27 April, 2015 SIM SBFC · 2016-09-17 · Ara, GlcA, Ac) are hypothesized to control non‐covalent...

1‐2: Characterization of Plant Cell Wall P ti C t ib ti t I d RProperties Contributing to Improved Reponses

to Alkaline Pretreatment and Hydrolysis

Feedstocks I – Genetics and Recalcitrance

27 April, 2015

37th SIM SBFCJacob Crowe Muyang Li Daniel WilliamsJacob Crowe, Muyang Li, Daniel Williams,

Guilong Yan, and David Hodge

www.glbrc.org

Cell Wall Diversity

Monocots Woody

Dicots

Herbaceous

DiversityImportant pimplications for cell wall deconstruction

SorghumSorghum((Sorghum bicolorSorghum bicolor))((Sorghum bicolorSorghum bicolor))

Corn/Maize Corn/Maize ((ZeaZea maysmays subspsubsp. . maysmays))

Switchgrass(Panicum virgatum)

Arabidopsis thaliana

Black Cottonwood (Populus trichocarpa)

Cell Wall Diversity

Monocots Woody

Dicots

Herbaceous

DiversityImportant G lp

implications for cell wall

Goals• Understanding interaction between

d l ll ll ideconstructionpretreatments and plant cell wall properties

SorghumSorghum((Sorghum bicolorSorghum bicolor))

• Identifying cell wall phenotypes with improved responses to pretreatment + enzymatic hydrolysis

((Sorghum bicolorSorghum bicolor))

Corn/Maize Corn/Maize ((ZeaZea maysmays subspsubsp. . maysmays))

Switchgrass(Panicum virgatum)

Arabidopsis thaliana

Black Cottonwood (Populus trichocarpa)

Cell Wall Properties Impacting Pretreatability, Ruminant Digestibility, and Cellulolytic Enzyme Hydrolyzability

Structural DifferencesGenotype Phenotype

– Relative abundance of cell types?

• Epidermisl hEnvironmental/agronomic • Sclerenchyma

• Vascular bundle zone cells• Pith parenchyma

C ll ll thi k

Environmental/agronomic– Harvest time/maturity

– N, water, environment,…– Cell wall thickness, composition, accessibility,…

Composition80

Unassigned

– Lignin, structural polysaccharides

pCA and FA30

sition

(wt %)

Water+EtOH ExtractivesAcetate

Lignin

Uronic Acids

Galactan

Mannan

Arabinan

MaizeSwitchgrass

– pCA and FA

– Acetyl 0

Corn stover Switchgrass

Arabinan

Glucan

(Pioneer hybrid 36H56) (cv. Cave-in-Rock)

• Understanding impacts of lpretreatments on plant

cell wall properties

• Identifying phenotypes y g p ypthat respond well to cellulolytic enzymes or y ypretreatment/hydrolysis

Li et al., (2015). J Exp Bot. 66(14):4305‐4315

Overview of Work

1. Response of diverse cell types in sorghum to alkaline pretreatmentsorghum to alkaline pretreatment

2. Impact of cell wall properties in diverse poplar on alkaline anddiverse poplar on alkaline and alkaline‐oxidative pretreatment

3 Impact of xylan o‐acetylation on3. Impact of xylan o acetylation on cell wall nanoscale porosity in Arabidopsisp

4. Impact of alkaline pretreatments on cell wall water sorption in pmaize and switchgrass

Overview of Work

2. Impact of cell wall properties in diverse poplar on alkaline and

Work performed by Dr. Muyang Li and Dr. diverse poplar on alkaline and

alkaline‐oxidative pretreatment3 Impact of xylan o‐acetylation on

In collaboration with John Mullet, TAMU

Guilong Yan

3. Impact of xylan o acetylation on cell wall nanoscale porosity in Arabidopsis

See poster: M28 – Tissue fractionation, extraction and characterization of energy p

4. Impact of alkaline pretreatments on cell wall water sorption in

sorghum and the development of a counter‐current extraction and alkaline pretreatment for high‐titer mixed sugar production (Tonight!)p

maize and switchgrass

Sorghum Physical Fractionation, Pretreatment, and HydrolysisPretreatment, and Hydrolysis

Energy Energy SorghumSorghum

Goals of this Study : • Comparison of within‐plant

diff i ll ll ti SorghumSorghum

Sweet Sweet SorghumSorghum

differences in cell wall properties in two sorghum cultivars

• Identify responses to alkaline d h d l i

Grain SorghumGrain Sorghum

Image : Bill Rooney TexasImage : Bill Rooney Texas A&MA&M

pretreatment and hydrolysis TX08001/ES5200• Photoperiod‐sensitive “energy

Cell Wall

Fructose

Glucose

Sucrose

Image : Bill Rooney, Texas Image : Bill Rooney, Texas A&MA&MPhotoperiod sensitive energy sorghum” hybrid

• Delayed flowering Extended vegetative growth

uble Sug

ar / g

Increased biomass yields

Della• Commercial sweet sorghum cultivar

TX08001 Della

luCommercial sweet sorghum cultivar• Mid‐season variety, matures early• Stalk height 10–11 ft.

Energy Sorghum Stem Anatomy

Image: Tesfamichael Kebrom and John Mullet, Texas A&M

Pith Parenchyma: 30%Internal “Fiber” (Vascular Bundles): 15%Rind “Fiber”: 50%Epidermis: 5%

Industrial Physical Fractionation of Grass Stems

• Depithing performed prior to chemical pulping of non‐wood f d t k ( b )feedstocks (sugarcane bagasse)

• Industrial depithingB d h illi i– Based on hammermilling‐screening

– Integration with inorganics removal from the biomasso t e b o ass

– 1500 dry tonnes/day moist depithing of sugarcane bagasse

• Potential for integration intoPotential for integration into cellulosic biofuels technologies?

Image Source: FMW, GmbH

Physical Fractionation of Sorghum Stemsof Sorghum Stems

TX08001 or Della

Bottom

MiddleTop

Bottom

Middle

Ep. + Outer R

VB + Inne

Ep. + Outer R

VB + In

Ep. + Outer R

VB + In

Internode Cross‐Section Epidermis + Outer Rind Vascular Bundles + Inner Rind Pith

Confocal Microscopy of Sorghum Stem Fractions

Outer rind/EpidermisOuter rind/Epidermis100 μm

SclerenchymaSclerenchyma cellscells, , collenchymacollenchyma cells, vascular bundlescells, vascular bundlesCollenchymaCollenchyma cellscells

Inner rind / Vascular bundles Inner rind / Vascular bundles 100 μm

Vessel cellsVessel cells Fiber cellsFiber cells

PithPith100 μm

LowLow‐‐lignin lignin parenchymaparenchyma cellscells

UnseparatedUnseparated bundle bundle sheath (fiber cells)sheath (fiber cells)HighHigh‐‐lignin parenchyma cellslignin parenchyma cells

Comparison of Della vs. TX08001: Yields

• Substantial differences in hydrolysis yields

• General trend for recalcitrance: Hydrolysis:CTec3: 15 mg protein/g glucan

Ep+OR > VB+IR > PithCTec3: 15 mg protein/g glucanHTec3: 7.5 mg protein/g glucan

Bottom pith samplesBottom pith samples were most completely“defibered”

Hydrolysis OnlyHydrolysis Only

Epidermis + Outer Rind

Vascular Bundles + Inner Rind

TX08001

Comparison of Della vs. TX08001: Yields G l t d f l it• General trends for recalcitrance:

Ep+OR > VB+IR, Pith Pretreatment: 0.10 g/g NaOH; 80°C; 1 hr

Top > Middle > Bottom Hydrolysis:CTec3: 15 mg protein /g glucanHTec3: 7.5 mg protein /g glucan

Pretreatment + Hydrolysis Pretreatment + Hydrolysis

Hydrolysis Only Hydrolysis Only

TX08001

Comparison of Della vs. TX08001: Properties

• Diverse range of properties

• Comparable tissues have comparable compositions and

responses to hydrolysis and pretreatment + hydrolysis

Acetyl (mg/g)

Xylan (%)

Glucan (%)

Lignin (%)

ella ) Hydrolysis only

Pretreatment + Hydrolysis

la Com

posit Lignin (%)

rolysis Y

72‐hr H

00 20 40 60 80

TX08001 Composition

0% 20% 40% 60% 80% 100% 120%

72‐hr Hydrolysis Yields (TX08001)

Cell Wall Properties Contributing to Differences in Sorghum Stem Internode Fractionse e ces So g u Ste te ode act o s• Most properties correlated to each other, e.g.:

Xylan Acetyl, Xylan 1/LigninXylan Acetyl, Xylan 1/Lignin

• Lignin strongest single property predictor of hydrolysis yields in untreated and pretreated sorghum fractions

1.2R = ‐0.596p = 0.0090

yields in untreated and pretreated sorghum fractions

30 Ac:Xyl = 0.764 mol/mol

TX08001: Solid data pointsDella: Open data points

olysis Yields

R = 0 85220

Top Pith

72‐hr H

Hydrolysis onlyPretreatment

R = ‐0.852p = 7.3 x 10‐6

ll Wall Xy

Ac:Xyl = 0.543 mol/mol

00 5 10 15 20 25 30

Cell Wall Lignin Content (%)

Pretreatment + Hydrolysis

0 2 4 6 8 10

Cell Wall Acetyl Content (%)

Overview of Work

3 Impact of xylan o‐acetylation on3. Impact of xylan o acetylation on cell wall nanoscale porosity in ArabidopsisIn collaboration with Wellington

Work performed by Dr. AdityaBhalla

p4. Impact of alkaline pretreatments

on cell wall water sorption in

Muchero and Gerry Tuskan, ORNL

pmaize and switchgrass

Cell Wall Property Diversity in Populus trichocarpa

ent (%)

in Populus trichocarpa• Wild‐type P. trichocarpa genotypes

isolated from geographically and

ignin Co

nteisolated from geographically and

environmentally diverse sites – Exhibit wide phenotypic diversity

150 1 2 3 4 5

Lignin S:G Ratio• Subset selected for diversity in

S:G ratio and lignin content

m CuFeMn

• Substantial diversity in cell wall‐associated inorganics as well

• Goal: Identify cell wall properties

10Metal Con• Goal: Identify cell wall properties

contributing to recalcitrance for: – No pretreatment

2H 15S

303H 39H 2S

105H 15H

121H 77S

Diverse poplar genotypes

– Mild NaOH pretreatment– Mild alkaline‐oxidative pretreatment

Correlating Properties to Yields: Alkaline Pretreatment

Alkaline PretreatmentR = ‐0.349p = 0.04

Hydrolysis Y

ield (

Hydrolysis Y

ield (

Hydrolysis Y

ield (

NaOH Pretreatment

R = ‐0.697

Glucose H

Hydrolysis Only

R 0.697p = 2.3x10‐6

• Significant negative correlation for lignin content

015.0 17.5 20.0 22.5 25.0 27.5

00.0 1.0 2.0 3.0 4.0 5.0

Lignin S:G Ratio

00.0 10.0 20.0 30.0

Cell Wall Transition Metal Content (ppm)

• Significant negative correlation for lignin content

• S:G and metals are not significant either i di id ll i bi ti ith li i t tindividually or in combination with lignin content

Correlating Properties to Yields: Alkaline‐Oxidative Pretreatment

Alkaline‐Oxidative PretreatmentAlkaline‐Oxidative Pretreatment

R = ‐0.446 R = 0 287R = 0.363

ydrolysis Y

ield (%

ydrolysis Y

ield (%

Hydrolysis Y

ield (

R = ‐0 697

p = 0.006R = 0.287p = 0.055

p = 0.029

Glucose Hy

40Glucose Hy

Glucose H

Hydrolysis Only

R = ‐0.697p = 2.3x10‐6

015.0 17.5 20.0 22.5 25.0 27.5

00.0 1.0 2.0 3.0 4.0 5.0

Lignin S:G Ratio

00.0 10.0 20.0 30.0

Cell Wall Transition Metal Content (ppm)

• All three properties significant (both individual and in combination with each other) for alkaline‐oxidative pretreatment

See talk: 5‐1 Effective copper‐catalyzed alkaline‐oxidative pretreatment of woody biomass.

• First identification of the impact of differences native cell wall‐associated metals on hydrolysis yields

(Tuesday, 8:00)

Summary of Correlations• Combination of 3 properties provides good prediction• 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

0.9Hydrolysis Only

• S:G ratio and cell wall positively correlated to yields for alkaline‐oxidative pretreatment

) NaOH

Hydrolysis OnlyAlkaline‐OxidativeNaOH Pretreatment

ysis Yield (%

Alkaline‐Oxidative Pretreatment

NaOHPretreatment

ameter Str

ose Hydroly Pretreatment

‐0.45

‐0.3

‐0.15 Lignin S:G Metals

lative Para

edicted Gluco

Hydrolysis OnlyLinear model:

‐0.9

‐0.75

‐0.6Rel

0 20 40 60 80 100

Actual Glucose Hydrolysis Yield (%)

Y = Xβ + ε

Overview of Work

2. Impact of cell wall properties in diverse poplar on alkaline and

In collaboration with Markus P l B k l E

Work performed by Jacob Crowe

diverse poplar on alkaline and alkaline‐oxidative pretreatment

3 Impact of xylan o‐acetylation on

Pauly, Berkeley‐Energy Biosciences Institute

3. Impact of xylan o acetylation on cell wall nanoscale porosity in Arabidopsisp

Water‐Cell Wall InteractionsWh l k t t ?• Why look at water?

Understand cell wall porosity and polysaccharide accessibility in the context of water swellingaccessibility in the context of water swelling

Improved water penetration into cell walls yields improved enzyme accessibility to polysaccharidesimproved enzyme accessibility to polysaccharides

• Methods used: DSC for water freezing– DSC for water freezing point depression

– Water Retention Value (WRV)

Williams and Hodge, (2014). Williams and Hodge, ( 0 4).Cellulose. 21(1):221‐235.

Impact of Xylan o‐Acetylation on Alkaline Pretreatment and Hydrolysis

• Identification of esk1/tbl29 as a xylano‐acetyl transferase in Arabidopsis

(Xiong et al., 2013. Mol Plant. 6(4):1373‐1375)

– Reduced size, lower cell wall glucancontent, collapsed xylem cellscontent, 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

Xiong et al., 2015. Mol Plant. DOI: 10.1016/j.molp.2015.02.013

between polysaccharides– Impacts cell wall rigidity‐porosity?

• Goal: Quantify differences in cellGoal: Quantify differences in cell wall‐associated water

Impact of Xylan o‐Acetylation on Alkaline Pretreatment and Hydrolysis

• Identification of esk1/tbl29 as a xylano‐acetyl transferase in Arabidopsis

(Xiong et al., 2013. Mol Plant. 6(4):1373‐1375)

– Reduced size, lower cell wall glucancontent, collapsed xylem cellscontent, 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 polysaccharidesbetween polysaccharides– Impacts cell wall rigidity‐porosity?

• Goal: Quantify differences in cellGoal: Quantify differences in cell wall‐associated water

Water Properties in Xylan o‐Acetylation‐Deficient ArabidopsisMutants

• No quantifiable differences in water retention value (WRV)• Lower content of nanoscale pore‐constrained water

Deficient ArabidopsisMutants

Lower content of nanoscale pore constrained water– Less porous cell walls due to tighter association between xylanand cellulose?

/g biom

Overview of Work

3 Impact of xylan o‐acetylation onWork performed by Dr. Dan Williams3. Impact of xylan o acetylation on

cell wall nanoscale porosity in Arabidopsis

In collaboration with Dr. Rebecca Garlock(MSU‐GLBRC)p

Grass Responses to Alkaline PretreatmentsSwitchgrassSwitchgrass(cv. Cave‐In‐Rock)• Goals:

– Relate differences in cell ll ti t i ldwall properties to yields

• Composition• Water sorption

Corn stover

Water sorption

• Pretreatments:• Pretreatments:– AFEX pretreatment

• Varying NH3:H2O loading,Varying NH3:H2O loading, temperature

– Alkaline hydrogen id (AHP)peroxide (AHP)

• Varying H2O2 loading

Correlating Yields to Composition• Xylan and lignin strong predictors of hydrolysis yields following alkaline‐oxidative delignificationyields following alkaline oxidative delignification

eld100%

elds 100%

eld100%

Corn Stover: Solid data pointsSwitchgrass: Open data points

drolysis Yie

Glucose Hyd

0%0.0 0.1 0.2 0.3 0.4

Cell Wall Xylan Content (g/g)

0%0.00 0.05 0.10 0.15 0.20 0.25 0.30

Cell Wall Lignin Content (g/g)

0%0.0 0.1 0.2 0.3 0.4

0%0.00 0.05 0.10 0.15 0.20 0.25 0.30

• No obvious trends for AFEX pretreatment

eld100%

elds 100%

drolysis Yie

Glucose Hyd

0%0.0 0.1 0.2 0.3 0.4

0%0.00 0.05 0.10 0.15 0.20 0.25 0.30

0%0.0 0.1 0.2 0.3 0.4

• No obvious trends for AFEX pretreatment

eld100%

drolysis Yie

Glucose Hyd

0%0.0 0.1 0.2 0.3 0.4

0%0.00 0.05 0.10 0.15 0.20 0.25 0.30

Cell Wall‐Constrained Water in Delignified Grasses• Water freezing point depression by DSC for AHP‐g p p ydelignified corn stover and switchgrass

• Clear trend of increasing cell wall‐associated water with increasing lignin removal– Corresponds to increasing hydrolysis yields

Corn Stover SwitchgrassCorn Stover Switchgrass

Correlating Properties to Water Sorption• WRV strongly correlated to hydrolysis yields and lignin content following alkaline‐oxidative d li ifi tidelignification

Yield Corn Stover: Solid data points

Switchgrass: Open data points

t (60%

Hydrolysis

t (60%

Hydrolysis

Wall Lignin

r Glucose H

Wall Lignin

r Glucose H

1.0 1.5 2.0 2.5 3.0 3.5

WRV (g/g)

0%1.0 1.5 2.0 2.5 3.0 3.5

WRV (g/g)

1.0 1.5 2.0 2.5 3.0 3.5

WRV (g/g)

0%1.0 1.5 2.0 2.5 3.0 3.5

WRV (g/g)

• Different trends for hydrolysis yields following AFEX t t t d d t NH l di

(g/g) Corn Stover: Solid data points

AFEX pretreatment – dependent on NH3 loading

t (60%

Hydrolysis

Wall Lignin

r Glucose H

Wall Lignin

1.0 1.5 2.0 2.5 3.0 3.5

WRV (g/g)

0%1.0 1.5 2.0 2.5 3.0 3.5

WRV (g/g)

0%1.0 1.5 2.0 2.5 3.0 3.5

WRV (g/g)

1.0 1.5 2.0 2.5 3.0 3.5

WRV (g/g)

AFEX pretreatment – dependent on NH3 loading

Hydrolysis Y

r Glucose H

0%1.0 1.5 2.0 2.5 3.0 3.5

WRV (g/g)

Summary Water swelling a good predictor of hydrolysis

AFEX pretreatment – dependent on NH3 loadingyields for AHP‐ and AFEX‐pretreated grasses

Water swelling not correlated to any property

Hydrolysis Y Water swelling not correlated to any property

other than hydrolysis yields for AFEX‐pretreated grasses

r Glucose H pretreated grasses

0%1.0 1.5 2.0 2.5 3.0 3.5

WRV (g/g)

SummarySorghum• Substantial differences in cell composition

and response to pretreatment and hydrolysis• Lignin strongest predictor of yieldsLignin strongest predictor of yields

Poplar• Lignin, S:G ratio, transition metals show

strong correlations to hydrolysis yields for alkaline‐oxidative pretreatment

ArabidopsisArabidopsis• Low‐acetate Arabidopsis cell walls contain

less pore‐constrained water

Corn Stover / Switchgrass• Substantial differences in cell wall response

to AFEX vs alkaline‐oxidative delignificationto AFEX vs. alkaline oxidative delignification• Water sorption is strong predictor of yields

for all pretreatments/feedstocks

AcknowledgementsCollaborators

Research Group:Dr. Muyang Li, Dr. Dan Williams, Dr. Aditya Bhalla, Dr. Ryan Stoklosa,

Collaborators• John Mullet, Texas A&M• Markus Pauly, Berkeley• Wellington Muchero ORNLy , y ,

Jacob Crowe, Lisaura Maldonado, Thanaphong Phongpreecha, DhruvGambhir, Nick Ferringa, Henry Pan

• Wellington Muchero, ORNL• Shi‐You Ding, MSU• Rebecca Garlock, MSU• Eric Hegg, MSUc egg, SU

Funding:Funding:• DOE, BER DE‐FC02‐07ER64494• NSF CBET‐1336622

ThankThank You!You!Questions?Questions?

27 April, 2015 SIM SBFC · 2016-09-17 · Ara, GlcA, Ac) are hypothesized to control non‐covalent...

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