2 nd Bioethanol Technology Meeting Detmold, Germany Specific Yeasts Developed for Modern Ethanol Production Mike Knauf Ethanol Technology 25 April 2006
Presentation Outline Start with the Alcohol Production Fermentor Yeast Strains Yeast Product Formulations Yeast Management Options Commercial Yeast Production, Quality Control, Technical Support Summary & Conclusions
Start with the Alcohol Production Fermentor This is where the yeast does its work! What are YOUR Objectives? Check one: Efficiency (or Yield) Throughput Consistency Choose & Prioritize: Process control Minimal contamination Fast fermentation Complete fermentation Minimal input costs Foolproof/Automate
Process Design Considerations Fermentation: A. Continuous (e.g., cascade) B. Batch Complexity Yeast Management: 1. Propagation (yeast biomass production) Batch Continuous 2. Conditioning (metabolic acclimitization) 3. Rehydration 4. Direct Pitch
Yeast Metabolic Pathways
From: Pretorius, 2000, Boulton et al., 1996 Substrates Thousands of other enzymes are also located in the cell. Glycolysis: The degradation of sugar to alcohol with production of energy (ATP) for cell growth. End products 12 Enzymes convert ~90% of available glucose to ethanol and CO 2 under conditions of controlled oxidation/reduction balance.
What Is a Yeast Strain? Species: A fundamental category of taxonomic classification consisting of organisms capable of interbreeding. 600+ species of yeast identified in nature Saccharomyces cerevisiae most significant Strain: A group of organisms of the same species having distinctive characteristics. Unlimited number of strains possible Thousands of yeast strains already selected
The Big Picture Genotype Environment Phenotype Production Method Product Form Plant Handling Inoculation Technique Fermentation Conditions Strain Characteristics Fermentation Performance
Yeast Strains: Saccharomyces cerevisiae Engineered Performance Wild Selected II Selected I Time
Strain Selection and Attributes Strain Selection and Development Techniques Isolation from fermentations Screening from culture collections Mutation and selection Breeding (hybridization) Spheroplast fusion Cloning How do Strains Differ? Temperature, ph, sugar, and alcohol tolerance Flocculation and foam production Respiratory growth rate, yield, and stability Killer factor and killer resistance Enzyme activity and carbohydrate utilization
Strain Comparison in a Maize Mash Fermentation 18 EtOH Concentration (%, v/v) 16 14 12 10 8 6 4 2 0 0 20 40 60. Time, h Zhang 2005
Fermentation Conditions Dose Rate Nutrition Stress Contamination
Dose Rate: Effect on Performance Zhang 2005 (2)
Nutrition For optimal fermentation, yeast require: Substrates (C, N, P, S) in optimal ratios Minerals (K, Na, Mg, Ca, Zn, Fe, Mn, Cu, Co, etc) Vitamins (B1, B5, B6, Biotin, etc) Oxygen (for production of unsaturated fatty acids)
Free Amino Nitrogen (mg/l mash @ 22% solids) Available from fermentation feedstock Thomas, Ingledew
Stress Contamination Ingledew 2003
Commercial Yeast Products Formulations & Performance ADY = Active Dry Yeast Fresh Cake Yeast Liquid Yeast (2)
Yeast Performance: Active Dry Yeast Acceptable Fermentation Performance Good viability Need for rehydration Acclimitization often recommended Storage and Handling Refrigerated storage not required Typically 2 year shelf life Obvious choice for irregular order patterns Lowest cost storage Must handle solid product in bags or boxes
Yeast Performance: Fresh Cake Yeast Excellent Fermentation Performance High viability No need for rehydration Minimal acclimitization requirement Storage and Handling Limited Shelf-Life Sealed, Refrigerated: Stable for at least 6 weeks Must handle solid product in bags or boxes
Dry vs Fresh Cake Yeast, Grain Mash Fermentation 7 EtOH Concentration (v/v) 6 5 4 3 2 1 0 0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 Time (h) ADY Fresh Cake
Yeast Performance: Fresh Liquid Yeast Superior Fermentation Performance Highest viability No need for rehydration Acclimitization not required Storage and Handling Poor ambient temperature storage stability Refrigerated, unstabilized: Shelf life = 1-2 weeks MAX Refrigerated, stabilized: 3+ month shelf life demonstrated Additional handling and cost for refrigeration Liquid product amenable to automated addition through DCS (Distributed Control System)
Yeast Form Comparison Active Dry Yeast Optimized for stability Does not require refrigeration Good for intermittent use Good for small volume strains Fresh Cake Yeast Optimized for activity Does not require rehydration Shorter lag phase in fermentor Good for difficult to dry strains Availability Stabilized Liquid Yeast Better stability than fresh yeast Higher activity than fresh or dry Amenable to automated dosing Suitable for direct pitch to fermentor Availability
Yeast Management Options 1. Propagation (yeast biomass production) Continuous Batch 2. Conditioning (metabolic acclimitization) 3. Rehydration 4. Direct Pitch Objective To supply to the Fermentor yeast with the following properties: Adequate numbers High Viability High Vitality Low Contamination In Log growth At the correct time in fermentation
Yeast Propagation Lesson #1: In-Plant Yeast Propagation is NEVER Completely Successful!! Typical Goals: Increase yeast cell numbers: 2-4X CFU/ml achievable Reduce lag times: From 6 hours to 2 hours Increase fermentation rates: Minimal impact (best rates with ~2 hours acclimitization) Decrease overall fermentation times: Not achieved (however, added nutrients will reduce fermentation time) Reduce costs: Not achieved (must factor cost of additional contamination, capital, labor, cleaning costs) True Propagation Requires Pure Yeast Culture, Sterile Conditions in Early Stages, Low Carbohydrate, High Oxygen Pure Yeast Culture not available in commercial quantities (cost prohibitive) Requires specialized equipment and expertise
Yeast Conditioning (or Metabolic Acclimitization) Objectives Reduce yeast use Reduce lag time Increase fermentation rate Problems Dry yeast lag time Insufficient oxygen Excess sugar Bacterial contamination Recommendations (if you decide to condition ) Batch is better than continuous Short time is better Nutrition is key (nutrient supplementation is more effective in the fermentor than during acclimitization) Bellissimi and Ingledew, 2004
Direct Pitch Commercial Yeast Production Fermentor 400 m 3 Advantages No conditioning Reduced contamination Disadvantages Increased yeast usage Dry yeast lag time 25 g/hl 2x10 10 CFU/g 100 kg yeast 48 72 hours 5x10 6 2x10 8 CFU/ml 4000 kg yeast
Commercial Yeast Production Proprietary Strain Storage
Commercial Yeast Production Overview Fresh cake yeast Active dry yeast Stabilized liquid yeast
Fermentation
Effect of Yeast Growth Rate on Performance Slow Growth During Yeast Production: More mature cells Higher carbohydrate Higher yield Lower initial activity Better stability Rapid Growth During Yeast Production: Less mature cells Higher protein and enzymes Lower yield Higher initial activity Lower stability
Separation and Drying
Yeast Production & Quality Control Consistent high performance depends on: Clean Fermentation Vial to seed flask propagation steps in sterile laboratory environment Extraordinary steps to ensure clean scale-up Contaminant organisms (usually bacteria) < 0.0001% of Yeast CFU Process control from seed to final formulation Process Control During Separation and Drying Final Product Analytical Laboratory Support (Quality Assurance) DNA karyotyping analysis ph, protein, solids/moisture Performance Quick test (also in-process); CO 2 production Fermentation performance test
Technical Support Alcohol Yeast = Specialty Products Each alcohol plant has unique objectives and constraints Each yeast strain + formulation combination is different Finding the best solution is not trivial Reputable yeast suppliers support their products Experts from the industry Regular visits and check-ups Develop understanding of plant process & management objectives Provide operator and staff training
Summary & Conclusions Start with the Alcohol Production Fermentor Yeast Strains Yeast Product Formulations Yeast Management Options Yeast Production, Quality Control, Technical Support
Acknowledgments Professor Mike Ingledew (U. Saskatchewan, Ethanol Technology Institute & The Alcohol School) Dr. Zhigen Zhang (Lallemand R&D) Kevin Kraus (Lallemand Specialties, Inc.) To learn more about yeast, and all aspects of alcohol production, attend this week-long education program! Chris Richards (Ethanol Technology, NA Technical Sales Manager) Dave Kelsall (Ethanol Technology, Technical Service Manager) May 22-26, Toulouse, France September 25-29, Montréal, Canada
Danke May 22-26, Toulouse, France September 25-29, Montréal, Canada