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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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Schematic (a) and photograph (b) of the prototype probe: commercialATR probe and the custom-built ultrasound accessory. (c) The two operatingmodes. The cells and the evanescent field are magnified by a factorof 10 in comparison to the other dimensions for visibility.
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fig1: Schematic (a) and photograph (b) of the prototype probe: commercialATR probe and the custom-built ultrasound accessory. (c) The two operatingmodes. The cells and the evanescent field are magnified by a factorof 10 in comparison to the other dimensions for visibility.

Mentions: The fiber optic probe was equipped with an in-house-designed ultrasoundaccessory that allowed for manipulation of particles in suspension(Figure 1a,b). The materials were chosen withconsideration of FDA regulations, i.e., standard materials used inbiotechnology were applied (body, stainless steel material no. 1.4571/DINX6CrNiMoTi17-12-2; screws, stainless steel A4 (1.4404) (Bossard, Zug,Switzerland); seals and O-rings, ZruElast and ZruElast FPM 75 shore(both Zrunek, Vienna, Austria)). The ultrasound composite transducerconsisted of a 10 mm PZT (lead zirconium titante, type PIC 181, PICeramics, Lederhose, Germany) disc with wrap-around silver electrodesglued to a Macor cylinder with a one-component epoxy resin (AralditeAV 171, Huntsman Advanced Materials, The Woodlands, TX, USA). Theassembled ultrasound-enhanced mid-IR ATR probe complies with the 3Drule to avoid turbulent flow stagnation, i.e., indentations were limitedto three times their respective widths, and it is autoclavable insitu (121 °C, 20 min). A modified (limited to 2.5 W max poweroutput) frequency power synthesizer (FPS 2540, SinePhase Instruments,Hinterbrühl, Austria) connected to the PZT was used for ultrasoundsignal generation and amplification; frequency and power were controlledusing a custom LabView script and GUI (National Instruments, Austin,TX, USA). The distance between the ultrasound transducer and ATR probewas adjusted to approximately 1.35 mm.


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)

Schematic (a) and photograph (b) of the prototype probe: commercialATR probe and the custom-built ultrasound accessory. (c) The two operatingmodes. The cells and the evanescent field are magnified by a factorof 10 in comparison to the other dimensions for visibility.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Schematic (a) and photograph (b) of the prototype probe: commercialATR probe and the custom-built ultrasound accessory. (c) The two operatingmodes. The cells and the evanescent field are magnified by a factorof 10 in comparison to the other dimensions for visibility.
Mentions: The fiber optic probe was equipped with an in-house-designed ultrasoundaccessory that allowed for manipulation of particles in suspension(Figure 1a,b). The materials were chosen withconsideration of FDA regulations, i.e., standard materials used inbiotechnology were applied (body, stainless steel material no. 1.4571/DINX6CrNiMoTi17-12-2; screws, stainless steel A4 (1.4404) (Bossard, Zug,Switzerland); seals and O-rings, ZruElast and ZruElast FPM 75 shore(both Zrunek, Vienna, Austria)). The ultrasound composite transducerconsisted of a 10 mm PZT (lead zirconium titante, type PIC 181, PICeramics, Lederhose, Germany) disc with wrap-around silver electrodesglued to a Macor cylinder with a one-component epoxy resin (AralditeAV 171, Huntsman Advanced Materials, The Woodlands, TX, USA). Theassembled ultrasound-enhanced mid-IR ATR probe complies with the 3Drule to avoid turbulent flow stagnation, i.e., indentations were limitedto three times their respective widths, and it is autoclavable insitu (121 °C, 20 min). A modified (limited to 2.5 W max poweroutput) frequency power synthesizer (FPS 2540, SinePhase Instruments,Hinterbrühl, Austria) connected to the PZT was used for ultrasoundsignal generation and amplification; frequency and power were controlledusing a custom LabView script and GUI (National Instruments, Austin,TX, USA). The distance between the ultrasound transducer and ATR probewas adjusted to approximately 1.35 mm.

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