Aceticlastic Methanogens Research Articles

Influence of monochlorophenols on methanogenic activity in granular sludge

The effects of monochlorophenols (MCPs) on volatile fatty acid degradation by methanogenic granular sludge from two full scale upflow anaerobic sludge blanket (UASB) reactors treating potato processing wastewater, were tested. Sludges 1 and 2 were derived from the first reactor, and sludge 3 from the second reactor. Methane production and utilization of acetate, propionate and butyrate were measured. Methane production by sludge 3 was more severely inhibited by MCPs (50% inhibition around 0.6mM or less) than were sludges 1 and 2 (50% inhibition above 1mM). Aceticlastic methanogens, propionate degrading consortia and butyrate degrading consortia of the two sludges had a different sensitivity. In sludge 3, the acetate utilization was most sensitive, while for the other sludges, butyrate utilization was inhibited most strongly. In an experiment with stored sludge, the inhibition was reversible, but depended on the exposure time. In addition, stored sludge was less active and more sensitive to MCPs compared to fresh sludge. An activation period after cold storage lowered the sensitivity of the sludge to 2-CP. These findings may be important when stored sludge is used to start up a reactor to treat wastewater that contains toxic compounds.

Methanocalculus halotolerans gen. nov., sp. nov., isolated from an oil-producing well.

Two irregular coccoid methanogens designated SEBR 4845T and FR1T were isolated from an oilfield in Alsace, France. Strain SEBR 4845T (T = type strain) is a hydrogenotrophic halotolerant methanogen, which grows optimally at 5% NaCI (w/v) and tolerates up to 12% NaCI. It does not use methylated compounds and therefore cannot be ascribed to any of the known genera of the halophilic methylotrophic methanogens. It differs from hydrogenotrophic members of the orders Methanococcales and Methanomicrobia les in the NaCI growth range (0-12% NaCI), which is the widest reported to data for any hydrogenotrophic methanogen. 16S rRNA gene sequence analysis indicated that strain SEBR 4845T is a novel isolate for which a new genus is proposed, Methanocalculus halotolerans gen. nov., sp. nov. (= OCM470T) that might be indigenous to the oilfield ecosystem. Strain FR1T (=OCM 471) is a moderately halophilic methanogen which growths optimally at 10% NaCI and tolerates up to 20% NaCI. It grows on trimethylamine and methanol as carbon and energy sources. The G+C content of its DNA is 43 mol%. It is therefore phenotypically and genotypically related to members of the genus Methanohalophilus. This report provides evidence that methylotrophic and hydrogenotrophic, but not aceticlastic methanogens are present in a saline subsurface oilfield environment, as already observed in surface saline to hypersaline environments.

Fermentation and methanogenic characteristics of leafy biomass feedstocks in a solid phase biogas fermentor

Biomass feedstocks, such as leaf litter, weeds and agroresidues, have been considered as alternative feedstocks to meet rural energy needs in India. Six different types of biomass substrates representing commonly available fresh and dry feedstocks were studied for their decomposition pattern and methanogenic activities in order to arrive at optimum design parameters for solid phase digestion. Broussenetia papyrifera, Parthenium hysterophorus, Synedrella nodiflora (fresh leaf biomass feedstocks), and paddy straw, underwent a rapid initial decomposition losing 30–40% volatile solids (VS) within 10 d. This decomposition pattern appeared to favour growth and colonization of hydrogenotrophic methanogens in the latter three feedstocks. Stable biogas production was found wherever approximately similar rates of aceticlastic and hydrogenotrophic methanogenic activity were recorded on decomposing biomass feedstocks. Inadequate colonization by aceticlastic methanogens was found to be the main cause of a poor start-up and lower daily gas production rates, especially in the presence of rapid VS destruction. Two dry feedstocks, cane trash and bagasse were found to have an acidogenesis-limited decomposition pattern with <40% VS destruction in 45 d. These results suggested that proper start-up procedures were needed to ensure adequate build-up of aceticlastic methanogens, and the use of a mixed biomass feedstock comprised of fresh and dry biomass had a better chance of stable biogas production, conversion efficiency and gas yield.

Inhibition of methanogenesis by methyl fluoride: studies of pure and defined mixed cultures of anaerobic bacteria and archaea

Methyl fluoride (fluoromethane [CH(inf3)F]) has been used as a selective inhibitor of CH(inf4) oxidation by aerobic methanotrophic bacteria in studies of CH(inf4) emission from natural systems. In such studies, CH(inf3)F also diffuses into the anaerobic zones where CH(inf4) is produced. The effects of CH(inf3)F on pure and defined mixed cultures of anaerobic microorganisms were investigated. About 1 kPa of CH(inf3)F, similar to the amounts used in inhibition experiments, inhibited growth of and CH(inf4) production by pure cultures of aceticlastic methanogens (Methanosaeta spp. and Methanosarcina spp.) and by a methanogenic mixed culture of anaerobic microorganisms in which acetate was produced as an intermediate. With greater quantities of CH(inf3)F, hydrogenotrophic methanogens were also inhibited. At a partial pressure of CH(inf3)F of 1 kPa, homoacetogenic, sulfate-reducing, and fermentative bacteria and a methanogenic mixed culture of anaerobic microorganisms based on hydrogen syntrophy were not inhibited. The inhibition by CH(inf3)F of the growth and CH(inf4) production of Methanosarcina mazei growing on acetate was reversible. CH(inf3)F inhibited only acetate utilization by Methanosarcina barkeri, which is able to use acetate and hydrogen simultaneously, when both acetate and hydrogen were present. These findings suggest that the use of CH(inf3)F as a selective inhibitor of aerobic CH(inf4) oxidation in undefined systems must be interpreted with great care. However, by a careful choice of concentrations, CH(inf3)F may be useful for the rapid determination of the role of acetate as a CH(inf4) precursor.

The bacterial numeration and an observation of a new syntrophic association for granular sludge

The bacterial numeration and microbial observation were made on granular sludge from laboratory single and two-phase UASB reactors. It was shown that the fermentative bacteria (group I), H2-producing acetogenic bacteria (group II) and methanogenic bacteria (group III) of granular sludge in single UASB reactor were 9.3 × 108−4.3 × 109, 4.3 × 107−4.3 × 108, 2.0−4.3 × 108, respectively, during the granulation process. The sludge of methanogenic reactor exhibited the similar results. That indicates there is no big difference between suspended and granular sludge, and bacterial population for three groups of anaerobic bacteria are similar. The formation of granular sludge depends mainly on the organization and arrangement of bacteria. An observation of granular sludge using electron microscope revealed that the fermentative bacteria and hydrogenotrophic methanogens existed on outer surface of granules, and aceticlastic methanogens and H2-producing acetogenic bacteria occupied the inner layer. A new syntrophic association between Methanosaeta sp. and Syntrophomonas sp. (even plus Methanobrevibacter sp.) was observed. Because Methanosaeta can effectively convert the acetate (the end product of propionate and butyrate) to methane, such a new syntrophic association is supposed to support the degradation of short fatty acids and high methanogenic activity of granular sludge. Based on structural pattern, a hypothesis on mechanism of granulation called “crystallized nuclei formation” is proposed.

The bacterial numeration and an observation of a new syntrophic association for granular sludge

The bacterial numeration and microbial observation were made on granular sludge from laboratory single and two-phase UASB reactors. It was shown that the fermentative bacteria (group I), H2-producing acetogenic bacteria (group II) and methanogenic bacteria (group III) of granular sludge in single UASB reactor were 9.3 × 108−4.3 × 109, 4.3 × 107−4.3 × 108, 2.0−4.3 × 108, respectively, during the granulation process. The sludge of methanogenic reactor exhibited the similar results. That indicates there is no big difference between suspended and granular sludge, and bacterial population for three groups of anaerobic bacteria are similar. The formation of granular sludge depends mainly on the organization and arrangement of bacteria. An observation of granular sludge using electron microscope revealed that the fermentative bacteria and hydrogenotrophic methanogens existed on outer surface of granules, and aceticlastic methanogens and H2-producing acetogenic bacteria occupied the inner layer. A new syntrophic association between Methanosaeta sp. and Syntrophomonas sp. (even plus Methanobrevibacter sp.) was observed. Because Methanosaeta can effectively convert the acetate (the end product of propionate and butyrate) to methane, such a new syntrophic association is supposed to support the degradation of short fatty acids and high methanogenic activity of granular sludge. Based on structural pattern, a hypothesis on mechanism of granulation called “crystallized nuclei formation” is proposed.

The Eastern and Western branches of the Wood/Ljungdahl pathway: how the East and West were won

Figure 1 is a transmission electron micrograph of Clostridium ljungdahlii [1]. It, like Acetobacterium woodii [2], was named after someone who made great contributions to the understanding of how anaerobes fix carbon dioxide. C. ljungdahlii was named after Lars Ljungdahl; A. woodii after Harland Wood. I had the good fortune to train under both Lars and Harland and have continued research in this area for approximately fifteen years, learning and gaining greater and greater appreciation for the art of anaerobes. This review introduces the concept of the Eastern and Western branches of the Wood/Ljungdahl pathway. The Eastern branch is important in one-carbon metabolism of all organisms; the Western branch is unique to anaerobic microorganisms that use the Wood/Ljungdahl pathway for fixing carbon dioxide or carbon monoxide to generate cell carbon or for synthesizing acetate (acetogens) or methane (aceticlastic methanogens) to generate energy. In focusing on the biochemistry and enzymology of this pathway, I, unfortunately, must risk leaving the important ecology and physiology of these organisms in the lurch. They are responsible for the turnover of a trillion tons of acetic acid per year. They convert a variety of compounds including sugars, aromatic compounds, and inorganic compounds like CO and H2 and CO2 into acetic acid which is then used by methane producing bacteria to make natural gas. They are important in soil microbiology and, in the biology of organisms that house them in their digestive system, like humans, termites and ruminants like cows and sheep [3–5]. The story of acetogens can be traced back to the ancient times when someone discovered that apple or grape juice could be fermented, first to alcohol and then to acetic acid, producing cider vinegar, which contains 3–6% acetic acid. The word acetum is the Latin word for vinegar. Acetic acid was isolated in pure form by Stahl in 1700. Over the years, it has been found to be an excellent solvent for many organic compounds and some inorganic compounds. It also is essential in the production of cellulose acetate and is used widely in the textile and rubber industries.

Influence of ammonia concentration on thermophilic anaerobic digestion of cattle manure in upflow anaerobic sludge blanket (UASB) reactors

Ammonia concentrations of 5 g N/litre or more inhibited thermophilic anaerobic digestion of cattle manure in upflow anaerobic sludge blanket (UASB) reactors. A stable digestion of cattle manure could be maintained with ammonia concentrations up to 7 g N/litre after 6 months of operation. However, the methane yield was reduced and the concentration of volatile fatty acids increased from 1 to 3 g/litre as acetic acid, compared to controls with an ammonia concentration of 3 g N/litre. The temporary strong inhibition following a one-step increase in ammonia concentration was reduced by applying a gradual increase. The specific methanogenic activity of ammonia-inhibited reactors (7 g N/litre) with acetate or hydrogen as substrate was reduced by 72 and 56%, respectively. Tests of ammonia toxicity on the acetate- and hydrogen-utilizing populations showed a higher sensitivity of the aceticlastic compared to the hydrogenotrophic methanogens; the specific growth rate for the aceticlastic methanogens was halved at ammonia concentrations of 4 g N/litre, compared to 7·5 g N/litre for the hydrogenotrophic methanogens.

Toxicity of kraft bleaching plant effluent to aceticlastic methanogens

The toxicity of kraft bleaching plant effluent to aceticlastic methanogens was investigated in batch assays, using an anaerobic serum bottle technique. The effluent was found to exhibit a strong inhibition of the anaerobic conversion of acetate to methane and carbon dioxide, expressed as a long lag-phase before methanogenesis started. The inhibitory compounds were shown to have a low molecular mass and to have their origin in the acidic part-stream of the bleaching plant effluent. The methanogens seemed not to be able to acclimate to the inhibitory compounds, but the toxicity was found to be bacteriostatic rather than bacteriocidal. The inhibitory compounds were found to be degraded slowly in the batch experiments, finally reaching non-toxic levels so that methane production could start. The toxicity of the chlorinated acetic acid analogues mono-, di- and trichloro-acetic acid were studied. These compounds were found not to contribute significantly to the methanogenic toxicity of kraft bleaching plant effluents.

The effect of sulfate and nitrate on methane formation in a freshwater sediment.

A freshwater sediment from a ditch of a peat grassland near Zegveld (Province of Utrecht, The Netherlands) was investigated for its potential methanogenic and syntrophic activity and the influence of sulfate and nitrate on these potential activities. Methanogenesis started after a 10 days lagphase. After 35-40 days aceticlastic methanogens were sufficiently enriched to cause a net decrease of acetate. In the presence of sulfate methane formation was only slightly affected. The addition of nitrate led to an outcompetition of aceticlastic methanogens by nitrate reducers. When inorganic electron acceptors were absent, substrates like propionate and butyrate were converted by syntrophic methanogenic consortia. Addition of inorganic electron acceptors resulted in an outcompetition of the syntrophic propionate and butyrate degrading consortia by the sulfate and nitrate reducers.

Methanogenesis in thermophilic biogas reactors

Methanogenesis in thermophilic biogas reactors fed with different wastes is examined. The specific methanogenic activity with acetate or hydrogen as substrate reflected the organic loading of the specific reactor examined. Increasing the loading of thermophilic reactors stabilized the process as indicated by a lower concentration of volatile fatty acids in the effluent from the reactors. The specific methanogenic activity in a thermophilic pilot-plant biogas reactor fed with a mixture of cow and pig manure reflected the stability of the reactor. The numbers of methanogens counted by the most probable number (MPN) technique with acetate or hydrogen as substrate were further found to vary depending on the loading rate and the stability of the reactor. The numbers of methanogens counted with antibody probes in one of the reactor samples was 10 times lower for the hydrogen-utilizing methanogens compared to the counts using the MPN technique, indicating that other non-reacting methanogens were present. Methanogens that reacted with the probe against Methanobacterium thermoautotrophicum were the most numerous in this reactor. For the acetate-utilizing methanogens, the numbers counted with the antibody probes were more than a factor of 10 higher than the numbers found by MPN. The majority of acetate utilizing methanogens in the reactor were Methanosarcina spp. single cells, which is a difficult form of the organism to cultivate in vitro. No reactions were observed with antibody probes raised against Methanothrix soehngenii or Methanothrix CALS-1 in any of the thermophilic biogas reactors examined. Studies using 2-14C-labeled acetate showed that at high concentrations (more than approx. 1 mM) acetate was metabolized via the aceticlastic pathway, transforming the methyl-group of acetate into methane. When the concentration of acetate was less than approx. 1 mM, most of the acetate was oxidized via a two-step mechanism (syntrophic acetate oxidation) involving one organism oxidizing acetate into hydrogen and carbon dioxide and a hydrogen-utilizing methanogen forming the products of the first microorganism into methane. In thermophilic biogas reactors, acetate oxidizing cultures occupied the niche of Methanothrix species, aceticlastic methanogens which dominate at low acetate concentrations in mesophilic systems. Normally, thermophilic biogas reactors are operated at temperatures from 52 to 56 degrees C. Experiments using biogas reactors fed with cow manure showed that the same biogas yield found at 55 degrees C could be obtained at 61 degrees C after a long adaptation period. However, propionate degradation was inhibited by increasing the temperature.

High rate performance and characterization of granular methanogenic sludges in upflow anaerobic sludge blanket reactors fed with various defined substrates

High rate granular methanogenic fermentations were performed in one-phase upflow anaerobic sludge blanket (UASB) reactors treating synthetic wastewaters containing starch, sucrose, ethanol, and butyrate plus propionate. All granules formed showed high settling velocities which enabled high cell mass retention and accommodation of high loading rates. The maximum COD removal rates (g COD/ l-reactor·d) obtained after 500-d operations were 7.6 for starch, 10.5 for sucrose, 32.1 for ethanol, and 42.6 for butyrate-propionate. Long-term growth on various defined substrates altered the population of bacterial trophic groups and overall characteristics of granules. The starch- and sucrose-grown granules were characterized by larger size and more abundant extracellular polymeric substances (EPS) than the ethanol- or fatty acids-grown granules. The fatty acids-grown granules contained a considerable amount of inorganic salts (ash content: 56 to 63%) but a small amount of EPS, and showed a denser ultrastructure than the other three types of granules. The granules grown on ethanol under slightly acidic conditions showed the lowest specific gravity and volatile suspended solids (VSS) density as well as ash content among all of the granules. As aceticlastic methanogens, Methanothrix spp. were predominant in the starch-, sucrose-, and fatty acids-grown granules, whereas comparable numbers of Methanosarcina spp. were observed only in the ethanol-grown granules. The populations of hydrogenotrophic methanogens were the largest of all bacterial trophic groups in the respective granules. The data confirm that the prevalence of Methanothrix spp. and high methanogenic activity for H 2 are general characteristics of methanogenic granucles and that EPS and inorganic deposits contribute chemically to the enhancement of structural stability and mechanical strength of granules.

Butyrate oxidation by Syntrophospora bryantii in co-culture with different methanogens and in pure culture with pentenoate as electron acceptor

Syntrophospora bryantii degraded butyrate in co-culture with methanogens that can use both H2 and formate for growth, but not in co-culture with methanogens that metabolize only H2, suggesting that in suspended cultures formate may be a more important electron carrier in the syntrophic degradation of butyrate than H2. Syntrophic butyrate oxidation was inhibited by the addition of 20 mm formate or the presence of 130 kPa H2. In the absence of methanogens, S. bryantii is able to couple the oxidation of butyrate to acetate with the reduction of pentenoate to valerate. Under these conditions, up to 300 Pa H2 was measured in the gas phase and up to 0.3 mm formate in the liquid phase. S. bryantii was unable to grow syntrophically with the aceticlastic methanogen Methanothrix soehngenii. However in triculture with Methanospirillum hungatei and Methanothrix soehngenii, S. bryantii degraded butyrate faster than in a biculture with only M. hungatei. Hydrogenase and formate dehydrogenase activities were demonstrated in cell-free extracts of S. bryantii.

Fermentative degradation of acetone by an enrichment culture in membrane-separated culture devices and in cell suspensions.

A mixed culture, WoAct, growing on acetone, consisted of two dominant morphotypes: a rod-shaped acetone-fermenting bacterium producing acetate, and an acetate-utilizing Methanosaeta species. Dense cell suspensions, largely free of the aceticlastic methanogen and supplemented with bromoethanesulfonate, were able to degrade acetone and grow in small volumes in membrane-separated culture devices in which the acetate produced could diffuse into a large volume of medium. Acetone degradation and growth halted when the acetate concentration reached about 10 to 12 mM. Cell suspensions were able to degrade acetone in the absence of active methanogenesis, but the addition of 10 mM acetate inhibited acetone metabolism. Addition of an active culture of Methanosaeta sp. greatly stimulated the rate of acetone degradation. The results show that acetate removal in the mixed culture is not a prerequisite for growth and acetone degradation by the acetone-fermenting bacterium.

Microbial colonization of different support materials used to enhance the methanogenic process

Macrobial colonization of the different support materials used to enhance methane production in anaerobic digestors is rapid and occurs in the first 24 h of sludge incubation. Scanning electron microscopy studies reveal a predominant presence of filamentous methanogenic forms, closely resemblingMethanosaeta (Methanothrix), which are located on the outer layer and in the bacterial framework of the biofilm. These findings are consistent with the results obtained from microbial counts using both the most probable number and epifluorescence microscopic techniques, which show an increase in the numbers of aceticlastic methanogens compared to other microbial groups involved, such as sulphate-reducing bacteria, the numbers of which are similar to those obtained under the initial conditions. Moreover, a sharp increase in the bacterial counts is observed by using the epifluorescence microscopic technique applied to homogenized samples, probably due to the count of bacteria released from the support materials.

A sensitive method for quantification of aceticlastic methanogens and estimation of total methanogenic cells in natural environments based on an analysis of ether-linked glycerolipids

A highly sensitive method for the quantification of methanogens in anaerobic digestor sludges was developed, based on an analysis of ether-linked glycerolipids. Core lipids were prepared from total lipids by HF treatment and mild methanolysis, and these core lipids were quantified as the corresponding 9-anthroyl derivatives by high-performance liquid chromatography with fluorescence detection. The amounts, in terms of cell carbon content, of Methanosaeta and Methanosarcina were proportional to the amounts of α-hydroxyarchaeol and β-hydroxyarchaeol, respectively. Moreover, the total amount of core lipids was well correlated with the cell mass of aceticlastic and H2/CO2-consuming methanogens. The limit of detection for Methanosaeta concilii was 17 ng of cell carbon when the signal/noise ratio was 3. This method allowed us to quantitate aceticlastic methanogens with high accuracy and to make a rough estimate of total methanogenic cells without any interference by the multifarious impurities that are present in anaerobic sludges. These results suggest that the present method will be a useful tool for investigations of methanogenic ecosystems.

A sensitive method for quantification of aceticlastic methanogens and estimation of total methanogenic cells in natural environments based on an analysis of ether-linked glycerolipids

A highly sensitive method for the quantification of methanogens in anaerobic digestor sludges was developed, based on an analysis of ether-linked glycerolipids. Core lipids were prepared from total lipids by HF treatment and mild methanolysis, and these core lipids were quantified as the corresponding 9-anthroyl derivatives by high-performance liquid chromatography with fluorescence detection. The amounts, in terms of cell carbon content, of Methanosaeta and Methanosarcina were proportional to the amounts of α-hydroxyarchaeol and β-hydroxyarchaeol, respectively. Moreover, the total amount of core lipids was well correlated with the cell mass of aceticlastic and H2/CO2-consuming methanogens. The limit of detection for Methanosaeta concilii was 17 ng of cell carbon when the signal/noise ratio was 3. This method allowed us to quantitate aceticlastic methanogens with high accuracy and to make a rough estimate of total methanogenic cells without any interference by the multifarious impurities that are present in anaerobic sludges. These results suggest that the present method will be a useful tool for investigations of methanogenic ecosystems.

Thermophilic anaerobic digestion of livestock waste: the effect of ammonia

Ammonia concentrations of 4 g N/l or more inhibited thermophilic digestion of cattle manure. A stable digestion of cattle manure could be maintained with ammonia concentrations up to 6 g N/l after 6 months of operation. However, the methane yield was reduced and the concentration of volatile fatty acids increased from 1 to 3 g/l as acetate, compared to controls with an ammonia concentration of 2.5 g N/l. The temporary strong inhibition following an one-step increase in ammonia concentration was reduced by applying a gradual increase. The specific methanogenic activity of ammonia-inhibited reactors (6 g N/l) with acetate or hydrogen as substrate was reduced by 73 and 52%, respectively. Tests of ammonia toxicity on the acetate- and hydrogen-utilizing populations showed a higher sensitivity of the aceticlastic compared to the hydrogenotrophic methanogens; the specific growth rate for the aceticlastic methanogens was halved at ammonia concentrations of 3.5 g N/l, compared to 7 g N/l for the hydrogenotrophic methanogens.

Comparison of acetate utilization among strains of an aceticlastic methanogen, Methanothrix soehngenii.

The kinetics of acetate utilization by concentrated suspensions of cells was examined in five strains of Methanothrix soehngenii. The rate of acetate utilization by all strains was dependent on the initial acetate concentration and followed simple Michaelis-Menten kinetics. The ability to utilize acetate differed among the various strains of M. soehngenii and was highest in the strain designated MTAS.

Enrichment of Thermophilic Propionate-Oxidizing Bacteria in Syntrophy with Methanobacterium thermoautotrophicum or Methanobacterium thermoformicicum

Thermophilic propionate-oxidizing, proton-reducing bacteria were enriched from the granular methanogenic sludge of a bench-scale upflow anaerobic sludge bed reactor operated at 55 degrees C with a mixture of volatile fatty acids as feed. Thermophilic hydrogenotrophic methanogens had a high decay rate. Therefore, stable, thermophilic propionate-oxidizing cultures could not be obtained by using the usual enrichment procedures. Stable and reproducible cultivation was possible by enrichment in hydrogen-pregrown cultures of Methanobacterium thermoautotrophicum DeltaH which were embedded in precipitates of FeS, achieved by addition of FeCl(2) to the media. The propionate-oxidizing bacteria formed spores which resisted pasteurization for 30 min at 90 degrees C or 10 min at 100 degrees C. Highly purified cultures were obtained with either M. thermoautotrophicum DeltaH or Methanobacterium thermoformicicum Z245 as the syntrophic partner organism. The optimum temperature for the two cultures was 55 degrees C. Maximum specific growth rates of cultures with M. thermoautotrophicum DeltaH were somewhat lower than those of cultures with M. thermoformicicum Z245 (0.15 and 0.19 day, respectively). Growth rates were even higher (0.32 day) when aceticlastic methanogens were present as well. M. thermoautotrophicum DeltaH is an obligately hydrogen-utilizing methanogen, showing that interspecies hydrogen transfer is the mechanism by which reducing equivalents are channelled from the acetogens to this methanogen. Boundaries of hydrogen partial pressures at which propionate oxidation occurred were between 6 and 34 Pa. Formate had a strong inhibitory effect on propionate oxidation in cultures with M. thermoautotrophicum. Inhibition by formate was neutralized by addition of the formate-utilizing methanogen or by addition of fumarate. Results indicate that formate inhibited succinate oxidation to fumarate, an intermediate step in the biochemical pathway of propionate oxidation.