OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)

OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)

goai VIETNAM NATIONAL UNIVERSITY, HANOI VNU UNIVERSITY OF SCIENCE FACULTY OF BIOLOGY Nguyen Thi Ngoc OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCC-B-51 TO ACHIEVE HIGH BIOMASS YIELD Submitted in partial fulfillment of the requirements for the degree of Bachelor of Science in Microbiology (International Standard Program) Hanoi – 2017 VIETNAM NATIONAL UNIVERSITY, HANOI VNU UNIVERSITY OF SCIENCE FACULTY OF BIOLOGY Nguyen Thi Ngoc OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCC-B-51 TO ACHIEVE HIGH BIOMASS YIELD Submitted in partial fulfillment of the requirements for the degree of Bachelor of Science in Microbiology (International Standard Program) Supervisor : MSc Hoang Van Thai Dr Pham The Hai Hanoi - 2017 Acknowledgements I would like to express my deepest appreciation to all those who have provided me the possibility to complete this research First and foremost, I would like to express my sincere gratitude to my advisors MSc Hoang Van Thai who directed me, guided me to take the first step in scientific research and he always made every effort in terms of equipment and chemicals in time Furthermore, my sincere thanks also go to Dr Pham The Hai who also gave me orientations, always willing to answer all my questions about the professional issues during my good thesis industry Besides, I am highly thankful to teachers, staff members and friends at Department of of Biology, Siblings in the Department of Biochemistry and Biochemistry Development, the teachers in the Institute of Microbiology Hanoi National University, VNU University of Science for their love and kindness, insightful comments and suggestions Last but not least, I would like to thank my family for encouraging and supporting me spiritually throughout my life Student Nguyen Thi Ngoc PREFACE Reasons for choosing themes Currently, in Vietnam as well as in the world, most people prefer bioproducts Bioproducts known for their use for controlling pathogens are a growing boom in developing countries, where products are cultivated or added to foods to enhance digestive balance Of particular interest is that biopreparations can be commercialized (Fuller, 1987) Therefore, among the microorganisms used for biological preparation, Bacillus subtilis is a popular example on the market today B subtilis has many good qualities They are food safety, have high economic benefits (in Japan and in European countries) B subtilis bacteria stimulate immunity, prevent bacterial pathogens from entering the body, Also they are functional foods to supplement beneficial bacteria Thus, it can be said that "B subtilis is a strain of probiotics that is safe for users" In addition, Vietnam is a very agricultural country with a great emphasis on livestock and farming In order to increase the productivity and quality of products based on practical needs and promote their strengths, I conduct research "Optimization of the fermentation medium for B subtilis strain VTCC-B-51 to achieve high biomass yield" Objectives of the study Study on the fermentation parameters of B subtilis on a 10 liter fermenter based on environmental factors and culture parameters (stirring speed, air, ) Abbreviation B subtilis LB T° Sv ODs OD h Bacillus subtilis Luria- Bertani Temperature Strain volume OD strain Optical density Hour List of tables List of figures Contents CHAPTER 1: LITERATURE REVIEWS 1.1 Overview for B subtilis 1.1.1 History of development B subtilis was first discovered in horse dung (1941) by the Nazi German medical organization At first, it was mainly used to prevent illness for German soldiers fighting in North Africa[7] Treatment had to wait until 1949-1957, when Henrry et al., obtaine the pure culture of B subtilis[16] Since then, the term "subtilistherapie" has been used to indicate the subtilis treatment of inflammatory bowel disease, colitis and diarrhea due to digestive disorders Today, this bacterium is widely applied, thanks to the development of biotechnology( as medicine, food and animal husbandry purposes) 1.1.2 Classification According to the classification of Bergey (1974), B subtilis belongs to: 1.1.3 - Kingdom: Bacteria - Division: Firmicutes - Class: Bacilli - Order: Bacillales - Family: Bacillaceae - Genus: Bacillus -Species: Bacillus subtils Distribution B subtilis belongs to the intestinal microflora of the intestinal tract, which is distributed almost exclusively in the wild such as hay, dust, soil Most of them exist in the soil, typically cropland soil containing about 10-100 million CFU / g In poor nutient soil in deserts, wastelands, B subtilis is very rare Water and mud at the estuaries as well as in seawater have the existence of B subtilis spores( Vu Thi Thu, 1996 )[7] 1.1.4 Cell morphology B subtilis is bacterium with small size and short rod-shaped cells each having two rounded tips of 0.5-1.0 μm x 1.5-3.0 μm; usually standing alone or making short strings The bacterium is a Gram-positive and has a capacity B subtilis is bacterium of producing spores that can be and between the cell The bacterium is developed by the germination Figure 1: Cells of B subtilis bacteria 1.1.5 Colony morphology After 24 hours implanted on agar medium, B subtilis usually produces clear colonies that are: dry, milky white, crease on the surface of the agar, creased and creamy lobes, in the middle with a knob small convex and often referred to as concentric rings and clinging to the agar surface Figure 2: Colony of B subtilis Figure 3: Colony of B subtilis 1.1.6 The process of spore formation Spores are a thickened solid biomass, the outermost of the spore is a membrane Crustaceans have many layers, which inhibit the penetration of water and water soluble substances Under the shell is the inner membrane of the spore and in the same is a homogeneous mass of cytoplasm[1] The outer membrane (Exosporium) Sheathed (Tunique externe) Covered in (Tunique interne) Shell (Cortex) Membrane biofilter spore center Spore wall Figure 4: Diagrams of internal structure bacterial spores (Nguyen Lan Dung)[13] 10 Table 2: Composition of the culture medium (for liter) Material Yeast extract Meat extract Pepton NaCl K2HPO4.3H2O Distilled water Mass(g) 5.0 2.0 5.0 1.0 1.9 1000ml 2.2 Methods 2.2.1 The physiological properties of B subtilis 2.2.1.1 Determination of pH suitable for growth medium The culture medium LB was distributed into triangular vases (60 ml/bottle) then H3PO4 (20%) and NaOH (0.5M) were used to adjust pH of the medium to different levels: Table 3: The pH of the culture medium tested for B subtilis pH requirements 4.0 pH after standard 3.99 20 pH after sterilization 4.01 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 4.48 5.01 5.51 5.98 6.49 7.02 7.52 8.01 4.50 5.02 5.52 6.01 6.49 7.01 7.51 8.00 Addition of ml of B subtilis culture medium (OD = 3.02) to the test tubes, cultured for 46-48 hours in a thermostatic shaker with 37 ° C, shaking 150 rpm During a 48-hour culture period of B subtilis at different pH levels, a growth test was performed by measuring the OD (by measuring the growth ability of the spectrophotometer) every hours from now 18 hours after adding microorganism 2.2.1.2 Determination of environmental temperature suitable for growth Prepare LB medium and then put in triangular vases with a volume of 60ml / bottle For ml B subtilis strain (OD = 2.96), each of the triangular vases of B subtilis was cultured within 48 hours at the corresponding temperature levels of 20; 25; 30; 35; 37; 40; 45; 50°C During 48 hours of culture, B subtilis at different temperatures was tested for growth by measuring OD (Spectrophotometer) 12 hours from now 24hours after adding microorganism 2.2.2 Determination of the cell density changes B subtilis strain ATCC was cultured on LB medium The culture was incubated in a shaking incubator at 37 ° C and at 150 rpm for 36h in a 250ml triangular vases containing 100ml of medium After 24h, B subtilis was cultured to collect centrifugal cells, removed fluid and add sterile saline to 1/10 of the original volume Since the diluted solution was diluted with salt, dilute 1/10,1/100,1/1000,1/10000, measured OD and cultured count[3] 21 2.2.3 Optimization of fermentation conditions on a 10 liter fermenter Through investigation and empirical studies, identification of optimal fermentation conditions for B subtilis strains will be made in 25 fermentation batches Parameters and batch rates were set for each batch to produce suitable parameters The following parameters were experimented: -Rating rate -Temperature -pH -The speed of the gas -Time of culture 2.2.4 Culturing steps Strains Propagation Culturing on a 10 liter fermenter Filtration/centrifugal translation Step 1: Propagation The strain was kepted under deep cooled conditions to be activated by multiplying in a 250 ml triangular vases volume containing 150 ml-200 ml LB culture medium, shaking at 150 rpm at 37 ° C for 18-24 hours After 18-24 hours, the sampling dye should been tested for the purity of the variety at the same time to measure OD and pH to prepare for culture in a 10 liter fermenter Note: When propagation, stir the implant on an alcohol lamp flame; limit communication to prevent 22 Step 2: Culturing on a 10 liter fermenter The LB medium was mixed into a 10 liter fermenter and checked the air inlet, inlet, cap, Sterilizing the 10 liter fermenter with LB medium at 110 ° C / 30 After sterilization, we were been cool down the 10 liter fermenter to 37°C The B subtilis strain was multiplied from the triangular vases into the 10 liter fermenter, set the stirring mode, temperature and air flow depending on the fermentation batch Sampling the OD and pH at the time of the test Samples was taken after 4-6 hours, stained for hours after screening, OD samples every hours When B subtilis was reached > = 80% of the spores, we were collected biomass Step 3: Filtration/centrifugal translation Concentrating solution by centrifugal method 23 Figure 9: Filtration system concentrates B subtilis biomass CHAPTER 3: RESULTS AND DISCUSSION 3.1 Results 3.1.1 Physiological properties of B subtilis 3.1.1.1 pH suitable for the growth of the strain During 48 hours of culture, B subtilis with different pH was tested for growth by measuring OD (spectrophotometer) hours from the 18 hours when adding the microorganism with the following table: Table 4: Test results of B subtilis culture in triangular flasks at different pH environments pH 0h (OD 600nm) 18h (OD 600nm) 24h (OD 600nm) 30h (OD 600nm) 24 36h (OD 600nm) 42h (OD 600nm) 48h (OD 600nm) pH after culture 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 0.14 0.20 0.17 0.12 0.16 0.18 0.19 0.22 0.18 0.13 0.15 0.14 1.74 2.35 2.39 2.09 2.11 1.40 0.17 0.14 0.04 1.79 2.48 2.74 2.23 2.38 1.91 0.05 0.12 0.13 1.86 2.62 2.82 2.44 2.54 2.38 0.08 0.12 0.16 1.95 2.92 3.14 2.91 2.99 2.43 0.22 0.15 0.24 2.18 3.10 3.18 3.01 3.04 2.56 0.27 0.13 0.57 2.24 3.12 3.21 3.05 3.08 2.87 4.03 4.53 5.02 6.96 7.26 7.66 7.46 7.74 7.63 Figure 10: Relationship between pH and growth capacity of B subtilis According to the results( figure 10) of the culture of B subtilis at different pH environments, it was found that B subtilis can grow at pH 5.5 to 8.0 and grows well at pHs from 6.0 to 7.5 This result was used for culturing the strain in 10 liters fermenter 3.1.1.2 Environmental temperature suitable for the growth of the strain The B subtilis strain was grown at different temperatures and OD (at 600 nm) was measured every 12 hours to establish Table 5: Test results for B subtilis culture at different temperatures levels Temperature 0h 24h 36h 25 48h pH after s (OD 600nm) (OD 600nm) (OD 600nm) (OD 600nm) culture ( C) 20 25 30 35 37 40 45 50 0.18 0.10 0.13 0.14 0.15 0.12 0.15 0.18 0.17 0.19 0.36 1.09 1.24 1.21 1.08 0.25 0.15 0.25 0.87 1.86 2.09 1.56 1.45 0.42 0.13 0.32 1.52 2.94 2.90 2.57 1.96 0.59 6.08 6.74 7.15 8.07 8.16 7.98 7.97 6.84 o Figure 11: Relationship between temperature and growth capacity of B subtilis The results (figure 6) showed that the B subtilis strain growed well at 35°C to 40°C We applied this condition to culture the strain in the 10 liter fermenter 3.2 Standard OD line for cell density measurement Table 6: Results of count culturing obtained at OD different levels OD measurement and B subtilis count OD (λ = 600nm) Density (cfu/ml) 4.09 x 1010 0.412 2.5 x 109 0.038 3.6 x 108 0.007 2.3 x 107 Figure 12: Standard line OD and density 3.3 Culture parameters for a 10 liter fermenter 25 batches of fermentation of the B subtilis were devided into main series to evaluate the culture parameters (table 7): +Series (i) with OD after fermentation ≤ 5.5 (OD≤ 5.5) 26 + Series (ii) with OD after fermentation satisfying 5.5 < OD < 7.0 +Series (iii) with OD after fermentation ≥ 7.0 (OD≥ 7.0) Table 7: Parameters and results of series (i) fermentation on a 10 liter fermenter Series Hours 12 17 21 23 24 T° Stir Gas (oC) (rpm) (l/m) Sv (ml) 12 24 36.9 38.1 36.9 130 150 180 100 14 37.0 36.8 120 150 150 22 16.5 20 19.5 23.2 12 20 14.5 23.5 12 24 14 23 37.1 36.2 36.3 37.1 36.8 36.7 37.2 180 130 150 180 130 150 170 7 ODs pH before pH afte r OD 8.54 5.1 8.53 5.2 8.39 4.8 8.44 5.5 8.47 5.0 8.35 5.4 6.98 4.3 7.02 2.9 100 7.01 150 6.98 3.2 Contaminated 36.5 36.9 36.8 37.0 37.1 36.9 130 150 180 120 150 170 100 7.01 3.1 100 6.99 4.7 Contaminated Comment: 27 From the results, we can see that series of culture (i) including: 1; 6; 7; 12; 17; 21; 23; 24 produced poor and unstable growth (OD ≤ 5.5): -Lowed rate of strains -Slow rised time As B subtilis is an aerobic bacterium, extending the duration of the latent phase and possibly kills the bacteria so low biomass was obtained Table 8: Parameters and results of batches (ii) fermentation on a 10 liter fermenter Serie s Hour s T° Stir Gas (oC) (rpm ) (l/m ) 16 23 12 20 16.2 24 16 23 12.5 23.5 14 24 37.1 36.9 36.9 37.0 36.9 36.8 36.8 36.8 36.5 35.9 36.8 36.9 36.8 36.9 37.1 37.1 36.9 37.0 150 180 200 130 150 200 130 180 200 110 150 180 150 180 200 130 170 200 7 8 7 10 14 15 28 Sv (ml) ODs 100 pH befor e 6.92 pH afte r OD 8.44 6.8 8.29 6.5 8.68 7.0 8.39 6.8 8.34 5.9 8.40 6.9 4.9 100 7.02 5.5 150 6.98 6.5 150 6.98 7.6 100 7.05 4.7 150 7.05 5.6 19 25 12 23.5 16 24 36.8 37.1 36.8 36.6 36.9 37.1 150 180 200 150 180 200 8 100 6.99 5.5 100 8.56 5.8 8.60 6.5 7.02 6.0 Comment: From the results, we can see that series of culture (ii) including: 3; 5; 9; 10; 14; 15; 19; 25 produced poor but stable growth (5.5 < OD < 7.0): -Lowed rate of strains -Slow rised time As B subtilis is an aerobic bacterium, extending the duration of the latent phase and possibly kills the bacteria so low biomass was obtained Table 9: Parameters and results of batches (iii) fermentation on a 10 liter fermenter Serie s Hour s 12 20 12.5 22 12.5 22 15.0 23.0 12.0 24 11 13 T° Stir Gas o ( C) (rpm ) (l/m ) 37.5 38.4 36.9 36.9 36.8 37.1 37.3 36.8 36.9 36.9 36.9 36.8 37.2 36.9 36.8 150 250 300 150 270 300 150 250 300 180 250 300 150 250 300 6 7 7 29 Sv (ml) ODs 150 pH befor e 7.00 pH afte r OD 8.44 7.4 8.45 7.6 8.62 7.1 8.36 7.6 8.37 7.8 4.69 200 7.02 5.6 200 6.98 6.0 150 6.95 6.94 150 7.04 6.0 16 18 20 22 12.5 22 12 24 15 23.5 12.5 24 36.5 37.1 36.7 36.9 37.0 36.9 36.8 37.1 37.0 36.9 37.0 36.9 150 250 300 150 250 300 150 270 300 150 250 300 7 7 200 7.02 6.5 150 8.73 7.7 8.64 7.9 8.35 7.8 8.50 7.7 6.98 6.6 150 7.03 6.8 200 7.00 6.9 Comment: From the results, we can see that series of culture (iii) including: 2; 4; 8; 11; 13; 16; 18; 20; 22 producef good and stable growth (OD ≥ 7.0): -Highed rate of breeding -Time to increased gas circulation properly From that it was brang economic benefits to producers while ensuring the efficiency high biomass Thus, with the temperature parameters considered to be determinable, the parameters of strain rate, gas flow rate and stirring rate (especially with respect to the time of the increase of gas) had been a significant effect on biomass yield obtained after fermentation 30 CONCLUSIONS AND PROPOSALS Conclusions During the study, operating on a 10 liter fermenter were successful in determining of the B subtilis strain with the following culture parameters: Temperature of culture: 35 - 37°C was the appropriate temperature range for breeding Speed of stirring: 150 rpm and - hour after breeding level increased from 250 to 300 rpm until collection Air flow: - l/m (increase with stirring speed) Collection time: 22 - 24 hours 31 pH of the medium: 6.5-7.0 Inoculant: 2.5- 3% by volume ODs: 2.5 - 2.8 (3-5x10^9 cfu/ml by density) Proposals In the coming time, it is recommended to continue to study and improve the parameters of culture of B subtilis on 10 liter fermenter with higher biomass efficiency and stable spore ≥ 95% REFERENCES VIETNAMESE Nguyễn Lân Dũng, Nguyễn Đình Quyến, Phạm Văn Ty, 2010 Vi sinh vật học Nxb Giáo dục Việt Nam Nguyễn Thành Đạt, 2007 Cơ sở sinh học vi sinh vật Nxb Đại học Sư Phạm Mai Thị Hằng, Đinh Thị Kim Nhung, Vương Trọng Hào, 2001 Thực hành vi sinh vật NXB Đại học Sư phạm Nguyễn Lân Dũng, Phạm Thị Trân Châu, Nguyễn Thanh Hiền, Lê Đình Lương, Đoàn Xuân Mượn, Nguyễn Đình Quyến, Phạm Văn Ty, 1978 Một số phương pháp nghiên cứu vi sinh vật học NXB khoa học kỹ thuật, tập 1,2 Trần Thị Thanh, 2011 Công nghệ vi sinh NXB Giáo dục Việt Nam Tống Thị Triêm, 2013 Tổng quan enzyme ngoại bào B subtilis 32 Nguyễn Duy Khánh, 2006 Khảo sát điều kiện nuôi cấy sinh bào tử vi khuẩn B subtilis Phạm Thị Ngọc Lan, Lê Thanh Bình, 2003 Đặc điểm phân loại chủng Lactobacillus probiotic CH123 CH 126 phân lập từ đường ruột gà Tuyển tập báo cáo Hội nghị Công nghệ Sinh học toàn quốc năm 2003, pp 101-105 Hoa NT, Baccigalupi L, Huxham A, Smertenko A, Van PH, Ammendola S, Ricca E, and Cutting SM (2000) Characterization of Bacillus species used for oral bacte- riotherapy and bacterioprophylaxis of gastrointestinal disorders Appl Environ Microbiol 66: 5241-5247 ENGLISH Aldrich, J, Baker, R 1970Biological control of Fusarium roseum f sp dianthi by Bacillus subtilis Plant Disease Reporter 54:446-448 11 Arima K, Kakinuma A, Tamura G (1968) Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of brin clot formation Biochem Biophys Res Commun 31(3): 488-494 12 Ciffo (1984) Determination of the spectrum of antibiotic resistance of the Bacillus subtilis strains of Enterogermina Chemioterapia 3:45–52 13 De Boer A S., Diderichsen B.(1991) On the safety of Bacillus subtilis and B amyloliquefaciens: a review Appl Microbiol Biotechnol 36:1–4 14 Kiss T., Gratwohl A., Frei R., Osterwalder B., Tichelli A., Spec 10 k B (1988)Bacillus subtilis infections Schweiz Rundsch Med Prax 77:1219–1223 15 Hong HA, Huang JM, Khaneja R, Hiep LV, Urdaci MC and Cutting SM (2008) The safety of and Bacillus indicus as food probiotics J Appl Microbiol 105:510-520 16 Hong HA, Khaneja R, Tam NK, Cazzato A, Tan S, Urdaci M, Brisson A, Gasbarrini A, Barnes I, Cutting SM (2009) Bacillus subtilis isolated from the human gastrointesti- nal tract Res Microbiol 160: 134-143 17 Huang JM, La Ragione RM, Nunez A, Cutting SM (2008) Immunostimulatory activity of Bacillus spores FEMS Immunol Med Microbiol 53: 195-203 33 Meroni PL, Palmieri R, Barcellini W, De Bartolo G, Zanussi C (1983) E ect of long-term treatment with Bacillus subtilis on the frequency of urinary tract infec- tions in older patients Chemioterapia 2: 142-144 19 Reva O N., Vyunitskaya V A., Reznik S R., Kozachko L A., Smirnov V V.(1995) Antibiotic susceptibility as a taxonomic characteristic of the genus Bacillus Int J Syst Bacteriol 45:409–411 20 Tam NK, Uyen NQ, Hong HA, Duc LH, Hoa TT, Serra CR, Henriques AO, and Cutting SM (2006) The intestinal life cycle of Bacillus subtilis and close relatives J Bacteriol.188(7): 2692–2700 21 Shen WY, Fu LL, Li WF, Zhu YR (2010) Effect of dietary supplementation with Bacillus subtilis on the growth, performance, immune response and antioxidant activities of the shrimp Aquac Res 41: 1691-1698 22 Velasco E.,De Sousa Martins C A., Tabak D., Bouzas L F (1992) Bacillus subtilis infection in a patient submitted to a bone marrow transplantation Rev Paul Med 110:116–117 18 WEBSITE 23 24 http://menvisinh.org/content/bacillus-subtilis http://intranet.tdmu.edu.ua/data/cd/disk2/ch015.htm 34 ... UNIVERSITY OF SCIENCE FACULTY OF BIOLOGY Nguyen Thi Ngoc OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCC-B-51 TO ACHIEVE HIGH BIOMASS YIELD Submitted in partial fulfillment of. .. quality of products based on practical needs and promote their strengths, I conduct research "Optimization of the fermentation medium for B subtilis strain VTCC-B-51 to achieve high biomass yield" ... accounts for 5-12% of the dry weight of a spore) The role of this acid is to make the spores resistant to high temperatures Water content in spores very low and exist in the form of link[2] A spore of