Description:Figure 1.1 : Wheat barn extract
Figure 1 2 : Cabbage leaves extract
Figure 1.3 : Agriculture waste extract placed in shaker incubator
Figure 1.4 : Removal of ammonium sulphate by dialysis

Bacillus thuringienses
Bio preparation based on spore crystals mixture of Bacillus thuringiensis seems to be good alternative for chemical pesticides. They are environment friendly, do not have any negative Influence on no target animals, including vertebrates and are effective in reducing the number of insect pests. Bacillus thuringiensis is a grampositive bacterium, which produces insecticidal crystal sporulation. It produces proteinaceous inclusion of Bacillus thuringiensis are called as crystals. These crystal proteins are activated by proteases in the alkaline condition of the midgut of insects. These activated toxin binds with the receptor on the brush border membranevesicle of the midgut epithelium and perforate the cell, which leads to ionic imbalance and insect death. Cry2 gene is isolated from Bacillus thuringiensis and used against pathogen from the cotton (Helicoverpaarmigera). Helicoverpaarmigerais a larva which feeds on a wide range of the plants including many important cultivated crops. It is major pest in cotton and one of the most polyphagous and cosmopolitan pest species. The greatest damage is caused to cotton, tomatoes, maize, peas and tobacco.
Biochemical test
TEST RESULT
INDOLE POSITIVE
VP POSITIVE
MR NEGATIVE
STARCH HYDROLYSIS POSITIVE
CITRATE UTILIZATION POSITIVE
CATALASE POSITIVE
Table- 1.a: Biochemical tests result for Bacillus thuringenesis

Bacterial delta-toxin
Bt toxins are a potential alternative to broad-spectrum insecticides. The toxicity of each Bt type is limited to one or two insect orders; it is nontoxic to vertebrates and many beneficial arthropods, because Bt works by binding to the appropriate receptor on the surface of mid gut epithelial cells. Any organism that lacks the appropriate receptors in its gut cannot be affected by Bt. A 2007 study funded by the European arm of Greenpeace suggested the possibility of a slight but statistically meaningful risk of liver damage in rats. The observed changes have been found to be of no biological significance by the European Food Safety Authority. Furthermore, the major advantage of this biocide is that risk of development of resistance of mosquitoes to B. t. based products is very low, due to its multi-toxin complex. The main criterion forB.thuringiensis differentiation from other soil-forming bacteria was crystal production by sporulation cultures.
Bacterial culturing media
In spite of many advantages of Bt based biopesticides, their application is limited due to high production costs. In conventional processes, Bt insecticide is commercially produced on synthetic medium comprising soybean meal, fishmeal, glucose, yeast extract, peptone and trace elements and sometimes more ingredients are added to enhance the sporulation process. The cost of Bt production depends on many factors; however, the raw material cost is one of the most important factors and may comprise 30–40% of the overall production cost . Therefore, selection of growth medium or raw material is critical for commercial production of this biopesticide. Severalraw materials (industrial and agriculture byproducts) have been tested as alternative culture media for the production of Btbiopesticides. Wastewater sludge can be a very good source of carbon, nitrogen, phosphorus, and other nutrients for many microbial processes that could add value to sludge by producing certain valuable metabolic products e.g. endotoxins. Recently, the expensive soya basedsynthetic media for Bt production have been successfully substituted with agro-industrial wastes (maize starch, wheat bran, rice straw, corn steep liquor, etc.) and wastewater sludge. The use of agricultural and vegetable wastes such as cabbage leaves, wheat barn, cactus extract etc is used as main carbon and nitrogen sources for the cultivation of Bacillus thuringiensis and then later the endotoxin is purified with no impurities as the end product.
Optimization of pH and Temperature
pH optimization
Five different pH media (6.5, 6.7, 6.9, 7.2, 7.4) pH were prepared by using NB (Nutrient Broth) glass tube, each one repeated three times, then inoculated with isolated Bacillus thuringiensis and incubated 24hr at 37˚C. The pH was controlled with pH meter by adding tiny drops of NaOH or HCl with constant stirring.
Temperature Optimization
Five NB glass tubes (7.2Ph) were incubated at different temperature (25, 27, 30, 33, 36) ˚C for 24hr. The growth was indicated by using spectrophotometer (600nm), and NB media as blank.
Media Selection
Material preparation
A 30g of wheat bran or rabbit feed were added to 1L distilled water (DW) and boiled for 10 min, then centrifuged at 3000 rpm for 10 min to have less turbid liquid. A 200g cabbage leave, potato, or cactuswas placed in 200 ml boiling DW for 20 min, and then centrifuged (3000rpm, 10min). A 1.0g MnCl2, 20.3g MgCl2, 10.2g CaCl2 was put to 100 ml DW to make 1% stock solution (7.2 pH). As shown in figure 1.1 and figure 1.2.
Media preparation
Collected mixtures of the previous liquid were tested as whole combinations of all trials. All the selected media had a constant pH and so temperature as it determined in the first place, which is 7.2 pH and 30˚C. The pH was controlled with the same procedure (NaOH, HCl, pH meter), and the temperature was set on the shaker incubator.
Sterilization and Cultivation
All media combination for the proper media selection was sterilized at 121˚C for 30 min, then when cooled were inoculated with 200 µl of B. thuringiensis in normal saline and cultivated for 24 hr at 30˚C using shaker incubator. As shown in figure 1.3.
Optimization of pH and Temperature

pH Optimization
The highest absorbance at (600nm) for the growth of the test organism at a temperature of 30˚C was at pH7.2.
Temperature optimization
Different absorbance values (600nm) for bacteria growth resulted for predetermined different temperature, and it was the 30˚C temperature that showed significantly highest one.
Extraction of endotoxin
Precipitation of Endotoxin using ammonium sulphatepurification of bacterial endotoxin using 4M ammonium sulphate solutions and stored for overnight at 40C for the precipitation reaction. The precipitation of certain proteins from a mixture by depends on the concentration of the salt. The Precipitate is obtained through centrifugation; the pellet were collected and taken up for dialysis. (Behn et al., 2001).
Purification of Endotoxin by Dialysis
The precipitated endotoxin obtained from Bacillus thuringenises were transferred to the activated dialysis bag tied after including some air and twisting the open end of the dialysis bag. The bags containing endotoxin contents were dialyzed against buffer S (10mM Phosphate buffer) without NaCl, pH 7.4 . The contents were dispensed into micro centrifuge tubes after dialysis and centrifuged in order to remove the remaining impurities (Behn et al., 2001). The endotoxin was further purified by Ion Exchange Chromatography (Jensenius et al., 1981). As shown in figure 1.4.
Conclusions
The selected media should contribute to a significant reduction of the cost of B. thuringiensisbioinsecticide production and utilization, which extremely expensive in the original media, thatprevent them from use as economical and biological source of insecticide. And also played a significant role for elimination of disease incidence such as mosquito that act as vector of Plasmodium which spread Malaria. In the other hand it consider as suitable alternative to the harmful chemical pesticide, that cause several side effects on the plants, animals and so human being.In our study, we developed a cheap, safe, productive media for optimum production of B. thuringiensis, based on using of locally cost-effective raw materials, that easily to extract and prepare, and most of them consider being waste product. We ended to select the “cactus and seawater” media to be the best media for bacterial growth. , C , C , C , Claims:We claim that,

1. We have developed a low cost media for the development of organic biopesticide using agricultural and vegetable wastes as main sources of carbon and nitrogen for the cultivation of Bacillus thuringiensis.
2. The endotoxin is purified with no impurities and the end product consist of highly purified protein for increasing the efficiency of the product.
3. Only agricultural and vegetables wastes have been used as the medium for the cultivation of Bacillus thuringiensis and no addition of any chemicals is present.
4. Totally natural product with highly purified protein which is non toxic, biodegradable, ecofriendly and with high specificity is developed.