Formation of dimethylsulfide and methanethiol from methoxylated aromatic compounds and inorganic sulfide by newly isolated anaerobic bacteria

Formation of gas and of methylated sulfur compounds was observed in anaerobic enrichment cultures with methoxylated aromatic compounds as substrates. Via direct dilution of mud samples in defined reduced media supplemented with trimethoxybenzoate or syringate two new strains of anaerobic homoacetogenic bacteria (strain TMBS4 and strain SA2) were obtained in pure culture. Both strains produced dimethylsulfide and methanethiol during growth on methoxylated aromatic compounds. Growth tests and determination of stoichiometries demonstrated that the volatile sulfur compounds were formed from the methyl group at the aromatic ring and the sulfide added as reducing agent to the medium (R = aromatic residue): 2 R - O - CH₃ + H₂ S 2 R - OH + (CH₃)₂SDimethylsulfide was the major organic sulfur compound formed, whereas methanethiol appeared only as intermediate in small quantities. The isolates grew also with trihydroxybenzenes such as gallate, phloroglucinol, or pyrogallol without formation of methylated sulfur compounds. The aromatic compounds were degraded to acetate. The freshwater strain TMBS4 also fermented pyruvate. Other aliphatic or aromatic compounds were not utilized. External electron acceptors (sulfate, nitrate, fumarate) were not reduced. Both strains were mesophilic and formed rod-shaped, non-motile, Gram-negative cells. Spore formation was not observed. Tentatively, both isolates can be affiliated to the genus Pelobacter.Abbreviations TMB 3,4,5-trimethoxybenzoate - MT methanethiol - DMS dimethylsulfide     

Anaerobic degradation of 3-aminobenzoate by a newly isolated sulfate reducer and a methanogenic enrichment culture

A new rod-shaped, gram-negative, non-sporing sulfate reducer, strain mAB1, was enriched and isolated from marine sediment samples with 3-aminobenzoate as sole electron and carbon source. Strain mAB1 degraded 3-aminobenzoate completely to CO₂ and NH₃ with stoichiometric reduction of sulfate to sulfide. Cells contained carbon monoxide dehydrogenase, cytochromes, and sulfite reductase P582. Strain mAB1 degraded also benzoate, 4-aminobenzoate, hydroxybenzoates, and some aliphatic compounds. Besides sulfates, also sulfite was reduced with 3-aminobenzoate as electron donor, but not thiosulfate, sulfur, nitrate, or fumarate. The strain grew in sulfide-reduced mineral medium supplemented with 7 vitamins. Strain mAB1 was tentatively affiliated with the genus Desulfobacterium. Experiments with dense cell supsensions showed benzoate accumulation during 3-aminobenzoate degradation under conditions of sulfate limitation or cyanide inhibition. 3-Aminobenzoate was activated to 3-aminobenzoyl-CoA by cell extracts in the presence of ATP, coenzyme A, and Mg²⁺. Acitivity of 3-aminobenzoyl-CoA synthetase was 16 nmol per min and mg protein, with a K_M for 3-aminobenzoate lower than 50 M. Cell extract of 3-aminobenzoate-grown cells activated also 3-hydroxybenzoate (31.7 nmol per min and mg protein) and benzoate (2.3 nmol per min and mg protein), but not 2-aminobenzoate or 4-aminobenzoate. In the presence of NADH of NADPH, 3-aminobenzoyl-CoA was further metabolized to a not yet identified reduced product.Freshwater enrichments with 3-aminobenzoate in the absence of an extenal electron acceptor led to a stable methanogenic enrichment culture consisting of three types of bacteria. 3-Aminobenzoate was degraded completely to CO₂ and stoichiometric amounts of CH₄, with intermediary acetate accumulation.     

Anaerobic degradation of 3-hydroxybenzoate by a newly isolated nitrate-reducing bacterium

A Gram-negative nitrate-reducing bacterium, strain Asl-3, was isolated from activated sludge with nitrate and 3-hydroxybenzoate as sole source of carbon and energy. The new isolate was facultatively anaerobic, catalase- and oxidase-positive and polarly monotrichously flagellated. In addition to nitrate, nitrite, N2O, and O2 served as electron acceptors. Growth with 3-hydroxybenzoate and nitrate was biphasic: nitrate was completely reduced to nitrite before nitrite reduction to N2 started. Benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, protocatechuate or phenyl-acetate served as electron and carbon source under aerobic and anaerobic conditions. During growth with excess carbon source, poly-beta-hydroxybutyrate was formed. These characteristics allow the affiliation of strain Asl-3 with the family Pseudomonadaceae. Analogous to the pathway of 4-hydroxybenzoate degradation in other bacteria, the initial step in anaerobic 3-hydroxybenzoate degradation by this organism was activation to 3-hydroxy-benzoyl-CoA in an ATP-consuming reaction. Cell extracts of 3-hydroxybenzoate-grown cells exhibited 3-hydroxybenzoyl-CoA synthetase activity of 190 nmol min-1 mg protein-1 as well as benzoyl-CoA synthetase activity of 86 nmol min-1 mg protein-1. A reductive dehydroxylation of 3-hydroxybenzoyl-CoA could not be demonstrated due to rapid hydrolysis of chemically synthesized 3-hydroxybenzoyl-CoA by cell extracts.     

Fermentation of L-tartrate by a newly isolated gram-negative glycolytic bacterium

Enrichments on L-tartrate from a freshwater lake sediment yielded a pure culture of anaerobic bacterium designated strain 16Lt1. The rod-shaped organism was motile, did not form spores, and had a gram-negative wall structure. No cytochromes were detected. The mol % G+C of the DNA was 58. The new strain was microaerotolerant, and grew optimally at 30°C and neutral pH in freshwater medium. A wide range of carbohydrates was fermented, with formate, acetate, ethanol, lactate and succinate being the end-products detected. L-tartrate and citrate were fermented to formate, acetate and CO₂. L-tartrate was fermented by the dehydratase pathway, and glucose by the Embden-Meyerhof-Parnas pathway. Fumarate was reduced, but nitrate, sulfate, sulfur and thiosulfate were not used as terminal electron acceptors. Glucose metabolism was constitutive, whereas L-tartrate-degrading activity was inducible. When glucose and L-tartrate were both present as substrates, growth was diauxic with glucose being metabolized first. The growth rate and growth yield were higher on glucose than on L-tartrate. Strain 16Lt1 has been deposited with the Deutsche Sammlung von Mikroorganismen as Bacteroides sp. DSM6268.     

Complete assimilation of cysteine by a newly isolated non-sulfur purple bacterium resembling Rhodovulum sulfidophilum (Rhodobacter sulfidophilus)

A rod-shaped, motile, phototrophic bacterium, strain SiCys, was enriched and isolated from a marine microbial mat, with cysteine as sole substrate. During phototrophic anaerobic growth with cysteine, sulfide was produced as an intermediate, which was subsequently oxidized to sulfate. The molar growth yield with cysteine was 103 g mol^–1, in accordance with complete assimilation of electrons from the carbon and the sulfur moiety into cell material. Growth yields with alanine and serine were proportionally lower. Thiosulfate, sulfide, hydrogen, and several organic compounds were used as electron donors in the light, whereas cystine, sulfite, or elemental sulfur did not support phototrophic anaerobic growth. Aerobic growth in the dark was possible with fructose as substrate. Cultures of strain SiCys were yellowish-brown in color and contained bacteriochlorophyll a, spheroidene, spheroidenone, and OH-spheroidene as major photosynthetic pigments. Taking the morphology, photosynthetic pigments, aerobic growth in the dark, and utilization of sulfide for phototrophic growth into account, strain SiCys was assigned to the genus Rhodovulum (formerly Rhodobacter) and tentatively classified as a strain of R. sulfidophilum. In cell-free extracts in the presence of pyridoxal phosphate, cysteine was converted to pyruvate and sulfide, which is characteristic for cysteine desulfhydrase activity (l-cystathionine γ-lyase, EC 4.4.1.1). Received: 15 December 1995 / Accepted: 1 April 1996     

Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities

Neocallimastix strain N1, an isolate from a ruminant (sheep), was cocultured with three Methanobacterium formicicum strains, Methanosarcina barkeri, and Methanobrevibacter smithii. The coculture with Methanobacterium formicicum strains resulted in the highest production of cellulolytic and xylanolytic enzymes. Subsequently four anaerobic fungi, two Neocallimastix strains (N1 and N2) from a ruminant and two Piromyces species from non-ruminants (E2 and R1), were grown in coculture with Methanobacterium formicicum DSM 3637 on filter paper cellulose and monitored over a 7-day period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Methanogens caused a shift in fermentation products to more acetate and less ethanol, lactate and succinate. Furthermore the cellulose digestion rate increased by coculture. For cocultures of Neoallimastix strains with Methanobacterium formicicum strains the cellulolytic and xylanolytic enzyme production increased. Avicelase, CMCase and xylanase were almost completely secreted into the medium, while 40–60% of the -glucosidase was found to be cell bound. Coculture had no significant effect on the location of cellulolytic and xylanolytic enzymes.     

Isolation and characterization of a thermophilic, acetate-utilizing methanogenic bacterium

Abstract A thermophilic acetate-decarboxylating methanogenic bacterium was isolated from a laboratory-scale 60°C sludge digestor. Cells form straight filaments with flat to blunted ends normally consisting of 2–3 cells held together by a sheath-like outer cell wall. The organism uses acetate, H₂-CO₂ and formate for methanogenesis and growth. With acetate as the sole methanogenic substrate, almost all of the radioactivity from methyl-labelled acetate appeared as methane. Acetate was converted to methane in equimolar amounts with a doubling time of 3 days.     

Anaerobic degradation of hydroaromatic compounds by newly isolated fermenting bacteria

Aerobic organisms degrade hydroaromatic compounds via the hydroaromatic pathway yielding protocatechuic acid which is further metabolized by oxygenase-mediated ring fission in the 3-oxoadipate pathway. No information exists on anaerobic degradation of hydroaromatics so far. We enriched and isolated from various sources of anoxic sediments several strains of rapidly growing gram-negative bacteria fermenting quinic (1,3,4,5-tetrahydroxy-cyclohexane-1-carboxylic acid) and shikimic acid (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid) in the absence of external electron acceptors. Quinic and shikimic acid were the only ones utilized of more than 30 substrates tested. The marine isolates formed acetate, butyrate, and H₂, whereas all freshwater strains formed acetate and propionate as typical fermentation products. Aromatic intermediates were not involved in this degradation. Characterization of the isolates, fermentation balances for both hydroaromatic compounds, and enzyme activities involved in one degradation pathway are presented.Abbreviations BV benzyl viologen (1,1-dibenzyl-4,4-bipyridinium dichloride) - CoA coenzyme A - CTAB cetyltrimethylammonium bronide - DCPIP 2,4-dichlorophenolindophenol - DTT 1,4-dithiotheriol - MV methyl viologen (1,1-dimethyl-4,4-bipyridinium dichloride) - Tricine N-[tris-(hydroxymethyl)-methyl]-glycine - Tris tris-(hydroxymethyl)-aminomethane     

Fermentation of methoxyacetate to glycolate and acetate by newly isolated strains of Acetobacterium sp.

Three strains of new mesophilic homoacetogenic bacteria were enriched and isolated from sewage sludge and from marine sediment samples with methoxyacetate as sole organic substrate in a carbonate-buffered medium under anoxic conditions. Two freshwater isolates were motile, Gram-positive, non-sporeforming rods. The marine strain was an immotile, Gram-positive rod with a slime capsula. All strains utilized only the methyl residue of methoxyacetate and released glycolic acid. They also fermented methyl groups of methoxylated aromatic compounds and of betaine to acetate with growth yields of 6–10 g dry matter per mol methyl group. H₂/CO₂, formate, methanol, hexamethylene tetramine, as well as fructose, numerous organic acids, glycerol, ethylene glycol, and glycol ethers were fermented to acetate as well. High activities of carbon monoxide dehydrogenase (0.4–2.2 U x mg protein^–1) were detected in all three isolates. The guanine-plus-cytosine-content of the DNA of the freshwater isolates was 42.7 and 44.4 mol %, with the marine isolate it was 47.7 mol %. The freshwater strains were assigned to the genus Acetobacterium as new strains of the species A. carbinolicum. One freshwater isolate, strain KoMac1, was deposited with the Deutsche Sammlung von Mikroorganismen GmbH, Braunschweig, under the number DSM 5193.     

Succinic acid production by a newly isolated bacterium

A new bacterial strain producing succinic acid was enriched from bovine rumen content. It is facultatively anaerobic, belongs to the family Pasteurellaceae and has similarity to the genus Mannheimia. In batch cultivations with D-glucose or sucrose the strain produced up to 5.8 g succinic acid l⁻¹ with a productivity and a yield of up to 1.5 g l⁻¹ h⁻¹ and 0.6 g g⁻¹, respectively. With crude glycerol up to 8.4 g l⁻¹, 0.9 g l⁻¹ h⁻¹ and 1.2 g g⁻¹ were obtained. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Anaerobic degradation of dimethylsulfoniopropionate to 3-S-methylmercaptopropionate by a marine Desulfobacterium strain

Dimethylsulfoniopropionate, an osmolyte of marine algae, is thought to be the major precursor of dimethyl sulfide, which plays a dominant role in biogenic sulfur emission. The marine sulfate-reducing bacterium Desulfobacterium strain PM4 was found to degrade dimethylsulfoniopropionate to 3-S-methylmercaptopropionate. The oxidation of one of the methyl groups of dimethylsulfoniopropionate was coupled to the reduction of sulfate; this process is similar to the degradation betaine to dimethylglycine which was described earlier for the same strain. Desulfobacterium PM4 is the first example of an anaerobic marine bacterium that is able to demethylate dimethylsulfoniopropionate.Abbreviations DMSP dimethylsulfoniopropionate - DMS dimethyl sulfide - MMPA 3-S-methylmercaptopropionate     

Fermentation of cysteate by a sulfate-reducing bacterium

We isolated a strictly anaerobic bacterium, strain GRZCYSA, from a sludge digestor for its ability to ferment cysteate (2-amino-3-sulfopropionate). The organism also fermented the organosulfonates isethionate (2-hydroxyethanesulfonate) and aminomethanesulfonate, but taurine (2-aminoethanesulfonate) was not a substrate. Strain GRZCYSA, a gram-negative, oxidase-negative and catalase-positive vibrio that could reduce sulfate and contained desulfoviridin, was tentatively identified as Desulfovibrio sp. Utilization of cysteate as a substrate for fermentative growth led to the formation of four products identified as acetate, ammonia, and equimolar amounts of sulfide and sulfate. The fermentation was in balance. Some reactions involved in this novel process were detected in cell-free extracts in which ammonia and acetate were formed from cysteate. Received: 10 March 1997 / Accepted: 14 May 1997     

Oxidation of ethanol by methanogenic bacteria

Methanogenium organophilum, a non-autotrophic methanogen able to use primary and secondary alcohols as hydrogen donors, was grown on ethanol. Per mol of methane formed, 2 mol of ethanol were oxidized to acetate. In crude extract, an NADP⁺-dependent alcohol dehydrogenase (ADH) with a pH optimum of about 10.0 catalyzed a rapid (5 mol/min·mg protein; 22°C) oxidation of ethanol to acetaldehyde; after prolonged incubation also acetate was detectable. With NAD⁺ only 2% of the activity was observed. F₄₂₀ was not reduced. The crude extract also contained F₄₂₀: NADP⁺ oxidoreductase (0.45 mol/min·mg protein) that was not active at the pH optimum of ADH. With added acetaldehyde no net reduction of various electron acceptors was measured. However, the acetaldehyde was dismutated to ethanol and acetate by the crude extract. The dismutation was stimulated by NADP⁺. These findings suggested that not only the dehydrogenation of alcohol but also of aldehyde to acid was coupled to NADP⁺ reduction. If the reaction was started with acetaldehyde, formed NADPH probably reduced excess aldehyde immediately to ethanol and in this way gave rise to the observed dismutation. Acetate thiokinase activity (0.11 mol/min·mg) but no acetate kinase or phosphotransacetylase activity was observed. It is concluded that during growth on ethanol further oxidation of acetaldehyde does not occur via acetylCoA and acetyl phosphate and hence is not associated with substrate level phosphorylation. The possibility exists that oxidation of both ethanol and acetaldehyde is catalyzed by ADH. Isolation of a Methanobacterium-like strain with ethanol showed that the ability to use primary alcohols also occurs in genera other than Methanogenium.Non-standard abbreviations ADH alcohol dehydrogenase - Ap₅ALi₃ P¹,P⁵-Di(adenosine-5-)pentaphosphate - DTE dithioerythritol (2,3-dihydroxy-1,4-dithiolbutane) - F₄₂₀ N-(N-l-lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8-dimethyl-8-hydroxy-5-deazariboflavin-5-phosphate - Mg. Methanogenium - OD₅₇₈ optical density at 578 nm - PIPES 1,4-piperazine-diethanesulfonic acid - TRICINE N-(2-hydroxy-1,1-bis[hydroxymethyl]methyl)-glycine - Tris 2-amino-2-hydroxy-methylpropane-1,3-diol - U unit (mol substrate/min)     

Fermentation of phenoxyethanol to phenol and acetate by a homoacetogenic bacterium

A strictly anaerobic gram-positive, rod-shaped bacterium, strain LuPhet1, was isolated from sewage sludge with phenoxyethanol as sole carbon and energy source, and was assigned to the genus Acetobacterium. The new isolate fermented the alkylaryl ether compound phenoxyethanol stoichiometrically to phenol and acetate, whereas phenoxyacetic acid was not degraded. In cell-free extracts of strain LuPhet1, cleavage of the ether linkage was shown, and acetaldehyde was detected as reaction product. Coenzyme A-dependent acetaldehyde: acceptor oxidoreductase, phosphate acetyltransferase, acetate kinase, and carbon monoxide dehydrogenase were measured in cell-free extracts of this strain. Our results indicate that the ether linkage of phenoxyethanol is cleaved by a shift of the hydroxyl group to the subterminal carbon atom, analogous to a corrinoid-dependent diol dehydratase reaction, to form an unstable hemiacetal that releases phenol and acetaldehyde. Obviously, phenoxyethanol is degraded by the same strategy as in anaerobic degradation of the alkyl ether polyethylene glycol.     

Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads

From various oxic or anoxic habitats several strains of bacteria were isolated which in the absence of molecular oxygen oxidized phenol to CO₂ with nitrate as the terminal electron acceptor. All strains grew in defined mineral salts medium; two of them were further characterized. The bacteria were facultatively anaerobic Gramnegative rods; metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as electron acceptor. The isolates were tentatively identified as pseudomonads. Besides phenol many other benzene derivatives like cresols or aromatic acids were anaerobically oxidized in the presence of nitrate. While benzoate or 4-hydroxybenzoate was degraded both anaerobically and aerobically, phenol was oxidized under anaerobic conditions only. Reduced alicyclic compounds were not degraded. Preliminary evidence is presented that the first reaction in anaerobic phenol oxidation is phenol carboxylation to 4-hydroxybenzoate.     

Transformation of nitroaromatic compounds by a methanogenic bacterium,Methanococcus sp. (strain B)

The transformation of several nitroaromatic compounds by a newly isolated methanogenic bacterium, Methanococcus sp. (strain B) was studied. The presence of nitroaromatic compounds (0.5 mM) viz., nitrobenzene, 2,4-dinitrobenzene, 2,4,6-trinitrobenzene, 2,4-dinitrophenol, 2,4-dinitrobenzene, and 2,6-dinitrotoluene in the culture medium did not inhibit growth of the isolate. The bacteria grew rapidly and reached stationary phase within seven days of incubation. All the nitroaromatic compounds tested were 80 to 100% transformed by the bacterium to amino compounds by a reduction process. The isolate did not use the nitroaromatic compounds as the sole source of carbon or nitrogen. The transformation of nitroaromatic compounds by this isolate was compared to that of other methanogenic bacteria. Out of five methanogens studied, only Methanococcus deltae and Methanococcus thermolithotrophicus could transform the nitroaromatic compounds; however, the transformation rates were significantly less than that of the new isolate Methanococcus sp. (strain B). The nitroaromatic compounds were not transformed by Methanosarcina barkeri, Methanobacterium thermoautotrophicum, and Methanobrevibacter ruminantium.Abbreviations NB Nitrobenzene - DNB 2,4-Dinitrobenzene - TNB 2,4,6-Trinitrobenzene - DNP 2,4-Dinitrophenol - 2,4-DNT 2,4-Dinitrotoluene - 2,6-DNT 2,6-Dinitrotoluene     

Degradation of methanol by a sulfate reducing bacterium

A sulfate reducing bacterium isolated from sewage sludge was capable of degrading methanol after growth on pyruvate, malate, or fumarate. ¹⁴C-Methanol was completely oxidized to carbon dioxide but not incorporated into the cellular material. The organism is a member of the genus Desulfovibrio.     

Enrichment and granulation of Anammox biomass started up with methanogenic granular sludge

A sequencing batch reactor (SBR) seeded with methanogenic granular sludge was started up to enrich Anammox (Anaerobic Ammonium Oxidation) bacteria and to investigate the feasibility of granulation of Anammox biomass. Research results showed that hydraulic retention time (HRT) was an important factor to enrich Anammox bacteria. When the HRT was controlled at 30 days during the initial cultivation, the SBR reactor presented Anammox activity at t = 58 days. Simultaneously, the methanogenic granular sludge changed gradually from dust black to brown colour and its diameter became smaller. At t = 90 days, the Anammox activity was further improved. NH₄⁺-N and NO₂⁻N were removed simultaneously with higher speed and the maximum removal rates reached 14.6 g NH₄⁺-N /(m³ _reactor·day) and 6.67 g NO₂⁻-N /(m³ _reactor·day), respectively. Between t = 110 days and t = 161 days, the nitrogen load was increased to a HRT of 5 days (70 mg/l NH₄⁺ and 70 mg/l NO₂), the removal rates of ammonium and nitrite were 60.6% and 62.5% respectively. The sludge changed to red and formed Anammox granulation with high nitrogen removal activity.     

Anaerobic mineralization of cholesterol by a novel type of denitrifying bacterium

A novel denitrifying bacterium, strain 72Chol, was enriched and isolated under strictly anoxic conditions on cholesterol as sole electron donor and carbon source. Strain 72Chol grew on cholesterol with oxygen or nitrate as electron acceptor. Strictly anaerobic growth in the absence of oxygen was demonstrated using chemically reduced culture media. During anaerobic growth, nitrate was initially reduced to nitrite. At low nitrate concentrations, nitrite was further reduced to nitrogen gas. Ammonia was assimilated. The degradation balance measured in cholesterol-limited cultures and the amounts of carbon dioxide, nitrite, and nitrogen gas formed during the microbial process indicated a complete oxidation of cholesterol to carbon dioxide. A phylogenetic comparison based on total 16S rDNA sequence analysis indicated that the isolated micro-organism, strain 72Chol, belongs to the β2-subgroup in the Proteobacteria and is related to Rhodocyclus, Thauera, and Azoarcus species. Received: 16 July 1996 / Accepted: 5 December 1996     

Fermentation of mandelate to benzoate and acetate by a homoacetogenic bacterium

A strictly anaerobic, Gram-positive, rod-shaped bacterium, strain AmMan1, was isolated from freshwater sediment with mandelate (-hydroxy-phenylacetate) as sole carbon and energy source, and was assigned to the genus Acetobacterium. Only the d-enantiomer of mandelate was degraded, and was fermented to acetate and benzoate. Non-aromatic growth substrates (pyruvate, lactate, malate, glycerol, ethylene glycol, and H₂/CO₂) were fermented to acetate as sole product. Methoxylated aromatics were demethoxylated to the corresponding phenols. The guanine-plus-cytosine content of the DNA was 36.5±1.5%. Carbon monoxide dehydrogenase, dichlorophenol indophenol-reducing lactate dehydrogenase, NAD-dependent mandelate dehydrogenase, phosphate acetyl transferase, acetate kinase, and pyruvate- or phenylglyoxylate-dependent benzylviologen reductase were measured in mandelate-and/or lactate-grown cells, respectively. A pathway of the homoacetogenic fermentation of mandelate is suggested as another example of incomplete substrate oxidation by homoacetogenic bacteria.