J. hist. Brew., July-August 1998, Vol. 104,/;/;.

221-228

STRAIN SPECIFIC RESPONSE OF BREWER'S YEAST STRAINS TO ZINC CONCENTRATIONS IN

CONVENTIONAL AND HIGH GRAVITY WORTS*

Bv Em/abkth M. R. Rees and Graham G. Stewart

(International Centre for Brewing and Distilling. Heriot-U'att University. Riccarton. Edinburgh. Scotland. EII14 4AS)

Recened 21 April 1998

The effect of zinc on a variety of yeast strains is extensively documented in the literature. However,
due to the varied experimental protocols employed in each study there is little opportunity to directly
compare the strain specificity of this ion. In the present study, the response of six yeast strains (three
Saccharomyces cerevisiae (ale type) and three S.cerevisiae (lager type)) to altered zinc concentrations,
in both high (1080 OG) and conventional (1048 OG) gravity worts, was investigated. Varying the initial
wort zinc concentration in both gravities had an effect on the ethanol production, rate of fermentation,
cell number and sugar uptake of all six strains studied. The extent of the response was found to be
dependent upon the zinc concentration, the strain employed and wort gravity employed.

Key Words: High gravity brewing, zinc, yeast, fermentation, wort.
Introduction
Metal ions play an important role in brewing in general and
yeast performance in particular, and a range are present in
wort, the concentration of which depends upon the raw
materials used and the method employed to produce the wort.
Yeast has a complex ionic nutritional requirement and a range
of ions are necessary to ensure efficient and complete
fermentation8-9. The requirement of trace elements by yeast is
strain dependent, but can be generally classed into three
categories: macroelements (which are required at a level of
0.1-1 mM): microelements (which are required in a concen
tration of 0.1-100 (iM) and inhibitors (which adversely effect
yeast growth at concentrations above 10-100 nM)6. Certain
ions appear to be present at suboptimal levels in brewer's wort
and it has been reported that zinc can become growth limiting
in such fermentations9". This could be due to several factors
including the introduction of stainless steel vessels and the low
level of zinc found in barley which is due to agronomic reasons.
In order to enhance the fermentation performance of yeast,
a greater understanding of the ionic nutrition of the yeast and
the interactions between metals and factors affecting their
bioavailability is necessary. Previous studies investigating
methods of optimizing yeast performance have shown that
modifying the metal ion levels in the wort can have a beneficial
effect on fermentation performance".
Zinc is rapidly absorbed from the wort prior to fermentation
and is taken into the yeast cell along the affinity series Mg:+,
Co:+, Zn2+>Mn2+>Ni:t>Ca:*>Sr' where it is stored in the
yeast vacuole8"-14. Zinc uptake into the yeast cell is via two
systems, a high affinity system that operates in zinc limited cells
and a low affinity system that operates in zinc replete cells,
these two systems are encoded by ZRT 1 and ZRT 2 genes
respectively18.
Zinc is one of the ions known to play a critical role in yeast
performance and is normally present in wort at a concentration
of 0.1-5 ppm, which unlike certain other metals ions e.g.
magnesium and calcium, is likely to be below the physiological
concentration required by yeast. This is due to the naturally
occurring chelating agents present in wort, decreasing the
concentration of bioavailable zinc;j:l4. The growth optimum
of zinc is strain dependent and ranges from 0.08-1.01 ppm,
with the glycolytic optimum being 1-2 ppm7-915.
*Parl of these results were presented ;U the 5th Aviemore conference.
25-29th Mav 1998.
Brewers regularly monitor wort zinc concentrations, as
concentrations less than 0.1 ppm can lead to sluggish and
incomplete fermentations, whilst if present in excess, it can have
a detrimental effect on fermentation parameters4. However, if
present in sufficient amount, zinc can have a positive effect on
protein synthesis, cell multiplication and fermentation rates, in
addition to improving yeast flocculation and beer head reten
tion2. It has also been reported that a high level of zinc, (above
0.18 mg zinc/g yeast), in the yeast crop can have a negative
effect on yeast vitality7".
The effect of zinc on certain yeast strains is well documented,
however there is little chance of comparing the strain specificity
of this ion due to the differing media, strains and conditions
employed in the individual studies. The present study investi
gates the effect of altering the initial zinc concentration in both
conventional and high gravity worts on certain fermentation
parameters of six strains of industrially employed brewers
yeast, under standard experimental conditions.
Materials and Methods
Yeast strains and maintenance
The six yeast strains employed in this study were industrially
used brewing strains of S. cerevisiae (ale type) and S. cerevisiae
(lager type). All strains were maintained on PYN agar slopes
and plates at 4C.
Media
The two all malt worts, 1048 OG (12P) and 1080 OG (20P)
original gravity, employed in this study were produced in the
ICBD 2hL pilot brewery, and were steam sterilized prior to use.
PYN medium was also used and comprised (g/1): peptone 3.5;
yeast extract 3.0; KH2PO4 2.0; (NH4);SO4 1.0; MgSO4.7H2O
1.0; glucose 100. The PYN medium was sterilized at 121C for
15 min prior to use.
Fermentations
Biomass production and pitching rates were determined as
previously described". Prior to pitching a filter sterilized solu
tion of zinc chloride was added to the wort to give the required
starting wort zinc concentrations of: 32.7; 65.5; 327.5; 655;
1310 ppm (corresponding to 0.5, 1, 5, 10 and 20 mM res
pectively). The control fermentations contained no added zinc.
All fermentations were carried out in duplicate, in 250 ml
conical flasks containing 150 ml of wort, which were shaken at
150 rpm. The lager fermentations were maintained at 13C and
the ale fermentations at 25C. Samples were taken every 24 h
until the end of fermentation to determine: wort sugar uptake

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 RHSI'ONSI; ()l BRIWIR'S VI AST STRAINS TO ZINC [J. Inst. Brew.

profile; wort gravity: ethanol concentration; cell number and

viability.
Figure 2 a

Cell and viable counts

Cell counts were determined with a Thoma heamocytometer.
Viable cells were assessed using the methylene blue stain.

Ethanol determination
Ethanol was measured using gas chromatography (Hewlitt
Packard series II), with a Poropaq Q packed column. Butanol
was used as the internal standard with H:/air as the carrier gas.

Sugar utilization
Sugar depletion during the fermentations was monitored by
Time (h)
measuring the decline in the wort gravity, using a densitometer.
The concentration of glucose, fructose, sucrose, maltose and
maltotriose in the samples was determined using Dionex High Figure 2 b
Performance Liquid Chromatography (HPLC). fitted with a
Dionex Carbopack PA104-mm guard column and a Dionex 1.06
PAD electrochemical detector, with deionised water and
500 mM sodium hydroxide as the eluents. 1.06

1.04

Figure 1 a 1.02

24 48 72 96 120 144 168 192

Figure 2 c

144
I

Figure 1 b

48 72 96 120 144 168 192

Time (h)

lrl(i. 2 a-c. The decrease in gravity by 5. cerevisim- (lager type) strain 1

(a), strain 2 (b) and strain 3 (c) in 20 (1080 OG) wort, with shaking,
in the presence of 0 (). 32.7 (). 65.5 ( ). 327.5 (X). 655 (*). or
l.'10() ppm /inc.

120
Rl-SUl.TS

Effect on fermentation rate

Figure 1 c S. cerevisiae (lager type)The attenuation time of all 3
strains, in the 1048 OG (12*P) wort, was unaffected by the
addition of 32.7, 65.5 or 327.5 ppm zinc (Fig. 1 a-c). However,
upon the addition of 655 ppm zinc, the fermentation rate
decreased, in all 3 strains, with the most marked effect found in
strain 1. The addition of 1310 ppm zinc brought about a
stalling of the fermentation at 1040 OG in 2 of the strains
(9 (strains land 3). S. cerevisiae (lager type) strain 2 exhibited the
greatest zinc tolerance at this concentration with the ferment
ation rate being drastically slowed down, however the gravity
dropped to 1024 after 120 h.
120 The addition of 32.7,65.5. 327.5,655.0,1310 ppm zinc to the
1080 OG (20"P) wort, resulted in an initial increase in the
fermentation rates of all 3 strains, which concluded in a
l-'ic. I a-c. The decrease in gravity by .V. rererisiiw (lager type) strain 1 decreased attenuation time for all 3 strains in the presence of
(a), strain 2 (b) and strain 3 (c) in 12'P (1040 OG) wort, with 32.7, 65.5, 327.5 and 655 ppm zinc (Fig. 2 a-c). Upon the
shaking, in the presence of 0 (). 32.7 (). 65.5 (). 327.5 (X). 655 addition of 1310 ppm zinc the attenuation time for all three
(*). or 1310 ()ppm zinc. strains was unaffected.

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Vol. 104. 1998] RI-SPONSI: <>l HRI WIR's VI AST STRAINS TO /I\C
Figure 3 a
Figure 3 b
Figure 3 c
1.06
I'Ki. 3 a-c. The decrease in gravity by S rcrevisiue (ale type) strain I (a),
strain 2 (b) and strain 3 (c) in I2l' (1(140 OG) wort, with shaking,
in the presence of 0 (). 32.7 (). 65.5 ( A ). 327.5 (X). 655 <*-). or
1310 () ppm /inc.
S. cerevisiac (ale type)In the lower gravity wort, the
fermentation rate of all 3 strains increased in the presence of
32.7 and 65.5 ppm zinc (Fig. 3 a-c). The addition of 327.5 ppm
zinc resulted in a decreased fermentation rate in one of the
strains (strain 2). whilst the fermentation rate of the remaining
2 strains (strains 1 and 3), were slightly increased or unaiTected
at this level. The addition of 655 ppm zinc resulted in a slightly
decreased fermentation rate in strain 3. whereas strain 1 was
unaffected with the final attentuation time the same with the
fermentation rate lagging slightly behind the control. However,
strain 2 was inhibited at this concentration of zinc with the wort
gravity at 1040 after 48 h. The addition of 1310 ppm zinc
caused the fermentation to stop fermenting at 1045 in all 3
strains 48 h after pitching.
The addition of 32.7, 65.5, 327.5 and 655 ppm zinc, to the
1080 OG (20'P) wort, resulted in a slightly increased initial
fermentation rates, with the final attenuation time remaining
unaffected in all 3 strains (Fig. 4 a-c). Upon the addition of
1310 ppm zinc, the fermentation capacity of 5. cerevisiae (ale
type) strains I and 2 decreased, with the gravity dropping to
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223
Figure 4 a
Figure 4 b
Figure 4 c
1.06
Fit;. 4 a-c. The decrease in gravity by S. terevhiiic (ale typel strain 1 (a),
strain 2 (b) and strain 3 (c) in 20'P (1080 OG) wort, with shaking,
in the presence of (I < ). .12.7 IB). 65.5 ( A). 327.5 (X). 655 (*). or
1310 () ppm /inc.
just 1070 after 96 h. However. S. cerevisiae (ale type) strain 3
showed the greatest tolerance of this concentration of zinc, with
the gravity reaching 1030 after 96 h.
To summarise the overall effect of the addition of a range of
zinc concentrations on these 6 strains. Figure 5 a and b shows
the time taken to reach 1020 gravity. In the lower gravity wort
(Fig. 5a), none of the six strains reached 1020 OG in the
presence of 1310 ppm zinc. At the higher gravity (Fig. 5b) all
three lager strains were able to reach 1020 OG in the presence
of 32.7-1310 ppm zinc, whereas the three ale strains did not
reach this gravity. In addition, the three lager strains reached
1020 OG faster in the presence of added zinc compared to the
control, whereas little variation was found between the time
taken by the ale strain controls to reach 1020 OG compared
with the presence of added zinc.
Effect on the sugar uptake profile
S. cerevisiae (lager type)In the 1048 OG (12P) wort, the
presence of up to 327.5 ppm zinc had little effect on maltose
uptake, however a stimulatory effect on maltotriose uptake was
224 rhsponsi: 01 iiri:\vi:r's vi:ast strains to zinc [J. Inst. Brew.

Figure 5 a

100

90

80

70

60

50

40

30

20

10

0
Lager strain Lager strain Lager strain Ale strain 1 Ale strain 2 Ale strain 3
1 2 3

Figure 5 b

180

160

140

120

. 100

| 80
60

40

20 |

0
Lager strain Lager strain Lager strain Ale strain 1 Ale strain 2 Ale strain 3
1 2 3

l:l<i. 5 a + b. The time taken by 3 lager and 3 ale .strains to reach 1020 gravity when grown
in 1048 OG wort (a) or 1080 OG wort (b| in the presence of 0 <). .12.7 ('[fl]). 65.5
327.5 (g). 655 Or 1310 <H> ppm zinc.

TABLE- I Percentage total uptake of mallosc (bold) and maltotriosc (iialic.s) by 3 lager
strains fermented in 12P or 20P wort, with shaking, in the presence of a zinc
concentration ranging from 32.7- 1.110 ppm

12Tort Control 32.7 ppm 65.5 ppm 327.5 ppm 655 ppm 1310 ppm

S. cvreri.shu' 1 89.9 91.7 94.8 90.9 70.4 28.9

73.6 .Sl.-t V0.I ,S2.6 .IS. 2 2.1.1
S. ceri'vixiai' 2 88.5 89.8 88.1 87.1 73.2 50.9
69.1 67.6 6(A7 .12.0 47.4 38.2
.V. tvrcmiiif 3 92.6 92.9 92.5 90.9 90.0 25.0
67J 7.V..V 70.2 68. a .17J 21.7

2(11' i>r(

.V. <CT<T/.W</<" / 86.3 92.8 95.5 88.7 88.5 88.2

62.2 65 1 67..V 58. V 53.5 56.6
S irivvisitic J 82.2 91.7 92.6 88.1 84.9 82.6
.17.X 66 .f 73.4 60.2 .18.2 53 6
S. ivrvvhiai'.' 87.7 91.4 92.7 93.4 84.1 84.2
5.V.J 62.4 60.4 .56.7 52.6 46. V

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Vol. 104, 1998] RESPONSE Ol BREWER'S YEAST STRAINS TO ZINC 225

TABLE II Percentage total uptake of maltose (bold) and maltotriose (italics) by 3 ale
strains fermented in 12P or 20P wort, with shaking, in the presence of a zinc
concentration ranging from 32.7-1310 ppm

12"P won Control 32.7 ppm 65.5 ppm 327.5 ppm 655 ppm 1310 ppm

S. cerevisiae 1 89.4 95.4 94.8 92.9 75.2 22.1

67.7 .17.7 55V 51.1 49.6 19.4
S. cerevisiae 2 92.2 92.7 91.5 92.5 38.6 6.7
69.0 77.6 7.?..? 62.5 J.V..V 11.0
S. cerevisiae 3 86.0 88.5 94.9 77.2 63.1 5.9
6S.9 68.0 62.0 50.1 4X.I 10.0

20T wort

S. cerevisiae 1 84.9 87.7 85.5 85.7 80.5 15.2

54.0 55./ 5V.X 34.9 28.5 13.6
S. cerevisiae 2 87.1 91.0 92.6 88.1 84.9 36.6
63.0 76.3 73.4 63.2 44.2 22.S
S. cerevisiae 3 80.8 78.4 87.7 83.0 83.1 70.2
57.X 56.4 60.4 46.7 40.1 42.6

observed in strains 1 and 3 (Table I). A decrease in the uptake
of both sugars was found in strains 1 and 2 upon the addition
of 655 ppm, however strain 3 exibited a slight decrease only on
the uptake of maltotriose.The addition of 1310 ppm zinc
caused a decreased uptake of both maltose and maltotriose,
with strains 1 and 3 being more affected than strain 2.
The addition of 32.7-1310 ppm zinc had little effect on the
maltose uptake of any of the three lager strains employed in this
study, at the higher gravity wort (Table I). An increase in
maltotriose uptake was observed, in all three strains, upon the
addition of 32.7 and 65.5 ppm, the increase being strain
dependent. A slight decrease in maltotriose uptake was
observed at 1310 ppm zinc.
S.eerevisiae (ale type)A strain dependent decrease in the
uptake of maltose and maltotriose was observed upon the
addition of 655 and 1310 ppm zinc to the conventional gravity
wort (strain 2 being the most affected) (Table II). The addition
of up to 327.5 ppm had a stimulatory effect on maltose uptake
in strain 1, no effect on strain 2, and a slightly negative effect
on strain 3. At the same zinc concentration range, maltotriose
uptake was adversely affected in strains 1 and 3, whereas strain
2 exhibited an increase in uptake upto 65.5 ppm, and a decrease
at 327.5 ppm.
With the higher gravity wort, the presence of 1310 ppm
resulted in a substantial decrease in maltose and maltotriose
uptake in strains 1 and 2 (strain I being more adversely affected
than strain 2), whereas in strain 3 the uptake of both sugars
only decreased slightly (Table II). The addition of up to
327.5 ppm zinc resulted in a slight increase in maltose uptake
in all three strains. Maltotriose uptake also increased slightly in
the presence of up to 65.5 ppm, with 327.5 ppm having no
effect uptake in strain 2, however a decrease at this zinc level
was found in strains 1 and 3 (19% and 11% respectively).
Effect on eihanol production
S. cerevisiae (lager type)In the lower gravity wort, all three
lager strains exhibited a decreased ethanol concentration at the
end of fermentation upon the addition of 1310 ppm zinc (Table
III). However, the extent of the decrease was strain dependent
with strain 1 exhibiting a reduction of 80%, strain 2. 33% and
strain 3, 88%. The zinc level evoking the highest ethanol
production was also found to be strain dependent, with strain

TABLE 111 Ethanol production (%) v/v by three lager strains grown in 12P wort (1048
OG) or 20!' (1080 OG), with shaking in the presence of a zinc concentration
ranging from 32.7 ppm to 1301 ppm

12T wort Control 32.7 ppm 65.5 ppm 327.5 ppm 655 ppm 1310 ppm

S. cercvi.siae 1 3.52 3.61 3.78 4.02 3.27 0.69

S. cerevisiae 2 3.54 4.09 3.86 3.41 3.60 2.36
S. cererisiae 3 3.66 3.96 3.58 3.89 3.41 0.44

20*P wort

S. cererisiae 1 5.84 5.73 6.41 5.55 5.47 5.10

S. cerevisiae 2 5.91 6.52 6.39 6.07 6.15 5.41
S. cerevisiae 3 6.31 6.49 7.25 7.11 6.74 5.74

TABLE IV Ethanol production (%) v/v by three ale strains grown in 12P wort (1048 OG)
or 20P (1080 OG). with shaking in the presence of a zinc concentration
ranging from 32.7 ppm to 1301 ppm

12P won Control 32.7 ppm 65.5 ppm 327.5 ppm 655 ppm 1310 ppm

S. cerevisiae 1 3.45 3.84 4.21 3.59 3.20 0.84

S. cerevisiae 2 3.21 3.24 3.52 3.04 1.38 0.72
S. cerevisiae 3 3.42 3.71 3.82 3.54 3.21 0.71

20'P wort

S. cerevisiae I 6.91 7.06 7.83 7.60 7.32 1.17

S. cerevisiae 2 6.16 6.87 6.94 6.72 6.24 1.33
S. cerevisiae 3 6.51 7.09 6.69 6.72 6.60 5.94

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226 RESPONSE <)l; IIRI-Wl-R S YEAST STRAINS TO ZINC [J. Inst. Brew.

Figure 6 a

100

90

80

70

60

50

40

30

20

10

0
Lager strain 1 Lager strain 2 Lager strain 3

Figure 6 b

100

Lager strain 1 Lager strain 2 Lager strain 3

;. 6a + b. The viability of .V. cen-risiue (lager type) strains 1-3 al the end of fermentation in
I2*P (HMO OG) (a) or 20T (I080OG) (b). wort in the presence of 0 Q). 32.7 (Hi. 65.5
327.5 (g). 655 Q)or 1310 (^) ppm /.inc.

1 producing the most ethanol upon the addition of 327.5 ppm
zinc (4.02 (%) v/v), whilst strains 2 and 3 produced the most
ethanol in the presence of 32.7ppm zinc (4.09 and 3.96 (%) v/v
respectively).
All 3 lager strains again exhibited a decreased ethanol
production upon the addition of 1310 ppm zinc to the 1080 OG
(20P) wort (Table III). However the decrease was far less
marked than that found in the conventional gravity wort, with
the ethanol concentration decreasing by 13% in strain 1, 9% in
strain 2 and 10% in strain 3. The zinc concentration resulting
in the highest ethanol concentration was again strain
dependent, with strains I and 3 requiring 65.5 ppm (producing
6.41 and 7.25 (%) v/v respectively), whilst strain 2 produced
6.52 (%) v/v in the presence of 32.7 ppm zinc.
S. cerevisiae(ale type)In the conventional gravity wort, the
ethanol production was again decreased in the presence of
1310 ppm zinc, with strain 1 production down by 75%, strain 2
by 78.5% and strain 3 by 80% (Table IV). The addition of
65.5 ppm zinc resulted in the highest ethanol value for all 3 ale
strains, however the extent of the response was strain
dependent with strain 1 exhibiting an increase (compared to the
control) of 22 %, whilst strains 2 and 3 exhibited an increase of
9.6 % and 11.7 % respectively. At 655 ppm zinc, a decreased
cthanol production by all 3 strains was also observed, again the
extent of the response was strain dependent, strain 1 showing a
decrease by 7%, strain 2 by 57% and strain 3 by 6%.
At 1080 OG (20"P), upon the addition of 1310 ppm zinc, a
decrease in ethanol production was once again observed (Table
IV). However the response of strains 1 and 2 (decreasing by
83% and 78% respectively) was more extreme than found in
strain 3 (8% reduction). The level of zinc resulting in the highest
production of ethanol also varied between strains. Strain 1 and
2 produced 7.83 (%) v/v and 6.94 (%) v/v respectively, in the
presence of 65.5 ppm zinc, whereas strain 3 produced its
highest ethanol concentration, 7.09 %, with the addition of
37.5 ppm zinc.
Effect on viability and cell number
S. cerevisiae (lager type)In the 1048 OG wort, the addition
of up to 65.5 ppm zinc had little effect on the viability of any of
the 3 lager strains employed in this study (Fig. 6a). The viability
of 5. cerevisiae (lager type) strain 3 was also unaffected in the
presence of 327.5 ppm zinc, but upon increasing the zinc
concentration to 655 ppm the viability of this strain fell to
79.6% at the end of fermentation. The addition of 1310 ppm
zinc to S. cerevisiae (lager type) strain 3 caused a decreased
viability (14.6%) at the end of fermentation. However, increas
ing the zinc concentration from 327.5 through to 1310 ppm had

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Vol. 104, 1998] RESPONSE OI-" BREWER'S Yl-AST STRAINS TO ZINC 227

Figure 7 a

Ale strain 1 Ale strain 2 Ale strain 3

Figure 7 b

5
.2

Ale strain 1 Ale strain 2 Ale strain 3

Flo. 7 a+b. The viability of S. cerevisiac (ale type) strains I-3 at the end of fermentation in
12*P( 1040 OG) (a) or 20'P (1080OG) (b). wort in the presence of 0 (), 32.7 ([J]). 65.5 Q).
327.5 (g). 655 Oor 1310 (gj) ppm zinc.

an increasingly negative effect on the viability of S. cerevisiae
(lager type) strains 1 and 2.
The addition of 327.5, 655 and 1310 ppm zinc, to the higher
gravity wort, had a negative effect on all 3 strains viability at the
end of fermentation (Fig. 6b). The presence of 32.7 and
65.5 ppm zinc had no effect on the viability of any of the 3 lager
strains.
S.eerevisiae (ale type)In the 1048 OG (12P) wort, the
addition of 655 and 1310 ppm zinc had a detrimental effect on
the viability of all 3 strains, at the end of fermentation. S.
cerevisiae (ale type) strain 2 exhibited the lowest viability at
655 ppm zinc (32%) (Fig. 7a). The presence of 327.5 ppm zinc
lowered the viability to 70%, in the worst affected yeast strain,
(strain 2) with the other 2 strains maintaining a viability above
80% at this concentration. The addition of 32.7 and 65.5 ppm
zinc seemingly had no effect on the viability of any of the 3 ale
strains.
The presence of 655 and 1310 ppm zinc, in the 1080 OG
(20P) wort, resulted in a decreased viability in all 3 strains, at
the end of fermentation, with strains 2 and 3 being more
adversely affected than strain 1 at 655 ppm (Fig. 7b). S.
cerevisiae (ale type) strains 2 also exhibited a decreased viability
in the presence of 327.5, whilst the viability of strains 1 and 3
was unaffected. The presence of 32.7 and 65.5 ppm zinc showed
no effect on the viability of any of the 3 ale strains.
In all six strains, the highest cell number (data not shown) in
both gravity worts was found upon the addition of 32.7 or
65.5 ppm zinc (causing a 14-32% increase compared to the
control in the lager strains and a 4-22% increase in the ale
strains). Whereas the lowest cell number for all 6 strains was
found upon the exposure to 1310 ppm zinc, which resulted in a
22-52% decrease in the lager strains, and a 26-73% decrease in
the ale strains.
Discussion
The essential influence of zinc on yeast growth has been
demonstrated in both synthetic/defined media and in
wort5-10-16. It has also been shown that altering the initial zinc
concentrations can result in modified fermentation charac
teristics. Skands el al., 199715, demonstrated 0.5 ppm zinc led
to a decreased attenuation time in 2 lager strains, accompanied
by reduced aldehyde levels and increased higher alcohol and
ester production. It has also been stated that the addition of up
to 500 ppm zinc has little toxic effect on yeast fermentation
ability proposing that the level at which a metal becomes toxic
is dependent on the concentration of other metal ions present
and the composition of the wort3. The study of Helin and
Slaughter (1977)5 strengthened this proposal by showing the
inhibitory effects of zinc were found to be dependent upon the

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228 response or brewer's yeast strains to zinc
concentration of manganese. Jones and Greenfield (1984)9 have
since reported that if more than 0.4 ppm Mn is present, a level
of upto 65.5 ppm zinc could be tolerated by a certain yeast
strain, whereas below 0.4 ppm Mn, only 2 ppm zinc could be
tolerated. Zinc has also been shown to have a stimulatory effect
on maltose and maltotriose uptake as well as playing a key role
in yeast vitality and viability9"17.
In the present study, applying uniform experimental con
ditions, the range of zinc additions produced a varied response
which was highly dependent on the strain employed, zinc
concentration and wort gravity used. The attenuation time
altered depending upon the zinc concentration, wort gravity
and strain employed. Interestingly in the higher gravity wort,
the attenuation time of all 3 lager strains was unaffected, even
upon the addition of 1310 ppm, however the fermentative
ability of 2 of the ale strains was drastically reduced at this level.
The tolerance by these three lager strains to elevated zinc levels
in the high gravity wort, reinforces the previous suggestion that
lager strains perform better than ale strains at higher gravities'.
All six strains also exhibited a strain dependent stimulation and
inhibition of maltose and/or maltotriose uptake depending on
the zinc concentration employed. The degree of the response
was strain dependent, with the uptake being less adversely
affected at the higher gravity wort.
In addition, ethanol production was also affected, with the
ale strains exhibiting a reduction of 75-80%, upon the addition
of 1310 ppm zinc to the conventional gravity wort, whereas the
lager concentrations fell by 33-80%. The zinc concentration
evoking the greatest ethanol production, at both gravities,
varied slightly between the strains with the extent of the
response being highly strain dependent. At the higher gravity
wort, the ethanol production by all 3 lager strains again
decreased when exposed to 1310 ppm zinc, however the
decrease was not as severe as found in the lower gravity wort.
Conversely, under the same conditions, the ethanol production
by 2 of the ale strains fell by 78-83% (the same as that found in
the 1048 OG wort), whereas strain 3 production fell by just 8%.
Again the optimal zinc concentration for ethanol production
was 32.7 or 65.5 ppm zinc, with the extent of the response
dependent upon the strain. At 655 and 1310 ppm zinc, the
viabilities of all 6 strains was negatively affected, at both
gravities, the extent being strain dependent. The concentration,
and degree of response which caused a decrease in viability was
reliant on the strain and wort gravity. The cell number was also
found to increase upon the addition of 32.7 and 65.5 ppm zinc,
which is contrary to the study of Bromberg et/.,' however this
may be due to the differences in experimental conditions
between the two studies.
The stimulatory effects observed in this study of certain zinc
levels on several fermentation parameters could be explained
by the essential role zinc plays in yeast metabolism including9:
the stabilization of proteins and membrane systems
acting as a catalytic centre of essential enzymes (e.g. alcohol
dehydrogenase, aldolase and acetaldehyde dehydrogenase)
enhancing riboflavin synthesis
activating acid and alkaline phosphatases
stimulating the uptake of maltose and maltotriose.
Conversely, if present in too high a concentration zinc can exert
a toxic effect. The strain dependent negative effects of differing
zinc levels on ethanol production, viability, cell number, sugar
uptake in all 6 strains employed in this study may be due to
several inter-related factors including8":
activation of degradative enzymes
suppression of secondary metabolism by which detoxifica
tion takes place
disruption of membrane structure leading to the leakage of
K* and UV absorbing materials
induction of (self) autolysis due to the stimulatory effect on
proteolytic activity
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[J. Inst. Brew.
blocking functional groups of important biomolecules, e.g.
enzymes
inhibition of transport systems for essential ions and
nutrients
displacement and substitution of essential metal ions
denaturation of enzymes.
Altering the initial zinc concentration in both gravity worts
led to changes in several fermentation characteristics in all six
strains employed in this study. The response and degree of
response was found to be highly strain specific. In general, it
seems that zinc may not be present in sufficient quantities, in
either gravity wort employed in this investigation, for optimal
yeast metabolism and hence growth and fermentation. In
addition, the fact that the corresponding yeast strains seem able
to endure a higher concentration of zinc at the higher gravity
wort suggests that either the zinc is present in a lower
concentration than in the conventional gravity wort, it has a
greater buffering capacity than that of the lower gravity wort or
another factor is alleviating the extent of zinc toxicity (e.g. the
concentration of manganese).
This study highlights the strain dependency to zinc levels in
both conventional and high gravity worts. In order to gain
maximum information about specific factors on yeast fer
mentative performance standard experimental conditions are
necessary to help elucidate the situation.
Acknowledgements. The authors would like to thank Suntory
Ltd, Osaka for funding this study. E. M. R. Rees would also like
to thank Graham McKernan for assistance with the HPLC
analysis.
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