Tailoring mixed microbial consortia for sustainable biodiesel: An attempt towards synergistic biomass production and cost-effective harvesting in pilot scale open cultivation

Biodiesel has emerged as a viable alternative to traditional fossil fuels due to the growing need for sustainable energy sources. Microbial mixed-culture system was developed to avoid sterile and extremely harsh conditions practised in open algal cultivation. A series of culture conditions, including the suitable supplementation, inoculum and C/N ratios, were optimised to establish a robust and stable consortia. Optimal conditions yielded a maximum of 3.17 ± 0.27 g/L and 2.98 ± 0.16 g/L (a 2.36- and 2.32-fold increase) in biomass with a lipid content of 26 % and 22 % (a 2.89- and 2.56-fold increase in lipid) using Chlorella minutissima and C. vulgaris mediated mixed cultures, respectively, in an open cultivation than in axenic cultures. The photosynthetic microalgae form a mutualistic relationship with heterotrophic Rhodococcus sp. and Aspergillus sp. through O₂/CO₂ exchange. Supportive growth of microbes in consortia was visualised through microscopic observations. The constructed consortium demonstrated a substantial increase in biomass; its efficacy under open-cultivation was systematically evaluated in a pilot-scale open pond of 20-L. The consortia enabled a significant reduction of 75–83 % in harvesting costs, 3.62- to 6.23-fold reduction compared to axenic cultures. This work presents a practical approach for large-scale, sustainable microalgal cultivation that leverages cost-efficient nutrient sources and enhanced process economics. Notably, the fatty acid content analysed through gas chromatography resulted in considerable alteration in methyl ester produced through axenic and mixed culture systems in both closed and open conditions, with the majority of composition being C₁₆-palmitic, C₁₈-stearic and C_18:1-oleic acids, validating its suitability for biodiesel application, matching the standard requirements.