Program Element 5: Assessment

Fundamental research in measuring and validating the biological and geochemical processes of bioremediation.

PROGRAM OBJECTIVES


To expand and validate innovative methods for measuring biodegradation rates, biotransformation processes, microbial community dynamics, electron flow, contaminant distributions, and hydrogeological and geochemical factors -- and to understand the implications of these measurements. The methods developed will focus on characterizing, monitoring (for process control), and defining end points essential to implementing biodegradation in the field, particularly those methods relevant to the transformation of metals and radionuclides.

BACKGROUND



Current methods for measuring and evaluating the effectiveness of bioremediation are inadequate and, in most cases, undeveloped.


Understanding the relationships between microbial community dynamics and the resulting biodegradation and transformation is key to evaluating and predicting the microbial processes that can be harnessed for bioremediation. However, current methods for measuring and evaluating the effectiveness of bioremediation are inadequate and, in most cases, undeveloped. The recently published evaluation by CISB (1993) suggests that "...demonstrating that in situ bioremediation is working should include three types of evidence:

1. Document[ed] loss of contaminants from the site,

2. Laboratory assays showing that microorganisms in site samples have the potential to transform the contaminants under expected site conditions, and

3. One or more pieces of evidence showing that the biodegradative potential is actually realized in the field."

Other appropriate evidence would include documented product accumulation and detoxification. The strongest case is made by having multiple lines of evidence that include documentation of electron flow towards bioremediation pathways.


Techniques that measure the electron flow critical to all the bioremediation processes are particularly important.


In today's practice, biological technologies applied during full-scale remediation rely on measuring the disappearance of parent compounds and analyzing the status of electron acceptors (O2, NO3, and SO4), selected nutrients, and microbial numbers. Research is needed to develop measurement tools to assess the transformation of complex wastes containing radionuclides and organic compounds and to interpret measurements of bioremediation. Techniques that measure the electron flow that is critical to all the bioremediation processes are particularly important. Environmental microbiology uses techniques largely adapted from clinical microbiology and chemical techniques designed to measure total chemical concentrations. Although useful, these techniques cannot adequately describe the identity, function, physiology, and activity of in situ organisms. Most importantly, populations, communities, and activities involving anaerobic environments, mixtures of contaminants, and in situ measurements of environmental conditions have not been examined. DNA probes and polymerase chain reaction (PCR) to amplify DNA have been used to assess the responses of individual biodegradative strains or various genera involved in bioremediation processes. But even these processes are limited when measuring community function under stress or competition.

Current chemical methodologies for assessing contamination rates and residuals are labor intensive, expensive, and provide indirect measures of in situ processes. They may also inaccurately relate total chemical measurements to bioavailability and risk to human health. This may be true even when the concentration of chemicals, e.g., metals, radionuclides, or chemical mixtures such as total petroleum hydrocarbons, are above statutory levels as commonly determined by analytical chemistry. Research is needed to determine the short- and long-term bioavailability of contaminants to the soil community at the micro and macro level. The limitations of existing techniques are most apparent when interpreting the results of end points of bioremediation for human health risk assessment. Leaching and other harsh methods for extracting chemical constituents from the soil for analytical determination may not adequately measure availability to human or ecological receptors. Surrogate methods that represent receptors, such as simulated gastrointestinal fluids, do exist, but they have not been validated for contaminant mixtures or residual environmental concentrations following bioremediation. Surrogate end points such as molecular, genetic, cellular, and other in vitro assays also exist, but they have not been validated against whole animal standards nor have they been applied to bioremediation.

Interference from physical or chemical components in the environment also constrains assessment methodologies. Techniques for documenting the loss of contaminants from a site have been confounded by a host of problems, including natural geologic heterogeneity, variable and unpredictable distributions of contaminants, and the practice of measuring total elemental compositions of soil, sediment, or groundwater samples. Improved methods for detecting the location of contaminants, both directly and indirectly, are needed. Moreover, the influence of geologic heterogeneity and contaminant transport processes on the distribution of contaminants needs to be better understood so that optimal strategies can be developed for locating sampling and monitoring sites. Improved methods for efficiently measuring the chemical form of metals and radionuclides (e.g., redox status, complexation, and sorption) are required to more effectively evaluate bioremediation potential and document biotransformation processes.

APPROACH


The Assessment element will focus on developing techniques for assessing the bioremedial activities of individual strains and functional groups within a community. It will validate existing and emerging laboratory and field techniques (diagnostics) and include data quality and utility. The element will also develop a suite of reliable, reproducible, timely, and economical monitoring techniques for evaluating bioremediation under laboratory and field conditions, and will examine issues in evaluating environmentally acceptable end points for bioremediation.

Solving the technical issues related to measuring, monitoring, and quantifying end points for bioremediation will require the integration of ecological, chemical, hydrogeological, geophysical, engineering, and information management disciplines. For assessing communities and biodegradation rates of parent compounds and intermediates, the emphasis must be on techniques that yield information on activities of consortia or on specific strains of bioremedial groups within the consortia. When assessing bioremediation end points, the focus must be on techniques that accurately reflect the impact on human and environmental health. In addition, an emphasis on near-real-time analysis and data acquisition will provide feedback and support to other program elements on complex reactions in heterogeneous matrices.

The Assessment element, therefore, will involve efforts at the field research centers and at appropriate satellite sites and will focus on these four subelements:

1. Structure rate and activity. Development of quantitative techniques for assessing bioremediation indicators relating to community structure rates of activity and types of microbial activity.

2. Geophysical, geochemical, and hydrogeological characterization. Development of techniques for measuring the geochemical and hydrogeologic factors that influence bioremediation.

3. Interpretive diagnostics. Development of innovative diagnostic tools for managing and interpreting bioremediation measurement information, including fatal-flaw-analysis methods.

4. Bioremediation end points. Development of surrogate methods that accurately reflect the effects of the residual products of bioremediation on human and environmental health.

Subelement 5.1: Structure, Rate, and Activity

Fundamental research in designing, developing, and measuring techniques to assess bioremediation activity, including community structure, biotransformation rates, and types of microbial and rhizosphere activity.

Objective


Develop a proven toolbox of measurement techniques for microbial activity, degradation rates, movement, community structure, degradative populations, and biotransformation products in situ that can provide timely information critical to the design, control, and evaluation of bioremediation efforts, including real-time measurement methods.

Goals


Three-Year

Adapt and improve existing molecular and biochemical techniques -- such as DNA and RNA molecular probes, bioreporters, biosensors, fluorescent stains, PCR techniques, and DNA and RNA fingerprinting analyses of gene products -- for application to field and laboratory measurements of microbial activity, community structure, and electron flow.

Initiate characterizations at field research centers using novel and conventional techniques.

Five-Year

Develop and evaluate new techniques -- such as isotopes of carbon, oxygen, and chloride to distinguish abiotic and biotic processes (e.g., to address mass balance and fate issues) -- for application to environmental in situ and ex situ measurements of microbial activity and community structure.

Ten-Year

Evaluate innovative techniques for cost-effective, real-time efficient laboratory field-monitoring capabilities and reliability.

Provide critical assessment of the degradative capabilities of microbial communities that degrade and transform complex mixtures of contaminants.

Subelement 5.2: Geochemical, Geophysical, and Hydrogeologic Characterization

Fundamental research in developing techniques for measuring important geological, hydrological, and geochemical parameters that influence the bioremediation of contaminants.

Objective


Develop tools and techniques that enable an accurate description of hydrogeologic and geochemical factors influencing bioremediation. These factors include the spatial distribution of key chemical species (e.g., electron acceptors and essential nutrients), the nature of the geological matrix (e.g., lithology, porosity, mineralogy, clay contact), and the mobility of the water, dissolved contaminants, nutrients, gases, and colloidal particles.

Goals


Three-Year

Evaluate the effectiveness of geophysical techniques, such as high-resolution seismic and electrical methods, for identifying contaminants, biodegradable products, and physical properties controlling fluid transport.

Identify and apply a suite of geophysical, geochemical, hydrological, and geological characterization techniques to describe the heterogeneity of the first field site.

Five-Year

Develop a suite of noninvasive geophysical techniques for locating contaminants and documenting losses.

Develop geophysical methods for monitoring biodegradation or biotransformation byproducts.

Develop hydrological methods for better characterization of transport properties for water, dissolved contaminants, and colloids, including bacteria-sized particles.

Ten-Year

Develop cost-effective and reliable geophysical technologies for designing and monitoring long-term bioremediation processes. Couple these technologies with new diagnostics tools to develop an approach to post-closure monitoring that complies with regulatory issues.

Subelement 5.3: Interpretive Diagnostics

Fundamental research in developing the diagnostics to interpret complex data sets, including temporal and spatial variability, in support of effective remediation management.

Objective


Develop innovative methods for analyzing data from in situ microbial biomass, biotransformation, and biodegradation activity, and other tools (including statistical methods) for analyzing bioremediation processes, in collaboration with the Biogeochemical Dynamics and System Integration, Prediction, and Optimization elements.

Goals


Three-Year

Review, evaluate, and develop methods and techniques for compiling, organizing, and analyzing data using analytic and statistical techniques.

Five-Year

Incorporate novel measurement techniques of community interaction and activity into an existing data-acquisition framework.

Develop methods for measuring and interpreting spatial heterogeneity in concentration and species of target compounds, biological communities, and hydrogeologic parameters in collaboration with the Biogeochemical Dynamics element.

Improve and validate mathematical and statistical methodologies for interpretation of data from the surveillance of large areas affected by complex contaminant mixtures over long time periods in collaboration with the System Integration, Prediction, and Optimization element.

Ten-Year

Develop diagnostic feedback models with associated hardware and software systems that can monitor bioremediation processes and provide feedback for the successful management of remediation systems.

Subelement 5.4: Bioremediation End Points

Fundamental research in developing surrogate methods that reflect the bioavailability and stability of contaminants, to help development of performance standards for remediation.

Objective


Assess the stability and bioavailability of the residual end products of bioremediation of complex contaminant mixtures.

Goals


Three-Year

In partnership with the Environmental Protection Agency (EPA) and other organizations sponsoring work on alternative end-points (e.g., GRI), develop methods and tools for estimating the bioavailability of residual contaminant mixtures.

Five-Year

Establish long-term monitoring programs to monitor the stability of sequestered or immobilized contaminants.

Ten-Year

Through collaborative efforts with the EPA, validate environmentally acceptable end points and performance standards for the bioremediation of complex contaminant mixtures.

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