Role of formate and hydrogen in the syntrophic degradation of propionate and butyrate

Xiuzhu Dong

Research output: Thesisinternal PhD, WU


<p>Under methanogenic conditions, complex organic matter is mineralized by fermentative, acetogenic and methanogenic bacteria. Propionate and butyrate are two important intermediates; they account for 35% and 8% of the total methane formation, respectively. Propionate and butyrate are converted to CH <sub><font size="-2">4</font></sub> and CO <sub><font size="-2">2</font></sub> by the syntrophic consortia of acetogenic and methanogenic bacteria. Both H <sub><font size="-2">2</font></sub> transfer and formate transfer were proposed as the possible mechanisms by which reducing equivalents are transferred from the acetogens to the methanogens. Since extreme low levels of H <sub><font size="-2">2</font></sub> and formate prevail in anaerobic ecosystems and cocultures, it is not known which of the two is more important.<p>The aim of this research was to clarify the relative importance of H <sub><font size="-2">2</font></sub> and formate transfer during syntrophic degradation of propionate and butyrate by means of physiological and biochemical approaches.<p>After succeeding in obtaining pure cultures of propionate oxidizer MPOB and butyrate oxidizer <em>Syntrophospora bryantii</em> by growing them on fumarate and crotonate, respectively, this study became possible. By constructing defined methanogenic cocultures, it was observed that propionate oxidation by MPOB and butyrate oxidation by <em>S.</em><strong></strong><em>bryantii</em> can only be driven by H <sub><font size="-2">2</font></sub> /formate- but not by H <sub><font size="-2">2</font></sub> -utilizing or aceticlastic methanogens (Chapter 2 and 3), indicating that formate transfer might be prevailing in these cultures. However, a triculture of MPOB with a <em>Desulfovibrio</em> strain, which converts formate into H <sub><font size="-2">2</font></sub> /CO <sub><font size="-2">2</font></sub> , and a H <sub><font size="-2">2</font></sub> -utilizing methanogen also degraded propionate, confirming that H <sub><font size="-2">2</font></sub> transfer is possible provided that low formate concentrations are maintained (Chapter 2).<p>MPOB is able to ferment fumarate to succinate and to couple propionate oxidation to fumarate reduction; while <em>S. bryantii</em> is able to couple butyrate oxidation to the reduction of pentenoate (Chapter 3). With a limiting amount of fumarate in a propionate degrading culture, the maximum H <sub><font size="-2">2</font></sub> and formate levels produced from propionate by MPOB were 6.8 Pa and 24 μmol, respectively; and with a limiting amount of pentenoate in a butyrate degrading culture, the maximum H <sub><font size="-2">2</font></sub> and formate levles produced by <em>S. bryantii</em> were 170 Pa and 280 μmol, respectively (Chapter 4). These results showed that both H <sub><font size="-2">2</font></sub> and formate indeed were formed and that the values which were reached were in the range which could be expected from thermodynamical consideration. Using a diffusion model it could be calculated that formate fluxes can be 100 times higher than H <sub><font size="-2">2</font></sub> fluxes in the methanogenic cocultures of MPOB and <em>S.</em><em>bryantii</em> suggesting that formate transfer is more important during syntrophic propionate and butyrate degradation.<p>Enzyme measurments (Chapter 5) showed that <em>S.</em><em>bryantii</em> contained very high hydrogenase activity and low formate dehydrogenase activity. The K <sub><font size="-2">m</font></sub> values of the two enzymes are about the same, 0.21 mM and 0.22 mM, respectively. Butyryl-CoA dehydrogenase and 3-hydroxybutyryl-CoA dehydrogenase, which are involved in the two oxidation reactions during butyrate oxidation, were determined as soluble enzymes. The hydrogenase was also determined as a cytoplasmic enzyme while formate dehydrogenase and CO <sub><font size="-2">2</font></sub> -reductase were membrane bound, likely located at the outer aspect of the cytoplasmic membrane. These observations suggest that during butyrate oxidation H <sub><font size="-2">2</font></sub> is formed intracellularlly, while formate is formed extracellularly. A reversed electron transfer mechanism was postulated to drive the endergonic oxidation of butyryl-CoA to crotonyl-CoA. Similar experiments were done with MPOB (Chapter 6). MPOB also contained relative high hydrogenase activities (1,5-3.7 μmol. min <sup><font size="-2">-1</font></SUP>.mg <sup><font size="-2">-1</font></SUP>) and low formate dehydrogenase activities (0.2-0.4 μmol. min <sup><font size="-2">-1</font></SUP>. mg <sup><font size="-2">-1</font></SUP>). The K <sub><font size="-2">m</font></sub> values are 0.05 mM of H <sub><font size="-2">2</font></sub> for hydrogenase and 0.3 mM of formate for formate dehydrogenase. Localization of the redox enzymes involved in propionate oxidation indicated that the fumarate reductase was membrane bound, likely located at the inner aspect of membrane; while malate dehydrogenase and pyruvate dehydrogenase were located in the cytoplasm. The hydrogenase is located partly in the periplasm, partly in the cytoplasm and partly in the membrane; while the formate dehydrogenase was determined partly in the periplasm and partly in the membrane. These results indicate that, during propionate oxidation, formate is formed outside of cytoplasmic membrane, However, where H <sub><font size="-2">2</font></sub> is produced is not clear yet. As with butyrate oxidation, a reversed electron transport mechanism was postulated to drive the oxidation of succinate (ΔG°'= +86.2 kJ/mol).<p>Although acetate is also a product of propionate and butyrate oxidation, removal of acetate by aceticlastic methanogen alone does not drive the oxidations by the acetogens MPOB and <em>S. bryantii.</em> However, addition of the aceticlastic methanogen <em>Methanothrix (Methanosaeta) soehngenii</em> increased the degradation rates of propionate and butyrate by the cocultures of MPOB and <em>S. bryantii.</em> with H <sub><font size="-2">2</font></sub> /formate-trophic methanogens (Chapter 2 and 3). The addition of 50 mM acetate decreased the butyrate degradation rate of the coculture of <em>S. bryantii.</em> and <em>Methanospirillum hungatei</em> by about 60% (Chapter 3). These results indicate that a low acetate concentration is beneficial for syntrophic degradation of propionate and butyrate.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Zehnder, A.J.B., Promotor
  • Stams, Fons, Co-promotor
Award date9 Dec 1994
Place of PublicationS.l.
Print ISBNs9789054853336
Publication statusPublished - 1994


  • microbial degradation
  • methanobacteriaceae
  • propionic acid
  • butyric acid


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