The potential sources and extent of microbial contamination of the bedrock cores, and the influence this contamination on the TCE-degrading microbial community structure, will be evaluated while drilling the first preliminary borehole. Using a variety of techniques simultaneously will allow us to compare the suitability of each method for assessing contamination. The first preliminary borehole will be drilled at a control (uncontaminated) site. The broad-based suite of analytical methods will be employed to address two specific drilling-associated questions.

Question 1. What are the sources and extent of drilling-associated bacterial contamination on the bedrock core fracture faces?

Three different tracers will be used in drilling the first preliminary borehole. An inorganic tracer, bromide, will be introduced into the drilling fluid to assess the overall level of drilling-associated contamination. A bacterial tracer, Chromobacterium violaceum, will be used to evaluate the extent of overburden contamination of the bedrock core fracture faces. Chromobacterium can be readily detected and distinguished from indigenous bacterial populations by its violet pigment (violacein). Dead cells of Pseudomonas syringae, an ice nucleation active (INA) bacterium, will be used to trace the extent of microbial contamination resulting from the drilling fluid. In addition to the tracers, semi-quantitative community-level physiological profiles (CLPP) and qualitative community-level phylogenetic profiles (CLGP) will be used to determine the extent of microbial contamination of the bedrock. CLPP and CLGP will be determined for the overburden (before and after introduction of the Chromobacterium tracer), drilling water (before and after introduction of the INA Pseudomonas tracer), porewater associated with the bedrock cores and the fracture surfaces of the bedrock. CLPP will be determined by submitting each sample to sole carbon source utilization testing. CLGP will be determined using denaturing gradient gel electrophoresis (DGGE). Finally, culturable and total bacterial cells will be enumerated from samples of the overburden, drilling water, porewater, and fracture surfaces using low-nutrient media and DAPI-staining, respectively.

Question 2. Does the drilling process contaminate the bedrock core fracture faces with TCE-degrading bacteria?

This question will be experimentally addressed by determining if the genetic potential for TCE biodegradation exists in the overburden, drilling water, and porewater, and on the bedrock core fracture faces. The genetic potential for TCE degradation will be evaluated by examining bacterial communities in samples for the presence of genes that encode for oxidative and reductive dehalogenating enzymes. The presence of the genes encoding for particulate methane monooxygenase, soluble methane monooxygenase, ammonia monooxygenase and/or TCE dehalogenase within a DNA extract from a specific sample would indicate that the genetic potential for TCE biodegradation exists within that bacterial community. In order to test this hypothesis, specific primers will be used to amplify these genes by PCR. Specific PCR products will be identified by their sizes on agarose gels.

If the genetic potential for TCE biodegradation is not introduced into the bedrock during drilling, then bacterial contamination associated with the drilling process will not impact our microbiological experimentation in Phase III. However, if this potential is introduced during drilling, then the broad-based approach we will use to determine the sources and extent of this contamination will be an integral component of our interpretation of data obtained in the Phase III microbiological experiments.