Crude oil samples from Sobhasan oil well # Y was collected with the help of O.N.G.C. authorities.

Physical and chemical properties of soil samples were analyzed. The samples were drawn with a hollow pipe, 3.5 cm in diameter. Air-dried and pulverized soil samples were analyzed for pH, temperature, moisture level, organic carbon, nitrogen, and available phosphorus with standard methods.

To prepare the inoculums, bacterial isolates were grown separately in 2-liter Erlenmeyer flasks containing 500 ml of mineral medium and molasses (2%) as the sole carbon and energy source. All the isolates were grown to mid-log phase (108CFU/ml) and they mixed in equal proportions. The mixed culture was used as the inoculum (5%) for large-scale culture in a bioreactor (Bioflow 3000, New Brunswick) with a working volume of 10 liters. The same minimal salt medium with molasses (2% wt/vol) as sole carbon source was used in the bioreactor for large-scale culture. The growth conditions in the bioreactor were as follows: temperature 32°C; aeration, 0.75 volume of air/volume of medium / min; agitation, 250 rpm; pH 7.0 (adjusted with 1N HCl-NaOH); and duration of growth, 15 h. Silicone oil was added to control excessive foaming in the bioreactor. After growth, the culture was immobilized onto the selected carrier material, corncob powder (a biodegradable agriculture residue) by simple mixing of carrier material and culture in ratio of 1:3. The carrier-based culture was dispensed into sterile reusable polythene bags (4 kg of culture immobilized onto carrier material in each 10 kg polythene bag) and stored at 4°C after the bag was aseptically sealed. In previous study, various carrier materials were screened to determine the survival of carrier-based culture by monitoring the CFU counts (on LBA plates) at 15-days intervals,upto 90 days (Mittal et al.,2005).

The total area (225 m2) of the feasibility study was divided into 20Blocks (four replicate Block for each treatment), 1 by 1 m each and separated by a gap of 2 m. The experimental design chosen was a completely randomized block design. The treatments were as follows: (i) Oil alone; control where no treatment was done. (ii) Oil +Indigenous Microflora (iii) Oil + Nutrients (iv) Oil + Nutrient (Fertilizer) + Tilling (Aeration) (v) Oil + Nutrient (Fertilizer) + Tilling (Aeration) + Developed consortium. In each Block garden soil was contaminated experimentally with Sobhasan crude oil (approximately 2% v/w), up to the height of 6 inches. The initial and final concentration of crude oil, soil pH, temperature, moisture level, organic carbon, nitrogen and available phosphorus were also determined over a period of 60 days. The soil used for microcosm study has a pH of 7.88, which is well within the range of optimal degradation. Therefore, no treatment for pH control is needed. The experiment was conducted in the premises of Kanya Gurukula Mahavidyalaya (G.K.U), Haridwar. The experiment was conducted during September to November months, in the year 2006. During the course of study, the following five sets of treatment were conducted.

Block 1-Oil alone: Soil was autoclaved at 121°C for 30 minutes and soil was treated with 2% HgCl₂ after one month time interval to destroy all microflora (Poisoned control). It is used to monitor abiotic hydrocarbon losses.

Block 2-Oil + Indigenous Microflora: It is essentially a no action control, wherein the initial state of the microcosm is left unmanipulated throughout the course of the investigation. In this Block no treatment was given except ½ to 1-liter water was added regularly.

Block 3-Oil + Nutrients: This microcosm was amended with inorganic nutrients in the form of Diammonium phosphate (DAP) and urea fertilizer, in order to have a C: N: P ratio of 120:10:1 as per Gibb’s formula (assuming crude oil is 78% carbon). Nutrients (fertilizer) were applied two times, after one-month time interval.

Block 4-Oil + Nutrient (Fertilizer) + Tilling (Aeration): It entails the two or three times per weekly tilling of the microcosm contents to promote soil aeration, in addition to nutrient amendment.

Block 5-Oil + Nutrient (Fertilizer) + Tilling (Aeration) + Developed consortium: The developed mixed culture consortium of hydrocarbon degraders (Pseudomonas strain PS-I, PSII, and PSIII) was also added in addition to nutrients and weekly tilling.

Throughout the course of study in field treatments were open to air and sufficient water was added to hydrate the soil to 50% of its water holding capacity. In biostimulation treatment, a dry mix fertilizer (N: P) was applied to select blocks (Block3, Block4 and Block5).

To assess the rate at which the TPH was being degraded, samples were collected at time Zero (just before initiating the bioremediation), 30 days later, and at the end of the study (60 days after initiating the process). The sample was weighed and mixed with anhydrous sodium sulfate in a 1:1 ratio and was then used for soxhlet extraction. Because the results were desired using dry-weight basis, the sample weight was adjusted to account for the moisture and this weight was used for analysis.

Total petroleum hydrocarbon from 10 gm soil was then consecutively extracted with hexane, methylene chloride and chloroform (100 ml each) for 16 hours in soxhlet apparatus. The extraction cycle around the thimble were approximately 6~8 cycles per hour. The solvent extract containing soluble oil from the soil was evaporated in Rota evaporator. The residual oil content was estimated.

The group type (saturate, aromatic and NSOs) analysis of whole oils has been carried out in chemito-1000 gas chromatography. For isolation of various compound class fractions (saturate, aromatic and NSOs), column chromatographic technique was used. The separation was carried out on dual packed activated silica-gel/alumina columns by successive elution with petroleum ether for saturates, benzene for aromatics and methanol for NSO compounds.

Aromatic fraction was further fractionated as follows:

Shimadzu VP-3 HPLC system using normal isocratic mode was used for the fractionation of aromatic hydrocarbons into mono, di and tri-aromatics on a silica amino (NH₂) column (30cmX4.5mm). Elution was carried out using n-hexane (HPLC grade) and the separation was monitored with UV-visible absorbance detector operated at 245 m wavelength. The flow rate was maintained at 6.5ml/min.

Survival of introduced bacterial consortium during the course of study was indicated indirectly by observing the increase in population of consortium in soil over the period of study. Soil samples (1 gm) were collected before and after the application of bacterial consortium i.e. after 15 Days, 30 Days, 45 Days and 60 Days, suspended in saline water (0.85% NaCl). The suspension after appropriate dilution was plated on Luria Bertani agar (LBA) media plates.

The first and foremost criterion for designing a bioremediation program is to study the native microflora of the system and to analyses the physico-chemical composition of soil. The soil sample taken for feasibility study (garden soil of Kanya Gurukula Mahavidyalaya, Haridwar) was analyzed for detection of different groups of microbes. The soil contains 8 X 108 /g of total heterotrophs, 4.5 X 105 /g of actinomycetes and 1.4 X 104 /g of fungi. The hydrocarbon degrader’s count is 2.2 X 104 /g of soil.

Physical and chemical properties of soil samples taken were analyzed for pH, temperature, moisture level, organic carbon, nitrogen and available phosphorus in each block at zero time intervals i.e. initially and after 60 days of bioremediation after application of microbial consortium.

In- situ bioremediation approach was adopted, after optimizing the factors under laboratory conditions. These optimized conditions were attempted to be maintained during feasibility study.

The soil used is sandy loam in texture and its pH was 7.88. Moisture content of soil was 7.30 %, while water holding capacity was 40.55. The temperature recorded during the study varied from 25°C- 33°C. Organic carbon content (TOC) of soil was 1.23%, nitrogen content - 0.126%, initially. After contamination with crude oil Organic carbon content (TOC) of soil reached 2.79% and amendment by fertilizer nitrogen content reached to 0.235%.

Figure 1

Table 1. Physical and chemical properties of soil at bioremediation site

The crude oil deployed for microcosm study (Sobhasan crude oil # y) was analyzed for its gross composition. Saturates, aromatics and NSO percentage was found to be 71.7%, 20.0% and 7.4% respectively.

Block 1-Oil alone: The hydrocarbon utilizing bacteria (HUB) count during the study was also found nil, though some heterotrophic bacterial activity in an open environment is not possible. It is because the soil was sterilized and 2%Hgcl₂ treatment also arrested the soil microbial activity. The minimum loss of TPH (5.02%) can be attributed to the abiotic losses like evaporation of low volatile fraction of crude oil and photo-oxidation etc. (Table-2). Several workers have reported hydrocarbon losses due to volatilization and photochemical oxidation (Smith et al., 1998; Dutta et al., 2002). The total organic carbon (TOC %) of original soil (before contamination) and contaminated soil was found to be 1.23% and 2.79% respectively. The organic carbon, nitrogen and phosphorous dropped down from 2.79%, 0.126% and 34.70 ppm (available phosphorous) to 2.70%, 0.121% and 34.63 ppm respectively in 60 days, in sterile control box. The pH of the soil dropped from 7.88 to 7.30 (Table-1). This could be due to that HgCl₂ solution is acidic in nature, which was used to hinder microbial activity. Bossert et al., (1984) also included poisoned controls (2% HgCl₂) to monitor abiotic hydrocarbon losses.

Block 2-Oil + Indigenous Microflora: The box -2, which shows the biodegradation by indigenous microflora, is containing 8 X 108 total heterotrophs and 2.2 X 104 hydrocarbon degraders. It has been reported previously that bioremediation is less if the population of hydrocarbon degrading microorganisms is less than 105 cfu/g in soil (Forsyth et al., 1995). This warranted that a bacterial consortium was needed for effective bioremediation.

The TPH decreased from 0.1885 gm to 0.1315 gm in 60 days, indicating 30.24% degradation of crude oil (Table-2). During the course of biodegradation, the pH dropped from 7.88 - 7.71. . Many other workers have also been reported the pH alteration during crude oil degradation (Zaidi et al., 1988; Kastner et al., 1998;Sims et al.,1992) (& Fig.1). The TOC content reduced from 2.79 to 1.942, which was due to biodegradation of crude oil present in soil. The nitrogen and phosphorous concentrations also decreased (Table-1, Fig.4.2).

Block 3-Oil + Nutrients: In this Box, loss in TPH was 49.89% in 60 days, which indicate that, fertilizer addition increases the rate of biodegradation as compared to box-1and box-2 in which no fertilizer treatment was given (Table-2). In Box-3, after 60 days pH dropped from 7.88 to 7.62. This lowering of pH can be attributed to the formation of various carboxylic acids during breakdown of hydrocarbons. Secondly, tap water (from over head tanks) that was being used for treatment of soil boxes had pH of 7.60.The source of water was deep tube well of Kanya Gurukula Vishwavidyalaya .If such a high pH water is being used, it will cause in drop of pH. The values of nitrogen and phosphorous determined during the study indicate that the soil was richly amended by Diammonium phosphate (DAP) and Urea fertilizer during study. Nitrogen content was depleted from 0.235gm to 0.201gm because the fertilizer was amended as per concentration of hydrocarbons remained in the soil after biodegradation. It can be concluded that microbial activity has consumed nitrogen amended in the form of fertilizer and nitrogen was available in plenty to the microbes at all the times. The concentration of available phosphorous was not dropped significantly because of addition of DAP fertilizer (Table 1& Fig.4.3). Roling (2002) reported that nutrient amendment over a wide range of concentrations significantly improved oil degradation, confirming the low concentration of nitrogen & phosphorous limited degradation.

Block 4-Oil + Nutrient (Fertilizer) + Tilling (Aeration): The loss of TPH is 54.16% (Table-2); the increased loss in this box as compared to other boxes was due to (i) aeration done weekly in this box (from the bottom) through out the soil matrix, which has helped in evaporation of volatile part of crude oil present in the whole soil system. (ii) Aeration does help in growth of microbes by overcoming the oxygen limitations in soil matrix leading to more degradation of crude oil.

The soil tilling stimulates the microbial activity due to breakdown of soil aggregates and better exposure and aeration of degradable material (Paul and Clark, 1989).

Block 5-Oil + Nutrient (Fertilizer) + Tilling (Aeration) + Developed consortium: Maximum of 79.16% loss of total petroleum hydrocarbon (TPH) was obtained in 60 days (Table-2). This treatment has been bioaugmented with potent hydrocarbon degraders improvised from soil microflora. This treatment has significantly increased the rate of degradation as the number of potential hydrocarbon utilizing bacteria (native of the soil) was artificially raised. The maximum loss of TPH could be attributed to the addition of laboratory grown inoculum of mixed consortium of native soil microflora (Hydrocarbon utilizers). (Eriksson et al., 1995, Lal et al., 1996). The organic carbon decreased from 2.79% to 0.566%, because of consumption of crude oil. Nitrogen and phosphorous content also decreased dramatically. It can be concluded that microbial activity has consumed nitrogen and phosphorous during crude oil degradation (Table 1 & Fig.4.5).

The temperature recorded during the study varied from 25°C - 33°C. Since maintenance of temperature in open soil system is not feasible, the bioremediation efforts should be concentrated during such a period of year when the temperature is suitable for treatment. Brady (1990) suggested that biodegradation rates increase with increasing soil temperature upto a certain maximum temperature, which would be optimum for indigenous hydrocarbon degraders (Barker and Neumann, 1993). Moisture content of soil is critical for microbial activities, since nutrients, organic constituents, Oxygen and metabolic wastes are transported to and from microbial cells in water. The optimum level of soil water for microbial activity is 10-13% i.e. 65–80% of water holding capacity. (Bossert and Bartha, 1984; Sims et al., 1989). To maintain this sufficient water was added regularly to hydrate the soil to 50% of its water holding capacity.

Figure 2

Table-2 Crude oil degradation in various treatments Boxes

Survival of the microorganisms in the soil after their application is a deciding factor in rate of degradation of hydrocarbons (Ramos et al., 1991). Continuous increase in microbial count, cfu/g soil indicated survival of introduced consortium in the soil. Before bioaugmentation the population of hydrocarbon degraders which was 2.2 X 104 increased and reached up to 9.20 X 108 during 60 days of bioremediation study. This increase in microbial count indirectly indicates survival of introduced consortium.

HPLC analysis of aromatic fractions of Sobhasan crude oil (deployed in microcosm study) was done after 60 days period only (fig.1). The effect of indigenous microflora as well as seeded consortium was observed. The widely accepted ratios of biodegradability di/tri aromatic and di / di + tri aromatic were calculated & are given in table 3. The results showed that the ratio of di / tri aromatics decreased from initial 0.63 to 0.25 with progressive treatment of nutrient addition, nutrient+tilling, nutrient+tilling+microbial seeding. Similar effect was observed in di / di+tri aromatics ratios which also decreased from 0.38 to 0.20.The results indicate that the developed consortium could help in degradation of higher aromatics, as ratio was decreased from 0.31 to 0.20 by bioaugmentation only. Thus developed consortium could help in detoxification of the harmful and carcinogenic aromatics specially polyaromatic hydrocarbons.

Figure 3

Table 3: Microcosm study: Degradation of Aromatics

Crude oil used: Sobhasan crude oil well # Y.

Figure 4

Fig1. HPLC analysis of aromatic fraction of Sobhasan crude oil obtained after 60 days of bioremediation from Block 5(Oil + Nutrient + Tilling + Developed consortium).

A carrier-based formulation made it easy to transfer the microorganisms from the laboratory to the field. Corncob powder (agricultural byproduct) was found to be the best carrier for this purpose as survival of 7.2 X 108 cfu/g was recorded during storage over 3 months. Corncob powder, being a good porous material facilitates growth and exposes a grater surface area to microbial action.

The present study clearly demonstrates that application of a carrier-based bacterial consortium, improvised after isolation of potent hydrocarbon degraders from the oil contaminated site, can be used to remediate soil contaminated with crude oil. Maintenance of proper environmental factors is an essential aspect for treating an oil spill by biotechnological methods. In view of synergistic and antagonistic relationship among the various strains of bacteria, bioremediation is considered as site-specific approach.