Triple-element compound-specific stable isotope analysis of 1,2-dichloroethane for characterization of the underlying dehalogenation reaction in two Dehalococcoides mccartyi strains.

Chlorinated ethanes belong to the most common groundwater and soil contaminants. Of these, 1,2-dichloroethane (1,2-DCA) is a man-made, persistent and toxic contaminant, released due to improper waste treatment at versatile production sites. This study investigated the anaerobic transformation of 1,2-DCA by Dehalococcoides mccartyi strain 195 and strain BTF08 using triple-element compound-specific stable isotope analysis of carbon, chlorine and hydrogen for the first time. Isotope fractionation patterns for carbon (εCBTF08 = -28.4 ± 3.7‰; εC195 = -30.9 ± 3.6‰) and chlorine (εClBTF08 = -4.6 ± 0.7‰; εCl195 = -4.2 ± 0.5‰) within both investigated D. mccartyi strains, as well as the dual-element analysis (ΛBTF08 = 6.9 ± 1.2; Λ195 = 7.1 ± 0.2), supported identical reaction mechanisms for dehalogenation of 1,2-DCA. Hydrogen isotope fractionation analysis revealed dihaloelimination as prevalent reaction mechanism. Vinyl chloride as major intermediate could be excluded by performing the experiment in deuterated aqueous media. Furthermore, evaluation of the derived apparent kinetic isotope effects (AKIECBTF08 = 1.029/AKIEC195 = 1.031; AKIEClBTF08 = 1.005/AKIECl195 = 1.004) pointed towards simultaneous abstraction of both involved chlorine-substituents in a concerted matter. It was shown that D. mccartyi strain BTF08 and strain 195 are capable of complete, direct dihaloelimination of 1,2-DCA to ethene

Temperature dependence of the single‐crystal elastic constants of Co‐rich Co–Fe alloys

The single-crystal elastic moduli, C11, C12, and C44 of three fcc cobalt-iron alloys (Co-6 at. % Fe, Co-8 at.% Fe, Co-10 at.% Fe) were measured in the range 0-315 °c. In addition C11 for the Co-6 at. % Fe alloy, and C' = (1/2)( C11 + C12 + 2 C44) for the three alloys are measured over the temperature range 0-1250 °c. Plots of the elastic moduli vs temperature exhibit a change in slope and deviation from linearity in the neighborhood of the Curie temperature. The temperature variation of the shear anisotropy in the fcc phase A fcc (=2 C44/ C11- C12) differs among the three alloys. A fcc exhibits a highly positive temperature dependence in the Co-10 at. % Fe alloy and a slight negative dependence in the Co-6 at. % Fe and Co-8 at. % Fe alloys. Previous statements in the literature that the hcp → ← fcc transformation in cobalt is preceded by a highly negative temperature dependence of the shear anisotropy ratio A (= C 44/ C 66) in the hcp phase between 523 °K and the transition at about 743 °K is not borne out by the present results. Rather it appears that the hcp → ← fcc transformation involves a change from A >1 in the hcp phase to A < 1 in the fcc phase

Mechanistic dichotomy in bacterial trichloroethene dechlorination revealed by carbon and chlorine isotope effects.

Tetrachloroethene (PCE) and trichloroethene (TCE) are significant groundwater contaminants. Microbial reductive dehalogenation at contaminated sites can produce nontoxic ethene but often stops at toxic cis-1,2-dichloroethene (cis-DCE) or vinyl chloride (VC). The magnitude of carbon relative to chlorine isotope effects (as expressed by Lambda(C/Cl), the slope of delta C-13 versus delta Cl-37 regressions) was recently recognized to reveal different reduction mechanisms with vitamin B-12 as a model reactant for reductive dehalogenase activity. Large Lambda(C/Cl) values for cis-DCE reflected cob(I)alamin addition followed by protonation, whereas smaller Lambda(C/Cl) values for PCE evidenced cob(I)alamin addition followed by Cl- elimination. This study addressed dehalogenation in actual microorganisms and observed identical large Lambda(C/Cl) values for cis-DCE (Lambda(C/Cl) = 10.0 to 17.8) that contrasted with identical smaller Lambda(C/Cl) for TCE and PCE (Lambda(C/Cl) = 2.3 to 3.8). For TCE, the trend of small Lambda(C/Cl) could even be reversed when mixed cultures were precultivated on VC or DCEs and subsequently confronted with TCE (Lambda(C/Cl) = 9.0 to 18.2). This observation provides explicit evidence that substrate adaptation must have selected for reductive dehalogenases with different mechanistic motifs. The patterns of Lambda(C/Cl) are consistent with practically all studies published to date, while the difference in reaction mechanisms offers a potential answer to the long-standing question of why bioremediation frequently stalls at cis-DCE

Towards improved accuracy in chlorine isotope analysis: Synthesis routes for in-house standards and characterization via complementary mass spectrometry methods.

Increasing applications of compound-specific chlorine isotope analysis (CSIA) emphasize the need for chlorine isotope standards that bracket a wider range of isotope values in order to ensure accurate results. With one exception (USGS38), however, all international chlorine isotope reference materials (chloride and perchlorate salts) fall within the narrow range of one per mille. Furthermore, compound-specific working standards are required for chlorine CSIA but are not available for most organic substances. We took advantage of isotope effects in chemical dehalogenation reactions to generate (i) silver chloride (CT16) depleted in 37Cl/35Cl and (ii) compound-specific standards of the herbicides acetochlor and S-metolachlor (Aceto2, Metola2) enriched in 37Cl/35Cl. Calibration against the international reference standards USGS38 (-87.90 ‰) and ISL-354 (+0.05 ‰) by complementary methods (gas chromatography-isotope ratio mass spectrometry, GC-IRMS, versus gas chromatography-multicollector inductively coupled plasma mass spectrometry, GC-MC-ICPMS) gave a consensus value of δ37ClCT16 = -26.82 ± 0.18 ‰. Preliminary GC-MC-ICPMS characterization of commercial Aceto1 and Metola1 versus Aceto2 and Metola2 resulted in tentative values of δ37ClAceto1 = 0.29 ± 0.29 ‰, δ37ClAceto2 = 18.54 ± 0.20 ‰, δ37ClMetola1 = -4.28 ± 0.17 ‰ and δ37ClMetola2 = 5.12 ± 0.27 ‰. The possibility to generate chlorine isotope in-house standards with pronounced shifts in isotope values offers a much-needed basis for accurate chlorine CSIA

New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography

Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens