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Carbohydrate Research 337 (2002) 1145– www.elsevier.com/locate/carres
Molecular interactions in bacterial cellulose composites studied by
1D FT-IR and dynamic 2D FT-IR spectroscopy
Marta Kacˇura´kova´,a^ Andrew C. Smith,a^ Michael J. Gidley,b^ Reginald H. Wilsona,^ *
a Norwich Laboratory , Institute of Food Research , Norwich Research Park , Colney Lane , Norwich NR 4 7 UA , UK b Unile er Research , Colworth House , Sharnbrook , Bedford MK 44 1 LQ , UK
Received 19 July 2001; received in revised form 21 March 2002; accepted 10 April 2002
Abstract
Specific strain-induced orientation and interactions in three Acetobacter cellulose composites: cellulose ( C ), cellulose/pectin ( CP ) and cellulose/xyloglucan ( CXG ) were characterized by FT-IR and dynamic 2D FT-IR spectroscopies. On the molecular level, the reorientation of the cellulose fibrils occurred in the direction of the applied mechanical strain. The cellulose-network reorientation depends on the composition of the matrix, including the water content, which lubricates the motion of macromolecules in the network. At the submolecular level, dynamic 2D FT-IR data suggested that there was no interaction between cellulose and pectin in CP and that they responded independently to a small amplitude strain, while in CXG , cellulose and xyloglucan were uniformly strained along the sample length. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords : Cellulose composites; FT-IR spectroscopy; Dynamic 2D FT-IR spectroscopy; Linear stretching
1. Introduction
Cellulose is composed of -D -glucopyranose units joined by (14)-glycosidic links, and is the primary structural element of the cell wall: it has a high molecu- lar weight and crystallinity.^1 Xyloglucan is the major hemicellulose component in primary cell walls, with chains of (14)--D -glucan with xylosyl units linked to the glucosyl units in the C-6 position. Pectin is a term for a group of heterogeneous polysaccharides whose backbone consists of (14)-linked -D -galacturonic acid repeating-units. Bacterial ( Acetobacter xylinum ) cellulose-based composites containing xyloglucan or pectin have been shown to possess organizational fea- tures similar to those observed in primary plant cell walls.2– Infrared (IR) spectroscopy has been extensively used in cellulose research and IR band assignment, orienta- tion data,5,6^ and structural details7,8^ have been pro- duced. Polymer stiffness is considerably enhanced by molecular orientation, and polarized IR spectroscopy
can be used to study orientation induced by mechanical strain and to characterize the segmental mobility of polymers under the influence of an external perturba- tion. 9 Dynamic 2D FT-IR spectroscopy has been used to unravel the IR bands affected by deformation and the orientation of submolecular groups in cellulose I in order to probe the relationship between hydrogen bonding and cellulose structure10–12^ in dry spruce-pulp samples. We have recently reported the first application of linear stretching FT-IR microscopy and 2D FT-IR spectroscopy^13 to functional cell walls in onion epider- mis. However, it is necessary to determine the 2D cross-correlation peak frequencies for pure cellulose and its composites with other cell-wall biopolymers in order to establish an interpretation of cross peaks in hydrated systems. Here we report the results of an FT-IR study of mechanically strained hydrated cellulose composites and interpret the data in terms of molecular mobility and interactions. Well-characterized model systems were used for detailed studies to allow interpretations to be applied to polymers of intact plant cell walls. Polarized IR spectra allow the determination of molec- ular alignment and contribute towards an understand- ing of the structure–function relationship in biopoly-
- Corresponding author. Tel.: +44-1603-255000. E - mail address : [email protected] (R.H. Wilson).
0008-6215/ 02 /$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 8 - 6 2 1 5 ( 0 2 ) 0 0 1 0 2 - 7
mer mixtures at different water contents. The analysis of the dynamic 2D FT-IR spectra and two-dimen- sional correlations are shown for samples measured with a polarized IR source.
2. Experimental
Materials .—The Acetobacter cellulose samples cellu- lose ( C ), cellulose/pectin ( CP , containing 20% apple pectin of 67% degree of methyl esterification), and cellulose/xyloglucan ( CXG , containing 35% of tamarind xyloglucan) were prepared as described pre- viously.^2 –^4 Fermentation duration of 24 h gave 10– 20 m thick films, which were still measurable with IR absorbance up to about 2 units in transmission, yet strong enough to be mounted in the clamps of the stretching device. The weight fraction of different cel- lulose morphologies in the C and CP samples has been reported as 57% I, 25% I crystalline, and 18% non-crystalline, 4 while in CXG the corresponding val- ues were 21% I, 32% I crystalline, and 47% non- crystalline. 2, Water content .—The water content was gravimetri- cally determined after the cellulose composites were equilibrated at discrete relative humidity (RH) val- ues.^13 The samples at 100% relative humidity (RH) showed (Table 1) water contents (w/w) of: 42% for C , 61% for CP , and 94% for CXG. Samples used for dynamic 2D and linear stretching experiments were measured at 97% RH (defined as wet samples, w-sam- ples) and certain samples were measured after partial drying at 84% RH (defined as moist, m-samples) as shown in Table 1. Samples dried below 76% RH were effectively dry, brittle, and not suitable for FT-IR me- chanical investigation. FT - IR spectroscopy .—A Bio-Rad FTS 6000 (Cam- bridge, MA) spectrometer, equipped with a liquid ni- trogen cooled MCT (HgCdTe) detector, was used to collect transmission spectra. In linear stretching exper-
iments, 128 scans were co-added before Fourier trans- formation with the IR beam polarized parallel or perpendicular to the stretching direction using a KRS 5 (TlBr and TlI mixed crystal) wire-grid polarizer (Graseby Specac, UK). The resolution was 8 cm−^1 and three to five replicates were measured in order to ensure reproducibility of measurement. The spectrom- eter was controlled by a computer running WIN-IR PRO (Bio-Rad) version 2.9. Linear stretching .—Polarized FT-IR spectra were collected from oriented samples. The composite sam- ples were washed in water, cut to 15 ×10 mm and mounted between the jaws of a Bio-Rad polymer stretcher. The sample was stretched ( l is the change of length) in steps of 3% strain, , ( l / l ) from 0 to 33%. Infrared dichroism .—Infrared dichroism reflects the mean orientation of the transition moments of the corresponding vibrational modes. Anisotropy follow- ing an applied deformation is characterized by the dichroic difference A =( A − A ) and/or dichroic ra - tio defined as R = A / A , where the A and A are the absorbances measured with parallel and perpen- dicular polarization.^14 –^16 The cellulose composites were studied as a function of uniaxial strain. For a polymer network, the segmental orientation ( F ) de- tected by IR spectroscopy^17 expressed in terms of the dichroic ratio is: F = C [( R −1)/( R +2)], where C = (2 cot^2 +2)/(2 cot 2 −1). For a given absorption band, is the angle between the transition moment vector of the vibrational mode and a directional vec- tor characteristic of the chain segment. The glycosidic (COC) link (band at 1162 cm−^1 ) is approximately co-aligned with the molecular long axis1,6^ and the stretching direction. In this case, = 0 ° and the equa- tion reduces to: F =( R −1)/( R +2). Data analysis .—In order to analyze the strain-in- duced IR spectral changes, A and F of selected bands were used to characterize the degree of orienta- tion of cellulose in the individual composites9,14,17^ in correlation with uniaxial strain. One of the effects of stretching is a change in the thickness of the polymer film. In order to minimize resultant error in A , each polarized spectrum was normalized to the correspond- ing unstretched IR absorption in the (CH) region at (1400–1300 cm−^1 ) where intensity is not orientation dependent.^18 For molecular orientation analysis A was used, in which case the bands from vibrational dipoles parallel to the polymer-chain axis appeared with positive intensity, whereas those that are perpen- dicular were of negative intensity.^15 Dynamic 2 D FT - IR spectroscopy .—In dynamic 2D FT-IR, a small-amplitude oscillatory strain is applied to a sample and the resulting spectral changes are measured as a function of time.^13 The dynamic per- turbation generates directional changes in the transi-
Table 1 Sorption isotherm data
Relative humidity (%) C CP CXG Samples
100 42 61 94 97 30 56 87 w 95 28 40 79 84 22 20 12 m 76 18 10 2
Water content (w/w%) in the cellulose composite samples: cellulose ( C ), cellulose/pectin ( CP ) and cellulose/xyloglucan ( CXG ).
C due to the contribution from -linked xyloglucan (Fig. 1). In the CP spectrum after subtraction of C , bands at 1738, 1243, 1146, 1097, 1019, 960, and 833 cm−^1 were seen, consistent with pectin^21 and in the CXG spectrum after subtraction of C , shoulders at 1130, 1079, 1045, and 944 cm−^1 corresponding to xyloglucan bands^22 (Table 2) were observed. Linear stretching and dichroic spectra. Wet sam - ples .—Linear stretching was first performed at the highest measurable water content (about 97% RH, Table 1) on cellulose ( C w), cellulose/pectin ( CP w ), and cellulose/xyloglucan ( CXG w ) samples (where subscript w denotes wet samples). The unstretched samples showed very small or zero A across the entire spec- trum. Dichroism of the cellulosic as(C-1OC-4) glyco- sidic link mode at 1162 cm−^1 was used as a measure of orientation.6,23^ Increased dichroism in the parallel direc- tion was observed with increasing strain (Fig. 2(a)), indicating that the polymer aligned along the direction of stretch. The bands at 1058 and 1033 cm−^1 in C w, assigned to vibrations of C3-HO3-H and C6- H 2 O6-H cellulose groups and pyranosyl-ring vibra- tions,^13 increased in intensity without significant frequency shift. In CP w (Fig. 2(b)), the intensity at 1162 cm−^1 was much higher relative to the two-ring vibra- tions, which broadened, reflecting a wider distribution of the intra- and intermolecular hydrogen bond-ener- gies compared to C w. In CXG w , the glycosidic 1062 cm−^1 band was the only one to show a significantly positive A , but relatively weak xyloglucan bands at 1075 and 1045 cm−^1 (Table 2) and also, frequency
shifts (Fig. 2(c)) in the 1070–1020 cm−^1 region were observed attributable to molecular deformation.^16 Moist samples .—The samples designated C m , CP m, and CXG m were measured at 84% RH, where the subscript m denotes moist samples which contained only about 12–22% water (Table 1). In the m samples, the spectral features were similar to those for the w-se- ries, but failure typically occurred at lower strain (18– 21%). Therefore, water plays an important role in the deformation of the sample, consistent with the com- ment that the mechanical properties of these systems depend on hydration level.^24 Pre - stretched sample .—An oriented (30% strain) C w sample was linearly stretched into a second dimension ( C ws), perpendicular to the initial sample orientation. At the starting point of stretching the molecular orien- tation ( A ) was observed to be perpendicular (Fig. 2(d)), but with increasing strain the negative bands at 1064, 1057, and 1037 cm−^1 tended to zero showing reorientation into the new stretching direction. This experiment demonstrated that the reorientation into the stretching direction occurs regardless of whether the initial orientation is random or partially orientated. The orientation is not perfect but it was certainly preferred. Orientation function .—The orientation function ( F ) of the cellulose glycosidic bond, a measure of distribu- tion of cellulose chains segments, is shown as a function of strain in Fig. 3. Like A , F increased 17,25^ in all the samples but was generally larger for w-samples than for m-samples (Fig. 3). The enhanced ability of cellulose to orient in a wetter environment can be interpreted in
Fig. 1. FT-IR spectra of dry cellulose composites C (upmost), CP (centre), CXG.
Fig. 2. Dichroic difference spectra ( A = A − A ) taken from transmission FT-IR spectra, of wet, C w (a), CP w (b), CXG w (c) cellulose composites and C ws (d) oriented in second dimension; at each step of displacement with 3% steps. Bottom to top spectrum, strains: 0, 3, 6, 9 (···), 12, 15, 18, 21 (—), 24, 27, 30, 33, 36 (-···-)%.
amplitude (100 m) sinusoidal displacement at 20 Hz frequency (i.e., C on Fig. 4(a)). Two-dimensional correlation spectra13,20^ were exam- ined in order to decide whether the network biopoly- mers were independent, since in synthetic polymer blends 19 such spectra have been shown to provide sub- molecular-level insight into the interactions between specific functional groups. The synchronous 2D cross- peak values for the studied samples are listed in Table
- C (Fig. 5(a)) gave cross peaks at 1162, 1112, 1062, and 1030 cm−^1 (assignment see Table 2), representing similar time-dependent movements of specific cellulose groups. CP (Fig. 5(b)) was essentially the same as for C and our data showed no evidence of connected motion between the two networks in the CP composite, consis-
tent with earlier evidence.^4 The dynamic spectra of the CXG exhibited bands at 1162 and 1080 cm−^1 in IP spectrum (not shown). In the 2D correlation maps (Table 3, Fig. 5(c)) the intense cellulose and xyloglucan cross-peaks appearing at the off-diagonal positions at 1162 and 1080 cm−^1 indicated a strong synchronous correlation between cellulose and xyloglucan (Table 3), thus providing evidence that the two macromolecules move collectively, as suggested earlier.^3 These cross- peaks are the same as in the completely parallel ori- ented dry spruce-pulp cellulose^12 model. Spectra of completely oriented cellulose sheets made of spruce pulp had shown^12 the band at 1064 cm−^1 with a shoulder at 1076 cm−^1 in parallel orientation. However, in the case of partially oriented CXG , the
Fig. 4. Dynamic IP (—), and Q (···) spectra of C measured at sample modulation frequency (SMF) of: 20 Hz (a) and 5 Hz (b). Sample modulation amplitude= 100 m.
Table 3 2D FT-IR cross-peaks as observed in 21% pre-stretched cellulose composites at 20 Hz SMF
Sample Wavenumbers (cm−^1 )
C 1162 – 1057 1115 1062 – 1055 1037 – 1025 CP 1170 – 1057 1115 1057 1030 CXG 1060 1115 1080 – 1070 1065 – 1055 1040 1035 – 1025
Fig. 5. 2D FT-IR synchronous correlation maps of C (a), CP (b) and CXG (c) measured at sample modulation frequency (SMF) of 20 Hz. Sample modulation amplitude= 100 m.
1080 cm−^1 band is a clearly defined peak and together with the 1040 cm−^1 band, already shown in the linear stretching experiment, they are assigned to the xyloglu- can contribution. These results are also in agreement with those from onion epidermis.^13 Wide-angle X-ray scattering (WAXS) has also shown that pectin does not bind to the cellulose whereas xyloglucan binds strongly to cellulose.^24 Sample modulation at different frequencies .—The w- and m-samples were measured at 5, 10, 16, 20, 25, and 50 Hz SMF. The dynamic IP and Q spectra were well resolved at 5–25 Hz in contrast to 50 Hz, where the signal to noise ratio was very low and the spectra gave no information about any response to the applied strain. This observation suggests that the molecular interaction may depend on the mechanical frequency and at high frequencies the motions in cellulose com- posites lose coherence. At low frequencies (5, 10 Hz, Fig. 4(b)), the intensity of the Q spectra was small relative to IP spectra in CP and CXG , showing that the polymers responded almost synchronously to the ap- plied strain indicating an elastic response of the com- posite networks to the applied small amplitude ( m) oscillation. It is interesting that earlier small defor- mation rheology of these systems showed E E , also indicating a dominance of the elastic over the viscous response. 3,4^ The intensity of Q spectra increased at higher frequencies (i.e., 20 Hz, Fig. 4) consistent with a loss of synchronicity. Cross peaks arising from the four cellulose groups were seen at all oscillation frequencies in C and CP. In CXG , xyloglucan and cellulose resulted in cross-peaks at 5–20 Hz but the 1120 cm−^1 cellulose peak was weak or absent suggesting a changed cellulose geometry at the O-2···O-6H hydrogen bond in the presence of xyloglucan. The presence of the xyloglucan 1070 cm−^1 peak at low frequencies showed that stress transmission occurred in both cellulose and hemicellulose. At 25 Hz,
cross peaks at 1030 and 1165 cm−^1 suggest that the cellulose glycosidic and the C-6H-2O-6H side groups interact at high frequencies. The results suggest that dynamic 2D FT-IR data can be linked to the viscous and elastic rheological properties of biopolymers. The results show that 1D FT-IR and dynamic 2D FT-IR spectroscopies can be used together or indepen- dently to characterize biopolymer systems depending on physical conditions, and are prospective in biological- research applications on a microscopic scale. Cellulose, pectin, and xyloglucan are major constituents of the plant cell wall and interactions within and between them shown by FT-IR spectroscopy are crucial in un- derstanding the structure of the plant cell wall.
Acknowledgements
This work was funded by a BBSRC competitive strategic grant. The authors gratefully acknowledge Dr E. Chanliaud (Unilever) for sample preparation, Dr N. Wellner (IFR) for technical advice with dynamic 2D FT-IR, Dr M.C. McCann (JIC, Norwich) and Dr K.W. Waldron (IFR) for helpful discussion.
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