Cavity-Enhanced Raman Spectroscopy in the Biosciences: In Situ, Multicomponent, and Isotope Selective Gas Measurements To Study Hydrogen Production and Consumption by Escherichia coli

High Resolution Image Download MS PowerPoint Slide 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 H 2 and simultaneously consume some of it. By introducing D 2 in 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 H 2 and CO 2 production and D 2 consumption were identified. Some of the D 2 consumed is converted back to H 2 via 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 D 2 oxidation and proton reduction occur are physically separated. Whereas in glucose supplemented cultures, addition of D 2 led to an increase in H 2 produced, while the yield of CO 2 remained unchanged; with glycerol, addition of D 2 led not only to increased yields of H 2, but also significantly increased CO 2 production, reflecting an impact on fermentation pathways. Addition of CO was found to completely inhibit H 2 production and significantly reduce D 2 oxidation, indicating at least some role for O 2 -tolerant Hyd-1 in D 2 consumption.