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

IntroductionLignocellulosic biomass

Subcritical water ?

ObjectiveEffect of subcritical water treatment

Experimental studySubcritical water treatment in continuous flow reactor

Enzymatic digestibility

Results

Conclusion

National biofuel action plan

3

New US Renewable Fuels Standard

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5

10

15

20

25

30

35

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2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022

Bill

ion

gallo

ns

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 :

Cellulose38 - 50%

23 - 32%

Lignin15 - 25%

Other 5 -15%

(Extractives, Ash etc)

Hemicellulose

http://www.nrel.gov

SwitchgrassCorn Stover Bagasse

Wood chips

Lignocellulosic Biomass

4

5

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

Pretreatment methods

6

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

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

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)

Enzymatic hydrolysis of cellulose by cellulase enzyme

9

Amorphous domain(Substrate for Endo-glucanase)

Reducing ends(Substrate for Exo-glucanase)

β-Glucosidase

Glucose

Cellulose

Cellobiose

Reducing end

Factors effecting enzymatic reactivity

Crystallinity of cellulose

Degree of polymerization

Accessibility

Polymorph of celluloseSix known polymorphs

Cellulose; I, II, III1, IIIII, IVI, and IVII

10

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)

11

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

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.

Results: Subcritical water treatment

15

0

15

30

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)

Effect on the crystallinity of cellulose after the treatment

16

75

77

79

81

83

85

0 3 6 9 12 15

Cry

stal

linity

(%)

lnRo

crystallinity for cellulose was determined using XRD pattern (Segal et al., 1959)

Removal of amorphous region increases crystallinity

Enzymatic reactivity at low enzyme loading

17

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

Total hydrolyzable cellulose at high enzyme loading

18

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

0

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 ?

Effect of temperature on degree of polymerization

19

332

296

247248

119

75

125

175

225

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

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

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

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

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.

23

Thank you !!

24

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)