Degradation and Crystallization of Cellulose in Hydrogen Chloride Vapor for High-Yield Isolation of Cellulose Nanocrystals.

Despite the structural, load-bearing role of cellulose in the plant kingdom, countless efforts have been devoted to degrading this recalcitrant polysaccharide, particularly in the context of biofuels and renewable nanomaterials. Herein, we show how the exposure of plant-based fibers to HCl vapor results in rapid degradation with simultaneous crystallization. Because of the unchanged sample texture and the lack of mass transfer out of the substrate in the gas/solid system, the changes in the crystallinity could be reliably monitored. Furthermore, we describe the preparation of cellulose nanocrystals in high yields and with minimal water consumption. The study serves as a starting point for the solid-state tuning of the supramolecular properties of morphologically heterogeneous biological materials.

The structure of Lactobacillus brevis surface layer reassembled on liposomes differs from native structure as revealed by SAXS.

The reassembly of the S-layer protein SlpA of Lactobacillus brevis ATCC 8287 on positively charged liposomes was studied by small angle X-ray scattering (SAXS) and zeta potential measurements. SlpA was reassembled on unilamellar liposomes consisting of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-3-trimethylammonium-propane, prepared by extrusion through membranes with pore sizes of 50nm and 100nm. Similarly extruded samples without SlpA were used as a reference. The SlpA-containing samples showed clear diffraction peaks in their SAXS intensities. The lattice constants were calculated from the diffraction pattern and compared to those determined for SlpA on native cell wall fragments. Lattice constants for SlpA reassembled on liposomes (a=9.29nm, b=8.03nm, and γ=84.9°) showed a marked change in the lattice constants b and γ when compared to those determined for SlpA on native cell wall fragments (a=9.41nm, b=6.48nm, and γ=77.0°). The latter are in good agreement with values previously determined by electron microscopy. This indicates that the structure formed by SlpA is stable on the bacterial cell wall, but SlpA reassembles into a different structure on cationic liposomes. From the (10) reflection, the lower limit of crystallite size of SlpA on liposomes was determined to be 92nm, corresponding to approximately ten aligned lattice planes.

Suppression of xylan endotransglycosylase PtxtXyn10A affects cellulose microfibril angle in secondary wall in aspen wood.

Certain xylanases from family GH10 are highly expressed during secondary wall deposition, but their function is unknown. We carried out functional analyses of the secondary-wall specific PtxtXyn10A in hybrid aspen (Populus tremula × tremuloides). PtxtXyn10A function was analysed by expression studies, overexpression in Arabidopsis protoplasts and by downregulation in aspen. PtxtXyn10A overexpression in Arabidopsis protoplasts resulted in increased xylan endotransglycosylation rather than hydrolysis. In aspen, the enzyme was found to be proteolytically processed to a 68 kDa peptide and residing in cell walls. Its downregulation resulted in a corresponding decrease in xylan endotransglycosylase activity and no change in xylanase activity. This did not alter xylan molecular weight or its branching pattern but affected the cellulose-microfibril angle in wood fibres, increased primary growth (stem elongation, leaf formation and enlargement) and reduced the tendency to form tension wood. Transcriptomes of transgenic plants showed downregulation of tension wood related genes and changes in stress-responsive genes. The data indicate that PtxtXyn10A acts as a xylan endotransglycosylase and its main function is to release tensional stresses arising during secondary wall deposition. Furthermore, they suggest that regulation of stresses in secondary walls plays a vital role in plant development.

Hydrophobin film structure for HFBI and HFBII and mechanism for accelerated film formation.

Hydrophobins represent an important group of proteins from both a biological and nanotechnological standpoint. They are the means through which filamentous fungi affect their environment to promote growth, and their properties at interfaces have resulted in numerous applications. In our study we have combined protein docking, molecular dynamics simulation, and electron cryo-microscopy to gain atomistic level insight into the surface structure of films composed of two class II hydrophobins: HFBI and HFBII produced by Trichoderma reesei. Together our results suggest a unit cell composed of six proteins; however, our computational results suggest P6 symmetry, while our experimental results show P3 symmetry with a unit cell size of 56 Å. Our computational results indicate the possibility of an alternate ordering with a three protein unit cell with P3 symmetry and a smaller unit cell size, and we have used a Monte Carlo simulation of a spin model representing the hydrophobin film to show how this alternate metastable structure may play a role in increasing the rate of surface coverage by hydrophobin films, possibly indicating a mechanism of more general significance to both biology and nanotechnology.

Cellulose structure and lignin distribution in normal and compression wood of the Maidenhair tree (Ginkgo biloba L.).

We studied in detail the mean microfibril angle and the width of cellulose crystals from the pith to the bark of a 15-year-old Maidenhair tree (Ginkgo biloba L.). The orientation of cellulose microfibrils with respect to the cell axis and the width and length of cellulose crystallites were determined using X-ray diffraction. Raman microscopy was used to compare the lignin distribution in the cell wall of normal/opposite and compression wood, which was found near the pith. Ginkgo biloba showed a relatively large mean microfibril angle, varying between 19° and 39° in the S2 layer, and the average width of cellulose crystallites was 3.1-3.2 nm. Mild compression wood without any intercellular spaces or helical cavities was observed near the pith. Slit-like bordered pit openings and a heavily lignified S2L layer confirmed the presence of compression wood. Ginkgo biloba showed typical features present in the juvenile wood of conifers. The microfibril angle remained large over the 14 annual rings. The entire stem disc, with a diameter of 18 cm, was considered to consist of juvenile wood. The properties of juvenile and compression wood as well as the cellulose orientation and crystalline width indicate that the wood formation of G. biloba is similar to that of modern conifers.

Structural and functional roles of carotenoids in chlorosomes.

Chlorosomes are large light-harvesting complexes found in three phyla of anoxygenic photosynthetic bacteria. Chlorosomes are primarily composed of self-assembling pigment aggregates. In addition to the main pigment, bacteriochlorophyll c, d, or e, chlorosomes also contain variable amounts of carotenoids. Here, we use X-ray scattering and electron cryomicroscopy, complemented with absorption spectroscopy and pigment analysis, to compare the morphologies, structures, and pigment compositions of chlorosomes from Chloroflexus aurantiacus grown under two different light conditions and Chlorobaculum tepidum. High-purity chlorosomes from C. aurantiacus contain about 20% more carotenoid per bacteriochlorophyll c molecule when grown under low light than when grown under high light. This accentuates the light-harvesting function of carotenoids, in addition to their photoprotective role. The low-light chlorosomes are thicker due to the overall greater content of pigments and contain domains of lamellar aggregates. Experiments where carotenoids were selectively extracted from intact chlorosomes using hexane proved that they are located in the interlamellar space, as observed previously for species belonging to the phylum Chlorobi. A fraction of the carotenoids are localized in the baseplate, where they are bound differently and cannot be removed by hexane. In C. tepidum, carotenoids cannot be extracted by hexane even from the chlorosome interior. The chemical structure of the pigments in C. tepidum may lead to π-π interactions between carotenoids and bacteriochlorophylls, preventing carotenoid extraction. The results provide information about the nature of interactions between bacteriochlorophylls and carotenoids in the protein-free environment of the chlorosome interior.

Distribution of counterions around lignosulfonate macromolecules in different polar solvent mixtures.

Lignosulfonate is a colloidal polyelectrolyte that is obtained as a side product in sulfite pulping. In this work we wanted to study the noncovalent association of the colloids in different solvents, as well as to find out how the charged sulfonate groups are organized on the colloid surface. We studied sodium and rubidium lignosulfonate in water-methanol mixtures and in dimethyl formamide. The number average molecular weights of the Na- and Rb-lignosulfonate fractions were 7600 g/mol and 9100 g/mol, respectively, and the polydispersity index for both was 2. Anomalous small-angle X-ray scattering (ASAXS) was used for determining the distribution of counterions around the Rb-lignosulfonate macromolecules. The scattering curves were fitted with a model constructed from ellipsoids of revolution of different sizes. Counterions were taken into account by deriving an approximative formula for the scattering intensity of the Poisson-Boltzmann diffuse double layer model. The interaction term between the spheroidal particles was estimated using the local monodisperse approximation and the improved Hayter-Penfold structure factor given by the rescaled mean spherical approximation. Effective charge of the polyelectrolyte and the local dielectric constant of the solvent close to the globular polyelectrolyte were followed as a function of the methanol content in the solvent and lignosulfonate concentration. The lignosulfonate macromolecules were found to aggregate noncovalently in water-methanol mixtures with increasing methanol or lignosulfonate content in a specific directional manner. The flat macromolecule aggregates had a nearly constant thickness of 1-1.4 nm, while their diameter grew when counterion association onto the polyelectrolyte increased. These results indicate that the charged groups in lignosulfonate are mostly at the flat surfaces of the colloid, allowing the associated lignosulfonate complexes to grow further at the edges of the complex.

Amorphous characteristics of an ultrathin cellulose film.

Swelling behavior and rearrangements of an amorphous ultrathin cellulose film (20 nm thickness) exposed to water and subsequently dried were investigated with grazing incidence X-ray diffraction, neutron reflectivity, atomic force microscopy, and surface energy calculations obtained from contact angle measurements. The film swelled excessively in water, doubling its thickness, but shrunk back to the original thickness upon water removal. Crystallinity (or amorphousness) and morphology remained relatively unchanged after the wetting/drying cycle, but surface free energy increased considerably (ca. 15%) due to an increase in its polar component, that is, the hydrophilicity of the film, indicating that rearrangements occurred during the film's exposure to water. Furthermore, stability of the films in aqueous NaOH solution was investigated with quartz crystal microbalance with dissipation monitoring. The films were stable at 0.0001 M NaOH but already 0.001 M NaOH partially dissolved the film. The surprising susceptibility to dissolve in dilute NaOH was hypothetically attributed to the lack of hierarchical morphology in the amorphous film.

Structural changes in microcrystalline cellulose in subcritical water treatment.

Subcritical water is a high potential green chemical for the hydrolysis of cellulose. In this study microcrystalline cellulose was treated in subcritical water to study structural changes of the cellulose residues. The alterations in particle size and appearance were studied by scanning electron microscopy (SEM) and those in the degree of polymerization (DP) and molar mass distributions by gel permeation chromatography (GPC). Further, changes in crystallinity and crystallite dimensions were quantified by wide-angle X-ray scattering and (13)C solid-state NMR. The results showed that the crystallinity remained practically unchanged throughout the treatment, whereas the size of the remaining cellulose crystallites increased. Microcrystalline cellulose underwent significant depolymerization in subcritical water. However, depolymerization leveled off at a relatively high degree of polymerization. The molar mass distributions of the residues showed a bimodal form. We infer that cellulose gets dissolved in subcritical water only after extensive depolymerization.

The lamellar spacing in self-assembling bacteriochlorophyll aggregates is proportional to the length of the esterifying alcohol.

Chlorosomes from green photosynthetic bacteria are large photosynthetic antennae containing self-assembling aggregates of bacteriochlorophyll c, d, or e. The pigments within chlorosomes are organized in curved lamellar structures. Aggregates with similar optical properties can be prepared in vitro, both in polar as well as non-polar solvents. In order to gain insight into their structure we examined hexane-induced aggregates of purified bacteriochlorophyll c by X-ray scattering. The bacteriochlorophyll c aggregates exhibit scattering features that are virtually identical to those of native chlorosomes demonstrating that the self-assembly of these pigments is fully encoded in their chemical structure. Thus, the hexane-induced aggregates constitute an excellent model to study the effects of chemical structure on assembly. Using bacteriochlorophyllides transesterified with different alcohols we have established a linear relationship between the esterifying alcohol length and the lamellar spacing. The results provide a structural basis for lamellar spacing variability observed for native chlorosomes from different species. A plausible physiological role of this variability is discussed. The X-ray scattering also confirmed the assignments of peaks, which arise from the crystalline baseplate in the native chlorosomes.

Changes in submicrometer structure of enzymatically hydrolyzed microcrystalline cellulose.

To understand the limitations occurring during enzymatic hydrolysis of cellulosic materials in renewable energy production, we used wide-angle X-ray scattering (WAXS), small-angle X-ray scattering (SAXS), X-ray microtomography, and transmission electron microscopy (TEM) to characterize submicrometer changes in the structure of microcrystalline cellulose (Avicel) digested with the Trichoderma reesei enzyme system. The microtomography measurements showed a clear decrease in particle size in scale of tens of micrometers. In all the TEM pictures, similar elongated and partly ramified structures were observed, independent of the hydrolysis time. The SAXS results of rewetted samples suggested a slight change in the structure in scale of 10-20 nm, whereas the WAXS results confirmed that the degree of crystallinity and the crystal sizes remained unchanged. This indicates that the enzymes act on the surface of cellulose bundles and are unable to penetrate into the nanopores of wet cellulose.

Structure of chlorosomes from the green filamentous bacterium Chloroflexus aurantiacus.

The green filamentous bacterium Chloroflexus aurantiacus employs chlorosomes as photosynthetic antennae. Chlorosomes contain bacteriochlorophyll aggregates and are attached to the inner side of a plasma membrane via a protein baseplate. The structure of chlorosomes from C. aurantiacus was investigated by using a combination of cryo-electron microscopy and X-ray diffraction and compared with that of Chlorobi species. Cryo-electron tomography revealed thin chlorosomes for which a distinct crystalline baseplate lattice was visualized in high-resolution projections. The baseplate is present only on one side of the chlorosome, and the lattice dimensions suggest that a dimer of the CsmA protein is the building block. The bacteriochlorophyll aggregates inside the chlorosome are arranged in lamellae, but the spacing is much greater than that in Chlorobi species. A comparison of chlorosomes from different species suggested that the lamellar spacing is proportional to the chain length of the esterifying alcohols. C. aurantiacus chlorosomes accumulate larger quantities of carotenoids under high-light conditions, presumably to provide photoprotection. The wider lamellae allow accommodation of the additional carotenoids and lead to increased disorder within the lamellae.

Interactions of hydrophobin proteins in solution studied by small-angle X-ray scattering.

Hydrophobins are a group of very surface-active, fungal proteins known to self-assemble on various hydrophobic/hydrophilic interfaces. The self-assembled films coat fungal structures and mediate their attachment to surfaces. Hydrophobins are also soluble in water. Here, the association of hydrophobins HFBI and HFBII from Trichoderma reesei in aqueous solution was studied using small-angle x-ray scattering. Both HFBI and HFBII exist mainly as tetramers in solution in the concentration range 0.5-10 mg/ml. The assemblies of HFBII dissociate more easily than those of HFBI, which can tolerate changes of pH from 3 to 9 and temperatures in the range 5 degrees C-60 degrees C. The self-association of HFBI and HFBII is mainly driven by the hydrophobic effect, and addition of salts along the Hofmeister series promotes the formation of larger assemblies, whereas ethanol breaks the tetramers into monomers. The possibility that the oligomers in solution form the building blocks of the self-assembled film at the air/water interface is discussed.

Effect of amylose content on physical and mechanical properties of potato-starch-based edible films.

The present study investigated the amylose content and the gelatinization properties of various potato starches extracted from different potato cultivars. These potato starches were used to prepare edible films. Physical and mechanical properties of the films were investigated. The crystallinity of selected native starches and edible films made of the same starches were determined by X-ray diffraction. The amylose content of potato starches varied between 11.9 and 20.1%. Gelatinization of potato starches in excess water occurred at temperatures ranging from 58 to 69 degrees C independently of the amylose content. The relative crystallinity was found to be around 10-13% in selected native potato starches with low, medium, and high amylose content. Instead, films prepared from the same potato starches were found to be practically amorphous having the relative crystallinity of 0-4%. The mechanical properties and the water vapor permeability of the films were found to be independent of the amylose content.

The relation between solution association and surface activity of the hydrophobin HFBI from Trichoderma reesei.

Hydrophobins are small fungal surface active proteins that self-assemble at interfaces into films with nanoscale structures. The hydrophobin HFBI from Trichoderma reesei has been shown to associate in solution into tetramers but the role of this association on the function of HFBI has remained unclear. We produced two HFBI variants that showed a significant shift in solution association equilibrium towards the tetramer state. However, this enhanced solution association did not alter the surface properties of the variant HFBIs. The results show that there is not a strong relationship between HFBI solution association state and surface properties such as surface activity.

Tailoring of the hierarchical structure within electrospun fibers due to supramolecular comb-coil block copolymers: polystyrene-block-poly(4-vinyl pyridine) plasticized by hydrogen bonded pentadecylphenol.

Previously we demonstrated hierarchical self-assembly and mesoporosity in electrospun fibers using selected polystyrene--poly(4-vinylpyridine) (PS--P4VP) diblock copolymers with hydrogen-bonded 3--pentadecylphenol (PDP), which rendered distorted spherical P4VP(PDP) domains within the PS matrix, internal lamellar order within the P4VP(PDP) domains, and allowed distorted spherical pores by removing PDP (. 2005, , 1048). Here we study whether the internal structure of electrospun fibers can be systematically tailored by varying the compositions of PS--P4VP(PDP). We expect these complexes to be feasible choices to combine electrospinning and self-assembly, as relatively high molecular weight block copolymers are useful for electrospinning, and enhanced structure formation due to plasticization by the amphiphilic PDP was expected. Not surprisingly, the self-assembled structures of the as-prepared fibers were less perfect than those in the corresponding well-annealed bulk materials. Compositions that show spherical self-assembly of P4VP(PDP) within the PS matrix in bulk lead to distinct and elongated worm-like P4VP(PDP) domains within the PS matrix in electrospun fibers. More symmetric compositions, which showed lamellar self-assembly in bulk, lead to structures where both PS and P4VP(PDP) domains were worm-like and elongated in a relatively symmetric manner. Finally, compositions which in bulk showed self-assembly of PS spheres within the P4VP(PDP) matrix, lead to separate distorted PS domains in the P4VP(PDP) matrix. Additionally, SAXS measurements suggest a lamellar structure within the P4VP(PDP) domains. As electrospinning is a facile method to prepare mesoscale fibers, and it is known that the amphiphiles can be removed from the hierarchical assemblies, the present method offers the potential to tune the internal porosity of the fibers for release and absorption purposes.

In muro deacetylation of xylan affects lignin properties and improves saccharification of aspen wood.

BACKGROUND: Lignocellulose from fast growing hardwood species is a preferred source of polysaccharides for advanced biofuels and "green" chemicals. However, the extensive acetylation of hardwood xylan hinders lignocellulose saccharification by obstructing enzymatic xylan hydrolysis and causing inhibitory acetic acid concentrations during microbial sugar fermentation. To optimize lignocellulose for cost-effective saccharification and biofuel production, an acetyl xylan esterase AnAXE1 from Aspergillus niger was introduced into aspen and targeted to cell walls. RESULTS: AnAXE1-expressing plants exhibited reduced xylan acetylation and grew normally. Without pretreatment, their lignocellulose yielded over 25% more glucose per unit mass of wood (dry weight) than wild-type plants. Glucose yields were less improved (+7%) after acid pretreatment, which hydrolyses xylan. The results indicate that AnAXE1 expression also reduced the molecular weight of xylan, and xylan-lignin complexes and/or lignin co-extracted with xylan, increased cellulose crystallinity, altered the lignin composition, reducing its syringyl to guaiacyl ratio, and increased lignin solubility in dioxane and hot water. Lignin-associated carbohydrates became enriched in xylose residues, indicating a higher content of xylo-oligosaccharides. CONCLUSIONS: This work revealed several changes in plant cell walls caused by deacetylation of xylan. We propose that deacetylated xylan is partially hydrolyzed in the cell walls, liberating xylo-oligosaccharides and their associated lignin oligomers from the cell wall network. Deacetylating xylan thus not only increases its susceptibility to hydrolytic enzymes during saccharification but also changes the cell wall architecture, increasing the extractability of lignin and xylan and facilitating saccharification.

Simulation of small-angle x-ray scattering from collagen fibrils and comparison with experimental patterns.

Simulation of small-angle x-ray scattering from collagen in healthy and cancerous breast tissue may reveal detailed information on the structural changes in collagen. Collagen fibril is modelled as a cylinder with axially periodic step-function electron density, and packing is approximated by placing the cylinders in small hexagonal bundles. The intensity from a bundle is calculated by summing analytical scattering amplitudes from the cylinders, and intensities from several bundles with varying lattice constants are averaged. Comparisons with more complex models are made to estimate the robustness of the model. The oscillations in the equatorial direction are not significantly affected by added complexity. The relative intensities of the Bragg peaks in the meridional direction can be tuned by modifying the axial electron density distribution. Tests with different fibril radius distributions show that the average radius can be determined with an accuracy of +/-0.5 nm but that the shape of the radius distribution cannot be accurately determined from the scattering patterns. The effect of multiple scattering and the detector point-spread function (PSF) is considered, and the PSF may make a significant contribution to the final slope of the scattering pattern. Comparisons with observed scattering indicate that the model is basically correct at the supra-molecular level.

Human breast cancer in vitro: matching histo-pathology with small-angle x-ray scattering and diffraction enhanced x-ray imaging.

Twenty-eight human breast tumour specimens were studied with small-angle x-ray scattering (SAXS), and 10 of those were imaged by the diffraction enhanced x-ray imaging (DEI) technique. The sample diameter was 20 mm and the thickness 1 mm. Two examples of ductal carcinoma are illustrated by histology images, DEI, and maps of the collagen d-spacing and scattered intensity in the Porod regime, which characterize the SAXS patterns from collagen-rich regions of the samples. Histo-pathology reveals the cancer-invaded regions, and the maps of the SAXS parameters show that in these regions the scattering signal differs significantly from scattering by the surrounding tissue, indicating a degradation of the collagen structure in the invaded regions. The DEI images show the borders between collagen and adipose tissue and provide a co-ordinate system for tissue mapping by SAXS. In addition, degradation of the collagen structure in an invaded region is revealed by fading contrast of the DEI refraction image. The 28 samples include fresh, defrosted tissue and formalin-fixed tissue. The d-values with their standard deviations are given. In the fresh samples there is a systematic 0.76% increase of the d-value in the invaded regions, averaged over 11 samples. Only intra-sample comparisons are made for the formalin-fixed samples, and with a long fixation time, the difference in the d-value stabilizes at about 0.7%. The correspondence between the DEI images, the SAXS maps and the histo-pathology suggests that definitive information on tumour growth and malignancy is obtained by combining these x-ray methods.

Influence of incorporated wild Solanum genomes on potato properties in terms of starch nanostructure and glycoalkaloid content.

Interspecific somatic hybrids produced by protoplast fusion between two wild Solanum species (S. acaule, acl; S. brevidens, brd) and cultivated potato Solanum tuberosum (tbr) were analyzed in terms of the starch nanometer-range structure and glycoalkaloid (GA) contents. The crystallinity of starch granules, the average size of starch crystallites, and the lamellar distances were obtained from tuber samples using wide-angle and small-angle X-ray scattering methods. These measurements showed that incorporation of wild genomes from either nontuberous (brd) or tuberous (acl) Solanum species caused no significant modifications of the nanostructure of potato starch. In contrast, the GA profiles of the hybrids, which were analyzed by LC-ESI-MS in both tuber and foliage samples, differed considerably from those of cultivated potato. Regardless of the low total tuber GA concentrations (approximately 9 mg/100 g of fresh weight), the somatic hybrids contained GAs not detected in the parental species. A high proportion of spirotype GAs consisting of 5,6-dihydrogenated aglycons, for example, alpha-tomatine and tomatidine bound with solatriose, and chacotriose were found in the hybrids. In conclusion, the foliage of interspecific hybrids contained a higher variation in the structures of GAs than did the tubers.