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

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.