Aerobic Degradation of Benzene by Escherichia spp. from Petroleum-contaminated Sites in Kolkata, West Bengal, India

Benzene is an omnipresent aromatic hydrocarbon of the environment and the pollution caused by it is a matter of great public concern. The study was designed to isolate, characterize and identify aerobic bacterial strains capable of benzene degradation from petroleum-contaminated soils of Kolkata, India. Three strains, designated as MKB1b, MKB2a and MKB2d were found to be able to degrade benzene, as the only source of carbon and energy. All the isolated strains had an optimal growth pH of 8.0, and grew best at 37°C to 40°C. Based on their molecular (16S rDNA sequence) characterization, all of the three bacterial strains were phylogenetically similar to the genus Escherichia. Strains MKB2a and MKB2d were identified as Escherichia coli, whereas Strain MKB1b was identified as Escherichia fergusonii; all three sequences were submitted to GenBank bearing accession numbers MK970556, MK970574, MK970557 respectively. This study is the first evidence of isolation, characterization and identification of aerobic benzene-degrading Escherichia spp. from petroleum-contaminated sites of an urban landscape in India. All of these isolated strains may be considered as potent candidates for the bioremediation of urban environment polluted with petroleum products.


INTRODUCTION
Benzene (formula C 6 H 6 , MW 78); a hydrophobic, volatile, colourless, highly flammable, aromatic hydrocarbon; is a preeminent component of crude as well as refined petroleum and most (98%) of it is commercially used as a resource for petrochemical and chemical industries, petroleum refining industries, and for manufacturing synthetic rubbers, tyres, gums, lubricants, pharmaceuticals, pesticides and other agricultural chemicals, plastics, dyes, polymers, resins, synthetic fibres and consumer products such as marking pens, glues, adhesives, thinners, shoe-polish, perfumes, after shave lotions and paints 1,2 . Benzene is a major component of petrol, diesel, gasoline and other automobile fuels. After glucosyl residues, benzene ring is globally regarded as the most extensively distributed chemical structure 3 .
Benzene enters into the human systems by cutaneous exposure or by inhalation 4 . The human population is exposed to benzene because of its presence in the proximity of benzene utilizing industries and gas stations, and in areas of high vehicular traffic. Cigarette smokers are also directly exposed to benzene 5,6 . The relation of benzene exposure and the concomitant risk of cancer formation in human tissues and organs have been well-documented 7 . The toxic effects of benzene have also been related to developing hematopoietic disorders like acute myeloid leukemia, lymphocytic leukemia and non-Hodgkin's lymphoma 8,9 , primarily caused by chromosomal alterations attributing to malignant transformation of the genome 9, 10 . It has been also suggested that a number of epigenetic alterations, mostly DNA methylation, may also play a role in formation of tumor 12 .
R e m e d i a t i o n o f g r o u n d w a t e r contaminated with benzene and other aromatic hydrocarbons has been proven to be an arduous task, due to the fact that these compounds are recalcitrant, water-insoluble and have the ability of diffusing rapidly once they are introduced into the aquifer system 13 . Mineralization of benzene has been a matter of priority for nearly three decades and microbial bioremediation of the same has been reported under aerobic conditions by genera, Nitrosomonas 14 , Pseudomonas 15,16,17 , Acinetobacter 18 , Lysinibacillus 19 as well as in the presence of other electron acceptors by genera, Geobacter 20 , Dechloromonas 21 , Desulfobacterium 22 . Studies on bacterial mineralization of aromatic compounds like benzene have been focused, and microorganisms capable of benzene biodegradation under aerobic conditions have been isolated from industrial areas, and areas adjoining oil fields and oil-refineries [22][23][24] . Exploration of aromatic hydrocarbon degrading microbial diversity in and around urban habitats in India as potential sources of bioremediation is severely lacking.
This study has been designed to explore the microbial diversity capable of benzene degradation, in pure culture, as the sole carbon and energy source in petroleum-contaminated sites located in Kolkata, India. Characterization of the microbes as well as assessment of environmental conditions favouring bioremediation of benzene were also studied.

MATERIALS AND METHODS Sample collection
Six gas stations situated in Kolkata, West Bengal, India (22.57° N, 88.36° E) were used as sites for collection of petroleum-mixed soil samples. Around 100 gm of soil from each site was collected in labeled, sterile glass containers from a depth of around 10 cm from top soil-surface and were aseptically transferred to the laboratory for further analysis. Microbiological investigations of all the samples were initiated strictly within 12 hours of collection.

Reagents and Culture Media
Microbial culture media mentioned in the experiments were procured from HIMEDIA (India), whereas, aromatic hydrocarbons were procured from Sigma Aldrich (USA)and were of HPLC grades. Pure cultures were obtained, and growth experiments were carried out with sterile nutrient agar and Luria agar medium used after sterilization at 120°C temperature and 105 K Pa pressure, for 15 7.0, was employed as a source of essential macro and micro-nutrients for culture of bacterial strains with aromatic hydrocarbons. Benzene was added to MSM, as and when required, after filter sterilization with 0.22µ bacteriological filter.

Isolation, purification and characterization of benzene-degrading strains
From each labeled sample, 5 gm of soil was inoculated in MSM (1L), supplemented with (1% v/v) n-hexadecane and incubated at 37°C temperature, under shaking condition (120 rpm) until the occurrence of visible growth. Inoculum from this MSM-n-hexadecane culture (10 -4 to 10 -8 dilutions) were then spread in MSM agar (2% w/v), supplemented with (2% v/v) n-hexadecane and once again incubated at 37°C temperature. After 72 hours of incubation, the bacterial isolate / colony was checked for purity by photomicrography with a Zeiss Axio Scope A1 Microscope, as well as spreading them on sterile Luria Agar plates. All the isolated and purified bacterial strains were stored both as nutrient agar stab and 15% glycerol stock at 4°C and -80°C, respectively. Strains capable of utilizing n-hexadecane as the sole carbon source were then screened for their aerobic benzene degradation ability by the following method. Each of the strains was inoculated into 15 ml MSM supplemented with benzene (1% v/v), and incubated at 37°C, at 120 rpm in an orbital shaker for 7 days. Cultures with visible growth were again spread onto MSM agar plates supplemented with benzene (1% v/v) in serially diluted suspensions (10 -4 to 10 -8 dilutions). Well-isolated colonies were chosen from these plates based on their cultural characteristics like, colour of surface, shape, margin, texture and elevation using single colony isolation procedure. An array of biochemical tests were performed to characterize the strains according to Bergey's Manual of Systematic Bacteriology 26 .

Antibiogram test
Antibiogram or antibiotic susceptibility of the isolated strains was executed by the disk diffusion method 27 on Mueller-Hinton agar (MHA) plates with antimicrobial susceptibility test disks (HiMedia) of 10 antimicrobial drugs. Bacterial isolates were analyzed for susceptibility, reduced susceptibility, or resistance towards an antimicrobial drug based on the inhibitory zone diameter matching the manufacturer's interpretive table's criteria, following the recommendations of the National Committee for Clinical Laboratory Standards 28 . Escherichia coli ATCC 25922 strain was used for quality control (qc).

Measurement of temperature and pH optima
Temperature and pH optima of the strains were tested to evaluate the culture conditions needed for most effective aerobic bioremediation of benzene. For testing the pH optima, each isolated strain was grown in six tubes each containing 5 ml of sterile nutrient broths (having pH varying from 2 to 9) and 15 x 10 7 ml -1 cells from overnight culture were added and incubated at 37°C temperature, at 120 rpm in an orbital shaker.
Temperature optima of each isolates were evaluated by inoculating 15 x 10 7 ml -1 cells from overnight cultures to six tubes each containing 5 ml of sterile nutrient broths (pH 7) and incubated at temperatures of 25°, 30°, 35°, 37°, 40° and 45°C in an orbital shaker at 120 rpm for 12 hours.
In both experiments, cell density of each tube, after 12 hours of incubation, was measured spectrophotometrically at 660 nm by a SYSTRONICS 2202 Double Beam Spectrophotometer.

Molecular identification of the isolated strains
Genomic DNA of the strains were isolated as described by Sambrook et. al., 2001 29 with the following modifications. Cells were harvested from 1.5 ml of the overnight Luria Broth culture and resuspended in 567µl TE Buffer (10mM Tris, 1mM EDTA), pH 8.0. After addition of 30µl 10% SDS and incubation for 1 hour at 37 °C, 100µl of 5M NaCl was added. After addition of 80µl of cetyltrimethyl ammonium bromide (CTAB)/NaCl, and after mixing vigorously, the mixture was incubated at 65°C for 10 minutes. After extraction with equal volume of chloroform: isoamyl alcohol (24:1) and another extraction with equal volume of phenol: chloroform: isoamyl alcohol, DNA was precipitated with isopropyl alcohol, washed with chilled 70% ethanol, dried and dissolved in appropriate volume of TE buffer, pH 8.0.

Phylogenetic analysis of the strains
For the identification and phylogenetic analyses of the strains the sequences obtained were queried in BLASTn software and top ten hits were selected for each strain for further downstream tests. CLUSTALW was used to align these sequences and for annotation purposes. The results were then subjected to phylogenetic analysis using Maximum Likelihood method and Jukes-Cantor model 30 . Initial trees for the heuristic search were obtained with the Neighbor-Joining method forming a pairwise distance matrix predicted using the Maximum Composite Likelihood (MCL) approach 31 . Branch lengths of the tree is determined by the number of base substitutions per site. All in silico analyses were done on MEGA X software 32 . The final sequences were then subjected to test for potential chimeras using DECIPHER chimera check software 33 and were then submitted to GenBank.

Benzene biodegradation assay
Aerobic degradation of benzene by the isolated bacterial strains along with their timecourse study were done as according to Mukherjee et. al., 2010 34 with following modifications. In sterile 15 ml Teflon-lined capped tubes, inocula (15x10 7 cells ml -1 from a 12 hour old culture, washed in sterile PBS, pH 7.0) were added into MSM overlaid with HPLC grade benzene (100µ Mole, added from filter-sterilized stock solutions) ensuring enough head-space for the maintenance of aerobic conditions, and incubated at 37°C temperature, 120 rpm in an orbital shaker-incubator. MSM with 100µM benzene supplemented with heatkilled (by 15 minutes incubation at 70°C) cells of each strains and 15x10 7 cells ml -1 of each strain in MSM, not supplemented with benzene were used at negative controls (Control 1 and Control 2 respectively). A week-long experiment was set in the manner and cell numbers were determined at a gap of 24 hours by centrifugation of the cultures at 8000 rpm for 10 min, washing cell pellets twice with sterile PBS, resuspension in sterile PBS and direct microscopic count under oil immersion.
After each 24 hours of biodegradation, samples of each tube were extracted with 10 ml of HPLC grade chloroform. Concentrations of benzene in each culture was evaluated by high performance liquid chromatography (HPLC) as described previously 21 , using a Waters model 600 HPLC system equipped with Waters 2996 Photodiode Array Detector (Milford, MA, USA), at 254 nm. HPLC analyses were done isocratically, at room temperature with a mobile phase of methanol: water 60:40 (v/v) at a flow rate of 1 mL min -1 , by an analytical column (Symmetry-C 18 5µm, 4.6mm i.d. X 25 cm long, Waters, USA).

Statistical analysis of data
Data values are expressed as [mean values ± standard deviation (S.D.)] from at least three replicates of experiments. Differences among sets were considered significant at (P≥ 0.05) level. SPSS 17.0 (SPSS Inc., Chicago, USA) was used to implement statistical analyses and to compare means by Student's t-test.

Identification of strains and determination of pH and temperature optima
Three bacterial strains were selected for further studies, among 16 isolates capable of utilizing n-hexadecane as sole carbon and energy source, due to their ability of growing in MSM, supplemented with benzene. Results of morphological and biochemical characterization of these three Gram-negative, rod-shaped facultative anaerobic bacterial strains are enlisted in Table  1. MKB1b was found to be the only non-motile bacteria among the three. The three strains exhibited nearly similar biochemical characteristics but differed in their ability in acid production from lactose and sucrose.
The three strains were identified and they all belong to the genus Escherichia. Strain MKB2a and MKB2d were identified as E. coli, whereas Strain MKB1b was identified as E. fergusonii. 16S Ribosomal DNA sequences of all three strains were submitted to the GenBank bearing accession numbers MK970556, MK970574, MK970557 respectively. The phylogenetic tree constructed is depicted in Fig. 1. The tree shows the phylogenetic position of the three strains when compared with their nearest neighbours from BLASTn hits. The three strains belong to the same lineage but differs in the strain level as elucidated by the difference in the branch lengths.
Strains MKB1b, MKB2a and MKB2d showed considerable growth within a range of pH 6.0 to 8.0, having the maximum growth at pH 8.0 (Fig. 2). All the strains exhibited better growth at an alkaline environment (pH 9.0) than in an acidic one (pH 4.0 and 2.0). All the three strains were able to grow at the range of temperature tested, from 25°C to 45°C. Optimum temperature for growth of the strains MKB1b and MKB2a was found to be at 37°C, whereas, MKB2d showed nearly equal growth pattern at both 37°C and 40°C (Fig. 3).

Aerobic biodegradation of Benzene
Aerobic benzene biodegradation of strains MKB1b, MKB2a and MKB2d were carried out at their optimum pH and temperature with 100µM benzene and 15x10 7 cells ml -1 respectively. Depletion of benzene in the heat-killed inoculum (Control 1) was negligible (P < 0.01), and 89.66 ± 0.87µM benzene remained undegraded in the culture, whereas, cell number (10.57x10 7 ± 1.89 Table 1. Morphological and biochemical characters of the three bacterial isolates obtained from petroleum contaminated soil samples in Kolkata.

MKB1b
MKB2a Journal of Pure and Applied Microbiology ml -1 ) in the culture without added benzene did not vary much (P < 0.01) over an incubation period of 7 days (Fig. 4)

DISCUSSION
E. coli is the most abundant and successful bacterial flora in the gut ecosystem of warmblooded animals and is a pathogen of the enteric, urinary, pulmonary, and nervous systems also 35 . However, this commensal can also thrive in extracorporeal environment 36 and has the ability to withstand ample range of physico-chemical conditions, enabling it as a successful survivor in various environments over long periods of time 37 . E. coli, can also survive and replicate in environments having an abundance of aromatic hydrocarbons; and can actually utilize the aromatic compounds as sources of carbon. On   the other hand, E. fergusonii has previously been documented to be able to degrade herbicides like, atrazine and diuron 38 and diesel oil 39,40 .
This is a novel report of aerobic degradation of benzene by E. coli and E. fergusonii strains, isolated from petroleum-contaminated sites in the densely populated city of Kolkata, West Bengal, India. Earlier reports of bacterial species capable of mineralizing benzene and other aromatic hydrocarbons, had been concentrated around oil-field and oil-refineries only [41][42][43] . In pure culture, benzene biodegradation have been reported by a number of aerobic bacterial genera like, Geobacter 44 , Desulfobacterium 45 , Dechloromonas 21 and Bacillus 46 ; and anaerobic genera like, Nitrosomonas 14 , Pseudomonas 16,17 and Acinetobacter 18 . In genus Escherichia, degradation of aromatic compounds like, hydroxyphenyl acetic acid, 3-hydroxyphenylpropionic acid; 3-hydroxycinnamic acid; and phenylacetic acid have been comprehensively studied 47 .
E. coli and E. fergusonii strains described in this study could degrade benzene as the sole carbon and energy source, in pure culture, up to a concentration of 71.52µM over a period of 7 days under aerobic conditions. In enrichment studies, all of the strains showed maximum proliferation at a temperature range of 37°C to 40°C and in alkaline pH 8.0, making them potential bio-remediators of benzene-contaminated soils. Further insights on the mechanism of benzene mineralization, characterization of the genes involved as well as extensive investigations of urban microbial diversity are warranted to utilize as proficient candidates against aromatic hydrocarbon pollution.