Biochemical and EPR-spectroscopic investigation into heterologously expressed vinyl chloride reductive dehalogenase (VcrA) from Dehalococcoides mccartyi strain VS.

Reductive dehalogenases play a critical role in the microbial detoxification of aquifers contaminated with chloroethenes and chlorethanes by catalyzing the reductive elimination of a halogen. We report here the first heterologous production of vinyl chloride reductase VcrA from Dehalococcoides mccartyi strain VS. Heterologously expressed VcrA was reconstituted to its active form by addition of hydroxocobalamin/adenosylcobalamin, Fe(3+), and sulfide in the presence of mercaptoethanol. The kinetic properties of reconstituted VcrA catalyzing vinyl chloride reduction with Ti(III)-citrate as reductant and methyl viologen as mediator were similar to those obtained previously for VcrA as isolated from D. mccartyi strain VS. VcrA was also found to catalyze a novel reaction, the environmentally important dihaloelimination of 1,2-dichloroethane to ethene. Electron paramagnetic resonance (EPR) spectroscopic studies with reconstituted VcrA in the presence of mercaptoethanol revealed the presence of Cob(II)alamin. Addition of Ti(III)-citrate resulted in the appearance of a new signal characteristic of a reduced [4Fe-4S] cluster and the disappearance of the Cob(II)alamin signal. UV-vis absorption spectroscopy of Ti(III)citrate-treated samples revealed the formation of two new absorption maxima characteristic of Cob(I)alamin. No evidence for the presence of a [3Fe-4S] cluster was found. We postulate that during the reaction cycle of VcrA, a reduced [4Fe-4S] cluster reduces Co(II) to Co(I) of the enzyme-bound cobalamin. Vinyl chloride reduction to ethene would be initiated when Cob(I)alamin transfers an electron to the substrate, generating a vinyl radical as a potential reaction intermediate.

Sequential reductive and oxidative biodegradation of chloroethenes stimulated in a coupled bioelectro-process.

This article for the first time demonstrates successful application of electrochemical processes to stimulate sequential reductive/oxidative microbial degradation of perchloroethene (PCE) in mineral medium and in contaminated groundwater. In a flow-through column system, hydrogen generation at the cathode supported reductive dechlorination of PCE to cis-dichloroethene (cDCE), vinyl chloride (VC), and ethene (ETH). Electrolytically generated oxygen at the anode allowed subsequent oxidative degradation of the lower chlorinated metabolites. Aerobic cometabolic degradation of cDCE proved to be the bottleneck for complete metabolite elimination. Total removal of chloroethenes was demonstrated for a PCE load of approximately 1.5 µmol/d. In mineral medium, long-term operation with stainless steel electrodes was demonstrated for more than 300 days. In contaminated groundwater, corrosion of the stainless steel anode occurred, whereas DSA (dimensionally stable anodes) proved to be stable. Precipitation of calcareous deposits was observed at the cathode, resulting in a higher voltage demand and reduced dechlorination activity. With DSA and groundwater from a contaminated site, complete degradation of chloroethenes in groundwater was obtained for two months thus demonstrating the feasibility of the sequential bioelectro-approach for field application.

Hydrogenase-independent uptake and metabolism of electrons by the archaeon Methanococcus maripaludis.

Direct, shuttle-free uptake of extracellular, cathode-derived electrons has been postulated as a novel mechanism of electron metabolism in some prokaryotes that may also be involved in syntrophic electron transport between two microorganisms. Experimental proof for direct uptake of cathodic electrons has been mostly indirect and has been based on the absence of detectable concentrations of molecular hydrogen. However, hydrogen can be formed as a transient intermediate abiotically at low cathodic potentials (<-414 mV) under conditions of electromethanogenesis. Here we provide genetic evidence for hydrogen-independent uptake of extracellular electrons. Methane formation from cathodic electrons was observed in a wild-type strain of the methanogenic archaeon Methanococcus maripaludis as well as in a hydrogenase-deletion mutant lacking all catabolic hydrogenases, indicating the presence of a hydrogenase-independent mechanism of electron catabolism. In addition, we discovered a new route for hydrogen or formate production from cathodic electrons: Upon chemical inhibition of methanogenesis with 2-bromo-ethane sulfonate, hydrogen or formate accumulated in the bioelectrochemical cells instead of methane. These results have implications for our understanding on the diversity of microbial electron uptake and metabolism.

Identification of a reductive tetrachloroethene dehalogenase in Shewanella sediminis.

The genome sequence of psychrophilic Shewanella sediminis revealed the presence of five putative reductive dehalogenases (Rdhs). We found that cell extracts of pyruvate/fumarate-grown S. sediminis cells catalysed reduced methyl viologen-dependent reductive dechlorination of tetrachloroethene (PCE) to trichloroethene (TCE) at a specific activity of approximately 1 nmol TCE min(-1) (mg protein)(-1). Dechlorination of PCE followed Michaelis-Menten kinetics with an apparent Km of 120 µM PCE. No PCE dechlorination was observed with heat-denatured extract or when cyanocobalamin was omitted from the growth medium; however, the presence of PCE in the growth medium increased PCE transformation rates. Analysis of mutants carrying in-frame deletions of all five Rdhs encoding genes showed that only deletion of Ssed_3769 resulted in the loss of PCE dechlorination activity suggesting that Ssed_3769 is a functional Rdh. This is the first study to show reductive dechlorination activity of PCE in a sediment-dwelling Shewanella species that may be important for linking the flux of organohalogens to organic carbon via reductive dehalogenation in marine sediments.

Application of electrolysis to stimulate microbial reductive PCE dechlorination and oxidative VC biodegradation.

A novel approach was applied to stimulate biodegradation of chloroethenes bya coupled bioelectro-process. In a flow-through column system, microbial dechlorination of tetrachloroethene to cis-dichloroethene, vinyl chloride, and ethene was stimulated by hydrogen produced by water electrolysis. Dechlorinating bacteria (Dehalococcoides spp. and Desulfitobacterium spp.) and also methanogens and homoacetogens were detected in the anaerobic column. Simultaneously, oxidative biodegradation of lower chlorinated metabolites (vinyl chloride) was stimulated by electrolytic oxygen formation in the corresponding aerobic column. The process was stable for more than 100 days and an average removal of approximately 23 micromol/d PCE and 72 micromo/d vinyl chloride was obtained with a current density of 0.05 mA/cm2. Abiotic electrochemical degradation of the contaminants was not observed. Microbial dechlorination correlated with the current densities in the applied range of 0.01-0.05 mA/cm2. The results are promising for environmental applications, since with electrolysis hydrogen and oxygen can be supplied continuously to chloroethene degrading microorganisms, and the supply rates can be easily controlled by adjusting the electric current.

Electromigration of microbial electron acceptors and nutrients: (I) transport in synthetic media.

Microbiological cleanup is a widely used in situ remediation strategy for organic soil and groundwater contaminations. However, often the availability of electron acceptors and nutrients are limiting factors for microbial pollutant degradation in the field. Electromigration represents a new approach for the transport of microbiological agents in soil. In this study, the electrokinetic transport of the microbial electron acceptors nitrate and sulfate and of the nutrients ammonium and phosphate was compared. All experiments were performed under standardized conditions, i.e. with constant voltage in demineralized water and a model sandy soil. Average transport rates for nitrate, sulfate, poly-phosphate, and ammonium were 1.34 cm/h, 1.91 cm/h, 0.48 cm/h, and 0.40 cm/h, respectively, in single compound studies. Transport velocities were dependent on applied voltage gradient but not on the initial ion concentration. Additionally, electrokinetic transport was studied with ion mixtures. The ion distribution in the soil was significantly influenced by the pH profile and the associated voltage gradient.

Electromigration of microbial electron acceptors and nutrients: (II) transport in groundwater.

Bioremediation in contaminated aquifers is often limited by the availability of microbial electron acceptors and nutrients. In this study, electromigration of the electron acceptor nitrate was assessed in sandy model soil mixed with groundwater from a contaminated site, and compared with previous results obtained in soil mixed with demineralized water. The specific nitrate transport rate in soil with groundwater at a voltage gradient of 2 V/cm and 0.5 g/L nitrate was 1.64 cm/h, and proved to be higher than in demineralized water (1.13 cm/h). The application of electrokinetic transport is a promising approach to enhance mass transfer in groundwater systems.