Why Does Clostridium acetireducens Not Use Interspecies Hydrogen Transfer for Growth on Leucine?

Clostridium acetireducens is the first reported anaerobic bacterium that is dependent on acetate as an electron acceptor for growth on branched-chain amino acids and alanine. The fermentation pathway of leucine and its deamination product α-ketoisocaproate were studied in this organism. Addition of Methanobacterium formicicum to pure cultures of C. acetireducens stimulated the degradation of α-ketoisocaproate but not the degradation of leucine, indicating that the electrons produced during the oxidative deamination of leucine were not transferred to hydrogen. This conclusion is supported by the observed low NAD(P)H ferredoxin reductase activity. Not only acetate but also crotonate proved to be an appropriate electron sink for the regeneration of NAD(P)⁺ in this bacterium. Interestingly, C. acetireducens was shown to form polyhydroxybutyrate during growth on leucine plus acetate. Received: 2 December 1996 / Accepted: 4 March 1997     

Cellulose Fermentation by a Rumen Anaerobic Fungus in Both the Absence and the Presence of Rumen Methanogens

The fermentation of cellulose by an ovine rumen anaerobic fungus in the absence and presence of rumen methanogens is described. In the monoculture, moles of product as a percentage of the moles of hexose fermented were: acetate, 72.7; carbon dioxide, 37.6; formate, 83.1; ethanol, 37.4; lactate, 67.0; and hydrogen, 35.3. In the coculture, acetate was the major product (134.7%), and carbon dioxide increased (88.7%). Lactate and ethanol production decreased to 2.9 and 19%, respectively, little formate was detected (1%), and hydrogen did not accumulate. Substantial amounts of methane were produced in the coculture (58.7%). Studies with [2-¹⁴C]acetate indicated that acetate was not a precursor of methane. The demonstration of cellulose fermentation by a fungus extends the range of known rumen organisms capable of participating in cellulose digestion and provides further support for a role of anaerobic fungi in rumen fiber digestion. The effect of the methanogens on the pattern of fermentation is interpreted as a shift in flow of electrons away from electron sink products to methane via hydrogen. The study provides a new example of intermicrobial hydrogen transfer and the first demonstration of hydrogen formation by a fungus.     

Isolation and characterization of an anaerobic syntrophic benzoate-degrading bacterium from sewage sludge

An anaerobic, motile, gram-negative, rod-shaped bacterium is described which degrades benzoate in coculture with an H₂-utilizing organism and in the absence of exogenous electron acceptors such as O₂, SO ₄ ⁼ or NO ₃ ^- . The bacterium was isolated from a municipal primary, anaerobic sewage digestor using anaerobic roll-tube medium with benzoate as the main energy source and in syntrophic association with an H₂-utilizing sulfate-reducing Desulfovibrio sp. which cannot utilize benzoate or fatty acids apart from formate as energy source. The benzoate utilizer produced acetate (3 mol/mol of substrate degraded) and presumably CO₂ and H₂, or formate from benzoate. In media without sulfate and with Methanospirillum hungatei (a methanogen that utilizes only H₂–CO₂ or formate as the energy source) added, 3 mol of acetate and 0.7 mol of methane were produced per mol of benzoate and CO₂ was probably formed. Low numbers of Desulfovibrio sp. were present in the methanogenic coculture and a pure coculture of the benzoate utilizer with M. hungatei was not obtained. The generation times for growth of the sulfate-reducing and methanogenic cocultures were 132 and 166h, respectively. The benzoate utilizer did not utilize other common aromatic compounds, C ₃ ^- –C₇ monocarboxylic acids, or C₄-C₆ dicarboxylic acids for growth, nor did it appear to use SO ₄ ⁼ , NO ₃ ^- or fumarate as alternative electron acceptors. Addition of H₂ inhibited growth and benzoate degradation.     

Metabolic interactions between anaerobic bacteria in methanogenic environments

In methanogenic environments organic matter is degraded by associations of fermenting, acetogenic and methanogenic bacteria. Hydrogen and formate consumption, and to some extent also acetate consumption, by methanogens affects the metabolism of the other bacteria. Product formation of fermenting bacteria is shifted to more oxidized products, while acetogenic bacteria are only able to metabolize compounds when methanogens consume hydrogen and formate efficiently. These types of metabolic interaction between anaerobic bacteria is due to the fact that the oxidation of NADH and FADH₂ coupled to proton or bicarbonate reduction is thermodynamically only feasible at low hydrogen and formate concentrations. Syntrophic relationships which depend on interspecies hydrogen or formate transfer were described for the degradation of e.g. fatty acids, amino acids and aromatic compounds.     

Cultivation of anaerobic fungi in a 10-l fermenter system for the production of (hemi-)cellulolytic enzymes

 Two species of anaerobic fungi, i.e. Piromyces strain E2 and Neocallimastix patriciarum strain N2, were cultivated in a 10-l batch fermenter with filter- paper cellulose as the carbon source. The accumulation of fermentation products, production of extracellular protein and (hemi-)cellulolytic enzymes were monitored during growth. Growth of Piromyces E2 in the fermenter resulted in a shift in the fermentation pattern to more acetate and formate and less ethanol, lactate, succinate and malate, possibly because of removal of hydrogen. The specific activities of Avicelase, endoglucanase, β-glucosidase and xylanase were up to threefold higher compared to small batch cultures. Enzyme activities produced per gram of cellulose were up to four times the values reported for Piromyces E2 grown in a semi-continuous coculture with the methanogen Methanobacterium formicicum. The performance of fermenter enzyme preparations from the anaerobic fungi with respect to hydrolysis of Avicel compared well to that of preparations of Trichoderma reesei. However, addition of exogenous β-glucosidase was indispensible with the latter preparation for the complete conversion to glucose. Received: 14 December 1995/Received revision: 19 March 1996/Accepted: 25 March 1996     

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.     

Thermophilic anaerobic amino acid degradation: deamination rates and end-product formation

Anaerobic thermophilic degradation of several amino acids was studied in batch cultures using an inoculum from a steady-state semicontinuous enrichment culture. Experiments were done in the presence and absence of methanogenesis and known electron acceptors in the Stickland reaction. Methanogenesis was found to be crucial for the degradation of amino acids known to be oxidatively deaminated (leucine, valine and alanine). Other amino acids (serine, threonine, cysteine and methionine) were degraded under both methanogenic and non-methanogenic conditions. Degradation rates for these four amino acids were 1.3 to 2.2 times higher in cases where methanogenesis was active. The degradation rates of serine, threonine, cysteine and methionine were about twice as high as the rates of leucine, valine and alanine under methanogenic conditions. Inclusion of different electron acceptors, known to work in the Stickland reaction, did not enhance the degradation rates of any amino acid used nor did they alter the degradation patterns. Glycine was oxidatively deaminated to acetate, carbon dioxide, hydrogen and ammonium.     

Characterization of Methanosarcina mazei N2M9705 Isolated from an Aquaculture Fishpond

A new methanogenic isolate, designated as strain N2M9705 (=OCM 668), was isolated from an aquaculture fishpond near Wang-gong, Taiwan. This strain grew on trimethylamine and methanol, but it did not catabolize H₂-CO₂, acetate, or formate. The cells were stained Gram-negative, nonmotile, irregular coccus 0.6–0.8 μm in diameter. Gas vacuoles were observed and cell aggregated to form various sizes of granules. Cells grew optimally at 32°–37°C with 1% NaCl. The pH range of growth was 6.2–7.4, and higher pH inhibited the cell growth. The cells grew well in minimal medium, but growth was greatly stimulated by yeast extract and peptone. A comparison of 16S rDNA sequences of this organism phylogenetically related to Methanosarcina mazei. This is the first report of methyltrophic methanogenic isolated from an aquaculture fishpond. Received: 16 March 1999 / Accepted: 16 April 1999     

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

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

The effect of low temperature (5–29 °C) and adaptation on the methanogenic activity of biomass

The influence of low temperature (5–29 °C) on the methanogenic activity of non-adapted digested sewage sludge and on temperature/leachate-adapted biomass was assayed by using municipal landfill leachate, intermediates of anaerobic degradation (propionate) and methane precursors (acetate, H₂/CO₂) as substrates. The temperature dependence of methanogenic activity could be described by Arrhenius-derived models. However, both substrate and adaptation affected the temperature dependence. The adaptation of biomass in a leachate-fed upflow anaerobic sludge-blanket reactor at approximately 20 °C for 4 months resulted in a sevenfold and fivefold increase of methanogenic activity at 11 °C and 22 °C respectively. Both acetate and H₂/CO₂ were methanized even at 5 °C. At 22 °C, methanogenic activities (acetate 4.8–84 mM) were 1.6–5.2 times higher than those at 11 °C. The half-velocity constant (K _s) of acetate utilization at 11 °C was one-third of that at 22 °C while a similar K _i was obtained at both temperatures. With propionate (1.1–5.5 mM) as substrate, meth‐anogenic activities at 11 °C were half those at 22 °C. Furthermore, the residual concentration of the substrates was not dependent on temperature. The results suggest that the adaptation of biomass enables the achievement of a high treatment capacity in the anaerobic process even under psychrophilic conditions. Received: 23 December 1996 / Received last revision: 18 June 1997 / Accepted: 23 June 1997     

The effect of cocultivation with hydrogen-consuming bacteria on xylanolysis byRuminococcus flavefaciens

The rate and extent of xylan utilization and the specific activities of extracellular polysaccharide-degrading enzymes formed byRuminococcus flavefaciens FD1 were increased by cocultivation withMethanobrevibacter smithii PS. As a consequence of interspecies hydrogen transfer interactions, the fermentation became acetogenic; methane, not hydrogen, was formed, less succinate was produced, and formate did not accumulate in the coculture. Accumulation of xylobiose and xylose released during xylanolysis was transient in the methanogenic coculture. The interaction ofR. flavefaciens and the hydrogen-utilizing acetogenAcetitomaculum ruminis also resulted in an acetogenic fermentation, higher polysaccharolytic enzyme activities, and increased xylan utilization; the effects of cocultivation ofR. flavefaciens withEubacterium limosum were not so pronounced.     

Regulatory O2 tensions for the synthesis of fermentation products in Escherichia coli and relation to aerobic respiration

In an oxystat, the synthesis of the fermentation products formate, acetate, ethanol, lactate, and succinate of Escherichia coli was studied as a function of the O₂ tension (pO₂) in the medium. The pO₂ values that gave rise to half-maximal synthesis of the products (pO_0.5) were 0.2–0.4 mbar for ethanol, acetate, and succinate, and 1 mbar for formate. The pO_0.5 for the expression of the adhE gene encoding alcohol dehydrogenase was approximately 0.8 mbar. Thus, the pO₂ for the onset of fermentation was distinctly lower than that for anaerobic respiration (pO_0.5≤ 5 mbar), which was determined earlier. An essential role for quinol oxidase bd in microaerobic growth was demonstrated. A mutant deficient for quinol oxidase bd produced lactate as a fermentation product during growth at microoxic conditions (approximately 10 mbar O₂), in contrast to the wild-type or a quinol-oxidase-bo-deficient strain. In the presence of nitrate, the amount of lactate was largely decreased. Therefore, under microoxic conditions, the pO₂ appears to be too high for (mixed acid) fermentation to function and too low for aerobic respiration by quinol oxidase bo. Received: 7 February 1997 / Accepted: 2 May 1997     

Fermentation of cellulose by Ruminococcus flavefaciens in the presence and absence of Methanobacterium ruminantium

The anaerobic cellulolytic rumen bacterium Ruminococcus flavefaciens normally produces succinic acid as a major fermentation product together with acetic and formic acids, H2, and CO2. When grown on cellulose and in the presence of the methanogenic rumen bacterium Methanobacterium ruminantium, acetate was the major fermentation product; succinate was formed in small amounts; little formate was detected; H2 did not accumulate; and large amounts of CH4 were formed. M. ruminantium depends for growth on the reduction of CO2 to CH4 by H2, which it can obtain directly or by producing H2 and CO2 from formate. In mixed culture, the methanobacterium utilized the H2 and possibly the formate produced by the ruminococcus and in so doing stimulated the flow of electrons generated during glycolysis by the ruminococcus toward H2 formation and away from formation of succinate. This type of interaction may be of significance in determining the flow of cellulose carbon to the normal rumen fermentation products.     

Interspecific hydrogen transfer during methanol degradation by Sporomusa acidovorans and hydrogenophilic anaerobes

In the presence of active hydrogenophilic sulfate-reducing bacteria, the homoacetogenic bacterium Sporomusa acidovorans did not produce acetate during methanol degradation. H₂S and presumably CO₂ were the only end products. Since the sulfate-reducer did not degrade methnol or acetate, the sulfidogenesis from methanol was related to a complete interspecific hydrogen transfer between both species.In coculture with hydrogenophilic methanogenic bacteria (Methanobacterium formicicum, Methanospirillum hungatei), the interspecific hydrogen transfer with S. acidovorans was incomplete. Beside CH₄ and presumably CO₂, acetate was produced. The results suggested that H₂-production and H₂-consumption were involved during anaerobic methanol degradation by S. acidovorans and the hydrogenophilic anaerobes play an important role during methanol degradation by homoacetogenic bacteria in anoxic environments.     

Anaerobic degradation of 1,3-propanediol by sulfate-reducing and by fermenting bacteria

Three strains of strictly anaerobic Gram-negative, non-sporeforming, motile bacteria were enriched and isolated from freshwater sediments with 1,3-propanediol as sole energy and carbon source. Strain OttPdl was a sulfate-reducing bacterium which grew also with lactate, ethanol, propanol, butanol, 1,4-butanediol, formate or hydrogen plus CO₂, the latter only in the presence of acetate. In the absence of sulfate, most of these substrates were fermented to the respective fatty acids in syntrophic cooperation with Methanospirillum hungatei. Sulfur, thiosulfate, or sulfite were reduced, nitrate not. The other two isolates degraded propanediol only in coculture with Methanospirillum hungatei. Strain OttGlycl grew in pure culture with acetoin and with glycerol in the presence of acetate. Strain WoAcl grew in pure culture only with acetoin. Both strains did not grow with other substrates, and did not reduce nitrate, sulfate, sulfur, thiosulfate or sulfite. The isolates were affiliated with the genera Desulfovibrio and Pelobacter. The pathways of propanediol degradation and the ecological importance of this process are discussed.     

On the Problem of Anaerobic Methane Oxidation

To clarify the biological mechanism of anaerobic methane oxidation, experiments were performed with samples of the Black Sea anaerobic sediments and with the aerobic methane-oxidizing bacterium Methylomonas methanica strain 12. The inhibition–stimulation analysis did not allow an unambiguous conclusion to be made about a direct and independent role of either methanogenic or sulfate-reducing microorganisms in the biogeochemical process of anaerobic methane oxidation. Enrichment cultures obtained from samples of water and reduced sediments oxidized methane under anaerobic conditions, primarily in the presence of acetate or formate or of a mixture of acetate, formate, and lactate. However, this ability was retained by the cultures for no more than two transfers on corresponding media. Experiments showed that the aerobic methanotroph Mm. methanica strain 12 is incapable of anaerobic methane oxidation at the expense of the reduction of amorphous FeOOH.     

Fermentation of cellulose by Ruminococcus flavefaciens in the presence and absence of Methanobacterium ruminantium.

The anaerobic cellulolytic rumen bacterium Ruminococcus flavefaciens normally produces succinic acid as a major fermentation product together with acetic and formic acids, H2, and CO2. When grown on cellulose and in the presence of the methanogenic rumen bacterium Methanobacterium ruminantium, acetate was the major fermentation product; succinate was formed in small amounts; little formate was detected; H2 did not accumulate; and large amounts of CH4 were formed. M. ruminantium depends for growth on the reduction of CO2 to CH4 by H2, which it can obtain directly or by producing H2 and CO2 from formate. In mixed culture, the methanobacterium utilized the H2 and possibly the formate produced by the ruminococcus and in so doing stimulated the flow of electrons generated during glycolysis by the ruminococcus toward H2 formation and away from formation of succinate. This type of interaction may be of significance in determining the flow of cellulose carbon to the normal rumen fermentation products.     

Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms

Strain SB^T is a new, strictly anaerobic, gram-negative, nonmotile, non-sporeforming, rod-shaped bacterium that degrades benzoate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms. Strain SB^T produced approximately 3 mol of acetate and 0.6 mol of methane per mol of benzoate in coculture with Methanospirillum hungatei strain JF1. Saturated fatty acids, some unsaturated fatty acids, and methyl esters of butyrate and hexanoate also supported growth of strain SB^T in coculture with Desulfovibrio strain G11. Strain SB^T grew in pure culture with crotonate, producing acetate, butyrate, caproate, and hydrogen. The molar growth yield was 17 ± 1 g cell dry mass per mol of crotonate. Strain SB^T did not grow with fumarate, iron(III), polysulfide, or oxyanions of sulfur or nitrogen as electron acceptors with benzoate as the electron donor. The DNA base composition of strain SB^T was 43.1 mol% G+C. Analysis of the 16 S rRNA gene sequence placed strain SB^T in the δ-subdivision of the Proteobacteria, with sulfate-reducing bacteria. Strain SB^T was most closely related to members of the genus Syntrophus. The clear phenotypic and genotypic differences between strain SB^T and the two described species in the genus Syntrophus justify the formation of a new species, Syntrophus aciditrophicus. Received: 2 June 1998 / Accepted: 16 November 1998     

Acetate Oxidation Is the Dominant Methanogenic Pathway from Acetate in the Absence of Methanosaetaceae

The oxidation of acetate to hydrogen, and the subsequent conversion of hydrogen and carbon dioxide to methane, has been regarded largely as a niche mechanism occurring at high temperatures or under inhibitory conditions. In this study, 13 anaerobic reactors and sediment from a temperate anaerobic lake were surveyed for their dominant methanogenic population by using fluorescent in situ hybridization and for the degree of acetate oxidation relative to aceticlastic conversion by using radiolabeled [2-¹⁴C]acetate in batch incubations. When Methanosaetaceae were not present, acetate oxidation was the dominant methanogenic pathway. Aceticlastic conversion was observed only in the presence of Methanosaetaceae.     

Occurrence of Methanogenic Archaea in Highly Polluted Sediments of Tropical Santos–São Vicente Estuary (São Paulo, Brazil)

Little is known about the ability of methanogens to grow and produce methane in estuarine environments. In this study, traditional methods for cultivating strictly anaerobic microorganisms were combined with Fluorescence in situ hybridization (FISH) technique to enrich and identify methanogenic Archaea cultures occurring in highly polluted sediments of tropical Santos–São Vicente Estuary (São Paulo, Brazil). Sediment samples were enriched at 30°C under strict anaerobic and halophilic conditions, using a basal medium containing 2% of sodium chloride and amended with glucose, methanol, and sodium salts of acetate, formate and lactate. High methanogenic activity was detected, as evidenced by the biogas containing 11.5 mmol of methane at 20 days of incubation time and methane yield of 0.138-mmol CH₄/g organic matter/g volatile suspense solids. Cells of methanogenic Archaea were selected by serial dilution in medium amended separately with sodium acetate, sodium formate, or methanol. FISH analysis revealed the presence of Methanobacteriaceae and Methanosarcina sp. cells.