Free Ammonia Stress Rewires Microbial Signaling Networks and Constrains Metabolic Cooperation in Oxygenic Photogranules.

Free ammonia (FA) is a prevalent chemical inhibitor in wastewater ecosystems, yet its ecological impacts on microbial communication and cooperation remain poorly understood. Here, we demonstrated that FA stress restructured the community function of oxygenic photogranules (OPGs) by rewiring signaling-associated regulatory networks and redirecting cellular energy allocation. Temporal profiling of extracellular signaling molecules and intracellular regulatory molecules, integrated with metagenomic and metatranscriptomic analyses, revealed a concentration-dependent reconfiguration of microbial signaling. Under low FA exposure (≤ 1.0 mg/L), diffusible signal factor (DSF)- and indole-3-acetic acid (IAA)-associated pathways were more active, coinciding with photogranules consolidation and efficient nitrogen removal. At moderate FA stress (5.0 mg/L), the regulatory landscape shifted toward acyl-homoserine lactone (AHL)-associated signaling and bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP)-mediated intracellular regulation, consistent with enhanced aggregation and stress adaptation. In contrast, severe FA stress (25.0 mg/L) broadly attenuated signaling-associated pathways, weakened metabolite cross-feeding networks, impaired energy generation, and increased maintenance-related energetic demands. Under these constraints, microbial populations appeared to shift from cooperative metabolism toward self-maintenance-oriented carbon metabolism, evidenced by activation of the carbon-efficient glyoxylate shunt. Collectively, these findings suggest that FA-induced signaling disruption constrains microbial cooperation through cellular energy limitation, highlighting energy allocation as a key determinant of microbial social stability in phototrophic wastewater microbiomes.