Optimization of Biosurfactant Production by Bacillus licheniformis DW3

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

2. Table of Contents

3. 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 Biosurfactants are biological surface active agents that are: • Amphiphilic • Biodegradable • Less toxic • Environmentally compatible • Highly selective • Specifically active • Reduce: • Surface tension • Critical Micelle Concentration (CMC) • Interfacial tensions • Improve: • Bioavailability of Hydrocarbons • Form: • Conditioning film at interface • Remove: • Lipopolysaccharide layer of microbes 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

4. To screen bacteria for biosurfactant production To maximize biosurfactant’s yield from B. licheniformisDW3 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 Objectives of the study

5. Study Plan

6. Materials and Methods

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. • Optimization of culture conditions was carried out. • Microorganism • Bacillus endophyticus MD1, Bacillus subtilis SNW3, Bacillus licheniformis DW3,Psychrobactersp. DW6, Pseudomonas putida SOL-10 and Bacillus sp. SS1 • Primary Screening • Oil Spread Method (JP05211892) • Oil Displacement Area (ODA) • Luria Bertani (LB) Agar • Secondary Screening • Emulsification Activity (E24) % • Drop-Collapse Test • Mineral Salts Medium (MSM) • Materials & Methods

8. Mineral Salts Medium (MSM) (g/L) Na2HPO4 2.2 KH2PO4 1.4 MgSO4.7H2O 0.6 FeSO4.7H2O 0.01 NaCl 0.05 CaCl2 0.02 Yeast Extract 0.02 and 0.1ml of trace element solution containing (g/L): ZnSO4.7H2O 2.32 MnSO4.4H2O 1.78 H3BO3 0.56 CuSO4.5H2O 1.0 NH4MoO4.2H2O 0.39 KI 0.66 EDTA 1.0 pH 7.0±0.2 + 1.5-2 % carbon and 0.1 % nitrogen sources separately sterilized. Luria Bertani (LB) Agar Tryptone 1% Sodium chloride 0.5% Yeast extract 0.5% Agar 1.5% pH7.0±0.2 Materials & Methods

9. Optimization parameters for Biosurfactant Production 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). Materials & Methods

10. Continued… 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 Materials & Methods

11. Analytical Methods

12. Oil Spread Method (JP05211892) A few drops of crude oil were dropped onto the solid surface of LB agar and uniformly spread and left for 24h. After 24h the plates were centrally inoculated with culturesto be screened, and incubated at 37 ºC for 24h. Halos of oil repellence were observed and halo size was then measured. Halo size was measured in cm.

13. Oil Displacement Activity (ODA) test(Rodrigueset al., 2006) The 50ml of distilled water was added to a large Petri dish (15 cm diameter). 20μl of crude oil is then added to the surface of water. 10μl of culture supernatant broth is then poured in center of the oil film. Zone of displacement is visualized and measured. ODA = 22/7 (radius)2 cm2

14. Emulsification Activity (E24) %(Techaoeiet al., 2007) 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. The E24 index is given as percentage of the height of emulsified layer (cm) divided by the total height of the liquid column (cm).

15. Drop Collapse Method (Krepskyet al., 2007) The drop of cell culture or culture supernatant is dropped onto a hydrophobic oil coated surface. The size and shape of the drop is anlyzed for biosurfactant production. If the drop contains surfactant it is collapsed and the size of drop is increased. Size of drop is measured in mm/μm.

16. Results

17. Primary Screening by oil spread technique Results

18. Secondary Screening by Emulsification Index (E24) % Results

19. Results Effect of Inoculum Size

20. Effect of Temperature Results

21. Effect of Agitation Results

22. Effect of pH Results

23. Effect of Carbon Sources Results

24. Effect of Carbon Source Concentration Results

25. Effect of Nitrogen Source Results

26. Effect of oil as an additional carbon source Results

27. Production of Biosurfactants under optimized conditions Results

28. Conclusion 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. Continued..... Agitation 150rpm 2% yeast extract as carbon source Highest emulsification index (E24%=62.43) was attained at optimized conditions Although theexperiments 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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