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.     

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.     

Carbon Monoxide, Hydrogen, and Formate Metabolism during Methanogenesis from Acetate by Thermophilic Cultures of Methanosarcina and Methanothrix Strains

CO and H₂ have been implicated in methanogenesis from acetate, but it is unclear whether they are directly involved in methanogenesis or electron transfer in acetotrophic methanogens. We compared metabolism of H₂, CO, and formate by cultures of the thermophilic acetotrophic methanogens Methanosarcina thermophila TM-1 and Methanothrix sp. strain CALS-1. M. thermophila accumulated H₂ to partial pressures of 40 to 70 Pa (1 Pa = 0.987 × 10^-5 atm), as has been previously reported for this and other Methanosarcina cultures. In contrast, Methanothrix sp. strain CALS-1 accumulated H₂ to maximum partial pressures near 1 Pa. Growing cultures of Methanothrix sp. strain CALS-1 initially accumulated CO, which reached partial pressures near 0.6 Pa (some CO came from the rubber stopper) during the middle of methanogenesis; this was followed by a decrease in CO partial pressures to less than 0.01 Pa by the end of methanogenesis. Accumulation or consumption of CO by cultures of M. thermophila growing on acetate was not detected. Late-exponential-phase cultures of Methanothrix sp. strain CALS-1, in which the CO partial pressure was decreased by flushing with N₂-CO₂, accumulated CO to 0.16 Pa, whereas cultures to which ca. 0.5 Pa of CO was added consumed CO until it reached this partial pressure. Cyanide (1 mM) blocked CO consumption but not production. High partial pressures of H₂ (40 kPa) inhibited methanogenesis from acetate by M. thermophila but not by Methanothrix sp. strain CALS-1, and 2 kPa of CO was not inhibitory to M. thermophila but was inhibitory to Methanothrix sp. strain CALS-1. Levels of CO dehydrogenase, hydrogenase, and formate dehydrogenase in Methanothrix sp. strain CALS-1 were 9.1, 0.045, and 5.8 μmol of viologen reduced min^-1 mg of protein^-1. These results suggest that CO plays a role in Methanothrix sp. strain CALS-1 similar to that of H₂ in M. thermophila and are consistent with the conclusion that CO is an intermediate in a catabolic or anabolic pathway in Methanothrix sp. strain CALS-1; however, they could also be explained by passive equilibration of CO with a metabolic intermediate.     

Syntrophic Association by Cocultures of the Methanol- and CO2-H2-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, H₂-CO₂, 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, CO₂, and H₂ from pectin. The coculture produced one-third more acetate, and butyrate and CO₂ were the only other significant end products. The results are discussed in relationship to microbial metabolic interactions and interspecies hydrogen transfer.     

Methanogenic Bacteria from the Bondyuzhskoe Oil Field: General Characterization and Analysis of Stable-Carbon Isotopic Fractionation

Selective enrichment culture techniques were employed to obtain mixed cultures of methanogenic rods and sarcina from surface flooding waters and deep subsurface (~1650 m) oil-bearing sedimentary rocks and formation waters sampled from an old oil field in the U.S.S.R. previously reported to display active biological methanogenesis. The methanogens were selectively isolated as colonies on agar petri dishes that were incubated in a novel container. The general cellular and growth features of three Methanobacterium isolates were determined. These strains grew optimally at 37 to 45°C in anaerobic pressure tube cultures with a doubling time of 16 to 18 h on H₂-CO₂ and proliferated as autotrophs. Acetate addition significantly enhanced the final cell yield. Growth of these strains was completely inhibited by either 0.6 g of sodium sulfide per liter or 31.0 of sodium chloride per liter, but growth was not inhibited by either 0.3 g of sodium sulfide per liter or 1.0 g of sodium sulfate per liter. One novel isolate, Methanobacterium sp. strain ivanov, was grown on H₂-CO₂, and the stable-carbon isotopic fractionations that occurred during synthesis of methane, cell carbon, and lipids were determined. The results of this study were used to examine the anomalous relationship between the isotopic and chemical compositions of natural gas occurring in the deep subsurface environment of the oil field.     

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.     

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.     

Propionate-Degrading Bacterium, Syntrophobacter wolinii sp. nov. gen. nov., from Methanogenic Ecosystems

A new genus and species of a nonmotile gram-negative rod, Syntrophobacter wolinii, is the first bacterium described which degrades propionate only in coculture with an H₂-using organism and in the absence of light or exogenous electron acceptors such as O₂, sulfate, or nitrate. It was isolated from methanogenic enrichments from an anaerobic municipal sewage digestor, using anaerobic roll tubes containing a medium with propionate as the energy source in association with an H₂-using, sulfate-reducing Desulfovibrio sp. which cannot utilize fatty acids other than formate. S. wolinii produced acetate and, presumably, CO₂ and H₂ (or formate) from propionate. In media without sulfate and with Methanospirillum hungatei, a methanogen that uses only H₂-CO₂ or formate as an energy source, acetate, methane, and, presumably, CO₂ were produced from propionate and only small amounts of Desulfovibrio sp. were present. Isolation in coculture with the methanogen was not successful. S. wolinii does not use other saturated fatty acids as energy sources.     

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.     

Control of Interspecies Electron Flow during Anaerobic Digestion: Significance of Formate Transfer versus Hydrogen Transfer during Syntrophic Methanogenesis in Flocs

Microbial formate production and consumption during syntrophic conversion of ethanol or lactate to methane was examined in purified flocs and digestor contents obtained from a whey-processing digestor. Formate production by digestor contents or purified digestor flocs was dependent on CO₂ and either ethanol or lactate but not H₂ gas as an electron donor. During syntrophic methanogenesis, flocs were the primary site for formate production via ethanol-dependent CO₂ reduction, with a formate production rate and methanogenic turnover constant of 660 μM/h and 0.044/min, respectively. Floc preparations accumulated fourfold-higher levels of formate (40 μM) than digestor contents, and the free flora was the primary site for formate cleavage to CO₂ and H₂ (90 μM formate per h). Inhibition of methanogenesis by CHCl₃ resulted in formate accumulation and suppression of syntrophic ethanol oxidation. H₂ gas was an insignificant intermediary metabolite of syntrophic ethanol conversion by flocs, and its exogenous addition neither stimulated methanogenesis nor inhibited the initial rate of ethanol oxidation. These results demonstrated that >90% of the syntrophic ethanol conversion to methane by mixed cultures containing primarily Desulfovibrio vulgaris and Methanobacterium formicicum was mediated via interspecies formate transfer and that <10% was mediated via interspecies H₂ transfer. The results are discussed in relation to biochemical thermodynamics. A model is presented which describes the dynamics of a bicarbonate-formate electron shuttle mechanism for control of carbon and electron flow during syntrophic methanogenesis and provides a novel mechanism for energy conservation by syntrophic acetogens.     

Formate production and utilization by methanogens and by sewage sludge consortia — interference with the concept of interspecies formate transfer

Pure cultures of H₂/CO₂- and formate-utilizing methanogens or mixed consortia of sewage sludge generated some formate from H₂/CO₂ at H₂ partial pressure in the gas phase above 200 kPa. At decreasing H₂ partial pressure the formate was taken up again and converted to methane. If methanogenesis was inhibited by bromoethanesulphonic acid (BESA) or a high redox potential (–180 to –200 mV), formate-utilizing methanogens produced high amounts of formate from H₂/CO₂. No formate was excreted by the species, which could only utilize H₂/CO₂ for methanogenesis. In contrast, H₂ formation from formate was observed in cultures of Methanobacterium thermoformicicum and M. formicicum. Measurable amounts were, however, only formed if its immediate utilization for methane production was inhibited by BESA. In the light of the data on formate formation from H₂/CO₂ and its re-utilization by all formate-utilizing methanogens, the concept of interspecies formate transfer of Thiele and Zeikus should be reconsidered. In pure cultures of methanogens or complex ecosystems with excess H₂, formate formation seemed to serve more as a means of disposal of surplus reducing power than for H₂ transfer. Correspondence to: J. Winter     

Growth and adaptive hydrogen production of Rhodospirillum rubrum (F1) in anaerobic dark cultures

Rhodospirillum rubrum (F₁) maintained electron balance mainly by producing propionate, formate and H₂ during fermentation metabolism. H₂ formation was inversely correlated with the production of propionate.In diluted, growing cultures high amounts of H₂ and only traces or no propionate were produced from pyruvate. In dense cultures or in resting cultures without (NH₄)₂SO₄, however, propionate was formed from pyruvate in relatively high amounts Cultures always produced much more propionate than H₂ from fructose in contrast to cells with pyruvate. Kinetic studies of growth and excretion of fermentation products indicated that the enzyme system for H₂ formation is adaptive. Chloramphenicol (3 μg/ml) completely inhibited the formation of H₂ if the cells were not adapted to fermentation metabolism. The production of propionate, on the other hand, was not prevented by chloramphenicol after shifting the cells from aerobic dark culture with malate to fermentation conditions with pyruvate.H₂ formation was not influenced by sodium ascorbate but it was significantly decreased by K₃[Fe(CN)₆].Poly(β-hydroxybutyric acid) was also synthesized by the cells during anaerobic dark metabolism especially in dense cultures, probably favoured by the rapid acidification of the medium. Formate can also accumulate in the fermentation metabolism, especially in young growing cultures.These results give an explanation for the differing reports in the literature on the fermentation metabolism of R. rubrum.     

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.     

Metabolic properties and kinetics of methanogenic granules

Two types of mesophilic methanogenic granules (R- and F-granules) were developed on different synthetic feeds containing acetate, propionate and butyrate as major carbon sources and their metabolic properties were characterized. The metabolic activities of granules on acetate, formate and H₂-CO₂ were related to the feed composition used for their development. These granules performed a reversible reaction between H₂ production from formate and formate synthesis from H₂ plus bicarbonate. Both types of granules exhibited high activity on normal and branched volatile fatty acids with three to five carbons and low activity on ethanol and glucose. The granules performed a reversible isomerization between isobutyrate and butyrate during butyrate or isobutyrate degradation. Valerate and 2-methylbutyrate were produced and consumed during propionate-butyrate degradation. The respective apparent K _m (mm) for various substrates in disrupted R- and F-granules was: acetate, 0.43 and 0.41; propionate, 0.056 and 0.038; butyrate, 0.15 and 0.19; isobutyrate, 0.12 and 0.19; valerate, 0.15 and 0.098. Both granules had an optimum temperature range from 40 to 50° C for H₂-CO₂ and formate utilization and 40° C for acetate, propionate and butyrate utilization and a similar optimum pH. Correspondence to: J. G. Zeikus     

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.     

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).     

Methanogenic Activity in Human Periodontal Pocket

Samples of subgingival dental tissues were examined for the presence of methanogenic activities. Using enrichment cultures, methanogenic activities were detected in 9 of 17 individuals. A mesophilic, Gram-positive, irregular coccoid methanogen, which showed close resemblance to a Methanosarcina sp., was isolated from one sample collected from a patient with type IV periodontal pocket (the periodontal pocket is a space bounded by the tooth on one side and by ulcerated epithelium lining the soft tissue wall on the other). The isolate used methanol, methylamine, acetate, and H₂-CO₂ as the sole source of carbon. However, the isolate was unable to use formate and trimethylamine as growth substrates. The organism had an optimum pH of 6.5 and an optimum temperature of 37°C. The isolate not only used ammonia, but also used nitrate as a nitrogen source. The niche of this methanogen in periodontal pockets may be to carry out terminal oxidation of simple organic compounds such as methanol and acetate produced by other obligate anaerobes present in periodontal pockets. This methanogen may also play a vital role in interspecies hydrogen transfer, as demonstrated by its use of H₂-CO₂ as a substrate. The isolate produced significant amount of methane in vitro. Received: 27 February 2002 / Accepted: 29 March 2002     

Kinetics of Formate Metabolism in Methanobacterium formicicum and Methanospirillum hungatei

The kinetics of formate metabolism in Methanobacterium formicicum and Methanospirillum hungatei were studied with log-phase formate-grown cultures. The progress of formate degradation was followed by the formyltetrahydrofolate synthetase assay for formate and fitted to the integrated form of the Michaelis-Menten equation. The K_m and V_max values for Methanobacterium formicicum were 0.58 mM formate and 0.037 mol of formate h⁻¹ g⁻¹ (dry weight), respectively. The lowest concentration of formate metabolized by Methanobacterium formicicum was 26 μM. The K_m and V_max values for Methanospirillum hungatei were 0.22 mM and 0.044 mol of formate h⁻¹ g⁻¹ (dry weight), respectively. The lowest concentration of formate metabolized by Methanospirillum hungatei was 15 μM. The apparent K_m for formate by formate dehydrogenase in cell-free extracts of Methanospirillum hungatei was 0.11 mM. The K_m for H₂ uptake by cultures of Methanobacterium formicicum was 6 μM dissolved H₂. Formate and H₂ were equivalent electron donors for methanogenesis when both substrates were above saturation; however, H₂ uptake was severely depressed when formate was above saturation and the dissolved H₂ was below 6 μM. Formate-grown cultures of Methanobacterium formicicum that were substrate limited for 57 h showed an immediate increase in growth and methanogenesis when formate was added to above saturation.     

Diffusion of the Interspecies Electron Carriers H2 and Formate in Methanogenic Ecosystems and Its Implications in the Measurement of Km for H2 or Formate Uptake

We calculated the potential H₂ and formate diffusion between microbes and found that at H₂ concentrations commonly found in nature, H₂ could not diffuse rapidly enough to dispersed methanogenic cells to account for the rate of methane synthesis but formate could. Our calculations were based on individual organisms dispersed in the medium, as supported by microscopic observations of butyrate-degrading cocultures. We isolated an axenic culture of Syntrophomonas wolfei and cultivated it on butyrate in syntrophic coculture with Methanobacterium formicicum; during growth the H₂ concentration was 63 nM (10.6 Pa). S. wolfei contained formate dehydrogenase activity (as does M. formicicum), which would allow interspecies formate transfer in that coculture. Thus, interspecies formate transfer may be the predominant mechanism of syntrophy. Our diffusion calculations also indicated that H₂ concentration at the cell surface of H₂-consuming organisms was low but increased to approximately the bulk-fluid concentration at a distance of about 10 μm from the surface. Thus, routine estimation of kinetic parameters would greatly overestimate the K_m for H₂ or formate.     

CO Metabolism in the Acetogen Acetobacterium woodii

The Wood-Ljungdahl pathway allows acetogenic bacteria to grow on a number of one-carbon substrates, such as carbon dioxide, formate, methyl groups, or even carbon monoxide. Since carbon monoxide alone or in combination with hydrogen and carbon dioxide (synthesis gas) is an increasingly important feedstock for third-generation biotechnology, we studied CO metabolism in the model acetogen Acetobacterium woodii. When cells grew on H₂-CO₂, addition of 5 to 15% CO led to higher final optical densities, indicating the utilization of CO as a cosubstrate. However, the growth rate was decreased by the presence of small amounts of CO, which correlated with an inhibition of H₂ consumption. Experiments with resting cells revealed that the degree of inhibition of H₂ consumption was a function of the CO concentration. Since the hydrogen-dependent CO₂ reductase (HDCR) of A. woodii is known to be very sensitive to CO, we speculated that cells may be more tolerant toward CO when growing on formate, the product of the HDCR reaction. Indeed, addition of up to 25% CO did not influence growth rates on formate, while the final optical densities and the production of acetate increased. Higher concentrations (75 and 100%) led to a slight inhibition of growth and to decreasing rates of formate and CO consumption. Experiments with resting cells revealed that the HDCR is a site of CO inhibition. In contrast, A. woodii was not able to grow on CO as a sole carbon and energy source, and growth on fructose-CO or methanol-CO was not observed.