Abstract
Fossil fuel reliance significantly contributes to climate change. Sustainable biofuels present a promising alternative. Among potential biofuels, microalgae stand out due to their high lipid content and environmental benefits. This study engineers a unique solution to further increase the lipid content in Chlorella vulgaris, an industrially relevant, lipid-rich microalgae strain. Although lipid yield is greater than terrestrial biofuel feedstocks, such as corn, soybean, and palm oil, algal biofuel remains a negative net energy investment. Several methods for improving algal lipid yields have been discovered including microalgal-bacterial co-culture and nitrogen deprivation. Nitrogen depletion forces microalgae into a stressed state with rapid lipid accumulation. Certain bacteria have symbiotic relationships with algae that can increase lipid content or biomass. Co-culture of algae with a nitrogen-fixing bacterium can supply low levels of bioavailable nitrogen supporting photosynthetic efficiency while keeping the algae in a stressed state. These effects can be stacked to enhance lipid production. The combination of these two approaches, however, had not previously been tested in an industrially relevant strain. This study combined lipid-rich C. vulgaris with nitrogen-fixing bacterium Azotobacter chroococcum in co-culture under nitrogen-deplete conditions. This novel pairing resulted in a significant 8.3-fold increase in lipid content per cell compared to nitrogen-replete monoculture algae. Additionally, a nearly 2-fold increase in algal lipids compared to a nitrogen-deprived control and an Escherichia coli co-culture nitrogen-deprived control was observed. These findings demonstrate that pairing nitrogen fixation with nitrogen deprivation can push algal lipid yields beyond the limits of stress induction alone, representing a meaningful step toward economically viable microalgal biodiesel.