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Characterization of yeast cultivations by steric sedimentation field-flow fractionation
ANALYTICAL
BIOCHEMISTRY
206,
300-308
(19%)
Characterization of Yeast Cultivations by Steric Sedimentation Field-Flow Fractionation Sabrina Hoffstetter-Kuhn,l Thomas Rbsler,* Markus Ehrat, and H. Michael Widmer Corporate Analytical Research, Ciba-Geigy Ltd., Base& Switzerland; and *Amendstrasse 51, BerEin, Germany
Received
June
15,1992
The characterization and quantification of biomass is often time consuming and dependent on the cultivation media and gives no detailed information between cell size and shape and their productivity. By monitoring the bioprocess with steric sedimentation field-flow fractionation (Sd/StFFF) in combination with laser light scattering, not only cell growth, but also the variation of cell size and shape during the cultivation, can be observed. In this work, the feasibility of separating and characterizing cell populations by steric sedimentation field-flow fractionation is demonstrated by its application to three different yeast cultivation broths. For this purpose samples which were collected at different cultivation times were injected into an FFF system. Fractograms were obtained in less than 4 min. Due to the relatively high resolution of the method, a cell sample could be fractionated in several subpopulations differing in their size as well as in their number of buds. 0 1992 Academic Press. Inc.
The possibility of controlling bioprocesses is strongly limited by the lack of reliable methods for the direct measurement of biomass and cell growth rate. Routine methods for the control of bioprocesses are based on physical or chemical parameters such as turbidimetry, glucose consumption, gas phase composition, or cell products correlated to cell growth, from which it is possible to only indirectly estimate the properties of the biomass (1,2). The budding yeast Saccharomyces cerevisiae is an eucaryotic microorganism widely used as a model organism of the cell cycle and of interest for the biotechnological production of biomass and ethanol. The progress of recombinant DNA technology allowing the transformation of yeast cells and the expression of heterologous
1 To whom 300
correspondence
should
be addressed.
proteins in yeast cultures with very high yields (3) has increased industrial interest in yeast cultivations and, thus, in useful methods to directly monitor and control biomass and growth rate during the bioprocess. This means one must look for analytical methods which are related to a physical property of the cell itself, such as its size, mass, or shape. Most of the techniques dealing with the determination of these cell characteristics are based on light scattering as in flow cytometry (4) and Coulter counting (5), where cells are not actually separated according to their differences in size, but only counted. Steric sedimentation field-flow fractionation (Sd/ StFFF)2 offers a new and exciting alternative for the characterization of yeast cells during a bioprocess by providing information about the size distribution via a fast and simple procedure. Sd/StFFF was introduced by Giddings and Meyers in 1978 (6) as a particular form of field-flow fractionation (FFF), a family of separation methods based on elution from a thin channel in which the separation is induced and controlled by an external force field acting in a direction perpendicular to laminar flow in the channel (7-10). Depending on the FFF subtechnique, the force field can be either a gravitational field (sedimentation FFF), *a temperature gradient (thermal FFF), or a crossflow (flow FFF). Under the influence of the field, particles are driven uniformely toward one channel wall, the accumulation wall, until their accumulation is balanced by diffusion. The steady-state concentration profile, which is exponentially decreasing from the wall to the middle of the channel, has a characteristic thickness and depends on the diffusion coefficients and hence on the mass of the particles. This leads to different profile thicknesses for particles differing in size. After the field has been applied and the sample has been injected, the carrier flow
2 Abbreviations steric sedimentation
used: FFF, field-flow
field-flow fractionation;
fractionation LS, light
Sd/StFFF, scattering.
0003-2697192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
FRACTIONATION
CHARACTERIZATION
-------FIG. 1.
Schematic
drawing
of the separation
in a steric
FFF
chan-
nel.
is stopped for a chosen time period to allow relaxation of the particles. This so-called stopflow time varies from a few seconds to several minutes. When the carrier flow is then turned on again, smaller particles will elute first with the carrier flow, because they are less compressed to the accumulation wall than larger ones and thus occupy on average faster stream lines. For a detailed description of FFF theory see Ref. (11). The most widely used subtechnique so far is sedimentation FFF (SdFFF), where particles are separated according to their differences in mass and density (12). For this purpose the FFF channel is placed into a centrifuge. Steric FFF is the high limit in respect to the size of normal FFF which is described above. Steric operation mode is realized, when diffusion of the particles becomes negligible. This is normally the case for particles with diameters above 1 pm, which sediment very easily. Figure 1 schematically shows the separation procedure in a steric sedimentation FFF channel. The particles extend into the flow stream primarily because of their finite size. Layer thickness is now determined by the diameter of the particle rather than by diffusion. The particles move at a velocity approximately equal to the carrier-flow velocity at their center of gravity. Larger particles elute in’this mode more rapidly than smaller particles. The flow velocity in steric FFF should be high enough to avoid strong adsorption at the wall and to ensure that the viscous forces dragging and rolling the particle along exceed the gravitational forces pulling the particles against the wall. High flow velocities lead to strong hydrodynamic lift forces that tend to drive the particles away from the wall. The influence of these lift forces and their mathematical description have been investigated extensively (13-15). They increase with increasing flow rates and can be counteracted by increasing the field strength. Smaller particles are more
OF
YEAST
301
affected by lift forces than larger ones. This can lead to a coelution of particles that would be separated under optimized conditions if the lift forces are too high. Thus, finding the optimum ratio of flow rate to field strength is very important to guarantee an efficient resolution in steric FFF. While size and density distributions can be obtained very easily in the normal mode of SdFFF on the basis of theoretical relationships without the need of calibration (16,17), an empirical calibration is necessary in steric FFF, because no theoretical expressions for the relationship between lift forces and particle migration have been described so far (15). If the particles of interest have all the same density, their size distribution can be obtained very easily by a calibration procedure based on density compensation (18). For particles differing in size as well as in density, a combination of Sd/StFFF and microscopy can be used to obtain both size and density distribution (19). Sd/StFFF has been successfully applied not only to the characterization of glass beads (18), chromatographic supports (19), metal particles (12), inorganic colloidal materials (20), and environmental samples (21), but also for the separation of various blood cells (22,23). The aim of this work is to examine the use of Sd/StFFF to monitor and control the growth rate of yeast cultivations during a bioprocess. MATERIALS
AND
METHODS
Reagents and Samples The carrier liquid was doubly distilled water containing 0.01% (v/v) Teepol “Shell” anionic detergent mixture (Fluka, Buchs, Switzerland). To optimize the system polystyrene particle standards of nominal diameters of 3.0,5.0, 7.0, 11.9, 14.9, and 25.7 pm (Serva, Heidelberg, Germany) were used. Three different types of yeast strains were investigated in this study. Samples of a synchronous culture of S. cereuisiae (strain ATCC 32167) several cycles after the initiation of synchronization were obtained from T. Munch (Institute of Biotechnology, ETH Zurich, Switzerland). The samples were collected every 10 min during cultivation, centrifugated, and resuspended in ethanol for fixation. They were stored at -18’C and resuspended in carrier solution before use. Experimental conditions of the cultivation of synchronous yeast can be found in Ref. (24). Additionally baker’s yeast obtained from Hefefabriken AG (Hindelbank, Switzerland) and a recombinant plasmid strain of S. cerevisiae (strain YB 18a1, CibaGeigy Ltd., Basel, Switzerland) producing acid phosphatase were cultivated in 250-ml shake flasks in a LabShaker (A. Kiihner AG, Birsfelden, Switzerland) under the following conditions: culture medium, He17; temperature, 30°C; pH 5.8. The culture medium He17 was a
302
HOFFSTETTER-KUHN
ET
AL.
modification of “yeast nitrogen base medium w/o amino acids” (DIFCO, Detroit, MI). It was sterilized by filtration through a 0.2-pm membrane filter (Millipore, Bedford, MA). Samples were collected every hour over a 9-h period.
Instrumentation The sedimentation system used in this work was the Model SlOl colloid/particle fractionator from FFFractionation, Inc. (Salt Lake City, UT). This instrument had a horizontal spin axis to prevent larger particles from settling to the edges of the channel during the run due to gravitational sedimentation. A channel of reduced dimensions was used for the steric runs. The width of the channel was 1.0 cm, the thickness 254 pm, and the tip-to-tip length 89 cm. The void volume as measured by a nonretained dyestuff is 2.3 ml. The outer channel wall was coated by fixing a polyimide tape to prevent excessive adsorption of the cells at the wall. Two Sykam Model S 1000 HPLC pumps (Stagroma, Wallisellen, Switzerland) were connected to the channel by means of a Model M 491 HPLC gradient mixer (Kontron Instruments, Birsfelden, Switzerland). Whereas one of the two pumps had an analytical pump head allowing defined flow rates from 0.1 to 10 ml/min, the other was equipped with a preparative one for flow rates between 10 and 20 ml/min. To avoid flow pulsation a Scientific Systems Model LP-21 pulse damper (Supelco, Bellefonte, PA) was added between the gradient mixer and the channel. This arrangement allowed for precise and pulse-free operation in the range of 0.1 to 30 ml/min. A Linear 206 PHD uv-vis detector (Spectra Physics, San Jose, CA) was utilized to monitqr the eluted polystyrene samples. For the detection of yeast cells a DAWNF multiangle light scattering detector (Wyatt Technologies, Santa Barbara, CA) was used. An HP Vectra computer (Hewlett-Packard, Widen, Switzerland) was applied for the control of rotation speed as well as for data acquisition using the FFF software purchased with the instrument. Additionally a Compac computer Model 386/20 (Dettwiler Informatik AG, Basel, Switzerland) served for data acquisition of the light scattering detector by using ASTRA Software from Wyatt Technologies. Fractions of eluted cells were collected with a Model 203 microfraction collector from Gilson Medical Electronics (Villiers-le-Bel, France). Micrographs of the collected fractions were made by a Polyvar MET microscope (Reichert-Jung, Wien, Austria) under transmission light with interference contrast. Optical density of the yeast samples was determined at 600 nm (OD,) using a Uvikon 810 photometer (Kontron Instruments, Birsfelden, Switzerland).
0.0
0.5
1.0
fractionation
1.5
time
2.0
[mini
FIG. 2. Fractogram of the separation of six different polystyrene microspheres under optimized conditions. Carrier, 0.01% Teepol; flow rate, 24 ml/min; channel volume, 2.3 ml; rotation speed, 1200 rpm resp. field strength, 2350 m/s2; detection, uv absorption at 254 nm.
Procedure
for SdIStFFF
After the channel was brought up to its final rotation speed, 20 ~1 of the sample were injected via an injection valve. Instead of choosing a stopflow time, the sample was injected at a very low flow rate (0.4 ml/min), but for a longer time than would be necessary to reach the channel inlet (slowflow injection). The sample components could sediment and relax during this slowflow time without coming into close contact with the wall. Thus, particle adsorption could be minimized by this procedure. After 35 s the flow was increased up to its final value by turning on the second pump. After each run the system was flushed with carrier for 5 min at 30 ml/min to elute adsorbed material and to avoid contamination of the channel. RESULTS
AND
DISCUSSION
Polystyrene latex particles are ideal model substances for optimizing an FFF system, because they are available in the size range from about 100 nm to 100 pm, thus covering the whole application range of SdFFF and flow FFF. Additionally they possess very low polydispersitivities, thus forming sharp peaks in a fractogram. Furthermore they are spherical and have all the same density of 1.05 g/ml. In this work, a model mixture of six polystyrene beads covering the same size range as yeast cells was used to optimize the system for the steric runs. Figure 2 shows the fractogram of their separation under optimized conditions. It illustrates not only the high resolution that can be achieved in Sd/StFFF, but also the
FRACTIONATION
cells
\ 0
FIG.
3.
CHARACTERIZATION
budding cells
\
Simplified
model
of the cell cycle.
very short analysis time due to the high flow rates used in this mode. The polystyrene beads were completely separated in only 90 s. To prevent interactions of the particles with themselves and with the wall, an anionic detergent mixture was added to the aqueous carrier. This experiment was repeated several times during this study to ensure the consistency of the FFF system. In the case of yeast cells, however, the situation is more complex. This is due to the fact that the cells differ not only in size, but also in density and shape. The shape of a single yeast cell is usually spherical to ellipsoidal. For ellipsoidal cells, a length of 5-10 pm and a width of 3-5 pm are common. Vegetative reproduction in yeast occurs mostly asexually by budding as depicted in Fig. 3. The bud grows until it has reached a size almost as large as the mother cell and then separates from it. If the daughter cells do not separate from the mother cells, yeasts form a pseudomycelium consisting of single or branched chains. All these different kinds of cells coexist in the culture medium. Moreover yeast cells undergo periodic fluctuations in density during the cell division cycle. Baldwin and Kubitschek (25) report that, for a typical strain of S. cereuisiae, the densities range from 1.1068 to 1.1168 g/ml with a mean density of 1.1126 g/ml and a maximum density difference of 0.01 g/ml. Densities are highest for cells with buds about one-fourt,h the diameter of their mother cells (midcycle, 0.4 generations) and lowest’ when bud diameters were about the same as their mother cells (end of the cycle, 0.9 generations). This means that the larger cells are less dense than the smaller ones. This can distort the elution order such that less dense, larger cells are coeluted with more dense, smaller cells. It has been shown earlier that even particles of approximately the same size can be separated in Sd/StFFF only due to their density difference of about 1.0 g/ml (26). The enormous diversitivity of yeast cells in a single sample is responsible for the fact that no sharp peaks can be obtained, but rather broad bands with one or several maxima. Because of the various shapes of the
OF
YEAST
303
cells and the additional density fluctuations, it is very difficult to determine the particle size distribution by one of the two methods mentioned above (l&19). Therefore, microscopic investigations were necessary to confirm the separation in each case. Since the densities of polystyrene and yeast cells are only slightly different, the running parameters optimized for the polystyrene beads should be easily adapted for the cell separations. However, because of the higher polydispersitivity in the case of yeast cells and the need for fraction collection, lower flow rates must be used. Therefore, the separation speed is slightly lower in the yeast fractionations. The detergent solution which was used for the polystyrene fractionation also gave reasonable results with respect to wall adsorption of cells compared with other carrier systems containing salts. Figure 4 shows the separation of a baker’s yeast sample. The light scattering (LS) intensity measured at an angle of 90” (Fig. 4a) is much higher than the uv signal obtained with a uv-vis detector (Fig. 4b), allowing the detection of far more diluted cell samples. The void peak (V,), which contains unretained material, is much more intense in the case of uv-vis detection and gives only a negligible LS signal. This proves that all particles are sediment under the chosen conditions. Unfortunately, light scattering does not show a linear dependence of the concentration. The larger the particles, the higher the light scattering signal for the same concentration. The positions of the collected fractions that were examined by microscopy as well as representative micrographs are also shown in Fig. 4. One can clearly see that the cells are successfully fractionated. Four different cell populations can be distinguished. The first consists of cells having two buds, the second contains only cells with one bud, and the later-eluting populations include single cells of different sizes. To find the best ratio of flow rate to field strength with respect to adsorption, resolution, and analysis time, several runs at different flow rates but constant field were made (Fig. 5). The fractionation pattern always looks the same, but less cells are adsorbed with increasing flow rate. This demonstrates that the lift forces do not affect resolution in the chosen range. But strong adsorption occurs if the flow rate is not high enough to force the particles away from the wall by means of the lift forces. In the following experiments a flow rate of 14 ml/min and a slightly lower field strength were used. The noise of the light scattering signal is influenced not only by the particle size, but also by the flow rate. It is highest for larger particles fractionated at low flow rates. In this case only a few particles with an intense scattering migrate through the flow cell per time unit, resulting in higher noise on the scattered light. To examine the use of Sd/StFFF for bioprocess monitoring two strains of S. cerevisiue which serve as models for two different types of cultivation were chosen. Since
304
HOFFSTETTER-KUHN
6
a)
ET
AL.
t
2 3 fractionation time [min] FIG. 4. Fractogram of a baker’s yeast sample showing the position of collected fractions and the corresponding micrographs. Cell concentration as determined by OD, , 19 g/liter. Carrier, 0.01% Teepol; flow rate, 14 ml/min; channel volume, 2.3 ml; rotation speed, 1200 rpm resp. field strength, 2350 m/s’; detection, light scattering intensity at 90” angle (a) and uv absorption at 600 nm (b).
FRACTIONATION
CHARACTERIZATION
*
6-
b)
8
d)
i?. I:
6
/;
0
2
4
6 fractionation
8 time
10 [min]
305
YEAST
the cells to the new environment in the medium. In a second adaptation phase between 50 and 60 h of cultivation, a second exponential growth rate could be observed. The cultivations were stopped after 68 h of growth. The same samples taken for the turbidimetric measurements were injected in the SdFFF instrument. Fig. 7 illustrates the changes in relative cell size and shape during the cultivation process of baker’s yeast. For each time a characteristic pattern was obtained. Only those fractograms in which the most obvious changes of the pattern could be seen are shown. The LS intensities, being a qualitative measure for the biomass, are continuously increasing with ongoing cultivation. The last two samples were diluted twice before injection. As confirmed by microscopy, cells with one bud were the predominant species in the beginning of the cultivation. After 20 h a second peak consisting of cells with two and three buds appeared. Single cells were visible in the fractogram after 26 h. The amount of single cells increased continuously, but at the end of the cultivation, again, cells with several buds were in the majority. The peak maxima shifted confirming again the great diversitivity of the cell sample. In every single stage of the bioprocess another cell distribution occurred. In Fig. 8, the most illustrative FFF runs made during the cultivation of the recombinant yeast are shown. The fractionation pattern is totally different from those of the baker’s yeast cultivation. A relatively low initial cell concentration of 0.1 g/liter and a long adaptation time
-1c,
&
OF
12
FIG. 5. Fractogram of a baker’s yeast sample at different flow rates. Cell concentration as determined by OD,, 19 g/l. Flow rates, 8 (a), 10 (b), 14 (c) and 18 ml/min (d). Other experimental conditions were the same as those given in the legend to Fig. 4. * indicates adsorbed material eluting out of the system, after the centrifuge was stopped.
baker’s yeast is already grown in the presence of nutrients other than glucose, all enzymes needed for the consumption of these nutrients are present within the cell. The recombinant yeast strain needs to adapt its metabolism to the new environment in a culture medium and needs some time to build all the enzymes that are necessary for consumption of first glucose and, after its depletion, other nutrients. The differences in their behavior of growth can roughly be seen in their growth curves, where the optical density at 600 nm is plotted versus the cultivation time (Fig. 6). Baker’s yeast grows without going through an exponential phase (Fig. 6a). When glucose is depleted in the culture medium, growth continues through the utilization of other nutrients. The growth of the recombinant yeast followed an exponential curve which starts after the first adaptation of
am 01 ' 0 20
I 20
'
8
-
I
40
1 80
b) .
z SW ”
'
60
. 1 10
..-
mm
1
0 I0
.
-t---. 20
cultivation
FIG. 6. baker’s
I
*
40 time
I 60
-
1 80
[min]
OD, as a function of cultivation yeast (a) and a recombinant strain
time for the cultivation of S. cereuisiue (b).
of
306
HOFFSTETTER-KUHN
ET
AL.
at the beginning of the cultivation were responsible for the very low LS signal in the samples taken before the start of the exponential phase after about 25 h. The first peak that can be seen in the fractograms from 42 until
“1
28 h
fractionation time [min] FIG.
8. Monitoring of a cultivation cereuisiae by Sd/StFFF. Experimental those given in the legend to Fig. 7.
of a recombinant conditions were
strain of S. the same as
68 h was eluted 30 s before the first peak in the baker’s yeast sample and contained mostly cells with more than two buds. Since the doubling time of the entire cell population differs from the individual generation time of a single cell growing and dividing to produce two cells, there is a 0 1 2 3 4 distribution of cells at various stages of the cell cycle in fractionation time [min] the culture. It is, however, possible to regulate a population so that all the individual cells are at the same stage FIG. 7. Monitoring of a baker’s yeast cultivation by Sd/StFFF. Rotation speed, 1600 rpm resp. field strength, 1635 m/s’. Other experi- of the cell cycle. These synchronous cultures are a convenient experimental tool for detailed study of, for exmental conditions were the same as those given in the legend to Fig. 4.
FRACTIONATION
CHARACTERIZATION
OF
307
YEAST
tern and the size distribution obtained by microscopy, the positions of the different kinds of cell are shown above the first fractogram. Since the size differences in the synchronous culture was not as high as those in the examined cultivation broths, this example illustrates the high resolution of Sd/StFFF even better than those in cultivation broths. At the beginning of the cell cycle single cells of all different sizes were predominant. The beginning of budding led to a displacement of the band to higher sizes. The number of single cells decreased during the budding phase. At the end of the cell cycle the band was displaced again to higher elution times.
c
CONCLUSIONS
It was shown that Sd/StFFF is capable of fractionating yeast cells in less than 4 min. The method offers high resolution and a high dynamic range. Since unretained as well as particulate materials are separated, the method is applicable independent of the composition of the culture medium. Additionally, infections can be seen very easily. By combining the data obtained by FFF with those of other measurements during the bioprocess, such as glucose consumption, ethanol production, pH, and gas phase composition, important information on the relationship between cell growth and productivity can be obtained. By connecting the FFF system directly to a bioprocess fermentor and using flow injection for sampling, it will be possible to monitor cell growth during a cultivation on line. ACKNOWLEDGMENT 1 -1POmin
,
,,lp\+,
1
2
3
We thank Teresa Olefirowicz for critical reading of the manuscript, Thomas Munch for providing samples of the synchronous yeast culture, and Morten Garn for helpful discussions. 4
5
REFERENCES fractionation FIG. 9. Fractograms of a synchronous yeast cles after the initiation of the synchronization. determined by OD,, 14-15 g/liter. Experimental same as those given in the legend to Fig. 4.
time [min] cultivation several cyCell concentration as conditions were the
ample, the cell cycle, because they provide an amplification of the individual cell characteristics. Figure 9 shows the fractograms of 10 samples which were taken during the synchronous cultivation covering exactly one cell cycle as demonstrated in Fig. 3. In this example about one-third of the cells were in the synchronous phase. The cell concentration during cultivation was kept constant. The culture contained only single and budding cells, but no higher cell aggregates. To give a crude image of the relationship between the fractogram pat-
1. Meyer, H.-P., Kappeli, O., and Fiechter, A. (1985) Annu. Reu. Microbial. 39, 299-319. 2. Cooney, L. C. (1981) inBiotechnology (Rehm, H. J., and Reed, G., Eds.), Vol. 1, pp. 73-113, Verlag Chemie, Weinheim. 3. Bitter, G. A. (1988) Deu. Znd. Microbial. 29, 25-31. 4. Alberghina, L., Ranzi, B. M., Porro, D., and Martegani, Biotechnol. Prog. 4, 299-304.
E. (1991)
5. Lines, R. W. (1987) Anal. Proc. 24, 272-276. 6. Giddings, J. C., and Myers, M. N. (1978) Sep. Sci. Technol. 637-645. 7. Giddings, J. C. (1966) Sep. Sci. 1, 123-125. 8. Giddings,
J. C. (1988)
C&n.
Eng. News
13,
66,34-45.
9. Caldwell, K. D. (1988) Anal. Chem. 60,959A-971A. 10. Janka, J. (1992) J. Chromatogr. Libr. A 61, A449-479. 11. Giddings, J. C. (1985) Sep. Sci. Technol. 19, 831-847. 12. Giddings, J. C., Myers, M. N., Moon, N. H., and Bhajendra, (1991) ACS Symp. Ser. 472, 198-216.
N. B.
308 13. Giddings, J. C., Chen, X., Wahlund, Anal. Chem. 59,1957-1962. 14. Koch, T., and Giddings, J. C. (1986)
HOFFSTE’M’ER-KUHN K.-G., Anal.
Myers, Chem.
M. N. (1987) 68,994-997.
15. Williams, P. S., Koch, T., and Giddings, J. C. (1992) Chem. Eng. Commurz. 111,121-147. 16. Yang, F. S., Caldwell, K. D., and Giddings, J. C. (1983) J. Colloid Interface Sci. 92, 81-91. 17. Koliadima, A., Dalas, E., and Karaiskakis, G. (1990) J. High Resolut. Chromatogr. 13, 338-342. 18. Giddings, J. C., Moon, H. M., Williams, P. S., and Myers, M. N. (1991) Anal. Chem. 63, 1366-1372. 19. Giddings, J. C., and Moon, H. M. (1991) Anal. Chem. 63,28692877. 20. Dalas, E., and Karaiskakis, G. (1987) Colloids Surf. 28, 169-183.
ET
AL.
21. Beckett, R., Hotchin, togr. 617.435-447.
D. M.,
and Hart,
22. Caldwell, K. D., Cheng, Z.-Q., Hradecky, (1984) Cell Biophys. 6, 233-251. 23. Giddings, J. C., Bhajendra, Symp. Ser. 464,128-144.
N. B., and
B. T. (1990)
J. Chroma-
P., and Giddings, Liu,
M.-K.
(1991)
J. C. ACS
24. Munch, T., Sonnleitner, B., and Fiechter, A. (1991) in Biochem. Engineering (Reuss, M., Chmiel, H., Gilles, E.-D., and Knackmuss, H.-J., Eds.), pp. 373-376, Gustav Fischer, Stuttgart/New York. 25. Baldwin, 701-704.
W. W., and Kubitschek,
26. Caldwell, K. D., Nguyen, (1979) Sep. Sci. Z’echnol.
H. E. (1984)
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14,935-946.
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M. N., and Giddings,
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BIOCHEMISTRY
206,
300-308
(19%)
Characterization of Yeast Cultivations by Steric Sedimentation Field-Flow Fractionation Sabrina Hoffstetter-Kuhn,l Thomas Rbsler,* Markus Ehrat, and H. Michael Widmer Corporate Analytical Research, Ciba-Geigy Ltd., Base& Switzerland; and *Amendstrasse 51, BerEin, Germany
Received
June
15,1992
The characterization and quantification of biomass is often time consuming and dependent on the cultivation media and gives no detailed information between cell size and shape and their productivity. By monitoring the bioprocess with steric sedimentation field-flow fractionation (Sd/StFFF) in combination with laser light scattering, not only cell growth, but also the variation of cell size and shape during the cultivation, can be observed. In this work, the feasibility of separating and characterizing cell populations by steric sedimentation field-flow fractionation is demonstrated by its application to three different yeast cultivation broths. For this purpose samples which were collected at different cultivation times were injected into an FFF system. Fractograms were obtained in less than 4 min. Due to the relatively high resolution of the method, a cell sample could be fractionated in several subpopulations differing in their size as well as in their number of buds. 0 1992 Academic Press. Inc.
The possibility of controlling bioprocesses is strongly limited by the lack of reliable methods for the direct measurement of biomass and cell growth rate. Routine methods for the control of bioprocesses are based on physical or chemical parameters such as turbidimetry, glucose consumption, gas phase composition, or cell products correlated to cell growth, from which it is possible to only indirectly estimate the properties of the biomass (1,2). The budding yeast Saccharomyces cerevisiae is an eucaryotic microorganism widely used as a model organism of the cell cycle and of interest for the biotechnological production of biomass and ethanol. The progress of recombinant DNA technology allowing the transformation of yeast cells and the expression of heterologous
1 To whom 300
correspondence
should
be addressed.
proteins in yeast cultures with very high yields (3) has increased industrial interest in yeast cultivations and, thus, in useful methods to directly monitor and control biomass and growth rate during the bioprocess. This means one must look for analytical methods which are related to a physical property of the cell itself, such as its size, mass, or shape. Most of the techniques dealing with the determination of these cell characteristics are based on light scattering as in flow cytometry (4) and Coulter counting (5), where cells are not actually separated according to their differences in size, but only counted. Steric sedimentation field-flow fractionation (Sd/ StFFF)2 offers a new and exciting alternative for the characterization of yeast cells during a bioprocess by providing information about the size distribution via a fast and simple procedure. Sd/StFFF was introduced by Giddings and Meyers in 1978 (6) as a particular form of field-flow fractionation (FFF), a family of separation methods based on elution from a thin channel in which the separation is induced and controlled by an external force field acting in a direction perpendicular to laminar flow in the channel (7-10). Depending on the FFF subtechnique, the force field can be either a gravitational field (sedimentation FFF), *a temperature gradient (thermal FFF), or a crossflow (flow FFF). Under the influence of the field, particles are driven uniformely toward one channel wall, the accumulation wall, until their accumulation is balanced by diffusion. The steady-state concentration profile, which is exponentially decreasing from the wall to the middle of the channel, has a characteristic thickness and depends on the diffusion coefficients and hence on the mass of the particles. This leads to different profile thicknesses for particles differing in size. After the field has been applied and the sample has been injected, the carrier flow
2 Abbreviations steric sedimentation
used: FFF, field-flow
field-flow fractionation;
fractionation LS, light
Sd/StFFF, scattering.
0003-2697192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
FRACTIONATION
CHARACTERIZATION
-------FIG. 1.
Schematic
drawing
of the separation
in a steric
FFF
chan-
nel.
is stopped for a chosen time period to allow relaxation of the particles. This so-called stopflow time varies from a few seconds to several minutes. When the carrier flow is then turned on again, smaller particles will elute first with the carrier flow, because they are less compressed to the accumulation wall than larger ones and thus occupy on average faster stream lines. For a detailed description of FFF theory see Ref. (11). The most widely used subtechnique so far is sedimentation FFF (SdFFF), where particles are separated according to their differences in mass and density (12). For this purpose the FFF channel is placed into a centrifuge. Steric FFF is the high limit in respect to the size of normal FFF which is described above. Steric operation mode is realized, when diffusion of the particles becomes negligible. This is normally the case for particles with diameters above 1 pm, which sediment very easily. Figure 1 schematically shows the separation procedure in a steric sedimentation FFF channel. The particles extend into the flow stream primarily because of their finite size. Layer thickness is now determined by the diameter of the particle rather than by diffusion. The particles move at a velocity approximately equal to the carrier-flow velocity at their center of gravity. Larger particles elute in’this mode more rapidly than smaller particles. The flow velocity in steric FFF should be high enough to avoid strong adsorption at the wall and to ensure that the viscous forces dragging and rolling the particle along exceed the gravitational forces pulling the particles against the wall. High flow velocities lead to strong hydrodynamic lift forces that tend to drive the particles away from the wall. The influence of these lift forces and their mathematical description have been investigated extensively (13-15). They increase with increasing flow rates and can be counteracted by increasing the field strength. Smaller particles are more
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affected by lift forces than larger ones. This can lead to a coelution of particles that would be separated under optimized conditions if the lift forces are too high. Thus, finding the optimum ratio of flow rate to field strength is very important to guarantee an efficient resolution in steric FFF. While size and density distributions can be obtained very easily in the normal mode of SdFFF on the basis of theoretical relationships without the need of calibration (16,17), an empirical calibration is necessary in steric FFF, because no theoretical expressions for the relationship between lift forces and particle migration have been described so far (15). If the particles of interest have all the same density, their size distribution can be obtained very easily by a calibration procedure based on density compensation (18). For particles differing in size as well as in density, a combination of Sd/StFFF and microscopy can be used to obtain both size and density distribution (19). Sd/StFFF has been successfully applied not only to the characterization of glass beads (18), chromatographic supports (19), metal particles (12), inorganic colloidal materials (20), and environmental samples (21), but also for the separation of various blood cells (22,23). The aim of this work is to examine the use of Sd/StFFF to monitor and control the growth rate of yeast cultivations during a bioprocess. MATERIALS
AND
METHODS
Reagents and Samples The carrier liquid was doubly distilled water containing 0.01% (v/v) Teepol “Shell” anionic detergent mixture (Fluka, Buchs, Switzerland). To optimize the system polystyrene particle standards of nominal diameters of 3.0,5.0, 7.0, 11.9, 14.9, and 25.7 pm (Serva, Heidelberg, Germany) were used. Three different types of yeast strains were investigated in this study. Samples of a synchronous culture of S. cereuisiae (strain ATCC 32167) several cycles after the initiation of synchronization were obtained from T. Munch (Institute of Biotechnology, ETH Zurich, Switzerland). The samples were collected every 10 min during cultivation, centrifugated, and resuspended in ethanol for fixation. They were stored at -18’C and resuspended in carrier solution before use. Experimental conditions of the cultivation of synchronous yeast can be found in Ref. (24). Additionally baker’s yeast obtained from Hefefabriken AG (Hindelbank, Switzerland) and a recombinant plasmid strain of S. cerevisiae (strain YB 18a1, CibaGeigy Ltd., Basel, Switzerland) producing acid phosphatase were cultivated in 250-ml shake flasks in a LabShaker (A. Kiihner AG, Birsfelden, Switzerland) under the following conditions: culture medium, He17; temperature, 30°C; pH 5.8. The culture medium He17 was a
302
HOFFSTETTER-KUHN
ET
AL.
modification of “yeast nitrogen base medium w/o amino acids” (DIFCO, Detroit, MI). It was sterilized by filtration through a 0.2-pm membrane filter (Millipore, Bedford, MA). Samples were collected every hour over a 9-h period.
Instrumentation The sedimentation system used in this work was the Model SlOl colloid/particle fractionator from FFFractionation, Inc. (Salt Lake City, UT). This instrument had a horizontal spin axis to prevent larger particles from settling to the edges of the channel during the run due to gravitational sedimentation. A channel of reduced dimensions was used for the steric runs. The width of the channel was 1.0 cm, the thickness 254 pm, and the tip-to-tip length 89 cm. The void volume as measured by a nonretained dyestuff is 2.3 ml. The outer channel wall was coated by fixing a polyimide tape to prevent excessive adsorption of the cells at the wall. Two Sykam Model S 1000 HPLC pumps (Stagroma, Wallisellen, Switzerland) were connected to the channel by means of a Model M 491 HPLC gradient mixer (Kontron Instruments, Birsfelden, Switzerland). Whereas one of the two pumps had an analytical pump head allowing defined flow rates from 0.1 to 10 ml/min, the other was equipped with a preparative one for flow rates between 10 and 20 ml/min. To avoid flow pulsation a Scientific Systems Model LP-21 pulse damper (Supelco, Bellefonte, PA) was added between the gradient mixer and the channel. This arrangement allowed for precise and pulse-free operation in the range of 0.1 to 30 ml/min. A Linear 206 PHD uv-vis detector (Spectra Physics, San Jose, CA) was utilized to monitqr the eluted polystyrene samples. For the detection of yeast cells a DAWNF multiangle light scattering detector (Wyatt Technologies, Santa Barbara, CA) was used. An HP Vectra computer (Hewlett-Packard, Widen, Switzerland) was applied for the control of rotation speed as well as for data acquisition using the FFF software purchased with the instrument. Additionally a Compac computer Model 386/20 (Dettwiler Informatik AG, Basel, Switzerland) served for data acquisition of the light scattering detector by using ASTRA Software from Wyatt Technologies. Fractions of eluted cells were collected with a Model 203 microfraction collector from Gilson Medical Electronics (Villiers-le-Bel, France). Micrographs of the collected fractions were made by a Polyvar MET microscope (Reichert-Jung, Wien, Austria) under transmission light with interference contrast. Optical density of the yeast samples was determined at 600 nm (OD,) using a Uvikon 810 photometer (Kontron Instruments, Birsfelden, Switzerland).
0.0
0.5
1.0
fractionation
1.5
time
2.0
[mini
FIG. 2. Fractogram of the separation of six different polystyrene microspheres under optimized conditions. Carrier, 0.01% Teepol; flow rate, 24 ml/min; channel volume, 2.3 ml; rotation speed, 1200 rpm resp. field strength, 2350 m/s2; detection, uv absorption at 254 nm.
Procedure
for SdIStFFF
After the channel was brought up to its final rotation speed, 20 ~1 of the sample were injected via an injection valve. Instead of choosing a stopflow time, the sample was injected at a very low flow rate (0.4 ml/min), but for a longer time than would be necessary to reach the channel inlet (slowflow injection). The sample components could sediment and relax during this slowflow time without coming into close contact with the wall. Thus, particle adsorption could be minimized by this procedure. After 35 s the flow was increased up to its final value by turning on the second pump. After each run the system was flushed with carrier for 5 min at 30 ml/min to elute adsorbed material and to avoid contamination of the channel. RESULTS
AND
DISCUSSION
Polystyrene latex particles are ideal model substances for optimizing an FFF system, because they are available in the size range from about 100 nm to 100 pm, thus covering the whole application range of SdFFF and flow FFF. Additionally they possess very low polydispersitivities, thus forming sharp peaks in a fractogram. Furthermore they are spherical and have all the same density of 1.05 g/ml. In this work, a model mixture of six polystyrene beads covering the same size range as yeast cells was used to optimize the system for the steric runs. Figure 2 shows the fractogram of their separation under optimized conditions. It illustrates not only the high resolution that can be achieved in Sd/StFFF, but also the
FRACTIONATION
cells
\ 0
FIG.
3.
CHARACTERIZATION
budding cells
\
Simplified
model
of the cell cycle.
very short analysis time due to the high flow rates used in this mode. The polystyrene beads were completely separated in only 90 s. To prevent interactions of the particles with themselves and with the wall, an anionic detergent mixture was added to the aqueous carrier. This experiment was repeated several times during this study to ensure the consistency of the FFF system. In the case of yeast cells, however, the situation is more complex. This is due to the fact that the cells differ not only in size, but also in density and shape. The shape of a single yeast cell is usually spherical to ellipsoidal. For ellipsoidal cells, a length of 5-10 pm and a width of 3-5 pm are common. Vegetative reproduction in yeast occurs mostly asexually by budding as depicted in Fig. 3. The bud grows until it has reached a size almost as large as the mother cell and then separates from it. If the daughter cells do not separate from the mother cells, yeasts form a pseudomycelium consisting of single or branched chains. All these different kinds of cells coexist in the culture medium. Moreover yeast cells undergo periodic fluctuations in density during the cell division cycle. Baldwin and Kubitschek (25) report that, for a typical strain of S. cereuisiae, the densities range from 1.1068 to 1.1168 g/ml with a mean density of 1.1126 g/ml and a maximum density difference of 0.01 g/ml. Densities are highest for cells with buds about one-fourt,h the diameter of their mother cells (midcycle, 0.4 generations) and lowest’ when bud diameters were about the same as their mother cells (end of the cycle, 0.9 generations). This means that the larger cells are less dense than the smaller ones. This can distort the elution order such that less dense, larger cells are coeluted with more dense, smaller cells. It has been shown earlier that even particles of approximately the same size can be separated in Sd/StFFF only due to their density difference of about 1.0 g/ml (26). The enormous diversitivity of yeast cells in a single sample is responsible for the fact that no sharp peaks can be obtained, but rather broad bands with one or several maxima. Because of the various shapes of the
OF
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cells and the additional density fluctuations, it is very difficult to determine the particle size distribution by one of the two methods mentioned above (l&19). Therefore, microscopic investigations were necessary to confirm the separation in each case. Since the densities of polystyrene and yeast cells are only slightly different, the running parameters optimized for the polystyrene beads should be easily adapted for the cell separations. However, because of the higher polydispersitivity in the case of yeast cells and the need for fraction collection, lower flow rates must be used. Therefore, the separation speed is slightly lower in the yeast fractionations. The detergent solution which was used for the polystyrene fractionation also gave reasonable results with respect to wall adsorption of cells compared with other carrier systems containing salts. Figure 4 shows the separation of a baker’s yeast sample. The light scattering (LS) intensity measured at an angle of 90” (Fig. 4a) is much higher than the uv signal obtained with a uv-vis detector (Fig. 4b), allowing the detection of far more diluted cell samples. The void peak (V,), which contains unretained material, is much more intense in the case of uv-vis detection and gives only a negligible LS signal. This proves that all particles are sediment under the chosen conditions. Unfortunately, light scattering does not show a linear dependence of the concentration. The larger the particles, the higher the light scattering signal for the same concentration. The positions of the collected fractions that were examined by microscopy as well as representative micrographs are also shown in Fig. 4. One can clearly see that the cells are successfully fractionated. Four different cell populations can be distinguished. The first consists of cells having two buds, the second contains only cells with one bud, and the later-eluting populations include single cells of different sizes. To find the best ratio of flow rate to field strength with respect to adsorption, resolution, and analysis time, several runs at different flow rates but constant field were made (Fig. 5). The fractionation pattern always looks the same, but less cells are adsorbed with increasing flow rate. This demonstrates that the lift forces do not affect resolution in the chosen range. But strong adsorption occurs if the flow rate is not high enough to force the particles away from the wall by means of the lift forces. In the following experiments a flow rate of 14 ml/min and a slightly lower field strength were used. The noise of the light scattering signal is influenced not only by the particle size, but also by the flow rate. It is highest for larger particles fractionated at low flow rates. In this case only a few particles with an intense scattering migrate through the flow cell per time unit, resulting in higher noise on the scattered light. To examine the use of Sd/StFFF for bioprocess monitoring two strains of S. cerevisiue which serve as models for two different types of cultivation were chosen. Since
304
HOFFSTETTER-KUHN
6
a)
ET
AL.
t
2 3 fractionation time [min] FIG. 4. Fractogram of a baker’s yeast sample showing the position of collected fractions and the corresponding micrographs. Cell concentration as determined by OD, , 19 g/liter. Carrier, 0.01% Teepol; flow rate, 14 ml/min; channel volume, 2.3 ml; rotation speed, 1200 rpm resp. field strength, 2350 m/s’; detection, light scattering intensity at 90” angle (a) and uv absorption at 600 nm (b).
FRACTIONATION
CHARACTERIZATION
*
6-
b)
8
d)
i?. I:
6
/;
0
2
4
6 fractionation
8 time
10 [min]
305
YEAST
the cells to the new environment in the medium. In a second adaptation phase between 50 and 60 h of cultivation, a second exponential growth rate could be observed. The cultivations were stopped after 68 h of growth. The same samples taken for the turbidimetric measurements were injected in the SdFFF instrument. Fig. 7 illustrates the changes in relative cell size and shape during the cultivation process of baker’s yeast. For each time a characteristic pattern was obtained. Only those fractograms in which the most obvious changes of the pattern could be seen are shown. The LS intensities, being a qualitative measure for the biomass, are continuously increasing with ongoing cultivation. The last two samples were diluted twice before injection. As confirmed by microscopy, cells with one bud were the predominant species in the beginning of the cultivation. After 20 h a second peak consisting of cells with two and three buds appeared. Single cells were visible in the fractogram after 26 h. The amount of single cells increased continuously, but at the end of the cultivation, again, cells with several buds were in the majority. The peak maxima shifted confirming again the great diversitivity of the cell sample. In every single stage of the bioprocess another cell distribution occurred. In Fig. 8, the most illustrative FFF runs made during the cultivation of the recombinant yeast are shown. The fractionation pattern is totally different from those of the baker’s yeast cultivation. A relatively low initial cell concentration of 0.1 g/liter and a long adaptation time
-1c,
&
OF
12
FIG. 5. Fractogram of a baker’s yeast sample at different flow rates. Cell concentration as determined by OD,, 19 g/l. Flow rates, 8 (a), 10 (b), 14 (c) and 18 ml/min (d). Other experimental conditions were the same as those given in the legend to Fig. 4. * indicates adsorbed material eluting out of the system, after the centrifuge was stopped.
baker’s yeast is already grown in the presence of nutrients other than glucose, all enzymes needed for the consumption of these nutrients are present within the cell. The recombinant yeast strain needs to adapt its metabolism to the new environment in a culture medium and needs some time to build all the enzymes that are necessary for consumption of first glucose and, after its depletion, other nutrients. The differences in their behavior of growth can roughly be seen in their growth curves, where the optical density at 600 nm is plotted versus the cultivation time (Fig. 6). Baker’s yeast grows without going through an exponential phase (Fig. 6a). When glucose is depleted in the culture medium, growth continues through the utilization of other nutrients. The growth of the recombinant yeast followed an exponential curve which starts after the first adaptation of
am 01 ' 0 20
I 20
'
8
-
I
40
1 80
b) .
z SW ”
'
60
. 1 10
..-
mm
1
0 I0
.
-t---. 20
cultivation
FIG. 6. baker’s
I
*
40 time
I 60
-
1 80
[min]
OD, as a function of cultivation yeast (a) and a recombinant strain
time for the cultivation of S. cereuisiue (b).
of
306
HOFFSTETTER-KUHN
ET
AL.
at the beginning of the cultivation were responsible for the very low LS signal in the samples taken before the start of the exponential phase after about 25 h. The first peak that can be seen in the fractograms from 42 until
“1
28 h
fractionation time [min] FIG.
8. Monitoring of a cultivation cereuisiae by Sd/StFFF. Experimental those given in the legend to Fig. 7.
of a recombinant conditions were
strain of S. the same as
68 h was eluted 30 s before the first peak in the baker’s yeast sample and contained mostly cells with more than two buds. Since the doubling time of the entire cell population differs from the individual generation time of a single cell growing and dividing to produce two cells, there is a 0 1 2 3 4 distribution of cells at various stages of the cell cycle in fractionation time [min] the culture. It is, however, possible to regulate a population so that all the individual cells are at the same stage FIG. 7. Monitoring of a baker’s yeast cultivation by Sd/StFFF. Rotation speed, 1600 rpm resp. field strength, 1635 m/s’. Other experi- of the cell cycle. These synchronous cultures are a convenient experimental tool for detailed study of, for exmental conditions were the same as those given in the legend to Fig. 4.
FRACTIONATION
CHARACTERIZATION
OF
307
YEAST
tern and the size distribution obtained by microscopy, the positions of the different kinds of cell are shown above the first fractogram. Since the size differences in the synchronous culture was not as high as those in the examined cultivation broths, this example illustrates the high resolution of Sd/StFFF even better than those in cultivation broths. At the beginning of the cell cycle single cells of all different sizes were predominant. The beginning of budding led to a displacement of the band to higher sizes. The number of single cells decreased during the budding phase. At the end of the cell cycle the band was displaced again to higher elution times.
c
CONCLUSIONS
It was shown that Sd/StFFF is capable of fractionating yeast cells in less than 4 min. The method offers high resolution and a high dynamic range. Since unretained as well as particulate materials are separated, the method is applicable independent of the composition of the culture medium. Additionally, infections can be seen very easily. By combining the data obtained by FFF with those of other measurements during the bioprocess, such as glucose consumption, ethanol production, pH, and gas phase composition, important information on the relationship between cell growth and productivity can be obtained. By connecting the FFF system directly to a bioprocess fermentor and using flow injection for sampling, it will be possible to monitor cell growth during a cultivation on line. ACKNOWLEDGMENT 1 -1POmin
,
,,lp\+,
1
2
3
We thank Teresa Olefirowicz for critical reading of the manuscript, Thomas Munch for providing samples of the synchronous yeast culture, and Morten Garn for helpful discussions. 4
5
REFERENCES fractionation FIG. 9. Fractograms of a synchronous yeast cles after the initiation of the synchronization. determined by OD,, 14-15 g/liter. Experimental same as those given in the legend to Fig. 4.
time [min] cultivation several cyCell concentration as conditions were the
ample, the cell cycle, because they provide an amplification of the individual cell characteristics. Figure 9 shows the fractograms of 10 samples which were taken during the synchronous cultivation covering exactly one cell cycle as demonstrated in Fig. 3. In this example about one-third of the cells were in the synchronous phase. The cell concentration during cultivation was kept constant. The culture contained only single and budding cells, but no higher cell aggregates. To give a crude image of the relationship between the fractogram pat-
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