Enhanced in situ anaerobic bioremediation involves the delivery of organic substrates into the subsurface to stimulate anaerobic degradation of contaminants in groundwater. Effective application of the technology depends primarily on the delivery of appropriate levels of organic substrate in the subsurface and the development of optimal geochemical and oxidation-reduction (redox) conditions for anaerobic degradation processes to occur.

Substrate loading rates are defined as the volume, concentration, and frequency of injection of organic substrates for in situ anaerobic bioremediation. Insufficient substrate loading rates or non-uniform delivery and mixing may result in areas of the aquifer that are not sufficiently reducing for complete dechlorination to occur, thereby increasing the potential for accumulation of regulated intermediate degradation products, for example, the potential accumulation of dechlorination products cis-1,2-dichloroethene (DCE), vinyl chloride (VC), or chloroethane (CA). The presence of excessive substrate may result in uncontrolled fermentation reactions, degradation of secondary water quality, and poor utilization of substrate for anaerobic degradation of the contaminants of concern. The ability for aquifer systems to recover to pre-injection redox conditions and the long-term impacts on groundwater quality after enhanced bioremediation are not well documented.

Given these effects, many enhanced anaerobic bioremediation applications fail to achieve performance expectations or develop unanticipated long-term compliance problems. The cost associated with poor performance or with compliance issues such as degradation of secondary water quality may greatly increase the life-cycle costs of full-scale applications. Therefore, determining an appropriate substrate loading rate and an effective distribution method for the various substrate types commonly applied is a critical design and operational objective.


The objectives of this project were to:

  1. Better understand the effects that substrate amendment loading rates have on substrate distribution and persistence (maintenance of the reaction zone)
  2. Determine how control of substrate loading rates affects amendment utilization and development of optimal geochemical and redox conditions
  3. Identify substrate loading rates that have adverse impacts on secondary water quality
  4. Evaluate the effect that differing substrate types or loading rates may have on hydraulic conductivity based on physical/chemical or biological (biomass) effects of the substrate amendment
  5. Develop practical guidelines for designing and optimizing substrate loading rates and injection scenarios for differing substrate types and for differing geochemical and hydrogeologic conditions based on observations from representative case studies.

To achieve these objectives, 15 case studies were evaluated. Quantitative and qualitative performance objectives were developed to evaluate the case studies and to identify limiting factors for enhanced in situ bioremediation.

Demonstration Results

A substrate estimating tool was developed to assist the practitioner in evaluating a site for an enhanced in situ bioremediation application. This tool was used during the case study evaluations to compare the substrate amendment designs and actual quantities used to the substrate requirements calculated by the tool using site-specific electron acceptor demand.

Six of the 15 case study sites exhibited issues with pH excursion. For all these sites, initial background pH values were below 6.5 and alkalinity was below 150 mg/L. Based on these observations from the case studies, a combination of pH below 6.0 to 6.5 and alkalinity below 300 mg/L indicates that modifications to buffer and control pH will be necessary. Sodium bicarbonate was the most common buffering compound used, typically at concentrations in excess of 10,000 mg/L. Sodium bicarbonate is a relatively weak buffering compound and may be most suitable for applications using frequent injections of soluble substrates. The use of stronger and more persistent buffering compounds (e.g., sodium carbonate or sodium phosphates) may be necessary for applications using slow release substrates, and further research and product development will be beneficial for sites with low buffering capacity.

The primary objective when selecting a substrate loading rate is to achieve a uniform distribution of substrate over time and space. The substrate requirement for each of the case studies was calculated using the substrate estimating tool. Based on these calculations and observations of case study performance, a conservative design factor on the order of three to seven times the estimated substrate requirement should be suitable for limiting the potential for insufficient substrate for slow release substrates injected in a one-time event. For soluble substrates, lower design factors on the order of two to three times the estimated substrate requirement are beneficial to avoid over-stimulating the aquifer and driving pH downward. The delivery methods for soluble substrates should target uniform substrate concentrations without excessive “spikes” in concentration.

Implementation Issues

The substrate estimating tool is useful to screen site conditions that will impact substrate delivery and utilization. The tool provides an estimate of total substrate required over the design life of the application, given a user specified design factor. The tool calculates a time-weighted average concentration of substrate by dividing the total volume of groundwater treated by the total substrate quantity.

The quantities and time weighted average substrate concentrations can be used for comparison to proposed or planned bioremediation applications as a check on the quantities of substrate being proposed or the performance targets for dissolved organic carbon. This should assist in avoiding application of either too little substrate or generating excessive substrate levels. Design tools are often provided by substrate vendors, and the estimated substrate quantity should always be compared to recommendations by the provider or with case studies in the literature.

Design tools that assist the practitioner with the configuration (well spacing) and injection volumes are being developed and should be incorporated into the design exercise. Examples include the Edible Oil Substrate tool being developed under ESTCP project ER-200626. The loading rates calculated by this (or any other design tool) should be compared to the recommended guidelines above to ensure that the input parameters to the design tool are producing realistic and appropriate calculations for substrate requirements.