title: Cavity-Enhanced Raman Spectroscopy in the Biosciences: In Situ, Multicomponent, and Isotope Selective Gas Measurements To Study Hydrogen Production and Consumption by Escherichia coli.
abstract: Recently we introduced cavity-enhanced Raman spectroscopy (CERS) with optical feedback cw-diode lasers as a sensitive analytical tool. Here we report improvements made on the technique and its first application in the biosciences for in situ, multicomponent, and isotope selective gas measurements to study hydrogen production and consumption by Escherichia coli. Under anaerobic conditions, cultures grown on rich media supplemented with d-glucose or glycerol produce H2and simultaneously consume some of it. By introducing D2in the headspace, hydrogen production and consumption could be separated due to the distinct spectroscopic signatures of isotopomers. Different phases with distinctly different kinetic regimes of H2and CO2production and D2consumption were identified. Some of the D2consumed is converted back to H2via H/D exchange with the solvent. HD was formed only as a minor component. This reflects either that H/D exchange at hydrogenase active sites is rapid compared to the rate of recombination, rapid recapture of HD occurs after the molecule is formed, or that the active sites where D2oxidation and proton reduction occur are physically separated. Whereas in glucose supplemented cultures, addition of D2led to an increase in H2produced, while the yield of CO2remained unchanged; with glycerol, addition of D2led not only to increased yields of H2, but also significantly increased CO2production, reflecting an impact on fermentation pathways. Addition of CO was found to completely inhibit H2production and significantly reduce D2oxidation, indicating at least some role for O2-tolerant Hyd-1 in D2consumption.
authors.affiliation: Department of Chemistry, University of Sheffield , Sheffield S3 7HF, United Kingdom.
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Smith
authors.affiliation: Department of Chemistry, University of Sheffield , Sheffield S3 7HF, United Kingdom.
authors.forename: Michael
authors.identifier: 0000-0002-3956-3922
authors.initials: M
authors.lastname: Hippler
mesh_terms: D000693:Anaerobiosis; D003470:Culture Media; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D005285:Fermentation; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000378:metabolism; D007554:Isotopes / Q000378:metabolism*; D007700:Kinetics; D054023:Lasers, Semiconductor; D057230:Limit of Detection; D013059:Spectrum Analysis, Raman / Q000295:instrumentation* / Q000379:methods*
chemical_list: D003470:Culture Media; D029968:Escherichia coli Proteins; D007554:Isotopes; D006859:Hydrogen; D006864:Hydrogenase; D005947:Glucose; D005990:Glycerol