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.