Evidence of reversed electron transport in syntrophic butyrateor benzoate oxidation by Syntrophomonas wolfei and Syntrophus buswellii Received: 2 March 1994 / Accepted: 20 April 1994 Abstract Syntrophomonas wolfei and Syntrophus buswellii were grown with butyrate or benzoate in a defined binary coculture with Methanospirillum hungatei. Both strains also grew independent of the partner bacteria with crotonate as substrate. Localization of enzymes involved in butyrate oxidation by S. wolfei revealed that ATP synthase, hydrogenase, and butyryl-CoA dehydrogenase were at least partially membrane-associated whereas 3-hydroxybutyryl-CoA dehydrogenase and crotonase were entirely cytoplasmic. Inhibition experiments with copper chloride indicated that hydrogenase faced the outer surface of the cytoplasmic membrane. Suspensions of butyrate-or benzoate-grown cells of either strain accumulated hydrogen during oxidation of butyrate or benzoate to a low concentration that was thermodynamically in equilibrium with calculated reaction energetics. The protonophore carbonylcyanide m-chlorophenyl-hydrazone (CCCP) and the proton-translocating ATPase inhibitor N,N'dicyclohexylcarbodiimide (DCCD) both specifically inhibited hydrogen formation from butyrate or benzoate at low concentrations, whereas hydrogen formation from crotonate was not affected. A menaquinone was extracted from cells of S. wolfei and S. buswellii grown syntrophically in a binary methanogenic culture. The results indicate that a proton-potential-driven process is involved in hydrogen release from butyrate or benzoate oxidation. Degradation of both compounds becomes feasible only at low hydrogen partial pressure (10-4-10 -5 atm; Schink 1992), which can be maintained by hydrogen-oxidizing anaerobes such as methanogenic bacteria (Zehnder 1978;Dolfing 1988). The pathways of butyrate and benzoate degradation in these bacteria have been at least tentatively elucidated (see Schink 1992). The energetically most difficult electron transfer steps are the oxidations of the saturated acid esters butyryl-CoA or glutaryl-CoA to the respective unsaturated compounds. The electrons released in the butyryl-CoA dehydrogenase reaction (E 0, = -125 mV; Gustafson et al. 1986) and glutaryl-CoA dehydrogenase, for which a similar redox potential is assumed, are used to reduce protons to molecular hydrogen (E 0" = -414 mV). Even at 10 .4 atm hydrogen, the redox potential of the couple 2 H+/H2 is still -295 mV and is much lower than that of the electron donor. It has been hypothesized, therefore, that part of the ATP gained by substrate level phosphorylation during butyrate oxidation has to be spent in a reversed electron transport step to shift these electrons to a lower redox potential (Thauer and Morris 1984). Similar problems arise with oxidation of saturated intermediates in syntrophic benzoate degradation, and involvement of reversed electron transport in this oxidation also has been postulated (Schink 1992). However, experimental evidence of such energy-driven processes in syntrophic ...
A new strain of syntrophically propionate-oxidizing fermenting bacteria, strain KoProp1, was isolated from anoxic sludge of a municipal sewage plant. It oxidized propionate or lactate in cooperation with the hydrogen-and formate-utilizing Methanospirillum hungatei and grew as well in pure culture without a syntrophic partner with propionate or lactate plus sulfate as energy source. In all cases, the substrates were oxidized stoichiometrically to acetate and CO 2 , with concomitant formation of methane or sulfide. Cells formed gas vesicles in the late growth phase and contained cytochromes b and c, a menaquinone-7, and desulforubidin, but no desulfoviridin. Enzyme measurements in cell-free extracts indicated that propionate was oxidized through the methylmalonyl CoA pathway. Protein pattern analysis by SDS-PAGE of cell-free extracts showed that strain KoProp1 differs significantly from Syntrophobacter wolinii and from the propionate-oxidizing sulfate reducer Desulfobulbus propionicus. 16S rRNA sequence analysis revealed a significant resemblance to S. wolinii allowing the assignment of strain KoProp1 to the genus Syntrophobacter as a new species, S. pfennigii. (1) ∆G 0´ = +76.0 kJ/mol propionate The hydrogen partial pressure has to be kept low by the partner organism to make the reaction energetically feasible, e.g., in syntrophic methanogenic propionate degradation: 4 CH 3 CH 2 COO -+ 2 H 2 O→4 CH 3 COO -+ CO 2 +3 CH 4 (2) ∆G 0´ = -26.5 kJ/mol propionate However, the amount of free energy liberated during syntrophic propionate oxidation is still low and just in the range of the minimum energy quantum needed for ATP formation by each partner bacterium (Schink 1990. So far, only one defined syntrophic propionate-degrading culture, Syntrophobacter wolinii, has been described; this culture contains a methanogenic bacterium and a sulfatereducing partner bacterium (Boone and Bryant 1980). It has been shown recently that the propionate-fermenting partner bacterium in this mixed culture could be grown in pure culture, either with pyruvate alone or with propionate plus sulfate as substrates (Wallrabenstein et al. 1994). Another propionate-degrading syntrophic anaerobe, strain MPOB, has been recently obtained in an enrichment culture with propionate plus fumarate as substrates, which were fermented to acetate, CO 2 , and succinate ); a thermophilic methanogenic propionate-oxidizing syntrophic enrichment culture has also been described (Stams et al. 1992). Key wordsThe present study reports on the isolation and characterization of a new strain of syntrophically propionate-oxidizing fermenting bacteria that grows also in pure culture by propionate-dependent sulfate reduction and represents a new species within the genus Syntrophobacter. Christina Wallrabenstein · Elisabeth Hauschild · Bernhard SchinkSyntrophobacter pfennigii sp. nov., new syntrophically propionate-oxidizing anaerobe growing in pure culture with propionate and sulfate Arch Microbiol (1995) 164 : 346-352
The syntrophically propionate-oxidizing bacterium Syntrophobacter wolinii was grown in binary methanogenic coculture with Methanospirillum hungatei as sole partner, free of contaminating Desulfovibrio vulgaris. Substrate tests revealed that S. wolinii could grow also without any syntrophic partner, either with pyruvate as sole substrate or with propionate plus sulfate. Pyruvate was fermented to acetate, propionate and presumably CO2; propionate in the presence of sulfate was oxidized incompletely to acetate and CO2, with stoichiometric sulfide formation. The pure culture contained cytochromes b and c and menaquinone-7. Desul-foviridin or desulforubidin could not be detected.
A new strain of syntrophically propionate-oxidizing fermenting bacteria, strain KoProp1, was isolated from anoxic sludge of a municipal sewage plant. It oxidized propionate or lactate in cooperation with the hydrogen-and formate-utilizing Methanospirillum hungatei and grew as well in pure culture without a syntrophic partner with propionate or lactate plus sulfate as energy source. In all cases, the substrates were oxidized stoichiometrically to acetate and CO 2 , with concomitant formation of methane or sulfide. Cells formed gas vesicles in the late growth phase and contained cytochromes b and c, a menaquinone-7, and desulforubidin, but no desulfoviridin. Enzyme measurements in cell-free extracts indicated that propionate was oxidized through the methylmalonyl CoA pathway. Protein pattern analysis by SDS-PAGE of cell-free extracts showed that strain KoProp1 differs significantly from Syntrophobacter wolinii and from the propionate-oxidizing sulfate reducer Desulfobulbus propionicus. 16S rRNA sequence analysis revealed a significant resemblance to S. wolinii allowing the assignment of strain KoProp1 to the genus Syntrophobacter as a new species, S. pfennigii. (1) ∆G 0´ = +76.0 kJ/mol propionate The hydrogen partial pressure has to be kept low by the partner organism to make the reaction energetically feasible, e.g., in syntrophic methanogenic propionate degradation: 4 CH 3 CH 2 COO -+ 2 H 2 O→4 CH 3 COO -+ CO 2 +3 CH 4 (2) ∆G 0´ = -26.5 kJ/mol propionate However, the amount of free energy liberated during syntrophic propionate oxidation is still low and just in the range of the minimum energy quantum needed for ATP formation by each partner bacterium (Schink 1990. So far, only one defined syntrophic propionate-degrading culture, Syntrophobacter wolinii, has been described; this culture contains a methanogenic bacterium and a sulfatereducing partner bacterium (Boone and Bryant 1980). It has been shown recently that the propionate-fermenting partner bacterium in this mixed culture could be grown in pure culture, either with pyruvate alone or with propionate plus sulfate as substrates (Wallrabenstein et al. 1994). Another propionate-degrading syntrophic anaerobe, strain MPOB, has been recently obtained in an enrichment culture with propionate plus fumarate as substrates, which were fermented to acetate, CO 2 , and succinate ); a thermophilic methanogenic propionate-oxidizing syntrophic enrichment culture has also been described (Stams et al. 1992). Key wordsThe present study reports on the isolation and characterization of a new strain of syntrophically propionate-oxidizing fermenting bacteria that grows also in pure culture by propionate-dependent sulfate reduction and represents a new species within the genus Syntrophobacter. Christina Wallrabenstein · Elisabeth Hauschild · Bernhard SchinkSyntrophobacter pfennigii sp. nov., new syntrophically propionate-oxidizing anaerobe growing in pure culture with propionate and sulfate Arch Microbiol (1995) 164 : 346-352
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