Impacts of riboflavin-overproducing engineered Escherichia coli towards Chlorella sorokiniana growth in co-cultivation approach

Riboflavin (vitamin B2), a cofactor for essential metabolic reactions and a facilitator of the electron transport chain, holds promise to enhance algal growth. In this study, the Escherichia coli BL21(DE3) strain was engineered to overproduce riboflavin, and its impact on algal growth were tested in a bacterial-algal co-cultivation system. To this end, the E. coli zwf (encodes glucose-6-phosphate-1-dehydrogenase) and fbp (encodes fructose-1,6-biphosphate) genes were co-expressed in BL21(DE3)/pETDuet-T7-zwf-T7-fbp, resulting in an increased riboflavin level to 9.$3\pm$0.2 mg L⁻¹ in 100 % bacterial exudate. Subsequently, this recombinant strain was co-cultivated with Chlorella sorokiniana at a bacteria: algae ratio of 5:1 (dry wt/wt) in BG11 medium supplemented with E. coli exudate (an equal-volume mixture of BG11 and exudate). The algal specific growth rate (0.9$\pm$0.02 d⁻¹), cell size (5.1$\pm$0.7 $\mu$m), pigment productivity (6.4$\pm$0.1 mg L⁻¹ d⁻¹), extracellular polysaccharide productivity (22.$4\pm$1.5 mg L⁻¹ d⁻¹), and lipid productivity (26.$3\pm$1.5 mg L⁻¹ d⁻¹) all showed at least a two-fold increase compared to the wild-type control. However, due to the limited bacterial growth, riboflavin's effect was most significant during the first two days, followed by cell adaptation phase (D2-D4), slower cell division phase (D4-D10), and metabolite accumulation phase (D6-D10). Our findings have highlighted a feasible strategy for enhanced microalgal growth beyond the conventional co-cultivation method.