Bioremediation - Anaerobic Design Considerations

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Bioremediation is the process by which contaminants in soil and/or groundwater are treated biologically, primarily by microorganisms or biomolecules generated and released by the cells. This article overviews key design considerations when planning and implementing a bioremediation remedy. This article focuses on enhanced in situ bioremediation (EISB) for the anaerobic biodegradation of organic contaminants, particularly chlorinated solvents, in soil and groundwater. However, much of the information provided is applicable to other contaminant types. There are numerous resources for design and implementation of bioremediation applications as well as detailed case studies and performance evaluations. This article provides a summary of key design considerations and parameters for anaerobic biodegradation for treatment of common organic contaminants.

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CONTRIBUTOR(S): Michaye McMaster and Leah MacKinnon, M.A.Sc., P.E.

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Key considerations for designing in situ anaerobic bioremediation applications include the conceptual site model (CSM) and remedial goals, which determine the remedial configuration, amendment types and dosage, longevity of amendments, and modes of delivery. Often, additional site characterization, laboratory microcosm studies, or small-scale field tests are necessary to evaluate the technology and support of the full-scale remedy.

Conceptual Site Model Interpretation for Design

Successful implementation of anaerobic bioremediation must consider the CSM to develop a robust site-specific design. A CSM is developed and refined during site investigation activities to describe the site conditions, contaminant sources and extent, and the risk they pose to receptors. The following components of a CSM are evaluated to develop the optimal remedial design:

  • Site geology and hydrogeology
  • Contaminant type, distribution, and concentrations, including source zones
  • Groundwater biogeochemical conditions
  • Site infrastructure (i.e., buildings, below-ground utilities and conduits)
  • Human health and ecological risks
  • Remedial goals
Figure 1. Amendment addition for biobarrier.

The remedial goals and contaminant distribution are key to defining the target treatment area and remedial configuration. The contaminant type, biogeochemistry, and hydrogeology (aquifer permeability) determines the amendment type and optimal dosage. The site infrastructure and hydrogeology also determine the delivery method, including spacing between injection points, volumes of injectate, and injection frequency.

Remedial Configurations

In-situ anaerobic bioremediation of contaminated soil and groundwater involves introducing amendments and microbial cultures, if bioaugmentation is used, into the saturated treatment zone. The two most common treatment configurations include:

  • A grid of injection and/or extraction points for targeted treatment of a source zone or plume, and
  • A linear treatment zone, referred to as a biobarrier (Fig. 1), treats contaminants as they flow through the biologically active zone to control plume migration. The biobarrier consists of a row(s) of injection wells/points or a trench filled with solid substrate.

The configuration is selected based on the remedial objectives, remedial timeframe, and site conditions and restrictions.

Delivery Modes

Amendments can be delivered into the subsurface using injection wells (e.g. batch injection, recirculation, push-pull), direct injections (e.g., direct push technology, hydraulic and pneumatic fracturing) or excavation and backfill. Only liquid amendments (quick release or slow release) can be emplaced through well screens. Direct injections can be used for delivery of all liquid amendments as wells as microscale-particulates (e.g., EHC®, ABC+®, which contain zero valent iron and solid phase carbon), while biobarriers containing mulch or compost are installed using trenches. The design spacing of direct injection points or wells are based on the estimated radius of influence, which is dependent on the site-specific geology and amendment characteristics.

Amendments can be applied in a passive or active manner to target the contaminant zones.

  • In a passive treatment approach, amendments are typically delivered through injection wells or direct injection points in one injection event. Alternatively, solid amendments may be installed via excavation and backfill, or direct injection. Natural flow of groundwater is then relied upon to deliver contaminated groundwater to biologically active areas where treatment occurs.
  • In a semi-passive approach, liquid or solid amendments are injected periodically, with intermittent periods of passive treatment between injection events. Recirculation may be employed during the active treatment periods, while during the passive treatment periods native flow of groundwater is relied upon for delivery.
  • Active bioremediation approaches for groundwater involve recirculation of dissolved amendments within the targeted saturated zone (Fig. 2). Recirculation may improve substrate distribution in the discrete targeted depth and area, provide hydraulic containment, enhance contaminant/substrate mixing, and accelerate treatment time.
Figure 2. Groundwater recirculation system.

Amendment Types

Amendments for anaerobic bioremediation include carbon-based electron donors, electron acceptors, nutrients, pH buffers, and microbial cultures. For each type of amendment that is used the critical design considerations in selection include the contaminant type, site geochemical/hydrogeological conditions, and delivery configuration/mode, as well as the amendment solubility, anticipated rate of consumption (or growth in the case of bioaugmentation), longevity, cost, and ability to be distributed in the subsurface. Bench scale testing may be used to confirm the most applicable amendment type and dose.

Electron Donors

Many materials have been used as electron donors for anaerobic reductive bioremediation applications for anaerobic reduction of contaminants such as chlorinated solvents, energetics and perchlorate. These materials are typically classified as quick release compounds (lactate, sodium benzoate, molasses, whey) or slow release compounds (emulsified vegetable oils, Hydrogen Release Compound [HRC®], EHC®, ABC+®, mulch, compost). Quick release compounds are often selected for active approaches to bioremediation where the electron donor may be continually replenished in the target treatment area. Slow release compounds are often selected for passive approaches where the greater longevity is desirable. In some cases, a mixture of amendments will be used; for example, a quick release compound may be used to provide initial rapid bacterial growth and a slow release compound may be used to provide a long-term source of electron donor.

Electron Acceptors

Electron acceptors such as nitrate, iron(III), or sulfate can be used as amendments for the anaerobic oxidative bioremediation of contaminants such as aromatic hydrocarbons, fuels, and some chloroethenes[3][4][5][6]. While these electron acceptors may promote slower kinetics compared to using oxygen, they may be chosen due to higher solubility, ease of delivery, and/or compatibility with existing geochemical conditions. Other considerations when using these electron acceptors include:

  • Nitrate is highly soluble in water and after oxygen, provides the most energy for the microbial reaction. However, the EPA’s maximum contaminant level (MCL) for nitrate is 10 mg/L in groundwater. Therefore, the concentration and migration of nitrate needs to be carefully managed.
  • Iron(III) is only slightly soluble in water, but gets reduced to iron(II) which is soluble in water. Water quality thresholds for iron include a secondary MCL guideline for iron of 0.3 mg/L for color, taste and staining effects.
  • Sulfate is very soluble in water and reduces to sulfide, which typically precipitates with the naturally occurring iron in the subsurface. However, under acidic conditions, the sulfide can become hydrogen sulfide gas, which is toxic to breathe. The secondary MCL of sulfate is 250 mg/L due to taste, but does not present a risk to human health. Some common sulfate amendments include magnesium sulfate (Epsom salts), calcium sulfate (gypsum), and commercially-available products such as Nutrisulfate®.


Under natural conditions, typical aquifers typically contain suitable amounts of trace nutrients for microbial growth, however substrate amendments may be used to provide additional nutrients such as nitrogen, phosphorous, sulfur, vitamin B12 and yeast extracts. While these nutrients may be valuable at some sites, there can be disadvantages to using nutrients, particularly at high levels, as the nutrients may precipitate with natural minerals and cause aquifer plugging. Nutrients may also compete for electron donors, when used. Additionally, nutrients such as vitamin B12 can be expensive to apply, and the cost-benefit of these additional amendments must be considered in the design process.

pH Buffers

The activity of the microorganisms that degrade the target compounds, and in particular the chlorinated volatile organic compound (cVOC)-dechlorinating microorganisms, can be inhibited at low pH (less than 6.0)[7]. Low groundwater pH can be a result of the geologic materials, contaminant impacts (i.e. acids), or can occur during EISB due to the fermentation of electron donors and/or dehalogenation of cVOCs. The addition of a 'buffer' (i.e., sodium bicarbonate, calcium carbonate) or base (i.e., magnesium hydroxide) may be required for some EISB applications, to neutralize pre-existing acidic groundwater conditions or to maintain pH > 6.0 during EISB. When buffering is required for an anaerobic bioremediation application it is critical to complete bench scale tests with both groundwater and aquifer solids to confirm the buffer capacity of the site materials.


Adding microbial cultures for bioaugmentation may be considered at a site when an appropriate population of anaerobic microorganisms is not present or sufficiently active to stimulate complete anaerobic degradation of the existing contaminants. For example, the presence of Dehalococcoides-related microorganisms has been linked to complete dechlorination of PCE and TCE to ethene in the field[8][9][10][11]. Commercially available bioaugmentation products that contain these microorganisms include KB-1®, SDC-9™, and Bio-Dechlor Inoculum® Plus.

Amendment Dose

Once the amendments for an application are selected, the quantity, concentration and frequency of amendment addition can be evaluated. To define the amendment dose, the site conditions are reviewed in terms of remedial objectives (i.e., treatment targets, longevity) to obtain the basis of dosing calculations including dimensions of the targeted subsurface zone and associated pore volume, groundwater velocity, concentrations of terminal electron acceptors (for electron donors), the type of contaminants and geochemical characteristics (e.g., initial redox state, pH). Although conservative designs are typically applied, the potential for producing methane gas or other gases (i.e. hydrogen sulfide) must also be considered, especially in shallow aquifers. As described above, the results from bench scale testing may be used to support this evaluation.

In the case of electron donors, dosing is based on “electron donor demand” which accounts for consumption of the amendment by treating the target constituent(s) as well as competing electron acceptors. An on-line Planning Aqueous Amendment Injection Systems Soluble Substrate Design Tool is available from ESTCP for estimating electron donor demand[12], and electron donor vendors may also have design tools to support EISB applications. These calculations can also be used to evaluate the need for and frequency of repeat injection events, which will then be confirmed based on performance monitoring. However, it is important to note that previous applications have shown that the treatment period may be extended beyond the longevity of the amended electron donor as a result of endogenic cell decay of biomass. Thus, the initial biomass growth stimulated by electron donor addition may serve as a secondary source of electron donor[13].

Milestones, Metrics, and Endpoints

Common parameters to evaluate the success of bioremediation applications include operational monitoring during implementation (e.g., amendment concentrations, injection rates, injection pressures, achieved volumes) and treatment performance monitoring. The performance monitoring program typically includes the following parameters to evaluate:

  • the distribution of amendments (e.g., conductivity, turbidity, total organic carbon, volatile fatty acids, methane)
  • the resulting changes in the geochemistry (e.g., redox, pH, anions, cations)
  • associated changes in desired microbiological populations (e.g., molecular and enzyme analyses) using molecular biological tools
  • influence on the target contaminants (i.e., cVOCs and their daughter products)
  • confirmation of degradation, and potentially degradation processes, through compound specific isotope analysis

Potential for adverse effects from amendments and/or their by-products (i.e., methane in soil gas, hydrogen sulfide, metals mobilization) is also evaluated on a site-specific basis. The monitoring plan should be adaptive to accommodate observed changes in groundwater quality at the site. Performance thresholds or triggers should be used to evaluate the performance monitoring results and identify when additional remedial contingencies may be needed to achieve the remedial objectives and/or when the remedy is complete.

Field Demonstrations and Performance

There have been numerous field demonstrations of anaerobic bioremediation documented in publicly available literature and reports, including:

  • U.S. EPA Contaminated Site Clean-Up Information (CLU-IN)[14]
  • In Situ Bioremediation of Chlorinated Ethene DNAPL Source Zones: Case Studies[15]
  • ESTCP Demonstrations: Cost and Performance Reports
    • ER-0008: Biodegradation of Dense Non-Aqueous Phase Liquids (DNAPLs) through Bioaugmentation of Source Areas - Dover National Test Site[16]
    • ER-0221: Edible Oil Barriers for Treatment of Chlorinated Solvent Groundwater[17]
    • ER-200219: Comparative Demonstration of Active and Semi-Passive In Situ Bioremediation Approaches for Perchlorate Impacted Groundwater: Active In Situ Bioremediation Demonstration (Aerojet Facility)[18]
    • ER-200627: Loading Rate and Impacts of Substrate Delivery for Enhanced Anaerobic Bioremediation[19]
  • Regulatory / Guidance Documents[7][12][15][18][19]


The design of a successful anaerobic bioremediation application depends on a strong understanding of the CSM and remedial goals for the site. Bioremediation can be adapted to work in a wide range of site conditions, and the amendment selection and delivery methods are designed to be effective for the site-specific hydrogeologic conditions, contaminants, biogeochemistry, and infrastructure constraints to satisfy the remedial goals.


  1. ^ USEPA, 2013. Introduction to In Situ Bioremediation of Groundwater. Report pdf
  2. ^ NAVFAC, 2015. Design considerations for Enhanced Reductive Dechlorination. TM-NAVFAC-EXWC-EV-1501. Report pdf
  3. ^ Edwards, E. A., Wills, L.E., Reinhard, M., Grbic-Galic, D., 1991. Anaerobic Degradation of Toluene and Xylene by Aquifer Microorganisms under Sulfate-Reducing Conditions. Applied and Environmental Microbiology 58:2663-2666.
  4. ^ Lovley, D.R., 1997. Potential for anaerobic bioremediation of BTEX in petroleum-contaminated aquifers. Journal of Industrial Microbiology and Biotechnology, 18(2-3), 75-81. doi 10.1038/sj.jim.2900246
  5. ^ Suflita, J.M. and Sewell, G.W., 1991. Anaerobic biotransformation of contaminants in the subsurface. Environmental Research Brief (USA).
  6. ^ Bradley, P.M. and Chapelle, F.H., 1997. Kinetics of DCE and VC mineralization under methanogenic and Fe (III)-reducing conditions. Environmental Science & Technology, 31(9), 2692-2696. doi 10.1021/es970110e
  7. ^ 7.0 7.1 Robinson, C., Barry, D.A., McCarty, P.L., Gerhard, J.I. and Kouznetsova, I., 2009. pH control for enhanced reductive bioremediation of chlorinated solvent source zones. Science of the Total Environment, 407(16), 4560-4573. doi:10.1016/j.scitotenv.2009.03.029
  8. ^ Parsons, 2004. Principles and Practices of Enhanced Anaerobic Bioremediation of Chlorinated Solvents. AFCEE, NFEC, ESTCP. Report pdf
  9. ^ Major, D.W., McMaster, M.L., Cox, E.E., Edwards, E.A., Dworatzek, S.M., Hendrickson, E.R., Starr, M.G., Payne, J.A. and Buonamici, L.W., 2002. Field demonstration of successful bioaugmentation to achieve dechlorination of tetrachloroethene to ethene. Environmental Science & Technology, 36(23), 5106-5116. doi 10.1021/es0255711
  10. ^ Hendrickson, E.R., Payne, J.A., Young, R.M., Starr, M.G., Perry, M.P., Fahnestock, S., Ellis, D.E. and Ebersole, R.C., 2002. Molecular analysis of Dehalococcoides 16S ribosomal DNA from chloroethene-contaminated sites throughout North America and Europe. Applied and Environmental Microbiology, 68(2), 485-495. doi 10.1128/aem.68.2.485-495.2002
  11. ^ Lendvay, J.M., Löffler, F.E., Dollhopf, M., Aiello, M.R., Daniels, G., Fathepure, B.Z., Gebhard, M., Heine, R., Helton, R., Shi, J., Krajmalnik-Brown, R., Major Jr., C.L., Barcelona, M.J., Petrovskis, E., Hickey, R., Tiedje, J.M., Adriaens, P., 2003. Bioreactive barriers: a comparison of bioaugmentation and biostimulation for chlorinated solvent remediation. Environmental Science & Technology, 37(7), 1422-1431. doi 10.1021/es025985u
  12. ^ 12.0 12.1 Borden, R.C., Cha, K.Y., Simpkin, T. and Lieberman, M.T., 2012. Development of a Design Tool for Planning Aqueous Amendment Injection Systems Soluble Substrate Design Tool (No. ER-200626). North Carolina State Univ. at Raleigh, NC. Report pdf
  13. ^ Adamson, D.T. and Newell, C.J., 2009. Support of source zone bioremediation through endogenous biomass decay and electron donor recycling. Bioremediation Journal, 13(1), 29-40. doi: 10.1080/10889860802690539
  14. ^ USEPA 2016. Anaerobic Bioremediation (Direct) Application.
  15. ^ 15.0 15.1 ITRC, 2007. In Situ Bioremediation of Chlorinated Ethene DNAPL Source Zones: Case Studies, BioDNAPL-2, 173 pgs. Report pdf
  16. ^ ESTCP, 2008. Biodegradation of Dense Non-Aqueous Phase Liquids (DNAPLs) through Bioaugmentation of Source Areas - Dover National Test Site, Dover, Delaware: ESTCP Cost and Performance Report. ESTCP Project ER-0008
  17. ^ Lieberman, M.T. and Borden, R.C., 2009. Edible Oil Barriers for Treatment of Chlorinated Solvent Contaminated Groundwater. Solutions Industrial and Environmental Services, Inc. Raleigh, NC. Report pdf
  18. ^ 18.0 18.1 Cox, E. and Krug, T., 2012. Comparative Demonstration of Active and Semi-Passive In Situ Bioremediation Approaches for Perchlorate Impacted Groundwater: Active In Situ Bioremediation Demonstration (Aerojet Facility) ESTCP Project ER-200219. ER-200219
  19. ^ 19.0 19.1 Henry, B., 2010. Loading Rate and Impacts of Substrate Delivery for Enhanced Anaerobic Bioremediation. ESTCP Project ER-200627, 90 pgs. ER-200627

See Also