3.1. Biomass production of P. tricornutum
Initial screening for measuring the growth and nutrient consumption in
photoautotrophic and mixotrophic conditions was performed. P.
tricornutum was grown with an initial biomass concentration of 0.1 g
L-1and attained a maximum biomass i.e. 0.77 g
L-1 (~2-fold higher) in M4 condition
in 10 days. Additionally higher growth rates were observed in the
mixotrophic cultures (Table 1) . Overall, the combined effect of
mixotrophy and urea feeding clearly show a higher growth rate and
biomass in P. tricornutum . Previous reports have shown the effect
of mixotrophy on growth of P. tricornutum which correlates with
our results [25,
27, 50,
51].
Substrate consumption reflects biomass productivity, thus nutrient
uptake rates were measured (Figure S1, S2, S3, S4, Supporting
information) . P. tricornutum consumed around 3 g
L-1 glycerol (10th day), 75 mg
L-1 NaNO3 (6th day),
and 26 mg L-1 urea (4th day)
(Figure S1, Supporting information ). Further, in the presence
of glycerol, consumption rate of nutrients increased resulting in the
exhaustion of NaNO3 by 4th day (M1)
and urea by 2nd day. Mixotrophic mode of P.
tricornutum enhanced the nutrient uptake rate was previously reported
by Villanova et al.,[51].
Interestingly, urea is completely consumed in the medium by
4th day (P2, P3, M2, M3), whereas
NaNO3 is exhausted by 6th (P1) day of
cultivation (Figure S2 , S3, Supporting information ).
This also justifies decrease in biomass and growth rate in P3 culture
conditions as compared to P1and P2. Hence, a strategy based on feeding
urea was designed to maintain a high biomass rate on a cost-effective
substrate (M4). Urea was fed into the medium at the early log phase at
36th hour (before urea depletion) according to the
12-hour urea consumption profile (Table S2 , Figure S4,
Supporting information) . Growth profile in M4 medium improved as
compared to the P1 and M3, highlighting it as effective nitrogen source
for the growth of P. tricornutum . Effect of different nitrogen
sources on growth of P. tricornutum , highlighted urea as the best
source due to the increase in biomass yield and lower substrate cost[28]. It can be
observed that P. tricornutum prefers urea as nitrogen source and
consumes it more efficiently than NaNO3 (Figure
S2, S4, Supporting information ).