By assessing whether the bacteria possess the genes that encode for various oxidative and reductive dehalogenating enzymes, we can decide what engineered approach(es) to bioremediation might be appropriate.

Currently, the major mechanism for identifying whether biodegradation of organic contaminants is occurring in situ is to monitor for the decrease in the parent compounds and the increase in their breakdown products. However, the metabolic process(es) responsible are oftentimes difficult to ascertain from such chemical data alone, particularly during biodegradation of chlorinated ethenes. In addition, it is impossible to know from this kind of an approach whether the in situ microorganisms have the genetic potential to use other more efficient metabolic process(es), if the appropriate electron donors or acceptors could be provided. There are several methods that have been developed to assess the capabilities of bacteria to degrade chlorinated ethenes, but they have never been used as a suite of tests to address the issue of what the current and potential in situ metabolic processes are (could be). We will use several new and traditional methods (multiplex and reverse transcriptase PCR with primers specific for genes encoding for TCE degrading enzymes, DGGE, and enrichment cultures) to evaluate the microorganisms obtained from our test boreholes (cores and groundwater). In addition, we propose to use hydrogen analyses of the groundwater to corroborate what anaerobic metabolic process(es) are operative in situ. By assessing whether the bacteria possess the genes that encode for various oxidative and reductive dehalogenating enzymes, we can decide what engineered approach(es) to bioremediation might be appropriate. For example, if the microbial community at a site does not possess genes that encode for methane monoxygenese, addition of methane and air would probably not be a successful bioremediation alternative.

Protists are known to impact the rate of bacterial degradation of organic contaminants in unconsolidated aquifer sediments (Kinner et al., 1997, 1998). There is no data available concerning whether protists exist in bedrock aquifers. This research will provide a first indication of whether they are present in situ. If they are, we will try to elucidate their role in the biodegradation process during Year II. Laboratory microcosms can be very useful in estimating the rates of reductive dechlorination occurring in the field as shown by Wilson et al. (1996). However, most microcosm methods have been developed for use with unconsolidated aquifer sediments. Yaeger et al. (1997) used groundwater alone and with sterile pulverized dolomite from a bedrock aquifer to conduct their microcosm studies. Other microcosm models for bedrock aquifers need to be developed and tested that also include the microbial communities associated with the fracture faces. This is especially important because it is often very difficult to compare contaminant and by-product concentrations along the groundwater flow path in bedrock aquifers because of the limited number of wells and the uncertainty regarding their connectivity. During Year I, we will develop four microcosm models that use rock faces or chips. During Year II, we will evaluate the models using fresh fracture material from Site 32 along with the groundwater microcosm used by Yaeger et al. (1997). This will allow us to recommend laboratory methods that will generate useable estimates of in situ biodegradation rates and microcosms that can be easily replicated to study the effects of in situ conditions (e.g., secondary mineralization of the rock) on biodegradation rates.

Fracture skins, the thin weathered layer on the surface of the bedrock in contact with the groundwater, can play an important role in solute transport and reactions (e.g., Robinson et al., 1998). For example, fracture skins can affect contaminant movement. Because the chemistry (mineralogy) of the rock can be important in understanding microbial reactions (e.g., Chapelle, 1993; Erlich, 1988), it is important to examine the rock composition, especially along the fractures. During Year I, we will begin petrographic analysis of the rock to determine what the mineralogy is in uncontaminated and differently-contaminated (e.g., TCE, DCE and VC) fractures. This examination will continue in Year II using XPS and SIMS. The data will help determine whether core samples can be used to infer the history of contamination and chemical/biochemical reactions at a specific location within the plume and how the presence of the contaminant(s) affects the secondary mineralization of the rock and the fate of potential electron donors or acceptors.

For subsequent years of the BBC's research, it is imperative that a series of hydraulically-connected wells be available within different biogeochemical regions of the plume (TCE, DCE, VC and uncontaminated zones). This will enable us to test new approaches to site characterization and bioremediation. As a result of the Year I drilling and geophysical and packer testing, we will have established such a network of wells for future in situ experiments at Site 32. The discrete fractures within these boreholes will be isolated to prevent cross-contamination between water masses from discrete fractures.