Enzymatic digestibility of Cellulose enhances after pretreatment for few seconds in subcritical water.

1. Enhancement of Enzymatic Digestibility
of Microcrystalline Cellulose by
Treatment in Subcritical Water
Sandeep Kumar, Rajesh Gupta, Y.Y. Lee, and Ram B. Gupta*
gupta@auburn.edu
Department of Chemical Engineering, Auburn University, Auburn, AL

2. Outline
Introduction
Lignocellulosic biomass
Subcritical water ?
Objective
Effect of subcritical water treatment
Experimental study
Subcritical water treatment in continuous flow reactor
Enzymatic digestibility
Results
Conclusion
2

3. Introduction : National biofuel action plan
New US Renewable Fuels Standard
Energy security and renewable fuel
40
35
30
Billion gallons
25
20
15
10
5
0
2008 2009 2010 201 201
1 2 201 201 201 201 201 201 201 2020 2021 2022
3 4 5 6 7 8 9
Co rn starch based Cellulo sic A ny o ther bio fuel
3
Frank D. Haagensen, Novozymes NA, Inc., Presentation in Auburn University, March 5th, 2008

4. Corn Stover Bagasse
Switchgrass Wood chips
Lignocellulosic Biomass
Other (Extractives, Ash etc)
5 -15%
Lignin
15 - 25% Cellulose
38 - 50%
Hemicellulose
23 - 32%
4
http://www.nrel.gov

5. Ethanol from lignocellulosics
Lignocellulosic Enzymatic
biomass Pretreatment hydrolysis
Fermentation Ethanol
Pretreatment to improve cellulose accessibility
Pretreatment enhances
•Rate of production of monomeric sugars
•Yield of monomeric sugars 5

6. Pretreatment methods
Physical Physio-chemical Chemical
1. Mechanical Comminution 1. Steam explosion 1. Acid / alkali
2. SO2 / CO2 Catalyzed Steam
2. Irradiation explosion 2. Organosolv
3. Ammonia fiber explosion 3. Subcritical / hot
compressed water
Critical point of water Subcritical water properties
Tc= 374 oC, Decreased Increased
Pc= 22.1 MPa, Density Ionization constant
ρ c= 0.375 g cm-3 Dielectric constant Diffusivity
Viscosity
Water is a non-toxic, environmentally benign and inexpensive
6

7. 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
7

8. Cellulose hydrothermal reaction pathway
Non reducing end
Reducing end
Water-soluble products (n = 2 to 8) (Oligomers, Cellobiose)
Hydrolysis
products
Glucose
Degradation products
(Glycoaldehyde, Anhydroglucose, HMF, Furfural, Organic acid etc)
8
. Bobleter, O., 1994. (Prog. Polym. Sci., )19, 797–841.

9. Enzymatic hydrolysis of cellulose by cellulase enzyme
Amorphous domain
(Substrate for Endo-glucanase)
Reducing end
Cellulose
Reducing ends
(Substrate for Exo-glucanase)
Cellobiose
β-Glucosidase
Glucose
9

10. Factors effecting enzymatic reactivity
Crystallinity of cellulose
Degree of polymerization
Accessibility
Polymorph of cellulose
Six known polymorphs
Cellulose; I, II, III1, IIIII, IVI, and IVII
10

11. Analytical techniques
Solids characterization Liquid products
Degree of polymerization Total organic carbon (TOC)
by viscosimetry High pressure liquid
X-ray diffraction (XRD) chromatography (HPLC)
Scanning electron microscope(SEM)
Fourier transform infra-red (FTIR)
Differential scanning calorimetry (DSC)
11

12. 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
12

13. Experimental set-up (subcritical)
Cellulose, size 20μm
Cellulose slurry input (reactor) = 2.7 wt%
13

14. 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)
Overend, R.P., Chornet, E., 1987. (Philosophical Transactions of the Royal Society of London )A321, 523-536. 14

15. Results: Subcritical water treatment
Conversion (%) with severity index (R0)
45
% Conversion
30
200 - 275 °C 300 - 315 °C
3.7 - 4.1 s 3.4 - 5.2 s
15
0
4 6 8 10 12
lnRo
Cellulose remained chemically stable upto 275 °C (t < 6.2 s) 15

16. Effect on the crystallinity of cellulose after the treatment
85
83
Crystallinity (%)
81
79
77
75
0 3 6 9 12 15
lnRo
Removal of amorphous region increases crystallinity
16
crystallinity for cellulose was determined using XRD pattern (Segal et al., 1959)

17. Enzymatic reactivity at low enzyme loading
1h 24 h
75 200-275°C
% Digestibility
47.2 42.2
50
25 7.9 6.2
0
0 4.1 4.5 7.6 7.9 9.1 9.4
lnRo
300-315°C 68.1
% Digestibility
75 54.6
47.2 48.5
50 22.0
25 7.9 11.1 13.0
0
0 10.7 11.3 11.7
lnRo
Digestibility increased for group II (300-315 C)samples only
17

18. Total hydrolyzable cellulose at high enzyme loading
% Digestibility 200-275 C
100 75.0 74.2
75
50
25
0
0 4.1 4.5 7.6 7.9 9.1 9.4
1h 24 h lnRo
300-315°C 90.6
100
% Digestibility
75.0
75 60.1
45.0
50
25
0
0 10.7 11.3 11.7
lnRo
Decrease in degree of polymerization ?
18
Transformation of cellulose structure ?

19. Effect of temperature on degree of polymerization
375
332 Residence time, 3.4 - 6.2 s
Degree of polymerization
325
296
275 247
248
225
175
125 119
75
180 200 220 240 260 280 300 320
Temperature (°C)
Sharp decline in degree of polymerization after 300 °C
19

20. XRD patterns of group II (300-315 °C) samples
New Peak
lnRo = 11.7
lnRo = 11.3
Intensity
lnRo = 10.7
Untreated
10 12 14 16 18 20 22 24 26 28
Angle (2θ)
Onset of cellulose II (Polymorph) peaks
20

21. SEM, FTIR, and DSC results
Untreated 300 °C
1µm 1µm
SEM image showing cracks and trenches in the treated sample
FTIR and DSC analysis
No significant changes in bonding arrangements
No changes in thermal properties
21

22. 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

23. 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.
Thank you !!
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24. Liquid product composition
302 °C, 5.2 s (lnRo = 11.3)
Other
compounds
28%
Hydrolysis
products
Degradation
8%
64%
Majority are the hydrolysis products in liquid
24