Metagenomic insights into salinity effect on diversity and abundance of denitrifying bacteria and genes in an expanded granular sludge bed reactor treating high-nitrate wastewater

•Complete nitrate removal and nitrite reduction recovery under decreasing NaCl.•High-throughput technique showed key role of Halomonas and Marinobacter in reactor.•Nitrite reduction under decreasing NaCl mainly by nirS gene carried by Halomonas.•Marinobacter and Halomonas for nitrate removal in high and low NaCl, respectively.

Increasing salinity can affect biological denitrification in bioreactors, but information is limited regarding the responses of microbial community and denitrification efficiency to the decrease of salt stress. This study characterized the alterations of microbial community structure, functional genes abundance and nitrogen metabolic pathways in an expanded granular sludge bed reactor treating high-nitrate wastewater under decreasing sodium chloride (NaCl) stress by using high-throughput sequencing and metagenomic analysis. Results showed that the reactor continuously had high nitrate removal (>99%) with the decrease of influent NaCl concentration from 11% to 0, and nitrite reduction was recovered after the stress disappeared. Pyrosequencing revealed that two halophilic genera, Halomonas and Marinobacter, contributed greatly to the denitrification. Functional annotation of the metagenomic datasets revealed that nitrate was mainly reduced by Marinobacter under 11% NaCl stress, and by Halomonas under lower NaCl stress. Various denitrifying genes had increased abundance under no NaCl stress, which is confirmed by quantitative real time PCR. Among the denitrifying genes, nitrate reductases genes had comparatively higher abundance in the bioreactor, and nitrite reductase gene nirS played a more important role in nitrite reduction than nirK. The decrease of NaCl stress enhanced the biodiversity of the denitrifying bacteria carrying the functional genes. The results may help to understand the molecular mechanisms of biological denitrification under saline stress and optimize the bioreactors treating the industrial wastewater containing varying levels of salt.