alexa Biodegradation of One Ring Hydrocarbons (Benzene and Toluene) and Two Ring Hydrocarbons (Acenapthene and Napthalene) by Bacterial Isolates of Hydrocarbon Contaminated Sites Located in Chhattisgarh: A Preliminary Study
ISSN:2157-7463
Journal of Petroleum & Environmental Biotechnology
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Biodegradation of One Ring Hydrocarbons (Benzene and Toluene) and Two Ring Hydrocarbons (Acenapthene and Napthalene) by Bacterial Isolates of Hydrocarbon Contaminated Sites Located in Chhattisgarh: A Preliminary Study

Jai Godheja*, Sudhir Kumar Shekhar and Dinesh Raj Modi
Department of Biotechnology, Babasaheb Bhimrao Ambedkar University, Lucknow-226025, India
Corresponding Author : Jai Godheja
Research Scholar, Department of Biotechnology
Babasaheb Bhimrao Ambedkar University
Lucknow-226025, India
Tel: 91-9300203648
E-mail: [email protected]
Received December 16, 2014; Accepted December 18, 2014; Published February 27, 2015
Citation: Godheja J, Shekhar SK, Modi DR (2015) Biodegradation of One Ring Hydrocarbons (Benzene and Toluene) and Two Ring Hydrocarbons (Acenapthene and Napthalene) by Bacterial Isolates of Hydrocarbon Contaminated Sites Located in Chhattisgarh: A Preliminary Study. J Pet Environ Biotechnol 6:202. doi:10.4172/2157-7463.1000202
Copyright: © 2015 Godheja J, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Aromatic hydrocarbons are common groundwater and soil contaminants associated with petroleum product releases. They are hydrophobic and can readily bio-accumulate in the environment. Some PAHs (Polyaromatic Hydrocarbons) are harmful and known to be carcinogenic, mutagenic or genotoxic. Soil samples from hydrocarbon contaminated sites of Chhattisgarh were taken aseptically and their physicochemical properties studied. Soil samples were cultured in BBH media from which a total of eight positive isolates were studied for utilization of four aromatic hydrocarbons viz. Benzene, Toluene, Acenapthene and Napthalene. After biochemical analysis the genera identified were Alacaligens, Arthobacter, Burkholderia, Psuedomonas, Ralstonia, Enterobacter, Micrococcus, Kluyvera. All the isolates except strains Burkholderia and Ralstonia were able to use all the aromatic hydrocarbons studied as sole source of carbon indicated by the color change of resazurine indicator. Strains Pseudomonas and Kluyvera were considered as best degraders since they degraded the broadest spectrum of tested aromatic hydrocarbons and they showed the highest microbial activity in the presence of hydrocarbons tested indicated by the rapidity of the color change of resazurine. Furthermore, strains Enterobacter and Micrococcus exhibited moderate microbial activity against all of the tested hydrocarbons. Isolate Ralstonia was able to utilize benezene, toluene and naphthalene but couldn’t utilize acenapthene. Isolate Burkholderia was able to utilize benzene, acenapthene and naphthalene, but their proliferation was not supported by toluene.

Keywords
Bioremediation; Aromatic hydrocarbons; BBH media; Resazurin
Introduction
Microorganisms, owing to their biodiversity and vast catabolic potential, have been enormously harnessed for the biodegradation of toxic pollutants for a long time [1]. Remediation work on a contaminated site, besides being an instrument for environmental and human health protection, is an important vector for social and economic development as it encourages the transformation of entire areas from nonproductive zones to zones of environmental, urban and economic redevelopment. The suitability of different remedial technologies must be assessed on the basis of a series of parameters connected with the pollutant type and its relative concentration, the characteristics of the contaminated matrix and the site typology, the specific environmental matrix, the chemical-physical characteristics of the pollutants, the age of the pollution and, finally, with the matrix microbial activity [2,3]. Production, refinery, transportation or storage of crude oil and its derivatives, through accidental leakages may lead to contamination of soils with BTEX compounds (benzene, toluene, ethylbenzene and xylenes), polycyclic aromatic hydrocarbons (PAHs), as well as aliphatic hydrocarbons. Oil wells, petroleum plants, distribution and storage devices, transportation equipments are the main sources of the contaminations [4]. Polycyclic aromatic hydrocarbons (PAHs), produced by incomplete combustion of fossil fuels, and also manufactured for use in the pesticide, pharmaceutical and dye making industry are ubiquitous and persistent in the environment [5,6]. They are hydrophobic and can readily bio-accumulate in the environment. Some PAHs are harmful and known to be carcinogenic, mutagenic or genotoxic [7-9]. Cases of lung, intestinal, liver, pancreatic and skin cancer, have been reported [10]. PAH exposure occurs by inhalation, ingestion and dermal contact and, as they are highly lipid-soluble, they are quickly absorbed through the gastrointestinal tract in mammals [11]. Most of the PAHs are used to conduct research. However, some of the PAHs are used to make dyes; plastics; detergent; fungicides and pesticides. Some are even used in medicines. One of the most common ways PAHs can enter the body is through breathing contaminated air. The PAHs get into the human lungs when they breathe them. In addition if the human eat or drink food and water that are contaminated with PAHs, they could be exposed. Exposure to PAHs can also occur if the skin touches PAH contaminated soil or products like heavy oils, coal tar, roofing tar or creosote [10].
A large number of microorganisms (bacteria, fungi and some algae) that are capable of using petroleum hydrocarbons as the sole source of carbon and energy have been described [12]. Since PAH are hydrophobic compounds with low solubility in water, they have a tendency to bind with organic matter or soil, limiting their availability to microorganisms. Despite these properties, many bacterial strains have been isolated for their ability to transform, degrade and utilize PAH as a source of carbon and energy [13]. The isolation of bacteria from mixed contaminated soils which display versatile catabolic activity to tolerate toxic metals and degrade PAHs could be a potential cost effective remediation strategy [14]. Significant bacterial communities with ability to degrade PAH in soil play a critical role in biodegradation in spite of their low bioavailability. Microorganisms inoculated into PAH-contaminated soil environments must find and mobilize PAH before degradation and hence motility and chemotaxis are thought to be desired properties [15].
Materials and Methods
Site description and soil sampling
Raipur (20.91° N, 82.00° E), Bilaspur (22.09° N, 82.15° E) and Bacheli (18.70° N, 81.25° E) are located in Chhattisgarh state. Soil was moderately acidic with sandy-loam texture, increased humus, nitrogen and phosphorus content (Table 1). Hydrocarbon-contaminated soil samples were taken in vicinity of aboveground, as well as underground diesel-oil storage units. Samples serving as negative controls were taken from adjacent non-contaminated areas, from the same soil types. Sample labels are summarized in Table 2. The top 10 cm of soil was collected using sterile spatula into sterile glass bottles closed with cotton plug for microbiological analysis and with rubber septa for chemical analysis. Samples were stored at 4°C until further processing (within 24 h). Soil pH of samples was determined by following the SR ISO 10390-1999 standard (Muntean and Rusu, 2011). Humus content of soils (expressed in %) was determined according to Damian et al. [16] while the amount of mobile forms of phosphorous and potassium in accordance with Lacatusu and Lacatusu [17] using spectrophotometry, respectively flame spectrometry in acetatelactate ammonium solution at pH 3.7. Nitrogen index was calculated on basis of total humus content and soil base saturation according to Budoi et al. [18].
Enrichment, purification and culturing of hydrocarbon degrading bacteria
Diesel-oil degrading bacteria were isolated using enrichment containing: 99 ml BBH mineral broth medium supplemented with 1% (v/v) diesel-oil and inoculated with 1 g of contaminated soil. After 2 weeks of incubation at 23°C, the enriched cultures were serially diluted and inoculated onto BBH agar plates. The lid of Petri-dishes contained 250 ml of sterile diesel-oil as sole source of carbon and energy. Colonies with different morphologies were selected as candidate petroleum hydrocarbon-degrading strains and were maintained on standard Nutrient Agar (HiMedia).
Grouping and identification of strains
A total of eight different plates Gram staining and biochemical analysis was done for identifying different strains. Biochemical tests were performed (Gram stain, Starch hydrolysis test, Urease test, Indole test, Methyl Red test, Voges Proskauer test, Citrate test, Catalase test, H2S test, Nitrate Reduction test, Cellulase test, Triple Sugar Iron, Caesinase, Gelatinase and Oxidase test). The genera of microorganisms were identified according to the data generated by abisonline.com.
Hydrocarbon degradation potential of isolates
Identified microorganisms were tested to degrade different hydrocarbons: benzene, toluene, acenapthene and naphthalene (Analytical Reagents-HiMedia). Test media contained 50 ml BBH mineral broth, supplemented with one of the filter sterilized (0.2 mm) hydrocarbons and resazurine (10 mg/L) as a redox indicator. In order to select strains that possess an increased hydrocarbon degradation potential the concentration of applied carbon sources was set to 0.5 g/L. Test solutions were inoculated with 250 ml strain culture solutions (OD600=0.5). In the case of hydrocarbon degradation the initial blue color of test solution changed to colorless via pink [19]. The test runs were incubated for a week in a rotary shaker at 145 rpm and 28°C. Samples with no degradation activity (blue color) were marked “-”, minimum microbial activity (bluish pink color) “+”, the medium activity pink samples by “++”, while samples showing increased hydrocarbon degradation activity (colorless) were marked “+++”.
Results
Grouping and identification of strains
Mixed culture colonies were obtained after inoculation of enriched cultures in nutrient agar (Figure 1). The morphological and biochemical results (Table 3) were used to identify bacterial genus through online method (www.abisonline.com) were Alacaligens, Arthobacter, Burkholderia, Psuedomonas, Ralstonia, Enterobacter, Micrococcus, Kluyvera (Figures 2-9).
Hydrocarbon degradation potential of isolates
All the isolates except strains Burkholderia and Ralstonia were able to use all the aromatic hydrocarbons studied as sole source of carbon (Table 4) indicated by the color change of resazurine indicator (Figures 10-12). Strains Pseudomonas and Kluyvera were considered as best degraders since they degraded the broadest spectrum of tested aromatic hydrocarbons and they showed the highest microbial activity in the presence of hydrocarbons tested indicated by the rapidity of the color change of resazurine. Furthermore, strains Enterobacter and Micrococcus exhibited moderate microbial activity against all of the tested hydrocarbons. Isolate Ralstonia was able to utilize benezene, toluene and naphthalene but couldn’t utilize acenapthene. Isolate Burkholderia was able to utilize benzene, acenapthene and naphthalene, but their proliferation was not supported by toluene.
Discussion
Identification results of isolated aerobic heterotrophic hydrocarbondegrading strains suggested the prevalence of the representatives of gamma-proteobacteria and beta-proteobacteria in the contaminated samples. This phenomenon is in accordance with results of several studies which established the dominance of Proteobacteria division in hydrocarbon polluted soils [20]. The dominant cultivable species in all samples were members of the genus Pseudomonas, these well known and widely investigated petroleum hydrocarbon-degrading organisms [21,22]. Genus Burkholderia was able to utilize Toluene only because of more complex nature of other hydrocarbons whereas Genus Ralstonia was not able to utilize Acenapthene because of its higher aromaticity. We have to mention however, that our results at least are influenced by the applied enrichment techniques, using BBH broth and diesel-oil as supplements. The quantitative analysis of hydrocarbon degradation will be carried out through GC-MS.
References

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