Isolation And Identification Of Crude Oil Degrading Bacteria From Soil Polluted With Automobile Lubricants

                                              MATERIALS AND METHODS

2.0. MATERIALS

2.2. SAMPLE COLLECTION

The soil contaminated with used motor oil collected from Mechanic Village, Nsukka, Enugu state. The lubricating oil was collected from total filling station, Nsukka, Enugu state. The kerosene was collected from Mobil filling state, Nsukka, Enugu state.

2.2. REAGENTS AND MEDIA

Nutrient agar, agar-agar, inorganic salts, casein, peptone, phenol red, sucrose, maltose. Lactose, dextrose, beef extract, mannitol, bromothymol blue, glucose, Kocac’s reagent, acetic acid, α-naphthalamine, suphanilinic acid, trypose, lubricating oil, distilled water, crystal violet, lugol’s iodine, ethanol and safranine.

2.3. APPARATUS

Gas cylinder, Bunsen burner, refrigerator, autoclave, syringe, detergent, test-tube rack, wire loop, spatula, glass rod, bijou bottles, petri dishes, test tubes, incubator, cotton wool, measuring cylinder, gloves, aluminum foil, durham tubes, slides, conical flask, weighing balance, filter paper, pressure pot, spectrophotometer, microscope, paper tape and maker.

2.4. METHODS

2,5, MEDIA PREPARATION

2.5.1. PREPARATION OF BUSHNELL HAAS BROTH (MINERAL SALT BROTH)

1.25g of (NH   4)2SO4 (ammonium sulphate), 1.95g of (NH4)2HPO4, 0.85g of KH2PO4 (potassium dihydrogen phosphate), 0.09g of MgSO4.7H2O (magnesium sulphate) and 0.01g of CaCl2 (calcium chloride) were all dissolved in 1000ml of distilled water, homogenized and sterilized at 121°C for 15 minutes.

2.5.2. PREPARATION OF BUSHNELL HAAS AGAR (MINERAL SALT AGAR)

0.50g of K2HPO4, 1.00g 0f NH4Cl, 2.00g of Na2SO4, 2.00g of KNO3, 1.oog of MgSO4, 0.01g of CaCl2 and 15g gram of agar-agar was dissolved in 1000ml of distilled water, homogenized and sterilized at 121°C for 15 minutes, then poured into petri dishes under aseptic condition.

2.5.3. PREPARATION OF NUTRIENT AGAR

23g of nutrient agar was dissolved in 1000ml of distilled water, homogenized and sterilized at 121°C for 15 minutes, then poured into petri dishes, allowed to solidify under aseptic condition.

2.6. ISOLATION OF CRUDE OIL DEGRADING BACTERIA

2.6.1. INNOCULATION OF SAMPLE INTO BUSHELL HAAS BROTH

The prepared Bushnell Haas broth was poured into four sterile conical flasks labeled A, B, C and D under aseptic condition, each conical flask containing 100ml each. 10g of the contaminated soil sample was added into each of the three conical flasks (A, B and C; where D served as control) under aseptic condition and incubated at room temperature for 7 days.

2.6.2. SERIAL DILLUTION

After 7 days, the Bushnell Hass broth containing the contaminated soil sample was serially diluted up to 10-9 fold dilution using 9ml of distilled water. This is done for the three conical flasks (A, B and C).

2.6.3. INNOCULATION INTO BUSHNELL HAAS AGAR

After 3 days condition into already prepared Bushnell Haas agar plates using spread plate method, these was done in duplicates. Filter papers were dipped into kerosene and attached to the cover of the plates aseptically to serve as the carbon source for the inoculated microorganism, and and then the plates were incubated at room temperature for 3 days.

2.6.4. VIABLE PLATE COUNT

Colonies observed were counted and the morphology was noted.

2.6.5. PURE CULTURE

 After 3 days, distinct colonies were picked from the Bushnell Haas agar and streaked on already prepared nutrient agar plates in aseptic condition and incubated at room temperature for 24 hours.

2.6.6. PREPARATION OF AGAR SLANT

Agar slant is done by preparing and sterilizing nutrient agar in bijou bottles at 121°C for 15 minutes and then kept in angle of 45° to get in order to form a slant. After which the bottles were incubated at room temperature for 24 hours to test for sterility.

2.6.7. INNOCULATION INTO AGAR SLANT

Ingle and distinct colonies from the nutrient agar plates were inoculated into the labeled nutrient agar slants and incubated at room temperature for 24 hours, after which they were stored in the refrigerator at 4°C.

2.7. IDENTIFICATION OF ISOLATES

2.7.1. MORPHOLOGICAL EXAMINATION AND IDENTIFICATION

2.7.1.1. MACROSCOPY

Macroscopy of the pure isolate was observed and identified based on the colour of the colonies, elevation, edge, texture, form and density.

2.7.1.2. MICROSCOPY

2.7.1.3. GRAM STAINING

Gram staining divides into two groups:

Gram positive and

Gram negative bacteria.

The two groups differ due to differences in cell wall organization. Gram positive bacteria have thicker peptidoglycan layer, they retain the crystal violet dye, they are not decolourized by alcohol while Gram negative bacteria lose the crystal violet, decolourized by alcohol and take of the second dye, safranin. Gram positive bacteria stain purple while the Gram negative bacteria stain red.

A drop of sterile water was added on a clean glass slide using a properly flamed wire loop, small amount of the culture was mixed with the sterile water on the glass slide and spread to make smear. The smear was allowed to air-dry. The smear was then heat fixed by passing it over the flame and allowed to cool. The smear was stained with 0.5% crystal violet solution, which is the primary stain for one min was then added to the ute and washed off. Lugol’s iodine was then added to the slide for one minutes and washed off.

The slide was decolourized with alcohol by adding it uniformly on the slide and washed of immediately. The smear was finally counter stained using safranin soulution for one minute and washed off. The slide was allowed to air-dry. Oil immersion was added to the slide and the slide was then viewed under X100 magnificaton objective lens of a microscope.

2.7.2. BIOCHEMICAL TESTS

2.7.2.1. CATALASE TEST

This test is used to determine the ability of the isolate to produce catalase enzyme. Catalase breaks down hydrogen peroxide and releases oxygen which detected as effervescence. The different isolates were picked separately with a flamed wire loop and placed on a different glass slides. A loop full of 3% hydrogen peroxide solution was added to the different slides. A positive result was indicated by appearance of effervescence after few secomds.

2.7.2.2. SUGAR FERMENTATION TEST

This test is used to check the ability of an organism to ferment various sugars.The test isolates were inoculated in peptone water broth containing 1% solution of the desired sugars(sucrose, lactose, maltose, dextrose and mannitol) in test tubes. Phenol red is added to the solution to serve as indicator of acid production. Inverted Durham tubes were inserted into the culture tube to check for gas production. The test culture was incubated at 37°C for 24 hours, A positive result was indicated by the production of acid by change in colour of the medium to red or pink and gas production by presence of gas bubbles in the Durham tubes.

2.7.2.3. NITRATE REDUCTION TEST

This test is used to detect the production of nitrate reductase enzyme by the test isolates. Nitrate reductase reduces nitrate to nitrite,  nitrous oxide, nitrogen and ammonia. The test isolates were inoculated into 5ml of nitrate broth, containing potassium nitrate, distilled water and peptone incubated at 37°C for 96 hours. After this, 1ml of α-naphthylamine reagent and 1ml of sulphanilamide reagent were added to the medium. Development of red colour with few minutes shows a positive result.

2.7.2.4. INDOLE TEST

This test is used to detect if the test isolate produce tryptophanase enzyme. This enzyme breaks down tryptophan into indole, pyruvic acid and ammonia. The isolates were inoculated into peptone water broth and incubated  at 37°C for 48 hours. 0.5ml of Kovac’s reagent was added to the medium and shaken gentily. Pink or red colour on top of the culture indicates a positive result.

2.7.2.5. STARCH HYDROLYSIS TEST

This test is used to determine the ability of test isolates to use starch as a source of carbon and energy for growth. The use of starch was achieved by the enzyme, alpha-amylase. The test isolates were streaked on a sterile plate of starch agar and incubated at 37°C for 24 hours. Iodine reagent was added to flood the growth on the plate. A positive result was indicated by the presence of clear halos around the colonies.

2.7.2.6. SIMMON CITRATE TEST

This test is used to examine the ability of an organism to utilize citrate present in Simmon’s medium as a source of carbon for growth.The test isolates were inoculated directly on different Simmon’s citrate agar slants. The medium contains bromothymol blue which selves as indicator and incubated at room temperature for 24 hours. A positive result was indicated by appearance of growth with blue colour from green colour.

2.7.2.7. METHYL RED TEST

This test is used to detect the organisms that are mixed fermenters. Some bacteria ferment glucose to produce large amount of lactic, acetic, succinic and formic acids plus carbon dioxide, ethanol and hydrogen. Mixed acid fermenters produce enzyme formic hydrogenlyase which split formic acid to hydrogen and carbon dioxide in equal parts.The test isolates were inoculated in glucose phosphate broth and incubated at 37°C for 3 days. After this, 5 drops of 0.004% of alcoholic methyl red solution was added to the medium and mixed very well. Bright red indicates a positive result while a yellow indicates a negative result.

2.7.2.8. VOGES-PROSKAUER TEST

This test is used for negative methyl red test culture. It is an indirect way of testing for 2, 3-butanediol. A negative methyl red indicates the production of a lot of 2, 3-butanediol and ethanol instead of acids by bacteria. An organism cannot be simultaneously Methyl Red and Voges Proskauer positive but can be Voges Proskauer and Methyl Red negative at the same time if it does not ferment or attack glucose.

1.5ml of 5% alcoholic α-naphtol was added to the test tubes containing the medium showing negative methyl red test. 0.5ml of 40% KOH was added. The test tubes were shaken and allowed to stand for 5 minutes. A positive result was indicated bt the pink or red colour. Faint pink colour developing late was scored negative.

2.7.2.9. MOTILITY TEST

The test is used to detect organisms that are motile. This test was carried out using culture method. The test isolates were different inoculated in a semi-solid agar containing agar, beef extract, sodium chloride and gelatin using a straight wire loop, making a single stab down the center of the tube to about half the depth of the medium and incubated at room temperature for 24 hours. Motile bacteria give diffuse, hazy growth that spread throughout the medium away from the line of stab while non-motile bacteria give growths along the line of stab.

2.8. DETERMINATION OF RATE OF UTILIZATION OF CRUDE OIL BY THE DIFFERENT ISOLATES

This is carried out using spectrophotometer. It is used to determine the ability of isolates to degrade crude oil and to select the degraders of crude oil. As the optical density increases, the medium becomes more turbid showing that the isolate is utilizing the crude oil as source of carbon and energy. The isolates were inoculated into 100ml of mineral salt broth containing 0.42g of MgSO4.7H2O, 0.29g of KCl, 0.85g of KH2PO4, 0.42g of NaNO3, 1.27g of K2HPO4 and 0.1g of NaCl, o.5ml of lubricating oil was added to the medium. This is done under aseptic condition. The medium was shaken vigorously. The medium is placed on a rotary shaker for continuous shaking. Reading were taken consecutively after every 24 hours for 7 days using spectrophotometer at a wavelength of 600nm starting from the zero reading, which is taken immediately after inoculation