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
Biomass conversion nrel.gov Cellulose Hemicellulose Enzymes Acid Hexoses Pentoses Inhibitors X Recombinant Yeast or Bacteria Fuel or Chemical
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
A microbiological approach to inhibitor abatement Cellulose Hemicellulose pretreat Hexoses Pentoses Inhibitors X recombinant yeast or bacterium Fuel or Chemical Bioabatement
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 64:125-131
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 64:125-131
Coniochaeta ligniaria NRRL3616 (Ascomycete)
Concentration (mm) Removal of furans from corn stover hydrolysate 15 1 Furfural 5 HMF 1 2 3 4 5 Time (hr) Lopez et al. Appl Microbiol Biotechnol 64:125 131
Bioabated hydrolysate 14 12 1 8 6 4 2 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 2 4 6 8 1 Time (H) Nichols et al. Enz Microbial Technol 42:624-63
Untreated corn stover hydrolysate 14 12 1 8 6 4 2 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 2 4 6 8 1 Time (H) Nichols et al. Enz Microbial Technol 42:624-63
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, 18-24 h. solids Saccharification & Fermentation
Ethanol (%) Glucose (%) Fermentation of glucose in corn stover 2 1.5 Treated 3 Untreated 1 2.5 Untreated 2 4 6 8 1 Time (H) 1 Treated 2 4 6 8 1 Time (H) Nichols et al. Appl Biochem Biotechnol 121 124:379-39
Sugars or EtOH (g/l) Wheat straw bioabatement: SSF 35 3 25 2 15 1 5 sugars ethanol 3.% ethanol.28 g ethanol/g wheat straw 83% of theoretical 25 5 75 1 125 15 Time (h) Saha et al. Bioresour Technol 175:17-22
Furfural (mm) Metabolism of furfural before glucose 7.5 6 5 4 3 2 1 Furfural Glucose.4.3.2.1 Glucose (%) 1 2 3 4 5 6 Time (H) Nichols et al. Appl Biochem Biotechnol 121 124:379-39
Furfural (mm) HMF (mm) Sugars % w/v Wheat straw--bioabatement 15 1 5 Total sugars Furfural HMF 2.5 2 1.5 1.5 1 2 3 4 5 Time (h) Saha et al. Bioresour Technol 175:17-22
Switchgrass 25 Preboot 3 Anthesis 25 Post frost mmol CO 2 2 15 1 5 2 4 6 8 Fermentation time (h) mmol CO 2 25 2 15 1 5 2 4 6 8 Fermentation time (h) mmol CO 2 2 15 1 5 2 4 6 8 Fermentation time (h) Fermentations bioabated untreated Nichols et al. Bioresource Technol 11:7545-755.
Xylose Utilization UV mutagenesis Recover in the presence of xylose + benomyl Plate Pick colonies & assay growth on xylose
Counterselection for xylose mutants Benomyl: Fungistatic not fungicidal Actively growing Viability Reduction + 6.1 x 1 4 X _ 1.8X Nichols et al. Biotechnol Prog 32:66-612
Xylose non-utilizing mutant WT C81
Growth Defect in C81 Carbon source WT C81 YPD 15 + 18 16 + 16 Glucose 184 + 21 179 + 21 Xylose 184 + 27 Arabinose 384 + 77 Furfural 42 + 148 454 + 194 *Generation Time (min.) Nichols et al. Biotechnol Prog 32:66-612
% w/v Xylose consumption by mutant 1.4 1.2 xylose 1.8.6.4 WT Mutant xylitol.2 1 2 3 4 5 6 7 Time (h) Nichols et al. Biotechnol Prog 32:66-612
Activity of Xylose Utilization Enzymes WT C81 % change XR XDH XK 28 + 16 332 + 84 31 + 11 82 + 15 99 + 66 34 + 11-71 -7 +9 (mu/mg protein) Nichols et al. Biotechnol Prog 32:66-612
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
Concentration (% w/v) Yield (g/g) Effect of temperature on xylitol production 3.5 3. 2.5 2. 1.5 1..5. 22 25 3 32 Temperature ( C) 1.8.6.4.2 Medium contained % xylose in.1x YP, ph 6.5 xylitol (%) xylose (%) yield (g/g)
Concentration ( % w/v) Yield (g/g) Effect of ph on xylitol production 2.5.8 2. 1.5.6.4 1..5.2. ph 4 5 6 7 8 ph Medium contained % xylose in.1x YP, at 32 C xylitol (%) xylose (%) yield (g/g)
Carbon Source --- Effect of fermentation inhibitors Xylose Consumed (% w/v) Xylitol Concentration (% w/v) Yield (g xylitol/g xylose consumed) 4.78 2.29.48 Benzoic acid 4.85 2.69.55 Furfural 5.7 2.48.49 HMF 4.73 2.31.49 (Cultures contained 5% w/v xylose in YP medium)
Effect of aeration on xylitol production Stirring rate (rpm) Xylitol Yield (g/g) Concentration (% w/v) 15.328 1.17 65.455 2.16 Cultures contained 5% w/v xylose in YP medium
Heterologous Xylose Reductase
Adding a Heterologous Xylose Reductase Cofactor NADPH NADH C8 (WT) 466 + 21 189 + 81 C81 (xylose -) 215 + 94 85 + 39 C81 + S. stipitis XYL1 44 + 237 162 + 81 mu/mg protein
Xylitol yield (g/g) Heterologous XR: Xylitol Yield.8.7.6.5.4.3 C81 + Cloning vector S. stipitis XYL1.2.1 YP (.1X) YP (1X) CSH Medium
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).7.95.7.22 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
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
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