Comparative Shotgun Proteomic Analysis of Wastewater-Cultured Microalgae: Nitrogen Sensing and Carbon Fixation for Growth and Nutrient Removal in <i>Chlamydomonas reinhardtii</i>

Chlamydomonas reinhardtii was batch-cultured for 12 days under continuous illumination to investigate nitrogen uptake and metabolic responses to wastewater processing. Our approach compared two conditions: (1) artificial wastewater containing nitrate and ammonia and (2) nutrient-sufficient control containing nitrate as sole form of nitrogen. Treatments did not differ in final biomass; however, comparison of group proteomes revealed significant differences. Label-free shotgun proteomic analysis identified 2358 proteins, of which 92 were significantly differentially abundant. Wastewater cells showed higher relative abundances of photosynthetic antenna proteins, enzymes related to carbon fixation, and biosynthesis of amino acids and secondary metabolites. Control cells showed higher abundances of enzymes and proteins related to nitrogen metabolism and assimilation, synthesis and utilization of starch, amino acid recycling, evidence of oxidative stress, and little lipid biosynthesis. This study of the eukaryotic microalgal proteome response to nitrogen source, availability, and switching highlights tightly controlled pathways essential to the maintenance of culture health and productivity in concert with light absorption and carbon assimilation. Enriched pathways in artificial wastewater, notably, photosynthetic carbon fixation and biosynthesis of plant hormones, and those in nitrate only control, most notably, nitrogen, amino acid, and starch metabolism, represent potential targets for genetic improvement requiring targeted elucidation.