INDEX Chapter No. Title Page No. Abstract 2 List of Tables 4 List of figure 5 Acknowledgement 6 Abbreviation 8 1. Introduction: Definition Origin, constitution and use Component of crude oil Behavior of petroleum in Marine environment 9 9 9 16 19 2. Aims & Objectives 22 3. Material & method: Collection of sample Culture media Biochemical reagents Methods 23 23 23 25 26 4. Results & Discussion: Physio-chemical characteristics of isolates. Biodegradation efficiency. Growth potential of isolates. Identification of petroleum degrading isolated strains 30 30 33 36 38 5. Conclusion 39 6. Appendixes: Appendix-1- Culture Medium Appendix-2- Stains & Reagents 40 40 44 7. References 45
ABBREVIATIONS BHM – Bushnell-Haas Media CaCl2 – calcium chloride D/W – Distilled water FeSO4 – Iron sulfate Gms – Grams H2O2 – Hydrogen peroxide H2S – Hydrogen sulfide HCl – Hydrochloric acid Inc. – Incubation K2HPO4 – Di-potassium hydrogen phosphate KH2PO4 – Mono potassium hydrogen phosphate KOH – Potassium hydroxide MgSO4 – Magnesium sulfate MnSO4 – Manganese sulfate M-R – Methyl red test Na2HPO4 – Disodium hydrogen phosphate NB/NA – Nutrient broth/Agar NaCl – Sodium chloride NaOH – Sodium hydroxide NH4Cl – Ammonium Chloride RPM – rotation per minutes SWA – Sea agar media Temp. – Temperature TMPD – V-P – Voges-Proskauer
Sphingomonas paucimobilis EPA505 Phenanthrene, Fluoranthene, Anthracene, Naphthalene Story et al., 2001, Mueller et al., 1990 Sphingomonas wittichii RW1 Chlorinated dibenzopdioxin Nam et al., 2006 Sphingomonas sp. KS14 Phenanthrene, Naphthalene Cho et al., 2001 Terrabacter sp.DBF63 Fluorene, Dibenzofuran, Chlorinated dibenzopdioxin, Chlorinated dibenzothophene Habe et al., 2004, Habe et al., 2001, Habe et al., 2002 Xanthamonas sp. Benzo[a]pyrene Pyrene, Carbazole Grosser et al., 1991 Table 2: Fungal genera capable of degrading PAHs (20):- Name of Fungus PAH Reference Phanerochaete chrysporium Anthracene Field et al.,1996 Bjerkandera sp. strain BOS55 Anthracene Field et al.,1996 Trametes versicolor Anthracene Collins et al., 1986 Cunninghamella elegansoxidizes Anthracene Cernigilia, 1997 P. chrysosporium Anthracene Hammel et al., 1991 Aspergillus flavus Benzo[a]pyrene Romero et al., 2010 Paecilomyces farinosus Benzo[a]pyrene Romero et al., 2010 Oil fields are not uniformly distributed around the but being in limited areas such as the Persian Gulf region. The world production of crude oil is more than three billion tons per year, and about the half of this is transported by sea. Consequently, the international transport of petroleum by tankers is frequent. All tankers take on ballast water which contaminates the marine environment when it is subsequently discharged. The recent spill of more than 200,000 barrels of crude oil from the oil tanker Exxon Valdez in Prince William Sound, Alaska, as well as smaller spills in Texas, Rhode
They were incubated at 30°C at 160rpm for 21 days. A control devoid of the bacterial isolate was prepared for each set of experiments.(17) Table-6 Biochemical Reagents Test Medium Reagent Observation 1.Carbohydrate fermentation test Glucose, maltose, Sucrose, Lactose, Mannitol, Xylose Phenol red Red yellow color ( Gas production) 2. Urea utilization test Urea broth, Phenol red Pinkish red color 3. H2S Production test 2% Peptone Lead acetate strip Blackish of paper 4. Gelatin hydrolysis test Nutrient gelatin broth – Liquefaction at 4°C 5. Citrate utilization test Simmons Citrate agar Bromothymole blue Green-Blue 6. Nitrate reduction test Peptone nitrate broth Sulfanylic acid + a-Naphylamine Red color 7. Oxidase test Nutrient Agar Slant Oxidase strip Violet 8. Catalase test Nutrient Agar Slant 3% H2O2 Formation of bubbles 9. M-R test Glucose Phosphate Methyl red Red color 10. V-P test Glucose Phosphate 40% KOH + a- Naphthol Pink 11. Iodole production test 1% Peptone Kovac`s reagent Red ring production 12.TSI slant Triple Sugar iron agar Slant – – 13. Macconkey`s Agar plate Macconkey’s agar plate – – 14. Gram`s stainining – Grams iodine, Crystal violet, Ethanol, D/W, Safranin Microscopic observation.
3. H2S Production test – – - 4. Gelatin hydrolysis test – – - 5 Citrate utilization test + + + (Blue color) 6. Nitrate reduction test - + + 7. Oxidase test + + + 8. Catalase test + + + 9. M-R test – – + 10. V-P test – – – 11. Iodole production test – – – 12. TSI slant No change No color change Slant/butt- Yellow No gas 13. Macconkey`s Agar plate No growth obtained Yellowish color colony Grown Pink colored colony With pink centre 14. Gram`s stainining Gram positive, Cocci Gram negative, Rod shape Gram negative, rod Shaped 15. Motility Non-motile Motile Non-motile Keys- + -- Positive test – -- Negative test Biodegradation efficiency:-
Inc. (Days) Control (Toluene) Test (Toluene) Control (Xylene) Test (Xylene) 0 Clear particles on top. Clear particles on top. Clear particles on top. Clear particles on top. 1 Same as above Same as above Same as above Same as above 5 Same as above Same as above Same as Same as above 10 Same as above Same as above Same as Same as above 15 Same as Medium become slightly cloudy Same as above Medium become cloudy 21 Same as above Same as above Same as above Same as above Table-9.3.1 By using A1 Bacterial culture Inc. period (Days) Control (Petrol) Test ( Petrol) Control (Diesel) Test (Diesel) 0 Clear particles of orange oil on top. Clear particles of orange oil on top. particles of orange oil on top. Clear particles of orange oil on top. 1 Same as above Same as Same as above Same as above 5 Same as above Medium become cloudy Same as above Medium become cloudy 10 Same as above Same as Same as above Same as above 15 Same as more cloudy Same as More cloudiness’ 21 Same as above Same as Same as above Become milky Table-9.3.2 By using A1 Bacterial culture Inc. period (Days) Control (Engine oil) Test (Engine oil) Control (Benzene) Test (Benzene) 0 Clear particles of orange oil on top. particles of orange oil on top. Clear particles on top. Clear particles on top. 1 Same as above Same as above Same as above Same as above 5 Same as above Medium become cloudy Same as above Medium become cloudy 10 Same as above Same as above Same as above Same as above 15 Same as above Increase cloudiness’ Same as above Same as above 21 Same as above Become milky Same as above more cloudiness’ Table-9.3.3 By using A1 Bacterial culture
Chapter-6 APPENDIXES Appendix-1 Culture media: Bushnell-Haas Media:- Components Amt. (Gms/Lit.) MgSo4 0.200 gm CaCl2 0.020 gm K2HPO4 1.0 gm KH2PO4 1.0 gm Ammonium Nitrate 1.0 gm Ferric Chloride 0.050 gm Final pH 7.0 ± 0.2 Directions- Suspend 3.270 grams in 1000 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Take 990 ml BHM +10 ml Hydrocarbon source (Oil, Petrol etc.) . Glucose Phosphate Broth:- Components Amt. (Gms/Lit.) Glucose 5.0 gm K2HPO4 5.0 gm Peptone 5.0 gm D/W 1000 ml Final pH 6.9-7.0 Directions- Suspend 15 in 1000 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Macconkeys Agar Media:- Components Amt. (Gm/Lit.) Peptone 17.0 gm
Protease peptone 3.0 gm Lactose 10.0 gm Bile salt 1.5 gm NaCl 5.0 gm Neutral red 0.03 gm Agar 20.0 gm Final pH 7.1 ± 0.2 Directions- Suspend 56.53 Gms in 1000 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Nutrient Agar Media:- Components Amt. (Gms/Lit.) Peptone 10 gm NaCl 5 gm Beef Extract 3 gm Agar 20 gm Final pH 7.4 ± 0.2 Directions- Suspend 38 gms in 1000 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Nutrient Gelatin broth:- Components Amt. (Gms/Lit.) Meat extract 3.0 gm Peptone 10.0 gm Gelatin 150.0 gm D/W 1000.0 ml Final pH 7.2 Directions- Suspend 163.0 Gms in 1000 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Nutrient sugar Broth:- Components Amt. 1% Peptone 90 ml