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Ultrasound-enhanced attenuated total reflection mid-infrared spectroscopy in-line probe: acquisition of cell spectra in a bioreactor.

Koch C, Brandstetter M, Wechselberger P, Lorantfy B, Plata MR, Radel S, Herwig C, Lendl B - Anal. Chem. (2015)

Bottom Line: Accumulation of storage carbohydrates (trehalose and glycogen) inside the cells was induced by a lack of a nitrogen source in the feed medium.Comparison of the cell spectra with spectra of trehalose, glycogen, glucose, and mannan, i.e., the major carbohydrates present in S. cerevisiae, and principal components analysis revealed that the changes observed in the cell spectra correlated well with the bands specific for trehalose and glycogen.This proves the applicability and capability of ultrasound-enhanced in-line ATR mid-IR spectroscopy as a real-time PAT method for the in situ monitoring of cellular biochemistry during fermentation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology , Getreidemarkt 9/164-UPA, 1060 Vienna, Austria.

ABSTRACT
This article presents a novel method for selective acquisition of Fourier transform infrared (FT-IR) spectra of microorganisms in-line during fermentation, using Saccharomyces cerevisiae as an example. The position of the cells relative to the sensitive region of the attenuated total reflection (ATR) FT-IR probe was controlled by combing a commercially available ATR in-line probe with contact-free, gentle particle manipulation by ultrasonic standing waves. A prototype probe was successfully constructed, assembled, and tested in-line during fed-batch fermentations of S. cerevisiae. Control over the position of the cells was achieved by tuning the ultrasound frequency: 2.41 MHz was used for acquisition of spectra of the cells (pushing frequency f(p)) and 1.87 MHz, for retracting the cells from the ATR element, therefore allowing spectra of the medium to be acquired. Accumulation of storage carbohydrates (trehalose and glycogen) inside the cells was induced by a lack of a nitrogen source in the feed medium. These changes in biochemical composition were visible in the spectra of the cells recorded in-line during the application of f(p) and could be verified by reference spectra of dried cell samples recorded off-line with a FT-IR microscope. Comparison of the cell spectra with spectra of trehalose, glycogen, glucose, and mannan, i.e., the major carbohydrates present in S. cerevisiae, and principal components analysis revealed that the changes observed in the cell spectra correlated well with the bands specific for trehalose and glycogen. This proves the applicability and capability of ultrasound-enhanced in-line ATR mid-IR spectroscopy as a real-time PAT method for the in situ monitoring of cellular biochemistry during fermentation.

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Second-derivative spectra of trehalose,glycogen, glucose, andmannan and PC1 of spectra acquired during N-limited growth.
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fig6: Second-derivative spectra of trehalose,glycogen, glucose, andmannan and PC1 of spectra acquired during N-limited growth.

Mentions: Theloading vector of PC1 and the second-derivative spectra oftrehalose, glycogen, glucose, and mannan show similar features (Figure 6). Spectral region A (1020–970 cm–1) of PC1 correlates with trehalose: the minimum is around 995 cm–1 for both, followed by a maximum at 980 cm–1 and a shoulder around 960 cm–1. This region alsovaries the most between spectra, reflected by the highest values ofthe first loading vector. The plateau at 1035 cm–1 followed by a minimum around 1025 cm–1 in PC1correlates with glycogen, superimposed with trehalose and glucose(thus, the shift of the minimum toward higher wavenumbers). In spectralregion B (1050 to 1103 cm–1), trehalose, glycogen,glucose, and PC1 show very similar features, and a clear attributioncannot be made. Around 1155 cm–1 (spectral regionC), the minimum in PC1 corresponds to a superposition of the minimaof both trehalose and glycogen.


Ultrasound-enhanced attenuated total reflection mid-infrared spectroscopy in-line probe: acquisition of cell spectra in a bioreactor.

Koch C, Brandstetter M, Wechselberger P, Lorantfy B, Plata MR, Radel S, Herwig C, Lendl B - Anal. Chem. (2015)

Second-derivative spectra of trehalose,glycogen, glucose, andmannan and PC1 of spectra acquired during N-limited growth.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4333607&req=5

fig6: Second-derivative spectra of trehalose,glycogen, glucose, andmannan and PC1 of spectra acquired during N-limited growth.
Mentions: Theloading vector of PC1 and the second-derivative spectra oftrehalose, glycogen, glucose, and mannan show similar features (Figure 6). Spectral region A (1020–970 cm–1) of PC1 correlates with trehalose: the minimum is around 995 cm–1 for both, followed by a maximum at 980 cm–1 and a shoulder around 960 cm–1. This region alsovaries the most between spectra, reflected by the highest values ofthe first loading vector. The plateau at 1035 cm–1 followed by a minimum around 1025 cm–1 in PC1correlates with glycogen, superimposed with trehalose and glucose(thus, the shift of the minimum toward higher wavenumbers). In spectralregion B (1050 to 1103 cm–1), trehalose, glycogen,glucose, and PC1 show very similar features, and a clear attributioncannot be made. Around 1155 cm–1 (spectral regionC), the minimum in PC1 corresponds to a superposition of the minimaof both trehalose and glycogen.

Bottom Line: Accumulation of storage carbohydrates (trehalose and glycogen) inside the cells was induced by a lack of a nitrogen source in the feed medium.Comparison of the cell spectra with spectra of trehalose, glycogen, glucose, and mannan, i.e., the major carbohydrates present in S. cerevisiae, and principal components analysis revealed that the changes observed in the cell spectra correlated well with the bands specific for trehalose and glycogen.This proves the applicability and capability of ultrasound-enhanced in-line ATR mid-IR spectroscopy as a real-time PAT method for the in situ monitoring of cellular biochemistry during fermentation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology , Getreidemarkt 9/164-UPA, 1060 Vienna, Austria.

ABSTRACT
This article presents a novel method for selective acquisition of Fourier transform infrared (FT-IR) spectra of microorganisms in-line during fermentation, using Saccharomyces cerevisiae as an example. The position of the cells relative to the sensitive region of the attenuated total reflection (ATR) FT-IR probe was controlled by combing a commercially available ATR in-line probe with contact-free, gentle particle manipulation by ultrasonic standing waves. A prototype probe was successfully constructed, assembled, and tested in-line during fed-batch fermentations of S. cerevisiae. Control over the position of the cells was achieved by tuning the ultrasound frequency: 2.41 MHz was used for acquisition of spectra of the cells (pushing frequency f(p)) and 1.87 MHz, for retracting the cells from the ATR element, therefore allowing spectra of the medium to be acquired. Accumulation of storage carbohydrates (trehalose and glycogen) inside the cells was induced by a lack of a nitrogen source in the feed medium. These changes in biochemical composition were visible in the spectra of the cells recorded in-line during the application of f(p) and could be verified by reference spectra of dried cell samples recorded off-line with a FT-IR microscope. Comparison of the cell spectra with spectra of trehalose, glycogen, glucose, and mannan, i.e., the major carbohydrates present in S. cerevisiae, and principal components analysis revealed that the changes observed in the cell spectra correlated well with the bands specific for trehalose and glycogen. This proves the applicability and capability of ultrasound-enhanced in-line ATR mid-IR spectroscopy as a real-time PAT method for the in situ monitoring of cellular biochemistry during fermentation.

Show MeSH