Introduction

Polycyclic aromatic hydrocarbon are a group of compounds containing carbon and hydrogen, composed of two or more fused aromatic rings in linear  angular and relatively insoluble in water. Polycyclic aromatic hydrocarbons are  ubiquitous pollutants have been identified as hazardous chemicals because of their toxin carcinogenic effects on living body¹ .

Engine oil contains metals and heavy polycyclic aromatic hydrocarbons that could contribute to chronic hazards including mutagenecity and carcinogenicity². Prolonged exposure to high oil concentration may cause the development of liver or kidney disease, possible damage to the bone marrow, and an  increased risk of cancer³.  Biodegradation has been successful for clean up of pentachlorophenol (PCP), a wood preservative and polycyclic aromatic hydrocarbon the advantages associated with fungal bioremediation lay primarily in the versatility of the technology and its cost efficiency compared to other degradation technologies⁴.Most recently, the deuteromycete Cladosporium species was isolated from a biofilter that had been used to remove toluene from contaminated air. This fungus can use toluene as the sole source of carbon an energy. These findings demonstrate that it is possible to isolated fungi that grow on aromatic hydrocarbons, provided adequate enrichment techniques are used⁵. Hydrocarbon compounds such as petroleum are essential elements of lift. In Iran is the first country in the oil – rich Middle East region to start oil operations with current production capacities of over 4 millions barrels of crude oil 80,000 millions m3/day diesel fuel. There are up to 1,500,000 cubic meters of soil contaminated with engine oil around Tehran refinery Iran. Spills and leaks of the petroleum hydrocarbons from storage facilities and distributions systems results in contaminations of soil and water systems world wide⁶.Oil biodegradation of subsurface does not require oxygen, it does require certain essential nutrients (e.g., nitrogen, phosphorus, potassium), which can be provided by dissolution of minerals in the water lake. Empirically, it has been noted that biodegraded oil accumulations occur in reservoirs that are at temperatures less than 80⁰C.

Toluene  is a aromatic hydrocarbon that is widely used as a industrial feed stock and as a solvent. Toluene sulphonyl iodide, in the presence of copper powder. Like other solvents, toluene is sometimes also used as an inhalant drug for its properties⁷. Pentachlorophenol is a chlorinated hydrocarbon insecticide and fungicide. It is used primarily to protect timber from fungal rot and wood – boring insects, this soil was contaminated with wood preserving wastes including composed primarily of polycyclic aromatic hydro carbons of Pentachlorophenol ⁸.

Materials And Methods

Sample Collection

Soil  samples  were  collected  randomly  from  engine oil  contaminated soil from an automobile workshop in Madukkur , Thanjavur District. The sample was exposed to fuel spills for more than 3 years . 100g of soil samples were obtained from the subsurface after removing the upper 3cm of the surface soil . It was thoroughly mixed , sieved through a 2mm pore size sieve and placed in polythene bags closed tightly and then stored in specific container .

Isolation of bacteria and fungi ⁹

After sample collection, serial dilution was performed for isolating microbial growth from the collected samples. For this, 1g of soil was added . The tube was vigorously vortexed for 3 minutes to  obtain uniform suspension of organisms. A series of tubes labeled as 10^-1 up to 10^-8 were filled with 9ml sterile distilled water and serial dilution was performed.  The bacterial plates were incubated  at 37⁰ C for 24 hrs  and the fungal plates were incubated  at 28 for  3 days.  After  collection of the incubation period  Isolated colonies were  observed and used for further use .

Identification of  bacteria

The colonies grown in the nutrient  agar medium  where subjected  to staining and other  biochemical  procedure  for  identifying  the bacteria¹⁰.

Isolation of efficient engine oil degraders of Fungi  ¹¹

0.1 ml of fungal culture were spread on potato dextrose agar plates. Then the plates were incubated at 28⁰C for 2 days same type of mycelium colonies were  developed on potato dextrose agar plates on third day. The plates were sprayed with 1% toluene and1% pentachlorophenol using acetone.     After spraying all plates appeared in mycelium growth. The pure culture of each of the two best potential strains were inoculated into 10 ml Bushnell – hass medium containing 1% toluene and 1% pentachlorophenol in conical flasks and incubated at 28⁰C on rotatory shaker for 7 days.

Isolation of efficient engine oil degraders of bacteria ¹²

0.1 ml of bacterial culture were spread on peptone glycerol phosphate agar plates. Then the plates were incubated at 37⁰C for 3 days same type of colonies was developed on peptone glycerol phosphate agar plates on third day. The plates were sprayed with 1% toluene and 1% pentachlorophenol using acetone.After spraying all plates appeared buff white color. The plates were further incubated at 37⁰C for the observation of zone of clearance around the colonies. After 2 days zone of clearance were observed. Colonies which shows maximum diameter of zone of clearance in 2 days were further inoculated into 10 ml Bushnell – hass medium containing 1% toluene and pentachlorophenol in conical flasks and incubated at 37⁰C on rotatory shaker for 9 days to determine percentage of degradation and bacterial density.

Estimation of bacterial  density

Cell biomass concentration was determined by optical density using photo colorimeter.

Estimation of  degradation percentage

The percentage of degradation was calculated with initial absorbance value, after inoculation by using the formula at 600 nm spectrophotometrically . Blank was made without addition of hydrocarbon¹³ .

Physico-Chemical properties ¹⁴

Determination of P^H and Temperature:

100 ml of Nutrient broth were prepared and separated into different conical flasks. Each flasks were adjusted to different p^H such as 4,5,6,7 and Temperatures at at 25⁰c, 35⁰c, 45⁰c and 55⁰c . After sterilization, 1% Bushnell-hass broth culture was added into different flasks containing medium. And the flasks were incubated for 48-72 hrs.

Determination of Alkalinity

5ml culture sample was taken and few drops of phenolphthalein indicator were added . Pink color solution was titrated against 0.01N H₂SO₄. To this colorless solution 2,3 drops of methyl orange indicator was added and titrated against 0.01N H₂SO₄ until orange colour was turned to yellow.

Determination of Chloride

Determination of chloride was also done by¹⁴

Statistical Analysis

Rondom sampling was used for the entire test. The  data of all values, were statistically analzsed  and expressed as mean ± standard deviation ¹⁵.

Result and Disscussion

In the present study hydrocarbons degrading bacterial and fungal species were isolated from the engine oil contaminated soil. There are engine oil degrading bacterial isolates were identified based on the biochemical characteristics. Different bacterial colonies were compared with Bergey’s manual of systemic bacteriology. Bacterial isolates were confirmed as P.alcaligenes, and Serratia marcescens.The  fungal colonies strains were identified by lacto phenol cotton blue staining.

After 9 days of  incubation the optical density of toluene degraders such as Pseudomonas alcaligenes was 0.32 IU/ml and Serratia marcescens 0.30 IU/ml.  optical density of pentachlorophenol degraders such as Pseudomonas  alcaligenes was 0.35 IU/ml and S.marcescens was 0.36U/ml. (Table 1)

Table 1 .Density of  Toluene and Pentachlorophenol Degraders (IU/MI)

Toluene degradation was carried out in 9 days by Pseudomonas alcaligenes and Serratia marcescens. The percentage of degradation of toluene was P.alcaligenes 52.36% and S.marcescens 48.66%. Pentachlorophenol degradation was carried out in 9 days by Pseudomonas alcaligenes and Serratia marcescens. The percentage of degradation of Pentachlorophenol was P.alcaligenes 51.48% and S.marcescens 47.88%. There organism degrade toluene and Pentachlorophenol with clear zone on liquid media with in 2 days(Table 2)

Table 2 .Percentage  of  Toluene and Pentachlorophenol Degraders (%)

Physico – chemical parameters

Determination of P^H and Temperature

Among the four different P^H and Temperature , maximum hydrocarbon degradation was observed in  P.alcaligenes at pH 7 (8.2±0.01 IU/ml) (Table 3) and Temperature was A.niger at 55⁰C  (8.7±0.01 IU/ml) (Table 4). 

Table 3: Determination of different P^H for toluene and Pentachlorophenol degraders.

 Values are mean + standard deviation

Table 4: Determination of different temperature for toluene and pentachlorophenol degraders.

 Values are mean + standard deviation

Determination of alkalinity and chloride :       

Alkalinity was estimated in Pseudomonas alcaligenes (3.2 Mg/l) and Chloride was estimated in Pseudomonas alcaligenes (2.7 Mg/l) (Table 5).

Table 5 .Determination of Chloride and Alkalinity (mg/l).

Polynuclear Aromatic Hydrocarbons (PAHs) degrading microbial strains were isolated from oil contaminated soil and characterized for specific features regarding toluene and pentachlorophenol degradation. The most efficient strains in terms of rapidity to degrade toluene and pentachlorophenol were identified was Pseudomonas species and Serratia species¹⁶.

Our study reports similar to the findings was also isolated more than 100 species representing 30 genera have been shown to be capable of utilizing hydrocarbons, the association of various bacterial and fungi with different hydrocarbon system viz. petrol, diesel, engine oil and kerosene appear to vary with reference to soil and water characteristics also the utilization potentials of these hydrocarbons by bacteria and fungi¹⁷.

To identify the degraded compounds of hydrocarbon further investigation is necessary. Moreover, bacterial and fungal stain can be used to recovery of hydrocarbons from oil contaminated site which field trial is also necessary.

Reference

1. Ruma Roy ., Raja Roy, Ranjana chowdheiry ., Pinaki Bhattacharya, (2007). Indian Journal of Biotechnology V(6) P: 107-113.
2. Keith,L.H., and Telliard ,W.A,(1979).Priority pollutions 1-A perspective view. Environmental science and technology,13:416-423.
3. Propst, T.L Lochmiller, R.L., Qualis, C.W., and M.C Bee,K.Jr(1999).Assesment of immune toxicity risks to small mammals inhabiting petrochemical waste sites Chemosphere 38: 1049-1067.
4. Andrea, R.C., Jania, A.A., Lucia, R.D.,(2001). Biodegradation of polycyclic aromatic hydrocarbons by soil fungi, Brazilian Journal  of  Microbiology,(32):1-4.
5. Weber, F.J., Hage,K.C and De Bont, J.A.M, (1995) .Groth of the fungus Cladosporium spp with Toluene as the sole carbon and energy source. Applied and environmental microbiology 6: 3562-3566.
6. Massol-deya, A.A., Rodrigez, Martivz, E.M and zhou, J., 2006. Microbial diversity and biodegradation of hydrocarbon contaminated aquifer. J.environ.Res.publi Health., 3(3) : 292.
7. Brown, E.J., Pignatello, J., Martinson, M and Crawford, R., 1986. Pentachlorophenol degradation. A pure bacterial culture and an epilithic microbial consortium. Applied and environmental microbiology 52: 92-97.
8. Jensen, V.(1975). Bacterial of soil after application of oily waste.Oikos 26:152-158.
9. James, N.(1958). Soil extract  in  soil  microbiology Microbiol., 4: 368-370.
10. Aneja, K.R.,2002. Experiment in microbiology. Plant pathology tissue culture and musroom production technology., 4^th edn :161-162.
11. T.,Cassidy,M.B.,Shaw,K.W.,Lee,H.,(1997). Pentachlorophenol biodegradation by Pseudomonas SPP .World journal of microbiology; 13:305-313.
12. Kumar ,M., Lenon, V., Materno,A.D and Iizins, O.A., (2006) . Enhancement of soil degradating and producing bacteria.J.microbial., 55(2): 139-146.
13. Thiyagarajan, V., Jeevan – (2003), Biodegradation of polycyclic aromatic hydrocarbons. Journal of microbial world 5(2) : 95-98.
14. (1998) Standard methods  for  examination  of  soil  and  waste  water  including  bottom  sediments  and  sludges (14^th edn). American  public  health  association, Washigton, D.C, Research science and technology, 3(5): 29-32.
15. Gupta,S. P,(1977). Measures of dispersion, In statistical methods. PP: 833-834.
16. Gutti L, and V.S Hamde (2009). Journal of microbial world 11(1) -59-63.
17. Jae Jun Jeong., Ji Hyun kim., Chi-kyung kim., Ingyu Hwang and Kyoung lee., (2003) 3and 4- alkyl phenol degradation pathway in Pseudomonas microbiology., 147: 1621-1630.