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Isolation and Characterization of Degrading Marine Bacteria

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Isolation and Characterization of (PAH) Biodegrading Marine Bacteria

Sulaiman Ali Alharbi1*, M.E.Zayed1, Arunachalam Chinnathambi1, Naiyf S. Alharbi1 and Milton Wainwright1,2

1Department of Botany and Microbiology, College of Science, King Saud University,

Riyadh, Kingdom of Saudi Arabia.

2Department of Molecular Biology and Biotechnology, University of Sheffield, S102TN, UK.

ABSTRACT

Polyaromatic hydrocarbons (PAH) are considered to be important and dangerous pollutants which cause serious health problems and/or genetic defects in humans, as well as harming the flora and fauna of affected habitats. In this study, we interested in determining if bacteria can be successfully used to bioremediate PAH pollution as an alternative to physical and chemical methods. The bacteria used in this study were isolated from three PAH polluted sites of Mediterranean Sea, off Alexandria, Egypt. The study is devoted to the isolation of bacteria that can degrade three low molecular weight PAHs and to determine the effect of pH on this process. Nine phenanthrene-degrading, seven-naphthalene-degrading and eight anthracene-degrading bacteria were isolated, by enrichment, from the marine water sample. All of the isolates grew on the PAHs (phenanthrene, anthracene and naphthalene) at varying rates and utilized them as sole source of carbon and energy.

Key words:  Polyaromatic hydrocarbons (PAH), Biodegrading marine bacteria, Environmental contamination, Marine water,

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are contaminants of aquatic and terrestrial ecosystems which generated continuously by the inadvertently incomplete combustion of organic matter, for instance in forest fires, home heating, traffic, and waste incineration1. PAHs constitute a large and diverse class of organic compounds and are generally described as molecules which consist of three or more fused aromatic rings in various structural configurations2. Polycyclic aromatic hydrocarbons are composed of fused, aromatic rings whose biochemical persistence arises from dense clouds of π-electrons on both sides of the ring structures, thereby making them resistant to nucleophilic attack3.

Environments contaminated with PAHs are deemed hazardous because of their carcinogenic, mutagenic and teratogenic effects4,5 and low molecular weight PAHs such as naphthalene (the simplest, containing two benzene rings), anthracene and phenanthrene (both of which contain three benzene rings) are also known to possess potentially hazardous health effects6.

A variety of techniques have been applied to the treatment of environments contaminated by PAH containing petroleum hydrocarbons, notably physical treatments using thermal or chemical processes7. However, these treatments are generally time consuming and expensive8,9. Microbial bioremediation however, provides a potentially cheap and effective means of bio-remediating PAH-contaminated environments10. The ability of microorganisms to degrade PAHs is well documented11, 12 and microbial degradation is a major environmental process affecting the fate of PAHs in both terrestrial and aquatic ecosystems13. Bioremediation using microbes converts toxic or persistent organic molecules into harmless end products, such as carbon dioxide and water 14.

Unfortunately PAHs possess physical properties, such as low aqueous solubility and high solid water distribution ratios, which militate against their rapid microbial utilization resulting in their accumulation in the terrestrial and aquatic environments1. It is imperative therefore that the factors which influence the bioavailability and decomposition of PAHs in the environment be studied and optimized7. The aim of the work described here was to screening environmental samples for bacteria that are capable of degrading PAHs and use them a sole carbon and energy source.

Materials and Methods

Isolation of bacteria:

Bacteria were isolated from three PAH-polluted sites of the Mediterranean Sea, Alexandria, Egypt, where PAH pollutants have been continually released. All isolates were preserved in 750 μl LB culture supplemented with 250 μl of 60% glycerol and maintained at -80oC.

Media used:

Bushnell-Haas (BH) medium, Luria-Bertani, Nutrient broth and Blood agar were used for the isolation of bacteria. All media were prepared using distilled water and sterilized by autoclaving at 120oC for 20 min. Medium- pH was adjusted as required before sterilization using  1N NaOH or 1N HCl.

Hydrocarbon stock solutions: Stock solutions of each PAH (100 mg/ml) were prepared in ethyl acetate and sterilized by filtration.

Isolation of phenanthrene, anthracene and naphthalene degrading bacteria:

Water samples were collected from three PAH-polluted sites in the Mediterranean Sea, off Alexandria, Egypt, where PAH pollutants have been continually released into these aquatic environments without any control. Water samples (50ml) were collected from the contaminated sites under aseptic conditions.

Phenanthrene, anthracene and naphthalene degrading bacteria were isolated from water samples by spreading onto solid medium; 100 µl of each water sample was spread over the surface Bushnell-Haas agar plates containing (100, 200, 300, 400, 500 and 600 mg/l) of either, phenanthrene, anthracene or naphthalene, as the sole carbon and energy source. The plates were then incubated at 30oC for 7 days.

Assay of phenanthrene, anthracene and naphthalene degradation:

Phenanthrene, anthracene and naphthalene degradation by the bacteria under a variety of concentrations was determined using 250 ml Erlenmeyer flasks containing 100 ml Bushnell-Haas broth supplemented with phenanthrene, anthracene and naphthalene in the concentration range,10, 30, 50, 70, 90, 130 to 150 mg/l.. The cultures were inoculated by transferring 1 ml of nutrient broth of pre culture medium of the strain under test. The cultures were then incubated at 30oC and 200 rpm; bacterial growth was daily evaluated by measuring the increase of OD600nm of the culture.

Determination of naphthalene, phenanthrene and anthracene residues in the culture medium:

The concentration of naphthalene and phenanthrene residues in the culture medium was determined by measuring the optical density at a wavelength of 254 nm and 275 nm respectively15. Extraction was carried out in a separator funnel, by mixing for two minutes an aliquot of the culture medium with an equal volume of hexane. The resulting organic phase was then used for the spectrophotometric readings. In some cases the sample was diluted with hexane in order to bring it out within the range of the calibration line (0.01-0.07 mg/ml for naphthalene and 0.001-0.1 mg/ml for phenanthrene).

For the determination of anthracene residue, aliquots of the culture medium were mixed for two minutes with an equal volume of ethyl acetate and the optical density of the resulting organic phase was measured at a wavelength of 254 nm using ethyl acetate as a blank. For preparation of standard curve of anthracene, a stock solution of 10 ppm in ethyl acetate was prepared and aliquots in the range of 0.2 to 1 ppm were separately measured at 254 nm16.

Utilization of carbon source:

All purified isolates were tested for growth on 0.01%, of either, naphthalene, phenanthrene, anthracene or phenol which were added as sole carbon sources to BH liquid medium. Sterilized BH medium containing the desired amount of hydrocarbon source was inoculated with the test strain and incubated in an orbital shaker at 200 rpm and 30oC for 72 h. Growth was tested by measuring the increase of OD600nm of the cultures.

Effect of pH on the degradation of naphthalene, phenanthrene and anthracene:

In order to determine the effect of pH on naphthalene, phenanthrene and anthracene degradation, 50 ml of BH broth cultures were first prepared at the following pH; 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11. After sterilization, 50 mg/l naphthalene, phenanthrene and anthracene was added to each flask, separately. 0.5 ml of Nutrient Broth overnight culture of bacterial strains (OD600 1.3-1.5) was added to each flask. The flasks were incubated at 30oC and 200 rpm for 72h. Bacterial growth was estimated using spectrophotometer at OD600nm of the cultures.

Results and Discussion

Isolation and selection of phenanthrene, naphthalene and anthracene biodegrading bacteria:

Phenanthrene, naphthalene and anthracene degrading bacteria were isolated from water samples.  A range of bacterial colony types were observed on the different carbon source-containing plates, nine isolates were found to utilize phenanthrene, seven  grew on the naphthalene plates and eight isolates grew on anthracene amended media.

Isolates Ph1, Ph2, Ph3, Ph4, Ph5 and Ph7 showed the ability to degrade phenanthrene as sole carbon sorce. The optical density (OD600)  for the bacterial isolates respective was was; 0.16, 0.512, 0.17, 0.482, 0.632 and 0.24. Isolates Ph6, Ph8 and Ph9 on the otherhand failed to utilize phenanthrene as sole carbon and energy source (Fig.1). Among the tested isolates only Ph5 isolate showed sustantial  growth rate on phenanthrene when compared with other tested isolates. According to the ioslates which showed no degrdation of the tetsed PAH, We assume that these isolates which did not degrade PAH may have lost this ability during preservation, or may have lost genes which control the utilization of this substrate; assumptions mirored in the work of  Zhao, et al., (2009)17, who reported that some phenanthrene degrading isolates lost their ability to degrade phenanthrene after a period of more than three weeks of preservation.

Figure 1. Degradation of phenanthrene (20 mg\l) by 9 bacterial isolates (named; Ph1-Ph9).    

In studies using naphthalene, only two isolates Na6 and Na7 showed dehradation  with the latter being the best naphthalene degrader (Fig.2). The other isolates, Na1, Na2, Na3, Na4 and Na5, in contrast, failed to use naphthalene as a sole carbon and energy source. Regarding the anthracene-degarding isolates, five- An1, An2, An3, An4 and An7 degraded the substrate with ODs respectively oft: 0.174, 0.614, 0.551, 0.482 and 0.164 (Fig.3). Isolate,  An2 isolate was the msot active anthracene degrader,while isolates An5, An6 and An8 were unable  to use anthracene as a sole source carbon and energy source.

Figure 2.  Degradation of naphthalene (20 mg\l) by 7 bacterial isolates (named; Na1-Na7).

Figure 3. Degradation of anthracene (20 mg\l) by the bacterial isolates (named; An1-An8).

Effect of pH on hydrocarbons degradation by the different bacterial isolates:

The effect of pH (2.0, 3.0, 4.0,  up to 11.0) on PAH degradation by the isolated bacteria Ph5, Na7 and An2 was investigated in BH medium containing 50 mg\l phenanthrene and 20 mg\l naphthalene or anthracene respectively.

In the case of phenanthrene, the optimum pH for bacterial growth and phenanthrene degradation was pH 7. At pH 6 and pH 8, bacterial growth and substrate degradation was markedly reduced (Fig.4). Shin et al.,(2008)18 reported that, relatively high mineralization rates of phenanthrene are found over a pH range of 6-8, with maximum mineralization rate occurring at pH 6 in a mineral salt medium. In the present study we found that the pH 7 is the optimum for obtaining a high mineralization rate of phenanthrene in BH medium; in agreement with Simarro, et al., (2011)19, our results confirm that the optimal pH value for the degradation of this substrate,  in BH medium, is pH7.

Figure 4. The ability of the selected Ph5 isolate to degrade 50 mg/l phenanthrene at different pH.

Growth and biodegradation depends on the type of PAHs used and the optimum pH range is very variable20. Some acid resistant Gram-positive bacteria, such as Mycobacterium sp., show better PAH degradation capabilities under acid conditions, largely because low pH seems to render such Mycobacteria more permeable to hydrophobic substrates21. However, other microorganisms belonging to Pseudomonas genus tend to prefer neutral pH conditions. In agreement with previous works19, our results confirm that neutral pH is optimum for the biodegradation of PAHs. Our results are also in agreement with Bisht, et al.(2010)22, who reported the ability of D. radiodurans SBA6 and B. circulans SBA12 to degrade naphthalene and anthracene over in the pH range of 5.0 to 11 (Fig.5). Othman et al.(2009)23 observed that changes in media pH can alter the electrical charge on various chemicals groups in enzymes molecules, thereby probably altering the enzyme's ability to bind its substrate and catalyze a given reaction. Imbalance of the electrical charges in very acidic and alkali conditions can also disrupt hydrogen bonds and other weak forces that maintain enzyme structure. Such disruption of enzyme structure is called denaturation, a phenomenon which leads to poor rates of biodegradation.

Figure 5. Determination of the ability of Na7 and An2 isolate to degrade 20 mg/l naphthalene and anthracene respectively, over a range of pH.

Utilization of different hydrocarbons by the same bacterium:

Phenol (a single ring compound) was also included in this experiment as an example of a low molecular weight PAHs to provide a comparison with the high molecular weights hydrocarbons e.g. naphthalene (two rings), anthracene, and phenanthrene (both three compounds).

Isolate Ph5 was shown to use phenol, naphthalene and anthracene as sole carbon and energy source (Fig.6). These results are slightly different from those reported by Zhao, et al. (2009)17, who reported that a bacterial isolate (ZP2) was able to degrade phenanthrene and naphthalene but failed to degrade anthracene as sole carbon source.

Figure 6. Assimilation of 0.01% of naphthalene, anthracene and phenol as a sole  carbon source in Bushnell-Haas medium by Ph5 isolate.

The same response was seen in the other two isolates Na7 and An2.  Isolate, Na7 rapidly mineralized phenol, phenanthrene and anthracene when added as sole carbon sources (Fig.7). Isolate An2 degraded phenol, naphthalene and phenanthrene as sole sources of energy (Fig.8). An isolate used in a study by Dean-Ross, et al. (2001)24 in contrasts was able to mineralize anthracene and phenanthrene but not naphthalene when grown under identical conditions.

Figure 7. Utilization of 0.01% of phenanthrene, anthracene and phenol as a sole  carbon source in Bushnell-Haas medium by the isolate Na7.

Figure 8. Consumption of 0.01% of phenanthrene, naphthalene and phenol as a sole  carbon source in Bushnell-Haas medium by An2 isolate.

Phenanthrene, naphthalene and anthracene are the main components of crude oil and ubiquitous in contaminated water and soil. These carbon-sources could be utilized by a range of living in these polluted environments. Hydrocarbon mineralization occurs in a variety of ways depending on the species of bacterium isolated and it use of a preferred pathway. In this study, the wide range carbon-source utilization of tested isolates confirms their ability to use potentially different degradation pathways. Al-Thani, et al.(2009)25, similarly reported that the acclimation of a microbial community to one substrate frequently results in the simultaneous acclimation to some, but not all structurally related molecules. As a result, individual microbial species have the ability to act on several structurally similar substrates and therefore more easily act on their analogues following initial exposure26,27.

Conclusion:

In this investigation, we fascinated in determining if bacteria can be successfully used to bioremediate PAH pollution as a substitute to physical and chemical methods.In conclusion, some of our isolates grew well on the tested 4 low molecular weight organic aromatic compounds and as also reported28, individual bacterial strains could degrade several PAHs, but tended to prefer a single one and also we establish that the pH 7 is the optimum for obtaining a high mineralization rate of phenanthrene in BH medium.

Acknowledgements

Authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project No. RGP-VPP-332.

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