Kreyenschulte Dirk, Paciok Eva, Regestein Lars, Blümich Bernhard, Büchs Jochen
AVT - Biochemical Engineering, RWTH Aachen University, Worringer Weg 1, 52074, Aachen, Germany.
Institute for Technical Chemistry and Macromolecular Chemistry, RWTH Aachen University, Aachen, Germany.
Biotechnol Bioeng. 2015 Sep;112(9):1810-21. doi: 10.1002/bit.25599. Epub 2015 May 12.
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.
对于学术界和工业界生物技术工艺的发展而言，需要有能够实现过程表征和控制在线监测的新技术。核磁共振（NMR）光谱法是一种很有前景的分析工具，已在离线过程分析中得到广泛应用。然而，在线监测的应用常常受到定制NMR生物反应器高度复杂性以及高场NMR仪器高昂成本（超过20万美元）的限制。因此，本研究在旁路系统中对低场¹H NMR进行了研究，用于实时观察发酵过程。该新技术在两种微生物系统中得到了验证。对于多形汉逊酵母，尽管培养基中存在大量复杂成分，甘油消耗仍能得到准确评估。在玉米黑粉菌的培养过程中，伴随着多种副产物的形成，可以对葡萄糖、衣康酸以及糖脂的相对含量进行定量分析。虽然与高场NMR相比，低场光谱的特点是光谱分辨率降低，但紧凑的设计与高时间分辨率（15秒至8分钟）的光谱采集相结合，使得能够对各个过程进行在线监测。这两个应用都清楚地表明，所研究的技术非常适合于不透明介质中的反应监测，同时它具有高度的稳健性和化学特异性。因此可以得出结论，低场NMR光谱法在研究和实际工业规模上对生物技术过程进行非侵入性在线监测具有巨大潜力。
