MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY ISOLATION, SELECTION AND IDENTIFICATION BACILLUS SUBTILIS FROM MUD-DREG OF BEER SUPERVISOR STUDENT Assoc. Prof. Dr. TRAN NHAN DUNG NGUYEN MINH THUY Student’s code: 3084035 Session: 34 Can Tho, 05/2013 APPROVAL SUPPERVISOR STUDENT Assoc. Prof. Dr. TRAN NHAN DUNG NGUYEN MINH THUY Can Tho, May, 2013 PRESIDENT OF EXAMINATION COMMITTEE Abstract Bacillus subtilis had widely used in many fields such as medicine, food processing, veterinary, aquaculture and especially in environmental treatment. It helps disintegrate dreg, mud ...In this study”Isolation selection and identification of Bacillus subtilis from “mud-dreg of beer” 21 strains of bacteria isolated from ” mud-dreg” and (Bs1, Bs2, Bs3, Bs4, Bs5, Bs6, Bs7, Bs8, Bs9) waste water of beer (BN1, BN2, BN3, BN4, BN5, BN6, BN7,BN8, BN9, BN10, BN11, BN12). There were sixteen strains selected because of Gram (Gram positive). After testing biochemical activities such as enzyme Catalase, Amylase, Protease and reaction with Methyl Red, all results were compared with Bacillus subtilis control from Biochemical Food laboratory. Eight strains (Bs1, Bs2, Bs4, BN1, BN2, BN5, BN9, BN12) are selected to do PCR (Polymerase chain reaction) with primer 16S-9F and 16S-1525R. The result of PCR perform size band of 8 strains and Bacillus subtilis control had 1500bp. Sequencing perform that 2 strains (Bs4 and BN5) were Bacillus subtilis (99% and 98% identical), they present both of sample “mud-dreg” and waste wate. Besides, Bs2 and BN9 also so found in “mud-dreg” and waste water, were sequenced as Bacillus megaterium. Key words: Bacillus subtilis, beer, isolation, mud-dreg, waste water ii CONTENTS Abstract ..................................................................................i Content ...................................................................................ii 1.Introduction .........................................................................1 2.Material and Method ...........................................................3 2.1.Material ......................................................................3 2.2.Method .......................................................................4 2.2.1.Isolation Method..................................................4 2.2.2.Observation shape and movement of bacteria ....5 2.2.3.Measured bacteria size ........................................5 2.2.4.Gram stained........................................................6 2.2.5.Spore stained Bacillus subtilis .............................7 2.2.6.Chemical reaction ................................................7 2.2.8.Polymerase chainreaction ....................................8 3.Result and Discussion .........................................................10 3.1.Bacteria Isolation and characteristic ..........................10 3.1.1.Bacteria Isolation .................................................10 3.1.2.Characteristic (size of cell, Gram, movement) ....12 3.1.3.Chemical reaction to test bacterial capacity create synthesize exoenzyme ............................................................14 iii 3.1.4.Catalase ...............................................................15 3.1.5.Amylase ...............................................................16 3.1.6.Protease ...............................................................16 3.1.7.Reaction with Methyl Red ...................................17 3.1.8.Spore stained .......................................................17 3.3.PCR product...............................................................18 3.4.Sequencing.................................................................19 4.Result and Sugestion ...........................................................20 Reference................................................................................21 iv 1. INTRODUCTION The genus Bacillus have a great potential for extracellular enzymes. Many of these enzymes are extracellular enzymes that hydrolyze large organic molecules. Thus, genus Bacillus has many application in different fields (R.Gupta et al., 2002), such as industrial manufacturing detergents, food industry, pharmaceuticals and leather processing, especially in minimize waste pollution (Outtrup H. Et al., 2002). Priest in 1977 had study demonstrated a Gram positive bacteria, spore forming. Bacillus subtilis is able to synthesize and produce protease, amylase, and a number of axoenzyme (extracellular enzyme)... The products nature of biotechnology, along with the ability to create and spore germination by Bacillus became hereditary system typical of Gram positive bacteria. (Le Thi Lan, 1997; Priest, 1993) Beer is a beverage products are increasingly popular and common in the holidays in Vietnam. In the process of brewing beer sludge is discharged into a nutritious environment for microorganisms to grow. Each day, about 2 tons of sludge discharged ”mud-dreg” from a beer brewing plant in Bac Lieu province that does not have the resolution, causing environmental pollution. Because of the biological properties of extracellular enzyme aforementioned Bacillus subtilis of the hydrolysis of large organic molecules. Beer dregs is a nutrient-rich environment and organic matter should be decided on the subject of Bacillus subtilis 1 isolated from waste water of beer and beer wallows in the brewing process. Objective: Isolation, selection and identification of Bacillus subtilis from beer dregs, that is done with the aim of isolating, identifying characteristic biochemical and morphological methods in molecular biology of Bacillus subtilis, which have the ability to stream selected synthetic protease and amylase enzyme highly active. For the purpose of beer dregs utilize as fertilizer for plants and micro-organisms to reduce environmental pollution. 2 2. MATERIALS AND METHODS 2.1. Materials - “Mud-dreg” 2kg; waste water 1 littre - Bacillus subtilis positive control from Food chemical labotary - Forward and reverse primer 16S-9F and 16S-1525r (JangSeu ki et al., 2009) 16S-9F 5’ GAG TTT GAT CCT GGC TAC G 3’ 16S-1525r 5’ AGA AAG GAG GTG ATC CAG CC 3’ -Medium: TSA agar, Luribernate liquid TSA (Tryptic Soy Agar) Chemical Concentration (g/l) Tryptone 15 Soytone 5 NaCl 5 Agarose 15 Starch 0.1%; NaCl 0.1%; Iod 0.02N; Machite Green 5%, safranin; H2O2 3%; Methyl Red; Iod; Fushin; Crystal violet; Ethanol 70%; BiH2O . Chemicals and equipments in Plant molecular laboratory 3 Medium SMA (Skim milk Agar) Chemical Concentrate (g/l) Pepton 5 Yeast extract 3 Skim milk 100 ml/l Agarose 20 2.2. Methods 2.2.1. Isolation method Identical samples: Add about 10g of “mud-dreg” to 90ml distilled water, these samples were shaked at 150round/per minute after that incubated in 80°C for 20 minutes. “Mud-dreg” suspension was dilluted into 5 concentrations such as 10-1, 10-2, 10-3, 10-4, 10-5. Waste water was diluted directly. After that spreaded samples on petri dish, incubated at 37°C for one or two days and observed the appearance of colonies. Cultured seperated colonies on medium until having isolate strains, cultured isolate strain to LB tube and storaged in refrigerator. 4 2.2.2. Observation shape and movement of bacteria After isolation and separation bacteria, observation the movement and shape in sterile distilled water. Preparation of baterial samples by pressure drop method. +Drip 5µl sterile distilled water to lam glass. +Sterilized wire loop on alcohol lamp. +Used wire loop to take a few colony and stretch it on the drop. +Took a lame cover on the drop. +Observed the specimen under optical microscope zoom 400. 2.2.3. Measure bacteria size Measured the diameter of the microscope's field of view. Using the low power objective, look through the microscope, and place the ruler under the field of view. Measure the diameter in millimeters. For example, you may find that the diameter of the field of view is five millimeters. Observed the bacteria under the microscope with low power. Place the bacteria slide on the stage of the microscope, and then bring it into focus using the fine course adjustment knobs. Locate a single bacterium. The bacteria slide will typically have more than one bacterium. Find one bacteria, and then estimate how many times it will fit across the field of view. For example, you may find that a single rod-shaped bacteria will fit across the field of view about three times. 5 Divide the diameter of the field of view by the number of times that the bacterium fits across the field of view. 2.2.4. Gram-stained Place a sample of a bacterial culture on a microscope slide. An inoculation loop can be used to transfer the bacteria to the slide. Dry the slide containing the bacterial culture. Pour crystal violet stain over the bacterial specimen on the slide. Let the slide stand for approximately 10 to 60 seconds depending on the thickness of the smear on the slide. Rinse the crystal violet stain off of the slide with water. Place Gram's iodine solution on the bacterial smear. Let the smear stand for another 10 to 60 seconds depending on smear thickness. Rinse off the extra Gram's iodine solution with more water. Add several drops of a decolorizer to the bacterial smear on the slide. Rinse the decolorizer off of the slide when the decolorizer is no longer colored by the previous stains as the decolorizer runs off of the slide. A typical time for this is approximately 5 seconds. Put a counterstain, such as basic fuchsin solution, on the slide over the bacterial smear. Allow the counterstain to remain on the smear for approximately 40 to 60 seconds and then rinse off the counterstain with water. Gram-positive bacteria will be colored purple, and Gram-negative bacteria will have a red or pink color. 6 2.2.5. spore-stain Bacillus subtilis Staining the spore of bacteria, the firt step like Gram staining, after that take machite green 5% on specimens. Clean up lam under water then keep it in safranin liquid about 1minute and clean up under water. Observing with lam in the oil. 2.2.6. Chemical reaction to identify Bacillus subtilis a. Catalase reaction: Drip one drop of H2O2 30% on the lam, take a amount of colony put into H2O2 30% drop. +Positive: bubble up +Negative: Not bubble up b. Methyl Red Test: Transfered 1ml of bacteria in LB medium into a tube. Drip 5 drop of Methyl Red into the tube. + Positive: turn into light red color + Negative: turn into yellow color c. A capacity to synthesize protease enzyme: Use micropipett to drop 5µl bacteria from LB liquid medium. Drip 5µl bacteria on SMA medium repeat 3 times and incubate in 40°C on 24 hours. Bacteria strains have protease enzyme will create halo round, measure thi round follow formula: Hydrolyze diameter = halo diameter – drop of bacteria diameter d. A capacity to synthesize amylase enzyme: 7 Take 1ml bacteria from LB medium in to the tube in which has 1ml NaCl 0.1%. After that, take 1ml starch and shake the tube, leave it at 30°C 30 minutes. Taking 1 drop of iod into the tube and observes the result for: + Bacteria have amylase enzyme: loose the color of starch + Bacteria do not have amylase enzyme: dark blue color 2.2.7. Electrophoresis PCR products PCR procedure: Primers 16S rRNA (16s-9F và 16S-1525r) (Jang-Seu ki et al.,2009) Forward primer: 16S-9F 5’GAG TTT GAT CCT GGC TAC G 3’ Reverse primer: 16S-1525r AGA AAG GAG GTG ATC CAG CC 3’ After DNA extracted, PCR reaction with primers above. BiH2O 9,5µl Buffer 10X 2µl MgCl2 25mM 1,6µl dNTPs 3,2µl Forward primer (diluted 10 times) 0,6µl Reverse primer (diluted 10 times) 0,6µl Taq polymerase 0,5µl 8 DNA 2µl Total 20µl *PCR cycle (repeat 35 cycles) Component of Gel: Agarose 1% 0,4g TE1X 45ml EtBr 0,8µl 9 3. RESULTS AND DISCUSSION 3.1. Bacteria Isolation and characteristic: 3.1.1.Bacteria Isolation: 21 isolates bacteria strains. 9 isolates from “mud-dreg” took 42,85% and 12 from waste water took 57,14%. No. isolate Sp Form Margin Elevation color Size cm 1 Bs1 Mud circular entire raised milky 3 2 Bs2 Mud circular entire umbonate white 3 3 Bs3 Mud circular curled umbonate white 2 4 Bs4 Mud circular entire raised milky 3 5 Bs5 Mud circular entire raised milky 1 6 Bs6 Mud circular curled umbonate milky 1.5 7 Bs7 Mud circular entire raised milky 2 8 Bs8 Bùn circular entire raised milky 4 9 Bs9 Bùn circular entire raised milky 1 10 Table 1a: 9 isolates bacteria strains from waste water Sp Form Margin Elevation N o Strain Color Size 1 BN1 W circular smooth raised milky 1 2 BN2 W circular smooth raised white 1 3 BN3 W circular curled raised white 1 4 BN4 W circular smooth raised milky 0.5 5 BN5 W circular curled umbonate milky 5 6 BN6 W circular smooth raised milky 0.5 7 BN7 W circular smooth raised milky 2 8 BN8 W circular curcled umbonate milky 2 9 BN9 W circular smooth raised milky 5.5 10 BN10 W circular smooth raised milky 5 11 BN11 W circular smooth umbonate milky 2 mm 11 12 BN12 W circular smooth umbonate milky 5 Table 1b: 12 isolates bacteria strains from waste water 3.1.2.Characteristic (size of cell, Gram, movement) No strain Shape mobility (rod) Gram length width test (µm) (µm) 1 Bs1 long + + 1.9 0.74 2 Bs2 short + + 1.0 0.68 3 Bs3 long + + 2.83 0.71 4 Bs4 short + + 1.65 0.74 5 Bs5 long + + 2.87 0.7 6 Bs6 long + - 1.86 0.65 7 Bs7 short + + 1.41 0.71 8 Bs8 long + + 2.86 0.74 9 Bs9 long + - 1.8 0.68 10 BN1 long + + 2.86 0.71 12 11 BN2 long + + 2.83 0.74 12 BN3 long + + 1.83 0.68 13 BN4 short + - 1.43 0.71 14 BN5 short + + 1.43 0.72 15 BN6 short + - 1.6 0.68 16 BN7 long + + 2.7 0.74 17 BN8 short + - 1.43 0.68 18 BN9 long + + 2.83 0.71 19 BN10 short + + 1.68 0.7 20 BN11 short + + 1.43 0.72 21 BN12 long + + 2.9 0.74 Table 2: Characteristic of baterial cell Notice:Gram(+): gram positive; move + : can move 13 The size of bacterial cell varies between 1.0 to 2.86µm and width in the range of 0.65 to 0.74µm consistent with the description of Nguyen Lan Dung (1997). After Gram stain 21 strains, obtained 16 strains had purple blue color from crystal violet, positive gram, accounting for 76,19%. Therefore, they suitable description of Bacillus subtilis are positive gram of Rahnman Sharmin (2007), excluding 5 strains had pink color from Fuchsin, gram negative bacteria. 3.2.Chemical reaction to test bacterial capacity create synthesize exoenzyme: No strain Catalase Amylase Protease Methyl Red spore 1 Bs1 +++ +++ + + + 2 Bs2 +++ +++ + + + 3 Bs3 - ++ None None None 4 Bs4 +++ ++ + + + 5 Bs5 + ++ - None - 6 Bs7 ++ + - None - 7 Bs8 - ++ - None - 14 8 BN1 +++ +++ + ++ + 9 BN2 +++ +++ + + + 10 BN3 - ++ None None None 11 BN5 ++ ++ + + + 12 BN7 - ++ None None None 13 BN9 ++ +++ + + + 14 BN10 +++ + - None - 15 BN11 - + None None None 16 BN12 +++ +++ + ++ + 17 control + +++ + + + Table 3: Bacterial chemical characteristic 3.2.1.Catalase: Seven strains had bubbling stream faster took 43.75%, 3 strains had bubbling at everage range at 18.75%, 1 strain 15 produced less bubbling (6.25%) and 4 strains without bubbling, that’s mean no catalase activity, accounting for 25%. Reaction 2H2O2 catalase 2H2O + O2 Bacillus subtilis are capable of producing enzyme catalase. When exposed to H2O2 is generated O2 bubbling in previous studies on the possibility of creating catalase by Nguyen Duc Luong and Nguyen Thi Thuy Duong (2003), Slepecky and Hemphill (2006), and Sharmin Rahman (2007). Therefore, selecting 12 strains to do the next reaction. 3.2.2.Amylase: In total 16 strains, 6 strains discoloration of starch and iodine immediately accounted for 37.5%, these strains are strong amylase activity, 7 strains of discoloration starch and iodine after a period accounted for 18.75%. All bacteria strain ha capable of synthesizing amylase consistent with the study by Nguyen Duc Luong and Nguyen Thi Thuy Duong (2003), Slepecky and Hemphill (2006). 3.2.3.Protease: After incubate at 37°C for 24h. 8 strains had halo round, can create protease enzyme, accounted for 66.7%. Besides, BN5 get highest halo diameter (1.25cm) and BN9 (1.2cm) took the second range. 16 Figure 10: diameter of Halo on skim milk agar In the same column mean values followed by the same letter are not significantly different at= 5% 3.2.4.Methyl Red When driped MR into 1ml of bacteria, making color changing 3 strains made medium turn into orange accounted for 25%, 8 strains made medium turm in to pale orange (50%). Another could not make change medium color. Therefore, 8 strains are chosen. Similar studies of Bacillus subtilis of Sharmin Rahman (2007) and Tran Linh Thuoc (2008). 3.2.5.Stained bacterial spore: When observed under the microscope at 400 times magnification, 8 strains bacterial spores form oval, accounting for 50%, they stared green of malachite green solution. This result the same with studies of Stainer et al.,(1999), Slepecky and 17 Hemphill (2006) and Luong Bich Dung (2008). The remaining 8 strains had red color from safranin (50%) 3.3. PCR product: After done bacterial chemical reaction. There are 8 strains suitable to do electrophoresis: No strain Catalase MR Amylase Protease spore 1 Bs1 +++ + +++ + + 2 Bs2 +++ + +++ + + 3 Bs4 +++ + ++ + + 4 BN1 +++ ++ +++ + + 5 BN2 +++ + +++ + + 6 BN5 ++ + ++ + + 7 BN9 ++ + +++ + + 8 BN12 +++ ++ +++ + + Table 4: Selecting 8 strains to do electrophoresis 18 1500bp Figure 13: DNA profiles of 9 well, others lanes are follows Bs1; Bs2; Bs4; BN1; BN2; BN5; BN9; BN12; control (+); control(-) 3.4.Sequencing: After sequencing by NUCLEOTIDE BLAST (http://www.ncbi.nlm.nih.gov/) . Two strains have been identified Bacillus subtilis (denoted as Bs4 and BN5) with 99% and 87%. The nucleotide sequence result can be seen in appensix. -Three Bacillus subtilis strains identical with Bs4 and BN5: +Bacillus subtilis strain S20 was identified by Mohammadou, B.A., Mbofung, C.M. and Barbier, G. in a research “Genotypic and phenotypic diversity among Bacillus species isolated”. It was published on Food engineering and 19 Quality journal in 2012, at University Institute of Technology, Cameroon, Africa. +Bacillus subtilis strain OSS 42 was sequenced in “Comparison of microbial communities native to three differently polluted elogical niches in the industrial site of Bagnoil” research, author: Sprocati, A.R., Alisi, C. And Tasso, F. and published on Modern multidisciplinary applied microbiology journal, in Italia (2006). +Bacillus subtilis strain E9-1was identified in “Isolation, identification, and characterrization of bacterial isolates degrading chlorophenols and absorbable organic halides”, author :Dhakephalkar, P.K., Lapsiya, K.L., Savant, D.V. and Ranade, D.R.. It was published on Microbial science Division in 2008, at India. -Bs2 and BN9 strains were consistent with Bacillus megaterium (99%). Bs2 sample is isolated from “mud-dreg” and BN9 from waste water. Bacillus megaterium is the one of bacteria that is commonly used in the field of environment treatment. 4.Conclusion and Suggestion: From “Mud-dreg” and waste water, there had 21 bacteria strains were isolated. Through out gram staning, spore staining, biochemistry test and biomolecular method, 2 strains are identified as Bacillus subtilis (99%, 87% identical). 20 Bs2 and BN9 strains were consistent with Bacillus megaterium (99%). Bacillus megaterium is the one of bacteria that commonly used in the field of environment treatment. Due to the limitation of time, several studies should be done in the future to determine the capacity of these isolated strains in degrading “mud-dreg” applies for fertilizer for plant. 21 REFERENCES Vietnamese Bùi Chí Bửu & Nguyễn Thị Lang (2004), Di truyền phân tử, NXB Nông nghiệp, TP. Hồ Chí Minh. triển Công nghệ Sinh học, Đại học Cần Thơ, tr.20-50. Cao Ngọc Điệp và Nguyễn Hữu Hiệp. 2002. 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(1999), “Characterization of two Bacillus probiotics”.Appl.Environ.Microbiol.65(9), pp.4288-4291. 23 24 [...]... -Bs2 and BN9 strains were consistent with Bacillus megaterium (99%) Bs2 sample is isolated from “mud-dreg” and BN9 from waste water Bacillus megaterium is the one of bacteria that is commonly used in the field of environment treatment 4.Conclusion and Suggestion: From “Mud-dreg” and waste water, there had 21 bacteria strains were isolated Through out gram staning, spore staining, biochemistry test and. .. industrial site of Bagnoil” research, author: Sprocati, A.R., Alisi, C And Tasso, F and published on Modern multidisciplinary applied microbiology journal, in Italia (2006) +Bacillus subtilis strain E9-1was identified in Isolation, identification, and characterrization of bacterial isolates degrading chlorophenols and absorbable organic halides”, author :Dhakephalkar, P.K., Lapsiya, K.L., Savant, D.V and Ranade,... with Bs4 and BN5: +Bacillus subtilis strain S20 was identified by Mohammadou, B.A., Mbofung, C.M and Barbier, G in a research “Genotypic and phenotypic diversity among Bacillus species isolated” It was published on Food engineering and 19 Quality journal in 2012, at University Institute of Technology, Cameroon, Africa +Bacillus subtilis strain OSS 42 was sequenced in “Comparison of microbial communities... DNA profiles of 9 well, others lanes are follows Bs1; Bs2; Bs4; BN1; BN2; BN5; BN9; BN12; control (+); control(-) 3.4.Sequencing: After sequencing by NUCLEOTIDE BLAST (http://www.ncbi.nlm.nih.gov/) Two strains have been identified Bacillus subtilis (denoted as Bs4 and BN5) with 99% and 87% The nucleotide sequence result can be seen in appensix -Three Bacillus subtilis strains identical with Bs4 and. .. chosen Similar studies of Bacillus subtilis of Sharmin Rahman (2007) and Tran Linh Thuoc (2008) 3.2.5.Stained bacterial spore: When observed under the microscope at 400 times magnification, 8 strains bacterial spores form oval, accounting for 50%, they stared green of malachite green solution This result the same with studies of Stainer et al.,(1999), Slepecky and 17 Hemphill (2006) and Luong Bich Dung... biomolecular method, 2 strains are identified as Bacillus subtilis (99%, 87% identical) 20 Bs2 and BN9 strains were consistent with Bacillus megaterium (99%) Bacillus megaterium is the one of bacteria that commonly used in the field of environment treatment Due to the limitation of time, several studies should be done in the future to determine the capacity of these isolated strains in degrading “mud-dreg”... Sankoff (1998), “The evolutionary relationships among known life forms”, J.Mol.Evol., 28, pp.98-112 Cutting, S.M & P.B Vander Horn 1990 “Genetic analysis” In: C.R Harwood and S.M Cutting Ed) Molecular biological methods for Bacillus, John Wiley & Sons Ltd., Chichester, England, pp.27-74 Jang-Seu Ki, Wen Zhang, Pei-Yuan Qian,” Discovery of marine Bacillus species by 16S rRNA and proB comparisons and. .. bubbling (6.25%) and 4 strains without bubbling, that’s mean no catalase activity, accounting for 25% Reaction 2H2O2 catalase 2H2O + O2 Bacillus subtilis are capable of producing enzyme catalase When exposed to H2O2 is generated O2 bubbling in previous studies on the possibility of creating catalase by Nguyen Duc Luong and Nguyen Thi Thuy Duong (2003), Slepecky and Hemphill (2006), and Sharmin Rahman... on the thickness of the smear on the slide Rinse the crystal violet stain off of the slide with water Place Gram's iodine solution on the bacterial smear Let the smear stand for another 10 to 60 seconds depending on smear thickness Rinse off the extra Gram's iodine solution with more water Add several drops of a decolorizer to the bacterial smear on the slide Rinse the decolorizer off of the slide when... spore-stain Bacillus subtilis Staining the spore of bacteria, the firt step like Gram staining, after that take machite green 5% on specimens Clean up lam under water then keep it in safranin liquid about 1minute and clean up under water Observing with lam in the oil 2.2.6 Chemical reaction to identify Bacillus subtilis a Catalase reaction: Drip one drop of H2O2 30% on the lam, take a amount of colony