Enrichment of Thermophilic Propionate-Oxidizing Bacteria in Syntrophy with Methanobacterium thermoautotrophicum or Methanobacterium thermoformicicum

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Abstract
Thermophilic propionate-oxidizing, proton-reducing bacteria were enriched from the granular methanogenic sludge of a bench-scale upflow anaerobic sludge bed reactor operated at 55°C with a mixture of volatile fatty acids as feed. Thermophilic hydrogenotrophic methanogens had a high decay rate. Therefore, stable, thermophilic propionate-oxidizing cultures could not be obtained by using the usual enrichment procedures. Stable and reproducible cultivation was possible by enrichment in hydrogen-pregrown cultures of Methanobacterium thermoautotrophicum ΔH which were embedded in precipitates of FeS, achieved by addition of FeCl2 to the media. The propionate-oxidizing bacteria formed spores which resisted pasteurization for 30 min at 90°C or 10 min at 100°C. Highly purified cultures were obtained with either M. thermoautotrophicum ΔH or Methanobacterium thermoformicicum Z245 as the syntrophic partner organism. The optimum temperature for the two cultures was 55°C. Maximum specific growth rates of cultures with M. thermoautotrophicum ΔH were somewhat lower than those of cultures with M. thermoformicicum Z245 (0.15 and 0.19 day-1, respectively). Growth rates were even higher (0.32 day-1) when aceticlastic methanogens were present as well. M. thermoautotrophicum ΔH is an obligately hydrogen-utilizing methanogen, showing that interspecies hydrogen transfer is the mechanism by which reducing equivalents are channelled from the acetogens to this methanogen. Boundaries of hydrogen partial pressures at which propionate oxidation occurred were between 6 and 34 Pa. Formate had a strong inhibitory effect on propionate oxidation in cultures with M. thermoautotrophicum. Inhibition by formate was neutralized by addition of the formate-utilizing methanogen or by addition of fumarate. Results indicate that formate inhibited succinate oxidation to fumarate, an intermediate step in the biochemical pathway of propionate oxidation.