Enrichment and Detection of Microorganisms Involved in Direct and Indirect Methanogenesis from Methanol in an Anaerobic Thermophilic Bioreactor

To gain insight into the microorganisms involved in direct and indirect methane formation from methanol in a laboratory-scale thermophilic (55°C) methanogenic bioreactor, reactor sludge was disrupted and serial dilutions were incubated in specific growth media containing methanol and possible intermediates of methanol degradation as substrates. With methanol, growth was observed up to a dilution of 10 8 . However, when Methanothermobacter thermoautotrophicus strain Z245 was added for H 2 removal, growth was observed up to a 10 10 -fold dilution. With H 2 /CO 2 and acetate, growth was observed up to dilutions of 10 9 and 10 4 , respectively. Dominant microorganisms in the different dilutions were identified by 16S rRNA-gene diversity and sequence analysis. Furthermore, dilution polymerase chain reaction (PCR) revealed a similar relative abundance of Archaea and Bacteria in all investigated samples, except in enrichment with acetate, which contained 100 times less archaeal DNA than bacterial DNA. The most abundant bacteria in the culture with methanol and strain Z245 were most closely related to Moorella glycerini . Thermodesulfovibrio relatives were found with high sequence similarity in the H 2 /CO 2 enrichment, but also in the original laboratory-scale bioreactor sludge. Methanothermobacter thermoautotrophicus strains were the most abundant hydrogenotrophic archaea in the H 2 /CO 2 enrichment. The dominant methanol-utilizing methanogen, which was present in the 10 8 -dilution, was most closely related to Methanomethylovorans hollandica . Compared to direct methanogenesis, results of this study indicate that syntrophic, interspecies hydrogen transfer-dependent methanol conversion is equally important in the thermophilic bioreactor, confirming previous findings with labeled substrates and specific inhibitors.