Metabolic pathways and microbial interactions in simultaneous removal of carbon, nitrogen and sulfur in anaerobic sequencing batch reactors (ASBRs) under high nitrite concentration

Simultaneous removal of carbon, nitrogen and sulfur is a challenging topic in wastewater treatment , with many available pathways. For high‑nitrogen wastewaters, the response of microbial community to nitrite-stress conditions, in autotrophic, heterotrophic and mixotrophic conditions is still an important area of study to develop a robust and synergic treatment. In this study, the denitrification performance and involved microbial community were evaluated regarding presence of nitrite and thiosulfate with a S/N ratio of 0.43, while varying the organic supplementation. This included anaerobic sequencing batch reactors (ASBRs) with no acetate addition (R 1 ) and C/N ratios of 0.33 (R 2 ), 0.67 (R 3 ) e 1.33 (R 4 ). With no acetate supplementation, the ASBRs could adapt to the stress condition after a lag phase with denitrification efficiencies close to 80 % and mean NO 2 − -N consumption rate similar to the ASBRs operated with organics presence. The organic supplementation in excess favored the heterotrophic pathway and reached the higher nitrite‑nitrogen efficiencies of around 99 %. The control of C/Nratios could allow the autotrophic and heterotrophic routes to cooperate improving nitrite‑nitrogen performance resulting in efficiencies close to 100 %. Pseudomonas , Pseudoxanthomonas and Lysobacter were likely the primary representatives of mixotrophic denitrification and may have outcompeted fermentative bacteria and methanogens in the ASBRs, particularly under limited organic substrate conditions. This study highlights an optimized mixotrophic strategy for cost-effective denitritation, enhancing carbon, nitrogen, and sulfur removal in high‑nitrogen wastewater. The findings suggest that a controlled mixotrophic approach offers an economical, efficient alternative to conventional denitrification, supporting the development of robust treatment systems for complex wastewater. • Mixotrophic denitrification could improve nitrogen removal under nitrite stress. • Organic supplementation led to a quick adaptation to nitrite toxicity. • N/C ratios affected the predominance of autotrophic or heterotrophic routes. • Facultative autotrophs were the main denitrifying representatives. • Simultaneous carbon, nitrogen and sulfur removal was obtained.