RESEARCH ARTICLE
Yeast species composition di¡ers between artisan bakery and
spontaneous laboratory sourdoughs
Gino Vrancken1, Luc De Vuyst1, Roel Van der Meulen1, Geert Huys2, Peter Vandamme2 & Heide-Marie
Daniel3
1
Research Group of Industrial Microbiology and Food Biotechnology, Faculty of Sciences and Bio-engineering Sciences, Vrije Universiteit Brussel,
Brussels, Belgium; 2Laboratory for Microbiology, Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium; and 3Mycothèque de
l’Université catholique de Louvain (MUCL), Member of the Belgian Coordinated Collection of Microorganisms (BCCM), Earth and Life Institute,
Mycology, Université catholique de Louvain, Louvain-la-Neuve, Belgium
Correspondence: Heide-Marie Daniel,
BCCM/MUCL, Earth and Life Institute,
Mycology, Université catholique de Louvain,
Croix du Sud 3, bte 6, B-1348 Louvainla-Neuve, Belgium. Tel.: 132 10 473 956;
fax: 132 10 451 501; e-mail: [email protected]
Received 9 September 2009; revised 2
December 2009; accepted 25 February 2010.
Final version published online 1 April 2010.
DOI:10.1111/j.1567-1364.2010.00621.x
Editor: Cletus Kurtzman
YEAST RESEARCH
Keywords
sourdough; yeast; fermentation; molecular
identification.
Abstract
Sourdough fermentations are characterized by the combined activity of lactic acid
bacteria and yeasts. An investigation of the microbial composition of 21 artisan
sourdoughs from 11 different Belgian bakeries yielded 127 yeast isolates. Also, 12
spontaneous 10-day laboratory sourdough fermentations with daily backslopping
were performed with rye, wheat, and spelt flour, resulting in the isolation of 217
yeast colonies. The isolates were grouped according to PCR-fingerprints obtained
with the primer M13. Representative isolates of each M13 fingerprint group were
identified using the D1/D2 region of the large subunit rRNA gene, internal
transcribed spacer sequences, and partial actin gene sequences, leading to the
detection of six species. The dominant species in the bakery sourdoughs were
Saccharomyces cerevisiae and Wickerhamomyces anomalus (formerly Pichia anomala), while the dominant species in the laboratory sourdough fermentations were
W. anomalus and Candida glabrata. The presence of S. cerevisiae in the bakery
sourdoughs might be due to contamination of the bakery environment with
commercial bakers yeast, while the yeasts in the laboratory sourdoughs, which
were carried out under aseptic conditions with flour as the only nonsterile
component, could only have come from the flour used.
Introduction
Sourdough fermentation has enjoyed an increasing popularity due to its beneficial effects on the flavour, texture,
shelf-life, and nutritional and health-promoting properties
of the resulting breads (Hansen, 2004; De Vuyst & Neysens,
2005). Sourdough develops by spontaneous fermentation of
yeasts and lactic acid bacteria in mixtures of cereal flour(s)
and water, with the lactic acid bacteria being responsible for
the acidification of the dough and the yeasts for the
leavening action via CO2 production. Usually, a stable
microbial community arises during periodic refreshments
of the flour/water mixture, closely depending on external
factors such as temperature and pH, propagation cycles, and
the type of cereal (De Vuyst & Vancanneyt, 2007; Vogelmann
et al., 2009).
FEMS Yeast Res 10 (2010) 471–481
Extensive research efforts have been directed towards the
study of the species diversity and identification of lactic acid
bacteria involved in sourdough fermentation processes
(Corsetti & Settanni, 2007; De Vuyst & Vancanneyt, 2007;
Vogel & Ehrmann, 2008). In contrast, fewer studies on
sourdough yeast species diversity and identification are
available (Vogel & Ehrmann, 2008). In no small measure,
this apparently lower interest in sourdough yeasts is due to
the fact that wild-type yeasts have almost been eliminated
from bakeries by the use of commercial bakers yeast,
Saccharomyces cerevisiae, as a starter culture for bread
production. However, a variety of yeasts can dominate a
spontaneous sourdough fermentation process, depending
on the flour type, process conditions, environment, and
location, all of which substantially add to the diversity and
originality of sourdough products (Rossi, 1996; Mantynen
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472
et al., 1999; Meroth et al., 2003; Succi et al., 2003; Pulvirenti
et al., 2004; Hammes et al., 2005).
The most frequently reported yeasts in both wheat and
rye sourdoughs are the species S. cerevisiae and Candida
humilis (synonym Candida milleri); other frequently reported species are Pichia kudriavzevii (formerly Issatchenkia
orientalis, anamorph Candida krusei) and Kazachstania
exigua [formerly Saccharomyces exiguus, anamorph Candida
(Torulopsis) holmii] (for reviews, see Rossi, 1996; De Vuyst &
Neysens, 2005, and further Gullo et al., 2003; Vernocchi
et al., 2004a, b; Garofalo et al., 2008; Vogelmann et al., 2009).
Less frequently reported species include, among others,
Wickerhamomyces anomalus (formerly Pichia anomala; Rocha & Malcata, 1999), Wickerhamomyces (formerly Pichia)
subpelliculosa (Barber et al., 1983), Candida glabrata (Meroth et al., 2003; Succi et al., 2003), Kazachstania unispora
(formerly Saccharomyces unisporus; Salovaara & Savolainen,
1984), and Torulaspora delbrueckii (Almeida & Pais, 1996).
Using culture-dependent and -independent approaches,
Meroth et al. (2003) could isolate S. cerevisiae, P. kudriavzevii, C. humilis, and C. glabrata from rye sourdough with
added bakers yeast, while they also detected Dekkera bruxellensis, Emericella nigrum, and unculturable ascomycetes by
culture-independent PCR-denaturing gradient gel electrophoresis (PCR-DGGE). In contrast, Gatto & Torriani (2004)
found no diversity in the yeast population and revealed the
dominance of S. cerevisiae in a traditional wheat sourdough
preparation.
A first reference point for accurate yeast identification is
the constantly growing D1/D2 large subunit (LSU) rRNA
sequence database, encompassing virtually all known yeast
species (Kurtzman & Robnett, 1998; Fell et al., 2000).
However, some distinct species such as W. anomalus and
Pichia myanmarensis (Nagatsuka et al., 2005) as well as
Kazachstania bulderi and Kazachstania barnettii (Middelhoven et al., 2000) show a low sequence divergence of two and
three substitutions in this region, a variation that can also be
observed among strains of the same species. Therefore, the
use of D1/D2 LSU sequences was complemented by PCR
amplification of repetitive DNA elements (e.g. Vassart et al.,
1987; Lieckfeldt et al., 1993; Groenewald et al., 2008) and by
determination of additional gene sequences such as the
internal transcribed spacer (ITS) region of the rRNA gene
cluster (Scorzetti et al., 2002) or protein-coding genes such
as the actin gene (ACT1; Daniel & Meyer, 2003), and the
mitochondrial cytochrome oxidase 2 (COX2) and cobalamin-independent methionine synthase (MET6) genes
(Gonzáles et al., 2006). The evaluation of conventional
classification criteria (Van der Walt & Yarrow, 1984; Kregervan Rij, 1987; Robert et al., 1997; Barnett et al., 2000) allows
the comparison of molecular with conventional identification results and conclusions on the physiological adaptation
of the yeast community to the investigated environment. In
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G. Vrancken et al.
recognition of the artificial nature of several yeast genera, a
series of taxonomic changes will gradually transform the
current phenotype-based classification towards a phylogeny-based classification. These changes concern, among
others, the species P. anomala, which has been shown by
multigene sequence analyses to be more appropriately
classified in a newly described genus as W. anomalus (Kurtzman et al., 2008).
This study aimed at characterizing the diversity of yeast
species in Belgian artisan sourdoughs, as well as gaining an
understanding of the contribution of naturally occurring
yeast species towards a stable microbiota during spontaneous sourdough fermentations. Therefore, both traditional
sourdoughs with daily propagation cycles as well as spontaneous laboratory sourdoughs, daily backslopped for 10 days,
were sampled. The results are discussed in relationship with
associated studies on the bacterial diversity of the same
samples (Scheirlinck et al., 2007; Van der Meulen et al.,
2007).
Materials and methods
Yeast isolates
A total of 127 yeast isolates were obtained from 21 artisan
sourdoughs collected from 11 Belgian bakeries, located in
seven of the 10 provinces of Belgium. The sampled sourdoughs were produced without the addition of bakers yeast
and were based on wheat, spelt, and rye flours, as well as
mixtures thereof (Table 1). The sampling and isolation
methods were described by Scheirlinck et al. (2007). A total
of 217 yeast isolates were obtained from 12 spontaneous
laboratory sourdough fermentations. The sampling and
isolation methods of these were described by Van der
Meulen et al. (2007). The laboratory sourdough fermentations were carried out in closed, presterilized fermentors
with rye, spelt, or wheat flour, derived from two different
mills in Belgium, as the only nonsterile component. Temperature, dough yield, and time between backsloppings
mimicked bakery sourdough conditions as closely as possible. In each case, up to 10 yeast colonies were isolated from
yeast extract–glucose agar (20 g L 1 glucose, 5 g L 1 yeast
extract, 20 g L 1 agar) supplemented with 0.1 g L 1 of chloramphenicol. Selected strains, representing each species from
each individual sourdough, were deposited in the Mycothèque de l’Université catholique de Louvain (MUCL), Belgium, under the numbers MUCL 51207–MUCL 51263.
M13 PCR-fingerprinting
To determine groups of isolates based on hypervariable
minisatellites, PCR-fingerprinting with a single primer derived from the core sequence within the protein III gene of
FEMS Yeast Res 10 (2010) 471–481
473
Yeast species composition in sourdough
Table 1. Yeast species distribution and main characteristics of 21 Belgian artisan sourdoughs
Log CFU g
Sample designation
Province
D01WW01T01
D02WR01T01
D02WR01T02
D02WW01T01
D03WW01T01
D04WW01T01
D05WW01T01
D06SS01T01
D06WW01T01
D07WR01T01
D07WR01T02
D07WR02T01
D08WW01T01
East Flanders
West Flanders
D09ME01T01
D10SS01T01
D10WW01T01
D10WR01T01
D11RR01T01
D11WW01T01
D11WW02T01
D11SS01T01
Flour
Age
Temp. Time
(years) ( 1C) (h)
Wheat
5
Wheat/rye
25
Wheat/rye
25
Wheat
2
West Flanders Wheat
6
Brabant Wallon Wheat
1
East Flanders
Wheat
1
Liège
Spelt
1.5
Wheat
12
Hainaut
Wheat/rye 1
0.8
Wheat/rye 2
0.8
Wheat/rye 3
0.01
Namur
First wheat,
15
then spelt
Namur
Wheat/rye/spelt 4
Luxemburg
spelt
3
Wheat
3
Wheat/rye
3
Namur
Rye
2
Wheat 1
2
Wheat 2
2
Spelt
2
1
pH
Yeasts LAB
30
10
AT
o 24
AT
22
AT
22
30
4
AT
0
28
24
21–23
2
21–23
2
26
13
26
13
AT
12
AT
6
3.83
4.01
3.8
3.83
4.12
5
3.63
5.04
4.75
3.83
3.86
3.78
3.8
7.68
4.2
3.89
5.72
7.57
3.12
7.14
4.55
4.77
7.46
7.46
6.98
6.84
8.9
8.86
9.02
9.15
8.97
8.29
8.6
7.67
7.5
8.67
8.19
8.9
7.99
o 24
4
4
4
12
12
12
12
3.78
4.28
4.09
3.96
3.79
3.72
3.85
3.83
7.54
5.63
5.94
5.92
7.67
7.26
7.62
7.29
8.68
8.03
8.3
8.14
9.25
9.08
9.17
9.02
AT
28
28
28
AT
AT
AT
AT
Yeast species identified (number of isolates)
S. cerevisiae
5
6
5
10
2
6
6
5
6
2
2
W. anomalus
Others
5
T. delbrueckii (1)
K. barnettii (5)
T. delbrueckii (1)
4
4
6
6
6
6
6
1
4
5
3
1
1
5
K. unispora (1)
1
Main characteristics are the region of origin (Belgian province), the flour, the time during which the sourdough starter culture has been maintained
(age), the sourdough fermentation temperature (temp.), the fermentation time since the inoculation of a fresh lot of flour with the maintained starter
culture, and pH of the sample. SDs of the CFU of yeasts and bacteria were reported in Scheirlinck et al. (2007).
Sample designations used in a complementary study on lactic acid bacteria (Scheirlinck et al., 2008).
AT, ambient temperature; LAB, lactic acid bacteria.
the bacteriophage M13 was used. DNA extraction and PCR
reactions were performed as described before (Groenewald
et al., 2008). For cluster analysis, the similarity among
digitized profiles was calculated using the Pearson correlation coefficient, and an unweighted pair group with arithmetic averages dendrogram was derived from the profiles
using BIONUMERICS version 5.1 (Applied Maths N.V., SintMartens-Latem, Belgium).
DNA sequencing
Ribosomal DNA regions and ACT1 gene fragments were
amplified and sequenced as described previously (Daniel
et al., 2009). Selected isolates of S. cerevisiae were further
characterized by COX2 gene sequences, which were amplified using the primers COII-3 and COII-5 (Belloch et al.,
2000), and by MET6 gene sequences, which were amplified
using the primers MET6-5 and MET6-3 (Gonzáles et al.,
2006). An approximately 600-bp fragment of both genes was
generated and sequenced using a CEQ 2000 XL capillary
automated sequencer (Beckman Coulter, Fullerton, CA).
After initial BLAST searches for the most similar sequences,
FEMS Yeast Res 10 (2010) 471–481
alignments were performed using BIOEDIT 7.0.5.3 (Hall,
1999) for comparisons with the corresponding sequences of
type strains and determination of substitutions and potential insertions or deletions (indels). To find gene sequences
of type strains, the CBS database (http://www.cbs.knaw.nl/
yeast/BioloMICS.aspx) was used as an additional resource.
The gene sequences obtained were deposited in the EMBL
databank (http://www.ebi.ac.uk/EMBL; Hinxton, UK)
with accession numbers FN393977–FN393993, FN435838
(D1/D2 LSU), FN393994–FN394008, FN435839 (ITS),
FN394009–FN394023, FN435840 (ACT1), FN394066–
FN394073 (MET6), and FN394074–FN394080 (COX2).
Physiological and morphological properties as
conventional classification criteria
Physiological and morphological profiles of representative
isolates were determined using the automated microplate
method Allev/Biolomics (BioAware SA, Hannut, Belgium)
of Robert et al. (1997), a yeast identification system based on
standard taxonomic criteria (Van der Walt & Yarrow, 1984;
Kreger-van Rij, 1987).
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474
Sourdough fermentation-related physiological
characterization
To investigate the physiological adaptation of the yeast
community to the sourdough fermentation environment,
the following physiological characteristics were determined
for a subset of strains: capacity to assimilate maltose,
glucose, fructose, and sucrose; low pH tolerance; and the
ability to grow in the presence of acetic acid. Tests were
carried out in duplicate on a 10-mL scale and were incubated aerobically with periodic agitation at 30 1C for 48 h,
except for the K. barnettii isolate D02WW01T02-1, which
was incubated at 23 1C. The test medium was composed of
6.2 g L 1 of yeast–nitrogen base (YNB; Difco, Basingstoke,
UK) and 5 g L 1 of carbohydrate (glucose, fructose, sucrose,
or maltose); YNB without an added carbon source was used
as a negative control. To test low pH tolerance, YNB
medium with 5 g L 1 of glucose was adjusted to pH 2.5, 3.5,
and 5.0 with 5 M HCl. To test the ability to grow in the
presence of acetic acid, YNB medium with 5 g L 1 of glucose
was supplemented with 1% w/v of acetic acid, after which
the pH was corrected to 5.0. All tubes were inoculated with
1% v/v of a yeast culture, which was grown at 30 1C (23 1C
for D02WW01T02-1) for 24 h. Growth was determined by
measurements of OD600 nm.
Results
Clustering of yeast isolates
In total, 344 yeast isolates were typed by M13 PCR-fingerprinting (Figs 1 and 2). Cluster analysis of the M13 PCRfingerprints resulted in six clusters and two single (ungrouped) isolates, referred to as A (n = 177), B1 (n = 91), B2
(n = 6), C (n = 5), D (n = 1), E1 (n = 61), E2 (n = 1), and F
(n = 2). Artisan sourdough isolates were grouped into five
clusters and one single isolate (Fig. 1), while spontaneous
laboratory sourdough isolates were grouped into three
clusters and one single isolate (Fig. 2).
Molecular identification of yeast isolates
Species identification using multiple gene sequences was
performed for 18 isolates, which were selected to represent
the six clusters and two single isolates found by PCRfingerprinting. This selection included isolates from all
subclusters and those with atypical profiles observed in the
combined similarity tree of bakery and laboratory sourdough isolates (not shown). For the selected isolates, 18 D1/
D2 LSU sequences, 16 ITS sequences, and 16 partial ACT1
gene sequences were generated (Supporting Information,
Table S1). Sequence variations detected within species, as
indicated by D1/D2 LSU, ITS, and ACT1 sequences, were
zero to one nucleotide differences in D1/D2 LSU sequences,
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G. Vrancken et al.
zero to nine nucleotide differences in ITS sequences, and
zero to four nucleotide differences in the ACT1 gene. The
fingerprint clusters of S. cerevisiae (B1, B2) and the cluster
and the single isolate of C. glabrata (E1, E2) were considered
as intraspecies variants, based on the low variation observed
in the ACT1 gene, which is evolving faster than ribosomal
sequences. The morphological and physiological characterization of representative isolates was in agreement with the
sequence-based species identification, with the exception of
the isolates representing cluster C, identified as K. barnettii,
which resembled S. cerevisiae and K. exigua physiologically
(Tables S1, S2).
To further characterize selected S. cerevisiae and
K. barnettii isolates, eight partial MET6 and seven partial
COX2 gene sequences were determined as additional protein-coding gene markers. The identification of the selected
K. barnettii isolate was confirmed by its COX2 sequence,
which was identical to that of the K. barnettii type strain,
and by its MET6 sequence, which was largely different from
S. cerevisiae. The MET6 sequences of S. cerevisiae isolates of
both clusters B1 and B2 were identical to S. cerevisiae, while
their COX2 sequences revealed the presence of two major
sequence types differing by 20–25 nucleotide substitutions.
The first sequence type was highly similar (two substitutions) to the type strain of S. cerevisiae and was only
represented by isolate D01WW01T01-2. The five isolates
belonging to the second sequence type showed nucleotide
substitutions in six positions among themselves. In comparison with the type strains of the most closely related species,
the five isolates showed 11–14 substitutions to Saccharomyces cariocanus, 17–21 substitutions to Saccharomyces
mikatae, and 18–23 substitutions to S. cerevisiae (Table S1).
This considerable variation between the second COX2
sequence type determined here and the type strains of
related and recognized species was contrasted by only one
to five substitutions in comparison with the COX2 sequence
of the Saccharomyces italicus type strain. Saccharomyces
italicus is considered a synonym of S. cerevisiae based on
high DNA reassociation values (96%; Vaughan Martini &
Kurtzman, 1985).
Physiological characterization
The physiological properties of representative yeast strains
as determined in microplates are reported in Table S2.
All yeast isolates tested for their sourdough-relevant
properties in tubes, marked in Figs 1 and 2, were capable of
assimilating glucose, fructose, and sucrose, and were capable
of growth at pH 3.5 and 5.0. Most isolates could assimilate
maltose, except for the seven C. glabrata isolates and the
single K. barnettii and K. unispora isolates tested. The
capacity to grow at pH 2.5 was variable, with one
W. anomalus isolate out of 23 tested (R11-96h3), nine out
FEMS Yeast Res 10 (2010) 471–481
475
100
90
80
60
70
40
50
30
10
20
Yeast species composition in sourdough
+
*
*
*
+
*
Saccharomyces cerevisiae
+
+
+
B1
+
+
+*
+
+
D01WW01T01-6 , D01WW01T01-9, D01WW01T01-5,
D03WW01T01-1*, D07WR01T01-6,
D07WR02T01-2*,
D07WR01T02-2*, D06SS01T01-2 , D07WR01T02-6,
D03WW01T01-2, D03WW01T01-4, D01WW01T01-2*,
D01WW01T01-3, D07WR01T01-1,
D07WR01T01-3,
D07WR01T01-4, D03WW01T01-6, D03WW01T01-8,
D03WW01T01-5, D03WW01T01-10, D06SS01T01-3,
D06SS01T01-4,
D06SS01T01-6,
D06SS01T01-5,
D03WW01T01-7, D03WW01T01-9, D02WR01T01-6 ,
D02WR01T02-5, D02WR01T02-2,
D02WR01T02-4,
D02WR01T02-6 , D06WW01T01-5, D06WW01T01-6,
D06SS01T01-1,
D02WR01T01-4,
D02WR01T02-1,
D02WR01T01-3, D02WR01T01-2,
D03WW01T01-3 ,
D11WW02T01-2, D11WW02T01-5, D11WW02T01-3,
D11WW02T01-4, D11SS01T01-1,
D11WW01T01-5 ,
D11WW01T01-3, D11SS01T01-2,
D11WW01T01-1,
D11SS01T01-4 , D02WR01T01-1,
D02WR01T01-5,
D06WW01T01-4, D04WW01T01-1, D04WW01T01-2,
D06WW01T01-1, D06WW01T01-3, D10SS01T01-3,
D11WW02T01-1 *, D10SS01T01-4,
D11WW01T01-2,
D10SS01T01-6,
D10SS01T01-7 , D10WW01T01-2,
D10WW01T01-1, D10WW01T01-3, D10WW01T01-4,
D10WW01T01-5, D10WW01T01-6b , D10WR01T01-4,
D10WR01T01-5, D10WR01T01-6 , D11RR01T01-3 ,
D10WR01T01-2, D10WR01T01-3,
D10WR01T01-1b,
D10SS01T01-1 , D10SS01T01-2,
D07WR01T01-4,
D07WR01T01-2, D07WR01T01-5
+
+
+
Kazachstania barnettii
+*
C
D02WW01T02-2, D02WW01T02-5, D02WW01T02-6,
D02WW01T02-1 *, D02WW01T02-3
Saccharomyces cerevisiae
*+*
B2
D05WW01T01-2,
D05WW01T01-5,
D
Kazachstania unispora
+
D05WW01T01-4, D05WW01T01-3,
D05WW01T01-6, D05WW01T01-1*
D11WW01T01-4 *
Wickerhamomyces anomalus
+
+
A
+
+
+*
F
D07WR02T01-3,
D01WW01T01-7,
D01WW01T01-4,
D07WR02T01-1,
D09ME01T01-1,
D09ME01T01-5,
D11RR01T01-5 ,
D11RR01T01-1,
D10WR01T01-1a,
D08WW01T01-5 ,
D11WW02T01-6,
D08WW01T01-1, D09ME01T01-6 ,
D01WW01T01-10, D01WW01T01-1,
D01WW01T01-8, D07WR01T02-5,
D09ME01T01-3, D09ME01T01-4,
D09ME01T01-2, D07WR01T02-1 ,
D07WR01T02-3, D07WR01T02-4,
D11RR01T01-6, D11RR01T01-4,
D11RR01T01-2, D10WW01T01-6a ,
D08WW01T01-3, D08WW01T01-4,
D08WW01T01-6, D08WW01T01-2,
D07WR02T01-5, D07WR02T01-6
Torulaspora delbrueckii
D02WR01T02-3*,
D06WW01T01-2
Fig. 1. Dendrogram generated by the unweighted pair-group method with arithmetic averages based on M13 PCR-fingerprints of yeasts isolated from
21 artisan sourdoughs, originating from 11 Belgian bakeries. Isolate numbers are given in the order of appearance of the profiles in the tree. Isolates that
were chosen for sourdough fermentation-related physiological characterization and sequence analysis are marked with a cross and an asterisk,
respectively.
FEMS Yeast Res 10 (2010) 471–481
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Published by Blackwell Publishing Ltd. All rights reserved
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476
90
100
80
70
60
40
50
20
30
10
G. Vrancken et al.
Candida glabrata
E1
R10-240h2, R10-240h5 , R10-96h5, R7-24h5*, R7-96h3, R14-48h,
R14-120-2, R10-48h3, R10-48h4,
R6-PR159-240h , R6-PR126-192h, R15-96-10,
R6-PR109-168h, R15-144h1, R15-240h , R15-144-2,
R14-168h2, R10-96h1, R14-24-2, R15-24-2, R7-96h5,
R7-240h3, R16-144-1, R16-240h, R17-120h, R14-120-3, R7-96h4,
R7-144h2, R17-48h1, R10-72h2, R17-24h1,
R17-24h2, R16-144-2, R17-?-2, R17-144h,
R17-240h , R17-96h, R15-48-2, R6-PR142-216h, R15-120h, R1772h, R10-24h4, R14-76h1, R6-PR110-168h, R7-240h2, R14-216h,
R14-240h , R14-168h1, R14-192h, R14-72h2, R14-96-3, R7240h5 , R7-96h1*, R7-144h1, R9-72h1,
R9-96h1, R9-48h4, R9-72h5, R9-96h3, R8R26-0h1,
R9-240h5
Saccharomyces cerevisiae
B1
R9-24h2 *, R14-24h2, R9-48h2, R9-96h2, R9-72h2 ,
R9-72h3, R14-72h3, R9-240h1 , R9-48h3, R6-PR48-72h, R9-96h5
Wickerhamomyces anomalus
A
R14-72-1, R14-120-1, R14-144h, R15-96-3, R15-96-4,
R10-240h1, R12-72h1, R6-PR36-48h, R7-24h2*,
R18R19-24h3, R11-240h2 , R6-PR124-192h, R11-240h1, R11-0h1 ,
R11-96h3 , R15-48-3, R16-48h, R11-72h3,
R16-24h, R6-PR48-72h, R11-48h5, R11-72h2,
R6-PR78-120h, R8R27-24h4, R15-144h1, R6-PR138-216h, R6PR140-216h, R12-95h1, R13-48-1, R13-72-1,
R6-PR47-72h, R6-PR19-24h, R14-24-1, R16-144-3,
R6-PR73-120h, R6-PR123-192h, R6-PR107-168h,
R6-PR108-168h, R6-PR35-48h, R12-240h1, R16-72h,
R15-48h1, R15-96-1, R15-24-1, R15-72h , R16-120h,
R12-144h1, R12-168h1, R12-120h1, R12-120h2,
R12-192h1, R12-216h1b, R12-216h1a, R15-96-5*,
R7-48h4, R10-48h5, R10-24h2, R10-24h3b, R10-24h3a, R10-48h1,
R10-48h2, R10-24h1, R7-72h3, R10-72h5,
R10-96h4, R10-240h3, R11-96h4, R10-96h3, R10-240h4, R10-72h3,
R13-24h1, R13-24h2, R7-72h2, R7-48h2,
R7-72h1, R7-48h, R7-48h3, R7-144h4, R7-144h5, R7-48h5, R772h4, R7-240h4, R7-144h3, R6-PR80-120h,
R6-PR125-192h, R6-PR61-96h, R6-PR122-192h,
R6-PR92-144h, R7-24h1*, R13-24h4, R13-48-2, R13-72-2, R1324h3, R10-72h4, R10-96h2, R6-PR141-216h,
R7-240h5, R7-240h1 R6-PR34-48h, R6-PR60-96h,
R8R28-24h3, R8R19-24h1, R8R19-24h2, R11-24h2 ,
R11-72h5, R11-96h2, R10-72h1, R9-24h3 , R9-24h4 ,
R9-24h5, R6-PR93-144h, R11-72h1, R9-24h1 , R11-96h5, R16-96h,
R6-PR18-120h, R9-240h2, R11-48h3,
R6-PR94-144h, R6-PR49-72h , R6-PR139-216h,
R11-48h4, R8R27-24h13 , R8R28-24h1 , R8R28-24h2,
R6-PR143-216h, R6-PR155-240h, R6-PR158-240h,
R6-PR160-240h, R6-PR156-240h , R6-PR157-240h, R8R27-24h2,
R8R27-24h1 , R11-24h3, R7-96h2 ,
R11-72h4 *, R11-240h5, R11-48h2, R11-96h1, R11-48h1, R6PR62-96h , R11-240h3, R11-240h4 , R15-192h *
Candida glabrata
E2
Fig. 2. Dendrogram generated by the unweighted pair-group method with arithmetic averages based on M13 PCR-fingerprints of yeasts isolated from
12 spontaneous laboratory sourdough fermentations. Isolate numbers are given in the order of appearance of the profiles in the tree. Isolates that were
chosen for sourdough fermentation-related physiological characterization and sequence analysis are marked with a cross and an asterisk, respectively.
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FEMS Yeast Res 10 (2010) 471–481
477
Yeast species composition in sourdough
of 17 S. cerevisiae tested (R9-24h2, R9-72h2, R9-96h5,
D01WW01T01-6,
D03WW01T01-3,
D06SS01T01-2,
D11WW01T01-5, D11WW02T01-1, and D11SS01T01-4),
and the single tested T. delbrueckii isolate (D06WW01T012) incapable of growing under these conditions. Most
isolates grew in the presence of 1% acetic acid, except for
three isolates of W. anomalus (R7-96h2, R9-24h3, and R15192h) and one isolate of S. cerevisiae (D10SS01T01-7).
Species distribution
The 127 artisan sourdough isolates were identified as
S. cerevisiae (68%), W. anomalus (26%), and K. barnettii
(4%). Kazachstania unispora and T. delbrueckii were represented by one and two isolates, respectively (Fig. 1). In
contrast, the 217 spontaneous laboratory sourdough isolates
were identified as W. anomalus (66%) and C. glabrata
(29%); S. cerevisiae (5%) was rarely detected (Fig. 2).
The 21 sourdoughs of 11 artisan bakeries in seven
provinces of Belgium were dominated by the presence of
S. cerevisiae (in 18 sourdoughs), followed by W. anomalus
(in nine sourdoughs) (Table 1). While 11 of the doughs led
to the isolation of only one yeast species, the other 10 were
comprised of two species. The species diversity was not
correlated with the flour type (Table 1). Some bakeries
maintained pure S. cerevisiae doughs and doughs with two
different yeast species in parallel; in some cases, the nonS. cerevisiae yeast even dominated a specific dough
(D07WR01T02, D07WR02T01, and D11RR01T01). However, pure W. anomalus doughs were only found in two
bakeries (D08 and D09), from which no S. cerevisiae doughs
were known and a pure K. barnettii dough was found in a
bakery (D02) that also maintained two S. cerevisiae-dominated doughs (Table 1). The yeast diversity did not indicate
a correlation with temperature, pH, time after refreshment,
or age (Table 1).
The 12 spontaneous laboratory sourdough fermentations
with flour from two different Belgian mills were dominated
by W. anomalus (found in 11 sourdoughs), followed by
C. glabrata (in nine sourdoughs), and the occasional detection of S. cerevisiae (in three sourdoughs). Although some
fermentations led to the isolation of only one yeast species,
the majority contained a combination of species, typically of
W. anomalus and C. glabrata. The yeast diversity did not
indicate a correlation with the mill or the flour (Table 2).
Discussion
Spontaneous sourdoughs usually develop a stable microbiological community (De Vuyst & Neysens, 2005; De Vuyst
& Vancanneyt, 2007), which was supported by the low
species diversity of yeasts identified during this study. To
our knowledge, this is the first study investigating the
spontaneously developing yeast microbiota in controlled
FEMS Yeast Res 10 (2010) 471–481
laboratory fermentations without the addition of a starter
culture. Meroth et al. (2003) have shown the dominance of
S. cerevisiae and C. humilis during controlled fermentations,
but these yeasts originated from the bakers yeast and
commercial sourdough starters used, respectively. Moreover,
this is the first study of Belgian artisan sourdoughs.
In the present study, the yeast species identification was
achieved by M13 PCR-fingerprinting, followed by multiple
gene sequencing and physiological characterization of representative isolates of each fingerprinting group. This
revealed the presence of primarily S. cerevisiae and
W. anomalus in artisan bakery sourdoughs and W. anomalus
and C. glabrata in spontaneous laboratory sourdoughs. The
clusters and the single isolate labelled with a common letter
and distinguished by subscript numbers (B1/B2, E1/E2) were
determined by DNA sequences to represent the same species
(S. cerevisiae and C. glabrata, respectively). The separation
of clusters B1 from B2 and of E1 from E2, as well as the
fingerprint variation within clusters, was attributed to
intraspecies variation, indicating the presence of genetically
different lineages among the sourdough yeast isolates.
Among these postulated genetic lineages, only the six
S. cerevisiae isolates in cluster B2 representing a single
sourdough from bakery D05 indicated the selection of a
particular strain in a bakery.
Most detected species, with the exception of K. barnettii
and K. unispora, are well known to occur in sourdough,
although some typically sourdough-associated species such
as K. exigua (anamorph C. holmii), C. humilis (synonym
C. milleri), and P. kudriavzevii (formerly I. orientalis, anamorph C. krusei) were not found (Pulvirenti et al., 2001;
Meroth et al., 2003; Succi et al., 2003; Vernocchi et al.,
2004b; Hammes et al., 2005). Apparently, many existing
studies have been based on Italian sourdoughs (Corsetti
et al., 2001; Pulvirenti et al., 2001; Succi et al., 2003;
Foschino et al., 2004; Vernocchi et al., 2004b) and significant
differences in the range of the detected yeast species may be
due to process-related differences.
During the current study, the species composition differed between bakery and laboratory sourdoughs. In Belgian
bakery sourdoughs, the frequently reported species S. cerevisiae (Corsetti et al., 2001; Pulvirenti et al., 2001; Meroth
et al., 2003; Succi et al., 2003; Vernocchi et al., 2004b) might
originate from the bakery’s environment in most cases, and
was rarely introduced into the process by the flour (Meroth
et al., 2003). As an additional difference, C. glabrata was
detected regularly and exclusively in the laboratory fermentations. Although documented before as an occasional
component (Succi et al., 2003; Vogelmann et al., 2009), the
present paper is, to our knowledge, the first report of a
sourdough fermentation dominated by C. glabrata. Its
growth relies mainly on glucose as an energy source.
Kazachstania barnettii, a maltose-negative yeast species
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478
G. Vrancken et al.
Table 2. Detection of yeast species during 12 spontaneous laboratory sourdough fermentations
Colony number obtained from sample collected at time (h):
Fermentation
Mill
Flour
Yeast species
24
48
72
96
120
144
168
192
216
240
R6
Spelt A
D12
Spelt
W. anomalus
C. glabrata
S. cerevisiae
0
2
3
2
3
3
3
3
2
4
1
5
1
5
1
4
2
7
3
1
1
2
1
1
1
2
1
5
4
1
4
1
R13
D12
Spelt
W. anomalus
C. glabrata
S. cerevisiae
2
R8
Wheat A
D12
Wheat
W. anomalus
C. glabrata
S. cerevisiae
R12
D12
Wheat
W. anomalus
C. glabrata
S. cerevisiae
R7
D12
Rye
W. anomalus
C. glabrata
S. cerevisiae
2
1
R14
D12
Rye
W. anomalus
C. glabrata
S. cerevisiae
1
1
1
1
1
2
1
1
2
5
5
5
1
1
1
1
R11
Spelt B
D13
Spelt
W. anomalus
C. glabrata
S. cerevisiae
1
R16
D13
Spelt
W. anomalus
C. glabrata
S. cerevisiae
1
R10
Wheat B
D13
Wheat
W. anomalus
C. glabrata
S. cerevisiae
4
1
3
2
4
1
3
2
R15
D13
Wheat
W. anomalus
C. glabrata
S. cerevisiae
1
1
2
1
1
4
1
W. anomalus
C. glabrata
S. cerevisiae
4
1
2
2
2
2
2
2
2
1
1
1
R9
R17
D13
D13
Rye
Rye
W. anomalus
C. glabrata
S. cerevisiae
3
2
1
2
3
3
1
2
1
1
1
5
1
1
2
1
3
2
1
1
2
1
1
1
1
1
1
1
2
Flour of different cereals processed at two different Belgian mills was used.
Sample designations used in a complementary study on lactic acid bacteria (Van der Meulen et al., 2007).
identified for the first time from sourdough, was the only
yeast isolated from the bakery sourdough D02W01T01. This
species might have been misidentified as S. cerevisiae or
K. exigua in studies using phenotypical identification methods. This same sourdough was the only bakery sourdough
dominated by a maltose-negative yeast species, which conforms to a mutualistic association with maltose-utilizing
Lactobacillus sanfranciscensis (Ottogalli et al., 1996). The
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best-known association of yeasts and lactic acid bacteria in
sourdough is that of the maltose-negative yeasts K. exigua or
C. humilis with the maltose-positive L. sanfranciscensis in
San Francisco sourdough and Panettone, respectively
(Gobbetti et al., 1994; Ottogalli et al., 1996). This stable
association has been attributed to the fact that, unlike
L. sanfranciscensis, K. exigua and C. humilis are unable to
ferment maltose, thereby avoiding nutritional competition
FEMS Yeast Res 10 (2010) 471–481
479
Yeast species composition in sourdough
and catabolite repression by glucose (Gobbetti et al., 1994).
This interaction is further enhanced by the reported excretion of glucose upon maltose consumption through maltose
phosphorylase activity by L. sanfranciscensis, which can then
serve as an energy source for the maltose-negative yeasts
(Gobbetti et al., 1994; Stolz et al., 1996). The species
K. unispora and T. delbrueckii can be considered as minor
components of the sourdough microbiota, as they were
represented by single isolates in doughs dominated by
S. cerevisiae.
Sequencing of two additional protein-coding genes
(MET6, COX2) allowed a better understanding of the
genetic variation in S. cerevisiae isolates. The observed large
intraspecies variation of the mitochondrial COX2 gene
sequences in S. cerevisiae has been noted before and might
be due to a high evolutionary rate of the COX2 gene in this
species (Kurtzman & Robnett, 2003), leading to conspecific
populations that developed distinctive COX2 genes. The
occurrence of the same COX2 gene sequences in isolates
from three different bakeries (D03WW01T01-1,
D05WW01T01-1, and D07WR02T01-2) indicated that a
common origin of the strains present in different bakeries
is possible.
The ability of all W. anomalus and S. cerevisiae isolates to
strongly assimilate maltose and sucrose, the major carbohydrates of flour, might be considered as an advantage of these
species, leading to their dominance in laboratory sourdough
fermentations and their frequent occurrence in bakery
sourdough fermentations. The known osmotolerance of
W. anomalus, but generally not of S. cerevisiae, may add to
the first species’ advantage, as invertase activity provided by
the yeast may lead to an increase of glucose from sucrose in
sourdough (Lues et al., 1993; Gobbetti et al., 1994). Saccharomyces cerevisiae showed the least tolerance to the lowest pH.
This may be another reason why this yeast species did not
dominate spontaneous laboratory sourdough fermentations,
as it could not outcompete more acid-tolerant strains of W.
anomalus. From only one laboratory fermentation with rye
flour, S. cerevisiae was isolated up to the end of the experiment, while this species was temporarily present in two other
fermentations. The isolate obtained at the end of the experiment was able to grow at a pH 2.5 in contrast to those
obtained at earlier time points of the same fermentation,
indicating an adaptation or selection towards acid tolerance.
No clear correlation could be established between the
yeast and lactic acid bacteria composition in artisan bakery
sourdoughs, although it is noteworthy that, among others,
the sourdoughs of two bakeries (D08 and D09), from which
W. anomalus was isolated as the sole yeast species, lack the
maltose-positive L. sanfranciscensis (Scheirlinck et al., 2008).
Previously, a difference in the lactic acid bacteria composition of sourdoughs based on different cereals has been
reported, namely that Lactobacillus fermentum is absent
FEMS Yeast Res 10 (2010) 471–481
from spelt sourdoughs, while present in rye and wheat
sourdoughs (Van der Meulen et al., 2007). Although the
same sourdoughs were included in the present study, no
such cereal-related difference could be observed for the
composition of the yeast population.
In laboratory fermentations, typically, the final species
composition was established after the first week, although
not all species could be cultured at all sampling times. The
bacterial populations and metabolite profiles of the four
fermentations analysed by Van der Meulen et al. (2007) had
stabilized after the same fermentation time.
To conclude, the 127 yeast isolates from artisan bakery
sourdoughs were dominated by S. cerevisiae (68%), followed
by W. anomalus (26%), whereas the 217 isolates from
spontaneous laboratory fermentations were dominated by
W. anomalus (66%), followed by C. glabrata (29%). This
study indicated a specific diversity of yeasts in artisan
Belgian sourdoughs that can partly be assigned to the local
flours as potential inoculum and the influence of the
environment and bakery practice on the growth of yeasts
during traditional sourdough fermentation, as evidenced by
the dominance of the yeast populations during spontaneous
laboratory sourdough fermentations.
Acknowledgements
The authors would like to acknowledge their financial
support from the Research Council of the Vrije Universiteit
Brussel (OZR, GOA, and IOF projects), the Fund for
Scientific Research – Flanders, the Institute for the Promotion of Innovation through Science and Technology in
Flanders, in particular, the SBO project ‘New Strategy for
the Development of Functional and Performant Starter
Cultures for Foods in Function of ‘‘Food Qualitomics’’’,
the Federal Research Policy, in particular, the project of the
Action for the Promotion of and Cooperation with the
Belgian Coordinated Collections of Microorganisms (contracts BCCM C3/10/003 and C4/00/001), and the European
Commission’s Marie Curie Mobility Actions contract
MIRG-CT-2005-016539. H.M.D. thanks M.-C. Moons and
P. Evrard for morphological and physiological analyses as
well as S. Huret and C. Bivort for sequence analyses.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Identification of sourdough isolates based on
DNA sequence comparisons and morphological and physiological characteristics, according to different M13 PCR
fingerprint (FP) clusters.
Table S2. Physiological/morphological profiles of 17 isolates
selected to represent the PCR-fingerprinting clusters.
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