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.