Anal. Chem. 2002, 74, 4496-4504
Steric-Hyperlayer Sedimentation Field Flow Fractionation and Flow Cytometry Analysis Applied to the Study of Saccharomyces cerevisiae Ramse´s Sanz,† Philippe Cardot,‡ Serge Battu,‡ and M. Teresa Galceran*,†
Departament de Quı´mica Analı´tica, Universitat de Barcelona, 1-11, Martı´ i Franque` s, E-08028 Barcelona, Spain, and Laboratoire de Chimie Analytique et Bromatologie, Faculte´ de Pharmacie, Universite´ de Limoges, 2, Dr. Marcland, F-87025 Limoges, France
Sedimentation field flow fractionation separation associated with flow cytometry has been used for the characterization of several commercial Saccharomyces cerevisiae yeasts used for wine production. A new type of channel 80 µm thick and new operating conditions, such as sample introduction when field and flow are established and a channel inlet connected to the accumulation wall, were used. Good repeatability (5% RSD) and reduced analysis time (2-10 min) were obtained. The avoidance of the stop-flow relaxation process in conjunction with the use of a channel of reduced thickness has demonstrated that an effective “steric-hyperlayer” mode driving to a major focusing effect of the species in the channel thickness is involved in the elution of the yeast cells. Flow cytometry analyses were performed, and the forward scattering and side scattering yeast characteristics correlation maps were obtained. Field flow fractionation and flow cytometry information obtained indicated that the fractogram profiles of the yeast cell depended not only on the size, but also on the shape and density. Saccharomyces cerevisiae yeast strains are widely used for brewing, baking, wine making, and biotechnological processes.1 In addition, there is evidence that S. cerevisiae can be involved in superficial and systemic diseases, such as vaginitis, pneumonia, and septicemia.2 In the particular case of wine manufacturing processes, yeast strains are commonly stored and prepared before use in special forms, that is, active dry wine yeast. Such types of conservation have made a large number of different yeast strains commercially available. Several procedures based on their physical, biochemical or morphological characteristics3 and, more recently, based on molecular techniques4 have been used for their characterization. As a result, for S. cerevisiae, a high number of substrains of different use have been characterized under specific * To whom correspondence should be addressed. Fax: +34 93 402 12 33. E-mail:
[email protected]. † Universitat de Barcelona. ‡ Universite´ de Limoges. (1) Kreger-van Rij, N. J. The yeasts- A Taxonomic Study, 3rd ed.; Elsevier: Amsterdam, 1984. (2) Murphy, A.; Kavanagh, K. Enzyme Microb. Techno. 1999, 25, 551-557. (3) Barnett, J. A.; Payne, R. W.; Yarrow, D. Yeasts-Characteristics and Identification; Cambridge University Press: Cambridge, 1983. (4) Querol, A.; Ramon, D. Trends Food Sci., Technol. 1996, 7, 73-78.
4496 Analytical Chemistry, Vol. 74, No. 17, September 1, 2002
names such as “Prise de Mousse, Premier Cuve´e, V1116, Fermivin, French Red”.5 A successful attempt to analyze yeast growing processes using a separation technique similar to chromatography, such as sedimentation field flow fractionation (SdFFF) at multigravitational fields, was made in the early nineties.6 Recently, SdFFF using simple gravity as external field, which led to a much more simplified separator design and operation methodology, was successfully applied to generate strain information.7 These results are of general interest, because some S. cerevisiae yeast strains cannot be accurately characterized using classical biochemical techniques,8 which warrants the systematic exploration of new characterization methods. The recent introduction of new SdFFF devices,9 the development of simplified analysis procedure,s10 and the setup of biocompatible separators11 make possible the optimization of FFF methods for yeast strain characterization. SdFFF is one of the versatile elution driven separation techniques invented by J. C. Giddings 30 years ago,12 which have had a constant but relatively limited development.13 The general field flow fractionation principle14 produces the separation of the sample components inside a narrow, ribbonlike channel in the form of a parallelepiped whose highly polished plane parallel walls are passed through perpendicularly by an externally generated field (the generic term is external field). A laminar, pressure-generated flow passing through the channel creates a parabolic flow profile in the ribbon thickness. Interactions of the sample with the field make it possible to concentrate or drive the components toward one wall (usually designated as the accumulation wall) in carrier flow streamlines (5) Johnston, J. R.; Baccari, C.; Mortimer, R. K. Res. Microbiol. 2000, 151, 583590. (6) Hofstetter-Kuhn, S.; Ro¨sler, T.; Ehrat, M.; Widmer, H. M. Anal. Biochem. 1992, 206, 300-308. (7) Sanz, R.; Puignou, L.; Reschiglian, P.; Galceran, M. T. J. Chromatogr. A 2001, 919, 339-347. (8) Richard, D.; Davis, T.; Josee, A.; Karen, M.; Andre, M.; Chantal, D. Am. J. Enol. Vitic. 1989, 40, 309-315. (9) Assidjo, E.; Chianea, T.; Dreyfuss, M. F.; Cardot, Ph. J. P. J. Chromatogr. B 1998, 709, 197-207. (10) Battu, S.; Hugon, J.; Cardot, Ph. J. P. BBA 2001, 1528, 89-96. (11) Cardot, Ph. J. P.; Battu, S.; Simon, A.; Delage, C. J. Chromatogr. B 2002, 768, 285-295. (12) Giddings, J. C. Science 1993, 260, 1456-1465. (13) Co ¨lfen, H.; Antonietti, M. Adv. Polym. Sci. 2000, 150, 67-187. (14) Giddings, J. C. In Field-Flow Fractionation Handbook; Schimpf, M. E., Caldwell, K., Giddings, J. C., Eds.; John Wiley & Sons: New York, 2000; Chapter 1. 10.1021/ac011234j CCC: $22.00
© 2002 American Chemical Society Published on Web 08/02/2002
of different velocities, consequently producing the separation. The versatility of the technique resides in part in the variety of the external fields and in their intensities. In descriptions in the literature, the field type generates the FFF subtechnique designation mode.13,14 Gravitational field, of limited cost, along with its subsequent FFF technique known as gravitational FFF (GFFF) belongs to the sedimentation FFF domain and has attracted growing interest for cell separations.7,15,16 Time-consuming separations and reduced field versatility with GFFF can be improved on using multigravitational fields, but much more sophisticated instrumentation is required.9,13,14 Sedimentation FFF techniques are remarkably well-suited for the separation of colloids and micrometer-sized species, mainly of biological-origin-like cells. The elution model for micrometer-sized species (diameter > 1 µm) is very specific, and it is generally described as “steric hyperlayer”.12,17,18 The aim of this work is to characterize yeast cells from the Saccharomyces genus and, in particular, of the cerevisiae species using SdFFF. Six different strains of commercial active dry wine yeast (ADWY) of particular importance in Spanish wine-making processes were systematically analyzed. For the first time in the FFF instrumental development, an ultrathin channel, associated with a submilliliter separator volume were used. The consequences are (i) a reduction in analysis time associated with a limited carrier phase volume and (ii) an enhancement of the separation properties. To correlate FFF data with physical yeast characteristics, a systematic control was performed using flow cytometry.19 THEORY “Steric-Hyperlayer” Elution Hypotheses in Field Flow Fractionation. In this work, important modifications of classical field flow fractionation instrumentation and working conditions were performed. The modifications were (i) the sample was introduced in the FFF separation system by means of a chromatographic injection loop when both the flow and the external field were established; (ii) In contrast with the commercially available instrumental set up used in SdFFF, the FFF channel inlet tube was connected to the accumulation wall of the channel; (iii) A channel of reduced thickness, 80 µm, was used to enhance the effect of the lifting forces as well as to reduce elution time and volume. These instrumental and working conditions led to some modifications of the steric-hyperlayer elution hypotheses classically described, but the general concept of the sample sterichyperlayer12,17,18,20,21 elution process can be conserved. As shown in Figure 1, sample particles in motion along the channel are focused by the balanced effect of both the force exerted on the particle by the external field and the force generated by the particle motion (hydrodynamic or “lifting” force). The retention ratio response to such a situation is only partially explained by (15) Bories, C.; Cardot, Ph. J. P.; Abramowski, V.; Pou ¨ s, C.; Merino-Dugay, A.; Baron, B.; Mougeot, G. J. Chromatogr. 1992, 579, 143-152. (16) Bernard, A.; Bories, C.; Loiseau, P. M.; Cardot, P. J. P. J. Chromatogr. 1995, 664, 444-448. (17) Chmelik, J. J. Chromatogr. A 1999, 845, 285-291. (18) Martin, M.; Williams, P. S. In Theoretical Advancement in Chromatography and Related Separation Techniques; Dondi, F., Guiochon, G., Eds.; Netherlands, 1992; p 513. (19) Me´te´zeau, Ph.; Ronot, X.; Le Noan-Merdrignac, G.; Ratinaud, M. H. In La Cytometrie en Flux; McGraw-Hill: Paris, 1988.
Figure 1. Field flow fractionation of micrometer-sized species: (a) particles in vicinity of the channel accumulation wall; (b) particles away from accumulation wall; (c) “average pseudoequilibrium” state; (d) size separation effect; v1, velocity of particle 1; v2, velocity of particle 2; (e) density separation effect.
the steric-hyperlayer elution hypotheses and depends on the size, density, rigidity, and shape of the particle. So far, the significance of each of these parameters can only be described in a qualitative way. Assuming nonturbulent flow injection, the elution starts in a nonstationary nonequilibrium particle state, the kinematics and time (or channel length required) of which is known only for standard spherical particles;20,21 nothing is known concerning nonspherical biological ones of high polydispersity. This kinematics toward an equilibrium position in the channel thickness cannot be modeled so far; however, an average pseudoequilibrium position can be deduced from the retention ratio according to the steric-hyperlayer model. The consequence of the hyperlayer focusing12,20,21 process driving to an equilibrium position in the channel thickness is that the retention ratio, measured as an indication of the sample average velocity along the channel,20,21 can be expressed as
Rr )
3x w
(1)
where x is the distance between the average particle gravity center to the accumulation wall as shown in Figure 1, and w is the channel thickness. The measured x value does not describe the real distance at equilibrium but can be considered an accurate evaluation of it, assuming that (i) in identical experimental conditions, the kinematics in the channel thickness of particles of analogous size, shape, and density are analogous; that (ii) flow injection reduces particle/wall interactions to negligible; and that (iii) lifting forces are so intense that no particle/wall retardation effects occur. From the general steric-hyperlayer theory,17,20,21 it can be deduced that in identical experimental conditions, particles of identical shape and density are retained in order of their size, the bigger being eluted first, as shown in Figure 1. Moreover, particles (20) Williams, P. S.; Koch, T.; Giddings, J. C. Chem. Eng. Comm. 1992, 111, 121-147. (21) Williams, P. S.; Lee, S.; Giddings, J. C. Chem. Eng. Comm. 1994, 130, 143166.
Analytical Chemistry, Vol. 74, No. 17, September 1, 2002
4497
of identical size could be separated according to their density,22,23 the more dense being more retained (Figure 1). Using the values of x and the size of the particle, which can be measured by an independent technique (flow cytometry, Coulter counter, or scanning electron microscopy), it is possible to deduce the distance of the particle from the accumulation wall (Figure 1).
δ)x-
d 2
(2)
where d is the average particle population diameter. The δ value was defined by Williams20,21 and is of major importance to assess the goodness of the steric-hyperlayer elution mode, the consequence of which is that δ must be at least twice the average particle radius. Flow Cytometry. In flow cytometry (FC), two principal cell characterization modes, forward scattering19,24,25 and side scattering detection,26 are used. At low angles ( d (d is the average diameter of the sample particles). Figure 5A, curves 1 and 2, corresponded to the smallest yeast strain, that is, Navarra 33 (2.65 µm measured by flow cytometry), and for this yeast, the retention ratios at identical flow rates decreased when the external field increased. The influence of the external field in the retention ratios is higher at high flow rates, and the slope of curve 1 was larger than that of curve 2 (0.34 versus 0.19), which is also in agreement with the elution mode in FFF. In this figure, particles of bigger average size, L1033, (2.85 µm measured by flow cytometry) systematically appeared more retained than the Navarra 33 strain. This result did not agree with the size-dependent elution order predicted by the steric hyperlayer mode and can only be explained if Navarra 33 yeast cells are of
Figure 5. (A) Effect of Flow rate on retention ratio: 2 Navarra 33 at 20 G, b Navarra 33 at 40 G, 9 L1033 at 40 G, / L1033 at 100 G. (B) Effect of low field strength at 0.4 mL/min for three different ADWY: 2 Navarra, b Awri 350, 9 L1033. (C) Effect of field on retention ratio: 2 Navarra 33 at 0.5 mL/min, b Navarra 33 at 1.0 mL/min, 9 L1033 at 1.0 mL/min, / L1033 at 0.5 mL/min. Other conditions as mentioned in Experimental Section.
lower density than L1033, taking into account that both have the same shape distribution (Figure 2B). Elution behavior observed for L1033 (Figure 5, curve 4) at very high external fields (100 G) suggested that even under these conditions, a steric-hyperlayer elution mode occurred. The “steric” elution mode calculation (δ ) 0) made it possible to evaluate the steric barrier. This parameter was calculated for L1033 using eq 1 and replacing x by the average radius given in Table 1 (flow cytometry) and was 0.107 expressed as the retention ratio and 3.569 µm expressed as δ. Because this δ value is higher than the average diameter of the yeast strain (Table 1), it can be deduced that the elution was done in a sterichyperlayer elution mode. External Field Effects. To evaluate the effect of the external field on the retention ratio, low external fields associated with low flow rate (Figure 5B) and high external fields associated with high flow rates (Figure 5C) were applied. In both cases, the retention ratio decreased when the external field increased, which is compatible with the steric-hyperlayer elution mode. The behavior of Navarra 33 and L1033 was studied at two different flow rates (0.5 and 1.0 mL/min, Figure 5C). In both cases, L1033 was retained more than Navarra 33. L1033’s lower retention ratio associated with a bigger average size can only be attributed to possible density differences. Retention ratio differences provoked by different field intensities appear to be more intense at low external fields than at high external ones (Figure 5B and 5C). It is observed that differences between the strains are higher at high external fields. Analytical Chemistry, Vol. 74, No. 17, September 1, 2002
4501
Table 2. FFF Elution Characteristics of Yeast Strains strain
t0 (min)a
tr (min)a
plates no.a
σ (min)a
skewa
excessa
Rra,b
t0 (min)c
tr (min)c
Rrc
∆R
Navarra 33 L1033 Intec Cerev. Killer D-47 Intec Red Awri 350
1.61 1.54 1.52 1.59 1.57 1.60
5.49 5.95 5.42 6.46 5.45 5.68
36.56 62.23 32.87 31.85 38.30 37.22
0.907 0.785 0.945 1.145 0.881 0.931
0.56 0.42 0.82 1.13 0.20 0.52
0.36 1.31 0.77 2.08 0.18 0.03
0.257 0.224 0.243 0.213 0.251 0.246
1.51 1.45 1.46 1.50 1.44 1.49
5.32 5.80 5.07 6.13 5.74 5.52
0.246 0.250 0.288 0.245 0.251 0.270
0.011 -0.026 -0.045 -0.031 0.000 -0.024
a Statistical moments (see ref 29). b Retention ratio ) (V - V )/(V - V ) with V , the void volume and V , the retention volume. V ) connecting 0 c r c 0 r c tube volume + 0.5(detection & injection volume). c Peak summit.
Figure 6. (A) SdFFF fractograms of monomodal yeast samples: (a) Navarra 33 (b) L1033 (c) Intec Cerevisiae. Field strength, 40 G; flow-rate, 0.5 mL/min; other conditions are given in the Experimental Section. (B) SdFFF fractograms of bimodal yeast samples: (a) Killer D-47, (b) Awri 350, (c) Intec Red under the same conditions as in Figure 6A.
Elution Characteristics of Yeast Strains. Under identical experimental conditions, six different yeast strains were eluted in SdFFF, and the fractograms are shown in Figure 6. Peak profiles and retention parameters calculated with different techniques are given in Table 2. It can be mentioned that the elution time was 8-9 min, and only 4 mL of mobile phase was necessary for the analysis. These values are lower than elution time and volume needed when gravitational FFF was used, 25 to 30 min and 5 to 8 mL, respectively.7 Therefore, the ultrathin channel design can be considered as one step in the direction of microFFF separators. Monomodal Yeast Strains. Retention ratios calculated using either the first statistical moment or peak summit mode appeared very well correlated; differences were less than 10%. It should be noticed, from Figure 6A and Table 2 data, that the L1033 strain is the most homogeneous population, which is in agreement with the bidimensional map obtained by flow cytometry (Figure 3). This correlation is not evident when analyzing forward-scattering PSD (Figure 2), which shows the importance of taking into 4502
Analytical Chemistry, Vol. 74, No. 17, September 1, 2002
account all the properties of the cells that are considered in SS patterns. Retention ratios of Table 2 compared to size given in Table 1 showed an inversion in the elution order of Navarra 33 and L1033, which, as has been previously commented, can be attributed to their density differences. Hofstetter-Kuhn et al.6 have also indicated that variations in elution order of yeast cells could be due to differences in density. For S. cerevisiae yeast strains, different density data, such as 1.1130 and 1.02 g/mL,31 have been reported, but for each strain, the density range is 0.01-0.02. These differences can play an important role on FFF separation, as has been demonstrated previously in the elution of red blood cells.23,32 Bimodal Yeast Strains. In these examples, the order or elution parameters obtained using calculation modes, statistical treatment, and peak summit were different (Table 2). This can be explained by the evident bimodality of the Intec Red strain that showed a shoulder in the peak front (Figure 6B, curve c) that produced an important calculation discrepancy between the maximum of the peak (peak summit) and the first statistical moment of the curve. Bimodal distribution patterns of the Awri 350 and Killer D-47 strains are less evident when Figure 6B curves are considered. Moreover, the Killer D-47 strain does not present a qualitatively very different shape from that of the monomodal L1033 (Figure 6A curve b). It is observed that retention ratios calculated by means of the peak summit method are well-correlated to the size for all the yeast strains. Average Position in the Channel Thickness. From the retention data of Table 2 (statistical moments) and using eq 1, the position (x) of the average particle gravity center in the channel thickness can be determined (Table 3). Assuming that the instrumental and injection contributions to band spreading are constant, it is possible to determine the theoretical equivalent position in the channel thickness of particles eluted at the peak front (gravity center elution time - 2 SD) and the peak tail (gravity center elution time + 2 SD); these values are given in Table 3. These hypothetical positions (x) allowed us to calculate δ values at peak front and peak tail for each strain under study. From these values, the hyperlayer thickness, ∆δ, can also be calculated. A “reduced hyperlayer thickness” can be obtained taking into account the average particle diameter. Most of the yeast strains gave values lower than 2, indicating an important effect of focusing in the ultrathin SdFFF channel used. There is, therefore, an experimental confirmation of the “hyperlayer” focusing concept. In fact, the real (30) Baldwin, W. W.; Kubitschek, H. E. J. Bacteriol. 1984, 158, 701-704. (31) Cooney, C. L. In Biotechnology; Rehm, H. J., Reed, G., Eds.; Verlag Chemie: Weinheim, 1981; Vol 1, p 73. (32) Cardot, Ph. J.; Launay, J. M.; Martin, M. J. LC RT 1997, 20 (16/17), 25432553.
Table 3. Hyperlayer Elution Mode, x and δ Values
strain
gravity center
x (µm)a peak front
Navarra 33 L1033 Intec Cerev. Killer D-47 Intec Red Awri 350
6.47 6.56 6.69 5.69 5.96 6.84
10.49 8.24 10.22 9.03 10.14 10.00
peak tail 5.08 4.67 4.73 4.15 4.99 4.88
δ (µm)b peak frontc peak taild 9.164 6.814 8.515 7.353 8.465 8.312
3.753 3.245 3.025 2.478 3.316 3.193
hyperlayer thickness ∆δ (µm)
reduced hyperlayer thickness (∆δ/diam)
5.411 3.569 5.490 4.875 5.149 5.119
2.039 1.251 1.606 1.457 1.535 1.519
a Calculated from data Table 2. b Calculated from x and data Table 1. c Front ) gravity center elution time - 2 SD. d Tail) gravity center elution time +2 SD.
Figure 7. (A) (I) FS SS correlation map for Killer D-47 crude sample. Y axis is given as an SS signal (gain 20); X axis is given as an FS signal (gain 15). (II) SEM photograph of the same sample. (III) SdFFF fractogram of the cumulated signal showing the two collected interval fractions (IV). Flow cytometry patterns as an SS-FS map for fraction 1. (V) Flow cytometry pattern as an SS-FS map for fraction 2. All conditions are given in the Experimental Section. (B) (I) FS pattern of the collected fractions for the same sample, as compared to the crude. (II) SS pattern of the collected fractions for the same sample, as compared to the crude. All of the experimental conditions are given in the Experimental Section.
focalization effect is even greater than the one calculated, because the experimental measurements of x and ∆δ did not take into account the effect of the sample injection volume. This major focusing effect can be the reason bimodality was not clearly observed for Killer D-47 yeast using SdFFF (Figure 6B). Killer D-47 Yeast Strain Analysis. Figure 7A I and II shows the bidimensional FS/SS correlation map as well as the SEM photograph of the crude sample, where different cell sizes and shapes can be observed. To analyze the bimodal aspect of Killer D-47 strain, four sample injections were performed, and the cumulative fractogram is shown in Figure 7A III. For every elution,
two different fractions, 1 and 2, were systematically collected and pooled. FS and SS correlation maps obtained for each fraction are given in Figure 7A IV,V. Very different qualitative patterns were obtained, and fraction 2 is more similar to the crude sample than fraction 1. FS and SS signals corresponding to the purified fractions are given in Figure 7B, and the quantitative data are presented in Table 4. Cells eluted in the first fraction have a higher average volume, and both fraction polydispersities are different from the one of the crude sample. The very different flow cytometry pattern of both cumulated fractions is a proof of the effective repeatability of SdFFF elution for biological species. Analytical Chemistry, Vol. 74, No. 17, September 1, 2002
4503
Table 4. Killer D-47 Collected Fraction Analysis fraction 1 2
flow cytometry collected vol flow cytometry polydispersity no. of cells mL × 4 av diam (µm) σ × 100/mean 99 603 99 572
0.46 0.54
3.50 ( 0.65 3.30 ( 0.58
17.55 18.57
The forward-scattering signal of fraction 2 compared to crude (Figure 7B I) showed an analogous size distribution. The major difference is the high enrichment of particles of greater size in fraction 1 and the decrease of these particles in fraction 2. Moreover, the side-scattering signal (Figure 7B II) showed that fraction 1 corresponded to particles with shape that is different from fraction 2 particles, which have a distribution similar to the general population. From Figure 7 and Table 4, it can be concluded that the separated fractions are not only different in size but that other characteristics, such as shape, surface properties, and internal composition mainly described by SS signal, are also different. SdFFF made it possible to separate fractions that corresponded to populations with mean size that differed by only 200 nm, although the nonequilibrium state produced by sample injection after the establishment of the flow rate did not permit observation of a bimodal distribution for sample Killer D-47. CONCLUSIONS A new SdFFF channel with a reduced thickness (80 µm) and reduced extrachannel volumes was built, expanding the domain of submilliliter FFF that permits cells fractionation and separation with a decrease in analysis time. As a consequence, a reduction of carrier-phase consumption as well as limited dilution of the sample were obtained. These advantages will make it possible soon to on-line couple SdFFF and characterization techniques, such as flow cytometry. However, FFF-FC hyphenated information is now available by means of appropriate fraction collections at the SdFFF outlet and subsequent FC analyses. The flow cytometry FS signal can be related after proper calibration to size or volume, but the complexity of the SS signal, linked to shape, surface properties, and cell composition can give only qualitative information.
4504
Analytical Chemistry, Vol. 74, No. 17, September 1, 2002
The simplified SdFFF instrumentation and analysis procedure with sample injection via the accumulation wall when field and flow are established have been used to study yeast strains and the fractogram profiles obtained coupled to FC analyses showed that the yeast cell population can be characterized. The remarkable repeatability of FFF experiments (5% of RSD) and their rapidity (2-10 min) make SdFFF suitable for use in quality control. It has also been demonstrated that for yeast strains, elution occurred according to the steric-hyperlayer mode, with a major focusing effect on the species in the channel thickness. Moreover, in SdFFF, cells with small differences in size can be separated, emphasizing other characteristics, such as density. Although accurate density determination of particles of mineral or organic origin, such as silica or latex beads, can be performed using appropriate gradient density measurements, it becomes more complicated for living species. This points to a new potentiality of SdFFF because, in addition to size distribution, information about shape and density could also be obtained from retention data, provided there is the proper calibration. ACKNOWLEDGMENT Acknowledgments go to Sophie Crouchet of LCAB (Universite´ de Limoges, France), Josep Guasch of the Group of Quı´mica Analı´tica Enolo`gica i dels Aliments (Universitat Rovira i Virgili, Tarragona, Spain), and Lluı´s Mestres of Tensum Ibe´rica, S.L. (Barcelona, Spain) for technical support and sample management. P. Reschiglian, Universita` di Bologna, is thanked for establishing connections between Universitat de Barcelona, Spain, and Universite´ de Limoges, France, resulting in a “yeast characterization network”. This work was supported in part by Region Limousin “Aide Re´gionale a` la Recherche Scientifique” Grant No. 2000 REC 4.1-S737. Ramse´s Sanz thanks the Universitat de Barcelona for a grant (Beca de Formacio´ en la Recerca i la Doce`ncia).
Received for review December 4, 2001. Accepted June 3, 2002. AC011234J
