RNA stable isotope probing and high‐throughput sequencing to identify active microbial community members in a methane‐driven denitrifying biofilm

Abstract

Aims Aerobic methane oxidation coupled to denitrification (AME-D) is a promising process for removing nitrate from groundwater and yet its microbial mechanism and ecological implications are not fully understood. This study used RNA stable isotope probing (RNA-SIP) and high-throughput sequencing to identify the micro-organisms that are actively involved in aerobic methane oxidation within a denitrifying biofilm. Methods and Results Two RNA-SIP experiments were conducted to investigate labelling of RNA and methane monooxygenase (pmoA) transcripts when exposed to 13C-labelled methane over a 96-hour time period and to determine active bacteria involved in methane oxidation in a denitrifying biofilm. A third experiment was performed to ascertain the extent of 13C labelling of RNA using isotope ratio mass spectrometry (IRMS). All experiments used biofilm from an established packed bed reactor. IRMS confirmed 13C enrichment of the RNA. The RNA-SIP experiments confirmed selective enrichment by the shift of pmoA transcripts into heavier fractions over time. Finally, high-throughput sequencing identified the active micro-organisms enriched with 13C. Conclusions Methanotrophs (Methylovulum spp. and Methylocystis spp.), methylotrophs (Methylotenera spp.) and denitrifiers (Hyphomicrobium spp.) were actively involved in AME-D. Significance and Impact of the Study This is the first study to use RNA-SIP and high-throughput sequencing to determine the bacteria active within an AME-D community.