Comparative analysis of crystallinity changes in cellulose I polymers using ATR-FTIR, X-ray diffraction, and carbohydrate-binding module probes

Cotton fiber cellulose is highly crystalline and oriented; when native cellulose (cellulose I) is treated with certain alkali concentrations, intermolecular hydrogen bonds are broken and Na-cellulose I is formed. At higher alkali concentrations Na-cellulose II forms, wherein intermolecular and intramolecular hydrogen bonds are broken, ultimately resulting in cellulose II polymers. Crystallinity changes in cotton fibers were observed and assigned using attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy and X-ray diffraction (XRD) subsequent to sodium hydroxide treatment and compared with an in situ protein-binding methodology using cellulose-directed carbohydrate-binding modules (CBMs). Crystallinity changes observed using CBM probes for crystalline cellulose (CBM2a, CBM3a) and amorphous cellulose (CBM4-1, CBM17) displayed close agreement with changes in crystallinity observed with ATR-FTIR techniques, but it is notable that crystallinity changes observed with CBMs are observed at lower NaOH concentrations (2.0 mol dm(-3)), indicating these probes may be more sensitive in detecting crystallinity changes than those calculated using FTIR indices. It was observed that the concentration of NaOH at which crystallinity changes occur as analyzed using the CBM labeling techniques are also lower than those observed using X-ray diffraction techniques. Analysis of crystallinity changes in cellulose using CBMs offers a new and advantageous method of qualitative and quantitative assessment of changes to the structure of cellulose that occur with sodium hydroxide treatment.     

Microscopic structure and properties changes of cassava stillage residue pretreated by mechanical activation

This study has focused on the pretreatment of cassava stillage residue (CSR) by mechanical activation (MA) using a self-designed stirring ball mill. The changes in surface morphology, functional groups and crystalline structure of pretreated CSR were examined by using scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) under reasonable conditions. The results showed that MA could significantly damage the crystal structure of CSR, resulting in the variation of surface morphology, the increase of amorphous region ratio and hydrogen bond energy, and the decrease in crystallinity and crystalline size. But no new functional groups generated during milling, and the crystal type of cellulose in CSR still belonged to cellulose I after MA.     

Relative crystallinity of plant biomass: studies on assembly, adaptation and acclimation

Plant biomechanical design is central to cell shape, morphogenesis, reproductive performance and protection against environmental and mechanical stress. The cell wall forms the central load bearing support structure for plant design, yet a mechanistic understanding of its synthesis is incomplete. A key tool for studying the structure of cellulose polymorphs has been x-ray diffraction and fourier transform infrared spectroscopy (FTIR). Relative crystallinity index (RCI) is based on the x-ray diffraction characteristics of two signature peaks and we used this technique to probe plant assembly, adaptation and acclimation. Confocal microscopy was used to visualize the dynamics of cellulose synthase in transgenic Arabidopsis plants expressing a homozygous YFP::CESA6. Assembly: RCI values for stems and roots were indistinguishable but leaves had 23.4 and 21.6% lower RCI than stems and roots respectively. Adaptation: over 3-fold variability in RCI was apparent in leaves from 35 plant species spanning Ordovician to Cretaceous periods. Within this study, RCI correlated positively with leaf geometric constraints and with mass per unit area, suggestive of allometry. Acclimation: biomass crystallinity was found to decrease under conditions of thigmomorphogenesis in Arabidopsis. Further, in etiolated pea hypocotyls, RCI values also decreased compared to plants that were grown in light, consistent with alterations in FTIR cellulose fingerprint peaks and live cell imaging experiments revealing rapid orientation of the YFP::cellulose synthase-6 array in response to light. Herein, results and technical challenges associated with the structure of the cell wall that gives rise to sample crystallinity are presented and examined with respect to adaptation, acclimation and assembly in ecosystem-level processes.     

Diurnal difference in the amount of immunogold-labeled glucomannans detected with field emission scanning electron microscopy at the innermost surface of developing secondary walls of differentiating conifer tracheids

The differences between cell wall formation at night, when the tangential strain used as an index of the volumetric changes in differentiating cells is high, and in the day, when the tangential strain is low, were investigated in Cryptomeria japonica D. Don. Samples containing differentiating xylem were collected at 0500 hours and 1400 hours. The innermost surface of developing secondary walls in differentiating tracheids was observed by field emission scanning electron microscopy. In the specimens collected at 0500 hours, an amorphous material was observed covering the cellulose microfibrils. The cell wall surface was immunogold-labeled with an anti-glucomannan antiserum. After chlorite treatment, the amorphous material disappeared, and immunogold labeling was rarely observed. In the specimens collected at 1400 hours, cellulose microfibrils were clearly evident, and amorphous material and immunogold labeling were rarely observed. We thus confirmed that much amorphous material containing glucomannans is observed at night, when differentiating tracheids are turgid due to the increase in their volume, while the amorphous material was rarely observed during the day when cellulose microfibrils are clearly observed.     

How Does Hemicelluloses Removal Alter Plant Cell Wall Nanoscale Architecture and Correlate with Enzymatic Digestibility?

Thorough understanding of how hemicelluloses removal influences cell wall nanoscale architecture and cellulose digestion is of crucial importance for enabling low-cost industrial conversion of lignocellulosic biomass to renewable biofuels. In this work, delignified poplar cell walls, after various degrees of hemicelluloses removal, were characterized by Fourier transform infrared imaging spectroscopy and atomic force microscopy to evaluate enhancement in cell wall digestibility. There was a gradual decrease in hemicelluloses content with dilute alkali treatment, which resulted in alterations in the nanoscale architecture and crystallinity of cell walls. Removal of hemicelluloses did not disrupt the integrity of microfibrils but resulted in exposure of microfibrils and a decrease in the diameter of microfibrils. X-ray analysis indicated that the increase in crystallinity beyond natural variations in the crystallinity of cellulose was mainly attributable to removal of hemicelluloses. In conclusion, alterations in the architecture and crystallinity of cell walls facilitated enzymatic digestion of delignified poplar, enhancing cellulose conversion from 68.24 to 75.16 %.     

Hemicelluloses negatively affect lignocellulose crystallinity for high biomass digestibility under NaOH and H2SO4 pretreatments in Miscanthus

ABSTRACT: BACKGROUND: Lignocellulose is the most abundant biomass on earth. However, biomass recalcitrance has become a major factor affecting biofuel production. Although cellulose crystallinity significantly influences biomass saccharification, little is known about the impact of three major wall polymers on cellulose crystallization. In this study, we selected six typical pairs of Miscanthus samples that presented different cell wall compositions, and then compared their cellulose crystallinity and biomass digestibility after various chemical pretreatments. RESULTS: A Miscanthus sample with a high hemicelluloses level was determined to have a relatively low cellulose crystallinity index (CrI) and enhanced biomass digestibility at similar rates after pretreatments of NaOH and H2SO4 with three concentrations. By contrast, a Miscanthus sample with a high cellulose or lignin level showed increased CrI and low biomass saccharification, particularly after H2SO4 pretreatment. Correlation analysis revealed that the cellulose CrI negatively affected biomass digestion. Increased hemicelluloses level by 25% or decreased cellulose and lignin contents by 31% and 37% were also found to result in increased hexose yields by 1.3-times to 2.2-times released from enzymatic hydrolysis after NaOH or H2SO4 pretreatments. The findings indicated that hemicelluloses were the dominant and positive factor, whereas cellulose and lignin had synergistic and negative effects on biomass digestibility. CONCLUSIONS: Using six pairs of Miscanthus samples with different cell wall compositions, hemicelluloses were revealed to be the dominant factor that positively determined biomass digestibility after pretreatments with NaOH or H2SO4 by negatively affecting cellulose crystallinity. The results suggested potential approaches to the genetic modifications of bioenergy crops.     

Brittle Culm1, a COBRA-Like Protein,Functions in Cellulose Assembly through Binding Cellulose Microfibrils

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.     

Temporal changes in wood crystalline cellulose during degradation by brown rot fungi

The degradation of wood by brown rot fungi has been studied intensely for many years in order to facilitate the preservation of in-service wood. In this work we used X-ray diffraction to examine changes in wood cellulose crystallinity caused by the brown rot fungi Gloeophyllum trabeum, Coniophora puteana, and two isolates of Serpula lacrymans. All fungi increased apparent percent crystallinity early in the decay process while decreasing total amounts of both crystalline and amorphous material. Data also showed an apparent decrease of approximately 0.05 Å in the average spacing of the crystal planes in all degraded samples after roughly 20% weight loss, as well as a decrease in the average observed relative peak width at 2θ = 22.2°. These results may indicate a disruption of the outer most semi-crystalline cellulose chains comprising the wood microfibril. X-ray diffraction analysis of wood subjected to biological attack by fungi may provide insight into degradative processes and wood cellulose structure.     

Correlation between X-ray diffraction measurements of cellulose crystalline structure and the susceptibility to microbial cellulase

Chemical and physical treatments of cotton cellulose have been studied in order to elucidate the relationship between the degree of crystallinity of cellulose and the susceptibility of cellulose to cellulase. Cotton cellulose powder was treated with the following solvents: 60% H₂SO₄, Cadoxen, and DMSO-p -formaldehyde. The dissolved celluloses were recovered at high yield of over 97% by addition of nine volumes of cold acetone. X-ray diffraction for measurements of relative crystallinity showed that the crystalline structure of cellulose declined in quantity and perfection by the dissolving treatment and changed to an amorphous form that is highly susceptible to enzymatic hydrolysis. These reprecipitated celluloses were hydrolyzed almost completely within 48 hr by Aspergillus niger cellulase containing mainly 1,4-β-glucan glucanohydrolase (EC 3.2.1.4), without action of 1,4-β-glucan cellobiohydrolase (EC 3.2.1. 91). On the other hand, cryo-milled cellulose (below 250 mesh) still had a crystalline structure, was resistant to cellulase, and gave a low percentage of saccharification. These results indicate that in pure cellulose there are good correlations between x-ray diffractograms and susceptibility to microbial cellulase.     

Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

The mechanism of formation of Cellulose-like microfibrils in a cell-free system from Acetobacter xylinum

The mechanism of formation of cellulose-like microfibrils by a non-soluble, particulate enzyme and uridine diphosphoglucose (UDPG) in a cell-free system from Acetobacter xylinum was studied by transmission electron microscopy and X-ray diffraction. The suspension of particles to which the enzyme is adsorbed is composed of whole, dense ovoids, 50–250 nm long when wet, of fragments of the ovoids, and amorphous substance. There is a typical unit membrane around each ovoid but initially there is no trace of fibrillar material in the suspension. When the suspension of particles is incubated with UDPG, linear wisps of fibrils are produced which associate rapidly to form longer and wider threads, especially in 0.01 M NaCl. There is no visible attachment of the wisps to the particles. After 20 min incubation, threads with the typical morphology of cellulose microfibrils are formed that later tend to become entangled in clumps. The microfibrils are insoluble in hot, aqueous, alkaline solutions and resistant to the action of trypsin, but may be degraded by glusulase. After treatment with 1 M NaOH at 100° C or with cold 18% NaOH they show an X-ray diffraction pattern which resembles that of Cellulose II from mercerized, authentic bacterial cellulose. Incorporation of radioactive glucose into the insoluble residue is enhanced by drying of the cellulose microfibrils before alkaline digestion and especially by the addition of a gross excess of carrier cellulose after incubation. In this system there is no evidence for participation of linear, axial, synthesizing sites on the cell wall of the bacterium or for ordered, organized granules in the assembly of the microfibrils. That is, cellulose-like microfibrils may be formed in a cell-free system without the action of any of the previously suggested cell organelles. In addition, these observations are consistent with a previously described notion of a transient, hydrated, nascent, bacterial cellulose microfibril. The possibility that cellulose microfibrils of green plants may be formed in the same way is considered.N.R.C.C. 18314     

The effect of the cellulose-binding domain from Clostridium cellulovorans on the supramolecular structure of cellulose fibers

The cellulose-binding domain (CBD) is the second important and the most wide-spread element of cellulase structure involved in cellulose transformation with a great structural diversity and a range of adsorption behavior toward different types of cellulosic materials. The effect of the CBD from Clostridium cellulovorans on the supramolecular structure of three different sources of cellulose (cotton cellulose, spruce dissolving pulp, and cellulose linters) was studied. Fourier-transform infrared spectroscopy (FTIR) was used to record amides I and II absorption bands of cotton cellulose treated with CBD. Structural changes as weakening and splitting of the hydrogen bonds within the cellulose chains after CBD adsorption were observed. The decrease of relative crystallinity index of the treated celluloses was confirmed by FTIR spectroscopy and X-ray diffraction (XRD). X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) were used to confirm the binding of the CBD on the cellulose surface and the changing of the cellulose morphology.     

一株产细菌纤维素菌株的筛选鉴定及其产物分析

从红茶菌液中筛选获得一株产细菌纤维素的菌株BC-41,经生理生化分析和分子生物学鉴定,现证实该菌株为中间葡糖酸醋杆菌(Gluconacetobacter intermedius)。对该菌株所产生的细菌纤维素进行了物理特性的表征和分析,获得以下数据:BC-41所产的纤维素纯度达到91.32%,湿纤维素膜含水率达99.16%,每克干纤维素膜能吸水28.59 g;扫描电子显微镜观察,显示该纤维素具有网状结构,且纤维束宽度分布在40-100 nm之间;X射线衍射分析,证实该纤维素的晶型为纤维素I型,结晶指数为48.8%;通过黏度测定法,得出该纤维素的平均聚合度达2 100。     

Enhancement of the nanofibrillation of wood cellulose through sequential periodate-chlorite oxidation

Sequential regioselective periodate-chlorite oxidation was employed as a new and efficient pretreatment to enhance the nanofibrillation of hardwood cellulose pulp through homogenization. The oxidized celluloses with carboxyl contents ranging from 0.38 to 1.75 mmol/g could nanofibrillate to highly viscous and transparent gels with yields of 100-85% without clogging the homogenizer (one to four passes). On the basis of field-emission scanning electron microscopy images, the nanofibrils obtained were of typical widths of approximately 25 ± 6 nm. All of the nanofibrillar samples maintained their cellulose I crystalline structure according to wide-angle X-ray diffraction results, and the crystallinity index was approximately 40% for all samples.     

X-ray microbeam and electron diffraction experiments on developing xylem cell walls

This paper describes the first successful application of the novel technique of X-ray microbeam diffraction to the study of wood cell walls in developing xylem tissue. The method enabled us to obtain quantitative diffraction diagrams from single cell walls. It was further combined with selected area electron diffraction. We find that the crystal size of cellulose is increasing from the primary wall (P, <19 A) over the outer layer (S(1), 19 A) to the middle layer of the secondary wall (S(2), 24 A). In particular, the cellulose crystals formed in the primary wall consist of an extremely low-crystalline state of cellulose I. We suggest the more applicable concept of microfibrils with increasing lateral disorder, similar to cellulose IV(I).     

Polymorphism of cellulose I family: reinvestigation of cellulose IVI

Polymorphs of cellulose I, III(I), and IV(I) have been investigated by X-ray diffraction, FT-IR, and solid-state (13)C NMR spectroscopy. Highly crystalline cellulose III(I) samples were prepared by treating cellulose samples in supercritical ammonia at 140 degrees C for 1 h, and conventional cellulose III(I) samples were prepared by liquid ammonia treatment. The cellulose IV(I) sample of highest crystallinity was that prepared from Cladophora cellulose III(I) in supercritical ammonia, followed by the sample treated in glycerol at 260 degrees C for 0.5 h, whereas the lowest crystallinity was observed in ramie cellulose prepared by conventional liquid ammonia treatment followed by glycerol annealing. In general, the perfection of cellulose IV(I) depends on the crystallinity of the original material: either of the starting cellulose I or of the cellulose III(I) after ammonia treatment. The product thus obtained was analogous to cellulose I(beta), which is what it should be called rather than cellulose IV(I). If the existence of the polymorph cellulose IV(I) is not accepted, the observations on which it has been based may be explained by the fact that the structure termed cellulose IV(I) is cellulose I(beta) which contains lateral disorder.     

棉纤维发育过程中细胞壁超微结构变化的研究

应用X射线衍射法研究了棉纤维发育过程中细胞壁超微结构变化的动态规律.其结果是:次生壁S_2层的平均微纤丝螺旋角随花后生长天数的增加而逐渐变小:纤维素微晶粒间的取向度逐渐变大;花后5—14天内,结晶度缓慢增加,14—17天内陡然增加,17天后缓慢趋向最大值.     

Dimeric calcium complexes of arabinan-rich pectic polysaccharides from Olea europaea L. cell walls

The study carried out in this work concerns the pectic polysaccharides of olive cell walls as present in olive pulp and that remained entrapped in the cellulosic residue after sequential extraction of the cell wall material (CWM) with imidazole, carbonate and KOH aqueous solutions. These polymers, obtained after neutralisation and dialysis of an aqueous suspension of the residue (sn-CR fraction), extracted with 4 M KOH, were arabinan-rich pectic polysaccharides. They accounted for 11–19% of the total pectic polysaccharides found in the olive pulp cell walls of fruits collected in two years and in three stages of ripening (green, cherry and black). The analysis by powder X-ray diffraction highlighted the existence, in all sn-CR fractions, of crystalline phases related with the presence of calcium-pectic polysaccharide complexes (CPPC) occurring in an amorphous carbohydrate network. The relative crystallinity of the CPPC varied linearly with the Ca²⁺/GalA molar ratio until a maximum of 0.57. Size-exclusion chromatography showed that sn-CR fractions possessed a bimodal molecular weight distribution. The lower molecular weight fraction of sn-CR (M_w = 70–135 kDa) was independent on the ripening stage of olive fruit, whereas the higher molecular weight fraction showed values of 1.1, 0.6–0.9 and 0.5–0.7 MDa, respectively, for green, cherry and black olives. Treatment of the sn-CR pectic polysaccharides with a 2 M imidazole solution disrupted the CPPC crystalline network showing the loss of low molecular weight galacturonan-rich material during dialysis (12–14 kDa cut off) and the decrease of molecular weight of the polymers to roughly half. These results allowed to infer the presence of oligogalacturonides held within cell walls by calcium ions and that the pectic polysaccharides of sn-CR fraction occurred in olive pulp cell walls as calcium bridged macrodimers.     

Enzymatic hydrolysis and recrystallization behavior of initially amorphous cellulose

Cellulose samples from cotton and wood pulps with varying low degrees of crystallinity (mechanically decrystallized) were studied. The influence of initial cellulose crystallinity on sugar yield after enzymatic hydrolysis was determined by two different methods. As expected, samples with low crystallinity were much more accessible to enzymatic attack and glucose yields were higher than were samples of high initial crystallinity. Hydrolysis of cellulose seems more dependent on cellulose crystallinity than on the source of cellulose. It is known that decrystallized or amorphous cellulose can recrystallize under proper conditions, e.g., during acid hydrolysis. The data reported here also reveal some recrystallization during enzymatic hydrolysis which probably occurs simulataneously with a selective enzymatic attack on the amorphous regions of cellulose. In all cases, the amorphous celluloses recrystallized in the original lattice form, that of native cellulose.     

Screening of the common culture conditions affecting crystallinity of bacterial cellulose

By analyzing with X-ray diffraction and FT-IR spectroscopy, the main effective factors among common culture conditions on crystallinity index and I _α fraction of cellulose produced by Gluconacetobacter xylinus were examined with Plackett–Burman design experiment. Varying pH value in the medium by adjusting the composition of citrate buffer or by adding HCl/NaOH solution indicates it is the content of citrate buffer rather than its function of pH buffering that gives the influence on crystallinity. Further experiment reveals that Na⁺ concentration of 0.174 mol/l in medium with citrate buffer added would decrease the crystallinity index significantly. Comparison of carbon sources shows that fructose leads to a higher crystallinity index than glucose, which suggests a relationship between crystallinity and production speed of bacterial cellulose affected by carbon sources. An interesting phenomenon was that a longer period of cultivation would decrease the crystallinity of bacterial cellulose. The reason is assumed to be the dense network of cellulose formed by bacterial cells that restrict the motion of themselves as the incubation period extends. Though the effect of inoculum age is still unclear, the influence on crystallinity of bacterial cellulose caused by variation of some ordinary culture conditions can be drawn out from data of this work.