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

George D Metcalfe,Michael Hippler,Thomas W Smith

doi:10.1007/s00216-020-02865-5

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.

Highlights

Microbial fermentation is a fascinating and important process with broad relevance to the fields of biotechnology, environmental science, and medicine
We demonstrate that Raman monitoring of the two major phosphate species present in the growth medium allows the spectroscopic determination of the pH of the solution, an important parameter in fermentation processes, and we derive the theoretical basis of this analysis
Characteristic features are peak A of HPO42− at 989 cm−1 and peak B of H2PO4− at 1076 cm−1; outside the range displayed, H2PO4− has an additional peak of similar intensity at 876 cm−1 [36]

Summary

Introduction

Microbial fermentation is a fascinating and important process with broad relevance to the fields of biotechnology, environmental science, and medicine. We describe and characterise the performance of a combined non-invasive FTIR and Raman approach in the gas phase and solution, respectively, for monitoring and controlling microbial fermentation, with the mixed acid fermentation of E. coli serving as a relevant example.

Results

Conclusion