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.     

Effect of H2-CO2 on Methanogenesis from Acetate or Methanol in Methanosarcina spp

Growth of Methanosarcina sp. strain 227 and Methanosarcina mazei on H₂-CO₂ and mixtures of H₂-CO₂ and acetate or methanol was examined. The growth yield of strain 227 on H₂-CO₂ 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 CO₂ was rapidly reduced to CH₄ in the presence of H₂, and little acetate was used for methanogenesis until H₂ 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 H₂-CO₂ and methanol, and less methanol oxidation occurred in the presence of H₂. In M. mazei the aceticlastic reaction was also inhibited by the added H₂, but since the cultures did not immediately metabolize H₂, the duration of the inhibition was much longer.     

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).     

Kinetic study and mathematical modeling of methanogenesis of acetate using pure cultures of methanogens

Kinetics of methanogenesis from acetate was studied using pure cultures of Methanosarcina barkeri and Methanosarcina mazei. Methane formation was found to be associated with cell growth. Nearly equimolar methane was produced from acetate during the methanogenic growth, and about 1.94 g of cells were formed from each mole of acetate consumed. Cell growth can be estimated from methane production. Significant substrate inhibition was found when acetate concentration was higher than 0.12 M. Among the three methanogenic strains studied, M. mazei strain S6 had the highest specific growth rate at all acetate concentrations studied and was least sensitive to environmental factors investigated (e.g., acetate concentration). The maximum specific growth rate found for strain S6 was 0.022 hr(-1) at acetic acid concentration around 7 g/L. The other two strains studied were M. barkeri strain 227 and strain MS. Growth of M. barkeri was completely inhibited at sodium acetate concentrations higher than 0.24 M. The maximum specific growth rate found for strains 227 and MS was 0.019 and 0.021 h(-1) at acetic acid concentrations of 3.6 and 6.8 g/L, respectively. A kinetic model with substrate inhibition was developed and can be used to simulate the methane formation from M. mazei strain S6 grown on acetate at 35 degrees C, pH 7.     

Influence of corrinoid antagonists on methanogen metabolism.

Iodopropane inhibited cell growth and methane production when Methanobacterium thermoautotrophicum, Methanobacterium formicicum, and Methanosarcina barkeri were cultured on H2-CO2. Iodopropane (40 microM) inhibited methanogenesis (30%) and growth (80%) when M. barkeri was cultured mixotrophically on H2-CO2-methanol. The addition of acetate to the medium prevented the observed iodopropane-dependent inhibition of growth. The concentrations of iodopropane that caused 50% inhibition of growth of M. barkeri on either H2-CO2, H2-CO2-methanol, methanol, and acetate were 112 +/- 6, 24 +/- 2, 63 +/- 11, and 4 +/- 1 microM, respectively. Acetate prevented the iodopropane-dependent inhibition of one-carbon metabolism. Cultivation of M. barkeri on H2-CO2-methanol in bright light also inhibited growth and methanogenesis to a greater extent in the absence than in the presence of acetate in the medium. Acetate was the only organic compound examined that prevented iodopropane-dependent inhibition of one-carbon metabolism in M. barkeri. The effect of iodopropane and acetate on the metabolic fates of methanol and carbon dioxide was determined with 14C tracers when M. barkeri was grown mixotrophically on H2-CO2-methanol. The addition of iodopropane decreased the contribution of methanol to methane and cell carbon while increasing the contribution of CO2 to cell carbon. Regardless of iodopropane, acetate addition decreased the contribution of methanol and CO2 to cell carbon without decreasing their contribution to methane. The corrinoid antagonists, light and iodopropane, appeared most specific for methanogen metabolic reactions involved in acetate synthesis from one-carbon compounds and acetate catabolism.     

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.     

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H(2) on Acetate Degradation

When grown in the absence of added sulfate, cocultures of Desulfovibrio desulfuricans or Desulfovibrio vulgaris with Methanobrevibacter smithii (Methanobacterium ruminantium), which uses H(2) and CO(2) for methanogenesis, degraded lactate, with the production of acetate and CH(4). When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H(2)-CO(2) and acetate for methanogenesis, lactate was stoichiometrically degraded to CH(4) and presumably to CO(2). During the first 12 days of incubation of the D. desulfuricans-M. barkeri coculture, lactate was completely degraded, with almost stoichiometric production of acetate and CH(4). Later, acetate was degraded to CH(4) and presumably to CO(2). In experiments in which 20 mM acetate and 0 to 20 mM lactate were added to D. desulfuricans-M. barkeri cocultures, no detectable degradation of acetate occurred until the lactate was catabolized. The ultimate rate of acetate utilization for methanogenesis was greater for those cocultures receiving the highest levels of lactate. A small amount of H(2) was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H(2), but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH(4) from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H(2)-CO(2) as the methanogenic substrate produced CH(4) from acetate at a rate equivalent to that observed for acetate-grown inocula when grown in a rumen fluid-vitamin-based medium but not when grown in a yeast extract-based medium. The results suggest that H(2) produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.     

Sodium dependence of acetate formation by the acetogenic bacterium Acetobacterium woodii.

Growth of Acetobacterium woodii on fructose was stimulated by Na+; this stimulation was paralleled by a shift of the acetate-fructose ratio from 2.1 to 2.7. Growth on H2-CO2 or on methanol plus CO2 was strictly dependent on the presence of sodium ions in the medium. Acetate formation from formaldehyde plus H2-CO by resting cells required Na+, but from methanol plus H2-CO did not. This is analogous to H2-CO2 reduction to methane by Methanosarcina barkeri, which involves a sodium pump (V. Müller, C. Winner, and G. Gottschalk, Eur. J. Biochem. 178:519-525, 1988). This suggests that the reduction of methylenetetrahydrofolate to methyltetrahydrofolate is the Na+-requiring reaction. A sodium gradient (Na+ out/Na+ in = 32, delta pNa = -91 mV) was built up when resting cells of A. woodii were incubated under H2-CO2. Acetogenesis was inhibited when the delta pNa was dissipated by monensin.     

Isolation and characterization of an h(2)-oxidizing thermophilic methanogen

A thermophilic methanogen was isolated from enrichment cultures originally inoculated with sludge from an anaerobic kelp digester (55 degrees C). This isolate exhibited a temperature optimum of 55 to 60 degrees C and a maximum near 70 degrees C. Growth occurred throughout the pH range of 5.5 to 9.0, with optimal growth near pH 7.2. Although 4% salt was present in the isolation medium, salt was not required for optimal growth. The thermophile utilized formate or H(2)-CO(2) but not acetate, methanol, or methylamines for growth and methanogenesis. Growth in complex medium was very rapid, and a minimum doubling time of 1.8 h was recorded in media supplemented with rumen fluid. Growth in defined media required the addition of acetate and an unknown factor(s) from digester supernatant, rumen fluid, or Trypticase. Cells in liquid culture were oval to coccoid, 0.7 to 1.8 mum in diameter, often occurring in pairs. The cells were easily lysed upon exposure to oxygen or 0.08 mg of sodium dodecyl sulfate per ml. The isolate was sensitive to tetracycline and chloramphenicol but not penicillin G or cycloserine. The DNA base composition was 59.69 mol% guanine plus cytosine.     

Production and Consumption of H(2) during Growth of Methanosarcina spp. on Acetate

Methanosarcina sp. strain TM-1 and Methanosarcina acetivorans produced and consumed H(2) to maintain H(2) partial pressures of 16 to 92 Pa in closed cultures during growth on acetate. Strain TM-1 produced H(2) continuously when H(2) was continuously removed from the culture. The potential physiological significance of H(2) in acetate metabolism to methane is discussed.     

Syntrophic Association by Cocultures of the Methanol- and CO(2)-H(2)-Utilizing Species Eubacterium limosum and Pectin-Fermenting Lachnospira multiparus During Growth in a Pectin Medium

Lachnospira multiparus grew very well in an anaerobic 0.2% pectin medium, whereas Eubacterium limosum, which utilizes methanol, H(2)-CO(2), and lactate, did not. Cocultures of the two species grew at a somewhat more rapid growth rate than did L. multiparus alone and almost doubled the amount of growth as measured by optical density. In model experiments with cultures transferred once a day with a 2-day retention time, L. multiparus produced mainly acetate, methanol, ethanol, formate, lactate, CO(2), and H(2) from pectin. The coculture produced one-third more acetate, and butyrate and CO(2) were the only other significant end products. The results are discussed in relationship to microbial metabolic interactions and interspecies hydrogen transfer.     

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.     

Adaptation of the acetogen Clostridium thermoautotrophicum to minimal medium.

Clostridium thermoautotrophicum was adapted to minimal medium and cultivated at the expense of glucose, methanol, or H2-CO2. No supplemental amino acids were required for growth of the adapted strain, and nicotinic acid was the sole essential vitamin. Neither N2 nor nitrate could replace ammonium as the nitrogen source, and biotin was preferentially stimulatory for glucose cell lines. Growth in minimal medium yielded substantially higher acetate concentrations per unit of biomass formed than did growth in undefined medium.     

Additional characteristics of one-carbon-compound utilization by Eubacterium limosum and Acetobacterium woodii.

Growth characteristics of Eubacterium limosum and Acetobacterium woodii during one-carbon-compound utilization were investigated. E. limosum RF grew with formate as the sole energy source. Formate also replaced a requirement for CO2 during growth with methanol. Growth with methanol required either rumen fluid, yeast extract, or acetate, but their effects were not additive. Cultures were adapted to grow in concentrations of methanol of up to 494 mM. Growth occurred with methanol in the presence of elevated levels of Na+ (576 mM). The pH optima for growth with methanol, H2-CO2, and carbon monoxide were similar (7.0 to 7.2). Growth occurred with glucose at a pH of 4.7, but not at 4.0. The apparent Km values for methanol and hydrogen were 2.7 and 0.34 mM, respectively. The apparent Vmax values for methanol and hydrogen were 1.7 and 0.11 mumol/mg of protein X min-1, respectively. The Ks value for CO was estimated to be less than 75 microM. Cellular growth yields were 70.5, 7.1, 3.38, and 0.84 g (dry weight) per mol utilized for glucose, methanol, CO, and hydrogen (in H2-CO2), respectively. E. limosum was also able to grow with methoxylated aromatic compounds as energy sources. Glucose apparently repressed the ability of E. limosum to use methanol, hydrogen, or isoleucine but not CO. Growth with mixtures of methanol, H2, CO, or isoleucine was not diauxic. The results, especially the relatively high apparent Km values for H2 and methanol, may indicate why E. limosum does not usually compete with rumen methanogens for these energy sources.(ABSTRACT TRUNCATED AT 250 WORDS)     

Additional characteristics of one-carbon-compound utilization by Eubacterium limosum and Acetobacterium woodii

Growth characteristics of Eubacterium limosum and Acetobacterium woodii during one-carbon-compound utilization were investigated. E. limosum RF grew with formate as the sole energy source. Formate also replaced a requirement for CO2 during growth with methanol. Growth with methanol required either rumen fluid, yeast extract, or acetate, but their effects were not additive. Cultures were adapted to grow in concentrations of methanol of up to 494 mM. Growth occurred with methanol in the presence of elevated levels of Na+ (576 mM). The pH optima for growth with methanol, H2-CO2, and carbon monoxide were similar (7.0 to 7.2). Growth occurred with glucose at a pH of 4.7, but not at 4.0. The apparent Km values for methanol and hydrogen were 2.7 and 0.34 mM, respectively. The apparent Vmax values for methanol and hydrogen were 1.7 and 0.11 mumol/mg of protein X min-1, respectively. The Ks value for CO was estimated to be less than 75 microM. Cellular growth yields were 70.5, 7.1, 3.38, and 0.84 g (dry weight) per mol utilized for glucose, methanol, CO, and hydrogen (in H2-CO2), respectively. E. limosum was also able to grow with methoxylated aromatic compounds as energy sources. Glucose apparently repressed the ability of E. limosum to use methanol, hydrogen, or isoleucine but not CO. Growth with mixtures of methanol, H2, CO, or isoleucine was not diauxic. The results, especially the relatively high apparent Km values for H2 and methanol, may indicate why E. limosum does not usually compete with rumen methanogens for these energy sources.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sulfate-Dependent Interspecies H(2) Transfer between Methanosarcina barkeri and Desulfovibrio vulgaris during Coculture Metabolism of Acetate or Methanol

We compared the metabolism of methanol and acetate when Methanosarcina barkeri was grown in the presence and absence of Desulfovibrio vulgaris. The sulfate reducer was not able to utilize methanol or acetate as the electron donor for energy metabolism in pure culture, but was able to grow in coculture. Pure cultures of M. barkeri produced up to 10 mumol of H(2) per liter in the culture headspace during growth on acetate or methanol. In coculture with D. vulgaris, the gaseous H(2) concentration was 相似文献   

Isolation and Characterization of a Thermophilic Bacterium Which Oxidizes Acetate in Syntrophic Association with a Methanogen and Which Grows Acetogenically on H(2)-CO(2)

We previously described a thermophilic (60 degrees C), syntrophic, two-membered culture which converted acetate to methane via a two-step mechanism in which acetate was oxidized to H(2) and CO(2). While the hydrogenotrophic methanogen Methanobacterium sp. strain THF in the biculture was readily isolated, we were unable to find a substrate that was suitable for isolation of the acetate-oxidizing member of the biculture. In this study, we found that the biculture grew on ethylene glycol, and an acetate-oxidizing, rod-shaped bacterium (AOR) was isolated from the biculture by dilution into medium containing ethylene glycol as the growth substrate. When the axenic culture of the AOR was recombined with a pure culture of Methanobacterium sp. strain THF, the reconstituted biculture grew on acetate and converted it to CH(4). The AOR used ethylene glycol, 1,2-propanediol, formate, pyruvate, glycine-betaine, and H(2)-CO(2) as growth substrates. Acetate was the major fermentation product detected from these substrates, except for 1,2-propanediol, which was converted to 1-propanol and propionate. N,N-Dimethylglycine was also formed from glycine-betaine. Acetate was formed in stoichiometric amounts during growth on H(2)-CO(2), demonstrating that the AOR is an acetogen. This reaction, which was carried out by the pure culture of the AOR in the presence of high partial pressures of H(2), was the reverse of the acetate oxidation reaction carried out by the AOR when hydrogen partial pressures were kept low by coculturing it with Methanobacterium sp. strain THF. The DNA base composition of the AOR was 47 mol% guanine plus cytosine, and no cytochromes were detected.     

Analysis of the anaerobic microbial community capable of degrading p-toluene sulphonate

Three strains of Clostridium sp., 14 (VKM B-2201), 42 (VKM B-2202), and 21 (VKM B-2279), two methanogens, Methanobacterium formicicum MH (VKM B-2198) and Methanosarcina mazei MM (VKM B-2199), and one sulfate-reducing bacterium, Desulfovibrio sp. SR1 (VKM B-2200), were isolated in pure cultures from an anaerobic microbial community capable of degrading p-toluene sulfonate. Strain 14 was able to degrade p-toluene sulfonate in the presence of yeast extract and bactotryptone and, like strain 42, to utilize p-toluene sulfonate as the sole sulfur source with the production of toluene. p-Toluene sulfonate stimulated the growth of Ms. mazei MM on acetate. The sulfate-reducing strain Desulfovibrio sp. SR1 utilized p-toluene sulfonate as an electron acceptor. The putative scheme of p-toluene sulfonate degradation by the anaerobic microbial community is discussed.     

Changes in concentrations of coenzyme F420 analogs during batch growth of Methanosarcina barkeri and Methanosarcina mazei

Coenzyme F420 has been assayed by high-performance liquid chromatography with fluorimetric detection; this permits quantification of individual coenzyme F420 analogs whilst avoiding the inclusion of interfering material. The total intracellular coenzyme F420 content of Methanosarcina barkeri MS cultivated on methanol and on H2-CO2 and of Methanosarcina mazei S-6 cultured on methanol remained relatively constant during batch growth. The most abundant analogs in M. barkeri were coenzymes F420-2 and F420-4, whilst in M. mazei coenzymes F420-2 and F420-3 predominated. Significant changes in the relative proportions of the coenzyme F420 analogs were noted during batch growth, with coenzymes F420-2 and F420-4 showing opposite responses to each other and the same being also true for coenzymes F420-3 and F420-5. This suggests that an enzyme responsible for transferring pairs of glutamic acid residues may be active. The degradation fragment FO was also detected in cells in late exponential and stationary phase. Coenzyme F420 analogs were present in the culture supernatant of both methanogens, in similar proportions to that in the cells, except for FO which was principally located in the supernatant.