- Email: [email protected]
Saccharomyces uvarum, a distinct group within Saccharomyces sensu stricto
FEMS Microbiology Letters 177 (1999) 177^185
Saccharomyces uvarum, a distinct group within Saccharomyces sensu stricto Sandra Rainieri a; *, Carlo Zambonelli a , John E. Hallsworth b , Andrea Pulvirenti c , Paolo Giudici c a
Dipartimento di Protezione e Valorizzazione Agroalimentare (DIPROVAL), Universita© di Bologna, Villa Levi, via F.lli Rosselli 107, Reggio Emilia 42100, Italy b Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa c Istituto di Industrie Agrarie, Universita© di Catania, via S. So¢a 98, Catania 95125, Italy Received 16 February 1999; received in revised form 10 May 1999; accepted 17 May 1999
Abstract A natural subgroup (that we refer to as Saccharomyces uvarum) was identified, within the heterogeneous species Saccharomyces bayanus. The typical electrophoretic karyotype, interfertility of hybrids between strains, distinctive sugar fermentation pattern, and uniform fermentation characteristics in must, indicated that this subgroup was not only highly homogeneous, but also clearly distinguishable from other species within the Saccharomyces sensu stricto group. Investigation of the S. bayanus type strain and other strains that have been classified as S. bayanus, confirmed the apparent lack of homogeneity and, in some cases, supported the hypothesis that they are natural hybrids. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Phenotypic characteristics; Saccharomyces bayanus; Saccharomyces sensu stricto; Saccharomyces uvarum; Taxonomy
1. Introduction The classi¢cation of Saccharomyces species, including those in the Saccharomyces sensu stricto group that are commonly used in the fermentation industries, has always been problematic at species level. According to the latest classi¢cation, the S. sensu stricto group is made up of four species: Saccharomyces bayanus, Saccharomyces cerevisiae, Sac* Corresponding author. Institute for Wine Biotechnology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa. Tel.: +27 (21) 808 3770; Fax: +27 (21) 808 3771; E-mail: [email protected]
charomyces paradoxus and Saccharomyces pastorianus [1]. The identi¢cation of these species was based mainly on DNA hybridisation analyses and the classi¢cation seems natural, with the sole exception of S. bayanus. It was recently suggested that S. bayanus can be divided into two subgroups: a bayanus group (including the type strain) and a uvarum group (not currently recognised as a distinct species). This grouping was based on PCR ampli¢cation and restriction polymorphism of the non-transcribed spacer 2 in the ribosomal DNA unit, and on electrophoretic karyotypes [2]. In 1970, S. uvarum was considered a distinct species according to a taxonomic study by
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 5 9 - 1
FEMSLE 8852 15-7-99
178
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185
van der Walt, based on variation in physiological characteristics [3]. In a later classi¢cation, all members of the S. sensu stricto group were attributed to S. cerevisiae and the term `uvarum' was used to describe one of the 17 physiological races into which S. cerevisiae had been divided [4]. By contrast, the most recent classi¢cation considers most of the strains that were classi¢ed as S. uvarum [3] indistinct from the species S. bayanus [1]. Recent taxonomic studies found that many Saccharomyces strains isolated from cold-stored grape juice (that ferment vigorously at low temperatures) produce an electrophoretic karyotype that is consistent with that of strains classi¢ed as uvarum by many [2], but distinct from that of the type strain of S. bayanus [5,6]. Such strains are quite widespread in nature, and are also commonly found in wine and other fermented beverages [7^9]. They exhibit a typical fermentation pro¢le in grape must that is clearly di¡erent from that of S. cerevisiae strains; they produce lower amounts of acetic acid, higher amounts of glycerol and succinic acid, and synthesise malic acid rather than degrading it [10,11]. This study was carried out to establish whether strains that produce an electrophoretic karyotype consistent with that of strains that were attributed to the uvarum group [2] make up a homogeneous and distinct group in terms of phenotypic characteristics. These included such as sugar fermentation pattern (i.e., ability to ferment speci¢c sugars), fermentation pro¢le (i.e., production of glycerol, succinic acid, acetic acid and ethanol, e¡ect on ¢nal malic acid concentration, fermentation vigour, and amount of residual sugars), and ability to produce fertile hybrids. Such information could then be used as a basis for examination of strains using molecular techniques.
2. Materials and methods 2.1. Yeast strains The yeast strains that were used are listed in Table 1. All the wine strains were obtained from the DIPROVAL collection (Dipartimento di Protezione e Valorizzazione Agroalimentare, University of Bologna, Reggio Emilia, Italy). Some of these, DIPROV-
AL 11204, DIPROVAL 12233, DIPROVAL C31 and DIPROVAL M12, represent a group of strains isolated from cold-stored grape juice (at di¡erent times and places), and were described in previous studies [8,11]. 2.2. Media Strains were maintained on Sabouraud dextrose agar (Oxoid, UK) and sporulated in acetate agar (sodium acetate 0.1%, w/v; agar 2%, w/v). Growth was studied over a range of temperatures in Sabouraud dextrose broth (Oxoid), and spore extraction and conjugation were conducted in yeast peptone dextrose broth (YPD: yeast extract 2%, w/v; peptone 2%, w/v; dextrose, 4%, w/v). Some yeasts carry out fermentation very slowly, and cannot exhaust sugars in synthetic media with a high sugar content ( s 10%, w/v). In the present study, therefore, grape juice was used as the fermentation medium. Grape juice provides a balance of nutrients that produce consistent and reproducible results for strain characterisation, despite slight variation in composition between batches. Fermentations were carried out in must produced from the Vitis vinifera cultivar Trebbiano (containing 210 g sugar l31 ) after dilution with a 1% yeast extract solution (w/v) to reduce the sugar concentration to 140 g l31 . 2.3. Preparation of chromosomal DNA and pulsed-¢eld gel electrophoresis (PFGE) Chromosome extraction was performed using the Schwartz and Cantor technique [12], as modi¢ed by Vaughan-Martini et al. [13]. PFGE was carried out with a contour-clamped homogeneous electric ¢eld (CHEF) mapper system (Bio-Rad, USA). The gel was stained for 1 h using ethidium bromide (1 Wg l31 ) at 25³C, decolourised in tris-borate EDTA bu¡er for 24 h (at 4³C), and then photographed. A more detailed description of the procedure has been given previously [6]. 2.4. Physiological characteristics Ability to ferment di¡erent sugars was assessed according to the methods of Kreger van Rij [14].
FEMSLE 8852 15-7-99
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185
179
The optimum temperature for growth was determined using a temperature gradient incubator constructed as described by Packer et al. [15]. This method was used to establish the temperature at which the maximum cell multiplication rate occurs, by 15 to 20 h from inoculation.
3.11 Base System (SPSS Inc., USA). The fermentations were carried out in triplicate, and mean values for glycerol, succinic acid, acetic acid, malic acid, residual sugar, and ethanol concentrations are listed in Table 3.
2.5. Fermentation tests
3. Results
Fermentations were carried out in a 25³C incubator in must (100 ml) that had been heated to 90³C for 20 min to eliminate the natural micro£ora. Fermentation was assessed by determining the weight loss caused by CO2 release at frequent intervals, until a constant weight was attained. This weight loss, determined during a 48 h period, represented fermentation vigour.
3.1. Optimum temperature for growth
2.6. Analysis of fermentation samples, post-fermentation
All the strains with an upper temperature limit for growth that was greater than 37³C had an optimum growth temperature that was greater than 30³C. The strains with a temperature limit for growth below 37³C had an optimum growth temperature that was below 30³C, and these were all strains of either S. bayanus or S. pastorianus (data not shown). 3.2. Electrophoretic karyotypes
Determinations of pH, and ethanol and sugar concentration were carried out using standard methods for wine analysis [16]. Glycerol, succinic acid, acetic acid and malic acid were quanti¢ed using enzymatic kits (Boehringer Mannheim, Germany). Glycerol, succinic acid and acetic acid concentrations were expressed as g compound 100 ml31 ethanol so that the results obtained were comparable, even for strains that could not ferment all the sugar provided. 2.7. Sporulation, spore viability and hybridisation Cultures were sporulated using the methods described by Kreger-van Rij [14]. Spore germination tests were carried out in an oil chamber, asci were broken and individual spores separated using a micromanipulator. Single spores were then transferred to microdrops of YPD. Germination and culture formation from spores were determined by light-microscopic examination after 1 to 2 days at 25³C. Hybrids were obtained by spore conjugation [17]. 2.8. Replication and statistical analyses Data relating to fermentation performance and fermentation byproducts were subjected to principal component analysis using SPSS 6.1 for Windows
The electrophoretic karyotypes for all the strains studied that cannot grow at temperatures above 37³C, and of the S. cerevisiae type strain, are shown in Figs. 1 and 2 (the other strains, classi¢ed unequivocally as S. cerevisiae or S. paradoxus, were not of primary interest in the present study). A karyotype characterised by only two bands in the region between 365 and 225 kb was produced by all the wine strains isolated from cold-stored juice (i.e. DIPROVAL 11204, DIPROVAL 12233, DIPROVAL C31, DIPROVAL M12), and from strains CBS 395, CBS 1604 and CBS 7001 (Fig. 1, lanes 2^5; Fig. 2, lanes 7^10). By contrast, most Saccharomyces strains produce karyotypes with three or more bands in this region [6]. The remaining strains tested exhibited a di¡erent electrophoretic pro¢le (Fig. 1, lanes 6^9; Fig. 2, lanes 2^6, respectively). The band pattern obtained from the type strains of S. pastorianus and S. bayanus (Fig. 1, lane 6 and Fig. 2, lane 2, respectively) did not typify those obtained from the other strains of each species, and the pro¢les were not uniform within these taxonomic groups. 3.3. Ability to ferment di¡erent sugars In 1984, S. sensu stricto strains were all grouped under the speci¢c name S. cerevisiae, and according
FEMSLE 8852 15-7-99
180
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185
to the fermentation of a range of sugars, S. cerevisiae was divided into 17 physiological races [4]. This method was used to con¢rm the identity of the strains examined in the present study, as shown in Table 2. Strains currently classi¢ed as S. bayanus and S. pastorianus [1] were found to vary in their ability to ferment sugars. Nevertheless, within S. bayanus there was a number of strains that exhibited the typical sugar fermentation pattern of the former physiological race uvarum (Table 2) [4]. These strains Table 1 List of Saccharomyces strains used in the present study Original speci¢c name a
S. bayanus S. uvarumb S. globosus S. heterogenicus S. tubiformis S. inusitatus S. bayanus S. abuliensis S. bayanus S. pastorianusa S. pastorianus S. carlbergensis S. paradoxusa S. paradoxus S. paradoxus S. cerevisiaea S. cerevisiae S. cerevisiae Wine strains S. bayanus S. bayanus S. bayanus S. bayanus S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae
CBS no.
Other culture collection nos.
380 395 424 425 431 1546 1604 7001
CLIB 181, DBVPG 6171 CLIB 251 CLIB 250 CLIB 255 CLIB 389 CLIB 252 CLIB 253, DBVPG 6259 CLIB 283 CCY 21-6-1, CLIB 271 CLIB 281, DBVPG 6047 DBVPG 6257 CLIB 176, DBVPG 6033 CLIB 228, DBVPG 6411 DBVPG 6466 DBVPG 6304, UCD 51^186 DBVPG 6173 DBVPG 6292 DBVPG 6295
1538 1260 1513 432 5829 1171 4054 5635
DIPROVAL DIPROVAL DIPROVAL DIPROVAL DIPROVAL DIPROVAL DIPROVAL DIPROVAL DIPROVAL
11204 12233 C31 M12 6167 7070 10003 11052 29001
CBS: Centraalbureau voor Schimmelcultures, Baarn, Netherlands. CCY : Czechoslovak Collection of Yeasts, Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia. CLIB: Collection de Levures d'Interet Biotechnologique, Thiverval-Grignon, France. DPVPG : Dipartimento di Biologia Vegetale, Universita© di Perugia, Perugia, Italy. DIPROVAL : Dipartimento di Protezione e Valorizzazione Agroalimentare, Universita© di Bologna, Reggio Emilia, Italy. UCD: Department of Viticulture and Enology, University of California, Davis, CA, USA. a Type strains. b Former type strain from the time when S. uvarum was recognised as a species [3].
Fig. 1. Contour clamped homogeneous electric ¢eld (CHEF) banding patterns of chromosomal DNA of Saccharomyces strains not growing above 37³C, and that of the S. cerevisiae type strain CBS 1171. Lane 1: S. bayanus strain CLIB 271, lanes 2 to 5: S. uvarum wine strains DIPROVAL 12233, DIPROVAL 11204, DIPROVAL C31 and DIPROVAL M12 respectively, lane 6: S. pastorianus type strain CBS 1538, lanes 7 and 8: S. pastorianus strains CBS 1260 and CBS 1513, lane 9: S. cerevisiae type strain CBS 1171, lane 10: S. cerevisiae yeast chromosomal DNA size standard (strain YPH80, BioLabs, USA).
were all the wine strains isolated from cold-stored must, and strains CBS 395, CBS 1604, and CBS 7001. 3.4. Fermentation properties in grape juice The results of the fermentations carried out in grape juice (containing 140 g sugar l31 ) are shown in Table 3. The strains classi¢ed as S. cerevisiae and S. paradoxus [1] produced a homogeneous fermentation pro¢le. They were vigorous, generally resistant to ethanol, and gave higher ethanol yields from the sugar fermented (approximately 60%, v/w). The mean amount of malic acid was reduced and the mean amount of glycerol produced was 5.4 g 100 ml31 ethanol, of succinic acid 0.8 g 100 ml31 ethanol, and of acetic acid 0.3 g 100 ml31 ethanol. Surprisingly, the least vigorous and least ethanol-resistant strain was the type strain of S. cerevisiae (CBS 1171). Strains belonging to S. bayanus and S. pastorianus produced a heterogeneous fermentation pro¢le. The wine strains DIPROVAL 11204, DIPROVAL 12233, DIPROVAL C31 and DIPROVAL M12 resembled strains CBS 1604 and CBS 7001, in terms of both their sugar fermentation pattern (Table 2), which was typical of S. cerevisiae physiological race uvarum [4], and their electrophoretic pro¢le (Figs. 1
FEMSLE 8852 15-7-99
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185
181
levels of succinic acid (on average 0.92 g 100 ml31 ethanol), and small amounts of acetic acid (on average 0.16 g 100 ml31 ethanol) (Table 3). The strain CBS 395, possessing the same karyotype as the above-mentioned strains, showed a fermentation pro¢le which was not consistent with that obtained from these strains, but resembled that of S. cerevisiae strains (Table 3). Although the S. pastorianus strains studied represented a fairly homogeneous group, the fermentation characteristics were close to those of S. cerevisiae strains (Table 3). Strains of S. pastorianus degraded malic acid and produced less glycerol, succinic acid and acetic acid than S. cerevisiae strains. Two of the S. pastorianus strains examined did not ferment all the sugar provided. None of the remaining strains could be ascribed to either of the above-mentioned groups. Indeed, although they had all been classi¢ed as S. bayanus [1] they form a heterogeneous group, suggesting that S. bayanus has been used as a `dustbin' taxon. In particular, strains CBS 380 and CBS 1505 exhibited some of the traits typical of the cold-isolated wine strains that were mentioned above (these include the synthesis of malic acid and the production of low
Fig. 2. Contour clamped homogeneous electric ¢eld (CHEF) banding patterns of chromosomal DNA of Saccharomyces strains not growing above 37³C. Lane 1: S. cerevisiae DNA size standard (strain YPH80) ; lane 2: S. bayanus type strain CBS 380, lane 3: S. intermedius strain CBS 1505, lane 4: S. globosus strain CBS 424, lane 5: S. inusitatus strain CBS 1546, lane 6: S. heterogenicus strain CBS 425, lane 7: S. uvarum strain CBS 395, lane 8: S. bayanus strain CBS 1604, lane 9: S. tubiformis strain CBS 431, lane 10: S. abuliensis strain CBS 7001.
and 2). All these strains exhibited a uniform fermentation pro¢le in grape juice, which is distinctive for this group: they fermented all the sugar provided, synthesised malic acid, produced high amounts of glycerol (on average 6.92 g 100 ml31 ethanol), high
Table 2 Ability to ferment di¡erent sugars for strains with an upper temperature limit for growth of 37³C Strain
Glucose
Galactose
Maltose
Melibiose
Ra¤nose
Sucrose
Physiological racea
CBS 380 CBS 395 CBS 424 CBS 425 CBS 431 CBS 1260 CBS 1505 CBS 1513 CBS 1538 CBS 1546 CBS 1604 CBS 7001 CLIB 271 DIPROVAL DIPROVAL DIPROVAL DIPROVAL
+ + + + + + + + + + + + + + + + +
3 + + 3 + + + + + 3 + + + + + + +
+ + 3 + + + + + + + + + + + + + +
3 + 3 3 3 + 3 3 3 +c + + 3 + + + +
+ + 3 3 + + + + + + + + + + + + +
+ + 3 + + + + 3 + + + + + + + + +
bayanus uvarum globosus heterogenicus cerevisiae uvarumb cerevisiae cerevisiae cerevisiae inusitatus uvarum uvarum cerevisiae uvarum uvarum uvarum uvarum
11204 12233 C31 M12
The strains that grew above 37³C belong unequivocally to S. cerevisiae or S. paradoxus, and were not of primary interest in this study. a According to Yarrow [4], who classi¢ed all S. sensu stricto strains as S. cerevisiae. b Recently, this strain has been found to resemble uvarum strains only in terms of sugar fermentation pattern, and it is now classi¢ed as S. pastorianus [1] (Table 3). c Poor growth.
FEMSLE 8852 15-7-99
182
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185
Table 3 Composition of wine produced from must containing 140 g sugar l31 Straina S. uvarumc CBS 395 CBS 1604 CBS 7001 DIPROVAL 11204 DIPROVAL 12233 DIPROVAL C31 DIPROVAL M12 S. bayanus CBS 380 CBS 424 CBS 425 CBS 431 CBS 1505 CBS 1546 CLIB 271 S. pastorianus CBS 1538 CBS 1260 CBS 1513 S. paradoxus CBS 432 CBS 5829 UCD 51^186 S. cerevisiae CBS 1171 CBS 4054 CBS 5635 DIPROVAL 6167 DIPROVAL 7070 DIPROVAL 10003 DIPROVAL 11052 DIPROVAL 29001
Glycerolb
Succinic acidb
Acetic acidb
Malic acid (g l31 )
Residual sugars (g l31 )
Alcohol (%, v/v)
5.75 6.68 6.62 7.13 7.96 6.46 6.68
0.582 0.803 0.876 0.980 1.047 0.907 0.938
0.460 0.192 0.289 0.158 0.123 0.077 0.165
1.183 1.476 1.641 1.951 2.036 1.476 1.663
61 6.36 61 61 61 61 61
7.8 8.1 7.8 7.8 7.8 7.6 7.6
5.64 3.98 3.93 4.74 5.65 8.81 3.08
0.613 0.516 0.513 0.425 0.749 0.589 0.404
0.027 0.216 0.005 0.527 0.006 0.768 0.643
1.471 1.183 1.308 1.256 1.618 1.202 1.179
6.36 21.67 11.87 3.78 61 3.15 10.86
7.3 6.0 6.6 7.6 7.9 7.6 7.1
3.33 5.84 3.33
0.508 0.567 0.508
0.007 0.482 0.135
1.285 1.148 1.223
10.3 61 8.28
7.8 7.9 6.8
5.35 4.70 5.49
0.894 0.654 0.847
0.007 0.130 0.130
1.001 1.148 0.864
61 61 61
7.9 7.9 7.8
5.97 4.39 3.79 6.46 5.23 5.80 5.96 5.97
0.404 1.030 0.750 0.821 0.775 1.127 0.912 0.859
0.444 0.351 0.419 0.259 0.472 0.358 0.297 0.334
1.039 1.152 0.845 1.157 1.195 1.020 1.091 1.025
10.01 61 61 61 61 61 61 61
7.4 8.2 8.2 8.4 8.3 7.9 8.0 8.3
The values listed are means of triplicate analyses. Current or former type strains are listed ¢rst for each species. Strains are listed as S. bayanus, S. pastorianus, S. paradoxus and S. cerevisiae according to [1]. b Values were expressed as g 100 ml31 ethanol. c These strains, that have been referred to variously as S. cerevisiae physiological race uvarum, S. bayanus or S. bayanus subgroup uvarum, are listed here as S. uvarum. a
amounts of acetic acid). On the other hand, some traits were characteristic of S. cerevisiae, such as the amounts of glycerol and succinic acid produced. Strains CBS 395, CBS 424, CBS 425, CBS 431, CBS 1546 and CLIB 271, that are all classi¢ed as S. bayanus [1], did not synthesise malic acid, but degraded it (Table 3). They produced average amounts of glycerol and succinic acid, and varying amounts of acetic acid. These strains did not ferment vigorously and were unable to complete fermentation, leaving high
amounts of residual sugar. In addition, these strains gave a pro¢le of fermentation very similar to that of S. pastorianus. 3.5. Sporulation ability and fertility Strains belonging to the group exhibiting the sugar fermentation pattern of S. cerevisiae physiological race uvarum, except for strain CBS 395, sporulated very well and produced numerous four-spore asci.
FEMSLE 8852 15-7-99
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185
Spore germination was close to 100% for these strains (data not shown). Hybridisation experiments resulted in the production of four hybrids: DIPROVAL 11204U12233, DIPROVAL 11204UC31, DIPROVAL 11204UM12 and DIPROVAL 12233UC31. These hybrids sporulated well, were fertile and produced viable spores. The type strain of S. bayanus (CBS 380), as well as strains CBS 1505, CBS 1546 and CLIB 271, did not sporulate. Strains CBS 395, CBS 424, CBS 425 and CBS 431 sporulated, but very slightly. These four strains were sterile due either to their inability to sporulate or because of the production of non-viable spores. In some cases the number of asci formed during sporulation was so low that germination tests could not be carried out. All of the S. pastorianus strains studied were sterile. 3.6. Principal component analysis Principal component analysis of the fermentation performance and fermentation byproducts indicated that the ¢rst two principal components accounted for 70.05% of variability. Principal component 1 (PC1) accounted for 43.78%; it was positively correlated with ethanol production and fermentation vigour, and negatively correlated with the residual sug-
Fig. 3. Principal component analysis of the fermentation performance and wine composition (especially the associated acidity) of the strains studied: S. cerevisiae (S); S. paradoxus (b); S. pastorianus (E); S. uvarum (F) according to the present study; and S. bayanus (a). Principal component 1 (PC1) re£ects the ¢tness of each strain for fermentation. Principal component 2 (PC2) re£ects the production of malic acid and the generation of an acidic environment.
183
ars, glucose and fructose. Component 1 e¡ectively represents overall fermentation ¢tness. This component separated vigorous, ethanol-tolerant strains able to complete the fermentation process from all the other strains. The vigorous ethanol-tolerant group was represented by all the S. cerevisiae strains except for the type strain CBS 1171, the strains isolated from cold-stored grape juice, the three S. paradoxus strains and one S. bayanus strain (CLIB 271) (Fig. 3). Principal component 2 (PC2) accounted for 26.27% of the variability. Component 2 could be interpreted as a measure of the associated acidity, being positively correlated with malic acid and negatively with pH. A slight correlation with glycerol production was also detected. Component 2 separated S. uvarum strains from those of S. cerevisiae and other species (Fig. 3). The other strains were dispersed and appeared both above and below the x-axis in the principal component analysis plot shown in Fig. 3.
4. Discussion The present study demonstrated the existence of a natural and homogeneous, but currently unrecognised group of strains within S. sensu stricto (that had been previously referred to as S. bayanus subgroup uvarum [2]). These strains exhibit (i) the typical sugar fermentation pattern of the former S. cerevisiae physiological race uvarum [4], which used to be considered a species in itself [3]; (ii) a typical electrophoretic karyotype, characterised by the presence of two bands in the area between 365 and 225 kb; (iii) a clearly de¢ned fermentation pro¢le in grape juice which di¡ers from that of S. cerevisiae and S. paradoxus strains in the amount of glycerol, succinic acid, and acetic acid produced, as well as whether they synthesise or degrade malic acid; and (iv) a high sporulation ability, and the spores are characterised by a high viability. The homogeneity of this group was con¢rmed by the fact that the strains studied produce intraspeci¢c fertile hybrids. Strains DIPROVAL 11204 and CBS 7001, the ancient type strain of S. abuliensis (reclassi¢ed within S. bayanus subgroup uvarum [2]), were the most representative strains of the group. According to the data obtained in the present study, and that obtained in
FEMSLE 8852 15-7-99
184
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185
earlier work [2,3], we refer to this group as S. uvarum. The remaining strains that are classi¢ed as S. bayanus [1] do not constitute a homogeneous group; they di¡er amongst themselves and are also distinct from the S. uvarum strains mentioned above. The S. bayanus type strain (CBS 380) exhibits a fermentation pro¢le similar to that of the hybrids between S. cerevisiae and S. uvarum [17,23] and, like these, produces a karyotype with a higher number of bands than that of S. cerevisiae or other S. bayanus strains [18,19]. The existence of natural hybrids between different species, or within the same species, has already been suggested by several authors [20^22]; the characteristics of strains CBS 380, CBS 1505 and CLIB 271 support the hypothesis that they are the result of hybridisation. Investigations carried out at a molecular level are also consistent with a hybrid origin (H.V. Nguyen and C. Gaillardin, personal communication). The other S. bayanus strains, as well as the strains of S. pastorianus that were studied, all demonstrated fermentation properties similar to those of S. cerevisiae strains, but not the fermentation vigour. Considering their sterility, the higher number of bands in their karyotypes and the non-homogeneous sugar fermentation pattern, a hybrid origin for these strains seems likely. Studies of phenotypic characteristics and electrophoretic karyotypes can be used to identify a natural taxon, and molecular techniques can then be employed to con¢rm or re¢ne the classi¢cation. The present study demonstrated that S. uvarum is a natural group within S. sensu stricto, and this is not acknowledged in the contemporary classi¢cation [1]. We think that a re-evaluation of the strains currently classi¢ed as S. bayanus, and a re-consideration of the type strain, is now needed to gain a more natural classi¢cation of the Saccharomyces genus.
Acknowledgements We are indebted to Professor C. Gaillardin and Dr. H.V. Nguyen (INRA-INA, Theverval-Grignon, France) for providing CLIB strains, and to H.V. Nguyen for sharing molecular typing results as well as for helpful discussion. Financial support (a MURST 40% award) was received from the Minis-
tero dell' Universita© e Ricerca Scienti¢ca e Tecnica, Italy.
References [1] Vaughan-Martini, A. and Martini, A. (1998) Saccharomyces Meyen ex Reess. In: The Yeasts, A Taxonomic Study (Kurtzman, C.P. and Fell, J.W., Eds.), 4th Edn., pp. 358^371. Elsevier, Amsterdam. [2] Nguyen, H.V. and Gaillardin, C. (1997) Two subgroups within the Saccharomyces bayanus species evidenced by PCR ampli¢cation and restriction polymorphism of the non-transcribed spacer 2 in the ribosomal DNA unit. Syst. Appl. Microbiol. 20, 286^294. [3] van der Walt, J.P. (1970) Genus 16 Saccharomyces Meyen emend. Reess. In: The Yeasts. A Taxonomic Study (Lodder, J., Ed.), pp. 555^718. North-Holland Publishing Company, Amsterdam. [4] Yarrow, D. (1984) Genus Saccharomyces Meyen ex Rees. In: The Yeasts. A Taxonomic Study (Kreger-van Rij, N.J.W., Ed.), 3rd Edn., pp. 379^396. Elsevier, Amsterdam. [5] Naumov, G., Naumova, E. and Gaillardin, C. (1993) Genetic and karyotypic identi¢cation of wine Saccharomyces bayanus yeasts isolated in France and Italy. Syst. Appl. Microbiol. 16, 274^279. [6] Giudici, P., Caggia, C., Pulvirenti, A. and Rainieri, S. (1998) Karyotyping of Saccharomyces strains with di¡erent temperature pro¢les. J. Appl. Microbiol. 84, 811^819. [7] Kusewics, D. (1975) Characteristics of fermentation abilities in some varieties of wine yeast of cryophilic type. Acta Alim. Polonica 25, 235^245. [8] Castellari, L., Pacchioli, G., Zambonelli, C., Tini, V. and Grazia, L. (1992) Isolation and initial characterisation of cryotolerant Saccharomyces strains. Ital. J. Food Sci. 3, 179^223. [9] Kishimoto, M., Shinohara, T., Soma, E. and Goto, S. (1993) Selection and fermentation properties of cryophilic wine yeasts. J. Ferment. Bioeng. 75, 451^453. [10] Kishimoto, M., Shinohara, T., Soma, E. and Goto, S. (1993) Identi¢cation and enological characteristics of cryophilic wine yeasts. J. Brew. Soc. Jpn. 88, 708^713. [11] Castellari, L., Ferruzzi, A., Magrini, A., Giudici, P., Passarelli, P. and Zambonelli, C. (1994) Unbalanced wine fermentation of cryotolerant vs non-cryotolerant Saccharomyces strains. Vitis 33, 49^52. [12] Schwartz, D.H. and Cantor, C.R. (1984) Separation of yeast chromosome-sized DNAs by pulsed-¢eld gradient gel electrophoresis. Cell 37, 67^75. [13] Vaughan-Martini, A., Martini, A. and Cardinali, G. (1993) Electrophoretic karyotyping as a taxonomic tool in the genus Saccharomyces. Antonie van Leeuwenhoek 62, 145^156. [14] Kreger-van Rij, N.J.W. (1984) The Yeasts. A Taxonomic Study, 3rd Edn. Elsevier, Amsterdam. [15] Packer, G.J.K., Prentice, G.A. and Clegg, L.F.L. (1973) Design of a temperature gradient incubator. J. Appl. Bacteriol. 36, 173^177.
FEMSLE 8852 15-7-99
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185 [16] Ough, C.S. and Amerine, M.A. (1988) Methods for Analysis of Musts and Wines. John Wiley and Sons, New York. [17] Zambonelli, C., Passarelli, P., Rainieri, S., Bertolini, L., Giudici, P. and Castellari, L. (1997) Technological properties and temperature response of interspeci¢c Saccharomyces hybrids. J. Sci. Food Agric. 74, 7^12. [18] Jolly, N.P., Janse, B.J.H., Van Rooyen, T.J. and Louw, J.H. (1993) Hybridization and typing of yeasts used in sparkling wine fermentations. Am. J. Enol. Vitic. 44, 217^ 226. [19] Giudici, P., Caggia, C., Pulvirenti, A., Zambonelli, C. and Rainieri, S. (1998) Electrophoretic pro¢le of hybrids between cryotolerant and non-cryotolerant Saccharomyces strains. Lett. Appl. Microbiol. 27, 31^34.
185
[20] Vaughan-Martini, A. and Martini, A. (1987) Three newly delimited species of Saccharomyces sensu stricto. Antonie van Leeuwenhoek 53, 77^84. [21] Naumov, G.I., Naumova, E. and Korhola, M. (1992) Genetic identi¢cation of natural Saccharomyces sensu stricto yeasts from Finland, Holland and Slovakia. Antonie van Leeuwenhoek 61, 237^243. [22] Masneuf, I., Hansen, J., Groth, C., Piskur, J. and Dubourdieu, D. (1998) New hybrids between Saccharomyces sensu stricto yeast species found among wine and cider production strains. Appl. Environ. Microbiol. 64, 3887^3892. [23] Wingen, O. and Laustsen, O. (1938) Arti¢cial species hybridisation in yeast. Comp. Ren. Trav. Lab. Carlsberg Ser. Physiol. 22, 235^244.
FEMSLE 8852 15-7-99
Saccharomyces uvarum, a distinct group within Saccharomyces sensu stricto Sandra Rainieri a; *, Carlo Zambonelli a , John E. Hallsworth b , Andrea Pulvirenti c , Paolo Giudici c a
Dipartimento di Protezione e Valorizzazione Agroalimentare (DIPROVAL), Universita© di Bologna, Villa Levi, via F.lli Rosselli 107, Reggio Emilia 42100, Italy b Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa c Istituto di Industrie Agrarie, Universita© di Catania, via S. So¢a 98, Catania 95125, Italy Received 16 February 1999; received in revised form 10 May 1999; accepted 17 May 1999
Abstract A natural subgroup (that we refer to as Saccharomyces uvarum) was identified, within the heterogeneous species Saccharomyces bayanus. The typical electrophoretic karyotype, interfertility of hybrids between strains, distinctive sugar fermentation pattern, and uniform fermentation characteristics in must, indicated that this subgroup was not only highly homogeneous, but also clearly distinguishable from other species within the Saccharomyces sensu stricto group. Investigation of the S. bayanus type strain and other strains that have been classified as S. bayanus, confirmed the apparent lack of homogeneity and, in some cases, supported the hypothesis that they are natural hybrids. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Phenotypic characteristics; Saccharomyces bayanus; Saccharomyces sensu stricto; Saccharomyces uvarum; Taxonomy
1. Introduction The classi¢cation of Saccharomyces species, including those in the Saccharomyces sensu stricto group that are commonly used in the fermentation industries, has always been problematic at species level. According to the latest classi¢cation, the S. sensu stricto group is made up of four species: Saccharomyces bayanus, Saccharomyces cerevisiae, Sac* Corresponding author. Institute for Wine Biotechnology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa. Tel.: +27 (21) 808 3770; Fax: +27 (21) 808 3771; E-mail: [email protected]
charomyces paradoxus and Saccharomyces pastorianus [1]. The identi¢cation of these species was based mainly on DNA hybridisation analyses and the classi¢cation seems natural, with the sole exception of S. bayanus. It was recently suggested that S. bayanus can be divided into two subgroups: a bayanus group (including the type strain) and a uvarum group (not currently recognised as a distinct species). This grouping was based on PCR ampli¢cation and restriction polymorphism of the non-transcribed spacer 2 in the ribosomal DNA unit, and on electrophoretic karyotypes [2]. In 1970, S. uvarum was considered a distinct species according to a taxonomic study by
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 5 9 - 1
FEMSLE 8852 15-7-99
178
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185
van der Walt, based on variation in physiological characteristics [3]. In a later classi¢cation, all members of the S. sensu stricto group were attributed to S. cerevisiae and the term `uvarum' was used to describe one of the 17 physiological races into which S. cerevisiae had been divided [4]. By contrast, the most recent classi¢cation considers most of the strains that were classi¢ed as S. uvarum [3] indistinct from the species S. bayanus [1]. Recent taxonomic studies found that many Saccharomyces strains isolated from cold-stored grape juice (that ferment vigorously at low temperatures) produce an electrophoretic karyotype that is consistent with that of strains classi¢ed as uvarum by many [2], but distinct from that of the type strain of S. bayanus [5,6]. Such strains are quite widespread in nature, and are also commonly found in wine and other fermented beverages [7^9]. They exhibit a typical fermentation pro¢le in grape must that is clearly di¡erent from that of S. cerevisiae strains; they produce lower amounts of acetic acid, higher amounts of glycerol and succinic acid, and synthesise malic acid rather than degrading it [10,11]. This study was carried out to establish whether strains that produce an electrophoretic karyotype consistent with that of strains that were attributed to the uvarum group [2] make up a homogeneous and distinct group in terms of phenotypic characteristics. These included such as sugar fermentation pattern (i.e., ability to ferment speci¢c sugars), fermentation pro¢le (i.e., production of glycerol, succinic acid, acetic acid and ethanol, e¡ect on ¢nal malic acid concentration, fermentation vigour, and amount of residual sugars), and ability to produce fertile hybrids. Such information could then be used as a basis for examination of strains using molecular techniques.
2. Materials and methods 2.1. Yeast strains The yeast strains that were used are listed in Table 1. All the wine strains were obtained from the DIPROVAL collection (Dipartimento di Protezione e Valorizzazione Agroalimentare, University of Bologna, Reggio Emilia, Italy). Some of these, DIPROV-
AL 11204, DIPROVAL 12233, DIPROVAL C31 and DIPROVAL M12, represent a group of strains isolated from cold-stored grape juice (at di¡erent times and places), and were described in previous studies [8,11]. 2.2. Media Strains were maintained on Sabouraud dextrose agar (Oxoid, UK) and sporulated in acetate agar (sodium acetate 0.1%, w/v; agar 2%, w/v). Growth was studied over a range of temperatures in Sabouraud dextrose broth (Oxoid), and spore extraction and conjugation were conducted in yeast peptone dextrose broth (YPD: yeast extract 2%, w/v; peptone 2%, w/v; dextrose, 4%, w/v). Some yeasts carry out fermentation very slowly, and cannot exhaust sugars in synthetic media with a high sugar content ( s 10%, w/v). In the present study, therefore, grape juice was used as the fermentation medium. Grape juice provides a balance of nutrients that produce consistent and reproducible results for strain characterisation, despite slight variation in composition between batches. Fermentations were carried out in must produced from the Vitis vinifera cultivar Trebbiano (containing 210 g sugar l31 ) after dilution with a 1% yeast extract solution (w/v) to reduce the sugar concentration to 140 g l31 . 2.3. Preparation of chromosomal DNA and pulsed-¢eld gel electrophoresis (PFGE) Chromosome extraction was performed using the Schwartz and Cantor technique [12], as modi¢ed by Vaughan-Martini et al. [13]. PFGE was carried out with a contour-clamped homogeneous electric ¢eld (CHEF) mapper system (Bio-Rad, USA). The gel was stained for 1 h using ethidium bromide (1 Wg l31 ) at 25³C, decolourised in tris-borate EDTA bu¡er for 24 h (at 4³C), and then photographed. A more detailed description of the procedure has been given previously [6]. 2.4. Physiological characteristics Ability to ferment di¡erent sugars was assessed according to the methods of Kreger van Rij [14].
FEMSLE 8852 15-7-99
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185
179
The optimum temperature for growth was determined using a temperature gradient incubator constructed as described by Packer et al. [15]. This method was used to establish the temperature at which the maximum cell multiplication rate occurs, by 15 to 20 h from inoculation.
3.11 Base System (SPSS Inc., USA). The fermentations were carried out in triplicate, and mean values for glycerol, succinic acid, acetic acid, malic acid, residual sugar, and ethanol concentrations are listed in Table 3.
2.5. Fermentation tests
3. Results
Fermentations were carried out in a 25³C incubator in must (100 ml) that had been heated to 90³C for 20 min to eliminate the natural micro£ora. Fermentation was assessed by determining the weight loss caused by CO2 release at frequent intervals, until a constant weight was attained. This weight loss, determined during a 48 h period, represented fermentation vigour.
3.1. Optimum temperature for growth
2.6. Analysis of fermentation samples, post-fermentation
All the strains with an upper temperature limit for growth that was greater than 37³C had an optimum growth temperature that was greater than 30³C. The strains with a temperature limit for growth below 37³C had an optimum growth temperature that was below 30³C, and these were all strains of either S. bayanus or S. pastorianus (data not shown). 3.2. Electrophoretic karyotypes
Determinations of pH, and ethanol and sugar concentration were carried out using standard methods for wine analysis [16]. Glycerol, succinic acid, acetic acid and malic acid were quanti¢ed using enzymatic kits (Boehringer Mannheim, Germany). Glycerol, succinic acid and acetic acid concentrations were expressed as g compound 100 ml31 ethanol so that the results obtained were comparable, even for strains that could not ferment all the sugar provided. 2.7. Sporulation, spore viability and hybridisation Cultures were sporulated using the methods described by Kreger-van Rij [14]. Spore germination tests were carried out in an oil chamber, asci were broken and individual spores separated using a micromanipulator. Single spores were then transferred to microdrops of YPD. Germination and culture formation from spores were determined by light-microscopic examination after 1 to 2 days at 25³C. Hybrids were obtained by spore conjugation [17]. 2.8. Replication and statistical analyses Data relating to fermentation performance and fermentation byproducts were subjected to principal component analysis using SPSS 6.1 for Windows
The electrophoretic karyotypes for all the strains studied that cannot grow at temperatures above 37³C, and of the S. cerevisiae type strain, are shown in Figs. 1 and 2 (the other strains, classi¢ed unequivocally as S. cerevisiae or S. paradoxus, were not of primary interest in the present study). A karyotype characterised by only two bands in the region between 365 and 225 kb was produced by all the wine strains isolated from cold-stored juice (i.e. DIPROVAL 11204, DIPROVAL 12233, DIPROVAL C31, DIPROVAL M12), and from strains CBS 395, CBS 1604 and CBS 7001 (Fig. 1, lanes 2^5; Fig. 2, lanes 7^10). By contrast, most Saccharomyces strains produce karyotypes with three or more bands in this region [6]. The remaining strains tested exhibited a di¡erent electrophoretic pro¢le (Fig. 1, lanes 6^9; Fig. 2, lanes 2^6, respectively). The band pattern obtained from the type strains of S. pastorianus and S. bayanus (Fig. 1, lane 6 and Fig. 2, lane 2, respectively) did not typify those obtained from the other strains of each species, and the pro¢les were not uniform within these taxonomic groups. 3.3. Ability to ferment di¡erent sugars In 1984, S. sensu stricto strains were all grouped under the speci¢c name S. cerevisiae, and according
FEMSLE 8852 15-7-99
180
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185
to the fermentation of a range of sugars, S. cerevisiae was divided into 17 physiological races [4]. This method was used to con¢rm the identity of the strains examined in the present study, as shown in Table 2. Strains currently classi¢ed as S. bayanus and S. pastorianus [1] were found to vary in their ability to ferment sugars. Nevertheless, within S. bayanus there was a number of strains that exhibited the typical sugar fermentation pattern of the former physiological race uvarum (Table 2) [4]. These strains Table 1 List of Saccharomyces strains used in the present study Original speci¢c name a
S. bayanus S. uvarumb S. globosus S. heterogenicus S. tubiformis S. inusitatus S. bayanus S. abuliensis S. bayanus S. pastorianusa S. pastorianus S. carlbergensis S. paradoxusa S. paradoxus S. paradoxus S. cerevisiaea S. cerevisiae S. cerevisiae Wine strains S. bayanus S. bayanus S. bayanus S. bayanus S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae
CBS no.
Other culture collection nos.
380 395 424 425 431 1546 1604 7001
CLIB 181, DBVPG 6171 CLIB 251 CLIB 250 CLIB 255 CLIB 389 CLIB 252 CLIB 253, DBVPG 6259 CLIB 283 CCY 21-6-1, CLIB 271 CLIB 281, DBVPG 6047 DBVPG 6257 CLIB 176, DBVPG 6033 CLIB 228, DBVPG 6411 DBVPG 6466 DBVPG 6304, UCD 51^186 DBVPG 6173 DBVPG 6292 DBVPG 6295
1538 1260 1513 432 5829 1171 4054 5635
DIPROVAL DIPROVAL DIPROVAL DIPROVAL DIPROVAL DIPROVAL DIPROVAL DIPROVAL DIPROVAL
11204 12233 C31 M12 6167 7070 10003 11052 29001
CBS: Centraalbureau voor Schimmelcultures, Baarn, Netherlands. CCY : Czechoslovak Collection of Yeasts, Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia. CLIB: Collection de Levures d'Interet Biotechnologique, Thiverval-Grignon, France. DPVPG : Dipartimento di Biologia Vegetale, Universita© di Perugia, Perugia, Italy. DIPROVAL : Dipartimento di Protezione e Valorizzazione Agroalimentare, Universita© di Bologna, Reggio Emilia, Italy. UCD: Department of Viticulture and Enology, University of California, Davis, CA, USA. a Type strains. b Former type strain from the time when S. uvarum was recognised as a species [3].
Fig. 1. Contour clamped homogeneous electric ¢eld (CHEF) banding patterns of chromosomal DNA of Saccharomyces strains not growing above 37³C, and that of the S. cerevisiae type strain CBS 1171. Lane 1: S. bayanus strain CLIB 271, lanes 2 to 5: S. uvarum wine strains DIPROVAL 12233, DIPROVAL 11204, DIPROVAL C31 and DIPROVAL M12 respectively, lane 6: S. pastorianus type strain CBS 1538, lanes 7 and 8: S. pastorianus strains CBS 1260 and CBS 1513, lane 9: S. cerevisiae type strain CBS 1171, lane 10: S. cerevisiae yeast chromosomal DNA size standard (strain YPH80, BioLabs, USA).
were all the wine strains isolated from cold-stored must, and strains CBS 395, CBS 1604, and CBS 7001. 3.4. Fermentation properties in grape juice The results of the fermentations carried out in grape juice (containing 140 g sugar l31 ) are shown in Table 3. The strains classi¢ed as S. cerevisiae and S. paradoxus [1] produced a homogeneous fermentation pro¢le. They were vigorous, generally resistant to ethanol, and gave higher ethanol yields from the sugar fermented (approximately 60%, v/w). The mean amount of malic acid was reduced and the mean amount of glycerol produced was 5.4 g 100 ml31 ethanol, of succinic acid 0.8 g 100 ml31 ethanol, and of acetic acid 0.3 g 100 ml31 ethanol. Surprisingly, the least vigorous and least ethanol-resistant strain was the type strain of S. cerevisiae (CBS 1171). Strains belonging to S. bayanus and S. pastorianus produced a heterogeneous fermentation pro¢le. The wine strains DIPROVAL 11204, DIPROVAL 12233, DIPROVAL C31 and DIPROVAL M12 resembled strains CBS 1604 and CBS 7001, in terms of both their sugar fermentation pattern (Table 2), which was typical of S. cerevisiae physiological race uvarum [4], and their electrophoretic pro¢le (Figs. 1
FEMSLE 8852 15-7-99
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185
181
levels of succinic acid (on average 0.92 g 100 ml31 ethanol), and small amounts of acetic acid (on average 0.16 g 100 ml31 ethanol) (Table 3). The strain CBS 395, possessing the same karyotype as the above-mentioned strains, showed a fermentation pro¢le which was not consistent with that obtained from these strains, but resembled that of S. cerevisiae strains (Table 3). Although the S. pastorianus strains studied represented a fairly homogeneous group, the fermentation characteristics were close to those of S. cerevisiae strains (Table 3). Strains of S. pastorianus degraded malic acid and produced less glycerol, succinic acid and acetic acid than S. cerevisiae strains. Two of the S. pastorianus strains examined did not ferment all the sugar provided. None of the remaining strains could be ascribed to either of the above-mentioned groups. Indeed, although they had all been classi¢ed as S. bayanus [1] they form a heterogeneous group, suggesting that S. bayanus has been used as a `dustbin' taxon. In particular, strains CBS 380 and CBS 1505 exhibited some of the traits typical of the cold-isolated wine strains that were mentioned above (these include the synthesis of malic acid and the production of low
Fig. 2. Contour clamped homogeneous electric ¢eld (CHEF) banding patterns of chromosomal DNA of Saccharomyces strains not growing above 37³C. Lane 1: S. cerevisiae DNA size standard (strain YPH80) ; lane 2: S. bayanus type strain CBS 380, lane 3: S. intermedius strain CBS 1505, lane 4: S. globosus strain CBS 424, lane 5: S. inusitatus strain CBS 1546, lane 6: S. heterogenicus strain CBS 425, lane 7: S. uvarum strain CBS 395, lane 8: S. bayanus strain CBS 1604, lane 9: S. tubiformis strain CBS 431, lane 10: S. abuliensis strain CBS 7001.
and 2). All these strains exhibited a uniform fermentation pro¢le in grape juice, which is distinctive for this group: they fermented all the sugar provided, synthesised malic acid, produced high amounts of glycerol (on average 6.92 g 100 ml31 ethanol), high
Table 2 Ability to ferment di¡erent sugars for strains with an upper temperature limit for growth of 37³C Strain
Glucose
Galactose
Maltose
Melibiose
Ra¤nose
Sucrose
Physiological racea
CBS 380 CBS 395 CBS 424 CBS 425 CBS 431 CBS 1260 CBS 1505 CBS 1513 CBS 1538 CBS 1546 CBS 1604 CBS 7001 CLIB 271 DIPROVAL DIPROVAL DIPROVAL DIPROVAL
+ + + + + + + + + + + + + + + + +
3 + + 3 + + + + + 3 + + + + + + +
+ + 3 + + + + + + + + + + + + + +
3 + 3 3 3 + 3 3 3 +c + + 3 + + + +
+ + 3 3 + + + + + + + + + + + + +
+ + 3 + + + + 3 + + + + + + + + +
bayanus uvarum globosus heterogenicus cerevisiae uvarumb cerevisiae cerevisiae cerevisiae inusitatus uvarum uvarum cerevisiae uvarum uvarum uvarum uvarum
11204 12233 C31 M12
The strains that grew above 37³C belong unequivocally to S. cerevisiae or S. paradoxus, and were not of primary interest in this study. a According to Yarrow [4], who classi¢ed all S. sensu stricto strains as S. cerevisiae. b Recently, this strain has been found to resemble uvarum strains only in terms of sugar fermentation pattern, and it is now classi¢ed as S. pastorianus [1] (Table 3). c Poor growth.
FEMSLE 8852 15-7-99
182
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185
Table 3 Composition of wine produced from must containing 140 g sugar l31 Straina S. uvarumc CBS 395 CBS 1604 CBS 7001 DIPROVAL 11204 DIPROVAL 12233 DIPROVAL C31 DIPROVAL M12 S. bayanus CBS 380 CBS 424 CBS 425 CBS 431 CBS 1505 CBS 1546 CLIB 271 S. pastorianus CBS 1538 CBS 1260 CBS 1513 S. paradoxus CBS 432 CBS 5829 UCD 51^186 S. cerevisiae CBS 1171 CBS 4054 CBS 5635 DIPROVAL 6167 DIPROVAL 7070 DIPROVAL 10003 DIPROVAL 11052 DIPROVAL 29001
Glycerolb
Succinic acidb
Acetic acidb
Malic acid (g l31 )
Residual sugars (g l31 )
Alcohol (%, v/v)
5.75 6.68 6.62 7.13 7.96 6.46 6.68
0.582 0.803 0.876 0.980 1.047 0.907 0.938
0.460 0.192 0.289 0.158 0.123 0.077 0.165
1.183 1.476 1.641 1.951 2.036 1.476 1.663
61 6.36 61 61 61 61 61
7.8 8.1 7.8 7.8 7.8 7.6 7.6
5.64 3.98 3.93 4.74 5.65 8.81 3.08
0.613 0.516 0.513 0.425 0.749 0.589 0.404
0.027 0.216 0.005 0.527 0.006 0.768 0.643
1.471 1.183 1.308 1.256 1.618 1.202 1.179
6.36 21.67 11.87 3.78 61 3.15 10.86
7.3 6.0 6.6 7.6 7.9 7.6 7.1
3.33 5.84 3.33
0.508 0.567 0.508
0.007 0.482 0.135
1.285 1.148 1.223
10.3 61 8.28
7.8 7.9 6.8
5.35 4.70 5.49
0.894 0.654 0.847
0.007 0.130 0.130
1.001 1.148 0.864
61 61 61
7.9 7.9 7.8
5.97 4.39 3.79 6.46 5.23 5.80 5.96 5.97
0.404 1.030 0.750 0.821 0.775 1.127 0.912 0.859
0.444 0.351 0.419 0.259 0.472 0.358 0.297 0.334
1.039 1.152 0.845 1.157 1.195 1.020 1.091 1.025
10.01 61 61 61 61 61 61 61
7.4 8.2 8.2 8.4 8.3 7.9 8.0 8.3
The values listed are means of triplicate analyses. Current or former type strains are listed ¢rst for each species. Strains are listed as S. bayanus, S. pastorianus, S. paradoxus and S. cerevisiae according to [1]. b Values were expressed as g 100 ml31 ethanol. c These strains, that have been referred to variously as S. cerevisiae physiological race uvarum, S. bayanus or S. bayanus subgroup uvarum, are listed here as S. uvarum. a
amounts of acetic acid). On the other hand, some traits were characteristic of S. cerevisiae, such as the amounts of glycerol and succinic acid produced. Strains CBS 395, CBS 424, CBS 425, CBS 431, CBS 1546 and CLIB 271, that are all classi¢ed as S. bayanus [1], did not synthesise malic acid, but degraded it (Table 3). They produced average amounts of glycerol and succinic acid, and varying amounts of acetic acid. These strains did not ferment vigorously and were unable to complete fermentation, leaving high
amounts of residual sugar. In addition, these strains gave a pro¢le of fermentation very similar to that of S. pastorianus. 3.5. Sporulation ability and fertility Strains belonging to the group exhibiting the sugar fermentation pattern of S. cerevisiae physiological race uvarum, except for strain CBS 395, sporulated very well and produced numerous four-spore asci.
FEMSLE 8852 15-7-99
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185
Spore germination was close to 100% for these strains (data not shown). Hybridisation experiments resulted in the production of four hybrids: DIPROVAL 11204U12233, DIPROVAL 11204UC31, DIPROVAL 11204UM12 and DIPROVAL 12233UC31. These hybrids sporulated well, were fertile and produced viable spores. The type strain of S. bayanus (CBS 380), as well as strains CBS 1505, CBS 1546 and CLIB 271, did not sporulate. Strains CBS 395, CBS 424, CBS 425 and CBS 431 sporulated, but very slightly. These four strains were sterile due either to their inability to sporulate or because of the production of non-viable spores. In some cases the number of asci formed during sporulation was so low that germination tests could not be carried out. All of the S. pastorianus strains studied were sterile. 3.6. Principal component analysis Principal component analysis of the fermentation performance and fermentation byproducts indicated that the ¢rst two principal components accounted for 70.05% of variability. Principal component 1 (PC1) accounted for 43.78%; it was positively correlated with ethanol production and fermentation vigour, and negatively correlated with the residual sug-
Fig. 3. Principal component analysis of the fermentation performance and wine composition (especially the associated acidity) of the strains studied: S. cerevisiae (S); S. paradoxus (b); S. pastorianus (E); S. uvarum (F) according to the present study; and S. bayanus (a). Principal component 1 (PC1) re£ects the ¢tness of each strain for fermentation. Principal component 2 (PC2) re£ects the production of malic acid and the generation of an acidic environment.
183
ars, glucose and fructose. Component 1 e¡ectively represents overall fermentation ¢tness. This component separated vigorous, ethanol-tolerant strains able to complete the fermentation process from all the other strains. The vigorous ethanol-tolerant group was represented by all the S. cerevisiae strains except for the type strain CBS 1171, the strains isolated from cold-stored grape juice, the three S. paradoxus strains and one S. bayanus strain (CLIB 271) (Fig. 3). Principal component 2 (PC2) accounted for 26.27% of the variability. Component 2 could be interpreted as a measure of the associated acidity, being positively correlated with malic acid and negatively with pH. A slight correlation with glycerol production was also detected. Component 2 separated S. uvarum strains from those of S. cerevisiae and other species (Fig. 3). The other strains were dispersed and appeared both above and below the x-axis in the principal component analysis plot shown in Fig. 3.
4. Discussion The present study demonstrated the existence of a natural and homogeneous, but currently unrecognised group of strains within S. sensu stricto (that had been previously referred to as S. bayanus subgroup uvarum [2]). These strains exhibit (i) the typical sugar fermentation pattern of the former S. cerevisiae physiological race uvarum [4], which used to be considered a species in itself [3]; (ii) a typical electrophoretic karyotype, characterised by the presence of two bands in the area between 365 and 225 kb; (iii) a clearly de¢ned fermentation pro¢le in grape juice which di¡ers from that of S. cerevisiae and S. paradoxus strains in the amount of glycerol, succinic acid, and acetic acid produced, as well as whether they synthesise or degrade malic acid; and (iv) a high sporulation ability, and the spores are characterised by a high viability. The homogeneity of this group was con¢rmed by the fact that the strains studied produce intraspeci¢c fertile hybrids. Strains DIPROVAL 11204 and CBS 7001, the ancient type strain of S. abuliensis (reclassi¢ed within S. bayanus subgroup uvarum [2]), were the most representative strains of the group. According to the data obtained in the present study, and that obtained in
FEMSLE 8852 15-7-99
184
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185
earlier work [2,3], we refer to this group as S. uvarum. The remaining strains that are classi¢ed as S. bayanus [1] do not constitute a homogeneous group; they di¡er amongst themselves and are also distinct from the S. uvarum strains mentioned above. The S. bayanus type strain (CBS 380) exhibits a fermentation pro¢le similar to that of the hybrids between S. cerevisiae and S. uvarum [17,23] and, like these, produces a karyotype with a higher number of bands than that of S. cerevisiae or other S. bayanus strains [18,19]. The existence of natural hybrids between different species, or within the same species, has already been suggested by several authors [20^22]; the characteristics of strains CBS 380, CBS 1505 and CLIB 271 support the hypothesis that they are the result of hybridisation. Investigations carried out at a molecular level are also consistent with a hybrid origin (H.V. Nguyen and C. Gaillardin, personal communication). The other S. bayanus strains, as well as the strains of S. pastorianus that were studied, all demonstrated fermentation properties similar to those of S. cerevisiae strains, but not the fermentation vigour. Considering their sterility, the higher number of bands in their karyotypes and the non-homogeneous sugar fermentation pattern, a hybrid origin for these strains seems likely. Studies of phenotypic characteristics and electrophoretic karyotypes can be used to identify a natural taxon, and molecular techniques can then be employed to con¢rm or re¢ne the classi¢cation. The present study demonstrated that S. uvarum is a natural group within S. sensu stricto, and this is not acknowledged in the contemporary classi¢cation [1]. We think that a re-evaluation of the strains currently classi¢ed as S. bayanus, and a re-consideration of the type strain, is now needed to gain a more natural classi¢cation of the Saccharomyces genus.
Acknowledgements We are indebted to Professor C. Gaillardin and Dr. H.V. Nguyen (INRA-INA, Theverval-Grignon, France) for providing CLIB strains, and to H.V. Nguyen for sharing molecular typing results as well as for helpful discussion. Financial support (a MURST 40% award) was received from the Minis-
tero dell' Universita© e Ricerca Scienti¢ca e Tecnica, Italy.
References [1] Vaughan-Martini, A. and Martini, A. (1998) Saccharomyces Meyen ex Reess. In: The Yeasts, A Taxonomic Study (Kurtzman, C.P. and Fell, J.W., Eds.), 4th Edn., pp. 358^371. Elsevier, Amsterdam. [2] Nguyen, H.V. and Gaillardin, C. (1997) Two subgroups within the Saccharomyces bayanus species evidenced by PCR ampli¢cation and restriction polymorphism of the non-transcribed spacer 2 in the ribosomal DNA unit. Syst. Appl. Microbiol. 20, 286^294. [3] van der Walt, J.P. (1970) Genus 16 Saccharomyces Meyen emend. Reess. In: The Yeasts. A Taxonomic Study (Lodder, J., Ed.), pp. 555^718. North-Holland Publishing Company, Amsterdam. [4] Yarrow, D. (1984) Genus Saccharomyces Meyen ex Rees. In: The Yeasts. A Taxonomic Study (Kreger-van Rij, N.J.W., Ed.), 3rd Edn., pp. 379^396. Elsevier, Amsterdam. [5] Naumov, G., Naumova, E. and Gaillardin, C. (1993) Genetic and karyotypic identi¢cation of wine Saccharomyces bayanus yeasts isolated in France and Italy. Syst. Appl. Microbiol. 16, 274^279. [6] Giudici, P., Caggia, C., Pulvirenti, A. and Rainieri, S. (1998) Karyotyping of Saccharomyces strains with di¡erent temperature pro¢les. J. Appl. Microbiol. 84, 811^819. [7] Kusewics, D. (1975) Characteristics of fermentation abilities in some varieties of wine yeast of cryophilic type. Acta Alim. Polonica 25, 235^245. [8] Castellari, L., Pacchioli, G., Zambonelli, C., Tini, V. and Grazia, L. (1992) Isolation and initial characterisation of cryotolerant Saccharomyces strains. Ital. J. Food Sci. 3, 179^223. [9] Kishimoto, M., Shinohara, T., Soma, E. and Goto, S. (1993) Selection and fermentation properties of cryophilic wine yeasts. J. Ferment. Bioeng. 75, 451^453. [10] Kishimoto, M., Shinohara, T., Soma, E. and Goto, S. (1993) Identi¢cation and enological characteristics of cryophilic wine yeasts. J. Brew. Soc. Jpn. 88, 708^713. [11] Castellari, L., Ferruzzi, A., Magrini, A., Giudici, P., Passarelli, P. and Zambonelli, C. (1994) Unbalanced wine fermentation of cryotolerant vs non-cryotolerant Saccharomyces strains. Vitis 33, 49^52. [12] Schwartz, D.H. and Cantor, C.R. (1984) Separation of yeast chromosome-sized DNAs by pulsed-¢eld gradient gel electrophoresis. Cell 37, 67^75. [13] Vaughan-Martini, A., Martini, A. and Cardinali, G. (1993) Electrophoretic karyotyping as a taxonomic tool in the genus Saccharomyces. Antonie van Leeuwenhoek 62, 145^156. [14] Kreger-van Rij, N.J.W. (1984) The Yeasts. A Taxonomic Study, 3rd Edn. Elsevier, Amsterdam. [15] Packer, G.J.K., Prentice, G.A. and Clegg, L.F.L. (1973) Design of a temperature gradient incubator. J. Appl. Bacteriol. 36, 173^177.
FEMSLE 8852 15-7-99
S. Rainieri et al. / FEMS Microbiology Letters 177 (1999) 177^185 [16] Ough, C.S. and Amerine, M.A. (1988) Methods for Analysis of Musts and Wines. John Wiley and Sons, New York. [17] Zambonelli, C., Passarelli, P., Rainieri, S., Bertolini, L., Giudici, P. and Castellari, L. (1997) Technological properties and temperature response of interspeci¢c Saccharomyces hybrids. J. Sci. Food Agric. 74, 7^12. [18] Jolly, N.P., Janse, B.J.H., Van Rooyen, T.J. and Louw, J.H. (1993) Hybridization and typing of yeasts used in sparkling wine fermentations. Am. J. Enol. Vitic. 44, 217^ 226. [19] Giudici, P., Caggia, C., Pulvirenti, A., Zambonelli, C. and Rainieri, S. (1998) Electrophoretic pro¢le of hybrids between cryotolerant and non-cryotolerant Saccharomyces strains. Lett. Appl. Microbiol. 27, 31^34.
185
[20] Vaughan-Martini, A. and Martini, A. (1987) Three newly delimited species of Saccharomyces sensu stricto. Antonie van Leeuwenhoek 53, 77^84. [21] Naumov, G.I., Naumova, E. and Korhola, M. (1992) Genetic identi¢cation of natural Saccharomyces sensu stricto yeasts from Finland, Holland and Slovakia. Antonie van Leeuwenhoek 61, 237^243. [22] Masneuf, I., Hansen, J., Groth, C., Piskur, J. and Dubourdieu, D. (1998) New hybrids between Saccharomyces sensu stricto yeast species found among wine and cider production strains. Appl. Environ. Microbiol. 64, 3887^3892. [23] Wingen, O. and Laustsen, O. (1938) Arti¢cial species hybridisation in yeast. Comp. Ren. Trav. Lab. Carlsberg Ser. Physiol. 22, 235^244.
FEMSLE 8852 15-7-99