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22 Anaerobic Metabolism
and its Regulation

MICHAEL J. MCINERNEY
Norman, OK, USA

1 Introduction 456
2 Trophic Structure of Anaerobic Ecosystems 456
3 Methanogens 458
3.1 Physiology of Methanogens 458
3.2 Effect of Sulfate on Methanogenesis 461
4 Regulation of Fermentative Metabolism 461
5 Regulation of Syntrophic Metabolism 463
6 Environmental Factors 467
6.1 Temperature 467
6.2 Effect of pH and Volatile Acid Toxicity 468
7 Types of Reactors 469
7.1 Conventional Stirred Anaerobic Reactor 469
7.2 Phase Separated Systems 470
7.3 Fixed Film or Anaerobic Filter Reactors 470
7.4 Fluidized/Expanded Bed Reactors 470
7.5 Anaerobic Rotating Biological Contactor 470
7.6 Anaerobic Baffle Reactor 471
7.7 Upflow Anaerobic Sludge Blanket Reactor (UASB) 471
8 Concluding Remarks 471
9 References 472

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22 Anaerobic Metabolism and its Regulation

1 Introduction
Methanogenesis is an important terminal
electron accepting process in many anaerobic
environments where the supply of oxygen,
nitrate, oxidized forms of sulfur, iron, and
manganese are limited (FERRY, 1993). Examples of such environments include freshwater
and some marine sediments, flooded soils, wet
wood of trees, tundra, landfills, and sewage digestors. In these environments, a complex microbial community consisting of many interacting microbial species completely degrades
natural polymers such as polysaccharides, proteins, nucleic acids, and lipids to methane and
carbon dioxide. Because of the large amounts
of organic matter that are degraded in the natural environments, methanogenesis is an important process in cycling of carbon and other
elements in nature and may be responsible for
up to 60% of atmospheric methane (CONRAD,
1996). The amount of energy released during
methanogenesis is relatively low compared to
other terminal electron accepting processes.
For example, the conversion of hexoses to
methane and carbon dioxide releases only
15% of the energy that would be released by
the aerobic mineralization of hexose (SCHINK,
1997). Thus, the amount of biomass produced
per unit of substrate degraded is much less
than that of other terminal electron accepting
processes. For this reason, methanogenesis has
been used as the treatment of choice for
sewage and other complex wastes since sludge
yields are low and most of the energy in the
original substrates is retained in the energyrich fuel, methane. Anaerobic digestion is often a net energy producer, resulting in significantly lower operating costs compared to
aerobic treatment (LETTINGA, 1995).
Although the low cell yields associated with
anaerobic digestion make it attractive for
wastewater treatment, it is also one of its main
disadvantages because large reactor volumes
and long retention times are needed to achieve
the required treatment efficiency (MCCARTY,
1971). Thus, only relatively high strength
wastes such as sewage are treated by anaerobic digestion. In addition, anaerobic digestion is
often perceived as being an unstable process
(ANDERSON et al., 1982; MCCARTY, 1971;

SPEECE, 1983). However, great advances have

been made in the past 20 years in our understanding of the biochemistry and energetics of
anaerobic metabolism. This has allowed the
delineation of the most sensitive steps in the
process and the development of strategies to
enhance the operational stability of anaerobic
reactors. The result has been the development
of novel reactors where the slow growing organisms are retained in the reactor even at
high volume loadings.With these advances, it is
now believed that almost any type of waste can
be treated (LETTINGA, 1995).
This chapter will provide a brief overview of
the microbiology of anaerobic digestion and
the factors that regulate this process. The constraints that thermodynamics places on key
reactions will be discussed to illustrate its role
in the control of the flow of carbon and energy
during anaerobic digestion. In addition, the
properties of microorganisms involved will be
discussed to understand how population dynamics influences the process. There is a number of excellent reviews and books where
these issues are treated in greater detail
(DOLFING, 1988; FERRY, 1993; HOBSON et al.,
1974; LETTINGA, 1995; LOWE et al., 1993; MALINA and POHLAND, 1992; MCCARTY and SMITH,
1986; MCINERNEY and BRYANT, 1981; SCHINK,
1988, 1997; SPEECE, 1983).

2 Trophic Structure of
Anaerobic Ecosystems
In anaerobic digestion, organic matter is
completely degraded to methane and carbon
dioxide in discrete steps by the concerted action of several different metabolic groups of
microorganisms (Fig. 1) (MCINERNEY and
BRYANT, 1981). In the first step, fermentative
bacteria hydrolyze the polymeric substrates
such as polysaccharides, proteins, and lipids
and ferment the hydrolysis products to acetate
and longer-chain fatty acids, CO2, formate, H2,
P
NHc
4 and HS . Acetogeneic bacteria O-demethylate pectins and low molecular weight
ligneous materials and ferment hydroxylated

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2 Trophic Structure of Anaerobic Ecosystems

457

Fig. 1. Schematic representation of

the different metabolic steps involved
in the complete degradation of organic matter to methane and carbon
dioxide (modified from MCINERNEY
and BRYANT, 1981).

and methoxylated aromatic compounds with

the production of acetate (DOLFING, 1988). In
the next step, a group of organisms, usually
called syntrophic or proton reducing acetogenic bacteria, degrades propionate and
longer-chain fatty acids, alcohols, and some
amino acids and aromatic compounds to the
methanogenic substrates, H2, formate, and acetate. The degradation of these compounds with
H2 production is thermodynamically unfavorable unless the concentration of H2 (or formate) is kept low by H2 using bacteria such as
methanogens (MCINERNEY and BRYANT, 1981).
Because of the diverse number of organisms
involved in these reactions and their ability to
perform other types of metabolisms such as
fermentation or sulfate reduction (SCHINK,
1997; MCINERNEY, 1992), the organisms that
participate at this second step will be called
syntrophic metabolizers. The final step involves two different groups of methanogens,
the hydrogenotrophic methanogens which use
the H2 and formate produced by other bacteria to reduce CO2 to CH4 and the aceto-

trophic methanogens which metabolize acetate to CO2 and CH4.

In the gastrointestinal tract of animals, organic matter is only partially converted to CO2
and CH4 (WOLIN, 1982). Acetate and longerchain fatty acids accumulate and are absorbed
and used by the animal as energy sources. Only
fermentative bacteria and H2 or formate using
methanogens seem to be involved in this partial methane fermentation. In termite guts
(BREZNAK, 1994), the cecum of rodents and
the large intestine of most humans (MILLER
and WOLIN, 1994), the reduction of carbon
dioxide to acetate by acetogenic bacteria
seems to be the dominant electron accepting
reaction. In these latter ecosystems, the community structure appears to be similar to that
of the rumen, except that H2 using acetogenic
bacteria replace the H2 using methanogens as
the terminal electron accepting group.
In ecosystems with high levels of sulfate,
such as marine and estuarine sediments and
petroleum reservoirs, sulfate reduction rather
than methanogenesis is the terminal electron

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22 Anaerobic Metabolism and its Regulation

accepting reaction. Organic matter is completely oxidized to CO2 with the reduction of
sulfate to sulfide. Again, the degradation process involves the concerted efforts of several
metabolic groups of bacteria with the sulfate
reducing bacteria apparently performing the
functions of the syntrophic metabolizers and
the hydrogenotrophic and acetotrophic methanogens (MCINERNEY, 1986; OUDE ELFERINK
et al., 1994; STAMS, 1994) since the degradation
of propionate and longer-chain fatty acids to
carbon dioxide in marine sediments does not
require interspecies H2 transfer (BANAT and
NEDWELL, 1983). However, it is likely that the
use of H2 by sulfate reducers influences product formation of fermentative bacteria in a
manner analogous to that found in methanogenic environments.
This brief survey of the trophic structure of
anaerobic ecosystems reveals that, in general,
anaerobic metabolism proceeds in a stepwise
manner where several metabolic groups of
bacteria interact in the mineralization process.
This is in contrast to metabolism under aerobic
and denitrifying conditions where a single species is usually able to mineralize completely a
compound when the electron acceptor (e.g.,
oxygen or nitrate) is in excess. The degree of
mutual interdependence between the different
trophic groups in anaerobic communities varies considerably depending on the genetic capabilities of the organisms and the constraints
that kinetics and thermodynamics place on
key reactions. For some interactions, energy
limitations are such that neither partner can
operate without the activity of the other organism. Perturbations may result in the accumulation of intermediates such as H2 and acetate that exceed the narrow limits that are
needed for the degradation of key intermediates such as fatty acids to be thermodynamically favorable. Other perturbations may stimulate fermentative metabolism or enhance the
growth of certain fermentative populations,
resulting in the production of acetate or other
organic acids at rates faster than the other
trophic groups can degrade these acids. Thus,
the efficiency of anaerobic digestion will depend on the dynamics and kinetics of key
populations within the reactor as well as on
thermodynamic limitations. The challenge of
anaerobic digestion is to maintain the appro-

priate balance between the different groups of

bacteria so that rates of production of key
intermediates match consumption rates and
the pool sizes are and within the narrow limits
that thermodynamics places on these reactions.

3 Methanogens
3.1 Physiology of Methanogens
Methanogens are a taxonomically and phylogenetically diverse group of microorganisms
(BOONE et al., 1993; JONES et al., 1987; ZINDER,
1993) that all gain energy for growth from the
reactions that lead to the production of methane.As a group, methanogens use a small number of compounds, H2 or one-carbon atom
compounds (BOONE et al., 1993; ZINDER,
1993). This specialization makes methanogens
dependent on other organisms for the supply
of substrates in most anaerobic environments.
Without methanogens, effective degradation
of organic matter would cease due to the accumulation of nongaseous products of fermentation which have almost the same energy content as the original substrate.
The ability of methanogens to use H2 plays a
key regulatory role that controls the types of
products made by fermentative bacteria and
sets the thermodynamic conditions required
for the degradation of fatty and aromatic acids.
The favorable thermodynamics of H2 use by
methanogens (Tab. 1) allows them to metabolize H2 to very low partial pressures. Methanogens are able to metabolize H2 down to H2
partial pressures ranging from 37 Pa (LOVLEY,
1985; CORD-RUWISCH et al., 1988). Methanogens also have a high affinity for H2 use, with
apparent KM values in the range of 513 mM
(6701,700 Pa) (Tab. 2) (ROBINSON and TIEDJE, 1984; WARD and WINFREY, 1985; ZINDER,
1993). With H2 partial pressures ranging from
21,200 Pa in digestors (CORD-RUWISCH et al.,
1997; GUWY et al., 1997; MOSEY and FERNANDEZ, 1989; STRONG and CORD-RUWISCH, 1995),
this means that the methanogens are undersaturated with respect to H2 use. Thus, in-

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3 Methanogens

459

Tab. 1. Reactions Involved in Syntrophic Metabolisma

DG0b
[kJ per reaction]

Reaction
Methanogenic reactions
c
4 H2 c HCOP
3 cH
P
Acetate c H2O

]
]

CH4 c 3 H2O
CH4 c HCOP
3

P135.6
P 31.0

Syntrophic reactions without H2 use by methanogens

c
] AcetateP c HCOP
c 2 H2
LactateP c 2 H2O
3 cH
] AcetateP c Hc c 2 H2
Ethanol c H2O
] 2 AcetateP c Hc c 2 H2
ButyrateP c 2 H2O
c
] AcetateP c HCOP
c 3 H2
PropionateP c 3 H2O
3 cH
] 3 AcetateP c HCOP
c
3
Hc c 3 H2
BenzoateP c 7 H2O
3
c
AcetateP c 4 H2O
] 2 HCOP
c 4 H2
3 cH

P 4.2
c 9.6
c 48.3
c 76.1
c 70.6b
c104.6

Syntrophic reactions with H2 use by methanogens

c
] 2 AcetateP c HCOP
2 LactateP c H2O
3 c CH4 c H
P
c
]
2
Acetate
c
H
O
c
H
c
CH
2 Ethanol c HCOP
3
2
4
] 4 AcetateP c Hc c CH4
2 ButyrateP c HCOP
3 c H2O
P
P
c
P
] 4 Acetate c HCO 3 c H c 3 CH4
4 Propionate c 3 H2O
c
] 12 AcetateP c HCOP
c 3 CH4
4 BenzoateP c 19 H2O
3 c9H

P143.6
P116.4
P 39.4
P102.4
P124.4

a
b

Calculated from the data in THAUER et al. (1977).

Calculated using the free energy of formation for benzoate given in KAISER and HANSELMANN (1982).

Tab. 2. Selected Kinetic Data for the Use of Hydrogen, Formate and Acetate by Methanogenic Bacteria
Substrate

Organisms

Apparent Km Reference

Threshold

Reference

Hydrogen most methanogens

313 mM

KRISTIJANSSON et al.
(1982), ROBINSON
and TIEDJE (1984)

312 Pa

CORD-RUWISCH et al.
(1988), LOVLEY (1985),
LOVLEY et al. (1984)

Formate

many methanogens

5580 mM

LOVLEY et al. (1984),

SCHAUER et al. (1982)

1526 mM

SCHAUER et al. (1982)

Acetate

Methanosarcina sp.

3.04.5 mM

Methanosaeta sp

SCHNHEIT et al.
(1982), WESTERMANN
et al. (1989)
F0.11.2 mM HUSER et al. (1982),
MIN and ZINDER
(1989), OHTSUBO
et al. (1992), AHRING
and WESTERMANN
(1987a)

creases in H2 production may not result in

higher H2 partial pressures because the H2
consumption rate by methanogens would also
increase (KASPAR and WUHRMANN, 1978 a;
ROBINSON and TIEDJE, 1982; SHEA et al., 1968;

0.61.2 mM WESTERMANN et al.

(1989), FUKUZAKI et al.
(1990a)
575 mM
WESTERMANN et al.
(1989), MIN and
ZINDER (1989),
AHRING and WESTERMANN (1987a),
JETTEN et al. (1990)

STRAYER and TIEDJE, 1978). Because of the

large capacity for H2 use by methanogens, H2
concentrations are normally very low in welloperated anaerobic digestors, even though
large amounts of H2 are produced. As will be

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22 Anaerobic Metabolism and its Regulation

discussed in Sects. 4 and 5, the ability of methanogens to maintain low levels of H2 affects
the types of products formed by fermentative
bacteria and is essential for the degradation of
fatty and aromatic acids by syntrophic associations.
Formate is a common fermentation product,
especially by bacteria that use pyruvateformate lyase in their metabolism, and may be an
essential intermediate for syntrophic metabolism (THIELE and ZEIKUS, 1988; DONG et al.,
1994). Many methanogens are able to use
formate and it serves as a source of electrons
for methane formation equivalent to H2. The
affinity for formate use varies for different
methanogens (Tab. 2). The apparent KM values
for formate use was 526 mM for two rumen
methanogens, 580 mM for Methanobacterium
formicicum, and 0.22 mM for Methanospirillum hungatei (LOVLEY et al., 1984; SCHAUER et
al., 1982). SCHAUER et al. (1982) reported that
the lowest concentration of formate metabolized was 26 mM for M. formicicum and 15 mM
for M. hungatei.
Acetate is a major product of fermentative
metabolism and is quantitatively the most important substrate for methane production.
About 6090% of the methane produced in
anaerobic digestors are derived from the
methyl group of acetate (BOONE, 1982;
MACKIE and BRYANT, 1981; MOUNTFORT and
ASHER, 1978; SMITH and MAH, 1966). At thermophilic temperatures or at high ammonium
levels, the oxidation of the methyl group of
acetate to H2 may be the predominant route
for acetate metabolism (see Sect. 5). Acetate
using methanogens include members of the
genera Methanosarcina and Methanosaeta
(BOONE et al., 1993). Methanosarcina sp. have
faster growth rates, higher apparent KM values
for acetate use, and higher threshold acetate
values than Methanosaeta sp. (Tab. 2) (ZINDER,
1993). The differences in the apparent KM values for acetate use have been attributed to
differences in respective enzymes used to activate acetate (JETTEN et al., 1990). Since Methanosarcina sp. and Methanosaeta sp. have
different threshold values for acetate use but
use the same reaction for acetate metabolism
(Tab. 1), the threshold values cannot represent
a thermodynamic limitation. Acetate threshold values may result when a critical or in-

hibitory concentration of undissociated acetic

acid is reached which, for Methanosarcinca sp.,
is between 4 and 7 mM (FUKUZAKI et al.,
1990a).
Consistent with the known growth characteristics of the acetotrophic methanogens, a
drop in acetate concentrations below 1 mM
was correlated with a displacement of Methanosarcina sp. by Methanosaeta sp. in a thermophilic digestor (ZINDER et al., 1984). Generally, high-rate anaerobic sludge blanket reactors usually have granules composed of Methanosaeta sp. rather than Methanosarcina sp.
(GROTENHUIS et al., 1991; WU et al., 1993).
Interestingly, RASKIN et al. (1996) found that
Methanosaeta sp. and Methanosarcina sp. were
present at approximately equal levels in glucose degrading anaerobic biofilm reactors
even though acetate levels were below the
threshold values for Methanosarcina sp. The
maintenance of Methanosarcina in these biofilms could not be attributed to the utilization
of other substrates such as methanol, suggesting that there may be Methanosarcina sp. that
have a lower threshold value than has been reported in the literature. STRAYER and TIEDJE
(1978) found that acetate was converted to
methane at or near the maximal rate in
eutrophic lake sediments since the addition of
acetate to these sediments did not result in a
corresponding increase in the rate of acetate
utilization or methane production. This suggests that there is little additional capacity to
metabolize acetate if the acetate production
rates increase. A similar conclusion was
reached by HICKEY and SWITZENBAUM (1991)
for anaerobic sewage digestors. This is probably the reason why acetate concentrations in
digestors increase when organic or volume
loading rates increase.
Methanol, methylamine and trimethylamine
also serve as substrates for Methanosarcina
sp. and other methylotrophic methanogens
(BOONE et al., 1993).These compounds may be
important substrates for methanogenesis in
marine systems (OREMLAND and POLCIN,
1982). Some hydrogenotrophic methanogens
can oxidize secondary alcohols to ketones and
primary alcohols to carboxylic acids (WIDDEL,
1986). Additional methanogenic substrates are
discussed by ZINDER (1993) and BOONE et al.
(1993).