specific yeasts developed for modern ethanol production · specific yeasts developed for modern...

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


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


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


Yeast Metabolic Pathways


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.


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


The Big Picture

EnvironmentGenotype Phenotype

Production MethodProduct FormPlant Handling

Inoculation TechniqueFermentation Conditions

Strain Characteristics Fermentation Performance


Yeast Strains: Saccharomyces cerevisiae

EngineeredEngineered

Selected Selected –– IIII

Selected Selected –– II

WildWild

TimeTime

Per

form

ance

Per

form

ance


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


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


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

Ingledew 2003

Contamination


Commercial Yeast Products

Formulations & PerformanceADY = Active Dry YeastFresh ‘Cake’ YeastLiquid Yeast (2)


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


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


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


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)


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


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


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


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


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


Commercial Yeast Production

Proprietary Strain Storage


Commercial Yeast Production Overview

Fresh cake yeast

Activedry yeast

Stabilized liquid yeast


Fermentation


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


Separation and Drying


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


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


Summary & Conclusions

Start with the Alcohol Production Fermentor

Yeast Strains

Yeast Product Formulations

Yeast Management Options

Yeast Production, Quality Control, Technical Support


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!


Danke

May 22-26, Toulouse, FranceSeptember 25-29, Montréal, Canada

specific yeasts developed for modern ethanol production · specific yeasts developed for modern...

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