In this work, we studied a novel algae cultivation strategy, mixotrophic microalgae biofilm, to improve the productivity and cost-efficiency of algal biofuel production. In contrast to previous methods, this improved approach can achieve high productivity at low cost by harnessing the benefits of mixotrophic growth’s high efficiency, i.e., capable of subsisting on inorganic and organic carbons thus unaffected by limited light, and microalgae biofilm’s low harvesting cost. Our results, as one of the first studies of this type, proved that microalgae biofilms under mixotrophic condition exhibited significantly higher productivity and quality of biofuel feedstock: 2–3 times higher of biomass yield, 2–10 times higher of lipid accumulation, and 40–60% lower of ash content when compared to microalgae biofilms under autotrophic condition. In addition, we investigated the impact of cell-surface properties (hydrophobicity and roughness) on the growth activities of microalgae biofilms and found that the productivity of mixotrophic biofilms was significantly correlated with the surface hydrophobicity. Finally, our work demonstrated the applicability of integrating this novel cultivation method with wastewater for maximum efficiency. This study opens a new possibility to solve the long-lasting challenges of algal biofuel feedstock production, i.e., low productivity and high cost of algal cultivation.

This work uses response surface methodology (RSM) to study the co-cultivation of symbiotic indigenous wastewater microalgae and bacteria under different conditions (inoculum ratio of bacteria to microalgae, CO₂, light intensity, and harvest time) for optimal bioenergy feedstock production. The findings of this study demonstrate that the symbiotic microalgae-bacteria culture not only increases total microalgal biomass and lipid productivity, but also enlarges microalgal cell size and stimulates lipid accumulation. Meanwhile, inoculum ratio of bacteria to microalgae, light intensity, CO_2, and harvest time significantly affect biomass and lipid productivity. CO₂ concentration and harvest time have significant interactive effect on lipid productivity. The response of microalgal biomass and lipid productivity varies significantly from 2.1 × 10⁵ to 1.9 × 10⁷ cells/mL and 2.8 × 10² to 3.7 × 10¹² Total Fluorescent Units/mL respectively. Conditions for optimum biomass and oil accumulation are 100% of inoculation ratio (bacteria/microalgae), 3.6% of CO₂ (v/v), 205.8 µmol/m²/s of light intensity, and 10.6 days of harvest time. This work provides a systematic methodology with RSM to explore the benefits of symbiotic microalgae-bacteria culture, and to optimize various cultivation parameters within complex wastewater environments for practical applications of integrated wastewater-microalgae systems for cost-efficient bioenergy production.