Assessing Biodegradation Susceptibilities of Selected Petroleum Hydrocarbons at Contaminated Soils

Assessing Biodegradation Susceptibilities of Selected Petroleum Hydrocarbons at Contaminated Soils (MH Langsa): The susceptibility to biodegradation of selected saturated hydrocarbons (SHCs), polycyclic aromatic hydrocarbons (PAHs) and asphaltenes in a Barrow crude oil and extracts isolated from soils contaminated with the Barrow crude oil at day 0 and 39 was determined. Soil samples were contaminated with a Barrow crude oil across the surface (5% w/w) as part of a mesocosm experiment in order to mimic similar conditions in the environment. The extent of biodegradation of the Barrow oil extracted from the contaminated soils at day 0 and day 39 was assessed by GC-MS analyses of SHCs and PAHs fractions. Changes in the relative abundances of n-alkanes (loss of low-molecular- weight hydrocarbons) and pristane relative to phytane (Pr/Ph) and their diastereoisomers were determined. Changes in the diastereoisomer ratios of Pr and Ph relate to the decrease in abundance of the phytol-derived 6(R),10(S) isoprenoids with increasing biodegradation. The percentage change in abundances of each of selected alkylnaphathalenes with time (day 0 to 39) was determined, enabling an order of susceptibility of their isomers to biodegradation. It was established that the 2-methylnaphthalene isomers (2-MN) is more susceptible to microbial attack than 1-MN isomer indicated by decreasing in percent abundance from day 0 to 39 for the 2-MN isomer. The GC-MS analyses of the original Barrow oil indicated the oil had not undergone biodegradation. When this oil was used in the soil mesocosm experiments the oil was shown to biodegrade to about a level 2 -3 based on the biodegradation susceptibility of the various SHCs and PAHs described above.


INTRODUCTION
The impact that an oil spill has on the environment depends on many factors, including the chemical and physical nature of the oil or refined product, the local environmental conditions (i.e. wind currents, temperature, salinity and biota). Some oil spills are quickly bioremediaded whereas other spills can have long-term effects spanning several years (Fisher et al. 1998;Peters and Moldowan 1993).
Bioremediation is a process that encourages microbial populations to degrade hydrocarbon contaminants (Barron 2004). Microbial degradation is one of the most effective methods to remediate oil from soil or sediment after a spill. The toxicity of the contaminants is reduced because bioremediation transforms xenobiotics introduced into the environment to less toxic or innocuous forms, and/or mineralises them to CO 2 , H 2 O, O 2 and other inorganic products. Thus migration of contaminants is usually prevented and ecological recovery is usually promoted.
Numerous studies also have been made to measure the effects of biodegradation on aromatic hydrocarbon distributions in soils (e.g., Bispo et al. 1999;Budzinski et al. 1998;Chang et al. 2002;Henner et al. 1999;Loehr 1991;Pospisil et al. 1996;Sauer et al. 1998;Skiba et al. 1992). Similar results to those reported above for reservoired oils have been observed for soils. For example, the rate of aromatic family biodegradation decreases with increasing aromatic rings (or molecular weight or boiling point), similar to what would generally be predicted through evaporation. Naphthalenes degrades faster than the . phenanthrenes and dibenzothiophenes and chrysene is one of the most resistant to biodegradation (Sauer et al. 1998). Within individual compound classes, some isomers are more susceptible to biodegradation than others, and this is controlled by the position of the alkyl substituent's on the aromatic ring (Huang et al. 2004). In a soil studied by Pospisil et al. (1996), it was found that the concentration of the aromatic hydrocarbons with three and four-rings decreased to about 0.5% of the original oil.
Barrow Island is a region where the climate is arid and the island experiences very high temperatures exceeding 30 o C, which means bacterial biodegradation is a slow process and the oil pollutants in that area will take a significantly longer period of time to degrade (Davie 2004). It has been recognised that because of these climatic conditions, action needs to be taken in order to optimise the bacterial activity. In a study by Watkins (1996) fertilizers were added to the soil to act as a carbon source for the bacteria and to promote bioremediation in that area.
The main objective of this study is to observe the rate of biodegradation of the Barrow crude oil in soil after production flowline leaks, including polycyclic aromatic hydrocarbons (PAHs) and saturated hydrocarbons (SHCs). This will be performed by a mesocosm experiment. GC-MS analyses of the saturates and aromatics fractions from the crude oils before and after contamination will be carried out and changes in abundances of compounds will be rigorously assessed. By testing the effects of crude oil on soil composition, this research will represent how production flowline leaks are affecting the soils on Barrow Island. These results may then be used as a basis for further detailed research and also provide clues on better management of contaminated sites upon the island.

Soil Samples
Soil samples provided by the Centre of Land Rehabilitation Laboratory UWA were collected from the top 20 cm of the soil profile on Barrow Island. Several physical properties of the soil were measured by UWA's laboratory. These properties include water holding capacity and soil moisture content. A vessel of contaminated soil was prepared in a 500 mL sealed glass jar containing 100 g of soil. The soil holding water capacity was adjusted to around 50%. The appropriate amount of oil (5% w/w) was applied to obtain an even distribution of oil across the surface of the soil to imitate the similar conditions in the field. Two replicates were used. The vessels were sealed after adding the oil and left to incubate for 39 days at 25 o C. The % of extractable hydrocarbons (saturated, aromatic and polar hydrocarbons, NSOs) was measured on day 0 and day 39.

Extraction Soil Contaminated Oil
Soil samples were dried at room temperature. The ground soils were extracted by using an accelerated solvent extractor using 9:1 mixture of dichloromethane and methanol. Extracts were dried under a nitrogen purge and weighed.

Crude Oil
Crude oil was provided by the Centre of Land Rehabilitation Laboratory UWA. The oil was collected from the production flowlines on Barrow Island. The whole crude has a specific gravity of 0.8363, a sulphur content of 500 mg kg -1 and a nitrogen content of 320 mg kg -1 . The crude oil is composed of 95% hydrogen and carbon, with smaller quantities of additional elements including sulphur, oxygen, nitrogen and metals.

Separation of Saturated, Aromatic and Polar Hydrocarbons (NSO) by Silica-Gel Chromatography
In a typical small-scale separation, the crude oil or maltenes fraction (20 mg ~ 2 drops) was applied to the top of a small column (6 cm x 0.4 cm) of activated (120 o C, overnight) silica gel pre-eluted with pentane. The aliphatic hydrocarbon (saturate) fraction was eluted with pentane (2 mL); the aromatic fraction with a solution of dichlromethane (DCM) in pentane (2 mL, 30%); and the polar fraction with a solution of equal parts of DCM and methanol (2mL). The resulting saturate (10 drops) and aromatic fractions (20 drops) were diluted with hexane for analysis by GC-MS.

Identification of Naphthalene and Alkylnaphthalenes
Naphthalene and alkylnaphthalenes are some of the major components of the aromatic fraction. These compounds were identified using their molecular ion mass chromatograms as shown in Figur e 1. Naphthalene (m/z 128) was assigned based on its mass spectrum and by comparison of retention time with that of a reference sample The alkyl isomers, i.e. methyl-(m/z 142), dimethyl-(m/z 156), trimethyl-(m/ z 170) and tetramethylnaphthalenes (m/z 184) were identified by comparing retention times and mass spectra with those reported in the chemical literature (Brown and Maher 1992;Jiang 1998;Volkman et al. 1984).

The Effect of Biodegradation on Individual Alkylnaphthalene Isomers of Extracted Oil from Soil
Selected mass chromatograms reveal the presence of naphthalene, the MNs, the DMNs, TMNsand TeMNs in the aromatic hydrocarbon fraction from the original oil, extracted oil at day 0 and 39 shown in Figure 2. Inspection of these chromatograms depicts that only naphthalene is almost completely removed from the oil extracted from biodegraded oil contaminated for 39 days whereas the MNs, the DMNs, the TMNs and TeMNs isomers are slightly altered. The peak areas for the alkylnaphthalene isomers were determined and plotted as % change in relative abundance calculated from the contribution of each alkylnaphthalene isomer to the total sum of the isomers. The results for these treatments are presented graphically in Figure 3. It is evident that 2-MN is decreasing in % abundance from day 0 to 39 whereas 1-MN is increasing in % abundance. Thus the 2-MN is easily biodegraded and therefore most susceptible to biodegradation.

The Effect of Biodegradation on Individual Alkylnaphthalene Isomers of Extracted Oil from Soil
Selected mass chromatograms reveal the presence of naphthalene, the MNs, the DMNs, TMNs and TeMNs in the aromatic hydrocarbon fraction from the original oil, extracted oil at day 0 and 39 shown in Figure 2. Inspection of these chromatograms depicts that only naphthalene is almost completely removed from the oil extracted from biodegraded oil contaminated for 39 days whereas the MNs, the DMNs, the TMNs and TeMNs isomers are slightly altered.
The peak areas for the alkylnaphthalene isomers were determined and plotted as % change in relative abundance calculated from the contribution of each alkylnaphthalene isomer to the total sum of the isomers. The results for these treatments are presented graphically in Figure 3. It is evident that 2-MN is decreasing in % abundance from day 0 to 39 whereas 1-MN is increasing in % abundance. Thus the 2-MN is easily biodegraded and therefore the most susceptible to biodegradation. As can be seen from Figure 3(b) that 2,6-DMN is also decreasing in % abundance to a greater extent than the other DMNs suggested that this isomer is far more susceptible to biodegradation than the other DMNs. 1,3-DMN, 1,5-DMN and 1,6-DMN appear to be fairly resistant to biodegradation indicated by increasing in % abundances, while 1,2-DMN and 1,4-DMN are intermediate. Susceptibility in ENs is also evident with 2-EN appears to have been easily removed relative to 1-EN shown by increasing in % abundances of this isomer after being contaminated with the soil for 39 days.
The results from the analysis of the TeMN are presented in Figure 3(e). It is evident that most of the isomers remain relatively unaltered indicated by almost parallel the bar graph of the isomers from the oil samples. The only slight decrease in % abundance is for 1,3,5,7-TeMN perhaps indicating this isomer is easily removed by biodegradation. 1,2,3,6-TeMN, 1,2,5,6-TeMN, 1,2,5,7-TeMN, and 2,3,6,7-TeMN are relatively constant while 1,2,3,7-TeMN is slightly increasing in % abundance showing that this compound is most resistant to biodegradation.   summary of susceptibility to biodegradation of the various alkylnaphthalene isomers.

Saturated Biomarkers
The total ion chromatograms of saturated hydrocarbons isolated from the Barrow crude oil and contaminated soils (day 0 and 39) are shown in Figure 4.
The effects of biodegradation on the diastereomers of pristane and phytane were also determined based on changes in relative abundances ratio. This ratio the diastereomers of pristane and phytane are presented in the Table 3.2. Generally, the ratio decreases consistently for samples at day 0 and day 39 due to the decrease in abundances of pristane.
The abudance of pr istane and phytane diasterioisomers is shown in Figure 6. The earlier eluting component (phytane) contains both RRR and RRS configurations as well as the corresponding enantiomers SSS and SSR, the second eluting component contains phytane with SRS and SRR configurations (Table 1. 1. n3, n4 and n5). For pristane the second eluting peak is a meso-form (RS = SR), while the For the samples there is a slight decrease in stereoisomericratio of pristane and phytane, which represents the decrease in relative abundances of RR and SS of pristane and RRR and RRS in phytane as biodegradation has proceeded.
The effects of biodegradation on the diastereomers of pristane and phytane were also determined based on changes in relative abundances ratio. This ratio the diastereomers of pristane and phytane are presented in the Table 2. Generally, the ratio decreases consistently for samples at day 0 and day 39 due to the decrease in abundances of pristane.
For the samples there is a slight decrease in stereoisomericratio of pristane and phytane, which represents the decrease in relative abundances of RR and SS of pristane and RRR and RRS in phytane as biodegradation has proceeded.
Assessing the rate of biodegradation of the crude oil spilled on soils is a tremendous technique for soil 15 recovering. Oil bioremediation as a result of microbial activity was the greatest in the agricultural soil as the soil has experienced regular incorporation of crop residues and fertilizers. It was expected that the greater microbial respiration found in the agricultural soil would be reflected in the size of the microbial population. Therefore, microbes would utilize the crude oil as a source of food. Further investigation on nutrients requirements for soils hopefully will improve the remediation of spill sites which was not covered in this study.
soil could be determined on the basis of changes in the composition of the saturated hydrocarbons and aromatic hydrocarbon distributions over 39 days by GC-MS analyses. These changes were accompanied by the alteration of the polycyclic aromatic hydrocarbons, in particular the alkylnapthalenes. It was evident that the 2-MN was more susceptible to biodegradation than the 1-MN. The depletion of the n-alkanes particularly the lower molecular weight n-C 6 -n-C 12 were removed at an early stage of biodegradation. As biodegradation proceeded, changes in pristine/phytane ratio occurred. The decrease in the PrDR/PhDR ratio was due to the loss of the RR and SS of pristane as a result of biodegradation.
As a result of the above observations, the biodegradation level of oil from the contaminated soils at day 0 and day 39 was assigned at 2-3.