FEMS Microbiology Ecology 28 (1999) 193^202
MiniReview
Contribution of hydrogen to methane production and control of
hydrogen concentrations in methanogenic soils and sediments
R. Conrad *
Max-Planck-Institut fuër terrestrische Mikrobiologie, Karl-von-Frisch-Str., D-35043 Marburg, Germany
Received 28 May 1998; received in revised form 29 July 1998; accepted 27 August 1998
Abstract
Hydrogen is, with acetate, one of the most important intermediates in the methanogenic degradation of organic matter and
serves as substrate for methanogenic archaea. Hydrogen should theoretically account for 33% of total methanogenesis when
carbohydrates or similar forms of organic matter are degraded. Many methanogenic environments show both much lower and
much higher contributions of H2 to CH4 production than is considered normal. While the lower contributions are relatively
easily explained (e.g. by the contribution of homoacetogenesis), the mechanisms behind higher contributions are mostly
unclear. In methanogenic environments H2 is rapidly turned over, its concentration being the result of simultaneous production
by fermenting plus syntrophic bacteria and consumption by methanogenic archaea. The steady-state concentration observed in
most methanogenic environments is close to the thermodynamic equilibrium of H2 -dependent methanogenesis. The threshold
is usually equivalent to a Gibbs free energy of 323 kJ mol31 CH4 that is necessary to couple CH4 production to the generation
of 1/3 ATP. Methanogenesis from H2 is inhibited if the H2 concentration decreases below this threshold. Concentrations of H2
can only be decreased below this threshold if a H2 -consuming reaction with a lower H2 threshold (e.g. sulfate reduction) takes
over at a rate that is equal to or higher than that of methanogenesis. The instantaneous and complete inhibition of H2 dependent CH4 production that is often observed upon addition of sulfate can only be explained if a comparably high sulfate
reduction potential is cryptically present in the methanogenic environment. z 1999 Federation of European Microbiological
Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : H2 ; CH4 ; Acetate; Fermentation ; Syntrophy; Methanogenesis; Homoacetogenesis; Threshold; Gibbs free energy; Km
1. Introduction
Methanogenic archaea utilize only a limited number of substrates, the most important ones being
acetate and H2 /CO2 (or formate) [1]. Most methanogenic archaea are able to utilize H2 /CO2 and such
methanogens can be found in every methanogenic
* Tel.: +49 (6421) 178 801; Fax: +49 (6421) 178 809;
E-mail: [email protected]
environment. Indeed, H2 is a ubiquitous compound
in anaerobic environments where it exhibits a fast
turnover but usually occurs at only very low concentration [2^4]. Low H2 concentrations are a thermodynamic prerequisite for the degradation of alcohols
and fatty acids by H2 -producing syntrophic bacteria
[5]. In methanogenic environments where inorganic
electron acceptors other than CO2 are not available,
consumption of H2 is only possible by methanogenic
archaea and homoacetogenic bacteria. There, degra-
0168-6496 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 8 6 - 5
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194
R. Conrad / FEMS Microbiology Ecology 28 (1999) 193^202
Fig. 1. Pathway of anaerobic degradation of organic matter to methane.
dation of alcohols and fatty acids is usually accomplished by syntrophy between H2 -producing syntrophic bacteria and H2 -consuming methanogenic archaea [5].
In this MiniReview I will address the following
two questions. (1) What is the percentage contribution of H2 to the production of CH4 ? (2) How is the
H2 concentration and methanogenesis controlled by
competition? I do not address the possibility that
formate may replace H2 in many of the processes
[6] which, however, should have no consequences
for the principal conclusions.
2. Contribution of H2 to methanogenesis
Hydrogen is a product of the anaerobic degradation of organic matter by fermenting and syntrophic
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R. Conrad / FEMS Microbiology Ecology 28 (1999) 193^202
bacteria. The most abundant source of dead organic
matter in natural environments is usually plant material consisting of lignin and polysaccharides. Some
aquatic sediments receive a large input of dead crustaceans consisting of chitin. Lignin is largely recalcitrant under anaerobic conditions [7], but methanol
may be released from the methoxy groups and thus
may support methanogenesis to a limited extent. In
general, however, we may assume that the anaerobic
degradation process is largely driven by carbohydrates as the dominant substrate. This assumption
is valid for aquatic sediments, peat, other wetlands,
ruminants, arthropods feeding on plant material,
and for many types of sewage sludge.
The anaerobic degradation pathway of dead organic matter is in principle well known [1]. Di¡erent
groups of microorganisms participate in the degradation which basically proceeds in three steps. (1)
Fermenting bacteria excrete enzymes that hydrolyze
organic polymers (e.g. polysaccharides) and catabolize the resulting monomers to alcohols, fatty acids
and H2 . (2) Syntrophic bacteria further degrade the
alcohols and fatty acids to acetate, H2 (alternatively
formate) and CO2 . (3) Acetate and H2 (alternatively
formate) plus CO2 ¢nally serve as substrates for
methanogens. Alternatively, many of the monomers
(e.g. sugars) can be catabolized by homoacetogenic
195
bacteria to acetate which then serves as substrate for
acetotrophic methanogens converting it to CH4 and
CO2 (Fig. 1).
Using the degradation of glucose as an example,
most of the standard Gibbs free energy content is
utilized during the ¢rst stage, i.e. the fermentation
to alcohols and fatty acids (Figs. 1 and 2; Table
1). The next stage, i.e. the syntrophic degradation
of alcohols and fatty acids to acetate and H2 , is
usually endergonic under standard conditions (Table
1) and is only possible when combined with H2 -consuming methanogenesis. Less than half of the Gibbs
free energy content of glucose is available for the
syntrophic degradation of the alcohols and fatty
acids to CH4 and CO2 (Fig. 2; Table 2) and this
energy has to be shared among the syntrophs and
the methanogens. Only if the fermentation step is
homoacetogenesis (reaction 1.5), the residual free energy (about a quarter of the total) is exclusively
available for acetotrophic methanogenesis (Fig. 2).
In fact, there is no thermodynamic reason why homoacetogenic degradation of carbohydrates coupled
to acetotrophic methanogenesis should not be a major pathway in anoxic environments. At the moment,
however, the role of homoacetogenesis in methanogenic environments is unclear.
Hydrogen can be produced in the ¢rst fermenta-
Table 1
Standard Gibbs free energies (vG³P) of de¢ned stages in the degradation of glucose to CH4 (calculated after [38] using CO2 in gaseous
state)
#
1.1
1.2
1.3
1.4
1.5
2.1
2.2
2.3
2.4
1-2
3
1-3
4
1-4
vG³P (kJ mol31 substrate)
Reaction
Fermentation
C6 H12 O6 C2 CH3 CHOHCOOH
C6 H12 O6 C2 CH3 CH2 OH+2 CO2
C6 H12 O6 C2/3 CH3 CH2 CH2 COOH+2/3 CH3 COOH+2 CO2 +8/3 H2
C6 H12 O6 C4/3 CH3 CH2 COOH+2/3 CH3 COOH+2/3 CO2 +2/3 H2 O
C6 H12 O6 C3 CH3 COOH
Syntrophy
CH3 CHOHCOOH+H2 OCCH3 COOH+CO2 +2 H2
CH3 CH2 OHCCH3 COOH+2 H2
CH3 CH2 CH2 COOH+2 H2 OC2 CH3 COOH+2 H2
CH3 CH2 COOH+2 H2 OCCH3 COOH+CO2 +3 H2
C6 H12 O6 +2 H2 OC2 CH3 COOH+2 CO2 +4 H2
Hydrogenotrophic methanogenesis
4 H2 +CO2 C2 H2 O+CH4
C6 H12 O6 C2 CH3 COOH+CO2 +CH4
Acetotrophic methanogenesis
CH3 COOHCCO2 +CH4
C6 H12 O6 C3 CO2 +3 CH4
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3198.1
3235.0
3248.0
3311.4
3311.2
348.7
+9.6
+48.3
+31.8
3216.1
332.7
3346.8
335.6
3418.1
196
R. Conrad / FEMS Microbiology Ecology 28 (1999) 193^202
Fig. 2. Residual standard Gibbs free energy (vG³P) after each reaction stage in the methanogenic degradation of glucose utilizing
di¡erent glucose fermentation reactions (equation numbers from
Table 1 in parentheses).
tive degradation stage (e.g. reaction 1.3), and it is
obligatorily formed in the second syntrophic stage
of organic matter degradation. The syntrophic stage
is sensitive to inhibition by H2 for thermodynamic
reasons. The maximum amount of H2 relative to
acetate that can be produced from the degradation
of carbohydrates is 4 mol H2 plus 2 mol acetate per
mol glucose (reaction sum 1-2), i.e. a ratio of H2 /
acetate of 2:1. Any contribution of homoacetogenesis (reaction 1.5) decreases this ratio. Degradation of
chitin (monomer = N-acetylglucosamine) results in
one more acetate (ratio of H2 /acetate of 4:3) than
in the case of the degradation of glucose and thus
decreases the possible contribution of H2 .
Since 4 H2 , but only 1 acetate, are required to
produce 1 CH4 , the contribution of H2 to methanogenesis during anaerobic degradation of carbohydrates can maximally be 33% of the total CH4
formed. Indeed, this percentage is consistent with
data obtained from many studies of methanogenic
environments (Table 2). However, lower contributions are also found in some methanogenic environments. Usually, they are easily explained. In marine
sediments the low contribution of H2 can be due to
the dominance of sulfate reduction for degradation
of organic matter, while methanogenesis depends on
non-competitive precursors such as trimethylamine
[8,9]. In acidic lake sediments the low contribution
of H2 may be explained by a larger contribution of
homoacetogenesis [10]. In Lake Constance sediment,
CH4 production occurs exclusively from acetate.
This observation is explained by sulfate reducers
which consume H2 in the upper sediment layers. Because of the lack of acetotrophic sulfate reducers
[11], acetate is not consumed, and thus it di¡uses
into deeper layers where it is consumed by methanogens [12]. In methanogenic rice ¢eld soil, the contribution of H2 decreases when the temperature is
shifted to lower values (30 to 15³C), so that CH4 is
then mainly produced from acetate [3,13]. Most
probably, homoacetogenesis becomes the main fermentation reaction under this condition.
There are many studies in the literature which report much higher contributions of H2 than the expected 33%. Conceivable explanations for these exceptions include (i) additional sinks of acetate, (ii)
additional sources of H2 , or (iii) measurements under
non-steady-state conditions. Additional sinks of acetate are not uncommon, e.g. in the rumen, acetate is
largely absorbed into the blood stream of the host,
leaving H2 as the predominant source for methanogenesis [14]. Similar observations were made in microbial mats where acetate is assimilated by the phototrophs [15]. Transient phenomena must occur
when H2 and acetate are sequentially produced or
utilized. For example, the low amounts of CH4 produced immediately after £ooding of paddy soil are
mainly due to H2 -dependent methanogenesis, since
Table 2
Examples of the contribution of H2 to CH4 production in di¡erent methanogenic sediments
Environment
Kichier Lake
Lake Mendota
Lake Washington
Anoxic paddy soil
Colne Pt. Salt marsh
Knaack Lake
Lake Constance
Kuznechika lake
Octopus Spring mat
Blelham Tarn
Cape Lookout Bight
Kings Lake Bog
Bunger Hills, Antarctica
Lake Baikal, deep sediment
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Contribution (%)
Contribution normal
32^46
36^46
15^39
17^31
Contribution low
8
4
0
Contribution high
97
74^86
76^82
71^80
100
95^97
99^100
Ref.
[39]
[40]
[41]
[42]
[9]
[10]
[12]
[39]
[15]
[43]
[44]
[45]
[46]
[47]
R. Conrad / FEMS Microbiology Ecology 28 (1999) 193^202
197
tron balances for CH4 and its precursors acetate and
H2 do not exist and thus it is unclear, for instance,
whether the preferential production of CH4 from H2 /
CO2 is balanced by an equivalent accumulation of
acetate or by non-methanogenic consumption of acetate. Clearly, more research is required to explain the
high contribution of H2 to CH4 production in these
anoxic environments.
3. Control of the environmental H2 concentration
Fig. 3. E¡ect of sulfate addition on the H2 partial pressure, the
Gibbs free energy (vG) of H2 -dependent methanogenesis and the
accumulation of CH4 in slurries of anoxic Italian rice ¢eld soil
(adapted from [24]).
the H2 -dependent methanogens apparently become
active before the acetotrophic ones [16]. Eventually,
however, steady state is reached and H2 then contributes about 30% to CH4 production as theoretically expected (Table 2).
In most cases, however, where H2 /CO2 -dependent
methanogenesis dominates (up to 100%) CH4 production in sediments of lakes, marine bights and
peat bogs (Table 2), an explanation for elevated contributions of H2 to methanogenesis is more di¤cult
to ¢nd. Additional sources of H2 are as yet undescribed except where there is a geological input of
H2 , such as in Lake Kivu [17]. In most of the deep
sediments and peat bogs, detailed carbon and elec-
Hydrogen is an intermediate in the methanogenic
degradation of organic matter and is rapidly turned
over (turnover times of minutes [3,4]). Any change of
H2 concentration (C) is caused by a change of either
its rate of production (p) or utilization (u):
dC=dt ˆ p3u
…1†
Steady state is reached if p = u. If p s u, H2 concentration will increase, thus also resulting in increased
H2 utilization. Assuming Michaelis-Menten kinetics
(with umax and Km as parameters) the new and higher
steady-state H2 concentration will then be given by:
C ˆ pKm =…umax 3p†
…2†
However, this higher H2 steady state will not persist for long, since the H2 utilizers will eventually
start to increase their biomass X, e.g. according to
the Monod equation:
dX=dt ˆ X…CWmax †=…Ks ‡ C†
…3†
Table 3
Gibbs free energies of H2 -dependent methanogenesis under steady-state conditions in various environments and at the threshold of H2
consumption in methanogens
Methanogenic system
3vG (kJ mol31 CH4 )
Reference
Sewage sludge
Lake Mendota; Knaack Lake
Wetwood
Canal with detritus and leaves
Alder swamp
Littoral sediment, Lake Constance
Profundal sediment, Lake Constance
Upland soils turned methanogenic
Italian rice ¢eld soil
Methanobacterium bryantii
Other methanogenic archaea
28^32
27^35
42
8^18
12^19
33^39
23^34
25^50
24^38
29^37
29^50
[2,48]
[2]
[2]
[30]
[30]
[49]
[12]
[50]
[13]
[27,28]
[27,28]
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R. Conrad / FEMS Microbiology Ecology 28 (1999) 193^202
(with Wmax = maximum growth rate; Ks = H2 concentration at Wmax /2). Since
umax ˆ Xvmax
…4†
(with vmax = speci¢c maximum H2 utilization rate),
this adaptation will return the H2 concentration to
the original value that existed before the increase of
H2 production. In other words, the H2 steady-state
concentration is basically under the control of the H2
utilizers and their kinetic characteristics [18].
The parameters Wmax , vmax , Ks and Km are speci¢c
for a given microorganism. Thus, it has been proposed that the parameters of competing H2 utilizers
should determine which organism ¢nally wins
the competition. Indeed, it was shown that sulfate
reducers utilize H2 faster than methanogens
because of their lower Km [19]. Similarly, it was
shown that sulfate reducers have a lower Ks (H2
concentration at half-maximum growth rate W)
than methanogens and thus are able to outgrow
the latter [19].
Indeed, it has repeatedly been demonstrated that
H2 -dependent CH4 production is inhibited in the
presence of sulfate [19,20]. This inhibition has usually been explained by the more e¤cient H2 utilization kinetics in sulfate reducers than in methanogens.
However, this model provides no explanation of why
the resident methanogens should not continue H2
utilization, albeit at a reduced rate. Complete inhibition can only be achieved after the methanogenic
population has been outgrown by the sulfate reducers [19,20]. Thus, methanogenic populations may be
replaced by sulfate reducers, iron reducers or nitrate
reducers in systems that have been exposed to sulfate, Fe(III) or nitrate for a long time, e.g. aquatic
sediments or aquifers. These environments are
largely in steady state with respect to concentrations
of sulfate, Fe(III) and nitrate and consequently exhibit H2 concentrations that are characteristic for
methanogenesis, sulfate reduction, iron reduction,
etc. [21]. However, the kinetic model does not provide an explanation for the instantaneous and complete inhibition of H2 -dependent CH4 production
that has been observed in some methanogenic environments upon addition of sulfate [22^24]. An alternative model, one which incorporates a threshold
concept, on the other hand, does provide such an
explanation [21,25^27].
The threshold concept of anaerobic H2 utilization
assumes that there is a certain H2 concentration below which utilization is no longer possible because of
thermodynamic constraints. Theoretically, the H2
threshold should be given by the conditions at which
reactants and products are in thermodynamic equilibrium (vG = 0). Thus, the H2 threshold should be
de¢ned by the equilibrium constant (K):
K ˆ exp…3vG o =RT†
…5†
For example, the H2 threshold partial pressure (pH2 )
of H2 -dependent methanogenesis is given by the
equilibrium constant and the partial pressures of
CO2 and CH4 :
pH2 ˆ ‰pCH4 =…pCO2 K†Š1=4
…6†
Indeed, it has been found that H2 thresholds for
various anaerobic H2 -utilizing reactions and bacteria
decrease with decreasing vG³ (increasing K) of the
H2 -utilizing reaction [21,26,28]. In reality, however,
the H2 thresholds were found to be slightly higher
than those indicated by the equilibrium constant
[27,28]. Obviously, H2 utilization stops at a value
which still allows for a small negative Gibbs free
energy, the critical Gibbs free energy (vGc ). This
critical value is probably explained by the coupling
to the energy-generating system of the cell which has
a threshold of about 1/3 ATP or approximately 323
kJ mol31 of the energy-generating reaction [5]. Interestingly, the values of vGc increase (less negative) in
the order sulfate reducers s methanogens s homoacetogens, indicating that sulfate reducers need
more free energy than homoacetogens to allow H2
utilization [28].
Reaction kinetics close to the thermodynamic
equilibrium become increasingly reversible. Therefore, they are not well described by Michaelis-Menten kinetics which are based on irreversible reactions.
Hoh and Cord-Ruwisch [29] recently modi¢ed the
Michaelis-Menten model. Their equilibrium model
takes into account the relative di¡erence of the actual H2 concentration to that at the thermodynamic
equilibrium by amending the Michaelis-Menten
equation with the term y/K:
u ˆ umax C…13y=K†=‰Km ‡ C…1 ‡ y=K†Š
…7†
with y = 2 (actual concentration of products)/2 (ac-
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R. Conrad / FEMS Microbiology Ecology 28 (1999) 193^202
tual concentration of reactants), and K = 2 (concentration of products at equilibrium)/2 (concentration
of reactants at equilibrium).
Thus, y is equivalent to the equilibrium constant,
but uses the actual concentrations instead of the concentrations at thermodynamic equilibrium. The authors were able to show that their model ¢tted experimental data well for both H2 -producing
reactions (e.g. propionate degradation by syntrophs)
and H2 -utilizing reactions (e.g. homoacetogenesis
and methanogenesis) [29]. An important result of
this modeling approach is that the H2 conversion
rates at environmentally relevant H2 concentrations
are much more sensitive to the thermodynamic conditions in the environment (i.e. y/K) than to the kinetic parameters of the microorganisms (i.e., vmax
and Km ). This response is because the H2 concentrations are much closer to the thermodynamic equilibrium than to the microbial Km values. The model
of Hoh and Cord-Ruwisch [29] may be further improved by using y/Kc instead of y/K, where Kc is the
equilibrium constant based on vGc rather than vG³
to account for the fact that H2 utilization (also H2
production) stops short of the thermodynamic equilibrium.
In contrast to the Michaelis-Menten model, the
threshold concept easily explains why a H2 -utilizing
process is rapidly and completely outcompeted when
another process with a lower threshold becomes
possible. As soon as the H2 concentration decreases
below the threshold for a process, activity stops.
Measurements in methanogenic environments indicate that in situ H2 concentrations correspond to
vG values of approximately 323 kJ mol31 CH4 ,
i.e. equivalent to the energetic threshold of 1/3
ATP, or less (Table 3). Only one study found vG
values that were much higher than 320 kJ mol31
CH4 [30]. In many cases, H2 -dependent methanogenesis obviously operates at its thermodynamic threshold. If we assume that the steady-state concentration
of H2 in methanogenic environments is identical to
the H2 threshold of the resident methanogenic £ora,
then we can consider what would happen if a second
H2 utilization process becomes active, e.g. H2 -dependent sulfate reduction after addition of sulfate.
Let the rates of methanogenic and sulfate-reducing
H2 utilization be um and us . Then, the steady-state
199
conditions (dC/dt = 0) would change from the methanogenic H2 utilization:
p ˆ um
…8†
to the simultaneous utilization by methanogenesis
and sulfate reduction:
p 6 um ‡ us
…9†
and the steady-state H2 concentration would consequently decrease below the threshold of the methanogens, so that CH4 production would stop. Now,
the H2 production would have to be balanced by the
sulfate reducers (us alone). Such a balance is only
possible if the instantaneous potential of H2 -dependent sulfate reduction is equal to or higher than that
of H2 -dependent methanogenesis (us v um ). If this is
not the case, then p s us , and consequently, H2 concentrations will increase again until H2 -dependent
methanogenesis resumes and balances H2 production. Then the same cycle would repeat itself. Macroscopically, this chain of events should result in a
partial but instantaneous inhibition of methanogenesis without any concomitant decrease of the H2
concentration. Only much later, the population of
the sulfate reducers would have eventually grown
up. Increasing X of sulfate reducers would result in
increasing us (Eqs. 3 and 4) until us = p, then also
resulting in decreasing H2 concentrations until a
new steady state characteristic of sulfate reducers
would be attained.
One example which may ¢t this pattern is that
of sediment of Lake Mendota where 2 days of incubation were required for a decrease of the H2 concentration although the partial inhibition of H2 dependent methanogenesis was immediate [31].
Methanogenic rice ¢eld soil, on the other hand, on
sulfate addition shows an instantaneous and complete inhibition of H2 -dependent methanogenesis
with concomitant decrease of the H2 concentration
to values that are thermodynamically no longer permissive for methanogens (Fig. 3). Similar results
have also been obtained with Lake Wintergreen sediment [22]. The instantaneous and rapid decrease of
H2 concentration indicates that the potential for H2 dependent sulfate reduction must be as high as that
of CH4 production.
Plentiful evidence indicates that most H2 -depend-
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R. Conrad / FEMS Microbiology Ecology 28 (1999) 193^202
ent methanogenesis operates in microbial aggregates
in which H2 producers are juxtaposed to H2 consumers [3,4]. It has been proposed that sulfate reducers
may act as syntrophic H2 producers in the absence of
sulfate, e.g. during syntrophic degradation of lactate,
ethanol or propionate [31]. The syntrophic propionate oxidizers that have so far been isolated are all
able to reduce sulfate (e.g. [32]). Addition of sulfate
would switch these bacteria from acting as syntrophs
to acting as sulfate reducers, stop the production of
H2 , and starve the juxtaposed methanogens. Also,
H2 concentrations would decrease where H2 production by sulfate reducers was one of the main H2
sources. Interestingly, circumstantial evidence indicates that sulfate reducers may indeed be involved
in the syntrophic propionate degradation in methanogenic rice ¢eld soils [33], where a rapid decrease of
H2 concentrations has been observed upon addition
of sulfate.
Analogously to addition of sulfate, addition of
ferrihydrite or nitrate should also inhibit methanogenesis by competition for H2 . Indeed, H2 concentrations decrease and CH4 production is inhibited
when ferrihydrite or nitrate are added to methanogenic rice ¢eld soil [23,34]. However, the microbes
utilizing Fe(III) or nitrate as electron acceptors probably compete not only for H2 and acetate, but also
for fermentation products that are precursors for H2
and acetate production and probably also for carbohydrates directly. Therefore, the e¡ects of these electron acceptors on H2 turnover and methanogenesis
are not comparable to those of sulfate. In addition,
the e¡ects of nitrate on methanogenesis were shown
to be due to toxicity of denitri¢cation products (nitrite, NO and/or N2 O) to the methanogens in rice
¢eld soils [35].
4. Control of the environmental acetate concentration
Another question which is currently unresolved is
to what extent acetate turnover follows similar principles as H2 turnover. Most experiments show that
addition of sulfate, ferrihydrite or nitrate also inhibited acetate-dependent methanogenesis. As in the
case of H2 , this inhibition is thought to be due to
sulfate, iron and nitrate reducers competing successfully for acetate [20,36]. The threshold concept has
occasionally been applied to acetate utilization but
less rigorously than in the case of H2 . Methanogens
have dramatically di¡erent thresholds for acetate due
to di¡erent activation mechanisms. Thus, Methanosarcina species, which activate acetate (input of 1
ATP) with an acetate kinase, have a much higher
threshold (0.2^1.2 mM) for acetate than Methanosaeta species (7^70 WM), which activate acetate (input of
2 ATP) with an acetyl-CoA synthetase [37]. If the
acetate steady-state concentration observed in methanogenic environments is equivalent to the threshold
of the resident methanogenic population, then inhibition of methanogenesis upon addition of sulfate,
iron or nitrate does not necessarily require an instantaneous decrease of the acetate concentration (see
conjecture above). Indeed, in experiments with anoxic rice ¢eld soil, such a decrease has not been
observed, although acetate-dependent CH4 production was inhibited [24,34]. The observed inhibition
would be consistent with an acetate-utilizing potential of the sulfate, iron and nitrate utilizers that is
lower than that of the acetate-utilizing methanogens.
More research is needed to con¢rm this possible conclusion.
Acknowledgments
I thank H. Scholten for critically reading the
manuscript.
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