Linking Genome-Centric Metagenomics to Kinetic Analysis Reveals the Regulation Mechanism of Hydroxylamine in Nitrite Accumulation of Biological Denitrification

Given hydroxylamine accumulation in various nitrification systems and its potential mechanism in regulating the subsequent denitrification process were unraveled in this study. Hydroxylamine (>0.5 mgN/L) immediately induced nitrite accumulation of activated sludge by inhibiting the activities of nitrite reductases and their electron transport modules (Complex III and cytochrome c). Moreover, long-term exposure to 0.5–2.5 mgN/L hydroxylamine accelerated the functional transformation from denitrification to denitratation under low C/N conditions. However, genome-centric metagenomics indicated that a genotypic complete rather than truncated denitrifier Thauera aminoaromatica TJ127 was enriched and mainly responsible for acetate storage and nitrate reduction of the denitratation community. Interestingly, its enrichment resulted in nitrite production and reduction sequentially but reduced nitrate only to nitrite under carbon-limited conditions (C/N ≤ 3.0). Thus, it showed higher tolerance to hydroxylamine than the concurrent phenotype denitrifiers in activated sludge. Moreover, due to its higher anoxic storage capability in the feast phase, this enrichment became highly specialized by decreasing the feast/famine ratio, and thus a satisfactory denitratation performance was still maintained without hydroxylamine. These results suggested that the transient release of hydroxylamine from nitrification may interfere with subsequent denitrification metabolism, but its continuous accumulation is beneficial for achieving denitratation, which could steadily provide nitrite for mainstream anammox.