Dose-dependent effects of biochar on low-temperature anammox: reactor performance, community variation, and functional potential.

Low temperature is a major constraint on the practical application of anaerobic ammonium oxidation (anammox). Although biochar has been reported to improve low-temperature anammox, the effect of dosage remains insufficiently understood. In this study, mature anammox sludge was amended with 0, 3, 7, and 9 g/L bamboo-derived biochar and operated under a stepwise temperature decrease from 35 to 15°C, followed by low-temperature operation for 70 d. Reactor performance, extracellular polymeric substances (EPS), microbial community composition, and metagenomic functional potential were analyzed to clarify the dose effect of biochar. Among the tested dosages, 7 g/L biochar achieved the highest nitrogen removal efficiency (48.6%) at 15°C, which was 12.8 percentage points higher than the control value of 35.8%. Biochar-amended reactors also showed higher EPS contents than the control, and the 7 g/L group better maintained the PN/PS ratio under low-temperature stress. Community analysis indicated a higher relative abundance of Candidatus Brocadia in the biochar-amended groups, especially at 7 g/L. Metagenomic analysis further showed higher abundance of genes associated with nitrogen metabolism, carbon metabolism, and EPS-related precursor synthesis in the 7 g/L group. These results suggest that an appropriate biochar dosage can improve low-temperature anammox performance and is associated with EPS stabilization, enrichment of key functional taxa, and enhanced functional potential. This study provides guidance for biochar dosage optimization in low-temperature anammox systems.