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. 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

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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 ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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 FEMS Yeast Res && (2014) 1–12

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Cell-to-cell heterogeneity in acetic acid tolerance

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. FEMS Yeast Res && (2014) 1–12

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 ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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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 ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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 FEMS Yeast Res && (2014) 1–12

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Cell-to-cell heterogeneity in acetic acid tolerance

(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.

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(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

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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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Cell-to-cell heterogeneity in acetic acid tolerance

(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 FEMS Yeast Res && (2014) 1–12

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 ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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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

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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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Cell-to-cell heterogeneity in acetic acid tolerance

(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

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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.

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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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