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Manufacture Analytical Science, Bioprocessing - Upstream & Downstream, Bioprocessing - Upstream & Downstream

From UV to MIR: the biomass monitoring spectrum

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Infrared spectroscopy includes spectral areas of near infrared (NIR, 740 nm to 1300 nm) and mid-infrared (MIR, up to 15000 nm). In general, IR light excites different vibrational modes of molecules. Each organic and inorganic compound has a special spectral IR signature from these vibrations. IR spectroscopy offers very fast, robust and sensitive multi-analyte information from the culture broth of bioprocesses. It is a non-invasive process analytical technology, applied in-line by direct beam or optical fiber.

MIR radiation excites fundamental rotational vibrations of functional groups from organic compounds. Molecules such as glucose, lactate, fructose, acetic acid, ammonia, and even antibodies (17) have a characteristic absorption spectrum which can be used to identify single components in bioprocesses quantitative, sensitive, and specific.

A high degree of water absorption appears in MIR spectra. However, in-line measurement in aqueous solutions is possible using appropriate fiber optic probes that incorporate attenuated total reflection (ATR) technology and Fourier transformation (18–20). The measurement principle of ATR probes results in a very short (only few µm) path length and cells cannot be detected because they are too large to enter the measuring zone.

NIR spectroscopy is also based on different vibrational modes, overtone and combination vibrations after excitation. Important targets are the O-H, C-H and N-H bonds. The NIR range is thus suitable for monitoring of substrates such as glucose and lactate, biomass, and the products of a bioprocess (17, 21). As a result of the lower energy of the NIR and the resulting overtone vibrations, the bands are much broader, often overlapping, and not as specific as in MIR spectroscopy (20). Thus, NIR spectroscopy has a more qualitative character, compared to the more precise and quantitative MIR spectroscopy. NIR spectroscopy offers a more global view to a bioprocess, e.g. by batch trajectory (22).

Due to its higher robustness, NIR spectroscopy is better applied for monitoring industrial production processes. MIR spectroscopy is well suited for process development and optimization due to its multiplexing technology and the fact that fragile ATR fibers are used.

UV/Vis spectroscopy uses ultraviolet and visible light (10–740 nm) to excite electrons of molecules, the observable transitions taking place at unsaturated bonds, such as in aromatics (11). A variety of analytes, substrates, metabolites, and products can be determined with UV/Vis spectroscopy, which has high sensitivity, and high resolution spectrophotometers can be compact, inexpensive, and robust, making these instruments interesting for industrial process applications (21). However, UV/Vis spectroscopy does not currently play a major role in bioprocess monitoring (23) despite the use of CCDs or photodiode arrays making UV/Vis spectroscopy even more attractive.

Using fluorescence spectroscopy,  many important molecules for bioprocesses can be monitored and controlled, including proteins with aromatic amino acids (tryptophan), NAD(P)H (biomass), ATP, pyruvate, vitamins, pyridoxines, coenzymes, and flavins (12, 21, 24–27). Each fluorescence-active compound has a specific pair of excitation and emission wavelengths. Simultaneous measurement of several different fluorophores in the culture broth is possible by 2-D fluorescence spectroscopy (13, 24, 28–31).

Raman spectroscopy is another form of vibrational spectroscopy. It is based on shifted wavelength scattering of molecules, after excitation by monochromatic light, usually produced by adjustable lasers (32). Several analytes, including glucose, lactate, acetate, formate, glutamine, and glutamate, can be measured (1, 15, 21, 33–37). The use of Raman spectroscopy is limited by the strong fluorescence activity of several biological molecules in the culture broth (34). The fluorescence signals overlay the Raman bands. To avoid fluorescence, low energy lasers can be used, but then heating effects can occur (21, 24, 38).

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About the Author
Dörte Solle, Philipp Biechele, Christoph Busse and Thomas Scheper

Dörte Solle, Philipp Biechele, Christoph, Busse, and Thomas Scheper are all based at the Institute of Technical Chemistry, Leibniz University, Hannover, Germany.

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