Chlorinated solvents, such as tetrachloroethene (PCE), trichloroethene (TCE), and chlorobenzenes, are the most prevalent groundwater contaminants at Department of Defense (DoD) and Department of Energy (DOE) sites. Anaerobic bioremediation is a promising treatment technology for cost-effective removal of these contaminants at a large scale. With natural attenuation, practitioners need information about the natural rates of biodegradation, which is difficult to obtain because of the process’s slow rate. With engineered bioremediation, rates tend to be much faster, but often when applied, the process does not work as planned because of a variety of problems, such as lack of suitable organisms, strong competition for substrate by other organisms, lack of appropriate nutrients, insufficient supply of electron donor (reductant), inhibitory conditions, etc. Thus, biomarkers are needed to help assess critical biological parameters at problem sites such as whether or not (i) the key microorganisms are present, (ii) the key genes are present, and (iii) the key genes are active (induced). The rate of hydrogen release, as well as knowledge of competing hydrogen-consuming reactions, are critical for the practitioner to guide successful remediation efforts. Monitoring the presence, abundance, and expression status of diverse hydrogenase as well as reductive dehalogenase genes should provide semi-quantitative markers to predict in situ rates of reductive dehalogenation.

The specific objectives of this project were to:

1. Develop and test quantitative molecular biomarkers for the hydrogen‐producing and hydrogen-consuming microbial population present in groundwater and sediment material.
2. Correlate the quantitative data obtained by molecular biomarkers with experimentally determined transformation rates, and test and refine a mathematical model with these data.

This project developed a novel and broad‐ranging approach to the characterization of complex microbial communities by analyzing the abundance and expression of key functional genes using a tiling DNA microarray. A test hybridization showed that the detection limit for the microbial community system was between approximately 1% and 10% of the total microbial community. This molecular tool is useful for monitoring the presence, shift in abundance, and expression status of hydrogenase as well as reductive dehalogenase genes and for semi-quantitatively predicting hydrogen flux in complex microbial communities. This tool also allowed shifts in populations of dehalogenating microorganisms, as well as evolution of novel lineages, to be observed.

Simulation of the anaerobic dehalogenation of TCE in a continuous stirred tank reactor (CSTR) experiment under excess and limited electron donor addition was carried out. Formate and TCE were fed to 2‐L and 5‐L CSTRs containing the Evanite culture (EV). To simulate the results of the two experiments, a non-steady‐state model was developed that coupled thermodynamic and kinetic models to include CSTR flow conditions. The model includes inhibition kinetics for the dehalogenation reaction and permits the growth of homoacetogens and dehalogenators as competing hydrogen consuming processes. Three microbial communities were simulated in the CSTR, dechlorinators that transform TCE to cis-dichloroethene (cDCE), dechlorinators that transform cDCE to ethane and homoacetogens. Hydrogen is assumed to be in equilibrium with formate. The system of model equations was solved numerically using COMSOL 3.5a. Vinyl chloride (VC) dehalogenation kinetics were determined in batch experiments using cells harvested from the CSTR. The model simulated well the steady-state and transient performance of the CSTR when excess and limiting electron donor were added. The batch-determined kinetic parameters did a good job of capturing the observed chlorinated aliphatic hydrocarbons, hydrogen, acetate, and biomass concentration in both the CSTR and in separate batch experiments. VC dehalogenation was rate limited when electron donor was in excess and stoichiometry limited when electron donor was not in excess. Simulations required a hydrogen threshold for homoacetogens of 40 nM to match CSTR observations, which is lower than reported in the literature. The agreement of the model with the experimental results provides confidence in the kinetic parameters being used and the ability to simulate these complex processes that include the competition for hydrogen.

This project demonstrated for the first time the proof of concept of using semi‐quantitative monitoring of key genes and their expression. This in conjunction with selecting a more efficient electron donor such as formate can make bioremediation of chloroethene-contaminated sites more efficient and less expensive.

Azizian, M.F. and L. Semprini. 2016. Simultaneous Anaerobic Transformation of Tetrachloroethene and Carbon Tetrachloride in a Continuous Flow Column. Journal of Contaminant Hydrology, 190:58-68.

Marshall, I.P.G., M.F. Azizian, L. Semprini and A.M. Spormann. 2013. Inferring Community Dynamics of Organohalide-respiring Bacteria in Chemostats by Covariance of rdhA Gene Abundance. FEMS Microbiology Ecology, 87:428-440.