6.2.1 Organism, media and growth conditions
The strain FC2 was grown under same cultivation conditions as mentioned in the section 5.2.1. However, the optimized BG11 medium obtained from the previous experiments (section 5.3.1) was used which comprises (in g L-1) urea 1.8, K2HPO4 0.076, MgSO4.7H2O0.124, CaCl2.2H2O 0.065, Na2CO3 0.038, citric acid 0.002, ferric ammonium citrate 0.01, EDTA 0.001, and A5 + Co solution (1 mL L-1) that consists of H3BO3, 2.86;
MnCl2.H2O, 1.81; ZnSO4.7H2O, 0.222; CuSO4.5H2O, 0.079; Na2MoO4.2H2O, 0.390; and Co(NO3)2.6H2O, 0.049 for the growth of FC2. All the chemicals were purchased from SRL Pvt. Ltd., India unless otherwise mentioned. All the experiments were conducted in triplicate and the data were expressed as mean ± standard error.
6.2.2 Experimentation
Three different experiments were conducted: (i) fed-batch cultivation with intermittent feeding of urea and phosphate at constant illumination of 250 µE m-2 s-1; (ii) fed-batch cultivation with intermittent feeding of urea and phosphate with dynamic increase in light intensity from 250-450 µE m-2 s-1 and finally (iii) decoupling of growth and lipid production phases by a two stage photoautotrophic cultivation.
6.2.2.1 High cell density cultivation of FC2 under fed-batch mode
The fed-batch processes started with the optimized BG11 medium at the optimal concentrations of urea and phosphate 1.8 g L-1 and 0.076 g L-1 respectively as their initial concentrations. These optimal concentrations of urea and phosphate were obtained from the medium optimization studies carried out in Chapter 5. The concentration of urea and phosphate in the fermentation medium was maintained at their optimal concentration (not less than 90 % of its initial concentration) by intermittent feeding after every light cycle of
the experiment thereby avoiding substrate limitation. In case of fed-batch mode with constant illumination, intermittent feeding of nutrients were performed at a constant illumination of 250 µE m-2 s-1 at 16:8 h light: dark cycle throughout the experiment. In case of fed-batch with dynamic increase in light intensity, a step-wise increase in light intensity was employed with an illumination of 250 µE m-2 s-1 for initial 5 days (112 h) of cultivation period followed by 350 µE m-2 s-1 till 10th day (232 h) of cultivation and then a constant light intensity of 450 µE m-2 s-1 was maintained till the end of the batch.
6.2.2.2 Two phase photoautotrophic cultivation of FC2: high cell density biomass formation in fed-batch mode and lipid enrichment via nitrogen starvation
A two phase cultivation strategy was employed to achieve lipid-rich biomass with high density of FC2 (Fig. 6.1).
Fig. 6.1 Schematic representation of the two phase cultivation process designed for high cell density lipid rich photoautotrophic cultivation of Chlorella sp. FC2 IITG in a 3.0 L automated photobioreactor. Phase I represents the high cell density cultivation of FC2 through intermittent feeding of urea and phosphate with dynamic increase in light intensity.
Phase II represents lipid enrichment phase via nitrogen starvation. In both the phases cells were grown at 28°C, 400 rpm and aerated with 1% (v/v) CO2
The first phase represents the growth phase in which high cell density cultivation of FC2 was achieved via intermittent dosing of urea and phosphate along with dynamic
increase in light intensity provided (as detailed in section 6.2.2.1). The second phase of the cultivation was the lipid enrichment phase. In the second phase, high density FC2 cells obtained from the phase 1 were collected through centrifugation and re-suspended in same volume of nitrogen free medium (devoid of urea). Ammonium ferric citrate in the optimized BG11 medium was also replaced by ferric chloride and citrate at their equimolar concentrations in order to ensure the absence of any nitrogen source in the medium.
Intermittent feeding of phosphate was given when the concentration of phosphate went below 90 % of its initial concentration, in order to avoid phosphate starvation or limitation in the study. Sampling was performed at regular intervals to obtain dynamic profiles of growth, intracellular lipid accumulation and substrates utilization.
6.2.3 Analysis of growth, substrate utilization and FAME composition
Fig. 6.2 Correlation graph between the dry cell weight and absorbance measured at 690 nm in a spectrophotometer under optimal nutrient sufficient growth condition under photoautotrophic cultivation
Cell density was obtained by the method as detailed in Section 4.2.4, Chapter 4. For photoautotrophic optimal, nutrient sufficient condition: one cell density corresponded to
0.232 g dry cells L-1 with R2 of 0.99 (Fig. 6.2) and for nutrient starved condition: one cell density corresponded 0.22 g dry cells L-1 with R2 value of 0.98 (Fig 4.1, Section 4.2.4). The biomass productivity (𝑃𝐵, mg L-1 day-1) under different cultivation conditions were calculated as per the equation 4.1 shown in section 4.2.4.1.
The urea concentration in the medium were estimated using diacetyl monoxime method as prescribed by Wybenga et al. (1971) as detailed in Section 5.2.4.1. The phosphate concentration in the media was obtained through the ascorbic acid method suggested by Parsons et al. (1984) (details in section 4.2.4.3). The two-step direct transesterification method prescribed by Kumar et al. (2014) was used to convert the intracellular lipids in to fatty acid methyl ester. The formed FAME was analyzed in GC equipped with FID detector (detailed protocol in section 4.2.4.8). The lipid productivity (𝑃𝐿, mg L-1 day-1) was calculated as detailed in equation 4.6 (section 4.2.4.8). The light intensity available per cell was calculated as suggested by Imaizumi et al. (2014) with the following equation:
𝐿 = 𝐼𝑋×𝑉0𝐻𝐷 (6.1)
where, L- represents the light intensity per cell (µE g-1 cell s-1); 𝐼0- represents the incident light intensity on the reactor surface (250 to 350 to 450 µE m-2 s-1); H – represents the height of the reactor (m); D- represents the diameter of the bioreactor (m); V- represents the volume of the bioreactor (L) and X – represents the cell density (g L-1).