Valorizing Agro-Industrial By-Products for Sustainable Cultivation of Chlorella sorokiniana: Enhancing Biomass, Lipid Accumulation, Metabolites, and Antimicrobial Potential

<b>Background/Objectives:</b> Mixotrophic cultivation of microalgae using agro-industrial by-products as supplements offers a sustainable strategy to enhance biomass production and bioactive compound synthesis. This study aimed to evaluate the effects of different agro-industrial by-products-orange peel extract, <i>Cladophora glomerata</i> macroalgal hydrolysate, and solid-state fungal fermentation hydrolysate-on the growth and bioactivity of <i>Chlorella sorokiniana</i>. <b>Methods:</b> Microalgae were cultivated under mixotrophic conditions with different agro-industrial by-products as organic carbon sources. Biomass accumulation was monitored through dry weight measurements. Lipid extraction was carried out using dimethyl carbonate. The antimicrobial activity of the extracted compounds was assessed against <i>Escherichia coli</i>, <i>Bacillus megaterium</i>, and <i>Bacillus subtilis</i> by determining the minimal inhibitconcentrations. <b>Results:</b> Orange peel extract supplementation resulted in the highest biomass production. It increased dry weight by 13.86-fold compared to autotrophic conditions. <i>Cladophora glomerata</i> macroalgal hydrolysate followed with a 5.79-fold increase, and solid-state fungal fermentation hydrolysate showed a 4.14-fold increase. The lipophilic fraction extracted from microalgal biomass showed high yields. Orange peel extract supplementation achieved the highest extraction yield (274.36 mg/g DW). Antimicrobial activity varied based on the supplement used: biomass cultivated with orange peel extract exhibited superior activity against <i>E. coli</i>, whereas <i>Cladophora glomerata</i> macroalgal hydrolysate biomass demonstrated potent activity against <i>B. subtilis</i> (MIC: 5.67 g/mL). <b>Conclusions</b>: These findings underscore the potential of agro-industrial by-products for enhancing microalgal biomass and metabolite production. The observed antimicrobial properties highlight the application of microalgal-derived compounds in sustainable bioprocesses, supporting their use in pharmaceutical and biotechnological applications.