Bioremediation of Petroleum and Petroleum Products

Preface

Biodegradation is a natural process, and with enough time, microorganisms can eliminate many components of petroleum oil from the environment. The concern is whether or not bioremediation technologies can accelerate this natural process enough to be considered practical, and, if so, whether they might find a niche as replacements for, or adjuncts to, other petroleum spill response technologies.

Petroleum (crude oil) is a complex mixture of thousands of different chemical compounds. In addition, the composition of each accumulation of oil is unique, varying in different producing regions, and even in different unconnected zones of the same formation. The composition of petroleum also varies with the amount of refining. Significantly, the many constituents of petroleum differ markedly in volatility, solubility, and susceptibility to biodegradation: some constituents are susceptible to microbial biodegradation, while others are non-biodegradable. Furthermore, the biodegradation of different petroleum constituents occurs simultaneously, but at very different rates. This leads to the sequential disappearance of individual components of petroleum over time and, because different species of microbes preferentially attack different compounds, to successional changes in the degrading microbial community. Thus, to evaluate the effectiveness of biodegradation through the application of bioremediation technologies, it is necessary to know the molecular effects of the process, starting with the molecular composition of the contaminants.

This book introduces the reader to the science and technology of biodegradation, a key process in the bioremediation of petroleum and petroleum based contaminants at spill sites. The contaminants of concern in the molecularly-variable petroleum and petroleum products can be degraded under appropriate conditions. But the success of the process depends on the ability to determine the necessary conditions and establish them in the contaminated environment.

Although the prime focus of the book is to determine the mechanism, extent, and efficiency of biodegradation, it is necessary to know the composition of the original petroleum or petroleum product. The laws of science dictate what can or cannot be done with petroleum and petroleum products to insure that biodegradation (hence, bioremediation) processes are effective. The science of the composition of petroleum and petroleum products is at the core of understanding the chemistry of biodegradation and bioremediation processes. Hence, inclusion of petroleum analyses and properties, along with petroleum product analyses and properties, is a necessary part of this text.

As a result, the book is divided into chapters that guide the reader through the composition of petroleum and petroleum products, as well as processes involved in the biodegradation/bioremediation of petroleum: Chapter 1: Introduction to Bioremediation; Chapter 2: Petroleum Composition and Properties; Chapter 3: Refinery Products and By-Products; Chapter 4: Composition and Properties of Gaseous Fractions; Chapter 5: Composition and Properties of Liquid Fractions; Chapter 6: Composition and Properties of Solid Fractions; Chapter 7: Sample Collection and Preparation; Chapter 8: Analytical Methods; Chapter 9: Biodegradation of Petroleum; Chapter 10: Biodegradation of Naphtha and Gasoline; Chapter 11: Biodegradation of Kerosene and Diesel; Chapter 12: Biodegradation of Fuel Oil; Chapter 13: Biodegradation of Lubricating Oil; Chapter 14: Biodegradation of Residua and Asphalt; Chapter 15: Bioremediation Methods for Oil Spills; and Chapter 16: The Future of Bioremediation.

Each chapter includes a copious reference section, and the book is further improved by the inclusion of an extensive Glossary.

James G. Speight PhD, DSc

Laramie, Wyoming, USA

Karuna K. Arjoon, MPhil

California, Trinidad and Tobago

Chapter 1

Introduction to Bioremediation

1 Introduction

One of the major and continuing environmental problems is hydrocarbon contamination resulting from activities related to petroleum and petroleum products. Soil contamination with hydrocarbons causes extensive damage of local systems, since accumulation of pollutants in animals and plant tissue may cause death or mutations.

However, not all petroleum products are harmful to health and the environment. There are records of the use of petroleum spirit for medicinal purposes. This was probably a higher boiling fraction than naphtha or a low boiling fraction of gas oil that closely resembled the modern-day liquid paraffin, for medicinal purposes. In fact, the so-called liquid paraffin has continued to be prescribed up to modern times, as a means for miners to take in prescribed doses to lubricate the alimentary tract and assist coal dust, taken in during the working hours, in passing though the body.

There are, however, those constituents of petroleum that are extremely harmful to health and the environment. Indeed, petroleum constituents, either in the pure form or as the components of a fraction, have been known to belong to the various families of carcinogens and neurotoxins. Whatever the name given to these compounds, they are extremely toxic.

As a result, once a spill has occurred, every effort must be made to rid the environment of the toxins. The chemicals of known toxicity range in degree of toxicity from low to high, and represent considerable danger to human health, and must be removed (Frenzel et al., 2009). Many of these chemicals substances come in contact with, and are sequestered by, soil or water systems. While conventional methods to remove, reduce, or mitigate the effects of toxic chemical in nature are available, including (1) pump and treat systems, (2) soil vapor extraction, (3) incineration, and (4) containment, each of these conventional methods of treatment of contaminated soil and/or water suffers from recognizable drawbacks, and may involve some level of risk. In short, these methods, depending upon the chemical constituents of the spilled material, may have limited effectiveness and can be expensive (Speight, 1996; Speight and Lee, 2000; Speight, 2005).

Although the effects of bacteria (microbes) on hydrocarbons have been known for decades, this technology (now known as bioremediation) has shown promise and, in some cases, high degrees of effectiveness for the treatment of these contaminated sites, since it is cost-effective and will lead to complete mineralization. Bioremediation functions basically on biodegradation, which may refer to complete mineralization of the organic contaminants into carbon dioxide, water, inorganic compounds, and cell protein, or transformation of complex organic contaminants to other simpler organic compounds that are not detrimental to the environment. In fact, unless they are overwhelmed by the amount of the spilled material or the material is toxic, many indigenous microorganisms in soil and/or water are capable of degrading hydrocarbon contaminants.

The United States Environmental Protection Agency (US EPA) uses bioremediation because it takes advantage of natural processes and relies on microbes that occur naturally or can be laboratory cultivated; these consist of bacteria, fungi, actinomycetes, cyanobacteria, and, to a lesser extent, plants (US EPA, 2006). These microorganisms either consume and convert the contaminants, or assimilate within them all harmful compounds from the surrounding area, thereby rendering the region virtually contaminant-free. Generally, the substances that are consumed as an energy source are organic compounds, while those that are assimilated within the organism are heavy metals. Bioremediation harnesses this natural process by promoting the growth and/or rapid multiplication of these organisms that can effectively degrade specific contaminants and convert them to nontoxic by-products.

The capabilities of micro-organisms and plants to degrade and transform contaminants provide benefits in the cleanup of pollutants from spills and storage sites. These remediation ideas have provided the foundation for many ex situ waste treatment processes (including sewage treatment), and a host of in situ bioremediation methods that are currently in practice.

Thus, bioremediation - the use of living organisms to reduce or eliminate environmental hazards resulting from accumulations of toxic chemicals and other hazardous wastes - is an option that offers the possibility to destroy or render harmless various contaminants using natural biological activity (Gibson and Sayler, 1992). In addition, bioremediation can also be used in conjunction with a wide range of traditional physical and chemical technology to enhance their effectiveness (Vidali, 2001).

In the current context, bioremediation of petroleum and petroleum fractions (or products) is the cleanup of petroleum spills or petroleum product spills by the use of microbes to breakdown the petroleum constituents (or other organic contaminants) into less harmful (usually lower molecular weight) and easier-to-remove products (biodegradation). The microbes transform the contaminants through metabolic or enzymatic processes, which vary greatly, but the final product is usually harmless and includes carbon dioxide, water, and cell biomass. Thus, the emerging science and technology of bioremediation offers an alternative method to detoxify petroleum-related soil and water contaminants.

Briefly and by means of clarification, biodegradation (biotic degradation, biotic decomposition) is the chemical degradation of contaminants by bacteria or other biological means. Organic material can be degraded aerobically (in the presence of oxygen) or anaerobically (in the absence of oxygen). Most bioremediation systems operate under aerobic conditions, but a system under anaerobic conditions may permit microbial organisms to degrade chemical species that are otherwise non-responsive to aerobic treatment, and vice versa.

Thus, biodegradation is a natural process (or a series of processes) by which spilled petroleum hydrocarbons, or other organic waste material, are broken down (degraded) into nutrients that can be used by other organisms. As a result, the ability of a chemical to be biodegraded is an indispensable element in understanding the risk posed by that chemical on the environment.

Biodegradation is a key process in the natural attenuation (reduction or disposal) of chemical compounds at hazardous waste sites. The contaminants of concern in crude oil are able to degrade under appropriate conditions, but the success of the process depends on the ability to determine these conditions and establish them in the contaminated environment. Thus, important site factors required for success include (1) the presence of metabolically capable and sustainable microbial populations, (2) suitable environmental growth conditions, such as the presence of oxygen, (3) temperature, which is an important variable: keeping a substance frozen or below the optimal operating temperature for microbial species can prevent biodegradation, and most biodegradation occurs at temperatures between 10 and 35°C (50 and 95°F), (4) the presence of water, (5) appropriate levels of nutrients and contaminants, and (6) favorable acidity or alkalinity (Table 1.1).

Table 1.1 Essential factors for microbial bioremediation.

In regard to the last parameter, soil pH is extremely important because most microbial species can survive only within a certain pH range: generally the biodegradation of petroleum hydrocarbons is optimal at a pH 7 (neutral), and the acceptable (or optimal) pH range is on the order of 6 to 8. Furthermore, soil (or water) pH can affect availability of nutrients.

Thus, through biodegradation processes, living microorganisms (primarily bacteria, but also yeasts, molds, and filamentous fungi) can alter and/or metabolize various classes of compounds present in petroleum. Furthermore, biodegradation also alters subsurface oil accumulations of petroleum (Winters and Williams, 1969; Speight, 2007). Shallow oil accumulations, such as heavy oil reservoirs and tar sand deposits, where the reservoir temperature is low-to-moderate (<80°C, <176°F) are commonly found to have undergone some degree of biodegradation (Winters and Williams, 1969; Speight, 2007).

Temperature influences the rate of biodegradation by controlling the rate of enzymatic reactions within microorganisms. Generally, the rate of an enzymatic reaction approximately doubles for each 10°C (18°F) rise in temperature (Nester et al., 2001). However, 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 mesophile organisms, which have an optimum working temperature range on the order of 25 to 45°C (77 to 113°F) (Nester et al., 2001). Thermophilic bacteria (those that survive and thrive at relatively high temperatures) that 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°C (140°F). This indicates the potential for natural attenuation in cool soils through thermally enhanced bioremediation techniques (Perfumo et al., 2007).

In order to enhance and make favorable the parameters presented above to ensure microbial activity, there are two other bioremediation technologies that offer useful options for cleanup of spills of petroleum and petroleum products: (1) fertilization and (2) seeding.

Fertilization (nutrient enrichment) is the method of adding nutrients, such as phosphorus and nitrogen, to a contaminated environment to stimulate the growth of the microorganisms capable of biodegradation. Limited supplies of these nutrients in nature usually control the growth of native microorganism populations. When more nutrients are added, the native microorganism population can grow rapidly, potentially increasing the rate of biodegradation.

Seeding is the addition of microorganisms to the existing native oil-degrading population. Some species of bacteria that do not naturally exist in an area will be added to the native population. As with fertilization, the purpose of seeding is to increase the population of microorganisms that can biodegrade the spilled oil.

Thus, biodegradation is an environmentally acceptable, naturally occurring process that takes place when all of the nutrients and physical conditions involved are suitable for growth. The process allows for the breakdown of a compound to either fully oxidized or reduced simple molecules, such as carbon dioxide/methane, nitrate/ammonium, and water. However, in some cases, where the process is not complete, the products of biodegradation can be more harmful than the substance degraded.

Intrinsic bioremediation is the combined effect of natural destructive and non-destructive processes to reduce the mobility, mass, and associated risk of a contaminant. Non-destructive mechanisms include sorption, dilution, and volatilization. Destructive processes are aerobic and anaerobic biodegradation.

Intrinsic aerobic biodegradation is well-documented as a means of remediating soil and groundwater contaminated with fuel hydrocarbons. In fact, intrinsic aerobic degradation should be considered an integral part of the remediation process (McAllister et al., 1995; Barker et al., 1995). There is growing evidence that natural processes influence the immobilization and biodegradation of chemicals such as aromatic hydrocarbons, mixed hydrocarbons, and chlorinated organic compounds (Ginn et al., 1995; King et al., 1995).

Phytoremediation is the use of living green plants for the removal of contaminants and metals from soil, and is essentially an in situ treatment of pollutant contaminated soils, sediments, and water: terrestrial, aquatic and wetland plants, and algae can be used for the phytoremediation process under specific cases and conditions of hydrocarbon contamination (Brown, 1995; Nedunuri et al., 2000; Radwan et al., 2000; Siciliano et al., 2000; Magdalene et al., 2009). It is best applied at sites with relatively shallow contamination of pollutants that are amenable to the various subcategories of phytoremediation: (1) phytotransformation, the breakdown of organic contaminants sequestered by plants, (2) rhizosphere bioremediation, the use of rhizosphere microorganisms to degrade organic pollutants, (3) phytostabilization, a containment process using plants, often in combination with soil additives, to assist plant installation, mechanically stabilize the site, and reduce pollutant transfer to other ecosystem compartments and the food chain, (4) phytoextraction, the ability of some plants to accumulate metals/metalloids in their shoots, (5) rhizofiltration, and/or (6) phytovolatilization/rhizovolatilization, processes employing metabolic capabilities of plants and associated rhizosphere microorganisms to transform pollutants into volatile compounds that are released to the atmosphere (Korade and Fulekar, 2009).

These technologies are especially valuable where the contaminated soils are fragile, and prone to erosion. The establishment of a stable vegetation community stabilizes the soil system, and prevents erosion. This aspect is especially relevant to certain types of soil where removal of large volumes of soil destabilizes the soil system, which leads to extensive erosion. However, when the above parameters are not conducive to bacterial activity, the bacteria (1) grow too slowly, (2) die, or (3) create more harmful chemicals.

Phytotransformation and rhizosphere bioremediation are applicable to sites that have been contaminated with organic pollutants, including pesticides. It is a technology that should be considered for remediation of contaminated sites because of its cost effectiveness, aesthetic advantages, and long-term applicability (Brown, 1995).

Plants have shown the capacity to withstand relatively high concentrations of organic chemicals without toxic effects, and they can uptake and convert chemicals quickly to less toxic metabolites in some cases. In addition, they stimulate the degradation of organic chemicals in the rhizosphere by the release of root exudates, enzymes, and the buildup of organic carbon in the soil.

Micro-organisms degrade or transform contaminants by a variety of mechanisms. Petroleum hydrocarbons (particularly alkanes), for example, are converted to carbon dioxide and water:

Or the hydrocarbon may be used as a primary food source by the bacteria, which use the energy to generate new cells.

Some contaminants, such as chlorinated organic or high aromatic hydrocarbons, are generally resistant to microbial attack. They are degraded either slowly or not at all, and hence, it is not easy to predict the rates of cleanup for a bioremediation exercise; there are no rules to predict if a contaminant can be degraded.

When the hydrocarbons are chlorinated (as might occur in several additives to improve the performance of petroleum products) the degradation takes place as a secondary or co-metabolic process, rather than a primary metabolic process. In such a case, enzymes, which are produced during aerobic utilization of carbon sources such as methane, degrade the chlorinated compounds. Under aerobic conditions, a chlorinated solvent, such as trichloroethylene (ClCH=CCl2), which may have been mixed with the petroleum product during processing or during use, can be degraded through a sequence of metabolic steps, where some of the intermediary by-products may be more hazardous than the parent compound (e.g., vinyl chloride, CH2=CHCl).

Since many of the contaminants of concern in petroleum and petroleum-related products oil are readily biodegradable under the appropriate conditions, the success of oil-spill bioremediation depends mainly on the ability to establish these conditions in the contaminated environment, using the technology to optimize the total efficiency of the microorganisms.

Over the past two decades, opportunities for applying bioremediation to a much broader set of contaminants have been identified. Indigenous and enhanced organisms have been shown to degrade industrial solvents, polychlorinated biphenyls (PCBs), explosives, and many different agricultural chemicals. Pilot, demonstration, and full-scale applications of bioremediation have been carried out on a limited basis. However, the full benefits of bioremediation have not been realized, because processes and organisms that are effective in controlled laboratory tests are not always equally effective in full-scale applications. The failure to perform optimally in the field setting stems from a lack of predictability, due in part to inadequacies in the fundamental scientific understanding of how and why these processes work.

2 Principles of Bioremediation

As already stated, bioremediation is an environmentally friendly technique used to restore soil and water to its original state by using indigenous microbes to break down and eliminate contaminants. Biological technologies are often used as a substitute to chemical or physical cleanup of oil spills, because bioremediation does not require as much equipment or labor as other methods: therefore, it is usually cheaper. It also allows cleanup workers to avoid contact with polluted soil and water.

The microorganisms used for bioremediation may be indigenous to a contaminated area, or they may be isolated from elsewhere and brought to the contaminated site. Contaminants 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, it is a process known as bioaugmentation.

For bioremediation to be effective, microorganisms must convert the pollutants 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. However, as is the case with other technologies, bioremediation has its limitations, and there are several disadvantages that must be recognized (Table 1.2).

Table 1.2 Advantages and disadvantages of bioremediation.

The control and optimization of bioremediation processes are 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; and the environment factors (type of soil, temperature, pH, the presence of oxygen or other electron acceptors, and nutrients).

One of the important factors in biological removal of hydrocarbons from a contaminated environment is their bioavailability to an active microbial population, which is the degree of interaction of chemicals with living organisms or the degree to which a contaminant can be readily taken up and metabolized by a bacterium (Harms et al., 2010). Moreover, the bioavailability of a contaminant is controlled by factors such as the physical state of the hydrocarbon in situ, its hydrophobicity, water solubility, sorption to environmental matrices such as soil, and diffusion out of the soil matrix. When contaminants have very low solubility in water, as in the case of n-alkanes and polynuclear aromatic hydrocarbons, the organic phase components will not partition efficiently into the aqueous phase supporting the microbes.

In the case of soil, the contaminants will also partition to the soil organic matter, and become even less bioavailable. Two-phase bioreactors containing an aqueous phase and a non-aqueous phase liquid (NAPL) have been developed and used for bioremediation of hydrocarbon-contaminated soil to address this very problem, but the adherence of microbes to the NAPL-water interface can still be an important factor in reaction kinetics. Similarly, two-phase bioreactors, sometimes with silicone oil as the non-aqueous phase, have been proposed for biocatalytic conversion of hydrocarbons like styrene (Osswald et al., 1996) to make the substrate more bioavailable to microbes in the aqueous phase. When the carbon source is in limited supply, its availability will control the rate of metabolism, and hence, biodegradation, rather than catabolic capacity of the cells or availability of oxygen or other nutrients.

In the case of the biomediation of waterways, similar principles apply. Under enhanced conditions, (1) certain fuel hydrocarbons can be removed preferentially over others, but the order of preference is dependent upon the geochemical conditions, and (2) augmentation and enhancement via electron acceptors accelerate the biodegradation process. For example, with regard to the aromatic benzene-toluene-ethylbenzene-xylenes (BTEX): (1) toluene can be preferentially removed under intrinsic bioremediation conditions, (2) biodegradation of benzene is relatively slow, (3) augmentation with sulfate can preferentially stimulate biodegradation of o-xylene, and (4) ethylbenzene may be recalcitrant under sulfate-reducing conditions, but readily degradable under denitrifying conditions (Cunningham et al., 2000).

3 Bioremediation and Biodegradation

In the current context, bioremediation (biodegradation) is a method for dealing with contamination by petroleum, petroleum products, and petroleum waste streams. The process typically occurs through the degradation of petroleum or a petroleum product through the action of microorganisms (biodegradation).

The method utilizes indigenous bacteria (microbes), compared to the customary (physical and chemical) remediation methods. Also, the microorganisms engaged are capable of performing almost any detoxification reaction. Biodegradation studies provide information on the fate of a chemical or mixture of petroleum-derived chemicals (such as oil spills and process wastes) in the environment, thereby opening the scientific doorway to develop further methods of cleanup by (1) analyzing the contaminated sites, (2) determining the best method suited for the environment, and (3) optimizing the cleanup techniques which lead to the emergence of new processes.

3.1 Natural Bioremediation

Natural biodegradation/bioremediation typically involves the use of molecular oxygen (O2), where oxygen (the terminal electron acceptor) receives electrons transferred from an organic contaminant:

In the absence of oxygen, some microorganisms obtain energy from fermentation and anaerobic oxidation of organic carbon. Many anaerobic organisms (anaerobes) use nitrate, sulfate, and salts of iron (III) as practical alternates to oxygen acceptor, as, for example, in the anaerobic reduction process of nitrates, sulfates, and salts of iron (III):

3.2 Traditional Bioremediation Methods

Methods for the cleanup of pollutants have usually involved removal of the polluted materials and their subsequent disposal by land filling or incineration (so-called dig, haul, bury, or burn methods) (Speight, 1996; Speight and Lee, 2000; Speight, 2005). Furthermore, available space for landfills and incinerators is declining. Perhaps one of the greatest limitations to traditional cleanup methods is the fact that, in spite of their high costs, they do not always ensure that contaminants are completely destroyed.

Conventional bioremediation methods that have been, and are still, used are (1) composting, (2) land farming, (3) biopiling, and (4) use of a bioslurry reactor (Speight, 1996; Speight and Lee, 2000; Semple et al., 2001).

Composting is a technique that involves combining contaminated soil with nonhazardous organic materials, such as manure or agricultural wastes; the presence of the organic materials allows the development of a rich microbial population and elevated temperature characteristic of composting. Land farming is a simple technique, in which contaminated soil is excavated and spread over a prepared bed and periodically tilled until pollutants are degraded. Biopiling is a hybrid of land farming and composting: it is essentially engineered cells that are constructed as aerated composted piles. A bioslurry reactor can provide rapid biodegradation of contaminants, due to enhanced mass transfer rates and increased contaminant-to-microorganism contact. These units are capable of aerobically biodegrading aqueous slurries created through the mixing of soils or sludge with water. The most common state of bioslurry treatment is batch; however, continuous-flow operation is also possible.

The technology selected for a particular site will depend on the limiting factors present at the location. For example, where there is insufficient dissolved oxygen, bioventing or sparging is applied, and biostimulation or bioaugmentation are suitable for instances where the biological count are low. On the other hand, application of the composting technique, if the operation is unsuccessful, will result in a greater quantity of contaminated materials. Land farming is only effective if the contamination is near the soil surface, or else bed preparation is required. The main drawback with slurry bioreactors is that high-energy input is required to maintain suspension and the potential needed for volatilization.

Other techniques are also being developed to improve the microbe-contaminant interactions at treatment sites, so as to use bioremediation technologies at their fullest potential. These bioremediation technologies consist of monitored natural attenuation, bioaugmentation, biosimulation, surfactant addition, anaerobic bioventing, sequential anaerobic/aerobic treatment, soil vapor extraction, air sparging, enhanced anaerobic dechlorination, and bioengineering (Speight, 1996; Speight and Lee, 2000).

The use of traditional methods of bioremediation continues, but there is also method evolution, which may involve the following steps:

1. Isolating and characterizing naturally-occurring microorganisms with bioremediation potential.

2. Laboratory cultivation to develop viable populations.

3. Studying the catabolic activity of these microorganisms in contaminated material through bench scale experiments.

4. Monitoring and measuring the progress of bioremediation through chemical analysis and toxicity testing in chemically-contaminated media.

5. Field applications of bioremediation techniques using either/both steps: (1) in-situ stimulation of microbial activity by the addition of microorganisms and nutrients and the optimization of environmental factors at the contaminated site itself, and/or (2) ex-situ restoration of contaminated material in specifically designated areas by land-farming and composting method.

3.3 Enhanced Bioremediation Treatment

Enhanced bioremediation is a process in which indigenous or inoculated microorganisms (e.g., fungi, bacteria, and other microbes) degrade (metabolize) organic contaminants found in soil and/or ground water, and convert the contaminants to innocuous end products. The process relies on general availability of naturally occurring microbes to consume contaminants as a food source (petroleum hydrocarbons in aerobic processes) or as an electron acceptor (chlorinated solvents, which may be waste materials from petroleum processing). In addition to microbes being present, in order to be successful, these processes require nutrients, such as carbon, nitrogen, and phosphorus.

Enhanced bioremediation involves the addition of microorganisms (e.g., fungi, bacteria, and other microbes) or nutrients (e.g. oxygen, nitrates) to the subsurface environment to accelerate the natural biodegradation process.

3.4 Biostimulation and Bioaugmentation

Biostimulation is the method of adding nutrients, such as phosphorus and nitrogen, to a contaminated environment to stimulate the growth of the microorganisms that break down oil. Additives are usually added to the subsurface through injection wells, although injection well technology for biostimulation purposes is still emerging. Limited supplies of these necessary nutrients usually control the growth of native microorganism populations. Thus, addition of nutrients causes rapid growth of the indigenous microorganism population, thereby increasing the rate of biodegradation.

It is to be anticipated that the success of biostimulation is case-specific and site-specific, depending on oil properties, the nature of the nutrient products, and the characteristics of the contaminated environments. When oxygen is not a limiting factor, one of keys for the success of oil biostimulation is to maintain an optimal nutrient level in the interstitial pore water. Several types of commercial biostimulation agents are available for use in bioremediation (Zhu et al., 2004).

Bioaugmentation is the addition of pre-grown microbial cultures to enhance microbial populations at a site to improve contaminant clean up and reduce clean up time and cost. Indigenous or native microbes are usually present in very small quantities and may not be able to prevent the spread of the contaminant. In some cases, native microbes do not have the ability to degrade a particular contaminant. Therefore, bioaugmentation offers a way to provide specific microbes in sufficient numbers to complete the biodegradation (Atlas, 1991).

Mixed cultures have been most commonly used as inocula for seeding because of the relative ease with which microorganisms with different and complementary biodegradative capabilities can be isolated (Atlas, 1977). Different commercial cultures were reported to degrade petroleum hydrocarbons (Compeau et al., 1991; Leavitt and Brown, 1994; Chhatre et al., 1996; Mangan, 1990; Mishra et al., 2001; Vasudevan and Rajaram, 2001).

Microbial inocula (the microbial materials used in an inoculation) are prepared in the laboratory from soil or groundwater, either from the site where they are to be used, or from another site where the biodegradation of the chemicals of interest is known to be occurring. Microbes from the soil or groundwater are isolated, and are added to media containing the chemicals to be degraded. Only microbes capable of metabolizing the chemicals will grow on the media. This process isolates the microbial population of interest. One of the main environmental applications for bioaugmentation is at sites with chlorinated solvents. Microbes called Dehalococcoides nethenogenes usually perform reductive dechlorination of solvents such as perchloroethylene and trichloroethylene.

Bioaugmentation adds highly concentrated and specialized populations of specific microbes to the contaminated area, while biostimulation is dependent on appropriate indigenous microbial population and organic material being present at the site.

3.5 In Situ and Ex Situ Bioremediation Techniques

Bioremediation can be used as a cleanup method for both contaminated soil and water. Its applications fall into two broad categories: in situ or ex situ. In situ bioremediation treats the contaminated soil or groundwater in the location in which it was found, while ex situ bioremediation processes require excavation of contaminated soil or pumping of groundwater before they can be treated.

In situ technologies do not require excavation of the contaminated soils, so they may be less expensive, create less dust, and cause less release of contaminants than ex situ techniques. Also, it is possible to treat a large volume of soil at once. In situ techniques, however, may be slower than ex situ techniques, may be difficult to manage, and are only most effective at sites with permeable soil.

The most effective means of implementing in situ bioremediation depends on the hydrology of the subsurface area, the extent of the contaminated area, and the nature (type) of the contamination. In general, this method is effective only when the subsurface soils are highly permeable, the soil horizon to be treated falls within a depth of 8–10 m, and shallow groundwater is present at 10 m or less below ground surface. The depth of contamination plays an important role in determining whether or not an in situ bioremediation project should be employed. If the contamination is near the groundwater, but the groundwater is not yet contaminated, then it would be unwise to set up a hydrostatic system. It would be safer to excavate the contaminated soil and apply an on-site method of treatment away from the groundwater.

The typical time frame for an in situ bioremediation project can be in the order of 12 to 24 months, depending on the levels of contamination and depth of contaminated soil. Due to the poor mixing in this system, it becomes necessary to treat for long periods of time to ensure that all the pockets of contamination have been treated.

In situ bioremediation is a very site-specific technology that involves establishing a hydrostatic gradient through the contaminated area by flooding it with water carrying nutrients and possibly organisms adapted to the contaminants. Water is continuously circulated through the site until it is determined to be clean.

In situ bioremediation of groundwater speeds the natural biodegradation processes that take place in the water-soaked underground region that lies below the water table. One limitation of this technology is that differences in underground soil layering and density may cause re-injected conditioned groundwater to follow certain preferred flow paths. On the other hand, ex situ techniques can be faster, easier to control, and used to treat a wider range of contaminants and soil types than in situ techniques. However, they require excavation and treatment of the contaminated soil before, and sometimes after, the actual bioremediation step.

In situ bioremediation is the preferred method for large sites, and is used when physical and chemical methods of remediation may not completely remove the contaminants, leaving residual concentrations that are above regulatory guidelines. This method has the potential to provide advantages such as complete destruction of the contaminant(s), lower risk to site workers, and lower equipment/operating costs. In situ bioremediation can be used as a cost-effective secondary treatment scheme to decrease the concentration of contaminants to acceptable levels, or as a primary treatment method, which is followed by physical or chemical methods for final site closure.

Finally, evidence for the effectiveness of petroleum bioremediation and petroleum product bioremediation should include: (1) faster disappearance of oil in treated areas than in untreated areas, and (2) a demonstration that biodegradation was the main reason for the increased rate of oil disappearance. To obtain such evidence, the analytical procedures must be chosen carefully, and careful data interpretation is essential, but there are disadvantages and errors when the method is not applied correctly (Chapter 9) (Speight, 2005).

4 Mechanism of Biodegradation

Biodegradation involves chemical transformations mediated by microorganisms that: (1) satisfy nutritional requirements, (2) satisfy energy requirements, (3) detoxify the immediate environment, or (4) occur fortuitously, such that the organism receives no nutritional or energy benefit (Stoner, 1994).

Mineralization is the complete biodegradation of organic materials to inorganic products, and often occurs through the combined activities of microbial consortia rather than through a single microorganism (Shelton and Tiedje, 1984).

Co-metabolism is the partial biodegradation of organic compounds that occurs fortuitously and that does not provide energy or cell biomass to the microorganisms. Co-metabolism can result in partial transformation to an intermediate that can serve as a carbon and energy substrate for microorganisms, as with some hydrocarbons, or can result in an intermediate that is toxic to the transforming microbial cell, as with trichloroethylene and methanotrophs.

4.1 Chemical Reactions

Biodegradation of petroleum constituents can occur under both aerobic (oxic) and anaerobic (anoxic) conditions (Zengler et al., 1999), albeit by the action of different consortia of organisms. In the subsurface, oil biodegradation occurs primarily under anoxic conditions, mediated by sulfate reducing bacteria (e.g., Holba et al., 1996) or other anaerobes, using a variety of other electron acceptors as the oxidant. Thus, two classes of biodegradation reactions are (1) aerobic biodegradation and (2) anaerobic biodegradation.

Aerobic biodegradation involves the use of molecular oxygen (O2), where oxygen (the terminal electron acceptor) receives electrons transferred from an organic contaminant:

Thus, the organic substrate is oxidized (addition of oxygen), and the oxygen is reduced (addition of electrons and hydrogen) to water (H2O). In this case, the organic substrate serves as the sources of energy (electrons) and the source of cell carbon used to build microbial cells (biomass). Some microorganisms (chemo-autotrophic aerobes or litho-trophic aerobes) oxidize reduced inorganic compounds (NH3, Fe²+, or H2S) to gain energy and fix carbon dioxide to build cell carbon:

At some contaminated sites, as a result of consumption of oxygen by aerobic microorganisms and slow recharge of oxygen, the environment becomes anaerobic (lacking oxygen), and mineralization, transformation, and co-metabolism depend upon microbial utilization of electron acceptors other than oxygen (anaerobic biodegradation). Nitrate (NO3), iron (Fe³+), manganese (Mn⁴+), sulfate (SO4), and carbon dioxide (CO2) can act as electron acceptors if the organisms present have the appropriate enzymes (Sims, 1990).

Anaerobic biodegradation is the microbial degradation of organic substances in the absence of free oxygen. While oxygen serves as the electron acceptor in aerobic biodegradation processes forming water as the final product, degradation processes in anaerobic systems depend on alternative acceptors, such as sulfate, nitrate or carbonate, yielding, in the end, hydrogen sulfide, molecular nitrogen, and/or ammonia and methane (CH4), respectively.

In the absence of oxygen, some microorganisms obtain energy from fermentation and anaerobic oxidation of organic carbon. Many anaerobes use nitrate, sulfate, and salts of iron (III) as practical alternates to oxygen acceptor. The anaerobic reduction process of nitrates, sulfates and salts of iron is an example:

Anaerobic biodegradation is a multistep process performed by different bacterial groups. It involves hydrolysis of polymeric substances like proteins or carbohydrates to monomers and the subsequent decomposition to soluble acids, alcohols, molecular hydrogen, and carbon dioxide, Depending on the prevailing environmental conditions, the final steps of ultimate anaerobic biodegradation are performed by denitrifying, sulfate-reducing or methanogenic bacteria.

In contrast to the strictly anaerobic sulfate-reducing and methanogenic bacteria, the nitrate-reducing microorganisms, as well as many other decomposing bacteria, are mostly facultative anaerobic, insofar as these microorganisms are able to grow and degrade organic substances under aerobic as well as anaerobic conditions. Thus, aerobic and anaerobic environments represent the two extremes of a continuous spectrum of environmental habitats which are populated by a wide variety of microorganisms with specific biodegradation abilities.

Anaerobic conditions occur where vigorous decomposition of organic matter and restricted aeration result in the depletion of oxygen. Anoxic conditions may represent an intermediate stage where oxygen supply is limited, while still allowing a slow (aerobic) degradation of organic compounds.

In a digester, the various bacteria also have different requirements to the surrounding environment. For example, acidogenic bacteria need pH values from 4 to 6, while methanogenic bacteria need values from 7 to 7.5. In batch tests, the dynamic equilibrium is often interrupted because of an enrichment of acidogenic bacteria as a consequence of lacking substrate in- and outflow.

On a structural basis, the hydrocarbons in crude oil are classified as alkanes (normal or iso), cycloalkanes, and aromatics. Alkenes are rare in petroleum, but occur in many refined petroleum products as a consequence of the cracking process (Speight, 2007). Increasing carbon numbers of alkanes (homology), variations in carbon chain branching (iso-alkanes), ring condensations, and interclass combinations, such as phenyl alkanes, account for the high numbers of hydrocarbons that occur in crude oil.

In addition, smaller amounts of oxygen-containing compounds (phenol derivatives, naphthenic acids), nitrogen-containing compounds (pyridine derivatives, pyrrole derivatives, indole derivatives), sulfur-containing compounds (thiophene derivatives), and the high molecular weight polar asphalt fraction also occur in petroleum, but not in refined petroleum products (Speight, 2007).

The inherent biodegradability of these individual components is a reflection of their chemical structure, but is also strongly influenced by the physical state and toxicity of the compounds. As an example, while n-alkanes as a structural group are the most biodegradable petroleum hydrocarbons, the C5 to C10 homologs have been shown to be inhibitory to the majority of hydrocarbon degraders. As solvents, these homologs tend to disrupt lipid membrane structures of microorganisms. Similarly, alkanes in the C20 to C40 range are hydrophobic solids at physiological temperatures. Apparently, it is this physical state that strongly influences their biodegradation (Bartha and Atlas, 1977).

Primary attack on intact hydrocarbons requires the action of oxygenases, and, therefore, requires the presence of free oxygen. In the case of alkanes, mono-oxygenase attack results in the production of alcohol. Most microorganisms attack alkanes terminally, whereas some perform sub-terminal oxidation. The alcohol product is oxidized finally into an aldehyde. Extensive methyl branching interferes with the beta-oxidation process, and necessitates terminal attack or other bypass mechanisms. Therefore, n-alkanes are degraded more readily than iso alkanes.

Cycloalkanes are transformed by an oxidase system to a corresponding cyclic alcohol, which is dehydrated to ketone, after which a mono-oxygenase system lactonizes the ring, which is subsequently opened by a lactone hydrolase. These two oxygenase systems usually never occur in the same organisms and hence, result in the frustrated attempts to isolate pure cultures that grow on cycloalkanes (Bartha, 1986b). However, synergistic actions of microbial communities are capable of dealing with degradation of various cycloalkanes quite effectively.

As in the case of alkanes, the monocyclic compounds, cyclopentane, cyclohexane, and cycloheptane have a strong solvent effect on lipid membranes, and are toxic to the majority of hydrocarbon degrading microorganisms. Highly condensed cycloalkane compounds resist biodegradation, due to their relatively complex structure and physical state (Bartha, 1986a).

Condensed polycyclic aromatics are degraded, one ring at a time, by a similar mechanism, but biodegradability tend to decline with the increasing number of rings and degree of condensation (Atlas and Bartha, 1998). Aromatics with more than four condensed rings are generally not suitable as substrates for microbial growth, though they may undergo metabolic transformations. The biodegradation process also declines with the increasing number of alkyl substituents on the aromatic nucleus.

Asphaltic constituents of petroleum (Speight, 2007) tend to increase during biodegradation in relative and sometimes absolute amounts. This would suggest that they not only tend to resist biodegradation, but may also be formed de novo by condensation reactions of biodegradation and photo-degradation intermediates.

In crude petroleum as well as in refined products, petroleum hydrocarbons occur in complex mixtures and influence each others’ biodegradation. The effects may go in negative as well as positive directions. Some iso-alkanes are apparently spared as long as n-alkanes are available as substrates, while some condensed aromatics are metabolized only in the presence of more easily utilizable petroleum hydrocarbons, a process referred to as co-metabolism (Wackett, 1996).

Finally, a word on the issue of adhesion as it affects biodegradation and, hence, bioremediation.

Adhesion to hydrophobic surfaces is a common strategy used by microorganisms to overcome limited bioavailability of hydrocarbons (Bouchez-Naïtali et al., 1999). Intuitively, it may be assumed that adherence of cells to a hydrocarbon would correlate with the ability to utilize it as a growth substrate, and conversely, that cells able to utilize hydrocarbons would be expected to be able to adhere to them. However, species like Staphylococcus aureus and Serratia marcescens, which are unable to grow on hydrocarbons, adhere to them (Rosenberg et al., 1980). Thus, adherence to hydrocarbons does not necessarily predict utilization (Abbasnezhad et al., 2011).

Biodegradation of poorly water-soluble liquid hydrocarbons is often limited by low availability of the substrate to microbes. Adhesion of microorganisms to an oil—water interface can enhance this availability, whereas detaching cells from the interface can reduce the rate of biodegradation. The capability of microbes to adhere to the interface is not limited to hydrocarbon degraders, nor is it the only mechanism to enable rapid uptake of hydrocarbons, but it represents a common strategy. The general indications are that microbial adhesion can benefit growth on and biodegradation of very poorly water-soluble hydrocarbons, such as n-alkanes and large polycyclic aromatic hydrocarbons dissolved in a non-aqueous phase. Adhesion is particularly important when the hydrocarbons are not emulsified, thereby giving limited interfacial area between the two liquid phases.

When mixed communities are involved in biodegradation, the ability of cells to adhere to the interface can enable selective growth and enhance bioremediation with time. The critical challenge in understanding the relationship between growth rate and biodegradation rate for adherent bacteria is to accurately measure and observe the population resides at the interface of the hydrocarbon phase.

4.2 Kinetic Aspects

The kinetics for modeling the bioremediation of contaminated soils can be extremely complicated. This is largely due to the fact that the primary function of microbial metabolism is not for the remediation of environmental contaminants. Instead, the primary metabolic function, whether bacterial or fungal in nature, is to grow and sustain more of the microorganism. Therefore, the formulation of a kinetic model must start with the active biomass and factors, such as supplemental nutrients and oxygen source, that are necessary for subsequent biomass growth (Cutright, 1995; Ron evi et al., 2005; Pala et al., 2006).

Studies of the kinetics of the bioremediation process proceed in two directions: (1) the first is concerned with the factors influencing the amount of transformed compounds with time, and (2) the other approach seeks the types of curves describing the transformation, and determines which of them fits the degradation of the given compounds by the microbiologic culture in the laboratory microcosm, and sometimes in the field. However, studies of biodegradation kinetics in the natural environment are often empiric, reflecting only a basic level of knowledge about the microbiologic population and its activity in a given environment (Maleti equation et al., 2009).

One such example of the empirical approach is the simple (perhaps over-simplified) model:

C is the concentration of the substrate, t is time, k is the degradation rate constant of the compound, and n is a fitting parameter (most often taken to be unity) (Hamaker, 1972; Wethasinghe et al., 2006). Using this model, one can fit the curve of substrate removal by varying n and k until a satisfactory fit is obtained. It is evident from this equation that the rate is proportional to the exponent of substrate concentration. First order kinetics are the most often used equation for representation of the degradation kinetics (Heitkamp et al., 1987; Heitkamp and Cerniglia, 1987; Venosa et al., 1996; Seabra et al., 1999; Holder et al., 1999; Winningham et al., 1999; Namkoonga et al., 2002; Grossi et al., 2002; Hohener et al., 2003; Collina et al., 2005; Ron equation evi equation et al., 2005; Pala et al., 2006).

However, researchers involved in kinetic studies do not always report whether the model they used was based on theory or experience, and whether the constants in the equation have a physical meaning or if they just serve as fitting parameters (Bazin et al., 1976; Ron equation evi equation et al., 2005).

4.3 Effect of Salt

Salt is a common co-contaminant that can adversely affect the bioremediation potential at sites such as flare pits and drilling sites (upstream sites) contaminated with saline produced formation water, or at oil and gas processing facilities contaminated by refinery wastes containing potassium chloride (KCl) and sodium chloride (NaCl) salts (Pollard et al., 1994). Because of increasing emphasis and interest in the viability of intrinsic bioremediation as a remedial alternative, the impact of salt on these processes is of interest.

The effect of salinity on microbial cells varies from disrupted tertiary protein structures and denatured enzymes to cell dehydration (Pollard et al., 1994), with different species having different sensitivities to salt (Tibbett et al., 2011). A range of organic pollutants, including hydrocarbons, has been shown to be mineralized by marine or salt-adapted terrestrial microorganisms that are able to grow in the presence of salt (Margesin and Schinner, 2001; Oren et al., 1992; Nicholson and Fathepure, 2004). In naturally saline soils, it has been shown that bioremediation of diesel fuel is possible at salinities up to 17.5% w/v (Riis et al., 2003; Kleinsteuber et al. (2006).

However, an inverse relationship between salinity and the biodegradation of petroleum hydrocarbons by halophilic enrichment cultures from the Great Salt Lake (Utah) has been observed (Ward and Brock, 1978). These cultures were unable to metabolize petroleum hydrocarbons at salt concentrations above 20% (w/v) in this hyper-saline environment. An inhibitory effect of salinity at concentrations above 2.4% (w/v) NaCl was found to be greater for the biodegradation of aromatic and polar fractions than for the saturated fraction of petroleum hydrocarbons in crude oil incubated with marine sediment (Mille et al., 1991). This represents ex-situ petroleum hydrocarbon degradation by salt-adapted terrestrial microorganisms.

Furthermore, the effects of salt as a co-contaminant on hydrocarbon degradation in naturally non-saline systems has been described (deCarvalho and daFonseca, 2005). The results showed that in the degradation of C5 to C16 hydrocarbons at 28°C (82°F) in the presence of 1.0, 2.0 or 2.5% (w/v) NaCl by the isolate Rhodococcus erythropolis DCL14, the lag phase of the cultures increased and growth rates decreased with increasing concentrations of sodium chloride. In a similar study (Rhykerd et al., 1995), soils were fertilized with inorganic nitrogen and phosphorus, and amended with sodium chloride at 0.4, 1.2, or 2% (w/w). After 80 days at 25°C (77°F), the highest salt concentration had inhibited motor oil mineralization.

However, investigation of the combinations of factors limiting biodegradation of petroleum hydrocarbon contamination at upstream oil and gas production facilities has received relatively little attention. A laboratory solid-phase bioremediation study reported that high salinity levels reduced the degradation rate of flare pit hydrocarbons (Amatya et al., 2002), and more recently it has been observed that addition of sodium chloride to a petroleum-contaminated Arctic soil decreased hexadecane mineralization rates in the initial stages of bioremediation and increased lag times, but the final extent of mineralization was comparable over a narrow range of salinity from 0 to 0.4% w/w (Børresen and Rike, 2007).

Continuing investigations are necessary to determine whether the effects observed in the laboratory are site-specific or contaminant-specific, or are applicable more broadly to sub-surface hydrocarbon bioremediation. Further research using more sites, including those previously having been impacted by sodium chloride, may allow inference of salt tolerance at upstream oil and gas sites. Particularly important is the impact of sodium chloride on anaerobic hydrocarbon degradation, which should be investigated.

Field evidence is sparse with respect to anaerobic biodegradation at salt contaminated upstream oil and gas sites. Before embarking on anaerobic microcosm tests, field evidence of indicators of anaerobic biodegradation, including changes in terminal electron acceptors, presence of metabolites, and isotopic analysis, would be a reasonable way to initiate the research (Ulrich et al., 2009).

5 Bioremediation Methods

Bioremediation technology exploits various naturally occurring mitigation processes: (1) natural attenuation, (2) biostimulation, and (3) 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, and 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.

In recent years, in situ bioremediation concepts have been applied in treating contaminated soil and ground water. Removal rates and extent vary based on the contaminant of concern and site-specific characteristics. Removal rates also are affected by variables such as contaminant distribution and concentration; co-contaminant concentrations; indigenous microbial populations and reaction kinetics; and parameters such as pH, moisture content, nutrient supply, and temperature. Many of these factors are a function of the site and the indigenous microbial community and, thus, are difficult to manipulate. Specific technologies may have the capacity to manipulate some variables, and may be affected by other variables as well (US EPA, 2006).

During bioremediation, microbes utilize chemical contaminants in the soil as an energy source and, through oxidation-reduction reactions, metabolize the target contaminant into usable 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.

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. 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 chlorocarbons or chlorohydrocarbons, is dependent on outcomes of research that searches for natural or genetically improved strains of microorganisms to degrade such contaminants into less toxic forms.

In summary, bioremediation is increasingly viewed as an appropriate remediation technology for hydrocarbon-contaminated polar soils. As for all soils, the successful application of bioremediation depends on appropriate biodegradative microbes and environmental conditions in situ. Laboratory studies have confirmed that hydrocarbon-degrading bacteria typically assigned to the genera Rhodococcus, Sphingomonas, or Pseudomonas are present in contaminated polar soils. However, as indicated by the persistence of spilled hydrocarbons, environmental conditions in situ are sub-optimal for biodegradation in polar soils.

Therefore, it is likely that ex situ bioremediation will be the method of choice for ameliorating and controlling the factors limiting microbial activity, i.e., low and fluctuating soil temperatures, low levels of nutrients, and possible alkalinity and low moisture. Care must be taken when adding nutrients to the coarse-textured, low-moisture soils prevalent in continental Antarctica and the high Arctic, because excess levels can inhibit hydrocarbon biodegradation by decreasing soil water potentials. Bioremediation experiments conducted on site in the Arctic indicate that land farming and biopiles may be useful approaches for bioremediation of polar soils (Aislabie et al., 2006; Nugroho et al., 2010).

5.1 Method Parameters

Several factors that affect the decision of which method is chosen are (1) the nature of the contaminants, (2) the location of contaminated site, (3) the time allotted to the cleanup, (4) effects on humans, animals and plants, and last but by no means least (5) the cost of the cleanup. Sometimes when one method is no longer effective and efficient, another remediation method can be introduced into the contaminated soil.

Oil spills introduce large amounts of toxic compounds into the environment, and though different methods of bioremediation have been successful in remediating soils and water contaminated with the lighter organics, the high-viscosity crude oils (heavier oils) are still less susceptible to these techniques.

Conventional bioremediation methods used are biopiling, composting, land farming, and bioslurry reactors, but there are limitations affecting the applicability and effectiveness of these methods (Speight and Lee, 2000). With the application of the composting technique, if the operation is unsuccessful, it will result in a greater quantity of contaminated materials. Land farming is only effective if the contamination is near the soil surface, or else bed preparation needs to take place. The main drawback with slurry bioreactors is that high-energy inputs are required to maintain suspension and the potential needed for volatilization.

For a bioremediation method to be successful in soil and water cleanup, the physical, chemical, and biological environment must be feasible. Parameters that affect the bioremediation process are (1) low temperatures, (2) preferential growth of microbes obstructive to bioremediation, (3) high concentrations of chlorinated organics, heavy metals, and heavy oils poisoning the microorganisms, (4) preferential flow paths severely decreasing contact between injected fluids and contaminants throughout the contaminated zones, and (5) the soil matrix prohibiting contaminant-microorganism contact.

Since most of the contaminants of concern in crude oil are readily biodegradable under the appropriate conditions, the success of oil spill bioremediation depends mainly on the ability to establish these conditions in the contaminated environment using the above new developing technologies to optimize the microorganisms’ total efficiency. The technologies used at various polluted sites depend on the limiting factor present at the location. For example, where there is insufficient dissolved oxygen, bioventing or sparging is applied, while biostimulation or bioaugmentation is suitable for instances where the biological count is low.

The bioventing process combines an increased oxygen supply with vapor extraction. A vacuum is applied at some depth in the contaminated soil, which draws air down into the soil from holes drilled around the site, and sweeps out any volatile organic compounds. The development and application of venting and bioventing for in situ removal of petroleum from soil have been shown to remediate hydrocarbons by venting and biodegradation (van Eyk, 1994).

Even though a particular technology may have reports of improving biodegradation efficiency (for example, surfactant addition), this may not be the case at times, depending on the sample.

5.2 In Situ and Ex Situ Bioremediation

Bioremediation applications fall into two broad categories: (1) in situ or (2) ex situ. In situ bioremediation is used when physical and chemical methods of remediation may not completely remove the contaminants, leaving residual concentrations that are above regulatory guidelines. Bioremediation can be used as a cost-effective secondary treatment scheme to decrease the concentration of contaminants to acceptable levels. In other cases, bioremediation can be the primary treatment method, and followed by physical or chemical methods for final site closure. Also, it is the preferred method for very large sites.

5.3 Biostimulation and Bioaugmentation of Contaminated Sites

The primary advantage of biostimulation is that bioremediation will be undertaken by already present native microorganisms that are well suited to the subsurface environment, and are well distributed spatially within the subsurface, but the main disadvantage is that the delivery of additives in a manner that allows the additives to be readily available to subsurface microorganisms is based on the local geology of the subsurface.

Bioaugmentation is the addition of pre-grown microbial cultures to enhance microbial populations at a site to improve contaminant cleanup and reduce cleanup time and cost. Indigenous or native microbes are usually present in very small quantities, and may not be able to prevent the spread of the contaminant. In some cases, native microbes do not have the ability to degrade a particular contaminant. Therefore, bioaugmentation offers a way to provide specific microbes in sufficient numbers to complete the biodegradation.

Bioaugmentation adds highly concentrated and specialized populations of specific microbes to the contaminated area, while biostimulation is dependent on appropriate indigenous microbial population and organic material being present at the site. Therefore, one might be led to believe that bioaugmentation is more effective than biostimulation.

5.4 Monitored Natural Attenuation

The term monitored natural attenuation refers to the reliance on natural attenuation to achieve site-specific remedial objectives within a time frame that is reasonable compared to that offered by other more active methods.

The natural attenuation processes that are at work in such a remediation approach include a variety of physical, chemical, or biological processes that, under favorable conditions, act without human intervention to reduce the mass, toxicity, mobility, volume, or concentration of contaminants in soil or groundwater. These in situ processes include biodegradation, dispersion, dilution, sorption, volatilization, and chemical or biological stabilization, transformation, or destruction of contaminants. A study of any contaminated site must first be performed to decide whether natural attenuation would make a positive input,