Specific Yeasts Developed for Modern Ethanol Production
Mike KnaufEthanol Technology
25 April 2006
2nd Bioethanol Technology MeetingDetmold, Germany
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
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
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Start with the Alcohol Production FermentorThis is where the yeast does its work!This is where the yeast does its work!
What are YOUR Objectives?Check one:
Efficiency (or Yield)ThroughputConsistency
Choose & Prioritize:― Process control― Minimal contamination― Fast fermentation― Complete fermentation― Minimal input costs― Foolproof/Automate
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Process Design Considerations
Fermentation:A. Continuous (e.g., cascade)B. Batch
Yeast Management:1. Propagation (yeast biomass
production)• Batch• Continuous
2. Conditioning (metabolic acclimitization)
3. Rehydration4. Direct Pitch
Com
plex
ity
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Yeast Metabolic Pathways
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
From: Pretorius, 2000, Boulton et al., 1996
Glycolysis: The degradation of sugar to alcohol with production of energy (ATP) for cell growth.
End products
Substrates
12 Enzymes convert ~90% of available glucose to ethanol and CO2 under conditions of controlled oxidation/reduction balance.
Thousands of other enzymes are also located in the cell.
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
What Is a Yeast Strain?Species:A fundamental category of taxonomic classification consisting of organisms capable of interbreeding.
600+ species of yeast identified in natureSaccharomyces cerevisiae most significant
Strain:A group of organisms of the same species having distinctive characteristics.
Unlimited number of strains possibleThousands of yeast strains already selected
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
The Big Picture
EnvironmentGenotype Phenotype
Production MethodProduct FormPlant Handling
Inoculation TechniqueFermentation Conditions
Strain Characteristics Fermentation Performance
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Yeast Strains: Saccharomyces cerevisiae
EngineeredEngineered
Selected Selected –– IIII
Selected Selected –– II
WildWild
TimeTime
Per
form
ance
Per
form
ance
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Strain Selection and AttributesStrain Selection and Development Techniques
Isolation from fermentationsScreening from culture collectionsMutation and selectionBreeding (hybridization)Spheroplast fusionCloning
How do Strains Differ?Temperature, pH, sugar, and alcohol toleranceFlocculation and foam productionRespiratory growth rate, yield, and stabilityKiller factor and killer resistanceEnzyme activity and carbohydrate utilization
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Strain Comparison in a Maize Mash Fermentation
0
2
4
6
8
10
12
14
16
18
0 20 40 60
Time, h
EtO
H C
once
ntra
tion
(%, v
/v)
.
Zhang 2005
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Fermentation Conditions
Dose Rate
Nutrition
Stress
Contamination
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Dose Rate: Effect on Performance
Zhang 2005 (2)
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
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)
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Free Amino Nitrogen (mg/L mash @ 22% solids)
Available from fermentation feedstock
Thomas, Ingledew
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Stress
Ingledew 2003
Contamination
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Commercial Yeast Products
Formulations & PerformanceADY = Active Dry YeastFresh ‘Cake’ YeastLiquid Yeast (2)
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Yeast Performance: Active Dry YeastAcceptable Fermentation Performance
Good viabilityNeed for rehydrationAcclimitization often recommended
Storage and HandlingRefrigerated storage not requiredTypically 2 year shelf lifeObvious choice for irregular order patternsLowest cost storageMust handle solid product in bags or boxes
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Yeast Performance: Fresh Cake Yeast
Excellent Fermentation PerformanceHigh viabilityNo need for rehydrationMinimal acclimitization requirement
Storage and HandlingLimited Shelf-LifeSealed, Refrigerated: Stable for at least 6 weeks Must handle solid product in bags or boxes
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Dry vs Fresh Cake Yeast, Grain Mash Fermentation
0
1
2
3
4
5
6
7
0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0
Time (h)
EtO
HC
once
ntra
tion
(v/v
)
ADY Fresh Cake
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Yeast Performance: Fresh Liquid YeastSuperior Fermentation Performance
Highest viabilityNo need for rehydrationAcclimitization not required
Storage and HandlingPoor ambient temperature storage stabilityRefrigerated, unstabilized: Shelf life = 1-2 weeks MAXRefrigerated, stabilized: 3+ month shelf life demonstratedAdditional handling and cost for refrigerationLiquid product amenable to automated addition through DCS (Distributed Control System)
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Yeast Form Comparison
Active Dry YeastOptimized for stabilityDoes not require refrigerationGood for intermittent useGood for small volume strains
Fresh Cake YeastOptimized for activityDoes not require rehydrationShorter lag phase in fermentorGood for ‘difficult to dry’ strainsAvailability
Stabilized Liquid YeastBetter stability than fresh yeastHigher activity than fresh or dryAmenable to automated dosingSuitable for direct pitch to fermentorAvailability
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Yeast Management
Options1. Propagation (yeast
biomass production)• Continuous• Batch
2. Conditioning (metabolic acclimitization)
3. Rehydration4. Direct Pitch
ObjectiveTo supply to the Fermentor yeast
with the following properties:Adequate numbersHigh ViabilityHigh VitalityLow ContaminationIn Log growthAt the correct time in fermentation
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Yeast PropagationLesson #1: InLesson #1: In--Plant Yeast Propagation is NEVER Plant Yeast Propagation is NEVER Completely Successful!!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
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Yeast Conditioning (or Metabolic Acclimitization)
ObjectivesReduce yeast useReduce lag timeIncrease fermentation rate
ProblemsDry yeast lag timeInsufficient oxygenExcess sugarBacterial contamination
Recommendations(if you decide to ‘condition’)
Batch is better than continuousShort time is betterNutrition is key (nutrient supplementation is more effective in the fermentor than during acclimitization)
Bellissimi and Ingledew, 2004
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Direct Pitch
ProductionFermentor
400 m3
CommercialYeast
48 – 72 hours5x106 → 2x108CFU/ml
4000 kg yeast
25 g/hl2x1010 CFU/g100 kg yeast
AdvantagesNo conditioningReduced contamination
DisadvantagesIncreased yeast usageDry yeast lag time
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Commercial Yeast Production
Proprietary Strain Storage
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Commercial Yeast Production Overview
Fresh cake yeast
Activedry yeast
Stabilized liquid yeast
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Fermentation
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Effect of Yeast Growth Rate on PerformanceSlow Growth During Yeast Production:
More mature cellsHigher carbohydrate
Higher yieldLower initial activityBetter stability
Rapid Growth During Yeast Production:Less mature cellsHigher protein and enzymes
Lower yieldHigher initial activityLower stability
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Separation and Drying
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Yeast Production & Quality ControlConsistent high performance depends on:
Clean FermentationVial to seed flask propagation steps in sterile laboratory environmentExtraordinary steps to ensure clean scale-upContaminant organisms (usually bacteria) < 0.0001% of Yeast CFU Process control from seed to final formulation
Process Control During Separation and DryingFinal Product Analytical Laboratory Support (Quality Assurance)
DNA karyotyping analysispH, protein, solids/moisturePerformance
• Quick test (also in-process); CO2 production• Fermentation performance test
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
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 productsExperts from the industryRegular visits and check-upsDevelop understanding of plant process & management objectivesProvide operator and staff training
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
Summary & Conclusions
Start with the Alcohol Production Fermentor
Yeast Strains
Yeast Product Formulations
Yeast Management Options
Yeast Production, Quality Control, Technical Support
AN INTRODUCTION TO ETHANOL TECHNOLOGYAN INTRODUCTION TO ETHANOL TECHNOLOGYYeasts for Modern Ethanol ProductionYeasts for Modern Ethanol Production
AcknowledgmentsProfessor Mike Ingledew (U. Saskatchewan,
Ethanol Technology Institute & The Alcohol School)
Dr. Zhigen Zhang (Lallemand R&D)
Kevin Kraus (Lallemand Specialties, Inc.)
Chris Richards (Ethanol Technology, NA Technical Sales Manager)
Dave Kelsall (Ethanol Technology, Technical Service Manager)
May 22-26, Toulouse, FranceSeptember 25-29, Montréal, Canada
To learn more about yeast, and all aspects of
alcohol production, attend this week-long education
program!