1. Maximizing Efficient Sugar Consumption Minimizing organic acid production Reducing glycerol production

2. Reducing Glycerol Production Metabolic Cycle of Glycerol Production Calculations on Measuring Glycerol Glycerol Causing Stress Factors and Minimizing StressorsMinimizing Organic Acid Production Bacterial Descriptions and Calculations Organic Acid Dissociation Troubleshooting Bacterial ContaminationsMaximizing Efficient Sugar Consumption Utilization of all Available Sugar Optimizing Fermentation Rate Sugar to Yeast Mass Calculations 3. Glycerol is a waste product produced by stressed yeast. It sends a signal out to stop working because the end is near. This inhibits the yeast from doing their job. Lactic acid from lactic acid producing bacteria affects a fermentation because it inhibits the yeast also. 4. The regeneration of NAD is necessary to maintain the redox balance of the cell and prevent the stalling of glycolysis (Step 6). The alternative means of replenishing NAD is the pathway to the formation of glycerol. Glycerol is quantitatively one of the most important products of fermentation behind ethanol and carbon dioxide. C3H6O3 + 2H C3H8O390(dihydroxyacetone)92 (glycerol)C3H4O3 CO2 + CH3CHO + 2H CH3CH2OH 88(pyruvate)4446 (ethanol) 5. How much ethanol is sacrificed to glycerol production? C6H12O6 + (2H)2 2 C3H8O3 180(glucose)C6H12O6 180(glucose)184 (glycerol) 2 CO2 + 2 C2H5OH 8892 (ethanol)= 180Starting with 1 mole glucose, metabolism results in a 1:1 ratio of either glycerol or ethanol as 2 moles of either product results. HPLC measures in weight, not molecules 6. Glycerol 2:1 on a weight basis; for every 0.1% w/v glycerol produced, 0.05% w/v ethanol is lost. Reducing glycerol from a 1.8% to 1.6% w/v theoretically results in 0.1% w/v increase in ethanol. 13.0 13.1% w/v increase = ~1,000,000 gal additional EtOH/year (100,000 MMGY)800,000 X 16.12% v/v = 128,960 gal EtOH/ferm 800,000 X 16.24% v/v = 129,952 gal EtOH/ferm ~1000 gal EtOH increase per ferm; 3 ferms per day, 360 operating days per year = 1,080,000 gal additional EtOH produced 184/92 = 2 ----- Thus glycerol:ethanol is 2:1 by wt 7. 1. Osmotic Stress (high glucose concentration i.e. sugar shock) 2. Water Potential Deficiency (high salt concentrations such as sodium and sulfates) 3. Elevated pH 4. Moderately High Temperatures 5. Moderately High Organic Acid Concentrations 6. High Ethanol Concentrations 8. Yeast stress factors are synergistic; while yeast can tolerate one or two moderate stress factors, several stress factors working together will add infinitely more stress to a yeast culture. 9. Saccharomyces cerevisiae is non-osmotolerant Glucose passes directly into the yeast cell with nothing stopping available glucose from entering cell; high levels of glucose place the cell wall and membrane under extreme stressPhysiological Response: Glycerol is the major by-product resulting from osmotic stress; this is the most efficient way for the yeast cell to regulate and rebalance the yeast cell Glycerol production helps to: restore cell volumerestore cell water balanceenables intracellular enzyme activity to continue 10. Practical Application 1) Restrict glucose availability in propagation Glucose should remain as low as possible without starving yeast (always greater than 0.5% wt/vol). Target 0.5-2.0% at prop send. Depending on how much control you have over the plant should determine how much of a cushion is needed at the end of propagation. 11. 2) Restrict glucose availability early in fermentation Glucose should remain as low as reasonably possible. Glucoamylase dosing should be done to ensure complete conversion of DP4+ to glucose but yet keep glucose from spiking too high in early fermentation Target glucose at 8-10% wt/vol maximum at any given time. Lower is better to stimulate yeast growth. Maximum levels to get substantial yeast growth are more likely around 4% wt/vol glucose. The longer yeast are exposed to high concentrations of glucose the more detrimental it is to yeast growth. Continuous dosing of glucoamylase is ideal for keeping a moderate and steady amount of glucose present in fermentation. 12. Physiological Response Sodium/Sulfates/Salts Cellular Water Imbalance Salts cause osmotic dehydration (low water potential) S. cerevisiae moderately tolerant to low water potential Results in loss of cell turgor pressure and decrease in cell volume and cytoplasmic space Restoring cell volume and stabilizing the cell is done by production of glycerol and trehalose 13. Practical Applications Sodium/Sulfates/Salts At times, there is nothing that can be done to combat this Never send waste CIP or distillate for cleaning through the beerwell or whole stillage where sodium and salts will end up in the backset If these become a problem, try to reduce backset inclusion by increasing production and discharge 14. pH range suitable for yeast/fermentation: 3.8-5.8 Yeast maintain an intracellular pH of 5.2 pH can be much higher/lower before causing toxicity During metabolism a hydrogen ion is excreted which helps control internal pH and subsequently reduces substrate pHPhysiological Response High pH denatures proteins especially within the cell wall; this causes loss of cellular activity and cell death Drives yeast away from ethanol production toward metabolic paths of glycerol and organic acid production (see following slide) 15. Practical Application Try to start fermentation at a moderate pH (< pH 5.8) When using anhydrous ammonia for a nitrogen source in fermentation, a lower mash pH may be necessary (through the addition of sulfuric acid or low slurry pH) Anhydrous ammonia addition to fermentation should also be added over as long of a time period as possible ALWAYS ensure that residual caustic has been removed 16. Practical Application: Glycerol Production??? Elevated fermentation temperatures cause increased yeast activity and faster rate of fermentation THIS is mostly responsible for increased glycerol production Elevated fermentation temperatures are a yeast stressor anything over ~80F is a stress upon yeast which makes yeast more susceptible to other stress factors Temperature Staging has a profound effect on reducing fermentation stress and reducing glycerol production 17. Presence of elevated organic acids result in a compromised stress tolerance to any other stress factor Lactic Acid MIC: >0.8% w/v Can see negative fermentations at 0.3% and higher due to cumulative stress factorsAcetic Acid MIC: >0.05% w/v Generally see levels up to 0.1% without any negative effects 18. Growth inhibitor, not necessarily metabolic inhibitor Ethanol removed from cells via passive diffusion so there is very little accumulation of ethanol within yeast cell Membrane structure and function primary targets of ethanol stress, not cellular/metabolic balance issue Elevated ethanol late if fermentation simply compounds other stress factors which can increase glycerol production. Try to reduce cumulative stress factors late in fermentation to protect yeast against stress. i.e. Temperature staging, low organic acids, low salt compounds, etc. 19. During fermentation, carbohydrates, ethanol, and organic acids are monitored in order to insure that the fermentation process is occurring normally and to insure that undesirable bacteria are kept under control. Also, the mash temperature is kept in a range of 90F to 95F. 20. Typical end fermenter process parameters are:Ethanol / Lactic acid / acetic acid / pH /max cell count/ viable count % 13.6