ABSTRACT
One of the world’s highest consumed resource and most dangerous pollutants are petroleum hydrocarbons. Petroleum hydrocarbons are known as the major antagonist in a number of various water, land and environmental disasters. Because of its highly destructive nature which is a direct consequence of its toxicity, resistance and degradation, the proper treatment and cleaning of this substance is of paramount importance. One potential solution to this problem is bioremediation which is an ongoing solution that involves the use of various microorganisms which are able to naturally biodegrade the petroleum in these dangerously contaminated regions. The purpose of this study was to observe degradation rate of the total petroleum hydrocarbons (TPH) in two samples of soil that were gathered from two different locations. Soil A was a compost soil sample while Soil B was collected from a Coles express petrol station that is under renovation. The combination of using cell counts, gas chromatography and bioinformatics for analysis were utilised to observe how effective degradation was in each of the soil types. Following this, bio stimulation effectiveness was measured by using an NP solution. The overall results following the four-week testing period indicated that Soil B was more successful in degrading petroleum molecules when directly compared to Soil A. The utilisation of bio stimulation in this study drastically increased the overall effectiveness of microorganisms in Soil A. In essence from this study it can be said that indigenous microorganisms and properties of the soils directly affected the overall rate of degradation in the soil.
INTRODUCTION
Oil spills pose a dangerous threat that can potentially lead to severe environmental damage. The Deepwater Horizon oil spill is one of history’s more recent examples, in which over 780,000m^3 of oil being discharged, this is one of the largest and most detrimental oil spills recorded in history. Oil spills that occur in aquatic environments cause a thin layer of oil over the surface of the water called ‘sheen’, the combination of wind and tidal movement, cause the spread of the oil for hundreds of meters , ultimately blocking sunlight from reaching beneath the water, which has a damaging effect on aquatic ecosystems as producers are left unable to use sunlight for photosynthesis hence potentially damaging the fragile ecosystems that thrive in these waters. In addition, this is harmful to both marine coastal wildlife as animals may be poisoned by ingestion or the spill may destroy natural habitats. Ingesting oil can also affect an animals’ ability to produce offspring.
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Bioremediation is a common procedure used for media treatment which includes contaminated water or soil. Bioremediation involves altering certain environmental conditions to encourage the growth of various microorganisms that degrade the targeted pollutants. Bioremediation is lower in cost and far more sustainable than biological treatment which is an alternate method of remediation in which a similar type of procedure is utilised to treat solid waste, industrial waste and wastewater. An adequate examination of the soil conditions in the treatment area is required in order to determine whether the organic contaminants can be biodegraded by soil microorganisms and if the residuals contaminants levels are acceptable (Vidali, 2001). If the conditions are favourable, introducing the microorganisms will completely metabolise the organic contaminants and successfully convert them into nontoxic by products such as carbon dioxide and water or organic acids and methane (US-EPA,1991)
Bioremediation can be categorised into in situ and ex situ with ex situ bioremediation referring to reactions that are performed outside the organism’s natural habitat while in situ refers to reactions performed inside the reaction mixture. Ex situ bioremediation characterises an increase in microbial activity though aeration and irrigation. In addition, it increases degradation by the transformation of polluted soils use of water and minerals. Furthermore, ex situ utilises bioreactors to speed up and enhance the biological reactions occurring in the microorganisms to decrease bioremediation time. On the other hand, in situ bioremediation involves bio venting, slurping and sparging which involves the use of moisture and various nutrients to further the transformation rate of harmful pollutants, using pumping techniques to apply oxygen and water and pushing air into the soil to stimulate microbial bioremediation.
These organisms have evolved over time to utilise hydrocarbons and organic compounds as a source of energy and by doing this they se molecular transfer mechanisms to denature toxins. The organisms anaerobic and aerobic properties allow them to respire and ferment compounds, hence resulting in the transformation of toxins into innocuous compounds which have more stable Ph levels, increased water solubility and are less aggressive molecularly. (Fiorenza ,2003) .The microorganism operates in a procedural manner when breaking down and metabolising the petroleum components by first acting on the linear alkanes, then the branched alkanes, following this is the small aromatic compounds and concluding with the cyclic alkanes.
DISCUSSION
This study focuses on evaluating the effectiveness of the various microorganisms that are present in the bioremediation procedures utilised for the degradation of petroleum hydrocarbons. At the start of this study it was hypothesised that Soil Type A would be have a larger number of colony forming units, this is believed to be due to the nutrients and bacterium’s diverse nature, however this was found to result in lower degradation rates of petroleum hydrocarbons. The reason for this was because the bacteria’s adaptations and resulted in petroleum being the resource with the most abundance for carbon.
Figure 1 illustrates that soil type B with oil had a higher normalised peak overall when compared to soil type A with oil. The only instances where soil A was greater is seen in retention time (32.3) and retention time (40.6). When analysing the spread and drop plates, it was observed that after a seven-day period of inoculation, the soil with the higher number of CFU was the compost soil and not the soil from the petrol station.
The petroleum hydrocarbons within the soil samples were identified by using gas chromatography. Table 8 depicts the results of the gas chromatography showcasing the hydrocarbons in each soil sample. When compared, both sample have similar contents with the exception of the compost soil sample having two additional hydrocarbons; Tetratriacontane and Docosane. Figure 2 illustrates the degradation of hydrocarbons and indicates the successful degradation of harmful hydrocarbons. The Np solution was highly effective in degrading hydrocarbons as it can be seen to have significantly lower levels of contents when compared to the soil+oil and autoclaved soil+oil samples.
A study conducted by the Indian institute of advanced research found that hydrocarbon degrading microorganisms in ecosystems that were unpolluted made up under 0.1% of the microbial community and in ecosystems polluted by petroleum hydrocarbons these organisms made up 1-10% of the microbial community (Varjani 2016). This is important as reinforces the importance of these microorganisms in the degradation of petroleum hydrocarbons. The future for bioremediation rests in further research that will indicate new design ideas for more efficient and feasible bioreactors and implementing systems that could potentially remove 100% of the polluting agents from the environments along with using the by products for other projects (Abbasi , 2014)
During the course of this study there were some errors that occurred that made the results less accurate. The first of which was the error in counts for flask 2 Soil A, it is believed that the sample was contaminated, resulting in no results being drawn from Flask A. Similarly, there was also contamination that occurred in flask 4. Due to flask 4 representing the autoclaved sample and being the abiotic control mechanism, hypothetically a positive CFU count should not have occurred. This contamination may have been caused by a human error that occurred after the autoclaving procedure, possibly because of the sample not being adequately sealed or due to the lack of aseptic techniques when handling the sample. To ensure this error does not occur again future studies, supervision and additional training may be necessary, in addition this section of the method could have been repeated to ensure reliability and duplicates could have been made ensuring that any flasks that appeared to have contamination were to be properly recorded and then discarded. This would ensure accurate results by the end of the study.
CONCLUSION
In essence the results following the four-week testing period indicated that Soil B was more successful in degrading petroleum molecules when directly compared to Soil A. The utilisation of bio stimulation in this study drastically increased the overall effectiveness of microorganisms in Soil A. In essence from this study it can be concluded that microorganisms and properties of the soils directly affected the overall rate of degradation in the soil.
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