Journal of Applied Microbiology 2003, 95, 325330

Effect of different starvation conditions on the flocculation

of Saccharomyces cerevisiae

E.V. Soares1 and A. Vroman1,2

1
CIEA Departamento de Engenharia Qumica, Instituto Superior de Engenharia do Instituto Politecnico do Porto, Rua Dr Antonio
Bernardino de Almeida, Porto, Portugal, and 2KdGH-Antwerpen,Departement Industriele Wetenschappen en Technologie, Campus
KIHA, Salesianenlaan, Hoboken, Belgium

2002/453: received 11 November 2002, revised 20 February 2003 and accepted 19 March 2003

ABSTRACT
E . V . S O A R E S A N D A . V R O M A N . 2003.
Aims: To study the effect of different starvation conditions on the flocculation of an ale brewing yeast of
Saccharomyces cerevisiae NCYC 1195.
Methods and Results: Flocculation was assessed by a micro-flocculation technique (Soares and Mota 1997).
Carbon-starved cells of a NewFlo phenotype strain did not lose flocculation during a 48 h period. Cells
incubated only in the presence of fermentable carbon sources (glucose, galactose and maltose at 2%, w/v),
showed a progressive flocculation loss. The incubation of cells in 4% (v/v) ethanol did not induce a flocculation loss.
The simultaneous incubation of cells in the presence of 2% (w/v) glucose and 15 lg ml)1 cycloheximide
hindered flocculation loss. The presence of 01 mmol l)1 PMSF or 10 mmol l)1 EDTA prevented partially or
completely, respectively, the loss of flocculation in the presence of glucose.
Conclusions: Fermentable sugars induced a flocculation loss, which seems to require de novo protein synthesis
and the involvement of different proteases.
Significance and Impact of the Study: The findings reported here contribute to the elucidation of the role
of nutrients on the physiological control of yeast flocculation.

Keywords: brewers yeast, carbon source, flocculation, Flo1 phenotype, NewFlo phenotype, proteases, protein
synthesis, starvation.

and Orton 1973; Miki et al. 1982). Although this property

INTRODUCTION
Yeast flocculation can be defined as an asexual aggregation
process of yeast cells into clumps with subsequent fast
sedimentation in the medium in which they are suspended
(Stewart 1975; Johnson et al. 1988; Stratford 1992). These
flocs are reversibly dispersed by the action of specific sugars
(Eddy 1955; Taylor and Orton 1978; Miki et al. 1982;
Stratford and Assinder 1991, Masy et al. 1992), salts (Taylor
and Orton 1973; Stewart and Goring 1976; Miki et al. 1982;
Nishihara et al. 1982, Stratford 1989) and EDTA (Taylor
Correspondence to: E.V. Soares, CIEA-Departamento de Engenharia Qumica,
Instituto Superior de Engenharia do Instituto Politecnico do Porto, Rua Dr Antonio
Bernardino de Almeida, 431, 4200-072 Porto, Portugal (e-mail: evs@isep.ipp.pt).
2003 The Society for Applied Microbiology
can be used in different fields of biotechnology as it is a
natural, easy and cheap method of cell separation from
media at the end of fermentation, flocculation is particularly
known for its use and importance in the brewing industry.
For the brewer, flocculation characteristics of the yeast
strain are of great importance as the number of yeast cells
suspended in wort during both primary or secondary
fermentations affects fermentation speed, beer flavour,
maturation and filtration (Stewart and Russell 1981; Jin
and Speers 1999).
Different hypotheses have been proposed to explain the
mechanism of flocculation in Saccharomyces cerevisiae.
Today, the most recognized hypothesis is the lectin-like
theory of flocculation (Miki et al. 1982). According to this
326 E . V . S O A R E S A N D A . V R O M A N
model, a specific protein (lectin) present only in flocculent
cells is secreted from the cell wall and the N-terminal part of
this protein binds mannose residues present in the cell walls
of neighbouring flocculent and non-flocculent yeast cells
(Bidard et al. 1995; Bony et al. 1998; Kobayashi et al. 1998;
Patelakis et al. 1998). In this process, calcium ions seem to
be necessary for the activation of the lectins (Miki et al.
1982; Stratford 1989).
Flocculation is a highly complex phenomenon affected by
many genetic (Stratford 1994; Teunissen and Steensma
1995; Teunissen et al. 1995), physiological and environ-
mental factors (Soares et al. 1994; Soares and Mota 1996; Jin
and Speers 1999, 2000).
Taking into account pH and sugar inhibition or the
requirement of ethanol to induce flocculation, different
phenotypes have been proposed: Flo1 phenotype, whose
flocculation is only inhibited by mannose; NewFlo pheno-
type, whose flocculation is inhibited by mannose and glucose;
mannose insensitive flocculation and ethanol dependent
flocculation (Stratford and Assinder 1991; Masy et al. 1992;
Dengis and Rouxhet 1997). The majority of brewing yeasts
belong to NewFlo phenotype, whose flocculation exhibits a
cyclic behaviour, flocculating in the stationary phase of
growth, while Flo1 phenotype strains, are constitutively
flocculent (Stratford and Assinder 1991; Stratford and Carter
1993; Soares and Mota 1996; Patelakis et al. 1998).
The reasons and advantages behind yeast flocculation have
long been debated. Flocculation may enhance the survival of
yeast cells in starvation conditions (B.F. Johnsons personal
communication to Stewart and Russell 1981). Flocculation is
an important characteristic in an environment with scarce
nutrients because the death and posterior autolysis of the cells
inside the flocs can provide further nutrients to surrounding
cells (Stewart and Russell 1981). According to this suppos-
ition, highly flocculent cells of NewFlo phenotype strains lost
flocculation in the early period of growth, in the presence of
nutrients, and recovered it towards the end of the exponential
phase of growth coinciding with the depletion of nutrients
(Smit et al. 1992; Soares and Mota 1996).
In this work, the effect of different starvation conditions
(complete absence of nutrients, carbon source starvation and
starvation of all nutrients except carbon source) on the
flocculation of a NewFlo phenotype ale brewing yeast strain
was investigated. Additionally, the role of protein synthesis
as well as proteases in the loss of flocculation of starved cells
was studied.
M A T E R I A LS A N D M E T H O D S
Strains
Two flocculent strains of Saccharomyces cerevisiae were used
in this work. They were NCYC 1195, an ale brewing yeast
2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 325330
characterized as NewFlo phenotype and S646-1B (MATa/
aHO/HO FLO1/FLO1 ade1/ade1), characterized as Flo1
phenotype (Stratford and Assinder 1991). The original
strain NCYC 1195 was obtained from the National Collec-
tion of Yeast Culture (NCYC), UK; the strain S646-1B was
obtained as a gift from Dr Malcolm Stratford of Unilever
Research (Colworth Laboratory, UK).
Medium and culture conditions
Yeasts were routinely maintained at 4C on YEPD slopes
containing: 1% (w/v) yeast extract, 2% (w/v) peptone, 2%
(w/v) agar, (Difco) and 2 % (w/v) glucose, (Merck).
Precultures were prepared in 40 ml of YEPD broth in
100-ml Erlenmeyer flasks. Cells were incubated at 25C on
an orbital shaker (Braun Certomat S, Braun Biotech
International GmbH, Melsungen, Germany), at 150 rpm,
during 48 h. Cultures were prepared by inoculating 04 l of
YEPD broth in 1-l Erlenmyer flasks with 8 ml from the
precultures and incubated during 48 h in the same condi-
tions of the precultures.
Starvation conditions
After growth, flocculent cells were harvested by centrifuga-
tion (2000 g, 5 min) and washed twice with 30 mmol l)1
EDTA solution to ensure floc dispersion. Finally, cells were
washed and suspended in deionized water.
The cells were transferred to various media indicated
below to a final concentration of ca. 2 106 cells ml)1 and
incubated with shaking (150 rpm) at 25C. The media were
as follows: YNB, 067% (w/v) yeast nitrogen base (Difco);
glucose, galactose and maltose (Merck, Darmstadt,
Germany), 2% (w/v) of each respectively; ethanol (Merck),
4% (v/v); glucose + Cyh, 2% (w/v) glucose + 15 lg ml)1
cycloheximide (Sigma); Glucose + EDTA, 2% (w/v)
glucose + 10 mmol l)1 EDTA (Merck); Glucose + PMSF,
2 % (w/v) glucose plus 01 mmol l)1 phenylmethylsulphonyl
fluoride (PMSF), Pierce.
Measurement of flocculation ability
Flocculation ability of the strains was monitored in
standard conditions, using a microflocculation technique
previously described (Soares and Mota 1997). At defined
times, yeasts were harvested by centrifugation (2000 g,
5 min), washed and resuspended on ice-cold deionized
water and then washed twice in 30 mmol l)1 EDTA
solution. Subsequently, cells were washed twice and
resuspended in deionized water at a final concentration
near 2 109 cells ml)1. Cell suspensions were placed in
citrate buffer (50 mmol l)1, pH 40) containing CaCl2
(8 mmol l)1), in test tubes of 15 mm diameter and 50 mm
 STARVATION AND LOSS OF FLOCCULATION 327

of height, at a final concentration of 5 107 cells ml)1. 100

The final volume of the suspension was 20 ml. The tubes
were sealed, stirred vigorously in a vortex for 10 s and
agitated in a horizontal position for 4 h on an orbital 80

Cells flocculated (%)

shaker at 100 rpm. After agitation, the tubes were allowed
to stand undisturbed for 60 s, in a vertical position, after
60
which, samples (50200 ll) were taken from just below

Cells flocculated (%)

100
the meniscus and dispersed in 100 mmol l)1 EDTA solution.
The number of cells was determined spectrophotometrically 40
50
(Helios c, Unican, UK) at 600 nm; a calibration curve
(number of cells vs absorbance) was previously constructed.
The percentage of flocculated cells was calculated by 20 0
0 12 24 36 48
subtracting the fraction of cells remaining in suspension Time (h)
(after 60 s of shaker stopping) from the total cell count.
0

8
Growth
Growth was monitored spectrophotometrically or by direct
microscopic counting, using a counting chamber, after 6
appropriate dilution in 100 mmol l)1 EDTA solution to
Cells 106 ml1
prevent cell aggregation.

Protein determination
Protein concentration, in the filtrates of cellular suspensions,
was determined by the micro-method described by Bradford
(1976), using bovine serum albumin (Calbiochem, La Jolla,
CA, USA) as standard.
RESULTS
In this work, high flocculent cells of S. cerevisiae NCYC
1195 were exposed to different starvation conditions during
a period of 48-h. Thus, cells were incubated in: (i) water, i.e.
in the complete absence of nutrients; (ii) carbon source
starved conditions, 067% (w/v) YNB, which contains
mainly ammonium sulphate (nitrogen and sulphur sources)
and other minor nutrients (amino acids, vitamins, trace
elements and salts) and (iii) 2% (w/v) of D-glucose in
deionized water, in which cells were deprived of all nutrients
except the carbon source. In all cases, cells were incubated in
a medium that did not contain all nutrients to support
growth. The NewFlo phenotype strain of S. cerevisiae
NCYC 1195 when incubated in water or in YNB (in the
absence of carbon source), remained fully flocculent and no
increase in the number of cells was observed (Fig. 1). On the
contrary, when cells were deprived of all nutrients except
glucose, cells progressively lost their flocculation ability and
cell population increased about two times. Flo1 phenotype
strain S646-1B, a constitutively flocculent strain retained its
flocculation ability during the starvation period of 48 h (or
96 h, data not shown), in the presence of 2% (w/v) glucose
(Fig. 1, inset).
2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 325330
4
2
0
0 8 16 24 32 40 48
Time (h)
Fig. 1 Effect of different starvation conditions on the flocculation and
number of cells of a strain of Saccharomyces cerevisiae NCYC 1195.
Flocculent cells were incubated in deionized water (triangles),
2% (w/v) glucose (circles) or in 067% (w/v) YNB (squares). At
periods of time indicated in the figure, cells were collected, washed and
suspended in citrate buffer (pH 40, 50 mmol l)1) containing Ca2+
(8 mmol l)1) for measuring flocculation ability. The number of cells
was evaluated spectrophotometrically or by direct microscopic count-
ing. For more details see material and methods. Inset: Effect of glucose
on the flocculation of Flo1 phenotype strain of S. cerevisiae S646-1B.
Each point represents the mean of two independent experiments
performed in duplicate; standard deviations are presented (n 4)
The effect of the presence of other carbon sources
(fermentable and non-fermentable), in the absence of other
nutrients, in the flocculation of the strain NCYC 1195 was
carried out by incubating flocculent cells in 2% (w/v)
galactose (a slowly metabolizable sugar), 2% (w/v) maltose
and 4% (v/v) ethanol. As it can be seen in Fig. 2, the strain
NCYC 1195 lost flocculation in the presence of fermentable
sugars (galactose and maltose) and remained fully flocculent
in the presence of ethanol. The flocculation loss in the
328 E . V . S O A R E S A N D A . V R O M A N
100
80
Cells flocculated (%)
60
40
20
0 0
0 8 16 24 32 40
Time (h)
Fig. 2 Effect of different starvation conditions on the flocculation of
the strain of Saccharomyces cerevisiae NCYC 1195. Flocculent cells
were inoculated in the following carbon sources: 2% (w/v) galactose
(j), 2% (w/v) maltose (s) and 4% (v/v) ethanol (n). Each point
represents the mean of two independent experiments performed in
duplicate; standard deviations are presented (n 4)
presence of maltose was similar to glucose (about 80%),
while galactose induced a slower (about 55%) flocculation
reduction during the starvation period of 48 h (Fig 2). A
96 h starvation period did not induce an increase of
flocculation loss in the cells incubated in water or ethanol,
where as in glucose, galactose and maltose, the flocculation
loss was 85% (data not shown).
The requirement of protein synthesis on the loss of
flocculation of the strain NCYC 1195, glucose-induced, was
evaluated by exposing flocculent cells simultaneously to
glucose and Cyh. The presence of Cyh hindered the
reduction of flocculation, indicating that glucose inducing
flocculation loss is dependent of protein synthesis (Fig 3).
No detectable amount of proteins was found in the
filtrates of cell suspensions incubated in glucose. However,
proteins could be present in an amount less than the
detection limit (13 lg ml)1, final volume assay). Thus, the
influence of proteases in the mechanism of flocculation loss
was tested by incubating flocculent cells in the presence of
glucose and 10 mmol l)1 EDTA or 01 mmol l)1 PMSF.
EDTA was used as several proteases are metalloenzymes
and PMSF is a known inhibitor of serine proteases (Walker
2001). The presence of 10 mmol l)1 EDTA prevented the
loss of flocculation in the strain NCYC 1195, while
the presence of 01 mmol l)1 PMSF partially impaired the
reduction of flocculation induced by glucose (Fig. 3). As
the stock solution of PMSF was prepared in ethanol (01%,
v/v), control experiments with flocculent cells were exposed
to the simultaneous presence of glucose (2%, w/v) and
2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 325330
100
80
Cells flocculated (%)
60
40
20
48 0 8 16 24 32 40 48
Time (h)
Fig. 3 Effect of the addition of 15 lg ml)1 cycloheximide (s),
10 mmol l)1 EDTA (j) and 01 mmol l)1 PMSF (n) on the
flocculation of the strain of Saccharomyces cerevisiae NCYC 1195
incubated in 2% (w/v) of glucose. Each point represents the mean of
two independent experiments performed in duplicate; standard
deviations are presented (n 4)
ethanol (01%, v/v); a comparable loss of flocculation was
obtained (data not shown) to that observed with glucose
(2%, w/v) alone.
DISCUSSION
Flocculent ale brewing yeasts, belonging to the NewFlo
phenotype, incubated in a culture medium (richYEPD or
chemically defined-YNB with 2% (w/v) glucose) with all
nutrients necessary for growing, lost rapidly (within the first
8 h of growth) flocculation ability (Soares and Mota 1996;
Soares and Seynaeve 2000); on the other hand, under
nitrogen or carbon source starved conditions, these strains
did not lose flocculation in the same period of time (Soares
and Duarte 2002). However, the addition of nutrients to
nitrogen or carbon source-deprived cells triggered a fast (in
the first 810 h after nutrients addition) loss of flocculation
(Soares and Duarte 2002). Here, we observed that a
prolonged starvation (48 h) in the presence of fermentable
carbon sources (glucose, maltose and galactose) induced a
slow (comparing with growing cells) but progressive loss of
flocculation while the other nutrients (nitrogen and sulphur
sources or other minor nutrients) were unable to do this.
These results suggest that the loss of flocculation is a process
that requires energy. Starved cells in the presence of a
gluconeogenic carbon source (ethanol) did not lose floccu-
lation. Interestingly, Flo1 phenotype strain (S646-1B),
whose flocculation ability is insensitive to the presence of
nutrients, did not lose flocculation when incubated in the

presence of glucose during a period of 48 h. This fact clearly
shows the importance of the carbon source in the loss of
flocculation of NewFlo phenotype strains.
Starvation of the brewing yeast NCYC 1195 in water did
not result in a reduction of flocculation. This is in agreement
with a previous work that reported a very slight loss of
flocculation (<10%) in an ale brewing strain starved in water
(Rhymes and Smart 1996).
It was proposed that the loss of flocculation of cells
grown in rich medium in the early growth was the result
of two combined effects: the dismantling of the floccula-
tion mechanism of the cells coming from the inoculum
and the non-flocculent state of the new cells produced
after growth was started (Soares and Mota 1996).
Although, the molecular mechanism that leads to the
flocculation loss, induced by fermentable carbon sources in
starved conditions, is not known, it seems to involve
protein synthesis. This is supported by the fact that the
incubation of cells in the presence of Cyh, which inhibits
cytoplasmatic protein synthesis at the ribosomal level, in
eukaryotics, hindered the flocculation loss. Additionally,
proteases also seem to be involved in the flocculation loss
as the presence of a protease inhibitor (PMSF) or a
chelator (EDTA, which removes bivalent ions, inactivating
metalloenzymes), prevented partially or completely,
respectively, flocculation loss induced by glucose in the
absence of other nutrients. PMSF is an inhibitor of serine
proteases, like trypsin and chymotrypsin. PMSF partially
impairs the reduction of flocculation induced by glucose,
which suggests that other proteases that are not inhibited
by PMSF can be implicated on the dismantling of yeast
flocculation mechanism.
In conclusion, in this work it was shown that water or
carbon source starved cells of a brewing NewFlo phenotype
strain did not lose flocculation during a period of 48 h. On
the contrary, starved cells of all nutrients except fermentable
carbon source lost progressively flocculation ability. The loss
of flocculation, in starved conditions, seems to be an energy-
dependent process, influenced by carbon source metabolism
and requires de novo protein synthesis; additionally, differ-
ent proteases seems to be involved on the dismantling of
flocculation mechanism of starved cells.
ACKNOWLEDGEMENTS
This work was supported by Fundo de Apoio a` Investigacao
from Instituto Politecnico do Porto (Project P24/96) and by
Programa Plurianual de Unidades de I & DCIEA/ISEP.
Ann Vroman (KdGH-Antwerpen, Belgium) wishes to thank
Dr Rob Gille for the opportunity to participate in the
ERASMUS bilateral agreement program between KdGH-
Antwerpen (Belgium) and ISEP (Portugal).
2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 325330
STARVATION AND LOSS OF FLOCCULATION 329
REFERENCES
Bidard, F., Bony, M., Blondin, B., Dequin, S. and Barre, P. (1995)
The Saccharomyces cerevisiae FLO1 flocculation gene encodes for a
cell surface protein. Yeast 11, 809822.
Bony, M., Barre, P. and Blondin, B. (1998) Distribution of the
flocculation protein, Flop, at the cell surface during yeast growth:
the availability of Flop determines the flocculation level. Yeast 14,
2535.
Bradford, M.M. (1976) A rapid and sensitive method for the
quantification of microgram quantities of protein utilizing the
principle of protein-dye-binding. Analytical Biochemistry 72, 248
254.
Dengis, P.B. and Rouxhet, P.G. (1997) Flocculation mechanisms of top
and bottom fermenting brewing yeast. Journal of the Institute of
Brewing 103, 257261.
Eddy, A.A. (1955) Flocculation characteristics of yeasts II. Sugars as
dispersing agents. Journal of the Institute of Brewing 61, 313317.
Jin, Y.-L. and Speers, R.A. (1999) Flocculation of Saccharomyces
cerevisiae. Food Research International 31, 421440.
Jin, Y.-L. and Speers, R.A. (2000) Effect of environmental conditions
on the flocculation of Saccharomyces cerevisiae. Journal of American
Society of Brewing Chemistry 58, 108116.
Johnson, B.F., Walker, T., Calleja, G.B. and Seligy, V.L. (1988)
Sexual co-flocculation and asexual self-flocculation in budding and
fission yeast: experimental establishment of a fundamental differ-
ence. Canadian Journal of Microbiology 34, 11051107.
Kobayashi, O., Hayashi, N., Kuroki, R. and Sone, H. (1998) Region of
Flo1 proteins responsible for sugar recognition. Journal of Bacterio-
logy 180, 65036510.
Masy, C.L., Henquinet, A. and Mestdagh, M.M. (1992) Flocculation
of Saccharomyces cerevisiae: inhibition by sugars. Canadian Journal of
Microbiology 38, 12981306.
Miki, B.L.A., Poon, N.H., James, A.P. and Seligy, V.L. (1982)
Possible mechanism for flocculation interactions governed by gene
FLO1 in Saccharomyces cerevisiae. Journal of Bacteriology 150, 878
889.
Nishihara, H.T., Toraya, T. and Fukui, S. (1982) Flocculation of cell
walls of brewers yeast and effects of metal ions, protein-denaturants
and enzyme treatments. Archives of Microbiology 131, 112115
Patelakis, S.J.J., Ritcey, L.L. and Speers, R.A. (1998) Density of lectin-
like receptors in the Flo1 phenotype of Saccharomyces cerevisiae.
Letters in Applied Microbiology 26, 279282.
Rhymes, M.R. and Smart, K.A. (1996) Effect of starvation on the
flocculation of ale and lager brewing yeasts. Journal of American
Society of Brewing Chemistry 54, 5056.
Smit, G., Straver, M.H., Lugtenberg, B.J.J. and Kijne, J.W. (1992)
Flocculence of Saccharomyces cerevisiae cells is induced by nutrient
limitation, with cell surface hydrophobicity as a major determinant.
Applied and Environmental Microbiology 58, 37093714.
Soares, E.V. and Mota, M. (1996) Flocculation onset, growth phase,
and genealogical age in Saccharomyces cerevisiae. Canadian Journal of
Microbiology 42, 539547.
Soares, E.V. and Mota, M. (1997) Quantification of yeast flocculation.
Journal of the Institute of Brewing 103, 9398.
Soares, E.V. and Seynaeve, J. (2000) The use of succinic acid, as pH
buffer, expands the potentialities of utilisation of a chemically
330 E . V . S O A R E S A N D A . V R O M A N
defined medium in Saccharomyces cerevisiae flocculation studies.
Biotechnology Letters 22, 859863
Soares, E.V. and Duarte, A.A. (2002) Addition of nutrients induce a
fast loss of flocculation in starved cells of Saccharomyces cerevisiae.
Biotechnology Letters 24, 19571960.
Soares, E.V., Teixeira, J.A. and Mota, M. (1994) Effect of cultural and
nutritional conditions on the control of flocculation expression in
Saccharomyces cerevisiae. Canadian Journal of Microbiology 40, 851
857.
Stewart, G.G. (1975) Yeast flocculation. Practical implications and
experimental findings. Brewing Digest 50, 4262.
Stewart, G.G. and Goring, T.E. (1976) Effect of some monovalent and
divalent metal ions on the flocculation of brewers yeast strains.
Journal of the Institute of Brewing 82, 341342.
Stewart, G.G. and Russell, I. (1981) Yeast flocculation. In Brewing
Science, Vol. 2 ed. Pollock, J.R.A. pp. 6192. Toronto: Academic
Press.
Stratford, M. (1989) Yeast flocculation: calcium specificity. Yeast 5,
487496.
Stratford, M. (1992) Lectin-mediated aggregation of yeasts yeast
flocculation. Biotechnology and Genetic Engineering Reviews 10,
283341.
2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 325330
Stratford, M. (1994) Genetic aspects of yeast flocculation: in particular,
the role of FLO genes in the flocculation of Saccharomyces cerevisiae.
Colloids and Surfaces B: Biointerfaces 2, 151158.
Stratford, M. and Assinder, S. (1991) Yeast flocculation: Flo1 and
NewFlo phenotypes and receptor structure. Yeast 7, 559574.
Stratford, M. and Carter, A.T. (1993) Yeast flocculation: lectin
synthesis and activation. Yeast 9, 371378.
Taylor, N.W. and Orton, W.L. (1973) Effect of alkaline-earth metal
salts on flocculence in Saccharomyces cerevisiae. Journal of the
Institute of Brewing 79, 294297.
Taylor, N.W. and Orton, W.L. (1978) Aromatic compounds and
sugars in flocculation of Saccharomyces cerevisiae. Journal of the
Institute of Brewing 84, 113114.
Teunissen, A.W.R.H. and Steensma, H.Y. (1995) Review: the
dominant flocculation genes of Saccharomyces cerevisiae constitute a
new subtelomeric gene family. Yeast 11, 10011013.
Teunissen, A.W.R.H., Van Den Berg, J. and Steensma, H.Y. (1995)
Transcriptional regulation of flocculation genes in Saccharomyces
cerevisiae. Yeast 11, 435446.
Walker, J. (2001) Protein structure, purification and characterization. In
Principles and Techniques of Practical Biochemistry ed. Wilson, K. and
Walker, J. pp. 312356. Cambridge: Cambridge University Press.