Formation of N,N-Dimethylglycine, Acetic Acid, and Butyric Acid from Betaine by Eubacterium limosum

Two bacterial strains that grow anaerobically on betaine were isolated from enrichment cultures and identified as strains of Eubacterium limosum. In a mineral medium supplemented with yeast extract and Casitone, the doubling time of E. limosum strain 11A on betaine was 6 h at 37°C. The molar growth yield amounted to 9 g of dry cell mass per mol. Betaine was fermented in accordance with the following equation: 7 betaine + 2 CO₂ → 7 N,N-dimethylglycine + 1.5 acetate + 1.5 butyrate. E. limosum also grew on methanol and choline. The former was converted to acetate and butyrate, and the latter was converted to N,N-dimethylethanolamine, acetate, and butyrate. The conditions for the quantitative determination of N,N-dimethylglycine by capillary tube isotachophoresis have been determined.     

Eubacterium acidaminophilum sp. nov., a versatile amino acid-degrading anaerobe producing or utilizing H2 or formate

An obligately anaerobic, rod-shaped bacterium was isolated on alanine in co-culture with H₂-scavenging Desulfovibrio and obtained in pure culture with glycine as sole fermentation substrate. The isolated strain, al-2, was motile by a polar to subpolar flagellum and stained Gram-positive. The guanine plus cytosine content of the DNA was 44.0 mol%. Strain al-2 grew in defined, reduced glycine media supplemented with biotin. The pure culture fermented 4 mol glycine to 3 mol acetate, 4 mol ammonia and 2 mol CO₂. Under optimum conditions (34°C, pH 7.3), the doubling time on glycine was 60 min and the molar growth yield 7.6 g cell dry mass. Serine was fermented to acetate, ethanol, CO₂, H₂ and ammonia. In addition, betaine, sarcosine or creatine served as substrates for growth and acetate production if H₂, formate or e.g. valine were added as H-donors. In pure culture on alanine under N₂, strain al-2 grew very poorly and produced H₂ up to a partial pressure of 3.6 kPa (0.035 atm). Desulfovibrio species, Methanospirillum hungatei and Acetobacterium woodii served as H₂-scavengers that allowed good syntrophic growth on alanine. The co-cultures also grew on aspartate, leucine, valine or malate. Alanine and aspartate were stoichiometrically degraded to acetate and ammonia, whereas the reducing equivalents were recovered as H₂S, CH₄ or newly synthetized acetate, respectively. Growth of strain al-2 in co-culture with the hydrogenase-negative, formate-utilizing Desulfovibrio baarsii indicated that a syntrophy was also possible by interspecies formate transfer. Growth on glycine, or on betaine, sarcosine or creatine (plus H-donors) depended strictly on the addition of selenite (0.1 M); selenite was not required for fermentation of serine, or for degradation of alanine, aspartate or valine by the co-cultures. Cell-free extracts of glycine-grown cells contained active glycine reductase, glycine decarboxylase and reversible methyl viologen-dependent formate dehydrogenase in addition to the other enzymes necessary for an oxidation to CO₂. In all reactions NADP was the preferred H-carrier. Both formate and glycine could be synthesized from bicarbonate. Serine-grown cells did not contain serine hydroxymethyl transferase but serine dehydratase and other enzymes commonly involved in pyruvate metabolism to acetate, CO₂ and H₂. The enzymes involved in glycine metabolism were repressed during growth on serine. By its morphology and physiology, strain al-2 did not resemble described amino acid-degrading species. Therefore, the new isolate is proposed as type strain of a new species, Eubacterium acidaminophilum.     

Fructose degradation byDesulfovibrio sp. in pure culture and in coculture withMethanospirillum hungatei

In a mineral medium containing sulfate, the sulfate-reducing bacteriumDesulfovibrio sp. strain JJ degraded 1 mol of fructose stoichiometrically to 1 mol of H₂S, 2 mol of acetate, and presumably 2 mol of CO₂. The doubling time was 10 h, and the yield was 41.6 g dry weight/mol fructose degraded. In the absence of sulfate, the hydrogenophilic methanogenMethanospirillum hungatei replaced sulfate as hydrogen sink. In such cocultures, 1 mol of fructose was converted to acetate, methane, succinate, and presumably CO₂ in varying concentrations. The growth yield of the H₂-transferring association was 33 g dry weight/mol fructose. In the absence of sulfate,Desulfovibrio strain JJ slowly fermented 1 mol of fructose to 1 mol of succinate, 0.5 mol of acetate, and 0.5 mol of ethanol. The results are compared with those of other anaerobic hexose-degrading bacteria.     

Anaerobic degradation of betaine by marine Desulfobacterium strains

From enrichment cultures with betaine (20 mM) and sulfate (20 mM) as the substrates and intertidal mud as an inoculum, a betaine-oxidizing, sulfate-reducing bacterium (strain PM4) was isolated. Strain PM4 was an oval to rod-shaped, Gram-negative, motile bacterium, which was able to oxidize lactate completely to CO₂ and contained, during growth on betaine and sulfate, high activities of key enzymes of the acetyl CoA/CO dehydrogenase pathway (carbon monoxide dehydrogenase and formate dehydrogenase), but not of 2-oxo-glutarate dehydrogenase, a key enzyme of the citric acid cycle. On the basis of its morphological and physiological characteristics, strain PM4 was identified as a Desulfobacterium strain. Desulfobacterium PM4 grew on betaine with a doubling time of approximately 20 h at 30°C and produced N, N-dimethylglycine (in a 1:1 ratio) and sulfide as products. In this type of betaine metabolism one of the methyl groups of betaine is oxidized to CO₂ and the reducing equivalents generated are used for the reduction of sulfate. Desulfobacterium autotrophicum (DSM 3382) grew also on betaine and sulfate with the formation of N,N-dimethylglycine, sulfide and CO₂.     

Metabolism of Aspartate by Propionibacterium freudenreichii subsp. shermanii: Effect on Lactate Fermentation

More than 90% of the aspartate in a defined medium was metabolized after lactate exhaustion such that 3 mol of aspartate and 1 mol of propionate were converted to 3 mol of succinate, 3 mol of ammonia, 1 mol of acetate, and 1 mol of CO₂. This pathway was also evident when propionate and aspartate were the substrates in complex medium in the absence of lactate. In complex medium with lactate present, about 70% of the aspartate was metabolized to succinate and ammonia during lactate fermentation, and as a consequence of aspartate metabolism, more lactate was fermented to acetate and CO₂ than was fermented to propionate. The conversion of aspartate to fumarate and ammonia by the enzyme aspartase and subsequent reduction of fumarate to succinate occurred in the five strains of Propionibacterium freudenreichii subsp. shermanii studied. The ability to metabolize aspartate in the presence of lactate appeared to be related to aspartase activity. The specific activity of aspartase increased during and after lactate utilization, and the levels of this enzyme were lower in cells grown in defined medium than levels in those cells grown in complex medium. Under the conditions used, no other amino acids were readily metabolized in the presence of lactate. The possibility that aspartate metabolism by propionibacteria in Swiss cheese has an influence on CO₂ production is discussed.     

Anaerobic degradation of sorbic acid by sulfate-reducing and fermenting bacteria: pentanone-2 and isopentanone-2 as byproducts

Strictly anaerobic bacteria were enriched and isolated from freshwater sediment sources in the presence and absence of sulfate with sorbic acid as sole source of carbon and energy. Strain WoSo1, a Gram-negative vibrioid sulfate-reducing bacterium which was assigned to the species Desulfoarculus (formerly Desulfovibrio) baarsii oxidized sorbic acid completely to CO₂ with concomitant stoichiometric reduction of sulfate to sulfide. This strain also oxidized a wide variety of fatty acids and other organic compounds. A Gram-negative rod-shaped fermenting bacterium, strain AmSo1, fermented sorbic acid stoichiometrically to about equal amounts of acetate and butyrate. At concentrations higher than 10 mM, sorbic acid fermentation led to the production of pentanone-2 and isopentanone-2 (3-methyl-2-butanone) as byproducts. Strain AmSo1 fermented also crotonate and 3-hydroxybutyrate to acetate and butyrate, and hexoses to acetate, ethanol, hydrogen, and formate. The guanine-plus-cytosine content of the DNA was 41.8±1.0 mol%. Sorbic acid at concentrations higher than 5 mM inhibited growth of this strain while strain WoSo1 tolerated sorbic acid up to 10 mM concentration.     

Fermentation of mannitol by Clostridium butyricum: role of acetate as an external hydrogen acceptor

Summary The continuous fermentation of mannitol (pH 6, dilution rate (D)=0.087 h^-1) by Clostridium butyricum LMG 1213t1 was investigated under several conditions. Mannitol was readily fermented when glucose or acetate were added in the in-flow medium as co-substrate. Butyrate, CO₂ and H₂ were the major fermentation products. In mannitol-glucose mixtures (ratios 4 or 8) the amount of mannitol fermented depended upon the amount of glucose in the in-flow medium. In mannitol-acetate mixtures, 1 mol of acetate was needed for the fermentation of approximately 5.5 mol mannitol. We detected d-mannitol-1-phosphate dehydrogenase activity, responsible for the generation of supplementary reduced nicotine adenine dinucleotide (NADH) as a source for extra H₂ gas. Fermentation of mannitol-acetate in the presence of [¹⁴C]-labelled acetate revealed butyrate as the only labelled fermentation end-product.     

Fermentative degradation of dipicolinic acid (pyridine-2,6-dicarboxylic acid) by a defined coculture of strictly anaerobic bacteria

Degradation of dipicolinic acid (pyridine-2,6-dicarboxylic acid) under strictly anaerobic conditions was studied in enrichment cultures from marine and freshwater sediments. In all cases, dipicolinic acid was completely degraded. From an enrichment culture from a marine sediment, a defined coculture of two bacteria was isolated. The dipicolinic acid-fermenting bacterium was a Gram-negative, non-sporeforming strictly anaerobic short rod which utilized dipicolinic acid as sole source of carbon, energy, and nitrogen, and fermented it to acetate, propionate, ammonia, and 2CO₂. No other substrate was fermented. This bacterium could be cultivated only in coculture with another Gram-negative, non-sporeforming rod from the same enrichment culture which oxidized acetate to CO₂ with fumarate, malate, or elemental sulfur as electron acceptor, similar to Desulfuromonas acetoxidans. Since this metabolic activity is not important in substrate degradation by the coculture, the basis of the dependence of the dipicolinic acid-degrading bacterium on the sulfur reducer may be sought in the assimilatory metabolism.     

Degradation of hydroxyhydroquinone by the strictly anaerobic fermenting bacterium Pelobacter massiliensis sp. nov.

A new rod-shaped, gram-negative, non-sporeforming, strictly anaerobic bacterium (strain HHQ7) was enriched and isolated from marine mud samples with hydroxyhydroquinone (1,2,4-trihydroxybenzene) as sole substrate. Strain HHQ7 fermented hydroxyhydroquinone, pyrogallol (1,2,3-trihydroxybenzene), phloroglucinol (1,3,5-trihydroxybenzene) and gallic acid (3,4,5-trihydroxybenzoate) to 3 mol acetate (plus 1 mol CO₂ in the case of gallic acid) per mol of substrate. Resorcinol accumulated intermediately during growth on hydroxy-hydroquinone. No other aliphatic or aromatic substrates were utilized. Sulfate, sulfite, sulfur, nitrate, and fumarate were not reduced with hydroxyhydroquinone as electron donor. The strain grew in sulfide-reduced mineral medium supplemented with 7 vitamins. The DNA base ratio was 59% G+C. Strain HHQ7 is classified as a new species of the genus Pelobacter, P. massiliensis. Experiments with dense cell suspensions of hydroxyhydroquinone-and pyrogallol-grown cells showed different kinetics of hydroxyhydroquinone and pyrogallol degradation, as well as different patterns of resorcinol accumulation, indicating that these substrates are metabolized by different transhydroxylation reactions.     

Anaerobic degradation of methoxylated aromatic compounds by Clostridium methoxybenzovorans and a nitrate-reducing bacterium Thauera sp. strain Cin3,4

The coupling of growth of the o-demethylating bacterium, Clostridium methoxybenzovorans SR3, with a nitrate-reducing bacterium able to degrade aromatic compounds, Thauera sp. Cin3,4, allowed complete mineralization of poorly oxidizable methoxylated aromatic compounds such as vanillate, isovanillate, vanilline, anisate, ferulate and veratrate. C. methoxybenzovorans o-demethylated these aromatic compounds to their corresponding hydroxylated derivatives and fermented the side chains to acetate and butyrate. The hydroxylated compounds and the fermentation end-products in the C. methoxybenzovorans spent growth medium were then completely metabolized to CO₂ on inoculation with the Thauera strain. Kinetic studies with veratrate indicated that C. methoxybenzovorans initially o-demethylated the substrate to vanillate and then further to protocatechuate together with the production of acetate and butyrate from the demethylated side chains. Protocatechuate, acetate and butyrate were then utilized as a carbon source by the Thauera strain aerobically or anaerobically in the presence of nitrate. The results therefore suggest that mono- or dimethoxylated aromatic compounds can be completely mineralized by coupling the growth of a fermentative bacterium with a nitrate-reducing bacterium, and a metabolic pathway for this is proposed.     

The new facultatively chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium Desulfovermiculus halophilus gen. nov., sp. nov., isolated from an oil field

The new mesophilic, chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium strain 11-6, could grow at a NaCl concentration in the medium of 30–230 g/l, with an optimum at 80–100 g/l. Cells were vibrios motile at the early stages of growth. Lactate, pyruvate, malate, fumarate, succinate, propionate, butyrate, crotonate, ethanol, alanine, formate, and H₂/CO₂ were used in sulfate reduction. Butyrate was degraded completely, without acetate accumulation. In butyrate-grown cells, a high activity of CO dehydrogenase was detected. Additional growth factors were not required. Autotrophic growth occurred, in the presence of sulfate, on H₂/CO₂ or formate without other electron donors. Fermentation of pyruvate and fumarate was possible in the absence of sulfate. Apart from sulfate, sulfite, thiosulfate, and elemental sulfur were able to serve as electron acceptors. The optimal growth temperature was 37°C; the optimum pH was 7.2. Desulfoviridin was not detected. Menaquinone MK-7 was present. The DNA G+C content was 55.2 mol %. Phylogenetically, the bacterium represented a separate branch within the cluster formed by representatives of the family Desulfohalobiaceae in the class Deltaproteobacteria. The bacterium was assigned to a new genus and species, Desulfovermiculus halophilus gen. nov., sp. nov. The type strain is 11-6^T (= VKM B-2364), isolated from the highly mineralized formation water of an oil field.     

Investigation of the catabolism of acetate and peptides in the new anaerobic thermophilic bacterium Caldithrix abyssi

This work is concerned with the metabolism of Caldithrix abyssi—an anaerobic, moderately thermophilic bacterium isolated from deep-sea hydrothermal vents of the Mid-Atlantic Ridge and representing a new, deeply deviated branch within the domain Bacteria. Cells of C. abyssi grown on acetate and nitrate, which was reduced to ammonium, possessed nitrate reductase activity and contained cytochromes of the b and c types. Utilization of acetate occurred as a result of the operation of the TCA and glyoxylate cycles. During growth of C. abyssi on yeast extract, fermentation with the formation of acetate, propionate, hydrogen, and CO₂ occurred. In extracts of cells grown on yeast extract, acetate was produced from pyruvate with the involvement of the following enzymes: pyruvate: ferredoxin oxidoreductase (2.6 μmol/(min mg protein)), phosphate acetyltransferase (0.46 μmol/(min mg protein)), and acetate kinase (0.3 μmol/(min mg protein)). The activity of fumarate reductase (0.14 μmol/(min mg protein)), malate dehydrogenase (0.17 μmol/(min mg protein)), and fumarate hydratase (1.2 μmol/(min mg protein)), as well as the presence of cytochrome b, points to the formation of propionate via the methyl-malonyl-CoA pathway. The activity of antioxidant enzymes (catalase and superoxide dismutase) was detected. Thus, enzymatic mechanisms have been elucidated that allow C. abyssi to switch from fermentation to anaerobic respiration and to exist in the gradient of redox conditions characteristic of deep-sea hydrothermal vents.     

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.     

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.     

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.     

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

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

Isolation and characterization of a new,methylotrophic, acidogenic anaerobe,the marburg strain

The cellular characteristics of a new methylotrophic, acidogenic, anaerobic bacterium that was first isolated from a sewage digestor in Marburg, Federal Republic of Germany, is described. The Marburg strain is a mesophilic, Gram-positive, nonmotile, pleomorphic rod that performs homoacetic, homobutyric, or heteroacidic fermentations. Cell morphology varies from single or paired straight rods to rudimentary branched rods, club-shaped cells, or oval refractile cells. Cell heat resistance correlated with the presence of a few refractile cells. Electron micrographs of thin sections revealed a thick monolayered cell wall and an atypical spore structure. The DNA base composition was 48.8±0.2 mol% guanosine plus cytosine. Growth required factors in yeast extract; methanol, H₂/CO₂, glucose, fructose, lactate, and pyruvate were fermented as energy sources. Corrinoid levels varied from 0.35±0.16 to 7.9±1.6 μg/mg cell dry weight when cells 0.1% yeast extract, N₂/CO₂, 100 mM methanol, and 50 mM Na acetate displayed a 20h doubling time, finalA ₅₄₀ of 0.9, butyric acid yield of 25 mM, and ≈stoichiometry of 3 mol butyrate formed per 10 mol methanol fermented. The nameButyribacterium (emend.)methylotrophicum sp. nov. is proposed for the Marburg strain.     

A new 3-hydroxybutyrate fermenting anaerobe,Ilyobacter polytropus,gen. nov. sp. nov., possessing various fermentation pathways

From marine anoxic mud, a new strictly anaerobic, Gram-negative, non-sporeforming bacterium was isolated with 3-hydroxybutyrate as substrate. 3-Hydroxybutyrate and crotonate were fermented to acetate and butyrate. Glycerol was fermented to 1,3-propanediol and 3-hydroxypropionate. Acetate and formate were the only products of pyruvate or citrate fermentation. Glucose and fructose were fermented to acetate, formate and ethanol. Malate and fumarate were fermented to acetate, formate and propionate. Neither sulfate, sulfur, nor nitrate was reduced. The DNA base ratio was 32.2±0.5 mol% guanine plus cytosine. Strain CuHbu1 is described as type strain of a new genus and species, Ilyobacter polytropus gen. nov. sp. nov., in the family Bacteroidaceae.     

Simultaneous heterolactic and acetate fermentation in the marine cyanobacterium Oscillatoria limosa incubated anaerobically in the dark

The marine nitrogen-fixing cyanobacterium Oscillatoria limosa, strain 23 (Oldenburg) was investigated with respect to its dark anaerobic metabolism. As soon as the cells were incubated anaerobically in the dark, they started to ferment. Glycogen was presumably degraded via the heterolactic fermentative pathway. Glycogen-glucose was degraded to equimolar amounts of lactate, ethanol and carbon dioxide. The disaccharide trehalose, which serves as an osmoprotectant in O. limosa, was also catabolized. Most probably, this compound was fermented almost exclusively to acetate. Some hydrogen was produced as well. In the presence of elemental sulfur, fermentative hydrogen production ceased and sulfide was produced instead. The presence of elemental sulfur had no effect on the amounts and ratios of the fermentation products produced.     

Ethanol Production by Thermophilic Bacteria: Fermentation of Cellulosic Substrates by Cocultures of Clostridium thermocellum and Clostridium thermohydrosulfuricum

The fermentation of various saccharides derived from cellulosic biomass to ethanol was examined in mono- and cocultures of Clostridium thermocellum strain LQRI and C. thermohydrosulfuricum strain 39E. C. thermohydrosulfuricum fermented glucose, cellobiose, and xylose, but not cellulose or xylan, and yielded ethanol/acetate ratios of >7.0. C. thermocellum fermented a variety of cellulosic substrates, glucose, and cellobiose, but not xylan or xylose, and yielded ethanol/acetate ratios of ~1.0. At nonlimiting cellulosic substrate concentrations (~1%), C. thermocellum cellulase hydrolysis products accumulated during monoculture fermentation of Solka Floc cellulose and included glucose, cellobiose, xylose, and xylobiose. A stable coculture that contained nearly equal numbers of C. thermocellum and C. thermohydrosulfuricum was established that fermented a variety of cellulosic substrates, and the ethanol yield observed was twofold higher than in C. thermocellum monoculture fermentations. The metabolic basis for the enhanced fermentation effectiveness of the coculture on Solka Floc cellulose included: the ability of C. thermocellum cellulase to hydrolyze α-cellulose and hemicellulose; the enhanced utilization of mono- and disaccharides by C. thermohydrosulfuricum; increased cellulose consumption; threefold increase in the ethanol production rate; and twofold decrease in the acetate production rate. The coculture actively fermented MN300 cellulose, Avicel, Solka Floc, SO₂-treated wood, and steam-exploded wood. The highest ethanol yield obtained was 1.8 mol of ethanol per mol of anhydroglucose unit in MN300 cellulose.