Glycerol and nitrate utilisation by marine microalgae Nannochloropsis salina and Chlorella sp and associated bacteria during mixotrophic and heterotrophic growth

Mixotrophic growth is an ability of microalgae to concurrently assimilate CO2 and organic carbon. In the present study, the use of glycerol to enhance microalgal biomass and lipid productivities was investigated in relation to nitrate availability. Under nitrate sufficient conditions, the biomass productivities of Nannochloropsis salina (CCMP 1776) and a marine Chlorella sp. were 1.7 and 1.9 times higher in mixotrophic culture than under strictly photoautotrophic conditions, respectively. Both algae required light to assimilate glycerol. No significant algae growth was observed under heterotrophic conditions, despite apparent utilisation of both nitrate and glycerol. Under nitrate deplete conditions both species took up only minimal amounts of glycerol thereby indicating the importance of the combined effects of carbon and nitrogen. Additionally, a carbon mass balance was conducted based on glycerol utilisation and biomass production in N. salina cultures, which revealed a net release of CO2 during mixotrophic growth. In the presence of glycerol, the proliferation of bacteria was consistently observable in these non-axenic cultures. 16S rRNA sequencing confirmed the presence of two phylogenetically distinct Gram-negative bacterial isolates belonging to alpha- and gamma-subclass of Proteobacteria (Paracoccus sp. MBIC4036, and Marinobacter alkaliphilus NBSL03 and Marinobacter lipolyticus SM-19, respectively) in the cultures of N. salina. Among these strains, M. alkaliphilus was shown to have denitrifying capabilities, which allowed it to oxidise glycerol using nitrate as a terminal electron acceptor. This provides an explanation for the utilisation of nitrate and glycerol that was observed in heterotrophic cultures despite the absence of microalgal growth. These results provide a better understanding of the interactions between bacteria and algae in mixotrophic growth that may enable the development of strategies to better utilise nitrogen and carbon for algae production. The present study has demonstrated the possibility of reutilisation of glycerol, a biodiesel by-product, to promote algal growth and lipid accumulation.