Helix Vol.

2:105-111 (2012)

Identification and Isolation of Hydrocarbon Degrading Bacteria by Molecular Characterization

Jyothi K*, K Surendra Babu*, Nancy Clara. K *, Amit Kumar*
Received January 27, 2012; Accepted February 25, 2012; Published March 01, 2012

Abstract:
Extensive hydrocarbons exploration activities often result in the pollution of the environment, which could lead to disastrous consequences for the biotic and abiotic components of the ecosystem if not restored. Remediation of hydrocarbon-contaminated system could be achieved by either physicochemical or biological methods. The present work was undertaken to assess, isolate and identify the hydrocarbon degrading bacteria associated with environmental samples collected from soil near petrol, diesel pumps and water samples from Husain sagar in Hyderabad and chlorine water from swimming pool. The samples were analyzed microbiologically using standard microbiological techniques. These organisms were further studied to determine their biodegrading activities on hydrocarbons (diesel and petrol) as the sole carbon source using enrichment medium. The microbial growths were determined using calorimeter blanked at 595 nm. The test on the degrading activity of isolates on hydrocarbon from environmental samples revealed that bacteria Bacillus megaterium, Bacillus cereus, Micrococcus luteus, Staphylococcus aureus, Lactobacillus acidophillus, Neisseria flavescence and Corynebacterium xerosis were the potent degraders of hydrocarbons (petrol and diesel). The identified bacteria by biochemical tests were further confirmed by 16s rDNA sequencing. The ability of these isolates to degrade hydrocarbons is clear evidence that their genome harbors the relevant degrading gene. These bacteria were screened for the presence of one of the hydrocarbon degrading enzyme catechol 2, 3 dioxygenase by DNA isolation, PCR amplification of gene using specific primers and sequencing of gene (C23O). The organisms with catechol 2, 3 dioxygenase enzyme were identified as Bacillus megaterium, Bacillus cereus, Micrococcus luteus, & Lactobacillus acidophillus with an 216bp amplification using C23O specific primers. The sequence has similarity about 90% to catechol 2, 3 dioxygenase gene. Keywords: Isolation, Identification, Biodegradation, Soil, Waste water, Diesel and Petrol.

INTRODUCTION
Many microorganisms have the ability to utilize hydrocarbons as sole sources of carbon as energy for metabolic activities .and these micro organisms are Omni present and widely distributed in the nature. The microbial utilization of hydrocarbons depends on the chemical nature of the compounds within the petroleum mixture and on environmental determinants [1]. Hydrocarbons enter into the environment through waste disposal, accidental spills, as pesticides and via losses during transport, storage, and use. Hydrocarbon (petroleum)degrading bacteria are reportedly ubiquitous in the environment and were widely distributed in marine, freshwater, soil habitats and their use in bioremediation of hydrocarbon-contaminated soils, which exploits their ability to degrade and/or detoxify organic contaminants, has been established as an efficient, economical, versatile and environmentally sound treatment [4,5,]Due to extensive increase in environmental pollution, numerous biodegradative bacteria have been isolated in the past, and their physiology, biochemistry, and genetics have been Intensively studied. Biodegradation, which is the destruction of organic compounds by microorganisms, is carried out largely by diverse bacterial populations, mostly by Pseudomonas species [9, 14] The extensive use of petroleum products leads to the contamination of almost all compartments of the environment, and biodegradation of the hydrocarbons by natural populations of microorganisms has been reported to be the main process acting in the depuration of hydrocarbon-polluted environments [10], the mechanism of which has been extensively studied and reviewed [3]. The fuel is a complex mixture of normal, branched and cyclic alkanes, and aromatic compounds obtained from the middle-distillate fraction during petroleum separation. Bioremediation process rely on the ability of microorganisms present naturally which are highly

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efficient due to their simplicity and cost-effectiveness when compared to other technologies. Bioremediation is a mineralization of organic chemicals, leading ultimately to the formation of CO2, H2O and biomass. Hydrocarbon utilizing microorganisms are ubiquitously distributed in the marine environment following oil spills [13] The study of identification of bacteria for the biodegrading capabilities is important in microbial ecology, especially with molecular techniques. In particular, analysis of the microbial communities that take part in in-situ hydrocarbon biodegradation activities has been a challenge to microbiologist. Interest in this area has been catalyzed by the rapid advancement of molecular ecological methodologies. The purpose of this research was to identify the feasibility of bacteria growing in soils near petrol and diesel pumps, waste water from Husain Sagar Lake and chlorine water from swimming pools to degrade hydrocarbons like petrol and diesel and to detect the presence of catechol 2,3 dioxygenase enzyme coding gene in the identified bacteria. sole carbon source (1% liquid petrol and diesel). The medium contains K2HPO4 (1.8 g/L); NH4Cl (4 g/L); MgSO4.7H2O (0.2 g/L); NaCl (0.1 g/L); Na2SO4.7H2O (0.01 g/L); agar (20 g/L); carbon source (1% petrol , diesel); and distilled water (1L) with pH 7.2. The medium without hydrocarbons was sterilized by autoclaving at 121C for 15 min. The medium was supplemented with 1% (v/v) filter sterilized hydrocarbons (petrol, diesel) to serve as the only source of carbon and energy [9]. The medium was incubated at 370C for 5-10 days. After the incubation period the bacterial colonies that were grown on the medium were identified by Grams staining and biochemical characterization according to Bergys manual.

Determination of Bacterial Biodegradative Activity by Turbidometry:

Turbidometry is to determine the bacterial growth by utilizing the hydrocarbons (1% petrol and diesel given as carbon source in MSM broth. This shows whether the bacterium possess the degrading activity of hydrocarbons like phenol, petrol and diesel. The degrading activities of each isolates were obtained by using Mineral salt broth (MSB) in which 1% of each hydrocarbon (petrol and diesel) was added and incubated at room temperature for 15 days. The growth of the bacterium was measured by taking the O.D readings at 595nm from 0hrs- 15 days at regular intervals of 2 days against mineral salt medium as blank.

Materials and Methodology:

Sample Collection: The study includes three types of samples to isolate the hydrocarbon degrading bacteria. Soil sample extending from the ground surface to a depth of 1020 cm were collected from petroleum-contaminated areas near petrol station, refining areas, and water samples such as chlorine water from swimming pool, wastewater from Husain sagar (tank bund, Hyderabad). Samples were then transported to laboratory under sterile conditions.

Growth and Isolation of Bacterial Cultures:

The bacteria were isolated from the collected samples by spreading the sample on nutrient agar medium. From the numerous colonies obtained on the NAM plates, they were screened for the hydrocarbon degrading bacteria.

Isolation of genomic DNA from bacteria:

DNA was extracted from 1ml of bacterial culture. the culture was pelleted by centrifuging at 12,000rpm for 2 min. the pellet was treated with lysis solution and proteinase k and incubated at 600C for 30min. Nucleic acids were precipitated with isopropanol by centrifuging at 10,000 rpm for 10 min, washed with 1 ml of a 70% (v/v) ethanol solution and dissolved in 0.1 ml of a TE buffer. The purity and quantity of DNA were examined by recording its UV absorption spectrum and running on 1% agarose gel electrophoresis.

Isolation of Hydrocarbon Degrading Bacteria:

The bacteria were isolated by inoculating the soil and water samples on enrichment medium that contains the autoclaved mineral salt medium (MSM) supplemented with single hydrocarbon compound as

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Helix Vol. 2:105-111 (2012) Sequence determination of 16s rDNA:

The DNA isolated was amplified using 16s rDNA universal primers and sequenced for the identification of bacterial strain at molecular level. Amplification of the PCR products of expected size was confirmed by electrophoresis. The sequence of the 16S rDNA was determined with a Dye terminator sequencing kit (Applied Biosystems), and the product was analyzed with an ABI Prism DNA sequencer (ABI). The gene sequences of each isolate obtained in this study were compared with known 16s rRNA gene sequences in the GenBank database. polymerase. Amplification of the PCR products of expected size was confirmed by electrophoresis, 1.5% (w/v) agarose gel in a TAE buffer.

Sequence determination of PCR amplified product:

The sequence of the PCR amplified product was determined with a Dye terminator sequencing kit (Applied Biosystems), and the product was analyzed with an ABI Prism DNA sequencer (Applied Biosystems 3500).

Amplification of Hydrocarbon Degrading Eenzyme Catechol 2, 3 dioxygenase coding Gene:

The bacteria were screened for the presence of catechol 2, 3 dioxgyenase enzyme that involved in hydrocarbon degradation. PCR primer design: PCR primers for amplifying catechol 2, 3 dioxygenase gene were constructed based upon conserved nucleotide regions of 17 known C23O genes listed in the gen bank. The C23OF 5CGACCTGATC(AT)(CG)CATGACCGA-3 and C23OR 5-T(CT)AGGTCA(GT)(AC)ACGGTCA-3 primers were synthesized at the Bioserve Biotechnologies India Pvt.Ltd, Hyderabad. PCR conditions were optimized using lab net thermal cycler. The PCR temperature program began with an initial 5-min denaturation step at 94C; 35 cycles of 94C for 45sec , 55C for 1 min, and 72C for 1 min; and a final 10-min extension step at 72C. All reaction mixtures were held at 4C until analyzed. The PCR reaction mixture contains 10XPCR buffer, 25 mM, Magnesium chloride, 2.5mM dNTPs , 10pm/l primer concentrations template DNA concentrations 100ng and and 1 U of Taq DNA tests were done for the isolated thirteen bacteria, one of them is found to be Gram-negative and the rest of them were Gram-positive. Different biochemical results were evident for catalase, glucose, starch, mannitol, Voges-Proskauer test and 6.5 % Nacl (Table 1). After morphological identification and number of biochemical tests, these isolates were identified as Staphylococcus aureus, Lactobacillus species, Bacillus species, Micrococcus luteus, Corynebacterium xerosis and Neisseria species.

Results and Discussion: Isolation and Identification of the Bacteria from Wastewater, Swimming Pool and Contaminated Soil
The bacteria were isolated from three different types of samples on nutrient agar medium. Further the samples were screened for the presence of hydrocarbon degrading bacteria on mineral salt medium with 1% of the hydrocarbons as the sole carbon source namely petrol and diesel individually. Hydrocarbons are needed as a carbon source but it can be toxic to microorganisms due to the solvent effects of diesel and petrol that could destroy bacterial cell membrane. Many biodegradation studies were reported on diesel are carried out using lesser diesel concentrations ranging from 0.5 to 1.5%. But M.Y Shukor et al., (2009) reported degradation of diesel by microorganisms at 3.5% and 6% diesel by Kwapisz et al., (2008). It has been found that degradation is generally unfavorable at [7, 15] concentrations higher than 1 or 1.5% . Number of colonies on mineral salt medium is lower when compared to the mother plate without hydrocarbons (Fig 1). This result showed that the bacteria grown on enriched medium were able to degrade the hydrocarbon source. Gram stain and biochemical

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Table 1: Morphological and biochemical characteristics of bacteria isolated from oil polluted soil and waste water.

Fig.1b: MSM + petrol medium

Bacillus cereus respectively. These results highlight the different group of bacterial genera involved in hydrocarbon degradation. Many scientists studied the petroleum degradation by various Pseudomonas species and by Bacillus species (2, 12). Boboye, B et al., (2010) reported the degradation of hydrocarbons form petroleum polluted areas by Staphylococcus aureus, Micrococcus luteus, Lactobacillus acidophillus and Bacillus species. Likewise Ojo (2006) reported hydrocarbons-utilizers in his research such as Bacillus megaterium, Pseudomonas putida, Micrococcus luteus, Bacillus brevis, Bacillus pumilis and Enterobacter aerogenes. Fig 2: DNA isolation from the isolated hydrocarbon degrading bacteria

Org-organism; +ve positive; -ve negative; A- Acid producer G- gas producer Fig 1: Growth of bacterial colonies on NAM and MSM with petrol.

Fig.2a

Fig.2b

Fig .1a : NAM medium

16S RDNA GENE SEQUENCE

The isolates were further confirmed by 16s rDNA sequencing. Based on DNA extracts of isolate (Fig 2), 16S rDNA which amplified by PCR using 35 cycles and primers 16sF and 16sR was got sequence result and listed in Table 2. The bacterial 16s rDNA sequences were aligned with Blast search of NCBI databases. The sequence aligned gave 99% similarity with Staphylococcus aureus, Micrococcus luteus, Lactobacillus acidophillus Corynebacterium xerosis, Neisseria flavescens, Bacillus megaterium and

It is evident from this study that when the environment was contaminated with petroleum and diesel components the proportion of hydrocarbondegrading microorganisms increases rapidly. High numbers of certain hydrocarbon-degrading microorganisms from an environment implies that those organisms are the active degraders of that environment [12]. The presence of oil-degrading organisms in the polluted soil and water is clear indication that the indigenous microbes were carrying out their metabolic activity. The activities of these microorganisms could be responsible for the bioremediation of the environment.

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Table 2: Identification of bacteria by 16s r DNA sequencing Orga nism 1 2 3 Sample Source water Soil and water Soil and water soil Soil Soil Soil and water Potent hydrocarbon degrader on petrol diesel petrol and diesel petrol and diesel petrol and diesel petrol and diesel petrol Identified by !6s sequencing Micrococcus luteus Staphylococcus aureus, Bacillus cereus microorganisms isolated from the soil and water samples were able to degrade hydrocarbons. The cells were able to multiply within the days of study, indicating that they were able to degrade and utilize the oil for their growth and development, hence the concomitant increase in the concentration of the broth (turbidity). This gradual increase in the concentration of the broth indicates bacterial growth hence degradation of hydrocarbons, mostly between days 5 and 12 and gradual decline in the concentration of the broth suggests decrease in the bacterial population and that the hydrocarbon has been degraded, mostly between days 13 and 15. The petrol and diesel degrading organisms isolated from the contaminated soil were Staphylococcus aureus, Lactobacillus acidophillus, Corynebacterium xerosis, Neisseria flavescens, Bacillus megaterium and Bacillus cereus and from water were Staphylococcus aureus, Micrococcus luteus, Lactobacillus acidophillus, and Bacillus cereu.

4 5 6 7

Bacillus maegaterium Corynebacterium xerosis Neisseria flavescence Lactobacillus acidophillus,

Biodegrading Activity of Each Isolates on Hydrocarbons by Turbidometry

Table 3 shows the O.D readings of biodegrading activity of each isolates on hydrocarbons (petrol and diesel). The O.D readings based on the turbidity of MSM broth at regular intervals of 2 days give the degradative activity on hydrocarbons by bacteria. The results demonstrated that Micrococcus luteus and Bacillus megaterium have the greatest ability to degrade petrol while Bacillus megaterium and Corynebacterium xerosis demonstrated the greatest ability to degrade diesel. The graphs based on the O.D readings at various time intervals of incubation period on the degrading activity of the oil-degrading bacteria are also illustrated in the figures 4&5. Our results showed that all the organisms maximally utilized all the hydrocarbon substrates (petrol and diesel) when supplied as the sole source of carbon and energy although, the level of utilization differs from one microbe to another (due to differences in their growth) and from one hydrocarbon substrate to the other, due to the obvious differences in their molecular sizes. The bacterium with the least degrading activities on petrol and diesel was Lcatobacillus acidophilus and Staphylococcus aureus respectively. These degrading capabilities on different hydrocarbons revealed that the

Amplification of Hydrocarbon Degrading Enzyme Catechol 2, 3 Dioxygenase Coding Gene The ability of an organism to degrade a specific substrate is clear evidence that its genome harbors the relevant degrading gene [11]. The previous studies [8] on hydrocarbon degradation by bacteria reveal that catechol 2, 3 dioxygenase is the one of the enzyme that involved in hydrocarbon degradation. The presence of this C23O enzyme in these identified hydrocarbon degrading bacteria by amplifying the gene coding the enzyme using C23O specific primers the primers used, gave the 216bp PCR product in three of the isolates. There is no amplification in the rest of the isolates indicating the absence of catechol 2, 3 dioxygenase enzyme activities for the hydrocarbon degradation. This indicates that C23O gene is involved in hydrocarbon degradation for Bacillus megaterium, Micrococcus luteus and Lactobacillus acidophilus bacteria isolated from the wastewater and the contaminated soils. However, it is true that the presence of a single gene does not ensure that the entire catabolic pathway will be present or that these genes will be expressed. The amplified product was sequenced and the aligned sequence gave 97% similarity with the known C23O gene sequence of Pseudomonas species

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Table 3: Growth curve readings at 595nm for 15 days of incubation.

Fig.5: Amplification of catechol 2,3, dioxygenase gene in seven identified hydrocarbon degrading bacteria. Lane1shows1Kbladder, Lane2:Micrococcus.luteus, Lane3:Bacillus.megaterium, Lane4:Staphylococcus.aureus,, Lane5: Lactobacillus acidophilus.

The above Table Shows the Optical Density of bacteria grown in Diesel containing Media Fig: 3 Graph showing the growth curve (O.D values) of bacteria in hydrocarbon (diesel) degrading broth for a period of 15 days of incubation

Series 1: Staphylococcus aureus, Series 2: Neisseria flavescence, Series 3: Bacillus cereus, Series 4: Bacillus megaterium, Sereis 5: Corynebacterium xerosis Fig: 4 Graph showing the growth curve (O.D values) of bacteria in hydrocarbon (petrol) degrading broth for a period of 15 days of incubation

Conclusion: Bioremediation is one of the most rapidly growing areas of environmental biotechnology, which has been used for the cleaning up of pollutants. This is because of its low costs and its public acceptability. Degradation of hydrocarbons by environmental microflorae involves microorganisms having specialized metabolic capacities. In polluted environments, specialized microorganisms are abundant because of the adaptation of the microflorae to pollutant. It is evident from this study that, hydrocarbon degrading organisms are ubiquitous in environment and they can be isolated from hydrocarbon polluted sites and waste water. It has also been shown that four bacterial strains isolated from waste water and seven bacterial strains from contaminated soil can be good petrol and diesel degraders. This study can focus on more cost effective applications of native bacterial strains for petrol and diesel degradation at large scale in industries, where it pose an alarming problem due to its detrimental health effects on different organisms and human beings. The degrading ability demonstrated by the microorganisms is a clear indication that they possess a gene that is used in hydrocarbon degradation. This study revealed that catechol 2, 3 dioxygenase gene was present in three

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