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].