ACS BIOT 148 Inhibitors final mar 14 16 - purdue.edu · BIOT 2016 251st National ACS Meeting, San...
Transcript of ACS BIOT 148 Inhibitors final mar 14 16 - purdue.edu · BIOT 2016 251st National ACS Meeting, San...
BIOT 2016251st National ACS Meeting, San Diego, CA
Liquid Hot Water Pretreatment InhibitorsEduardo Ximenes1, Youngmi Kim2, Cristiane Farinas3
Michael R. Ladisch1
1Laboratory of Renewable Resources EngineeringPurdue University
2Agricultural Engineering TechnologyU. Wisconsin, River Falls
3Embrapa InstrumentacaoSao Carlos, Brazil
March 14, 2016
BIOT 153: Biofuel and Biobased Chemical Production: Biomass Pretreatment and Hydrolysis Presiding: Marcus Foston and Michael Resch
Acknowledgements
Purdue University Colleges of Agriculture and Engineering Agricultural and Biological EngineeringHatch Act Projects 10646 and 10677
US Department of Energy Cooperative Agreement GO18103, GO17059‐16649, 0012846; DE‐SC0000997
Embrapa: Dr. Cristiane Farinas (U. Sao Carlos, Brazil)
Types and Sources of Biomass
a. Agricultural residuesGlobal, US Midwest, China, Brazil
b. WoodUpper Midwest US, Canada (hardwoods)Southeast US (softwoods)Europe (softwoods, hardwoods)
c. Purposely grown energy crops (in the future)Brazil (energy cane and sugar cane)US (switchgrass, poplar)Africa (grasses, energy crops)
Composition of Lignocellulosic Biomass
Glucan44%
Xylan17%
Lignin32%
Acetyl groups3%
Ash2%
Extractive2%
Hardwoods Similar Compositions:
Corn residueSugar Cane BagasseSwitch grass
Lignin a big factor
Pretreatment and Cost Effective Enzymes are KeyPretreatment
makes substrate susceptibleincreases yield of fermentable sugars (and ethanol)in theory, reduces required enzyme dose for hydrolysis
Mosier et al, 2005
Liquid Hot Water Pretreatment vs Steam Explosion
Both use water (liquid or steam)Steam explosion
- may add acid (to hydrolyze xylan)- releases pressure through explosive decompression
Liquid hot water (LHW) cooking- carried out under pressure (heat up to cool down)- conditions keep water in liquid phase- pH at 4 to 7; lignocelluloses self-buffer to this pH- no chemicals added- temperatures between 120 and 215 C
Pretreatment also releases or forms inhibitors
Released: xylo‐oligosaccharides, phenols, tannic & acetic acids Formed: aldehydes (furfural, hydroxy‐methyl furfural)
Degree
of Inh
ibition
Pretreatment Type
Acid Neutral AlkalineNovo, 2009
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00 6 12
8g/L Ethanol
Fermentation Time
Hydrolyzed XO only
Hydrolyzed xylo‐oligosaccharides (XO)and bio‐abatement
Control –no treatment
Bio‐abatement only
Bioabatement removes acetic acid, furfural, hydroxymethylfurfural and phenolicsHydrolysis with Bio‐mimetic removes xylo‐oligomers (XO)
Fermentation of glucose to ethanol by yeast
Cao et al., 2013; 2015; Nichols, Kim et al., 2016
Enzyme Inhibitors Correlate to Color
Wash Liquid from Liquid Hot Water Pretreated Hardwood
Methanol and Acetone Samples for LC analysis
Retention Time
AU at 2
80 nm
Reverse Phase Chromatography of Compounds Recovered by XAD‐7 and Back‐extracted into Methanol
Kim et al, 2013
Tannic acid
O
OCH3
OH
VanillinO
OH
H3CO OCH3
Syringaldehyde
OH
OH
O
4-hydroxybenzoic acid
O
OH
Transcinnamic acidO
OH
HO
ρ-coumaric acidO
OH
HO
CH3O
Ferulic acid
Phenolic molecules inhibit enzyme hydrolysis of cellulose
Mechanisms of inhibition by phenols
Oligomeric phenolics may inactivate cellulases by reversibly complexing with them;
Enzyme‐tannic acid interactions show distinct specificity even in the same family of cellulases;
Kinetic analysis : inhibition of a GH11 endo‐β‐1,4‐xylanase from Thermobacillusxylanilyticus followed a “multi‐site,” non‐competitive inhibition mechanism, indicating that more than one aromatic molecule interacts with the enzyme molecule to induce its complete inactivation
Boukari et al. J. Mol. Catal. B: Enzym., 72, 130–138 (2011)Tejirian and Xu. Enzyme Microb. Technol., 48, 239–247 (2011)Olsen et al. Enzyme Microb. Technol., 49, 353–359 (2011)
0
20
40
60
80
100
Dea
ctiv
atio
n (%
)
p-NPGase activity
Cellobiase activity
Tannic acid
Gallic acid
Cinnamicacid
Ferulicacid
ρ-Coumaricacid Sinapic
acid
VanillinSyringaldehyde
4-Hydroxybenzoicacid
0
20
40
60
80
100
Dea
ctiv
atio
n (%
) p-NPGase activity
Cellobiase activity
Tannic acid
Gallicacid
Cinnamicacid
Ferulicacid
ρ-Coumaricacid
Sinapicacid
VanillinSyringaldehyde
4-Hydroxybenzoicacid
β‐glucosidase in A. niger (Novozyme 188)
β‐glucosidase in T. reesei (Spezyme CP)
Phenols are also major deactivators of β‐glucosidases
Ximenes et al, 2010, 2011
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Loss of β‐G suppresses cellulose hydrolysis
Cellulose Cellobiose Glucose
Cellulase(CBH, EG) β‐glucosidase
a cascade of enzyme inhibition is triggered by β‐glucosidase depletion due to its binding onto lignin
Cellobiose builds up and inhibits cellulase activity
Ladisch et al, 1976; Gong et al. (1977), Hong et al. (1981)
1. Liquid hot water pretreatment modifies lignin structure and exposes it to a greater extent
2. Lignin binds cellulase non‐productively
3. Control of non‐productive binding enhances the enzymatic hydrolysis of cellulose
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Hypotheses for Inhibition by solid lignin
A
B
C
D
E
F
5 µm 5 µm
4 µm
5 µm
4 µm 2 µm
Untreated log Ro = 11.56 log Ro = 12.51
Morphological changes of pretreated wood18
Syringyl‐Guaiacyl (S‐G) type hardwood lignin → G‐rich type (Softwood like)
• Increase in AIL/ASL ratio• Increase in glass transition temperature• Decrease in methoxyl (OCH3)groups
Study of modifying lignin structure through genetic engineering:• Decrease in lignin content (Chen and Dixon, 2007) • S/G ratio regulation (Chapple et al. 2007; Bonawitz et al. 2014) • Cellulose hydrolysis in LHW pretreated transgenic plant with S‐rich lignin > G‐
rich lignin (Li et al. 2010, Bonawitz et al, 2014)
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Pretreatment modifies lignin structure
S‐lignin G‐lignin
Lignin(from Kim severity)a
EmaxMaximum capacity(mg‐protein/g‐lignin)
KAffinity
(mL/mg‐protein)
Kp = Emax× KPartition coefficient
(mL/g‐lignin)
10.44 37.0 2.8 105.111.39 44.8 2.9 130.211.56 44.5 3.0 132.912.51 36.6 4.9 178.2
Lignin from higher severity pretreatments adsorbs enzymes more strongly
E ads = adsorbed enzyme (mg/g‐lignin)E free = free enzyme in supernatantE max = maximum adsorption capacityK = Affinity constant
aKim et al. (2013)
Glass transition temperature (Tg)
Lignin severity Tg (°C) Range (°C)
10.44 171.9 ± 0.1 150‐18811.39 170.8 ± 0.1 150‐18511.56 174.2 ± 0.5 155‐18812.51 179.7 ± 0.1 160‐205
Differential scanning calorimetry (DSC)
Scanning temperature range: 0‐220°C
Heating rate: 10°C/min
Tg: a mid‐point of heat capacity changes
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MwEnzyme control
Free protein in supernatant
SDS‐PAGE analysis of proteins in supernatant after contact with lignin 22
Shows adsorption of β‐glucosidases (78‐94 kDa)
CBH I (54 kDa)
CBH: Cellobiohydrolase, EG: Endoglucanase, XYN: Xylanase
CBH II, EG I (48‐50 kDa)EG II (44 kDa)
EG III‐V, XYN I‐III (24‐38 kDa)
Enzyme: Ctec2
Addition of BSA to Enzyme High Yield at Lower Enzyme Loading and High Severity
BSA Added
No BSA Added
No Pretreatment
Cellic Ctec2 of 5 FPU (8 mg protein)/g glucan, pH 4.8, in 50 mM citrate buffer, 50°C, 200 rpm for 168 hrs. Equivalent to 3.5 mg/g total solids prior to pretreatment
Kim et al, 2015
24Diluting Enzyme with Non Catalytic Protein Increases Yield
As specific activity decreases, conversion increases
Cellulase loading fixed at 1.8 FPU / g glucan, equivalent to 1.3 FPU / g pretreated solids
Kim et al, 2015
Summary
LHW pretreatment modifies lignin structure to be more condensed and S‐deficient form
Enzyme is lost due to non‐productive binding onto lignin
‐ More condensed lignin (higher Tg) adsorbs enzymes more strongly
‐ Loss of β‐glucosidase from enzyme mixture triggers severe inhibition
‐ Blocking lignin surface decreases enzyme adsorption and improves
glucose yield
‐ Enzyme required for hydrolysis is as low as 1.3 FPU / g solids
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Conclusions
Cellulose surface area made accessible by pretreatment enhances hydrolysis yield
Lignin shields cellulose from hydrolysis and interferes with enzyme action both before and after pretreatment
Inhibition / deactivation varies with pretreatment severity.
Major reductions in amounts of enzyme needed for cellulose hydrolysis are possible by:
blocking enzyme adsorption by lignin and removing soluble inhibitors derived from lignin