1. Creating a Bio-refinery Based on Pulping Technology Don Guay – Dept. of Paper Science & Engineering Eric Singsaas – Dept of Biology UW – Stevens Point

2. Biofuel barriers Feedstock Grains are not a long-term solution Cellulosic feedstocks must come from many sources Cellulose processing Low cellulose hydrolysis yield Cellulase enzymes are costly Requires caustic and energy-intensive pretreatment Many steps required for bioprocessing Products (ethanol) Low energy density compared with petroleum fuels Corrosive Transport issues Market saturation

3. Lee R Lynd, Mark S Laser, David Bransby, Bruce E Dale, Brian Davison, Richard Hamilton, Michael Himmel, Martin Keller, James D McMillan, John Sheehan & Charles E Wyman Nature Biotechnology 26, 169 - 172 (2008)doi:10.1038/nbt0208-169

4. Availability Existing forest harvest 142 million tons Residues and non-marketable biomass 226 million tons Incompletely pulped material 1-10 tons/day per mill Miscanthus potential yield 10-15 tons/acre (UIUC) 6-10 tons/acre (ORNL) Unrecyclable paper

5.  Softwood pulp  Hardwood pulp  Corn stover  Miscanthus grass http://www.energy.iastate.edu/becon/tour/tourimages/02-corn_stover.jpg

6. SSF Size reduction Remove Hemicellulose Remove Lignin Catalyst Fermentation Enzymatic Hydrolysis

13. Treated softwood pulp Untreated softwood pulp

14. Direct Bioproduction of Energy-Rich Fuels This breakthrough, high-payoff opportunity focuses on microbes for direct production of hydrophobic alternative fuels (i.e., alkanes, longer-chain alcohols, and fatty acids). This would overcome one limitation of nearly all bioconversions—they result in dilute aqueous mixtures. Typical industrial product concentrations are 100 to 150 g/L for ethanol and other such products as organic acids. This limitation imposes separation requirements that increase process and energy costs. New fermentation systems would be highly desirable to allow significant increases in product concentration, new types of products, and new processes for product recovery. Strong increases in efficiency also could be achieved by developing continuous processes. U.S. DOE. 2006. Breaking the Biological Barriers to Cellulosic Ethanol: A Joint Research Agenda, DOE/SC/EE-0095, U.S. Department of Energy Office of Science and Office of Energy Efficiency and Renewable Energy, http://doegenomestolife.org/biofuels/.. Adapted from M. Himmel and J. Sheehan, National Renewable Energy Laboratoryhttp://doegenomestolife.org/biofuels/

15. Ethanol Methyl- butenol Isoprene Formula C2H6OC2H6OC2H6OC2H6O C 5 H 10 O C5H8C5H8C5H8C5H8 Water solubility mol m -3 ∞117011.7 Henry’s law coeff. Pa m 3 mol -1 0.5071.567780 K O/W mol mol -1 0.4917.8263 Energy content BTU gal -1 85,000106,000135,000 T boil (°C / °F) 78.4 / 173 98.6 / 209 33.9 / 92.9 Potential Uses FuelFuel Rubber products Pharmaceuticals *BTU/gal. Gasoline energy content ranges 111,000-125,000

16. Tomohisa KUZUYAMA, “Mevalonate and Nonmevalonate Pathways for the Biosynthesis of Isoprene Units”, Biosci. Biotechnol. Biochem., Vol. 66, 1619-1627 (2002). Glucose Methyl butenol Isoprene ispS MBO synthase The MEP pathway 2-C-methyl- D -erythritol 4-phosphate

18. StructureClassExampleUses C5HemiterpeneIsoprene Industrial ? C5+OHemiterpenoid Methyl butenol Biofuel ? C10Monoterpeneß-pinene C10+OMonoterpenoid Pyrethrin -I Pesticide C15 (+O) SesquiterpeneGossypol Male contraceptive Modified sesquiterpenoid Artemisinin Anti-malarial drug C20 (+O) Diterpenoids (modified) TaxolAnti-cancer C30 (+O) TriterpenoidsPhytosterolsNutrient/medical C40Tetraterpenoidsß-caroteneLycopeneNutrient Isoprenoids & their potential uses

19. Enabling technologies Metabolic control analysis of the MEP pathway Synthetic MEP pathway operon MBO synthase gene Bioreactor experiments

20. Inputs Waste paper and Mill sludge Papermill pulp Energy crops Forest products Stover Products Methyl butenol Isoprene Ethanol Other products? http://www.ornl.gov/sci/besd/highlights.shtml Accessed 11-15-07

21. Pulp Mills in Our region

22. Research in progress – Benchtop Pulping Optimize catalyst and enzyme dosage Analyze product streams Develop microorganisms Consolidate bioprocessing Economic analysis

23. Cellulose Sciences, Inc. Others

24. Our sincerest thanks to: WiSys Maliyakal John Lisa Murray Bill Adolfsen UWSP administration Chancellor Linda Bunnell Christine Thomas Randy Champeau Lance Grahn Charles Clark Hydrite Technologies Chuck Krier Marc Baures Students Brent Rivard Aaron Stieve Becky Slatterley Collaborators Tom Sharkey, MSU Amy Wiberley, MSU