U.S. EPA Contaminated Site Cleanup Information (CLU-IN)


U.S. Environmental Protection Agency
U.S. EPA Technology Innovation and Field Services Division

Bioremediation

Cometabolic Aerobic and Anaerobic Bioremediation

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 SolventsAdobe PDF Logo (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 SoilAdobe PDF Logo as well as the Battelle Memorial Institute Principles and Practices of Bioventing (Leeson and Hinchee 1995), Volume IAdobe PDF Logo and Volume IIAdobe PDF Logo.

The AFCEE IRP Aerobic Cometabolic In Situ Bioremediation Technology Guidance Manual and Screening Software User's GuideAdobe PDF Logo 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 Monitoring Parameters for Cometabolic Biodegradation

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.



References:

American Public Health Association (APHA), American Water Works Association, and Water Environment Federation. 1992. Standard Methods for the Examination of Water and Wastewater, 18th Edition. Standard Methods commercial Web site


Adobe PDF LogoEarth Technology Corp. 1998. IRP Aerobic Cometabolic In Situ Bioremediation Technology Guidance Manual and Screening Software User's Guide
AFCEE, Report No: AFRL-ML-TY-TR-1998-4530. NTIS Order No: ADA359333/XAB. 84 pp, June 1998


EPA. SW-846 On-Line: Test Methods for Evaluating Solid Waste, Physical/Chemical Methods
U.S. EPA, Office of Resource Conservation and Recovery.


Adobe PDF LogoEPA. 2000. Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents: Fundamentals and Field Applications. EPA 542-R-00-008


EPA. 2004a. How to Evaluate Alternative Cleanup Technologies for Underground Storage Tank Sites: A Guide for Corrective Action Plan Reviewers. EPA 510-R-04-002.


Adobe PDF LogoEPA. 2006. Engineering Forum Issue Paper: In Situ Treatment Technologies for Contaminated Soil. EPA 542/F-06/013.


Adobe PDF LogoHazen, T.C. 2010. Cometabolic Bioremediation. In Handbook of Hydrocarbon and Lipid Microbiology, p 2505-2514. Springer-Verlag Berlin Heidelberg, 2010.


Adobe PDF LogoHunkeler, D., R.U. Meckenstock, B. Sherwood-Lollar, T.C. Schmidt, and J.T. Wilson. 2008. A Guide for Assessing Biodegradation and Source Identification of Organic Ground Water Contaminants Using Compound Specific Isotope Analysis (CSIA). EPA 600-R-08-148, 82 pp,


Adobe PDF LogoITRC. 1998. Technical and Regulatory Requirements for Enhanced In Situ Bioremediation of Chlorinated Solvents in Groundwater. Interstate Technology and Regulatory Cooperation Workgroup.


Adobe PDF LogoITRC. 2007. In Situ Bioremediation of Chlorinated Ethene DNAPL Source Zones: Case Studies
Interstate Technology & Regulatory Council. 173 pp.


Adobe PDF LogoLeeson, Andrea and Robert E. Hinchee. 1996. Principles and Practices of Bioventing Volume I: Bioventing Principles


Adobe PDF LogoLeeson, Andrea and Robert E. Hinchee. 1996. Principles and Practices of Bioventing Volume II: Bioventing Design


Adobe PDF LogoPope, D., S. Acree, H. Levine, S. Mangion, J. van Ee, K. Hurt, and B. Wilson. 2004. Performance Monitoring of MNA Remedies for VOCs in Ground Water. EPA 600-R-04-027, 92 pp.


Powell, C.L., G. Nogaro, and A. Agrawal. 2011. Aerobic Cometabolic Degradation of Trichloroethene by Methane and Ammonia Oxidizing Microorganisms Naturally Associated with Carex Comosa Roots. Biodegradation, Vol. 22(3), p. 527-538.


Adobe PDF LogoRui, Lingyun, Young Man Kwon, Kenneth F. Reardon, and Thomas K. Wood. 2004. Metabolic pathway engineering to enhance aerobic degradation of chlorinated ethenes and to reduce their toxicity by cloning a novel glutathione S-transferase, an evolved toluene o-monooxygenase, and γ-glutamylcysteine synthetase. Environmental Microbiology, Vol. 6 (5), p. 491-500.


Adobe PDF LogoSipkema, E. Marjin, Wim de Koning, Klaassien J. Ganzeveld, Dick B. Janssen, and Antonie A.C.M. Beenackers. 2000. NADH-Regulated Metabolic Model for Growth of Methylosinus trichosporium OB3b. Cometabolic Degradation of Trichloroethene and Optimization of Bioreactor System Performance. Biotechnol. Prog., Vol. 16, p. 189-198.


Adobe PDF LogoSemprini, Lewis, Mark E. Dolan, Gary D. Hopkins, and Perry L. McCarty. 2005. Development of Effective Aerobic Cometabolic Systems for the In Situ Transformation of Problematic Chlorinated Solvent Mixtures. Strategic Environmental Research and Development Program. FINAL REPORT: ER-1127.


USGS. National Field Manual for the Collection of Water-Quality Data. U.S. Geological Survey Techniques of Water-Resources Investigations (Book 9, Chapters A1-A9).

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