Understanding and Enhancing Alkaline and Oxidative Chemical Pretreatments for theOxidative Chemical Pretreatments for the Production of Biofuels through Improved
CharacterizationCharacterization
David Hodgea d odgeAssistant Professor
Chemical Engineering, Michigan State Universityg g, g y
3rd International Symposium on y pBioenergy and Biotechnology
Wuhan, Hubei, P.R. China
16 October, 2012
Plant Tissues are Diverse and Highly HeterogeneousStorage Tissues: Food Crops Structural Tissues: Plant Cell Walls
Starch Granules in Corn Endosperm Source: USDA/ARS/ERRC
Arabidosis stem PNAS. 107(51): 22338–22343
Corn Stem Source: John Sedbrook, U. Illinois
Arabidopsis OleosomesNature Methods 3, 47 ‐ 53 (2006)
Softwood secondary xylem (tracheids) http://www.cb.uu.se/
Hardwood secondary xylem (vessel elements and fibers)Plant Physiol 112:1479‐1490.
ts ous)
Commelinoid
Diversity of Cell Wall PolymersPrimary Cell Wall Secondary Cell Wall
3
Mon
ocot
(Herbaceo Commelinoid
(Grasses)
Non‐commelinoidpe
rms β‐GlucansGAX GMGAX
S GS G H FA FA
Herbaceous Dicot
commelinoid
Ang
iosp
XyGPP GX GMGX
S G
Dicots
Dicot
Woody Dicot
yPP
S GS G H
Woody Dicot(Hardwood)
ms
XyGPP GX GM GX
SHGSHG
SoftwoodGAX: glucuronoarabinoxylansGX: glucuronoxylans
mno
sper
m
SG Guaiacyl
Syringyl
Stem cross-sections
XyG: xyloglucansGMs: glucomannansPP: pectic polysaccharides
Gym H p-hydroxyphenyl
FA Ferulates
4
Monocot Lignins pCA
Lignin
S
Differences in composition and structural organization relative to herbaceous and woody dicots or
Li i
Sherbaceous and woody dicots or gymnosperm lignins
Ferulates and p‐coumarate can comprise a significant fraction of grass lignins Lignin
FA
significant fraction of grass ligninsEster crosslinksHighly condensed (~85%)High phenolic hydroxyl content
High alkali solubility
SGH pCA FA
p-coumaryl alcohol(H Lignins)
coniferyl alcohol(G Lignins)
sinapyl alcohol(S Lignins)
p-coumaricacid
ferulic acid
Barriers to the (Biological) Utilization of Plant Cell Wall Componentsof Plant Cell Wall Components
Targets/problems Science 315, 804 (2007)
Ultrastructure of plant cell walls needs to be broken downdown
Lignin prevents access to carbohydrate fraction
At the molecular level:Chemical modifications
Crystalline cellulose resists depolymerization, blocks access to internally packed
Chemical modifications
Depolymerization
Solubilizationaccess to internally packed polymers
How pretreatments
Solubilization
At the structural level:Physical redistribution ofHow pretreatments
address this problemPhysical redistribution of components
Hemicellulose
Ch i l P t t tEnzymatic Depolymerization of
Cellulose
Chemical Pretreatment Polysaccharides
Biochemical Conversion of Plant Cell Wall Polysaccharides
Lignocellulose Feedstock (Plant Cell Walls)
yto Biofuels
Biological Conversion of Monosaccharides
Microbial MetabolitesEthanol, Butanol, Carboxylic Acids, Alkanes, Isoprenoids, …
Biological Mechanism 1: Secreted set of ll l
Oxidative Deconstruction of LignocelluloseT. terrestris GH61EBiochemistry 2010,
cellulases• Individual proteins with CBM, flexible
linker, and catalytic domain (e.g. Trichoderma reesei)
49, 3305–3316
c ode a eese )
Biological Mechanism 2: Brown rot fungi• Deconstruction of cellulose and lignin• Deconstruction of cellulose and lignin
using biologically‐generated ∙OH
Bi l i l M h i 3 Whit t f iBiological Mechanism 3: White rot fungi• Peroxidases and oxidases for lignin
degradation Lignin Peroxidase from P. chrysosporium
Laccase from M. albomyces
Science 333:762‐765.Images source: http://chemistry.umeche.maine.edu/CHY431/Wood4.html
Alkaline Hydrogen Peroxide PretreatmentBased on existing alkaline hydrogenBased on existing alkaline hydrogen peroxide pulp bleaching stages in the paper industry
Alkaline‐oxidative pretreatments as either standalone pretreatments ORdelignifying “finishing” post‐delignifying finishing postpretreatment step
Unique advantagesWell‐suited for grasses
Current Challenges:
Alkaline hydrogen peroxide bleaching tower >1000 tpd capacity at Smurfit‐K K f li Pi å S d ( h
Process integration
Economics
Water use/recycleKappa Kraftliner, Piteå, Sweden (photo courtesy: Outokumpu Oy)
/ y
Alkaline Hydrogen Peroxide PretreatmentBased on existing alkaline hydrogenBased on existing alkaline hydrogen peroxide pulp bleaching stages in the paper industry
Agricultural Residues:• Corn Stover/Corn Fiber• Wheat Straw
Alkaline‐oxidative pretreatments as either standalone pretreatments ORdelignifying “finishing” post‐
Wheat Straw• Sugar Cane Bagasse• Sorghum Bagasse• Rice Strawdelignifying finishing post
pretreatment step
Unique advantages
• Rice Straw
Well‐suited for grasses
Current Challenges: Bioenergy Grasses:• Switchgrass
Process integration
Economics
Water use/recycle
g• Miscanthus sp. • Reed Canary Grass
/ y
Alkaline Hydrogen Peroxide PretreatmentBased on existing alkaline hydrogenBased on existing alkaline hydrogen peroxide pulp bleaching stages in the paper industry
Alkaline‐oxidative pretreatments as either standalone pretreatments ORdelignifying “finishing” post‐
Improved Processes?Catalytic approaches?
delignifying finishing postpretreatment step
Unique advantages70
80
90
ass
Upper Theoretical Limit
Well‐suited for grasses
Current Challenges: 40
50
60OH / ton
Bioma
Switchgrass
Process integration
Economics
Water use/recycle 0
10
20
30
Gal EtO Corn Stover
/ y$0.00 $2.00 $4.00 $6.00
Pretreatment Chemical Cost / Gal EtOH
11Alkaline Hydrogen Peroxide PretreatmentPretreatment Liquefaction/Saccharificationq
90%
100%
Unquantified Solids
h
90%
100%
Unquantified Solids
A hSolids transferred to Solids transferred to the
50%
60%
70%
80%
onen
t Fraction
Ash
Water+EtOH Extractives
Acetate
Uronic Acids
Galactan50%
60%
70%
80%
onen
t Fraction
Ash
Water+EtOH Extractives
Acetate
Uronic Acids
GalactanInsoluble Insoluble
Solids transferred to the liquid phase
Solids transferred to the liquid phase (hydrolysate)
10%
20%
30%
40%
Compo
Galactan
Mannan
Arabinan
Xylan
Glucan10%
20%
30%
40%
Compo
Galactan
Mannan
Arabinan
Xylan
Glucan
FractionFraction
0%
0 3 6 12 18 30 36 42 48
Pretreatment Time (h)
Lignin (Klason)
ASL
0%
0 3 6 12 18 30 36 42 48
Pretreatment Time (h)
Lignin (Klason)
ASL0 4 8 12 16 20 24 28 32 36 40 44 48
Hydrolysis Time (h)Banerjee et al. (2012). BiotechnolBioeng. 109(4):922‐931.
Pretreatment, 0 hPretreatment, 0 hIntegration of AHP Pretreatment,
Hydrolysis, and Fermentation
on o
f ne
nts
y y ,
70
80
90
Glc Xly
Y87 (pH5.5) OD 1
6
8
Pretreatment, 48 hPretreatment, 48 h
solu
biliz
atio
all c
ompo
n
30
40
50
60
conc
. (g/
l)
y EtOH Xylitol glycerol OD 600
4
OD
600
Xylose-fermenting Saccharomyces
Hydrolysis, 0 Hydrolysis, 0 hrhr
crea
sing
san
t cel
l wa
0 50 100 150 200 2500
10
20
0
2
Inc
pla
1.67 g/L YNB w/o ammonium sulfate and 2 27 g/L of urea
0 50 100 150 200 250
time (h)
Hydrolysis, 24 Hydrolysis, 24 hrhrand 2.27 g/L of urea
Filter Sterilize
Fermentation
Sterilize(0.22 μm)
Liu et al. (in preparation).
Relating Cell Wall Properties to RecalcitranceUnderstanding cell wall properties impacting recalcitrance and relationship of these properties to enzymatic digestibility
Using pretreatment of plant cell walls with diverse phenotypes to t l ith di l it tigenerate samples with diverse recalcitrance properties
Analytical ToolProperty Impacting Recalcitrance
Water adsorption/retention
Potentiometric titration
Cell wall hydrophilicity
Cellulose oxidation
Glycome Profiling
Klason Lignin
Polysaccharide accessibility
Lignin content
HPLC
Pyrolysis‐GC/MS
Ferulate content
S/G ratio
Thioacidolysis‐GC/MS
HSQC NMR
Lignin condensation
Diverse Cell Wall Properties: BiomassMonocot grasses
Corn Stovers (Zea mays)• Pioneer hybrid 36H56
• Inbred brown midrib bm1Inbred brown midrib bm1
• Inbred brown midrib bm3
Switchgrass(Panicum virgatum cv. Cave‐in‐Rock)
Miscanthus (Miscanthus x giganteus )
Woody dicotHybrid poplar
(Populus nigra var Charkoviensis x Caudina)
Herbaceous dicot
(Populus nigra var. Charkoviensis x Caudina)
Goldenrod (Solidago sp.)dicot
Diverse Cell Wall Properties: Pretreatments 15
AHP
Soda pulping
Liquid Hot Water + qAHP post‐treatment
Cu(bpy)‐catalyzed AHP t t tAHP pretreatment
Cell Wall – Water Interactions ObjectivesHow can cell wall–water interactions Property: Cell impact deconstruction
Water Retention Value
i l S li
p yWall Composition
Quantifiable Particle Settling
Water Activity
15% Solids15% Solids
Lignin Content
Polysaccharide Content
Qmetrics
15% Solids15% Solids
Accessible vs. Amorphous
Corn StoverCorn Stover SwitchgrassSwitchgrass Inaccessible
Property: CellO t
vs. Crystalline
Property: Cell Wall Hydrophilicity
Outcomes:Water Penetration
Enzyme Penetration Property: Cell Wall Porosity
Enzyme Penetration
Polysaccharide Hydrolysis
Introduction of Carboxyl Groups with Oxidative Pretreatments?
?
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOHCOOHCOOHCOOH
COOH
Interactions Between Water and Cell WallWater retention value (WRV)
Step 1: Filter wash Step 2: Centrifuge Step 3: Weighing
Water retention value (WRV)
Determine Moisture Content of Pad
MwaterMdrypad
WRV =
Filter wash using 200mesh screen to
“Cookie Cutter” pad onto 200 mesh disk from filter
Weigh wet pad; Dry in oven at 105oC for ~2hrs
then weigh dry pad200mesh screen to ~ 80% moisture
200 mesh disk from filter funnel; Centrifuge at
900xg for 15min
Interactions Between Water and Cell WallB th t t ti l (WRV) Settling height (ht)Both water retention value (WRV)
and swelling volume are increased with pretreatment
Settling height (ht) Total height (Ht)
WRV is a very good predictor of
More water confined in cell wall
htHt
digestibility
80%
100%
on
LHW=Liquid Hot Water pretreated
1.00
ght
ht
40%
60%
80%
can
Con
vers
io
0.80
eigh
t/Tot
al h
eig
CS UntreatedCS 25% H2O2SG UntreatedSG 25% H2O2AHP
IncreasesS lli
20%
40%
7 D
ay G
lu SG
LHW SG
CS
LHW CS0.40
0.60S
ettli
ng h
e Swelling
0%1.4 2.0 2.6 3.2 3.8 4.4
WRV
2.0% 4.0% 6.0% 8.0% 10.0% 12.0%
Solids Concentration (w/w)Williams et al. (in preparation).
Interactions Between Water and the Cell Wall
30
Pretreated corn stover can adsorb more water in the cell wall Dynamic vapor
25
30
)
Corn StoverQuantifiable by decreased water
i i
sorption/desorption isotherms
15
20C
onte
nt (%
)activity at a moisture content
i e more water is
10
Moi
stur
e Ci.e. more water is
present in a constrained
i t
0
5environment
0 0.25 0.5 0.75 1
Water Activity (aw) = Relative Humidity (hr) of vaporWilliams et al. (in preparation).
Interactions Between Water and the Cell Wall
30
Pretreated corn stover can adsorb more water in the cell wall Dynamic vapor
25
30
)
Corn StoverAHP Pretreated
Quantifiable by decreased water
i i
sorption/desorption isotherms
15
20C
onte
nt (%
)Lower hr for AHP Corn
activity at a moisture content
i e more water is
10
Moi
stur
e Ci.e. more water is
present in a constrained
i t
0
5environment
0 0.25 0.5 0.75 1
Water Activity (aw) = Relative Humidity (hr) of vaporWilliams et al. (in preparation).
Hydrolysis Results for AHP Pretreatment of Diverse Cell Wall Phenotypes
Grasses: Increasing response to AHP
of Diverse Cell Wall Phenotypes
response to AHPpretreatment
Herbaceous Dicot: Conversion saturates
Cell wall properties?
brid
pl
ar
nrod
over
6) ov
er
) tove
r 3)hu
s
rass
Hyb
Pop
Gol
den
Cor
n St
o(3
6H56
Cor
n St
o(b
m1
Cor
n St
(bm
3
Mis
cant
Switc
hgr
Dicots MonocotsLi et al. (in preparation)
Correlating Digestibility to Plant Cell Wall Properties: Lignin RemovalWall Properties: Lignin Removal
Digestibility and lignin content correlated
100%Hybrid Stover
Extracted with alkali or AHP pretreated
Negative relationship
70%
80%
90%
ty
Hybrid Stover
Inbred bm1 stover
Inbred bm3 Stover
Hybrid Poplar
g pwith “threshold” value for lignin removal
40%
50%
60%
ucan
Digestibilit Miscanthus
Critical level for cell wall hydrophobicity?
10%
20%
30%Glu
Properties associated with lignin removal?
0%0 0.05 0.1 0.15 0.2 0.25
Klason Lignin (Mass Fraction of Cell Wall)
g
24Lignin Analytical Methods: Pyrolysis GC-MS
High‐throughput destructive characterization of complete cell wallsBased on characterization of volatilized pyrolysis productspy y pPyrolysis for 10 s at 600oC, heated transfer line to GC/MS at 300oC
3.0
4.0
5.0
(x10,000,000)TICTIC
GC-MS
5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5
1.0
2.0
Lignin Analytical Methods: Pyrolysis GC-MSCorrelating aromatic products to monomers in the lignin polymer
25
70%
75%
80%
85%
acol o
rcrease
Correlating aromatic products to monomers in the lignin polymerOnly represents a fraction of original lignin
• Poor yield on condensed ligninsUnique products for each lignin monomer?
50%
55%
60%
65%
70%
Yrolytic 4‐vinylguai
4‐vinylphe
nol de
c
Ferulate
p‐coumarateH lignins ???
q p gpCA and FA yield clearly identifiable markers
• 4‐vinylphenol and 4‐vinylguaiacol
40%
45%
50%
0.00% 0.25% 0.50% 0.75% 1.00% 1.25%
PY
Quantified ferulate or p‐coumarate released
p‐coumarateH-lignins
p-coumaric???
(percentage of cell wall)acid
G-lignins???
FerulicAcid
???
S-lignins
Lignin Analytical Methods: Pyrolysis GC-MSCorrelating aromatic products to monomers in the lignin polymer
26
Correlating aromatic products to monomers in the lignin polymerOnly represents a fraction of original lignin
• Poor yield on condensed ligninsUnique products for each lignin monomer?
100% Syringyl Lignins
q p gpCA and FA yield clearly identifiable markers
• 4‐vinylphenol and 4‐vinylguaiacol Li et al. (2012). BiotechnolBiofuels. 5(1)38.
70%
80%
90%
zed by
Pyrolysis 3,4,5‐Trimethoxytoluene
Syringol
Syringaldehyde
Methylsyringol
MethoxyeugenolFerulic acid
Guaiacyl Lignins
Sy gy g s
5 pools of lignin monomers identifiable
30%
40%
50%
60%
of Aromatics Volatiliz
Guaiacol
Eugenol
Creosol
Acetoveratrone
(E)‐Isoeugenol
4 i l i l
Ferulic acid (or guaiacyl lignin)
monomers identifiable
Can be used to:• Estimate S/G
0%
10%
20%
Switchgrass (cv. Pioneer Hybrid Inbred bm1 Inbred bm3 Sugar Maple
Fraction
o 4‐vinylguaiacol
Phenol
4‐vinylphenol
p-coumaric acid (or other lignin)
• Characterize changes to lignin during pretreatment
Cave‐In‐Rock) Stover Stover Stover (Acer saccharum)
Herbaceous Monocots (Grasses) Woody dicot
S l bili d f l i l d
Correlating Digestibility to Plant Cell Wall Properties
Solubilized ferulate is correlated to glucan digestibility
Pyrolytic 4‐vinylguaiacolPyrolytic 4 vinylguaiacol (Ferulate/Lignin): Correlated to lignin content and glucandigestibilitydigestibility
800000
28
G monomer S monomerDetermine relative quantities of (0.38)(0.26)(0.99)(0.86)(S/G)
Lignin Methods: Thioacidolysis GC/MS
500000
600000
700000Determine relative quantities of G, S, H lignins
Foster et al., 2010. J Vis Exps, 37
bm1bm3
0 6
0.7
0.8
0.9
1.0
Mon
omers Re
leased
lysis
p‐Hydroxyphenyl (H)
Guiacyl (G)
Syringyl (S)
( )( )( )( )( )
100000
200000
300000
400000
Cleavage of alkyl‐aryl ethers in ligninQuantification of derivatized 0.2
0.3
0.4
0.5
0.6
ive Fraction
of Lignin M
by Thioacido
08.05 8.15 8.25 8.35 8.45 8.55 8.65 8.75
fragments by GC‐MSProblematic in grasses?
• Highly condensed ligninsGC MS
Time (minutes)
0.0
0.1
Pioneer Hybrid Stover
bm1 Inbred Stover
bm3 Inbred Stover
Switchgrass (cv. Cave‐In‐Rock)
Relati
GC-MS quantification
BSTF
CPG-SM
EtSH, BF3
β-O-4 linkageLi et al. (2012). BiotechnolBiofuels. 5(1)38.
29Quantitative ThioacidolysisDetermines the fraction of lignin involved in only ether linkagesDetermines the fraction of lignin involved in only ether linkagesThioacidolysis 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/wLi et al. (2012). BiotechnolBiofuels. 5(1)38.
100%Cu(bpy)-Catalyzed AHP 30
Hybrid PoplarCatalyst significantly improves subsequent
80%
No AHP, no xylanaseAHP, no xylanaseAHP, xylanaseAHPwith Cu(bpy) no xylanase
Catalyst significantly improves subsequent enzymatic conversion of cellulose
60%
version
AHP with Cu(bpy), no xylanaseCu(bpy) AHP, xylanase
40%
Glucan Co
nv
20%
0%24 48 72
Hydrolysis time (h)Li et al. (accepted 2012). Biotechnol Bioeng.
Oxidative Depolymerization of CelluloseO R
COOHOH
R HO
AHP depolymerizes cellulose (filter paper)
OO
COOHOHHO
C6 oxidation
?(filter paper)
Introduces ‐COOHO
OO
R
OHOH
OHOH
RHO
HO
Cellulose
O
HO
R
OH
OH
HO
on+
O
OHRHO
?
?
OOH
OH
R HO
COOH
C1 oxid
ation+OH OH
O R
OH
OH
HO
OC4 oxidation
+?
Li et al. (accepted 2012). Biotechnol Bioeng.
www.glbrc.org
SummaryW t d ti d t ti b l t d
32
Water adsorption and retention can be correlated to enzymatic conversion
Correlating digestibility to plant cell wall propertiesNegative relationship between Klason lignin and di ibili di ll lldigestibility across diverse cell wall typesFerulate content quantified by Py‐GC/MS in residual cell wall and LC/MS in hydrolysates can be correlated towall and LC/MS in hydrolysates can be correlated to lignin removal and digestibility
Oxidative cellulose depolymerization and –COOH p yintroduction demonstratedImpact on digestibility improvement?
AcknowledgementsResearch Group:
Dan WilliamsMarc Hansen(†) Ryan Stoklosa
Dr. Tongjun LiuCharles Chen
Collaborators:Eric Hegg, MSUChris Saffron MSU
David Hodge Muyang LiAlex Smith(†)y
Zhenglun LiChris Saffron, MSUJonathan Walton, MSUMichael Hahn, U. GeorgiaTrey Sato U WisconsinTrey Sato, U. WisconsinArt Ragauskas, Georgia
Funding:DOE, BER DE‐FC02‐07ER64494,DOT, NE Sun Grant InitiativeNSF, DUE #0757020
Not pictured: pElizabeth Häggbjer, Natassa Christides, Genevieve Gagnier
