Cavity-Enhanced Raman and Helmholtz Resonator Photoacoustic Spectroscopy to Monitor the Mixed Sugar Metabolism of E. coli

Abstract

We introduce and compare two powerful new techniques for headspace gas analysis above bacterial batch cultures by spectroscopy, Raman spectroscopy enhanced in an optical cavity (CERS), and photoacoustic detection in a differential Helmholtz resonator (DHR). Both techniques are able to monitor O2 and CO2 and its isotopomers with excellent sensitivity and time resolution to characterize bacterial growth and metabolism. We discuss and show some of the shortcomings of more conventional optical density (OD) measurements if used on their own without more sophisticated complementary measurements. The spectroscopic measurements can clearly and unambiguously distinguish the main phases of bacterial growth in the two media studied, LB and M9. We demonstrate how 13C isotopic labeling of sugars combined with spectroscopic detection allows the study of bacterial mixed sugar metabolism to establish whether sugars are sequentially or simultaneously metabolized. For E. coli, we have characterized the shift from glucose to lactose metabolism without a classic diauxic lag phase. DHR and CERS are shown to be cost-effective and highly selective analytical tools in the biosciences and in biotechnology, complementing and superseding existing conventional techniques. They also provide new capabilities for mechanistic investigations and show a great deal of promise for use in stable isotope bioassays.

On-line analysis and in situ pH monitoring of mixed acid fermentation by Escherichia coli using combined FTIR and Raman techniques

“…We would also like to take advantage of the spectral distinction of isotopomers to perform isotope labelling studies to elucidate reaction mechanisms. In future work, we want to include our recently introduced technique of cavity-enhanced Raman spectroscopy [11,20,33,34] to monitor gas-phase species by Raman spectroscopy, including H 2 . In conclusion, spectroscopic monitoring of bioprocesses has great potential to supplement or replace traditional sampling techniques.…”

“…Acetaldehyde and ethanol partial pressures were determined by FTIR spectroscopy using the Beer-Lambert law and reference spectra; partial pressures can then be converted into concentration in the solution using Henry's law (see also refs. [11,19,20]).…”

“…This allows an estimation of molar concentrations of CO 2 , ethanol, and acetaldehyde in the bacterial suspension from the FTIR measurements in the headspace (see also refs. [11,19,20]). Henry's law constants for aqueous solutions at 37°C are calculated as 0.026 mM mbar −1 for CO 2 , 82 mM mbar −1 for ethanol, and 6.3 mM mbar −1 for acetaldehyde [47].…”

“…OD is a conventional and widely employed technique to distinguish bacterial growth phases and estimate bacterial density (but also compare ref. [20]). In the experiment, the OD started quite low (ca.…”

“…Using Henry's law, the partial pressures of volatiles and gaseous products determined in the headspace can be converted to their dissolved concentrations (see also refs. [11,19,20]).…”