Understanding and Enhancing Alkaline and. Production of Biofuels through Improved

Transcription

1 Understanding and Enhancing Alkaline and Oxidative Chemical Pretreatments for the Production of Biofuels through Improved Characterization David Hodge Assistant Professor Chemical Engineering, g, Michigan State University 3 rd International Symposium on Bioenergy and Biotechnology Wuhan, Hubei, P.R. China 16 October, 2012

2 Plant Tissues are Diverse and Highly Heterogeneous Storage Tissues: Food Crops Starch Granules in Corn Endosperm Source: USDA/ARS/ERRC Arabidopsis Oleosomes Nature Methods 3, (2006) Structural Tissues: Plant Cell Walls Corn Stem Source: John Sedbrook, U. Illinois Arabidosis stem PNAS. 107(51): Softwood secondary xylem (tracheids) Hardwood secondary xylem (vessel elements and fibers) Plant Physiol 112:

3 Angiosp perms Gym mnosperm ms Monocot ts (Herbaceo ous) Dicots Diversity of Cell Wall Polymers Stem cross-sections Commelinoid (Grasses) Non commelinoid Herbaceous Dicot Woody Dicot (Hardwood) Primary Cell Wall Secondary Cell Wall GAX β Glucans GAX S G S G H FA FA GM PP XyG GX GX S G H S G PP XyG GX GM GX G Softwood GAX: glucuronoarabinoxylans GX: glucuronoxylans XyG: xyloglucans GMs: glucomannans PP: pectic polysaccharides H S G S H G H Guaiacyl Syringyl GM p-hydroxyphenyl FA Ferulates S 3

4 Monocot Lignins pca 4 Differences in composition and structural organization relative to 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 H G S pca FA p-coumaryl alcohol (H Lignins) coniferyl alcohol (G Lignins) sinapyl alcohol (S Lignins) p-coumaric acid ferulic acid

5 Barriers to the (Biological) Utilization of Plant Cell Wall Components Targets/problems Ultrastructure of plant cell walls needs to be broken down Lignin prevents access to carbohydrate fraction Crystalline cellulose resists depolymerization, blocks access to internally packed polymers How pretreatments address this problem Science 315, 804 (2007) At the molecular level: Chemical modifications Depolymerization Solubilization At the structural level: Physical redistribution of components

6 Hemicellulose 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

7 Oxidative Deconstruction of Lignocellulose Biological Mechanism 1: Secreted set of cellulases l Individual proteins with CBM, flexible linker, and catalytic domain (e.g. Trichoderma reesei) ) Biological Mechanism 2: Brown rot fungi Deconstruction of cellulose and lignin using biologically generated OH Biological i l Mechanism 3: White rot fungi Peroxidases and oxidases for lignin degradation Laccase from M. albomyces Lignin Peroxidase from P. chrysosporium T. terrestris GH61E Biochemistry 2010, 49, Images source: Science 333:

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 >1000 tpd capacity at Smurfit Kappa Kraftliner, Piteå, Sweden (photo courtesy: Outokumpu Oy)

9 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 Agricultural Residues: Corn Stover/Corn Fiber Wheat Straw Sugar Cane Bagasse Sorghum Bagasse Rice Straw Bioenergy Grasses: Switchgrass Miscanthus sp. Reed Canary Grass

10 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 80 Unique advantages 70 Well suited for grasses Gal EtO OH / ton Bioma ass Current Challenges: 40 Process integration Economics 10 Water use/recycle Improved Processes? Catalytic approaches? Upper Theoretical Limit Switchgrass Corn Stover $0.00 $2.00 $4.00 $6.00 Pretreatment Chemical Cost / Gal EtOH

11 Alkaline Hydrogen Peroxide Pretreatment Pretreatment Liquefaction/Saccharification % 100% Compo onent Fraction 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Solids transferred to the liquid phase Insoluble Fraction Pretreatment Time (h) Unquantified Solids Ah Ash 90% Solids transferred to the liquid phase (hydrolysate) 80% Water+EtOH Extractives 70% Acetate 60% Uronic Acids 50% Galactan 40% Mannan 30% Arabinan 20% Xylan Glucan 10% 0% 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. (2012). Biotechnol Bioeng. 109(4):

12 Integration of AHP Pretreatment, Hydrolysis, y and Fermentation conc. (g/l) Y87 (ph5.5) OD 1 Xylose-fermenting Saccharomyces Glc Xly EtOH Xylitol glycerol OD time (h) 1.67 g/l YNB w/o ammonium sulfate and g/l of urea OD 600 on of nents solubilizatio all compon creasing s ant cell wa Inc pla Pretreatment, 0 h Pretreatment, 48 h Hydrolysis, 0 hr Hydrolysis, 24 hr Fermentation Filter Sterilize (0.22 μm) Liu et al. (in preparation).

13 Relating Cell Wall Properties to Recalcitrance Understanding cell wall properties impacting recalcitrance and relationship of these properties to enzymatic digestibility Using pretreatment of plant cell walls with diverse phenotypes to generate samples with diverse recalcitrance lit properties Property Impacting Recalcitrance Cell wall hydrophilicity Cellulose oxidation Polysaccharide accessibility Lignin content Ferulate content S/G ratio Lignin condensation Analytical Tool Water adsorption/retention Potentiometric titration Glycome Profiling Klason Lignin HPLC Pyrolysis GC/MS Thioacidolysis GC/MS HSQC NMR

14 Diverse Cell Wall Properties: Biomass Corn Stovers (Zea mays) Pioneer hybrid 36H56 Inbred brown midrib bm1 Inbred brown midrib bm3 Switchgrass (Panicum virgatum cv. Cave in Rock) Miscanthus (Miscanthus x giganteus ) Hybrid poplar Monocot grasses Woody dicot (Populus nigra var. Charkoviensis x Caudina) Goldenrod (Solidago sp.) Herbaceous dicot

15 Diverse Cell Wall Properties: Pretreatments 15 AHP Soda pulping Liquid Hot Water + AHP post treatment Cu(bpy) catalyzed AHP pretreatment t t

16 Cell Wall Water Interactions Objectives How can cell wall water interactions impact deconstruction Water Retention Value Particle il Settling Water Activity 15% Solids Quantifiable metrics Property: Cell Wall Composition Polysaccharide Content Lignin Content Corn Stover Switchgrass Accessible vs. Amorphous Inaccessible vs. Crystalline Outcomes: Water Penetration Enzyme Penetration Polysaccharide Hydrolysis Property: Cell Wall Hydrophilicity Property: Cell Wall Porosity

17 Introduction of Carboxyl Groups with Oxidative Pretreatments?? COOH COOH COOH COOH COOH COOH COOH COOH COOH COOH

18 Interactions Between Water and Cell Wall Water retention value (WRV) Step 1: Filter wash Step 2: Centrifuge Step 3: Weighing Determine Moisture Content of Pad WRV = M water M drypad Filter wash using 200mesh screen to ~ 80% moisture Cookie Cutter pad onto 200 mesh disk from filter funnel; Centrifuge at 900xg for 15min Weigh wet pad; Dry in oven at 105 o C for ~2hrs then weigh dry pad

19 can Conversio on 7 Day Glu 100% 80% 60% 40% 20% Interactions Between Water and Cell Wall Both water retention ti value (WRV) and swelling volume are increased with pretreatment More water confined in cell wall WRV is a very good predictor of digestibility LHW=Liquid Hot Water pretreated SG LHW SG CS LHW CS 0% WRV Settling he eight/total heig ght Settling height (ht) Total height (Ht) Ht ht AHP Increases Swelling CS Untreated CS 25% H2O2 SG Untreated SG 25% H2O % 4.0% 6.0% 8.0% 10.0% 12.0% Williams et al. (in Solids Concentration (w/w) preparation).

20 Interactions Between Water and the Cell Wall Pretreated corn stover can adsorb more water in the cell wall Quantifiable by decreased water activity ii at a moisture content ie i.e. more water is present in a constrained environment Moisture Content (%) Corn Stover Dynamic vapor sorption/desorption isotherms Williams et al. (in preparation) Water Activity (a w ) = Relative Humidity (h r ) of vapor

21 Interactions Between Water and the Cell Wall Pretreated corn stover can adsorb more water in the cell wall Quantifiable by decreased water activity ii at a moisture content ie i.e. more water is present in a constrained environment Moisture Content (%) Dynamic vapor sorption/desorption Corn Stover isotherms AHP Pretreated Lower h r for AHP Corn Williams et al. (in preparation) Water Activity (a w ) = Relative Humidity (h r ) of vapor

22 Hydrolysis Results for AHP Pretreatment of Diverse Cell Wall Phenotypes Grasses: Increasing response to AHP pretreatment Herbaceous Dicot: Conversion saturates Cell wall properties? Li et al. (in preparation) Hyb brid Pop plar Golden nrod Dicots hus Miscant Switchgr rass Corn Sto over (36H56 6) Corn Sto over (bm1 ) Monocots Corn St tover (bm3 3)

23 Correlating Digestibility to Plant Cell Wall Properties: Lignin Removal Digestibility and lignin content correlated Extracted with alkali or AHP pretreated Negative relationship with threshold value for lignin removal Critical level for cell wall hydrophobicity? Properties associated with lignin removal? Glu ucan Digestibilit ty 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Hybrid Stover Inbred bm1 stover Inbred bm3 Stover Hybrid Poplar Miscanthus Klason Lignin (Mass Fraction of Cell Wall)

24 Lignin Analytical Methods: Pyrolysis GC-MS 24 High throughput destructive characterization of complete cell walls Based on characterization of volatilized pyrolysis y products Pyrolysis for 10 s at 600 o C, heated transfer line to GC/MS at 300 o C GC-MS (x10,000,000) 5.0 TIC TIC

25 Lignin Analytical Methods: Pyrolysis GC-MS 25 Correlating aromaticproducts to monomers in the lignin polymer Only represents a fraction of original lignin Poor yield on condensed lignins Unique products for each lignin monomer? pca and FA yield clearly identifiable markers 4 vinylphenol and 4 vinylguaiacol H-lignins p-coumaric acid?????? acol or crease PY Yrolytic 4 vinylguai 4 vinylphenol dec 85% 80% 75% 70% 65% 60% 55% 50% 45% Ferulate p coumarate 40% 0.00% 0.25% 0.50% 0.75% 1.00% 1.25% Quantified ferulate or p coumarate released (percentage of cell wall) G-lignins Ferulic Acid?????? S-lignins

26 Lignin Analytical Methods: Pyrolysis GC-MS 26 Fraction of Aromatics Volatiliz zed by Pyrolysis Correlating aromaticproducts to monomers in the lignin polymer Only represents a fraction of original lignin Poor yield on condensed lignins Unique products for each lignin monomer? pca and FA yield clearly identifiable markers 4 vinylphenol and 4 vinylguaiacol Li et al. (2012). Biotechnol Biofuels. 5(1) % Syringyl Lignins 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Guaiacyl Lignins Ferulic acid (or guaiacyl lignin) p-coumaric acid (or other lignin) Switchgrass (cv. Pioneer Hybrid Inbred bm1 Inbred bm3 Sugar Maple Cave In Rock) Stover Stover Stover (Acer saccharum) Herbaceous Monocots (Grasses) Woody dicot 3,4,5 Trimethoxytoluene Syringol Syringaldehyde Methylsyringol Methoxyeugenol Guaiacol Eugenol Creosol Acetoveratrone (E) Isoeugenol 4 vinylguaiacol i l Phenol 4 vinylphenol 5 pools of lignin monomers identifiable Can be used to: Estimate S/G Characterize changes to lignin during pretreatment

27 Correlating Digestibility to Plant Cell Wall Properties Solubilized ferulate is correlated to glucan digestibility Pyrolytic 4 vinylguaiacol (Ferulate/Lignin): Correlated to lignin content and glucan digestibility

28 Lignin Methods: Thioacidolysis GC/MS Determine relative quantities of G, S, H lignins Foster et al., J Vis Exps, 37 Cleavage of alkyl aryl ethers in lignin (S/G) Monomers Released lysis ive Fraction of Lignin M by Thioacido Quantification of derivatized 0.2 fragments by GC MS Problematic in grasses? Highly condensed lignins Relati G monomer (0.86) (0.99) S monomer (0.26) (0.38) 28 bm1 bm3 p Hydroxyphenyl (H) Guiacyl (G) Syringyl (S) Pioneer Hybrid bm1 Inbred bm3 Inbred Switchgrass (cv. Time (minutes) Stover Stover Stover Cave In Rock) GC-MS quantification EtSH, BF 3 BSTF β-o-4 linkage CPG-SM Li et al. (2012). Biotechnol Biofuels. 5(1)38.

29 Quantitative Thioacidolysis Determines the fraction of lignin involved in only ether linkages Thioacidolysis yield decreased from 5 12% w/w to 1% w/w after pretreatment AHP pretreatment solubilizing 50% lignin Fraction of condensed lignin increased from 88 95% w/w to 99% w/w Li et al. (2012). Biotechnol Biofuels. 5(1)38. 29

30 30 Cu(bpy)-Catalyzed AHP 100% Catalyst significantly improves subsequent enzymatic conversion of cellulose Glucan Conv version 80% 60% 40% Hybrid Poplar No AHP, no xylanase AHP, no xylanase AHP, xylanase AHP with Cu(bpy), no xylanase Cu(bpy) AHP, xylanase 20% 0% Li et al. (accepted 2012). Biotechnol Bioeng Hydrolysis time (h)

31 Oxidative Depolymerization of Cellulose COOH AHP depolymerizes cellulose (filter paper) Introduces COOH Li et al. (accepted 2012). Biotechnol Bioeng. R HO OH OH O + R HO R HO OH O C4 oxidation O HO OH OH O OH HO O C6 oxidation OH O? R OH HO O OH O COOH? Cellulose OH O OH R R R? O OH HO + OH HO HO COOH OH R O OH C1 oxidation on

32 Summary Wt Water adsorption and retention ti can be correlated ltd to enzymatic conversion Correlating digestibility to plant cell wall properties Negative relationship between Klason lignin and digestibility ibili across diverse cell wall types Ferulate content quantified by Py GC/MS in residual cell wall and LC/MS in hydrolysates can be correlated to lignin removal and digestibility Oxidative cellulose depolymerization and COOH introduction demonstrated Impact on digestibility improvement? 32

33 Acknowledgements Research Group: Dan Williams Dr. Tongjun Liu Marc Hansen ( ) Ryan Stoklosa Charles Chen David Hodge Alex Smith ( ) Muyang Li Zhenglun Li Collaborators: Eric Hegg, MSU Chris Saffron, MSU Jonathan Walton, MSU Michael Hahn, U. Georgia Trey Sato, U. Wisconsin Art Ragauskas, Georgia Not pictured: p Elizabeth Häggbjer, Natassa Christides, Genevieve Gagnier Funding: DOE, BER DE FC02 07ER64494 DOT, NE Sun Grant Initiative NSF, DUE #

34 Thank You! Questions? edu 34