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Optimization of Biosurfactant Production by Bacillus licheniformis DW3 Muneer Ahmed Qazi, Maria Abid, Abdul Hameed and Safia Ahmed Department of Microbiology.

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Presentation on theme: "Optimization of Biosurfactant Production by Bacillus licheniformis DW3 Muneer Ahmed Qazi, Maria Abid, Abdul Hameed and Safia Ahmed Department of Microbiology."— Presentation transcript:

1 Optimization of Biosurfactant Production by Bacillus licheniformis DW3 Muneer Ahmed Qazi, Maria Abid, Abdul Hameed and Safia Ahmed Department of Microbiology Quaid i Azam University, Islamabad. Title: Author(s): Affiliation:

2 Introduction Objectives of Study Plan of Study Materials and Methods Screening Optimization of Biosurfactant Production Results Screening Optimization of Biosurfactant Production

3 General Applications of Biosurfactants Classification of Biosurfactants Why Biosurfactants? Mechanism of Action What are Biosurfactants? Merits  Biodegradability  Generally low toxicity  Biocompatibility and digestibility  Availability of raw materials  Acceptable production economics  Use in environmental control  Specificity  Vast application fields Challenges  Expensiveness at large scale  High grade purity  Low productivity  Use of expensive media  Poor understanding of synthesis regulation  Foam formation Two classification systems:  On the basis of Molecular mass Low-molecular-mass molecules High-molecular-mass molecules  On the basis of Polar nature Anionic Cationic Neutral Amphoteric Biosurfactants are biological surface active agents that are:  Amphiphilic  Biodegradable  Less toxic  Environmentally compatible  Highly selective  Specifically active  Enhanced Oil Recovery (EOR)  Bioremediation and Biodegradation of hydrocarbons  Pharmaceuticals  Cosmetics  Food industry  Textile industry  Detergents and cleaners  Herbicide and Pesticide formulations  Leather and Paper industries  Agriculture  Bioleaching of Metals  Immunological molecules  Biomedical field Introduction Reduce: Surface tension Critical Micelle Concentration (CMC) Interfacial tensions Improve: Bioavailability of Hydrocarbons Form: Conditioning film at interface Remove: Lipopolysaccharide layer of microbes

4  To screen bacteria for biosurfactant production  To maximize biosurfactant’s yield from B. licheniformis DW3 by optimizing different cultural and environmental conditions, such as:  Inoculum size  Temperature  pH  Carbon sources  Carbon source concentration  Nitrogen source  Agitation speed  Oil as additional Carbon Source

5 Production and Optimization Experiments Production-basedGrowth-based Screening Experiments Primary (Agar Plate-based) Secondary (Fermentation Broth-based)

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7  Microorganism: Best Bioemulsifier producer strain was further studied for Production and Optimization experiments.  Inoculum Preparation: A 5% of seed culture of the bacterial strain grown in nutrient broth at 30 ºC, 150rpm, for 18-24hours was used as inoculum.  Production Medium: The Mineral Salts Medium (MSM) in addition with Carbon and Nitrogen sources separately sterilized was used as production medium.Mineral Salts Medium (MSM) Optimization of culture conditions was carried out.culture conditions Microorganism Bacillus endophyticus MD1, Bacillus subtilis SNW3, Bacillus licheniformis DW3, Psychrobacter sp. DW6, Pseudomonas putida SOL-10 and Bacillus sp. SS1 Primary Screening Oil Spread Method (JP ) Oil Displacement Area (ODA) Luria Bertani (LB) Agar Secondary Screening Emulsification Activity (E 24 ) % Drop-Collapse Test Mineral Salts Medium (MSM)

8 Media and their composition Luria Bertani (LB) Agar Tryptone 1% Sodium chloride 0.5% Yeast extract 0.5% Agar 1.5% pH 7.0±0.2 Luria Bertani (LB) Agar Tryptone 1% Sodium chloride 0.5% Yeast extract 0.5% Agar 1.5% pH 7.0±0.2 Mineral Salts Medium (MSM) (g/L) Na2HPO4 2.2 KH2PO41.4 MgSO4.7H2O0.6 FeSO4.7H2O0.01 NaCl0.05 CaCl20.02 Yeast Extract0.02 and 0.1ml of trace element solution containing (g/L): ZnSO4.7H2O 2.32 MnSO4.4H2O1.78 H3BO CuSO4.5H2O1.0 NH4MoO4.2H2O0.39 KI0.66 EDTA1.0 pH 7.0± % carbon and 0.1 % nitrogen sources separately sterilized. Mineral Salts Medium (MSM) (g/L) Na2HPO4 2.2 KH2PO41.4 MgSO4.7H2O0.6 FeSO4.7H2O0.01 NaCl0.05 CaCl20.02 Yeast Extract0.02 and 0.1ml of trace element solution containing (g/L): ZnSO4.7H2O 2.32 MnSO4.4H2O1.78 H3BO CuSO4.5H2O1.0 NH4MoO4.2H2O0.39 KI0.66 EDTA1.0 pH 7.0± % carbon and 0.1 % nitrogen sources separately sterilized.

9  Inoculum's size (1, 2, 3, 4, 5, 7, and 10%).  Temperature (25, 30,37,45 and 50°C).  Agitation speed (0, 100, 150 and 200rpm).  pH (3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,8.5,9,9.5 and 10).

10  Carbon sources  Peptone, malt extract, corn oil, glucose, yeast extract, olive oil, used oil, soyabean oil  Carbon source concentration  0.5, 1, 1.5 and 2%  Nitrogen sources  NaNO3, NaNO2, NH4NO3, and Urea  Oil as additional Carbon Source  0.5,1% soyabean and 0.5,1% used oil

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12 1. A few drops of crude oil were dropped onto the solid surface of LB agar and uniformly spread and left for 24h. 2. After 24h the plates were centrally inoculated with cultures to be screened, and incubated at 37 ºC for 24h. 3. Halos of oil repellence were observed and halo size was then measured. 4. Halo size was measured in cm.

13 1. The 50ml of distilled water was added to a large Petri dish (15 cm diameter) μl of crude oil is then added to the surface of water μl of culture supernatant broth is then poured in center of the oil film. 4. Zone of displacement is visualized and measured. 5. ODA = 22/7 (radius)2 cm2

14 1. Equal volumes of kerosene and cell-free supernatant in test tube were vortexed at high speed for 2 min and allowed to stand for 24h. 2. The E 24 index is given as percentage of the height of emulsified layer (cm) divided by the total height of the liquid column (cm).

15 1. The drop of cell culture or culture supernatant is dropped onto a hydrophobic oil coated surface. 2. The size and shape of the drop is anlyzed for biosurfactant production. 3. If the drop contains surfactant it is collapsed and the size of drop is increased. 4. Size of drop is measured in mm/μm.

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17 S. No.Bacterial sp.Halo size (cm) 1Bacillus subtilis SNW32.5 ± 0.2 2Pseudomonas putida SOL ± 0.2 3Bacillus endophyticus MD12.0 ± 0.2 4Psychrobacter sp. DW61.5 ± 0.2 5Bacillus licheniformis DW34.5 ± 0.2 6Bacillus sp. SS10.5 ± 0.2

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28  B. licheniformis DW3 is a potent biosurfactant producer  Optimum parameters for maximum production of biosurfactant were found as:  Inoculum's size 5%  Temperature 30ºC  pH 8.0

29  Agitation 150rpm  2% yeast extract as carbon source  Highest emulsification index (E 24 %=62.43) was attained at optimized conditions  Although the experiments with different nitrogen sources and oil as an additional carbon source revealed some negative effects on biosurfactant production, they had positively supported heavy growth.

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