Bioremediation

By Nathan Ford

Submitted to: Dr. Jones 4/5/1999

Table of Contents
List of Figures and Tables..........................................................................................iii Abstract ....................................................................................................................iv I. Introduction .......................................................................................................... 1 II. Definition and History of Bioremediation ............................................................. 1 III. Principles of Bioremediation ............................................................................... 2 IV. Types of Bioremediation .................................................................................... 4 Ground Water Bioremediation..................................................................... 6 Vadose Zone Bioremediation....................................................................... 8 V. Evaluating (Proving) Bioremediation ................................................................... 9 VI. Advantages and Disadvantages of Bioremediation .............................................13 VII. Conclusion.......................................................................................................13 VIII. Sources ..........................................................................................................15

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List of Figures
Figure 1: Bioremediation Triangle ............................................................................... 2 Figure 2: Water Circulation System ............................................................................. 7 Figure 3: Air Injection System ..................................................................................... 8 Figure 4: Vadose Zone Bioremediation System............................................................ 9

List of Tables
Table 1: Compounds Degraded by Anaerobic Conditions............................................. 3 Table 2: Relationship of Available Oxygen to Bacteria Numbers .................................. 6

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Abstract
The purpose of this paper is to define bioremediation and describe the history of this technology. This paper explains the difference between intrinsic and engineered bioremediation, how bioremediation operates, its current uses for aquifer and vadose zone bioremediation, and its advantages and disadvantages.

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INTRODUCTION Bioremediation is becoming an increasingly popular technology for remediating contaminated sites. The purpose of this paper is to define bioremediation and describe the history of this technology. In addition, this paper will explain the principle role that microbes play in destroying or demobilizing contaminants and what contaminants are susceptible to bioremediation. Next, the current uses of bioremediation will be discussed. However, bioremediation is an extremely broad field so all aspects of this technology will not be considered. This paper will explain the differences between intrinsic bioremediation and engineered bioremediation but will focus primarily on engineered bioremediation for aquifers and the vadose zone. After discussing the current uses of bioremediation, the steps to prove that it is working will be presented along with an example. Next, a discussion of the advantages and disadvantages will be given before a conclusion is reached on the topic of bioremediation. DEFINITION AND HISTORY OF BIOREMEDIATION Bioremediation has been described as a treatability technology that uses biological activity to reduce the concentration or toxicity of a pollutant. It commonly uses processes by which microorganisms transform or degrade chemicals in the environment (King 1). This use of microorganisms (mainly bacteria) to destroy or transform hazardous contaminants is not a new idea. Microorganisms have been used since 600 B.C. by the Romans and others to treat their wastewater. Although this same technology is still used today to treat wastewater it has been expanded to treat an array of other contaminants. In fact, bioremediation has been used commercially for almost 30 years. The first commercial use of a bioremediation system was in 1972 to clean up a Sun Oil pipeline spill

in Ambler, Pennsylvania (National Research Council 47). Since 1972, bioremediation has become a well-developed way of cleaning up different contaminants. A survey prepared by the Environmental Protection Agency in 1992 received information on 240 cases of bioremediation in the United States (Alexander 249). Most of these cases involved treating contaminated soil or groundwater. PRINCIPLES OF BIOREMEDIATION

Figure 1: Bioremediation Triangle (adapted from King 20) There are three essential components needed for bioremediation. These three components are microorganisms, food, and nutrients. These three main components shown in Figure 1 are known as the bioremediation triangle. Microorganisms are found almost everywhere on earth with the exception of active volcanoes. So a lack of food and nutrients are usually the missing ingredients that prevent successful bioremediation. Microorganisms find the food they eat in the soil or water where they live. However, if a contaminant is present it can become an additional food source for the microorganisms. The contaminant serves two useful purposes for the

microbes. First, the contaminant provides a source of carbon needed for growth. Second, the microbes obtain energy by breaking chemical bonds and transferring electrons away from the contaminant. This is known as an oxidation-reduction reaction. The contaminant that loses electrons is oxidized and the chemical that gains the electrons (electron acceptor) is reduced. The energy gained from the electron transfer is used along with the carbon and some electrons to produce more cells. Microbes generally use oxygen as an electron acceptor but nitrate, sulfate, iron, and CO2 are also commonly used. The use of oxygen as an electron acceptor is called aerobic respiration. The major byproducts of aerobic respiration are carbon dioxide, water, and an increase in the microbe population. Anaerobic respiration uses nitrate, sulfate, iron, or CO2 as the electron acceptor instead of oxygen. Anaerobic respiration can occur after the oxygen has been depleted by aerobic respiration or where there is not sufficient oxygen in the first place. The process of anaerobic degradation has been ignored for many years. However, recently it has been gaining more attention. Table 1 shows several contaminants that can be degraded using this anaerobic respiration process. Table 1: Compounds Degraded by Anaerobic Conditions (Alexander 219)

Carbon Tetrachloride Chloroform Vinyl Chloride DDD DDT TCE Tetrachlorethane

Tetrachloroethylene Phenols Benzoates Toluene Ethylbenzene Xylene PCBs

There are also several nutrients that must be accessible to the microorganisms for bioremediation to be successful. These include moisture, nitrogen, phosphorus, and other trace elements. Microorganisms like other organisms need moisture to survive and grow. In addition, microbes depend on the moisture to transport food to them since they do not have mouths. The optimal moisture content for microbes in the vadose zone has been determined to be between 10 and 25% (King 16). Besides moisture, nitrogen (ammonia) and phosphorus (orthophosphate) are two major nutrients needed for the microorganisms. The microorganisms also require minor elements such as sulfur, potassium, magnesium, calcium, manganese, iron, cobalt, copper, nickel, and zinc (King 19). However, these minor elements are usually available in the environment in sufficient amounts where nitrogen and phosphorus may be lacking and need to be added. There are many contaminants susceptible to bioremediation. Petroleum hydrocarbons, in particular, benzene, toluene, ethylbenzene, and xylene (BTEX), the major components of gasoline, have been biodegraded using this technology. In addition, alcohols, ketones, and esters are well established as being biodegradable by microorganisms. Many other contaminants are emerging as treatable using bioremediation such as halogenated aliphatics, halogenated aromatics, polychlorinated biphenyls, and nitroaromatics (National Research Council 30-31). TYPES OF BIOREMEDIATION Bioremediation can be broken into two main types: intrinsic and engineered. Intrinsic bioremediation is also known as natural attenuation or passive bioremediation. Intrinsic bioremediation is preferred to engineered bioremediation primarily because the cost is much lower. Intrinsic bioremediation consists of allowing the natural occurring

microorganisms to degrade the contaminants without implementing any engineered steps to enhance the process. There are four main requirements that must be met for intrinsic bioremediation to be successful. These four requirements are 1. 2. 3. 4. Sufficient microorganisms that can biodegrade the contaminant. Required nutrients are available. Good environmental conditions exist. The time to allow the natural process to degrade the contaminant. This method is different from doing nothing because intrinsic bioremediation must prove that the microorganisms are degrading the contamination faster than the plume is migrating. This requires a network of monitoring devices to determine the location and concentration of the contaminant, the number of microbes, and other appropriate parameters. Engineered bioremediation is also known as enhanced bioremediation. Engineered bioremediation is a process that adds to or enhances the natural process of degradation. Generally it is used when any one of the four necessary conditions for intrinsic bioremediation is not available or when the process needs to be completed faster. An example of engineered bioremediation is to install wells to circulate fluids and nutrients to stimulate the microorganisms. Engineered bioremediation often must add an electron acceptor for the microorganisms to be stimulated. Generally, this electron acceptor is oxygen. Research has shown that there is a strong correlation between the amount of oxygen and the number of bacteria that can grow as shown in Table 2. This increase in the population of bacteria corresponds to a greater quantity of contamination being degraded. Also a larger

population has the ability to degrade the contamination much faster than a smaller population. Table 2: Relationship of Available Oxygen to Bacteria Numbers (Flathman 313) Bacterial Population Density [colony forming units (cfu)/g dry soil] Maximum Available Oxygen (ppm) 8 (air) 40 (O2) 112 (250 ppm H2O2) 200 (500 ppm H2O2) Correlation Coefficient, r Total Heterotrophic Bacteria 5 x 104 5.5 x 106 7.5 x 107 2.1 x 108 .97 Gasoline-Utilizing Bacteria 1 x 102 7 x 105 2.7 x 107 3.1 x 107 .93

There are two basic ways to provide oxygen for bioremediation. These two ways are physically and chemically. The physical method forces air or pure oxygen into the contaminated soil or ground water. The chemical method provides oxygen through the introduction of another substance such as hydrogen peroxide, which then is converted into oxygen. Ground Water Bioremediation There are two main types of bioremediation systems for treating ground water: water circulation and air injection systems. Water circulation systems work by circulating water that contains nutrients and other substances needed to help the microorganisms grow between the injection and recovery wells. Generally as much of the free product as

possible is removed before this process begins. This system injects nutrients such as nitrogen and phosphorus and an electron acceptor (often hydrogen peroxide, H2O2) into the contaminated soil and ground water. The microorganisms biodegrade the contaminants and then the water is removed using a recovery well. The recovered water is then treated with an air stripper to remove any remaining volatile contaminants. In addition, this method has the option of providing an additional above ground treatment facility. The recovered water can be injected into the system again or it can be placed somewhere else and uncontaminated water can be used for injection. This type of system is shown in Figure 2.

Figure 2: Water Circulation System (National Research Council 54) Air injection systems or air sparging is another way to treat contaminated ground water. One of the greatest advantages of this ground water treatment technique is that water does not have to be pumped. This process involves the injection of air directly into the ground

water below the contaminant plume. The air displaces the water in the ground providing the microorganisms with an electron acceptor needed for bioremediation. The air also helps to remove the volatile contaminants that can be captured by using a soil vapor recovery system. If nutrients or water are not present in sufficient quantities they can be provided using an injection well. This system works because air movement helps to mix and distribute the nutrients to the microorganisms as shown in Figure 3.

Figure 3: Air Injection System (National Research Council 58) Vadose Zone Bioremediation Vadose zone bioremediation is similar to an air injection system for treating contaminated ground water. Vadose zone bioremediation differs from a ground water air injection system by injecting the air above the water table instead of below the water table and the contaminant plume. The injection of moisture is also more important since the soil

can become dry due to the circulating air. However, too much moisture can transport the contaminant deeper into the ground and possibly into the ground water. A diagram of a vadose zone bioremediation system is shown in Figure 4.

Figure 4: Vadose Zone Bioremediation System (National Research Council 52) EVALUATING (PROVING) BIOREMEDIATION Proving that a bioremediation project is working requires evidence that the contaminant has decreased due to the microorganisms. Without this evidence the contaminant may have just volatilized, migrated off site, stuck to the soil solids, or changed due to an abiotic reaction. There are several things that help indicate that biodegradation is eliminating the contaminant. However, due to the complex nature of all the processes involved, it is difficult to be absolutely sure that biodegradation is what is destroying the

contaminants. This section focuses on three main strategies that help establish the occurrence of bioremediation. These three strategies are 1. documented loss of contaminants, 2. lab results showing microorganisms have the potential to destroy contaminants, and 3. evidence showing this potential is realized in the field (National Research Council 64). The first two strategies are relatively easy to prove. Soil and water samples from the monitored site will indicate if the contamination is decreasing or not. The laboratory results will also show if the microorganisms can degrade the contaminants under conditions similar to the conditions at the site. The third strategy is harder to prove, but there are several tests that help show that bioremediation is occurring. These include testing soil and water samples to determine the number of bacteria, the number of protozoan, bacterial adaptation, the inorganic carbon concentration along with the carbon isotope ratios, and the electron acceptor concentration. Although there are also other tests, a combination of these tests help indicate that bioremediation is occurring in the field. Whatever tests are used it is important to realize that soil samples are the best because the microorganisms tend to be attached to the solid surfaces. Water samples can be several orders of magnitude lower because they usually only contain the microorganisms that have been dislodged or that are easily transported in the water. Contaminants are an extra food source for the microorganisms. This extra food source frequently causes the microorganisms to reproduce more rapidly. A comparison of the number of bacteria at a similar non-contaminated site to the contaminated site will indicate if the bacteria are reproducing at a greater rate. An increase in the number of bacteria

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along with a decrease in the contaminant concentration indicates that bioremediation may be occurring. Protozoan feed on bacteria. Consequently their population fluctuates with the population of the bacteria. This often means an increase in the number of bacteria will result in an increase in the number of protozoan. This is similar to the relationship that exists between the contaminant and bacteria. An increase in the protozoan generally indicates that the bacteria population has also increased. Bacterial adaptation is also a sign that bioremediation is working. This occurs when the bacteria adapt due to the presence of the contaminant. Often this adaptation allows the bacteria to degrade a contaminant that it previously could not. Bacteria produce inorganic carbon when they degrade organic contaminants. This inorganic carbon can be present in the form of gaseous CO2, dissolved CO2, or HCO3- as a result of the microbial activity. Therefore samples having a higher than normal level of inorganic carbon often indicate that biodegradation is happening. The 13C/12C will help determine the source of this inorganic carbon increase. Since this ratio varies for contaminant degradation, degradation of other organic matter or mineral dissolution. The process of bioremediation consumes electron acceptors as it breaks down the contaminants. So another good indication that there is bioremediation is a decrease in the electron acceptors, usually O2, NO3-, or SO42-. However, this method is only valid for intrinsic bioremediation because engineered bioremediation often adds electron acceptors. These three strategies were applied to an oil and fuel spill in Denver, Colorado. A temporary holding tank under a service garage had leaked crankcase oil, diesel fuel, and gasoline. This leak contaminated the surrounding soil and created a BTEX plume in the

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ground water. The tank was removed and an engineered bioremediation system was implemented in 1989. This system injected oxygen (hydrogen peroxide), phosphorus (phosphate), and nitrogen (ammonium chloride) to encourage bioremediation. After three years most of plume had been eliminated but a small layer of BTEX had been trapped in the aquifer. Although this engineered bioremediation system was not able to eliminate all the contaminant it did eliminate a large portion. The three strategies were able to provide proof that bioremediation was responsible for the decrease in concentration of the contaminant. The three strategies and how they proved bioremediation for this case study: 1. Documented loss of contaminants: The BTEX concentration at the monitoring well closest to the nutrient injection well dropped from 2030 g/l before bioremediation to 6 g/l three years later. Other monitoring wells showed a concentration drop more than an order of magnitude to less than 46 g/l (National Research Council 71). 2. Lab results showing microorganisms have the potential to destroy contaminants: Lab studies showed the microorganisms could consume 7 mg/l of oxygen per day while destroying 2 mg/l of hydrocarbons. The lab results for BTEX were not as extensive since the destruction of BTEX by bioremediation is already well established. 3. Evidence showing this potential is realized in the field: Two tests were performed at this site. The first test showed that the oxygen consumption rate of the microorganisms was the greatest near the contaminants. This suggests that the microorganisms nearest the contaminants had been stimulated by the bioremediation system. The second test compared the ratio of BTEX to total petroleum hydrocarbons (TPH). The ratio was lower in the bioremediated areas than the contaminant source. Past research has shown that microorganisms prefer BTEX to other components of

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TPH. This means the microorganisms leave a TPH residual low in BTEX after successful bioremediation (National Research Council 72). ADVANTAGES AND DISADVANTAGES OF BIOREMEDIATION There are many advantages to bioremediation. The first advantage is the remediation is performed in situ so there are no excavation or transport costs. This is advantageous because the contaminant is destroyed. It is not just moved from one place to another. This minimizes the long-term liability of the party responsible for the contamination. Another advantage is that bioremediation is a natural process and is environmentally friendly. It is also generally cost-effective and can compete with other treatment methods. Bioremediation does have some disadvantages. Bioremediation cannot be used on all contaminated areas because some contaminants are not susceptible to microbial biodegradation. Also, there are some sites where the necessary ingredients for the process cannot be made available to the microorganisms due to the site conditions. Often the greatest problem is that bioremediation involves microorganisms which people cannot see and often do not understand. CONCLUSION: Bioremediation is a powerful tool available to clean up contaminated sites. The idea of bioremediation has a long history. However, other applications are relatively new and many other applications are emerging or being developed. Bioremediation occurs when there are microorganisms present that can biodegrade the given contaminant and the necessary nutrients such as nitrogen, phosphorus, electron acceptors, and trace elements. This process can be aerobic or anaerobic depending on the microorganisms and the electron acceptors available. This process may be natural (intrinsic bioremediation) or it

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may be enhanced by man (engineered bioremediation). Regardless of which aspect of bioremediation that is used; this technology offers an efficient and cost effective way to treat contaminated ground water and soil. Its advantages generally outweigh the disadvantages, which is evident by the number of sites that choose to use this technology and its increasing popularity.

SOURCES Alexander, Martin. Biodegradation and Bioremediation. San Diego, Academic Press, 1994.

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Alvarez-Cohen, Lisa. Engineering Challenges of Implementing In Situ Bioremediation. University of California, Berkeley, California, 1993. Flathman, P., D.E. Jerger, J.H. Exner. Bioremediation Field Experience. Boca Raton, Lewis Publishers, 1994. King, R.B., G.M. Long, and J.K. Sheldon. Practical Environmental Bioremediation The Field Guide. Boca Raton, Lewis Publishers, 1998. National Research Council. In Situ Bioremediation When Does it Work? Washington, D.C., National Academy Press, 1993.

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