Dilution rate caused nutrient limitation effects on the biomass composition and proteomic level of continuously cultivated Spirulina in recirculating aquaculture system wastewater

Recirculating aquaculture system (RAS) wastewater from Nile tilapia cultivation can negatively impact aquatic ecosystems . Continuous cultivation provides a steady state for Spirulina growth and nutrient removal. This study investigated the optimal dilution rate of the RAS wastewater to maximize biomass production and nutrient removal in continuous Spirulina cultivation. Moreover, changes in the biomass composition and proteome under nutrient–limiting conditions were investigated. Continuous cultivation was performed at four different dilution rates of 0.20, 0.25, 0.35, and 0.50 d −1 using two different culture mediums, RAS N50 and RAS N80 , which were prepared from raw RAS wastewater containing nitrate concentrations of 50 mg N/L and 80 mg N/L, respectively. The optimal dilution rates for RAS N50 and RAS N80 were 0.35 d −1 and 0.20 d −1 , respectively. Under these conditions, Spirulina could maintain high removal efficiencies of over 80 % for nitrate and over 99 % for phosphate while achieving effective biomass productivities ranging from 0.18 to 0.22 g DW/L/d. However, the biomass exhibited decreased protein content and increased carbohydrate content when grown in RAS N50 at a dilution rate of 0.20 d −1 , indicating nutrient–limiting conditions, particularly for nitrogen and phosphorus. Under these conditions, the protein content in the biomass was reduced to 42.0 ± 3.3 % due to phycocyanin degradation. Meanwhile, Spirulina enhanced glucose synthesis through carbon fixation and gluconeogenesis, resulting in an increase in carbohydrate content up to 48.4 ± 2.9 %. In contrast, biomass produced under other conditions exhibited protein and carbohydrate contents ranging from 55.8 to 62.9 % and 14.4 to 17.1 %, respectively. • RAS wastewater was used for continuous Spirulina cultivation. • Spirulina cultivation achieved nutrient removal efficiencies of over 80 %. • Optimal dilution rate for RAS wastewater with 80 mg NO 3 -N/L was 0.20 d −1 . • Nutrient limitation occurred at 0.20 d −1 in RAS wastewater with 50 mg NO 3 -N/L. • Carbon fixation and gluconeogenesis increased under nutrient limitation.