Executive Summary

The United States is investing billions of dollars in cleaning up polluted ground water and soils, yet this large investment may not be producing the benefits that citizens expect. Recent studies have revealed that because of limitations of ground water cleanup technologies, there are almost no sites where polluted ground water has been restored to a condition fit for drinking. While soil cleanup efforts have come closer to meeting regulatory goals, the technologies typically used to decontaminate soils often increase the exposure to contaminants for cleanup crews and nearby residents.

The limitations of conventional ground water cleanup technologies and the hazards of conventional soil treatment methods—along with the high costs of both—have spurred investigations into alternative cleanup technologies, including in situ bioremediation. In situ bioremediation uses microorganisms to destroy or immobilize contaminants in place. The technology already has achieved a measure of success in field tests and commercial scale cleanups for some types of contaminants.

Proponents of in situ bioremediation say the technology may be less costly, faster, and safer than conventional cleanup methods. Yet despite mounting evidence in support of the technology, bioremediation is neither universally understood nor trusted by those who must approve its use. Bioremediation is clouded by controversy over what it does and how well it works, partly because it relies on microorgan-



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Executive Summary The United States is investing billions of dollars in cleaning up polluted ground water and soils, yet this large investment may not be producing the benefits that citizens expect. Recent studies have revealed that because of limitations of ground water cleanup technologies, there are almost no sites where polluted ground water has been restored to a condition fit for drinking. While soil cleanup efforts have come closer to meeting regulatory goals, the technologies typically used to decontaminate soils often increase the exposure to contaminants for cleanup crews and nearby residents. The limitations of conventional ground water cleanup technologies and the hazards of conventional soil treatment methods—along with the high costs of both—have spurred investigations into alternative cleanup technologies, including in situ bioremediation. In situ bioremediation uses microorganisms to destroy or immobilize contaminants in place. The technology already has achieved a measure of success in field tests and commercial scale cleanups for some types of contaminants. Proponents of in situ bioremediation say the technology may be less costly, faster, and safer than conventional cleanup methods. Yet despite mounting evidence in support of the technology, bioremediation is neither universally understood nor trusted by those who must approve its use. Bioremediation is clouded by controversy over what it does and how well it works, partly because it relies on microorgan-

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isms, which cannot be seen, and partly because it has become attractive for "snake oil salesmen" who claim to be able to solve all types of contamination problems. As long as the controversy remains, the full potential of this technology cannot be realized. In this report the Committee on In Situ Bioremediation communicates the scientific and technological bases for in situ bioremediation, with the goal of eliminating the mystery that shrouds this highly multidisciplinary technology. The report presents guidelines for evaluating in situ bioremediation projects to determine whether they will or are meeting cleanup goals. The Committee on In Situ Bioremediation was established in June 1992 with the specific task of developing such guidelines, and it represents the span of groups involved in bioremediation: buyers of bioremediation services, bioremediation contractors, environmental regulators, and academic researchers. Included with the report are seven background papers, authored by committee members, representing the range of perspectives from which bioremediation may be viewed. PRINCIPLES OF BIOREMEDIATION 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 organ-

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isms 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. THE CURRENT PRACTICE OF BIOREMEDIATION 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 cleanup 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 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

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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 Bioremediation 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, the effectiveness of intrinsic bioremediation must be continually monitored by analyzing the fate of the contaminants and other reactants and products indicative of biodegradation. 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.

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Integration of Bioremediation with Other Technologies 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 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: documented loss of contaminants from the site, laboratory assays showing that microorganisms from site samples

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have the potential to transform the contaminants under the expected site conditions, and 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 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

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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. When growth-stimulating materials such as oxygen and nutrients are added to one subsite within the contaminated area but not another, the relative rate of contaminant loss should increase in the stimulant-amended subsite. The contrast in contaminant loss between enhanced and unenhanced subsites can be attributed to bioremediation. Measuring the stimulant uptake rate. Growth-stimulating materials, such as oxygen, can be added to the site in pulses to determine the rate at which they are consumed. Relatively rapid loss of oxygen or other stimulants in the contaminated area compared to an uncontaminated area suggests successful bioremediation.

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Monitoring conservative tracers. Tracer compounds that are not biologically reactive can be added to the site to determine how much contaminant (or growth-stimulating material) is disappearing through nonbiological pathways and how much is being consumed by microorganisms. Labeling contaminants. Contaminants can be labeled with chemical elements that appear in metabolic end products when the contaminants are degraded, providing another mechanism for determining whether biodegradation is responsible for a contaminant's disappearance. Modeling Experiments A final set of techniques for evaluating whether bioremediation is occurring in the field uses models—sets of mathematical equations that quantify the contaminant's fate. Modeling techniques provide a framework for formally deciding what is known about contaminant behavior at field sites. When modelers have a high degree of confidence that the model accurately represents conditions at the site, modeling experiments can be used to demonstrate field biodegradation. There are two general strategies for using models to evaluate bioremediation. The first strategy, useful when biodegradation is the main phenomenon controlling the contaminant's fate, is to model the abiotic processes to determine how much contaminant loss they account for. Bioremediation is indicated when the concentrations of contaminant actually found in field sites are significantly lower than would be expected from predictions based on abiotic processes (such as dilution, transport, and volatilization). The second strategy involves directly modeling the microbial processes to estimate the biodegradation rates. Direct modeling, while the intellectually superior approach, requires quantitative information about the detailed interactions between microbial populations and site characteristics. Because this information may be difficult to obtain, direct modeling is primarily a topic of academic research and is seldom a routinely applied procedure. 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.

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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. 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. While uncertainties should be minimized, it is important to recognize that no strategy can entirely eliminate the uncertainties, even for the best-designed systems. Given today's knowledge base, it is not possible to fully understand every detail of whether and how bioremediation is occurring. The goal in evaluating in situ bioremediation is to assess whether the weight of evidence from tests such as those described above makes a convincing case for successful bioremediation. CONCLUSIONS: FUTURE PROSPECTS FOR BIOREMEDIATION 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

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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. Recommended Steps In Research The committee recommends research in the following areas to improve evaluations of bioremediation: Evaluation protocols. Protocols for putting the three-part evaluation strategy into practice need to be developed and field tested through co-ordinated efforts involving government, industry, and academia. Innovative site characterization techniques. Rapid, reliable, and inexpensive site characterization techniques would simplify many of the evaluation techniques this report describes. Examples of relevant site measurements include distribution of hydraulic conductivities, contaminant concentrations associated with solid or other nonaqueous phases, native biodegradation potential, and abundance of different microbial populations. Improved models. Improvements in mathematical models would increase the ability to link chemical, physical, and biological phenomena occurring in the subsurface and to quantify how much contaminant loss occurs because of biodegradation. Recommended Steps in Education Steps need to be taken to improve the understanding of what bioremediation is and what it can and cannot do. The committee recommends three types of educational steps: Training courses that selectively extend the knowledge bases of the technical personnel currently dealing with the uses or potential uses of in situ bioremediation. This step explicitly recognizes that practitioners and regulators who already are dealing with complicated applications of bioremediation need immediate education about technical areas outside their normal expertise. Formal education programs that integrate the principles and practices for the next generation of technical personnel. This step explicitly recognizes the need to educate a new generation of technical personnel who have far more interdisciplinary training than is currently available in most programs.

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Means for effective transfer of information among the different stakeholders involved in a project. Effective transfer requires that all types of stakeholders participate, that all are invested in achieving a common product (such as a design, a report, or an evaluation procedure), and that sufficient time is allocated for sharing perceptions and achieving the product. This step may involve more time and more intensive interactions than have been the norm in the past. In summary, in situ bioremediation is a technology whose full potential has not been realized. As the limitations of conventional ground water and soil cleanup technologies become more apparent, research into alternative cleanup technologies will intensify. Bioremediation is an especially attractive alternative because it is potentially less costly than conventional cleanup methods, it shows promise for reaching cleanup goals more quickly than pump-and-treat methods, and it results in less transfer of contaminants to other media. However, bioremediation presents a unique technological challenge. The combination of the intricacies of microbial processes and the physical challenge of monitoring both microorganisms and contaminants in the subsurface makes bioremediation difficult to understand, and it makes some regulators and clients hesitant to trust bioremediation as an appropriate cleanup strategy. The inherent complexity involved in performing bioremediation in situ means that special attention must be given to evaluating the success of a project. Whether a bioremediation project is intrinsic or engineered, the importance of a sound strategy for evaluating bioremediation will increase in the future as the search for improved cleanup technologies accelerates.