Reviewing YAN and Hydrogen Sulfide: Part 1
By: Denise M. Gardner
Yeast assimilible nitrogen (YAN) is the sum of the amino acid and ammonium concentrations available in the grape juice at the start of fermentation. Typically, the amino acid proline is not included in the reported amino acid content as it is not readily utilizable by yeast cells.
The amino acid component of YAN is often referred to as the “organic” YAN form. In contrast, the ammonium ion content is referred to as the “inorganic” YAN form and may be written in its ionic abbreviation: NH4+. Due to the fact that ammonium is only connected to a series of protons (H+ ions), it tends to be easier to move in and throughout the yeast cell to be consumed during fermentation (Mansfield, 2014). When these two components (organic + inorganic) are added together, the resultant value is the YAN, written with the units: mg N/L.
The winemaking challenge associated with YAN is the fact that it is quite variable, and current research has not identified ways to change the YAN, predictively, in fruit through the manipulation of vineyard practices. YAN varies by vintage year, grape variety, cultivar, and with the use of various vineyard management practices. In Penn State’s research vineyards, ~1 acre in size and containing 20 different wine grape varieties, YAN values ranged dramatically each vintage year amongst the various wine grape varieties. On any given vintage year YAN values ranged from low (<100 mg N/L) to high (>300 mg N/L) amongst the varieties grown in that one site.
The variability associated with YAN provides a secondary challenge to winemakers: the lack of predictability associated with hydrogen sulfide formation during primary fermentation due to unfulfilled nitrogen needs by wine yeasts.
What does YAN have to do with Hydrogen Sulfide?
Winemakers often talk about YAN in relation to hydrogen sulfide (H2S) as the two have been associated with one another throughout primary fermentation. Although there are several potential causes of hydrogen sulfide formation during wine production, some of which we will talk about in our Part 2 series, nitrogen imbalance has been one of the factors that winemakers can influence through production. Unfortunately, there is no way to ensure that a wine will not produce hydrogen sulfide by the end of fermentation, but treating wines with proper nutrient supplementation can help minimize the incidence of hydrogen sulfide production during primary fermentation.
Hydrogen sulfide is produced by the yeast cell via the sulfate reduction pathway (Figure 1). While I know this figure looks scientifically daunting, we can try to simplify its purpose to discuss how hydrogen sulfide is released into wine. Sulfate (SO42-), naturally abundant in grape juice (Eschenbruch 1974), is transported into the yeast cell for amino acid (cysteine and methionine) development, which are naturally lacking in concentration in grape juice (Bell and Henschke, 2005). Energy is used by the yeast (represented as ATP in Figure 1) to chemically alter the structure of sulfate in order to make it useable by the yeast cell. This useable form can be seen as sulfide (S2-) in the image below. Using nitrogen, which is required to make an amino acid, the sulfide content is depleted as cysteine and methionine amino acids get produced. Therefore, as sulfide reserves are depleted, cysteine and methionine contents generally increase to be used for building proteins that will be needed by the existing or new yeast cells.
Sulfur dioxide (SO2) plays a role in the sulfate reduction pathway in that it bypasses the transport mechanism required to bring sulfur into the yeast cell. It other words, it can diffuse across the cell membrane and into the internal parts of the yeast cell. Sulfur dioxide will get chemically altered to be made into the useable sulfide , S2-, form as well. Therefore, fermentations that contain a high concentration of sulfur dioxide at the start of fermentation have the potential to increase the utilization of sulfur dioxide during yeast metabolism.
These processes function normally until a depletion of nitrogen (from the nitrogen pool) or an accumulation of sulfide develops in the yeast cell.
If there is not enough nitrogen (low YAN fermentations) available to make the sulfur-containing amino acids (cysteine and methionine) then, eventually, the yeast cell will not be able to continue manufacturing these amino acids. In this situation, the sulfide concentration generally starts to increase within the yeast cell.
The chemical form sulfide, however, is toxic to the yeast cell and thus, the yeast will try to eliminate it from its internal structures. Therefore, when sulfide concentrations get too high, the yeast will diffuse this across its cell membrane into the surrounding media: the fermenting juice. When hydrogen sulfide concentrations get high enough in the fermenting juice, winemakers can often sense the rotten or hardboiled egg aroma associated with the compound.
What if there is too much nitrogen?
In contrast, too much nitrogen (high YAN fermentations) can also be problematic. Higher concentrations of the inorganic component of YAN can lead to a high initial biomass (population) of yeast. The rapid increase in yeast populations can lead to nutrient starvation by a majority of the yeast when the wine is about almost finished completing fermentation. With a large biomass of yeast incapable of obtaining the proper nutrient (nitrogen) content to grow and reproduce, hydrogen sulfide development can result. This is due to the fact that there is a large population of yeast in situations in which there is not enough nitrogen to support their growth (i.e., there is not a lot of food to go around for all of the yeast cells). With hydrogen sulfide development occurring late in primary fermentation, it is obvious that the winemaker would become concerned with hydrogen sulfide retention by the time fermentation is fully complete.
Too much nitrogen can also cause other quality problems. Due to the excess amount of available nutrients, yeast can grow and reproduce quickly, which often leads to very rapid or very hot fermentations. The speed of fermentation, of course, can affect the aromatics and quality of the wine (i.e., fast fermentations often lead to simpler aroma and flavor profiles). This may not be an issue with some styles of wine, but for many white wine or fruit (other than grapes)-based fermentations, aromatic retention is often a priority by the winemaker.
Due to the fact the initial YAN is so high, all of the nitrogen contents may not be utilized by the yeast population by the end of fermentation, and could remain in suspension in the finished wine. As yeasts begin to autolyze, all of their inner components, including the remaining nitrogen content, will become available in the wine. The excess “food” could be available for other microorganisms (like acetic acid bacteria, lactic acid bacteria, or Brettanomyces), which could potentially lead to spoilage problems if the wine is not properly stabilized. Such spoilage is, obviously, detrimental to wine quality and undesirable by the winemaker. Alternatively, remaining nutrients could be utilized by malolactic bacteria or those wines that will be given tirage for sparkling production (Bell and Henschke, 2005).
Finally, higher YAN concentrations can lead to an increased risk of ethyl carbamate production in wine; ethyl carbamate is a known carcinogen that can give susceptible individuals headaches, or even respiratory illness. Ethyl carbamate is produced in a reaction between ethanol and urea (Bell and Henschke, 2005). The heavy use of DAP has also been linked to a higher potential risks of ethyl carbamate due to the fact that DAP inhibits the transport of amino acids into the yeast cells, and therefore, leaves a higher concentration of amino acids available that can potentially be altered into urea, a precursor for ethyl carbamate (Bell and Henschke, 2005).
The fact that excess nitrogen can be problematic during wine production should provide insight to winemakers to avoid over-supplementing their fermentations. Hence, it is often recommended to that winemakers measure and identify their starting concentration of YAN and supplement accordingly.
Nitrogen (nutrient) management and supplementation is not uncommon during primary fermentation as nutrients are an important component of yeast cell growth and metabolism. In the yeast cell, nitrogen is a required nutrient in the synthesis of amino acids and to build proteins that are used in the yeast cell walls and organelles, as discussed above. Without protein development, the yeast cell cannot live.
Winemakers can supplement their fermentations with nitrogen by adding nutrient supplements in the form of:
- Hydration nutrients (e.g., GoFerm, Nutriferm)
- Complex nutrients (e.g., Fermaid K, Nutriferm)
- Diammonium phosphate (DAP)
DAP is considered an inorganic form of nitrogen, while the complex nutrients may contain additional organic yeast components that contribute organic forms of nitrogen. Recall, above, that the inorganic form of nitrogen is more readily consumed by yeast, and it can be easily absorbed by yeast cells even as alcohol concentrations rise during primary fermentation. Amino acids, on the other hand, require energy expenditure in order to be brought into the cell through transport proteins located on the cell membrane. The presence of both alcohol and ammonium ions inhibit the transfer of amino acids from the juice into the yeast cell (Santos, 2014). Therefore, it is often recommended to avoid the addition DAP or products that contain DAP (i.e., Fermaid K, Nutriferm Advance) at inoculation and until after yeasts have the opportunity to best absorb amino acids. If you are looking for some guidance on when to add nutrients to your fermentation, please refer to our Wine Made Easy fact sheet on the Penn State Extension website.
Starting YAN Concentrations
Nonetheless, nutrient supplementation strategies are often based on starting YAN concentrations in the fruit. Due to the regular variability of YAN concentrations, winemakers are encouraged to measure YAN for each lot of grapes every year. This is often problematic for winemakers whom do not have the time to run the appropriate analyses associated with YAN or the financial resources to send samples to an analytical lab. Such challenges force many winemakers into a situation in which all fermentation lots are treated with the same repeated nutrient supplementation regardless of the starting concentration of YAN.
In previous Extension workshops, research from Cornell University on Riesling wine grapes found that they could accurately predict the harvest YAN when good field samples were taken within 2 weeks from harvest (Nisbet et al., 2013). In 2016, Cornell released a second publication that focused on YAN prediction models for Cabernet Franc, Chardonnay, Merlot, Noiret, Pinot Noir, Riesling, and Traminette. While the prediction models were not recommended for regions outside of the Finger Lakes (where the data was sourced from for this study), they found that in some cases, YAN data could be obtained within 5 weeks of harvest (Nisbet et al., 2014). This extra flexibility in time can aid in obtaining accurate YAN results before the grapes reach the crush pad, which ultimately helps winemakers prepare for nutrient supplementation before the start of fermentation.
Until further research can provide predictive modeling for other wine regions, it is generally accepted that winemakers should measure YAN at or as close to harvest as possible.
YAN can be measured using the following the analytical procedures:
- Enzymatic methods for both primary amino acids and ammonium.
- Probe for ammonium ions.
- Formol titration
While the Formol titration is often preferred by many small wineries due to the lower start-up investment, the use of formaldehyde, a known carcinogen and lung irritant, in this protocol does require some consideration for laboratory safety. Additionally, the proper disposal of formaldehyde, a hazardous substance, can be an issue for many wineries.
Enzymatic methods by spectrophotometer definitely require a bit of experience in order to become more efficient in their use, which can be problematic for those operations that find measuring YAN too timely. Additionally, enzymatic kits have to be purchased fresh and have a small shelf life. The advantage of investing in a spectrophotometer, however, is that other enzymatic kits can be purchased to measure additional wine components including residual sugar, malic acid, and acetic acid.
Nonetheless, measuring YAN should be a consideration for wineries that struggle with hydrogen sulfide aromas by the end of primary fermentation. It is through the starting numerical value that winemakers can better manage and adjust nutrient supplementation strategies to help minimize the reoccurrence of hydrogen sulfide at the end of fermentation.
Nutrient availability during primary fermentation is only one potential contributor to hydrogen sulfide formation in wines. In the next blog post, we’ll explore other potential causes of hydrogen sulfide formation and how to best mediate the problem when it exists.
Eschenbruch. R. 1974. Sulfite and sulfide formation during winemaking – a review. Am. J. Enol. Vitic. 25(3): 157-161.
Bell, S.-J. and P.A. Henschke. 2005. Implications of nitrogen nutrition for grapes, fermentation and wine. Aust. J. Grape and Wine Res. 11:242-295.
Mansfield, A.K. Are you feeding your yeast?: The importance of YAN in healthy fermentation. Webinar. Feb. 2014.
Nisbet, M.A., T.E. Martinson, and A.K. Mansfield. 2013. Preharvest prediction of yeast assimilable nitrogen in Finger Lakes Riesling using linear and multivariate modeling. Am. J. Enol. Vitic. 64(4): 485-494.
Nisbet, M.A., T.E. Martinson, and A.K. Mansfield. 2014. Accumulation and prediction of yeast assimilible nitrogen in New York winegrape cultivars. Am. J. Enol. Vitic. 65(3): 325-332.
Santos, J. Getting Ready for Harvest: Yeast Nutritional Needs. Workshop Seminar. July 2014.