Metabolism of Formate in Methanobacterium formicicum

Methanobacterium formicicum strain JF-1 was cultured with formate as the sole energy source in a pH-stat fermentor. Growth was exponential, and both methane production and formate consumption were linear functions of the growth rate. Hydrogen was produced in only trace amounts, and the dissolved H₂ concentration of the culture medium was below 1 μM. The effect of temperature or pH on the rate of methane formation was studied with a single fermentor culture in mid-log phase that was grown with formate under standard conditions at 37°C and pH 7.6. Methane formation from formate occurred over the pH range from 6.5 to 8.6, with a maximum at pH 8.0. The maximum temperature of methanogenesis was 56°C. H₂ production increased at higher temperatures. Hydrogen and formate were consumed throughout growth when both were present in saturating concentrations. The molar growth yields were 1.2 ± 0.06 g (dry weight) per mol of formate and 4.8 ± 0.24 g (dry weight) per mol of methane. Characteristics were compared for cultures grown with either formate or H₂-CO₂ as the sole energy source at 37°C and pH 7.6; the molar growth yield for methane of formate cultures was 4.8 g (dry weight) per mol, and that of H₂-CO₂ cultures was 3.5 g (dry weight) per mol. Both formate and H₂-CO₂ cultures had low efficiencies of electron transport phosphorylation; formate-cultured cells had greater specific activities of coenzyme F₄₂₀ than did H₂-CO₂-grown cultures. Hydrogenase, formate dehydrogenase, chromophoric factor F₃₄₂, and low levels of formyltetrahydrofolate synthetase were present in cells cultured with either substrate. Methyl viologen-dependent formate dehydrogenase was found in the soluble fraction from broken cells.     

Isolation and Characterization of a Thermophilic Bacterium Which Oxidizes Acetate in Syntrophic Association with a Methanogen and Which Grows Acetogenically on H2-CO2

We previously described a thermophilic (60°C), syntrophic, two-membered culture which converted acetate to methane via a two-step mechanism in which acetate was oxidized to H₂ and CO₂. 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 CH₄. The AOR used ethylene glycol, 1,2-propanediol, formate, pyruvate, glycine-betaine, and H₂-CO₂ 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 H₂-CO₂, 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 H₂, 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.     

Diversity and Structure of the Methanogenic Community in Anoxic Rice Paddy Soil Microcosms as Examined by Cultivation and Direct 16S rRNA Gene Sequence Retrieval

A dual approach consisting of cultivation and molecular retrieval of partial archaeal 16S rRNA genes was carried out to characterize the diversity and structure of the methanogenic community inhabiting the anoxic bulk soil of flooded rice microcosms. The molecular approach identified four groups of known methanogens. Three environmental sequences clustered with Methanobacterium bryantii and Methanobacterium formicicum, six were closely related but not identical to those of strains of Methanosaeta concilii, two grouped with members of the genus Methanosarcina, and two were related to the methanogenic endosymbiont of Plagiopyla nasuta. The cultivation approach via most-probable-number counts with a subsample of the same soil as an inoculum yielded cell numbers of up to 10⁷ per g of dry soil for the H₂-CO₂-utilizing methanogens and of up to 10⁶ for the acetate-utilizing methanogens. Strain VeH52, isolated from the terminal positive dilution on H₂-CO₂, grouped within the phylogenetic radiation characterized by M. bryantii and M. formicicum and the environmental sequences of the Methanobacterium-like group. A consortium of two distinct methanogens grew in the terminal positive culture on acetate. These two organisms showed absolute 16S rRNA gene identities with environmental sequences of the novel Methanosaeta-like group and the Methanobacterium-like group. Methanosarcina spp. were identified only in the less-dilute levels of the same dilution series on acetate. These data correlate well with acetate concentrations of about 11 μM in the pore water of this rice paddy soil. These concentrations are too low for the growth of known Methanosarcina spp. but are at the acetate utilization threshold of Methanosaeta spp. Thus, our data indicated Methanosaeta spp. and Methanobacterium spp. to be the dominant methanogenic groups in the anoxic rice soil, whereas Methanosarcina spp. appeared to be less abundant.     

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.     

Sulfate-Dependent Interspecies H2 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 μmol of H₂ per liter in the culture headspace during growth on acetate or methanol. In coculture with D. vulgaris, the gaseous H₂ concentration was ≤2 μmol/liter. The fractions of ¹⁴CO₂ produced from [¹⁴C]methanol and 2-[¹⁴C]acetate increased from 0.26 and 0.16, respectively, in pure culture to 0.59 and 0.33, respectively, in coculture. Under these conditions, approximately 42% of the available electron equivalents derived from methanol or acetate were transferred and were utilized by D. vulgaris to reduce approximately 33 μmol of sulfate per 100 μmol of substrate consumed. As a direct consequence, methane formation in cocultures was two-thirds that observed in pure cultures. The addition of 5.0 mM sodium molybdate or exogenous H₂ decreased the effects of D. vulgaris on the metabolism of M. barkeri. An analysis of growth and carbon and electron flow patterns demonstrated that sulfate-dependent interspecies H₂ transfer from M. barkeri to D. vulgaris resulted in less methane production, increased CO₂ formation, and sulfide formation from substrates not directly utilized by the sulfate reducer as electron donors for energy metabolism and growth.     

Effect of Monensin on Growth and Methanogenesis of Methanobacterium formicicum

Monensin inhibited methanogenesis from formate but not from H₂-CO₂ by resting-cell suspensions of Methanobacterium formicicum. The antibiotic severely inhibited growth on formate. The lag phase of H₂-CO₂-grown cultures was prolonged by monensin, but these cultures recovered from the initial inhibition. The recovery did not result from the development of a monensin-resistant population or inactivation of the antibiotic.     

Isolation and Characterization of a Fast-Growing, Thermophilic Methanobacterium Species

A thermophilic, autotrophic methanogen (strain CB12, DSM 3664) was isolated from a mesophilic biogas digestor. This bacterium used H₂-CO₂ or formate as a substrate and grew as short rods, sometimes in pairs and in crooked filaments. Motility was not observed. Its optimum temperature (56°C) was lower than that of other thermophilic members of the genus Methanobacterium. The maximum observed specific growth rate was 0.564 h⁻¹ (74-min doubling time).     

Acetate Synthesis from H2 plus CO2 by Termite Gut Microbes

Gut microbiota from Reticulitermes flavipes termites catalyzed an H₂-dependent total synthesis of acetate from CO₂. Rates of H₂-CO₂ acetogenesis in vitro were 1.11 ± 0.37 μmol of acetate g (fresh weight)⁻¹ h⁻¹ (equivalent to 4.44 ± 1.47 nmol termite⁻¹ h⁻¹) and could account for approximately 1/3 of all the acetate produced during the hindgut fermentation. Formate was also produced from H₂ + CO₂, as were small amounts of propionate, butyrate, and lactate-succinate. However, H₂-CO₂ formicogenesis seemed largely unrelated to acetogenesis and was believed not to be a significant reaction in situ. Little or no CH₄ was formed from H₂ + CO₂ or from acetate. H₂-CO₂ acetogenesis was inhibited by O₂, KCN, CHCl₃, and iodopropane and could be abolished by prefeeding R. flavipes with antibacterial drugs. By contrast, prefeeding R. flavipes with starch resulted in almost complete defaunation but had little effect on H₂-CO₂ acetogenesis, suggesting that bacteria were the acetogenic agents in the gut. H₂-CO₂ acetogenesis was also observed with gut microbiota from Prorhinotermes simplex, Zootermopsis angusticollis, Nasutitermes costalis, and N. nigriceps; from the wood-eating cockroach Cryptocercus punctulatus; and from the American cockroach Periplaneta americana. Pure cultures of H₂-CO₂-acetogenic bacteria were isolated from N. nigriceps, and a preliminary account of their morphological and physiological properties is presented. Results indicate that in termites, CO₂ reduction to acetate, rather than to CH₄, represents the main electron sink reaction of the hindgut fermentation and can provide the insects with a significant fraction (ca. 1/3) of their principal oxidizable energy source, acetate.     

Sulfate Reduction Relative to Methane Production in High-Rate Anaerobic Digestion: Technical Aspects

The effect of different substrates and different levels of sulfate and sulfide on methane production relative to sulfate reduction in high-rate anaerobic digestion was evaluated. Reactors could be acclimated so that sulfate up to a concentration of 5 g of sulfate S per liter did not significantly affect methanogenesis. Higher levels gave inhibition because of salt toxicity. Sulfate reduction was optimal at a relatively low level of sulfate, i.e., 0.5 g of sulfate S per liter, but was also not significantly affected by higher levels. Both acetoclastic and hydrogenotrophic methane-producing bacteria adapted to much higher levels of free H₂S than the values reported in the literature (50% inhibition occurred only at free H₂S levels of more than 1,000 mg/liter). High levels of free H₂S affected the sulfate-reducing bacteria only slightly. Formate and acetate supported the sulfate-reducing bacteria very poorly. In the high-rate reactors studied, intensive H₂S formation occurred only when H₂ gas or an H₂ precursor such as ethanol was supplied.     

Desulfotomaculum and Methanobacterium spp. Dominate a 4- to 5-Kilometer-Deep Fault

Alkaline, sulfidic, 54 to 60°C, 4 to 53 million-year-old meteoric water emanating from a borehole intersecting quartzite-hosted fractures >3.3 km beneath the surface supported a microbial community dominated by a bacterial species affiliated with Desulfotomaculum spp. and an archaeal species related to Methanobacterium spp. The geochemical homogeneity over the 650-m length of the borehole, the lack of dividing cells, and the absence of these microorganisms in mine service water support an indigenous origin for the microbial community. The coexistence of these two microorganisms is consistent with a limiting flux of inorganic carbon and SO₄²⁻ in the presence of high pH, high concentrations of H₂ and CH₄, and minimal free energy for autotrophic methanogenesis. Sulfide isotopic compositions were highly enriched, consistent with microbial SO₄²⁻ reduction under hydrologic isolation. An analogous microbial couple and similar abiogenic gas chemistry have been reported recently for hydrothermal carbonate vents of the Lost City near the Mid-Atlantic Ridge (D. S. Kelly et al., Science 307:1428-1434, 2005), suggesting that these features may be common to deep subsurface habitats (continental and marine) bearing this geochemical signature. The geochemical setting and microbial communities described here are notably different from microbial ecosystems reported for shallower continental subsurface environments.     

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 H₂. 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 H₂-CO₂ 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 H₂-CO₂ 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 H₂-CO₂.     

A study of mixed continuous cultures of sulfate-reducing and methane-producing bacteria

Ecological relationships between sulfate-reducing and methane-producing bacteria in mud of Lake Vechten have been studied by continuous culture studies using the chemostat technique. The maximum specific growth rate (μ _max) and saturation constant (K _s) were, respectively, 0.36 hr⁻¹ and 0.047 mM for lactate-limited growth ofDesulfovibrio desulfuricans and 0,011 hr⁻¹ and 0.17 mM for acetate-limited growth ofMethanobacterium sp. Calculated values for the true molar growth yieldsY _G) and maintenance coefficients (m) were 30.6 g bacterial mass/mole of lactate and 0.53 g substrate/g dry wt hr forD. desulfuricans and 37.8 g bacterial mass/mole of acetate and 0.54 g substrate/g dry wt hr forMethanobacterium. No growth ofMethanobacterium was observed at apS²⁻ value (the hydrogen sulfide potential) of more than 11 and there was no effect on the growth atpS²⁻ values above 13. In mixed continuous culture experiments the concentration of acetate decreased in the secondstage growth vessel, whereas that of methane increased stoichiometrically. If the substrate concentration in the reservoirs (S _r) was increased from 0.1 to 0.5 mg/ml, the population ofDesulfovibrio increased and that ofMethanobacterium was washed out of the culture vessel, since the concentration of hydrogen sulfide reached apS²⁻ value of 10.5. From the mixed continuous culture experiments a commensalism between the two species can be described, i.e., the acetate-fermentingMethanobacterium benefits from the acetate released byDesulfovibrio which is, in turn, not affected in the presence of the former.     

Effect of Fall Turnover on Terminal Carbon Metabolism in Lake Mendota Sediments

The carbon and electron flow pathways and the bacterial populations responsible for the transformation of H₂-CO₂, formate, methanol, methylamine, acetate, ethanol, and lactate were examined in eutrophic sediments collected during summer stratification and fall turnover. The rate of methane formation averaged 1,130 μmol of CH₄ per liter of sediment per day during late-summer stratification versus 433 μmol of CH₄ per liter of sediment per day during the early portion of fall turnover, whereas the rate of sulfate reduction was 280 μmol of sulfate per liter of sediment per day versus 1,840 μmol of sulfate per liter of sediment per day during the same time periods, respectively. The sulfate-reducing population remained constant while the methanogenic population decreased by one to two orders of magnitude during turnover. The acetate concentration increased from 32 to 81 μmol per liter of sediment while the acetate transformation rate constant decreased from 3.22 to 0.70 per h, respectively, during stratification versus turnover. Acetate accounted for nearly 100% of total sedimentary methanogenesis during turnover versus 70% during stratification. The fraction of ¹⁴CO₂ produced from all ¹⁴C-labeled substrates examined was 10 to 40% higher during fall turnover than during stratification. The addition of sulfate, thiosulfate, or sulfur to stratified sediments mimicked fall turnover in that more CO₂ and CH₄ were produced. The addition of Desulfovibrio vulgaris to sulfate-amended sediments greatly enhanced the amount of CO₂ produced from either [¹⁴C]methanol or [2-¹⁴C]acetate, suggesting that H₂ consumption by sulfate reducers can alter methanol or acetate transformation by sedimentary methanogens. These data imply that turnover dynamically altered carbon transformation in eutrophic sediments such that sulfate reduction dominated over methanogenesis principally as a consequence of altering hydrogen metabolism.     

Effect of pH on Anaerobic Mild Steel Corrosion by Methanogenic Bacteria

Methanogens can use H₂ produced by cathodic depolarization-mediated oxidation of elemental iron to produce methane. Thermodynamic consideration of the cathodic depolarization mechanism predicts more oxidation of Fe⁰ at lower pH. Methanogenic responses to pH by Methanococcus deltae, Methanococcus thermolithotrophicus, and Methanosarcina barkeri were examined. When grown on H₂-CO₂, these bacteria had pH optima from 6.2 to 7.0, but when all H₂ was supplied from Fe⁰, methanogenic pH optima were lower, 5.4 to 6.5. Corrosion was monitored with and without cultures and at various pHs; more corrosion occurred when cultures were present, biologically induced corrosion was greatest at the pH optima for methanogenesis from Fe⁰, and corrosion without cultures increased with a drop in pH.     

Influence of pH on Terminal Carbon Metabolism in Anoxic Sediments from a Mildly Acidic Lake

The carbon and electron flow pathways and the bacterial populations responsible for transformation of H₂-CO₂, formate, methanol, methylamine, acetate, glycine, ethanol, and lactate were examined in sediments collected from Knaack Lake, Wis. The sediments were 60% organic matter (pH 6.2) and did not display detectable sulfate-reducing activity, but they contained the following average concentration (in micromoles per liter of sediment) of metabolites and end products: sulfide, 10; methane, 1,540; CO₂, 3,950; formate, 25; acetate, 157; ethanol, 174; and lactate, 138. Methane was produced predominately from acetate, and only 4% of the total CH₄ was derived from CO₂. Methanogenesis was limited by low environmental temperature and sulfide levels and more importantly by low pH. Increasing in vitro pH to neutral values enhanced total methane production rates and the percentage of CO₂ transformed to methane but did not alter the amount of ¹⁴CO₂ produced from [2-¹⁴C]acetate (~24%). Analysis of both carbon transformation parameters with ¹⁴C-labeled tracers and bacterial trophic group enumerations indicated that methanogenesis from acetate and both heterolactic- and acetic acid-producing fermentations were important to the anaerobic digestion process.     

Comparison of Assimilatory Organic Nitrogen, Sulfur, and Carbon Sources for Growth of Methanobacterium Species

Experiments document the ability of two species of autotrophic methanogens to assimilate and utilize organic substrates as the nutrient sulfur or nitrogen source and as a carbon source during growth on H₂-CO₂. Methanobacterium thermoautotrophicum strain ΔH and the mesophilic species Methanobacterium sp. strain Ivanov grew with glutamine as the nitrogen source or cysteine as the sulfur source. M. thermoautotrophicum also utilized urea as the nitrogen source and as a carbon precursor for methane and cell synthesis. Methanobacterium sp. strain Ivanov grew with methionine as the sulfur source. The growth rate of two different Methanobacterium species was lower on an organic N or S source than on ammonium or sulfide. ³⁵S and ¹⁴C tracer studies demonstrated that amino acid or urea assimilation correlated with time and amount of growth. The rate of [³⁵S]cysteine incorporation was similar in strain ΔH (34 nmol h⁻¹ mg of cells⁻¹) and strain Ivanov (23 nmol h⁻¹ mg of cells⁻¹). However, the rate of [¹⁴C]acetate incorporation was dramatically different (17 versus 208 nmol h⁻¹ mg of cells⁻¹ in strains ΔH and Ivanov, respectively). [¹⁴C]acetate accounted for 1.3 and 21.2% of the total cell carbon synthesized by strains ΔH and Ivanov, respectively. Amino acids and urea were mainly assimilated into the cell protein fraction, but accounted for less than 2.0% of the total cell carbon synthesized. The data suggest that a biochemical-genetic approach to understanding cell carbon synthesis in methanogens is feasible; mutants that are auxotrophic for either acetate, glutamine, cysteine, or methionine are suggested as future targets for genetic studies.     

A metagenomic window into carbon metabolism at 3?km depth in Precambrian continental crust

Subsurface microbial communities comprise a significant fraction of the global prokaryotic biomass; however, the carbon metabolisms that support the deep biosphere have been relatively unexplored. In order to determine the predominant carbon metabolisms within a 3-km deep fracture fluid system accessed via the Tau Tona gold mine (Witwatersrand Basin, South Africa), metagenomic and thermodynamic analyses were combined. Within our system of study, the energy-conserving reductive acetyl-CoA (Wood-Ljungdahl) pathway was found to be the most abundant carbon fixation pathway identified in the metagenome. Carbon monoxide dehydrogenase genes that have the potential to participate in (1) both autotrophic and heterotrophic metabolisms through the reversible oxidization of CO and subsequent transfer of electrons for sulfate reduction, (2) direct utilization of H₂ and (3) methanogenesis were identified. The most abundant members of the metagenome belonged to Euryarchaeota (22%) and Firmicutes (57%)—by far, the highest relative abundance of Euryarchaeota yet reported from deep fracture fluids in South Africa and one of only five Firmicutes-dominated deep fracture fluids identified in the region. Importantly, by combining the metagenomics data and thermodynamic modeling of this study with previously published isotopic and community composition data from the South African subsurface, we are able to demonstrate that Firmicutes-dominated communities are associated with a particular hydrogeologic environment, specifically the older, more saline and more reducing waters.     

Acclimation of Two Tomato Species to High Atmospheric CO(2): II. Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Phosphoenolpyruvate Carboxylase

Lycopersicon esculentum Mill. cv Vedettos and Lycopersicon chmielewskii Rick, LA 1028, were exposed to two CO₂ concentrations (330 or 900 microliters per liter) for 10 weeks. The elevated CO₂ concentrations increased the initial ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity of both species for the first 5 weeks of treatment but the difference did not persist during the last 5 weeks. The activity of Mg²⁺-CO₂-activated Rubisco was higher in 900 microliters per liter for the first 2 weeks but declined sharply thereafter. After 10 weeks, leaves grown at 330 microliters per liter CO₂ had about twice the Rubisco activity compared with those grown at 900 microliters per liter CO₂. The two species showed the same trend to Rubisco declines under high CO₂ concentrations. The percent activation of Rubisco was always higher under high CO₂. The phosphoenolpyruvate carboxylase (PEPCase) activity measured in tomato leaves averaged 7.9% of the total Rubisco. PEPCase showed a similar trend with time as the initial Rubisco but with no significant difference between nonenriched and CO₂-enriched plants. Long-term exposure of tomato plants to high CO₂ was previously shown to induce a decline of photosynthetic efficiency. Based on the current study and on previous results, we propose that the decline of activated Rubisco is the main cause of the acclimation of tomato plants to high CO₂ concentrations.     

Characterization of aMethanosarcina strain isolated from goat feces,and that grows on H2-CO2 only after adaptation

AMethanosarcina species, designated strain ChGul, was isolated from goat feces; this is the first fully described pure culture ofMethanosarcina obtained from feces. Antigenic fingerprinting suggests that isolate ChGul is a new immunotype. The mol% G + C content of DNA was 42.2%. Strain ChGul grew on methanol, methylamines, and acetate in a minimal salts medium. It grew on H₂-CO₂ only after adaptation. Growth occurred as a milky-white suspension and contained cells mostly in doublets and quadruplets of irregular cocci; many cells contained phase bright spots typical of gas vacuoles. The isolate did not grow on formate, or CO₂ plus isopropanol, ethanol, or acetone as substrates and did not produce methane from formate. The optimum growth temperature was 35–37°C, and optimum pH was 6.2–6.8. ChGul is unusually sensitive to sulfide and has low tolerance for NaCl. Optimal levels of total sulfide and NaCl for growth were 0.5 mM and 20–40 mM, respectively. Since ChGul requires adaptation for growth on H₂-CO₂ and cannot use formate, it may be restricted to methylotropic or acetoclastic methanogenesis in the rumen, a function not observed in previously isolated rumen methanogens that use H₂-CO₂ and formate. Our work suggests that improper NaCl and sulfide concentrations, and cell lysis, may have made isolation of rumenMethanosarcina difficult in the past. It also underscores the need to evaluate feed compositions and media components for most probable number studies, with respect to NaCl and sulfide levels, to understand the role ofMethanosarcina in the rumen.     

Hydrogenotrophic Methanogenesis by Moderately Acid-Tolerant Methanogens of a Methane-Emitting Acidic Peat

The emission of methane (1.3 mmol of CH₄ m⁻² day⁻¹), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH₄ (0.4 to 1.7 μmol g [dry wt] of soil⁻¹ day⁻¹) under anoxic conditions. At in situ pH, supplemental H₂-CO₂, ethanol, and 1-propanol all increased CH₄ production rates while formate, acetate, propionate, and butyrate inhibited the production of CH₄; methanol had no effect. H₂-dependent acetogenesis occurred in H₂-CO₂-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H₂ that were subsequently consumed. The accumulation of H₂ was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H₂; these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H₂-utilizing methanogens. A total of 10⁹ anaerobes and 10⁷ hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H₂-CO₂-supplemented, fatty acid-enriched defined medium. CH₄ production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae were retrieved from the highest CH₄-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H₂ that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H₂ is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae.