Michael J. Dybas

Generation and initial characterization of Pseudomonas stutzeri KC mutants with impaired ability to degrade carbon tetrachloride

Abstract Under iron-limiting conditions, Pseudomonas stutzeri KC secretes a small but as yet unidentified factor that transforms carbon tetrachloride (CT) to CO2 and nonvolatile products when activated by reduction at cell membranes. Pseudomonas fluorescens and other cell types activate the factor. Triparental mating was used to generate kanamycin-resistant lux::Tn5 recombinants of strain KC. Recombinants were streaked onto the surface of agar medium plugs in microtiter plates and were then screened for carbon tetrachloride degradation by exposing the plates to gaseous 14C-carbon tetrachloride. CT+ recombinants generated nonvolatile 14C-labeled products, but four CT– recombinants did not generate significant nonvolatile 14C-labeled products and had lost the ability to degrade carbon tetrachloride. When colonies of P. fluorescens were grown next to colonies of CT+ recombinants and were exposed to gaseous 14C-carbon tetrachloride, 14C-labeled products accumulated around the P. fluorescens colonies, indicating that the factor secreted by CT+ colonies had diffused through the agar and become activated. When P. fluorescens was grown next to CT– colonies, little carbon tetrachloride transformation was observed, indicating a lack of active factor. Expression of lux reporter genes in three of the CT– mutants was regulated by added iron and was induced under the same iron-limiting conditions that induce carbon tetrachloride transformation in the wild-type.

Evaluation of Three Electron-Donor Permeable Reactive Barrier Materials for Enhanced Reductive Dechlorination of Trichloroethene

ABSTRACT Understanding the fate of complex electron-donor materials is important for developing efficient biostimulation strategies to treat ground water contamination by chlorinated ethenes (CEs). The fermentation product distributions and H2 production of common permeable reactive barrier (PRB) carbon substrates (dairy whey, sodium lactate syrup, and Hydrogen Release Compound [HRC]) were monitored as measures of substrate efficiency in aquifer microcosms spiked with trichloroethene (TCE). In long-term experiments, the fermentation of PRB substrates to slow-degrading organic acids maintained low H2 partial pressures (≤ 10−3.5) that, as previous studies suggest, may give competitive advantage to dechlorinators over hydrogenotrophic methanogens. Whey-amended and lactate-amended microcosms exhibited faster complete dechlorination and, according to organic acid carbon flow, higher rates of fermentation to acetate. In HRC-amended microcosms, propionate appeared to serve as a carbon sink that prolonged dechlorination. Upon complete dechlorination, whey microcosms contained the highest percentage of organic acid carbon. Native Dehalococcoides populations increased by 3 orders of magnitude (per g sediment) in whey-amended microcosms. Wheys efficiency improved in microcosms prepared with aquifer sediment and water from within a downgradient whey PRB. Results suggested whey loading values of 0.2 kg/m3 may be appropriate under sufficiently reducing conditions to efficiently stimulate hydrogenotrophic and potentially actetotrophic dechlorinating populations. Renewal of whey PRBs may, however, be required. Implications for further long-term study of cost-efficiencies are discussed.

Bioaugmentation with Pseudomonas Stutzeri KC for Carbon Tetrachloride Remediation

Bioaugmentation with denitrifying Pseudomonas stutzeri strain KC is a field-demonstrated strategy to treat groundwater contaminated with carbon tetrachloride (CT) to concentrations below current regulatory criteria. The key to this strategy is that strain KC secretes a compound (pyridine-2,6-bis-thiocarboxylate, or PDTC) that promotes chemical dechlorination of CT outside the cell. Strain KC is a highly motile (chemotactic toward nitrate) facultative aerobe capable of complete denitrification. Unlike other denitrifiers, however, strain KC degrades CT without producing chloroform (CF) and does so faster than competing microbial populations that produce CF, thus minimizing or avoiding CF formation. Because bioaugmentation with strain KC is accomplished with nitrate in a denitrifying environment and requires relatively low amounts of added organic carbon, it avoids side effects that result from biostimulation with large amounts of organic carbon, minimizing accumulation of volatile fatty acids, hydrogen sulfide (H2S), ferrous iron, carbon disulfide, ammonium, methane and biomass. This chapter summarizes the rationale behind this unique bioaugmentation strategy, and reviews the laboratory and field research that led to its development and validation.