Sulfur taint in wine production: Genesis and exodus

A winemaker is continuously on guard to ensure the earliest possible detection of issues that could have potentially negative effects on the quality of in-process or finished wine. One issue that is treated with the utmost respect, and attended to with some alacrity, is sulfur taint, the primarily olfactory manifestation of sulfur compounds in the wine. Sulfur taint is the bane of the winemaker because ( Lansing, Wine defects during fermentation, Wine Business Monthly, April 2011):

• It is generally associated with negative aromas
• It has a low threshold for sensory detection
• It has high chemical reactivity
• It is difficult to mask and/or remove.

The sulfur compounds associated with sulfur taint, and the population of odors associated therewith, are illustrated in the figure below.

In this post I will examine the origins of sulfur in must, the creation of sulfur compounds during winemaking, and strategies for minimizing the incidence and/or removing these compounds from the medium.

Sulfur taint has its origins in either the vineyard, the cellar, or both. In the vineyard, elemental sulfur is sprayed on the vines to combat the potential effects of powdery mildew. If this spraying is conducted too close to harvest, portions of the sulfur will remain on the grapes and make its way into the fermentation process. An example of sulfur-like off odors created in the cellar is the case of hydrogen sulfide production by the yeast to synthesize the sulfur-containing amino acids methionine and cysteine. This process is facilitated by the reduction of sulfates via the sulfur-reduction pathway. A lack of intracellular nitrogen will not curtail the process and the excess hydrogen thus created cannot be incorporated into the amino acid. Rather, it is secreted into the medium (Kennedy and Reid, Yeast nutrient management in winemaking, The Australian and New Zealand Grapegrower and Winemaker, 537, October 2008).

A listing of the sources of sulfur-like off odors is presented in Table 1.

Table 1: Sources of sulfur taint in wine production.

Environment	Source	Action	Impact
Vineyard	Elemental sulfur used as fungicide	Reduction during fermentation
	Sulfur-containing pesticides	do.
	Excess of metal ions
	Vine stress
	Unsound fruit
Cellar	Cold soaking	Growth of yeasts such as Kloeckera	Depletion of amino acids and micronutrients
	Native Yeasts	High hydrogen sulfide production	Compete against other yeasts for dominance of fermentation
	Excess hydrogen sulfide from sulfate reduction	Hydrogen sulfide used to synthesize	Absence of nitrogen causes produced hydrogen sulfide  to be secreted into the medium
	High levels of sulphur dioxide added to must at crush	Allows sulphur dioxide to bypass the sulfate reduction system	Sulfur dioxide enters the yeast cell directly
	Vitamin shortage in high YAN musts
	Nitrogen limitation	Produces sulfur-like off odors	Production begins 30 minutes after ammonia starvation initiates

Source: Compiled from Lansing and Kennedy and Reid)

The timing of the production of sulfur-like off odors is shown in Table 2 below.

Table 2. Production timing of sulfur taint by sulfur class.

Sulfur Class	Production Timing	Source
Hydrogen Sulfide	Early in fermentation (2 - 4 days)	Nitrogen/vitamin deficiency
	Fermentation end	Degradation of sulfur-containing compounds
	Sur lie aging	Autolysis
	In bottle	Generally under screw cap
Higher Sulfides	Late in fermentation/Sur lie aging	Release of compounds by metabolically active yeasts Degradation of sulfur-containing amino acids Degradation of cell compounds during autolysis

Source: Compiled from Lansing

There are a number of precautionary steps that can be taken to minimize the potential for sulfur taint (Lansing; Kennedy and Reid):

• Minimize the use of sulfur in the vineyards and cellar
• In the vineyard, ensure adequate time spacing between application and harvest
• Press stressed fruit separately
• Provide adequate nutrition to support the yeast during alcoholic fermentation
• The less assimilable nitrogen in the must, the greater the production of hydrogen sulfide
• Keep yeast cells suspended in the tank during fermentation
• Allows an even distribution of fermentation
• Allows the yeast full access to distributed nutrients
• Manage fermentation temperatures
• Hydrogen sulfide tends to form more commonly in hot, fast fermentations
• Mix the tank contents to prevent stratification
• An especial risk in tall, narrow-diameter tanks
• Remove wine from lees at the first hint of trouble
• Smell, smell, smell.

In the cases where the odors are manifested in the wine, remedies include (i) blowing it off through volatility; (ii) inert gas sparging; (iii) precipitating with copper additions; and (iv) fining.


©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