Effect of Nitrate and Sulfate on Dechlorination by a Mixed Hydrogen-Fed Culture 225 1058-8337/02/$.50 © 2002 by CRC Press LLC Bioremediation Journal 6(3):225–236 (2002) Effect of Nitrate and Sulfate on Dechlorination by a Mixed Hydrogen-Fed Culture D. K. Nelson, R. M. Hozalski, L. W. Clapp, M. J. Semmens, and P. J. Novak The Department of Civil Engineering, University of Minnesota, 122 Civil Engineering Building, 500 Pillsbury Drive S.E., Minneapolis, MN 55455–0220 Abstract: A novel hollow-fiber membrane remediation technology developed in our laboratory for hydrogen delivery to the subsurface was shown to support the dechlorination of perchloroethene (PCE) to cis-dichloroethene. In previous research, the presence of nitrate or sulfate has been observed to inhibit biological reductive dechlo- rination. In this study hollow-fiber membranes were used to supply hydrogen to a mixed culture to investigate whether adequate hydrogen could be added to support dechlorination in the presence of alternative electron acceptors. By continuously supplying hydrogen through the membrane, the hydrogen concentrations within the reactor were maintained well above the hydrogen thresholds reported to sustain reductive dechlorination. It was hypothesized that by preventing nitrate and sulfate reducers from decreasing hydrogen concentrations to below the dehalorespirer threshold, the inhibition of PCE dechlorination by nitrate and sulfate might be avoided and dechlorination could be stimulated more effectively. Enough membrane-fed hydrogen was supplied to completely degrade the alternative electron acceptors present and initiate dechlorination. Nevertheless, nitrate and sulfate inhibited dechlorinating activity even when hydrogen was not limiting. This suggests that competition for hydrogen was not responsible for the observed inhibition. Subsequent microcosm experiments demonstrated that the denitrification intermediate nitrous oxide was inhibitory at 13 μM. Key Words: dechlorination, hollow-fiber membranes, hydrogen, nitrate, sulfate, electron donor. Introduction The contamination of aquifers with chlorinated solvents such as perchloroethene (PCE) has emerged as a major problem over the last few decades. One technique for remediating these contaminated aquifers is in situ anaero- bic biological reductive dehalogenation. Indigenous mi- croorganisms are capable of mediating the reduction of chlorinated solvents such as PCE via either a gratuitous process or via a process termed dehalorespiration, where microorganisms are able to conserve the energy pro- duced during PCE reduction (Holliger, 1993; Neumann et al., 1994). The reduction of chlorinated solvents is often limited in situ by a lack of electron donor. Based on this concept, several technologies have been used to supply excess electron donor to contaminated sites to stimulate biological reductive dehalogenation. These include the addition of fermentable substrates (Carr and Hughes, 1998; Fennell et al., 1997; Fennell and Gossett, 1998) and direct sparging of hydrogen gas (H 2 ) (Hughes et al., 1997). One novel technology that has been developed recently is the use of gas-permeable hollow-fiber mem- brane modules to continuously supply H 2 directly to groundwater (Fang et al., 2002; Muenzner et al., ac- cepted). Membrane modules can be designed to pro- vide a large surface area for passive gas transfer while presenting minimal resistance to hydraulic flow. This innovative gas transfer technology overcomes the prob- lems of conventional gas transfer devices: inefficiency, nonuniform delivery, and gas loss, by dissolving H 2 directly into the water without bubble formation (Côté et al., 1988; Côté et al., 1989; Ahmed and Semmens, 1992a). These membranes are capable of achieving 100% gas transfer efficiency (Ahmed and Semmens, 1992a; Ahmed and Semmens, 1992b). In addition, the resulting dissolved H 2 concentration in the water can be controlled precisely by adjusting membrane surface area and gas pressure (Semmens and Gantzer, 1993). Thus, gas-permeable membranes appear to be a suit- able method for providing H 2 as an electron donor for in situ biological dehalogenation.