CO2 membrane aeration enhances urine valorization by coordinating carbon-nitrogen metabolic coupling in Chlorella vulgaris.

Source-separated urine is a promising resource for nutrient recovery and microalgal biomass production, yet efficient valorization is frequently constrained by insufficient inorganic carbon and a limited mechanistic understanding of carbon-nitrogen (C-N) metabolic coordination. Here, a bubbleless CO2 membrane aeration strategy was employed to regulate inorganic carbon supply during Chlorella vulgaris cultivation in fresh and hydrolyzed urine, and integrated high-throughput qPCR, transcriptomics, and metabolomics were used to elucidate the underlying metabolic responses. Controlled CO2 delivery increased biomass productivity by 25% while enhancing total nitrogen and phosphorus removal by 28% and 112%, respectively. Multi-omics analyses revealed that membrane-mediated CO2 supply reprogrammed intracellular metabolism by upregulating genes associated with carbon fixation (acsA and acsE) and nitrogen assimilation (ureC and gdhA). Fresh urine supplied with CO2 exhibited strong metabolic coupling, indicating that balanced carbon availability and gradual nitrogen release synergistically promoted photosynthetic carbon fixation and nutrient assimilation. Our findings demonstrate that bubbleless CO2 membrane aeration enhances urine valorization not merely through improved carbon availability, but by strengthening intracellular C-N metabolic coordination. This work provides mechanistic insight into carbon-regulated nutrient recovery and establishes a molecular basis for developing microalgae-based wastewater treatment systems that simultaneously achieve carbon utilization and resource recovery.