April 2016 Issue of Wines & Vines
 
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Spontaneous Fermentations

A case study in the Finger Lakes explores where 'wild' yeast comes from

 
by Anna Katharine Mansfield and Camila Tahim
 
 
NY Riesling Sampled
 
The authors sampled this New York Riesling for microflora.

The complex microbial reactions that transform grape juice into wine involve a sequential evolution of different yeast species. Since the first (likely accidental) fermentation of grape juice, winemakers have relied on whatever yeast happened along to do the work of sugar-to-alcohol conversion. Though Dr. Hermann Müller-Thurgau first used a pure yeast culture as an inoculate in the 1890s, it wasn’t until the 1970s that commercial cultures were widely available. Winemakers turned to these products to increase predictability in fermentation speed and wine quality, especially in regions with a limited history of wine production.

More recently, the notion that wine should be terroir-driven, and that commercial yeast somehow obscures terroir effects, has prompted a resurgence of spontaneous (or “wild”) fermentations in New World wine regions. This trend seems to be driven by the perception that uninoculated fermentations produce wines of higher quality, or at least more sensory complexity. Growing consumer interest also has prompted new research into spontaneous fermentation; consequently, advances in microbial and sensory methods are shedding new light on an age-old processing method.

Microbial ecology
One question taunting winemakers and researchers alike relates to origin. Namely, where do the yeast and bacteria that bring about spontaneous fermentation come from? There are three potential sources: Organisms can originate in the vineyard, exist as resident winery microflora and be transferred through the movement of picking bins and equipment, and even move about via insects like social wasps or fruit flies. The composition of the microbial community at each source is influenced by many factors. On grape berry surfaces, populations are impacted by physical damage to the fruit, disease pressure, rainfall and use of pesticides. In the winery, practices like sulfur dioxide use, cleaning and sanitization programs, and degree of juice clarification further modify the active players in spontaneous fermentations.

Yeast in the vineyard
Saccharomyces cerevisiae, the yeast that almost universally completes wine fermentations, always was assumed to move into must from the waxy cuticle coating grapes. More recently, studies have shown that S. cerevisiae is rare on healthy fruit and can be isolated from just one per 1,000 undamaged berries. Indigenous vineyard yeast are more commonly non-Saccharomyces species, including Kloeckera apiculata/Hanseniaspora uvarum, Candida, Cryptococcus, Debaryomyces, Hansenula, Issatchenkia, Kluyveromyces, Metschnikowia, Pichia and Rhodotorula.

When S. cerevisiae strains have been identified in vineyards, they are often commercial strains that have been used in wineries associated with the site. In effect, commercial strains take up residence in the winery and migrate outward, populating the vineyard in numbers somewhat proportional to the distance traveled from the winery. This is not surprising, as 95% of strains associated with wine production have been found to share a unique common ancestry, suggesting that yeasts adapted to winemaking developed and spread with human activity. In other words, though there is evidence for region-specific subpopulations in vineyards with a long history of wine production, they are likely the result of localized gene flow between regional wild yeast and strains that evolved in the early days of wine production and were spread by humans.

A recent study in Missouri provided evidence that genetic information was exchanged between members of the so-called “grape/wine” S. cerevisiae population common to winemaking areas and the indigenous regional population that thrives on oak trees. These “wild” oak tree strains are generally unable to survive the levels of SO2 and alcohol that occur in wine fermentation. In theory, such merging of “grape/wine” and “oak tree” populations could result in the development of yeast strains in the vineyard that retain some wild characteristics but are capable of successful fermentation.

On the whole, however, there is little evidence that a population of wine-ready yeasts is lurking in every corner of the globe, just waiting for a “natural” winemaker to give it a fermentation of its own. It is probable that wine yeast are more similar to domesticated dogs that share a common ancestor but have evolved to display breed differences based on regional work conditions.

    KEY POINTS
     

     
  • Sources for the yeast and bacteria for spontaneous fermentations can come from the vineyard, from microflora resident in the winery, or from equipment, bins or insects.
     
  • Because the mix of microorganisms in spontaneous fermentations is not known, it is difficult for winemakers to manage the nutritional requirements.
     
  • The Cornell Enology Extension Lab tested the YAN requirements and microflora in five spontaneous fermentations of Riesling grapes at two Finger Lakes wineries.
     

Yeast in the winery
Several researchers have argued that the winery, rather than the vineyard, is the primary source of S. cerevisiae in spontaneous fermentations. In some studies they have been found to completely displace any yeast strains originating in the vineyard. The concept of “resident microflora” in the winery has been described frequently, and in many cases commercial yeasts have been found to adapt to winery conditions and colonize equipment for several years, even surviving typical cleaning methods.

If present, these yeast strains are often the most competitive in a given fermentation and have been found to coexist with other strains or take over spontaneous fermentations in the facility. It’s important to remember, however, that yeast reproduces rapidly and readily accepts genetic material, so that resident winery yeast often show genetic variance from their commercial strain of origin and may naturally evolve enough over time to show significant difference in activity and, consequently, wine sensory characteristics.

Sensory impact of mixed cultures
Whether spontaneous fermentations are a pure expression of vineyard micro-terroir or simply a history of previous yeasts used in a winery, there is no doubt that their varying microbial populations produce a unique assortment of wine aromas and flavors. Most microorganisms have lower tolerance for SO2, CO2 and alcohol than commercial yeast cultures, so typical inoculation methods that include post-crush SO2 treatment and pitching with robust commercial yeast severely limit the number of players that can live, die and contribute secondary metabolites (aroma and flavor compounds) in the early stages of fermentation.

In fact, mixed microbial colonies are quite literally death matches, with each species battling for limited nutritional resources and using all possible weapons to stunt the growth of all others. Defensive actions can include the release of targeted toxins (as with the infamous “killer yeasts”), organic acids or fatty acids and enzymes that, by happy coincidence, increase the variety and concentration of aroma compounds in wine.

Non-saccharomyces strains produce different acids and more enzymes (esterases, glycosidases, β-glucosidases, proteases, etc.) than most S. cerevisiae, producing different aromatic profiles and potentially enhancing mouthfeel. In most cases, the high ethanol tolerance and anaerobic capability of S. cerevisiae strains leaves them as final victors in the fermentation battle, though some other species (such as Torulaspora and Pichia) are occasionally able to survive to the bitter end. While it’s important to note that bacteria—both desirable and spoilage—also play a role in microbial interactions, the influence of various environmental factors on bacterial communities in grapes and wine is currently understudied and poorly understood.

Yeast assimilable nitrogen
Nitrogen management is another major challenge for winemakers conducting spontaneous fermentations. Existing research has established rough guidelines for yeast assimilable nitrogen (YAN) concentration for a number of commercial yeast strains and initial Brix levels, but the variables governing YAN requirements are poorly understood. In spontaneous fermentations, the mix of microorganisms is generally unknown, making the prediction of nutritional needs difficult.

Non-Saccharomyces yeast strains may cripple S. cerevisiae by depleting individual amino acids early in fermentation, and Torulaspora delbrueckii has been reported to exhaust nitrogen supplies in the first 48 hours. On the other hand, the early death and autolysis of some short-lived non-Saccharomyces yeasts, and the proteolytic action of others, may increase the pool of nutrients available for S. cerevisiae during mid- to late-stage fermentation. (After alcoholic fermentation, the subsequent death and autolysis of S. cerevisiae provides nutrients for spoilage organisms if appropriate preventative steps aren’t taken.)

Some producers argue that indigenous YAN concentration is another feature of terroir. Though that point is debatable, it is certain that the sheer number of variables impacting nitrogen consumption in spontaneous fermentation makes nutritional requirements hard to predict and manage.

Why the long fermentation?
Spontaneous fermentations have been observed to drag on for weeks or months longer than comparable inoculated juices, either getting off to a sluggish start or hanging on to the last few grams of residual sugar. A long lag phase (the period before fermentation takes off) can result from low initial YAN, nitrogen depletion by non-Saccharomyces yeasts (as discussed above) or a low initial yeast biomass (compared to that added in a commercial inoculation), compounded by the usual impacts of sub-optimal environmental parameters like temperature, pH, etc. Toxins released by “killer” yeast and some non-Saccharomyces strains also can inhibit fermentation progression.

Notably, a 2014 study by Jarosz et al. identified an inheritable metabolic change induced in yeast by common wine bacteria that allows affected yeast to metabolize non-glucose carbon sources and reduce ethanol production. This change is mutually beneficial to yeast and bacteria, prolonging their co-existence in fermentation media, but results in stuck or sluggish fermentation problematic to winemakers. The particulars of this metabolic shift are still under investigation, and its impact on nitrogen consumption is unknown.

A New York case study
In the New York Finger Lakes, YAN concentrations (especially in Riesling) are usually lower than the 140 mg of nitrogen per liter (N/L) considered the bare minimum for successful fermentation. As the popularity of spontaneously fermented Riesling has grown in recent years, the Cornell Enology Extension Lab conducted an initial assessment of YAN requirements and microflora in five spontaneous fermentations at two commercial Finger Lakes wineries during the 2014-15 season. To assess microbial populations before fermentation, grape samples were collected from each vineyard before harvest, and the surface of winery equipment was sampled using sterile cotton swabs. Once grapes were received and crushed, researchers monitored fermentation kinetics, YAN consumption and microbial populations in an effort to understand the interaction of microflora and nitrogen requirements.

As expected, fermentation times for all Rieslings were considerably longer than inoculated fermentations, though kinetics differed by winery. Winery 1’s fermentations started quickly, then slowed considerably and were ultimately stopped by the winemaker around 90 days post-inoculation. In contrast, Winery 2’s fermentations had very long lag phases, but once fermentation started, sugars were consumed at a steady rate. Differences in kinetics may be partially explained by different juice YAN concentrations or different microbial populations, so these factors were examined in greater detail.

Initial YAN concentrations were low in four of the five juices (ranging from 68-144 mg N/L), only surpassing the standard recommended level of 200 mg N/L in one juice (231 mg N/L). As in inoculated fermentations, inorganic nitrogen was consumed faster than organic nitrogen, and ammonium was exhausted from all fermentations except the one with the highest YAN. In contrast, primary amino nitrogen (PAN) concentration never reached zero; in fact, a slight increase in PAN was observed in the final stages of all fermentations. Notably, YAN consumption (calculated as [initial YAN]-[final YAN]) in all fermentations was at or below 140 mg/L.

One factor that may contribute to this seemingly small nitrogen demand is residual sugar (RS) concentration. In studies that measure nitrogen requirements, RS in successful fermentations is usually less than 1 g/L, while the wines monitored in this study retained an average 8.6 and 23.5 g/L RS (in Wineries 1 and 2, respectively) for stylistic reasons. Another, as discussed above, is the release of PAN following the death of non-Saccharomyces yeast in mid- and late fermentation. As this activity is hard to measure accurately, it complicates robust assessment of total YAN consumption.

The winery-specific fermentation profiles are partially explained by differences in microbial ecology. Hanseniaspora uvarum and species of Kluyveromyces were isolated in both vineyard samples, but the distinctions in resident winery microflora were more marked. Winery 1 had overall greater yeast counts than Winery 2, and S. cerevisiae was identified in some of the isolates. Although two Candida and one unknown yeast species were identified on equipment in Winery 2, no yeast or bacteria was detected on clarification and fermentation tanks.

The presence of S. cerevisiae at Winery 1 may help explain the rapid commencement of fermentations there, as winery microflora would be well adapted to the environment, and S. cerevisiae strains rapidly dominated all fermentations. Total non-Saccharomyces population was lower and quickly declined. The long lag phases (averaging 28 days) in Winery 2 may have been due to lower initial S. cerevisiae populations, as the non-Saccharomyces population in those wines was found to be more diverse and peaked at the same population as the S. cerevisiae.

Among the eight S. cerevisiae isolates, five commercial strains were identified, most of which had been used previously in the winery in which it was found. Different strains were isolated from different fermentation stages, suggesting that no single strain dominated fermentation. Some of the non-Saccharomyces species identified in the vineyard, equipment and fermentation samples were detected through advanced stages of fermentation. H. uvarum was isolated when fermentations had 5° Brix of residual sugar, or approximately 7.7% ethanol; Kluyveromyces was detected at the end of fermentation (10.2% ethanol) in one wine. Other non-Saccharomyces yeasts were present in fermenting musts, including Pichia. fermentans, which survived until approximately 5% ethanol, unknown Hanseniaspora and Torulaspora spp., P. anomala and Dekkera anomala. All wines were of commercial quality and have been released for sale in their respective tasting rooms.

Future…spontaneity?
The current reexamination of spontaneous fermentation methods and effects have greatly increased understanding of underlying processes, but plenty of questions—both scientific and stylistic—remain. The sheer number of yeasts and bacteria that can colonize in vineyards and wineries ensures that microflora will vary by region, winery and even grape cultivar, with each mix as variable as the harvest season that spawns it. Research examining yeast-yeast, yeast-bacteria and bacteria-bacteria interactions continues, as do investigations of the nutritional needs of mixed-colony fermentations.

At the same time, advanced techniques in yeast genotyping are providing new insight into the origin of S. cerevisiae, the concept of “microbial terroir” and the role yeast play in “natural” winemaking. One thing is for certain: Spontaneous fermentations are likely to produce a wider palette of sensory characteristics than are usually found in inoculated fermentations, but whether those characteristics are positive or negative is dictated by factors we don’t yet fully understand.


Anna Katharine Mansfield, PhD., is an associate professor of enology in the food science department at Cornell University’s New York State Agricultural Experiment Station in Geneva, N.Y., and Camila Tahim is a recent master’s degree graduate of the same department.

Resource Note
A thorough list of yeasts that can contribute to wine sensory characteristics is available from the University of California, Davis. Visit wineserver.ucdavis.edu/industry/enology/winemicro/wineyeast/index.html.

 
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