Heterotrophic sulfide-oxidizing nitrate-reducing bacteria enables the high performance of integrated autotrophic-heterotrophic denitrification (IAHD) process under high sulfide loading.

Micro-aerobic enhancement technology has been developed as an effective tool to enhance simultaneous removal of sulfide, nitrate and organic carbon during the integrated autotrophic-heterotrophic denitrification (IAHD) process under high loading; however, its mechanism of enhancement for functional bacteria remains ambiguous. In this study, we discovered that heterotrophic sulfide-oxidizing nitrate-reducing bacteria (h-soNRB) are responsible for enhancing IAHD performance under micro-aerobic conditions with high sulfide loading. In a continuous IAHD bioreactor, aeration rate of 2.6 mL min-1·L-1 promoted 2 to 4 times higher removal efficiencies of sulfide, nitrate and acetate with an influent sulfide concentration of 18.75 mmol/L. Metagenomic analysis revealed that trace oxygen stimulated the abundance of genes responsible for sulfide oxidation (sqr, glpE, pdo, sox and cysK), which were upregulated by 15.2%-129.9%, and the genes encoding nitrate reductase were up-regulated by 67.4%. The increased acetate removal efficiency was attributed to upregulation of ack, pta and TCA cycle related genes. The h-NRB Pseudomonas, Azoarcus, Thauera and Halomonas were detected and regarded as h-soNRB in our bioreactor. According to Illumina MiSeq sequencing, these genera were absolutely dominant in the micro-aerobic microbial community at relative abundances ranging from 82.72% to 90.84%. The sulfide, nitrate and acetate removal rates of Pseudomonas C27, a typical h-soNRB, were at least 10 times higher under micro-aerobic conditions than under anaerobic conditions. Besides, the sulfur, nitrogen and carbon metabolic network was constructed based on the Pseudomonas C27 genome. The pdo and cysK genes found in this strain may be the most advantageous for autotrophic sulfide oxidizing nitrate reducing bacteria (a-soNRB), which are closely related to the high-efficiency sulfide, nitrate and acetate removal performance under high sulfide concentrations and a limited oxygen supply. In addition, after micro-aerobic cultivation, the anaerobic sulfide loading tolerance of the IAHD bioreactor increased from 18.75 to 37.5 mmol/L with sulfide, nitrate and acetate removal efficiencies increasing 1.5 to 3 times, which suggests that intermittent micro-aeration might be a more economical and efficient regime for high-sulfide IAHD regulation.