IAA as a microbial activator for enhanced low-temperature nitrogen removal: mechanistic insights and pilot-scale...

Cold-induced nitrification failure remains a bottleneck for municipal wastewater treatment plants. Activation of microbial quorum sensing (QS) has emerged as a central regulatory mechanism for restoring nitrification under cold stress. However, conventional acyl-homoserine lactone signals are hindered by high synthesis costs and enzymatic instability, limiting their engineering application. Here, we identify the phytohormone indole-3-acetic acid (IAA) as a novel microbial activator to alleviate cold stress. Functional screening of endogenous phytohormones showed that 2 μM IAA increased specific nitrification activity by 65.3% at 5 °C. Mechanistically, IAA enhanced microbial resistance to cold stress by increasing membrane fluidity and promoting ROS scavenging, accompanied by a 47.2% increase in intracellular ATP level. Meanwhile, IAA activated quorum-sensing-mediated regulatory networks in Nitrosomonas and Nitrospira, upregulating ammonium- and nitrite-transporter expression. Consequently, the abundances of key nitrogen-metabolism genes, including amoABC, hao, and nxrB, increased by 4.0-, 2.2-, and 1.9-fold, respectively, ultimately restoring activated sludge nitrification under low-temperature conditions. The strategy was validated in a 14.4 m³/d pilot-scale anaerobic/anoxic/oxic (A/A/O) system treating real municipal wastewater, which maintained stable nitrogen removal with an average effluent NH₄⁺-N of 1.1 mg/L under winter conditions. Life-cycle assessment showed a 31% reduction in total carbon footprint without burden shifting among environmental impact categories. This study provides a low-carbon and efficient strategy for wastewater treatment under low-temperature conditions.