Metagenomic and metatranscriptomic insights into the complex nitrogen metabolic pathways in a single-stage bioreactor coupling partial denitrification with anammox

Combined partial-denitrification and anammox process has received growing interest for treating wastewaters with high nitrate concentrations, but the underlying molecular mechanisms are currently poorly understood due to the complexity in bacterial community composition and functionality of the coupled system. This study applied metagenomic and metatranscriptomic approaches to gain comprehensive and deep insights into the nitrogen metabolic pathways in a lab-scale partial-denitrification/anammox bioreactor showing stable nitrogen removal. Stoichiometric analysis showed that most of nitrogen was removed by the coupled system (similar to 92.1%), but dissimilatory denitrification also contributed to nitrogen gas production. The latter was further evidenced by nosZ gene transcripts in the metatranscriptome. Metagenomic and metatranscriptomic analyses identified Candidatus Jettenia caeni as the most abundant and active anammox bacteria with relative abundance and mRNA expression of over 5% and 27%, respectively, while Thauera appeared as the major denitrifying bacteria in the system (relative abundance and mRNA expression exceeded 8% and 10%, respectively). mRNA levels of hdh/hzo and hzsA/B/C genes in Candidatus Jettenia caeni and Candidatus Brocadia sinica were much higher than those of other functional genes, confirming the major roles of the anammox bacteria in nitrogen metabolism. In addition, nrfA/B/C/D genes were mainly expressed in anammox bacteria, implying their involvement in that dissimilatory nitrite reduction to ammonia (especially for species Candidatus Brocadia sinica). Nitrate reduction process in Thauera was mainly achieved by the activation of the napA/B/C genes instead of narG/H genes. This study extablished the nitrogen metabolic network based on the comprehensive analyses of the abundance and mRNA expression of functional genes, which might improve our understanding of the nitrogen metabolic mechanisms of the combined partial-denitrification and anammox process.