The multi-scale architecture of cellulose in plant cell wall systems investigated by small angle scattering techniques

Marta Martinez-Sanz mmartinez@iata.csic.es

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

Isotropic behaviour radial average

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

Deuterated BC hydrogels

SANS SAXS

Ribbon Microfibril

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

D-BC composite hydrogels

D-CH D-CH-AX D-CH-XG D-CH-MLG

Native hydrogels

Air-dried samples

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

Acknowledgements

ANSTO Elliot Gilbert Christine Rehm Liliana de Campo UQ Mike Gidley Patricia Lopez-Sanchez Deirdre Mikkelsen Bernadine Flanagan Dongjie Liu Si-Qian Chen Dongjie Wang