Tensile deformation of bacterial cellulose composites

The polymeric basis for the mechanical properties of primary plant cell walls has been investigated by forming analogous composites based on fermentation of the bacterium Acetobacter xylinus, either alone or in the presence of xyloglucan or pectin. Simultaneous small-angle X-ray scattering and uniaxial deformation experiments has shown how the cellulose microfibrils reorient during deformation. Despite very different stress/strain curves, the reorientation behaviour is similar, regardless of the presence or absence of xyloglucan or pectin. A simple theory has been developed to predict the orientation behaviour. This is qualitatively similar to the measured behaviour, but differs quantitatively.     

Characterization of the crystalline structure of cellulose using static and dynamic FT-IR spectroscopy

The cellulose structure is a factor of major importance for the strength properties of wood pulp fibers. The ability to characterize small differences in the crystalline structures of cellulose from fibers of different origins is thus highly important. In this work, dynamic FT-IR spectroscopy has been further explored as a method sensitive to cellulose structure variations. Using a model system of two different celluloses, the relation between spectral information and the relative cellulose Ialpha content was investigated. This relation was then used to determine the relative cellulose Ialpha content in different pulps. The estimated cellulose I allomorph compositions were found to be reasonable for both unbleached and bleached chemical pulps. In addition, it was found that the dynamic FT-IR spectroscopy technique had the potential to indicate possible correlation field splitting peaks of cellulose Ibeta.     

Competitive binding of pectin and xyloglucan with primary cell wall cellulose

Assemblies of pectin, xyloglucan and cellulose were studied in vitro using two ternary systems. In the first one, xyloglucan concentration varied, while pectin amount was kept constant. In the second one, pectin concentration varied, whereas xyloglucan amount was fixed. The use of ternary systems allowed to put forward the hypothesis that pectin/cellulose and xyloglucan/cellulose associations may exist together or separately, depending on the proportion of non-cellulosic polysaccharides in cell walls. It can be hypothesized that pectin plays a double role within primary cell walls: (i) pectin loosely bound to cellulose, in xyloglucan-rich cell walls, (ii) pectin associated with cellulose, in xyloglucan-poor cell walls.     

The mechanical properties and molecular dynamics of plant cell wall polysaccharides studied by Fourier-transform infrared spectroscopy

Polarized one- and two-dimensional infrared spectra were obtained from the epidermis of onion (Allium cepa) under hydrated and mechanically stressed conditions. By Fourier-transform infrared microspectroscopy, the orientation of macromolecules in single cell walls was determined. Cellulose and pectin exhibited little orientation in native epidermal cell walls, but when a mechanical stress was placed on the tissue these molecules showed distinct reorientation as the cells were elongated. When the stress was removed the tissue recovered slightly, but a relatively large plastic deformation remained. The plastic deformation was confirmed in microscopic images by retention of some elongation of cells within the tissue and by residual molecular orientation in the infrared spectra of the cell wall. Two-dimensional infrared spectroscopy was used to determine the nature of the interaction between the polysaccharide networks during deformation. The results provide evidence that cellulose and xyloglucan associate while pectin creates an independent network that exhibits different reorientation rates in the wet onion cell walls. The pectin chains respond faster to oscillation than the more rigid cellulose.     

Mechanical properties of primary plant cell wall analogues

Mechanical effects of turgor pressure on cell walls were simulated by deforming cell wall analogues based on Acetobacter xylinus cellulose under equi-biaxial tension. This experimental set-up, with associated modelling, allowed quantitative information to be obtained on cellulose alone and in composites with pectin and/or xyloglucan. Cellulose was the main load-bearing component, pectin and xyloglucan leading to a decrease in modulus when incorporated. The cellulose-only system could be regarded as an essentially linear elastic material with a modulus ranging from 200 to 500 MPa. Pectin incorporation modified extensibility properties of the system by topology/architecture changes of cellulose fibril assemblies, but the cellulose/pectin composites could still be described as a linear elastic material with a modulus ranging from 120 to 250 MPa. The xyloglucan/cellulose composite could not be modelled as a linear elastic material. Introducing xyloglucan into a cellulose network or a cellulose/pectin composite led to very compliant materials characterised by time-dependent creep behaviour. Modulus values obtained for the composite materials were compared with mechanical data found for plant-derived systems. After comparing bi-axial and uni-axial behaviour of the different composites, structural models were proposed to explain the role of each polysaccharide in determining the mechanical properties of these plant primary cell wall analogues.     

FT-IR study of the Chara corallina cell wall under deformation

Fourier-transform infrared (FT-IR) microspectroscopy was used to investigate both the chemical composition of, and the effects of an applied strain on, the structure of the Chara corallina cell wall. The inner layers of the cell wall are known to have a transverse cellulose orientation with a gradient through the thickness to longitudinal orientation in the older layers. In both the native state and following the removal of various biopolymers by a sequential extraction infrared dichroism was used to examine the orientation of different biopolymers in cell-wall samples subjected to longitudinal strain. In the Chara system, cellulose microfibrils were found to be aligned predominantly transverse to the long axis of the cell and became orientated increasingly transversely as longitudinal strain increased. Simultaneously, the pectic polysaccharide matrix underwent molecular orientation parallel to the direction of strain. Following extraction in CDTA, microfibrils were orientated transversely to the strain direction, and again the degree of transverse orientation increased with increasing strain. However, the pectic polysaccharides of the matrix were not detected in the dichroic difference spectra. After a full sequential extraction, the cellulose microfibrils, now with greatly reduced crystallinity, were detected in a longitudinal direction and they became orientated increasingly parallel to the direction of strain as it increased.     

Classification and identification of Arabidopsis cell wall mutants using Fourier-Transform InfraRed (FT-IR) microspectroscopy

We have developed a novel procedure for the rapid classification and identification of Arabidopsis mutants with altered cell wall architecture based on Fourier-Transform Infrared (FT-IR) microspectroscopy. FT-IR transmission spectra were sampled from native 4-day-old dark-grown hypocotyls of 46 mutants and the wild type treated with various drugs. The Mahalanobis distance between mutants, calculated from the spectral information after compression with the Discriminant Variables Selection procedure, was used for alpha hierarchical cluster analysis. Despite the completely unsupervised nature of the classification procedure, we show that all mutants with cellulose defects appeared in the same cluster. In addition, mutant alleles of similar strength for several unrelated loci were also clustered, which demonstrates the sensitivity of the method to detect a wide array of cell wall defects. Comparing the cellulose-deficient cluster with the cluster that contained wild-type controls led to the identification of wave numbers that were diagnostic for altered cellulose content in the context of an intact cell wall. The results show that FT-IR spectra can be used to identify different classes of mutants and to characterize cell wall changes at a microscopic level in unknown mutants. This procedure significantly accelerates the identification and classification of cell wall mutants, which makes cell wall polysaccharides more accessible to functional genomics approaches.     

WAXS and 13C NMR study of Gluconoacetobacter xylinus cellulose in composites with tamarind xyloglucan

- Model composites, produced using cellulose from stationary cultures of the bacterium Gluconoacetobacter xylinus and tamarind xyloglucan, were examined by wide-angle X-ray scattering (WAXS) and CP/MAS solid-state (13)C NMR spectroscopy. The dominant crystallite allomorph of cellulose produced in culture media with or without xyloglucan was cellulose I(alpha) (triclinic). The presence of xyloglucan in the culture medium reduced the cross-section dimensions of the cellulose crystallites, but did not affect the crystallite allomorph. However, when the composites were refluxed in buffer, the proportion of cellulose I(beta) allomorph increased relative to that of cellulose I(alpha). In contrast, cellulose I(alpha) remained the dominant form when cellulose, produced in the absence of xyloglucan, was then heated in the buffer. Hence the presence of xyloglucan has a profound effect on the formation of the cellulose crystallites by G. xylinus.     

Sequential cell wall transformations in response to the induction of a pedicel abscission event in Euphorbia pulcherrima (poinsettia)

Alterations in the detection of cell wall polysaccharides during an induced abscission event in the pedicel of Euphorbia pulcherrima (poinsettia) have been determined using monoclonal antibodies and Fourier transform infrared (FT-IR) microspectroscopy. Concurrent with the appearance of a morphologically distinct abscission zone (AZ) on day 5 after induction, a reduction in the detection of the LM5 (1→4)-β- d -galactan and LM6 (1→5)-α- l -arabinan epitopes in AZ cell walls was observed. Prior to AZ activation, a loss of the (1→4)-β- d -galactan and (1→5)-α- l -arabinan epitopes was detected in cell walls distal to the AZ, i.e. in the to-be-shed organ. The earliest detected change, on day 2 after induction, was a specific loss of the LM5 (1→4)-β- d -galactan epitope from epidermal cells distal to the region where the AZ would form. Such alteration in the cell walls was an early, pre-AZ activation event. An AZ-associated de-esterification of homogalacturonan (HG) was detected in the AZ and distal area on day 7 after induction. The FT-IR analysis indicated that lignin and xylan were abundant in the AZ and that lower levels of cellulose, arabinose and pectin were present. Xylan and xyloglucan epitopes were detected in the cell walls of both the AZ and also the primary cell walls of the distal region at a late stage of the abscission process, on day 7 after induction. These observations indicate that the induction of an abscission event results in a temporal sequence of cell wall modifications involving the spatially regulated loss, appearance and/or remodelling of distinct sets of cell wall polymers.     

Characterization of bioscoured cotton fabrics using FT-IR ATR spectroscopy and microscopy techniques

The effects of bioscouring were investigated by characterizing the chemical and physical surface changes of cotton fabrics using a purified pectinase enzyme from Bacillus subtilis strain WSHB04-02. Fourier-transform infrared (FT-IR) attenuated total-reflectance (ATR) spectroscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM) techniques were employed. FT-IR ATR spectroscopy provided a fast and semi-quantitative assessment of the removal of pectins and/or waxes on the cotton surface by comparing the changes in intensity of the carbonyl peak induced by HCl vapor treatment at around 1736 cm(-1). The bioscoured surface could be clearly distinguished from those of untreated and alkali-treated cotton fibers using a combination of SEM and AFM. The images produced using these techniques revealed that the surface morphography and topography of the cotton fibers were shaped by the etching action mode of pectinases during bioscouring. These findings demonstrated that AFM is a useful supplement to SEM in characterizing cotton surfaces.     

Pea Xyloglucan and Cellulose: VI. Xyloglucan-Cellulose Interactions in Vitro and in Vivo

Since xyloglucan is believed to bind to cellulose microfibrils in the primary cell walls of higher plants and, when isolated from the walls, can also bind to cellulose in vitro, the binding mechanism of xyloglucan to cellulose was further investigated using radioiodinated pea xyloglucan. A time course for the binding showed that the radioiodinated xyloglucan continued to be bound for at least 4 hours at 40°C. Binding was inhibited above pH 6. Binding capacity was shown to vary for celluloses of different origin and was directly related to the relative surface area of the microfibrils. The binding of xyloglucan to cellulose was very specific and was not affected by the presence of a 10-fold excess of (1→2)-β-glucan, (1→3)-β-glucan, (1→6)-β-glucan, (1→3, 1→4)-β-glucan, arabinogalactan, or pectin. When xyloglucan (0.1%) was added to a cellulose-forming culture of Acetobacter xylinum, cellulose ribbon structure was partially disrupted indicating an association of xyloglucan with cellulose at the time of synthesis. Such a result suggests that the small size of primary wall microfibrils in higher plants may well be due to the binding of xyloglucan to cellulose during synthesis which prevents fasciation of small fibrils into larger bundles. Fluorescent xyloglucan was used to stain pea cell wall ghosts prepared to contain only the native xyloglucan:cellulose network or only cellulose. Ghosts containing only cellulose showed strong fluorescence when prepared before or after elongation; as predicted, the presence of native xyloglucan in the ghosts repressed binding of added fluorescent xyloglucan. Such ghosts, prepared after elongation when the ratio of native xyloglucan:cellulose is substantially reduced, still showed only faint fluorescence, indicating that microfibrils continue to be coated with xyloglucan throughout the growth period.     

Localization of Cell Wall Polysaccharides during Kiwifruit (Actinidia deliciosa) Ripening

The localization of pectin, cellulose, xyloglucan, and callose was compared in kiwifruit (Actinidia deliciosa [A. Chev.] C. F. Liang and A. R. Ferguson var. deliciosa "Hayward") at harvest, at the end of the first phase of softening, and when ripe. Pectin was visualized using three different methods: labeling of galacturonic acid residues, labeling of negatively charged groups, and labeling with JIM 5 (nonesterified residues) and JIM 7 (methyl-esterified) monoclonal antibodies. Labeling of pectin gave different results depending on the detection system used. Differences related to patterns of change during ripening and to spatial distribution of label intensity. Cell wall pectin was available for labeling at all stages of fruit softening, but no clear differentiation of the middle lamella region was seen, although JIM 5 binding predominated where the middle lamellae joined the intercellular spaces in unripe fruit. Negatively charged groups (cationic gold labeling) and, to a lesser extent, galacturonic acid residues (Aplysia depilans gonad lectin labeling) were preferentially located near the cell wall/plasma membrane boundary. The lack of strong binding of the JIM antibodies indicated that the reactive groups were inaccessible. Cellulose remained intact and labeled densely across the wall at all stages of fruit ripening. Distribution of xyloglucan was patchy at harvest but was scattered throughout the wall later in ripening. Alterations to labeling of xyloglucan indicated that some epitopes were differentially exposed. Plasmodesmatal regions were clearly different in composition to other wall areas, showing an absence of cellulose labeling, specific pectin labeling, and callose presence. A similar predominance of pectin labeling compared with cellulose also occurred at the middle lamella wedge near intercellular spaces.     

Characterisation of Ca- and Al-pectate gels by thermal analysis and FT-IR spectroscopy

Thermal analysis (TG-DTA) and FT-IR spectroscopy have been performed on calcium-pectate membranes to investigate their structure and the consequent variation caused by aluminium sorption. Calcium-polygalacturonate (Ca-PG) membranes, model systems of the soil-root interface, were exposed to aluminium solutions at different concentrations (25-800 microM). Three different pHs (3.50, 4.00 and 4.50) were chosen to study the influence of different aluminium species, such as [Al(H2O)6]3+, [Al(OH)(H2O)5]2+ and [Al(OH)2(H2O)(4)]+, on the structure of the Ca-PG membrane. The DTA profiles and FT-IR spectra showed how aluminium sorption induces structural modifications leading to a reorganisation of the chain aggregates and a weakening of the structure. Higher pH, that is, 4.00 and 4.50, and thus hydrolytic aluminium species and related higher calcium content maintain a more regular structure than at pH 3.50. At pH 3.50, both the effect of [Al(H2O)6]3+ and a major calcium release had a greater impact and thus induced a greater weakening of the structure.     

Nascent pectin formed in Golgi apparatus of pea epicotyls by addition of uronic acids has different properties from nascent pectin at the stage of galactan elongation

Microsomal membranes were prepared from etiolated pea (Pisum sativum L.) epicotyls and used to form nascent [Uronic acid-14C]pectin. The enzyme products were characterized by selective enzymic degradation, gel permeation chromatography and analysis of cellulose binding properties. The product obtained had a molecular weight of around 40 kDa, which was significantly lower than that of nascent [Gal-14C]pectin prepared from the same tissues. It is composed mainly of polygalacturonan and perhaps also rhamnogalacturonan (RG-I). Evidence was obtained for the presence of a protein attached to the nascent [Uronic acid-14C]pectin, but it was unaffected by endoglucanase and did not bind to cellulose. Hence, no xyloglucan appeared to be attached to the nascent [Uronic acid-14C]pectin. A model is proposed in which xyloglucan is attached to nascent pectin after formation of homogalacturonan, but before the pectin leaves the Golgi apparatus.     

Celery (Apium graveolens) parenchyma cell walls: cell walls with minimal xyloglucan

The primary walls of celery ( Apium graveolens L.) parenchyma cells were isolated and their polysaccharide components characterized by glycosyl linkage analysis, cross-polarization magic-angle spinning solid-state ¹³C nuclear magnetic resonance (CP/MAS ¹³C NMR) and X-ray diffraction. Glycosyl linkage analysis showed that the cell walls consisted of mainly cellulose (43 mol%) and pectic polysaccharides (51 mol%), comprising rhamnogalacturonan (28 mol%), arabinan (12 mol%) and galactan (11 mol%). The amounts of xyloglucan (2 mol%) and xylan (2 mol%) detected in the cell walls were strikingly low. The small amount of xyloglucan present means that it cannot coat the cellulose microfibrils. Solid-state ¹³C NMR signals were consistent with the constituents identified by glycosyl linkage analysis and allowed the walls to be divided into three domains, based on the rigidity of the polymers. Cellulose (rigid) and rhamnogalacturonan (semi-mobile) polymers responded to the CP/MAS ¹³C NMR pulse sequence and were distinguished by differences in proton spin relaxation time constants. The arabinans, the most mobile polymers, responded to single-pulse excitation (SPE), but not CP/MAS ¹³C NMR. From solid-state ¹³C NMR of the cell walls the diameter of the crystalline cellulose microfibrils was determined to be approximately 3 nm while X-ray diffraction of the cell walls gave a value for the diameter of approximately 2 nm.     

傅立叶变换红外光谱技术对两株大肠杆菌的鉴别

【目的】应用傅立叶变换红外光谱(Fourier transform infrared spectroscopy,FT-IR)技术对两株不同来源的大肠杆菌进行鉴别。【方法】用FT-IR技术对两株不同来源的大肠杆菌进行指纹图谱数据采集,用化学计量学分析方法对光谱进行分析。【结果】建立了基于主成分分析(Principal component analysis,PCA)和分级聚类分析(Hierarchical cluster analysis,HCA)两种聚类分析模型,均可将两株大肠杆菌进行成功区分。【结论】傅立叶变换红外光谱分析方法简便、快速、易操作,结果重现性好,可用于区分不同来源的同种细菌。     

Structural differences between TSEs strains investigated by FT-IR spectroscopy

Strain diversity in transmissible spongiform encephalopathies (TSEs) has been suggested to be "enciphered" in the structure of the misfolded prion protein isoform PrP(Sc). We have recently demonstrated the strain typing potential of the FT-IR spectroscopy technique, analyzing four different TSE agents adapted to Syrian hamsters [A. Thomzig, S. Spassov, M. Friedrich, D. Naumann and M. Beekes, Discriminating scrapie and BSE isolates by infrared spectroscopy of pathological prion protein J. Biol. Chem. 279 (2004) 33847-33854.] [1]. In the present paper, we have extended the FT-IR study, exploring the secondary structure, temperature stability, and hydrogen-deuterium exchange characteristics of PrP27-30, from the TSE agents 263K, ME7-H, 22A-H, and BSE-H. The strain differentiation capacity of the FT-IR approach was objectively proven for the first time by multivariate cluster analysis. The second derivative FT-IR spectra obtained from dried protein films or samples hydrated in H(2)O or D(2)O consistently exhibited strain-specific infrared characteristics in the secondary structure sensitive amide I region, complemented by strain dependent spectral traits in the amide II and amide A absorption regions, and the different H/D-exchange behaviour of the various PrP27-30 samples. FT-IR spectra of PrP27-30 samples from 263K, ME7-H and 22A-H exposed to increasing temperature (up to 90 degrees C) showed that a strain-specific response to heat treatment is associated with strain specific thermostability of distinct secondary structure elements, providing additional means for TSEs strain discrimination.     

Structural characterization, degree of esterification and some gelling properties of Krueo Ma Noy (Cissampelos pareira) pectin

Pectins extracted from Krueo Ma Noy (Cissampelos pareira) leaves mainly consisted of galacturonic acid with trace amount of neutral sugars. The dominant structure of Krueo Ma Noy pectin was established as a 1,4-linked -D-galacturonan by a combination of carboxyl reduction and methylation analysis, and confirmed by FT-IR spectroscopy. The degree of esterification of Krueo Ma Noy pectins was 41.7 and 33.7% for crude and dialyzed pectins, respectively. Krueo Ma Noy pectin has an average molecular weight of 55 kDa, radius of gyration of 15.2 nm and intrinsic viscosity of 2.3 dl/g. Krueo Ma Noy pectin exhibited gelling properties in aqueous solutions at 0.5% (w/v) at 5 °C. Gels were formed at concentrations of 1.0% (w/v) and above even at room temperature. The gel strength, melting point, and melting enthalpy of Krueo Ma Noy pectin increased with polysaccharide concentration.     

Use of Fourier transform infrared (FT-IR) spectroscopy as a tool for pollen identification

Airborne pollen are largely studied to obtain information about the atmospheric content of natural allergens. Aerobiological monitoring networks have been established to provide reliable data that facilitate the timely initiation of preventive actions aimed at minimizing allergic symptoms. Airborne pollen are usually identified and counted using an optical microscope, but as such procedures are extremely time-consuming, more expedient options are being explored. We have assessed the potential of Fourier transform infrared (FT-IR) spectroscopy as an alternative method for the rapid and reliable identification of allergenic pollen using six well-known allergenic pollen taxa and obtaining the respective FT-IR spectra. In doing this, a first IR spectral library has been created. The spectra of unknown pollen were compared to those of the reference library, and two pollen taxa of a mixed sample were identified.     

Quantification of polymeric mannose in wine extracts by FT-IR spectroscopy and OSC-PLS1 regression

FT-IR spectroscopy has being a widespread technique in the agro-industry for the quick assess of food components, including the wine. Using the region of wavenumbers 1200–800 cm⁻¹ of the FT-IR spectra wine polysaccharides, Partial Least Squares Regression (PLS1) independent calibration models were built for mannose quantification in complex matrices from white and in red wine extracts. With PLS1 it was not possible to build a calibration model that included both white and red wine extracts. However, a predictive ability of the model for quantification of mannose from mannoproteins based on this FT-IR spectral region was achieved by the application of orthogonal signal correction (OSC)-PLS1.