Determination of C/N Ratio and development of Bioinsecticide Production by Bacillus thuringiensis Using Tofu waste Cultivation Media

Abstract

Development and application of local product biopesticide, especially based on B. thuringiensis (Bt) still has been considerable potential. This is supported by the local availability of Bt local strains and agro-based raw materials are abundant and not expensive. Research toward utilization of industrial waste of tofu as a medium of cultivation of Bt has been done. In the early stages the Bt strains are isolated and selected. Bt B2 bacteria from the culture collection, protein crystals form bipiramid and cubes, are strains with the highest toxicity against C. binotalis. The optimization of media formulation is performed on a scale of 500 ml with a variation of C/N ratio 3:1, 5:1, 7:1, 9:1 and 11:1. B2 strains provides optimum toxicity value at C/N ratio 7:1 with LC50 value of 0.90 mg/L or 8356 IU/mg. The results of cultivation on the bioreactor 3 L shows substrate utilization efficiency of 67.77 % with LC50 value of 3.56 g/ml or 2112 IU/mg. Characterization of δ-endotoxin with SDS-PAGE indicates protein pattern at all media formulations contain genes Cry 1 (144 kD) and Cry 2 (66.80 kD and 55.51 kD) and Cyt1Aa (25.27). Amino acid δ-endotoxin in 500 ml scale cultivation and 3 L bioreactor show the same composition as many as 15 kinds of amino acids but the quantity of amino acids on the 3 L bioreactor scale smaller than which on 500 ml. Determination of physical factors for the increasing scale on the 40 L bioreactor using a base power per unit volume (Pg/V) still provide power value per unit volume (Pg/V) of 0.037 HP/m3 (weak level of agitation impellers), aeration requirement is 0.7 VVM with agitation speed 111.57 rpm. Cultivation at 40 L bioreactor run at a pH range from 6.5 to 7.0 where the total number of maximum cells is 10.35± 0.03 x 10 8 cfu/ml with a maximum cell growth rate of 0.48 ± 0.01 hours-1, while the number of the highest spore of 3.90±0.06 x 108 cfu/ml with a maximum rate of spore growth is 0.25 ± 0.01 hours-1. Substrate utilization efficiency at 68.21 % with LC50 values is 3.19 g/ml or 2357 IU/mg.