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 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.     

Relationship of Intracellular Coenzyme F420 Content to Growth and Metabolic Activity of Methanobacterium bryantii and Methanosarcina barkeri

The use of F₄₂₀ as a parameter for growth or metabolic activity of methanogenic bacteria was investigated. Two representative species of methanogens were grown in batch culture: Methanobacterium bryantii (strain M.o.H.G.) on H₂ and CO₂, and Methanosarcina barkeri (strain Fusaro) on methanol or acetate. The total intracellular content of coenzyme F₄₂₀ was followed by high-resolution fluorescence spectroscopy. F₄₂₀ concentration in M. bryantii ranged from 1.84 to 3.65 μmol · g of protein⁻¹; and in M. barkeri grown with methanol it ranged from 0.84 to 1.54 μmol · g⁻¹ depending on growth conditions. The content of F₄₂₀ in M. barkeri was influenced by a factor of 2 depending on the composition of the medium (minimal or complex) and by a factor of 3 to 4 depending on whether methanol or acetate was used as the carbon source. A comparison of F₄₂₀ content with protein, cell dry weight, optical density, and specific methane production rate showed that the intracellular content of F₄₂₀ approximately followed the increase in biomass in both strains. In contrast, no correlation was found between specific methane production rate and intracellular F₄₂₀ content. However, qCH₄(F₄₂₀), calculated by dividing the methane production rate by the coenzyme F₄₂₀ concentration, almost paralleled qCH₄(protein). These results suggest that F₄₂₀ may be used as a specific parameter for estimating the biomass, but not the metabolic activity, of methanogens; hence qCH₄(F₄₂₀) determined in mixed populations with complex carbon substrates must be considered as measure of the actual methanogenic activity and not as a measure of potential activity.     

Comparison of unitrophic and mixotrophic substrate metabolism by acetate-adapted strain of Methanosarcina barkeri

We examined the unitrophic metabolism of acetate and methanol individually and the mixotrophic utilization of these compounds by using detailed ¹⁴C-labeled tracer studies in a strain of Methanosarcina barkeri adapted to grow on acetate as the sole carbon and energy source. The substrate consumption rate and methane production rate were significantly lower on acetate alone than during the unitrophic or mixotrophic metabolism of methanol. Cell yields (in grams per mole of substrate) were identical during exponential growth on acetate and exponential growth on methanol. During unitrophic metabolism of acetate, the methyl moiety accounted for the majority of the CH₄ produced, but 14% of the CO₂ generated originated from the methyl moiety. This correlated with the concurrent reduction of equivalent amounts of the C-1 of acetate to CH₄. ¹⁴CH₄ was also produced from added ¹⁴CO₂, although to a lesser extent than from reduction of the C-1 of acetate. During mixotrophic metabolism, methanol and acetate were catabolized simultaneously. The rates of ¹⁴CH₄ and ¹⁴CO₂ generation from [2-¹⁴C]acetate were logarithmic and higher in mixotrophic than in unitrophic cultures at substrate concentrations of 50 mM. A comparison of the oxidoreductase activities in cell extracts of the acetate-adapted strain grown on acetate and of strain MS grown on methanol or on H₂ plus CO₂ indicated that the pyruvate, α-ketoglutarate, and isocitrate dehydrogenase activities remained constant, whereas the CO dehydrogenase activity was significantly higher (5,000 nmol/min per mg of protein) in the acetate-adapted strain. These results suggested that a significant intramolecular redox pathway is possible for the generation of CH₄ from acetate, that energy metabolism from acetate by M. barkeri is not catabolite repressed by methanol, and that the acetate-adapted strain is a metabolic mutant with derepressed CO dehydrogenase activity.     

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H2 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 H₂ and CO₂ for methanogenesis, degraded lactate, with the production of acetate and CH₄. When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H₂-CO₂ and acetate for methanogenesis, lactate was stoichiometrically degraded to CH₄ and presumably to CO₂. 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 CH₄. Later, acetate was degraded to CH₄ and presumably to CO₂. 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 H₂ was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H₂, but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH₄ from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H₂-CO₂ as the methanogenic substrate produced CH₄ 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 H₂ produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.     

Effects of Hydrogen Pressure during Growth and Effects of Pregrowth with Hydrogen on Acetate Degradation by Methanosarcina Species

Methanosarcina barkeri 227 and Methanosarcina mazei S-6 grew with acetate as the substrate; we found little effect of H₂ on the rate of aceticlastic growth in the presence of various H₂ pressures between 2 and 810 Pa. We used physical (H₂ addition or flushing the headspace to remove H₂) and biological (H₂-producing or -utilizing bacteria in cocultures) methods for controlling H₂ pressure in Methanosarcina cultures growing on acetate. Added H₂ (ca. 100 Pa) was removed rapidly (a few hours) by M. barkeri and slowly (within a day) by M. mazei. When the H₂ produced by the aceticlastic methanogens was removed by coculturing with an H₂-using Desulfovibrio sp., the H₂ pressure was about 2.2 Pa. Under these conditions the stoichiometry of aceticlastic methanogenesis did not change. H₂-grown inocula of M. barkeri grew with acetate as the sole catabolic substrate if the inoculum culture was transferred during logarithmic growth to acetate-containing medium or if the transfer was accomplished within 1 or 2 days after exhaustion of H₂. H₂-grown cultures incubated for 4 or more days after exhaustion of H₂ were able to grow with H₂ but not with acetate as the sole catabolic substrate. Addition of small quantities of H₂ to acetate-containing medium permitted these cultures to initiate growth on acetate.     

Methanosarcina mazei LYC, a New Methanogenic Isolate Which Produces a Disaggregating Enzyme

A methanogenic coccoid organism, Methanosarcina mazei LYC, was isolated from alkaline sediment obtained from an oil exploration drilling site. The isolate resembled M. mazei S-6 by exhibiting different morphophases during its normal growth cycle. It differed from M. mazei S-6 by undergoint a spontaneous shift from large, irregular aggregates of cells to small, individual, irregular, coccoid units. In batch cultures at pH 7.0, M. mazei LYC grew as aggregates during the early growth stage. As the batch culture began exponential growth, the cell aggregates spontaneously dispersed: the culture liquid became turbid, and myriads of tiny (diameter, 1 to 3 μm) coccoid units were observed under phase-contrast microscopy. Disaggregation apparently was accomplished by the production of an enzyme which hydrolyzed the heteropolysaccharide component of the cell wall; the enzyme was active on other Methanosarcina strains as well. Although the enzyme was active when tested at pH 6.0, it apparently was not produced at that pH: when strain LYC was grown at pH 6.0, only cell aggregates were present throughout batch growth. Individual coccoid cells of M. mazei LYC were sensitive to sodium dodecyl sulfate, but the large aggregates of cells were not. Strain LYC rapidly used H₂-CO₂, in addition to methanol, and mono-, di-, and trimethylamine as methanogenic substrates; acetate was used very slowly. Its optimum growth temperature was 40°C, and its optimum pH was 7.2.     

Energetics of Growth of a Defined Mixed Culture of Desulfovibrio vulgaris and Methanosarcina barkeri: Interspecies Hydrogen Transfer in Batch and Continuous Cultures

Interspecies hydrogen transfer was studied in Desulfovibrio vulgaris-Methanosarcina barkeri mixed cultures. Experiments were performed under batch and continuous growth culture conditions. Lactate or pyruvate was used as an energy source. In batch culture and after 30 days of simultaneous incubation, these organisms were found to yield 1.5 mol of methane and 1.5 mol of carbon dioxide per mol of lactate fermented. When M. barkeri served as the hydrogen acceptor, growth yields of D. vulgaris were higher compared with those obtained on pyruvate without any electron acceptor other than protons. In continuous culture, all of the carbon derived from the oxidation of lactate was recovered as methane and carbon dioxide, provided the dilution rate was minimal. Increasing the dilution rate induced a gradual accumulation of acetate, causing acetate metabolism to cease at above μ = 0.05 h⁻¹. Under these conditions all of the methane produced originated from carbon dioxide. The growth yields of D. vulgaris were measured when sulfate or M. barkeri was the electron acceptor. Two key observations resulted from the present study. First, although sulfate was substituted by M. barkeri, metabolism of D. vulgaris was only slightly modified. The coculture-fermented lactate produced equimolar quantities of carbon dioxide and methane. Second, acetogenesis and methane formation from acetate were completely separable.     

Effect of trimethylamine on acetate utilization by Methanosarcina barkeri

During growth of Methanosarcina barkeri strain Fusaro on a mixture of trimethylamine and acetate, methane production and acetate consumption were biphasic. In the first phase trimethylamine (33 mmol x l^-1) was depleted and some acetate (11–14 from 50 mmol x l^-1) was metabolized simultaneously. In the second phase the remaining acetate was cleaved stoichiometrically into CH₄ and CO₂. Kinetic experiments with (2-¹⁴C)acetate revealed that only 2.5% of the methane produced in the first phase originated from acetate: 18% of the acetate metabolized was cleaved into CH₄ and CO₂, 23% of the acetate was oxidized, and 55% was assimilated. Methane produced from CD₃–COOH in the first phase consisted of CD₂H₂ and CD₃H in a ratio of 1:1.     

Fermentation of Cellulose to Methane and Carbon Dioxide by a Rumen Anaerobic Fungus in a Triculture with Methanobrevibacter sp. Strain RA1 and Methanosarcina barkeri

The fermentation of cellulose by a rumen anaerobic fungus in the presence of Methanobrevibacter sp. strain RA1 and Methanosarcina barkeri strain 227 resulted in the formation of 2 mol each of methane and carbon dioxide per mol of hexose fermented. Coculture of the fungus with either Methanobrevibacter sp. or M. barkeri produced 0.6 and 1.3 mol of methane per mol of hexose, respectively. Acetate, formate, ethanol, hydrogen, and lactate, which are major end products of cellulose fermentation by the fungus alone, were either absent or present in very low quantities at the end of the triculture fermentation (≤0.08 mol per mol of hexose fermented). During the time course of cellulose fermentation by the triculture, hydrogen was not detected (<1 × 10⁻⁵ atm; <0.001 kPa) and only acetate exhibited transitory accumulation; the maximum was equivalent to 1.4 mol per mol of hexose at 6 days which was higher than the total acetate yield of 0.73 in the fungus monoculture. The effect of methanogens is interpreted as a shift in the flow of electrons away from the formation of electron sink products lactate and ethanol to methane via hydrogen, favoring an increase in acetate, which is in turn converted to methane and carbon dioxide by M. barkeri. The maximum rate of cellulose degradation in the triculture (3 mg/ml per day) was faster than previously reported for bacterial cocultures and within 16 days degradation was complete. The triculture was used successfully also in the production of methane from cellulose in the plant fibrous materials, sisal (fiber from leaves of Agave sisalona L.) and barley straw leaf.     

Spontaneous Disaggregation of Methanosarcina mazei S-6 and Its Use in the Development of Genetic Techniques for Methanosarcina spp

When monomethylamine was the growth substrate, spontaneous disaggregation of Methanosarcina mazei S-6 commenced at the mid-exponential phase and resulted in the formation of a suspension containing 10⁸ to 10⁹ free cells per ml. Free cells were osmotically fragile and amenable to extraction of DNA. Hypertonic media for the manipulation and regeneration of free cells into aggregates were developed, and plating efficiencies of 100% were achieved for M. mazei S-6 and LYC. Free cells of strain S-6 required MgCl₂ (10 mM) for growth, whereas aggregates did not. Specific growth rates of strains S-6 and LYC were increased by MgCl₂. Treatment with pronase caused sphere formation and removal of the protein wall of cells of strain S-6, but protoplasts could not be regenerated. The disaggregating enzyme produced by strain S-6 facilitated the preparation of suspensions of free cells of some strains of Methanosarcina barkeri. Although this provided a means of extracting high-molecular-weight DNA from M. barkeri, less than 0.1% of free cells were viable.     

Metabolic Activity of Fatty Acid-Oxidizing Bacteria and the Contribution of Acetate, Propionate, Butyrate, and CO2 to Methanogenesis in Cattle Waste at 40 and 60°C

The quantitative contribution of fatty acids and CO₂ to methanogenesis was studied by using stirred, 3-liter bench-top digestors fed on a semicontinuous basis with cattle waste. The fermentations were carried out at 40 and 60°C under identical loading conditions (6 g of volatile solids per liter of reactor volume per day, 10-day retention time). In the thermophilic digestor, acetate turnover increased from a prefeeding level of 16 μM/min to a peak (49 μM/min) 1 h after feeding and then gradually decreased. Acetate turnover in the mesophilic digestor increased from 15 to 40 μM/min. Propionate turnover ranged from 2 to 5.2 and 1.5 to 4.5 μM/min in the thermophilic and mesophilic digestors, respectively. Butyrate turnover (0.7 to 1.2 μM/min) was similar in both digestors. The proportion of CH₄ produced via the methyl group of acetate varied with time after feeding and ranged from 72 to 75% in the mesophilic digestor and 75 to 86% in the thermophilic digestor. The contribution from CO₂ reduction was 24 to 29% and 19 to 27%, respectively. Propionate and butyrate turnover accounted for 20% of the total CH₄ produced. Acetate synthesis from CO₂ was greatest shortly after feeding and was higher in the thermophilic digestor (0.5 to 2.4 μM/min) than the mesophilic digestor (0.3 to 0.5 μM/min). Counts of fatty acid-degrading bacteria were related to their turnover activity.     

Uncoupling of Methanogenesis from Growth of Methanosarcina barkeri by Phosphate Limitation

Production of methane by Methanosarcina barkeri from H₂-CO₂ was studied in fed-batch culture under phosphate-limiting conditions. A transition in the kinetics of methanogenesis from an exponentially increasing rate to a constant rate was due to depletion of phosphate from the medium. The period of exponentially increasing rate of methanogenesis was extended by increasing the initial concentration of phosphate in the medium. Addition of phosphate during the constant period changed the kinetics to an exponentially increasing rate of methanogenesis, indicating the reversibility of phosphate depletion. The relation between methanogenesis and growth of M. barkeri was investigated by measuring the incorporation of phosphorus, supplied as KH₂³²PO₄, in the medium. At a low (1 μM) initial concentration of phosphate in the medium and during the constant period of methanogenesis, there was no net cell growth. At a higher (10 μM) initial concentration of phosphate, cell growth proceeded linearly with time after phosphate had been removed from the medium by uptake into cells.     

Nutritional Requirements of Methanosarcina sp. Strain TM-1

Methanosarcina sp. strain TM-1, an acetotrophic, thermophilic methanogen isolated from an anaerobic sludge digestor, was originally reported to require an anaerobic sludge supernatant for growth. It was found that the sludge supernatant could be replaced with yeast extract (1 g/liter), 6 mM bicarbonate-30% CO₂, and trace metals, with a doubling time on methanol of 14 h. For growth on either methanol or acetate, yeast extract could be replaced with CaCl₂ · 2H₂O (13.6 μM minimum) and the vitamin p-aminobenzoic acid (PABA, ca. 3 nM minimum), with a doubling time on methanol of 8 to 9 h. Filter-sterilized folic acid at 0.3 μM could not replace PABA. The antimetabolite sulfanilamide (20 mM) inhibited growth of and methanogenesis by Methanosarcina sp. strain TM-1, and this inhibition was reversed by the addition of 0.3 μM PABA. When a defined medium buffered with 20 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid was used, it was shown that Methanosarcina sp. strain TM-1 required 6 mM bicarbonate-30% CO₂ for optimal growth and methanogenesis from methanol. Cells growing on acetate were less dependent on bicarbonate-CO₂. When we used a defined medium in which the only organic compounds present were methanol or acetate, nitrilotriacetic acid (0.2 mM), and PABA, it was possible to limit batch cultures of Methanosarcina sp. strain TM-1 for nitrogen at NH₄⁺ concentrations at or below 2.0 mM, in marked contrast with Methanosarcina barkeri 227, which fixes dinitrogen when grown under NH₄⁺ limitation.     

Pyruvate — a novel substrate for growth and methane formation in Methanosarcina barkeri

Methanosarcina barkeri strain Fusaro was found to grow on pyruvate as sole carbon and energy source after an incubation period of 10–12 weeks in the presence of high pyruvate concentrations (100 mM). Growth studies, cell suspension experiments and enzymatic investigations were performed with pyruvate-utilizing M. barkeri. For comparison acetate-adapted cells of M. barkeri were analyzed.

1. Pyruvate-utilizing M. barkeri grew on pyruvate (100 mM) with an initial doubling time of about 25 h (37 °C, pH 6.5) up to cell densities of about 0.8 g cell dry weight/l. The specific growth rate was linearily dependent on the pyruvate concentration up to 100 mM indicating that pyruvate was taken up by passive diffusion. Only CO₂ and CH₄ were detected as fermentation products. As calculated from fermentation balances pyruvate was converted to CH₄ and CO₂ according to following equation: Pyruvate^-+H⁺+0.5 H₂O » 1.25 CH₄+1.75 CO₂. The molar growth yield (Ych ₄) was about 14 g dry weight cells/mol CH₄. In contrast the growth yield (Ych ₄) of M. barkeri during growth on acctate (Acetate^-+H⁺ » CH₄+CO₂) was about 3 g/mol CH₄.
2. Cell suspensions of pyruvate-grown M. barkeri catalyzed the conversion of pyruvate to CH₄, CO₂ and H₂ (5–15 nmol pyruvate consumed/min x mg protein). At low cell concentrations (0.5 mg protein/ml) 1 mol pyruvate was converted to 1 mol CH₄, 2 mol CO₂ and 1 mol H₂. At higher cell concentration less H₂ and CO₂ and more CH₄ were formed due to CH₄ formation from H₂/CO₂. The rate of pyruvate conversion was linearily dependent on the pyruvate concentration up to about 30 mM. Cell suspensions of acetate-grown M. barkeri also catalyzed the conversion of 1 mol pyruvate to 1 mol CH₄, 2 mol CO₂ and 1 mol H₂ at similar rates and with similar affinity for pyruvate as pyruvate-grown cells.
3. Cell extracts of both pyruvate-grown and acetate-grown M. barkeri contained pyruvate: ferredoxin oxidoreductase. The specific activity in pyruvate-grown cells (0.8 U/mg) was 8-fold higher than in acetate-grown cells (0.1 U/mg). Coenzyme F₄₂₀ was excluded as primary electron acceptor of pyruvate oxidoreductase. Cell extracts of pyruvate-grown M. barkeri contained carbon monoxide dehydrogenase activity and hydrogenase activity catalyzing the reduction by carbon monoxide and hydrogen of both methylviologen and ferredoxin (from Clostridium).

This is the first report on growth of a methanogen on pyruvate as sole carbon and energy source, i.e. on a substrate more complex than acetate.     

Selective Inhibition by 2-Bromoethanesulfonate of Methanogenesis from Acetate in a Thermophilic Anaerobic Digestor

The effects of 2-bromoethanesulfonate, an inhibitor of methanogenesis, on metabolism in sludge from a thermophilic (58°C) anaerobic digestor were studied. It was found from short-term experiments that 1 μmol of 2-bromoethanesulfonate per ml completely inhibited methanogenesis from ¹⁴CH₃COO⁻, whereas 50 μmol/ml was required for complete inhibition of ¹⁴CO₂ reduction. When 1 μmol of 2-bromoethanesulfonate per ml was added to actively metabolizing sludge which was then incubated for 24 h. it caused a 60% reduction in methanogenesis and a corresponding increase in acetate accumulation; at 50 μmol/ml it caused complete inhibition of methanogenesis and accumulation of acetate. H₂, and ethanol.     

Effect of Substrate Concentration on Carbon Isotope Fractionation during Acetoclastic Methanogenesis by Methanosarcina barkeri and M. acetivorans and in Rice Field Soil

Methanosarcina is the only acetate-consuming genus of methanogenic archaea other than Methanosaeta and thus is important in methanogenic environments for the formation of the greenhouse gases methane and carbon dioxide. However, little is known about isotopic discrimination during acetoclastic CH₄ production. Therefore, we studied two species of the Methanosarcinaceae family, Methanosarcina barkeri and Methanosarcina acetivorans, and a methanogenic rice field soil amended with acetate. The values of the isotope enrichment factor (ɛ) associated with consumption of total acetate (ɛ_ac), consumption of acetate-methyl (ɛ_ac-methyl) and production of CH₄ (ɛ_CH4) were an ɛ_ac of −30.5‰, an ɛ_ac-methyl of −25.6‰, and an ɛ_CH4 of −27.4‰ for M. barkeri and an ɛ_ac of −35.3‰, an ɛ_ac-methyl of −24.8‰, and an ɛ_CH4 of −23.8‰ for M. acetivorans. Terminal restriction fragment length polymorphism of archaeal 16S rRNA genes indicated that acetoclastic methanogenic populations in rice field soil were dominated by Methanosarcina spp. Isotope fractionation determined during acetoclastic methanogenesis in rice field soil resulted in an ɛ_ac of −18.7‰, an ɛ_ac-methyl of −16.9‰, and an ɛ_CH4 of −20.8‰. However, in rice field soil as well as in the pure cultures, values of ɛ_ac and ɛ_ac-methyl decreased as acetate concentrations decreased, eventually approaching zero. Thus, isotope fractionation of acetate carbon was apparently affected by substrate concentration. The ɛ values determined in pure cultures were consistent with those in rice field soil if the concentration of acetate was taken into account.Methane (CH₄) is the most abundant reduced gas in the earth''s atmosphere and is an important greenhouse gas with a high global-warming potential (7). It is presently a matter of discussion whether the contribution of CH₄ to the greenhouse effect will increase in the future (3, 23). This has made it necessary and more urgent to understand natural processes that lead to the production of CH₄.Methanogenesis, the microbial formation of CH₄, is the final step in the degradation of organic matter in anoxic environments like natural wetlands, lake sediments, and flooded rice fields. The most important precursors for the production of CH₄ are acetate (equation 1) and CO₂ (equation 2) with the following reactions (8): (1) (2)Acetate is the most important substrate since it contributes more than 67% to microbial methanogenesis during anoxic degradation of polysaccharides. In methanogenic environments only two genera of archaea, Methanosaeta and Methanosarcina, are capable of using acetate (2). While Methanosaeta can be considered a specialist that uses only acetate, Methanosarcina can use a wide range of substrates besides acetate, for example, H₂/CO₂, methanol, methylamines, and methylated sulfides. Among methanogens, Methanosarcinaceae also display the largest environmental distribution. They can be found in freshwater sediments and soil, marine habitats, landfills, and animal gastrointestinal tracts (46).Additionally, differences between Methanosarcina and Methanosaeta were found for isotope fractionation of stable carbon. The fractionation factor (α) or, equivalently, the enrichment factor (ɛ) during acetoclastic methanogenesis in Methanosarcina barkeri strains typically ranges from an α of 1.021 to 1.027 or an ɛ of −27‰ to −21‰ (14, 27, 48), whereas isotope fractionation in Methanosaeta spp. is weaker, i.e., an α of 1.007 (ɛ = −7‰) for Methanosaeta thermophila (43) and an α of 1.010 (ɛ = −10‰) for Methanosaeta concilii (34). It is suggested that the two archaeal genera differ in isotope fractionation due to differences in their biochemical activation of acetate to acetyl-coenzyme A (acetyl-CoA) (34). However, isotopic data for acetoclastic methanogens are rare. For instance, all data for Methanosarcina refer to only one species, namely M. barkeri.Hence, in this study we investigated whether differences in carbon isotope fractionation within the genus Methanosarcina occur. Therefore, we determined isotope ratios of stable carbon in cultures of the acetoclastic species M. barkeri and Methanosarcina acetivorans. Second, we were interested if these data, obtained from pure cultures, could also be applied to understand natural environments. For that reason, we determined isotope fractionation during acetoclastic methanogenesis in the model system rice field soil. Furthermore, we discuss the effect of substrate concentration on carbon isotope fractionation and the importance of monitoring isotope fractionation during the course of acetate consumption.     

Factors Affecting the Methanogenic Activity of Methanothrix soehngenii VNBF

Methane production by Methanothrix soehngenii VNBF grown on acetate (50 mM) as the sole carbon and energy source was influenced by the addition of Fe, trace elements, and pesticides. The addition of Fe and trace elements significantly enhanced the rate of CH₄ production. The addition of pesticides in the early growth phase caused complete inhibition. However, less inhibition was noted when pesticides were added during early exponential growth phase. Addition to culture tubes of Co, Ni, or Mo at 2 μM produced 64, 41, or 17%, respectively, more CH₄ than that produced in tubes lacking the corresponding trace element. A concentration of more than 5 μM of these trace elements in the medium resulted in decreased CH₄ production, presumably because of toxic effects.     

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.     

Distribution of Sulfate-Reducing and Methanogenic Bacteria in Anaerobic Aggregates Determined by Microsensor and Molecular Analyses

Using molecular techniques and microsensors for H₂S and CH₄, we studied the population structure of and the activity distribution in anaerobic aggregates. The aggregates originated from three different types of reactors: a methanogenic reactor, a methanogenic-sulfidogenic reactor, and a sulfidogenic reactor. Microsensor measurements in methanogenic-sulfidogenic aggregates revealed that the activity of sulfate-reducing bacteria (2 to 3 mmol of S²⁻ m⁻³ s⁻¹ or 2 × 10⁻⁹ mmol s⁻¹ per aggregate) was located in a surface layer of 50 to 100 μm thick. The sulfidogenic aggregates contained a wider sulfate-reducing zone (the first 200 to 300 μm from the aggregate surface) with a higher activity (1 to 6 mmol of S²⁻ m⁻³ s⁻¹ or 7 × 10⁻⁹ mol s⁻¹ per aggregate). The methanogenic aggregates did not show significant sulfate-reducing activity. Methanogenic activity in the methanogenic-sulfidogenic aggregates (1 to 2 mmol of CH₄ m⁻³ s⁻¹ or 10⁻⁹ mmol s⁻¹ per aggregate) and the methanogenic aggregates (2 to 4 mmol of CH₄ m⁻³ s⁻¹ or 5 × 10⁻⁹ mmol s⁻¹ per aggregate) was located more inward, starting at ca. 100 μm from the aggregate surface. The methanogenic activity was not affected by 10 mM sulfate during a 1-day incubation. The sulfidogenic and methanogenic activities were independent of the type of electron donor (acetate, propionate, ethanol, or H₂), but the substrates were metabolized in different zones. The localization of the populations corresponded to the microsensor data. A distinct layered structure was found in the methanogenic-sulfidogenic aggregates, with sulfate-reducing bacteria in the outer 50 to 100 μm, methanogens in the inner part, and Eubacteria spp. (partly syntrophic bacteria) filling the gap between sulfate-reducing and methanogenic bacteria. In methanogenic aggregates, few sulfate-reducing bacteria were detected, while methanogens were found in the core. In the sulfidogenic aggregates, sulfate-reducing bacteria were present in the outer 300 μm, and methanogens were distributed over the inner part in clusters with syntrophic bacteria.