WRHSRC Research Program:
Anaerobic Biological Processes

Focus Group Leader:

Mark Dolan, Oregon State University

Technical Description of Research Focus:

Investigation of anaerobic reductive processes focus on the remediation of high concentrations of chlorinated solvents associated with NAPL source zones and on basic research to develop more effective anaerobic processes for the treatment of halogenated organic contaminants. Center researchers have been actively involved with:

  1. Understanding the fundamentals of basic research related to anaerobic transformations.
  2. Performing several of the first field demonstrations on enhanced in-situ transformations (Semprini et al., 1992; Dybas et al., 1998).
  3. Implementing demonstrations of intrinsic transformations (Semprini et al., 1995).

Research is directed towards improving the efficiency of halorespiration processes at concentrations close to the solubility limits in water. Recent work of Center investigators has shown that halorespiration can be highly effective for chlorinated solvent treatment (Yang and McCarty, 1998; Rosner et al., 1997).


Project 1-OSU-01:

Developing and Optimizing Biotransformation Kinetics for the Bio-remediation of Trichloroethylene at NAPL Source Zone Concentrations

Principal Investigators:

Lewis Semprini and Mark Dolan, Oregon State University

Year Initiated: 2002
Project Summary:

This project aims to develop a mixed anaerobic culture that is effective at transforming PCE and TCE via halorespiration at elevated concentrations representative of those associated with NAPL contamination. The specific objectives of this project are to:

  1. develop a culture with the ability to reductively dechlorinate TCE to ethylene at very high concentrations (above 1,000 mM) and in the presence of DNAPL;
  2. characterize microbial growth and measure maximum substrate utilization rates and half velocity coefficients for successive dechlorinations of TCE to ethylene;
  3. and characterize the microbial consortium by investigating molecular methods to evaluate the diversity of the mixed culture developed in the kinetic studies.

Over the past year kinetic parameters were determined for each step in the dehaogenation process, with two mixed cultures and a binary culture (a mixture of the two cultures) to describe the reductive dechlorination of chlorinated ethylenes. Kinetics of the inhibition of the CAHs was also studied. Model simulations of the sequential transformation of PCE and TCE to VC and ethene matched well the results of batch kinetic experiments over a factor of 30 change in concentration, using the independently determined kinetic parameters. Molecular characterization of the two cultures was also performed.

Diamgram illustrating the chemical reactions that transform TCE to Ethene.

This diagram illustrates the chemical reactions that transform TCE to Ethene.

  • TCE/ cis-DCE/ VC serve as electron acceptors
  • Carbon and an energy source (electron donor) are required to support the reductive dechlorination reaction.
  • Fermentation of organic compounds produces H2 which serves as the electron donor for dechlorination (Gossett et al., 1997).

Project 1-SU-01:

Strategies for In-Situ Mixing of Contaminants and Additives

Principal Investigators:

Peter Kitanidis and Craig Criddle, Stanford University

Year Initiated: 2002
Project Summary:

This project is focused on developing strategies for cost-effective in situ mixing of contaminants and additives in bioremediation. Such methods will employ recirculation units, pairs of extraction-injection wells, sparging systems, biocurtains, and time- and space-sequenced operations. Over the past year (2003), the researchers have focused on the design of an effective chemical delivery and mixing scheme for in situ bioremediation of Uranium (VI) at Oak Ridge National Laboratory (ORNL). Mathematical models of flow, transport and biogeochemistry have been developed and predictions compared with the results of experiments and field tests. Software has been developed for the delineation of injection, extraction, and recirculation zones; the efficient determination of breakthrough curves; the application of travel-time methods of modeling transport; and biogeochemical modeling using PHREEQC in conjunction with hydrogeological modeling within the MATLAB computational environment.

Computer model images of groundwater circulation.

The images above illustrate how three-dimensional flow induced by two recirculation units can be used to speed up in-situ bioremediation.

Link to another illustration of in-situ mixing at a demonstration site at Edwards Air Force Base in California.

Project 2-OSU-07:

Continuous-Flow Column Studies of Reductive Dehalogenation with Two Different Enriched Cultures: Kinetics, Inhibition, and Monitoring of Microbial Activity

Principal Investigators:

Lewis Semprini, Oregon State University, Mark Dolan, Oregon State University, Alfred Spormann, Stanford University

Year Initiated: 2004
Project Summary:

The objective of this study is to conduct continuous-flow column studies with the dehalogenating enrichment cultures that have been studied in our current WRHSRC grant. Detailed kinetic and inhibition constants have been determined for these cultures and preliminary molecular characterization has been performed as well. This work is intended to build on previous results to permit evaluation of the different cultures performance under flow conditions more representative of in-situ remediation. The work will include an evaluation of the sequential transformation process, the microbial populations that develop and their activity both spatially and temporally in continuous-flow columns. The long term sustainability of the process will also be evaluated. Concentrations of PCE and TCE will be tested up to solubility limits corresponding to NAPL source zone levels. Sequencing-batch-reactor studies will also be used to evaluate the analytical and molecular methods prior to use in the continuous-flow column studies. The column studies will evaluate whether kinetic values that have been generated in previous studies can be used in a transport model to predict both the spatial and temporal sequential transformation of chlorinated ethenes. Through collaborations with OSU and Stanford University molecular methods such as Real-time PCR and fluorescence in situ hybridization (FISH) analyses will be used to track and enumerate the dehalogenating population that develops in the columns. Specific gene expression assays employing mRNA-based methods that have been developed at Stanford University will also be employed to evaluate dehalogenating enzyme activity spatially and temporally in the columns. Dechlorinating activity will also be evaluated by monitoring influent chlorinated ethenes and the lesser chlorinated transformation products that are produced, as well as conducting spatially discreet activity-based assays using fluorinated analogs of the chlorinated ethenes. As the project evolves we hope to address inhibitory and toxic effects on the transformation process. Chloroform, for example, is thought to produce a toxic effect on dehalogenators while acetylene has been shown to be a reversible inhibitor of dechlorination. Ultimately, the results of the various activity tests and microbial population estimates will be compared with model simulations to determine the applicability of using kinetic parameters developed in batch systems to predict temporal and spatial transformations in a continuous-flow system.

Project Update Slide (November 2004):

Summary slide of project update, November 2004.

To download this slide, right click here.