Process Simplification for Conversion of Lignocellulosic Biomass into Renewable Fuels and Chemicals Agricultural and woody residues,bagasse, energy crops and trees Supported by DOE Basic Energy Sciences, DOE EERE, USDA, Univ. of Florida, Florida State Legislature, Buckeye Technologies Myriant Technologies, and BP-Vercipia Lonnie O. Ingram ingram@ufl.edu
Woody Biomass ~60-70% Carbohydrate Corn ~70% starch Black thermoplastic that can be burned to provide energy for ethanol production. Lignin Nature s plastic glue (thermoplastic) Cellulose Hemicellulose Primarily pentoses, 5-carbon sugars also 6-C sugars (Dilute phos acid Inhibitors) Homopolymer of glucose, hexose, a 6-carbon sugar. (Enzymes) Composition of Lignocellulosic Biomass
HEXOSES (C6) + PENTOSES (C5) Microbial Platform Embden-Meyerhof-Parnas Entner-Doudoroff Pentose Phosphate + ATP + NADH Succinate X PEP PYRUVATE (Zymomonas mobilis) Lactate Dehydrogenase 7.2 mm (ldha) Lactate X Acetyl-CoA + Formate X X Ethanol Acetate X Pyruvate Formate-Lyase 2 mm (pflb) CO 2 H 2 BP Biofuels (Verenium/Vercipia) - NADH Pyruvate Decarboxylase 0.4mM (pdc) Acetaldehyde + CO 2 Alcohol Dehydrogenase (adha, adhb) Ethanol (95% Yield) UF Cellulosic Tequilla based process
Fermentative Metabolism No external electron acceptor Low cell mass Low ATP yield High Product Yield Selecting for faster growth and higher cell densities co-selects for higher productivity and titer (and resistance) Sean York & Lorraine Yomano 6 ATP 6 ADP+Pi 6 ADP+Pi Cell Growth 6 Xylose 10 Pyruvate 10 NAD+ 10 NADH 10 CO 2 + 10 acetaldehyde pdc adha adhb 10 NADH 10 NAD+ 10 ethanol + CO 2 5% Carbon cell mass 95% Carbon product ~1 ATP/xylose
Metabolic Evolution minimal salts + 2% Xylose minimal salts 5-14% Xylose + 1 mm Betaine 16 hours <16 hours 24 hours 24 hours 24 hours 37 C 120 rpm seed flask transfer #1 T #2 37 C, ph 6.5, 150 rpm Inocula: 1:100 to 1:350 dilutions
Current Results with Bagasse and hydrolysate resistant biocatalysts: 65 85 gal ethanol per dry ton bagasse 70 60 Lab sugar and mineral salts medium Xylose to EtOH 37C Corn Process: 50 85-90 gal ethanol per ton corn (~33 bu) EtOH, g/l 40 30 Improved strain 1 Improved strain 2 KO11 20 10 Mineral Salts + 15% xylose Inoculum = 3 mg dcw/l 37C, 150 rpm, ph 6.5 0 15 40 65 90 115 140 Hours
Comparison of conversion processes A. Sulfuric Lignocellulose Process Lignocellulose Dilute Acid Hydrolysis (Zirconium Hydrolyzer) Liquid/solid Separation cc-washing Hemicellulose Syrup & inhibitors Cellulose +Lignin Fermentation Cellulose+ Cellulase CapX Side products of hydrolysis (hydrolysate inhibitors) drive process complexity! Hemicellulose Syrup Detox Hemicellulose Fermentation Purification Gypsum B. Phosphoric Lignocellulose Process (goal) Lignocellulose Dilute Acid Hydrolysis (Stainless Steel Hydrolyzer) L+SScF Process (eliminates steps) Liquefaction + cellulase & hemicellulase Fermentation + cellulase & hemicellulase CapX Purification Co-products C. Mature Corn to Ethanol Industry CapX Corn Steam Cooker (Stainless Steel Cooker) Liquefaction + amylase & glucoamylase Fermentation + amylase & glucoamylase Purification Co-products
Research Advances Required for Process Simplification: 1. Develop biocatalysts with improved resistance to hemi toxins. 2. Replace Sulfuric with less aggressive acid Phosphoric Eliminate Zirconium Lower the level of inhibitors Gypsum piles replaced with crop fertilizer (borrowed) 3. Solve mixing and pumping issues with high fiber solids 10-20% dw CSTR -- Liquefaction tank with 6 h mean residence time 4. Develop process using only fertilizer chemicals (N,P,K,Mg,S,trace metals) Coproduct New crops Eliminated fiber washing, detox, separate fermentations, & developed co-product value for all process materials.
Phase I: State Board of Regents -Legislature/DOE EERE Campus Biofuels Lab - Unit Ops (50 lb batches)
190C, 5 min 0.5% phosphoric dry bagasse basis
Alcohols catechol coniferyl dihydrosynapil guaiacol synapil syringol vanillyl Acids caproic coumaric ferulic gallic gentisic hydroxybenzoic protocatechuic synapic syringic vanillic Aldehydes cinnamaldehyde hydroxybenzaldehyde syringaldehyde vanillin Lignin (aromatics) 10-20% Pectin (polygalacturonate) 2-20% Other 8 % Hemicellulose (xyl, ara, man, glu, gal) 20-40% Cellulose (glu) 20-50% Potential Inhibitors from Dilute Acid Pretreatment Furfural Hydroxymethylfurfural Acetic acid Formic acid Levulinic acid Inhibitors can be our friends. What does a microbiologist do with an inhibitor?
(Isolate a mutant.) Furan Resistant Mutants of Escherichia coli Furfural (xylose, arabinose) 5-Hydroxymethylfurfural (fructose, glucose, mannose, galactose, etc.) BACKGROUND: Elliot Miller Multiple furfural reductase(s) NADPH dependent None identified or well-characterized Furfurol (alcohol) is less toxic than the aldehyde. Over 10 beneficial genes so far. Laura Jarboe Berenice Jaramillo Mike Mullinnix Pete Turner Phi Do
Mixing & Pumping: Liquefaction with Cellulases Pumps that did not work! Rotary lobe Gear Diaphragm Progressive cavity Centrifugal Peristaltic Viscocity (centipoise,cp) 20500 20000 19500 12000 10000 8000 6000 60 C 0.5-10% BioW phz 10% Solids Shredded Biomass 0.25 0.5% FPU/g fiber 0.50 1% FPU/g fiber 5.0 10% FPU/g fiber 5 FPU 99% reduction in viscosity <5% digestion of cellulose The velcro and marbles model 4000 2000 0 0 1 2 3 4 5 6 23 24 Time (h)
Model CSTR 6 hr residence Velcro hooks and loops After liquefaction (cellulase) http://www.architerials.com/
Process Development Woody Biomass Dilute Acid Impregnation High Temp Steam Distillation Purification Fermentation (C5+C6) Liquefaction Enzymes Ethanol & other chemicals Fiber for boiler No substrate fractionation No liquid/solid separation No toxin cleanup No purification of sugars No Zirconium or exotic metal Fermentation broth = fertilizer
Seed Cultures are grown in hydrolysate supplemented with ammonia, MgSO4, and trace metals. Flask T1 T2 T3 Flask T1 T2 T3 3.6 million fold expansion of seed
Phase I - Scaling up the L+SScF Process Bagasse 0.27 g E/g bag 27 g Ethanol/L 342 liters/tonne ~90 gal/tonne Furfural Gluc Xyl EtOH ~82 gal/ US ton 65-82 gal Ismael Nieves Claudia Geddes Mike Mullinix Ralph Hoffmann
N, P, K, Mg S, Fe, Ca Borrowing water and nutrients Day 0 Seed Propagation Day 1 Pretreatment Liquefaction Fermentation Day 1-4 Fermentation Day 4-5 Beerwell Day 4-5 Distillation Decanter
Engineered Bacteria for Plastics and Fuel 2 ADP 2 ATP Sugar 2 NADH (5 glucose= 6 xylose) 2 NAD CO 2 1.7 succinate 2 Pyruvate ldha or ldhd ldhl 2 D(-)-lactate (>90%) (Purac) 2 L(+)-lactate 2 ethanol + 2 CO 2 (50% yield by wt)
Buckeye Technologies, Perry, Florida (dissolving pulp) UF Stan Mayfield Biorefinery Pilot Plant Time to scale up!
UF Stan Mayfield Biorefinery Pilot Plant hosted by Buckeye Technologies, Perry FL Woody Residues Woody Crops and Trees BKI materials Size: 3 tons of cellulosic biomass/day for Bagasse; ~ 5 tons/day for Wood Sorghum syrup Partial Saccharification CO 2 Fuel Ethanol Lignin Fertilizer Co-products Hemi streams Sweet Sorghum and Related sugar crops Maximum Capacity per Day (lignocellulose) 400 gal ethanol/day or 5,000 lb of organic acids Process borrows water and nutrients that are used to grow new energy crops. Organic acids, Plastics
UF Stan Mayfield Biorefinery Pilot Plant 8,500 sq. ft. process 3,500 sq. ft. service labs 6,000 sq. ft. client space chiller 18,000 sq. ft. Distillation
Goals for the Stan Mayfield Biorefinery Pilot Plant Facilitate the deployment of biobased companies in Florida Validate and demonstrate Florida Biomass feedstocks Attract and support corporate R&D in Florida Develop co-products for biobased fuels and chemicals Validate and improve integration of bioconversion processes Develop new biobased processes and products Educate and train scientist and engineers Increase employment opportunities
UF, Stan Mayfield Biorefinery Pilot Plant VP Agriculture Florida Center for Renewable Chemicals and Fuels Steering Committee (UF, BKI, +?) Scientific/Technical Advisory Committee Chief Scientist-LOI 14-20 directly involved in operation Laboratory Director-GL Operating Committee (LOI, GL, RH, IN, LF) Manager of Operations-RH Senior Process Engineer-IN Contract Maintenance Support needed Admin/reception 3 Biomass loading 3 Plant Operators 2 New Post Docs: Troy (Eng), Marco (Eng) Continuing Technician: Mike Mullinix (Dec); Joe Sauvage (BS Engineer, May) Buckeye 1 Analytical Chemist, 1 Process Engineer Unit Ops (50 lb/day) Biofuels Pilot Plant Strain Development Lab 2 lab helpers, half time each. Additional Post Docs?
Unique Features of UF Stan Mayfield Biorefinery 1. University based, open to academia, industry, and public 2. Fully integrated process, biomass to ethanol to co-product 3. Phosphoric and fertilizer based chemistry 4. No exotic metals; Reduction in process steps 5. Vapor phase membrane permeation (replaces molecular sieve, reducing energy) 6. Paper Mill partner, wood focus, but substrate flexible 7. Presteamer to soften tissues and displace air 8. Hemicellulose hydrolysate used to grow seed train 9. Capacity of 3-5 ton/day; 3 each 10,000 gal fermenters 10. Florida Biomass: Sweet sorghum, perrenial grasses, Eucalyptus, Poplar, Cottonwood
Funding the Stan Mayfield Biorefinery Pilot Plan 1. Base level of support - UF, State, DOE, USDA, BKI 2. Anchor bio-industry 3. Multiple industry clients: materials, services, testing 4. Federal, state, and industry Research Grants 5. USDA Regional Biomass Program? 6. Foreign companies? South America, Asia, Europe Operating costs depends on activities. Estimated at $ 2 mil - $3 mil per yr
Biomass Handling Feed Bin Pretreatment Reversing Conveyor Pre-Steam Pretreatment C5- Stor age Tank Prep Tank Product Storage Distillation Distillation Disposal C6-Bin Screw-Press Prop 3A Prop 3B Prop 2A Prop 2B Prop 1A Pro p 1B Propagation Deca nter Tank Liquefaction ph Adjust -ment Ferme n-tor A Ferme n-tor B Ferme n-tor C Beer Well Decanter Centrifuge (C5+C6) Liquefaction Fermentation Heat Kill Fiber Solids Stillage Tank CIP Overall Process Buckeye Waste Treatment Heat Kill
Cellulosic Biorefinery
Gravity is our friend.
Gravity helps.
UF Biofuel Crop Studies (Erickson) Sweet Sorghum Energy cane and others Perennial grasses (8-12 dry tons/yr) John Erickson JErickson@ufl.edu
EA at Citra 8 mos EA at Valdosta 8 yrs EA at Valdosta 5 yrs Don Rockwood DLRock@ufl.edu
SRWC Summaries for Florida and the SE E. grandis E. amplifolia P. deltoides C. torelliana Dry TAY 16.1 11.2 8.9? Rotation 2-4 3-5 4-6 8 Location Land Type C and S FL; N FL Infertile Sands to Fertile Clays Don Rockwood DLRock@ufl.edu C and N FL; Lower SE Alkaline, Ag Lands C and N FL; SE Moderate, Ag Lands C and S FL Infertile Sands to Moderate, Ag Lands Propaga tion Seedlings, Cultivars Seedlings, Varieties Clones Seedlings
Gravity! Lonnie O. Ingram Univ of Florida Ingram@ufl.edu Dr. Nieves Dr. Geddes Mr. Hoffmann 352/392-8176 Thank you
Process for Cellulosic Ethanol Steam Pretreatment + Phosphoric Acid (presteamer, hydrolyzer and blow tank) Process Development Similar to Liquifaction (enzymes, ph) Borrowing water and nutrients Corn Dry Milling N, P, K, Mg S, Fe, Ca Sized Bagasse Or Wood Single Vessel Fermentation Distillation Ethanol Heat - kill Cleanup Stillage Centrifugation Waste treatment (energy crops) Ca-Phos Coproducts Boiler & Coproducts Lignin enriched fiber