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Anthracene biodegradation capacity of newly isolated rhizospheric bacteria Bacillus cereus S13

Anthracene biodegradation capacity of newly isolated rhizospheric bacteria Bacillus cereus S13 Biodegradation of hazardous pollutants is of immense importance for maintaining a clean environment. However, the concentration of such contaminants/pollutants can be minimized OPENACCESS with the help of microorganisms that has the ability to degrade the toxic pollutants into non- Citation: Bibi N, Hamayun M, Khan SA, Iqbal A, toxic metabolites. In the current study, 23 bacterial isolates were purified from the rhizo- Islam B, Shah F, et al. (2018) Anthracene spheric soil of Sysimbrium irio, growing as a wild plant in the vicinity of gas filling stations in biodegradation capacity of newly isolated Peshawar city. The isolated strains were initially screened on solid nutrient agar and further rhizospheric bacteria Bacillus cereus S . PLoS ONE 13(8): e0201620. https://doi.org/10.1371/ purified by culturing it on anthracene amended mineral media (PNR). The bacterial growth journal.pone.0201620 and anthracene disappearance were observed by calculating optical density (OD). The iso- Editor: Andrea Franzetti, Universita degli Studi di lates showed a concentration-dependent growth on anthracene amended PNR media at Milano-Bicocca, ITALY 30ÊC and pH7. Also, an increase in bacterial OD from 0.351 to 1.80 with increased shaking Received: December 26, 2017 speed was noticed. On the contrary, alternate carbon sources (glucose, fructose, sucrose) or nitrogen sources (KNO , NaNO , NH NO and CaNO ) posed inhibitory effect on bacte- 3 3 4 3 3 Accepted: July 18, 2018 rial growth during anthracene degradation. The recorded efficiency of anthracene degrada- Published: August 2, 2018 23 -1 tion by the selected bacterial isolate (1.4×10 CFUmL and 1.80 OD) was 82.29%, after Copyright:© 2018 Bibi et al. This is an open access 120 h of incubation. The anthracene was degraded to 9, 10, dihydroxy-anthracene and article distributed under the terms of the Creative anthraquinone, detected through GC-MS. The efficient bacterial isolate was identified as Commons Attribution License, which permits unrestricted use, distribution, and reproduction in S , a new strain of Bacillus cereus, using 16S rRNA analysis, showing 98% homology. The any medium, provided the original author and isolated bacterial strain S may be used as a potential tool for bioremediation of toxic hydro- source are credited. carbons and to keep the environment free from PAH pollutants. Data Availability Statement: All relevant data are within the paper. Funding: Research was supported by the Agenda Program, Rural Development Administration, Background Republic of Korea (Project No. PJ01228603 to In- A major portion of petroleum mixture contains fuse-ringed aromatic compounds, the poly- Jung Lee). The funders had no role in study design, data collection and analysis, decision to publish, or aromatic hydrocarbons (PAHs). The PAHs are the most abundant contaminants in the atmo- preparation of the manuscript. sphere and are kept on top of the pollutants list by US Environmental Protection Agency [1, 2]. These contaminants can be found abundant around the industrial sites, such as gas produc- Competing interests: The authors have declared that no competing interests exist. tion sites and wood preservation industries, and release through automobile exhausts [3] that PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 1 / 18 Anthracene degradation by B. cereus S Abbreviations: PAHs, Polyaromatic hydrocarbons; are consistent threats to human. PAHs along with their derivatives are the major factors caus- LMW, Low-molecular weight; OD, Optical density; ing anaemia, asthma, splenomegaly and various types of cancer in humans [4]. Some of the HPLC, High performance liquid chromatography; PAHs with low molecular weights have caused reproductive abnormalities and even death in GC-MS, Gas chromatography±mass spectrometry; aquatic animals [5, 6]. DNA, Deoxyribonucleic acid; rRNA, Ribosomal Anthracene is used as a signature compound for the detection of PAHs contamination, as it ribonucleic acid; NCBI, National Centre for Biotechnology Information; UV, Ultraviolet light; is an integral part of many carcinogenic PAHs. Due to its hydrophobicity and potential to BLAST, Basic Local Alignment Search Tool; CFU, bioaccumulate, it is used as model compound [7] to define factors affecting bioavailability and Colony forming units; KH PO , Potassium 2 4 rate of degradation of PAHs in environment. In spite of structural identity between anthracene phosphate monobasic; (NH ) SO , Ammonium 4 2 4 and phenanthrene regarding the number of aromatic rings, anthracene differs in degradation sulfate; NaOH, Sodium Hydroxide; MgSO .7H O, 4 2 due to the hydrophobic nature [4, 8, 9]. The presences of these compounds in the environment Magnesium Sulfate Heptahydrate; FeSO .7H O, 4 2 have to be taken seriously, because of their broad toxic effects on living organisms [3, 10]. Iron(II) Sulfate Heptahydrate; HCl, Hydrogen chloride; KNO , Potassium nitrate; NaNO , Sodium 3 3 PAHs in contaminated soils must be treated to avoid any possible noxious effect on environ- nitrate; CaNO , Calcium nitrate; NH NO , 3 4 3 ment and human health. In this regard, microbes can play key role in degradation/bioremedia- Ammonium nitrate. tion of the toxic PAHs into non-toxic compounds [4]. From the last few decades, bioremediation is getting importance day by day, because it is cheap, feasible and safe to clean the contaminated localities [6, 11]. The efficient microbial strains, unlike physical and chemical treatments can completely mineralize the PAHs present on the soil surface or soil sediments [11]. Previously, it has been discovered that certain starins of fungi, bacteria and algae can feed on the harmful PAHs and produce harmless compounds out of them [12]. Haleyur, Shahsavari (13) has demonstrated that some of the microorganisms (including, Rhodococcus sp., Achromobacter sp., Oerskovia paurometabola, Pantoea sp., Sejongia sp., Microbacterium maritypi- cum and Arthrobacter equi) exhibits catechol 1,2-dioxygenase activity. The presence of catechol 1,2-dioxygenase activity enables the above mentioned microorganisms to grow on PAHs and degrade catechol [13]. Alfaalfa, rape, vetch, mulberry and mustard rhizoremediation have been reported to grow and flourish in PAHs rich soil. In fact, this might be due to the presence of exten- sive population of microbes, mainly PAHs degrading bacteria around the roots of those plants [14± 18], which enables them to grow normally. Also, pea straw has been found to be one of the most useful sources in decreasing PAHs concentration in the soil [19]. Metagenomic analysis proved that the effect of pea straw was indirect. In fact, pea straw has biostimulated the PAHs biodegrader (Pseudoxanthomonas spp. and Alcanivorax spp) in the soil that were lying latent prior to the addi- tion of pea straw to the soil [19]. The ability of many microorganisms to degrade hydrocarbons [20±22] through metabolism or co-metabolism have been reported over the years [5]. However, there is still a space to discover microbial strains that can serve as a potential source to be used in bioremediation of PAHs. The present study was also focused on the exploration of prospective strains of beneficial microorganism that can degrade toxic chemicals and provide stress free envi- ronment. For this purpose research was conducted to (a) isolate Anthracene degrading rhizospheric bacteria from soil; (b) identification of potential PAHs degrading strain(s) in contaminated soils. Methods Nutrient agar media and mineral salts media were used for initial isolation and screening of rhizospheric bacteria. The composition of PNR and PNRG (PNR+5 mM glucose) per liter of -1 -1 distilled water [23, 24], is PN (20x) 50 mL used as 50 mLL : KH PO 13.6% (wv ), (NH ) SO 2 4 4 2 4 -1 -1 -1 -1 2.4% (wv ), NaOH 2.5% (wv ) and R salts used as 7 mLL , MgSO .7H O 8% (wv ), FeS- 4 2 -1 -1 O .7H O 0.2% (wv ), HCl 0.4% (wv ), Agar (2%) was used as solidifying agent. 4 2 Collection of samples Sysimbrium irio was found in a wild, i.e. growing in oil contaminated soil at an operational gas filling station in Peshawar. The texture of the soil was sandy loam with pH = 7.0 ± 0.2; soil PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 2 / 18 Anthracene degradation by B. cereus S moisture = 9.6%, maximum water holding capacity = 43.9% and electrical conductivity of 3.24 -1 ds m , respectively. The plants from the contaminated soil were dug as a whole, identified and placed in plastic zipper bag. The samples were immediately brought to the microbiology research laboratory at the University of Peshawar and stored at 4ÊC till further processing. Isolation of bacteria Soil samples were sieved through 2 mm mesh to collect uniform sized sample. After grading, 1 g of soil sample was serially diluted in a distilled water and then followed the spread plate method as described by Alias S [25]. The plates were incubated at 28ÊC for 3±4 days until appearance of the colonies. The developed colonies were carefully picked and further cultured in a fresh nutrient agar plates, the inoculation step was repeated till achieving the pure culture. After obtaining the pure cultures, they were stored in anthracene slants at 4ÊC. Screening of the isolated strains on solid media The purified bacterial isolates were screened for their capability to utilize anthracene as a sole source of carbon and energy (required for biodegradation). A confirmatory spray-plate assay was used to check the efficiency of isolated bacterial strains to grow on media supplemented with anthracene [26, 27]. Anthracene was initially dissolved in acetone and sprayed on the plates containing bacterial culture. Acetone was then allowed to evaporate leaving anthracene on the surface of the plates to be digested by the bacteria. Screening of the isolated strains in liquid media Screening in liquid medium was performed using 250 ml flasks containing 100 ml PNR media, -1 10% of bacterial inoculum and 1000 mgL anthracene. The media was incubated at 28ÊC and the bacterial growth was monitored at every 24 h interval till 120 h. Spectrophotometric analy- sis of bacterial growth and disappearance of anthracene was observed in PNR media at 600 nm and 540 nm, respectively. Optimization of growth conditions for the isolated strains Different parameters, like concentration of anthracene, incubation temperature and pH of the growth media were optimized. Range of anthracene concentration was (100, 150, 500 and 1000 ppm), temperature (28, 30, 35, 40, 45 and 50ÊC) and pH used were (4, 5, 6, 7, 8 and 9). Effect of shaking speed and inoculum size was quantified using speed of (0, 120, 150, 180, 200 and 220 rpm) and (0, 8, 9, 10, 11, 12, 13%). Alternate carbon source than anthracene used were different sugars, like sucrose, glucose and fructose for their effect on the growth of bacte- ria at the expense of selected PAH [28, 29]. The bacterial isolate S inoculum was exposed to UV-light for 15 minutes and added to media containing different concentrations of anthra- cene [30]. After every 24 h, one ml of culture broth was aseptically collected to check OD, while one ml was collected and stored at 4ÊC to test for degradation capability. CFU was calcu- lated at 24 h interval till 120 h in order to check the viability of the bacterial isolate. All the experiments were performed in triplicate. Biodegradation experiment The biodegradation experiment was performed using 250 ml flasks containing 100 ml PNR -1 media, 10% of bacterial inoculum and 1000 mgL anthracene dissolved in acetone. Acetone was allowed to evaporate, 100 mL media was poured to the flask containing different concentration PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 3 / 18 Anthracene degradation by B. cereus S of anthracene and 10% bacterial inoculum was added. The flasks were incubated at 30ÊC and 1 mL of sample was drawn for HPLC analysis after 24 hours interval for 5 days [31]. Extraction of anthracene for GC-MS analysis For GC-MS analysis, Shimadzu fused silica capillary column was used. The column tempera- -1 -1 ture was set to 100ÊC for 1 min, 15ÊC min to 160ÊC and 5ÊCmin to 300ÊC for 7min. The GC injector was held isothermally at 280ÊC with a splitless period of 3 min. Helium was used -1 as the carrier gas, at a flow rate of 1 mL min by using electronic pressure control. The GC± MS interface temperature was maintained at 280ÊC [32]. Plasmid curing, isolation and agarose gel electrophoresis Plasmid DNA was isolated from 18±24 hours old culture grown in nutrient broth. For curing experiment the culture was exposed to high temperature of 45ÊC and DNA isolation was done according to standard protocols as described earlier [33, 34]. DNA isolation, molecular identification and phylogenetic analysis of S Isolation of genomic DNA was carried out using standard phenol/chloroform extraction protocol [35, 36]. Isolated DNA was run on agarose gel to check its purity. It was stored at -4ÊC till further use [37]. Bacterial primers cloning of nearly full length 16S rDNA and sequencing were performed according to the methods described previously [37, 38]. The 16S rRNA gene sequence of the strains was analysed at NCBI (National Centre for Biotechnology Information) using BLAST tool and compared to the corresponding neighbour sequences from GenBank-NCBI database. Consensus sequence was imported into the Multalin program and multiple alignments were performed with related species (GenBank-NCBI database). Sequences were compared to those present in the data bank using blast and aligned with the ClustalW program. The results obtained were further imp- orted into the MEGA-7 software for the construction of a phylogenetic tree using Bootstrap analy- sis and maximum likelihood with 500 replicates, the substitution method used was the Kimura 2-Parameter model and the statistical method used was maximum likelihood [39]. Results Isolation of bacteria from collected samples A total of 25 bacterial strains were isolated from rhizospheric soil samples collected from S. irio. Theses strains were cultured on solid and liquid media amended with anthracene. Screening of isolated strains on anthracene amended solid media Out of 25 bacterial isolates grown on anthracene amended nutrient agar media, 23 strains were found to utilize anthracene as a main source for energy, when cultured on PNR media amended with anthracene (Table 1). Screening of isolated strains in liquid media The isolates that performed best on PNR-anthracene media were further screened in liquid media. Out of 23 bacterial isolates 12 isolates having highest OD were selected for further study (Table 2). In order to confirm the bacterial growth (24 h interval till 120 h) at the expense of anthracene in liquid media, spectrophotometric analysis of bacterial growth and disappearance of anthracene was observed in PNR media at 600 nm and 540 nm, respectively (Fig 1). The results of an optimiza- tion study of parameters including anthracene concentration, temperature, pH of the media, PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 4 / 18 Anthracene degradation by B. cereus S Table 1. Screening of bacterial isolates from S. irio on anthracene amended PNR media. No Isolate Anthracene Concentration in ppm 25 50 100 200 300 400 500 600 700 800 900 1000 1100 1200 1. S +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ 2. S +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ 3. S +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ 4. S +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ 4. S +++ ++ +++ +++ +++ +++ +++ ++ + + + - - - 5. S +++ +++ +++ +++ +++ +++ +++ ++ + + + - - - 6. S +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ 7. S +++ +++ +++ +++ +++ +++ +++ ++ +++ ++ + + - - 8. S +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ + + 9. S +++ +++ +++ +++ +++ +++ +++ ++ +++ ++ ++ ++ + + 10. S +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ ++ + 10. S +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ ++ ++ 11. S +++ ++ +++ +++ +++ +++ +++ ++ ++ ++ + + + - 12. S +++ +++ ++ +++ +++ +++ +++ +++ ++ + + + - - 13. S +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ + + - - 14. S +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ + - 15. S +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ + - 16. S +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ + + + 17. S +++ ++ +++ +++ +++ +++ +++ +++ +++ ++ ++ - - - 18. S +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ + + + 19. S +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ + - - 20. S +++ +++ +++ +++ ++ ++ ++ + + + + - - - 21. S +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ ++ + + 22. S +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ + + - 23. S +++ +++ +++ ++ ++ ++ + + + + + - - - +++ = Rich growth ++ = Medium growth + = Less growth (-) = No growth https://doi.org/10.1371/journal.pone.0201620.t001 Table 2. Bacterial growth on anthracene after 3-days incubation in large test tube (600 ppm) PNR. S. No Strain OD 600nm 1. S 0.199 2. S 0.321 3. S 0.211 4. S 0.159 5. S 0.425 6. S 0.200 7. S 0.269 8. S 0.223 9. S 0.265 10. S 0.169 11. S 0.243 12. S 0.231 https://doi.org/10.1371/journal.pone.0201620.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 5 / 18 Anthracene degradation by B. cereus S Fig 1. UV-spectrophotometric analysis of bacterial growth and anthracene disappearance. The OD of bacterial growth was observed at 600 nm; the OD of anthracene concentration was observed at 540 nm. Each data point represents the mean of triplicated data with ±S.E. The data points with similar letters are not significantly different at P < 0.05. https://doi.org/10.1371/journal.pone.0201620.g001 alternate carbon and energy source, effect of nitrate salts and UV-light, shaking speed and inocu- lum size are given below. Optimization of anthracene concentration and bacterial inoculum The optimized concentration of anthracene was 1000 ppm for isolate S during this study (Fig -1 2A) and inoculum concentration was 10% vv for maximum degradation as shown in Fig 2B. Optimization of temperature, pH and shaking speed for anthracene degradation Maximum growth of bacterial isolate S was observed at 30ÊC (Fig 2C), at optimized pH 7 as shown in Fig 2D. Maximum growth was observed at 180 rpm as shown in Fig 2E. Effect of different carbon and nitrogen sources on growth of isolated S Both the carbon sources and nitrate salts used were inhibitory on the isolate growth except potassium nitrate, with enhanced growth of our isolate (Fig 3A and 3B). Effect of UV-light induced mutation on anthracene utilization The UV-light treated S isolate gave better growth results with increasing concentration of anthracene as compared to control experiment, not exposed to UV-light, as shown in Fig 3C. -1 CFU mL of isolated bacteria The viability of bacteria was determined by a CFU study of samples drawn after every 24 h intervals for 120 h as shown in Table 3. The cells showed an increase in growth from initial 6 23 1.5×10 per ml to 1.4×10 after 120 h. PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 6 / 18 Anthracene degradation by B. cereus S Fig 2. Optimization of conditions for the growth of bacterial isolates S . (A) represents optimization of anthracene concentration vs growth of isolate S ; (B) 13 13 represents optimization temperature vs growth of isolate S ; (C) represents optimization of media pH vs growth of isolate S ; (D) represents optimization of 13 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 7 / 18 Anthracene degradation by B. cereus S agitation speed vs growth of isolate S ; (E) represents optimization of inoculum concentration (%) vs growth of isolate S . Each bar represents the mean of 13 13 triplicated data with ±S.E. The bars with similar letters are not significantly different at P < 0.05. https://doi.org/10.1371/journal.pone.0201620.g002 Biodegradation of anthracene Biodegradation of anthracene and bacterial growth OD are shown in Fig 4. The isolate S degraded 82.29% anthracene in 120 h. Strain S degraded the anthracene effectively during the incubation period of 24 to 96 h, whereas the growth of the bacterial isolate reached to its maximum (OD = 1.15) at 120 h. Increase in the growth of S was observed with an OD value of 0.598±1.15 between 96±120 h at the expense of only 36.19% anthracene. Confirmation of anthracene biodegradation by bacterial isolate S was sought by GC-MS analysis. The identi- fied products included 9, 10-dihydroxyanthracene, anthraquinone, benzene acetic acid and catechol, respectively (Fig 5). Fig 3. Effect of UV and different media on the growth of of bacterial isolate S (A) represents the effect of different carbon sources on the growth of isolate S ; 13. 13 (B) represents the effect of different nitrogen source on the growth of isolate S ; (C) represents the effect of UV light and anthracene concentration on the growth of isolate S incubated for 96 h. Each bar represents the mean of triplicated data with ±S.E. The bars with similar letters are not significantly different at P < 0.05. https://doi.org/10.1371/journal.pone.0201620.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 8 / 18 Anthracene degradation by B. cereus S -1 Table 3. CFUmL of the bacterium isolate S . Time (h). 0 24 48 72 96 120 -1 6 11 14 17 20 23 CFUmL 1.5×10 2.9×10 3.8×10 1.8×10 3.2×10 1.4×10 https://doi.org/10.1371/journal.pone.0201620.t003 Plasmid curing and isolation The results from agarose gel electrophoresis and plasmid curing suggested that anthracene deg- radation is certainly plasmid associated. The isolated plasmid from bacterial culture without curing treatment and gel electrophoresis indicated the presence of 7 plasmid bands of different sizes (Figs 6 and 7). Cured culture gave negative results for the plasmid presence and was unable to grow on anthracene amended media (Fig 8). Identification of bacterial isolate S Initially the bacterial isolate was identified biochemically. The result showed it was gram posi- tive, motile, rod shaped, catalase and urease positive and was capable of starch hydrolysis, while it was negative for citrate, casein hydrolysis and indole production (Table 4). Colony morphology on agar plate showed off white/creamy color colonies with irregular margins, thus identified as Bacillus sp. Molecular identification and phylogenetic analysis of isolate S The strain isolated from the rhizospheric soil samples collected from S. irio was identified by ITS rDNA region sequencing analysis. Phylogenetic analysis was carried out through MEGA 7.0 software for the construction of a phylogenetic tree using Bootstrap analysis and maximum likelihood with 500 replicates. A total of 20 sequences were downloaded from BLAST data that were showing the maximum relatedness with our isolate. Results of BLAST search showed Fig 4. Anthracene disappearance by bacterial isolate S . Each bar represents the mean of triplicated data with ±S.E. The bars with similar letters are not significantly different at P < 0.05. https://doi.org/10.1371/journal.pone.0201620.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 9 / 18 Anthracene degradation by B. cereus S Fig 5. Biodegradation pathway of anthracene. Bold squares show the intermediates detected using GC-MS analysis of biodegraded samples by bacterial isolate S . https://doi.org/10.1371/journal.pone.0201620.g005 highest sequence similarity (98%) between the bacterial isolate S , capable of anthracene utili- zation as carbon and energy and Bacillus cereus RNS-1, Bacillus cereus strain LP20-03. The strain also showed 87% similarity with Bacillus thurengensis strain 13. On the basis of sequence homology and phylogenetic analysis, the isolated bacterial strain was identified as S strain of B. cereus (Fig 9). Discussion Polycyclic aromatic hydrocarbons are the main concern for the world environment that causes great damage to humans, plants and animals wellbeing. In the current study, we have identi- fied an isolate S from rhizospheric soil samples collected from S. irio with high activity against anthracene. Furthermore, the observed bacterial isolate had achieved higher growth at an increased level of anthracene from 100±1000 ppm. Similar results have been reported in the past where higher growth was attained by the bacterial strain in a medium enriched with anthracene [31]. Other factors that can be detrimental to bacterial growth and activity to digest PAHs include temperature, pH, aeration and the presence of nutrients in the medium. PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 10 / 18 Anthracene degradation by B. cereus S Fig 6. Plasmid bands from bacterial isolate S . 1 Kb ladder is on the left side, whereas S plasmid is on the right side of 13 13 the figure. https://doi.org/10.1371/journal.pone.0201620.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 11 / 18 Anthracene degradation by B. cereus S Fig 7. Cured plasmid sample from bacterial isolate S against 1 Kb ladder. No band can be seen (Left). https://doi.org/10.1371/journal.pone.0201620.g007 PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 12 / 18 Anthracene degradation by B. cereus S Fig 8. Degradation of anthracene by bacterial isolate S after plasmid curing. The OD of bacterial growth was observed at 600 nm; the OD of anthracene concentration was observed at 540 nm. Each data point represents the mean of triplicated data with ±S.E. The data points with similar letters are not significantly different at P < 0.05. https://doi.org/10.1371/journal.pone.0201620.g008 Rise in temperature can affect both solubility [40] and degradation of PAHs by bacteria. For instance, high temperature can make the PAHs more soluble and bioavailable, whereas it also decreases the solubility of oxygen that can mainly affect the activity of aerobic bacteria. Therefore, most of the previous researches tend to focus on moderate temperatures rather high or low temperatures. Likewise, in the present study, high anthracene degradation has been noticed at 30ÊC, which can be attributed to the optimal growth conditions of the selected strain. The optimal growth conditions can allow the MO to secrete a vast array of enzymes in the surroundings that can degrade the toxic compounds in question. Unsuitable temperatures can deter the enzyme action by blocking its access to substrate due to insolubility (low temper- ature) or effecting the confirmation of the enzymes (high temperature). Similarly, all MO can perform its activity at certain pH range, i.e. minimal, maximal and an optimal pH, where at optimal pH the activity of the MO is significantly high. Any drastic changes in pH can interfere with cell wall and cell contents of the MO, thus affecting its growth and metabolism [3]. The result of this study also revealed that at pH7 the growth and activity of the bacterial isolate S was high due to balanced ionic distribution inside and outside of the cells. However, changes in pH can disturb the ionic balance and disrupt the growth and metab- olism of the bacterial isolate S , resulting in low degradation of PAHs. Shaking speed also proved to be an important factor in the aerobic degradation of PAHs that needed to be optimized in order to achieve optimum bacterial growth and degradation of the pollutants. Proper aeration has considerably improved the growth of S in the anthracene supplemented medium. Faster agitation could result in a higher degradation rate, which can Table 4. Biochemical tests for the identification of isolate S . Microscopy Biochemical tests Rods Gram's Test Catalase Starch hydrolysis Citrate Urease +ive +ive +ive +ive +ive https://doi.org/10.1371/journal.pone.0201620.t004 PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 13 / 18 Anthracene degradation by B. cereus S Fig 9. Phylogenetic analysis of strain S . The evolutionary history was inferred by using the Tamura-Nei model Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA7. https://doi.org/10.1371/journal.pone.0201620.g009 be credited to sufficient supply of oxygen and dissolution of solute in the solvent to be taken up by microorganism [41]. Also, the degradation of anthracene by S might be facilitated by oxygenase enzyme. The level of enzyme production and activity might enhance in the presence of molecular oxygen that can lead to a complete degradation of anthracene. Certainly, ample supply of oxygen could efficiently incorporate it enzymatically in the aromatic ring of the anthracene, a rate regulating step in the biodegradation of PAHs [26, 42, 43]. Some microorganisms can consume PAHs as a source of carbon and energy [44], but the presence of glucose in the form of additional carbon source might effect it [45]. This has been confirmed by the present study, where S failed to digest the anthracene completely as a sole carbon source in the presence of glucose, fructose and sucrose. This means that supplementa- tion of media with any alternate carbon sources can influence the degradation of anthracene. The readily available carbon sources may negate the degradation of anthracene due to prior PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 14 / 18 Anthracene degradation by B. cereus S assimilation, thus causing inhibition of enzymes responsible for anthracene degradation [46]. Correspondingly, supplementation of the growth media with different nitrogen sources (KNO , NaNO , CaNO and NH NO ) has no significant effect on growth and degradation 3 3 3 4 3 activity of anthracene. The negative effect of different energy and nitrogen sources in this study might be linked to its role as a competitor to anthracene, making anthracene less avail- able to be degraded. Anthracene is hydrophobic due to its cyclic structure and lack of highly hydrophilic hydroxyl groups (-OH) that might be the reason for the slow rate of biodegradation by the MO. The rate of bioremediation of a pollutant mainly depends on the number and nature of degrading organism, intrinsic and extrinsic factors, solvent and chemical structure of the com- pound to be degraded. Algae, fungi and bacteria have the capabilities to degrade PAHs into less complex substances through biotransformation mechanisms [16]. Though, MO needs to adapt the conditions first that allow the fast growth of microbial populations with the ability to degrade PAHs [47]. Additionally, bioavailability-induced adaptations are equally important for microbial populations to build an interaction with contaminants and make it more bioac- cessible [48]. Similarly, the presence of other contaminants can affect the efficiency of micro- bial degradation, which is critical in terms of biodegradation and bioremediation. Despite well-studied phenomenon, there remains limited understanding of many fundamental aspects of plant-microbe interactions during PAHs phytoremediation [49, 50]. In the present study, it was noticed that 82.92% anthracene was degraded in 120 h in PNR media contrary to the past th reports. Previously, 74.8% anthracene degradation was recorded in BSM media on the 10 day of incubation, whereas the complete degradation of added anthracene to autoclaved soil by Burkholderia sp. has taken 20 days [10, 51]. In fact, the chemical structure and nature of a com- pound (including, molecular weight, water solubility and lipophobicity) and nature of MO would affect the bioaccumulation and the rate of degradation of the compound by MO. The results of this study have confirmed this argument, where bacterial isolate S has actively degraded anthracene (degradation rate = 82.29%) within six days, contrary to previous reports (89% degradation of three ring PAHs within seven weeks) [52, 53]. Conclusion Bacillus cereus S can be used for biodegradation of anthracene, which is the main pollutants of incomplete organic combustion produced by petroleum and coal industry. We isolated novel anthracene biodegrading bacterium. Our isolate used anthracene as a sole source of car- bon and it can be utilized for bioremediation of other PAHs. Bacillus cereus S can be a poten- tial tool for bioremediation of toxic hydrocarbons and to keep the environment free from PAH pollutants. Though, the development of precise and effective technology for the treat- ment of complex PAHs mixtures is still needed. Author Contributions Conceptualization: Muhammad Hamayun, Sumera Afzal Khan, In-Jung Lee. Data curation: Amjad Iqbal. Formal analysis: Muhammad Hamayun, Sumera Afzal Khan, Amjad Iqbal. Funding acquisition: In-Jung Lee. Investigation: Nadia Bibi, Badshah Islam, Farooq Shah, Muhammad Aaqil Khan. Project administration: In-Jung Lee. Resources: Muhammad Hamayun, In-Jung Lee. PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 15 / 18 Anthracene degradation by B. cereus S Supervision: Muhammad Hamayun, In-Jung Lee. Writing ± original draft: Muhammad Hamayun, Sumera Afzal Khan, Amjad Iqbal. Writing ± review & editing: Muhammad Hamayun, Sumera Afzal Khan, Amjad Iqbal, In- Jung Lee. References 1. Jacques RJS, Santos EC, Bento FM, Peralba MCR, Selbach PA, Sa ELS, et al. Anthracene biodegra- dation by Pseudomonas sp. isolated from a petrochemical sludge landfarming site. Int Biodeter Biodegr. 2005; 56(3):143±50. http://dx.doi.org/10.1016/j.ibiod.2005.06.005. 2. Othman N HN, Talib SA. Degradation of polycyclic aromatic hydrocarbon by pure strain isolated from municipal sludge: Synergistic and cometabolism phenomenon. International Conference on Environ- ment. 2010:86±90. 3. Nour SEG MY, Habib SA, Ali S. Evaluation of Corynebacterium variabilis Sh42 as a degrader for differ- ent poly aromatic compounds. 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Anthracene biodegradation capacity of newly isolated rhizospheric bacteria Bacillus cereus S13

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Abstract

Biodegradation of hazardous pollutants is of immense importance for maintaining a clean environment. However, the concentration of such contaminants/pollutants can be minimized OPENACCESS with the help of microorganisms that has the ability to degrade the toxic pollutants into non- Citation: Bibi N, Hamayun M, Khan SA, Iqbal A, toxic metabolites. In the current study, 23 bacterial isolates were purified from the rhizo- Islam B, Shah F, et al. (2018) Anthracene spheric soil of Sysimbrium irio, growing as a wild plant in the vicinity of gas filling stations in biodegradation capacity of newly isolated Peshawar city. The isolated strains were initially screened on solid nutrient agar and further rhizospheric bacteria Bacillus cereus S . PLoS ONE 13(8): e0201620. https://doi.org/10.1371/ purified by culturing it on anthracene amended mineral media (PNR). The bacterial growth journal.pone.0201620 and anthracene disappearance were observed by calculating optical density (OD). The iso- Editor: Andrea Franzetti, Universita degli Studi di lates showed a concentration-dependent growth on anthracene amended PNR media at Milano-Bicocca, ITALY 30ÊC and pH7. Also, an increase in bacterial OD from 0.351 to 1.80 with increased shaking Received: December 26, 2017 speed was noticed. On the contrary, alternate carbon sources (glucose, fructose, sucrose) or nitrogen sources (KNO , NaNO , NH NO and CaNO ) posed inhibitory effect on bacte- 3 3 4 3 3 Accepted: July 18, 2018 rial growth during anthracene degradation. The recorded efficiency of anthracene degrada- Published: August 2, 2018 23 -1 tion by the selected bacterial isolate (1.4×10 CFUmL and 1.80 OD) was 82.29%, after Copyright:© 2018 Bibi et al. This is an open access 120 h of incubation. The anthracene was degraded to 9, 10, dihydroxy-anthracene and article distributed under the terms of the Creative anthraquinone, detected through GC-MS. The efficient bacterial isolate was identified as Commons Attribution License, which permits unrestricted use, distribution, and reproduction in S , a new strain of Bacillus cereus, using 16S rRNA analysis, showing 98% homology. The any medium, provided the original author and isolated bacterial strain S may be used as a potential tool for bioremediation of toxic hydro- source are credited. carbons and to keep the environment free from PAH pollutants. Data Availability Statement: All relevant data are within the paper. Funding: Research was supported by the Agenda Program, Rural Development Administration, Background Republic of Korea (Project No. PJ01228603 to In- A major portion of petroleum mixture contains fuse-ringed aromatic compounds, the poly- Jung Lee). The funders had no role in study design, data collection and analysis, decision to publish, or aromatic hydrocarbons (PAHs). The PAHs are the most abundant contaminants in the atmo- preparation of the manuscript. sphere and are kept on top of the pollutants list by US Environmental Protection Agency [1, 2]. These contaminants can be found abundant around the industrial sites, such as gas produc- Competing interests: The authors have declared that no competing interests exist. tion sites and wood preservation industries, and release through automobile exhausts [3] that PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 1 / 18 Anthracene degradation by B. cereus S Abbreviations: PAHs, Polyaromatic hydrocarbons; are consistent threats to human. PAHs along with their derivatives are the major factors caus- LMW, Low-molecular weight; OD, Optical density; ing anaemia, asthma, splenomegaly and various types of cancer in humans [4]. Some of the HPLC, High performance liquid chromatography; PAHs with low molecular weights have caused reproductive abnormalities and even death in GC-MS, Gas chromatography±mass spectrometry; aquatic animals [5, 6]. DNA, Deoxyribonucleic acid; rRNA, Ribosomal Anthracene is used as a signature compound for the detection of PAHs contamination, as it ribonucleic acid; NCBI, National Centre for Biotechnology Information; UV, Ultraviolet light; is an integral part of many carcinogenic PAHs. Due to its hydrophobicity and potential to BLAST, Basic Local Alignment Search Tool; CFU, bioaccumulate, it is used as model compound [7] to define factors affecting bioavailability and Colony forming units; KH PO , Potassium 2 4 rate of degradation of PAHs in environment. In spite of structural identity between anthracene phosphate monobasic; (NH ) SO , Ammonium 4 2 4 and phenanthrene regarding the number of aromatic rings, anthracene differs in degradation sulfate; NaOH, Sodium Hydroxide; MgSO .7H O, 4 2 due to the hydrophobic nature [4, 8, 9]. The presences of these compounds in the environment Magnesium Sulfate Heptahydrate; FeSO .7H O, 4 2 have to be taken seriously, because of their broad toxic effects on living organisms [3, 10]. Iron(II) Sulfate Heptahydrate; HCl, Hydrogen chloride; KNO , Potassium nitrate; NaNO , Sodium 3 3 PAHs in contaminated soils must be treated to avoid any possible noxious effect on environ- nitrate; CaNO , Calcium nitrate; NH NO , 3 4 3 ment and human health. In this regard, microbes can play key role in degradation/bioremedia- Ammonium nitrate. tion of the toxic PAHs into non-toxic compounds [4]. From the last few decades, bioremediation is getting importance day by day, because it is cheap, feasible and safe to clean the contaminated localities [6, 11]. The efficient microbial strains, unlike physical and chemical treatments can completely mineralize the PAHs present on the soil surface or soil sediments [11]. Previously, it has been discovered that certain starins of fungi, bacteria and algae can feed on the harmful PAHs and produce harmless compounds out of them [12]. Haleyur, Shahsavari (13) has demonstrated that some of the microorganisms (including, Rhodococcus sp., Achromobacter sp., Oerskovia paurometabola, Pantoea sp., Sejongia sp., Microbacterium maritypi- cum and Arthrobacter equi) exhibits catechol 1,2-dioxygenase activity. The presence of catechol 1,2-dioxygenase activity enables the above mentioned microorganisms to grow on PAHs and degrade catechol [13]. Alfaalfa, rape, vetch, mulberry and mustard rhizoremediation have been reported to grow and flourish in PAHs rich soil. In fact, this might be due to the presence of exten- sive population of microbes, mainly PAHs degrading bacteria around the roots of those plants [14± 18], which enables them to grow normally. Also, pea straw has been found to be one of the most useful sources in decreasing PAHs concentration in the soil [19]. Metagenomic analysis proved that the effect of pea straw was indirect. In fact, pea straw has biostimulated the PAHs biodegrader (Pseudoxanthomonas spp. and Alcanivorax spp) in the soil that were lying latent prior to the addi- tion of pea straw to the soil [19]. The ability of many microorganisms to degrade hydrocarbons [20±22] through metabolism or co-metabolism have been reported over the years [5]. However, there is still a space to discover microbial strains that can serve as a potential source to be used in bioremediation of PAHs. The present study was also focused on the exploration of prospective strains of beneficial microorganism that can degrade toxic chemicals and provide stress free envi- ronment. For this purpose research was conducted to (a) isolate Anthracene degrading rhizospheric bacteria from soil; (b) identification of potential PAHs degrading strain(s) in contaminated soils. Methods Nutrient agar media and mineral salts media were used for initial isolation and screening of rhizospheric bacteria. The composition of PNR and PNRG (PNR+5 mM glucose) per liter of -1 -1 distilled water [23, 24], is PN (20x) 50 mL used as 50 mLL : KH PO 13.6% (wv ), (NH ) SO 2 4 4 2 4 -1 -1 -1 -1 2.4% (wv ), NaOH 2.5% (wv ) and R salts used as 7 mLL , MgSO .7H O 8% (wv ), FeS- 4 2 -1 -1 O .7H O 0.2% (wv ), HCl 0.4% (wv ), Agar (2%) was used as solidifying agent. 4 2 Collection of samples Sysimbrium irio was found in a wild, i.e. growing in oil contaminated soil at an operational gas filling station in Peshawar. The texture of the soil was sandy loam with pH = 7.0 ± 0.2; soil PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 2 / 18 Anthracene degradation by B. cereus S moisture = 9.6%, maximum water holding capacity = 43.9% and electrical conductivity of 3.24 -1 ds m , respectively. The plants from the contaminated soil were dug as a whole, identified and placed in plastic zipper bag. The samples were immediately brought to the microbiology research laboratory at the University of Peshawar and stored at 4ÊC till further processing. Isolation of bacteria Soil samples were sieved through 2 mm mesh to collect uniform sized sample. After grading, 1 g of soil sample was serially diluted in a distilled water and then followed the spread plate method as described by Alias S [25]. The plates were incubated at 28ÊC for 3±4 days until appearance of the colonies. The developed colonies were carefully picked and further cultured in a fresh nutrient agar plates, the inoculation step was repeated till achieving the pure culture. After obtaining the pure cultures, they were stored in anthracene slants at 4ÊC. Screening of the isolated strains on solid media The purified bacterial isolates were screened for their capability to utilize anthracene as a sole source of carbon and energy (required for biodegradation). A confirmatory spray-plate assay was used to check the efficiency of isolated bacterial strains to grow on media supplemented with anthracene [26, 27]. Anthracene was initially dissolved in acetone and sprayed on the plates containing bacterial culture. Acetone was then allowed to evaporate leaving anthracene on the surface of the plates to be digested by the bacteria. Screening of the isolated strains in liquid media Screening in liquid medium was performed using 250 ml flasks containing 100 ml PNR media, -1 10% of bacterial inoculum and 1000 mgL anthracene. The media was incubated at 28ÊC and the bacterial growth was monitored at every 24 h interval till 120 h. Spectrophotometric analy- sis of bacterial growth and disappearance of anthracene was observed in PNR media at 600 nm and 540 nm, respectively. Optimization of growth conditions for the isolated strains Different parameters, like concentration of anthracene, incubation temperature and pH of the growth media were optimized. Range of anthracene concentration was (100, 150, 500 and 1000 ppm), temperature (28, 30, 35, 40, 45 and 50ÊC) and pH used were (4, 5, 6, 7, 8 and 9). Effect of shaking speed and inoculum size was quantified using speed of (0, 120, 150, 180, 200 and 220 rpm) and (0, 8, 9, 10, 11, 12, 13%). Alternate carbon source than anthracene used were different sugars, like sucrose, glucose and fructose for their effect on the growth of bacte- ria at the expense of selected PAH [28, 29]. The bacterial isolate S inoculum was exposed to UV-light for 15 minutes and added to media containing different concentrations of anthra- cene [30]. After every 24 h, one ml of culture broth was aseptically collected to check OD, while one ml was collected and stored at 4ÊC to test for degradation capability. CFU was calcu- lated at 24 h interval till 120 h in order to check the viability of the bacterial isolate. All the experiments were performed in triplicate. Biodegradation experiment The biodegradation experiment was performed using 250 ml flasks containing 100 ml PNR -1 media, 10% of bacterial inoculum and 1000 mgL anthracene dissolved in acetone. Acetone was allowed to evaporate, 100 mL media was poured to the flask containing different concentration PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 3 / 18 Anthracene degradation by B. cereus S of anthracene and 10% bacterial inoculum was added. The flasks were incubated at 30ÊC and 1 mL of sample was drawn for HPLC analysis after 24 hours interval for 5 days [31]. Extraction of anthracene for GC-MS analysis For GC-MS analysis, Shimadzu fused silica capillary column was used. The column tempera- -1 -1 ture was set to 100ÊC for 1 min, 15ÊC min to 160ÊC and 5ÊCmin to 300ÊC for 7min. The GC injector was held isothermally at 280ÊC with a splitless period of 3 min. Helium was used -1 as the carrier gas, at a flow rate of 1 mL min by using electronic pressure control. The GC± MS interface temperature was maintained at 280ÊC [32]. Plasmid curing, isolation and agarose gel electrophoresis Plasmid DNA was isolated from 18±24 hours old culture grown in nutrient broth. For curing experiment the culture was exposed to high temperature of 45ÊC and DNA isolation was done according to standard protocols as described earlier [33, 34]. DNA isolation, molecular identification and phylogenetic analysis of S Isolation of genomic DNA was carried out using standard phenol/chloroform extraction protocol [35, 36]. Isolated DNA was run on agarose gel to check its purity. It was stored at -4ÊC till further use [37]. Bacterial primers cloning of nearly full length 16S rDNA and sequencing were performed according to the methods described previously [37, 38]. The 16S rRNA gene sequence of the strains was analysed at NCBI (National Centre for Biotechnology Information) using BLAST tool and compared to the corresponding neighbour sequences from GenBank-NCBI database. Consensus sequence was imported into the Multalin program and multiple alignments were performed with related species (GenBank-NCBI database). Sequences were compared to those present in the data bank using blast and aligned with the ClustalW program. The results obtained were further imp- orted into the MEGA-7 software for the construction of a phylogenetic tree using Bootstrap analy- sis and maximum likelihood with 500 replicates, the substitution method used was the Kimura 2-Parameter model and the statistical method used was maximum likelihood [39]. Results Isolation of bacteria from collected samples A total of 25 bacterial strains were isolated from rhizospheric soil samples collected from S. irio. Theses strains were cultured on solid and liquid media amended with anthracene. Screening of isolated strains on anthracene amended solid media Out of 25 bacterial isolates grown on anthracene amended nutrient agar media, 23 strains were found to utilize anthracene as a main source for energy, when cultured on PNR media amended with anthracene (Table 1). Screening of isolated strains in liquid media The isolates that performed best on PNR-anthracene media were further screened in liquid media. Out of 23 bacterial isolates 12 isolates having highest OD were selected for further study (Table 2). In order to confirm the bacterial growth (24 h interval till 120 h) at the expense of anthracene in liquid media, spectrophotometric analysis of bacterial growth and disappearance of anthracene was observed in PNR media at 600 nm and 540 nm, respectively (Fig 1). The results of an optimiza- tion study of parameters including anthracene concentration, temperature, pH of the media, PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 4 / 18 Anthracene degradation by B. cereus S Table 1. Screening of bacterial isolates from S. irio on anthracene amended PNR media. No Isolate Anthracene Concentration in ppm 25 50 100 200 300 400 500 600 700 800 900 1000 1100 1200 1. S +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ 2. S +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ 3. S +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ 4. S +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ 4. S +++ ++ +++ +++ +++ +++ +++ ++ + + + - - - 5. S +++ +++ +++ +++ +++ +++ +++ ++ + + + - - - 6. S +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ 7. S +++ +++ +++ +++ +++ +++ +++ ++ +++ ++ + + - - 8. S +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ + + 9. S +++ +++ +++ +++ +++ +++ +++ ++ +++ ++ ++ ++ + + 10. S +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ ++ + 10. S +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ ++ ++ 11. S +++ ++ +++ +++ +++ +++ +++ ++ ++ ++ + + + - 12. S +++ +++ ++ +++ +++ +++ +++ +++ ++ + + + - - 13. S +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ + + - - 14. S +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ + - 15. S +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ + - 16. S +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ + + + 17. S +++ ++ +++ +++ +++ +++ +++ +++ +++ ++ ++ - - - 18. S +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ + + + 19. S +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ + - - 20. S +++ +++ +++ +++ ++ ++ ++ + + + + - - - 21. S +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ ++ + + 22. S +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ + + - 23. S +++ +++ +++ ++ ++ ++ + + + + + - - - +++ = Rich growth ++ = Medium growth + = Less growth (-) = No growth https://doi.org/10.1371/journal.pone.0201620.t001 Table 2. Bacterial growth on anthracene after 3-days incubation in large test tube (600 ppm) PNR. S. No Strain OD 600nm 1. S 0.199 2. S 0.321 3. S 0.211 4. S 0.159 5. S 0.425 6. S 0.200 7. S 0.269 8. S 0.223 9. S 0.265 10. S 0.169 11. S 0.243 12. S 0.231 https://doi.org/10.1371/journal.pone.0201620.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 5 / 18 Anthracene degradation by B. cereus S Fig 1. UV-spectrophotometric analysis of bacterial growth and anthracene disappearance. The OD of bacterial growth was observed at 600 nm; the OD of anthracene concentration was observed at 540 nm. Each data point represents the mean of triplicated data with ±S.E. The data points with similar letters are not significantly different at P < 0.05. https://doi.org/10.1371/journal.pone.0201620.g001 alternate carbon and energy source, effect of nitrate salts and UV-light, shaking speed and inocu- lum size are given below. Optimization of anthracene concentration and bacterial inoculum The optimized concentration of anthracene was 1000 ppm for isolate S during this study (Fig -1 2A) and inoculum concentration was 10% vv for maximum degradation as shown in Fig 2B. Optimization of temperature, pH and shaking speed for anthracene degradation Maximum growth of bacterial isolate S was observed at 30ÊC (Fig 2C), at optimized pH 7 as shown in Fig 2D. Maximum growth was observed at 180 rpm as shown in Fig 2E. Effect of different carbon and nitrogen sources on growth of isolated S Both the carbon sources and nitrate salts used were inhibitory on the isolate growth except potassium nitrate, with enhanced growth of our isolate (Fig 3A and 3B). Effect of UV-light induced mutation on anthracene utilization The UV-light treated S isolate gave better growth results with increasing concentration of anthracene as compared to control experiment, not exposed to UV-light, as shown in Fig 3C. -1 CFU mL of isolated bacteria The viability of bacteria was determined by a CFU study of samples drawn after every 24 h intervals for 120 h as shown in Table 3. The cells showed an increase in growth from initial 6 23 1.5×10 per ml to 1.4×10 after 120 h. PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 6 / 18 Anthracene degradation by B. cereus S Fig 2. Optimization of conditions for the growth of bacterial isolates S . (A) represents optimization of anthracene concentration vs growth of isolate S ; (B) 13 13 represents optimization temperature vs growth of isolate S ; (C) represents optimization of media pH vs growth of isolate S ; (D) represents optimization of 13 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 7 / 18 Anthracene degradation by B. cereus S agitation speed vs growth of isolate S ; (E) represents optimization of inoculum concentration (%) vs growth of isolate S . Each bar represents the mean of 13 13 triplicated data with ±S.E. The bars with similar letters are not significantly different at P < 0.05. https://doi.org/10.1371/journal.pone.0201620.g002 Biodegradation of anthracene Biodegradation of anthracene and bacterial growth OD are shown in Fig 4. The isolate S degraded 82.29% anthracene in 120 h. Strain S degraded the anthracene effectively during the incubation period of 24 to 96 h, whereas the growth of the bacterial isolate reached to its maximum (OD = 1.15) at 120 h. Increase in the growth of S was observed with an OD value of 0.598±1.15 between 96±120 h at the expense of only 36.19% anthracene. Confirmation of anthracene biodegradation by bacterial isolate S was sought by GC-MS analysis. The identi- fied products included 9, 10-dihydroxyanthracene, anthraquinone, benzene acetic acid and catechol, respectively (Fig 5). Fig 3. Effect of UV and different media on the growth of of bacterial isolate S (A) represents the effect of different carbon sources on the growth of isolate S ; 13. 13 (B) represents the effect of different nitrogen source on the growth of isolate S ; (C) represents the effect of UV light and anthracene concentration on the growth of isolate S incubated for 96 h. Each bar represents the mean of triplicated data with ±S.E. The bars with similar letters are not significantly different at P < 0.05. https://doi.org/10.1371/journal.pone.0201620.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 8 / 18 Anthracene degradation by B. cereus S -1 Table 3. CFUmL of the bacterium isolate S . Time (h). 0 24 48 72 96 120 -1 6 11 14 17 20 23 CFUmL 1.5×10 2.9×10 3.8×10 1.8×10 3.2×10 1.4×10 https://doi.org/10.1371/journal.pone.0201620.t003 Plasmid curing and isolation The results from agarose gel electrophoresis and plasmid curing suggested that anthracene deg- radation is certainly plasmid associated. The isolated plasmid from bacterial culture without curing treatment and gel electrophoresis indicated the presence of 7 plasmid bands of different sizes (Figs 6 and 7). Cured culture gave negative results for the plasmid presence and was unable to grow on anthracene amended media (Fig 8). Identification of bacterial isolate S Initially the bacterial isolate was identified biochemically. The result showed it was gram posi- tive, motile, rod shaped, catalase and urease positive and was capable of starch hydrolysis, while it was negative for citrate, casein hydrolysis and indole production (Table 4). Colony morphology on agar plate showed off white/creamy color colonies with irregular margins, thus identified as Bacillus sp. Molecular identification and phylogenetic analysis of isolate S The strain isolated from the rhizospheric soil samples collected from S. irio was identified by ITS rDNA region sequencing analysis. Phylogenetic analysis was carried out through MEGA 7.0 software for the construction of a phylogenetic tree using Bootstrap analysis and maximum likelihood with 500 replicates. A total of 20 sequences were downloaded from BLAST data that were showing the maximum relatedness with our isolate. Results of BLAST search showed Fig 4. Anthracene disappearance by bacterial isolate S . Each bar represents the mean of triplicated data with ±S.E. The bars with similar letters are not significantly different at P < 0.05. https://doi.org/10.1371/journal.pone.0201620.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 9 / 18 Anthracene degradation by B. cereus S Fig 5. Biodegradation pathway of anthracene. Bold squares show the intermediates detected using GC-MS analysis of biodegraded samples by bacterial isolate S . https://doi.org/10.1371/journal.pone.0201620.g005 highest sequence similarity (98%) between the bacterial isolate S , capable of anthracene utili- zation as carbon and energy and Bacillus cereus RNS-1, Bacillus cereus strain LP20-03. The strain also showed 87% similarity with Bacillus thurengensis strain 13. On the basis of sequence homology and phylogenetic analysis, the isolated bacterial strain was identified as S strain of B. cereus (Fig 9). Discussion Polycyclic aromatic hydrocarbons are the main concern for the world environment that causes great damage to humans, plants and animals wellbeing. In the current study, we have identi- fied an isolate S from rhizospheric soil samples collected from S. irio with high activity against anthracene. Furthermore, the observed bacterial isolate had achieved higher growth at an increased level of anthracene from 100±1000 ppm. Similar results have been reported in the past where higher growth was attained by the bacterial strain in a medium enriched with anthracene [31]. Other factors that can be detrimental to bacterial growth and activity to digest PAHs include temperature, pH, aeration and the presence of nutrients in the medium. PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 10 / 18 Anthracene degradation by B. cereus S Fig 6. Plasmid bands from bacterial isolate S . 1 Kb ladder is on the left side, whereas S plasmid is on the right side of 13 13 the figure. https://doi.org/10.1371/journal.pone.0201620.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 11 / 18 Anthracene degradation by B. cereus S Fig 7. Cured plasmid sample from bacterial isolate S against 1 Kb ladder. No band can be seen (Left). https://doi.org/10.1371/journal.pone.0201620.g007 PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 12 / 18 Anthracene degradation by B. cereus S Fig 8. Degradation of anthracene by bacterial isolate S after plasmid curing. The OD of bacterial growth was observed at 600 nm; the OD of anthracene concentration was observed at 540 nm. Each data point represents the mean of triplicated data with ±S.E. The data points with similar letters are not significantly different at P < 0.05. https://doi.org/10.1371/journal.pone.0201620.g008 Rise in temperature can affect both solubility [40] and degradation of PAHs by bacteria. For instance, high temperature can make the PAHs more soluble and bioavailable, whereas it also decreases the solubility of oxygen that can mainly affect the activity of aerobic bacteria. Therefore, most of the previous researches tend to focus on moderate temperatures rather high or low temperatures. Likewise, in the present study, high anthracene degradation has been noticed at 30ÊC, which can be attributed to the optimal growth conditions of the selected strain. The optimal growth conditions can allow the MO to secrete a vast array of enzymes in the surroundings that can degrade the toxic compounds in question. Unsuitable temperatures can deter the enzyme action by blocking its access to substrate due to insolubility (low temper- ature) or effecting the confirmation of the enzymes (high temperature). Similarly, all MO can perform its activity at certain pH range, i.e. minimal, maximal and an optimal pH, where at optimal pH the activity of the MO is significantly high. Any drastic changes in pH can interfere with cell wall and cell contents of the MO, thus affecting its growth and metabolism [3]. The result of this study also revealed that at pH7 the growth and activity of the bacterial isolate S was high due to balanced ionic distribution inside and outside of the cells. However, changes in pH can disturb the ionic balance and disrupt the growth and metab- olism of the bacterial isolate S , resulting in low degradation of PAHs. Shaking speed also proved to be an important factor in the aerobic degradation of PAHs that needed to be optimized in order to achieve optimum bacterial growth and degradation of the pollutants. Proper aeration has considerably improved the growth of S in the anthracene supplemented medium. Faster agitation could result in a higher degradation rate, which can Table 4. Biochemical tests for the identification of isolate S . Microscopy Biochemical tests Rods Gram's Test Catalase Starch hydrolysis Citrate Urease +ive +ive +ive +ive +ive https://doi.org/10.1371/journal.pone.0201620.t004 PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 13 / 18 Anthracene degradation by B. cereus S Fig 9. Phylogenetic analysis of strain S . The evolutionary history was inferred by using the Tamura-Nei model Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA7. https://doi.org/10.1371/journal.pone.0201620.g009 be credited to sufficient supply of oxygen and dissolution of solute in the solvent to be taken up by microorganism [41]. Also, the degradation of anthracene by S might be facilitated by oxygenase enzyme. The level of enzyme production and activity might enhance in the presence of molecular oxygen that can lead to a complete degradation of anthracene. Certainly, ample supply of oxygen could efficiently incorporate it enzymatically in the aromatic ring of the anthracene, a rate regulating step in the biodegradation of PAHs [26, 42, 43]. Some microorganisms can consume PAHs as a source of carbon and energy [44], but the presence of glucose in the form of additional carbon source might effect it [45]. This has been confirmed by the present study, where S failed to digest the anthracene completely as a sole carbon source in the presence of glucose, fructose and sucrose. This means that supplementa- tion of media with any alternate carbon sources can influence the degradation of anthracene. The readily available carbon sources may negate the degradation of anthracene due to prior PLOS ONE | https://doi.org/10.1371/journal.pone.0201620 August 2, 2018 14 / 18 Anthracene degradation by B. cereus S assimilation, thus causing inhibition of enzymes responsible for anthracene degradation [46]. Correspondingly, supplementation of the growth media with different nitrogen sources (KNO , NaNO , CaNO and NH NO ) has no significant effect on growth and degradation 3 3 3 4 3 activity of anthracene. The negative effect of different energy and nitrogen sources in this study might be linked to its role as a competitor to anthracene, making anthracene less avail- able to be degraded. Anthracene is hydrophobic due to its cyclic structure and lack of highly hydrophilic hydroxyl groups (-OH) that might be the reason for the slow rate of biodegradation by the MO. The rate of bioremediation of a pollutant mainly depends on the number and nature of degrading organism, intrinsic and extrinsic factors, solvent and chemical structure of the com- pound to be degraded. Algae, fungi and bacteria have the capabilities to degrade PAHs into less complex substances through biotransformation mechanisms [16]. Though, MO needs to adapt the conditions first that allow the fast growth of microbial populations with the ability to degrade PAHs [47]. Additionally, bioavailability-induced adaptations are equally important for microbial populations to build an interaction with contaminants and make it more bioac- cessible [48]. Similarly, the presence of other contaminants can affect the efficiency of micro- bial degradation, which is critical in terms of biodegradation and bioremediation. Despite well-studied phenomenon, there remains limited understanding of many fundamental aspects of plant-microbe interactions during PAHs phytoremediation [49, 50]. In the present study, it was noticed that 82.92% anthracene was degraded in 120 h in PNR media contrary to the past th reports. Previously, 74.8% anthracene degradation was recorded in BSM media on the 10 day of incubation, whereas the complete degradation of added anthracene to autoclaved soil by Burkholderia sp. has taken 20 days [10, 51]. In fact, the chemical structure and nature of a com- pound (including, molecular weight, water solubility and lipophobicity) and nature of MO would affect the bioaccumulation and the rate of degradation of the compound by MO. The results of this study have confirmed this argument, where bacterial isolate S has actively degraded anthracene (degradation rate = 82.29%) within six days, contrary to previous reports (89% degradation of three ring PAHs within seven weeks) [52, 53]. Conclusion Bacillus cereus S can be used for biodegradation of anthracene, which is the main pollutants of incomplete organic combustion produced by petroleum and coal industry. We isolated novel anthracene biodegrading bacterium. Our isolate used anthracene as a sole source of car- bon and it can be utilized for bioremediation of other PAHs. Bacillus cereus S can be a poten- tial tool for bioremediation of toxic hydrocarbons and to keep the environment free from PAH pollutants. Though, the development of precise and effective technology for the treat- ment of complex PAHs mixtures is still needed. Author Contributions Conceptualization: Muhammad Hamayun, Sumera Afzal Khan, In-Jung Lee. Data curation: Amjad Iqbal. Formal analysis: Muhammad Hamayun, Sumera Afzal Khan, Amjad Iqbal. Funding acquisition: In-Jung Lee. Investigation: Nadia Bibi, Badshah Islam, Farooq Shah, Muhammad Aaqil Khan. Project administration: In-Jung Lee. Resources: Muhammad Hamayun, In-Jung Lee. 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