3.2. Biochemical analysis
The volumetric concentration of biochemical constituent were estimated
to understand the influence of various growth conditions on cellular
constituents of P. tricornutum (Table 1 ). Biochemical
constituents were significantly enhanced in M4 condition. Protein titer
in M4 condition was maximum i.e., ~362 mg
L-1 (~2.5-fold increase) and with
higher biomass productivity (Figure 1) . In addition, increased
protein concentrations were observed in photoautotrophic cultures with 2
nitrogen sources and combined mixotrophic medium supplemented with mixed
nitrogen source (P2 and M2) (Table 1) . Microalgae under the
influence of nitrogen rich environment (feed cultures) channelize the
photosynthetically fixed carbon to protein synthesis to support growth
and division, however, exactly an opposite trend is seen in nitrogen
deprived conditions, where cell division ceases and fraction of carbon
allocation to storage molecules increases at the expense of protein
synthesis [28].
The maximum lipid titer was 227 mg L-1 and 208 mg
L-1 in M4 and M3 cultures respectively (Table
1) . Our study highlights that glycerol and urea supplementation
enhanced lipid productivity which is quite interesting, as many
microalgae are known to accumulate lipids in response to N limitation[52]. This can be
justified by the addition of glycerol; P. tricornutum in lipid
accumulation phase uses glycerol as a carbon source and hence channelize
the carbon directly towards lipid production, moreover feeding nitrogen
leads to more biomass, which in turn reflects in increasing the
productivity of biochemical constituents. The result clearly indicates
that the strategy of urea recharge via feeding in glycerol
supplemented medium can effectively produce biomass and lipids.
Cultures supplemented in M3 condition showed higher carbohydrate
production i.e., 164 mg L-1, and was enhanced to 231
mg L-1 (~1.5-fold increase) in M4
(Table 1) . Carbohydrate are primary storage reserves of
diatoms, one the first photosynthetic product of the Calvin–Benson
cycle and serves as a precursor for various cellular components[53]. Therefore,
carbohydrate accumulation and conversion into other metabolites is
necessary for cell survival. It was observed that addition of glycerol
switches the cellular metabolism towards carbohydrate production inP. tricornutum[52]. Compared to
lipid production, the carbohydrate content was less in glycerol
supplemented cultures, which clearly demonstrates the channeling of
carbon flux towards TAG production[54]. In this
context, feeding P. tricornutum with a cost-effective nitrogen
source like urea, resulted in higher protein, lipid, carbohydrate and
biomass production. Highlighting the potential of our substrates to
enhance the overall productivity of P. tricornutum and to further
use in a biorefinery-based system.