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Ultrasound-Enhanced Attenuated Total Reflection Mid-infrared Spectroscopy In-Line Probe: Acquisition of Cell Spectra in a Bioreactor
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† Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-UPA, 1060 Vienna, Austria
‡ Institute of Chemical Engineering, Vienna University of Technology, Gumpendorferstraße 1a, 1060 Vienna, Austria
⊥ Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, Institute of Chemical Engineering, Vienna University of Technology, Getreidemarkt 9/166, 1060 Vienna, Austria
*E-mail: [email protected]
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Analytical Chemistry

Cite this: Anal. Chem. 2015, 87, 4, 2314–2320
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https://doi.org/10.1021/ac504126v
Published January 12, 2015

Copyright © 2015 American Chemical Society. This publication is licensed under CC-BY.

Abstract

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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 fp) 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 fp 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.

Copyright © 2015 American Chemical Society

Subjects

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Fourier transform infrared (FT-IR) spectroscopy is an established method for at-line, online, and in-line bioprocess analysis and monitoring, especially in the mid-IR region (400–4000 cm–1). Because water, a strong infrared absorber, is the most abundant solvent and maximum interaction lengths are a few tens of micrometers, attenuated total reflection (ATR) configurations are the most commonly used. The light is totally reflected within an ATR element, resulting in an evanescent field that interacts with the sample, with typical penetration depths of approximately 1 to 2 μm. (1) Multiple reflections lead to enhanced interaction with the sample without having to face the challenges encountered when measuring transmission (clogging, path length changes due to pressure drops, etc.). Multivariate data analysis can be used to extract chemical information from data (i.e., chemometrics); this makes quantification of spectroscopic measurements possible. Lourenço et al. (2) gave a good general overview of optical spectroscopy methods in combination with chemometrics used for bioreactor monitoring, including mid-IR spectroscopy, whereas a mini-review by Landgrebe et al. (3) summarizes recent applications of near- and mid-IR spectroscopy for bioprocess monitoring. A frequently used chemometric method is principal component analysis (PCA). ATR mid-IR spectroscopy in combination with PCA has been, e.g., successfully used for the classification and identification of chemical differences of edible fats and oils, (4)Robusta and Arabica coffees, (5) and injured and intact Listeria monocytogenes. (6)
For various bioprocesses, spectra of media components and excreted secondary metabolites have been recorded at-line, (7-10) online, and in-line. Online bypass systems were successfully used to monitor media composition and products (11-13) and for pH control (14) in different types of bioprocesses. In-line ATR probes are realized using fiber optics (typically, silver halide) or light conduits and are commercially available. Doak and Phillips reported the first use of a conduit probe to monitor Escherichia coli fermentation in 1999; (15) since then, numerous reports of in-line, i.e., in situ (the more commonly used term in bioprocess engineering), monitoring of fermentation have been published. (16-20)
Spectra of microorganisms are acquired at-line or off-line and allow for differentiation between physiological states, reflected by biochemical composition and the quantification of secondary metabolites accumulated inside cells. In most studies, transmission spectra of washed, dried cells were measured, either by FT-IR microspectroscopy (21, 22) or with the dedicated microplate reader accessory HTS-XT for high-throughput analysis (Bruker Optics). (23-27) Transmission and ATR FT-IR microspectroscopy were successfully used for the analysis and differentiation of physiological states in the yeast Saccharomyces cerevisiae. (28-31) Jarute et al. presented the first online setup for the quantification of poly beta-hydroxybutyic acid (PHB) in E. coli using a horizontal ATR in combination with a stopped-flow system. (32) When the flow was on, spectra of the medium were acquired; subsequently, the flow was switched off, and the cells sedimented onto the ATR surface and thus spectra of the cells could be acquired. Biofouling was avoided by chemical cleaning of the ATR as necessary.

Ultrasound Particle Manipulation

Ultrasonic standing wave fields, built up between an ultrasound transducer and a reflector, are a contact-free, gentle method for manipulation of small particles in suspension. Particle sizes between one and a few tens of micrometers require ultrasound frequencies between 1 and 10 MHz. The axial acoustic radiation force, i.e., in the direction of sound propagation, acting on the particles depends on particle volume, the ultrasound frequency, the acoustic energy density, i.e., the power that is transmitted into the medium, and the acoustic contrast factor. (33-35) The force results in a pattern of planes that are half of a wavelength apart, into which the particles are driven. These planes are either the pressure nodal planes (i.e., the planes of minimum acoustic pressure) or the pressure antinodal planes of the standing wave field, depending on the acoustic contrast factor between the particles and the carrier medium. This, in turn, depends on the ratio of the sound speeds and mass densities between the particles and the medium; dense particles, like cells, are driven into the pressure nodes of the standing wave field. Ultrasound particle manipulation is not harmful to cells (36, 37) and is a state-of-the-art method for perfusion filtering of mammalian cell cultures, where it is used to hold cells against a continuous flow of medium. (38-40)
Ultrasound particle manipulation was used to increase the robustness of online cell spectra acquisition (S. cerevisiae) in a setup similar to that of Jarute et al., (32) by using the radiation force to lift the cells off of the horizontal ATR surface, which acted as ultrasound reflector. (41) Furthermore, the settling speed, and thus the measurement intervals, could be increased by the formation of cell aggregates in the pressure nodal planes, which sedimented faster than single cells once the ultrasound and the flow were switched off. A major drawback of bypass setups are sterility issues. This can be overcome by taking the system in-line. (42) Radel et al. showed in 2010 that the combination of ultrasound particle manipulation and a fiber optic ATR probe made acquisition of mid-IR spectra of PTFE particles in stirred suspensions possible. (43) Here, the ATR element was oriented vertically, and the particles were pushed against it by tuning the ultrasound frequency such that a net acoustic radiation force acted in the direction of the ATR element. In 2013, we showed that spectra of S. cerevisiae could be acquired with a prototype in-line applicable probe. (44)
This article presents a major step forward in this development: an autoclavable, in-line ultrasound-enhanced fiber optic mid-IR ATR probe constructed according to FDA guidelines and its application during S. cerevisiae fermentation in-line in a semi-industrial fermenter. Within fed-batch fermentation, the cells were deliberately nitrogen-limited by the design of the media and therefore started to accumulate storage carbohydrates, i.e., trehalose and glycogen, which was investigated. We recently showed the possibility of off-line quantification of these carbohydrates in whole S. cerevisiae by FT-IR microspectroscopy. (31) Here, we investigated the possibility of monitoring these biochemical changes in-line by comparing them to off-line reference spectra and principal component analysis.

Experimental Section

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Ultrasound-Enhanced Fiber Optic ATR FT-IR Probe

Spectrometer and In-Line Probe

FT-IR spectra were acquired in-line with the process spectrometer ReactIR 15 equipped with an attenuated total reflection DS DiComp (diamond) 9.5 mm probe connected via a 1.5 m AgX (silver halide) fiber (all Mettler Toledo, Greifensee, Switzerland). The spectrometer was equipped with a liquid nitrogen cooled mercury–cadmium–telluride (MCT) detector. Spectra were acquired with resolutions of 4 and 8 cm–1, respectively, in the spectral region from 700 to 2800 cm–1 as the co-addition of 256 scans. iC IR 4.2 software (Mettler Toledo, Greifensee, Switzerland) was used for acquisition and basic data manipulation such as single-point baseline offset correction (1840 cm–1 set to zero). The DS DiComp probe was chosen due to its flat top, which makes it especially suitable as a reflector for the ultrasonic wave.

Ultrasound Technology: Material and Design Considerations

The fiber optic probe was equipped with an in-house-designed ultrasound accessory that allowed for manipulation of particles in suspension (Figure 1a,b). The materials were chosen with consideration of FDA regulations, i.e., standard materials used in biotechnology were applied (body, stainless steel material no. 1.4571/DIN X6CrNiMoTi17-12-2; 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 transducer consisted of a 10 mm PZT (lead zirconium titante, type PIC 181, PI Ceramics, Lederhose, Germany) disc with wrap-around silver electrodes glued to a Macor cylinder with a one-component epoxy resin (Araldite AV 171, Huntsman Advanced Materials, The Woodlands, TX, USA). The assembled ultrasound-enhanced mid-IR ATR probe complies with the 3D rule to avoid turbulent flow stagnation, i.e., indentations were limited to three times their respective widths, and it is autoclavable in situ (121 °C, 20 min). A modified (limited to 2.5 W max power output) frequency power synthesizer (FPS 2540, SinePhase Instruments, Hinterbrühl, Austria) connected to the PZT was used for ultrasound signal generation and amplification; frequency and power were controlled using a custom LabView script and GUI (National Instruments, Austin, TX, USA). The distance between the ultrasound transducer and ATR probe was adjusted to approximately 1.35 mm.
The working principle is shown in the inset in Figure 1c: At retracting frequency fr, cells are held in the pressure nodal planes away from the evanescent field of the ATR probe, whereas they are pushed against the ATR surface and thus into the evanescent field when the pushing frequency fp is applied.

Figure 1

Figure 1. Schematic (a) and photograph (b) of the prototype probe: commercial ATR probe and the custom-built ultrasound accessory. (c) The two operating modes. The cells and the evanescent field are magnified by a factor of 10 in comparison to the other dimensions for visibility.

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Laboratory FT-IR Spectrometers: Reference Spectra

Reference spectra of glucose, trehalose, and mannan solutions and glycogen mixed with H2O were acquired with a Tensor 37 FT-IR spectrometer equipped with a DTGS (deuterated triglycine sulfate) detector using a Platinum diamond ATR (all Bruker Optics, Ettlingen, Germany). Instrument control and spectrum acquisition were performed with OPUS 7 software (Bruker Optics). Spectral resolution was set to 2 cm–1, and spectra were recorded as the co-addition of 32 scans.
Sampling, pretreatment of the off-line samples, and acquisition of reference spectra of dried yeast samples drawn during fermentation have been described elsewhere. (31)

S. cerevisiae Cultivations

Fermentation of S. cerevisiae (strain CBS8340) was carried out in two different, fully automated and controlled stirred bioreactors: a 10 L glass/steel bioreactor (Chemap, Switzerland) and a 15 L steel bioreactor (Infors, Bottmingen, Switzerland). Both were equipped with 25 mm Ingold ports for coupling with the ultrasound-enhanced mid-IR ATR probe. Media preparation and cultivation conditions are described in detail in the Supporting Information (Experimental, page S2, and Table S1, page S2); the stirring speed was varied (800–1200 rpm), and the pO2 level was always kept above 80% by adjusting the inflow of air. CO2 content of the off-gas was measured using an off-gas analyzer (Bluesens, Snirgelskamp, Germany). Biomass was determined off-line by measuring the optical density at 600 nm with a Genesys 20 visible spectrophotometer (Thermo Scientific, Waltham, MA, USA).
After the batch phase on glucose was completed, the bioprocess was carried on as a C-limited fed-batch with an exponential feed strategy. The feed medium was deficient in a nitrogen source. Under C-limited feeding conditions, no excess carbon source was present in the medium. The nitrogen limitation led to the synthesis and accumulation of storage carbohydrates, i.e., trehalose and glycogen, inside the cells.

Experimental Procedure for Ultrasound-Enhanced Mid-IR Spectroscopy

During fermentations, fp was 2.410 MHz at 1.8–1.9 W true electrical power input (t.e.p.i.), and fr was 1.870 MHz at 1.6 W t.e.p.i.

Data Analysis

Matlab (Mathworks, Natick, MA, USA) and the PLS toolbox (Eigenvector Research, Wenatchee, WA, USA) were used for data manipulation and PCA. For PCA, the spectra were area normalized in the carbohydrate region (938–1186 cm–1) prior to calculation of the second derivate (Savitzky-Golay, second order, window width 9 or 11 depending on spectral resolution). Single-analyte spectra were normalized and differentiated using the same settings for comparison.

Results and Discussion

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In-Line Procedure

Spectra were acquired in-line during fermentation, alternating between pushing and retracting frequency every 10 min. Three FT-IR spectra were acquired at each ultrasound frequency; they were subsequently averaged for further data analysis, resulting in a single, average, spectrum for each frequency application. The background was acquired in batch medium with the probe in its final position in the bioreactor after autoclaving. This was used as the background for the absorbance spectra of the medium, i.e., spectra recorded during the application of the retracting frequency. Absorbance spectra of the cells were calculated as difference spectra between a spectrum acquired during the application of the pushing frequency and the respective previous media spectrum (Figure 2). This was necessary to compensate for changes in the medium composition, as there is always medium in the probed volume and analyte concentrations are higher (and vary stronger over the course of the fermentation) in the medium than in the cells themselves. Samples were drawn from the bioreactor at different times for biomass determination and the acquisition of cell reference spectra.

Figure 2

Figure 2. In-line spectra acquired during S. cerevisiae fermentation when applying retracting frequency (top) or pushing frequency (middle) and their difference spectrum showing typical spectral features of yeast cells.

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Optimization of Experimental Conditions

In total, four fermentations were conducted; the influence of the ultrasound frequency for pushing and retracting, stirring speed, and spectral and temporal resolution were investigated, and the optima were subsequently used. The highest yeast-related absorption values in difference spectra were found using a pushing frequency of 2.410 MHz and a power of 1.8–1.9 W t.e.p.i. (fp). For retracting the particles from the evanescent field of the ATR element, the ultrasound transducer was driven around fr 1.870 MHz (at 1.6 W t.e.p.i.). Stirring speeds between 600 and 1200 rpm were tested: an increase in stirring speed led to a decrease of absorption values of the recorded spectra due to Stokes drag (data not shown). As a compromise between sufficient stirring and satisfactory signal of the mid-IR spectra of cells, a stirring speed of 800 rpm was identified. Comparison of spectral resolutions of 4 and 8 cm–1 showed that the broad absorption bands of S. cerevisiae were sufficiently resolved by the latter (data not shown). Temporal resolution was governed by spectrum acquisition time (i.e., 1 min at 8 cm–1 resolution and co-addition of 256 scans), sufficient data redundancy (in case of erroneous spectra and to increase the signal-to-noise ratio), and maximum handleable file size. Spectra were acquired every 2 min; thus, as mentioned before, three spectra were available for averaging during alternating application of the ultrasound frequencies for 10 min each. These parameters were determined during the first fermentation and were subsequently used. The spectra recorded during the four fermentations showed similar trends, and an exemplary one will be presented here.

Influence of Growth Conditions on Yeast Cells

In order to study changes on the (bio)chemical composition of the cells, the fermentation was carried out in two parts: a batch phase, with an excess of glucose (C-source) and ammonium (N-source), and subsequently a C-limited fed-batch phase during which no additional N-source was fed and trehalose and glycogen were accumulated inside the cells.

Batch Phase

At the beginning of the batch phase, glucose was present in excess in the medium, leading to the production of ethanol as a main metabolite in addition to biomass (Crabtree effect, overflow metabolism). The cells exhibited characteristic diauxic growth: first, glucose was metabolized to ethanol in parallel with biomass formation (0–12 h) and then, after a short lag phase, the extracellular ethanol was metabolized as a carbon source (12–20 h). This was reflected by the CO2 off-gas profile and the FT-IR spectra of the medium (Figure 3). Characteristic spectral features of glucose were predominant at first (Figure 3b, dark red spectra, bands at 1150, 1110, 1080, and 1037 cm–1) followed by a superposition of glucose and ethanol (light red spectra). Once the glucose was depleted, ethanol dominated the FT-IR spectra, with its characteristic bands at 1045 and 1080 cm–1 (blue spectra), which gradually decreased until the end of the batch phase (light blue spectrum).

Figure 3

Figure 3. (a) CO2 off-gas and biomass over the course of the batch phase. (b) ATR FT-IR spectra of the medium (i.e., retracting frequency applied) over the course of the batch phase.

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During batch phase, yeast cell composition did not change; only biomass growth occurred, which was reflected by an increase in absorbance over time, whereas spectral features remained similar (see Supporting Information Figure S1).

Fed-Batch Phase

The spectra of dried S. cerevisiae fed-batch samples acquired off-line (Figure 4a) show spectral features typical for the constituents of biological cells (from refs 45 and 46): The C═O stretching of ester groups in lipids leads to an absorption around 1730 cm–1. Proteins lead to absorptions around 1655 cm–1 (amide I band, mainly from C═O stretching and, to a lesser extent, N–H bend and C–N stretch), around 1550 cm–1 (amide II band, N–H bend, and C–N stretch), and around 1300 cm–1 (amide III band, complex combination of different vibrations). In the region between 1450 and 1200 cm–1, numerous C–H and O–H deformation vibrations contribute to the spectrum (lipids, carbohydrates). Antisymmetric PO2– stretching vibrations of DNA, RNA, and phospholipids lead to absorptions around 1240 cm–1. The region between 950 and 1200 cm–1 (hereafter, carbohydrate region) is dominated by C–O stretching originating from carbohydrates, with contributions around 1100 cm–1 from symmetric PO2– stretching (again, DNA, RNA, and phosopholipids).
The changes in carbohydrate content induced by N-limited growth are reflected by changes in the shape of the spectra acquired off-line (Figure 4a, normalized to the amide I band). The relative carbohydrate content increases with fermentation time, and the shape of the carbohydrate region changes as trehalose and glycogen are formed in the cells. The carbohydrate bands of the difference spectra of the cells acquired in-line at the time points of sampling (Figure 4b, offset baseline corrected) are in good agreement with the off-line spectra (Figure 4a). Differences can be attributed to matrix influence, band shifts due to acquisition in ATR mode, and the lower signal-to-noise ratio for in-line spectra.

Figure 4

Figure 4. (a) Spectra of dried S. cerevisiae acquired off-line using a laboratory FT-IR microscope (normalized to amide I band). (b) Spectra of S. cerevisiae acquired in-line using the ultrasound-enhanced mid-IR ATR probe at the times when samples for off-line analysis were drawn.

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A major difference between the off- and in-line measurements was observed in the amide I and amide II regions: here, H2O absorbs strongly (H–O–H bending at approximately 1640 cm–1) and therefore the intensity reaching the detector is low, leading to a high noise level. The protein bands (amide I and II) are only weakly visible in the in-line spectra, most likely because the H2O present when the fr spectrum is acquired is simply replaced by the protein present in S. cerevisiae when fp is applied, leading to approximately the same absorption. Furthermore, when fp is applied, H2O is, for the most part but never completely, replaced by yeast cells (even assuming close packing of equal spheres, approximately 26% of the space would still be occupied by H2O).
Since changes associated with switching to nitrogen-limited growth are mainly visible in the carbohydrate region of the FT-IR spectra, further analysis was focused on this region. The main carbohydrates of yeast were studied: mannan, a cell wall polysaccharide composed of mannose units, and intracellular carbohydrates trehalose, a disaccharide of glucose, glycogen, a polysaccharide of glucose, and glucose. ATR FT-IR reference spectra of the major carbohydrates (area normalized as described in the Experimental Section) present in yeast cells are shown in Figure 5a. At 994 cm–1, trehalose, one of the storage carbohydrates accumulated during N-limited growth, has an intense and specific absorption. In the spectral region between 1025 and 1175 cm–1, trehalose and glucose show bands at similar positions; their relative intensities are, however, different. Glucose absorbs strongest at 1035 cm–1, whereas the relative absorption maximum of trehalose in this region is shifted toward slightly higher wavenumbers and is lower in intensity. At around 1155 cm–1, trehalose and glycogen have a common absorption band. Glycogen absorbs broadly between 970 and 1170 cm–1, with a distinct maximum at 1022 cm–1 and smaller maxima at 1080 and 1152 cm–1. Mannan has a broad absorption band between 1000 and 1150 cm–1, with a distinct maximum at 1060 cm–1. The exact positions of absorption maxima are even clearer when looking at second derivatives of the spectra: maxima in the spectra are minima here (Figure 5b, top).

Figure 5

Figure 5. (a) Spectra of major carbohydrates present in yeast cells (off-line ATR, in solution) and spectra of yeast cells acquired in-line over the course of the fermentation. (b) Second-derivative spectra of major carbohydrates present in yeast cells and of yeast cells acquired over the course of the fermentation, respectively. In both graphs, carbohydrate spectra are offset for better visibility.

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In the yeast, changes in the carbohydrate composition can be observed by spectra acquired in-line as difference spectra over the course of the N-limited fed-batch fermentation (Figure 5a, bottom). The shape of the carbohydrate band changed with fermentation time, most notably between 1050 and 950 cm–1. The blue spectra, which were acquired toward the end of the fermentation, show higher absorption around 990 cm–1, correlating well with the absorption spectra of trehalose. This becomes even clearer when investigating second-derivate spectra: The decrease around 994 cm–1 and the increase around 970 cm–1 in the spectra acquired toward the end of the fermentation (blue) correlate well with the trehalose second-derivative spectrum (gray). Between 1030 and 1000 cm–1, the slope of the second-derivative yeast spectra steepens with fermentation time. The minimum at ∼1150 cm–1, which further decreases with fermentation time, correlates with an increase in both glycogen and trehalose. Since this minimum is less pronounced for trehalose compared to the one at 994 cm–1, it can be concluded that glycogen mainly contributes to this change.

PCA

We have recently shown that quantitative determination of carbohydrates is possible from off-line yeast spectra; (31) from the in-line difference spectra, only qualitative information on the changes in carbohydrate composition could be observed. Normalization of the spectra proved to be difficult because changes in biomass and in the carbohydrate content occurred simultaneously, and this correlation could not be broken. To evaluate if the trends observed in the in-line difference spectra are statistically detectable and related to carbohydrate features, PCA was performed on the second-derivative spectra.
Individual PCA of the four fermentations resulted in very similar principal component 1 (PC1), which explained between 53 and 73% of the variation in the respective fermentation spectra. A PCA of the combined spectra from all four fermentations again resulted in a PC1 with very similar features; however, only 40% of the variance was explained. This can be attributed to interfermentation variations leading a smaller contribution from changes induced by N-limited growth. To concentrate on the biochemical changes inside the cells, the following discussion is based on the PCA of a single fermentation.
The loading vector of PC1 and the second-derivative spectra of trehalose, 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 also varies the most between spectra, reflected by the highest values of the first loading vector. The plateau at 1035 cm–1 followed by a minimum around 1025 cm–1 in PC1 correlates with glycogen, superimposed with trehalose and glucose (thus, the shift of the minimum toward higher wavenumbers). In spectral region B (1050 to 1103 cm–1), trehalose, glycogen, glucose, and PC1 show very similar features, and a clear attribution cannot be made. Around 1155 cm–1 (spectral region C), the minimum in PC1 corresponds to a superposition of the minima of both trehalose and glycogen.

Figure 6

Figure 6. Second-derivative spectra of trehalose, glycogen, glucose, and mannan and PC1 of spectra acquired during N-limited growth.

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Conclusions and Outlook

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The presented method allows for the selective acquisition of mid-IR spectra of either dissolved analytes (media components) or suspended particles (cells). This is, to the best of our knowledge, the first report on the acquisition of mid-IR spectra of microorganisms (S. cerevisiae) inside a semi-industrial bioreactor during fermentation without the necessity of drawing a sample. The cell spectra acquired in-line are in good agreement with spectra of dried cell samples that were recorded off-line. Trends in the changes of the biochemical composition of the cells during N-limited fed-batch fermentation were visible, showing the potential of ultrasound-enhanced ATR mid-IR spectroscopy to be used as a PAT (process analytical technology) probe to detect the cells’ physiological response to nutritional limitations. Quantitative analysis proved to be difficult, as the number of cells present in the evanescent field is hard to estimate and the amide I band, which could be used for normalization off-line, was not accessible in-line. Furthermore, the signal-to-noise ratio of the difference spectra still needs to be improved to make quantitative analysis by, e.g., partial least-squares regression possible. More insight into the spatial distribution of the cells in the evanescent field could potentially be gained using the gel method proposed by Gherardini et al.; (47) with a better understanding of the number of cells held in the evanescent field at a given time under different conditions, the before-mentioned challenges will be addressed in a follow-up project.
The described novel method is generic and suitable for (ideally spherical) cells within a size range of 1 to approximately 40 μm, as is often employed in white and red biotechnology and biorefinery. It delivers real-time information on the chemical composition of cells in situ, thus aiding the optimization of process conditions and media composition. Moreover, physiological responses of cells can be investigated, helping to obtain a basic understanding of cell physiology/metabolism. In industrial fermentation, the method could serve as a quality control tool: here, complex media are often employed, for which exact determination of nutrient concentrations would usually require extensive and laborious chemical analyses or is not possible at all. By measuring the cell’s biochemical composition, potential limitations could be detected, and appropriate measures to ensure the desired quality could be taken early in the process.
Apart from bioprocesses, the presented method can, for example, be applied to gain insight into crystallization processes.

Supporting Information

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Experimental details on media preparation and cultivation conditions. Table S1: Overview of the setup and conditions of the fed-batch fermentations. Figure S1: Spectra of S. cerevisiae acquired in-line during batch phase fermentation. This material is available free of charge via the Internet at http://pubs.acs.org.

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Author Information

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  • Corresponding Author
    • Bernhard Lendl - Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-UPA, 1060 Vienna, Austria;  Email: [email protected]
  • Authors
    • Cosima Koch - Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-UPA, 1060 Vienna, Austria
    • Markus Brandstetter - Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-UPA, 1060 Vienna, Austria
    • Patrick Wechselberger - Institute of Chemical Engineering, Vienna University of Technology, Gumpendorferstraße 1a, 1060 Vienna, Austria;  Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, Institute of Chemical Engineering, Vienna University of Technology, Getreidemarkt 9/166, 1060 Vienna, Austria
    • Bettina Lorantfy - Institute of Chemical Engineering, Vienna University of Technology, Gumpendorferstraße 1a, 1060 Vienna, Austria
    • Maria Reyes Plata - Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-UPA, 1060 Vienna, Austria
    • Stefan Radel - Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-UPA, 1060 Vienna, Austria
    • Christoph Herwig - Institute of Chemical Engineering, Vienna University of Technology, Gumpendorferstraße 1a, 1060 Vienna, Austria;  Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, Institute of Chemical Engineering, Vienna University of Technology, Getreidemarkt 9/166, 1060 Vienna, Austria
  • Notes
    The authors declare no competing financial interest.

Acknowledgment

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The authors express their gratitude to Lukas Strobl for realization of the Labview control of the ultrasound frequency generator and Gerhard Fritsch for assistance in the assembly of the prototype probe and with preliminary experiments. Partial financial support provided by the Austrian Research Promotion Agency (FFG) under the scope of the COMET program within the research network “Process Analytical Chemistry (PAC)” (contract no. 825340) (C.K. and M.B.) and the Austrian Science Fund FWF (project no. P24154-N17) (C.K. and S.R.) is gratefully acknowledged. C.K. gratefully acknowledges partial financial support by the AB-Tec graduate school (TU Wien). European Social Fund from European Union and Junta de Comunidades de Castilla-La Mancha are gratefully acknowledged for supporting given through a Postdoctoral research contract for M.R.P.

References

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    A review. Biotechnol. processes are crucial to the development of any economy striving to ensure a relevant position in future markets. The cultivation of microorganisms in bioreactors is one of the most important unit operations of biotechnol. processes, and real-time monitoring of bioreactors is essential for effective bioprocess control. In this review, published material on the potential application of different spectroscopic techniques for bioreactor monitoring is critically discussed, with particular emphasis on optical fiber technol., reported for in situ bioprocess monitoring. Application examples are presented by spectroscopy type, specifically focusing on UV-visible, near-IR, mid-IR, Raman, and fluorescence spectroscopy. The spectra acquisition devices available and the major advantages and disadvantages of each spectroscopy are discussed. The type of information contained in the spectra and the available chemometric methods for extg. that information are also addressed, including wavelength selection, spectra pre-processing, principal component anal., and partial least-squares. Sample handling techniques (flow and sequential injection anal.) that include transport to spectroscopic sensors for ex-situ online monitoring are not covered in this review.
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    Bioprocess optimization is often limited by its inability to measure biomass, nutrients and product concns. in a time frame that allows process adjustments. IR spectroscopic techniques can potentially be used to measure each of these components within about 3 min of sampling, of an unprocessed whole broth sample. In the present study, the use of Fourier transform mid IR (FT-MIR), near IR red (NIR), and Fourier transform (FT)-Raman spectroscopic techniques for at-line (rapid off-line) monitoring of biomass, glucose, and lactic acid in a lactic acid fermn. by Lactobacillus casei was investigated. Suitable spectral wavenumber regions were selected for principal least square (PLS) regression and principal component regression (PCR) and calibration models were developed. The std. error of calibration for the PLS-1st deriv. calibration models for glucose, lactic acid, and optical cell d. were 2.5, 0.7 g/l, and 0.23, resp. Prediction errors were high for NIR and FT-Raman methods. Results indicated that FT-MIR spectroscopy could be used for rapid detection of glucose, lactic acid, and optical cell d. during lactic acid fermn.
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    Mazarevica, Gunta; Diewok, Josef; Baena, Josefa R.; Rosenberg, Erwin; Lendl, Bernhard
    Applied Spectroscopy (2004), 58 (7), 804-810CODEN: APSPA4; ISSN:0003-7028. (Society for Applied Spectroscopy)
    A new method for online monitoring of fermns. using mid-IR (MIR) spectroscopy has been developed. The method was used to predict the concns. of glucose and EtOH during a bakers' yeast fermns. A completely automated flow system was employed as an interface between the bioprocess under study and the Fourier transform IR (FT-IR) spectrometer, which was equipped with a flow cell housing a diamond attenuated total reflection (ATR) element. By using the automated flow system, exptl. problems related to adherence of CO2 bubbles to the ATR surface, as well as formation of biofilms on the ATR surface, could be efficiently eliminated. Gas bubbles were removed during sampling, and by using rinsing steps any biofilm could be removed from the ATR surface. In this way, const. measuring conditions could be guaranteed throughout prolonged fermn. times (∼8 h). As a ref. method, high-performance liq. chromatog. (HPLC) with refractive index detection was used. The recorded data from different fermns. were modeled by partial least-squares (PLS) regression comparing 2 different strategies for the calibration. On the one hand, calibration sets were constructed from spectra recorded from either synthetic stds. or from samples drawn during fermn. On the other hand, spectra from fermn. samples and synthetic stds. were combined to form a calibration set. Differences in the kinetics of the studied fermn. processes used for calibration and prediction, as well as the precision of the HPLC ref. method, were identified as the main chemometric sources of error. The optimal PLS regression method was obtained using the mixed calibration set of samples from fermns. and synthetic stds. The root mean square errors of prediction in this case were 0.267 and 0.336 g/L for glucose and EtOH concn., resp.
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    A simple method to monitor and control methanol feeding of Pichia pastoris fermentations using mid-IR spectroscopy
    Schenk, Jonas; Marison, Ian W.; von Stockar, Urs
    Journal of Biotechnology (2007), 128 (2), 344-353CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)
    Mid-IR FTIR spectroscopy is an efficient tool for the monitoring of bioprocesses, since it is fast and able to detect many compds. simultaneously. However, complex and time-consuming calibration procedures are still required, and have inhibited the spreading of these instruments. A simple and quick method to calibrate a FTIR instrument was developed for the control of fed-batch fermns. of the methylotrophic yeast Pichia pastoris. Based on the assumptions that (1) only substrate concn. may change significantly during a fed-batch process and (2) absorbance can be considered as proportional to concn., a linear two-point calibration was implemented. Long-term instability of the instrument had to be addressed in order to get accurate results: two fixed points, on both sides of substrate absorbance peak, were used to perform online a linear correction of the signal drift. Fed-batch expts. at const. methanol (substrate) concn. ranging from 0.8 to 15 g l-1 were carried out. Off-line HPLC control anal. showed a good agreement with online FTIR data, with std. error of prediction values <0.12 g l-1. Even though methanol acts both as carbon source and inducer of protein expression, no significant effect was obsd. on the level of protein expression in the recombinant strain used.
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    In situ monitoring of an Escherichia coli fermentation using a diamond composition ATR probe and mid-infrared spectroscopy
    Doak, Denise L.; Phillips, Janice A.
    Biotechnology Progress (1999), 15 (3), 529-539CODEN: BIPRET; ISSN:8756-7938. (American Chemical Society)
    A diamond compn. ATR probe was used in situ to obtain IR spectra on replicate Escherichia coli fermns. involving a complex medium. The probe showed excellent stability over a 6-mo operating period and was unaffected by either agitation or aeration. The formation of an unknown was obsd. from IR spectra obtained during the sterilization; subsequent expts. proved this to be a reaction product between yeast ext. and the phosphates used as buffer salts. Partial-least-squares-based calibration/prediction models were developed for both glucose and acetate using in-process samples. The resulting models had prediction errors of ±0.26 and ±0.75 g/L for glucose and acetic acid, resp., errors which were statistically equiv. to the estd. exptl. errors in the ref. measurements. Relative concn. profiles for the unknown formed during sterilization could be generated either by tracking peak height at an independent wavelength or by self-modeling curve resoln. of the spectral region overlapping that of glucose. These profiles indicated that this compd. was metabolized simultaneously with glucose; upon depletion of the glucose, when the microorganism switched to consumption of acetic acid, utilization continued but at a lower rate. The data presented provide an extensive characterization of the performance characteristics of this in situ anal. and clearly demonstrate its utility not just in the quant. measurement of multiple known species but in the qual. evaluation of unknown species.
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    Real-time analyte monitoring of a fungal fermentation, at pilot scale, using in situ mid-infrared spectroscopy
    Pollard, D. J.; Buccino, R.; Connors, N. C.; Kirschner, T. F.; Olewinski, R. C.; Saini, K.; Salmon, P. M.
    Bioprocess and Biosystems Engineering (2001), 24 (1), 13-14CODEN: BIENEU; ISSN:1615-7591. (Springer-Verlag)
    Robust in situ biochem. monitoring is essential for the development of substrate feed control to optimize fermn. processes. The scale up of the fermn. for the fungus Glarea lozoyensis can benefit from such technol. to improve the yield of the pharmaceutically important pneumocandin of interest and control the levels of unwanted analogs. A new in situ probe, using a diamond attenuated total reflection element, was evaluated at pilot scale for the quant. measurement of fermn. analytes using Fourier transform mid-IR spectrometry. The new technol. was shown to be stable, unaffected by reactor operation conditions of agitation, airflow, and backpressure, but sensitive to temp. control. Both glucose and phosphate were simultaneously monitored during a seed fermn. at 280 L pilot scale using complex medium with detection to 0.1 g/L for both analytes. Fructose, glutamate, and proline were monitored at 75 L scale using prodn. media with detection limits of 0.1, 0.5, and 0.5 g/L resp. Partial least squares calibration/prediction models were created for analytes of interest using off-line ref. measurements and specific spectral regions. Good fits were obtained between off-line measurements and those predicted by in situ mid-IR. Std. errors of prediction (SEP) for glucose (range 18-0.1 g/L) and phosphate (range 11-7.5 g/L) were 0.16 and 1.8 g/L resp. with mean percentage errors (MPEs) around 2.5%. SEP values for the prodn. process: fructose (range 20-0.1 g/L), glutamate (8-0.5 g/L), and proline (12-0.5 g/L) were 0.44, 0.6, and 0.5 g/L resp. with MPEs of 2.2, 5.3, and 10.1%. The technol. effectively demonstrates quant. multicomponent anal. of fermn. processes using in situ monitoring.
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    Monitoring and control of Gluconacetobacter xylinus fed-batch cultures using in situ mid-IR spectroscopy
    Kornmann, Henri; Valentinotti, Sergio; Duboc, Philippe; Marison, Ian; von Stockar, Urs
    Journal of Biotechnology (2004), 113 (1-3), 231-245CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)
    A partial least-squares calibration model, relating mid-IR spectral features with fructose, ethanol, acetate, gluconacetan, phosphate and ammonium concns. has been designed to monitor and control cultivations of Gluconacetobacter xylinus and prodn. of gluconacetan, a food grade exopolysaccharide (EPS). Only synthetic solns. contg. a mixt. of the major components of culture media have been used to calibrate the spectrometer. A factorial design has been applied to det. the compn. and concn. in the calibration matrix. This approach guarantees a complete and intelligent scan of the calibration space using only 55 stds. This calibration model allowed std. errors of validation (SEV) for fructose, ethanol, acetate, gluconacetan, ammonium and phosphate concns. of 1.16 g/l, 0.36 g/l, 0.22 g/l, 1.54 g/l, 0.24 g/l and 0.18 g/l, resp. With G. xylinus, ethanol is directly oxidized to acetate, which is subsequently metabolized to form biomass. However, residual ethanol in the culture medium prevents bacterial growth. Online spectroscopic data were implemented in a closed-loop control strategy for fed-batch fermn. Acetate concn. was controlled at a const. value by feeding ethanol into the bioreactor. The designed fed-batch process allowed biomass prodn. on ethanol. This was not possible in a batch process due to ethanol inhibition of bacterial growth. In this way, the productivity of gluconacetan was increased from 1.8 × 10-3 [C-mol/C-mol substrate/h] in the batch process to 2.9 × 10-3 [C-mol/C-mol substrate/h] in the fed-batch process described in this study.
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    The application of micro-FTIR spectroscopy to analyze nutrient stress-related changes in biomass composition of phytoplankton algae
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    Micro-Fourier transform IR (FTIR) spectroscopy was used to study changes in spectral features of 3 species of Cyanobacteria (Microcystis aeruginosa, Chroococcus minutus, and Nostoc sp.) and 2 Bacillariophyceae (Cyclotella meneghiniana, and Phaeodactylum tricornutum) in response to nutrient stress. The change of physiol. state of the cells was followed during a 4-wk starvation period on the basis of physiol. key parameters and by means of FTIR spectroscopy. Changes in the integrated FTIR bands of cell spectra assigned to proteins, lipids, carbohydrates and silicate were used to calc. relative biomass compn. The results show that short-term acclimatization becomes visible at first in pigmentation and photosynthetic efficiency, whereas changes in biomass compn. reflect long term modulation in the metab. Simultaneous monitoring of short term and long term stress acclimatization showed evidence that the metabolic strategies to cope with increasing nutrient limitation are highly species-specific. This species-specificity can only be resolved in natural phytoplankton samples by single cell techniques. The results show that the FTIR technique has the potential to become applicable for the detn. of single cell biomass compn. from natural phytoplankton communities.
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    Dean, Andrew P.; Sigee, David C.; Estrada, Beatriz; Pittman, Jon K.
    Bioresource Technology (2010), 101 (12), 4499-4507CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
    In this study Fourier transform IR micro-spectroscopy (FTIR) was used to det. lipid and carbohydrate content over time in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus subspicatus grown in batch culture in limiting concns. of nitrogen (N). Both algae exhibited restricted cell division and increased cell size following N-limitation. FTIR spectra of cells in N-limited media showed increasing lipid:amide I and carbohydrate:amide I ratios over time. The use of lipid- and starch-staining dyes confirmed that the obsd. ratio changes were due to increased lipid and carbohydrate synthesis. These results demonstrate rapid metabolic responses of C. reinhardtii and S. subspicatus to changing nutrient availability, and indicate the efficiency of FTIR as a reliable method for high-throughput detn. of lipid induction.
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    Wagner, Heiko; Liu, Zhixin; Langner, Uwe; Stehfest, Katja; Wilhelm, Christian
    Journal of Biophotonics (2010), 3 (8-9), 557-566CODEN: JBOIBX; ISSN:1864-063X. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A mid-IR spectroscopic method was developed for the simultaneous and quant. detn. of total protein, carbohydrate and lipid contents of microalgal cells. Based on a chemometric approach, measured FTIR (Fourier transform IR) spectra from algal cells were reconstructed by a partial least square algorithm, using the spectra of the ref. substances to det. their relative contribution to the overall cell spectrum. From this specific absorption, abs. macromol. cell compn. [pg cell-1] can be calcd. using calibration curves, which have been validated by independent biochem. methods. The future potential of this method for photosynthesis research is shown by its application to follow time-resolved changes in the cellular compn. of microalgae during an illumination period of several hours. We show how the macromol. compn. can be investigated by FTIR spectroscopy methods. This can substantially increase the efficiency of screening processes like bioreactor monitoring and may be beneficial in metabolic engineering of algal cells.
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    Analytical and Bioanalytical Chemistry (2011), 399 (1), 387-401CODEN: ABCNBP; ISSN:1618-2642. (Springer)
    Fourier transform IR (FT-IR) spectroscopy was employed as a rapid high-throughput phenotypic typing technique to generate metabolic fingerprints of Escherichia coli MG1655 pDTG601A growing in fed-batch culture, during the dioxygenase-catalyzed biotransformation of toluene to toluene cis-glycol. With toluene fed as a vapor, the final toluene cis-glycol concn. was 83 mM, whereas the product concn. was only 22 mM when the culture was supplied with liq. toluene. Multivariate statistical anal. employing cluster anal. was used to analyze the dynamic changes in the data. The anal. revealed distinct trends and trajectories in cluster ordination space, illustrating phenotypic changes related to differences in the growth and product formation of the cultures. In addn., partial least squares regression was used to correlate the FT-IR metabolic fingerprints with the levels of toluene cis-glycol and acetate, the latter being an indicator of metabolic stress. The authors propose that this high-throughput metabolic fingerprinting approach is an ideal tool to assess temporal biochem. dynamics in complex biol. processes, as demonstrated by this redox biotransformation. Moreover, this approach can also give useful information on product yields and fermn. health indicators directly from the fermn. broth without the need for lengthy chromatog. anal. of the products.
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    Vibrational Spectroscopy (2008), 47 (2), 139-147CODEN: VISPEK; ISSN:0924-2031. (Elsevier B.V.)
    The present paper reports the first application of FTIR microspectroscopy in the mid-IR range to study the major biochem. changes assocd. with autolysis in yeast cells. Measurements were done both in transmission and in attenuated total reflection (ATR) mode on cells of Saccharomyces cerevisiae strain EC1118 before and after induction of the autolytic process in a model wine medium and in a Chardonnay base wine. Unsupervised multivariate statistical anal. (Hierarchical Cluster Anal. and Principal Component Anal.), as well as accurate spectral anal. based on curve fitting, were applied to the acquired spectra. The spectral behavior of S. cerevisiae in the model and base wines was found to be the same. A detailed interpretation of absorption bands was given by ref. to the literature and through comparison of transmission and ATR spectra. It was shown that FTIR microspectroscopy is a rapid and accurate tool to simultaneously probe the major biochem. events assocd. with the autolytic process. Moreover, the intrinsically higher sensitivity of ATR with respect to transmission spectra in analyzing autolysis was also demonstrated.
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    A fast and simple method to control variations in carbohydrate compn. of Saccharomyces cerevisiae, baker's yeast, during fermn. was developed using mid-IR (mid-IR) spectroscopy. The method allows for precise and accurate detns. with minimal or no sample prepn. and reagent consumption based on mid-IR spectra and partial least squares (PLS) regression. The PLS models were developed employing the results from ref. anal. of the yeast cells. The ref. analyses quantify the amt. of trehalose, glucose, glycogen, and mannan in S. cerevisiae. The selection and optimization of pretreatment steps of samples such as the disruption of the yeast cells and the hydrolysis of mannan and glycogen to obtain monosaccharides were carried out. Trehalose, glucose, and mannose were detd. using high-performance liq. chromatog. coupled with a refractive index detector and total carbohydrates were measured using the phenol-sulfuric method. Linear concn. range, accuracy, precision, LOD and LOQ were examd. to check the reliability of the chromatog. method for each analyte.
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  • Abstract

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    Figure 1

    Figure 1. Schematic (a) and photograph (b) of the prototype probe: commercial ATR probe and the custom-built ultrasound accessory. (c) The two operating modes. The cells and the evanescent field are magnified by a factor of 10 in comparison to the other dimensions for visibility.

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    Figure 2

    Figure 2. In-line spectra acquired during S. cerevisiae fermentation when applying retracting frequency (top) or pushing frequency (middle) and their difference spectrum showing typical spectral features of yeast cells.

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    Figure 3

    Figure 3. (a) CO2 off-gas and biomass over the course of the batch phase. (b) ATR FT-IR spectra of the medium (i.e., retracting frequency applied) over the course of the batch phase.

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    Figure 4

    Figure 4. (a) Spectra of dried S. cerevisiae acquired off-line using a laboratory FT-IR microscope (normalized to amide I band). (b) Spectra of S. cerevisiae acquired in-line using the ultrasound-enhanced mid-IR ATR probe at the times when samples for off-line analysis were drawn.

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    Figure 5

    Figure 5. (a) Spectra of major carbohydrates present in yeast cells (off-line ATR, in solution) and spectra of yeast cells acquired in-line over the course of the fermentation. (b) Second-derivative spectra of major carbohydrates present in yeast cells and of yeast cells acquired over the course of the fermentation, respectively. In both graphs, carbohydrate spectra are offset for better visibility.

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    Figure 6

    Figure 6. Second-derivative spectra of trehalose, glycogen, glucose, and mannan and PC1 of spectra acquired during N-limited growth.

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      Mazarevica, Gunta; Diewok, Josef; Baena, Josefa R.; Rosenberg, Erwin; Lendl, Bernhard
      Applied Spectroscopy (2004), 58 (7), 804-810CODEN: APSPA4; ISSN:0003-7028. (Society for Applied Spectroscopy)
      A new method for online monitoring of fermns. using mid-IR (MIR) spectroscopy has been developed. The method was used to predict the concns. of glucose and EtOH during a bakers' yeast fermns. A completely automated flow system was employed as an interface between the bioprocess under study and the Fourier transform IR (FT-IR) spectrometer, which was equipped with a flow cell housing a diamond attenuated total reflection (ATR) element. By using the automated flow system, exptl. problems related to adherence of CO2 bubbles to the ATR surface, as well as formation of biofilms on the ATR surface, could be efficiently eliminated. Gas bubbles were removed during sampling, and by using rinsing steps any biofilm could be removed from the ATR surface. In this way, const. measuring conditions could be guaranteed throughout prolonged fermn. times (∼8 h). As a ref. method, high-performance liq. chromatog. (HPLC) with refractive index detection was used. The recorded data from different fermns. were modeled by partial least-squares (PLS) regression comparing 2 different strategies for the calibration. On the one hand, calibration sets were constructed from spectra recorded from either synthetic stds. or from samples drawn during fermn. On the other hand, spectra from fermn. samples and synthetic stds. were combined to form a calibration set. Differences in the kinetics of the studied fermn. processes used for calibration and prediction, as well as the precision of the HPLC ref. method, were identified as the main chemometric sources of error. The optimal PLS regression method was obtained using the mixed calibration set of samples from fermns. and synthetic stds. The root mean square errors of prediction in this case were 0.267 and 0.336 g/L for glucose and EtOH concn., resp.
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    13. 13
      Schenk, J.; Marison, I. W.; von Stockar, U. J. Biotechnol. 2007, 128, 344– 353
      13
      A simple method to monitor and control methanol feeding of Pichia pastoris fermentations using mid-IR spectroscopy
      Schenk, Jonas; Marison, Ian W.; von Stockar, Urs
      Journal of Biotechnology (2007), 128 (2), 344-353CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)
      Mid-IR FTIR spectroscopy is an efficient tool for the monitoring of bioprocesses, since it is fast and able to detect many compds. simultaneously. However, complex and time-consuming calibration procedures are still required, and have inhibited the spreading of these instruments. A simple and quick method to calibrate a FTIR instrument was developed for the control of fed-batch fermns. of the methylotrophic yeast Pichia pastoris. Based on the assumptions that (1) only substrate concn. may change significantly during a fed-batch process and (2) absorbance can be considered as proportional to concn., a linear two-point calibration was implemented. Long-term instability of the instrument had to be addressed in order to get accurate results: two fixed points, on both sides of substrate absorbance peak, were used to perform online a linear correction of the signal drift. Fed-batch expts. at const. methanol (substrate) concn. ranging from 0.8 to 15 g l-1 were carried out. Off-line HPLC control anal. showed a good agreement with online FTIR data, with std. error of prediction values <0.12 g l-1. Even though methanol acts both as carbon source and inducer of protein expression, no significant effect was obsd. on the level of protein expression in the recombinant strain used.
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    14. 14
      Schenk, J.; Marison, I. W.; von Stockar, U. Biotechnol. Bioeng. 2008, 100, 82– 93
      There is no corresponding record for this reference.
    15. 15
      Doak, D. L.; Phillips, J. A. Biotechnol. Prog. 1999, 15, 529– 539
      15
      In situ monitoring of an Escherichia coli fermentation using a diamond composition ATR probe and mid-infrared spectroscopy
      Doak, Denise L.; Phillips, Janice A.
      Biotechnology Progress (1999), 15 (3), 529-539CODEN: BIPRET; ISSN:8756-7938. (American Chemical Society)
      A diamond compn. ATR probe was used in situ to obtain IR spectra on replicate Escherichia coli fermns. involving a complex medium. The probe showed excellent stability over a 6-mo operating period and was unaffected by either agitation or aeration. The formation of an unknown was obsd. from IR spectra obtained during the sterilization; subsequent expts. proved this to be a reaction product between yeast ext. and the phosphates used as buffer salts. Partial-least-squares-based calibration/prediction models were developed for both glucose and acetate using in-process samples. The resulting models had prediction errors of ±0.26 and ±0.75 g/L for glucose and acetic acid, resp., errors which were statistically equiv. to the estd. exptl. errors in the ref. measurements. Relative concn. profiles for the unknown formed during sterilization could be generated either by tracking peak height at an independent wavelength or by self-modeling curve resoln. of the spectral region overlapping that of glucose. These profiles indicated that this compd. was metabolized simultaneously with glucose; upon depletion of the glucose, when the microorganism switched to consumption of acetic acid, utilization continued but at a lower rate. The data presented provide an extensive characterization of the performance characteristics of this in situ anal. and clearly demonstrate its utility not just in the quant. measurement of multiple known species but in the qual. evaluation of unknown species.
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    16. 16
      Pollard, D.; Buccino, R.; Connors, N.; Kirschner, T.; Olewinski, R.; Saini, K.; Salmon, P. Bioprocess Biosyst. Eng. 2001, 24, 13– 24
      16
      Real-time analyte monitoring of a fungal fermentation, at pilot scale, using in situ mid-infrared spectroscopy
      Pollard, D. J.; Buccino, R.; Connors, N. C.; Kirschner, T. F.; Olewinski, R. C.; Saini, K.; Salmon, P. M.
      Bioprocess and Biosystems Engineering (2001), 24 (1), 13-14CODEN: BIENEU; ISSN:1615-7591. (Springer-Verlag)
      Robust in situ biochem. monitoring is essential for the development of substrate feed control to optimize fermn. processes. The scale up of the fermn. for the fungus Glarea lozoyensis can benefit from such technol. to improve the yield of the pharmaceutically important pneumocandin of interest and control the levels of unwanted analogs. A new in situ probe, using a diamond attenuated total reflection element, was evaluated at pilot scale for the quant. measurement of fermn. analytes using Fourier transform mid-IR spectrometry. The new technol. was shown to be stable, unaffected by reactor operation conditions of agitation, airflow, and backpressure, but sensitive to temp. control. Both glucose and phosphate were simultaneously monitored during a seed fermn. at 280 L pilot scale using complex medium with detection to 0.1 g/L for both analytes. Fructose, glutamate, and proline were monitored at 75 L scale using prodn. media with detection limits of 0.1, 0.5, and 0.5 g/L resp. Partial least squares calibration/prediction models were created for analytes of interest using off-line ref. measurements and specific spectral regions. Good fits were obtained between off-line measurements and those predicted by in situ mid-IR. Std. errors of prediction (SEP) for glucose (range 18-0.1 g/L) and phosphate (range 11-7.5 g/L) were 0.16 and 1.8 g/L resp. with mean percentage errors (MPEs) around 2.5%. SEP values for the prodn. process: fructose (range 20-0.1 g/L), glutamate (8-0.5 g/L), and proline (12-0.5 g/L) were 0.44, 0.6, and 0.5 g/L resp. with MPEs of 2.2, 5.3, and 10.1%. The technol. effectively demonstrates quant. multicomponent anal. of fermn. processes using in situ monitoring.
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    17. 17
      Kornmann, H.; Rhiel, M.; Cannizzaro, C.; Marison, I.; von Stockar, U. Biotechnol. Bioeng. 2003, 82, 702– 709
      There is no corresponding record for this reference.
    18. 18
      Kornmann, H.; Valentinotti, S.; Duboc, P.; Marison, I.; von Stockar, U. J. Biotechnol. 2004, 113, 231– 245
      18
      Monitoring and control of Gluconacetobacter xylinus fed-batch cultures using in situ mid-IR spectroscopy
      Kornmann, Henri; Valentinotti, Sergio; Duboc, Philippe; Marison, Ian; von Stockar, Urs
      Journal of Biotechnology (2004), 113 (1-3), 231-245CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)
      A partial least-squares calibration model, relating mid-IR spectral features with fructose, ethanol, acetate, gluconacetan, phosphate and ammonium concns. has been designed to monitor and control cultivations of Gluconacetobacter xylinus and prodn. of gluconacetan, a food grade exopolysaccharide (EPS). Only synthetic solns. contg. a mixt. of the major components of culture media have been used to calibrate the spectrometer. A factorial design has been applied to det. the compn. and concn. in the calibration matrix. This approach guarantees a complete and intelligent scan of the calibration space using only 55 stds. This calibration model allowed std. errors of validation (SEV) for fructose, ethanol, acetate, gluconacetan, ammonium and phosphate concns. of 1.16 g/l, 0.36 g/l, 0.22 g/l, 1.54 g/l, 0.24 g/l and 0.18 g/l, resp. With G. xylinus, ethanol is directly oxidized to acetate, which is subsequently metabolized to form biomass. However, residual ethanol in the culture medium prevents bacterial growth. Online spectroscopic data were implemented in a closed-loop control strategy for fed-batch fermn. Acetate concn. was controlled at a const. value by feeding ethanol into the bioreactor. The designed fed-batch process allowed biomass prodn. on ethanol. This was not possible in a batch process due to ethanol inhibition of bacterial growth. In this way, the productivity of gluconacetan was increased from 1.8 × 10-3 [C-mol/C-mol substrate/h] in the batch process to 2.9 × 10-3 [C-mol/C-mol substrate/h] in the fed-batch process described in this study.
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    19. 19
      Dahlbacka, J.; Weegar, J.; von Weymarn, N.; Fagervik, K. Biotechnol. Lett. 2012, 34, 1009– 1017
      There is no corresponding record for this reference.
    20. 20
      Koch, C.; Posch, A. E.; Goicoechea, H. C.; Herwig, C.; Lendl, B. Anal. Chim. Acta 2014, 807, 103– 110
      There is no corresponding record for this reference.
    21. 21
      Kansiz, M.; Billman-Jacobe, H.; McNaughton, D. Appl. Environ. Microbiol. 2000, 66, 3415– 3420
      There is no corresponding record for this reference.
    22. 22
      Stehfest, K.; Toepel, J.; Wilhelm, C. Plant Physiol. Biochem. 2005, 43, 717– 726
      22
      The application of micro-FTIR spectroscopy to analyze nutrient stress-related changes in biomass composition of phytoplankton algae
      Stehfest, Katja; Toepel, Joerg; Wilhelm, Christian
      Plant Physiology and Biochemistry (Amsterdam, Netherlands) (2005), 43 (7), 717-726CODEN: PPBIEX; ISSN:0981-9428. (Elsevier B.V.)
      Micro-Fourier transform IR (FTIR) spectroscopy was used to study changes in spectral features of 3 species of Cyanobacteria (Microcystis aeruginosa, Chroococcus minutus, and Nostoc sp.) and 2 Bacillariophyceae (Cyclotella meneghiniana, and Phaeodactylum tricornutum) in response to nutrient stress. The change of physiol. state of the cells was followed during a 4-wk starvation period on the basis of physiol. key parameters and by means of FTIR spectroscopy. Changes in the integrated FTIR bands of cell spectra assigned to proteins, lipids, carbohydrates and silicate were used to calc. relative biomass compn. The results show that short-term acclimatization becomes visible at first in pigmentation and photosynthetic efficiency, whereas changes in biomass compn. reflect long term modulation in the metab. Simultaneous monitoring of short term and long term stress acclimatization showed evidence that the metabolic strategies to cope with increasing nutrient limitation are highly species-specific. This species-specificity can only be resolved in natural phytoplankton samples by single cell techniques. The results show that the FTIR technique has the potential to become applicable for the detn. of single cell biomass compn. from natural phytoplankton communities.
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    23. 23
      Dean, A. P.; Sigee, D. C.; Estrada, B.; Pittman, J. K. Bioresour. Technol. 2010, 101, 4499– 4507
      23
      Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae
      Dean, Andrew P.; Sigee, David C.; Estrada, Beatriz; Pittman, Jon K.
      Bioresource Technology (2010), 101 (12), 4499-4507CODEN: BIRTEB; ISSN:0960-8524. (Elsevier Ltd.)
      In this study Fourier transform IR micro-spectroscopy (FTIR) was used to det. lipid and carbohydrate content over time in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus subspicatus grown in batch culture in limiting concns. of nitrogen (N). Both algae exhibited restricted cell division and increased cell size following N-limitation. FTIR spectra of cells in N-limited media showed increasing lipid:amide I and carbohydrate:amide I ratios over time. The use of lipid- and starch-staining dyes confirmed that the obsd. ratio changes were due to increased lipid and carbohydrate synthesis. These results demonstrate rapid metabolic responses of C. reinhardtii and S. subspicatus to changing nutrient availability, and indicate the efficiency of FTIR as a reliable method for high-throughput detn. of lipid induction.
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    24. 24
      Wagner, H.; Liu, Z.; Langner, U.; Stehfest, K.; Wilhelm, C. J. Biophotonics 2010, 3, 557– 566
      24
      The use of FTIR spectroscopy to assess quantitative changes in the biochemical composition of microalgae
      Wagner, Heiko; Liu, Zhixin; Langner, Uwe; Stehfest, Katja; Wilhelm, Christian
      Journal of Biophotonics (2010), 3 (8-9), 557-566CODEN: JBOIBX; ISSN:1864-063X. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A mid-IR spectroscopic method was developed for the simultaneous and quant. detn. of total protein, carbohydrate and lipid contents of microalgal cells. Based on a chemometric approach, measured FTIR (Fourier transform IR) spectra from algal cells were reconstructed by a partial least square algorithm, using the spectra of the ref. substances to det. their relative contribution to the overall cell spectrum. From this specific absorption, abs. macromol. cell compn. [pg cell-1] can be calcd. using calibration curves, which have been validated by independent biochem. methods. The future potential of this method for photosynthesis research is shown by its application to follow time-resolved changes in the cellular compn. of microalgae during an illumination period of several hours. We show how the macromol. compn. can be investigated by FTIR spectroscopy methods. This can substantially increase the efficiency of screening processes like bioreactor monitoring and may be beneficial in metabolic engineering of algal cells.
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    25. 25
      Winder, C. L.; Cornmell, R.; Schuler, S.; Jarvis, R. M.; Stephens, G. M.; Goodacre, R. Anal. Bioanal. Chem. 2011, 399, 387– 401
      25
      Metabolic fingerprinting as a tool to monitor whole-cell biotransformations
      Winder, Catherine L.; Cornmell, Robert; Schuler, Stephanie; Jarvis, Roger M.; Stephens, Gill M.; Goodacre, Royston
      Analytical and Bioanalytical Chemistry (2011), 399 (1), 387-401CODEN: ABCNBP; ISSN:1618-2642. (Springer)
      Fourier transform IR (FT-IR) spectroscopy was employed as a rapid high-throughput phenotypic typing technique to generate metabolic fingerprints of Escherichia coli MG1655 pDTG601A growing in fed-batch culture, during the dioxygenase-catalyzed biotransformation of toluene to toluene cis-glycol. With toluene fed as a vapor, the final toluene cis-glycol concn. was 83 mM, whereas the product concn. was only 22 mM when the culture was supplied with liq. toluene. Multivariate statistical anal. employing cluster anal. was used to analyze the dynamic changes in the data. The anal. revealed distinct trends and trajectories in cluster ordination space, illustrating phenotypic changes related to differences in the growth and product formation of the cultures. In addn., partial least squares regression was used to correlate the FT-IR metabolic fingerprints with the levels of toluene cis-glycol and acetate, the latter being an indicator of metabolic stress. The authors propose that this high-throughput metabolic fingerprinting approach is an ideal tool to assess temporal biochem. dynamics in complex biol. processes, as demonstrated by this redox biotransformation. Moreover, this approach can also give useful information on product yields and fermn. health indicators directly from the fermn. broth without the need for lengthy chromatog. anal. of the products.
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    26. 26
      Scholz, T.; Lopes, V. V.; Calado, C. R. C. Biotechnol. Bioeng. 2012, 109, 2279– 2285
      There is no corresponding record for this reference.
    27. 27
      Corte, L.; Rellini, P.; Roscini, L.; Fatichenti, F.; Cardinali, G. Anal. Chim. Acta 2010, 659, 258– 265
      There is no corresponding record for this reference.
    28. 28
      Burattini, E.; Cavagna, M.; Dell’Anna, R.; Malvezzi Campeggi, F.; Monti, F.; Rossi, F.; Torriani, S. Vib. Spectrosc. 2008, 47, 139– 147
      28
      A FTIR microspectroscopy study of autolysis in cells of the wine yeast Saccharomyces cerevisiae
      Burattini, E.; Cavagna, M.; Dell'Anna, R.; Malvezzi Campeggi, F.; Monti, F.; Rossi, F.; Torriani, S.
      Vibrational Spectroscopy (2008), 47 (2), 139-147CODEN: VISPEK; ISSN:0924-2031. (Elsevier B.V.)
      The present paper reports the first application of FTIR microspectroscopy in the mid-IR range to study the major biochem. changes assocd. with autolysis in yeast cells. Measurements were done both in transmission and in attenuated total reflection (ATR) mode on cells of Saccharomyces cerevisiae strain EC1118 before and after induction of the autolytic process in a model wine medium and in a Chardonnay base wine. Unsupervised multivariate statistical anal. (Hierarchical Cluster Anal. and Principal Component Anal.), as well as accurate spectral anal. based on curve fitting, were applied to the acquired spectra. The spectral behavior of S. cerevisiae in the model and base wines was found to be the same. A detailed interpretation of absorption bands was given by ref. to the literature and through comparison of transmission and ATR spectra. It was shown that FTIR microspectroscopy is a rapid and accurate tool to simultaneously probe the major biochem. events assocd. with the autolytic process. Moreover, the intrinsically higher sensitivity of ATR with respect to transmission spectra in analyzing autolysis was also demonstrated.
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    29. 29
      Cavagna, M.; Dell’Anna, R.; Monti, F.; Rossi, F.; Torriani, S. J. Agric. Food Chem. 2010, 58, 39– 45
      There is no corresponding record for this reference.
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      Kuligowski, J.; Quintás, G.; Herwig, C.; Lendl, B. Talanta 2012, 99, 566– 573
      There is no corresponding record for this reference.
    31. 31
      Plata, M. R.; Koch, C.; Wechselberger, P.; Herwig, C.; Lendl, B. Anal. Bioanal. Chem. 2013, 405, 8241– 8250
      31
      Determination of carbohydrates present in Saccharomyces cerevisiae using mid-infrared spectroscopy and partial least squares regression
      Plata, Maria R.; Koch, Cosima; Wechselberger, Patrick; Herwig, Christoph; Lendl, Bernhard
      Analytical and Bioanalytical Chemistry (2013), 405 (25), 8241-8250CODEN: ABCNBP; ISSN:1618-2642. (Springer)
      A fast and simple method to control variations in carbohydrate compn. of Saccharomyces cerevisiae, baker's yeast, during fermn. was developed using mid-IR (mid-IR) spectroscopy. The method allows for precise and accurate detns. with minimal or no sample prepn. and reagent consumption based on mid-IR spectra and partial least squares (PLS) regression. The PLS models were developed employing the results from ref. anal. of the yeast cells. The ref. analyses quantify the amt. of trehalose, glucose, glycogen, and mannan in S. cerevisiae. The selection and optimization of pretreatment steps of samples such as the disruption of the yeast cells and the hydrolysis of mannan and glycogen to obtain monosaccharides were carried out. Trehalose, glucose, and mannose were detd. using high-performance liq. chromatog. coupled with a refractive index detector and total carbohydrates were measured using the phenol-sulfuric method. Linear concn. range, accuracy, precision, LOD and LOQ were examd. to check the reliability of the chromatog. method for each analyte.
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    32. 32
      Jarute, G.; Kainz, A.; Schroll, G.; Baena, J. R.; Lendl, B. Anal. Chem. 2004, 76, 6353– 6358
      There is no corresponding record for this reference.
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      King, L. V. Proc. R. Soc. London, Ser. A 1934, 147, 212– 240
      There is no corresponding record for this reference.
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      Yosioka, K.; Kawasima, Y. Acustica 1955, 5, 167– 173
      There is no corresponding record for this reference.
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      Gor’kov, L. P. Phys.–Dokl. 1962, 6, 773– 775
      There is no corresponding record for this reference.
    36. 36
      Radel, S.; McLoughlin, A. J.; Gherardini, L.; Doblhoff-Dier, O.; Benes, E. Ultrasonics 2000, 38, 633– 637
      There is no corresponding record for this reference.
    37. 37
      Wiklund, M. Lab Chip 2012, 12, 2018– 2028
      37
      Acoustofluidics 12: Biocompatibility and cell viability in microfluidic acoustic resonators
      Wiklund, Martin
      Lab on a Chip (2012), 12 (11), 2018-2028CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
      A review. Manipulation of biol. cells by acoustic radiation forces is often motivated by its improved biocompatibility relative to alternative available methods. On the other hand, it is well known that acoustic exposure is capable of causing damage to tissue or cells, primarily due to heating or cavitation effects. Therefore, it is important to define safety guidelines for the design and operation of the utilized devices. This tutorial discusses the biocompatibility of devices designed for acoustic manipulation of mammalian cells, and different methods for quantifying the cell viability in such devices.
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    38. 38
      Trampler, F.; Sonderhoff, S. A.; Pui, P. W. S.; Kilburn, D. G.; Piret, J. M. Nat. Biotechnol. 1994, 12, 281– 284
      There is no corresponding record for this reference.
    39. 39
      Gorenflo, V. M.; Smith, L.; Dedinsky, B.; Persson, B.; Piret, J. M. Biotechnol. Bioeng. 2002, 80, 438– 444
      There is no corresponding record for this reference.
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      Gorenflo, V. M.; Ritter, J. B.; Aeschliman, D. S.; Drouin, H.; Bowen, B. D.; Piret, J. M. Biotechnol. Bioeng. 2005, 90, 746– 753
      There is no corresponding record for this reference.
    41. 41
      Radel, S.; Schnöller, J.; Gröschl, M.; Benes, E.; Lendl, B. IEEE Sens. J. 2010, 10, 1615– 1622
      41
      On chemical and ultrasonic strategies to improve a portable FT-IR ATR process analyzer for online fermentation monitoring
      Radel, Stefan; Schnoeller, Johannes; Groeschl, Martin; Benes, Ewald; Lendl, Bernhard
      IEEE Sensors Journal (2010), 10 (10), 1615-1622CODEN: ISJEAZ; ISSN:1530-437X. (Institute of Electrical and Electronics Engineers)
      A setup for online fermn. monitoring was tested with suspensions contg. yeast cells. A flow cell was equipped with a horizontal attenuated total reflection (ATR) unit for measurements of mid-IR spectra. The stopped flow principle was employed to sep. assess the liq. and the dispersed cells: dissolved components in the supernatant can be assessed while pumping the fermn. broth through the flow cell. Upon stopping the flow the culture can be monitored as the microorganisms settle onto the horizontal ATR diamond. Due to the surface sensitivity of ATR spectroscopy cleanness of the optical element is of particular importance. For yeast fermns. the formation of biofilms on the ATR surface was identified as limitation in regard to long-term stability. Initial expts. showed that the effective removal of residues was impossible by rinsing with water or a NaHCO3 soln. Therefore, various cleaning agents (2%) have been tested for their ability to clear off the biofilm. The problem of biofilm formation was addnl. addressed by the exploitation of forces exerted on suspended particles within an ultrasonic standing wave (USW). The USW (∼2 MHz) was built up between the ATR element and an ultrasonic transducer facing it. This technique of ultrasonic particle manipulation was applied to actively lift the sedimented material from the ATR after the IR measurement, thus have the rinsing stream carry it away more effectively. Among the studied reagents surfactants and oxidizing agents showed to be most effective, the use of an USW was found to be feasible to remove a biofilm.
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    42. 42
      Lendl, B.; Radel, S.; Brandstetter, M. Device for FTIR Absorption Spectroscopy. PCT/AT2010/000006, 2010.
      There is no corresponding record for this reference.
    43. 43
      Radel, S.; Brandstetter, M.; Lendl, B. Ultrasonics 2010, 50, 240– 246
      There is no corresponding record for this reference.
    44. 44
      Koch, C.; Brandstetter, M.; Lendl, B.; Radel, S. Ultrasound Med. Biol. 2013, 39, 1094– 1101
      44
      Ultrasonic manipulation of yeast cells in suspension for absorption spectroscopy with an immersible mid-infrared fiberoptic probe
      Koch Cosima; Brandstetter Markus; Lendl Bernhard; Radel Stefan
      Ultrasound in medicine & biology (2013), 39 (6), 1094-101 ISSN:.
      Recent advances in combining ultrasonic particle manipulation with attenuated total reflection infrared spectroscopy of yeast suspensions are presented. Infrared spectroscopy provides highly specific molecular information about the sample. It has not been applicable to in-line monitoring of cells during fermentation, however, because positioning cells in the micron-thin measurement region of the attenuated total reflection probe was not possible. Ultrasonic radiation forces exerted on suspended particles by an ultrasonic standing wave can result in the buildup of agglomerates in the nodal planes, hence enabling the manipulation of suspended cells on the microscopic scale. When a chamber setup and a prototype in-line applicable probe were used, successful control over the position of the yeast cells relative to the attenuated total reflection sensor surface could be proven. Both rate of increase and maximum mid-infrared absorption of yeast-specific bands during application of a pushing frequency (chamber setup: 1.863 MHz, in-line probe: 1.990 MHz) were found to correlate with yeast cell concentration.
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      Microbiological characterizations by FT-IR spectroscopy
      Naumann D; Helm D; Labischinski H
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      Infrared signals of microorganisms are highly specific fingerprint-like patterns that can be used for probing the identity of microorganisms. The simplicity and versatility of Fourier-transform infrared spectroscopy (FT-IR) makes it a versatile technique for rapid differentiation, classification, identification and large-scale screening at the subspecies level.
      >> More from SciFinder ®
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      Gherardini, L.; Radel, S.; Sielemann, S.; Doblhoff-Dier, O.; Gröschl, M.; Benes, E.; McLoughlin, A. Bioseparation 2001, 10, 153– 162
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      A study of the spatial organisation of microbial cells in a gel matrix subjected to treatment with ultrasound standing waves
      Gherardini L; Radel S; Sielemann S; Doblhoff-Dier O; Groschl M; Benes E; McLoughlin A J
      Bioseparation (2001), 10 (4-5), 153-62 ISSN:0923-179X.
      Retention and manipulation of microbial cells through exploitation of ultrasonic forces has been reported as a novel cell immobilisation technique. The spatial ordering of yeast cells, within suspensions subjected to an ultrasonic standing wave field, was analysed for the first time. A technique, based on 'freezing' the spatial arrangement using polymer gelation was developed. The resultant gel was then sectioned and examined using microscopic techniques. Light Microscopy confirmed the presence of specific regions in the ultrasonic field, where the cells are organised into bands corresponding to the standing waves' pressure nodal planes. Computer Image Analysis measurement of several physical parameters associated with this cell distribution matched the values derived from the theoretical model. The spatial cell-cell re-arrangement within each band and uneven distribution along the nodal planes have been analysed by Scanning Electron Microscopy. These results complement the ongoing study of the process of immobilisation of microbial cells by ultrasound standing waves.
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  • Supporting Information

    Supporting Information

    Experimental details on media preparation and cultivation conditions. Table S1: Overview of the setup and conditions of the fed-batch fermentations. Figure S1: Spectra of S. cerevisiae acquired in-line during batch phase fermentation. This material is available free of charge via the Internet at http://pubs.acs.org.


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