Over the next 30 years, the EPA has estimated that 350,000 contaminated sites in the United States will require remediation at a projected cost of $250 billion. Halogenated pollutants are one of the most common sources of contamination at these sites. Microorganisms, called (de)halorespirers, have been identified that can reduce toxic chlorinated organics to less- or non-toxic compounds using chlorinated organics as electron acceptors for growth and energy. Although halorespirers have been shown to be marginally effective for bioremediation at some contaminated sites, the potential that these microorganisms hold remains unrealized. The PI recently discovered that Dehalococcoides-like microorganisms exist in uncontaminated soils at high numbers and that their concentration in the environment statistically correlates to the amount of natural chlorinated organic matter present. This discovery raises questions regarding the links between uncontaminated and contaminated environments. The research proposed herein will begin to address these links by examining the ability of organisms in uncontaminated soils to halorespire different chlorinated compounds, investigating the transcription of reductive dehalogenase (rdh) genes from uncontaminated soils during halorespiration, and determining how the rdh genes, gene transcripts, and bacterial communities change in uncontaminated soils upon exposure to anthropogenic chlorinated compounds. It is hypothesized that halorespirers from uncontaminated soils can respire both chlorinated xanthones and trichloroethylene and that the reductive dehalogenase enzymes used for the respiration of these two different compounds are homologous. This research will further our understanding of halorespiration, particularly in uncontaminated environments. This has critical implications for the remediation field, ecology, and geochemical cycling.
The proposed research will provide a direct educational benefit for one graduate student and one undergraduate student, developing their experimental and critical thinking skills and helping them develop as engineers/scientists. In addition, results obtained in this research will be incorporated into the lecture notes of several classes, including Introduction to Environmental Engineering, a required course for undergraduate Civil Engineers at the University of Minnesota, and Environmental Microbiology, an upper-level undergraduate and lower-level graduate laboratory course. In particular, results from this project will be incorporated into lecture material on bioremediation and elemental cycling. Finally, if successful, this research could result in more rapid and successful remediation through the discovery of natural agents for use in bioaugmentation and biostimulation at contaminated sites.
Bacteria exist that are able to use halogenated compounds, including many chlorinated organic pollutants, for energy generation in essentially the same way that humans use oxygen. They are called organohalide respirers. Because these bacteria are able to dechlorinate and detoxify chlorinated organic pollutants through this respiration process, they have been of interest to scientists and engineers. Indeed, these bacteria hold great promise for the bioremediation of sites contaminated with toxic chlorinated organic pollutants, such as polychlorinated biphenyls (PCBs), dioxins, and chlorinated ethenes, compounds that have been listed by the EPA as "Priority Pollutants." Unfortunately, this bioremediation process can often be slow or in some cases does not occur. If more is known about these organisms, it is likely that engineers and scientists will be able to better-support and stimulate their growth and they can be utilized more effectively for contaminant dechlorination and detoxification. Interestingly, recent research had shown that it was possible that these organohalide respirers might also play a role in uncontaminated environments where they were able to grow on chlorinated natural organic matter (NOM). At the time that this research began, little to nothing was known about organohalide respirers that lived in uncontaminated environments and whether (1) those organisms might be exploited for the dechlorination of chlorinated organic pollutants, or (2) chlorinated NOM might be able to stimulate the dechlorination of chlorinated organic pollutants. The broad goal of this NSF-supported research was to understand the niche and activity of organohalide respiring bacteria in uncontaminated environments such that new insights might be gained into the physiology of these important organisms. The activities sponsored by this grant, described below, have indeed been successful toward reaching this overarching goal, resulting in two published manuscripts and a third manuscript to be submitted shortly. This research showed for the first time that Dehalococcoides-like bacteria have a niche in uncontaminated environments respiring chlorinated NOM. In brief, this research showed that not only were Dehalococcoides-like bacteria detected in multiple uncontaminated soils, but their numbers were found to correlate with the chlorinated NOM content of the soil as well. To further characterize the link between these organohalide respirers and chlorinated NOM, additional experiments were performed. In these experiments NOM was chlorinated enzymatically (as occurs in the environment) and this chlorinated NOM was added to bottles containing uncontaminated forest soil. The number of Dehalococcoides-like bacteria grew in these bottles, with a simultaneous increase in free chloride. A natural sulfur gradient that occurs geographically in lake waters across the Upper Midwest was used to determine if biogeochemical factors other than organochloride content affected the abundance and diversity of organohalide respiring bacteria. In this study, we found that both the number of Dehalococcoides-like Chloroflexi and their diversity were negatively impacted in lake sediments with higher concentrations of sulfur. This finding is significant in that it quantified and characterized organohalide respiring populations in uncontaminated sediment and it highlighted a potential naturally occurring inhibitor of organohalide respiring populations in uncontaminated sediments. Additionally, it highlighted an important biogeochemical link between high sulfur content and bioremediation and chlorine and carbon cycling. Finally, microcosms were operated to determine if a specific class of chlorinated NOM, chlorinated xanthones, could support the growth of organohalide respiring bacteria. Chlorinated xanthones have a similar chemical structure to several organic pollutants: dioxins and PCBs. It is therefore possible that chlorinated xanthones could stimulate contaminant degraders. Two chloroxanthones were studied. Five soils and sediments were tested for dechlorination; four successfully dechlorinated both chlorinated xanthones. A novel group of bacteria (referred to as the "Gopher group") were identified in which their growth correlated to the dechlorination of 2,7-dichloroxanthone. This research is significant in that it shows that (1) a novel, and believed-to-be-common group of natural organochlorines can serve as substrates for dechlorination, and that (2) a unique group of unisolated Firmicutes are putative 2,7-dichloroxanthone dechlorinators. The second finding is particularly important because similar organisms have also been found to dechlorinate PCBs, chlorinated ethenes, and other chlorinated organic pollutants. This could lead to the development of novel bioremediation applications. One student supported on this grant earned his Ph.D. This student also continued the broader objectives of the project for 1 year of post-doctoral study and gained additional professional experience during this time. He recently accepted an offer as a tenure-track Assistant Professor in Civil and Environmental Engineering, demonstrating the benefits gained from working on this project. This project has also provided valuable research experience for an undergraduate student who is now a Ph.D. student at another university. Project results were incorporated into three courses: Introduction to Environmental Engineering, Environmental Microbiology, and Environmental Microbiology Laboratory. Results from this research were used to describe the diversity of bacteria, communicate principles of evolution, demonstrate the value of combined culture-based and culture-independent techniques in environmental microbiology, and advance understanding of bioremediation techniques and principles.
