Metagenomic insights into nitrite accumulation in sulfur-based denitrification systems utilizing different electron donors: functional microbial communities and metabolic mechanisms

Abstract

Sulfur-based autotrophic denitrification (SADN) offers new pathway for nitrite supply. However, sequential transformation of nitrogen and sulfur forms, and the functional microorganisms driving nitrite accumulation in SADN with different reduced inorganic sulfur compounds (RISCs), remain unclear. Desirable nitrite accumulation was achieved using elemental sulfur (S⁰-group), sulfide (S^2--group) and thiosulfate (S₂O₃^2--group) as electron donors. Under equivalent electron supply conditions, S₂O₃^2--group exhibited a superior nitrate conversion rate (NCR) of 0.285 kg N/(m³·d) compared to S^2--group (0.103 kg N/(m³·d)). Lower NCR in S^2--group was attributed to sulfide strongly inhibiting energy metabolism process of TCA cycle, resulting in reduced reaction rates. Moreover, the NCR of S⁰-group (0.035 kg N/(m³·d)) was poor due to the chemical inertness of S⁰. Specific microbial communities were selectively enriched in phylum level, with Proteobacteria increasing to an astonished 96.27-98.49%. Comprehensive analyses of functional genus, genes, and metabolic pathways revealed significant variability in the active functional genus, with even the same genus showed significant metabolic differences in response to different RISCs. In S⁰-group, Thiomonas (10.0%) and Acidithiobacillus (5.1%) were the primary contributor to nitrite accumulation. Thiobacillus was the most abundant sulfur-oxidizing bacteria in S^2--group (43.84%) and S₂O₃^2--group (18.92%). In S^2--group, it contributed to nitrite accumulation, while in S₂O₃^2--group, it acted as a complete denitrifier (NO₃^--N→N₂). Notably, heterotrophic denitrifying bacteria, Comamonas (12.52%), were crucial for nitrite accumulation in S₂O₃^2--group, predominating NarG while lacking NirK/S. Overall, this work advances our understanding of SADN systems with different RISCs, offering insights for optimizing nitrogen and sulfur removal.