Bioconversion Of Wastes Into Bacillus Thuringiensis Based Biopesticides Under Submerged Fermentation

Bacillus thuringiensis（Bt） is the most widely and successfully used bio-insecticide. It has the characteristics of easily production , specificity and no toxicity to vertebrates , comparing to chemical pesticides which caused“3R”problems when used. However,production of Bt formulations using existing expensive raw material and fermentation technology incurs heavy expenditure. There is one of the primary causes why the use of these biopesticides has limitations. Hence,it is imperative to develop a cheaper media for culturing Bt and providing a feasible technical route,which would facilitate the production of biopesticides in a cost effective manner. The quantity of wastewater generated from brewery is large and is characterized by a high pollution load,and its sustainable management via value-addition is an alternative efficacious route to other disposal options,namely,biological wastewater treatment processes. So far, there is no report about that Bt-based biopesticide production from brewery wastewater and its castoff. In this study, brewery wastewater and its castoff were used as a novel medium to cultivate Bt based on clean-up option as well as augmentation path for commercialization of economical biopesticides.The wastewater formed flow and COD variations days/months of sewage from urban sewage-digestive plant and brewery wastewater-digestive plant were extensively investigated. In the year,the average COD of sewage from brewery wastewater-digestive plant is 1342.0 mg/L which is 8 folds higher than that of sewage from urban sewage-digestive plant. The variability coefficient of months for urban sewage-digestive plant and brewery wastewater is 16.7% and 57.3% respectively, and 42-52.4% and 184.4-391.3% for variation days. Wastewater from brewery wastewater-digestive plant possesed relatively invariable COD and with abundance nutrition and sanitation.Brewery raw wastewater/acidified waste water+yeast fluid （brewery-waste-medium） from different working procedure were found to be suitable for culturing Bacillus thuringiensis strain BRC-WLY1,which could produce spores up to 10⁹cfu/mL and high crystal compound as well as short fermentation cycle （around 20h）. The optium combination is 4/5 （1/2 acidified wastewater + 1/2 brewery raw wastewater）+ 1/5 pretreatmented yeast fluid. Supply carbon and nitrogen is favor to BRC-WLY₁ production. Due to brewery wastewater/sludge/ waste yeast slurry is a cheaper raw material and very easy to obtain,it would be a good material for the industrialisation production of Bt-based bioinsecticide.In order to improve the utilization ratio of brewery wastes and to ease the sterilization with high pressure steam,the methods to pretreat sludge/sewage used in environment engineering were selected to pretreat brewery sludge /sewage. It showed that method A is the top choice. An orthogonal design was furthur used to optimize the condition, including dilution ratio, pH, pretreatment time, and/or the time of sterilization with high pressure steam, for the pretreament of brewery wastewater and sludge, and waste yeast slurry. The optimal condition comprised 6 times diluent of the waste yeast slurry ,pH at 5.0,pretreatment time for 2 min and/or the time of sterilization with high pressure steam for 45min was obtained. By the optimal pretreatment condition, the produce ofα-amino nitrogen after pretreatment improved up to 73%, 64.2% and 198% respectively for brewery wastewater, sludge and waste yeast slurry, and the harvest rate of theα-amino nitrogen for waste yeast slurry is 4.37%.The sewages collected from the different process stages and sewage-digestive plants were used to isolated Bt strains, and 2 strains （named BRC-WLY₁ and BRC-WLY2） were obtained. Besides the investigation of the morphology, biochemical characteristics and fermentation performance, the gene and protein types of the above two strains were analysis by PCR-RFLP with 14 couples specific primers and by SDS-PAGE respectively. The results indicated that both BRC-WLY₁ and BRC-WLY2 harbored the genes of cry1（cry1Ag,cry1Ba,cry1Gb,cry1La） , cry2（cry2Ac） , vip3A and aiiA , and the protein with 65KD. Compared to the two control strains HD-1 and 8010, BRC-WLY₁ and BRC-WLY2 possess shorter fermentation cycle（nearly cut 20%-30%）. The minimum fermentation cycle of BRC-WLY₁ is 15h and its hybrid of crystal and sporangium in the ferment liquid is 0.1312g/25mL. Bioassay results showed that the correction mortalitie at 48h of BRC-WLY₁, BRC-WLY2, 8010 and HD-1 was 96.6%, 100%, 89.7% and 93.1% respectively for 2-3rd instar larvae of Plutella xylostella. To seek the nutritional deficiency factors in brewery-waste-medium for BRC-WLY1,8 nutritional factors were optimized by single factor experiments. Based on the above result, the Plackett-Burman （PB）Methods and the response surface methodology（RSM） were used to further optimize the fermentation medium.The optimal brewery-waste-medium comprised 0.2% of glucose,0.1% of （NH4）2SO₄,0.05% of ZnSO₄,0.6% of NaCl,0.15% of Tuween80,0.4% of material A,30% of pretreatmented yeast fluid , 0.12% of KH₂PO₄ and 1/2brewery waste water+1/2 acidified wastewater was obtained, which could produce spores up to 10.86×10⁸ cfu/mL resulted in a 5.1 times increase of sporangium yield in comparison with the original medium. OD595 increased to 0.258 from 0.194 that of fermentation without optimized.The single factor experiment were used to optimize fermentation conditions and the optimal conditions comprised initial pH at 7.5-8,80 mL of liquid volume in the 500mL flask,fermentation temperature at 30-34℃and 5% of inoculation volume was obtained . The optimal fed batch mode is fedding 10%（V/V） of yeast fluid after culturing for 8 h,which resulted in spores yield, crystal weightiness, production intensity and yield of units sugar increased 8, 1.78, 0.98 and 3.07 times than those of fermentation without fed batched.In conclusion,BRC-WLY1 and BRC-WLY2 isolated from sewage/sludge are suitable to grow in brewery-wastewater-medium. It suggested that brewery wastewater could be used for the industrialisation production of Bt based bioinsecticide,which would effectively utilize the bio-organic waste materials from the environment,on the one hand, and reduce the cost the Bt formulation on the other.