In the production of surfactin from Bacillus sp. , two types of Bacillus subtilis strains were used in isolating the wanted lipopeptides; surfactin fot this study. Bacillus subtilis ATCC 21332 and Bacillus subtilis 3M was selected to produce surfactin provided by Faculty of Sciences and Technology, Universiti Sains Islam Malaysia. 3. 2. 1. 2 Culture Media Both Bacillus subtilis ATCC 21332 and Bacillus subtilis 3M was first subcultured individually on nutrient agar preparing by diluting the nutrient agar powder with distilled water; 28g producing 1 litre or nutrient agar.
The quantity of nutrient agar produced was according to the packaging of the manufacturing. For the first and second stages of the fermentation, the seed cultured from the nutrient agar were transferred into a defined mineral salts medium (MSM) developed by Cooper et al, 1981 known as Cooper`s media. The media consists of 0. 03M KH2PO4, 0. 04M Na2HPO4, 0. 05M NH4NO3, 7. 0 x 10-6 M CaCl2, 4. 0 x 10-6M FeSO4. 7H20, 4. 0 x 10-6M EDTA, 8. 0 x 10-4M MgSO4 and 4%(w/v) glucose.
All chemicals were diluted with distilled water and sterilized in autoclave at 121?C for 15 minutes except for 4. 0 x 10-6M FeSO4. 7H20 which after diluting with distilled water was sterilized using syringe filter. 3. 2. 1. 3 Inoculum and culture conditions Culture of Bacillus subtilis ATCC 21332 and Bacillus subtilis 3M were taken from -80?C frozen stock and agar slant stock respectively for precultured onto the nutrient agar. The nutrient agar streaked with the Bacillus subtilis ATCC 21332 and Bacillus subtilis 3M were incubated at 37?C for overnight.
Two loops of cells were then inoculated into 100mL of Cooper`s media in 250mL Erlenmeyer flask and incubated in a rotator shaker at 37?C with 200rpm agitation for 24hours as the first stage of fermentation or for the preparation of seed culture. The second stage of the fermentation was done by transferring 5mL of seed culture into 245mL of Cooper`s media in 500mL Erlenmeyer flask and further incubated in a rotator shaker at 37?C for 144hours with 200rpm agitation. For every 24hours, samples were collected for further analysis of bacterial growth determination and surfactin concentration. . 2. 2 Analytical measurement 3. 2. 2. 1 Measurement of Bacterial Growth Each of the samples taken during the time interval was used to determine the bacterial growth during the cultivation period of 144hours. A biophotometer with a wavelength of 600nm was used in reading the absorbance of each of the sample.
The reading was done triplicate. A graph of ODnm of each of the sample versus time interval will ne plotted using the result obtained from the biophotometer. The method was done both for the sample taken from the fermentation of B. ubtilis ATCC 21332 and B. subtilis 3M strains. 3. 2. 2. 2 Surfactin Concentration Analysis Each of the culture sample taken for every 24 hours for 144 hours(23h, 48h, 72h, 96h, 120h, and 144h) were withdrawn and centrifuged using the mini centrifuged (Eppendorf) at 8000rpm for 10 minutes to separated the biomass impurities and supernatant. The supernatant was then filtered using a 0. 45µm Nylon membrane filter and collected in six labelled screw capped-vials for each of the time interval respectively.
The supernatant samples were analyzed using an HPLC (Agilent Technologies, 1100 Series, USA) equipped with C-18, 250mm x 4. 6 mm, 5µm), and detected at 205nm with a Variable Wavelength Detector(VWD). The system was operated at a flow rate of 1. 5mL/min with solvents 3. 8mM trifluoroacetic acid (TFA) in 80% acetonitrile (ACN) under isocratic mode. Surfactin purified purchased from Sigma served as standard. The area of the peaks eluting between 7 to 37 minutes were identified as having the same retention times as those peaks eluting from the standard surfactin.
The total surfactin peak area was obtained by summing the area under the peaks that eluting identical to the standard. The value was further used in determining the surfactin concentration in fermentation broth of both B. subtilis ATCC 21332 and B. subtilis 3M, as well as samples from the recovery procedure. 3. 2. 2. 3 Surfactin Calibration Curve Standard solution was prepared in several dilutions (10 mg/L, 50mg/L, 100mg/L, 200mg/L, 500mg/L, 600mg/L and 800mg/L) from the stock solution diluted from the surfactin powder purchased from Sigma(ST.
Loius, MO, USA). The dilution series of standard surfactin was prepared with methanol HPLC grade as the solvent and further analyse by using a High Performance Liquid- Chromatography, HPLC (Agilent Technologies, 1100 Series, USA) equipped with C-18 column (Agilent Zorbax Eclipse C18, 250mm x 4. 6mm, 5µm) and detected at 205nm with the Variable Wavelength Detector (VWD). The system was operated at the flow rate of 1. 5mL/min with the solvents 3. 8 mM trifluoroacetic acid (TFA) in 80% acetonitrile under isocratic mode.
Surfactin calibration curve was constructed by plotting graph of the total peak area (TPA) against surfactin concentration. 3. 2. 2. 4 Recovery of surfactin The remaining liquor after 144 hours of fermentation was centrifuged at 10,000 x g to removw biomass impurities (Centrifuge, Beckman). The supernatant was called raw broth was further treated by acid precipitation following the method proposed by Chen et. al (2007) with a modification. 1M HCl was added into the raw broth to a pH of around 2 and further centrifuged at 10,000 x g for 15 minutes.
The yellowish precipitate was obtained and dried at 37?C for 2 days. The dried precipitate was then diluted with methanol (HPLC grade) and filtered through 0. 45µm Nylon membrane filter to be analyzed by HPLC for surfactin concentration. 3. 2. 3 Antibacterial Activity 3. 2. 3. 1 Preparation of Test Organism There are 8 microorganisms that have been used for this study. Four of them were gram-positive bacteria; Bacillus subtilis ATCC 21332, Bacillus cereus ATCC 13061, Streptococcus faecalis ATCC 29212 and Staphylococcus epidermidis ATCC 12228.
Another four of them were gram-negative bacteria; Shigella dysenteriae ATCC 11311, Serratia marcerscens ATCC 13830, Salmonella typhimurium ATCC 13311 and Klebsialle pneumonia ATCC 13883. All the bacteria strains were grown from Microbiology Laboratory, Faculty of Science and Technology, Universiti Sains Islam Malaysia. Test organisms were prepared based on the method proposed by Andrews (2001). The bacteria were cultured in Nutrient Broth (NB) for 24 hours at 37?C until the visible turbidity is equal or greater than the 0. 5 McFarland standard. 3. 2. 3. 2 Preparation of Stock Solution and Test Samples
In this study, the antibacterial activity of surfactin obtained from B. subtilis ATCC 21332 and B. subtilis 3M were compared to the antibacterial activity obtained from the surfactin standard purchased from Sigma, USA. Stock solution of standard surfactin was prepared with 100% methanol analysis grade. From the stock solution a serial dilution was performed with methanol to produced four different concentrations ranging from 100. 0mg/mL to 25. 0mg/mL. For comparing the antibacterial activity of standard surfactin, an antibacterial agent or antibiotic (Streptomycin sulfate) was used as a control.
Antibiotic solution was prepared by diluting 10. 0mg of antibiotic powder with 10mL of sterilized distilled water producing a solution with 10. 0mg/mL concentration. The solution was further sterilized through filtration with 0. 2µm membrane. A sterile dilution was prepared for surfactin obtained both from B. subtilis ATCC 21332 and B. subtilis 3M. The obtained precipitated after acid precipitation and drying was weighed and further diluted with methanol producing a stock solution each for the surfactin from both of the Bacillus sp. strain 3. 2. 4 Antibacterial Assay
The antibacterial activities of surfactin against four gram-positive bacteria (Bacillus subtilis ATCC 21332, Bacillus cereus ATCC 13061, Streptococcus faecalis ATCC 29212 and Staphylococcus epidermidis ATCC 12228) and against four gram-negative bacteria (Shigella dysenteriae ATCC 11311, Serratia marcerscens ATCC 13830, Salmonella typhimurium ATCC 13311 and Klebsialle pneumonia ATCC 13883) were evaluated by using two methods which were microdilution test to determine Minimum Inhibition Concentration (MIC) value, microdilution test to determine Minimum Bactericidal Concentration (MBC) value.
The tests were done by using four different concentrations in seriel dilution both for MIC and MBC. 3. 2. 4. 1 Determination of Minimum Inhibition Concentration (MIC) value by Microdilution Method. The minimum inhibition concentration (MIC) of surfactin against the bacteria will be determined by microdilution method to determine the lowest concentration of the test sample that showed no bacterial growth. In this experiment, the 96-well of sterilized microtitre plate (Biofil, India) was used.
A 25µL of surfactin (100. 0, 75. 0, 50. 0, 25. 0 mg/mL) was added into 96-well microtitre plate in decreasing order of concentration then 25µLmof an overnight incubated bacterial inoculums was added into the well that contain the test sample solution. The microtitre paltes were then incubated for overnight at 37?C. The inhibition of bacteria by the test sample were identified using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). 0µL of MTT was added into each of the test wells and the microtitre plates were further incubated at the temperature 37?C for 30 minutes. The absorbance at 630nm was measured by using the ELISA reader (Biotex, Winooski, USA) to determine the optical density value. The MIC value was determined from the lowest dilution showing no bacterial growth. 3. 2. 4. 2 Determination of Minimum Bactericidal Concentration (MBC) Value Determination of minimum bactericidal concentration (MBC) value was done based on the method proposed by Igbinosa et al. 2009) with modifications. In this method, 25µL of overnight bacterial inoculums was added with 25µL of test sample (serial dilution) in each 96 wells of microtitre plates. The plate was then incubated at 37?C for overnight. Each of the samples from the test wells were then streaked onto the nutrient agar (NA) plates. An observation was done after the incubation period. The MBC were taken as the lowest concentration of the sample that did not any visible bacteria colony on agar plate after the period of incubation.