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Production of Succinic Acid from Lignocellulosic Materials

Production of Succinic Acid from Lignocellulosic Materials N. P. Nghiem, M. I. Donnelly*, and B. H. Davison Oak Ridge National Laboratory *Argonne National Laboratory. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY. Product Opportunities for Succinic Acid Derivatives.

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Production of Succinic Acid from Lignocellulosic Materials

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  1. Production of Succinic Acid from Lignocellulosic Materials N. P. Nghiem, M. I. Donnelly*, and B. H. Davison Oak Ridge National Laboratory *Argonne National Laboratory OAK RIDGE NATIONAL LABORATORY U.S.DEPARTMENT OF ENERGY

  2. Product Opportunities for Succinic Acid Derivatives Tetrahydrofuran (THF) - 255MM lbs 1,4-butanediol (BDO) - 150MM lbs MaleicAnhydride - 450MM lbs Gamma-butyrolactone (GBL) - 105MM lbs FumaricAcid - 35MM lbs 2-pyrrolidinone Succinic Acid - 6 MM lbs Glucose MalicAcid - 20MM lbs n-methyl- 2-pyrrolidinone (NMP) - 80MM lbs DibasicEsters - 50MM lbs 4,4-polyesters U. S. Market lb/yr - SRI Report, 9/94

  3. Succinic acid from Lignocellulosics • Goal: to test existing and new strains for lignocellulosic mixed sugar fermentation • Outline- • prior patent strain results on glucose • develop and test two new derived strains • on mixed sugars • on actual hydrolyzates

  4. Feedstock Sources for Biobased Products • Corn starch: Glucose • Lignocellulosic materials: glucose and xylose

  5. Rice Straw Composition Feedstock component Dry wt% Glucan 38.9 Mannan 0.0 Galactan 0.5 Xylan 20.4 Arabinan 3.4 Lignin 13.5 Extractives 5.3 Ash 18.0 We will use a 2/1 glucose/xylose ratio and actual rice straw hydrolyzate from Arkenol’s concentrated acid and separation technology

  6. Fermentation by E. coli Wild Type AFP 111

  7. Lineage of AFP111A mutant E. coli that produces succinic acid as its major fermentation product W1485 (wild type) FMJ123 (pfl::Cam) NZN111 (pfl::Cam, ldh::Kan) AFP111 (pfl::Cam, ldh::Kan, ptsG) (ATCC 202021; US Pat # 5770435)

  8. Succinic Acid Fermentation Process Description • Two-stage fed-batch process * Aerobic growth stage * Anaerobic production stage • Inexpensive industrial medium * Light steep water at 25% (v/v) or corn steep solids at 2% (w/v) as nutrient source * Simple inorganic salts (in g/L): K2HPO4 1.4 KH2PO4 0.6 (NH4)2SO4 2.0 MgSO4 0.2

  9. AFP 191 • Spontaneous mutant derived from AFP 111 • Selected for ability to grow on xylose

  10. Lineage of AFP184A mutant E. coli that produces succinic acid at high yields from both glucose and xylose C600 (near wild type, xylose fermenter) AFP182 (ptsG21) AFP183 (ptsG21 DpflB::Cam) AFP184 (ptsG21, DpflB::Cam, ldhA::Kan) (Patent application)

  11. Calculated Results of AFP 184 Expts Here (Productivity, Yields, etc.)

  12. Succinic Acid from Lignocellulosic Hydrolyzates - Highlights • Production of succinic acid from lignocellulosic materials • All AFP three strains can produce SA from xylose/glucose mixtures • AFP 184 is best in yield and concentration • ptsG mutant is key in shift to good SA production • All can grow on actual Arkenol hydrolyzates without inhibition • Xylose fermentation is linked to glucose fermentation

  13. Acknowledgements Funding provided by U.S. Department of Energy - Office of Industrial Technologies - Chemicals Team. Hydrolysate: Arkenol, Inc. Experiments: ORNL Fermentations: Cathy McKeown, Robin Kirkegard, Tim Cofer ANL gene manipulation:Cynthia Sanville Millard Succinic Acid Commercialization partner: Applied Carbochemicals, Inc

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