CONTENT

INTRODUCTION PRINCIPLE OF BIOREMEDIATION FECTOR OF BIOREMEDIATION MICROBIAL POPULATIONS FOR BIOREMEDIATION

PROCESSES ENVIRONMENTAL FACTOR OF BIOREMEDIATION ADVANTAGES OF BIOREMEDIATION DISADVANTAGES OF BIOREMEDIATION USE OF BIOREMEDIATION BIOREMEDIATION OF SOIL BIOREMEDIATION OF OIL POLLUTANTS BIOREMEDIATION OF AIR POLLUTANTS REFERENCE

INTRODUCTION

The quality of life on Earth is linked inextricably to the overall quality of the environment. In early times, we believed that we had an unlimited abundance of land and resources; today, however, the resources in the world show, in greater or lesser degree, our carelessness and negligence in using them. The problems associated with contaminated sites now assume increasing prominence in many countries. Contaminated lands generally result from past industrial activities when awareness of the health and environmental effects connected with the production, use, and disposal of hazardous substances were less well recognized than today. The problem is worldwide, and the estimated number of contaminated sites is significant. It is now widely recognized that contaminated land is a potential threat to human health, and its continual discovery over recent years has led to international efforts to remedy many of these sites, either as a response to the risk of adverse health or environmental effects caused by contamination or to enable the site to be redeveloped for use.The conventional techniques used for remediation have been to dig up contaminated soil and remove it to a landfill, or to cap and contain the contaminated areas of a site. The methods have some drawbacks. The first method simply moves the contamination elsewhere and may create significant risks in the excavation, handling, and transport of hazardous material. Additionally, it is very difficult and increasingly expensive to find new landfill sites for the final disposal of the material. The cap and contain method is only an interim solution since the contamination remains on site, requiring monitoring and maintenance of the isolation barriers long into the future, with all the associated costs and potential liability. A better approach than these traditional methods is to completely destroy the pollutants if possible, or at least to transform them to innocuous substances. Some technologies that have been used are high-temperature incineration and various types of chemical decomposition (e.g., base-catalyzed dechlorination, UV oxidation). They can be very effective at reducing levels of a range of contaminants, but have several drawbacks, principally their technological complexity, the cost for small-scale application, and the lack of public acceptance, especially for incineration that may increase the exposure to contaminants for both the workers at the site and nearby residents.Bioremediation is an option that offers the possibility to destroy or render harmless various contaminants using natural biological activity. As such, it uses relatively low-cost, low-technology techniques, which generally have a high public acceptance and can often be carried out on site. It will not always be suitable, however, as the range of contaminants on which it is effective is limited, the time scales involved are relatively long, and the residual contaminant levels achievable may not always be appropriate. Although the methodologies employed are not technically complex, considerable experience and expertise may be required to design and implement a successful bioremediation program, due to the need to thoroughly assess a site for suitability and to optimize conditions to achieve a satisfactory result.Because bioremediation seems to be a good alternative to conventional clean-up technologies research in this field, especially in the United States, rapidly increasing.

Bioremediation has been used at a number of sites worldwide, including Europe, with varying degrees of success. Techniques are improving as greater knowledge and experience are gained, and there is no doubt that bioremediation has great potential for dealing with certain types of site contamination. Unfortunately, the principles, techniques, advantages, and disadvantages of bioremediation are not widely known or understood, especially among those who will have to deal directly with bioremediation proposals, such as site owners and regulators. Here, we intended to assist by providing a straightforward, pragmatic view of the processes involved in bioremediation, the pros and cons of the technique, and the issues to be considered when dealing with a proposal for bioremediation.Some tests make an exhaustive examination of the literature of bioremediation of organic [2–4] and inorganic pollutants, and another test takes a look at pertinent field application case histories.

PRINCIPLES OF BIOREMEDIATION Environmental biotechnology is not a new field; composting and wastewater treatments are familiar examples of old environmental biotechnologies. However, recent studies in molecular biology and ecology offer opportunities for more efficient biological processes. Notable accomplishments of these studies include the clean-up of polluted water and land areas.Bioremediation is defined as the process whereby organic wastes are biologically degraded under controlled conditions to an innocuous state, or to levels below concentration limits established by regulatory authorities.By definition, bioremediation is the use of living organisms, primarily microorganisms, to degrade the environmental contaminants into less toxic forms. It uses naturally occurring bacteria and fungi or plants to degrade or detoxify substances hazardous to human health and/or the environment.The microorganisms may be indigenous to a contaminated area or they may be isolated from elsewhere and brought to the contaminated site. Contaminant compounds are transformed by living organisms through reactions that take place as a part of their metabolic processes. Biodegradation of a compound is often a result of the actions of multiple organisms. When microorganisms are imported to a contaminated site to enhance degradation we have a process known as bioaugmentation.For bioremediation to be effective, microorganisms must enzymatically attack the pollutants and convert them to harmless products. As bioremediation can be effective only where environmental conditions permit microbial growth and activity, its application often involves the manipulation of environmental parameters to allow microbial growth and degradation to proceed at a faster rate. Like other technologies, bioremediation has its limitations. Some contaminants, such as chlorinated organic or high aromatic hydrocarbons, are resistant to microbial attack. They are degraded either slowly or not at all , hence it is not easy to predict the rates of clean-up for a bioremediation exercise; there are no rules to predict if a contaminant can be degraded. Bioremediation techniques are typically more economical than traditional methods such as incineration, and some pollutants can be treated on site, thus reducing exposure risks for clean-up personnel, or potentially wider exposure as a result of transportation accidents. Since bioremediation is based on natural attenuation the public considers it more acceptable than other technologies.

Most bioremediation systems are run under aerobic conditions, but running a system under anaerobic conditions may permit microbial organisms to degrade otherwise recalcitrant molecules.

FACTORS OF BIOREMEDIATION

The control and optimization of bioremediation processes is a complex system of many factors. These factors include: the existence of a microbial population capable of degrading the pollutants; the availability of contaminants to the microbial population; the environment factors (type of soil, temperature, pH, the presence of oxygen or other electron acceptors, and nutrients).

MICROBIAL POPULATIONS FOR BIOREMEDIATION PROCESSES

Microorganisms can be isolated from almost any environmental conditions. Microbes will adapt and grow at subzero temperatures, as well as extreme heat, desert conditions, in water, with an excess of oxygen, and in anaerobic conditions, with the presence of hazardous compounds or on any waste stream. The main requirements are an energy source and a carbon source. Because of the adaptability of microbes and other biological systems, these can be used to degrade or remediate environmental hazards. We can subdivide these microorganisms into the following groups:Aerobic. In the presence of oxygen. Examples of aerobic bacteria recognized for their degrada-tive abilities are Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus, and Mycobacterium. These microbes have often been reported to degrade pesticides and hydrocarbons, both alkanes and polyaromatic compounds. Many of these bacteria use the contaminant as the sole source of carbon and energy.Anaerobic. In the absence of oxygen. Anaerobic bacteria are not as frequently used as aerobicbacteria. There is an increasing interest in anaerobic bacteria used for bioremediation of polychlorinated biphenyls (PCBs) in river sediments, dechlorination of the solvent trichloroethylene (TCE), and chloroform.Ligninolytic fungi. Fungi such as the white rot fungus Phanaerochaete chrysosporium have theability to degrade an extremely diverse range of persistent or toxic environmental pollutants. Common substrates used include straw, saw dust, or corn cobs.Methylotrophs. Aerobic bacteria that grow utilizing methane for carbon and energy. The initialenzyme in the pathway for aerobic degradation, methane monooxygenase, has a broad substrate range and is active against a wide range of compounds, including the chlorinated aliphatics trichloroethylene and 1,2-dichloroethane.An overview of the microbiological aspects of the application of microorganisms is given in [9].For degradation it is necessary that bacteria and the contaminants be in contact. This is not easily achieved, as neither the microbes nor contaminants are uniformly spread in the soil. Some bacteria are mobile and exhibit a chemotactic response, sensing the contaminant and moving toward it. Other microbes such as fungi grow in a filamentous form toward the

contaminant. It is possible to enhance the mobilization of the contaminant utilizing some surfactants such as sodium dodecyl sulphate (SDS).

ENVIRONMENTAL FACTORSNutrientsAlthough the microorganisms are present in contaminated soil, they cannot necessarily be there in the numbers required for bioremediation of the site. Their growth and activity must be stimulated. Biostimulation usually involves the addition of nutrients and oxygen to help indigenous microorganisms. These nutrients are the basic building blocks of life and allow microbes to create the necessary enzymes to break down the contaminants. All of them will need nitrogen, phosphorous, and carbon. Carbon is the most basic element of living forms and is needed in greater quantities than other elements. In addition to hydrogen, oxygen, and nitrogen it constitutes about 95% of the weight of cells Phosphorous and sulfur contribute with 70% of the remainders. The nutritional requirement of carbon to nitrogen ratio is 10:1, and carbon to phosphorous is 30:1 Microbial growth and activity are readily affected by pH, temperature, and moisture. Although microorganisms have been also isolated in extreme conditions, most of them grow optimally over a narrow range, so that it is important to achieve optimal conditions. If the soil has too much acid it is possible to rinse the pH by adding lime. Temperature affects biochemical reactions rates, and the rates of many of them double for each 10 °C rise in temperature. Above a certain temperature, however, the cells die. Plastic covering can be used to enhance solar warming in late spring, summer, and autumn. Available water is essential for all the living organisms, and irrigation is needed to achieve the optimal moisture level. The amount of available oxygen will determine whether the system is aerobic or anaerobic. Hydrocarbons are readily degraded under aerobic conditions, whereas chlorurate compounds are degraded only in anaerobic ones. To increase the oxygen amount in the soil it is possible to till or sparge air. In some cases, hydrogen peroxide or magnesium peroxide can be introduced in the environment. Soil structure controls the effective delivery of air, water, and nutrients. To improve soil structure, materials such as gypsum or organic matter can be applied. Low soil permeability can impede movement of water, nutrients, and oxygen; hence, soils with low permeability may not be appropriate for in situ clean-up techniques.

BIOREMEDIATION STRATEGIES

Different techniques are employed depending on the degree of saturation and aeration of an area. In situ techniques are defined as those that are applied to soil and groundwater at the site with minimal disturbance. Ex situ techniques are those that are applied to soil and groundwater at the site which has been removed from the site via excavation (soil) or pumping (water). Bioaugmentation techniques involve the addition of microorganisms with the ability to degrade pollutants.

In situ bioremediation

These technique are generally the most desirable options due to lower cost and less disturbance since they provide the treatment in place avoiding excavation and transport of contaminants. In situ treatment is limited by the depth of the soil that can be effectively treated. In many soils effective oxygen diffusion for desirable rates of bioremediation extend to a range of only a few centimeters to about 30 cm into the soil, although depths of 60 cm and greater have been effectively treated in some cases.The most important land treatments are:Bioventing is the most common in situ treatment and involves supplying air and nutrients through wells to contaminated soil to stimulate the indigenous bacteria. Bioventing employs low air flow rates and provides only the amount of oxygen necessary for the biodegradation while minimizing volatilization and release of contaminants to the atmosphere. It works for simple hydrocarbons and can be used where the contamination is deep under the surface.In situ biodegradation involves supplying oxygen and nutrients by circulating aqueous solutions through contaminated soils to stimulate naturally occurring bacteria to degrade organic contaminants. It can be used for soil and groundwater. Generally, this technique includes conditions such as the infiltration of water-containing nutrients and oxygen or other electron acceptors for groundwater treatment.Biosparging- Biosparging involves the injection of air under pressure below the water table to increase groundwater oxygen concentrations and enhance the rate of biological degradation of contaminants by naturally occurring bacteria. Biosparging increases the mixing in the saturated zone and there by increases the contact between soil and groundwater. The ease and low cost of installing small-diameter air injection points allows considerable flexibility in the design and construction of the system.Bioaugmentation. Bioremediation frequently involves the addition of microorganisms indigenous or exogenous to the contaminated sites. Two factors limit the use of added microbial cultures in a land treatment unit: 1) nonindigenous cultures rarely compete well enough with an indigenous population to develop and sustain useful population levels and 2) most soils with long-term exposure to biodegradable waste have indigenous microorganisms that are effective degrades if the land treatment unit is well managed.

Ex situ bioremediation

These techniques involve the excavation or removal of contaminated soil from ground.Landfarming is a simple technique in which contaminated soil is excavated and spread over a prepared bed and periodically tilled until pollutants are degraded. The goal is to stimulate indigenous biodegradative microorganisms and facilitate their aerobic degradation of contaminants. In general, the practice is limited to the treatment of superficial 10–35 cm of soil. Since landfarming has the potential to reduce monitoring and maintenance costs, as well as clean-up liabilities, it has received much attention as a disposal alternative.Composting is a technique that involves combining contaminated soil with nonhazardous organic amendants such as manure or agricultural wastes. The presence of these organic materials supports the development of a rich microbial population and elevated temperature characteristic of composting.Biopiles are a hybrid of landfarming and composting. Essentially, engineered cells are con-

structed as aerated composted piles. Typically used for treatment of surface contamination with petroleum hydrocarbons they are a refined version of landfarming that tend to control physical losses of the contaminants by leaching and volatilization. Biopiles provide a favorable environment for indigenous aerobic and anaerobic microorganisms.Bioreactors. Slurry reactors or aqueous reactors are used for ex situ treatment of contaminated soil and water pumped up from a contaminated plume. Bioremediation in reactors involves the processing of contaminated solid material (soil, sediment, sludge) or water through an engineered containment system. A slurry bioreactor may be defined as a containment vessel and apparatus used to create a three-phase (solid, liquid, and gas) mixing condition to increase the bioremediation rate of soil bound and water-soluble pollutants as a water slurry of the contaminated soil and biomass (usually indigenous microorganisms) capable of degrading target contaminants. In general, the rate and extent of biodegradation are greater in a bioreactor system than in situ or in solid-phase systems because the contained environment is more manageable and hence more controllable and predictable. Despite the advantages of reactor systems, there are some disadvantages. The contaminated soil requires pre treatment (e.g., excavation) or alternatively the contaminant can be stripped from the soil via soil washing or physical extraction (e.g., vacuum extraction) before being placed in a bioreactor

Advantages of bioremediation

•Bioremediation is a natural process and is therefore perceived by the public as an acceptable waste treatment process for contaminated material such as soil. Microbes able to degrade the contaminant increase in numbers when the contaminant is present; when the contaminant is degraded, the biodegradative population declines. The residues for the treatment are usually harmless products and include carbon dioxide, water, and cell biomass.•Theoretically, bioremediation is useful for the complete destruction of a wide variety of contaminants. Many compounds that are legally considered to be hazardous can be transformed to harmless products. This eliminates the chance of future liability associated with treatment and disposal of contaminated material.•Instead of transferring contaminants from one environmental medium to another, for example, from land to water or air, the complete destruction of target pollutants is possible.•Bioremediation can often be carried out on site, often without causing a major disruption of normal activities. This also eliminates the need to transport quantities of waste off site and the potential threats to human health and the environment that can arise during transportation.•Bioremediation can prove less expensive than other technologies that are used for clean-up of hazardous waste.

Disadvantages of bioremediation

•Bioremediation is limited to those compounds that are biodegradable. Not all compounds are susceptible to rapid and complete degradation.•There are some concerns that the products of biodegradation may be more persistent or toxic than the parent compound.

•Biological processes are often highly specific. Important site factors required for success include the presence of metabolically capable microbial populations, suitable environmental growth conditions, and appropriate levels of nutrients and contaminants.•It is difficult to extrapolate from bench and pilot-scale studies to full-scale field operations.•Research is needed to develop and engineer bioremediation technologies that are appropriate for sites with complex mixtures of contaminants that are not evenly dispersed in the environment.Contaminants may be present as solids, liquids, and gases.

Bioremediation often takes longer than other treatment options, such as excavation and removal of soil or incineration.•Regulatory uncertainty remains regarding acceptable performance criteria for bioremediation. There is no accepted definition of “clean”, evaluating performance of bioremediation is difficult, and there are no acceptable endpoints for bioremediation treatments.

PHYTOREMEDIATIONAlthough the application of microbe biotechnology has been successful with petroleum-based constituents, microbial digestion has met limited success for widespread residual organic and metals pollutants. Vegetation- based remediation shows potential for accumulating, immobilizing, and transforming a low level of persistent contaminants. In natural ecosystems, plants act as filters and metabolize substances generated by nature. Phytoremediation is an emerging technology that uses plants to remove contaminants from soil and water [14–16]. The term “phytoremediation” is relatively new, coined in 1991. Its potential for encouraging the biodegradation of organic contaminants requires further research, although it may be a promising area for the future.We can find five types of phytoremediation techniques, classified based on the contaminant fate:phytoextraction, phytotransformation, phytostabilization, phytodegradation, rhizofiltration, even if a combination of these can be found in nature.Phytoextraction or phytoaccumulation is the process used by the plants to accumulate contami-nants into the roots and aboveground shoots or leaves. This technique saves tremendous remediation cost by accumulating low levels of contaminants from a widespread area. Unlike the degradation mechanisms, this process produces a mass of plants and contaminants (usually metals) that can be transported for disposal or recycling.Phytotransformation or phytodegradation refers to the uptake of organic contaminants from soil, sediments, or water and, subsequently, their transformation to more stable, less toxic, or less mobile form. Metal chromium can be reduced from hexavalent to trivalent chromium, which is a less mobile and noncarcinogenic form.Phytostabilization is a technique in which plants reduce the mobility and migration of contami-nated soil. Leachable constituents are adsorbed and bound into the plant structure so that they form a stable mass of plant from which the contaminants will not reenter the environment.Phytodegradation or rhizodegradation is the breakdown of contaminants through the activity existing in the rhizosphere. This activity is due to the presence of proteins and enzymes produced by the plants or by soil organisms such as bacteria, yeast, and fungi. Rhizodegradation

is a symbiotic relationship that has evolved between plants and microbes. Plants provide nutrients necessary for the microbes to thrive, while microbes provide a healthier soil environment.

Phytoremediation is well suited for use at very large field sites where other methods of remediation are not cost effective or practicable; at sites with a low concentration of contaminants where only polish treatment is required over long periods of time; and in conjunction with other technologies where vegetation is used as a final cap and closure of the site. There are some limitations to the technology that it is necessary to consider carefully before it is selected for site remediation: long duration of time for remediation, potential contamination of the vegetation and food chain, and difficulty establishing and maintaining vegetation at some sites with high toxic levels.

Methanotrophic Bacteria: Use in Bioremediation

Microbial communities are highly diverse and capable of conducting an extensive range of metabolic activities. Irrespective of depth or geological formation, subsurface microorganisms carry out all the major nutrient cycling, i.e., carbon, sulfur, nitrogen, manganese, iron and phosphorus. Although each geological formation appears to have its own microbial structure, sandy formations that are highly permeable to air or water flow have higher microbial activity. Considering a generally large subsurface microbiota there is considerable interest for the prospect of degrading hazardous contaminants in situ by stimulating selective bacterial populations (biostimulation) or by the addition of organisms to contaminated sites (bioaugmentation). Stimulation of an indigenous population of methanotrophs by methane is likely to enrich for species that are well adapted to their environment, whereas the deliberate addition of more microorganisms into such an environment may be compromised since the introduced organisms are not as likely to be able to compete.

Defining of temporal and spatial relationships and population dynamics/interactions of selected microorganisms such as methanotrophs in the natural setting is important for the evaluation of bioremediation potential and its effectiveness. It has become increasingly evident that indigenous microbial systems are able to facilitate the degradation and mineralization of a wealth of compounds that twenty years ago were thought to be biologically recalcitrant. This realization has necessitated technologies whereby defined microbial types can be followed in situ in real time by techniques that are designed to be selective, sensitive and easily applicable to soils, sediments and groundwater.

Methanotrophs are physiologically versatile in their ability to exist in a variety of habitats and live in hostile environments having a wide range of pH, temperature, heavy metal concentrations, oxygen concentrations, barometric pressures, salinity and radiation. Under these diverse conditions a number of methanotrophs have been isolated that facilitate the degradation of TCE and its daughter products.

Evaluation, characterization, and utilization of microbial communities associated with in situ bioremediation of subsurface and groundwater contamination is a technological necessity for environmental restoration and assessment. In the past sixty years both industrial and government

nuclear production and waste management facilities have generated a significant quantity of organic wastes. These wastes have found their way into the vadose zones and groundwater resulting in unacceptable environmental impacts. The adaptability and manageability of indigenous microorganisms such as methanotrophs make them ideal for the remediation of hazardous environmental wastes under a diverse range of habitats.

Figure 1. Oxidation of methane by the methane monooxygenase enzymeand associated cometabolism of trichloroethylene.

BIOREMEDIATION OF CONTAMINATED SOILMany substances known to have toxic properties have been introduced into the environment through human activity. These substances range in degree of toxicity and danger to human health. Many of these substances either immediately or ultimately come in contact with and are sequestered by soil. Conventional methods to remove, reduce, or mitigate toxic substances introduced into soil or ground water via anthropogenic activities and processes include pump and treat systems, soil vapor extraction, incineration, and containment. Utility of each of these conventional methods of treatment of contaminated soil and/or water suffers from recognizable drawbacks and may involve some level of risk. The emerging science and technology of bioremediation offers an alternative method to detoxify contaminants. Bioremediation has been demonstrated and is being used as an effective means of mitigating:

hydrocarbons halogenated organic solvents halogenated organic compounds non-chlorinated pesticides and herbicides nitrogen compounds metals (lead, mercury, chromium) radionuclides

Bioremediation technology exploits various naturally occurring mitigation processes: natural attenuation, biostimulation, and bioaugmentation. Bioremediation which occurs without human intervention other than monitoring is often called natural attenuation. This natural attenuation relies on natural conditions and behavior of soil microorganisms that are indigenous to soil. Biostimulation also utilizes indigenous microbial populations to remediate contaminated soils. Biostimulation consists of adding nutrients and other substances to soil to catalyze natural attenuation processes. Bioaugmentation involves introduction of exogenic microorganisms (sourced from outside the soil environment) capable of detoxifying a particular contaminant, sometimes employing genetically altered microorganisms (Biobasics, 2006).During bioremediation, microbes utilize chemical contaminants in the soil as an energy source and, through oxidation-reduction reactions, metabolize the target contaminant into useable energy for microbes. By-products (metabolites) released back into the environment are typically in a less toxic form than the parent contaminants. For example, petroleum hydrocarbons can be degraded by microorganisms in the presence of oxygen through aerobic respiration. The hydrocarbon loses electrons and is oxidized while oxygen gains electrons and is reduced. The result is formation of carbon dioxide and water (Nester et al., 2001). When oxygen is limited in supply or absent, as in saturated or anaerobic soils or lake sediment, anaerobic (without oxygen) respiration prevails. Generally, inorganic compounds such as nitrate, sulfate, ferric iron, manganese, or carbon dioxide serve as terminal electron acceptors to facilitate biodegradation (State of Mississippi, Department of Environmental Quality, 1998). Three primary ingredients for bioremediation are: 1) presence of a contaminant, 2) an electron acceptor, and 3) presence of microorganisms that are capable of degrading the specific contaminant. Generally, a contaminant is more easily and quickly degraded if it is a naturally occurring compound in the environment, or chemically similar to a naturally occurring compound, because microorganisms capable of its biodegradation are more likely to have

evolved (State of Mississippi, Department of Environmental Quality, 1998). Petroleum hydrocarbons are naturally occurring chemicals; therefore, microorganisms which are capable of attenuating or degrading hydrocarbons exist in the environment. Development of biodegradation technologies of synthetic chemicals such DDT is dependent on outcomes of research that searches for natural or genetically improved strains of microorganisms to degrade such contaminants into less toxic forms.Microorganisms have limits of tolerance for particular environmental conditions, as well as optimal conditions for pinnacle performance. Factors that affect success and rate of microbial biodegradation are nutrient availability, moisture content, pH, and temperature of the soil matrix. Inorganic nutrients including, but not limited to, nitrogen, and phosphorus are necessary for microbial activity and cell growth. It has been shown that “treating petroleum-contaminated soil with nitrogen can increase cell growth rate, decrease the microbial lag phase, help to maintain microbial populations at high activity levels, and increase the rate of hydrocarbon degradation” (Walworth et al., 2005). However, it has also been shown that excessive amounts of nitrogen in soil cause microbial inhibition. Walworth et al. (2005) suggest maintaining nitrogen levels below 1800 mg nitrogen/kg H2O for optimal biodegradation of petroleum hydrocarbons. Addition of phosphorus has benefits similar to that of nitrogen, but also results in similar limitations when applied in excess (State of Mississippi, Department of Environmental Quality, 1998). All soil microorganisms require moisture for cell growth and function. Availability of water affects diffusion of water and soluble nutrients into and out of microorganism cells. However, excess moisture, such as in saturated soil, is undesirable because it reduces the amount of available oxygen for aerobic respiration. Anaerobic respiration, which produces less energy for microorganisms (than aerobic respiration) and slows the rate of biodegradation, becomes the predominant process. Soil moisture content “between 45 and 85 percent of the water-holding capacity (field capacity) of the soil or about 12 percent to 30 percent by weight” is optimal for petroleum hydrocarbon degradation (US EPA, 2006, “Landfarming”). Soil pH is important because most microbial species can survive only within a certain pH range. Furthermore, soil pH can affect availability of nutrients. Biodegradation of petroleum hydrocarbons is optimal at a pH 7 (neutral); the acceptable range is pH 6 – 8 (US EPA, 2006, “Landfarming”; State of Mississippi, Department of Environmental Quality, 1998).Temperature influences rate of biodegradation by controlling rate of enzymatic reactions within microorganisms. Generally, “speed of enzymatic reactions in the cell approximately doubles for each 10 oC rise in temperature” (Nester et al., 2001). There is an upper limit to the temperature that microorganisms can withstand. Most bacteria found in soil, including many bacteria that degrade petroleum hydrocarbons, are mesophiles which have an optimum temperature ranging from 25 degree C to 45 degree C (Nester et al., 2001). Thermophilic bacteria (those which survive and thrive at relatively high temperatures) which are normally found in hot springs and compost heaps exist indigenously in cool soil environments and can be activated to degrade hydrocarbons with an increase in temperature to 60 degree C. This finding “suggested an intrinsic potential for natural attenuation in cool soils through thermally enhanced bioremediation techniques” (Perfumo et al., 2007).Contaminants can adsorb to soil particles, rendering some contaminants unavailable to microorganisms for biodegradation. Thus, in some circumstances, bioavailability of

contaminants depends not only on the nature of the contaminant but also on soil type. Hydrophobic contaminants, like petroleum hydrocarbons, have low solubility in water and tend to adsorb strongly in soil with high organic matter content. In such cases, surfactants are utilized as part of the bioremediation process to increase solubility and mobility of these contaminants (State of Mississippi, Department of Environmental Quality, 1998). Additional research findings of the existence of thermophilic bacteria in cool soil also suggest that high temperatures enhance the rate of biodegradation by increasing the bioavailability of contaminants. It is suggested that contaminants adsorbed to soil particles are mobilized and their solubility increased by high temperatures (Perfumo et al., 2007).Soil type is an important consideration when determining the best suited bioremediation approach to a particular situation. In situ bioremediation refers to treatment of soil in place. In situ biostimulation treatments usually involve bioventing, in which oxygen and/or nutrients are pumped through injection wells into the soil. It is imperative that oxygen and nutrients are distributed evenly throughout the contaminated soil. Soil texture directly affects the utility of bioventing, in as much as permeability of soil to air and water is a function of soil texture. Fine-textured soils like clays have low permeability, which prevents biovented oxygen and nutrients from dispersing throughout the soil. It is also difficult to control moisture content in fine textured soils because their smaller pores and high surface area allow it to retain water. Fine textured soils are slow to drain from water-saturated soil conditions, thus preventing oxygen from reaching soil microbes throughout the contaminated area (US EPA, 2006, “Bioventing”). Bioventing is well-suited for well-drained, medium, and coarse-textured soils.In situ bioremediation causes minimal disturbance to the environment at the contamination site. In addition, it incurs less cost than conventional soil remediation or removal and replacement treatments because there is no transport of contaminated materials for off-site treatment. However, in situ bioremediation has some limitations: 1) it is not suitable for all soils, 2) complete degradation is difficult to achieve, and 3) natural conditions (i.e. temperature) are hard to control for optimal biodegradation. Ex situ bioremediation, in which contaminated soil is excavated and treated elsewhere, is an alternative. Ex situ bioremediation approaches include use of bioreactors, landfarming, and biopiles. In the use of a bioreactor, contaminated soil is mixed with water and nutrients and the mixture is agitated by a mechanical bioreactor to stimulate action of microorganisms. This method is better-suited to clay soils than other methods and is generally a quick process (US EPA, 2006, “Guide”). Landfarming involves spreading contaminated soil over a collection system and stimulating microbial activity by allowing good aeration and by monitoring nutrient availability (US EPA, 2006, “Landfarming”). Biopiles are mounds of contaminated soils that are kept aerated by pumping air into piles of soil through an injection system (US EPA, 2006, “Biopiles”). In each of these methods, conditions need to be monitored and adjusted regularly for optimal biodegradation. Use of landfarming and biopiles also present the issue of monitoring and containing volatilization of contaminants. Like in situ methods, ex situ bioremediation

techniques generally cost less than conventional techniques and apply natural methods. However, they can require a large amount of land and, similar to in situ bioremediation, complete degradation is difficult to achieve, and evaporation of volatile components is a concern (US EPA, 2006, “Landfarming”; US EPA, 2006, “Biopiles”). If the challenges of bioremediation, particularly of in situ techniques, can be overcome, bioremediation has potential to provide a low cost, non-intrusive, natural method to render toxic substances in soil less harmful or harmless over time. Currently, research is being conducted to improve and overcome limitations that hinder bioremediation of petroleum hydrocarbons. On a broader scope, much research has been and continues to be developed enhance understanding of the essence of microbial behavior as microbes interact with various toxic contaminants. Additional research continues to evaluate conditions for successful introduction of exogenic and genetically engineered microbes into a contaminated environment, and how to translate success in the laboratory to success in the field (US DOE, 2006).

BIOREMEDIATION OF OIL POLLUTANTSAn oil spill is a release of a liquid petroleum hydrocarbon into the environment due to human activity, and is a form of pollution. The term often refers to marine oil spills, where oil is released into the ocean or coastal waters. Oil spills include releases of crude oil from tankers, offshore platforms, drilling rigs and wells, as well as spills of refined petroleum products (such as gasoline, diesel) and their by-products, and heavier fuels used by large ships such as bunker fuel, or the spill of any oily refuse or waste oil. Spills may take months or even years to clean up.Oil also enters the marine environment from natural oil seeps. Public attention and regulation has tended to focus most sharply on seagoing oil tankers.The oil penetrates into the structure of the plumage of birds, reducing its insulating ability, thus making the birds more vulnerable to temperature fluctuations and much less buoyant in the water. It also impairs birds' flight abilities to forage and escape from predators. As they attempt to preen, birds typically ingest oil that covers their feathers, causing kidney damage, altered liver function, and digestive tract irritation. This and the limited foraging ability quickly causes dehydration and metabolic imbalances. Hormonal balance alteration including changes in luteinizing protein can also result in some birds exposed to petroleum.Most birds affected by an oil spill die unless there is human intervention. Marine mammals exposed to oil spills are affected in similar ways as seabirds. Oil coats the fur of Sea otters and seals, reducing its insulation abilities and leading to body temperature fluctuations and hypothermia. Ingestion of the oil causes dehydration and impaired digestions. Because oil floats on top of water, less sunlight penetrates into the water, limiting the photosynthesis of marine plants and phytoplankton. This, as well as decreasing the fauna populations, affects the food chain in the ecosystem.There are three kinds of oil-consuming bacteria. Sulfate-reducing bacteria (SRB) and acid-producing bacteria are anaerobic, while general aerobic bacteria (GAB) are aerobic. These bacteria occur naturally and will act to remove oil from an ecosystem, and their biomass will tend to replace other populations in the food chain.

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