4. Discussion
Microalgae derived HVRs are progressively playing a significant role in the production of cosmeceuticals, medicinal alternatives and high-value foods [58].P. tricornutum as a cell factory for marketable products which majorly includes omega 3 PUFAs like EPA and light harvesting pigment fucoxanthin, [8]. Due to their high pharmaceutical and nutritional relevance, these HVRs currently have a high market value for example the market size of omega 3 ingredients in 2019 exceeded USD 2.3 billion and is estimated to grow at over 7.2% compound annual growth rate (CAGR) between 2020 and 2026 (www.gminsights.com). Moreover, revenue generated from fucoxanthin was USD 100 million in 2019, and is expected to reach USD 123 million by 2025, with a CAGR of 3.5% (www.wboc.com).
Sustainable co-production of these HVRs is a challenging task in terms of economic feasibility. Therefore, to enhance production, modification in the cultivation parameters can be employed as an effective strategy. Our preliminary analysis highlighted the effect of different substrates (glycerol, urea, and NaNO3) and their different combination on growth and biochemical constituents. Optimization of medium with glycerol and urea (M4) addition is a suitable, sustainable and economic approach, due to its beneficial effect on cellular metabolism, biomass and HVRs production enhanced. Furthermore, since the nitrate is depleted from the medium in the early phases of growth, employing a strategy to feed urea in culture medium before exhaustion proved to be more feasible in terms of enhancing HVR production.
Screening highlighted M4 as a suitable condition to achieve higher biomass and better growth rate in P. tricornutum . A combined effect of mixotrophy and urea feeding resulted in higher biomass compared to cultures without urea feeding. Mixotrophy metabolism reportedly engages both respiration and photosynthesis at the same time[26]. Transport activities between the chloroplast and mitochondria, as well as the physical connection between the two organelles, have an intense energetic exchange during mixotrophy in P. tricornutum[52]. Moreover, mixotrophy enhances the nutrient uptake rate as observed in glycerol-supplemented (M1 and M3) medium, which enhances nitrogen uptake (Figure S1, S2, S3, Supporting information) .
The increase in biomass reflects the assimilation of C and N in cellular biochemical components. Higher protein content was obtained in the medium supplemented with M2 and M4 combinations depicting conversion of excess nitrogen into cellular proteins. Currently microalgal proteins are in demand as functional foods due to its high nutritional value, and health benefits. Additionally utilization of whole algal biomass as super foods or healthy foods is promoted worldwide in order to maintain a balance diet[59]. Therefore higher protein production in P. tricornutum makes it a suitable candidate for nutraceutical applications. Moreover, carbohydrate productivities reached to 231 mg L-1 in M4 condition (Table 1) . Anticancer activity of polysaccharides derived fromP. tricornutum along with antibacterial, antioxidant, and antiviral properties is well demonstrated[60, 61]. Polysaccharides from P. tricornutum have a high commercial potential and a wide range of applications in different industrial sectors.
Biodiesel is considered as a desirable energy source, an exceptional alternative to fossil fuels. According to the previous reports the FAME, profile of P. tricornutum meets the requirements of international biodiesel standards, showing that it could be a good alternative for biodiesel production[2]. Our results are in correlation with these studies showing a high percentage of C16, C16:1, C18:1 (%TFA). Moreover, higher FAME yields obtained in P. tricornutum on cost effective substrates, indicates de novofatty acid synthesis in presence of glycerol. These fatty acids are main components of storage lipids, which justifies its higher accumulation in the form of TAGs in the mixotrophic conditions[26, 56]. Feeding additional nitrogen shifted the fatty acid metabolism towards membrane lipid synthesis i.e., EPA showing a higher percentage of PUFAs in feed conditions (Table 2 ).
The expense of nutrients is an inevitable liability in the production of algal biomass (4-8 % of total cost)[62]. As a result, the usage of recovered nutrients from secondary streams, such as glycerol from the biodiesel sector and urea, ammonia from wastewater, may be included into the biorefinery framework for the long-term synthesis of HVRs. Although previous reports highlights the use of glycerol as a C source for microalgae production[63] and the recovery of nutrients (N and P) by digestate pretreatment[64], lesser data is available on the combined effect of replacing both C and N with secondary streams. The validity of such a strategy has been introduced in our study by applying it on production of HVRs.
In M4 condition, higher EPA (27 mg L-1) was accumulated along with TAG (92 mg L-1) (Figure 2A, Table 2). Indicating that glycerol triggers metabolic changes resembling not only nitrogen depletion but also promote growth[56]. An increase in EPA content on initial growth days (Figure 2A) indicate the necessity of structural lipids in log phase but as nitrogen depletes in the medium EPA content did not change (Figure 2A) in the respective conditions, though urea-fed cultures maintain the EPA pool throughout the experiment. EPA being one of the main structural lipids required during the growth supporting conditions, whereas is incorporated into TAGs when the cells are under nitrogen stress. Hence, to maintain high EPA content in stress condition use of glycerol as carbon source can be ideal, additionally feeding with the nitrogen source can enhance the productivity via ., higher biomass yields.
Along with EPA, fucoxanthin content highlights a significant increase in the productivity especially during feed conditions. Fucoxanthin is associated with photosynthetic machinery and is enhanced as the growth increases. Nitrogen on the contrary in the form of urea positively regulates the photosynthetic machinery in feed condition (M4) reflected by the higher biomass production. Hence, in the condition supplemented with nitrogen substrates (P1, P2, and P3) fucoxanthin content was higher (Figure 2B) as compared to glycerol-supplemented cultures (M1, M2, and M3). Glycerol is found to be directly entering glycolysisvia glycerol kinase producing dihydroxyacetone phosphate and thus channelizing flux towards lipid synthesis rather than carotenoid production [52].
Our findings highlight the effect of feeding nitrogen to the glycerol-supplemented cells (M4), higher nitrogen content results in increasing the biomass and thus enhancing growth related metabolites like EPA and fucoxanthin. Presence of glycerol in the medium upregulates the EPA biosynthesis via regulating enzyme like stearoyl desaturase [56] and the effect of feeding strategies of various nutrients on biomass, and lipid productivities[65-67] is mentioned in previous reports. In conclusion, this study successfully highlights an alternative application of feeding nutrients in mixotrophic mode to increase HVRs like EPA and fucoxanthin. Further, the ability of P. tricornutum to grow mixotrophically using glycerol as the main carbon source and urea as an additional nitrogen source can be applied in the biorefinery approach for recovering carbon and nitrogen from waste effluents to produce HVRs.