Biodegradation of selected hydrocarbons by novel bacterial strains isolated from contaminated Arabian Gulf sediment

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.


Scientific Reports
| (2020) 10:21846 | https://doi.org/10.1038/s41598-020-78733-0 www.nature.com/scientificreports/ persistent in the environment 30 . 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 biodegradation 28 . 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 weeks 29 . However, optimum pyrene degradation tends to occur above room temperatures 31,32 . In related study carried by Singh et al. 33 confirmed 60% maximum degradation of pyrene at 37 °C. Al-Thukair and Malik 23 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 Minerals 23,44 . 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 PAHs 23,44 .
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 loss 23,44 . 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 coating 34 . The SPME instrument was soaked with agitation in the samples and allowed to stabilize for 30 min 35 .

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 Malik 23 . 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. 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 lowmolecular-weight metabolism of naphthalene by Geobacillus strain was realized to be completely degraded in 72 h [36][37][38] . 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 www.nature.com/scientificreports/ 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 °C 39,40 . 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.

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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 environments 7,8 . 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 www.nature.com/scientificreports/ an initial concentration of 500 ppm) in 8 days 41 . 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. 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 days 29 . It was also reported that the initial pyrene concentration inhibited the full biodegradation process 42 . Table 2 summarizes the concentrations of unused pyrene found in bacterial cultures and control.  (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 3-11 37 . Degradation of naphthalene occurred in extremely acidic pH 2 soils with 50% mineralization in 28 days 21 . 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%      www.nature.com/scientificreports/ 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.
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 biometabolism [42][43][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.

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.   Percentage residual pyrene (%) 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.