Degradation of Petroleum Fractions in Soil Under Natural Environment: A Gravimetric and Gas Chromatographic Analysis

The modern civilization depends on exploration, distribution and use of petroleum and thousands of products derived from it. The environment all throughout the world has been very badly damaged due to these chemicals. While pollution of the atmosphere has been caused almost totally by burning of the petroleum products, the leaks, spillages and accidental fallout of petroleum products and the crude petroleum itself have greatly affected land and water resources. The oil field areas always receive a large amount of effluents rich in crude oil and land degradation is a common phenomenon. While using different fractions of petroleum (gasoline, kerosene, diesel etc.) spills and leakages cannot be avoided and in industrial areas, garages and other places, a large amount of it flows to the nearby areas. All types of oil have amazing spreading power and once a leak occurs, the oil may spread horizontally as well as vertically depending upon soil conditions, moisture, temperature etc. The lighter fraction being volatile are easily removed by evaporation and other physical processes, but the heavy components such as aromatics – simple and polycyclic, Hopanes etc. remain in the soil for a very long time unless biodegraded by soil micro organisms. The detection of presence of some carcinogenic hydrocarbons viz. PAH has made the situation a matter of serious concern. In most cases; the pollutant oil exerts its detrimental effects before it degrades into harmless and simple compounds. Hence a study on the type of degradation mechanism, the measures which can expedite the process of bioremediation and how the soil parameters are influenced by oil pollutants and their degradation is demanded by situation. The present investigation intends to gains some true knowledge on the matter based on experimental findings.


Introduction
The modern civilization depends on exploration, distribution and use of petroleum and thousands of products derived from it. The environment all throughout the world has been very badly damaged due to these chemicals. While pollution of the atmosphere has been caused almost totally by burning of the petroleum products, the leaks, spillages and accidental fallout of petroleum products and the crude petroleum itself have greatly affected land and water resources. The oil field areas always receive a large amount of effluents rich in crude oil and land degradation is a common phenomenon. While using different fractions of petroleum (gasoline, kerosene, diesel etc.) spills and leakages cannot be avoided and in industrial areas, garages and other places, a large amount of it flows to the nearby areas. All types of oil have amazing spreading power and once a leak occurs, the oil may spread horizontally as well as vertically depending upon soil conditions, moisture, temperature etc. The lighter fraction being volatile are easily removed by evaporation and other physical processes, but the heavy components such as aromatics -simple and polycyclic, Hopanes etc. remain in the soil for a very long time unless biodegraded by soil micro organisms. The detection of presence of some carcinogenic hydrocarbons viz. PAH has made the situation a matter of serious concern. In most cases; the pollutant oil exerts its detrimental effects before it degrades into harmless and simple compounds. Hence a study on the type of degradation mechanism, the measures which can expedite the process of bioremediation and how the soil parameters are influenced by oil pollutants and their degradation is demanded by situation. The present investigation intends to gains some true knowledge on the matter based on experimental findings.

Soil is a depleting natural resource
Human race is dependent on a number of gifts of the nature. One such gift of nature is the Soil resource. It has been defined as the thin layer of the earth's crust in which biological activities take place. The beneficial activities of soil are multidimensional. Animal life is absolutely dependent on plant kingdom for food. Plants absorb nutrients from soil and convert it into a form acceptable to animal kingdom. Thus it is one of the best supporters of www.intechopen.com life. Soil is made of weathered rock material and organic matter. Weathering, the physical and chemical breakdown of rocks is the first step in the soil forming process. The process of soil formation is a very long and slow geological process. The process is cyclic, because the rocks are changed to unconsolidated particles, which may be eventually be cemented together by other chemical and physical mechanisms to yield new rocks. The slowness of this soil producing process indicates that soil is a depleting natural resource when subjected to erosion, loss of fertility or pollution. Soil fertility, the ability of the soil to supply the plants with their essential nutrient elements, directly determines what crops can be grown on a soil and the nutritional value of these crops for man and animals.

Land degradation due to hydrocarbon pollutants
By virtue of the properties such as a portable, dense energy source and as the base of many industrial chemicals; Petroleum is one of the world's most important commodities. Today about 90% of fuel needs are met by oil. The presence of the oil industry has significant environmental impacts. Accidents and routine activities such as seismic exploration, drilling, and generation of polluting wastes affect the society. Oil extraction is often environmentally damaging. Leaks, spillages and accidental fallouts of petroleum and its various fractions are of frequent occurrence. Offshore exploration and extraction of oil disturbs the surrounding marine environment. Crude oil and refined fuel spills from tanker, underground pipelines, and ship accidents and have damaged soil resource, ecosystem in many parts of the world.
Land degradation due to this is a common phenomenon in the modern world. Since the early part of the twentieth century, the damaging effect of the hydrocarbons from oil has been known. Many workers reported the damaging effect of petroleum and its various fractions on soil and water resources (Young, 1935). It was reported that crude oil can sterilise soils and prevent crop growth for various period of time. The duration of the damaging effect depends largely on the degree and depth to which the soil is saturated with the oil. The damage that the oil does is due mostly to the prevention of the plant from obtaining sufficient moisture and from ramifying its roots very little is due to toxicity, as such (Plice, 1948).
Out of various processes of disappearance of oils from polluted soil, microbial degradation of pollutants is one of the most important ones (Davies & Westlaki, 1979). Crude petroleum and its fractions are converted into soil organic matter by bacteria and fungi. During the conversion, the organisms, which are free lives, fix fairly large amount of atmosphere nitrogen in the soil. Later this nitrogen becomes available for plant growth and the organic matter improves soil physical conditions. Based on general principle of hydrocarbon degradation, a programme of rehabilitation such as liming, fertiliser addition, and frequent tilling is considered to be broadly applicable for all types of mineral oils and experiments showed that these are really effective measures (Dibble & Bartha, 1979). Dry micro organisms contain approximately 14% N, 3% P and 1% S in the form of proteins, nucleic acids, polysaccharides and low molecular weight compounds. On the other hand, Petroleum products are composed of hydrogen and sulphur and essentially no phosphorous in their environment in order to grow on hydrocarbons as their carbon and energy sources (Rosenberg, 1993). This application of nutrients especially that of Phosphorous seems to be an effective means of rehabilitation and experiment showed that this is really so (Reynolds & Walworth, 2000). On the contrary, studies showed that the presence of Polychlorinated Biphenyls negatively impact fossil fuel degradation ( Hoeppel et.al, 1995).Out of several methods to expedite the process of degradation of Petroleum Hydrocarbons and to rehabilitate an oil inundated soil such as Bioremediation, Polyencapsulation, Vapour Transport, Land Farming, Alcohol Flooding etc. Bioremediation is considered effective and environmentally benign. Before taking up remediation measures emergency clean up operation is an important activity.

Methodology
Soil sample having no history of oil pollution were taken, polluted by known quantity of oil using emulsifier-some with nutrients, some with oxidising agents and some without any additive and the stated experiment was done by placing indoor in polyethene bags and maintaining the conditions necessary for microbial activities (Table-1). Series with B, C, D and E are similar to series A except that the oil is replaced by Kerosene oil, Diesel oil, Lubricating oil and Residual oil respectively. Gravimetric determination was done by withdrawing 20 g polluted soil sample from each bag and by recovering the oil by soxhlet extraction method using Petroleum ether as solvent after definite time interval. Gas Liquid Chromatography (GLC/GC) is the method of choice for rapidly and accurately analysing the volatile substances. It is also applicable to non volatile ones due to availability of higher column temperatures. Different compounds maintain similar sequence in the chromatogram analyzed under same system of column and identical conditions.

Sample
No.

Mass of Soil
Total mass

Results and discussions
A few important parameters of the soil such as Texture, pH, Hydraulic Conductivity, Bulk Density, Water Holding Capacity, Soil Organic Carbon, NPK nutrients etc. were determined before the start, in between, and at the end of the experiment (Sing et al, 2000) ( Table-2 Remains of hydrocarbons and their oxygenated derivatives in the above mentioned soil bags were extracted by soxhlet extraction using Petroleum Ether as solvent and gravimetric determination was done (Table-3). It was found that the NPK supplementation is an effective measure of rehabilitation of oil inundated soil. Supply of nutrients to the soil helps micro organisms to grow abundantly, consequently the degradation becomes faster. Hydrogen peroxide decomposes some of the organic matter present in the polluted soil and it also contributes towards the degradation to some extent. It has been found that more than 50% of the applied mass undergoes degradation in average in all the three sets of crude oil experiment during the first two months of placement. The activities of micro organisms are highly dependent on temperature. Ambient temperature was also recorded during the period of the experiment.  (Sarma et al, 2005a). Hydrogen Peroxide decomposes organic compounds from soil. Hence a good number of volatile components under GC conditions have been found in the 3A5 sample. Later these components escape from soil as carbon dioxide and a fraction fixes with soil. In the mass determination of the remaining hydrocarbons, it has been found that degradation in the second set i.e. set having applied NPK is highest. It means that it has already degraded and given away more compounds from the polluted soil sample. It has been seen from the chromatograms that there are no components with retention time in the range of 16.315 -43.247 minutes, 17.682 -40.729 minutes and 11.589 -42.677 minutes respectively in1A5, 2A5 and 3A5. A large number of components with minute differences in their retention time are seen in the chromatograms. Table-5 represents some of such peaks between 1A5 and 2A5. The corresponding peaks in 2A5 appeared with an advance retention time of 0.452 minute in average belonging to the range of 0.571 to 0.392 minute. There are no corresponding peaks in 2A5 for five compounds present in 1A5 (Table-6). Probably these are the compounds degraded completely as a result of profuse microbial activities due to application of nutrients.  Table 9. List of peaks found in 1A5 giving no corresponding peak in 3A5.
hydrogen peroxide. Table-10 represents the list of peaks which are found in 3A5 but not in 1A5. These are the peaks of compounds formed due to higher microbial activities that occurred on the compounds. Number of such components are 25 and these compounds occupied a minute area (average 1.84%) of the chromatogram in the range of 0.06 to15.19%.
GC analysis of the oil, recovered after 1 month and 2 months in the sample 2A3 gave a total of 17 and 24 peaks respectively. There is no peaks during12.217 to 47.277 minutes and during 15.303 to 44.055 minutes in the samples after 1 month and after 2 months respectively. Out of the 17 compounds obtained after I month, it appears that 14 compounds are still present in oil recovered after two months. It means that these components could resist degradation in the second month. These are placed in table ( 72.525 and 81.026 minutes with area percent report of 25.47, 1.85 and 1.17 respectively in the sample after 1 month got disappeared in the second month. It has also been seen that another 10 peaks due to 10 new compounds appear in the sample after 2 months (Table-12).
These compounds seem to be the degradation products of various hydrocarbons. These peaks have area percent in the range of 0.11 to 11.00 with an average of 1.69%.

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In the GC analysis of the sample 3A3 after 1 month and after 2 months, it has been seen that there are 11 and 33 components respectively including that for the solvent. There are no peaks appear in between 20.082 to 46.835 and in between 11.662 to 42.728 minutes in the two  Table 12. List of peaks found in the sample 2A3 after 2 months but not in after 1 month.
samples respectively. As many as 8 numbers of peaks of components present after 1 month in 3A3 can be linked to same number of components present in the extract obtained after 2 months.  Table 13. List of Similar Peaks in GC analysis report of 3A3 extracted after 1 and 2 months.
The 17 and 11 peaks found respectively in the GC chromatogram of samples 2A3 and 3A3 after 1 month of placement, possess 9 peaks ( Gas Chromatographic analysis of recovered oil from kerosene oil polluted soil (B-set) after 2 months of placement showed that number of components at 1B5 and 3B5 ( Plate-2) became 17 and 35 respectively. Gravimetric determination shows that mass of oil recovered in the samples having applied Hydrogen Peroxide is less, whereas number of components in the same is more than that of the sample without Hydrogen Peroxide (Table-17). It indicates that Hydrogen Peroxide removes the pollutants by degrading into smaller compounds.
There are no peaks appeared in between the retention time ranges 4.838 to 32.566 minute in1B5 and in the range of 9.611 to 32.669 minute in 3B5. It appears that for every peak in the 1B5 sample, there is a corresponding peak in the 3B5 sample (  Table 19. List of peaks found in the sample 3B5 but not in 1B5.

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Against 50 compounds present in 1C3, there are 42 and 27 compounds present in the samples 2C3 and 3C3, where nutrients and Hydrogen Peroxide were applied respectively. It implies that nutrients and Hydrogen Peroxide have a positive role in degradation of higher hydrocarbons into lower ones and subsequent removal of them from soil. A list of similar peaks in GC analysis report of 1C3, 2C3 and 3C3 are given in table (Table-20). It has been seen that the peaks due to same compounds appear in the chromatogram of 2C3 and 3C3 after an average delay in retention time of 0.078 and 0.123 minutes compared to the peaks of 1C3 in a range of 0.018 to 0.194 and 0.087 to 0.232 minutes respectively. This minute difference in retention time is probably due to minor change in experimental conditions during transit. There two peaks in the chromatogram of 2C3 and 3C3 at 54.466 and 54.552 minute with area % 0.53 and 0.22 respectively are probably due to same compound, which is a degradation product. The other two such compounds appear at retention time 61.654 and 71.422 minute in the 2C3 and 3C3 reports are also due to degradation products.
Plate 3. GC Chromatogram of Oil recovered after 3 months of placement of the sample 2C3.
In the GC report of the extracted oil samples from 1C5, 2C5 and 3C5 after three months of placement, number of peaks seen are 21, 34 and 40 respectively. From gravimetric analysis it has been found that extent of degradation in corresponding C5 samples are higher than C3 samples (Table-21). Thus it seems that number of components increases against increase of degradation at a certain stage and then the components gradually disappear. A list of peaks due to same compounds is given in table (Table-22). The peaks at 2C5 appear with a negligible average delay of 0.012 minute in retention time in the range of -0.036 to 0.055. The same for 3C5 peaks is 0.007 minute in the range of -0.070 to 0.034 minute. The existence of these compounds in all the samples indicates that these compounds could resist degradation during the initial period of three months. The other compounds present in 2C5 and 3C5 are some of the degradation products. In the same way similar peaks have been found in 1C3 and 1C5, 2C3 and 2C5, and between 3C3 and 3C5.
The chromatograms obtained from the extracts of Lubricating oil polluted samples 1D1, 1D2 and 1D3 (Plate-4) are represented in plates. Number of components which persists degradation and number of components produced as a result of microbial degradation taken together has been found to be 64, 53 and 67 respectively, including solvent peak after lapse of 3 months (Sarma & Bhattacharyya ,2010). From the gravimetric analysis, it appears that the extent of degradation is highest in the samples with nutrients. The extent of degradation in presence of hydrogen peroxide is moderate. It seems that hydrogen peroxide increases number of components and later these degradation products disappear from soil. There are some peaks which seem to be due to same compounds (Table-23).
-1-Sl No.  Table 23. List of Similar Peaks in GC Analysis Report of 1D1, 1D2 and 1D3.
It means that these common compounds are the compounds which are resistant to degradation. The number of such compounds in between 1D1 and 1D2 is 48, between 1D1 Plate 4. GC Chromatogram of oil recovered after 3 months of placement of sample 1D3.
www.intechopen.com and 1D3 is 41and among the three is 33 .The common compounds appear with a uniform difference in their retention time. 1D2 peaks appear with an average delay of 0.194 minute in the range of 0.049 to 0.404 minute and 1D3 peaks appear with an average advance retention time of 0.318 in the range of 0.019 to 0.441 minute. The other peaks of 1D2 and 1D3 are probably due to some degradation products. Number of such compounds in 1D2 is 5 and in 1D3 are 26. It indicates that Hydrogen Peroxide decomposes the higher hydrocarbons first and then the degraded components gradually disappear.
G.C. analysis of the recovered oil from residual oil polluted sample 1E5 after 1,2 ( Plate-5) and 3 months gave 37, 65 and 52 peaks due to the undegraded components, derivatives and degradation products respectively. Out of the 37 components obtained after 1 month; 33 and 29 could resist degradation in the second and third months respectively, as revealed from their peaks appeared at a uniformly different retention time ( Sarma et al,2004a) ( Table-24). The stated 33 peaks appeared at a uniform advance retention time in the range of 0.914 to 1.247 minute with an average of 1.046. The stated 29 peaks appeared at a uniform advance retention time in the range of 0.914 to 1.247 minute with an average of 1.233. The remaining 4 and 4 components appeared in chromatograms obtained after 1 and 2 months respectively seems to be lost from the soil due to microbial activities. The number of peaks appeared in the chromatogram taken after 2 months but not in the previous chromatogram are 32. These peaks, and the corresponding peaks present in the last chromatogram are given in table (Table -25). Here only 17 peaks could resist complete degradation. These 17 peaks appeared at a uniform advance retention time in the range of 0.038 to 0.403 minutes with an average advance retention time of 0.196 minutes. The other 15 components seem to be lost from the soil due to microbial activities during the third month. Out of total 52 peaks appeared in the last chromatogram, only 6 peaks are due to new compounds (Table-26). These are the degradation products of the oil in the third month. Thus it can be concluded that degradation in the initial stage is more vigorous. The process slows down gradually. During degradation the higher compounds produce some fragments or some derivatives as a result of which number of components increases. Later on these compounds gradually disappear. Table-27 shows number of components detected by GC analysis of a few samples of some of the mineral oils viz. Crude oil, Kerosene oil and Diesel oil-without agents, with nutrients, and with hydrogen peroxide. It appears that Hydrogen Peroxide increases the number of components in the sample and thus degraded the higher compounds. Application of NPK also increases the same, but lesser than Hydrogen Peroxide. For example, in the A5 samples, number of components after 3 months becomes 22 in 1A5 (without agent), 37 in 2A5 (with nutrients) and 39 in 3A5 (with hydrogen peroxide).The following table (Table 28) shows number of components detected by GC analysis of a few samples under identical conditions after one, two and three months. It appears that number of components increases initially and then decreases. It means that higher compounds give some smaller compounds and then escape from soil.
A good number of common components can be identified in the GC chromatogram of different samples (Table-29). These common components appear in the chromatogram after maintaining a uniform difference in their retention time. This difference is probably due to minor difference in the experimental conditions. In between the peaks at serial number 1and 2, the average difference in the retention time is 1.311 minute in the range of 1 In order to make an attempt to identify different components those are persistent for a stipulated time in the soil samples, an experiment with some standard sample solutions were done (Sarma & Devi, 2009 Benzophenone, Cinnamic Acid, Anthracene and Benzoic Acid respectively. Out of these Anthracene and Naphthalene are tricyclic and bicyclic aromatic hydrocarbons respectively and others are oxygenated derivatives. The presence of these compounds in the polluted soil samples cannot be ruled out. That the polycyclic aromatic hydrocarbons, which are of great concern due to their toxicity and suspected carcinogenicity; are resistant to biodegradation was reported by many workers at different point of time. The influence of applied phosphorous on bioremediation is positive. Since the phosphorous cycle is a sedimentary one, its fixation rate was studied. It has been found that a sample of sandy loam which possess 1.987µg g -1 available phosphorous fixes 99.85% phosphorous against addition of 50 g and 100 g of commercial single super phosphate fertiliser during progressive remediation from Lubricating oil pollution in a 120 days experiment ( Sarma et al, 2008).
Bioremediation improves the soil physical conditions of petroleum polluted soil. During such remediation the amount of Soil Organic Carbon remarkably increases. A maximum of 111.51% and 65.20% increase of SOC was found in two samples of Sandy Clay and Sandy soil at an applied concentration of 20,000 ppm crude oil pollutant in an indoor experiment of 346 days. The increase of SOC in the samples where degradation was carried out in presence of added NPK was less and in those with added Hydrogen Peroxide was more than in the samples without NPK and Hydrogen Peroxide (Sarma & Sadhanidar , 2007).
It has been found that the pH and Electrical Conductivity of a remediating soil decreases. For example, in the first 6 months in the 1A5 sample pH decreases from 6.86 to 6.77; 6.73; 6.64; 6.59; 6.40 and 6.14 in regular monthly interval. Similarly, the EC decreases from 0.43 mS cm -1 to 0.41; 0.30; 0.27; 0.18; 0.14 and 0.11 mS cm -1 respectively in regular monthly intervals (Sarma et al, 2003a). Similar results were found when Lubricating oil is the pollutant (Sarma et al, 2003b(Sarma et al, , 2004b(Sarma et al, , 2005b. This is due to formation of some oxygenated derivatives from hydrocarbons, which are weakly acidic as the microbial degradation is a process of oxidation.

Conclusion
It has been found that under identical conditions a suitable soil sample degrades petroleum fractions to different extent. The extent of degradation of kerosene oil is highest, followed by crude oil, diesel oil, lubricating oil and residual oil. The disappearance of hydrocarbon is more in the initial stage and gradually it becomes a slow process. Complete recovery from hydrocarbon pollutants is not achieved during the experimental period of one year. The application of nutrients expedites the process of degradation. The action of hydrogen peroxide is moderate. The number of compounds in the recovered oil increases up to a period and then gradually decreases. In most cases the number of components generated is more in samples where hydrogen peroxide is applied. A good number of peaks formed probably due to same compounds can be pointed out and these appear in the chromatogram by maintaining an almost uniform difference in retention time. Parameters pH and Electrical