Dr. Carien Coetzee
30 August 2023
Hydrogen sulphide (H2S) is a powerful aroma associated with the so-called “reductive” aroma present in some wines. Descriptors commonly used to describe H2S in wine include “rotten egg” and “sewage”. The presence of H2S in wine is generally considered a wine fault, and winemakers aim to minimize its occurrence.
Multiple previous blog posts addressed the topic of latent H2S formation in wine (H2S formation after alcoholic fermentation), however, a significant amount of H2S is produced by yeast during fermentation. It is commonly assumed that H2S production during fermentation is linked to the nitrogen status of the juice with low yeast assimilable nitrogen (YAN) juices resulting in higher H2S formation1. Therefore, the most common strategy to limit the formation of H2S during fermentation is to provide adequate YAN to ensure sufficient availability of nitrogen sources.
H2S is formed in concentrations up to hundreds of μg/L during fermentation2,3. It is assumed that high H2S-forming fermentations will result in high final concentrations of H2S. However, a clear relationship has never been established. To what extent this fermentation-derived H2S can contribute to residual H2S in the finished wine is explored in the current blog post.
A study4 performed by the Australian Wine Research Institute investigated the contribution of yeast strain and nitrogen supplementation to H2S formation during fermentation and its consequent occurrence in the resulting wines. The aim was to investigate:
- The role of yeast strain in H2S production and residual H2S
- The role of juice nitrogen supplementation in H2S production and residual H2S
- The relationship between H2S production during fermentation and the H2S present in the finished wine
Materials and Methods
Five commercial Saccharomyces cerevisiae yeast strains (with different H2S production characteristics) were used to ferment a juice which initially contained 110 mg/L of YAN (low). Prior to inoculation, this juice was adjusted (supplemented with di-ammonium phosphate (DAP)) to increase the YAN concentration to either moderate (260 mg/L) or high (410 mg/L) YAN levels. Each treatment was performed in triplicate and all fermentations reached dryness (residual sugars <1 g/L).
- Five yeast strains
- Three YAN levels (110, 260 and 410 mg/L)
The H2S concentration was monitored throughout fermentation and then measured in the finished wines.
Effect of YAN on the fermentation
- Increased initial YAN concentration in the juice increased the fermentation rate as well as the maximum yeast cell density and population. Increased initial YAN therefore resulted in reduced fermentation duration and a shorter time to reach maximum cell density.
Timing of H2S production during fermentation
- For the low YAN juice (110 mg/L YAN), H2S formation started when approximately 25% of the available sugars were fermented. For the moderate and high YAN juices (260 and 410 mg/L YAN) a longer lag phase was observed between the beginning of alcoholic fermentation and the onset of H2S production when compared to the low YAN juice. H2S formation was therefore delayed when more nitrogen was available.
- For most of the yeasts tested, the majority of H2S formation took place during the cell growth phase. A very high rate of H2S formation was observed when the YAN was depleted. However, this was not the case for all of the yeast strains tested. For example, one of the yeast strains produced H2S during both the exponential and stationary phases irrespective of the initial YAN content of the juice.
- Relatively little H2S was produced during the final stages of fermentation.
Total H2S production during fermentation
- Results showed that the yeast strain was the most important factor responsible for the observed differences in total H2S production. The yeasts tested in the study showed a wide range of H2S production capabilities with certain yeasts producing more (or less) H2S across the YAN levels tested.
- The initial YAN concentration of the juice also influenced the total H2S production during fermentation, however, the nitrogen status was not as important contributor compared to the yeast strain.
- Interestingly, the high YAN juice (410 mg/L) generally resulted in total H2S production similar to or lower than the low YAN juice (110 mg/L), while the moderate YAN juice (260 mg/L) resulted in a 2-3 fold increase in total H2S formed during fermentation. This result is in contrast with the generally accepted paradigm that increased YAN in the juice reduces the formation of H2S during fermentation. Here it is important to consider the specific YAN concentrations tested in the study as well as the selected yeast strains used.
- Yeast and YAN interactions were also significant. Therefore, different yeasts might behave differently depending on the YAN level of the must. This would suggest that the amount of H2S produced by the yeast in response to YAN levels is highly dependent on the genetic background of different strains. Therefore, H2S production in fermentation, although strain-dependent, is significantly affected by the composition of the medium.
Residual H2S concentration in the finished wines
- In general, the residual H2S concentration in all the fermented wines were relatively low (< 1 μg/L).
- For the residual H2S present in the finished wines, it seems that the initial YAN concentration of the juice was a more important contributor compared to the yeast strain that was used to conduct the fermentation.
- Low (YAN = 110 mg/L) and moderate (YAN = 260 mg/L) nitrogen supplementation resulted in wines with significantly higher residual H2S compared to wines from high (YAN = 410 mg/L) nitrogen supplementation.
- Yeast and YAN interactions were shown to be even more important compared to yeast strain and YAN levels alone. This again highlights the complex response of individual yeast strains to nitrogen supplementation.
- The total H2S generated during fermentation was not directly related to the H2S concentration in the finished wines. Despite each yeast producing significantly increased total H2S during fermentation for the moderate (YAN = 260 mg/L) nitrogen supplementation, there was very little difference between the final H2S concentration in the low and moderate YAN fermented wines. The highest nitrogen-supplemented juice (YAN = 410 mg/L) resulted in wines with decreased residual H2S concentrations irrespective of yeast strain or H2S produced during fermentation.
The amount of H2S generated during fermentation was affected by the yeast as well as the nitrogen status of the must. In contrast to the widely reported decrease in H2S production in response to nitrogen supplementation, a non-linear relationship was found in the current study as moderate nitrogen supplementation resulted in a remarkable increase in H2S formation during fermentation by each of the five wine yeasts.
The interaction between the specific yeast (with a unique genetic background) and the nitrogen status of the must was a significant contributor for both H2S production during fermentation as well as the residual H2S content present after fermentation. Regulation of H2S metabolism has been shown to be genetically complex and highly variable between strains5.
Interestingly, no correlation was observed between the concentration of H2S produced during fermentation and the H2S concentration in the finished wines and low H2S-forming fermentations often delivered wines with relatively high final H2S concentrations and vice versa. However, the final H2S concentration in the high YAN fermented wines was significantly lower than in the low and moderate YAN fermented wines even though similar amounts of H2S were formed during the fermentation of both the high and low YAN juices.
The management of H2S formation through nitrogen supplementation requires knowledge of the initial YAN content and the yeast H2S characteristics. Nitrogen supplementation can strongly affect H2S production during fermentation, however, the yeast strain that is used to conduct the fermentation might be a more important contributor compared to the nitrogen availability. The addition of DAP during stinky ferments, might, therefore not always reduce the amount of H2S produced.
(1) Vos, P. J. A.; Gray, R. S. The Origin and Control of Hydrogen Sulfide during Fermentation of Grape Must. Am J Enol Vitic 1979, 30 (3), 187–197. https://doi.org/10.5344/ajev.19220.127.116.11.
(2) Ugliano, M.; Henschke, P. A. Comparison of Three Methods for Accurate Quantification of Hydrogen Sulfide during Fermentation. Anal Chim Acta 2010, 660 (1–2), 87–91. https://doi.org/10.1016/j.aca.2009.09.049.
(3) Park, S. K.; Boulton, R. B.; Noble, A. C. Formation of Hydrogen Sulfide and Glutathione During Fermentation of White Grape Musts. Am J Enol Vitic 2000, 51 (2), 91–97. https://doi.org/10.5344/ajev.2000.51.2.91.
(4) Ugliano, M.; Kolouchova, R.; Henschke, P. A. Occurrence of Hydrogen Sulfide in Wine and in Fermentation: Influence of Yeast Strain and Supplementation of Yeast Available Nitrogen. J Ind Microbiol Biotechnol 2011, 38 (3), 423–429. https://doi.org/10.1007/s10295-010-0786-6.
(5) Linderholm, A. L.; Findleton, C. L.; Kumar, G.; Hong, Y.; Bisson, L. F. Identification of Genes Affecting Hydrogen Sulfide Formation in Saccharomyces Cerevisiae. Appl Environ Microbiol 2008, 74 (5), 1418–1427. https://doi.org/10.1128/AEM.01758-07.
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