3.4. Enhancement of EPA and fucoxanthin production in P. tricornutum.
Total EPA and fucoxanthin content is represented in Figure 2A & B. The EPA content increased from 9 mg L-1 to 27 mg L-1 in M4 condition within 10 days (Table 3) , which accounts for a ~3-fold increase compared to P1 control and ~ 2-fold increase in compare to M3 conditions. Content wise on 6th day, EPA was higher in initial log phase accounting to 37 mg g-1 in the M2 condition, whereas M1 and M3 condition showed 34 mg g-1 of EPA (Figure 2A) . Furthermore, EPA content was higher in culture supplemented with only urea (P3) when compared to NaNO3 (P1) on 4th and 6th day (Figure 2A) . Increase in EPA content in the initial growth phase was reported earlier in the presence of urea[28] and glycerol along with urea in P. tricornutum[29, 30, 56].
Along with EPA, fucoxanthin content was measured in various culture conditions as depicted in Figure 2B. Higher fucoxanthin production was observed in M4 conditions i.e., 11 mg L-1 on 10th day of experiment (Table 3) . This showed a ~ 4-fold increase in fucoxanthin productivity compared to P1 and M3 conditions. Fucoxanthin production was 6.62 mg L-1 in medium supplemented with P2 on 10th day, whereas highest content was 21.9 mg g-1 on 6th day of cultivation in the P1 condition (Figure 2B) . Addition of glycerol limited the nitrogen content in the medium, and thus a decrease in the fucoxanthin content is observed in glycerol supplementation (M1 and M3) (Figure 2B) , although higher production is observed in M2 condition compared to M1 and M3 conditions, which can be justified by the presence of two nitrogen source and residual N in the medium until 10th day (Figure S1, S2, S3, Supporting information) . Fucoxanthin being a primary carotenoid is highly influenced by nitrogen content in the medium[57]. Previous reports highlights higher fucoxanthin content in P.tricornutumcultivated in nitrate-enriched medium[7, 16], thus feeding cultures with urea, increased biomass and the biomass associated metabolites i.e. EPA and fucoxanthin.