Who Is the Rock Miner and Who Is the Hunter? The Use of Heavy-Oxygen Labeled Phosphate (P18O4) to Differentiate between C and P Fluxes in a Benzene-Degrading Consortium.

Phosphorus availability and cycling in microbial communities is a key determinant of bacterial activity. However, identifying organisms critical to P cycling in complex biodegrading consortia has proven elusive. Here we assess a new DNA stable isotope probing (SIP) technique using heavy oxygen-labeled phosphate (P18O4) and its effectiveness in pure cultures and a nitrate-reducing benzene-degrading consortium. First, we successfully labeled pure cultures of Gram-positive Micrococcus luteus and Gram-negative Bradyrhizobium elkanii and separated isotopically light and heavy DNA in pure cultures using centrifugal analyses. Second, using high-throughput amplicon sequencing of 16S rRNA genes to characterize active bacterial taxa (13C-labeled), we found taxa like Betaproteobacteria were key in denitrifying benzene degradation and that other degrading (nonhydrocarbon) inactive taxa (P18O4-labeled) like Staphylococcus and Corynebacterium may promote degradation through production of secondary metabolites (i.e., "helper" or "rock miner" bacteria). Overall, we successfully separated active and inactive taxa in contaminated soils, demonstrating the utility of P18O4-DNA SIP for identifying actively growing bacterial taxa. We also identified potential "miner" bacteria that choreograph hydrocarbon degradation by other microbes (i.e., the "hunters") without directly degrading contaminants themselves. Thus, while several taxa degrade benzene under denitrifying conditions, microbial benzene degradation may be enhanced by both direct degraders and miner bacteria.