Response Surface Methodology for Optimization of Biopesticides Production using Industrial Wastewater as Substrate

GOICOECHEA, HÉCTOR C; AUGUSTO MOREIRA, GUILHERME; ALEJANDRO BECCARIA,

Campinas

Congreso; 10th Internacional Conference on Chemometrics in Analytical Chemistry (CAC 2006); 2006

CAC

Insecticides based on entomopathogens are generally specific and present low or no toxicity to vertebrates. One of the leads to successful production of biopesticides is the development of the medium composition. The objective of this work was to optimize the parasporal crystals production of Bacillus thuringiensis var. kurstaki using an experimental desing of the medium components (substrates) coupled with Response Surface Methodology (RSM) followed by multiple variable response optimization through a desirability function. The strain Bt var. kurstaki HD-1 was provided by Dra. Graciela Benintende, IMYZA, Instituto Nacional de Tecnología Agropecuaria, Argentina, was grown on Tryptic Soy Agar slants (Britania, Argentina) and stored at 4°C. The substrates used were sugar cane molasses (30º Brix in DI water), reduced-fat milk (2.624g.L-1 in DI water with Biochemical Oxygen Demand equivalent to 1915.5mg.L-1) and brewery wastewater (with Chemical Oxygen Demand equivalent to 3473.0mg.L-1 and pH 10.2). Effluents proportion was analysed by using a simplex lattice {3,2} augmented with the overall centroid and axial points. This design has 10 points, with four of these points in the interior of the simplex. Additional replicates and a random point were added in order to increment the number of experiments for modelling purposes. A culture medium TSB was employed as control culture. The responses were analysed by counting, in a calibrated microscope, vegetative cells, spores and parasporal crystals (fixed by flame and coloured by gentian violet) in a 0.28 cm2 slide. Design Expert™ version 6.0.10 trial (Stat-Ease, Inc., Minneapolis, USA) was used for performing experimental design and data analysis. We found that the F values (F tests performed for the responses prediction models) are lower than expected, causing not significance of the models (cubic models). We also found that the F for LOF (lack-of-fit) is also low (p>0.05) which means that instead of the low reliability of the models they still can be fit in order to get an approximation of the optimum medium composition. For this purpose, we used the desirability function (D) for crystals and vegetative cells. The value for D was found to be 0.979 (the maximum value for D is equal to 1) which gives a prediction for the best composition (76.5 % of reduced-fat milk, 22.0 % of brewery wastewater and 1.5 % of sugar cane molasses). The results are the following: 4.5E+8 crystals/mL, 3.3E+8 vegetative cells/mL, 6.4E+6 vegetative cells/mL. Further analysis with Artificial Neural Networks (ANNs) resulted in an optimal mixture as follows: 74% reduced-fat milk, 26% brewery wastewater, 0% sugar cane molasses (D = 0.896). The predicted response for this mixture is: 4.1E+8 crystals/mL, 2.9E+8 vegetative cells/mL, 9.0E+7 vegetative cells/mL. We found an optimal mixture of effluents defined by RSM, although the fitted model presented low reliability. Further analysis made with ANNs showed an optimal composition consistent with the one obtained by RSM but analytically more reliable. This probably was due to the higher complexity of the bioprocesses, the strong interaction between the components and high experimental noise level. We achieved good Bt var. kurstaki growth and good parasporal crystals production, according to our objectives. However, complementary studies should be made to evaluate the efficiency and potency of the products in target larvae. The study could be enhanced with a new screening of combinations of other effluents by using this methodology.   Acknowledgment. Universidad Nacional del Litoral and CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas) are acknowledged for financial support. G.A. Moreira. thanks AUGM for a fellowship. G.A. Micheloud thanks CONICET for a fellowship.