Bioremediation uses the metabolic abilities of microorganisms to remove pollutants from contaminated environments. For over 45 years microorganisms have been used in the bioremediation of terrestrial, freshwater and marine environments. [1] Microorganisms are increasingly being used to treat pollution, because of the high likelihood that this bioremediation method, will prove as effective in the environment as laboratory investigations have encountered. Bioremediation can be classified as either in-situ or ex-situ. This essay will focus on the in-situ bioremediation where by the pollutant is treated at the source, as compared to ex-situ bioremediation where the contaminant is treated externally from the source.
Firstly there are numerous ways in which contaminated terrestrial environments can be treated. Among the many techniques employed, in situ bioremediation using indigenous microorganisms is by far the most widely used. This approach to treating contaminated land reduces the threat to groundwater and enhances the rate of biodegradation. [2] The rate of hydrocarbon biodegradation is determined by many important factors; the populations of indigenous hydrocarbon degrading microorganisms, the physiological capabilities of those populations, the physical state of the hydrocarbons and numerous abiotic factors. Intrinsic bioremediation is the natural removal of contaminants from the environment. In-situ intrinsic bioremediation is only an appropriate option for oil spill clean up after a risk assessment has been carried out. The results of which must show that it is unlikely for an adverse effect on humans, plants, animals and natural ecosystems to occur. [3] In addition to which the hydrology of the subsurface area must be known as if a contaminant is near groundwater or an aquifer then a safer option is the extract the contaminant for ex-situ remediation. However when intrinsic bioremediation is selected this process often faces limiting factors such as oxygen & nutrient levels. This can be overcome via biostimulation which is the modification of environmental conditions, to stimulate the microorganisms that carry out bioremediation.
Through the addition of nitrogen and phosphorus containing fertilisers the metabolic rate of bioremediators increases. The same can be seen for the addition of oxygen which is essential for oxygenase enzyme activity in the oxidation of a hydrocarbon, both usually via injection wells into the subsurface of soil. [4] Linking in with oxygen levels is the amount of moisture present which is an important factor. At 100 % saturation aerobic biodegradation is inhibited due to the lack of oxygen. Moisture levels in the range between 20-80% allow suitable biodegradation to occur. PH also plays an important role in the rate of biodegradation, which is most efficient in slightly alkaline conditions of 7.5 pH. However through the metabolic processes which occur during biodegradation of hydrocarbons the pH can be significantly lowered and thus requires monitoring and appropriate adjustments should be made to keep an optimal pH.
Soil particle size and distribution also affects microbial growth and plays a crucial role in the speed of biodegradation. Soil with open structure will encourage aeration and thus the rate of biodegradation will increase. In addition to which oil will infiltrate into the open soil structure and aid in the prevention of evaporation of volatile hydrocarbons. [5] The physical state of petroleum hydrocarbons is yet another factor which influences the success of biodegradation. When concentrations are low hydrocarbons will be soluble in water. However the large proportion of oil spill incidents release concentrations in greater quantities of the solubility limit. A greater dispersal of oil in lower concentration offers a greater surface area for hydrocarbon-degrading microbes to colonise. [6] Other abiotic factors such as temperature do have an impact on the rate of biodegradation however these are difficult to influence, resulting in a larger focus towards the previously stated conditions. [5] With the correct environmental conditions up to 80% of the non-volatile components of oil can be oxidised within a year of a spillage occurring. [7]
Under optimal conditions in the presence of a pollutant such as oil or petroleum, Oil-oxidising microorganisms' activity will rapidly increase. [7] Oleophilic bacteria are microorganisms which naturally use oils present in the environment as a carbon source. Oil is comprised of mostly hydrocarbons which act as a rich source of carbon and thus microorganisms will readily attack the aliphatic or light aromatic fractions of the oil. It has been shown that straight chained alkanes are the easiest to degrade, where as 6-member rings such as benzene show low levels of degradability only occurring at low rates when more preferred carbon sources are not available. [6] The hydrocarbons are broken down into fatty acids or carboxylic acid. These are then further degraded supplying the microbes with a source of carbon which is used in the citric acid cycle to produce energy; eventually leading to the formation of non-toxic products of carbon, carbon dioxide and water. [8] Communities which are exposed to hydrocarbons become adapted and will exhibit genetic changes, resulting in the proportion of hydrocarbon-degrading bacteria containing plasmids encoding hydrocarbon catabolic genes to increase. As a result rates of biodegradation will increase providing environmental conditions are maintained at the optimum levels. [6]
Microorganisms are not confined to the in-situ bioremediation of terrestrial environments but also play a crucial role in marine oil spills. Crude oil spills will naturally biodegrade in marine environments by indigenous microbes as seen in the same manner as terrestrial environments. However the time taken for biodegradation to occur is often long-term. With short-term major impacts on marine environment and the organisms within occurring after oil spill, allowing natural biodegradation is not an option. Crude oil in its self is not considered a hazardous waste although the coating of marine organisms is usually fatal. [6] One of first application of marine oil spill cleanup by 'oil-eating microbes' was in 1990 after the mega-borg oil spill. The microorganisms are grown in a catalytic solution for rapid reproduction until trillions are produced and stored in a powered form for application. The cost of cleanup via OEM equates to 1/10th of the price of alternative clean up techniques and thus economically is an attractive choice of oil-spill remediation. Close monitoring followed the application of the microbes, the result of which no negative environmental impact was observed.
Another example of marine bioremediation is that of the Exxon Valdez spill in 1989 which became the world's largest bioremediation project at the time. The Exxon Valdez spill has been classed as one of the worst human-caused environmental disasters being the largest oil spill in U.S waters until the Deepwater Horizon oil spill in 2010. The remediation process was criticised for being slow and began with dispersants which resulted in more problems than it solved. Overall only 14% of the oil had been removed via methods other than bioremediation through microbes. [9]As a result of treatment methods proving to be relatively ineffective the shorelines of Alaska were treated with oleophilic liquid fertilizer containing nitrogen and phosphorus. This was done to increase the metabolic ability of naturally occurring microorganisms that are capable of biodegrading hydrocarbons, in a slow releasing granulated form. This fertiliser was one of three which were trailed after the Exxon Valdez spill and produced the best results. As a result optimum levels were applied to the marine coast which resulted in a desirable level of biodegradation, without the undesirable impact of eutrophication from algal blooms and preventing toxicity to invertebrates and fish. Within 10 days oil-blackened shoreline rocks were deemed oil-free after treatment. The overall results of which proved that bioremediation was an effective method of oil spill cleanup. The choice of bioremediation as a technique proved successful and gave a better understanding and highlighted the potential for future use. [10]
In-situ bioremediation of oil spills of terrestrial and marine environments is not only limited to the indigenous microorganisms present in the contaminated area, but the addition of genetically engineered microbes. These microbes have been altered to enhance the speed of oil biodegradation and the ability to degrade fractions of oil with higher molecular weights, which are not a favourable carbon source of microbes as compared to low molecular weighted alkanes. Several genetically engineered microbes have been produced to contain multiple hydrocarbon degrading plasmids. In the late 1970's Dr. Chackrobarty was first to produce a 'superbug' for the biodegradation of hydrocarbons. With expanding knowledge of genetic engineering a multiplasmid-containing Pseudomonas strain in 2003 was produced with the capability of oxidising aliphatic, aromatic, terpenic and polyaromatic hydrocarbons. Followed by another GM bacterium, Pseudomonas putida, capable of biodegrading lower molecular weighted alkanes and aromatics. [9] Genetic engineering microbes for the use of bioremediation can be extended further than just the incorporation of multiple hydrocarbon degrading plasmids. Microbes can be modified to deal with extreme abiotic conditions such as temperature, nutrient & oxygen levels. As a result biostimulation may be all together avoidable, or to a lesser extent than currently.
However these microbes have yet to be used due regulations and public concern of genetically engineered microorganisms being used in the process of bioremediation. These microbes are not the only example; other cases of genetically engineered microbes have been developed but remain unused in the process of bioremediation. Deinococcus radiodurans is the most radio-resistant organism and was successfully genetically engineered to degrade toluene. However for the same reasons as Pseudomonas, this microbe has failed to reach commercialisation. The use of genetically engineered microbes for bioremediation will firstly have to overcome several hurdles before application is granted. The major concern currently lies with the possibility of gene transfer of toxic-degrading genes between the applied GM microbes, to neighbouring pathogenic bacteria which would be incapable of feeling on such pollutants. Alongside this public concern lies with the persistence of genetically engineered microbes after the pollutant has been removed. The containment of microbes once applied into the environment faces a challenge; however a solution to this has arisen. Once all pollutants have been degraded and microbes no longer express genes for the biodegradation, this in turn actives genes which result in the death of the microbes. However the concern underlying gene transfer to pathogenic or indigenous microbes still has yet to be resolved fully. [11]
Another oil spill remediation method is the use of petroleum remediation products, designed by NASA, which are rapidly becoming the world standard method for smaller scale oil spillages. Petroleum remediation products are able to float and thus can be applied to both land and water in stocks or a powdered form. PRP consists of natural bee's wax which is processed into microscopic hollow spheres. This encourages naturally occurring microorganisms to consume the PRP including the hydrocarbons which have been absorbed and contained within. [12] Petroleum remediation products offer a good solution to the bioremediation of oil spills without having to introduce genetically engineered microbes, but lead to the successful containment of the oil spills and the biodegradation.
Overall bioremediation proves to be an effective method of treating both terrestrial and marine oil spills. Until the hurdles of genetically engineered microbes have been overcome, for now the combination of intrinsic bioremediation alongside other techniques shows positive results under the right environmental conditions. With technologies such as PRP's developing which harness the metabolic abilities of microbes, future oil spills should see a reduction in the negative impact caused as a result, alongside a reduction in remediation cleanup time.
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