Journal of IndustriaI Microbiology, 4 (I989) 315-324

Elsevier 315

SIM00189

Sugar utilization by yeast during fermentation

T o n y D ' A m o r e , Inge Russell and G r a h a m G. Stewart

Research Department, Labatt Brewing Company Limited, London, Ontario, Canada

Received 8 August 1988

Accepted 16 December 1988

Key words: Sugar uptake; Yeast; Brewer's wort

SUMMARY

When glucose and fructose are fermented separately, the uptake profiles indicate that both sugars are
utilized at similar rates. However, when fermentations are conducted in media containing an equal concentra-
tion of glucose and fructose, glucose is utilized at approximately twice the rate of fructose. The preferential
uptake of glucose also occurred when sucrose, which was first rapidly hydrolyzed into glucose and fructose by
the action of the enzyme invertase, was employed as a substrate. Similar results were observed in the fer-
mentation of brewer's wort and wort containing 30% sucrose and 30% glucose as adjuncts. In addition, the
high levels of glucose in the wort exerted severe catabolite repression on maltose utilization in the Saccharo~
myces uvarum (carlsbergensis) brewing strain. Kinetic analysis of glucose and fructose uptake in Saccharo-
myces cerevisiae revealed a Km of 1.6 mM for glucose and 20 mM for fructose. Thus, the yeast strain has a
higher affinity for glucose than fructose. Growth on glucose or fructose had no repressible effect on the uptake
of either sugar. In addition, glucose inhibited fructose uptake by 60% and likewise fructose inhibited glucose
uptake by 40%. These results indicate that glucose and fructose share the same membrane transport compo-
nents.

INTRODUCTION transport systems [2,3]. Low affinity transport ap-

pears to occur by constitutive facilitated diffusion
The glucose and fructose transport system of whereas the high affinity transport system is de-
yeast has remained controversial over the last two pendent upon the activity of phosphorylating en-
decades [1,7]. Recent studies indicate that the up- zymes. Although extensive studies have not been
take of glucose and fructose in Saccharomyces cere- performed, it is generally accepted that glucose and
visiae is mediated by both high and low affinity fructose compete for the same membrane carrier
[4,7]. Since many commercial substrates (eg., brew-
Correspondence: T. D'Amore, Research Department, Labatt
er's wort) contain a mixture of sugars, it is impor-
Brewing Company Limited, 150 Simcoe Street, London, Onta- tant to examine the role of these sugars on hexose
rio, N6A 4M3, Canada. transport and their effect on the uptake of more

0169-4146/89/$03.50 9 1989 Society for Industrial Microbiology

316

complex sugars such as maltose and maltotriose. charomyces uvarum (carlsbergensis) brewing lager
Sucrose is frequently used in many parts of the strain 3021. The yeast cells were subcultured in
world as a carbohydrate adjunct by breweries and PYN medium which consisted of: peptone, 3.5 g;
other fermentation-based industries employing yeast extract, 3.0 g; KH2PO4, 2.0 g; (NH~)2SO4, 1.0
yeast. It is a disaccharide composed of D-glucose g; MgSO4 9 7H20, 1.0 g; glucose, 100 g; all dis-
and D-fructose linked by an c~-1,4 glycosidic bond. solved in 1 litre of distilled water and adjusted to
In the initial stages of fermentation, sucrose is rap- pH 5.6.
idly hydrolyzed into glucose and fructose by the ac-
tion of the periplasmic enzyme invertase, prior to Fermentation conditions
the sugars being transported across the cell mem- Fermentations were conducted in PYN medium
brane [4,10]. Glucose was observed in this case to be containing varying concentrations of sugar as de-
taken up preferentially over the other hydrolysis scribed in the Results section. Fermentations were
product fructose [8.9]. carried out at 30~ in 300 ml Erlenmeyer shake
Growth of Saccharomyces cerevisiae on a medi- flasks containing 100 ml of medium and with con-
um consisting of a mixture of glucose and fructose stant agitation of 150 rpm. Fermentations with 16~
also results in the preferential uptake of glucose Plato brewer's wort were conducted in 30 1 batches
[4,8,9]. However, when glucose and fructose were in stainless steel fermentors without stirring at
added separately, the uptake profile for each sugar 15~ The yeast inoculum used in all cases was 3.5 g
was very similar [8,9]. On the other hand, other re- wet weight cells/l.
ports suggest that even when glucose and fructose
are fermented separately, glucose is utilized at a fas- Estimation of sugar uptake during fermentation
ter rate than fructose [4]. These differences may be At specified times during fermentation, l0 ml of
attributed to the concentration of sugar used in the cell suspension was withdrawn. The samples were
medium. centrifuged at 4000 g for 10 rain. The supernatant
The present manuscript reports on the sugar up- was subjected to HPLC analysis for determining
take profiles in two brewing yeast strains grown on sugar concentrations. A Spectra-Physics model
either glucose, fructose or sucrose, on an equimolar SP8100 high performance liquid chromatograph in-
mixture of glucose and fructose and on brewer's corporating a Bio-Rad oligosaccharide column
wort supplemented with various carbohydrate ad- (Aminex HPX-42A) for glucose, fructose and malt-
juncts. In addition, the kinetics of glucose and fruc- ose analysis and an HPX-87P column for sucrose
tose uptake were also investigated. analysis was employed. The eluant used was double
distilled deionized water and the flow rate was 0.6
ml/min. Both operated in conjunction with a Spec-
MATERIALS AND METHODS tra-Physics model SP6040 XR refractive index de-
tector and a Spectra-Physics model SP4270 com-
Chemicals puting integrator.
D-[U-14C]Glucose (270 mCi/mmol) and D-
[U-14C]fructose (267 mCi/mmol) were obtained Uptake studies
from ICN Biomedicals (Irvine, CA). All other Late-exponential grown yeast cells (16 h) were
chemicals were obtained from commercial sources harvested by centrifuging at 4000 z g for 10 rain at
and were of the highest available purity. 4~ The cells were washed twice with ice-cold 100
mM potassium phosphate buffer pH 6.6 and sus-
Yeast strains and growth medium pended in the same buffer at room temperature to a
The yeast strains used in this study were, with cell density of 5 mg dry weight/ml. Uptake studies
their Labatt Culture Collection numbers, Saccharo~ were initiated by addition of 1 ml of cell suspension
myces cerevisiae brewing ale strain 3001, and Sac~ to 2 ml of radioactive substrate (1.0 #Ci) to the de-
 317

sired final concentration and with constant stirring of glucose was observed with each yeast strain (Fig.
at 23~ At 15 s intervals, 200 #1 of cell suspension 2). It can be calculated that glucose is taken up at
were withdrawn, filtered through 0.45 #m nitrocel- about twice the rate of fructose in both strains, al-
lulose filters and washed with 5 ml of ice-cold phos- though strain differences are apparent for the actual
phate buffer. The filters were dried, solubilized in rates of glucose and fructose uptake. These experi-
liquid scintillation fluid and radioactivity deter- ments were repeated with several non-brewing yeast
mined on a liquid scintillation counter. strains and the results were very similar to those
above (data not shown).
When fermentations were conducted with 20%
RESULTS sucrose, the sucrose was first rapidly hydrolyzed in-
to glucose and fructose by the action of the enzyme
Sugar uptake in synthetic media invertase prior to the uptake of either sugar (Fig. 3).
The uptake profiles of glucose and fructose were Again, the results indicate a preferential uptake of
studied with two brewing yeast strains [Saccharo- glucose over fructose for both yeast strains when
myces cerevisiae 3001 and Saccharomyces uvarum sucrose was employed as a substrate.
(carlsbergensis) 3021]. In the first series of experi-
ments sugar uptake studies in PYN media contain- Sugar uptake in brewer's wort
ing either 20% glucose or 20% fructose as substrate The effect of carbohydrate adjuncts on sugar uti-
were carried out in shake flasks at 30~ Fig. 1 il- lization profiles in brewer's wort was examined in a
lustrates that the uptake rates of glucose and fruc- Saccharomyces uvarum (carlsbergensis) lager brew-
tose with a particular strain were very similar. How- ing strain. Fig. 4 illustrates the effect of employing
ever, when fermentations were conducted in media 30% glucose and 30% sucrose as wort adjuncts and
containing an equal concentration of glucose and also illustrates the situation with corn adjunct in
fructose (10% of each sugar), a preferential uptake wort. In these experiments the initial wort gravity

240 A 2,0] B
200 200

-r 160 -r
,=.,
160

E E
.r 120 9~ 120

03
80 " 80

40- 40

0 1'2 2'4 3'6 4'8 6r0 72 8'4 9'6 0 1'2 24 36 4'8 6'0 "I2 84 9'6
Time (hours) Time (hours)
Fig. l. Glucose and fructose uptake profiles in brewing yeast strains. Fermentations were conducted in PYN media containing 20%
~lucose (O) or 20% fructose(A) with Saccharomycescerevisiae3001 (A) and Saceharomycesuvarum (carlsbergensis)3021 (B) in 300 ml
Erlenmeyershake flasks at 30~ Inoculum used was 3.5 g wet weight/i.
318

120-
A '~~1 B
100~ 1001

80- .~
,i-,
80
.J
D

E E I
9~ 60 9~ 60
J L ~i .
O~

=
if) 40- g~ 40

20 9 20

1'2 24 3'6 4'8 60 72 0 12 24 36 48 60 72

Time (hours) Time (hours)

Fig. 2. Simultaneous uptake of glucose and fructose in brewing yeast strains. Fermentations were conducted in PYN media containing
10% glucose ( 9 and 10% fructose (A) with Saccharomyces cerevisiae 3001 (A) and Saccharomyces uvarum (carlsbergensis) 3021 (B) in
300 ml Erlenmeyer shake flasks at 30~ Inoculum used was 3.5 g wet weight/1.

,oo , ,ool
I/ ~ 16o

12011 ~ 1201[-I1

all }li
00t 4 0 ~ 4

0 12 24 60 72 12
Time (hours) Time (hours)

Fig. 3. Sucrose uptake profile in brewing yeast strains. Fermentations were conducted in PYN media containing 20% sucrose (D) with
Saccharomyces cerevisiae 3001 (A) and Saccharornyces uvarum (carlsbergensis) 3021 (B) in 300 ml Erlenmeyer shake flasks at 30~
Glucose ( 9 and fructose (A) concentrations. Inoculum used was 3.5 g wet weight/1.
 319

A 6C
~7

2
0 24 48 72 96 1~0 144 16~80 24
~ ~ 4'8 - 72 96 120 144
~ ~8; 24 48 72 96 120 144
Fermentation time (hours) Fermentation time (hours) Fermentation time (hours)

Fig. 4. Uptake of sugars during fermentation of a 16 degree Plato wort by Saccharomyces uvarum (carlsbergensis) 3021. Fermentations
were conducted with 30% corn adjunct (A), 30% glucose adjunct (B) and 30% sucrose adjunct (C) in 30 1 batches in stainless steel
fermentors without stirring at 15~ Glucose (O), sucrose (A), fructose ( 9 and maltose ( B ) concentrations. Inoculum used was 3.5 g
wet weight/1.

was maintained constant at 16~ Plato and brewery Transport studies

fermentation conditions were employed. It can be The results of 0.2 mM glucose and 0.2 mM fruc-
seen in the corn adjunct wort fermentation (Fig. tose uptake studies in both glucose and fructose
4A) that glucose was preferentially utilized, fol- grown Saccharomyees cerevisiae 3001 and Saccha-
lowed by fructose and then maltose, with some romyces uvarum (carlsbergensis) 3021 are presented
overlap. When the glucose concentration in the in Table 1. They indicate that growth of yeast on
wort was increased, an inhibition of fructose and glucose or fructose has no effect on the uptake of
maltose uptake rates was observed, with glucose be- either sugar. In addition, it can be seen that in both
ing rapidly utilized (Fig. 4B). When sucrose was em- yeast strains examined glucose is taken up at a fas-
ployed as a wort adjunct, an increase in glucose and ter rate than fructose. In the case of Saccharomyces
fructose concentration in the fermentation wort was cerevisiae 3001, glucose is taken up at about three-
initially observed as the result of the rapid hydroly- times the rate of fructose and in the case of Saccha~
sis of sucrose by the enzymatic action of invertase romyces uvarum (carlsbergensis) 3021, glucose is
(Fig. 4C). Furthermore, glucose was again observed taken up at about twice the rate of fructose. In sup-
to be taken up at a faster rate than fructose, with port of these results, Fig. 5 illustrates the kinetics of
maltose being utilized at a much slower rate. glucose and fructose uptake in glucose grown Sac-

Table I
Effect of growth substrate on glucose and fructose uptake by Saccharomyces cerevisiae and Saecharomyces uvarum (earlsbergensis)

Strain Growth v (nmol/mg dry weight cells/min)"

substrate
0.2 m M Glucose 0.2mMFructose

Saccharomyces cerevisiae Glucose 1.0 4- 0.10 0.39 • 0.12

Fructose 1.05 • 0.16 0.44 • 0.14
Saccharomyces uvarum (carlsbergensis) Glucose 0.093 + 0.005 0.066 4- 0.014
Fructose 0.I1 • 0.01 0.07 • 0.02

a The results are the average of four trials.

320

10 The inhibitory effect o f glucose and fructose on

each others uptake rate is shown in Table 2. In these
8
experiments, 2 m M o f the competing sugar was
6 used to determine its effect on 0.2 m M sugar uptake
~4 in glucose grown cells. It can be seen that for both
yeast strains, fructose results in approximately a
38% inhibition o f glucose uptake, whereas glucose
results in approximately a 56% and 74% inhibition
-'i 0 "i 2 3 4 5
1/[Sugar] (1/mM) o f fructose uptake in Saccharomyces cerevisiae and
Saccharomyces uvarum ( carlsbergensis ), respective-
Fig. 5. Double reciprocal plot of glucose and fructose uptake in
ly. These results provide additional evidence that
Saecharomyces cerevisiae 3001. Rates of glucose (O) and fruc-
tose (• uptake in the concentration range of 0.2 mM - - 2.0 mM the yeast strains studied have a higher affinity for
were determined as described in the Materials and Methods sec- glucose.
tion.

DISCUSSION
charomyces cerevisiae. The kinetics for glucose up-
take revealed a Km o f approximately 1.6 m M and a The sugar uptake profiles indicate that when glu-
Vmax o f approximately 3 n m o l / m g dry weight/rain. cose and fructose are fermented separately, both
On the other hand, fructose was taken up with a Km sugars are taken up and utilized at similar rates
o f approximately 20 m M and a Vmax o f approxi- (Fig. 1). However, when fermentations are conduct-
mately 5.5 n m o l / m g dry weight/rain. These results ed in media containing an equal concentration of
clearly demonstrate that the yeast has higher affin- glucose and fructose, glucose is preferentially uti-
ity for glucose than fructose and thus would trans- lized over fructose. In fact, for both yeast strains
port glucose at a faster rate. examined, glucose was utilized at approximately

Table 2
Inhibitory effects of glucose and fructose

Strain v (nmol/mg dry weight cells/min)"

A. Inhibition of Glucose Uptake by Fructose

0.2 mM Glucose 0.2 mM Glucose + % Inhibition
2 mM Fructose

Saccharomyces cerevisiae 1.0 • 0.01 0.62 :t: 0.05 38

Saccharomyces uvarum (carlsbergensis) 0.093 4- 0.005 0.058 • 0.002 38

B. Inhibition of Fructose Uptake by Glucose

0.2 mM Fructose 0.2 mM Fructose + % Inhibition
2 mM Glucose

Saccharomyces cerevisiae 0.39 4- 0.12 0.17 • 0.03 56

Saccharomyces uvarum (carlsbergensis) 0.066 + 0.014 0.017 + 0.006 74

" The yeast strains were grown in glucose medium. The results are the average of four trials.
 321

twice the rate of fructose (Fig. 2). The preferential uptake delayed when compared to the wort employ-
uptake of glucose also occurred when sucrose was ing corn as an adjunct (i.e., compare Fig. 4A with
employed as the substrate (Fig. 3). Sucrose was first 4C). Indeed, in the sucrose adjunct wort, maltose
rapidly hydrolyzed into glucose and fructose by the uptake did not commence until 48 h into the fer-
action of the enzyme invertase prior to the uptake mentation and even after 72 h of fermentation some
of either sugar. Again, glucose is taken up at ap- maltose remained unfermented. The problem of in-
proximately twice the rate of fructose. These results complete fermentation in worts containing in excess
are similar to those previously reported by this and of 25% of their fermentable sugar as sucrose has
other laboratories [4,8,9]. been reported previously and is thought to be due
The sugar uptake patterns in a typical North to glucose repression continuing well into the fer-
American brewer's wort where corn, either as grits mentation, thus inhibiting the uptake of repressible
or as syrup, is routinely employed as an adjunct is sugars such as maltose and maltotriose [4,14]. The
illustrated in Fig. 4A. In the normal situation, brew- effect of high concentrations of glucose in a wort
ing yeast strains (of the species Saccharomyces cere- was even more accentuated when the fermentation
visiae and Saccharomyces uvarum (carlsbergensis)) pattern of wort containing 30% glucose as adjunct
utilize glucose, fructose, maltose and maltotriose in was studied (Fig. 4B). In this situation the uptake of
this approximate sequence although some overlap maltose was severely repressed and only 55% of the
does occur, leaving the dextrins unfermented. For maltose was utilized by the yeast. In addition, mal-
the sake of simplicity the results of only the glucose, totriose utilization was also severely repressed (data
fructose and maltose uptake profiles for Saccharo- not shown). On the other hand, a recent study in-
myces uvarum (carlsbergensis) are presented. It has dicated that glucose did not exert catabolite repres-
been shown that the major limiting factor in the sion on the maltose membrane transport and uti-
fermentation of brewer's wort is the repressing in- lization systems in a particular brewing strain of
fluence of glucose upon maltose and maltotriose Saccharomyces cerevisiae [4]. In their study, up to
uptake [5,11,12]. For example, in Fig. 4A, only 8% glucose was used as adjunct in wort. This is
when approximately 60% of the wort glucose was considerably lower than the 30% glucose adjunct
taken up by the yeast did the uptake of maltose employed in this study and the reduced glucose con-
commence. In most strains of Saccharomyces cere- centration may account for the differences observ-
visiae and Saccharomyces uvarum (carIsbergensis) ed. In addition, yeast strain variation may also ac-
maltose utilization is subject to control by glucose count for the differences in the results.
or catabolite repression [5,11,12]. Kinetic analysis of glucose and fructose uptake in
In a number of countries (e.g., Australia, South Saccharomyces cerevisiae revealed that the Km val-
Africa and Mauritius) sucrose is extensively em- ues for glucose and fructose uptake were 1.6 mM
ployed as a wort adjunct [13]. The use of this sugar and 20 raM, respectively (Fig. 5). This demonstrates
significantly modifies the initial wort sugar spec- that the yeast has a much higher affinity for glucose
trum and also the pattern of sugar uptake during than fructose. These results are similar to those ob-
fermentation. Employing 30% sucrose as adjunct in served in other studies [2,3]. However, in most cases
a 16~ Plato wort, hydrolysis of this disaccharide two transport systems were observed for glucose
commenced almost immediately upon inoculation and fructose uptake i a low affinity transport system
of the yeast resulting in accumulation of its hydro- which appears to occur by constitutive facilitated
lytic products, glucose and fructose in the medium diffusion and a high affinity transport system which
(Fig. 4C). Uptake of glucose and fructose com- is dependent on the activity of phosphorylating en-
menced early in the fermentation with glucose being zymes [2,3]. In this study, only one transport system
taken up at a faster rate than fructose. In addition, was observed for the uptake of glucose and fructose
when sucrose was employed as a wort adjunct, which would correspond to the high affinity system
maltose utilization was significantly repressed and [2,3]. This, however, may be due to the limited con-
322

centration range of substrates employed in per- ACKNOWLEDGEM ENTS

forming the kinetic studies. Investigations are in
progress utilizing a wider concentration range of The authors are grateful to J. Campbell, E. Leis-
substrates to establish the presence of two transport ka, R. Hoover and R. McKee, of the Process and
systems for glucose and fructose in these particular Product Development Department, for helpful ad-
brewing strains. vice and expert technical assistance with the wort
Since the yeasts were observed to have a higher fermentations. The authors would also like to thank
affinity for glucose than fructose, it is not surprising I. Hancock, W. Henderson and G. Celotto for ex-
that the uptake of 0.2 mM glucose occurred at ap- pert technical assistance.
proximately two to three times the rate of 0.2 mM
fructose for both glucose and fructose grown Sac-
charomyces cerevisiae and Saccharomyces uvarum
(carlsbergensis) brewing strains (Table 1). How- REFERENCES
ever, this is in contrast to the fermentation results
1 Bisson, L.F. and D.G. Fraenkel. 1983. Involvement ofkinas-
which indicate that glucose and fructose, when fer- es in glucose and fructose uptake by Saccharomyces cere~
mented separately, are utilized at approximately the visiae. Proc. Natl. Acad. Sci. U.S.A. 80: 1730-1734.
same rate (Fig. 1). The differences in these results 2 Bisson, L.F. and D.G. FraenkeL 1984. Expression ofkinase-
are most likely due to the concentration of sugars dependent glucose uptake in Saccharomyces cerevisiae. J.
Bacteriol. 159: 1013-1017.
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3 Bisson, L.F., L. Neigeborn, M. Carlson and D.G. Fraenkel.
in the fermentation studies which corresponds to a 1987. The SNF3 gene is required for high-affinity glucose
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differing rates of fructose and glucose utilization in Saccha-
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the other hand, the uptake of 0.2 mM sugar is below charomyces eerevisiae. J. Gen. Microbiol. 121: 479-482.
the Kmvalue of either sugar and the uptake profile is 6 Hopkins, R.H. and R.H. Roberts. 1935. Kinetics of alcoholic
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surprising that glucose is taken up faster than fruc- 70rlowski, J.H. and J.P. Barford. 1987. The mechanism of
tose, especially at the lower concentrations. In sup- uptake of multiple sugars by Saccharomyees cerevisiae in
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8 Panchal, C.J. and G.G. Stewart. 1982. The influence of media
below 1%, fructose was utilized more slowly than conditions on the utilization of monosaccharides by a strain
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by brewer's yeast strains. MBAA Tech. Quart. 16: 543-549.
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(carIsbergensis), respectively, whereas fructose re- paper. In: Ethanol from Biomass (Duckworth, H.E. and
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strains under the conditions employed (Table 2). 11 Stewart, G.G., I. Russell and A.M. Sills. 1983. Factors that
These results add support to the conclusion that control tire utilization of wort carbohydrates by yeast.
glucose and fructose compete for the same mem- MBAA Tech Quart. 20: 1-8.
brane carrier [4,7]. 12 Stewart, G.G., R.M. Jones and [. Russell. 1985. The use of
 323

derepressed yeast mutants in the fermentation of brewing Conv. (Aust. and N.Z. Sect.), 20, in press.
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20th Congress, Helsinki, pp. 243-250, IRL Press, Oxford. ing adjunct. Inst. Brew. Proc. Conv. (Aust. and N.Z. Sect.)
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