ABSTRACT
During alcoholic fermentation, Saccharomyces cerevisiae is exposed to a host of environmental and physiological stresses. Extremes of fermentation temperature have previously been demonstrated to induce fermentation arrest under growth conditions that would otherwise result in complete sugar utilization at “normal” temperatures and nutrient levels. Fermentations were carried out at 15°C, 25°C, and 35°C in a defined high-sugar medium using three Saccharomyces cerevisiae strains with diverse fermentation characteristics. The lipid composition of these strains was analyzed at two fermentation stages, when ethanol levels were low early in stationary phase and in late stationary phase at high ethanol concentrations. Several lipids exhibited dramatic differences in membrane concentration in a temperature-dependent manner. Principal component analysis (PCA) was used as a tool to elucidate correlations between specific lipid species and fermentation temperature for each yeast strain. Fermentations carried out at 35°C exhibited very high concentrations of several phosphatidylinositol species, whereas at 15°C these yeast strains exhibited higher levels of phosphatidylethanolamine and phosphatidylcholine species with medium-chain fatty acids. Furthermore, membrane concentrations of ergosterol were highest in the yeast strain that experienced stuck fermentations at all three temperatures. Fluorescence anisotropy measurements of yeast cell membrane fluidity during fermentation were carried out using the lipophilic fluorophore diphenylhexatriene. These measurements demonstrate that the changes in the lipid composition of these yeast strains across the range of fermentation temperatures used in this study did not significantly affect cell membrane fluidity. However, the results from this study indicate that fermenting S. cerevisiae modulates its membrane lipid composition in a temperature-dependent manner.
INTRODUCTION
Industrial fermentation processes are ever-changing environments that produce numerous and diverse stresses on microorganisms that must adapt to changes in osmolarity, increasing levels of toxic by-products of metabolism, and thermal fluctuations to grow, reproduce, and survive. Strains of Saccharomyces cerevisiae are industrially important microorganisms that are utilized in processes ranging from bread making to automobile fuel production due to their ability to thrive in these hostile environments. Indeed, many strains of S. cerevisiae can withstand ethanol concentrations as high as 19% (vol/vol), while half this level of ethanol would prove lethal for most organisms (1). While yeast and many other microorganisms possess the innate ability to convert sugar to ethanol, under certain circumstances some fermentations will stop due to a number of environmental and physiological factors, such as yeast strain, nutrient availability, alcohol level, and temperature (2). Extremes of fermentation temperature have been demonstrated to result in fermentation arrest under growth conditions that would otherwise result in complete sugar utilization at “normal” temperatures and nutrient levels (3). A major contributing factor to fermentation arrest is the inability of the yeast strain to tolerate or adapt to increasing ethanol concentrations, and exposure to temperature extremes can exacerbate this effect (1, 2). Knowledge of how yeasts adapt to temperature changes and how temperature contributes to fermentation slowing and arrest in the presence of ethanol could lead to the development of methods for the prediction and mitigation of problem fermentations. Furthermore, understanding the traits that contribute to ethanol tolerance and temperature adaptation would allow them to be introduced into potential production strains that have other desirable characteristics, for example, the ability to simultaneously utilize five- and six-carbon sugars.
Yeast strains commonly employed in alcoholic fermentations have developed physiological mechanisms that involve complex signal transduction and genomic pathways that allow this organism to adapt and survive in the dynamic and hostile environment of a fermentation (4). The fermentation temperature has been shown to have a profound effect on the growth and fermentation abilities of S. cerevisiae in wine and other high-potential alcohol fermentations (5, 6). Furthermore, the heat shock response in yeast at elevated fermentation temperatures exhibits a number of similar features and functional overlap with the ethanol stress response in S. cerevisiae (7). Under the circumstance of increased ethanol concentration or elevated temperatures, the induction of the yeast stress response employs heat shock elements that stabilize membrane-associated proteins that attempt to maintain cellular homeostasis to overcome increased permeability of the cell membrane (7). Indeed, the yeast plasma membrane appears to be a primary target of the perturbing effects of ethanol exhibited by impacts on membrane integrity, as well as membrane-associated processes (1, 2, 7–9). Furthermore, there is significant evidence that the lipid composition of the strain contributes to its tolerance of increasing quantities of self-produced ethanol (10–13). However, it is less clear if the lipid composition of the yeast cell membrane contributes to the thermotolerance in S. cerevisiae.
The yeast cell membrane is a semipermeable barrier that allows the cell to maintain an internal environment conducive to the myriad chemical reactions that are essential for successful growth, reproduction, and survival in the inhospitable environment of an alcoholic fermentation. The cellular membranes of yeast are composed primarily of phospholipids, sterols, sphingolipids, and membrane-associated proteins (14). The principal sterol in Saccharomyces cerevisiae is ergosterol, and the principal phospholipids have been shown to be phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and phosphatidylcholine with fatty acid chains that are predominantly oleic acid (C18:1) and palmitoleic acid (C16:1), with smaller amounts of palmitic acid (C16:0) and stearic acid (C18:0) (12, 13, 15, 16). Variations in the fatty acid moieties esterified to the glycerol backbone of phospholipids yield hundreds of different molecules that yeast cells utilize to maintain cellular function and adapt to their environment (17–19). Owing to the complex composition of these membranes, little is known about the physical responses of these lipid bilayers to increasing temperatures and/or ethanol concentrations. However, work with model membrane systems composed of phosphatidylcholines and sterols has demonstrated that lipid composition and structure can have a protective effect on membrane bilayers in the presence of ethanol by mitigating the membrane-thinning effect of ethanol (20–24). Furthermore, it has previously been demonstrated that the membrane thinning effect of ethanol is exacerbated by temperature in model membranes (21). Ethanol-induced changes in the membrane thickness of fermenting yeast cells could potentially interfere with membrane-associated protein function (25–28), e.g., proteins involved in sugar, nitrogen, or ion transport, as well as signal transduction. However, until this point, the high-resolution lipid data necessary for understanding how yeast cell membrane composition varies with fermentation temperature and its association with thermotolerance have not been available.
In the present work, we utilize three yeast strains that we have previously shown to have diverse fermentation characteristics (12) to examine lipid compositional changes that occur in these strains when fermenting at different temperatures. We describe growth, sugar utilization, and ethanol production characteristics of these strains undergoing alcoholic fermentation at 15°C, 25°C, and 35°C. Lipid samples were collected and extracted from each strain during early- and late-stationary-phase metabolism at the same ethanol concentrations. Fluorescence anisotropy measurements were performed concurrently with lipid sampling to ascertain yeast cell membrane fluidity of the strains at similar temperature and ethanol concentrations. We describe how different fermentation temperatures and metabolic phases affect membrane lipid composition, as well as the utilization of the multivariate statistical analysis method, principal component analysis (PCA), to examine correlations of individual lipids with each yeast strain and fermentation temperature. Finally, we discuss the potential role of these lipids in ethanol tolerance and thermotolerance.
MATERIALS AND METHODS
Materials.All chemicals were acquired from Sigma-Aldrich (St. Louis, MO), and all Nanopure water used was obtained from a Milli-Q Synthesis A-10 water purification system (Millipore, Billerica, MA) unless noted otherwise. Internal standards (ISTDs) were used during method development and to construct standard curves for quantitation. They were chosen based on their not being endogenous and not having the same molecular weight (isobaric) as lipids previously identified in the S. cerevisiae lipidome (15, 16) or reference (REF) lipids representing each lipid class being analyzed. Specifically, these were 1,2-dilauroyl-sn-glycero-3-phosphate (PA 12:0-12:0) (ISTD), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (PE 18:2-18:2) (ISTD), l-phosphatidylinositol (liver, bovine) (PI 18:0-20:4) (ISTD), 1,2-dilinoleoyl-sn-glycero-3-phospho-l-serine (PS 18:2-18:2) (ISTD), 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (PC 18:2-18:2) (ISTD), cholesterol (plant derived) (ISTD), 1,2-di-heptadecanoyl-sn-glycero-3-phosphate (PA 17:0-17:0) (REF), 1,2-dihep-tadecanoyl-sn-glycero-3-phosphoethanolamine (PE 17:0-17:0) (REF), l-phosphatidylinositol (soy) (PI 16:0-18:2) (REF), 1,2-diheptade-canoyl-sn-glycero-3-phospho-l-serine (PS 17:0-17:0) (REF), and 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (PC 15:0-15:0) (REF), and they were purchased from Avanti-Polar Lipids (Alabaster, AL). Ergosterol (REF) was purchased from Sigma-Aldrich. Lipid extraction solvents were high-pressure liquid chromatography (HPLC)-grade chloroform with 0.5 to 1.0% (vol/vol) ethanol stabilizer, HPLC-grade methanol, and HPLC-grade water. Mobile-phase and gas chromatography (GC) solvents and standards were HPLC-grade hexanes, absolute ethanol, isopropanol, and water and liquid chromatography-mass spectrometry (LC-MS)-grade formic acid and triethanolamine. Yeast extract-peptone-dextrose (YEPD) agar plates were prepared using 10 g of Bacto yeast extract/liter, 20 g of Bacto peptone/liter, 20 g of glucose (dextrose)/liter, and 20 g of Bacto agar (Becton, Dickinson, Sparks, MD)/liter according to the method of Amberg et al. (29). Gases necessary for instrumentation operation were medical-grade nitrogen and helium and hydrogen of 99.99% grade (Praxair, Danbury, CT).
Low-sugar (250 g/liter; ∼25.0 °Brix; a 1:1 mixture of glucose [125 g/liter] and fructose [125 g/liter]) synthetic grape juice medium (minimal must medium [MMM]) was prepared according to the method of Giudici and Kunkee (30) as previously reported by Henderson et al. (12).
Lipid extraction solvent A was composed of 2.5:1:1 methanol-chloroform-water, extraction solvent B was composed of 1:1 methanol-chloroform, and extraction solvent C was composed of 5% (vol/vol) formic acid in water. The injection solvent and mobile phase A were composed of 7:3 hexane-isopropanol with 0.5% (vol/vol) formic acid and 0.5% (vol/vol) triethylamine, and mobile phase B was composed of 92:8 isopropanol-water with 0.5% (vol/vol) formic acid and 0.5% (vol/vol) triethylamine.
Yeast strains and inoculation.All S. cerevisiae strains were acquired from the UC Davis Enology Culture Collection and are listed in Table 1. The strains were stored and plated on YEPD plates for single-colony isolation according to the method of Amberg et al. (31). Yeast strains were stored at 4°C on plates for no longer than 1 month. The optical density at an absorbance wavelength of 600 nm (OD600) of the yeast culture was determined using a UV-1201 spectrophotometer (Shimadzu Scientific Instruments, Inc., Kyoto, Japan) as previously described by Henderson et al. (12).
Origins of S. cerevisiae strains used in this study
To inoculate experimental cultures, a preculture was prepared by transferring a single colony from the YEPD agar plate to 15 ml of MMM and was allowed to grow aerobically for 24 to 48 h at 25°C in an orbital incubator (New Brunswick Scientific, Edison, NJ). Next, an aliquot was taken from this preculture to inoculate 50 ml of MMM in a 250-ml Erlenmeyer flask to an OD600 of 0.1. This inoculum was grown anaerobically in an orbital incubator at 25°C until a corrected OD600 of 3 was reached. Finally, a volume of the inoculum to give an initial OD600 of 0.15 (∼15 ml) was added to 400 ml of MMM in a 500-ml Erlenmeyer flask fitted with a Ferm-Rite vented silicone stopper (The Boswell Company, San Rafael, CA).
Fermentation sampling.Fermentations were performed in triplicate on orbital incubators (New Brunswick Scientific, Edison, NJ) at three fermentation temperatures: 15°C, 25°C, and 35°C. All sugar concentrations (dissolved solids) are reported in °Brix, such that 1 °Brix is equivalent to 1 g sucrose in 100 g of solution, or approximately 10 g sugar in l liter of solution. Initial cell concentration determinations (OD600) and Brix measurements were carried out at the beginning of fermentation and subsequently every 24 h until the fermentation achieved dryness (≤−1.0 °Brix) or the °Brix remained constant for three consecutive measurements, at which point the fermentations were stopped. Brix measurements were performed on unclarified supernatants using a DMA 35 N densitometer (Anton Paar USA Inc., Ashland, VA). Yeast cells were harvested via centrifugation (4,000 × g, 10 min) from a volume of fermenting medium that yielded a 150-mg cell pellet (∼50 ml [wet weight]) at early and late stationary phases. For lipid analysis, the harvested cells were washed three times in Nanopure water, and the cell pellet was stored at −80°C. For fluorescence anisotropy measurements, the yeast cells were washed three times in phosphate-buffered saline (PBS) buffer, pH 7.4, and resuspended in PBS buffer at an OD600 of 0.75. The supernatants were sterile filtered with 0.45-μm-pore-size syringe filters (Millipore) and stored at −20°C.
Ethanol analysis.The volume percent ethanol concentration of the fermentations at early and late stationary phase was determined utilizing GC coupled to a flame ionization detector (GC-FID) according to the method of Zoecklein et al. (32), using isopropanol as the quantitative standard. The separation of ethanol and isopropanol was achieved using a Hewlett-Packard 5890 GC equipped with a 7673A autosampler on a Restek SilcoSmooth (2 m by 2 mm [inner diameter]) and 5% Carbowax (20 m) on an 80/120 mesh CarboBlack B support-packed column (Restek Corp., Bellefonte, PA). The carrier gas was nitrogen at a column head pressure of 35 lb/in2, with a corresponding flow rate of ∼30 ml/min. The air and hydrogen gas flows for the FID were 300 and 30 ml/min, respectively. The injector and detector temperatures were held at 150°C. The initial oven temperature was 85°C, with no hold. Oven temperature was ramped at 65°C per min to 150°C with a 180-s hold and approximately 60 s to return to 85°C, for a total run time of ∼5 min. Clarified and filtered supernatants were diluted 1:99 in 0.20% isopropanol prior to analysis, and the ethanol/isopropanol peak area ratios were correlated to a standard curve constructed using quantitatively prepared standards containing between 0 and 20 volume percent of absolute ethanol. The weight percent of ethanol was calculated based upon the ratio of the density of ethanol (0.789 g/cm3) to the density of pure water (33).
Lipid extraction and sample preparation.The lipid extraction procedure was a modified Bligh-Dyer method adopted from Weckwerth et al. (34) with the addition of a 5% formic acid extraction step to improve recovery of acidic phospholipids as previously described (12). The lipid extracts stored under N2 (gas) were removed from −20°C and allowed to warm to room temperature prior to analysis. The samples were then resuspended in the appropriate injection solvent and loaded into the LC autosampler (Agilent Technologies, Santa Clara, CA) for analysis.
LC-MS analysis of phospholipids.Quantitative analysis of phospholipids was performed using a normal-phase LC-MS method previously described (12) on an Agilent 1260 series Rapid Resolution HPLC equipped with a temperature-programmable column compartment and a temperature-controlled autosampler (Agilent Technologies) equipped with a YMC 2.0- by 150-mm column packed with PVA-Sil (5-μm particle size) and a guard column of the same material (Waters Corp., Milford, MA) at a temperature of 45°C. The samples were kept at a temperature of 10°C prior to injection (5 μl). A binary gradient of mobile phases A and B sequentially eluted the polar lipids according to the following schedule: (i) from 0 to 10 min, mobile phase B increased from 12.5 to 15% at a flow rate of 400 μl/min; (ii) from 10 to 15 min, mobile phase B was increased to 100% at the same flow rate; (iii) from 15 to 15.5 min, mobile phase B remained at 100%, and the flow rate was decreased to 350 μl/min; (iv) from 15.5 to 25 min, mobile phase B remained at 100%; (v) from 25 to 26 min, mobile phase B returned to 12.5%; (vi) from 26 to 31 min, mobile phase B and the flow rate remained unchanged; and (vii) from 31 to 36 min, mobile phase B was kept at 12.5%, and the flow rate was returned to 400 μl/min. The total chromatographic cycle between injections was 36 min. The column eluent was directed to a microsplitter valve (IDEX Health and Science, Oak Harbor, WA) to reduce solvent flow to the electrospray ionization (ESI) source operating in negative-ion detection mode to 50 μl/min on an Agilent 6440 series triple-quadrupole (QqQ) mass spectrometer. The ESI source was operated in negative-ion detection mode. The QqQ MS was tuned and calibrated using an ESI calibration standard from Agilent Technologies. The MS settings were as follows: the capillary voltage was 3.5 kV, the nebulizer pressure was 25.0 lb/in2, and the dry gas flow rate and temperature were 5 liters/min and 350°C, respectively. Nitrogen gas was utilized as the nebulizing and drying gas and for collision-induced dissociation (CID). The QqQ MS was operated in selected ion monitoring (SIM) mode. The Fragmentor voltage was optimized using Optimizer software (Agilent Technologies), and instrument control and HPLC-MS data were collected and analyzed using MassHunter software (version B04.00; Agilent Technologies).
Phospholipid characterization was performed utilizing LC-MS on an Agilent 6330 series ion trap MS using the same chromatographic scheme described above on an Agilent 1100 series capillary HPLC system. Flow splitting and ESI source conditions for the ion trap MS were identical to those described above for the QqQ LC-MS method. The ion trap MS was tuned and calibrated using an ESI calibration standard from Agilent Technologies. The MS scan range was m/z 100 to 900, operating in the UltraScan scan mode (m/z 21,000/s). The nebulizing and drying gas was nitrogen, and the CID and ion-cooling gas was helium. Instrument control and HPLC-MS and HPLC-multistage tandem mass spectrometry (MSn) data were collected and analyzed using the ChemStation software package that accompanied the instrument (Agilent Technologies).
Sterol analysis using flow injection analysis.The analysis of squalene, lanosterol, and ergosterol from yeast extracts was adapted from the method of Toh et al. (35) as previously described (12). Sample injections of 10 μl were carried out using an Agilent 1200 series HPLC binary pump system (Agilent Technologies) at a flow rate of 400 μl/min, without a chromatographic column. The total time between injections was 6 min. The samples were kept at a temperature of approximately 25°C. The sample/carrier solvent was directed to the atmospheric pressure chemical ionization (APCI) source operating in positive-ion detection mode on an Agilent 6100 series MS (Agilent Technologies) operating in full-scan mode covering an m/z range from 300 to 500. The instrument was tuned and calibrated using an APCI calibration standard from Agilent Technologies. The MS settings were as follows: corona current, 4,000 nA; capillary voltage, 3.5 kV; nebulizer pressure, 60.0 lb/in2; dry gas flow rate, 5 liters/min; and dry and vaporizer temperatures, 350 and 400°C, respectively. The nebulizing and drying gas was nitrogen. The instrument control and FIA-MS data were collected and analyzed using the ChemStation software package that accompanied the instrument (Agilent Technologies).
Lipid quantitation.Standard curves for quantification of lipids using MS data were generated as follows. Multiple samples were made, varying the final concentration of REF lipids from 1 to 1,000 μM, and were extracted, prepared for analysis, and analyzed in the same manner as for the yeast lipid extracts. Lipid standard data were plotted as the ratio of the concentrations (μM) of the REF standard to that of the ISTD (i.e., REF/ISTD) on the ordinate axis and the ratio of molecular ion intensity (i.e., peak areas) of the REF standard to that of the ISTD (i.e., MIAREF/MIAISTD) on the abscissa, and the least-squares linear models and R2 values of these data were generated in Excel, as previously described (12). All lipid levels are reported in mol%, i.e., the fraction of moles of one lipid component in the total number of moles detected in the sample.
Fluorescence anisotropy measurements.Fluorescence anisotropy measurements were performed as previously described (36) with modifications. Yeast cells were harvested from triplicate fermentations at 15°C, 25°C, and 35°C at approximately equivalent ethanol concentrations during early and late stationary phase. Cells were washed as described above, followed by the addition of 10 μl of 12 mM diphenylhexatriene (DPH) dissolved in tetrahydrofuran to the PBS-buffered cell suspension and incubated for 30 min at room temperature. Anisotropy measurements were carried out at 25°C using the L-format on a PerkinElmer LS 55 fluorescence spectrometer (PerkinElmer, Inc., Waltham, MA). Samples containing DPH were excited at 360 nm, and emission intensities at 440 nm were used to determine anisotropy values. Emission spectra of samples were examined from 420 to 460 nm to ensure that instrument saturation from fluorescence signals was averted. Band passes of 3 nm and 5 nm were used on the excitation and emission monochromators, respectively, with a scanning speed of 180 nm/min and an integration time of 4 s. Each anisotropy value reported is the average of two measurements of samples examined in triplicate (six measurements total). The instrumental G factor was determined for each sample to account for polarization bias in the monochromators.
Data analysis methods.Fermentation kinetic data were entered into Microsoft Excel 2010 (Microsoft Corp., Bellevue, WA) for statistical analysis using the Analysis ToolPak, and SigmaPlot version 12.0 (Systat Software, Inc., San Jose, CA) was used to plot Brix and the cell culture OD600 at 24-h intervals until the fermentation achieved dryness (°Brix ≤ −1.0) or the °Brix remained constant for three consecutive measurements, at which point sampling was ceased. Unless otherwise noted, Student t tests were carried out as two-sample, two-tailed tests assuming unequal variances, and analysis of variance (ANOVA) was performed as single-factor ANOVA.
Ion trap MS (MSn and m/z profiles) and chromatography data were exported in mzXML format and imported into MZmine version 2.10 (37) for MS feature extraction and processing. Phospholipid identification was performed using a chromatographic and MSn database that was constructed using elution times, m/z values, and expected fragmentation patterns and/or characteristic losses following MS2 and MS3 determined from ISTD and REF standards, as well as values that had been previously reported (38). Triple-quadrupole MS and chromatography data were processed using MassHunter software to integrate selected m/z ratio peak areas, including ISTDs, and the processed MS data were exported to Excel for quantitative analysis. Sterol quantitation was performed based upon the m/z values ascertained using cholesterol, squalene, lanosterol, and ergosterol standards with flow injection analysis on an APCI-MS (12). Lipid composition data were entered into Excel for statistical analysis and SigmaPlot version 12.0 to generate plots of the lipid data.
Principal component analysis (PCA) of lipid data.The quantitative lipid data (X-block) were imported into MATLAB (version 7.11.0; MathWorks, Natick, MA) for PCA using the PLS toolbox (version 4.0; Eigenvector Research, Inc., Wenatchee, WA). Principal component analysis was performed using a singular value decomposition (SVD) algorithm, and preprocessing of the X-block consisted of autoscaling followed by mean centering. Scores and loadings plots were generated at the 95% confidence interval.
RESULTS
Fermentation kinetics and membrane sampling points.The three S. cerevisiae strains used in this study were chosen based on our previous experience with these strains. In previous work, our isolate of Prise de Mousse had relatively weak fermentation characteristics, exhibited as weak growth and low ethanol tolerance, compared to that of Sake A18 and Enoferm T306 (12). Fermentations were carried out in triplicate in the same defined medium at three temperatures, 15°C, 25°C, and 35°C, and the lipid composition and membrane fluidity (fluorescence anisotropy) of these three strains were measured at two points during fermentation, as outlined in Table 2. These points corresponded to two distinct stages during fermentation, at approximately 15 °Brix, early stationary phase, when the ethanol concentration was relatively low, and between 6.1 and 11.3 °Brix, which corresponded to late stationary phase, when ethanol was approaching its highest levels, but not before cessation of fermentation. Prise de Mousse fermentations failed to achieve Brix levels below 15 at 15°C and 35°C; therefore, lipid profiles for this strain were limited to ethanol concentrations of between 3 and 4 weight percent (Table 2). Fermentations utilizing Sake A18 and Enoferm T306 were measured between 3 and 4 weight percent of ethanol early in fermentation and between 4.5% and 7.5% ethanol (wt/wt) during late stationary phase.
Fermentation characteristics of S. cerevisiae strains when lipid composition and fluorescence anisotropy were measured
Of the three yeast strains used in this study, Prise de Mousse demonstrated the poorest fermentation characteristics, experiencing stuck fermentations at all three temperatures (Fig. 1). Fermentations carried out at 15°C and 35°C became stuck at approximately 15 °Brix (∼150 g/liter glucose and fructose), while the 25°C fermentation stuck around 6 °Brix. This strain achieved similar biomass (OD600) levels relative to the other strains fermenting at 15°C and 25°C (Fig. 1A and B); however, Prise de Mousse had significantly less biomass fermenting at 35°C (Fig. 1C). Sake A18 fermentations at 15°C and 35°C became stuck around 10 °Brix and 6 °Brix, respectively (Fig. 1). However, Sake A18 did reach the lowest Brix levels of the three yeast strains fermenting at 35°C. Fermenting at 25°C, this strain became stuck at approximately 2 °Brix (Fig. 1B). Enoferm T306 fermented to dryness at 25°C (approximately −1.0 °Brix), while finishing at approximately 9 °Brix at 35°C. It appears that this strain fermenting at 15°C became sluggish, and the fermentation was stopped at approximately 6 °Brix due to the volume of the fermentation becoming too low to allow for proper sampling (Fig. 1C). Enoferm T306 achieved the lowest Brix levels (highest ethanol concentration) of all strains fermenting at 15°C and 25°C (Fig. 1).
Fermentation kinetics for the three yeast strains used in this study at 15°C (A), 25°C (B), and 35°C (C). In each, the top plot represents biomass accumulation (optical density at 600 nm) versus time and the bottom plot depicts sugar concentration (measured in °Brix) versus time.
Lipid profiles of strains fermenting at different temperatures.The method used in this study quantitatively analyzed 61 phospholipids and sterols in yeast to determine lipid compositional changes that occur in this organism to adapt to different fermentation temperatures (Fig. 2, 3, 4). The dynamic range of lipid concentrations ranged from less than 0.5 mol% to over 25 mol%. In all of the strains used in this study, elevated concentrations of phosphatidylethanolamine were observed during both early- and late-stationary-phase metabolism in fermentations at 15°C, while the relative levels of this lipid species were lowest in fermentations at 35°C (Fig. 2, 3, and 4). Conversely, phosphatidylinositol concentrations were extremely elevated in these three strains when fermenting at 35°C, particularly in the low-abundance species, PI 18:0-18:0, PI 18:1-18:1, and PI 34:2 (Fig. 2A, 3A, and 4A). Concentrations of phosphatidylinositol continued to increase into late stationary phase (Fig. 2B, 3B, and 4B). However, Prise de Mousse exhibited the highest phosphatidylinositol concentrations of these strains in early stationary phase (Fig. 2A, 3A, and 4A).
Fermentation lipid profiles for Prise de Mousse at early-stationary-phase (A) and late-stationary-phase (B) metabolism. The upper and lower panes represent high- and low-abundance lipids, respectively.
Fermentation lipid profiles for Sake A18 at early-stationary-phase (A) and late-stationary-phase (B) metabolism. The upper and lower panes represent high- and low-abundance lipids, respectively.
Fermentation lipid profiles for Enoferm T306 at early-stationary-phase (A) and late-stationary-phase (B) metabolism. The upper and lower panes represent high- and low-abundance lipids, respectively.
Overall, phosphatidylcholine concentrations decreased as the fermentations progressed in stationary-phase metabolism (Fig. 2, 3, and 4). The concentrations of phosphatidylcholine were lowest in Prise de Mousse at all fermentation temperatures and sampling points. However, like Sake A18 and Enoferm T306, PC 34:1 was the most abundant PC regardless of temperature (Fig. 2, 3, 4). The concentrations of PC 18:1-18:1 and PC 16:0-18:0 were highest in ferments at 25°C and 35°C, while PC 16:1-18:1 and PC 16:0-16:1 were most concentrated in the 15°C fermentations. Interestingly, PC 16:1-16:1 were extremely elevated in fermentations at 15°C. Quantities of phosphatidylserine were relatively low during fermentation, regardless of temperature, with the exception of PS 16:0-18:1, which was extremely elevated in Prise de Mousse at 15°C (Fig. 2A).
Concentrations of ergosterol were highest in Prise de Mousse and Sake A18 fermenting at 15°C, in both early and late stationary phase (Fig. 2, 3, and 4). The concentration of squalene was elevated in these strains at 25°C and 35°C (Fig. 2A and 3A). For Prise de Mousse fermenting at 25°C, ergosterol concentrations increased from ∼18 mol% to over 25 mol%, while quantities of lanosterol and squalene did not significantly change (Fig. 2A and B). Ergosterol concentrations in Sake A18 fermenting at 15°C increased from ∼16 mol% to ∼24 mol%, and ferments at 35°C also had a slight but significant increase in late stationary phase (Fig. 3A and B).
Interestingly, concentrations of the ergosterol precursors, lanosterol and squalene, demonstrated a temperature-dependent increase at both early- and late-stationary-phase metabolism in this strain. Enoferm T306 had the lowest ergosterol concentrations and did not experience significant changes in its concentration at 15°C and 25°C but did in ferments at 35°C (Fig. 4A and B). At 25°C, the ergosterol precursor lanosterol increased, while squalene decreased as stationary phase progressed.
Correlations of specific lipids to yeast strains using PCA.Principal component analysis was performed on lipid data sets from early- and late-stationary-phase metabolism as a means to objectively identify the relationships that can be observed in Fig. 2 to 4 between specific lipids and fermentation temperature with individual yeast strains. In the early-stationary-phase lipid composition data, PCA yielded two principal components that captured 79.51% of the variation in the lipid composition of the yeast strains (Fig. 5A). In the first PC, representing 56.90% of the variation in the lipid composition data, score plots indicate that the strains fermenting at 35°C were anticorrelated to those at 15°C. The loading plot indicated that PI 36:1, PI 34:1, PS 16:1-18:1, and squalene were the most closely associated with the 35°C fermentations, with Prise de Mousse exhibiting the strongest association with these lipids (Fig. 5B). Conversely, a number of PC and PE lipids, as well as PS 16:0-18:1 and ergosterol, were more strongly associated with the 15°C ferments. The second PC, capturing 22.61% of the variation in the lipid data, indicates that the PC and PE lipids were associated with Enoferm T306 and Sake A18 in the 15°C fermentations. Ergosterol and PS 16:0-18:1 were very strongly correlated with Prise de Mousse (Fig. 5B).
Correlations of specific lipids with yeast strains during early stationary phase indicated by score plot (A) and loading plot (B). Strain designations: PdM, Prise de Mousse; Sake, Sake A18; EnfT306, Enoferm T306. Fermentation stage designation: ESP, early stationary phase; LSP, late stationary phase. The fermentation temperature is indicated between the strain and fermentation stage.
In the late-stationary-phase lipid composition data, PCA yielded two principal components that captured 88.02% of the variation in the lipid composition of the yeast strains (Fig. 6A). Principal component analysis of late-stationary-phase lipid data again indicated that in the first PC of the score plot, representing 53.51% of the variation in the data, Sake A18 and Enoferm T306 ferments at 15°C and 25°C were anticorrelated with their counterparts fermenting at 35°C. Recall that Prise de Mousse became stuck very early in fermentation and is not present in this analysis. The Sake A18 and Enoferm T306 fermentations at 35°C were very strongly associated with that of PI 36:1 and PI 34:1 in the first PC of the loadings plot (Fig. 6B). These strains fermenting at lower temperatures were also associated with many of the same PE and PC lipids that they were associated with earlier in fermentation. In the second PC, which captured 34.51% of the data, Prise de Mousse fermenting at 25°C was correlated with ergosterol, squalene, lanosterol, PS 16:0-18:1, and PS 16:1-18:1. This is in contrast to Sake A18 and Enoferm T306 fermenting at both 15°C and 25°C, which were associated with a number of PC and PE phospholipid species, including several membrane structural lipids, such as PC 16:1-16:1, PC 16:1-18:1, and PC 18:1-18:1 (Fig. 6B).
Correlations of specific lipids with yeast strains during late stationary phase indicated by scores plot (A) and loading plot (B). Strain designations: PdM, Prise de Mousse; Sake, Sake A18; EnfT306, Enoferm T306. Fermentation stage designation: ESP, early stationary phase; LSP, late stationary phase. The fermentation temperature is indicated between the strain and fermentation stage.
Measuring yeast membrane fluidity during fermentation using fluorescence anisotropy.Fluorescence anisotropy measurements were performed at the same early- and late-stationary-phase time points used for LC-MS analysis. Fluorescence anisotropy is an optical technique that measures the mobility of DPH in the membrane microenvironment (39). Low anisotropy values are associated with fluid membranes, whereas higher anisotropy levels indicate gel-like membranes. In the fermentations performed at 15°C, the anisotropy values for Sake A18 and Enoferm T306 decreased over the course of stationary-phase metabolism (Fig. 7A). The fluorescence anisotropy value of Prise de Mousse during early stationary phase at 15°C was not significantly lower than the other strains at this same fermentation point and temperature (P > 0.05). At 25°C, the anisotropy of Enoferm T306 did not significantly change (P < 0.05), while Prise de Mousse and Sake A18 both experienced decreased anisotropy (increased fluidity) as fermentation progressed (Fig. 7B). During early stationary phase in fermentation at 35°C, Enoferm T306 and Prise de Mousse had similar anisotropy values, while those of Sake A18 were significantly higher (Fig. 7C). Later in these fermentations, the anisotropy values of Sake A18 remained unchanged and Enoferm T306 experienced a significant increase in anisotropy (P < 0.05).
Fluorescent anisotropy values at different fermentation temperatures of 15°C (A), 25°C (B), and 35°C (C). Values were measured at both early and late stationary phase for all strains except Prise de Mouse, which became stuck early when fermenting at 15°C and 35°C.
DISCUSSION
The results presented here are in agreement with the observations of others that fermentation temperature can have a profound effect on yeast cell growth and its ability to convert sugar to ethanol in a strain-dependent manner. The yeast strains used in this study were chosen due to their diverse fermentation characteristics. Prise de Mousse experienced stuck fermentations at both temperature extremes (15°C and 35°C), while exhibiting significantly different maximum OD600 levels. This indicates that the mechanisms for fermentation arrest at 15°C are likely different than those at 35°C. Regardless, Prise de Mousse was extremely temperature sensitive. Both Sake A18 and Enoferm T306 had significantly lower maximum OD600 levels at 35°C than at 15°C or 25°C, and both strains experienced stuck fermentations at 35°C. During the low-temperature fermentation, Enoferm T306 appeared to experience a sluggish fermentation, whereas Sake A18 became stuck. Therefore, these strains represent a spectrum of fermentation capabilities under the growth and temperature conditions used in this study.
Fermentation temperature and rising ethanol concentrations are known factors that lead to fermentation arrest (1, 2, 8, 9). The cell biomembrane is a susceptible target to both of these stress factors, and this has been demonstrated using both model lipid bilayers (20, 22–24, 40) and at the whole-cell level (8, 9, 25). It has previously been demonstrated that at lower fermentation temperatures, yeast viability was enhanced by higher levels of shorter-chain (C14 to C16), unsaturated fatty acids in yeast cell membranes (5, 41). However, these previous studies did not measure which specific glycerophospholipid classes were most affected by these fatty acid compositional changes. The results presented here represent the first evidence of changes in individual phospholipid composition of multiple S. cerevisiae strains with temperature.
The lipid profile data and PCA indicate that different fermentation temperatures elicited changes in the membrane concentration of several glycerophospholipids during early and late stationary phase. The phosphatidylinositol concentrations were greatest in the 35°C fermentations that reached the lowest biomass levels of all three fermentation temperatures and the lowest ethanol production. In particular, two phosphatidylinositol species (PI 36:1 and PI 34:1) were highly correlated with elevated fermentation temperatures. We previously reported that higher phosphatidylinositol levels in yeast early in fermentation were associated with lower biomass and stuck fermentations (12). Elevated phosphatidylinositol levels in S. cerevisiae are generally observed at the end of the exponential growth phase, when nitrogen and other nutrients have been depleted and the yeasts transition into stationary-phase metabolism (42). Analysis comparing the temperature-induced stress response to the ethanol-induced stress response in S. cerevisiae indicates that there is significant genetic and functional overlap in these responses, particularly the expression of heat shock proteins known to stabilize membrane-associated proteins (7), though other explanations may be possible. The observation of high levels of phosphatidylinsitol early in fermentation, which is associated with low biomass levels and ethanol production, may be an early indicator of problem fermentations (13).
Phosphatidylethanolamine is a non-bilayer-forming lipid that, due to its smaller headgroup than that of phosphatidylcholine, induces curvature in membranes by partitioning in the inner leaflet of the bilayer of vesicles and other highly curved membranes (43). Both the lipid profiles and PCA demonstrated that the yeast strains analyzed in this study exhibited higher concentrations of phosphatidylethanolamine in their membranes when fermenting at 15°C. Furthermore, these data also indicate that at lower temperatures, medium-chain phosphatidylcholines (e.g., PC 16:1-16:1) were at higher concentrations than the long-chain phosphatidylcholines (e.g., PC 18:1-18:0). Taken together, this may indicate that the yeast cell membrane is in a more gel-like state (i.e., tighter acyl chain packing) at lower fermentation temperatures, necessitating a lipid composition that maintains proper cell membrane mechanics (25, 28, 44). Ultimately, however, it is unclear from these data what necessitated higher concentrations of phosphatidylethanolamine in yeast strains fermenting at lower temperatures.
Ergosterol is an essential component of yeast cellular membranes that must be present to initiate yeast cell growth (45, 46) and for proper function of H+-ATPases and other membrane-associated proteins (47). Indeed, this sterol has been deemed such an integral component of yeast's fermentation ability that it has been referred to as a “survival factor” (2, 46). For the Prise de Mousse and Sake A18 strains utilized in this study, membrane concentrations of ergosterol were highest in the 15°C fermentations, while they were lowest in Enoferm T306 at this temperature. Indeed, ergosterol concentrations in Prise de Mousse and Sake A18 were over two times that observed in Enoferm T306 at 15°C. Enoferm T306 experienced the lowest ergosterol levels observed at any of the three fermentation temperatures used in this study. A number of studies have concluded that higher ergosterol concentrations do not correlate with ethanol tolerance and fermentation completion (12, 48, 49), and the results presented here are in agreement with these observations.
Fluorescence anisotropy (FA) is a commonly utilized optical method used to measure membrane dynamics in lipid bilayers using the lipophilic dye molecule, diphenylhexatriene (50). Alexandre et al. (51), and more recently Huffer et al. (36), utilized FA in Saccharomyces cerevisiae and other microorganisms to determine how membrane fluidity was influenced by ethanol exposure. They reported that the membrane fluidity of S. cerevisiae remained relatively constant compared to that of other less alcohol-tolerant microorganisms when exposed to ethanol. Furthermore, Huffer et al. (36) reported that growth rate and membrane fluidity in microorganisms with the greatest ethanol tolerance maintained their membrane fluidity upon exposure to ethanol, even at ethanol concentrations that significantly reduced cell growth. The fluorescence anisotropy data for the strains analyzed in this study also did not indicate dramatic changes. Furthermore, fermentation temperature, which is known to modulate the membrane-perturbing effect of ethanol (21), did not appear to significantly affect the FA of these yeast strains. Unfortunately, data for Prise de Mousse is incomplete, and further work is necessary to determine if the changes in lipid composition observed at different fermentation temperatures are an attempt to maintain membrane fluidity.
The three yeast strains examined in this study were chosen based upon their diverse fermentation kinetics to determine how their ability to grow and convert sugar to ethanol would be affected by fermentation temperature and to determine if their lipid composition changes to adapt to different temperatures. The results of these analyses indicate that Saccharomyces cerevisiae experiences significant changes in its lipidome in a temperature-dependent manner. Utilizing mass spectrometry-based methodologies, we obtained structural data that allowed us to determine that phosphatidylcholines with shorter-chain, unsaturated fatty acids were at significantly higher concentrations in yeast fermenting at 15°C. Principal component analysis confirmed these finding, as well as the relationship between elevated phosphatidylethanolamine levels in cooler fermentations. Furthermore, these data confirmed results that elevated membrane concentrations of phosphatidylinositol early in fermentation are associated with low biomass and stuck fermentations (12). Additionally, higher membrane levels of ergosterol were not associated with fermentation completion or thermotolerance in these yeast strains. Fluorescence anisotropy measurements of the yeast cell membrane during fermentation indicated that these strains did not experience dramatic changes in membrane fluidity, even at different fermentation temperatures. These data demonstrate the dynamic nature of the yeast cellular membrane, as well as underscore the need for further investigation into the physiological and environmental factors that influence lipid composition in Saccharomyces cerevisiae.
ACKNOWLEDGMENTS
This project was supported by National Research Initiative grant 2007-35504-18332 from the U.S. Department of Agriculture-Cooperative State Research, Education, and Extension Service (USDA-CSREES), the American Vineyard Foundation, the California Competitive Grant Program for Research in Viticulture and Enology, and the Ernest Gallo Endowed Chair in Viticulture and Enology.
We are grateful to Anita Oberholster, Atul Parikh, and Sue Ebeler for use of their experimental facilities.
FOOTNOTES
- Received 9 April 2013.
- Accepted 24 June 2013.
- Accepted manuscript posted online 28 June 2013.
- Copyright © 2013, American Society for Microbiology. All Rights Reserved.
