7 Biodegradation

7 BIODEGRADATION/BIOTRANSFORMATION
Biodegradation/biotransformation is biologically mediated reactions - aerobic or anaerobic - that convert harmful organic compounds to carbon dioxide or to less harmful substance. Two main type of reactions are discussed here: 1) organic compound is an electron donator (e.g. BTEX compounds and other aromatic hydrocarbons) and 2) organic compound is electron acceptor (e.g. many chlorinated solvents - PCE, TCE,..). 1) BTEX-compounds (Benzene, Toluene, Ethylbenzene and Xylene) are aromatic hydrocarbons which are present in petroleum fuels and are often indicated as the major contaminants from underground storage tank releases. These compounds are also widely used as solvents, as raw materials for pharmaceutical, agrochemical, polymer, and explosive production. They are also often identified as ground-water contaminants at manufacturing facilities and in landfill leachate. Both aerobic and anaerobic degradation are discussed. 2) For chlorinated ethenes and ethanes (perchloroethene PCE, trichloroethene TCE, dichloroethene DCE, vinyl chloride, TCA trichloroethane, dichloroethane DCA and chloroethane CA) biotransformation via reductive dechlorination is the dominant biotransformation process at most chlorinated solvent sites. Reductive dechlorination is assumed to occur under anaerobic conditions and dissolved solvent degradation is assumed to follow a sequential first-order decay process. Biodegradation of BTEX-compounds: Naturally occurring biological processes can significantly enhance the rate of organic mass removal from contaminated ground water aquifers. Biodegradation of BTEX-compounds is a reaction where the contaminant (e.g. benzene C6H6) is transformed to carbon dioxide CO2 and water. Biologically mediated degradation reactions are oxidation/reduction (redox) reactions, involving the transfer of electrons from the organic contaminant compound to an electron acceptor. Oxygen is the electron acceptor for aerobic metabolism whereas nitrate, ferric iron, sulphate and carbon dioxide serve as electron acceptors for alternative anaerobic pathways. Early conceptual models of natural attenuation were based on the assumption that the anaerobic degradation pathways were too slow to have any meaningful effect on the overall natural attenuation rate at most sites. Accordingly, most field programs focused only on the distribution of oxygen and contaminants, and did not measure the indicators of anaerobic activity such as depletion of anaerobic electron acceptors or accumulation of anaerobic metabolic by-products. Recent research suggests that hydrocarbons are degraded both aerobically and anaerobically in subsurface environments (Rafai et al., 1998). Figs below list the redox reactions for benzene, toluene, ethyl benzene, and xylene (BTEX). In the presence of organic substrate and dissolved oxygen, microorganisms capable of aerobic metabolism will predominate over anaerobic forms. However, dissolved oxygen is rapidly consumed in the interior of contaminant plumes, converting these areas into anoxic (low oxygen) zones. Under these conditions, anaerobic bacteria begin to utilize other electron acceptors to metabolize dissolved hydrocarbons. The principle factors influencing the utilization of the various electron acceptors include: 1) the relative biochemical energy provided by the reaction; 2) the availability of individual or specific electron acceptors at a particular site; and 3) the kinetics (rate) of the microbial reaction associated with the different electron acceptors.
Last update 18.12.2009. Original material by Tuomo Karvonen, 2002. Department of Civil and Environmental Engineering, Helsinki University of Technology http://civil.tkk.fi
7.1 Benzene
The oxidation/reduction reactions for benzene are shown below (Rafai et al. 1998): Oxidation reaction:
Reduction reaction:
Overall reaction:
7.2 Toluene
The oxidation/reduction reactions for toluene are shown below (Rafai et al. 1998): Oxidation reaction:
Reduction reaction:
Overall reaction:
7.3 Ethylbenzene and xylene

The oxidation/reduction reactions for ethylbenzene and xylene are shown below (Rafai et al. 1998): Oxidation reaction:
Reduction reaction:
Overall reaction:
The transfer of electrons during the redox reaction releases energy which is utilized for cell maintenance and growth. The biochemical energy associated with alternative degradation pathways can be represented by the redox potential of the alternative electron acceptors: the more positive the redox potential, the more energetically favourable is the reaction utilizing that electron acceptor. With everything else being equal, organisms with more efficient modes of metabolism grow faster and therefore dominate over less efficient forms. ________________________________________________________________________________ Table 7-1.
Based solely on thermodynamic considerations, the most energetically preferred reaction should proceed in the plume until all of the required electron acceptor is depleted. At that point, the next most-preferred reaction should begin and continue until that electron acceptor is gone, leading to a
pattern where preferred electron acceptors are consumed one at a time, in sequence. Based on these principles, one would expect to observe monitoring well data with "no-detect" results for the more energetic electron acceptors, such as oxygen and nitrate, in locations where evidence of less energetic reactions is observed (e.g. monitoring well data indicating the presence of ferrous iron). In practice, however, it is unusual to collect samples from natural attenuation monitoring wells that are completely depleted in one or more electron acceptors. Two processes are probably responsible for this observation (Rafai et al. 1998): 1. Alternative biochemical mechanisms having very similar energy potentials (such as aerobic oxidation and nitrate reduction) may occur concurrently when the preferred electron acceptor is reduced in concentration, rather than fully depleted. Similarly noting the nearly equivalent redox potentials for sulphate and carbon dioxide (-220 volts and -240 volts, respectively) one might expect that sulphate reduction and methanogenic reactions may also occur together. 2. Standard monitoring wells, having 1.5 to 3 m screened intervals, will mix waters from different vertical zones. If different biodegradation reactions are occurring at different depths, then one would expect to find geochemical evidence of alternative degradation mechanisms occurring in the same well. If the dissolved hydrocarbon plume is thinner than the screened interval of a monitoring well, then the geochemical evidence of electron acceptor depletion or metabolite accumulation will be diluted by mixing with clean water from zones where no degradation is occurring.
7.4 Kinetics of the Biodegradation Reactions

Aerobic biodegradation can be simulated as an "instantaneous" reaction that is limited by the amount of electron acceptor (oxygen) that is available. The microbial reaction is assumed to occur at a much faster rate than the time required for the aquifer to replenish the amount of oxygen in the plume. Although the time required for the biomass to aerobically degrade the dissolved hydrocarbons is on the order of days, the overall rate that groundwater is replenished in most plumes is on the order of years or tens of years. For example, microcosm data presented by Davis et al. (1994) show that microbes that have an excess of electron acceptors can degrade concentrations of dissolved benzene (~1 mg/L) very rapidly. In the presence of a surplus of oxygen, aerobic bacteria can degrade dissolved benzene in about 8 days, which can be considered "instantaneous" compared to years required for flowing ground water to replenish the plume area with oxygen. Recent results from the Air Force Natural Attenuation Initiative indicate that the anaerobic reactions, which were originally thought to be too slow to be of significance in ground water, can also be simulated as instantaneous reactions (Newell et al., 1995). For example, Davis et al. (1994) also ran microcosms with sulphate reducers and methanogens that indicated that benzene could be degraded within a couple of weeks time frame (after acclimation). When compared to the time required to replenish electron acceptors, the anaerobic reactions can also be considered to be instantaneous at many sites. This conclusion is supported by observing the pattern of anaerobic electron acceptors and by-products along the plume at natural attenuation research sites.
7.5 Biodegradation of chlorinated solvents (chlorinated ethenes and ethanes)

"Natural Attenuation" refers to naturally-occurring processes in soil and ground-water environments that act without human intervention to reduce the mass, toxicity, mobility, volume, or concentration of contaminants in those media. Biotransformation can often be a dominant process in the natural attenuation of chlorinated solvents. At chlorinated solvent contaminated sites, most of the solvent degradation occurs by reductive dechlorination (U.S.EPA, 1998). Reductive dechlorination is a microbially-mediated reaction whereby a chlorine atom on the chlorinated solvent is replaced by a hydrogen atom (Vogel and McCarty, 1987). In many bioremediation processes, an organic contaminant (such as benzene) acts as an electron donor and another substance (such as oxygen, nitrate, etc.) acts as the electron acceptor. However, during reductive dechlorination, hydrogen acts as the electron donor and halogenated compounds, such as chlorinated solvents, act as electron acceptors and thus become reduced, as shown in the following half reaction (Aziz & al. 2000): R-Cl + H+ + 2e- R-H+ ClFigure 7-1 shows the reductive transformation pathways for the common chlorinated aliphatics.
Fig. 7-1. Reductive dechlorination pathways for common chlorinated aliphatic hydrocarbons (after Vogel and McCarty, 1985; Vogel and McCarty, 1987).
Fig. 7-2. Reductive dechlorination of perchloroethene PCE to trichloroethene TCE.

Fig. 7-3. Reductive dechlorination of chlorinated ethanes. For biological reductive dechlorination to occur, the following conditions must exist: 1. The subsurface environment must be anaerobic and have a low oxidation-reduction potential (ORP). 2. Chlorinated solvents that are amenable to reductive dechlorination must be present. 3. A population of dechlorinating bacteria must be present. 4. An adequate supply of fermentation substrates to produce dissolved hydrogen must be present. The environmental chemistry and the ORP of a site play an important role in determining whether reductive dechlorination will occur. Based on thermodynamic considerations, reductive dechlorination will occur only after both oxygen and nitrate have been depleted from the aquifer, because oxygen and nitrate are more energetically favourable electron acceptors than chlorinated solvents when hydrogen is the electron donor (U.S. EPA, 1998). According to Rafai et al. (1998), the role of hydrogen as an electron donor during reductive dechlorination is now widely recognized as a key factor governing the dechlorination of chlorinated compounds. The hydrogen is produced in the terrestrial subsurface by the fermentation of a wide variety of organic compounds including anthropogenic compounds such as petroleum hydrocarbons and natural organic matter. Hydrogen is then used by the dechlorinating bacteria as an electron donor. Although the chlorinated solvents degrade primarily via reductive dechlorination, which occurs under highly reduced anaerobic conditions, some of the chlorinated solvents may degrade under aerobic conditions. TCE, c-DCE and VC degrade cometabolically (McCarty and Semprini, 1994) and VC (Hartmans et al., 1985; Hartmans and de Bont, 1992) and possibly c-DCE (Bradley and Chapelle, 1998) can be directly
oxidized to carbon dioxide under aerobic conditions. PCE has not been found to degrade aerobically (McCarty and Semprini, 1994).
7.6 Modelling of biodegradation of BTEX-compounds

Biodegradation Models for BTEX-compounds To apply an electron-acceptor limited kinetic model, such as the instantaneous reaction model, the amount of biodegradation that the groundwater that moves through the source zone can support must be calculated. The conceptual model is that: 1. Ground water upgradient of the source contains electron acceptors; 2. As the upgradient ground water moves through the source zone, hydrocarbons in NAPLs (Non-Aqueous Phase Liquids) and contaminated soil release dissolved hydrocarbons (in the case of petroleum sites, BTEX); 3. The biological reactions continue until the available electron acceptors are consumed 4. The total amount of available electron acceptors available for biological reactions can be estimated by: a) calculating the difference between the upgradient wells and source zone wells for oxygen, nitrate, and sulphate; and b) measuring the production of by-products ferrous iron and methane in the source zone; 5. Using stoichiometry, a utilization factor can be developed to convert the mass of oxygen, nitrate, and sulphate consumed to the mass of dissolved hydrocarbon that is used in the biodegradation reactions. Similarly, utilization factors can be developed to convert the mass of metabolic by-products that are consumed to the mass of dissolved hydrocarbon that are used in the biodegradation reactions. 6. For a given background concentration of an individual electron acceptor, the potential contaminant mass removal or "biodegradation capacity" depends on the "utilization factor" for that electron acceptor. Dividing the background concentration of an electron acceptor by its utilization factor provides an estimate (in concentration units) of the assimilative capacity of the aquifer by that mode of biodegradation. When the available electron acceptor/byproduct concentrations (Step 4) are divided by the appropriate utilization factor (Step 5), an estimate of the "biodegradation capacity" of the groundwater flowing through the source zone and plume can be developed. Example: Utilization factor for benzene: Oxygen: 1 mole benzene reacts with 7.5 moles oxygen or (6x12 + 6) grams of benzene react with (7.5x32) grams of oxygen; 78 gms benzene react with 240 gms of oxygen Utilization Factor = 240/78 = 3.08 Nitrate 1 mole benzene reacts with 6 moles nitrate: Utilization Factor = 372.06/78 = 4.77
7.7 Conceptual Model for Biodegradation (BIOPLUME III as an example; Rafai et al. 1999)
The main electron acceptors include oxygen for aerobic biodegradation and nitrate, iron (III), sulphate, and carbon dioxide for anaerobic biodegradation. Manganese has also been identified as an anaerobic electron acceptor; however, manganese has not been incorporated into the current version of BIOPLUME III. The conceptual model used in BIOPLUME III to simulate these biodegradation processes tracks six plumes simultaneously: hydrocarbon, oxygen, nitrate, iron (II), sulphate, and carbon dioxide. Iron (III) is input as a concentration matrix of ferric iron in the formation. Once ferric iron is used for biodegradation, BIOPLUME III simulates the production and transport of ferrous iron. Biodegradation occurs sequentially in the following order: Oxygen Nitrate Iron (III) Sulphate Carbon Dioxide The biodegradation of hydrocarbon in a given location using nitrate, for example, can only occur if oxygen has been depleted to its threshold concentration at that location. Three different kinetic expressions can be utilized for the biodegradation reaction for each of the electron acceptors: 1. First-order decay 2. Instantaneous reaction 3. Monod kinetics The first-order decay model implemented e.g. in BIOPLUME III (Rafai et al.) for any of the electron acceptors is limited by the availability of the electron acceptor in question. In other words, the model allows the first-order reaction to take place up to the point that the electron acceptor concentration available in the aquifer has been depleted. The Monod kinetic model assumes a constant microbial population for each of the aerobic and anaerobic reactions and does not simulate the growth, transport and decay of the microbial population in the subsurface.
7.7.1 Monod kinetics (Michaelis-Menten kinetics)

A popular expression for simulating biological processes (e.g. biodegradation) is the Monodfunction referred also to as Michaelis-Menten kinetics: =MAXC/(Kc+C) (7-1)
where is the growth rate of biomass [1/d], MAX is the maximum growth rate [1/d], C is concentration of growth-limiting substrate [mg/l] and K c is half-saturation coefficient [mg/l] which is the concentration which allows the microorganism to grow at half the maximum growth rate. In groundwater, the Monod growth function is related to the rate of decrease of an organic compound. The reduction of contaminant concentrations using Monod kinetics can be expressed as (Wiedemeier et al. 1999): dC/dt = Mt MAXC/(Kc+C) (7-2)
where C is the contaminant concentration, Mt is the total microbial concentration, MAX is the maximum contaminant utilization per unit mass microorganisms and Kc is the contaminant halfsaturation constant.
7.7.2 Limitations
The limitations/difficulties involved in the modelling of biodegradation of BTEX-compounds 1. It is difficult to take into account the selective or competitive biodegradation of the hydrocarbons. This means that hydrocarbons are generally simulated as a lumped organic which represents the sum of benzene, toluene, ethyl benzene or xylene. If a single component is to be simulated, the user would have to determine how much electron acceptor would be available for the component in question. 2. The conceptual models for biodegradation are simplifications of the complex biologically mediated redox reactions that occur in the subsurface. Exercise 7-1 Consider biodegradation of benzene in saturated groundwater. Biodegradation follows first-order reaction: dC/dt = -kBC and initial concentration C(t=0) = 2000 g/l Biodegradation coefficient is assumed to be constant; k B = 0.005 d-1. Other processes are neglected here. Exact solution is C(t) = exp(-kBt)*C(0) a) How long time does it take that concentration falls below the drinking water limit 5 g/l? b) Degradation equation can be solved numerically by approximating the differential equation as follows: [Ct+1 - Ct]/ t = - kB[ Ct+1 + (1- )Ct] (7-3)
where Ct+1 is the unknown value at time t+t, Ct is the known value at time t is the coefficient of implicity; if =0, the solution is explicit, if =0.5, the approximation is called Crank-Nicolson and if =1, the solution is fully implicit. What is the error (%) of the numerical solution after 3600 days as compared to the analytical solution when a) explicit, b) Crank-Nicolson and c) fully implicit solution is used. Time step t=30 d. c) Optimal value for the weighting coefficient OPT can be calculated using the method given in section 5.2.6 Computation of first-order reaction (biodegradation). 1) What is the optimal value for if input data from b) are used? 2) What is the optimal value for if time step is 300 d? Show that the numerical solution method gives accurate solution if optimal value for is used for both t=30 d, and t=300 d. Exercise 7-2 Calculate utilization factors for toluene both in aerobic degradation and in anaerobic degradation reactions (nitrate, manganese, iron(III), sulphate and methanogenic reaction).
7.8 Sequential model for chlorinated solvents

Reductive dechlorination can be modelled as a sequential first-order decay process. This means that a parent compound undergoes first-order decay to produce a daughter product and that product undergoes first-order decay and so on. Generally, the more highly chlorinated the compound, the more rapidly it is reduced by reductive dechlorination (Vogel and McCarty, 1985; Vogel and McCarty, 1987). Kinetics of Sequential First Order Decay Although the chlorinated ethenes primarily degrade biologically, chlorinated ethanes can degrade both biologically and abiotically. For chloroethane (CA), abiotic decay to ethanol occurs much more rapidly than biotransformation to ethane. The abiotic decay of 1,1-DCA is slow relative to biotransformation. 1,1,1-TCA can degrade abiotically to both acetic acid (by hydrolysis) and to 1,1DCE (by elimination) (Vogel and McCarty, 1987).
7.9 Chlorinated Ethenes

The reaction rate equations describing the sequential first order decay of the chlorinated ethenes are shown below:
(7-4) where 1, 2, 3, 4 and E, are the first order biotransformation rate coefficients, y1, y2, y3, and y4 are the daughter: parent compound molecular weight ratios and CPCE, CTCE, CDCE, CVC and CETH are the aqueous concentration of PCE, TCE, DCE, vinyl chloride VC, and ethene, respectively. From these expressions, it is clear that TCE, DCE, and VC are simultaneously being produced and degraded, which often results in net accumulation before observed degradation. Furthermore, these reaction expressions cause the reactive transport equations to be coupled to each other as discussed. General transport equation for chlorinated solvents:
(7-5) so that r1=rPCE, r2=rTCE, r3=rDCE, r4=rVC, r5=rETH. Transport of chlorinated ethenes can be calculated by solving a set of coupled partial differential equations to describe the reactive transport of chlorinated solvent species, such as PCE, TCE, DCE, VC and ETH, in saturated ground-water systems. The equations describe one-dimensional advection, three-dimensional dispersion, linear sorption, and sequential, first-order
biotransformation. All equations, except the first, are coupled to a parent species equation through the reaction term as shown below:
(7-6) where C1, C2, C3, C4 and C5 concentrations of PCE, TCE, DCE, VC, and ETH, respectively [mg/L], Dx , Dy and Dz are the hydrodynamic dispersion coefficients [m2/d], vs is the seepage velocity [m/d], i, i=1,..,5 are the first-order degradation coefficients [1/d], yi, i=1, ,5 are the yield coefficients (dimensionless values, for example, y1 would represent the mg of TCE produced per unit mg of PCE destroyed); and Ri, i=1, ,5 are respective retardation factors.
7.10 Chlorinated Ethanes

The following are the rate expressions for the degradation of the chlorinated ethanes.
(7-7) where 5, 6, and 7 are the first order biotransformation rate coefficients, A is the abiotic rate coefficient for chloroethane, y5 and y6 are the daughter: parent compound molecular weight ratios and CTCA, CDCA and CCA and the concentration of 1,1,1-trichloroethane, 1,1-dichloroethane and chloroethane, respectively. Yield constants (i.e., y1, y2, y6) are incorporated to account for molecular weight differences between parent and daughter compounds. The constants are necessary because kinetic expressions are valid on a molar basis only. Exercise 7-3 Compute the yield coefficients of chlorinated ethenes and ethanes y1,..y6.
Exercise 7-4 Use EXCEL to create a model for calculating the transport and sequential biodegradation of chlorinated ethenes. Compute advection, adsorption (linear isotherm) and biodegradation. Assume 1-D case, neglect dispersion in all directions (Dx=Dy=Dz=0) and use only one storage to describe the aquifer (i.e. an ordinary differential equation is used for each compound). Input data: Pore water velocity v = 0.5 m/d Aquifer length dx=15 m Retardation factor R same for all compounds; R=3.5 Biodegradation coefficients: PCE = 0.0055 1/d, TCE = 0.0041 1/d, DCE =0.0022 1/d, VC = 0.0018 1/d and ETH=0.0. Concentration is zero for all compounds at time t=0 Source area concentrations (constant) are: CPCE = 10 mg/l, CTCE = 40 mg/l, CDCE = 100 mg/l, CVC = 5 mg/l, CETH = 0.1 mg/l Yield coefficients y1,..y4 as computed in Ex. 7-3. Calculate a period of five years using explicit solution method and select a time step length that ensures numerical stability of the solution. Note! Input concentration of the aquifer is the source area concentration.
7.11 Remediation by Natural Attenuation (RNA)

7.11.1 Fundamentals of Natural Attenuation (Intrinsic Remediation)
Naturally occurring biological processes can significantly enhance the rate of organic mass removal from contaminated aquifers. Biodegradation research performed in USA by universities, government agencies, and other research groups has identified several main themes that are crucial for future studies of natural attenuation (Rafai et al. 1998): 1. The relative importance of groundwater transport vs. microbial kinetics is a key consideration for developing workable biodegradation expressions in models. Results from the United Creosote site (Texas) and the Traverse City Fuel Spill site (Michigan) indicate that biodegradation is better represented as a macro-scale wastewater treatment-type process than as a micro-scale study of microbial reactions. 2. The distribution and availability of electron acceptors control the rate of in-situ biodegradation for most petroleum release site plumes. Other factors (e.g., population of microbes, pH, temperature, etc.) rarely limit the amount of biodegradation occurring at these sites. Naturally-occurring processes in soil and ground-water environments reduce the mass, toxicity, mobility, volume, or concentration of contaminants in those media. These in-situ processes include biotransformation, dispersion, dilution, adsorption, volatilization, and chemical or biological stabilization or destruction of contaminants (U.S. EPA, 1998).
7.11.2 How to use models in Remediation by Natural Attenuation?

Models are generally used to answer a number of questions regarding natural attenuation: 1. How long will the plume extend if no engineered/source controls are implemented? 2. How long will the plume persist until natural attenuation processes completely dissipate the contaminants? 3. How long will the plume extend or persist if some engineered controls or source reduction measures are undertaken (for example, free phase removal or residual soil contamination removal)? The model can also be used to simulate bioremediation of BTEX-hydrocarbons in ground water by injecting electron acceptors (except for iron(III)) and can also be used to simulate air sparging for low injection air flow rates. Finally, the model can be used to simulate advection, dispersion, and sorption without including biodegradation. The models will predict the maximum extent of dissolved-phase plume migration, which may then be compared to the distance to potential points of exposure (e.g. drinking water wells, ground-water discharge areas, or property boundaries). Models can be used in RNA studies in two ways: 1. As a screening-level model to determine if RNA is feasible at sites contaminated with BTEX-compounds or with chlorinated solvents. Model is intended to be used as a screening-level model to determine if natural attenuation is occurring at sufficient rates at a site to warrant a full natural attenuation study. Ideally, site-specific biotransformation rate constants should be employed, but literature values can be used if measured rate constants are unavailable. 2. As an RNA ground-water model to address selected chlorinated solvent problems A mathematical model is an appropriate tool at sites where simplifying assumptions (e.g. uniform ground-water flow, a vertical plane source, first-order decay) can be made so that the resulting simulations provide useful information for the problem being addressed. In any modelling study, it is recommended that proper care should be used to select the model that is best suited to 1) the source, hydrogeology, and biotransformation processes present at the site and, 2) the type of problem being addressed (e.g. screening of alternatives, providing supporting evidence of natural attenuation, developing detailed design information). A screening model has the following limitations: 1. It assumes simple ground-water flow conditions. The model should not be applied where pumping systems create a complicated flow field. In addition, the model should not be applied where vertical flow gradients affect contaminant transport. 2. A screening tool assumes uniform hydrogeologic and environmental conditions over the entire model area. Moreover, it assumes constant source, hydrogeological, and biological property values for the entire model area and, therefore, simplifies actual site conditions. For this reason, the screening model should not be applied where extremely detailed, accurate results that closely match site conditions are required. More comprehensive numerical models should be applied in such cases.
7.11.3 Natural Attenuation Lines of Evidence

To support remediation by natural attenuation, it must be scientifically demonstrated that attenuation of the site contaminants is occurring at rates sufficient to be protective of human health and the environment. According to the "Technical Protocol for Evaluating Natural Attenuation of Chlorinated Solvents in Ground Water" (U.S. EPA, 1998), three lines of evidence can be used to support natural attenuation of chlorinated solvents including: 1. Observed reductions in contaminant concentrations along the flow path downgradient from the source of contamination 2. Documented loss of contaminant mass at the field scale using: a) Chemical and geochemical analytical data including decreasing parent compound concentration, increasing daughter compound concentrations, depletion of electron acceptors and donors, and increasing metabolic by-product concentrations; and/or b) A rigorous estimate of residence time along the flow path to document contaminant mass reduction and to calculate biological decay rates at the field scale. 3. Laboratory microcosm or field data that support the occurrence of biotransformation and give rates of biotransformation. At a minimum, the investigator must obtain the first two lines of evidence or the first and third lines of evidence. The second or third line of evidence is crucial because it provides biotransformation rate constants. These rate constants can be used in conjunction with other fate and transport parameters to predict contaminant concentration and to assess risk at a downgradient point of exposure (U.S. EPA, 1998). Compared to fuel hydrocarbon plumes, use of natural attenuation as a stand-alone remedy for chlorinated solvent plumes is appropriate for a much lower percentage of plumes, because of their longer plume lengths.
7.12 Ground Water Characterization measurements (According to Rafai et al., 1998)

Dissolved Oxygen Dissolved oxygen is the most thermodynamically favoured electron acceptor used in the biodegradation of fuel hydrocarbons. Dissolved oxygen concentrations are used to estimate the mass of contaminant that can be biodegraded by aerobic processes. As a rule, the stoichiometric ratio of dissolved oxygen consumed by microbes to destroyed BTEX compound is 1.0 mg/L of dissolved oxygen consumed to approximately 0.32 mg/L of BTEX compounds destroyed. During aerobic biodegradation, dissolved oxygen levels are reduced as aerobic respiration occurs. Also, anaerobic bacteria (obligate anaerobes) generally cannot function at dissolved oxygen levels greater than about 0.5 mg/L. Therefore, higher values of dissolved oxygen indicate that aerobic biodegradation is likely at work. Redox potential The oxidation/reduction (redox) potential of ground water (EH) is a measure of electron activity and is an indicator of the relative tendency of a solution to accept or transfer electrons. Redox reactions in ground water are usually biologically mediated and therefore, the redox potential of a ground water system depends upon and influences rates of biodegradation. Knowledge of the redox potential of ground water is also important because some biological processes only operate within a prescribed range of redox conditions. Knowledge of the redox potential of ground water can be used as an indicator of certain geochemical activities such as sulphate reduction. The redox potential of ground water generally ranges from -400 millivolts [mV] to 800 mV. Redox potential can be used to provide real time data on the location of the contaminant plume, especially in areas undergoing anaerobic biodegradation. Mapping the redox potential of the ground water while in the field allows
the field scientist to determine the approximate location of the contaminant plume. To map the redox potential of the ground water while in the field it is important to have at least one redox measurement (preferably more) from a well located upgradient of the plume. The redox potential of a ground water sample taken inside the contaminant plume should be somewhat lower than that taken in the upgradient location. Redox potential measurements should be taken during well purging and immediately before and after sample acquisition using a direct-reading meter. Because most well purging techniques can allow aeration of collected ground water samples (which can affect redox potential measurements), it is important to minimize potential aeration. pH, Temperature, and Conductivity Because the pH, temperature, and electric conductivity of a ground water sample can change significantly within a short time following sample acquisition, these parameters must be measured in the field in unfiltered, unpreserved, "fresh" water collected by the same technique as the samples taken for laboratory analyses. The measurements should be made in a clean glass container separate from those intended for laboratory analysis and the measured values should be recorded in the ground water sampling record. The pH of ground water has an effect on the presence and activity of microbial populations in ground water. This is especially true for methanogens which may be active after all aerobic, sulphate reduction, and nitrate reduction degradation has taken place. Microbes capable of degrading petroleum hydrocarbon compounds generally prefer pH values varying from 6 to 8 standard units. Electric conductivity is a measure of the ability of a solution to conduct electricity. For ground water, conductivity is directly related to the concentration of ions in solution, increasing as ion concentration increases. Like chloride, conductivity is used to ensure that ground water samples collected at a site are representative of the water comprising the saturated zone in which the dissolved-phase contamination is present. If the conductivities of samples taken from different sampling points are radically different, then the waters may be from different hydrogeologic zones. Ground water temperature directly affects the solubility of oxygen and other geochemical species. The solubility of dissolved oxygen is temperature dependent, being more soluble in cold water than in warm water. Ground water temperature also affects the metabolic activity of bacteria. Rates of hydrocarbon biodegradation roughly double for every 10C increase in temperature ("Q"10 rule) over the temperature range between 5 and 25C. Ground water temperatures less than about 5C tend to inhibit biodegradation, and slow rates of biodegradation are generally observed in such waters. Alkalinity The total alkalinity of a ground water system is indicative of water capacity to neutralize acid. s Alkalinity is defined as the net concentration of strong base in excess of strong acid with a pure CO2 -water system as the point of reference (Domenico and Schwartz, 1990). Alkalinity results from the presence of hydroxides, carbonates, and bicarbonates of elements such as calcium, magnesium, sodium, potassium, or ammonia. These species result from the dissolution of rock (especially carbonate rocks), the transfer of CO2 from the atmosphere, and respiration of microorganisms. Alkalinity is important in the maintenance of ground water pH because it buffers the ground water system against acids generated through both aerobic and anaerobic biodegradation processes. Nitrate In the hierarchical order of processes occurring in the microbiological treatment zone, after dissolved oxygen has been depleted, nitrate may be used as an electron acceptor for anaerobic biodegradation. Nitrate concentrations are used to estimate the mass of contaminant that can be biodegraded by denitrification processes. By knowing the volume of contaminated ground water, the background nitrate concentration, and the concentration of nitrate measured in the contaminated area, it is possible to estimate the mass of BTEX lost to biodegradation. Stoichiometrically, each 1.0
mg/L of ionic nitrate consumed by microbes results in the destruction of approximately 0.21 mg/L of BTEX compounds. Nitrate concentrations are a direct input parameter to the Platform. Sulphate and Sulphide Sulphur After dissolved oxygen and nitrate have been depleted in the microbiological treatment zone, sulphate may be used as an electron acceptor for anaerobic biodegradation. This process is termed sulphanogenesis and results in the production of sulphide. Sulphate concentrations are used as an indicator of anaerobic degradation of fuel compounds. By knowing the volume of contaminated ground water, the background sulphate concentration, and the concentration of sulphate measured in the contaminated area, it is possible to estimate the mass of BTEX lost to biodegradation through sulphate reduction. Stoichiometrically, each 1.0 mg/L of sulphate consumed by microbes results in the destruction of approximately 0.21 mg/L of BTEX. Sulphate concentrations are a direct input parameter for the Platform. Ferrous Iron Ferric iron is also used as an electron acceptor during anaerobic biodegradation of petroleum hydrocarbons after nitrate or sulphate depletion, or some times in conjunction with them. During this process, ferric iron is reduced to the ferrous form which may be soluble in water. Ferrous iron concentrations are used as an indicator of anaerobic degradation of fuel compounds. By knowing the volume of contaminated ground water, the background ferrous iron concentration, and the concentration of ferrous iron measured in the contaminated area, it is possible to estimate the mass of BTEX lost to biodegradation through ferric iron reduction. Stoichiometrically, the degradation of 1 mg/L of BTEX results in the production of approximately 21.8 mg/L of ferrous iron during ferric iron reduction. Iron concentrations are used as a direct input parameter to the Platform. The equivalent amount of Ferric Iron is estimated from the measured Ferrous Iron, which is used as model input. BIOPLUME III simulates the hydrocarbon reduction and the corresponding Ferric Iron depletion. Carbon Dioxide Metabolic processes operating during biodegradation of fuel hydrocarbons result in the production of carbon dioxide (CO2). Accurate measurement of CO2 produced during biodegradation is difficult because carbonate in ground water (measured as alkalinity) serves as both a source and sink for free CO2. If the CO2 produced during metabolism is not removed by the natural carbonate buffering system of the aquifer, CO2 levels higher than background may be observed. Comparison of empirical data to stoichiometric calculations can provide estimates of the degree of microbiological activity and the occurrence of in situ mineralization of contaminants. Methane During methanogenesis (an anaerobic biodegradation process), carbon dioxide (or acetate) is used as an electron acceptor, and methane is produced. Methanogenesis generally occurs after oxygen, nitrate, and sulphate have been depleted in the treatment zone. The presence of methane in ground water is indicative of strongly reducing conditions. Because methane is not present in fuel, the presence of methane in ground water above background concentrations in contact with fuels is indicative of microbial degradation of fuel hydrocarbons. Methane concentrations can be used to estimate the amount of BTEX destroyed in an aquifer. By knowing the volume of contaminated ground water, the background methane concentration, and the concentration of methane measured in the contaminated area, it is possible to estimate the mass of BTEX lost to biodegradation through methanogenesis reduction. The degradation of 1 mg/L of BTEX results in the production of approximately 0.78 mg/L of methane during methanogenesis. Methane concentrations are used as an indirect input parameter to the Platform. The equivalent amount of CO2 is estimated from the
measured methane, which is used as model input. BIOPLUME III simulates the hydrocarbon reduction and the corresponding CO2 depletion. Chloride Chloride is used to ensure that ground water samples collected at a site are representative of the water comprising the saturated zone in which the dissolved-phase contamination is present (i.e. to ensure that all samples are from the same ground water flow system). If the chloride concentrations of samples taken from different sampling points are radically different, then the waters may be from different hydrogeologic zones. Total Petroleum Hydrocarbons and Aromatic Hydrocarbons These analytes are used to determine the type, concentration, and distribution of fuel hydrocarbon in the aquifer. Of the compounds present in most gasolines and jet fuels, the BTEX compounds generally represent the regulatory contaminants of concern. For this reason, these compounds are generally of significant interest in the fate and transport analysis. At a minimum, the aromatic hydrocarbon analysis (Method SW8020) must include the BTEX compounds and the trimethylbenzene and tetramethylbenzene isomers. The combined dissolved-phase concentrations of BTEX, trimethylbenzene, and tetra-methylbenzene should not be greater than about 30 mg/L for a JP4 spill. If these compounds are found in concentrations greater than 30 mg/L then sampling errors such as emulsification of NAPL in the ground water sample have likely occurred and should be investigated. Exercise 7-5 Landfill bottom liner is composed of a clay soil: the thickness of the layer is 1 m, hydraulic conductivity is K=1.0*10-9 m/s, porosity n=0.5, dry bulk density = 1.5 kg/dm3 and combined diffusion/dispersion coefficient for benzene is D=2.5*10-10 m2/s. The hydraulic gradient is assumed to be unity, i.e. dH/dz=1.0. Adsorption isotherm for benzene is linear. See also section 4.5 Case Study: landfill bottom liner. Transport of benzene through the clay liner into the underlying groundwater zone is computed by solving numerically the advection -diffusion/dispersion - adsorption - biodegradation equation:
(7-8) Initial and boundary conditions: C(x,0) = 0.0 C(0,t) = 100 g/l (the limiting value of benzene in drinking water is 5 g/l) dC(L,t)/dz = (v/R)C (L = 100 cm) (advection outflow) The adsorption is taken into account using the linear isotherm, i.e. S = KdC which leads to the final partial differential equation to be solved:
(7-9)
where In this case the distribution coefficient Kd is 0.67 l/kg. The equation was solved numerically and after 15 years a steady-state situation was attained. In steady-state the penetration of benzene was stopped and the influence of advection, diffusion and dispersion equals the biodegradation rate. In the numerical solution the nodal distance is dz=2.0 cm. The table shown below lists the steady-state concentration distribution as a function of depth z between 0..49 cm. Problem: calculate an estimate of the biodegradation coefficient k B based on the available data (unit of kB is d-1).

7 Biodegradation

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7 Biodegradation

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7 BIODEGRADATION/BIOTRANSFORMATION

7.3 Ethylbenzene and xylene

7.4 Kinetics of the Biodegradation Reactions

7.5 Biodegradation of chlorinated solvents (chlorinated ethenes and ethanes)

Fig. 7-2. Reductive dechlorination of perchloroethene PCE to trichloroethene TCE.

7.6 Modelling of biodegradation of BTEX-compounds

7.7.1 Monod kinetics (Michaelis-Menten kinetics)

7.8 Sequential model for chlorinated solvents

7.9 Chlorinated Ethenes

7.10 Chlorinated Ethanes

7.11 Remediation by Natural Attenuation (RNA)

7.11.2 How to use models in Remediation by Natural Attenuation?

7.11.3 Natural Attenuation Lines of Evidence

7.12 Ground Water Characterization measurements (According to Rafai et al., 1998)

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