iTRAQ quantitative proteomic analysis reveals the pathways for methanation of propionate facilitated by magnetite

Methanation of propionate requires syntrophic interaction of propionate-oxidizing bacteria and hydrogenotrophic methanogens, which is referred to as interspecies electron transfer. The present study showed that 10 mg/L conductive magnetite enhanced the methane production rate from propionate by around 44% in batch experiments, and both direct interspecies electron transfer and interspecies H₂ transfer were thermodynamically feasible with the addition of magnetite. The methanation of propionate facilitated by magnetite was also demonstrated in a long-term operated continuous reactor. The methane production rate from acetate by the enriched mixed culture with magnetite was higher than that without magnetite, while similar methane production rates were found from H₂/CO₂ by the enriched mixed culture with and without magnetite. The ability to utilize molecular H₂ indicated interspecies H₂ transfer played a role in the enriched culture with magnetite, and propionate-oxidizing bacteria relating with interspecies H₂ transfer were also detected by metagenomic sequencing. Metagenomic sequencing analysis also showed that Thauera, possibly relating with direct interspecies electron transfer, were enriched with the addition of magnetite. iTRAQ quantitative proteomic analysis, which was used in mixed culture for the first time, showed that magnetite induced the changes of protein expression levels involved in various pathways during the methanation of propionate. The up-regulation of proteins involved in propionate metabolism were found, and they were mainly originated from propionate-oxidizing bacteria which were not reported to be capable of direct interspecies electron transfer until now. Cytochrome c oxidase was also revealed as the possible protein relating with direct interspecies electron transfer considering its up-regulation with the addition of magnetite and origination from Thauera. Most of the up-regulated proteins in methane metabolism were originated from Methanosaeta, while most of the enzymes with down-regulated proteins were originated from Methanosarcina. However, the up-regulated proteins relating with hydrogenotrophic methanogenesis were originated from neither Methanosaeta nor Methanosarcina, indicating they were not involved in direct interspecies electron transfer. The hydrogenotrophic methanogens, e.g. Methanospirillum, Methanosphaerula et al., might be involved in direct interspecies electron transfer. Overall, the present study showed that both direct interspecies electron transfer and interspecies H₂ transfer were present during methanation of propionate facilitated by magnetite.