FEMS MicrobiologyReview 75 (1990) 125-138
Published by Elsevier
125
FEMSRE 00134
Conversion of acetic acid to methane by thermophiles
Stephen H. Z i n d e r
Department of Microbiology. SwcktngHall, Cornell UnR'ersily,Ithaca, N Y, U.S.A.
Key word~: Methanogenesis; Acetate; Thermophiles, Interspecics hydrogen transfer
1. SUMMARY
Acetate is the precursor of approximately twothirds of the methane produced in mesophilic (3040oC) and thermophilic (45-65°C) anaerobic
bioreactors. In the past decade, several thermophilic aeetotrophic methanoganic cultures have
been isolated including Methanosarcina thermophila, Methanothrix sp. strain CALS-1, the T A M
organism, and a symrophic acetate-oxidizing coculture. The thermophilic Methanosarcina and
Methanothrix cultures physiologically and morphologically resemble their mesophilie homologues, except that they grow two to five times as
rapidly. This more rapid growth offers a partial
explanation for the higher rates of methanogenesis
and lower minimum retention times often found
in thermophilic anaerobic bioreactors when compared with mesophilic bioreactors. The thermophilic Methanothrix shows a much lower minimum threshold for acetate utilization (12-20 #M)
than does a thermophilic Methanosarcina (1-2.5
raM), consistent with ecological evidence indicating that low acetate concentrations favor
Methanothrix. The thermophilic Methanothrix cultures have a maximum growth temperature near
70 ° C, while that for thermophilie Methanosarcina
cultures is near 60"C, and there is evidence that
bioreactors in which Methanothrix predominates
Correspondence to: S.H. Zinde¢, Department of Microbiology.
Stocking Hall. Corndl University,Ithaca, NY 14853, U.S.A.
are more tolerant to temperatures over 60 o C. The
rod-shaped T A M organism grows optimally at
60°C, and may represent a new genus. A thermophilic syntrophic coculture converts acetate to
methane by a two-step process in which acetate is
first oxidized to H 2 and CO2 by an acetate oxidizing rod (AOR) followed by methanogenesis from
H e and CO z by a thermophific Methanobaeterium.
The A O R has been isola~d in pure culture and
has been found to be an acetogen capable of
converting H2-CO2 to acetate, the opposite of the
reaction it carries out in syntrophic coculture. The
partial pressures of hydrogen in the acetate oxidizing coeulture must be poised such that beth
organisms are able to conserve energy. It was
found that entropy effects must be taken into
account to make an accurate prediction of H 2
partial pressures at 60°C, and it is predicted that
H 2 partial pressures will be generally about five to
ten-fold higher in thermophilic anaerobic bioreactots than in mesophilic ones. The presently known
upper temperature limit for methanogenesis from
acetate is 70°C, despite attempts by the author
and others to isolate more thermophilic strains
from geothermal environments.
2. I N T R O D U C T I O N
The use
(45-65°C)
one of the
philos. The
0168-6445/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
of thermophilic anaerobic digestion
to ccnvert organic wastes to C H 4 was
first bioteclmological uses of thermofeasibility of the thermophilic process
126
was first demonstrated by Rudolfs and Heukelekian [1] in 1930. Since then, there have been numerous studies, using both laboratory and pilot-scale
reactors, demonstrating that a wide variety of
agricultural, industrial, and domestic wastes can
be treated by thermophilic anaerobic digestion,
and full scale thermophilic sewage sludge digesters
have been operated for many years. Thermophilic
anaerobic digestion has been reviewed by Buhr
and Andrews [2] and, more recently, by Zinder [3].
A major advantage of thermophilic digestion is
that metabolic rates are usually higher than the
corresponding mesophilic process, and that the
retention time can be two-fold lower. For example, Wiegant and de Man [4] found that sludge
granules in thermophilie sludge blanket bioreaetors produced methane at greater than twice the
rate of similar granules from mesophilic reactors.
An example of the advantage of shorter retention
times is a study on full-scale sewage digesters in
the Chicago area [5]. It was shown that by increasing the temperature from 3 5 ° C to 52-55°C, the
retention time could be halved from 14 to 7 days,
thereby doubling the capacity of the digester and
saving the city the capital costs of building a new
digester. As will be discussed, these greater rates
can b¢ partially explained by the faster growth
and metabolism of thermophilic acetate utilizing
methanogans, as well as other organisms carrying
out rate-limiting processes.
Another advantage of the thermophilic process
is that water has a lower viscosity at high temperature, making mixing and sludge dewatering easier.
Also, pathogens are essentially pasteurized, makhag the finished product more sanitary. The obvious disadvantage of thcrmopinlic anaerobic digestion is that more energy must he used to mainrain higher temperatures. In some cases, such as
industries in which heat is essentially a waste
product, this is not a problem. The full-scale thormophilic digcsturs in Chicago previously mentioned [5] were energy self-sufficient in that they
produced enough methane to provide for their
heating needs when it was combnsted. However,
this greater energy requirement, the greater need
for temperature control at high temperatures, and
reports of greater instability than mesophilic reactors. must b¢ taken into account before a thermo-
COMPLEX POLYMERS
(prole]ns,potysacchmides,etc.)
le
MONO AND OLIQOMERS
(sugars, amino acids, peptldes)
@1
PROPIONATE
BUTYRATE ETC.
(IonQ-ch~in fedty acids)
H2 + CO2
X
l®
ACETATE
Fig. 1. Flow of carbon to methane and carbon dioxide in an
anaerobic bioreactor. Adapted from [6].
philic process is to be considered practical.
In recent years) there has been a much better
understanding of the microbiological processes involved in conversion of organic matter to methane
in both thermophilic and mesophilic environments. In this brief review 1 will describe the
thermophilic anaerobic digestion process, the importance of methanogenesis from acetate in these
reactors, and the properties of thermophilic microorganisms which convert acetate to methane, with
an emphasis on organisms studied in our laboratory. I will focus mainly on their responses to
temperature, and how they may effect performance of thermophilic anaerobic bioreactors.
3. CARBON FLOW T O M E T H A N E IN ANAEROBIC BIOREACTORS
Fig. 1 is a typical representation of carbon flow
to methane in anaerobic bioreactors [61. Methanegenie bacteria earl use only a limited number of
127
TABLE t
Summary of properties of mesophili¢acetotrophic methanogens
Genus
Morphology
Melhanosarcina
Large packets of
coccoid cells
Methanothrix
Sheathed
filamentousrod
Other substrates
used
Methanol, CO.
melhylamines,
H2-CO2(±)
None
Minimum td
on acetate
1-2 days
Apparettt Km
for acetate
3 rnM
3.5-9 days
0.7 mM
"
Some cultures do not use H2-CO2+
simple substrates (H2-CO 2, acetate, formate,
methanol, methylamines, etc.), and are dependent
on non-methanogens to break down complex
organic matter to substrates they can use. Therefore, an anaerobic food-chain is required '.o convert most anaerobic substrates to methane. Polymeric substrates such as cellulose or protein, if
present, must be broken down by hydrolytic enzymes to soluble monomers and oligomers before
fermentative organisms (Group 1) can use them.
Generally, the products of fermentation are
organic acids, H2, and COz. Fatty acids longer
than acetate (mainly propionate and butyrate) and
other fermentation products, such as ethanol and
lactate, are oxidized to acetate by a group termed
the hydrogen-producing (proton-reducing) acetogenie bacteria [7]. The reactions these organisms
carry out are only thermodynamically feasible if
the hydrogen partial pressure is maintained below
10 -3 arm (approx. 100 Pa) by hydrogen-consuming organisms, such as methanogens or sulfate
reducers.
The net result of the first two groups is to
convert complex organic material to the two
primary substrates for methanogenesis: H2-CO2
and acetate. Hydrogen-consuming (hydrogenotrophic) methanogans (Group 3) compete for the
same substrates as hydrogen-consuming acatogens
(Group 4), and usually win that competition because they are capable of using hydrogen at lower
partial pressures (minimum threshold = 2-10 Pa)
than acetogens (minimum threshold = 40-100 Pa)
(see below). Acetotrophic methanogens (Group 5)
decarboxylate acetate to CH4 and 'CO2. It has
been found that roughly two-thirds of all methane
is derived from acetate decarboxylation while
about one-third is from CO2 reduction, with small
amounts coming from other snbstrates, such as
methanol or methylamines, depending on the substrate. Formate may replace H 2 as an electron
donor for CO 2 reduction to CH 4 [8].
it is important that all groups function propcdy for balanced metabolism of substrates to
methane. In mesophilic systems, acid-producing
fermentative bacteria are capable of growth on
many soluble substrates with doubting times of
less than an hour, while acid-consuming b~cteria+
the hydrogen-producing acetogens (Group 3) and
the acetotrophic methanogens (Group 5), usually
have doubting times of 1-9 days. Therefore it is
easy for acid producers to overtake acid consumers after a perturbation, thereby causing acid
build-up. Also, the slow growth of the acid consumers fimits the rate of the digestion process, in
that in completely mixed systems the hydraulic
retention times must be considerably greater than
the organisms doubting time to prevent washout.
The slow growth of acid-consuming organisms
also explains the current interest in attached film
and sludge blanket reactors in which the organisms
are retai~ed so that they are no longer subject to
washout. For a more complete discussion of
anaerobic digestion, the reader is referred to the
excellent review by Schink [9].
Studies on carbon flow in thermophilic
anaerobic bioreactors [10,11] indicate that the
overall flow of carbon to methane in them resembles that in mesophitic bioreactors, with acetotrophic methanogens accounting for approximately two-thirds of the methanogencsis.
128
4. MESOPHILIC ACETOTROPHIC
ANOGENS
METH-
Two genera of mesophilic methanogens are
known to use acetate: Methanosarcina and
Methanothrix. Their properties are summarized in
Table 1. These two genera cluster together in one
branch of the Methanomierobiales [12] with other
methylotrophic (methanol and methylamine-utilizing) methanogens including Methanococcoides and
Methanolobus. This suggests that the ability to use
acetate only evolved once in the methanogens. The
two acetotrophic genera apparently have different
strategies for growth on acetate. Methanosardna
appears to be a generalist, capable of growing on
several different substrates in addition to acetate
including methanol, methylamines, and usually
Hz-CO 2. Methanothrix, on the other hand, is a
specialist and is only known to use acetate. Since
Methanosarcina can grow more rapidly on acetate
than does Methanothrix, one may wonder how
Methanothrix exists at all. At least part of the
answer is that Methanothrix is favored by low
concentrations of acetate. For example, Schfinhait
et al. [13| reported an apparent K m near 3 m~l for
acetate utilization by Methanosarcina barkeri strain
Fusaro, while Huser ¢ t a l . [14] estimated an apparent K m for growth of Methanothrix soehngeng
strain Opfikon near 0.7 raM. Ecological evidence
has been obtained showing predominance of
Methanosarcina at high acetate concentrations and
Methanothrix at low concentrations [4,11].
5. T H E R M O P H I L I C METHANOSARCINA
The first thermophilic acetotrophie methanogen
isolated was Methanosarcina sp. strain TM-1,
which was described by myself and R.A. Mah in
1979 [15]. Now known as Methanosarcina thermophila strain TM-1 [161, this culture grew optimally
at 5 0 ° C with a doubling time, when using acetate
as methanogenic substrate, near 12 h, while the
doubling time when using methanol plus acetate
was 4.5 h. The culture was derived from the fulb
scale bioreaetors at the Hyperion sewage treatment plant ~a Los Aatg~l~s> CA, in which autofluOreace~t Methanosarcina-llke clumps (most moth-
anngens contain large amounts of the fluorescent
cofactor F420 [12]) were numerous in samples examined by epifluorescence microscopy. Interestingly, it was reported that acids accumulated in
the bioreaetor if the temperature was increased
beyond 5 2 ° C [17], in agreement with the effect of
temperature on growth we had found in the pure
culture of strain TM-1. Strain TM-1 used acetate,
methanol, or methylamines as methanoganic substrates and the original culture was unable to use
H2-CO 2 [15], but a variant that can use Ha-CO 2
was subsequently isolated [16]. Sludge supematant
in the original growth medium [15] could be replaced [18] by trace metals, including nickel and
calcium, p-aminobenzoic acid, and CO2/bicarbonate.
The rapid growth of M. thermophila strain
TM-1 makes it well suited for biochemical and
genetic studies on methanogcnesis from acetate.
J.G. Ferry and colleagues have purified a CO
dehydrogenase complex [19], a thermostable
acetate kinase [20] and a thermostable ferredoxin
[21] from strain TM-L Potential genetic studies on
strain TM-1 have been hampered by its growth as
large aggregates containing many cells, complicating the isolation of genetically altered clones. Recently, Sowers a n d Gunsalus [22] showed that
strain TM-1, which was originally cultured in
medium with low salinity, could grow in marine
growth medium as long as the growth temperature
was 45 ° C or lower. The marine-adapted cells had
lost their outer heteropolysaccharide (methanocondroitin [2.3]) layer, and grew mainly as single
cells. The marine-adapted strains could grow at
saiinities as high as 1.2 M NaCl, and would lyse
when transferred to low ionic strength medium,
thereby allowing isolation of high molecular weight
D N A and RNA. Apparently, the heteropolysaccharidv layer plays a role in both osmotic and
temperature stabilization. Some strains of the
mesophiles M. barkeri and M. mazei also showed
a similar response to salinity, indicating that this
phenomenon may be widespread and used for
genetic studies of other Methanosareina strains.
Other thermophillic strains of Methanosareina
have been isolated which grow optimally at 55 ° C
rather than 50 o C, including strains CHTI-S5 [24]
and CALS-1 [25]. These strains can grow at 60°C,
129
a temperature at which strain TM-1 cannot grow.
However, all thermophilic Methanosarcina cultures described to date grow much more slowly at
6 0 ° C than at 55°C, and cannot grow at all beyond 620C. D N A - D N A hybridization studies by
Touzel (unpublished) indicate that these strains
are closely related to Methanosarcina thermophila
strain TM-1 and should be considered the same
species.
6. THERMOPHILIC M E T H A N O T H R I X
In 1983, Nozhevnikova and Yagodina [26l described a thermophilic enrichment culture which
converted acetate to methane, and which bore a
strong morphological resemblance to Methanothrix soehngeniL The culture was derived from a
thermal lake located in Kamchatka, U.S.S.R., and
similar cultures were obtained from thermophilic
anaerobic bioreactors. A purified enrichment was
subsequently named Methanothrix thermoacetophila [27]. It had an optimum growth temperature of 65°C, and only used acetate as a substrate for methanogenesis, similar to M. soehngenii.
The culture had a striated sheath and cross-walls
identical with M. soehngenii, and also gas vesicles.
We independently observed a similar organism
in a thermophilie anaerobic bioreactor and obtained it in enrichment culture [11]. Subsequently,
wc isolated the culture [28] using serial dilution
into liquid medium and treatments with vancomycin, and have named it Methanothrix sp. strain
CALS-I. It showed the typical morphology of
Methanothrix except that it too, had gas vesicles.
It is interesting that thermophilic Methanothrix
cultures isolated on opposite sides of the world
both have gas vesicles, while these structures have
not been reported in mesophilic Methanothrix cultures. Cells of strain CALS-I had more vesicles
after they had consumed all of the acetate i:t the
growth medium, and occasionally bands of cells
were seen floating at the top of stationary phase
cultures, leading me to speculate that numbers of
gas vesicles per cell may be regulated by nutrient
availability.
The only substrate tested used for methanogen°sis by Methanothrix sp. strain CALS-1 was acetate
[28]. The doubling time at 6 0 ° C was near 24 h,
considerably shorter than mesophilic strains.
Strain CALS-I had an optimum growth temperature near 60°C with good growth occurring at
65°C, and no growth at 7 0 ° C or 37°C. The
optimum pH value for growth was near 6.5, with
no growth occurring at pH 5.5 or 8.4. Strain
CALS-I grew in a chemically defined medium
with biotin as the only organic nutrient supplement.
From results of D N A - D N A hybridization
studies (J.P. Touzel, personal communication) and
rRNA sequencing (P. Rouviere, personal communication), as well as immunological studies [29],
it appears that strain CALS-I is only distantly
related to mesophilic Methanothrix strains, and
may be considered a separate genus. Its relationship with the Methanothrix thermoacetophila culture has yet to be determined.
7. COMPETITION BETWEEN THERMOPHILIC M E T H A N O S A R C I N A AND METHA N O T H R I X : I N F L U E N C E O F ACETATE
CONCENTRATION AND TEMPERATURE
The general model in which high acetate concentrations favor the faster growing Methanosar¢ina while low acetate concentrations favor
Methanothrix appears to be true in thermophific
systems. Wiegant and de Man [4] found that when
they fed acetate to thermophilic (55°C) sludge
blanket reactors such that effluent acetate concentrations never dropped below 11 raM, then
Melhanosarcina predominated, while Methanothrix dominated if the concentration was kept below
2.5 mM. In our own studies [11] we found that
Methanosarcina predominated in a 58°C anaerobic blot°actor fed a cellulosic substrate daily (10
day retention time) during the initial start-up
phase, when acetate concentrations were several
millimolar. Methanothrix predominated several
months later, when acetate concentrations stabilized to less than 1 mM just prior to feeding [11[.
We interpreted these findings to indicate that an
ecological succession occurred in which Methanosarcina lowered that acetate concentration to the
point where Methanothrix could begin to outcompete it for substrate.
130
osarcioa
200
"7¢: 100
. . . . . . . .
Acetate [raM)
Fig. 2. Effect of acetate concentration on rates of acetate
utilizationby washed and concentrated cellsof Methanosarcina
sp. strain CAL$-I, and Methanothrix sp. strain CALS-I. Because of the scale, the thr~hold of fl.0l5 mM for Melhanolhrix
appears to be zero. Data from [30].The results for Methanothrix sp. strain CAL$-I were extrapolated past I mM on the
basis of earlier experiments [28] showing that the rate was
constant at higheracetate concentrations.
We recently examined the kinetics of acetate
utilization in washed and concentrated cells of two
thermophilic aeatotrophic methanogens isolated
from the same bioreactor: Methanosarcina sp.
strain CALS-I and Methanothrix
sp. strain
CALS-1 [30]. Methanosarcina used acetate at a
constant rate at concentrations above 5 mM (Fig.
2), and acetate utilization ceased at concentrations
below 1 mM. We found minimum thresholds of
0.8 to 2.5 mM acetate for acetate utilization by
Methanosarcina sp. strain CALS-1, depending on
the experiment. Growing cultures incubated for 40
days still contained 0.2-0.7 mM acetate. For
Methanofhrix sp. strain CALS-I, acetate was used
at a constant rate at concentrations greater than
100 /tM, with thresholds of 12-21 /~M detected
(Fig. 2). The maximum rate of acetate utilization
by Me&anosareina (210 nmol acetate consumed
rain -1 [mg protein] 1) was neatly double that for
Methanothrix 110 nmol acetate consumed rain -1
frog protein]-l), in rough proportion to their
growth rates. The results fit the general model of
interaction between the two genera, hut acetate
utilization did not follow Monod kinetics in either
organism. Thresholds for acetate utilization have
also been described for mesophilic Methanosarcina
and Melhanothrix cultures by Westcrmann et al.
1311.
The optimum and maximum growth temperatures for Methanothrix sp. strain CALS-1 are 5 1 0 ° C higher than those for Melhanosureina sp.
strain CALS-1 (Fig. 3), which can not grow above
600C. The growth curve for Methanobacterium
strain CALS-1, a hydrogenotrophic methanogen
isolated from the same reactor, was biphasic with
an optimum near 7 0 ° C , which is typical for
Melhanobacterium thermoautotrophicum [12]. We
found slightly higher temperature optima for natural methanosenie populations in a 5 8 ° C reactor
when we incubated them for short periods with
14C-labelled acetate, i.e. an optimum for methanogenesis from acetate near 5 8 ° C when Methanosarcina predominated and near 6 5 ° C when
Methanothrix predominated [25].
Since there are many fermentative anaerobes
capable of good growth at 6 0 ° C and higher [3], it
could be predicted that Methanosarcina was the
population most likely to be adversely affected by
increasing the temperature to greater than 6 0 0 C.
In an "inadvertent experiment" [25] which occurred when Methanosarcina was still predomi-
100
E=
Msarc[na
Mthrix
Mbact
60
7-0
8O
E_
=~ 6o
2O
4O
50
~C
Fig. 3. Effectof temperature on growth of Metham~arcina sp.
strain CALS-I, Methanothrlx sp. strain CALS-I. and Math.
anobacterJum sp. strain CALS-LDala from [25.28].
131
nant, we found that after the temperature of our
5 8 ° C bioreaetor was accidentally increased to
6 4 ° C for about 20 h, there was an immediate
drop in gas production, accompanied by a rapid
rise in acetate concentration. This indicates that
the acetate consuming population, i.e. Methanosarcina, was the one most sensitive to an upward
temperature shift. After about a week, the reactor
recovered and the acetate concentration decreased
to its normal value (approx. 3 mM). When
Methanothrix was predominant, the same sort of
experiment was repeated by incubating sludge at
different temperatures in vials [25], and in this
ease, there was normal methanogenesis and no
build-up of acetate in samples incubated 24 h at
65 ° C. The prediction that thermophilic anaerobic
bioreactors would he more tolerant to higher temperature when Methanothrix is predominant appeared to be borne out by experiment.
8. T H E T AM O R G A N I S M
In 1984, Ahring and Westermann [32,33] described a thermophilic rod-shaped acetotrophie
methanogen which they called the TAM (thermophilie acetate-utilizing methanogan) organism.
W h i l e the culture superficially resembles
Methanothrix, there are some significant differences. It was capable of growth on H2-CO 2 and
formate, and its doubling time on acetate, nearly 3
days, was considerably higher than for Methanorhrix sp. strain CALS-1. The T A M organism has a
wavy outer layer and shows a constriction near the
division plane, instead of a rigid sheath and a
cross wall like Methanothrix. Therefore, the TAM
organism probably represents a novel genus. The
optimal growth temperature for the T A M
organism was near 6 0 ° C with slow growth occurring at 70°C. The TAM organism grew in medium
without yeast extract or rumen fluid, but these
additions decreased the lag time from 3 weeks to 1
week. Ahring and Westermann [34] examined the
kinetics of acetate utilization of the T A M organism
when it was a member of a thermophilie
butyrate-catabolizing consortium. They found that
uptake was linear down to concentrations near 0.6
mM, and that there was a minimum threshold of
25-75 ~M. These results, obtained before ours,
resemble those obtained for thermophilie
Methanosarcina and Methanothrix [30] and place
the affinity for acetate utilization by the T A M
organism closer to that for Methanothrix.
9.
THERMOPHILIC SYNTROPH1C
TATE-OXIDIZING COCULTURE
ACE-
The previously discussed cultures convert
acetate to methane by decarboxylation of acetate.
However, Barker [35] originally proposed a twostep mechanism in which acetate is first oxidized
to CO z and H z, followed by reduction of CO 2 to
CH 4 using H 2 (Table 2). In 1984, we described a
thermophilic syntrophic two-memhered culture
which converted acetate to methane by this oxidation mechanism [36]. The first step was carried out
by an organism called the acetate-oxidizing rod
(AOR), while the second step was carried out by a
hydrogenotrophic methanogen, Methanobacterium
sp. strain THF, which also could use formate, in
this mutualistic symbiosis, the AOR is dependent
on the methanogeu to keep H , partial pressures
low enough so that its reaction is exergonic while
the methanogen is dependent on the A O R for its
growth substrate. The two-membered coculture
TABLE2
Reaction mechanisms for methanogenesis from acetate
Mechanism
Reactants
Products
AG o'(kl/Rxn)
l
iI(a)
It(b)
llta+ b)
CH3COO - +
H20
CH3COO- +4H20
4H2 + HCO3- + H +
CH3COO- +H20
CH4 + HCO32HCO3- +4H 2 + H +
CH4 + 3H20
CH4 + HCO3
- 31
+ 105
- 136
-31
132
had a doubling time of 36 h at its optimum
temperature of 60°C. There is evidence that
methanogenic acetate oxidation occurs in anaerobic marine sediments below the sulfate reducing
zone [37] and in certain mesophilic anaerobic binreactors [38]. No mesophilic acetate oxidizing cocultures have been described to date, however.
We were able to culture the A O R in the absence of a methanogen by using ethylene glycol as
a growth substrate [39] and this allowed us to
isolate it. We found that axenic cultures of the
A O R could use a small number of substrates,
including pyruvate, bctaine, and most interestingly, H2-CO 2 and formate, both of which are
converted to acetate. Therefore, the A O R is an
acetogan and whether it produces acetate or
oxidizes it depends on the concentrations of reactants and products in the growth medium. We
have also found that both acetogenic cultures and
acetate-oxidizing cocultures have high activities of
the key enzyme carbon monoxide dehydrogenase
and low amounts of the citric acid cycle enzyme
isocitrate dehydrogenase [40] indicating that the
A O R oxidizes acetate using a pathway resembling
a reversal of the acetate synthesis pathway in
acetogens [41], in agreement with a prediction we
originally made [36] on the basis of rapid isotopic
exchange between the earboxyl group of acetate
and CO 2 detected in acetate-oxidizing cultures.
Use of a reversed acetogenesis pathway for acetate
oxidation has now been demonstrated in certain
acetate-oxidizing sulfate-reducing bacteria [42,43].
On the basis of analysis of the sequence of its 16S
rRNA (B. White and D. Stahl, personal communication), the AOR groups with the Cram-positive
eubaeteria, consistent with its acetogenic phenotype.
In obligately syntrophic cocultures carrying out
interspecies hydrogen transfer, the hydrogen partial pressure must be poised such that reactions of
both partners are thermodynamically favorable. If
it is too low, then the reaction for the hydrogen
consumer becomes unfavorable (/iG > 0), while if
it is too high, then the reaction of the hydrogen
produces becomes unfavorable. These boundaries
form a 'window" for the hydrogen partial pressure,
with the actual value found in between, since both
organisms must conserve energy from the reac-
tion. This concept is consistent with the recent
finding that hydrogen-consuming anaerobes show
minimum thresholds [44,45] below which hydrogen consumption does not occur. The values of
these thresholds correlate with the free energies of
reactions for the various electron accepters [45]
such that the threshold for an organism reducing
nitrate to ammonia was near 0.003 Pa, that for a
sulfate reducer was near 1 Pa, those for methanogens were 2-10 Pa, while for acetogens they were
40-90 Pa. These threshold values are all greater
than the partial pressure at which AG = 0 for
those organisms, thereby allowing them to conserve energy.
Our initial calculations [36], bascd on the free
energy form of the Nernst equation adjusted for
6 0 ° C [6], indicated a minimum H 2 partial pressure near 0.9 Pa, and a maximum near 17 Pa, and
a geometric mean value near 4 Pa (approx. 4 x
10 -5 atm or 24 riM). When we measured H 2
parlial pressures in cocultures actively oxidizing
acetate [46[, we found tbat the H 2 was actually
near 20-40 Pa (Fig. 4), reaching a minimum
threshold near 14 Pa, after acetate was consumed.
This discrepancy lead us to correct our free energy
values from 2 5 ° C to 6 0 ° C using the equation
A G ffi ~ i l l - T/iS. Using this equation, we predicted minimum and maximum values of 2.6 Pa and
75 Pa respectively, which is in much better agreement with the experimental data. Using this equation and the Nernst equation, we predicted the
effect of temperature on the boundary conditions
for H 2 for syntrophic acetate oxidizing cocultures
(Fig. 5). We found that temperature has a profound effect on both reactions with both varying
250-fold over the range 0 - 8 0 ° C . A similar calculation was performed by Zehnder and Stumm [47].
Since there are presently no mesophilic homologueS of the AOR, this result can not be tested
experimentally for acetate oxidation. Butyrate
oxidation, which has somewhat better energetics
than acetate oxidation, shows a similar effect over
the temperature range of 2 5 - 6 0 ° C (Fig. 5), and
recently there have been measurements of the
hydrogen partial pressures in mesophilic and thermophilic butyrate oxidizing cocultures. Some of
these values have been plotted in Fig. 5 to show
that they follow the general trend predicted by the
133
100
in rice p a d d y soils d r o p p e d f r o m 8.5 Pa to 0.9 Pa
w h e n the t e m p e r a t u r e was decreased f r o m 3 0 ° C
to 1 7 ° C .
These d a t a are all consistent with increasing
t e m p e r a t u r e s c a u s i n g a n increase in h y d r o g e n partial pressure. T h i s mode l then predicts that h y d r o gen partial pressures will b e h i g h e r in high-tern-
5O
CH4
B0
40
~o
~
4
,000I
Butyrate
. l~ oxidation
.,yy
100
20
10
120
10
D_
5
enesis
Days 5
20
Fig. 4. H z partial pressure and methane production by a
thermophilic acetate oxidizing cocuhure during growth on 40
mM sodium acetate. Figure from 146l.
I
oi
0.01
t h e r m c d y n a m i c calculations. D w y e r et a1. [48]
f o u n d tha; the h y d r o g e n p a r t i a l pressure at the
p o i n t during, g r o w t h at which the acetate a n d
b u t y r a t e c o n c e n t r a t i o n s were equal w a s n e a r 20
Pa, while H 2 a c c u m u l a t e d to approx. 100 Pa w h e n
m e t h a n o g e n e s i s w a s inhibited with b r o m o e t h a n e
sulfonate, r e p r e s e n t i n g the u p p e r limit for h y d r o g e n p r o d u c t i o n b y the b u ty r ate oxidizer. A h r i n g
a n d W e s t e r m a n n [491 f o u n d n o i n h i b i t i o n of
butyrate oxidation when a thermophilie eocuhure
w a s b u b b l e d with g a s c o n t a i n i n g 75 Pa H 2, whi l e
p a r t i a l pressures n e a r 3000 Pa H 2 were required
for c o m p l e t e inhibition. T h g s e values m a y be
s o m e w h a t higher th~n the actual c o n c e n t r a t i o n s in
l i q u i d if g a s tr an sf er into the liquid p h a s e was
linfiting. Ho w ev er , they are c o n s i d e r a b l y higher
t h a n the c o r r e s p o n d i n g values for m e s o p h i l i c cultures. T h e m i n i m u m H z thresholds for m e s o p h i l i c
methanogens vary from 2-10 Pa [45], while that
for the thcrmophile Methanobacterium sp. strain
THF was 14 Pa [44]. in terms of ecological data,
Conrad et al. [50] found that H 2 partial pressure
. i . i
1o 20
.
i
30
.
i
40
.
i
50
.
i
60
.
i
70
.
i
80
=C
Fig. 5. Effect of temperature on the hydrogen partial pressure
at which ZIG" = 0 for various reactions under conditions similar to ~e midpoint of growth of a symrophie coculturP- The
reactions are: methanoge;lesls (4H 2 + HCOj- + H + ~ CH 4 43HzO); acetaL¢ oxidation (acetate +4HzO~2HCO 3- -b4H z
+ H + ); butyrate oxidation l h u t y r a t c - + 2 H 2 0 ~ 2 acctatc +
2H z + H + ). For acetate oxidation and methanogenesis, the
concentrations of the products and reactants are: acetate, 20
mM: HCO3, 30 raM: CH 4. 0.35 arm. For butyrate oxidation
the concentrations of products and reactants are: butyrate. 10
mM, acetate. 10 mM. Adapted from [46]. t. H a partial pres,
sure at the point at which (acetate} = Ibutyrate) i,, a mesophilic
butyrate oxidizing coculture [48]. 2. H 2 partial pressure in a
mesophilie butyrate oxidizing coculture in which methanogenesis was inhibited with bromocthane sulfonate [48]. 3. H z partial pressure which caused complete inhibition when bubbled
through a thermophilic butyrate oxidizing cocuhure [49]. 4. H 2
partial pressure which caused complete inhibition when bubbled through a thermophilic butyrate oxidizing coculture 149].
~;. Range of minimum H 2 thresholds in several mesophilic
hydrogenotrophic metbanogens [45]. 6. Minimum H z threshold
in a thermophilic anaerobic acetate-oxidizing cocuhure [46]. 7.
H2 partial pressure during the midpoint of growth of a thermophilic acctat¢ oxidizing coculturc.
134
perature environments in which interspecies hydrogen transfer is occurring than in corresponding
low-temperature environments. Specifically it predicts about a ten-fold increase in hydrogen partial
pressure for each increase of 3 0 ° C so that hydrogen partial pressures would be expected to be
about five to ten-fold higher in thermophilic
anaerobic digesters than in corresponding
mesophilic ones. This increase in hydrogen pool
size affects the turnover rate of the hydrogen pool
and the diffusion of hydrogen in these envlronments.
10. UPPER TEMPERATURE LIMIT
METHANOGENESIS FROM ACETATE
FOR
The upper temperature limit for the TAM
organism and Methanothrix cultures is near 70 o C.
It is likely that a hyperthermophilic acetotrophic
methanogen would grow faster than those presently described, and would potentially increase
the present maximum temperature of 650C for
thermophilic anaerobic digestion [2,3]. There is no
a priori reason why acetotrophic methanogens
which can grow over 7 0 ° C shouldn't exist. There
are hydrogenotrophic methanogens, such as
Methanothermus feroidus and Methanococcus jannaschii [12], which can grow at temperatures in
excess of 80°C. There are many different
fermentative anaerobes found in geothermal
habitats capable of growth at temperatures in
excess of 70 oC. Most of these organisms produce
acetate as a major fermentation product. Therefo,.e, there should be high-temperature habitats in
which acetate-utilizing methanogens would be
selected for.
Over the years, I have initiated enrichment
cultures for thermophilic acetotrophic methanogens from a variety of geothermal habitats including California hot springs, California geothermal
wells, Yellowstone National Park hot springs, and
New Zealand hot springs without any success.
Fermentative heterotrophs and hydrogenotrophic
methanogens were always easily enriched from
these samples when attempted. Enrichments using
acetate as a substrate by Karl Stetter and eelleagues (personal communication) have also been
1.5
1.0
0.5
°.°2° so ,0
so '0'0 ',o
0o . .90. . 100
Temperature {"C)
Fig. 6. Effect of temperature on Ihe rate of acetate activation
in crude extracts of Mezhanothrix sp. strain C A L S - t as measured using the hydroxamale assay [5It.
unsuccessful. Obviously, this does not prove that
they do not exist, and Methanothrix thermoacetophila was enriched from a geothermal area in
the U.S.S.R. We have recently found that one key
enzyme apparently involved in acetate activation
by Methanothrix sp. strain CALS-I, acetyI-CoA
synthetase, shows nearly optimal activity at 80 ° C
(Fig. 6). Another key enzyme, carbon monoxide
dehydrogenase (G. Alien and S. Zinder, unpublished), is even more thermotolerant and shows
increasing activity at temperatures up to 95oc,
and has a half life of 10 rain when incubated
anaerobically at 100 ° C. Certainly neither of these
enzymes limits the possibility of a higher temperature thermophilic acetotropbic methanogen.
11. C O N C L U D I N G REMARKS
The past decade has seen a vast increase in our
knosvledge about methanogenesis from acetate by
thermophiles. Table 3 summarizes the properties
of some presently known thermophilic acetotrophic methanogens. Except for their more rapid
growth rate and the presence of gas vesicles in the
thermophilic Metkanothrix cultures, thermophiiic
Methanosarcina and Methanothrix cultures closely
resemble their mesophilic counterparts. It is quite
135
TABLE 3
Summary of properties of thermophihe acetotrophic methanogenie cultures
Culture
Morphology
Top=
( o C)
Minimum t a
on acetate (h)
Acetate
threshold
Methanosa~ina
Large packets
50-55
12
Mezhanothrix
Sheathed
filament
60
24
12-20 ,aM
Acetate-oxidizing
cocuhure
Two rods
60
36
ND ~
Ethanol.
ethylene glycol.
betaine, etc.
TAM organism
Rod
60
60
25-75 p-M
H2-CO2, formate,
1-2.5 mM
Other substrates
used
Methanol,
methylamines,
H 2-CO2( + )
"
Not determined
likely that the faster g r o w t h rates of t h e r m o p h i l i c
aeetotrophie m e t h a n o g e n s c o n t r i b u t e to the m o r e
r a p i d rates o f m e t h a n o g e n e s i s a n d shorter retention t i m e s p o ssib le in t h e r m o p h i l i c a n a e r o b i c bioreactors. T h e T A M o r g a n i s m requires further
c h a r a c t e r i z a t i o n b e f o r e it can b e classified. T h e
acetate-oxidizing eoeulture represents a novel
s y m b i o s i s a n d a novel m e c h a n i s m for m e t h a n o genesis f r o m acetate, a n d has yet to have a
m e s o p h i l i e c o u n t e r p a r t . It is o f interest that the
eubacterial AOR
and the archaebacterial
m e t h a n o g e n s b o t h use a c a r b o n m o n o x i d e de hyd r o g e n a s e p a t h w a y for d i s s i m i l a t i o n of acetate.
T h e s e f i n d in g s h a v e increased o u r u n d e r s t a n d i n g
of c o n v e r s i o n of acetate to m e t h a n e in t h e r m o phiIic bioreactors, a n d a b o u t m e t h a n o g e n e s i s f r o m
acetate in general. It will b e interesting to see w h a t
the next deP,.ade h a s in store.
ACKNOWLEDGEMENTS
T h e research o f the a u t h o r in this area h a s been
s u p p o r t e d b y g r an t D E - F G 0 2 - 8 5 E R 1 3 3 7 0 f r o m
the U.S. D e p a r t m e n t o f Energy. T h e a u t h o r wishes
to a c k n o w l e d g e the essential c o n t r i b u t i o n s of his
collaborators, i n c l u d i n g T. A n g u i s h , S. Cardwell,
M . K o c h , M . Lee, A. L o b o , R. M a h , H. Min, a n d
P. M u r r a y . H e also wishes to t h a n k colleagues for
s h a r i n g u n p u b l i s h e d data, especially J. Touzel.
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