Proses Produksi Bioinsektisida Bacillus thuringiensis Isolat Lokal Menggunakan Limbah Agroindustri pada Kultivasi Media Padat.

Abstract

Due to increased awareness of the need for safe insecticide, currently the use of bioinsecticide is increasingly in demand by farmers who want to produce vegetables and fruits safe for consumption. Also, it is used in the control of mosquito larvae that can be vectors of several dangerous diseases. The use of safe insecticides is needed, so as not to pollute the soil, water and other living creatures. The advantages of using microbial bioinsecticide are safe, environmentally friendly, specific to certain insect pests, not harmful to pets, do not result in residues on crops and soil. The purpose of this research was to develop the production process of bioinsecticide (Bacillus thuringiensis) using local isolates with starter and cultivation media of agroindustrial by products, using solid state cultivation, in order to obtain bioinsecticide that is cheap and easy to produce. The purpose of each stage are to obtained isolate characteristics suitable with the solid state cultivation and suitable with climatic environment in Indonesia, development of starter from agroindustrial waste, design the condition of solid state cultivation production process using agroindustrial waste, and for evaluation and analysis of efficacy in the target insects. The stages of research began from isolate isolation from Attacus atlas and Bombyx mori caterpillar carrions. Isolates further obtained were then characterized and identified to obtain B. thuringiensis, from both isolates obtained, it was then selected one of the most suitable for solid state cultivation. The next stage was the selection of starter medium from agroindustrial waste, followed by a bioinsecticide production using the mixed media of sago lees and iles-iles lees by solid state cultivation. Bioinsecticide produced was characterized by amino acid content protein profiles and toxicity ability against A. aegepty larvae dan C. pavonana larvae. B. thuringiensis producing endotoxins that served as a bioinsecticide active ingredient could be isolated from A. atlas and B. mori caterpillar carrions from Bogor, Indonesia. The isolate characteristics of A. atlas caterpillar are the bacteria belonging to Gram-positive, having flagella, punctiform shaped colonies, slimy, white, rod-shaped vegetative cells with a length of approximately 1700 nm and a width of about 830 nm. The protein crystals yielded are spherical, with a diameter of 470-770 nm, and diameter of spore of 550-750 nm. The isolate characteristics of B. mori caterpillar are, belonging to Gram-positive bacteria, motile, circular, white and slimy, long and round vegetative cells with a length approximately of 3900-9500 nm and a width approximately of 830 nm. The protein crystals yielded are spherical, bipyramid and cuboid with a size of 300-700 nm, and diameter of spore of 500-750 nm. Philogenetic tree reconstruction showed that both isolates are in the same cluster with B. thuringiensis. Of two selected isolates, for bioinsecticide production by solid state cultivation is isolates from A. atlas. Selection of isolate is based on the nature of higher motility characteristic, cell growth, the number of spores produced and their toxicity against the test insects. Starter media is selected based on the growth parameters of B. thuringiensisin tofu whey media, tofu whey plus urea, coconut water, and nutrient broth. It is obtained the highest results in tofu whey with the maximum number of cells of 10.51 (log CFU/mL), the maximum number of spores of 8.67 (log CFU/mL), and the maximum cell growth rate of 0.052 hour-1. Starter media uses tofu whey suitable to be used as the starter media of B. thuringiensis. During cultivation, media thickness and aeration provision effect pH, cell growth, the number of spores, and substrate consumption. increasing thickness of substrate causes pH decrease, and resulting decrease in cell growth, spores and substrate consumption. Like wise, the ability toxicity is decreasing. Aeration provision gives a better effect because it can increase the number of cells, the number of spores and amino acids formed. Toxicity to mosquito larvae and caterpillar larvae are not affected by the number of cells, where as the number of spores shows that the higher the spore is the higher the toxicity value is. It means that the formation of spores is in line with the formation of crystalprotein. Bioinsecticide produced has a toxicity against C. pavonanalarvae (Lepidoptera group) and A. aegypti (Diptera groups), but it is more effective against A. aegypti larvae. The ability of the highest toxicity against A. aegypti larvae is equal to LC50 0.03 mg/mL and against C. pavonana is equal to LC50 0.33 mg/mL.