Microbial characterization of thermophilicArchaea isolated from the Guaymas Basin hydrothermal vent

Extremely thermophilic bacteria were isolated from sediments collected at the Guaymas Basin hydrothermal vent located in the Gulf of California. One isolate, (FC89) is a hydrogenotrophic methanogen with an optimal growth temperature of 85°C; this isolate appears to be closely related to the previously describedMethanococcus jannaschii. Thermophilic isolates TY and TYS are heterotrophic, sulfur-reducing archaea that differ from other thermophilic heterotrophic strains in physiological and molecular properties. Both heterotrophic isolates fermented carbohydrates and proteinaceous substrates; acetate was the primary product of carbohydrate fermentation, whereas acetate and a mix of organic acids were primary products of proteinaceous substrate fermentation. A detailed microbiological characterization of the isolates and a profile of fermentable substrates and fermentation products are described.     

Bacterial sulfur reduction in hot vents

Abstract: Elemental sulfur can be reduced through different microbial processes, including catabolically significant sulfur respiration and reduction of sulfur in the course of fermentation. Both of these processes are found in thermophilic microorganisms inhabiting continental and submarine hot vents, where elemental sulfur is one of the most common sulfur species. Among extreme thermophiles, respresented mainly by Archaea, sulfur-respiring bacteria include hydrogen-utilizing lithoautotrophs and heterotrophs, oxidizing complex organic substrates. Some marine heterotrophic sulfur-reducing Archaea were found to ferment peptides and polysaccharides, using elemental sulfur as an electron sink and thus avoiding the formation of molecular hydrogen which is highly inhibiting. Moderately thermophilic communities contain eubacterial sulfur reducers capable of lithotrophic and heterotrophic growth. Total mineralization of organic matter is carried out by a complex microbial system consisting of fermentative heterotrophs, which use elemental sulfur as an electron sink, and sulfur-respiring bacteria of the genus Desulfurella , which oxidize other fermentation products, yielding only CO_f2 and H_f2S. The most remarkable thermophilic microbial community is the thermophilic cyanobacterial mat found in the Uzon caldera, Kamchatka, which contains elemental sulfur among the layers. Organic matter produced by the thermophilic Oscillatoria is completely and rapidly mineralized by means of sulfur reduction.     

Extremely Thermophilic Fermentative Archaebacteria of the Genus Desulfurococcus from Deep-Sea Hydrothermal Vents

Two strains of extremely thermophilic, anaerobic bacteria are described that are representative of isolates obtained from a variety of oceanic hydrothermal vent sites at depths from 2,000 to 3,700 m. The isolates were similar in their requirements for complex organic media, elemental sulfur, and seawater-range salinities (optimum, 2.1 to 2.4%); their high tolerance for sulfide (100 mM) and oxic conditions below growth-range temperatures (50 to 95°C); and their archaebacterial characteristics: absence of murein, presence of certain diand tetraethers, and response to specific antibiotics. The two strains (S and SY, respectively) differed slightly in their optimum growth temperatures (85 and 90°C, optimum pHs for growth (7.5 and 7.0), and DNA base compositions (52.01 and 52.42 G+C mol%). At their in situ pressure of about 250 atm (25,313 kPa), growth rates at 80 and 90°C were about 40% lower than those at 1 atm (101.29 kPa), and no growth occurred at 100 and 110°C, respectively, at either pressure. In yeast extract medium, only 2% of the organic carbon was used and appeared to stem largely from the proteinaceous constituents. According to physiological criteria, the isolates belong to the genus Desulfurococcus.     

Involvement of thermophilic archaea in the biocorrosion of oil pipelines

Two thermophilic archaea, strain PK and strain MG, were isolated from a culture enriched at 80°C from the inner surface material of a hot oil pipeline. Strain PK could ferment complex organic nitrogen sources (e.g. yeast extract, peptone, tryptone) and was able to reduce elemental sulfur (S°), Fe(3+) and Mn(4+) . Phylogenetic analysis revealed that the organism belonged to the order Thermococcales. Incubations of this strain with elemental iron (Fe°) resulted in the abiotic formation of ferrous iron and the accumulation of volatile fatty acids during yeast extract fermentation. The other isolate, strain MG, was a H(2) :CO(2) -utilizing methanogen, phylogenetically affiliated with the genus Methanothermobacter family. Co-cultures of the strains grew as aggregates that produced CH(4) without exogenous H(2) amendment. The co-culture produced the same suite but greater concentrations of fatty acids from yeast extract than did strain PK alone. Thus, the physiological characteristics of organisms both alone and in combination could conceivably contribute to pipeline corrosion. The Thermococcus strain PK could reduce elemental sulfur to sulfide, produce fatty acids and reduce ferric iron. The hydrogenotrophic methanogen strain MG enhanced fatty acid production by fermentative organisms but could not couple the dissolution Fe° with the consumption of water-derived H(2) like other methanogens.     

Growth and Phylogenetic Properties of Novel Bacteria Belonging to the Epsilon Subdivision of the Proteobacteria Enriched from Alvinella pompejana and Deep-Sea Hydrothermal Vents

Recent molecular characterizations of microbial communities from deep-sea hydrothermal sites indicate the predominance of bacteria belonging to the epsilon subdivision of Proteobacteria (epsilon Proteobacteria). Here, we report the first enrichments and characterizations of four epsilon Proteobacteria that are directly associated with Alvinella pompejana, a deep sea hydrothermal vent polychete, or with hydrothermal vent chimney samples. These novel bacteria were moderately thermophilic sulfur-reducing heterotrophs growing on formate as the energy and carbon source. In addition, two of them (Am-H and Ex-18.2) could grow on sulfur lithoautrotrophically using hydrogen as the electron donor. Optimal growth temperatures of the bacteria ranged from 41 to 45°C. Phylogenetic analysis of the small-subunit ribosomal gene of the two heterotrophic bacteria demonstrated 95% similarity to Sulfurospirillum arcachonense, an epsilon Proteobacteria isolated from an oxidized marine surface sediment. The autotrophic bacteria grouped within a deeply branching clade of the epsilon Proteobacteria, to date composed only of uncultured bacteria detected in a sample from a hydrothermal vent along the mid-Atlantic ridge. A molecular survey of various hydrothermal vent environments demonstrated the presence of two of these bacteria (Am-N and Am-H) in more than one geographic location and habitat. These results suggest that certain epsilon Proteobacteria likely fill important niches in the environmental habitats of deep-sea hydrothermal vents, where they contribute to overall carbon and sulfur cycling at moderate thermophilic temperatures.     

Growth and phylogenetic properties of novel bacteria belonging to the epsilon subdivision of the Proteobacteria enriched from Alvinella pompejana and deep-sea hydrothermal vents

Recent molecular characterizations of microbial communities from deep-sea hydrothermal sites indicate the predominance of bacteria belonging to the epsilon subdivision of Proteobacteria (epsilon Proteobacteria). Here, we report the first enrichments and characterizations of four epsilon Proteobacteria that are directly associated with Alvinella pompejana, a deep sea hydrothermal vent polychete, or with hydrothermal vent chimney samples. These novel bacteria were moderately thermophilic sulfur-reducing heterotrophs growing on formate as the energy and carbon source. In addition, two of them (Am-H and Ex-18.2) could grow on sulfur lithoautrotrophically using hydrogen as the electron donor. Optimal growth temperatures of the bacteria ranged from 41 to 45 degrees C. Phylogenetic analysis of the small-subunit ribosomal gene of the two heterotrophic bacteria demonstrated 95% similarity to Sulfurospirillum arcachonense, an epsilon Proteobacteria isolated from an oxidized marine surface sediment. The autotrophic bacteria grouped within a deeply branching clade of the epsilon Proteobacteria, to date composed only of uncultured bacteria detected in a sample from a hydrothermal vent along the mid-Atlantic ridge. A molecular survey of various hydrothermal vent environments demonstrated the presence of two of these bacteria (Am-N and Am-H) in more than one geographic location and habitat. These results suggest that certain epsilon Proteobacteria likely fill important niches in the environmental habitats of deep-sea hydrothermal vents, where they contribute to overall carbon and sulfur cycling at moderate thermophilic temperatures.     

Extremophiles,Thermophily section,species description <Emphasis Type="Italic">Thermococcus atlanticus </Emphasis>sp. nov., a hyperthermophilic Archaeon isolated from a deep-sea hydrothermal vent in the Mid-Atlantic Ridge

An extremely thermophilic archaeon, strain MA898, was isolated from a deep-sea hydrothermal vent on the Mid-Atlantic Ridge. This strain is a strictly anaerobic coccus of approximately 0.7-1.2 microm in diameter. Optimal temperature, pH, and NaCl concentration for growth are around 85 degrees C, pH 7, and 3%, respectively. Strain MA898 grows preferentially in the presence of elemental sulfur, polysulfur, cystine, or L-cysteine. The microorganism requires rich proteinaceous substrates. BHI-S medium supports rapid growth, with a final concentration of more than 1.2 x 10(9) cells ml(-1), but strain MA898 exhibits poor growth on 2216S medium (yeast/peptone) and poor growth on starch. Growth is inhibited by rifampicin and chloramphenicol at a concentration of 100 microg/ml. The DNA G+C content is 50 mol%. Sequencing of the 16S rRNA gene indicates that strain MA898 belongs to the Thermococcusgenus, and from DNA/DNA hybridization data it is proposed as a new species: Thermococcus atlanticus. The deposition numbers are CIP-107420T and DSM15226.     

Thiosulfate reduction and alanine production in glucose fermentation by members of the genus Coprothermobacter

Coprothermobacter platensis is an anaerobic, proteolytic, thermophilic bacterium, which is phylogenetically related to the genera Fervidobacterium and Thermotoga. The organism was found to reduce thiosulfate to sulfide during growth on carbohydrates and proteinaceous substrates. Growth on glucose was inhibited by hydrogen, but this inhibition was overcome by thiosulfate reduction, stirring, increasing the headspace volume and coculturing with a hydrogen-consuming methanogen. Alanine was detected during glucose fermentation, its formation was influenced by the hydrogen concentration in the gas phase suggesting an electron sink mechanism, as was previously reported for the phylogenetically related Thermotogales and the archaeal hyperthermophile Pyrococcus furiosus.     

Occurrence of Sulfolobus acidocaldarius,an extremely thermophilic acidophilic bacterium,in New Zealand hot springs

Several hot springs in the Rotorua-Taupo regions, North Island, New Zealand, were tested for the presence of extremely thermophilic acidophilic bacteria. In the majority of the springs, ranging in temperature from 43–96°C and in pH from 2.1–6.9, direct microscopic observations revealed the presence of both rod-shaped and spherical bacteria. Isolations were attempted at 70°C and pH 2.0 and 7.0, with either yeast extract for heterotrophic growth, or elemental sulfur as the sole source of energy for autotrophic growth. Eight of the samples produced grwoth at pH 2.0 with either yeast extract or sulfur, but none of the samples grew at pH 7.0. All the isolates obtained, resembled Sulfolobus acidocaldarius, a thermophilic acidophilic bacterium which has previously been reported from various regions in the Northern Hemisphere. Immunofluorescence examination of six of these isolates revealed varying degrees of cross reactions with two already characterized Sulfolobus isolates from the Yellowstone National Park, U.S.A. This paper is the first published record of Sulfolobus from the Southern Hemisphere.     

Isolation and phylogenetic diversity of members of previously uncultivated epsilon-Proteobacteria in deep-sea hydrothermal fields

We report the successful cultivation and partial characterization of novel members of epsilon-Proteobacteria, which have long been recognized solely as genetic signatures of small subunit ribosomal RNA genes (rDNA) from a variety of habitats occurring in deep-sea hydrothermal fields. A newly designed microhabitat designated 'in situ colonization system' was used for enrichment. Based on phylogenetic analysis of the rDNA of the isolates, most of these represent the first cultivated members harboring previously uncultivated phylotypes classified into the Uncultivated epsilon-Proteobacteria Groups A, B, F and G, as well as some novel members of Group D. Preliminary characterization of the isolates indicates that all are mesophilic or thermophilic chemolithoautotrophs using H(2) or reduced sulfur compounds (elemental sulfur or thiosulfate) as an electron donor and O(2), nitrate or elemental sulfur as an electron acceptor. The successful cultivation will enable the subsequent characterization of physiological properties and ecological impacts of a diversity of epsilon-Proteobacteria in the deep-sea hydrothermal environments.     

Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence of Methanobacterium thermoautotrophicum.

The fermentation of cellulose and cellobiose by Clostridium thermocellum monocultures and C. thermocellum/Methanobacterium thermoautotrophicum cocultures was studied. All cultures were grown under anaerobic conditions in batch culture at 60 degrees C. When grown on cellulose, the coculture exhibited a shorter lag before initiation and growth and celluloysis than did the monoculture. Cellulase activity appeared earlier in the coculture than in the monoculture; however, after growth had ceased, cellulase activity was greater in the monoculture. Monocultures produced primarily ethanol, acetic acid, H2 and CO2. Cocultures produced more H2 and acetic acid and less ethanol than did the monoculture. In the coculture, conversion of H2 to methane was usually complete, and most of the methane produced was derived from CO2 reduction rather than from acetate conversion. Agents of fermentation stoppage were found to be low pH and high concentrations of ethanol in the monoculture and low pH in the coculture. Fermentation of cellobiose was more rapid than that of cellulose. In cellobiose medium, the methanogen caused only slight changes in the fermentation balance of the Clostridium, and free H2 was produced.     

南大西洋深海热液区可培养硫氧化微生物多样性及其硫氧化特性

【目的】探索南大西洋热液环境中的硫氧化细菌多样性并研究其硫氧化特性。【方法】通过富集培养和分离纯化获得硫氧化细菌,利用变性梯度凝胶电泳(DGGE)分析富集菌群组成结构,采用离子色谱法对获得的硫氧化细菌硫氧化特性进行检测。【结果】从南大西洋深海环境样品中共分离到48株菌,分属于alpha-Proteobacteria(28株,58.3%)、Actinobacteria(11株,22.9%)和gama-Proteobacteria(9株,18.8%)共3个门,其中Thalassospira、Martelella和Microbacterium为优势属。DGGE结果表明深海热液环境样品中微生物多样性丰富且不同站位存在差异。硫氧化特性研究结果表明,约60%的分离菌株具有硫氧化能力,可以氧化S_2O_3~(2–)生成SO_4~(2–)。获得一株硫氧化能力较强的潜在新种L6M1-5,在实验条件下可高效氧化S_2O_3~(2–),最大氧化速率可达0.56 mmol/(L·h)。【结论】南大西洋深海热液环境中可培养硫氧化细菌多样性丰富,为研究热液环境中的硫循环过程提供了实验材料和理论参考;同时高效硫氧化菌的获得,为工业化含硫废水的处理提供了良好的菌种资源。     

Dissimilatory Oxidation and Reduction of Elemental Sulfur in Thermophilic Archaea

The oxidation and reduction of elemental sulfur and reduced inorganic sulfur species are some of the most important energy-yielding reactions for microorganisms living in volcanic hot springs, solfataras, and submarine hydrothermal vents, including both heterotrophic, mixotrophic, and chemolithoautotrophic, carbon dioxide-fixing species. Elemental sulfur is the electron donor in aerobic archaea like Acidianus and Sulfolobus. It is oxidized via sulfite and thiosulfate in a pathway involving both soluble and membrane-bound enzymes. This pathway was recently found to be coupled to the aerobic respiratory chain, eliciting a link between sulfur oxidation and oxygen reduction at the level of the respiratory heme copper oxidase. In contrast, elemental sulfur is the electron acceptor in a short electron transport chain consisting of a membrane-bound hydrogenase and a sulfur reductase in (facultatively) anaerobic chemolithotrophic archaea Acidianus and Pyrodictium species. It is also the electron acceptor in organoheterotrophic anaerobic species like Pyrococcus and Thermococcus, however, an electron transport chain has not been described as yet. The current knowledge on the composition and properties of the aerobic and anaerobic pathways of dissimilatory elemental sulfur metabolism in thermophilic archaea is summarized in this contribution.     

Analysis of lipids from a hydrothermal vent methanogen and associated vent sediment by supercritical fluid chromatography

Lipids from a thermophilic methanogen and the associated hydrothermal vent sediment (Guaymas Basin, Gulf of California) were analyzed by gas chrmotography-mass spectrometry (GC-MS) and supercritical fluid chromatography (SFC). The neutral lipids of the thermophilic methagonen consisted of straight chain alkanes (nC₂₂ to nC₃₆), with nC₂₄, nC₂₈, nC₃₂ and nC₃₆ predominating and C₂₅, C₃₀ and C₃₅-isoprenoids and hydroisoprenoids. The squalene (C₃₀) series was the most abundant (95.6%). The backbone structure of the novel C₃₅-isoprenouds was tentatively identified as 2,6,10,14,19,23,27-heptamethyloctacosane. Polar lipids of the thermophilic methanogen were analyzed by SFC and consisted fo diphytanyl glyceril diether (61.6%), macrocyclic glycerol diether (15.3%), dibiphytanyl diglycerol tetraether (11.8%) and an unidentified component (11.4%).Biomarker analysis of the Guaymas Basin sediment revealed the presence of small amounts of polyunsaturated C₃₀-isoprenoids, with a distribution similar to the C₃₀-isoprenoids of the thermophilic methanogen. In addition, the sediment contained ‘free’ diphytanyl glycerol diether as predominant ether lipid. Low levels of polar ether lipids, indicative of ‘active’ archaebacteria, were also detected. Results suggest that Guaymas Basin sediment recently contained active microbial populations with a lipid profile similar to the isolated thermophilic methanogen.     

Oxidation of inorganic sulfur compounds by obligatory organotrophic bacteria

New data obtained by the author and other researchers on two different groups of obligately heterotrophic bacteria capable of inorganic sulfur oxidation are reviewed. Among culturable marine and (halo)alkaliphilic heterotrophs oxidizing sulfur compounds (thiosulfate and, much less actively, elemental sulfur and sulfide) incompletely to tetrathionate, representatives of the gammaproteobacteria, especially from the Halomonas group, dominate. Some of denitrifying species from this group are able to carry out anaerobic oxidation of thiosulfate and sulfide using nitrogen oxides as electron acceptors. Despite the low energy output of the reaction of thiosulfate oxidation to tetrathionate, it can be utilized for ATP synthesis by some tetrathionate-producing heterotrophs; however, this potential is not always realized during their growth. Another group of marine and (halo)alkaliphilic heterotrophic bacteria capable of complete oxidation of sulfur compounds to sulfate mostly includes representatives of the alphaproteobacteria most closely related to nonsulfur purple bacteria. They can oxidize sulfide (polysulfide), thiosulfate, and elemental sulfur via sulfite to sulfate but neither produce nor oxidize tetrathionate. All of the investigated sulfate-forming heterotrophic bacteria belong to lithoheterotrophs, being able to gain additional energy from the oxidation of sulfur compounds during heterotrophic growth on organic substrates. Some doubtful cases of heterotrophic sulfur oxidation described in the literature are also discussed.     

Comparison of thermophilic methanogens from submarine hydrothermal vents

An extremely thermophilic methanogen was isolated from hydrothermal vent sediment (80°–120° C) collected from the Guaymas Basin, Gulf of California, at a depth of approximately 2000 m. The isolate was a characteristic member of the genus Methanococcus based on its coccoid morphology, ability to produce methane from CO₂ and H₂, and DNA base composition (31.4 mol% G+C); it is distinguished from previously described extremely thermophilic vent methanogens by its ability to grow and produce methane from formate and in the composition of membrane lipids. The temperature range for growth was 48°–94° C (optimum near 85° C); the pH optimum was 6.0. The isolate grew autotrophically but was stimulated by selenium and growth nutrients supplied by yeast extract and trypticase. Extracted polar lipids consisted primarily of diphytanyl glycerol diether (62%), macrocyclic glycerol diether (15.3%), and dibiphytanyl glycerol tetraether (11.8%). Neutral lipids were dominated by a series of C₃₀ isoprenoids; in addition, a novel series of C₃₅ isoprenoids were detected. The isolate appears to be a close relative of the previously described Methanococcus jannaschii, isolated from the East Pacific Rise hydrothermal vent system. From the frequency of isolation, it appears that extremely thermophilic methanococci are the predominant representatives of the methanogenic archaebacteria occurring at deep sea hydrothermal vents.     

Oxidation of Inorganic Sulfur Compounds by Obligately Organotrophic Bacteria

New data obtained by the author and other researchers on two different groups of obligately heterotrophic bacteria capable of inorganic sulfur oxidation are reviewed. Among culturable marine and (halo)alkaliphilic heterotrophs oxidizing sulfur compounds (thiosulfate and, much less actively, elemental sulfur and sulfide) incompletely to tetrathionate, representatives of the gammaproteobacteria, especially from the Halomonas group, dominate. Some denitrifying species from this group are able to carry out anaerobic oxidation of thiosulfate and sulfide using nitrogen oxides as electron acceptors. Despite the low energy output of the reaction of thiosulfate oxidation to tetrathionate, it can be utilized for ATP synthesis by some tetrathionate-producing heterotrophs; however, this potential is not always realized during their growth. Another group of marine and (halo)alkaliphilic heterotrophic bacteria capable of complete oxidation of sulfur compounds to sulfate mostly includes representatives of the alphaproteobacteria which are most closely related to nonsulfur purple bacteria. They can oxidize sulfide (polysulfide), thiosulfate, and elemental sulfur via sulfite to sulfate but neither produce nor oxidize tetrathionate. All of the investigated sulfate-forming heterotrophic bacteria belong to lithoheterotrophs, being able to gain additional energy from the oxidation of sulfur compounds during heterotrophic growth on organic substrates. Some doubtful cases of heterotrophic sulfur oxidation described in the literature are also discussed.     

Phylogenetic and metabolic diversity of Planctomycetes from anaerobic, sulfide- and sulfur-rich Zodletone Spring, Oklahoma

We investigated the phylogenetic diversity and metabolic capabilities of members of the phylum Planctomycetes in the anaerobic, sulfide-saturated sediments of a mesophilic spring (Zodletone Spring) in southwestern Oklahoma. Culture-independent analyses of 16S rRNA gene sequences generated using Planctomycetes-biased primer pairs suggested that an extremely diverse community of Planctomycetes is present at the spring. Although sequences that are phylogenetically affiliated with cultured heterotrophic Planctomycetes were identified, the majority of the sequences belonged to several globally distributed, as-yet-uncultured Planctomycetes lineages. Using complex organic media (aqueous extracts of the spring sediments and rumen fluid), we isolated two novel strains that belonged to the Pirellula-Rhodopirellula-Blastopirellula clade within the Planctomycetes. The two strains had identical 16S rRNA gene sequences, and their closest relatives were isolates from Kiel Fjord (Germany), Keauhou Beach (HI), a marine aquarium, and tissues of marine organisms (Aplysina sp. sponges and postlarvae of the giant tiger prawn Penaeus monodon). The closest recognized cultured relative of strain Zi62 was Blastopirellula marina (93.9% sequence similarity). Detailed characterization of strain Zi62 revealed its ability to reduce elemental sulfur to sulfide under anaerobic conditions, as well as its ability to produce acids from sugars; both characteristics may potentially allow strain Zi62 to survive and grow in the anaerobic, sulfide- and sulfur-rich environment at the spring source. Overall, this work indicates that anaerobic metabolic abilities are widely distributed among all major Planctomycetes lineages and suggests carbohydrate fermentation and sulfur reduction as possible mechanisms employed by heterotrophic Planctomycetes for growth and survival under anaerobic conditions.     

Continuous culture of Methanococcus jannaschii, an extremely thermophilic methanogen

Methanococcus jannaschii, an extremely thermophilic methanogen isolated from a deep-sea hydrothermal vent, was grown at 80 degrees C in continuous culture on a mineral salts medium gassed with H(2) and CO(2) at three different flow rates. The maximum specific growth rate was 0.56 h(-1), and the maximum specific methane productivity was 0.32 (mol g(-1) h(-1)). Uncoupling of growth and methane production was evidenced by an increase in teh non-growth-associated rate of methane formation, beta, with increasing gaseous input. The specific hydrogenase activity exhibited growth-assiciated behaviour at low growth rates, but showed no dependence on growth at higher growth rates. The growth dependence of hydrogenase activity is consistent with the pressure dependence of hydrogenase activity measured in previous experiments. In contrast, the specific protease activity was independent of the growth rate over the entire range of dilution rates studied. (c) 1994 John Wiley & Sons, Inc.     

Degradation of proteins and amino acids by Caloramator proteoclasticus in pure culture and in coculture with Methanobacterium thermoformicicum Z245

This study investigated the degradation of proteins and amino acids by Caloramator proteoclasticus, an anaerobic thermophilic (55 °C) fermentative bacterium isolated from an anaerobic bioreactor. Experiments were performed in the presence and absence of Methanobacterium thermoformicicum Z245, a methanogen that can use both hydrogen and formate for growth. Higher production rates and yields of the principal fermentation products from gelatin were observed in methanogenic coculture. The specific proteolytic activity in coculture tripled the value obtained in pure culture. C. proteoclasticus fermented glutamate to acetate, formate, hydrogen and alanine. In methanogenic coculture, a shift towards higher amounts of acetate and hydrogen with no alanine production was observed. Extracts of glutamate-grown cells possessed high activities of β-methylaspartase, a key enzyme of the mesaconate pathway leading to acetate. The presence of two enzymes (alanine-α-ketoglutarate aminotransferase and NADH-dependent alanine dehydrogenase) usually involved in the biosynthesis of alanine from pyruvate was also detected. The fermentation of amino acids known to be oxidatively deaminated (leucine and valine) was improved in the presence of both methanogenesis and glycine, a known electron acceptor in the Stickland reaction. Culture conditions seem to be very important in the way C. proteoclasticus disposes of reducing equivalents formed during the degradation of amino acids. Received: 29 March 1999 / Received revision: 2 July 1999 / Accepted: 1 August 1999