Unraveling of Chlorella-associated bacterial load, diversity, and their imputed functions at high- and low-yield conditions through metagenome sequencing

Chlorella-associated bacteria can have a significant influence on facilitating higher Chlorella biomass yield due to their symbiotic relationship. In this study, non-axenic Chlorella was cultivated in an airlift photobioreactor at high and low-yield conditions. The associated bacterial diversity was analyzed using 16S rRNA metagenome sequencing. At high-yield conditions, the bacterial load was observed in the range of 10⁸ -10¹⁰ CFU · mL^-1 , whereas at low-yield conditions, bacteria were more dominant and observed in the range of 10¹⁴ -10¹⁵  CFU · mL^-1 . The majority of the bacterial species associated with Chlorella at high-yield conditions belongs to Proteobacteria and Bacteroidetes. Further, Bacteroidetes levels were decreased at low-yield conditions and were highly diversified with Planctomycetes, Firmicutes, and 18 others. Predicted functional genes indicated that Chlorella-associated bacteria have the enzymes involved in the metabolism and biosynthesis of B-complex vitamins (i.e., vitamin B₁₂ , thiamin, biotin, pyridoxine, and riboflavin). A critical evaluation revealed that vitamin biosynthesis genes were more abundant at low-yield conditions; however, vitamin B₁₂ transport genes (B₁₂ transport ATP-binding protein, B₁₂ substrate-binding transportation, and B₁₂ permease protein) were less abundant, indicating even though vitamins production occurs, but their availability to Chlorella was limited due to the lack of vitamin transport genes. Further, at high yield, Chlorella-associated bacteria enabled higher growth by supplementing the vitamins. In contrast, at low-yield condition-an increased bacterial load, diversity, and limited vitamin transport functional genes affected the Chlorella yield. It can be inferred that Chlorella yield was significantly affected by three factors: associated bacterial load, diversity, and transport functional genes of vitamins.