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 environment. Indeed, H2 is a ubiquitous compound

Methanogenic archaea utilize only a limited num-
ber of substrates, the most important ones being
acetate and H2 /CO2 (or formate) [1]. Most methano-
genic 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: conrad@mailer.uni-marburg.de
in anaerobic environments where it exhibits a fast
turnover but usually occurs at only very low concen-
tration [2^4]. Low H2 concentrations are a thermo-
dynamic 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 accom-
plished by syntrophy between H2 -producing syntro-
phic bacteria and H2 -consuming methanogenic ar-
chaea [5].
In this MiniReview I will address the following
two questions. (1) What is the percentage contribu-
tion 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 degrada-
tion 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 ma-
terial consisting of lignin and polysaccharides. Some
aquatic sediments receive a large input of dead crus-
taceans consisting of chitin. Lignin is largely recalci-
trant 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 carbohy-
drates 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 or-
ganic matter is in principle well known [1]. Di¡erent
groups of microorganisms participate in the degra-
dation which basically proceeds in three steps. (1)
Fermenting bacteria excrete enzymes that hydrolyze
organic polymers (e.g. polysaccharides) and catabo-
lize 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
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)
# Reaction
Fermentation
1.1 C6 H12 O6 C2 CH3 CHOHCOOH
1.2 C6 H12 O6 C2 CH3 CH2 OH+2 CO2
1.3 C6 H12 O6 C2/3 CH3 CH2 CH2 COOH+2/3 CH3 COOH+2 CO2 +8/3 H2
1.4 C6 H12 O6 C4/3 CH3 CH2 COOH+2/3 CH3 COOH+2/3 CO2 +2/3 H2 O
1.5 C6 H12 O6 C3 CH3 COOH
Syntrophy
2.1 CH3 CHOHCOOH+H2 OCCH3 COOH+CO2 +2 H2
2.2 CH3 CH2 OHCCH3 COOH+2 H2
2.3 CH3 CH2 CH2 COOH+2 H2 OC2 CH3 COOH+2 H2
2.4 CH3 CH2 COOH+2 H2 OCCH3 COOH+CO2 +3 H2
1-2 C6 H12 O6 +2 H2 OC2 CH3 COOH+2 CO2 +4 H2
Hydrogenotrophic methanogenesis
3 4 H2 +CO2 C2 H2 O+CH4
1-3 C6 H12 O6 C2 CH3 COOH+CO2 +CH4
Acetotrophic methanogenesis
4 CH3 COOHCCO2 +CH4
1-4 C6 H12 O6 C3 CO2 +3 CH4
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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 -con-
suming 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 en-
ergy (about a quarter of the total) is exclusively
available for acetotrophic methanogenesis (Fig. 2).
In fact, there is no thermodynamic reason why ho-
moacetogenic degradation of carbohydrates coupled
to acetotrophic methanogenesis should not be a ma-
jor pathway in anoxic environments. At the moment,
however, the role of homoacetogenesis in methano-
genic environments is unclear.
Hydrogen can be produced in the ¢rst fermenta-
vG³P (kJ mol31 substrate)
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

CH4 production occurs exclusively from acetate.

This observation is explained by sulfate reducers
which consume H2 in the upper sediment layers. Be-
cause 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 methano-
gens [12]. In methanogenic rice ¢eld soil, the contri-
bution 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 fer-
mentation reaction under this condition.
There are many studies in the literature which re-
Fig. 2. Residual standard Gibbs free energy (vG³P) after each re-
action stage in the methanogenic degradation of glucose utilizing
port much higher contributions of H2 than the ex-
di¡erent glucose fermentation reactions (equation numbers from pected 33%. Conceivable explanations for these ex-
Table 1 in parentheses). ceptions include (i) additional sinks of acetate, (ii)
additional sources of H2 , or (iii) measurements under
non-steady-state conditions. Additional sinks of ace-
tive degradation stage (e.g. reaction 1.3), and it is tate are not uncommon, e.g. in the rumen, acetate is
obligatorily formed in the second syntrophic stage largely absorbed into the blood stream of the host,
of organic matter degradation. The syntrophic stage leaving H2 as the predominant source for methano-
is sensitive to inhibition by H2 for thermodynamic genesis [14]. Similar observations were made in mi-
reasons. The maximum amount of H2 relative to crobial mats where acetate is assimilated by the pho-
acetate that can be produced from the degradation totrophs [15]. Transient phenomena must occur
of carbohydrates is 4 mol H2 plus 2 mol acetate per when H2 and acetate are sequentially produced or
mol glucose (reaction sum 1-2), i.e. a ratio of H2 / utilized. For example, the low amounts of CH4 pro-
acetate of 2:1. Any contribution of homoacetogene- duced immediately after £ooding of paddy soil are
sis (reaction 1.5) decreases this ratio. Degradation of mainly due to H2 -dependent methanogenesis, since
chitin (monomer = N-acetylglucosamine) results in
one more acetate (ratio of H2 /acetate of 4:3) than Table 2
in the case of the degradation of glucose and thus Examples of the contribution of H2 to CH4 production in di¡er-
decreases the possible contribution of H2 . ent methanogenic sediments
Since 4 H2 , but only 1 acetate, are required to Environment Contribution (%) Ref.
produce 1 CH4 , the contribution of H2 to methano- Contribution normal
genesis during anaerobic degradation of carbohy- Kichier Lake 32^46 [39]
drates can maximally be 33% of the total CH4 Lake Mendota 36^46 [40]
formed. Indeed, this percentage is consistent with Lake Washington 15^39 [41]
Anoxic paddy soil 17^31 [42]
data obtained from many studies of methanogenic
Contribution low
environments (Table 2). However, lower contribu- Colne Pt. Salt marsh 8 [9]
tions are also found in some methanogenic environ- Knaack Lake 4 [10]
ments. Usually, they are easily explained. In marine Lake Constance 0 [12]
sediments the low contribution of H2 can be due to Contribution high
Kuznechika lake 97 [39]
the dominance of sulfate reduction for degradation
Octopus Spring mat 74^86 [15]
of organic matter, while methanogenesis depends on Blelham Tarn 76^82 [43]
non-competitive precursors such as trimethylamine Cape Lookout Bight 71^80 [44]
[8,9]. In acidic lake sediments the low contribution Kings Lake Bog 100 [45]
of H2 may be explained by a larger contribution of Bunger Hills, Antarctica 95^97 [46]
Lake Baikal, deep sediment 99^100 [47]
homoacetogenesis [10]. In Lake Constance sediment,

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 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 ace-
tate. 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

Hydrogen is an intermediate in the methanogenic

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 con-
tributes about 30% to CH4 production as theoreti-
cally expected (Table 2).
In most cases, however, where H2 /CO2 -dependent
methanogenesis dominates (up to 100%) CH4 pro-
duction in sediments of lakes, marine bights and
peat bogs (Table 2), an explanation for elevated con-
tributions of H2 to methanogenesis is more di¤cult
to ¢nd. Additional sources of H2 are as yet unde-
scribed 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-
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 concen-
tration 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 per-
sist 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 28^32 [2,48]
Lake Mendota; Knaack Lake 27^35 [2]
Wetwood 42 [2]
Canal with detritus and leaves 8^18 [30]
Alder swamp 12^19 [30]
Littoral sediment, Lake Constance 33^39 [49]
Profundal sediment, Lake Constance 23^34 [12]
Upland soils turned methanogenic 25^50 [50]
Italian rice ¢eld soil 24^38 [13]
Methanobacterium bryantii 29^37 [27,28]
Other methanogenic archaea 29^50 [27,28]

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 198 R. Conrad / FEMS Microbiology Ecology 28 (1999) 193^202
(with Wmax = maximum growth rate; Ks = H2 concen-
tration at Wmax /2). Since
umax ˆ Xvmax …
(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 pro-
posed 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 usu-
ally been explained by the more e¤cient H2 utiliza-
tion 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 inhib-
ition can only be achieved after the methanogenic
population has been outgrown by the sulfate reduc-
ers [19,20]. Thus, methanogenic populations may be
replaced by sulfate reducers, iron reducers or nitrate
reducers in systems that have been exposed to sul-
fate, 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 ex-
hibit H2 concentrations that are characteristic for
methanogenesis, sulfate reduction, iron reduction,
etc. [21]. However, the kinetic model does not pro-
vide an explanation for the instantaneous and com-
plete inhibition of H2 -dependent CH4 production
that has been observed in some methanogenic envi-
ronments upon addition of sulfate [22^24]. An alter-
native model, one which incorporates a threshold
concept, on the other hand, does provide such an
explanation [21,25^27].
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The threshold concept of anaerobic H2 utilization
assumes that there is a certain H2 concentration be-
low which utilization is no longer possible because of
4†
thermodynamic constraints. Theoretically, the H2
threshold should be given by the conditions at which
reactants and products are in thermodynamic equi-
librium (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]. Inter-
estingly, the values of vGc increase (less negative) in
the order sulfate reducers s methanogens s homo-
acetogens, 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. There-
fore, they are not well described by Michaelis-Men-
ten 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 ac-
tual 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-
 R. Conrad / FEMS Microbiology Ecology 28 (1999) 193^202
tual concentration of reactants), and K = 2 (concen-
tration 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 con-
centrations at thermodynamic equilibrium. The au-
thors were able to show that their model ¢tted ex-
perimental 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 con-
ditions in the environment (i.e. y/K) than to the ki-
netic parameters of the microorganisms (i.e., vmax
and Km ). This response is because the H2 concentra-
tions are much closer to the thermodynamic equi-
librium than to the microbial Km values. The model
of Hoh and Cord-Ruwisch [29] may be further im-
proved 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 equi-
librium.
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 indi-
cate 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 methanogen-
esis obviously operates at its thermodynamic thresh-
old. 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 -de-
pendent sulfate reduction after addition of sulfate.
Let the rates of methanogenic and sulfate-reducing
H2 utilization be um and us . Then, the steady-state
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199
conditions (dC/dt = 0) would change from the meth-
anogenic 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 conse-
quently decrease below the threshold of the metha-
nogens, 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 -depend-
ent 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 con-
centrations will increase again until H2 -dependent
methanogenesis resumes and balances H2 produc-
tion. Then the same cycle would repeat itself. Macro-
scopically, this chain of events should result in a
partial but instantaneous inhibition of methanogen-
esis 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 in-
cubation were required for a decrease of the H2 con-
centration 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 com-
plete inhibition of H2 -dependent methanogenesis
with concomitant decrease of the H2 concentration
to values that are thermodynamically no longer per-
missive for methanogens (Fig. 3). Similar results
have also been obtained with Lake Wintergreen sedi-
ment [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-
 200 R. Conrad / FEMS Microbiology Ecology 28 (1999) 193^202
ent methanogenesis operates in microbial aggregates
in which H2 producers are juxtaposed to H2 consum-
ers [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 propio-
nate 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 produc-
tion by sulfate reducers was one of the main H2
sources. Interestingly, circumstantial evidence indi-
cates that sulfate reducers may indeed be involved
in the syntrophic propionate degradation in metha-
nogenic 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 methano-
genesis by competition for H2 . Indeed, H2 concen-
trations decrease and CH4 production is inhibited
when ferrihydrite or nitrate are added to methano-
genic rice ¢eld soil [23,34]. However, the microbes
utilizing Fe(III) or nitrate as electron acceptors prob-
ably compete not only for H2 and acetate, but also
for fermentation products that are precursors for H2
and acetate production and probably also for carbo-
hydrates directly. Therefore, the e¡ects of these elec-
tron 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 (ni-
trite, 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 prin-
ciples as H2 turnover. Most experiments show that
addition of sulfate, ferrihydrite or nitrate also inhib-
ited acetate-dependent methanogenesis. As in the
case of H2 , this inhibition is thought to be due to
sulfate, iron and nitrate reducers competing success-
fully for acetate [20,36]. The threshold concept has
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FEMSEC 972 8-3-99
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, Methano-
sarcina 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 Methanosae-
ta 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 meth-
anogenic environments is equivalent to the threshold
of the resident methanogenic population, then inhib-
ition of methanogenesis upon addition of sulfate,
iron or nitrate does not necessarily require an instan-
taneous decrease of the acetate concentration (see
conjecture above). Indeed, in experiments with an-
oxic rice ¢eld soil, such a decrease has not been
observed, although acetate-dependent CH4 produc-
tion was inhibited [24,34]. The observed inhibition
would be consistent with an acetate-utilizing poten-
tial 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 con-
clusion.
Acknowledgments
I thank H. Scholten for critically reading the
manuscript.
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