IDENTIFICATION OF HYDROCARBON DEGRADERS IN A CRUDE OIL POLLUTED VESSEL AND POSSIBLE GROWTH INDUCING POTENTIAL OF AZADIRACHTA INDICA

Abstract

Polycyclic aromatic hydrocarbons (PAH) are a class of organic compounds that have accumulated in the natural environment mainly, as a result of anthropogenic activities such as the combustion of fossil fuels. Some microorganisms, mainly from the genera Pseudomonas and Mycobacteria were found to be capable of transforming and degrading PAHs. Bioremediation is one approach that has been used to remediate contaminated soil and waters, and at the same time, promotes the natural attenuation of the contaminants, using microbial community of the site. However, the aim of this study was to investigate the possible effect of Azadirachta indica leaves on hydrocarbon degraders in crude oil polluted cotton wool vessel. The study was carried out using pumpkin seeds, Azadirachta indica, and crude oil polluted cotton wool vessel. The result from this study showed that, the vessel with the highest quantity of Azadirachta indica leaves had the highest growth of hydrocarbon degrading microorganism.

Headnote

ABSTRACT

Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds that have accumulated in the natural environment mainly as a result of anthropogenic activities such as the combustion of fossil fuels. Some microorganisms, mainly from the genera Pseudomonas and Mycobacteria were found to be capable of transforming and degrading PAHs. Bioremediation is one approach that has been used to remediate contaminated soil and waters, and at the same time promotes the natural attenuation of the contaminants using microbial community of the site. However, the aim of this study was to investigate the possible effect of Azadirachta indica leaves on hydrocarbon degraders in crude oil polluted cotton wool vessel. The study was carried out using pumpkin seeds, Azadirachta indica, and crude oil polluted cotton wool vessel. The result from this study showed that the vessel with the highest quantity of Azadirachta indica leaves had the highest growth of hydrocarbon degrading microorganism.

KEYWORDS: Azadirachta indica, Hydrocarbon degraders, polycyclic aromatic hydrocarbons.

INTRODUCTION

Petrochemical industries and petroleum refineries generate large amounts of priority pollutants. The major pollutants found in these industries are petroleum hydrocarbons (Chavan and Mukherji, 2008).

Petroleum is a complex mixture of hydrocarbons derived from the geologic transformation and decomposition of plants and animals that lived hundreds of millions of years ago. Petroleum consists mostly of hydrocarbon molecules. Crude oil and natural gas are the most important primary fossil fuels. Polycyclic aromatic hydrocarbons (PAHs), also known as poly-aromatic hydrocarbons or polynuclear aromatic hydrocarbons, are potent atmospheric pollutants that consist of fused aromatic rings and do not contain hetero-atoms or carry substituent (Fetzer, 2000).

Bioremediation is the process whereby organic wastes are biologically degraded under controlled conditions to an innocuous state. The main principle of this technique is to remove pollutants from the natural environment or convert the pollutants to a less harmful product using the indigenous microbiological community of the contaminated environment (Mueller et al., 1997). Interest in the microbial biodegradation of pollutants has intensified in recent years as mankind strives to find sustainable ways to clean up contaminated environments. These bioremediation and biotransformation methods use the naturally occurring, microbial catabolic diversity to degrade, transform or accumulate a huge range of compounds including hydrocarbons, polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), pharmaceutical substances, radionuclide's and metals. The concentration of PAHs in the environment varies widely depending on the level of industrial development, proximity of the contaminated sites to the production source and the mode of PAHs transport. Reported soil and sediment PAHs contaminations range from 1 |ig/kg to over 300 g/ kg (Kanaly, 2000).

Anaerobic metabolism of PAHs is thought to occur via the hydrogenation of the aromatic ring. The basis of this mechanism is the oxidation of the aromatic ring, followed by the systematic breakdown of the compound to PAH metabolites or carbon dioxide.

PAH-degrading microorganisms are ubiquitously distributed in the natural environment, such as in soils (bacteria and non- ligninolytic fungi) and woody materials (ligninolytic fungi). Many PAH contaminated soils and sediments host active populations of PAH-degrading bacteria (Tam et al., 2002).

Medicinal plants are part and parcel of human society to combat diseases, from the dawn of civilization. Azadirachta indica A. Juss (syn. Melia azadirachta) is well known in India and its neighbouring countries for more than 2000 years as one of the most versatile medicinal plant showing a wide spectrum of biological activity. A. indica A. Juss and M. azedarach are two closely related species of the family Meliaceae. The former is popularly known as Indian neem (margosa tree) or Indian lilac, and the latter is known as Persian lilac. Neem is an evergreen tree, cultivated in various parts of the Indian subcontinent. Every part of the tree has been used as traditional medicine for household remedy against various human ailments, from antiquity (Chatterjee and Pakrashi, 1994).

This study is therefore aimed at using grounded neem tree leaves to remediate crude oil polluted vessel. The result will ascertain the effectiveness of these leaves in bioremediation process.

MATERIALS AND METHODS

Chemicals

Crystal violet, Lugol iodine, Ethanol, Hydrogen peroxide, Urea solution, K2HPO4, Methyl red, a -naphtol, KOH, Methyl-P- phenylene diamine hydrochloride, Mineral salt medium, Sabouraud dextrose agar, Nutrient agar, Urea agar, Citrate agar, Nutrient broth, and Peptone were all purchased from Sigma Aldrich, USA.

Experimental design:

Vessel 1 - Pumpkin seeds + water only

Vessel 2 - Pumpkin seeds + 200 ml of crude oil + water

Vessel 3 - Pumpkin seeds + 200 ml of crude oil + 150 g of grounded Azadirachta indica Leaves + water

Vessel 4 - Pumpkin seeds + 200 ml of crude oil + 300 g of grounded Azadirachta indica Leaves + water

The pumpkin seeds were planted and monitored for eight weeks using sterilized cotton wool as soil. After which the cotton wool polluted with crude oil was collected and used for the microbiological assay.

Isolation of hydrocarbon utilizing organisms from cotton wool polluted with crude oil.

30 g of each polluted cotton wool was soaked in saline water (100 ml) for 1 hour, after which 1 ml from each of the soaked container was pipette and mixed with 9 ml of sterile water in a test tube and the dilution were prepared up to 10-5 dilution.

Enumeration of crude oil utilizing fungi

This was done using the surface spreading technique (Mbagwu, 1992)

1 ml from 10-5 of each sample was used to seed each sterile plate in triplicates. 20 ml of the mineral salt agar medium at 45oC supplemented with 1% Lactic acid was poured into the seeded plates and swirled.

Enumeration of crude oil utilizing bacteria

20 ml of mineral salt medium was supplemented with nystatin was poured in the seeded plates and swirled. These plates were left on the bench to set. Filter sterile paper were soaked in sterile crude oil and placed on the lids of the cultured plates, after which the plates were incubate for 14 days at room temperature. Colony that was developed on the plates was counted with colony counter and was recorded as colony forming unit per gram (Okpowasili et al., 1988).

Identification of bacteria

The procedure of Hamamura et al., (2006) was used.

Pure culture that was obtained was sub- cultured from the primary culture on Nutrient agar plates and was incubated at room temperature for 24 hours. Discrete colonies were stocked in nutrient agar slants and labeled accordingly. Further characterization and identification were carried out base on microscopic examination, gram staining and some biochemical assays.

A smear of the culture was placed on a clean grease-free slide, which was air-dried and fixed. The slide was then stained with crystal violet solution (primary stain) for 1 minute, after which it was washed with tap water and well drained. Iodine solution (mordant) was used to flood the slide for 30seconds, after which the slide was washed with tap water. Decolourization was done using acetone and washed with water. The slide was flooded with safranin (counter staining) for 60 secs, after which the slide was washed and allowed to dry. It was examined under oil immersion objective. Gram positive organisms retained the colour of the primary stain (purple) while the gram negative retained the secondary stain (pink) (Fowole and Oso, 1988).

Biochemical test

The biochemical test for identification of bacteria isolates were carried out as described by Prescott et al., (2005)

Catalase test

A drop of 30% H2O2 was placed on a glass slide using a wire loop, a little inoculums was removed and mixed with the H2O2 on the slide. A positive test was indicated by bubbling; the enzyme present on catalase positive organisms degrades hydrogen peroxide and releases O2 which is detected as effervescence.

Motility test

This test was used to determine the presence or absence of Flagella. The motility medium in tubes were inoculated by making a fine stab of the isolates with an inoculating needle to the depth of about 1-2 cm short of the tubes bottom and were incubated for 24 hours at 37oC.

Motile organisms grow outside the line of stabbing, while the non-motile organisms grow on the line of stabbing.

Urease test

This test is used to study the ability of an organism to utilize Citrate present in Simmon's media as a sole source of carbon for growth. In order to identify an organism that is able to use citrate as carbon source, the test organisms were inoculated into Simmon citrate agar slant and incubated for 24-72 hours. The development of a blue colour indicates a positive test.

Triple sugar iron test (TSI)

This test is based on the fermentation of the sugars, lactose, dextrose and the production of gas and hydrogen sulphide. A sterilized wire loop was used to pick the inoculums and stab the butt of the triple sugar iron agar medium with the test organism; these tubes were inoculated for 24 hour.

Oxidase test

The test indicates the presence of cytochrome oxidase which catalyses the oxidation of reduced cytochrome by oxygen. It indicates the ability of microbes to oxidize amine.

A freshly prepared 1% solution of oxidase reagent was soaked on a piece of filter paper and with a sterile loop the test organisms were smeared on the area impregnated with the oxidase reagent. Deep purple coloration after a few seconds shows a positive test.

Screen test of isolates for ability to utilize hydrocarbons

Bacterial and fungal isolates were tested for their ability to utilize hydrocarbon using turbidity method. Bacterial isolates were cultured on Nutrient broth and incubated for 24 hours at 28oC. Fungal isolates were inoculated into malt extract broth and incubated at room temperature (Nweke and Okpowasili, 2011).

1 ml and 1 g each from bacterial and fungal isolates were inoculated into mineral salt broth and 1ml of sterile crude oil was added into the inoculated tubes .The control tubes were incubated at room temperature under stationary condition for 7 days. The growths of the inoculates were determined by visual observation of the mineral salt broth medium turbidity as compared with the un-inoculated control tubes.

RESULTS

Effect of Azadirachta indica Leaves on the Levels of Bacterial Hydrocarbon Degraders in Crude Oil Polluted Cotton Wool Vessel.

As shown in Figure 1 below, the bacterial counts of the crude oil polluted cotton wool medium was significantly affected by bioremediation with the A. indica leaves in a concentration dependent manner. At all concentrations of A. indica the bacterial total hydrocarbon degraders levels were significantly higher (p<0.05) compared to the levels in the control. However, at 150 g of neem leaves, there was no significant difference (p > 0.05) in the bacterial counts (2.0 x 105cfu/g) compared to the vessel with plant and crude oil only (2.1 x 105cfu/g).

Effect of Azadirachta indica Leaves on the Levels of Fungal Hydrocarbon Degraders in Crude Oil Polluted Cotton Wool Vessel.

Figure 2 shows that the fungal counts of the crude oil polluted cotton wool was significantly improved by bioremediation with the leaves of A. indica in a concentration dependent manner. At all concentrations the fungal total hydrocarbon degraders levels were significantly higher (p < 0.05) in the A. indica treated medium compared to the levels in the control (1.0 x 104cfu/g).

DISCUSSION

The results of the present study confirmed the matter that many of bacterial strains, especially gram-negative bacteria were found to degrade poly aromatic hydrocarbons (PAHs) compounds at various extents (Frick et al., 1999; Cerniglia 1992 'Sutherland et al., 1995).this explains the significant growth of these degraders as seen in vessels rich in crude oil compared to control that received normal water. All of PAHs degrading bacterial strains which have, been identified in the present study were gram negative, which agreed with the results indicated that most efficient of the PAHs degrading bacteria were belong to the genus Pseudomonas fFrick et al, 1999). Microorganisms and plants have complementary roles in phyto-remediation of the polluted soil.

Phyto-remediation refers to the use of plants to clean contaminated soil. Increase in biodegradation of organic contaminants in the rhizosphere soil, the zone of soil directly adjacent to and under the influence of plant roots has been reported (Frick et al, 1999).

For successful phyto-remediation, both plants and microorganisms must survive and grow in crude oil contaminated soil. Phyto- remediation can be applied at moderate contamination levels or after the application of other remediation measures as a polishing step to further degrade residual hydrocarbons and improve soil quality (Frick et al., 1999).

From the result in figure 1 and 2, leaves of Azadirachta indica has been able to enhance the growth of hydrocarbon degraders according to the work of Frick et al., (1999) showing their complementary role in bioremediation. There was high microbial growth in vessel 4 with 300 g of Azadirachta leaves compared to other vessels. The biostimulation potential of Azadirachta Indica leaves in increasing the microbial population in crude oil contaminated vessel maybe due to the nitrogen and phosphorus content of the leaves. Okpkwasilli (1994), observed that the use of NPK fertilizer, urea fertilizer and poultry droppings effectively stimulated bacterial growth into utilization of crude oil. The high total hydrocarbon degraders (THD) bacteria in vessel 4 with 300 g of grounded leaves (3-2 x 105cfu/g) and that of total fungal count(2.0 x 105cfu/g) compared to control (1.0 x 104cfu/g) respectively suggests its effectiveness in bioremediation process.

CONCLUSION

The biodegradation of polycyclic aromatic hydrocarbons in the environment is a complex process, microorganisms such as bacteria and fungi are the key agents of bioremediation, with bacteria assuming the dominant role in marine ecosystems and fungi becoming more important in freshwater and terrestrial environments (Leahy and Colwell, 1990). The result from this study shows that the vessel with the highest quantity of Azadirachta indica leaves in it had the highest growth of hydrocarbon degraders in it, which means that the presence of A. Indica leaves enhanced the growth of the hydrocarbon degraders. It therefore suggests that A. indica leaves could act as growth inducers to enhance the growth of hydrocarbon degraders (bacteria and fungi) for effective bioremediation process in a hydrocarbon polluted environment.

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Cite this article:

Etim Okon E, Udosen Christiana I, Akara Priestine O N J (2013), IDENTIFICATION OF HYDROCARBON DEGRADERS IN A CRUDE OIL POLLUTED VESSEL AND POSSIBLE GROWTH INDUCING POTENTIAL OF AZADIRACHTA INDICA, Global J Res. Med. Plants & Indigen. Med., Volume 2(7): 492-498

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