On-line analysis and in situ pH monitoring of mixed acid fermentation by Escherichia coli using combined FTIR and Raman
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RESEARCH PAPER
On-line analysis and in situ pH monitoring of mixed acid fermentation by Escherichia coli using combined FTIR and Raman techniques George D. Metcalfe 1 & Thomas W. Smith 1,2 & Michael Hippler 1 Received: 27 May 2020 / Revised: 23 July 2020 / Accepted: 5 August 2020 # The Author(s) 2020
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. Keywords Bioanalytical methods . Biological samples . IR spectroscopy/Raman spectroscopy . pH measurements . Biosensors
Introduction Microbial fermentation is a fascinating and important process with broad relevance to the fields of biotechnology, environmental science, and medicine. In the absence of terminal electron Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02865-5) contains supplementary material, which is available to authorized users. * Michael Hippler [email protected] 1
Department of Chemistry, University of Sheffield, Sheffield S3 7HF, UK
2
Water and Environmental Engineering Group, Faculty of Engineering and Physical Sciences, University of Southampton, Southampton SO17 1BJ, UK
acceptors, such as oxygen, various forms of fermentation are carried out by many facultative
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