Isolation, screening, identification and characterization of microalgae for neutral lipid accumulation
4.3 Results and Discussion
4.3.2 Characterization of growth and lipid productivity of Chlorella sp. FC2 IITG under nitrate and phosphate starvation
lipid accumulation in the microalgal strains (Illman et al., 2000; Feng et al., 2012; Sforza et al., 2012). In the present study, a similar trend was observed between nitrate or phosphate utilization and neutral lipid induction. Therefore, we hypothesized that the higher induction of neutral lipid under photoautotrophic growth may be due to phosphate starvation whereas under mixotrophic condition it may be due to phosphate starvation and nitrate limitation. In order to prove this hypothesis the organism was further tested under phosphate and nitrate starvation.
These characterization experiments have showed mixotrophic conditions as the best nutritional mode for the growth and lipid productivity from FC2. However, the use of organic carbon sources for the growth of microalgae under mixotrophic condition has restricted its use in open pond systems and therefore, all further experiments were conducted under photoautotrophic conditions. As a future prospect on availability of suitable waste water rich in organic carbon sources, mixotrophic growth condition can be considered as a viable option for the biodiesel production from FC2.
4.3.2 Characterization of growth and lipid productivity of Chlorella sp. FC2 IITG
initial 24 hours of nitrate starvation phase and reached a maximum neutral lipid content of 54.4 % (w/w, DCW) on 7th day of starvation (Fig. 4.7B). In case of phosphate starvation maximum neutral lipid accumulation of 28.60 % (w/w, DCW) was recorded on 7th day of cultivation under starved condition (Fig. 4.8B). These results point towards the significance of nitrate and phosphate starvation as triggers for neutral lipid accumulation in the cell mass.
Fig. 4.7 Dynamic profiles for growth, neutral lipid accumulation and substrate utilization of the strain Chlorella sp. FC2 IITG grown under nutrient sufficient and nitrate starvation conditions: (A) phosphate utilization and feeding; (B) growth (●) and neutral lipid percentage in the biomass (○); (C) nitrate (○) and phosphate utilization (●). The figure in the inset of (B) shows the total lipid productivity (TL) and neutral lipid productivity (NL) in mg L-1 day-1 during the starvation phase
A significant increase in lipid accumulation was reported for Nannochloropsis sp.
(Rodolfi et al., 2009) and Chlorella sp. BUM11008 (Praveenkumar et al., 2012) when the cultures were transferred from nitrate sufficient to nitrate depleted condition. Further, an
elevated accumulation of intracellular lipid content was reported for C. zofingiensis grown under phosphate starvation (Feng et al., 2012) and for Monodus subterraneus grown under phosphate limitation (Khozin-Goldberg and Cohen, 2006). Neutral lipid productivity of 71.9 mg L-1 day-1 and 60.8 mg L-1 day-1 was obtained for growth (7 days in starvation) of FC2 under phosphate and nitrate starvation respectively. The total lipid productivity of 85.56 mg L-1 day-1 and 63.45 mg L-1 day-1 was obtained under phosphate starved and nitrate starved conditions, respectively.
Fig. 4.8 Dynamic profiles for growth, neutral lipid accumulation and substrate utilization of the strain Chlorella sp. FC2 IITG grown under nutrient sufficient and phosphate starvation conditions: (A) phosphate utilization and feeding; (B) growth (●) and neutral lipid percentage in the biomass (○); (C) nitrate (○) and phosphate utilization (●). The figure in the inset of (B) shows the total lipid productivity (TL) and neutral lipid productivity (NL) in mg L-1 day-1 during the starvation phase
Comparison of lipid productivity under nutritional stress conditions for our strain with the available literatures revealed that this organism can serve as a superior platform for lipid overproduction in comparison to majority of the algal strains. However, the lipid productivity of the FC2 was lower in comparison to few other algal strains reported in the literatures (Table T2, Appendix). It is important to note that these strains were grown under higher CO2 concentration, higher light intensity and continuous light cycle.
Optimization of the cultivation conditions for FC2 will further improve biomass and lipid productivity which may be comparable with these lipid overproducing strains. Under phosphate or nitrate starvation, carbon flux from some of the intracellular components such as protein, carbohydrate and pigments is redirected towards lipid biosynthesis (Rodolfi et al., 2009). These lipids are higher energy yielding compounds than carbohydrate and hence, serve as an efficient energy reserve for the cell (Rodolfi et al., 2009). A significant change in the intracellular composition of FC2 was observed while transferred from nutrient sufficient condition to the nutrient starved condition (Table 4.2).
Table 4.2 Intracellular composition of macromolecules expressed in % (w/w) DCW of the strain Chlorella sp. FC2 IITG grown under nutrient sufficient and starved phases
Condition
Sufficient Phase (8th day) Starvation Phase (14th day) Carbohydrate Protein Neutral
Lipid
Carbohydrate Protein Neutral Lipid Nitrate 45.57±1.2 41.21±1.6 1.19±0.6 18.32±1.3 22.31±1.1 54.41±0.7 Phosphate 47.35±1 40.65±1.3 1.0 32.21±1.4 27.19±0.7 28.60±0.5 Redirection of carbon flux from carbohydrate and protein fractions of the biomass towards accumulation of neutral lipid was also evident from changes in macromolecular composition of the cells during the transition from exponential phase to the stationary phase of growth under two different trophic conditions (Fig. 4.9). Similar metabolic shift in terms of biomass composition was reported for photoautotrophic growth of Neochloris oleoabundans and Chlorella vulgaris under nitrogen starvation where reduction in intracellular protein content was coupled with increase in neutral lipid accumulation
(Pruvost et al., 2011). Further, Pseudochlorococcum sp. was found to accumulate starch as the major energy storage compound during nitrogen sufficient condition whereas under nitrogen depleted condition, the strain accumulated neutral lipids with significant reduction in the carbohydrate content (Li et al., 2011). Increased carbohydrate catabolism and subsequent rise in acetyl CoA pool under nitrogen starved condition for Micractinium pusillum revealed that carbohydrate forms the major carbon source for triacyl glycerol synthesis rather than photosynthesis which supports the finding in the present study (Li et al., 2012).
Fig. 4.9 Intracellular composition of macromolecules in the strain Chlorella sp. FC2 IITG grown under different trophic modes: (A) Carbohydrate content; (B) protein content; (C) neutral Lipid content and (D) chlorophyll content. The strain was grown under photoautotrophic (■) and mixotrophic (●) condition on BG11 medium at 28°C and 400 rpm in a 3 L automated bioreactor