Online monitoring of fermentation processes via non‐invasive low‐field NMR

Abstract

For the development of biotechnological processes in academia as well as in industry new techniques are required which enable online monitoring for process characterization and control. Nuclear magnetic resonance (NMR) spectroscopy is a promising analytical tool, which has already found broad applications in offline process analysis. The use of online monitoring, however, is oftentimes constrained by high complexity of custom-made NMR bioreactors and considerable costs for high-field NMR instruments (>US$200,000). Therefore, low-field (1) H NMR was investigated in this study in a bypass system for real-time observation of fermentation processes. The new technique was validated with two microbial systems. For the yeast Hansenula polymorpha glycerol consumption could accurately be assessed in spite of the presence of high amounts of complex constituents in the medium. During cultivation of the fungal strain Ustilago maydis, which is accompanied by the formation of several by-products, the concentrations of glucose, itaconic acid, and the relative amount of glycolipids could be quantified. While low-field spectra are characterized by reduced spectral resolution compared to high-field NMR, the compact design combined with the high temporal resolution (15 s-8 min) of spectra acquisition allowed online monitoring of the respective processes. Both applications clearly demonstrate that the investigated technique is well suited for reaction monitoring in opaque media while at the same time it is highly robust and chemically specific. It can thus be concluded that low-field NMR spectroscopy has a great potential for non-invasive online monitoring of biotechnological processes at the research and practical industrial scales.

Benchtop NMR for Online Reaction Monitoring of the Biocatalytic Synthesis of Aromatic Amino Alcohols

“…For correct quantification, the time between two pulse experiments (spectrum scans) should be at least 5‐ to 7‐times the value of the relaxation time to ensure that all spins (99.9 % in the case of 7×T 1 ) have returned to the initial state before starting the next pulse sequence. If the time between the scans is less than 5‐ to 7‐times T 1 , the signal intensity will successively decrease and is then no longer directly correlated with the concentration of the component.…”

“…In addition to the above‐mentioned advantages, NMR in general enables reaction components to be determined which can be extremely challenging to quantify with other analytical techniques, but which frequently serve as reaction components in biocatalytic synthesis, e. g. isopropylamine or acetaldehyde . The distinct advantages mentioned above, are expected to lead to an increasing relevance of NMR for applications in biotechnological systems as already indicated by the first few publications in this field …”

Desktop NMR and Its Applications From Materials Science To Organic Chemistry

“…External locks are known from high‐field NMR spectroscopy. They are standard in some compact NMR spectrometers and enable monitoring complex biochemical reactions over times longer than a day …”

NMR mit Tischgeräten und deren Anwendungen von der Materialwissenschaft bis zur organischen Chemie

“…Externe Locks sind aus der Hochfeld‐NMR‐Spektroskopie bekannt. Sie sind bei einigen kompakten NMR‐Spektrometern Standard und ermöglichen die Überwachung komplexer biochemischer Reaktionen über längere Zeiträume von mehr als einem Tag …”