Cultivating microbial dark matter in benzene‐degrading methanogenic consortia

Summary The microbes responsible for anaerobic benzene biodegradation remain poorly characterized. In this study, we identified and quantified microbial populations in a series of 16 distinct methanogenic, benzene‐degrading enrichment cultures using a combination of traditional 16 S rRNA clone libraries (four cultures), pyrotag 16 S rRNA amplicon sequencing (11 cultures), metagenome sequencing (1 culture) and quantitative polymerase chain reaction ( qPCR ; 12 cultures). An operational taxonomic unit ( OTU ) from the D eltaproteobacteria designated ORM 2 that is only 84% to 86% similar to S yntrophus or D esulfobacterium spp. was consistently identified in all enrichment cultures, and typically comprised more than half of the bacterial sequences. In addition to ORM 2, a sequence belonging to P arcubacteria (candidate division OD 1) identified from the metagenome data was the only other OTU common to all the cultures surveyed. Culture transfers (1% and 0.1%) were made in the presence and absence of benzene, and the abundance of ORM 2, OD 1 and other OTUs was tracked over 415 days using qPCR . ORM 2 sequence abundance increased only when benzene was present, while the abundance of OD 1 and other OTUs increased even in the absence of benzene. D eltaproteobacterium   ORM 2 is unequivocally the benzene‐metabolizing population. This study also hints at laboratory cultivation conditions for a member of the widely distributed yet uncultivated P arcubacteria ( OD 1).