6.3 Results and discussion
6.3.1 High cell density biomass formation in fed-batch mode with constant and dynamic light intensity
Increasing the light intensity up to 250 µE m-2 s-1 has showed increased consumption of urea and phosphate leading to phosphate depletion at 96 h of growth (Fig. 5.4B & 5.4C, Section 5.3.2) in the optimized growth medium. Low biomass titer is often attributed to limited substrate availability and decreased light availability through dense culture (Imaizumi et al., 2014). Therefore to achieve high cell density cultivation, fed-batch process with intermittent feeding of the rate limiting nutrients urea and phosphate was designed.
Further, the effect of dynamic increase in light intensity was also evaluated by conducting two different fed-batch experiments, one with a constant light intensity of 250 µE m-2 s-1 and other with the step-wise increase in light intensity from 250 to 450 µE m-2 s-1.
The fed-batch processes started with the initial concentration of urea and phosphate which was set at their optimum value 1.8 g L-1 and 0.076 g L-1 respectively. Intermittent feeding of urea and phosphate was required to maintain their concentration above 90 % of initial value after every light cycle of the photoperiod regime. The fed-batch with constant light intensity was started with 250 µE m-2 s-1 and maintained constant throughout the experiment. Whereas in the fed-batch with step-wise increase in light intensity, initial 5 days (112 h) of cultivation period was provided with 250 µE m-2 s-1, 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 throughout the experiment. Final biomass titer of 7.75 g L-1 and the biomass
productivity of 516.67 mg L-1 day-1 was obtained in the fed-batch with constant illumination, which was 27 % higher than the productivity obtained from the batch cultivation (Fig. 6.3A).
The fed-batch with dynamic increase in light intensity resulted in maximum biomass titer of 13.5 g L-1 and biomass productivity of 675 mg L-1 day-1 (Fig. 6.4A). The optimum light intensity for the growth of algal cells varies with the cell density and growth conditions.
With a constant light intensity supplied to the reactor, the available light to the cells will decrease with increasing cell density attributed to cell shading effect. Thus, a single optimum light intensity may not be suitable for growth of microalgae under photoautotrophic condition. Therefore, the light intensity supplied to the reactor needs to be increased simultaneously with increase in the cell density to achieve a sufficient light availability per cell (Imaizumi et al., 2014).
In the present study optimum nutritional conditions resulted in rapid growth of the organism which in turn led to early crowding of the cells in bioreactor and associated light attenuation. Even though light intensity of 450 µE m-2 s-1 was found to be detrimental for growth of FC2 (Fig. 5.5), providing the light intensity at higher cell numbers has supported the growth to the maximum attributed to the increased light availability per cell. The changes in light availability per cell are depicted in the table 6.1. This shows that dynamic increase in light intensity from 250 to 450 µE m-2 s-1 has increased the light availability per cell up to 84.2 % when compared with the constant light intensity with 250 µE m-2 s-1. Thus, feeding of the urea and phosphate, have resulted in significant increase in biomass productivity. Similar feeding of the rate limiting nutrients in the medium enhanced the biomass productivity in other Chlorella sp. (Hseih and Wu, 2009).
Fig. 6.3 Dynamic profiles for growth and substrate utilization profiles of FC2 grown under fed-batch mode with intermittent feeding of urea and phosphate and with constant light intensity. (A) biomass formation (●) and neutral lipid production (○); (B) intermittent feeding and utilization of urea; (C) intermittent feeding and utilization of phosphate.
Intermittent feeding is represented by. The experiment was conducted in an automated bioreactor of 3.0 L volume at 28 °C, 400 rpm aerated with 1% (v/v) CO2 and constant light intensity of 250 µE m-2 s-1 for a light: dark cycle of 16:8 h
Fig. 6.4 Dynamic profiles for growth and substrate utilization profiles of FC2 grown fed- batch with intermittent feeding and dynamic increase in light intensity under photoautotrophic condition supporting high cell density cultivation: (A) biomass formation (●); (B) intermittent feeding and utilization of urea; (C) intermittent feeding and utilization of phosphate; (D) step-wise increase in light intensity. The experiments were conducted in an automated bioreactor of 3.0 L volume at 28 °C, 400 rpm aerated with 1% (v/v) CO2 and dynamic increase in light intensity from 250 to 450 µE m-2 s-1 for a light: dark cycle of 16:8 h
Table 6.1 Light availability per cell (µE g cells-1 s-1) calculated using eq. 6.1 for the fed- batch with dynamic change in light intensity and intermittent feeding of nutrients. The light availability per cell was calculated assuming a constant light intensity of 250 µE m-2 s-1 and for the dynamic change in light intensity from 250 to 450 µE m-2 s-1 with the changing biomass density
Age (h)
Biomass (g L-1)
Light intensity (µE m-2 s-1)
Light availability per cell (µE g-1 cell s-1)
Constant light intensity at 250 µE m-2 s-1
0 0.0139 250 187.05
120 3.4676 250 0.75
136 4.4099 250 0.59
232 7.9785 250 0.33
240 7.91 250 0.33
480 13.5 250 0.19
Variable light intensity up to 450 µE m-2 s-1
0 0.0139 250 187.05
120 3.4676 250 0.75
136 4.4099 350 0.83
232 7.9785 350 0.46
240 7.91 450 0.59
480 13.5 450 0.35
With the dynamic increase in light intensity, the frequency of feeding required for maintaining the concentration of nutrients was also found high in comparison to the constant light fed-batch (Fig. 6.3 & 6.4). The concentration of the limiting nutrients and their utilization are dependent on the available light intensity per cell. Higher initial concentration of the rate limiting nutrients leads to high cell densities resulting in associated light attenuation during the growth phase. Therefore, to achieve high cell density cultivation dynamic increase in the light intensity is required along with intermittent feeding of the limiting nutrients. Thus, changes in light intensity have significantly influenced growth and the substrate utilization capability of the organism. Similar observation showing an increase in experimental biomass yield per phosphorus with increasing light intensity was reported for the microalga Scenedesmus sp. LX1 (Wu et al., 2014).