Fermentation of glutamate and other compounds by Acidaminobacter hydrogenoformans gen. nov. sp. nov., an obligate anaerobe isolated from black mud. Studies with pure cultures and mixed cultures with sulfate-reducing and methanogenic bacteria

From mud from the Ems-Dollard estuary (The Netherlands) an L-glutamate-fermenting bacterium was isolated. The isolated strain glu 65 is Gram-negative, rodshaped, obligately anaerobic, non-sporeforming and does not contain cytochromes. The G+C content of its DNA is 48 mol percent.Pure cultures of strain glu 65 grew slowly on glutamate (_max 0.06 h^-1) and formed acetate, CO₂, formate and hydrogen, and minor amounts of propionate. A more rapid fermentation of glutamate was achieved in mixed cultures with sulfate-reducing bacteria (Desulfovibrio HL21 or Desulfobulbus propionicus) or methanogens (Methanospirillum hungatei or Methanobrevibacter arboriphilicus AZ). In mixed culture with Desulfovibrio HL21 a _max of 0.10 h^-1 was observed. With Desulfovibrio or the methanogens propionate was a major product (up to 0.47 mol per mol glutamate) in addition to acetate.Extracts of glutamate-grown cells possessed high activities of 3-methylaspartase, a key enzyme of the mesaconate pathway leading to acetate, and very high activities of NAD⁺-dependent glutamate dehydrogenase, an enzyme most likely involved in the pathway to propionate.The following other substrates allowed reasonable to good growth in pure culture: histidine, -ketoglutarate, serine, cysteine, glycine, adenine, pyruvate, oxaloacetate and citrate. Utilization in mixed cultures was demonstrated for: glutamine, arginine, ornithine, threonine, lysine, alanine, valine, leucine and isoleucine (with Desulfovibrio HL21) and malate (with Methanospirillum).The shift in the fermentation of glutamate and the syntrophic utilization of the above substrates are explained in terms of interspecies hydrogen transfer.Strain glu 65 is described as the type strain of Acidaminobacter hydrogenoformans gen. nov. sp. nov.     

Non-aceticlastic methanogenesis from acetate: acetate oxidation by a thermophilic syntrophic coculture

Methanogenesis from acetate by a rod-shaped enrichment culture grown at 60° C was found to require the presence of two organisms rather than a single aceticlastic methanogen. A thermophilic Methanobacterium which grew on H₂/CO₂ or formate was isolated from the enrichment. Lawns of this methanogen were used to co-isolate an acetate oxidizer in roll tubes containing acetate agar. The rod-shaped acetate oxidizer was morphologically distinct from the methanogen and did not show F₄₂₀ autofluorescence. The coculture completely degraded 40 mol/ml acetate, and produced nearly equal quantities of methane, and methanogenesis was coupled with growth. The doubling time for the coculture at 60°C was 30–40 h and the yield was 2.7±0.3 g dry wt/mol CH₄. Studies with ¹⁴C-labelled substrates showed that the methyl group and the carboxyl group of acetate were both converted primarily to CO₂ by the coculture and that CO₂ was concurrently reduced to CH₄. During growth, there was significant isotopic exchange between CO₂ and acetate, especially with thecarboxyl position of acetate. These results support a mechanism for methanogenesis from acetate by the coculture in which acetate was oxidized to CO₂ and H₂ by one organism, while H₂ was subsequently used by a second organism to reduce CO₂ to CH₄. Since the H₂ partial pressure must be maintained below 10^-4 atm by the methanogen for acetate oxidation to be thermodynamically feasible, this is an example of obligate interspecies hydrogen transfer. This mechanism was originally proposed for a single organism by Barker in 1936.     

Desulfacinum subterraneumsp. nov., a New Thermophilic Sulfate-Reducing Bacterium Isolated from a High-Temperature Oil Field

A new thermophilic sulfate-reducing bacterium isolated from the high-temperature White Tiger oil field (Vietnam) is described. Cells of the bacterium are oval (0.4–0.6 by 0.6–1.8 m), nonmotile, non-spore-forming, and gram-negative. Growth occurs at 45 to 65°C (with an optimum at 60°C) at NaCl concentrations of 0 to 50 g/l. In the course of sulfate reduction, the organism can utilize lactate, pyruvate, malate, fumarate, ethanol, salts of fatty acids (formate, acetate, propionate, butyrate, caproate, palmitate), yeast extract, alanine, serine, cysteine, and H₂+ CO₂(autotrophically). In addition to sulfate, the bacterium can use sulfite, thiosulfate, and elemental sulfur as electron acceptors. In the absence of electron acceptors, the bacterium can ferment pyruvate and yeast extract (a yet unrecognized capacity of sulfate reducers) with the formation of acetate and H₂. The G+C content of DNA is 60.8 mol %. The level of DNA–DNA hybridization of the isolate (strain 101^T) and Desulfacinum infernum(strain BG1^T) is as low as 34%. Analysis of the nucleotide sequence of 16S rDNA places strain 101^Tin the phylogenetic cluster of the Desulfacinumspecies within the sulfate reducer subdivision of the delta subclass of Proteobacteria. All these results allowed the bacterium studied to be described as a new species, Desulfacinum subterraneumsp. nov., with strain 101 as the type strain.     

Isolation and characterization of a thermophilic sulfate-reducing bacterium Desulfotomaculum thermoacetoxidans sp. nov.

An obligately anaerobic thermophilic sporeforming sulfate-reducing bacterium, named strain CAMZ, was isolated from a benzoate enrichment from a 58°C thermophilic anaerobic bioreactor. The cells of strain CAMZ were 0.7 m by 2–5 m rods with pointed ends, forming single cells or pairs. Spores were central, spherical, and caused swelling of the cells. The Gram stain was negative. Electron donors used included lactate, pyruvate, acetate and other short chain fatty acids, short chain alcohols, alanine, and H₂/CO₂. Lactate and pyruvate were oxidized completely to CO₂ with sulfate as electron acceptor. Sulfate was required for growth on H₂/CO₂, and both acetate and sulfide were produced from H₂/CO₂-sulfate. Sulfate, thiosulfate, or elemental sulfur served as electron acceptors with lactate as the donor while sulfite, nitrate, nitrite, betaine, or a hydrogenotrophic methanogen did not. The optimum temperature for growth of strain CAMZ was 55–60°C and the optimum pH value was 6.5. The specific activities of carbon monoxide dehydrogenase of cells of strain CAMZ grown on lactate, H₂/CO₂, or acetate with sulfate were 7.2, 18.1, and 30.8 mol methyl viologen reduced min^–1 [mg protein]^–1, respectively, indicating the presence of the CO/Acetyl-CoA pathway in this organism. The mol%-G+C of strain CAMZ's DNA was 49.7. The new species name Desulfotomaculum thermoacetoxidans is proposed for strain CAMZ.     

Methanogenic degradation of acetone by an enrichment culture

An anaerobic enrichment culture degraded 1 mol of acetone to 2 mol of methane and 1 mol of carbon dioxide. Two microorganisms were involved in this process, a filament-forming rod similar to Methanotrix sp. and an unknown rod with round to slightly pointed ends. Both organisms formed aggregates up to 300 m in diameter. No fluorescing bacteria were observed indicating that hydrogen or formate-utilizing methanogens are not involved in this process. Acetate was utilized in this culture by the Methanothrix sp. Inhibition of methanogenesis by bromoethanesulfonic acid or acetylene decreased the acetone degradation rate drastically and led to the formation of 2 mol acetate per mol of acetone. Streptomycin completely inhibited acetone degradation, and neither acetate nor methane was formed. ¹⁴CO₂ was incorporated exclusively into the C-1 atom of acetate indicating that acetone is degraded via carboxylation to an acetoacetate residue. It is concluded that acetone is degraded by a coculture of an eubacterium and an acetate-utilizing methanogen and that acetate is the only intermediate transferred between both. The energetical problems of the eubacterium converting acetone to acetate are discussed.     

Carbon monoxide pathway enzyme activities in a thermophilic anaerobic bacterium grown acetogenically and in a syntrophic acetate-oxidizing coculture

We recently isolated an acetate-oxidizing rodshaped eubacterium (AOR) which was capable of oxidizing acetate to CO₂ when grown in coculture with the hydrogenotrophic methanogen Methanobacterium sp. strain THF. The AOR was also capable of growing axenically on H₂CO₂ which it converted to acetate. Previous results for the acetate oxidizing coculture showed isotopic exchange between acetate and CO₂, suggesting that the AOR was using a pathway for acetate oxidation resembling a reveral of the acetogenic (carbon monoxide) pathway. In this study, it was found that production of ¹⁴CO₂ from ¹⁴CH₃COO^- by the coculture was inhibited by 200 M cyanide, while methanogenesis from H₂–CO₂ was unaffected, implying the involvement of carbon monoxide dehydrogenase (CODH) in acetate oxidation. CODH was present at 0.055 mol methyl viologen reduced min^-1 mg^-1 protein in extracts of Methanobacterium sp. strain THF, but was present in higher levels in the acetate oxidizing coculture and in the AOR grown axenically and on H₂–CO₂ (2.0 and 6.4 mol min^-1 mg^-1 protein respectively). Anaerobic activity stains for CODH in native polyacrylamide gels from the AOR coculture showed components co-migrating with bands from both organisms, as well as an additional band in extracts of the coculture. Formate dehydrogenase (FDH) was present in both the AOR coculture and monoculture but not in extracts of H₂–CO₂ grown cells of Methanobacterium sp. strain THF. Formyltetrahydrofolate (FTHF) synthetase was not detectable in extracts of the AOR monoculture or coculture, although it was found in high amounts in extracts of H₂–CO₂ grown cells of the thermophilic acetogen Acetogenium kivui. Extracts of H₂–CO₂ grown cells of the AOR showed a fluorescence spectrum typical of pterin derivatives. Bioassay for folates showed levels to be at anabolic rather than catabolic levels. It is possible that the AOR uses pterins distinct from folate for catabolism. Isocitrate dehydrogenase, a citric acid cycle enzyme, was also present in the AOR, but at anabolic levels and -ketoglutarate dehydrogenase was not detectable.Abbreviations (AOR) acetate-oxidizing rod - (CODH) carbon monoxide dehydrogenase - (FDH) formate dehydrogenase - (FTHF) formyltetrahydrofolate     

Reductive cleavage of sarcosine and betaine by Eubacterium acidaminophilum via enzyme systems different from glycine reductase

The obligate anaerobe Eubacterium acidaminophilum metabolized the glycine derivatives sarcosine (N-monomethyl glycine) and betaine (N-trimethyl glycine) only by reduction in a reaction analogous to glycine reductase. Using formate as electron donor, sarcosine and betaine were stoichiometrically reduced to acetate and methylamine or trimethylamine, respectively. The N-methyl groups of the cosubstrates or of the amines produced were not transformed to CO₂ or acetate. Under optimum conditions (formate/acceptor ratio of 1 to 1.2, 34°C, pH 7.3) the doubling times were 4.2 h on formate/sarcosine and 3.6 h on formate/betaine. The molar growth yields were 8.15 and 8.5 g dry cell mass per mol sarcosine and betaine, respectively. The assays for sarcosine reductase and betaine reductase were optimized in cell extracts; NADPH was preferred as physiological electron donor compared to NADH, dithioerythritol was used as artificial donor; no requirements for AMP and ADP could be detected. Growth experiments mostly revealed diauxic substrate utilization pattern using different combinations of glycine, sarcosine, and betaine (plus formate) and inocula from different precultures. Glycine was always utilized first, what coincided with the presence of glycine reductase activity under all growth conditions except for serine as substrate. Sarcosine reductase and betaine reductase were only induced when E. acidaminophilum was grown on sarcosine and betaine, respectively. Creatine was metabolized via sarcosine. [⁷⁵Se]-selenite labeling revealed about the same pattern of predominant labeled proteins in glycine-, sarcosine-, and betaine-grown cells.Abbreviations DTE dithioerythritol - TES N-Tris (hydroxymethyl) methyl-2-amino-ethane sulfonic acid     

Pyruvate fermentation in Rhodospirillum rubrum and after transfer from aerobic to anaerobic conditions in the dark

The fermentative metabolism of Rhodospirillum rubrum (strain Ha, F₁, S₁) was studied after transfering the cells from aerobic to anaerobic dark culture conditions. Pyruvate was metabolized mainly to acetate and formate, and to a lesser extent to CO₂ and propionate, by all strains. Therefore, pyruvate formate lyase would appear to be the characteristic key enzyme of the dark anaerobic fermentation metabolism in R. rubrum. Strain F₁ and S₁ metabolized the formate further to H₂ and CO₂. It is concluded that this cleavage was catalysed by a formate hydrogen lyase system. Strain Ha was unable to metabolize formate. The cleavage of formate and the synthesis of poly--hydroxy-butyric acid were increased by a low pH value (6.5). Fermentation equations and schemes of the pyruvate metabolism are discussed.     

Growth yields and energy generation by Campylobacter sputorum subspecies bubulus during growth in continuous culture with different hydrogen acceptors

Campylobacter sputorum subspecies bubulus was grown in continuous culture with excess of l-lactate or formate, and growth-limiting amounts of oxygen, fumarate, nitrate or nitrite. l-Lactate was oxidized to acetate, fumarate was reduced to succinate, and nitrate and nitrite were reduced to ammonia. The Y _lactate values (g dry weight bacteria/g mol lactate) for the respective hydrogen acceptors were much higher than the Y _formate values. Steady state cultures on formate and nitrite could only be obtained at a low dilution rate and low nitrite concentrations in the growth medium. In H⁺/2e measurements with lactate-grown cells proton ejections were observed with lactate or pyruvate as a hydrogen donor, and oxygen or hydrogen peroxide as a hydrogen acceptor. Proton ejection was also observed with pyruvate and nitrate. Proton ejection did not occur with lactate and nitrate, neither with lactate or pyruvate and fumarate or nitrite. With formate as a hydrogen donor acidification occurred with all hydrogen acceptors mentioned. It has been concluded that during growth on lactate and fumarate or nitrite substrate level phosphorylation at acetate formation is the sole ATP-generating system. Growth on formate and fumarate or nitrite is explained by a proton gradient generated as a result of oxidation of formate at the periplasmic side of the cytoplasmic membrane. With oxygen and nitrate additional ATP is formed by electron transport-linked phosphorylation. The low molar growth yields with formate are explained by the observation that formate-grown cells had a great permeability to protons.Abbreviations H⁺/2e value number of protons ejected per electron pair transported in the respiratory system - P/2e value mol of ATP formed per electron pair transported in the respiratory system - CCCP carbonyl cyanide m-chlorophenyl-hydrazone     

Growth yields of Desulfotomaculum orientis with hydrogen in chemostat culture

Desulfotomaculum orientis (strain Singapore 1) was grown autotrophically with H₂+CO₂ and sulfate, thiosulfate or sulfite as electron acceptor in sulfide- and pH-controlled continuous culture. Under sulfate-limiting conditions real growth yields of up to 9.7 g cell dry mass per mol sulfate were obtained. Electron acceptor limitation resulted in the excretion of up to 14.5 mmol acetate per liter, formed by reduction of CO₂ with H₂. Acetate production was not coupled to an increase of growth yields: under hydrogen-limiting conditions only 1.6 mmol acetate per liter was produced, and even higher growth yields of up to 12,4 g cell dry mass per mol sulfate were obtained. With thiosulfate or sulfite as electron acceptor growth yields increased up to 17.9 g cell dry mass per mol electron acceptor. Growth yields were not simply correlated with the growth rate, and did not allow the determination of maintenance coefficients and the extrapolation to maximal yields at infinite growth rate (Y ^max). The maximal growth rates (_max) with sulfate and thiosulfate were 0.090 and 0.109 h^-1, respectively, if cells were grown continuously in sulfidostat culture under nonlimiting conditions.The net energy yield of sulfate reduction and the energy requirement for the activation of sulfate by Desulfotomaculum orientis are discussed.     

Energetics and regulations of formate and hydrogen metabolism by Methanobacterium formicicum

Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium formicicum species on H₂–CO₂. Hydrogen was also produced during formate metabolism by M. formicicum. The amount of formate accumulated in the medium or the amount H₂ released in gas phase was influenced by the bicarbonate concentration. The formate hydrogenlyase system was constitutive but regulated by formate. When methanogenesis was inhibited by addition of 2-bromoethane sulfonate, M. formicicum synthesized formate from H₂ plus HCO _inf3 ^sup- or produced H₂ from formate to a steady-state level at which point the Gibbs free energy (G) available for formate synthesis or H₂ production was approximately -2 to -3 kJ/reaction. Formate conversion to methane was inhibited in the presence of high H₂ pressure. The relative rates of conversion of formate and H₂ were apparently controlled by the G available for formate synthesis, hydrogen production, methane production from formate and methane production from H₂. Results from ¹⁴C-tracer tests indicated that a rapid isotopic exchange between HCOO^- and HCO _inf3 ^sup- occurred during the growth of M. formicicum on H₂–CO₂. Data from metabolism of ¹⁴C-labelled formate to methane suggested that formate was initially split to H₂ and HCO _inf3 ^sup- and then subsequently converted to methane. When molybdate was replaced with tungstate in the growth media, the growth of M. formicicum strain MF on H₂–CO₂ was inhibited although production of methane was not Formate synthesis from H₂ was also inhibited.     

Acetonema longum gen.nov.sp.nov., an H2/CO2 acetogenic bacterium from the termite,Pterotermes occidentis

A previously undescribed, H₂-oxidizing CO₂-reducing acetogenic bacterium was isolated from gut contents of the wood-feeding termite, Pterotermes occidentis. Cells of representative strain APO-1 were strictly anaerobic, Gram-negative, endospore-forming motile rods which measured 0.30–0.40×6–60 m. Cells were catalase positive, oxidase negative, and had 51.5 mol percent G+C in their DNA. Optimum conditions for growth on H₂+CO₂ were at 30–33°C and pH (initial) 7.8, and under these conditions cells formed acetate according to the equation: 4 H₂+2 CO₂CH₃COOH+2 H₂O. Other energy sources supporting good growth of strain APO-1 included glucose, ribose, and various organic acids. Acetate and butyrate were major fermentation products from most organic compounds tested, however propionate, succinate, and 1,2-propanediol were also formed from some substrates. Based on comparative analysis of 16S rRNA nucleotide sequences, strain APO-1 was related to, but distinct from, members of the genus Sporomusa. Moreover, physiological and morphological differences between strain APO-1 and the six known species of Sporomusa were significant. Consequently, it is proposed herewith that a new genus, Acetonema, be established with strain APO-1 as the type strain of the new species, Acetonema longum. A. longum may contribute to the nutrition of P. occidentis by forming acetate, propionate and butyrate, compounds which are important carbon and energy sources for termites.     

Acetate and carbon dioxide assimilation by Desulfovibrio vulgaris (Marburg), growing on hydrogen and sulfate as sole energy source

Desulfovibrio vulgaris (Marburg) was grown on hydrogen plus sulfate as sole energy source and acetate plus CO₂ as the sole carbon sources. The incorporation of U-¹⁴C acetate into alanine, aspartate, glutamate, and ribose was studied. The labelling data show that alanine is synthesized from one acetate (C-2 + C-3) and one CO₂ (C-1), aspartate from one acetate (C-2 + C-3) and two CO₂ (C-1 + C-4), glutamate from two acetate (C-1–C-4) and one CO₂ (C-5), and ribose from 1.8 acetate and 1.4 CO₂. These findings indicate that in Desulfovibrio vulgaris (Marburg) pyruvate is formed via reductive carboxylation of acetyl-CoA, oxaloacetate via carboxylation of pyruvate or phosphoenol pyruvate, and -ketoglutarate from oxaloacetate plus acetyl-CoA via citrate and isocitrate. Since C-5 of glutamate is derived from CO₂, citrate must have been formed via a (R)-citrate synthase rather than a(S)-citrate synthase. The synthesis of ribose from 1.8 mol of acetate and 1.4 mol of CO₂ excludes the operation of the Calvin cycle in this chemolithotrophically growing bacterium.     

Selenomonas acidaminovorans sp. nov., a versatile thermophilic proton-reducing anaerobe able to grow by decarboxylation of succinate to propionate

A moderately thermophilic anaerobic bacterium (strain Su883), which decarboxylated succinate to propionate, was isolated from granular methanogenic sludge. The bacterium appeared to ferment a number of amino acids including glutamate, histidine, arginine, ornithine, citrulline, and threonine to propionate, acetate and hydrogen. Propionate was formed via the oxidative decarboxylation of -ketoglutarate to succinyl-CoA. In addition, the strain degraded glucose, fructose, glycerol, pyruvate, serine, alanine, citrate and malate to acetate, carbon dioxide and hydrogen, and branched-chain amino acids to branched-chain fatty acids. With all single substrates solely hydrogen was formed as reduced fermentation product. Mixed cultures of strain Su883 and Methanobacterium thermoautotrophicum H showed a more rapid conversion of substrates and with some substrates a shift from acetate to propionate formation.Strain Su883 is a motile, gram-negative, non-sporeforming, slightly curved rod with a DNA base ratio of 56.5 mol% guanine-plus-cytosine. Selenomonas acidaminovorans Su883 is proposed as type strain for the new species within the genus Selenomonas.     

Autotrophic growth and CO2 fixation of Chloroflexus aurantiacus

Chlorofluexus aurantiacus OK-70 fl was grown photoautotrophically with hydrogen as the electron source. The lowest doubling time observed was 26 h.The mechanism of CO₂ fixation in autotrophically grown cells was studied. The presence of ribulose-1,5-bis-phosphate carboxylase and phosphoribulokinase could not be demonstrated. Carbon isotope fractionation (¹³C) was small, and alanine and aspartate but not 3-phosphoglycerate were the major labelled compounds in short term ¹⁴CO₂ labelling. Thus CO₂ is not fixed by the Calvin cycle.Fluoroacetate (FAc) completely inhibited protein synthesis in cultures and caused a slight citrate accumulation. However, CO₂ fixation continued and increased polyglucose formation occurred. Under these conditions added acetate was metabolized to polyglucose, as were glycine, serine, glyoxylate and succinate, but to a lesser extent; little or no formate or CO was utilised.Glyoxylate inhibited CO₂ fixation in vivo, indicating that pyruvate is formed from acetyl-CoA and CO₂ by pyruvate synthase. Two key enzymes of the reductive TCA cycle, citrate lyase and -ketoglutarate synthase were not detected in cell free extracts, but pyruvate synthase and phosphoenolpyruvate carboxylase were demonstrated. It is concluded that acetyl-CoA is a central intermediate in the CO₂ fixation process, but the mechanism of its synthesis is not clear.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase - TCA cycle tricarboxylic acid cycle - FAc monofluoroacetate - PEP phosphoenolpyruvate - MV methyl viologen - TTC triphenyltetrazolium chloride - PMS phenazine methosulfate     

Amino acid and glucose fermentation by Treponema denticola

Summary Treponema denticola was grown in serum-containing media to which ¹⁴C-labelled compounds were added. Determinations of radioactivity in the products formed indicated that the organism fermented alanine, cysteine, glycine, serine, and glucose. Fermentation products included acetate, lactate, succinate, formate, pyruvate, ethanol, CO₂, H₂S, and NH₃. The products formed from glucose constituted a small portion of the total products. Assays of enzymatic activities in cell extracts indicated that the organism degraded glucose via the Embden-Meyerhof pathway. T. denticola possessed a coenzyme A-dependent CO₂-pyruvate exchange activity associated with a clostridial-type clastic system for pyruvate metabolism. Phosphotransacetylase and acetate kinase activities were present in cell extracts. Acetyl phosphate formation and benzyl viologen reduction were detected when cell extracts were incubated with pyruvate, serine or cysteine. The data indicate that T. denticola is an amino acid fermenter and that it possesses the enzymes needed for the fermentation of glucose. However, glucose does not serve as the primary substrate when the organism grows in media including both this carbohydrate and amino acids.     

Growth parameters (K s, μmax,Y s) of Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum was grown on a mineral salts medium in a fermenter gassed with H₂ and CO₂, which were the sole carbon and energy sources. Under the conditions used the bacterium grew exponentially. The dependence of the growth rate () on the concentration of H₂ and CO₂ in the incoming gas and the dependence of the growth yield ( ) on the growth rate were determined at pH 7 (the pH optimum) and 65° C (the temperature optimum).The curves relating growth rate to the H₂ and CO₂ concentration were hyperbolic. From reciprocal plots apparent K _s values for H₂ and CO₂ and _max were obtained: app. = 20%; app. = 11%; = 0.69 h^-1; t (max)=1 h. was 1.6 g mol^-1 and almost independent of the growth rate, when the rate of methane formation was not limited by the supply of either H₂ or CO₂. The yield increased to near 3 g mol^-1 when H₂ or CO₂ were limiting. These findings indicate that methane formation and growth are less tightly coupled at high concentrations of H₂ or CO₂ in the medium than at low concentrations. The physiological significance of these findings is discussed. K _s: H₂ and CO₂ concentration supporting 0.5 _max; _max: specific growth rate at infinite substrate concentration; Y _s:growth yield (g dry weight/mol substrate); t : doubling time     

Growth yield increase linked to caffeate reduction in Acetobacterium woodii

Growth yields were determined with Acetobacterium woodii strain NZva 16 on hydrogen and CO₂, formate, methanol, vanillate, ferulate and fructose in mineral medium in the absence and presence of 0.05% yeast extract. Yeast extract was not essential for growth but enhanced growth yields by 25–100% depending on the substrate fermented. Comparison of yields on formate or methanol allowed calculation of an energy yield in the range of 1.5–2 mol ATP per mol acetate formed during homoacetate fermentation of A. woodii. In the presence of 6 mM caffeate, growth yields were determined with the substrates formate or methanol. Caffeate was reduced to hydrocaffeate and increased growth yields were obtained. An ATP yield of about 1 mol per mol of caffeate reduced was calculated. Cytochromes were not detectable in cell free extracts or membrane preparations.     

Mixotrophy in the termite gut acetogen,Sporomusa termitida

Cell suspensions of H₂/CO₂-grown Sporomusa termitida catalyzed an H₂-supported synthesis of acetate from CO₂ at rates of about 1 mol acetate x h^-1 x mg protein^-1. Cells pre-grown on methanol, mannitol, lactate, or glycine also displayed H₂-supported acetogenesis from CO₂, although at rates 5–85% that of H₂/CO₂-grown cells. With methanol-grown cell suspensions: the presence of methanol greatly stimulated the rate of H₂-supported conversion of ¹⁴CO₂ to ¹⁴C-acetate (which became labeled mainly in the COOH-group); and like-wise the presence of H₂ stimulated the conversion of ¹⁴CH₃OH+CO₂ to ¹⁴C-acetate (which became labeled mainlyan the CH₃-group). Analogous stimulatory effects were observed for cell suspensions pre-grown on methanol + CO₂+H₂. Furthermore, when H₂ (+CO₂) was included as a growth substrate with either methanol or lactate: both substrates were used simultaneously; there was no diauxie in the growth of cells or in acetate production; and the molar growth yield of S. termitida was close to that predicted from summation of the yields observed when grown with each substrate alone. These data indicated that S. termitida can grow by mixotrophy, i.e. by the simultaneous use of H₂/CO₂ and organic compounds for energy. Results are discussed in light of the ability of H₂/CO₂ acetogens to outprocess methanogens in H₂ consumption in the hindgut fermentation of wood-feeding termites.