Robust and high-efficient nitrogen removal from real sewage and waste activated sludge (WAS) reduction in zero-external carbon PN/A combined with in-situ fermentation-denitrification process under decreased temperatures.

Despite the advantages of the combined anammox and fermentation-driven denitrification process in nitrogen removal and energy consumption, stable performance at decreased temperatures remains a challenge. In this study, a robust and high-efficient nitrogen removal efficiency (95.0-93.1 ∼ 86.8-93.4%) with desirable effluent quality (3.0-4.1 ∼ 7.9-4.9 mg/L) under long-term decreased temperatures (30 °C→25 °C→20 °C) was achieved in a zero-external carbon Partial Nitritation/Anammox combined with in-situ sludge Fermentation-Denitrification process treating sewage. Excellent sludge reduction averaged at 14.9% assuming no microbial growth. Increased hzsB mRNA (2.2-fold) and reduced E (80.9 kJ/mol) proved resilient anammox to lower temperature. RT-qPCR tests revealed increased NarG/NirK (5.1) and NarG/NirS (4.9) mRNA at 20 °C, suggesting higher NO→NO over NO→N pathway. Metagenomics unraveled dominant anammox bacteria (Candidatus_Brocadia, 2.27%), increased denitritation bacteria containing more NarG (Hyphomicrobium, 0.8%), fatty acid biosynthesis and CAZymes genes. Enhanced denitritation with recovered organics from sludge reserved nitrite for anammox and facilitated higher anammox contribution to N removal at 20 °C (42.4%) than 30 °C (39.5%). This study proposed an innovative low-temperature strategy for in-situ sludge fermentation, and demonstrated stability of advanced municipal wastewater treatment and sludge disposal through energy savings and carbon recovery under decreased temperatures.