Article

Cite This: Macromolecules XXXX, XXX, XXX−XXX pubs.acs.org/Macromolecules

Quantifying Localized Macromolecular Dynamics within Hydrated

Cellulose Fibril Aggregates
Pan Chen,†,§ Camilla Terenzi,∥ Istvań Furo,́ †,‡ Lars A. Berglund,† and Jakob Wohlert*,†
†
Wallenberg Wood Science Center and ‡Department of Chemistry, KTH Royal Institute of Technology, SE-10044 Stockholm,
Sweden
§
Beijing Engineering Research Center of Cellulose and its Derivatives, School of Materials Science and Engineering, Beijing Institute
of Technology, 5 South Zhongguancun Street, Haidian District, Beijing 100081, China
∥
Laboratory of Biophysics, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands
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*
S Supporting Information

ABSTRACT: Molecular dynamics (MD) simulations of 13C

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NMR longitudinal relaxation (T1) distributions were recently

established as a powerful tool for characterizing moisture
adsorption in natural amorphous polymers. Here, such
computational−experimental synergy is demonstrated in a
system with intrinsically high structural heterogeneity, namely
crystalline cellulose nanofibrils (CNFs) in highly hydrated
aggregated state. In such a system, structure−function
properties on the nanoscale remain largely uncovered by
experimental means alone. In this work, broadly polydispersed
experimental 13C NMR T1 distributions could be successfully reproduced in simulations and, for the first time, were
decomposed into contributions from distinct molecular sources within the aggregated CNFs, namely, (i) the core and (ii) the
less-accessible and accessible surface regions of the CNFs. Furthermore, within the surface groups structurally different sites
such as (iii) residues with different hydroxymethyl orientations and (iv) center and origin chains could be discerned based on
their distinct molecular dynamics. The MD simulations unravel a direct correlation between dynamical and structural
heterogeneity at an atomistic-level resolution that cannot be accessed by NMR experiments. The proposed approach holds the
potential to enable quantitative interpretation of NMR data from a range of multicomponent high-performance nanocomposites
with significantly heterogeneous macromolecular structure.

■ INTRODUCTION
High-strength fibers constitute one of the most important
contributions of polymer science to the development of
lightweight macromolecular structures for industrial use.
Poly(p-phenylene terephthalamide) (Kevlar) is the most
well-known example,1 and poly(p-phenylene-2,6-benzobisox-
azole) (Zylon) possibly has the best mechanical properties
among commercially available fibers.2 Interestingly, cellulose,
the linear β-(1,4) linked homopolymer of glucose, has similar
intrinsic mechanical properties as Kevlar but is biologically
synthesized and can be disintegrated into its nanoscale
components for subsequent engineering into man-made
materials.3 Both natural and synthetic functional polymers
typically exhibit strongly heterogeneous nanostructures due,
for instance, to the coexistence of ordered and disordered
regions that react differently to the ingress of moisture or to
chemical treatments. Cellulose is no exception to this. The
elementary units of its native structure are micrometer long
cellulose nanofibrils (CNFs) that are packed into larger fibril
aggregates (FA). The CNFs are structurally heterogeneous
such that they have a highly crystalline core surrounded by
noncrystalline surface polymers as well as less ordered domains
occurring intermittently along the length of the nanofibril.4
The structural heterogeneity of native cellulose is carried over
to synthetic cellulose-based biomaterials, both where the
hierarchical structure of the woody tissue is conserved, such as
in transparent wood,5,6 or where the CNFs have been
disintegrated from the plant cell wall.
The strong nanostructural heterogeneity that exists in both
man-made and natural fibers is undoubtedly a key factor
responsible for the complex structure−function relationships of
the final polymeric product. Hence, being able to quantitatively
describe these materials with nanoscale spatial and chemical
resolution would be of great practical importance for a better
control of the design of new functional materials with finely
tuned responses. The physicochemical structure of fibers has
been studied by using advanced structural characterization
techniques, such as synchrotron X-ray and neutron scattering.
These methods provide valuable structural information about
the crystalline regions but are poorly informative about
disordered domains that yield excessively broad diffraction
peaks.7 Particularly in the case of highly heterogeneous
Received: March 8, 2019
Revised: September 9, 2019

© XXXX American Chemical Society A DOI: 10.1021/acs.macromol.9b00472

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Figure 1. Hierarchical structure of the cellulose fibril aggregate (FA) model. (A) The four nanofibrils are arranged in antiparallel configuration, with
the direction defined by the direction of the glucan chains within each fibril. (B) The model assumes a square cross section made up of 36 glucan
chains. It distinguishes between chains located in surfaces and in the nanofibril core. Surfaces are further classified as either accessible or less
accessible, with respect to water, and chains located at fibril corners. (C) The cellulose Iβ unit cell contains two nonequivalent chains, named center
and origin. This distinction is inferred also on the noncrystalline surfaces, which further can be classified as (110) or (1−10) depending on which
crystal plane they reside upon. (D) Molecular graphics showing the hydrated FA model used in the MD simulations. Surfaces are red, the core is
black, and water molecules are shown in gray. Accessible and less-accessible surfaces are fully and partially hydrated, respectively. (E) Atomic
structure of the glucan chain, going from the nonreducing (left) to reducing end (right), with the atom naming used herein. Note that the 2-fold
screw symmetry makes the exocyclic hydroxymethyl group (C6−OH6) point in alternating directions.

materials like cellulose, 13C solid-state NMR spectroscopy has
contributed significantly to supramolecular structure elucida-
tion, thanks to its specific capability to partially resolve ordered
and disordered regions.8−12 In addition, spectrally resolved 13C
longitudinal relaxation time (T1) measurements have enabled
qualitative assessment of the local reorientational dynamics of
the 13C−1H bonds.8,13,14
Recently, Terenzi et al.15 have experimentally studied by 13C
CP-MAS T1 measurements the effect of varying the moisture
content on the polymer dynamics in cellulose and xyloglucan.
Other earlier similar 13C CP-MAS NMR works8,9,16,17 typically
reported only one single average T1 value per carbon site and/
or focused only on C4 atoms by means of measurements that
were often undersampled due to excessively long experiment
duration typical of 13C CP-MAS. A remarkable exception is the
work of Fernandes et al.,13 who observed a bimodal relaxation
behavior in SP-MAS measurements that, however, are not
designed for probing long T1 ranges and could not be further
decomposed into multiple dynamic modes due to low signal-
to-noise ratio. As a significant step forward, Terenzi et al.15
introduced site-specific 13C T1 distributions, obtained by
inverse Laplace transformation (ILT) of raw data acquired
with sufficient signal-to-noise ratio and over long enough spin
evolution times (see Figure S1) under CP-MAS conditions
that enable sampling both short and long T1 ranges. In full
analogy to widely explored water 1H T1 distributions, under
suitable experimental conditions the polymer 13 C T 1
distributions enable conjointly probing both local macro-
molecular dynamics and its heterogeneity,15 which cannot be
described adequately in the framework of just a single-8 or two-
component13 dynamic model per carbon site. Indeed, at each
hydration level, the experimental 13C T1 distributions of CNFs
covered a very broad range, namely between 10−2 and 103 s,
suggesting the existence of distributions of both fast and slow
molecular motions on the nanosecond time scale. Fast and
B DOI: 10.1021/acs.macromol.9b00472
slow dynamics were also observed by Fernandes et al.13 in
isolated cellulose: ring carbons were reported to be the most
rigid ones, and C6 signals yielded several groups of 13C T1
values that were attributed to varying polymer mobility over
different hydroxymethyl conformers (see the Results and
Discussion section). Yet, linking the complex macromolecular
dynamics with local structural heterogeneity by experimental
means only remains somewhat conjectural and is practically
still an open challenge. To address this challenge, Chen et al.18
recently demonstrated that MD simulations of 13C T1
distributions of the amorphous polysaccharide xyloglucan
enable reproducing experimental NMR results and interpreting
them at an atomistic level that cannot be accessed by NMR
measurements alone.15
Inspired by that significant advance, the same methodology
is here further developed with the aim to study the more
complex hydrated structure of CNFs. Specifically, we set out to
demonstrate the capability of MD simulations in disentangling,
with unprecedented nanoscale spatial and chemical resolution,
localized macromolecular motions within core or surface
regions of the heterogeneous CNF structures at high degree of
hydration. To this end, 13C T1 distributions are calculated
directly from MD simulation trajectories and compared to the
experimental ILT results. Significant differences in the
macromolecular dynamics could be observed among structur-
ally distinct regions of CNFs, such as locations of individual
polymer chains, surfaces, and interfaces. In a longer term
perspective, the present methodology will enable a more
quantitative understanding of structure−function relationships,
in both natural and synthetic high-strength fibers with well-
controlled hydration conditions. Ultimately, this robust
approach will contribute to support future efforts toward the
development of improved high-performance polymeric materi-
als.
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Table 1. Average 13C T1 Values (in seconds) Obtained from Simulated and Experimental T1 Distributions as Well as from
Fitting of Experimental Raw Data before ILT Processing (See Also Figure S1)a
C1b C235c C4 (core)d C4 (surface)e C6 (core)f C6 (surface)g
total
experimental (ILT) 50 34 105 25 34 1.6
experimental (fit) 49 33 106 20 28 1.8
MD 77 69 161 49 20 2.1
core (simulated values)
inner 167 148 193 27
intermediate 136 121 152 18
surface (simulated values)
accessible 52 50 58 1.8
less accessible 61 53 64 4.0
corner 22 20 21 0.8
accessible surfaces (simulated values)
center 36 39 45 1.4
origin 73 64 76 2.2
110 57 54 60 2.3
1−10 46 47 57 1.4
C6 exterior 39 40 43 0.2
C6 interior 68 63 79 14
a
The average spread of simulated T1 values is 2%, and in all cases it is <6%, while the error in the experimental values from ILT, due to
measurement reproducibility, is within 10%; the corresponding fitting error on the raw data is within 7−15%. Simulated values are further
decomposed into average contributions from various distinct sites within the model (see Figure 1E and text). b105.5 and 102.6 ppm. c75.6 and 72.9
ppm. d89.5 ppm. e84.3 ppm. f65.6 ppm. g62.5 ppm (see 13C CP-MAS spectrum in Terenzi et al.15).

■ METHODS
The presence of cellulose FA in the secondary cell wall and in
chemically processed wood fibers (pulp) is well established.19 A
model FA was therefore built based on the X-ray and neutron
diffraction results for highly crystalline cellulose Iβ samples.20 In the
present model, the elementary unit within the structure, the CNF,
contained 36 chains, each 40 anhydroglucose units long, arranged in a
square cross section with the crystallographic (110) and (11̅0)
surfaces exposed. Recent advances in both experimental character-
ization of fibril dimensions and understanding of plant cellulose
synthesis show that the wood nanofibril may contain as few as 24, or
even 18, cellulose chains. The chains can further be arranged in
various cross sections, commonly assumed to be either hexagonal or
rectangular, that differ in which crystallographic planes are exposed to
the surroundings.13,21,22 Although this can be assumed to affect the
physicochemical properties of the fibrils, regardless of which structural
model is assumed, they all share the same basic structural hierarchy:
partially disordered surface-accessible chains surrounding a crystalline
core, albeit in slightly different ratios. They also share the other
structural features considered here (Figure 1), which means that the
36-chain model used in this work in this respect is qualitatively similar
to any of the smaller fibril models.
The model fibril was subsequently used to build a model FA. Here,
one faces the choice of aligning the CNFs in either parallel or
antiparallel configuration. There are a number of experimental
observations in favor of an antiparallel arrangement. Swelling of
wood pulp fibers in concentrated NaOH aqueous solution followed
by regeneration in water (mercerization) inevitably produces cellulose
II. In this crystal allomorph, the individual glucan chains are known to
be antiparallel.23,24 These facts can hardly be reconciled without
having antiparallel entities in close proximity in the fibril aggregate. In
fact, partial conversion was also observed in intact wood cell walls,25
which agrees with the direct observation of cellulose synthases moving
in opposite direction in native Arabidopsis.26 Furthermore, antiparallel
arrangement of fibrils was also observed in Valonia by using
transmission electron microscopy.27−29 The notion of antiparallel
fibrils was also recently used to explain the conversion of wood
cellulose into a pseudo-orthorhombic structure during hydrothermal
treatment.30 Based on these observations, the model FA used here
consisted of four cellulose nanofibrils assembled in an antiparallel
manner (Figure 1A). In reality, the situation is complicated by the
possibility of having a mixture of both antiparallel and parallel fibrils
as well as other factors that may influence the properties of the fibril−
fibril interface. Such factors include twisting of individual nanofibrils,
which would prevent aggregation of crystallographic planes13 and,
furthermore, based on the observation that native aggregate sizes
depend on both lignin and hemicellulose concentration,31 the
possibility of having cell wall matrix material also inside the FA.
The FA was initially positioned in a periodic simulation box with
dimensions 10 × 10 × 22 nm3, with a distance between two adjacent
fibrils of 0.3 nm. A total of 14585 water molecules, corresponding to
the moisture content15 measured experimentally at a relative humidity
(RH) of 92%, were subsequently inserted in the system; out of these,
around 1500 water molecules were added in between the fibrils, at the
less-accessible surfaces. This amount corresponds to experimental
indications.32
MD simulations were run for 300 ns using GROMACS version
2016.3.33 Potential parameters for cellulose were taken from the
GLYCAM06 force field,34 and water was modeled by using TIP3P.35
Distributions of 13C NMR relaxation times and their means were
calculated from the Fourier transform of the rotational autocorrelation
function of the corresponding C−H vector,36 as described in more
detail in the Supporting Information. Relaxation contributions from
dipole−dipole interactions other than that with directly bonded
hydrogens or from any spin couplings other than dipole−dipole
interactions are neglected. Enhanced relaxation effects for carbons
having more than one directly bound hydrogens (nH > 1) are
represented by simply multiplying by nH the relaxation rate yielded by
the behavior of a single C−H bond. This is a conventional
approximation,37,38 well suited for experiments with strong dipolar
decoupling18 and carbon sites, such as the exocyclic carbon C6, where
there is no symmetry between the two involved hydrogens.
All production runs were performed in the NVT ensemble, with
the temperature maintained at 300 K using the velocity-rescaling
method.39 The cutoff for Lennard-Jones interactions and the real-
space part of the electrostatic interactions was 1 nm. Long-range
electrostatics was included using PME.40 The equations of motion
were integrated using a leapfrog algorithm with a basic time step of 2
fs. Constraints were applied to all bonds involving hydrogen atoms by
using P-LINCS.41

C DOI: 10.1021/acs.macromol.9b00472
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Figure 2. Comparison of experimental (dashed line) and simulated (solid line) distributions of 13C T1 relaxation times for selected carbon atoms.
The experimental curves were adapted from Terenzi et al.15 Within each graph, simulated and experimental distributions are normalized to the
same value.
■ RESULTS AND DISCUSSION
The 13C NMR spectrum of CNFs is partially resolved with
respect to signals from the chemically nonequivalent carbons
C1, C4, and C6 (Figure 1E), while signals from C2, C3, and
C5 atoms exhibit significant overlap.42 The 13C chemical shift
values for cotton cellulose have been reported and assigned in
the literature as follows:10,43 C1 (106.3, 105.2 ppm), C4 (89.8,
85.1 ppm), C2, C3, C5 (72.4, 73.5, 75.3 ppm), and C6 (66.2,
63.8 ppm). These chemical shift values are consistent with the
experimental results of Terenzi et al.,15 here summarized in
Table 1. We note that both the signals of C4 and C6 partially
split into two peaks: these are assigned to glucan chains that
belong either to nanofibril surfaces or to the crystalline
nanofibril interior.16,44
The reason for the splitting of the C4 and C6 peaks can be
traced to the conformations of the hydroxymethyl groups. The
hydroxymethyl group in cellulose, as in all hexopyranoses,
adopts three more or less stable conformations, named tg, gg,
and gt. These letters stand for trans and gauche, respectively,
where the first letter refers to the rotation of the C5−C6 bond
with respect to O6 and O5 and the second one refers to the
C5−C6 bond rotation with respect to O6 and C4.45 From 13C
D DOI: 10.1021/acs.macromol.9b00472
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NMR data on known crystal structures of cello-oligomers,46 it
was shown that tg has higher chemical shift than both gg and gt,
which was assumed to be the cause for splitting of the C6
signal also in the 13C NMR spectrum of cellulose. This
assignment was recently corroborated by density functional
theory calculations, where it was also noted that the
hydroxymethyl conformation affects the chemical shift of C4
in a similar manner as for C6, which can explain the splitting of
that peak as well.47 Thus, since tg dominates in the crystalline
regions of native cellulose,20 the gg/gt signals for both C6 and
C4 become significative of fibril surfaces. However, the
assignment of individual chemical shifts to location within
the fibril assembly is complicated by the fact that there is no
unambiguous one-to-one mapping between hydroxymethyl
conformation and structural entity (e.g., surface or core), as
they all possess a distribution of both chemical shifts48 and
conformation.49
By performing spectrally resolved 13C T1 measurements, it is
possible to obtain information about local C−H dynamics at
each carbon site. This has, for example, unravelled that the
exocyclic carbon C6 is more mobile; i.e., it has shorter 13C T1
than the ring carbons C1 to C5.15 This can be explained by the
larger conformational degree of freedom of C6 as compared to
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the ring carbons and the resulting faster rotational dynamics of
the hydroxymethyl group, whereas relaxation times for C1-to-
C5 sites to a larger extent reflects the more restricted glucan
backbone dynamics. Furthermore, the shorter T1 for C4 and
C6 surface chains, when compared to those for the respective
crystalline peaks, can be understood in terms of the less
constrained mobility of surface chains14 as compared to that
for the chains in the fibril interior.
Such link between dynamics and structural properties has
certainly been instrumental for improving our understanding of
the nature of cellulose supramolecular organization. However,
the resolution of 13C NMR spectroscopy is limited by chemical
identity of carbon sites and by the need for multicomponent
spectral deconvolution to resolve partially overlapping
contributions from surfaces and core regions.8,16 MD
simulations do not suffer from such limitations and can
therefore be used as a powerful tool to probe dynamics in the
range from picoseconds up to hundreds of nanoseconds, with a
resolution down to individual atoms. A crucial element of the
work here is to compare detailed experimental 13C relaxation
time distributions, to corresponding distributions obtained
from simulations. Specifically, 13C T1 and their distributions
were simulated for the different chemically nonequivalent
carbons (Figure 1E) in a hydrated cellulose FA, at conditions
closely corresponding to those in the experimental study of
Terenzi et al.15 The good quantitative match between
experimental and simulated mean relaxation times and
distributions lends credibility to the simulation results, in
terms of both model and interaction parameters. Furthermore,
the excellent consistency between the mean experimental T1
values obtained either from the T1 distributions, after ILT
processing, or from the original raw data, by biexponential
fitting (see Figure S1 and Table 1), confirms the reliability of
the experimental data here used as a reference for the MD
simulations. Based on this method validation, our combined
NMR-MD approach is here exploited to interpret the results
based on the structural details revealed in the simulations.
Mean 13C T1 Values: Site-Specific Macromolecular
Dynamics. Outside the extreme narrowing regime, such as in
the present case, short T1 values correspond to short motional
correlation times and thus to high C−H mobility.50 Here, the
simulations reproduce the important trends in the mean
experimental 13C T1 relaxation times. Indeed, we find (see
Table 1) that C6 has shorter T1 values than the ring carbons,
and surface chains have shorter T1 than the nanofibril interior.
We also find that the C−H bond mobility for C2, C3, and C5
is higher than for C1 (and C4, average not shown in Table 1).
This can be rationalized by considering that the effect on C−H
bond orientations from ring puckering motions is larger for C2,
C3, and C5. This particular contribution to the relaxation is
relatively more important in the core than in surfaces, where
the dynamics around glycosidic bonds is less restricted.
Simulations resulted in average 13C T1 values that were
systematically larger, by up to a factor of 2, than the
experimental ones (Table 1). Although this discrepancy may
seem large, it is in reality a good agreement, considering how
sensitive the dynamics is to the exact parametrization of, for
example, the dihedral potentials involved. Those are usually
optimized to reproduce equilibrium distributions,51 with little
attention paid to the kinetics. This may not be the only reason,
however, and this point will be further discussed below.
13
C T1 Distributions: Intrinsic Dynamical Heteroge-
neity. In the following, the discussion of T1 distributions
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focuses solely on the different behavior of C1, C4, and C6,
while ring carbons C2, C3, and C5 were all found to follow a
similar behavior as C1. Figure 2 shows the simulated and
experimental T1 distributions for C4 and C6, separately for
core and surface chains. The first important evidence is that
both experimental and simulated distributions are very broad,
even after core and surface contributions were disentangled,
and cover, in each plot, approximately the same number of
decades along the T1-axis. Specifically, the simulated C4
distributions cover the ranges 10−2000 s and 1−1000 s, for
core and surface respectively. The simulated C6 distributions
are respectively about 2 and 1 decade broader toward shorter
T1 values, covering about 0.2−1000 s (core) and 0.4−500 s
(surface). The T1 ranges covered by the experimental
distributions are basically the same as those seen in the
respective simulations, with excellent agreement at short T1
values, while a small narrowing effect, by a factor of up to 2−
2.5 for surface chains, is observed at long T1 values. We also
note that the polymers underlying the surface chains, termed
“intermediate core” in Table 1 and identified as having
conformations distinguishable from that of the chains in the
innermost core,48 exhibit dynamics that is similarly distinguish-
able from the dynamics in the innermost core.
This substantially good agreement between NMR and MD
distributions, in terms of their overall T1 range, enables safely
neglecting possible artifactual excessive broadening due to ILT
processing52,53 of natural abundance 13C NMR data if, for
example, those are acquired with too low signal-to-noise ratio
and/or insufficient data sampling. This proved not to be the
case of our similar measurements of amorphous xyloglucan,15
where a comparably good agreement with MD simulations was
previously observed.18 For completeness, we also note that in
the experimental data and for both C4 and C6 sites there is
always some degree of spectral overlap between peaks
belonging to crystalline (interior) and surface chains, which
does not exist in the MD simulations. Yet, by varying the
integration range of the NMR spectral peaks (data not shown
here), no appreciable variation in the resulting T1 distributions
was observed.
We can thus conclude that an intrinsically large variation in
terms of polymer dynamics exists in CNFs, even over moieties
that are identical, both chemically and in their basic spatial
classification. We also note that the dynamical heterogeneity of
C6 core and surface sites, as seen by both NMR and MD
simulations, is much more pronounced than in the respective
C4 domains.
Yet, some important differences between MD and NMR in
the shape of the respective distributions are noticeable in
Figure 2, and to explain those discrepancies the main factor to
be taken into account is the role of cellulose processing and
sample preparation. Indeed, a biexponential fitting analysis (see
text and Figure S1) of experimental raw data, before the ill-
posed ILT procedure was applied, confirmed the non-
monodispersed character observed in the experimental
distributions.
In the case of C4, the experimental distributions present, at
short T1 values, a higher signal density population than the
simulated ones and a correspondingly lower intensity at long
T1 values. It is likely that the real sample contains a fraction of
highly mobile glucan chains54 that could, for instance, be
connected to naturally occurring disordered regions in wood
CNFs or to defects induced during processing, e.g., during
disintegration from the wood fiber cell wall, that are not
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present in the model. Furthermore, in the real CNF sample
measured by NMR a fraction of residual hemicellulose was
present54 in the amount of 12.3 wt %. Both defects and
residual hemicellulose are expected to behave like a hydrated
amorphous material. As previously seen in hydrated
xyloglucan, which has mean T1 values at 92% RH that are
even lower than for cellulose surface chains (e.g., 8 s in the case
of xyloglucan C4 compared to 25 s in cellulose),18 such a
mobile polymer phase would exhibit a relatively higher signal
density population at short T1 values. As fast motional modes
become more abundant, slow modes at long T1 values are
expected to become correspondingly less populated, as
compared to a system devoid of defects and residual
hemicellulose such as the model used here for the simulations.
This is indeed what is observed in Figure 2.
In addition, we note that the 13C NMR chemical shifts for
the implicated disordered and/or defect-rich regions in C4 are
expected to be closer to those of surface chains than to those
for the crystalline core chains. Hence, any contribution from
disordered regions or hemicellulose in the real sample should
add to the experimental signal assigned to surface chains.
Indeed, the existence of a larger difference between the shapes
of the NMR and MD distributions for the surface chains than
between those for the interior chains supports this hypothesis.
Based on this interpretation, the presence of disordered/
defective regions and residual hemicellulose, alongside the
choice of potentials in MD calculations, can also explain well
the larger difference between experimental and simulated mean
T1 values (see Table 1) for the surface chains (on average, by
more than a factor of 2) as compared to those for the interior
chains (∼40% average difference).
The simulated T1 distributions of C6 are bimodal, with two
well-separated peaks positioned around 0.4−1 and 40 s toward
the edges of the T1 axis. This is most clearly visible for the case
of surface chains, where the short-T1 peak is more intense. The
experimental distributions do not display such neat bimodality,
although they cover the same decades along the T1-axis.
In the case of C6, we cannot interpret differences between
experiments and simulations in terms of defects because those
affect mainly the backbone carbon sites; instead, the effect of
residual hemicellulose is expected to be similar to that
observed for C4. Other sources of artifacts, e.g., due to ILT
processing, can be safely discarded here, as it was anticipated
above. We then note that in our previous work on hydrated
xyloglucan18 we argued that the inherently longer time scales
for experiments, as compared to simulations, could lead to a
more efficient motional averaging of dynamical differences in
the random and loose molecular network of xyloglucan, which
was detected as a symmetric narrowing of the experimental
relaxation distributions. However, in cellulose the chain
positions are fixed, and thereby such an effect is expected to
be negligible. On the other hand, the high-amplitude molecular
motions of C6, associated with conformational transitions as
described in detail in the Results and Discussion section, are
expected to be strongly sensitive to the accuracy of the
representation of dihedral potentials in the simulations.51 This
effect could explain the pronounced discrepancy observed for
core C6 sites in the long-T1 range.
Having demonstrated the intrinsic dynamical heterogeneity
in cellulose, in the next section we shall attempt to identify the
underlying structural causes for it by performing selective
decomposition of the MD results.
F DOI: 10.1021/acs.macromol.9b00472
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Nanoscale Structural Heterogeneity in CNFs. To gain
insight into the structural effects associated with the
polydispersed dynamical modes within CNFs, we resort to
simulations and to their ability to decompose the 13C T1
distributions, calculated on a per-atom basis, into basically any
imaginable deconvolution criteria. One illustrative example of
this is shown in Figure 2 where in full analogy to C4 and C6
data the T1 distribution for C1 is decomposed into its
respective contributions from fibril interior and surface chains.
We note that such a distinction could not be achieved for
NMR experimental data due to the lack of sufficient chemical
shift contrast between the overlapping core and surface
populations. As expected, the width, shape, and T1 location
of the core and surface distributions for C1 resemble both the
simulated and experimental results for C4 (Figure 2).
Nanofibril Core: The Crystalline Region. As demonstrated
in Figure 2, the 13C T1 distributions remain broad even when
the core regions are investigated separately. This is surprising
since the core region has a high degree of order, as also
indicated by its far slower average mobility (see Table 1). The
source of that broadening can be investigated by, first, further
dividing the core region into (i) an intermediate layer, which
contains the chains immediately below the surface chains, and
(ii) the inner core. The former, intermediate layer, which could
be expected to have properties in between that of a highly
crystalline and a disordered surface layer, has been proposed to
be the source of the paracrystalline signal that was used in the
spectral decomposition of the 13C spectrum.8,16 Although the
distribution for the intermediate layer is shifted to shorter T1
values as compared to that for the inner core (see Figure S2),
this shift is not significant compared to the width of the
individual distributions, whose shape is also quite similar.
Hence, the question remains: what is the molecular origin of
the wide T1 distribution for highly ordered regions of CNFs,
such as their crystalline core? A preliminary indication is
provided by a spatial mapping of high/low T1 values as a
function of glucose positions within the FA model (Figure 3).
As expected, a higher concentration of high-mobility sites in
the intermediate layer (the outermost layer in Figure 3) is
visible, which indicates that the inner core is the most
constrained part of the crystalline region. But it also shows that
the more mobile regions are not homogeneously distributed
along the length of the fibrils, but instead exhibit a degree of
clustering. Two possible structural causes for this heterogeneity
are stresses either caused by (i) nanofibril−nanofibril contacts
and/or (ii) varying degree of hydration over the less-accessible
surfaces. As such stresses may influence the response of
cellulose to, for example, chemical modification, this feature
deserves further studies in the future.
As indicated in Table 1, the exocyclic C6 is more mobile
than other carbons within the core. Rotational mobility around
the C5−C6 bond involves transitions between the three
conformational states discussed above. In the core (as opposed
to the surfaces), most (>96%) hydroxymethyl groups are in the
tg state, as expected for crystalline cellulose Iβ.20 Yet, our
simulations also indicate rare conformational transitions into
the minor gg and gt states with a mean transition time of about
4 ns. Although due reservations must be retained considering
the large sensitivity of those transition times to the potentials
involved, the existence of such conformational transitions can
certainly cause appreciable broadening of the C6 T 1
distributions.
Macromolecules XXXX, XXX, XXX−XXX
Macromolecules
Figure 3. Simulation snapshots depicting C1 atoms whose T1 values
are (i) <50 s and are therefore in the most mobile segments (red
spheres), (ii) >400 s are therefore in the most rigid segments (blue
spheres), and (iii) small black dots indicating C1 atoms in segments
with roughly average mobility. The FA is viewed along the fibril
direction (A) and from the side (B).
Accessible and Less-Accessible Surfaces. Native and
processed cellulose typically exists as FA. As shown in Figure
1B, aggregation gives rise to a possible distinction between
nanofibril surfaces located at the exterior of a FA and those
within. In wet systems, the former ones are readily accessible to
water, whereas the latter ones, although still hydrated to some
extent, are in very slow exchange with bulk water and exhibit
more restricted and slower molecular dynamics.16,32,55 In the
following, these distinct regions are termed accessible or less-
accessible surfaces, respectively.
Interestingly, as can be seen in Figure 4, the C6 T1
distributions for both surfaces retain a bimodal character,
although as expected the short-T1 part of the distribution is
more suppressed for the less-accessible regions. This part of
the distribution is likely to have its origin in the large-
amplitude motions connected to rotation of the hydroxymethyl
group, which is naturally more restricted at the less-accessible
surfaces. Nevertheless, the effect is small, particularly so if we
G DOI: 10.1021/acs.macromol.9b00472
Article
Figure 4. Decomposition of simulated 13C T1 distributions of surface
chains into contributions from accessible surfaces, less-accessible
surfaces, and corner chains.
consider that local mobility in an amorphous polysaccharide,
such as xyloglucan,18 has been shown to be very strongly
dependent on the global level of hydration, a parameter that
locally differs a lot between accessible and less-accessible
surfaces. The local order imposed by the nanofibril surfaces,
which is lacking in hydrated xyloglucan, presumably dampens
the sensitivity of chain dynamics to the presence of water.
Wickholm et al.16 reported surface-specific estimates of C4
T1 values based on a small (0.2−0.8 ppm) difference between
the C4 chemical shifts for accessible and less-accessible
surfaces in combination with spectral deconvolution. They
indicated mean T1 values for less-accessible surfaces in cotton
linters that were roughly twice as long (39 s) as for the
accessible surfaces therein (11−18 s). This result is not
reproduced in the present model, which gives approximately
identical mean values for those two surfaces. We have two
possible explanations to offer. First, all nanofibril−nanofibril
contacts are antiparallel in the model used here, separated by at
most a single layer of water molecules, whereas in the
experimental system there is certainly a possibility for parallel
arrangement as well, which would permit cocrystallization.
Second, when two fibrils come into contact, the process
involved for attaining the lowest energy conformation may be
slow compared to the MD time scales. Thus, there is an
inevitable risk that the FA model may not have reached
equilibrium in this regard. In both cases, fibril−fibril
interactions at the less-accessible surfaces could be under-
estimated in the model, yielding a too-high mobility close to
that for accessible surfaces. To investigate this, the number of
hydrogen bonds formed between fibrils was calculated based
on standard geometrical criteria: a donor−acceptor distance
<0.3 nm and a hydrogen donor−acceptor angle <35°. In
equilibrium, this number is expected to be constant, though
fluctuating, over long time scales. However, the simulations did
indeed exhibit a small drift in fibril−fibril hydrogen bonds,
increasing by ∼5% during the full 250 ns run (Figure S3). In
addition, as mentioned in the Introduction, there is a range of
other factors that could potentially contribute to the
heterogeneity in real FAs. This includes twisting of nanofibrils
as well as the possibility of having hemicellulose present inside
Macromolecules XXXX, XXX, XXX−XXX
Macromolecules
Figure 5. Decomposition of simulated 13C T1 distributions of accessible surfaces into contributions from (A) glucose residues with hydroxymethyl
orientations either exterior or interior and (B) chain identity (center or origin).
the aggregates, both of which would lead to increased disorder
in the less-accessible surfaces.
Regardless of which fibril cross section is assumed, there will
be chains at the corner between different surfaces, which makes
the assignment of these surfaces with respect to their
accessibility to water ambiguous (see Figure 1B). These chains
are also the least constrained by neighboring chains. From the
simulations it was found that corner chains exhibit a broad T1
distribution, shifted toward short T1 values as compared to the
total distribution, meaning that corner chains are more mobile
than the average surface chain. This is an illustration of the
level of detail possible with the present methodology.
Role of Surface Orientation for Hydroxymethyl Groups.
Throughout surface chains, the glucose unit alternatively has
its hydroxymethyl group pointing outward, to the exterior of
the fibril, or inward, to the interior of the fibril (Figure 1E).
When the distributions for surface C6 carbons were
decomposed based on this distinction, a clear trend was
found. For C6, the population of interior groups accounted for
the long T1 values, while exterior groups accounted for the
short T1 values (Figure 5A and Table 1). It comes as no
surprise that this orientation influences the T1 of C6, since the
motions of hydroxymethyl groups oriented toward the fibril
interior are obviously restricted compared to the exterior ones.
As noted above, crystalline cellulose have its hydroxymethyl
groups almost exclusively in tg, whereas disordered chains (e.g.,
surfaces) primarily exhibit gt and/or gg. This distinction
between core and surfaces with respect to the hydroxymethyl
conformation was seen in MD simulations already 20 years
ago.56 It is also reproduced in the present work, where the core
chains exhibits >96% tg, the rest having gg conformation.
For surfaces, the relative population in tg is 37%, but when
also the orientation is taken into account, it is found that for
exterior surface hydroxymethyl groups the population is
distributed as (tg:gg:gt) 7:27:66, and for interior groups
H DOI: 10.1021/acs.macromol.9b00472
Article
62:37:1, which is in qualitative agreement with earlier
simulations using the same force field.57 On the basis of
NMR measurements on CNF that was repeatedly TEMPO-
oxidized followed by alkali extraction, Funahashi et al.58
concluded that gg was the dominating conformer of the
exterior surface C6 groups, while gt was dominating for the
interior ones. The same assignment was also made by
Fernandes et al.13 However, Phyo et al.49 reported based on
2D 13C NMR that the gt conformer is the dominating one in
surface chains. Their conclusion correlates well with the results
presented here for the exterior surface C6, although the
simulated population in gg is higher than what was detected in
the experiments. The distribution for interior groups, on the
other hand, is closer to what is expected for the core, with tg
being the dominant conformer.
The mean transition time between conformations is 0.5 ns
for the exterior C6, 2 ns for interior, and, as mentioned above,
4 ns for the crystalline core. Obviously this is much shorter
than the NMR time scales and also sufficiently fast to
contribute to 13C relaxation times. This contributes to the
complexity of the C6 region of the 13C NMR spectrum,
presumably consisting of three populations which are all
substantially overlapping: one almost exclusively in tg (core),
one with almost no tg at all (surface exterior), and one in
between these two (surface interior). This distinction has to be
accounted for a correct interpretation of the relative signal
intensities.59 Importantly, the short transition time among
different conformers indicates that experimental NMR results
that have been obtained in a given finite chemical shift region
cannot be assumed to arise from unique distinct conformers.
Instead, also considering that line widths are quite significant
as compared to the chemical shift differences between distinct
conformers, one cannot exclude a significant admixture of
other conformational states in any data recorded in a set
Macromolecules XXXX, XXX, XXX−XXX
Macromolecules
chemical shift range. Hence, the results of Phyo et al.49 may
permit a small yet nonvanishing tg population in surface chains.
The hydroxymethyl populations in the accessible surfaces
were stable in the simulations, but in the less-accessible
surfaces, just as for the fibril−fibril hydrogen bonds, the
simulations exhibited a small drift, where gt was slowly
increasing, at the expense of tg, a further indication of the
extremely long time scales needed to bring the fibril−fibril
interface to equilibrium (Figure S4). In addition, the
hydroxymethyl orientation also affects the local mobility of
the backbone carbons (C1 and C4) within the same glucose
unit. Although the distributions are overlapping to a large
extent (Figure 4), their means differ by a factor of 2 (Table 1).
This result stands in contrast with a previous study where the
C6 orientation did not affect simulated mean T1 for C460 at all.
In that same study, a significant difference was detected at full
hydration for mean C4 T1 values in surface chains on top of
the crystallographic (110) or (11̅0) planes. This difference is
not reproduced here. As one explanation, one has to keep in
mind that the model used in that study was far less detailed
and realistic both as concerning atomic (such as no explicit
inclusion of aliphatic hydrogens) and nanofibril (a semi-infinite
crystal model instead of a nanofibril aggregate) arrangements.
Although equilibrium properties are less affected, coarse-
grained schemes are known to influence dynamic properties,
primarily by smoothing out the potential energy surface, which
in turn leads to a speed-up of the kinetics.
Center and Origin Chains. In addition to the structural
distinctions discussed above, the cellulose Iβ structure contains
two distinguishable molecules that are present in the crystal
unit cell, termed center and origin chains, respectively,20 where
one chain is located at the origin of the unit cell and the other
at its center (Figure 1C). This implies that half of the molecules
in CNF are origin chains and the other half are center chains.
Experiments indicate that these chains show differences in
terms of structural parameters, such as puckering of the
pyranose rings, conformation of glycosidic linkages, and
orientation of the hydroxymethyl group as well as hydrogen-
bonding pattern.20,51 These structural differences impart small
variations in the 13C NMR chemical shifts of C1, C4, and C6
in highly crystalline samples.61 The most important geo-
metrical differences between center and origin chains are also
reproduced in simulations with the GLYCAM06 force field.57
Yet, within an infinite crystal one would expect the difference
in dynamics between center and origin chains to be rather
small, since the coordination of the glucose units in them is
very similar. However, this is not valid anymore when the
chains are located at fibril surfaces. From the present
simulations, we find that average 13C T1 values from surface
origin chains are consistently larger than the corresponding
values from surface center chains (Table 1). The underlying
reason can be seen in Figure 5B, which shows that for the case
of C1 and C4 center and origin chains give rise to two distinct
subpopulations in the T1 of accessible surfaces. For C6, on the
other hand, the differences are negligible, presumably because
faster dynamical modes dominate.
Interestingly, when considering also the orientation of C6,
one can see that for residues with C6 oriented to the exterior,
the difference between center and origin chains observed for
C1 and C4 relaxation becomes stronger, while for those with
C6 oriented toward the interior, the difference disappears
(Figure S5). This means that there is a difference in backbone
dynamics between center and origin chains that is tangible
I DOI: 10.1021/acs.macromol.9b00472
Article
when the hydroxymethyl group in the same residue is free to
rotate. This is further strengthened by the observation that
there is no significant difference in C1 and C4 relaxation
between center and origin chains at less-accessible surfaces,
where also exterior hydroxymethyl groups are more con-
strained. We tentatively identify center and origin chains as
those two distinct populations of surface chains that have been
termed f and g by Wang et al.,48 which have been observed to
exhibit different average conformations but to appear well-
mixed and hydrated.
■
CONCLUSIONS
In the present work, 13C NMR T1 distributions in a model
cellulose fibril aggregate were calculated from MD simulation
trajectories. Good agreement between simulated and exper-
imental distributions was demonstrated for the spectrally
resolved carbons C1, C4, and C6 separately. Both simulated
and experimental distributions were broad, indicating a large
variation in dynamics over chemically equivalent 13C groups.
The results showed that this dynamical heterogeneity to a large
extent is a consequence of the structural heterogeneity in
cellulose. Utilizing the full atomistic resolution of MD
simulations, it was possible to reach beyond the mere
distinction between surface and core chains, which can also
be made in experiments, and investigate effects from more
subtle structural variation such as orientation of the
hydroxymethyl group and differences between center and
origin chains. This demonstrated the strength of the present
methodology since these are features that cannot be resolved
by using experiments alone.
From a materials perspective, nanocellulose is unique
compared with other high-strength fibers and other nano-
particles in that there is substantial dynamics in the reinforcing
phase. Today, there are many types of nanocelluloses available.
They originate from different sources and disintegration
procedures, which give them different fibril size distributions
and degrees of order. These nanocellulose materials typically
undergo various chemical modifications and also have different
hygrothermal histories (e.g., in terms of drying and rewetting),
which severely affects the state of aggregation. Even common
chemical pulp consists of cellulose fibril aggregates, with a
structural heterogeneity that is dependent on its history. Here,
the combination of spectrally resolved 13C NMR relaxometry
and MD simulations is shown to be a excellent tool for learning
about supramolecular structure and organization, and its effect
on the state of macromolecular mobility in the presence of
moisture. Specifically, we show that the interplay between
structure and dynamics is highly complex and that even the
seemingly homogeneous crystalline core exhibits heteroge-
neous dynamics that varies over an order of magnitude. The
methodology can be easily adapted to study other natural
fibers, e.g., chitin, polymer matrices in nanocomposites, native
hemicellulose/cellulose systems in plant cell walls, or synthetic
fibers, with a level of detail that goes far beyond the
experimental limit.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.macro-
mol.9b00472.
Macromolecules XXXX, XXX, XXX−XXX
Macromolecules
Experimental site-resolved T1 buildup curves with
respective biexponential fitting results; computational
details for calculation of NMR relaxation times;
decomposed T1 distributions for the core, accessible
surfaces, and less-accessible surfaces; number of fibril−
fibril hydrogen bonds; conformation of hydroxymethyl
groups in less-accessible surfaces (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: jacke@kth.se.
ORCID
Pan Chen: 0000-0003-3794-717X
Camilla Terenzi: 0000-0003-3278-026X
István Furó: 0000-0002-0231-3970
Lars A. Berglund: 0000-0001-5818-2378
Jakob Wohlert: 0000-0001-6732-2571
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was partially supported by the Wallenberg Wood
Science Center (WWSC) funded by the Knut and Alice
Wallenberg Foundation, the Swedish Research Council, VR
(I.F.), and the Beijing Institute of Technology Research Fund
for Young Scholars (P.C.). Computational resources were
provided by the Swedish National Infrastructure for Comput-
ing (SNIC) at the PDC Center for High Performance
Computing.
■
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K DOI: 10.1021/acs.macromol.9b00472
Macromolecules XXXX, XXX, XXX−XXX