Post on 18-Oct-2020
transcript
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
-8
-7
-6
-5
-4
-3
-2
-1
0
1
2
0 10 20 30 40 50 60 70 80Time / min
-8
-7
-6
-5
-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
-8
-7
-6
-5
-4
-3
-2
-1
0
0 10 20 30 40 50 60 70 80Time / min
-8
-7
-6
-5
-4
-3
-2
-1
0
0 10 20 30 40 50 60 70 80Time / min
Mas
sx10
3 / g
m- 2
-8
-7
-6
-5
-4
-3
-2
-1
0
0 10 20 30 40 50 60 70 80Time / min
-8
-7
-6
-5
-4
-3
-2
-1
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
-8
-7
-6
-5
-4
-3
-2
-1
0
1
2
0 10 20 30 40 50 60 70 80Time / min
-8
-7
-6
-5
-4
-3
-2
-1
0
1
2
0 10 20 30 40 50 60 70 80Time / min
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)
-8
-7
-6
-5
-4
-3
-2
-1
0
1
2
0 10 20 30 40 50 60 70 80
Time / min
Mas
sx10
5/ g
m-2
Batch mode
0.00056%
0.00167%
0.005%
-8
-7
-6
-5
-4
-3
-2
-1
0
1
2
0 10 20 30 40 50 60 70 80
Time / min
Mas
sx10
5/ g
m-2
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
-8
-7
-6
-5
-4
-3
-2
-1
0
1
2
Mas
sx10
5/ g
m-2
0 10 20 30 40 50 60 70 80Time / min
28 °C
33 °C38 °C
Batch mode
-8
-7
-6
-5
-4
-3
-2
-1
0
1
2
Mas
sx10
5/ g
m-2
pH (0.005%, 38°C)
-8
-7
-6
-5
-4
-3
-2
-1
0
1
2
0 10 20 30 40 50 60 70 80
Time / min
Mas
sx10
5/ g
m-2
Batch mode
pH 10
pH 7
pH 4.5
-8
-7
-6
-5
-4
-3
-2
-1
0
1
2
0 10 20 30 40 50 60 70 80
Time / min
Mas
sx10
5/ g
m-2
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
-40
-30
-20
-10
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