The case for spontaneous fermentation of wine grapes

Saccharomyces cerevisiae (SC) is the yeast species which completes the alcoholic fermentation process in both inoculated and spontaneous fermentations but the role of non-Saccharomyces (non-S) yeasts in the process should not be discounted. Spontaneous fermented wines carry a higher risk of spoilage but bring along the benefits of increased complexity, improved mouthfeel, and a higher degree of flavor integration (Jolly, et al.). This post, drawing heavily on the work of Jolly, et al., takes a closer look into the functioning of non-S yeasts in alcoholic fermentation.

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). Wine-associated yeasts are identified in the table below.

The general consensus was that all non-S yeasts died shortly after the beginning of alcoholic fermentation but, according to Jolly, et al., that is not borne out by more recent reasearch. Rather, the progression, they say, is as follows:

• H. uvarum is usually present in the highest numbers initially, followed by various Candida spp.
• The majority of the non-S yeasts disappear during the early stages of a vigorous fermentation
• May be a result of :
• Slow growth
• Inhibition of the combined effects of SO2, low pH, high ethanol, oxygen deficiency, nutrient limitation, and size or dominance of SC inoculants
• Non-S yeasts that do survive and are present till the end of the fermentation (Z. bailii, Pichia spp.)may have a higher tolerance to ethanol.

Jolly, et al., have conducted a literature survey which has identified a number of benefits that accrue to the practitioners of spontaneous fermentation:

• Lower ethanol yields -- as the authors point out, these yields "are sometimes the result of wines with higher residual sugar"
• A range of metabolic products to include terpenoids, esters, higher alcohols, glycerol, acetaldehyde, acetic acid, and succinic acid
• Hydrolization of glycosolated flavorless precursors by the enzyme ß-glucosidase to form free volatiles that can improve the flavor and aroma of wine 
• Several flavor and aroma compounds are present in the grapes as glycosolated flavorless precursors
• Enzyme ß-glucosidase not encoded by the SC genome
• Several of the non-S yeasts possess varying degrees of the enzyme
• Contribution to flavor production -- Non-S yeasts can be divided into neutral and flavor-producing yeasts. P. anomala, K. apiculata, and Candida pulcherima are flavor-producing non-S species, with the latter being known as a high producer of esters.
• Some non-S yeasts can consistently produce high glycerol concentrations during fermentation
• Glycerol important for regulating cell redox potential during fermentation
• Glycerol also contributes to smoothness, sweetness, and complexity of wine
• Glycerol production can also be associated with increased acetic acid production.

In summary, there are risks associated with spontaneous fermentation but those risks seem to be more than offset by the benefits that accrue to the user. And the number of great wines in France and Italy that utilize this approach attest to the fact that most of the leading producers have arrived at this conclusion.

Bibliography
E.J. Bartowsky, Bacterial spoilage of wine and approaches to minimize it, Letters in Applied Microbiology.
Neil P. Jolly, et al., Not your ordinary yeast; non-Saccharomyces yeasts in wine production uncovered, FEMS Yeast Research, 14 (2).
Loureiro and Malfeito-Ferreira (Spoilage yeasts in the wine industry, International Journal of Food Microbiology 86, 2003).

©Wine -- Mise en abyme

Nitrogen as a yeast nutrient in alcoholic fermentation

Wine is a result of using yeasts in an anaerobic (oxygen-free) environment to convert sugars from pressed grape juice into ethanol in the two-step process illustrated in the figure below. The first step -- glycolysis -- results in the 6-carbon glucose being split into two 3-carbon pyruvate molecules. In the next step --- alcoholic fermentation (AF) - four atoms of oxygen and two atoms of carbon leave the pyruvate, resulting in acetaldehyde, which is subsequently converted into ethanol.

Alcoholic fermentation (Source: http://alcoholicfermentation.net/)

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. The yeast that receives most of the credit -- and does most of the work -- in alcoholic fermentations 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.). Yeasts require nutritive support to allow the performance of the above functions in the hostile environment (ethanol-rich, acidic) of the fermentation tank. It is the nitrogenous aspect of that support that is the focus of this blog post.

Proteins are used by the yeast as (i) enzymes for the glycolytic pathway (indicated above), (ii) permeases in the cell membrane responsible for the transportation of compounds into the cells, (iii) cellular constituents (Kennedy and Reid, Yeast nutrient management in winemaking, The Australian and New Zealand Grapegrower and Winemaker, 537, October 2008). These proteins are synthesized by the yeast and nitrogen (N) is a key component in that process. According to Kennedy and Reid, "Efficient protein synthesis is needed for efficient sugar transport and overall yeast metabolism."

According to Schwarcz and Schoeninger (Stable Isotope Analysis in Human Nutrition, Yearbook of Physical Anthropology 34, pp. 293-321), almost 100% of exchangeable nitrogen is found in the atmosphere or dissolved in the world's oceans and is transferred from these environments into the biological system through the processes illustrated in the figure below.  Grape vine plants receive their nitrogen through this terrestrial nitrogen cycle.

Source: http://tolweb.org/notes/?note_id=3920

The nitrogen content of grapes are affected by variety, rootstock, climatic conditions, soil composition, vineyard management practices, fertilization, irrigation, rot incidence, and grape maturity (Kennedy and Reid). The yeast cells extract yeast assimilable nitrogen (YAN) from the grape must in the form of ammonia (preferred source of nitrogen for yeast growth as most easily assimilated) and amino acids and these are stored in the cell walls for later use. This extraction and storage of YAN is front-loaded in the AF process.

Nitrogen is required throughout the fermentation process with larger amounts being utilized during the exponential growth phase of the yesats and small amounts during the stationary phase. In some cases the grape must does not provide adequate amounts of assimilable nitrogen and is supplemented by added nitrogen in the form of diammonium phosphate (DAP). Juice levels of < 25 mg/L ammonia or < 150 mg N/L (measured as YAN) is considered nitrogen-deficient (UCDavis). Insufficient nitrogen can result in sluggish/stuck fermentations or sulfide formation (sulfur-like off-odors, mercaptans, and sulfur-containing acetic esters; the less assimilable nitrogen in the must, the more hydrogen sulfide will be produced). Supplements are best added incrementally and proportional to yeast growth (UCDavis).

Excessive nitrogen in the must can lead to elevated levels of ethyl carbamate (a supposed carcinogen) or urea excretion. The levels of nitrogen required for a successful AF is dependent on (Kennedy and Reid):

• Initial must YAN
• Yeast strain
• Fermentation temperature
• Initial grape sugar
• Other factors.

Amino acids are the building blocks of proteins and, when brought into the yeast cell, can be incorporated as is, transformed into a different amino acid, or broken down as a source of nitrogen or sulfur. The amino acids taken up by the yeasts from the grape must is primarily stored in the vacuole to be used for protein synthesis during yeast growth. Once access to inorganic nitrogen becomes difficult, the yeast begins to break down the stored amino acids to provide nitrogen for protein synthesis. The most important amino acids taken up by the yeasts are shown in the table below.

Amino Acid	Characteristics
Proline	Not metabolized appreciably by yeasts under winemaking conditions One of the predominant amino acids along with Arginine and Glutamine Main amino acid from low-fertilization vineyards
Arginine	One of the predominant amino acids Breakdown results in formation of urea and ammonia (During wine storage, urea can react with ethanol to form ethyl carbamate, a carcinogen). Located mostly in grape skin so processing practices could influence content in juice Main amino acid from low-fertilization vineyards
Glutamine	One of the predominant amino acids Favored by yeasts because it can be broken down to glutamate and ammonia

The less-important amino acids taken up by the yeast cells are alanine, serine, and theronine.

©Wine -- Mise en abyme

Characteristics of high-quality wine grapes

As the starting point for the production of a quality wine, it is imperative that high-quality grapes be delivered to the cellar door. I posit that high quality grapes are a funcntion of both what is within and what is without. Further, what is within can be divided into two broad classes: objective and subjective.

The objective criteria revolve around the components of grape juice at maturity, with soluble solids (major component being sugar), acid, and pH being the primary indicators based on (i) abundance and (ii) ease of measurement.

• Soluble solids -- 6-carbon sugars (glucose and fructose; 90 - 95% of total), non-fermentable sugars (inclusive of 5-carbon pentose sugars), pectins, acids and their salts, tannins, pigments, and dry extract.
• Acid – primarily tartaric acid (5 - 10 g/l in grapes) but also includes malic (2 - 4 g/l), citric, and other acids.
• pH – measure of the amount of hydrogen ions in a solution. Regarded as the acidic strength of the solution.. Increases with increases in sugar concentration. According to Wynboer, not a reliable measure on its own. Should be between 3.1 and 3.3 for white grapes and 3.3 - 3.5 for red grapes.

  Table 1. Summary of objective grape quality measures (Source: UCDavis)

Wine Type	Soluble Solids (%)	Tartaric Acid (g/L)	pH
Sparkling	18 - 20	7 - 9	2.8 – 3.2
White Table	19 - 23	7 - 8	3 – 3.3
Red Table	20 - 25	6 – 7.5	3.2 – 3.4
Sweet Table	22 - 25	6.5 - 8	3.2 – 3.4
Dessert	23 - 26	5 – 7.5	3.3 – 3.7

Soluble solids are reported in Brix, specific gravity, Baume, or Oeschle, depending on preferences, and are measured by refractometry (juice in the vineyard) or hydrometry (during fermentation). Anthocyanin and total phenolic levels are measured using Near-infrared and Spectrophotometry, equipment that is not readily available to most wineries. Titratable acidity (TA) is measured by acid-base titrations while pH is measured using a pH meter.

It should be noted that, depending on winemaker preferences or climatic conditions, some of the soluble solids and acidity level metrics may be over- or under-shot and is then adjusted during the winemaking process by acid/sugar addition or subtraction.

The subjective criteria for quality are as follows: color; ease of removal of berries from pedicel; texture; aroma; and flavor. These tests should be conducted by the winemaker during vineyard walk-throughs. Of the objective tests mentioned, the test for flavor is the most important. Bisson (UCDavis) sees optimal maturity as assessable only by monitoring flavorants themselves but such a task is laborious and expensive and has to be approximated through tasting.

But all of the quality elements associated with the grape are not subcutaneous. 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). 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 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).

It is the yeast element of this microflora that natural-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.

Wine-associated yeasts are identified in the table below.

©Wine -- Mise en abyme

Potential contributors to "stuck" alcoholic fermentations

In the recent past, I wrote a post on stuck fermentations and identified a number of contributory factors. In this post I expand on a number of those factors. 

Let us begin with some definitions. According to Bisson (Stuck and Sluggish Fermentations, AJEV 50(1) 1999), “Incomplete or stuck enological fermentations are defined as those leaving a higher than desired residual sugar content in the wine at the end of the alcoholic fermentation.” Arrested fermentations are generally the result of conditions in the grape juice that lead to cell death and a concomitant reduction in viable biomass and sugar conversion. Some of the contributors to the aberrant conditions are listed below:

• High sugar concentration – According to Jackson (Wine Science: Principles and Applications, 3rd edition, Elsevier, New York, 2008), at sugar concentrations in excess of 25 – 30%, the potential for stuck fermentations increases significantly. High sugar content can cause cells to lose water through osmosis and contract, resulting in slow or incomplete fermentation. High sugar content also increases the production of acetic acid and its esters.

• Nutrient limitation – According to Bisson, lack of macronutrients such as nitrogen and phosphate can lead to stuck fermentations; as will an imbalance of  pH and potassium ions. Adequate levels of nitrogen are required in both the growth and stationary phases. In the stationary phase, it is required as: (i) an energy source, (ii) amino acid for synthesis of actively degraded proteins, and (iii) compounds needed to minimize the inhibitory effects of ethanol. Nitrogen is especially important in the re-synthesis of sugar transporters and the supply must be available early enough to be stored in the cell vacuole rather than have its translocation inhibited by later high ethanol concentrations. According to Allison Crowe (Avoiding Stuck Ferments, Wine Business Monthly, August 2007), macronutrients, micronutrients, and long chain fatty acids and sterols are all “important for healthy yeast growth, reproduction and survival as heat and alcohol increase in fermentation.” 

• Ethanol toxicity -- According to Jackson, S. cerevisiae is highly tolerant of alcohol toxicity but increasing alcohol buildup eventually inhibits fermentation by suppression of sugar uptake.

• Low pH -- Cerevisiae is tolerant to low pH. According to Bisson, it can grow in the range of 2.8 to 4.2 but growth and fermentation are significantly affected at levels below 2.8.

• Temperature extremes – According to Jackson, the growth rate of yeast cells is strongly influenced by temperature, with experiments showing significant reductions in cell division for every 10 degrees C increase in temperature. Excessively high temperature affects membrane fluidity and, therefore, transporter performance, and may induce stuck fermentations. Red wines are typically fermented between 24 and 27 degrees C. Dr. Murli Dharmadhikari (Lactic Acid Bacteria and Wine Spoilage, extension.iastate.edu) admonishes us to not allow fermentation temperatures to rise above 30℃. According to A.P. Haynk (The control of temperature in wine fermentation, July 1897), “For normal must with a normal yeast, the death point is 98-100℉ (36.6℃+). Before this point, however, the yeast stops working and bacteria begins to grow.

• Zymostatic and zymocidal toxins – Killer factors, molds, organic and medium-chain fatty acids, and fungicides and pesticides used in the vineyard are all cited by Bisson as potentially toxic to the yeast during fermentation. 

• Microbiological activity – This is the biggest source of volatile acidity. Could be triggered by wild yeasts, malolactic bacteria and acetic bacteria (Wynboer.co.za). Fermentations which include high populations of non-Saccharomyces yeast and bacteria are at risk of arrest because of the competition for nutrients plus the production of inhibitory factors. According to Bisson, some Saccharomyces strains are especially subject to inhibition by malolactic bacteria. An arrested fermentation of this type requires that the compromised biomass be removed in order to attempt re-starting the fermentation.

• Acetic acid -- There seems to be some confusion in the literature as to the role of acetic acid in stuck fermentations. According to Eglinton and Henschke, “Acetic acid toxicity has been suggested as a cause of stuck fermentation because it can inhibit the growth and fermentation activity of S. cerevisiae.” Rasmussen et al. (Acetic Acid as a Causative Agent in Producing Stuck Fermentations, AJEV 46(2), 1995), showed that removal of acetic acid from stuck lots with a Vinovation process resulted in prompt re-initiation of fermentation. Adding inoculants to the lots further “advanced “ the re-fermentation process. On the other side of the coin, Dr. Dharmadhikari notes “serious spoilage” occurring in musts with stuck fermentations because lactic acid bacteria can attack sugar (according to Dr. Daharmadhikari, lactic acid bacteria metabolizes sugars such as glucose and sucrose and produces lactic and acetic acid as by-products) and increase the VA levels in the wine. Bisson and Butzke see elevated levels of acetic acid leading to a stuck fermentation but they see that as a result of a problem fermentation rather than as the cause. In other words, the problem fermentation has some underlying cause which itself leads to elevated levels of acetic acid production and a stuck fermentation. In that case, getting rid of the acetic acid will still not have solved the underlying problem.

According to Bisson and Butzke, “The consensus strategy for re-initiation of stuck fermentations involves first removal of the settled yeast biomass followed by re-inoculation with a starter ‘adapted’ to the arrested environment.” Removal of the settled yeast biomass is accomplished by racking the wine off the lees and this will, in some cases, lead to a spontaneous restart of the fermentation. The conditions which are alleviated by racking off the lees are (Bisson and Butzke):

• Sterol or fatty acid limitation
• Exposure to temperature extremes. 

If there are no other contributing limitations (and, in the case of extreme temperature, once the temperature is restored to an acceptable range), racking will, in some cases, lead to a spontaneous re-initiation of stuck ferments. If, for example, one of the above limitations is combined with nitrogen or micronutrient limitations, racking alone will not suffice (Bisson and Butzke). 

©Wine -- Mise en abyme

The risks of sluggish and stuck fermentations when utilizing natural yeasts

I have previously characterized the risks associated with natural-yeast fermentations thusly: (i) stuck fermentations; (ii) yeasts washed off grapes during inclement weather; (iii) spoilage yeast contamination; (iv) lengthy fermentation times; and (v) persistence of negative characteristics.  I have begun a process of exploring these perceived risks in greater detail -- beginning with a recent post on spoilage yeast contamination -- and will continue on that path with the current post on the risks of sluggish and stuck fermentations.

Slow or sluggish fermentation is characterized by low sugar utilization by the attendant yeasts while incomplete (stuck) fermentations occur when a higher-than-desired level of residual sugar remains at the conclusion of alcoholic fermentation (Klaus A. Sutterlin, Fructophilic yeasts to cure stuck fermentations in alcoholic beverages, PhD dissertation, Stellenbosch University, March 2010).  Sluggish or stuck fermentations are the second most significant enological problem faced by winemakers (2003 American Vineyard Foundation survey and 1996 Association for the Development of Wine Biotechnology survey, both cited in Sutterlin 2010) and, with more than 60% of respondents  admitting to having experienced one or both of these problems, the economic costs are perceived as being enormous.

According to Bisson and Butzke (Diagnosis and Rectification of Stuck and Sluggish Fermentations, American Journal of Enology and Viticulture 51(2), 2000), there are four types of fermentations which deviate from the "normal" fermentation profile: (i) sluggish initiation with the rate eventually becoming normal; (ii) normal initiation becoming sluggish; (iii) sluggish throughout the entire process; and (iv) abrupt arrest late in the fermentation.

Bisson and Butzke report that slow fermentation initiation (Type (i) in the foregoing) can occur in both natural and inoculated fermentations and, in the case of natural fermentations, "the sluggish start may simply be due to low numbers of yeasts in the must and not reflect any particular problem other than an initial low biomass."  Based on their research, the authors aver that fermentation will go to completion, depending on juice conditions and the presence of other organisms, with as little as 100 viable Saccharomyces cells/mL present at initiation.

The risk of deficient (< 100 viable cells/mL) Saccharomyces populations leading to a problem fermentation is highest in the earliest portions of the crush.  As crush is prolonged, Saccharomyces bacteria will colonize the winery equipment such that juice and must passing through said equipment will have their levels of Saccharomyces elevated.

In the cases where there are low initial levels of Saccharomyces, holding juice at low temperatures is risky as it (i) encourages the growth of Kloeckera apiculata and (ii) is injurious to the existing Saccharomyces yeasts.  If the Saccharomyces yeasts are able to dominate, the fermentation will proceed to completion; if not, the fermentation will arrest.  It should be noted that the lower the level of the initial Saccharomyces population, the greater the growth requirements of the juice.  Bisson and Butzke estimate that it will take 13 generations to get from an initial level of 100 cells/mL to a typical innoculum level of  10⁶ cells/mL.

According to Malherbe, stuck fermentations can be caused by glucose/fructose ratio imbalance, nutritional limitations of the must (nitrogen deficiency, oxygen deficiency, mineral deficiency, vitamin deficiency), inhibitory substances (ethanol, toxic acids, the effects of sulphites, killer toxins, fungicide/pesticide residues), and a number of physical factors (excessive must clarification, temperature extremes, excessive use of Sulphur Dioxide).  When compared to this range of potential stuck-fermentation causative factors, the risk of low initial yeast population in natural fermentations does not seem that stark.  Bisson and Butzke has shown that fermentation can conclude successfully even beginning with yeast levls as low as 100 cells/mL and if the fermentation continues in a sluggish manner, or gets stuck, it is a biomass rather than a starter problem.  Further, the risk of sluggish initiation is not restricted to natural yeast fermentations.  According to Bisson and Butzke, poor starter culture can lead to sluggish initiation in the case of inoculated fermentations.

To conclude then, sluggish/stuck fermentations is an issue that a winemaker always has to be cognizant off and has to constantly monitor against.  There are many opportunities for this curse to be visited upon the winemaker and one of those cases is at the initiation of the fermentation where it will register as a sluggish start.  Such a manifestation could be apparent whether the fermentation is natural or inoculated.  All things being equal in the biomass, the natural yeast fermentation should right itself and proceed to completion.  There does not seem to be an outsized and determinative risk of stuck fermentations if one practices natural yeast fermentation.  There is an obvious lag phase however, as the yeast levels build up from cellar-entry levels to standard starter inoculate levels.

©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

Indigenous- versus inoculated-yeast fermentation: The pros and the cons

There is an ongoing battle between natural-wine proponents and pragmatists as to the types of yeast strains that provide the "best" results in the alcoholic fermentation of wine grapes, a battle, according to Isak Pretorius (The Power of Yeast, TONG #12) that is far from new.  According to Pretorius, once Louis Pasteur was able to show that some wild yeasts could spoil wine, the debate began as to whether pasteurization or the addition of sulphur dioxide should be utilized to kill off the spoilage agents or whether inoculated ferments should should be used in lieu of indigenous ferments.  This post looks at both sides of this continuing argument.

As described in a previous post, 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 (indigenous, wild, feral, and spontaneous used interchangeably throughout this post) 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 only species left standing.

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).

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.

According to Fugelsang, the first commercial yeast strain was introduced in 1965 by Red Star Yeast and, since that time, over 100 cultures have been commercially produced.  And winemakers continue to take advantage of these commercial strains in order to improve the capabilities of their wines. According to Suárez-Lepe and A. Marota and Pretorius, winemakers are continually on the lookout for yeast strains that can improve the technological and sensorial properties of their wines.

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.

In a future post I will treat the topic of trends in yeast selection.

©Wine -- Mise en abyme