BIOREMEDIATION: Biotransformation of toxic wastes to harmless products

Mrs. Lakshmi Sivasubramaniam

The rapid expansion and increasing sophistication of the chemical industries in the past century and particularly over the last thirty years has meant that there has been an increasing amount and complexity of toxic waste effluents. At the same time, fortunately, regulatory authorities have been paying more attention to problems of contamination of the environment. Industrial companies are therefore becoming increasingly aware of the political, social, environmental and regulatory pressures to prevent escape of effluents into the environment.

The occurrence of major incidents (such as the Exxon Valdez oil spill, the Union-Carbide (Dow) Bhopal disaster, large-scale contamination of the Rhine River, the progressive deterioration of the aquatic habitats and conifer forests in the Northeastern US, Canada, and parts of Europe, or the release of radioactive material in the Chernobyl accident, etc.) and the subsequent massive publicity due to the resulting environmental problems has highlighted the potential for imminent and long-term disasters in the public's conscience.¹⁰

Even though policies and environmental efforts should continue to be directed towards applying pressure to industry to reduce toxic waste production, biotechnology presents opportunities to detoxify industrial effluents. Bacteria can be altered to produce certain enzymes that metabolize industrial waste components that are toxic to other life, and also new pathways can be designed for the biodegradation of various wastes. Since waste management itself is a well-established industry, genetics and enzymology can be simply "bolted-on" to existing engineering expertise.¹⁰

 PRINCIPLES OF BIOREMEDIATION^{9, 10, 11, 12}

The most important principle of bioremediation is that microorganisms (mainly bacteria) can be used to destroy hazardous contaminants or transform them to less harmful forms. The microorganisms act against the contaminants only when they have access to a variety of materials—compounds to help them generate energy and nutrients to build more cells. In a few cases the natural conditions at the contaminated site provide all the essential materials in large enough quantities that bioremediation can occur without human intervention—a process called intrinsic bioremediation More often, bioremediation requires the construction of engineered systems to supply microbe-stimulating materials-a process called engineered bioremediation Engineered bioremediation relies on accelerating the desired biodegradation reactions by encouraging the growth of more organisms, as well as by optimizing the environment in which the organisms must carry out the detoxification reactions.

A critical factor in deciding whether bioremediation is the appropriate cleanup remedy for a site is whether the contaminants are susceptible to biodegradation by the organisms at the site (or by organisms that could be successfully added to the site). Although existing microorganisms can detoxify a vast array of contaminants, some compounds are more easily degraded than others. In general, the compounds most easily degraded in the subsurface are petroleum hydrocarbons, but technologies for stimulating the growth of organisms to degrade a wide range of other contaminants are emerging and have been successfully field tested.

The suitability of a site for bioremediation depends not only on the contaminant's biodegradability but also on the site's geological and chemical characteristics. The types of site conditions that favor bioremediation differ for intrinsic and engineered bioremediation For intrinsic bioremediation, the key site characteristics are consistent ground water flow throughout the seasons; the presence of minerals that can prevent pH changes; and high concentrations of either oxygen, nitrate, sulfate, or ferric iron. For engineered bioremediation, the key site characteristics are permeability of the subsurface to fluids, uniformity of the subsurface, and relatively low (less than 10,000 mg/kg solids) residual concentrations of nonaqueous-phase contaminants.

When deciding whether a site is suitable for bioremediation, it is important to realize that no single set of site characteristics will favor bioremediation of all contaminants. For example, certain compounds can only be degraded when oxygen is absent, but destruction of others requires that oxygen be present. In addition, one must consider how the bioremediation system may perform under variable and not perfectly known conditions. A scheme that works optimally under specific conditions but poorly otherwise may be inappropriate for in situ bioremediation

Examination of effluents from the chemical and petrochemical industries shows that such effluents typically contain either one or a limited range of major toxic components. In some cases other considerations (such as aesthetic ones) can be important for removal of certain components (such as dyes). This means that in general one industry may apply one or a few genetically modified bacterial strains to get rid of its major toxic waste. However, it may be important to contain the "waste-eating" bacteria within the manufacturing plant, and not release these with the waste water. In such cases, filter installations will have to be built to separate the bacteria from the effluent.

Microorganisms have also been successfully applied during the removal of the Exxon Valdez oil spill. A number of microorganisms can utilize oil as a source of food, and many of them produce potent surface-active compounds that can emulsify oil in water and facilitate the removal of the oil. Unlike chemical surfactants, the microbial emulsifier is non-toxic and biodegradable. Also, "fertilizers" have been utilized to increase the growth rate of the indigenous population of bacteria that are able to degrade oil.

Use of microbes for bioremediation is not limited to detoxification of organic compounds. In many cases, selected microbes can also reduce the toxic cations of heavy metals (such as selenium) to the much less toxic and much less soluble elemental form. Thus, bioremediation of surface water with significant contamination by heavy metals can now be attempted.

Types of Bioremediation^{9, 12}

Ø In-Situ Bioremediation

In-situ bioremediation treats the contaminated soil or groundwater in the location in which it is found.

Ø Ex -Situ Bioremediation

Ex-situ bioremediation processes require excavation of contaminated soil or pumping of groundwater before they can be treated.

Types of in-situ bioremediation 

1. In-Situ Bioremediation of Soil⁷

 In-situ techniques do not require excavation of the contaminated soils so 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 most effective at sites with permeable (sandy or uncompacted) soil.

The goal of aerobic in-situ bioremediation is to supply oxygen and nutrients to the microorganisms in the soil. Aerobic in-situ techniques can vary in the way they supply oxygen to the organisms that degrade the contaminants.

Two such methods are bioventing and injection of hydrogen peroxide. Oxygen can be provided by pumping air into the soil above the water table (bioventing) or by delivering the oxygen in liquid form as hydrogen peroxide. In-situ bioremediation may not work well in clays or in highly layered subsurface environments because oxygen cannot be evenly distributed throughout the treatment area. In-situ remediation often requires years to reach cleanup goals, depending mainly on how biodegradable specific contaminants are. Less time may be required with easily degraded contaminants.

Bioventing

Bioventing systems deliver air from the atmosphere into the soil above the water table through injection wells placed in the ground where the contamination exists. The number, location, and depth of the wells depend on many geological factors and engineering considerations. An air blower may be used to push or pull air into the soil through the injection wells. Air flows through the soil and the oxygen in it is used by the microorganisms. Nutrients may be pumped into the soil through the injection wells. Nitrogen and phosphorous may be added to increase the growth rate of the microorganisms.

Injection of Hydrogen Peroxide

This process delivers oxygen to stimulate the activity of naturally occurring microorganisms by circulating hydrogen peroxide through contaminated soils to speed the bioremediation of organic contaminants. Since it involves putting a chemical (hydrogen peroxide) into the ground (which may eventually seep into the groundwater), this process is used only at sites where the groundwater is already contaminated. A system of pipes or a sprinkler system is typically used to deliver hydrogen peroxide to shallow contaminated soils. Injection wells are used for deeper contaminated soils.

1. In-Situ Bioremediation of Groundwater

 In-situ groundwater bioremediation is a technology that encourages growth and reproduction of indigenous microorganisms to enhance biodegradation of organic constituents in the saturated zone. In-situ groundwater bioremediation can effectively degrade organic constituents, which are dissolved in groundwater and adsorbed onto the aquifer matrix.¹

In-situ bioremediation of groundwater can be combined with other saturated zone remedial technologies (e.g., air sparging) and vadose zone remedial operations (e.g., soil vapor extraction, bioventing).

Application

In-situ groundwater bioremediation can be effective for the full range of petroleum hydrocarbons. While there is some notable exceptions (e.g., MTBE) the short-chain, low molecular weight, more water-soluble constituents are degraded more rapidly and to lower residual levels than are long-chain, high molecular weight, less soluble constituents. Recoverable free product should be removed from the subsurface prior to operation of the in-situ groundwater bioremediation system. This will mitigate the major source of contaminants as well as reduce the potential for smearing or spreading high concentrations of contaminants.^1,2

System Design^{4, 5, 7}

 In-situ bioremediation can be implemented in a number of treatment modes, including: Aerobic (oxygen respiration); anoxic (nitrate respiration); anaerobic (non-oxygen respiration); and co-metabolic. The aerobic mode has been proven most effective in reducing contaminant levels of aliphatic (e.g., hexane) and aromatic petroleum hydrocarbons (e.g., benzene, naphthalene) typically present in gasoline and diesel fuel. In the aerobic treatment mode, groundwater is oxygenated by one of three methods: Direct sparging of air or oxygen through an injection well; saturation of water with air or oxygen prior to re-injection; or addition of hydrogen peroxide directly into an injection well or into reinjected water. Whichever method of oxygenation is used, it is important to ensure that oxygen is being distributed throughout the area of contamination. Anoxic, anaerobic, and co-metabolic modes are sometimes used for remediation of other compounds, such as chlorinated solvents, but are generally slower than aerobic respiration in breaking down petroleum hydrocarbons.

The key parameters that determine the effectiveness of In-situ groundwater bioremediation are:

• Hydraulic conductivity of the aquifer, which controls the distribution of electron acceptors and nutrients in the subsurface;
• Biodegradability of the petroleum constituents, which determines both the rate and degree to which constituents will be degraded by microorganisms; and
• Location of petroleum contamination in the subsurface. Contaminants must be dissolved in groundwater or adsorbed onto more permeable sediments within the aquifer.

In general, the aquifer medium will determine hydraulic conductivity. Fine-grained media (e.g., clays, silts) have lower intrinsic permeability than coarse-grained media (e.g., sands, gravels). Bioremediation is generally effective in permeable (e.g., sandy, gravelly) aquifer media. However, depending on the extent of contamination, bioremediation also can be effective in less permeable silty or clayey media. In general, an aquifer medium of lower permeability will require longer cleaning up than a more permeable medium. Soil structure and stratification are important to in-situ groundwater bioremediation because they affect groundwater flow rates and patterns when water is extracted or injected. Structural characteristics such as micro fracturing can result in higher permeabilities than expected for certain soils (e.g., clays). In this case, however, flow will increase in the fractured media but not in the unfractured media. The stratification of soils with different permeabilities can dramatically increase the lateral flow of groundwater in the more permeable strata while reducing the flow through less permeable strata. This preferential flow behavior can lead to reduced effectiveness and extended remedial times for less-permeable strata.

The biodegradability of a petroleum constituent is a measure of its ability to be metabolized (or co-metabolized) by hydrocarbon-degrading bacteria or other microorganisms. The chemical characteristics of the contaminants will dictate their biodegradability. For example, heavy metals are not degraded by bioremediation. The biodegradability of organic constituents depends on their chemical structures and physical/chemical properties (e.g., water solubility, water/octanol partition coefficient). Highly soluble organic compounds with low molecular weights will tend to be more rapidly degraded than slightly soluble compounds with high molecular weights. The low water solubilities of the more complex compounds render them less bioavailable to petroleum-degrading organisms. Consequently, the larger, more complex chemical compounds may be slow to degrade or may even be recalcitrant to biological degradation (e.g., asphaltenes in No. 6 fuel oil).

The location, distribution, and disposition of petroleum contamination in the subsurface can significantly influence the likelihood of success for bioremediation. This technology generally works well for dissolved contaminants and contamination adsorbed onto higher permeability sediments (sands and gravels). However, if the majority of contamination is (1) in the unsaturated zone; (2) trapped in lower permeability sediments, or (3) outside the "flow path" for nutrients and electron acceptors, this technology will have reduced impact or no impact.

Excessive calcium, magnesium, or iron in groundwater can react with phosphate, which is typically supplied as a nutrient in the form of tripolyphosphate, or with carbon dioxide, which is produced by microorganisms as a by-product of aerobic respiration. The products of these reactions can adversely affect the operation of an in-situ bioremediation system. When calcium, magnesium, or iron reacts with phosphate or carbon dioxide, crystalline precipitates or "scale" is formed. Scale can constrict flow channels and can also damage equipment, such as injection wells and sparge points. In addition, the precipitation of calcium or magnesium phosphates ties up phosphorus compounds, making them unavailable to microorganisms for use as nutrients. This effect can be minimized by using tripolyphosphates to acta as sequestering agents to keep the magnesium and calcium in solution (i.e., prevent the metal ions from precipitating and forming scale).

When oxygen is introduced to the subsurface as a terminal electron acceptor, it can react with dissolved iron [Fe (II)] to form an insoluble iron precipitate, ferric oxide. This precipitate can be deposited in aquifer flow channels, reducing permeability. The effects of iron precipitation tend to be most noticeable around injection wells, where oxygen concentration in groundwater is highest and can render injection wells inoperable.

Extreme pH values (i.e., less than 5 or greater than 10) are generally unfavorable for microbial activity. Typically, optimal microbial activity occurs under neutral pH conditions (i.e., in the range of 6 8). The optimal pH is site specific. For example, aggressive microbial activity has been observed at lower pH conditions outside of this range (e.g., 4.5 to 5) in natural systems. Because indigenous microorganisms have adapted to the natural conditions where they are found, pH adjustment, even toward neutral, can inhibit microbial activity. If man-made conditions (e.g., releases of petroleum) have altered the pH outside the neutral range, pH adjustment may be needed. If the pH of the groundwater is too low (too acid), lime or sodium hydroxide can be added to increase the pH. If the pH is too high (too alkaline), then a suitable acid (e.g., hydrochloric, muriatic) can be added to reduce the pH. Changes to pH should be closely monitored because rapid changes of more than 1 or 2 units can inhibit microbial activity and may require an extended acclimation period before the microbes resume their activity.

Microorganisms require carbon as an energy source to sustain their metabolic functions, which include growth and reproduction. The metabolic process used by bacteria to produce energy requires a terminal electron acceptor (TEA) to enzymatically oxidize the carbon source (organic matter) to carbon dioxide. Microorganisms are classified by the carbon and TEA sources they use to carry out metabolic processes. Bacteria that use organic compounds as their source of carbon are called heterotrophs; those that use inorganic carbon compounds such as carbon dioxide are called autotrophs. Bacteria that use oxygen as their TEA are called aerobes; those that use a compound other than oxygen (e.g., nitrate, sulfate) are called anaerobes; and those that can utilize both oxygen and other compounds as TEAs are called facultative. For in-situ groundwater bioremediation applications directed at petroleum products, bacteria that are both aerobic (or facultative) and heterotrophic are most important in the degradation process.

Extraction wells are generally necessary to achieve hydraulic control over the plume to ensure that it does not spread contaminants into areas where contamination does not exist or accelerate the movement toward receptors. Placement of extraction wells is critical, especially in systems that also use nutrient injection wells or infiltration galleries. These additional sources of water can alter the natural groundwater flow patterns, which can cause the contaminant plume to move in an unintended direction or rate. Without adequate hydraulic control, this situation can lead to worsening of the original condition and complicate the cleanup or extend it.

Nutrient injection systems may not be necessary at all, if the groundwater contains adequate amounts of nutrients, such as nitrogen and phosphorus. Microorganisms require inorganic nutrients such as nitrogen and phosphate to support cell growth and sustain biodegradation processes. Nutrients may be available in sufficient quantities in the aquifer but, more frequently, nutrients need to be added to maintain adequate bacterial populations.

THE CURRENT PRACTICE OF BIOREMEDIATION¹²

Few people realize that in situ bioremediation is not really a "new" technology. The first in situ bioremediation system was installed 20 years ago to clean up an oil pipeline spill in Pennsylvania, and since then bioremediation has become well developed as a means of cleaning up easily degraded petroleum products. What is new is the use of in situ bioremediation to treat compounds other than easily degraded petroleum products on a commercial scale. The principles of practice outlined here were developed to treat petroleum-based fuels, but they will likely apply to a much broader range of uses for bioremediation in the future.

Engineered Bioremediation^{9, 12}

Engineered bioremediation may be chosen over intrinsic bioremediation because of time and liability. Where an impending property transfer or potential impact of contamination on the local community dictates the need for rapid pollutant removal, engineered bioremediation may be a more appropriate remedy than intrinsic bioremediation. Because engineered bioremediation accelerates biodegradation reaction rates, it requires less time than intrinsic bioremediation. The shorter time requirements reduce the liability for costs required to maintain and monitor the site.

Since many petroleum hydrocarbons require oxygen for their degradation, the technological emphasis of engineered bioremediation systems in use today has been placed on oxygen supply. Bioremediation systems for soil above the water table usually consist of a set of vacuum pumps to supply air (containing oxygen) and infiltration galleries, trenches, or dry wells to supply moisture (and sometimes specific nutrients). Bioremediation systems for ground water and soil below the water table usually consist of either a set of injection and recovery wells used to circulate oxygen and nutrients dissolved in water or a set of compressors for injecting air. Emerging applications of engineered bioremediation, such as for degradation of chlorinated solvents, will not necessarily be controlled by oxygen. Hence, the supply of other stimulatory materials may require new technological approaches even though the ultimate goal, high biodegradation rates, remains the same.

Intrinsic Bioremediation¹¹

Intrinsic bioremediation is an option when the naturally occurring rate of contaminant biodegradation is faster than the rate of contaminant migration. These relative rates depend on the type and concentration of contaminant, the microbial community, and the subsurface hydrogeochemical conditions. The ability of native microbes to metabolize the contaminant must be demonstrated either in field tests or in laboratory tests performed on site-specific samples. In addition, analyzing the fate of the contaminants and other reactants and products indicative of biodegradation must continually monitor the effectiveness of intrinsic bioremediation.

In intrinsic bioremediation the rate-controlling step is frequently the influx of oxygen. When natural oxygen supplies become depleted, the microbes may not be able to act quickly enough to contain the contamination. Lack of a sufficiently large microbial population can also limit the cleanup rate. The microbial population may be small because of a lack of nutrients, limited availability of contaminants resulting from sorption to solid materials or other physical phenomena, or an inhibitory condition such as low pH or the presence of a toxic material.

Integration of Bioremediation with Other Technologies^{6, 11}

Bioremediation frequently is combined with nonbiological treatment technologies, both sequentially and simultaneously. For example, when soil is heavily contaminated, bioremediation may be implemented after excavating soils near the contaminant source—a process that reduces demand on the bioremediation system and the immediate potential for ground water contamination. Similarly, when pools of contaminants are floating on the water table, these pools may be pumped to the surface before bioremediation of residual materials. Bioremediation may follow treatment of the ground water with a conventional pump-and-treat system designed to shrink the contaminant plume to a more manageable size. Bioremediation may also be combined with a vapor recovery system to extract volatile contaminants from soils. Finally, it is possible to follow engineered bioremediation, which cleans up most of the contamination, with intrinsic bioremediation, which may be used for final polishing and contaminant containment.

EVALUATING IN SITU BIOREMEDIATION^1,5

The inherent complexity of performing bioremediation in situ means that special attention must be given to evaluating the success of a project. The most elemental criterion for success of an in situ bioremediation effort is that the microorganisms are mainly responsible for the cleanup. Without evidence of microbial involvement, there is no way to verify that the bioremediation project was actually a bioremediation—that is, that the contaminant did not simply volatilize, migrate off site, sorb to the soil, or change form via abiotic chemical reactions. Simply showing that microbes grown in the lab have the potential to degrade the contaminant is not enough. While bioremediation often is possible in principle, the more relevant question is, "Are the biodegradation reactions actually occurring under site conditions?" No one piece of evidence can unambiguously prove that microorganisms have cleaned up a site. Therefore, the Committee on In Situ Bioremediation recommends an evaluation strategy that builds a consistent, logical case for bioremediation based on converging lines of independent evidence. The strategy should include three types of information:

1.Documented loss of contaminants from the site

2. Laboratory assays showing that microorganisms from site samples have the    potential to transform the contaminants under the expected site conditions, and

3.One or more pieces of information showing that the biodegradation potential is actually realized in the field.

Every well-designed bioremediation project, whether a field test or full-scale system, should show evidence of meeting the strategy's three requirements. Regulators and buyers of bioremediation services can use the strategy to evaluate whether a proposed or ongoing bioremediation project is sound; researchers can apply the strategy to evaluate the results of field tests.

The first type of evidence—documented loss of contaminants from the site-is gathered as part of the routine monitoring that occurs (or should occur) at every cleanup site. The second type of evidence requires taking microbes from the field and showing that they can degrade the contaminant when grown in a well-controlled laboratory vessel. The most difficult type of evidence to gather is the third type—showing that microbes in the field are actively degrading the contaminant. There are two types of sample-based techniques for demonstrating field biodegradation: measurements of field samples and experiments run in the field. In most bioremediation scenarios a third technique, modeling experiments, provides an improved understanding of the fate of contaminants in field sites. Because none of these three techniques alone can show with complete certainty that biodegradation is the primary cause of declining contaminant concentrations, the most effective strategy for demonstrating bioremediation usually combines several techniques.

Measurements of Field Samples^{4, 5, 7}

The following techniques for documenting in situ bioremediation involve analyzing the chemical and microbiological properties of soil and ground water samples from the contaminated site:

• Number of bacteria. Because microbes often reproduce when they degrade contaminants, an increase in the number of contaminant-degrading bacteria over usual conditions may indicate successful bioremediation

• Number of protozoans. Because protozoans prey on bacteria, an increase in the number of protozoans signals bacterial population growth, indicating that bioremediation may be occurring.

• Rates of bacterial activity. Tests indicating that bacteria from the contaminated site degrade the contaminant rapidly enough to effect remediation when incubated in microcosms that resemble the field site provide further evidence of successful bioremediation.

• Adaptation. Tests showing that bacteria from the bioremediation zone can metabolize the contaminant, while bacteria from outside the zone cannot (or do so more slowly), show that the bacteria have adapted to the contaminant and indicate that bioremediation may have commenced.

• Carbon isotopes. Isotopic ratios of the inorganic carbon (carbon dioxide, carbonate ion, and related compounds) from a soil or water sample showing that the contaminant has been transformed to inorganic carbon are a strong indicator of successful bioremediation.

• Metabolic byproducts. Tests showing an increase in the concentrations of known byproducts of microbial activity, such as carbon dioxide, provide a sign of bioremediation

• Intermediary metabolites. The presence of metabolic intermediates—simpler but incompletely degraded forms of the contaminant—in samples of soil or water signals the occurrence of biodegradation.

• Growth-stimulating materials. A depletion in the concentration of growth-stimulating materials, such as oxygen, is a sign that microbes are active and may indicate bioremediation

• Ratio of nondegradable to degradable compounds. An increase in the ratio of compounds that are difficult to degrade to those that are easily degraded indicates that bioremediation may be occurring.

Experiments Run in the Field

The following methods for evaluating whether microorganisms are actively degrading the contaminant involve conducting experiments in the field:

• Stimulating bacteria within subsites.

• Measuring the stimulant uptake rate.

• Monitoring conservative tracers.

• Labeling contaminants.

Modeling Experiments

Four different types of models have been developed:

• Saturated flow models. These models describe where and how fast the water and dissolved contaminants flow through the saturated zone.

• Multiphase flow models. These models characterize the situation in which two or more fluids, such as water and a nonaqueous-phase contaminant or water and air, exist together in the subsurface.

• Geochemical models. These models analyze how a contaminant's chemical speciation is controlled by the thermodynamics of the many chemical and physical reactions that may occur in the subsurface.

• Biological reaction rate models. These models represent how quickly the microorganisms transform contaminants.

Because so many complex processes interact in the subsurface, ultimately two or more types of models may be required for a complete evaluation.

Advantages and Disadvantages

Limitations Inherent in Evaluating In Situ Bioremediation

Although microorganisms grown in the laboratory can destroy most organic contaminants, the physical realities of the subsurface—the low fluid flow rates, physical heterogeneities, unknown amounts and locations of contaminants, and the contaminants' unavailability to the microorganisms—make in situ bioremediation a technological challenge that carries inherent uncertainties. Three strategies can help minimize these uncertainties: (1) increasing the number of samples used to document bioremediation, (2) using models so that important variables are properly weighted and variables with little influence are eliminated, and (3) compensating for uncertainties by building safety factors and flexibility into the design of engineering systems. These strategies should play important roles in evaluating bioremediation projects.

CONCLUSIONS: FUTURE PROSPECTS FOR BIOREMEDIATION^{9, 12}

Bioremediation integrates the tools of many disciplines. As each of the disciplines advances and as new cleanup needs arise, opportunities for new bioremediation techniques will emerge. As these new techniques are brought into commercial practice, the importance of sound methods for evaluating bioremediation will increase. The fundamental knowledge base underlying bioremediation is sufficient to begin implementing the three-part evaluation strategy the committee has recommended. However, further research and better education of those involved in bioremediation will improve the ability to apply the strategy and understanding of the fundamentals behind bioremediation.

References

1. Brubaker, G.R. 1993. "In-situ Bioremediation of Groundwater." in D.E. Daniel, ed., Geotechnical Practice for Waste Disposal. London/New York: Chapman & Hall.

2. Kinsella, J.V. and M.J.K. Nelson. 1993. "In-situ Bioremediation: Site Characterization, System Design and Full-Scale Field Remediation of Petroleum Hydrocarbon- and Trichloroethylene-Contaminated Groundwater." in P.E. Flathman and D.E. Jerger, eds. Bioremediation Field Experience. Boca Raton, FL: CRC Press.

3. Norris, R.D. 1994. "In-situ Bioremediation of Soils and Groundwater Contaminated with Petroleum Hydrocarbons." in R.D. Norris, R.E. Hinchee, R.A. Brown, P.L. McCarty, L. Semprini, J.T. Wilson, D.H. Kampbell, M. Reinhard, E.J. Bower, R.C. Borden, Handbook of Bioremediation. Boca Raton, FL: CRC Press.

4. Norris, R.D., R.E. Hinchee, R. Brown, P.L. McCarty, and L. Semprini. 1993. In-situ Bioremediation of Groundwater and Geologic Material: A Review of Technologies. Washington, DC: U.S. Environmental Protection Agency, EPA/600/R-93/124, (NTIS: PB93-215564/XAB).

5.Norris, R.D. and K.D. Dowd. 1993. "In-situ Bioremediation of Petroleum Hydrocarbon-Contaminated Soil and Groundwater in a Low-Permeability Aquifer." in P.E. Flathman and D.E. Jerger, eds., Bioremediation Field Experience. Boca Raton, FL: CRC Press.

6.Sims, J.L., J.M. Suflita, and H.H. Russell. 1992. In-situ Bioremediation of Contaminated Groundwater. Washington, DC: U.S. Environmental Protection Agency, EPA/540/S-92/003, (NTIS: PB92-224336/XAB), February.

7. Staps, S.J.J.M. 1990. International Evaluation of In-Situ Biorestoration of Contaminated Soil and Groundwater. Washington, DC: U.S. Environmental Protection Agency, Office of Emergency and Remedial Response. EPA 540/2 90/012.

8.Van der Heijde, P.K.M., and O.A. Elnawawy. 1993. Compilation of Groundwater Models. Washington, DC: U.S. Environmental Protection Agency, Office of Research and Development, EPA/600/R 93/118, (NTIS: PB93-209401), May.

9. In situ bioremediation, year of clean water, 2003

10.The best information source for ground water and bioremediation, Ground water Bioremediation and modeling center

11.Bioremediation (Chapter 13)

12.Bioremediation and its applications

About Authors

Madhumathi Seshadri^b, Neha Shah^c and Mrs. Lakshmi Sivasubramaniam ^a, *

^{* a} Lecturer, Department of Pharmaceutical Analysis, College of Pharmacy, SRM Institute of

Science and Technology

^b  Department of Chemistry, Pharmaceutical Chemistry unit, Vellore Institute of Technology,

Vellore-632 014, India

^c Bio medical Genetics, Department of Bio sciences, Vellore Institute of Technology,

Vellore-632 014, India

^{* a} Author for Correspondence: Lakshmi Sivasubramaniam, Lecturer, Department of Pharmaceutical Analysis, College of Pharmacy, SRM Institute of Science and Technology, Deemed University, Katangulathur, Chennai, India

E mail: laxmisiva@rediffmail.com