The link between the microbial ecology, gene expression, and biokinetics of denitrifying polyphosphate-accumulating systems under different electron acceptor combinations.

The emission of the greenhouse gas nitrous oxide (NO) can occur during biological nutrient removal. Denitrifying enhanced biological phosphorus removal (d-EBPR) systems are an efficient means of removing phosphate and nitrogen, performed by denitrifying polyphosphate-accumulating organisms (d-PAOs). The aim of this work was to study the effect of various combinations of electron acceptors, nitrate (NO), nitrite (NO), and NO, on the denitrification pathway of a d-EBPR system. Batch tests were performed with different electron acceptor combinations, to explore the denitrification pathway. Reverse transcriptase-qPCR (RT-qPCR) and high-throughput sequencing, combined with chemical analysis, were used to study gene expression, microbial diversity, and denitrification kinetics. The potential for NO production was greater than the potential for its reduction in most tests. A strong correlation was observed between the NO reduction rate and the relative gene expression of nitrous oxide reductase per nitrite reductase (nosZ/(nirS + nirK)), suggesting that the expression of denitrifying marker genes is a strong predictor of the NO reduction rate. The d-EBPR community maintained a core population with low variations throughout the study. Furthermore, phylogenetic analyses of the studied marker genes revealed that the organisms actively involved in denitrification were closely related to Thauera sp., Candidatus Accumulibacter phosphatis, and Candidatus Competibacter denitrificans. Moreover, Competibacter-related OTUs seem to be important contributors to the NO reduction capacity of the system, likely scavenging the NO produced by other organisms. Overall, this study contributes to a better understanding of the microbial biochemistry and the genetics involving biological denitrification removal, important to minimize NO emissions in wastewater treatment plants.