Nghiên cứu tuyển chọn vi khuẩn bacillus thuringiensis phục vụ tạo chế phẩm diệt côn trùng bộ hai cánh (diptera) tt tiếng anh

MINISTRY OF EDUCATION MINISTRY OF AGRICULTURE AND TRAINING AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES PHAM THUY DUONG STUDY ON SELECTION OF BACILLUS THURINGIENSIS STRAIN FOR DIPTERAN INSECTIDAL PREPARATION PRODUCTION Major: Biotechnology Major code: 42 02 01 SUMMARY OF THE AGRICULTURAL DISSERTATION Ha Noi – 2019 The work is completed at: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES Science instructors: Academic advisor 1: Assoc Prof Ngo Dinh Binh Academic advisor 2: Dr Le Duc Khanh Reviewer Reviewer Reviewer The dissertation will be defended before the State-level thesis judge council meets at … ……………………………………………………………… ……………………………………………………………… The thesis can be found at the library: - Vietnam National Library - Library of Vietnam Academy of Agricultural Sciences INTRODUCTION The urgency of the research Bacillus thuringiensis (B thuringiensis) is soil, positive-gram bacteria.The ability of this bacteria is to produce toxic crystal proteins against insects but not human, ecology and usful organism.Studying on pesticidal activites against dipteral species of B thuringinensis isolated in Vietnam is essential to find the potential isolates with the native, strong toxic gene to produce the biological product against dipteral species The insect species belonged to dipteral order is one of the largest order in the insect class with the variety of numbers and species Diptera order is very diverse in terms of ecology and mainly harmful agents in agriculture (fruit flies) or healthy like flies, mosquitoes They are also the vector of many diseases between people and people or between animals and people Mosquito is highly lethal transmission such as Anopheles gambiae is the vector of malaria Aedes aegypti transmits dengue fever Culex tritaeniorhynchus infects Japanese encephalitis while flies are the transmitting agents about 100 diseases but mainly dangerous diseases such as polio, pain trachoma, hepatitis (A, E), Rickettsiale regression fever, dysentery, cholera, typhoid, Streptococcus and Staphyloccocus Today, there are many different methods for control this kind of insects which were applied The most commonmethod is the insecticide However, these drugs are quite expensive, are produced from chemical agents causing pollution and bad health effects on human, animals but not eradicating the pathogen Starting from the above practice we conduct research on the topic: “Study on selection of Bacillus thuringiensis strain for Dipteran insectidal preparation production” Objects The main purpose of this topic is to increase the knowledge of two species P heterotremus and P westermani, provide a scientific basis for diagnosis and prevention of lung fluke dieasease, contributie to public health protection The specific objects: 2.1 General goal Selection of indigenous Bacillus thuringiensis strains produce Toxic crystalline proteins against insects belonging Diptera order; Expression, purificationcrystal proteinin E coli bacteria for intensive research; And produce biological production of Bacillus thuringiensis against house flies (Musca domestica) from brewery wastes Screening and collection of Bacillus thuringiensis strains which are capable of producing cry2A coding genesagainst dipteral species soil and leaf samples in some provinces of Vietnam and identify Express and purify recombinant protein (rCry2A) in E.coli BL 21 (DE3) Find out the method of treating beer malt waste to produce culture environment for B thuringiensis bacteria and study of factors affecting grow and Crystalline protein synthesis of B thuringiensis when using malt beer as a culture medium at the laboratory scale At the same time, preliminarily evaluatethe effect of insecticidal insecticide of BT product against dipteral Research content - Isolating and identification biological characteristics of some strains B thuringiensis (Bt) has insecticidal activity - Cloning and sequencing the cry2 gene of Bt strains, isolated from soil, leaves, having the pesticide activity against dipteral insect - Expression Cry2 protein from strain of B thuringiensis selected - Studying methods for beer malt waste treatmentto make the culture medium for B thuringiensis fermentation Optimizing the culture medium for fermenting B thuringiensis from beer malt - Study the factors affecting growth and cry protein biosynthesis of B thuringiensis when using beer malt ascultural medium at the laboratory scale - BT fermentation to of biological products against dipteral and evaluated the insecticidal effectiveness against dipteral insect in laboratory and field New contributions of the topic - This study is one of the systematic studies about genes cry2A of B thuringiensis in Vietnam - Cloning and successful expression of cry2A gene in E coli BL21 (DE3) - Using beer malt wastewhich is waste of beverage manufacturing industry as materials to provide nutrients (carbon, nitrogen, minerals ) for B thuringiensis fermentation to production BT bio-pesticides Optimizing the cultural condition for bacteria B thuringiensis from the brewer's malt waste by response surface methodology to produce biological production against dipteral insects Scientific and practical meaning of the topic 5.1 Scientific meaning: A systematic study of the cry2 gene is a reliable theoretical basisfor further studies on B thuringiensis in common and cry2 gene in particular Based on the evaluation of the effect of the gene cry2 protein on insecticidal ability against diptera like flies, this study was paved the way to create biological products 5.2 Practical meaning: The results of the thesis contribute to the effective exploitation of microbiological gene sources in general and the cry2A gene of B thuringiensis in particular contributed produce biological productionagainst dipteral insects On the other hand applying Beer malt a waste of industrial manufacturing asthe material for the fermentation process is needed This research is to directly improve the value of beer malt waste and produce the environmentally friendly products for sustainable agricultural production Thesis structure Thesis is 149 pages (excluding references and appendices) computer with A4 size with 23 tables and 43 figures The thesis consists of parts: Introduction (04 pages); literatural review (40 pages); Materials and methods (19 pages); Result and discussion (60 pages); Conclusion and prepective (02 pages) Referenced 146 documents including 20 Vietnamese documents and 126 English documents CHAPTER OVERVIEWAND BASIS OF SCIENCE OF THE THESIS The thesis has consulted and summarized the English and Vietnamese documents related to issue: Overview of B thuringiensis; overview gene cry2; Overview of E coli expression system; Overview of test insects experience; B thuringiensis bio-pesticide ; Overview of Malt beer; and the situation of research and application of B thuringiensis bacteria in Vietnam In it, Bacillus thuringiensis (B thuringiensis) is soil, positive gram bacteria During the development process, B thuringiensis is to capably produce main insecticidal toxin including Cry (crystal endotoxin ), Cyt (cytolysin) and Vip (vegetative insecticidal protein) The cry2 genes encode crystal proteins of about 65 - 71 kDa, produced by some subspecies of B thuringiensis such as : kurstaki (HD-1, HD-263, NRD-12 and 14 other lines), thurigiensis, tolwwarthi, israelensis, kenyae (Dwu et al., 1991; Ohba and Aizawa, 1986; Yamamoto, 1983) So far, more than 80 cry2 genes have been identity (http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/ B thuringiensis /toxins2.html ) Flies and mosquitoes are parasitic arthropods, belonging to the class of insects (Insecta), order Diptera They are many intermediaries vector of many dangerous diseases between people and people or between animals and people Further, weather conditions and climate in Vietnam are very favorable for their development There are many different methods applied to control the harmful insects The most common method is using chemical toxin against flies and mosquito However, these drugs are usually too expensive It is also easy to pollute the environment and affect to the health of humans, animals, but not eradicating the pathogen These key points are presented in Chapter Thereby, it is easy to found that the study about pesticidal activities against dipteral larva of B thuringiensis strains isolated in Vietnam is necessary Furthermore, finding out strains that carry natural genes having strong activity is essential to produce biologicalproducts against dipteral insects CHAPTER MATERIAL, LOCATION AND METHODS 2.1 Material and location Soil and leaf samples used to isolate Bacillus thuringiensis were taken from Dien Bien, Hanoi, Nghe An, Nha Trang (Khanh Hoa) and Lam regions Dong, Ho Chi Minh City, Kien Giang Beer malt is taken from the Saigon - Hanoi brewery belonging to the public park Small and medium enterprises in Bac Tu Liem district Larvae of house flies age 2; Fruit flies received from the Insect Department - Plant Protection Institute Anopheles minimus mosquito larvae , Aedes aegypti, Culex quinquefasciatus provided by the Central Institute of Malaria parasites E coli strains DH5α for gene cloning, E.coli BL21 for the expression of cry2A gene from The strain is same provided by Seed Breeding Center and Conservation of Microbiological Genetic Resources, Public Institute Biology Sequence of cry2A amplifiers Location and practice time The experiments were performed at the Seed Center laboratory and Preservation of microbial gene sources, Institute of Biotechnology, Department of Microbiology School - Institute of Environmental Technology, Department of Insects - Institute of Real Protection Materials, Laboratory of Department of Biotechnology and Environment-School Oriental University Experimental implementation period from June / 2012-December / 2016 2.2 Methods 2.3.1 Microbiological method The method of isolating Bacillus thuringiensis B thuringiensis bacteria were isolated soil samples and leaf samples collected according to the method of Thiery and Frachon (1997) Classification of B thuringiensis bacteria by free serum method Translate 2.3.2 Biological activity testing method Quantify spore and crystal density The isolated Bt strain was planted with carpet on a peptri containing lips MPA school The number of spores is calculated according to the formula (Ohba et al., 1986) Determine LC50 for test insects Testing the ability to kill the larvae Two wings on the larvae Musca domestica housefly, fruit fly larvae age of two, mosquito larvae under Thiery and Frachon's methods at two concentrations are 10 and 10 spores / ml with house flies, fruit flies and 10 and 10 spores / ml with mosquitoes Each concentration is tested Experiment with plastic cups (10 cups per each cup) The rate of dead larvae is calculated according to Abbott formula (Abbott, 1925) 2.3.3 Molecular biological method Method of extracting plasmid DNA from bacteria Bacterial plasmid DNA is extracted and purified by kit GenJET TM Plasmid Miniprep (Fermentas) PCR method for amplifying cry2A gene DNA biosynthesis was conducted by PCR (Polymerase technique) Chain Reaction) (Sambrook, 2001) Method of isolating the cry2 gene stream Cut DNA by cutting enzyme restriction DNA pairing with T4 DNA ligase Transform recombinant plasmid into E coli DH5 α Electrophoresis on agarose gel DNA was detected and relatively quantified by the above electrophoresis techniqueagarose gel according to the method of Sambrook & Russel, 2001 Purify DNA from agarose gel The DNA product is purified from agarose gel with GenJET kit TM Gel Extraction (Fermentas) according to the manufacturer's instruction manual Method of determining the nucleotide sequence of the cloned gene segment The DNA sequence is determined according to the method of Sanger and his colleagues DNA samples were submitted for reading at Microgen, Korea using primers M13 Processing sequencing results with Bioedit software, comparing gene sequences separated the lines with the sequences published in the International GenBank only online: https://blast.ncbi.nlm.nih.gov/Blast.cgi Expression vector design Use pET22b (+) vector as an expression vector The cry2A gene is given Enter pET22b (+) in the position BamHI and Xho I The amino acid sequence of the protein recombinant includes the entire amino acid sequence of the natural cry2A gene by 6amino Histidine acid at the beginning of C and 36 amino acids at the beginning of N Genetic expression method Activating a colony in LB environment with ampicillin supplemented The final concentration is 50 μ g / ml, shaking at 37 o C overnight Then transplant 1% into 20 ml of MPB medium supplemented with ampicillin antibiotic concentration of 50 μ g / ml and shaking at 37 o C for OD to 0.5-0.8 IPTG sensor (100 mM) reached 1mM, raised at 37 o C for hours Electrophoresis on polyacrylamide gel 12.6% for product testing Expressive products 12.6% polyacrylamide gel electrophoresis method according to the method by Hoefer et al (1994) Western blot method Protein samples after testing with SDS-PAGE on acryamide 12% gel will transferred to PVDF film Protein samples will be incubated with specific antibodies and antibody detection Then detected by solution containing substrate peroxidase (Sigma) Recombinant protein purification method Recombinant protein is purified by chromatography Ni2+ column agarose according to rules Guidelines for Invitrogen 2.3.4 Optimization of fermentation environment according to the surface response method Prepare to breed Bacteria are raised on sterile MTCS environment Shaking at 30 o C, 200 cycles / minute, breeding time 8-10 hours Cultured culture (contains cells being in the growth stage) is used to breed for experiments next (Adjiable et al., 2009; Yezza et al., 2006) Microbial fermentation method Bacillus thuringiensis in the triangle Ferment in 500 ml triangle flask at a temperature of 30 ± 0.1, shaking speed 200 round / minute, 48-hour culture period (Yezza et al., 2006) Fermentation in a 10-liter fermentation device Put 10 liters of medium into the fermentation tank, sterilize at 121 o C, at, in Within 30 minutes, cool to room temperature, insert the fermentation vessel into the device yeast Set up fermentation parameters: temperature 30 ± 0, o C, pH7 ± 0,1, stirring speed 300 rpm, DO> 25 mg / l, air flow rate of 2-4 liters / minute (Luong Duc Pham, 2008; Avigone et al, 1992) Hydrolysis of beer residues The beer residue after being taken to the laboratory is milled and processed Microbial culture materials by combined pyrolysis method pH (Nguyen Thi Van Trang et al, 2012; Valo et al, 2004) Experimental layout The hydrolysis of beer residue after treatment is taken to analyze the chemical composition follow standard methods In which TOC is determined by method SMEWW 5310B-2005; TN, TP is determined by method EPA-352.1 and EPA-365.2, metal components are determined by mode France SMEWW 3125-2005 (Adjiable et al, 2009) Study the effect of solids concentration on the growth process and crystalline protein biosynthesis of MSS8.4 strain Determine the additional nutrient source for the hydrolysis of beer residue Evaluate the independent impact of nutritional factors on growth and the production of crystalline proteins Optimize the environmental composition by surface response response method The description is done according to the method of Rajesh et al (2012) After determine the impact range of each element on the growth and total fertility protein combination of MSS8.4, import data into expert Design software 10.1 Data processing CHAPTER RESULT AND DISCUSSION 3.1 Isolation and selection of efficacy resistant strains of double-winged insects Bacillus thuringiensis strains 3.1.1 Isolation of Bacillus strains For isolation and selection the Bt strain having insecticidal activity against Diptera, 317 soil and leaf samples were collected in provinces from regions Vietnam These sites have never applied Bt product before In 317 soil samples and leaves, 198 samples carried Bacillus bacteria and 119 samples were not isolated Bt Thus the frequency of Bacillus appears 62.4% Location with high rates of Bacillus in the sample included Kien Giang (75.0%), Dien Bien (72.8%) and Hanoi (72.8%); The lowest bt isolation rate is Nghe An (33.3%) Of the 1,020 colonies having characteristics specific for genus Bacillus, there are 440 Bt group colonies are based on the ability to form crystals (accounting for 43%) This ratio depends on the number of samples collected in the provinces, the highest in the collected samples in Hanoi (54%) and Ho Chi Minh City (53%), the lowest in the sample collect in Lam Dong (25%), Dien Bien and Kien Giang provinces for similar ratios each other (42% and 44%) Soil samples were collected in geographic regions of Vietnam with Bt frequency higher than previous publications (Martin and Travers, 1989; Infantry and plus The, 2005) The frequency of Bt in this study was 62.4% in accordance with previous publications by Martin and Travers (1989), in the soil sample New Zealand (Chilcott and Wigley, 1993), in the Korean soil (Lee and partner, 1995) and in the Japanese sample (Ohba and Aizawa, 1986) The appearance of Bt is almost everywhere in the living environment, including: soil agriculture, forest land, urban land, mangrove land, even desert (Dulmage and Aizawa, 1982; Martin and Travers, 1989; Zhang et al, 2000; Uribe and plus The, 2003) In this study, the appearance of Bt was concentrated mainly in Hanoi urban land and high population density This result is much higher than research of Quesada-Moraga (Quesada-Moraga et al., 2004), appropriate with the results of Ayyasamy (2012) According to Ayyasamy, the ratio of Bt in urban land is 80%, however, the highest frequency appears in agricultural land (Ayyasamy and partner, 2012) Thus, compared to the data published by the above authors, the frequency appears Bt and the Bt index that the topic isolates are quite high It means that the number of Bt species in Vietnam is very rich This finding indicated that Bt collected in Vietnam can provide many precious genetic resources 3.1.2 Research on crystalline proteins The strains of B Thuringiensis isolated in geographical regions of Vietnam Male (198 strains), was cultured on MPA environment After days this samples were staining with fushin dye and directly observated on optical microscopes in oil glass objectsto determine the crystal shape of micro strains bacteria Results from 196 isolates have the ability to produce crystals in all shapes format There are strains capable of producing 1-3 types of crystals Specifically, 186 strains pyramid shaped crystals accounted for 42.2%; 193 spherical crystals accounting for 43.8%; 46 sets BamHI and XhoI were also indicated that the Cry2A structure were successfully constructed 3.2.3.2 Expression cry2A in E coli Preliminary expression of cry2A gene Result of total protein electrophoresis on SDS-PAGE gel showed that the induced clone E coli BL21 (DE3) containing expression vector pET22b(+)-cry2A generated a bold protein band with size approximate 70 kDa, completely coinciding with the expected size of fusion form of Cry2A protein Thus, it can be concluded that the expression of cry2A gene was successful Confirm recombinant Cry2A by Western blot analysis The results of Figure 3.17 indicated that the fusion protein was expressed a band with the precise size (about 70 kDa) This can be assured that Cry2A protein was successfully expressed in pET22b (+) Figure 3.17 Analysis of expression of Cry2A protein by Western blot M: Protein marker (Biobasic); 1: Positive control (Sigma); 2-4: Samples after induction at 1h, 2h, 5h; 5: Empty vector In order to be able to produce Cry2A products that have insecticidal activity against Diptera, protein products need to be further processed and evaluate insecticidal activity on insects Table 3.3 Bioassay of the insecticidal activity of rCry2A and crystal protein from B thuringiensis with M domestica larvae CI 95% Larvae Protein LC50 (g) Min Max M 4D4 442,5 11,3 56,5 domestica MSS8.4 283,5 17,6 100 rCry2A 264,7 28,3 66,1 MSS8.4+rCry2A 268,6 27,8 59,3 Bioassay of insecticidal activity after 72 hours showed that recombinant protein was highly active with M domestica larvae (LC 50 = 264.7 µg) Simultaneously, the activity of the natural Cry2A protein of MSS8.4 (LC 50 = 283.5 µg) was also higher than that of the 4D4 standard strain (LC 50 = 442.5 µg) The difference in insecticidal ability can be found in the MSS8.4 strain not only form Cry2A toxin but also from Cry1A toxin 3.2.4 Study onoptimal gene expression conditions 3.2.4.1 Optimizing the temperature in cry2A gene expression E coli BL21 cells were inoculated at different temperature conditions: 28, 30, 37, 40oC After hours of culture, cellswere harvested, run electrophoresis on 12.6% polyacrylamide gel The results showed that at 37°C, the cell expressed the darkest protein band Therefore,the temperature of 37oC was chosen for rCry2A expression 3.2.4.2 Optimizing the concentration of inducer in cry2A geneexpression Study the concentration of inducer IPTG at 0.1; 0.2; 0.5; 1; 1.5 mM The results showed that the most optimal level of Cry2A protein was produced when induced at 1mM 3.2.4.3 Optimizingthe inducition time in cry2A gene expression Fixed the induction temperature at 37oC, an IPTG-induced concentration of mM and investigated recombinant protein expression by time (1, 2, 3, 4, hours) Samples were treated and run electrophoresis on 12.6% polyacrylamide gel As a result, the amount of recombinant protein produced was increasedgradually by time, and got the highest levelafter and hours Therefore, the induction time of hours was chosen to harvest samples to ensure product quality and fermentation costs 3.2.5 Purification of recombinant protein by Probond Nikel Resin affinity chromatography column Recombinant strain was expressed at 37oC, mM IPTG, and harvesed after hours The cell biomass was then collected by centrifugation, treated and purified SDS-PAGE electrophoresis result showed that recombinant Cry2A protein was purified with size of 70 kDa 3.2.6 The insecticidal activity against Diptera of rCry2A and the strain MSS8.4 Table 3.4 Insecticidal activity of crystalline protein from MSS8.4 strain and recombinant rCry2A protein Larvae M domestica A minimus A aedes Protein LC50 (g)* CI 95% Min Max 442.5 11.3 56.5 MSS8.4 283.5 17.6 100 rCry2A 264.7 28.3 66.1 4D4 9.42 53.33 70 MSS8.4 7.34 56.67 83.33 rCry2A 7.03 70 93.33 4D4 19.05 6.67 63.33 MSS8.4 14.52 56.67 73.33 rCry2A 13.33 63.33 80 19.05 36.67 46.67 MSS8.4 17.6 70 90 rCry2A 56.67 86.67 4D4 Cx quinquefasciatus 4D4 14.52 The insecticidal activity of crystalline proteins was determined based on the ability of killingDiptera larvae: M domestica, A minimus, A aedes, Cx quinquefasciatus Tested at concentrations ranging from 0.5 to 500 µg/g, the results were determined and analyzed by Probit analysis Bioassay results were displayed in Table 3.4 Thus, the recombinant Cry2A protein has been successfully expressed in E coli and revealed insecticidal activity against Diptera larvae higher than the natural protein as well as the standard strain did.This resultswere much higher than that of Misra et al., purified Cry2Aa4 protein by affinity chromatography hadLC50 values in the range of 100-500 mg (Misra et al., 2002); Yilmaz et al (2017) indicated that Cry2Aa18 expressed from cry2A gene of B thuringiensis SY49-1 can kill C pipiens larvae (LC50 = 630 µg/ml) Compared to the study of Reyaz and Arulsel (2016), recombinant Cry2AI1protein after purification was active for Spodoptera litura and Helicoverpa armigera (LC50 = 2,448 µg/ml) and rCry2A activity was much lower for Helicoverpa armigera (LC50 = 3.374μg/ml) 3.3 Environmental selection and optimal fermentation increase the ability of crystal protein formation of MSS8.4 strains from beer waste 3.3.1 Treatment beer waste as raw material for culture of MSS8.4 3.3.1.1 Influence of pre-treatment methods of beer wasteas raw material for fermentation of MSS8.4 When used as raw material for fermenting bacteria MSS8.4, beer wastewas grounded and adjusted to a concentration of 2% solids The experiment was carried out in a triangle flask at 30°C, 48 hours and shaking rate of 200 rpm.The analyses were presented in Table 3.5 The analytical results showed that using acid hydrolyzate generated the highest density of TCC, SC and crystal protein, the cell density was reached 4.9x108 CFU/ml; meanwhile, an experiment using sterile beer waste (TN3), the TCC only reached 107 CFU/ml Thus, pretreatment of beer waste by heat acid and alkaline methods has increased the concentration of nutrients in the medium which helps MSS8.4 grow better This results were also suitable to that of Brar and colleagues, when hydrolyzing organic compounds at high temperatures, it will increase the nutrition contents in the medium Therefore, the cell density and spores of MSS8.4 bacteria on the heat-acid-method-hydrolyzed beer wastemedium were higher than in other media (Brar et al, 2005) The ability of producing toxic crystals ofMSS8.4also differed significantly in different experiments In the experiment using sterile beer waste, after 48 hours of fermentation of MSS8.4, delta-endotoxin concentrations reached 319 mg/l Meanwhile, in the experiment of using pretreatment beer waste, delta-endotoxin concentration reached 428 mg/l, which 1.3 times higher than the experiment using sterile beer waste Therefore, the heat acid method was chosen as the pre-treatment method of beer waste as the culture medium of MSS8.4 3.3.1.2 Analysis of the composition of the beer waste by using heat acid method In order to evaluate the content of substances in the hydrolysis of beer waste, the beer manure hydrolysis by heat acid method was sent to the Department of Environmental Quality Analysis, Institute of Environmental Technology for analysis The analytical results show that in the hydrolysis of beer waste by heat acid method, the contents of C and N (the two most important substrates in the growth of bacteria) were much higher than that of beer waste hydrolyzed by the heat alkaline or sterile method This result was completely consistent with the above observation, given that the acid agent in high temperature condition facilitated to increase the ability of decomposing high molecular compounds, increasing the amount of organic compounds readily absorbed in hydrolyzate Mineral elements are indispensable factors during the growth and development of bacteria in general and Bt strains in particular The metal ions such as Mg, Mn, Fe, Zn, Ca, etc have important regulating effects on growth, formation of spores as well as biosynthesis of insecticidal crystal proteins Therefore, the synthesis medium of bacteria fermentation is often supplemented some minerals with the concentration as follows: 0.3% MgSO4.7H2O; 0.02%o MnSO4.7H2O; 0.02%o FeSO4.7H2O; 0.2%o ZnSO4.7H2O and 1.0%o CaCO3 (Ngo Dinh Binh, 2000) Considering this mineral needs of bacteria, no element in all three experiments meets the mineral needs Therefore, additional minerals must be added to the beer waste hydrolysis to improve fermentation medium quality for bacteria MSS8.4 However, according to the study of Ozkan and colleagues: at a concentration of 10-6M, Mn is a key factor which affects the biosynthesis of Bt bacteria without impacting to other cell processes In the beer manure hydrolyzed by heat acid method, Mn concentration of 0.136 mg/l (2.7x10 M/l) is the concentration in a suitable rangefor fermenting Bt bacteria to obtain delta endotoxin toxin (Ozkan et al, 2003) According to a research by Iỗgen and colleagues, the growth, formation of spores and biosynthesis of crystal proteins of Bt bacteria depend not only on carbon and nitrogen nutritional factors but also greatly influenced by the mineral factors Specifically, when supplementing Mg with the concentration from 8x10-5M to 4x10-3M, cell density and crystal protein concentration increased strongly (Iỗgen et al., 2002) According to the analysis results, the concentration of Mg in beer waste hydrolyzed by heat acid method was 12 mg/l (2.9x10-3M).It is the concentration of Mg in the range suitable for growth and producing toxins of Bt bacteria follow the study of Iỗgen 3.3.1.3 Determine the concentration of beer waste for fermenting bacteria MSS8.4 The fermentation experiments of MSS8.4 using beer waste were carried out at the following concentrations: 1% TS; 1.5% TS; 2% TS; 2.5% TS; 3% TS; 3.5% TS The research results show that the percentage of dry matter of beer waste in the range of 2-3 was suitable for the development of Bt At low concentrations (1% TS) the bacterial density in the sample after 48 hours of fermentation was only 2.9x107 CFU/ml, while at the concentration of 2.0 - 3.0% TS, cell density after 48 hours reached over 108 CFU/ml In the experiment 6, when increasing the concentration of beer waste to 3.5%, the density of cells obtained after 48 hours of fermentation was not high, only 107 CFU/ml Crystal protein is produced simultaneously with spore formation and is a desirable product in the fermentation of MSS8.4 bacteria Therefore, this is a critical indicator of product quality Results in Table 3.6 showed that at the concentration of 2.5% beer waste, the crystal protein concentration achieved the highest Thus, in terms of overall, the concentration of 2.5% TS was the most suitable for fermenting MSS8.4 to collect toxins againstneonatal fly larvae The concentration of 2.5% TS will be used for all subsequent studies 3.3.2 Assessment of the independent effects of nutritional factors added to the hydrolysis of beer waste to the growth and toxic crystal production of bacteria MSS8.4 3.3.2.1 Determine the nutrient source added to the beer waste medium The experiment was carried out in a flask, fermentation time of 48 hours, shaking speed of 200 rpm Determination of cell density, spore and crystal protein concentration after 48 hours of fermentation The results of assessing the effect of additional organic compounds on the hydrolysis of beer waste showed that rice bran and soybean powder were two factors that have positive effects on growth as well as the formation of toxic crystals of the strain MSS8.4 According to research of Nguyen Thi Hoai Tram and colleagues, soybean powderwas the best source of nutrition for the growth of Bt to produce toxins (Nguyen Hoai Tram, 2004) This result is also consistent with the results reported by Nguyen Thi Hoa and colleagues, when adding rice bran and soybean powder to hydrolysis of bio mud waste as a medium for fermenting Bt bacteria to obtain toxin (Hoa et al., 2014) In two nutrient sources of rice bran and soybean powder, soybean powder has better solubility, so soybean powderwas chosen as an additional nutrient source for hydrolysis of beer waste When considering the crystal protein content in fermentation of MSS8.4 after 48 hours of culture, there was a clear difference when adding MgSO4.7H2O and MnSO4 to the hydrolysis of beer waste In two additional experiments with 0.5 g/l MgSO4.7H2O and 0.05 g/l MnSO4, the amount of crystal protein obtained after 48 hours of fermentation increased about 10% compared to the control of hydrolysis of beer waste, deta endotoxin content reached 469 mg/liter and 461 mg/liter, respectively This research result is completely consistent with the results of Braun's study in 2000, Icgen and colleagues when studying the role of Mg2+ in the growth and biosynthesis of crystal proteins These authors have shown that the deficiency of Mg2+ in the fermentation medium will reduce the ability of growth and spore formation in fermenting fluid, but the most significant effect is reduced ability to synthesize Crystal toxin Mg2+ is the core that controls enzyme activity, the presence of Mg will affect spore formation and crystal protein biosynthesis (Braun S, 2000; Icgen and colleagues, 2002) The results displayed on Table 3.9 showed that Mn2+ is one of two factors that strongly influence to the ability of crystal protein synthesis This result is also consistent with the judgment of Ozkan and colleagues when it is suggested that Mn is a key factor affecting the biosynthesis of crystal protein in Bt without affecting other cellular processes (Ozkan et al, 2003) According to research by Subbiah and colleagues, when studying the production of mosquito-killing products from B thuringiensis subsp Israelensis, the addition of Mn2+ to the feathered nutrient medium significantly increased the crystalprotein content of fermented Bt broth (Subbiah et al., 2012) Based on the above research results, soybean powder, MgSO4.7H2O and MnSO4 are selected elements as nutritional factors to improve the fermentation medium of MSS8.4 on the basis of hydrolysis of beer waste 3.3.2.2 Evaluation of independent effects of selected factors on growth and biosynthesis of endotoxin delta of MSS8.4 After assessing the independent effect of selected factors on the growth, spore formation and crystal protein biosynthesis, the manganese concentration in a range of - 0.1 g/l, magnesium from - 1,0 g/l; soybean powder from 0-5 g/l has a strong effect on the growth, biosynthesis of crystal protein of MSS8.4 when added to hydrolysis of beer waste 3.3.3 Optimizing the fermentation medium for MSS8.4 strains from beer waste 3.3.3.1 Building the optimal model via response surface methodology The composition of supplementary substances is determined by response surface methodology with the survey areas of the influencing factors as follows: concentration of Mn (A) 0.0 - 0.1 g / l; concentration of Mg (B) - 0.8 g / l; concentration of soybean meal (C) - g / l By Design expert software ver 10.1 obtained a matrix of 20 experiments for influencing factors The experiments were repeated times to get the average results of the repetitions put into statistical processing by expert design software software 10.1 The results of regression analysis of the model show that: p value of