Methanogenesis in the sediments of Rio Tinto, an extreme acidic river

Río Tinto (Iberian Pyritic Belt, SW Spain) is well known for its low pH (mean pH 2.3), high redox potential (> +400 mV) and high concentration of heavy metals. In this work we describe and analyse the presence of methanogenic archaea in the extreme acidic and oxidizing environment of the Tinto basin. Methane formation was measured in microcosms inoculated with sediments from the Rio Tinto basin. Methanol, formate, volatile fatty acids and lactate stimulated the production of methane. Methane formation was associated with a decrease of redox potential and an increase in pH. Cores showed characteristic well-defined black bands in which a high acetate concentration was measured among the otherwise reddish-brown sediments with low acetate concentration. Methanosaeta concilii was detected in the black bands. In enrichment cultures, M. concilii (enriched with a complex substrate mixture), Methanobacterium bryantii (enriched with H(2)) and Methanosarcina barkeri (enriched with methanol) were identified. Our results suggest that methanogens thrive in micro-niches with mildly acidic and reducing conditions within Rio Tinto sediments, which are, in contrast, immersed in an otherwise extremely acidic and oxidizing environment.     

Hydrogen production by methanogens under low-hydrogen conditions

Hydrogen production was studied in four species of methanogens (Methanothermobacter marburgensis, Methanosaeta thermophila, Methanosarcina barkeri, and Methanosaeta concilii) under conditions of low (sub-nanomolar) ambient hydrogen concentration using a specially designed culture apparatus. Transient hydrogen production was observed and quantified for each species studied. Methane was excluded as the electron source, as was all organic material added during growth of the cultures (acetate, yeast extract, peptone). Hydrogen production showed a strong temperature dependence, and production ceased at temperatures below the growth range of the organisms. Addition of polysulfides to the cultures greatly decreased hydrogen production. The addition of bromoethanesulfonic acid had little influence on hydrogen production. These experiments demonstrate that some methanogens produce excess reducing equivalents during growth and convert them to hydrogen when the ambient hydrogen concentration becomes low. The lack of sustained hydrogen production by the cultures in the presence of methane provides evidence against "reverse methanogenesis" as the mechanism for anaerobic methane oxidation.     

Methanogenesis in a Thermophilic (58 degrees C) Anaerobic Digestor: Methanothrix sp. as an Important Aceticlastic Methanogen

Aceticlastic methanogens and other microbial groups were enumerated in a 58 degrees C laboratory-scale (3 liter) anaerobic digestor which was fed air-classified municipal refuse, a lignocellulosic waste (loading rate = 1.8 to 2.7 g of volatile solids per liter per day; retention time = 10 days). Two weeks after start-up, Methanosarcina sp. was present in high numbers (10 to 10 CFU/ml) and autofluorescent Methanosarcina-like clumps were abundant in sludge examined by using epifluorescence microscopy. After about 4 months of digestor operation, numbers of Methanosarcina sp. dropped 2 to 3 orders of magnitude and large numbers (most probable number = 10 to 10/ml) of a thermophilic aceticlastic methanogen morphologically resembing Methanothrix sp. were found. Methanothrix sp. had apparently displaced Methanosarcina sp. as the dominant aceticlastic methanogen in the digestor. During the period when Methanothrix sp. was apparently dominant, acetate concentrations varied between 0.3 and 1.5 mumol/ml during the daily feeding cycle, and acetate was the precursor of 63 to 66% of the methane produced during peak digestor methanogenesis. The apparent K(m) value obtained for methanogenesis from acetate, 0.3 mumol/ml, indicated that the aceticlastic methanogens were nearly saturated for substrate during most of the digestor cycle. CO(2)-reducing methanogens were capable of methanogenesis at rates more than 12 times greater than those usually found in the digestor. Added propionate (4.5 mumol/ml) was metabolized slowly by the digestor populations and slightly inhibited methanogenesis. Added n-butyrate, isobutyrate, or n-valerate (4.5 mumol/ml each) were broken down within 24 h. Isobutyrate was oxidized to acetate, a novel reaction possibly involving isomerization to n-butyrate. The rapid growth rate and versatile metabolism of Methanosarcina sp. make it a likely organism to be involved in start-up, whereas the low K(m) value of Methanothrix sp. for acetate may cause it to be favored in stable digestors operated with long retention times.     

Solution (sup13)C Nuclear Magnetic Resonance Spectroscopic Analysis of the Amino Acids of Methanosphaera stadtmanae: Biosynthesis and Origin of One-Carbon Units from Acetate and Carbon Dioxide

We found that general pathways for amino acid synthesis of Methanosphaera stadtmanae, a methanogen that forms CH(inf4) from H(inf2) and methanol, resembled those of methanogens that form CH(inf4) from CO(inf2) or from the methyl group of acetate. We determined the incorporation of (sup14)C-labeled CO(inf2), formate, methanol, methionine, serine, and acetate into cell macromolecules. Labeling of amino acid carbons was determined by solution nuclear magnetic resonance spectroscopy after growth with (sup13)C-labeled acetate, CO(inf2), serine, and methanol. The (alpha) and (beta) carbons of serine and alanine were formed from carboxyl and methyl carbons of acetate, respectively, and the amino acid carboxyl groups were formed from CO(inf2). This indicates that pyruvate was formed by reductive carboxylation of acetate. Labeling of the methyl carbon of methionine indicated that the major route of synthesis was from the hydroxymethyl carbon of serine that arises from the methyl carbon of acetate. Methanol was a minor source of the methyl of methionine. Unambiguous assignment was made of the sources of all carbons of histidine. Labeling of the histidine 7 position ((epsilon) carbon) was consistent with formation from the C-2 of the purine ring of ATP and the origin of the C-2 from a formyl unit derived from the hydroxymethyl carbon of serine.     

Kinetics of the methanogenic fermentation of acetate

Inhibition of the fermentation of acetate to methane and carbon dioxide by acetate was analyzed with an acetate-acclimatized sludge and with Methanosarcina barkeri Fusaro under mesophilic conditions. A second-order substrate inhibition model, q(ch(4) ) = q(m)S/[K(s) + S + (S/K(i))], where S was the concentration of undissociated acetic acid, not ionized acetic acid, could be applicable in both cases. The analysis resulted in substrate saturation constants, K(s), of 4.0 muM for the acclimatized sludge and 104 muM for M. barkeri. The threshold concentrations of undissociated acetic acid when no further acetate utilization was observed were 0.078 muM (pH 7.50) for the acclimatized sludge and 4.43 muM (pH 7.45) for M. barkeri. These kinetic results suggested that the concentration of undissociated acetic acid became a key factor governing the actual threshold acetate concentration for acetate utilization and that the acclimatized sludge in which Methanothrix spp. appeared dominant could utilize acetate better and survive at a lower concentration of undissociated acetic acid than could M. barkeri.     

Immobilization patterns and dynamics of acetate-utilizing methanogens immobilized in sterile granular sludge in upflow anaerobic sludge blanket reactors

Sterile granular sludge was inoculated with either Methanosarcina mazeii S-6, Methanosaeta concilii GP-6, or both species in acetate-fed upflow anaerobic sludge blanket (UASB) reactors to investigate the immobilization patterns and dynamics of aceticlastic methanogens in granular sludge. After several months of reactor operation, the methanogens were immobilized, either separately or together. The fastest immobilization was observed in the reactor containing M. mazeii S-6. The highest effluent concentration of acetate was observed in the reactor with only M. mazeii S-6 immobilized, while the lowest effluent concentration of acetate was observed in the reactor where both types of methanogens were immobilized together. No changes were observed in the kinetic parameters (Ks and mumax) of immobilized M. concilii GP-6 or M. mazeii S-6 compared with suspended cultures, indicating that immobilization does not affect the growth kinetics of these methanogens. An enzyme-linked immunosorbent assay using polyclonal antibodies against either M. concilii GP-6 or M. mazeii S-6 showed significant variations in the two methanogenic populations in the different reactors. Polyclonal antibodies were further used to study the spatial distribution of the two methanogens. M. concilii GP-6 was immobilized only on existing support material without any specific pattern. M. mazeii S-6, however, showed a different immobilization pattern: large clumps were formed when the concentration of acetate was high, but where the acetate concentration was low this strain was immobilized on support material as single cells or small clumps. The data clearly show that the two aceticlastic methanogens immobilize differently in UASB systems, depending on the conditions found throughout the UASB reactor.     

Conservation of structure in the human gene encoding argininosuccinate synthetase and the argG genes of the archaebacteria Methanosarcina barkeri MS and Methanococcus vannielii.

The DNA sequences of the argG genes of Methanosarcina barkeri MS and Methanococcus vannielii were determined. The polypeptide products of these methanogen genes have amino acid sequences which are 50% identical to each other and 38% identical to the amino acid sequence encoded by the exons of the human argininosuccinate synthetase gene. Introns in the human chromosomal gene separate regions which encode amino acids conserved in both the archaebacterial and human gene products. An open reading frame immediately upstream of argG in Methanosarcina barkeri MS codes for an amino acid sequence which is 45 and 31% identical to the sequences of the large subunits of carbamyl phosphate synthetase in Escherichia coli and Saccharomyces cerevisiae, respectively. If this gene encodes carbamyl phosphate synthetase in Methanosarcina barkeri, this is the first example, in an archaebacterium, of physical linkage of genes that encode enzymes which catalyze reactions in the same amino acid biosynthetic pathway.     

Novel, acid-labile, hydroxydiether lipid cores in methanogenic bacteria

Polar ether lipids extracted from 15 methanogenic bacteria, representative of seven genera, were screened by nuclear magnetic resonance and thin layer chromatography for the presence of hydroxyl groups on the C20-phytanyl moieties. Major amounts of hydroxydiether core lipid were confirmed for Methanosaeta concilii and discovered in two Methanosarcina species, Methanococcus voltae, and tentatively in several Methanobacterium species. Signals at 1.24 and 1.8-1.9 ppm in 1H NMR spectra are characteristic of Methanosaeta concilii lipids hydroxylated on carbon-3 (sn-3 chain). Related signals, which were shifted slightly, appeared in spectra of the polar lipids extracted from both Methanosarcina species. Following mild hydrolysis to remove the polar head groups, only two chromatographically distinct core lipids were found in significant amounts in Methanosarcina barkeri (and Methanosarcina mazei) consisting of 43% 2,3-di-O-phytanyl-sn-glycerol (C20,20-diether) and 57% C20,20-hydroxydiether. This latter core lipid differed from the hydroxydiether from M. concilii by hydroxylation, on carbon-3, of the phytanyl chain in ether linkage to the sn-2 carbon of glycerol. The structural assignment was based on identification of the novel hydroxydiether core and its methylation products by 1H NMR, 13C NMR, and mass spectroscopy. The hydroxy core lipid degraded to various products during standard methanolic HCl and sulfuric acid procedures, including a methoxy derivative (methanolic HCl) and the 3-mono-O-phytanyl-sn-glycerol.     

Characterization of a heme-dependent catalase from Methanobrevibacter arboriphilus.

Recently it was reported that methanogens of the genus Methanobrevibacter exhibit catalase activity. This was surprising, since Methanobrevibacter species belong to the order Methanobacteriales, which are known not to contain cytochromes and to lack the ability to synthesize heme. We report here that Methanobrevibacter arboriphilus strains AZ and DH1 contained catalase activity only when the growth medium was supplemented with hemin. The heme catalase was purified and characterized, and the encoding gene was cloned. The amino acid sequence of the catalase from the methanogens is most similar to that of Methanosarcina barkeri.     

Microbial populations associated with fixed- and floating-bed reactors during a two-stage anaerobic process

Microbial populations associated with methanogenic fixed- or floating-bed bioreactors used for anaerobic digestion of lignocellulosic waste were investigated. Fluorescent in situ hybridization (FISH) was used to characterize microorganisms in samples obtained from different heights in the reactors, which were operated in a semi-continuous manner (feeding and mixing once every 2 days). The FISH results showed that Methanosaeta concilii cells were most numerous at the bottom of both reactors. M. concilii cells were more abundant in the fixed-bed reactor (FXBR), which performed better than the floating-bed reactor (FLBR). Species of the Methanosarcina genera (mainly M. barkeri and M. mazei) were also observed in the FLBR but rarely in the FXBR. Methane production in each of the reactors ranged from 0.29 to 0.33 m3 CH(4)/kg COD(rem) (chemical oxygen demand removed). The removal of volatile fatty acids (VFA; 70-75 h) in the FXBR was more efficient than in the FLBR.     

Membrane ATPase from the aceticlastic methanogen Methanothrix thermophila.

A new isolate of the aceticlastic methanogen Methanothrix thermophila utilizes only acetate as the sole carbon and energy source for methanogenesis (Y. Kamagata and E. Mikami, Int. J. Syst. Bacteriol. 41:191-196, 1991). ATPase activity in its membrane was found, and ATP hydrolysis activity in the pH range of 5.5 to 8.0 in the presence of Mg2+ was observed. It had maximum activity at around 70 degrees C and was specifically stimulated up to sixfold by 50 mM NaHSO3. The proton ATPase inhibitor N,N'-dicyclohexylcarbodiimide inhibited the membrane ATPase activity, but azide, a potent inhibitor of F0F1 ATPase (H(+)-translocating ATPase of oxidative phosphorylation), did not. Since the enzyme was tightly bound to the membranes and could not be solubilized with dilute buffer containing EDTA, the nonionic detergent nonanoyl-N-methylglucamide (0.5%) was used to solubilize it from the membranes. The purified ATPase complex in the presence of the detergent was also sensitive to N,N'-dicyclohexylcarbodiimide, and other properties were almost the same as those in the membrane-associated form. The purified enzyme revealed at least five kinds of subunits on a sodium dodecyl sulfate-polyacrylamide gel, and their molecular masses were estimated to be 67, 52, 37, 28, and 22 kDa, respectively. The N-terminal amino acid sequences of the 67- and 52-kDa subunits had much higher similarity with those of the 64 (alpha)- and 50 (beta)-kDa subunits of the Methanosarcina barkeri ATPase and were also similar to those of the corresponding subunits of other archaeal ATPases. The alpha beta complex of the M. barkeri ATPase has ATP-hydrolyzing activity, suggesting that a catalytic part of the Methanothrix ATPase contains at least the 67- and 52-kDa subunits.     

The genome characteristics and predicted function of methyl-group oxidation pathway in the obligate aceticlastic methanogens, Methanosaeta spp

In this work, we report the complete genome sequence of an obligate aceticlastic methanogen, Methanosaeta harundinacea 6Ac. Genome comparison indicated that the three cultured Methanosaeta spp., M. thermophila, M. concilii and M. harundinacea 6Ac, each carry an entire suite of genes encoding the proteins involved in the methyl-group oxidation pathway, a pathway whose function is not well documented in the obligately aceticlastic methanogens. Phylogenetic analysis showed that the methyl-group oxidation-involving proteins, Fwd, Mtd, Mch, and Mer from Methanosaeta strains cluster with the methylotrophic methanogens, and were not closely related to those from the hydrogenotrophic methanogens. Quantitative PCR detected the expression of all genes for this pathway, albeit ten times lower than the genes for aceticlastic methanogenesis in strain 6Ac. Western blots also revealed the expression of fwd and mch, genes involved in methyl-group oxidation. Moreover, (13)C-labeling experiments suggested that the Methanosaeta strains might use the pathway as a methyl oxidation shunt during the aceticlastic metabolism. Because the mch mutants of Methanosarcina barkeri or M. acetivorans failed to grow on acetate, we suggest that Methanosaeta may use methyl-group oxidation pathway to generate reducing equivalents, possibly for biomass synthesis. An fpo operon, which encodes an electron transport complex for the reduction of CoM-CoB heterodisulfide, was found in the three genomes of the Methanosaeta strains. However, an incomplete protein complex lacking the FpoF subunit was predicted, as the gene for this protein was absent. Thus, F(420)H(2) was predicted not to serve as the electron donor. In addition, two gene clusters encoding the two types of heterodisulfide reductase (Hdr), hdrABC, and hdrED, respectively, were found in the three Methanosaeta genomes. Quantitative PCR determined that the expression of hdrED was about ten times higher than hdrABC, suggesting that hdrED plays a major role in aceticlastic methanogenesis.     

Acetogens and acetoclastic methanosarcinales govern methane formation in abandoned coal mines

In abandoned coal mines, methanogenic archaea are responsible for the production of substantial amounts of methane. The present study aimed to directly unravel the active methanogens mediating methane release as well as active bacteria potentially involved in the trophic network. Therefore, the stable-isotope-labeled precursors of methane, [(13)C]acetate and H(2)-(13)CO(2), were fed to liquid cultures from hard coal and mine timber from a coal mine in Germany. Guided by methane production rates, samples for DNA stable-isotope probing (SIP) with subsequent quantitative PCR and denaturing gradient gel electrophoretic (DGGE) analyses were taken over 6 months. Surprisingly, the formation of [(13)C]methane was linked to acetoclastic methanogenesis in both the [(13)C]acetate- and the H(2)-(13)CO(2)-amended cultures of coal and timber. H(2)-(13)CO(2) was used mainly by acetogens related to Pelobacter acetylenicus and Clostridium species. Active methanogens, closely affiliated with Methanosarcina barkeri, utilized the readily available acetate rather than the thermodynamically more favorable hydrogen. Thus, the methanogenic microbial community appears to be highly adapted to the low-H(2) conditions found in coal mines.     

Effects of Temperature on Methanogenesis in a Thermophilic (58 degrees C) Anaerobic Digestor

The short-term effects of temperature on methanogenesis from acetate or CO(2) in a thermophilic (58 degrees C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with C-labeled methane precursors (CH(3)COO or CO(2)). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60 degrees C and was completely inhibited at 65 degrees C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58 degrees C and did not grow or produce methane at 65 degrees C. An accidental shift of digestor temperature from 58 to 64 degrees C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from CH(3)COO was optimal at 65 degrees C and completely inhibited at 75 degrees C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70 degrees C. Methanogenesis from CO(2) in the sludge was optimal at 65 degrees C and still proceeded at 75 degrees C. A CO(2)-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75 degrees C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65 degrees C produced more methane than sludge incubated at 60 degrees C, and no acetate accumulated at 65 degrees C. Methanogenesis was severely inhibited in sludge incubated at 70 degrees C, but since neither acetate nor H(2) accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.     

Biosynthetic pathways in Methanospirillum hungatei as determined by 13C nuclear magnetic resonance

The main metabolic pathways in Methanospirillum hungatei GP1 were followed by using 13C nuclear magnetic resonance, with 13C-labeled acetate and CO2 as carbon sources. The labeling patterns found in carbohydrates, amino acids, lipids, and nucleosides were consistent with the formation of pyruvate from acetate and CO2 as the first step in biosynthesis. Carbohydrates are formed by the glucogenic pathway, and no scrambling of label was observed, indicating that the oxidative or reductive pentose phosphate pathways are not functioning at significant rates. The pathways for amino acid biosynthesis are the usual ones, with the exception of that for isoleucine. The tricarboxylic acid pathway is incomplete and operates in a reductive direction to form alpha-ketoglutarate. The phytanyl chains of lipids are synthesized from acetate via mevalonic acid.     

PCR-based DGGE fingerprinting and identification of methanogens detected in three different types of UASB granules

Methane is produced by various methanogenic bacteria present in upflow anaerobic sludge blanket (UASB) bioreactors. Methane can be used to predict and improve UASB bioreactor efficiency. The methanogen population in the granules can be influenced by the composition of the substrate. The aim of this study was to fingerprint and identify the methanogens present in three different types of UASB granules that had been used to treat winery, brewery and peach-lye canning effluents. This was done using polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis. The DGGE fingerprints obtained from the methanogen reference cultures of Methanosaeta concilii, Methanosaeta thermophila, Methanosarcina barkeri, Methanosarcina mazeii and Methanobacterium formicicum were compared to the DGGE profiles of the Archaea in the different granules. The positions of the DGGE bands that did not correspond well to the bands of the known species were sequenced and compared to sequences available on GenBank using the Blastn search option. The aligned DNA sequences were used to construct a phylogenetic tree. Based on the data obtained, a DGGE marker was constructed which was used to provide a quick method to identify the Archaeal members of the microbial consortium in UASB granules.     

Isolation of methanogens from Arabian sea sediments and their salt tolerance

Abstract Sea sediments in tropical regions have been less studied for methanogenesis and methanogens present therein. Three species of methanogens viz. Methanobacterium bryantii, Methanococcus voltae and Methanosarcina barkeri were isolated from Arabian sea sediments collected near the west coast of India. Maximum methane was formed by M. voltae at 3.0% (w/v) NaCl and other two methanogens at 0.06% (w/v) NaCl. M. bryantii and M. barkeri tolerated 2.5 and 3.0% (w/v) NaCl respectively due to which these methanogens must have survived in salt conditions of the sea sediments.     

Metabolic Pathways in Methanococcus jannaschii and Other Methanogenic Bacteria

Eleven strains of methanogenic bacteria were divided into two groups on the basis of the directionality (oxidative or reductive) of their citric acid pathways. These pathways were readily identified for most methanogens from the patterns of carbon atom labeling in glutamate, following growth in the presence of [2-¹³C]acetate. All used noncyclic pathways, but members of the family Methanosarcinaceae were the only methanogens found to use the oxidative direction. Methanococcus jannaschii failed to incorporate carbon from acetate despite transmembrane equilibration comparable to other weak acids. This organism was devoid of detectable activities of the acetate-incorporating enzymes acetyl coenzyme A synthetase, acetate kinase, and phosphotransacetylase. However, incorporation of [1-¹³C]-, [2-¹³C]-, or [3-¹³C]pyruvate during the growth of M. jannaschii was possible and resulted in labeling patterns indicative of a noncyclic citric acid pathway operating in the reductive direction to synthesize amino acids. Carbohydrates were labeled consistent with glucogenesis from pyruvate. Leucine, isoleucine, phenylalanine, lysine, formate, glycerol, and mevalonate were incorporated when supplied to the growth medium. Lysine was preferentially incorporated into the lipid fraction, suggesting a role as a phytanyl chain precursor.     

Hydrogen-using bacteria in a methanogenic acetate enrichment culture

A rcher , D.B. 1984. Hydrogen-using bacteria in a methanogenic acetate enrichment culture. Journal of Applied Bacteriology 56 , 125–129.
In a study of the anaerobic utilization of acetate, an enrichment culture of sewage sludge organisms was initiated with calcium acetate as the sole carbon and energy source. A mixed bacterial population became established from which 14 anaerobic species were isolated. Two of the isolates were methanogenic bacteria but only one of these, Methanosarcina barkeri , utilised acetate as an energy source in axenic culture. The other methanogenic isolate, a Methanobacterium sp., utilised H₂/CO₂ but not acetate. A third methanogen, which was morphologically identical to Methanothrix soehngenii , was detected in the enrichment but was not obtained in monoculture. 2-Bromoethanesulphonate, a specific inhibitor of methanogenesis. completely inhibited the enrichment at a concentration of 10 μmol/1. Addition of H₂ formate or methanol to the enrichment did not affect the rate of methanogenesis. An H₂-utilizing Desulfovibrio sp. was also isolated from the enrichment.     

Reduction of Fe(III) oxide by methanogens in the presence and absence of extracellular quinones

Five methanogens (Methanosarcina barkeri MS, Methanosphaera cuniculi 1R7, Methanobacterium palustre F, Methanococcus voltaei A3 and Methanolobus vulcani PL-12/M) were investigated for their ability to reduce Fe(III) oxide and the soluble quinone anthraquinone-2,6-disulphonate (AQDS). Two species (M. barkeri and M. voltaei) reduced significant amounts of Fe(III) oxide using hydrogen as the electron donor, and 0.1 mM AQDS greatly accelerated Fe(III) reduction by these organisms. Although Fe(III) appeared to inhibit growth and methanogenesis of some strains, hydrogen partial pressures under donor-limited conditions were much lower (<0.5 Pa) in the presence of Fe(III) than in normal media (1-10 Pa) for all species except for M. vulcani. These results demonstrate that electrons were transferred to Fe(III) by hydrogen-utilizing methanogens even when growth and methanogenesis were inhibited. All species except the obligate methylotroph M. vulcani were able to reduce AQDS when their growth substrates were present as electron donors, and rates were highest when organisms used hydrogen as the electron donor. Purified soil humic acids could also be reduced by the AQDS-reducing methanogens. The ability of methanogens to interact with extracellular quinones, humic acids and Fe(III) oxides raises the possibility that this functional group of organ-isms contributes to Fe(III) and humic acid reduction under certain conditions in the environment and provides an alternative explanation for the inhibition of methanogenesis in some Fe(III)-containing ecosystems.