enzyme and microbial conversions for … and microbial conversions for transportation fuels joy...

Enzyme and Microbial Conversions for Transportation Fuels

Joy Doran Peterson, Chair Biofuels, Biopower, and Biomaterials Initiative

Latin American Federation of Chemical Associations (FLAQ)July 29, 2008 San Juan, Puerto Rico

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Biofuels, Biopower, Biomaterials B3 Initiative (B3I)

Visit our Website at www.uga.edu/bioenergy

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Outline

Cellulosic EthanolBiochemical PlatformUsing sugarbeets and woody biomass as examples• 6C versus 5C• Novel enzymes and Biocatalysts• Inhibitors

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Status of Cellulosic Ethanol

Commercial=700 tons feedstock/d for 10-20 MGY FuelDemonstration= 70 tons feedstock/d for 1 MGYPilot = smallerCapacity ~629.5 MGY potential 995 MGY

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DOE and National Renewable Energy Laboratory Biomass Conversion Technology “Platforms”

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Cellulosic Biomass Processing:Biochemical Route

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Biomass

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Sugarbeet Carbohydrates

Pectin24%

(Galact-uronic acid)

Lignin2% Ash

11%

Sucrose1% Cellulose

24%(glucose)

Hemicellulose20-30%

(arabinose, rhamnose)

Protein8%

15-20% Sucrose

Sugarbeet Pulp

Sugarbeet

http://upload.wikimedia.org/wikipedia/commons/b/b3/Sucrose-inkscape.svg

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Sugar Beet Biorefinery

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Plant Structural Carbohydrates

Carpita and Gibeaut, 1993

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Lignocellulose degrading enzymes

endo-β-glucanase

cellobiohydrolase

β-glucosidase

cellobiohydrolase (GH7)

[β-1,4-glucosyl]

CelluloseCellobiase

[α-1,4-galacturonosyl]x + [(α-1,2-rhamnosyl)1+ (α-1,4-galacturonosyl)1]y

[various sugars]

pectin lyase + endo-polygalacturonase

α-rhamnosidase

α-arabinofuranosidaseα-galactosidase Pectin

Turner et al. 2007. Microbial Cell Factories.

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Biomass ≠ Biomass ≠ Biomass

15-20% Sucrose = 20-30 gallons ethanol/ton

Sugarbeet

Pectin24%

(Galact-uronic acid)

Lignin2% Ash

11%

Sucrose1% Cellulose

24%(glucose)

Hemicellulose20-30%

(arabinose, rhamnose)

Protein8%

Saccharomyces uses sucrose and glucosevery efficiently, but cannot use galacturonic acid nor arabinose. = 36 gal/ton

http://upload.wikimedia.org/wikipedia/commons/b/b3/Sucrose-inkscape.svg

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Hexoses and Pentoses

Embden-Meyerhof-Parnas Entner-Doudoroff Pentose Phosphate

Succinate

Lactate

Acetate

Acetyl-CoA + Formate

COCO22 H2EthanolEthanol

Lactate Dehydrogenase7.2 mM (ldhA)

PyruvateFormate-Lyase2 mM (pfl)

Pyruvate Decarboxylase0.4mM (pdc)

Acetaldehyde + COCO22

Alcohol Dehydrogenase(adhB)

Ethanol (95% Theo. Yield)Ethanol (95% Theo. Yield)

Zymomonas mobilisZymomonas mobilisPYRUVATEPYRUVATE

Microbial PlatformMicrobial Platform

PEP

L.O. Ingram Univ. Florida

X

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Improved Biocatalysts for Pectin-Rich Fermentations (+ Cellobiase)

CelluloseHemicellulose Pectin

LY40A

Ethanol

cellobiase

Pyruvate decarboxylase & alcohol dehydrogenase

Henriksen, Yomano, Ingram, Doran-Peterson, 2008.

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Ethanol Production from Sugarbeet Pulp Using Yeast or Pentose/Hexose Fermenting Biocatalyst with Enzymes

from Trichoderma and Aspergillus

Saccharomycesonly used the glucose and residual sucroseNeed ability to ferment arabinose(5C) and galacturonic acid for increased yields

Peterson, Int. Sugar J. 2006

10 Filter Paper Units of Cellulase/g dw pulp

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Discovery of novel enzymes and biocatalysts

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Fermentation

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Overview of proposed biorefinery (broken lines). Pulp mill operations are indicated by solid lines. Modified from Art Ragauskas, Georgia Institute of Technology

Integrating Biofuels and Pulp Production

Ethanol

Ethanol Sludge Bioethanol

Bioethanol

Wood Hemicellulose

Bioethanol

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Wood Composition and Theoretical Ethanol Yield for Each Component

Component Wood Ethanol (g/ton dw)

Pine (S) Birch (H) Pine (S) Birch (H)

Cellulose 6C 38-44 40-41 69.1 69.1

Glucomannan6C Total Ethanol

11-20 2-5 26.895.9

675.1

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Wood Composition and Theoretical Ethanol Yield for Each Component

Component Wood Ethanol (g/ton dw)

Pine (S) Birch (H) Pine (S) Birch (H)

Cellulose 6C 38-44 40-41 69.1 69.1

Glucomannan 6C 11-20 2-5 26.895.9

675.1

Xylan 5C 7-10 20-25 15 39.8

Other Carbs 0-5 0-4 4.4 3.5

TOTAL ETHANOL 115.3 118.4

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Composition of pulp and papermill sludge (variable due to amount of recycled and colored materials)

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Sludge Cellulose Hemicellulose Lignin Theoretical Yield Ethanol (gal/ton)

1 56.3 4.7 14.7

18.1

20.7

25.3

17.0

8.3

10.0

12.0

2 56.0 13.9

103 (47)

3 48.8 13.5

4 38.5 10.2

5 57.0 9.0

6 55.5 11.5

7* 69.7 8.6

8 34.6 8.2

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Improved Biocatalysts for Cellulose Fermentations (+ Cellobiase)

CelluloseHemicellulose Pectin

Ethanol

LY40A

Pyruvate decarboxylase & alcohol dehydrogenase

cellobiase

Henriksen, Yomano, Ingram, Doran-Peterson, 2008.

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Elemental Analysis of Paper Mill Sludge Before and After Enzyme Digestion

0.01

0.1

1

10

100

1000

10000

100000

Al Ca Ti Cr Fe Co Ni Cu Zn Se Pd Cd Sn Pb

Metals

Before enzymatic digestion

After enzymatic digestion(20FPU)

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Composition of pulp and papermill sludge (variable due to amount of recycled and colored materials)

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Sludge Cellulose Hemicellulose Lignin Theoretical Yield Ethanol (gal/ton)

1 56.3 4.7 14.7

18.1

20.7

25.3

17.0

8.3

10.0

12.0

2 56.0 13.9

103 (47)

115.4 (70)

135.6 (115)

3 48.8 13.5

4 38.5 10.2

5 57.0 9.0

6 55.5 11.5

7* 69.7 8.6

8 34.6 8.2

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SummaryCellulosic Ethanol Biochemical Platform approach converts biomass

to sugars, followed by fermentation to produce ethanol.

Sugarbeets, flexible process:• Yeast work well if fermenting sucrose• Need a 5C fermenter if fermenting pulp• Adding enzyme activities to fermenting organism can provide

benefit and increase ethanol yield

Woody biomass, integrated pulp and paper mill refinery • Know the substrate composition• 6C versus 5C fermenter depending upon the composition• Inhibitors may have significant impact on fermentation yields• Cost effective removal may yield additional coproducts

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AcknowledgementsPI: Dr. Joy Doran PetersonLab members: Dana Cook

Amruta JangidWhitney BolandBrian GardnerTyler RogersErica MillerKate Brandon Sean SuggsZeynep Cvetkovich

Collaborators:UGA Department of Microbiology

Dr. Eric StabbDr. Jeff BoseDawn Adin

UGA Complex Carbohydrate Research Center

UGA Mass Spectroscopy FacilityDr. Dennis Phillips

UF Department of MicrobiologyDr. Lonnie IngramLorraine Yomano

USDA Wyndmore, PADr. Arland HotchkissDr. Kevin Hicks

Funding:ASM Southeastern Branch:Henry Aldrich Student Research Grant, Beet Sugar Development Foundation, DOE, USDA

enzyme and microbial conversions for … and microbial conversions for transportation fuels joy...

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