Production of S-containing Compounds (H

2S, SO

2, mercaptans, DMS) by Yeasts

The issue at hand....

can occur at any stage of the ferment

can occur in all must types: red, white, rose, sparkling or botrytis-infected

can occur in 'under turbid' or 'overly turbid' wine or juice

causes are rarely determined because of the difficulty of prediction or diagnosis in the complex matrices like grape juice and wine

In short, it's a battle we must all take on in our careers.

INTRODUCTION OF TERMS and DESCRIPTORS

H2S - Hydrogen Sulfide rotten eggs

SO2 - Sulfur Dioxide

SO4 - Sulfate

HSO3 - Sulfite

Also consider that Salts containing the HSO3− ion are termed bisulfites

Disulphides = represent the 'latent' form of sulphides in wine

More terms..........

Mercaptans – also known as thiols – if hydrogen sulfide isn't removed quickly can result in mercaptans – cabbagey, burnt rubber, struck flintSulfur analogues of alcohols, -SH.Named by adding -thiol to alkane name.The -SH group is called mercapto.

DMS – dimethyl sulfide (CH3)2S - cabbage like smell - dimethyl sulfate (CH3O)2SO2. - onion-like odour in high concn.

VOSC - Volatile Organic Sulfur compound - normally effective at very low concn (ppb or less)

Examples: 3-(methylthio)-1-propanolmethanethioldimethyl disulfideDimethyl trisulfideAllyl methyl sufide

SH

What's happening......

Under 'normal conditions' H2S production is

from the reduction of sulphate via the sulphate reduction pathwaythe dissimilatory reduction of sulfate seems to be strictly an anaerobic process as all the microbes capabable of carrying it out grow in environments devoid of oxygen, i.e. yeasts sulphate reduction pathway = Sulfate (SO4

-2) is reduced to sulfide (S-2), typically in the form of hydrogen sulfide (H2S). Eight electrons are added to sulfate to make sulfide

acetate + SO4-2 + 3H+ +---------------> 2CO2 + H2S + 2H2O

or........

WINE YEASTS......

use this hydrogen sulphide to synthesize the sulphur-containing amino acids

methionine – is a sulphur containing amino acids and is a very special amino acid because it is the "start" amino acid in the process of protein synthesis

cysteine – inter-related to cystine and required in the manufacturing of amino acid taurine

The demand for Nitrogen at this growth stage is high and in the absence of an intracellular nitrogen pool (nutrient depleted), sulphate and sulphite reduction continues forming excess H2S that cannot be used in the amino acids and then released into the must

Which is why adding DAP early in the ferment can solve this problem

YAN (yeast available/assimilable nitrogen) counts are very important. Low YAN musts leads to a deficit of C-N precursor compounds.

YAN is measured in parts per million. The YAN number is a combination of ammonia and alpha amino acids

Other causes....

High levels of SO2 added at the crusher (greater than 80ppm) can up the

production of sulphur-like odours by allowing the SO2 to enter the yeast cell

directly (by passing the sulphate reduction system)

Agrochemicals with elementary Sulphur

metal ions (chemical reduction)

Turbidity of juice (measured in NTU)

Cold soaking or wild ferments – b/c of nutrient depletion from Kloeckera spp.

Breakdown of methionine to obtain the amino group, a pantothenate (Vitamin B5) an essential nutrient. Shortage in high YAN musts.

Thioacetates (reacting thiol with acetal) are unstable in acidic conditions and can hydrolyse to release the respective mercaptan.

All of these play a lesser role in H2S production

Management

Yeast growth

late-phase or non-growth H2S growth

Extended and throughout ferment

Early stage growth(solid)Prolonged H2S growth (dotted)

MGMT Most of the recent research suggests that residual H2S in wine depends less on the amount produced and more on the stage of fermentation at which it is produced (Ugliano et al.)

For this reason the previous graph is so very important. At the early stages of the ferment production of H

2S doesn't phase most winemakers. During

yeast growth phase the associated high fermentation vigour leads to rapid loss of the highly volatile H

2S by entrainment with CO

2

But later in the ferment when the vigour is low, any H2S that forms is less efficiently removed from the wine, and less so in large fermentors.

This is why mgmt of the problem early is so crucial

But it is still not well understood whether the H2S reacts with other

compounds to form the more complex VSCs (disulfides, thiazoles, etc).

H2S Production in the late phase of ferment

- occurs after yeast growth has finished, late in ferment, usually when sugar levels are below 50-100g/L- varies among varieties: Syrah can be bad, Cab can be good

- varies among yeast strains- typically unresponsive to DAP additions- can respond well to aeration or vitamins, though only lowered and not eliminated- it is thought that it this time the yeast cells are trying to get N for cell maintenance activities by mobilizing reserves of org. Sulphur compounds (previously mentioned methionine, cysteine and also glutathione). And again, breaking these down results in H

2S production.

- aeration isn't the simple answer as it can lead to the formation of disulfides if mercaptans are also present. (good winemakers have good palates)-disulfides are less volatile but can revert into mercaptans in the right 'conditions'

Recent studies show that early H2S production ceased b/f midpoint of ferment it was

not detected in final wine (cold settled, no copper addition)

Yeast's role

Strain of yeast seems to be an important factor in production of VSCs, but not yet understood in the winemaking environment.

But what of indigenous yeasts or genetically modified yeasts?

Indigenous 'wild' and other yeasts

Older literature and possibly a myth about indigenous yeasts causing more H2S production seems to be just that, a myth. That is, no more or less a factor than with most inoculated yeasts. That also applies to the idea that wild yeasts are the cause of H

2S production in wild yeasts.

There are only theories and no real literature on the role of non-Saccharomyces yeasts effects on the ferment. General knowledge is that these yeasts are in decline by the middle stages of the ferment it is not considered a factor, again relating to the time of the H

2S production.

More Yeast issues:

Some yeasts are known to act as a source of mercaptans and related VSCs.

Different results in different musts in relation to DAP additions. Once again, grape must and wine is a complex matrix. Some yeasts are known to cause issues in certain types of musts. More research is being done.

DAP addition

- varies within yeast strain and conditions

- can effect varietal characteristics such as 3MH and 3MHA in SB because synthesis only occurs in conditions when DAP might be needed

- can only delay H2S production in some yeast strains and leave residual levels in the wine, because once again timing is crucial. So bumping up the YAN levels with DAP in low nutrient musts can actually hurt the wine in some cases.And this biochemical mechanism is unclearBut more additions later on can help this out showing that different patterns of H2S production can result from DAP additions.

- yeasts that respond well to DAP when the initial juice is YAN is about 250mg/L but just getting the YAN level up to that level is not that simple. Remember, DAP is only 20% N. So what's the rest?...

And what if DAP or other N supplements fail to cease H2S productions or worse yet increase H2S production? First a test can be done but then......

The mgmt techniques....

Vitamins

- vitamin deficiency can be directly related to H2S production (Henschke et al.)

- sound, healthy fruit is a key issue. Can relate to vineyard disease but also handling during harvest, transport, destemming/crushing, etc.

- avoid microbial growth or wild yeast growth prior to inoculation to avoid depletion of thiamine, leading to sluggish ferments

- use a careful balance of SO2 during transport and handling of fruit.

- use of yeast vitamin preparations at or before inoculation along with a small amount of DAP can control H2S production

Commercial nutrients

There are many of these on the market and many wineries use these during yeast inoculation (starter culture) and during the ferment.

However, some of these do not contain sufficient amounts of Nitrogen to help with production of H2S.

Copper additions- Copper sulphate can effectively remove H2S and mercaptans, it does not remove disuphides- Also can strip wine of varietal characters such as other important thiols

For instance, the 3MH and 3MHA, which can be more important to the wine than the presence of some other unwanted thiols

Another 'underlying' issue is that of closure type

We don't want the 'new cork taint'

Though consumers may be more accepting of mercaptans in their wines, the rate of 2.2% of

screwcapped wines is alarming.

Clearly why AWRI has ongoing research trials.

References:

Goode, J. Mercaptans and other volatile sulfur compounds in wine. Retrieved 26 July 2010 from http://www.wineanorak.com/mercaptansinwine.htm

Jackson, R. Wine Science.3rd Edition 2008.

Theron, C. Wynboer Dec 2009 The practical management of Hydrogen Sulphide

Moore, D. Various course handouts. Structural and Metabolic Biochemistry. Eastern Institute of Technology.

Ugliano, M., Winter, G., Coulter, A.D., Henschke, P.A. The Australian & New Zealand Grapegrower and Winemaker. Annual Technical Issue 2009. Practical management of hydrogen sufide during fermentation – an update.

Various resources on www.wikipedia.org

Dobson, J., 2010. personal communications.