Shifts in microbial communities and antibiotic resistance genes in activated sludge driven by varying heterotrophic nitrifying aerobic denitrifying bacteria ratios

Abstract

Activated sludge systems experience reduced microbial activity under low-temperature conditions, which adversely affects the biological nitrogen removal process. Heterotrophic nitrifying aerobic denitrifying bacteria (HNADB), with their strong tolerance to low temperatures, present a promising solution through bioaugmentation. This study explored the bio-enhancement of activated sludge systems using HNADB to improve nitrogen removal, and evaluated microbial community structure, functional genes, and antibiotic resistance genes (ARGs) through batch experiments with varying inoculation ratios (from 0 % to 100 %) of HNADB-enriched sludge. Higher inoculation ratios (80 % and 100 %) significantly enhanced total nitrogen (TN) removal by 32.75 % and 43.62 %, respectively, though nitrification efficiency declined. Community diversity increased, while richness decreased under higher inoculation. Abundant taxa (AT) constituted the dominant proportion in all systems and were further enriched in the high-inoculation treatments. High inoculation ratios enriched denitrifying bacteria (DNB), HNADB (Thauera, Hyphomicrobium, Rhodococcus), and their related functional genes, while decreasing the abundance of nitrifying microorganisms and genes. The ARGs diversity increased but abundance decreased over time. DNB and HNADB may serve as hosts for ARGs, while nitrifying microorganisms show higher sensitivity to antibiotics. This study highlighted the potential of HNADB-based inoculation to enhance nitrogen removal performance and community resilience under low-temperature conditions, providing an effective biological enhancement strategy for low-temperature wastewater treatment.