Experimental evaluation of the metabolic reversibility of ANME-2d between anaerobic methane oxidation and methanogenesis

The reverse methanogenesis hypothesis as the metabolic pathway of AOM has recently been supported in the novel ANME lineage ANME-2d in denitrifying anaerobic methane oxidation (DAMO). However, no previous studies have experimentally evaluated the reversal of methane oxidation and methane production in this archaea. In the present study, the metabolic reversibility of ANME-2d from AOM to methanogenesis was evaluated using H-2/CO2 and acetate as substrates. The results showed that the system produced methane from H-2/CO2 but not from acetate. However, the clone library and real-time PCR analysis of the culture showed that both the percentage and quantity of ANME-2d decreased significantly under this condition, while methanogen abundance increased. Further high-throughput sequencing results showed that the archaea community did not change at the fourth day after H-2/CO2 was supplied, but changed profoundly after methanogenesis took place for 3 days. The percentage of DAMO archaea in the total archaea decreased obviously, while more methanogens grew up during this period. Comparatively, the bacteria community changed profoundly at the fourth day. These results indicated that ANME-2d might not reverse its metabolism to produce methane from H-2/CO2 or acetate. After archaea were returned to DAMO conditions, DAMO activity decreased and the amount of ANME-2d continued to fall, implying that the lineage had suffered from severe injury and required a long recovery time.