Sunday, May 25, 2014

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, 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 ( 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

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