To date, the most commonly used MBTs are polymerase chain reactionMakes copies of a specific DNA sequence within a target gene of microorganisms that can be further analyzed. (PCR), quantitative PCR (qPCR) and reverse transcriptase-qPCR (RT-qPCR), and DNA microarrays. However other MBTs have uses as well, including fluorescence in situ hybridization (FISH)Detects the presence of targeted genetic material in an environmental sample and estimates the number of specific microorganisms or groups of microorganisms., enzyme activity probes (EAPs)Transformation of surrogate compounds (probes) resembling contaminants produces a fluorescent (or other distinct) signal in cells which is then detected using a microscope. and stable isotope probing (SIP)A synthesized form of the contaminant containing a stable isotope (e.g., ¹³C label) is added. If biodegradation is occurring the isotope will be detected in biomolecules (e.g., phospholipids, DNA).. There are also several potentially useful microbial fingerprinting techniques, including phospholipid fatty acid (PLFA) analysisA laboratory analytical techniques that differentiate microorganisms or groups of microorganisms based on quantifying PLFA groups., denaturing gradient gel electrophoresis (DGGE)Type of gel electrophoresis used to separate mixtures of PCR products based on the melting point which is reflective of the DNA sequence.  DGGE is used to generate a genetic fingerprint of the microbial community and potentially identify dominant microorganisms., and terminal restriction fragment length polymorphism (T-RFLP)A nucleic acid (DNA or RNA)-based technique used to generate a genetic fingerprint of the microbial community and potentially identify dominant microorganisms..

EMDs have application in each phase of environmental site management, including site characterization, remediation, monitoring, and closure activities (See Figure ES-1). EMDs can provide unique information valuable in conjunction with more conventional data.

Figure ES-1. Overview of EMDs.

Figure ES-2. EMD projects by state.

EMDs can provide the following benefits:

• Improve the management of contaminated sites
• Determine whether biotic or abiotic degradation is occurring at a site
• Identify specific contaminant sources or reveal whether multiple sources of contamination are present (for instance, isotopic fractionationSome processes (for example, those which involve breaking chemical bonds) have slightly different rates for different isotopes, leading to a more rapid consumption of one isotope over the other. This characteristic is manifested in a change in the isotopic ratio of the residual compound.)
• Improve evaluation and decision making for remediation strategies
• Identify degradation pathways and their degree of completion
• Identify the need for enhancements such as chemical amendments or bioaugmentationThe introduction of cultured microorganisms into the subsurface environment for the purpose of enhancing bioremediation of organic contaminants (USEPA 2011)
• Estimate degradation rates, for instance, CSIA using the Rayleigh equation (see Appendix C.12)
• Aid in monitoring program decisions
• Provide complementary data to support site closure and other site management decisions

Figure ES-3 describes potential uses of EMDs in each phase of site management.

Figure ES-3. Potential uses of EMDs in site management.

The key information available from such tools includes:

• Microbial presence (and abundance in some cases)
• Microbial cellular activityRefers to when a microorganism performs a specific function (e.g., sulfate reduction, metabolism of benzene) (e.g., transcriptionThe first step in activation of a biochemical pathway where a complementary RNA copy is synthesized from a DNA sequence.)
• Biodegradation activity
• Direct evidence of contaminant biodegradationA process by which microorganisms transform or alter (through metabolic or enzymatic action) the structure of chemicals introduced into the environment (USEPA 2011).

To select the appropriate EMDs, it is important to understand the connections between the information provided by DNA-based analysis, RNA analysis, and stable isotope-based analysis. In general, the genes of microbes (and other organisms) are composed of deoxyribonucleic acid, or DNA. DNA can be transcribed into RNA, and ultimately translated into enzymesAny of numerous proteins or conjugated proteins produced by living organisms and facilitating biochemical reactions (based on USEPA 2004a). (and other proteins) that degrade the contaminant (see Figure ES-4). Thus DNA-based analyses (such as PCR, FISH, some fingerprinting methods, and microarrays) can determine if microorganisms with the potential to biodegrade target contaminants are present at a site, and in some cases (notably qPCR), the abundance of the target microorganisms. RNA-based analyses (such as RT-qPCR, FISH, microarrays, and some fingerprinting methods) or analyses that identify the end product of specific enzymes (such as EAPs) can show that biodegrading organisms are actively expressing biodegradation genes.

One of the most frequent uses of EMDs is to verify that natural or enhanced biodegradation can occur, or in fact is occurring, in situ. Some EMDs can be used to estimate biodegradation rates. CSIA in particular can be very useful for this purpose. Care must be taken, however, in extrapolating rates both spatially and temporally. Additionally, EMDs that indicate number of geneA segment of DNA containing the code for a protein, transfer RNA, or ribosomal RNA molecule (based on Madigan et al. 2010). copies, or count the number of organisms in a given sample, could potentially be used to infer degradation rates, or at least to conclude that useful rates are occurring or that native or added organisms are increasing in number over time. However, there is currently no method to calculate degradation rates from the number of organisms or gene copies in a sample.

Figure ES-4. Flow of information within cells.

EMD plans and analyses should be based on a sufficient number of samples, taken at appropriate locations and times, using appropriate techniques, and including appropriate documentation and QA/QC controls. The number of samples needed for a given site will be a function of site conditions including geology, hydrogeology, geochemistry, and contaminant distribution. A successful sampling program will incorporate these parameters in a site-specific sampling plan. Generally, vadose zone biology is more location-specific because microbial transport (and thus distribution) depends on excess water. With less water available, more samples may be required to characterize a smaller area. Since microbial diversityMicrobial diversity can have many definitions but in this context generally refers to the number of different microbial species and their relative abundance in an environmental sample (Nannipieri et al. 2003). in soils and sediments can vary on a millimeter scale, homogenization and multiple samples are desirable.

Samples may be collected by either active or passive sampling techniques, from groundwater or solid materials. For some bacteria (e.g., DehalococcoidesDehalococcoides is a genus of organohalide-respiring bacteria (for example, bacteria that use chlorinated solvents as metabolic electron acceptors) within the phylum Chloroflexi, in the domain Bacteria, and currently represented by a single species, Dehalococcoides mccartyi (Dhc). This species is the only one known with strains that dechlorinate dichloroethenes (DCEs) and vinyl chloride (VC) to ethene and inorganic chloride., perchlorate degraders) active sampling of groundwater alone will be useful because a significant fraction of the bacteria can be found in the aqueous phase (see Section 10.4.2). However, other bacteria may be primarily attached to the aquifer solids, and groundwater analysis alone may not be appropriate. When sampling groundwater, most EMDs require filtration of the sample to collect concentrated biomass for analysis. Various biomass extraction/filtration approaches are available for collecting active microbial biomass from environmental media. Passive microbial sampling devices are groundwater sampling tools (for example, biofilm coupons, in situ microcosms, groundwater dialysis chambers, porous beads, Bio-Trap® samplers) that facilitate colonization of subsurface microorganisms onto a retrievable matrix. Passive microbial sampling devices may be very useful for assessing activity in situ, but the results may only be semi-quantitative, since it is difficult to relate microbial concentrations in the groundwater or aquifer matrix to those detected on the passive microbial sampling device (See EMD Sampling Methods Fact Sheet and Section 10.4.3).

This ITRC document includes several QA/QC considerations that should be part of any plan for the use of EMDs. However, if EMDs are to be used at a site, it is important that the regulator be involved as early as possible, to allay such QA/QC concerns. During the initial meeting, a draft work plan should be available, and should include:

1. The current conceptual site model, based upon the results of existing conventional methods
2. The EMDs to be used, with an explanation of how EMD data can complement the existing data
3. The life cycle stage of the environmental cleanup process for which the EMD is to be used
4. The sample locations and frequencies
5. Specific data quality objectives.
6. Any permitting requirements necessary for the use of the EMD.

Finally, EMD team members and most of those surveyed agree that education is the key to more widespread use of EMDs in the environmental site management field. Figure ES-5 includes the results from the respondents to the survey when asked about their EMD experience.

Figure ES-5. Experience with EMDs survey results.

As regulators become better educated and more comfortable with their use, guidance and regulations specific to EMD use will be developed. Until state documents are developed, this ITRC document and the related Fact Sheets developed by this team will serve as the most comprehensive resources available for regulators, consultants, and the general public.