==== Front Sci Rep Sci Rep Scientific Reports 2045-2322 Nature Publishing Group UK London 78733 10.1038/s41598-020-78733-0 Article Biodegradation of selected hydrocarbons by novel bacterial strains isolated from contaminated Arabian Gulf sediment Al-Thukair Assad Ahmed thukair@kfupm.edu.sa 1 Malik Karim 2 Nzila Alexis 1 1 grid.412135.00000 0001 1091 0356Life Sciences, King Fahd University of Petroleum and Minerals, Dhahran, Saudi Arabia 2 grid.268252.90000 0001 1958 9263Department of Geography and Environmental Studies, Wilfrid Laurier University, Waterloo, Canada 14 12 2020 14 12 2020 2020 10 218469 8 2020 11 11 2020 © The Author(s) 2020Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.Three strains of novel bacteria were isolated from oil-contaminated sediment from the Arabian Gulf (Brevibacillus brevis T2C2008, Proteus mirabilis T2A12001, and Rhodococcus quinshengi TA13008). The isolated strains were tested for their degrading efficacy of low and high molecular hydrocarbon (naphthalene and pyrene). The efficacy of the two-hydrocarbon degradation by the isolates bacterial was determined at a temperature of 25 °C and 37 °C and pH of 5.0 and 9.0. In inoculated media at 37 °C, Rhodococcus qinshengi fully metabolized naphthalene and degrade 56% of pyrene. Brevibacillus brevis break down over 80% of naphthalene at room temperatures (25 °C). However, it was found that P. mirabilis and R. qinshengi biodegraded nearly 94% of naphthalene in the incubated media. The capacity for pyrene and naphthalene degradation in varying pH and temperature conditions was shown to be significant in Rhodococcus qinshengi because of its mineralization exceeding 50% across the tested pH and temperature. This implies that the isolated strains are ideal for biodegradation of contaminated sediment with naphthalene and pyrene. Subject terms MicrobiologyEnvironmental sciencesissue-copyright-statement© The Author(s) 2020 ==== Body Introduction Contamination of the natural environment with oil, including polycyclic aromatic hydrocarbons (PAHs), is a widespread concern because of the health risks associated with these potential cancerous and mutagenic compounds1,2. The health risk associated with PAHs is primarily due to their lipophilic nature and inherent tendency to bioaccumulate and eventually undergo bioconcentration and biomagnification in food chains3,4. Microbial degradation potential of oil components seems to decline from: n-alkanes, branched alkanes, monoaromatics, cyclic alkanes, polycyclic aromatic hydrocarbons, and ultimately asphalt and resin5. As in most environments, bacteria are the predominant candidates responsible for the natural dissipation of PAHs. Pseudomonas, Rhodococcus, Paenibacillus, and Ralstonia species are among the most intensively investigated bacteria with respect to PAH biodegradation6–10. In addition to the bacteria’s active role in biodegradation, a wide variety of yeast and filamentous fungi, algae, cyanobacteria, and some protozoan organisms could degrade different types of hydrocarbons11–15. For example, it was reported that pyrene biodegradation mediated by the genera Rhodococcus in contaminated environments16,17. Wong et al.18 in his study highlighted the effects of pH on PAH bioremediation and observed the exponential bacterial growth at pH ranges of 5.5–7.5. In general, most PAHs can get optimal bacterial degradation at pH 7.519. However, several bacterial strains are sensitive to low pH values, which leads to reduced biodegradation20. PAH degradation was reported in extremely acidic soils (pH 2)21. However, pyrene was shown to biodegrade at pH values above 7.022. Al-Thukair and Malik23 have documented improved degradation of pyrene under alkaline medium. The bioavailability of PAHs at contaminated sites is a critical determinant of the biodegradation kinetics. Naphthalene is the simplest of PAHs and the most soluble and bioavailable hydrocarbon to be degraded by microbes. The ease with which naphthalene is solubilized by micelles in liquid media enhances its bioavailability and biological bacterial attack24. Pseudomonas aeruginosa for example can make use of naphthalene as its sole source of carbon25 and Streptomyces sp. is known to grow effectively on naphthalene, using it as the main source of carbon26. In addition, Bacillus fusiformis can mineralize close to 100% naphthalene within 96 h at 30 °C27. For most hydrocarbon-degrading microbes, the ideal temperature ranges from 25 to 30 °C28. Thermophilic bacteria Bacillus sp. and Thermus brockii efficiently metabolized pyrene and benzo (a) pyrene at 60 °C and 70 °C29. Pyrene comprises peri-fused benzene rings, low solubility, and poor bioavailability; hence, it is highly persistent in the environment30. It was found that in 20 days, Pseudomonas sp. WJ6 could metabolize 19.5% of the provided pyrene. As the initial concentration of pyrene seems to substantially affect the rate and magnitude of biodegradation28. For example, when Mycobacterium sp. JS19b1 inoculated to an initial concentration of 40 ppm of pyrene, it was able effectively to degrade 100% of the pyrene in 2 weeks29. However, optimum pyrene degradation tends to occur above room temperatures31,32. In related study carried by Singh et al.33 confirmed 60% maximum degradation of pyrene at 37 °C. Al-Thukair and Malik23 demonstrated over 50% pyrene degradation by B. fungorum at 37 °C. This study investigates the degradation of naphthalene and pyrene by novel bacterial isolates including; Brevibacillus brevis (T2C2008), Proteus mirabilis (T2A12001), and Rhodococcus quinshengi (TA13008). The three novel bacterial isolates were tested to determine their degrading ability at different temperatures and pH values. The selected temperatures (25 °C, 37 °C) and pH (5, 9) were used to test the hypothesis that the degradation rate is influenced by temperatures and pH. As there is no available literature to the best of the authors’ knowledge describing the biodegradation of naphthalene and pyrene involving bacterial isolates from the Arabian Gulf. Therefore, it is of interest to assess the efficiency of PAH degradation of the isolated strains and the optimal conditions for their biodegradation. This study will contribute to identifying competent bacteria isolates for utilization in the clean-up process of hydrocarbon-polluted sites in the future. Material and methods Materials and microorganisms Previously isolated from oil-contaminated sites, the bacterial strains Brevibacillus brevis, Proteus mirabilis, and Rhodoccus quinshengi were identified via 16S RNA. The above isolates were described, identified, and preserved in previous research carried out at the Life Sciences Department of the King Fahd University of Petroleum and Minerals23,44. Pre-culture of the strains in nutrient agar (yeast extract, 2.0 g/L; meat extract, 1.0 g/L; peptone, 5 g/L; NaCl, 5.0 g/L; and agar, 15 g/L) naphthalene and pyrene were of scientific quality (99%). The following ingredients (g/L) were used in Bushnell–Haas mineral medium (BH): MgSO4, 0.2; CaCl2, 0.02; KH2PO4, 1.0; K2HPO4, 1.0; NH4NO3, 1.0 and FeCl3, 0.05. Using phosphate-buffered solution the pH was modified to approximately 7.0 ± 0.2. By autoclaving at 121 °C. The culture media were sterilized for 15 min to remove the biotic agents' propensity to biodegrade the PAHs23,44. Biodegradation experiment We focused mainly on two physical parameters (temperature and pH) in our experimental design. In comparison to those observed in our sampling site, two temperatures (37 °C and 25 °C) and pH (5.0 and 9.0) values were chosen. At mesothermic and room temperatures (37 °C and 25 °C), the cultures mixed with naphthalene and pyrene were incubated. Media pH was set to 5.0 and 9.0, and the samples had been incubated. Erlenmeyer flasks (250 mL) held liquid media (BH) with mineral salts of 100 mL for incubation at the temperatures and pH values selected. The flask furnished with 1 mL of naphthalene and pyrene, and 2 ml of inoculum suspension; thus, the initial hydrocarbon concentration in each culture flask was 100 ppm. For 18 days in shake-incubators, flasks were incubated using a Wise Cube Fuzzy System (WIS-20 model) with continuous shaking at 120 rpm. The system included a control and two replicates. The autoclaved bacterial cultures constituted monitors to compensate for the hydrocarbon abiotic loss23,44. Sample extraction and preparation Residual pyrene and naphthalene analyses were performed to determine the degradation potentials of each bacterial isolate in specified intervals of 3 days. Solid-phase micro-extraction (SPME) using poly (dimethylsiloxane)-coated fibers (PDMS) was used for sample extraction and preparation before the residual PAH analysis. SPME is an equilibrium extraction technique where fibers are immersed in the sample, and target analytes are extracted from aqueous samples as the analytes partition between the aqueous matrix and the fiber coating34. The SPME instrument was soaked with agitation in the samples and allowed to stabilize for 30 min35. GC–MS analysis Residual pyrene and naphthalene analysis and detection are performed using gas chromatographic-mass spectrometry (GC/MS). The requirements of the GC device model are as follows: Agilent Technologies (6890N series), Injector Unit (7683B series), and MS System; Inert XL EI/CIMSD (5975B series). The SPME fiber was inserted into the hot injection port after the extraction of residual PAHs that achieved equilibrium where the analytes were desorbed into the GC column. King et al.35 adapted the GC/MS system, with slight modifications. An improved analyte removal from the PDMS-coated fiber was used in a desorption time of 10 min. After successive extractions, the fiber was conditioned (blank desorption after each extraction) to restrict analyte carryover effects. The temperature system for GC/MS was as follows: inlet temperature set at 220 °C to maximize desorption, oven temperature set at 50 °C, and ramped at 15 °C/min to 100 °C, kept for 2 min, ramped at 7.5 °C/min to 200 °C and gradually increased to 300 °C. Light and scanning electron microscope (SEM) Morphologies of cellular and colony structures on culture plates and prepared slides were examined using light and scanning microscopes (SEM). Preparation and examination of bacterial samples for SEM have followed the procedure described by Al-Thukair and Malik23. The prepared slides were coated with a thin layer of gold and examined in JEOL JSM-6460LV. Statistical analysis Sigma Plot software 11.1 was used to analyze the experimental data collected from biodegradation over the 18-day incubation period. One-way variance analysis (ANOVA) was used to a value of 0.05. Results and discussion SEM bacterial strains analysis Scanning electron microscopy (SEM) was conducted to differentiate the various types of bacterial cells. The SEM allowed to create and confirm the existence of bacteria in the media in which they were cultivated. For growing strain, the strains were morphologically classified using light microscopy as rod forms on nutrient agar plates with varying cell sizes. SEM images showed clearly that the strains of bacteria were rod-shaped with various cell sizes (Fig. 1A1,A2,B1,B2,C1,C2).Figure 1 SEM images and photographs of bacteria on nutrient agar plates of P. mirabilis (A1,A2), B. brevis (B1,B2) and R. quinshengi (C1,C2). Assessing existing PAHs At the end of the biodegradation trials, we subjected samples spiked with naphthalene and pyrene to GC/MS analysis. Figures 2 and 3 show GC chromatograms and naphthalene and pyrene mass spectra, respectively. GC pyrene elution time was estimated to be 18.51 min. The molecular ion had 202.1 m/z, while the fragment ions had 101.0 and 174.0 m/z. Naphthalene was estimated at 9.53 min retention time; molecular ion was at 128.0 m/z, with large fragment ions at 102.0 and 64.0 m/z values.Figure 2 GC Chromatogram (a1) and mass spectra (a2) of naphthalene. Figure 3 GC Chromatogram (b1) and mass spectra (b2) of pyrene. Effects of temperature on microbial degradation Naphthalene biodegradation As simpler PAH is known, naphthalene has substantial solubility and bioavailability with 3–5 fused benzene rings in comparison to other PAHs. Naphthalene was undetected at 18 days of incubation under 37 °C for flasks inoculated with R. qinshengi (Fig. 4a1). One logical inference is that biodegradation occurred at 100%. In previous work the low-molecular-weight metabolism of naphthalene by Geobacillus strain was realized to be completely degraded in 72 h36–38.Figure 4 Naphthalene degradation curves at (a1) 37 °C and (a2) 25 °C; Control experiments (solid sphere), B. brevis (solid squares), P. mirabilis (diamond), and R. qinshengi (triangle). The P. mirabilis strain seemed to have biodegraded naphthalene by over 95%. Nevertheless, B. brevis decomposed the lowest rates, dissipating 87% of naphthalene. Bacterial metabolism of naphthalene did not decrease significantly at room temperature (25 °C) (Fig. 4a2). For the strains tested, naphthalene was degraded by more than 80% (Fig. 5). Interestingly, P. mirabilis and R qinsheng had the highest mineralization observed in the inoculated flasks (5.95 ± 0.37 and 5.63 ± 0.35). The strains biodegraded naphthalene by no less than 94%. A comparison of residual naphthalene in the abiotic controls, P. mirabilis, B. brevis, and R. quinshengi is shown in Fig. 5. Efficient naphthalene mineralization at near-room temperatures was highlighted in several studies, such as Bacillus sp. SBER3 which biodegrade 75.1% naphthalene at 27 °C39,40. Under mesothermal conditions, compared with biodegradation, B. brevis remain the strain with reduced metabolic activity. Table 1 summarizes the amounts of available naphthalene found in bacterial cultures and control.Figure 5 Residual naphthalene in cultures at 37 °C and 25 °C after 18 days, vertical bar represents error bar. Table 1 Levels of unmetabolized naphthalene (ppm) in bacterial cultures at day 18 with varying temperatures. Bacterial strains Temperature (37 °C) Temperature (25 °C) Mean ± SD Mean ± SD R. quinshengi ND 5.63 ± 0.35 P. mirabilis 5.4 ± 0.15 5.95 ± 0.37 B. brevis 12.73 ± 0.42 17.84 ± 0.89 Abiotic control 81.05 ± 0.91 90.12 ± 0.85 ND not detected, SD standard deviation. Pyrene biodegradation Because of its comparatively low solubility and bioavailability, pyrene, containing four bonded benzene rings, is extraordinarily resistant to degradation. Few bacteria can metabolize this hydrocarbon altogether. However, pyrene biodegradation has been documented in oil-polluted environments7,8. Degradation of the pyrene at 37 °C is shown in Fig. 6c1 and Table 2. Nearly 52% of pyrene was removed by R. qinshengi at 25 °C and 37 °C. The level of unmetabolized pyrene measured (Mean ± SD) at 25 °C and 37 °C were 46.09 ± 0.62 and 41.88 ± 0.23 respectively. It was found that Mobacterium sp. KR2 capable to mineralized nearly 65% of pyrene (at an initial concentration of 500 ppm) in 8 days41. Singh et al.33 observed a cumulative degradation of 60% at 37 °C in a related experiment. P. miribilis biodegraded about the same quantity of hydrocarbon (Fig. 7). The strain biodegrades 44% of pyrene at an initial concentration of 100 ppm. It was found that B. brevis strain biodegraded pyrene by less than 36% due to its structural complexity. Xia et al.30 reported that Pseudomonas sp. WJ6 biodegrade 19.5% of pyrene in 20 days.Figure 6 Pyrene degradation curves at (c1)37 °C and (c2)25 °C; Control experiments (solid sphere), B. brevis (solid squares), P. mirabilis (diamond), and R. qinshengi (triangle). Table 2 Levels of unmetabolized pyrene (ppm) in bacterial cultures at day 18 with varying temperatures. Bacterial strains Temperature (37 °C) Temperature (25 °C) Mean ± SD Mean ± SD R. quinshengi 41.88 ± 0.23 46.09 ± 0.62 P. mirabilis 55.66 ± 0.67 62.47 ± 0.85 B. brevis 64.69 ± 1.49 61.16 ± 0.23 Abiotic control 90.01 ± 1.03 90.32 ± 0.97 SD standard deviation. Figure 7 Residual pyrene for the 3 culture strains at 37 °C and 25 °C after 18 days, vertical bar represents error bar. The metabolic activity of the strains has been studied at 25 °C (Fig. 6c2). P. Mirabilis, B. Brevis biodegrade between 36 and 39%. B. Brevis exhibited the least potential for mineralization at 37 °C and 25 °C as levels of unmetabolized pyrene measured at 37 °C and 25 °C were 64.69 ± 1.49 and 61.16 ± 0.23 as shown in Table 2. Figure 7 provides a comparison for the three cultured strains of P. mirabilis, B. Brevis, and R. quinshengi and their percentage of the remaining pyrene in abiotic controls. At an initial concentration of 40 ppm of pyrene, Mycobacterium sp., JS19b1 was found to be fully degraded in 14 days29. It was also reported that the initial pyrene concentration inhibited the full biodegradation process42. Table 2 summarizes the concentrations of unused pyrene found in bacterial cultures and control. Effects of pH on microbial biodegradation Naphthalene biodegradation Biodegradation of naphthalene decreased by many orders of magnitude under acidic conditions (Fig. 8b1, Table 3). However, the highest degradation was reported in R. qinshengi-inoculated flasks; in which 93% naphthalene was metabolized by this strain. The mean and standard deviation (Mean ± SD) of residual naphthalene in abiotic control (94.01 ± 0.91), P. mirabilis (39.13 ± 1.27), B. Brevis (29.99 ± 1.47), and R. quinshengi (6.90 ± 0.21) are shown in Fig. 9 and Table 3. It was reported that biodegradation of low-molecular-weight PAH particularly at pH 3–1137. Degradation of naphthalene occurred in extremely acidic pH 2 soils with 50% mineralization in 28 days21. Microbial metabolism in alkaline samples followed a trend of reduction comparatively like that seen in acidic medium. However, extreme pH is known to inhibit metabolic activity in the bacteria but did not stop biodegradation altogether. The degradation of naphthalene in the alkaline media is shown in Fig. 8b2. The maximum biodegradation of 95.5% occurred in R. qinshengi-incubated media. P. mirabilis used about 75% of naphthalene quantitatively. Table 3 summarizes the levels of unutilized naphthalene detected in bacterial cultures and abiotic control.Figure 8 Naphthalene degradation curves at (b1) pH 5.0 and (b2) pH 9.0; Control experiments (solid sphere), B. brevis (solid squares), P. mirabilis (diamond), and R. qinshengi (triangle). Table 3 Levels of unmetabolized naphthalene (ppm) in bacterial cultures at day 18 with varying pH. Bacterial strains pH (9.0) pH (5.0) Mean ± SD Mean ± SD R. quinshengi 9.52 ± 0.73 6.90 ± 0.21 P. mirabilis 39.13 ± 1.27 24.81 ± 0.33 B. brevis 29.99 ± 1.47 33.31 ± 1.42 Abiotic control 94.01 ± 0.91 91.06 ± 0.85 SD standard deviation. Figure 9 Residual naphthalene in cultures at pH 5.0 and pH 9.0 after 18 days, vertical bar represents error bar. Pyrene biodegradation Bacterial metabolism of pyrene appeared to have decreased under acidic medium for all the strains studied (Fig. 10, Table 4). Whereas this statement excludes R. qinshengi due to a marginal increase in the metabolic activities of the strain resulting in nearly 61% biodegradation of the initial 100 ppm pyrene. Unlike P. mirabilis, and B. brevis degraded the pyrene by no less than 26%. In an alkaline medium (pH 9.0), all the strains degraded approximately 30% pyrene. It is essential to reiterate that while biodegradation declined for R. quinshengi (71.34 ± 1.53), it increased marginally for P. mirabilis (70.89 ± 0.028) and B. brevis (71.81 ± 0.40). Increases in the solubility of PAHs by biological-producing bacteria can be due to the comparatively enhanced degradation of alkaline media, which improves biometabolism42–44. Figure 11 illustrates a percentage comparison of residual naphthalene in abiotic controls, P. mirabilis, B. Brevis, and R. quinshengi. Table 4 summarizes the concentrations of unused pyrene found in bacterial and abiotic control cultures. Figure 10 Pyrene degradation curves at (d1) pH 5.0 and (d2) pH 9.0; Control experiments (solid sphere), B. brevis (solid squares), P. mirabilis (diamond), and R. qinshengi (triangle). Table 4 Levels of unmetabolized pyrene (ppm) in bacterial cultures at day 18 with varying pH. Bacterial strains pH (9.0) pH (5.0) Mean ± SD Mean ± SD R. quinshengi 71.34 ± 1.53 39.16 ± 1.20 P. mirabilis 70.89 ± 0.028 74.1 ± 0.49 B. brevis 71.81 ± 0.40 74.05 ± 0.86 Abiotic control 83.23 ± 0.93 85.62 ± 1.13 SD standard deviation. Figure 11 Residual pyrene in culture for the 3 strains at pH 5.0 and pH 9.0 after 18 days, vertical bar represents error bar. Conclusion This paper demonstrates that R. qinshengi metabolized fully naphthalene and biodegrade 56% of pyrene at mesothermal temperatures (37 °C). B. Brevis metabolized naphthalene by more than 80% at room temperature (25 °C). P. mirabilis and R. qinshengi were noted for the highest metabolism of about 94% of naphthalene. Also, R. qinshengi under varying pH conditions, displayed a significant degradation potential for both naphthalene and pyrene. The pyrene mineralization of this strain over the pH spectrum was over 50%. The only exemption was in an alkaline condition where R. qinshengi metabolism fell marginally below 30%. P. mirabilis, and B. Brevis, are both documented for their biodegradation activity on textile dyes, but not polyaromatic hydrocarbon (PAHs). They are found to actively mediate the degradation of PAHs, especially naphthalene and pyrene. R. qinshengi biodegraded naphthalene and pyrene at varying temperatures and pH over the half-life of the initial 100 ppm concentration. Therefore, the three isolated strains are ideal for remediation of contaminated sediment with naphthalene and pyrene. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. These authors contributed equally: Assad Ahmed Al-Thukair and Karim Malik. Acknowledgements This study was funded by the National plan for Science, Technology and Innovation (MAARIFA), the King Abdulaziz City for Science and Technology, and the Unit for Science and Technology at King Fahd Petroleum & Minerals University (KFUPM), Kingdom of Saudi Arabia, award number (08-ENV458-4). Author contributions A.A.A.-T. and K.M. wrote the manuscript and prepared the figures and tables. A.A.A.-T. and K.M. carry out laboratory experiments, collect data and interpretation A.N. review the manuscript and list of references. Competing interests The authors declare no competing interests. ==== Refs References 1. Ronald WP Toxicity of polyaromatic hydrocarbons other than benzo(A)pyrene: a review J. Toxicol. Cutan. Ocul. Toxicol. 2000 19 55 67 10.3109/15569520009051478 2. Meyer T Lei YD Wania F Transport of polycyclic aromatic hydrocarbons and pesticides during snowmelt within an urban watershed Water Res. 2011 45 1147 1156 10.1016/j.watres.2010.11.004 21112607 3. Froehner S Maceno M Assessment of bioaccumulation of biphenyls in the trophic chain of a coastal area of Parana, Brazil Environ. Monit. Assess. 2010 164 189 198 10.1007/s10661-009-0884-y 19353284 4. Manoli E Samara C Konstantinou I Albanis T waters of Northern Greece Chemosphere 2000 41 1845 1855 10.1016/S0045-6535(00)00134-X 11061306 5. Van Hamme JD Singh A Ward OP Recent advances in petroleum microbiology Microbiol. Mol. Biol. Rev. 2003 67 503 549 10.1128/MMBR.67.4.503-549.2003 14665675 6. Roling WF Bacterial community dynamics and hydrocarbon degradation during a field-scale evaluation of bioremediation on a mudflat beach contaminated with buried oil Appl. Environ. Microbiol. 2004 70 2603 2613 10.1128/AEM.70.5.2603-2613.2004 15128509 7. Ghosal D Ghosh S Dutta TK Ahn Y Current state of knowledge in microbial degradation of polycyclic aromatic hydrocarbons (PAHs): a review Front. Microbiol. 2016 7 1369 27630626 8. Haritash AK Kaushik CP Biodegradation aspects of polycyclic aromatic hydrocarbons (PAHs): a review J. Hazard. Mater. 2009 169 1 15 10.1016/j.jhazmat.2009.03.137 19442441 9. Nzila A Update on the cometabolism of organic pollutants by bacteria Environ. Pollut. 2013 178 474 482 10.1016/j.envpol.2013.03.042 23570949 10. Farhadian M Duchez D Vachelard C Larroche C Monoaromatics removal from polluted water through bioreactors—a review Water Res. 2008 42 1325 1341 10.1016/j.watres.2007.10.021 18023838 11. Abed RMM Al-Thukair A De Beer D Bacterial diversity of a cyanobacterial mat degrading petroleum compounds at elevated salinities and temperatures FEMS Microbiol. Ecol. 2006 57 290 301 10.1111/j.1574-6941.2006.00113.x 16867146 12. Abed RM Zein B Al-Thukair A de Beer D Phylogenetic diversity and activity of aerobic heterotrophic bacteria from a hypersaline oil-polluted microbial mat Syst. Appl. Microbiol. 2007 30 319 330 10.1016/j.syapm.2006.09.001 17056222 13. Al-Thukair AA Abed RM Mohamed L Microbial community of cyanobacteria mats in the intertidal zone of oil-polluted coast of Saudi Arabia Mar. Pollut. Bull. 2007 54 173 179 10.1016/j.marpolbul.2006.08.043 17045306 14. Roberts PH Thomas KV The occurrence of selected pharmaceuticals in wastewater effluent and surface waters of the lower Tyne catchment Sci. Total Environ. 2006 356 143 153 10.1016/j.scitotenv.2005.04.031 15936058 15. Paul D Pandey G Pandey J Jain RK Accessing microbial diversity for bioremediation and environmental restoration Trends Biotechnol. 2005 23 135 142 10.1016/j.tibtech.2005.01.001 15734556 16. Dean-Ross D Moody JD Freeman JP Doerge DR Cerniglia CE Metabolism of anthracene by a Rhodococcus species FEMS Microbiol. Lett. 2001 204 205 211 10.1111/j.1574-6968.2001.tb10886.x 11682202 17. Song X Isolation, characterization of Rhodococcus sp. P14 capable of degrading high-molecular-weight polycyclic aromatic hydrocarbons and aliphatic hydrocarbons Mar. Pollut. Bull. 2011 62 2122 2128 10.1016/j.marpolbul.2011.07.013 21871639 18. Wong JWC Lai KM Wan CK Ma KK Fang M Isolation and optimization of PAH-degradative bacteria from contaminated soil for PAHs bioremediation Water Air. Soil Pollut. 2002 139 1 13 10.1023/A:1015883924901 19. Pawar R The effect of soil pH on bioremediation of polycyclic aromatic hydrocarbons (PAHS) J. Bioremed. Biodegrad. 2015 6 291 20. Kastner M Breuer-Jammali M Mahro B Impact of inoculation protocols, salinity, and pH on the degradation of polycyclic aromatic hydrocarbons (PAHs) and survival of PAH-degrading bacteria introduced into soil Appl. Environ. Microbiol. 1998 64 359 362 10.1128/AEM.64.1.359-362.1998 9435090 21. Stapleton RD Savage DC Sayler GS Stacey G Biodegradation of aromatic hydrocarbons in an extremely acidic environment Appl. Environ. Microbiol. 1998 64 4180 4184 10.1128/AEM.64.11.4180-4184.1998 9797263 22. Ping L Isolation and characterization of pyrene and benzo[a]pyrene-degrading Klebsiella pneumonia PL1 and its potential use in bioremediation Appl. Microbiol. Biotechnol. 2014 98 3819 3828 10.1007/s00253-013-5469-6 24389667 23. Al-Thukair AA Malik K Pyrene metabolism by the novel bacterial strains Burkholderia fungorum (T3A13001) and Caulobacter sp. (T2A12002) isolated from an oil-polluted site in the Arabian Gulf Int. Biodeterior. Biodegrad. 2016 110 32 37 10.1016/j.ibiod.2016.02.005 24. Liu Z Jacobson AM Luthy RG Biodegradation of naphthalene in aqueous nonionic surfactant systems Appl. Environ. Microbiol. 1995 61 145 151 10.1128/AEM.61.1.145-151.1995 7887597 25. Agarry SE Aremo MO Batch equilibrium and kinetic studies of simultaneous adsorption and biodegradation of naphthalene by orange peels immobilized Pseudomonas aeruginosa NCIB 950 J. Bioremed. Biodegrad. 2012 3 138 10.4172/2155-6199.1000138 26. Balachandran C Duraipandiyan V Balakrishna K Ignacimuthu S Petroleum and polycyclic aromatic hydrocarbons (PAHs) degradation and naphthalene metabolism in Streptomyces sp. (ERI-CPDA-1) isolated from oil contaminated soil Bioresour. Technol. 2012 112 83 90 10.1016/j.biortech.2012.02.059 22425516 27. Lin C Gan L Chen Z-L Biodegradation of naphthalene by strain Bacillus fusiformis (BFN) J. Hazard. Mater. 2010 182 771 777 10.1016/j.jhazmat.2010.06.101 20643503 28. Ruberto L Vazquez S Balbo A Mac Cormack W Psychrotolerant hydrocarbon-degrading Rhodococcus strains isolated from polluted Antarctic soils Antarct. Sci. 2005 17 47 56 10.1017/S0954102005002415 29. Feitkenhauer H Muller R Markl H Degradation of polycyclic aromatic hydrocarbons and long chain alkanes at 6070 °C by Thermus and Bacillus spp. Biodegradation 2003 14 367 372 10.1023/A:1027357615649 14669867 30. Xia W Biosurfactant produced by novel Pseudomonas sp. WJ6 with biodegradation of n-alkanes and polycyclic aromatic hydrocarbons J. Hazard. Mater. 2014 276 489 498 10.1016/j.jhazmat.2014.05.062 24929788 31. Li S Li X Zhao H Cai B Physiological role of the novel salicylaldehyde dehydrogenase NahV in mineralization of naphthalene by Pseudomonas putida ND6 Microbiol. Res. 2011 166 643 653 10.1016/j.micres.2011.01.003 21376550 32. Teh ZC Hadibarata T Enhanced degradation of pyrene and metabolite identification by Pleurotus eryngii F032 Water Air Soil Pollut. 2014 225 1909 10.1007/s11270-014-1909-x 33. Singh S Kumari B Upadhyay S Mishra S Kumar D Bacterial degradation of pyrene in minimal salt medium mediated by catechol dioxygenases: enzyme purification and molecular size determination Bioresour. Technol. 2013 133C 293 300 10.1016/j.biortech.2013.01.068 34. Pawliszyn J Theory of solid-phase microextraction J. Chromatogr. Sci. 2000 38 270 278 10.1093/chromsci/38.7.270 10901412 35. King AJ Readman JW Zhou JL Determination of polycyclic aromatic hydrocarbons in water by solid-phase microextraction–gas chromatography–mass spectrometry Anal. Chim. Acta 2004 523 259 267 10.1016/j.aca.2004.07.050 36. Mohanty G Mukherji S Biodegradation rate of diesel range n-alkanes by bacterial cultures Exiguobacterium aurantiacum and Burkholderia cepacia Int. Biodeterior. Biodegrad. 2008 61 240 250 10.1016/j.ibiod.2007.06.011 37. de Carvalho CCCR Adaptation of Rhodococcus erythropolis cells for growth and bioremediation under extreme conditions Res. Microbiol. 2012 163 125 136 10.1016/j.resmic.2011.11.003 22146587 38. Zhang J Zhang X Liu J Li R Shen B Isolation of a thermophilic bacterium, Geobacillus sp. SH-1, capable of degrading aliphatic hydrocarbons and naphthalene simultaneously, and identification of its naphthalene degrading pathway Bioresour. Technol. 2012 124 83 89 10.1016/j.biortech.2012.08.044 22985850 39. Andreolli M Lampis S Zenaro E Salkinoja-Salonen M Vallini G Burkholderia fungorum DBT1: a promising bacterial strain for bioremediation of PAHs-contaminated soils FEMS Microbiol. Lett. 2011 319 11 18 10.1111/j.1574-6968.2011.02259.x 21388438 40. Bisht S Utilization of endophytic strain Bacillus sp. SBER3 for biodegradation of polyaromatic hydrocarbons (PAH) in soil model system Eur. J. Soil Biol. 2014 60 67 76 10.1016/j.ejsobi.2013.10.009 41. Rehmann K Noll HP Steinberg CE Kettrup AA Pyrene degradation by Mycobacterium sp. strain KR2 Chemosphere 1998 36 2977 2992 10.1016/S0045-6535(97)10240-5 9734274 42. Chen K Microcalorimetric investigation of the effect of non-ionic surfactant on biodegradation of pyrene by PAH-degrading bacteria Burkholderia cepacia Ecotoxicol. Environ. Saf. 2013 98 361 367 10.1016/j.ecoenv.2013.08.012 24011930 43. Lovaglio R Rhamnolipids production by a Pseudomonas eruginosa LBI mutant: solutions and homologs characterization Tenside Surf. Deterg. 2014 51 397 405 10.3139/113.110321 44. Karim, M. Investigation of Various Bacterial Isolates in Degradation of Specific Hydrocarbons. Thesis (M.Sc.), King Fahd University of Petroleum and Minerals, 135 (2015).