Evaluation of Pretreatments in the Enzymatic Hydrolysis of Cellulose
Cellulose hydrolysis in subcritical water
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Enhancement of Enzymatic Digestibility of Microcrystalline Cellulose by Treatment in Subcritical Water
Sandeep Kumar, Rajesh Gupta, Y.Y. Lee, and Ram B. Gupta*
Department of Chemical Engineering, Auburn University, Auburn, AL
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Outline
IntroductionLignocellulosic biomass
Subcritical water ?
ObjectiveEffect of subcritical water treatment
Experimental studySubcritical water treatment in continuous flow reactor
Enzymatic digestibility
Results
Conclusion
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National biofuel action plan
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New US Renewable Fuels Standard
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2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022
Bill
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gallo
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Corn starch based Cellulosic Any other bio fuel
Frank D. Haagensen, Novozymes NA, Inc., Presentation in Auburn University, March 5th, 2008
Energy security and renewable fuel
Introduction :
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Cellulose38 - 50%
23 - 32%
Lignin15 - 25%
Other 5 -15%
(Extractives, Ash etc)
Hemicellulose
http://www.nrel.gov
SwitchgrassCorn Stover Bagasse
Wood chips
Lignocellulosic Biomass
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Lignocellulosic
biomass PretreatmentEnzymatic hydrolysis
Ethanol
Pretreatment enhances
•Rate of production of monomeric sugars
•Yield of monomeric sugars
Pretreatment to improve cellulose accessibility
Ethanol from lignocellulosics
Fermentation
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Pretreatment methods
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Physical Physio-chemical Chemical
1. Mechanical Comminution
2. Irradiation
1. Steam explosion2. SO2 / CO2 Catalyzed Steam
explosion
3. Ammonia fiber explosion
1. Acid / alkali
2. Organosolv
3. Subcritical / hot compressed water
Water is a non-toxic, environmentally benign and inexpensive
Critical point of water Tc= 374 oC, Pc= 22.1 MPa, ρ c= 0.375 g cm-3
DecreasedDensityDielectric constantViscosity
Increased Ionization constantDiffusivity
Subcritical water properties
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Objective
Effect of temperature and residence time on
cellulose structure in a subcritical water treatment
process
Changes in enzymatic reactivity after subcritical
water treatment
Factors affecting enzymatic reactivity of cellulose
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Cellulose hydrothermal reaction pathway
Water-soluble products (n = 2 to 8)
Glucose
Hydrolysis products
Degradation products
.
Non reducing end
Reducing end
Bobleter, O., 1994. (Prog. Polym. Sci., )19, 797–841.
(Glycoaldehyde, Anhydroglucose, HMF, Furfural, Organic acid etc)
(Oligomers, Cellobiose)
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Enzymatic hydrolysis of cellulose by cellulase enzyme
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Amorphous domain(Substrate for Endo-glucanase)
Reducing ends(Substrate for Exo-glucanase)
β-Glucosidase
Glucose
Cellulose
Cellobiose
Reducing end
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Factors effecting enzymatic reactivity
Crystallinity of cellulose
Degree of polymerization
Accessibility
Polymorph of celluloseSix known polymorphs
Cellulose; I, II, III1, IIIII, IVI, and IVII
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Analytical techniques
Solids characterization
Degree of polymerization
by viscosimetry
X-ray diffraction (XRD)
Scanning electron microscope(SEM)
Fourier transform infra-red (FTIR)
Differential scanning calorimetry (DSC)
Liquid products
Total organic carbon (TOC)
High pressure liquid
chromatography (HPLC)
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Enzymatic Digestibility
NREL Laboratory Analytical Procedure (LAP #. 009)
Cellulase enzyme (brand name: Spezyme CP)
Enzyme loadings
Low enzyme loading (3.5 FPU/g of glucan), and
High enzyme loading (60 FPU/g of glucan)
pH 4.8 substrate buffer
Temperature 50 °C, 140 rpm
Samples collected after 1 hr and 24 hrs
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Experimental set-up (subcritical)
Cellulose slurry input (reactor) = 2.7 wt%
Cellulose, size 20μm
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Experimental conditions
At constant pressure (27.6 MPa) in continuous flow
Group I
200 - 275 °C and residence time(t), 3.7 to 6.2 s
Group II
300 - 315 °C and residence time, 3.4 to 5.2 s
Severity index (Ro)
14Overend, R.P., Chornet, E., 1987. (Philosophical Transactions of the Royal Society of London )A321, 523-536.
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Results: Subcritical water treatment
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0
15
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45
4 6 8 10 12
% C
onve
rsio
n
lnRo
300 - 315 °C3.4 - 5.2 s
200 - 275 °C3.7 - 4.1 s
Cellulose remained chemically stable upto 275 °C (t < 6.2 s)
Conversion (%) with severity index (R0)
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Effect on the crystallinity of cellulose after the treatment
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Cry
stal
linity
(%)
lnRo
crystallinity for cellulose was determined using XRD pattern (Segal et al., 1959)
Removal of amorphous region increases crystallinity
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Enzymatic reactivity at low enzyme loading
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0
25
50
75
0 4.1 4.5 7.6 7.9 9.1 9.4
7.9 6.2
47.2 42.2
% D
iges
tibili
ty1 h 24 h
200-275°C
lnRo
0
25
50
75
0 10.7 11.3 11.7
7.9 11.1 13.022.0
47.2 48.5 54.668.1
% D
iges
tibili
ty
300-315°C
lnRo
Digestibility increased for group II (300-315 C)samples only
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Total hydrolyzable cellulose at high enzyme loading
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0255075
100
0 4.1 4.5 7.6 7.9 9.1 9.4
75.0 74.2
% D
iges
tibili
ty
1 h 24 h
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25
50
75
100
0 10.7 11.3 11.7
45.060.1
75.090.6
% D
iges
tibili
ty
300-315°C
200-275 C
lnRo
lnRo
Decrease in degree of polymerization ?
Transformation of cellulose structure ?
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Effect of temperature on degree of polymerization
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247248
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275
325
375
180 200 220 240 260 280 300 320
Deg
ree
of p
olym
eriz
atio
n
Temperature (°C)
Residence time, 3.4 - 6.2 s
Sharp decline in degree of polymerization after 300 °C
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XRD patterns of group II (300-315 °C) samples
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10 12 14 16 18 20 22 24 26 28
Inte
nsity
Angle (2θ)
Untreated
lnRo = 11.3
lnRo = 11.7
Onset of cellulose II (Polymorph) peaks
New Peak
lnRo = 10.7
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SEM, FTIR, and DSC results
SEM image showing cracks and trenches in the treated sample
FTIR and DSC analysis
No significant changes in bonding arrangements
No changes in thermal properties21
Untreated
1µm 1µm
300 °C
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Conclusions
Subcritical water can be used as an effective pretreatment medium
for biomass without degrading or changing properties of cellulose
Cellulose maintained crystallinity untill it dissolved
Cellulose conversion to water soluble products starts above 275 °C
in continuous flow reactor (short residence time)
Presence of cellulose II polymorph was confirmed in the cellulose
treated at 300 - 315 °C, and degree of polymerization decreased
substantially at 315 °C
For highly crystalline cellulose (> 80%), enzymatic reactivity
improved only for group II samples (300 - 315 °C)22
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Acknowledgements
National Science Foundation
(grant NSF-CBET-0828269)
Alabama Center for Paper and Bioresource Engineering
Rajeev Kumar (CE-CERT, University of California,
Riverside) for help in DPv analyses.
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Thank you !!
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Hydrolysis products
64%Degradation
8%
Other compounds
28%
Liquid product composition
302 °C, 5.2 s
Majority are the hydrolysis products in liquid
(lnRo = 11.3)