Syntrophic oxidation of butyrate and ethanol

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Syntrophic bacteria live at the thermodynamic limit of growth. The biochemistry of those secondary fermenters is not fully understood yet. In this thesis, two model systems of syntrophic organisms growing on two difficult to degrade substrates were investigated: Syntrophomonas wolfei in coculture with Methanospirillum hungatei converting butyrate to acetate and methane and Pelobacter carbinolicus or P. acetylenicus in coculture with M. hungatei fermenting ethanol to acetate and methane.
All prominent membrane-bound and soluble proteins expressed in S. wolfei specifically during syntrophic growth with butyrate were examined by 1D- and 2D-PAGE. A membrane-bound, externally oriented formate dehydrogenase complex was expressed at high level. Soluble hydrogenases were expressed at high levels specifically during growth with the pure-culture substrate crotonate. The results were confirmed by native PAGE, by formate dehydrogenase and hydrogenase-activity staining, and by analysis of formate dehydrogenase and hydrogenase activities in intact cells and cell-free extracts. In S. wolfei, electrons derived from butyryl-CoA are hypothesized to be transferred through a membrane-bound EtfAB:quinone oxidoreductase to a menaquinone cycle and further via a b-type cytochrome to an externally oriented formate dehydrogenase. Hence, an ATP hydrolysis-driven proton-motive force across the cytoplasmatic membrane would provide the energy input for the electron potential shift necessary for formate formation.
The membrane-associated and cytoplasmic enzymes of P. carbinolicus and partially also P. acetylenicus were investigated with 1D- and 2D-PAGE, enzyme tests and cultivation experiments as well. In the cytoplasmic fraction of ethanol-grown P. carbinolicus cells, acetaldehyde was oxidized mainly to acetyl-CoA. Hence, substrate-level phosphorylation appeared to be the dominant mode of energy conservation. Additionally, a ferredoxin-dependent, non-acetylating acetaldehyde dehydrogenase activity could be detected. Comparative 2D-PAGE revealed enhanced expression of tungsten-dependent acetaldehyde:ferredoxin oxidoreductases and formate dehydrogenase. Proteomics allowed the detection of an Rnf subunit once and several putatively Na+-dependent ATP synthase subunits in membrane preparations. Protein purification allowed the enrichment and identification of P. carbinolicus ferredoxin Pcar_2544 which had 44% sequence identity to Clostridium pasteurianum ferredoxin. Comproportionating hydrogenases and formate dehydrogenase were expressed constitutively and are probably involved in interspecies electron transfer. The hydrogenase activity depended on flavin mononucleotide. In ethanol-grown cocultures, the maximum hydrogen partial pressure was about 1,000 Pa (1 mM) while 2 mM formate was produced. The redox potentials of hydrogen and formate released during ethanol oxidation were calculated to be EH2 = -358 ± 12 mV and EHCOOH = -366 ± 19 mV, respectively. Both hydrogen and formate contributed to interspecies electron transfer. Both Pelobacter species could be cultivated axenically on acetaldehyde, yielding energy from its disproportionation to ethanol and acetate. The early assumption that acetaldehyde is a central intermediate in Pelobacter metabolism has now been proven biochemically.
One of the first scientists publishing (in 1916) on microbial methanogenesis from butyrate and ethanol was V. L. Omeljanskij (1867-1928). Although he was an early pioneer of methanogenesis research, his lifework was never presented to an international audience. He founded a school of thought based on his teacher’s S. N. Vinogradskij’s discoveries and theories in Russia and the USSR. Omeljanskij published the first Russian textbook on microbiology, “Principles of microbiology” (“Основы микробиологии“) in 1909. He published internationally in German, French and English, thus representing a cross-border type of scientist.

Zusammenfassung in einer weiteren Sprache

Syntroph lebende Bakterien können durch die Vergärung einfachster organischer Stoffe nur so wenig Energie gewinnen, dass Wachstum thermodynamisch gerade noch möglich ist. Die Biochemie der syntrophen Butansäure- und Ethanolgärung ist noch nicht vollständig entschlüsselt. In dieser Doktorarbeit wurden daher modellhaft zwei syntrophe Cokulturen näher untersucht: Syntrophomonas wolfei mit Methanospirillum hungatei (Oxidation von Butyrat zu Acetat und Methan), sowie Pelobacter carbinolicus bzw. P. acetylenicus mit M. hungatei (Oxidation von Ethanol zu Acetat und Methan).
Die am höchsten exprimierten Proteine der Membran und des Cytoplasmas von Butyrat-gewachsenem S. wolfei wurden mit 1D- und 2D-PAGE untersucht. Eine membranständige, nach außen ausgerichtete Formiat-Dehydrogenase war stark überexprimiert. Lösliche Hydrogenasen waren ebenfalls stark überexprimiert, wenn S. wolfei in Reinkultur mit Crotonsäure wuchs. Die PAGE-Ergebnisse wurden durch nicht-denaturierende PAGE, anoxische Aktivitätsfärbung und entsprechende Enzymtests mit Zellextrakten und ganzen Zellen überprüft. Die Ergebnisse ermöglichten ein neues Verständnis des Elektronenflusses in S. wolfei. Die Elektronen der Butyryl-CoA-Oxidation werden wahrscheinlich mittels einer membranständigen EtfAB:Chinon-Oxidoreduktase in einen Menachinonzyklus eingespeist. Die Elektronen werden durch ein Cytochrom b an eine nach außen orientierte Formiat-Dehydrogenase weitergeleitet.
In P. carbinolicus und teilweise auch in P. acetylenicus wurden ebenfalls membranständige und cytoplasmatische Proteine mit 1D-und 2D-PAGE, Enzymtests und Kultivierungsexperimenten untersucht. In der Cytoplasmafraktion Ethanol-gewachsener P. carbinolicus Zellen wird Acetaldehyd aus Ethanol gebildet und hauptsächlich zu Acetyl-CoA weiter oxidiert. Dieses dient in der Substratstufenphosphorylierung zur Synthese von ATP. Zusätzlich wurde eine Ferredoxin-abhängige, nichtacetylierende Acetaldehyd-Dehydrogenase gefunden. Vergleichende 2D-PAGE konnte zeigen, dass diese Acetaldehyddehydrogenase und eine Formiatdehydrogenase abhängig vom Spurenelement Wolfram ist. Weiterhin wurde ein einziges Mal eine Untereinheit eines Rnf-Komplexes und verschiedene ATP-Synthase-Untereinheiten in der Membran identifizert. Durch Proteinreinigung gelang auch die Anreicherung und die Identifizierung des P. carbinolicus Ferredoxins Pcar_2544, das zu 44% sequenzidentisch zum Ferredoxin aus Clostridium pasteurianum ist. Die Enzyme des Interspecies-Elektronentransfers, Formiatdehydrogenase und Hydrogenase, waren komproportionierend und sind konstitutiv exprimiert. Die Hydrogenase-Aktvität war abhängig von Riboflavin als Cofaktor. In Ethanol-gewachsenen Cokulturen war der maximale Wasserstoffpartialdruck 1.000 Pa (entspricht 1 mM) und die maximale Formiatkonzentration 2 mM. Das Redoxpotenzial der Freisetzung von Wasserstoff und Formiat konnte als EH2 = -358 ± 12 mV bzw. EHCOOH = -366 ± 19 mV berechnet werden. Wasserstoff und Formiat trugen gleichberechtigt zum Elektronentransfer zwischen den syntrophen Partnern bei. Beide Pelobacter-Spezies konnten in Reinkultur Acetaldehyd als Wachstumssubstrat nutzen. Dabei disproportionierten sie Acetaldehyd zu Ethanol und Acetat. Dies ist der biochemische Beweis, dass Acetaldehyd ein zentrales Intermediat im Metabolismus von Pelobacter ist.
Als einer der ersten publizierte V. L. Omeljanskij (1867-1928) zum Thema der mikrobiellen Methanbildung aus Butansäure und Ethanol. Sein Lebenswerk blieb jedoch bislang unerforscht und einem internationalen Publikum unzugänglich. Er verfasste 1909 das erste russische, mikrobiologische Lehrbuch, „Grundlagen der Mikrobiologie“ (“Основы микробиологии“) und verbreitete auf diese Weise die Entdeckungen und Theorien seines Lehrers S. N. Vinogradskij in Russland und der UdSSR. Omeljanskij veröffentlichte seine Arbeiten international und grenzüberschreitend in deutscher, französischer und englischer Sprache.

Fachgebiet (DDC)
570 Biowissenschaften, Biologie
Schlagwörter
Syntrophie, syntrophy, Sytrophomonas wolfei, Pelobacter, Ethanol, Butyrat
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ISO 690SCHMIDT, Alexander, 2014. Syntrophic oxidation of butyrate and ethanol [Dissertation]. Konstanz: University of Konstanz
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@phdthesis{Schmidt2014Syntr-29510,
  year={2014},
  title={Syntrophic oxidation of butyrate and ethanol},
  author={Schmidt, Alexander},
  address={Konstanz},
  school={Universität Konstanz}
}
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December 17, 2014
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Konstanz, Univ., Diss., 2014
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