Published on: 1 st June 2012 STUDY OF BACTERIAL DIVERSITY OF CRUDE OIL DEGRADING BACTERIA ISOLATED FROM CRUDE OIL CONTAMINATED SITES

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1 Published on: 1 st June 2012 STUDY OF BACTERIAL DIVERSITY OF CRUDE OIL DEGRADING BACTERIA ISOLATED FROM CRUDE OIL CONTAMINATED SITES CHARUSHEELA AFUWALE 1 * AND H. A. MODI 2 1*- DEPT. OF MICROBIOLOGY, SMT. S. M. PANCHAL SCIENCE COLLEGE, TALOD. 2. DEPT. OF LIFE SCIENCES, SCHOOL OF SCIENCES, GUJARAT UNIVERSITY, AHMEDABAD. charudevang@gmail.com ABSTRACT: Hydrocarbon utilizing microorganisms are ubiquitously distributed in the environment following oil spills. These microorganisms can naturally degrade numerous contaminating petroleum hydrocarbons and cleans the oceans of oil pollutants. As the usage of petroleum hydrocarbon products increases, soil contamination with diesel and engine oils is becoming one of the major environmental problems. To investigate the counter measure to remediate soils contaminated with oils, bioremediation provides an effective and efficient strategy to speed up the clean-up processes.36 bacterial isolates capable of utilizing crude oil as a carbon source were isolated from various contaminated soils using the enrichment technique. Five of them were characterized based on morphological, cultural and biochemical tests. Further, the screening was done by estimation of whole cell protein and growth in terms of turbidity when crude oil is supplied as the sole source of carbon. The degradation efficiency was confirmed by a rapid sheen screen test using 2, 6, DCPIP reduction test. KEY WORD: Bacterial diversity, Crude oil, Hydrocarbons, Bioremediation, 2, 6, DCPIP. INTRODUCTION: In natural environments, microbes occur almost always in a mixed population composed of a multitude of different strains and species. For analyzing the properties of a defined organism out of such a mixed population, a pure culture is required. Apart from direct isolation of strains by diluting and plating, enrichment cultures with hydrophobic substrates are very promising for the isolation of microbes. An efficient screening strategy is the key to success in isolating new and interesting microbes or their variants, because a large number of strains need to be characterized. A complete strategy for screening of any strain consists of three steps: sampling, isolation of strains and investigation of strains. PEER-REVIEWED Page 13

2 The principle of enrichment culture is to provide growth conditions that are very favorable for the organisms of interest and as unfavorable as possible for competing organisms. Hence, the microbes of interest are selected and enriched ( Huy et al., 1999). Biodegradation by natural population of micro-organisms is the most reliable mechanism by which thousands of xenobiotic pollutants including crude oil are eliminated by the environment. There are so many bacterial strains that can degrade or transform the components of crude oil products to the non-toxic, non- hazardous, biodegradable and environmentally friendly compounds. This action is known as a biodegradation. The biodegradation of crude oil by microorganisms is one of the primary ways for eliminating crude oil from contaminated sites and appears to be the most environmentally friendly method of removal oil pollutant. (Korda et al., 1997; Kapley et al., 1999; Del Arco and De Franca, 2001; Bharathi and Vasudevan, 2001). Bioremediation, which is accomplished by adding exogenous microbial populations or stimulating indigenous ones, attempts to raise the rates of degradation found naturally to significantly higher rates (Watanabe, 2001). Many oil-degrading bacteria have been isolated and their degradation potential is investigated. Most of bioremediation studies have been carried out using pure-cultures and the roles of these bacteria in a natural environment remain substantially unknown (Harayama et al., 1999; Lies Indah, 2007). A wide range of studies have dealt with biotransformation, biodegradation, and bioremediation of petroleum hydrocarbons and interest in exploiting crude oil-degrading organisms for environmental clean-up has become central to petroleum microbiology. There are so many of bacteria and fungi with this ability (biodegradation of oil pollution) and these organisms are widely distributed in marine, freshwater and soil habitats (Head and Swannell, 1999), but scientists reported that indigenous and adapted microorganisms are more efficient for biodegradation of oil pollutant. The adapted organism degrades oil pollution normally but rate of this action is critically depending on different factors include microbial composition, contaminant type, geology of polluted site and chemical conditions at the contaminated site (Sepahi A. et al., 2008). Soil is a rich source of micro-organisms which promote the microbial degradation of hydrocarbons and residual oil (Atlas and Bartha 1999). It has been found that soils receiving hydrocarbons like the area of oil sludge, oil fields etc. have significant higher population of hydrocarbon degrading micro-organisms. Several methods of isolating and enumerating petroleum degrading bacteria have been reported including plating on oil agar plating on silica gel oil media and inoculating liquid media containing hydrocarbons by the most probable number method. Hence in this investigation, indigenous bacteria which degrade petroleum hydrocarbons (crude oil) are isolated from various oil contaminated sites and screened for their hydrocarbon degradation efficiency. They were further characterized by morphological, cultural and biochemical techniques. PEER-REVIEWED Page 14

3 MATERIALS AND METHODOS: Collection of soil samples: Total 14 soil samples from garages, refinery waste, petrol pumps, service stations which are contaminated by petroleum refinery products like diesel, petrol and lubricant oil and from near the oil drilling sites and wells were collected in duly labeled pre-sterilized bags from the depth of 0.5 to 1.0 cm surface and subsurface. All the samples were then carefully transferred to laboratory and stored at 4 o C before analysis. Crude oil sample: The crude oil was collected from the oil drilling site of O.N.G.C. (Chandkheda) Well no. 2. (Fig 2.1) This oil was used as a sole source of carbon in entire study. It was collected in sterilized air tight bottle and stored at cool and dark place. Fig 1:Oil collection site at O.N.G.C Chandkheda with oil drilling machinery Isolation and screening: Samples from various contaminated sites were inoculated into Mineral salt Medium Bushnell and Haas Medium Hi- Media (BHM) in 250 ml Erlenmeyer flasks with 2% crude oil as sole source of carbon. The flasks were incubated on rotary shaker at 200 rpm for 7 days at room temperature, after which the cells were transferred to fresh media with crude oil as the sole carbon source. After four to five such subsequent transfers, the suspensions from each flask were streaked on Nutrient Agar plates to obtain isolated colonies (Santhini, et al., 2009). Characterization of selected isolates: Each isolate was examined for its size, shape, margin, consistency, opacity, elevation, pigmentation, Gram reaction and cell morphology as described in Bergey s Manual of Determinative Bacteriology (Holt, 1994).The isolates were characterized by using Microexpress identification kit for Gram positive as well as Gram negative bacteria. Biochemical properties tested include, production of catalase, indole, urease, oxidative fermentation of sugars, methyl red test, Voges Proskauer test and citrate utilization test, PEER-REVIEWED Page 15

4 gelatin utilization/ liquefaction test, dehydrogenase test, starch utilization test, Casein utilization test, lipid hydrolysis test etc ( Santhini et al., 2009; Holt, 1994, and Koneman, 1992). Estimation of crude oil degradation Unless otherwise mentioned, all shake flask experiments to assess crude oil degradation and factors affecting crude oil concentration by bacterial isolates were carried out in 250ml Erlenmeyer flasks with 50ml BHM and 1% crude oil as the sole source of carbon. In all cases 1.0 ml of crude oil- grown stationary phase culture was used as inoculum( A 600 =1). The flasks were incubated on rotary shaker (200rpm 30±2 0 C) for 3 to 5 days. The degradation of this crude oil by selected isolates was assessed by two methods. 1. Absorbance at 600 nm (A 600 ) was measured, because the increase in cell mass in terms of turbidity directly indicates the utilization of crude oil as the sole source of carbon. Inoculated BHM medium without hydrocarbon served as control. Cultures without any increase in turbidity over the initial O.D of test and control were scored as no growth (-), culture with slight increase over initial O.D significantly greater than the control O.D were scored as poor growth (+). Cultures with growth well above the initial were scored as moderate (++), while cultures with luxuriant growth were scored as heavy growth (+++) and (++++). (Santhini et al., 2009) 2. Estimation of whole cell protein by Folin Lowry s method (Lowry et al., 1951). Bacterial growth estimation: The estimation of growth of screened bacteria in terms of whole cell protein was done by hydrolyzing 1.0 ml of cell suspension with 1N NaOH at C for 10 min followed by quantitative estimation of protein by Folin Lowry s method (Hanson, 1993). Confirmative Screening of hydrocarbon degraders by 2,6- Dichlorophenol Indophenol (2,6-DCPIP) oxidation test Hanson et al (1993) method was used for screening hydrocarbon degrading isolates. This method consisted of incorporating into the medium an electron acceptor dye such as 2,6-dichlorophenolindophenol (DCPIP) to test the ability of the microorganism to utilize the hydrocarbon substrate by observing the color change of DCPIP from blue (oxidized) to colorless (reduced).this is one of the quick and reliable method for the screening of crude oil degrading bacteria. The cells were grown in nutrient broth and washed in saline to obtain cell density 3.5 mg dry wt/ml by adjusting A 600 = 1.0. The 25µl of this preparation was added in wells of micro titre plates. 150µl of BHM and 10µl of crude oil were then added to these wells.1.5 µg DCPIP was then added finally and plates were incubated at 30 0 C and observed for the de-colorization of dye at the interval of 1 hour for the 24 hours (Hanson 1993). PEER-REVIEWED Page 16

5 RESULTS AND DISCUSSION: Isolation of crude oil degrading Bacteria: 25 soil and sludge samples were analyzed for the isolation and screening of hydrocarbon degradation. In all cases, 2% crude oil was used as the sole source of carbon. 36 number of isolates were obtained from all the samples. (Table 1). The isolates were then grown individually on MSM and BHM media. In almost all cases, BHM medium was found to be more selective for the screening of hydrocarbon degrading bacteria so in consequent studies only BHM was used as the medium of choice. Table 1: Isolation of crude oil degrading bacteria from different sampling sites S. No. Sample source No. of isolates 1. Soil from Petrol pump Soil from near oil drilling well of O.N.G.C, Chandkheda, Ahmedabad Soil from the service station Residue from tanker Soil sludge from Jamnagar refinery Soil from oil spillage site Soil from near oil drilling well of O.N.G.C, Mehsana Fertile Garden soil 03 Total 36 Thus, the distribution of bacterial isolates obtained from various sampling sources indicates the common occurrence of metabolically active strains in areas that are contaminated with hydrocarbons strongly suggesting the ability of these bacteria to utilize these hydrocarbons as their carbon and energy source. The distribution of 36 isolates indicates the versatility and abundance of petroleum hydrocarbon degraders in terrestrial environments having constant output of hydrocarbon source. This study also proves that population of microorganisms found in a polluted environment degrades petroleum differently and at a different rate than microorganisms in a relatively clean environment (Obire and Okudo, 1997). Petroleum hydrocarbon utilizing bacteria can tolerate oil contaminated environments because they possess the PEER-REVIEWED Page 17

6 capability to utilize oil as energy sources (Iliyana et al., 1990). Other species may not and are gradually eliminated. Characterization of selected strains of bacteria: Out of 36 isolates obtained, after initial screening and enrichment, 7 isolates were characterized for morphological (Table 2.), cultural (Table 3.) and biochemical characteristics (Table 4) and identified at the genus level. Table 2. Morphological characteristics of selected bacterial isolates Sr.no Code no. of isolate Size Shape Gram s reaction Motility 1. Bn 1 Very small Small Cocci Gram Positive Non-Motile 2. Bn 2 Very small Small Cocci Gram Positive Non-Motile 3. Ps 1 Small Rods Gram Negative Motile 4. Ps 2 Small Rods Gram Negative Motile 5. Ac 1 Small Coccobacilli Gram Negative Non-Motile 6. Bs 1 Big Rods Gram Positive Non-Motile 7. St 1 Small Cocci Gram Positive Non-Motile Table 3: Cultural characteristics of selected isolates Sr.No Code no. of isolates Size Shape Elevation Margin Opacity Texture Pigment 1. Bn 1 Pin point Circular Raised Entire Opaque Smooth Orange 2. Bn 2 Pin point Circular Raised Entire Opaque Smooth Dark Yellow 3. Ps 1 Big Irregular Slightly raised 4. Ps 2 Big Irregular Slightly raised Irregular Opaque Irregular Opaque Smooth Bluish green Smooth Bluish green PEER-REVIEWED Page 18

7 5. Ac1 Very small Circular Convex Entire Slightly opaque smooth Colorless 6. Bs 1 Very big Irregular, fused Flat Irregular Opaque Rough White 7. St 1 Small Circular Raised Entire Opaque Smooth Golden yellow Table 4: Biochemical characteristics of selected isolates Name Biochemical test of Code no. of isolates Bn1 Bn 2 Ps 1 Ps 2 Ac 1 Bs 1 St 1 Citrate Utilization + + V V _ + _ Urease Production V V _ Nitrate Reduction + + V + _ H 2 S Production _ Oxidase Production _ + + Catalase Production Phenyl Alanine Deamination N.D. _ + Gelatin Utilization + + _ Starch Hydrolysis _ + _ Casein Hydrolysis _ + _ Indole Production _ N.D. N.D M. R. test + + _ V. P. test _ Lipid hydrolysis Glucose Utilization T T Sucrose Utilization + + _ T T PEER-REVIEWED Page 19

8 Lactose Utilization _ T T Xylose Utilization T T Maltose Utilization + _ + + T T T Mannitol Utilization _ T T T V- Variable, N.D Not detected, T- only turbidity observed. The preliminary identification of all the seven strains (Table 5) was done on the basis of above described morphological, cultural and biochemical characteristics. The ability of these isolates to utilize crude oil as estimated in terms of increase in turbidity indicated a variety of results ranging from luxuriant growth to moderate growth. The other method of screening for hydrocarbon degrading efficiency was estimation of whole cell protein. This method directly indicates that increase in whole cell protein implies the ability of bacteria to grow on crude oil as the sole carbon and energy source (Hanson, 1996). TABLE 5: Diversity and crude oil utilization ability of some isolates at the end of 48 hours with crude oil as the sole source of carbon Code no. of Isolates Gram s reaction Name Turbidity due to utilization of crude oil Whole cell protein( g/ml) Bn1 Gram +ve Cocci Micrococcus sp Bn2 Gram +ve Cocci Micrococcus sp Ps1 Gram -ve rods Pseudomonas sp Ps2 Gram -ve rods Pseudomonas sp Ac1 Gram -ve rods Acinetobacter sp Bs1 Positive big rods Bacillus spp St1 Gram +ve Cocci Staphylococcus sp Where + indicates poor growth, ++ indicates moderate growth, +++ and ++++ growth in terms of turbidity. indicates luxuriant Out of the isolates Ps1 and Ps 2 obtained indigenously from the soil in the vicinity of oil drilling well were found to be most efficient crude oil utilizers as observed by the visual turbidity and whole cell protein. So, these two bacterial strains were used for further investigation henceforth. PEER-REVIEWED Page 20

9 Rapid confirmative Screening of hydrocarbon - degraders by 2,6- Dichlorophenol Indophenol (2,6- DCPIP) oxidation test Hydrocarbons are highly reduced substrates requiring an electron acceptor for the initial oxidation step (Atlas, 1984). A rapid miniaturized screening technique using a redox indicator dye 2, 6, Dichlorophenol indophenol (2, 6, DCPIP) a redox indicator dye was used to assess the potential of most efficient isolates to degrade oil. Of the 36 isolates tested, only two (Ps 1 and Ps 2) were selected because they completely discolored the dye within 8 h of incubation at 30 C as shown in fig. 2.3.The other 5 isolates discolored the dye after periods of 48 h and 72 hrs, (not shown in figure 2.3) indicating slow response to the biological oxidation (Hanson, et al., 1993). The rate and extent of color change of dye from blue (oxidized) to colorless (reduced) with time indicated the potential of the bacteria to degrade crude oil. As seen in the figure 2.3 complete decolorization of the dye occurred within 8 hours in wells inoculated with two bacterial isolates Ps 1 and Ps 2. Dye containing medium without crude oil but with inoculum I.C(Inoculum control) and medium without crude oil but with inoculum S.C(Substrate control) were kept as controls which showed no decolorization. The faster and complete decolorization of dye 2, 6, DCPIP by isolates Ps 1 and Ps 2indicates their high potential for crude oil degradation. Thus, based on growth (assessed in terms of Whole cell protein) and rate of crude oil oxidation (based on 2, 6, DCPIP decolorization), the isolates Ps 1 and Ps 2 indicate their high potential for crude oil degradation. Fig 2. : A micro titre plate showing time course decolorization of 2.6, DCPIP in BHM with crude oil by isolates Ps1 and Ps 2. The last vertical row shows Substrate control (S.C) where no crude oil is added while last horizontal row shows inoculum control (I.C) where no inoculum is added. The gradual decolorization of dye is witnessed in each row with time. CONCLUSIONS: In order to develop environmental technologies for crude oil degradation it is necessary to isolate and characterize specific microbial species for evaluation of their efficacy in utilization of hydrocarbons before application to field conditions. PEER-REVIEWED Page 21

10 Initially 36 bacterial strains were isolated from 14 samples of soil originating from three different districts of Gujarat which were contaminated with petroleum hydrocarbons. The morphological, cultural and biochemical characterization yielded the primary identification of strains on genus level. Hence, the following study indicates a good bacterial diversity from soil samples exposed to crude oil due to different consequences. Overall, 5 species of bacteria (Micrococcus, Pseudomonas, Acinetobater, Staphylococcus and Bacillus,) were isolated from 14 contaminated sites and cultivated on BHM medium with crude oil as the sole source of carbon in order to screen them for their hydrocarbon degrading efficiency after the morphological, cultural and biochemical characterization. Following this, a rapid screening procedure was performed to assess the dye (2, 6, DCPIP) decolorization efficiency of selected isolates for confirmation of hydrocarbon degradation. REFERENCES: Atlas, R. M, (Ed), (1984). Petroleum Microbiology, Macmillan Co., New York. Atlas, R.M. and R. Bartha, (1998). Microbial Ecology: Fundamentals and Applications. 4th Edn., Benjamin/Cummings Publishing Co. Inc., California, USA., pp: Bharathi, S. and N. Vasudevan (2001). Utilization of petroleum hydrocarbons by Pseudomonas fluorescens isolated from petroleum contaminated soil. Environ. Int., 26, Del Arco JP and De Franca FP (2001). Influence of oil contamination levels on hydrocarbon biodegradation in sandy sediment. Environ. Pollut. 110: Hanson, K. G., Desai, D.; Desai, A. J. (1993), A rapid and simple screening technique for potential crude oil degrading microorganisms. Biotechnol. Techn., 7, Harayama S., Kishira, H., Kasai, Y., Shutsubo K., (1999). Petroleum Biodegradation in Marine Environments, J. Mol.Microbiol. Biotechnol., 1 (1): Head, I. M., Swannell, R. P., (1999). Bioremediation of petroleum hydrocarbon contaminants in marine habitats. Curr. Opin. Biotechnol., 10: Holt, J.G., N.R. Krieg, P.H.A. Sneath, J.T. Staley and S.T. Williams, (1994). Bergey s Manual of Determinative Bacteriology. 9th Ed. Williams and Wilkins.Baltimore, pp: Huy N, Jin S, Amada K. et al (1999). Characterization of petroleum-degrading bacteria from oilcontaminated sites in Vietnam. J Biosci Bioeng.;88(1): Ilyina, A., M.I. Castillo Sanchez, J.A. Villarreal Sanchez, G. Ramirez Esquivel and J. Candelas Ramirez, (2003). Isolation the soil bacteria for bioremediation of hydrocarbon contamination. Afr. J. Biotechnol., 41(6): 135 Kapley A, Heman J, Chhatre JP, Shanker R, Chakrabarti Khann P (1999). Osmotolerance and hydrocarbon degradation by a genetically engineered Microbial consortium. Bioresour. Technol. 61: PEER-REVIEWED Page 22

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