Statistical elicitor optimization studies for the enhancement of azadirachtin production in bioreactor Azadirachta indica cell cultivation

Suspension culture of Azadirachta indica produces the biopesticide azadirachtin. Some elicitors (salicylic acid, chitosan, jasmonic acid, methyl jasmonate, yeast extract and yeast extract carbohydrate fraction) at different concentrations were added in shake flask suspension culture of A. indica. Chitosan, salicylic acid and jasmonic acid stimulated the highest increase in azadirachtin content, which ranged from 2 to 3-fold greater than the control. The combined effect of these elicitor(s) on azadirachtin content was then studied by Response Surface Methodology. A synergistic effect of these elicitor(s) on azadirachtin production resulted in 5-fold higher azadirachtin production (15.9 mg/g DCW versus 3.2 mg/g in control cultures). Exposure time studies with elicitor(s) addition on 8th day revealed that highest azadirachtin accumulation reached after 48 h of combined addition of elicitor(s) (17.4 mg/g). Cultivation of A. indica cells was also carried out with combined (statistically optimized) elicitors addition on 8th day in Stirred Tank Bioreactor. This led to more than 3-fold greater azadirachtin accumulation (161.1 mg/l) as opposed to control bioreactor with no elicitor addition (50 mg/l) in 10 days of cultivation period. The present study not only identifies the elicitor(s) and their respective concentrations for enhanced azadirachtin synthesis but also establishes the role of combined elicitors to improve secondary metabolite production of plant cell cultures more efficiently.