Overview

Some microorganisms fortuitously degrade contaminants while oxidizing or reducing other compounds (metabolites) for energy and carbon (EPA 2006). Cometabolic bioremediation is the breakdown of a contaminant by an enzyme or cofactor that is produced during microbial metabolism of another compound (EPA 2000). This type of bioremediation is highly targeted because it ensures that only the microbes that can degrade the contaminant of concern are stimulated (Hazen 2010). An important advantage of cometabolic bioremediation is that biodegradation can be stimulated at low contaminant concentrations, below concentrations that would provide a carbon or energy benefit to the microorganisms if a direct aerobic or anaerobic bioremediation route was taken. Therefore, contaminants can be reduced to undetectable (parts per trillion) concentrations (Hazen 2010).

Cometabolic bioremediation may be aerobic or anaerobic. In aerobic cometabolic bioremediation, the contaminant is oxidized by an enzyme or cofactor produced during microbial metabolism of another compound with oxygen. In anaerobic cometabolic bioremediation, the contaminant is reduced by an enzyme or cofactor produced during microbial metabolism of another compound in an environment devoid of oxygen. In these cases, biodegradation of the contaminant does not yield any energy or growth benefit for the microbe mediating the reaction (EPA 2000).

Cometabolic bioremediation has been used on a variety of contaminants, including polycyclic aromatic hydrocarbons (PAHs), explosives, dioxane, polychlorinated biphenyls (PCBs), pesticides, methyl tertiary-butyl ether (MTBE), chlorinated alkenes, and halogenated aliphatic and aromatic hydrocarbons (Hazen 2010). Chlorinated contaminants that have been observed to be oxidized cometabolically under aerobic conditions include trichloroethene (TCE), dichloroethene (DCE), vinyl chloride (VC), trichloroethane (TCA), dichloroethane (DCA), chloroform (CF), and methylene chloride (MC). Reductive dechlorination (cometabolic) has been observed for tetrachloroethene (PCE), TCE, DCE, VC, DCA, and carbon tetrachloride (CT) under anaerobic conditions (EPA 2000).

Cometabolic bioremediation requires a suitable substrate to stimulate the appropriate reactions. The electron donors observed in cometabolic aerobic oxidation include methane, ethane, ethene, propane, butane, aromatic hydrocarbons (such as toluene and phenol), and ammonia (EPA 2000). Methanol, glucose, acetate, lactate, sulfate, or pyruvate can serve as substrates during cometabolic anaerobic reduction (Hazen 2010). Enzymes (or cofactors) are produced in response to microbial degradation of these substrates. The bacteria involved in cometabolic degradation do not receive a carbon or energy benefit from the contaminants. In addition, intermediate products that act as inhibitors of microbial metabolism can be produced during cometabolic bioremediation (Powell et al. 2011, Rui et al. 2004, Sipkema et al. 2000, Semprini et al. 2005).

Cometabolic bioremediation systems may involve bioaugmentation, which is performed by injection of non-native microorganisms into the substrate (Parsons 2004). For guidance on bioaugmentation, refer to the Environmental Security Technology Certification Program document on Bioaugmentation of Chlorinated Solvents (2005). The table below summarizes the types of contaminants that can be broken down by cometabolic bioremediation and the typical substrates needed to produce enzymes involved in biodegradation.

	Cometabolic Bioremediation Conditions
	Aerobic	Aerobic	Aerobic	Anaerobic	Anaerobic
Contaminants	TCE DCE VC PAHs PCBs MTBE Creosote >300 other compounds	TCE DCE VC TNT	TCE DCE VC 1,1-DCE 1,1,1-TCA MTBE	PCE TCE DCE VC Hexachlorocyclohexane	BTEX PCE PAHs Atrazine TNT
Substrates	Methane Methanol Propane Propylene	Ammonia Nitrate	Toluene Butane Phenol Citral Cumin Aldehyde Cumene Limonene	Methanol	Glucose Acetate Lactate Sulfate Pyruvate
Microoganism(s)	Methylosinus	Nitrosomonas Nitrobacter	Rhodococcus Pseudomonas Arthrobacter	Pseudomonas Streptomyces Corynebacterium	Dehalococcoides Methanogens Desulfovibrio Clostridium Geobacter Clavibacter
Enzyme(s) produced	Methane monooxygenase Methanol dehydrogenase Alkene mono-oxygenase Catechol dioxygenase	Ammonia monooxygenase	Toluene monooxygenase Toluene dioxygenase	Alcohol dehydrogenases	Dehalogenase AtzA Dichloromethane Dehalogenase

Modified from Hazen (2010)

Electron donor or electron acceptor substrate, pulsing may be needed to reduce competitive inhibition between use of substrate and contaminant by the microorganisms. For example, methane pulsing was found to improve rates of TCE degradation by methanomorphs (Hazen 2010). Overall, the amount of substrate required for a specific area of remediation depends on site conditions. The type of substrate is generally determined through preliminary laboratory studies (AFCEE 1998).

Substrates are generally introduced through injection wells. See the CLU-IN focus page on Anaerobic Bioremediation for information on injection methods. Cometabolic bioventing is another possibility and involves injecting air into the subsurface along with a suitable gaseous substrate to promote cometabolic reactions with the target compound. A suitable substrate should be determined in the laboratory but may include methane, ethane, propane, butane, and pentane. The delivery system is similar to other bioventing technologies and subject to many of the same limitations. Cometabolic bioventing is applicable to contaminants, such as TCE, TCA, ethylene dibromide, and DCE, that resist direct aerobic degradation. This technology is not applicable to PCE. Because experience with cometabolic bioventing is limited, laboratory and pilot-scale studies are recommended to evaluate effectiveness, select a cometabolite, identify needs for acclimation periods, design the system, and estimate treatment times. For further guidance on cometabolic bioventing, refer to the EPA Engineering Forum Issue Paper: In Situ Treatment Technologies for Contaminated Soil as well as the Battelle Memorial Institute Principles and Practices of Bioventing (Leeson and Hinchee 1995), Volume I and Volume II.

The AFCEE IRP Aerobic Cometabolic In Situ Bioremediation Technology Guidance Manual and Screening Software User's Guide provides specific guidance on well placement and technology design in general.

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Performance Monitoring

The table below provides a menu of performance parameters for chemicals that can be degraded cometabolically. It lists parameters that can be used to understand the type of biodegradation that is taking place under a certain set of geochemical conditions, as well as whether degradation is occurring. The type of information needed and the associated parameters should be determined in the site-specific data quality objectives process.

Remedial progress or success with source zone mass removal also can be estimated using flux techniques and tracers. For nonspecific performance monitoring techniques that might be useful, see Remediation Measurement Tools.

Performance Parameter	Method	Data Use	Performance Expectation	Recommended Frequency of Analysis
Chemicals of Concern (CoCs)	EPA SW-846: 8260B (VOC) or 8270D (SVOC) (laboratory) Field gas chromatography (GC) or GC/mass spectroscopy (MS)	Used to determine background and source/plume concentrations of target analytes for subsequent comparison with target analyte concentrations following treatment. Also used to determine if there are any non-target compounds that may inhibit the cometabolic process.	CoCs and degradation products typically are expected to decline to below regulatory compliance levels within the treatment zone after substrate addition.	Baseline and recommended for each groundwater sampling round.
Primary Substrate	EPA SW-846: 8260B (VOC) or 8270D (SVOC) (laboratory) Field gas chromatography (GC) or GC/mass	Used to determine the extent and availability of substrate for consumption by cometabolic bacteria.	Downward trend in substrate concentrations should track downward trend in CoC concentrations.	Site specific— Baseline and all sampling events thereafter.
Appropriate Cometabolic Degrading Micro-organisms	Quantified by quantitative molecular biological techniques such as polymerase chain reaction.- specialty laboratory	Used to determine presence and quantity of appropriate microorganism at baseline period or after bioaugmentation.	Appropriate microorganisms will be detected and increase as a consequence of adding electron acceptors and/or donors.	Baseline prior to remedy initiation and quarterly based on the numbers achieved. Once a high titer is measured and growth is ensured, the test can be continued but is not critical.
Microcosm Study	Various	1) Used to determine degradation products by analyzing carbon labeled CoC.	Cometabolic degradation indicated if the expected byproducts carry the same relative amounts of radioisotope.	Bench scale before field deployment and after field deployment to confirm bench study results.
		2) Used to determine what substrate is most appropriate for native microorganisms and CoCs.	Cometabolic enzyme degrades target compounds without undo interaction with non-target compounds.	Site specific.
Oxygen	Bacharach Fyrite® Gas Analyzer (soil gas)   DO meter calibrated in the field according to the manufacturer's specifications (APHA et al. 1992: 4500-O G) (field). Should be done with downhole probe or flow-through cell to avoid atmospheric contact (groundwater).	For aerobic cometabolism determines aerobic conditions exist. For anaerobic cometabolism determines absence of oxygen.	Determines if consumption of oxygen requires supplementation or if increase in oxygen will require introduction of a substrate to reduce it.	Site specific— Baseline and as appropriate thereafter.
pH	Field probe with direct-reading meter calibrated in the field according to the manufacturer's specifications (APHA et al. 1992: 4500-H+ B)	Used to confirm pH conditions are stable and suitable for microbial bioremediation or to identify trends of concern (EPA 2004).	The optimum pH for bacterial growth is approximately 7. Enhanced aerobic bioremediation can be effective over a pH range of 5 to 9 pH units (EPA 2004).	For active systems daily for the startup phase (7-10 days) and weekly to monthly thereafter (EPA 2004).
Carbon Dioxide	Draeger Tube (soil gas) Bacharach Fyrite® Gas Analyzer (soil gas)	Used as an indicator that microbial activity has been stimulated.	Indicator parameter.	For active systems daily for the startup phase (7-10 days) and weekly to monthly thereafter (EPA 2004).
	APHA et al. 1992: 4500-CO2  C (titrimetric) or 4500-CO2 D (calculation requiring known values for total alkalinity and  pH) ISE for field measurement			Optional for active system (ITRC 2008).
Oxidation Reduction Potential (ORP)	Direct-reading meter, A2580B, or USGS A6.5 (field)	Used to provide data on whether or not aerobic or anaerobic conditions are present. Used in conjunction with other geochemical parameters to determine whether or not groundwater conditions are optimal for aerobic or anaerobic biodegradation.	Positive ORP values (>0.0 mV) in conjunction with elevated levels of DO and the absence of TOC/DOC can indicate that additional substrate is required to promote anaerobic biodegradation.	Baseline and typically measured at the wellhead using a flow-through cell to protect samples from exposure to oxygen.

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