Comparative metabolic footprinting of a large number of commercial wine yeast strains in Chardonnay fermentations

Authors


Correspondence: Chandra L. Richter, E. & J. Gallo Winery, P.O. Box 1130, Modesto, CA 95353, USA. Tel.: 209 341 8429; fax: 209 341 4541; e-mail: chandra.richter@ejgallo.com

Abstract

Wine has been made for thousands of years. In modern times, as the importance of yeast as an ingredient in winemaking became better appreciated, companies worldwide have collected and marketed specific yeast strains for enhancing positive and minimizing negative attributes in wine. It is generally believed that each yeast strain contributes uniquely to fermentation performance and wine style because of its genetic background; however, the impact of metabolic diversity among wine yeasts on aroma compound production has not been extensively studied. We characterized the metabolic footprints of 69 different commercial wine yeast strains in triplicate fermentations of identical Chardonnay juice, by measuring 29 primary and secondary metabolites; we additionally measured seven attributes of fermentation performance of these strains. We identified up to 1000-fold differences between strains for some of the metabolites and observed large differences in fermentation performance, suggesting significant metabolic diversity. These differences represent potential selective markers for the strains that may be important to the wine industry. Analysis of these metabolic traits further builds on the known genomic diversity of these strains and provides new insights into their genetic and metabolic relatedness.

Introduction

Evidence for the production of fermented beverages by Saccharomyces yeasts, mostly S. cerevisiae, dates at least as far back as 7000 bc, making it one of the world's oldest biotechnological processes (Cavalieri et al., 2003; McGovern, 2003; McGovern et al., 2004). Fermentation technologies, and their associated yeasts, have comigrated with humans and agriculture to spread throughout the world. Saccharomyces yeasts, originally present as wild strains in soil, fruits, or tree sap, were transported by humans, along with their associated sugar source, that is, grape or grain. This likely imposed a selective pressure on these domesticated yeasts to functionally specialize for the production of specific alcoholic beverages on differing substrates and/or the production of particular desirable flavor compounds. There is now a sizable collection of domesticated strains of S. cerevisiae associated with, and specialized for, specific industrial habitats, and strains isolated from a given habitat are often genetically similar to each other (Legras et al., 2005, 2007; Camarasa et al., 2011; Sicard & Legras, 2011).

Grape juice (or must), which has a low pH (2.9–3.8) and high osmolarity (sugars of 200–300 g L−1), is an environment that is highly unfavorable for the survival of most microorganisms, but to which wine yeasts are well adapted. In addition, SO2 is routinely added at the winery to concentrations of 40–100 mg L−1 to prevent oxidation and microbial spoilage, also contributing to the harsh conditions (Pizarro et al., 2007). The onset of fermentation makes the condition even less hospitable for most organisms: the environment becomes anaerobic, ethanol concentrations begin to rise, and nutrients are depleted. However, due to being well adapted to this changing environment, S. cerevisiae rapidly becomes the dominant species in the fermentation.

Evolutionary adaptation of the stress response to enable S. cerevisiae to better survive in the harsh environment of fermenting grape juice is not well understood, but has likely resulted in the divergence of S. cerevisiae wine strains from nonwine S. cerevisiae strains (Legras et al., 2007; Dunn et al., 2012). Because winemaking has been practiced for several millennia (Frankel, 1998; Cavalieri et al., 2003; McGovern, 2003), there has been ample opportunity for divergence of wine yeast strains from other industrial and nonindustrial S. cerevisiae strains. Indeed, large-scale genomic studies have shown this divergence from other industrial, laboratory, and pathogenic strains of S. cerevisiae (Legras et al., 2007; Borneman et al., 2011; Dunn et al., 2012). It is likely these genomic differences allow wine yeast to maintain metabolic activity throughout the entire winemaking process. To date, there have been few studies examining the fermentation performance and metabolic diversity within wine yeast during wine fermentation.

Starting approximately 50–60 years ago, wine yeast strains began to be isolated from various geographical regions and sold commercially. Today, most commercial wine fermentations are inoculated with a pure culture of a single commercial yeast strain rather than allowing a natural or wild (noninoculated) fermentation to occur. These strains, likely derived from strains domesticated hundreds or thousands of years earlier, were isolated from fermentations with certain desirable sensory traits (metabolic footprint) and/or predictable behavior (fermentation kinetics) (Pretorius, 2000). Currently, there are more than 200 commercially produced wine yeast strains available to winemakers. Domestication, commercialization, and subsequent dissemination by humans have likely influenced the genetic structure of wine yeast strains (Pretorius, 2000; Dunn et al., 2005, 2012; Legras et al., 2007).

Genomic differences can lead to phenotypic differences between strains, resulting in unique fermentation behavior and/or sensory characteristics [reviewed in (Pretorius et al., 2012)]. The sensory perception of wine is due to a specific metabolite profile, that is, the small molecules that contribute to the aroma, flavor, and mouthfeel. Grape juice is composed primarily of water, sugar, organic acids, and minor components, which can be used as a source of nutrients for yeast during fermentation. The wine resulting from fermentation of juice is composed primarily of water, ethanol, glycerol, organic acids, and many minor compounds contributing to flavor, aroma, and mouthfeel (Pizarro et al., 2007). These flavor, aroma, and mouthfeel compounds either are present in the grape and remain unmodified during fermentation or are synthesized during the winemaking process by yeast, typically from precursors in the grape juice.

The effect of a specific yeast strain's cellular metabolism on its environment results in a unique set and concentration of metabolites; the concentration of many of these metabolites can be measured, giving a ‘metabolic footprint’ or ‘metabolite profile’. For a given starting juice, this footprint can change due to the yeast strain used or the environmental conditions of the fermentation. Previous studies have shown that different S. cerevisiae wine yeast strains fermenting the same juice under identical conditions can yield very different wines due to differences in the metabolic footprint (Gardner et al., 1993; Romano et al., 1994, 2003; Benitez et al., 1996; Mortimer, 2000; Remize et al., 2000; Torrea & Ancin, 2002; Fleet, 2003). However, most of these studies compared only a small number of yeast (< 10) and/or a small number of metabolites (< 20), often in synthetic must or media, thereby not giving a true representation of a grape fermentation.

In this study, we determined, in triplicate, the fermentation profiles and metabolic footprints (a total of 29 attributes) of 69 commercial wine yeast strains in Chardonnay grape juice under identical fermentation conditions. This allowed us to better understand the phenotypic relatedness of these strains, compared with the genomic relatedness we previously reported (Dunn et al., 2012). We observed variation in both the fermentation kinetics and metabolic footprints for these strains. We found groups of strains with related phenotypes (both fermentation and metabolite attributes) and were able to correlate these similarities to genomic similarities, providing insight into the diversity of this unique class of industrial strains.

Materials and methods

Yeast strains

The S. cerevisiae wine yeast strains used in this study, listed in Table 1, were obtained from commercial yeast suppliers. Single colonies for each were isolated and then preserved at −80 °C for reuse, to ensure pure cultures were used for each strain and that the same single colony isolate was used for all inoculations.

Table 1. Origins and sources of Saccharomyces cerevisiae wine strains studied. Origins were obtained from yeast supplier's catalog. A single-colony isolate was collected from each commercial yeast preparation and preserved at −80 °C for reuse; the same single-colony isolate was used to inoculate all fermentations
Yeast strainCatalog nameSupplierOrigin
43Uvaferm 43LallemandInter Rhone
228228AnchorInter Rhone
4F9Fermicru 4F9DSMNantes, France
58W358W3VinquiryAlsace, France
71BLalvin 71BLallemandNarbonne, France
AWRI 350AWRI 350MaurivinAWRI
AWRI 796AWRI 796MaurivinSouth Africa
AWRI R2Maurivin R2MaurivinBordeaux, France
BA11BA11LallemandEstacao Vitivinicola de Baraida
BDXEnoferm BDXLallemandPastuer Institute, Paris, France
BGYBurgundyLallemandBurgundy, France
BM45Lalvin BM45 BrunelloLallemandUniversity of Siena
BP 725BP 725MaurivinFrance
BRL97BRL97 BaroloLallemandUniversity of Torino
CSMCSMLallemandITV Bordeaux
CY3079Lalvin CY3079LallemandBourgogne
D254Lalvin ICV-D254LallemandICV, Rhone Valley
D47Enoferm IVC-D47LallemandCotes du Rhone
D80Lalvin ICV-D80LallemandCote Rotie, Rhone Valley
DV10DV10LallemandChampagne
EC1118Lalvin EC-1118LallemandChampagne
EleganceMaurivin EleganceMaurivinPortugal
EpernayIIMaurivin EP 2MaurivinEpernay, France
F15Zymaflore F15LaffortMedoc
F33Actiflore C (F33)Scott Labs/LaffortLaffort Research Laboratory
FA1FA1Scott Labs/Lallemand 
FermichampFermichampDSMAlsace, France
ICV-GRELalvin ICV-GRELallemandRhone Valley
IOC 18-2007IOC 18-2007Epernay 
K1Lalvin V1116LallemandMontpellier
L2056Rhone L2056LallemandCotes du Rhone
L2226Enoferm L2226LallemandCotes du Rhone
L2323Lalvin L2323LallemandRhone Valley
Lalvin ACLalvin ACLallemandLoire
LVCBFermicru LVCBDSMCasablanca Valley, Chile
N96N96AnchorSouth Africa
NT112NT 112AnchorStellenbosch, South Africa
NT116NT 116AnchorStellenbosch, South Africa
NT202NT 202AnchorStellenbosch, South Africa
NT45NT 45AnchorSouth Africa
NT50NT 50AnchorStellenbosch, South Africa
PCPremier CuveeLesaffreFrance
PDMMaurivin PDMMaurivin 
PrimeurMaurivin PrimeurMaurivinINRA Narbonne, France
QA23Enoferm QA23LallemandUTAD in Portugal
R2R2LallemandSauternes region of Bordeaux
RC212Lalvin RC212LallemandBurgundy
Rhone 4600Rhone 4600LallemandCotes du Rhone
S-101St. Georges S-101Lesaffre/SpringerBeaujolais
S-102C.K. S-102Lesaffre/SpringerVal de Loire
S-325U.C.L.M. S-325Lesaffre/SpringerSpain
S-377U.C.L.M. S-377Lesaffre/SpringerSpain
SAUV L3Sauvignon L3MaurivinBordeaux, France
Simi WhiteSimi WhiteLallemand 
SYRSyrahLallemandCotes du Rhone
T306T306LallemandHunter Valley, NSW Australia
T73T73LallemandLa Universidad de Velencia of Spain
UCD522-LLallemand UCD 522LallemandPastuer Institute, Paris, France
UCD522-MMauri UCD522MaurivinUC Davis
VIN13VIN 13AnchorStellenbosch, South Africa
VIN7VIN 7AnchorStellenbosch, South Africa
VL1Zymaflore VL1Scott Labs/LaffortBordeaux Institute of Oenology
VL2Zymaflore VL2Scott Labs/Laffort 
VL3CZymaflore VL3Scott Labs/LaffortBordeaux Institute of Oenology
VR5Fermicru VR5DSMBurgundy, France
W372WE 372AnchorStellenbosch, South Africa
WE14WE 14AnchorSouth Africa
Williams SelyemWilliams SelyemVinquirySonoma, CA
X5Zymaflore X5Scott Labs/LaffortLaffort Research Laboratory

Preparation of juice

Chardonnay juice (at 24 Brix or 240 g L−1 glucose and fructose) was collected during harvest 2007 and stored frozen in 60-L drums until use. Each 60-L drum was thawed for 4 days at 4 °C. After thawing, the juice was pad-filtered (2 μm; 3M Purification, Inc, Tustin CA) and sterile-filtered (0.2 μm; Pall Corp, Covina, CA). The filtered juice was added to sterile spinner flasks (Bellco, Vineland, NJ) in 1.5 L volumes.

Preparation of fermentation starter cultures

A small amount of the single-colony isolate of each commercial yeast strain was transferred from its frozen stock to an YPD plate (1% Bacto yeast extract, 2% Bacto peptone, 2% glucose, 1.5% agar) and incubated for 2 days at 28 °C. One colony from the plate was used to inoculate 5 mL YPD broth, for a total of three 5-mL cultures, and incubated for 2 days at 28 °C. After 2 days, the 5-mL cultures were used to inoculate three different flasks each with 100 mL YPD broth and incubated for 2 days at 28 °C. The cells were harvested by centrifugation, washed in 200 mL sterile 0.9% sodium chloride solution, and resuspended in 25 mL of 0.9% sodium chloride. Cell concentration of the yeast slurry was obtained using a Z2 Coulter Particle Counter (Beckman Coulter, Hialeah, FL), and the juice was inoculated with the amount of yeast slurry to give an initial concentration of 5 × 106 cells mL−1.

Fermentation protocol

Fermentations were performed in spinner flasks in 1.5 L volumes at 18 °C with a constant agitation of 25 r.p.m. Samples were collected daily to measure cell concentration (Z2 Coulter Particle Counter) and four chemical attributes: sugar (glucose and fructose UV Method, Randox, Antrim, UK), ethanol, and two measurements of yeast assimilable nitrogen (YAN): NOPA (nitrogen by OPA) and ammonia (Ammonia Enzymatic UV, Randox, Antrim, UK).

Analytical methods

Upon completion of fermentation, the wines were cold-settled (48 h at 1.7 °C/35 °F) and analyzed for standard wine chemical attributes, including the following: residual sugar, ethanol (GC column 80/120 Carbopack B AW 5% Carbowax 20M, Supelco, Bellefonte, PA), pH, malic acid (L-Malic Acid, Megazyme, County Wicklow, Ireland), volatile acidity (Bergmeyer & Mollering, 1974), glycerol (Glycerol Assay, Megazyme, County Wicklow, Ireland), total SO2 (FIAstar 5000, Foss, Hoganas, Sweden), and free SO2 (FIAstar 5000, Foss, Hoganas, Sweden). The finished wines were analyzed for yeast-produced metabolites, including the following: fusel alcohols, acetaldehyde, and ethyl acetate (A.O.A.C., 1997). To measure additional aroma compounds, samples were analyzed using 6890 Series Gas Chromatograph with a J&W Scientific DB-5MS High-Resolution Gas Chromatography Column [adapted from (Soleas et al., 2002)].

Screening for polygalacturonase activity

Select strains were screened for polygalacturonase activity using a plate assay described by Louw et al. (Louw et al., 2010). Vin7, Epernay II, NT45, and NT50, grown overnight in YPD (1% yeast extract, 2% peptone and 2% dextrose), were spotted on polygalacturonase plates (1.25% polygalacturonic acid (Sigma-Aldrich), 0.67% yeast extract, 1% dextrose, 20% agar) and incubated at 28 °C for 3 days. Degradation was visualized by washing colonies off with distilled water and staining plates with 6M HCl.

Statistical analysis

Statistical analyses, including principal component analysis (PCA) and agglomerative hierarchical clustering, were performed using xlstat software, version 2012.4.03 (Addinsoft, New York, NY) and The unscrambler, version 10.1 (Camo Software, Woodbridge, NJ).

Results

To investigate phenotypic differences among 69 commercial wine yeast strains, we characterized their fermentation performance and metabolic footprints in filter-sterilized Chardonnay juice (Table 1). The fermentations were conducted using standard winemaking practices in triplicate for each strain, using a single juice source to minimize variation based on juice chemistry. We measured fermentation kinetics as well as concentrations of metabolic byproducts and volatile organoleptic compounds that are either produced or volatilized by the yeast.

Fermentation kinetics

We observed significant variation in the fermentation kinetics between the different strains. The length of fermentation, measured from the time of yeast inoculation to the point at which all fermentable sugars were consumed, varied from 5 to 18 days (Fig. 1, Table 2). The length of time required to ferment 50% of the available sugar (120 g L−1) was similar among all strains tested, ranging from 2.3 to 4.2 days (Fig. 1, Table 2). However, the range became larger during the next stage of fermentation, with the consumption of 83% of the sugars (40 g L−1) ranging from 4.0 to 11.3 days (Fig. 1, Table 2). Variability among the strains was the most pronounced during consumption of the final 17% of sugars (dryness, < 2 g L−1).

Table 2. Fermentation kinetics for 69 wine yeast strains. Fermentations were carried out in triplicate in Chardonnay grape juice containing 230–250 g L−1 sugar, 390–420 mg L−1 YAN, 5.6–5.8 g L−1 TA, and 27–31 mg L−1 SO2. Fermentation rate is displayed as time to consume sugar: Time to 120 g L−1 sugar is the time (in days) for each strain to consume 50% of the available sugar, Time to 40 g L−1 sugar is the time for each strain to consume 83%, and Time to Dryness is the time for each strain to consume all available sugar (where dryness is < 2.0 g L−1 sugar). Maximum cell concentration was the maximum concentration observed through daily tracking. Percentage of YAN and malic acid consumed during fermentation is also shown
YeastDays to 120 g L−1 sugarDays to 40 g L−1 sugarDays to drynessMax cell density wine × 106 cells mL−1% YAN consumed% Malic consumedAlcohol%v/v
433.14 ± 0.024.64 ± 0.076.17 ± 0.26138 ± 380.5 ± 0.136.7 ± 0.514.51 ± 0.03
2283.47 ± 0.187.57 ± 0.5411.37 ± 0.69153 ± 1085.7 ± 3.718.7 ± 2.014.42 ± 0.09
4F92.64 ± 0.074.87 ± 0.407.85 ± 0.90138 ± 1881.2 ± 2.620.0 ± 0.614.39 ± 0.05
58W32.67 ± 0.015.28 ± 0.1810.23 ± 0.32121 ± 576.7 ± 1.217.1 ± 0.714.58 ± 0.05
71B3.12 ± 0.105.24 ± 0.369.69 ± 0.44125 ± 564.6 ± 1.235.7 ± 0.814.64 ± 0.01
AWRI R22.72 ± 0.035.00 ± 0.148.84 ± 0.51142 ± 980.6 ± 2.622.9 ± 0.414.57 ± 0.00
AWRI3504.20 ± 0.148.25 ± 0.7518.65 ± 2.3456 ± 658.3 ± 1.09.6 ± 0.614.37 ± 0.05
AWRI7962.82 ± 0.015.68 ± 0.249.58 ± 0.48148 ± 277.5 ± 0.921.6 ± 0.714.39 ± 0.07
BA113.20 ± 0.058.13 ± 0.5714.12 ± 1.19133 ± 473.7 ± 2.328.2 ± 1.014.55 ± 0.07
BDX3.38 ± 0.025.66 ± 0.078.58 ± 0.14120 ± 1571.3 ± 2.326.4 ± 1.414.55 ± 0.04
BGY3.82 ± 0.068.45 ± 0.3012.55 ± 1.68118 ± 1074.6 ± 0.540.0 ± 1.214.55 ± 0.03
Bm452.68 ± 0.014.92 ± 0.039.73 ± 0.11110 ± 379.0 ± 0.520.7 ± 0.314.49 ± 0.02
BP7252.61 ± 0.054.51 ± 0.196.81 ± 0.75142 ± 379.0 ± 3.38.8 ± 1.314.61 ± 0.07
BRL973.30 ± 0.147.66 ± 1.7814.08 ± 3.81119 ± 972.8 ± 4.620.5 ± 1.714.39 ± 0.12
CSM2.88 ± 0.096.71 ± 0.5710.93 ± 1.30109 ± 1574.8 ± 3.130.1 ± 1.414.55 ± 0.07
CY30792.97 ± 0.026.37 ± 0.0510.29 ± 0.13136 ± 875.0 ± 0.717.0 ± 1.114.73 ± 0.03
D2542.77 ± 0.036.41 ± 0.1011.03 ± 0.2993 ± 877.1 ± 1.127.6 ± 1.114.63 ± 0.02
D472.82 ± 0.035.65 ± 0.499.98 ± 1.00141 ± 781.6 ± 0.133.4 ± 0.614.54 ± 0.03
D802.89 ± 0.036.71 ± 0.2511.62 ± 0.81119 ± 473.3 ± 1.513.5 ± 0.214.65 ± 0.03
DV102.98 ± 0.065.03 ± 0.289.02 ± 0.89138 ± 877.0 ± 2.014.8 ± 0.314.61 ± 0.06
EC11182.96 ± 0.114.44 ± 0.497.44 ± 1.40148 ± 1575.4 ± 6.321.2 ± 0.614.62 ± 0.07
Elegance2.87 ± 0.074.51 ± 0.437.58 ± 0.75138 ± 1477.8 ± 3.020.8 ± 0.814.54 ± 0.06
EPII3.38 ± 0.1211.29 ± 1.6617.46 ± 1.85105 ± 956.1 ± 2.633.3 ± 1.214.54 ± 0.16
F152.69 ± 0.025.47 ± 0.1710.86 ± 1.24107 ± 577.4 ± 2.025.7 ± 0.414.70 ± 0.05
F332.60 ± 0.044.45 ± 0.207.52 ± 1.13134 ± 1286.5 ± 3.514.6 ± 2.114.43 ± 0.07
FA12.80 ± 0.116.35 ± 1.3210.09 ± 1.43136 ± 881.7 ± 2.518.9 ± 0.414.49 ± 0.03
Fermichamp2.86 ± 0.034.60 ± 0.116.33 ± 0.41115 ± 1080.8 ± 0.76.8 ± 0.914.32 ± 0.02
ICV-GRE2.80 ± 0.046.31 ± 0.3912.62 ± 0.6291 ± 273.1 ± 1.021.1 ± 0.614.52 ± 0.04
IOC-18-20072.61 ± 0.034.90 ± 0.128.23 ± 0.62149 ± 783.2 ± 0.919.7 ± 0.314.36 ± 0.04
K12.87 ± 0.065.71 ± 0.1810.98 ± 0.95148 ± 1377.0 ± 1.925.8 ± 0.914.70 ± 0.04
L20562.77 ± 0.014.81 ± 0.128.11 ± 0.33132 ± 772.9 ± 2.222.5 ± 1.414.55 ± 0.05
L22262.70 ± 0.064.66 ± 0.227.43 ± 0.63119 ± 176.0 ± 0.536.8 ± 1.314.56 ± 0.04
L23232.84 ± 0.115.74 ± 0.819.27 ± 1.38152 ± 1378.0 ± 4.722.2 ± 0.914.75 ± 0.07
Lalvin AC2.63 ± 0.055.04 ± 0.159.47 ± 0.65131 ± 177.6 ± 1.410.7 ± 0.414.62 ± 0.05
LVCB2.93 ± 0.094.63 ± 0.316.94 ± 1.13150 ± 1374.3 ± 4.316.4 ± 1.814.45 ± 0.07
N962.64 ± 0.054.85 ± 0.268.48 ± 0.96132 ± 379.8 ± 1.820.6 ± 0.314.42 ± 0.04
NT1123.15 ± 0.075.64 ± 0.498.96 ± 0.16164 ± 1073.9 ± 1.418.9 ± 0.214.56 ± 0.01
NT1163.14 ± 0.065.27 ± 0.158.17 ± 0.46136 ± 1885.5 ± 1.118.8 ± 0.914.46 ± 0.01
NT2022.92 ± 0.054.76 ± 0.136.40 ± 0.43161 ± 1190.0 ± 0.827.0 ± 2.114.40 ± 0.08
NT453.03 ± 0.085.02 ± 0.126.98 ± 0.25148 ± 485.6 ± 3.026.7 ± 0.614.44 ± 0.06
NT502.80 ± 0.044.02 ± 0.265.42 ± 0.52117 ± 1690.7 ± 4.525.14 ± 2.214.13 ± 0.11
PC2.98 ± 0.154.95 ± 0.438.35 ± 1.36141 ± 2678.7 ± 3.818.8 ± 5.114.47 ± 0.11
PDM3.08 ± 0.045.63 ± 0.119.93 ± 0.18149 ± 2273.3 ± 0.426.4 ± 0.214.53 ± 0.04
Primeur3.60 ± 0.066.04 ± 0.3411.95 ± 0.95112 ± 560.5 ± 1.633.4 ± 1.214.53 ± 0.07
QA232.99 ± 0.145.19 ± 0.348.69 ± 1.12146 ± 1178.3 ± 1.216.1 ± 0.814.55 ± 0.08
R22.68 ± 0.035.43 ± 0.2310.94 ± 0.90121 ± 377.6 ± 2.017.1 ± 1.814.42 ± 0.17
RC2122.98 ± 0.106.98 ± 1.3312.80 ± 2.68122 ± 877.5 ± 4.419.2 ± 1.914.43 ± 0.06
Rhone 46002.94 ± 0.024.92 ± 0.068.43 ± 0.49136 ± 176.5 ± 0.821.5 ± 0.414.49 ± 0.06
S1013.04 ± 0.105.41 ± 0.1111.18 ± 1.76123 ± 469.5 ± 1.013.3 ± 0.114.18 ± 0.01
S1022.84 ± 0.025.46 ± 0.169.32 ± 0.45136 ± 378.3 ± 1.023.3 ± 0.714.57 ± 0.02
S3253.44 ± 0.058.36 ± 0.3115.98 ± 0.2989 ± 466.3 ± 3.122.3 ± 2.014.52 ± 0.03
S3772.99 ± 0.048.96 ± 0.6118.48 ± 1.62114 ± 271.7 ± 1.932.5 ± 0.414.73 ± 0.01
SauvL32.73 ± 0.085.36 ± 0.478.80 ± 0.90136 ± 1177.4 ± 2.817.7 ± 1.114.68 ± 0.04
Simi White3.02 ± 0.047.14 ± 0.1413.28 ± 0.17119 ± 677.4 ± 1.016.9 ± 1.814.58 ± 0.04
SYR2.82 ± 0.025.29 ± 0.078.62 ± 0.06113 ± 376.7 ± 0.114.6 ± 0.814.51 ± 0.12
T3063.26 ± 0.037.02 ± 0.8813.84 ± 0.61146 ± 473.1 ± 0.917.0 ± 0.214.51 ± 0.05
T733.06 ± 0.107.10 ± 0.7811.60 ± 1.12120 ± 973.3 ± 2.719.9 ± 1.414.64 ± 0.04
UCD5222.26 ± 0.044.36 ± 0.538.22 ± 1.30125 ± 388.5 ± 4.026.3 ± 1.514.41 ± 0.07
UDC522-Mauri2.47 ± 0.044.63 ± 0.158.90 ± 1.07121 ± 780.9 ± 1.917.8 ± 1.014.61 ± 0.05
Vin132.49 ± 0.034.55 ± 0.137.08 ± 0.26137 ± 783.4 ± 2.022.6 ± 0.414.49 ± 0.01
VIN72.91 ± 0.064.78 ± 0.217.67 ± 0.7674 ± 174.8 ± 1.715.8 ± 0.714.56 ± 0.01
VL12.67 ± 0.024.92 ± 0.128.22 ± 0.45120 ± 380.7 ± 1.116.1 ± 1.514.48 ± 0.01
VL22.85 ± 0.045.25 ± 0.238.65 ± 0.89135 ± 678.1 ± 1.821.2 ± 0.914.52 ± 0.11
VL3C2.90 ± 0.056.17 ± 0.2310.09 ± 0.36129 ± 1771.8 ± 1.715.2 ± 0.614.75 ± 0.02
VR52.67 ± 0.065.22 ± 0.289.11 ± 1.28136 ± 281.9 ± 2.916.8 ± 1.214.40 ± 0.04
W3723.11 ± 0.045.64 ± 0.159.22 ± 0.48144 ± 477.6 ± 1.615.9 ± 0.414.36 ± 0.04
WE143.18 ± 0.255.57 ± 0.4410.83 ± 0.94125 ± 678.3 ± 3.323.0 ± 0.914.46 ± 0.08
Williams Selyem3.02 ± 0.094.56 ± 0.166.40 ± 0.04132 ± 381.2 ± 0.729.3 ± 1.114.48 ± 0.02
X53.00 ± 0.016.46 ± 0.0110.09 ± 0.04144 ± 972.2 ± 0.021.6 ± 0.414.59 ± 0.05
Figure 1.

Comparison of fermentation rates for 69 Saccharomyces cerevisiae wine yeast strains fermenting Chardonnay grape juice. Fermentation rate is displayed as time to consume sugar; Time 120 sugar is the time for each strain to consume 50% of the available sugar, Time 40 g L−1 sugar is the time for each strain to consume 83%, and Time Dryness is the time for each strain to consume all available sugar (dryness < 2.0 g L−1 sugar); note that this data are presented in numerical form in Table 2. Fermentations were performed in triplicate. Error bars represent one standard deviation. EPII: Epernay II.

In the early stages of fermentation, the yeasts were actively dividing, increasing in cell concentration until day three or four, at which point cell division ceased and the cell concentration remained stable for the remainder of the fermentation (≤ 10% reduction in the final fermentation phase; data not shown). Most strains reached a maximum cell concentration of 1.3 × 108 cells mL−1, although concentrations ranged from 6 × 107 to 1.6 × 108 cells mL−1 (Table 2). There was a weak positive correlation between maximum cell concentration and the duration of fermentation (r = 0.49). There are likely several factors contributing to this observation; for example, cell concentration does not always correlate to total cell biomass or dry weight (Prescott, 1975). The cell biomass was not measured in this study but may have had a stronger influence on fermentation rate than maximum cell concentration.

In addition to consuming sugars, yeast will consume nitrogen in the form of free amino acids and ammonia, the combination of which is referred to as YAN. Assimilable nitrogen is required for S. cerevisiae growth in a grape juice fermentation. Although there are many sources of nitrogen in grape juice, only ammonia and nonproline amino acids can be assimilated. Nitrogen assimilation is important for yeast growth and completion of fermentation; individual yeast strains have been shown to have unique assimilation patterns (Crépin et al., 2012). The strains used in this study consumed 55–90% of the available YAN, with a mean of 76.8% (Table 2). Nitrogen consumption contributes, in part, to total biomass, and there was a weak positive correlation (r = 0.46, P < 0.001) between the maximum cell concentration and the percentage of YAN consumed by each strain.

Metabolite footprinting

Large differences were seen among the strains in metabolite production. We measured 29 yeast-derived metabolic byproducts in the wine. These metabolites represent the exo-metabolome, with each compound contributing to the complex aroma and flavor of wine, including higher alcohols, ethyl esters, and acetate esters, among others (Table 3). The majority of the traits measured centered symmetrically around the mean, rather than having bimodal or multimodal distributions (Fig. 2). Camarasa et al. (2011) have previously measured 18 metabolic byproducts produced by yeast during fermentation and identified ranges of 2- to 15-fold. In this study, we measured many additional fermentation attributes and metabolic byproducts and observed differences of up to 1000-fold between strains (Fig. 2, Table 3). The impact of yeast on wine flavor is determined by the differential production of these metabolic compounds, several of which are volatile and also influence the aroma of the wine (Guth, 1997; Ferreira et al., 2000).

Table 3. Comparison of phenotypic variables comprising yeast metabolic footprint. Fermentations were carried out in triplicate in Chardonnay grape juice. Concentrations were determined in the final wine product
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430.64 ± 0.0231 ± 07.3 ± 0.231 ± 214 ± 0132 ± 111 ± 10 ± 034 ± 141 ± 114 294 ± 4450 ± 05617 ± 1410 ± 00 ± 0373 ± 6308 ± 19955 ± 130 ± 0925 ± 110 ± 00 ± 058 ± 295 ± 2343 ± 78593 ± 193560 ± 360 ± 062 ± 10
2280.18 ± 0.0225 ± 07.0 ± 0.130 ± 825 ± 0127 ± 330 ± 10 ± 043 ± 241 ± 117 395 ± 5270 ± 0143 ± 350 ± 00 ± 0475 ± 120 ± 01224 ± 1240 ± 01203 ± 1380 ± 0106 ± 3558 ± 070 ± 1205 ± 137295 ± 554402 ± 260 ± 053 ± 2
4F90.10 ± 0.0143 ± 16.1 ± 0.144 ± 321 ± 0156 ± 219 ± 14 ± 663 ± 941 ± 111 415 ± 1670 ± 04935 ± 1750 ± 00 ± 0498 ± 21988 ± 651634 ± 1100 ± 02093 ± 5996 ± 8219 ± 2351 ± 075 ± 3244 ± 1014 243 ± 478401 ± 50 ± 00 ± 0
58W30.35 ± 0.0140 ± 26.2 ± 0.641 ± 523 ± 1144 ± 922 ± 10 ± 037 ± 143 ± 118 881 ± 14820 ± 011 100 ± 4680 ± 00 ± 0569 ± 45676 ± 621292 ± 11982 ± 211797 ± 13965 ± 00 ± 063 ± 749 ± 1264 ± 306494 ± 577299 ± 270 ± 00 ± 0
71B0.64 ± 0.0017 ± 16.8 ± 0.234 ± 119 ± 1138 ± 319 ± 13 ± 118 ± 140 ± 112 255 ± 20940 ± 05158 ± 3100 ± 00 ± 0388 ± 2628 ± 931327 ± 3864 ± 01712 ± 2980 ± 0170 ± 00 ± 057 ± 1274 ± 1514 889 ± 1344416 ± 710 ± 00 ± 0
AWRI R20.34 ± 0.0238 ± 26.2 ± 0.546 ± 523 ± 1135 ± 627 ± 10 ± 036 ± 141 ± 116 182 ± 1820 ± 012 920 ± 4510 ± 00 ± 0461 ± 11282 ± 191430 ± 430 ± 01662 ± 980 ± 00 ± 057 ± 550 ± 1248 ± 55954 ± 210358 ± 60 ± 053 ± 0
AWRI3500.60 ± 0.0312 ± 16.8 ± 0.254 ± 1219 ± 3138 ± 619 ± 10 ± 0150 ± 843 ± 010 718 ± 7690 ± 03021 ± 1900 ± 02782 ± 157420 ± 22499 ± 3712 553 ± 1070 ± 01306 ± 400 ± 0207 ± 9843 ± 5454 ± 1196 ± 153360 ± 287175 ± 80 ± 00 ± 0
AWRI7960.53 ± 0.0118 ± 17.3 ± 0.028 ± 221 ± 1148 ± 722 ± 10 ± 067 ± 343 ± 014 875 ± 4220 ± 09254 ± 7220 ± 00 ± 0579 ± 35185 ± 321440 ± 9073 ± 72194 ± 2810 ± 00 ± 00 ± 061 ± 2291 ± 98310 ± 479431 ± 150 ± 00 ± 0
BA110.35 ± 0.0134 ± 36.2 ± 0.148 ± 824 ± 1134 ± 231 ± 20 ± 028 ± 241 ± 018 089 ± 10290 ± 013 823 ± 1430 ± 00 ± 0480 ± 30461 ± 421244 ± 40 ± 01569 ± 1890 ± 073 ± 3655 ± 049 ± 3239 ± 115134 ± 360353 ± 220 ± 059 ± 1
BDX0.50 ± 0.0113 ± 26.9 ± 0.347 ± 1336 ± 2130 ± 655 ± 60 ± 042 ± 342 ± 026 030 ± 5510 ± 012 849 ± 5580 ± 00 ± 0359 ± 2990 ± 52906 ± 470 ± 0810 ± 670 ± 00 ± 00 ± 072 ± 3390 ± 128359 ± 577915 ± 550 ± 056 ± 2
BGY0.97 ± 0.0416 ± 15.6 ± 0.241 ± 618 ± 1133 ± 633 ± 40 ± 025 ± 141 ± 120 493 ± 17920 ± 014 520 ± 13290 ± 00 ± 0320 ± 1151 ± 01077 ± 470 ± 0960 ± 1190 ± 051 ± 179 ± 2054 ± 2211 ± 104867 ± 269374 ± 170 ± 059 ± 5
Bm450.38 ± 0.01149 ± 47.5 ± 0.2121 ± 1019 ± 1130 ± 924 ± 10 ± 088 ± 343 ± 142 349 ± 12410 ± 03085 ± 3190 ± 00 ± 0572 ± 4375 ± 151559 ± 1558 ± 01940 ± 800 ± 00 ± 0180 ± 2462 ± 2301 ± 106412 ± 131378 ± 120 ± 00 ± 0
BP7250.53 ± 0.0219 ± 26.9 ± 0.225 ± 628 ± 2139 ± 939 ± 30 ± 029 ± 243 ± 018 094 ± 11170 ± 013 032 ± 6540 ± 00 ± 0559 ± 22461 ± 1951200 ± 6257 ± 01470 ± 3966 ± 170 ± 00 ± 067 ± 4403 ± 1511 794 ± 543603 ± 280 ± 056 ± 0
BRL970.34 ± 0.0120 ± 35.7 ± 0.533 ± 417 ± 2132 ± 1516 ± 20 ± 0154 ± 2341 ± 118 468 ± 17800 ± 012 563 ± 350 ± 00 ± 0293 ± 28194 ± 971295 ± 660 ± 01215 ± 1530 ± 0139 ± 3257 ± 646 ± 1179 ± 53640 ± 163249 ± 130 ± 055 ± 4
CSM0.65 ± 0.0324 ± 26.5 ± 0.228 ± 323 ± 1129 ± 838 ± 30 ± 014 ± 1542 ± 212 039 ± 7280 ± 0194 ± 520 ± 00 ± 0452 ± 14345 ± 691374 ± 3171 ± 72155 ± 4262 ± 60 ± 059 ± 244 ± 3191 ± 54653 ± 211290 ± 40 ± 00 ± 0
CY30790.39 ± 0.0134 ± 25.8 ± 0.045 ± 619 ± 0124 ± 320 ± 13 ± 511 ± 139 ± 113 336 ± 30420 ± 05140 ± 2470 ± 00 ± 0489 ± 16226 ± 1141137 ± 12851 ± 01486 ± 120 ± 0144 ± 053 ± 054 ± 1260 ± 614 747 ± 1759404 ± 870 ± 00 ± 0
D2540.38 ± 0.0127 ± 16.5 ± 0.125 ± 323 ± 0149 ± 120 ± 00 ± 00 ± 040 ± 18566 ± 10600 ± 05567 ± 2860 ± 00 ± 0479 ± 21577 ± 601226 ± 8757 ± 91627 ± 740 ± 00 ± 072 ± 1061 ± 3226 ± 116 696 ± 1466292 ± 260 ± 00 ± 0
D470.64 ± 0.0231 ± 07.3 ± 0.231 ± 214 ± 0132 ± 111 ± 10 ± 034 ± 141 ± 114 294 ± 4450 ± 05617 ± 1410 ± 00 ± 0373 ± 6308 ± 19955 ± 130 ± 0925 ± 110 ± 00 ± 058 ± 295 ± 2343 ± 78593 ± 192560 ± 360 ± 062 ± 10
D800.40 ± 0.0026 ± 25.5 ± 0.136 ± 1323 ± 6125 ± 3724 ± 70 ± 031 ± 836 ± 1210 947 ± 11370 ± 05976 ± 5950 ± 00 ± 0396 ± 42459 ± 1071267 ± 14283 ± 121610 ± 1790 ± 0178 ± 8508 ± 9734 ± 12237 ± 294996 ± 506257 ± 140 ± 00 ± 0
DV100.14 ± 0.0045 ± 16.3 ± 0.338 ± 220 ± 2153 ± 519 ± 10 ± 081 ± 342 ± 115 620 ± 4040 ± 06780 ± 1510 ± 00 ± 0540 ± 55732 ± 3881852 ± 378181 ± 1341914 ± 377160 ± 3374 ± 0127 ± 660 ± 1300 ± 2018 391 ± 1555530 ± 280 ± 00 ± 0
EC11180.25 ± 0.0346 ± 16.7 ± 0.256 ± 714 ± 1149 ± 811 ± 12 ± 032 ± 939 ± 112 964 ± 1670 ± 06402 ± 3000 ± 00 ± 0601 ± 22725 ± 622008 ± 26898 ± 121848 ± 129112 ± 750 ± 089 ± 865 ± 3343 ± 718 196 ± 461489 ± 140 ± 00 ± 0
Elegance0.28 ± 0.0239 ± 37.3 ± 0.238 ± 518 ± 1161 ± 619 ± 00 ± 048 ± 941 ± 114 407 ± 9150 ± 05622 ± 2210 ± 00 ± 0619 ± 30995 ± 1381847 ± 127339 ± 572074 ± 179562 ± 640 ± 0101 ± 260 ± 1326 ± 413 575 ± 525380 ± 820 ± 00 ± 0
EPII0.65 ± 0.0330 ± 26.4 ± 0.260 ± 2918 ± 2115 ± 923 ± 30 ± 00 ± 041 ± 112 555 ± 1570 ± 05676 ± 2230 ± 00 ± 04287 ± 8543 ± 421638 ± 430 ± 01412 ± 1030 ± 0148 ± 0186 ± 1753 ± 2317 ± 79446 ± 590209 ± 60 ± 00 ± 0
F150.25 ± 0.0248 ± 66.2 ± 0.341 ± 428 ± 8149 ± 522 ± 10 ± 031 ± 143 ± 012 884 ± 23410 ± 03360 ± 28680 ± 00 ± 00 ± 0550 ± 2292025 ± 1270 ± 01742 ± 317124 ± 062 ± 6150 ± 2759 ± 2330 ± 7421 177 ± 4009468 ± 580 ± 00 ± 0
F330.18 ± 0.0225 ± 07.0 ± 0.130 ± 825 ± 0127 ± 330 ± 10 ± 043 ± 241 ± 117 395 ± 5270 ± 0143 ± 350 ± 00 ± 0475 ± 120 ± 01224 ± 1240 ± 01203 ± 1380 ± 0106 ± 3558 ± 070 ± 1205 ± 137295 ± 554402 ± 260 ± 053 ± 2
FA10.34 ± 0.0238 ± 26.2 ± 0.546 ± 523 ± 1135 ± 627 ± 10 ± 036 ± 141 ± 116 182 ± 1820 ± 012 920 ± 4510 ± 00 ± 0461 ± 11282 ± 191430 ± 440 ± 01662 ± 980 ± 00 ± 057 ± 550 ± 1248 ± 55954 ± 210358 ± 60 ± 053 ± 0
Fermichamp0.054 ± 0.0123 ± 08.6 ± 0.628 ± 318 ± 1148 ± 217 ± 10 ± 050 ± 144 ± 122 402 ± 7110 ± 01003 ± 2160 ± 00 ± 0530 ± 38729 ± 311599 ± 136101 ± 21501 ± 103375 ± 40 ± 078 ± 172 ± 2323 ± 247302 ± 620686 ± 5256 ± 00 ± 0
ICV-GRE0.34 ± 0.0122 ± 25.5 ± 0.339 ± 224 ± 1134 ± 228 ± 10 ± 035 ± 143 ± 012 524 ± 16010 ± 01974 ± 2590 ± 00 ± 0545 ± 14617 ± 511829 ± 58103 ± 92030 ± 1510 ± 0133 ± 29730 ± 3243 ± 1188 ± 44239 ± 124251 ± 310 ± 00 ± 0
IOC-18-20070.16 ± 0.0239 ± 15.9 ± 0.242 ± 319 ± 1159 ± 419 ± 13 ± 459 ± 340 ± 114 203 ± 1160 ± 06905 ± 2540 ± 00 ± 0582 ± 14857 ± 501943 ± 65224 ± 72089 ± 76366 ± 80 ± 091 ± 474 ± 2393 ± 1421 765 ± 237584 ± 90 ± 00 ± 0
K10.41 ± 0.0184 ± 46.1 ± 0.672 ± 825 ± 8162 ± 521 ± 10 ± 042 ± 242 ± 013 307 ± 10010 ± 0714 ± 10560 ± 00 ± 00 ± 0302 ± 461646 ± 1730 ± 01439 ± 3590 ± 00 ± 0165 ± 6055 ± 0342 ± 3221 064 ± 2389465 ± 130 ± 00 ± 0
L20560.50 ± 0.0113 ± 26.9 ± 0.347 ± 1336 ± 2130 ± 655 ± 60 ± 042 ± 342 ± 026 030 ± 5510 ± 012 849 ± 5580 ± 00 ± 0359 ± 2990 ± 52906 ± 470 ± 0810 ± 670 ± 00 ± 00 ± 072 ± 3390 ± 128359 ± 577915 ± 550 ± 056 ± 2
L22260.97 ± 0.0416 ± 15.6 ± 0.241 ± 618 ± 1133 ± 633 ± 40 ± 025 ± 141 ± 120 493 ± 17920 ± 014 520 ± 13290 ± 00 ± 0320 ± 1151 ± 01077 ± 470 ± 0960 ± 1190 ± 051 ± 179 ± 2054 ± 2211 ± 104867 ± 269374 ± 170 ± 059 ± 5
L23230.43 ± 0.0342 ± 35.9 ± 0.448 ± 622 ± 2141 ± 921 ± 21 ± 115 ± 340 ± 19715 ± 3580 ± 05355 ± 1420 ± 00 ± 0399 ± 7599 ± 94998 ± 754 ± 41406 ± 470 ± 00 ± 050 ± 057 ± 2264 ± 313 706 ± 732357 ± 210 ± 00 ± 0
Lalvin AC0.47 ± 0.0144 ± 16.5 ± 0.643 ± 521 ± 2130 ± 1121 ± 10 ± 029 ± 243 ± 114 765 ± 23850 ± 012 360 ± 11760 ± 00 ± 0553 ± 24882 ± 691293 ± 65116 ± 101884 ± 11176 ± 230 ± 054 ± 552 ± 33057 ± 216812 ± 343315 ± 4552 ± 00 ± 0
LVCB0.34 ± 0.0120 ± 35.7 ± 0.533 ± 417 ± 2132 ± 1516 ± 20 ± 0154 ± 2341 ± 118 468 ± 17800 ± 012 563 ± 350 ± 00 ± 0293 ± 28194 ± 971295 ± 660 ± 01215 ± 1530 ± 0139 ± 3257 ± 646 ± 1179 ± 53640 ± 163249 ± 130 ± 055 ± 4
N960.12 ± 0.0136 ± 16.0 ± 0.234 ± 318 ± 0160 ± 216 ± 00 ± 055 ± 341 ± 113 604 ± 8060 ± 05541 ± 2120 ± 00 ± 0606 ± 33683 ± 931867 ± 243182 ± 61849 ± 279352. ± 120 ± 0144 ± 3480 ± 1365 ± 4220 783 ± 2687488 ± 570 ± 00 ± 0
NT1120.36 ± 0.01202 ± 47.2 ± 0.3153 ± 914 ± 1119 ± 312 ± 11 ± 0169 ± 1440 ± 234 945 ± 6100 ± 02600 ± 2420 ± 00 ± 0392 ± 17342 ± 211199 ± 410 ± 01175 ± 650 ± 00 ± 0150 ± 3880 ± 2247 ± 174046 ± 202254 ± 90 ± 00 ± 0
NT1160.13 ± 0.0137 ± 48.3 ± 0.634 ± 333 ± 19155 ± 215 ± 10 ± 0112 ± 644 ± 311 708 ± 10310 ± 0609 ± 1000 ± 00 ± 0481 ± 1528 ± 631255 ± 5851 ± 01273 ± 17060 ± 00 ± 0130 ± 9107 ± 3444 ± 2029 627 ± 3707696 ± 600 ± 00 ± 0
NT2020.09 ± 0.00022 ± 18.6 ± 0.229 ± 127 ± 4201 ± 3025 ± 30 ± 070 ± 043 ± 144 864 ± 67180 ± 011 434 ± 3710 ± 00 ± 0540 ± 76574 ± 781152 ± 20064 ± 91570 ± 19480 ± 320 ± 0272 ± 2687 ± 6441 ± 5014 162 ± 1126581 ± 220 ± 00 ± 0
NT450.09 ± 0.0123 ± 38.4 ± 0.230 ± 523 ± 1174 ± 723 ± 10 ± 065 ± 343 ± 133 759 ± 40650 ± 010 529 ± 12710 ± 00 ± 0574 ± 60636 ± 551416 ± 1183 ± 121682 ± 207107 ± 180 ± 0175 ± 3092 ± 1463 ± 2214 077 ± 812565 ± 730 ± 00 ± 0
NT500.09 ± 0.0474 ± 810.0 ± 0.882 ± 433 ± 3160 ± 128 ± 40 ± 075 ± 442 ± 116 127 ± 42070 ± 06933 ± 1410 ± 00 ± 0401 ± 66339 ± 1751364 ± 2640 ± 01374 ± 2300 ± 00 ± 0156 ± 6792 ± 13367 ± 3728 456 ± 5260684 ± 1370 ± 00 ± 0
PC0.21 ± 0.0545 ± 236.9 ± 0.446 ± 1921 ± 3164 ± 1320 ± 20 ± 080 ± 1543 ± 327 802.5 ± 15 658115 ± 1410 500 ± 30762135 ± 00 ± 0599 ± 81814 ± 2741662 ± 200259 ± 1671889 ± 2581349 ± 13281603 ± 46189 ± 17870 ± 5281 ± 337884 ± 1119524 ± 4651 ± 062 ± 11
PDM0.24 ± 0.0049 ± 16.8 ± 0.149 ± 1220 ± 4173 ± 4422 ± 50 ± 086 ± 2046 ± 1113 372 ± 6190 ± 06831 ± 3250 ± 00 ± 0630 ± 99735 ± 551831 ± 297194 ± 171749 ± 63882 ± 51132 ± 0430 ± 9066 ± 15265 ± 466576 ± 1051406 ± 100 ± 00 ± 0
Primeur0.61 ± 0.0224 ± 17.0 ± 0.252 ± 438 ± 1398 ± 322 ± 10 ± 056 ± 242 ± 010 0527 ± 4070 ± 05965 ± 3460 ± 00 ± 00 ± 0335 ± 451450 ± 1560 ± 01018 ± 10550 ± 00 ± 089 ± 354 ± 2560 ± 18518 178 ± 3351397 ± 210 ± 00 ± 0
QA230.24 ± 0.0238 ± 26.5 ± 0.435 ± 521 ± 4175 ± 1722 ± 10 ± 074 ± 543 ± 015 867 ± 16140 ± 06559 ± 7420 ± 00 ± 0531 ± 0863 ± 1461803 ± 109281 ± 01657 ± 284644 ± 4030 ± 0150 ± 2359 ± 1349 ± 4718 561 ± 357491 ± 500 ± 00 ± 0
R20.33 ± 0.0240 ± 46.2 ± 0.637 ± 724 ± 1134 ± 424 ± 20 ± 034 ± 143 ± 112 197 ± 8420 ± 05434 ± 4600 ± 00 ± 00 ± 0525 ± 871757 ± 3450 ± 01473 ± 3100 ± 055 ± 2181 ± 1948 ± 1402 ± 12818 825 ± 2765385 ± 420 ± 00 ± 0
RC2120.52 ± 0.0236 ± 35.6 ± 0.537 ± 423 ± 2128 ± 1730 ± 40 ± 034 ± 443 ± 19840 ± 8430 ± 04514 ± 1370 ± 00 ± 0474 ± 42471 ± 1311297 ± 18685 ± 71558 ± 19258 ± 0115 ± 7659 ± 8047 ± 3202 ± 164252 ± 202251 ± 300 ± 00 ± 0
Rhone 46000.24 ± 0.0139 ± 17.3 ± 0.035 ± 519 ± 1153 ± 220 ± 10 ± 075 ± 343 ± 149 609 ± 18800 ± 013 158 ± 3090 ± 00 ± 0693 ± 87960 ± 441845 ± 84300 ± 752239 ± 171928 ± 1770 ± 0229 ± 1970 ± 3307 ± 78748 ± 612588 ± 50 ± 00 ± 0
S1010.23 ± 0.0014 ± 07.9 ± 0.736 ± 120 ± 0138 ± 021 ± 00 ± 062 ± 143 ± 013 849 ± 860 ± 02436 ± 780 ± 00 ± 0500 ± 19596 ± 321367 ± 6278 ± 11484 ± 32106 ± 10184 ± 8615 ± 3552 ± 1185 ± 105302 ± 183333 ± 60 ± 00 ± 0
S1020.43 ± 0.0167 ± 16.4 ± 0.153 ± 422 ± 0168 ± 121 ± 10 ± 037 ± 143 ± 055 786 ± 94790 ± 0116 ± 00 ± 00 ± 0572 ± 19157 ± 291202 ± 3993 ± 01548 ± 1720 ± 00 ± 0241 ± 2661 ± 1334 ± 3111 690 ± 601646 ± 920 ± 00 ± 0
S3250.34 ± 0.0147 ± 36.3 ± 0.265 ± 1320 ± 1137 ± 521 ± 10 ± 037 ± 143 ± 146 822 ± 15180 ± 010 163 ± 2280 ± 00 ± 0522 ± 11497 ± 91531 ± 3764 ± 81939 ± 290 ± 069 ± 5295 ± 3346 ± 1235 ± 135580 ± 246329 ± 160 ± 00 ± 0
S3770.41 ± 0.0125 ± 15.9 ± 0.246 ± 1127 ± 9183 ± 1123 ± 10 ± 044 ± 245 ± 111 346 ± 12060 ± 05629 ± 2560 ± 00 ± 0322 ± 105258 ± 41925 ± 30756 ± 01254 ± 1080 ± 0100 ± 16469 ± 16541 ± 1188 ± 704442 ± 1404281 ± 180 ± 00 ± 0
SauvL30.46 ± 0.0243 ± 46.4 ± 0.248 ± 617 ± 1126 ± 619 ± 20 ± 011 ± 240 ± 19266 ± 1870 ± 05950 ± 1530 ± 00 ± 0429 ± 8597 ± 1481353 ± 480 ± 01858 ± 730 ± 0196 ± 90 ± 055 ± 2284 ± 613 289 ± 762382 ± 90 ± 00 ± 0
Simi White0.56 ± 0.0118 ± 16.8 ± 0.249 ± 1422 ± 2137 ± 1126 ± 30 ± 034 ± 243 ± 017 533 ± 766151 ± 411 738 ± 4540 ± 00 ± 0459 ± 42410 ± 971215 ± 10453 ± 11665 ± 14755 ± 30 ± 066 ± 747 ± 1236 ± 165482 ± 258252 ± 100 ± 00 ± 0
SYR0.50 ± 0.0260 ± 16.6 ± 0.251 ± 425 ± 1151 ± 326 ± 10 ± 038 ± 043 ± 147 537 ± 19560 ± 09955 ± 1840 ± 00 ± 0591 ± 55498 ± 621663 ± 18751 ± 01856 ± 660 ± 00 ± 0197 ± 3363 ± 1404 ± 3310 898 ± 915559 ± 190 ± 00 ± 0
T3060.40 ± 0.0139 ± 15.5 ± 0.257 ± 217 ± 1125 ± 421 ± 10 ± 034 ± 143 ± 19524 ± 5110 ± 0807 ± 760 ± 02757 ± 39510 ± 24444 ± 311389 ± 7776 ± 101622 ± 1120 ± 0219 ± 16496 ± 5749 ± 1192 ± 133495 ± 191199 ± 80 ± 00 ± 0
T730.48 ± 0.0155 ± 26.0 ± 0.366 ± 620 ± 1142 ± 918 ± 10 ± 027 ± 143 ± 144 931 ± 27930 ± 09945 ± 12530 ± 00 ± 0538 ± 48188 ± 1201624 ± 16353 ± 11911 ± 1630 ± 068 ± 14235 ± 744 ± 1234 ± 247353 ± 992326 ± 190 ± 00 ± 0
UCD5220.32 ± 0.0341 ± 106.5 ± 0.340 ± 127 ± 4152 ± 1930 ± 65 ± 420 ± 139 ± 123 693 ± 41660 ± 06195 ± 750 ± 00 ± 0546 ± 4237 ± 711231 ± 420 ± 01383 ± 760 ± 00 ± 0209 ± 5973 ± 2479 ± 1630 390 ± 7171040 ± 1540 ± 00 ± 0
UDC522-Mauri0.17 ± 0.0159 ± 45.8 ± 0.251 ± 728 ± 1143 ± 1026 ± 10 ± 032 ± 043 ± 116 687 ± 4570 ± 011 557 ± 350 ± 00 ± 0625 ± 51828 ± 661342 ± 13288 ± 251931 ± 16369 ± 90 ± 085 ± 764 ± 3361 ± 711 734 ± 517526 ± 2556 ± 00 ± 0
Vin130.15 ± 0.0130 ± 36.4 ± 0.132 ± 419 ± 0128 ± 314 ± 12 ± 425 ± 140 ± 19785 ± 7090 ± 00 ± 00 ± 00 ± 0458 ± 20606 ± 611391 ± 540 ± 01729 ± 410 ± 0227 ± 320 ± 0106 ± 1379 ± 322 630 ± 1547635 ± 19208 ± 180 ± 0
VIN70.66 ± 0.0121 ± 16.2 ± 0.222 ± 134 ± 19190 ± 3232 ± 80 ± 033 ± 1394 ± 8976 221 ± 51040 ± 09992 ± 1030 ± 00 ± 0630 ± 22681 ± 411636 ± 9498 ± 52195 ± 88121 ± 400 ± 0357 ± 464 ± 2302 ± 127546 ± 190823 ± 460 ± 00 ± 0
VL10.39 ± 0.0241 ± 16.4 ± 0.237 ± 520 ± 1131 ± 222 ± 10 ± 030 ± 143 ± 143 059 ± 33370 ± 00 ± 00 ± 00 ± 0585 ± 33503 ± 711377 ± 9768 ± 192203 ± 2500 ± 00 ± 00 ± 058 ± 0324 ± 158067 ± 159466 ± 380 ± 00 ± 0
VL20.57 ± 0.0169 ± 14.0 ± 0.358 ± 420 ± 1152 ± 621 ± 10 ± 038 ± 243 ± 049 219 ± 42690 ± 00 ± 00 ± 00 ± 0501 ± 46203 ± 451310 ± 8452 ± 01799 ± 450 ± 00 ± 00 ± 048 ± 3297 ± 247790 ± 996422 ± 330 ± 00 ± 0
VL3C0.51 ± 0.0150 ± 16.0 ± 0.246 ± 216 ± 1116 ± 317 ± 11 ± 229 ± 140 ± 19056 ± 1240 ± 06119 ± 2070 ± 00 ± 0421 ± 12476 ± 451283 ± 153 ± 41646 ± 210 ± 00 ± 052 ± 050 ± 1257 ± 1211 943 ± 311326 ± 50 ± 00 ± 0
VR50.35 ± 0.0142 ± 52.6 ± 0.339 ± 426 ± 3146 ± 1325 ± 30 ± 033 ± 243 ± 047 878 ± 38280 ± 09912 ± 5380 ± 00 ± 0637 ± 5608 ± 1311327 ± 8164 ± 121952 ± 2030 ± 00 ± 00 ± 058 ± 3277 ± 48359 ± 801460 ± 480 ± 00 ± 0
W3720.60 ± 0.0220 ± 17.2 ± 0.220 ± 119 ± 1148 ± 820 ± 10 ± 060 ± 343 ± 044 768 ± 16280 ± 010 571 ± 1390 ± 00 ± 0648 ± 7172 ± 1361322 ± 7152 ± 01669 ± 1940 ± 00 ± 00 ± 064 ± 2321 ± 48649 ± 253434 ± 180 ± 00 ± 0
WE140.40 ± 0.0138 ± 26.6 ± 0.240 ± 832 ± 9168 ± 631 ± 20 ± 039 ± 243 ± 114 881 ± 11930 ± 0627 ± 1540 ± 00 ± 0435 ± 3172 ± 01331 ± 3671 ± 01812 ± 28186 ± 066 ± 058 ± 446 ± 1250 ± 96957 ± 209377 ± 340 ± 00 ± 0
Williams Selyem0.27 ± 0.0279 ± 76.3 ± 0.662 ± 109 ± 1149 ± 69 ± 00 ± 082 ± 943 ± 034 381 ± 9070 ± 0141 ± 00 ± 00 ± 0477 ± 10494 ± 311360 ± 7858 ± 91307 ± 83194 ± 550 ± 00 ± 072 ± 2295 ± 127341 ± 254350 ± 210 ± 00 ± 0
X50.35 ± 0.0134 ± 16.5 ± 0.237 ± 817 ± 1111 ± 317 ± 10 ± 064 ± 940 ± 115 387 ± 4950 ± 012 387 ± 3990 ± 00 ± 0456 ± 41521 ± 211125 ± 6975 ± 51495 ± 960 ± 065 ± 058 ± 052 ± 0287 ± 355923 ± 177284 ± 80 ± 00 ± 0
Figure 2.

Comparison of phenotypic variables (fermentation performance attributes and wine metabolite concentrations) for 69 Saccharomyces cerevisiae wine yeast strains fermenting Chardonnay grape juice. (a) Pie graph showing the breakdown of types of attributes measured in this study. (b) Distribution frequency graphs for maximum cell density and six representative wine metabolites, for 69 S. cerevisiae wine strains. Maximum cell density was the maximum concentration observed during the course of fermentation as monitored by daily measurement. Final total SO2, acetaldehyde, ethyl acetate, glycerol, 2-phenyl ethanol, and ethyl hexanoate concentrations were measured in the finished wine.

Alcohols

In a grape juice fermentation, ethanol yields are 90–95% of theoretical, with the remaining 5–10% explained by both conversion of glucose to biomass and loss of ethanol through evaporation (Konig et al., 2009). The range of ethanol produced was 14.18–14.75%v/v (Table 2). During the course of fermentation, yeast will also produce higher alcohols (also called fusel alcohols, containing > 2 carbon atoms) as secondary metabolites from amino acid synthesis. These higher alcohols are the most abundant volatile components in wine (Sumby et al., 2010). In this study, we measured active amyl alcohol, iso-amyl alcohol, iso-butyl alcohol, n-butyl alcohol, and n-propyl alcohol. These alcohol metabolites can have both positive and negative impacts on the wine, depending on the concentration (Konig et al., 2009). Total higher alcohol concentrations of 400 mg L−1 or greater can result in a pungent, solvent aroma, whereas concentrations of < 300 mg L−1 are often described as imparting a fruity aroma (Swiegers & Pretorius, 2005). Approximately 10% of the strains produce total higher alcohols > 300 mg L−1; none of the strains were observed to produce > 400 mg L−1. Several of the higher alcohols correlated positively with each other. Active amyl alcohol and iso-butyl alcohol are two branched-chain alcohols synthesized through the Ehrlich pathway that we found to be positively correlated (r = 0.67, P < 0.001, Fig. 3). Strains producing high amounts of both were BDX, L2056, Vin7, and BP725. Strains producing low amounts of both compounds were 43, D47, EC1118, NT112, and VL3C. Although not as abundant as active amyl alcohol, n-propyl alcohol and the total concentration of higher alcohols were also positively correlated (r = 0.76, P < 0.001, Fig. 3). Methanol, which is not a higher alcohol, was also measured. All strains, except one, had approximately 40 mg L−1 methanol in the finished wine, similar to levels that have been reported previously (Rossouw et al., 2008). Vin7-produced wine contained over twofold more methanol than any other strain. Methanol can be released into wine through pectinase activity (Louw et al., 2010; Eschstruth & Divol, 2011). Although most S. cerevisiae have little to no pectinase activity, a few strains and several hybrid yeast strains have been shown to possess pectinase activity (Louw et al., 2010; Eschstruth & Divol, 2011). Vin7 is a S. cerevisiae/S. kudriavzevii hybrid and was found to have increased pectinase activity suggesting that Vin7 may possess the ability to degrade pectin thereby releasing methanol (Supporting Information, Fig. S1).

Figure 3.

Relationships between metabolic variables. (a) Correlation matrix of entire dataset including fermentation kinetics and wine metabolites. (b) Relationship between specific pairs of phenotypic variables: time to dryness and time to 40 g L−1 sugar; total SO2 concentration and acetaldehyde; n-propyl alcohol and total higher alcohols; active amyl alcohol and iso-butyl alcohol; 2-phenethyl acetate and hexyl acetate; hexyl acetate and isoamyl acetate. r values labeled on graphs.

Sulfur dioxide

During fermentation, yeast produces sulfur dioxide, the amount of which has been shown to be strain dependent (Swiegers & Pretorius, 2007). Sulfur dioxide can help minimize oxidation events that may ruin wine, but it can also result in problematic secondary processing steps if present at levels above 100 mg L−1 (Konig et al., 2009). SO2 production among the strains in this study varied by 10-fold, ranging from 20 to 200 mg L−1. The majority of the strains (86%) produced < 50 mg L−1; however, 16% produced > 50 mg L−1, and two strains, BM45 and NT112, produced greater 100 mg L−1.

Volatile acidity

Volatile acidity (VA) is the measurement of volatile acids, including acetic acid, lactic acid, succinic and propionic acid, which are important in winemaking because of flavor impacts as well as legal regulations. Yeast will produce these acids through metabolic pathways. High levels of acetic acid can cause the wine to become pungent and smell like vinegar; however, low levels can improve the quality. Legally, the VA must be below 1.1 g L−1 in white wine. The yeasts in this study produced a range of 0.1–1.0 g L−1 VA (Table 3). The mean VA produced was 0.4 g L−1; however, we did observe a few strains that produced 1.0 g L−1, very close to the legal limit. In addition to producing acids, yeast can metabolize acid. Several groups have observed that yeast will consume malic acid during fermentation in a strain-dependent manner (Pretorius, 2000; Konig et al., 2009). In this study, the yeast strains consumed anywhere from 10 to 40% of the available malic acid (Table 2).

Glycerol

Glycerol, the third major component in dry wines after ethanol and water, is the final product in the glyceropyruvic fermentation pathway that results in the regeneration of NAD+ (Moreno-Arribas & Polo, 1993). The glycerol concentrations produced by these strains varied from 0.6 to 10 g L−1, with an average of 6.5 g L−1 (Table 3). Only one strain, NT50, reached the 10 g L−1 threshold that is thought to confer a desirable sweetness flavor and positive mouthfeel sensation (Remize et al., 1999). NT50 is a S. cerevisiae/S. kudriavzevii hybrid (Dunn et al., 2012) that may have elevated glycerol production due to its S. kudriavzevii heritage (see Discussion).

Acetaldehyde

Acetaldehyde is produced by S. cerevisiae through the oxidation of ethanol by alcohol dehydrogenase II (encoded by ADH2) (Jackowetz et al., 2011). The yeast strains characterized in this study produced between 20 and 150 mg L−1 (Table 3). Acetaldehyde will continue to increase as the wine ages through oxidation of ethanol and can reach concentrations of 400 mg L−1 (Konig et al., 2009). Acetaldehyde contributes a nutty, sherry-like quality to the wine and is not desirable in most wine styles. Yeast strains BM45 and NT112 both produced acetaldehyde in concentrations > 100 mg L−1 (Table 3). These concentrations are above the sensory threshold for acetaldehyde, and further increases may negatively affect the wine flavor. Two strains, Vin7 and W372, were very low producers (< 25 mg L−1, Table 3).

Esters

Esters are produced by yeast during fermentation and typically impart a fruity aroma to the wine. Esters are the second most abundant volatile compounds in wine and are produced by microbial esterases, alcohol acetyltransferases, and lipases (Sumby et al., 2010). Different commercial yeast strains produce variable amounts of esters and, therefore, a variety of wine aroma profiles (Lambrechts & Pretorius, 2000; Vilanova et al., 2007; Cadiere et al., 2011). We measured 14 esters important for Chardonnay varietal characteristics to investigate yeast strain differences. Ethyl acetate is the most common ester in wine and is readily formed by a reaction between ethanol and acetic acid (Sumby et al., 2010). At low levels (< 100 mg L−1), ethyl acetate contributes to a fruity aroma in the wine; at concentrations above 100 mg L−1, ethyl acetate is described as solvent/nail polish. Most strains in this study produced < 100 mg L−1 ethyl acetate, with a mean of 62 mg L−1 (Table 3), while Vin13 and NT116 both produced over 100 mg L−1 ethyl acetate. Factors such as nitrogen content and grape esters impact the final concentration. Specific grape varieties are associated with specific yeast-derived esters, likely due to different amino acid profiles in grape (Ferreira et al., 2000).

Correlation between phenotypic traits

We used the Pearson's product moment method to perform correlation analysis between the phenotypic traits assayed in this study (Fig. 3a). Consistent with previous studies, most of the variables were independent (Camarasa et al., 2011). However, a few of the variables were strongly correlated (Fig. 3). Not surprisingly, Time to 40 g L−1 sugar was strongly correlated with Time to Dryness (r = 0.91, P < 0.001). The production of SO2 and acetaldehyde was also positively correlated (r = 0.89, P < 0.001, Fig. 3b) and highly variable among strains (Moreno-Arribas & Polo, 1993). Aldehydes are typically unstable compounds; however, SO2 will bind acetaldehyde creating the stable adduct acetaldehyde hydroxysulfonate (Moreno-Arribas & Polo, 1993). Therefore, high-sulfite-producing strains are acetaldehyde stabilizing strains, resulting in a correlation between these two metabolites.

The total fusel alcohol concentration was positively correlated with n-propyl alcohol concentration (r = 0.76, Fig. 3b). n-Propyl alcohol is the second most abundant fusel alcohol identified in this study (Table 3). It is produced as a byproduct in sterol biosynthesis (Rossouw et al., 2008). Active amyl alcohol and isobutyl alcohol are also positively correlated (r = 0.68, Fig. 3b). Active amyl alcohol and isobutyl alcohol are produced through catabolism of iso-leucine and valine, respectively (Rossouw et al., 2008).

Additionally, we found correlations between some acetate esters (Fig. 3b). Phenethyl acetate (honey, rose) correlated with hexyl acetate (fruity; r = 0.68), and isoamyl acetate (banana) correlated with hexyl acetate (fruity; r = 0.69). Phenethyl acetate is produced through catabolism of phenylalanine, and isoamyl acetate is produced through the catabolism of leucine (Rossouw et al., 2008). Hexyl acetate is produced through C6 compounds, such as hexanol, which are neutral compounds commonly found in grapes that influence wine aroma (Dennis et al., 2012).

Hierarchical clustering and PCA

To better understand the phenotypic relatedness between all 69 strains, we performed an agglomerative hierarchical clustering of the metabolic footprints of the yeasts. This analysis included all of the quantitative phenotypic traits, that is, the 29 yeast-produced metabolites. This clustering resulted in four groups of yeast strains, with each group containing from 6 to 30 strains (Fig. 4a). The dendrogram for each of the four groups is flat, suggesting homogeneity within the group (Fig. 4a). The compounds with the largest variability (based on standard deviation) were as follows: glycerol, ethyl hexanoate, ethyl octanoate, 2-phenyl ethanol, isoamyl acetate, and 4-vinyl guaiacol (Fig. 2 and data not shown). These compounds are all important for the aroma and mouthfeel of wine and are produced as byproducts of glycolysis (glycerol), fatty acid metabolism (ethyl hexanoate and ethyl octanoate), amino acid catabolism (2-phenyl ethanol and isoamyl acetate), and grape derived phenolic precursors (4-vinyl guaiacol).

Figure 4.

Phenotypic relationships among wine yeast strains. (a) Agglomerative hierarchical clustering (Ward's method) of the metabolite profiles (using all 29 metabolites measured in this study) of the 69 wine yeast strains was performed to generate a dendrogram. Unique clusters are highlighted in a separate color: Group 1, pink; Group 2, green; Group 3, blue; and Group 4, brown. (b) Principal component analysis of metabolite profile dataset. The first two principal components are shown and explain 93% of the data. Strains are shown as dots colored according to groups identified in the dendrogram in A. (c) Total ethyl and acetate esters (excluding ethyl acetate) by groups identified in the dendrogram in A. The calculated ratios of acetate esters to ethyl esters are as follows: Group 1, 4.4; Group 2, 2.0; Group 3, 1.7; and Group 4, 2.4.

A PCA using the same 29 yeast-derived metabolites was also performed. This analysis explained 77% and 16% of the variance in the first two discriminant axes, respectively. The metabolites most responsible for the variation are 2-phenyl ethanol (a volatile phenol) and glycerol (an alcohol) along PC1 and isoamyl acetate (an acetate ester) and 4-vinyl guaiacol (a volatile phenol) along PC2. Groups one and three were spread across PC2 axis, whereas groups two and four were spread across PC1 (Fig. 4b). Several strains were shown to be phenotypically similar by both hierarchical clustering and PCA.

One distinguishing characteristic between the groups was the difference in ratios of classes of aroma compounds (Fig. 4c). There are two types of esters in wine: acetate esters and ethyl esters. Variation in the ratios of these two classes of compounds was observed between the groups. Group 1 showed the highest acetate to ethyl ester ratio, whereas Group 3 showed the lowest. Ethyl acetate, the most abundant ester in wine, was excluded from the total acetate ester concentration due to the high concentrations.

Included in this study is a distinct group of 14 commercial wine yeast strains commonly known among winemakers and yeast producers to be related; in addition, these strains have been shown to be genetically similar (Dunn et al., 2012). These strains belong to the Prise de Mousse (PDM) family (LVCB, DV10, Elegance, 4F9, Rhone4600, EC1118, QA23, NT116, NT112, NT202, N96, IOC-18-2007, Pris De Mousse, and Premier Cuvee) and were found to be genetically distinct from other wine yeast strains, suggesting that they may be an ancestral population of closely related wine yeast (Rossouw et al., 2008, 2009, 2010, 2012; Rossouw & Bauer, 2009; Dunn et al., 2012). In this study, the PDM family spreads across the phenotypic landscape with representatives in each of the four clusters; however, the majority (eight of the 14) of the strains are found in cluster one (Fig. 4a). The remaining six strains are spread evenly across the three other clusters.

Discussion

Continuous selection, over potentially thousands of years, has led to a set of yeast strains optimized for winemaking. An understanding of how these yeasts influence the properties of wine flavor, aroma, and mouthfeel provides the basic knowledge necessary for appropriate strain selection. This study provides a description of the phenotypic differences for 69 yeast strains, including fermentation kinetic differences and the strains’ metabolic footprints, during the course of a wine fermentation using identical juice. The fermentation time was highly variable, ranging from 5 to 18 days suggesting that there may be differences in the tolerances of yeast to the extreme conditions that are present toward the latter stages of fermentation (high ethanol levels, nutrient limitations, low pH, etc.). However, all strains were eventually able to ferment all consumable sugars, a condition termed ‘dryness’ in the wine industry (sugar ≤ 2 g L−1), suggesting they are able to survive throughout the winemaking process. Grape varietals have a significant impact on the formation of yeast metabolites. For example, grape varieties are composed of distinct amino acid profiles and, therefore, have differential precursor levels for formation of fusel alcohols and esters. We selected Chardonnay for this study because it is primarily characterized by a suite of yeast-derived esters (Moreno-Arribas & Polo, 1993). In addition, Chardonnay is the top-selling varietal wine by volume in the United States, so there are clear economic benefits to better understanding how differences in metabolite production can contribute to differences in Chardonnay aroma and mouthfeel.

One impact yeast have on mouthfeel is through the production of glycerol. Glycerol is an important compound because not only it protects the cells against high osmotic pressures early in alcoholic fermentation, but at high concentrations (> 10 g L−1) it can confer sweetness and a positive mouthfeel sensation. For these reasons, a strain that produces elevated levels (> 10 g L−1) may be desirable (Remize et al., 1999). The highest producer of glycerol was strain NT50, producing 10 g L−1. NT50 is an incomplete hybrid between S. cerevisiae and S. kudriavzevii (Dunn et al., 2012), and indeed, S. cerevisiae/S. kudriavzevii hybrid strains have been shown to produce moderately higher concentrations of glycerol than S. cerevisiae strains (González et al., 2007). NT50 contains a complete set of S. cerevisiae chromosomes in addition to a copy of S. kudriavzevii chromosome 8. Yeast strain NT45 is genetically very similar to NT50, containing a complete set of S. cerevisiae chromosomes, plus S. kudriavzevii chromosome 8, and additionally S. kudriavzevii chromosome 14. The S. cerevisiae genome contained both NT45 and NT50 has been shown to be very similar to that of the PDM family of strains (Dunn et al., 2012), possibly indicating that these two strains share a common ancestor. We found that NT45 produced the third highest concentration of glycerol among all strains (8.4 g L−1). Note, though, that the two other S. cerevisiae/S. kudriavzevii hybrid strains included in our study (Vin7 and EPII) did not produce higher-than-average glycerol levels, although both of these strains contain significantly more of the S. kudriavzevii genome than NT45 and NT50 (Dunn et al., 2012), making it difficult to directly compare the strains.

The S. cerevisiae/S. kudriavzevii hybrid yeast strain Vin7 contains a complete set of chromosomes from both lineages (Borneman et al., 2012). Vin7 was the only strain in the study to show significant differences in methanol concentration, twice that of any other strain. Hybrid yeast strains are often used in the wine industry to introduce new or unique aroma profiles (Belloch et al., 2009; Bellon et al., 2011; Perez-Traves et al., 2012). As described above for glycerol, our results support the idea that a yeast strain with a unique genetic background can be used to create a wine with a unique metabolite profile.

The yeast metabolic footprint is primarily composed of fusel alcohols and esters. Chardonnay is typically characterized by ethyl esters (ethyl butanoate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate), and the acetate ester, hexyl acetate. Each of these compounds is associated with a specific fruity aroma; the fact that we see variation among yeast strains in these compounds indicates that each strain may yield a unique metabolic footprint, resulting in a unique wine sensory profile. We found considerable variability in metabolic footprints and fermentation characteristics. Ethyl butanoate, ethyl decanoate, and n-propyl alcohol were compounds determined to have the variability, whereas the lower variability was seen in ethanol production, maximum cell concentration, and nitrogen consumption. Each of these qualities has important industrial implications.

Previous studies have shown that commercial wine yeasts are genomically and phenotypically distinct from other industrial yeast strains (beer, bread, and sake), as well as laboratory strains, pathogenic strains, and ‘wild’ yeast strains (Legras et al., 2007; Camarasa et al., 2011; Dunn et al., 2012). Our work focused on the differences between commercial wine yeast strains, rather than industrial S. cerevisiae yeast as a whole. Although it has been appreciated that there are phenotypic differences among wine strains, this is the first study to perform an extensive analysis of fermentation characteristics and metabolite production of wine yeast in a grape juice fermentation. Hierarchical clustering of our metabolite data identified four clusters of wine yeast strains, where each cluster is distinguished by patterns in the metabolic footprint; strains within each cluster perform similarly in Chardonnay fermentations. For example, each cluster displays a distinct ratio of acetate to ethyl esters (Fig. 4c). The two classes of esters are formed through discrete sets of precursor compounds through the action of specific enzymes. Overexpression of ATF1 causes an increase in acetate esters, whereas overexpression of FAS1 or FAS2 increases the concentration of ethyl esters (Furukawa et al., 2003; Varela et al., 2012). Groups 1 and 4 have the highest acetate ester to ethyl ester ratio, providing insight into important phenotypic and potential genetic differences between the clusters of strains.

It has been suggested that wine yeast strains have co-evolved with the advance of agricultural technologies and have spread throughout the world with grapevines (Pretorius, 2000). It has been reported that genetic lineages by continent can be distinguished in wild yeast strains, whereas commercialized wine yeast strains segregate with the strains of European origin (Legras et al., 2007; Camarasa et al., 2011; Schuller et al., 2012). The genetic diversity seen in ‘wild’ wine yeast strains, or between yeast strains associated with different industries, has been somewhat lost through the commercialization of strains (Liti et al., 2009), with wine yeast strains being commercially produced since the 1960s (Pretorius, 2000). In the 50 years since, commercial wine yeast strains have been in close proximity to each other, introducing the potential for mixing and mating, creating the admixed population that we see today (Dunn et al., 2012). The continued practice of winemaking has resulted in the ‘selection’ of strains with ideal fermentation properties. In this study, we have focused on the differences among commercial wine yeast under winemaking conditions, providing insight into the diversity of one economically and culturally important class of yeast strains. This work, in combination with recent genetic studies, may help us begin to correlate the genetic signature associated with wine yeast to metabolic profiles influencing industrially important traits.

Acknowledgements

We thank Jessica Parsons, Ivonne Dresser and all members of the Analytical Laboratory at E. & J. Gallo for technical assistance; Nick Dokoozlian and Mike Cleary for helpful discussions; and Caleb Richter for critical reading of the manuscript.

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