Research article

Received: 10 January 2020 Revised: 15 April 2020 Accepted: 8 July 2020 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jib.621

Effect of the respiro-fermentative balance

during yeast propagation on fermentation and
wort attenuation
Maria E. Moutsoglou* and Ashley C. Dearden
Breweries use different yeast strains to create beers with different flavours and aromas. Yeast propagation must produce yeast that
performs consistently from the first fermentation to harvesting and re-pitching in subsequent fermentations. Breweries propagate
yeast in wort leading to low efficiency fermentative growth in Crabtree-positive yeast. There is limited knowledge on the impact on
beer production when fermenting with yeast propagated in sugar limited and nutrient supplemented wort. It was hypothesised
that propagating yeast in this way would have a positive impact on subsequent fermentation performance. Saccharomyces
cerevisiae was propagated at the laboratory scale in standard wort with a high carbon to nitrogen (C:N) ratio (850) or in modified
wort supplemented with yeast extract to achieve a low C:N ratio (100) and at varying sugar concentrations. Propagation in low
C:N wort with 2°P sugar yielded a 27% decrease in fermentation efficiency and a 46% increase in cell production compared to
2°P high C:N wort. This suggests nitrogen is critical to the respiro-fermentative balance during growth. Yeast propagated in
standard wort resulted in slower fermentations and significant under-attenuation compared to yeast grown in the modified wort
with low sugar and high nitrogen. The results of this study suggest the nitrogen and sugar content drive the respiro-fermentative
balance during yeast propagation. The metabolism of yeast during propagation induces significant downstream impacts on the
subsequent fermentation performance and wort attenuation. © 2020 The Institute of Brewing & Distilling

Additional supporting information may be found online in the Supporting Information section at the end of the article.

Keywords: yeast; propagation; fermentation; metabolism; quality

Introduction under fermentative conditions allow yeast to out-compete other

Shifting market demographics and the diversity of new products
require the brewing industry to innovate to meet changing
consumer demands. Using yeast for improved process efficiency
and bioflavouring can lead to new innovations in brewing (1–4).
However, breweries cannot leverage yeast for innovation if not
propagated appropriately to achieve desirable results and
facilitate new product development. As the link between yeast
quality and fermentation performance has been established (5, 6),
propagation must yield viable, physiologically ‘tuned’ yeast for serial
repitching or for single use fermentation. Additionally, yeast must
produce a consistent ethanol content and organoleptic profile
between propagations, especially if blending is not available for
quality control. Propagations that fail to generate adequate yeast
or produce yeast with poor fermentation performance can result
in costly waste of raw materials, labour, and product.
Saccharomyces yeasts used in brewing are Crabtree-positive (7),
simultaneously generating adenosine triphosphate (ATP) from
respiration and fermentation pathways depending on the
environmental conditions (8). Regulation of this respiro--
fermentative balance is controlled through the composition of
the medium used for propagation. High sugar concentrations shift
the metabolic flux towards fermentation, even in an aerobic
environment where oxygen requirements are met (9). Respiration
is a high energy yielding pathway that generates up to 38 ATP per
mole of glucose. Fermentation has a lower energy yield (2 ATP per
mole of glucose), does not require oxygen, and produces CO2
and ethanol as by-products. Despite low ATP production, it has
microorganisms (7, 10, 11). The purpose of yeast propagation is cell
production, not ethanol formation. Accordingly, using a medium that
promotes respiration would be a more efficient yeast production
strategy. Indeed, industrial producers of yeast (baking, distilling,
etc.) employ aerobic fed-batch propagation to maximise respiratory
yeast growth without fermentation. However, in breweries,
propagation remains an aerobic (to varying degrees) batch process
with inefficient yeast growth through solely fermentation.
In the brewing industry, batch yeast propagation is a volumetric
scale-up process that begins in the laboratory with agar slants (6).
The medium used for propagation directly impacts the yeast
phenotype, ultimately impacting fermentation performance (12).
Laboratory scale cultivation of yeast is often performed in a
glucose-limited, nutrient-rich medium for yeast to grow under
conditions that promote more aerobic growth. Such ingredients
are cost prohibitive at large scale and are inconvenient compared
to using wort to propagate yeast. (13, 14) Wort provides the
necessary carbon and nutrient content for yeast to ferment sugars,
with the occasional exception of zinc and lipids (15). The sugar
concentration in wort is high and drives the metabolic flux to
fermentation with any oxygen supplied used by yeast for lipid
synthesis (16).
* Correspondence to: Maria E. Moutsoglou, Molson Coors Beverage Company,
3939 W. Highland Blvd. Milwaukee, WI 53208 USA. Email: maria.
moutsoglou@molsoncoors.com
Sierra Nevada Brewing Company, Research and Development, 1075 East 20
th

been proposed that the faster growth rate and ethanol production St. Chico, CA 95926 USA
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 M. E. Moutsoglou and A. C. Dearden

The nitrogen content in wort can impact biomass production Composition of wort and synthetic media
and sugar attenuation (17, 18). Assimilable nitrogen in wort is
Brewery wort (100% malted barley) with an original gravity of
referred to as free amino nitrogen (FAN). Although the literature
13.17 ± 0.10°P with 8.79 ± 0.87°P (n = 264 worts) fermentable
cites the minimum FAN concentration for healthy fermentation
sugars was used for yeast propagation and for subsequent
(19), there is little consideration of how the FAN concentration
fermentation. Wort was obtained from Sierra Nevada Brewery in
may need to change depending on the fermentable sugar
Chico, CA, USA. Wort was collected midway during transfer from
content and the ratio of carbon to nitrogen (C:N). The nitrogen
whirlpool to fermenter. Synthetic yeast complex media (YCM)
requirements for fermentation have been well studied (20), but
was prepared with 1% (w/v) yeast extract (yeast extract 19512,
nitrogen requirements for brewer’s yeast propagation have not
OrganoTechnie S.A.S., France), 1% (w/v) malt extract (CBW®
been reported. Standard wort may be deficient in available
Sparkling Amber Dry, Briess Industries, USA), and 2°P D-(+)-maltose
nitrogen content and the sugar concentration may exceed that
monohydrate (Penta International Corporation, USA). The yeast
needed for efficient cell growth.
extract used in this study supplied 2% (w/w) free amino nitrogen.
This study builds on research showing the positive impact on
Wort was diluted with deionised, distilled water and
fermentation quality of using yeast propagated under oxidative
supplemented as reported in Table 1. Dilution and nutrient
conditions with mannitol as the carbon source (21, 22). However,
supplementation were performed prior to autoclaving. Zinc was
instead of mannitol, standard wort and yeast extract were used
supplemented to 36.1 μM (2.4 mg/L) using a 0.1 M solution of
to develop a propagation scheme that could be translated to a
Zn2+ from zinc sulphate heptahydrate (Millipore Sigma, USA).
brewing setting. Accordingly, the effect of nitrogen and sugar in
Yeast extract (YE) was added to achieve the desired FAN
wort based media for the propagation of a brewing yeast was
concentration. Media was sterilised by autoclaving at 121°C at 15
evaluated together with the effect on subsequent laboratory scale
psi for 45 minutes.
fermentations. Wort was diluted to various sugar concentrations to
The fermentability of wort was determined by accelerated
modulate the respiro-fermentative balance of carbohydrate
(forced) fermentation, with S. cerevisiae added at a ratio of 1 g yeast
metabolism during propagation. To understand how the carbon
to 4 g of wort and incubated with shaking at 185 rpm at room
to nitrogen ratio impacted on performance, standard wort (850
temperature for 24 hours. Yeast was removed by centrifugation
C:N ratio) was supplemented with yeast extract to a ratio of 100
at 3500 x g for 10 minutes at 4°C. The residual density (real extract,
C:N to mimic a synthetic medium. Yeast were propagated in each
RE) was determined using an Alcolyzer Beer Analyzing System
medium and monitored for growth kinetics, growth efficiency,
(Anton-Paar GmbH, Germany).
and fermentation efficiency. Propagated yeast was pitched into
wort and fermentation kinetics assessed at the bench scale. Wort
attenuation and fermentation efficiency were compared to Elemental analysis
industrial scale fermentations using harvested (non-propagated)
yeast in the same wort. This study aimed to determine if yeast The elemental profiles of YCM and wort without YE were
propagated in nutrient supplemented, low sugar wort would determined using inductively coupled plasma optical emission
improve the subsequent fermentation performance. The findings spectrometry (5100 ICP-OES, Agilent Technologies, USA) coupled
indicate both wort sugar and nitrogen content in the propagation with an SPS 4 Autosampler (Agilent Technologies). Prior to
medium have significant impact on the fermentation quality and elemental analysis, wort was supplemented with zinc to 36.1 μM
wort attenuation. and autoclaved. Emissions for calcium (422.7 nm), chromium
(267.7 nm), copper (327.4 nm), iron (238.2 nm), potassium (769.9
nm), magnesium (285.2 nm), manganese (257.6 nm), sodium
Materials and methods (589.6 nm), phosphorous (213.6 nm), sulphur (182.0 nm), silicon
(251.6 nm), and zinc (213.9 nm) were monitored at their respective
Yeast strain
emission wavelengths, and element concentration was determined
The yeast used for all experiments was an industrial strain of using a standard curve fit using linear regression (R2 ≥ 0.999) (ICP
Saccharomyces cerevisiae provided by Sierra Nevada Brewing Expert Software, Agilent Technologies). The concentrations of
Company. elements for diluted and supplemented wort were calculated

Table 1. Media properties for yeast complex media and wort based media at high (H) and low (L) C:N ratios. H5 is standard wort.

Media Type Media Fermentable Sugars Original Gravity Media FAN C:N Yeast Extract Added
Identifier (°P) (°P) pH ( g/L) ratio ( g/L)
synthetic base YCM 2.0 3.2 6.13 0.200 100 10
wort base, high C:N H1 2.0 3.1 5.18 0.024 850 0
H2 3.5 5.5 5.15 0.041 850 0
H3 5.0 7.8 5.09 0.059 850 0
H4 6.5 10.2 5.05 0.076 850 0
H5 8.5 13.3 5.00 0.100 850 0
wort base, low C:N L1 2.0 3.3 5.77 0.200 100 9
L2 3.5 6.8 5.61 0.350 100 16
L3 5.0 9.8 5.51 0.500 100 22
L4 6.5 12.7 5.35 0.650 100 29
L5 8.5 16.1 5.50 0.850 100 38
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Yeast propagation metabolism impacts fermentation quality
based on dilution factor, and/or the amount of supplemented
yeast extract or zinc.
Free amino nitrogen
FAN was determined using the ninhydrin method (23). A
glycine standard curve fit using linear regression (R2 ≥ 0.99)
was used to determine FAN concentration, where the absorbance
of amine-bound ninhydrin at 570 nm is proportional to the
concentration of FAN.
Yeast growth
Yeast was stored in 25% (v/v) glycerol at -78°C. Yeast was recovered
from glycerol stocks and streaked on YEG plates containing 1%
(w/v) yeast extract, 2% (w/v) D-glucose anhydrous (Penta), and
1.8% (w/v) agar (Fisher Scientific, USA). Plates were incubated for
48 hours at 30°C and stored for no more than three weeks at 4°C.
Five to ten colonies from a YEG plate were inoculated into 1.5 mL
YCM cultures and incubated at 20 ± 2°C for 24 hours with shaking
at 250 rpm. Precultures were inoculated into 25 mL of media in a
250 mL baffled Erlenmeyer flask (10% v/v fill). Initial cell analysis
samples (~50 μL) were collected after at least 30 seconds shaking
to ensure homogeneity. Subsequent samples were collected
until stationary phase was established. Samples were stored at 4°C
until analysis.
Yeast cell concentration, viability, and average diameter
Cell concentration and the percentage of viable cells were
analysed using brightfield and fluorescence imaging (emission
525 nm/excitation 595 nm), respectively, using a Cellometer X2
(Nexcelom Bioscience LLC, USA). Samples were diluted in double
distilled water to a cell concentration below 1x106 cells/mL to
ensure accurate counting. Samples were then diluted 1:1 with
propidium iodide (Nexcelom), a nuclear stain impermeable to live
cell membranes, and applied to CHT4-xD100 slides (Nexcelom). A
minimum of 500 cells per sample were analysed to determine
average diameter. The minimum and maximum average cell
diameter thresholds were 4 and 10 μm, respectively. Fluorescence
exposure time was set to 5,000 ms, and fluorescence imaging
conducted using optics module XB-595-501. A decluster edge factor
and decluster threshold factor of 0.6 and 1.0 were used, respectively.
Cell concentration, viability, and average diameter were quantified
using Cellometer AutoX4hm software (Nexcelom).
Cell production and fermentation efficiency post-cultivation
For propagation trials, sugar utilisation and ethanol production
were quantified in stationary phase after growth ceased. Yeast
were cultured in duplicate as described above, inoculated into 25
mL of media, and incubated until late log phase was reached
based on growth curves (Table S1). Cultures were then inoculated
into 125 mL of media in a 1 L baffled Erlenmeyer flask and
incubated at room temperature at 250 rpm. The incubation time
was determined by multiplying the time to reach late log phase
by 1.25 (Table S1) to ensure complete sugar utilisation. Cultures
were centrifuged at 3,500 x g for 10 minutes at 4°C, then analysed
for real extract and ethanol using an Alcolyzer Beer Analyzing
System (Anton-Paar). The amount of sugar utilised was calculated
from the difference between original and final densities.
J. Inst. Brew. 2020; 126: 289–297 © 2020 The Institute of Brewing & Distilling
Fermentation efficiency was calculated using the ratio of
ethanol produced to sugar utilised. Percent alcohol by volume
was converted to g/L where the density of ethanol = 0.7893
g/mL. Cell production was determined using the difference
between the average cell concentration (n = 4) in stationary phase
and the inoculum at time zero from at least two independent
growth profiles. Cell production efficiency was calculated from
the ratio of cell production (cells/mL) to sugar utilised ( g/L).
Anaerobic fermentation
Yeast propagated in yeast complex media or wort based media
were pitched into brewery wort to determine sugar utilisation
and ethanol production. Brewery wort (370 mL) was added to
sterilised 450 mL jars. Dissolved oxygen in the wort was 6 mg/L,
determined using a HQ30D portable dissolved oxygen meter with
a field luminescent DO sensor (Hach, Loveland, CO, USA). The
pitching rate was 0.5x106 cells/mL/OG°P. Yeast cultured in YCM
were included as a control in separate fermentations with yeast
cultivated in high or low C:N media. Yeast were cultured in
duplicate as described above into 25 mL of media until late log
phase, and the viable cell concentration for each culture was
determined. The volume of cells needed to meet the target pitching
rate were centrifuged at 3,000 x g for 5 minutes at 22°C. The yeast
pellet was reconstituted in 30 mL of wort and pitched into
fermenters for a total wort volume of 400 mL. Inoculated jars were
capped with a lid containing a ~1 mm diameter opening for gas
exchange and statically incubated at 20.5 ± 2.0°C. Fermentation
progress was monitored by measuring weight loss over time,
which reflects the production of CO2. The fermenters were chilled
to 4°C for 24 hours once the weight loss decreased by less than
0.1 g over 24 hours, then processed for sugar utilisation and ethanol
concentration as described above.
Regression and statistical analyses
GraphPad Prism 8.1.1 (224) for macOS (GraphPad Software, Inc.,
La Jolla, CA) was used to perform regression analysis and
obtain growth and fermentation parameters from the fit. Cell
concentration from growth profiles and CO2 production from
fermentation profiles (y data) with time (t, x data) were fit to a four
parameter logistic function
ðy max  y min Þ
y ðtÞ¼y min þ
n ; (1)
1 þ κt
where ymin and ymax are the minimum and maximum plateau
values for y, respectively; κ is the midpoint in units of time; and n
is the slope at κ.
Standard deviation in growth profile parameters from fits were
determined from the 95% confidence intervals using
C:I:upper  C:I:lower pffiffiffiffi
S:D:¼ N; (2)
3:92
where C.I.upper and C.I.lower are the upper and lower values for each
residual and N is the number of independent data sets analysed
to generate the curve. Cell concentration and CO2 production
data were interpolated for a standard set of time points using
parameters from the fit (Tables S2 - S4). The derivative of this data
set was used to determine the rate of change in cell concentration
and CO2 production with time. The maximum of the derived cell
concentration data and the absolute value of the maximum of
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the derived fermenter mass data yielded the maximum absolute
growth rate (AGRmax) in cell concentration and maximum rate of
change in CO2 production (rCO2max), respectively.
Lag time λ for cell growth profiles during cultivation were
determined using parameters from fit (Eq. 1) using
y ðt Þ y ðt Þ
λ¼t  ln
y 0 ðt  Þ y ðt¼0Þ
where t* is the time at maximum absolute growth rate, and y’(t) is
the derivative of the fit to the growth curve (Eq. 1) (24).
The efficiency of cell production with respect to sugar
concentration for wort based media at a low C:N ratio was fitted
to a one phase decay using y = (y0  yplateau) * exp(K * x)+yplateau,
where y is cell production efficiency, y0 is the initial cell production
efficiency, yplateau is the low plateau cell production efficiency
value, x is sugar concentration, and K is the rate constant.
Fermentation efficiency with respect to sugar concentration for
wort based media at a low C:N ratio was fitted to a one-phase
association using y = y0+(yplateau  y0) * (1  exp(K * x)), where
y is fermentation efficiency, y0 is the initial fermentation efficiency,
yplateau is the low plateau fermentation efficiency, x is sugar
concentration, and K is the rate constant.
GraphPad Prism was also used for linear regression and
correlation analysis. Where appropriate for wort based media,
linear correlations and their significance between kinetic and
metabolic parameters and media sugar concentration were
determined using the Pearson correlation, where Pearson’s
correlation coefficient was designated as r. The unpaired t-test was
used to determine significance between parameters from wort
based media against YCM. p ≤ 0.05 was considered significant.
Results and discussion
Wort supplemented with yeast extract is a nutrient rich
medium for yeast propagation
Synthetic media like yeast complex media supports the
biosynthetic needs for yeast propagation by providing
carbohydrates, nitrogen, and other macro and microelements
necessary for growth (25). Yeast extract (YE) is the principal trace
nutrient and nitrogen source in yeast complex media (YCM), and
contains FAN, peptides, B vitamins, purine and pyrimidine bases
(26). YE is a cost effective source of organic nitrogen and provides
critical amino acids that produce higher biomass yields than
inorganic nitrogen (27, 28). YCM is not suitable for large scale
yeast propagation due to high substrate cost (i.e. maltose or
maltotriose). Brewery wort is readily available to use for industrial
scale propagation but has some differences in nutrient availability
compared to YCM. The primary difference between wort and YCM
is the availability of nitrogen. Yeast complex media had a C:N ratio
of 100 compared to 850 in standard wort (Table 1). Additionally,
there were 4.25x more available sugars in wort than YCM. Elemental
analysis showed YCM was around 10x higher in chromium and 7x
higher in manganese, while wort was around 10x higher in calcium
(Table S8). Few studies focus on the concentration and elemental
profile needed for the propagation of brewing yeast. The results
presented here may provide some guidance in future studies
to identify specific concentrations of elements critical to both
propagation and subsequent fermentation performance.
Three media types were compared in laboratory scale
propagations: yeast complex media, standard brewery wort, and
standard wort supplemented with YE to decrease the carbon
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M. E. Moutsoglou and A. C. Dearden
to nitrogen ratio. To determine the impact on propagation of
wort sugar content, the two wort based media were diluted to
varying sugar concentrations as shown in Table 1. Maltose was
selected as the fermentable sugar in YCM as it is the primary
fermentable sugar in wort (29). Conversely, cells cultivated with
glucose can have reduced maltose and maltotriose utilisation in
(3) subsequent fermentations due to the repression of maltose
transport genes (30, 31).
To determine the impact of nitrogen limitation, wort based
media with a high C:N ratio was compared to wort based media
supplemented with YE to reduce the C:N ratio to 100. Differences
in trace elements increased as standard wort (Table 1, H5 media)
was diluted to reduce fermentable sugars. As yeast extract was
added to wort to decrease the C:N ratio, trace elements increased
accordingly. Low C:N wort diluted to 2°P of fermentable sugars (L1
media) most closely resembled YCM in nitrogen, trace elements,
and sugar content. While the added nutrients and metal ions from
yeast extract could have some impact on performance compared
to standard wort, the interpretation of results in this study is
focused on the differences in available sugar and nitrogen.
Sugar and C:N ratio drive growth kinetics
The growth kinetics of S. cerevisiae were determined during
propagation in YCM and wort media. Yeast viability remained
above 95% during propagation (Fig. S1) and cell size profiles are
shown Fig. S2. Growth was monitored during two independent
propagations, and the pooled data was fitted to a four-parameter
logistic model using non-linear regression (Fig. S3 – S4). Growth
curve fits provided the fermentation midpoint κ and maximum
absolute growth rate (AGRmax). Parameters from fits were used to
calculate the lag time λ. To determine the relationship between
sugar concentration, C:N ratio, and growth kinetics, the kinetic
metrics were plotted against the fermentable sugar concentration
of the media (Fig. 1).
Yeast can slow or cease growth when adapting to environmental
changes (temperature, pH, ethanol) or nutrient changes (nitrogen,
sugar). At the start of cell growth this is termed the lag phase
(32–35). The lag phase is dependent on the physiological impact
on yeast from environmental changes (33). Yeast grown in wort
with low C:N had similar lag times of around three hours with all
sugar concentrations (Fig. 1A). Under nitrogen limitation, there
was a significant positive correlation between lag time and sugar
content, which was not observed at low C:N. The osmolarity stress
at higher sugar concentrations may have increased lag times for
yeast under nitrogen limitation. YE supplementation could improve
the environmental stress response from inoculation into new
media regardless of sugar concentration, explaining the shorter
lag times in low C:N wort.
The midpoint correlates to total growth time (Fig. 1B). As sugar
concentration increased beyond 2°P, a significant positive linear
correlation between midpoint and sugar concentration was found
in high C:N wort (Fig. 1B). These results suggest nitrogen limitation
caused slower cell growth as the respiro-fermentative balance
shifted to fermentation at higher sugar concentrations.
The Crabtree effect has been shown to produce higher growth
rates for S. cerevisiae at higher sugar concentrations, in addition
to influencing the selection of the fermentative pathway for
ATP generation and ethanol production (8, 36). In this study, a
significant positive correlation between sugar concentration and
growth rate was observed only in media with a high C:N (Fig. 1C).
J. Inst. Brew. 2020; 126: 289–297
Yeast propagation metabolism impacts fermentation quality
Figure 1. Effect of media sugar and C:N ratio on S. cerevisiae propagation growth
kinetics. (A) Lag time plotted against the propagation media sugar content for YCM
(black) or wort with a low (red) or high (blue) C:N ratio. Linear regression (solid line)
is shown with shading representing 95% confidence interval of fit. The Pearson
correlation coefficient r and p-value are shown. A consistent colouring scheme is used
throughout. (B) The average midpoint of cell growth for conditions in (A). Error bars
represent S.D. from fit using non-linear regression to at least n = 2 pooled growth
profiles. (C) The maximum absolute growth rate for conditions in (A). [Colour figure
can be viewed at wileyonlinelibrary.com]
Modulating the respiro-fermentation balance with nitrogen
and sugar
Fermentation and cell production were determined for yeast
propagated in each medium. The fermentation or cell production
efficiency is defined as the total cell number or quantity of
ethanol produced per total sugar consumed at the completion of
growth. Increased fermentation and lower cell production indicate
higher utilisation of the fermentation pathway. Significant linear
correlations were observed between sugar and fermentation/cell
production for yeast cultivated in high C:N wort (Fig. 2). However,
these linear relationships were not strictly observed for yeast
propagated in low C:N wort. At 2°P sugar, fermentation efficiency
decreased by 23% and cell production efficiency increased by
J. Inst. Brew. 2020; 126: 289–297 © 2020 The Institute of Brewing & Distilling
46% compared to 2°P high C:N wort (Fig. 2). The total cell
count in low C:N wort with 2°P sugar was 301 ± 53 x 106 cells/mL
(Table S7). For a standard wort (13°P) and a pitching rate of 6.5
x106 cells/mL, the target inoculum could be met with a 2% (v/v)
transfer. This is markedly more efficient than the common brewery
scale-up strategy that uses a 10% or more (v/v) transfer (14).
The balance between respiration and fermentation may be
modulated by the enzyme costs associated with each pathway.
Cells undergo a cost-benefit analysis that balances ATP and
protein production to promote maximum fitness, as has been
shown for Escherichia coli (37, 38). Similarly, for S. cerevisiae, flux
balance analysis has shown that fermentation is more catalytically
efficient than respiration, producing more ATP with fewer enzymes
and reducing the overall protein cost (39). The results presented
here suggest that even at lower sugar concentrations,
Crabtree-positive yeast shift towards a more cost effective, lower
yield pathway (fermentation) if the nitrogen costs for the protein
expense are not met. Here, the respiro-fermentative balance
shifted to the protein inexpensive, fermentation pathway as sugar
concentration increased under nitrogen limitation.
The fermentation efficiency for industrial scale fermentations of
the same wort using the same (serially repitched) yeast in
this study was 0.51 ± 0.01 (n = 400 independent fermentations).
Therefore, growing yeast in media with > 5°P sugar promoted
almost complete utilisation of the fermentation pathway (Fig. 2A),
regardless of nitrogen. Breweries typically aerate yeast
propagation tanks (13, 14, 40, 41), but the oxygen only supports
lipid synthesis and not aerobic respiration due to the ‘repressing’
concentration of fermentable sugar.
Fermentation quality is dependent on the yeast propagation
medium
The principle goal of optimising propagation media is to generate
high quality yeast for consistent fermentation and the production
of high quality beer. The control of fermentation kinetics is critical
in industrial beer production, as the fermentation cycle time
impacts on both beer quality and brewery plant efficiency. Yeast
metabolism during fermentation has a direct impact on the
production of flavour active metabolites important to beer quality,
including higher alcohols and esters (42) and diacetyl (19). Using
yeast of good physiological quality improves yeast dependent
beer quality (43). Slow, sluggish fermentations impact on yeast
recovery, beer quality and packaging schedules. Further, stuck
fermentations can present issues by not meeting ethanol
specification, which can be exacerbated if blending is not an
option. Accordingly, beer quality and brewery efficiency rely on
high quality yeast and consistent fermentation cycle times.
Here, the fermentation performance of yeast propagated in
yeast complex media or wort with high or low C:N ratios was
evaluated based on CO2 production, wort attenuation, and the
efficiency of ethanol production at the bench scale. CO2 production
profiles are shown in Fig. S5 – S6. To determine the relationship
between sugar concentration, C:N ratio, and fermentation kinetics,
the fermentation midpoint (κ) and the maximum rate of CO2
production (rCO2max) were plotted against the sugar concentration
of the propagation media (Fig. 3). Two rounds of fermentation using
yeast propagated in high or low C:N wort were performed in wort
collected on separate dates and included YCM as a control in each
round. In the following discussion, overall kinetic performance of
yeast cultivated in wort at high or low C:N is considered based on
a comparison of the YCM control included in each group.
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 M. E. Moutsoglou and A. C. Dearden

Figure 2. Effect of media sugar and C:N ratio on S. cerevisiae sugar metabolism during propagation. (A) Average fermentation efficiency plotted against the propagation media
sugar content for YCM (black) or wort with a low (red) or high (blue) C:N ratio. Error bars represent S.D. from n = 2 independent propagations. Linear regression (high C:N) or fit to
one-phase association (low C:N) is displayed with shading representing 95% confidence interval. The Pearson correlation coefficient r and p-value are provided for the linear fit.
(B) The average cell production efficiency for conditions in (A). Linear regression (high C:N) or fit to one-phase decay (low C:N) is shown with shading representing 95% C.I.
The Pearson correlation coefficient r and p-value are shown for linear fit. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3. Effect of the propagation media sugar and C:N ratio on subsequent S. cerevisiae fermentation kinetics. (A) The average fermentation midpoint from n = 2 independent
fermentations plotted against the propagation media sugar content for YCM (black) or wort with a low (red) C:N ratio. Error bars represent S.D. from fit. Linear regression
(solid line) is displayed with shading representing 95% confidence interval of fit. The Pearson correlation coefficient r and p-value are shown. (B) The average fermentation
midpoint from n = 2 independent fermentations plotted against the propagation media sugar content for YCM (black) or wort with a high (blue) C:N ratio. (C) Average maximum
rate of CO2 production from conditions in (A). (D) Average maximum rate of CO2 production from conditions in (B). A consistent colouring scheme is used throughout. [Colour
figure can be viewed at wileyonlinelibrary.com]

At 2°P sugar, there was no significant difference in the
fermentation midpoint between low C:N (p = 0.1144) or high C:N
(p = 0.6107) wort (Fig. 3A-3B). However, as the sugar increased,
the midpoints linearly increased in all conditions, suggesting
overall fermentation time is dependent on the concentration of
sugars used in the propagation media. The rate of CO2 production
(fermentation rate) was impacted by both the sugar and nitrogen
content of the propagation medium (Fig. 3C – 3D). There was no
significant difference in rCO2max between yeast propagated in YCM
(p = 0.2558) and 2°P low C:N wort, whereas there was a significant
294
rate decrease for yeast propagated in high C:N wort (p = 0.0021).
Nitrogen limitation during propagation universally reduced
the fermentation rate independently of sugar concentration.
Conversely, the sugar concentration in nitrogen sufficient media
was significantly correlated to fermentation rate.
The results indicate fermentation kinetics depend strongly on
the yeast propagation environment. Yeast grown under nitrogen
limitation had significantly reduced fermentation performances
independent of sugar concentration. Yeast propagated with higher
nitrogen exhibited overall improved fermentation performance

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Yeast propagation metabolism impacts fermentation quality

that was subject to regulation by sugar concentration. Based on
these results, the conventional ideology correlating propagation
growth rate to fermentation performance (36, 44) may only be
appropriate under certain environmental conditions.
The more efficient fermentation performance for yeast
propagated in low C:N media may be due to the enhanced
capability of yeast to produce and store critical nutrients,
including trace elements and proteins. Research has shown
reserves assist in yeast adaptation to new environments (45),
including glycogen and trehalose, which are important in cellular
responses to environmental stress (46–48). Future studies into
stress response and reserve nutrient accumulation of yeast
propagated in high or low C:N wort at varying respiro-fermentative
balances would yield insight into the observed trends in
fermentation performance.
Propagating yeast under nitrogen deficiency leads to wort
under attenuation
Achieving the ethanol specification in finished beer depends
on the conversion of sugar to ethanol (fermentation efficiency)
and the utilisation of available fermentable sugars in wort
(attenuation). Both fermentation efficiency and wort attenuation
have direct impact on product quality. Although yeast cultivated
with low sugar content had lower fermentation efficiency
than with high sugar (Fig. 2A), there was no induced loss of
fermentation efficiency in the subsequent fermentation (Tables S5
and S6).
Wort attenuation was significantly impacted by yeast
propagation conditions. Fig. 4 shows the effect of the propagation
media on the total sugar remaining in beer post fermentation.
Residual sugar in beer using propagated yeast was compared
to industrial fermentations of the same wort by the same
serially repitched yeast. For both laboratory scale fermentation
groups, yeast propagated in yeast complex media were able
to attenuate wort consistently with industrial fermentations
using harvested yeast (Fig. 4A – 4B). A significant increase in
residual sugars in beer was observed using yeast propagated in
high C:N wort which linearly correlated to the concentration
of media sugar (r = 0.9826, p = 0.0028) (Fig. 4A). Residual
sugars using yeast propagated in low C:N wort was not
significantly different to using harvested yeast (Fig. 4B). Yeast
propagated in standard wort with 8.5°P fermentable sugars
(high C:N) left three times the amount of sugar in beer than in
industrial scale fermentations using harvested yeast. While
performance in fermentation using first generation yeast is shown,
the generational stability of the yeast in subsequent fermentations
remains unknown.
These results suggest nitrogen limitation during yeast propagation
induced significant defects in subsequent wort attenuation.
Although the concentration of nitrogen present in wort may be
satisfactory for serially repitched yeast to completely utilise sugars
during fermentation, the nutritional requirements of propagated
yeast may be different. Nitrogen limitation has been shown to have
significant impact on fermentation rate and attenuation by
(S. cerevisiae) wine yeast (49, 50) and brewing yeast (51). The ability
of yeast to respond to nitrogen limitation during fermentation
may be impacted by the propagation environment through
induced changes in the TOR (Target Of Rapamycin) signal
transduction cascade, implicated in regulating cellular response to
nutrients (52, 53). Nitrogen starvation occurs more rapidly in a
nitrogen limiting environment and inactivates the sugar transport
catabolite system in S. cerevisiae (54), leading to incomplete and
slow fermentations.
Here, inactivation of sugar transport systems during yeast
propagation carried through to subsequent fermentations.
Future research can build on these results by investigating why
yeast cultivated in standard wort with a high C:N ratio leads to
sluggish and stuck fermentations. If wort was supplemented
with FAN in the subsequent fermentation, could yeast overcome
the deficiencies generated during propagation? Additionally, the
scalability of this propagation protocol should be assessed to
determine the impact on large scale fermentation performance.
Finally, questions remain on the impact on the organoleptic
quality of beer produced with yeast propagated under different
conditions.

Figure 4. Effect of sugar and C:N ratio of the propagation media on wort attenuation of subsequent bench-scale fermentations compared to industrial fermentations pitched with
harvested yeast. (A) The average residual sugar remaining in beer from fermentations pitched with harvested yeast, yeast propagated in YCM, or yeast propagated in high C:N wort with
varying fermentable sugar concentrations. Error bars represent S.D. Significance established against harvested yeast, where significance is designated by * p ≤ 0.05, ** p ≤ 0.005, and
*** p ≤ 0.0005 (α = 0.05). (B) The average residual sugar remaining in beer from fermentations pitched with harvested yeast, yeast propagated in YCM, or yeast propagated in low
295

C:N wort with varying fermentable sugar concentrations. Error bars represent S.D. Significance established against harvested yeast.

J. Inst. Brew. 2020; 126: 289–297 © 2020 The Institute of Brewing & Distilling wileyonlinelibrary.com/journal/jib

Conclusions
A viable propagation medium is described to promote efficient
yeast growth, producing yeast with a physiological advantage for
subsequent fermentations and beer production. The sugar
concentration of the propagation medium caused a significant
impact on growth kinetics only under nitrogen limiting conditions.
Propagating yeast in a nitrogen limited environment had a
significant negative impact on subsequent fermentation kinetics.
Using standard wort as the propagation medium increased the risk
of low ethanol content in the finished beer. This work suggests
yeast propagated in wort supplemented with yeast extract to a
100 C:N ratio and diluted to 2°P of fermentable sugars yields high
quality fermentations and promotes complete wort attenuation.
Author contributions
M.E.M. and A.C.D. designed and performed the experiments. M.E.
M. performed data analysis and wrote the original draft. M.E.M.
and A.C.D. reviewed and edited the manuscript.
Acknowledgements
The authors gratefully acknowledge William F. Cayler for ICP-OES
analysis, Andrew R. Reyes for helpful statistics discussions, Anders
MacCarthy for production support, the Sierra Nevada Quality
Assurance-Analytical Department for Beer Alcolyzer analysis, and
Sierra Nevada Brewing Company for supporting this work.
Conflict of interest
The authors declare there are no conflicts of interest.
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Supporting information
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
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