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OVERVIEW OF PROBLEM FERMENTATIONS Linda F. Bisson Department of Viticulture and Enology, UCD Types of Problem Fermentations •Lack of fermentation progression •Off-character production PROBLEMS IN FERMENTATION PROGRESSION Stuck and Sluggish Fermentations • Characterized by failure of yeast to consume all available sugar • Multiple causes • Can be difficult to treat as cells have arrested metabolism • Leads to reduced wine quality Typical Fermentation Profile 5 1 Brix 2 3 4 Time 1: lag time; 2: max fermentation rate; 3: transition point; 4: post-transition fermentation rate; 5: overall time to dryness Stages of Fermentation • Stage 1: Lag due to need to adapt then grow to maximize population levels • Stage 2: Fastest rate of fermentation: largest population of cells supported by medium, absence of inhibitory ethanol • Stage 3: Transition: at this point ethanol has become inhibitory to continued maximal fermentation; cells are adjusting plasma membrane composition and internal membranes and proteins to an ethanol tolerant form • Stage 4: Maximal fermentation rate of ethanol-adapted cells • Stage 5: Complete metabolic consumption of sugar, cells enter decline or death phase Fermentation Progression Assessment • Lag time • Duration? • Maximum fermentation rate • Rate value? • Duration? • Transition point • At what Brix level? • How sharp? • Post-transition fermentation rate • Value relative to max fermentation rate? • Length of time? • Brix/ethanol/nitrogen level at which it occurs? • Overall time to dryness Fermentation Capacity Is a Function of: • Yeast Biomass Concentration • Fermentative Ability of Individual Cells Fermentation Capacity Is a Function of: • Yeast Biomass Concentration • Fermentative Ability of Individual Cells • Both are dependent upon: • Adequate nutrition • Absence of inhibitors • Permissive growth conditions • Permissive fermentation (energy generation) conditions Causes of Stuck/Sluggish Fermentations • Nutrient limitation Nutrient Limitation • Nitrogen: most often limiting • Phosphate can also be limiting • Depending upon circumstances, micronutrient limitation may also be a problem • Pitching yeast • Deficiency in vineyard • Microbial blooms prior to fermentation • Loss during rehydration Causes of Stuck/Sluggish Fermentations • Nutrient limitation • Ionic imbalance Ionic Imbalance Ratio of K+:H+ • Must be at least 25:1 • Needs to be adjusted early in fermentation • Probably important in building an ethanol tolerant membrane Causes of Stuck/Sluggish Fermentations • Nutrient limitation • Nutrient imbalance • Ethanol toxicity Ethanol Toxicity • Ethanol disrupts membrane component interactions affecting transporter activity and ability to remove toxic substances like free protons from the cell • Cells adapt to ethanol by changing the composition of the membrane and by changing the species of the proteins present • Adaptation requires the ability to make new proteins and membrane components (lipids, sterols) • If conditions do not enable development of ethanol tolerance, fermentation rates will decrease Causes of Stuck/Sluggish Fermentations • Nutrient limitation • Nutrient imbalance • Ethanol toxicity • Presence of toxic substances Presence of Toxic Substances • Toxins may arise from the metabolic activity of other microbes • Toxins may arise from sub-optimal metabolic activity of Saccharomyces (such as acetic acid production) • Toxins may have arisen in vineyard, but are not inhibitory until ethanol has accumulated The Most Common Toxins • Acetic acid • Higher organic acids (C2 – C4) • Medium chain fatty acids/fatty acid esters • Acetaldehyde • Fungicide/Pesticide residues • Higher alcohols • Higher aldehydes • Killer factors • Sulfur dioxide Causes of Stuck/Sluggish Fermentations • Nutrient limitation • Nutrient imbalance • Ethanol toxicity • Presence of toxic substances • Poor adaptation of strain Poor Adaptation of Strain • Strain may not display sufficient ethanol tolerance • Strain may have high nitrogen/vitamin requirements that are not being met • Strain may be a poor fermentor, but capable of dominating the fermentation • Rehydration/Implantation issues: Strain not rehydrated properly, stored correctly or is being used past its expiration date • Temperature shock effects during rehydration and pitching into the tank Causes of Stuck/Sluggish Fermentations • Nutrient limitation • Nutrient imbalance • Ethanol toxicity • Presence of toxic substances • Poor adaptation of strain • Low pH pH • pH is reduced by metabolism of Saccharomyces • Low pH musts (below pH 3.0) may drop to an inhibitory level (pH 2.7) • Dependent upon K+ concentration Causes of Stuck/Sluggish Fermentations • Nutrient limitation • Nutrient imbalance • Ethanol toxicity • Presence of toxic substances • Poor adaptation of strain • Low pH • Temperature shock Temperature Shock • Superheating or supercooling • Will impact cell membrane functionality and ability to tolerate ethanol • Main issue is a ratcheting down of maximal level of ethanol tolerance Problem Fermentation Profiles Types of Problem Fermentations 30 Normal Long Lag 25 Becoming Sluggish Sluggish Throughout Brix 20 Abrupt Arrest 15 10 5 0 0 50 100 150 200 -5 Time (hours) 250 300 350 Types of Problem Fermentations 30 Normal Long Lag 25 Becoming Sluggish Sluggish Throughout Brix 20 Abrupt Arrest 15 10 5 0 0 50 100 150 200 -5 Time (hours) 250 300 350 Causes of Long Lag • Poor health of starter culture • Presence of inhibitors • Poor grape quality • Mold infestation • Premature initiation of fermentation Poor Health of Starter Culture • Active Dry Yeast: • Past expiration date • Not hydrated properly • Not stored properly • Natural Fermentation: • Yeast numbers low • Inhibitory microbes present (heavy rot of fruit) • Poor yeast strain present (high N requirements, lack of ethanol tolerance) Presence of Inhibitors • Sulfur dioxide concentration too high • Sulfur dioxide added improperly • Microbial competition for nutrients • Microbial activity generating inhibitory compounds • Enzymatic competition for oxygen • Pesticide/fungicide residues on grapes at harvest • Temperature of must/juice too high/low Poor Grape Quality • Infected grapes: loss of micronutrients • Infected grapes: high microbial loads • Loss of free oxygen Types of Problem Fermentations 30 Normal Long Lag 25 Becoming Sluggish Sluggish Throughout Brix 20 Abrupt Arrest 15 10 5 0 0 50 100 150 200 -5 Time (hours) 250 300 350 Causes of Slow Rate Over Entire Time Course • Failure to reach maximum cell density • Nutrient (growth factor) limitation • Strain a poor choice for conditions • Inhibitory fermentation conditions: temperature, pH, ionic imbalances, organic acids Types of Problem Fermentations 30 Normal Long Lag 25 Becoming Sluggish Sluggish Throughout Brix 20 Abrupt Arrest 15 10 5 0 0 50 100 150 200 -5 Time (hours) 250 300 350 Causes of a Decrease in Rate • Poor ethanol tolerance • Loss of viability • Loss of fermentative capacity • Nutrient (survival factor) limitation • Poor strain Types of Problem Fermentations 30 Normal Long Lag 25 Becoming Sluggish Sluggish Throughout Brix 20 Abrupt Arrest 15 10 5 0 0 50 100 150 200 -5 Time (hours) 250 300 350 Causes of an Abrupt Stop • Temperature shock • Rapid build up of inhibitors: acetic/organic acids: generally caused by addition of active ML culture • pH decreases too much • Strain very ethanol sensitive Most Common Causes of Stuck/Sluggish Fermentations • Temperature extreme • Ethanol intolerance • Nutrient deficiency • Deficient yeast strain • Microbial incompatibility • Presence of an inhibitory substance • Poor fermentation management decisions Why are stuck fermentations difficult to treat? • Cells adapt to adverse conditions by reducing fermentation capacity • Biological adaptation difficult to reverse • Diagnosis of cause of fermentation problem difficult • Conditions that cause stuck fermentations are also conducive to cell death • New inocula respond to cell death by arresting activities Best Advice • Pay attention to strain selection: match strain to fermentation conditions • Provide adequate nutrient levels • For active dry yeast: follow packet instructions for rehydration and adaptation to juice • For native fermentations: be aware of microbiota issues, heed historical observations for nutrient requirements and ethanol tolerance • Temperature Shock: should be avoided but if it happens consider racking and re-inoculation with unshocked yeast • Adjust juices of marginal pH (too high or too low) • Limit activity of inhibitory microbes (wild lactics)