Echoes of resilience: functional prioritization of stress-response pathways through relative metabolic upregulation in activated sludge

Workflow from activated sludge sampling at an industrial wastewater treatment plant through RNA extraction, cDNA library preparation, sequencing, and generation of metatranscriptomic data. Global warming and pollution have intensified environmental stress, yet activated sludge bacteriota continue to efficiently remove pollutants. Unlike prior laboratory studies focusing on controlled stressors, this research investigates a full-scale industrial wastewater treatment plant operating under persistent and diverse stresses. The facultative reactor in the industrial wastewater treatment plant studied herein operates under fluctuating oxygen conditions, which differ from those in conventional activated sludge reactors, providing a unique perspective on microbial adaptation. The industrial influent reveals microbial resilience under diverse stresses, while metatranscriptomics captures real-time functional responses. Longitudinal metatranscriptomic data, spanning two years and four months and comprising 32 samples, reveal stress response transcripts involved in various metabolic pathways. The top stress-related transcripts include DnaK, AhpC, DGC, CspA, and soxA, which play key roles in stress response and cellular protection. By combining metatranscriptomic profiling with random forest feature importance analysis, we identified sulfate as the environmental factor most strongly correlated with transcriptional reprogramming and characterized patterns of relative metabolic upregulation that appear to support homeostasis under fluctuating conditions. This integrative approach offers a framework for exploring the factors linked to microbial functional resilience in engineered ecosystems.