Stefan Radel

Ultrasonic separation of suspended particles

The forces on suspended particles in acoustic fields are reviewed briefly and the theoretical modelling of ultrasonic separators based on piezoelectrically excited layered resonators is described. Two flow-through resonator chamber concepts for ultrasonic particle (bio-cell) separation are investigated: (a) the coagulation or sedimentation approach, (b) the so-called h-shaped ultrasonic separator. The h-shaped ultrasonic separator is analysed by combining for the first time the mathematical modelling of the laminar flow with the acoustic force based velocity field of the particles relative to the suspension medium. This allows a complete modelling of the resonators particle separation performance. Examples for separation chamber designs optimized by use of the mathematical model are presented and the calculated particle traces in the h-resonator are shown and compared with experimental results. For direct comparison of different ultrasonic flow through separator concepts a separation performance figure is introduced and its value is given for the two investigated separator concepts for the sample suspensions of polystyrene spheres, yeast and spirulina cells in (salt) water. The presented results are of importance for the state of the art design of acoustic cell filters for perfusion type bioreactors, as recently launched at the biotechnology market, as well as for the ultrasonic separation of plant (algae) cells under low gravity conditions, where the sedimentation concept fails.

Forthcoming Lab on a Chip tutorial series on acoustofluidics: Acoustofluidics—exploiting ultrasonic standing wave forces and acoustic streaming in microfluidic systems for cell and particle manipulation

Forthcoming lab on a chip tutorial series on acoustofluidics : Acoustofluidics - Exploiting ultrasonic standing wave forces and acoustic streaming in microfluidic systems for cell and particle manipulation

Observation of particles manipulated by ultrasound in close proximity to a cone-shaped infrared spectroscopy probe

The presented investigations aimed to enhance surface sensitive infrared spectroscopy for chemical analysis by ultrasonic particle manipulation. The combination of these techniques has the potential for new measurement concepts for use in the chemical analysis of suspensions. Local increases of particle concentration brought about by ultrasound could facilitate measurements of molecular-specific infrared spectra of the suspending phase and particles independently. By changing the frequency of an ultrasonic standing wave around 2 MHz it was possible to control the position of particles in respect to the optically sensitive region of the infrared spectroscope. Results obtained with a set-up that enabled us to explore the application of an ultrasonic standing wave to push suspended particles at or into mum distances of the sensing element of an in-line fiber optic probe and subsequently retract them from there are presented. Light micrographs suggested, that the task was successfully accomplished with polystyrene beads suspended in methanol, aggregates were manipulated to and from the cut surface of the truncated, cone-shaped fibre probe tip by changes of the ultrasonic frequency between 1.85 and 1.87 MHz. Feasibility was confirmed by infrared absorption spectra recorded when PTFE particles suspended in tetrahydrofuran were used.

Breakdown of immobilisation/separation and morphology changes of yeast suspended in water-rich ethanol mixtures exposed to ultrasonic plane standing waves

Some physiological/morphological changes have been reported before, when suspended yeasts have been irradiated with well-defined ultrasonic standing, as well as propagating, plane waves around 2.2 MHz, as used in ultrasonic coagulation, e.g., for cell filtering. Thus we used yeast as a biological model to explore the reasons for both those morphology changes and some unusual macroscopic behaviour in the case of water-rich ethanol mixtures when used as carrier liquid. When the cells were suspended in 12% (v/v) ethanol–water mixture separation was greatly reduced; the yeast cells were not retained in the pressure nodal planes of the standing wave, but mixed turbulently through the separation system. How this behaviour alters the efficiency of retention/immobilisation was measured. As the viability of the yeast was decreased as well the morphology of the cells was examined using transmission electron microscopy. Two effects, according to the type of assessment, were evident; a disruption of the cells vacuole and also damage to the cell wall/membrane complex. The extent of the alterations in vacuole structure with sonication time, utilising a fluorescent vacuole membrane dye, was measured. Transient cavitation was not detected and thus could be excluded as being responsible for the observed effects. Other possible reasons for the disruption of the intracellular compartments may be acoustic pressure, displacement or other, secondary effects like (sub) harmonic cavitation. The investigations contribute to a better understanding of the physical conditions experienced when a cell is stressed in a high-frequency ultrasonic wave in the MHz range.

Ultrasound-Enhanced Attenuated Total Reflection Mid-infrared Spectroscopy In-Line Probe: Acquisition of Cell Spectra in a Bioreactor

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.

Ultrasonic standing wave accelerates on-line measurement and prevents coating of a FTIR ATR flow cell

It has been shown elsewhere that infrared (IR) spectroscopy can be successfully employed for the on-line monitoring of bio-processes. A horizontal attenuated total reflection (ATR) unit connected to a portable IR-cube was used here to measure the IR absorption spectra of supernatant and microorganisms separately. The common problem of bio-film formation on the ATR was addressed before, e.g. by chemical means. We present a novel method employing the principles of ultrasonic particle manipulation to avoid and potentially remove this coating brought about by the use of fermentation broth. A novel flow cell for a horizontal ATR was developed that decreases measurement time and the undesired formation of bio-films on the ATR surface. An ultrasonic standing wave (/spl sim/2 MHz) is built up between a horizontal transducer and the ATR crystal. Yeast cells in suspension were agglomerated within certain regions by the ultrasound field and therefore settled about 3-4 times faster on the ATR when the field was switched off compared to the slow sedimentation of freely dispersed cells. After the IR spectrum had been measured, the same sound field was used to actively lift the settled material from the optical sensitive surface which therefore could be rinsed away more effectively.

Ultrasonic particle manipulation exploited in on-line infrared spectroscopy of (cell) suspensions

SummaryAn ultrasonic standing wave field of 2 MHz was used to manipulate yeast cells in an on-line flow cell for Fourier transformed mid-infrared spectroscopy (FT-IR) purposes. Results show, that a significant decrease of the undesired formation of biofilms on the attenuated total reflection (ATR) surface was achieved. In addition the time resolution was more than doubled.ZusammenfassungEin 2-MHz-Ultraschallstehwellenfeld wurde verwendet, um Hefezellen in einer Online-Durchflusszelle zu Zwecken der Fourier-transformierten Infrarotspektroskopie (FT-IR) anzuordnen. Die Resultate zeigen, dass eine signifikante Reduktion unerwünschten Biofilms auf der totalreflektierenden (ATR) Oberfläche erreicht wurde. Darüber hinaus konnte die Zeitauflösung mehr als verdoppelt werden.

On Chemical and Ultrasonic Strategies to Improve a Portable FT-IR ATR Process Analyzer for Online Fermentation Monitoring

A setup for online fermentation monitoring was tested with suspensions containing yeast cells. A flow cell was equipped with a horizontal attenuated total reflection (ATR) unit for measurements of mid-infrared spectra. The stopped flow principle was employed to separately assess the liquid and the dispersed cells: dissolved components in the supernatant can be assessed while pumping the fermentation 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 fermentations the formation of biofilms on the ATR surface was identified as limitation in regard to long-term stability. Initial experiments showed that the effective removal of residues was impossible by rinsing with water or a NaHCO3 solution. Therefore, various cleaning agents (2%) have been tested for their ability to clear off the biofilm. The problem of biofilm formation was additionally 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 infrared 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.

Influence of biomass, throughput and true electric power input on the separation efficiency of a 60 mL acoustic filter

ZusammenfassungEin seriennahes Separationssystem wurde verwendet, um den Einfluss bestimmter Prozessparameter auf die Separationsleistung eines auf dem Ultraschall Enhanced Settling-Prinzip basierenden akustischen Filters zu ermitteln. Messungen der Zellkonzentration der ersten 80 mL Hefesuspension, die das System verließen, wurden bei verschiedenen Durchflussraten und verschiedenen elektrischen Wirkleistungseinträgen durchgeführt. Darüber hinaus wurde die anfängliche Zellkonzentration über zwei Größenordungen variiert. Die Resultate legen einen Maximalpegel der Wirkleistung, über dem keine Verbesserung der Separationseffizienz mehr stattfindet, sowie eine notwendige Minimalzellkonzentration unterhalb der prinzipiell zu wenig Zellen verfügbar sind, nahe.SummaryA close-to-production separation system was used to assess the influence of certain process parameters on the separation performance of an acoustic filter based on the Ultrasonically Enhanced Settling technique. Measurements of the cell concentration of the first 80 mL of yeast suspension that left the filter were conducted at various throughputs and true electrical power inputs. Moreover, the number of cells/mL was ranging over two orders of magnitude. Results delivered an upper limit for the true electrical power input from which an increase does not improve the separation efficiency and a lower threshold of cells/mL below which too less cells were available for the principle to work.

Raman spectroscopy of particles in suspension concentrated by an ultrasonic standing wave

SummaryAn ultrasonic standing wave field of 2MHz was used to manipulate yeast cells and theophylline crystals in suspension to control the local concentration of solid material in the focus of a Raman spectroscope to improve the signal quality of such measurements for suspensions. In case of agglomerated yeast cells the signal-to-noise ratio was significantly increased in comparison to Raman spectra of the suspension. The spectra corresponded well with yeast dried on glass used as reference. Measurements of theophylline suspensions confirmed these results. Furthermore, it was possible to show the absence of theophylline crystals between the pressure nodal planes of the ultrasonic standing wave. This opens the possibility to specifically measure the solid fraction and the pure liquid of a suspension almost simultaneously by only changing the optical focus of the Raman microscope in respect to the location of the ultrasonic standing waves nodes.ZusammenfassungEin 2-MHz-Ultraschallstehwellenfeld wurde verwendet, um suspendierte Hefezellen und Theophylline Kristalle im optischen Fokus eines Raman-Spektroskops zu konzentrieren, mit dem Ziel, die Signalqualität solcher Messungen für Suspensionen zu verbessern. Das Signal-Rausch-Verhältnis konnte für die Raman-Spektren von agglomerierten Hefezellen im Vergleich zu Hefesuspensionen signifikant verbessert werden. Die Spektren zeigten auch eine hohe Übereinstimmung mit Referenzspektren von auf Glas getrockneten Zellen. Diese Resultate bestätigten sich bei Messungen mit Theophylline Suspensionen. Hier war es zudem möglich, die Abwesenheit von Theophylline Kristallen zwischen den Druckknoten der Ultraschallstehwelle zu zeigen. Dies eröffnet die Möglichkeit, die Partikel und die reine Flüssigkeit einer Suspension beinahe gleichzeitig spezifisch zu messen, indem man den optischen Fokus des Raman-Mikroskops relativ zum Ort der Knoten des Ultraschallstehwellenfeldes verschiebt.