2.2 Experimental design
Different substrates and various combinations along with concentrations used for initial experiments are mentioned in Table S1 (Supporting information) . Glycerol and urea concentrations were selected based on previous literature[29, 30]. Inoculation was carried out using the inoculum in linear growth phase as described earlier with a biomass of 0.1 g L-1. The organic nutrients (urea and glycerol) were sterilized by filtration through 0.2 µm membrane filters and added to the autoclaved medium in sterile conditions. Subsequently the effect of urea feed was investigated by cultivating P. tricornutum on glycerol + urea or M3. Urea was added as feed according to the consumption profile, before its complete exhaustion from the medium (Table S2, Supporting information) . Sampling was carried out at regular time intervals of 2 days for further analysis and growth was monitored by cell count using hemocytometer[36] and dry weight analysis (DCW). Parameters such as specific growth rate and doubling time were calculated as described previously[37, 38].