John T. Wilson
Scissortail Environmental Solutions, LLC
Dr. John T. Wilson has extensive experience in natural attenuation processes and bioremediation. Dr. Wilson served as research microbiologist for U.S. EPA at the R.S. Kerr Environmental Research Center in Ada, Oklahoma for 36 years. His research for EPA was primarily on natural attenuation of BTEX compounds, fuel additives, and chlorinated solvents, as well as in-situ bioremediation of chlorinated solvents. Dr. Wilson is currently the Principal Scientist with Scissortail Environmental Solutions, LLC. He provides consulting services to evaluate site monitoring data to determine the contribution of biological and abiotic degradation to natural attenuation of organic contaminants in ground water. He also provides consulting services on the use of Compound Specific Isotope Analysis (CSIA) to identify biotic and abiotic degradation of organic compounds.
SESSION KEYNOTE PRESENTER – Site and Remedy Diagnostic Tools: Doctor, Doctor, Give Me the News
Getting Diagnostic Tools to Tell Us More: Moving Towards Quantitative Predictions
Molecular Biological Tools are widely used in our industry, but they are often restricted to a supporting role. Although the assays and tests return a quantitative measure of the abundance of a marker, the interpretation is qualitative. We should do more to relate the abundance of the MTB marker to a quantitative estimate of activity. A series of enzyme activity probes (EAP) are available that are diagnostic for aerobic TCE co-oxidation. Several qPCR assays are available for enzymes known to co-oxidize TCE. To move these diagnostic tools toward more quantitative predictions, the probes and the qPCR assays were evaluated against a direct measurement of the rate of TCE co-oxidation in groundwater samples.
The traditional carbon 14 assay for biodegradation was redesigned and reconfigured to measure TCE co-oxidation. The rate of co-oxidation was estimated as the rate of accumulation of label that could not be purged from the water sample. To account of autolysis of the radioactive TCE, rates were also estimated in water samples that had been filtered to remove microorganisms. The rate of TCE co-oxidation was determined in nineteen wells distributed over five different sites. Co-oxidation was detected in eight of the wells at rate constants that exceeded the rate constants for autolysis at 95% confidence. The rate constants in the filtered water were subtracted from the overall rate constants to provide a rate constant for biological co-oxidation. The first order rate constants for co-oxidation varied from 2.6 per year to 0.006 per year. In the other eleven wells, the overall rate constant for TCE degradation was not different from the rate constant in the filtered control that contained no bacteria. In these eleven wells, none of the overall rate constants were above 0.02 per year.
The rate constants were compared to the abundance of cells that reacted with the probes 3-hydroxyphenylacetylene, phenylacetylene or trans-cinnamonitrile to accumulate a fluorescent product. In general, as the abundance of reactive cells increased, the rate constant increased in the same proportion. In six wells where the abundance of reactive cells was ≥ 103 per mL and a rate constant for co-oxidation of TCE could be determined, the rate constants were equal to or greater than 0.02 per year. The maximum abundance of 4 X 104 reactive cells per mL was associated with a rate constant of 0.5 per year. However, in eight wells where the overall rate constant for TCE degradation could not be distinguished from the rate constant for autolysis the abundance of reactive cells was also ≥ 103 per mL. An abundance of EAP reactive cells ≥ 103 per mL was not unequivocally associated with TCE co-oxidation. As a result, the EAP provides a useful indication of the possibility for co-oxidation, but the EAP should not be used as the sole criterion to assign a rate constant.
The rate constants were also compared to the abundance of gene copies of Phenol Hydroxylase (PHE), and Toluene Monooxygenase (RMO and RDEG). As a general trend, as the abundance of PHE, RMO and RDEG increased, the rate constants for TCE co-oxidation increased in the same proportion. In four wells the abundance of gene copies amplified by the PHE, RMO or RDEG primers was ≥ 103 per mL and a rate constant for co-oxidation of TCE could be determined. The rate constants in these four wells were equal to or greater than 0.06 per year. The maximum abundance of 5 X 104 gene copies per mL was associated with a rate constant of 2.7 per year. However, in two wells where the overall rate constant for TCE degradation could not be distinguished from the rate constant for autolysis the abundance of gene copies was also ≥ 103 per mL. As was the case with the enzyme activity probes, an abundance of gene copies ≥ 103 per mL should not be used as the sole criterion to assign a rate constant.