Study of Saccharomyces Cerevisiae Yeast Cells by Field Flow Fractionation and Image Analysis

Copyright © IFAC Computer Applications in Biotechnology. Gannisch-Partenkirchen. Gennany. 1995

STUDY OF SACCHAROMYCES CEREVISIAE YEAST CELLS BY FIELD FLOW FRACTIONATION AND IMAGE ANALYSIS A. Litzen a .c, G.M. Kresbach a , M.N. Pons bl , M. Ehrat a , H. Vivierb

2Ciba-Geigy Ltd. Corporate Analytical Research CH-4002 Basel. Switzerland bLaboratoire des Sciences du Genie Chimique. CNRS-ENSIC-INPL 1. rue Grandville BP 451. F-54001 Nancy Cedex. France cPresent address: Astra-Hassle. S-43183 Molndal. Sweden

Abstract: Asymmetrical flow FFF has been applied to characterize Saccharomyces cerevisiae cells. the morphology of which has been assessed by image analysis. during fermentation on glucose medium. Full cells. budding or non-budding. are eluted according to the sterichyperlayer modes. while macromolecules and other cell debris are eluted according to the normal mode. Keywords : Field flow fraction nation - Image analysis - Yeast cells - Fermentation monitoring - Cell lysis

I. INTRODUCTION

Field-flow fractionation (FFF) is a family of separation techniques suitable for the fractionation of molecules and particles in the size range from about 1000 in molecular weight to 50 ~m in particle diameter. The FFF methods are based on the concept of a laminar flow profile in a channel through which a carrier liquid is pumped . A field is applied perpendicular to the channel and interacts with the sample components. depending upon their characteristics. In asymmetrical flow FFF. the retaining force is created by a crossflow. perpendicular to the axial flow. through the porous lower wall of a thin flat channel. The opposite upper wall is made out of a glass plate (Fig. la and b). The selectivity is based on differences in the diffusion coefficient and the samples are separated according to size and shape. Depending upon the particle size. two different phenomena take place in the chamber: the Brownian normal mode applies to the smaller « about 1 ~m) 1 Corresponding author

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particles. and the steric-hyperlayer mode to the larger particles. The normal mode (Fig. I a) is based on the balance reached between crossflow induced convection. driving particles or molecules towards the accumulation wall and molecular diffusion on the opposite direction . Under this mode the sample components elute in order of decreasing diffusion coefficient. i.e. the smallest components elute first. The steric-hyperlayer effect (Fig. 1b) is taking place when the average distance from the accumulation wall. as predicted by the convection-diffusion balance. is exceeded by the particle radius . The particles are transported by the bulk flow : their velocity increases with their diameter. As a result. under this mode. the largest particles elute first. Samples containing a wide range of particle sizes may be subject to both mechanisms. Flow FFF has been used to separate biomaterials of very different nature. such as proteins (Giddings et al.. 1992). viruses (Giddings et al.. 1977). antibodies (Litzen et al .• 1993). red blood cells (Barman et al .• 1993). plasmids or unicellular algae (Wahlund and Liaen. 1989). In the latter case. fractograms of mixed

populations were obtained, but the identity of the microorganisms in the different peaks could not be fully determined. (0.)

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Matrox board on a Datacube 486-2x33MHz computer. Visilogn.! 4.1.3 . (Noesis, Orsay, France) image analysis software was used under Windows. Additional software was written in C. Images were intermediately stored on tapes (VIP Archive) for later analysis . A graticule was used for pixel size calibration . Image analysis of yeast cells has been previously described (Pons et al.. 1993). Its purpose is to compute the number of cells (single, double cells. agglomerate). their size (equivalent diameter Deq A/1t where A is the projected area) and their shape. A macroscopic shape descriptor, circularity p2/( 41tA). was used.

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Figure 1: Principle of FFF: a: Normal mode, b : Steric-hyperlayer mode The purpose of this contribution is to examine whether asymmetrical flow FFF is suitable to characterize Saccharomyces cerevisiae cells (diameter == 10 Ilm) during fermentations on glucose, using optical microscopy and image analysis to elucidate the obtained fractograrns . 2. MATERIALS AND METHODS

2.1. Culture conditions A wild strain of Saccharomyces cerevlszae LBGH I 022 was obtained from Botanisches Institut, Basel University and stored in liquid nitrogen in cryovials . Cells were cultivated in a 250 mL Erlenmeyer flask, containing 100 mL of culture medium (medium D 20/c according to Hug et al. (1974) and equipped with a gauze stopper and placed on a shaker under a thermostated hood. Initial glucose concentration was 12 .5 gIL . Samples were taken regularly. Optical density was measured on a Lambda2 spectrophotometer (Perkin-Elmer. Uberlingen, Germany). Glucose, acetaldehyde and acetic acid were determined by enzymatic assays (refs 716 251, 668 613 and 148 261 respectively from Boehringer Mannheim, Mannheim, Germany). Cells debris were obtained by vigorous sonication of full cells (Branson Sonifier 250, Branson, Danbury, C. USA). Viability was checked by methylene blue exclusion and cell counting.

2.2./mage analysis Cells, from full broth and FFF eluate fractions (those were concentrated by centrifugation before examination), were observed by phase contrast light microscopy (Zeiss Labor Mikroskop, Zeiss, Oberkochen, Germany) (magnification x 100). A monochrome CCTV Hitachi video camera was fitted to the microscope. Images (256 lines of 256 pixels) on 256 grey levels were grabbed via a PIPlO24

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An asymmetrical flow FFF channel was constructed in house. It was similar to that described by Litzen et al. (1993) . The separation channel has a rectangular geometry with a length of 28 .5 cm and a breadth of 1.5 cm. The ultrafiltration membrane making up the accumulation wall was cellulosic (type NADIR UFC-1O from Hoechst AG, Wiesbaden. Germany) . The experimental set-up was based on commercially available and slightly modified HPLC components and similar to that described in Litzen et al. (1993). A PBS pH 7.0 carrier was used for cell samples fractionation . For calibration polystyrene latex beads (Seradyn , Indianapolis, IN, USA) were fractionated uSing a (O.19c SDS + 0.029c NaN3) carrier. Cells fractograms were characterized by their mean time (T mean). mode time eT mode). variation coefficient (= standard deviatiOn/mean), kurtosis and skewness. time of maximal value in the range 0 .6 - 3 min after injection . 3. RESULTS In order to test the separation performance of the FFF channel. polystyrene latex beads were fractionated using baSIcally the same experimental conditions. The beads were easily separated and that the resolution increases with decreasing diameter. The order of elution was checked by separate experiments. Fig . 2a. presents the general fermentation kinetics, while the results of image analysis obtained on the full broth and the characteristics of FFF fractograms are given In Figs 2b. and 2c. respectively. Up to t = 20 hrs, the fractograms statistical characteristics (mean time , mode time and vanation coeffiCIent) are constant , with equal values for the mean and mode times . DUring that period, the fractions of cells considered as single (non-budding cells and mother cells exhibiting a small bud) and double (mother cells exhibiting a large bud) are also constant (659c and 30O/C respectively). It was found that the average diameter for all type of cells cells was almost identical and that the double cells were only slightly

bigger (Fig 2b). As flow-FFF separation is based on effective diameter. a good separation of single. double and triple cells cannot be expected. Glucose concentration is not limiting and almost no acetate or acetaldehyde are produced . After 20 hrs . the fractogram variation coefficient increases suddenly and the mean time becomes smaller than the mode time: this indicate a broadening of the fractograms . During that period. the double cells fraction decreases. indicating a decrease of growth. and production of acetate and acetaldehyde is noticeable. After 25 hrs. no glucose can be found in the broth. [a]

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Although the viability given by methylene blue exclusion ranged between 80% and 900/,. it became evident that there was a relationship between the appearance of the second peak at shon elution times. and the cells lysis. When the cells are dying. acetaldehyde and acetate are released in larger amounts in the broth. either due to a increase of membrane permeability or to complete membrane breakage . When the cell membrane is disrupted. membrane fragments and various organelles and macromolecules are released in the broth . To confirm thiS interpretation. a sample of fully growing cells was taken and the cells were submitted to sonication. until only cells debris could be seen under the microscope . The corresponding FFF fractogram produced with a single peak around 1.2 min. as the various components. of small size. are eluted according to the normal mode .

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hyperlayer mode. However both single and double cells are eluted in the same time and there is. at least under the operation conditions used here. no full separation between budding and non-budding cells. After 20 hrs. a second peak is noticeable on the fractograms at shoner elution times (= 1.2 min). No yeast cells could be detected on fractions taken at that moment but very small objects could be observed.

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Figure 2: a: General kinetics : • : optical density (a.u .) x 10; 0 : glucose (glL) x 10; + : acetaldehyde (mglL); 0 : acetate (mglL); A : ethanol (glL) x 10; b: Image analysis results : (0) % and (0) Deq single cells; (A) 0/, and (+) Deq double cells; (l1) % and (.) Deq triple cells; c : FFF results: . : variation coefficient. [) : T mean; + : T mode Fig . 3 gives a closer look of some of the fractograms obtained during fermentation L 7. Until 20 hrs they are characterized by a single. rather narrow peak (elution time range 1.75 - 2.6 min) . Examination of different peak fractions by image analysis indicate that larger cells are eluted first: the equivalent diameter decreases as the elution time increases. Therefore the cells are eluted according to the steric-

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The yeast fractogram mean times (T mean) were plotted versus the equivalent diameter derived from image analysis . Values obtained from polystyrenelatex particles (fractionated using a different carrier) were added to this plOL The data obtained for the yeast cells were in coarse correspondence with those expected for particles of the same size . Several factors could account for the deviations . Particle-membrane interaction is not totally negligible in flow FFF and the largely different surface characteristics of yeast cells and of polystyrene latex particles will have a strong impact on that interaction. In contrary to the spherical and monodisperse polystyrene latex particles. the cell populations exhibit a continuous distribution of shape and size factors: single and double cells show an average circularity of 1.15 and 1.3 respectively . 4. CONCLUSION Samples of full cultivation broth of Saccharomyces cerevisiae during different stages of growth on

glucose containign media were separated and analyzed using asymmetrical flow FFF and image analysis. Due to their size. intact cells are separated according to the steric hyperlayer mode; macromolecules expressed into the medium during later stages of cultivation as well as cellular debris are separated according to the normal mode. Interference of these two classes during the separation did not occur. A complete separation between single and double cells. however. could not be avhieved under the conditions chosen here . Image analysis data showed the continuous spread of shapes; the relatively low population of double cells as well as the minute differences between the diameter ranges of single and couble cells explain the apparently low resolution of the FFF separation under the chosen cultivation conditions. Although the method needs refinements to improve cell samples fractionation. asymmetrical flow FFF is a fast technique of reasonable cost (similar to HPLC) which could be used to monitor ceUlysis and separate different types of cells under near native conditions. which could permit their reculture later on.

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REFERENCES Bannan. B.N .. E.R . Ashwood and l .C. Giddings. (1993 ) Separation and size distribution of red blood cells of diverse size . shape and origin by flow hyperlayer field flo w fract ionation. Anal. Biochem .• 2 12 . 3542 . Giddmgs . l .C. . F.J . Yang and M.N. Myers (1977 ). Flow field -flow fractionation : New method for separating. purifYIng and characterizing the diffusivity of viruses . 1. Virol ogy . 21. 131-138 . Giddings . l .C. . M .A. Benincasa. M.K. Liu and P. Li . ( 1992). Separation of watersoluble synthetic and biological macromolecules by flow FFF . 1. Liq. Chromatog . . 15 (10). 1729- 1747 . Hug. H .. H.W . Blanch and A. Fiechter (1974 ). The functional role of lipids in hydrocarbon assimilation . Biotechnol. Bioeng .. 16. 965-985 . Litzen . A . . l .K . Waiter. H. Krischollek and K.G. Wahlung (1993 ). Separation and quantitation of monoclonal antibody aggregates by as ymmetrical flow field-flow fractionation and comparison to gel penneation chromatography . Anal. Biochem .. 212 . 469-480 Pons . M.N .. H. Vi vier. J.F. Remy and J.A. Dodds (1993 ) Morphology characterization of yeast by image anal ysIs dunng alcoholic fennentation . Biotechnol. Bioeng . . 42. 1352-1359. Wahlund. K.G . and A. Litzen (1989) Application of an asymmetrical flow field -flow fractionation channel to the separation and characterization of proteins. plasmids . plasmid fragments. polysaccharides and unicellular algae. 1. Chromatograph),. 461. 73-87 .

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