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The multi-scale architecture of cellulose in plant cell wall systems investigated by small angle scattering techniques
Marta Martinez-Sanz [email protected]
The plant cell wall (PCW) is the structural component covering plant cells, providing a number
of functions:
Strength to support the plant
Rigidity to fix cell shape
Flexibility to enable tissue growth
Porosity, protection against environmental stress, signaling and sensing…
Shape, growth rate and resistance of plants
The Plant Cell Wall
Hierarchical assembly of cellulose in PCWs
Need to combine different characterisation techniques to cover the whole size range
Cellulose crystallites
(dimensions, allomorph Iα/Iβ)
Cellulose microfibrils
(cross-section, crystalline, amorphous
and paracrystalline cellulose
localization)
Microfibril aggregates
(bundles/ribbons)
(cellulose interactions with PCW
components)
Cellulose in the plant cell wall
Microscopy techniques involve specimen preparation (often drying) possible structural changes
Scattering techniques powerful tool to characterise native PCW (partially hydrated)
Cellulose in the plant cell wall
Based on interactions between incident radiation (light, X-ray, neutrons) and particles
By analysing the scattered radiation we can obtain information about size, shape,
orientation and particle correlations.
Wide angle scattering (WAS): High q d ~ 0.1-1 nm
Small angle scattering (SAS): Low q d=1-100s nm
Ultra-small angle scattering (USAS): Low q d=100s nm-10 µm
Modification of scattering angle (θ) q range size range
Incident-Scattered radiation
Scattering vector:
Real-space dimension
Characterization of PCWs by SAS techniques
SANS SAXS Size range 0.004 Å-1 < q < 0.7 Å-1 (d ≈ 1-150 nm) 0.02 Å-1 < q < 0.3 Å-1 (d ≈ 2-30 nm)
ρ crystalline cellulose (1010 cm-2) 1.87 14.46
ρ paracrystalline cellulose (1010 cm-2) 1.77 13.65
ρ amorphous cellulose (1010 cm-2) 1.73 13.38
ρ D2O exchanged cellulose (1010 cm-2) 3.66 14.19
ρ Arabinoxylan (1010 cm-2) 1.62 12.64
ρ Xyloglucan (1010 cm-2) 1.62 12.65
ρ crystalline d-cellulose (1010 cm-2) 7.59 13.62
ρ H2O (1010 cm-2) -0.56 9.47
ρ D2O (1010 cm-2) 6.38 9.37
physical
density
Characterization of PCWs by SAS techniques
SANS enables modification of cellulose ρ by H/D exchange
- Soaking samples in D2O or H2O/ D2O mixtures (labile OH groups replaced by OD)
- Deuterium labelling (C6H10O5 C6D10O5)
PCW structure deconstruction: Progressive removal of PCW components and cellulose
isolation.
Application: Lignocellulosic biomass for the production of biofuels
Bottom-up approach: Use of PCW analogues to mimic the biosynthesis process
Bacterial cellulose as a model system. Incorporation of PCW components into the culture media
Application: Investigation of the biosynthesis process and roles of different PCW components
Approaches to study the structure of PCW
Investigation of the interaction mechanism of cellulose with PCW
matrix components:
- BC hydrogels with AX and XG
- BC hydrogels with pectins (solutions and Ca2+ gels)
- dBC hydrogels with AX, XG and MLG
MODEL SYSTEMS: Highly hydrated (~98-99% H2O) layer of cellulose
synthesised by bacteria (G. xylinus) CELLULOSE HYDROGELS
PCW SYSTEMS:
Mature cotton fibres
Food-extracted CWs
Characterization of PCWs by SAS techniques
BC BC-AX BC-XG
AX
BC hydrogels with AX and XG
Martinez-Sanz et al. Cellulose (2015) 22, 1541-1563
SAXS
XRD
XG affects the arrangement of cellulose microfibrils within the ribbon
XG promotes the crystallization of Iβ allomorph (typically found in plants)
Ribbons’ CORE
- 70-80% bound solvent (40-30% non-exchanged H2O)
- Partially exchanged cellulose (crystalline + paracrystalline)
- XG domains strongly interacting with cellulose microfibrils
Ribbons’ SHELL
- > 90% bound solvent + Fully exchanged paracrystalline
cellulose
- Surface AX/XG domains (non-specific adsorption
interaction mechanism)
BC hydrogels with AX and XG
Martinez-Sanz et al. Soft Matter, (2016)
12, 1534-1549
SANS Contrast variation experiments CORE-SHELL model
Non-interacting pectin (60-80%) Removed after washing. Leads to phase separation upon
drying. Mainly located filling in the pores between the ribbons Denser hydrogels
“Bound” pectin (20-40%) Remains after washing. Reduces the XC slightly but does not affect
the crystalline configuration. Interacts directly with cellulose µfibrils (without affecting
crystallisation process) forming domains of 10-12 nm
BC hydrogels with Pectins (a) Hydrogels prepared in the presence of high DM pectin solutions with different viscosities
Lopez-Sanchez et al. Carbohyd.Polym., (2016) 153, 236-245
H-CH D-CH
Ф=32 ± 12 nm Ф=27 ± 11 nm
Deuterated BC hydrogels
Aim: Increase neutron SLD contrast by replacing H atoms in cellulose with D
Martinez-Sanz et al. Carbohyd.Polym., (2016) 147, 542-555
Molecular structure : C6D5H5O5
- Similar XC (97-98%) and crystallite cross-sections
- Predominant cellulose Iα allomorph
PROPOSED BIOSYNTHESIS MECHANISM vs. INVESTIGATED STRUCTURAL FEATURES
Deuterated BC hydrogels
Brown R.M., J.Macromol.Sci. (1996) 33, 1345-1373
Cellulose µfibrils are synthesised by TC sub-units
Proximity of TC sub-units Association of adjacent µfibrils
Interaction of µfibrils with strongly bound H2O through H bonding network Ribbon
~ 5 cm Larger samples required
Extending the q range by using SANS (4 config.) + USANS
Martinez-Sanz et al. Polymer, (2016) 105, 449-460 D-BC composite hydrogels
D-CH-AX
D-CH-MLG
D-CH
D-CH-XG
Ribbon cross-links
Small nodules
13% AX
39% XG 32% MLG
C6D5H5O5
D-CH crystallinity (84%) decreases with
the presence of MLG (68%) and XG
(59%). Only XG promotes Iβ crystallisation
D-BC composite hydrogels
- Core-shell + Beaucage model to fit the entire
q range New structural features
CORE-SHELL RIBBON STRUCTURE
- D-CH slower synthesis rate less dense ribbon
- AX and MLG modify shell properties
- XG affects core and shell properties
LONGITUDINAL STRUCTURE
- Individual µfibril Crystalline cellulose domains
- Ribbon Periodical twisting
1.6 nm
140-180 nm
1.4-1.5 µm
Approach to investigate the structure of native cellulose hydrogels: Combination of small
angle scattering techniques with diffraction, microscopy and spectroscopy
Hierarchical architecture of cellulose modelled by multi-scale core-shell formalism
SAXS microfibril structure, SANS ribbon structure (H/D exchange process)
Partial deuteration of cellulose enhances the appearance of SANS structural features
Extending the q range with USANS Scattering features likely related to cellulose
longitudinal structure
Elucidation of the distinct interaction mechanism of PCW matrix polysaccharides with cellulose
To summarize
- Interferes with cellulose
crystallisation
- Promotes Iβ allomorph
- Interfibrillar domains cross-
linking and interspacing µfibrils
- Surface domains cross-linking
ribbons
XG
- Interferes with cellulose
crystallisation but does not create
a network of cross-linked µfibrils
- Surface domains cross-linking
ribbons
MLG
Surface interactions via non-
specific adsorption mechanism
AX
Pectin
(i) non-interacting fraction filling in
ribbon pores
(ii) Interacting fraction coating
cellulose µfibrils
Extraction of different lignocellulosic fractions from different algae and seaweed species
Investigation of the cellulose architecture and structural roles of matrix polysaccharides by
means of scattering techniques (SAXS/WAXS synchrotron experiments
Application of extracted carbohydrates as encapsulation matrices, bioactive materials and
reinforcing agents for packaging structures
Ongoing work