KEIO knockout collection reveals metabolomic crosstalk in Chlorella spp.-Escherichia coli co-cultures.

The interdependence between microalgae and bacteria has sparked scientific interest over years, primarily driven by the practical applications of microalgal-bacteria consortia in wastewater treatment and algal biofuel production. Although adequate studies have focused on the broad interactions and general behavior between the two entities, there remains a scarcity of study on the metabolic role of symbiotic bacteria in promoting microalgal growth. Here, we use the KEIO Knockout Collection, an Escherichia coli gene knockout mutant library, to systematically screen for genes involved in the interdependence of Chlorella sorokiniana and E. coli. By co-cultivating C. sorokiniana and E. coli knockout mutants in 96-well microplates (200 μL medium per well) under white light at 25°C, 31 potential algal growth-promoting and 56 growth-inhibiting genes out of 3985 genes were identified that enhanced (≥1.25-fold) and diminished (≤0.8-fold) the production of algal chlorophyll-a content, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping of these growth-regulating genes suggested a metabolic symbiosis involving bacteria-derived cobalamin (cobU, cobC), biotin (bioB, bioF, bioC, bioD, fabF, fabH), riboflavin (fbp, guaB, gnd, guaA, zwf, purA), and 2,3-butanediol (fumB, adhE, mdh, frdB, pta, sdhC). The effects of these metabolites were further validated by supplementing the agents into the axenic algal cultures; Dose-dependent trends were observed for each metabolite, with a maximum four-fold increase in algal biomass productivity over the control. The specific growth rate of algae was increased by ≥1.27-fold and doubling time was shortened by ≥22.5%. The present results, obtained through genome-wide analyses of interdependence between microalgae and bacteria, reveals multiple interactions between organisms via metabolites.