Benjamin D. Bice, Donald Busalacchi, RagothamanAvanasi Narasimhan, Rebecca J. Breuer, Trey K. Sato, David B. Hodge AIChE Annual Meeting

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Hydrolysate Fermentation Characterization for

Derived From Directed Evolution for Improved

Tongjun Liu, Daniel L. Williams, Lucas S.

Bice, Donald Busalacchi, RagothamanAvanasi

1 Hydrolysate Fermentation Characterization for Xylose-Fermenting Saccharomyces Cerevisiae Strains Derived From Directed Evolution for Improved Xylose Utilization and Tolerance to Inhibitors Tongjun Liu, Daniel L. Williams, Lucas S. Parreiras, Li Qin, Benjamin D. Bice, Donald Busalacchi, RagothamanAvanasi Narasimhan, Rebecca J. Breuer, Trey K. Sato, David B. Hodge AIChE Annual Meeting September 28, 212

2 Other Talks from Our Group Tuesday, 12:3 pm. Room 334 (26a) Characterization of Carbohydrate Accessibility and Enzyme Adsorption Capacity for Diverse Cell Wall Phenotypes Subjected to Alkaline Hydrogen Peroxide Pretreatment. 2 Tuesday, 12:55 pm. Room 334 (26b) Lignin Structural Changes Associated with Oxidative Pretreatment Catalyzed by Cu-Diimine Complexes. Wednesday, 12:3 pm. Room 335 (498a) Hydrolysate Fermentation Characterization for Xylose-Fermenting Saccharomyces Cerevisiae Strains Derived From Directed Evolution for Improved Xl Xylose Utilization i and Tolerance to Inhibitors. Wednesday, 2:1 pm. Room 33 (471e) Characterization of Solubilized Biopolymers Fractionated From Alkali Pulping Liquors. Thursday, 12:55 pm. Room 335 (667b) Phenotypic and Multi-Omic i Approaches to Address Molecular l Bottlenecks in the Fermentation of Lignocellulose Into Ethanol by Saccharomyces Cerevisiae.

3 3 Background: Pretreatment Xylose fermentation Hydrolysate inhibitors Rationale/Goals Results Summary Outline

4 Hemicellulose 4 Chemical Pretreatment t t Lignocellulose Feedstock (Plant Cell Walls) Cellulose Biochemical Conversion of Plant Cell Wall Polysaccharides to Biofuels Enzymatic Depolymerization of Polysaccharides Microbial Metabolites Ethanol, Butanol, Carboxylic Acids, Alkanes, Isoprenoids, Biological Conversion of Monosaccharides

5 Plant Cell Wall Matrix Polymers Lignin (1 35%) Heteropolysaccharides (25 45%) Cellulose (35 55%)

6 Composition of Plant Cell Walls Pentose utilization is important Xylose is the 2 nd most abundant sugar in biosphere For lignocellulose ll l conversion to biofuels, pentose fermentation is an attractive trait Other components in plant cell walls can be problematic for fermentation Monocots (Grasses) Hardwoods Softwoods Woody Plants Source: Rydholm S, Pulping Processes. Wiley Interscience.

7 Microorganism Background: Xylose Utilization by S. cerevisiae Challenge: Convert all lignocellulose polysaccharide sugars to ethanol at high yields, titers, and productivities Two approaches for xylose fermentation Bacteria (and Piromyces): Xylose isomerase (XI) Yeast: Xylose reductase (XR) + xylitol dehydrogenase (XDH) Potential xylitol accumulation due to redox imbalance using XR, XDH pathway (NAD + /NADH vs. NADP + /NADPH) NAD(P)H NAD(P) + NAD + NADH ATP ADP Xylose XR Xylitol XI XDH Xylulose XK Xylulose -5- Phosphate Pentose Phosphate Pathway Glycolysis Fermentation Ethanol

Alkaline Hydrogen Peroxide Pretreatment Basedonexisting

the paper industry Alkaline oxidative pretreatments as either

pretreatment step Unique advantages Well suited for grasses

use/recycle Alkaline hydrogen peroxide bleaching tower >1 tpd

8 Alkaline Hydrogen Peroxide Pretreatment Basedonexisting existing alkalinehydrogen peroxide pulp bleaching stages in the paper industry Alkaline oxidative pretreatments as either standalone pretreatments OR delignifying finishing post pretreatment step Unique advantages Well suited for grasses Current Challenges: Process integration Economics Water use/recycle Alkaline hydrogen peroxide bleaching tower >1 tpd capacity at Smurfit Kappa Kraftliner, Piteå, Sweden (photo courtesy: Outokumpu Oy)

Alkaline Hydrogen Peroxide Pretreatment Pretreatment Liquefaction/Saccharification 9 1% 1%

Insoluble Fraction 3 6 12 18 3 36 42 48 Pretreatment Time (h) Unquantified Solids Ah Ash 9%

Uronic Acids 5% Galactan 4% Mannan 3% Arabinan 2% Xylan Glucan 1% % Lignin (Klason) 4 8 12

Fraction Unquantified Solids Ashh Water+EtOH Extractives Acetate Uronic Acids Galactan

9 Alkaline Hydrogen Peroxide Pretreatment Pretreatment Liquefaction/Saccharification 9 1% 1% Compo onent Fraction 9% 8% 7% 6% 5% 4% 3% 2% 1% % Solids transferred to the liquid phase Insoluble Fraction Pretreatment Time (h) Unquantified Solids Ah Ash 9% Solids transferred to the liquid phase (hydrolysate) 8% Water+EtOH Extractives 7% Acetate 6% Uronic Acids 5% Galactan 4% Mannan 3% Arabinan 2% Xylan Glucan 1% % Lignin (Klason) ASL Hydrolysis Time Pretreatment (h) Time (h) Compo onent Fraction Unquantified Solids Ashh Water+EtOH Extractives Acetate Uronic Acids Galactan Mannan Arabinan Xylan Glucan Lignin (Klason) ASL Banerjee et al. (212). Biotechnol Bioeng. 19(4):

10 Fermentation Inhibitors 1 >1% of plant cell wall 1-15% of plant cell wall pca p-coumaric acid Compon nent Fraction 1% 9% 8% 7% 6% 5% 4% 3% 2% 1% % FA Ferulic acid 2-5% of plant cell wall Acetate Low MW Fraction Solids transferred to the liquid phase (prehydrolysate) Insoluble Fraction Pretreatment Time (h) Other aromatics Extractives: 8-15% of plant cell wall Oxidative degradation products of sugars, lignin, extractives? >1% Unquantified Solids Ah Ash Water+EtOH Extractives Acetate Uronic Acids Galactan Mannan Arabinan Xylan Glucan Lignin (Klason) ASL High MW Fraction Lignin/aromatics (+ Hemicellulose?) Other Polymers (?) Lignin (Klason) Glucan Low Mol. Wt. Hemicelluloses (?) (+ Pectin,starch,..?) Arabinan Xylan Polymers in hydrolysate Hemicellulose Aggregates (minimal lignin?) ass Abundance Polymer M

$fractionof of grass$

11 Monocot Lignins pca 11 Monomer composition and structural organization significantly different than herbaceousandwoody and dicots or gymnosperm lignins Ferulates and p coumarate can comprise a significant fractionof of grass lignins Ester crosslinks Highly condensed (~85%) High phenolic hydroxyl content High alkali solubility S Lignini FA Lignin

12 Quantification of Solubilized p-hydroxycinnamic Acids in Hydrolysates p y y y y 12 Identification and quantification of ferulic acid and p coumaric acid in corn stover and switchgrass hydrolysates by LC MS Can represent more the 1.5% of the cell wall Potential to reach concentrations of >1. g/l pca and >.5 g/l FA in hydrolysates from 2% solids pretreatment Inhibitory to fermentation 3.5E+7 Coumaric Acid Hydroxycinnamic Accid Concentration (g//l) 4.E+7 Ferulic Acid Intensity (cps) 3.E+7 2.5E+7 2.E+7 pca FA 1.5E+7 1.E+7 5 E 6 5.E+6.E+ SG Ferulic Acid 1.% SG pcoumaric Acid NaOH only ph 11.5 AHP (12.5%Acid H2O2) CS Ferulic.8% 1.2 ph 11.5 AHP (25% H2O2)Acid CS pcoumaric ph 11.5 AHP (5% H2O2).6%.8.4% 187 m/z amu.4.2% Time (min) % Hydrogen Peroxide Loading (g H 2O2 /g Biomass).25 Hydroxycinnamic Acid H d Yield on Biomass (gg/g) 1.6

13 13 Outline Background: Rationale/Goals Generate, screen, isolate Saccharomyces strains with: Xylose fermenting capacity Increased tolerance to AHP inhibitors Results Summary

14 Strain Development 14 Screening of >1 WT Saccharomyces strains for tolerance to AHP hydrolysate inhibitors Na +, Acetate, p coumaric acid (pca), ferulic acid (FA) Parallel screening by OD measurement in microtiter plates Quantified as specific growth rate (μ) Relative Specific Growth Rate High Low Sa accharomc cyes stra ains Acetate Na 2 SO 4 pca and FA Norma lized cell density Norma lized cell density Normalized cell density p-coumaric and Ferulic Acids YB21 YIIc17_E5 CEN.PK2 S288c Time (hrs) Acetate Time (hrs) Na 2 SO Time (hrs)

15 Strain Development 15 Evolution for improved Wild type tpe YB21 xylose fermentation Subsequent evolution in presence of p coumaric acid and ferulic acid for improved hydrolysate tolerance Y35 Y56 Y6 Y73 S. cerevisiae Chromosomal integration of xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK) from Pichia stipitis Ferments xylose Aerobic evolution on YP xylose media for improved xylose utilization Improved xylose fermentation Y83 Y84 Y85 Y86 Aerobic evolution on YP xylose media with p hydroxycinnamic hd i i acids for improved hydrolysate tolerance Improved tolerance to AHP hydrolysate inhibitors

16 16 Outline Background: Rationale/Goals Results Strain performance on inhibitors, hydrolysates y Characterization of hydrolysates Summary

17 Performance of Xylose Fermenting Yeasts OD Growth curve on YNB medium Growth Y35 Y56 Y Ethanol Production time (h) Specific xylose uptake rate, Qxyl (g/g/h) Biomass yield (g/g) Y56 Y73 Xylose Consumption Anaerobic growth in YNB media Evolution improves xylose and ethanol rates Ferments xylose c. (g/l) EtOH con Y35 Y56 Y73 xylose conc c. (g/l) Y35 Y56 Y73 Y73 Y56 Y35 Improved xylose fermentation Improved tolerance to AHP hydrolysate inhibitors time (h) time (h)

18 Impact of Individual Inhibitors on Y73 18 All inhibitors impact biomass yield on glucose Differing impacts on xylose uptake Y73 Ferments xylose Improved xylose fermentation Improved tolerance to AHP hydrolysate inhibitors

19 iability (%) Cell Vi Performance of Xylose-Fermenting Yeasts Validation by improved growth and viability in rich media, microaerobic conditions Significant improvement in tolerance for xylose media hrs 2 hrs 25 hrs 44 hrs 49 hrs Y73 Ferments xylose Improved xylose fermentation Improved tolerance to AHP hydrolysate inhibitors 25 Y73 YPD YPD Y73 YPX YPX Glucose Xylose

20 Generation and Characterization of High Solids AHP Hydrolysates Three corn stover and two switchgrass hydrolysates Goal: Fermentable hydrolysates at sugar concentrations >1 g/l 2 1 Con ncentration (g/l) Ara 8% Xyl 7% Glc Conve rsion to Mo nomers 6% 5% 4% 3% 2% 1% Glc Xyl SG1 SG2 CS1 CS2 CS3 % SG1 SG2 CS1 CS2 CS3

21 Generation and Characterization of High Solids AHP Hydrolysates Quantification of fermentation inhibitors Important inhibitor pools: Inorganics, aliphatic acids, phenolic acids SG Na+ (mm) SG2 CS1 CS2 CS3 Co oncentration (g/l) (g/l) Con ncentration urry g solids / g sl Na+ Formate Acetate Ferulate p coumarate Solids to Hydrolysis

22 Effect of ph on Undetoxified High Sugar Corn Stover Hydrolysate Fermentation Engineered yeasts capable of fermenting high sugar, undetoxified AHP hydrolysates y (CS2) to 4% ethanol Minimal supplementation: 1.67 g/l YNB g/l of urea High ph is critical 22 (g/l) conc. ph 5. ph 5.5 (ph5.) OD 1 8 Glc Xly EtOH Xylitol 6 glycerol OD OD 6 conc. (g/l) (ph5.5) OD 1 Glc Xly EtOH Xylitol glycerol OD OD time (h) time (h)

23 ted (g/l) Metabolite Generat Ethanol Glycerol Estimating Yields for Y73 and : Diverse AHP Hydrolysates ted (g/l) Metabolite Generat Ethanol Xylitol Glycerol 23 Metabolite Yields: Relatively unaffected by inhibitors Glucose Consumed (g/l) Xylose Consumed (g/l) Cell Mass Generated (g DCW/L) Undetoxified SG2, CS2, CS3, ph 5.5 Detoxified CS1 or SG1, ph 5. Undetoxified CS1 or SG1, ph 5. Pure Glucose ll Mass Generated (g DCW/L) Cel Detoxified CS1 or SG1, ph 5. Undetoxified SG, ph 5.5 Pure Xylose y =.32x R² =.698 Cell Mass Yields: Strongly affected by inhibitors Glucose Consumed (g/l) Xylose Consumed

24 Impacts of AHP Hydrolysates on Fermentation: Rates and Yields Inhibition of rates Strong impact on xylose rate shows improved xylose fermentation in hydrolysates Ferments xylose Rate (g/g/h) Spec cific Xylose Uptake Y Abso olute Xylose Uptake Rate (g/l/h) Y73 24 Y73 Improved xylose fermentation Improved tolerance to AHP hydrolysate inhibitors SG1 CS1 CS2 YNB G+X.7 Inhibition of biomass yields 6.6 AHP hydrolysates impact biomass yield ATP driven Mechanisms: efflux of H, Na, or phenolic acids decreases anaerobic ATP availability for growth? omass Yield on Glu ucose (g/g) Bi SG1 CS1 CS2 YNB G+X Y73 SG1 CS1 CS2 YNB G+X

25 Summary / Conclusions 25 Strains initially selected for improved hydrolysate inhibitor tolerance Strains can completely ferment glucose and xylose in AHP hydrolysates y with no detoxification to > 4% ethanol Hydrolysate y inhibitors impact both biomass yields and xylose consumption rates Strains evolved on phenolic acids show improved xylose fermentation ate (g/l/h) Absolute Xylose Uptake Ra Y73 se (g/g) Biomass Yield on Glucos Y73 rates in hydrolysates SG1 CS1 CS2 YNB G+X SG1 CS1 CS2 YNB G+X

26 Research Group: Acknowledgements Collaborators: Trey Sato U. Wisc. Dan Williams Dr. Tongjun Liu Marc Hansen ( ) Ryan Stoklosa Charles Chen David Hodge Alex Smith ( ) Muyang Li Zhenglun Li N t i t d Not pictured: Elizabeth Häggbjer, Natassa Christides, Genevieve Gagnier Funding: Department of Energy, BER DE FC2 7ER64494

27 Thank You! Questions? 27

Understanding and Enhancing Alkaline and. Production of Biofuels through Improved

Understanding and Enhancing Alkaline and Oxidative Chemical Pretreatments for the Production of Biofuels through Improved Characterization David Hodge Assistant Professor Chemical Engineering, g, Michigan