Orlando J. Rojas Department of Forest Biomaterials Sci.& Eng.,

NC State University, Raleigh, NC

Crystallinity and Hydrolysis of Cellulose Nanofilms

2008 International conference on Nanotechnologyfor the Forest Product Industry

The Generation and Stability of Organic Films on Surfaces Nonwovens Cooperative Res. Center

Boundary Layer LubricationNational Textile Center

Electrokinetic Behavior of Polyelectrolytes and Surfactants throughout Tortuous Micro/NanoporesACS - Petroleum Research Fund,Nippon paper, USP

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0

1

2

0 10 20 30 40 50 60 70 80Time / min

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-4

-3

-2

-1

0

1

2

0 10 20 30 40 50 60 70 80Time / min

Mas

sx10

3 / g

m-2

Himmel et al., 2000

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0

0 10 20 30 40 50 60 70 80Time / min

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0 10 20 30 40 50 60 70 80Time / min

Mas

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3 / g

m- 2

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0 10 20 30 40 50 60 70 80Time / min

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0

0 10 20 30 40 50 60 70 80Time / min

Mas

sx10

3 / g

m- 2 Enzymatic Activity via

Piezoelectric SensorsNC Biotech Center, Novozymes, USDA

Surface modification(ATRP, TEMPO)

USDA

Lignocellulosics as Precursors of Biopolymer Structures

USDA-NRI

Enzyme activity

Polyampholytes

Gang Hu

Deusanilde Silva

Youssef Habibi

Justin Zoppe

Xiaomeng Liu

Junlong

Kelley Spence

Maria Peresin

Ning

Hongyi Liu

Fei Shen (Carbonell)

Takashi Yamagushi

Ingrid Hoeger

Wood impregnation with complex fluids

Sun Grant

Cellulose nanocrystals and MFC

Hofmann Fellowship and USDA

Lignin and composites -electrospinning

Cellulose Tribology

Adsorption & Electrokinetics

Cellulose nano-structures and

applications

Surface phenomena

Surface Functionalization

Impregnation Interfaces

Colloids

Biomate

rials

Forest Interactions

10- 1

10- 2

10- 3

10- 4

0 0.4 0.8

INTE

RFA

CIA

L TE

NSI

ON

(mN

/m)

1.2 1.6 2.0 2.4 2.8 3.2 0.8

10- 1

10- 2

10- 3

10- 4

0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 0.8

Formulation Variable

gmogmw

Water surface tension

b)

Stimuli-responsive Surfaces and Pathogen Detection National Center for Food Protection and Defense

25 °C

Dis

sipa

tion

(x10

6 )PN

IPAM

bru

shes

1.4

1.6

1.8

2.0

2.2

2.4

0 100 200 300 400Time, s

100mM NaCl

20mM NaCl

Xavier Turon

Colloids and Interfaces Groupwww4.ncsu.edu/~ojrojas

Conclusions

HOOH

H3

OOH

OCH3

OCH3

O

OCH3

H3CO

O

O

OC

O

OCH3

OCH3

OCH3

OH

O

HO

H3CO

HO

HO

H3CO

OCO

O

OH

OCH3

OCH3

OCH3

HOOH

H3

OOH

OCH3

OCH3

O

OCH3

H3CO

O

O

OC

O

OCH3

OCH3

C 3

OH

O

HO

H3CO

HO

O

HO

H3CO

HO

OO

OH

H3

OO

OH

H3

O H

HO

H3CO

OCO

O

OH

OCH3

OCH3

HO

H3CO

OCO

O

OH

OCH3

OCH3

OH

H3

OH

H3OCO

O

OCH3OCO

O

OCH3

OH

H3

OH

H3

O H

HO

H3CO

OCO

O

OH

OCH3

OCH3

HO

H3CO

OCO

O

OH

OCH3

OCH3

Lignin

Cellulose

& hemicelluloses

Energy

Cryo-fracture deep-etch EMC. Haigler, NCSU

Cellulose

Nanofiber

bundles

6 Assembly proteins (rosette) which produces cellulose nanofibers

~28nm

Top-down (deconstruction)

Objective

Quantification (in-situ and real time)…

• Interactions enzyme – substrate

(binding and hydrolysis rates)

Cellulosic FibersTopographicheterogeneity!

Chemicalheterogeneity!

Conclusions

Ligno-cellulose

Ultrathin polymer

films

Adsorption

Swelling

Langmuir, 24(8), 3880-3887 (2008)Bioresources 3(1): 270-294 (2008)Materials, Chemicals, and Energy from Forest Biomass, ACS Symposium Series 954, 478-494 (2007).

Hydrolysis

Substrate: Cellulose model surfacesSubstrate: Cellulose model surfaces

• Nanofibrillar cellulose, NFC (native cellulose):Cellulose nanofibrils disintegrated from delignified SW sulphite pulp (high-pressure fluidizer)..

• Langmuir film, LF (regenerated cellulose from TMSC). Layer by Layer structure.

• Spin coated film, SC (Regenerated cellulose): Microcrystalline cellulose dissolved in NMMO.

• Cellulose nanocrystals, CNx (crystalline cellulose). From hydrolysis of filter paper.

Thin Films of Cellulose

2x2 μm

Spin coated cellulose film, SC

Cellulose nanocrystals CNx

Thin Films of Cellulose

5x5 μm

1x1 μm5x5 μm

30x30 μm

2x2 μm

5x5 μm

Nanofibrillar cellulose NFCSpin coated cellulose film, SC

Langmuir-Blodgett cellulose film, LBElectrospun cellulose nonwoven

Conclusions

Sensor

~Adsorption

Time →Det

ecte

d si

gnal

Adsorption occurs

resonant coupling to surface plasmons

~Adsorption

Time →

Cry

stal

s Le

ngth

Adsorption occurs

Quartz Crystal MicrobalanceQuartz Crystal Microbalance

Quartz Crystal MicrobalanceQuartz Crystal Microbalance

~Viscoelasticity

Alternating potential

Time →Cry

stal

Len

gth

Circuit opened

Piezoelectric Sensor• quartz / gold

• quartz / gold / silica

active electrode

counter electrode

quartz disc

active side

contact side

active side

contact sideCellulose on silica/goldLignin on silica (LB, SC)Electrospun fibers

Mass Sensitivity

in air (1 bar) ~0.2 ng/cm2

in water (25 °C) ~0.9 ng/cm2

(picture from Q-sense)

Enzymatic Activity via Piezoelectric Sensors

Enzyme used in this work was a commercial cellulase mixture (CelluclastTM) which is available as an aqueous solution (≥700 U/g). It is from Trichoderma reesei fungus and contains endoglucanases exoglucanases, cellobiohydrolases, and β-glucosidases. It is used for the efficient saccharification of lignocellulosic materials with maximum activity in mild acidic conditions (pH of ca. 5), and temperatures between 50-60°C

Enzyme used in this work was a commercial cellulase mixture (CelluclastTM) which is available as an aqueous solution (≥700 U/g). It is from Trichoderma reesei fungus and contains endoglucanases exoglucanases, cellobiohydrolases, and β-glucosidases. It is used for the efficient saccharification of lignocellulosic materials with maximum activity in mild acidic conditions (pH of ca. 5), and temperatures between 50-60°C

(Himmel’s group)

0

20

40

60

80

100

120

20 40 60Time (min)

Freq

uenc

y (f 3

/3)

-20

Enzymatic Activity via Piezoelectric Sensors

Quartz crystalQuartz crystal

Quartz crystalQuartz crystal

Cellulose filmCellulose film

Cellulose filmCellulose film

Cellulose filmCellulose film

Quartz crystalQuartz crystal

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Mas

sx10

3/ g

m-2

Enzymatic Activity via Piezoelectric Sensors

Quartz crystalQuartz crystal

Quartz crystalQuartz crystal

Cellulose filmCellulose film

Cellulose filmCellulose film

Cellulose filmCellulose film

Quartz crystalQuartz crystal

nfC

nf

fnfft

m qqqq Δ−=Δ−

=Δ−

=Δ 20 0

2

νρρ17.8 ng cm−2 Hz−1quartz density

Quartz shear wave velocitythickness of the quartz crystal

Enzyme Concentration (pH 4.5, 38°C)

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Batch mode

0.00056%

0.00167%

0.005%

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Time / min

Mas

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Batch mode

0.00056%

0.00167%

0.005%

Temperature (0.005%, pH 4.5)

T: Criquet, S. J. Microbiological Methods 50: 165 (2002)Adsorption: Kim and Hong, Biotechnol. Lett. 22: 1337 (2000)

0 10 20 30 40 50 60 70 80Time / min

28 °C

33 °C38 °C

Batch mode

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0 10 20 30 40 50 60 70 80Time / min

28 °C

33 °C38 °C

Batch mode

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pH (0.005%, 38°C)

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Mas

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Batch mode

pH 10

pH 7

pH 4.5

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Mas

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Batch mode

pH 10

pH 7

pH 4.5

-50

0

50

100

150

200

250

-3

-2

-1

0

1

2

3

4

3

ΔD3 X 10

6

0 50 100 150 200 250 300Time (min)

ΔF/3

(Hz)

V50

A B

C

C

B

A

Energy Dissipation Signature

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21=Δ

ΔD-Δf Plot

Conclusions

Effects of Substrate in Enzyme ActivityEffects of Substrate in Enzyme Activity

Nanofibrillar cellulose, NFC Nanofibrillar cellulose, NFC

Native celluloseAmorphous and crystalline regionsResidual Hemicelluloses3D nanofiber web

5x5 μm 1x1 μm

Langmuir film, LFLangmuir film, LF

Regenerated celluloseAmorphous, some crystalline regions (cellulose II)Low CrI, but highly structured self-assembled layers Flat, reduced roughness

5x5 μm 1x1 μm

Spin coated film, SCSpin coated film, SC

Regenerated celluloseAmorphous with some crystalline regionsLow CrI, random structureReduced roughness (higher than LB)

5x5 μm 1x1 μm

Cellulose nanocrystals, CNxCellulose nanocrystals, CNx

HCl hydrolyzed filter paperCrystalline structure (cellulose I)High CrIRough surface

5x5 μm 1x1 μm

Substrates (before incubation)Substrates (before incubation)

NFC

Spin-coated (NMMO) Nanocrystals, CNx (cast on PVAm)

LB

5×5 μm

Substrates (after incubation)Substrates (after incubation)

NFC

Nanocrystals, CNx (cast on PVAm)

LB

Spin-coated (NMMO)5×5 μm

QCM Fingerprints: effect of the nature of the substrate ΔF3/3 (Hz)

-50

0

50

100

150

200

250

0 1 2 3 4 5 6Time (min)

-2

0

2

4

6

8

10D (10-6)

a)

ΔF3/3 (Hz)

-50

0

50

100

150

200

250

0 5 10 15Time (min)

D (10-6)

b)

-2

0

2

4

ΔF3/3 (Hz)

-50

0

50

100

150

200

250

0 10 20 30 40 50 60Time (min)

D (10-6)

c)

70-2

0

2

4

ΔF3/3 (Hz)

-50

0

50

100

150

200

250

0 60 120 180 240 300 360Time (min)

D (10-6)

d)

-2

0

2

4

NFC LB

SC CNx

Dynamics of Binding and Hydrolysis (Δf)

1⎟⎟⎟⎟

⎠

⎞

⎜⎜⎜⎜

⎝

⎛

−−=Δ τt

eMf MAX

-50

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0

0 1 2 3 4Time (min)

ΔF3/3

(Hz)

-50

-40

-30

-20

-10

0

0 1 2 3 4Time (min)

ΔF3/3

(Hz)

1

50

⎟⎟⎠

⎞⎜⎜⎝

⎛+

−+=Δ−CtV

e

ABAf

020406080

100

120140160180200

0 20 40 60 80 100 120Time (min)

ΔF3/3

(Hz)

020406080

100

120140160180200

0 20 40 60 80 100 120Time (min)

ΔF3/3

(Hz)

Enzyme dose & Binding (NFC case)

C (%) 0.50% 0.25% 0.05%

MMAX -95 -70 -34

τ 0.7 1.3 1.7

NFC:

Saturation

-100

-80

-60

-40

-20

0

0.00% 0.25% 0.50%

Δf

Enzyme concentration

Topography & Binding

• NFC: 3-D network. High surface area

• LB: Flat.

• SC: Flat (rougher than LB).

• CNx: 3-D (rods). Surface area?

Film NFC LB SC CNx

MMAX -95 -52 -31 -29.6

τ 0.7 0.7 1.9 0.5

Crystallinity effect?

MFC

SC CNx

LB

Substrate Hydrolysis: Thickness (& total film mass)

Film NFC LB SC CNx

“B” 115 142 232 120

Incomplete hydrolysis (AFM)

“Sensed”thickness

“Actual”thickness

Max (Hz)

Film thick. NFC LB SC CNx

(nm) 12 15 24 13

(nm) <10 ~15 20-30 20-30 (50)

Substrate Hydrolysis: Hydrolysis rate

Time to exhaustion – overall kinetics

Hydrolysis rate

Film NFC LB SC NCx1/C x 100

(hydrolysisrate) 220 63 16 0.8

ΔF3/3 (Hz)

-50

0

50

100

150

200

250

0 60 120 180 240 300 360Time (min)

CNx

SC

LS

NFC

Current work: Purified enzymes are being tested to investigate further the effect of enzyme composition.

Acknowledgments-Collaborators in Helsinki University of Technology: Monika Osterberg, Susanna Ahola, & Janne Laine.

- North Carolina Biotechnology Center

- Novozymes of North America

-USDA National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant # 2007-3550418290