually being formed. Sulfate concentra-
tions >500 mg L–1 can reduce methane production and generate up
to 4% hydrogen sulfide in the biogas. The hydrogen sulfide will form
insoluble compounds with heavy metals. Therefore, as long as hydro-
gen sulfide and heavy metals are present in equivalent proportions,
then hydrogen sulfide production will cause no problems and, in fact,
be beneficial by removing heavy metals that may cause inhibition.
However, two problems can occur: (i) hydrogen is consumed by sul-
fate reduction and is no longer available for methane formation,
which is inhibited, and (ii) hydrogen sulfide itself has a direct toxic
effect on the methanogenic bacteria being toxic at concentrations
174 Anaerobic Wastewater Treatment

>200 mg L–1 (Mosey, 1976). Remedial action involves precipitating

the excess hydrogen sulfide out of the solution as ferric sulfide by the
addition of an iron salt, such as ferric chloride or ferric oxide, but not,
of course, ferric sulfate (Sec. 8.2).
Nitrates can also cause problems because if denitrification occurs
within the digester, there will be a shift in the redox potential, which
will suppress methane production (Akunna et al., 1994; Klüber and
Conrad, 1998). Methanogens are strict anaerobes and can be com-
pletely inhibited by dissolved oxygen concentrations as low as 0.01
mg L–1 (Wolfe, 1971). They require a reduced environment with a
redox potential within the range of –200 to –420 mv. In a bench scale
experiment, Sheng et al. (2013) observed that a total ammonia nitro-
gen (TAN) concentration of <1.0 g L–1 had no effect on anaerobic
digestion and was, in fact, beneficial. The subsequent addition of
nitrate reduced methane production by 12 and 51% at NO3-N con-
centrations of 1.0 and 1.5 g L–1, respectively, although at 0.5 g L–1,
methane production was enhanced by 12% over the control with no
nitrate giving a maximum methane yield of 315 mL g–1 VS. This has
been a serious problem at many WWTPs in the UK and has been
remedied by allowing denitrification to occur before the sludge enters
the digester, with the nitrate being converted to nitrogen gas under
anoxic conditions. The advent of aerobic-anaerobic-anoxic biological
nutrient removal has greatly reduced this problem.

Inhibition testing
Laboratory screening to test the biodegradability of wastewaters and
assess the possible inhibitory effects of wastewaters and chemicals
should always be carried out prior to anaerobic digestion (HMSO,
1987; 1989).
Screening tests fall into two categories: biodegradability and inhibi-
tion/toxicity, with both relying on gas production. Standard tests for
anaerobic digestion have been produced by all the main standards
organizations, including the International Organization for
Standardization (ISO), British Standards Institution (BSI), American
Society for Testing Materials (ASTM), Deutsches Institut für Normung
(DIN), Comité Européen de Normalisation (CEN), Organization for
 Sludge Digesters 175

Economic Cooperation and Development (OECD), and European

Chemical Industry Ecology and Toxicology Centre (ECETOC).
Although, in practice, they are all quite similar, individual countries
tend to use their own standard methods, with the ISO and OECD
procedures the most widely used. There is also an array of research-
based tests, some of which are more rapid than the conventional stand-
ard test methods (Kawahara et al., 1999; Astals et al., 2015).
Anaerobic biodegradability can be measured using the test
method of either the ISO (ISO 11734:1995), the OECD (OECD
311:2006), or ASTM (ASTM E1192-92) (ISO, 1995; ASTM, 1992;
OECD, 2006). Two tests have been developed by the ISO for deter-
mining the inhibition of gas production by test chemicals, using either
a concentrated anaerobic digesting sludge (ISO, 13641-1: 1003) or
diluted sludge (ISO 13641-2: 2003) (ISO, 2003a; 2003b). A similar
test has been published by the OECD (OECD 224: 2007). In this
test, samples of actively digesting anaerobic sludge, with a total solids
concentration of between 20 and 40 g L–1, are mixed with a readily
degradable soluble substrate, to which a range of different concentra-
tions of the test chemical or wastewater is added and then incubated
at 35 ± 2°C in a sealed bottle for 3 days (Fig. 5.14). The gas pro-
duced, both methane and carbon dioxide, is measured using a pres-
sure gauge to monitor the increase in pressure in pascals (Pa). The
percentage difference between the gas production at different con-
centrations of the test compound to the control allows the percentage
inhibition to be plotted against the Log concentration of the test
compound, from which the EC50 can be calculated (OECD, 2007).
Test methods have been reviewed by Müller et al. (2004) and
Rozzi and Remigi (2004), while Gartiser et al. (2007) give an excel-
lent example of using ISO tests to assess the anaerobic inhibition and
biodegradation of antibiotics.

5.4.2 M
 ethod of Sludge Addition to Digester and
Commissioning
Digesters can be operated either as batch or continuous processes that
may incorporate the recycling of solids, gas, or both. For optimum
performance, sludge should be added to the digester as frequently as
176 Anaerobic Wastewater Treatment

Figure 5.14. Test bottle for anaerobic biodegradability and toxicity testing used in
ISO testing.

possible in order to avoid fluctuations in gas production or problems

with scouring the active bacteria from the primary digester. It is not
generally possible to feed sludge directly from a sedimentation tank on
a continuous basis, as it requires consolidation or thickening before it
is suitable for use. Although the majority of digesters are fed with raw
sludge at least once a day, two or three times a day is preferable.
Commissioning digesters is a topic of some controversy, with two
approaches generally used. One is to fill the tank with settled sewage
and seed sludge; the other is to fill the tank with primary sludge and
add a seed if available. In the first method, the digester is filled with
settled sewage to a level where circulation and heating can be oper-
ated. While the settled sewage quickly becomes anaerobic, it is advan-
tageous if the headspace and gas piping are purged with nitrogen gas
 Sludge Digesters 177

to exclude all oxygen. Once the digester has reached its operating
temperature, the seed sludge can be added, equivalent to a minimum
of 5% of the total digester volume, with the sludge feed then added
slowly over several weeks. The advantage is that the amount of vola-
tile acids is limited, ensuring correct operating pH and alkalinity.
However, if the seed sludge becomes too diluted, the correct balance
of microorganisms is not maintained, resulting in incomplete diges-
tion and producing unpleasant odors. Although it is possible to use
raw sludge, which has been stored for several months as a seed, it is
best to use sludge from another digester, preferably from the same
plant. The quality of the seed sludge is very important and should
only be taken from an operating digester with a good methanogenic
population and biogas production rate. The ratio of seed sludge to
raw sludge should be between 1:10 and 1:5. Once started, the per-
centage of seed can be increased, but care must be taken to prevent
excessive acid production from inhibiting the development of metha-
nogens. There will be little biogas production for the first 4–6 weeks,
so an alternative source of heating the reactor during commissioning
will be necessary. Examples of full-scale digester commissioning using
this method have been detailed by Hatzigeorgiou et al. (2006) and
experimentally without using a seed sludge by Dohdoh and
Aboullfotoh (2017), who achieved a volatile fatty acid/alkalinity
(V/A) ratio of 0.2 after 55 days, and stable operation after 73 days,
taking much longer than when a seed was used.
The alternative method is to gradually fill the digester with dilute
primary sludge together with the seed sludge. The digester is then
heated and mixed (i.e., with a sludge circulation pump and heat
exchanger, as there will be no digester gas to operate a gas mixing
system), which can take up to 4 days to reach optimal mesophilic
temperature. Once at the required temperature, seed sludge can be
added if available. The volatile solids concentration, pH, and alkalinity
should be monitored throughout. Thickened sludge can be slowly
added, ensuring that the <5% new volatile solids are added each day.
An increase in the loading rate is governed by the process parameters,
and if they fall outside the recommended range, feeding should be
temporarily reduced or stopped. Once digester gas becomes available,
it can then be used for internal mixing and heating via CHP. Normal
178 Anaerobic Wastewater Treatment

loading is achieved after 30 days when a seed has been used, or 60–90
days without the addition of a seed. It is not recommended to gradu-
ally fill the digester with sludge as it is not possible to commence
circulation or heating until the reactor is full, which can lead to prob-
lems in starting up due to the compaction of solids at the base.
A range of methods can be found in USEPA (1976).
Key process control parameters should be regularly monitored dur-
ing commissioning and during normal operation (Bajpai, 2017). The
pH should be as close to neutral as possible — if the pH drops, this
indicates an increase in volatile acid production. VFA concentration of
the digesting sludge should decrease as methanogenesis begins; if it
rises, this is an indicator of problems. Biogas composition should show
a slow decrease in carbon dioxide composition and a rise in methane; if
the opposite starts, again, this indicates a problem. Temperature must
stay constant and any instability will impact methanogenesis. Monitoring
should include the flow rate (m3d–1) and solids concentration (%) of the
feed sludge. Digester efficiency can be measured by calculating the
reduction in volatile solids, which should be 40–60%, although overall
gas production per kg VS consumed is also used. The V/A ratio is cal-
culated as the volatile acids concentration in digesting sludge (mg L–1)
divided by the alkalinity (mg L–1) and should be <0.1. It is an important
parameter that can indicate process instability during commissioning, in
particular. If the ratio increases, the pH will drop as the volatile acids
build up, more CO2 is produced in the biogas, and methane formation
declines due to the inhibition of methanogenesis (Sec. 5.4.5).

5.4.3 Internal Mixing and Circulation

The purpose of mixing and circulation within the digester is: (i) to
promote close contact between the raw and digesting sludges; (ii) to
maintain a uniform temperature and solids mixture throughout
the tank and prevent localized accumulation of inhibitory substances;
(iii) to discourage scum formation and settlement of grit and dense
solids; and, (iv) most important of all, to encourage the release of gas
from the sludge in the lower regions of the digester. Poor mixing will
lead to stratification within the digester and will result in partially
 Sludge Digesters 179

digested sludge being withdrawn. Efficient mixing turns the conven-

tional plug-flow reactor into a high-rate digestion process by making
use of the total reactor volume and ensuring a faster reaction rate due
to the removal of mass transfer limitations of food and microorgan-
isms. Mixing can be done either mechanically or by the recirculation
of biogas. Mixing is enhanced by the circulation of sludge through
the heat exchanger that operates continuously. The evolution of gas,
which reaches a maximum of 2–3 hours after the addition of raw
sludge, will also supplement the mixing effect. The actual mixing
mechanism may not be operated continuously at all plants.
The need for optimal mixing to maximize digestion has led to
alternative reactor designs. In small to medium-sized digesters, this has
resulted in the development of egg-shaped reactors that can achieve
near-perfect mixing, thereby avoiding stagnant areas of settled debris
and sludge as well as reducing scum formation (Fig. 5.15). Small
to medium-sized conventional tank digesters are very susceptible to
particulate settlement and scum formation, seriously reducing mixing

Figure 5.15. Design of an egg-shaped anaerobic digester and heat exchange system
(Subramanian et al., 2015). Reproduced with permission of Elsevier Publishing.
180 Anaerobic Wastewater Treatment

and operational efficiency (Figs. 5.16 and 5.17). This problem has
become worse as the diameter of shallow digesters is increased, with
small ovoid-shaped mixing zones formed within the tanks where the
reaction between the liquid sludge and biomass occurs. In contrast,
egg-shaped digesters ensure the entire area of the reactor is utilized for
treatment (Dichtl, 1997).
Scum, which is composed of grease, oil, and soaps with floating
material entrained, will tend to form on the surface of the digestion tank
if mixing is not adequate. It causes a number of problems, such as

Figure 5.16. Mixing patterns in conventional and egg-shaped digesters showing

potential for scum and sediment accumulation (Ditchtl, 1997). Reproduced with
permission of the Chartered Institution of Water and Environmental Management,
London.
 Sludge Digesters 181

Figure 5.17. Cross-section through a conventional (American) digester (Ditchtl,

1997). Reproduced with permission of the Chartered Institution of Water and
Environmental Management, London.

reducing the effective capacity and thus the retention time of the reac-
tor, interfering with mixing and the internal circulation of the sludge,
interfering with the movement of the floating gas holder, and restricting
the evolution of gas. Most mixing systems withdraw the sludge from the
base of the tank and spray it over the surface, preventing scum forma-
tion, and screw pumps can be reversed to carry any scum down into the
body of the tank. Prevention is best, however, and the sludge should be
adequately screened before entry to the digester, and excessive grease
and oil should be removed prior to discharge to the sewer. There is also
a number of design features aimed at reducing scum formation, includ-
ing scum removal devices, which disperse sludge by discharging raw or
digested sludge into the surface layer via jets or nozzles.

5.4.4 Solids Retention Time

The period that solids are retained in the digester is a crucial factor
affecting performance. In flow-through systems, the residence time of
the waste (HRT) and microbial biomass (MCRT) will be the same. If
complete digestion is to be achieved in heated mesophilic digesters,
182 Anaerobic Wastewater Treatment

then minimum retention times of more than 15 days are required to

ensure that the methanogenic bacteria, which are slow-growing, are
able to accumulate to a sufficient population density. The design of
digesters normally aims at a minimum retention period of 25–30
days, allowing for some loss in digester capacity due to accumulated
non-biodegradable solids within the reactor. Clearly, any increase in
the water content of the raw sludge will increase the HRT, and in
flow-through systems, this will cause an increased wash-out rate of the
microbial biomass and an associated reduction in performance. As
mentioned earlier, the minimum MCRT is dependent on the operat-
ing temperature of the reactor.

5.4.5 Temperature
Anaerobic digestion can occur over a wide temperature range (Table 5.6),
which has generally been subdivided into three separate ranges: psychro-
philic (5–25°C), mesophilic (25–38°C), and thermophilic (50–70°C)
digestion. The rate of anaerobic digestion and gas production is temper-
ature-dependent (Fig. 5.18), with optimum gas production at the
higher temperature ranges. Therefore, the warmer the reactor, the
shorter the MCRT needs to be for complete digestion (Table 5.7).

Table 5.6. Examples of optimum ranges for pH and

temperature for different methanogen genera.
Genus pH Temperature (°C)
Methanothermus 6.5 40–85
Methanogenium 6.5–7.8 30–35
Methanolacinia 6.6–7.2 35–40
Methanospirillium 6.6–7.4 37
Methanomicrobium 6.7–7.0 36–38
Methanosphaera 6.8 30–40
Methanolobus 7.0–7.2 37
Methanococcoides 7.0–7.5 30–35
Methanohalobium 7.0–7.5 40-55
Methanothrix 7.1–7.8 37
 Sludge Digesters 183

Table 5.7. Suggested mean cell residence

times (MCRT) for the anaerobic digestion
of sewage sludge at various temperatures.
Temperature (°C) MCRT (days)
18 28
20 22
25 18
30 14
35 10
40 8–10

Figure 5.18. Effect of temperature on gas production.

Septic tanks, Imhoff tanks, sludge lagoons, and unheated sludge

digesters, which are located mainly at smaller treatment plants, fall
into the psychrophilic range. At ambient temperatures in temperate
climates, the rate of digestion is so low that the residence time of the
bacteria needs to be of the order of 3–12 months. Suitable bacteria
for seeding such systems acclimatized to these low temperatures can
be obtained from marshlands. The majority of heated sewage sludge
digesters operate in the mesophilic range, usually between 30
and 32°C, with maximum residence times of 20–40 days. Sludge
184 Anaerobic Wastewater Treatment

residence time (Sec. 5.4.6) is significantly reduced where mixing is

enhanced and where the sludge is pre-treated (Sec. 5.3). Thermophilic
digestion is technically feasible, and a number of plants have been
built and operated successfully. However, the rate of gas production
does not increase continuously with temperature but rapidly declines
after reaching an optimum at 55°C. A similar situation is found in the
mesophilic range at 35°C (Fig. 5.18), indicating that different micro-
bial populations are responsible for thermophilic and mesophilic
digestion rather than thermo-tolerant mesophilic species. It appears
that between these optimal temperatures, erratic gas production will
be encountered. Inhibition of methanogenic archaea occurs at about
63°C (Pfeffer, 1979). The effect of temperature is not great on the
first stage of anaerobic digestion, as so many different species are
involved that the operating temperature will always fall within the
optimum range of some of the microorganisms present. However, the
acetogenic and methanogenic bacteria are particularly sensitive to
temperature, with even a 2–3°C drop in a mesophilic digester
adversely affecting biogas production.
Once operational, a heated digester will be adversely affected if
the temperature is allowed to fluctuate by more than just a few
degrees. It is general practice to ensure that the operating tempera-
ture is near the top of the preferred range before the onset of winter.
However, a fall in temperature can be caused by a number of factors,
including inadequate heating capacity, scaling of the heat exchanger
surfaces, and the raw sludge having a low solids concentration. The
temperature can be raised by reducing heat losses from the whole
digestion unit by adequate insulation, increasing the heat input,
reducing the water content of the raw sludge, and thus reducing the
total volume to be heated, and by descaling heat-exchanger surfaces.
Biological hydrolysis can be used as part of a two-stage digestion
process, with hydrolysis taking place in the first (thermophilic) digester,
followed by mesophilic digestion, a process known as temperature-
phased anaerobic digestion (TPAD) (Sec. 5.3.1). The first stage has a
short retention time of 2–5 days at 55–65°C, with the subsequent
digestion phase operated at 35°C for 10–30 days (Ge et al., 2010;
 Sludge Digesters 185

Wang et al., 2018). Studies on TPAD have provided insight into the
effect of temperature on methanogenesis, in particular, with changes in
temperature causing significant changes in methanogen abundance,
with different mechanisms of methanogenesis also reported at different
temperatures. For example, Ho et al. (2014) found Methanosarcina, an
acetoclastic methanogen, dominated at 55–60°C, while at 65°C, the
hydrogenotrophic methanogen Methanothermobacter dominated.
Chen and Chang (2020) also studied the changes in methanogen
diversity and abundance from mesophilic to thermophilic conditions.
With increasing temperature, there is a discernible shift from acetoclas-
tic methanogenesis to hydrogenotrohic methanogenesis dominance,
with changes in diversity discernible. This was confirmed by Steiniger
et al. (2023) noting a change from acetoclastic (e.g., Methanosarcina
and Methanothrix/Methanosaeta) to hydrogenotrohic/methylotrophic
methanogenesis (e.g., Methanothermobacter/Methanoculleus), with
temperatures in laboratory digesters operated at 37, 43, 47, and 53°C,
with a loss in species diversity for both bacteria and archaea. The bacte-
rial families Caldatribacteriaceae and Coprothermobacteraceae increased
with temperature while the Anaerolineaceae decreased while
Synergistaceae dominated at the mid-temperature range (Fig. 5.19).
The methanogen dominance changed with temperature from
Methanosaeta (68%) at 37°C to Methanosarcina (97%) at 43°C and
continued to dominate at 47°C (92%) and 53°C (69%), with
Methanothermobacter (30%) also common at the highest temperature
studied (Fig. 5.20). This study showed that both bacteria and Archaea
had a high degree of adaptability over the temperature range between
the normal mesophilic and thermophilic operating ranges, intriguingly
offering the possibility of successful operation at temperatures between
the two. However, acetoclastic methanogenesis can dominate thermo-
philic digesters under certain conditions, such as high pH or high
ammonium concentrations. While numbers of thermophilic methano-
gens are recorded at mesophilic temperatures, they only dominate
under thermophilic conditions. de Vrieze et al. (2015) identified three
distinct clusters of bacteria with ammonia and temperature. Bacteria in
mesophilic digesters are dominated by the phyla Firmicutes,
186 Anaerobic Wastewater Treatment

Figure 5.19. Bacterial families detected in biogas reactors operated at four differ-
ent temperatures after 65, 70, and 72 days. The color code indicates the relative
abundance within each sample. Families with an abundance <1% were excluded
(Steiniger et al., 2023). Reproduced under the Creative Commons Attribution (CC
BY) license (https://creativecommons.org/licenses/by/4.0/).

Proteobacteria, Bacteriodetes, and Chloroflexi, while in thermophilic

digesters, Firmicutes, Proteobacteria, Chloroflexi, and Actinobacteria
tend to dominate.

5.4.6 pH and Alkalinity

Most anaerobic treatment systems have problems with pH control,
which arises from differences in the growth rate of the synergistic
microbial populations. The activity of the acid-producing bacteria
tends to reduce the pH of digesting sludge from the ideal 6.8–7.4
range required by methanogenic archaea, with optimal activity
observed at pH 7.0–7.2 (Table 5.6).
 Sludge Digesters 187

Figure 5.20. Genera of archaea detected in biogas reactors operated at four differ-
ent temperatures after 65, 70, and 72 days. The color code indicates the relative
abundance within each sample. Families with an abundance <1% were excluded
(Steiniger et al., 2023). Reproduced under the Creative Commons Attribution (CC
BY) license.

Alkalinity is the key driver of pH, so under normal operating con-

ditions, once a stable population of each of the groups has been estab-
lished, an equilibrium is maintained by the buffering action of
ammonium bicarbonate (the bicarbonate alkalinity); hence, no exter-
nal pH control is required. The bicarbonate ions are derived from
carbon dioxide in the digester gas, and the ammonium ions derive
from the degradation of proteins in the raw sludge. So, the pH is
closely linked to the carbon dioxide concentration in the biogas, which
is removed by methanogen metabolism sustaining alkalinity and pH
while also improving biogas quality at the same time. Carbon dioxide
can form carbonic acid, bicarbonate, or carbonate alkalinity. The
three, as well as ammonia and ammonium ions, are in equilibrium
with their dominance depending on the pH within the digester, with
bicarbonate alkalinity the major source of carbon for methanogens.
188 Anaerobic Wastewater Treatment

Figure 5.21. The effect of pH on biogas production.

However, the digesting sludge has a tendency to become acidic,

especially if the methanogens are inhibited, or the digester is over-
loaded, which results in an excessive accumulation of VFAs. Under
these conditions, the buffering capacity may be exceeded, with the pH
rapidly decreasing to 6.0, causing the process to fail, resulting in a sud-
den decline in gas production (Fig. 5.21). Methanogenic bacteria
exhibit a negative response when the pH shifts toward the acid region,
as they do when the temperature falls. Growth of methanogens is
inhibited below pH 6.2, although the fermentative bacteria will con-
tinue to function until the pH has dropped to 4.5–5.0. It should be
remembered that the high concentrations of VFAs that are likely to
occur in digesters are not toxic to methanogenic bacteria in themselves
— it is the pH that is inhibitory, with concentrations of acids up to
11,800 mg L–1 observed as non-toxic to methanogens (Velsen and
Lettinga, 1979; Newell, 1982). Therefore, any continuous downward
trend in pH or alkalinity is an important warning sign requiring imme-
diate attention. The measurement of pH must be done rapidly, as
samples of digesting sludge, once exposed to the atmosphere, will
rapidly lose carbon dioxide and cause erroneously high pH values. So
wherever possible, monitoring systems should be within the reactor
itself, giving real-time data and linked to a SCADA system.
As long as the sludge has a fairly high alkalinity, which is especially
the case with thick sludges, an increase in acid production will initially
produce little effect on the pH. Therefore, in practice, the measure-
ment of volatile acids is a better control factor of the buffering
 Sludge Digesters 189

Figure 5.22. Effect of volatile acid production on the pH in a digester.

capacity within an operating digester. Any change in the loading of

the digester must be gradual to ensure that the concentration of VFAs
does not exceed the normal buffering capacity of the system. Normal
VFA concentrations in sewage sludge digesters are between 250 and
1000 mg L–1, with values in excess of 1800–2000 mg L–1, indicating
a problem (Fig. 5.22). Determination of individual VFAs is also very
useful, as a shift to higher VFAs, such as acetic to butyric, is a sign of
instability, and remedial action is required. Provided adequate buffer-
ing capacity is available, higher VFA concentrations may be tolerated,
although this will lead to incomplete conversion of biodegradable
material to gas, with a subsequent increase of the BOD5 of the
digester effluent. Depending on the chemical nature of the sludge, it
is possible to have rapid and effective digestion at all pH values
between 6.2 and 7.8, although certain trade and sewage wastes
restrict rapid digestion to much smaller ranges. Mosey (1983), in his
excellent review on anaerobic processes, gives two interesting exam-
ples. Ferrous carbonate can become sufficiently soluble below pH 6.4
to release inhibitory concentrations of ferrous ions into solution,
whereas, above pH 7.5, an increasing percentage of ammonium ions
will be converted to toxic dissolved ammonia gas. The latter example
is particularly important in relation to strong sewage and farm wastes.
A loss of alkalinity can also be caused by toxic compounds in the
feed sludge, which inhibit the methanogens, or a shock load of organic
acids, which can occur in some industrial and food processing
190 Anaerobic Wastewater Treatment

scenarios. Proteinaceous wastes tend to produce high alkalinities due to

the formation of ammonia and carbon dioxide, while in comparison,
non-proteinaceous wastes produce carbon dioxide only. Foaming can
also reduce alkalinity, leading to the inhibition of methanogenesis.
The pH value can be neutralized within the reactor by the addition
of an alkali. The cheapest and most widely used alkali in full-scale
digesters is probably calcium hydroxide (lime). Lime is extremely effi-
cient at increasing the pH to about 6.4–6.5. However, further increases
in pH can only be achieved by precipitating most of the dissolved car-
bonates as calcium carbonate. Therefore, at pH >7.0, the lime reacts
with carbon dioxide, which not only results in serious scale formation
but reduces the pressure of the gas phase above the sludge by removing
the carbon dioxide, which could seriously affect the structural stability
of the reactor. Bicarbonates or carbonates of either sodium or potas-
sium are generally used to raise the pH from 6.5 to 7.0, although
excessive use of these salts can result in partial or severe inhibition at
concentrations of 3,500–5,500 and >8,000 mg Na L–1 and 2,500–
4,000 and >2,000 mg K L–1, respectively. Other alkalis are less suitable;
for example, sodium hydroxide also removes carbon dioxide from the
solution, and the use of ammonia or ammonium ions can inhibit the
process. For laboratory-scale digestion units, the most useful reagents
for adjusting the pH are hydrochloric acid and sodium bicarbonate.
If the sludge contains heavy metals in solution at concentrations
likely to cause inhibition, the addition of alkali to raise the pH to
7.5–8.0 will precipitate most of the metal ions out of solution, usually
as carbonates, thus reducing the inhibitory effect. Sulfide can also be
used to precipitate the heavy metal ions without causing a significant
pH change. Apart from chemical buffering, pH control can also be
achieved by using other wastes or by increasing the sludge recycle rate
where the reactor design allows, such as fluidized beds or downflow
biofilters (Chap. 6) (Wheatley, 1985).

5.4.7 Foaming
As anaerobic digestion is increasingly employed as an intergral part of
a carbon net-zero plan for WWTPs, concerns over foaming have
 Sludge Digesters 191

increased globally due to potentially serious impacts on process effi-

ciency and operational costs (Ganidi et al., 2009). The effects of
foaming can be extremely costly, with a loss of methane generation
leading to an increase in energy costs to operate digesters as well as
applying remediation procedures. The main effects of foaming are
inefficient gas recovery; an inverse solids profile, resulting with the
densest sludge layer at the top of the digester; creation of dead zones,
which leads to a reduction of the active volume of digesters, resulting
in poor stabilization of sludges; blockages of gas mixing devices and
sludge recirculation pipes; possible egress of foam from between the
floating covers and the digester wall; and possible damage to the cov-
ers of certain designs of digesters. Gas production in full-scale digest-
ers can be reduced by between 30 and 50% during foaming, with 90%
reductions in extreme conditions.
Foaming is where the biogas formed during digestion does not
escape from the liquid phase to the gas space at the top of the digester;
rather, it becomes entrained within the sludge (Yang et al., 2021).
Foam is the dispersion of gas bubbles in a liquid or solid. In digesters,
the bubbles are dispersed within the sludge, and when they rise to the
surface, the water layer (lamella) surrounding the bubble thins so that
it bursts, releasing the gas. Surface active agents, commonly referred
to as surfactants, stabilize the foam by their molecules becoming
attracted to the bubbles, making the lamella stronger so that it must
become much thinner than normal before rupturing. So, the presence
of surfactants will result in stronger bubbles and more stable foams.
Hydrophobic particles also form foams. These particles are poorly
wetted by water, producing complex foams made up of dispersed
phases of hydrophobic particles and air within the water phase. The
particles congregate at the gas–liquid interface, making the foam even
stronger. Jenkins et al. (1993) described how hydrophobic particles,
if they are large enough, bridge the water film between bubbles, cre-
ating what is, in essence, a dam that prevents water from draining
away, thus preventing the film from thinning and rupturing (Fig. 5.23).
As gas bubbles are formed, they become coated with surface active or
hydrophobic substances, which increase as they rise and then become
attached to solids and microorganisms. The foam slowly expands
192 Anaerobic Wastewater Treatment

(a)

(b)

(c)

Figure 5.23. Various types of headspace foams in anaerobic digesters. (a) No sur-
face active material is present. (b) Surface active materials present that accumulate at
the gas–water interface and stabilize it. (c) Hydrophobic solids present that accumu-
late at the interface and stabilize it, with larger solid particles forming bridges across
the gas bubbles, preventing the drainage of water from it.

upward where it then blocks the gas outlet, which can lead to an
increase in pressure, causing the digestate to overflow and even reac-
tor damage, or, in the worst case, an explosion. The foam causes the
microbial biomass to become concentrated within the foam, reducing
free mixing and contact between the substrate (sludge) and the bio-
mass, leading to a decrease in methanogenic activity and biogas pro-
duction. When the entrained bubbles cannot rise to the surface due
to the viscosity of the sludge, the foam is then found throughout the
reactor rather than just at the surface. This phenomenon was first
described by Chapman and Krugel (2011), who called it rapid volume
expansion, as the foam expands evenly and quickly, similarly to how
yeast dough expands (Fig. 5.24). It is generally found in reactors
where digestate viscosity is high, as found with food wastes, with the
volume of the sludge expanding rapidly, with digestate overflow
occurring within a very short time frame, generally less than an hour.
 Sludge Digesters 193

(a) (b)

Figure 5.24. Artistist’s impression of (a) conventional foaming and (b) rapid vol-
ume expansion in anaerobic digesters (Yang et al., 2021). Reproduced with permis-
sion of Elsevier Publishing.

Numerous causes of foaming have been suggested, including organic

overload, accumulation of acetic acid, inadequate mixing, tempera-
ture fluctuations, shock loadings, and the presence of hydrophobic
and extracellular polymeric substances (EPS). While all these factors
are known to interfere with the operational efficiency of digesters, the
presence of microbial hydrophobic filaments appears to be the major
cause being directly associated with foam structure, including its for-
mation and stability, although the proliferation of foam-causing
microorganisms does require specific abiotic conditions.
Microbial foams produced by surfactant-producing and hydro-
phobic organisms are more viscous and stable than chemical ones.
Foaming is a common phenomenon in the activated sludge process
(Gray, 2023), and is associated with the digestion of sludges compris-
ing waste activated sludge (WAS). Two common activated sludge
foaming species were identified as causing foaming in digesters in the
1990s: Giordonia (formally Nocardia) spp. (Hernandez and Jenkins,
1994; Pagilla et al., 1997) and Microthrix parvicella filaments
(Westlund et al., 1998), and since then, increasing evidence has
emerged to support hydrophobic filamentous microorganisms as a
key causative factor for anaerobic foaming. Microthrix and Gordonia
are known to only survive under strict anaerobic conditions for a few
days (Kirkegaard et al., 2017) with foaming, in this case, linked to the
194 Anaerobic Wastewater Treatment

continuous addition of organisms via the WAS. So, dealing with

foaming at the activated sludge stage is probably the best approach to
the control of foaming in digesters. EPSs can be either hydrophobic
or hydrophilic, with the former directly correlated to anaerobic foam-
ing and the latter contributing to foaming by increasing sludge viscos-
ity. Foam stability is linked to hydrophobic proteins, which reduce
surface tension, while the role of humic substances, which contain
functional groups (e.g., amino hydroxyl), increases the viscosity of the
bubble lamella (Yang et al., 2022; 2023). However, not all the fila-
mentous organisms, both bacteria and archaea, isolated from anaero-
bic foams, have their origin from activated sludge, with a number
found only in strictly anaerobic environments. Jiang et al. (2021)
studied 13 mesophilic digesters at 11 different WWTPs in Denmark
and identified the organisms responsible for foaming, using 16S
rRNA gene amplicon sequencing with species-level resolution and
fluorescence in situ hybridization (FISH) for visualization (Fig. 5.25).
Foaming was categorized into three categories: non-foaming
(Group 1), unstable foams (Group 2), and stable foams (Group 3).
They found a significant difference in species composition between
non-foaming (Group 1) and foaming (Group 3) sludges. Groups 2
and 3 foams were both dominated by microorganisms producing
surfactants (e.g., archaeal species Methanospirillum) while Group 3
was also rich in microorganisms with hydrophobic cell surfaces (e.g.,
Microthrix). Non-activated sludge species associated with foaming
include Brevefilum fermentans, Tetrasphaera, Rikenellaceae,
Anaerolineae, Actinomyces, Corynebacterium, and Lactobacillus and
the archaea genera Methanospirillum and Methanothrix (formerly
Methanosaeta). Community structure is also distinctly different
between mesophilic and thermophilic digesters (Kirkegaard et al.,
2017). Subramanian et al. (2015) developed a simple foaming poten-
tial test using an air diffuser fitted in the base of a 2 L graduated
cylinder into which 200 mL of digested sludge is added. Air is added
at 1.5 L min–1, which expands the sludge with bubbles. The maxi-
mum height the foam attains is an indicator of the foam potential.
Unstable foams collapse after the diffuser is closed, while stable foams
remain expanded (Eqs. (5.6) and (5.7)):
 Sludge Digesters 195

Figure 5.25. Heat map of the 25 most abundant bacterial amplicon sequence vari-
ants (ASVs) at 13 mesophilic digesters in Demark, characterized by the foaming
propensity test. Non-foaming (Group 1), producing an unstable foam (Group 2), or
foaming (Group 3). Taxonomic classification levels presented are phylum, genus,
species, and ASV (Jiang et al., 2021). Reproduced with permission of Elsevier
Publishing.

maximum foam height (mL )

Unstable foam potential = (5.6)
Initial height of sludge (mL )

The unstable foam potential gives an indication of the operational

or working foam potential, which the authors categorized as: 0–1
(indicating non-foaming), 1–2 (mild foaming), and >2 (average to
severe foaming).

Settled foam height (mL )

Stable foam potential = (5.7)
Initial height of sludge (mL )
196 Anaerobic Wastewater Treatment

The stable foam potential is a measure of permanent foam, in

which a value of 0–0.2 is indicative of non-foaming, 0.2–0.5 is indica-
tive of mild foaming, and >0.5 is indicative of average to severe foam-
ing. As with activated sludge foam potential tests, these values will
vary between reactor design and operational conditions and so should
be used for measuring foam variation at individual plants rather than
as a comparative parameter (Gray, 2023).
Foam potential tests are widely used to assess foaming potential
and stability in activated sludge, with similar tests used to screen
sludge fed into digesters (Jiang et al., 2018). Jiang et al. (2021)
recorded a strong correlation between foaming potential and the
abundance of Microthrix spp. in the sludge feed originating from
WAS. A similar relationship was seen with the abundance of several
true anaerobes that are common in digesters and associated with
foaming, including Brevefilum fermentans, Tetrasphaera,
Rikenellaceae, and the archaeal genus Methanospirillum (Fig. 5.26).
It is difficult to actually see or monitor foaming within closed
digesters, so apart from looking for the presence of indicator micro-
organisms, the foam potential test is a useful indicator of foaming
potential and stability. As in activated sludge, organic overloading
may also be a causative factor in foaming due to the accumulation
of VFAs, ammonium, and TN (total nitrogen), which can be used
as an alternative indicator to the foam potential test as they are all
positively and strongly correlated (Jiang et al., 2021). Alfaro et al.
(2014) observed a direct relationship between foaming and micro-
bial filament density. Gordonia amarae was recorded as causing
significant foaming in full-scale egg-shaped digesters by Subramanian
et al. (2015), who found that while excessive induced mixing exac-
erbated foaming, it could be controlled by reducing mixing or turn-
ing it off for short periods, which did not affect overall performance.
They were also able to identify a foaming threshold for the actino-
mycete of 106 intersections mg–1 VSS in the mixed liquor feed to the
digester. The presence of G. amarae was found to be seasonal and
closely linked to the temperature in the activated sludge reactor,
with foaming in the digesters only problematic when the threshold
value was exceeded (Fig. 5.27).
 Sludge Digesters 197

(a) (d)

(b)

(c)

(e)

(f)

Figure 5.26. Composite FISH micrographs of potential foam-forming microbes

isolated in Demark by Jiang et al. (2021). (a) Ca. Brevefilum spp. (b) Ca. Brevefilum
spp. (c) Tetrasphaera spp. (d) Ca. Microthrix spp. (e) midas_g_141 members. (a) to
(e) The EUBmix probe set was applied to target most bacteria. Target populations
appearing magenta (red + blue), and other bacterial cells appear blue. (f)
Methanospirillum spp. The Methanospirillum cells appear magenta. Scale bar =
10 μm. Reproduced with permission of Elsevier Publishing.

Foaming control is achieved either by preventing foam generation

or by controlling foam once it has formed. The best preventative
method is to ensure the digester is operated at suitable mixing
rates, temperature, and organic loading. Alternatively, the feed can be
pre-treated by thermal or ultrasonic hydrolysis, which can destroy
those filamentous bacteria associated with activated sludge foaming
(e.g., M. parvicella) (Alfaro et al., 2014). Emulsification and saponi-
fication of lipid-rich substrates have also proven to be effective
198 Anaerobic Wastewater Treatment

Figure 5.27. Variation of filament density and temperature of Gordonia amarae in

the activated sludge reactors forming the feed to anaerobic digesters. Foaming in the
digesters was triggered when the threshold filament density was reached, which is
represented by the dotted line (Subramanian et al., 2016). Reproduced with permis-
sion Elsevier Publishing.

(Diamantiset al., 2021). Trace elements have also been successfully

added, which are used as co-factors of enzymes that speed up the
metabolism of intermediate metabolites. These reduce the viscosity of
the digestate, which, in turn, helps to reduce the foam (Moestedt et
al., 2016). Foam control methods are not fully effective and are often
long-term, making them an expensive option. This involves destabi-
lizing the foam structure, which can be done chemically or physically.
Initially, a mechanical approach is taken to disperse the foam, which
usually takes the form of either increasing mixing/stirring rates or
injecting water under high pressure. The objective is to increase shear
stress within the digester, which will weaken the surface tension of the
bubble wall, or by removing some of the surfactants at the bubble
surface, weakening the bubble and causing them to collapse.
However, the addition of large volumes of water lowers the total sol-
ids content of the digester, reducing performance. Mechanical solu-
tions are unlikely to solve the problem, which will require either a
change in feed or the operating conditions of the digester. The most
widely adopted approach is to use anti-foaming or defoaming chemicals,
 Sludge Digesters 199

which act directly on the surface of the bubbles, reducing the surface
tension or removing the surface surfactants, encouraging the bubbles
to rupture and thereby destabilizing the foam structure. There is a
wide range of commercial, often synthetic, defoaming chemicals avail-
able, although natural oils and buffering agents are also used.
Defoaming chemicals are not always successful, and when they are,
they can significantly interfere with the performance of the digester in
terms of biogas formation. The presence of silicones in synthetic
defoamers should be avoided due to the formation of siloxanes in the
biogas (Sec. 3.6). Due to the cost of commercial products, organic
oils such as vegetable oil or biodiesel are often used in their place.
However, over time, the microorganisms present adapt to using them
as a substrate, and so their effectiveness quickly declines, requiring
increasingly larger dosages for effective control. Lindorfer and
Demmig (2016) have suggested that this problem can be overcome
by alternating the use of vegetable oil, a commercial defoamer, and
biodiesel so that microorganisms do not become too acclimatized to
the natural oils as a food source.
Buffering agents include a wide range of compounds and salts,
including urea, sodium carbonate, sodium bicarbonate, and ammo-
nium chloride. Generally, large quantities of these compounds are
required, with effects taking several days to appear (Kougias et al.,
2014).
Biological control is achieved by identifying the main foaming
microorganism and targeting it by altering operational conditions,
removing it from the feed, or reducing substrate addition, leading to
starvation conditions that reduce gas production so that the foaming
problem is gradually reduced. In practice, changing the operational
conditions of the digester is probably the best and cheapest way that
foaming can be controlled; for example, by altering the organic load-
ing by diluting the feed, which also alters the viscosity of the sludge
within the reactor (Lindorfer and Demmig, 2016).
The mechanisms of foam formation, assessment, and control have
been reviewed by Ganidi et al. (2009) and, more recently, by Yang
et al. (2021).
200 Anaerobic Wastewater Treatment

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 Section C

Contact (High-rate) Anaerobic

Wastewater Treatment Systems
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 6
Biofilm (Attached Growth) Systems

6.1 Introduction to Contact Systems

Because of the difficulties involved in degrading particulate organic
matter and the slow growth of anaerobic bacteria, and of methano-
gens in particular, digester design has been traditionally based on
flow-through stirred tanks with long retention times. Unlike sludge,
the organic matter in wastewater is often in solution and is far more
amendable to treatment. However, the primary problem with treating
these wastes anaerobically has been how to retain sufficient biomass
within the reactor and prevent wash-out of the bacteria and archaea.
Contact anaerobic systems are specifically designed to treat weaker
sludges with low solids concentrations and strong effluents. Unlike
the conventional continuously stirred digester, the hydraulic retention
time (HRT) and solids retention time (SRT) are independent of each
other, with the biomass either retained within the reactor or recycled
after separation (Chap. 7). Many contact systems have been devel-
oped (Table 6.1) based around anaerobic static media filters, fluidized
media, activated sludge, and sludge blanket systems. Data on over
400 anaerobic plants, mainly treating vegetable-processing wastewa-
ters, was collated by Demuynck et al. (1983), who gave details on
many contact systems, with reactor volumes ranging from 2,000 to
20,000 m3. Anaerobic contact systems have been a popular choice for
industrial wastewater treatment for over 40 years, with an example of
the range of applications summarized by Wheatley et al. (1997).

213
214 Anaerobic Wastewater Treatment

Table 6.1. List of anaerobic contact systems used for wastewater treatment.
Reactor group Example of reactor design Abbreviation
Anaerobic biofilter Submerged anaerobic reactor SMAR
Up-flow anaerobic filter ANFIL
Anaerobic up-flow filter AUF
Anaerobic downflow stationary bed reactor ADSR
Downflow stationary fixed film reactor DSFF
Up-flow stationary fixed film filter USFF
Hybrid anaerobic bed reactor
Anaerobic biological Anaerobic biological contactors AnRBC
contactors
Anaerobic moving bed Anaerobic moving bed biofilm reactors AnMBBR
biofilm reactors
Expanded and Static or porous medium bed reactor AnPBR
Fluidized beds Expanded bed reactor AnEBR
Fluidized bed reactor AnFBR
Anaerobic ammonium oxidation Anammox

These included food wastewaters such as distilleries, sugar refiners,

potato processing, breweries, starch refiners, milk processing, abat-
toirs, yeast processing, canneries, fruit processing, soft drinks, and
non-food wastewaters, such as paper, pharmaceuticals, chemicals, and
textiles. At that time, up-flow anaerobic sludge blanket (UASB) reac-
tors (48%) were the most popular, followed by anaerobic filters (23%).
While anaerobic treatment systems, especially UASB reactors and
anaerobic filters, are very efficient and cost-effective at treating a wide
variety of wastewaters, including sewage, the final effluent still has
relatively high concentrations of chemical oxygen demand (COD),
nutrients, and suspended solids. This requires further treatment, usu-
ally by an aerobic bioreactor, before final discharge to receiving
waters. Aerobic treatment is normally used as a post-treatment pro-
cess for both carbonaceous and nutrient removal to achieve modern
discharge standards. All aerobic processes are employed, including
activated sludge (Nmñez and Martinez, 2001), biological aerated
filters (BAFs) (Lacelle et al., 2001), constructed wetlands (Denny,
1997; Sousa et al., 2001), land treatment (Chernicharo et al., 2001),
maturation ponds (Dixo et al., 1995; Catunda and van Haandel,
 Biofilm (Attached Growth) Systems 215

1996; Cavalcanti et al., 2001), percolating filters (Chernicharo and

Nascimento, 2001), submerged aerated filters (SAFs) (Gonçalvez
et al., 1999), and sequencing batch reactors (SBRs) (Sousa and
Foresti, 1996; Zaloum and Abbott, 1997).

6.2 Anaerobic Biofilters or Static (Fixed) Bed

Reactors
Anaerobic reactors using fixed or static media to retain the active
biomass are generally known as anaerobic biofilters (Fig. 6.1). There
are a number of different anaerobic biofilter designs employing a
fixed medium, including the submerged anaerobic reactor (SMAR),
the up-flow anaerobic filter (ANFIL) and anaerobic up-flow filter
(AUF), the anaerobic downflow stationary bed reactor (ADSR), and
the downflow stationary fixed-film reactor (DSFF). The differences in
these designs can be quite small, so it is useful to compare two sys-
tems: the up-flow stationary fixed film filter (USFF), which is
operated with the influent wastewater passing through the filter in an
up-flow mode (Fig. 6.1) while (ii) DSFF systems operate with
the influent passing through the medium in a down-flow direction
(Fig. 6.2). This creates two very different reactor environments, even
though both have fixed medium on which the microbial community

Figure 6.1. Schematic of a typical anaerobic up-flow stationary fixed film filter
(USFF) design.
216 Anaerobic Wastewater Treatment

Figure 6.2. Schematic of an anaerobic downflow stationary fixed film reactor

(DSFF) design.

develops. In USFF reactors, the majority of the active biomass is

found within the interstices of the medium, while in DSFFs, the bio-
mass is predominantly present as attached biomass. While both can
treat medium to strong wastewaters, the DSFF system tends to use
media with large interstices, often orientated vertically, which allows
wastewaters with high suspended solids concentrations to be treated,
which would normally block USFF media. In DSFF reactors, there is
also a counter-movement of gas and liquid, resulting in intense mix-
ing, which makes them better suited to wastewaters with higher con-
centrations of suspended solids. Another biofilm system is the
anaerobic baffled reactor (AnBR), which is used as an upgrade for
septic tanks treating domestic wastewater, especially in Asia (Sec. 7.7).
Normally, AnBRs use granular sludge, but a modified version, the
packed anaerobic baffled reactors (PABR), includes a fixed medium
or carrier to increase biomass concentration to achieve COD removal
of up to 90%. Small carriers, such as bamboo sections, are suspended
in bunches on rods that sit on the up-flow section of each chamber
(normally 4–6 chambers per reactor) (Fig. 7.9).
In design, anaerobic biofilters are submerged trickling filters,
full of wastewater so that no oxygen can enter the reactor and are
 Biofilm (Attached Growth) Systems 217

operated using a wide variety of media, from mineral to random and

modular plastic filter media. The biomass comprises a thin bacterial
film that is firmly attached to this support media. Apart from being
operated as an anaerobic process, with some form of biogas collection
device, submerged filters can also be operated sequentially or in series
aerobically using aeration units or anoxically with a separate carbon
input where necessary (Gray, 2021).
The design of anaerobic biofilters varies, although the shape and
diameter of both the reactor and the media can have important effects
on the stability of the attached film (Berg and Lentz, 1980). Other
factors, such as gas production, can also dislodge the film, although
its scouring effect may also reduce the chance of clogging within the
filter medium. In cylindrical reactors, the most satisfactory diameter-
to-height ratio is 1:4. The entire reactor can be filled with the
medium, and most anaerobic filters now use plastic media (Anderson
et al., 1984), or it may be restricted to the upper part of the reactor
only, with the detached biomass settling to the lower chamber,
thus providing a dual mixed and fixed system (i.e., hybrid systems)
(Fig. 6.3).
Various media have been used. Of the mineral media, porous
stones, gravel, glass, and pottery fragments appear to be the favorites,
although these have a large bulk density and relatively low surface
areas. Mineral media also require strong reactor designs to take the

Figure 6.3. Schematic of a hybrid anaerobic biofilter comprising a dual mixed and
fixed reactor area.
218 Anaerobic Wastewater Treatment

Figure 6.4. COD removal efficiencies of anaerobic filters in relation to different

types of media: 1. Large modular; 2. Small modular; 3. Pall rings; 4. Spheres (Young
and Dahab, 1982). Reproduced with permission IWA Publishing.

added weight of the medium. In contrast, plastic media have a much

greater specific surface area (90–350 m2m–3) and lower density (50–
100 kg m–3), with a voidage in excess of 90% (Characklis and Trlear,
1982). Different media result in very different performance charac-
teristics, and unlike the traditional trickling filter, removal efficiency
is not directly related to specific surface areas of the media (Young
and Dahab, 1983; Wilkie et al., 1983) (Fig. 6.4). This is because in
up-flow filters, a significant portion of the active biomass will be pre-
sent not as attached film, but as unattached dispersed growths in the
interstices (voids) of the medium. The suspended solids flocculate as
they travel up through the filter, forming larger particles that finally
settle back down the filter column. Therefore, provision must be
made for periodic desludging, usually once a year (Young, 1991).
The movement of solids and gas bubbles being released introduces an
element of mixing into this essentially plug-flow system. When oper-
ated in the down-flow mode, all the dispersed solids are washed out
of the reactor, leaving only the attached biomass. In these reactors,
the media must ensure stable film development as well as prevent
 Biofilm (Attached Growth) Systems 219

excessive solids accumulation. Therefore, modular tube or crossflow

media, as opposed to random media, are recommended (Wilkie et al.,
1983; Bonastre and Paris, 1989). As all the biomass is attached in
DSFFs, the performance in this case is generally directly related to the
specific surface area of the medium. In practice, filters employing
porous media perform better than those using non-porous material,
due not only to higher specific surface areas but to better surfaces for
the adhesion and retention of biomass (Fox et al., 1990; Picanço
et al., 2001). Needle punched polyester (NPP) medium is widely
employed in DSFFs due to its high specific surface area. Biofilter
media has been extensively explored in an earlier book in this series
on fixed-film reactors (Gray, 2021).
There is increasing interest in the use of biochar as a filter medium
in anaerobic biofilters as well as an amendment to digesters and floc-
based anaerobic treatment systems. Biochar, which is highly porous
with strong adsorptive properties (Gęca et al., 2022), promotes more
rapid establishment of the biofilm as well as increasing resistance to
inhibitory substances, common in leachate for example, as well as
improving overall effluent quality and methane production. It is more
commonly used as a secondary stage to another anaerobic process,
such as a UASB or a leach bed reactor (LBR) (Collins et al., 2023).
Paritosh and Kesharwani (2024) explain that biochar encourages
interspecies electron transfer, cell immobilization, and gene level
alternations at the microbial level within the biofilm.
Anaerobic biofilters require an initial inoculum of bacteria to
speed up the colonization of bacteria and archaea. Although expen-
sive, a start-up inoculum of 10% v/v from a similar unit is recom-
mended, limiting organic loading to <0.1 kg COD kg VSS–1 d–1, with
a minimum HRT >1 day to limit washout of the biomass. Once
established, anaerobic biofilters can achieve 75–85% COD removal
when loaded at between 1 and 10 kg COD m3d–1 with HRTs between
18–24 hours. Anaerobic biofilters are ideal for relatively cold and
dilute wastes operated in temperate areas, as they have an extremely
high MCRT:HRT ratio with an MCRT (mean cell residence time)
>100 days. This gives the process a high degree of stability, excellent
resistance to inhibitory compounds, and satisfactory performance,
220 Anaerobic Wastewater Treatment

Table 6.2. Comparison of features of various anaerobic reactors. ANFLOW,

anaerobic flow-through digester without sludge recycle; ANCONT, contact digester
with sludge recycle; ANBIOL, anaerobic biofilters; FANBIOF, fluidized anaerobic
biofilter; UASB, upflow anaerobic sludge bed reactor. The more asterisks, the better
the process. Adapted from (Oleskiewicz and Olthof, 1982).
Feature ANFLOW ANCONT ANBIOF FANBIOF UASB
Ease of start-up * ***** **** *** **
Ease of operation after ** ** ***** *** ***
proper acclimatization
Good process control * ***** *** *** ***
possible under
transient influent
conditions
Resistance to shocks
due to:
temperature * *** ***** ***** ****
toxics * *** ***** ***** ****
high organic load * **** ***** ***** *****
Tolerance for influent * **** ** **** *****
quality variations
Can tolerate high influent **** *** * ** **
solids fluctuations
May incorporate sludges ***** **** * * *
from pretreatment
and aerobic polishing

even at low temperatures (Table 6.2). They can treat similar organic
loadings as anaerobic activated sludge systems, even though the
MCRT is much higher (Table 6.3). Successful applications include:
treating undiluted sewage, starch, whey, cellulose, distillery, pharma-
ceutical, piggery, and fish-processing wastewaters (Duff and van den
Berg, 1982; Genung et al., 1982; Rittman et al., 1982; Sachs et al.,
1982; Bedogni et al., 1983; Mathur et al., 1986; Elmitwalli et al.,
2001), with an average COD removal rate of between 80 and 90% at
a typical organic loading of 3.5 kg m–3d –1. DSFF technology is ideal
for treating sugar refinery wastewaters as well as producing high
biogas yields. In a comparative study using polyvinyl chloride pipe
Table 6.3. Features of various reactors compared with aerobic counterparts. Adapted from (Oleskiewicz and Olthof, 1982).
The key is given in Table 6.2.
Anaerobic processes Aerobic processes

Activated
Feature ANFLOW ANCONT ANBIOF FANBIOF UASB sludge Trickling filter
Loads practised 0.5–3 2–8 2–10 0.5–12 1–15 0.5–2 1–3 roughing
(kg COD m–3 d–1)
Loads used in 0.5–10 0.5–100 0.5–25 1–40 1–60 1–10 2–15
experimental scale
(kg m–3d–1)

Biofilm (Attached Growth) Systems 221

HRT used (days) 8+ 0.2–8 0.2–4 0.15–3 0.15–8 1–5 0.05–0.2
SRT resulting (days) 8+ 15–80 20–300 20–100 30–200 10–30 ~30(+)
Temperatures used (°C) 35, 55 35, 55 15–35 35 5–35 15–25(+) 15–25(+)
COD removals attained 60 90+ 90+ 90+ 90+ 90+ 60–80 in 1 stage.
at practised loads: Multi-stages
similar waste typical
assumed (%)
222 Anaerobic Wastewater Treatment

pieces as a medium, Pradeep et al. (2014) found that the BOD

and COD removal increased with decreasing HRT (48 to 12 hours),
reaching an optimal 79 and 82% removal respectively, at 12 hours,
with optimal biogas production but declining at an HRT of
6 hours. A DSFF unit containing fired pottery clay medium with a
surface area of 157 m2m–3 was used to treat piggery waste at 35°C
(Kennedy and Berg, 1982). It was found to be able to treat higher
loadings and have higher rates of methane production than partially
or fully mixed reactors and also plug-flow reactors. The support
medium was evenly coated with a thin microbial film 2–4 mm thick.
Reactor performance, based on the amount of microbial film present,
was between 1.1 and 1.4 g COD removed per g of film per day, which
is similar to that reported by Berg and Lentz (1980) for other types
of media. Canovas-Diaz and Howell (1986) used a modified DSFF
reactor to treat cheese whey, which was initially operated flooded as a
standard DSFF system, but then the liquid height was reduced by
two-thirds. The authors found that partially exposing the biofilm
increased gas production while increasing the resistance of the metha-
nogenic archaea to high VFA concentrations (i.e., 3,000 mg L–1).
COD removal of up to 90% was achieved using DSFF by Duff and
van den Berg (1982) for the treatment of high-protein fish processing
wastewater at loading >10 kg COD m3d–1.
However, USFF systems are limited to treating substrates with
relatively low solids, as the interstices of the medium, especially min-
eral media, can easily become blocked. This is why they are often
recommended to be operated in series, with an up-flow anaerobic
sludge blanket reactor, as the effluent from this process will be largely
free from suspended solids, thus making it ideal for further anaerobic
treatment using a USFF. Loading rates are compared to other anaero-
bic systems in Table 6.4.

6.3 Rotating Biological Contactors

Conventional anaerobic filters require the influent wastewater
to be pumped either into or up through the reactor, which
requires significant energy for pumping. Also, there is a risk of ponding
 Biofilm (Attached Growth) Systems 223

Table 6.4. Comparison of loading rates used in anaerobic

filters, UASB, and expanded beds (Hobson and Wheatley,
1993). Reproduced with permission of Elsevier Publishing.
Type of unit Range
Anaerobic filter
Organic load (kg COD/m3.d) 2–10
Retention period (h) 10–15
COD removal (%) 70–80
Critical solids concentration in feed (mg/1) 450–1,050

Upflow anaerobic sludge blanket reactor

Load (kg COD/m3. d) 2–15
Retention period (h) 10–50
COD removal (%) 70–90

Expanded bed
Load (kg COD/m3.d) 2–50
Retention period (h) 0.5–24
COD removal (%) 70–80

(i.e., clogging) of the medium, causing short-circuiting, reduced

loading rates, and HRTs. In contrast, anaerobic rotating biological
contactors (AnRBCs) have a very small head loss, requiring minimal
pumping and low energy requirement, and are simple to operate with
no risk of clogging.
The AnRBC is based on standard RBCs, which consist of a series
of flat or corrugated discs 2.0 to 3.5 m in diameter and between 10
and 20 mm thick on which the biofilm develops. The discs are
mounted on a horizontal shaft, 20 mm apart, which is driven mechan-
ically, usually by a small electric motor, so that the discs rotate at a
right angle to the flow of wastewater (Fig. 6.5). The discs are nor-
mally fabricated in corrugated polythene, MDPE, PVC, or glass
reinforced plastic, although other materials are occasionally used.
The surface area of the medium varies between 150 and 200 m2m–3.
224 Anaerobic Wastewater Treatment

Figure 6.5. Schematic view of disc assembly in a rotating biological contactor

showing support rods, used in many designs, that ensure exact spacing between discs
(CIWEM, 2000). Reproduced with permission of the Chartered Institution of Water
and Environmental Management.

The discs are designed to fit very closely to the outer shell of the tank,
in which they are placed to prevent short-circuiting. The majority of
RBCs are operated aerobically, with 35–40% of the disc area sub-
merged in the wastewater, which is slowly but continuously rotated,
with oxygen transfer taking place while the biofilm passes through the
air space above before being re-immersed in the wastewater below.
The design, construction, and operation of RBCs have been reviewed
by Gray (2021) in the volume on fixed-film reactors in this series. In
contrast, the discs of AnRBCs are fully submergered, with the gas
space above the water level anaerobic.
While RBCs had been developed in the 1920s, it was not until
1965 that they became commercially available. The AnRBC was
developed in the late 1970s and pioneered by the early experimental
work of Tait and Friedman (1980). The basic design is the same as
the standard aerobic RBC, except that the reactor is airtight and the
discs are generally fully submerged, with any biogas collected by a
 Biofilm (Attached Growth) Systems 225

(a)

(b)

Figure 6.6. Schematic design of an anaerobic rotating biological contactor (AnRBC):

(a) side view and (b) cross-section.

series of pipework (Fig. 6.6). The discs are grouped together into sets
or stages, which may be housed with separated baffled areas, which
minimizes short circuiting while also allowing the development of
different microbial consortia to utilize the wastewater as it changes
composition as it passes through the reactor. This design particularly
lends itself to anaerobic operation, where the different stages of diges-
tion require different consortia of anaerobic bacteria and archaea.
This is the design used by Tait and Friedman (1980), who operated
their laboratory AnRBCs at 35°C to successfully treat a strong organic
wastewater with a COD of 8,500 mg L–1 (BOD 5,250 mg L–1). There
have been a number of successful AnRBC laboratory and pilot trials.
For example, Laquidora et al. (1986) achieved COD removal rates of
between 10 and 140 g m–2d–1 while treating wastewater similar to that
from the food processing industry, while Satyanarayan et al. (1987)
achieved 60–81% COD removal rates treating dairy wastewater. The
226 Anaerobic Wastewater Treatment

performance of AnRBCs follows a simple Monod-type expression

(Deshpande et al., 1991), with most researchers opting to use a
modified Stover–Kincannon model for design and performance esti-
mation. The final effluent quality and reactor volume required can be
calculated as:
Final effluent quality (Se)

U maxS0
S e = S0 − (6.1)
 S0 
KB 
 HRT 

Reactor liquid volume required (V)

QS0 (6.2)
V=
U maxS0 
 S −S  − KB
 0 e

where Q is the flow rate (L d–1), V is the reactor liquid volume (L), S0
is the influent COD (mg L–1), Se is the effluent COD (mg L–1), Umax
is the maximum substrate removal rate (mg L–1 d–1), and KB is the
saturation value constant (mg L–1 d–1). Ebrahimi et al. (2018) deter-
mined Umax and KB as 7.77 and 8.57 mg L–1 d–1, respectively.
The most complete study on AnRBCs was carried out by Lu et al.
(1995), who recorded an average COD and BOD removal rate of 71
and 76%, respectively, while treating a high-strength organic wastewa-
ter at a loading rate of 13.33 kg COD m–3d–1 (equivalent to a surface
loading rate of 111.4 g COD m2d–1) using a bench-scale reactor
(Fig. 6.7). The discs were completely submerged with a rotational
speed of 12 rpm and a HRT of 21.6 hours. Biogas was produced from
all four baffled sections of the AnRBC, with the highest rate recorded
in the first section, which also had the highest biodegradation rate as
shown by COD and BOD removal, showing that there were sufficient
methanogenic archaea throughout the unit and not just restricted to
the final section. Detailed modeling of their system was also carried
out by the authors. Disc submergence, rotation speed, and HRT are
all significant factors affecting performance.
 Biofilm (Attached Growth) Systems 227

Figure 6.7. Laboratory-scale AnRBC developed by Lu et al. (2015). Reproduced

with permission of Elsevier Publishing.

There are very few examples of full-scale AnRBCs, with anaerobic

biofilters or anaerobic moving bed biofilm reactors being preferred
(Sec. 6.4). A full-scale system was used to treat brewery wastewater
comprising an AnRBC using random-packed caged medium followed
by an aerobic RBC section. The system performed well under fluctu-
ating organic loading and variable pH, but the medium in the AnRBC
failed to establish a significant biofilm, thought to be due to variable
operating conditions and excessive turbulence exerted on the medium
within the cage. Suspended biomass in the anaerobic unit was identi-
fied as an important factor in treatment (Ware and Pescod, 1998). An
AnRBC followed by an aerobic MBBR to treat a strong organic
wastewater (3,500–20,000 mg COD L–1) was tested by Ebrahimi
et al. (2018). The optimum performance of the AnRBC was achieved
using an HRT of 3 days at an organic loading rate of 3.33 kg COD
228 Anaerobic Wastewater Treatment

m–3d–1, using a disk rotational speed of 7 rpm to achieve a removal

efficiency of 81.5%, with a methane yield of 0.309 L CH4 g–1 COD.
The effect of HRT, disc rotational speed, and organic loading rate is
illustrated in Fig. 6.8 and shows the importance of optimizing these
parameters at full-scale operation. Renewed interest in AnRBC tech-
nology has arisen in the potential treatment of high carbohydrate
wastewaters for the production of hydrogen using dark fermentation
(Mohammadi et al., 2019; 2022) (Sec. 9.1.1). Similarly, the potential
of AnRBCs to precipitate metal sulfides from weak mining wastewater
by sulfate reducing bacteria (SRB) was successfully demonstrated by
Kiran et al. (2017). They found that influent metal loading concen-
trations in excess of 3.64 mg Cu(II) L–1 h–1 and 1.87 mg L–1 h–1 for
Fe(III), Pb(II), Ni(II), Zn(II), and Cd(II) inhibited SRB activity with
optimum removal at an HRT of 48 hours. At metal loading rates of
Cu (II) of 100–150 mg L–1 and 50–90 mg L–1 for the other metals,
maximum removal rates of 97% were recorded for Cu(II), 90% for
Cd(II), and >77% for the other metals, with removal efficiencies in
the order Cu > Cd > Pb > Fe > Zn > Ni (Fig. 6.9). High sulfate and
COD removals were also recorded at 53–77 and 68–82%, respec-
tively, at an HRT of 48 hours. Shorter HRTs resulted in reduced
removal efficiency. The results offer a potential solution for treating a
wide range of metal-contaminated wastewaters (Sec. 5.4.1).

6.4 Anaerobic Moving Bed Biofilm Reactors

Anaerobic moving bed biofilm reactors (AnMBBRs) utilize small
plastic media, normally referred to as carriers in these types of reac-
tors. They are normally manufactured from polyethylene and are
10–20 mm in diameter and of a density close to that of water (0.90–
0.95 g cm–3), which allows them to move freely within the reactor, on
which biofilm readily develops (Fig. 6.10). The media is retained
within the reactor by a perforated steel barrier, allowing treated efflu-
ent and associated solids to pass freely into a settlement tank. Except
for hybrid systems, AnMBBRs do not have sludge return, as all the
required biomass is retained on the media, although the suspended
biomass also plays a role in treatment. In contrast to other fixed-film
systems, the reactor is completely mixed so that the whole reactor
 Biofilm (Attached Growth) Systems 229

(a)

(b)

(c)

Figure 6.8. The effect of different operating parameters on an anaerobic rotating

biological contactor treating a strong organic wastewater: Effect of (a) HRT, (b) disk
rotational speed, and (c) organic loading rate on COD removal and methane produc-
tion (Ebrahimi et al., 2018). Reproduced with permission of the American Society of
Civil Engineers, Reston, VA.
230 Anaerobic Wastewater Treatment

(a) (b)

(c) (d)

(e) (f)

Figure 6.9. Removal rate of heavy metals as a function of inlet loading rate in
a laboratory-scale AnRBC operated by Kiran et al. (2017): (a) Cd(II), (b) Cu(II),
(c) Ni(II), (d) Fe(III), (e) Pb(II), and (f) Zn(II). Reproduced with permission of
Elsevier Publishing.

volume is biologically active, resulting in higher biomass activity

(Ødegaard et al., 1994), with no clogging problems and a lower head
loss than other contact systems.
Originally developed in Norway, with the first reactor installed in
Lardnal in 1989, moving bed biofilm reactors are extremely flexible
and can be operated as aerobic, anoxic, or anaerobic systems, either
as standalone processes, in series, or sequentially within a single tank
 Biofilm (Attached Growth) Systems 231

Figure 6.10. Example of a moving bed biofilm reactor carrier used in AnMBBRs,
although foam pads are also widely employed. Reproduced with permission of
Headworks Inc. Houston, Texas.

offering the full range of carbonaceous, nitrification, denitrification,

and phosphorus removal options. All MBBR systems have the same
advantages over other biological systems, in that they generally have
a reduced footprint, low maintenance costs, are robust, can withstand
variable loadings, and are easily retrofitted into existing systems.
AnMBBRs are generally used as a pretreatment step for stronger
wastewaters and biogas generation (Fig. 6.11).
AnMBBRs use the same media as conventional aerobic and anoxic
MBBR systems (Table 6.5), with the commercially available MBBR
media shown in the review by di Biase et al. (2019). Ødegaard et al.
(2000) analyzed the influence of media size and shape on performance
and found that MBBRs should be designed based on surface
area loading rates (g COD m–2d–1), while the shape and size of the
media is not a significant factor as long as an effective surface area is
232 Anaerobic Wastewater Treatment

(a)

(b)

(c)

Figure 6.11. Comparison between aerobic (MBBR) and anaerobic moving bed
biofilm reactors (AnMBBR). While aerobic systems rely on aeration to keep the car-
riers in suspension, AnMBBRs rely on base-fed influent, biogas recycling, or a motor-
ized propellor to keep the carriers suspended.
 Biofilm (Attached Growth) Systems 233

Table 6.5. Characteristics of a typical

plastic carrier (Flocor RMP™) used in mov-
ing bed biofilm reactors, although there are
a wide variety of other types of carriers used
in AnMBBRs.
Polypropylene
Material (density = 0.94 g cm3)
Shape Corrugated cylinder
Dimensions Length: 20–30 mm
Diameter: 15–20 mm
Specific surface 160 m2/m3
Filling rate 70%

Figure 6.12. The new profiled AnoxKalnes Z-200 carriers developed for AnMBBRS
by Veolia (2024). Reproduced with permission of Veolia, Lund, Sweden.

provided. There are several commercial AnMBBRs available, includ-

ing Veolia’s Anoxthane™ Process, which uses the new AnoxK™Z car-
riers, and Headworks EnergyCell™ Process using ActiveCell ™carriers.
AnoxK™Z carriers are unusual in that they do not have holes or gaps
in their structure but are rather like potato crisps, flat and oval in
shape, and slightly curved, with each side embossed with a raised
grid of square cells 225 × 225 µm in size (Fig. 6.12). The biofilm in
AnMBBRs develops very slowly, compared to aerobic systems, and so
234 Anaerobic Wastewater Treatment

it is important to prevent sloughing to maintain optimum biomass

density and diversity. According to the manufacturer, the anaerobic
biofilm develops within the surface cells of AnoxK™Z carriers — the
depth of the cell wall controlling the overall depth of the biofilm with
the excess continuously scoured off. Two cell depths are available at
200 and 400 µm. Flat surface carriers like these that control biofilm
development ensure a constant maximum weight of the individual car-
riers so that they are easily maintained in suspension, also ensuring a
fixed reactor biomass density. In contrast, carriers with holes can result
in excessive biofilm development and entrapped solids, making them
heavy and less easy to maintain in suspension, which requires the car-
riers to be mechanically cleaned periodically. Where the biofilm devel-
opment is uncontrolled and irregular, there is a possibility of variable
removal efficiencies, although in practice, both types of media work
well. This was shown by Arabgol et al. (2020; 2022), who compared
both AnoxK™Z carriers with a more conventional carrier (AnoxK™K5)
under aerobic conditions. They found that the conventional carrier
had a significantly higher carbonaceous removal and lower solids pro-
duction but higher biofilm thickness, which was less dense than both
AnoxK™Z carriers. They also noted there was no significant difference
between the efficiency of the different cell depths (i.e., 200 and 400 µm),
although the thinner grid depth of the AnoxK™Z-200 carrier had
maximum density in relation to thickness and was the lightest
carrier, making them easier to keep in suspension (Aragol et al., 2022)
(Fig. 6.13). However, in AnMBBRs, continuous biofilm depth con-
trol, as achieved with the AnoxK™Z carriers, is advantageous where
mixing is dependent on mechanical means and shear stress on the car-
riers is minimal. While the majority of carriers are made out of poly-
ethylene, high-density polyethylene, or polypropylene, Jirasaranporn
et al. (2022) experimented with PVA gel beads, which are known to
provide excellent surfaces for microbial development, in a pilot-scale
AnMBBR treating fish canning wastewater. Gel beads are very porous
and have a very high specific surface area of 2,500 m2m–3 (i.e., 1,000
m2m–3 surface plus 1,500 m2m–3 internal effective surface area)
compared to other carrier materials, which have surface areas between
200 and 1,200 m2m–3. They found that the beads achieved >70%
removal at loading rates between 3.0 and 8.7 kg COD m3d–1, with the
 Biofilm (Attached Growth) Systems 235

Figure 6.13. Comparison of total biomass, biofilm thickness, and density in

identically loaded and operated AnMBBRs using a standard carrier similar to that in
Fig. 6.10 (K5) and AnoxKalnes Z-200 and Z-400 (Arabgol et al., 2022). Reproduced
with permission of Elsevier Publishing.

acetoclastic methanogen Methanosaeta predominating with a relative

abundance of 24.0–28.9% and the hydrogenotrophic methanogen
Methanobacterium (7.7–14.9%), ensuring good biogas production.
The latter methanogen is extremely slow-growing, so it appears the
very long residence time associated with the gel beads facilitated their
establishment. Among the advantages they observed were that the
beads had a specific gravity close to water, making them easily sus-
pended in the wastewater, minimizing energy input for mixing. They
also reduced biomass loss by sloughing due to the retention of the
biomass within the beads, which also protected biological activity
from toxic shocks. The authors provide some very interesting 16S
rRNA gene amplicon sequencing data on the seed sludge used col-
lected from a UASB using granules (Figs. 6.14 and 6.15) and the
AnMBBRs. The dominant phyla in both the seed and the AnMBBRs
were Bacteroidota (7.1–10.0% of total sequences), Desulfobacterota
(14.1–22.7%), Euryarchaeota (3.5–45.9%), Firmicutes (5.9–10.15%),
and Synergistota (4.5–8.9%).
Maximum organic loading rates of 20 kg COD m–3d–1 were
recorded in laboratory-scale AnMBBRs treating a synthetic protein-
based wastewater, achieving 80% COD removal with a maximum
236 Anaerobic Wastewater Treatment

Figure 6.14. Microbial phyla recovered in the seed sludge and on the PVA gel beads
used in pilot-scale AnMBBR treating fish canning wastewater. Samples were taken at
two different depths of 0.75 m (S) and 1.25 m (T) height within the AnMBBRs, which
were operated at organic loading rates of (P1) 4.14 kg COD m–3d–1; (P2) 5.38 kg
COD m–3d–1; (P3) 7.54 kg COD m–3d–1; and finally (P4) 4.14 kg COD m–3d–1.
Reproduced from Jirasaranporn et al. (2022) under the Creative Commons Attribution
license (CC BY 4.0) (http://creativecommons.org/licenses/by/4.0/).

biogas production of 9.25 m3 CH4 m–3, equivalent to an average

methane yield of 0.36 m3 CH4 kg–1 COD removed, with a biogas
methane content of 60–70% (di Baise et al., 2016). They used
HeadworksBio cubic AC920 plastic carriers and compared two reac-
tors operating at 25 and 35% of the reactor volume, respectively,
although no significant difference in performance was recorded
between the two reactors. Seed was taken from a sludge digester, with
optimum biofilm development occurring after eight weeks.
Interestingly, they observed that only 2.5% of COD removal was due
to the suspended biomass, with nearly all biodegradation carried out
by the carrier biofilm. Shahzad et al. (2021) compared the use of an
 Biofilm (Attached Growth) Systems 237

Figure 6.15. The most abundant genera recovered in the seed sludge and on the PVA
gel beads used in pilot-scale AnMBBR treating fish canning wastewater. Samples were
taken at two different depths of 0.75 m (S) and 1.25 m (T) height within the
AnMBBRs, which were operated at organic loading rates of (P1) 4.14 kg COD m–3d–1;
(P2) 5.38 kg COD m–3d–1; (P3) 7.54 kg COD m–3d–1; and finally (P4) 4.14 kg COD
m–3d–1. Reproduced from (Jirasaranporn et al., 2022) under the Creative Commons
Attribution (CC BY 4.0) license (http://creativecommons.org/licenses/by/4.0/).

AnMBBR and an up-flow anaerobic hybrid reactor (UAHR) for the

treatment of textile desizing wastewater and found optimum loadings
of 6 and 8 kg COD m3d–1, respectively. By increasing the operating
temperature from 35 to 55°C, the COD removal, biogas production,
and methane content all increased in the AnMBBR by 1.2, 2.6, and
1.1 times, respectively, with similar increases observed for the UAHR.
Laboratory scale trials to assess the removal of pharmaceuticals during
municipal wastewater treatment using an AnMBBR coupled with a
secondary aerobic MBR (Iliopoulou et al., 2023). The plant was
operated at two different temperature phases at 35°C (A) and 20°C (B).
Together, they produced excellent removal efficiencies (Table 6.6),
with the AnMBBR having higher biogas production at 35°C but
 238 Anaerobic Wastewater Treatment
Table 6.6. Experimental conditions (pH, temperature, TSS, and biogas production) of each reactor in both phases and removal
efficiency of COD, BOD, NH4–N, and TP (Iliopoulou et al., 2023). Reproduced with permission of Elsevier Publishing.
Methanogenic moving bed biofilm reactor (AnMBBR)

Biogas
MLSS Production CODdis BOD NH4–N Tp removal
Phase pH T (°C) (mg/L) (mL/Lreactord) removal (%) removal (%) removal (%) (%)
Phase A 6.9 ± 0.5 35.0 ± 0.5 265 ± 190 176 ± 86 39.6 ± 16.0 51.8 ± 17.0 5.7 ± 9.9 <5%
Phase B 6.7 ± 0.9 20.0 ± 0.5 431 ± 171   68 ± 16 42.3 ± 17.7 54.3 ± 12.2 7.0 ± 10.0 <5%

Aerobic membrane bioreactor (AeMBR)

MLSS a
Total CODdis a
Total BOD a
Total NH4-N a
Total TP
Phase pH T (°C) (mg/L) removal (%) removal (%) removal (%) removal (%)
Phase A 7.4 ± 0.6 18.7 ± 1.3 1942 ± 650 86.4 ± 12.5 91.6 ± 5.3 96.4 ± 4.9 <5%
Phase B 7.6 ± 0.2 17.7 ± 1.7 3321 ± 1177 90.9 ± 12.0 95.6 ± 3.9 95.5 ± 6.0 64.4 ± 15.3

Total removal of CODdis, BOD, NH4–N and TP achieved in the AnMBBR-AeMBR system.
a
 Biofilm (Attached Growth) Systems 239

better COD and BOD removal rates at 20°C. Percentage removal

ranged from 65 to 100% for metronidazole (MTZ), trimethoprim
(TMP), sulfamethoxazole (SMX), and valsartan (VAL). Carbamazepine
(CBZ) and diclofenac (DCF) removal efficiencies were much less at
<30%. Interestingly, the AnMBBR was solely responsible for the
removal of MTZ, TMP, and CBZ, while it also contributed to the
removal of SMX and VAL mainly by sorption (Fig. 6.16). Azo dyes
are widely used in a wide variety of industries, including paper print-
ing, carpet and textile manufacture, tanneries, polymers, and cosmet-
ics, and are highly polluting. Congo red, for example, is carcinogenic,
teratogenic, and mutagenic in animals, including humans, making it
a priority-listed pollutant. Maurya et al. (2021) used a laboratory
scale AnMBBR employing polyurethane and polypropylene foam

Figure 6.16. Removal (%) of target pharmaceuticals in each experimental phase

and reactor of the studied system. The temperature of the Anaerobic Moving
Bed Biofilm Reactor (AnMBBR) was set at 35°C at Phase A and 20°C at Phase B
(Iliopoulou et al., 2023). MTZ: Metronidazole; TMP: Trimethoprim; SMX:
Sulfamethoxazole; CBZ: Carba­ mazapine; VAL: Valsartan; DCF: Diclofenac.
Reproduced with permission of Elsevier Publishing.
240 Anaerobic Wastewater Treatment

carriers into which the bacterium Lysinibacillus fusiformis, which is

able to biodegrade Congo red dye, had been immobilized. Removal
rates of between 58 and 89% were achieved at different loading rates
and under different operating conditions. The study opens up the
possibility of employing AnMBBRs using immobilized bacteria for
the biodegradation of highly toxic waste streams.
AnMBBRs are especially suited for temperatures found in temper-
ate areas, although the optimum temperature range is between 20
and 36°C, making them ideal for food processing wastewaters, which
are composed of soluble and highly degradable organic matter.
AnMBBRSs have been successfully employed in the dairy industry,
sugar beet processing and sugar refining, and vegetable and fruit pro-
cessing, including brewing, distilling wine production, and general
drink and beverage industries. All strong organic wastewaters are
amenable to treatment with AnMBBRs, including pulp and paper
wastewaters and those from the chemical, textile, oil, and gas indus-
tries. The combination of compact, low odor, low maintenance and
energy costs, and carbon recovery through biomethane production
makes the AnMBBR process an ideal choice for all these industries.
Many authors have modeled AnMBBRs and have generally found the
Grau second-order and modified Stover-Kincannon models the best
fit (Maurya et al., 2021; Shahazad et al., 2021). MBBR technology,
in general, is reviewed by Ødegaard (2006) and di Biase et al. (2019)
and carriers specifically by Zhao et al. (2019).

6.5 Expanded and Fluidized Bed Reactors

Anaerobic expanded and fluidized bed reactors (Fig. 6.17) are modi-
fications of the anaerobic biofilter in which fine-grained inert (sand)
or reactive (activated carbon or zeolite) random carriers (i.e., media)
are used (Yoda et al., 1989). The carrier, which is coated with bacteria
and archaea, is expanded or fluidized by applying a high-mixing
velocity. However, it is difficult to maintain the optimum mixing
velocity in order to ensure the carrier remains in suspension without
causing shear forces strong enough to strip off the majority of the
accumulated biomass. Therefore, mixing is achieved within the
 Biofilm (Attached Growth) Systems 241

Figure 6.17. Schematic comparison of a porous medium bed reactor (AnPBR),

expanded bed reactor (AnEBR), and an anaerobic fluidized bed reactor (AnFBR).

reactor by applying high rates of gas or effluent recycle, along with

the influent wastewater, from the base of the reactor. The carrier par-
ticles can be fully mixed (fluidized), which increases the volume of the
reactor occupied by the medium by 20–25%, or just expanded, which
increases the reactor volume occupied by the medium by <10%
(Switzenbaun, 1983; Iza, 1991).
When influent wastewater or recycled biogas passes upward
through the voids (i.e., interstices) in a layer of an appropriate carrier
settled at the base of a reactor without disturbing the carriers, this is
known as a static, fixed, or porous medium bed reactor (AnPBR).
When the rate of flow through the settled carriers is increased, the
carriers begin to be suspended within the reactor, the buoyancy bal-
ancing the gravitational and drag forces exerted on the induvial carrier
particles, increasing the size of the interstices (i.e., expanded bed reac-
tor — AnEBR). As the flow rate increases even further, the minimum
fluidization velocity is reached, and the bed of the medium expands
and becomes fluidized (i.e., the solids-liquid mixture behaves as a
liquid), the density decreases, and the bed voidage rapidly increases
(i.e., fluidized bed reactor — AnFBR) (Fig 6.17). The required mini-
mum fluidization velocity decreases as the size of the medium
decreases, so less energy is required where finer carrier particles are
used. The hydrodynamics and mass transfer characteristics of fluidized
beds are reviewed by Sharma et al. (2021).
The particle size of carriers ranges from 0.3 to 1.0 mm, depend-
ing on the type of carrier used. The decrease in carrier particle size has
242 Anaerobic Wastewater Treatment

resulted in a significant increase in the specific surface area available

for biological growth, making such systems highly efficient. However,
smaller sizes are readily lost from the reactor, while larger-sized carri-
ers are difficult to fluidize. Due to the difficulty of maintaining an
optimum mixing velocity to ensure the medium remains in suspen-
sion without causing biomass stripping due to excessive shear, it may
be that expanded bed reactors are the more effective system. In
expanded beds, the biofilm-coated carrier remains on the floor of the
reactor and is expanded by the movement of the liquid forced up
through the medium. Fluidized beds are normally much taller, with a
height-to-diameter ratio of 5–6:1. Although difficult to start up, flu-
idized beds are highly resistant to temperature, toxic compounds, and
high organic loadings. They are also tolerant to wide fluctuations in
influent quality including high solids concentrations (Table 6.2).
Expanded and fluidized beds have large solids-liquid contact areas, no
clogging, a compact design with a small footprint, and no moving
parts or internal structures, making them an attractive option for
more complex and strong wastewaters, especially at ambient tempera-
tures (10–20°C), although expanded beds are also highly efficient in
treating settled sewage. Details on the design and operation of fluid-
ized bed reactors are given by (Heijnen et al., 1989; Iza, 1991; Marín
et al., 1999). Typical loading rates are given in Table 6.4.

6.5.1 Anaerobic Expanded Bed Reactors

Anaerobic expanded beds reactors (AnEBRs) were developed in the
early 1970s, and Jewell et al. (1981) successfully used a laboratory
scale unit operated at 20°C to treat settled municipal wastewater.
They found that even at HRTs of <30 minutes, the majority of the
biodegradable organic matter was removed, producing a final effluent
of 40 mg COD L–1 and 5 mg SS L–1 (Fig. 6.18). This is a much shorter
HRT compared to similar anaerobic systems. They recorded opti-
mum removal efficiencies up to a maximum loading of 4 kg BOD
m–3d–1 and that the AnEBR was able to withstand significant tempera-
ture differences as well as rapid changes in organic loading and flow
rate, making it a very versatile and robust system.
 Biofilm (Attached Growth) Systems 243

Figure 6.18. Influence of HRT on COD and suspended solids (SS) removal of an
anaerobic expanded bed reactor treating settled domestic wastewater (Jewell et al.,
1981). Reproduced with permissions of the Water Environment Federation,
Alexandria, VA.

AnEBRs have been successfully used to treat a wide range of

industrial and agricultural wastewaters (Sharma et al., 1999), offering
compact and efficient COD reduction prior to aerobic treatment.
They have also been successfully employed to remove trace micropol-
lutants from drinking water using a carbon source such as methanol,
although it may be possible to use the natural humic acids in the
water as the electron acceptor, with very short HRTs of 1–2 hours, a
process known as reductive dechlorination (Carter and Jewell, 1993).
244 Anaerobic Wastewater Treatment

The long sludge retention times mean that the biomass often becomes
naturally granular in nature, offering better settleability and treatment
potential. Much research was carried out in the 1990s to understand
how granulation could be stimulated and maintained (Ye et al.,
2005), and today, expanded beds have now been largely superseded
by expanded granular sludge bed (EGSB) technology, which is a
hybrid between a UASB and AnEBR. They are available commer-
cially, for example, the BioBed™ EGSB reactor from Veolia (Sec. 7.2).

6.5.2 Anaerobic Fluidized Bed Reactors

Primary and thickened waste-activated sludges were successfully
treated in mesophilic fluidized bed reactors (AnFBRs) using zeolite as
media, achieving almost double the treatment rates achieved by con-
ventional digestion (Mustafa et al., 2014). At an HRT of 9 days,
COD and VSS removal was 85 and 88%, respectively, for primary
sludge at an organic loading rate of 4.2 COD m–3d–1 and a 68 and
69% reduction in COD and VSS, respectively, for the waste-activated
sludge at the same HRT and loading rate. Methane yields were 0.47
and 0.51 L CH4 g–1 VSS removed for the primary and waste-activated
sludges, respectively. Andalib et al. (2012) successfully used a zeolite
medium (diameter of 425–610 mm and specific surface area of 26.5
m2 g–1) to treat the byproduct of corn bioethanol production (i.e.,
stillage) with a COD of 130,000 mg L–1 and suspended solids con-
centration of 47,000 mg L–1 in an AnFBR at a loading rate of 29 kg
COD m–3d–1 with an HRT of 3.5 days. The system achieved an 88 and
78% reduction in COD and TSS (total suspended solids), respectively,
with a methane yield of 0.31 L CH4 g–1 COD (equivalent to 40 L
CH4 L–1 stillage d–1). They subsequently compared this with primary
sludge and found similar results with an 82% reduction in both COD
and TSS at a loading rate of 9.5 kg COD m–3d–1 and a maximum
methane yield of 0.25 L CH4 g–1 COD at an HRT of 4 days (Andalib
et al., 2014).
The design and operation of AnFBRs are explained in detail by
Deng et al. (2016), who give details of how the key design and operat-
ing parameters are selected and calculated, including HRT, organic
loading rate, sludge loading rate, inclined settler dimensions, sludge
 Biofilm (Attached Growth) Systems 245

discharge frequency, and liquid and gas recycle ratios, and then trans-
lated to a full-scale design. They give an example of the design and
operating parameters for a full-scale AnFBR for treating 300 tonnes of
synthetic dyeing wastewater per day. The influent to the AnFBR con-
tains 100 mg L–1 (ppm) of the dye Reactive Blue 250 and a COD of
1,000 mg L–1, with a COD/BOD ratio of 0.42. The wastewater has a
pH of 5.5–8.0, a temperature of 20–23°C, and a total nitrogen and
phosphorus content of 50 mg N L–1 and 20 mg P L–1. The design is
required to achieve a 90% color reduction and 80% COD removal.
The reactor design is shown in Fig. 6.19 and the full-scale design and
operating conditions are given in Table 6.7. Carriers for AnFBRs
should be porous, have a large surface area, be non-reactive but able

Figure 6.19. Schematic design for an AnFBR treating a synthetic dyeing wastewa-
ter (Deng et al., 2016). Details of design values are given in Table 6.7. Reproduced
with permission of Elsevier Publishing.
246 Anaerobic Wastewater Treatment

Table 6.7. Design and operational parameters for a full-scale AnFBR treating a
synthetic dyeing wastewater (Deng et al., 2016). Reproduced with permission of
Elsevier Publishing.
Influent flowrate, Q0 300 m3/d
Hydraulic retention time, HRT 14h
Organic loading rate, OLR 1.46 kgCOD/m3 d
Sludge loading rate, SLR 0.29 kgCOD/kgVSS d
Percentage of bioparticles volume 15%
Safety factor 1.5
Volume of the reaction region, V 309 m3
Number of reactors, N 1
Reactor height, H 12.1 m
Length of reaction region, LR 5.5 m
Reactor width, W 4.6 m
Time between bioparticles discharge, td 67 days
Biogas production rate 112.7 m3/d
Liquid recycle ratio 5.8
Biogas recycle ratio 4.8
Length of the inclined channel filled with suspension, L 1.2 m
Width of the inclined channel, w 0.05 m
Spacing between the channels, b 0.05 m
Angle of inclination of the channel from the vertical, θ 30°
Number of inclined channels, NC 2376
Arrangement of inclined channels in the inclined settler 33 × 72
Length of the separation region, LS 1.8 m

to adsorb pollutants, be ideally between 0.1 and 0.7 mm, and have a
density of 1,500 to 2,500 kg m3 (Heijnen et al.,1989). Granular acti-
vated carbon (GAC) is an ideal carrier and can reduce the start up-time
from 100 to 15 hours when used instead of sand (Wright and Raper,
1996). Typical loading values and HRT based on maximum influent
COD and BOD/COD ratio have been summarized by Deng et al.
(2016) (Table 6.8).
As the COD is consumed, a portion is used for biomass produc-
tion, approximately 1.42 kg COD per kg biomass, and the remainder
 Biofilm (Attached Growth) Systems 247

Table 6.8. Typical operating values for the solids loading rate (SLR), organic load-
ing rate (OLR), hydraulic retention time (HRT), and volume of bioparticles for
AnFBRs based on influent BOD/COD ratio and maximum COD (Deng et al.,
2016). Reproduced with permission of Elsevier Publishing.
Initial Maximum SLR OLR Percentage
BOD/ influent (kgCOD/ (kgCOD/ HRT of bioparticles
COD COD (ppm) kgVSS d) m3 d) (h) volume in reactor
≤0.3 ≤1500 0.05–0.3 <3.0 >12 5–20
0.3–0.5 ≤2000 0.1–0.55 1–8 8–48 5–20
>0.5 ≤15,000 0.6–2 5–40 3–24 5–20

for biogas production. The amount of methane in biogas can be

approximated as 65%, with a production rate of 0.35 m3 methane per
kg COD consumed. Therefore, the amount of methane generated
(Gmethane) can be calculated as the amount of COD removed by sub-
tracting the amount of COD used for biomass production:

Gmethene = 0.35(Q 0(S0 – S) – 1.42mt)(6.3)

where Q 0 is the influent flow rate (m3d–1), S0 is the influent COD

concentration (kg m–3), S is the effluent COD concentration (kg m–3),
and mt is the amount of biomass produced per day (kg VSS d–1). The
parameter mt is calculated by measuring the amount of biomass at the
start of degradation to final discharge:
V (Mt d − M i )
mt = (6.4)
td
where V is the total volume of the reaction region of the reactor (m3),
Mtd is the biomass concentration at the point of discharge (kg VSS
m–3), Mi is the initial biomass concentration in the reactor (kg VSS
m–3), and td is the time between each bioparticles discharge (d).
Carriers will need to be recovered and biomass removed by washing
before the carriers are returned to maintain the optimum sludge load-
ing rate (Table 6.8). The biogas (Gbiogas) production rate is calculated
as:

G methane
Gbiogas = (6.5)
0.65
248 Anaerobic Wastewater Treatment

This is further explained by van Lier et al. (2008) and Deng et al.
(2016).
In Cuba, fluidized beds are used to treat vinasse, the final waste
from the alcohol distillation process from sugar cane molasses. With
organic loading rates of up to 10 kg COD m–3d–1 and COD removals
>70%, the waste produces methane-rich biogas but with sulfide con-
centrations >1% (Conde et al., 1993). However, sulfide concentra-
tions can be controlled by using either granular activated carbon
(GAC) or zeolites as carriers. Other researchers have also observed
the simultaneous removal of sulfate and ammonia in anaerobic GAC
fluidized beds treating vinasse (Fdz-Polanco et al., 2000; 2001).
This resulted in high concentrations of molecular nitrogen in the
biogas but low concentrations of hydrogen sulfide. They also identi-
fied an unusual anaerobic nitrogen removal mechanism, resulting in
the loss of nitrogen in the liquid phase and the presence of nitrogen
in the biogas, with up to 55% of the total Kjeldahl nitrogen removed
by this process, which was identified as the anammox process reac-
tion. The removal mechanism of sulfur is less clear, with elemental
sulfur found within the reactor and associated with the effluent sus-
pended solids. The function of the GAC appears to be to increase the
localized concentration of nitrogenous and sulfur species by adsorp-
tion close to the biofilm, encouraging the biochemical reactions to
occur.
The overall reaction has been postulated as:

  SO4= + 2NH4+ → S + N2 + 4H2O (∆G° = –47.8 kJ mol–1)(6.6)

Fdz-Polanco et al. (2001) have suggested that this reaction com-

prises three separate reactions:

3SO4= + 4NH4+ → 3S= + 4NO2– + 4H2O + 8H+(6.7)

3S= + 2NO2– + 8H+ → 3S + N2 + 4H2O(6.8)

2NO2– + 2NH4+ → 2N2 + 4H2O  (6.9)

The last equation being the Anammox reaction.

 Biofilm (Attached Growth) Systems 249

6.5.3 Anammox Process

Although both nitrification and denitrification were observed in the
19th century, anammox was not discovered until the 1990s. The reac-
tion is carried out by anaerobic ammonium oxidation (anammox)
bacteria, where nitrite (the electron acceptor) is converted to nitrogen
gas by autotrophic bacteria using ammonium as the electron donor:

NH4+ + NO2 → N2 + 2H2O(6.10)

As this is an autotrophic reaction, no external carbon source (e.g.,

methanol) is required (Jetten et al., 1997), with activity inhibited at
oxygen concentrations >0.5% (Strous et al., 1997a; 1997b). To facilitate
the anammox reaction, nitrite must be present and can be produced by
a separate nitritation process known as a partial nitritation (PN). Most
commercial anammox systems employ PN as an integrated part of the
main process (Lackner et al., 2014), including when used as a tertiary
step for nitrate removal from domestic wastewater treatment plants
(Cao et al., 2019). The process is normally used in conjunction with the
PN SHARON® process (Gray, 2021), which produces an ammonium-
nitrite feed effluent ideal for the anammox process (Sec. 4.4.7).
The autotrophic bacteria responsible for the oxidation of ammo-
nia and the simultaneous reduction of nitrite belong to the widely
distributed phylum Planctomycetes. Not all the bacteria in this phylum
are able to oxidize ammonium, this being restricted to the anammox-
Planctomycetes, which have a characteristic membrane-bound cell
compartment called the anammoxosome (Fuerst and Sagulenko,
2011). Ammonium is used as an energy source by the anammox bac-
teria and carbon dioxide as a carbon source, with NO2– , and NO3–, if
present, used as electron acceptors. Kartel et al. (2011) have described
the process in detail where the enzyme nitrite oxidoreductase reduces
nitrite to NO, which then reacts with ammonium to form hydrazine
(N2H2), enabled by the enzyme hydrazine synthase, which is oxidized
by hydrazine dehydrogenase to nitrogen. The growth rate (doubling
time 11 days) and growth yield (0.11 g VSS g–1 NH4-N) for these
microorganisms are very low, resulting in low sludge production.
250 Anaerobic Wastewater Treatment

Therefore, efficient biomass retention is required to maintain suitable

anammox biomass concentrations within reactors, favoring up-flow
fixed bed reactors but especially expanded and fluidized systems, and
also AnMMBRs (Strous et al., 1997b). Anammox reactors are very
compact due to a high maximum specific nitrogen consumption rate
(0.82 g N g–1 VSS d–1), a high affinity for ammonium and nitrite, and
granular growth, allowing efficient biomass retention. They use far
less energy (60%) than alternative nitrification-denitrification pro-
cesses and do not require a separate organic carbon source, making
them a more cost-effective treatment option for ammonia-rich efflu-
ents (Lackner et al., 2014). Also, as nitrous oxide and carbon dioxide
are not an intermediate reaction product in the anammox reaction,
greenhouse gas emissions are minimized.
A key disadvantage to the system is the sensitivity of the anammox
bacteria to changes in environmental conditions, in particular, the
nature of the wastewater feed. While organic matter, nutrients,
sulfide, and salinity concentrations are all possible sources of inhibi-
tion, it is the toxic nature of ammonium and nitrite that can act syn-
ergistically in terms of toxicity, which is most often the cause of
process inhibition and failure. Therefore, the feed ratio must be care-
fully controlled, as well as the pH. It has been established by Zhang
et al. (2016) that influent nitrogen concentrations of below 320 mg
N L–1 guaranteed stable conditions are maintained in expanded bed
reactors used for anammox. The development of the anammox gran-
ule can take a very long time and have a settling velocity in the region
of 100–150 m h–1 (Fig. 6.20).

(a) (b) (c)

Figure 6.20. Structure of the carrier used by Zhang et al. (2016) in their anaerobic
expanded bed anammox reactor. (a) The carrier, (b) the carrier with anammox bio-
film development (1.0–1.2 mm depth), and (c) the actual anammox granule (average
size 2.4 mm) with a high settling velocity of >200 m h–1. Reproduced with permis-
sion of Elsevier Publishing.
 Biofilm (Attached Growth) Systems 251

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 7
Floc and Granular-based Systems
(Dispersed Growth)

The drive toward more sustainable and low-carbon wastewater

treatment has focused attention on the exploitation of processes uti-
lizing anaerobic granulation. Show and Lee (2016) compared the
major advantages that such systems have over their more common
aerobic treatment options. These included: (a) higher organic load-
ings enabling the treatment of high-strength wastewaters, (b) lower
operational costs due to no aeration, (c) tall column or rectangular-
shape reactor designs requiring much lower bioreactor footprint and
land space, (d) no noise, aerosol, and odor problems, (e) higher flex-
ibility in the range of wastewaters that can be treated in terms of
strength, toxicity, etc., (f) significantly lower sludge volume genera-
tion leading to significant cost sludge production, resulting in reduc-
tion in sludge treatment and disposal costs, (g) high carbon capturing
potential through biogas production coupled with high chemical
oxygen demand (COD) removal capabilities, and (h) anaerobic gran-
ules can be stored unfed and without need of aeration for long peri-
ods of time without loss of viability.
Granular anaerobic sludge has opened up new opportunities
for sustainable, low-cost, zero-carbon wastewater treatment, from
the early up-flow anaerobic sludge bed (UASB) reactors (Sec. 7.1)
through the transition to expended granular bed reactors (EGBRs)
(Sec. 7.2) and now the third-generation anaerobic systems such as
Internal Circulation (IC) (Sec. 7.3). There are many other designs

259
260 Anaerobic Wastewater Treatment

using granular sludge, including anaerobic continuously stirred tank

reactors (AnCSTRs) (Sec. 7.4), anaerobic sequencing batch reactors
(AnSBRs) (Sec. 7.5), anaerobic baffled reactors (AnBRs) (Sec. 7.6),
anaerobic migrating blanket reactors (Sec. 7.7), and hybrid reactors
(AnHRs) (Sec. 7.8). All these systems are commercially available with
many different often-patented modifications.

7.1 Up-flow Anaerobic Sludge Blanket Reactors

In the UASB system, the biomass is retained in the reactor by floc-
culation, using a similar process technology to that used in sludge
blanket clarification in potable water production. Originally devel-
oped in the Netherlands (Lettinga et al., 1980), it is now the most
popular type of anaerobic digester design for industrial wastewater
treatment. A heavily flocculated sludge develops within the special
reactor (Fig. 7.1), which acts as a separate fluidized bed, able to with-
stand high mixing forces. It should be stressed that no support
medium (carrier) is added to the reactor and that the biomass that is
produced flocculates to produce dense flocs or granules up to several

Figure 7.1. Schematic design of an up-flow sludge blanket reactor (UASB) with a
simplified gas-liquid-solids (GLS) separator.
 Floc and Granular-based Systems (Dispersed Growth) 261

millimeters in diameter and a settling velocity of 10 m h–1 (Laguna

et al., 1999). Granules are composed of a wide range of proteins and
polysaccharides (extracellular polymeric substances (EPS)), both bacteria
and methanogenic archaea (e.g., Methanothrix, Methanobacterium,
Methanobrevibacter, Methanosarcina) as well as calcium precipitates
(Wu et al., 1987; Vissier et al., 1991; Brummeler et al., 1985).
Granules have three distinct layers, with the exact microbial composi-
tion dependent on the growth substrate (Hulshoff-Pol et al., 1982;
1983; 1986; Grotenhuis et al., 1991: Harmsen et al., 1996). The
external layer is a heterogenous layer of hydrogen-producing micro-
organisms consisting of rods, cocci, and filaments. The middle layer
contains predominantly bacterial rods, including hydrogen-producing
acetogens and hydrogen-consuming organisms. In the center is a
homogenous bacterial core with a large number of cavities. It consists
of Methanothrix-like cells that are thought to act as the nucleation
centers for granule development (MacLeod et al., 1990) (Sec. 9.2.1).
The reactor comprises the sludge bed, the sludge blanket, and a
series of gas-liquid-solids (GLS) separators at the top of the reactor
(Fig. 7.1). The biomass takes up 40–60% of the reactor volume, with
a solids concentration range of 40–100 g L–1. While the sludge is
expanded by the passage of influent wastewater from a distribution
system at the base of the reactor, forming the sludge bed at an up-
flow velocity of 0.5 to 1.0 m h–1, mixing is also caused by gas produc-
tion bubbles, which can attach to granules carrying them upward
along with smaller sludge particles forming the sludge blanket, which
occupies between 15 and 25% of the reactor volume.
The GLS separator provides a series of inclined plates or baffles
on which the particles rising to the surface come into contact, causing
the gas bubbles to be released, thereby allowing the granules to fall
back to the sludge bed. The gas that is constantly being released from
the sludge bed, as well as the bubbles detached by the GLS separator,
are subsequently collected by an inverse hopper at the top of the reac-
tor and passed to the biogas storage facility. The effluent will contain
some fine particulate matter, mainly flocs, as well as small granules,
which are removed in a separate settlement zone, allowing them
to fall back into the UASB reactor. COD removal efficiencies of
262 Anaerobic Wastewater Treatment

70–85% are possible at 20–25°C with organic loadings of up to 10 kg

COD m–3d–1 and an hydraulic retention time (HRT) of 4–20 hours,
depending on wastewater characteristics. All wastewaters must first
pass through screens and grit separation, and also, if necessary, pri-
mary sedimentation. Design and loading rates are given by Lettinga
and Holshoff-Pol (1991).
Mixing is achieved by pumping the feed wastewater through the
base of the reactor up through the sludge blanket. Above the blanket,
finer particles flocculate, and in the upper settlement zones, they set-
tle as sludge back to the blanket, thus preventing the washout of
biomass. The settlement zones occur below the gas collection bowls
that slope at an angle of 50°, which promotes the return of the sludge
from the settlement areas. The process is characterized by very high
mean cell residence times (MCRT), which are even higher than
anaerobic filters, and a low HRT (<1 day). It takes up to six months
for a suitable granular sludge to develop from a soluble wastewater,
so a suitable inoculum is required for rapid start-up. The process is
dependent entirely on the ability of the biomass to form granules
using particular wastewaters and is widely used for strong industrial
wastewaters with low suspended solids content, such as creamery
wastes and the waste from the manufacture of soft drinks. Reactors
are typically 3–5 m in height. A 200 m3 UASB successfully treated
sugar beet waste at a rate of 16 kg COD m–3d–1, achieving a 90%
removal at an HRT of only 4 hours. UASB reactors have been used
to treat a variety of complex wastewaters (Imai et al., 2000), including
wastewaters from fish canning (Puñal and Lema, 1999), fiberboard
manufacture (Fernández et al., 1995; Puñal et al., 1999), landfill lea-
chate (Chang, 1989; Borzacconi et al., 1999), agro-industries
(Driessen and Yspeert, 1999), chemical industry wastewaters such as
polyethylene terephthalate (PET) (Fdz-Polanco et al., 1999), malting
and brewing (Fang et al., 1990; Yan and Tay, 1996; Martínez et al.,
2001), slaughterhouses (Manjunath et al., 2000; Del Nery et al.,
2001), and papermills (van Lier et al., 2001). Compared to contact
processes, it is difficult to operate and maintain the structure of the
sludge blanket (Lettinga and Hulshoff-Pol, 1991) (Table 6.2).
Details of loading rates are summarized in Table 6.4. Sludge blanket
 Floc and Granular-based Systems (Dispersed Growth) 263

reactors have been used as a separate methanogenic phase, with the

first two stages of the digestion process being carried out in a separate
reactor with increased biogas production (Morris and Burgess, 1984).
The most well-known commercial system is the Biothane® UASB
process. A development of the UASB reactor is the expanded granular
sludge bed (EGSB) reactor, which is designed to treat low-strength
wastewaters at high velocities at temperatures of 10°C and less (Kato
et al., 1994; Rebac et al., 1995; Jeison and Chamy, 1999; van Lier
et al., 2001) (Sec. 7.2).
The structure of anaerobic granules was captured by Wiegant
and de Man (1986) in a series of remarkable images (Fig. 7.2), and

(a) (b)

(c) (d)

Figure 7.2. Scanning electron micrographs of thermophilic granular sludge grown

on acetate from digested sewage sludge as inoculum. (a) Large granule showing
several cavities through which the biogas is excreted. (b) Detail of (a) showing one
such cavity. (c) Detail showing blunt-ended filaments typical of the acetoclastic
methanogen Merhanorhrix. (d) Large magnification showing the dense structure of
the granule (Wiegant and de Man, 1986). Bars indicate 100, 10, 1, and 10 pm,
respectively. Reproduced with permission of John Wiley and Sons Inc.
264 Anaerobic Wastewater Treatment

while the process of granulation remains only partially understood, a

number of different hypotheses have been proposed (Show et al.,
2020) (Sec. 9.2.1). An amazing in-depth study of the granulation —
regeneration cycle in the anammox process, including the formation
and structure of granules and the role of the different mechanisms
involved including quorum sensing, has been carried out by Xue et al.
(2023).

7.2 Expanded Granular Sludge Bed Reactors

The development of EGSB technology arose from the need to treat
chemical industry wastewaters that were inhibitory or toxic to UASBs
(Zoutberg and de Been, 1997). Like the UASB, EGSB technology
relies on granular sludge development, although when compared to
the UASB reactor, they are much taller: 12–18 m compared to 5–7
m, with a height:width ratio of 4–5:1 and increased up-flow velocities
at 6–10 m h–1, with high recycle ratios (van Lier, 2008). This ensures
a truly expanded bed of biomass with a high degree of mixing, maxi-
mizing wastewater–granule contact to achieve high COD removal
rates, and biogas production (Fig. 7.3). The increased recycling rates
ensure a high degree of dilution so that toxic compounds can be
safely treated. For example, an EGSB successfully treated a chemical
wastewater containing formaldehyde and methanol, from which the
former was being produced from the latter, at concentrations of up to
10 g L–1 and 20 g L–1, respectively, achieving removal efficiencies of
>98% for both compounds (Zoutberg and de Been, 1997). Organic
loading rates of 30 kg m–3d–1 are achievable with both low and high-
strength wastewaters able to be treated, including normal municipal
wastewaters at low temperatures (Lettinga et al., 1997). High biogas
production can be achieved when treating food processing wastewa-
ters, such as brewery, yeast, sugar, fruit and vegetable processing, and
corn ethanol production wastewaters.
The higher loading rates, simpler operation, and lower opera-
tional costs have seen a shift from the popular UASB to EGSB systems
for industrial treatment (van Lier, 2008). The relative loading rates of
different reactor types are compared in Fig. 7.4. Biothane systems
 Floc and Granular-based Systems (Dispersed Growth) 265

Figure 7.3. Schematic design of an expanded granular bed reactor (EGBR) with a
simplified GLS separator.

have developed a commercial EGSB process called Biobed®, operat-

ing at high liquid (10 m h–1) and gas velocities (7 m h–1) (Zoutberg
and de Been, 1997).
A down-flow version of the EGBB reactor has been developed by
Professor Eyyup Ellis in the United States (Mach, 2000) and has been
shown to be effective in treating a wide range of wastewaters, includ-
ing municipal wastewater, which achieved a final effluent with a BOD
of <30 mg L–1 at an HRT of 24 hours (Evans, 2004). The static
granular bed reactor (SGBR) is simpler in operation to a standard
266 Anaerobic Wastewater Treatment

Figure 7.4. Comparison of major floc-based anaerobic wastewater treatment sys-

tems in terms of loading capacity (Show et al., 2020). Reproduced with permission
of Elsevier Publishing.

EGBR or UASB, requiring significantly less energy input, except for

occasional backwashing to remove solids accumulation, with no gran-
ule washout in the effluent. Debrik and Coskun (2009) successfully
used a SGBR to treat poultry wastewater, achieving a 95% COD
reduction using laboratory-scale reactors producing 0.25 L CH4 g–1
COD removed at an operating temperature of 22°C (Fig. 7.5). The
process followed modified Stover–Kincannon and Grau second-order
models. Debrik also tested the system on leachate at a loading rate of
15kg COD m–3 d–1, recording >90% COD removal (Debrik et al.,
2005). However, the long-term operation of the process, especially in
maintaining the structure of the granular sludge, remains less clear.

7.3 Internal Circulation Systems

Internal Circulation (IC) has been pioneered in the Netherlands by
the company Paques and comprises two reactor chambers, one on top
of the other, each with its own GLS separator (Fig. 7.8). Essentially,
the IC process is two UASB reactors: the lower unit treating a high
organic loading and the upper acting as a polishing unit treating a low
organic loading. Enhanced GLS separation results in better biomass
retention and enhanced treatment, resulting in high-quality effluents
(Mao et al., 2015). The reactors are more complex than either stand-
ard UASB or EGSB reactors but with a smaller footprint and the
 Floc and Granular-based Systems (Dispersed Growth) 267

Figure 7.5. The laboratory scale static granular bed reactor (SGBR) used by
Debrik and Coskun (2009). Reproduced with permission of Elsevier Publishing.

ability to treat much higher organic loadings of between 20 and

50 kg COD m–3d–1. They are very tall compared to other anaerobic
contact systems, being 2–3 times higher than normal UASBs and
cheaper to construct in terms of treatment capacity (Wang et al.,
2018). The influent enters the base of the reactor and is mixed with
the granular sludge via the distribution system. Biogas produced from
the organic matter in the lower chamber is collected by the first GLS
separator, resulting in a gas lift effect, which pulls the biomass and
partially treated wastewater upward via the riser tube into the gas-
liquid separator at the very top of the reactor (Fig. 7.6). Here, the
268 Anaerobic Wastewater Treatment

Figure 7.6. Paques BIOPAQ®IC internal circulation (IC) reactor comprising

two reactor chambers on top of each other, each with its own GLS separator:
1. Wastewater enters the reactor and mixes with granular biomass via the distribution
system, 2. Organic fraction converted to biogas, 3. Biogas collected in a lower separa-
tor, creating a gas lift, 4. Liquid rises upward in the riser with biogas, 5. Biogas leaves
the reactor in a liquid–gas separator, 6. Water returns to the distribution system via
the downer, 7. Effluent undergoes further treatment (polishing) in the upper reactor
chamber, 8. Biogas collected from the upper chamber by the upper phase separator,
9. Effluent leaves the reactor, 10. Biogas leaves the reactor. https://cdn.opptylab.
com/hg/assets/brochurebiopaqic.pdf. Reproduced with permission of Paques
Global, https://www.paquesglobal.com/.

biogas is removed and the liquid returned to the lower chamber via
the downer tube into the distribution system, causing effective mix-
ing and ensuring good expansion of the granules in the lower cham-
ber, hence the concept of internal circulation. The second chamber
acts as a polishing area for the effluent, with any biogas removed by
the second GLS separator, after which the effluent is discharged
(Fig. 7.6). IC is described as a third-generation anaerobic treatment
 Floc and Granular-based Systems (Dispersed Growth) 269

process, being a development from the UASB and EGSB processes.

It is slowly replacing the UASB as the first choice for the treatment
of strong and complex wastewaters, including leachate (Habets and
Driessen, 2007). It is marketed by Paques under the trade name
BIOPAQ® IC (https://www.paquesglobal.com/products/biopaq-ic),
with numerous successful plants in operation globally treating a wide
range of industrial wastewaters. A full-scale IC reactor was success-
fully employed to treat leachate from a municipal solid waste incinera-
tion plant. At an organic loading rate (OLR) of 21–25 kg COD
m–3d–1, high COD removals were recorded at between 89 and 93%
and biogas yields of 0.42–0.50 m3 kg–1 COD (Wang et al., 2018).
The zeta potential and hydrophobicity of the granular sludge were
correlated to the protein/polysaccharide ratio of the EPS, with the
protein content increasing over time, showing that protein was an
important part of EPS development and the process granulation
(Figs. 7.7 and 7.8).

Figure 7.7. Development of protein and polysaccharide content in EPS as granules

mature in an IC reactor (Wang et al., 2018). Reproduced with permission of Elsevier
Publishing.
270 Anaerobic Wastewater Treatment

(a)

(b)

Figure 7.8. Relationship between zeta potential, cell surface hydrophobicity, and
EPS during sludge granulation in an IC reactor treating leachate (Wang et al., 2018).
Reproduced with permission of Elsevier Publishing.
 Floc and Granular-based Systems (Dispersed Growth) 271

All anaerobic processes are susceptible to inhibition due to incon-

sistency in loading or organic overloading or the presence of toxic and
inhibitory compounds. It is important to ensure real-time monitoring
to ensure consistency of operation; this is especially so with IC sys-
tems (Jimenez et al., 2015; Irizar et al., 2018).

7.4 Anaerobic Continuously Stirred

Tank Reactors
The simplest of all contact systems is the anaerobic continuously
stirred tank reactor (AnCSTR), which is used for traditional sludge
digestion. The design ensures excellent mixing of the biomass and
the wastewater, ensuring complete breakdown and good biogas pro-
duction. It is especially well-adapted to treating wastes with high
FOG (fats, oil, and grease) content, as foaming is minimized by stir-
ring rather than by the expansion of the sludge blanket. Biomass
retention is low due the completely mixed nature of the liquid/bio-
mass, so a solids separation step ensures biomass concentration is
maintained, ensuring operational stability even under variable loading
conditions (Fig. 7.9). Internal baffles can also be installed to enhance
retention of the granular sludge or a membrane separator (Sec. 9.2.3).
Loading rates depend on the nature of the substrate but are normally
between 2 and 5 kg COD m–3d–1. The AnCSTR is often operated in
series with another bioreactor, such as an AnBR, and can be used for
all types of strong organic wastewaters and unthickened sludges, such
as waste-activated sludge and flotation sludges with high lipid con-
tents. For example, Lutze and Engelhart (2020) explored the use of
an AnCSTR to treat waste-activated sludge and lipid-rich flotation
sludge from milk processing, which achieved a maximum biogas yield
of 280 L kg–1 COD at a SRT of 20 days as well as good COD remov-
als, although biogas yields were affected by lipid concentration
requiring longer sludge ages as lipid content increased. Lipid contents
>0.8 kg CODlipid kg–1 COD required a minimum SRT of 15 days,
while those at <0.8 kg CODlipid kg–1 COD were effectively treated at
an SRT of 10 days. Among the commercially available AnCSTRs are
the ADI® CSTR from Evoqua and Veolia’s Biobulk-CSTR.
272 Anaerobic Wastewater Treatment

Figure 7.9. Schematic diagram of an anaerobic continuously stirred tank reactor

(AnCSTR).

7.5 Anaerobic Sequencing Batch Reactor

The original activated sludge process was developed as a single-unit
process batch reactor where aeration and settlement occurred within
the same tank (Gray, 2023). Today, a number of sequencing batch
reactors are used in parallel so while one tank is filling, another is in
the aeration stage and another is in the settlement/decanting phase,
thus allowing for continuous loading. Coordinating the phases to
ensure that influent wastewater can always enter a reactor requires
computer-aided technology.
In anaerobic sequencing batch reactors (AnSBRs), the aeration
phase is omitted and replaced with a reaction time during which
sludge granules adsorb and biodegrade the organic fraction of the
wastewater under anaerobic conditions. Mixing is usually achieved
by a paddle, although gas and/or liquid recycling can be used. While
the mixing stage is longer than the aeration phase seen in activated
sludge, settlement is much faster due to the granular nature of the
sludge. In practice, it is far more difficult to synchronize cycle time
between AnSBR reactors, permitting continuous loading, especially
where hydraulic or organic rates vary, so they are generally operated
as single batch reactors treating waste streams with consistent charac-
teristics and predictable flow patterns or which have been stored in an
equalization basin (Akil and Jayanthi, 2012). This makes them ideal
for the pharmaceutical industry, where manufacturers rely on small
 Floc and Granular-based Systems (Dispersed Growth) 273

batch processes based on predictable work-shift patterns and more

complex wastewaters, such as those from the dye and textile industries
and leachate, which generally require longer reaction times (Timur
and Özturk, 1999; Kumar et al., 2022). It has been suggested
that AnSBRs are not as efficient as UASBs at high loading rates,
although while COD removal is comparable, more biomass is pro-
duced (Angenent and Sung, 2001). The time for a suitable granular
sludge to develop depends on the seed used and the time required for
the acclimatization of the imported seed sludge to the wastewater
being treated, which can be as long as five months (Wirtz and Dague,
1996). The design is very simple and does not include a feed distribu-
tion or GLS separation system as in UASB, with no up-flow hydraulic
pattern (Fig. 7.10).
Co-digestion of wastewaters has many benefits, such as optimiz-
ing buffering capacity and C:N ratio, diluting inhibitory and toxic
compounds, improving COD removal and methane production,
increasing operational stability, and reducing costs. For example, the
treatability of tannery wastewaters was improved by mixing 50:
50 with dairy wastewater (Berhe and Leta, 2023). Tannery wastewa-
ter is difficult to treat anaerobically due to its high alkalinity and
low COD:TN ratio, leading to ammonification and an increase in
pH, both of which inhibit methanogenesis. In contrast, dairy waste-
water has a low alkalinity content, which quickly becomes acidified,

Figure 7.10. Four phases of an anaerobic sequencing batch reactor.

274 Anaerobic Wastewater Treatment

increasing volatile fatty acid (VFA) concentration that lowers the pH

but with a high COD:TN (total nitrogen) ratio.
Berhe and Leta (2023) also used two-stage AnSBRs to treat their
wastewaters in order to split acidogenesis and methanogenesis, with
the working volume split at a ratio of 2:3, respectively, which further
increased methane yields and also improved operational stability.
Separating phases in this way increases the plug flow nature of the
system, which is already a feature of the AnSBR that promotes a more
diverse microbial community structure, allowing optimum control of
each reactor for the key consortia of bacteria and archaea present,
such as preventing intermediate products such as VFAs or inhibitory
compounds to accumulate that may affect methanogenesis. A final
effluent COD removal rate of 85.6% was achieved at an HRT of
15 days, with a methane yield of 892 mL CH4 g–1 COD at a biogas
methane content of 72%. A similar problem was identified by Deng
et al. (2024) in treating high-strength slaughterhouse wastewater,
which is rich in protein and FOG, which accumulate in the reactor,
causing foaming or sludge flotation and other problems. The typical
strength of the wastewater is COD (4,700­–6,500 mg L–1), proteins
(500–1,100 mg L–1), FOG (100–600 mg L–1), TSS (1,100–3,700 mg
L–1), and NH4+-N (175–420 mg L–1). Proteins are hydrolyzed to
peptides and amino acids, then deaminated to ammonium and VFAs
(i.e., ammonification), while FOG is hydrolyzed into glycerol and
long-chain fatty acids (LCFAs). Interestingly, the degradation of
LCFAs involves a syntrophic relationship between acetogenic
β-oxidizing bacteria and acetate and hydrogen-utilizing methano-
genic archaea. Lipid hydrolysis occurs far more quickly than LCFA
degradation and so can result in elevated concentrations of LCFAs,
which can inhibit both hydrogenotrophic and acetolactic metha­
nogens as well as the syntrophic β-oxidizing bacteria (Sousa et al.,
2009). So, where protein-rich wastewaters are treated, high ammo-
nium concentrations can occur, with ammonium inhibiting methano-
gens at concentrations >1,500 mg L–1, with VFAs also increasing in
concentration, causing acidification. Similar problems are seen when
treating wastewaters rich in FOG, which has high methane-producing
potential. In practice, carbohydrates are degraded much more quickly
 Floc and Granular-based Systems (Dispersed Growth) 275

than either proteins or FOG, so where they are constantly being

added to a completely mixed reactor, protein degradation is reduced,
leading to protein accumulation similarly with FOG. To a large
extent, this is overcome by the feast-famine regime associated with
batch reactors, such as AnSBRs, which ensures all are fully degraded
each cycle. The AnSBR system used by Deng et al. (2024) employed
a buffer tank before the AnSBR, which increased the rate of ammoni-
fication, achieved a 90 and 80% removal of COD and protein, respec-
tively, and a COD to methane conversion rate of >70% at an optimum
OLR of 6.2 kg COD m–3 d–1. While all operating parameters play
a part in determining the microbial community structure within
the reactor, the feeding frequency and OLR were identified by Zeils
et al. (2017) as the most important. Fly ash has been used as an
enhancer in AnSBRs, treating winery wastewater, and was found to
increase biogas yield by 12% and COD removal by 42% (Lauzurique
et al., 2023).
The problem of maintaining a granular sludge is overcome
by using carriers in AnSBRs, which also reduces or eliminates the
time required for settlement, reducing overall cycle time, and reduces
solids loss as well as increasing both COD removal and biogas
generation per unit reactor volume. Anaerobic sequencing batch bio-
film reactors (AnSBBRs) were compared to a standard AnSBR in
laboratory tests, using synthetic wastewater as feed and granular acti-
vated carbon (GAC) and zeolites as carriers (Fig. 7.11). Operating a
24-hour cycle of 12 minutes of feeding, 23 hours of anaerobic bio-
degradation, 30 minutes of settling, and 15 minutes of decanting at
35°C, gas production was greater in the AnSBBRs, with an increase
over the AnSBR of 7% with GAC only as a carrier, 4% using only
zeolite as a carrier, and 21% using combined GAC and Zeolite carri-
ers; while mean COD removals were 89.5, 92.3, 96.5, and 96.5%,
respectively (Dutta et al., 2014). They have been used successfully to
treat a wide range of wastewaters. Sarti et al. (2007) used a pilot-scale
AnSBBR to treat domestic wastewater with polyurethane foam cubes
with 50 mm sides as a carrier in an 8-hour cycle (feeding (2 hours),
reaction (5 hours), discharge (0.5 hours), and idle (0.5 hours)),
although the mean COD removal was only 66%. No settlement
276 Anaerobic Wastewater Treatment

(a) (b)

Figure 7.11. Scanning electron micrograph of granular activated carbon (GAC)

and zeolite carriers used in an anaerobic sequencing batch reactor treating synthetic
municipal wastewater: (a) GAC outer surface (top) and inside (bottom) and (b) zeo-
lite outer surface (top) and inside (bottom) (Dutta et al., 2014). Reproduced with
permission of Elsevier Publishing.

period was used during the cycle, with the carriers held within a per-
forated wall against the reactor wall (Fig. 7.12). An unusual example
is a full-scale pilot AnSBBR used to successfully treat sulfate-rich
wastewaters by a consortium of acidogenic bacteria, acetoclastic
methanogenic archaea, and sulfate reducing bacteria, dominating the
biofilm that developed on a bed of mineral coal (40–80 mm diame-
ter), which acted as a medium (Sarti et al., 2011). The wastewater was
batch-fed into the AnSBBR from a storage tank and then continu-
ously recycled through the bed during the entire reaction period.
Using a cycle time of 48 hours (feeding (1 hour), reaction (46 hours),
and discharge (1 hour)), sulfate concentrations of 0.58 to 5.2 g SO42–
L–1 mixed with domestic wastewater achieved a biological sulfate
removal rate efficiency of 99%.
 Floc and Granular-based Systems (Dispersed Growth) 277

(a) (b)

Figure 7.12. Pilot-scale AnSBBR used to treat domestic wastewater: (a) image and
(b) schematic diagram, with all dimensions in mm. The carrier (polyurethane foam
cubes) is held in an outer chamber, with the wastewater able to pass freely from the
central mixed zone to the biofilm on the carriers via the perforated wall (Sarti et al.,
2007). Reproduced with permission of Elsevier Publishing.

Both AnSBR and AnSBBRs have been successfully employed in

producing methane and biohydrogen (Sec. 9.1.1).

7.6 Baffled Reactors

The concept of incorporating baffles in anaerobic reactors first
occurred early in the 1980s when septic tanks were modified to
increase COD removal. Bachman et al. (1982; 1985) created the first
anaerobic baffled reactor AnBR as a dedicated secondary treatment
step rather than a combined settlement/treatment process, such as a
septic or Imhoff tank, utilizing anaerobic granulated sludge. The rec-
tangular reactor incorporates a number of vertical baffles, creating
chambers where the wastewater passes from one chamber to another
under one baffle and over the next, ensuring maximum HRT and
biomass contact with the wastewater and minimizing the loss of
278 Anaerobic Wastewater Treatment

Figure 7.13. Schematic diagram of a five-chambered anaerobic baffled reactor

(AnBR).

granules (Fig. 7.13). This is often referred to as a push-flow pattern

(Reynaud and Buckley, 2016). There is no GSL separation system as
in EGSBs, making them far simpler to operate (Uyanik et al., 2002).
Granulation occurs relatively quickly in AnBRs, with a diverse range
of bacteria and archaea developing. The plug-flow nature of the reac-
tor caused by multiple chambers ensures that there is a substrate
concentration gradient through the system so that biogas production
and sludge activity tend to fall off toward the end of the reactor. The
series of chambers allows a large degree of separation of acidogenic
and methanogenic activity (Zhu et al., 2015). AnBRs are widely used
for domestic wastewaters in rural China (Cui and Liang, 2019; Zha
et al., 2019), achieving a 62–71% COD removal at loadings ranging
from 0.06 to 0.61 kg COD d–1 (Da Silva et al., 2017). AnBRs have
good biosolids retention, are able to withstand shock and intermittent
loading, and have low energy consumption, being easy to operate
with low maintenance (Soh et al., 2020).
AnBRs utilizing granular sludge have been successfully used to
treat problematic waste streams. For example, Zang et al. (2024)
were able to successfully treat strong wastewater (COD 4,000 mg L–1)
containing 12.47 mg L–1 of allicin, a sulfur-containing compound
derived from garlic processing, which is highly bactericidal. This was
achieved by using a step feed system with 40% loading to chamber 1,
30% to chamber 2, 20% to chamber 3, and 10% to chamber 4, and an
effluent recycling rate of 200% to ensure adequate sludge expansion
 Floc and Granular-based Systems (Dispersed Growth) 279

within the chambers as well as diluting the wastewater, thereby reduc-

ing toxicity. This achieved an 89 and 90% reduction in the COD and
allicin, respectively. The loading and recycling regime controlled the
pH to within 6.5–8.0, which was optimal for methanogens.
Packed anaerobic baffled reactors (PABRs) include fixed media in
the up-flow channels to increase biomass concentration, increasing
COD removals to 90% (Renuka et al., 2016; Feng et al., 2018)
(Fig. 7.14). Zheng et al. (2023) tested a laboratory-scale PABR to
compare performance under summer (32 ± 1°C) and winter tempera-
tures (16 ± 1°C), using different HRTs of 36, 48, 60, 72, and 90
hours. The study showed the greater the HRT, the better the removal
of COD, reaching 85 and 52% during summer and winter conditions,
respectively (Fig. 7.15), recommending a minimum HRT of 72
hours. As with AnBRs, the COD removal rates fell in the different
chambers as the wastewater passed through. The authors observed
variations in the bacterial and archaeal flora between chambers. In
the four-chambered system, the abundance of hydrogen trophic
methanogens decreased progressively with flow direction, while the

Figure 7.14. Schematic diagram of the laboratory-scale packed anaerobic baffled

reactors (PABR) used by Zheng et al. (2023). Reproduced with permission of
Elsevier Publishing.
280 Anaerobic Wastewater Treatment

100 10 20

NH4+–N removal rate (%)

31°C-33°C 31°C-33°C 18 31°C-33°C
COD removal rate (%)

TN removal rate (%)

80 15°C-17°C 5 15°C-17°C 16 15°C-17°C
14
60 0 12
10
40 8
–5 6
20 4
–10 2
0 0
96 72 60 48 36 96 72 60 48 36 96 72 60 48 36
HRT (h) HRT (h) HRT (h)
(a) (b) (c)

Figure 7.15. Effect of temperature and HRT on the removal of: (a) COD,
(b) ammonia (NH4+-N), and (c) total nitrogen (TN) in experimental PABRs (Zheng
et al., 2023). Reproduced with permission of Elsevier Publishing.

abundance of acetic acid trophic methanogens progressively increased

with flow direction (Fig, 7.16). Overall, the most abundant phyla in
the PABR were Chloroflexi (53.2%), Bacteroidota (9.8%), Proteobacteria
(9.3%), Actinobaciota (6.8%), Firmicutes (6.3%), Pattescibateria
(3.7%), Acidobacteriota (1.5%), and Caldisericota (1.3%). Similar
microbial results were reported by Zha et al. (2019), who used a
PABR to treat black water (i.e., water only from toilets) and found the
optimum HRT after testing over a range of 24–72 hours to be 48
hours, with mean reductions of 94% COD, 2.9% TN, 14% NH4+-N,
and 33% TP (total phosphorus) over the sampling period of 112 days.
A wide range of carriers are used either suspended as bunches, on
rods, or as strings (Fig. 7.17). The optimum number of chambers
should be kept to between 4 and 6 per unit (Xu et al., 2016), with
a width ratio of 1:3 for the downward and upward flow sections of
each chamber, with the bottom section of the baffle inclined at 45°.
Renuka et al. (2016) only incorporated carriers in the final two cham-
bers of their PABR, operating the first three chambers as an AnBR.
The potential of AnBRs for rural wastewater treatment and biogas
production offers huge opportunities for sustainable sanitation and
energy production at the local level, with research also showing the
potential of AnBRs for hydrogen production from specific wastewa-
ters rich in starch, such as potatoes and tapioca (Sec. 9.1.1).
 Floc and Granular-based Systems (Dispersed Growth) 281

Figure 7.16. Structure of the archaeal community in the four different compart-
ments in an experimental PABR (Fig. 7.14). Biomass samples were taken from the
top middle and base of each compartment and mixed to give a single sample. Three
replicates (1–3) were taken from each compartment (A–D) (Zheng et al., 2023).
Reproduced with permission of Elsevier Publishing.

Figure 7.17. A wide range of carriers are used in PABRs, including the standard
anaerobic biofilter carrier. However, local natural materials are often employed, such
as small spheres woven from bamboo fibers used by Feng et al. (2018). Reproduced
with permission of Elsevier Publishing.
282 Anaerobic Wastewater Treatment

7.7 Anaerobic Migrating Blanket Reactor

The anaerobic migrating blanket reactor was patented in 1999 and
is similar to AnBRs, as it comprises a number of compartments
inducing an up-flow down-flow pattern through the tank (Angenent
and Dague, 1995; Angenent and Sung, 2001) (Fig. 7.18). Waste is
continuously fed to the reactor, with biodegradation and biogas
production by granular sludge. Movement is created by the influent
wastewater, although mechanical mixers are used intermittently, as
required when the flow rate is not sufficient to keep granules
expanded within the compartments. While there is no GLS separa-
tor, feed distribution system, or effluent recycling, the need for
expensive stirrers in each compartment of the reactor makes them
more expensive than other designs. Like the baffled reactor, the
compartmentalized design with the wastewater entering one end
and leaving at the far end means that a substrate gradient is formed
so that there is very little COD removal or biogas production in
the final compartment, which is often used as a settlement zone pre-
venting biomass loss. Inevitably, granules accumulate in the final
compartment. This is overcome by operating the reactor with the
hydraulic flow reversed, with the first compartment now operating
as the settlement zone. Operationally, this is quite difficult in order
to maintain constant removal efficiency, so the feed may be split
between the first and penultimate compartment just before reversing
the flow in order to condition the granules in the final compartment
for higher organic loading. Anaerobic migrating bed reactors require
a minimum of three compartments, preferably four or five. Compared

Figure 7.18. Schematic diagram of an anaerobic migrating blanket reactor

(Angenent and Sung, 2001). Reproduced with permission of Elsevier Publishing.
 Floc and Granular-based Systems (Dispersed Growth) 283

to UASBs, the granules that form are smaller and denser, helping to
retain them within the tank. Like AnBRs, building an anaerobic
migrating bed reactor on site is complex due to the baffles, so pre-
fabricated units are easier to construct and install. Some authors use
the abbreviation AnMBR for anaerobic migrating bed reactors; how-
ever, this is far more widely used for anaerobic membrane reactors
(Sec. 9.2.3).

7.8 Hybrid Reactors

A major drawback of UASB reactors is the potential to lose biomass,
which has resulted in many innovative GLS separator designs and
configurations. The anaerobic hybrid reactor (AnHR) overcomes this
to a certain extent by incorporating an anaerobic biofilter within the
UASB, creating a hybrid sludge blanket-biofilm reactor (Fig. 7.19).
Confusingly, AnHRs are also widely known as hybrid up-flow anaero-
bic sludge blanket (HUASB) reactors. The AnHB is constructed with
the top 20–30% of the reactor volume packed with a carrier, either a
modular medium or a standard random biofilter carrier, such as polyu-
rethane foam or plastic units (Figs. 6.10 and 6.12). The medium or
carriers provide surface area for biomass development as well as trap-
ping suspended biomass displaced from the lower section. AnHRs are
well suited for treating complex industrial wastewaters, especially those
that may contain more recalcitrant compounds as well as more readily
biodegradable materials. Good COD removals have been reported
due to the biofilter section acting as a polishing step as well as adding
significantly to the total biomass within the reactor, with excellent
biogas yields containing up to 87% methane achievable. A wide range
of wastewaters can be successfully treated in AnHRs, with good biogas
yields including chemical, petrochemical, pharmaceutical (Mullai
et al., 2024), antibiotic production (Mullai et al., 2018), biopesticides
(e.g., azadirachtin) (Mullai et al., 2022), pulp paper processing
(Hemalatha and Keerthinarayanna, 2017), food processing, brewery,
distillery, leather, and heavy metals (Murugesan and Selvarasu, 2017).
Comparing an AnHR with a UASB treating phenolic wastewater,
Ramakrishnan and Surampalli (2012) observed that the hybrid
284 Anaerobic Wastewater Treatment

Figure 7.19. Schematic design of an experimental anaerobic hybrid reactor used to

treat pharmaceutical wastewater (Oktem et al., 2007). Reproduced with permission
of Elsevier Publishing.
 Floc and Granular-based Systems (Dispersed Growth) 285

reactor was able to achieve effective granulation within 45 days com-

pared to 58 days in the UASB and was able to withstand far greater
shock loadings. The AnBR achieved a slightly higher percentage of
COD removal at 88% (85% UASB) and phenolics removal at 93%
(91% UASB) but similar biogas production rates of 0.33 m–3 kg–1
COD removed with 67 and 60% methane content in the AnHR and
UASB, respectively. Although AnHRs operate well under normal
temperatures (12–20°C), many studies on industrial wastewaters have
recorded optimal performance in the mesophilic range (30–35°C),
especially for biogas production, and so where wastewaters are not
already at the correct temperature, heating of the lower chamber may
be advantageous.
Treating fermentation-based antibiotic wastewater (COD 15,000
to 25,000 mg L–1 and BOD 5,000 to 9,020 mg L–1) in a laboratory-
scale AnHR, Mullai et al. (2018) recorded optimum biogas produc-
tion at an organic loading rate of 16 kg COD m–3 d–1, with COD
removal of 83% at an HRT of 6 hours. The authors successfully used
the Stover–Kincannon and Van der Meer and Heertjes models to
predict biogas production in full-scale systems. A loss of granulation
was noted in an experimental AnHR treating olive oil processing
wastewater due to phenolic compounds present, although a reduced
performance was maintained by the biomass carriers in the upper sec-
tion of the reactor, with the hydrogenotroph Methanobacterium bei-
jingense the dominant methanogen (Zakoura et al., 2022). In
contrast, a UASB using flocculent biomass performed well using the
same wastewater.
The major phyla in the AnHR were Bacteroidetes (8.4–57.9%),
Firmicutes (0.7–34.9%), Chloroflexi (0.5–40.5%), Proteobacteria (2.3–
13.0%), Planctomycetes (0.6–30.2%), Actinobacteria (0.3–6.3%), and
Synergistetes (0.2–14.8%), with Prevotella and Longilinea, the domi-
nant allicin resistant genera. Using a laboratory scale AnHR, Oktem
et al. (2007) describe the acclimatization procedure to treat pharma-
ceutical wastewater (6,000 mg L–1). Optimum treatment and gas
production was achieved at an OLR of 8 kg COD m–3 d–1, at an HRT
of 2 days, and a reactor up-flow velocity of <12 m d–1 producing 72%
COD removal and 200 L CH4 kg–1 VS d–1. They observed that COD
286 Anaerobic Wastewater Treatment

removal fell with an increased percentage of pharmaceutical wastewa-

ter added, with 99, 96, 91, and 85% COD removals at 10, 30, 70,
and 100% pharmaceutical wastewater treated at an OLR of 3 kg COD
m–3d–1 and a reactor up-flow velocity of 4.8 m d–1.

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 Section D

Biogas Production Systems

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 8
Biogas Production and Cleaning

8.1 Methane Production and Collection

There are three products of anaerobic digestion: the digested sludge,
waste liquor, and a mixture of gases. Digested sludge is different from
either primary or secondary sludge in a number of ways. It is patho-
gen-free, stabilized, and far less offensive, having a tarry smell and
drying to an inert friable condition, thus making it ideal for disposal
to agricultural land. The waste liquor from anaerobic digesters has a
high suspended solids (500–1,000 mg L–1) and biochemical oxygen
demand (BOD) concentration (400–800 mg L–1) due to all the solu-
ble organics present. However, the BOD of the liquor can be very
high indeed, reaching up to 10,000 mg L–1, although up to 60% of
the BOD is due to the suspended solids fraction. Because of the deg-
radation of organic nitrogen, the liquor may have high concentrations
of soluble nitrogen present. The characteristics and strength of the
liquor make it difficult to dispose of or treat separately, and it is
returned to the works inlet, where it is diluted by the incoming sew-
age and treated in admixture aerobically.
Sludge or digester gas is more commonly referred to as biogas. Gas
production is the most direct and sensitive measure of the rate of
anaerobic digestion, a decrease in production being the first indication
that the process is unstable. Apart from trace amounts of water vapor,
hydrogen sulfide, hydrogen, nitrogen, unsaturated hydrocarbons, and
other gases, biogas is predominantly a mixture of carbon dioxide and
methane, the exact proportions of which determine its calorific value.

297
298 Anaerobic Wastewater Treatment

A typical biogas contains between 65% and 70% methane by volume,

with the remaining being largely carbon dioxide. Although hydrogen
is an important precursor of methane formation in digestion, the con-
centration of hydrogen, which can be measured within the reactor, is
usually very low, which suggests an immediate uptake by the metha-
nogens. When burnt, biogas produces water, which complicates the
determination of its calorific value. The net calorific value for combus-
tion is where the water formed remains in the vapor phase, whereas the
gross calorific value is where the water formed is condensed. The calo-
rific values are calculated using the percentage of methane (M) present
as the net calorific value = (334 × M) kJ m–3, or the gross calorific value
= (370 × M) kJ m–3, both at 15.5°C and 1 atmosphere pressure. For
normal biogas, the gross calorific value is between 24,000 and 26,000
kJ m–3, with the net value being some 10% less.
The production of biogas can be related to the amount and type
of organic matter utilized. For example, the removal of 1 kg of chemi-
cal oxygen demand (COD) yields 0.35 m3 of biogas at standard tem-
perature and pressure (STP), whereas 1 kg of organic carbon would
yield 1.87 m3 at STP. The exact gas yield per kg of volatile solids
removed depends on the composition of the waste but is approxi-
mately 1.0 m3. Some food-processing industries produce a pure sub-
strate waste with 80–95% of the organic (volatile) matter removed,
whereas only 40–50% of the organic matter in sewage sludge, and
even slightly less in animal slurries, will be utilized with a proportion-
ately lower gas yield in terms of m3 of gas produced per kg of sub-
strate supplied. Therefore, for a mixed primary and secondary sludge
from a purely domestic sewage treatment works, the gas production
will be of the order of 0.5 m3 kg–1 organic (volatile) matter, or about
0.375 m3 kg–1 of total (dry) solids, added to the digester. The compo-
sition and quantity of gas produced by complete digestion can be
theoretically determined using the equation:

CcHhOoNnSs + 1/4(4c – h – 2o + 3n + 2s)H2O

= 1/8(4c – h + 2o + 3n + 2s)CO2 + 1/8(4c + h – 2o – 3n – 2s)CH4
+ nNH3 + sH2S(8.1)

where c, h, o, n, and s are the number of atoms of carbon, hydrogen,

oxygen, nitrogen, and sulfur, respectively. This equation replaces the
 Biogas Production and Cleaning 299

earlier equation developed by Buswell and Mueller (1952), which

ignored the nitrogen and sulfur components of the waste that are
largely utilized within the digester.
Thus, by neglecting the very small volume of other gases formed
and using the simplified formula, carbohydrates will produce 73%
CO2 and 27% CH4, lipids 52% CO2 and 48% CH4, and protein 73%
CO2 and 27% CH4. The total volume of biogas produced from each
of these basic substrates is 0.75, 1.44, and 0.98 m3 kg–1 dry matter,
respectively (Buswell and Mueller, 1952). Actual values of gas yield
and composition from the basic substrates have been given by
Konstandt (1976). Lipids are only slowly degraded anaerobically, and
the measurement of their removal provides a very useful quality con-
trol parameter for the process.
A more accurate estimation of methane production per unit time
can be obtained by using the equation:

G = 0.35 (L – l.42 St)(8.2)

where G is the volume of methane produced (m3d–1), L is the mass of

ultimate BOD removed (kg d–l), and St is the mass of volatile solids
accumulated (kg d–1). St can be estimated as:

St = aL/1 + bts(8.3)

where a is the mass of volatile solids synthesized per kg of ultimate

BOD removed, b is the endogenous respiration constant, and ts is
the solids retention time (Speece and McCarty, 1964). Methane is
the most valuable byproduct of anaerobic wastewater treatment, and
it is useful to express the yield as m3 CH4 kg–1 organic matter removed,
or m3 CH4 kg–1 COD removed. Typical methane yields from meso-
philic digesters operated at 35oC are 0.86 m3 kg–1 organic matter
removed for mixed sewage sludge, 0.55 m3 kg–1 for dairy manure, and
0.42 m3 kg–1 for pig manure. Under continuous reactor operation, a
mesophilic digester will produce between 12 and 16 L of methane per
capita per day for primary sludge, rising to about 20 L ca–1 d–1 for
combined primary and secondary sludges.
The simplest use of biogas is to use it on-site either to produce
heat directly for the anaerobic reactors, which normally operate in the
300 Anaerobic Wastewater Treatment

mesophilic range using gas boilers, or to generate electricity using

modified diesel (dual fuel) engines to drive alternators, with the cool-
ing water from these engines used to heat the reactors, a system
known as combined heat and power (CHP) generation. Some plants
now use biogas for vehicle fuel, either in the form of biomethane or
LPG, while larger plants and those treating organic wastewaters, espe-
cially food and agricultural wastes, can generate enough biogas to
make it viable for it to be cleaned to the high standard required for
supply to the natural gas grid for domestic and industrial use.
Biomethane is different from biogas methane, as it requires further
processing, as discussed below. Hydrogen can also be produced via
digestion and can be used as a vehicle fuel or for household heating
(Sec. 9.1.1). Smaller digestion units are used widely in the Third
World for cooking and lighting.
With the increase in the size of anaerobic digesters and the cen-
tralization of sludge treatment, large quantities of biogas can be gen-
erated enough for sale, either to national gas networks for home and
industrial use or for the large-scale production of biomethane for
vehicle fuel. However, the drawback is that the biogas contains a wide
range of contaminants that need to be removed, as well as increasing
the methane content to match the calorific value of natural gas. The
precise allowable concentrations of contaminants allowable in biom-
ethane for injection into the gas network vary slightly from country
to country. Gas Networks Ireland, which is the key distributor of
natural gas in Ireland, has produced a technical specification data
sheet for biomethane suppliers who wish to sell their gas to the net-
work (Table 8.1). There are a number of European Standards for
biomethane. While standard EN16726-1 provides gas quality stand-
ards for natural gas, EN16723-1 deals specifically with biomethane to
be injected into natural gas networks, and EN16726-2 deals with its
use as vehicle fuel (BS EN, 2016; 2017).
Gas supplied to the grid must have an odor in order to identify
leaks. While this is normally added by the supplier, even after the most
effective desulfurization, trace amounts of hydrogen sulfide will
remain, and with an odor threshold of 0.0047 ppm, odor detection is
unlikely to be a problem.
 Biogas Production and Cleaning 301

Table 8.1. Technical specifications for maximum allowable contaminant

concentrations for biomethane supplied to Gas Networks Ireland.
Parameter Units Limit value
Hydrogen sulfide content mg m–3 ≤5
Total S content including H2S mg m –3
≤50
Hydrogen content % mol mol–1 <0.1
Oxygen content % mol mol–1 ≤1.0
Hydrocarbon dewpoint o
C ≤ –2
Water (moisture) content mg m –3
≤50
–3
Wobbe index (high limit) Mj m 51.41
–3
Wobbe index (low limit) Mj m 47.2
Incomplete combustion factor — <0.48
Sooting index — <0.6
–3
Gross calorific value (CV high limit) Mj m 42.3
–3
Gross calorific value (CV low limit) Mj m 36.9
–1
Carbon dioxide content % mol mol <2.5
–3
Organo-halides mg m <1.5
–1
Radioactivity Becquerels g <5
–1
Ethane % mol mol <12
Nitrogen % mol mol –1
≤5
Ammonia content mg m–3 10
–3
Siloxanes content mg m 0.3
–3
Amines mg m 10
–1
Carbon monoxide % mol mol 0.1

8.2 Hydrogen Sulfide Control and Removal

Any sulfate present in the waste stream will be readily converted to
sulfide by sulfur reducing bacteria (SRB) during anaerobic treatment
(Fig. 8.1). Sulfate is common in many wastewaters from concentrations
between 20 and 200 mg L–1 in domestic and up to 35,000 mg L–1 in
industrial wastewaters. The sulfide can be present in anaerobic reactors
in a number of forms, either in solution (S2–, HS–, and H2S) or gaseous
(H2S) form, which contaminates the biogas (Jung et al., 2022). The
302 Anaerobic Wastewater Treatment

(a) (b)

Figure 8.1. Sulfide is formed during anaerobic biodegradation by sulfate reducing

bacteria through the dissimilatory sulfate-reduction pathway. (a) The stages at which
sulfate is formed, and (b) the key enzymes and bacteria involved in sulfide formation
(Jung et al., 2020). Reproduced with permission of Elsevier Publishing.

form in which the sulfide is found is dependent largely on the pH and

temperature but is toxic to methanogens at concentrations between
100 and 800 mg total dissolved sulfide L–1 (Krayzelova et al., 2015;
Wang et al., 2020). Hydrogen sulfide is a hazardous gas with a wide
range of environmental and health effects as well as corrosion problems,
which are explored in detail in Secs. 3.3.2 and 3.3.3, respectively.
Biogas contains, on average, 0.2% H2S (typical range 0.08–5.5%)
and there are a number of different approaches for directly removing
it from the biogas, such as metal precipitation, chemical oxidation,
micro-aeration, and most commonly, adsorption and absorption
(Table 8.2), although preventing hydrogen sulfide production during
anaerobic treatment (i.e., in-situ technologies) should be part of
good operational practice (Skerman et al., 2012; Jung et al., 2022).
Comparative cost analysis of the different desulfurization methods is
given by Allegue and Hinge (2014).
The control of hydrogen sulfide is critical for stable anaerobic
operation and is increasingly important, as biogas is used off-site for
energy generation and other commercial uses. Post-treatment is rela-
tively expensive, especially when sulfur-rich substrates are used as feed
to digesters. There is also real potential in recovering not only ele-
mental sulfur (S0) but also metals as sulfide compounds (Fu and
Wang, 2011). In theory, this is most economically done by
 Biogas Production and Cleaning 303

Table 8.2. List of current desulfurization methods for biogas (Kapłan et al., 2021).
Reproduced under the Creative Commons Attribution (CC BY) license.
Range of biogas
Process or desulfurization
Method technique Application efficiency
Physical Absorption (wet) – scrubbing water — differences >94%
in solubility of CO2 and CH4
in polar solvents are used;
– absorbent:
(a) methanol (Rctisol),
(b) poly(ethylene glycol)
dimethyl ether (Selexol,
Genosorb),
(c) propylene carbonate
(Fluor),
(d) sulfolane (Sulfinol).
Absorption (dry) – adsorbent: up to 95%
(a) carbon (activated
carbon),
(b) mineral,
(c) mineral and carbon.
Absorption (wet) – dissolution of H2S in water >96%
or organic solvents:
(a) Genosorb,
(b) Rectisol,
(c) Ifpex.
Cryogenic – very low temperatures down up to 99%
separation to –100°C and high pressure
of about 40 bar.
Chemical Absorption – iron compounds: up to 90%
(a) bog iron ore (porous
sedimentary rock).
Membrane (porous – chemical potential of up to 99%
or diffusive) solutions or mixtures.
Absorption (wet) – caustic soda solution; (95 ÷ 99)%
– aqueous alkanolamine
solutions;
– ferrous chloride solution.
Biological Oxidation – supplying air to biogas. over 95%
Microbiological – bacteria of the family (80 ÷ 99)%
reaction Thiobacillus producing
elemental sulfur.
304 Anaerobic Wastewater Treatment

controlling sulfur loadings to digesters, by mixing different waste

streams, or by in-situ treatment (Table 8.3), with many new tech-
nologies, such as electrochemical oxidation, iron cycling, and sulfide
oxidation coupled with denitrification and stimulation of electric-
syntropy in development (Table 8.4). Post-treatment of sulfides is
generally more expensive although relatively simple and effective
(Jung et al., 2022). All anaerobic treatment methods seem amenable
to at least one or more in-situ sulfide control technologies.

Table 8.3. The advantages and challenges of using conventional in-situ desul­
furization methods (Jung et al., 2022). Reproduced with permission of Elsevier
Publishing.
Technology Advantages Challenges
Metal sulfide High sulfide removal Need for continuous supply of
precipitation efficiency chemicals
Simple operation Loss of digester working volume
by precipitates
Low toxicity Increase in salinity
Chemical High sulfide removal Need for continuous supply of
oxidation efficiency chemicals
Low energy consumption Toxicity of chemical oxidants to
methanogens
Simple operation Non-selective oxidation of
organic matter
Microaeration High sulfide removal Disturbance of anaerobic
efficiency metabolism
Enhanced substrate Biogas dilution (i.e., reduced
hydrolysis energy content)
Applicable with high sulfide Energy consumption for aeration
loads
Recovery of elemental sulfur Risk of explosion
Adsorption High sulfide removal Limited adsorption capacity
efficiency
Availability of low-cost Non-selective adsorption of
adsorbents organics or ions
Process simplicity Deactivation of adsorbents by
biofilm formation
Need for periodic replacement or
regeneration of adsorbents
 Biogas Production and Cleaning 305

Table 8.3. (Continued)

Technology Advantages Challenges

Suppression of Reduced formation of toxic Transient suppression of sulfate-
sulfidozenesis hydrogen sulfide Relief reducing activity Potential
of competitive inhibition inhibition of beneficial
of methanogens microorganisms
Reduced burden on biogas Additional competitive reactions
upgrading process to methanogenesis Possible
formation of undesirable
compounds

Using different substrates, Peu et al. (2012) were able to develop a

methodology to determine the hydrogen sulfide content of biogas dur-
ing anaerobic digestion. A wide range of sulfur contents were recorded,
ranging from 1 to 29.6 mg S kg–1 TS (total solids) (Table 8.5), and
using the biochemical methanogenic potential, biochemical biogas
potential, and the sulfur content of the substrates, tested a model that
was developed to predict the hydrogen sulfide content of the raw
biogas. From this, a clear relationship between the C:S ratio and the
concentration of hydrogen sulfide in biogas was established. Two
methods were used to calculate the predicted H2S–S biogas (PSB)
content (%): PSBmax and PSBbio (Fig. 8.2), with the former overes-
timating the actual hydrogen sulfide concentration in biogas while the
latter closer to the actual measured values. However, it is recom-
mended that using PSBmax is a better safety indicator for digester
operation and biogas management and is simpler to calculate. The
model showed that hydrogen sulfide in biogas could be kept to <2%
(v/v) if the C:S ratio was maintained at 40 or above.

8.2.1 Adsorption
Desulfurization of biogas is generally achieved by adsorption onto hydrated
ferric oxide, although a wide range of other adsorbents is available.
Suitable adsorbents have a high specific surface area and can operate
at low temperatures, such as activated carbon, which is highly effec-
tive and widely used but has high operation costs. Currently, research
is being carried out into low-cost and natural alternatives, including
wood, coal, peat, and lignite or industrial byproducts such as fly
306 Anaerobic Wastewater Treatment

Table 8.4. The advantages and challenges of using advanced in-situ desulfurization
methods (Jung et al., 2022). Reproduced with permission of Elsevier Publishing.
Technology Advantages Challenges
Electrochemical Chemical-free and Passivation of electrode
oxidation cost-effective surfaces by S°
Faster than chemical or Regeneration of sulfide from
biological oxidation S° by electrode biofilms
High energy efficiency Need to develop sustainable
and cost-effective
electrode materials
Iron cycling Possibility of elemental sulfur Lack of study in organic-rich,
recovery active methanogenic
Easy regeneration and reuse of environments
iron granules Little understanding of how
Increased removal capacity of iron granules influence
regenerated granules microbial redox reactions
Additional sulfide removal by and sulfur metabolism
FeS precipitation
Suppression of sulfate-
reducing activity
Sulfide oxidation Simultaneous removal of Need to remove S° from
coupled with sulfide, nitrogen, and digesters continually to
denitrification carbon prevent sulfide
Mitigation of inhibitory effects regeneration
of sulfide and nitrate High sensitivity to
Minimal material requirements environmental variations
Simple operation Low biomass yield
Stimulation of Possibility of recovering S° Very limited information
electric Possible enhancement of Little understanding of the
syntrophy methanogenesis underlying mechanism
Simple operation Unpredictable complexity of
electro-syntrophic
associations in
methanogenic systems

ash, slag, bauxite sludge, and sludge ash (Żarczyński et al., 2014).
However, ferric oxide and activated carbon remain the commonest
desulfurization absorbents.
Ferric oxide is widely available as a commercially prepared media
using Fe2O3.H2O. The media reacts with the biogas, which is usually
moist, to remove the hydrogen sulfide as ferric sulfide:
 Biogas Production and Cleaning 307

Figure 8.2. Predicted H2S–S biogas (PSB) content (%) over a range of different
C:S ratios (g/g) using two different methods, PSBmax (solid line) and PBSbio (dot-
ted line) (see Table 8.5), based on a wide range of different substrates tested by Peu
et al. (2012). Reproduced with permission of Elsevier Publishing.

Fe2O3.H2O + 3H2S → Fe2S3 + 3H2O(8.4)

Any light mercaptans (methyl and ethyl) present are also removed:

Fe2O3.H2O + 6RSH → Fe2(RS)3 + 3H2O(8.5)

The optimum temperature for desulfurization using ferric oxides

is 25–50oC at pH 8.0–8.5, and as the reaction requires water, the
biogas should not be too dry. Stoichiometrically, 1 kg Fe2O3.H2O
absorbs 0.64 kg H2S, although, in practice, the process is about 90%
efficient. Biogas hydrogen sulfide concentrations of <1 ppm are
achievable, with 99.98% removal possible even at raw hydrogen sulfide
gas concentrations of 1,000 ppm. Once exhausted, the absorbent is
regenerated using air or oxygen to oxidize the Fe2S3 back to its original
state, although this is a highly exothermic reaction:

2 Fe2S3 + 3O2 → Fe2O3.H2O + 6S (8.6)

Eventually, adsorption sites will be fully taken up with elemental S,

and the absorbent will then need to be replaced. Commercial oxide
Table 8.5. Characteristics of 37 different substrates (feedstocks) for anaerobic digestion used in the calculation of potential hydro-

308 Anaerobic Wastewater Treatment

gen sulfide biogas content, with details of total solids content (TS), volatile solids content (VS), total sulfur, total carbon, crude
proteins (CP), biochemical methanogenic potential (BMP), biochemical biogas potential (BBP), carbon biodegradability (CB),
carbon to sulfur ratio (C/S), and predicted H2S–S biogas content (PBS), with PBSmax measured using total S analysis and biogas
production and PBSbio determined using the molar ratio between total S and total C using the method of Peu et al. (2012).
Reproduced with permission of Elsevier Publishing.
TS VS Total S Total C BMP BBP
(g/kg (g/kg (g S/ (g C/ CP (NLCH4/ (NL Biogas/ CB C/S PBSmax PBSbio
fd) fd) kgTS) kg TS) (g/kg TS) kg VS) kg fd) (%) (g/g) (%) (%)
Feedstock from urban wastewater treatment plant
  Primary sludge 52.8 44.5 4.5 339.7 nd* nd nd nd 74.7 — —
  Biological sludge 57.0 45.8 8.6 505.3 493.4 255.5 18.6 34.6 58.6 1.9 0.6
  DAF sludge 47.6 36.3 8.4 576.5 154.0 806.1 39.4 76.9 68.7 0.7 0.5

Feedstock from agricultural resources

Cover crops
  Camelina crops 213.0 178.0 4.0 394.4 132.9 234.3 84.2 53.7 98.6 0.7 0.4
  White mustard crops 228.0 194.0 3.3 390.4 100.9 222.5 86.4 52.0 117.6 0.6 0.3
  Brown mustard crops 173.0 154.0 3.7 514.5 98.3 264.1 87.5 52.7 137.9 0.5 0.3
  Radish crops 301.0 126.0 3.4 245.8 62.3 236.7 67.6 49.0 73.2 1.0 0.5
  Soybean crops 249.0 84.0 2.4 152.6 78.8 305.0 58.2 82.0 63.3 0.7 0.6
Animal wastes
  Farm manure 225.2 209.9 1.2 297.3 71.3 271.5 103.0 82.4 245.3 0.2 0.2
  Dairy cow slurry (1) 31.0 26.0 2.8 477.4 141.1 326.9 13.4 48.5 170.5 0.5 0.2
  Dairy cow slurry (2) 106.9 79.2 3.8 429.3 140.9 238.7 28.3 33.0 112.7 1.0 0.3
  Pig faeces (1) 362.0 280.0 3.1 360.0 147.8 304.0 135.1 55.5 115.4 0.6 0.3
  Pig faeces (2) 390.0 320.0 3.9 398.0 124.8 328.0 166.6 57.5 102.4 0.6 0.4
  Pig slurry (1) 43.2 30.4 8.0 349.3 172.2 230.0 9.8 34.8 43.6 2.5 0.9
  Pig slurry (2) 38.5 25.0 8.0 411.9 204.0 140.2 5.5 18.6 51.5 3.9 0.7

Feedstock from agri-food facilities

Industrial wastewater treatments
  Biological sludge PS** 60.3 47.8 8.3 553.0 130.3 259.5 18.0 28.9 66.6 1.9 0.6
  Biological sludge DMI*** 69.2 40.0 7.0 319.4 445.2 282.8 16.9 41.0 45.8 2.0 0.8
  Biological sludge PI ****
61.6 41.3 4.7 348.4 456.8 205.9 13.7 34.2 74.2 1.5 0.5
  Screening refusal PS 328.6 310.8 5.1 500.9 338.7 545.7 247.7 80.6 97.9 0.5 0.4
  Screening refusal CS***** 176.2 163.2 2.8 531.4 227.5 490.2 119.2 68.2 189.5 0.3 0.2
Fatty effluents

Biogas Production and Cleaning 309

  Grease trap waste CS 237.7 231.6 1.0 765.9 42.6 863.4 271.7 79.9 797.9 0.1 0.0
  Grease trap waste PI 361.3 350.0 1.5 702.3 67.7 1011.5 454.8 96.0 478.9 0.1 0.1
  Grease trap waste PS 557.1 552.6 1.5 609.2 102.5 902.9 648.3 102.3 412.0 0.1 0.1
Vegetable residues
  Carrot pulp 177.1 166.7 1.1 447.2 93.1 330.6 107.7 72.8 389.0 0.1 0.1
  Onion pulp 211.5 194.6 3.0 434.5 122.5 416.7 153.1 89.2 142.6 0.3 0.3
  Shallot pulp 226.7 212.7 3.4 456.0 144.6 413.3 169.9 88.0 134.8 0.3 0.3
(Continued)
 Table 8.5. (Continued)

310 Anaerobic Wastewater Treatment

TS VS Total S Total C BMP BBP
(g/kg (g/kg (g S/ (g C/ CP (NLCH4/ (NL Biogas/ CB C/S PBSmax PBSbio
fd) fd) kgTS) kg TS) (g/kg TS) kg VS) kg fd) (%) (g/g) (%) (%)
Others
  Rumen content CS 166.3 155.4 1.5 493.6 124.2 341.1 82.7 54.0 325.6 0.2 0.1
  Meat waste PI 330.3 293.9 4.9 626.5 332.2 871.0 346.9 89.8 127.3 0.3 0.3
  Bovine blood CS 155.8 145.1 6.8 505.1 925.0 454.9 92.3 62.8 74.5 0.8 0.5
  Pig mucus PS 190.5 169.1 7.7 526.0 695.1 579.5 141.6 75.7 68.6 0.7 0.5
  Pig bristles PS 285.5 278.4 26.0 492.5 904.2 277.3 105.5 40.2 18.9 4.9 2.0
  Fish viscera 418.0 380.0 4.6 568.6 269.6 657.9 403.0 90.8 122.6 0.3 0.3

Feedstock from municipal waste

  Canteen refusals 271.3 257.5 3.6 545.1 244.2 570.8 249.6 90.4 151.4 0.3 0.2
  Dehydrated canteen 975.4 827.1 4.0 464.2 305.0 395.3 535.9 63.4 116.2 0.5 0.3
refusals
  Harvested red seaweeds 200.9 120.2 23.3 276.2 136.9 133.1 25.6 24.7 11.9 12.8 3.2
  Harvested green 222.0 128.0 29.6 203.4 141.0 132.8 26.0 30.8 6.9 17.7 5.5
seaweeds
  Lawn mowing 238.2 187.5 5.5 438.2 220.4 289.6 86.7 44.5 79.2 1.1 0.5
*
Not determined.
**
Pig slaughterhouse.
***
Dairy milk industry.
****
Pig industry.
*****
Cattle slaughterhouse.
 Biogas Production and Cleaning 311

pellets are widely available and can be used in a variety of different

desulfurization reactor designs. Wood or ceramic granules or pellets
are coated with Fe2O3 or Fe3O4 and used in low-pressure vessels, with
the biogas passing through in a down-flow direction. Ceramic-based
pellets are possibly better due to their stability and uniform size, opti-
mizing gas flow contact and ease of handling. Examples of commer-
cial desulfurization adsorbents are SulfaTreat 410-HP, Sulfa-Bind,
Sulfur-Rite, and BioBond. Regeneration is generally achieved by
blowing air through the media, in which case at least two units will
be required for continuous operation as one unit is taken out of
operation (i.e., batch regeneration). Alternatively, a small amount of
air (1%) can be added to the biogas so long as the hydrogen sulfide
concentration is not too high (i.e., continuous regeneration). Where
high hydrogen sulfide concentration is present, batch regeneration is
required. Some media are non-flammable, such as Sulfa-Bind, elimi-
nating the risk of combustion during the regeneration phase.
Dry scrubbers are used for small to medium biogas flows and
hydrogen sulfide contents, with the media housed in compact vertical
cylindrical vessels made out of HDPE or stainless steel. Chemical or
biological scrubbers are generally used for higher biogas flow rates.
The dry scrubbing process is often referred to iron sponge scrubbing,
and some basic design criteria are given in Table 8.6 (Zicari et al.,
2003). Some research has suggested that the spent media could be
used as a soil amendment or in brick manufacture or road construc-
tion. Currently, the main disposal route is landfill.
Activated carbon is extremely effective in removing hydrogen
sulfide by adsorption while also allowing its oxidation to elemental
sulfur or sulfate by exposing the carbon to air or oxygen.

2H2S + O2 → 0.25S8 + 2H2O(8.7)

Normal practice is to inject a small amount of oxygen into the

biogas stream, although the activated carbon can be periodically
removed and left exposed to the air for oxidation to occur. Biogas
must be free from dust and moisture before treatment. Improved
efficiency in desulfurization by activated carbon can be achieved by
using alkaline or oxide coatings, which can increase hydrogen sulfide
312 Anaerobic Wastewater Treatment

removal capacity from 10 to 20 kg H2S m–3 for normal carbon to

120–140 kg H2S m–3 for coated carbon. However, it is not feasible
to regenerate all coated activated carbon medium, which has to be
landfilled. SULFUSORB® GAC, manufactured by Calgon Carbon
Corporation, is impregnated with copper oxide, and like other metal-
oxide-coated granular activated carbon (GAC), it is possible to regen-
erate the medium by heat treatment.

Table 8.6. Basic design criteria for an iron sponge scrubber adapted from (Zicari
et al., 2003).
Design parameter Recommendation
Tower/column Stainless steel construction; two columns in series
preferred
Biogas feed Downward flow to maintain bed moisture
Use column with the most contaminated medium first
Residence time Biogas residence time >60 seconds
Temperature 18–40°C to optimize reaction kinetics; prevent
medium drying
Tower/column height Minimum 3 m for H2S removal; 6 m for mercaptans
Superficial gas velocity Liner velocity 0.6–3 m min–1
Surface mass loading rate <10 g S min–1 m–2
Humidity Optimum 40% (range 25–55%)
pH 8–10; use sodium carbonate supplement
Pressure Minimum 140 kPa

8.2.2 Absorption
Hydrogen sulfide can also be removed from biogas by absorption in a
solvent, most commonly water, but also methanol, propylene carbon-
ate, and ethers of polyethylene glycol, preferably under alkaline condi-
tions. Two columns are operated in series: the absorber or scrubber,
followed by the regeneration vessel. Here, the hydrogen sulfide is
stripped from the biogas into the solvent, usually water, which then
requires it to be treated, which makes it ideal for use at wastewater
treatment plants. High biogas flow rates can be treated at 100–10,000
m3h–1, with up to 99% H2S removal. The resultant contaminated waste
stream may contain up to 20–30 g m–3. Where water scrubbing is
 Biogas Production and Cleaning 313

Figure 8.3. Water scrubber media is designed to maximize hydrogen sulfide and
carbon dioxide absorption into water. Shown here is the Tellerette® 2R medium
(70 × 25 mm). Reproduced with permission of Verantis Environmental Solutions
Group, OH, USA. https://www.verantis.com/products/tellerette-packing/tellerette-
packing

used, the hydrogen sulfide is removed with pressurized water, which

also removes the carbon dioxide present. Water is ideal as a solvent, as
both hydrogen sulfide and carbon dioxide are more soluble than
methane. To ensure maximum contact between the biogas moving
upward through the column and the water moving downward, the
column is packed with a medium, ensuring a large surface area for
gaseous absorption to take place. For example, Tellerette™ media
come in a range of sizes from 46 mm (width) × 19 mm (height) up to
105 mm × 56 mm (Fig. 8.3) and is designed to create numerous drop-
lets that collide with the next piece of media, causing it to burst, creat-
ing new surfaces and maximizing water gas contact, hence absorption.
The structural design of the media is more important than the actual
surface area, which is more important in biofilm reactors. If the water
is to be reused, which is often not the case, where the final effluent is
used at wastewater treatment plants (WWTPs), then it must go
through an air or steam stripper to remove the contaminants.
314 Anaerobic Wastewater Treatment

Figure 8.4. Schematic layout of the LO-CAT® system, which removes hydrogen
sulfide from biogas using chemical absorption by catalytic oxidation using a chelated
iron salt solution. https://merichemtech.com/sulfur-recovery-with-lo-cat/

8.2.3 Catalytic Scrubbing

Chelated iron solutions are widely used in a range of patented liquid
redox systems to convert hydrogen sulfide to elemental sulfur. They
can operate at low temperatures and employ continuous regeneration,
with removal efficiencies >99.9%. The system uses a proprietary
catalyst solution, which is then regenerated — an example of which is
the LO-CAT®system from Merichem Technologies. Iron ions are
bound to organic-chelating agents, which remove hydrogen sulfide
by the reduction of ferric chelated iron (Fe3+) into ferrous chelated
iron (Fe2+), both of which are in aqueous solution. The ferrous iron
is subsequently re-oxidized to ferric chelated iron in the regeneration
stage by air stripping (Fig. 8.4).

Purification: H2S + 2[Fe3+] → 2[Fe2+] + S + 2H+(8.8)

Regeneration: 2[Fe2+] + 0.5O2 +2H+ → 2[Fe3+] + H2O(8.9)

8.2.4 Biological Oxidation

Where hydrogen sulfide is in low concentrations, then it can
be removed in situ by bacterial action using microaeration. Sulfur
oxidizing bacteria (SOB) are predominantly chemoautotrophs, Gram-
negative, and of the genera Thiobacillus, Thiothrix, Sulfolobus,
 Biogas Production and Cleaning 315

Thermothrix, and Beegiatoa, which oxidize hydrogen sulfide to ele-

mental sulfur, which is then deposited into their cells, forming charac-
teristic granules that are easily seen using phase contrast microscopy.

H2S + 0.5O2 → S0 + H2O ∆G0′ = –218 kJ mol–1(8.10)

The energy produced by this reaction is used to fix the carbon

dioxide required for cell growth. SOBs are microaerophilic, which
means that they are found in areas of very low oxygen tension, ensur-
ing abundant reduced compounds but just enough oxygen for metab-
olism. Most favorable growth conditions mimic their natural habitat,
such as marsh sediments. SOB species vary in their optimum pH and
temperature and so can work successfully in many different treatment
scenarios. Filamentous forms include Beggiatoa and Thiothrix, while
the rod-shaped SOBs belong to the genus Thiobacillus. In 2000, a
reclassification of the species belonging to the genus Thiobacillus was
carried out, with some reclassified into the new genera Acidithiobacillus
and Halothiobacillus (Kelly and Wood, 2000), so Thiobacillus thioox-
idans is now Acidithiobacillus thiooxidans. While all SOB are obligate
aerobes, Acidothiobacillus denitrificans is actually a facultative anaer-
obe, which can replace oxygen with nitrate as its terminal electron
acceptor to convert hydrogen sulfide to sulfate.
Biological desulfurization falls into three different treatment pro-
cesses: biofilters, biotrickling filters, and bioscrubbers (Fig. 8.5), with
major advantages over other treatment desulfurization methods,
including lower operational costs and reduced use of chemicals
(although nutrients are sometimes required), no regeneration step,
and no polluting end products requiring disposal. There are a number
of different biological outcomes using SOB, with the oxygen satura-
tion concentration controlling the production of elemental sulfur or
sulfate ions (Pudi et al., 2022):

H2S + H2O → HS– + H3O+(8.11)

H2S + 2O2 → SO42– + 2H+(8.12)

HS– + 0.5O2 → S0 + H2O(8.13)

S0 + H2O + 0.5O2 → SO42– + 2H+(8.14)

316 Anaerobic Wastewater Treatment

Occasional Continous
Depolluted irrigation Depolluted trickling
air air
Nutrient
solution
Bed made Nutrient
Bed made
from inert solution
from
materials,
organic
inoculated
materials

Polluted
Polluted air Waste solutions,
air Possible recycling possible recycling
of waste solutions
(a) (b)

Aqueous
Depolluted
solution
air

Activated sludge,
suspended in a
Absorption nutrient solution
column
Bioreactor

Polluted
air
Waste solutions
containing the polluants
(c)

Figure 8.5. Schematic view of biofiltration systems used for the desulfurization of
biogas: (a) biofilter, (b) biotrickling filter, and (c) bioscrubber (Pudi et al., 2022).
Reproduced with permission of Elsevier Publishing.

Biofiltration
Both biofilters and biotrickling filters employ a random or modular
medium on which a biofilm develops, comprised primarily of SOB.
Air must be made available as the bacteria are aerobic, and a con-
trolled amount is normally injected into the biogas stream to give 4%
dissolved oxygen saturation. Biofilters use organic media, which pro-
vides the necessary nutrients (Fig. 8.5a), whereas biotrickling filters
use plastic media, requiring nutrients to be added separately in the
 Biogas Production and Cleaning 317

recycled process water (Fig. 8.5b). A wide variety of other materials

has been used as media, including compost, wood chips, charcoal,
larva, zeolites, and ceramics (Pokorna et al., 2015).
As the hydrogen sulfide is broken down into sulfate, H2SO4 is also
formed. While the reactor is generally operated under acidic condi-
tions (pH 1–3), some buffering is required, which, in the case of
biofilters, is achieved by adding alkaline material to the bed or using
a medium that is alkaline. In biotrickling filters, pH control is easier
as the recycled process water can be buffered by chemical addition.
Among the commercial units available is the Biopuric® process from
Biothane and various systems from BioGlasclean, such as QSR®(quick
sludge remover), MBR (moving bed reactor), and MUW® (make-up
water). Generally, the removal efficiency of hydrogen sulfide by bio-
filters and biotrickling filters is between 90 and 99%. The majority of
the hydrogen sulfide is oxidized to elemental sulfur, which builds up
in the biomass and requires the medium to be flushed every few
months. To reach the required standards for biomethane, the treated
biogas may need polishing by passing through an activated carbon
adsorption vessel (Allegue and Hinge, 2014).

Bioscrubbing
A bioscrubber consists of an absorption tower where hydrogen sulfide
is absorbed into water or alkaline solution, followed by an aerobic
treatment phase, where the sulfur is removed with the effluent recy-
cled back to the absorption tower. The key advantages are that there
is no injection of air or oxygen into the biogas and that bioscrubbing
has high hydrogen sulfide removal efficiency (99%) (Fig. 8.5c).
The Biogasclean MBR™ is a moving biological reactor where the
gas bubbles ensure that the floating medium is kept in suspension and
that the digested wastewater used as the liquid phase is kept fully
mixed. The biogas is compressed, cooled, and injected with air and
released via diffusers at the base of the reactor, where it mobilizes the
media-rich in SOB, oxidizing the hydrogen sulfide to elemental sul-
fur. The process is specifically designed for treating biogas from etha-
nol distilling, which has a high hydrogen sulfide concentration of
between 20,000 and 30,000 ppm and uses significantly less oxygen
318 Anaerobic Wastewater Treatment

Processed Flash Gas

Gas Out to flare Air
(Shell-Paques
only) Circulation
Pump

Scrubber
Aerobic
Bioreactor
Settling
Sour pH = 8.2–9 Tank
Effluent
Gas P = 1 bar Bleed;
Water and
sodium salts

Flash Vessel Sulfur

Vacuum
(Shell-Paques only) Air Product
Filter Press
NaOH Nutrients

Figure 8.6. (a) Layout of a chemical scrubber to remove the hydrogen sulfide
using a counter flow of alkaline wash water. (b) Example of a chemical scrubber using
a counter flow of sodium hydroxide. Reproduced with permission of Biogas Products
Ltd. www.biogasproducts.co.uk

(25%) than biofilters. Biological desulfurization techniques and the

microbiology of SOB have been reviewed by Pudi et al. (2022).
Caustic scrubbers remove the hydrogen sulfide using a counter
flow of alkaline wash water (pH 8.2–9.0). The biogas enters the bot-
tom of a tower packed with medium to ensure maximum liquid-gas
contact, while recirculated NaOH is sprayed downward through the
medium, absorbing the hydrogen sulfide (Fig. 8.6a). The wastewater
leaving the scrubber enters the completely mixed biological reactor,
where sulfur bacteria convert the dissolved sulfide into elemental sul-
fur and sodium hydroxide, thus restoring the caustic nature of the
wash water (Fig. 8.6b).

Scrubber: NaOH + H2S → NaHS + H2O(8.15)

Bioreactor: NaHS + 0.5O2 → S0 + NaOH (8.16)

A second reaction also occurs in the bioreactor, with <5% of the

NaHS converted to sodium sulfate:

2NaHS + 4O2 → NaHSO4 → Na2SO4 + H2SO4(8.17)

 Biogas Production and Cleaning 319

This requires a small addition of sodium hydroxide to maintain

the optimum alkaline pH. An example of a commercial system is
THIOPAQ™ by Paques.

8.2.5 Metal Sulfide Precipitation

Hydrogen sulfide in biogas can be employed to remove and recover met-
als from waste streams as highly insoluble salts even at very low concentra-
tions, offering the potential not only for enhanced environmental
protection but also the recovery of currently lost valuable materials.
Metal wastewaters are among the most difficult to treat, having
low pH, low COD, and high concentration of metals, requiring
expensive physicochemical treatment, principally ion exchange,
adsorption, and membrane filtration, with chemical precipitation,
coagulation-filtration, electrolysis, flotation, or reverse osmosis also
used (Fu and Wang, 2011). Therefore, they are often discharged
into the sewer, with the resultant metals ending up in the sludge.
Metals are also a common component of sewage often associated
with personal care products. There is increasing interest in the recov-
ery of metals from a wide range of wastewaters, especially those with
high concentrations, as part of the circular economy approach to
waste management.
The commonest metals in wastewater are zinc, copper, nickel,
lead, chromium, cadmium, and mercury. While copper and zinc are
both essential trace elements for human health, excessive amounts are
toxic. All these metals can cause severe health problems, with nickel
carcinogenic, mercury a neurotoxin, and lead causing damage to the
central nervous system. They all have both acute and chronic toxici-
ties to a wide range of aquatic species.
Compared to the physicochemical treatment options, sulfide pre-
cipitation using SRB offers a range of advantages, including the ability
to form insoluble metal sulfides at low pH (2.5­–3.0), high settling
velocities of the sulfides, excellent thickening and dewatering of metal
sludges, and low capital and operational costs. Selective metal recovery
is possible by controlling the pH (Table 9.7), although this is generally
used when treating acid mine drainage rather than municipal wastewa-
ters (Bijmans et al., 2009; Sampaio et al., 2009). SRB use sulfate ions
320 Anaerobic Wastewater Treatment

as terminal electron acceptors under anaerobic conditions, utilizing

simple organic compounds (CH2O) as an electron donor (Eq. (9.3)).
Sulfides are produced, which react with metals (M2+) present to form
insoluble metal sulfides (MS), which are very stable (Eq. (9.4)). During
these reactions, alkalinity is released into the wastewater (Eq. (9.5)).
It is important that metal sulfides are fully recovered from waste
streams and not allowed to enter the watercourse, where they can be
environmentally damaging. The sulfide precipitate is made up of very
small particles, which, like nanoparticles, have high specific surface
areas and are very reactive (Sec. 9.2.2).
Sulfate reduction and metal precipitation can be carried out,
either in single reactors or multi-stage reactors where these steps are
separated. The latter is more efficient as direct contact between metals
and the biomass is avoided. Down-flow fluidized beds and air lift
reactors are most efficient, with removal efficiencies >98% for most
metals (Table 9.9) (Kumar et al., 2021).

8.2.6 In-situ Sulfide Control

Metal salts (chlorides, phosphates, and oxides) can be added to reac-
tors, mainly digesters, to control hydrogen sulfide in biogas. They
react with the hydrogen sulfide to form insoluble iron sulfides,
thereby removing a significant portion of soluble hydrogen sulfide
(Kiilerich et al., 2017). Although zinc and copper can be used, iron
salts are predominately employed and, in particular, ferrous chloride
(FeCl2), which is highly effective and has a low toxicity:

FeCl2 + H2S → FeS + 2HCl (8.18)

Ferrous chloride

2FeCl3 + 3H2S → 2FeS + S + 6HCl (8.19)

Ferric chloride

2Fe(OH)3 + H2S → 2Fe(OH)2 + S + 2H2O(8.20)

Ferric hydroxide

Fe(OH)2 + H2S → FeS + H2O(8.21)

Ferrous hydroxide
 Biogas Production and Cleaning 321

FeO + H2S → FeS + H2O(8.22)

Ferrous oxide

CuO + H2S → CuS + H2O(8.23)

Cupric oxide

ZnO + H2S → ZnS + H2O(8.24)

Zinc oxide

Zero-valent iron has also been found to be highly effective (Al

Mamam and Torii, 2015; Su et al., 2015). However, metal salts used
in situ cannot reduce hydrogen sulfide concentrations low enough to
meet current standards for national grid or vehicle specifications
on their own, so an additional desulfurization step will be required.
The constant addition of metal salts is expensive and an additional
operational factor, although savings can be made by reducing the
amount of hydrogen sulfide that ultimately ends up in the biogas,
requiring further treatment (Table 8.3).
There is growing interest in iron dosing of the sewer network or
inlet works of WWTPs that reduces hydrogen sulfide formation,
enhances phosphate removal, and improves sludge dewaterability. In
a major study, Kulandaivelu et al. (2020) compared the effects of dos-
ing the wastewater at the WWTP inlet (Trail 1) and within the sewer
network (Trail 2) with ferrous chloride. They found that COD
removal in the primary settlement phase was increased by 49 and 47%
in Trials 1 and 2, respectively, with 76 and 53% increase in phosphate
removal in the biological nutrient removal activated sludge system,
respectively. Hydrogen sulfide was reduced by 36 and 45% during
anaerobic digestion in Trials 1 and 2, respectively, with sludge dewa-
terability improved by 10% in both trials. The head space in the sewer
network showed significant reductions in hydrogen sulfide concentra-
tion. The idea of using iron salts for multiple functions within the
wastewater treatment cycle rather than simply for desulfurization for
biogas upgrading is an example of the modern sustainability approach
to WWTP operation and management.
Air or oxygen dosing to the biogas digester headspace or gas stor-
age tank allows biological aerobic oxidation of the hydrogen sulfide
to occur, with 80–99% reductions recorded. The process is known as
322 Anaerobic Wastewater Treatment

microaeration and has already been explored above in conjunction

with other systems. Oxygen acts as an electron acceptor for the oxida-
tion of hydrogen sulfide to elemental sulfur, sulfate, or thiosulfate:

H2S + O2 → S0 + 2OH–(8.25)

H2S + 2O2 → SO42– + 2H+(8.26)

4H2S + 5O2 → 2S2O3 + 4H2O(8.27)

Thiosulfate is the main product of anaerobic sulfide oxidation,

which can subsequently be reduced to sulfide again by sulfate reducing
bacteria. This tends to occur at high hydrogen sulfide concentrations
due to the inhibition of SOB. Elemental sulfur is the main product of
biotic activity by SOB occurring at hydrogen sulfide concentrations of
<5 mM. Microaeration is an effective technology, and although five
times more air is required than pure air, using air increases the risk of
explosion when mixed with methane. Mixing is very important to
ensure maximum contact between oxygen and the sulfide, so the loca-
tion of the air or oxygen inlet in the reactor is critical (Jung et al.,
2022). However, residual oxygen levels of 0.5 to 1.8% remain, which
may exceed required biomethane standards, with the methane content
of biogas also diluted due to the presence of oxygen. Oxygen is a well-
documented inhibitor of methanogenesis as well as increasing the risk
of explosion.
Other less successful methods of in-situ treatment include adsorp-
tion and the suppression of SOB by, for example, operating at a more
alkaline pH (Table 8.3), but neither are as reliable or successful as the
previous methods. Current research on alternative in-situ methods of
desulfurization has focussed on electrochemical oxidation, iron
cycling, and sulfide oxidation coupled with denitrification and stimu-
lation of electric syntrophy (Table 8.4), all of which have been
reviewed by Jung et al. (2022).

8.3 Carbon Dioxide Removal Technologies

Biogas contains, on average, 35% carbon dioxide (typical range
14–48%), with some removed during desulfurization. However, to
 Biogas Production and Cleaning 323

optimize the calorific value of the biogas, most of the carbon dioxide
must be removed to <2.5% (Table 8.1). There are numerous carbon
dioxide removal methods, including membrane filtration, pressure
swing adsorption (PSA), absorption, both with and without chemical
reaction, cryogenic removal, and by adding propane (Hagen et al.,
2001).
Membrane separation takes advantage of the differences in per-
meability of methane and carbon dioxide as well as hydrogen sulfide,
which can also be removed by a membrane, usually in the form of
hollow fibers made from polymers, such as acetate-cellulose. Operating
at a partial pressure of between 25 and 40 bar, the retained enriched
methane is also at high pressure, usually eliminating the need for fur-
ther compression before supply to the gas grid. The permeability of
hydrogen sulfide is 60 times greater than methane, while carbon
dioxide is 20 times higher, making membrane filtration ideal for both
desulfurization and carbon dioxide removal. However, as methane
purity increases, the methane yield falls. Methane purity can be
increased by using a number of membranes in series, but this also
coincides with methane permeating through the membrane (Fig. 8.7).
To a certain extent, this can be minimized by recirculating a portion
of the permeate (i.e., the carbon-dioxide-enriched gas) through the
last membrane for a second time. Alternatively, only one or two mem-
branes can be used, and the lower quality is brought up to standard
by the addition of propane.
PSA employs four adsorber tanks filled with zeolites or granular
activated carbon, with each tank operating alternating cycles of
(i) adsorption, (ii) depressurization regeneration (desorption under
vacuum), and (iii) repressurization. The biogas enters the vessel at the
base, and as it passes through the medium, carbon dioxide, oxygen,
and nitrogen are all adsorbed, and the biogas collected from the top
has methane purity in excess of 97%. Before purity levels fall, the
adsorber is closed, and the untreated biogas enters the next vessel,
where the medium has already been regenerated and repressurized.
The first vessel is now depressurized to atmospheric pressure and then
evacuated by a vacuum pump, regenerating the medium. The vessel is
then repressurized, ready to commence the cycle once again. By using
tanks in series, a continuous process can be maintained (Fig. 8.8).
324 Anaerobic Wastewater Treatment

Figure 8.7. The effect of using membranes in series to improve methane purity
(blue line) on methane yield (red line) using recirculation (Hagen et al., 2001).
Reproduced with permission of the Swedish Gas Center, Malmo, Sweden. info@sgc.
se/[email protected]

The most common absorption method is using a counterflow

water scrubber packed with a random medium to ensure maximum
gas–water contact. The gas is pressurized before entering the base
of the scrubber to increase the solubility of the carbon dioxide;
however, methane is also partially soluble in pressurized water, so
some will be lost in the wash water. This is recovered using a flash

Figure 8.8. Pressure swing adsorption (PSA) is widely used for the removal of
carbon dioxide from biogas.
 Biogas Production and Cleaning 325

tank, where the wash water is partially depressurized, so that when

the methane comes out of the solution, it can be returned to the
incoming biogas stream. The wash water is then fully depressurized
to atmospheric pressure and passed downward through the desorp-
tion (regeneration) column, with air pumped up from the base,
which strips out the carbon dioxide. The water is then reheated to
the required temperature before recycling back to the absorption
column (Fig. 8.9). Desulfurization absorption using water also
removes carbon dioxide, but the reverse only works with low hydro-
gen sulfide concentrations and requires a different operational
approach to regeneration due to the build-up of sulfur on media.

Figure 8.9. Water scrubbers are used to reduce carbon dioxide concentration in
biogas. The system comprises an absorption column, followed by a regeneration
column comprising a flash separator to recover any dissolved methane before the
desorption section, where carbon dioxide is recovered or vented (Hagen et al.,
2001). Reproduced with permission of the Swedish Gas Center, Malmo.
326 Anaerobic Wastewater Treatment

One advantage of the process is that treated effluent can be used

instead of clean water without the need to treat and recirculate. This
can be advantageous in some treatment processes where recircula-
tion is normally employed. However, the methane removed by the
pressurized water during absorption will have to be recovered by
depressurization in a flash tank before return to the WWTP. Carbon
dioxide can also be removed by chemical action, or cryogenically
(Hagen et al., 2001).

8.4 Removal of Water

Biogas is saturated with water vapor, approximately 5% at 35oC, and
much of this condensates in the pipework as it cools to ambient tem-
perature. It can be collected by ensuring the pipework has a very small
slope (10 mm m–1), allowing the condensate to fall back into drip
traps. If moisture remains a problem, then chilling may be required,
lowering the dew point to 0.5–1.0oC. Hydrogen sulfide is also par-
tially removed in the condensate. There are alternative adsorption and
absorption methods using drying agents, but these tend to be expen-
sive and are not always successful.

8.5 Removal of Other Impurities

Other gases found in biogas are hydrogen (0–5%), carbon monoxide
(0–2.1%), nitrogen (0.6–7.5%), and oxygen (<0.1%) although gener-
ally found in trace amounts and so require no removal unless they
exceed required thresholds. However, ammonia, volatile organic
compounds (VOCs), including terpenes, and siloxanes may need to
be removed when biogas is upgraded to biomethane for off-site
energy use (Table 8.1). Particulate matter is removed using simple
filters.

Ammonia: Ammonia is generally removed during desulfurization

using adsorption or a water scrubbing process. Activated carbon is
widely used for ammonia removal, but large-scale industrial units rely
on acid washing with weak sulfuric or nitric acid.
 Biogas Production and Cleaning 327

Oxygen: Oxygen is normally only found in trace amounts in biogas,

although it can be introduced during desulfurization. If it is a prob-
lem, it can be removed by membrane separation or PSA.

Terpenes: The removal efficiency of terpenes (Sec. 3.5.2) varies sig-

nificantly between methods, with PSA achieving 100% removal, while
water scrubbers achieve higher removal efficiencies (83–96%) than
amine scrubbers (61–85%) (Arrhenius et al., 2017).

Siloxanes: Currently, siloxane (Sec. 3.6) is removed from the biogas

using adsorption, usually activated carbon, although a wide range of
other absorbents has been used, such as metal oxides, zeolites, and
polyurethane foams (Paglini et al., 2022; Werkneh, 2022). For high
biogas volumes, or high siloxane concentrations, adsorption can be
improved by employing an initial refrigeration/condensation step
before adsorption, with efficiencies of >99% removal possible, allow-
ing for concentrations <1.0 mg m–3 (de Arespacochaga et al., 2015).
Some siloxanes are also removed during biogas chilling to control the
moisture content.

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Section E

The Future
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 9
Sustainability and Future of
Anaerobic Wastewater Treatment

9.1 Sustainability and the Circular Economy

Achieving sustainable wastewater treatment is one of the most impor-
tant engineering challenges facing society, as the need to mitigate the
dual problems of population growth and global warming becomes
critical. The traditional roles of protecting surface and ground waters,
preserving biodiversity, and preventing pathogen transfer have all
become more challenging as wastewaters become more complex with
new chemicals that are increasingly inhibitory or toxic to treatment.
To fulfill the needs of sustainability, circularity has become a defining
layer on top of our existing environmental and health priorities, with
carbon harvesting, recovery of metals and other useful products, and,
of course, reuse of water — all achievable expectations. Traditionally,
wastewater treatment has relied on separation and aerobic processes,
with a large portion of the carbon in the wastewater lost as carbon
dioxide through aerobic metabolism, with anaerobic treatment occa-
sionally used for sludge stabilization. In order to achieve sustainabil-
ity, the emphasis of treatment has to change to primarily anaerobic
treatment, with aerobic treatment used for effluent polishing where
filtration technologies are unable to achieve the final effluent quality,
although many natural treatment options are available (Gray, 2022).
Traditionally, anaerobic digestion was a slow process, operated at
low microbial concentrations, requiring large reactors, and subject to

333
334 Anaerobic Wastewater Treatment

many operational problems. But today’s modern designs and a greater

understanding of the microbial basis of anaerobic treatment, espe-
cially of the methanogenic archaea, have seen a rapid development of
compact, rapid, and highly efficient processes, many utilizing granular
sludges.
Modern wastewater treatment has embraced the circular economy
ideology, becoming increasingly sustainable. Among the huge range
of valuable byproducts that can be generated by fermentation are
biomethane, biohydrogen, bioplastics, biodiesel, and volatile fatty
acids (VFAs). VFAs are a valuable raw material used in the food,
chemical, and pharmaceutical sectors as well as the carbon source for
a wide range of bio-based compounds (Atasoy et al., 2018).

9.1.1 Biohydrogen and Dark Fermentation

While hydrogen is an ideal alternative to fossil fuels, its success in
terms of carbon footprint depends on how it is produced.
Thermochemical processes can be a source of carbon and other pol-
lutants, so the possibility of using anaerobic fermentation to produce
biohydrogen is extremely attractive. Key operational factors that
affect the maintenance of hydrogen-producing bacterial communities
include pH, temperature, organic loading rate, substrate concentra-
tion, and characteristics. Show et al. (2007) operated a laboratory-
scale anaerobic continuously stirred tank reactor (AnCSTR) using a
granular sludge to successfully produce biohydrogen, with butyrate,
acetate, and ethanol, the major metabolic byproducts. Using glucose
as a substrate, the optimum solids loading rate (SLR) was between
0.6 and 0.8 g glucose g–1 VSS h–1, with a maximum biohydrogen pro-
duction of 3.26 L L–1 h–1, a yield of 1.84 mol H2 mol–1 glucose at an
organic loading rate of 20 g glucose L–1 h–1, with a hydraulic retention
time (HRT) of 0.5 hours. The relationship between HRT and influ-
ent substrate concentration on biohydrogen production is shown in
Fig. 9.1, with a yield affected at concentrations >20 g L–1 or an HRT
<0.5 hours.
Food crops such as potatoes and tapioca are a vital part of the diet
of billions of people, with starch products resulting in significant
 Sustainability and Future of Anaerobic Wastewater Treatment 335

Figure 9.1. The effect of hydraulic retention time and influent substrate concentra-
tion on hydrogen yield (mol H2 mol–1 glucose) (Show et al., 2007). Reproduced with
permission of Elsevier Publishing.

water pollution in many countries but particularly in Asia (Thanwised

et al., 2012). Traditional treatments, such as stabilization ponds
including anaerobic lagoons, result in significant odor production as
well as the uncontrolled release of huge quantities of greenhouse
gases (Rajbhandari and Annachhatre, 2004). Closed anaerobic treat-
ment of such wastes by contact treatment systems can harvest the
carbon in these wastewaters to produce biogas. Many of the starch
products manufactured need to be dried, making them energy inten-
sive; therefore, using anaerobic treatment processes such as anaerobic
baffled reactors (AnBRs), anaerobic fixed film reactors (AnFFRs), and
up-flow sludge blanket reactors (UASBs) close the production loop,
resulting in the biogas produced being able to offset energy require-
ments by up to 75%. For example, tapioca production results in 20 m3
of wastewater from each ton of starch produced, and with an average
chemical oxygen demand (COD) of 16,000 mg L–1, it can yield up to
336 Anaerobic Wastewater Treatment

10 m3 of biogas per m3 of wastewater treated with significant COD

reductions. Starch-rich wastewaters have also been used to generate
biohydrogen for energy use (Wang et al., 2008). For example, the
treatment of wastewaters from tapioca, cassava, and molasses process-
ing resulted in biohydrogen yields of 13, 54, and 46 L H2 kg–1 COD,
respectively, under ideal operating conditions using an AnBR, a
UASB, and a different AnBR system, although COD removal was
poor (>30%) (Thanwised et al., 2012). Some methane can be gener-
ated, which is minimized by controlling the HRT, with up to 70–80%
of the biogas comprising carbon dioxide, so a lot of the embedded
carbon is lost.

Dark fermentation
Hydrogen is an alternative to fossil fuels, which can be used as a sub-
stitute for natural gas or as a replacement fuel for propane and natural
gas household boilers, many of which are already able to use hydro-
gen with minor modifications. Biohydrogen (Bio-H2) offers a sustain-
able manufacturing pathway, producing a zero-carbon,
combustion-high gravimetric energy density (120 kJ g–1) fuel. It has
been mainly generated using hydrocarbons or gas, and there are
increasing opportunities to use anaerobic fermentation to produce
Bio-H2. Currently, photo-fermentation or direct or indirect photoly-
sis is the major method for producing Bio-H2, although an exciting
alternative is dark fermentation. Unlike the former methods, dark
fermentation does not require light nor a wide range of nutrients and
is able to be produced by a range of standard anaerobic treatment
systems (e.g., anaerobic completely stirred reactor (AnCSTR), anaer-
obic membrane reactor (AnMBR), upflow anaerobic sludge bed reac-
tor (UASB), and anaerobic sequencing batch reactor (AnSBR)) using
mixed cultures of both facultative and obligate anaerobes, with a wide
range of potential substrates, both liquid and solids.
Dark fermentation can be carried out over a wide temperature
range of 20 to 80oC, and, like normal anaerobic breakdown, carbon
dioxide and hydrogen sulfide are also produced. The Bio-H2 is pro-
duced primarily from glucose molecules in carbohydrates with VFAs
 Sustainability and Future of Anaerobic Wastewater Treatment 337

Figure 9.2. Main breakdown pathways of glucose during dark fermentation

(Ahmed et al., 2024). Reproduced with permission of Elsevier Publishing.

or alcohols produced as byproducts. The key operational factors are

pH, HRT, and partial pressure of the gas — the latter directing the
metabolic pathway and electron flows so that acetate, butyrate, pro-
pionate, and alcohols are the main byproducts, along with hydrogen
(Fig. 9.2). The main pathways are acetate and butyrate:

Acetate pathway: C6H12O6 + 2H2O → 2CH3COOH + 2CO2

+ 4H2 ∆Go –126 kJ mol–1(9.1)

Butyrate pathway: C6H12O6 → CH3CH2CH2COOH + 2CO2

+ 2H2 ∆Go –214 kJ mol–1(9.2)
338 Anaerobic Wastewater Treatment

The anaerobic reactor can be operated to control how the Bio-H2

is generated, although the acetate pathway is preferred as it produces
twice as much Bio-H2 as butyrate, while lactic acid bacteria suppress
Bio-H2 production. Methanogenesis must be suppressed to improve
Bio-H2 production and prevent methane from being produced.
Unlike methanogenesis, the microbial consortia involved in hydro-
gen production are constantly changing, leading to a variation in Bio-
H2 production and process instability. Hydrogen-producing species fall
into three broad groups, the main group comprising spore-
forming obligate anaerobes, then the non-spore-forming obligate
anaerobes, and, finally, facultative anaerobes. The spore-forming obli-
gate anaerobes are classed as main hydrogen producers and include the
most efficient hydrogen-producing species, with Clostridium spp., the
best-known genera producing 1.5–3.0 mol H2 mol–1 hexose. Although
they are sensitive to low pH (pH < 5.0), they are abundant in anaerobic
reactors and can also be used as a biocatalyst. In theory, hydrogen yield
is restricted to just 4 moles per mol of glucose.(Eq. (9.1)) This is
known as the Thauer limit and restricts the potential output from dark
fermentation. However, exciting new research by Ergal et al. (2020),
who used an artificial consortia of Clostridium acetobutylicum and a
facultative anaerobe Enterobacter aerogenes to increase the yield beyond
the Thauer limit to 5.6 mol mol–1 glucose. Non-spore-forming obligate
anaerobes are classed as auxiliary functioning species and include
Ethanoligenens harbinense (Pylum Bacillota), which produces ethanol as
well as Bio-H2 and carbon dioxide, Acetanaerobacterium elongatum
(Order: Clostridiales) with Bio-H2 yields of 1.55 mol mol–1 glucose,
Megasphaera spp. (Phylum: Firmicutes) that utilize lactate to produce
hydrogen, Acidaminococcus sp. (Phylum: Firmicutes), and Prevotella
sp. (Phylum: Bacteroidetes), which is able to use a wide range of sub-
strates and actively reduce methane production by diverting the hydro-
gen flow in glycolysis away from methanogenesis. The final group
comprises the facultative anaerobes, which are classed as challenged
producers and include a wide range of genera, including Citrobacter sp.,
Klebsiella sp., Enterobacter sp., Bacillus sp., and also Shewanella onei-
densis and Pseudomonas stutzeri (Dahiya et al., 2021).
Supplements have been shown to improve dark fermentation,
with Fe, Ni, and Mg ions playing an important role in the formation
 Sustainability and Future of Anaerobic Wastewater Treatment 339

of cofactors for enzymes, being inhibitory at low concentrations.

Nanoparticles (NPs) have been widely used experimentally to enhance
hydrogen-producing microorganisms during dark fermentation in
terms of production rate and efficiency by improving metabolic activ-
ity. For example, NPs reduce the dependence on NADH and improve
the formation of formate hydrogen lactase, an enzyme used to
decompose formate and improve electron adsorption and transport
between hydrogen-producing bacteria and hydrogenase. There have
been many studies on the effect of NPs on dark fermentation, the
majority of which show a large increase in Bio-H2 production; for
example, FeO NPs specifically enhance acetate synthesis and reduce
ethanol formation, thereby increasing Bio-H2 yield. These studies and
the role of supplements to enhance dark fermentation have been
reviewed by Ahmed et al. (2024).
The concept of a closed-loop integrated biorefinery able to deal
with a wide range of liquid and solid wastes has been proposed by
Venkata Mohan et al. (2020), with waste VFAs from acidogenesis
being used for the production of a wide range of products from alco-
hols to bioplastics (Fig. 9.3). However, dark fermentation faces many
challenges before it can be fully scaled-up to become a major indus-
trial producer of hydrogen (Sarkar et al., 2021; Dahiya et al., 2021).

9.1.2 VFA Production

In terms of resource recovery, there are many waste streams that offer
opportunities to produce valuable byproducts. One of the most valu-
able waste streams is that produced by the dairy industry, being rich
in carbohydrates, proteins, and fats with 2,250,000 m3 produced
worldwide daily. Using the Eurostat website (https://ec.europa.eu/
eurostat/web/main/home), it is estimated that 9.15 Mt acetate,
5.39 Mt butyrate, and 6.47 Mt propionate could be recovered from
that waste stream alone, far in excess of current global demand. Of
the three, acetic acid is the most widely used, being a key building
block in the manufacture of paint, rubber, plastics, synthetic fibers,
textile finishes, pesticides, polymer emulsions, and paper coatings, and
in the food industry as a flavor, acidity regulator, and preservative
(Cheryan, 2009). Current manufacturing costs for VFAs vary, as does
340 Anaerobic Wastewater Treatment

Figure 9.3. Details of a closed-loop biorefinery incorporating dark fermentation

for the production of biohydrogen (Dahiya et al., 2021). Reproduced with permis-
sion of Elsevier Publishing.

demand (Table 9.1). VFAs are easy to transport and store, have a high
market value, and have a very low carbon footprint when produced
using anaerobic fermentation.
Currently, the vast majority of VFAs (90%) are produced syntheti-
cally from petrochemical derivatives (Agnihotri et al., 2022). While
VFAs can also be produced by fermentation, the development of
large-scale biorefineries is still some way away, but with increasing
markets and the move away from fossil fuels, it is inevitable that the
change will be made with wastewater, a key substrate (Bhatia and
Yang, 2017; Venkata Mohan et al., 2019). Within all anaerobic treat-
ment processes, VFA can be promoted by inhibiting methanogens.
While, in theory, this could be done using a methanogenic inhibitor,
the normal practice is to reduce the HRT and solids retention time
(SRT) and adjust the pH to <6.0 or >8.0, which effectively inhibits
Table 9.1. Uses, production methods, and market for volatile fatty acids (Atasoy et al., 2018). Reproduced with permission of
Elsevier Publishing.
Chemical Market size Market price
VFAs formula (kton/year) (€/ton) Usage/application Production methods References

Sustainability and Future of Anaerobic Wastewater Treatment 341

Acetic 14,000–17,000 400–800 Vinyl acetate monomer Chemical synthesis Bhatia and
acid (polymers, adhesives, (carboxylation of Yang
dyes), food additive, methanol) and (2017)
solvent, vinegar, ester microbial fermentation
production, chemicals (oxidative and
anaerobic)
Butyric 90–105 1,500–1,650 Animal and human food Chemical synthesis Zigová and
acid additive, chemical (oxidation of Šturdík
intermediate, solvent, butyraldehyde), (2000)
flavoring agent extraction from butter,
microbial fermentation
Propionic 350–470 2,000–2,500 Esters used food industry Chemical Synthesis Cheryan
acid as aroma additive, (ethylene hydro (2009)
food additive, formylation,
flavoring, carboxylation of
pharmaceuticals, ethylene, direct
animal feed oxidation of
supplement, fishing hydrocarbons), by
bait additive product of acetic acid
manufacturing,
microbial fermentation
342 Anaerobic Wastewater Treatment

the methanogens, allowing fermentative bacteria to flourish. Maximum

VFA production is achieved under alkaline conditions (pH 10). The
key bacterial species employed for VFA production varies; for exam-
ple, for propionic acid, Propionibacterium freudenreichii, P. acidipro-
pionici, P. thoenii, Propionibacterium shermanii, and P. jensenii; for
acetic acid fermentation, Acetobacter spp. Gluconacetobacter spp., and
Gluconobacter spp.; and for butyric acid fermentation, Clostridium
butyricum, C. kluyveri, C. beijerinckii, C. barkeri, C. acetobutylicum,
C. thermobutyricum, C. thermopalmarium, C. pasteurianum,
Butyribacterium spp., Sarcina spp., Megasphaera spp., Fusobacterium
nucleatum, Peptococcus asacelarolyticus, Butyrivibrio fibrisolvens,
Pseudobutyrivibrio ruminis, and Eubacterium limosum. Atasoy et al.
(2020) used cheese-processing wastewater in both a batch reactor and
an AnSBR to produce VFAs with yields of 0.97 and 0.95 g–1 VS COD
g–1 SCOD, respectively. While a wide range of VFAs was produced,
the dominant type was lactic acid (48%) in the batch and propionic
acid (80%) in the AnSBR. Certain bacterial families were correlated
with different VFAs, for example, butyric acid (Gracilibacteraceae,
Desulfovibrionaceae), propionic acid (Desulfovibrionaceae, Syner­
gistaceae), and lactic acid (Pseudomonadaceae, Rhodocyclaceae)
(Zigová and ŠturdÍk, 2000). A similar study using cheese whey as a
substrate and an AnSBR compared performance using a range of
HRT and SRT scenarios and observed optimum process efficiency at
longer SRTs and shorter HRT operating conditions (Lagora-Costa
et al., 2020). The degree of acidification rose from 0.73 to 0.83 when
the SRT was increased from 5 to 15 days, with hydrolytic bacteria
replaced by acidogenic bacteria, and the HRT reduced from 3 to
1 days (Table 9.2). Other waste streams, such as chicken manure, have
huge potential for energy recovery, with globally approximately 3.85
billion egg-laying hens and 50 billion broilers producing hundreds of
millions of tonnes of manure annually (Sakar et al., 2009). Using an
AnMBR, Yin et al. (2022) were able to produce a high yield of VFA
of 0.90 g VFA g–1 VS from chicken manure without any pH control,
with acetic acid the major VFA produced (>50%). Another untapped
waste stream for acidogenic fermentation is olive mill wastewater, with
acetic acid the dominant VFA produced. Yarimtepe et al. (2017)
 Sustainability and Future of Anaerobic Wastewater Treatment 343

Table 9.2. Performance of an AnSBR treating cheese whey (a) at different solids
residence times (SRTs) of of 5, 10, and 15 days and (b) at different hydraulic resi-
dence times (HRTs) of 1, 2, and 3 days (Lagora-Costa et al., 2020). Reproduced
with permission of Elsevier Publishing.
(a)
SRT = 5d SRT = 10d SRT = 15d
Operation time (d) 51 34 38
Feed concentration (g COD L ) –1
11.90 (± 0.53) 12.06 (± 0.44) 11.54 (± 0.35)
HRT (d) 2 2 2
OLR (g COD L d ) –1 –1
6.01 (± 0.32) 6.03 (± 0.22) 5.77 (± 0.17)
VSS (g VSS L )–1
4.19 (± 0.39) 5.36 (± 0.52) 7.38 (± 0.66)
F/M ratio (g COD g VSS d ) –1 –1
1.46 (± 0.14) 1.13 (± 0.09) 0.81 (± 0.04)
Total VFA (g COD-VFA L ) –1
9.72 (± 0.66) 9.44 (± 0.40) 10.23 (± 0.34)
Acetic acid (%) 37.39 (± 2.95) 25.83 (± 3.36) 35.71 (± 3.43)
Propionic acid (%) 20.34 (± 3.69) 7.52 (± 1.46) 10.99 (± 1.42)
Butyric acid (%) 22.13 (± 4.35) 59.62 (± 2.71) 48.40 (± 4.10)
Valeric add (%) 4.11 (± 2.24) 5.46 (± 2.20) 4.90 (± 1.32)
Lactic acid (%) 16.03 (± 7.37) 1.57 (± 1.86) 0.00
DA a
0.73 (± 0.05) 0.75 (± 0.03) 0.83 (± 0.04)
YVFA/S (g COD-VFA g COD- 0.78 (± 0.05) 0.78 (± 0.03) 0.87 (± 0.04)
Lactose–1)b
Productivity (g COD-VFA L–1 d–1) 4.45 (± 0.29) 4.51 (± 0.20) 4.54 (± 0.48)
DA: Degree of acidification.
a

YVFA/S: Acidification yield.

b

(b)
HRT = ld HRT = 2d HRT = 3d
Operation time (d) 51 43 66
Feed concentration (g COD L–1) 5.58 (± 0.17) 11.54 (± 0.35) 17.13 (± 0.79)
OLR (g COD L–1 d–1) 5.58 (± 0.17) 5.77 (± 0.17) 5.71 (± 0.26)
SRT (d) 15 15 15
VSS (g VSS L )–1
7.99 (± 0.56) 7.38 (± 0.66) 7.18 (± 0.44)
F/M ratio (g COD g VSS–1 d–1) 0.69 (± 0.05) 0.81 (± 0.04) 0.80 (± 0.06)
Total VFA (g COD-VFA L ) –1
5.15 (± 0.36) 10.23 (± 0.34) 14.93 (± 0.66)
Acetic acid (%) 27.97 (± 3.08) 35.71 (± 3.43) 23.33 (± 2.98)

(Continued)
344 Anaerobic Wastewater Treatment

Table 9.2. (Continued)

(b)

HRT = ld HRT = 2d HRT = 3d

Propionic acid (%) 8.84 (± 4.88) 10.99 (± 1.42) 6.83 (± 2.10)
Butyric acid (%) 53.97 (± 7.93) 48.40 (± 4.10) 60.91 (± 4.24)
Valeric acid (%) 8.20 (± 4.06) 4.90 (± 1.32) 7.90 (± 2.28)
Lactic add (%) 1.02 (± 0.87) 0.00 (± 0.00) 1.04 (± 1.66)
DAc 0.83 (± 0.03) 0.83 (± 0.04) 0.79 (± 0.05)
YVFA/S(g COD-VFA g COD- 0.90 (± 0.05) 0.87 (± 0.03) 0.86 (± 0.06)
Lactose–1)d
Productivity (g COD-VFA L–1 d–1) 4.65 (± 0.15) 4.54 (± 0.48) 4.53 (± 0.34)
DA: Degree of acidification.
c

YVFA/S: Acidification yield.

d

successfully operated a laboratory-scale AnSBR using olive mill waste-

water, pre-treated by electrocoagulation to reduce its toxicity and
solids content, at a pH of 5.5 with an HRT of 2 days, with an acidifica-
tion rate of 68% at a maximum effective loading of 20 g sCOD L–1 d–1.
The greatest challenge for commercial anaerobic fermentation
production is the recovery of VFAs and, more specifically, separating
the individual VFAs. There are a number of methods currently
employed, including gas stripping with absorption, adsorption, elec-
trolysis, solvent extraction, nanofiltration, reverse osmosis, and PTFE
membranes. These have been reviewed by Atosoy et al. (2018).

9.2 Biotechnology and the Future of Anaerobic

Wastewater Treatment
9.2.1 Granulation
Under certain operating conditions, including plug flow conditions,
high shear stress, and long sludge ages, aerobic and anaerobic bacte-
rial flocs can develop into dense, much larger and rounded granules.
Granules rapidly separate from suspension, ensuring high biomass
retention while offering much higher rates of biodegradation activity,
including methane production. There are a number of theories as to
how granules are formed, although the role of quorum sensing (QS)
 Sustainability and Future of Anaerobic Wastewater Treatment 345

and signal molecules are clearly critical in their formation, especially

in promoting and accelerating the synthesis of extra cellular polymers
(ECPs), more recently referred to as extracellular polymeric sub-
stances (EPSs), cellular metabolism, and improving the surface
attachment capability of granules (see the section Quorum Sensing
below). Apart from rapid settlement, granules offer a number of
advantages over flocs, which include protection against toxic and
inhibitory compounds, defense against extreme pH, and high flow
rates, ensuring close contact between the various groups of anaerobic
microorganisms and rapid breakdown from hydrolysis through to
methanogenesis, high concentrations of microorganisms, and the
ability to withstand shock and variable loadings. Each granule matrix
supports a diverse syntrophic community of bacteria and archaea,
creating what is essentially an independent treatment unit.
Starting up a granular sludge from scratch can take up to four to
six months, which is why biofilm carriers are so widely used as an
alternative. Seeding directly with granules from another reactor is a
more rapid option, if available, but can be expensive due to transpor-
tation and does not always work due to sudden changes in food
sources and other operating conditions. There is a wide range of
physicochemical factors that affect granulation, such as temperature,
pH, substrate type, HRT, up-flow liquid velocity, organic loading
rate, availability of trace elements, and nutrients. These have been
evaluated by Tay et al. (2006) and summarized in Table 9.3.
The development, structure, and microbiology of aerobic granu-
lar sludge have been discussed at length in an earlier book in this
series (Gray, 2023).

Theory and life cycle

The formation of granules is not accurately understood, and to
explain the process, many models have been proposed, which include
both physicochemical and structural models. Physicochemical models
include the inert nuclei model, selection pressure model, multi-
valence positive ion-bonding model, EPS bonding model, synthetic
and natural polymer-bonding model, secondary minimum adhesion
model, local dehydration and hydrophobic interaction model, and the
346 Anaerobic Wastewater Treatment

Table 9.3. Optimum conditions to support and improve granulation adapted from
Tay et al. (2006) and Show et al. (2020). Reproduced with permission of Elsevier
Publishing.
Key operating factors
Biological · Seed sludge for a start-up should be acclimatized with the same
wastewater to be treated. If this is not possible, then the seed must
comprise as much diversity of bacterial populations as possible.
· Microbial loading for a start-up should not be excessive (<0.6 kg
COD kg–1 VSS d–1).
Chemical · Divalent compounds should be supplemented in the feed.
· Granulation will progress sooner on unacidified wastewaters than
on acidified feeds. The pH of the reactor should be kept close to
neutral.
· Chemicals such as caustic soda, soda ash, or sodium bicarbonate
can be added to maintain alkalinity and buffering capacity in the
reactor.
· Essential growth nutrients such as N, P, S and trace elements
(Fe, Ni, Co) should be present in sufficient amounts.
Physical · Reactor design and operating temperature can affect the liquid
mixing, the liquid, and gas up-flow velocities and even the size
and shape of the granule.
· Temperature also affects the biochemical reaction rates. The
optimal temperatures for treatment are in the range of mesophilic
30–38°C and thermophilic 50–60°C.
Feed · Lower concentration of wastewater feed should be used. It was
found that the lower the feed concentration, the faster the granule
will be developed. The concentration should, however, be
sufficient to sustain bacterial growth. A minimum concentration
was suggested at 500 mg COD L–1.
· The presence of dispersed suspended solids in the feed may retard
or even deter granulation. These substances should be avoided or
be removed in a pretreatment.
· Toxic substances should be avoided. If toxic wastewater is to be
treated, the inoculum microbes should be well acclimatized.

surface tension model. Structural models comprise the Capetown’s

model, spaghetti theory, syntrophic microcolony model, multi-layer
model, cellular automaton model, and the cell-to-cell communication
model. All these models, including the proton translocation-dehydration
theory, have been fully explored by Liu et al. (2004) and more
recently by Show et al. (2020).
 Sustainability and Future of Anaerobic Wastewater Treatment 347

Granulation comprises a number of stages — it shares the first two

with flocculation, while the last two require specific operating condi-
tions, relating in particular to the feeding regime and sheer stress as
described above.
Initially, bacteria have to make contact with each other by passive
or active movement and become attached by physicochemical forces
(e.g., electrostatic, van der Waals forces, hydrophobic effect, etc.),
which are reversible. As bacteria are generally negatively charged, they
have a hydrophobic surface. They also possess fimbriae or attachment
pili, which are different from flagella, which are used for locomotion,
being both thinner (3–8 nm) and shorter, and also very numerous
(up to 1,000 per cell). Fimbriae are also important in adhesion to
conditioned inert surfaces as well as in the formation of biofilms
(Lettinga et al., 1980; Hulshoff-Pol, 1989), probably by overcoming
the electrostatic repulsion between the cell wall and medium surface.
Their role in floc formation is thought to be similar to attaching to
inert particles or fibers, as well as other cells. However, the surface
properties of individual bacterial cells, such as the presence of EPS,
lipo-polysaccharides, proteins, and mycolic acid, all increase hydro-
phobicity and hence adhesion potential (Wilschut and Hoekstra,
1984).
EPSs are not food reserves, like poly-b-hydroxybutrate, and are
not easily decomposed. EPS is a polymer that can be composed of a
number of organic compounds, such as polysaccharides, amino poly-
saccharides, and proteins (Sato and Ose, 1980). Lipids may also be
present with minor amounts of nucleic acids and other biopolymers,
but the exact nature of these flocculating polymers will depend on the
species of bacteria. Each polymer will have varying surface properties
and charges that will influence not only the settling characteristics but
also, importantly, the water-binding properties of the floc (Wilén
et al., 2003). The polymer not only gives the floc components cohe-
sion and an open porous structure, but it also allows suspended par-
ticles in the wastewater to bind to the floc by adsorption. Cations and
anions, including phosphorus and a range of pollutants and toxic
compounds, are also adsorbed by such polymers. The EPS matrix is a
dynamic system that enables cells in flocs to function in a manner
348 Anaerobic Wastewater Treatment

similar to multicellular organisms. They have unique sorption proper-

ties and Decho (2000) has suggested that the sorption of heavy met-
als may be a strategy to protect the bacteria against toxic effects.
The next stage is the irreversible attachment of the bacteria to
each other, which is achieved by the secretion of EPS that creates
strong chemical bonds between the cells. The bacteria continue to
produce EPS, creating a matrix in which bacteria can divide and
new bacteria are attracted and attached, thus creating an active floc.
It is at this stage that methanogens start to become enmeshed into
flocs.
The bonding of particles in solution and the formation of
agglomerates can be explained by Derjaguin–Landau–Verwey–
Overbeek (DLVO) forces (van der Waals and electrostatic forces),
non-DLVO forces (bridging, hydrophobic forces), and physical
enmeshment. So, flocs are held together largely by electrostatic
forces. At neutral pH, divalent cations, in particular Ca2+, bridge the
negatively charged functional groups on EPS produced by the floc-
forming bacteria, which are referred to as the divalent cation bridging
theory (DCBT), enhancing floc aggregation and structural stability.
This bridging also occurs between other materials enhancing enmesh-
ment (Higgins et al., 2004b) (Fig. 9.4).

Covalent bonds
(e.g., linking of glycoprotein subunits)

Hydrogen bonds
(e.g., linking of polysaccharide chains)

Van der Waals forces

(e.g., linking of polysaccharide chains)
Ionic bonds
(e.g., linking with charged ions)

Intermingling
(e.g., entanglement of large
molecules)

Hydrophobic interacons
(e.g., interacon of lipohilic groups)

Figure 9.4. Representation of attachment and aggregation processes (Tansel,

2018). Reproduced with permission of Elsevier Publishing.
 Sustainability and Future of Anaerobic Wastewater Treatment 349

The importance of divalent cations in floc formation, strength,

and settleability is well known with calcium ions, which are preferen-
tially bound to EPS, having an important role in floc formation and
size through the construction of calcium bridges (Schmidt and
Ahring, 1996; Schmidt and Ahring, 1994; Shen et al., 1993). High
concentrations of sodium ions in wastewaters can displace the divalent
cations from within the floc, reducing bridging and leading to the
weakening of the floc structure (Bruus et al., 1992: Higgins and
Novak, 1997a) (Fig. 9.5), with flocculation efficiency that can be

Figure 9.5. Within the activated sludge floc, Ca2+ ions create bridges that can be
displaced by Na+ ions, weakening the floc and leading to possible deflocculation.
Within each floc are two pools of calcium, Ca2+, involved in bridging (DCBT-Ca) and
precipitated calcium solids. Al3+ exchanges with DCBT-Ca while Ca-solids are dis-
solved at low pH (Peeters et al., 2011). Reproduced with permission of Elsevier
Publishing.
350 Anaerobic Wastewater Treatment

estimated by the ratio of monovalent to divalent cations in meq L–1,

known as the M+/D++ ratio (Higgins et al., 2004a). The total quan-
tity of Ca2+ ions in flocs is split between those required for bridge
formation (i.e., DCBT-Ca) and precipitated Ca-solids that become
enmeshed within the floc. Total Ca2+ in flocs varies over time, with
approximately half the DCBT-Ca fraction depending on the actual
sludge and wastewater characteristics (e.g., whether the wastewater
arises from a hard or soft water catchment) (Bruus et al., 1992).
Figure 9.5 shows the nature of divalent bridging in a floc and that at
low pH, Ca-solids are leached from flocs. At low pH with Al3+ ions
present, both exchangeable (DCBT-Ca) and enmeshed (Ca) solids
are leached.
Once this initial agglomeration of cells is complete, the floc then
begins to go through a series of developmental stages. Early develop-
ment is often referred to as expansion, with the floc becoming
increasingly larger and developing a unique structure. While flocs can
still attract new bacteria, both the same and different species, most of
their growth is by cell division. Unlike biofilms, the structure that
develops is largely spherical and simple. This is not random but
depends again on cells communicating with each other through QS.
The voids have an important function, allowing nutrients to penetrate
from the liquid phase deep into the center of the floc and, at the same
time, allowing methane and other gases, as well as biodegradable
materials, to be removed. It is assumed that only the bacteria in the
outer surface layer actively reproduce, while those within the floc
remain viable and actively metabolizing. If this is not the case, then
the floc or granule would continue to grow from the inside out, forc-
ing it to break up. Adsorption of organic and inert material takes place
both on the EPS matrix as well as on adsorption sites on the bacterial
cell walls. In this way, the EPS matrix is creating a digestive system,
to quote Flemming and Wingender (2010), retaining extra-cellular
enzymes within the floc where they can release metabolized material
for absorption by other bacteria and archaea. This is just one example
of how the floc community works together, creating a massive advan-
tage over those microorganisms that live as individuals, with up and
down-regulation of genes controlling the release of gene products,
 Sustainability and Future of Anaerobic Wastewater Treatment 351

which govern the actions of individuals or layers of bacteria pivotal in

community organization.
Granular sludge comprises much larger, denser, and rounded
flocs, referred to as granules. The use of inert particles or nucleating
agents to increase the rate of granulation and to increase stability has
had some success. These include the addition to the substrate of metal
ions (Ca2+, Mg2+, and Fe3+) and carriers, such as biochar and nanopar-
ticles. Biochar stimulates EPS secretion, thus promoting granulation
(Zhang et al., 2017b). However, granulation can be achieved without
the use of any nucleating agents. The main difference between flocs
and granules is the larger amount of EPS produced in granules, which
also has a stronger adhesive nature. Under normal operation condi-
tions, the EPS in granules is found as a gel, which is not the case with
normal flocs (Seviour et al., 2009), with the hydrogel matrix key to
the greater strength and stability of granules.
The precise mechanism of granule formation is not fully under-
stood, although Nancharaiah and Sarvajith (2019) have explored the
current theories, including the role of QS, where granulation and
granule stability appear related to ATP-dependent N-acyl-homoserine
lactone (AHLs). Current research is increasingly highlighting the role
of QS and signal molecules in promoting and accelerating the synthe-
sis of extracellular polymers and improving the surface attachment
capability of microorganisms (Sarma et al., 2017; Zhang et al., 2019),
both critical attributes for successful granulation and granule stability
(i.e., the AHL-based granulation strategy).
Steps 3 and 4 are unique to granulation and require specific oper-
ating conditions, namely, a feast-famine regime associated with plug
flow systems, which encourages the secretion of EPS that creates
aggregates and a sufficient up-flow velocity. Granulation requires a
separation stage, usually built into reactors, which allows weaker-
formed flocs to be lost with the final effluent, while the denser flocs
are retained due to the more rapid terminal settling velocities (Qin
et al., 2004). High up-flow velocities subject aggregates to a high-
shear stress environment within the reactor that helps to create the
dense granules. Therefore, correct operational conditions are critical
not only for granularization but to sustaining the granules in their
352 Anaerobic Wastewater Treatment

optimal condition. The process of granulation and the parameters

affecting it are discussed by Hamza et al. (2022). The addition of Ca2+
mg and Al2+ ions has been shown to help granulation in UASBs,
although excessive Ca concentrations (>500 mg L–1) have the oppo-
site effect (Yu et al., 2001).
The surface nature and shape of granules alter as the sludge
matures. Initially, they have rough, irregular shapes that gradually
become smother, increasingly spherical, and develop a fine-grained
porous structure. This is shown by Xiao et al. (2023), who, by study-
ing granulation in detail using scanning electron microscopy, were
able to show the transition from flocs to mature granules. Initially, the
seed flocs comprised filaments that became small compact granules
after 40 days of operation. By day 190, the granules were larger with
discernible pores, allowing biogas to escape — the filamentous bacte-
ria had disappeared after day 40 but returned by day 190 on the
surface of the granules, which were largely composed of cocci and
rod-shaped cells. The authors speculated that the filaments acted as
docking stations for other microbes as well as giving granules struc-
tural stability, although their role in adsorption, as seen in filaments,
is also a possibility. The fluorescent staining shows that the cells
within the granules remained active (Fig. 9.6).

Quorum sensing
Much research has been done on the role of quorum sensing (QS), a
molecular communication system, in the creation of complex micro-
bial structures (i.e., biofilms, flocs, and granules) with a high degree
of organization and cooperation between bacterial cells. Bacteria can
sense the density of other cells close by, and when a threshold number
(or quorum) is achieved, they begin to work together, using a range
of physicochemical and biological processes to form a floc, biofilm, or
granule.
QS is a continuous process where individual bacteria produce
signaling molecules or autoinducers (AI) that diffuse through the cel-
lular membrane into the liquid phase. So, the higher the density of
bacteria in an area, the higher the concentration of AI molecules
 Sustainability and Future of Anaerobic Wastewater Treatment 353

(a)

(b)

(c)

(d)

Figure 9.6. Scanning electron microscopy (SEM) and confocal laser scanning
microscopy (CLSM) images of anaerobic sludge granules at different stages. (a) Seed
sludge. (b) Granular sludge on day 40. (c) Granular sludge on day 190. (d) CLSM
images of granular sludge on day 190 stained with live (green) and dead (red) stain-
ing kit; D1 — outer layer of the granules; D2 — the inner part of granules; and
D3 — the composite 3-D image of granules (Xiao et al., 2023). Reproduced with
permission of Elsevier Publishing.

released, which eventually triggers a collective behavioral change in all

the bacteria. The secreted AIs are a mixture of homoserine lactones
and specific peptides, which control specific repressor or activator
sequences in the bacterial DNA responsible for the production of
mRNA. This forms new protein, which includes, among others, the
genes for the formation of a floc or other structure. Where this gene
is absent, then an organized floc is not produced.
Not all bacteria produce AIs or chemical signals responsible for
cell-to-cell communication, although over 100 Proteobacteria species
354 Anaerobic Wastewater Treatment

are able to produce the QS molecule AHL thought responsible for

flocculation, EPS production, and general intraspecies communica-
tion (Tan et al., 2014). Details of all the common QS AIs produced
by bacteria have been summarized by Maddela et al. (2019), includ-
ing AHLs, autoinducing peptides (AIPs), autoinducers-2 (AI-2),
pseudomonas quinolone signal (PQS), diffusible signal factor (DSF),
and autoinducers-3 (AI-3). AHLs are primarily produced by Gram-
negative bacteria, as are DSF. AIPs are produced by Gram-positive
bacteria, while AI-2 signaling molecules are found in both Gram-
positive and negative bacteria. The high density of microorganisms in
wastewater treatment systems results in high concentrations of these
chemicals and of AHLs in particular (Chong et al., 2012), with the
highest concentrations found within and around the aggregated bac-
teria, indicating high QS activity (Song et al., 2014). Many com-
monly occurring wastewater bacteria are known to produce signaling
molecules, for example, Acinetobacter, Aeromonas, Chitinimonas,
Citrobacter, Enterobacter, Klebsiella, Leclercia, Malikia, Raoultella,
Pseudomonas, and Serratia (Maddela et al., 2019), including many
obligate anaerobic bacteria and archaea such as Methanosaeta harun-
dinacea 6Ac (Li et al., 2015).
QS signaling molecules are produced by specific genes, and those
responsible for cell aggregation have been identified as BtaI1/BtaR1
(homologs of LuxI/LuxR), which mediate C8-HSL signaling
(Chandler et al., 2009). Producing signals are generated by AHLs
synthase genes (luxI homolog), while regulatory genes (luxR
homolog) perceive and respond to AHLs. The main signaling mole-
cules in anaerobic processes, which are very common, are AHLs AI-2
and DSF, with DSF mainly located within granules and AI-2 in the
liquid phase. Feng et al. (2014) noted that AHLs with long acyl
chains tended to diffuse into granules; those with short to medium
acyl chains were found in the liquid. Ma et al. (2018) examined ten
full-scale industrial anaerobic granular bioreactors and were able to
detect four different AHLs, with C8-HSL and C10-HSL both facili-
tating granulation through EPS synthesis, which they appear to con-
trol and were produced by 12 different microorganisms. These two
AHLs seem to be universally associated with granulation. Although
 Sustainability and Future of Anaerobic Wastewater Treatment 355

the ability to produce AHLs is not universal, it may be that the major-
ity of bacteria respond to them. Li et al. (2021) studied the recovery
of a UASB reactor after a shock loading by increasing the organic
loading rate (OLR) by stages and observing a change in microbial
community and endogenous AHLs during recovery. In particular,
C14-HSL improved aggregation through increasing protein synthesis
of EPS. The metabolism of acidogenic fermentation bacteria was
improved by C4-HSL, C8-HSL, and 3-oxoC14-HSL. Interestingly,
the authors concluded that 3-oxoC6-HSL was a key signal molecule
in enhancing methanogenesis.
The role of QS in anaerobic treatment has been reviewed in detail
by He and Zhu (2024), with their key functions in the regulation of
EPS secretion, promoting biofilm formation, acceleration of gran­
ulation, and the promotion of bacterial metabolism and symbiosis
(Fig. 9.7). AHLs play a significant role at every stage of digestion
from, increasing the activity of hydrolytic acid-producing bacteria,
thereby increasing acid production to regulate the expression of
methane-related genes in methanogens, increasing methane produc-
tion. Hue and Zhu use the term “controlling social behavior,” and
that is what the signaling molecules are doing, controlling the interac-
tions within the four stages of anaerobic digestion. They have also
compiled a useful list of QS studies in different anaerobic treatment
processes (Table 9.4). QS signals are disrupted by quorum quenching
(QQ) molecules, which effectively turn off that particular function
(Song et al., 2014). QS molecules have a significant impact on floc and
granule structure, composition, and functions, as do QQ molecules
with the development of operational strategies to utilize them at the
core of making anaerobic processes more efficient and sustainable.

9.2.2 Role of Metals

Trace metals
Trace elements and, in particular, metals are essential for optimal
functionality of microorganisms in the anaerobic process (Table 9.5a).
These and other micronutrients need to be present in sufficient
356 Anaerobic Wastewater Treatment

Figure 9.7. Graphical representation of key roles of quorum sensing in anaerobic

treatment (He and Zhu, 2024). Reproduced with permission of Elsevier Publishing.

quantities to support both growth and metabolic activity, although it

is essential that they are not only present but in a bioassimilable form
(Zandvoort et al., 2006). Methanogens are particularly sensitive to
metal concentrations being inhibited at either high or low concentra-
tions (Table 9.5b). The essential metals for anaerobes are Fe, Zn, Ni,
Co, Cu, Mn, V, Mo, Se, and W (Fermoso et al., 2009).
The most important and essential metal is Fe, which is used as a
co-enzyme, oxidation-reduction agent, and terminal acceptor, with a
positive correlation between the acetate utilization rate and iron
Table 9.4. Examples of signaling molecules and their specific functions in different systems based on case studies. Details of indi-
vidual case studies are given by He and Zhu (2024). Reproduced with permission of Elsevier Publishing.
Types of signaling
Systems Function Regulatory performance molecules References
Bacteria EPS Syntrophic and methanogenic bacteria can 3OC6-HSL, C10-HSL (Li et al., 2019)
bi-directionally regulate their EPS production

Sustainability and Future of Anaerobic Wastewater Treatment 357

through the QS system to construct a syntrophic
colony and improve the methane fermentation
efficiency of propionic acid and butyric acid.
Vital movement The methanogenic archaeon Methanosaeta — (Zhang et al.,
harundinacea 6Ac encodes an active community 2012)
sensing system for regulating cell assembly and
carbon metabolic fluxes.
Metabolism (1) C14-HSL dominates the phase I of SBOB, and (1) C4-HSL, C6-HSL, (Chen et al.,
C12-HSL dominates the phase IV of SBOB; C10-HSL, 3OC10- 2023; Li
C4-HSL dominated the whole growth process of HSL, C12-HSL, et al., 2019;
SEOB, and C12-HSL dominated the phase IV of C14- HSL, Zhang et al.,
SEOB; C6 dominates phase I and IV of SPOB, 3OC14-HSL 2019)
and C12-HSL secretion increases from phase I to
III; The concentrations of C4-HSL, C6-HSL,
and C12-HSL were the highest in the first two
phase of AM (phase I and II); C4-HSL, C10-
HSL, OXOC10-HSL, and OXOC14-HSL are the
highest in the first two phases of HM (phase I
and II), and C12-HSL is the dominant signaling
molecule in phase III and IV.a
(Continued)
 Table 9.4. (Continued)

358 Anaerobic Wastewater Treatment

Types of signaling
Systems Function Regulatory performance molecules References
(2) Intraspecies regulation was mediated by OXOC6- (2) 3OC6-HSL,
HSL and C10-HSL for SPOB and HM, C10-HSL
respectively. Metabolic coordination between
SPOB and AM was bi-directionally regulated
under the mediation of the enjoyment signaling
molecule, whereas metabolic coordination of
SPOB by HM was unidirectionally regulated.a
(3) C4-HSL can promote the growth of butyric acid- (3) C4-HSL
producing bacteria at 15 g L–1 NaCl.
Anaerobic EPS Adding C6-HSL, C8-HSL can increase the C6-HSL, C8-HSL (Lv et al., 2018)
granular concentration of TB-PN.
sludge
Metabolism (1) The addition of AHLs changed the microbial (1) C4-HSL, C6-HSL, (Lv et al., 2018;
community structure of bacteria and C8-HSL, 3OC6- Dang et al.,
methanogens to different degrees, and improved HSL, 3OC8-HSL 2022; Li
organic matter removal and methanogenesis. et al., 2021a;
(2) The addition of C6-HSL, C8-HSL, and 3OC6- (2) C4-HSL, C6-HSL, 2021b;
HSL induced the enrichment of actinomycetes C8-HSL, 2021c)
and changed the community structure; the 3OC6-HSL
addition of C6-HSL, 3OC6-HSL, and C4-HSL
promoted the potential methanogenic metabolic
pathways of granules.
(3) The addition of 3OC10-HSL, C6-HSL, and (3) 3OC10-HSL,
C8-HSL increased the abundance of acetyl coenzyme C6-HSL, C8-HSL
A and acetyltransferase in the system, resulting in a
more active fatty acid production reaction.
 Sludge (1) AI-2 promoted the formation of LE-EPS when (1) AHLs, DSF, AI-2 (Ding et al.,
granulation alkaline; AI-2 and DSF jointly regulated sludge 2015; 2016)
granulation when acidic, neutral, and weakly
alkaline.
(2) C4-HSL, C6-HSL, AI-2, and DSF jointly regulated
to maintain the stability of sludge particles.

Sustainability and Future of Anaerobic Wastewater Treatment 359

Calcification of C6-HSL and 3OC6-HSL can improve the C6-HSL, 3OC6-HSL (Li et al., 2021b;
granular calcification of anaerobic granular sludge and Fu et al.,
sludge reduce the production of calcium carbonate. 2022a;
2022b)
UAnMBR Bioabundance Ten types of AHLs were detected, with C10-HSL C4-HSL, 3OC4-HSL, C6- (Tabraiz et al.,
Reactor being the predominant AHL, followed by HSL, 3OC6-HSL, 2020)
C4-HSL, and C8-HSL. Biostatistical analyses C8-HSL, 3OC8-
showed that C10-HSL was associated only with HSL, C10-HSL,
bacteria, while the other types of AHLs were 3OC10-HSL, C12-
associated with both bacteria and archaea. HSL, 3OC12-HSL
AnMBR Metabolism (1) Starvation may lead to an increase in medium- (1) AHLs, AI-2 (Shah et al.,
Reactor and short chain AHLs and AI-2 during long-term 2022; Xiao
operation of the AnMBR. et al., 2019)
(2) Lower concentrations of AI-2 may reduce the (2) AI-2
occurrence of acetic acid and favor the occurrence
of hydrogenated methane, thus reducing the accum­
ulation of VFA and increasing methane production.
EGSB Metabolism and C4-HSL and C8-HSL enhance anaerobic sludge C4-HSL, C8-HSL,3OC6- (Yan et al.,
Reactor Sludge granulation by stimulating the secretion of HSL, 3OC14-HSL 2021b)
granulation proteins into EPS, and 3OC6-HSL and 3OC14-
HSL contribute to methanogenesis.
(Continued)
 Table 9.4. (Continued)

360 Anaerobic Wastewater Treatment

Types of signaling
Systems Function Regulatory performance molecules References
UASB EPS C14-HSL improves protein synthesis in EPS. C14-HSL (Li et al., 2021a)
Reactor
Sludge (1) C6-HSL can increase the abundance and (1) C6-HSL (Zhang et al.,
granulation metabolic activity of acetate crumb methanogens, 2022b; Ma
promote EPS secretion, and reassemble et al., 2018a,
decomposed sludge into large particles. 2018b)
(2) C8-HSL and C10-HSL were involved in EPS (2) C8-HSL, C10-HSL
production and granulation process.
Metabolism (1) C4-HSL, C8-HSL and 3OC14-HSL all promoted (1) C4-HSL, C8 HSL, (Li et al., 2021a;
the metabolism of acid-producing fermenters; 3OC14-HSL Yan et al.,
3OC6-HSL promoted methanogenesis. 2021b)
(2) The dominant signaling molecule C4-HSL, which (2) C4-HSL, 3OC6-
promotes extracellular polysaccharide synthesis HSL, 3OC12-
and aspects of acidogenic metabolism, regulates HSL, C14-HSL
metabolism at low alkalinity (700–1,800 mg L–1); 3 C6-HSL,
3OC6-HSL, 3OC12-HSL, and C8-HSL, C10-HSL
C14-HSL regulate metabolism at alkalinity
of 1,800–2,200 mg L–1.
(3) The introduction of C6-HSL, C8-HSL, C10- (3) OC6-HSL
HSL and 3OC6-HSL signalling molecules in
UASB increased the overall number of bacteria
and archaea and favored methanogenesis.
a
Phase I is the lag phase, Phase II is the logarithmic phase, Phase III is the third phase, and Phase IV is the logarithmic phase. SPOB — syntrophic
propionate-oxidizing bacteria, SBOB — syntrophic butyrate-oxidizing bacteria, SEOB — syntrophic ethanol-oxidizing bacteria , HA — homoace-
togens, AM — methanogens including aceticlastic methanogens, and HM — hydrogenotrophic methanogens.
Table 9.5. (a) The application of trace metals in anaerobic processes. (b) The optimum, stimulatory, and inhibiting concentrations
of trace elements and their corresponding metal-rich enzymes on key anaerobic microorganisms (Nakhate et al., 2023). Reproduced
by permission of Elsevier Publishing.
(a)
TEs Anaerobic digestion Stimulatory Inhibitory Conclusion
Fe Promotes organic degradation, stabilizes food 1.4–2.3 g/L NA Stimulation for substrate hydrolysis

Sustainability and Future of Anaerobic Wastewater Treatment 361

waste, and reacts with H2S to form Fe-S
Ca Enhances methane production, prevents 120–200 mg/L 7,000–8,000 mg/L Helps in maintenance of microbial
foaming and granulation of anaerobic community
sludge
Mg Enhances methane production and prevents 720 mg/L 300–400 mg/L Inhibits acetoclastic methanogenesis
foaming at a higher concentration
Co Cofactor of methyltransferase and carbon 0.02 mg/L >250 mg/L Supply in trace amount is beneficial
monoxide dehydrogenate (CODH) Boosts Suppress
acidogenesis methanogenesis
Ni Component of cofactor F430 required for 0.5uM –0.1 mg/L 1 mg/L Fix VFA accumulation problem
methyl reductase activity during the last VFA oxidation Hampers
step of methanogenesis acetoclastic
methanogenesis
Ni and Stimulate both biogas production and Ni: 0.6 mg/kg and Ni: 250 mg/L Co: Acetoclastic methanogenesis
Co methane content of biogas Co: 0.05 mg/kg 250 mg/L pathway
Se and Tolerate high ammonia concentration and NA 6 ng Se/g COD and Accelerate microbial metabolism
W preventing VFA accumulation 14 ng W/g COD
Cu Cofactor for microbial enzymes and coenzymes 10 mg/L 30–50 mg/L Enhances conversion of organics
involved in the methane production
Mo Stable reactor operation at high OLR 0–1.6 mg/L NA Essential micronutrient for
methnogens

(Continued)
 Table 9.5. (Continued)

362 Anaerobic Wastewater Treatment

(b)
Optimum /
Trace Stimulatory Inhibitory
element Steps Microbes concentration concentration Enzymes
Ni Acetogenesis Syntrophs 0.03–27 mg/L 35–1,600 mg/L CODH, Methylreductase,
Hydrogenase
Methanogenesis Methanosarcina barkei 0.0059 mg/L NA MCR/F430
Methanobacterium smithii 0.059 mg/L NA
Methanobacterium bryantii 0.96 g/L NA
Co Methanogenesis Methanococcus voltae 0.001 g/L 0.1289 g/L Methyltransferase /Factor III
Fe Acetogenesis and NA <0.33 mg/L NA Formylmethanofuran
methanogenesis dehydrogenase, CODH, ACS,
Hydrogenase
Methanogenesis Methanobacterium 0.073 g/L NA Formylmethanofuran
thermoautotrophicum dehydrogenase
Mo Methanogenesis Methanobacterium >0.002 g/L NA Formylmethanofuran
autotrophicum dehydrogenase
Methanosarcina barkeri 0.048 mg/L NA Formylmethanofuran
dehydrogenase
Methanospirillum hungatei 0.0064 g/L NA FDH
Se Methanogenesis Methanococcus voltae 0.64 g/L NA NA
Methanococcus maripaludis 0.0157 g/L NA NA
 Methanococcus vannielii 0.79 g/L NA NA

w Methanogenesis Methanococcus vannielii 0.75 g/ L NA Formylmethanofuran

dehydrogenase
Mo Methanogenesis Syntrophs 0.79 mg/L NA FDH
Se — — — — —

Sustainability and Future of Anaerobic Wastewater Treatment 363

W — — — — —
NA: Not Available.
364 Anaerobic Wastewater Treatment

availability, as well as being essential for methanogen growth. Cobalt

is often cited as the limiting metal due to its low concentration in
most wastewaters and sludges, with all methanogens requiring cobalt,
nickel, and iron. The role of cobalt and other major metals are sum-
marized in Table 9.6.
While anaerobic wastewater treatment processes can be inhibited
by excessive concentrations of metals, cellular function, and, in

Table 9.6. Role of some essential trace metals in the production of key enzymes
required in anaerobic metabolism (Fermoso et al., 2009). Reproduced by permission
of Elsevier Publishing.
Element Functions Element Functions
Cu • Superoxide dismutase Ni • CO-dehydrogenase
• Hydrogenase (facultative • Acetyl-CoA synthase
anaerobes) • Methyl-CoM reductase
• Nitrite reductase (F430)
• Acetyl-CoA synthase • Urease
• Stabilize DNA. RNA
• Hydrogenase
Co • B12-enzymes Se • Hydrogenase
• CO-dehydrogenase • Formate dehydrogenase
• Methyltransferase • Glycin reductase
Fe • Hydrogenase W • Formate dehydrogenase
• CO-dehydrogenase • Formylmethanofuran-
• Methane monooxygenase dehydrogenase
• NO-reductase • Aldehyde-oxydoreductase
• Superoxide dismutase • Antagonist of Mo
• Nitrite and nitrate
reductase
• Nitrogenase
Mn • Stabilize methyltransferase Zn • Hydrogenase
in methane-producing • Formate dehydrogenase
bacteria • Superoxide dismutase
• Redox reactions
Mo • Formate dehydrogenase V • Nitrogenase
• Nitrate reductase • Chloroperoxydase
• Nitrogenase • Bromineperoxydase
 Sustainability and Future of Anaerobic Wastewater Treatment 365

particular, biogas production can be affected when they are in short

supply or not in an assimilable form. For example, Fermoso et al.
(2008a) demonstrated how a low assimilable Ni concentration inhib-
ited methanogenic activity in a UASB reactor. They observed that this
could be rectified by using a metal supplement. However, this was not
the case when Zn was the limiting metal, with a supplement failing to
readjust the pH caused by excessive acidification due to reduced
methanogenic activity (Fermoso et al., 2008b). For supplements to
work effectively, they must be in a bioavailable form, which depends
on the metal speciation. This requires the metals to present as free
forms or bound to organic ligands, and must be added at a suitable
rate so that they are not precipitated or removed by complexation
before they can be utilized by the microorganisms (Roussel, 2012).
Metal solubility in reactors is affected by the pH, with solubility
increasing as the pH falls, as well as affecting the retention of metals
in the biofilm or granules. The most used inorganic form of metal
supplement is either as a metal chloride or sulfate due to their solu­
bility, but they can be readily precipitated by the hydrogen sulfide
present into insoluble metal sulfides. Metals can also be effectively
removed by phosphate or carbonate precipitation or biomass chela-
tion. By using a metal-organic compound as a supplement, precipita-
tion by hydrogen sulfide is largely prevented, although the metal is
less readily available to microorganisms.
Three chelators (i.e., aminopolycarboxylic chelating ligands) are
often used to maximize metal bioavailability: ethylene diamine tetra
acetic acid (EDTA), ethylenediamine-N N’-disuccinic acid (EDDS),
and nitrilotriacetic acid (NTA) (Chauhan et al., 2015; Zhang et al.,
2015). For example, cobalt was found to be limiting in a two-stage
anaerobic digestion system by Li et al. (2023a). While the acidogenic
phase was unaffected, the methanogens were inhibited by the lack of
Co2+. The optimum concentration of 2 mg L–1 was restored by using
the chelating agent EDDS for Co2+ addition, which increased the
levels of both vitamin B12 and the important methanogen coenzyme
F420, restoring normal metabolic activity and biogas production.
366 Anaerobic Wastewater Treatment

The abundance of the key methanogens Methanofollis and

Methanosarcina were enhanced.
In order to optimize anaerobic treatment processes, especially in
terms of biogas production, optimum nutrient balances must be
maintained in reactors, which is especially important with industrial
wastewaters, which are generally deficient in one or more micronutri-
ents or trace elements. The behavior of metals in anaerobic digesters
has been the subject of an interesting study by Jimmy Roussel at
Birmingham University and gives an insight as to how metal supple-
ments work (Roussel, 2012), while Zandvoort et al. (2006) give an
excellent overview of the importance of trace metals in enzyme reac-
tions, metal speciation, and mobility (Fig. 9.8) and the need for sup-
plement dosing.

Figure 9.8. Schematic representation of metal speciation in granular anaerobic

sludge as found in UASB reactors (Zandvoort et al., 2006). Reproduced with per-
mission of John Wiley and Sons.
 Sustainability and Future of Anaerobic Wastewater Treatment 367

Heavy metal recovery

Using sulfate reducing bacteria (SRB) to recover metals from waste-
waters is set to play an important role in safeguarding our future sup-
plies of important metals as well as protecting the environment from
heavy metal pollution. Recovering and the reuse of useful materials,
in this case, metals, from waste streams is a central strand in the cir-
cular economy concept. The key advantage to using anaerobic treat-
ment in the recovery of metals is that even at very low concentrations,
they can be effectively recovered, producing highly insoluble salts at
low capital and operating costs.
SRB use SO42– as a terminal electron acceptor under anaerobic
conditions to oxidize simple organic compounds (CH2O), which act
as an electron donor:

2CH2O + SO42– → H2S + 2HCO3(9.3)

The hydrogen sulfide produced reacts with dissolved metals (M2+)

to form a metal sulfide, which is highly insoluble and so readily pre-
cipitates out of solution.

H2S+ M2+ → MS + 2H+(9.4)

The reaction is completed, causing an increase in alkalinity.

HCO3– + H+ → CO2 + H2O(9.5)

The pH is important not only for successful sulfate reduction but

also for metal recovery, as different metals precipitate at specific pHs
(Table 9.7), leading to selective metal recovery. The effective recovery
of the metal sulfide precipitate is also important, as they can naturally
contain metal sulfide particles of various sizes, including those that fall
into the NP size range. These metallic NPs (MeNPs) are very small,
have a large surface area, and are very reactive, making them very toxic
to a wide range of organisms. Polycrystalline metal NPs are generally
different in their size range for each metal: FeS and PbS <10 nm,
CdS and CuS <14 nm, and NiS and ZnS <17 nm, with smallest parti-
cles produced when treating acid mine drainage (AMD) 2–10 nm.
368 Anaerobic Wastewater Treatment

Table 9.7. Effective pH for

metal recovery by precipitation
by hydrogen sulfide.
Metal pH
Fe ~6
Cu 2.8 and 3
Zn 5.5–6.0
Ni 7.5–8.0
Cd 4.5–5.0
Pb 7.5–8.5
Mn 9.42 and 10.34

However, metal sulfide NPs have a wide range of potentially very useful
uses, including the manufacture of electronics (e.g., batteries, LEDs,
solar cells), the use in environmental remediation, biomedicine (e.g.,
drug therapy, fluorescent probes), antifungal agents in agriculture,
electroplating, and catalytic reactions, such as water splitting, to gener-
ate hydrogen (Table 9.8) (Kumar et al., 2021; Carmona et al., 2023).
A wide range of reactors can be used, both attached and sus-
pended biomass systems, with sulfate reduction and metal precipita-
tion occurring together in single reactor systems, while in multi-stage
systems, the two processes can be separated, making the recovery of
metal sulfide precipitation easier and more effective (Fig. 9.9). Kumar
et al. (2021) compared the various reactor types and found inverse or
down-flow fluidized beds (AnIFBs) the best in terms of metal recov-
ery (Table 9.9). In a study by Villa-Gomez et al. (2011), a single-
stage AnIFB was able to recover between 91 and 99% of the common
wastewater metals from an influent containing just 5 mg L–1 and at a
COD/sulfate ratio of 1 at neutral pH. The problem of single-stage
systems is that heavy metals can inhibit the SRB as they can only toler-
ate short-term exposure.

9.2.3 Membrane Technology

One of the drawbacks of anaerobic wastewater treatment is the need
for tertiary treatment, either in the form of aerobic treatment and
Table 9.8. The synthesis method, size, and shape and the main applications of metal sulfide nanoparticles (kumar et al., 2021).
Reproduced by permission of elsevier publishing.
Metal sulfide
Nanoparticles
(MeS NPs) Synthesis methods Size and shape Applications
Iron (Fe) Surfactant assisted hot-injection method Cubic shape with good uniformity Photovoltaic devices, labeling of

Sustainability and Future of Anaerobic Wastewater Treatment 369

(chemical synthesis), high-energy and size distribution in the biological materials, transformer
milling (mechanical method) range 60–200 nm core materials, environmental
remediation and deactivation of
radioactive heavy metals
Zinc (Zn) Lewis acid-base reaction (chemical Spherical crystals with an average In vitro imaging, light emitting diodes,
synthesis), Thio-urea hydrolysis diameter of 5.5 nm bioactive coatings, fuel cells
(hydrothermal synthesis), metal-
organic framework strategy
Copper (Cu) Biological synthesis, sonochemical Different types — spherical particles Dye degradation, solar cells, immuno­
synthesis using ultrasonicator, in-situ of average size 4 nm; triangular sensors, drug-therapy, agriculture,
growth by solvothemral method, plates of average size 12 nm; lithium ion batteries, wound
microwave irradiation, surfactant- distorted spherical to oval shape dressings, catalyst (click reaction),
based synthesis, enzymatic treatment with slight aggregations and size air filtration technology
(green synthetic method) 8–14 nm.
Cadmium (Cd) Chemical precipitation method, Varies from a cubic to hexagonal Solar cells, fluorescent probe, water
sonochemical method. crystalline structure, with size purification
ranging from 2.4 nm to 6.2 nm
Lead (Pb) Chemical precipitation method, Uniform nanowires of average Solar cells, telecommunications
ultrasonic irradiation. diameter 40 nm and length 5m.
Nickel (Ni) Hydrothermal synthesis, solvothemral Sphere-like structure with an Dye-sensitized solar cells, infrared
method using a domestic microwave average crystallite size of 22 nm detectors.
oven.
370 Anaerobic Wastewater Treatment

(a)

(b)

Figure 9.9. Schematic layout of single and multistage systems for metal sulfide
recovery from wastewater (Kumar et al., 2021). Reproduced with permission of
Elsevier Publishing.

Table 9.9. Advantages and disadvantages of anaerobic reactors for biological sul-
fate reduction (Kumar et al., 2021). Reproduced by permission of Elsevier Publishing.
Bioreactor type Advantages Disadvantages
Continuous stirred Consistent and reliable High solids retention time
tank reactor performance (SRT) requires a large
reactor volume;
biomass washout
Gas lift reactor High mass transfer efficiency, High pressure needed to
very good mixing pump gas inside the
reactor
Packed bed High SRT results in lower Clogging; high pressure
reactor reactor volume than CSTR; for pumping the flow
can be operated in up-flow
or down-flow mode
Membrane No need of sedimentation High cost to overcome
bioreactor basin; high biomass the trans-membrane
retention results in high pressure; requires
substrate degradation rate; periodic backwash of
avoids direct contact the membrane
between metals and SRB
 Sustainability and Future of Anaerobic Wastewater Treatment 371

Table 9.9. (Continued)

Bioreactor type Advantages Disadvantages

Up-flow anaerobic No clogging; good biomass Biomass washout; high
sludge blanket settling capacity; no susceptibility to
(UASB) requirement of support influent characteristics
reactor material
Bubble column Good mixing; used for turbid Not available (NA)
reactor medium and shear sensitive
cells
Fluidized bed No channeling or clogging, Energy needed for carrier
reactor biomass retention is good; fluidization; shear
biomass activity and force can detach
treatment efficiency are biomass; less volume
high available for biomass
compared to the
UASB reactor due to
the inert biomass
carrier
Anaerobic filter Low shear force; sludge Channeling of flow can be
reactor retention time is more than possible; pressure
that for CSTR; H2S gradients can be large
stripping is effective in
downflow mode
Anaerobic digester Excellent biomass holding Biomass disintegrates into
capacity as compared with flocs and sludge
the CSTR
Anaerobic hybrid Less vulnerable to clogging Not available
reactor compared with the AFR;
sludge removal easier than
in the AFR; biomass
retention better than in the
UASB
Anaerobic baffled High sludge retention time; Not available
reactor reduced costs due to no
biomass carrier; good
tolerance toward shocks of
hydraulic and organic
loading
372 Anaerobic Wastewater Treatment

sedimentation or filtration. Membrane technology has improved

the efficiency of anaerobic treatment by employing membranes to
retain solids and biomass within the reactor while also ensuring a
high-quality final effluent (i.e., permeate), which is particle-free with
extremely low pathogen and antibiotic gene concentrations (Ozgun
et al., 2013). By separating the SRT from the HRT, membranes can
significantly reduce treatment times, required reactor volume, start-
up times, and improve final effluent quality. They can also be used for
upgrading biogas. AnMBRs are ideal where slow-growing microor-
ganisms are utilized, especially where municipal wastewaters are
treated where its low strength limits growth, and for treating inhibi-
tory contaminants (Ji et al., 2020; Li et al., 2023b).
AnMBRs were developed in the 1980s and employ either ultrafil-
tration or microfiltration membranes within the bioreactor, often as
a submerged hollow fiber membrane or as a crossflow membrane at
the effluent outlet; alternatively, both can be used as a separate side
stream process (Fig. 9.10). In crossflow systems, the liquid flows

Biogas scouring Biogas

Effluent Biogas
Effluent
Influent Influent
Biogas recycle for
membrane scouring
Membrane

(a) (b)

Biogas

Influent Effluent

Membrane

(c)

Figure 9.10. There are three common anaerobic membrane reactor configura-
tions: (a) the membrane is submerged within the reactor, (b) external side stream
hollow filter membrane, or (c) an external cross flow membrane (Chang, 2014).
Reproduced under the Creative Commons Attribution (CC BY) license (https://
creativecommons.org/licenses/by/4.0/).
 Sustainability and Future of Anaerobic Wastewater Treatment 373

along the membrane surface at high velocity, creating sufficient shear

to control fouling and for the permeate to pass the membrane. These
are generally used with UASB and AnCSTR reactors and require extra
energy for pumping. In submerged systems, the membrane is located
within the reactor, with a vacuum used to draw the permeate, which
reduces energy consumption. Three membrane types are used: tubu-
lar, hollow-fiber, and flat sheet membranes, all of which come in the
form of cassettes or units, which are easier to handle. In this case,
fouling is controlled by scouring using biogas (Fig. 9.10). The types
of membranes available are fully explained in an earlier volume in this
series (Gray, 2023).
All wastewater anaerobic bioreactors can employ membrane sepa-
ration, including sludge digesters treating sewage sludges from con-
ventional aerobic treatment (Liao et al., 2006). However, one of the
limiting factors in AnMBR technology is the fouling of the mem-
branes, preventing the passage of the permeate, with increased energy
costs as permeability declines. Soluble microbial products and EPS are
both key factors in membrane fouling, both becoming more prob-
lematic at lower temperatures. While liquid or biogas scouring is
generally sufficient, occasional chemical cleaning may be necessary.
While AnMBRs can be operated successfully at ambient tempera-
tures (5–30oC), seasonal temperature fluctuations have been reported as
causing operational difficulties, with both COD removal and methane
yield falling as temperatures fall. The reported performance of AnMBRs
treating wastewater varies, with COD removal between 69 and 93%, a
methane yield of 0.07–0.244 L CH4 g–1 COD (Table 9.10a), and when
used for sewage sludge digestion, methane yields range from 0.19 to
0.31 L CH4 g–1 VS, with COD removal at 97–99% (Table 9.10b).
Upgrading biogas by converting the carbon dioxide present to
methane by introducing hydrogen to improve methanogenesis is con-
sidered highly sustainable and can be achieved in AnMBRs by improv-
ing hydrogen transfer to the liquid phase. This can be achieved using
submerged hollow-fiber or tubular membrane cassettes placed in the
digestate, although this method can result in the unwanted accumula-
tion of VFAs in the reactor due to excess hydrogen, so a separate unit
is more reliable but more expensive, both in capital and operating
costs (Fig. 9.6). This has been reviewed by Sun et al. (2022) and Li
et al. (2023b).
Table 9.10. Performance of anaerobic membrane reactors for (a) wastewater treatment and (b) sludge digestion (Li et al., 2023).

374 Anaerobic Wastewater Treatment

Reproduced under the Creative Commons Attribution license.
(a) Wastewater treatment bioreactors

System Scale Temperature CODinfluent HRT SRT Methane yield COD removal
configuration* (L) (°C) (mg/L) (h) (d) (L/g-COD) efficiency (%)
S-FS-AnMBR 40 18/24 428–477 48–12 50 — 69–89
S-HF-AnMBR 20 15 412 6–24 21–491 0.21–0.23 90.5
S-HF-AnMBR 1,650 25 762 11–21 100 0.2 92
ES-HF-AnMBR 34,400 10–27 755–1,403 25–41 70 0.07–0.17 93
S-HF-AnMBR 5,000 15–25 203–490 8 20–71 0.205–0.244 77–93
ES-TM-AnMBR/UASB 160 + 150 18 892 7–17 — 0.199–0.235 73–90
*S: submerged; ES: external submerged; FS: flat sheet; HF: hollow fiber; TM: tubular membrane.

(b) Sludge treatment systems

System Temperature OLR HRT COD

configuration* Scale (L) (°C) (g-COD/Ld) (d) SRT (d) Methane yield removal (%)
ES-HF-AnMBR/CSTR 15 35 1.72–3.72 15–30 25–60 0.239 L/g-VS 99.0
ES-HF-AnMBR/CSTR 15 55 4.31 15 — 0.31 L/g-VS 96.7
ET-AnMBR 500 24, 35, 55 3.4–3.7 6.9–7.3 22–39 0.19–0.34 L/g-VS —
ET-HF-AnMBR/CSTR 2.37 + 0.1 37 0.21–0.27 30–33 102–107 0.19–0.24 L/g-VS —
ET-CM-AnMBR/CSTR 25 35 & 55 4.8–10.4 3–7 30–50 0.213–0.245 L/g-COD —
*ES: external submerged; HF: hollow fiber; ET: external tubular; CM: ceramic membrane.
 Sustainability and Future of Anaerobic Wastewater Treatment 375

9.2.4 The Future

Conventional (aerobic) biological wastewater treatment treats the
majority of domestic wastewaters and a significant proportion of
industrial wastewaters with anaerobic processes, usually limited to
sludge digestion. Aerobic treatment has been the vanguard for over a
hundred years in the improvement of public health and environmen-
tal protection, especially in the improvement of river water quality.
However, the high-energy demand for aerobic processes and their
comparatively high-carbon footprint, combined with increasing
needs to better control nutrients and pathogens and to conserve
resources, including water, has led water companies to increasingly
look toward anaerobic wastewater treatment to provide a more sus-
tainable alternative. Also, the core change in the concept from a dis-
posal facility to a resource recovery center embracing the concepts of
the circular and green economies has forced us as an industry to alter
our traditional expectations of what wastewater treatment is. Physical
and chemical treatment methods remain core process options, but
how we deal with the biological treatment of wastewater, where we
employ microorganisms to breakdown the biodegradable fraction of
our waste streams, is where the main opportunities lie for the recovery
of carbon, nutrients, and many other resources, rather than simply
discharging them back into the environment as oxidized and still pol-
luting end-products (e.g., CO2, NO2, NO3, PO4).
Innovative use of combining different biological processes can
yield exciting results. For example, combining dark and photo fer-
mentation can produce high-quality effluents with nutrient removal
but capturing most of the available carbon as methane (Elsayad et al.,
2024).
A major advancement in anaerobic wastewater treatment is
the incorporation of membranes, which has made the treatment of
municipal wastewaters anaerobically a feasible option, although
limited post-treatment will still be required, especially for nutrient
removal. The main problem with AnMBRs is the energy balance, with
energy required in particular to control membrane fouling. Energy
demand by an AnMBR is estimated at 1–2 kWh m–3, although this
376 Anaerobic Wastewater Treatment

varies widely in the literature, depending on reactor configuration

and wastewater type. However, methane production will easily cancel
out this energy demand, usually with a significant surplus that can be
used elsewhere on-site or exported to the grid. Key problems with
membrane systems, apart from fouling, is the build-up of inhibitory
substances, including salinity (Aslam et al., 2022). One recent innova-
tion to control membrane fouling is to use a low-voltage electric field,
a process known as the electrochemical field-assisted anaerobic mem-
brane reactor (AnEMBR), which also improves pollutant removal and
overall methane generation (Hu et al., 2024).

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Figure 9.11. Comparison of performance efficiency of decentralized wastewater

treatment technologies, in terms of percentage removal. The anaerobic membrane
reactor (AnMBR) is compared to aerobic MBRs (AeMBRs), activated sludge (AS),
constructed wetland (CW), septic tanks (STs), anaerobic baffled reactors (AnBRs),
aerated fixed-bed reactors (AFBRs), septic tank-constructed wetland (ST-CW), and
stabilization ponds (SPs) using the following parameters: (a) TSS, (b) COD, (c)
BOD, (d) TN, (e) TP, (f) NH4+–N, (g) E. coli, and (h) fecal coliforms (Zhang et al.,
2023). Reproduced under the Creative Commons Attribution (CC BY) license
(https://creativecommons.org/licenses/by/4.0/).
 Sustainability and Future of Anaerobic Wastewater Treatment 377

Zhang et al. (2023) compared the performance of AnMBRs to a

range of other similar aerobic and anaerobic technologies (Fig. 9.11).
AnMBRs perform similarly to aerobic MBRs, except that AnMBRs
are unable to remove nutrients. In terms of the removal of suspended
solids, including pathogens, MBRs are exceptional at achieving
removal rates of 98–99%, compared to activated sludge (86%), con-
structed wetland (77%), septic tanks (84%), AnBRs (50%), aerated
fixed-bed reactors (89%), septic tank-constructed wetland (87%), and
stabilization ponds (67%). AnMBRs performed equally well as aerobic
MBRs in terms of COD (89% and 90%, respectively) and BOD
removal (91% and 92%). In terms of the loading rate, an AnMBR can
operate at 1.92 Kg COD m–1d–1, compared to 0.67, 0.56, and 0.8 for
aerobic MBRs, activated sludge, and AnBRs, respectively (Fig. 9.11).
It is estimated that between 3 and 4% of global electricity supplies
are consumed by wastewater treatment plants (WWTPs) being pri-
marily used for aeration systems, pumping, and sludge dewatering.
The objective of wastewater sustainability is to obtain net zero in
terms of energy consumption and, if possible, even become net posi-
tive. However, as effluent standards and loadings increase, more
energy is required. Carbon neutrality is only possible using natural
treatment systems or anaerobic processes, the latter being the only
option for centralized wastewater treatment systems. The primary
objective of sustainable wastewater treatment is to ensure that green-
house gases are not released directly from unit processes. For exam-
ple, carbon dioxide from aerobic processes or methane from anaerobic
digestion released to the air, or unstabilized organic matter discharged
to water or land, where it subsequently rapidly degrades (i.e., direct
carbon emissions). A secondary objective is to reduce carbon emis-
sions derived from energy consumption and other operational activi-
ties (i.e., indirect carbon emissions). Zhang et al. (2024) define a
carbon-neutral anaerobic-based WWTP as being “simplified in pro-
cess, such as one-stage or integrated design, and has a high removal
efficiency for different concentration levels, and should be capable
realizing energy self-sufficiency for either high-strength or low-
strength wastewater.” Among enhancement options suggested by the
authors are: (a) co-digestion to ensure an optimum C:N ratio.
Co-digestion can correct the substrate balance, optimizing pH,
378 Anaerobic Wastewater Treatment

alkalinity, and prevent excessive VFA production and free ammonia

nitrogen causing inhibition. (b) Ensuring adequate biodiversity of
bacteria and archaea, which may require the addition of a co-cultured
consortium of microbes, especially where industrial wastewaters are
treated. (c) The disadvantage of methane production is that carbon
dioxide is produced when it is utilized. This can be overcome by pro-
ducing hydrogen instead, which, when used, produces only water;
and finally, (d) wastewaters often contain recalcitrant organic matter,
which can be pre-treated using chemical addition to make them more
amenable to anaerobic breakdown by destroying the functional
groups within the organic molecules.
Biochar can also be added, which improves microbial activity by
providing a stable habitat, allowing high densities of bacteria and
archaea to thrive. Biochar also helps regulate the alkalinity within the
reactor (Wang et al., 2018) (Fig. 9.12). Conductive materials, both
iron and carbon-based, have also been shown to be effective in the
treatment of antibiotic wastewaters. Apart from biochar, conductive
materials include zero-valent iron, magnetite, activated carbon, gra-
phene, and combinations of iron and organic materials. They work by
ensuring rapid adsorption and biodegradation of antibiotics, as well as
reducing inhibitory effects and preventing the expression of antibiotic
resistant genes (ARGs). All these materials are also electron carriers,
which accelerate interspecies electron transfer, facilitating organic
matter breakdown and methanogenesis (Song et al., 2024; Zhao
et al., 2024).
Anaerobic treatment has many advantages, including the ability to
treat high organic loading rates, low production rates of stabilized
sludge, excellent potential for bioenergy and byproduct transforma-
tion, and more stable and reliable degradation capability as well as
requiring a fraction of the energy that a comparable aerobic system
would require (Table 9.11). We tend to think of nutrient removal as
requiring an aerobic step, but today, both nitrogen and phosphorus
can also be effectively treated using anaerobic technologies, including
anaerobic ammonium oxidation (anammox) and sulfur-based auto-
trophic denitrification (SAD) (Zhang et al., 2024) (Fig. 9.12).
Nitrogen removal is normally carried out using a combination
of aerobic and anoxic biological treatment and requires extra energy
 Sustainability and Future of Anaerobic Wastewater Treatment 379

(a)

(b)

Figure 9.12. Visual summary of (a) enhancement strategies for anaerobic treat-
ment and (b) carbon-neutral biological nitrogen removal technologies (Zhang et al.,
2024). Reproduced with permission of Elsevier Publishing.

for the aeration of wastewaters to achieve sufficient oxygen for the

nitrifying bacteria. Another source of global warming is caused by
incomplete nitrification — denitrification producing nitrous oxide, a
greenhouse gas three hundred times more powerful than carbon
dioxide. Strong wastewaters associated with anaerobic treatment
are often rich in protein amino acids, amines, and amides, all rich in
Table 9.11. Comparison of modern anaerobic wastewater treatment systems treating both low and high-strength wastewaters

380 Anaerobic Wastewater Treatment

at a range of different organic loading rates and hydraulic retention times (Zhang et al., 2024). Reproduced with permission of
Elsevier Publishing.
High-strength OLR (kg Removal Methane yield
Process wastewater COD (mg/L) COD/m3/d) HRT (h) COD (%) (L CH4/g COD)
UASB Swine 4,939–5,124 10.3–20.1 6–12 89.1–93.8 0.03–0.261
Distillery 21,000 5–33 2.67–0.54 (d) 84.52 ± 3.68 10 (L/L)
Slaughterhouse 3,500–32,000 1.75–16 24 68–95 0.04–0.28
Dimethylformamide 3,000 1.63–4.22 18–48 92–97 0.557–1.270 (L/L)
Cassava 18,000–20,000 15 N.A. 92.5 0.328
Tapioca starch 16,700 ± 3,503 7.5–25 N.A. 70–82 0.23–0.34 (biogas)
EGSB Cheese whey 46,348 ± 3,200 5.74–7.76 6–8 (d) 90–92 0.328–0.334
Cephalosporin 5,981–91,870 3.60–9.96 24–25 72 0.24–0.33
Corn alcohol 20,720 1.4 1.4–8 (d) 95.7 10.72 (L/L)
Winery 1,250–1,327 15.32 26 70 ± 2 0.31 ± 0.07
Leachate 4,909–7,200 14.8–21,8 8 70.8–91.6 0.07
Piggery 780–2,970 0.32–6.13 12 86.2 0.215–0.299
Tetracycline 6,000 5.06 24 67–79 0.099
IC Pharmaceutical 16,500–18,950 4.3–6.3 3–4 (d) 82–92 0.3
Dyeing 2,000–4,000 4.8–9.6 10 75–87 1.65 (L/L)
Synthetic molasses 5,000–12,500 2.50–18.94 0.66–2 85–90 0.44–4.49 (L/L)
 Medicine 11,000 14.67 18 95 0.33
Leachate 43,045–65,000 21.06–25.16 2.4 (d) 84.6–93.4 0.42–0.50 (biogas)
AnMBR Municipal wastewater 755–1,403 0.6–1.28 25–41 86.7–93.1 0.07–0.169
367–481 0.37–1.84 6–24 89.5–93.2 0.16–0.26 (biogas)
576–6,510 0.25–1.96 2.4–3.6 90–96 0.214–0.322

Sustainability and Future of Anaerobic Wastewater Treatment 381

250.6–272.7 0.82–3.01 2–8 70.6–77.3 0.08–0.12
300–600 0.41–0.53 16–24 90.5 0.21–0.23
200–400 N.A. 6–12 85 0.31
365 ± 101 0.72–1.52 6–12 88.7–89.3 0.21–0.24
610 ± 260 1.3 ± 0.5 11 ± 3 88 ± 7 0.14 ± 0.06
382 Anaerobic Wastewater Treatment

nitrogen, so that during anaerobic degradation, they are normally

transformed into ammonia. Exciting new developments in anaerobic
biotechnology can now provide far more sustainable denitrification of
ammonia, which includes SAD and the anammox processes, which
also require either partial nitritation or nitrification-partial denitrifica-
tion as pre-steps (Fig. 9.12). Nitrate or nitrite is removed from waste-
water using sulfur compounds as electron donors and carbon dioxide
as a carbon source. The SAD process can be quickly established, is
easy to operate, and is a very stable and reliable system. It is ideal for
wastewaters rich in NO3 or NO2, including leachate, and has also
been successfully employed for treating groundwater (Liu et al.,
2021). The anammox process is a unique system that is highly effi-
cient and cost-effective, which occurs under anoxic conditions where
nitrite is converted to nitrogen gas by anammox bacteria using
ammonium as an electron donor:

NH4+ + NO2 → N2 + 2H2O(9.6)

Most commercial anammox processes utilize a separate nitritation

process called partial nitritation (PN), where ammonium is oxidized to
nitrite in order to ensure sufficient nitrogen dioxide for the process.
So, while partial nitrification-anammox (PN/A) reduces some oxygen,
which is reduced by over 60%, no extra carbon is required (Fig. 9.13).
The most widely used PN system is the SHARON® process,
which is an acronym for Single reactor system for High activity
Ammonium Removal Over Nitrite, and is a process that produces the
exact feed required for the Anammox® process, with the anammox
bacteria housed in a separate reactor. The partial nitrification process
provides the exact mixture of ammonium and nitrite (approximately
50:50 ammonium and nitrite), with pH control used to adjust the
balance (van Donogen et al., 2001).

NH4+ + HCO3– + 0.75 O2 → 0.5 NH4+ + 0.5 NO2– + CO2 + 1.5 H2O
 (9.7 )

The process is used to treat ammonia-rich effluents from anaero-

bic sludge digestion that is usually returned to the inlet works. The
 (a) (b) (c)

Sustainability and Future of Anaerobic Wastewater Treatment 383

(d) (e)

Figure 9.13. Conventional nitrification/denitrification (N/DN) and four anammox-based nitrogen removal pathways: (a)
Conventional N/DN process, (b) partial nitrification/anammox (PN/A), (c) partial denitrification anammox (PD/A),
(d) PN/A and PN/D combined with denitrification (DN), and (e) PN/A-sulfur autotrophic denitrification (SADN) (Deng et al.,
2025). Reproduced with permission of Elsevier Publishing.
384 Anaerobic Wastewater Treatment

stoichiometry implies that no extra addition of a base is required

since sludge liquor from anaerobic digestion generally contains suffi-
cient alkalinity in the form of bicarbonate to compensate for acid
production, where only 50% of the ammonia is oxidized. The process
operates without any biomass retention, so the SRT equals the
HRT. Effluent concentration is only dependent on the growth rate
(1/SRT) of the bacteria involved and is independent of the influent
concentration. Fast-growing ammonium oxidizers are selected by the
process when operated at temperatures >25oC. As these microorgan-
isms have a low affinity for ammonia (affinity constant 20–40 mg
NH4-N L–1), final effluents will always have a relatively high ammonia
concentration (50–100 mg N L–1). Thus, the SHARON® process is
only applicable to wastewaters with high ammonia concentrations
(>500 mg N L–1) and where final effluent quality is non-critical. The
microorganisms involved are diverse, with Nitrosomonas spp. identi-
fied using a 16S rRNA-targeted fluorescent oligonucleotide probe.
The commonest species is very similar to Nitrosomonas eutrophia,
which is known to be a fast-growing nitrifier able to grow at high
ammonia and nitrite concentrations (Logemann et al., 1998). When
treating sludge digester effluents, the SHARON® process is operated
at 30–40oC without any biomass retention. The dilution rate is set at
such a rate that the ammonium oxidizers grow fast enough to stay
within the reactor while the slower-growing nitrite oxidizers are
washed out (Mulder et al., 2001). The ratio of ammonium to nitrite
can be altered by changing the reactor pH over the range of 6.5–7.5
(van Dongen et al., 2001). Typical design data for full-scale reactors
are given in Table 9.12.
The growth rate (doubling time: 11 days) and growth yield (0.11
g VSS g–1 NH4-N) of anammox bacteria are very low, resulting in
low sludge production. Therefore, efficient biomass retention is
required to maintain suitable anammox biomass concentrations
within reactors, therefore favoring AnSBR systems (Strous et al.,
1997). Anammox reactors are very compact due to a high maximum
specific nitrogen consumption rate (0.82 g N g–1 VSS d–1), a high
affinity for ammonium and nitrite, and granular growth, allowing
efficient biomass retention. The autotrophic bacteria responsible for
the oxidation of ammonia and the simultaneous reduction of nitrite
 Sustainability and Future of Anaerobic Wastewater Treatment 385

Table 9.12. Dimensions of a full-scale SHARON®–Anammox® process for three

different cases (van Dongen et al., 2001). Reproduced with permission of IWA
Publishing.
Reactor Parameter Unit Case 1 Case 2 Case 3
General N-load kg N/day 1,200 1,200 1,200
NH4-N concentration kg N/m 3
500 1,200 2,000
Influent flow m /day
3
2,400 1,000 600
SHARON-reactor Volume m 3
3,120 1,300 780
Oxygen demand kg O2/day 2,181 2,181 2,181
Air demand Nm3/day 56,000 56,000 56,000
Moving bed Volume m 3
450 450 450
Anammox-reactor HRT Hour 4.5 11 18
Granular sludge Volume m 3
75 75 75
Anammox-reactor HRT Hour 0.75 1.8 3

belong to the widely distributed phylum Planctomycetes. Not all the

bacteria in this phylum are able to oxidize ammonium, with this being
restricted to the Anammox-Planctomycetes, which have a characteristic
membrane-bound cell compartment called the anammoxosome
(Fuerst and Sagulenko, 2011). Ammonium is used as an energy
source by the anammox bacteria and carbon dioxide as a carbon
source, with NO2– , and NO3– , if present, used as electron acceptors.
Kartel et al. (2011) have described the process in detail where the
enzyme nitrite oxidoreductase reduces nitrite to nitrogen oxide,
which then reacts with ammonium to form hydrazine (N2H2),
enabled by the enzyme hydrazine synthase, which is oxidized by
hydrazine dehydrogenase to nitrogen. The bacteria are extremely
slow-growing and strict anaerobes, unable to function if the dissolved
oxygen concentration rises above 0.5 mg L–1 (Sec. 4.4.7).
The Anammox® process has been used successfully at the
Dokhaven Wastewater Treatment Plant in Rotterdam, where it is
operated as a granular sludge AnSBR process. Ammonium conversion
rates to nitrogen gas exceed 80% at loading rates of 1.2 kg N m–3 d–1
(van Dongen et al., 2001). Typical design data for three different
loading rates is given in Table 9.12.
386 Anaerobic Wastewater Treatment

Granulation creates inert centers on which bacteria can adhere. It

is possible to operate the Anammox® process to simultaneously
remove phosphorus, creating a complete nutrient removal process.
Phosphorus is removed by biomineralization forming hydroxyapatite
(HAP — Ca10(PO4)6(OH)2), which accumulates within the granules.

10Ca2+ + 6PO43– +2OH– → Ca10(PO4)6(OH)2(9.8)

During the anammox reaction, H+ ions are consumed, increasing

the pH locally within the granule, so by adding calcium, calcium
phosphate precipitation occurs, reaching supersaturation, causing
HAP to become deposited within the granule. Over time, as the gran-
ule increases in size, the density of anammox bacteria within the
granules declines due to the reduced substrate concentration and a
high pH, causing an increase in calcium phosphate precipitation. This
results in the creation of anammox-HAP granules, which have a
unique structure, with an anammox biofilm surface layer for nitrogen
removal and an inner inert HAP core for the biomineralization of
phosphorus (Xue et al., 2022; 2023). Apart from removing nitrogen
from solution, the excess granule sludge provides a high-quality, easy-
to-use phosphorus fertilizer.
There is no doubt that there will be a shift away from energy-
hungry greenhouse gas-producing conventional BNR systems (AOA)
to low-carbon footprint combined AnMBRs and anammox-HAP
technology, enabling a whole range of byproduct recovery, especially
biomethane and biohydrogen. The technology itself is resilient and
reliable and is able to deliver the required treatment capacity currently
unavailable at most centralized WWTPs, which are struggling to cope
with ever-increasing flow rates, more complex wastewaters, increasing
operational costs, and the inability to meet carbon-neutral or carbon-
zero targets. Our increasing understanding of the role of QS signaling
molecules in anaerobic treatment will be critical in enhancing overall
performance and reactor stability, especially in relation to granulation
and methanogenesis, whether this is by adding exogenous signaling
molecules, adding QS microorganisms, or by the use of conductive
materials. The key areas where QS will be able to make a significant
difference are in promoting: (a) EPS development through regulation
 Sustainability and Future of Anaerobic Wastewater Treatment 387

of amino acid synthesis and gluconeogenesis, (b) enzyme synthesis for

all stages of anaerobic digestions (i.e., hydrolysis, acidogenesis, and
methanogenesis), and (c) interspecies electron transfer by controlling
electron carrier synthesis (Chen et al., 2024). Performance data for a
wide range of anaerobic wastewater treatment systems have been
summarized by Stazi and Tomei (2018), who also discuss future pros-
pects for anaerobic processes.
It is inevitable as we move toward using stricter sustainability
indicators to measure WWTP performance, the role of anaerobic sys-
tems will begin to dominate future domestic as well as industrial
WWTP designs, especially in relation to carbon footprint and nutrient
and pathogen removal. This may also include eco-labeling specific
processes using a tool such as ISO 14045 (ISO, 2024), as demon-
strated by Lorenzo-Toja et al. (2016) and fully discussed in an earlier
book in this series (Gray, 2022). However, Li et al. (2022) have
shown that there is a significant difference in the way we assess
WWTPs in terms of zero-carbon and the reality of wastewater treat-
ment in terms of carbon neutrality. They propose that carbon foot-
print analysis should be carried out throughout the whole collection,
treatment, and disposal cycle and proposed three boundaries for sepa-
rate analysis: the actual wastewater treatment plant as defined by its
boundary fence, the urban infrastructure as related to WWTP, and the
human society and ecological system (Fig. 9.14). Within-the-fence
boundary accounting for carbon emissions is restricted to when
the wastewater starts its treatment and ends at the point it leaves the
site as a discharge or sludge. This is the normal method of assessing
the carbon footprint of wastewater treatment but fails to take into
account the upstream and downstream impacts. So, using a secondary
boundary, the urban infrastructure related to WWTPs, emissions
could be included from the sewer network and also where other
wastes are transported to the WWTP for treatment, including other
materials such as food waste used in co-digestion. For example,
anaerobic digestion is increasingly used to degrade food waste, which
is diverting organic matter from landfills to produce useful biogas and
digestion products. In the European Union alone, 88 million tonnes
of food waste is produced, representing 55% of the total waste going
 388 Anaerobic Wastewater Treatment
Figure 9.14. Proposed system for carbon accounting of wastewater treatment using three boundaries: within-the-fence of WWTPs
(yellow-dashed line), urban infrastructure related to WWTPs (pink-dashed line), and human society and ecological system (blue-
dashed line). Since the operation of most facilities in boundaries 1 and 2 requires energy and chemicals, their inputs are simplified
as flows pointing into the boundary. Direct carbon emissions are presented by specific symbols. Indirect carbon emissions are implied
in the flows of energy/chemicals consumption and recycled products, which represent positive and negative contributions to carbon
emissions, respectively (Li et al., 2022). Reproduced under the Creative Commons Attribution (CC BY) license (https://crea-
tivecommons.org/licenses/by/4.0/).
 Sustainability and Future of Anaerobic Wastewater Treatment 389

to landfill, while China, India, and the United States produce 108,
72, and 35 million tonnes of food waste, respectively, each year (Thi
et al., 2015). This represents an enormous opportunity to reduce
greenhouse gas emissions through the generation of bioenergy. The
third boundary, human society and ecological system, creates an
inventory of how the treated wastewater can be used. For example,
some wastewater effluents are reused for irrigation or for supply pur-
poses, while the sludge can be used as a source of energy, nutrients,
or soil conditioner. Recovered resources also benefit society by pro-
viding an alternative to high-energy production systems. When
anaerobic systems are used instead of traditional aerobic-based treat-
ment processes, the advantages can be directly related to significant
reductions in carbon footprint and takes us ever closer to real carbon
neutrality.

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 Index

Acetate, see Acetic acid Anaerobic baffled reactor,

Acetate pathway, 337 see AnBR
Acetic acid, 6–9, 30–32, 339–344 Anaerobic baffled septic tank, 101
Acetoclastic, 45, 185, 235, 274 Anaerobic biofilter, 103–104,
Acetogenesis, 7–9, 32–33, 355 213–222
Acidogenesis, 7–9, 29–32 Anaerobic biological rotating
Adenosine triphosphate, see ATP contactor, see AnRBC
ADSR, 214 Anaerobic breakdown, 6–9, 25
Aerotolerant anaerobes, 22 Anaerobic continuously stirred tank
Alkalinity, 186–190 reactor, see AnCSTR
American Type Culture Collection, Anaerobic downflow stationary bed
23 reactor, see ADSR
Ammonia, 63–65, 168–172, Anaerobic expanded bed, see
248–250 AnEBR
Biogas, 326 Anaerobic expanded granular bed
Forms, 64 reactor, see EGBR
Lagoons, 124–127 Anaerobic fluidized bed, see
Removal, 170–172, 378–386 AnFBR
Stripping, 170–172, 378–386 Anaerobic hybrid reactor, see
Anaerobic, AnHR
Biodegradability test, 175 Anaerobic lagoons, see Lagoons
Biofilters, 103–104, 213–222 Anaerobic microorganisms in
Filter, 4, 13 digester, 46
Inhibition testing, 175 Anaerobic microbial process, 5–9
Anaerobic ammonium oxidation, Anaerobic migrating blanket
see Anammox reactor, 282–283

401
402 Anaerobic Wastewater Treatment

Anaerobic moving bed biofilm Archaea, 185–187, 195, 235–237,

reactor, see AnMBBR 261, 280, 285
Anaerobic moving biofilm reactor, Orders, 35–36, 44–45
13 ARGs, 106
Anaerobic pathways, 47 ATP, 22, 25, 44–46
Anaerobic sequencing biofilm Attached growth systems, 11,
batch reactor, 13 213–250
Anaerobic treatment, AUF, 214
Advantages/disadvantages, 5
Comparison with aerobic Bacteria, 185–186, 193–195,
systems, 5–6 235–237, 261, 280, 285, 338
Energy requirement, 4 Acetogenic, 7–9, 185–186,
Role, 12, 16–19 193–195, 274
Anaerobic unit processes, 10–18 Fermentative, 7–9, 185–186,
Anaerobic up-flow filter, 193–195
see AUF Hydrolytic , 7–9, 185–186,
Anammox, 133, 248–250, 193–195
378–386 Biochar, 219, 378
Anammox-HAP, 386 Biodegradability test, 175
AnBR, 216, 335, 371 Biofilm systems, 213–250
AnCSTR, 4, 11, 13, 271–272, Biofilters, 103–104, 213–222
334, 336, 370 Biofiltration,
AnEBR, 210–214 Sulfide removal, 316–317
AnEMBR, 376 Biogas, see Methane, 297–327
AnFBR, 240–242, 244, 371 Lagoons, 133–136
AnFFR, 335, 371–373 Odor, 300
AnFIB, 368 Biogas contaminants, 45–72
AnFIL, 216 Ammonia, 63–65, 326
AnHR, 217, 277–281, 283–286, Carbon dioxide, 322–326
371 Hydrogen sulfide, 56–59,
AnMBB, 228–240 301–302
AnMBBR, 238 Oxygen, 326
AnMBR, 4, 13, 336, 368–374 Siloxanes, 72–73, 326
AnPBR, 214 Terpenes, 70–72, 326
AnRBC, 222–228 Volatile organic compounds,
AnSBR, 272–277, 336, 342–344 65–72
Antibiotic resistant genes, see Water, 326
ARGs Biohydrogen, 334
Antibiotic wastewater, 285 Biological hydrolysis, 155–157
 Index 403

Biological pre-hydrolysis, see BPH Comparison to aerobic treatment,

Biological sulfide recovery, 5–6
367–371 Contact systems, 11, 213–250
Biomethane, 12, 300–301 Covered lagoons, 133–136
Standards, 300–301 Co-digestion, 273–274, 377
Bio-CNG, 43 Co-treatment, 375
Bio-LNG, 43 Crown corrosion, 59–63
BPH, 156–157
BTEX suite, 72–73 Dark fermentation, 336–339
Butyrate pathway, 337 Denitrification, 132–133, 174, 378
Butyric acid, 6–9, 30–31, 33, Desulfurization,
339–344 Absorption, 312–314
Activated carbon, 311
Carbohydrates, 15, 28, 30–31, Adsorption, 305–312
299 Biological, 314–322
Carbon, Bioscrubbing, 317–318
Accounting, 387–389 Catalytic scrubbing, 314
Emissions, 17, 55–56, 377, Caustic scrubbers, 318
387–389 Dry scrubbers, 311
Harvesting, 333 In-situ sulfide control, 320
Mass balance, 6 Metal sulfide precipitation,
Carbon dioxide removal from 319–320
biogas, 322–326 Microaeration, 321–322
Carriers, see Media Zero-valent iron, 321, 378
Cellulolytic bacteria, 29 Detergents, 162–164
Cesspools, 85–86 Detergents Ingredients Database,
Chelation, 365 163–164
Chlorinated hydrocarbons, Digesters, 143–197, 371
164–165 Alkalinity, 186–190
Chloroform, 164–165 Ammonia, 168–172
CHP, 12, 43, 157–158, 300 Biogas, 297–327
Circular economy, 333 Chlorinated hydrocarbons,
COD:N ratio, 274 164–165
COD:N:P ratio, 161, 168 Chloroform, 164–165
Combined heat and power CHP, 157–158
eneration, see CHP COD:N:P ratio, 161, 168
Combined systems, 81–136 Commissioning, 175–178
Commissioning digesters, 175–178 Comparison of raw sludge, 160
Comparison of raw sludge, 160 Conventional, 145–147
404 Anaerobic Wastewater Treatment

Design, 143–154 Energy requirements, comparison,

Detergents, 162–164 4, 375
EPS, 155–157, 161, 193–194 Enzymatic hydrolysis, 155–157
Foaming, 190–200 EPS, 155–157, 161, 193–194,
Heating, 150–152 269, 386–388
Heavy metals, 166–168, 190 Degradation, 155–157
High-rate, 145–147 Foaming, 193–194
Hydrolysis, 154–159 Quorum sensing, 345,
Inhibition, 161, 164–168 347–452
Inhibition tests, 174–175 EU Wastewater Directive, 17
Microbiology, 46, 169–170,
185–186, 193–197 Facultative anaerobes, 21, 29
Mixing, 178­–181 Fats and oils, 15, 27, 271, 275,
Modern designs, 150, 179 299
Number, 12 Fermentation of amino acids,
Operational management, 31–32
159–195 Fermentation of monosaccharides,
Organic loading, 153–154 30–31
Other ions, 173 Floc-based systems, see Granular
pH, 186–190 systems
Plug-flow, 145–147 Flow-through systems, 10
Secondary digester tanks, 154 Fluidized bed reactor, 4, 13,
Single-stage, 144 240–242, 244
SRT, 181–182 Foam, 190–200, 271
Surfactants, 162–164 Control, 197–200
Temperature, 182–186 Formation, 190–200
Two-stage, 144 Microorganisms, 193–197
Digestion, liquor, 297 Potential tests, 195–196
Dispersed growth systems, Food waste, 387–389
259–286 Future of anaerobic treatment,
Downflow stationary fixed film 375–389
reactor, see DSFF
DSFF, 215–217, 220 Gas-liquid-solids separator, see
DSMZ, see Leibniz Institute GLS separator
GHG emissions, 17, 55–56
Eco-labeling, 387 GLS separator, 261, 265,
EGBR, 264–266 266–267, 283
Electrical field-assisted anaerobic Granular systems, 259–286
membrane reactor (AnEMBR), Granules,
376 Anammox, 386
 Index 405

Formation, 344–352 Lactic acid, 30–31, 339–344

Life cycle, 345–352 Lagoons, 111–136
Structure, 261, 263–264 Ammonia, 124–127
Application of, 118–121,
Heating digesters, 150–152 133–136
Heavy metals, 166–168, 190, 228 Covered, 133–136
HRT, 11, 213, 218, 221, 227, Denitrification, 132
247, 380 Design, 111–114, 121–124
Hybrid anaerobic bed reactor, see Methane, 124–127
AnHR Microbiology, 114
Hybrid reactor, 4, 13 Odor, 127–132
Hybrid up-flow anaerobic sludge Operating conditions, 118,
blanket (HUASB), 283 120–121
Hydraulic residence time, see HRT ORP, 118
Hydrogen sulfide, Process, 111–114
Corrosion, 59–63 VFA, 118, 127–132
Desulfurization, 305–322 LCFAs, 14, 30–31, 274
Environmental effects, 58 Leibniz Institute, 23
Form, 301 Lipid hydrolysis, 274, 299
Formation, 56–58, 129–132,
301–302 Mass balance of carbon utilization,
Health effects, 58–59 6
Inhibition of digesters, MCRT:HRT ratio, 219
173–174 Media, 18
Removal methods, 302–305 Coal, 276
Hydrogenotrophic, 45, 185, 235, Contact systems, 217–219,
274 228, 231–238
Hydrolysis, 7–9, 25–29, 154–159 GAC, 275–276
PABR, 280–281
IC, 266–271 Septic tank, 103–104
Imhoff tanks, 18, 86, 106–111, Zeolites, 275–276
131 Membrane technology, 368–374
Design, 108–111, 131 Metals, 166–168, 190, 228,
Microbiology, 107–108 319–320, 355–368
Inhibition, 161, 164–168, 273 Recovery, 367–368
Inhibition testing, 174–175 Role, 355–367
Integrated up-flow septic tank, 102 Methane, 6–9, 11–12, 44–56
Internal circulation, see IC AnMBR, 373
Inverse fluidized bed (AnIFB), 368 Calorific value, 298
406 Anaerobic Wastewater Treatment

Fermentation steps, 47 General, 5–9

Formation, 44–56 Granules, 261, 263
Health effects, 54 Imhoff tank, 107–108
Hydrogen sulfide, 302 Lagoons, 114
Lagoons, 124–127, 133–136 Methane, 45
Metabolism, 45 Quorum sensing, 354
Microbiology, 45 Septic tank, 104–106
Production, 47–54, 297 VFA production, 339–344
Quality, 298–301 MIDAS, 23
Septic tank, 89 Mixing in digesters, 178–118
Yield, 12, 47–54, 298–299, Modeling, 12–16
380 Monod function, 13
Methanogen, 169–173, 235–236,
366 Nanoparticles,
Ammonia, 168–173 Biohydrogen production, 339
Contact systems, 235–236 Metallic, 367
Foaming, 193–195 Natural gas grid, 43, 300–301
Inhibition, 161, 164–165, Net zero, 387
168–173
MRCT, 182 Obligate anaerobes, 21
Orders, 45 Odor,
Septic tank, 104–106 Biogas, 300
Temperature, 182 Lagoons, 127–132
Methanogenesis, 7–9, 33–36 Number, 128
Methylotrophic, 45, 185 OLR, 275, 286, 374, 380
Microbial Database for Activated Operational parameters, 4, 13
Sludge, see MIDAS Organic loading to digesters,
Microbial foams, 193–197 153–154
Microbiology, 5–9, 104, 114, ORP, 36–39, 45, 118
169–170, 185–186, 169–171, Other ions, 173
193–197, 235–237, 261, 263,
280–281 PABR, 216, 279–281
Baffled reactors, 280–281 Packed anaerobic baffled reactor,
Biohydrogen, 338–339 see PABR
Contact systems, 235–237 Partial nitritation (PN), 382
Digester, 46, 169–170, Pasteurization, 157–159
185–186, 193–197 Pathogens,
Effects of ammonia, 169–171 Agricultural land, 120
Foaming, 193–197 Septic tank, 106
 Index 407

pH, 186–190 SMAR, 216

Pharmaceutical treatment, SOB, see Sulfur oxidizing bacteria
237–239 Solids residence time, see SRT
Propionic acid, 6–9, 30–31, SRB, see Sulfate reducing bacteria
339–344 SRT, 11, 181–182, 213, 221,
Protein/polysaccharide ratio, 269 374
Proteins, 15, 25, 31–32, 299 Stable foam potential, 195–196
Static granular bed reactor
Quorum sensing molecules, (SGBR), 265–266
352–255, 386 Static or porous medium bed
reactor, see AnPBR
Reactive oxygen species (ROS), 22 Steps in anaerobic decomposition,
Redox potential, see ORP 7–9,
Submerged anaerobic reactor, see
Secondary digester tanks, 154–195 SMAR
Septic tanks, 18, 81–106 Sulfate reducing bacteria, 57–58,
Commissioning, 90 129–132, 367
Design, 86–93 Sulfur based autotrophic
Development, 81–83 denitrification (SAD), 378
Effluent quality, 86 Sulfur oxidizing bacteria, 60–63
Faults, 92–93, 100 Sulfur removal, 248
Maintenance, 95–100 Surfactants, 162–164
Media, 103–104 Sustainability, 333, 387
Methane production, 89 Syntrophic fermentation, 274
Microbiology, 104
Modifications, 100–104 Temperature phased anaerobic
Operation, 93–100 digestion, see TPAD
Pathogen removal, 106 Temperature, 182–186
Sludge accumulation, 94–100 Thermal hydrolysis, 159
Standards, 84 TPAD, 184
Upgrading, 92
Sewage sludge, UASB, 4, 13, 214, 221, 223,
Chemical composition, 24 260–264, 266, 285, 372–375
Sewer, UASBST, 101
Iron dosing, 321 Unstable foam potential, 195–196
Sewer corrosion, 59–63 Up-flow anaerobic filter, see
SHARON process, 382–386 AnFIL
Siloxanes, 72–73 Up-flow anaerobic sludge bed, see
Sludge digestion, see Digesters UASB
408 Anaerobic Wastewater Treatment

Up-flow anaerobic sludge blanket, Lagoons, 118, 120–121

see UASB Production, 339–344
Up-flow septic tank, see UST Uses, 339–341
Up-flow stationary fixed film filter, VOCs, 65–72
see USFF Volatile fatty acids, see VFAs
USFF, 215–217, 222 Volatile organic compounds, see
UST, 101 VOCs

Valeric acid, 339–344 Zero-valent iron, 321, 378

VFAs, 6–9, 15, 25, 147, 188–189,
274, 334