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Wastewater Microbiology Series

The Microbiology
of Anaerobic Digesters

MICHAEL H. GERARDI

The Microbiology of
Anaerobic Digesters

WASTEWATER MICROBIOLOGY SERIES

Editor

Michael H. Gerardi

Nitrification and Denitrification in the Activated Sludge Process
Michael H. Gerardi

Settleability Problems and Loss of Solids in the Activated Sludge
Process

Michael H. Gerardi

The Microbiology of Anaerobic Digesters
Michael H. Gerardi

The Microbiology of
Anaerobic Digesters

Michael H. Gerardi

iWILEY-
INTERSCIENCE

A John Wiley & Sons, Inc., Publication

Published by John Wiley & Sons, Inc., Hoboken, New Jersey.
Published simultaneously in Canada.

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Library of Congress Cataloging-in-Publication Data:

Gerardi, Michael H.
The microbiology of anaerobic digesters / Michael H. Gerardi.

p. cm.
Includes bibliographical references and index.
ISBN 0-471-20693-8 (cloth)

1. Sewage sludge digestion. 2. Anaerobic bacteria. I. Title.
TD769 .G47 2003
628.3 , 5— dc21

2003007454

Printed in United States of America

10 987654321

Mom and Dad

The author extends his sincere appreciation to
joVanna Gerardi for computer support

and
Cristopher Noviello for artwork used in this text.

PART I OVERVIEW

1 Introduction

Contents

Preface ix

2 Bacteria 11

3 Methane-forming Bacteria 17

4 Respiration 31

5 Anaerobic Food Chain 39

6 Fermentation 43

7 Anaerobic Digestion Stages 51

PART II SUBSTRATES, PRODUCTS, AND BIOGAS 59

8 Substrates and Products 61

9 Biogas 73

PART III OPERATIONAL CONDITIONS 77

10 Introduction to Operational Conditions 79

■ ■

VII

VIII CONTENTS

11 Start-up 81

12 Sludge Feed 85

13 Retention Times 87

14 Temperature 89

15 Nutrients 93

16 Alkalinity and pH 99

17 Toxicity 105

18 Mixing 117

PART IV PROCESS CONTROL AND TROUBLESHOOTING 121

19 Upsets and Unstable Digesters 123

20 Foam and Scum Production and Accumulation 127

21 Supernatant 133

22 Monitoring 135

PARTV DIGESTERS 141

23 Types of Anaerobic Digesters 143

24 Anaerobic Digesters versus Aerobic Digesters 153

References 161

Abbreviations and Acronyms 165

Chemical Compounds and Elements 167

Glossary 171

Index 175

Preface

Completely mixed anaerobic digesters are the most commonly used treatment
system in North America for the degradation of municipal sludges. Although these
suspended-growth systems are not used as commonly at industrial wastewater treat-
ment plants, more and more industrial plants are using fixed-film anaerobic digesters
for the treatment of soluble organic compounds in their wastewaters.

Anaerobic digesters perform most of the degradation of organic compounds at
wastewater treatment plants. However, digesters often experience operational
problems that result in process upsets and increased operational costs. Examples
of process upsets and operational problems include foam and scum production,
decanting and dewatering difficulties, loss of treatment efficiency, toxic upsets, and
"souring" of the digester. Poorly operating anaerobic digesters often contribute to
operational problems in other treatment units such as the activated sludge process,
gravity thickener, clarifiers, and sludge dewatering facilities.

Because of the importance of anaerobic digesters in wastewater treatment
processes, a review of the microbiology of the bacteria and the operational condi-
tions that affect their activity is of value in addressing successful and cost-effective
operation. This book provides an in-depth review of the bacteria, their activity, and
the operational conditions that affect anaerobic digester performance. The identifi-
cation of operational problems and troubleshooting and corrective measures for
process control are presented.

This book is prepared for an audience of operators and technicians who are
responsible for the daily operation of anaerobic digesters. It presents troubleshoot-
ing and process control measures to reduce operational costs, maintain treatment
efficiency, and prevent system upsets.

The Microbiology of Anaerobic Digesters is the third book in the Wastewater
Microbiology Series by John Wiley & Sons. This series is designed for operators and
technicians, and it presents a microbiological review of the organisms involved in
wastewater treatment processes and provides biological techniques for monitoring
and regulating these processes.

Michael H. Gerardi
Linden, Pennsylvania

Parti

Overview

Introduction

The organic content of sludges and soluble wastes can be reduced by controlled bac-
terial activity. If the bacterial activity is anaerobic, the reduction in organic content
is achieved through sludge digestion. If the bacterial activity is aerobic, the reduc-
tion in organic content is achieved through sludge stabilization.

Anaerobic digesters having suspended bacterial growth are commonly used at
municipal wastewater treatment plants to degrade (digest) sludges (Figure 1.1). With
the development of anaerobic digesters having fixed-film bacterial growth (Figure
1.2), more and more industrial wastewater treatment plants are using anaerobic
digesters to degrade soluble organic wastes. Anaerobic digesters represent catabolic
(destructive) processes that occur in the absence of free molecular oxygen (0 2 ).

The goals of anaerobic digesters are to biologically destroy a significant portion
of the volatile solids in sludge and to minimize the putrescibility of sludge. The main
products of anaerobic digesters are biogas and innocuous digested sludge solids.
Biogas consists mostly of methane (CH 4 ) and carbon dioxide (C0 2 ).

Primary and secondary sludges are degraded in anaerobic digesters (Figure 1.3).
Primary sludge consists of the settled solids from primary clarifiers and any colloidal
wastes associated with the solids. Secondary sludge consists mostly of waste-
activated sludge or the humus from trickling filters. The mixture of primary and
secondary sludges contains 60% to 80% organic matter (dry weight) in the forms
of carbohydrates, fats, and proteins.

The mixture of primary and secondary sludges is an ideal medium for bacterial
growth. The sludges are rich in substrates (food) and nutrients and contain a large
number and diversity of bacteria required for anaerobic digestion.

The anaerobic digester is well known as a treatment process for sludges that
contain large amounts of solids (particulate and colloidal wastes). These solids

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

INTRODUCTION

Biogas Withdrawal

Inlet

§£gifr:£^$£:

Supernatant

Active Biomass

Outlet

Stabilized Solids

Solids Withdrawal

Figure 1.1 Suspended growth anaerobic digesters are commonly used at municipal wastewater
treatment plants for the degradation of primary and secondary sludges. These digesters produce
several layers as a result of sludge degradation. These layers are from top to bottom: biogas, scum,
supernatant, active biomass or sludge, and stabilized solids.

CH 4 + CO

Fixed Film Media

Figure 1.2 Fixed film anaerobic digesters employ the use of a medium such as plastic or rocks on
which bacteria grow as a biofilm. Wastewater passing over the medium is absorbed and adsorbed by
the biofilm and degraded.

INTRODUCTION

Primary
Clarifier

Aeration
i Tank

Secondary
Clarifier

x°--

-►

V V

Anaerobic Digester

Figure 1.3 Primary and secondary sludges typically are degraded in suspended growth anaerobic
digesters at municipal wastewater treatment plants. The sludges contain relatively large quantities of
particulate and colloidal wastes.

require relatively long digestion periods (10-20 days) to allow for the slow bacte-
rial processes of hydrolysis and solubilization of the solids. Once solubilized, the
resulting complex organic compounds are degraded to simplistic organic com-
pounds, mostly volatile acids and alcohols, methane, new bacterial cells (C 5 H 7 2 N),
and a variety of simplistic inorganic compounds such as carbon dioxide and hydro-
gen gas (H 2 ).

With the development of fixed-film bacterial growth in anaerobic digesters, many
soluble organic wastes can be digested quickly and efficiently. Because the wastes
are soluble, time is not required for hydrolysis and solubilization of the wastes.

When sludges are digested, the organic content of the sludges is decreased as
volatile materials within the sludges are destroyed, that is, the volume and weight
of the solids are reduced. The volatile content for most anaerobic digested sludges
is 45%-55% (Figure 1.4).

Anaerobic digesters (Figure 1.5) degrade approximately 80% of the influent
organic waste of a conventional municipal wastewater treatment plant. Nearly 30%
of the waste is removed by primary clarifiers and transferred to anaerobic digesters,
and approximately 50% of the waste is synthesized or transformed into new bacte-
rial cells or solids [mixed-liquor volatile suspended solids (MLVSS) or trickling filter
humus]. These synthesized solids also are transferred to anaerobic digesters through
the wasting of secondary solids.

Because of the relatively large quantity of organic wastes placed on the anaero-
bic digestion process, a review of the bacteria, their activity, and the operational
factors that influence their activity are critical. This review provides for proper
maintenance of digester performance and cost-effective operation and helps to
ensure adequate monitoring, troubleshooting, and process control of anaerobic
digesters.

Anaerobic sludge digestion consists of a series of bacterial events that convert
organic compounds to methane, carbon dioxide, and new bacterial cells. These
events are commonly considered as a three-stage process.

The first stage of the process involves the hydrolysis of solids (particulate and
colloidal wastes). The hydrolysis of these wastes results in the production of

INTRODUCTION

Digester Feed Sludge 100 kg, 70% Volatile Solids

Volatile Solids,
70 kg

Inert Solids,
30 kg

V V

Biogas
(CH 4 + C0 2 ),

40 kg

Volatile Solids,
30 kg

Inert Solids,
30 kg

Digested Sludge,

60 kg,

50% Volatile Solids

Figure 1.4 The digestion of sludges in anaerobic digesters results in significant reduction in the
volatile content of the sludges as well as the volume and weight of the sludges.

Primary
Clarifier

Aeration
Tank

Secondary
Clarifier

^UL

2U r

Anaerobic Digester

30% of influent
organic waste

50% of influent
organic waste

Figure 1.5 Most of the influent organic wastes of a wastewater treatment plant are degraded in an
anaerobic digester. Settled solids in the primary clarifier represent approximately 30% of the influent
organic wastes, while secondary solids represent approximately 50% of the influent organic wastes.
In the activated sludge process much of the organic waste is converted to bacterial cells. These cells
represent organic wastes, i.e., upon their death; they serve as a substrate for surviving bacteria.

INTRODUCTION 7

simplistic, soluble organic compounds (volatile acids and alcohols). The second stage
of the process, acetogenesis, involves the conversion of the volatile acids and alco-
hols to substrates such as acetic acid or acetate (CH 3 COOH) and hydrogen gas that
can be used by methane-forming bacteria. The third and final stage of the process,
methanogenesis, involves the production of methane and carbon dioxide.

Hydrolysis is the solubilization of particulate organic compounds such as cellu-
lose (Equation 1.1) and colloidal organic compounds such as proteins (Equation
1.2) into simple soluble compounds that can be absorbed by bacterial cells. Once
absorbed, these compounds undergo bacterial degradation that results in the pro-
duction of volatile acids and alcohols such as ethanol (CH 3 CH 2 OH) and propionate
(CH 3 CH 2 COOH). The volatile acids are converted to acetate and hydrogen gas.
Methane production occurs from the degradation of acetate (Equation 1.3) and the
reduction of carbon dioxide by hydrogen gas (Equation 1.4).

cellulose + H 2 — hydrolysis — > soluble sugars (1.1)

proteins + H 2 — hydrolysis — > soluble amino acids (1.2)

CH3COOH -* CH 4 + C0 2 (1.3)

C0 2 + 4H 2 -* CH 4 + 2H 2 (1.4)

In addition to the reduction in volume and weight of sludges, anaerobic digesters
provide many attractive features including decreased sludge handling and disposal
costs and reductions in numbers of pathogens (Table 1.1). The relatively high tem-
peratures and long detention times of anaerobic digesters significantly reduce the
numbers of viruses, pathogenic bacteria and fungi, and parasitic worms. This reduc-
tion in numbers of pathogens is an extremely attractive feature in light of the
increased attention given by regulatory agencies and the general public with respect
to health risks represented by the use of digested sludges (biosolids) for agricultural
and land reclamation purposes.

Although anaerobic digesters offer many attractive features, anaerobic digestion
of sludges unfortunately has an unwarranted reputation as an unstable and difficult-
to-control process. This unwarranted reputation is due to several reasons, including
a lack of adequate knowledge of anaerobic digester microbiology and proper
operational data (Table 1.2).

TABLE 1.1 Attractive Features of Anaerobic Digesters

Able to degrade recalcitrant natural compounds, e.g., lignin

Able to degrade xenobiotic compounds, e.g., chlorinated aliphatic hydrocarbons

Control of some filamentous organisms through recycling of sludge and supernatant

Improved dewaterability of sludge

Production of methane

Use of biosolids as a soil additive or conditioner

Suitable for high-strength industrial wastewater

Reduction in malodors

Reduction in numbers of pathogens

Reduction in sludge handling and disposal costs

Reduction in volatile content of sludge

8 INTRODUCTION

TABLE 1.2 Reasons Contributing to the Unwarranted
Reputation of the Anaerobic Digester as an Unstable
Process

Lack of adequate knowledge of anaerobic digester

microbiology
Lack of commercial interest
Lack of operator training
Lack of proper operational performance data for installed

digesters
Lack of research and academic status
Regrowth needed for industrial toxicity episodes

TABLE 1.3 Examples of Significant Differences
Between Aerobic Stabilization and Anaerobic Digestion
of Wastes

Feature Anaerobic Aerobic

Digestion Stabilization

Process rate Slower Faster

Sensitivity to toxicants Higher Lower

Start-up time Slower Faster

Until recently, little information was available that reviewed the bacteria and
their requirements for anaerobic digestion of solids. The difficulty in obtaining ade-
quate data was caused by the overall complex anaerobic digestion process, the very
slow generation time of methane-forming bacteria, and the extreme "sensitivity" of
methane-forming bacteria to oxygen. Therefore, it was not uncommon for opera-
tors to have problems with digester performance.

These problems, the development and use of aerobic "digesters," and the use of
relatively cheap energy for aerobic stabilization of wastes contributed to the lack
of interest in anaerobic digesters. Although aerobic stabilization, that is, the use of
aerobic digesters, and anaerobic digestion of wastes are commonly used at waste-
water treatment process, significant differences exist between these biological
processes (Table 1.3).

Methane production under anaerobic conditions has been occurring naturally for
millions of years in such diverse habitats as benthic deposits, hot springs, deep ocean
trenches, and the intestinal tract of cattle, pigs, termites, and humans. Methane pro-
duction also occurs in rice paddies.

More than 100 years ago, anaerobic digesters were first used in Vesoul, France
to degrade domestic sludge. Until recently, anaerobic digesters were used mostly to
degrade municipal sludges and food-processing wastewater. Municipal sludges
and food-processing wastewater favor the use of anaerobic digesters, because the
sludges and wastewater contain a large diversity of easily degradable organics and
a large complement of inorganics that provide adequate nutrients and alkalinity that
are needed in the anaerobic digestion process.

INTRODUCTION

TABLE 1.4
Digestion

Chemical Wastes Amenable to Anaerobic

Acetone

Acrylates

Alcohols

Aldehydes

Amino acids

Anilines

Catechols

Cresol

Formaldehyde

Formate

Glycerol

Glycols

Ketones

Methyl acetate

Nitrobenzene

Organic acids

Phenols

Quinones

TABLE 1.5
Digestion

Industrial Wastes Amenable to Anaerobic

Alcohol stillage

Pectin

Bean

Petroleum

Beverage production

Pharmaceutical

Brewery

Potato

Canning

Pulp and paper

Cheese

Seafood and shellfish

Chemical

Slaughterhouse and meat packing

Corn

Sugar

Dairy

Vegetable

Distillery

Wheat and grain

Egg

Winery

Fruit

Wool scouring

Leachate

Yeast

A better understanding of the microbiology of anaerobic digesters and process
modifications, particularly fixed-film processes, have permitted the use of anaerobic
digesters for dilute wastewaters and a large variety of industrial wastes (Tables 1.4
and 1.5). This understanding and these process modifications, together with the need
to pretreat industrial wastewaters and sludges and the attractive features of anaer-
obic digesters, have generated renewed interest in their use in degrading not only
municipal sludges but also industrial wastewaters.

The number of wastes that are amenable to anaerobic digestion is quite large.
Examples of industrial wastes include acetone, butanol, cresol, ethanol, ethyl
acetate, formaldehyde, formate, glutamate, glycerol, isopropanol, methanol, methyl
acetate, nitrobenzene, pentanol, phenol, propanol, isopropyl alcohol, sorbic acid,
te/t-butanol, and vinyl acetate. Because many industrial wastes can be treated
anaerobically, the feasibility of anaerobic digestion of an industrial waste is deter-
mined by several factors. These factors include the concentration of the waste, the
temperature of the waste stream, the presence of toxicants, biogas and sludge pro-
duction, and expected treatment efficiency.

The development of the fixed film filter was a significant achievement in anaero-
bic technology (Figure 1.6). The filter provides relatively long solids retention time
(SRT). Increased retention time makes it possible to treat moderately low-strength

INTRODUCTION

Effluent

Influent Wastewater

Figure 1.6 In an anaerobic filter, wastewater flows from bottom to top or top to bottom of the treat-
ment unit. The wastewater passes over media that contains a fixed film of bacteria growth that
degrades the organic wastes in the wastewater.

[2000-20,000 mg/1 chemical oxygen demand (COD)] soluble organic industrial
waste. Because of the highly concentrated bacterial population of the filter, a highly
stable digestion process can be achieved even during significant variations in oper-
ating conditions and loadings. Therefore, interest in anaerobic biotechnology for
treating industrial waste streams has grown considerably.

Bacteria

At least 300 different species of bacteria are found in the feces of a single individ-
ual. Most of these bacteria are strict anaerobes. The majority of the remaining bac-
teria are facultative anaerobes. Escherichia coli is a common facultative anaerobe
in feces.

Bacteria from fecal wastes as well as hundreds of soil and water bacteria that
enter a conveyance system through inflow and infiltration (I/I) are found in the influ-
ent of municipal wastewater treatment processes. For the purpose of this text, bac-
teria that are commonly found in wastewater treatment processes are divided into
groups according to 1) their response to free molecular oxygen (0 2 ) and 2) their
enzymatic ability to degrade substrate in the anaerobic digester.

RESPONSE TO FREE MOLECULAR OXYGEN

Bacteria may be divided further into three groups according to their response to
free molecular oxygen (Table 2.1). These groups are 1) strict aerobes, 2) facultative
anaerobes, and 3) anaerobes, including the methane-forming bacteria.

Strict aerobes are active and degrade substrate only in the presence of free
molecular oxygen. These organisms are present in relatively large numbers in
aerobic fixed-film processes, for example, trickling filters, and aerobic suspended-
growth processes, for example, activated sludge. In the presence of free molecular
oxygen they perform significant roles in the degradation of wastes. However, strict
aerobes die in an anaerobic digester in which free molecular oxygen is absent.

Facultative anaerobes are active in the presence or absence of free molecular
oxygen. If present, free molecular oxygen is used for enzymatic activity and the

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

BACTERIA

TABLE 2.1 Groups of Bacteria According to Their Response to Free Molecular Oxygen

Group

Example

Significance

Strict aerobes Haliscomenobacter hydrossis

Nitrobacter sp.
Nitrosomonas sp.
Sphaerotilus natans

Facultative
anaerobes

Anaerobes

Zoogloea ramigera

Escherichia coli

Bacillus sp.

Desulfovibrio sp.
Methanobacterium formicium

Degrades soluble organic compounds; contributes

to filamentous sludge bulking
Oxidizes NOi to NOg
Oxidizes NH} to NO2
Degrades soluble organic compounds; contributes

to filamentous sludge bulking
Degrades soluble organic compounds; contributes

to floe formation
Degrades soluble organic compounds; contributes

to floe formation; contributes to denitrification or

clumping
Degrades soluble organic compounds; contributes

to denitrification or clumping
Reduces SOf~ to H 2 S
Produces ChL

TABLE 2.2 Groups of Anaerobic Bacteria

Group

Example

Significance

Oxygen tolerant
Oxygen intolerant

Desulfovibrio sp.
Desulfomarculum sp.
Methanobacterium formicium
Methanobacterium propionicium

Reduces SO|" to H 2 S
Reduces SOf" to H 2 S
Produces CH 4
Produces CH 4

degradation of wastes. If free molecular oxygen is absent, another molecule, for
example, nitrate ion (NO3), is used to degrade wastes such as methanol (CH 3 OH)
(Equation 2.1). When nitrate ions are used, denitrification occurs and dinitrogen gas
(N 2 ) is produced.

6N(X + 5CH.OH -* 2N ? + 5C0 9 + 7H ? + 6OH

(2.1)

Most bacteria within fixed-film processes and suspended growth processes are
facultative anaerobes, and these organisms also perform many significant roles in
the degradation of wastes. Approximately 80% of the bacteria within these aerobic
processes are facultative anaerobes. These organisms are found in relatively large
numbers not only in aerobic processes but also in anaerobic processes.

During the degradation of wastes within an anaerobic digester, facultative an-
aerobic bacteria, for example, Enter obacter spp., produce a variety of acids and
alcohols, carbon dioxide (C0 2 ), and hydrogen from carbohydrates, lipids, and pro-
teins. Some organisms, for example, Escherichia coli, produce malodorous com-
pounds such as indole and skatole.

Anaerobes are inactive in the presence of free molecular oxygen and may be
divided into two subgroups: oxygen-tolerant species and oxygen-intolerant species
or strict anaerobes (Table 2.2). Some anaerobes are strong acid producers, such as,
Streptococcus spp., whereas other anaerobes, such as Desulfomarculum spp., reduce
sulfate (SO|~) to hydrogen sulfide (H 2 S) (Equation 2.2). Although oxygen-tolerant
anaerobes survive in the presence of free molecular oxygen, these organisms cannot

RESPONSE TO FREE MOLECULAR OXYGEN 13

perform normal cellular activities, including the degradation of substrate, in the
presence of free molecular oxygen. Strict anaerobes, including methane-forming
bacteria, die in the presence of free molecular oxygen.

CH3COOH + SOf -> 2C0 2 + 2H 2 + H 2 S (2.2)

Numerous acid-forming bacteria are associated with methane-forming bacteria.
These organisms include facultative anaerobes that ferment simple, soluble organic
compounds and strict anaerobes that ferment complex proteins and carbohydrates.

The products of fermentation vary greatly depending on the bacteria involved in
the fermentative process. Therefore, changes in operational conditions that result in
changes in dominant bacteria also result in changes in the concentrations of acids
and alcohols that are produced during fermentation. Changes in the concentrations
of acids and alcohols significantly change the substrates available for methane-
forming bacteria, their activity, and, consequently, digester performance.

Most strict anaerobes are scavengers. These organisms are found where anaero-
bic conditions exist in lakes, river bottoms, human intestinal tracts, and anaerobic
digesters. Anaerobes survive and degrade substrate most efficiently when the
oxidation-reduction potential (ORP) of their environment is between -200 and -400
millivolts (mV). Any amount of dissolved oxygen in an anaerobic digester raises the
ORP of the sludge and discourages anaerobic activity including hydrolysis, aceto-
genesis, and methanogenesis. Therefore, sludges and wastewaters fed to an anaero-
bic digester should have no molecular oxygen. Settled and thickened sludges usually
do not have a residual dissolved oxygen concentration. These sludges typically have
a low ORP (-100 to -300 mV).

The ORP of a wastewater or sludge can be obtained by using an electrometric
pH meter with a millivolt scale and an ORP probe. The ORP of a wastewater or
sludge is measured on the millivolt scale of the pH meter.

The ORP is a measurement of the relative amounts of oxidized materials, such
as nitrate ions (NO 3) and sulfate ions (SO 4"), and reduced materials, such as ammo-
nium ions (NH4) (Table 2.3). At ORP values greater than +50 mV, free molecular
oxygen is available in the wastewater or sludge and may be used by aerobes and
facultative anaerobes for the degradation of organic compounds. This degradation
occurs under an oxic condition.

At ORP values between +50 and -50 mV, free molecular oxygen is not available
but nitrate ions or nitrite ions (NO?) are available for the degradation of organic
compounds. The degradation of organic compounds without free molecular oxygen
is an anaerobic condition. The use of nitrate ions or nitrite ions occurs under an
anoxic condition and is referred to as denitrification, clumping, and rising sludge in
the secondary clarifier of an activated sludge process.

At ORP values less than -50 mV, nitrate ions and nitrite ions are not available
but sulfate ions are available for the degradation of organic compounds. This degra-
dation also occurs without free molecular oxygen. When sulfate is used to degrade
organic compounds, sulfate is reduced and hydrogen sulfide is formed along with a
variety of acids and alcohols.

At ORP values less than -100 mV, the degradation of organic compounds pro-
ceeds as one portion of the compound is reduced while another portion of the com-
pound is oxidized. This form of anaerobic degradation of organic compounds is

BACTERIA

TABLE 2.3 Oxidation-reduction Potential (ORP) and Cellular Activity

Approximate

Carrier Molecule for

Condition

Respiration

ORP, mV

Degradation of Organic
Compounds

>+50

o 2

Oxic

Aerobic

+50 to -50

NOi or NO2

Anaerobic

Anoxic

<-50

soj-

Anaerobic

Fermentation, sulfate reduction

<-100

Organic Compound

Anaerobic

Fermentation, mixed acid
production

<-300

C0 2

Anaerobic

Fermentation, methane
production

Insoluble substrate

Exocellular slime

Cell membrane

Cell wall

Figure 2.1 There are two types of enzymes that are used by bacteria to degrade substrate. Exoen-
zymes are produced in the cell and released through the cell membrane and cell wall to hydrolyze
insoluble substrate that is adsorbed to the exocellular slime. Soluble wastes enter the bacterial cell
and are degraded by endoenzymes.

commonly known as mixed-acid fermentation because a mixture of acids, for
example, acetate, butyrate, formate, and propionate, are produced. A mixture of
alcohols is also produced during fermentation.

At ORP values less than -300 mV, anaerobic degradation of organic compounds
and methane production occur. During methane production, simple organic com-
pounds such as acetate are converted to methane, and carbon dioxide and hydro-
gen are combined to form methane.

ENZYMATIC ABILITY TO DEGRADE SUBSTRATE

Bacteria degrade substrate through the use of enzymes. Enzymes are proteinaceous
molecules that catalyze biochemical reactions. Two types of enzymes are involved
in substrate degradation — endoenzymes and exoenzymes (Figure 2.1).

Endoenzymes are produced in the cell and degrade soluble substrate within the
cell. Exoenzymes also are produced in the cell but are released through the "slime"
coating the cell to the insoluble substrate attached to the slime. Once in contact with
the substrate the exoenzyme solubilizes particulate and colloidal substrates. Once

ACETATE-FORMING BACTERIA

TABLE 2.4 Exoenzymes and Substrates

Substrate to be
Degraded

Exoenzyme
Needed

Example

Bacterium

Product

Polysaccharides

Proteins

Lipids

Saccharolytic

Proteolytic

Lipolytic

Cellulase
Protease
Lipase

Cellulomonas

Bacillus

Mycobacterium

Simple sugar
Amino acids
Fatty acids

solubilized, these substrates enter the cell and are degraded by endoenzymes. The
production of exoenzymes and solubilization of particulate and colloidal substrates
usually take several hours.

All bacteria produce endoenzymes, but not all bacteria produce exoenzymes. No
bacterium produces all the exoenzymes that are needed to degrade the large variety
of particulate and colloidal substrates that are found in sludges and wastewaters
(Table 2.4). Each exoenzyme as well as each endoenzyme degrades only a specific
substrate or group of substrates. Therefore, a large and diverse community of bac-
teria is needed to ensure that the proper types of exoenzymes and endoenzymes
are available for degradation of the substrates present.

The relative abundance of bacteria within an anaerobic digester often is greater
than 10 16 cells per milliliter. This population consists of saccharolytic bacteria
(~10 8 cells/ml), proteolytic bacteria (~10 6 cells/ml), lipolytic bacteria (~10 5 cells/ml),
and methane-forming bacteria (~10 8 cells/ml).

There are three important bacterial groups in anaerobic digesters with respect
to the substrates utilized by each group. These groups include the acetate-forming
(acetogenic) bacteria, the sulfate-reducing bacteria, and the methane-forming
bacteria. The acetate-forming bacteria and sulfate-reducing bacteria are reviewed
in this chapter, and the methane-forming bacteria are reviewed in Chapter 3.

ACETATE-FORMING BACTERIA

Acetate-forming (acetogenic) bacteria grow in a symbiotic relationship with
methane-forming bacteria. Acetate serves as a substrate for methane-forming bac-
teria. For example, when ethanol (CH 3 CH 2 OH) is converted to acetate, carbon
dioxide is used and acetate and hydrogen are produced (Equation 2.3).

CH.CHoOH + CO? -> CH.COOH + 2H:

(2.3)

When acetate-forming bacteria produce acetate, hydrogen also is produced. If
the hydrogen accumulates and significant hydrogen pressure occurs, the pressure
results in termination of activity of acetate-forming bacteria and lost of acetate
production. However, methane-forming bacteria utilize hydrogen in the production
of methane (Equation 2.4) and significant hydrogen pressure does not occur.

CO, + 4H 9 -> CH. + 2H.O

(2.4)

Acetate-forming bacteria are obligate hydrogen producers and survive only at
very low concentrations of hydrogen in the environment. They can only survive if
their metabolic waste — hydrogen — is continuously removed. This is achieved in

BACTERIA

SOA

Sulfate-reducing
bacteria

Methane- forming
bacteria

HoS

Figure 2.2 Many different groups of bacteria within the anaerobic digester often compete for the
same substrate and electron acceptor. An example of this competition is the used of acetate and
hydrogen by sulfate-reducing bacteria and methane-forming bacteria. Acetate is used by as a sub-
strate by both groups of bacteria. Methane is produced by methane-forming bacteria and a variety of
acids and alcohols are produced by sulfate reducing bacteria. Hydrogen is used with sulfate (SOt)
by sulfate-reducing bacteria and hydrogen sulfide (H 2 S) is produced.

their symbiotic relationship with hydrogen-utilizing bacteria or methane-forming
bacteria. Acetogenic bacteria reproduce very slowly. Generation time for these
organisms is usually greater than 3 days.

SULFATE-REDUCING BACTERIA

Sulfate-reducing bacteria also are found in anaerobic digesters along with acetate-
forming bacteria and methane-forming bacteria. If sulfates are present, sulfate-
reducing bacteria such as Desulfovibrio desulfuricans multiply. Their multiplication
or reproduction often requires the use of hydrogen and acetate — the same sub-
strates used by methane-forming bacteria (Figure 2.2).

When sulfate is used to degrade an organic compound, sulfate is reduced to
hydrogen sulfide. Hydrogen is needed to reduce sulfate to hydrogen sulfide. The
need for hydrogen results in competition for hydrogen between two bacterial
groups, sulfate-reducing bacteria and methane-producing bacteria.

When sulfate-reducing bacteria and methane-producing bacteria compete for
hydrogen and acetate, sulfate-reducing bacteria obtain hydrogen and acetate more
easily than methane-forming bacteria under low-acetate concentrations. At sub-
strate-to-sulfate ratios <2, sulfate-reducing bacteria out-compete methane-forming
bacteria for acetate. At substrate-to-sulfate ratios between 2 and 3, competition is
very intense between the two bacterial groups. At substrate-to-sulfate ratios >3,
methane-forming bacteria are favored.

The hydrogen sulfide produced by sulfate-reducing bacteria has a greater
inhibitory effect at low concentrations on methane-forming bacteria and acetate-
forming bacteria than on acid-forming bacteria.

Methane-forming Bacteria

Methane-forming bacteria are known by several names (Table 3.1) and are a mor-
phologically diverse group of organisms that have many shapes, growth patterns,
and sizes. The bacteria can be found as individual rods, curved rods, spirals, and cocci
(Figure 3.1) or grouped as irregular clusters of cells, chains of cells or filaments, and
sarcina or cuboid arrangements (Figure 3.2). The range in diameter sizes of
individual cells is 0.1-15 jam. Filaments can be up to 200 jam in length. Motile and
nonmotile bacteria (Figure 3.3) as well as spore-forming and non-spore-forming
bacteria can be found.

Methane-forming bacteria are some of the oldest bacteria and are grouped in the
domain Archaebacteria (from arachae meaning "ancient") (Figure 3.4). The domain
thrives in heat. Archaebacteria comprise all known methane-forming bacteria, the
extremely halophilic bacteria, thermoacidophilic bacteria, and the extremely ther-
mophilic bacteria. However, the methane-forming bacteria are different from all
other bacteria.

Methane-forming bacteria are oxygen-sensitive, fastidious anaerobes and are
free-living terrestrial and aquatic organisms. Although methane-forming bacteria
are oxygen sensitive, this is not a significant disadvantage. Methane-forming bacte-
ria are found in habitats that are rich in degradable organic compounds. In these
habitats, oxygen is rapidly removed through microbial activity. Many occur as sym-
bionts in animal digestive tracts. Methane-forming bacteria also have an unusually
high sulfur content: Approximately 2.5% of the total dry weight of the cell is sulfur.

The of methane-forming bacteria are classified in the domain Archaebacteria
because of several unique characteristics that are not found in the true bacteria
or Eubacteria. These features include 1) a "nonrigid" cell wall and unique cell
membrane lipid, 2) substrate degradation that produces methane as a waste, and 3)

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

METHANE-FORMING BACTERIA

TABLE 3.1 Commonly Used Names for Methane-
forming Bacteria

Methanogenic bacteria
Methanogens
Methane-forming bacteria
Methane-producing bacteria

specialized coenzymes. The cell wall lacks muramic acid, and the cell membrane
does not contains an ether lipid as its major constituent (Figure 3.5). Coenzymes
that are unique to methane-forming bacteria are coenzyme M and the nickel-
containing coenzymes F 42 o and F 430 . Coenzyme M is used to reduce carbon dioxide
(C0 2 ) to methane. The nickel-containing coenzymes are important hydrogen
carriers in methane-forming bacteria.

The coenzymes are metal laden organic acids that are incorporated into enzymes
and allow the enzymes to work more efficiently. The coenzymes are components of
energy-producing electron transfer systems that obtain energy for the bacterial cell
and remove electrons from degraded substrate (Figure 3.6).

•* j

(a)

(c)

o 9

'":•. 'to-

(d)

Figure 3. 1 Common shapes of methane-forming bacterial cells. Commonly occurring shapes of
methane-forming bacteria include rod or bacillus (a), curved rod (b), spiral (c), and coccus or spheri-
cal (d).

METHANE-FORMING BACTERIA

(a)

Figure 3.2 Common growth patterns of methane-forming bacterial cells. Commonly occurring growth
patterns of methane-forming bacteria include an irregular cluster (a) and a filamentous chain (b).

Figure 3.3 Non-motile and motile methane-forming bacteria. Methane-forming bacteria may be non-
motile (a) or motile (b, c, and d). Motile bacteria possess a flagellum or several flagella. The flagel-
lum or flagella may be located at one end of the cell or on the entire surface of the bacterial cell.

METHANE-FORMING BACTERIA

Eucarya

Animals

Bacteria

Plants

Spirochetes

Archaea

Methanogens

Gram-positive
bacteria

Thermophiles

Figure 3.4 Location of methane-forming bacteria on the phylogenetic tree. The phylogenetic tree
(the historical development of different life forms) contains old farachaej life forms closest to the base
of the tree, while new life forms closest to the end of the branches. The tree contains the domains
Thermopiles, Archaea, Eubacteria (true bacteria), and the Eucarya (higher life forms). The methane-
forming bacteria are found closest to the base of the tree.

METHANE-FORMING BACTERIA 21

Cell
wall

capsule

(a)

Cell
wall

]Q)^^6&«*&&&ft*bfe6^

capsule

muramic
acid

P&99S?

fiV

^^M^fc^

(b)

cell membrane

Figure 3.5 Cell wall of methane-forming bacteria. The cell wall of methane-forming bacteria (a) does
not contain muramic acid, while the cell of other bacteria (b) contains varying amounts of muramic
acid.

Cell Wall Cell Membrane

Figure 3.6 Electrons (e) released from broken chemical bonds of substrates inside a bacterial cell
are removed through the used of electron transport systems. These systems employ the use of pro-
teins that contain co-enzymes such as metals and vitamins.

METHANE-FORMING BACTERIA

TABLE 3.2 Examples of Methane-forming Bacteria with
and without a Protective Envelope

Genus

Envelope

Methanobacterium

Methanobrevibacter

Methanosarcina

Methanococcus

Methanogenium

Methanomicrobium

Methanospirillum

Absent

Present

(b)

Figure 3.7 Presence of an envelope on some methane-forming bacteria. Some methane-forming
bacteria possess an envelope (a) that provides added protection for the bacterial cell. Methane- forming
bacteria that do not possess an envelope (b) are easily lyzed in the presence of surfactants.

The unique chemical composition of the cell wall makes the bacteria "sensitive"
to toxicity from several fatty acids. Also, many methane-forming bacteria lack a
protective envelope around their cell wall (Table 3.2). Surfactants or hypotonic
shock easily lyse methane-forming bacteria that do not have this envelope
(Figure 3.7).

All methane-forming bacteria produce methane. No other organism produces
methane. Methane-forming bacteria obtain energy by reducing simplistic com-
pounds or substrates such as carbon dioxide and acetate (CH 3 COOH). Some
methane-forming bacteria are capable of fixing molecular nitrogen (N 2 ).

Methane-forming bacteria are classified according to their structure, substrate
utilization, types of enzymes produced, and temperature range of growth. There are
approximately 50 species of methane-forming bacteria that are classified in three
orders and four families (Table 3.3).

Methane-forming bacteria grow as microbial consortia, tolerate high concentra-
tions of salt, and are obligate anaerobes. The bacteria grow on a limited number of

METHANE-FORMING BACTERIA 23

TABLE 3.3 Groups of Methane-forming Bacteria

Order Family

Methanobacteriales Methanobacteriaceases

Methanococcales Methanococcaceae

Methanomicrobials Methanomicrobiaceas

Methanosarcinaceae

substrates. Methanobacterium formicium, for example, grows on formate, carbon
dioxide, and hydrogen and is one of the more abundant methane-forming bacteria
in anaerobic digesters. Methanobacterium formicium performs a significant role
in sludge digestion and methane production. Methanobacterium formicium and
Methanobrevibacter arboriphilus are two of the dominant methane-forming bacte-
ria in anaerobic digesters. The activity of these organisms and that of all methane-
forming bacteria is usually determined by measuring changes in volatile acid
concentration or methane production.

In nature, methane-forming bacteria perform two very special roles. They partic-
ipate in the degradation of many organic compounds that are considered biorecalci-
trant, that is, can only be degraded slowly, and they produce methane from the
degradation of organic compounds. Methane is poorly soluble in water, inert under
anaerobic conditions, non-toxic, and able to escape from the anaerobic environment.

Methane-forming bacteria are predominantly terrestrial and aquatic organisms
and are found naturally in decaying organic matter, deep-sea volcanic vents, deep
sediment, geothermal springs, and the black mud of lakes and swamps. These
bacteria also are found in the digestive tract of humans and animals, particularly
the rumen of herbivores and cecum of non-ruminant animals.

The rumen is a special organ in the digestive tract in which the degradation of
cellulose and complex polysaccharides occurs. Cows, goats, sheep, and deer are
examples of ruminant animals. The bacteria, including methane-forming bacteria,
that grow in the digestive tract of ruminant animals are symbionts and obtain most
of their carbon and energy from the degradation of cellulose and other complex
polysaccharides from plants. Ruminants cannot survive without the bacteria. The
bacteria and substrates produced by the bacteria through their fermentative
activities provide the ruminants with most of their carbon and energy.

Methane-forming bacteria grow well in aquatic environments in which a strict
anaerobic condition exists. The anaerobic condition of an aquatic environment
is expressed in terms of its oxidation-reduction potential or ORP (Table 3.4).
Methane-forming bacteria grow best in an environment with an ORP of less than
-300 mV. Most facultative anaerobes do well in aquatic environments with an ORP
between +200 and -200 mV.

There are Gram-negative and Gram-positive methane-forming bacteria that
reproduce slowly. Gram stain results (negative, positive, and variable) are different
within the same order of methane-forming bacteria because of their different types
of cell walls (Figure 3.8).

The reproductive times or generation times for methane-forming bacteria range
from 3 days at 35° C to 50 days at 10° C. Because of the long generation time of
methane-forming bacteria, high retention times are required in an anaerobic
digester to ensure the growth of a large population of methane-forming bacteria for

METHANE-FORMING BACTERIA

TABLE 3.4 Oxidation-Reduction Potential (ORP) and Cellular Activity

Approximate
ORP Values, mV

Molecule Used for
Degradation of Substrate

Type of Degradation or Respiration

>+50

Oxygen (0 2 )

+50 to -50

Nitrite (NO£) or nitrate (NOi)

<-50

Sulfate (SOJ-)

<-100

Organic (CHO)

<-300

Organic (CHO), C0 2 , CO, H 2

Oxic (aerobic)

Anoxic (anaerobic)

Sulfate reduction (anaerobic)

Fermentation (mixed acids and alcohol

production)
Fermentation (methane production)

Order of
Reagent

Reagent

Color of
Gram-positive
Bacteria

Color of
Gram- negative
Bacteria

Primary Stain

Crystal Violet

Violet

Mordant

Iodine

Violet

Decoloring Agent

95% Alcohol

Violet

Colorless

Counter Stain

Safranin

Violet

Red

Figure 3.8 Gram staining is a laboratory technique that separates bacteria into two grous, Gram-
positive and Gram-negative, depending on the response of bacteria to the stains Crystal violet and
Safranin. The technique was developed in 1884 by the Danish bacteriologist Christian Gram. Although
the technique was developed as a procedure for detecting pathogenic bacteria, it is used for taxo-
nomic (classification) and identification purposes.

The response of bacteria to the Gram stain is determined by microscopic examination of bacteria
that have been successively stained with a basic dye (Crystal violet), treated with an iodine solution
or mordant, and rinsed with an organic solvent such as acetone or alcohol. Gram-positive bacteria
retain the violet stain and are violet under microscopic examination. Gram-negative bacteria are decol-
orized by the solvent. The colorless, Gram-negative bacteria are stained with the counter stain Safranin
to impart a pink or red color.

the degradation of organic compounds. At least 12 days are required to obtain
a large population of methane-forming bacteria.

Methane-forming bacteria obtain their energy for reproduction and cellular
activity from the degradation of a relatively small number of simple substrates
(Table 3.5). These substrates include hydrogen, 1-carbon compounds, and acetate as
the 2-carbon compound. One-carbon compounds include formate, methanol, carbon
dioxide, carbon monoxide (CO), and methylamine.The most familiar and frequently
acknowledged substrates of methane-forming bacteria are acetate and hydrogen.
Acetate is commonly split to form methane while hydrogen is combined with carbon
dioxide to form methane. The splitting of acetate to form methane is known as
aceticlastic cleavage.

Each methane-forming bacterium has a specific substrate or group of substrates
that it can degrade (Table 3.6). Hydrogen can serve as a universal substrate for

METHANE-FORMING BACTERIA

TABLE 3.5
Bacteria

Substrates Used by Methane-forming

Substrate

Chemical Formula

Acetate

Carbon dioxide

Carbon monoxide

Formate

Hydrogen

Methanol

Methylamine

CH3COOH

C0 2

HCOOH

H 2

CH3OH

CH 3 NH 2

TABLE 3.6 Species of Methane-forming Bacteria and Their Substrates

Species

Substrate

Methanobacterium formicium
Methanobacterium thermoantotrophicum
Methanococcus frisius
Methanococcus mazei
Methanosarcina bakerii

Carbon dioxide, formate, hydrogen
Hydrogen, carbon dioxide, carbon monoxide
Hydrogen, methanol, methylamine
Acetate, methanol, methylamine
Acetate, carbon dioxide, hydrogen, methanol,
methylamine

methane-forming bacteria, and carbon dioxide functions as an inorganic carbon
source in the forms of carbonate (COf") or bicarbonate (HCO3). Carbon dioxide
also serves as a terminal acceptor of electrons released by degraded substrate.

Other 1-carbon compounds that can be converted to substrates for methane-
forming bacteria include dimethyl sulfide, dimethylamine, and trimethylamine.
Several alcohols including 2-propanol and 2-butanol as well as propanol and butanol
may be used in the reduction of carbon dioxide to methane.

The majority of methane produced in an anaerobic digester occurs from the use
of acetate and hydrogen by methane-forming bacteria. The fermentation of sub-
strates such as acetate (aceticlastic cleavage) results in the production of methane
(Equation 3.1), and the reduction of carbon dioxide also results in the production
of methane (Equation 3.2).

CH3COOH -> CH 4 + C0 2
CO9 + 4H 9 -> CH. + 2H.O

(3.1)

(3.2)

Aceticlastic cleavage of acetate and reduction of carbon dioxide are the
two major pathways to methane production. Fermentation of propionate
(CH 3 CH 2 COOH) and butyrate (CH 3 CH 2 CH 2 COOH) are minor pathways to
methane production. However, the fermentation of propionic acid to methane
requires two different species of bacteria and two microbial degradation steps
(Equations 3.3 and 3.4). In the first reaction, methane and acetate are produced
from the fermentation of propionate by a volatile acid-forming bacterium (Syntro-
phobacter wolinii) and a methane-forming bacterium. In the second reaction,
methane is produced from the cleavage of acetate by a methane-forming bacterium.
These reactions occur only if hydrogen and formate are kept low (used) by

26 METHANE-FORMING BACTERIA

methane-forming bacteria. Accordingly, the accumulation of propionate is a
common indicator of stress in an anaerobic digester.

4CH 3 CH 2 COOH + 2H 2 -> 4CH 3 COOH + C0 2 + 3CH 4 (3.3)

4CH 3 COOH -> 4CH 4 + 4C0 2 (3.4)

Butyrate also is degraded to methane through two microbial degradation steps
(Equations 3.5 and 3.6). The degradation steps again are mediated by two different
bacteria. In the first reaction, methane and acetate are produced from the fermen-
tation of butyrate by a volatile acid-forming bacterium and a methane-forming bac-
terium. In the second reaction, methane is produced from the cleavage of acetate
by a methane-forming bacterium. Because butyrate can be used indirectly by
methane-forming bacteria, its accumulation is an indicator of stress in an anaerobic
digester.

CH 3 CH 2 CH 2 COOH + 2H 2 -> 4CH 3 COOH + C0 2 + CH 4 (3.5)

4CH 3 COOH -> 4CH 4 + 4C0 2 (3.6)

No species of methane-forming bacteria can utilize all substrates. Therefore,
successful fermentation of substrates in an anaerobic digester requires the presence
of not only a large number of methane-forming bacteria but also a large diversity
of methane-forming bacteria.

There are three principal groups of methane-forming bacteria. These groups are
1) the hydrogenotrophic methanogens, 2) the acetotrophic methanogens, and 3) the
methylotrophic methanogens. The term "trophic" (from trophe, "nourishment")
refers to the substrates used by the bacteria.

GROUP 1 HYDROGENOTROPHIC METHANOGENS

The hydrogenotrophic methanogens use hydrogen to convert carbon dioxide to
methane (Equation 3.7). By converting carbon dioxide to methane, these organisms
help to maintain a low partial hydrogen pressure in an anaerobic digester that is
required for acetogenic bacteria.

C0 2 + 4H 2 -> CH 4 + 2H 2 (3.7)

GROUP 2 ACETOTROPHIC METHANOGENS

The acetotrophic methanogens "split" acetate into methane and carbon dioxide
(Equation 3.8). The carbon dioxide produced from acetate may be converted by
hydrogenotrophic methanogens to methane (Equation 3.7). Some hydrogeno-
trophic methanogens use carbon monoxide to produce methane (Equation 3.9).

4CH 3 COOH -> 4C0 2 + 2H 2 (3.8)

4CO + 2H 2 -> CH 4 + 3C0 2 (3.9)

GROUP 3 METHYLOTROPHIC METHANOGENS 27

The acetotrophic methanogens reproduce more slowly than the hydrogeno-
trophic methanogens and are adversely affected by the accumulation of hydrogen.
Therefore, the maintenance of a low partial hydrogen pressure in an anaerobic
digester is favorable for the activity of not only acetate-forming bacteria but also
acetotrophic methanogens. Under a relatively high hydrogen partial pressure,
acetate and methane production are reduced.

GROUP 3 METHYLOTROPHIC METHANOGENS

The methylotrophic methanogens grow on substrates that contain the methyl group
(-CH 3 ). Examples of these substrates include methanol (CH 3 OH) (Equation 3.10)
and methylamines [(CH 3 ) 3 -N] (Equation 3.11). Group 1 and 2 methanogens
produce methane from C0 2 and H 2 . Group 3 methanogens produce methane
directly from methyl groups and not from C0 2 .

3CH 3 OH + 6H -> 3CH 4 + 3H 2 (3.10)

4(CH 3 ) 3 - N + 6H 2 -> 9CH 4 + 3C0 2 + 4NH 3 (3.11)

The use of different substrates by methane-forming bacteria results in different
energy gains by the bacteria. For example, hydrogen-consuming methane produc-
tion results in more energy gain for methane-forming bacteria than acetate degra-
dation. Although methane production using hydrogen is the more effective process
of energy capture by methane-forming bacteria, less than 30% of the methane
produced in an anaerobic digester is by this method. Approximately 70% of the
methane produced in an anaerobic digester is derived from acetate. The reason
for this is the limited supply of hydrogen in an anaerobic digester. The majority
of methane obtained from acetate is produced by two genera of acetotrophic
methanogens, Methanosarcina and Methanothrix.

Reproduction of methane-forming bacteria is mostly by fission, budding, con-
striction, and fragmentation (Figure 3.9). Methane-forming bacteria reproduce
very slowly. This slow growth rate is due to the relatively small amount of energy
obtained from the use of their limited number of substrates. Therefore, a relatively
large quantity of substrates must be fermented for the population of methane-
forming bacteria to double, that is, a relatively small quantity of cells or sludge
is produced for a relatively large quantity of substrate degraded. Therefore,
anaerobic digesters produce relatively small quantities of bacteria cells or sludge
(solids).

Under optimal conditions, the range of generation times of methane-forming
bacteria may be from a few days to several weeks. Therefore, if solids retention time
(SRT) is short or short-circuiting or early withdrawal of digester sludge occurs, the
population size of methane-forming bacteria is greatly reduced. These conditions
decrease the time available for reproduction of methane-forming bacteria, that is,
the bacteria are removed from the digester faster than they can reproduce. This
results in poor digester performance or failure of the digester.

With increasing retention time the production of new methane-forming bacteria
gradually decreases as a result of increased energy requirements of the cells in order

28 METHANE-FORMING BACTERIA

(b)

Figure 3.9 Modes of reproduction for methane-forming bacteria. Methane-forming bacteria repro-
duce very slowly. Generation time for these organisms is usually greater than 3 days. Reproduction
is asexual and may occur through fission (a), budding (b), fragmentation (c), and constriction (d).

TABLE 3.7 Optimal Growth Temperature of Some
Methane-forming Bacteria

Genus

Temperature Range, °C

Methanobacterium

Methanobrevibacter

Methanosphaera

Methanothermus

Methanococcus

Methanocorpusculum

Methanoculleus

Methanogenium

Methanoplanus

Methanospirillum

Methanococcoides

Methanohalobium

Methanohalophilus

Methanolobus

Methanosarcina

Methanothrix

37-45
37-40
35-40
83-88
35-40
65-91
30-40
35-40
20-40
30-40
35-40
30-35
50-55
35-45
35-40
30-40
50-55
35-50

GROUP 3 METHYLOTROPHIC METHANOGENS 29

to maintain cellular activity (more degradation of substrate). Therefore, increasing
retention time of a properly operated anaerobic digester results in decreased sludge
production. Increasing retention time produces a large consumption of substrate by
slowing reproducing bacteria as an energy requirement of old cells (sludge) for the
maintenance of cellular activity.

Most methane-forming bacteria are mesophiles or thermophiles, with some
bacteria growing at temperatures above 100°C (Table 3.7). Mesophiles are those
organisms that grow best within the temperature range of 30-35°C, and ther-
mophiles are those organisms that grow best within the temperature range of
50-60°C. Some genera of methane-forming bacteria have mesophilic and ther-
mophilic species.

It is difficult to grow methane-forming bacteria in pure culture. Standard labo-
ratory enumeration techniques are not suitable for methane-forming bacteria. This
difficulty is caused by 1) their extreme obligate anaerobic nature and the probabil-
ity that they are killed rapidly by relatively short time exposures to air compared
with other anaerobes and 2) their limited number of substrates. To correct for the
oxygen sensitivity of methane-forming bacteria in laboratory experiments with pure
cultures, the "Hungate" technique is used. Growth or cell masses of methane-
forming bacteria may be gray, green, greenish black, orange-brown, pink, purple,
yellow, or white.

Respiration

Respiration is one of many cellular processes. For the purpose of this text, respira-
tion is considered to be the degradation of substrate to obtain cellular nourishment.
During respiration large compounds of high energy content are broken down to
small compounds of low energy content (Figure 4.1). Much of the energy lost by
the large compounds is captured by the respiring organisms. This capture results in
a gain in the amount of useful energy.

Two types of nourishment are obtained from the degradation of substrate —
carbon and energy. Carbon is required for the synthesis of cellular materials for
growth and reproduction. Energy is required for cellular activity including
reproduction. Bacteria may obtain their nourishment from one substrate or two
substrates. The energy substrate may be organic or inorganic.

Most bacteria use organic compounds to obtain carbon and energy. These organ-
isms are known as organotropic. The term "troph" comes from the Greek trophe,
meaning "nourishment." Organotropic obtain their carbon and energy from the
degradation of organic compounds such as glucose (C^nOe)- An example of an
organotroph is Zoogloea ramigera. This bacterium is a floe former that degrades
soluble organic compounds in the activated sludge and trickling filter processes.
Another example of an organotroph is Pseudomonas aeruginosa. This bacterium
degrades soluble organic compounds in activated sludge and trickling filter
processes and anaerobic digesters.

Some bacteria use inorganic compounds to obtain carbon and energy. These
organisms are known as chemoautotrophs. They obtain their carbon from carbon
dioxide (C0 2 ) and their energy from inorganic compounds. An example of a
chemoautotroph is Nitrobacter winogradski. This bacterium oxidizes nitrite ions
(NO2) to nitrate ions (NO2) to obtain energy and uses carbon dioxide in the form

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

RESPIRATION

Glucose,
high energy compound

fermentation

lower energy compounds

Acetate

HoO

Ethanol

Figure 4. 1 The degradation of organic compounds results in the production of small compounds that
contain less energy than the degraded compound. Inorganic compounds as well as organic com-
pounds are produced from the degradation of organic compounds.

of bicarbonate alkalinity (HCO3) as its carbon source. Nitrobacter winogradski is
found in activated sludge and trickling filter processes.

When substrate is degraded in a bacterial cell, energy is obtained from the elec-
trons that are released from the broken chemical bonds of the substrate (Figure
4.2). The electrons released from the substrate are transferred along a series of elec-
tron carrier molecules — an electron transport system (Figure 4.3). As the electrons
are transferred from one carrier molecule to another, some of the energy from the
electrons is taken up by the carrier molecules to form high-energy phosphate bonds
in the molecule adenosine triphosphate, or ATP (Figure 4.4). Phosphate bonds are
the energy "currency" of the cell. When the cell needs energy, energy is "withdrawn"
by breaking a phosphate bond. When this occurs, ATP is converted to adenosine
diphosphate, or ADP. When the cell stores energy, energy is "deposited" by pro-
ducing a phosphate bond. When this occurs, ADP is converted to ATP. Energy
storage and release are based on a coupling and uncoupling of phosphate groups
(POl).

Eventually, the electrons are removed from the cell by a final electron carrier
molecule. This molecule takes the electrons from the electron transport system and
releases the electrons to the surrounding environment (Figure 4.5). Several final
electron carrier molecules may be used by bacteria (Table 4.1). The molecule used
by bacteria determines the form of respiration (Table 4.2).

The final electron carrier molecule used by the bacteria is dependent on several
factors. These factors include 1) the presence or absence of the molecule, 2) the pres-
ence or absence of the necessary bacterial enzymes to use the molecule, and 3) the
oxidation-reduction potential (ORP) of the wastewater or sludge that harbors the
molecule and the bacteria (Table 4.3).

RESPIRATION

Cell Wall

Cell Membrane

Energy from the electron is captured in an electron transport system.

Figure 4.2 Energy from degraded organic compounds is obtained by the capture of released elec-
trons from broken chemical bonds. The captured electrons are transported along an electron trans-
port system. The electrons release energy as they move along the transport system.

used to make high energy
phosphate bonds

to final electron
** transport molecule, e.g.,

2 , NO3-, so 4 2 -
and removed from the cell

Figure 4.3 The electron transport system consists of a series of interlocking, electron transport mol-
ecules that pass the electrons from one molecule to another. As the electrons are passed along the
transport system, energy from the electrons is released and captured by the bacterial cell. The capture
energy is used to form high energy phosphate bonds.

For a final electron carrier molecule to be used by a bacterium, the molecule must
be available and the bacterium must have the ability (enzymes) to use the mole-
cule. Finally, the ORP of the bacterial environment (wastewater or sludge) deter-
mines the order or sequence of utilization of the final electron carrier molecules.

Respiration may be complete or incomplete. Complete respiration results in the
transfer of the carbon in the organic substrate to carbon dioxide and new bacterial

RESPIRATION

High Energy Bonds

Adenine

Ribose

Phosphate

Thosphate

Adenosine diposphate (ADP)

Adenosine triphosphate (ATP)

Figure 4.4 Energy captured by bacterial cells by their electron transport system is used to form high
energy phosphate bonds. When bonds are formed, ATP is produced. When the bonds are broken,
energy is released and ADP is produced.

Cell Wall

Cell Membrane

02 or NO3- or S04 2-

H2O or N2 or H2S

Figure 4.5 Electrons released from the degradation of organic wastes are removed from the bacte-
rial cell by a final, electron transport molecule such as free molecular oxygen, nitrate ion, and sulfate
ions. The choice of final, electron transport molecule determines the form of respiration.

RESPIRATION

TABLE 4.1 Final Electron Carrier Molecules in Order of Sequence of Utilization at
Wastewater Treatment Plants

Order of Sequence

Electron

Occurrence

Reduced Product

of Utilization

Carrier

(Example)

o 2

Aeration tank

H 2

NOi

Denitrification tank and secondary
clarifier

N 2) N 2

sof-

Secondary clarifier and thickener

S 2 - (H 2 S)

CH 2 0*

Thickener and anaerobic digester

Volatile organic acids

C0 2

Anaerobic digester and sewer
system

CH 4

Organic compound

TABLE 4.2 Forms of Respiration

Respiration

Biochemical Reaction

Complete/Incomplete Respiration

Aerobic or oxic

Anaerobic: anoxic

(denitrification)
Anaerobic: fermentation

(sulfate reduction)

Anaerobic: fermentation
(mixed acids and alcohol)

Anaerobic: fermentation
(methane production)

CH 2 + 2 -^ C0 2 + H 2 +

cells
CH 2 + NO3 -* C0 2 + H 2 +

N 2 + N 2 + cells
CH 2 + SOf- -^ C0 2 + H 2 +

H 2 S + acids + alcohols +

cells
CH 2 -> C0 2 + H 2 + acids

+ alcohols + cells
CH 2 + C0 2 -^ H 2 + CH 4 +

cells

Complete
Complete
Incomplete with exceptions

Incomplete
Incomplete

TABLE 4.3 Oxidation-Reduction Potential and Respiration

Approximate
Millivolts (mV)

Final Electron
Carrier Molecule

Respiration Occurring

>+50

o 2

+50 to

-50

NOi

<-50

sor

<-100

CH 2
Organic molecule

<-300

C0 2 (carbonate, CO|")*

Aerobic or oxic

Anaerobic or anoxic

Anaerobic or sulfate reduction

Anaerobic or mixed acids and alcohol fermentation

Anaerobic or methane fermentation

Carbon dioxide as carbonate

cells. Incomplete respiration results in the transfer of the carbon in the organic sub-
strate to carbon dioxide, new bacterial cells, and organic products such as simple
acids and alcohols.

The sequence of utilization for the carrier molecules is: 2 , NO3, SOl", CH 2 0, and
C0 2 . By using 2 to degrade the organic compounds, bacterial cells obtain more
energy from the organic compounds than through the use of any other carrier mol-
ecule (Table 4.4). With more energy, more bacterial growth (reproduction) or sludge
is produced (Table 4.4). If 2 is not available for bacterial use and NO3 is available,

36 RESPIRATION

TABLE 4.4 Final Electron Carrier Molecule, Energy Yield, and Cell (Sludge) Production

Final Electron

Form of

Energy

Pound of Cells Produced

Carrier Molecule

Respiration

Yield Rank

per
Pound of COD Degraded

o 2

Aerobic or oxic

-0.4-0.6

NOi

Anaerobic or anoxic

-0.4

sof-

Anaerobic:
sulfate reduction

0.04-0.1

Organic molecule

Anaerobic:

mixed acids and
alcohol

0.04-0.1

co 2

Anaerobic:

methane production

0.02-0.04

NO3 is used next, if the bacteria have the enzymatic ability to use nitrate ions. The
use of NO3 provides the second-largest energy yield for bacterial cells and the
second-largest yield in bacterial growth (sludge production). Because of decreasing
yields in energy and bacterial growth with different carrier molecules, there is a
sequential order with respect to the choice of final electron carrier molecules. This
order is determined by the ORP of the bacterial environment.

ORP is an indicator of the capacity of the molecules in the wastewater or sludge
to release or gain electrons (oxidation or reduction, respectively). This measurement
also is an indicator of the form of respiration that may occur (Table 4.3).

Generally, at values greater than +50 mV aerobic respiration may occur and from
+50 to -50 mV anoxic respiration (denitrification) may occur. At values less than
-100 mV, anaerobic respiration may occur. At values less than -50 mV sulfate (SOj)
reduction (also known as fermentation) may occur. At values less than -100 mV,
mixed acids and alcohol fermentation may occur. Methane fermentation may start
at values less than -200 mV. However, in a mixed culture of fermenting organisms
as would exist in an anaerobic digester, methane fermentation or the growth of
methane-forming bacteria does not occur until the ORP is less than -300 mV. This
is due to the inability of the methane-forming bacteria to successfully compete with
other fermenting organisms at values greater than -300 mV.

The use of 2 (Equation 4.1) and NOj (Equation 4.2) as final electron carrier
molecules results in complete degradation of CH 2 0. In complete degradation, all of
the carbon in the CH 2 is assimilated into new bacterial cells and C0 2 . However,
the use of NO3 results in a smaller production of bacterial cells and a greater
production of C0 2 (Table 4.4).

CH 2 + 2 -> cells + C0 2 + H 2 (4.1)

CH 2 + NO3 -> cells + C0 2 + H 2 + N 2 + N 2 (4.2)

The use of nitrate ions by bacteria to degrade carbonaceous compounds is
known as anoxic respiration or denitrification. The occurrence of denitrification
in secondary clarifiers of activated sludge processes is known as rising sludge or
clumping. Many different groups of bacteria are capable of using nitrate ions to

RESPIRATION 37

TABLE 4.5 Significant Organic Compounds Produced
During Anaerobic Fermentation

Name Formula

Acetate CH 3 COOH

Acetone CH 3 COCH 3

Acetaldehyde CH 3 CHO

Butanol CH 3 (CH 2 ) 2 CH 2 OH

Butanone C 2 H 5 COCH 3

Butyraldehyde C 2 H 5 CHO

Caproic acid CH 3 (CH 2 ) 4 COOH

Formaldehyde CH 2

Formate HCOOH

Ethanol CH 3 CH 2 OH

Lactate CH 3 CHOHCOOH

Methane CH 4

Methanol CH 3 OH

Propanol CH 3 CH 2 CH 2 OH

Propionate CH 3 CH 2 COOH

Valeric acid CH 3 (CH 2 ) 3 COOH

TABLE 4.6 Groups of Chemolithotrophs Found in
Wastewater Treatment Plants

Group

Substrate

Product

Ammonium oxidizers

NH;

N0 2

Hydrogen bacteria

H 2

H +

Iron bacteria

Fe 2+

Fe 3+

Nitrite oxidizers

N0 2

NOi

Sulfur bacteria

H 2 S

S°

sor

degrade carbonaceous compounds. These bacteria include facultative and anaero-
bic bacteria.

With exceptions, all other forms of respiration (anaerobic fermentation) are
incomplete. During these forms of respiration, the carbon within the CH 2 is
assimilated into new bacterial cells, C0 2 , and a variety of simplistic, soluble organic
molecules, mostly acids and alcohols (Table 4.5). Because some of the carbon
from the CH 2 is assimilated into a variety of organic molecules, the produc-
tion of bacterial cells is greatly reduced (Table 4.4). However, several sulfate-
reducing bacteria are capable of complete respiration. These sulfate-reducing
bacteria provide the exceptions for complete respiration under anaerobic
respiration.

For most obligate anaerobic bacteria to grow, the absence of free molecular
oxygen and a low redox potential are required. Methane-forming bacteria only grow
in anaerobic digester sludge with a redox potential less than -300 mV. Also, the
digester sludge must have thiol group-containing (-SH) compounds. These com-
pounds produce a reducing environment.

38 RESPIRATION

Sulfates, carbonates (COf"), and bicarbonates are the primary electron carrier
molecules for facultative anaerobic and anaerobic bacteria. If sulfate is used as the
final electron carrier molecule, dissimilatory sulfate reduction occurs (Equation 4.2).
During dissimilatory sulfate reduction, sulfate serves as the electron acceptor and
hydrogen sulfide (H 2 S) is produced. Only a relatively small number of genera of
bacteria are capable of dissimilatory sulfate reduction. Desulfovibrio is the pre-
dominant genus responsible for the conversion of sulfate to hydrogen sulfide.
Desulfotomaculum also is capable of reducing sulfate. Conversely, in an oxidizing
environment, sulfides (HS~) are oxidized to sulfate. Genera of bacteria containing
species of sulfide-oxidizing bacteria are Thiobacillus, Thio bacterium, and Thiospira.

S0 4 2 ~ + CH 2 -> H 2 S + C0 2 + H 2 (4.2)

In the absence of an inorganic final electron carrier molecule, an organic
compound may be used to achieve respiration. If an organic compound is used,
mixed-acid fermentation occurs.

The substrate degraded or electron-releasing compound used during respiration
may be organic, for example, glucose, or inorganic, for example, ammonium ions
(NH4). Bacteria that respire by using organic substrates are organotropic, whereas
bacteria that respire by using inorganic substrates are chemolithotrophs. Several
important groups of chemolithotrophs are found in wastewater treatment processes
(Table 4.6). These groups include ammonium oxidizers, hydrogen bacteria, iron
bacteria, nitrite oxidizers, and sulfur bacteria.

Anaerobic Food Chain

In natural habitats that are void of free molecular oxygen and nitrate ions, insolu-
ble and complex organic compounds are degraded by different groups of bacteria
through a variety of anaerobic or fermentative biochemical reactions. These reac-
tions result in the production of soluble and simplistic organic compounds. These
compounds do not accumulate in natural habitats.

As one group of bacteria produces soluble compounds they are quickly degraded
as substrate by another group of bacteria. The bacteria form a chain — an anaerobic
food chain — in which large, complex compounds are degraded to more simplistic
compounds as they are passed along the food chain (Figure 5.1).

In freshwater habitats, methane fermentation is the terminal link in the anaero-
bic food chain. Here, complex organic compounds have been degraded or reduced
to methane, carbon dioxide, and minerals. Some of the carbon dioxide produced
during the degradation of organic compounds is reduced to form methane.

For organic compounds to be degraded through the food chain, the compounds
must be degraded to simplistic organic and inorganic compounds that can be used
as substrate by methane-forming bacteria. These compounds include the organics
formate, methanol, methylamine, and acetate and the inorganics hydrogen and
carbon dioxide.

Methane is produced by methane-forming bacteria from organic compounds
such as acetate (Equation 5.1) or from the combination of the inorganics carbon
dioxide [as bicarbonate (HCO3) or carbonate (COf - )] with hydrogen (H 2 ) (Equa-
tions 5.2 and 5.3).

CH3COOH -> CH 4 + C0 2 (5.1)

4H 2 + HCO3 + H + -> CH 4 + 3H 2 (5.2)

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

ANAEROBIC FOOD CHAIN

Complex Substrates,
Carbohydrates, Lipids, Proteins

Simple Substrates,
Sugars, Fatty Acids, Amino Acids

Acids and
Alcohols

CO2 + H2

Acetate

Formate

Methanol,
Methylamine

Figure 5. 1 The anaerobic food chain consists of several groups of facultative anaerobes and anaer-
obes that degrade and transform complex organic compounds into simplistic organic compounds. The
final organic compound produced in the anaerobic food is methane. This compound is the most
reduced form of carbon.

4H 2 + CO f + 2H + -> CH 4 + 3H 2

(5.3)

Methane is the most reduced organic compound. The production of methane is
the final step of the anaerobic food chain. Methane-forming bacteria are respon-
sible for this step, and therefore they are of critical importance for the success of
the anaerobic food chain.

Organic compounds that cannot be used directly as substrate by methane-
forming bacteria can be used indirectly if they are converted to compounds such as

ANAEROBIC FOOD CHAIN 41

acetate. Examples of compounds that can be converted to acetate include butyrate
and propionate.

Within the anaerobic food chain there are syntrophic relationships between bac-
teria. In these relationships at least two different bacteria are involved and the activ-
ity of one organism is dependent on the activity of another organism. An example
of a syntrophic relationship in the anaerobic food chain is the association between
hydrogen-producing bacteria and hydrogen-consuming bacteria. In this association
hydrogen-producing bacteria degrade organic compounds to more simplistic
compounds and hydrogen (Equation 5.4).

glucose + 4H 2 -> 2 acetate + 2HCO3 + 2H + + 4H 2 (5.4)

However, the degradation of organic compounds by hydrogen-producing bacte-
ria occurs only if the partial pressure of hydrogen is kept low, that is, <10~ 4 atmos-
pheres. Therefore, it is essential that hydrogen does not accumulate to a partial
pressure >10"* atmospheres. In the anaerobic food chain, hydrogen is consumed and
hydrogen partial pressure is maintained at a low value by hydrogen-consuming
bacteria, including methane-forming bacteria. These organisms combine hydrogen
with carbon dioxide to produce methane.

As long as the hydrogen partial pressure is maintained at a low level, hydrogen-
producing bacteria continue to degrade organic compounds and the anaerobic food
chain continues to function. Fermentation under low partial pressure of hydrogen
helps to ensure that fermentation products other than methane and carbon dioxide
do not accumulate.

The partial pressure of hydrogen in the rumen, in mud, and in anaerobic digesters
is kept low by the microbial activity of methane-forming bacteria. This favors the
organisms that produce hydrogen and acetate. The maintenance of a low hydrogen
pressure is necessary for proper microbial activity within the anaerobic food chain.

Acetate is the most important organic compound in the anaerobic food chain.
Acetate is the substrate most commonly used by methane-forming bacteria and may
be degraded in the absence of sulfate. In the presence of sulfate, acetate is not split
to methane and carbon dioxide.

Fermentation

The term "fermentation" was first used by Pasteur to define respiration in the
absence of free molecular oxygen. Fermentation can be broadly defined as respira-
tion that occurs in the dark (no photosynthesis) and does not involve the use of free
molecular oxygen, nitrate ions, or nitrite ions as the final electron acceptors of
degraded organic compounds. Therefore, respiration may occur through several
fermentative pathways including sulfate reduction, mixed acid production, and
methane production.

Fermentation is a form of anaerobic respiration. The bacteria that perform fer-
mentation are facultative anaerobes and anaerobes. Fermentation involves the
transformation of organic compounds to various inorganic and organic products.
During fermentation a portion of an organic compound may be oxidized while
another portion is reduced. It is from this oxidation-reduction of organic compounds
that fermenting bacteria obtain their energy and produce numerous simplistic and
soluble organic compounds.

Fermentative bacteria are capable of performing a variety of oxidation-reduction
reactions involving organic compounds, carbon dioxide, carbon monoxide (CO),
molecular hydrogen, and sulfur compounds. Fermentative bacteria include faculta-
tive anaerobes, aerotolerant anaerobes, and strict anaerobes. Some fermenta-
tive bacteria such as the Clostridia (Table 6.1) and Escherichia coli (Table 6.2)
produce a large variety of products, whereas other fermentative bacteria such as
Acetobacterium produce a very small number of products. As environmental or
operational conditions change, for example, pH and temperature, the bacteria that
are active and inactive also change. These changes in activity are responsible for
changes in the types and quantities of compounds that are produced through
fermentation.

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

44 FERMENTATION

TABLE 6.1 Fermentative Products of Clostridia

Organic Inorganic

Acetate Carbon dioxide

Acetone Hydrogen

Butanol

Butyrate

Ethanol

Lactate

TABLE 6.2 Fermentative Products of Escherichia coli

Organic Inorganic

Acetate Carbon dioxide

2,3-Butanediol Hydrogen

Ethanol

Formate

Lactate

Succinate

Some products of fermentative bacteria such as acetate and formate can be used
as substrate for methane-forming bacteria. Some products of fermentative bacteria
such as butyrate and propionate may be used as substrate for methane-forming bac-
teria only if they are converted to compounds such as acetate and formate. Some
products of fermentative bacteria cannot be used as substrate by methane-forming
bacteria. Therefore, changes in operational conditions of an anaerobic digester such
as pH and temperature determine which fermentative bacteria are dominant and
consequently which fermentative products are dominant. These products in turn sig-
nificant influence the activity of methane-forming bacteria and the efficiency of the
anaerobic digester process.

A relatively large variety of organic compounds and inorganic compounds are
produced through fermentation. The compounds obtained through fermentation are
dependent on the compounds fermented, the bacteria involved in the fermentation
process, and the operational conditions that exist during fermentation. There are
several types of fermentation, which are classified according to the major end prod-
ucts obtained in the fermentation process (Equation 6.1). The types of fermenta-
tion include acetate, alcohol (ethanol), butyrate, lactate, mixed acid, mixed acid and
butanediol, propionate and succinate, sulfide, and methane (Figure 6.1).

reactants — > products (6.1)

ACETATE FERMENTATION

Acetate is produced in several fermentative pathways. A large diversity of
bacteria, collectively known as acetogenic or acetate-forming bacteria, produces

ACETATE FERMENTATION

Hexose, e.g.,
Glucose, Fructose

Lactate
Ethanol, C02

Butyrate, Butanol,

Isopropanol,

Ethanol,

C02

Propionate,
Acetate,
C0 2

Lactate

Alcohol

Fermentation

Butyrate

Butanediol

Fermentation

Propionate

Mixed Acid

Ethanol,
CO2

Butanediol,
C0 2

Fermentation

Acetate, Ethanol,
Formate, CO2

Figure 6.1 There are numerous types of fermentation. The type of fermentation that occurs is clas-
sified or named after the major product(s) obtained in the fermentation process.

nongaseous acetate. These organisms include bacteria in the genera Aceto bacterium,
Clostridium, and Sporomusa. Some acetogenic bacteria are thermophilic.

Several biochemical reactions are used by acetogenic bacteria to produce acetate.
Most acetogenic bacteria produce acetate from H 2 and C0 2 (Equation 6.2), while
some produce acetate from H 2 and carbon monoxide (Equation 6.3). Some
acetogenic bacteria produce acetate from C0 2 and methanol (Equation 6.4), and
often six-carbon sugars or hexoses are degraded to acetate (Equation 6.5). Even
propionate is converted to acetate.

4H 2 + 2C0 2 -> CH3COOH + 2H 2

4CO + 2H 2 -> CH3COOH + 2C0 2

4CH 3 OH + C0 2 -> 3CH 3 COOH + 2H 2

C 6 H 12 6 -> 3CH 3 COOH

(6.2)
(6.3)
(6.4)
(6.5)

46 FERMENTATION

ALCOHOL (ETHANOL) FERMENTATION

Although alcohol fermentation is the domain of yeast (mostly Saccharomyces),
alcohol is produced by several species of bacteria in the genera Erwinia, Sarcina,
and Zymomonas. These organisms produce ethanol from the anaerobic degrada-
tion of hexoses such as glucose (Equation 6.6). At relatively low pH values (<4.5),
alcohol is produced by bacteria in the genera Enter obacter and Serratia.

C 6 H 12 6 -> 2C 2 H 5 OH + 2C0 2 (6.6)

BUTYRATE FERMENTATION

Butyrate (CH 3 CH 2 CH 2 COOH) is a major fermentative product of many bacteria.
Strict anaerobes in the genera Clostridium and Butyrivibrio ferment a variety of
sugars to produce butyrate (Equation 6.7). Under low pH values (<4.5), several
Clostridia produce small amounts of acetone and n-butanol. rc-Butanol is highly toxic
to bacteria because of its interference with cellular membrane functions.

hexose -> CH 3 CH 2 CH 2 COOH (6.7)

LACTATE FERMENTATION

A common product of many fermentative reactions is lactate. The production
of lactate is achieved by the aerotolerant, strictly fermentative lactate-forming
bacteria (Table 6.3). Lactate-forming bacteria are highly saccharolytic.

There are three biochemical reactions for lactate production from sugars such as
glucose (Equations 6.8, 6.9, and 6.10). In addition to glucose, other sugars fermented
by lactate-forming bacteria include fructose, galactose, mannose, saccharose, lactose,
maltose, and pentoses.

glucose — > 2 lactate (6.8)

glucose — > lactate + ethanol + C0 2 (6.9)

2 glucose — > 2 lactate + 3 acetate (6.10)

TABLE 6.3 Major Genera of Lactate-forming Bacteria

Bifidobacterium

Lactobacillus

Leuconostoc

Pediococcus

Sporola c toba cillus

Streptococcus

METHANE FERMENTATION 47

TABLE 6.4 Major Genera of Propionate-forming
Bacteria and Succinate-forming Bacteria

Bacteroides

Clostridium

Peptostreptococcus

Ruminococcus

Selenomonas

Succinivibrio

Veillonella

PROPIONATE AND SUCCINATE FERMENTATION

Anaerobic propionibacteria or propionate-forming bacteria (Table 6.4) ferment
glucose and lactate (Equations 6.11 and 6.12). Lactate, the major end product of
lactate fermentation, is the preferred substrate of propionate-forming bacteria.
Although succinate (HOOCCH 2 CH 2 COOH) usually is an intermediate product of
fermentation, some succinate is produced as an end product.

1.5 glucose — > 2 propionate + acetate + C0 2 (6.11)

3 lactate — > 2 propionate + acetate + C0 2 (6.12)

Propionate is a major substrate of acid fermentation that can be converted to
acetate and then used in methane production. Propionate increases to relatively
high concentrations under adverse operational conditions.

SULFIDE FERMENTATION

Sulfate is reduced to sulfide by bacteria for two purposes. First, bacteria use sulfate
as the principal sulfur nutrient. This is done by enzyme systems that reduce sulfate
to sulfide. The reduction of sulfate to sulfide and its incorporation as a nutrient into
cellular material is termed assimilatory sulfate reduction. Second, during sulfide fer-
mentation or desulfurication, sulfate is reduced to sulfide as organic compounds are
oxidized. Because the sulfide produced through fermentation is released to the envi-
ronment and not incorporated into cellular material, sulfide fermentation is also
known as dissimilatory sulfate reduction.

There are two groups of sulfate-reducing bacteria — incomplete oxidizers and
complete oxidizers (Table 6.5). Incomplete oxidizers degrade organic compounds
to new bacterial cells, carbon dioxide, and acetate, ethanol, formate, lactate, and pro-
pionate, whereas complete oxidizers degrade organic compounds to new bacterial
cells and carbon dioxide.

METHANE FERMENTATION

Three types of methane-forming bacteria achieve methane production — two groups
of obligate chemolithotrophic methanogens and one group of methylotrophic

FERMENTATION

TABLE 6.5 Genera of Sulfate-reducing Bacteria

Genus

Species of Incomplete Oxidizers

Species of Complete Oxidizers

Desulfobacter

Desulfobulbus

Desulfococcus

Desulfonema

Desulfosarcina

Desulfotomaculum

Desulfovibrio

X
X

X
X
X
X

methanogens. Chemolithotrophic methanogens produce methane from carbon
dioxide and hydrogen (Equation 6.13) or formate (Equation 6.14). Carbon monox-
ide also may be used by some chemolithotrophic methanogens in the production
of methane (Equation 6.15). Methylotrophic methanogens produce methane by
using methyl group (-CH 3 ) -containing substrates such as methanol (Equation 6.16),
methylamine (Equation 6.17), and acetate (Equation 6.18). These organisms
produce methane directly from the methyl group and not via carbon dioxide.

CO, + 4H 9 -> CH 4 + 2H.O

2HCOOH -> CH 4 + CO

4CO + H 2 -> CH 4 + 3C0 2

3CH 3 OH + 3H 2 -> 3CH 4 + 3H 2

4 (CH 3 ) 3-N + 6H 2 -> 9CH 4 + 3C0 2 + 4NH 3

CH3COOH -> CH 4 + C0 2

(6.13)
(6.14)
(6.15)
(6.16)
(6.17)
(6.18)

MIXED-ACID FERMENTATION AND MIXED-ACID AND
BUTANEDIOL FERMENTATION

A large variety of bacteria in the genera Enterobacter, Escherichia, Erwinia,
Salmonella, Serratia, and Shigella are responsible for mixed acid fermentation.
These organisms ferment sugars to a mixture of acids — acetate, formate, lactate, and
succinate. Carbon dioxide, hydrogen, and ethanol also are produced. The prevalence
of acids among the products of mixed-acid fermentation account for the name of
the fermentation process.

Bacteria in the genera Enterobacter and Erwinia also produce 2,3-butanediol in
addition to acids. Production of butanediol increases with decreasing pH (<6).

In anaerobic digesters, acid production (Equation 6.19) takes place simultane-
ously with methane production (Equation 6.20). Although several acids are pro-
duced during acid fermentation, acetate is the primary substrate used for methane
production in an anaerobic digester.

polysaccharides — > glucose — Escherichia — > acetate
acetate — Methano coccus —> methane + carbon dioxide

(6.19)
(6.20)

MIXED-ACID FERMENTATION AND MIXED-ACID AND BUTANEDIOL FERMENTATION 49

Cellulose

glucose

Figure 6.2 Cellulose is an insoluble starch or particulate organic waste. Cellulose must be hydrolyzed
before it can be degraded. Exoenzymes releases by specific hydrolytic bacteria such as Cellulomonas
add water to the chemical bonds between the glucose units that make up cellulose. Once the chem-
ical bonds are hydrolyzed, glucose goes into solution and is absorbed by numerous bacteria and
degraded inside the bacterial cells.

For methane production to occur in municipal anaerobic digesters, complex,
insoluble organic compounds must be converted to simplistic, soluble compounds
that can enter bacterial cells. For example, cellulose is converted through bacterial
action to numerous small and soluble molecules of glucose (Figure 6.2). Through
acid fermentation, glucose is converted to acetate and, finally, acetate is converted
to methane. The majority of large, complex, and insoluble organic compounds in
municipal anaerobic digesters consist of three basic substrates — carbohydrates,
lipids, and proteins.

The fermentation of organic compounds by acid-forming bacteria and methane-
forming bacteria also results in the growth of new bacterial cells or sludge. However,
the energy obtained by the bacteria during fermentation (anaerobic respiration)
is relatively small (compared with aerobic respiration) and this small quantity
of energy results in production of a relatively small quantity of cells or sludge
(Table 6.6).

50 FERMENTATION

TABLE 6.6 Sludge Production or Yield (kg VSS/kg
COD) for Volatile Acid-forming and Methane-forming
Bacteria

Bacterial Group Yield (kg VSS/kg COD)

Volatile acid-forming bacteria 0.15

Methane-forming bacteria 0.03

The production of acetate through fermentation is accompanied by the produc-
tion of hydrogen. Acetate-forming bacteria produce acetate — the major substrate
used by methane-forming bacteria — as long as the hydrogen partial pressure is low.
A low partial hydrogen pressure is maintained in the digester as long as methane-
forming bacteria use hydrogen to form methane. By removing hydrogen, acetate
production is favored.

Anaerobic Digestion Stages

The anaerobic digestion process and production of methane is divided into stages.
Three stages often are used to illustrate the sequence of microbial events that occur
during the digestion process and the production of methane (Figure 7.1). These
stages are hydrolysis, acid forming, and methanogenesis. The critical biochemical
reactions within these stages are presented in Figure 7.2.

The anaerobic digestion process proceeds efficiently if the degradation rates
of all three stages are equal. If the first stage is inhibited, then the substrates for
the second and third stages will be limited and methane production decreases. If the
third stage is inhibited, the acids produced in the second stage accumulate. The inhi-
bition of the third stage occurs because of an increase in acids and, consequently,
loss of alkalinity and decrease in pH. The most common upsets of anaerobic
digesters occur because of inhibition of methane-forming bacteria — the third stage.

The anaerobic digestion process contains different groups of bacteria. These
groups work in sequence, with the products of one group serving as the substrates
of another group. Therefore, each group is linked to other groups in chainlike
fashion, with the weakest links being acetate production and methane production.

STAGE 1— HYDROLYSIS STAGE

In the anaerobic digester complex insoluble compounds such as particulate and
colloidal wastes undergo hydrolysis. Particulate and colloidal wastes consist of
carbohydrates, fats, and proteins. These wastes are polymeric substances, that is,
large insoluble molecules consisting of many small molecules joined together by
unique chemical bonds. The small molecules are soluble and quickly go into solu-

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

ANAEROBIC DIGESTION STAGES

Complex Substrates

Hydrolysis,

performed by hydrolytic bacteria
(facultative anaerobes and anaerobes)

Simple Substrates

Acid production, including

acetogenesis

(facultative anaerobes and anaerobes)

Acetate, Formate, CO2, CO, H2,

Methanol,
Methyl Amine, Propionate, Butyrate

Methane production (methanogenesis)

CH4 + C02

Figure 7.1 There are three basic stages of the anaerobic digestion process and production of
methane. These stages include the solublization of complex organic compounds or hydrolysis, the
production of simplistic acids or acid production, and the formation of methane or methane production.

tion once the chemical bonds are broken. Hydrolytic bacteria or facultative anaer-
obes and anaerobes that are capable of performing hydrolysis achieve breakage of
these unique bonds. Hydrolysis is the splitting (lysis) of a compound with water
(hydro). An example of an insoluble compound that undergoes hydrolysis in an
anaerobic digester is cellulose (Figure 7.3).

Cellulose [(C 6 H 12 6 ) n ] is an insoluble starch that is commonly found in primary
and secondary municipal sludges. Cellulose may make up approximately 15% of the
dry weight of the sludges. Cellulose consists of many sugar units or mers of glucose
(C 6 H 12 6 ) joined together by unique chemical bonds. Although glucose is soluble

STAGE 1— HYDROLYSIS STAGE

Hydrolysis

Complex carbohydrates > Simple sugars

Complex lipids > Fatty acids

Complex proteins > Amino acids

Acid Production

Simple sugars + fatty acids + amino acids > organic acids, including acetate + alcohols

Acetogenesis (acetate production)

Organic acids + alcohols > acetate

Methane production: acetoclastic methanogenesis

Acetate

CH4 + CO2

Methane production: hydogenotrophic methanogenesis

H2 + CO2

Methane production: methyltrophic methanogenesis

Methanol

CH4 + H2O

Figure 7.2 The critical biochemical reactions in the anaerobic digestion process and production of
methane include hydrolysis, acid production, acetogenesis, and methane production. Methane pro-
duction may occur through the use of acetate, hydrogen and carbon dioxide, and methanol.

in water, the joining of the many mers of glucose by unique chemical bonds results
in the production of the insoluble polymer cellulose.

When cellulose is hydrolyzed in an anaerobic digester, many molecules of soluble
glucose are released (Equation 7.1). Cellulose is hydrolyzed by the hydrolytic
bacterium Cellulomonas . The bacterium is able to hydrolyze cellulose because it
processes the enzyme cellulase, which is capable of breaking the bonds between the
mers of glucose.

(C 6 H 12 6 ) n + H 2 -> nC 6 H 12 (

(7.1)

Anaerobic digesters at industrial wastewater treatment plants that degrade sim-
plistic, soluble organic compounds such as glucose do not experience hydrolysis or
stage 1. However, complex, soluble organic compounds such as table sugar (sucrose)
must be hydrolyzed. Table sugar is a disaccharide consisting of two 6-carbon sugars,
glucose and fructose, that are bonded together. Although soluble in water, table
sugar is too complex to enter a bacterial cell where it can be degraded. Table sugar

ANAEROBIC DIGESTION STAGES

Cellulose

Figure 7.3 Cellulose is an insoluble starch or particulate organic waste. Cellulose must be hydrolyzed
before it can be degraded. Exoenzymes releases by specific hydrolytic bacteria such as Cellulomonas
add water to the chemical bonds between the glucose units that make up cellulose. Once the chem-
ical bonds are hydrolyzed, glucose goes into solution and is absorbed by numerous bacteria and
degraded inside the bacterial cells.

must be hydrolyzed to glucose and fructose (Equation 7.2). After hydrolysis glucose
and fructose can enter a bacterial cell and be degraded (Figure 7.4).

sucrose + H 2 — > glucose + fructose

(7.2)

STAGE 2— ACID-FORMING STAGE

In the acid-forming stage, soluble compounds produced through hydrolysis or dis-
charged to the digester are degraded by a large diversity of facultative anaerobes
and anaerobes through many fermentative processes. The degradation of these com-
pounds results in the production of carbon dioxide, hydrogen gas, alcohols, organic

STAGE 2— ACID-FORMING STAGE

Table sugar (sucrose)

Glucose

Exoenzymes

Fructose

Saccharolytic bacterium

Glucose

Figure 7.4 Although table sugar is soluble in water, table sugar is too large and complex to enter a
bacterial cell. In order for bacteria to degrade table sugar, the sugar must be hydrolyzed to its indi-
vidual units, glucose and fructose. Once hydrolyzed, glucose and fructose can enter the bacterial cell
and be degraded. Hydrolysis of table sugar is achieved through exoenzymes, while degradation is
achieved through endoenzymes.

acids, some organic-nitrogen compounds, and some organic-sulfur compounds.
(Table 7.1). The most important of the acids is acetate.

Acetate is the principal organic acid or volatile acid used as a substrate by
methane-forming bacteria. Carbon dioxide and hydrogen can be converted directly
to acetate or methane. The presence of organic-nitrogen compounds and organic-
sulfur compounds is due to the degradation of amino acids and proteins. The con-
version of large soluble organic compounds to small soluble organic compounds

56 ANAEROBIC DIGESTION STAGES

TABLE 7.1 Major Acids and Alcohols Produced
Through Fermentation Processes in Anaerobic
Digesters

Name Formula

Acetate CH 3 COOH

Butanol CH 3 (CH 2 ) 2 CH 2 OH

Butyrate CH 3 (CH 2 ) 2 CH 2 COOH

Caproic acid CH 3 (CH 2 ) 4 COOH

Formate HCOOH

Ethanol CH 3 CH 2 OH

Lactate CH 3 CHOHCOOH

Methanol CH 3 OH

Propanol CH 3 CH 2 CH 2 OH

Propionate CH 3 CH 2 COOH

Succinate HOOCCH 2 CH 2 COOH

TABLE 7.2 Alcohols, Organic-nitrogen Compounds,
and Organic Acids Used as Substrates by Methane-
forming Bacteria

Substrate Chemical Formula

Acetate CH 3 COOH

Formate HCOOH

Methanol CH 3 OH

Methylamine CH 3 NH 2

TABLE 7.3 Alcohol and Organic Acids Used Indirectly
as Substrates by Methane-forming Bacteria

Substrate Chemical Formula

Ethanol CH 3 CH 2 OH

Butyrate CH 3 CH 2 CH 2 COOH

Propionate CH 3 CH 2 COOH

results in little change in the organic strength of the compounds. Some of the organic
compounds are converted to organic acids and alcohols, and some are converted to
new bacterial cells. It is only in methane formation or the methanogenic stage that
degradable organics are removed as methane and carbon dioxide.

Within the pool of organic acids, alcohols, and organic-nitrogen compounds, there
are those that can be used directly as a substrate by methane-forming bacteria
(Table 7.2) and those that can be used indirectly (Table 7.3) if they are degraded to
acetate by fermentative bacteria. If the methane-forming bacteria do not degrade
the products of the second stage, the products will accumulate and produce an acid
medium.

Acetate can be produced not only through the fermentation of soluble organic
compounds but also through acetogenesis. Acetogenesis occurs in the acid-forming
stage. Here, many of the acids and alcohols, for example, butyrate, propionate, and
ethanol, produced during the acid-forming stage may be degraded to acetate that

STAGE 3—METHANOGENESIS STAGE 57

can be used as a substrate by methane-forming bacteria. The production of acetate
is accomplished through the activity of acetogenic or acetate-forming bacteria.

STAGE 3—METHANOGENESIS STAGE

In the methanogenic stage, methane is formed mostly from acetate and carbon
dioxide and hydrogen gas. Methane is also formed from some organic compounds
other than acetate (Table 7.2). Therefore, all other fermentative products must be
converted to compounds that can be used directly or indirectly by methane-forming
bacteria. Acids, alcohols, and organic-nitrogen compounds that are not degraded by
methane-forming bacteria accumulate in the digester supernatant. The accumula-
tion of these compounds is responsible for the relatively high organic strength or
carbonaceous biochemical oxygen demand (cBOD) of the supernatant.

As long as the "working velocity" of acid-producing bacteria and methane-
forming bacteria are roughly the same, the metabolic activity of the methanogenic
stage is safeguarded. If the methanogenic stage is safeguarded, the acids are broken
down and a slightly alkaline medium is achieved from the overall process because
of the formation of ammonia (NH 3 ) from amino groups (-NH 2 ) that are released
through the degradation of proteins and amino acids.

Ammonia released in the sludge often reacts with carbon dioxide and water,
resulting in the production of ammonium carbonate that provides alkalinity to the
system (Equation 7.3). The ammonium carbonate is available to react with the
volatile acids that are present in the sludge. This reaction results in the production
of volatile acid salts (Equation 7.4).

NH 3 + C0 2 + H 2 -* NH4HCO3 (7.3)

NH4HCO3 + RCOOH* -* RCOONH4 + H + + HCO3 (7.4)

*R represents the non-carboxyl (-COOH) portion of the volatile acid.

The decomposition of complex organic compounds to methane proceeds as
rapidly as the compounds can be converted to substrates that are capable of being
used by methane-forming bacteria. Within the anaerobic conversions and degrada-
tions of organic compounds, the production of acetate is the rate-limiting step or
"bottleneck" in the final degradation of complex organic compounds. For organic
compounds that are poorly biodegradable, the hydrolysis stage may become the
rate-limiting step.

Part II

Substrates, Products,

and Biogas

Substrates and Products

In chemical reactions there are reactants and products (Equation 8.1). During chem-
ical reactions, reactants (chemical compounds) undergo change and often release
energy (heat) to the environment. The changes that occur to the reactants result in
the formation of products (new chemical compounds). Often, a catalyst may be
involved in a chemical reaction. The catalyst accelerates the rate of the chemical
reaction and may be changed or consumed.

reactants — catalyst — > products (8.1)

Chemical reactions that occur inside bacterial cells are known as biochemical
reactions. In biochemical reactions, reactants or substrates undergo change as bac-
terial cells degrade them. As the substrates are degraded, energy is released and
new compounds (products) are formed (Equation 8.2).

substrates — > products (8.2)

Some of the energy released by the substrates is captured by the bacterial cells
and stored in high-energy phosphate bonds for use in cellular activity. Energy that
is not captured by the bacterial cells is lost to the environment as heat. New bacte-
rial cells and carbon dioxide are products of biochemical reactions that involve
organic compounds (Equation 8.3).

substrates (organic compounds) — > bacterial cells + carbon dioxide (8.3)

Catalysts are involved in biochemical reactions. These catalysts are known as
enzymes. Enzymes are large proteinaceous molecules that greatly accelerate the

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

62 SUBSTRATES AND PRODUCTS

TABLE 8.1 The Three Stages of Anaerobic Digestion of Solids

Stage Activity Enzymes Used

First Hydrolysis: Exoenzymes

Solubilization of particulate and colloidal wastes
Second Acid forming: Endoenzymes

Conversion of soluble organic acids and alcohols to
acetate, carbon dioxide, and hydrogen
Third Methanogenesis: Endoenzymes

Production of methane and carbon dioxide

rate of biochemical reactions. However, enzymes, unlike chemical catalysts, are not
altered or consumed during the reaction (Equation 8.4).

substrates — bacteria and bacterial enzymes — >

bacterial cells + carbon dioxide (8.4)

During some biochemical reactions, intermediate products or "intermediates" are
formed (Equation 8.5). Intermediates usually are short-lived, that is, they do not
accumulate. However, specific environmental or operational conditions such as a
change in pH or temperature may permit the accumulation of intermediates. The
presence of some intermediates may result in operation problems in an anaerobic
digester.

substrates — intermediates — > bacterial cells + carbon dioxide (8.5)

Initial substrates for bacteria in municipal anaerobic digesters include carbohy-
drates, lipids, and proteins. These substrates are found as particulates such as the
carbohydrate cellulose and as colloids such as proteins.

The degradation process or digestion of solids within an anaerobic digester con-
sists of three stages (Table 8.1). The first stage is the hydrolysis of particulate and
colloidal wastes to soluble wastes in the form of organic acids and alcohols. The
second stage is the conversion of the organic acids and alcohols to acetate, carbon
dioxide, and hydrogen. The third stage is the production of gases, mostly methane,
and new bacterial cells or sludge from acetate and hydrogen. Because a great diver-
sity of bacteria are required in an anaerobic digester to perform hydrolysis, produce
acetate and hydrogen, and produce methane, the substrate feed to the digester
should contain a great diversity of wastes.

The net results of anaerobic digestion of solids are significant decreases in
percent solids and percent volatile solids in digester sludge. The first and second
stages of anaerobic digestion are achieved through the activities of facultative
anaerobes and anaerobes, whereas the third stage is achieved through the activity
of only anaerobes, the methane-forming bacteria.

Hydrolysis rates for particulate and colloidal wastes vary greatly according to
the waste to be degraded and the operational conditions at the time of hydrolysis.
Substrates hydrolyzed in the first stage consist of carbohydrates, lipids, and
proteins. These substrates may be wasted to the digester from primary and second-
ary sludges.

CARBOHYDRATES

Carbohydrates are synthesized in the green leaves of plants by the conversion of
carbon dioxide into glucose during photosynthesis. Carbohydrates are macromole-
cules or polymers that contain numerous monomers of sugars (Figure 8.1). The
range of lengths of the polymers or carbohydrates varies greatly.

Within the digester all carbohydrates are degraded inside the cell of facultative
anaerobes and anaerobes. Carbohydrates too large to enter the cell, that is, in an
insoluble or complex soluble form, must be hydrolyzed into smaller, soluble sugars

Mer or monomer, monosaccharide

2 mers or disaccharide

3-7 carbon sugar

Many mers or polysaccharide

3-7 carbon sugar

Figure 8. 1

SUBSTRATES AND PRODUCTS

outside the cell through the use of exoenzymes (Figure 8.2). Once hydrolyzed, the
smaller, soluble sugars enter the cell, where they are degraded by endoenzymes.

The monomers of carbohydrates are simple sugars (Table 8.2). These sugars are
known as monosaccharides, and they contain three to seven carbon units. The
common generic formulae for monosaccharides are (CH 2 0) 3 - (CH 2 0) 7 .The major
monomers or monosaccharides in our diet are fructose and glucose. Although many

3-7 carbon sugar

Exoenzymes

3-7 carbon sugar

Saccrolytic bacterium

3-7 carbon sugar

Insoluble polysaccharide

3-7 carbon sugar

3-7 carbon sugar
Z

Endoenzymes

3-7 carbon sugar

Figure 8.2 Large, complex, and insoluble carbohydrates or polysaccharides must be hydrolyzed by
sacchrolytic bacteria with the use of exoenzymes. Once solublized, the individual sugar units of the
polysaccharides can enter the bacterial cells and can be degraded by endoenzymes.

CARBOHYDRATES 65

TABLE 8.2 Common Monosaccharides or Simple
Sugars

Monosaccharide Carbon Units Formula

Deoxyribose

C 5 H 10 O 5

Glucose (dextrose)

UeH^Og

Galactose

OgH^Oe

Fructose (levulose)

L>q\~\^2^6

Ribose

C 5 H 10 O 5

Mannose

OeH^Oe

TABLE 8.3 Common Disaccharides

Disaccharide Composition of Monosaccharides

Lactose Galactose-Glucose

Maltose Glucose-Glucose

Sucrose Glucose-Fructose

Cellobiose Galactose-Galactose

TABLE 8.4 Common Polysaccharides

Agar

Amylopectin (starch)

Amylose (starch)

Cellulose

Fiber

Glycogen

Pectin

Vegetable gum

monomers have identical chemical formulae, for example, glucose (CeH^C^) and
fructose (C6H12O6), they are structurally different (Figure 8.3).

When two monomers are linked together, disaccharides are formed (Table 8.3),
and when numerous monosaccharides are linked together, polysaccharides are
formed (Table 8.4). Major disaccharides in our diet are sucrose (table sugar) and
lactose (milk sugar). Disaccharides are carbohydrates composed of monosaccha-
rides linked by an acetal bond (-C-O-C-). Polysaccharides often are referred to as
complex carbohydrates. The largest digestible polysaccharide in our diet is starch.
This polysaccharide is found in grains such as wheat and rice, root vegetables such
as potatoes, and legumes such as beans and peas.

Although some sugars contain nitrogen and phosphorus, all sugars contain
carbon, hydrogen, and oxygen. The basic chemical formula for sugars is (CH 2 0) x .
The word "carbohydrate" was used originally to describe glucose — the hydrate of
carbon (CH 2 0) or C 6 (H 2 0) 6 .

Monosaccharides are water soluble and are quickly and easily transported across
the cell wall and cell membrane into the bacterial cell. Disaccharides also are water
soluble but must be converted or hydrolyzed to monosaccharides before they can
enter the bacterial cell.

Polysaccharides are very large, complex insoluble sugars that have a high molec-
ular weight. These sugars require the presence of specific exoenzymes and, usually,

SUBSTRATES AND PRODUCTS

CH20H

B-

-C

-OH

-H

B-

-OH

-C

-OH

B-

-OH

B-

-OH

CH2OH

Glucose

CH2OH

Fructose

Figure 8.3

TABLE 8.5 Acids and Alcohols Produced from
Monosaccharide Degradation

Genus

End Product from Monosaccharide
Degradation

Clostridium

Enterobacter

Escherichia

Lactobacillus

Streptococcus

Propionibacterium

Butanol, butyrate, isopropanol

Butanediol, ethanol, formate, lactate

Acetate, ethanol, lactate, succinate

Lactate

Acetate, propionate

several enzymatic steps to ensure their hydrolysis and degradation. Because of their
insoluble nature, complex structure, and need for specific exoenzymes and numer-
ous enzymatic steps, polysaccharides are degraded slowly.

When disaccharides and polysaccharides are hydrolyzed, monosaccharides are
released. When monosaccharides are degraded in an anaerobic digester, organic
acids and alcohols are produced (Table 8.5). Many of these compounds are further
degraded to volatile acids.

LIPIDS

Lipids are naturally occurring organic molecules found in animal and plant tissues.
Lipids do not dissolve in water, that is, lipids are extracted from animal and plant
tissues with nonpolar organic solvents such as ether.

LIPIDS

-C

-OH

-C

-OH

-C

-OH

H
Figure 8.4

CH3(CH2)l4COOH

saturated palmitic acid

CH3(CH2)7CH=CH(CH2)7COOH

unsaturated oleic acid

Figure 8.5

TABLE 8.6 Some Common Fatty Acids

Name

Carbon Units

Saturated

Unsaturated

Number of Double Bonds

Laurie

Myristic

Palmitic

Stearic

Linoleic

Linolenic

Oleic

2
3
1

There are numerous groups of lipids. The lipids most often wasted to a munici-
pal anaerobic digester include fats and oils. These compounds are derived from glyc-
erol (Figure 8.4). Glycerol combined with three fatty acids produces a triglyceride
or fat.

Fatty acids are straight-chain carbon compounds containing a terminal carboxylic
acid group (-COOH) (Figure 8.5). There are approximately 40 naturally occurring
fatty acids (Table 8.6). Fatty acids without a double bond (=) between carbon units
are known as saturated fatty acids, for example, palmitic acid. Fatty acids with a
double bond between carbon units (-C=C-) are known as unsaturated fatty acids,
for example, oleic acid. Fatty acids with two or more double bonds between carbon

68 SUBSTRATES AND PRODUCTS

TABLE 8.7 Composition of Some Common Fats and Oils

Fat/Oil

Animal

Vegetable

Principle Saturated

Principle Unsaturated

Fat

Oil

Fatty Acid

Butter

Palmitic

Oleic

Lard

Palmitic

Oleic

Corn

Palmitic

Oleic

Olive

Palmitic, stearic

Oleic

Peanut

Palmitic

Oleic

Soybean

Palmitic

Linoleic

units are known as polyunsaturated fatty acids, for example, linoleic acid. Palmitic
acid and stearic acid are the most abundant saturated fatty acids, and oleic acid and
linoleic acid are the most abundant unsaturated fatty acids.

Animal fats and vegetable fats or oils are the most abundant lipids in nature.
Examples of animal fats include butter and lard, and examples of vegetable oils
include corn, olive, peanut, soybean, and sunflower oils. All fats and oils have similar
chemical structures. They are triglycerides. The three fatty acids of a triglyceride are
not necessarily the same (Table 8.7).

Large and complex fatty acids, fats, and oils are hydrolyzed in an anaerobic
digester. The resulting small and simplistic molecules obtained from hydrolysis are
degraded further to organic acids.

In anaerobic digesters fats undergo degradation through two principal steps.
First, the fats are hydrolyzed to glycerol and fatty acids. Lipase enzymes are used
by bacteria to hydrolyze the fats. Glycerol is degraded, and the fatty acids released
through hydrolysis are degraded two carbon units at a time.

PROTEINS

The principal nitrogenous wastes in municipal sludges are proteins. Proteins are
complex, high molecular-weight compounds. These molecules have a relatively large
surface area and do not dissolve in wastewater or settle out of wastewater. Proteins
are made of amino acids that are either straight-chain (aliphatic) or ring-shaped
(cyclic) in structure (Figure 8.6). There are 20 different amino acids. Regardless of
their structure, all amino acids contain an amino group (-NH 2 ) and a carboxyl group
(-COOH). The carboxyl group is the "acid" portion of the amino acid.

Amino acids are joined together by peptide bonds to form proteins (Figure 8.7).
Proteins consist of long chains of amino acids. Each protein has a unique composi-
tion and sequence of amino acids in its chain. The complex proteins formed from
peptide bonds cannot be transported into bacterial cells. The use of exoenzymes,
namely, proteases or peptidases, by bacteria to hydrolyze peptide bonds permits
the release of individual amino acids that are transported into bacteria cells (Figure
8.8). Once inside the cell, amino acids undergo additional degradation resulting in
the production of organic acids. Examples of amino acids fermented in anaerobic
digesters include alanine (Equation 8.6), arginine, glutamate, glycine, and lysine
(Table 8.8).

PROTEINS

CH3CH2COOH

NH2

Alanine (aliphatic amino acid)

H2CHCOOH

NH 2

Phenylalanine (cyclic amino acid)
Figure 8.6

H 2

H 2 N-R

amino acid-amino acid
peptide bond

Figure 8.7 Amino acids are joined together to form proteins. Amino acids are joined together through
the production of peptide bonds. The bonds are produced by joining the hydroxy! group (-OH) in the
carboxyl group (-COOH) of one amino acid with the amino group (-NH 2 ) of other amino acid. When
the peptide bond is formed, water is produced.

TABLE 8.8 Amino Acids Commonly Fermented in
Anaerobic Digesters

Amino Acid

Fermenting Bacteria

Alanine
Arginine

Glutamate

Glycine

Lysine

Clostridium propionicium
Clostridium spp.
Streptococcus spp.
Clostridium tetanomorphium
Peptostreptococcus micros
Clostridium sticklandii

SUBSTRATES AND PRODUCTS

Amino acid

Peptide bonds

Amino acid

Figure 8.8 Large, complex, and colloidal proteins must be hydrolyzed by bacteria with the use of
exoenzymes. Once solublized, the individual amino acids of the proteins can enter the bacterial cells
and can be degraded by endoenzymes.

4H,NCH 9 COOH + 2H.O -> 4NH, + 2CO? + 3CH.COOH

(8.6)

The degradation of amino acids results in the production of a variety of organic
acids including acetate and butyrate. Ammonia is released during the degradation
of amino acids. Acetate and butyrate serve as substrates for methane-forming bac-
teria, whereas ammonia increases digester alkalinity or may contribute to ammonia
toxicity.

VOLATILE ACIDS 71

Proteins can be classified as simple or conjugated according to their chemical
composition. Simple proteins are those that release only amino acids and no other
compounds on hydrolysis. Blood serum albumin is an example of a simple protein.
Conjugated proteins are more common than simple proteins and release amino
acids and non-protein substances on hydrolysis. Glycoproteins that contain carbo-
hydrates and lipoproteins that contain lipids are examples of conjugated proteins.

VOLATILE ACIDS

Some organic acids are known also as volatile acids or volatile fatty acids. These
acids occur as substrates and products in the anaerobic digester. Many serve as sub-
strate for methane-forming bacteria, and they are the products of the fermentative
activities of facultative anaerobes and anaerobes.

Volatile acid production in an anaerobic digester results in the production of
methane. Although volatile acids vary in length, most volatile acids produced in
an anaerobic digester are low-molecular-weight, short-chain acids, for example,
formate (1 carbon unit), acetate (2 carbon units), propionate (3 carbon units), and
butyrate (4 carbon units) (Table 8.9).

These short-chain acids are known as volatile acids because they can vaporize or
evaporate at atmospheric pressure. Of these acids, acetate is the predominant acid
produced in an anaerobic digester. Approximately 85% of the volatile acid content
of an anaerobic digester is acetate. All volatile acids are soluble in water

As wastes are degraded, new bacterial cells or sludge are produced. The cellular
growth or amount of sludge produced is expressed as net biomass yield [as per-
centage of chemical oxygen demand (COD) removed]. Growth yield for several
wastes is presented in Table 8.10.

TABLE 8.9 Volatile Acids Commonly Found in
Anaerobic Digesters

Volatile Acid

Number of
Carbon Units

Formula

Formate

HCOOH

Acetate
Propionate
Butyrate
Valeric acid

2
3
4
5

CH3COOH
CH 3 CH 2 COOH
CH 3 (CH 2 ) 2 COOH
CH 3 (CH 2 ) 3 COOH

Isovaleric acid
Caproic acid

5
6

(CH 3 ) 2 CHCH 2 COOH
CH 3 (CH 2 ) 4 COOH

TABLE 8.10 Growth Yields (as % of COD removed)

Substrate (waste) Yield

Alcohols 0.06-0.08

Carbohydrates 0.08-0.15

Organic acids 0.02-0.04

Proteins 0.03-0.06

72 SUBSTRATES AND PRODUCTS

The higher the volatile solids feed to the digester, the larger the amount of
volatile acids formed in the digester. The larger the amount of volatile acids in the
digester, the greater the impact of volatile acids on digester alkalinity and pH. There-
fore, sludges that have a high volatile content should be transferred slowly to an
anaerobic digester.

Bio gas

Anaerobic digestion of municipal sludges results in the production of a mixture of
gases (Figure 9.1). Collectively, these gases are referred to as digester gas or biogas.
The only gas of economic value that is produced in an anaerobic digester is methane.
In a properly operating digester most of the gas produced from a day's feed sludge
appears within 24 hours.

Methane can be used as a source of fuel. It is a natural flammable gas. Methane
is odorless and burns cleanly (Equation 9.1). Pure methane has a heat value of
1,000 Btu/ft 3 . When methane is mixed with carbon dioxide that is produced in an
anaerobic digester, its heat value decreases significantly.

CH 4 + 20 2 -* C0 2 + 2H 2 (9.1)

Typically, biogas production in municipal anaerobic digesters is between 10 and
25 ft 3 per pound of volatile solids degraded (cu ft/lb VS) or 0.75-1 .0m 3 /kg VS. The
heat value of biogas is approximately 500-600 Btu/ft 3 , much lower than that of
methane because of the dilution of methane by carbon dioxide. With increasing
quantities of carbon dioxide in biogas, decreasing heat values of biogas occur. If the
carbon dioxide content of biogas becomes too large, biogas will not allow for a
self-sustained burn and supplemental fuel will be required. If the carbon dioxide
fraction in the biogas increases above 30%, the acid concentration in the sludge
increases and the pH drops below 7.0. At pH values below 7.0, significant acid fer-
mentation occurs.

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

BIOGAS

CH4, C02,
CO,H2,H2S,NH3,

N2, N2O

Figure 9. 1

Sewer main

Wastewater

Biofilm

Sediment

Figure 9.2 Anaerobic respiration occurs in the sewer main. Anaerobic respiration occurs in the biofilm
lining the inside of the sewer main and in the sediment.

Numerous gases are produced in an anaerobic digester. The gases produced in
largest quantities are methane and carbon dioxide. By volume, methane is 60% to
65%, and carbon dioxide is 35 to 40%. Most municipal wastewater treatment plants
use biogas to heat digesters to 32-35° C (90-95°F). The biogas also may be used to
heat buildings. Biogas not used to heat digesters is simply flamed.

When anaerobic digestion of sludges and wastewaters is interrupted by changes
in operational conditions, numerous insoluble and volatile compounds are pro-
duced. These compounds may be released wherever anaerobic digestion of organic

BIOGAS

Sediment

Figure 9.3 Within the sewer main aerobic respiration and anaerobic respiration occur. Bacteria on
the surface of the biofilm that are exposed to free molecular oxygen use aerobic respiration. Bacteria
beneath the surface of the biofilm that do not receive free molecular oxygen use anaerobic respira-
tion using sulfate ions or mixed acid fermentation. Bacteria at the bottom of the sediment use anaer-
obic respiration and produce methane. Because nitrate ions and nitrite ions are seldom found in sewer
mains, anoxic respiration does not occur.

compounds is interrupted. Many of these compounds are malodorous and often are
released in sewer mains (Figures 9.2 and 9.3), lift stations, secondary clarifier sludge
blanket, thickener, and anaerobic digester. The organic and inorganic compounds
produced are listed in Tables 9.1 and 9.2.

The organic compounds (Table 9.1) include methane and volatile organic com-
pounds (VOC). The VOC contain volatile fatty acids (VFA), nitrogen-containing
compounds, and volatile sulfur compounds (VSC). The production of nitrogen-
containing VOC and VSC is usually due to the degradation of proteinaceous
wastes.

Of the inorganic gases (Table 9.2) produced in an anaerobic digester, hydrogen
sulfide (H 2 S) is the most undesirable. If biogas contains too much hydrogen sulfide,
the gas may damage digester equipment. Hydrogen sulfide can be scrubbed from
biogas, but scrubbing is expensive and often cost-prohibitive for small wastewater
treatment plants. Excess production of hydrogen sulfide is due to the excess of
sulfur-containing wastes such as proteinaceous compounds that are transferred to
the digester.

BIOGAS

TABLE 9.1 Organic Gases Produced Through
Microbial Activity in Anaerobic Digesters

Name

Formula

VFA

vsc

Acetate

CH3COOH

Butyrate

CH 3 (CH 2 ) 2 CH 2 COOH

Caproic acid

CH 3 (CH 2 ) 4 COOH

Formate

HCOOH

Propionate

CH 3 CH 2 COOH

Succinate

CH3CHOHCOOH

Valeric acid

CH 3 (CH 2 ) 3 COOH

Methane

CH 4

Cadaverine

H 2 N(CH 2 ) 5 NH 2

Dimethylamine

CH 3 NHCH 3

Ethylamine

C 3 H 5 NH 2

Indole

C 8 H 13 N

Methylamine

CH 3 NH 2

Putrescine

H 2 N(CH 2 ) 4 NH 2

Propylamine

CH 3 CH 2 CH 2 NH 2

Pyridine

C 5 H 6 N

Skatole

C 9 H 9 N

Trimethylamine

CH 3 NCH 3 CH 3

Allyl mercaptan

CH 2 =CHCH 2 oH

Benzyl mercaptan

C6n5CH 2 SH

Dimethyl sulfide

(CH 3 ) 2 S

Ethyl mercaptan

C 2 n5oH

Methyl mercaptan

CH 3 SH

Thiocresol

CH 3 C6n4SH

Thioglycolic acid

HSCH 2 COOH

TABLE 9.2 Inorganic Gases Produced Through
Microbial Activity in Anaerobic Digesters

Name

Formula

Ammonia
Carbon dioxide
Carbon disulfide
Carbon monoxide
Hydrogen sulfide
Nitrogen
Nitrous oxide

NH 3

C0 2

CS 2

H 2 S

N 2

The inorganic gases molecular nitrogen (N 2 ) and nitrous oxide (N 2 0) are pro-
duced through anoxic respiration (denitrification) in the anaerobic digester. Anoxic
respiration can occur with the transfer of nitrate ions (NO3) to the digester with
sludges or the addition of nitrate-containing compounds such as sodium nitrate
(NaN0 3 ) to increase digester alkalinity.

Part III

Operational Conditions

Introduction to
Operational Conditions

The rate-limiting reaction in anaerobic digestion is usually the conversion of volatile
acids to methane. Methane-forming bacteria obtain very little energy from the
degradation of volatile acids. Most of the energy released from the volatile acids is
transferred to the methane.

Because of the low energy yield obtained from volatile acids by methane-forming
bacteria, their growth rate is restricted, that is, the amount of substrate utilization
per unit of organisms is high. Therefore, bacterial growth or sludge production is
low and optimum operational conditions must be maintained for satisfactory rates
of solids destruction and methane production. These factors are responsible for the
rate-limiting reaction of the conversion of volatile acids to methane. However, if the
substrates fed to the anaerobic digester were mostly slowly degrading particulate
materials, then the rate-limiting reaction would be the hydrolysis of the particulate
material.

Methane-forming bacteria are strict anaerobes and are extremely sensitive to
changes in alkalinity, pH, and temperature. Therefore, operational conditions in the
digester must be periodically monitored and maintained within optimum ranges. In
addition to alkalinity, pH, and temperature, several other operational conditions
should be monitored and maintained within optimum ranges for acceptable activ-
ity of methane-forming bacteria. These conditions are gas composition, hydraulic
retention time (HRT), oxidation-reduction potential (ORP), and volatile acid con-
centration (Table 10.1).

Process control of anaerobic digesters is often difficult, because numerous oper-
ational conditions are interrelated and changes in one condition may directly or
indirectly affect others. Also, the relatively low concentrations of solids and short
solids retention times (SRTs) maintained in completely mixed digesters render the

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

INTRODUCTION TO OPERATIONAL CONDITIONS

TABLE 10.1 Operational Conditions for Acceptable Activity of Methane-forming Bacteria and
Methane Production

Condition

Optimum

Marginal

Alkalinity, mg/l as CaC0 3

Gas composition
Methane, % volume
Carbon dioxide, % volume

Hydraulic retention time, days

Temperature, mesophilic
Temperature, thermophilic
Volatile acids, mg/l as acetic acid

1 500-3000

65-70
30-35
10-15
6.8-7.2
30-35°C
50-56°C
50-500

1 000-1 500
3000-5000

60-65 & 70-75
25-30 & 35-40
7-1 & 1 5-30
6.6-6.8 & 7.2-7.6
20-30° & 35-40°C
45-50° & 57-60°C
500-2000

process susceptible to toxic upsets and shock loadings. Another difficulty in achiev-
ing proper digester operation is the presence of different bacterial groups that
have different optimum values or ranges of values for operational conditions. For
example, there are two optimal temperatures for anaerobic digestion of solids. The
acid-forming bacteria have an optimum temperature at 30° C, and the mesophilic,
methane-forming bacteria have an optimum temperature at 35°C.

Start-up

Primary and secondary sludges that provide the substrates for an anaerobic digester
also provide the bacteria needed for the hydrolysis and degradation of these com-
pounds and the production of methane. Both facultative anaerobes and anaerobes
including methane-forming bacteria are needed in an anaerobic digester. Faculta-
tive anaerobes and anaerobes are needed for 1) the hydrolysis of particulate and
colloidal compounds and 2) the degradation of soluble organic compounds to
volatile acids. Methane-forming bacteria are needed for the degradation of volatile
acids and the production of methane.

To seed an anaerobic digester with an adequate population of facultative anaer-
obes and anaerobes including methane-forming bacteria, a ratio of 1 : 10 of second-
ary sludge to primary sludge may be used. Although the amount of secondary sludge
is much less compared with primary sludge, the secondary sludge is highly con-
centrated with facultative anaerobes. The primary sludge provides not only some
facultative anaerobes but also many anaerobes including methane-forming bacte-
ria and many organic particulates.

Because methane-forming bacteria are strict anaerobes and die quickly in an
activated sludge process, an anaerobic digester cannot be successfully seeded with
secondary sludge alone. Therefore, primary sludge that contains an abundant pop-
ulation of methane-forming bacteria is needed. Primary sludge performs three
important roles during anaerobic digester start-up. These roles consist of seeding
the digester with 1) methane-forming bacteria, 2) facultative anaerobes and anaer-
obes, and 3) organic particulates.

Once an anaerobic digester has been seeded properly and is operating efficiently,
the digester can be fed secondary sludge alone. Secondary sludge contains numer-
ous facultative anaerobes and many particulate and colloidal organics. Primary

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

START-UP

sludge contains only a relatively small number of facultative anaerobes that would
not adequately replace the bacteria wasted from the digester during routine solids
pumping and dewatering operations.

Because the successful operation of an anaerobic digester requires the activity
of an abundant and diverse population of methane-forming bacteria, seeding the
digester that is heated to 35°C with fresh cow manure may be helpful. Seeding with

TABLE 11.1 Chemicals Commonly Used for pH
Adjustment and Alkalinity Addition

Chemical

Formula

Ammonia, anhydrous

Caustic soda

Lime, quick

Lime, hydrated

Soda ash

Sodium bicarbonate

NH 3

NaOH

CaO

Ca(OH) 2

Na 2 C0 3

NaHC0 3

NH 2

Figure 11.1 Cationic, polyacrylamide polymers are commonly used at wastewater treatment plants
for solids capture and sludge thickening and dewatering. Often these polymers may be found in
relatively large concentrations in anaerobic digesters. The degradation of the polymers results in the
release of amino groups from the aery lam ide component of the polymer. The amino group is quickly
converted to ammonia and then ammonium ions in the anaerobic digester. The conversion of ammonia
to ammonium ions is pH dependent.

START-UP

cow manure can be practiced during start-up or when the efficiency of the digester
deteriorates, for example, when the digester goes "sour." Approximately 5 gallons
of fresh cow manure should be added to the digester for every 100,000 gallons of
digester sludge. The manure should be added daily until successful start-up or
improved efficiency is obtained.

Numerous methane-forming bacteria are alive and active deep within cow
manure. Care should be taken not to expose the bacteria within the cow manure to
the atmosphere. Methane-forming bacteria die quickly in the presence of free
molecular oxygen. Difficulties during start-up of an anaerobic digester also can be
overcome by inoculating a digester with previously digested sludge.

During start-up, loading to the digester should proceed slowly. Careful monitor-
ing and control of pH and alkalinity are essential. This is especially true when good
seed is not available. The digester pH should be maintained within the optimum
range of 6.8 to 7.2. The pH within this range is required for acceptable activity of

Percent
NH 3

100 —

Percent

NH4

100

pH at 35°C

Figure 11.2 The quantities of the reduced forms of nitrogen — ammonia and ammonium ion — in an
anaerobic digester are pH dependent. Increasing pH results in the production of more ammonia, while
decreasing pH results in the production of more ammonium ions.

84 START-UP

methane-forming bacteria; it also helps to ensure that adequate buffering capacity
or alkalinity is present to neutralize the acids within the digester.

Anaerobic digester start-up should proceed smoothly, and the time between
initial digester feed sludge and stable operation should be as short as possible.
Approximately 1 month will be required to achieve a steady-state condition or an
efficiently operating digester. This condition is reflected by the production of burn-
able biogas and a stable volatile acid-to-alkalinity ratio.

Several chemicals can be added to an anaerobic digester to maintain proper pH
and alkalinity (Table 11.1). The choice of chemical is dependent on cost, handling,
safety, storage, and requirements for feeding the chemical to the digester. If the pH
within the digester is greater than the optimum range, ammonia toxicity may occur.

Ammonium ions (NH4) are a natural component of a municipal anaerobic
digester. The ions are produced in the digester as a result of bacterial degradation
of amino acids and proteins. Ammonium ions may be added to the digester in sec-
ondary sludge as a result of protein degradation or in primary and secondary sludges
that contain cationic polyacrylamide polymers. These polymers contain amino
groups (-NH 2 ) that are released through bacterial activity. Once released, these
groups form NH4 (Figure 11.1).

Ammonia in the digester may be in the form of ammonium ions (ionized
ammonia — NH4) or dissolved ammonia gas (nonionized ammonia — NH 3 ).The two
forms are in equilibrium, and the relative concentration of each form is dependent
on the digester pH (Figure 11.2). When the digester pH is 7.2 or lower, the presence
of NH4 is favored. When the digester pH is greater than 7.2, the presence of NH 3
is favored. Dissolved ammonia gas or NH 3 is toxic to bacteria, especially methane-
forming bacteria.

Ammonia toxicity can be avoided if the digester pH is maintained within the
optimum range of 6.8 to 7.2 and the ammonia-nitrogen concentration does not
increase into the range of 1500 to 3000 mg/1. An additional problem related to an
increase in ammonia-nitrogen or alkalinity is foam and scum production. This
problem often occurs during digester start-up.

Sludge Feed

Because the predominant application of anaerobic digesters is the degradation of
particulate and colloidal wastes, sludge feed or organic loading rates to digesters
usually are expressed in terms of volatile solids (VS). Designed and recommended
loadings for anaerobic digesters that are mixed and heated are 200-450 lb VS/
1000ft 3 /day (3.2-7.2 kg VS/m7day). However, loading rates of 30-501b VS/
1000ft 3 /day (0.5-0.6 kg VS/m 3 /day) are typical. Higher loading rates could be treated
if a more concentrated sludge could be fed to the digester.

The volatile solids loadings to anaerobic digesters are controlled in most waste-
water treatment plants by the efficiency of the primary and secondary clarifiers in
removing and concentrating sludge. Therefore, the thickening of sludge is an impor-
tant operational factor affecting digester performance.

Typically, raw sludges or feed sludges having low solids content are transferred
to municipal anaerobic digesters . These sludges often contain 3-6% solids. These
dilute feed sludges adversely impact digester operation. They reduce hydraulic
retention time (HRT), reduce volatile solids destruction, and reduce methane
production.

The blending of primary and secondary sludges may be helpful in improving
anaerobic digester performance (Figure 12.1). Primary sludge may be blended with
thickened excess activated sludge, or blended primary and secondary sludges may
be thickened.

The percentage of primary sludge in feed sludge may influence VS reduction in
the anaerobic digester. Generally, with increasing percent primary sludge in digester
feed sludge, an increase in VS reduction can be expected.

The HRT of anaerobic digesters is affected by not only the quantity of feed
sludge but also the quantities of digested sludge, supernatant, and grit. Digested

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

SLUDGE FEED

Scum

Primary clarifier

Scum

Secondary clarifier

Primary sludge

Secondary sludge

' ^N

-►

Anaerobic
Digester

Thick

ener

Figure 12.1 Digester performance or treatment efficiency is affected by the blending of primary and
secondary sludges.

sludge and supernatant must be withdrawn on a routine basis, and grit must be
removed as needed to ensure adequate retention time. Common operational prob-
lems associated with anaerobic digesters are overpumping of raw sludge and exces-
sive withdrawal of digested sludge.

Retention Times

There are two significant retention times in an anaerobic digester. These are solids
retention time (SRT) and hydraulic retention time (HRT). The SRT is the average
time that bacteria (solids) are in the anaerobic digester. The HRT is the time that
the wastewater or sludge is in the anaerobic digester. The SRT and the HRT are the
same for a suspended-growth anaerobic digester that has no recycle. If recycle of
solids is incorporated in the operation of the digester, then the SRT and HRT may
vary significantly.

Because the generation time, that is, the time required for a population of bac-
teria to double in size, of methane-forming bacteria is relatively long compared with
aerobic bacteria and facultative anaerobic bacteria (Table 13.1), typical SRTs for
anaerobic digesters are >12 days. Detention times <10 days are not recommended.
At detention times <10 days significant washout of methane-forming bacteria
occurs. This indicates that SRT, not HRT, is the more important retention time. The
SRT is not greatly affected by the nature of the wastewater or sludge under treat-
ment, unless the wastewater or sludge is toxic to the bacteria.

Anaerobic digesters that utilize fixed-film media for the growth of bacteria favor
the development of a concentrated mass (biomass) of bacteria that are attached to
the media. The biomass prevents the washout of large numbers of bacteria and
permits high SRT values.

High SRT values are advantageous for anaerobic digesters. High SRT values
maximize removal capacity, reduce required digester volume, and provide buffer-
ing capacity for protection against the effects of shock loadings and toxic com-
pounds in wastewaters and sludges. High SRT values also help to permit biological
acclimation to toxic compounds. High SRT values may be achieved through two
measures. First, the volume of the digester may be increased. Second, the concen-
tration of the bacteria (solids) may be increased.

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

RETENTION TIMES

TABLE 13.1 Approximate Generation Times of Important Groups of Wastewater Bacteria

Bacterial
Group

Function

Approximate
Generation Time

Aerobic
organotrophs

Facultative
anaerobic
organotrophs

Nitrifying bacteria

Methane-forming
bacteria

Floe formation and degradation of soluble organics
in the activated sludge and trickling filter processes

Floe formation and degradation of soluble organics
in the activated sludge and trickling filter processes,
hydrolysis and degradation of organics in the
anaerobic digester

Oxidation of NH 4 + and N0 2 " in the activated sludge
and trickling filter processes

Production of methane in the anaerobic digester

15-30min

2-3 days
3-30 days

The conversion of volatile solids to gaseous products in an anaerobic digester is
controlled by the HRT. The design of the HRT is a function of the final disposition
of the digested sludge. The HRT may be relatively high or low, if the digested sludge
is to be land applied or incinerated, respectively. However, increases in detention
time >12 days do not contribute significantly to increased destruction of volatile
solids.

HRT values affect the rate and extent of methane production. Of all the opera-
tional conditions within an anaerobic digester, for example, temperature, solids
concentration, and volatile solids content of the feed sludge, HRT is perhaps the
most important operational condition affecting the conversion of volatile solids to
gaseous products.

Temperature

Common recurring problems associated with anaerobic digesters are loss of heating
capability and maintenance of optimum digester temperature. An acceptable and
uniform temperature should be maintained throughout the digester to prevent
localized pockets of depressed temperature and undesired bacterial activity.
Variations in temperature of even a few degrees affect almost all biological activity
including the inhibition of some anaerobic bacteria, especially methane-forming
bacteria. Adequate mixing of the digester content prevents the development of
localized pockets of temperature variation.

Most methane-forming bacteria are active in two temperature ranges. These
ranges are the mesophilic range from 30 to 35°C and the thermophilic range from
50 to60°C. At temperatures between 40°C and 50°C, methane-forming bacteria are
inhibited. Digester performance falters somewhere near 42°C, as this represents the
transition from mesophilic to thermophilic organisms.

Although methane production can occur over a wide range of temperatures
(Figure 14.1), anaerobic digestion of sludge and methane production at municipal
wastewater treatment plants is performed in the mesophilic range, with an optimum
temperature of approximately 35° C (Table 14.1).

Whenever digester temperature falls below 32° C, close attention should be paid
to the volatile acid-to-alkalinity ratio. Volatile acid formation continues at depressed
temperatures, but methane production proceeds slowly. Volatile acid production can
continue at a rapid rate as low as 21 °C, whereas methane production is essentially
nonexistent. Therefore, 32° C is the minimum temperature that should be main-
tained, and 35° C is the preferred temperature.

Although methane-forming bacteria are active and grow in several tempera-
ture ranges (Table 14.2), most methane-forming bacteria are mesophiles. Some

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

TEMPERATURE

Thermophilic methane-forming bacteria

Mesophilic methane- forming bacteria

Digestion time, days

Figure 14.1 Methane production occurs over a relatively large range of temperature values. Most
anaerobic digesters, especially those at municipal wastewater treatment plants, operate in the
mesophilic range of temperatures.

TABLE 14.1 Temperature Range for Methane
Production for Municipal Anaerobic Digesters

Temperature, °C

Methane Production

32-34
21-31
<21

Optimum

Minimum

Little, digester going "sour"

Nil, digester is "sour"

TABLE 14.2 Optimum Temperature Ranges for the
Growth of Methane-forming Bacteria

Bacterial Group

Temperature Range, °C

Psychrophiles
Mesophiles
Thermophiles
Hyperthermophiles

5-25
30-35
50-60
>65

methane-forming bacteria are psychrophiles, thermophiles, and hyperthermophiles
or stearothermophiles. Anaerobic sludge digestion in the psychrophilic range
usually is confined to small-scale treatment units such as Imhoff tanks, septic tanks,
and sludge lagoons. Here the digestion process is not heated, and the temperature
of the digester sludge is approximately equal to the outside environment. There-
fore, the rate of digestion of sludge varies from season to season. Because of the

TEMPERATURE 91

TABLE 14.3 Comparison of Mesophilic and Thermophilic Digesters

Feature

Mesophilic Digester

Thermophilic Digester

Loading rates

Lower

Higher

Destruction of pathogens

Lower

Higher

Sensitivity to toxicants

Lower

Higher

Operational costs

Lower

Higher

Temperature control

Less difficult

More difficult

depressed temperature of the digester sludge, the sludge retention time (SRT) is
usually long, often greater than 12 weeks.

Methane production in the thermophilic range is usually performed at industrial
wastewater treatment plants that are able to heat wastewaters or sludges. A com-
parison of advantages and disadvantages of mesophilic and thermophilic digesters
is presented in Table 14.3. The greater destruction of pathogens by thermophilic
digesters has drawn attention to their use to satisfy existing and proposed regula-
tions for the disposal and reuse of municipal sludges.

The rate of anaerobic digestion of sludge and methane production is proportional
to digester temperature, that is, the higher the temperature the greater the destruc-
tion rate of volatile solids and the production of methane. The rate of anaerobic
digestion of sludge and methane production is considerably faster in thermophilic
digesters than in mesophilic digesters.

Although 25% to 50% more activity occurs in thermophilic digesters than in
mesophilic digesters, there are several significant microbiological characteristics
associated with thermophilic anaerobes and thermophilic digestion that may
adversely affect digester performance. These characteristics include 1) the low bac-
terial growth or yield (increase in population size) of these anaerobes, 2) the high
endogenous death rates of these bacteria, and 3) the lack of diversity of these anaer-
obes. These characteristics are responsible for 1) relatively high residual values
of volatile acids, for example, >1000mg/l, and 2) inconsistent treatment of sludge
during continuously shifting operational conditions. Also, thermophilic anaerobes
are very sensitive to rapid changes in temperature. Therefore, fluctuations in digester
temperature should be as small as possible, that is, <1°C per day for therm ophiles
and 2-3°C per day for mesophiles.

Temperature is one of the most important factors affecting microbial activity
within an anaerobic digester, and methane production is strongly temperature
dependent. Fluctuations in temperature affect the activity of methane-forming bac-
teria to a greater extent than the operating temperature.

Temperature influences not only methane-forming bacteria but also volatile
acid-forming bacteria. Therefore, fluctuations in temperature may be advantageous
to certain groups and disadvantageous to other groups. For example, a 10° C tem-
perature increase can stop methane production or methane-forming bacterial activ-
ity within 12 hours, while volatile acid production increases. Changes in the activity
of different groups of volatile acid-forming bacteria result in changes in the relative
quantities of organic acids and alcohols produced during fermentation. Changes in
the quantities of organic acids and alcohols that are used directly and indirectly as
substrates by methane-forming bacteria affect overall digester performance.

92 TEMPERATURE

The effect of temperature on hydrolysis of particulate and colloidal wastes is not
very great. Hydrolytic bacteria are not as sensitive to temperature change as the
acetate-forming bacteria and methane-forming bacteria.

Temperature affects biological activity. This effect is due mostly to the impact of
temperature on enzymatic activity or reactions. Therefore, increases in temperature
result in more enzymatic activity whereas decreases in temperature result in less
enzymatic activity. Because of the impact of temperature on enzymatic activity, SRT
within digesters should increase with decreasing temperatures.

Although anaerobic bacteria can be acclimated to operating temperatures
outside their optimum range, biomass activity and digester performance may be
adversely affected. Because methane-forming bacteria grow slowly and are very
sensitive to small changes in temperature, acclimation must proceed very slowly.

Nutrients

Although nutrient needs for bacteria in aerobic and anaerobic biological treatment
processes may be grouped as macronutrients and micronutrients, there are signi-
ficant differences in nutrient requirements between these two treatment processes.
These differences are due to the unique needs of methane-forming bacteria and the
lower cell (sludge) yield of fermentative bacteria as compared to aerobic bacteria.
Macronutrients, for example, nitrogen and phosphorus, are nutrients that are
required in relatively large quantities by all bacteria. Micronutrients, for example,
cobalt and nickel, are nutrients that are required in relatively small quantities by most
bacteria. The inorganic nutrients critical in the conversion of acetate to methane — the
rate-limiting reaction in an anaerobic digester — are the macronutrients nitrogen and
phosphorus and the micronutrients cobalt, iron, nickel, and sulfur.

MACRONUTRIENTS

Macronutrient requirements for anaerobic biological treatment processes are much
lower than the requirements for aerobic biological treatment processes such as
activated sludge and trickling filter processes. The reduced requirement for macro-
nutrients in anaerobic processes is due to lower cell (sludge) yield compared with
aerobic processes from the degradation of equal quantities of substrate.

The two macronutrients of concern in any biological treatment process are
nitrogen and phosphorus. These nutrients are made available to anaerobic bac-
teria, including methane-forming bacteria, as ammonical-nitrogen (NH4-N) and
orthophosphate-phosphorus (HPO4-P). These nutrients, like all nutrients, are avail-
able to bacteria only in a soluble form.

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

94 NUTRIENTS

Although NH4-N is the preferred nitrogen nutrient for methane-forming bacte-
ria, some methane-forming bacteria can obtain nitrogen from other sources. Some
are able to fix molecular nitrogen (N 2 ), and some are able to use the amino acid
alanine (CH 3 CHNH 2 COOH). Orthophosphate-phosphorus is the preferred phos-
phorus nutrient.

The amount of nitrogen and the amount of phosphorus needed to satisfy anaer-
obic bacterial activity and maintain acceptable digester performance may be deter-
mined by one of two methods. The first method consists of calculating the amount
of nutrients that must be present in the digester feed sludge and, if necessary, adding
the nutrient. In the second method, adequate residual concentrations of soluble
nutrients must be found in the digester effluent. If these residual concentrations are
not found, the nutrients must be added.

Because carbonaceous biochemical oxygen demand (cBOD) is measured under
aerobic conditions over a rather short test period (5 days) compared with relatively
long digester retention times (>12 days), the resulting cBOD tends to underesti-
mate the total oxygen demand present in a sample of sludge or wastewater. Also,
oxygen is not used in an anaerobic digester to degrade organic compounds. There-
fore, under anaerobic conditions of degradation of substrates in which oxygen is not
used and hydrolysis of substrates occurs, cBOD underestimates the total strength
of a sample of sludge or wastewater. These discrepancies have led to the use of
chemical oxygen demand (COD) for characterization of the strength of a sample
of sludge or wastewater.

The amount of nitrogen and the amount of phosphorus that must be available
in the digester can be determined from the quantity of substrate or COD of the
digester feed sludge. Nutrient requirements for anaerobic digesters vary greatly at
different organic loading rates (Figure 15.1). Generally, COD:N:P of 1000:7:1 and
350:7:1 have been used for high-strength wastes and low loadings, respectively.
These ratios have a C/N value of at least 25:1 that is suggested for optimal gas
production. If either of these ratios is used, it is assumed that nitrogen is ap-
proximately 12% of the dry weight of bacterial cells or sludge and phosphorus
is approximately 2% of the dry weight of bacterial cells or sludge (Table 15.1).
These ratios are based on the common empirical formula for cellular material,
C 2 H 7 2 N.

By assuming that 10% of the COD fed to the digester is converted to new bac-
terial cells (C 2 H 7 2 N), that is, growth yield of 0.1 kg VSS/kg COD removed, the
amount of nitrogen and phosphorus that are needed can be calculated. For example,
if the COD of the digester feed sludge is 10,000 mg/1, and 80% of the COD is
degraded, then the amount of nitrogen and the amount of phosphorus that are
needed are 96 mg/1 and 16 mg/1, respectively (Figure 15.2).

By ensuring residual values of ammonical-nitrogen and orthophosphate-
phosphorus in the digester effluent, nitrogen and phosphorus should not be limited
in the digester. Residual values of 5 mg/1 of NH4-N and 1-2 mg/1 of HPO4-P are
commonly recommended.

Adequate nutrient needs for anaerobic digesters may be determined by ensur-
ing at least a minimum amount of a nutrient as a percentage of the COD loading
to the digester. Table 15.2 lists some nutrient needs.

If nutrient addition is required for nitrogen or phosphorus, several chemicals may
be used. For nitrogen addition, ammonium chloride, aqueous ammonia, and urea

MACRONUTRIENTS

-d

• •

2500:7

2000:7

1500:7

1000:7

500:7

0.5

1.0

1.5

kg COD/kg VSS per day

Figure 15.1 Nutrient needs of an anaerobic digester are determined by the loading or the COD.N
and COD.P in the feed sludge. With increasing COD loading there is a corresponding increase in
nutrient needs for nitrogen and phosphorus.

Influent COD

10,000 mg/1

Treatment efficiency
COD removed

Biomass growth (0.1 X 8,000)
Nitrogen required (0.12 X 800)
Phosphorus required (0.02 X 800)

Figure 15.2

8,000 mg/1

= 800mgVSS/l

96 mg/1

16 mg/1

TABLE 15.1 Elementary Composition of Bacterial Cells
(Dry Weight)

Element

Approximate Percent Composition

Carbon

Oxygen

Nitrogen

Hydrogen

Phosphorus

Sulfur

Potassium

Others

2
1
1
6

NUTRIENTS

TABLE 15.2 Significant Nutrient Needs for Anaerobic Digesters

Nutrient

icronutrient

acronutrient

inimum Recommended
(% of COD)

Cobalt

0.01

Iron

0.2

Nickel

0.001

Nitrogen

3-4

Phosphorus

0.5-1

Sulfur

0.2

may be used. For phosphorus addition, phosphate salts and phosphoric acid may be

used.

MICRONUTRIENTS

Because methane-forming bacteria possess several unique enzyme systems, they
have micronutrient requirements that are different from those of other bacteria.
The need for several micronutrients, especially cobalt, iron, nickel, and sulfide, is
critical. Additional trace elements on which enzymes of methane-forming bacteria
are dependent include selenium and tungsten. The incorporation of micronutrients
in enzyme systems is essential to ensure not only proper degradation of substrate
but also efficient operation of the digester. Cobalt, iron, nickel, and sulfide are
obligatory micronutrients, because they are required by methane-forming bacteria
to convert acetate to methane. Therefore, attention to macronutrient needs alone is
grossly inadequate for methane-forming bacteria.

Molybdenum, tungsten, and selenium may be obligatory micronutrients. Addi-
tional micronutrients of concern are barium, calcium, magnesium, and sodium.
Deficiencies for micronutrients in anaerobic digesters often have been mistaken for
symptoms of toxicity.

Although these micronutrients are usually present in sufficient quantities in
municipal wastewater, the digester effluent should be analyzed to ensure that
residual soluble quantities of these nutrients exist, especially in industrial waste-
water treatment plants. The presence of adequate nutrients, especially micronu-
trients, helps to minimize digester upsets caused by the accumulation of volatile fatty
acids.

Methane-forming bacteria are able to easily remove or "harvest" micronutrients
from the bulk solution. The harvesting of micronutrients is accomplished through
the production and excretion of extracellular "slime" that chelates and transports
the nutrients into the cell (Figure 15.3). The use of extracellular slime permits
"luxury" uptake of micronutrients, that is, the removal and storage of nutrients
beyond the quantity that is needed.

If it is necessary to add micronutrients to an anaerobic digester, yeast extract
can be used. Yeast extract contains numerous amino acids, minerals, and vitamins,
including the B vitamins biotin and folic acid. The addition of 1.5kg/m 3 of yeast
extract at all loading rates should provide adequate micronutrients.

NICKEL

Slime

Cell wall

Figure 15.3 Relatively large quantities of micronutrients are removed from the bulk solution by
methane-forming bacteria through their adsorption to the slime that coats the bacterial cells. Once
adsorbed the nutrients are then absorbed by the bacterial cells.

COBALT

Cobalt is required as an activator of enzyme systems in methane-forming bacteria.
The incorporation of cobalt into enzyme systems provides for more efficient
conversion of acetate to methane.

IRON

Although methane-forming bacteria have a relatively high iron requirement,
and iron usually exists in high concentrations in the environment, it is difficult for
methane-forming bacteria as well as anaerobic bacteria in general to assimilate iron.
For iron to be assimilated, it must be in solution. Unfortunately, this requirement is
usually not satisfied in the environment of methane-forming bacteria and other
anaerobic bacteria.

NICKEL

Nickel is a unique micronutrient requirement for methane-forming bacteria,
because nickel is generally not essential for the growth of most bacteria. For
example, the F 430 enzyme in methane-forming bacteria contains nickel. The addition
of nickel can increase acetate utilization rate of methane-forming bacteria.

The requirement for nickel has long been overlooked because of the high back-
ground level or presence of nickel in bacterial growth media. However, the lack of
adequate usable nickel in the bulk solution of an anaerobic digester results in a

NUTRIENTS

CH3

CH2

NH2

COOH

Methionine

Figure 15.4

significant decrease in the rate of methane production, that is, decreased enzymatic
ability to convert acetate to methane.

SULFIDE

Sulfide is the principle source of sulfur for methane-forming bacteria. For sulfide to
enter a bacterial cell, it must exist as nonionized hydrogen sulfide (H 2 S). This form
of sulfide occurs in a relatively high concentration within the pH range of 6.8 to 6.9,
which is also near the pH of normal anaerobic digester operation.

Additional sulfur sources for methane-forming bacteria are the amino acids cys-
teine and methionine (Figure 15.4). These amino acids contain sulfur (S) or the thiol
group (-SH), which releases sulfur on degradation of the amino acids.

Although sulfide is considered a micronutrient for methane-forming bacteria, the
sulfide content of these bacteria is relatively high. On a dry weight basis, approxi-
mately 2.5% of the bacterial cell is sulfide. This quantity of sulfide also is approxi-
mately 50% greater than the phosphorus content of the cell.

Although sulfide is required in relatively high concentrations and is considered
an obligate micronutrient, relatively high concentrations of sulfide present two sig-
nificant problems for successful anaerobic digester operation. Sulfide presents oper-
ational problems by precipitating trace metals or micronutrients and causing toxicity
at high concentrations.

Alkalinity and pH

Sufficient alkalinity is essential for proper pH control. Alkalinity serves as a buffer
that prevents rapid change in pH. Enzymatic activity or digester performance is
influenced by pH. Acceptable enzymatic activity of acid-forming bacteria occurs
above pH 5.0, but acceptable enzymatic activity of methane-forming bacteria does
not occur below pH 6.2. Most anaerobic bacteria, including methane-forming bac-
teria, perform well within a pH range of 6.8 to 7.2.

The pH in an anaerobic digester initially will decrease with the production of
volatile acids. However, as methane-forming bacteria consume the volatile acids and
alkalinity is produced, the pH of the digester increases and then stabilizes. At
hydraulic retention times >5 days, the methane-forming bacteria begin to rapidly
consume the volatile acids.

In a properly operating anaerobic digester a pH of between 6.8 and 7.2 occurs
as volatile acids are converted to methane and carbon dioxide (C0 2 ).The pH of an
anaerobic system is significantly affected by the carbon dioxide content of the
biogas.

Digester stability is enhanced by a high alkalinity concentration. A decrease in
alkalinity below the normal operating level has been used as an indicator of pending
failure. A decrease in alkalinity can be caused by 1) an accumulation of organic
acids due to the failure of methane-forming bacteria to convert the organic acids to
methane, 2) a slug discharge of organic acids to the anaerobic digester, or 3) the
presence of wastes that inhibit the activity of methane-forming bacteria. A decrease
in alkalinity usually precedes a rapid change in pH.

The composition and concentration of the feed sludge directly influence the alka-
linity of the digester. For example, large quantities of proteinaceous wastes trans-
ferred to the anaerobic digester are associated with relatively high concentrations

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

100 ALKALINITY AND pH

of alkalinity. The alkalinity is the result of the release of amino groups (-NH 2 ) and
production of ammonia (NH 3 ) as the proteinaceous wastes are degraded. Also,
thickened sludges have relatively high alkalinity. This alkalinity is due to the
increased feed rate of proteins within the thickened sludges.

Alkalinity is present primarily in the form of bicarbonates that are in equilibrium
with carbon dioxide in the biogas at a given pH. When organic compounds are
degraded, carbon dioxide is released. When amino acids and proteins are degraded,
carbon dioxide and ammonia are released.

The release of carbon dioxide results in the production of carbonic acid, bicar-
bonate alkalinity, and carbonate alkalinity (Equation 16.1). The release of ammonia
results in the production of ammonium ions (Equation 16.2).

C0 2 + H 2 <-> H 2 C0 3 o H + + HCO3 ^ H + + COf" (16.1)

NH 3 + H + ^ NH 4 + (16.2)

The equilibrium between carbonic acid, bicarbonate alkalinity, and carbonate
alkalinity as well as ammonia and ammonium ions is a function of digester pH
(Figure 16.1). Bicarbonate alkalinity is the primary source of carbon for methane-
forming bacteria.

Significant changes in alkalinity or pH are introduced in an anaerobic digester
by substrate feed or the production of acidic and alkali compounds, such as organic
acids and ammonium ions, respectively, during the degradation of organic com-
pounds in the digester.

Alkalinity in an anaerobic digester also is derived from the degradation of
organic-nitrogen compounds, such as amino acids and proteins, and the production
of carbon dioxide from the degradation of organic compounds. When amino acids
and proteins are degraded, amino groups (-NH 2 ) are released and alkalinity is
produced. When amino groups are released, ammonia is produced. The ammonia

Protein degradation (C,N,0,N,S) >

(CH4, RCOOH, H2S)
CO2 < — - > H2CO3 < — - > H+ + HCO3- < — - > H+ + CO3 2 -

NH3 < — - > NH4+ + OH-

Figure 16.1

ALKALINITY AND pH 101

dissolves in water along with carbon dioxide to form ammonium bicarbonate
(NH4HCO3) (Equation 16.3).

NH 3 + H 2 + C0 2 o NH4HCO3 (16.3)

However, the degradation of organic compounds produces organic acids that
destroy alkalinity. For example, as a result of the degradation of glucose, acetate is
formed (Equation 16.4). This acid destroys alkalinity, for example, ammonium bicar-
bonate (Equation 16.5), and the alkalinity is not returned until methane fermenta-
tion occurs (Equation 16.6).

C 6 H 12 6 -> 3CH 3 COOH (16.4)

3CH 3 COOH + 3NH4HCO3 -> 3CH 4 COONH 4 + 3H 2 + 3C0 2 (16.5)

3CH 3 COONH 4 + + 3H 2 -> 3CH 4 + 3NH 4 HC0 3 (16.6)

Although anaerobic digester efficiency is satisfactory within the pH range of
6.8 to 7.2, it is best when the pH is within the range of 7.0 to 7.2. Values of pH below
6 or above 8 are restrictive and somewhat toxic to methane-forming bacteria
(Table 16.1). To maintain a stable pH, a high level of alkalinity is required.

If the feed sludge to the anaerobic digester does not contain alkali compounds
or precursors of alkali compounds, alkalinity must be added to the digester to main-
tain stable and acceptable values for alkalinity and pH. The quantity of alkalinity
to be added should be based on the anticipated organic acid production capacity of
the sludge feed (1 g of volatile acids per gram of volatile solids). Also, if the rate of
acid production exceeds the rate of methane production, alkalinity must be added.
A higher rate of volatile acid production than methane production usually occurs
during start-up, overload, loss of adequate temperature, and inhibition.

Alkalinity also may be lost or "washed out" of the digester. When increased
wastewater temperature occurs, increased microbial activity within an activated
sludge process occurs and buoyant sludge is usually produced. Increased pumping
from the activated sludge process or thickener to the anaerobic digester occurs
because of the presence of buoyant sludge. Increased pumping produces decreased
digester hydraulic retention time (HRT) and "washout" of digester alkalinity.

TABLE 16.1 Optimum Growth pH of Some Methane-
forming Bacteria

Genus pH

Methanosphaera 6.8

Methanothermus 6.5

Methanogenium 7.0

Methanolacinia 6.6-7.2

Methanomicrobium 6.1-6.9

Methanospirillium 7.0-7.5

Methanococcoides 7.0-7.5

Methanohalobium 6.5-7.5

Methanolobus 6.5-6.8

Methanothrix 7.1-7.8

102 ALKALINITY AND pH

TABLE 16.2 Chemicals Commonly Used for Alkalinity
Addition

Chemical

Formula

Buffering
Cation

Sodium bicarbonate

NaHC0 3

Na +

Potassium bicarbonate

KHCO3

K +

Sodium carbonate (soda ash)

Na 2 C0 3

Na +

Potassium carbonate

K 2 C0 3

K +

Calcium carbonate (lime)

CaC0 3

Ca 2+

Calcium hydroxide (quick lime)

Ca(OH) 2

Ca 2+

Anhydrous ammonia (gas)

NH 3

NH 4+

Sodium nitrate

NaN0 3

Na +

Several chemicals can be used to adjust alkalinity and pH in an anaerobic digester
(Table 16.2). Because methane-forming bacteria require bicarbonate alkalinity,
chemicals that release bicarbonate alkalinity directly are preferred. Of these chem-
icals, sodium bicarbonate and potassium bicarbonate are perhaps the best chemi-
cals of choice because of their desirable solubility, handling, and minimal adverse
impacts within the digester. For example, overdosing of these chemicals does not
cause the pH of the digester to quickly rise above the optimum. Also, of all the
cations released by the alkali chemicals used for alkalinity addition, sodium and
potassium are the least toxic to the bacteria in the digester. Chemicals that release
hydroxide alkalinity, for example, caustic soda, are not effective in maintaining
proper alkalinity in the digester because of the bicarbonate alkalinity requirement
of methane-forming bacteria.

Lime (CaC0 3 ) may be used to increase digester pH to 6.4, and then either bicar-
bonate or carbonate salts (sodium or potassium) should be used to increase the pH
to the optimum range. Lime increases pH quickly and dramatically, but lime does
not significantly increase alkalinity. Overdosing with lime may easily cause the pH
to exceed the optimum pH range.

Caution should be used when using hydrated lime or quick lime [calcium hydrox-
ide (Ca(OH) 2 )] and soda ash [sodium carbonate (Na 2 C0 3 )] to increase alkalinity.
Calcium hydroxide and sodium carbonate first react with soluble carbon dioxide in
the sludge (Equations 16.7 and 16.8, respectively). If carbon dioxide is removed too
rapidly or in too large a quantity from the sludge, then carbon dioxide from the
biogas will replace the carbon dioxide lost from the sludge. When carbon dioxide is
lost from the biogas, a partial vacuum condition develops under the digester dome.
This condition may cause the digester cover to collapse. Also, as the concentration
of alkalinity increases in the anaerobic digester, the continued use of quick lime
results in the precipitation of calcium carbonate (Equation 16.9).

Ca(OH) 2 + 2C0 2 -> Ca(HC0 3 ) 2 (16.7)

Na 2 C0 3 + H 2 + C0 2 -* 2NaHC0 3 (16.8)

Ca(OH) 2 + C0 2 -* CaC0 3 + H 2 (16.9)

Anhydrous ammonia also may be used to adjust alkalinity and pH. Ammonia
reacts with carbon dioxide and water, resulting in the production of ammonium

ALKALINITY AND pH 103

bicarbonate (Equation 16.10). Ammonium carbonate adds alkalinity and is avail-
able to react with volatile acids, resulting in the production of volatile acid salts
(Equation 16.11).

NH 3 + C0 2 + H20 -> NH4HCO3 (16.10)

NH4HCO3 + RCOOH* -> RCOONH 4 + H + + HCO3 (16.11)

*R represents the non-carboxyl (-COOH) portion of the volatile acid.

Anhydrous ammonia also may help to dissolve scum layers. Although the
addition of anhydrous ammonia has several benefits for an anaerobic digester, there
are some concerns. Anhydrous ammonia may produce a negative pressure in the
digester by reacting with carbon dioxide. In addition, at elevated pH values excess
ammonia gas may cause toxicity.

If pH and alkalinity both must be increased in an anaerobic digester, sodium
carbonate may be used to increase pH if it drops below 6.5. Sodium carbonate also
replenishes alkalinity. If sodium bicarbonate, sodium carbonate, or sodium nitrate
is added too rapidly to an anaerobic digester, a foaming problem may develop.
Sodium bicarbonate and sodium carbonate release carbon dioxide on addition,
whereas sodium nitrate releases molecular nitrogen (N 2 ) and nitrous oxide (N 2 0)
upon addition.

Caution also should be used when adding sodium nitrate, because the release
of nitrate ions (NO3) increases the oxidation-reduction potential (ORP) of the
digester. The ORP of the digester should not be allowed to increase above -300 mV,
for example, -250 mV, because methane-forming bacteria cannot produce methane
at ORP values greater than -300 mV in a mixed culture.

Any chemical selected for addition to the digester should be added slowly to
prevent any adverse impact on the bacteria due to rapid changes in alkalinity, pH,
ionic strength, or ORP.

Caution should be exercised in the choice of the chemical used for pH/alkalin-
ity adjustments. The precipitation of CaC0 3 creates unwanted solids, and the large
quantities of a single cation, for example, Na + , presents the potential for alkali metal
toxicity. Therefore, it may be preferable to use mixtures of cations, for example, Ca 2+
from Ca(OH) 2 , Na + from NaOH, and K + from KOH, for pH/alkalinity control.

Although the pH of the digester is more easily and quickly determined than the
alkalinity of the digester, the pH is only an indication of what has already happened
in the digester, whereas changes in alkalinity indicate what is happening in the
digester. The alkalinity of the digester indicates whether alkalinity addition or cor-
rective measures are needed.

Excessive alkalinity in the digester should be avoided. Excess alkalinity can be
destroyed or neutralized with the addition of ferric chloride or citrate.

Toxicity

A variety of inorganic and organic wastes can cause toxicity in anaerobic digesters
(Table 17.1). Many toxic wastes are removed in primary clarifiers and transferred
directly to the anaerobic digester. Heavy metals may be precipitated as hydroxides
in primary sludge, and organic compounds such as oils and chloroform are removed
in primary scum and sludge, respectively. Industrial wastewaters often contain
wastes that are toxic to anaerobic digesters.

Although guideline values or ranges of values exist at which toxicity occurs for
specific inorganic wastes (Table 17.2) and organic wastes (Table 17.3), methane-
bacteria often can tolerate higher values by acclimating to the wastes. When toxic
values of specific wastes for anaerobic digesters are assessed, the toxic value is deter-
mined by several factors. These factors include 1) the ability of the bacteria to adapt
to a constant concentration of toxic waste, 2) the absence or presence of other toxic
wastes, and 3) changes in operational conditions.

Toxicity in an anaerobic digester may be acute or chronic. Acute toxicity results
from the rapid exposure of an unacclimated population of bacteria to a relatively
high concentration of a toxic waste. Chronic toxicity results from the gradual and
relatively long exposure of an unacclimated population of bacteria to a toxic waste.

The population of bacteria may acclimate under chronic toxicity by two means.
First, they may repair damaged enzyme systems in order to adjust to the toxic wastes
or degrade the toxic organic compound. Second, they may grow a relatively large
population of bacteria that is capable of developing the enzyme systems necessary
to degrade the toxic organic compounds. The time of chronic toxicity in an anaer-
obic digester is determined by 1) the time of contact between the toxic waste and
the bacteria and 2) the ratio of toxic waste to the bacterial population (biomass or
solids).

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

105

106 TOXICITY

TABLE 17.1 Inorganic and Organic Toxic Wastes to
Anaerobic Digesters

Alcohols (isopropanol)

Alkaline cations (Ca 2+ , Mg 2+ , K + , and Na + )

Alternate electron acceptors, nitrate (NO3) and sulfate

(SOf)
Ammonia

Benzene ring compounds
Cell bursting agent (lauryl sulfate)
Chemical inhibitors used as food preservatives
Chlorinated hydrocarbons
Cyanide

Detergents and disinfectants
Feedback inhibition
Food preservatives
Formaldehyde
Heavy metals
Hydrogen sulfide

Organic-nitrogen compounds (acrylonitrile)
Oxygen

Pharmaceuticals (monensin)
Solvents
Volatile acids and long-chain fatty acids

TABLE 17.2 Toxic Values for Selected Inorganic
Wastes

Waste Concentration (mg/l) in

Influent to Digester

Ammonia 1500

Arsenic 1.6

Boron 2

Cadmium 0.02

Chromium (Cr 6+ ) 5-50

Chromium (Cr 3+ ) 50-500

Copper 1-10

Cyanide 4

Iron 5

Magnesium 1000

Sodium 3500

Sulfide 50

Zinc 5-20

TABLE 17.3 Toxic Values for Selected Organic Wastes

Waste Concentration (mg/l) in

influent to digester

Alcohol, allyl 100

Alcohol, octyl 200

Acrylonitrile 5

Benzidine 5

Chloroform 10-16

Carbon tetrachloride 10-20

Methylene chloride 100-500

1,1,1-Trichloroethane 1

Trichlorofluoromethane 20

Trichlorotrifluoroethane 5

AMMONIA TOXICITY 107

Indicators of toxicity in an anaerobic digester may appear rapidly or slowly
depending on the type of toxicity and the concentration of the toxic waste. Indicators
of toxicity include the disappearance of hydrogen, the disappearance of methane,
decreases in alkalinity and pH, and an increase in volatile acid concentration.

Wastes that are toxic to anaerobic digesters are numerous and diverse. Perhaps
the three most commonly reviewed types of toxicity are ammonia, hydrogen sulfide,
and heavy metals. Additional types of toxic wastes are listed in Table 17.1 and may
be found in simple household detergents and complex anthropogenic organic
compounds. Household detergents that contain the dispersing agent lauryl sulfate
burst the cell walls of bacteria. Anthropogenic organic compounds include solvents
and pesticides. These compounds are either highly chlorinated or contain cyanide
(CN).

AMMONIA TOXICITY

Ammonical-nitrogen (NH4-N) or ammonium ions (NHJ), a reduced form of nitro-
gen, may be transferred to an anaerobic digester or may be produced during the
anaerobic degradation of organic nitrogen compounds such as amino acids and pro-
teins. Reduced nitrogen exits in two forms, the ammonium ion and free or nonion-
ized ammonia (NH 3 ).The effects of ammonical-nitrogen/ammonia in the anaerobic
digester are positive and negative (Table 17.4). Ammonium ions are used by bac-
teria in the anaerobic digester as a nutrient source for nitrogen. Free ammonia is
toxic.

The amount of each form of reduced nitrogen in an anaerobic digester is deter-
mined by the digester pH, and the forms are in relatively equal amounts at pH 9.3
(Equation 17.1). With increasing pH, the amount of free ammonia increases. With
decreasing pH, the amount of ammonium ions increases. At pH 7, free ammonia
accounts for approximately 0.5% of the total reduced nitrogen.

NH 4 + o NH 3 + H + (17.1)

Free ammonia is toxic to methane-forming bacteria. The toxic effects of ammonia
as well as cyanide and hydrogen sulfide are determined by digester pH. All are toxic
in their undissociated (nonionized) form, that is, NH 3 , HCN (Equation 17.2), and
H 2 S (Equation 17.3). The pH effect on ammonia is direct, that is, with increasing pH
ammonia is produced in large quantities. The pH effect on cyanide and hydrogen
sulfide is indirect, that is, with decreasing pH cyanide and hydrogen sulfide are pro-
duced in large quantities. Although methane-forming bacteria can acclimate to free
ammonia, unacclimated methane-forming bacteria can be inhibited at free ammonia
concentrations >50mg/l.

TABLE 17.4 Effects of Ammonical-nitrogen/Ammonia in an Anaerobic Digester

Ammonical-nitrogen (NH^/Dissolved Ammonia (NH 3 ), N Effect

50-200 mg/l Beneficial

200-1 000 mg/l No adverse effect

1500-3000 mg/l Inhibitory at pH > 7

108 TOXICITY

HCN <-> CN" + H + (17.2)

H 2 S <r± HS~ + H + (173)

Concentrations of ammonia >50mg/l can be tolerated by methane-forming bac-
teria if the bacteria have been acclimated. If methane-forming bacteria cannot be
acclimated to free ammonia, digester pH can be decreased or digester feed sludge
can be diluted to prevent ammonia toxicity.

The toxic effects of free ammonia may be confined to methane-forming bacte-
ria, and the precise concentration at which free ammonia is toxic remains uncertain.
However, anaerobic digesters with acclimated populations of methane-forming
bacteria can tolerate several hundred milligrams per liter of free ammonia.
Ammonia concentrations >1500mg/l at high pH may result in digester failure. At
concentrations above 3000 mg/1, free ammonia becomes toxic enough to cause
digester failure.

Variations in concentrations of free ammonia toxicity result from several opera-
tional factors. These factors include digester alkalinity or buffering capacity, tem-
perature, and sludge loading rates.

Although relatively high concentrations of free ammonia, for example, 1500-
3000 mg/1, can be inhibitory to methane-forming bacteria, ammonia inhibition may
be "self-correcting." Because methane-forming bacteria are inhibited by free
ammonia, volatile acid concentration increases. With an increase in digester volatile
acids, the pH of the digester drops. The drop in pH converts much of the free
ammonia to ammonium ions.

A shock load of free ammonia (a concentration greater than the digester design
limit) causes a rapid and large accumulation of volatile acids and a rapid and sig-
nificant drop in pH. Besides volatile acid accumulation, loss of alkalinity, and drop
in pH, a decrease in methane production also is indicative of ammonia toxicity.

Ammonium ions perform several important roles in an anaerobic digester.
Ammonium ions are the preferred bacterial nutrient for nitrogen. They also provide
buffering capacity in an anaerobic digester. However, although ammonium bicar-
bonate acts as a buffer, high ammonium bicarbonate concentrations resulting from
the degradation of amino acids, proteins, and highly concentrated sludges may cause
free ammonia toxicity.

A common cause of digester failure is the presence of an unacclimated po-
pulation of methane-forming bacteria at high ammonia concentrations. Therefore,
methane-forming bacteria should be gradually acclimated to increasing concentra-
tions of ammonia.

HYDROGEN SULFIDE

Bacterial cells need soluble sulfur as a growth nutrient and satisfy this need by using
soluble sulfide (HS~). However, excessive concentrations of sulfides or dissolved
hydrogen sulfide gas (H 2 S) cause toxicity.

Hydrogen sulfide is one of the compounds most toxic to anaerobic digesters. The
methane-forming bacteria are the bacteria that are most susceptible to hydrogen
sulfide toxicity. Hydrogen-consuming methane-forming bacteria are more sus-
ceptible to hydrogen sulfide toxicity than acetoclastic methane-forming bacteria.
Acid-forming bacteria also are susceptible to hydrogen sulfide toxicity.

HYDROGEN SULFIDE

109

H2S

more problematic with
increasing pH

NH4+

Cell wall

Cell membrane

Figure 17.1 The toxicity of hydrogen sulfide, hydrogen cyanide, and ammonia are pH dependent. In
the non-ionized forms (H 2 S, HCN, and NH 3 ) toxicity can occur. In the non-ionized forms these mole-
cules are capable of easily entering the bacterial cell and attacking enzyme systems.

Soluble hydrogen sulfide toxicity occurs because sulfide inhibits the metabolic
activity of anaerobic bacteria. Although the mechanism by which sulfide inhibits
anaerobic bacteria is not completely understood, toxicity can occur at concentra-
tions as low as 200mg/l at neutral pH. Because diffusion through a cell membrane
is required to exert toxicity and non-ionized hydrogen sulfide diffuses more rapidly
across a cell membrane than sulfide, hydrogen sulfide toxicity is pH dependent
(Figure 17.1).

Hydrogen sulfide is formed in anaerobic digesters from the reduction of sulfate
and the degradation of organic compounds such as sulfur-containing amino acids
and proteins. The amino acids cystine, cysteine, and methionine that are incorpo-
rated into many proteins contain sulfur in a thiol group (-SH) that is released during
the degradation of the amino acids (Figure 17.2).

Sulfate is relatively non-inhibitory to methane-forming bacteria. Sulfate is
reduced to hydrogen sulfide by sulfate-reducing bacteria (SRB). For each gram of
chemical oxygen demand (COD) degraded by SRB 1.5 grams of sulfate are reduced
to hydrogen sulfide.

Several genera of anaerobic bacteria reduce sulfate or sulfur to hydrogen sulfide.
The genus name of these bacteria begins with the prefix "Desulf." The genera
include Desulfuromonas, Desulfovibrio, and Desulfomonas. SRB are similar to
methane-forming bacteria with respect to habitat and cellular morphology or
structure.

The presence of hydrogen sulfide also can be due to the reduction of elemental
sulfur. An additional source of sulfides is sulfate salts present in wastewaters from
metallurgical industries.

110

TOXICITY

CH3

CH 2

NH 2

COOH

Methionine

Figure 17.2

Sulfide in an anaerobic digester may be in the soluble or insoluble form. In the
insoluble form such as lead sulfide (PbS) and iron sulfide (Fe 2 S 3 ), sulfide does not
exert toxicity. Insoluble sulfide cannot enter bacterial cells. A common operational
practice to prevent sulfide toxicity in anaerobic digesters is to add iron. This prac-
tice precipitates the sulfide as iron sulfide, which gives the treated sludge a black
color. Dissolved sulfide can react with any heavy metal except chromium.

Although some of the sulfide leaves the digester sludge as free hydrogen sulfide
gas, and some is precipitated as heavy metal salts, a portion of the sulfide remains
dissolved. Concentrations of dissolved hydrogen sulfide above 200mg/l are toxic and
should be reduced.

Free hydrogen sulfide gas can be removed from digester sludge by the rapid
production of carbon dioxide, hydrogen, and methane. Treatment measures that can
be used to reduce soluble hydrogen sulfide include 1) diluting the sulfides, 2) se-
parating and treating the sulfate/sulfide waste stream, 3) precipitating the sulfide as
a metal salt, and 4) scrubbing and recirculating digester biogas.

Sulfide toxicity is most likely to occur under low organic loadings. Under these
conditions, insufficient biogas is produced. This deficiency in biogas production
results in poor stripping of sulfide from the sludge.

HEAVY METALS

Numerous heavy metals such as cobalt (Co), copper (Cu), iron (Fe), nickel (Ni), and
zinc (Zn) are found in wastewaters and sludges and are transferred to anaerobic
digesters. These metals are referred to as "heavy" because of their undesired impact
on wastewater treatment processes and operational costs including their accumula-
tion in sludges. High concentrations of metals in sludges affect sludge disposal
options and costs.

HEAVY METALS

111

COO"

coo-

Key functional group

Cell membrane
Fibril

Cell wall

Figure 17.3 Heavy metals cause toxicity in the soluble form. The metals are adsorbed to the surface
of the negatively charged, bacterial fibrils that extend into the bulk solution from the cell membrane
through the cell wall. The fibrils are negatively charged by the ionization (loss of hydrogen) from key
functional groups such as carboxyl (-COOH) and hydroxy! (-OH). Once adsorbed the metals are then
absorbed by the bacterial cells. Inside the cells the metals attack enzyme systems.

Although some heavy metals (cobalt, molybdenum, and nickel) at trace concen-
trations serve as additives or activators that enhance enzymatic activity of methane-
forming bacteria, heavy metals in moderate to excessive concentrations may cause
toxicity in anaerobic digesters.

Soluble heavy metals are removed from wastewaters and sludges through their
adsorption to the surface of bacterial cells (Figure 17.3). Once absorbed, heavy
metals exert toxicity by inactivating enzymatic systems. Inactivation occurs when
the metals bind to the thiol groups in enzymes. Inactivation of enzymes results in
digester failure. The concentration at which heavy metals exert toxicity is depend-
ent on the composition of the digester feed sludge.

Although heavy metals often are present in relatively high concentrations in
anaerobic digesters, these metals usually do not cause toxicity. Most heavy metals
are combined — not free — therefore, they cannot be adsorbed or absorbed by bac-
teria, and toxicity cannot occur.

Heavy metals can be combined through several mechanisms. Metal ions may be
bonded to a variety of naturally occurring chelating compounds that are found in
domestic and municipal wastewaters. Chelated metals cannot enter bacterial cells.
Many metals in anaerobic digesters are present in the form of insoluble salts or pre-
cipitates of oxides, hydroxides, sulfides, and carbonates. At pH values >7.5 signifi-
cant precipitation of the salts of carbonate and sulfides occurs. Precipitated metals
cannot enter bacterial cells. Metal salts in the form of chlorides and nitrates are
soluble and undergo ionization that releases soluble heavy metal ions.

1 12 TOXICITY

Heavy metal ions that are very toxic to methane-forming bacteria at relatively
low concentrations are copper, nickel, and zinc. These ions are soluble in anaerobic
digesters. Reacting the ions to precipitate as metal sulfides can reduce the toxicity
of these ions. Approximately 2 mg/1 of ions are precipitated as metal sulfides by
1 mg/1 of sulfide.

ALTERNATE ELECTRON ACCEPTORS

The presence of nitrate ions (NO3) or sulfate ions (SOf) may inhibit methane-
forming bacteria. Nitrate ions and sulfate ions may be found in relatively high con-
centrations in industrial wastewaters or aerated and nitrified municipal wastewaters
and sludges.

Both ions adversely impact the activity of methane-forming bacteria by increas-
ing the redox value within the anaerobic digester. Low redox values (less than
-300 mV) are required for proper activity of methane-forming bacteria.

Because SRB can out-compete methane-forming bacteria for substrates (acetate,
alcohols, formate, hydrogen, and carbon dioxide) that are used for methane produc-
tion, hydrogen sulfide production predominates over methane production. Here,
organic compounds are oxidized to carbon dioxide and sulfate is reduced to hydrogen
sulfide.

ALKALINE CATIONS

Four cations are associated with alkali compounds. These cations or metals are
calcium (Ca), magnesium (Mg), potassium (K), and sodium (Na).The salts of these
metals, for example, sodium hydroxide (NaOH), often are added to anaerobic
digesters to increase alkalinity and pH.The cations also may be transferred to anaer-
obic digesters from industrial wastes.

The cations have stimulatory and inhibitory effects on anaerobic digesters. At
relatively low concentrations (100-400 mg/1) the cations are desirable and enhance
anaerobic bacterial activity. At concentrations >1500 mg/1 the cations begin to exhibit
significant toxicity. Diluting the cation concentration can prevent cation toxicity.

BENZENE RING COMPOUNDS

Methane-forming bacteria are inhibited by a variety of benzene ring compounds
(Figure 17.4). These compounds include benzene, pentachlorophenol, phenol, phe-
nolic compounds, and toluene.

Phenolic compounds include chlorophenols,nitrophenols, and tannins. Tannins are
naturally occurring phenolic compounds found in fruits and vegetables, for example,
apples, bananas, beans, cereals, and coffee. Tannins may exert toxicity at 700 mg/1.

CHLORINATED HYDROCARBONS

Chlorinated hydrocarbons are toxic to methane-forming bacteria (Table 17.4). Chlo-
roform, for example, is toxic at a concentrations of 15 mg/1. However, methane-
forming bacteria can acclimated to many chlorinated hydrocarbons.

FEEDBACK INHIBITION 1 13

Benzene (CftH^)

PH3

Toluene (C6H5CH3)

vOH

Phenol (C6H5OH)
Figure 17.4

CYANIDE

Cyanide (-CN) and cyanide-containing compounds (cyano-compounds) are com-
monly found in industrial wastewaters from metal cleaning and electroplating
firms. In the metal finishing industry they are used in plating baths. Cyanide and
cyano-compounds are toxic to methane-forming bacteria. Toxicity occurs at cyanide
concentrations >100mg/l. Cyanide prevents methane production from acetate,
but it may not prevent methane production from carbon dioxide and methanol.
However, cyanide toxicity is reversible. The reversibility of toxicity is dependent
on the concentration of cyanide and its time in the digester as well as the con-
centration of solids (bacteria) in the digester, solids retention time (SRT), and
temperature.

FEEDBACK INHIBITION

Fermentation often results in the production of several intermediates such as hydro-
gen and volatile fatty acids that are toxic. The presence of toxicity that is caused by
the production of hydrogen and volatile fatty acids is referred to as feedback
inhibition.

Excess hydrogen production and accumulation results in increased partial hydro-
gen pressure. This increased pressure inhibits acetate-forming bacteria. Excess
volatile fatty acid production and accumulation inhibits methane-forming bacteria
through direct toxicity such as that caused by propionate or decreased alkalinity
and pH.

114

TOXICITY

CH4, C02

Sludge feed

Volatile acid
production

Methane
production

Figure 17.5

TABLE 17.5 Chlorinated Hydrocarbons that are Toxic
to Methane-forming Bacteria

Chloroform

Hexachlorocyclopentadiene
Hexachloroethane
Hexachloro-1 ,3-butadiene
2,4-Dichlorophenol

Feedback inhibition may be overcome by using a two-phase anaerobic digester
system (Figure 17.5). This system separates volatile acid production and methane
production. The system also provides improved stability and increased resistance to
toxic wastes. Long SRTs also allow the bacteria to increase in number and permit
the bacteria to acclimate to toxic wastes.

FORMALDEHYDE AND PHENOLIC WASTES

Formaldehyde (H 2 CO) is an example of an organic compound that is degradable at
low concentrations but toxic at high concentrations. Phenolic wastes are additional
examples (Table 17.5).

Formaldehyde is toxic to methane-forming bacteria. Toxicity occurs at concen-
trations >100mg/l. The inhibited activity of methane-forming bacteria recovers at
lower concentrations.

VOLATILE ACIDS AND LONG-CHAIN FATTY ACIDS

The presence of a relatively high concentration of short-chain (1-3 carbon units),
nonionized volatile acids such as acetate, butyrate, and propionate causes a decrease
in the concentration of alkalinity and a drop in pH. Propionate is perhaps the most
toxic of the volatile acids and may exert toxicity at concentrations <5 mg/1.

Toxicity is exerted at near-neutral pH values and occurs in populations of acid-
forming bacteria and methane-forming bacteria. The presence of an excess concen-
tration of volatile acids can be corrected with the addition of an alkaline compound.

RECALCITRANT COMPOUNDS

115

TABLE 17.6 Phenolic Wastes That Are Toxic to
Methane-forming Bacteria

Nitrobenzene
2-Nitrophenol
4-Nitrophenol

TABLE 17.7 Long-Chain Fatty Acids That Inhibit Methane Production from Acetate

Fatty Acid

Carbon
Units

Saturated/
Unsaturated

Formula

Caprylic (octanoic)
Capric (decanoic)
Laurie (dodecanoic)
Myristic (tetradecanoic)
Oleic (c/s-9-octadecanoic)

Saturated

CH 3 (CH 2 ) 6 COOH

Saturated

CH 3 (CH 2 ) 8 COOH

Saturated

CH 3 (CH 2 ) 10 COOH

Saturated

CH 3 (CH 2 ) 12 COOH

Unsaturated

CH 3 (CH 2 ) 7 CH=CH(CH 2 ) 7 COOH

Because the chemical composition and structure of several long-chain fatty acids
are similar to those of the lipid components in the cell wall of acetoclastic bacteria
and methane-forming bacteria, the fatty acids dissolve in the cell wall. Once
dissolved in the cell wall, the acids inhibit the activity of the bacteria at very low
concentrations.

Long-chain fatty acids of concern include capric, caprylic, lauric, myristic, and
oleic acids (Table 17.6). The acids contain carbon chains of 8 to 18 units. Although
lauric acid is the most toxic of the long-chain fatty acids, combinations of these acids
produce a synergetic effect. Wastewaters that contain significant quantities of
long-chain fatty acids include domestic, edible oil refinery, palm oil processing,
slaughterhouse, and wool scouring (coning oil). Long-chain fatty acids concentra-
tions >500g/l may cause toxicity in anaerobic digesters.

RECALCITRANT COMPOUNDS

Difficult to degrade or recalcitrant compounds in anaerobic digesters may cause
toxicity to methane-forming bacteria. Examples of these compounds include
aliphatic hydrocarbons and some chlorinated compounds such as lignin, humic sub-
stances, and chlorinated biphenyls. The recalcitrant compounds become even more
difficult to degrade when they contain alkyl groups, halogens, nitro groups, and sulfo
groups.

Mixing

Anaerobic digester content should be mixed. Mixing enhances the digestion process
by distributing bacteria, substrate, and nutrients throughout the digester as well as
equalizing temperature. The metabolic activities of acetate-forming bacteria and
methane-forming bacteria require that they be in close spatial contact. Slow, gentle
mixing ensures that contact. Also, mixing provides for efficient hydrolysis of wastes
and production of organic acids and alcohols by acid-forming bacteria. For example,
insoluble starches are kept from clumping by mixing action. This allows the
hydrolytic bacteria to attack a much larger surface area of the starches and provides
for their rapid hydrolysis.

Mixing minimizes the settling of grit and reduces the buildup of scum. Over
lengthy periods of operation, solids accumulation can reduce digester performance
as the reactor hydraulics become restricted by localized dead volumes and
short-circuiting of sludge flow. The advantages of mixing digester content are listed
in Table 18.1.

Mixing can be accomplished through mechanical methods or gas recirculation.
These methods include external pumps, gas injection or recirculation from the floor
or roof of the digester, propellers or turbines, and draft tubes. Mechanical mixers
are more effective than gas recirculation, but they often become clogged or fouled
with digester solids.

Mixing methods may be grouped into two modes. An intermediate mode incor-
porates heating with limited mixing achieved through the recycle of sludge in a
heat exchanger (Figure 18.1). A rapid mode or high rate (Figure 18.2) incorporates
heating and complete mixing and provides significant volatile solids destruction
(Figure 18.2).

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

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118

MIXING

TABLE 18.1 Advantages of Mixing Digester Content

Eliminating or reducing scum buildup

Eliminating thermal stratification or localized pockets of depressed temperature
Maintaining digester sludge chemical and physical uniformity throughout the tank
Rapid dispersion of metabolic wastes (products) produced during substrate digestion
Rapid dispersion of any toxic materials entering the tank (minimizing toxicity)
Prevent deposition of grit

Recirculated sludge

Heat exchanger

Figure 18.1

Heat

exchanger

Feed sludge

Digested sludge

Figure 18.2

MIXING 119

Sludge recirculation can be used for mixing digester content, but this method
generally is not used. When the method is used, sludge is recirculated through heat
exchangers and modest mixing is achieved. Sludge recirculation often is used when
no mixing equipment is available.

Mixing need not be continuous to achieve acceptable volatile solids destruction.
Continuous mixing is costly and requires a facility that will enhance the separation
of digested solids from the liquid phase. Routine mixing of digester content, for
example, three to six periods of mixing per day of 1- to 3-hour duration for each
mixing period, may be an efficient alternate to continuous mixing.

Methane-forming bacteria are very sensitive to rapid mixing. If rapid mixing
continuously washes out methane-forming bacteria in the effluent, then retention
periods of <7 days are not realistic.

Part TV

Process Control and

Troubleshooting

Upsets and
Unstable Digesters

Under steady-state conditions the anaerobic digester operates without difficulty.
Adequate mixing and proper, uniform temperature contribute to a steady-state
condition. However, interruptions of this condition do occur, resulting in upsets and
unstable digesters.

Seven basic conditions are responsible for upsets or unstable anaerobic digesters
(Table 19.1). Many of the conditions are directly or indirectly related and include
hydraulic overload, organic overload, pH changes, temperature fluctuations, toxic-
ity, large withdrawal of sludge, and sudden changes. Air contamination (presence of
oxygen) is possible.

There are several indicators of unstable anaerobic digesters (Table 19.2). These
indicators are either increases or decreases in specific operational values. The indi-
cators include decreases in biogas production and methane production, decreases
in alkalinity and pH, decrease in volatile solids destruction, and increases in volatile
acid concentration and percent carbon dioxide in the biogas.

With respect to indicators of unstable digesters, several comments are worth
noting. Biogas production is not as meaningful as methane production, because only
methane production represents final degradation of organic compounds. Although
a decrease in methane production is associated with an unstable digester, a decrease
in methane production also may be associated with a change (less substrate) in the
composition of the feed sludge.

Methane production and alkalinity may be correlated, and this correlation may
be used as an indicator of an unstable digester. A decrease in methane production
and a decrease in alkalinity indicate toxicity occurring in methane-forming bacte-
ria. A decrease in methane production and no significant change in alkalinity indi-
cate toxicity occurring in methane-forming bacteria and acid-forming bacteria. An

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

123

124

UPSETS AND UNSTABLE DIGESTERS

TABLE 19.1 Conditions Responsible for Upsets and Unstable Anaerobic Digesters

Condition

Example

Hydraulic overload

Organic overload

pH changes

Temperature fluctuations

Toxicity

Large withdrawal of sludge

Sudden changes

Overpumping of dilute feed sludge

Overpumping of concentrated feed sludge

Drop in pH (<6.8) and loss of alkalinity

Overpumping of feed sludge

Specific inorganic and organic wastes

Excess withdrawal of sludge and reduced retention time

Rapid increase in nitrate ion concentration

TABLE 19.2
Digesters

Indicators of Unstable Anaerobic

Indicator

Decrease

Increase

Biogas production

Methane production

Alkalinity

Volatile solids destruction

Volatile acid concentration

Percent C0 2 in biogas

increase in effluent volatile solids also will take place if toxicity to both groups of
bacteria occurs. However, this increase will take at least one hydraulic retention
time (HRT).

HYDRAULIC OVERLOAD

Hydraulic overload is defined as occurring when HRT is reduced to a value at which
the methane-forming bacteria cannot reproduce fast enough to avoid washout.
Hydraulic overload may be the result of the transfer of too large a quantity of
dilute sludge, sludge production exceeding digester capacity, or reduction in digester
volume. Grit accumulation and scum formation contribute to decreased digester
capacity. A washout of alkalinity accompanies a hydraulic overload.

The washout of alkalinity results in a loss of digester buffering capacity and the
buildup of organic acids. The buildup of organic acids is commonly referred to as a
"sour" digester. Neutralizing some of the acids with alkali or caustic compounds
may accelerate recovery of a sour digester.

Additional concerns related to a hydraulic overload include:

• Increased heating requirements

• Increased sludge dewatering and disposal costs

• Decreased methane production

• Decreased volatile solids destruction.

Dilute raw sludges are produced through several operational conditions. The
sludges may be the result of clarifier design, sludge removal equipment, and sludge

ORGANIC OVERLOAD 125

pumping schedules, especially in warm wastewater temperatures. Concerns that are
related to the production of malodors during warm wastewater temperatures often
dictate that dilute sludges be pumped to the digester before adequate thickening
can occur.

ORGANIC OVERLOAD

An organic overload is usually accompanied by a relatively high concentration
of nitrogenous wastes in municipal wastewater treatment plants. The release of
ammonia during the degradation of the nitrogenous wastes may result in ammonia
toxicity.

Foam and Scum

Production and

Accumulation

The production and accumulation of foam is a common problem experienced by
many anaerobic digesters. Foam production is caused by several operational condi-
tions (Table 20.1). Foam first appears in the annular space between the floating
cover and the digester wall and may completely coat the floating cover and spill
over the coping of the digester wall. Foam presents safety, housekeeping, and
malodor concerns as well as maintenance and operational problems.

Operational problems associated with foam production and accumulation
include reduced sludge feed pumping and inversion of digester solids profile, that
is, thick solids are located at the top of the digester and dilute solids are located at
the bottom of the digester. Maintenance problems associated with foam production
and accumulation include fouling of gas collection compressors and recirculating
pipes and gas binding of sludge recirculating pumps.

Foam occurs when gas bubbles become entrapped in a liquid matrix. Gases com-
monly associated with anaerobic digester foam include carbon dioxide, hydrogen
sulfide, methane, and nitrogen. Foaming occurs because the surface tension of the
liquid or sludge is reduced, resulting in the accumulation of solids over entrapped
gas bubbles. Solids within the foam usually are 2-5% by weight, and the specific
gravity of the foam is <1.0.

Adequate mixing must be ensured throughout the digester to reduce the amount
of entrapped gases and to homogenize digester contents. Inadequate mixing may
result in stratification of digester contents and insufficient stripping of gases pro-
duced during fermentation of wastes. Pockets of undigested and stratified wastes
near the surface of the digester generate volatile acids, resulting in the production
and accumulation of foam.

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi
ISBN 0-471-20693-8 Copyright © 2003 by John Wiley & Sons, Inc.

127

128 FOAM AND SCUM PRODUCTION AND ACCUMULATION

TABLE 20.1 Operational Conditions Associated with Foam Production

Condition Contributing Factor

Alkalinity increase Lysis of large numbers of strict aerobic bacteria including Nocardioforms

High percentage of activated sludge feed
Carbon dioxide increase Change in digester fermentation reactions
Fatty acid increase Excess grease

Excess triglycerides

Lysis of large numbers of strict aerobic bacteria including Nocardioforms
Mixing Insufficient stripping of gases

Excessive entrapment of gas from fine bubble mixing
Polymers Excess cationic polymers from dewatering units

Excess cationic polymers from thickening units
Solids, fine Excessive particulate surfactants

Solids, total Low level of total solids

Temperature fluctuations Intermittent feeding of sludge

Slug feeding of sludge
Scum Rapid breakdown of scum in mature digester

Feed sludge High organic content

Vigorous mixing or excessively high mixing rates and fine bubble gas mixing
enhance the entrapment of gas and the production of foam. Coarse bubble gas
mixing and mechanical mixing do not enhance the production of foam as much as
fine bubble gas mixing.

Foaming episodes usually occur in anaerobic digesters during start-up, system
imbalance, and overloading. Common operational conditions associated with
foaming include changes in loading or concentration of alkalinity and fatty acids,
cationic polymers, carbon dioxide, temperature, fine solids, and low total solids.
Foaming is usually worse shortly after sludge feeding or overloading of the digester.

START-UP

During start-up, the volatile acid-forming bacteria are numerous and very active
whereas the methane-forming bacteria are just becoming established. The differ-
ence in the relative abundance and activity of these two bacterial groups is a result
of the short generation time of the volatile acid-forming bacteria compared with
the long generation time of the methane-forming bacteria. Because of the large
quantity of volatile acids present during start-up and the resulting reduction in
surface tension of the sludge, carbon dioxide and methane released from the fer-
mentation of organic wastes become entrapped in the sludge. This results in foam
production. The foam is usually a light black froth that dissipates as the concentra-
tion of volatile acids decreases. Foam production in a mature digester is usually thick
and black.

ALKALINITY AND FATTY ACIDS

Alkalinity is inversely proportional to surface tension, that is, as alkalinity increases
within digester sludge the surface tension of the sludge decreases. The sludge
becomes more surface active and has a greater propensity to foam. Increasing

CARBON DIOXIDE 129

alkalinity also may serve as an indicator of other operational factors that contribute
to foam production. High alkalinity changes the surface tension of anaerobic
digester sludge in a similar fashion as biosurfactants from dead Nocardioforms
change the surface tension of activated sludge.

Alkalinity within an anaerobic digester increases because of significant changes
in specific operational conditions. Operational conditions that result in an increase
in alkalinity include increased alkalinity loading (ammonium ions, amino acids,
proteins, and cationic polymers), death of large numbers of strict aerobic bacteria
resulting in the release of large quantities of amines, and decreased alkalinity
destruction within the digester.

Because wasteactivated sludge contains alkalinity (ammonium ions, amino
acids, and proteins) and increases the alkalinity of the digester sludge, the alka-
linity of the activated sludge should be closely monitored and regulated to control
digester foam. This is especially true in warm wastewater temperatures, when
increased bacterial activity in the activated sludge results in the release of ammo-
nium ions from nitrogenous wastes. An increase in alkalinity also may serve as an
indicator of an adverse operational condition, for example, change in wastewater
composition.

Excess fatty acids within an anaerobic digester enhance foam production. Fatty
acids are surfactants and decrease the surface tension of the sludge. Again, the
reduced surface tension of the sludge results in foam production. The presence of
excess fatty acids is usually associated with grease or animal fat (triglycerides) and
the death of large numbers of bacteria. Phospholipids also released after the death
of bacteria serve as surface-active agents that favor foam production.

Excess grease transferred to an anaerobic digester presents two significant oper-
ational problems. First, the quantity of grease may increase the solids loading rate
to the digester and may adversely affect retention time. Second, the degradation
of grease may result in an increase in volatile fatty acids. The fatty acids would
negatively impact the buffering capacity, pH, and methane gas production of the
digester.

Grease may be removed upstream of the digester and treated aerobically with
appropriate bioaugmentation products. Bioaugmentation products also may have
some value in the control of scum blankets and accelerating the recovery of an
anaerobic digester that has experienced an upset condition.

CARBON DIOXIDE

Carbon dioxide (C0 2 ) is one of several gases found in foam. With increased carbon
dioxide production in an anaerobic digester, the amount of carbon dioxide within
the sludge also increases. An increase in carbon dioxide within the sludge promotes
foam production.

Carbon dioxide content within the digester can be reduced by bubbling digester
gas through a potassium hydroxide (KOH) solution or introducing natural gas into
the gas system to dilute the carbon dioxide content. A decrease in carbon dioxide
content results in an increase in digester pH and a more favorable volatile acid-
to-alkalinity ratio.

130 FOAM AND SCUM PRODUCTION AND ACCUMULATION

POLYMERS

Cationic polymers used upstream of an anaerobic digester for sludge thickening and
cationic polymers found in centrates and filtrates from sludge dewatering units have
been suspected in the production of foam. Cationic polyacrylamide polymers
contain numerous amino groups that are released as the polymers are degraded.
Once released, the amino groups form ammonium ions that increase sludge alka-
linity. The presence of additional ammonium ions and increased alkalinity within
the sludge change the surface tension of the sludge, resulting in foam production.

SOLIDS, FINE AND LOW TOTAL

The accumulation of fine solids in the digester often is associated with foam
production. Accumulation of fine solids may be due to the presence of particulate
surfactants found in centrates, filtrates, and supernatants. The presence of low
total solids in the digester reduces the surface tension of the sludge, resulting in the
production of foam.

STRUVITE

Struvite is a cottony-white substance that mimics foam and is sometimes pro-
duced in anaerobic digesters. This substance is magnesium ammonium phosphate
(MgNH 4 P0 4 ).

TEMPERATURE

Fluctuations in digester temperature significantly affect the activity of volatile acid-
forming bacteria and methane-forming bacteria and the concentration of products
formed by the bacteria. The production of foam may occur with temperature
fluctuations as small as 2°C. Slug feeding and intermittent feeding may cause
temperature fluctuations.

Foaming episodes as affected by temperature fluctuations occur more frequently
in thermophilic digesters than in mesophilic digesters. Because of a higher bacterial
activity and the die-off of large numbers of bacteria, thermophilic digesters have
higher concentrations of alkalinity and volatile acids.

SCUM

Digester scum consists of floating materials such as grease and vegetable matter
with a specific gravity <1.0. Plastics, hair, and rubber products are commonly found
in scum.

Scum may have entrapped bubbles of gas. If scum is broken or dissipated
by mixing over a very short period of time, the rapid breakdown of the grease and
vegetable matter results in a buildup of volatile acids. These acids at the surface
of the digester are responsible for the production of foam.

SCUM

131

TABLE 20.2

Control Measures for Foam and Scum Production

Measure

Description

Activated sludge
Digester foam/scum

Mixing

Primary clarifier scum

Solids loading

Temperature

Feed sludge to one digester at a relatively low feed rate, e.g., <0.05lb

VS/ft 3 /day
Manually remove foam and scum

Treat foam and scum with an appropriate defoaming agent
Break bubbles by passing foam through impeller
Produce homogenized sludge, i.e., prevent stratification of solids
Do not waste to digester, i.e., find alternate means of disposal
Treat with bioaugmentation products or enzymatic products to degrade

lipids
Avoid high or slug loadings
Avoid intermittent loadings
If possible, feed continuously
Maintain stable temperature
Avoid temperature fluctuations >2°C

Blankets of scum may form during a start-up or in a mature system. During start-
up a thin zone of high volatile acid content may become localized at the digester
surface. Under a mature system a thick, black layer of grease, vegetable matter, and
concentrated activated sludge may cover the entire surface.

Under a mature system the breakdown of scum at the surface of the digester
may cause foaming. When scum degrades, a pocket of high volatile acid content
develops. The acids produce a condition that is similar to start-up. Stratification of
non-digested solids near the surface causes foaming.

If foam and scum production in an anaerobic digester is a severe and frequent
problem, numerous control measures are available (Table 20.2). These measures
address activated sludge feed, foam and scum accumulation, adequate mixing,
primary clarifier scum, solids loading to the digester, and temperature control.

Often a combination of control measures rather than just one measure may be
needed to control foam and scum production and accumulation. It should be noted
that the most effective measures to control foam production might not be the most
effective measures to control scum production and vice versa.

Supernatant

When sludge is allowed to settle in a digester, a supernatant develops. Anaerobic
digester supernatant is commonly returned to the head of wastewater treatment
plants and mixed with the influent (Figure 21.1). Although the supernatant is
relatively small in volume, it contains dissolved and suspended organic and
inorganic materials. These materials add suspended solids, nutrients (nitrogen and
phosphorus), and organic compounds to the influent.

The returned materials may cause a variety of operational problems (Table 21.1).
Therefore, wastewater treatment plants that are not achieving significant liquid-
solid separation in anaerobic digesters should consider discontinuing the practice
of returning supernatant to the headworks of the plant.

Increased chlorine demand may occur because of the presence of excess ammo-
nium ions in the supernatant. Volatile fatty acids, volatile organic compounds,
volatile sulfur compounds, and hydrogen sulfide released from the supernatant
in the turbulent headworks of a wastewater treatment plant may contribute to
malodor problems.

Sludge bulking in the activated sludge process may occur through the weaken-
ing of floe particles by excess total dissolved solids (TDS) or the rapid and unde-
sired growth of filamentous organisms. The presence of sulfides in the supernatant
may trigger the growth of sulfide-loving filamentous organisms such as Beggiatoa
spp. and Thiothrix spp., whereas the presence of readily degradable organic com-
pounds may trigger the growth of foam-producing filamentous organisms such as
Microthrix parvicella and Nocardioforms.

133

134

SUPERNATANT

Influent

Primary
Clarifier

Aeration
Tank

Secondary
Clarifier

Effluent

Return activated Sludge

Primary sludge

Waste sludge

Supernatant

Anaerobic
Digester

Digested sludge

Figure 21.1

TABLE 21.1 Operational Problems Associated with the
Return of Digester Supernatant to the Head of a
Wastewater Treatment Plant

Increased chlorine demand
Malodor problems
Sludge bulking

Undesired impact of high concentrations of nitrogen and
phosphorus

Several operational problems are associated with the presence of high con-
centrations of nitrogen and phosphorus. These problems include possible permit
violations, nitrification, denitrification, and excess growth of algae in secondary
clarifiers.

Monitoring

Extensive analytical monitoring of anaerobic digesters has not been a common prac-
tice at many wastewater treatment plants. Lack of adequate and timely monitoring
has resulted in numerous digester failures. Lack of monitoring often is due to the
relatively large amount of time required to perform the many analytical tests.

To monitor the activity of the bacteria in an anaerobic digester and evaluate and
troubleshoot digester operations, several analytical tests should be performed on a
periodic basis. These tests include analyses of digester content (Table 22.1) and
digester feed sludge (Table 22.2). The frequencies of analyses of several common
analytical tests are presented in Table 22.3. During start-up and upsets analytical
tests should be performed more frequently. Because digester sludge contains a
relatively large quantity of inert solids, analysis of solids is not a meaningful
measure of the amount of biomass and biomass activity.

ALKALINITY

Adequate buffering capacity or alkalinity is needed in an anaerobic digester for
maintenance of proper pH. Alkalinity is produced in the digester through the degra-
dation of some wastes, for example, cationic polymers, amino acids, and proteins,
and alkalinity is lost in the digester through the production of volatile acids.

Acceptable alkalinity concentrations are normally 1000-2000 mg/1 in a primary
digester and 1500-3000 mg/1 in a secondary digester. If a deficiency in alkalinity
exists, the amount of alkalinity needed can be estimated on the basis of excess
volatile acids.

135

136

MONITORING

TABLE 22.1 Recommended Analytical Tests for Anaerobic

Digester Content

Test

V Desired Monitoring Frequency

Daily

Weekly

As Needed

Alkalinity

Ammonical-nitrogen

Chemical oxygen demand (COD)

Gas composition

Gas production

Grease

Organic-nitrogen

Orthophosphate-phosphorus

Settleable solids, supernatant

Temperature

Total solids

Toxicity

Volatile acids-to-alkalinity

Volatile solids

Volume level

TABLE 22.2 Recommended Analytical Tests for Anaerobic Digester Feed Sludge

Test

V Desired Monitoring Frequency

Daily

Weekly

As Needed

Alkalinity

Ammonical-nitrogen

Chemical oxygen demand (COD)

Grease

Organic-nitrogen

Total solids

Volatile acids

Volatile solids

Volume, gallons

pH 137

TABLE 22.3 Frequencies of Analyses of Several Anaerobic Digester Content Tests

Test Frequency Sample Type

Alkalinity 1 time/week Composite

Ammonical-nitrogen 1 time/week Composite

Chemical oxygen demand (COD) 1 time/day Composite

Micron utrients 1 time/month Composite

pH 1 time/day Composite

Orthophosphate-phosphorus 1 time/week Composite

Volatile acids 1-2 times/week Composite

Alkalinity may be added to a digester through chemical addition or changes in
operational measures. These measures include transferring secondary digester alka-
linity to the primary digester, increasing mixing and heating times, and decreasing
the amount of primary and secondary sludges that is wasted to the digester.

BIOGAS/METHANE PRODUCTION

Gas production, especially methane, increases with increasing organic loading to the
digester until methane-forming bacteria are no longer capable of degrading volatile
acids. The volume, rate, and composition of the biogas produced are indicative
of digester performance. An acceptable or normal range of biogas production is
10-25 ft 3 /lb volatile suspended solids (VSS) destroyed or 0.4-0.6 1/g of chemical
oxygen demand (COD) converted at 35°C. A decrease in volume of biogas, rate of
biogas production, or percent methane composition is an early indicator of digester
failure.

Treatability of wastes or substrate by anaerobic digesters is usually determined
by monitoring biogas production. The rate and volume of methane produced during
anaerobic digestion of a waste can be used to determine its relative rate of conver-
sion. The more rapid and the larger quantity of biogas produced, the more easily
the waste is treated in an aerobic digester.

When volatile acid production occurs more rapidly than volatile acid consump-
tion, that is, methane production, an upset condition occurs in an anaerobic digester.
The digester becomes acidic or "sour." Because methane-forming bacteria are very
sensitive to acidic conditions, methane production decreases as volatile acid con-
centration increases. Methane production usually terminates when the digester pH
drops below 6.0.

The pH of an anaerobic digester is mostly the result of the volatile acid-to-
alkalinity ratio, but the pH is usually the last indicator to change when a digester
is upset. Adjusting the volatile acid-to-alkalinity ratio or adding alkalinity impacts
digester pH.

An acceptable range of pH values for a primary digester is 6.6 to 7.0. An accept-
able range of pH values for a secondary digester is 6.8 to 7.2.

138 MONITORING

TEMPERATURE

Change in temperature has the most significant impact on the activity of anaerobes
and the efficiency of digester operation. Change in temperature also affects the
quality and quantity of products obtained through fermentation. These products
may or may not be readily available substrates for methane-forming bacteria. There-
fore, a change in temperature >2°C per day should not be permitted and the tem-
perature throughout the digester should be consistent. An acceptable range of
temperatures for mesophilic digesters is 30-35° C.

SETTLEABLE SOLIDS— SUPERNATANT

The characteristics of digester supernatant vary greatly according to the type of
sludge feed to the digester and the type of digester used. Solids in the supernatant
that are discharged to the head of the treatment plant represent particulate organic
loading and solids loading on the primary clarifier and secondary treatment process.
To maintain low loadings, the settleable solids in the supernatant should be <50ml
after 4-5 hours of testing. Low levels of loading may be obtained by ensuring proper
digester operation and maximum settling time.

TOTAL SOLIDS— SUPERNATANT

Total solids within the supernatant that are discharged to the head of the plant also
represent particulate organic and solids loadings. Total solids <5000mg/l are accept-
able. However, supernating should begin when solids are 2000 mg/1. Ensuring proper
digester operation and maximum settling time also may reduce total solids in the
supernatant.

TOTAL SOLIDS— SLUDGE FEED

Total solids should be 1.5-3.0% in primary sludge and 4.0-8.0% in secondary sludge.
Heavy solids or solids greater than 3.0% in primary sludge may be reduced by
decreasing retention time in primary clarifiers. Thin solids or solids <4.0% in
secondary sludge may be increased by extending wasting intervals or adding
primary sludge.

VOLATILE ACIDS

An increase in volatile acid concentration without an increase in alkalinity is an
indicator of an adverse operational condition within an anaerobic digester. Acetate
is a precursor for most of the methane produced in an anaerobic digester.

VOLATILE ACID-TO-ALKALINITY RATIO 139

Butyrate and propionate are important intermediates or precursors of methane
production. The accumulation of these acids or an increase in volatile acid concen-
tration can be associated with digester instability or stress.

Acceptable volatile acid concentrations are usually 50-200 mg/1 in a primary
digester and 50-500 mg/1 in a secondary digester. An increase in volatile acid
production or decrease in pH or alkalinity usually is caused by a change in
bacterial activity, that is, an increase in the activity of volatile acid-forming
bacteria or a decrease in activity of methane-forming bacteria. Optimizing
mixing, maintaining proper pH/alkalinity and temperature, and ensuring acceptable
sludge feed and withdrawal rates promote the activity of methane-forming
bacteria.

VOLATILE ACID-TO-ALKALINITY RATIO

The range of acceptable volatile acid-to-alkalinity ratios is 0.1-0.2. An acceptable
ratio may be obtained by adjusting volatile acid concentration, alkalinity concen-
tration, or both concentrations. Reducing or terminating feed sludge to the digester
also helps to lower the volatile acid-to-alkalinity ratio. If feed sludge cannot be
reduced or terminated, the use of chemicals for alkalinity adjustment is required.

An unacceptable volatile acid-to-alkalinity ratio is usually the first warning of an
adverse operational condition. Significant deviations from an acceptable ratio may
be caused by shock loading or excess sludge withdrawal

The volatile acid-to-alkalinity ratio is a key control parameter. Perhaps the best
method of maintaining a properly operating anaerobic digester is to ensure an
acceptable volatile acid-to-alkalinity ratio. A ratio of 0.07-0.08 is a good working
ratio, whereas a ratio >0.5 is indicative of digester upset and possible failure.

PartV

Digesters

Types of Anaerobic

Digesters

Anaerobic digesters are capable of treating insoluble wastes and soluble waste-
waters. Insoluble wastes such as particulate and colloidal organics are considered
to be high-strength wastes and require lengthy digestion periods for hydrolysis and
solubilization. Digester retention times of at least 10-20 days are typical for
high-strength wastes.

High-rate anaerobic digesters are used for the treatment of soluble wastewaters.
Because these wastewaters do not require hydrolysis and solubilization of wastes,
much faster rates of treatment are obtained. High-rate anaerobic digesters usually
have retention times of less than 8 hours.

High-strength wastes are usually treated in suspended growth systems, whereas
soluble wastewaters are usually treated in fixed-film systems. Several anaerobic
digester processes and configurations are available for the treatment of insoluble
wastes and soluble wastewaters (Table 23.1). Each configuration impacts solids
retention time (SRT) and hydraulic retention time (HRT). Minimal HRT is desired
to reduce digester volume and capital costs. Maximal SRT is desired to achieve
process stability and minimal sludge production.

BACTERIAL GROWTH— SUSPENDED

In suspended growth systems, the bacteria are suspended in the digester through
intermittent or continuous mixing action (Figure 23.1). The mixing action distrib-
utes the bacteria or biomass throughout the digester.

Because completely mixed anaerobic digesters do not incorporate a means
for retaining and concentrating the biomass, the SRT is the same as the HRT.

143

144

TYPES OF ANAEROBIC DIGESTERS

Feed
sludge

Digested
sludge

Figure 23. 1

TABLE 23.1 Types of Anaerobic Digesters

Characteristic

Application

Bacterial growth system
Temperature

Configuration

Suspended
Fixed film
Psychrophilic
Mesophilic
Thermophilic
Single-stage (phase)
Two-stage (phase)

TABLE 23.2 Advantages and Disadvantages of Suspended Growth Anaerobic Digesters

Advantages

Disadvantages

Suitable for the treatment of particulate, colloidal, and soluble wastes
Toxic wastes may be diluted

Uniform distribution of nutrients, pH, substrate, and temperature
Large digester volume required to provide necessary SRT
Treatment efficiency may be reduced due to loss of particulate and colloidal
wastes and bacteria in digester effluent

Completely mixed anaerobic digesters are designed for relatively long HRTs. Feed
sludge can be added to the digester on a continuous or intermittent basis. Advan-
tages and disadvantages of completely mixed suspended growth digesters are listed
in Table 23.2.

BACTERIAL GROWTH— FIXED FILM

Anaerobic fixed-film (sludge blankets) systems provide a quiescent environment for
the growth of an agglutinated mass of bacteria (Figure 23.2). Because bacterial
growth requires relatively long periods of time to develop, the media used in

BACTERIAL GROWTH— FIXED FILM

145

Influent

Figure 23.2

fixed-film systems hold the bacteria in the digester for relatively long periods and
provide for long SRTs and short HRTs.

The bacteria grow as fixed films of dendritic or "stringlike" masses on the sup-
portive media or as clumps of solids within the openings or voids of the supportive
media. Fixed-film systems usually use gravel, plastic, and rock as the supportive
media. The openings make up approximately 50% or more of the media.

Fixed-film systems operate as flow-through processes, that is, wastewater passes
over and through a bed of fixed film of bacteria growth and through entrapped
clumps of bacterial growth (Figure 23.3). Soluble organic compounds in the waste-
water are absorbed (diffuse into) by the bacteria, whereas insoluble organic com-
pounds are adsorbed (attach) to the surface of the bacteria. The flow of wastewater
through fixed-film systems may be from the bottom to the top (upflow) or from the
top to the bottom (downflow) (Figure 23.4).

Because the bacteria (solids) in fixed-film systems remain in the digester for long
SRTs, the systems allow methane-forming bacteria to acclimate to toxicants such as
ammonia, sulfide, and formaldehyde. Therefore, anaerobic fixed-film systems with
long SRTs and short HRTs may be used to treat industrial wastewater containing
toxicants.

Numerous fixed-film systems are available for use in the digestion of municipal
and industrial wastewaters and sludges (Table 23.3 and Figure 23.5). These systems
are capable of treating a variety of wastewaters and sludges, provide good contact

146

TYPES OF ANAEROBIC DIGESTERS

Biogas

Effluent

Influent

Figure 23.3

Batch

feed

Bioga

Effluent

}

' V

o o

▲

Two-stage, sludge bed filter
Figure 23.4

TABLE 23.3 Anaerobic Fixed-Film Systems

Baffled reactor

Expanded bed

Expanded microcarrier bed (MCB)

Fluidized-bed reactor

Fully packed upflow

Hybrid flow

Modular

Rotating biological contactor

Thin-film bioreactor

Upflow anaerobic sludge blanket (UASB)

TEMPERATURE— PSYCHROPHILIC

147

Biogas

Influent wastewater

Upflow anaerobic
sludge blanket

Upflow anaerobic

sludge blanket and

clarifier

Biogas

Influent wastewater

Recycle

Influent wastewater

Recycle

Downflow packed bed
anaerobic digester

Baffled anaerobic digester

Figure 23.5

between the wastes and the bacteria, and can treat wastewaters and sludges over a
relatively large range of temperature values (4-55°C) (Table 23.4).

TEMPERATURE— PSYCHROPHILIC

Psychrophilic sludge digestion and methane production occur at a relatively low
temperature range (5-20° C). Because of less than optimal activity of the anaerobic
bacteria in the psychrophilic temperature range, digestion of sludge is limited to
small-scale operations such as Imhoff tanks, septic tanks, and sludge lagoons. The

148 TYPES OF ANAEROBIC DIGESTERS

TABLE 23.4 Examples of Wastewaters and Sludges Treated by Anaerobic Fixed-Film
Digesters

Airport deicing fluids

Contaminated groundwater

Industrial wastewaters containing high concentrations of carbohydrates

Industrial wastewaters containing high concentrations of nitrogenous compounds

Low-strength wastewaters (<600mg/l COD) at relatively short HRTs (<6 hours)

temperature of the digester contents is approximately the temperature of its sur-
rounding environment and varies from season to season. Because temperatures in
psychrophilic digesters are relatively low, the SRTs of these digesters are greater
than 100 days.

TEMPERATURE— MESOPHILIC

Mesophilic sludge digestion and methane production occur at a moderate temper-
ature range (30-35° C). Mesophilic anaerobic digestion of sludge is commonly used
at municipal and industrial wastewater treatment process and offers two practical
advantages of operation compared with psychrophilic and thermophilic anaerobic
digestion. First, there are more anaerobic mesophiles in nature than there are psy-
chrophiles and thermophiles. Second, it is less expensive to maintain mesophilic
temperatures in digesters than it is to maintain thermophilic temperatures. Most
anaerobic digesters in North America operate in the mesophilic range.

TEMPERATURE— THERMOPHILIC

Thermophilic sludge digestion and methane production occur at a high tempera-
ture (50-60°C). Thermophilic anaerobic digestion of sludge is more often used at
industrial wastewater treatment plants, where process heat or steam is available to
heat digesters to the thermophilic range.

Because of the high operating temperature of these digesters, sludge digestion
and methane production occur rapidly and significant destruction of pathogens is
achieved. However, in addition to high operation costs, thermophilic digesters do
have some significant microbiological concerns with respect to their use in degrad-
ing sludges. The number of thermophilic methane-forming bacteria is very limited,
the bacterial growth is slow, and the bacterial population experiences a high endoge-
nous death rate. Also, the bacteria are very sensitive to fluctuations in digester
temperature.

CONFIGURATION— SINGLE STAGE

A typical single-stage digester consists of only one tank or reactor. Digester
operations consist of sludge addition and withdraw, mixing, heating, gas collecting,
and supernating. These operations are possible because of stratification of the

CONFIGURATION— SINGLE STAGE

149

Biogas Withdrawal

Gas

Inlet

: :S£um :

Supernatant

Active Digester Sludge

Outlet

Digested Sludge and Grit

Solids Withdrawal

Figure 23.6

Sludge feed

Biogas

Methane
production

Figure 23.7

digester content. Stratification results in the following layers from top to bottom of
the digester: gas, scum, supernatant, active digester sludge, and digested sludge and
grit (Figure 23.6).

Single-stage digesters are more easily upset than two-stage digesters. This is
because of the presence of the simultaneous activities of two groups of bacteria, the

150

TYPES OF ANAEROBIC DIGESTERS

Biogas

Sludge feed

First stage

Second stage

Sludge digestion and
methane production

Thickening and dewatering

Figure 23.8

Biogas

Sludge feed

Figure 23.9

acid-forming bacteria and the methane-forming bacteria. Because acid-forming bac-
teria grow more quickly than methane-forming bacteria and are more tolerant
of fluctuations in operational conditions, an imbalance between acid production rate
and methane production rate often occurs. This imbalance may cause a decrease in
alkalinity and pH that results in digester failure.

CONFIGURATION— TWO STAGE

Two-stage digester systems consist of at least two separate tanks or reactors. A
limited variety of two-stage systems are available. A two-stage system yields
improved efficiency and stability over a single-stage system. A two-stage system is

CONFIGURATION— TWO STAGE 151

capable of obtaining methane production and solids reduction similar to those of a
single-stage system at a smaller HRT. Also, toxicants are removed in the first stage.

In some two-stage systems acid production occurs in the first stage or tank and
methane production occurs in the second stage (Figure 23.7). In some two-stage
systems, sludge digestion and methane productions occur simultaneously and con-
tinuously in one tank and sludge thickening and storage occur in the other tank
(Figure 23.8). In this configuration the first stage is continuously mixed and heated
for sludge digestion, whereas stratification is permitted in the second stage, where
sludge thickening and storage occur.

Other two-stage systems consist of temperature-phased anaerobic digestion of
sludges or wastewaters. These systems are a combination of thermophilic and
mesophilic anaerobic digestion (Figure 23.9). These systems provide for improved
dewaterability of sludges and reduction in numbers of pathogens.

Anaerobic Digesters versus

Aerobic Digesters

Aerobic and anaerobic digesters can degrade organic compounds. The aerobic
process consists of a large variety of bacteria working side by side to degrade
the organic compounds, whereas the anaerobic process consists of a large variety of
bacteria working in sequence, that is, one after the other (Figure 24.1).

During aerobic degradation of organic compounds, aerobes and facultative
anaerobes use free molecular oxygen to completely degrade organic compounds
such as proteins to C0 2 , H 2 0, new bacterial cells (sludge), and inorganic compounds
such as NH4, HPO|~, and SO|~ (Equation 24.1). The aerobic degradation of organic
compounds is enhanced by the activity of higher life-forms — ciliated protozoa,
rotifers, and free-living nematodes (Figure 24.2).

organic compound (protein) + 2 — > C0 2 + H 2

+ cells + NH 4 + + HPOj- + SOj (24.1)

Nitrification occurs in the aerobic process. During nitrification strict aerobic, nitri-
fying bacteria, Nitrosomonas and Nitrobacter, oxidize NH4 to NO3 (Equations 24.2
and 24. 3). The sources of ions that are oxidized in an aerobic digester include ammo-
nium ions, amino acids, proteins, cationic polymers, and surfactants that were not
oxidized or degraded in an upstream treatment process, for example, activated
sludge or trickling filter.

NH^ + 1.50 2 — Nitrosomonas -> N0 2 + 2H + + H 2 + energy (24.2)
N0 2 + 0.5O 2 — Nitrobacter -> NO3 + energy (24.3)

753

154

ANAEROBIC DIGESTERS VERSUS AEROBIC DIGESTERS

Volatile Solids (Sludge)

Hydro lytic bacteria
(facultative anaerobes and anaerobes)

Soluble organic compounds
(amino groups, fatty acids, sugars)

Acid-producing bacteria
(facultative anaerobes and anaerobes)

Acids and alcohols

Acetate-forming bacteria

Acetate

Anaerobic

methane-forming

bacteria

Other
compounds

Anaerobic
methane- forming
bacteria

Figure 24. 1

During aerobic degradation of organic compounds, the carbon from the com-
pounds is degraded completely and is incorporated in the end products C0 2 and
new bacterial cells (sludge). This is complete oxidation of the organic compounds
or substrate.

Complete oxidation of organic compounds also can occur with nitrate ions (NO3)
instead of free molecular oxygen (Equation 24.4). If nitrate ions are used, molecu-
lar nitrogen is produced. During complete oxidation of organic compounds with
nitrate ions, the carbon from the compounds is degraded completely and is incor-
porated in the end products C0 2 and new bacterial cells (sludge).

NO3 + I.8CH3OH + H + -> 0.065C 5 H 7 O 2 N
+ 0.47N 2 + 0.76CO 2 + 2.44H 2

(24.4)

ANAEROBIC DIGESTERS VERSUS AEROBIC DIGESTERS

155

(b)

(d)

Figure 24.2 Higher life forms in aerobic digesters. The degradation of organic wastes in aerobic
digesters is enhanced through the activities of higher life forms such as the free-swimming ciliate
Paramecium (a), the crawling ciliate Euplotes, the stalked ciliates Epistylis (c), the rotifer (d), and the
free- living nematode (e).

If free molecular oxygen or nitrate ions are not available to the aerobic process,
nitrification stops and facultative anaerobes and aerotolerant anaerobes degrade
the organic compounds. The organic compounds such as proteins then are degraded
through fermentative reactions to C0 2 , H 2 0, new bacterial cells, inorganic com-
pounds including hydrogen, and a variety of smaller compounds such as organic
acids and alcohols (Equation 24.5). During anaerobic degradation of organic
compounds in an aerobic digester that has no free molecular oxygen or nitrate ions,
all of the carbon in the organic compounds is not degraded completely. Although
some of the carbon is incorporated in the end products C0 2 and new bacterial

156

ANAEROBIC DIGESTERS VERSUS AEROBIC DIGESTERS

cells (sludge), some of the carbon remains in the end products organic acids and
alcohols.

organic compound (protein) — fermentation — > C0 2 + H 2
+ cells + NH4 + HPOj" + H 2 S + organic acids and alcohols

(24.5)

These fermentative reactions result in incomplete oxidation of the organic com-
pounds because some, often much, of the carbon in the degraded organic com-
pounds is not incorporated in C0 2 and new bacterial cells. Some of the carbon is
incorporated in the fermentative products, organic acids and alcohols. These prod-
ucts still contain much energy. These products cannot be degraded further to
methane because the strict anaerobic methane-forming bacteria were destroyed in
the presence of free molecular oxygen in the aerobic digester.

The degradation of organic compounds in anaerobic digesters is not enhanced
by the activity of ciliated protozoa, rotifers, and free-living nematodes. Anaerobic
protozoa usually are not found in large numbers in anaerobic digesters, and rotifers
and free-living nematodes are strict aerobes that die in an anaerobic digester.

Nitrogenous wastes in an anaerobic digester consist of ammonium ions, amino
acids, proteins, cationic polymers, and surfactants that were not degraded upstream
of the digester. Another component of the nitrogenous wastes in anaerobic digesters
is the nitrogen-containing compounds released by dead bacteria and higher life-
forms. Strict aerobic bacteria including Nitrosomonas and Nitrobacter die in the
absence of free molecular oxygen. Because of the die-off of these two genera of
nitrifying bacteria, nitrogenous wastes cannot be nitrified in an anaerobic digester,
that is nitrite ions (NO?) and nitrate ions cannot be produced.

There are significant microbiological (Table 24.1) and operational differences
between the degradation of organic compounds by aerobic and anaerobic digesters.
Microbiological differences include the types of bacteria involved in the degrada-
tion process, the final electron carrier of degraded compounds, the quantity of new
bacterial cells or sludge produced, and the products obtained from the degradation
process.

TABLE 24.1 Significant Microbiological Differences Between Aerobic and Anaerobic
Digesters

Microbiological Feature

Aerobic Digester

Anaerobic Digester

Significant bacteria

Final electron carrier

Number of cells produced
Products from reactions

Higher life forms

Nitrification

Strict aerobic, including
nitrifying bacteria
Facultative anaerobic

Free molecular oxygen

Higher

C0 2 , H 2 0, cells, NHJ, NO3,

SOih HPO4
Numerous, ciliated protozoa,

metazoa
Yes

Facultative anaerobic, anaerobic,
including methane-forming

Organic compounds, hydrogen,
sulfur compounds, carbon
dioxide

Fewer

C0 2 , H 2 0, cells, NHJ, CH 4 , H 2 , H 2 S

Few, ciliated protozoa
No

ANAEROBIC DIGESTERS VERSUS AEROBIC DIGESTERS

157

TABLE 24.2 Advantages and Disadvantages of
Aerobic and Anaerobic Digesters

Feature

Digeste

Aerobic

Anaerobic

Alkalinity additional

If nitrifying

Yes

Degradation rate of organics

Rapid

Slow

Degradation of xenobiotics

Yes

Design and construction costs

Higher

Lower

Heating requirement

Yes

Malodor production

Yes

Methane production

Yes

Nutrient requirements

Higher

Lower

Operating costs

Higher

Lower

Oxygen requirement

Yes

Pathogen destruction

Less

Sensitivity to changes

Less

Sludge disposal costs

Higher

Lower

Sludge production

Higher

Lower

Solids retention time

Lower

Higher

Start-up time

Lower

Higher

Operational differences between aerobic and anaerobic digesters include the
types and strengths of sludge or wastewaters that can be treated, start-up time, sen-
sitivity to changes in operational conditions, operational costs, and nutrient require-
ments. The differences in operational conditions can be described as advantages and
disadvantages (Table 24.2)

The carbon in organic compounds is used for cellular growth and reproduction
by bacteria. The new cells produced from the carbon are referred to as solids or
sludge. The carbon within organic compounds is available for bacterial use in
primary and secondary sludges. When complete oxidation of organic compounds
occurs, more cells (sludge) are produced compared with incomplete oxidation of
organic compounds.

Because of the relatively high solids retention time (SRT) and fermentative path-
ways of anaerobic digesters, properly operated anaerobic digesters are capable of
achieving significant reduction in quantity of sludge and percent volatile content of
sludge. The high SRT permits the solubilization and degradation of particulate and
colloidal compounds. The fermentative pathways permit low cellular reproduction
(sludge yield) and the degradation of organic acids and alcohols to methane, hydro-
gen, and carbon dioxide. A significant advantage of anaerobic digesters is the low
growth rate of bacteria or synthesis of sludge. However, during start-up, toxicity, and
recovery from toxicity this low growth rate is a disadvantage.

As organic compounds are degraded in an anaerobic digester more bacterial cells
(sludge) are produced. However, because more of the carbon and energy in the
degraded compounds goes into waste products (organic acids and alcohols) during
anaerobic digestion compared with aerobic digestion, anaerobic digesters produce
less sludge than aerobic digesters. Much of the carbon and energy from organic
compounds that are degraded in anaerobic digesters can be found in methane.
Approximately 50% of the organic carbon from degraded compounds in aerobic
digesters can be found in new bacterial cells or sludge, whereas approximately 5%

158

ANAEROBIC DIGESTERS VERSUS AEROBIC DIGESTERS

'ell synthesis
2

Bacterial cells or

sludge

(0.5 pounds)

Organic

wastes

(1 pound)

Aerobic
Oxidation

Respiratory

wastes or

end products

(0.5 pounds)

ell synthesis
CH 2

Bacterial cells or
sludge
(0.05 pounds)

Organic

wastes

(1 pound)

Anaerobic
Respiration

Respiratory
wastes or
end products
(0.95 pounds)

Figure 24.3 Aerobic respiration (top) produces more bacterial cells or sludge from one pound of
organic waste than does anaerobic respiration from the same pound of organic waste.

of the organic carbon from degraded compounds in anaerobic digesters can be
found in new bacterial cells or sludge (Figure 24.3).

Besides the significant difference in sludge production between aerobic and
anaerobic digesters, there are other differences (advantages and disadvantages)
between aerobic and anaerobic digesters (Table 24.2). Of importance is the ability
of anaerobic digesters to destroy pathogens. Numerous pathogens are present in
wastewaters and sludges and consequently enter digesters. Because of the high tem-
perature and long detention time of anaerobic digesters compared with aerobic
digesters, significant reduction in the number of viable pathogens occurs.

Anaerobic digester sludge that has a significant reduction in pathogens as
well as malodors and reduced volatile content may be used as a soil conditioner or
additive. The anaerobically digested sludge contains nitrogen and phosphorus and
other nutrients that can be used to improve the fertility and texture of soils.

Another advantage of the anaerobic digester is the production of methane. This
gas is a usable source of energy. The energy within methane is in excess of that

ANAEROBIC DIGESTERS VERSUS AEROBIC DIGESTERS

159

Trichloroethylene

Irichloromethane

Figure 24.4

required to maintain digester temperature at most wastewater treatment plants.
Methane may be used to heat the digester, heat buildings, and generate electricity.

Anaerobic digesters are capable of efficient performance over a relatively wide
range of operating conditions and are capable of degrading xenobiotic compounds
and recalcitrant compounds. Examples of xenobiotic compounds include chlori-
nated aliphatic hydrocarbons such as trichloroethylene and trihalomethanes (Figure
24.4). An example of a natural recalcitrant compound is lignin.

The principal disadvantages of anaerobic digesters include the high capital costs,
the long SRTs, and the quality of the supernatant. High capital costs occur because
of the need for large, covered tanks, sludge feed and circulating pumps, heating
equipment, and gas mixing equipment. Long SRTs are required to grow a large and
active population of methane-producing bacteria. The quality of the digester super-
natant often is poor. The supernatant may contain relatively high concentrations
of suspended solids, soluble organic compounds, and nutrients (nitrogen and
phosphorus).

Despite these disadvantages, there is renewed interest in the use of anaerobic
digesters. As regulatory agencies require digesters to reduce the number of viable
pathogens significantly and produce more stable and less odorous sludges, a variety
of anaerobic digesters are being used to satisfy these requirements.

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Abbreviations and

Acronyms

ADP

Adenosine diphosphate

ATP

Adenosine triphosphate

BOD

Biochemical oxygen demand

BTU

British thermal unit

Celsius

COD

Chemical oxygen demand

Day

Fahrenheit

ft 3

Cubic feet

HRT

Hydraulic retention time

I/I

Inflow and infiltration

Kilogram

mg/1

Milligrams per liter

MLVSS

Mixed liquor volatile suspended solids

Millivolt

ORP

Oxidation-reduction potential

SRT

Solids retention time

sp.

(one) species

spp.

(two or more) species

SRB

Sulfate-reducing bacteria

TDS

Total dissolved solids

jim

Micron

YFA

Volatile fatty acids

VOC

Volatile organic compounds

Volatile solids

YSC

Volatile sulfur compounds

YSS

Volatile suspended solids

165

Chemical Compounds

and Elements

CaC0 3

-CH 3

CH 4

CH 3 CH 2 CH 2 COOH

CH 3 CH 2 OH

C 2 H 5 CHO

CH 3 CH 2 CH 2 OH

CH 3 (CH 2 ) 2 CH 2 OH

CH 3 CH 2 CH 2 NH 2

CH 2 =CHCH 2 SH

CH 3 CHNH 2 COOH

CH 3 CO

CH 3 CH 2 COOH

CH 3 (CH 2 ) 3 COOH

(CH 3 ) 2 CHCH 2 COOH

CH 3 C(5H4SH

C6H5CH 2 SH

CH 3 (CH 2 ) 4 COOH

CH 3 CHOHCOOH

CH 3 COCH 3

CH 3 COOH

C 5 H 6 N

C 9 H 9 N

C 8 H 13 N

Calcium

Calcium carbonate

Methyl group

Methane

Butyrate

Ethanol

Butyraldehyde

Propanol

Butanol

Propylamine

Allyl mercaptan

Alanine

Acetaldehyde

Propionate

Valeric acid

Isovaleric acid

Thiocresol

Benzyl mercaptan

Caproic acid

Lactate

Acetone

Acetate

Pyridine

Skatole

Indole

167

168

CHEMICAL COMPOUNDS AND ELEMENTS

CH3NCH3CH3

Trimethylamine

CH 2

Formaldehyde

CH3OH

Methanol

C5H40O5

Deoxyribose

C 6 H 12 6

Glucose

C 5 H 7 2 N

Bacterial cells

CH 3 NH 2

Methyl amine

C 3 H 5 NH 2

Ethylamine

CH3NHCH3

Dimethylamine

(CH 3 ) 2 S

Dimethyl sulfide

CH 3 CH

Methyl mercaptan

C 2 H 5 SH

Ethyl mercaptan

Cyanide

Cobalt

Carbon monoxide

co 2

Carbon dioxide

-c-o-c-

Acetal bond

-COOH

Carboxylic acid group

CS 2

Carbon disulfide

Copper

Iron

Fe 2 S 3

Iron sulfide

H +

Hydrogen proton

H 2

Hydrogen gas

HCOOH

Formate

HPO4 2 -

Orthophosphate

HS-

Sulfide

H 2 S

Hydrogen sulfide

HSCH 2 COOH

Thioglycolic acid

Potassium

KOH

Potassium hydroxide

Magnesium

N 2

Molecular nitrogen

Sodium

NaN0 3

Sodium nitrate

NaOH

Sodium hydroxide

-NH 2

Amino group

NH 3

Ammonia

NH 4 +

Ammonium ion

NH4HCO3

Ammonium carbonate

H 2 N(CH 2 ) 4 NH 2

Putrescine

H 2 N(CH 2 ) 5 NH 2

Cadaverine

Nickel

N 2

Nitrous oxide

N0 2 -

Nitrite ion

N0 3

Nitrate ion

o 2

Free molecular oxygen

OH-

Hydroxyl ion

CHEMICAL COMPOUNDS AND ELEMENTS 169

PbS Lead sulfide

P0 3 2 ~ Phosphate

S Sulfur

-SH Thiol group

SO4 2 - Sulfate ion

Zn Zinc

Glossary

absorb Penetration of a substance into the body of an organism

aceticlastic cleavage Conversion of acetate to methane by methane-forming
bacteria

acetogenesis Production of acetate by acetate-forming bacteria

acetotrophic Use of acetate by bacteria as a substrate

activator Metal or vitamin incorporated into an enzyme that improves the
efficiency of enzymatic activity

acute Having a sudden onset and short course

adsorb The taking up of one substance at the surface of an organism

aerotolerant Anaerobes that can survive in the presence of free molecular oxygen

aldehyde A compound containing the CO- radical attached to both a hydrogen
atom and a hydrocarbon radical, i.e., R-CHO

aliphatic Chainlike pattern of carbon units bonding together

amino acid A group of organic acids in which a hydrogen atom of the hydrocar-
bon (alkyl) radical is exchanged for the amino group; used in the production of
proteins

anaerobic An environment in which bacteria do not use free molecular oxygen

anoxic An environment in which bacteria use nitrite ions or nitrate ions

anthropogenic Produced under the influence of human activity

bioaugmentation The addition of commercially prepared cultures of bacteria to a
wastewater treatment process to improve operational conditions

biochemical A chemical reaction occurring inside a living cell

171

172 GLOSSARY

biomass The quantity or weight of all organisms within the treatment process

biorecalcitrant A compound that is degraded slowly by organisms

biosolids Thickened and dewatered sludge obtained from a digester

biosurfactant A compound released by an organism that reduces the surface
tension of wastewater or sludge and permits the production of foam

carbonaceous A compound that is organic or contains carbon and hydrogen

catabolic Destructive or degradative biochemical reactions

catalyst A substance that accelerates a chemical reaction

catechol A phenolic compound found in vegetable matter and coal tar

cellulose A polysaccharide consisting of numerous glucose molecules linked
together to form an insoluble starch

centrate The liquid and its content that are discharged from a centrifuge

chronic Having a long term or duration

Clostridia Anaerobes in the bacterial genus Clostridium

coenzyme An activator added to an enzyme

colloid Suspended solid with a large surface area that cannot be removed by
sedimentation alone

consortium Many organisms grouping together in beneficial relationship

denitrification The use of nitrite or nitrate ions by facultative anaerobes to
degrade substrate

desulfurication The use of sulfate ions by anaerobes to degrade substrate

disaccharide Two sugar units (mers) or monosaccharides joined together

electron A fundamental particle with negative charge; electrons are grouped
around the nuclei of atoms in several possible orbits

endoenzyme An enzyme used inside the cell to degrade substrate

endogenous The degradation of internal reserve substrate

enumerate To count

eubacteria True bacteria

exoenzyme An enzyme used outside the cell to hydrolyze substrate

facultative anaerobe Bacterium capable of using free molecule oxygen or other
carrier molecule to degrade substrate

fermentation A mode of energy-yielding metabolism that involves a sequence of
oxidation-reduction reactions to degrade organic substrates

filtrate The liquid and its content that pass through filter paper or a belt filter press

free-living Living or moving independently

generation time The time required for the cell population or biomass to double

halophile Freshwater organisms capable of surviving in salt water

humic substance Complex organic substances occurring in soil

hydrocarbon A general term for organic compounds that contain only carbon and
hydrogen

hydrolysis The biochemical process of decomposition involving the splitting of a
chemical bond and the addition of water

GLOSSARY 173

hydrogenotroph The use of hydrogen by bacteria as a substrate

hyperthermophile Organisms that grow at very high temperatures

intermediate A compound produced during a biochemical reaction that usually is
short lived; a compound that usually does not accumulate

lignin A mixture of substances produced by certain cells of plants

lipolytic An enzyme that attacks or degrades lipids

lysis To break open; namely, on the death of bacterial cells, the content of the cells
is released to the environment

macromolecule A very large molecule with much surface area

macronutrient A nutrient required in a relatively large quantity by all bacteria

monosaccharide One sugar mer or unit having three to seven carbon units

mer Unit

metabolism Pertaining to cellular activity, such as the degradation of substrate

methylotrophic The use of methyl groups by bacteria as a substrate

micro nutrient A nutrient required in a relatively small quantity by most bacteria

molecule Smallest part of a compound that exhibits all the chemical properties of
that specific compound

morphologic Structural features

niche The role performed by an organism in its environment

nitrification The oxidation of ammonium ions to nitrite ions or the oxidation of
nitrite ions to nitrate ions

Nocardioform A group of highly branched and specialized bacteria that produce
viscous chocolate brown foam in the activated sludge process

obligate Required

organic Compound containing carbon and hydrogen

organic-nitrogen Compound containing carbon, hydrogen, and nitrogen

organic-sulfur Compound containing carbon, hydrogen, and sulfur

oxic An environment in which bacteria use free molecular oxygen to degrade
substrate

oxidation The biological or chemical addition of oxygen to a compound or the
removal of electrons from a compound

pathogenic Disease-causing

phospholipid Lipid containing phosphorus

photosynthesis Biochemical reaction performed by green plants in which carbon
dioxide is fixed to form sugar

product Chemical compounds produced from the degradation of substrate

proteinaceous Containing proteins

proteolytic An enzyme that attacks or degrades proteins

psychrophile An organism that grows under cold temperatures (<20°C)

putrescibility Decomposition of plants and animals after death resulting in the
production of obnoxious substances

quinone A compound derived from benzene

174 GLOSSARY

reduction The biological or chemical removal of oxygen from a compound or the
addition of electrons to a compound

respiration The degradation of substrate; a mode of energy-yielding metabolism
that requires a final electron carrier for substrate oxidation

rumen The first division of the stomach in ruminants, being an expansion of the
lower end of the esophagus

saccharolytic An enzyme that attacks or degrades sugars

sarcina Small package

solubilization To place particulate or colloid materials in solution

substrate Compounds that are used by bacteria to obtain carbon and energy

supernatant The liquid above the settled solids

surfactant Soap or detergent; a compound that alters the surface tension of waste-
water or sludge

thermoacidophile Organisms that grow at a high temperature and low pH

volatile Changing readily to a vapor

xenobiotic A synthetic product that is not formed by natural biosynthetic
processes; a foreign substance or poison

Index

absorption of wastes; 4, 7, 49, 111, 145
acetate; 7, 14, 15, 16, 22, 24, 25, 26, 27, 32, 37,

39, 40, 41, 44, 45, 47, 48, 50, 51, 52, 55, 56,

57, 62, 70, 71, 76, 93, 96, 97, 101, 112, 113,

114, 115, 138, 154
acetate -forming bacteria; 15, 16, 27, 45, 50,

92, 113, 117, 154
acetogenesis; 6, 13, 52, 53, 56
acetotrophic methanogens; 26, 27, 53
activated sludge; 3, 6, 11, 31, 88, 93, 129, 131,

133, 153
ADP; 32, 34

adsorption of wastes; 4, 97, 111, 145
alkaline cations; 106, 112
alkalinity; 8, 32, 51, 57, 70, 72, 76, 80, 82, 83,

84, 99-103, 107, 108, 112, 113, 114, 123,

124, 128, 129, 130, 135-137, 138, 139, 150,

157
alternate electron acceptors; 106, 112
ammonia/ammonium ions; 13, 38, 57, 70, 82,

84, 88, 100, 102, 103, 106, 107-108, 109,

129, 130, 133, 153, 156
anoxic condition; 13, 14, 24, 35, 36, 76
ATP; 32, 34

benzene ring compounds; 106, 112, 113
bicarbonates; 25, 38, 39, 100, 102, 103

bioaugmentation; 129, 131

biofilm; 4, 74, 75

biogas; 3, 4, 6, 9, 73-76, 100, 102, 110, 123,

124, 137
biorecalcitrant; 23
biosolids; 7
BOD; 57, 94

carbohydrates; 12, 13, 40, 49, 51, 62, 63-66
carbonates; 25, 35, 38, 39, 57, 100, 102, 103,

111
catabolic processes; 3
cellulose; 7, 20, 23, 49, 52, 53, 54
chelating compounds; 111
chlorinated hydrocarbons; 106, 112, 114,

159
COD; 10, 36, 94, 95, 96, 109, 136, 137
coenzymes; 18, 21
colloidal wastes; 3, 5, 7, 14, 15, 51, 62, 70, 81,

85, 92, 143, 144, 157
cyanide; 106, 107, 108, 113

denitrification; 13, 36, 76, 134

electron transfer systems; 18, 21, 32, 33
endoenzymes; 14, 15, 55, 64, 70
exoenzymes; 14, 15, 55, 64, 66, 68, 70

175

176

INDEX

feedback inhibition; 106, 113
filamentous organisms; 7, 12, 17, 19, 133
fixed-film bacteria/digesters; 3, 4, 5, 9, 10, 11,

12, 87, 143, 144-147, 148
foam; 84, 127-131, 133
formaldehyde; 106, 114
free-molecular oxygen; 11, 12, 13, 17, 36, 37,

39, 43, 106, 123, 153, 154, 155, 156, 157

Gram staining; 24

heavy metals; 105, 106, 107, 110-112
high-rate anaerobic digesters; 143
HRT; 79, 80, 85, 87, 88, 99, 101, 124, 143,

144, 145, 151
hydraulic overload; 123, 124
hydrogen pressure; 15, 27, 41, 50, 113
hydrogen sulfide/sulfides; 12, 16, 38, 44, 47,

75, 98, 106, 107, 108-109, 111, 112, 127,

133
hydrogen-utilizing bacteria; 16, 41
hydrolysis; 5, 7, 13, 49, 51, 52, 53, 54, 55, 57,

62, 63, 64, 66, 68, 79, 81, 88, 92, 94, 117,

143
hydrogenotrophic methanogens; 26, 53

inhibition; 16, 51, 89, 99, 112, 115
intermediates; 62, 138

lactate -forming bacteria; 46

lipids; 12, 15, 18, 40, 49, 51, 62, 66-68, 115

long-chain fatty acids; 106, 114, 115

malodors; 7, 12, 125, 127, 133, 134, 157, 158
mesophiles; 29, 80, 89, 90, 91, 148, 150
methane; 3, 7, 12, 14, 16, 17, 22, 23, 24, 26,
36, 37, 39, 40, 47, 48, 49, 50, 51, 52, 53, 55,
57, 62, 73, 75, 79, 81, 85, 88, 89, 90, 91, 93,
96, 99, 101, 107, 108, 110, 114, 123, 124,
127, 128, 129, 137, 138, 148, 151, 157,
158
methane -forming bacteria; 7, 8, 11, 13, 15,
16, 17-29, 36, 39, 41, 44, 49, 50, 56, 57, 62,
71, 73, 74, 76, 79, 80, 81, 83, 84, 87, 88, 89,
90, 92, 93, 94, 96. 97, 98, 99, 100, 101, 102,
105, 107, 108, 109, 111, 112, 113, 114, 115,
117, 119, 123, 124, 128, 130, 137, 138, 145,
148, 150, 154, 159
methanogenesis; 7, 13, 51, 57, 62
methylotrophic methanogens; 27, 47, 48, 53
mixing; 117 -119, 123, 127, 128, 131, 137, 143
monitoring; 135-139

nematodes; 153, 155, 156
nitrification; 153
Nocardioforms; 128, 129, 133
nutrients; 3, 8, 93-98, 107, 117, 133, 144, 157,
159

organic overload; 123, 124, 125
oxic condition; 13, 14, 24, 35, 36
oxidation-reduction potential (ORP); 13,

14, 23, 24, 32, 33, 35, 36, 37, 79, 103, 112

particulate wastes; 3, 5, 14, 115, 49, 51, 54,

62, 79, 85, 92, 143, 144, 157
pathogens; 7, 9, 148, 151, 157, 158, 159
pH; 43, 44, 46, 51, 62, 72, 73, 79, 80, 82, 83,

84, 98, 99-103, 107, 108, 109, 112, 113,

114, 123, 124, 129, 135, 136, 137, 138, 144,

150
phenolic wastes; 112, 114, 115
polysaccharides; 15, 23, 48, 63, 64, 65, 66
propionate -forming bacteria; 47
proteins; 12, 13, 15, 21, 40, 49, 51, 57, 61, 62,

68-71, 75, 84, 99, 100, 107, 109, 129, 135,

153, 155, 156
protozoa; 153, 155, 156
putrescibility of sludge; 3

recalcitrant compounds; 7, 115, 159
respiration; 31-38, 43, 49, 74, 75, 158
rotifers; 153, 155, 156

scum; 4, 84, 103, 117, 118, 124, 127-131,

149
shock loading; 80, 87, 139
single-stage digester; 144, 148
sludge feed/loading; 85-86, 108
sludge production; 9, 27, 29, 35, 36, 50, 71,

79,93
sludge stabilization; 3, 8
solubilization; 5, 7, 14, 62, 64, 70, 143, 157
sour digester; 83, 124, 137
SRT; 9, 27, 79, 87, 91, 92, 113, 114, 143, 144,

145, 148, 157, 159
start-up; 8, 81-84, 128, 131, 135, 157
struvite; 130
sulfate; 12, 13, 14, 16, 24, 33, 34, 36, 38, 41,

47,74,109,112,156
sulfate-reducing bacteria; 15, 16, 37, 47, 48,

109
supernatant; 4, 57, 85, 86, 133-134; 138, 149,

159
surfactants; 22, 128, 129, 130, 153, 156

INDEX

177

temperature; 7, 9, 28, 29, 43, 44, 62, 74, 79,
80, 82, 83, 88, 89-92, 108, 113, 117, 118,
123, 124, 128, 130, 131, 136, 137, 138, 144,
147, 148, 151, 158

thermopiles; 29, 80, 89, 90, 91, 148, 150

three-stage process; 5, 51

toxicity; 8f, 9, 22, 46, 70, 80, 84, 87, 96, 98,
103, 105-115, 118, 123, 124, 125, 136, 144,
145, 151, 157

trickling filter; 3, 5, 11, 31, 88, 93, 153

two-phase anaerobic digesters; 114, 144,
149, 150-151

unstable digesters; 123-125
upsets; 51, 99, 123-125, 135

volatile acid-forming bacteria; 26, 50, 91,

128, 130, 138
volatile acid-to-alkalinity ratio; 84, 89, 99,

129, 136, 137, 139
volatile acids; 5, 7, 23, 57, 71-72, 79, 80, 81,

89, 91, 96, 101, 103, 106, 107, 108, 113, 114,

123, 124, 127, 128, 130, 131, 135, 137, 138,

139

xenobiotic compounds; 7, 157, 159

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