The surface structure of well-ordered native cellulose fibrils in contact with water

Article abstract of DOI:10.1016/j.carres.2009.10.020

CP/MAS ¹³C NMR spectroscopy was used in combination with spectral fitting to examine the surface structure of hydrated cellulose I fibrils from Halocynthia and Gluconoacetobacter xylinus. To increase the spectral intensities and minimize signal overlap, G. xylinus celluloses site-specifically enriched in ¹³C either on C4 or on both C1 and C6 were examined. The experimental data showed multiple C4 and C6 signals for the water accessible fibril surfaces in the highly crystalline celluloses. These signal multiplicities were attributed to structural features in the surface layers induced by the fibril interior, and could not be extracted by spectral fitting in celluloses with a lower degree of crystallinity such as cellulose from cotton.

Full text of DOI:10.1016/j.carres.2009.10.020

Carbohydrate Research 345 (2010) 97–100
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
The surface structure of well-ordered native cellulose fibrils in contact
with water
Erik Malm a, Vincent Bulone a, Kristina Wickholm b, Per Tomas Larsson b, Tommy Iversen b,
*
a Royal Institute of Technology (KTH), School of Biotechnology, AlbaNova University Centre, SE-106 91 Stockholm, Sweden
b INNVENTIA AB, PO Box 5604, SE-114 86 Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
CP/MAS 13C NMR spectroscopy was used in combination with spectral fitting to examine the surface
structure of hydrated cellulose I fibrils from Halocynthia and Gluconoacetobacter xylinus. To increase
the spectral intensities and minimize signal overlap, G. xylinus celluloses site-specifically enriched in
13C either on C4 or on both C1 and C6 were examined. The experimental data showed multiple C4
and C6 signals for the water accessible fibril surfaces in the highly crystalline celluloses. These signal
multiplicities were attributed to structural features in the surface layers induced by the fibril interior,
and could not be extracted by spectral fitting in celluloses with a lower degree of crystallinity such as
cellulose from cotton.
Article history:
Received 1 June 2009
Received in revised form 14 October 2009
Accepted 23 October 2009
Available online 30 October 2009
Keywords:
Cellulose
CP/MAS 13C NMR
Spectral fitting
Halocynthia,
Ó 2009 Elsevier Ltd. All rights reserved.
Gluconoacetobacter xylinus
1. Introduction
signal at d 83.8 ppm has been attributed to cellulose at water
inaccessible surfaces formed either by interior distortions or
The surface characteristics of cellulose fibrils have important
consequences for the utilization of cellulose as a raw material for
refinement to yield various polymeric and monomeric products.
Fibril dimensions and the structure of cellulose chains at fibril
surfaces can influence the reactivity in chemical and enzymatic
hydrolysis, as well as physical phenomena such as adsorption and
adhesion.
Our knowledge of the molecular arrangement at the surface of
cellulose fibrils is based largely on data obtained from atomic force
microscopy (AFM) applied to systems composed of highly ordered
cellulose fibrils of large lateral dimensions. AFM images of the
surface of cellulose fibrils from Valonia ventricosa immersed in
aggregation of fibrils, and a broad cluster of C6 signals in the
region of d 62 ppm arises from the hydroxymethyl groups of the
glucose residues present at the fibril surface.3–9
In the present study we have reinvestigated the surface structure
of
a set of hydrated cellulose I samples from Halocynthia,
Gluconoacetobacter xylinus and cotton, using 13C enrichment
and CP/MAS 13C NMR spectroscopy in combination with spectral
fitting.
2. Experimental
2.1. Samples
water—and rich in the I
with triclinic organization (I
a
allomorph—indicate the presence of areas
like), whereas areas typical of a
a
Cellulose samples from cotton linters and Halocynthia sp. were
obtained as described previously.8
Colonies of G. xylinus were cultivated on Hestrin–Schramm (HS)
solid medium for 7 days at 27 °C.10,11 Liquid cultures were ob-
tained by inoculating 3 mL of fresh HS medium with single
monoclinic organization (Ib like) have been observed for the cellu-
lose from Valonia macrophysa.1,2 The surface structure of less
ordered fibrils of smaller lateral dimensions such as wood and cot-
ton cellulose fibrils has been examined utilizing CP/MAS 13C NMR
spectroscopy. In the spectra obtained from these plant celluloses,
two C4 signals at d 83.3 ppm and d 84.3 ppm have been assigned
to cellulose at fibril surfaces in contact with water, one broad C4
colonies. The liquid HS medium contained either
D-glucose,
D
-[4-13C]glucose or -[6-13C]glucose, and the cultures were grown
D
for 14 days at 27 °C under static conditions. The cellulose film was
collected and bacteria were removed by treatment with 0.1 M
NaOH (aq) at 80 °C for 3 h, followed by extensive washing with
de-ionized water.
* Corresponding author. Tel.: +46 8 6767210.
E-mail address: tommy.iversen@innventia.com (T. Iversen).
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.10.020

98
E. Malm et al. / Carbohydrate Research 345 (2010) 97–100
Table 1
2.2. NMR spectroscopy
Non-linear least-squares fittings of the C4 regions of the CP/MAS 13C NMR spectra of
different celluloses
All spectra were recorded on never-dried samples (>35% water
content) packed uniformly in a zirconium oxide rotor. CP/MAS 13C
NMR spectra were recorded at ambient temperature on a Bruker
Avance AQS 300 WB instrument operating at 7.04 T. The MAS rate
was in the 4–5 kHz range. A 7 mm double air-bearing probe was
used. Acquisition was performed with a CP pulse sequence, using
Cellulose source
I
a
Ib
(%)
Cr
(%)
Fibril
widtha
(nm)
Fibril
(%)
aggregate
widtha (nm)
Gluconoacetobacter
xylinus
Gluconoacetobacter
xylinus 13C enriched
in C4
Gluconoacetobacter
xylinus 13C enriched
in C6b
56
(1)
48
(1)
22
(1)
33
(1)
78
(2)
80
(1)
10 (1)
27 (1)
28 (2)
11 (0.4)
a 4.3 ls proton 90° pulse, a 800 ls ramped (100–50%) falling con-
tact pulse and a 2.5 s delay between repetitions. A TPPM15 pulse
sequence was used for 1H decoupling. Glycine was used for the
Hartmann–Hahn matching procedure, as well as an external stan-
dard for calibration of the chemical shift scale relative to tetra-
methylsilane ((CH3)4Si). The data point of maximum intensity in
56
22
78
10 (0.4)
28 (2)
(3)
(2)
(4)
Halocynthia sp.
nd
69
69
11 (0.7)
7 (0.1)
38 (5.5)
40 (1.3)
(1.2) (1.2)
38
Cotton linters
32
6
the glycine carbonyl line was assigned
176.03 ppm.
a chemical shift of
(0.6) (0.3) (0.8)
Cr = total allomorph content; nd = not detectable. Values given in parentheses are
standard errors.
2.3. Spectral fitting
a
Fibril and fibril aggregate widths were calculated assuming square cross-
sections.
Spectral fitting was performed as detailed elsewhere.8,9 The
software based on the Levenberg-Marquardt method12 was devel-
oped at STFI-Packforsk.
b
The fitting was from the C6 region.
no cellulose Ia is detectable. In our hands it has not been possible
3. Results and discussion
to obtain consistent results for cellulose samples of different ori-
gins without the use of mixed line shapes for the spectral fitting
procedure. Theoretical support for the need of multiple line shapes
exist when spectral fitting is performed on CP/MAS spectra re-
corded on semi-crystalline solids such as cellulose I.14 The cotton
sample is representative for celluloses isolated from higher plants,
that is, characterized by a lower allomorph content and slightly
thinner fibrils (width of about 7 nm) compared to the other two
celluloses examined. Table 1 compiles the lateral dimensions, fibril
widths and fibril aggregate widths, as well as the proportion of cel-
lulose allomorphs present in the different samples.
We previously described a procedure for analyzing molecular
ordering in cellulose I fibrils, based on CP/MAS 13C NMR spectra
of celluloses in combination with spectral fitting of the C4 re-
gion.5,8,9 The cross-sectional dimensions (fibril width) and bulk
arrangement of the cellulose chains in fibrils from different biolog-
ical sources exhibit large variations. This variability influences the
features of the CP/MAS 13C NMR spectra of the hydrated celluloses
examined, as illustrated in Figure 1a–e. The cellulose from G. xyli-
nus is composed of large well-ordered fibrils (width of about
10 nm) containing cellulose Ia and Ib as the major and minor allo-
morphs, respectively. The cellulose of Halocynthia sp. also consists
of large (width of about 11 nm), well-ordered fibrils, but cellulose
Ib is the single detectable allomorph. For the Halocynthia cellulose
3.1. Spectral fitting of the C4 region
The spectral C4 region representing the cellulose chains located
in the fibril interior (d 86–92 ppm) typically yields signals of high
signal-to-noise ratios (Fig. 1). However, the large cross-sectional
dimensions of the Gluconoacetobacter and Halocynthia celluloses
we have previously reported the presence of about 10% cellulose I
a
from spectral fitting based on Lorentzian signals only.13 However,
in our refined method using both Gaussian and Lorentzian lines
a
b
c
d
e
110
100
90
80
70
60
13C chemical shift (ppm)
Figure 1. CP/MAS 13C NMR spectra of cellulose samples from (a) cotton, (b) Halocynthia, (c) Gluconoacetobacter xylinus, (d) Gluconoacetobacter xylinus enriched in 13C at the C4
position, and (e) Gluconoacetobacter xylinus enriched in 13C at the C1 and C6 positions.

E. Malm et al. / Carbohydrate Research 345 (2010) 97–100
99
result in very low intensities for the signals in the C4 region orig-
inating from the cellulose chains located at fibril surfaces (d 80–
86 ppm). A further complication is the presence of fibril aggregates.
The fibril aggregates contain fibril-to-fibril contact surfaces which,
together with internal fibril distortions, give surfaces that are inac-
cessible to water and appear as a broad Gaussian signal at or close
to d 84 ppm upon spectral fitting. Spectral fitting of the C4 surface
region of the well-ordered cellulose from Halocynthia, which
contains only the Ib allomorph, demonstrates a pattern of four
signals (d 83.3, d 83.7, d 84.3 and d 84.9 ppm) of nearly equal inten-
sity from water accessible surfaces, using Gaussian line shapes
(Fig. 2). This multiplicity may be related to the monoclinic organi-
zation (Ib like) of surface cellulose, as observed in AFM images of
V. macrophysa fibrils.1 The surface chains in the Halocynthia cellu-
lose are in contact with the crystalline interior and could be
arranged in a manner resembling a two-chain monoclinic crystal-
line phase with cellobiose repeating units.
a loss of the fine structure in the C4 surface signals.5,8 This behavior
is expected if the C4 signals of water accessible surfaces in hy-
drated cellulose fibrils reflect in part the underlying structure in
the fibril interior. However, in a highly ordered system lacking
any detectable presence of para-crystalline cellulose surface chains
reside directly on top of a crystalline lattice resulting in a more re-
solved signal pattern. This is the case observed for surface signals
from well-ordered cellulose fibrils such as those from G. xylinus,
which contain substantial quantities of both the Ia and Ib allo-
morphs (Table 1). Hence the different spectral features of the sur-
face signals detected in cotton cellulose and in cellulose from G.
xylinus can be explained in terms of different system properties
mediated by the same mechanism. Although inhomogeneous line
broadening is the most probable cause for the structural effects
on spectra, similar influences have recently been observed for the
dynamic properties of fibril surface chains by analyzing experi-
mental 13C T1 relaxation times using molecular dynamics (MD)
simulations.16 The MD simulations could explain the different
relaxation times observed for the two surface signals in cotton lin-
ters (d 83.2 and d 84.3 ppm) in terms of the different underlying
crystallographic planes.
The well-ordered cellulose fromG. xylinus enriched in 13C on the
C4 position, and containing both cellulose I
a and Ib, exhibits five
13C NMR signals originating from the accessible surfaces (Fig. 2).
Most of the signals reproduce, within experimental error, the sig-
nal position and relative intensities found for the C4 surface signals
in the Halocynthia cellulose. The main difference is an additional
signal of relatively high intensity at d 83.5 ppm for the G. xylinus
cellulose. We have previously observed such a high intensity signal
3.2. Spectral fitting of the C6 region
To investigate the C6 surface signals in more detail, G. xylinus
in spectra of cellulose samples rich in the I
a
allomorph from the
was cultivated in the presence of
labeling from C6 to C1 during biosynthesis of cellulose in G. xylinus
grown in the presence of
-[6-13C]glucose has been reported ear-
D
-[6-13C]glucose. Transfer of 13C
algal species V. ventricosa and Cladophora sp.5,15 An explanation
of the relatively broad (FWHH 1.5 ppm) signal at d 83.5 ppm could
be related to a triclinic organization (one chain) of surface cellu-
lose, as observed earlier in AFM images of V. ventricosa fibrils rich
D
lier.11 In our case, nearly half of the original 13C-6 labeling was
found on the C1 position after culture (Fig. 1). However, the spec-
trum gave a low noise signal cluster of C6 signals from interior and
surface chains. It should be noted that spectral fitting of the C-6
signals resulted in the same average lateral fibril and fibril aggre-
gate dimensions and allomorph compositions as spectral fitting
of the C4 signals in the unlabeled G. xylinus cellulose (Table 1). In
Figure 3, the C-6 signals corresponding to water accessible surfaces
as obtained by spectral fitting are shown. For the rest of the cellu-
loses investigated, the C-6 signals could not be separated by spec-
tral fitting but it is apparent from the spectral features that there
exist signal intensities at positions similar to those observed in
the G. xylinus cellulose (Fig. 4). Analysis of the spectrum corre-
sponding to the 13C6-enriched G. xylinus cellulose reveals three sig-
nals of nearly identical intensities at d 61.0, d 61.2, and d 61.7 ppm.
The positions of these signals are all within the range 60–62 ppm
proposed for the gauche–gauche conformation of the hydroxy-
in the I
organization of the surface cellulose is further substantiated by
a
allomorph.2 A combination of monoclinic and triclinic
comparison of the Ia/Ib ratios which are 1.5 and 2.6, respectively,
in the fibril interior and surface cellulose of 13C4-enriched cellulose
from G. xylinus. Even if this does not give an exact agreement in the
ratios, the trend is the same, that is, a dominance of the I
a
allomorph.
In the less ordered cotton cellulose the C4 accessible surface
signals coalesce into two signals or signal clusters at d 83.3 and d
84.3 ppm.5 In the cotton cellulose, the content of the I
a
(6%) and
Ib (32%) allomorphs in the fibril is too low to give a completely
crystalline interior which theoretically would require a I
a and Ib
content near 70%, based on an average fibril width of 7 nm.5 In-
stead we observe a substantial amount of para-crystalline cellulose
(30%) probably also in contact with the surface chains, resulting in
a
b
c
86
85.5
85
84.5
84
83.5
83
82.5
82
Chemical shift (ppm)
Figure 2. Spectral fitting of the C4 surface region from (a) cotton, (b) Gluconoacetobacter xylinus enriched in 13C at the C4 position, and (c) Halocynthia.

100
E. Malm et al. / Carbohydrate Research 345 (2010) 97–100
be attributed to the gauche–trans conformer of the hydroxymethyl
group. In the cotton cellulose that contains substantial amounts of
para-crystalline cellulose (30%) a major part of the fine structure of
both the C4 and C6 signal clusters is missing.
4. Conclusions
The structure of the surface of well-ordered hydrated cellulose
fibrils such as those from Halocynthia and G. xylinus can be de-
scribed by a model involving both water accessible surface signals,
which partially reflect the underlying interior allomorph structure
(Ia and Ib), and a distorted water inaccessible C4 signal, which
arises as a consequence of fibril aggregation and/or defects in the
fibril interior when such are present. For the C6 signal cluster,
water accessible and inaccessible surface signals are observed,
but the fine structure is given by the conformation of the hydroxy-
methyl group and dominated by three signals in the range
61–62 ppm recently attributed to gauche–gauche conformers. In
the less ordered cotton cellulose containing a substantial amount
of para-crystalline cellulose, a major part of the fine structure of
both the C4 and C6 signal clusters is lost.
63
62
61
60
13C chemical shift (ppm)
Figure 3. Spectral fitting of the C6 surface region from Gluconoacetobacter xylinus
cellulose enriched in 13C at the C6 position. Only the fitted functions corresponding
to water accessible surfaces are shown, the function corresponding to inaccessible
fibril surfaces is omitted for clarity.
Acknowledgment
This work was supported by the Swedish Centre for Biomimetic
Fibre Engineering (www.biomime.org).
a
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