| Application of DNA Testing for Dehalococcoides and other specific bacteria to Evaluating Bioremediation |
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During the last 10-15 years, the use of nucleic acid based methods (DNA and RNA), most notably quantitative polymerase chain reaction (qPCR), has grown substantially in the environmental restoration industry. The reasons for this enthusiasm are simple: DNA can be extracted directly from groundwater, soil, or sediment samples avoiding the biases associated with traditional cultivation techniques (plate counts) and qPCR assays quantify specific bacteria (i.e. dehalococcoides) or specific biological processes responsible for contaminant biodegradation providing a more direct, accurate, and sensitive method to evaluate bioremediation as a corrective action. QPCR is a molecular method whereby a multitude of copies of a specific gene are generated. As each gene copy is made, a fluorescent marker is released, measured, and used to quantify the number of target genes present in the sample. The gene copied during this process (target gene) is determined by short segments of DNA called “primers” which are added to the reaction mixture. Since different primers can be designed for different target genes, qPCR assays can be developed to quantify organisms capable of an extremely broad variety of biological functions including metabolism of subsurface contaminants.Chlorinated solvents including tetrachlorethene (PCE) and trichloroethene (TCE) were widely used in a variety of industrial/commercial applications and are now prominent groundwater contaminants. Under anaerobic conditions, PCE and TCE are susceptible to biological reductive dechlorination through the daughter products cis-dichloroethene (DCE) and vinyl chloride (VC) to ethene, an innocuous end product. Thus, anaerobic bioremediation is an attractive treatment measure at PCE/TCE-contaminated sites particularly in Florida where aquifers are often naturally anaerobic. A number of bacterial cultures are capable of transforming PCE and TCE, however, Dehalococcoides species may be the most important because they are the only bacterial group that has been isolated to date which is capable of complete reductive dechlorination of PCE to ethene. In fact, the presence of Dehalococcoides has been associated with the full dechlorination of PCE to ethene at sites across North America and Europe. Thus, qPCR monitoring of the abundance of Dehalococcoides allows site managers to evaluate the feasibility of complete reductive dechlorination under monitored natural attenuation (MNA) conditions and the effectiveness of biostimulation (electron donor injection) to promote growth of key reductive dechlorinating bacteria and enhance bioremediation. Although an attractive treatment alternative, anaerobic bioremediation of PCE and TCE can be hindered by a few site-specific factors. The accumulation of the daughter product vinyl chloride can be problematic at PCE/TCE sites because VC is generally considered more carcinogenic than the parent compounds. Therefore, qPCR assays targeting vinyl chloride reductase genes have been developed to more definitively confirm the potential for biodegradation of VC. Reductive dechlorination can also be hindered by competing biological processes and chemical compounds. Provided with an electron donor, sulfate reducing bacteria will produce hydrogen sulfide which can inhibit reductive dechlorination. Considering the relatively high concentrations of sulfate in some Florida groundwaters, qPCR monitoring of sulfate reducing bacteria may be beneficial to assess their impact on reductive dechlorination particularly at sites undergoing biostimulation. Finally, trichloroethanes (TCAs) were extensively used in industrial applications as degreasers and are common co-contaminants at PCE/TCE impacted sites. The presence of 1,1,1-TCA is especially problematic at PCE impacted sites due to inhibition of reductive dechlorination of chlorinated ethenes. Members of the bacterial genus Dehalobacter have the somewhat unique ability to utilize chlorinated ethanes including 1,1,1-TCA and 1,1,2-TCA. Therefore, qPCR monitoring of Dehalobacter at sites co-contaminated by TCAs can be used to assess whether TCAs will be biodegraded or whether inhibition of reductive dechlorination of chlorinated ethenes is likely. As with chlorinated solvents, bioremediation is frequently the preferred corrective action at sites impacted by petroleum products like gasoline and diesel fuel. At gasoline sites, the monoaromatic hydrocarbons benzene, toluene, ethylbenzene, and xylenes (BTEX) and the additive methyl tert-butyl ether (MTBE) are often the contaminants of principal concern. Under aerobic conditions, the first step in BTEX biodegradation is the incorporation of oxygen which is catalyzed by aromatic oxygenases. Quantification of aromatic oxygenase genes encoding enzymes directly responsible for BTEX biodegradation provides a direct route to assess the feasibility of MNA and the effectiveness of engineered remedial approaches such as air sparging or injection of oxygen releasing materials. Likewise, qPCR enumeration of Methylibium petroleiphilum PM1, one of the few bacteria isolated which is capable of growth on MTBE, can be used to assess aerobic MTBE bioremediation. BTEX compounds are also biodegraded under anoxic and anaerobic conditions. Although the pathways for anaerobic BTEX biodegradation are not as well characterized as the aerobic pathways, benzylsuccinate synthase has been shown to be involved in the anaerobic biodegradation of toluene. Thus qPCR monitoring of the bssA gene encoding benzylsuccinate synthase can be used to assess anaerobic BTEX biodegradation. In summary, qPCR results, when coupled with site chemical/geochemical data, are an extremely powerful tool to assess bioremediation as a treatment technology and to guide site management decisions |