Abstract
Pelobacter carbinolicus and P. acetylenicus oxidize ethanol in syntrophic cooperation with methanogens. Cocultures with Methanospirillum hungatei served as model systems for the elucidation of syntrophic ethanol oxidation previously done with the lost “Methanobacillus omelianskii” coculture. During growth on ethanol, both Pelobacter species exhibited NAD+-dependent alcohol dehydrogenase activity. Two different acetaldehyde-oxidizing activities were found: a benzyl viologen-reducing enzyme forming acetate, and a NAD+-reducing enzyme forming acetyl-CoA. Both species synthesized ATP from acetyl-CoA via acetyl phosphate. Comparative 2D-PAGE of ethanol-grown P. carbinolicus revealed enhanced expression of tungsten-dependent acetaldehyde: ferredoxin oxidoreductases and formate dehydrogenase. Tungsten limitation resulted in slower growth and the expression of a molybdenum-dependent isoenzyme. Putative comproportionating hydrogenases and formate dehydrogenase were expressed constitutively and are probably involved in interspecies electron transfer. 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. Hydrogen and formate formation and degradation further proved that both carriers contributed to interspecies electron transfer. The maximum Gibbs free energy that the Pelobacter species could exploit during growth on ethanol was −35 to −28 kJ per mol ethanol. Both species could be cultivated axenically on acetaldehyde, yielding energy from its disproportionation to ethanol and acetate. Syntrophic cocultures grown on acetoin revealed a two-phase degradation: first acetoin degradation to acetate and ethanol without involvement of the methanogenic partner, and subsequent syntrophic ethanol oxidation. Protein expression and activity patterns of both Pelobacter spp. grown with the named substrates were highly similar suggesting that both share the same steps in ethanol and acetalydehyde metabolism. The early assumption that acetaldehyde is a central intermediate in Pelobacter metabolism was now proven biochemically.
Highlights
The genus Pelobacter embraces strictly anaerobic, Gram-negative delta-proteobacteria [1]
The specific enzyme activities detected in both P. acetylenicus and P. carbinolicus after syntrophic growth with ethanol were highly similar (Table 1)
Key enzymes of ethanol and acetaldehyde metabolism were assayed in two Pelobacter species, together with hydrogenase and formate dehydrogenase activities which act as electron carriers to the methanogenic partner organism
Summary
The genus Pelobacter embraces strictly anaerobic, Gram-negative delta-proteobacteria [1]. In contrast to other Desulfuromonadales, Pelobacter species are fermenting bacteria incapable of anaerobic respiration. Their fermenting capabilities are regarded as a secondary evolutionary event [2] since genes of key enzymes in their fermentative metabolism are likely to originate from lateral gene transfer [3]. The best investigated species within this genus are Pelobacter carbinolicus [4] and Pelobacter acetylenicus [5]. Both strains originate from different environments: brackish water sediment or freshwater sewage sludge, respectively. The metabolism of all these substrates includes acetaldehyde as central intermediate which was proposed to be the ecological specialisation or niche of these bacteria [1]
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