Comprehensive analyses of functional bacteria and genes in a denitrifying EGSB reactor under Cd(II) stress

A bench-scale expanded granular sludge bioreactor (EGSB) was continuously operated to treat synthesized high-nitrate industrial wastewater with increasing bivalent cadmium (Cd(II)) stress. The bioreactor showed nearly complete nitrate removal regardless of Cd(II) loadings, while nitrite accumulated in the effluent when influent Cd(II) loading was over 64 mg/L. Mi-seq sequencing of 16S rRNA gene amplicons elucidated that denitrifiers had decreasing abundances while biodiversity showed increasing trend as the Cd(II) loading increased. In the bioreactor, genera Halomonas, Thauera, Pseudomonas, and Zoogloea played major roles in the denitrification under lower Cd(II) loadings (< 32 mg/L), while Halomonas sp. KM-1 and Halomonas sp. BC04 acted as the crucial Cd-resistant denitrifiers under 128 mg/L Cd(II) loading. Metagenomic analyses and real-time quantitative PCR consistently indicated that napA encoding nitrate reductase was the predominant denitrifying gene, that could be mainly functioning on the efficient nitrate removal. Statistical analyses revealed the significantly positive correlation between Halomonas and nirS gene, both of which were functionally responsible for nitrite reduction. The obtained results may be practically useful for regulation and optimization of the biological processes to treat industrial wastewater containing high levels of nitrate and Cd(II).