Enzyme and Microbial Conversions for Transportation Fuels Joy Doran Peterson, Chair Biofuels, Biopower, and Biomaterials Initiative

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1 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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3 Outline Cellulosic Ethanol Biochemical Platform Using sugarbeets and woody biomass as examples 6C versus 5C Novel enzymes and Biocatalysts Inhibitors 3

4 Status of Cellulosic Ethanol Commercial=700 tons feedstock/d for MGY Fuel Demonstration= 70 tons feedstock/d for 1 MGY Pilot = smaller Capacity ~629.5 MGY potential 995 MGY 4

5 DOE and National Renewable Energy Laboratory Biomass Conversion Technology Platforms 5

6 Cellulosic Biomass Processing: Biochemical Route 6

7 Biomass 7

8 Sugarbeet Carbohydrates Sugarbeet Lignin 2% Ash 11% Protein 8% Sucrose 1% Cellulose 24% (glucose) 15-20% Sucrose Sugarbeet Pulp Pectin 24% (Galacturonic acid) Hemicellulose 20-30% (arabinose, rhamnose) 8

9 Sugar Beet Biorefinery 9

10 Plant Structural Carbohydrates Carpita and Gibeaut,

11 Cellobiase Lignocellulose degrading enzymes endo-β-glucanase β-glucosidase [β-1,4-glucosyl] cellobiohydrolase cellobiohydrolase (GH7) Cellulose α-galactosidase α-arabinofuranosidase Pectin pectin lyase + endo-polygalacturonase α-rhamnosidase [α-1,4-galacturonosyl] x + [(α-1,2-rhamnosyl) 1 + (α-1,4-galacturonosyl) 1 ] y [various sugars] 11

12 Biomass Biomass Biomass Sugarbeet Pectin 24% (Galacturonic acid) Lignin 2% Ash 11% Protein 8% Sucrose 1% Cellulose 24% (glucose) Hemicellulose 20-30% (arabinose, rhamnose) 15-20% Sucrose = gallons ethanol/ton Saccharomyces uses sucrose and glucose very efficiently, but cannot use galacturonic acid nor arabinose. = 36 gal/ton 12

13 L.O. Ingram Univ. Florida Hexoses and Pentoses Microbial Platform Embden-Meyerhof-Parnas Entner-Doudoroff Pentose Phosphate Succinate X PEP PYRUVATE Zymomonas mobilis Lactate Dehydrogenase 7.2 mm (ldha) Lactate Acetate Acetyl-CoA + Ethanol Pyruvate Formate-Lyase 2 mm (pfl) Formate CO 2 H 2 Pyruvate Decarboxylase 0.4mM (pdc) Acetaldehyde + CO 2 Alcohol Dehydrogenase (adhb) Ethanol (95% Theo. Yield) 13

14 Improved Biocatalysts for Pectin-Rich Fermentations (+ Cellobiase) LY40A Cellulose Hemicellulose Pectin cellobiase Ethanol Pyruvate decarboxylase & alcohol dehydrogenase 14 Henriksen, Yomano, Ingram, Doran-Peterson, 2008.

15 Ethanol Production from Sugarbeet Pulp Using Yeast or Pentose/Hexose Fermenting Biocatalyst with Enzymes from Trichoderma and Aspergillus 10 Filter Paper Units of Cellulase/g dw pulp Saccharomyces only used the glucose and residual sucrose Need ability to ferment arabinose (5C) and galacturonic acid for increased yields Peterson, Int. Sugar J

16 Discovery of novel enzymes and biocatalysts 16

17 Fermentation 17

18 Integrating Biofuels and Pulp Production Bioethanol Ethanol Wood Hemicellulose Bioethanol Ethanol Sludge Bioethanol Overview of proposed biorefinery (broken lines). Pulp mill operations are indicated by solid lines. Modified from Art Ragauskas, Georgia Institute of Technology 18

19 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 Glucomannan 6C Total Ethanol

20 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 Glucomannan 6C Xylan 5C Other Carbs TOTAL ETHANOL

21 . Composition of pulp and papermill sludge (variable due to amount of recycled and colored materials) Sludge Cellulose Hemicellulose Lignin Theoretical Yield Ethanol (gal/ton) (47) 7*

22 Improved Biocatalysts for Cellulose Fermentations (+ Cellobiase) LY40A Cellulose Hemicellulose Pectin cellobiase Ethanol Pyruvate decarboxylase & alcohol dehydrogenase 22 Henriksen, Yomano, Ingram, Doran-Peterson, 2008.

23 Elemental Analysis of Paper Mill Sludge Before and After Enzyme Digestion Before enzymatic digestion After enzymatic digestion(20 FPU) Al Ca Ti Cr Fe Co Ni Cu Zn Se Pd Cd Sn Pb Metals 23

24 . Composition of pulp and papermill sludge (variable due to amount of recycled and colored materials) Sludge Cellulose Hemicellulose Lignin Theoretical Yield Ethanol (gal/ton) (47) (70) 7* (115) 24

25 Summary Cellulosic 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 25

26 Acknowledgements PI: Dr. Joy Doran Peterson Lab members: Dana Cook Amruta Jangid Whitney Boland Brian Gardner Tyler Rogers Erica Miller Kate Brandon Sean Suggs Zeynep Cvetkovich Funding:ASM Southeastern Branch:Henry Aldrich Student Research Grant, Beet Sugar Development Foundation, DOE, USDA Collaborators: UGA Department of Microbiology Dr. Eric Stabb Dr. Jeff Bose Dawn Adin UGA Complex Carbohydrate Research Center UGA Mass Spectroscopy Facility Dr. Dennis Phillips UF Department of Microbiology Dr. Lonnie Ingram Lorraine Yomano USDA Wyndmore, PA Dr. Arland Hotchkiss Dr. Kevin Hicks 26