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| Resource Library > Technology Transfer > Programs and Initiatives > Enhanced In Situ Anaerobic Bioremediation > Technology Basics |
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Enhanced in situ anaerobic bioremediation can be an effective method of degrading various forms of chlorinated compounds dissolved in groundwater. When anaerobic degradation of chlorinated aliphatic hydrocarbons (CAHs) occurs naturally, it is considered a component of natural attenuation. Unfortunately, MNA alone is typically not sufficient to achieve remedial objectives in a timely manner at many sites contaminated with CAHs. The addition of an organic substrate to an aquifer has the potential to stimulate microbial growth and development, creating an anaerobic environment in which rates of anaerobic degradation of CAHs may be greatly enhanced. Therefore, a variety of organic substrates in various engineering designs have been applied to the subsurface to promote anaerobic degradation of CAHs to innocuous end products. In some cases, microorganisms also may need to be added (bioaugmentation), but only if the natural microbial population is incapable of performing the required transformations.
Remedial Objectives and Regulatory Acceptance
In general, the remedial objective of enhanced in situ anaerobic bioremediation is restoration of contaminated groundwater to pre-existing levels of beneficial use. In the case of drinking water aquifers, this is usually to federal or state established maximum contaminant levels (MCLs). In many cases, cleanup criteria may be less stringent if the impacted groundwater does not constitute a potable water supply. Exposure pathways such as surface water discharge or volatilization to soil vapor also may dictate cleanup criteria. Project- or site-specific remedial objectives may vary accordingly.
Regulatory acceptance of enhanced in situ anaerobic bioremediation has evolved over the last several years. Enhanced in situ anaerobic bioremediation has been implemented under various federal programs including the Comprehensive Environmental Response, Compensation and Liability Act (CERCLA) and the Resource Conservation and Recovery Act (RCRA). The technology has been applied in over 32 states, including under the jurisdiction of regulatory agencies such as the California Regional Water Quality Control Board and the Florida Department of Environmental Protection. While the use of enhanced in situ anaerobic bioremediation has been approved by the US EPA and the majority of the states, it has yet to gain widespread acceptance as a proven technology primarily due to a lack of consistency in achieving remedial objectives.
Applicable Contaminants (Chlorinated Solvents)
The most common chlorinated solvents released to the environment include tetrachloroethene (PCE, or perchloroethene), trichloroethene (TCE), trichloroethane (TCA), and carbon tetrachloride (CT). These chlorinated solvents are problematic because of their health hazards and their resistance to natural degradation processes. Because these compounds exist in an oxidized state, they are generally not susceptible to aerobic oxidation processes. However, oxidized compounds are susceptible to reduction under anaerobic conditions by either biotic (biological) or abiotic (chemical) processes. Enhanced anaerobic bioremediation is intended to exploit biotic anaerobic processes in order to degrade chlorinated solvents in groundwater.
Other common groundwater contaminants that are subject to reduction reactions are also susceptible to enhanced anaerobic bioremediation. While not addressed in this document, constituents that can also potentially be treated with this approach include the following:
- Chlorobenzenes;
- Chlorinated pesticides (e.g., chlordane), polychlorinated biphenyls (PCBs), and chlorinated cyclic hydrocarbons (e.g., pentachlorophenol);
- Oxidizers such as perchlorate and chlorate;
- Explosive and ordnance compounds;
- Dissolved metals (e.g., hexavalent chromium); and
- Nitrate and sulfate.
Many of the techniques described in AFCEE Technology Transfer Enhanced In Situ Anaerobic Bioremediation Initiative to create anaerobic reactive zones for chlorinated solvents may also be applicable to the design and implementation of enhanced in situ anaerobic bioremediation systems for the constituents listed above.
Degradation Processes
There are many potential reactions that may degrade CAHs in the subsurface, under both aerobic and anaerobic conditions (Table 1). Not all CAHs are amendable to degradation by each of these processes. However, anaerobic biodegradation processes may potentially degrade all of the common chloroethenes, chloroethanes, and chloromethanes.
Anaerobic reductive dechlorination is the degradation process targeted by enhanced anaerobic bioremediation. Through addition of organic substrates to the subsurface, enhanced anaerobic bioremediation converts naturally aerobic or mildly anoxic aquifer zones to anaerobic and microbiologically diverse reactive zones, making them conducive to anaerobic degradation of CAHs.
Biodegradation of an organic substrate depletes the aquifer of dissolved oxygen (DO) and lowers the oxidation-reduction potential (ORP) of groundwater, thereby stimulating conditions conducive to anaerobic biodegradation processes. After DO is consumed, anaerobic microorganisms typically use native electron acceptors (as available) in the following order of preference: nitrate, manganese and ferric iron oxyhydroxides, sulfate, and finally carbon dioxide. Anaerobic dechlorination has been demonstrated under nitrate- , iron-, and sulfate-reducing conditions, but the most rapid biodegradation rates, affecting the widest range of CAHs, occur under methanogenic conditions.
| Table 1. Potential Degradation Processes for CAHs |
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Anaerobic Reductive Dechlorination
The following three general reactions may degrade CAHs by reductive dechlorination:
- Direct Anaerobic Reductive Dechlorination is a biological reaction in which bacteria gain energy and grow as one or more chlorine atoms on a CAH molecule are replaced with hydrogen in an anaerobic environment. In this reaction, the chlorinated compound serves as the electron acceptor and hydrogen serves as the direct electron donor. Hydrogen used in this reaction is typically supplied by fermentation of organic substrates. This reaction may also be referred to as halorespiration or dehalorespiration.
- Cometabolic Anaerobic Reductive Dechlorination is a reaction in which a chlorinated compound is reduced by a non-specific enzyme or co-factor produced during microbial metabolism of another compound (i.e., the primary substrate) in an anaerobic environment. By definition, cometabolism of the chlorinated compound does not yield any energy or growth benefit for the microbe mediating the reaction. For the cometabolic process to be sustained, sufficient primary substrate is required to support growth of the transforming microorganisms.
- Abiotic Reductive Dechlorination is a chemical degradation reaction not associated with biological activity where a chlorinated hydrocarbon is reduced by a reactive compound. Addition of an organic substrate and creation of an anaerobic environment may create reactive compounds such as iron-monosulfides that can degrade CAHs. In this case, substrate addition may indirectly cause abiotic reductive dechlorination.
In practice, it may not be possible to distinguish between these three different reactions at the field scale. All three reactions may be occurring, and abiotic reductive dechlorination may be enhanced indirectly by substrate addition. As described, anaerobic dechlorination refers to the biotic processes of direct and cometabolic anaerobic reductive dechlorination.
In general, anaerobic dechlorination occurs by sequential removal of chloride ions. For example, the chlorinated ethenes are transformed sequentially from PCE to TCE to the dichloroethene (DCE) isomers (cis-DCE or trans-DCE) to vinyl chloride (VC) to ethene as illustrated on Figure 1:
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| Figure 1. Sequential Reduction of PCE to Ethene by Anaerobic Reductive Dechlorination |
In this reaction, hydrogen is the electron donor, which is oxidized. The chlorinated ethene molecule is the electron acceptor, which is reduced. While other fermentation products (e.g., acetate) may serve as an electron donor, hydrogen appears to be the most important electron donor for anaerobic dechlorination of CAHs.
Similar to the chlorinated ethenes, the common chloroethanes and chloromethanes are transformed sequentially by anaerobic dechlorination as follows:
- Chloroethanes: 1,1,1-TCA to 1,1-dichloroethane (DCA) to chloroethane (CA) to ethane.
- Chloromethanes: CT to chloroform (CF) to methylene chloride (MC) to chloromethane (CM) to methane.
Anaerobic reductive dechlorination of CAHs is dependent on many environmental factors (e.g., anaerobic conditions, presence of fermentable substrates, and appropriate microbial populations). The most thoroughly studied anaerobic dechlorination pathway is degradation of PCE to TCE to cis-DCE to VC, and finally to ethene and ethane.
Reductive dechlorination of chlorinated solvents affects each of the chlorinated compounds differently. For example, of the chlorinated ethenes, PCE and TCE are the most susceptible to anaerobic dechlorination because they are the most oxidized. Conversely, VC is the least susceptible to anaerobic dechlorination because it is the least oxidized of these compounds. Therefore, the potential exists for VC to accumulate in a treatment system when the rate at which it is generated is greater than the rate at which it degraded. This is a common concern because VC is considered more toxic than the other chlorinated ethenes. However, there are other degradation pathways for VC (Table 1), and the formation and persistence of large VC plumes (i.e., larger than the footprint of the initial CAH plume) is rarely observed in practice.
Molecular Hydrogen as a Direct Electron Donor
Researchers have recognized the role of hydrogen as the direct electron donor in the anaerobic dechlorination of CAHs. Laboratory cultures used to study direct anaerobic reductive dechlorination are typically mixed cultures, with at least two distinct strains of bacteria. One strain ferments the organic substrate to produce hydrogen, and another strain utilizes the hydrogen as an electron donor for anaerobic dechlorination.
Hydrogen is generated by fermentation of non-chlorinated organic substrates including naturally occurring organic carbon, accidental releases of anthropogenic carbon (fuel), or introduced substrates such as carbohydrates (sugars), alcohols, and low-molecular-weight fatty acids. As an example, lactate in the form of sodium lactate (a stable lactate salt) is commonly used as a substrate for enhanced anaerobic bioremediation. When added to the subsurface, sodium lactate first disassociates as follows:
(1) C3H5NaO3 Þ C3H5O3- + Na+
The lactate molecule may then be fermented by more than one process. For example, it may be fermented to acetate in the following fermentation reaction:
(2) C3H5O3- + 2H2O Þ C2H3O2- (acetate) + HCO3- (bicarbonate ion) + 5H+ + 4e-
The electrons produced in the above fermentation reaction cannot exist alone in solution, and must be part of molecules or atoms. Therefore, the hydrogen ions and the electrons produced will form molecular hydrogen as in the following half reaction:
(3) 5H+ + 4e- Þ H+ + 2H2
Furthermore, the bicarbonate ion and hydrogen may form carbon dioxide and water:
(4) HCO3- + H+ Þ CO2- + H2O
If one combines equations (2), (3), and (4), the fermentation of lactate to acetate and hydrogen can be written as the following balanced fermentation reaction:
(5) C3H5O3- + 2H2O Þ C2H3O2- + CO2- + H2O + 2H2
Thus, the fermentation of a single molecule of lactate to acetate produces two molecules of molecular hydrogen. The acetate produced in this reaction may be used directly as an electron donor for reduction reactions, or may be further fermented to produce hydrogen. Table 2 lists a few examples of some other fermentation reactions where the substrate (electron donor) is fermented to produce hydrogen.
Table 2. Examples of Fermentation Half Reactions Using
Organic Substrates as an Electron Donor to Yield Hydrogen |
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As hydrogen is produced by fermentative organisms, it is rapidly consumed by other bacteria including denitrifiers, iron-reducers, sulfate-reducers, methanogens, and dechlorinating microorganisms. As an example, consider the reduction of PCE. First, molecular hydrogen disassociates in the following half-cell reaction:
(6) H2 Þ 2H+ + 2e-
Then, PCE is reduced by the substitution of a chloride ion with a hydrogen ion and the transfer on one electron:
(7) C2Cl4 + 2H+ + 2e- Þ C2HCl3 + H+ + Cl-
The hydrogen ion and chloride ion produce may form hydrochloric acid as follows:
(8) H+ + Cl- Þ HCl
Combining and balancing equations (6), (7), and (8), the dechlorination of PCE using hydrogen as the electron donor can be written as follows:
(9) H2 + C2Cl4 Þ C2HCl3 + HCl
Table 3 lists a few examples of some common half reactions that utilize hydrogen as an electron donor for reduction of native electron acceptors and CAHs. The production of hydrogen through fermentation does not, by itself, guarantee that hydrogen will be available for reductive dechlorination of CAHs. For anaerobic reductive dechlorination to occur, dechlorinators must successfully compete against the other microorganisms that also utilize hydrogen.
| Table 3. Examples of Half Reactions Using Hydrogen as the Electron Donor |
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Research suggest that the competition for hydrogen is controlled primarily by the Monod half-saturation constant Ks(H2), which is the concentration at which a specific strain of bacteria can utilize hydrogen at half the maximum utilization rate. Ks(H2) values were measured for dechlorinators and methanogens of 100 nanomoles per liter (nmol/L) and 1,000 nmol/L, respectively. Based on this result, research suggests that dechlorinators would successfully compete for hydrogen only at very low hydrogen concentrations. This implies that the selection of an organic substrate whose fermentation results in a slow, steady, and low-level release of hydrogen (electron donor) over time could maximize dechlorination potential while minimizing methanogenic competition for the available hydrogen.
Research points out that competition for hydrogen also depends on additional factors including the bacterial growth rate (relative cell yields), temperature (higher temperatures (35°C) favor methanogens), and maximum hydrogen utilization rate. While it is concluded that dechlorinating bacteria may out-compete methanogens for hydrogen utilization at low hydrogen concentrations (Ks(H2) values of 9 to 21 nmol/L), it is also concluded that dechlorinators can compete successfully with methanogens up to a hydrogen partial pressure of 100 parts per million (ppm), or 50 nmol/L. Because hydrogen concentrations seldom exceed 50 nmol/L in methanogenic environments, dechlorinators should normally have an advantage. Researchers investigated the effect of limiting both electron donor (hydrogen) and electron acceptor (cis-DCE and VC) substrates on reaction kinetics using bacterium strain VS (shown to metabolize both cis-DCE and VC). Based on experimental data, the authors calculated a Ks(H2) value of 7 ± 2 nmol/L, which is similar to that found by others.
These studies suggest that attempts to limit hydrogen concentrations by limiting substrate availability in order to reduce competition for hydrogen (methanogenesis) and increase substrate utilization is unnecessary, and may result in significant portions of the treatment zone remaining insufficiently reducing for complete dechlorination to occur. This may result in sites "stalling" at intermediate dechlorination byproducts such as cis-DCE or VC. Even though a large percentage of substrate added to the subsurface may be utilized for sulfate reduction or methanogenesis, the stoichiometric relationships for the direct anaerobic dechlorination of CAHs are relatively favorable. High rates of anaerobic dechlorination and mass destruction may be achieved even with relatively low substrate utilization rates. Conversely, caution should be used that too much substrate is added to the subsurface in that other conditions may develop such as degradation of secondary water quality or adverse changes in pH.
Microbiology of Anaerobic Reductive Dechlorination
Anaerobic reductive dechlorination is carried out by only a few metabolic classifications of bacteria. These groups may behave very differently from one another, and include methanogens, sulfate-reducing bacteria, and dechlorinating bacteria. There are many classifications and strains of bacteria that can reduce PCE and TCE to cis-DCE, and these microorganisms appear to be ubiquitous in the subsurface environment. However, dechlorination of cis-DCE and VC to ethene may be limited to dechlorinating microorganisms which are not ubiquitous in the subsurface environment.
Some dechlorinators sequentially dechlorinate PCE to TCE, some to cis-DCE, and some to VC. Complete degradation of PCE to ethene has only been demonstrated for a single species, Dehalococcoides ethenogenes. Anaerobic dechlorination is typically carried out by mixed cultures of dechlorinators. Researchers demonstrated complete dechlorination of PCE to ethene with a mixed culture that did not contain the Dehalococcoides species, and found that at least two populations of dechlorinators were responsible for the sequential dechlorination of PCE to ethene observed. This suggests that mixtures of differing dechlorinating strains can achieve complete dechlorination without reliance on any one specific strain of bacteria. In addition, other degradation pathways exist for the less chlorinated compounds such as DCE and VC in both aerobic and anaerobic environments, which also may achieve the desired degradation endpoint. Dehalococcoides ethenogenes appear to be common, but not ubiquitous in the environment.
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