High Efficiency Production of Xylitol from Hemicellulose by Fermentation

High Efficiency Production of Xylitol from Hemicellulose by Fermentation

High Efficiency Production of Xylitol from Hemicellulose by Fermentation David Demirjian, Ph.D. World Congress of Industrial Biotechnology President & CEO June 17, 2013 Corporate Overview . Biotech R&D Group . Enabling Carbohydrate Technology . Platform of Proprietary Process Technologies and Know-How . Focus: . Food Ingredients/Specialty Chemicals . Fine Chemicals/Pharma . Biotech Company . Chicago - Headquarters . Peoria - Main Research Facility . Key Collaborators . U.S. Department of Agriculture National Center for Agriculture Utilization Research (NCAUR) Contributors . zuChem . University of Illinois . F. Mike Racine, Ph.D. Huimin Zhao, Ph.D. Ryan Woodyer, Ph.D . Paul Taylor, Ph.D. Ryan Sullivan, Ph.D. Nathan Wymer, Ph.D. Nick Nair, Ph.D. Shama Khan . Trevor Christ . Other Collaborators/Consultants . USDA - NCAUR . Ian Fotheringham, Ph.D. – Ingenza Ltd. Badal Saha, Ph.D. Yoshikiyo Sakaibura, Ph.D. David Dodds, Dodds & Associates . Douglas Antibus, Ph.D. Bill Dowd, Ph.D. – fmr VP R&D Dow . Greg Kennedy . David Ager, Ph.D. – Competence Mgr DSM Funding Biotechnology Research and Development Corporation a U.S. Department of Energy – Biomass Program National Science Foundation Improving Economics of Ethanol Production INPUT OUTPUT Ethanol Inexpensive Biomass Feedstock DDG, Fiber BY-PRODUCTS Bagasse ~$300/ton Value Added Chemicals e.g. Xylitol >>$3000/ton Polyol Market Specialty Sweeteners Sorbitol $750 Xylitol - 3X growth projected $125 $80 Mannitol $370 Erythritol and Others Xylitol Production Current Chemical Process Xylitol Purified Xylose expensive Chemical Polyol contaminants & limited supply Hydrogenation purification cost driven by xylose purity zuChem Bioprocess Hemicellulose Xylitol inexpensive abundant supply Fermentation No polyol contaminants Mixed C5/C6 is OK Simple purification Challenges and Process Goals • Little or no arabitol produced • Overcome Problems with Fermentation Inhibition – Furfurals and other inhibitors • Abundant Feedstock Supply • Tolerant to Feedstock Variability • Process Economics – Yield – 100 g/l – Purity – essentially free of sugar and polyol contaminants – Throughput - >3 g/L-h – Recovery - >85% Hemicellulose C5/C6 Composition C5 C6 D-Xylose L-Arabinose D-Glucose D-Galactose Other Birch Wood 89.3% 1% 1.4% - 8.3% Rice Bran 46% 44.9% 1.9% 6.1% 1.1% Wheat 65.8% 33.5% 0.3% 0.1% 0.1% Straw Corn Fiber 48-54% 33-35% - 5-11% 3-6% Sugar Cane 80-90% 5-10% 0-5% 0-2% 0-5% Bagasse Saha, B., J. Ind. Microbiol. Biotech. (2003) 30: 279-91, unpublished observations Conversion of Xylose to Xylitol XR in ara+ host Conversion of Xylose to Xylitol with XI / XDH 8 3.5 7 Glucose 3 6 D-Xylose Xylose 2.5 5 L-Arabinose Arabinose 2 4 Arabitol Arabitol 1.5 3 Xylitol 1 2 Xylitol Concentration (W/V) Concentration Concentration (W/V) Concentration 0.5 1 0 0 0 10 20 30 40 0 20 40 60 80 Hour Hour Hours Hours Xylose Specific Selection Strain Plate on L-arabinose/D-xylose No -XR growth + xdh araB LETHAL XR L-arabitol L-arabitol-5-phosphate + No Xylitol xdh growth D-xylulose + araB XR’ xdh xylitol D-xylulose Growth XR - active on L-arabinose and D-xylose XR’ - D-xylose specific enzyme Xylose Specific Mutants Specific Xylose D-Xylose/L-Arabinose Rate Ration WT MUT1 MUT2 MUT3 MUT4 1.2 1 o i t a 0.8 r e t a r e s o n i b 0.6 a r a - L / e s o l y 0.4 x - D 0.2 0 Pstip XR mut#1 mut#15 mut#16 mut#17 Conversion of Arabinose to Xylitol Testing on Hydrolysate Fermentation Test On Unremediated Hydrolysates 4 3.5 Slow growth on 3 hydrolysates due 2.5 to fermentation 2 inhibition 1.5 1 0.5 0 Fiber 1 Fiber 2 Fiber 3 Fiber 3 Stover Hardwood Purified O.D. 600 Remediation Tests Treatment Relative growth Absorbance 600 nm NaOH to pH 7 1.8 Ca(OH)2 to pH 7 2.1 Ca(OH)2 to pH 9.05 3.8 H2SO4 to pH 7 Ca(OH)2 to pH 10.5 6.2 H2SO4 to pH 7 Remediation Comparison Fermenter results confirmed that over-liming at high pH is best; charcoal also helps Xylitol Process Optimization Xylitol Production from Hydrolysate • Optimum pH (6.8) and 120 temperature (37°C) were determined 100 80 • Optimal nutrients Glucose identified 60 Xylose Arabinose • Fed-batch protocol 40 Arabitol developed Xylitol g/L Substrate or ProductSubstrate g/L 20 • Air/agitation 0 0 10 20 30 40 50 requirements were Hours determined Met Goals of Process • Conversion of xylose to xylitol was 98%. • Volumetric productivity 3.1g /L-h at 32 hours • Yield was >100 g/L • Complete conversion of sugars Summary • ~8 patents on file in four patent families - First patents now issued. • Piloting now underway with first Manufacturing Partner – Godavari Biorefineries • Sampling to Customers Expected this Summer • New Manufacturing facility expected to be operational by end of 2014 • Broad Licensing Strategy .

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