Isolation and Characterization of a Thermophilic Strain of Methanosarcina Unable to Use H(2)-CO(2) for Methanogenesis

A thermophilic strain of Methanosarcina, designated Methanosarcina strain TM-1, was isolated from a laboratory-scale 55 degrees C anaerobic sludge digestor by the Hungate roll-tube technique. Penicillin and d-cycloserine, inhibitors of peptidoglycan synthesis, were used as selective agents to eliminate contaminating non-methanogens. Methanosarcina strain TM-1 had a temperature optimum for methanogenesis near 50 degrees C and grew at 55 degrees C but not at 60 degrees C. Substrates used for methanogenesis and growth by Methanosarcina strain TM-1 were acetate (12-h doubling time), methanol (7- to 10-h doubling time), methanol-acetate mixtures (5-h doubling time), methylamine, and trimethylamine. When radioactively labeled acetate was the sole methanogenic substrate added to the growth medium, it was predominantly split to methane and carbon dioxide. When methanol was also present in the medium, the metabolism of acetate shifted to its oxidation and incorporation into cell material. Electrons derived from acetate oxidation apparently were used to reduce methanol. H(2)-CO(2) was not used for growth and methanogenesis by Methanosarcina strain TM-1. When presented with both H(2)-CO(2) and methanol, Methanosarcina strain TM-1 was capable of limited hydrogen metabolism during growth on methanol, but hydrogen metabolism ceased once the methanol was depleted. Methanosarcina strain TM-1 required a growth factor (or growth factors) present in the supernatant of anaerobic digestor sludge. Growth factor requirements and the inability to use H(2)-CO(2) are characteristics not found in other described Methanosarcina strains. The high numbers of Methanosarcina-like clumps in sludges from thermophilic digestors and the fast generation times reported here for Methanosarcina TM-1 indicate that Methanosarcina may play an important role in thermophilic methanogenesis.     

Growth and methanogenesis by Methanosarcina strain 227 on acetate and methanol.

Methanosarcina strain 227 exhibited exponential growth on sodium acetate in the absence of added H(2). Under these conditions, rates of methanogenesis were limited by concentrations of acetate below 0.05 M. One mole of methane was formed per mole of acetate consumed. Additional evidence from radioactive labeling studies indicated that sufficient energy for growth was obtained by the decarboxylation of acetate. Diauxic growth and sequential methanogenesis from methanol followed by acetate occurred in the presence of mixtures of methanol and acetate. Detailed studies showed that methanol-grown cells did not metabolize acetate in the presence of methanol, although acetate-grown cells did metabolize methanol and acetate simultaneously before shifting to methanol. Acetate catabolism appeared to be regulated in response to the presence of better metabolizable substrates such as methanol or H(2)-CO(2) by a mechanism resembling catabolite repression. Inhibition of methanogenesis from acetate by 2-bromoethanesulfonate, an analog of coenzyme M, was reversed by addition of coenzyme M. Labeling studies also showed that methanol may lie on the acetate pathway. These results suggested that methanogenesis from acetate, methanol, and H(2)-CO(2) may have some steps in common, as originally proposed by Barker. Studies with various inhibitors, together with molar growth yield data, suggest a role for electron transport mechanisms in energy metabolism during methanogenesis from methanol, acetate, and H(2)-CO(2).     

Nickel requirement and factor F430 content of methanogenic bacteria.

Methanobacterium thermoautotrophicum has been reported to require nickel for growth and to contain high concentrations of a nickel tetrapyrrole designated factor F430. In this communication it is shown that all methanogenic bacteria investigated incorporated nickel during growth and also synthesized factor F430. This was also true for Methanobrevibacter smithii, which is dependent on acetate as a carbon source, and for Methanosarcina barkeri growing on acetate or methanol as energy sources. Other bacteria, including Acetobacterium woodii and Clostridium thermoaceticum, contained no factor F430. It is further shown that two yellow nickel-containing degradation products were formed from factor F430 when heated at pH 7. This finding explains why several forms of factor F430 were found in methanogenic bacteria when a heat step was employed in the purification procedure.     

Effect of H(2)-CO(2) on Methanogenesis from Acetate or Methanol in Methanosarcina spp

Growth of Methanosarcina sp. strain 227 and Methanosarcina mazei on H(2)-CO(2) and mixtures of H(2)-CO(2) and acetate or methanol was examined. The growth yield of strain 227 on H(2)-CO(2) in complex medium was 8.4 mg/mmol of methane produced. Growth in defined medium was characteristically slower, and cell yields were proportionately lower. Labeling studies confirmed that CO(2) was rapidly reduced to CH(4) in the presence of H(2), and little acetate was used for methanogenesis until H(2) was exhausted. This resulted in a biphasic pattern of growth similar to that reported for strain 227 grown on methanol-acetate mixtures. Biphasic growth was not observed in cultures on mixtures of H(2)-CO(2) and methanol, and less methanol oxidation occurred in the presence of H(2). In M. mazei the aceticlastic reaction was also inhibited by the added H(2), but since the cultures did not immediately metabolize H(2), the duration of the inhibition was much longer.     

Control of the Life Cycle of Methanosarcina mazei S-6 by Manipulation of Growth Conditions

The morphology of Methanosarcina mazei was controlled by magnesium, calcium, and substrate concentrations and by inoculum size; these factors allowed manipulation of the morphology and interconversions between pseudosarcinal aggregates and individual, coccoid cells. M. mazei grew as aggregates in medium with a low concentration of catabolic substrate (either 50 mM acetate, 50 mM methanol, or 10 mM trimethylamine) unless Ca²⁺ and Mg²⁺ concentrations were high. Growth in medium high in Ca²⁺, Mg²⁺, and substrate (i.e., 150 mM acetate, 150 mM methanol, or 40 mM trimethylamine) converted pseudosarcinal aggregates to individual cocci. In such media, aggregates separated into individual cells which continued to grow exclusively as single cells during subsequent transfers. Conversion of single cells back to aggregates was complicated, because conditions which supported the aggregated morphology (e.g., low calcium or magnesium concentration) caused lysis of coccoid inocula. We recovered aggregates from coccoid cells by inoculating serial dilutions into medium high in calcium and magnesium. Cells from very dilute inocula grew into aggregates which disaggregated on continued incubation. However, timely transfer of the aggregates to medium low in calcium, magnesium, and catabolic substrates allowed continued growth as aggregates. We demonstrated the activity of the enzyme (disaggregatase) which caused the dispersion of aggregates into individual cells; disaggregatase was produced not only during disaggregation but also in growing cultures of single cells. Uronic acids, the monomeric constituents of the Methanosarcina matrix, were also produced during disaggregation and during growth as coccoids.     

Methanogenesis from acetate: a nonmethanogenic bacterium from an anaerobic acetate enrichment

A methanogenic acetate enrichment was initiated by inoculation of an acetate-mineral salts medium with domestic anaerobic digestor sludge and maintained by weekly transfer for 2 years. The enrichment culture contained a Methanosarcina and several obligately anaerobic nonmethanogenic bacteria. These latter organisms formed varying degrees of association with the Methanosarcina, ranging from the nutritionally fastidious gram-negative rod called the satellite bacterium to the nutritionally nonfastidious Eubacterium limosum. The satellite bacterium had growth requirements for amino acids, a peptide, a purine base, vitamin B12, and other B vitamins. Glucose, mannitol, starch, pyruvate, cysteine, lysine, leucine, isoleucine, arginine, and asparagine stimulated growth and hydrogen production. Acetate was neither incorporated nor metabolized by the satellite organism. Since acetate was the sole organic carbon source in the enrichment culture, organism(s) which metabolize acetate (such as the Methanosarcina) must produce substrates and growth factors for associated organisms which do not metabolize acetate.     

Methanogenesis from acetate: a nonmethanogenic bacterium from an anaerobic acetate enrichment.

A methanogenic acetate enrichment was initiated by inoculation of an acetate-mineral salts medium with domestic anaerobic digestor sludge and maintained by weekly transfer for 2 years. The enrichment culture contained a Methanosarcina and several obligately anaerobic nonmethanogenic bacteria. These latter organisms formed varying degrees of association with the Methanosarcina, ranging from the nutritionally fastidious gram-negative rod called the satellite bacterium to the nutritionally nonfastidious Eubacterium limosum. The satellite bacterium had growth requirements for amino acids, a peptide, a purine base, vitamin B12, and other B vitamins. Glucose, mannitol, starch, pyruvate, cysteine, lysine, leucine, isoleucine, arginine, and asparagine stimulated growth and hydrogen production. Acetate was neither incorporated nor metabolized by the satellite organism. Since acetate was the sole organic carbon source in the enrichment culture, organism(s) which metabolize acetate (such as the Methanosarcina) must produce substrates and growth factors for associated organisms which do not metabolize acetate.     

Growth substrate effects on acetate and methanol catabolism in Methanosarcina sp. strain TM-1.

When Methanosarcina sp. strain TM-1 is grown in medium in which both methanol and acetate are present, growth is biphasic, with methanol used as the primary catabolic substrate during the first phase. To better understand this phenomenon, we grew cells on methanol or on acetate or on both and examined the abilities of anaerobically washed cells to catabolize these substrates. Washed acetate-grown cells incubated with 10 mM acetate, 10 mM methanol, or both substrates together produced methane at initial rates of 325, 3, and 315 nmol min-1 mg of protein-1, respectively. Although the initial rate of methanogenesis from both substrates was nearly identical to the rate for acetate alone, after several hours of incubation the rate was greater for cells provided with both substrates. Studies with 14C-labeled methanol indicated that methanol was catabolized to methane at increasing rates by acetate-grown cells in a manner reminiscent of an induction curve, but only when cells were provided with acetate as a cosubstrate. Acetate was presumably providing energy and carbon for induction of methanol-catabolic enzymes. Methanol-grown cells showed a pattern of substrate utilization significantly different from that of acetate-grown cells, producing methane from 10 mM acetate, 10 mM methanol, or both substrates at initial rates of 10, 280, and 450 nmol min-1 mg of protein-1, respectively. There was significant oxidation of the methyl group of acetate during metabolism of both substrates. Cells grown on methanol-acetate and harvested before methanol depletion (methanol phase) showed catabolic patterns nearly identical to those of methanol-grown cells, including a low rate of methanogenesis from acetate. Cells harvested from methanol-acetate cultures in the acetate phase were capable of significant methanogenesis from either methanol or acetate alone, and the rate from both substrates together was nearly equal to the sum of the rates for the single substrates. When both 10 mM methanol and 10 mM acetate were presented to the acetate-phase cells, there was a preference for the methanol. These results are consistent with a model for regulation in Methanosarcina sp. strain TM-1 in which methanol represses acetate catabolism while methanol catabolism is inducible.     

Levels of coenzyme F420, coenzyme M, hydrogenase, and methylcoenzyme M methylreductase in acetate-grown Methanosarcina.

Methanosarcina barkeri strain 227 maintained on an acetate medium for 2 years was found to possess hydrogenase, methylcoenzyme M methylreductase, coenzyme F420, and coenzyme M. The levels of these constituents in acetate-grown cells were similar to those found in cells of the same strain grown on methanol or hydrogen and carbon dioxide.     

Levels of coenzyme F420, coenzyme M, hydrogenase, and methylcoenzyme M methylreductase in acetate-grown Methanosarcina

Methanosarcina barkeri strain 227 maintained on an acetate medium for 2 years was found to possess hydrogenase, methylcoenzyme M methylreductase, coenzyme F420, and coenzyme M. The levels of these constituents in acetate-grown cells were similar to those found in cells of the same strain grown on methanol or hydrogen and carbon dioxide.     

Disaggregation of Methanosarcina spp. and Growth as Single Cells at Elevated Osmolarity

The effect of medium osmolarity on the morphology and growth of Methanosarcina barkeri, Methanosarcina thermophila, Methanosarcina mazei, Methanosarcina vacuolata, and Methanosarcina acetivorans was examined. Each strain was adapted for growth in NaCl concentrations ranging from 0.05 to 1.0 M. Methanosarcina spp. isolated from both marine and nonmarine sources exhibited similar growth characteristics at all NaCl concentrations tested, demonstrating that these species are capable of adapting to a similar range of medium osmolarities. Concomitant with the adaptation in 0.4 to 1.0 M NaCl, all strains disaggregated and grew as single cells rather than in the characteristic multicellular aggregates. Aggregated cells had a methanochondroitin outer layer, while disaggregated single cells lacked the outer layer but retained the protein S-layer adjacent to the cell membrane. Synthesis of glucuronic acid, a major component of methanochondroitin, was reduced 20-fold in the single-cell form of M. barkeri when compared with synthesis in aggregated cells. Strains with the methanochondroitin outer cell layer exhibited enhanced stability at low (<0.2 M NaCl) osmolarity and grew at higher temperatures. Disaggregated cells could be converted back to aggregated cells by gradually readapting cultures to lower NaCl (<0.2 M) and Mg²⁺ (<0.005 M) concentrations. Disaggregated Methanosarcina spp. could also be colonized and replica plated with greater than 95% recovery rates on solidified agar basal medium that contained 0.4 to 0.6 M NaCl and either trimethylamine, methanol, or acetate as the substrate. The ability to disaggregate and grow Methanosarcina spp. as viable, detergent-sensitive, single cells on agar medium makes these species amenable to mutant selection and screening for genetic studies and enables cells to be gently lysed for the isolation of intact genetic material.     

Methanosarcina mutant unable to produce methane or assimilate carbon from acetate.

Mutants of Methanosarcina barkeri 227 resistant to monofluoroacetate were isolated from monofluoroacetate-treated cultures. Mutant strain FAr9 was 100 times more resistant to monofluoroacetate than the wild-type strain and was deficient in carbon uptake and CH4 and CO2 production from methyl-labeled acetate. Methanol was assimilated at increased levels. Strain FAr9 was unable to shift from using methanol to using acetate for growth and exhibited increased sensitivity to growth inhibition by NaCN in methanol-containing complex medium. Unlike parent strain 227, acetate addition to methanol-containing media did not prevent NaCN inhibition. The specific activities of enzymes of exogenous acetate assimilation, CO dehydrogenase, and enzymes of the tricarboxylic acid cycle were similar for mutant and parent strain cell extracts. Mutation to monofluoroacetate resistance did not confer simultaneous resistance to 2-bromoethanesulfonate or pyruvate or alter propionate uptake. We conclude that strain FAr9 is either an acetate permeability mutant or is defective in an activation step required for the catabolism and anabolism of acetate.     

Genetic, physiological and biochemical characterization of multiple methanol methyltransferase isozymes in Methanosarcina acetivorans C2A

Biochemical evidence suggests that methanol catabolism in Methanosarcina species requires the concerted effort of methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MtaB), a corrinoid-containing methyl-accepting protein (MtaC) and Co-methyl-5-hydroxybenzimidazolylcobamide:2-mercapto-ethanesulphonic acid methyltransferase (MtaA). Here we show that Methanosarcina acetivorans possesses three operons encoding putative methanol-specific MtaB and corrinoid proteins: mtaCB1, mtaCB2 and mtaCB3. Deletion mutants lacking the three operons, in all possible combinations, were constructed and characterized. Strains deleted for any two of the operons grew on methanol, whereas strains lacking all three did not. Therefore, each operon encodes a bona fide methanol-utilizing MtaB/corrinoid protein pair. Most of the mutants were similar to the wild-type strain, with the exception of the DeltamtaCB1 DeltamtaCB2 double mutant, which grew more slowly and had reduced cell yields on methanol medium. However, all mutants displayed significantly longer lag times when switching from growth on trimethylamine to growth on methanol. This indicates that all three operons are required for wild-type growth on methanol and suggests that each operon has a distinct role in the metabolism of this substrate. The combined methanol:CoM methyltransferase activity of strains carrying only mtaCB1 was twofold higher than strains carrying only mtaCB2 and fourfold higher than strains carrying only mtaCB3. Interestingly, the presence of the mtaCB2 and mtaCB3 operons, in addition to the mtaCB1 operon, did not increase the overall methyltransferase activity, suggesting that these strains may be limited by MtaA availability. All deletion mutants were unaffected with respect to growth on trimethylamine and acetate corroborating biochemical evidence indicating that each methanogenic substrate has specific methyltransfer enzymes.     

Features of rumen and sewage sludge strains of Eubacterium limosum, a methanol- and H2-CO2-utilizing species

Eubacterium limosum was isolated as the most numerous methanol-utilizing bacterium in the rumen fluid of sheep fed a diet in which molasses was a major component (mean most probable number of 6.3 X 10(8) viable cells per ml). It was also isolated from sewage sludge at 9.5 X 10(4) cells per ml. It was not detected in the rumen fluid of a steer on a normal hay-grain diet, although Methanosarcina, as expected, was found at 9.5 X 10(5) cells per ml. The doubling time of E. limosum in basal medium (5% rumen fluid) with methanol as the energy source (37 degree C) was 7 h. Acetate, cysteine, carbon dioxide, and the vitamins biotin, calcium-D-pantothenate, and lipoic acid were required for growth on a chemically defined methanol medium. Acetate, butyrate, and caproate were produced from methanol. Ammonia or each of several amino acids served as the main nitrogen source. Other energy sources included adonitol, arabitol, erythritol, fructose, glucose, isoleucine, lactate, mannitol, ribose, valine, and H2-CO2. The doubling time for growth on H2-CO2 (5% rumen fluid, 37 degree C) was 14 h as compared with 5.2 h for isoleucine and 3.5 h for glucose. The vitamin requirements for growth on H2-CO2 were the same as those for methanol; however, acetate was not required for growth on H2-CO2, although it was necessary for growth on valine, isoleucine, and lactate and was stimulatory to growth on glucose. Acetate and butyrate were formed during growth on H2-CO2, whereas branched-chain fatty acids and ammonia were fermentation products from the amino acids. Heat tolerance was detected, but spores were not observed. The type strain of E. limosum (ATCC 8486) and strain L34, which was isolated from the rumen of a young calf, grew on methanol, H2-CO2, valine, and isoleucine and showed the same requirements for acetate as the freshly isolated strains.     

Features of rumen and sewage sludge strains of Eubacterium limosum, a methanol- and H2-CO2-utilizing species.

Eubacterium limosum was isolated as the most numerous methanol-utilizing bacterium in the rumen fluid of sheep fed a diet in which molasses was a major component (mean most probable number of 6.3 X 10(8) viable cells per ml). It was also isolated from sewage sludge at 9.5 X 10(4) cells per ml. It was not detected in the rumen fluid of a steer on a normal hay-grain diet, although Methanosarcina, as expected, was found at 9.5 X 10(5) cells per ml. The doubling time of E. limosum in basal medium (5% rumen fluid) with methanol as the energy source (37 degree C) was 7 h. Acetate, cysteine, carbon dioxide, and the vitamins biotin, calcium-D-pantothenate, and lipoic acid were required for growth on a chemically defined methanol medium. Acetate, butyrate, and caproate were produced from methanol. Ammonia or each of several amino acids served as the main nitrogen source. Other energy sources included adonitol, arabitol, erythritol, fructose, glucose, isoleucine, lactate, mannitol, ribose, valine, and H2-CO2. The doubling time for growth on H2-CO2 (5% rumen fluid, 37 degree C) was 14 h as compared with 5.2 h for isoleucine and 3.5 h for glucose. The vitamin requirements for growth on H2-CO2 were the same as those for methanol; however, acetate was not required for growth on H2-CO2, although it was necessary for growth on valine, isoleucine, and lactate and was stimulatory to growth on glucose. Acetate and butyrate were formed during growth on H2-CO2, whereas branched-chain fatty acids and ammonia were fermentation products from the amino acids. Heat tolerance was detected, but spores were not observed. The type strain of E. limosum (ATCC 8486) and strain L34, which was isolated from the rumen of a young calf, grew on methanol, H2-CO2, valine, and isoleucine and showed the same requirements for acetate as the freshly isolated strains.     

Methanosarcina lacustris sp. nov., a new psychrotolerant methanogenic archaeon from anoxic lake sediments

A new psychrotolerant methanogenic archaeon strain ZS was isolated from anoxic lake sediments (Switzerland). The cells of the organism were non-motile cocci, 1.5-3.5 microm in diameter. The cells aggregated and formed pseudoparenchyma. The cell wall was Gram-positive. The organism utilized methanol, mono-, di-, trimethylamine and H2/CO2 with methane production. The temperature range for growth was 1-35 degrees C with an optimum at 25 degrees C. The DNA G+C content of the organism was 43.4. mol%. Analysis of the 16S rRNA gene sequence showed that strain ZS was phylogenetically closely related to members of the genus Methanosarcina, but clearly differed from all described species of this genus (95.6-97.6% of sequence similarity). The level of DNA-DNA hybridization of strain ZS with Methanosarcina barkeri and Methanosarcina mazei was 15 and 31%, respectively. Based on the results of physiological and phylogenetic studies strain ZS can be assigned to a new species of the genus Methanasarcina. The name Methanosarcina lacustris sp. nov. is proposed. The type strain is ZS (= DSM 13486T, VKM B-2268).     

Studies on an acetate-fermenting strain of Methanosarcina.

An acetate-fermenting strain of Methanosarcina was isolated from an acetate enrichment culture inoculated with anaerobic sludge from a waste treatment digestor. In pure culture, this organism fermented acetate in the absence of added hydrogen at rates comparable in magnitude to those found in digestor systems. This rate was significantly higher than previously obtained for pure cultures of this genus. Mineral components of yeast extract were highly stimulatory for cultures growing on methanol. Comparable stimulation was not observed for cultures growing on acetate. Labeling studies indicated that acetate was converted to methane and CO2 as predicted by previous studies on mixed cultures. Total oxidation or reduction of acetate was not the mechanism of conversion of acetate to methane by the pure culture. The ability of this strain to form colonies or to produce methane from acetate was apparently influenced by the choice of substrate and conditions used for growing the inoculum.     

Methanosarcina baltica,sp. nov., a novel methanogen isolated from the Gotland Deep of the Baltic Sea

A novel methanogen, Methanosarcina baltica GS1-AT, DSM 14042, JCM 11281, was isolated from sediment at a depth of 241 m in the Gotland Deep of the Baltic Sea. Cells were irregular, monopolar monotrichous flagellated cocci 1.5-3 microm in diameter often occurring in pairs or tetrads. The catabolic substrates used included methanol, methylated amines, and acetate, but not formate or H2/CO2. Growth was observed in a temperature range between 4 degrees and 27 degrees C with an optimum at 25 degrees C. The doubling time with methanol as substrate was 84 h at 25 degrees C, 120 h at 9 degrees C, and 167 h at 4 degrees C. The doubling time with acetate as substrate was 252 h at 25 degrees C and 425 h at 20 degrees C. After the transfer of methanol-grown cultures, long lag phases were observed that lasted 15-20 days at 25 degrees C and 25 days at 4 degrees -9 degrees C. The NaCl optimum for growth was 2%-4%, and the fastest growth occurred within a pH range of 6.5-7.5. Analysis of the 16S rDNA sequence revealed that the strain was phylogenetically related to Methanosarcina. The sequence similarity to described species of <95.7% and its physiological properties distinguished strain GS1-A(T) from all described species of the genus Methanosarcina.     

Methanosarcina acetivorans sp. nov., an Acetotrophic Methane-Producing Bacterium Isolated from Marine Sediments

A new acetotrophic marine methane-producing bacterium that was isolated from the methane-evolving sediments of a marine canyon is described. Exponential phase cultures grown with sodium acetate contained irregularly shaped cocci that aggregated in the early stationary phase and finally differentiated into communal cysts that released individual cocci when ruptured or transferred to fresh medium. The irregularly shaped cocci (1.9 ± 0.2 mm in diameter) were gram negative and occurred singly or in pairs. Cells were nonmotile, but possessed a single fimbria-like structure. Micrographs of thin sections showed a monolayered cell wall approximately 10 nm thick that consisted of protein subunits. The cells in aggregates were separated by visible septation. The communal cysts contained several single cocci encased in a common envelope. An amorphous form of the communal cyst that had incomplete septation and internal membrane-like vesicles was also present in late exponential phase cultures. Sodium acetate, methanol, methylamine, dimethylamine, and trimethylamine were substrates for growth and methanogenesis; H₂-CO₂ (80:20) and sodium formate were not. The optimal growth temperature was 35 to 40°C. The optimal pH range was 6.5 to 7.0. Both NaCl and Mg²⁺ were required for growth, with maximum growth rates at 0.2 M NaCl and 0.05 M MgSO₄. The DNA base composition was 41 ± 1% guanine plus cytosine. Methanosarcina acetivorans is the proposed species. C2A is the type strain (DSM 2834, ATCC 35395).     

Characterization of Methanosarcina mazei N2M9705 Isolated from an Aquaculture Fishpond

A new methanogenic isolate, designated as strain N2M9705 (=OCM 668), was isolated from an aquaculture fishpond near Wang-gong, Taiwan. This strain grew on trimethylamine and methanol, but it did not catabolize H₂-CO₂, acetate, or formate. The cells were stained Gram-negative, nonmotile, irregular coccus 0.6–0.8 μm in diameter. Gas vacuoles were observed and cell aggregated to form various sizes of granules. Cells grew optimally at 32°–37°C with 1% NaCl. The pH range of growth was 6.2–7.4, and higher pH inhibited the cell growth. The cells grew well in minimal medium, but growth was greatly stimulated by yeast extract and peptone. A comparison of 16S rDNA sequences of this organism phylogenetically related to Methanosarcina mazei. This is the first report of methyltrophic methanogenic isolated from an aquaculture fishpond. Received: 16 March 1999 / Accepted: 16 April 1999