Production of Biopesticides in an In Situ Cell Retention Bioreactor

Abstract

The seeds of Azadirachta indica contain azadirachtin and other limonoids, which can be used as a biopesticide for crop protection. Significant variability and availability of seed only in arid zones has triggered biotechnological production of biopesticides to cope up with its huge requirement. Batch cultivation of A. indica suspension culture was carried out in statistically optimized media (25.0 g/l glucose, 5.7 g/l nitrate, 0.094 g/l phosphate and 5 g/l inoculum) in 3 l stirred tank bioreactor. This resulted in 15.5 g/l biomass and 0.05 g/l azadirachtin production in 10 days leading to productivity of 5 mg l(-1) day(-1). Possible inhibition by the limiting substrates (C, N, P) were also studied and maximum inhibitory concentrations identified. The batch kinetic/inhibitory data were then used to develop and identify an unstructured mathematical model. The batch model was extrapolated to simulate continuous cultivation with and without cell retention in the bioreactor. Several offline computer simulations were done to identify right nutrient feeding strategies (with respect to key limiting substrates; carbon, nitrate and phosphate) to maintain non-limiting and non-inhibitory substrate concentrations in bioreactor. One such continuous culture (with cell retention) simulation was experimentally implemented. In this cultivation, the cells were propagated batch-wise for 8 days. It was then converted to continuous cultivation by feeding MS salts with glucose (75 g/l), nitrate (10 g/l), and phosphate (0.5 g/l) at a feed rate of 500 ml/day and withdrawing the spent medium at the same rate. The above continuous cultivation (with cell retention) demonstrated an improvement in cell growth to 95.8 g/l and intracellular accumulation of 0.38 g/l azadirachtin in 40 days leading to an overall productivity of 9.5 mg l(-1) day(-1).