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\begin{document}

\title{Methods of~ Identifying the Rigidity and solvation profile of Bacterial
Cellulose hydrogels~ and its hemicelluloic counterparts using Solid
state NMR Techniques.~~~~~~~~~~~~}



\author[1]{Haider Salam}%
\affil[1]{University of East Anglia}%


\vspace{-1em}



  
  \date{May 04, 2024}


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\selectlanguage{english}
\begin{abstract}
\hyperref[csl:1]{(Uhlin et al., 1995)}\hyperref[csl:2]{(Tzvi, 2001)}\hyperref[csl:3]{(MCCANN and CARPITA, 2008)}\hyperref[csl:4]{(Kaflak and Kolodziejski, 2008)}
The structure of Protiated, Deuterated and composite Cellulose hydrogels
with plant cell wall (PCW) polysaccharides has been investigated using
Solid state NMR. A combination of CP kinetics and Water polarisation
transfer (WPT) experiments enabled the identification of structural
features of Cellulose Crystallinity/rigidity and solvation profile
respectively. The ratio of amorphous to crystalline region increases by
15\% thereby decreasing the crystallinity of the Cellulose gel and this
connects with the fact that the rigidity of Cellulose-Xyloglucan
composite is significantly lower as compared with Bacterial Cellulose
Gel as evidenced by AFM studies. (by contrast, Arabinoxylan-Cellulose
composite experienced negligible differences in rigidity and
crystallinity). These results seem to be paradoxical with CP kinetics
results which show that Bacterial Cellulose gel and its Hemicelluloic
Composites all exhibit High and relatively similar degree of rigidity.
This indicates that the rigid components for all of the hydrogels remain
at the same degree of rigidity but ratio of rigid to non-rigid
components in the hydrogel changes and these changes of crystallinity at
the nanostructure have compounded effects on the rigidity of the
microstructure. These results highlight the potential Solid State NMR
has to probe the multi-scale architecture of Cellulose hydrogels and to
observe changes to that structure once hemicelluloses are added to it.~
\hyperref[csl:5]{(P{\'{e}}rez-Hern{\'{a}}ndez et al., 2012)}\hyperref[csl:6]{(Kolodziejski and Klinowski, 2010)}\hyperref[csl:7]{(Renard and Jarvis, 1999)}%
\end{abstract}%



\sloppy


\par\null

\section*{Introduction}

{\label{874460}}

\par\null

PCW are the world's most abundant type of biomaterials and have
applications ranging from dietary fibre and structuring the components
in foods, raw materials for bio refining and as a source of Polymers for
things such as gum and stabilisers. It also has an important role to
play from a drug delivery perspective in that it can be the precursor to
the formation of novel forms of biocapsules to used for a plethora of
issues ranging from~ delivering insulin to type 1 diabetes patients~ to
delivering cytotoxic drugs to patients who have cancer. These
biocapsules would be superior to more traditional forms of drug delivery
methods in that they would have far less contamination level and exhibit
very low degree of stomach degradation due to the ``recalcitrant
nature''\hyperref[csl:8]{(Atalla et al., n.d.)} of PCW while at the same time being
vulnerable to degradation in the small intestitine which is precisely
the position at which the biocapsule should be degraded for maximum
absorbtion.~ Studying the structural and mechanical properties of PCW~
is key to being able to utilise PCW for any of these functions.~ PCW are
composed of Cellulose microfibrils which consists of unbranched,
unsubstituted (1,4)-B-D-glucan chains that are able to form
microfibrills. These exist in several different levels of crystallinity
and length depending on site and function of cell. Furthermore there is
difference of crystallinity in the individual cell where Cellulose is
broken into layers of crystalline, paraccystaline and amorphous
Cellulose. PCW also contains Pectins which are wall polysaccharides that
are solubilized by aqueous buffers and dilute acidic solutions and
calcium chelators~\hyperref[csl:9]{(Cosgrove, 2015)}. Pectins are very important for PCW
as they form hydrated gels that push microfibrils apart, easing their
sideways slippage during cell growth, while also locking them in place
when growth ceases~\hyperref[csl:10]{(Lord and Mollet, 2002)}.~ Hemicelluloses are
heteropolymers that are present with Cellulose in almost all plant
cells, these are a wide variety of molecules which have a random
amorphous structure with little strength and they could be solubilised
by a strong alkali. These would include Xylan, Glucuronoxylan,
Arabinoxylan, Glucomannan and Xyloglucan. Different levels of these
polymers are found in different plant and in different locations around
the plant~\hyperref[csl:11]{(Carpita and Gibeaut, 1993)}.Further complexity is added by the fact
that plant cells normally have two cell walls. A primary cell wall
formed during cell expansion and a secondary cell wall that is deposited
once cell expansion has ceased. Cellulose and Lignin levels are
typically higher in secondary PCW than in primary PCW. Secondary cell
walls provide additional protection to cells and rigidity and strength
to the larger plant. These walls are constructed of layered sheaths of
Cellulose microfibrils, wherein the fibres are in parallel within each
layer.~The inclusion of~Lignin~makes the secondary cell wall less
flexible and less permeable to water than the primary cell
wall~\hyperref[csl:12]{(Evert and Eichhorn, 2013)} .In addition to making the walls more resistant
to degradation, the hydrophobic nature of Lignin within these tissues is
essential for containing water within the vascular tissues that carry it
throughout the plant.~ All these factors contribute to making PCW highly
complex systems that are very hard to analyse the individual parts
without damaging the structure and thus not understanding the structure
properly. Thus, PCW mimics were made in the form of Bacterial Cellulose
Hydrogels and were characterised by SSNMR ~\hyperref[csl:13]{(Suzuki and Itoh, 2001)} .~ These
hydrogels~are highly hydrated systems in which cellulose fibrillar
networks interact with interstitial water at different structural
levels. Several bacterial species such as Komagataeibacter xylinus
(formerly known as Gluconacetobacter xylinus)\hyperref[csl:14]{(Lin and Catchmark, 2012)}, are
able to synthesise cellulose hydrogels in the form of pellicles when
inoculated into a culture medium rich in carbohydrates or polyols. The
synthesised cellulose hydrogels present a high degree of purity and
hydration (ca. 99 wt\% H2O) and possess a complex structure in which
cellulose is hierarchically assembled to form different structural
features~\hyperref[csl:15]{(Mart{\'{\i}}nez-Sanz et al., 2016)}\hyperref[csl:16]{(Mart{\'{\i}}nez-Sanz et al., 2016)}. The incorporation of PCW
components into the bacterial culture Makes it possible to study the
effects of individual hemcelluloses on the strucutre of cellulose, this
is especially useful since it has been shown that the way hemecelluloses
get incorrporated in the gel is in similar form to the way they get
incorporated in the PCW. The incorporation of several PCW
polysaccharides such as XG, mannans, AX, MLG and pectins using this
approach is well reported in the literature {[}2,12,13,15e18,21e27{]}.
From some of these studies it has been inferred that only XG and mannans
are able to interfere with the cellulose crystallisation process,
reducing the crystallinity index and promoting the formation of the
cellulose Ib allomorph {[}14,15,17,18,23,26{]}. This indicates that a
certain fraction of XG (or mannan) is able to interact directly with the
individual cellulose microfibrils. In addition, a different fraction of
XG, which interacts with the surface of cellulose ribbons, has been
identified {[}14,15,17{]}. This fraction is thought to correspond to the
thin XG cross-bridges detected in the microscopy images from composite
hydrogels {[}14,15,17,26{]}.~ The aim of this work is to use SSNMR to
obtain data about the rigdity,Crystalinity,Solavation and relaxation
times of Cellulose Hydrogels and to identify the differences that happen
when either Xyloglucan or Arabinoxylan is added to it and to use a model
of I-S or I-I*-S to Interpret data obtained and to also to point out the
limitation that CP might have to probing the structure of Bacterial
cellulose and to present methods to tackle that problem.

\par\null\par\null

Introduction

PCW are the world's most abundant type of biomaterials and have
applications ranging from dietary fibre and structuring the components
in foods, raw materials for bio refining and as a source of Polymers for
things such as gum and stabilisers. It also has an important role to
play from a drug delivery perspective in that it can be the precursor to
the formation of novel forms of biocapsules to used for a plethora of
issues ranging from~ delivering insulin to type 1 diabetes patients~ to
delivering cytotoxic drugs to patients who have cancer. These
biocapsules would be superior to more traditional forms of drug delivery
methods in that they would have far less contamination level and exhibit
very low degree of stomach degradation due to the ``recalcitrant
nature''(Atalla) of PCW while at the same time being vulnerable to
degradation in the small intestitine which is precisely the position at
which the biocapsule should be degraded for maximum absorption.~
Studying the structural and mechanical properties of PCW~ is key to
being able to utilise PCW for any of these functions.~ PCW are composed
of Cellulose microfibrils which consists of unbranched, unsubstituted
(1,4)-B-D-glucan chains that are able to form microfibrills. These exist
in several different levels of crystallinity and length depending on
site and function of cell. Furthermore there is difference of
crystallinity in the individual cell where Cellulose is broken into
layers of crystalline, paraccystaline and amorphous Cellulose. PCW also
contains Pectins which are wall polysaccharides that are solubilized by
aqueous buffers and dilute acidic solutions and calcium chelators
(Cosgrove 2015). Pectins are very important for PCW as they form
hydrated gels that push microfibrils apart, easing their sideways
slippage during cell growth, while also locking them in place when
growth ceases (Lord 2002).~ Hemicelluloses are heteropolymers that are
present with Cellulose in almost all plant cells, these are a wide
variety of molecules which have a random amorphous structure with little
strength and they could be solubilised by a strong alkali. These would
include Xylan, Glucuronoxylan, Arabinoxylan, Glucomannan and Xyloglucan.
Different levels of these polymers are found in different plant and in
different locations around the plant (Carpita 1993).Further complexity
is added by the fact that plant cells normally have two cell walls. A
primary cell wall formed during cell expansion and a secondary cell wall
that is deposited once cell expansion has ceased. Cellulose and Lignin
levels are typically higher in secondary PCW than in primary PCW.
Secondary cell walls provide additional protection to cells and rigidity
and strength to the larger plant. These walls are constructed of layered
sheaths of Cellulose microfibrils, wherein the fibres are in parallel
within each layer. The inclusion of Lignin makes the secondary cell wall
less flexible and less permeable to water than the primary cell wall
(Evert 2013) .In addition to making the walls more resistant to
degradation, the hydrophobic nature of Lignin within these tissues is
essential for containing water within the vascular tissues that carry it
throughout the plant.~ All these factors contribute to making PCW highly
complex systems that are very hard to analyse the individual parts
without damaging the structure and thus not understanding the structure
properly. Thus, PCW mimics were made in the form of Bacterial Cellulose
Hydrogels and were characterised by SSNMR~ (Suzuki 2001) .~ These
hydrogels are highly hydrated systems in which cellulose fibrillar
networks interact with interstitial water at different structural
levels. Several bacterial species such as Komagataeibacter xylinus
(formerly known as Gluconacetobacter xylinus)(Lin 2012), are able to
synthesise cellulose hydrogels in the form of pellicles when inoculated
into a culture medium rich in carbohydrates or polyols. The synthesised
cellulose hydrogels present a high degree of purity and hydration (ca.
99 wt\% H2O) and possess a complex structure in which cellulose is
hierarchically assembled to form different structural features
(Mart\selectlanguage{ngerman}ínez-Sanz 2016)(Martínez-Sanz 2016a). The incorporation of PCW
components into the bacterial culture Makes it possible to study the
effects of individual hemcelluloses on the strucutre of cellulose, this
is especially useful since it has been shown that the way hemecelluloses
get incorrporated in the gel is in similar form to the way they get
incorporated in the PCW. The incorporation of several PCW
polysaccharides such as XG, mannans, AX, MLG and pectins using this
approach is well reported in the literature {[}2,12,13,15e18,21e27{]}.
From some of these studies it has been inferred that only XG and mannans
are able to interfere with the cellulose crystallisation process,
reducing the crystallinity index and promoting the formation of the
cellulose Ib allomorph {[}14,15,17,18,23,26{]}. This indicates that a
certain fraction of XG (or mannan) is able to interact directly with the
individual cellulose microfibrils. In addition, a different fraction of
XG, which interacts with the surface of cellulose ribbons, has been
identified {[}14,15,17{]}. This fraction is thought to correspond to the
thin XG cross-bridges detected in the microscopy images from composite
hydrogels {[}14,15,17,26{]}.~ The aim of this work is to use SSNMR to
obtain data about the rigdity,Crystalinity,Solavation and relaxation
times of Cellulose Hydrogels and to identify the differences that happen
when either Xyloglucan or Arabinoxylan is added to it and to use a model
of I-S or I-I*-S to Interpret data obtained and to also to point out the
limitation that CP might have to probing the structure of Bacterial
cellulose and to present methods to tackle that problem.

Materials and Methods

13C~ H2O or D2O~~ Bacterial Cellulose Gel was made~ by the method
provided by the Literature (Martínez-Sanz 2016a).~ They were made from
from Komagataeibacter xylinus which was fermended on Protiated and
deuterated Carbon Sources to produce Protiated and Deuterated Cellulose
hydrogels. The bacterial strainwas cultivated in Hestrin and Shramm (HS)
medium with the addition of glucose as the carbon source. Incubation was
performed at 30 °C for 72 h.

At the end of incubation, cellulose hydrogels were harvested and washed
in ice cold milliQ water with gentle agitation (150 rpm), with frequent
rinsing to wash off excess medium and dislodge loosely-associated
bacterial cells, until the harvested hydrogels were white in appearance.

Solid state MAS 13C NMR. For all resonance spectroscopy measurements a
Bruker 400 MHz spectrometer equipped with Topspin software (Version ?)
and a ?.? mm Probe. The MAS spinning rate was 6 KHz. Adamantane was used
as an external reference and the downfield peak as set to ?.? ppm.
Experiments were done in two temperatures (25 °C and 5~ °C ). These
general parameters were used for all the NMR experiments. The other
parameters (Contact time,Mixing time,D20,etc\ldots{}) were then adjusted
for each series of experiments as deemed appropriate

Relaxation time. Relaxation experiments conducted were done using CP
Saturation recovery to obtain the spin-Lattice (T1) relaxation time
under CP conditions and direct polarisation inversion recovery to obtain
the T1 with no CP conditions. The Parameters for the experiment were:
128 NS, 8 DS, D1 10 s and the P15 for the CPsatrec is 2000 us.~

CP Kinetics. Cross polarization is the transfer of magnetization from
the abundant spin I to the rare spin S using dipolar couplings. This is
done to enhance signal-to-noise of nuclei such as 13C (1.1\% of all
carbons). This is done by sending spin-lock pulses that are set to
achieve the Hartmann-Hahn Matching Condition \selectlanguage{greek}γ\selectlanguage{english}?\selectlanguage{english}1(1?)=\selectlanguage{greek}γ\selectlanguage{english}?\selectlanguage{english}1(?). When the
Hartmann-Hahn condition is met, the energy gaps between H and X spin
states are equal. Under these conditions, there is a transfer of
population from the abundant spin I to rare spin S by dipolar coupling.~
The time in which the Hartmann-Hahn condition is allowed to exist is
called the contact time (P15). The parameters for the experiment were:
Number of scans (NS) 512, Dummy scans (DS) 8,~ Recylce delay (d1) 10 s.
The P15 was arranged in sequence from 10 us to 6 Ms with very short
sequences early on (10,20,40,60,80,100) because most of the growth phase
in CP happens then. This is done to obtain a profile of CP efficiency in
regards to the Contact time permitted. This should allow us to
comprehend the rigidity of the system. That is because CP relies on
heteronuclear dipolar couplings between the source and target spins, its
efficiency is uniquely dependent on the concentration, distribution and
mobility of the nuclei involved. Therefore, the technique is remarkably
sensitive to the composition, structure, properties and molecular
dynamics in the solid(Kaflak 2008). Processing parameters made to be the
same for all graphs in order make them consistent with each other and
hence comparable.

CP/HPDEC normalisation. High powered decoupling (HPDEC) is applied to
remove the broading effects of heteronuclear coupling.The application of
high power irradiation at the frequency of the 1H resonance causes those
spins to undergo repeated transitions at a rate determined by the
amplitude of the irradiation.If the rate is rapid enough the desired
nuclei can be observed without the broadening effects of dipolar
coupling.HPDEC is normally useful in understanding the flexibility of
the sample and in being able to quantitatively analyze it. However. We
used HPDEC as a normalization against CP. This is so we can quantitate
the proportion of Crystalline to amorphous regions for each gel in the
sample.

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Fig. 1

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ CP Pulse
sequence

Water polarisation transfer (WPT). Water polarisation transfer is an NMR
technique that allows to obtain the hydration profile of polymer fibres.
It does so by using a T2 filter to remove the transverse magnetisation
of the surface of the fibres and leaving water polarisation to then be
transferred to the polymer by spin diffusion, chemical exchange and NOE.
By representing the normalised growth of water polarisation against the
[?] mixing time, the hydration profile of each carbon is obtained.~ The
parameters for the experiment were:~ NS was 256 scans, DS was8 scans, D1
was 10 s, P15 was 500 us. One spectrum was run with no T2 filter (D20)
to be used a reference sample. One spectra was ran with a T2 filter but
with no mixing time to check for effectiveness of T2 filter. The rest
were subsequently had a D20 of 2 Ms and a series of D8 that ranged from
1Ms to 324 Ms. All WPT experiments were conducted in 293 K.

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Fig.
2

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ WPT Pulse sequence

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Fig. 3

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Transfer of Water
polarisation

Data Fitting.~~~ The Equations used to fit the CP experiments are ones
that are used to understand the mobility~ of gel like systems that have
a high degree of water in them(P\selectlanguage{ngerman}érez-Hernández 2012) . The equations
used operate the I-I*-S model in which I* denotes protons in close
proximity to the S Nucleus.~ Within this model, source spins, such as
protons, do not have a common spin temperature. The I*-S spin pairs or
clusters can be viewed as isolated in a solid and exchanging
polarization in an oscillatory manner, damped by the subsequent
spin-diffusion contact with the remaining I spins.(Kolodziejski 2010)~
This model is especially useful in solids where the system is
anisotropic. The equation used for adjusting the experimental curves:

From this fit, it is possible to obtain the TCH values which represent
the efficiency of magnetisation transfer and that can be used as a guide
to describe the rigidity of the system (The higher the magnetisation
transfer the more rigid a system is).~ The software used for the fitting
of the graph was Origin.~

Results

Investingation of rigid compoents by CP kinetics. the CP done on BC gels
first allowed for a characterisation of them. it showed that there were
C1 at 105 ppm along and it had separate peaks for different allomorphs
of C1 (specifically C1 \textbackslash{}alpha\selectlanguage{greek}α \selectlanguage{english}and C1
\textbackslash{}beta\selectlanguage{greek}β\selectlanguage{english}).~ It managed to isolate C3 (75 ppm) from C2,5
(71-73 ppm). It managed to distinguish the amorphous C4 and C6 (85 and
62 ppm respectively) from The crystalline C4 and C6 (89 and 65 ppm
respectively).

Fig. 4

~ 1H-13C CP-MAS NMR, Unlabelled BC gel, SSNMR 400 MHz, 6 KHz, 25 \selectlanguage{ngerman}° C

~~~~~~~~ The CP kinetics result showed that the CP efficiency was very
high for all the carbons, with them reaching the peak of magnetisation
transfer around 1250-1000 us. The Carbon 6, both amorphous and
crystalline, had especially high CP efficiency with maximum
magnetisation reached at 250 us. The CP efficiency was relatively
unchanged between the BC gel and the BC- hemicellulose composites. The
change in temperature from 20 ° C to 5 ° C showed slight increase in CP
efficiency and effect was relatively the same for all the hydrogels.

~

~

Graph 4: CP kinetics of C1 XG under 25 °C and 5 °C

CP/HPDEC Data Fitting. The results of comparing CP to HPDEC showed that
there was a 14\% reduction in Crystallinity in BC-XG compared to BC gel
while the decrease was only 3\% in crystallinity in BC-AX compared to BC
gel. This level of Crystallinity is consistent with the current
literature on this subject (Citation)

Fig. 5

~~~~ Superimposition of HPDEC spectra of BC gel and BC-XG composite
under 25 °C

BC BC-XG BC-AX

Degree of Crystallinity 77\% 63\% 74\%

Investigation of hydration profile using WPT.~ The graphs obtained from
WPT show that all the Carbons under BC-XG exhibited significantly more
solvation than BC and BC-AX. BC-AX was slightly more solvated than BC.
Also interestingly, BC-XG has a curve that exhibits first order kinetics
as opposed to BC and BC-AX which seem to operate under zero order
kinetics. Also BC-XG clearly operates on three phases. An early rapid
phase in water polarisation, a more gradual increase around 100 (?) and
a plateau after wards.

Relaxation time measurement. The T1 of Carbon for the BC gel under CP
conditions was around 2.1 seconds and was homogenous for all carbons,
same result with BC-XG. The T1 of Carbon of BC gel fell from around 2.1
s under 25 °C to around 1.4 s under conditions of 5 °C.~ When the T1 was
measured under T1 IR, the results were far more heterogenous between
carbon to carbon in each gel (C6' had the lowest T1 for each gel). Also
differences arose between each gel. BC-XG had, in the range of 2-4
seconds, higher T1 than BC gel. In comparison, BC-AX had negligible
differences between it and BC gel.

Carbon BC BC-XG

C1 2.202 s 2.121 s

C4 2.175 s 2.163 s

C4' 1.961 s 2.186 s

C3 2.159 s 2.125 s

C2,5 2.149 s 2.167 s

C6 2.101 s 2.116 s

C6' 2.054 s 1.899 s

Fig. 6

~~~~~~~~~~~~~~~~~~~~~ Table of T1 CPsatrec of Carbon for BC and BC-XG
under 25 °C

Carbon 25 °C 5 °C

C1 2.202 s 1.423 s

C4 2.175 s 1.485 s

C4' 1.961 s 1.349 s

C3 2.159 s 1.395 s

C2,5 2.149 s 1.437 s

C6 2.101 s 1.426 s

C6' 2.054 s 1.020 s

Fig. 7

~~~~~~~~~~~~~~~~~~~~~ Table of T1 CPsatrec of Carbon for BC under 25 °C
and 5 °C

Carbon BC BC-XG BC-AX

C1 7.7 s 9.2 s 7.2 s

C4 8.5 s 10.4 s 8.0 s

C4' 4.7 s 8.9 s 5.0 s

C3 5.3 s 6.3 s 5.2 s

C2,5 6.6 s 6.9 s 6.6 s

C6 7.3 s 9.2 s 7.6 s

C6' 4.1 s 6.8 s 4.4 s

Fig. 8

~~~~~~~~~~~~~~~~~~~~~ Table of T1IR of Carbon for BC, BC-XG and BC-AX
under 25 °C

Discussion

The relaxation times of the gels were roughly similar between gels under
CP conditions. They were also similar between carbon to carbon as well.
When relaxation time was measured under direct polarisation differences
started to appear which showed that BC-XG had on average 2-4 s more T1
compared with BC and BC-AX. The experiment also showed that C6' had the
T1 on average in all three gels (that could be explained by low amount
of data from C6' as very little of it was detectable in T1IR). Also
another important part of the experiment is that it enabled the
identification at which point the BC carbons are situated at the T1
graph.~ T1 relaxation graph is approximately a parabola where on the
left side of the graph the less molecular motion the higher T1 because
the more rigid a molecule, the less favoured is the kinetic equilibrium
between the excited state and relaxed state. Thus making for longer
Relation time. There is a minima for molecules which are medium tumbling
molecules and then on the right side of the curve the smaller and faster
a molecule becomes the higher the T1 becomes because the smaller the
molecules becomes, the easier it is for something to stop its motion. In
a system where molecules are sufficiently small. The average of the
motion starts to decrease hence increasing T1. To know on which side of
the parabola BC gel is. The experimented was conducted in Both 25 °C and
5 °C. The experiments showed that as you move from 25 °C to 5 °C (hence
increasing rigidity) that there is a decrease in T1. Meaning that that
BC gel is situated on the right side of the T1 graph. That would
indicate that when BC-XG has its T1 increased, it does so because of it
being less rigid, freer and more disordered. However, Experiments of the
same type need to be conducted on BC-XG and BC-AX just to make sure that
the composites exhibit the same kind of behaviour as BC gel.

The CP kinetics for all three compounds have the same behaviour, meaning
that all three have the same CP efficiency. What is puzzling about this
sample is that our results suggest that the CP kinetics for all three
samples exhibit very high CP efficiency. This is surprising given that
the gels are 99.9 \% water. However the rigidity of the system might
explain why something that is 99.9\% water has solid-like rheological
properties.~ The decay of all three samples was slow meaning that proton
spin diffusion was high. That is also consistent with the fact the gel
is 99.9\% water. Also what is really strange is that the rigidity of C6
(both amorphous and crystalline) are the highest for the Cellulose~
hydrogel which is odd since that is the carbon normally most mobile.. CP
kinetics when done under lower temperature exhibits small change in
rigidity (becoming a bit more rigid). Meaning that the compound is
already quite rigid.

~~~~~~~~ The problem with these findings is that our other experiments
that we performed seem to have results that, at least superficially,
contradict the CP kinetics findings. First of all, the AFM studies that
were conducted on the BC-XG gel show that it is way softer and the
fibres are 10 times thinner. Also the Water polarisation studies that
BC-XG had a significantly higer hydration profile with different
behaviour c\hyperref[csl:17]{(Pongsawatmanit et al., 2006)}\hyperref[csl:18]{(Wise, 1955)}ompared to BC and BC-AX
(Altough BC-AX showed significant increase in hydration, the shape of
the curve was the same with BC gel).~ Hydration profile done under WPT
can be increased by two things. Chemical exchange, which is the same for
all compounds, and Coherent spin diffusion: mediated by distance- and
mobility-dependent 1H-1H dipolar couplings.There is no reason to suggest
that there is a decrease in distance 1H-1H dipolar couplings (the
proposed mechanisms of how Xylogl\hyperref[csl:1]{(Uhlin et al., 1995)}can and Arabinoxylan
interact with Cellulose don't mention a change in distance between
Cellulose and its surroundings Citations required). Therefore the only
possible explanation is that the mobility got increased in BC-XG and
BC-AX. Also the CPH/HPDEC normalised Data show that Xyloglucan had a
significantly lower crystallinity compared with BC gel and that would
also imply a lower rigidity. Now of Course there are other factors that
influence CP efficiency apart from rigidity and P15 (spin-lock power
levels, decoupling power and the rate of magic-angle spinning (MAS).
However we made sure during the experiment that these were consistent
throughout and the same for all the gels.

We propose, in this paper, two ways in which harmonise the findings from
CP kinetics with all our other findings and the wider literature. First,
we suggest a Model of how xyloglucan breaks down the interfibrillar
connections, increasing amount of amorphous regions in the cellulose
while also increasing the surface area. Thus increasing the amount of
water bound to cellulose at the surface. Water might be closer to the
surface of cellulose than it was when the cellulose was rigid because
the hydration profile for xyloglucan got better.~ This model is based on
the idea that the bundles normally found in cellulose due to the
interfibrillar connections are broken hence there is decrease in
rigidity of the macrostructure but not of the microscopic domains.
Xyloglucan may break interfibrillar connections by preventing Hydrogen
bonding between Cellulose microfibril. This may help each microfibril to
slide during cell enlargement, because xyloglucans have no mutual
affinity (13). This would also explain the increased flexibility found
in BC-XG composite.

Fig. 9

~~~~~~~~~~~~~~~~~~~~~ Model of how Xyloglucan and Cellulose Microfibril
interact (14)

Fig. 10

~~~~~~~~~~~~~~~~~~~~~ Model to explain how Xyloglucan composite can have
higher hydration

Second part of the explanation is in terms of CP limitations. The
limitation is that CP targets the rigid components of the culture but we
know cellulose is multi phased system with plenty of paraccystaline,
amorphous (with different meaning of amorphous) and crystalline. The~
fact that CP kinetics that we got are standard solid like behaviour even
though the sample is 99.99\% water points out that there are mobile
components that are not being detected by our experiments. This would
also explain the homogenising effect of CPsatrec and how if you take CP
out of the equation then you observe the heterogeneity found inside the
gel. It also explain why the HPDEC data gives so different results from
CP and explains why when we normalise CP with HPDEC we get results of
the gel's crystallinity that are consistent with the larger literature.
Now for a full understanding of the BC hydrogels, there needs to be
methods of identifying the disordered regions of the Compound. We
propose methods of circumnavigating the problems associated with CP.
CPSP (Cross polarisation with a single pulse (SP)) was developed to to
quantitatively analyse compositions of multiphase polymers with marked
difference in phase mobility. The quantification of CP spectrum is done
by measuring the enhancement factor of CP with a reciprocity relation,
while the quantification of SP is derived directly from the Boltzmann
distribution in thermo equilibrium. CPSP is done by using a conventional
CP combined with a SP sequence. The initial 13C 90° pulse rotates the
magnetization to the x-y plane in the rotating frame and the
magnetization is then spin-locked by the subsequent 13C CP pulse. Here,
the 13C spin-locking pulse plays two roles: one is to hold the
magnetization so that it will not dephase during the contact time t and
the other to transfer polarization from 1 H to 13C. (15)

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Fig. 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ CPSP pulse
sequence

Another experiment that is relevant is Cross Depolarisation experiment
(CDP). This experiment is needed for full quantification of CPSP(16).~
This is a method where, instead of the polarized S magnetization in the
standard CP-MASS experiment, the depolarized S magnetization is
monitored. The pulse sequence utilized is shown below. It is a
Conventional CP sequence with some modifications. Two additional
intervals ti; and tc 2 are inserted between the usual polarization time
tcI, and detection time td. This is very good at revealing the details
of cross relaxation process. With CP~ it is difficult to know the real
maximum polarized S signal and the exact time dependence of S signal to
be determined by cross polarization alone, because the creation of S
polarization depends not only on the cross relaxation process itself,
but also on some other factors, such as
T\textasciitilde{}\textasciitilde{} of I spin, and Hartmann-Hahn
matching condition. In the depolarization scheme, however, the finite
initial S magnetization at the beginning of t, 2 will certainly reach
zero at last and the turning point occurs during the first tens of usec.
This experiment allows for the quantitative expression of 13C
magnetisation as a function of depolarisation time using the following
formula(17):

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Msx is the polarized 13C magnetization for~ 13CH group, b is dipolar
coupling between ' 13C and its directly bonded proton, R is the spin
diffusion rate among protons, and t is the polarization time. Both CPSP
and CDP should tackle two problems. First, they should be able to allow
us to make quantitative judgements on CP experiments and second, to able
to target the amorphous regions of BC gel and its composites.

~~~~~~~~~~~~~~~~~~~~~~~

Fig. 11

Pulse sequence utilized in the depolarization experiment, where tci is
the polarization time for S spins, ti is the dephase time for I spins,
and tc 2 is the depolarization time for S spins. The development of the
5 magnetization is schematized by a solid line.

Conclusion

\selectlanguage{english}* To conclude, SSNMR has provided lots of insights into the structure of
BC Hydrogels. The implementation of CP kinetics and WPT along relaxation
time experiments showed that these were gels that have a high degree of
CP efficiency. That the CP efficiency is equivalent between Hydrogels
was equivalent and that a decrease in temperature has minimal effect on
the CP efficiency. The WPT show zero order kinetics for the graph of
both arabinoxylan and Bacterial Cellulose but higher hydration but also
a curve operating under 1st order kinetics for Cellulose-Xyloglucan
composite.~ The T1 of Bacterial cellulose and Arabinoxylan composites
are practically the same but the Xyloglucan composite has a difference
between 2-4 s between its T1 and the T1 of BC gel. There is also
difference in T1 between different carbons T1 suggesting that there are
different levels of mobility between each carbon in each sample. All
these insights into the Structure of BC Hydrogels was done using SSNMR
Techniques. Nevertheless, SSNMR has important limitations that need to
be considered. Namely, Poor acquisition time and reliance of CP for most
of the experiments and that makes the results qualitative and they also
target the rigid components of the system and has homogenising effect on
the less ordered components of it. Making them appear the same.~ We
propose methods of how to tackle these limitations through CPSP and CDP
which will make Cross polarisation experiments quantitative and will
allow for detection of disordered materials as well. With the goal of
formulating a model that incorporates both the crystalline regions and
the amorphous regions as well.

Acknowledgement

References

Sorted By Appearance

1. Rajai H. Atalla, John W. Brady, James F. Matthews, Shi-You Ding,
Michael E. Himmel. Structures of Plant Cell Wall Celluloses. 188--212 In
Biomass Recalcitrance. Blackwell Publishing Ltd. Link

2. Daniel J. Cosgrove. Plant cell wall extensibility: connecting plant
cell growth with cell wall structure mechanics, and the action of
wall-modifying enzymes. Journal of Experimental Botany 67, 463--476
Oxford University Press (OUP), 2015. Link

3. E. M. Lord, J.-C. Mollet. Plant cell adhesion: A bioassay facilitates
discovery of the first pectin biosynthetic gene. Proceedings of the
National Academy of Sciences 99, 15843--15845 Proceedings of the
National Academy of Sciences, 2002. Link

4. Nicholas C. Carpita, David M. Gibeaut. Structural models of primary
cell walls in flowering plants: consistency of molecular structure with
the physical properties of the walls during growth. The Plant Journal 3,
1--30 Wiley-Blackwell, 1993. Link

5. Ray F. Evert, Susan E. Eichhorn. Plants and People. 501--523 In Raven
Biology of Plants. Macmillan Learning, 2013. Link

6. Kiyoshi Suzuki, Takao Itoh. The changes in cell wall architecture
during lignification of bamboo Phyllostachys aurea Carr.. Trees 15,
137--147 Springer Nature, 2001.Link

7. Fang Lin, Jeffrey M Catchmark. Analysis of Gluconacetobacter xylinus
exopolysaccharides and its impacts on bacterial cellulose production and
crystallization. In2012 Dallas Texas, July 29 - August 1, 2012. American
Society of Agricultural and Biological Engineers, 2012. Link

8. Marta Martinez-Sanz, Michael J. Gidley, Elliot P. Gilbert.
Hierarchical architecture of bacterial cellulose and composite plant
cell wall polysaccharide hydrogels using small angle neutron scattering.
Soft Matter 12, 1534--1549 Royal Society of Chemistry (RSC), 2016. Link

9. Marta Martinez-Sanz, Deirdre Mikkelsen, Bernadine Flanagan, Michael
J. Gidley, Elliot P. Gilbert. Multi-scale model for the hierarchical
architecture of native cellulose hydrogels. Carbohydrate Polymers 147,
542--555 Elsevier BV, 2016. Link

10. Natalia Perez-Hernandez, Trung Quan Luong, Martin Febles, Carlos
Marco, Hans-Heinrich Limbach, Martina Havenith, Cirilo Perez, Maria
Victoria Roux, Ricardo Perez, Julio D. Martin. The Mobility of Water
Molecules through Hydrated Pores. The Journal of Physical Chemistry C
116, 9616--9630 American Chemical Society (ACS), 2012. Link

11. Waclaw Kolodziejski, Jacek Klinowski. ChemInform Abstract: Kinetics
of Cross-Polarization in Solid-State NMR: A Guide for Chemists.
ChemInform 33, no--no Wiley-Blackwell, 2010. Link

12. Agnieszka Kaflak, Waclaw Kolodziejski. Kinetics of1H
\textbackslash{}(\textbackslash{}rightarrow\textbackslash{})31P NMR
cross-polarization in bone apatite and its mineral standards. Magnetic
Resonance in Chemistry 46, 335--341 Wiley-Blackwell, 2008.

13. Hayashi, T. , Marsden, M. P. F. , Delmer, D. P. 1 987. Pea
xyloglucan and cellulose. V. Xyloglucan-cellulose interactions in vitro
and in vivo. Plant Physioi. 83:384-89

14. Hayashi, T. (1989). Xyloglucans In The Primary Cell Wall. Annual
Review of Plant Physiology and Plant Molecular Biology, 40(1), p.156.

15. Shu, J., Li, P., Chen, Q. and Zhang, S. (2010). Quantitative
Measurement of Polymer Compositions by NMR Spectroscopy:Targeting
Polymers with Marked Difference in Phase Mobility. Macromolecules,
43(21), pp.8993-8996.

16. Xiaoling, W., Shanmin, Z. and Xuewen, W. (1988). Two-stage feature
of Hartmann-Hahn cross relaxation in magic-angle sample spinning.
Physical Review B, 37(16), pp.9827-9829.

17. S. R. Hartmann and E. L. Hahn, Phys. Rev. 128, 2042 (1962)

18.\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.42\columnwidth]{figures/c0cp00644k-f2/c0cp00644k-f2}
\caption{{CP Pulse sequence
{\label{370662}}%
}}
\end{center}
\end{figure}

\textbf{Water polarisation transfer (WPT).~}Water polarisation transfer
is an NMR technique that allows to obtain the hydration profile of
polymer fibres. It does so by using a T2 filter to remove the transverse
magnetisation of the surface of the fibres and leaving water
polarisation to then be transferred to the polymer by spin diffusion,
chemical exchange and NOE. By representing the normalised growth of
water polarisation against the [?] mixing time, the hydration profile of
each carbon is obtained.~ The parameters for the experiment were:~ NS
was 256 scans, DS was8 scans, D1 was 10 s, P15 was 500 us. One spectrum
was run with no T2 filter (D20) to be used a reference sample. One
spectra was ran with a T2 filter but with no mixing time to check for
effectiveness of T2 filter. The rest were subsequently had a D20 of 2 Ms
and a series of D8 that ranged from 1Ms to 324 Ms. All WPT experiments
were conduced in 293 K.

\par\null\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.49\columnwidth]{figures/Picture1/Picture1}
\caption{{WPT Pulse seuquence
{\label{727064}}%
}}
\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.28\columnwidth]{figures/Capture/Capture}
\caption{{Transfer of Water polarisation
{\label{472226}}%
}}
\end{center}
\end{figure}

~\textbf{Data Fitting.~~ ~}The Equations used to fit the CP experiments
are ones that are used to understand the mobility~ of gel like systems
that have a high degree of water in
them\hyperref[csl:5]{(P{\'{e}}rez-Hern{\'{a}}ndez et al., 2012)}\textbf{~.~}The equations used operate the I-I*-S
model in which I* denotes protons in close proximity to the S Nucleus.~
Within this model, source spins, such as protons, do not have a common
spin temperature. The I*-S spin pairs or clusters can be viewed as
isolated in a solid and exchanging polarization in an oscillatory
manner, damped by the subsequent spin-diffusion contact with the
remaining I spins.\hyperref[csl:6]{(Kolodziejski and Klinowski, 2010)}~ This model is especially useful in
solids where the system is anisotropic. The equation used for adjusting
the experimental curves:~\(I\left(t\right)=I_{0^{\ }}\left(1-\frac{T_{CH}}{T_{1P^{ }}^{ }H}\right)^{-1}\left(\exp\left(-\frac{t}{T_{1P^{ }}^{ }H}\right)-\exp\left(-\frac{t}{T_{CH}}\right)\right)\)

From this fit, it is possible to obtain the T\textsubscript{CH~}values
which represent the efficiency of magnetisation transfer and that can be
used as a guide to describe the rigidity of the system (The higher the
magnetisation transfer the more rigid a system is).~ The software used
for the fitting of the graph was Origin.~

\par\null\par\null

\section*{Results~}

{\label{623779}}

\textbf{Investingation of rigid compoents by CP kinetics.~}the CP done
on BC gels first allowed for a characterisation of them. it showed that
there were C1 at 105 ppm along and it had separate peaks for different
allomorphs of C1 (specifically C1~\(\alpha\) and
C1~\(\beta\)).~ It managed to isolate C3 (75 ppm) from C2,5
(71-73 ppm). It managed to distinguish the amorphous C4 and C6 (85 and
62 ppm respectively) from The crystalline C4 and C6 (89 and 65 ppm
respectively).~

\par\null\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/capturee-1/capturee-1}
\caption{\selectlanguage{ngerman}{\textsuperscript{1}H-\textsuperscript{13}C CP-MAS NMR, Unlabelled BC
gel, SSNMR 400 MHz, 6 KHz, 25 ° C
{\label{787383}}%
}}
\end{center}
\end{figure}\selectlanguage{ngerman}

~ ~ ~ ~ ~The CP kinetics result showed that the CP efficiency was very
high for all the carbons, with them reaching the peak of magnetisation
transfer around 1250-1000 us. The Carbon 6, both amorphous and
crystalline, had especially high CP efficiency with maximum
magnetisation reached at 250 us. The CP efficiency was relatively
unchanged between the BC gel and the BC- hemicellulose composites. The
change in temperature from 20 ° C to 5~ ° C showed slight increase in CP
efficiency and effect was relatively the same for all the hydrogels.

\par\null

~

\par\null

\par\null

\textbf{Relaxation time measurement.~}The~ T1 of Carbon for the~

\par\null\par\null\par\null

\section*{Acknowledgements}

{\label{687807}}\par\null

\selectlanguage{english}
\FloatBarrier
\section*{References}\sloppy
\phantomsection
\label{csl:8}Atalla, R.H., Brady, J.W., Matthews, J.F., Ding, S.-Y., Himmel, M.E., n.d. {Structures of Plant Cell Wall Celluloses}, in: Biomass Recalcitrance. Blackwell Publishing Ltd., pp. 188–212. \url{https://doi.org/10.1002/9781444305418.ch6}

\phantomsection
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\phantomsection
\label{csl:9}Cosgrove, D.J., 2015. {Plant cell wall extensibility: connecting plant cell growth with cell wall structure mechanics, and the action of wall-modifying enzymes}. Journal of Experimental Botany 67, 463–476. \url{https://doi.org/10.1093/jxb/erv511}

\phantomsection
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\phantomsection
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\phantomsection
\label{csl:6}Kolodziejski, W., Klinowski, J., 2010. {{ChemInform} Abstract: Kinetics of Cross-Polarization in Solid-State {NMR}: A Guide for Chemists}. {ChemInform} 33, no--no. \url{https://doi.org/10.1002/chin.200221281}

\phantomsection
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\phantomsection
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\phantomsection
\label{csl:16}Mart{\'{\i}}nez-Sanz, M., Mikkelsen, D., Flanagan, B., Gidley, M.J., Gilbert, E.P., 2016. {Multi-scale model for the hierarchical architecture of native cellulose hydrogels}. Carbohydrate Polymers 147, 542–555. \url{https://doi.org/10.1016/j.carbpol.2016.03.098}

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\end{document}

