C5 fermentation; strain engineering for high level xylitol (and xylonate) production. Merja Penttilä VTT Technical Research Centre of Finland

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1 C5 fermentation; strain engineering for high level xylitol (and xylonate) production Merja Penttilä VTT Technical Research Centre of Finland

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2 CIMV ORGANOSOLV PROCESS: C5 SYRUP C6 cellulose pulp C5 hemicellulose syrup lignin Biotechnical products Increased value of the fraction in a multiproduct biorefinery 2

3 PRODUCT OPTIONS FROM XYLOSE Glucose Xylose Arabinose The CIMV C5 syrup is rich in xylose C5 syrup MICROBE Yeast Ethanol Itaconic acid Isopropanol Lactic acid, etc A traditional case: Pentose fermentation to ethanol from mixed sugar solutions Dry matter (%) 48 Total Xyl (%) 55 Total Glu (%) 4 Lignin (%) 0 Protein (%) 44 Ash (%) 75 3

4 PRODUCT OPTIONS FROM XYLOSE AIM: Specific conversion of the xylose-rich C5 syrup to a higher-value product with good yields and productivities Xylose Xylose MICROBE Yeast XR A single step reduction MICROBE Yeast XYD A single step oxidation Xylitol Xylonate 4

Xylose A renewable component for polyesters (with TA/sebacic acid, alkyd resins,

5 XYLITOL A natural sweetener with caries reducing properties. Sweet as sucrose, 33% less calories. Xylose A renewable component for polyesters (with TA/sebacic acid, alkyd resins, explosives) Today produced chemically from biomass streams (e.g. sulphite spent liquor) with nickel catalyst at high temperatures MICROBE Yeast XR A single step reduction Xylitol 5

Xylitol (g l - ) XYLITOL PRODUCTION Increases in xylitol production through production host development 50 00 Strain 2-3 copy Strain 2 - copy B67002 multi-copy B67002 2 copy B67002 copy B67002

6 Xylitol (g l - ) XYLITOL PRODUCTION Increases in xylitol production through production host development Strain 2-3 copy Strain 2 - copy B67002 multi-copy B copy B67002 copy B67002 Various yeast hosts engineered - baker s yeast Saccharomyces - Candida/Pichia Industrially relevant productivities of xylitol on pure xylose 50 Titer Rate Yield > 60 g/l > 2 g/l/h > 0.9 g/g xylose Time (h) Production on pure xylose Efficient production strains available Strain 2, P.kudriavzevii 6

7 Xylitol, residual xylose, formate, acetate (g L - ) C5 SYRUP CHALLENGES Formic acid concentration in the C5 syrup is high (50-00g/l; needs removal) Total xylose is ~ 200 g/l, but remains partly polymeric 20 70% v/v CIMV3 C5 in feed Feed of 70% CIMV C5 fraction Xylitol Xylose Formate Acetate Time (h) Titer Rate Yield > 00 g/l xylitol, > 0.82 g/l/h 0.89 g/g xylose Engineering yeast tolerance towards formic acid improves fermentation performance Fed-batch culture 7

8 XYLONATE Alternative to gluconic acid Production from lignocellulose sugar xylose Chelator, dispersant, clarifying agent Cement retardant Polyester, -amide or hydrogel modifier,2,4 butanetriol precursor MICROBE Yeast Xylose XYD A single step oxidation Xylonate 8

9 D-Xylonate (g/l) D-Xylonate (g l - ) XYLONATE PRODUCTION Various yeast hosts engineered - baker s yeast Saccharomyces - Candida/Pichia 0 strain enzyme condition Strain Strain 2 Strain 3 Strain 4 Strain 5 Strain 6 Strain 7 2 Yeast strain Increases in xylonate production through production host development Industrially relevant productivities of xylonate on pure xylose ph 5.5 (65 g/l) ph 5.5 (90 g/l) ph 3 (55 g/l) ph 3 (50 g/l) Titer Rate Yield > 60 g/l >.4 g/l/h > 0.9 g/g xylose Time (h) Production on pure xylose (amount of xylose provided in brackets) Good production at low ph (ph 3) Efficient production strains available 9

10 SUSTAINABILITY OF PRODUCT CHAINS Biomass BIOCORE biorefinery Pretreatment Climate change Xylitol / IA Advantages Disadvantages Material inputs Fractionation (Organosolv) Xylitol / ethanol Ethanol / IA Fuel C5 C6 Lignin SHF ethanol Power Heat / power generation Processing Processing Processing Processing t CO 2 eq. / t biomass (dry) Water Residues Xylitol Itaconic acid Lignin powder Acidification Xylitol / IA Advantages Disadvantages Ethanol Ethanol Xylitol / ethanol Ethanol / IA Fertiliser SHF ethanol Further conversion in chemical industry Distribution Distribution Distribution of products Use and end of life Distribution kg SO 2 eq. / t biomass (dry) Xylitol is a favourable product based on sustainability calculations

$obtainable specifically from the hemicellulose C5 fractions of$ biorefineries Biotechnical production offers industrially

production CIMV hemicellulose syrup can be used in production

tolerance towards toxic components Xylitol (and xylonate)

11 CONCLUSIONS Xylitol (and xylonate) provide biotech products obtainable specifically from the hemicellulose C5 fractions of biorefineries Biotechnical production offers industrially relevant productivities Yeasts are good and robust hosts for production CIMV hemicellulose syrup can be used in production process optimisation and host strain engineering to improve tolerance towards toxic components Xylitol (and xylonate) provide good options to increase the sustainability and economy of multiproduct biorefineries

12 THANK YOU! Thank you all Biocore partners for a nice collaboration! The European Commission is kindly acknowledged for funding this FP7 project under contract no FP ( )

The CIMV organosolv Process. B. Benjelloun

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