Physical analysis of soil
Physical soil properties like soil texture, bulk density, moisture and water holding capacity of all the four soil samples were determined. Soil texture was determined by using sieves of different sizes. Bulk density was determined gravimetrically, while water holding capacity was evaluated by flooding the soils with water. The weight of flooded soil was determined. After 24 hours of drying at 80ËšC temperature amount of soil remained was determined and the percentage of water evaporated was calculated. Moisture content was determined similarly. Soil chemical properties like pH, electrical conductivity, available phosphorus, nitrogen, organic matter, available carbonate and bicarbonate, chloride, nitrogen and phosphorus content were determined by using standard titrimetric procedures (Pandey and Sharma, 2003). Heavy metal jobs (Sodium, potassium, iron, manganese lead, Zinc, nickel etc) were analyzed by atomic absorption spectroscopy.

Total Petroleum hydrocarbons analysis
Total Petroleum hydrocarbons present in the sample were estimated with the help of column chromatography and gas chromatography. Aliphatic and Aromatic fractions present soil sample were separated by column chromatography, Silica get of 60-120 mesh size. Activated at 90ËšC was used for column packing. The flow rate was maintained at 1 ml/min. The aliphatic fractions were separated with hexane (petroleum fraction) as an eluant. The aromatic fractions were separated with toluene. Both of these fractions were analyzed using gas chromatograph- Mass spectroscopy. The GC/MS analyses were performed using a MS 5973 spectrometer coupled with 9 Hewlett Packard Model 6890, with a column ULBON HR-1 which is equivalent to Ov-1 fused silica capillary (0.25 mm*50 mm) with thickness of 0.25 micron; 1ml/min; pressure 18.5 psi and split ratio 20%. The solvent used in analysis was chloroform.

Results and Discussion:
Utilization of chemical contaminant present in the soil as source of carbon and energy by different bacterial communities leads to ameliorate a wide range of contaminants like petroleum and polyaromatic hydrocarbons.

In bioremediation process microbes metabolize the target contaminants to derive energy through enzyme driven oxidation-reduction reactions. The end products of bioremediation processes are non-toxic or relatively less toxic as compared to the parent contaminants.

Prevailing environmental conditions are among the most important limiting factors for optimum bioremediation. The factors affecting the success and rate of microbial bioremediation are nutrient availability, moisture content, soil reaction (pH), temperature, C/N ratio, soil texture etc. The present study was carried out to assess the quality of contaminated soils. These qualities determine the capability of bacterial isolates to biodegrade petroleum compounds and their wastes like oily sludge, a hazardous hydrocarbon waste generated by the petroleum industry. The bacterial communities are believed to adapt the local soil environment. These environmental factors play a vital role in the bioremediation of soil.

All soil microorganisms require moisture for growth and functioning. Moisture affects diffusion of water and soluble nutrients into and out of the microbial cells. In the present investigation the moisture content recorded during the experiments ranged from 13.18 % to 16.01%. A better degradation of PAH was recorded in soil sample PCS-1 having moisture content 16.01 % as compared to PCS-2 with 13.18% moisture content. Excess moisture, in saturated soil, is undesirable because it reduces the amount of available oxygen for aerobic respiration. The soil water holding capacity between 45 and 85 percent is optimal for petroleum hydrocarbon degradation (US-EPA, 2006). The water holding capacity of the contaminated soil samples under investigation was 58.56% in PCS-1 and 59.18% in PCS-2 (Table 1), i.e. in the range suggested to be optimal for bioremediation (US-EPA, 2006). It is also difficult to control moisture content in fine soil because their small pores and high surface area allow better water retention. In water saturated condition the movement of water is relatively slow in fine soils than the soils with higher texture. In such soils the movement of air and oxygen through soil profile is not adequate for microbial activities (US-EPA, 2006).
Soil reaction (pH) is a critical factor for microbial growth and survival. Different microbial strains exhibit their maximum growth potential in a limited pH range. A pH value of near neutral is suitable for growth of diverse bacterial populations. The most appropriate range for bioremediation has been suggested to be pH 6-8 (US-EPA, 2006). In current investigation, the soil reaction (pH) of all the soil samples falls well within the range suggested by US-EPA(2006) (Table 1).
The rate and extent of degradation of hydrocarbons greatly depends on nutrient composition of soil. The amount of various nutrients and ratio of particular nutrients like C, N and P is quite conceivable regarding success of a bioremediation process. The organic carbon content in all the contaminated soils was very high. This is attributed to the continuous input of petroleum hydrocarbons. The optimum C:N ratio closer to 12.5:1 by Hupe et al.,(1996).Relatively more PAH degradation has been noticed in soil samples with a C:N ratio closer to 12.5:1 (Luepromchai et al.,2007). In the present investigation, analysis of the soil samples revealed C: N ratio of 12.1:1 and 9.7:1 in PCS-1 and PCS-2, respectively (Table 1).

Chemical composition, quantity and toxicity of contaminants are also among the critical factors for microbial diversity of contaminated soils. Comparatively, higher amount of TPH (Total petroleum hydrocarbon) was recorded in PCS-2 soil sample. This suggests the probability of reduced microbial (bacterial) population in this soil sample. The PCS-1 soil sample had considerably less TPH content (11149 mg/kg) than PCS-2 (14244 mg/kg) (Table 1). The high amount of contaminants may be toxic to soil microorganisms (Luepromchai et al., 2007). Heavy metal concentration was found higher in both the contaminated soil samples as compared to their normal counterparts. Iron content was exceptionally higher in PCS-1. Presence of high concentration of metal ions attributes to greater electrical conductivity in both the contaminated samples. Higher electrical conductivity also indicates high salinity in the contaminated samples. In the present study, salinity of both the contaminated samples was much higher than their uncontaminated counterparts (Table 1).




Table1: Physico-chemical properties of petroleum contaminated soil and Normal soil:

Parameters Unit Petroleum contaminated soil 1 Petroleum contaminated soil 2 Normal soil 1 Normal soil 2
pH - 7.2 0.33 7.20 0.20 7.05 0.13 7.25 0.35
Electric conductivity
(EC) µs/cm 317 328 167 159
Total dissolved salt(TDS) ppm 177.3  188  0.59 70 0.16 73 0.27
Soil organic carbon(SOC) % 4.96 0.40 4.33 0.5 0.56 0.2 0.65 0.26
Available phosphorus mg/kg 3.6  0.16 1.5 0.64 5.5 0.33 5.8 0.44
Total nitrogen % 0.04  0.02 0.07 0.01 0.68  0.1 0.55 0.05
C/N Ratio - 12.15 0.68 9.71 0.55 1.03 0.01 1.33 0.1
Sodium ppm 863 17 498 3  235 18
Potassium ppm 185 5 (ns) 172 8(ns) 192 4 156 3
Sulphur ppm 760 11 824  14 373 16 296 15
Organic matter % 6.33 6.11 1.30 1.25
Salinity ppm 174  4 190 6 80 2 92 4
Carbonate
(CO3 -2 ) mg/100 gm 0.22 0.02 0.35 0.05 0.63 0.05 0.69 0.03
Biocarbonate (HCO3- ) mg/100 gm 1.68 0.33
(ns) 1.22  0.19 2.47 0.4 2.14 0.30
Chloride mg/100 gm 805.1  20 717 26 270 10 211 4.7
Iron mg/kg 715 0.05 660.1 0.07 215.18 6 ND
Manganese mg/kg 75.08 0.01 82.19 0.08 52.08 0.08 57.38 0.08
Lead mg/kg 0.60 0.03 0.73 0.04 0.3 0.02 0.19 0.06
Zinc mg/kg 0.72 0.02 0.61 0.01 0.3 0.016 0.26  0.02
Nickle mg/kg 0.65  0.02 0.33 0.03 0.13 0.02 0.08 0.01
Moisture content % 16.01  0.5 13.18 0.80 11.38 0.15 12.69 0.50
Water holding capacity % 58.56 0.30 59.18 0.36 54.12 0.07 53.18 0.35
Bulk density gm/ml 0.90  0.17 0.95 0.25 0.74 0.2 0.68 0.3
Porosity % 66.30 1.2 67.10 0.80 69.39 1.8 69.95 1.53
Total Petroleum Hydrocarbon (TPH) mg/kg 11149 133 14244 130 700 18 614 15


Soil physical properties like soil texture and bulk density have also been considered to be very important for bioremediation because several factors affecting the degradation process like soil aeration, movement of nutrients through soil pores, water holding capacity and several others are also under the direct or indirect influence of soil physical properties (Atlas, 1981; Luepromchai et al., 2007) (Table 1). In the present investigation PCS-1 had low clay percentage (23.9%) as compared to PCS-2 (27.9%), indicating possibility of a higher degree of hydrocarbon degradation in PCS-1. Increased ventilation has a direct impact on microbial growth which can enhance the biodegradation of petroleum compounds (Pathak et.al. 20100(Table 2).

Table 2: showing the Percentage of soil Texture in Petroleum contaminated Soil (PCS) and Normal Soil (NS)

Samples Sand % Slit % Clay %
PCS-1 35.2  2.2 40.6 1.6 23.9 1.6
PCS-2 37.09 1.5 36.05  1.2 27.06 1.8
NS-1 47.20 1.5 35.8 2.7 17.6 1
NS-2 42.28 1.3 39.90 1.2 17.22 1.7


From the above results, it could be concluded that hydrocarbon contamination adversely alters the soil properties. The water holding capacity and porosity which determines the extent of water retention and aeration in the soil, become reduced. Both of these properties have profound importance for the biological status of the soil. The high C: N ratio even makes the problem more critical for the proper sustenance of life. Presence of heavy metals and high concentration of petroleum hydrocarbons cause further decline in the soil quality. The altered properties makes the soil potent toxic and carcinogenic and hence very harmful for human beings. It is necessary to restore the contaminated sites and much is needed to be done in this direction.

About Author / Additional Info:
1. Bos RP, Theuws JLG, Leijdekkers CM, Henderson PT (1984) The presence of the mutagenic polycyclic aromatic hydrocarbons benzo- [a]pyrene and benz[a]anthracene in creosote P1. Mutat. Res. 130:153-158.
2. Boxall ABA, Maltby L (1997) The effects of motorway runoff on freshwater ecosystems. Toxicant