Sporomusa termitida sp. nov., an H2/CO2-utilizing acetogen isolated from termites

H₂-oxidizing CO₂-reducing acetogenic bacteria were isolated from gut contents of Nasutitermes nigriceps termites. Isolates were strictly anaerobic, Gram negative, endospore-forming, straight to slightly curved rods (0.5–0.8×2–8 m) that were motile by means of lateral flagella. Cells were oxidase negative, but catalase positive and possessed a b-type cytochrome(s) associated with the cell membrane. Cells grew anaerobically with H₂+CO₂ as energy source and catalyzed a total synthesis of acetate from this gas mixture. H₂ uptake by a representative isolate (strain JSN-2) displayed a K _m=6 M and V _max=380 nmol x min^-1 x mg protein^-1. Other substrates used as energy sources for growth and acetogenesis included CO, methanol, betaine, trimethoxybenzoate, and various other organic acids. Succinate was also fermented, but propionate was formed from this substrate instead of acetate. Of a variety of sugars and sugar alcohols tested, only mannitol supported growth. Cells grew optimally at 30° C and pH 7.2 and required yeast extract or a source of amino acids (e.g. Casamino acids) for good growth. During initial enrichment and isolation, cells appeared sensitive to various reducing agents commonly employed in media for anaerobes. The DNA base composition of strain JSN-2 was 48.6 mol% G+C. On the bases of cell morphology, substrate utilization spectrum, and DNA base composition, strain JSN-2 is here-with proposed as the type strain of the new species Sporomusa termitida.Journal article no. 12513 from the Michigan Agricultural Experiment Station     

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.     

Desulfurella multipotens sp. nov., a new sulfur-respiring thermophilic eubacterium from Raoul Island (Kermadec archipelago,New Zealand)

A new moderately thermophilic sulfur-reducing eubacterium was isolated from bottom deposits of Green Lake (Raoul Island, Kermadec archipelago, New Zealand). Cells are short rods, 1.5–1.8 by 0.5–0.7 m, single or in pairs, motile with one polar flagellum, gram-negative with S-layer of subunit structure. Growth occurred between 42 and 77°C with the optimum at 58–60°C and at pH from 6.0 to 7.2 with the optimum at 6.4–6.8. The bacterium was obligately anaerobic and obligately sulfur-respiring, and capable of lithoautotrophic growth on a mineral medium with S° and H₂/CO₂ gas phase. In addition to molecular hydrogen, a wide range of substrates can be utilized as energy source in the presence of elemental sulfur: pyruvate, acetate, butyrate, pentadecanate, palmitate, stearate. Products are CO₂ and H₂S. The G+C content of DNA is 33.5 mol%. DNA-DNA homology with the type species of the genus Desulfurella — Desulfurella acetivorans — is 69±2%. A new species, Desulfurella multipotens sp. nov., with the type strain RH-8 is described.     

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.     

Clostridium mayombei sp. nov., an H2/CO2 acetogenic bacterium from the gut of the African soil-feeding termite,Cubitermes speciosus

Clostridium mayombei sp. nov., a previously undescribed H₂-oxidizing CO₂-reducing acetogenic bacterium, was isolated from gut contents of the African soilfeeding termite, Cubitermes speciosus. Cells were anaerobic, Gram positive, catalase and oxidase negative, endospore-forming motile rods which measured 1×2 – 6 m and which had a DNA base composition of 25.6 mol% G+C (strain SFC-5). Optimum conditions for growth on H₂+CO₂ were at 33°C and pH 7.3, and under these conditions cells produced acetate according to the equation: 4 H₂+2 CO₂CH₃COOH+2 H₂O. Other substrates supporting good growth included carbohydrates (e.g. glucose, xylose, starch), sugar alcohols, and organic and amino acids, and with these substrates acetate was almost always the principle fermentation product. Comparative analysis of 16S rRNA nucleotide sequences confirmed that C. mayombei was closely related to various members of the genus Clostridium. However, morphological and physiological differences between C. mayombei and other homoacetogenic clostridia were deemed significant enough to warrant creation of a new taxon. Results are discussed in light of the diversity of H₂/CO₂ acetogens recently isolated from various termites, and in terms of the relative importance of H₂/CO₂ acetogenesis to termite nutrition.     

Desulfosporomusa polytropa gen. nov., sp. nov., a novel sulfate-reducing bacterium from sediments of an oligotrophic lake

Five strains of sulfate-reducing bacteria were isolated from the highest positive dilutions of a most probable number (MPN) series supplemented with lactate and inoculated with sediments from the oligotrophic Lake Stechlin. The isolates were endospore-forming and were motile by means of laterally inserted flagella. They stained Gram-negative and contained b-type cytochromes. CO difference spectra indicated the presence of P582 as a sulfite reductase. Phylogenetic analyses of the 16S rDNA sequences revealed that the isolates were very closely affiliated with the genus Sporomusa. However, sulfate and amorphous Fe(OH)₃, but not sulfite, elemental sulfur, MnO₂, or nitrate were used as terminal electron acceptors. Homoacetogenic growth was found with H₂/CO₂ gas mixture, formate, methanol, ethanol, and methoxylated aromatic compounds. The strains grew autotrophically with H₂ plus CO₂ in the presence or absence of sulfate. Formate, butyrate, several alcohols, organic acids, carbohydrates, some amino acids, choline, and betaine were also utilized as substrates. The growth yield with lactate and sulfate as substrate was 7.0 g dry mass/mol lactate and thus two times higher than in sulfate-free fermenting cultures. All isolates were able to grow in a temperature range of 4–37°C. Physiologically and by the presence of a Gram-negative cell wall, the new isolates resemble known Desulfosporosinus species. However, phylogenetically they are affiliated with the Gram-negative genus Sporomusa belonging to the Selenomonas subgroup of the Firmicutes. Therefore, the new isolates reveal a new phylogenetic lineage of sulfate-reducing bacteria. A new genus and species, Desulfosporomusa polytropa gen. nov., sp. nov. is proposed.Dedicated to Prof. H. G. Schlegel on the occasion of his 80th birthday.     

Slostridium homopropionicum sp. nov., a new strict anaerobe growing with 2-, 3-, or 4-hydroxybutyrate

From anoxic sewage sludge a new strictly anaerobic, spore-forming bacterium was isolated with 2-hydroxybutyrate as sole substrate. 2-, 3-, and 4-hydroxybutyrate, 4-chlorobutyrate, crotonate, vinylacetate, and pyruvate were fermented to acetate and butyrate. Fructose was converted to acetate, butyrate, butanol, and H₂. Lactate and acrylate were fermented to acetate and propionate. Cells pregrown with lactate fermented 2-hydroxybutyrate to butyrate, propionate and acetate. No inorganic electron acceptors were reduced. The DNA base ratio was 32.0±1.0 mol % and was similar to that of Clostridium propionicum, which was determined to be 35.3±0.5 mol %. Strain LuHBu1 is described as type strain of a new species, Clostridium homopropionicum sp. nov. Another isolate obtained from marine sediment degraded 2-and 3-hydroxybutyrate to acetate and butyrate and was in some respects similar to the known species Ilyobacter polytropus.     

Thermohydrogenium kirishiense gen. nov. and sp. nov., a new anaerobic thermophilic bacterium

Six strains of a new anaerobic thermophilic non-sporeforming bacterium were isolated in pure culture from industrial yeast biomass. Cells were rod-shaped (0.4–0.8×1.0–11.0 m), non-motile. They stained gram-negative, but outer membrane was not present. The growth occurred between 45–75 °C, the optimal temperature is 65°. Optimal pH value was 7.0–7.4. The bacterium utilized for growth several sugars, starch and yeast extract. The best source of nitrogen was peptone. The main fermentation products of glucose were ethanol, acetate, H₂ and CO₂. As minor products isopropanol, butanol, butyrate and lactate were found. Glucose was metabolized via the Embden-Meyerhoff pathway. Cytochromes and quinones were not found. DNA-base composition was 33.2–34.0 mol%. The DNA-DNA hybridization and 5S rRNA nucleotide sequences showed distantly related of isolated stains to phenotypical similar bacteria. It was proposed to consider the isolated bacterium as Thermohydrogenium kirishiense gen. nov. and sp. nov.     

Methanogenium liminatans spec. nov., a new coccoid,mesophilic methanogen able to oxidize secondary alcohols

A new mesophilic, coccoid methanogen, assigned as Methanogenium liminatans spec. nov. strain DSM 4140, was isolated from effluent of a reactor for the anaerobic treatment of industrial waste water. Cells of M. Liminatans formed irregular cocci, about 1.5 m in size, and occurred singly. The cell envelope was an S-layer with hexagonally arranged glycoprotein subunits (M_r=118000). The center-to-center spacings were 15.4 nm. The polar lipid pattern was similar to that of Methanogenium tationis, the polyamine content similar to that found in several Methanogenium species. Strain DSM 4140 grew with H₂/CO₂, formate, 2-propanol/CO₂, 2-butanol/CO₂ and cyclopentanol/CO₂. For growth with the different substrates acetate was required as an additional carbon source. Growth on H₂/CO₂ was stimulated by the addition of tungstate. The optimal concentration was 1–2 M Na₂WO₄. ¹⁸⁵WO _inf4 ^sup2- was incorporated into cells. Growth was not influenced by 0–600 mM NaCl, but no growth occurred in the presence of 800 mM NaCl. Increasing concentrations of KCl up to 100 mM were slightly inhibitory for growth. The optimal growth temperature was around 40°C. The G+C content of the DNA was 59.3 mol% (T_m) or 60.5 mol% (HPLC).Abbreviations HPLC = high performance liquid chromatography - PAS = periodic acid-Schiff reagent - sADH = secondary alcohol dehydrogenase - F₄₂₀ = 7,8-didemethyl-8-hydroxy-5-deazariboflavin     

Ilyobacter delafieldii sp. nov., a metabolically restricted anaerobic bacterium fermenting PHB

Enrichments from an estuarine sediment with crotonate as substrate resulted in the isolation of a motile, gram-negative, obligately anaerobic rod with pointed ends, designated strain 10cr1. The organism was asporogenous, did not reduce sulfur, sulfate, thiosulfate, nitrate, oxygen or fumarate, and had a mol %G+C ratio of 29. Strain 10cr1 was able to ferment crotonate, 3-hydroxybutyrate, lactate, pyruvate, and poly--hydroxybutyric acid (PHB). Acetate, propionate, butyrate, CO₂ and H₂ were the fermentation products. When grown on PHB there was accumulation of 3-hydroxybutyrate once growth had ceased, indicating degradation of PHB to the monomer. The 3-hydroxybutyrate formed during growth of the culture was fermented to acetate, butyrate and H₂. Experimental evidence suggested the production of an extracellular PHB depolymerase. The cells were not attached to the PHB granules. This is the first isolation of an anaerobic bacterium capable of degrading exogenous PHB. This strain is described as a new species, Ilyobacter delafieldii sp. nov., and strain 10cr1 (=DSM 5704) is designated as the type (and at present, only) strain.Abbreviations G+C guanine plus cytosine - OD optical density - PHB poly--hydroxybutyric acid - specific growth rate - HPLC high-performance liquid chromatography - YE yeast extract     

Thermoanaerobium lactoethylicum spec. nov. a new anaerobic bacterium from a hot spring of Kamchatka

A new anaerobic thermophilic Gram-positive, nonsporeforming bacterium strain ZE-1 was isolated from a hot spring of Kamchatka (USSR). The cells are rod-shaped, (0.5–0.8 · 2.0–20 m), non-motile. The bacterium can grow between 42 and 75°C; the optimal temperature is 65°C. The growth is possible between pH values 5.0 and 8.5; optimal pH is 7.0. The cultures grow on the media containing peptone, yeast extract, or casein hydrolysate as nitrogen sources in the presence of glucose or some other sugars, mannitol or starch. The main fermentation products of glucose are ethanol, acetate, lactate, H₂, CO₂; byproducts are propionic, butyric and isovaleric acids. Glucose is metabolized via Embden-Meyerhoff-Parnas pathway. Molecular hydrogen does not inhibit growth. The bacterium does not reduce aceton to isopropanol, but is able to form H₂S from elemental sulfur. The bacterium contains a soluble hydrogenase. This enzyme catalyzes both evolution and uptake of H₂ and is active in the presence of methyl viologen. The DNA-base composition is 34.6 mol%; the genome size 2.08x10⁹ D. The name proposed for the isolated bacterium strain ZE-1 is Thermoanaerobium lactoethylicum spec. nov.     

Catalysis of an isotopic exchange between CO2 and the carboxyl group of acetate by Methanosarcina barkeri grown on acetate

Cell suspensions of Methanosarcina barkeri (strain Fusaro) grown on acetate were found to catalyze the formation of methane and CO₂ from acetate (30–40 nmol/min·mg protein) and an isotopic exchange between the carboxyl group of acetate and ¹⁴CO₂ (30–40 nmol/min·mg protein). An isotopic exchange between [¹⁴C]-formate and acetate was not observed. Cells grown on methanol mediated neither methane formation from acetate nor the exchange reactions. The data indicate that the isotopic exchange between CO₂ and the carboxyl group of acetate is a partial reaction of methanogenesis from acetate. Both reactions were completely inhibited by low concentrations of cyanide (20 M) or of hydrogen (0.5% in the gas phase). Methane formation from acetate was also completely inhibited by low concentrations of carbon monoxide (0.2% in the gas phase) whereas only significantly higher concentrations of CO had an effect on the exchange reaction. In the concentration range tested KCN, H₂ and CO had no effect on methane formation from methanol or from H₂ and CO₂; however, cyanide (20 M) also affected methane formation from CO. The results are discussed with respect to proposed mechanisms of methane and CO₂ formation from acetate.     

Methanogenium marinum sp. nov., a H2-using methanogen from Skan Bay,Alaska, and kinetics of H2 utilization

A methanogen, strain AK-1, was isolated from permanently cold marine sediments, 38- to 45-cm below the sediment surface at Skan Bay, Alaska. The cells were highly irregular, nonmotile coccoids (diameter, 1 to 1.2 μm), occurring singly. Cells grew by reducing CO₂ with H₂ or formate as electron donor. Growth on formate was much slower than that on H₂. Acetate, methanol, ethanol, 1- or 2-propanol, 1- or 2-butanol and trimethylamine were not catabolized. The cells required acetate, thiamine, riboflavin, a high concentration of vitamin B₁₂, and peptones for growth; yeast extract stimulated growth but was not required. The cells grew fastest at 25 °C (range 5 °C to 25 °C), at a pH of 6.0 – 6.6 (growth range, pH 5.5 – 7.5), and at a salinity of 0.25 – 1.25 M Na⁺. Cells of this and other H₂-using methanogens from saline environments metabolized H₂ to a very low threshold pressure (less than 1 Pa) that was dependent on the methane partial pressure. We propose that the threshold pressure may be limited by the energetics of catabolism. The sequence of the 16S rDNA gene of strain AK-1 was most similar (98%) to the sequences of Methanogenium cariaci JR-1 and Methanogenium frigidum Ace-2. DNA–DNA hybridization between strain AK-1 and these two strains showed only 34.9% similarity to strain JR-1 and 56.5% similarity to strain Ace-2. These analyses indicated strain AK-1 should be classified as a new species within the genus Methanogenium. Phenotypic differences between strain AK-1 and these strains (including growth temperature, salinity range, pH range, and nutrient requirements) support this. Therefore, a new species, Methanogenium marinum, is proposed with strain AK-1 as type strain. This revised version was published online in August 2006 with corrections to the Cover Date.     

Improvement on laboratory fermentation equipment to study Saccharomyces cerevisiae metabolism

Beside being an ordinary fermenter, the present equipment was conceived to sample the medium, to store the samples and to record photographs of the yeasts. Ten sensors were used to measure gas exchanges. During the growth of ScM1 (a Saccharomyces cerevisiae strain) on glucose, we could observe two different linear decreases of CO₂ production rates (18.17±0.12 mmol CO₂ h^–2 (g biomass)^–1 and 8.67±0.12 mmol CO₂ h^–2 (g biomass)^–1), together with a sudden variation of slope during the respiro-fermentative phase. Nomenclature F_in InletairFlowl h ^–1 F_out OutletgasFlowl h ^–1 _in Inletairtemperature°C_out Outletgastemperature°CP _atm AtmosphericPressuremmHgP _in InletairOverPressuremmHgP _out OutletgasOverPressuremmHgDODissolvedO ₂ mg l^–1 pO₂ PartialPressureO ₂ in Outlet gas % (v/v) pCO₂ PartialPressureCO ₂ in Outlet gas % (v/v) Int(t) Whole number of hours     

New types of acetate-oxidizing,sulfate-reducing Desulfobacter species,D. hydrogenophilus sp. nov., D. latus sp. nov., and D. curvatus sp. nov.

Sulfate-reducing bacteria with oval to rod-shaped cells (strains AcRS1, AcRS2) and vibrio-shaped cells (strains AcRM3, AcRM4, AcRM5) differing by size were isolated from anaerobic marine sediment with acetate as the only electron donor. A vibrio-shaped type (strain AcKo) was also isolated from freshwater sediment. Two strains (AcRS1, AcRM3) used ethanol and pyruvate in addition to acetate, and one strain (AcRS1) grew autotrophically with H₂, sulfate and CO₂. Higher fatty acids or lactate were never utilized. All isolates were able to grow in ammonia-free medium in the presence of N₂. Nitrogenase activity under such conditions was demonstrated by the acetylene reduction test. The facultatively lithoautotrophic strain (AcRS1), a strain (AcRS2) with unusually large cells (2×5 m), and a vibrio-shaped strain (AcRM3) are described as new Desulfobacter species, D. hydrogenophilus, D. latus, and D. curvatus, respectively.     

Ectothiorhodospira halochloris sp. nov., a new extremely halophilic phototrophic bacterium containing bacteriochlorophyll b

A new bacteriochlorophyll b containing phototrophic bacterium was isolated from extremely saline and alkaline soda lakes in Egypt. Enrichment and isolation were performed using a synthetic medium with high contents of sodium carbonate, sodium sulfate and sodium chloride. Photoautotrophic growth occurred with hydrogen sulfide as photosynthetic electron donor. During oxidation of sulfide to sulfate extracellular elemental sulfur globules appeared in the medium. Cells were also capable to grow under photoheterotrophic conditions with acetate, propionate, pyruvate, succinate, fumarate or malate as carbon sources and electron donors. Under these conditions sulfate was assimilated. Optimal growth under the applied experimental conditions occurred at a total salinity of 14–27%, a pH-range between 8.1 and 9.1 and a temperature between 47°C and 50°C. The cells were 0.5–0.6 m wide and, depending on cultural conditions, 2.5–8.0 m long; they were spiral shaped, multiplied by binary fission and were motile by means of bipolar flagella. Intercytoplasmic photosynthetic membranes were present as stacks. Bacteriochlorophyll b was the main photosynthetic pigment; small amounts of carotenoids were mainly present as glucosides of rhodopin and its methoxy derivative. The new organism is described as Ectothiorhodospira halochloris.Dedicated to Professor C. B. van Niel on the occasion of his 80th birthday     

An ammonia-oxidizing bacterium,Nitrosovibrio tenuis nov. gen. nov. sp.

An ammonia-oxidizing, autotroph growing, slender, curved rod was isolated from the soil of Hawaii. It is well distinguishable from any other nitrifying bacteria thus far described by their morphology. The cells are 1.1–3.0 m long and 0.3–0.4 m wide. They are motile by means of 1–4 subpolar to lateral flagella. In contrast to most of the ammonia-oxidizing bacteria the isolated vibrio is void of an extensive cytomembrane system. To categorize this not yet described species we propose to create the new genus Nitrosovibrio and to classify the isolated strain as Nitrosovibrio tenuis.     

ATP formation coupled to caffeate reduction by H2 in Acetobacterium woodii NZva16

We have addressed the question, whether the reduction of caffeate in Acetobacterium woodii strain NZva16 is coupled to ATP synthesis by electron transport phosphorylation. The following results were obtained: 1. Cultures of A. woodii with H₂ and CO₂, grew to greater cell densities, when caffeate was also present. Caffeate was reduced to give hydrocaffeate and less acetate was formed. The cell yield based on the amount of caffeate reduced was approximately 1 g dry cells/mol. 2. Non-growing bacterial suspensions catalyzed the reduction of caffeate by H₂. The specific activity (0.2–1.0 mol · min^–1 · mg^–1 bacterial protein) was as high as expected for a catabolic reaction. 3. The ATP content of bacteria incubated, with H₂ increased from < 1 to about 7 mol per g cellular protein on the addition of caffeate. The ATP yield was calculated as 0.06 mol ATP · mol^–1 caffeate from the initial velocity of ATP formation and the activity of caffeate reduction. Valinomycin together with nigericin inhibited ATP formation and caused a 2–3-fold increase of the activity of caffeate reduction. Protonophores were without, effect. 4. Caffeate in the presence of H₂ caused the uptake of tetraphenylphosphonium cation by the bacteria. The uptake was abolished by valinomycin plus nigericin, and was considerably enhanced by monensin. Protonophores were without effect, even in the presence of monensin. It is concluded that caffeate reduction by H₂ is coupled to ATP formation by electron transport phosphorylation. However, the failure of protonophores to prevent phosphorylation and TPP uptake cannot be explained.Abbreviations Caffeate 3,4-Dihydroxycinnamate - Hydrocaffeate 3,4-dihydroxyphenylpropionate - TPP⁺ tetraphenylphosphonium cation - FCCP carbonylcyanide-4-trifluoromethoxyphenylhydrazone - TTGB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazol - TCS 3,5,3,4-tetrachlorosalicylanilide     

Ethane production by Methanosarcina barkeri during growth in ethanol supplemented medium

Methanosarcina barkeri strain 227 produced ethane during growth on H₂/CO₂ when ethanol was added to the medium in concentrations of 89–974 mM; ethane production varied from 14 to 38 nmoles per tube (20 ml gas phase, 5.7 ml liquid) with increasing ethanol concentrations. Cells grown to mid-logarithmic phase (A₆₀₀ 0.46, protein = 64 g/ml) on H₂/CO₂, thoroughly flushed with H₂/CO₂, then exposed to ethanol, produced maximal ethane levels (at 585 and 974 mM ethanol) of about 215 nmoles per tube, with an ethane/methane ratio of 1×10^-3. Mid-logarithmic-phase cultures of Methanosarcina barkeri strain Fusaro also produced ethane (up to 20 nmoles per tube) when exposed to ethanol. Cultures of strain 227 growing on methanol in the absence of H₂ produced 6 nmoles per tube of ethane when supplemented with ethanol whereas those lacking ethanol but containing H₂ and/or methanol produced 1.6 nmoles per tube. Cultures of Methanococcus deltae strains LH and RC, Methanospirillum hungatei or Methanobacterium thermoautotrophicum produced 5 nmoles ethane per tube when grown in medium containing ethanol. Ethanol concentrations of 177–886 mM were inhibitory to growth of all methanogens examined. Production of ethane by Methanosarcina was inhibited by >62 mM methanol, and both methanogenic inhibitors tested, CCl₄ and Br–CH₂–CH₂–SO _inf3 ^sup- , inhibited ethane and methane production concurrently. The data suggest that ethanol is converted to ethane by Methanosarcina species using the terminal portion of the methanol-to-methane pathway.     

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