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

Abstract

We introduce an experimental setup allowing continuous monitoring of bacterial fermentation processes by simultaneous optical density (OD) measurements, long-path FTIR headspace monitoring of CO2, acetaldehyde and ethanol, and liquid Raman spectroscopy of acetate, formate, and phosphate anions, without sampling. We discuss which spectral features are best suited for detection, and how to obtain partial pressures and concentrations by integrations and least squares fitting of spectral features. Noise equivalent detection limits are about 2.6 mM for acetate and 3.6 mM for formate at 5 min integration time, improving to 0.75 mM for acetate and 1.0 mM for formate at 1 h integration. The analytical range extends to at least 1 M with a standard deviation of percentage error of about 8%. The measurement of the anions of the phosphate buffer allows the spectroscopic, in situ determination of the pH of the bacterial suspension via a modified Henderson-Hasselbalch equation in the 6–8 pH range with an accuracy better than 0.1. The 4 m White cell FTIR measurements provide noise equivalent detection limits of 0.21 μbar for acetaldehyde and 0.26 μbar for ethanol in the gas phase, corresponding to 3.2 μM acetaldehyde and 22 μM ethanol in solution, using Henry’s law. The analytical dynamic range exceeds 1 mbar ethanol corresponding to 85 mM in solution. As an application example, the mixed acid fermentation of Escherichia coli is studied. The production of CO2, ethanol, acetaldehyde, acids such as formate and acetate, and the changes in pH are discussed in the context of the mixed acid fermentation pathways. Formate decomposition into CO2 and H2 is found to be governed by a zeroth-order kinetic rate law, showing that adding exogenous formate to a bioreactor with E. coli is expected to have no beneficial effect on the rate of formate decomposition and biohydrogen production.

Advanced spectroscopic analysis and 15N-isotopic labelling study of nitrate and nitrite reduction to ammonia and nitrous oxide by E. coli

“…21 We report a combined approach for characterizing DNRA and N 2 O production in anaerobic E. coli batch cultures using mostly non-invasive spectroscopic techniques. Sampling of the bacterial culture was only done for NO 2colorimetry and FTIR detection of 14 23 The White cell pathlength was adjustable between 4-8 m, with 6 m used for this work. Spectra were recorded every 5 minutes.…”

“…A multiplier equivalent to ethanol partial pressure was obtained by a least-squares fit of 1 ppmv ethanol and water reference spectra in the 2800-3100 cm -1 region. 23 Using Henry's law, all partial pressures could be converted into concentrations in solution.…”

“…15,16 Good sensitivities are observed in the condensed phase, but measuring headspace gases often suffers from low sensitivity, and special enhancement techniques are required such as Cavity Enhanced Raman Spectroscopy (CERS) [15][16][17][18][19][20][21][22] or long-path absorption White cells in FTIR spectroscopy. 23 Partial pressures in the headspace can be converted into concentrations in the solution via Henry's law. Quantum Cascade Laser (QCL) absorption spectroscopy has been applied to detect N 2 O and other trace gases; [24][25][26] while sensitive, the limited tuning range of QCLs over a single IR absorption band limits the dynamic range due to band saturation effects.…”

“…Fluorescence can be avoided by moving to longer excitation wavelengths or by using media free of fluorescent components, such as M9 minimal media. 23 Vibrational spectroscopic tools…”

Non-invasive Raman spectroscopy for time-resolved in-line lipidomics

“…Raman spectroscopy is a valuable tool in a process analytical chemist's toolbox. 15 It has been proven benecial for in-line monitoring of various organisms such as bacteria, 16,17 fungi, 18 yeast, [19][20][21][22] or mammalian cells. 23 Raman spectroscopy is a noninvasive, optical method that mirrors the chemical composition of the probed focal volume.…”