Non-Saccharomyces yeasts as antidotes to climate change: Fundamentals

In my post on the role of alcohol in wine balance I indicated that, in addition to unbalancing the wine, an excess of alcohol: makes the wine appear hot; will lead to a reduced perception of wine aroma; and can impart a sense of intoxication to the taster.  The winemaker may address the issue of excess alcohol by (i) reducing the concentration of sugar present in grapes or (ii) by removing "excess" alcohol from the wine.

Reducing grape sugar requires harvesting the grapes earlier than normal but this risks affecting wine composition and quality due to fewer aroma flavors, less color intensity, non-attainment of phenolic ripeness, and increased acidity. I have dealt with removing alcohol from the wine in a previous post and technologies/approaches employed include: (i) reverse osmosis, (ii) the spinning cone, and (iii) adding water to the wine. Reverse osmosis and the spinning cone are authorized in the U.S by wine regulation 27 CFR 24.248 Processes Authorized for the Treatment of Wine, Juice, and Distilling Materials.  Under this regulation the processes must be conducted at a Distilled Spirits Plant (DSP) or at a bonded winery that is authorized to alternate between a DSP and a bonded winery.

An additional alcohol-reducing approach has come to my attention by way of a recent Elin McCoy article which discusses how some Oregon producers were turning to non-Saccharomyces yeasts to aid in countering potential climate effects on the region's Pinot Noir wines. According to the article, wines made with the Pinot Noir grape, a famously finnicky variety, could become increasingly unbalanced in the future as the effects of climate change bring riper grapes (with higher potential alcohol) into the cellar.

Source: wineeconomist.com

According to Elin, Chapter 24, an Oregon-based producer was partnering with a professor at MIT to study the potential of non-Saccharomyces yeasts to reduce the alcohol content during fermentation without negatively affecting the aroma and flavor profile of the wines. I explore the science behind this effort in this series but begin with some background material on fermentation, Saccharomyces cerevisiae(SC), and non-Saccharomyces (non-SC) yeasts.

Wine is the result of applying yeasts to grape berries/must/juice in an anerobic environment in order to convert the resident sugars into alcohol.  The yeast that receives most of the credit -- and does most of the work -- is a species called Saccharomyces cerevisiae (SC) which is "specialized in metabolizing media with high sugar content and small quantities of nitrogenous compounds" (Suárez-Lepe and A. Marota, New trends in yeast selection for winemaking, Trends in Food Science and Technology 23 (2012), 39-50.).  According to Fugelsang (Overview of yeast selection and malolactic fermentation on aroma, flavor and phenols), the yeasts (i) extract compounds from the solids in the must/juice in order to form the "characteristic metabolites of fermentation (alcohols, esters, fatty acids, carbonyls, etc.) and (ii) cleave cysteine-containing precursors such that volatile thiols (aroma component of several varieties) can be released.  SC is the yeast species which completes the alcoholic fermentation process in both inoculated and spontaneous ferments.

Grapes in a vineyard are hosts to what Gourrand (Using non-Saccharomyces yeasts during alcoholic fermentations: taking advantage of yeast biodiversity) calls native microflora -- molds, lactic bacteria, acetic bacteria, Saccharomyces spp, and non-Saccharomyces yeasts (Pichia, Metchnikowia, Kloeckera, Kluyveromyces, Candida, Zygosaccharomyces, Torulaspora, Cryptoccus, Brettanomyces, and Hanseniaspora) -- and it is the yeast element of this microflora that the feral-yeast winemaking adherents seek to exploit.  Wild yeasts accumulate on the grapes from flowering through harvest with the presence of SC being pegged at 1 in 1000 grapes (Robert Mortimer, Vineyard Theory of Wild Yeast, UC Berkeley).  At harvest, SC is the least prevalent of the grape-resident yeast strains.

In the case of indigenous yeast fermentation, the process is kick-started and dominated initially by the "weakly fermentative" -- but numerically dominant -- non-Saccharomyces Kloeckera.  This initiation can take up to a week to begin due to the relatively small amount of wild yeasts present at startup (relative to the amount of yeast used to begin the process in the case of inoculated ferments).  For the first few days of fermentation, the weakly fermentative non-SC population dominates but is then replaced by more adaptive non-SC strains.  As the alcohol level continues to rise, the more alcohol-tolerant SC increases in number at a rapid rate such that at the end of the fermentation it is the dominant population.

Natural wine adherents assert that the progression from non-SC to SC fermentation in the vessel is an integral part of non-interventionist winemaking and adds complexity to the finished wine (Mortimer; Pretorius).  Critics of the approach see it as akin to Russian roulette because of the inherent risks (Ross; Pretorius): (i) the irregularity of natural fermentation and the associated risk of a stuck fermentation; (ii) in the event of rains around harvest time, the wild yeasts could be washed off the grapes; (iii) spoilage yeasts are often present in grape-derived yeasts; (iv) spontaneous ferments take longer to begin and longer to complete; and (v) while the positive characteristics of natural yeasts are not detectable after 6 or so months of aging, the negative characteristics tend to persist much longer.

For inoculated ferments, a large dose of SC is added to the juice/must in order to initiate fermentation.  The yeast strains utilized have traditionally been selected on the basis of the ability to start the fermentation quickly, the toleration of increasing alcohol levels, low acetic acid production, and resistance to sulfur dioxide (Ross; Suárez-Lepe and A. Marota).

The advantages that are perceived by "inoculants" are clear: (i) quick, effective, efficient fermentations: (ii) flexibility; (iii) lower risk production process; (iv) the ability to tailor the fermentation; and (v) the ability to take advantage of future advancements in commercially produced strains.  The disadvantage of the use of inoculation is, as perceived by the "naturalists," even more power placed into the hands of the winemaker to manipulate the dickens out of the wine; and the customer loses as a result.

As both Ross and Pretorius point out, the needs of large- and small-production wineries may lead to different emphasis in yeast-strain selection.  For the large producer, effective, efficient production and maintenance of quality is key and a strain that meets that need will be selected.  The smaller producer, on the other hand, is more likely to take advantage of varying yeast strains and temperature regimes as a means of enhancing the wine's aromatic and flavor characteristics.

To gain the benefits associated with both spontaneous and inoculated ferments, some winemakers are employing cocktails of strains hoping to get the "complexity of flavors ... without running the risk of contamination of spoilage yeasts" that comes along with the spontaneity.

©Wine -- Mise en abyme

The risk of spoilage yeast contamination during natural yeast fermentations

In a comment on my post on indigenous- versus inoculated-yeast fermentations, reader Ben opined that three of the five disadvantages of natural fermentation that I had listed were "discountable" but that the remaining two provided the basis for an "interesting debate."  Ben's comment has prompted me to provide readers with a fuller discussion of the mentioned disadvantages beginning with the current post on spoilage yeasts as a risk in indigenous fermentations.

Absent an inoculation, all yeasts found in grape must and wine will originate from one or more of the following sources: vineyard, grapes, or winery processing equipment (E.J. Bartowsky, Bacterial spoilage of wine and approaches to minimize it, Letters in Applied Microbiology).  According to Loureiro and Malfeito-Ferreira (Spoilage yeasts in the wine industry, International Journal of Food Microbiology 86, 2003), mature, healthy grapes harbor microbial populations (yeasts, lactic and acetic acid bacteria, filamentous molds) at levels of 10³ - 10⁵ CFU/g (colony forming unit -- a measure used in microbiology that indicates the number of micro-organisms present in a water sample (www.legionella.com/cfu)), levels that vary based on environmental conditions (rainfall. temperature, grape variety, the application of chemicals in the vineyard).  Yeasts resident on grape berries tend to congregate in areas where juice might escape (Loureiro and Malfeito-Ferreira).  Wine-associated yeasts are identified in the table below.

As seen from the foregoing, yeasts exist in the vineyard environment and on healthy grapes but, for our purposes, the intersection of yeasts and damaged grapes is of significance.  Grapes can be damaged in any number of ways (hail, birds, etc.) but it is the damage caused by phytopathogenic molds that is of greatest interest.  The effects of these molds on grapes and wine are spelt out in the table below.

Loureiro and Malfeito-Ferreira found a number of ascomycetous yeasts proliferating on grapes damaged by sour rot as well as Zygosaccharomyces spp and other spoilage yeasts such as Dekkera bruxellensis.  In a separate study on sour rot, Berata et al., isolated 17 ascomycetous species from sour-rot-damaged samples and only five from sound grapes.  The most significant find was the presence of Zygosaccharomyces bailii, a species which the authors describe as "acidophilic" and "one of the most dangerous wine spoilage yeasts."  Once introduced, this species was recovered from all of the alcoholic fermentation steps.  The authors conclude that "yeast species from sour rot grapes are an important contamination source of wineries and wines."  Loureiro and Malfeito-Ferreira make much the same argument: "For many of the most important wine spoilage species (Dekkera/Brettanomyces), the main entry to the winery is grapes affected by sour rot."  The consensus, then, is that sour rot is the primary vehicle for wine spoilage yeasts transiting from the vineyard to the winery.  Let us now turn our eyes to the winery environment.

According to Woolford et al., (Genome Survey Sequencing of the Wine Spoilage Yeast Dekkera (Brettanomyces) bruxellensis, Eukaryotic Cell 6(4), April 2007), Brettanomyces bruxellensis is a major microbial cause of wine spoilage worldwide and results in significant economic loss.  The yeast makes "the winery itself a primary habitat surviving in the walls ... interior surfaces of presses and fermentation tanks, or on the wood of barrels."  From these positions the microbe is well situated to "colonize the fermenting must or maturing wine."  Wines infected with Brettanomyces will exhibit aromas of mousiness, wet wool, burnt plastic, horse sweat, or barnyard.  According to the same authors, the severity and frequency of Brettanomyces has been on the uptick as winemaking has trended towads wines with higher levels of residual sugar and that are unsulfited, unfiltered, and aged on lees.

Brettanomyces is exceptionally dangerous because it has all of the characteristics of Saccharomyces cerevisiae (ethanol-tolerant, facultatively anaeorobic, can exist without mitochondrial DNA, ferments preferentially in the presence of high glucose under aerobic conditions) but extend beyond it in that, while slower growing, "it can assimilate a wider variety of carbon choices."  These conditions lead to the following progression in a Brettanomyces-contaminated alcohol fermentation (Woolford et al.):

• Saccharomyces cerevisiae dominates throughout primary fermentation then is replaced by D. bruxellensis during the maturation phase when ethanol concentration is high and minimal amounts of sugar remain.

It was long thought that Brettanomyces contamination was a result of poor hygiene in wineries but contamination persists even in the face of intensive hygiene efforts on their parts (Renouf et al., Interactions between Brettanomyces and other yeast species during the initial stages of winemaking, Journal of Applied Microbiology 100 (6), June 2006).  The research cited in this post seems to indicate that Brettanomyces can enter the winery through sour rot  and can then take up residence within the facility and contaminate batches of wine essentially at will.  Intensive hygiene efforts can clean up an infected location but re-contamination is potentially just another sour-rot affected batch away.  Vigilance in screening for sour rot would seem to be in the best interest of the winery.

Grape must is nutrient-rich and ethanol poor, a candy store for most micro-organisms.  The longer  those conditions exist, the more non-Saccharomyces yeasts will thrive. With the exception of Brettanomyces, most wine-spoilage yeasts are ethanol-intolerant so the quicker that high ethanol levels are obtained, the less opportuntiy for these micro-organisms to proliferate.  The issue with natural fermentations is that they start slowly and take longer to get up to levels in which S. cerevisiae thrives.  And it is in that period that the fermented wine becomes susceptible to the travails of wine-spoilage yeasts.

The remaining disadvantages of natural wine fermentation will be coverd in a number of upcoming posts.

©Wine -- Mise en abyme