1 2: Characterization of Plant Cell Wall. to Alkaline Pretreatment and Hydrolysis

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1 1 2: Characterization of Plant Cell Wall Properties Contributing ti to Improved dreponses to Alkaline Pretreatment and Hydrolysis Feedstocks I Genetics and Recalcitrance 27 April, th SIM SBFC Jacob Crowe, Muyang Li, Daniel Williams, Guilong Yan, and David Hodge

2 Cell Wall Monocots Herbaceous Dicots Woody Diversity Important implications for cell wall deconstruction Sorghum (Sorghum bicolor) Corn/Maize (Zea mays subsp. mays) Switchgrass (Panicum virgatum) Arabidopsis thaliana Black Cottonwood (Populus trichocarpa)

3 Cell Wall Monocots Herbaceous Dicots Woody Diversity Important Goals implications for Understanding cell wall interaction between pretreatments and plant cell wall properties deconstruction Identifying cell wall phenotypes with improved responses to pretreatment + enzymatic hydrolysis Sorghum (Sorghum bicolor) Corn/Maize (Zea mays subsp. mays) Switchgrass (Panicum virgatum) Arabidopsis thaliana Black Cottonwood (Populus trichocarpa)

4 Cell Wall Properties Impacting Pretreatability, Ruminant Digestibility, and Cellulolytic Enzyme Hydrolyzability Genotype Phenotype Environmental/agronomic Harvest time/maturity N, water, environment, Composition Lignin, structural polysaccharides pca and FA Acetyl Composition (wt %) Corn stover (Pioneer hybrid 36H56) Switchgrass (cv. Cave-in-Rock) Unassigned Ash Water+EtOH Extractives Acetate Lignin Uronic Acids Galactan Mannan Arabinan Xylan Glucan Structural Differences Relative abundance of cell types? Epidermis Sclerenchyma Vascular bundle zone cells Pith parenchyma Cll Cell wall thickness, composition, accessibility, Switchgrass Maize

5 Goals Understanding impacts of pretreatments on plant cell wall properties Identifying gphenotypes that respond well to cellulolytic enzymes or pretreatment/hydrolysis Li et al., (2015). J Exp Bot. 66(14):

6 Overview of Work 1. Response of diverse cell types in sorghum to alkaline pretreatment 2. Impact of cell wall properties in diverse poplar on alkaline and alkaline oxidative pretreatment 3. Impact of xylan o acetylation on cell wall nanoscale porosity in Arabidopsis 4. Impact of alkaline pretreatments on cell wall water sorption in maize and switchgrass

7 Overview of Work 1. Response of diverse cell types in sorghum to alkaline pretreatment 2. Impact of Work cell performed wall by properties in Dr. Muyang Li and Dr. diverse poplar Guilong Yanon alkaline and alkaline oxidative In collaboration with pretreatment John Mullet, TAMU 3. Impact of xylan o acetylation on cell See wall poster: nanoscale M28 Tissue fractionation, porosity in Arabidopsis extraction and characterization of energy sorghum and the development of a countercurrent extraction of and alkaline pretreatments for 4. Impact high titer mixed sugar production (Tonight!) on cell wall water sorption in maize and switchgrass

8 Sorghum Physical Fractionation, Pretreatment, and Hydrolysis Goals of this Study : Comparison of within plant differences in cell wall properties in two sorghum cultivars Identify responses to alkaline pretreatment and hd hydrolysis TX08001/ES5200 Photoperiod sensitive energy sorghum hybrid Delayed flowering Extended vegetative growth Increased biomass yields Della Commercialsweet sorghum cultivar Mid season variety, matures early Stalk height ft. Energy Sorghum Grain Sorghum Image : Bill Rooney, Texas A&M 1.00 Fructose 0.90 Glucose 0.80 Sucrose Cell Wall g Solu uble Sugar / g TX08001 Sweet Sorghum Della

9 Energy Sorghum Stem Anatomy Epidermis: 5% Rind Fiber : 50% Internal Fiber (Vascular Bundles): 15% Pith Parenchyma: 30% Image: Tesfamichael Kebrom and John Mullet, Texas A&M

10 Industrial Physical Fractionation of Grass Stems Depithing performed prior to chemical pulping of non wood feedstocks (sugarcane bagasse) Industrial depithing Based on hammermilling screening i Integration with inorganics removal from the ebo biomass 1500 dry tonnes/day moist depithing of sugarcane bagasse Potential for integration into cellulosic biofuels technologies? Image Source: FMW, GmbH

11 Physical Fractionation of Sorghum Stems TX08001 or Della Top Middle Bottom Ep. + Outer Rind VB + Inner Rind Ep. + Outer Rind VB + Inner Rind Ep. + Outer Rind VB + Inner Rind Top Pith Pith Pith Middle Bottom Internode Cross Section Epidermis + Outer Rind Vascular Bundles + Inner Rind Pith

12 Confocal Microscopy of Sorghum Stem Fractions 100 μm Outer rind/epidermis Collenchyma cells Sclerenchyma cells, collenchyma cells, vascular bundles 100 μm Inner rind / Vascular bundles Vessel cells Fiber cells 100 μm Pith High lignin parenchyma cells Low lignin parenchyma cells Unseparated bundle sheath (fiber cells)

13 Comparison of Della vs. TX08001: Yields Substantial differences in hydrolysis yields General trend for recalcitrance: Hydrolysis: Ep+OR > VB+IR > Pith CTec3: 15 mg protein/g glucan HTec3: 7.5 mg protein/g glucan Bottom pith samples were most completely defibered Hydrolysis Only Hydrolysis Only Epidermis + Outer Rind Vascular Bundles + Inner Rind Della Pith Epidermis + Outer Rind Vascular Bundles + Inner Rind TX08001 Pith

14 Comparison of Della vs. TX08001: Yields General ltrends for recalcitrance: lit Ep+OR > VB+IR, Pith Top > Middle > Bottom Pretreatment: 0.10 g/g NaOH; 80 C; 1 hr Hydrolysis: CTec3: 15 mg protein /g glucan HTec3: 7.5 mg protein /g glucan Pretreatment + Hydrolysis Pretreatment + Hydrolysis Hydrolysis Only Hydrolysis Only Epidermis + Outer Rind Vascular Bundles + Inner Rind Della Pith Epidermis + Outer Rind Vascular Bundles + Inner Rind TX08001 Pith

15 Comparison of Della vs. TX08001: Properties Diverse range of properties Comparable tissues have comparable compositions and responses to hydrolysis and pretreatment + hydrolysis 72 hr Hydr rolysis Yield 100% 80% s (Della )120% 60% 40% 20% 0% Hydrolysis only Pretreatment + Hydrolysis 0% 20% 40% 60% 80% 100% 120% 72 hr Hydrolysis Yields (TX08001) la Composit tion Del Acetyl (mg/g) Xylan (%) Glucan (%) Lignin (%) TX08001 Composition

16 Cell Wall Properties Contributing to Differences ee in Sorghum Stem Internode Fractions Most properties correlated to each other, e.g.: Xylan Acetyl, Xylan 1/Lignin Lignin strongest single property predictor of hydrolysis yields in untreated and pretreated sorghum fractions 72 hr Hydro olysis Yields R = p = R = p = 7.3 x 10 6 Hydrolysis only Pretreatment + Hydrolysis TX08001: Solid data points Della: Open data points Cell Wall Lignin Content (%) Cell Wall Xy ylan (%) 30 Ac:Xyl = mol/mol Top Pith Ac:Xyl = mol/mol Cell Wall Acetyl Content (%)

17 Overview of Work 1. Response of diverse cell types in sorghum to alkaline pretreatment 2. Impact of cell wall properties in diverse poplar on alkaline and alkaline oxidative pretreatment 3. Impact of xylan o acetylation on Work performed by Dr. Aditya cell wall nanoscale Bhalla porosity in Arabidopsis In collaboration with Wellington Muchero and Gerry Tuskan, ORNL 4. Impact of alkaline pretreatments on cell wall water sorption in maize and switchgrass

18 Cell Wall Property Diversity in Populus trichocarpa Wild type P. trichocarpa genotypes isolated from geographically and environmentally diverse sites Exhibit wide phenotypic diversity Subset selected for diversity in S:G ratio and lignin content Substantial diversity in cell wallassociated inorganics as well Goal: Identifycell wall properties contributing to recalcitrance for: No pretreatment Mild NaOH pretreatment Mild alkaline oxidative pretreatment 27 Li ignin Conte ent (%) Metal Con ntent (ppm m) S 1637S 2H Lignin S:G Ratio Cu Fe Mn 6 303S S 443S 297S 1637H 39S 166H 102H 15S 564H 303H 39H 2S 304S 564S 443H 304H 105S 319S S 105H 15H 193S 319H 121H 77S 77H 297H 193H Diverse poplar genotypes

19 Glucose Hydrolysis Yield (%) Correlating Properties to Yields: Alkaline Pretreatment NaOH Pretreatment Hydrolysis Only R = p = 0.04 R = p = 2.3x Cell Wall Lignin Content (%) Glucose Hydrolysis Yield (%) Lignin S:G Ratio Significant negative correlation for lignin content %) Glucose Hydrolysis Yield ( Cell Wall Transition Metal Content (ppm) S:G and metals are not significant either individually id or in combination with lignin i content t

20 Glucose Hy ydrolysis Yield (% %) Correlating Properties to Yields: Alkaline Oxidative Pretreatment Alkaline Oxidative Pretreatment Hydrolysis Only R = R = p = R = p = 2.3x Cell Wall Lignin Content (%) Glucose Hy ydrolysis Yield (% %) R = p = Lignin S:G Ratio %) Glucose Hydrolysis Yield ( R = p = Cell Wall Transition Metal Content (ppm) All three properties significant (both individual and in combination with each other) See for talk: alkaline oxidative 5 1 Effective coppercatalyzed alkaline oxidative pretreatment pretreatment of woody biomass. (Tuesday, 8:00) First identification of the impact of differences native cell wall associated metals on hydrolysis yields

21 Summary of Correlations Combination of 3 properties provides good prediction Lignin contribution negatively correlated to yields for all S:G ratio and cell wall positively correlated to yields for alkaline oxidative pretreatment ength Rel lative Para ameter Str Hydrolysis Only Alkaline Oxidative NaOH Pretreatment 0.15 Lignin S:G Metals Linear model: Y = Xβ + ε Pre edicted Gluco ose Hydroly ysis Yield (%) 90 )NaOH 80 Pretreatment Alkaline Oxidative Pretreatment Hydrolysis Only Actual Glucose Hydrolysis Yield (%)

22 Overview of Work 1. Response of diverse cell types in sorghum to alkaline pretreatment Work performed by Jacob Crowe 2. Impact of cell wall properties in diverse poplar In collaboration with Markus Pauly, Berkeley Energy on alkaline and Biosciences Institute alkaline oxidative pretreatment 3. Impact of xylan o acetylation on cell wall nanoscale porosity in Arabidopsis 4. Impact of alkaline pretreatments on cell wall water sorption in maize and switchgrass

23 Water Cell Wall Interactions Why look at water? Understand cell wall porosity and polysaccharide accessibility in the context of water swelling Improved water penetration into cell walls yields improved enzyme accessibility to polysaccharides Methods used: DSC for water freezing point depression Water Retention Value (WRV) Williams and Hodge, (2014). Williams and Hodge, ( 0 4). Cellulose. 21(1):

24 Impact of Xylan o Acetylation on Alkaline Pretreatment and Hydrolysis y Identification of esk1/tbl29 as a xylan o acetyl transferase in Arabidopsis (Xiong et al., Mol Plant. 6(4): ) Reduced size, lower cell wall glucan content, collapsed xylem cells Reduction in xylan o acetylation Substitutions on hemicelluloses (e.g. Ara, GlcA, Ac) are hypothesized to control non covalent cross linking between polysaccharides Impacts cell wall rigidity porosity? Goal: Quantify differences in cell wall associated water Xiong et al., Mol Plant. DOI: /j.molp

25 Impact of Xylan o Acetylation on Alkaline Pretreatment and Hydrolysis y Identification of esk1/tbl29 as a xylan o acetyl transferase in Arabidopsis (Xiong et al., Mol Plant. 6(4): ) Reduced size, lower cell wall glucan content, collapsed xylem cells Reduction in xylan o acetylation Substitutions on hemicelluloses (e.g. Ara, GlcA, Ac) are hypothesized to control non covalent cross linking between polysaccharides Impacts cell wall rigidity porosity? Goal: Quantify differences in cell wall associated water

26 Water Properties in Xylan o Acetylation Deficient Arabidopsis Mutants No quantifiable differences in water retention value (WRV) Lower content of nanoscale pore constrained water Less porous cell walls due to tighter association between xylan and cellulose? mass) H 2 O/g biom WRV (g H

27 Overview of Work 1. Response of diverse cell types in sorghum to alkaline pretreatment 2. Impact of cell wall properties in diverse poplar on alkaline and alkaline oxidative pretreatment Work performed by 3. Impact Dr. Dan Williams of xylan o acetylation on In collaboration with cell Dr. wall Rebecca nanoscale Garlock porosity in (MSU GLBRC) Arabidopsis 4. Impact of alkaline pretreatments on cell wall water sorption in maize and switchgrass

28 Grass Responses to Alkaline Pretreatments Goals: Relate differences in cell wall properties to yields ild Composition Water sorption Switchgrass (cv. Cave In Rock) Pretreatments: AFEX pretreatment Varying NH 3 :H 2 O loading, temperature Alkaline hydrogen peroxide (AHP) Varying H 2 O 2 loading Corn stover

29 Correlating Yields to Composition Xylan and lignin strong predictors of hydrolysis yields following alkaline oxidative delignification Glucose Hyd drolysis Yie elds 120% 100% 80% 60% 40% 20% Corn Stover: Solid data points Switchgrass: Open data points Glucose Hyd drolysis Yie eld100% 80% 60% 40% 20% 0% Cell Wall Lignin Content (g/g) 0% Cell Wall Xylan Content (g/g)

30 Correlating Yields to Composition Xylan and lignin strong predictors of hydrolysis yields following alkaline oxidative delignification No obvious trends for AFEX pretreatment Glucose Hyd drolysis Yie elds 120% 100% 80% 60% 40% 20% Corn Stover: Solid data points Switchgrass: Open data points Glucose Hyd drolysis Yie eld100% 80% 60% 40% 20% 0% Cell Wall Lignin Content (g/g) 0% Cell Wall Xylan Content (g/g)

31 Correlating Yields to Composition Xylan and lignin strong predictors of hydrolysis yields following alkaline oxidative delignification No obvious trends for AFEX pretreatment Glucose Hyd drolysis Yie elds 120% 100% 80% 60% 40% 20% Corn Stover: Solid data points Switchgrass: Open data points Glucose Hyd drolysis Yie eld100% 80% 60% 40% 20% 0% Cell Wall Lignin Content (g/g) 0% Cell Wall Xylan Content (g/g)

32 Cell Wall Constrained Water in Delignified Grasses Water freezing point depression by DSC for AHP delignified corn stover and switchgrass Clear trend of increasing cell wall associated water with increasing lignin removal Corresponds to increasing hydrolysis yields Corn Stover Switchgrass

33 Correlating Properties to Water Sorption WRV strongly correlated to hydrolysis yields and lignin content following alkaline oxidative dli delignificationifi r Glucose Hydrolysis 72 h Yield120% 100% 80% 60% 40% 20% 0% WRV (g/g) Wall Lignin n Content (g/g) Cell Corn Stover: Solid data points Switchgrass: Open data points WRV (g/g)

34 r Glucose Hydrolysis 72 h Correlating Properties to Water Sorption WRV strongly correlated to hydrolysis yields and lignin content following alkaline oxidative dli delignificationifi Different trends for hydrolysis yields following AFEX pretreatment t t dependent d on NH 3 loading Yield120% 100% 80% 60% 40% 20% 0% WRV (g/g) Wall Lignin n Content (g/g) Cell Corn Stover: Solid data points Switchgrass: Open data points WRV (g/g)

35 r Glucose Hydrolysis Y 72 h Correlating Properties to Water Sorption WRV strongly correlated to hydrolysis yields and lignin content following alkaline oxidative dli delignificationifi Different trends for hydrolysis yields following AFEX pretreatment t t dependent d on NH 3 loading Yield120% 100% 80% 60% 40% 20% 0% WRV (g/g) Corn Stover: Solid data points Switchgrass: Open data points

36 r Glucose Hydrolysis Y 72 h Correlating Properties to Water Sorption WRV strongly correlated to hydrolysis yields and lignin content following alkaline oxidative dli delignification Summary ifi Different Water swelling trends a for good hydrolysis predictor yields of hydrolysis following AFEX pretreatment t t dependent d on NH 3 loading Yield120% 100% yields for AHP and AFEX pretreated grasses Corn Stover: Solid data points Switchgrass: Open data points Water swelling not correlated to any property other than hydrolysis yields for AFEX pretreated grasses 80% 60% 40% 20% 0% WRV (g/g)

37 Summary Sorghum Substantial differences in cell composition and response to pretreatment and hydrolysis Ligninstrongest predictor ofyields Poplar Lignin, S:G ratio, transition metals show strong correlations to hydrolysis yields for alkaline oxidative pretreatment Arabidopsis Low acetate Arabidopsis cell walls contain less pore constrained water Corn Stover / Switchgrass Substantial differences in cell wall response to AFEX vs. alkaline oxidative delignification Water sorption is strong predictor of yields for all pretreatments/feedstocks

38 Acknowledgements Research Group: Dr. Muyang Li, Dr. Dan Williams, Dr. Aditya Bhalla, Dr. Ryan Stoklosa, Jacob Crowe, Lisaura Maldonado, Thanaphong Phongpreecha, Dhruv Gambhir, Nick Ferringa, Henry Pan Collaborators John Mullet, Texas A&M Markus Pauly, Berkeley Wellington Muchero, ORNL Shi You Ding, MSU Rebecca Garlock, MSU Eric Hegg, MSU Funding: DOE, BER DE FC02 07ER64494 NSF CBET

39 Thank You! Questions?