RESEARCH ARTICLE
The fraction of cells that resume growth after acetic acid
addition is a strain-dependent parameter of acetic acid
tolerance in Saccharomyces cerevisiae
ndez-Nin
~ o1, Daniel Gonza
lez-Ramos2,3, Antonius J. A. van Maris2,3 &
Steve Swinnen1, Miguel Ferna
1
Elke Nevoigt
1
School of Engineering and Science, Jacobs University gGmbH, Bremen, Germany; 2Department of Biotechnology, Delft University of Technology,
Delft, The Netherlands; and 3Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands
Correspondence: Elke Nevoigt, School of
Engineering and Science, Jacobs University
gGmbH, Campus Ring 1, 28759 Bremen,
Germany. Tel.: +49 421 200 3541;
fax: +49 421 200 3249;
e-mail: [email protected]
Received 9 December 2013; revised 12
March 2014; accepted 12 March 2014. Final
version published online 11 April 2014.
DOI: 10.1111/1567-1364.12151
Editor: Jens Nielsen
Keywords
Saccharomyces cerevisiae; yeast;
lignocellulose; acetic acid tolerance; cell-tocell heterogeneity; intraspecies diversity.
Abstract
High acetic acid tolerance of Saccharomyces cerevisiae is a relevant phenotype
in industrial biotechnology when using lignocellulosic hydrolysates as feedstock.
A screening of 38 S. cerevisiae strains for tolerance to acetic acid revealed considerable differences, particularly with regard to the duration of the latency
phase. To understand how this phenotype is quantitatively manifested, four
strains exhibiting significant differences were studied in more detail. Our data
show that the duration of the latency phase is primarily determined by the
fraction of cells within the population that resume growth. Only this fraction
contributed to the exponential growth observed after the latency phase, while
all other cells persisted in a viable but non-proliferating state. A remarkable
variation in the size of the fraction was observed among the tested strains differing by several orders of magnitude. In fact, only 11 out of 107 cells of the
industrial bioethanol production strain Ethanol Red resumed growth after
exposure to 157 mM acetic acid at pH 4.5, while this fraction was 3.6 9 106
(out of 107 cells) in the highly acetic acid tolerant isolate ATCC 96581. These
strain-specific differences are genetically determined and represent a valuable
starting point to identify genetic targets for future strain improvement.
YEAST RESEARCH
Introduction
The presence of compounds in lignocellulosic hydrolysates that are inhibitory to microorganisms is one hurdle
in the bioconversion of this renewable feedstock into
valuable products (Palmqvist & Hahn-H€agerdal, 2000).
Among these compounds, acetic acid derived from acetylated hemicelluloses is one of the most potent inhibitors,
particularly at low pH (Casal et al., 1996; Thomas et al.,
2002; Graves et al., 2006). Efficient bioconversion of lignocellulosic hydrolysates therefore requires either reduction of the acetic acid concentration, adjustment of the
pH, or engineering of microorganisms for improved acetic acid tolerance. The yeast Saccharomyces cerevisiae is a
popular microorganism in industrial biotechnology,
mainly due to its robustness in industrial processes and
the extensive toolbox available for engineering this organism (Nevoigt, 2008; Hong & Nielsen, 2012). Although
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S. cerevisiae has an innate tolerance to moderate concentrations of acetic acid (Abbott et al., 2009), this is not
sufficient for processes based on crude lignocellulosic
hydrolysates that contain substantially higher concentrations (Chandel et al., 2011; Zha et al., 2012; Demeke
et al., 2013).
Acetic acid is a weak acid (pKa = 4.76) and is therefore
mainly present in an undissociated state at the relatively
low pH values of typical industrial batch fermentations
using yeast without pH control. In contrast to the dissociated form of acetic acid, the undissociated form diffuses
across the plasma membrane into the cytosol, where it
dissociates into protons and acetate anions at physiological pH (Casal et al., 1996, 1998; Mollapour & Piper,
2007). The accumulation of protons during weak acid
stress can lead to decreased DNA and RNA synthesis
rates, reduced metabolic activity, and disrupted electrochemical proton gradients, while the accumulation of
FEMS Yeast Res 14 (2014) 642–653
643
Cell-to-cell heterogeneity in acetic acid tolerance
anions primarily results in increased turgor pressure and
oxidative stress (Pampulha & Loureiro-Dias, 1990; Piper
et al., 2001; Booth & Statford, 2003; Giannattasio et al.,
2012). The severity of how these physiological processes
are affected by acetic acid is a function of the undissociated acid concentration (Thomas et al., 2002).
Upon exposure to moderate acetic acid concentrations,
S. cerevisiae cells exhibit an extended period of growth
latency before entering exponential growth phase. During
this latency period, which has also been referred to as the
lag or adaptation period (Piper et al., 2001; Mira et al.,
2010b), cells are assumed to adapt to the adverse effects
exerted by acetic acid by modifying their cellular processes to ensure survival and resume growth, as comprehensively reviewed by Mira et al. (2010b) and
Giannattasio et al. (2013). Among these cellular adjustments is the increased translocation of protons across the
plasma membrane by H+-ATPases, which appears to be a
key event in the adaptation process. Indeed, increased
expression of H+-ATPases has been observed during the
adaptation period (Carmelo et al., 1997), and recovery of
intracellular pH is concomitant with entry into exponential growth phase (Lambert & Stratford, 1999; Ullah et al.,
2012). The toxic effect exerted by the accumulation of
acetate anions is counteracted by the activation of
drug : H+ antiporters, which translocate the anions across
the plasma membrane (Holyoak et al., 1999; Tenreiro
et al., 2000, 2002; Fernandes et al., 2005). Other cellular
defense mechanisms against acetic acid stress include a
reconfiguration of the cell wall and a saturation of the
plasma membrane (Mira et al., 2010c; Lindberg et al.,
2013). These changes are assumed to limit the entry of
undissociated acetic acid molecules into the cell.
The high energy demand that is concomitant with the
detoxification mechanisms mentioned above results in an
energy deficit. The need of the cell to compensate for this
deficit seems to be reflected in several experimental findings such as an increased sugar consumption rate during
weak acid stress (Pampulha & Loureiro-Dias, 2000; Bellissimi et al., 2009), as well as an increased activity of
enzymes involved in glycolysis and the Krebs cycle during
the latency period (Almeida et al., 2009; Mira et al.,
2010a). The observed transcriptional responses to acetic
acid are under the control of several transcription factors,
of which Haa1 seems to be of high relevance. Indeed,
approximately 80% of all genes upregulated during early
response to acetic acid (that is, 30 min after exposure to
50 mM acetic acid at pH 4.0) have been found to be
dependent on expression of HAA1 (Mira et al., 2010a).
Recent genome-wide studies, such as the analysis of gene
expression changes upon exposure to acetic acid (Kawahata
et al., 2006; Li & Yuan, 2010; Mira et al., 2010a) and the
screening of the single-gene deletion collection for mutants
FEMS Yeast Res 14 (2014) 642–653
sensitive to acetic acid (Kawahata et al., 2006; Mira et al.,
2010c), have been limited to laboratory strains of S. cerevisiae (predominately the BY series). In comparison with
industrial strains and natural isolates, these laboratory
strains are usually less robust (Argueso et al., 2009). As one
major motivation of our study was to identify targets for
industrial strain improvement, we reasoned that analyzing
strains different from laboratory strains might be more
straightforward. It is well known that there is, in general,
high phenotypic and genotypic diversity among different
strains of the species S. cerevisiae (Fay & Benavides, 2005;
Carreto et al., 2008; Kvitek et al., 2008; Liti et al., 2009;
Schacherer et al., 2009; Csoma et al., 2010; Wang et al.,
2012). This intraspecies diversity has also been recently
demonstrated for the phenotype of acetic acid tolerance
(Haitani et al., 2012). In the current study, we investigated
how acetic acid tolerance is quantitatively manifested in
different S. cerevisiae strains.
Materials and methods
Strains and cultivation conditions
The full list of the 38 S. cerevisiae strains that were
screened for acetic acid tolerance in this study can be
taken from Fig. 3. The four strains that were analyzed in
more detail are listed in Table 1.
Yeast cells taken from frozen stocks were grown on
YPD medium containing 1% (w/v) yeast extract, 2% (w/v)
peptone, and 2% (w/v) glucose to obtain single cell colonies. Cultivation of yeast cells was routinely carried out at
30 °C. Throughout this study, all experiments for assaying acetic acid tolerance were performed in synthetic
medium according to Verduyn et al. (1992) containing
2% (w/v) glucose as the carbon source and acetic acid at
the indicated concentrations. The pH of all synthetic
media (that is, with and without acetic acid) was adjusted
to 4.5 with 2 M potassium hydroxide. In order to prepare
solid medium, 2% (w/v) agar was added.
Acetic acid tolerance assay in liquid medium
using the Growth Profiler 1152, in static
cultures, and on solid medium
Acetic acid tolerance of S. cerevisiae strains is strongly
dependent on the physiological state of the culture. We
therefore included a figure giving a detailed overview of
the specific steps performed to prepare the cells for the
acetic acid tolerance assays (Fig. 1). For pre-culture, 3 mL
of synthetic medium in a glass tube was inoculated with
cells originating from a single colony on a YPD plate. The
cells were then incubated overnight in an orbital shaker at
200 r.p.m. The pre-culture was used to inoculate 3 mL of
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S. Swinnen et al.
644
Table 1. Saccharomyces cerevisiae strains analyzed in detail in this study
Strain
Description
Reference
ATCC 96581
Isolated from a spent sulfite liquor
fermentation plant
S. cerevisiae sensu lato
American Type Culture Collection
MUCL 11987
CEN.PK
Ethanol Red
Diploid strain obtained by mating
CEN.PK113-1A and CEN.PK113-7D
Strain widely applied in industrial
bioethanol production
fresh synthetic medium to an optical density (OD600 nm) of
0.2. This culture, which we refer to here as the intermediate
culture, was subsequently grown under the same conditions
as the pre-culture for 6–8 h until mid-exponential phase
was reached (OD600 nm between 1.0 and 1.5). An appropriate amount of cells from the intermediate culture was pelleted by centrifugation at 800 g for 5 min and resuspended
in synthetic medium containing acetic acid to obtain an
OD600 nm of 0.2. Aliquots from this sample were then
transferred immediately into wells of a white Krystal
24-well clear bottom microplate (Porvair Sciences, Leatherhead, UK; Fig. 1a). In detail, three aliquots (except indicated otherwise) of 750 lL each from the same culture
sample were transferred to three separate wells of a plate to
obtain technical replicates for each experiment. Growth
was recorded using the Growth Profiler 1152 (Enzyscreen,
Haarlem, the Netherlands) at 30 °C and orbital shaking at
200 r.p.m. The Growth Profiler 1152 was set to scan
the plate every 40 min. Based on this scan, the Growth
Profiler software allows calculating the density of the culture
in each single well of the plate (expressed as green value or
G-value). A calibration curve was generated to convert the
G-values into OD600 nm values (referred to here as
OD600 nm equivalents). The following equation was derived
from the calibration curve and used throughout this study:
OD600 nm equivalent = 6.1108.10 9 9 G-value3.9848.
In static cultures, the conditions were the same as
described above with the exception that the 24-well plate
was placed in a static incubator at 30 °C for 3 days
(Fig. 1b).
For acetic acid tolerance assays on solid medium, an
aliquot of the exponential phase intermediate culture was
serially diluted (dilution factors 10 1–10 4) in synthetic
medium without acetic acid and without glucose
(Fig. 1c). Exactly 250 lL of each dilution was streaked on
solid synthetic medium either with or without 157 mM
acetic acid (pH 4.5). Plates were incubated in a static
incubator at 30 °C. The incubation time was 2 days for
cells that were streaked on medium without acetic acid
and 4 days for cells streaked on medium with acetic acid.
Dilutions resulting in colony forming units (CFU) in the
range of 50–150 per plate were included for counting.
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Belgian Co-ordinated Collection
of Microorganisms
This study
Fermentis, France
Time course experiment to study the effects of
prolonged exposure to acetic acid
The pre- and intermediate cultivation as well as the transfer of the exponentially growing non-stressed cells into
acetic acid containing medium was performed in the
same way as described above, with the exception that larger culture volumes were used (that is, 20 mL for precultures, 50 mL for intermediate cultures, and 75 mL for
acetic acid containing cultures), and cultivations were
performed in shake flasks on an orbital shaker. Every 3 h,
samples from the acetic acid containing cultures were
taken in order to determine the optical density and prepare tenfold dilutions (Fig. 1c). These dilutions were subsequently streaked on solid synthetic medium without
acetic acid to determine the total number of viable cells
in the culture and on solid synthetic medium with
157 mM acetic acid (pH 4.5) to determine the fraction of
cells that proliferated in the presence of the acid.
Live cell imaging
Live imaging of single cell proliferation was performed using
the BioStation IM (Nikon, Germany). The BioStation IM
moves the objective lens instead of the stage, thereby minimizing culture vibration and thus enabling the monitoring
of the same cell population over time. Growth conditions
were the same as described for the acetic acid tolerance assay
in liquid medium with the exception that 1.5 mL was transferred to a l-Dish (35 mm, high) from Ibidi (Martinsried,
Germany; Fig. 1d). The incubator chamber was maintained
at 32 °C and 100% humidity. Phase contrast images were
acquired with 809 magnification during 17 h.
Results
Effect of increasing acetic acid concentrations
on the maximum specific growth rate and
latency phase of CEN.PK
A diploid CEN.PK strain obtained by mating the commonly used laboratory strains CEN.PK113-1A and
FEMS Yeast Res 14 (2014) 642–653
Cell-to-cell heterogeneity in acetic acid tolerance
645
significantly affected up to 70 mM acetic acid, whereas
any further increase strongly lengthened this phase up to
44 h at 157 mM acetic acid (Fig. 2b).
Saccharomyces cerevisiae intraspecies diversity
with regard to acetic acid tolerance
(a)
(b)
(c)
(d)
Fig. 1. Schematic representation of the different acetic acid tolerance
assays used in this study. Acetic acid tolerance was assayed in liquid
medium in order to record optical density using the Growth Profiler
1152 (a), in static cultures to follow the formation of single cell
colonies in liquid medium (b), and on solid medium to quantify the
formation of single cell colonies (c). Live imaging of single cell
proliferation was performed using the BioStation IM (d). aa, acetic
acid; SM, synthetic medium.
CEN.PK113-7D (van Dijken et al., 2000) was used to
create a reference point for quantifying the effect of acetic
acid on the maximum specific growth rate and latency
phase of different S. cerevisiae isolates. The term latency
phase is used throughout this study to refer to the time
period until exponential growth was detectable by optical
density measurement. As shown in Fig. 2a, the maximum
specific growth rate (lmax) of CEN.PK gradually
decreased from 0.45 to 0.21 h 1 when the acetic acid
concentration was increased from 17 to 157 mM at pH
4.5. No growth was observed at concentrations
of 175 mM or higher. The latency phase was not
FEMS Yeast Res 14 (2014) 642–653
Based on the data obtained with CEN.PK, we chose a concentration of 157 mM acetic acid at pH 4.5 (corresponding
to 9 g L 1 total or 101 mM undissociated acetic acid) to
screen 38 S. cerevisiae isolates for acetic acid tolerance. As
this was the highest concentration that allowed growth of
CEN.PK, with a growth rate reduced by 53% compared to
non-stress conditions and a latency phase of 44 h, strains
with a significantly higher acetic acid tolerance should be
detectable by a higher lmax and/or shorter latency phase.
The data obtained from the screening showed considerable
intraspecies diversity with regard to both lmax and duration of the latency phase (Fig. 3a). Interestingly, no correlation between these two parameters was found within the
collection of strains tested (correlation coefficient of 0.2;
Fig. 3b).
The screening of the 38 S. cerevisiae strains led to
another remarkable finding regarding the reproducibility
of technical replicates. In general, technical replicates that
are obtained from one biological replicate are expected to
result in very similar data. However, during the course of
our acetic acid tolerance screening, we observed a general
trend that strains with a short latency phase showed high
reproducibility in technical replicates, while strains with a
long latency phase showed low reproducibility. In extreme
cases, some technical replicates from a single strain even
did not show growth at all. For those strains, values
shown in Fig. 3 were calculated based only on the technical replicates that resulted in growth (strains are marked
with an asterisk).
To address the reproducibility problem in the technical replicates, four strains showing large differences in
the duration of the latency phase were analyzed in
more detail. In addition to CEN.PK, we selected strains
ATCC 96581, MUCL 11987, and Ethanol Red.
ATCC 96581 has been the subject of several studies
determining the performance of S. cerevisiae in lignocellulosic hydrolysates (Linden et al., 1992; Palmqvist
et al., 1999; Brandberg et al., 2004, 2007; Hou & Yao,
2012) and showed the shortest latency phase of all
strains included in our screening. Ethanol Red is a
widely used strain in bioethanol production and is
therefore of industrial relevance. This strain was
selected as a representative of those strains with an
extraordinarily long latency phase. MUCL 11987 and
CEN.PK showed latency phases that were intermediate
to those of ATCC 96581 and Ethanol Red. For each of
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S. Swinnen et al.
646
(a)
(b)
Fig. 2. Effect of increasing acetic acid concentrations on the lmax and latency phase of the diploid Saccharomyces cerevisiae strain CEN.PK. Cells
were cultivated in synthetic medium until exponential phase, then transferred to medium with identical composition but containing increasing
acetic acid concentrations from 17 to 210 mM (increments of 17.5 mM), and adjusted to pH 4.5. Growth of the cultures was recorded for
4 days using the Growth Profiler 1152. The growth curves were used to calculate lmax and latency phases. Mean values and standard deviations
were obtained from three biological replicates; each biological replicate is the mean value of three technical replicates.
the four strains, the growth curves of six technical replicates were analyzed in detail (Fig. 4a). ATCC 96581
and MUCL 11987 showed a relatively short latency
phase after acetic acid exposure and high reproducibility within technical replicates, while CEN.PK showed a
longer latency phase and slight variability. In clear contrast to these strains, Ethanol Red showed a conspicuously low reproducibility. In fact, some technical
replicates showed growth after an extremely long
latency phase, while others did not show growth at all.
The experiment was repeated two more times with similar results. The average values and standard deviations
of the lmax and latency phases obtained from the three
biological replicates are shown in Table 2.
The low reproducibility within technical replicates of
Ethanol Red and other strains exhibiting a long latency
phase led us to hypothesize that for those strains, only a
very small fraction of cells in the population may resume
growth after exposure to acetic acid. Accordingly, any
fortuitous variation in the size of this fraction among
technical replicates would result in significant differences
with regard to the duration of the latency phases. In a
first step, a theoretical assessment of the fraction of cells
that resume growth was carried out. For this calculation,
it was assumed that the cells that resumed growth started
growing immediately after exposure to acetic acid with a
growth rate equal to the lmax obtained after cells entered
the exponential growth phase. Furthermore, the number
of cells per milliliter was derived from the optical density
based on a calibration between OD600 nm and cell concentration. According to the theoretical assessment, seven
cells of Ethanol Red per 750 lL culture resumed growth
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after transfer to acetic acid containing medium. With
regard to strains ATCC 96581, MUCL 11987, and
CEN.PK, the calculated numbers were 70 000, 1600, and
300 cells per 750 lL culture, respectively.
The data obtained in the theoretical assessment were
experimentally validated by two approaches. In the first
approach, six technical replicates of ATCC 96581,
MUCL 11987, CEN.PK, and Ethanol Red were cultivated
under the same conditions as described before, with the
only difference that the cultures were not shaken so that
the formation of single cell colonies could be followed
even in liquid medium (Fig. 4b). ATCC 96581 showed a
high number of CFU; single cell colonies could not even
be distinguished from each other. MUCL 11987 and
CEN.PK formed significantly lower numbers of single
cell colonies, in which the number for MUCL 11987
was visibly higher compared to CEN.PK. In contrast to
the other strains and in line with the theoretical assessment, Ethanol Red showed a remarkably low number of
CFU. In fact, only one or two colonies were detected in
certain technical replicates, while others did not even
show a single colony. In the second approach, exponentially growing cells from the same intermediate culture
were streaked on solid medium containing acetic acid.
The acetic acid concentration was the same as used in
the liquid cultures, that is, 157 mM acetic acid at pH
4.5. Notably, the numbers of CFU were in the same
order of magnitude as the numbers obtained by the theoretical assessment (Fig. 4c). Taken together, our data
show that only a fraction of S. cerevisiae cells resumed
growth after exposure to acetic acid and that the size of
this fraction was highly variable between strains.
FEMS Yeast Res 14 (2014) 642–653
647
Cell-to-cell heterogeneity in acetic acid tolerance
(a)
(b)
Fig. 3. Phenotypic intraspecies diversity of Saccharomyces cerevisiae with regard to acetic acid tolerance. Acetic acid tolerance was dissected
here into two measurable parameters, that is, the duration of the latency phase and the maximum specific growth rate (lmax). (a) A total of 38
S. cerevisiae strains were screened for growth in synthetic medium containing 157 mM acetic acid at pH 4.5. Mean values and standard
deviations were obtained from at least two biological replicates. For each biological replicate, two technical replicates were cultivated. For those
strains that showed high reproducibility in technical replicates, the duration of the latency phase and lmax were calculated based on the average
growth curve of the two replicates. For those strains that showed a low reproducibility (marked by an asterisk), the duration of the latency phase
and lmax were calculated based on the growth curve of the replicate with the shortest latency phase. (b) Scatterplot of the duration of the
latency phase versus lmax for the 36 S. cerevisiae strains that showed growth during the course of the experiment.
The subpopulation that resumes growth after
acetic acid exposure is a result of phenotypic
cell-to-cell heterogeneity
The fact that only a subpopulation of cells resumes
growth after exposure to acetic acid may either originate
from phenotypic cell-to-cell heterogeneity, or be caused
by mutations that have naturally arisen in these cells.
These possibilities were investigated in more detail for
strains CEN.PK and Ethanol Red, for which only a small
fraction of cells resumed growth following exposure to
157 mM acetic acid at pH 4.5 (Fig. 4b and c). Three single cell colonies from CEN.PK and Ethanol Red obtained
on acetic acid containing solid medium were therefore
subjected to a second acetic acid tolerance assay. The cells
were first reverted to a non-stressed physiological state by
cultivating them on YPD medium. This is important
because it has been shown that S. cerevisiae cells can
FEMS Yeast Res 14 (2014) 642–653
adapt to acetic acid stress, that is, pre-adapted cells have
a significantly shorter latency phase upon exposure to
acetic acid (Piper et al., 2001). Comparison of the first
and second round of acetic acid tolerance assay showed
no difference in the latency phase in liquid acetic acid
containing medium, as well as no difference in the number of CFU on solid acetic acid containing medium (data
not shown). This data confirm that the small number of
CEN.PK and Ethanol Red cells that resumed growth in
the presence of acetic acid was the result of phenotypic
cell-to-cell heterogeneity.
Cells that do not resume growth die after
prolonged exposure to acetic acid
As only a fraction of cells resumed growth upon exposure
to acetic acid, the question arose whether the remaining
cells died. This was studied in more detail for strains
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S. Swinnen et al.
648
(a)
(b)
(c)
Fig. 4. The acetic acid-induced latency phase of a Saccharomyces cerevisiae strain is determined by the fraction of cells that resume growth.
(a) Cells from strains ATCC 96581, MUCL 11987, CEN.PK and Ethanol Red were harvested from exponential growth phase and transferred to
medium with identical composition but supplemented with 157 mM acetic acid (pH 4.5). From each of these four suspensions, six aliquots of
750 lL each (representing technical replicates) were cultivated in the Growth Profiler 1152. (b) Corresponding static liquid cultures prepared in
the same way as described in ‘a’ with the only difference that the cultures were grown in a static incubator allowing the formation of single cell
colonies in liquid medium. (c) Number of cells per 750 lL culture that were able to form colonies after streaking on solid synthetic medium
containing 157 mM acetic acid at pH 4.5 (white bars). In parallel, an aliquot of the same culture was streaked on medium without acetic acid to
determine the corresponding total number of viable cells (gray bars). See Fig. 1 for the experimental details.
Table 2. Maximum specific growth rate (lmax) and duration of the
latency phase of four selected Saccharomyces cerevisiae strains after
exposure to 157 mM acetic acid at pH 4.5
Strain
Latency phase (h)
ATCC 96581
MUCL 11987
CEN.PK
Ethanol Red*
15
25
44
53
2
1
3
8
lmax (h 1)
0.22
0.28
0.21
0.26
0.02
0.00
0.02
0.02
Mean values and standard deviations were obtained from three biological replicates.
*In order to calculate the lmax and duration of the latency phase for
strain Ethanol Red, only those technical replicates that showed
growth were used.
CEN.PK and Ethanol Red. In a first step, the effect of a
short-term exposure to acetic acid on cell viability was
determined. Synthetic medium with and without acetic
acid was inoculated with an equal number of cells from
an intermediate culture. After incubation for 15 min,
aliquots from both cultures were streaked on solid
medium without acetic acid to be able to count the number of viable cells. Similar numbers of CFU were obtained
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for both cultures (data not shown), indicating that all
cells in the population were alive and retained the ability to resume growth upon transition to non-stress
conditions.
Next, we investigated whether the non-proliferating
cells die after a prolonged incubation in acetic acid containing medium. This was pursued by recording the number of viable cells for a time period of 66 h. Notably, the
course of viable cells is influenced by dying, by viable but
non-proliferating and by proliferating cells. Nevertheless,
the fact that the total number of viable cells in the culture
significantly decreased only after approximately 20 h for
CEN.PK or 40 h for Ethanol Red implies that cells only
die after very long exposure to the stressful condition
(Fig. 5).
The actual lag or adaptation phase of cells that
resume growth is significantly shorter than the
phase detected by optical density
measurement
When performing the above experiment to record the
time course of viable cells, an aliquot of each culture was
FEMS Yeast Res 14 (2014) 642–653
Cell-to-cell heterogeneity in acetic acid tolerance
649
also streaked on solid medium with acetic acid to record
the time course of those cells that started to proliferate in
the presence of the acid (Fig. 5). The data showed that
the actual adaptation phase for these cells was significantly shorter when determined at the single cell level
(15 h for both CEN.PK and Ethanol Red) compared to
optical density measurement (41 h for CEN.PK and 51 h
for Ethanol Red). In fact, the optical density only
increased after the few cells that started to proliferate substantially added to the optical density at the point of
inoculation (Fig. 5).
The elongation of the latency phase that is
concomitant with increasing acetic acid
concentrations is also caused by a reduction in
the fraction of cells that resume growth
It is known that the duration of the acetic acid induced
latency phase of a S. cerevisiae strain depends on the
stringency of the acetic acid stress (Narendranath et al.,
2001). This dependency has also been demonstrated for
CEN.PK in the current study (Fig. 2b). The latency phase
of CEN.PK was not affected at acetic acid concentrations
up to 70 mM. Above this concentration, the latency
phase was directly affected by the acetic acid concentration in a dose-dependent manner. To investigate whether
the elongation of the latency phase is a function of the
fraction of cells that resume growth, CEN.PK cells were
exposed to different acetic acid concentrations (70, 79,
87, 96 and 105 mM), and proliferation of single cells was
followed by live cell imaging (Fig. 6). The data confirm
that, whereas almost all cells resume growth in the presence of acetic acid up to 79 mM, only a fraction of cells
resumes growth at 87 mM or higher concentrations. In
addition, the number of cells that resume growth was a
function of the acetic acid concentration (data not
shown), which is in agreement with the dose-dependent
effect of the acetic acid concentration on the duration of
the latency phase.
Discussion
This study shows that in acetic acid containing medium,
only a fraction of S. cerevisiae cells resumes growth, and
that the size of this fraction differs by orders of magnitude between different S. cerevisiae strains. In addition
the fraction of cells that resume growth is a parameter
that can be reproducibly quantified, implying that its size
is inheritable and thus genetically determined. The mere
fact that acetic acid tolerance is determined by a fraction
of highly resistant cells within the population is in accordance with two previous studies describing the effect of
FEMS Yeast Res 14 (2014) 642–653
Fig. 5. Prolonged exposure of cells from Saccharomyces cerevisiae
strains CEN.PK and Ethanol Red to acetic acid. Exponential phase cells
from CEN.PK and Ethanol Red were transferred to medium containing
157 mM acetic acid (pH 4.5). The cells were subsequently cultivated
on an orbital shaker for a time period of 66 h. Every 3 h, samples
were taken in order to determine the optical density (□), the
concentration of viable cells (●), and the concentration of cells that
resumed growth (▲). Data from one representative experiment out
of two biological replicates are shown.
other weak acids on S. cerevisiae (Viegas et al., 1998;
Stratford et al., 2013).
Phenotypic cell-to-cell heterogeneity within a clonal
population of cells is a well-known phenomenon that has
been described in many types of cells (Geiler-Samerotte
et al., 2013). It is assumed that this diversity at the single
cell level is a general survival strategy in nature, as it
allows a population of cells to overcome unanticipated
environmental changes. Based on the few known examples, it seems that cell-to-cell heterogeneity can be caused
by either deterministic factors (e.g., cell cycle, size or age,
or biological rhythms), or by spontaneous events (e.g.,
stochasticity in gene expression, or epigenetic modifications) (Raser & O’Shea, 2004; Avery, 2006). Cell-to-cell
heterogeneity has been shown to be of clinical relevance
with regard to antibiotic resistance of microorganisms
(Bishop et al., 2007) and chemoresistance of tumors
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Published by John Wiley & Sons Ltd. All rights reserved
650
(Roesch et al., 2010). The current study demonstrates
that this phenomenon can also affect the performance of
microorganisms in industrial fermentations.
The data presented in this study provide evidence that
the fraction of cells that resume growth after exposure to
acetic acid is the main determinant of the duration of the
acetic acid-induced latency phase. In fact, it was only this
fraction that was accountable for the exponential growth
phase following the latency phase. All other cells persisted
in a non-proliferating state and may have eventually died
after long-term exposure to the acid. The fact that only a
fraction of cells resumes growth implies that the traditional understanding of the lag or adaptation phase can
only be applied to this fraction.
All previous conclusions on the molecular mechanisms
determining acetic acid tolerance of S. cerevisiae have
been based exclusively on data obtained by examining
bulk populations. According to the data obtained in the
current study, these previously obtained data may need to
be re-evaluated depending on the yeast strain and acetic
acid concentration used. We have determined the threshold concentration of CEN.PK at which cell-to-cell heterogeneity becomes relevant. Microscopic time-lapse
experiments showed that the threshold concentration lies
between 79 and 87 mM acetic acid at pH 4.5 (that is, 51
and 56 mM undissociated acetic acid). However, most
studies on acetic acid tolerance have been performed with
the laboratory strain S288c, or its auxotrophic derivatives BY4741 and BY4742 (Kawahata et al., 2006; Mira
et al., 2010a, c). Although we did not determine the cellto-cell heterogeneity threshold for S288c, the diploid
S. Swinnen et al.
prototrophic variant of this strain was included in our
initial screening and its tolerance was lower than that of
CEN.PK (Fig. 3). Therefore, it is likely that the issue of
cell-to-cell heterogeneity for S288c becomes relevant at a
lower acetic acid concentration compared to CEN.PK.
Previously, the acetic acid tolerance of S. cerevisiae
strains had been compared by means of various measurable parameters including specific growth rate, lag phase
(referred to here as latency phase), biomass formation,
cell viability, as well as specific and volumetric ethanol
productivity (Palmqvist et al., 1999; Narendranath et al.,
2001; Thomas et al., 2002; Abbott & Ingledew, 2004; Garay-Arroyo et al., 2004; Graves et al., 2006). We demonstrate here that the fraction of cells that resume growth is
a novel measurable parameter of acetic acid tolerance and
that this fraction can be simply quantified by counting
CFU on solid acetic acid containing medium.
Uncovering the mechanisms underlying cell-to-cell heterogeneity in acetic acid tolerance is of high importance in
second-generation bioethanol production because it determines the duration of the latency phase, which in its turn
affects the volumetric ethanol productivity. Ethanol Red,
which is a commonly used commercial bioethanol production strain, showed a relatively long latency phase after
exposure to acetic acid, although after adaptation, it exhibited a lmax comparable to the best isolate ATCC 96581
(Table 2). The fact that the latency phase and lmax were differentially affected by acetic acid in the collection of strains
screened in this study implies that these two parameters are
independent aspects of acetic acid tolerance and therefore
most probably controlled by different molecular factors.
Fig. 6. Live imaging of the proliferation of
Saccharomyces cerevisiae CEN.PK cells after
exposure to increasing concentrations of acetic
acid. Images were recorded in phase contrast
with 809 magnification. For each acetic acid
concentration, one frame is shown for
different time points (that is, 1, 5, 7, 12, and
17 h after exposure to acetic acid). Because
cells might have slightly moved during the
course of the experiment, arrows were added
in each frame for an easier identification of
identical cells.
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
FEMS Yeast Res 14 (2014) 642–653
Cell-to-cell heterogeneity in acetic acid tolerance
In conclusion, the current study shows that cell-to-cell
heterogeneity within a population is an important factor
contributing to the quantitative manifestation of acetic
acid tolerance in S. cerevisiae. Crude lignocellulosic hydrolysates generally contain acetic acid concentrations in
the range of 1–8 g L 1 (Chandel et al., 2011; Zha et al.,
2012; Demeke et al., 2013). The concentrations used in
this study imply that the obtained data are of practical
relevance for lignocellulose-based bioethanol production
processes. The search for targets to improve acetic acid
tolerance of S. cerevisiae strains, which is the focus of
our ongoing research, should therefore be expanded to
genes that are involved in cell-to-cell heterogeneity.
Their identification will contribute to the understanding
of the molecular mechanisms underlying cell-to-cell heterogeneity with regard to acetic acid tolerance and facilitate the engineering of strains for more efficient
conversion of lignocellulosic hydrolysates during the production of second-generation bioethanol and other commodity chemicals using the yeast S. cerevisiae as a
biocatalyst.
Acknowledgements
This work was funded through the ERA-NET Scheme of
the 6th EU Framework Program (INTACT). MFN
received a personal stipend from Colciencias (Colombia).
We thank Heide-Marie Daniel (Belgian Co-ordinated
Collection of Microorganisms, Belgium) for kindly providing us with the strains MUCL 51248 and MUCL
52901 to 52909, Nina Gunde-Cimerman (University of
Ljubljana, Slovenia) for the strains from the Culture Collection of Extremophilic Fungi (EXF; Infrastructural Centre Mycosmo, Department of Biology, Biotechnical
Faculty, University of Ljubljana, Slovenia), and Johan
Thevelein (KU Leuven, Belgium) for all other strains used
in this study. We are also grateful to Ping-Wei Ho for
technical support and Mathias Klein for fruitful discussions.
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