Quantification of nosZ genes and transcripts in activated sludge microbiomes with novel group-specific qPCR methods validated with metagenomic analyses.

Substantial N2O emission results from activated sludge nitrogen removal processes. N2O-reducing organisms possessing NosZ-type N2O reductases have been recognized to play crucial roles in suppressing emission of N2O produced in anoxic activated sludge via denitrification; however, which of the diverse nosZ-possessing organisms function as the major N2O sink in situ remains largely unknown. Here, nosZ genes and transcripts in wastewater microbiomes were analyzed with the group-specific qPCR assays designed de novo combining culture-based and computational approaches. A sewage sample was enriched in a batch reactor fed continuous stream of N2 containing 20-10,000 ppmv N2O with excess amount (10 mM) of acetate as the source of carbon and electrons, where 14 genera of potential N2O-reducers were identified. All available amino acid sequences of NosZ affiliated to these taxa were grouped into five subgroups (two clade I and three clade II groups), and primers/probe sets exclusively and comprehensively targeting the subgroups were designed and validated with in silico PCR. Four distinct activated sludge samples from three different wastewater treatment plants in Korea were analyzed with the qPCR assays and the results were validated with the shotgun metagenome analysis results. With these group-specific qPCR assays, the nosZ genes and transcripts of six additional activated sludge samples were analyzed and the results of the analyses clearly indicated the dominance of two clade II nosZ subgroups (Flavobacterium-like and Dechloromonas-like) among both nosZ gene and transcript pools.