Considerations on the energy metabolism of Clostridium kluyveri

Summary On the basis of the known fermentation balance and of the enzyme activities reported in Clostridium kluyveri the ethanol-acetate fermentation of Clostridium kluyveri has been analyzed with respect to possible ATP-yielding reactions and to the significance of the evolution of hydrogen gas during the fermentation. The fermentation pathway presented allows the following conclusions: hydrogen gas is an essential end product of the ethanol-acetate fermentation. For each two moles of hydrogen gas evolved one mole of acetyl coenzyme A becomes available to the cells for ATP synthesis, and it is not necessary to assume that ATP is synthesized by Clostridium kluyveri by electron transport phosphorylation. Hydrogen gas must be formed in the dehydrogenation of acetaldehyde. Since Clostridium kluyveri contains a NAD reductase, less than one mole of hydrogen gas is formed per mole of acetaldehyde oxidized, thus explaining that acetate is required for the fermentation of ethanol.It could be demonstrated that growth of Clostridium kluyveri is slow in a hydrogen atmosphere as compared with growth in an argon atmosphere. The general fermentation equation constructed is in accordance with the experimental data of Bornstein and Barker and of Thauer et al.

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Zusammenfassung Auf der Grundlage bekannter Gärungsanalysen und der in zellfreien Extrakten nachgewiesenen Enzymaktivitäten wurde die Äthanol-Acetat-Gärung von Clostridium kluyveri auf mögliche Energie liefernde Reaktionen hin untersucht. Das entworfene Schema der Äthanol-Acetat-Gärung erlaubt folgende Schlußfolgerungen: Die Bildung von molekularem Wasserstoff während der Gärung its von elementarer Bedeutung für den Energiestoffwechsel von C. kluyveri. Je Mol freigesetzten Wasserstoffs stehen C. kluyveri 0,5 Mole Acetyl-Coenzym A für die Energiegewinnung zur Verfügung, und es ist überflüssig, eine ATP-Synthese durch Elektronentransport-Phosphorylierung anzunehmen. Der molekulare Wasserstoff muß bei der Dehydrogenierung des Acetaldehyds gebildet werden. Da hierbei weniger als ein Mol molekularer Wasserstoff je Mol Acetaldehyd entsteht und ein Teil des Wasserstoffs auf NAD übertragen wird, ist Acetat als Wasserstoffacceptor für die Vergärung des Äthanols notwendig.Die abgeleitete Gärungsgleichung stimmt mit den von Bornstein und Barker und von Thauer et al. ermittelten Gärungsbilanzen überein. Es konnte nachgewiesen werden, daß C. kluyveri in einer Wasserstoffatmosphäre sehr viel langsamer wächst als in einer Argonatmosphäre.

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Abbreviations ATP adenosine triphosphate - CoA coenzyme A - Fd_red reduced ferredoxin - NAD(P) nicotinamide-adenine-dinucleotide(phosphate)     

Characterization of crotonate grown Clostridium kluyveri by its assimilatory metabolism

Summary Considerable behavioral differences were observed during growth of Clostridium kluyveri on ethanol-acetate and on crotonate media. The identity of the crotonate grown Clostridium with the ethanol grown Clostridium kluyveri was therefore established by three characteristic biosynthetic routes: 1. ribose is synthesized from CO₂ and acetate via pyruvate, triose phosphate and a non-oxidative pentose phosphate pathway, 2. reduced one-carbon units are formed predominantly from CO₂ and not from serine as usual, and 3. glutamate biogenesis follows an atypical stereochemical course.This paper contains parts of the doctoral thesis of J. W., Medizinische Fakultät, Universität Freiburg, 1968.     

Synthetic co-culture of autotrophic Clostridium carboxidivorans and chain elongating Clostridium kluyveri monitored by flow cytometry

Syngas fermentation with acetogens is known to produce mainly acetate and ethanol efficiently. Co-cultures with chain elongating bacteria making use of these products are a promising approach to produce longer-chain alcohols. Synthetic co-cultures with identical initial cell concentrations of Clostridium carboxidivorans and Clostridium kluyveri were studied in batch-operated stirred-tank bioreactors with continuous CO/CO₂-gassing and monitoring of the cell counts of both clostridia by flow cytometry after fluorescence in situ hybridization (FISH-FC). At 800 mbar CO, chain elongation activity was observed at pH 6.0, although growth of C. kluyveri was restricted. Organic acids produced by C. kluyveri were reduced by C. carboxidivorans to the corresponding alcohols butanol and hexanol. This resulted in a threefold increase in final butanol concentration and enabled hexanol production compared with a mono-culture of C. carboxidivorans. At 100 mbar CO, growth of C. kluyveri was improved; however, the capacity of C. carboxidivorans to form alcohols was reduced. Because of the accumulation of organic acids, a constant decay of C. carboxidivorans was observed. The measurement of individual cell concentrations in co-culture established in this study may serve as an effective tool for knowledge-based identification of optimum process conditions for enhanced formation of longer-chain alcohols by clostridial co-cultures.     

Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri.

A pathway of succinate fermentation to acetate and butanoate (butyrate) in Clostridium kluyveri has been supported by the results of 13C nuclear magnetic resonance studies of the metabolic end products of growth and the detection of dehydrogenase activities involved in the conversion of succinate to 4-hydroxybutanoate (succinic semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase). C. kluyveri fermented [1,4-13C]succinate primarily to [1-13C]acetate, [2-13C]acetate, and [1,4-13C]butanoate. Any pathway proposed for this metabolism must account for the reduction of a carboxyl group to a methyl group. Succinic semialdehyde dehydrogenase activity was demonstrated after separation of the crude extracts of cells grown on succinate and ethanol (succinate cells) by anaerobic nondenaturing polyacrylamide gel electrophoresis. 4-Hydroxybutanoate dehydrogenase activity in crude extracts of succinate cells was detected and characterized. Neither activity was found in cells grown on acetate and ethanol (acetate cells). Analysis of cell extracts from acetate cells and succinate cells by sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that several proteins were present in succinate cell extracts that were not present in acetate cell extracts. In addition to these changes in protein composition, less ethanol dehydrogenase and hydrogenase activity was present in the crude extracts from succinate cells than in the crude extracts from acetate cells. These data support the hypothesis that C. kluyveri uses succinate as an electron acceptor for the reducing equivalents generated from the ATP-producing oxidation of ethanol.     

13C NMR studies of butyric fermentation in Clostridium kluyveri

The fermentation of 13C-labeled ethanol and acetate into butyrate and caproate by Clostridium kluyveri has been studied by using 13C NMR. The pathway involves the conversion of both ethanol and acetate into acetyl coenzymes A, two of which condense to form CoA-linked precursors of butyrate. If butyryl-CoA is involved in the condensation, caproate is the ultimate product. ATP is produced from acetyl-CoA via the reactions catalyzed by phosphotransacetylase and acetate kinase with acetate, a required carbon source, as a co-product. In spectra of whole cells incubated with the labeled carbon sources, label from ethanol appears rapidly in acetate, which then reaches a lower, steady-state concentration due to its re-entry into the pathway. The rapid initial production of acetate indicates equally rapid production of ATP. Label from acetate appears in ethanol only if ethanol is already present, indicating that this process is one of isotopic equilibration rather than net synthesis of ethanol from acetate. The ratio of butyrate to caproate produced depends strongly on the initial ratio of ethanol to acetate in the medium. The relative rates of utilization of ethanol and acetate vary as the fermentation proceeds. 13C-13C coupling in the butyrate and caproate produced from [1-13C]ethanol and [2-13C]acetate can be used to determine if the acetyl-CoA molecules arising from ethanol and acetate enter the same pool or if they remain separated. The data are consistent with random mixing of the acetyl-CoA produced from the two carbon sources.