Screening and optimization of elicitor molecules for lipid induction

Process development for high cell density lipid rich microalgae cultivation and enhanced lipid productivity

7.2 Materials and Methods

7.2.2 Screening and optimization of elicitor molecules for lipid induction

Though from our previous experiment sodium acetate was found as lipid inducer, further screening of lipid inducer was performed to identify effective combination of lipid inducers which supports maximum lipid induction without hampering growth. To that end the organism was grown on modified BG11 medium supplemented with individual elicitor molecule and 15 g L^-1 glucose as the primary carbon source for growth under mixotrophic condition. Nine different elicitor molecules sodium acetate, sodium chloride, glycerol, bovine serum albumin, magnesium chloride, hydrogen peroxide, trisodium citrate, ferric chloride and calcium chloride at four different concentrations of each (Table 7.1) were used for the characterization of the strain. It is important to note that in order to eliminate any possible effect of nutritional starvation on lipid induction the experiments were carried out under nutrient sufficient condition through intermittent feeding of nitrate, phosphate and glucose (Palabhanvi et al., 2014).

Table 7.1 Screening of elicitor molecules for lipid induction and also supports synchronized growth and lipid accumuation in Chlorella sorokinianaFC6 IITG

Inducers Concentrations (g L^-1)

Sodium acetate 5 10 15 20

Glycerol 5 10 15 20

Sodium chloride 5 10 15 20

Ferric chloride 0.05 0.10 0.15 0.20

Trisodium citrate 0.05 0.10 0.15 0.20

Magnesium chloride 0.05 0.10 0.15 0.20

Hydrogen peroxide 0.05 0.10 0.15 0.20

Bovine serum albumin 0.05 0.10 0.15 0.20

Calcium chloride 0.05 0.10 0.15 0.20

Feeding of these nutrients was done to maintain broth concentration above 50% of their initial value. All the screening experiments were conducted at shake flask under mixotrophic condition at 28°C, 150 rpm, and continuous light intensity of 30 µE m^-2 s^-1. Sampling was performed at regular time intervals for monitoring the growth and lipid accumulation in the strain.

Once, the effective elicitor molecules were screened, their combined effect on lipid productivity was assessed by growing the organism in presence of these elicitors at different concentration. Further, concentrations of these elicitor molecules were optimized via central composite design (CCD) of experiment and response surface methodology (RSM) with maximization of net lipid productivity as the objective function. A total of thirteen experiments were designed through CCD with five replications at the center values to evaluate the pure error. The response was measured in terms of maximum net lipid productivity (Y) on 16^th day batch. The behaviour of the system was determined by assuming a second order polynomial with linear, quadratic and interaction effects as shown by Eq.

(7.1).

𝑌 = 𝛽₀+ ∑^𝑘_𝑖=1𝛽_𝑖𝑋_𝑖 + ∑^𝑘_𝑖<1𝛽_𝑖𝑗𝑋_𝑖𝑋_𝑗+ ∑^𝑘_𝑗=1𝛽_𝑗𝑗𝑋_𝑗² (7.1) Where Y is the response; 𝑋₁and 𝑋₂, and are input variables; 𝛽₀is constant; 𝛽_𝑖 is the linear coefficient; 𝛽_𝑖𝑗is the interaction coefficient, and 𝛽_𝑗𝑗is the quadratic coefficient. Estimation

of regression coefficients and statistical tests were carried out in the MINITAB (Version 16, Minitab Inc., State College, PA, USA) statistical software based on RSM. Analysis of variance (ANOVA) was conducted on the variables to understand the effect of individual factors on lipid productivity. The optimized elicitor concentrations were further validated in shake flask. The conditions in the shake flask were kept similar to all the conditions as discussed in above section 7.2.2. The best combination of lipid inducers with their optimal concentration was used for lipid enrichment in process development for synchronized growth and lipid accumulation.

7.2.3 Process development for synchronized growth and neutral lipid accumulation in automated photobioreactor under fed-batch & chemostat operation mode

The inoculum was prepared by transferring two loops full of slant culture into 250 mL Erlenmeyer flask containing 100 mL modified BG11 medium supplemented with 15 g L^-1 glucose and incubated in an orbital shaker under mixotrophic condition at 28°C, 150 rpm, and continuous light intensity of 30 µE m^-2 s^-1. This concentration of glucose was found to be optimal for growth when FC6 was characterized under different concentration of glucose (5, 10, 15 and 20 g L^-1) under mixotrophic condition. Actively growing mid log phase seed culture of 1% (v/v) was used as inoculum in all the experiments. Two different experiments were conducted with the dual aim of: (i) simultaneous growth and lipid enrichment and (ii) maximization of neutral lipid productivity. These objectives will be achieved via supplementation of specific lipid inducers in the growth media for lipid enrichment and maintaining higher specific growth rate of the organism throughout the entire cultivation period to enhance the biomass and lipid productivity. To that end, in the first experiment the organism was grown under fed-batch mode of cultivation with intermittent feeding of nitrate, phosphate, glucose and sodium acetate. The broth concentrations of all the nutrients and inducers were maintained above 50% of their initial

concentration in the medium. In the second experiment the organism was grown in a chemostat with continuous feeding of modified BG11 media supplemented with glucose and lipid elicitor molecules (sodium chloride and sodium acetate). Initially the strain FC6 was grown under fed-batch mode of operation with intermittent feeding of limiting nutrients for a period of 5-6 days till 2.23 g L^-1 of biomass (equivalent to absorbance at 690 nm, A690

-10) was reached. Once the desired cell density was reached, the mode of operation was switched to chemostat. In continuous mode, the modified BG11 medium containing all the nutrients along with the optimal concentration of elicitors were fed at different dilution rates ranging from 0.9 day^-1 to 0.18 day^-1 resulting in sequential steady state of the culture. Two variable speed peristaltic pump with the maximum and minimum flow rate of 2.8 mL min^-1 and 0.28 mL min^-1 respectively was used for continuous feeding and removal of medium from the reactor. Sampling was performed at regular time intervals for monitoring the growth, lipid production and substrate utilization. These experiments were carried out in a 7.5 L automated bioreactor (Bioflo 115, New Brunswick Pvt. Ltd., USA) with working volume of 4 L at agitation speed of 250 rpm and continuous illumination of 350 µE m^-2 s^-1 provided by 23 W CFL lights (Havells Pvt. Ltd., India) arranged surrounding the reactor surface. The pH of the medium was maintained at 8 ± 0.2 via purging air mixed with 1-2%

(v/v) of CO2 at the flow rate of 4 L min^-1. In both the experiments, modified BG11 medium containing 15.0 g L^-1 glucose along with optimal concentration of both the lipid inducers were used as the starting media.