Title: Western Region Hazardous Substance Research Center Project 1-OSU-04
Development and Evaluation of Field Sensors for Monitoring Bioaugmentation with Anaerobic Dehalogenating Cultures for In Situ Treatment of TCE

Investigator: James D. Ingle, Oregon State University

Institution: Oregon State University

Research Category: Bioremediation, groundwater

Project Period: 2001-2003


Objectives: The overall objective of this study was to refine and use redox sensors based on redox indicators as monitoring tools for assessing and optimizing redox conditions for treatment of PCE and TCE with dehalogenating cultures. Specific objectives were to 1) deploy, evaluate, and refine redox indicators for on-line monitoring of the redox conditions in two collaborative situations involving a bioaugmentation approach, 2) understand the nature of the redox conditions under which dechlorination microbial processes occur.

Rationale: Better on-line monitoring techniques for redox status are needed for 1) the initial assessment of laboratory samples or models and of subsurface conditions at a site, 2) continued assessment of the progress of remediation, and 3) control of injections of amendments (e.g., substrates, nutrients) during remediation. We have shown that redox sensors based on redox indicators exhibit promise for monitoring environmental redox levels. Research is needed to1) understand the nature of the response of these indicators, 2) improve the monitoring devices for practical use, and 3) demonstrate that these devices can be employed for on-line monitoring of the status of anaerobic dehalogenating cultures in laboratory systems.

Approach: Redox indicators immobilized on transparent films have been shown to be able to differentiate between different microbial redox levels (Fe(III)-reducing, sulfate-reducing, methanogenic). These redox indicator flow sensors were deployed in two primary situations for calibration and demonstration of their applicability: 1) continuous monitoring of redox conditions of cultures inside bioreactors or microcosm bottles as a tool for the optimizing conditions for effective dechlorination of PCE and TCE with enriched halorespiratory cultures, 2) on-line monitoring of the redox status of the material in a physical aquifer model (PAM) bioaugmented with the developed dehalogenating cultures. The design and characteristics of the redox sensor monitoring systems were improved for low oxygen permeation and portability for easy operation in the field.

Status: We have refined the portable, immobilized redox monitoring system and used it to monitor sulfate-reducing and methanogenic conditions in a PAM containing wastewater slurry and also dechlorinating cultures in bioreactors and modified microcosm bottles. The enriched dechlorinating culture (Lew Semprini lab) was loaded into our bioreactors and microcosm bottles to calibrate the response of the redox indicators to the dechlorination of PCE. The indicator data support the concept the dechlorinating process is increasingly more reducing as PCE is dechlorinated, with the most reducing step in the process being the dechlorination of vinyl chloride to ethene. Specifically, the reduction of Thionine (THI) indicates degradation of PCE and formation of TCE and cis-DCE; whereas, ~50% reduction of Cresyl Violet (CV) correlates to the formation of vinyl chloride, and production of ethene is only observed when CV is nearly or fully reduced. We worked closely with Dr. Semprini and his students to address concerns about oxygen contamination during culture transfer steps and while monitoring with our redox indicator, flow monitoring system. Refining techniques for transfer of highly oxygen-sensitive cultures was critical for eventual column and PAM studies.

We developed a method to precipitate finely divided platinum particles into membranes with immobilized indicators through reduction of Pt2+ solutions. H2 levels as low as 0.01% by volume in the headspace do reduce the indicator in the platinum embedded membranes. The indicator Phenosafranine (PSaf) is useful for monitoring dechlorinating cultures because, without Pt, PSaf is not reduced by reductants in dechlorinating cultures. Reduction of a PSaf membrane with embedded Pt indicates active fermentation and H2 production necessary for dechlorination. The rate of reduction of the indicator changed with varying H2 concentrations. Although the results were preliminary, this approach could be the basis of a convenient and inexpensive method to determine if H2 concentrations in cultures are sufficient for effective dechlorination laboratory without the need to run expensive GC testing. This area of research was not further pursued.



Journal Articles

Cantrell, K. and J. D. Ingle, Jr. (2003). The SLIM Spectrometer. Anal. Chem., 75, 27-35.

Ruiz-Haas, P. and Ingle, J.D, Jr. (2007). Monitoring Redox Conditions with Flow-Based and Fiber Optic Sensors Based on Redox Indicators: Application to Reductive Dehalogenation in a Bioaugmented Soil Column. Geomicrobiology Journal, Vol. 24 (3/4) 365-378.


Conference Proceedings and Presentations

Cakin, D.and J. D. Ingle, Jr. (2003). Development of a Liquid Core Waveguide Based Field Instrument For Analysis of Iron. Second Black Sea Basin Conference on Analytical Chemistry, Istanbul-TURKEY.

Cakin, D. and J. D. Ingle, Jr. (2004). Design and Characterization of a Liquid Core Waveguide based Analytical Device for the Analysis of Anaerobic Systems. EPA/ORD/HSRC Superfund Research on Risk Characterization and Monitoring, Las Vegas, NV.

Cantrell, K and J. D. Ingle, Jr. (2004). Design of Miniature Spectrometers based on Light Emitting Diodes. 51st International Conference on Analytical Sciences and Spectroscopy (ICASS).

Ruiz-Haas, Peter, and J.D. Ingle, Jr. (2004). Evaluation of Redox Conditions with Redox-Indicator Based Sensors in Soil and Microcosms Bioaugmented with Reductive Dehalogenating Bacteria. EPA/ORD/HSRC Superfund Research on Risk Characterization and Monitoring, Las Vegas, NV.



Cantrell, Kevin (2002).The Development and Characterization of Miniature Spectrometers for Measuring the Redox Status of Environmental Samples. Ph.D., Oregon State University.

Prayoonpokarach, Sanchai (2003). Development and Evaluation of Sampling Techniques, Instrumentation, and Pyridine Derivative Reagents for Fluorometric Determination of Chloroform and TCE in Water with a Portable Fluorometer. Ph.D., Oregon State University.


Supplemental Keywords: biotransformation; characterization; VOCs; chlorinated solvents; bioremediation; environmental chemistry