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

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 degrees 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 DeltaH which were embedded in precipitates of FeS, achieved by addition of FeCl(2) to the media. The propionate-oxidizing bacteria formed spores which resisted pasteurization for 30 min at 90 degrees C or 10 min at 100 degrees C. Highly purified cultures were obtained with either M. thermoautotrophicum DeltaH or Methanobacterium thermoformicicum Z245 as the syntrophic partner organism. The optimum temperature for the two cultures was 55 degrees C. Maximum specific growth rates of cultures with M. thermoautotrophicum DeltaH were somewhat lower than those of cultures with M. thermoformicicum Z245 (0.15 and 0.19 day, respectively). Growth rates were even higher (0.32 day) when aceticlastic methanogens were present as well. M. thermoautotrophicum DeltaH 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.