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.

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, Fe3+, 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.

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.

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.

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