Boettcher, Gary & Nyer, Evan K. "In Situ Bioremediation " In Situ Treatment Technology Boca Raton: CRC Press LLC,2001 ©2001 CRC Press LLC CHAPTER 7 In Situ Bioremediation Gary Boettcher and Evan K. Nyer CONTENTS Introduction Microbiology and Biochemistry Basics Microorganisms Distribution and Occurrence of Microorganisms in the Environment Soil Groundwater Microorganism Biochemical Reactions Energy Production Oxidation and Reduction Aerobic Respiration Facultative Respiration Anaerobic Respiration Microbial Degradation and Genetic Adaptation Gratuitous Biodegradation Cometabolism Microbial Communities Community Interaction and Adaptation Genetic Transfer Growth Growth Cycle Important Environmental Factors that Affect Growth Water pH Temperature Hydrogen Ion Concentration ©2001 CRC Press LLC Oxygen Nutrition Toxic Environments that Affect Growth Microbial Degradation and Modification Degradation Rate Natural Attenuation Application of Natural Attenuation Advantages and Disadvantages of Natural Attenuation Lines of Evidence Site Characterization Petroleum Hydrocarbons and Chlorinated Hydrocarbons Biodegradation of Petroleum Hydrocarbons Biodegradation of Chlorinated Hydrocarbons Biodegradation of the Chlorinated Hydrocarbon Used as an Electron Donor (Carbon and Energy Source) Biodegradation of the Chlorinated Hydrocarbon Used as an Electron Acceptor (Reductive Dechlorination) Cometabolism Abiotic Degradation Natural Attenuation Data Collection and Evaluation Modeling Tools Case Histories Petroleum Hydrocarbon Site Application Chlorinated Hydrocarbon Site Application Geology and Hydrogeology Groundwater Quality Initial Evaluation of Remedial Technologies Evaluation of Natural Attenuation Geochemical Study Reduction of Contaminant Concentrations Plume Retardation Reduction of Contaminant Mass Conclusions Summary References INTRODUCTION In situ bioremediation is a true in situ technology. In this process, biochemical reactions destroy or chemically modify compounds such that these compounds are no longer a threat to human health or the environment. The reactions occur below ground, making this one of the few in place or in situ technologies used today. Chapters 3, 4, and 5 describe physical and chemical in situ technologies that are designed to actively remediate petroleum hydrocarbons, chlorinated hydrocarbons, pesticides, or inorganics (including heavy metals). As discussed in those chapters, ©2001 CRC Press LLC the air used as a carrier during those processes can also stimulate biological reactions. Bioremediation is an important element of most of these remedial solutions where groundwater and soil are impacted with organic or inorganic compounds. However, not all remedial solutions require active soil or groundwater remediation. In recent years, protocols have been developed that are designed to evaluate the environment’s ability to naturally attenuate impacts. This is a powerful remediation technology that will continue to be expanded, refined, and applied where soil and groundwater have been impacted with organic and inorganic compounds. This chapter is divided into two main sections in order to understand bioreme- diation and implement natural attenuation. The first section will focus on biological and chemical processes that are important to understand when designing or operating all in situ biological remediation systems. The second section will focus on natural attenuation processes whereby the environmental conditions and these processes are able to achieve the remediation goals. MICROBIOLOGY AND BIOCHEMISTRY BASICS Microorganisms in soil and groundwater complete biochemical reactions. These reactions often directly or indirectly destroy or modify organic and inorganic chem- icals. These microorganisms are living creatures, and as such, require favorable environmental conditions in order to complete their biochemical reactions and reme- diate organic and inorganic chemicals. Therefore, it is important to have a basic understanding of microorganisms, metabolism, growth, and microbial degradation processes in order to design or evaluate in situ bioremediation systems. This under- standing will allow design engineers to exploit biochemical reactions in soil and groundwater environments and avoid potentially inhibitory conditions. Microorganisms Free-living microorganisms that exist on earth include bacteria, fungi, algae, protozoa, and metazoa. Viruses are also prevalent in the environment; however, these particles can only exist as parasites in living cells of other organisms and will not be discussed in this text. Microorganisms have a variety of characteristics that allow survival and distribution throughout the environment. They can be divided into two main groups. The eucaryotic cell is the unit of structure that exist in plants, metazoa animals, fungi, algae, and protozoa. The less complex procaryotic cell includes the bacteria and cyanobacteria. Even though the protozoa and metazoa are important organisms that affect soil and water biology and chemistry, they do not perform important degradative roles. Therefore, this chapter will concentrate on bacteria and fungi. Bacteria are by far the most prevalent and diverse organisms on earth. There are over 200 genera in the bacterial kingdom (Holt 1981). These organisms lack nuclear membranes and do not contain internal compartmentalization by unit membrane systems. Bacteria range in size from approximately 0.5 micron to seldom greater than 5 microns in diameter. The cellular shape can be spherical, rod-shaped, fila- ©2001 CRC Press LLC mentous, spiral, or helical. Reproduction is by binary fission. However, genetic material can also be exchanged between bacteria. The fungi, which include molds, mildew, rusts, smuts, yeasts, mushrooms, and puffballs, constitute a diverse group of organisms living sometimes in fresh water and marine water, but predominantly in soil or on dead plant material. Fungi are responsible for mineralizing organic carbon and decomposing woody material (cel- lulose and lignin). Reproduction occurs by sexual and asexual spores or by budding (yeasts). Distribution and Occurrence of Microorganisms in the Environment Due to their natural functions, microorganisms are found throughout the envi- ronment. Habitats that are suitable for higher plants and animals to survive will permit microorganisms to flourish. Even habitats that are adverse to higher life forms can support a diverse microorganism population. Soil, groundwater, surface water, and air can support or transport microorganisms. Since this text focuses on in situ treatment, the following briefly describes the distribution and occurrence of micro- organisms in soil and groundwater only. Microorganisms found in soil or groundwater represent the part of the entire population that has flourished under the environmental conditions that are present during the time of sampling. If the environmental conditions are changed by natural or man-made influences, then the microbial population will change in response to the new environment. Chapters 8 and 9 will show how to manipulate the environment in order to change the microbial population and promote new types of biochemical reactions. We will limit this chapter to mainly discussing what is naturally found in the soil and groundwater. Soil Bacteria outnumber the other organisms found in a typical soil. These organisms rapidly reproduce and constitute the majority of biomass in soil. It is estimated that surficial soil can contain some 10,000 different microbial species and can have as many as 109 cells/gm of soil. In addition, cellular biomass can comprise up to approximately 4 percent of the soil organic carbon (Adriano et al. 1999). Micro- organisms generally adhere to soil surfaces by electrostatic interactions, London- van der Waals forces, and hydrophobic interactions (Adriano et al. 1999). Typically, microorganisms decrease with depth in the soil profile, as does organic matter. The population density does not continue to decrease to extinction with increasing depth, nor does it necessarily reach a constant declining density. Fluctuations in density commonly occur at lower horizons. In alluvial soils, populations fluctuate with textural changes; organisms are more numerous in silt or silty clay than in inter- vening sand or course sandy horizons. In soil profiles above a perched water table, organisms are more numerous in the zone immediately above the water table than in higher zones (Paul and Clark 1989). Most fungal species prefer the upper soil profile. The rhizosphere (root zone) contains the most variety and numbers of microorganisms. ©2001 CRC Press LLC Groundwater Microbial life occurs in aquifers. Many of the microorganisms found in soil are also found in aquifers and are primarily adhered to soil surfaces. Bacteria exist in shallow to deep subsurface regions but the origins of these organisms are unknown. They could have been deposited with sediments millions of years ago, or they may have migrated recently into the formations from surface soil. Bacteria tend not to travel long distances in fine soils but can travel long distances in course or fractured formations. These formations are susceptible to contamination by surface water and may carry pathogenic organisms into aquifer systems from sewage discharge, landfill leachate, and polluted water (Bouwer 1978). MICROORGANISM BIOCHEMICAL REACTIONS Microorganisms responsible for degradation of organic environmental impacts obtain energy and building blocks necessary for growth and reproduction from degrading organic compounds. Energy is conserved in the C-C bonds, and during degradation, the organics are converted to simpler organic compounds while deriving energy. Ultimately, the organic compounds are degraded (mineralized) to carbon dioxide or methane, inorganic ions, and water. During the process, microbes use portions of these compounds as building blocks for new microbial cells. As discussed above, microorganism populations can be numerous in soil and groundwater. These populations complete diverse biochemical reactions, and are able to thrive in wide ranges of environmental conditions. In addition, the presence of particular microorganisms and the biochemical reactions that they complete are influenced by the physical and chemical environment. These physical and chemical environments can be modified by organisms creating favorable conditions for a new consortium of organisms and biochemical reactions to occur. Often, different envi- ronmental conditions are created whereby new degradative pathways are induced resulting in the ability to biochemically degrade different organic pollutants, chem- ically modify inorganic compounds, or immobilize inorganic compounds such as heavy metals. The following sections describe important biochemical reactions that site inves- tigators and remediation design engineers should understand. Understanding these reactions will allow remediation teams to determine if biodegradation is likely occurring or if environmental conditions can be modified to create conditions favor- able to degrade or modify environmental pollutants. Failure to understand these concepts can result in remediation systems that limit biological processes, and therefore minimize effectiveness. Energy Production Microorganisms derive energy by degrading a wide variety of organic com- pounds including man-made (xenobiotic or anthropogenic) compounds. Enzymes are induced, respiration occurs, organic compounds are cleaved releasing energy, ©2001 CRC Press LLC intermediate compounds are produced, and growth and reproduction occurs. These processes allow microorganisms to thrive and contribute to the natural cycling of carbon throughout the environment (Figure 1). As seen in Figure 1, microorganisms perform a portion of the overall carbon cycling and it is this portion that bioreme- diation systems rely on to degrade or modify environmental pollutants. Oxidation and Reduction The utilization of chemical energy in microorganisms generally involves what are called oxidation-reduction reactions. For every chemical reaction, oxidation and reduction occurs. Oxidation of a compound corresponds to an oxygen increase, loss of hydrogen, or loss of electrons. Conversely, reduction corresponds to an oxygen decrease, an increase of hydrogen, or an increase in electrons. This process is coupled (half-reactions); if a target chemical is oxidized, another compound must be reduced. In this case, the reactant serves as the electron donor and becomes oxidized, while the other compound serves as the electron acceptor and becomes reduced. In terms of energy released, the electron donor is also an energy source (substrate), whereas the electron acceptor is not an energy source. Once the electron donor has been fully oxidized (lost all the electrons that it can loose) it is usually no longer an energy source but may now serve as an electron acceptor. This is an important concept to understand because biochemical reactions and the ability to degrade or modify compounds are usually dependant on the oxidation state of the target compounds and the predominant biochemical processes that are occurring in soil and groundwater. For example, organic compounds that are in a reduced state, such as aliphatic hydrocarbons, are more likely to be oxidized in the environment. Chemicals that are in an oxidized state, such as highly chlorinated Figure 1 Carbon cycle. ©2001 CRC Press LLC volatile organic compounds, are more likely to be reduced in the environment. In addition, because it is often difficult to directly confirm that degradation is occurring during remediation, it is often necessary to measure indicator parameters in order to determine the predominant biochemical processes that are occurring. The types of electron acceptors used by microorganisms affect the quantity of energy that is available from organics. The energy available from the oxidation- reduction reaction is expressed as the standard electrode potential (oxidation-reduc- tion potential [Eh]) (referenced to hydrogen at pH = 7). Common electron acceptors used to evaluate in situ bioremediation processes are shown in Figure 2. The electron accepting reactions are shown in order of decreasing energy availability. In addition, common organisms responsible for these reactions are also shown (Adriano et al. 1999 and Brock 1979). Aerobic Respiration Aerobic microorganisms have enzyme systems that are capable of oxidizing organic compounds. The organic compound serves as the electron donor and the electrons are transferred to molecular oxygen (O 2 ). This is the most efficient (less energy required) biochemical reaction whereby the electron donor (organic substrate) is degraded producing biomass, carbon dioxide (CO 2 ), water, and potentially other organics as depicted by: electron donor (organic substrate) + O 2 (electron acceptor) → biomass + CO 2 + H 2 O + metabolites + energy. Figure 2 Energy tower for different electron acceptors in biodegradation pH = 7 (Adriano et al. 1999) (Adapted from Brock, 1979). ©2001 CRC Press LLC Facultative Respiration In reduced or low molecular oxygen environments, facultative anaerobes are a class of microorganisms that are able to shift their metabolic pathways and use nitrate (NO 3 - ) as a terminal electron acceptor. This process is called denitrification and is generally depicted as follows: electron donor (organic substrate) + NO 3 - (electron acceptor) → biomass + CO 2 + H 2 O + N 2 + metabolites + energy. The reduction of NO 3 - to nitrogen gas (N 2 ) is completed through a series of electron transport reactions as follows: NO 3 - (nitrate) → NO 2 - (nitrite) → NO (nitic oxide) → N 2 O (nitrous oxide) → N 2 (nitrogen gas) Most denitrifiers are heterotrophic and commonly occur in soil such as Pseudomonas, Bacillus, and Alcaligenes genera. A large number of species can reduce nitritate to nitrite in the absence of oxygen, with a smaller number of species that can complete the reaction by reducing nitrous oxide to nitrogen gas. Anaerobic Respiration Anaerobic respiration is completed by different classes of microorganisms in the absence of molecular oxygen. The anaerobic organisms important to environmental remediation include iron and manganese reducing bacteria, and sulfanogenic and methanogenic bacteria. Anaerobic growth in the environment is not as efficient as aerobic growth (less energy produced per reaction); however, these organisms com- plete important geochemical reactions including bacterial corrosion, sulfur cycling, organic decomposition, and methane production. These reactions are more complex than aerobic respiration and often rely on a consortium of bacteria to complete the reactions. In addition, these classes of bacteria are also capable of either degrading organic pollutants and/or alter environmental conditions whereby chemical reactions can occur. The following depicts the generalized (and simplified) reactions these classes of organisms complete: Iron Reduction: organic substrate (electron donor) + Fe(OH) 3 (electron acceptor) + H 2 + → biomass + CO 2 + Fe 2+ + H 2 O + energy Manganese Reduction: organic substrate (electron donor) + MnO 2 (electron acceptor) + H 2 + → biomass + CO 2 + Mn 2+ + H 2 O + energy ©2001 CRC Press LLC Sulfanogenesis: organic substrate (electron donor) + SO 4 2- (electron acceptor) + H + → biomass + CO 2 + H 2 O + H 2 S + metabolites + energy Methanogenesis: organic substrate + CO 2 (electron acceptor) + H + (electron donor) → biomass + CO 2 + H 2 O + CH 4 + metabolites + energy More detailed information regarding these biochemical reactions can be obtained by reviewing mircobiological texts such as Brock 1979, Stanier, Adelberg, and Ingrahm 1979, and Paul and Clark 1989. Microbial Degradation and Genetic Adaptation In the preceding sections, the reactions associated with degradation and growth were discussed. However, the susceptibility of an environmental pollutant to micro- bial degradation is determined by the ability of the microbial population to catalyze the reactions necessary to degrade the organics. Readily degradable compounds have existed on earth for millions of years; therefore, there are organisms that can mineralize these compounds. Industrial chem- icals (xenobiotic or anthropogenic) have been present on earth for a short time on the evolutionary time scale. Many of these compounds are degradable, and many are persistent in the environment. Some xenobiotic compounds are similar to natural compounds and bacteria will degrade them easily. Other xenobiotic compounds will require special biochemical pathways in order to undergo biochemical degradation. Biodegradation of organic compounds (and maintenance of life sustaining pro- cesses) is reliant on enzymes. The best way to understand enzyme reactions is to think of them as a lock and key. Figure 3 shows how only an enzyme with the right shape (and chemistry) can function as a key for the organic reactions. The lock and key in the real world are three-dimensional. The fit between the two is precise. Organic compounds in the environment that are degradable align favorably with the active site of specific enzymes. The microorganism will not affect compounds that do not align favorably or compounds that do not bind with the active site of their enzyme. Degradation of these compounds requires that the microorganism population adapt in response to the environment by synthesizing enzymes capable of catalyzing degradation of these compounds. A few definitions would be helpful here in order to understand different levels of biological reactions. Biodegradation means the biological transformation of an organic chemical to another form with no extent implied (Grady 1985). Biodegra- dation does not have to lead to complete mineralization. Mineralization is the complete degradation of an organic compound to carbon dioxide or methane and inorganic ions. Recalcitrance is defined as inherent resistance of a chemical to any degree of biodegradation and persistence means that a chemical fails to undergo [...]... -OCH3, -CH3 Alcohols, Aldehydes, Acids, Esters, Amides, Amino Acids Insoluble in Water Relatively Large Many Functional Groups In Oxidized Environment In Reduced Environment Chemically by Man High Molecular Weight Alkanes Branched Chains Polyaromatic Hydrocarbons -F, -Cl, -NO2 -CF3, -SO3H, -NH2 Alkanes, Olefins, Ethers, Ketones, Dicarboxylic Acids, Nitriles, Amines, Chloroalkanes m- or o-position m-disubstituted... oxidants can inhibit microbial growth In addition, oxygen, water, and nutrients can be toxic if added in too high of concentrations Chemical agents such as heavy metals and halogens can disrupt cellular activity by interfering with protein function Mercury ions combine with SH groups in proteins, silver ions will precipitate protein molecules, and iodine will iodinate proteins containing tyrosine residues... hydrogen transfer (Lovley and Goodwin 1988) Nitrate- Fe(III )-, Mn(IV )-, sulfate-, and CO2-reducing (methanogenic) microorganisms exhibit different efficiencies in using the H2 that is continually produced Nitrate reducers are highly efficient H2 utilizers and maintain low steady-state H2 concentrations Fe(III) reducers are slightly less efficient and thus maintain somewhat higher H2 concentrations Sulfate reducers... provides guidance regarding interpreting lines of evidence as described below (USEPA 19 97) In general, more supporting information may be required to demonstrate the efficacy of monitored natural attenuation at those sites with contaminants which do not readily degrade through biological processes (e.g., most non-petroleum compounds, inorganics), at sites with contaminants that transform into more toxic and/or... the most predominant and important attenuation process; however, abiotic mechanisms must also be considered In order to evaluate if the impacts are being degraded, the second line of evidence is usually divided into two parts The first includes completing mass balance calculations which include determining the likely environmental conditions and respiratory pathways occurring, and correlating concentrations... Rings Substitutions on Organic Molecules Substitution Position Number of Hydroxy Group Biphenyl and Dioxins Halogenated Alkanes Substitution of Halogen Derivatives Less Easily Soluble in Water Relatively Small Fewer Functional Groups In Reduced Environment In Oxidized Environment Biologically Aliphatic up to 10 Cchains Straight Chains Aromatic Compounds with One or Two Nuclei -OH, -COOH, -CHO, -CO -OCH3,... Testing Materials, and many state and local regulatory agencies have developed protocols for evaluating natural attenuation as a groundwater remedial solution In addition, the Interstate Technology and Regulatory Cooperation Work Group, In Situ Bioremediation Work Team is a state led, national coalition of personnel from the regulatory and technology programs devoted to deploying and improving innovative... responsible for all treatment Therefore, three primary lines of evidence are used to evaluate if natural attenuation is effectively treating groundwater impacts as follows (USEPA 19 97 and 1998): 1 Historical groundwater and/or soil chemistry data that demonstrate a clear and meaningful trend of decreasing contaminant mass and/or concentration over time at appropriate monitoring or sampling points In the case... site The second line of evidence builds upon the first Once it has been determined that the dissolved contaminant plume is stable, no longer migrating, concentrations are decreasing, or the contaminant mass is decreasing, then the mechanism by which the attenuation is occurring must be determined Therefore, it is necessary to evaluate the likely mechanisms by which the contaminants are being destroyed... containing vitamins (thiamin, biotin, and lipoic acid) (Brock 1 979 ) Several enzymes including those involved in protein synthesis are activated by potassium Magnesium is required for activity of many enzymes, especially phosphate transfer and functions to stabilize ribosomes, cell membranes, and nucleic acids Calcium acts to stabilize bacterial spores against heat and may also be involved in cell wall stability . activity by interfering with protein function. Mercury ions combine with SH groups in proteins, silver ions will precipitate protein molecules, and iodine will iodinate proteins containing tyrosine residues. magnesium, cal- cium, and sodium. Sulfur is used to synthesize two amino acids, cysteine and methionine. Inorganic sulfate is also used to synthesize sulfur containing vitamins (thiamin, biotin, and. Gary & Nyer, Evan K. " ;In Situ Bioremediation " In Situ Treatment Technology Boca Raton: CRC Press LLC,2001 ©2001 CRC Press LLC CHAPTER 7 In Situ Bioremediation Gary