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 measure-ments 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.

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