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In-Situ Groundwater Bioremediation

The following description of In-Situ Groundwater Bioremediation is an excerpt from Chapter X of OUST's publication: How to Evaluate Alternative Cleanup Technologies for Underground Storage Tank Sites: A Guide for Corrective Action Plan Reviewers. (EPA 510-B-95-007). This publication also describes 9 additional alternative technologies for remediation of petroleum releases. You can download PDF files of every chapter of the document at: http://www.epa.gov/swerust1/pubs/tums.htm.

In-situ groundwater bioremediation is a technology that encourages growth and reproduction of indigenous microorganisms to enhance biodegradation of organic constituents in the saturated zone. In-situ groundwater bioremediation can effectively degrade organic constituents which are dissolved in groundwater and adsorbed onto the aquifer matrix.


In-situ groundwater bioremediation can be effective for the full range of petroleum hydrocarbons. While there are some notable exceptions (e.g., MTBE) the short-chain, low-molecular-weight, more water soluble constituents are degraded more rapidly and to lower residual levels than are long-chain, high-molecular-weight, less soluble constituents. Recoverable free product should be removed from the subsurface prior to operation of the in-situ groundwater bioremediation system. This will mitigate the major source of contaminants as well as reduce the potential for smearing or spreading high concentrations of contaminants.

In-situ bioremediation of groundwater can be combined with other saturated zone remedial technologies (e.g., air sparging) and vadose zone remedial operations (e.g., soil vapor extraction, bioventing).

Operation Principles

Bioremediation generally requires a mechanism for stimulating and maintaining the activity of these microorganisms. This mechanism is usually a delivery system for providing one or more of the following: An electron acceptor (oxygen, nitrate); nutrients (nitrogen, phosphorus); and an energy source (carbon). Generally, electron acceptors and nutrients are the two most critical components of any delivery system.

In a typical in-situ bioremediation system, groundwater is extracted using one or more wells and, if necessary, treated to remove residual dissolved constituents. The treated groundwater is then mixed with an electron acceptor and nutrients, and other constituents if required, and re-injected upgradient of or within the contaminant source. Infiltration galleries or injection wells may be used to re-inject treated water. In an ideal configuration, a "closed-loop" system would be established. All water extracted would be reinjected without treatment and all remediation would occur in situ. This ideal system would continually recirculate the water until cleanup levels had been achieved. If your state does not allow re-injection of extracted groundwater, it may be feasible to mix the electron acceptor and nutrients with fresh water instead. Extracted water that is not re-injected must be discharged, typically to surface water or to publicly owned treatment works (POTW).

System Design

In-situ bioremediation can be implemented in a number of treatment modes, including: Aerobic (oxygen respiration); anoxic (nitrate respiration); anaerobic (non-oxygen respiration); and co-metabolic. The aerobic mode has been proven most effective in reducing contaminant levels of aliphatic (e.g., hexane) and aromatic petroleum hydrocarbons (e.g., benzene, naphthalene) typically present in gasoline and diesel fuel. In the aerobic treatment mode, groundwater is oxygenated by one of three methods: Direct sparging of air or oxygen through an injection well; saturation of water with air or oxygen prior to re-injection; or addition of hydrogen peroxide directly into an injection well or into reinjected water. Whichever method of oxygenation is used, it is important to ensure that oxygen is being distributed throughout the area of contamination. Anoxic, anaerobic, and co-metabolic modes are sometimes used for remediation of other compounds, such as chlorinated solvents, but are generally slower than aerobic respiration in breaking down petroleum hydrocarbons.

The key parameters that determine the effectiveness of In-situ groundwater bioremediation are:

In general, the aquifer medium will determine hydraulic conductivity. Fine-grained media (e.g., clays, silts) have lower intrinsic permeability than coarse-grained media (e.g., sands, gravels). Bioremediation is generally effective in permeable (e.g., sandy, gravelly) aquifer media. However, depending on the extent of contamination, bioremediation also can be effective in less permeable silty or clayey media. In general, an aquifer medium of lower permeability will require longer to clean up than a more permeable medium. Soil structure and stratification are important to in-situ groundwater bioremediation because they affect groundwater flowrates and patterns when water is extracted or injected. Structural characteristics such as microfracturing can result in higher permeabilities than expected for certain soils (e.g., clays). In this case, however, flow will increase in the fractured media but not in the unfractured media. The stratification of soils with different permeabilities can dramatically increase the lateral flow of groundwater in the more permeable strata while reducing the flow through less permeable strata. This preferential flow behavior can lead to reduced effectiveness and extended remedial times for less-permeable strata.

The biodegradability of a petroleum constituent is a measure of its ability to be metabolized (or co-metabolized) by hydrocarbon-degrading bacteria or other microorganisms. The chemical characteristics of the contaminants will dictate their biodegradability. For example, heavy metals are not degraded by bioremediation. The biodegradability of organic constituents depends on their chemical structures and physical/chemical properties (e.g., water solubility, water/octanol partition coefficient). Highly soluble organic compounds with low molecular weights will tend to be more rapidly degraded than slightly soluble compounds with high molecular weights. The low water solubilities of the more complex compounds render them less bioavailable to petroleum-degrading organisms. Consequently, the larger, more complex chemical compounds may be slow to degrade or may even be recalcitrant to biological degradation (e.g., asphaltenes in No. 6 fuel oil).

The location, distribution, and disposition of petroleum contamination in the subsurface can significantly influence the likelihood of success for bioremediation. This technology generally works well for dissolved contaminants and contamination adsorbed onto higher permeability sediments (sands and gravels). However, if the majority of contamination is (1) in the unsaturated zone; (2) trapped in lower permeability sediments, or (3) outside the "flow path" for nutrients and electron acceptors, this technology will have reduced impact or no impact.

Excessive calcium, magnesium, or iron in groundwater can react with phosphate, which is typically supplied as a nutrient in the form of tripolyphosphate, or with carbon dioxide, which is produced by microorganisms as a by-product of aerobic respiration. The products of these reactions can adversely affect the operation of an in-situ bioremediation system. When calcium, magnesium, or iron reacts with phosphate or carbon dioxide, crystalline precipitates or "scale" is formed. Scale can constrict flow channels and can also damage equipment, such as injection wells and sparge points. In addition, the precipitation of calcium or magnesium phosphates ties up phosphorus compounds, making them unavailable to microorganisms for use as nutrients. This effect can be minimized by using tripolyphosphates to acta as sequestering agents to keep the magnesium and calcium in solution (i.e., prevent the metal ions from precipitating and forming scale).

When oxygen is introduced to the subsurface as a terminal electron acceptor, it can react with dissolved iron [Fe(II)] to form an insoluble iron precipitate, ferric oxide. This precipitate can be deposited in aquifer flow channels, reducing permeability. The effects of iron precipitation tend to be most noticeable around injection wells, where oxygen concentration in groundwater is highest and can render injection wells inoperable.

Extreme pH values (i.e., less than 5 or greater than 10) are generally unfavorable for microbial activity. Typically, optimal microbial activity occurs under neutral pH conditions (i.e., in the range of 6 8). The optimal pH is site specific. For example, aggressive microbial activity has been observed at lower pH conditions outside of this range (e.g., 4.5 to 5) in natural systems. Because indigenous microorganisms have adapted to the natural conditions where they are found, pH adjustment, even toward neutral, can inhibit microbial activity. If man-made conditions (e.g., releases of petroleum) have altered the pH outside the neutral range, pH adjustment may be needed. If the pH of the groundwater is too low (too acid), lime or sodium hydroxide can be added to increase the pH. If the pH is too high (too alkaline), then a suitable acid (e.g., hydrochloric, muriatic) can be added to reduce the pH. Changes to pH should be closely monitored because rapid changes of more than 1 or 2 units can inhibit microbial activity and may require an extended acclimation period before the microbes resume their activity.

Microorganisms require carbon as an energy source to sustain their metabolic functions, which include growth and reproduction. The metabolic process used by bacteria to produce energy requires a terminal electron acceptor (TEA) to enzymatically oxidize the carbon source (organic matter) to carbon dioxide. Microorganisms are classified by the carbon and TEA sources they use to carry out metabolic processes. Bacteria that use organic compounds as their source of carbon are called heterotrophs; those that use inorganic carbon compounds such as carbon dioxide are called autotrophs. Bacteria that use oxygen as their TEA are called aerobes; those that use a compound other than oxygen (e.g., nitrate, sulfate) are called anaerobes; and those that can utilize both oxygen and other compounds as TEAs are called facultative. For in-situ groundwater bioremediation applications directed at petroleum products, bacteria that are both aerobic (or facultative) and heterotrophic are most important in the degradation process.

Extraction wells are generally necessary to achieve hydraulic control over the plume to ensure that it does not spread contaminants into areas where contamination does not exist or accelerate the movement toward receptors. Placement of extraction wells is critical, especially in systems that also use nutrient injection wells or infiltration galleries. These additional sources of water can alter the natural groundwater flow patterns which can cause the contaminant plume to move in an unintended direction or rate. Without adequate hydraulic control, this situation can lead to worsening of the original condition and complicate the cleanup or extend it.

Nutrient injection systems may not be necessary at all, if the groundwater contains adequate amounts of nutrients, such as nitrogen and phosphorus. Microorganisms require inorganic nutrients such as nitrogen and phosphate to support cell growth and sustain biodegradation processes. Nutrients may be available in sufficient quantities in the aquifer but, more frequently, nutrients need to be added to maintain adequate bacterial populations.




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