Stress Tolerance in Group 1 and Group 2 Lager Yeast Strains

Pauline 1
Chabert , Tobias Fischborn 2 and Chris Powell1
1The University of Nottingham, Nottingham, UK. 2Lallemand Inc, Montreal, Canada.

1. Introduction
S. bayanus

Saccharomyces pastorianus strains are natural hybrids of S. cerevisiae and S. bayanus yeast and Low 72%
Group 1. Saaz

display properties particularly suited to the production of lager type beers. Recently it has been S. paradoxus Low S. pastorianus
Group 2. Frohberg

suggested that the S. pastorianus species may contain two subgroups, designated Group 1 (Saaz 50% 50%

type) and Group 2 (Frohberg type), arising as a result of separate mating events. These two S. cerevisiae

groups have been differentiated according to a number of key features including genome
The Saccharomyces Sensu Stricto group The Lager yeasts
rearrangements, gene copy number, DNA sequence polymorphisms and differences in ploidy
(Dunn and Sherlock, 2008).
Despite detailed genetic analysis, only limited studies have been performed to characterize the phenotypic properties of these strains. Recent data has
suggested that Group 2 strains may be less tolerant to desiccation related stress factors than Group 1 type yeast (Layfield, 2009). However analysis of the
response to multiple stress factors by a number of strains from each group has not yet been performed. Here we ascertain the response of 5 Saaz strains,
3 Frohberg yeast and a production lager strain to a number of industrially significant stress factors.

2. Methods 4. Propagation
Yeast Strains Ammonia Feed mL/h (Actual) Ammonia Feed mL/h (Recipe) Molasses Feed mL/h (Actual) Ammonia Feed mL/h (Actual) Ammonia Feed mL/h (Recipe) Molasses Feed mL/h (Actual)

Group 1 (Saaz): 1486, 1174 & 2440 (CBS, Netherlands), 6033 & 6261 (DBVPG, Italy) 180
Molasses Feed mL/h (Recipe)
Yeast Growth g @ 30% Solids
Ethanol ppm (Set Point) Ethanol ppm (Actual) 1600 180
Molasses Feed mL/h (Recipe)
Yeast Growth g @ 30% Solids
Ethanol ppm (Set Point) Ethanol ppm (Actual) 1600

Group 2 (Frohberg): 1260 & 1485 (CBS, Netherlands), 6282 (DBVPG, Italy) 160
1400
160
1400
Molasses/Ammonia feed rate (mL/h)

Molasses/Ammonia feed rate (mL/h)

Production Strain: LAL1

EtOH (ppm) and Yesat Growth (g)

EtOH (ppm) and Yeast Growth (g)

140 140
1200 1200

Osmotic Stress 120

1000
120
1000

Stationary phase yeast were stressed by suspending cells in 35% NaCl. Viability was assessed
100 100

800 800

by methylene blue staining after 24, 48 and 72 hours.

80 80

600 600

Oxidative Stress
60 60

400 400
40 40

Stationary phase yeast were stressed by suspending cells in 1% H2O2. Viability was assessed 20
200
20
200

by methylene blue staining over a time period of 1 hour. 0 0 0 0

Heat Stress 0 2 4 6 8 10 12 14 16 18 20 22 0 2 4

Fermentation Time (Hours - 2 Days)

6 8 10 12 14 16 18 20 22 0 0 2 4 6 8 10 12 14 16 18 20 22 0 2 4

Fermentation Time (Hours - 2 Days)

6 8 10 12 14 16 18 20 22 0

Stationary phase yeast were stressed by placing cells at 45°C. Total viable cells were
determined by cultivation on YPD agar and compared to a control sample maintained at 25°C. Group 1 (Saaz) strain 6003 Group 2 (Frohberg) strain 6282
Propagation • More difficult to propagate • Easier to propagate
Strain 6033 (Group 1) and 6282 (Group 2) were cultivated using a laboratory scale fed batch
propagation system under the standard regime employed by Lallemand Inc. • Tendency to produce alcohol • Growth simple to control

3. Response to Stress
100.0 100.0 2.50E+07

90.0 90.0

80.0 80.0 2.00E+07

70.0 1486 S 70.0 1486 S 1486 S

1174 S 1174 S 1174 S
Viable Cells/mL

60.0 60.0 1.50E+07

Viability (%)

Viability (%)

2440 S 2440 S 2440 S

50.0 6033 S 50.0 6033 S 6033 S

40.0
6261 S 40.0
6261 S 1.00E+07
6261 S
LAL1 LAL1 LAL1
30.0 30.0
1260 F 1260 F 1260 F
20.0 1485 F 20.0 1485 F 5.00E+06 1485 F
6282 F 6282 F 6282 F
10.0 10.0

0.0 0.0 0.00E+00

0 24 48 72 0 30 60 0 30 60 90 120
Time (Hours) Time (Hours) Time (Hours)

Oxidative Stress - 1% H2O2 Osmotic Shock - 35% NaCl Heat Shock - 45°C
• Variation in tolerance to stress • Variation in tolerance to stress • Group 1 (Saaz) yeast are rapidly
between strains between strains killed at high temperatures
• No trends observed between yeast • No trends observed between yeast • Group 2 (Frohberg) strains display
groups groups greater tolerance to heat

References and Acknowledgement 5. Conclusions

Dunn, B and Sherlock, G. 2008. Reconstruction of the genome origins and evolution • Individual strains respond differently to environmental stress factors
of the hybrid lager yeast Saccharomyces pastorianus. Genome Res. 18, 1610-23. • Frohberg (G2) strains are more resistant to heat than Saaz (G1) strains
Layfield, B. 2009. Characterization of hybrid strains of Saccharomyces pastorianus for
• Saaz (G1) strains may be more difficult to propagate than Frohberg (G2) yeast
desiccation tolerance and fermentation performance. MSc Thesis, NCSU.
• Interestingly, the majority of commercial lager strains are believed to be
Pauline Chabert would like to thank Lallemand Inc. for financial support Frohberg (G2) type
during her time at the University of Nottingham. • Yeast heritage may play a future role in selection for industrial yeast strains