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Principles of Bioremediation

The key players in bioremediation are bacteria—microscopic organisms that live virtually everywhere. Microorganisms are ideally suited to the task of contaminant destruction because they possess enzymes that allow them to use environmental contaminants as food and because they are so small that they are able to contact contaminants easily. In situ bioremediation can be regarded as an extension of the purpose that microorganisms have served in nature for billions of years: the breakdown of complex human, animal, and plant wastes so that life can continue from one generation to the next. Without the activity of microorganisms, the earth would literally be buried in wastes, and the nutrients necessary for the continuation of life would be locked up in detritus.

Whether microorganisms will be successful in destroying man-made contaminants in the subsurface depends on three factors: the type of organisms, the type of contaminant, and the geological and chemical conditions at the contaminated site. This chapter explains how these three factors influence the outcome of a subsurface bioremediation project. It reviews how microorganisms destroy contaminants and what types of organisms play a role in in situ bioremediation. Then, it evaluates which contaminants are most susceptible to bioremediation in the subsurface and describes the types of sites where bioremediation is most likely to succeed.



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2 Principles of Bioremediation The key players in bioremediation are bacteria—microscopic organisms that live virtually everywhere. Microorganisms are ideally suited to the task of contaminant destruction because they possess enzymes that allow them to use environmental contaminants as food and because they are so small that they are able to contact contaminants easily. In situ bioremediation can be regarded as an extension of the purpose that microorganisms have served in nature for billions of years: the breakdown of complex human, animal, and plant wastes so that life can continue from one generation to the next. Without the activity of microorganisms, the earth would literally be buried in wastes, and the nutrients necessary for the continuation of life would be locked up in detritus. Whether microorganisms will be successful in destroying man-made contaminants in the subsurface depends on three factors: the type of organisms, the type of contaminant, and the geological and chemical conditions at the contaminated site. This chapter explains how these three factors influence the outcome of a subsurface bioremediation project. It reviews how microorganisms destroy contaminants and what types of organisms play a role in in situ bioremediation. Then, it evaluates which contaminants are most susceptible to bioremediation in the subsurface and describes the types of sites where bioremediation is most likely to succeed.

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THE ROLE OF MICROBES IN BIOREMEDIATION The goal in bioremediation is to stimulate microorganisms with nutrients and other chemicals that will enable them to destroy the contaminants. The bioremediation systems in operation today rely on microorganisms native to the contaminated sites, encouraging them to work by supplying them with the optimum levels of nutrients and other chemicals essential for their metabolism. Thus, today's bioremediation systems are limited by the capabilities of the native microbes. However, researchers are currently investigating ways to augment contaminated sites with nonnative microbes—including genetically engineered microorganisms—specially suited to degrading the contaminants of concern at particular sites. It is possible that this process, known as bioaugmentation, could expand the range of possibilities for future bioremediation systems. Regardless of whether the microbes are native or newly introduced to the site, an understanding of how they destroy contaminants is critical to understanding bioremediation. The types of microbial processes that will be employed in the cleanup dictate what nutritional supplements the bioremediation system must supply. Furthermore, the byproducts of microbial processes can provide indicators that the bioremediation is successful. How Microbes Destroy Contaminants Although bioremediation currently is used commercially to cleanup a limited range of contaminants—mostly hydrocarbons found in gasoline—microorganisms have the capability to biodegrade almost all organic contaminants and many inorganic contaminants. A tremendous variety of microbial processes potentially can be exploited, extending bioremediation's utility far beyond its use today. Whether the application is conventional or novel by today's standards, the same principles must be applied to stimulate the right type and amount of microbial activity. Basics of Microbial Metabolism Microbial transformation of organic contaminants normally occurs because the organisms can use the contaminants for their own growth and reproduction. Organic contaminants serve two purposes for the organisms: they provide a source of carbon, which is one of the basic building blocks of new cell constituents, and they provide electrons, which the organisms can extract to obtain energy.

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FIGURE 2-1 Microbes degrade contaminants because in the process they gain energy that allows them to grow and reproduce. Microbes get energy from the contaminants by breaking chemical bonds and transferring electrons from the contaminants to an electron acceptor, such as oxygen. They "invest" the energy, along with some electrons and carbon from the contaminant, to produce more cells. Microorganisms gain energy by catalyzing energy-producing chemical reactions that involve breaking chemical bonds and transferring electrons away from the contaminant. The type of chemical reaction is called an oxidation-reduction reaction: the organic contaminant is oxidized, the technical term for losing electrons; correspondingly, the chemical that gains the electrons is reduced. The contaminant is called the electron donor, while the electron recipient is called the electron acceptor. The energy gained from these electron transfers is then "invested," along with some electrons and carbon from the contaminant, to produce more cells (see Figure 2-1). These two materials—the electron donor and acceptor—are essential for cell growth and are commonly called the primary substrates. (See Box 2-1 and the glossary for definitions of these and other key terms.) Many microorganisms, like humans, use molecular oxygen (O2) as the electron acceptor. The process of destroying organic compounds with the aid of O2 is called aerobic respiration. In aerobic respiration, microbes use O2 to oxidize part of the carbon in the contaminant to carbon dioxide (CO2), with the rest of the carbon used to produce new cell mass. In the process the O2 gets reduced, produc-

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BOX 2-1 KEY TERMS FOR UNDERSTANDING BIOREMEDIATION Microorganism: An organism of microscopic size that is capable of growth and reproduction through biodegradation of "food sources," which can include hazardous contaminants. Microbe: The shortened term for microorganism. Oxidize: The transfer of electrons away from a compound, such as an organic contaminant. The coupling of oxidation to reduction (see below) usually supplies energy that microorganisms use for growth and reproduction. Often (but not always), oxidation results in the addition of an oxygen atom and/or the loss of a hydrogen atom. Reduce: The transfer of electrons to a compound, such as oxygen, that occurs when another compound is oxidized. Electron acceptor: The compound that receives electrons (and therefore is reduced) in the energy-producing oxidation-reduction reactions that are essential for the growth of microorganisms and bioremediation. Common electron acceptors in bioremediation are oxygen, nitrate, sulfate, and iron. Electron donor: The compound that donates electrons (and therefore is oxidized). In bioremediation the organic contaminant often serves as an electron donor. Primary substrates: The electron donor and electron acceptor that are essential to ensure the growth of microorganisms. These compounds can be viewed as analogous to the food and oxygen that are required for human growth and reproduction. Aerobic respiration: The process whereby microorganisms use oxygen as an electron acceptor. Anaerobic respiration: The process whereby microorganisms use a chemical other than oxygen as an electron acceptor. Common "substitutes" for oxygen are nitrate, sulfate, and iron. Fermentation: The process whereby microorganisms use an organic compound as both electron donor and electron acceptor, converting the compound to fermentation products such as organic acids, alcohols, hydrogen, and carbon dioxide.

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Cometabolism: A variation on biodegradation in which microbes transform a contaminant even though the contaminant cannot serve as the primary energy source for the organisms. To degrade the contaminant, the microbes require the presence of other compounds (primary substrates) that can support their growth. Reductive dehalogenation: A variation on biodegradation in which microbially catalyzed reactions cause the replacement of a halogen atom on an organic compound with a hydrogen atom. The reactions result in the net addition of two electrons to the organic compound. Intrinsic bioremediation: A type of bioremediation that manages the innate capabilities of naturally occurring microbes to degrade contaminants without taking any engineering steps to enhance the process. Engineered bioremediation: A type of remediation that increases the growth and degradative activity of microorganisms by using engineered systems that supply nutrients, electron acceptors, and/or other growth-stimulating materials. ing water. Thus, the major byproducts of aerobic respiration are carbon dioxide, water, and an increased population of microorganisms. Variations on Basic Metabolism In addition to microbes that transform contaminants through aerobic respiration, organisms that use variations on this basic process have evolved over time. These variations allow the organisms to thrive in unusual environments, such as the underground, and to degrade compounds that are toxic or not beneficial to other organisms. Anaerobic Respiration. Many microorganisms can exist without oxygen, using a process called anaerobic respiration. In anaerobic respiration, nitrate (NO3-), sulfate (SO42-), metals such as iron (Fe3+) and manganese (Mn4+), or even CO2 can play the role of oxygen, accepting electrons from the degraded contaminant. Thus, anaerobic respiration uses inorganic chemicals as electron acceptors. In addition to new cell matter, the byproducts of anaerobic respiration may include nitrogen gas (N2), hydrogen sulfide (H2S), reduced forms of metals, and methane (CH4), depending on the electron acceptor.

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Some of the metals that anaerobic organisms use as electron acceptors are considered contaminants. For example, recent research has demonstrated that some microorganisms can use soluble uranium (U6+) as an electron acceptor, reducing it to insoluble uranium (U4+). Under this circumstance the organisms cause the uranium to precipitate, decreasing its concentration and mobility in the ground water. Inorganic Compounds as Electron Donors. In addition to organisms that use inorganic chemicals as electron acceptors for anaerobic respiration, other organisms can use inorganic molecules as electron donors. Examples of inorganic electron donors are ammonium (NH4+), nitrite (NO2-), reduced iron (Fe2+), reduced manganese (Mn2+), and H2S. When these inorganic molecules are oxidized (for example, to NO2-, NO3-, Fe3+, Mn4+, and SO42-, respectively), the electrons are transferred to an electron acceptor (usually O2) to generate energy for cell synthesis. In most cases, microorganisms whose primary electron donor is an inorganic molecule must obtain their carbon from atmospheric CO2 (a process called CO2 fixation). Fermentation. A type of metabolism that can play an important role in oxygen-free environments is fermentation. Fermentation requires no external electron acceptors because the organic contaminant serves as both electron donor and electron acceptor. Through a series of internal electron transfers catalyzed by the microorganisms, the organic contaminant is converted to innocuous compounds known as fermentation products. Examples of fermentation products are acetate, propionate, ethanol, hydrogen, and carbon dioxide. Fermentation products can be biodegraded by other species of bacteria, ultimately converting them to carbon dioxide, methane, and water. Secondary Utilization and Co-metabolism. In some cases, microorganisms can transform contaminants, even though the transformation reaction yields little or no benefit to the cell. The general term for such nonbeneficial biotransformations is secondary utilization, and an important special case is called co-metabolism. In co-metabolism the transformation of the contaminant is an incidental reaction catalyzed by enzymes involved in normal cell metabolism or special detoxification reactions. For example, in the process of oxidizing methane, some bacteria can fortuitously degrade chlorinated solvents that they would otherwise be unable to attack. When the microbes oxidize methane, they produce certain enzymes that incidentally destroy the chlorinated solvent, even though the solvent itself cannot support

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microbial growth. The methane is the primary electron donor because it is the organisms' primary food source, while the chlorinated solvent is a secondary substrate because it does not support the bacteria's growth. In addition to methane, toluene and phenol have been used as primary substrates to stimulate co-metabolism of chlorinated solvents. Reductive Dehalogenation. Another variation on microbial metabolism is reductive dehalogenation. Reductive dehalogenation is potentially important in the detoxification of halogenated organic contaminants, such as chlorinated solvents. In reductive dehalogenation, microbes catalyze a reaction in which a halogen atom (such as chlorine) on the contaminant molecule gets replaced with a hydrogen atom. The reaction adds two electrons to the contaminant molecule, thus reducing the contaminant. For reductive dehalogenation to proceed, a substance other than the halogenated contaminant must be present to serve as the electron donor. Possible electron donors are hydrogen and low-molecular-weight organic compounds (lactate, acetate, methanol, or glucose). In most cases, reductive dehalogenation generates no energy but is an incidental reaction that may benefit the cell by eliminating a toxic material. However, researchers are beginning to find examples in which cells can obtain energy from this metabolic process. Microbial Nutritional Requirements for Contaminant Destruction Regardless of the mechanism microbes use to degrade contaminants, the microbes' cellular components have relatively fixed elemental compositions. A typical bacterial cell is 50 percent carbon; 14 percent nitrogen; 3 percent phosphorus; 2 percent potassium; 1 percent sulfur; 0.2 percent iron; and 0.5 percent each of calcium, magnesium, and chloride. If any of these or other elements essential to cell building is in short supply relative to the carbon present as organic contaminants, competition for nutrients within the microbial communities may limit overall microbial growth and slow contaminant removal. Thus, the bioremediation system must be designed to supply the proper concentrations and ratios of these nutrients if the natural habitat does not provide them. How Microbes Demobilize Contaminants In addition to converting contaminants to less harmful products, microbes can cause mobile contaminants to be demobilized, a strat-

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egy useful for containing hazardous materials. There are three basic ways microbes can be used to demobilize contaminants: Microbial biomes can sorb hydrophobic organic molecules. Sufficient biomass grown in the path of contaminant migration could stop or slow contaminant movement. This concept is sometimes called a biocurtain. Microorganisms can produce reduced or oxidized species that cause metals to precipitate. Examples are oxidation of Fe2+ to Fe3+, which precipitates as ferric hydroxide (FeOH3(s)); reduction of SO42- to sulfide (S2-), which precipitates with Fe2+ as pyrite (FeS(s)) or with mercury (Hg2+) as mercuric sulfide (HgS(s)); reduction of hexavalent chromium (Cr6+) to trivalent chromium (Cr3+), which can precipitate as chromium oxides, sulfides, or phosphates; and, as mentioned previously, reduction of soluble uranium to insoluble U4+, which precipitates as uraninite (UO2). Microorganisms can biodegrade organic compounds that bind with metals and keep the metals in solution. Unbound metals often precipitate and are immobilized. Indicators of Microbial Activity In the process of degrading or demobilizing contaminants, microbes cause changes in the surrounding environment that are important to understand when evaluating bioremediation. Chemical Changes Bioremediation alters the ground water chemistry. These chemical changes follow directly from the physiological principles of microorganisms outlined above. Microbial metabolism catalyzes reactions that consume well-defined reactants—contaminants and O2 or other electron acceptors—converting them to well-defined products. The specific chemical reactants and products can be determined from the chemical equations for the reactions the microbes catalyze. These equations are familiar to anyone with a basic understanding of microbiology. For example, the chemical equation for the degradation of toluene (C7H8) is: Thus, when bioremediation is occurring, the concentration of inorganic carbon (represented by CO2) should increase as the concentrations of toluene and oxygen decrease. Another example is the dechlo-

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rination of trichloroethane (C2H3Cl3, or TCA) to dichloroethane (C 2H4Cl2, or DCA) by hydrogen-oxidizing anaerobic bacteria: Here, TCA and hydrogen (H2) decrease as DCA, hydrogen ion (H+), and chloride ion (Cl-) increase. The formation of hydrogen ion may cause the pH to decrease, depending on the ground water chemistry. In general, under aerobic conditions, one should expect to observe a drop in the O2 concentration when microbes are active. Similarly, under anaerobic conditions, concentrations of other electron acceptors— NO3-, SO42-, Fe3+, Mn4+—will decrease, with a corresponding increase in the reduced species of these compounds (N2, H2S, Fe2+, and Mn2+, respectively). Under both types of conditions the inorganic carbon concentration should increase, because organic carbon is oxidized. The inorganic carbon may take the form of gaseous CO2, dissolved CO2, or bicarbonate ion (HCO3-). Adaptation by Native Organisms In addition to producing chemical changes in the ground water, bioremediation can alter the metabolic capabilities of native microorganisms. Often, microorganisms do not degrade contaminants upon initial exposure, but they may develop the capability to degrade the contaminant after prolonged exposure. Several mechanisms have been proposed to explain metabolic adaptation, including enzyme induction, growth of biodegrading populations, and genetic change. However, these proposals remain largely speculative because methodological limitations usually preclude rigorous understanding of how microbial communities develop, both in laboratory tests and at field sites. Regardless of the mechanisms, adaptation is important because it is a critical principle in ensuring the existence of microorganisms that can destroy the myriad new chemicals that humans have created and introduced into the environment. Adaptation occurs not only within single microbial communities but also among distinct microbial communities that may evolve a co-operative relationship in the destruction of compounds. One community may partially degrade the contaminant, and a second community farther along the ground water flow path may complete the reaction. This type of coupling occurs naturally in anaerobic food chains that convert plant-derived organic compounds to methane. Such coupling has obvious applications for bioremediation of sites bearing contaminant compounds whose complete metabolism may require alternation between anaerobic and aerobic processes.

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Growth of Predators Although bacteria are the agents for biodegradation during bioremediation, other organisms that prey on bacteria also may grow as a result of bioremediation. Protozoa are the most common bacterial predators. Just as mammalian predators, such as wolves, can only be supported by certain densities of their prey, microbial protozoan predators flourish only when their bacterial prey are in large, rapidly replenished supplies. Thus, the presence of protozoa normally signifies that enough bacteria have grown to degrade a significant quantity of contaminants. Complicating Factors The basic principles of how microbes degrade contaminants are relatively straightforward. Yet many details of microbial metabolism are not yet understood, and the successful use of microbes in bioremediation is not a simple matter. A range of factors may complicate bioremediation. Some of the key complicating factors are the unavailability of contaminants to the organisms, toxicity of contaminants to the organisms, microbial preference for some contaminants or naturally occurring chemicals over other contaminants, partial degradation of contaminants to produce hazardous byproducts, inability to remove contaminants to very low concentrations, and aquifer clogging from excessive biomass growth. Unavailability of Contaminants to the Organisms Readily biodegradable contaminants may remain undegraded or be biodegraded very slowly if their concentrations in the ground water are too low. The problem of too low concentrations usually is caused by unavailability, in which the contaminant is sequestered from the microorganisms. Sequestering of organic contaminants can occur when the contaminant is dissolved in a nonaqueous-phase liquid—a solution that does not mix easily with water and therefore travels through the ground separately from the ground water. Sequestering of organic contaminants can also occur if the contaminant is strongly adsorbed to soil surfaces or is trapped in pores too small for circulating ground water to penetrate easily. In these cases, almost all of the contaminant is associated with the solid, the nonaqueous-phase liquid, or the pores, and the very small concentrations that dissolve in the water support very small or zero biodegradation rates.

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Sequestering of metals and other inorganic contaminants occurs most frequently when they precipitate. One possible strategy for overcoming the unavailability problem is to add chemical agents that mobilize the contaminants, causing them to move with the ground water. Such chemical agents are already used at some sites to increase the efficiency of conventional pump-and-treat ground water cleanup systems. However, their use to facilitate bioremediation is more complex than their use for pump-and-treat systems because the mobilizing agents not only affect the physical properties of the contaminants but may also affect the activity of the microorganisms. Organic contaminants can be mobilized by adding surfactants. When only small surfactant concentrations are applied, the surfactant molecules accumulate at solid surfaces, reduce the surface tension, and, in principle, increase the spreading of organic contaminants. This spreading might improve contaminant transfer to the water and thereby accelerate bioremediation, but evidence is not clear for actual subsurface conditions. When large concentrations of surfactant are added, the surfactant molecules join together in colloids, called micelles. Organic contaminants dissolve into the micelles and are transported with the water inside them. However, biodegradation usually is not enhanced by contaminant transfer into the micelles because the true aqueous-phase concentration is not increased. Metals can be mobilized by adding chemicals called complexing agents, or ligands, to which the metals bond. The formation of metalligand bonds dissolves precipitated metals, increasing their mobility. However, the effectiveness of strong ligands, such as ethylene-diaminetetra-acetic acid (EDTA), in enhancing biodegradation is not yet proven. One potential limitation of using ligands to mobilize metals is that microbes may degrade the ligands, releasing the metals and causing them to precipitate again. In some cases, bacteria produce their own surfactants and ligands that are useful in mobilizing trapped contaminants. In these cases the main purpose of the microorganisms is to produce mobilizing agents, not to biodegrade the contaminants. Bacterially mediated mobilization makes trapped contaminants more accessible for cleanup with pump-and-treat technology; it is potentially less costly than injecting commercial surfactants. Toxicity of Contaminants to the Organisms Just as contaminant concentrations that are too low can complicate bioremediation, high aqueous-phase concentrations of some con-

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FIGURE 2-2 The differences between intrinsic and engineered bioremediation. In intrinsic bioremediation, left, native subsurface microbes degrade the contaminants without direct human intervention. In the close-up view, the native microbes use iron (Fe3+) as an electron acceptor to degrade toluene (C7H8), a representative contaminant, and convert it to carbon dioxide (CO2). In engineered bioremediation, right, oxygen (O2), nitrogen (N), and phosphorus (P) are circulated through the subsurface via an injection and extraction well to promote microbial growth. In this case the microbes use oxygen as the electron acceptor, converting it to water (H2O) as they degrade the toluene. Note that, as pictured in the close-up view, considerably more microbes are present in the engineered bioremediation system than in the intrinsic system. Consequently, contaminant degradation occurs more quickly in the engineered system. Intrinsic bioremediation requires extensive monitoring to ensure that the contaminant does not advance more quickly than the native microbes can degrade it. mentation of the role of native microorganisms in eliminating contaminants via tests performed at field sites or on site-derived samples of soil, sediment, or water. Furthermore, the effectiveness of intrinsic bioremediation must be proven with a site-monitoring regime that routinely analyzes contaminant concentrations. The terms "natural," "passive," and ''spontaneous" bioremediation and "bioattenuation"

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BOX 2-2 INTRINSIC BIOREMEDIATION OF A CRUDE OIL SPILL— BEMIDJI, MINNESOTA In August 1979 an oil pipeline burst near Bemidji, Minnesota, spilling approximately 100,000 gallons of crude oil into the surrounding ground water and soil. In 1983 researchers from the U.S. Geological Survey (USGS) began monitoring the site carefully to determine the crude oil's fate. They discovered that, although components of the crude oil initially migrated a short distance, native microorganisms capable of degrading the oil have prevented widespread contamination of the ground water. The microbes went to work with no human intervention, showing that intrinsic bioremediation can be effective for containing spills of petroleum products. In the years following the spill, portions of the crude oil dissolved in the flowing ground water and moved 200 m from the original spill site. The undissolved crude oil itself migrated 30 m in the direction of ground water flow, and crude oil vapors moved 100 m in the overlying soil. However, the USGS researchers' detailed monitoring shows that the contaminant plume has not advanced since 1987, and the researchers have attributed this halt to intrinsic bioremediation. Three types of evidence convinced the researchers that intrinsic bioremediation was largely responsible for containing the crude oil. First, modeling studies showed that if the oil were not biodegradable, the plume would have spread 500 to 1200 m since the spill (see Figure 2-3). Second, the concentrations of Fe2+ and CH4 increased dramatically in the portion of the contaminant plume where oxygen was not present—evidence of an increase in activity by anaerobic organisms capable of degrading certain crude oil components, such as toluene. Third, concentrations of the crude oil components benzene and ethylbenzene, which are susceptible to aerobic degradation but less susceptible to anaerobic degradation, remained relatively stable in the anaerobic portion of the plume but decreased dramatically at the outer edges of the plume, where mixing with oxygenated water allowed aerobic degradation to occur. The evidence from this site shows that, in hydrologic settings where intrinsic bioremediation rates are fast relative to hydrologic transport rates, native microbes can effectively confine contaminants to near the spill source without further human intervention. However, it is essential for such sites to have detailed, long-term monitoring plans to ensure that the contamination is, indeed, contained. At some sites, the rates of hydrologic transport outpace the rates of intrinsic bioremediation, and additional engineering steps to contain or remove the contamination will be necessary.

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FIGURE 2-3 Concentrations of the crude oil components toluene, ethylbenzene, and benzene at various distances from the center of the Bemidji, Minnesota, oil spill. These concentrations have remained relatively stable at the levels shown here since 1987. Note that the contaminant concentrations are very high near the center of the plume but that they drop dramatically within 100 m of the spill. If the contaminants were not biodegradable, this concentration drop would not occur, and the contamination would have spread much farther, as shown by the hypothetical concentration of a nondegradable solute (called a "conservative solute") pictured here. Thus, at this site, intrinsic bioremediation has effectively confined the contamination to a small region. SOURCE: Baedecker et al. (in press), reprinted with permission. References Baedecker, M. J., D. I. Siegal, P. E. Bennett, and I. M. Cozzarelli. 1989. The Fate and Effects of Crude Oil in a Shallow Aquifer: I. The Distribution of Chemical Species and Geochemical Facies. U.S. Geological Survey Water-Resources Investigations Report 88-4220. Reston, Va.: U.S. Geological Survey. Baedecker, M. J., I. M. Cozzarelli, D. I. Siegal, P. E. Bennett, and R. P. Eganhouse. In press. Crude oil in a shallow sand and gravel aquifer: 3. Biogeochemical reactions and mass balance modeling in anoxic ground-water. Applied Geochemistry. Cozzarelli, I. M., R. P. Eganhouse, and M. J. Baedecker. 1991. Transformation of monoaromatic hydrocarbons to organic acids in anoxic ground-water environment. Environmental Geology and Water Sciences 16(2):135-141. Hult, M. F. 1984. Ground-Water Contamination by Crude Oil at the Bemidji, Minnesota Research Site. U.S. Geological Survey Water-Resources Investigations Report 84-4188. Reston, Va.: U.S. Geological Survey.

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have also been used to describe intrinsic bioremediation. Box 2-2 describes a Minnesota site where researchers documented that intrinsic bioremediation prevented the further spreading of crude oil contamination. Engineered bioremediation is the acceleration of microbial activities using engineered site-modification procedures, such as installation of wells to circulate fluids and nutrients to stimulate microbial growth. The principal strategy of engineered bioremediation is to isolate and control contaminated field sites so that they become in situ bioreactors. Other terms used to describe engineered bioremediation include "biorestoration" and "enhanced bioremediation." As summarized in Box 2-3 and described below, the site conditions that influence a bioremediation project's success differ for intrinsic and engineered bioremediation. Site Conditions for Engineered Bioremediation Because engineered bioremediation uses technology to manipulate environmental conditions, the natural conditions are less important for engineered than for intrinsic bioremediation. For engineered bioremediation, the critical property influencing success is how well the subsurface materials at the site transmit fluids. For systems that circulate ground water, the hydraulic conductivity (the amount of ground water that moves through a unit section of the subsurface in a given time) in the area containing the contaminant should be on the order of 10-4 cm/s or greater (the precise value is site specific). For systems that circulate air, the intrinsic permeability (a measure of how easily fluids flow through the subsurface) should be greater than 10-9 cm2. For both types of systems, the contaminated area will be much more difficult to treat if it has crevices, fractures, or other irregularities that allow channeling of fluids around contaminated material. Land near river deltas, floodplains of large rivers, and areas where sand and gravel aquifers were formed from the melting of glaciers can be uniform over large areas. On the other hand, braided stream channels can contain a substantial number of irregularities that complicate bioremediation system design. At high concentrations, contaminants (including petroleum products and chlorinated solvents) that form a nonaqueous-phase liquid can exclude water or air from pores in the subsurface. Nonaqueous-phase liquids restrict access of the remedial fluids and gases and complicate engineered bioremediation. In most cases such contaminants at residual concentrations of less than 8000 to 10,000 mg/kg of soil do not significantly affect water or air flow, because at this level

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BOX 2-3 SITE CHARACTERISTICS THAT FAVOR IN SITU BIOREMEDIATION Engineered bioremediation requires installing wells and other engineering systems to circulate electron acceptors and nutrients that stimulate microbial growth. Key site characteristics for engineered bioremediation are: Transmissivity of the subsurface to fluids: hydraulic conductivity greater than 10-4 cm/s (if the system circulates water) intrinsic permeability greater than 10-9 cm2 (if the system circulates air) Relatively uniform subsurface medium (common in river delta deposits, floodplains of large rivers, and glacial outwash aquifers) Residual concentration of nonaqueous-phase contaminants of less than 10,000 mg/kg of subsurface solids. Intrinsic bioremediation destroys contaminants without human intervention, as the population of native microbes capable of degrading the contaminant increases naturally. The process requires thorough site monitoring to demonstrate that contaminant removal is occurring. Key characteristics of sites amenable to intrinsic bioremediation are: Consistent ground water flow (speed and direction) throughout the seasons: seasonal variation in-depth to water table less than 1 m seasonal variation in regional flow trajectory less than 25 degrees Presence of carbonate minerals (limestone, dolomite, shell material) to buffer pH High concentrations of electron acceptors such as oxygen, nitrate, sulfate, or ferric iron Presence of elemental nutrients (especially nitrogen and phosphorus)

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the contaminants are essentially nonmobile and occupy much less pore space than the water. The specific concentration value at which nonaqueous-phase contaminants begin interfering with fluid circulation varies depending on the contaminant (the value is higher for denser contaminants) and the soil. Site Conditions for Intrinsic Bioremediation If intrinsic bioremediation is the only option, ambient site conditions must be accepted as constraints for meeting cleanup goals, because intrinsic bioremediation by definition occurs without adding anything to the site. Only a fraction of the contaminated sites offer naturally occurring hydrogeochemical conditions in which microorganisms can degrade contaminants quickly enough to prevent them from spreading without human intervention. The critical site characteristic for intrinsic bioremediation is predictability of ground water flow in time and space. Predictable water flow is essential for determining whether the native microbes will be able to act in all the places where the contaminant might travel in all seasons and for determining whether the microbes can act quickly enough to prevent the contamination from spreading with the flowing ground water. The hydraulic gradient and trajectory of ground water flow should be consistent through the seasons and from year to year. To ensure predictability of flow, the fluctuation in the water table should not vary more than about 1 m, although the precise number is site specific. In addition, the trajectory of regional flow should not change by more than about 25 degrees from the primary flow direction. These circumstances are more likely in upland landscapes with humid, temperate climates. In contrast, contaminant plumes in estuaries or the flood plains of large rivers often behave unpredictably. Another valuable characteristic is the presence in the aquifer of minerals such as carbonates to buffer pH changes that would otherwise result from biological production of carbon dioxide or other acids or bases. Carbonates in the aquifer matrix can be expected when limestone or dolomite are the parent material or when limestone dust or sand occurs in glacial outwash. Carbonates can also occur as shell material in beach deposits. Intrinsic bioremediation is more extensive when the ambient ground water surrounding the spill has high concentrations of oxygen or other electron acceptors. The importance of ambient concentrations of nitrate, sulfate, and ferric iron as potential electron acceptors that can stimulate microbial growth in the absence of oxygen is too often

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ignored. Most ground waters have more nitrate and sulfate than oxygen. This is particularly true in agricultural areas that have been overfertilized and in arid regions where gypsum is dissolved in the ground water. The concentration of electron acceptors required to ensure bioremediation varies with the contaminant's chemical characteristics and the amount of contamination. More soluble contaminants and large contaminant sources require larger electron acceptor concentrations. Natural ground water circulation conditions at the site also influence the required amount of electron acceptor. The circulation patterns should provide enough mixing between contaminated water and surrounding water that the organisms never consume all of the electron acceptors within the bioremediation region. If the electron acceptor supply becomes depleted, bioremediation will slow or cease. Also necessary for intrinsic bioremediation is the presence of the elemental nutrients that microbes require for cell building, especially nitrogen and phosphorus. Although nutrients must be present naturally for intrinsic bioremediation to proceed, the quantity of nutrients required is much less than the quantity of electron acceptors. Therefore, a nutrient shortage is less likely to limit intrinsic bioremediation than an inadequate electron acceptor supply. Impact of Site Heterogeneity on Bioremediation Observation of the geological cross section at a typical excavation site reveals a complex patchwork of layers, lenses, and fingers of different materials. Indeed, two overriding characteristics of the subsurface are that it is intricately heterogeneous and difficult to observe. The patterns of variability of the properties that govern the flow of water and the transport of chemicals are so complex that it is not possible to predict these properties quantitatively or even to interpolate them with accuracy from sparse observations. In practice, estimation of subsurface hydrogeochemical properties depends on site-specific measurements from water or soil samples and well tests. However, the inherent unobservability of the system means that there is usually insufficient information to characterize the site with certainty. A consequence of this complexity and heterogeneity, in combination with the poor observability of the subsurface, is that completely reliable prediction of chemical transport and fate is out of reach in most real-world cases. In evaluating a proposed intrinsic or engineered bioremediation scheme, one must consider how it may per-

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form 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. FURTHER READING While this chapter has briefly reviewed the principles underlying successful bioremediation, the references listed in Table 2-2 provide more thorough coverage of the key disciplines related to bioremediation. The list is not exhaustive. The references it provides were selected to represent the diversity of attitudes, perspectives, and paradigms that are pertinent to understanding bioremediation.

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TABLE 2-2 Recommended Sources for Obtaining In-Depth Information About the Disciplines Pertinent to Bioremediation Discipline Reference Synopsis Environmental microbiology Chapelle, F. H. 1993. Ground-Water Microbiology and Geochemistry. New York: John Wiley & Sons. Reviews how the growth, metabolism, and ecology of microorganisms affect ground water chemistry in both pristine and chemically contaminated aquifer systems.   Gibson, D. T. 1984. Microbial Degradation of Organic Compounds. New York: Marcel Dekker. Provides a detailed survey of how microorganisms metabolize organic compounds. Each chapter, written by a different expert, focuses on a different class of compounds.   Madsen, E. L. 1991. Determining in situ biodegradation: facts and challenges. Environmental Science and Technology 25:1661-1673. Reviews principles and limitations of environmental microbiology as they apply to determining in situ biodegradation. Proposes useful approaches, especially as applicable to academic research.   Madsen, E. L., and W. C. Ghiorse. 1993. Ground water microbiology: subsurface ecosystems processes. Pp. 167-213 in Aquatic Microbiology: An Ecological Approach, T. Ford, ed. Cambridge, Mass.: Blackwell Scientific Publishers. Reviews major concepts and methodological developments that determine our understanding of microorganisms and the processes they carry out in subsurface ecosystems.   VanLoosdrecht, M. C. M., J. Lyklema, W. Norse, and A. J. B. Zehnder. 1990. Influences of interfaces on microbial activity. Microbiologic Reviews 54:75-87. Provides a critical and cross-disciplinary review of how surfaces affect microbial activity and substrate availability.

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Hydrobiology Dominic, P. A., and F. W. Schwartz. 1990. Physical and Chemical Hydrogeology. New York: John Wily & Sons. Reviews principles and practice of ground water hydrology, with emphasis on environmental applications.   Freeze, R. A., and J. A. Cherry. 1979. Groundwater. Englewood Cliffs, NJ.: Prentice-Hall. Provides a comprehensive presentation of the theory, principles, and practice of hydrogeology. Environmental engineering McCarty, P. L. 1988. Bioengineering issues related to in situ remediation of contaminated soils and groundwater. Pp. 143-162 in Environmental Biotechnology: Reducing Risks from Environmental Chemicals Through Biotechnology, G. S. Omenn, ed. New York: Plenum Press. Discusses engineering issues relevant to in situ bioremediation.   Rittmann, B. E., A. J. Valocchi, E. Seagren, C. Ray, and B. Wrenn. 1992. A Critical Review of In Situ Bioremediation. Chicago: Gas Research Institute. Provides a comprehensive critical review of the microbiological, engineering, and institutional possibilities and restrictions for in situ bioremediation. Statistics ASCE Task Committee. 1990. Review of geostatistics in geohydrology, parts I and II. ASCE Journal of Hydraulic Engineering 116(5):612-658. Discusses geostatistical techniques and how they can assist in the solution of estimation problems, including interpolation, averaging, and network design.

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Discipline Reference Synopsis Contaminant fate and transport Fetter, C. W. 1993. Contaminant Hydrogeology. New York: Macmillan Publishing Co. Gives a comprehensive treatment of ground water contaminants and their transport, retardation, and transformation in the subsurface. Particularly strong on the chemistry of organic contaminants.   National Research Council. 1990. Ground Water Models. Washington, D.C.: National Academy Press. Provides a thorough review of the theory, use, limitations, and applications of computer modeling applied to the subsurface.   Sahwney, B. L., and K. Brown, eds. 1989. Reactions and Movement of Organic Chemicals in Soils. Madison, Wisc.: Soil Science Society of America. Provides a thorough review of sorption-desorption behavior of contaminants in soil. Includes chapters on contaminant movement and transformation. Commercial application Hinchee, R. E., and R. F. Olfenbuttel, eds. 1992. In Situ Bioreclamation: Applications and Investigations for Hydrocarbon and Contaminated Site Remediation. Boston: Butterworth-Heinemann. Papers in this compendium discuss field and research studies of in situ and on-site bioremediation.