Production of xylitol from biomass using an inhibitor-tolerant fungus

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1 Production of xylitol from biomass using an inhibitor-tolerant fungus Nancy Nichols National Center USDA ARS National Center for Agricultural Utilization Research Peoria IL USA Peoria, IL

2 Biomass conversion nrel.gov Cellulose Hemicellulose Enzymes Acid Hexoses Pentoses Inhibitors X Recombinant Yeast or Bacteria Fuel or Chemical

3 Inhibitors formed during hydrolysis O Pentoses Hemicellulose HO O HO OH OH O H 3 C OH Acetic O O HO Ferulic O OH CH 3 Galactose Glucose HO O OH OH HO OH Cellulose OH OH O H OH H H OH H OH Furfural HO O O HMF H 3 C O OH Formic O O Lignin Phenolics OH Levulinic

4 A microbiological approach to inhibitor abatement Cellulose Hemicellulose pretreat Hexoses Pentoses Inhibitors X recombinant yeast or bacterium Fuel or Chemical Bioabatement

5 Enrichment of detoxifying microbes Environmental sample: contaminated soil Enrichment in defined media Furfural 5-HMF Ferulic acid Enrichment in Hydrolysate Isolation and identification Microorganisms metabolize toxic compounds Microorganisms survive/grow in dilute acid hydrolysates Lopez et al. Appl Microbiol Biotechnol 64:

6 Identification of isolates A3, A6, B1-1: Pseudomonas sp. A1: Methylobacterium extorquens A5: Arthrobacter aurescens B1-1: Acinetobacter sp. C6: Flavobacterium indologenes C7: Stenotrophomonas maltophylia Coniochaeta ligniaria Lopez et al. Appl Microbiol Biotechnol 64:

7 Coniochaeta ligniaria NRRL3616 (Ascomycete)

8 Concentration (mm) Removal of furans from corn stover hydrolysate 15 1 Furfural 5 HMF Time (hr) Lopez et al. Appl Microbiol Biotechnol 64:

9 Bioabated hydrolysate Lactic acid Levulinic acid 2-Furoic acid 3,4-dihydroxybenzoic acid 3,5-dihydroxybenzoic acid 2,5-dihydroxybenzoic acid 3,4-dihydroxybenzaldehyde Salicylic acid 4-Hydroxybenzaldehyde Vanillic acid 4-Hydroxyacetophenone Caffeic aicd Syringic acid Vanillin Syringealdehyde 4-Hydroxycoumaric acid Ferulic acid Time (H) Nichols et al. Enz Microbial Technol 42:624-63

10 Untreated corn stover hydrolysate Lactic acid Levulinic acid 2-Furoic acid 3,4-dihydroxybenzoic acid 3,5-dihydroxybenzoic acid 2,5-dihydroxybenzoic acid 3,4-dihydroxybenzaldehyde Salicylic acid 4-Hydroxybenzaldehyde Vanillic acid 4-Hydroxyacetophenone Caffeic aicd Syringic acid Vanillin Syringealdehyde 4-Hydroxycoumaric acid Ferulic acid Time (H) Nichols et al. Enz Microbial Technol 42:624-63

11 General bioabatement scheme 1 % corn stover and.66% H 2 S 4 Steel pipe reactors fluidized heating bath, 18 C, 1 min. Adjust ph Wash solids Bioabatement: Inoculate with C. ligniaria cell pellet, incubate 3 C, shaking, h. solids Saccharification & Fermentation

12 Ethanol (%) Glucose (%) Fermentation of glucose in corn stover Treated 3 Untreated Untreated Time (H) 1 Treated Time (H) Nichols et al. Appl Biochem Biotechnol :379-39

13 Sugars or EtOH (g/l) Wheat straw bioabatement: SSF sugars ethanol 3.% ethanol.28 g ethanol/g wheat straw 83% of theoretical Time (h) Saha et al. Bioresour Technol 175:17-22

14 Furfural (mm) Metabolism of furfural before glucose Furfural Glucose Glucose (%) Time (H) Nichols et al. Appl Biochem Biotechnol :379-39

15 Furfural (mm) HMF (mm) Sugars % w/v Wheat straw--bioabatement Total sugars Furfural HMF Time (h) Saha et al. Bioresour Technol 175:17-22

16 Switchgrass 25 Preboot 3 Anthesis 25 Post frost mmol CO Fermentation time (h) mmol CO Fermentation time (h) mmol CO Fermentation time (h) Fermentations bioabated untreated Nichols et al. Bioresource Technol 11:

17 Xylose Utilization UV mutagenesis Recover in the presence of xylose + benomyl Plate Pick colonies & assay growth on xylose

18 Counterselection for xylose mutants Benomyl: Fungistatic not fungicidal Actively growing Viability Reduction x 1 4 X _ 1.8X Nichols et al. Biotechnol Prog 32:66-612

19 Xylose non-utilizing mutant WT C81

20 Growth Defect in C81 Carbon source WT C81 YPD Glucose Xylose Arabinose Furfural *Generation Time (min.) Nichols et al. Biotechnol Prog 32:66-612

21 % w/v Xylose consumption by mutant xylose WT Mutant xylitol Time (h) Nichols et al. Biotechnol Prog 32:66-612

22 Activity of Xylose Utilization Enzymes WT C81 % change XR XDH XK (mu/mg protein) Nichols et al. Biotechnol Prog 32:66-612

23 Xylitol 5-carbon sugar alcohol Low-calorie sweetener Used in pharmaceuticals, personal care products Alternative sweetener in gums, candies Chemical synthesis: metal catalyst, high-pressure hydrogenation

24 Concentration (% w/v) Yield (g/g) Effect of temperature on xylitol production Temperature ( C) Medium contained % xylose in.1x YP, ph 6.5 xylitol (%) xylose (%) yield (g/g)

25 Concentration ( % w/v) Yield (g/g) Effect of ph on xylitol production ph ph Medium contained % xylose in.1x YP, at 32 C xylitol (%) xylose (%) yield (g/g)

26 Carbon Source --- Effect of fermentation inhibitors Xylose Consumed (% w/v) Xylitol Concentration (% w/v) Yield (g xylitol/g xylose consumed) Benzoic acid Furfural HMF (Cultures contained 5% w/v xylose in YP medium)

27 Effect of aeration on xylitol production Stirring rate (rpm) Xylitol Yield (g/g) Concentration (% w/v) Cultures contained 5% w/v xylose in YP medium

28 Heterologous Xylose Reductase

29 Adding a Heterologous Xylose Reductase Cofactor NADPH NADH C8 (WT) C81 (xylose -) C81 + S. stipitis XYL mu/mg protein

30 Xylitol yield (g/g) Heterologous XR: Xylitol Yield C81 + Cloning vector S. stipitis XYL1.2.1 YP (.1X) YP (1X) CSH Medium

31 Yield and Productivity in Hydrolysate Candida tropicalis (Cheng et al.) Candida tropicalis (Huang et al.) Hydrolysate Corncobs: extensively detoxified Rice straw:.19 g/l furfural.19 g/l HMF 1.47 g/l acetate Yield (g/g) Productivit y (g/l h) Coniochaeta ligniaria C81 Corn stover:.92 g/l furfural.21 g/l HMF 2.3 g/l acetate.34 (.39 with Ss XR).1-.1

32 Summary A biological approach to inhibitor abatement Coniochaeta ligniaria NRRL3616 consumes inhibitors from hydrolyzed biomass Enhanced fermentation of biomass feedstocks: crop residues & energy crops Xylitol produced by a xylose-negative mutant Inhibitor tolerance, but low productivity compared to yeast

33 Acknowledgements USDA/ARS/NCAUR Greg Kennedy, Sarah Frazer, Ron Hector, Badal Saha Central Michigan University Steve Gorsich University of Almeria (Spain) Maria López, Gema Guisado Illinois State University Wade Nichols, Matt Szynkarek Baylor University Kevin Chambliss, Lekh Sharma

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