Research article Institute of Brewing & Distilling

Received: 7 June 2016 Revised: 8 August 2016 Accepted: 23 August 2016 Published online in Wiley Online Library: 16 November 2016

(wileyonlinelibrary.com) DOI 10.1002/jib.362

Lachancea thermotolerans as an alternative

yeast for the production of beer†
P. Domizio,1,2 J. F. House,3 C. M. L. Joseph,1 L. F. Bisson1 and C. W. Bamforth3*
The ability of Lachancea thermotolerans strains to ferment brewer’s wort has been investigated. Initial fermentations with three
L. thermotolerans strains compared the use of maltose and maltotriose, as well as production of glycerol and lactic acid and pH
evolution over the course of the fermentation. The most promising strain was subsequently tested for additional traits important
for beer production, including pitching rate, generational capacity, foam stability, hop tolerance, vicinal diketone production,
oxygen requirement and flocculation. These tests suggest that L. thermotolerans may be a good choice for producing sour beers
in a single fermentation step without the use of lactic acid bacteria. Copyright © 2016 The Institute of Brewing & Distilling

Keywords: Lachancea thermotolerans; sour beer; brewing; yeast

Introduction In addition to the production of lactic acid, L. thermotolerans

Saccharomyces species have traditionally been the primary
fermentation organisms for beer, wine and other alcoholic bever-
ages. In the last decade there has been an increasing interest in
the use of non-Saccharomyces yeasts in the production of some
wine styles owing to recognition that these organisms could
enhance aromatic profiles (1). There have been fewer studies of
the use of non-Saccharomyces yeast in the production of beers.
Kluyveromyces lactis and K. marxianus strains have been studied
in beer fermentations owing to their unique metabolic properties
relating to both flavour and preservation (2–4). Strains of
Torulaspora delbrueckii have also been considered for brewing
owing to their ability to enhance flavour and allow reduced etha-
nol content (1,4–6). Recently, Lachancea thermotolerans (formerly
Kluyveromyces thermotolerans) has been studied for its use in wine,
particularly for its ability to decrease pH through lactic acid pro-
duction. K. thermotolerans was separated from the other species
of Kluyveromyces and placed in Lachancea owing to distinct ge-
netic and metabolic differences (7) and genetic similarity to other
members of the Lachancea genus. To date, there has been little re-
search on the use of this organism in beer production. At the 2015
European Brewing Convention Congress, VTT Technical Research
from Finland presented a poster involving the use of Lachancea
fermentati for brewing purposes (8). That strain produced low alco-
hol levels and held little potential for alcoholic beer production.
Lachancea is unique among yeast genera for its ability to pro-
duce lactic acid, which can affect both flavour and mouthfeel (9).
As with commercial wine production, it could be used in addition
to Saccharomyces in brewing fermentations to produce a rapid
drop in pH (10). However, strains producing high levels of lactic
acid could potentially be used for sour beer production without
the addition of bacteria, thereby simplifying and shortening the
process. Sour beer production is accomplished in various ways,
typically involving multiple microorganisms, both yeast and bacte-
ria, and a long aging process (11). Lactic acid bacteria are sensitive
to hop iso-α-acids (12) so hop kettle additions must be minimized.
In addition, introducing lactic acid bacteria to a brewing environ-
ment complicates cleaning and sanitizing procedures if cross
contamination is to be avoided.
could also enhance other beer flavours. During wine fermentation
this non-Saccharomyces yeast gave an increase in levels of ethyl
lactate (10), which has a fruity aroma and has been found at high
levels in some Belgian beers (Lambic and Gueuze) (13) and
German Berliner Weisse (14). This latter characteristic could be of
particular interest in the production of other beers where unique
aromas are prized.
Most studies of L. thermotolerans in wine have focused on its
co-fermentation with Saccharomyces because of its lower
fermentation capacity (10,15–18). Although it has been proposed
that some non-Saccharomyces yeasts be used in mixed fermenta-
tions to enhance flavours in beers (5,6), co-fermentations are not
practical in brewing owing to the customary practice of re-using
yeast collected from a fermentation as an inoculum. For this
reason, this study focused on using L. thermotolerans in pure
culture in the primary fermentation.
Materials and methods
Yeast strains
Three yeast strains were used, belonging to the species L.
thermotolerans from the UC Davis Viticulture and Enology Wine
Yeast and Bacteria Collection (UCD VEN) and from the yeast culture
* Correspondence to: Bamforth, Charles, Department of Food Science and
Technology, University of California, Davis, CA 95616-8598, USA. E-mail:
cwbamforth@ucdavis.edu
†
A presentation of this work was made by Jennifer House to the Young
Scientists Symposium at Chico, CA, USA on 23 April 2016.
1
Department of Viticulture and Enology, University of California, Davis,
CA 95616-8598, USA
2
Dipartimento di Biotecnologie Agrarie, Università degli Studi di Firenze,
Via Donizetti, 6, 50144, Firenze, Italy
3
Department of Food Science and Technology, University of California, Davis,
CA 95616-8598, USA
599

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collection of the Department of Agricultural, Food and Forestry
Systems (GESAAF, University of Florence, Italy) and the Depart-
ment of the Polytechnic University of Marche SAIFET (Ancona, Italy;
Table 1). Two S. cerevisiae strains, UCD VEN 915, Anchor Ale and
White Labs WLP001 (White Labs, San Diego, CA, USA), were used
as reference strains for comparison.
Wort fermentation
For analysis of yeast properties during small-scale wort fermenta-
tion, 750 mL bottles were filled with 500 mL of wort of 13.5°P with
a pH of 5.66, produced from dry malt extract. All three L.
thermotolerans strains were evaluated, with UCD915 used for com-
parison. Each bottle was inoculated with a pre-culture of 48 h
grown at 25 °C on the same wort used for the subsequent fermen-
tation. The cell concentration, determined with hemocytometer,
was ~5 × 106 cells/mL. The fermentations were carried out at
14 °C for 3 weeks.
Analytical determinations during fermentation
Residual sugars, ethanol, glycerol and lactic acid were determined
by high-performance liquid chromatography (HPLC) using an
Agilent 1100 series HPLC system (Agilent, Santa Clara, CA, USA)
coupled with an HP-1047 A refractive index detector (Hewlett
Packard, Palo Alto, CA, USA) detector, according to the method
of Castellari et al. (19). Samples were diluted in water and filtered
through a 0.45 μm cellulose membrane filter prior to injection of
20 μL of each sample. Isocratic separation was performed at
45 °C on a (300 x 7.7 mm) Hi-Plex H column (Agilent, Santa Clara,
CA, USA). The mobile phase was a mixture of 0.0045 N sulfuric acid
and acetonitrile (6%, v/v) at a flow rate of 0.5 mL/min. The com-
pounds were identified and quantified by comparisons with exter-
nal standards. The areas of the peaks of interest were integrated
using the ChemStation Data Analysis System, version A.10.2
(1757) (Agilent, Santa Clara, CA, USA). All of the analyses were
carried out in duplicate.
Pitching rate
Wort of 12.75°P with a pH of 5.60 was produced from dry malt ex-
tract for this analysis. These five different pitching rates were eval-
uated in triplicate: 2.5 × 105, 5.0 × 105, 1.0 × 106, 2.0 × 106 and
3.0 × 106 cells/mL/°P. Aliquots of 300 mL of wort were added to
500 mL flasks sealed with silicone vent bungs (Ferm-Rite), allowing
the CO2 to escape. Flasks were gently agitated and stored in the
dark. Five millilitres of beer from each flask was aseptically sampled
daily in a biological safety cabinet and evaluated for pH using an
Accumet Basic AB15 pH meter (Fisher Scientific, Waltham, MA,
USA) and gravity/alcohol using an Alcolyzer Plus Beer/DMA
4100 M (Anton Paar USA, Ashland, VA). Samples were kept static,
Table 1. Strains used
Strain Collection
602 UCD VEN
1020 UCD VEN
101 GESAAF-SAIFET
915 UCD VEN
WLP001 White Labs
600
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P. Domizio et al.
except for gentle movements between the dark drawer and
sampling hood. At the end of fermentation, after 16 days for all
rates, cell viability was determined using a hemocytometer and
1% methylene blue staining (ASBC Method Yeast-3-A) (20).
Generational capacity
Wort was produced from dry malt extract to give a starting gravity
of 13.75°P, pH 5.14, and 43 bitterness units (BU). Aliquots of 300 mL
in 500 mL Erlenmeyer flasks were prepared and autoclaved. Flasks
were closed with breathable silicone bungs. Wort was stored at
2 °C until ready for use. Yeast recovered from the pitching rate
experiment that featured 2.0 × 106 cells/mL/°P was re-pitched into
flasks in triplicate. At the end of each fermentation, pH, gravity,
alcohol by volume (ABV), and percentage viability were evaluated
as in the pitching study above.
Hop tolerance
Wort from dry malt extract was produced with an original gravity
of 13.75°P and pH 5.48. At the start of the boil, an 8 g aliquot of
Hallertau hops (2.7% α-acids) was added to 16 L of wort to help
reduce foaming without contributing significant bitterness. Wort
was aliquoted into 1 L flasks and dosed with appropriate amounts
of 30% iso-α-acid ethanol extract (Hopsteiner, Yakima,
Washington, USA) to obtain ~30, 40, 50 and 60 BU wort. For each
sample in triplicate, 300 mL of wort was added to a 500 mL Erlen-
meyer flask and stoppered with breathable silicone bungs. Yeast
from the pitching rate experiment featuring 1.0 × 106 and
3.0 × 106 cells/mL/°P was combined and pitched into the samples
at a rate of 1.8 × 106 cells/mL/°P. Flasks were placed in a dark area
at room temperature (~22 °C) and fermented for 12 days. Wort was
analysed by ASBC method Beer 23-A (21) using an Evolution 201
spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA)
to confirm BU. Gravity, pH and ABV were periodically measured
throughout the fermentation as described above. After 12 days
of fermentation cell viability was assessed as described above.
Oxygen requirements
Wort was produced on a 19 L pilot system from all Pilsner malt. An
infusion mash was rested for 1 h at 65 °C, followed by a 10 min
vorlauf. Wort was boiled for 1 h with hop additions at the start
and at flame-out. After a 10 min whirlpool stand, the wort was
chilled and was split into two 9 L batches and bubbled with either
pure air or pure oxygen until saturation. Original gravity was 11°P,
pH 5.24, and ~50 BU. For an 11°P wort at 20 °C, saturation with
oxygen would yield about 37 ppm and saturated air would yield
about 7.8 ppm (22,23). Gravity, pH and ethanol content were eval-
uated after 48 h and monitored periodically for 14 days.
Genus and species Source
Lachancea thermotolerans Wine or must
Lachancea thermotolerans Unknown, Madrid, Spain
Lachancea thermotolerans Wine (Italy)
Saccharomyces cerevisiae Anchor Ale
Saccharomyces cerevisiae California Ale Yeast
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Lachancea thermotolerans in beer fermentation
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Flocculation
Flocculation was evaluated using the Helm Assay (ASBC Method
Yeast-11.A) (24). A 106–107 dilution of L. thermotolerans was pre-
pared in a 0.9% sterile saline solution and plated onto each of
three hopped wort agar plates. Plates were incubated at ~22 °C
on the benchtop for 3 days. Yeast was transferred to 200 mL of
sterile wort in 1 L flasks and allowed to grow for 3 days at room
temperature (~22 °C). Yeast was suspended in triplicate and
agitated in the calcium sulphate solution per the protocol and
the suspension volume was measured.
Foam stability
Wort made from 100% two-row malt grist was produced as per the
oxygen requirements study above, with hop additions at the start
of the boil, after 50 min, and at flame-out. After whirlpooling and
chilling, wort was split into two carboys. Initial wort parameters
were 11°P, pH 5.16 and 56 BU. One carboy was pitched with
WLP001 California Ale Yeast (White Labs, San Diego, CA, USA),
while the other was pitched with L. thermotolerans, and both were
allowed to ferment at 18 °C. Beers were cold crashed, racked and
vacuum filtered using a 0.45 μm CA filter (Corning, Corning, NY,
USA). Filtered beer was analysed for foam stability using the Rudin
method (25). Each sample was analysed in triplicate.
as compared with the S. cerevisiae strain UCD VEN 915 Anchor Ale
were evaluated (Fig. 1). The three strains of L. thermotolerans and S.
cerevisiae showed a similar ability to ferment maltose, with 93–94%
of the maltose consumed within 21 days. Maltotriose was not
fermented by the three strains of L. thermotolerans whereas the
S. cerevisiae strain used 64% of this sugar. The three L.
thermotolerans strains produced only 6–12% less ethanol when
compared with the S. cerevisiae strain.
The three strains of L. thermotolerans produced higher levels of
lactic acid compared with the S. cerevisiae strain (Fig. 2), confirming
that, as in wine, this behaviour is expressed (8). This feature was
highly strain dependent, with strain 101 producing the highest
level of lactic acid (0.24 g/L) by the tenth day of fermentation, this
corresponding to the lowest final pH (Fig. 3). No significant further
increase in lactic acid was observed for any of the yeasts during the
rest of fermentation. In the wort fermented by L. thermotolerans
strain 101, the pH dropped to 3.77. On the contrary, the wort
fermented by the other two strains of L. thermotolerans showed
similar pH values (pH 4.11 and pH 4.28) to that (pH 4.24) observed
in the wort fermented by the S. cerevisiae strain UCD 915, used as
reference. The discrepancy between the final pH achieved by
strain 101 reported in re-pitching and hop tolerance experiments

Vicinal diketones
Beers from the foam stability study were analysed mid-
fermentation when the Saccharomyces beer was producing
perceivable amounts of diacetyl. All four beers from the oxygen re-
quirement and foam stability studies were tested at the end of fer-
mentation. Additionally, the foam stability test beers were used to
compare the vicinal diketone (VDK) levels produced by L.
thermotolerans as compared with a widely used commercial ale
strain (WLP001). Beers were heated for 90 min at 60 °C in a water
bath. Beers were analysed on a 6890 N gas chromatograph with
a 5973 mass spectrometer detector and a 122–7032 DB-WAX col-
umn (0.25 mm × 30 m × 0.25 μm; Agilent, Santa Clara, CA, USA)
using an MPS2 autosampler (Gerstal, Linthicum Heights, MD,
Figure 1. Sugar consumption and alcohol production after 21 days by the three
USA) with the selective ion method (MW = 57, 71, 86, 100, 114). strains of L. thermotolerans (Lt 101, UCD 602, UCD 1020) compared with the S.
For each beer in duplicate, 10 mL of beer was added to a 20 mL cerevisiae strain (UCD 915). (■) Maltotriose; (□) maltose; ( ) ethanol.
amber glass vial with 3 g of NaCl and spiked with 10 μL of
100 ppm 2,3-hexanedione as the internal standard. Samples were
agitated at 40 °C for 5 min. The solid phase micro-extraction was
performed with a Light Blue 85 μm carboxen/PDMS fibre (Supleco,
St Louis, MO, USA). The fibre was inserted and compounds
adsorbed at 40 °C for 30 min and subsequently desorbed in the
GC inlet for 10 min at 250 °C. The initial oven temperature was
35 °C for 5 min and the oven was ramped to 250 °C at a rate of
7 °C/min and finally held at 250 °C for 5 min (total exposure
40.71 min). The carrier gas was helium at a total flow of 0.9 mL/min
and a constant pressure of 6.02 psi. Post-run analysis of all samples
was performed using enhanced MSD ChemStation software
(Agilent, Santa Clara, CA, USA).

Results
Wort fermentation
Figure 2. Production of lactic acid (□) and glycerol (■) after 10 and 21 days by the
The maltose and maltotriose levels and the ethanol production three strains of L. thermotolerans (Lt 101, UCD 602, UCD 1020) compared with the S.
after 21 days of fermentation by the three L. thermotolerans strains
601

cerevisiae strain (UCD 915).

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Table 3. Although the fermentation was slightly more complete in

the first fermentation, subsequent fermentations finished at about
the same gravity with the viability remaining high, >89%, in each
successive pitch. Bitterness did not appear to have any effect on
the fermentation, even up to 59.5 BU (Table 4).
In a test of the effect of oxygen, both beers fermented at the
same rates and yielded similar final pH and ABV values, regardless
of initial dissolved oxygen content (Fig. 4). Likewise there was no
significant difference in the foam stability for the beer fermented
with S. cerevisiae strain UCD VEN 915 and that fermented with
L. thermotolerans strain 101(data not shown).
In a test of flocculation after 10 min post-re-agitation in calcium
sulphate solution the suspension volume of strain 101 was an
average of 10.07 mL with a sediment volume of <0.1 mL. Accord-
ing to the Helm Assay protocol, this strain would be classified as
Figure 3. The pH decrease during fermentation of wort by the three strains of L. non-flocculent (Type II) (24)
thermotolerans (Lt 101, UCD 602, UCD 1020) compared with the strain of S. cerevisiae
At mid-fermentation the level of diacetyl was higher in the
(UCD 915). ( ) Lt101; ( ) UCD602; ( ) UCD1020; ( )
UCD915. Saccharomyces fermentation but in the L. thermotolerans fermen-
tation there were higher levels of 2,3-pentanedione. In the
finished beers the trend was similar for diacetyl but the 2,3-
Table 2. Impact of pitching rate of L. thermotolerans 101 pentanedione levels for L. thermotolerans were lower than for S.
cerevisiae. In a study comparing L. thermotolerans grown in
Cells/mL/°P pH °Plato ABV (%) Viability fermentations of wort saturated with either air or oxygen, the
(%) samples with pure oxygen had the higher levels of both
compounds (Table 5). Overall, the total VDK levels observed in
2.5 × 105 4.28 ± 0.01 1.60 ± 0.03 5.65 ± 0.03 93.4 the various fermentations were relatively low.
5.0 × 105 4.23 ± 0.03 1.57 ± 0.00 5.66 ± 0.02 91.5
1.0 × 106 4.20 ± 0.00 1.52 ± 0.00 5.67 ± 0.01 92.0
2.0 × 106 4.21 ± 0.01 1.46 ± 0.01 5.72 ± 0.02 89.5 Discussion
3.0 × 106 4.19 ± 0.02 1.50 ± 0.01 5.69 ± 0.03 88.8 Upon initial survey, several L. thermotolerans strains showed
potential for beer fermentation. While others have shown that
(see later) and in Fig. 3 reflects the experimental conditions not all strains are capable of utilizing maltose, the three surveyed
employed as described in Materials and Methods. This would then here were well suited for growth on maltose, although not
allow us to leave the tables in the current sequence. The relevant maltotriose. These strains also produced higher levels of glycerol
tables are 3 and 4. and lactic acid than did S. cerevisiae strain UCD 915. Only strain
The three L. thermotolerans strains also showed an increased 101 showed a large pH drop when compared with S. cerevisiae,
production of glycerol as compared with the S. cerevisiae strain. although the pH was still much higher than that of most sour beers
The percent increase was between 36 and 54% after 10 days of (11,26). Preliminary studies of other strains within the UCD VEN
fermentation and between 65 and 75%, after 21 days of fermenta- collection suggest that some strains produce significantly more
tion. Because of the higher levels of lactic acid production, lactic acid, dropping the pH of beer to <3.6 (data not shown). It
subsequent analyses focused on strain 101. is therefore likely that some strains could contribute a sour
character to beer. Lactic acid production by bacteria can be
difficult to control and the use of L. thermotolerans to standardize
lactic acid production may be of interest to the beer industry.
Investigation of the impact of fermentation variables
Strain 101 was selected for further analysis based on its ability to
A pitching rate of 2.0 × 106 cells/mL/°P for strain 101 gave the low- produce higher levels of lactic acid and for its positive contribution
est final sugar level and highest percentage of alcohol (Table 2). to beer aroma as assessed by sensory evaluation (data not shown).
The pH was not significantly different at any of the pitching rates This strain was capable of fermenting wort at various pitching rates
except for the lowest rate, where it was slightly higher. with optimal performance at a rate similar to S. cerevisiae (27).
A comparison of the re-pitching capacity of the L. Viability and vitality remained high after re-pitching for at least five
thermotolerans strain 101 over successive generations is shown in generations, making it well-suited for use in commercial

Table 3. Impact of successive re-pitching with L. thermotolerans 101

Generation pH °Plato ABV (%) Viability (%)

1 4.21 ± 0.01 1.46 ± 0.01 5.72 ± 0.02 89.5 ± 0.0
2 4.17 ± 0.10 1.70 ± 0.09 5.82 ± 0.08 93.0 ± 1.4
3 4.24 ± 0.02 1.78 ± 0.00 5.99 ± 0.04 94.7 ± 0.5
4 4.30 ± 0.02 1.66 ± 0.01 5.90 ± 0.13 90.7 ± 0.3
5 4.23 ± 0.00 1.66 ± 0.02 6.08 ± 0.02 89.1 ± 0.6
602

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Lachancea thermotolerans in beer fermentation
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Table 4. Hop tolerance of L. thermotolerans 101

BU Target BU Actual pH °Plato ABV (%) Viability (%)

30 33.4 4.18 ± 0.03 1.72 ± 0.01 5.88 ± 0.10 94.9 ± 0.8
40 44.95 4.22 ± 0.03 1.78 ± 0.02 5.92 ± 0.03 96.2 ± 0.2
50 52.7 4.22 ± 0.01 1.73 ± 0.01 5.93 ± 0.00 95.4 ± 0.5
60 59.5 4.22 ± 0.02 1.78 ± 0.01 5.97 ± 0.02 94.5 ± 0.3

Further research is necessary in order to determine the influence

of this non-Saccharomyces yeast on the overall aroma profile of
beer produced with it. At the lower temperature of fermentation
(14 °C), tasters described the beer made from strain 101 as fruity,
floral, sour, clove, melon and strawberry. It should be noted that
a commercial strain, Concerto™ (Chr. Hansen), was trialled in initial
studies, but performed poorly in fermentations and yielded strong,
unpleasant phenolic aromas, notably 4-ethylphenol. Preliminary
studies with other UC Davis strains yielded a variety of aromas
including floral and fruity, rotten fruit, minty, woody and cider-like.
Strain 101 has been shown to possess higher β-glucosidase activity
than Saccharomyces (18), indicating that it could be involved in the
release of glycosidically bound terpenoids extracted from hops
during the brewing process.

Conclusions
Figure 4. The impact of air (□) and oxygen (♦) saturation on wort fermentation by L. Pure culture beer fermentation by L. thermotolerans is achievable.
thermotolerans 101. Numerous strains of L. thermotolerans are capable of fermenting
wort to dryness (~2.5°P or less) within about 2 weeks, and they
lowered the pH more than what occurred in beers fermented
Table 5. Vicinal diketone levels in fermentations with S. with Saccharomyces. Basic quality analyses of beers made from
cerevisiae WLP001 and L. thermotolerans 101 one strain showed that it did not cause any detriment to beer
quality when compared with commercial ale strains. Strain 101
Diacetyl 2,3-Pentanedione successfully finished fermentations regardless of pitching rate,
(ppb) (ppb) bitterness, dissolved oxygen or generation. It proved to be a
robust strain for beer production, despite needing more time
Mid-fermentation 46.6 13.2
than a traditional ale yeast to finish fermentation. Additionally,
S. cerevisiae
physical properties of beer, including foam and clarity, were not
Mid-fermentation 30.8 33.8
negatively affected.
L. thermotolerans
Final S. cerevisiae 47.3 15.7
Acknowledgments
Final L. thermotolerans 29.3 4.5
O2 L. thermotolerans 31.3 7.5 Carolyn Doyle is thanked for her technical assistance.
Air L. thermotolerans 22.8 6.1
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