Isolation, screening, identification and characterization of microalgae for neutral lipid accumulation
4.3 Results and Discussion
4.3.1 Evaluation of the strain Chlorella sp. FC2 IITG under different trophic modes Biomass and lipid productivity of the organism FC2 was evaluated under
photoautotrophic and mixotrophic conditions. Among the two cultivation conditions, the highest biomass titer (1.03 g L-1) was achieved for mixotrophic condition (Fig. 4.5A). This biomass titer was ~1.5 fold higher than that achieved for photoautotrophic condition (Table 4.1). An improved biomass concentration was reported for C. sorokiniana (Wan et al., 2011), Nannochloropsis oculata and C. vulgaris (Heredia-Arroyo et al., 2011) when grown under mixotrophic condition in comparison to photoautotrophic condition which supports the present study. Mixotrophic cultivation provides an opportunity to the strain to follow both heterotrophic route and light dependent route of growth in simultaneous and independent manner (Sforza et al., 2012). Therefore, growth is not strictly limited by the availability of light or availability of carbon source in the medium as is the case for photoautotrophic and heterotrophic growth respectively.
Table 4.1 Kinetic parameters for growth and lipid formation of Chlorella sp. FC2 IITG cultivated under photoautotrophic and mixotrophic cultivation conditions
Parameters Cultivation Conditions
Photoautotrophic* Mixotrophic*
Specific growth rate µexp (day-1) 0.92 ± 0.01a 0.97 a
Dry cell mass (g L-1) 0.69 ± 0.03 1.03
Biomass productivity (mg L-1 day-1) 114 b 73b
Neutral Lipid (%, w/w DCW) 37.64 ± 0.8 44.83 ± 0.16
Neutral Lipid productivity (mg L-1 day-1) 28.88 ± 0.7c 35.37 ± 1.68c
Total Lipid (%, w/w DCW) 45.18 ± 3.1 68.75 ± 0.01
Total lipid productivity (mg L-1 day-1) 35.02 d 50.42 ± 0.01d
*values are mean ± SE (n=3)
aSpecific growth rate µexp was calculated as average in the exponential phase of growth (0-5 days of cultivation) for all the two cultivation conditions.
bData from 6 days of cultivation was used to calculate biomass productivity under photoautotrophic condition.
Data from 14 days of cultivation was used to calculate biomass productivity under mixotrophic condition.
c,d Lipid productivity was calculated based on the data from 9 days of cultivation under photoautotrophic growth, whereas for mixotrophic condition data from 14 days of cultivation was used.
Fig. 4.5 Dynamic profiles for growth, changes in intracellular neutral lipid content of the strain Chlorella sp. FC2 IITG under photoautotrophic and mixotrophic conditions: (A) growth; (B) neutral lipid percentage in the biomass. The strain was grown on BG11 medium at 28°C and 400 rpm in a 3 L automated bioreactor
The biomass productivity of the strain under mixotrophic condition (73 mg L-1 day-
1) was found to be lesser than photoautotrophic (114 mg L-1 day-1) condition (Table 4.1).
This was attributed to the longer cultivation period (14 days) required to achieve the maximum biomass concentration in case of mixotrophic condition as opposed to the photoautotrophic cultivation condition (6 days). The maximum achievable biomass concentration in photoautotrophic condition was restricted by the combined effect of phosphate exhaustion from the medium (Fig. 4.6A) and insufficient photon flux attributed to cell shading effect (Cheirsilp and Torpee, 2012).
Under mixotrophic condition, the organism was able to grow beyond 6th day of cultivation by utilizing glucose as the source of energy even under phosphate exhaustion (Fig. 4.6A) and photon limitation. This is evident from the glucose uptake profile showing utilization till 8th day of cultivation under mixotrophic condition with the requirement of two pulse additions (on 3rd day and 7th day) of glucose in the medium (Fig. 4.6C).
Fig. 4.6 Substrate utilization profile of the strain Chlorella sp. FC2 IITG grown under photoautotrophic (■) and mixotrophic (●): (A) phosphate utilization; (B) nitrate utilization and (C) glucose utilization for mixotrophic conditions. The arrow mark in (C) indicates the time of glucose feeding in case of mixotrophic conditions. The glucose feeding was performed when the concentration of glucose in the medium was less than 1.8 g L-1. The strain was grown on BG11 medium at 28°C and 400 rpm in a 3 L automated bioreactor with 20 µE m-2 s-1 light intensity for a light: dark cycle of 16:8 h
Intermittent addition of glucose for mixotrophic cultivation of Chlorella sp. and Nannochloropsis sp. was found to enhance the biomass productivity (Cheirsilp and Torpee, 2012). Given that the comparison of biomass productivity of the present strain with the available literatures may be difficult owing to markedly different growth conditions and cultivation systems, a detailed comparison of the biomass productivity of FC2 with other microalgae is given in the supplementary materials (Table T1, Appendix).
Difference in cultivation conditions resulted in significant variation in the neutral lipid productivity (28.88 mg L-1 day-1 to 35.37 mg L-1 day-1) and total lipid productivity (35.02 mg L-1 day-1 to 50.42 mg L-1 day-1) of the strain (Table 4.1). Neutral lipid accumulation under different cultivation condition was captured by Nile-red based assay method (Fig. 4.5B) and total lipid content in terms of FAME was obtained from GC analysis.
Highest neutral lipid productivity and neutral lipid content was obtained for mixotrophic culture followed by photoautotrophic condition (Table 4.1). The total lipid productivity of our strain under mixotrophic growth was found to be higher than most of the literature reported values, with exception of a few microalgal strains (Table T1, Appendix). In contrast to the present finding, no significant difference in lipid content was reported for four different strains tested under mixotrophic, heterotrophic and photoautotrophic conditions (Cheirsilp and Torpee, 2012). Hence, it is possible that the lipid accumulation property of the cells is strain dependent (Cheirsilp and Torpee, 2012).
The intracellular neutral lipid induction may be attributed to the exhaustion of nutrients during transition from growth phase to the stationary phase of the cultivations. For instance, utilization profile of the substrates under both the cultivation conditions (Fig.
4.6A) showed that phosphate was consumed completely within three days of cultivation and hence, creating nutritional stress condition for the cells. A large pool of literature has demonstrated that the phosphate and nitrate starvation (or limitation) serve as the drivers for
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