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Cellulose microfibrils, which form the mechanical framework of the plant cell wall, are synthesized by the cellulose synthase complex in the plasma membrane. Here, we introduced point mutations into the catalytic domain of cellulose synthase 6 (CESA6) in Arabidopsis to produce enhanced yellow fluorescent protein (EYFP)-tagged CESA6D395N, CESA6Q823E, and CESA6D395N+Q823E, which were exogenously produced in a cesa6 null mutant, prc1-1. Comparison of these mutants in terms of plant phenotype, cellulose content, cellulose synthase complex dynamics, and organization of cellulose microfibrils showed that prc1-1 expressing EYFP:CESA6D395N or CESA6D395N+Q823E was nearly the same as prc1-1, whereas prc1-1 expressing EYFP:CESA6Q823E was almost identical to wild type and prc1-1 expressing EYFP:WT CESA6, indicating that CESA6D395N and CESA6D395N+Q823E do not function in cellulose synthesis, while CESA6Q823E is still functionally active. Total internal reflection fluorescence microscopy and confocal microscopy were used to monitor the subcellular localization of these proteins. We found that EYFP:CESA6D395N and EYFP:CESA6D395N+Q823E were absent from subcellular regions containing the Golgi and the plasma membrane, and they appeared to be retained in the endoplasmic reticulum. By contrast, EYFP:CESA6Q823E had a normal localization pattern, like that of wild-type EYFP:CESA6. Our results demonstrate that the D395N mutation in CESA6 interrupts its normal transport to the Golgi and its eventual participation in cellulose synthase complex assembly., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Aims: Obtain varieties of Gluconacetobacter hansenii from original strain ATCC 23729 with greater efficiency to produce bacterial cellulose (BC) membrane with better dry mass yield for application as support of sustained antimicrobials' drug release., Methods and Results: Application of different chemical and physical conditions (pH, temperature and UV light exposure) to obtain different G. hansenii varieties with high capacity to produce BC membranes. Characterization of the G. hansenii variants was performed by scanning electron microscopy (SEM) and optical microscopy of the colony-forming units. BC membrane produced was characterized by SEM, infrared spectroscopy and X-ray diffraction. The BC produced by variants isolated after incubation at 35°C showed elevated dry mass yield and high capacity of retention and sustained release of ceftriaxone antibiotic with the produced BC by original G. hansenii ATCC 23769 strain subjected to incubation at 28°C and with commercial BC., Conclusion: The application of different chemical and physical conditions constitutes an important method to obtain varieties of micro-organisms with dissimilar metabolism advantageous in relation to the original strain in the BC production., Significance and Impact of the Study: These results demonstrate the importance of in vivo studies for the application, in medicine, of BC membranes as support for antimicrobial-sustained release for the skin wound treatment., (© 2018 The Society for Applied Microbiology.)

Structural organization of the plant cell wall is a key parameter for understanding anisotropic plant growth and mechanical behavior. Four imaging platforms were used to investigate the cell wall architecture of Miscanthus sinensis cv. internode tissue. Using transmission electron microscopy with potassium permanganate, we found a great degree of inhomogeneity in the layering structure (4-9 layers) of the sclerenchymatic fiber (Sf). However, the xylem vessel showed a single layer. Atomic force microscopy images revealed that the cellulose microfibrils (Mfs) deposited in the primary wall of the protoxylem vessel (Pxv) were disordered, while the secondary wall was composed of Mfs oriented in parallel in the cross and longitudinal section. Furthermore, Raman spectroscopy images indicated no variation in the Mf orientation of Pxv and the Mfs in Pxv were oriented more perpendicular to the cell axis than that of Sfs. Based on the integrated results, we have proposed an architectural model of Pxv composed of two layers: an outermost primary wall composed of a meshwork of Mfs and inner secondary wall containing parallel Mfs. This proposed model will support future ultrastructural analysis of plant cell walls in heterogeneous tissues, an area of increasing scientific interest particularly for liquid biofuel processing.

Aims: The influence of bacterial species/strains in agitated culture was investigated on the morphology and structure characteristics of bacterial cellulose., Methods and Results: Komagataeibacter nataicola Y19 and Gluconacetobacter entanii ACCC10215 were inoculated in Hestrin-Schramm (HS) medium and subjected to agitated cultivation. Different kinds of BCs were obtained including flocky asterisk-like BC by G. entanii ACCC10215 and solid sphere-like BC by K. nataicola Y19. The SEM results showed that the asterisk-like BC had larger pores than the solid sphere-like BC. The FT-IR and X-ray diffraction results showed the asterisk-like BC had lower crystallinity (81·43%), higher cellulose Iα mass fraction (79·74%) and smaller crystallite size., Conclusions: The different species/strains can influence the morphology and structure characteristics of BC in agitated culture., Significance and Impact of the Study: We examined the influence of different species/strains on the morphology, macro- and microstructure of BCs produced in agitated culture for the first time, which suggest that different BCs with potential applications could be obtained by choosing different species or strains and fermentation method., (© 2014 The Society for Applied Microbiology.)

A substantial proportion of the architecture of the plant cell wall remains unknown with a few cell wall models being proposed. Moreover, even less is known about the green algal cell wall. Techniques that allow direct visualization of the cell wall in as near to its native state are of importance in unravelling the spatial arrangement of cell wall structures and hence in the development of cell wall models. Atomic force microscopy (AFM) was used to image the native cell wall of living cells of Ventricaria ventricosa (V. ventricosa) at high resolution under physiological conditions. The cell wall polymers were identified mainly qualitatively via their structural appearance. The cellulose microfibrils (CMFs) were easily recognizable and the imaging results indicate that the V. ventricosa cell wall has a cross-fibrillar structure throughout. We found the native wall to be abundant in matrix polysaccharides existing in different curing states. The soft phase matrix polysaccharides susceptible by the AFM scanning tip existed as a glutinous fibrillar meshwork, possibly incorporating both the pectic- and hemicellulosic-type substances. The hard phase matrix producing clearer images, revealed coiled fibrillar structures associated with CMFs, sometimes being resolved as globular structures by the AFM tip. The coiling fibrillar structures were also seen in the images of isolated cell wall fragments. The mucilaginous component of the wall was discernible from the gelatinous cell wall matrix as it formed microstructural domains over the surface. AFM has been successful in imaging the native cell wall and revealing novel findings such as the 'coiling fibrillar structures' and cell wall components which have previously not been seen, that is, the gelatinous matrix phase.

Aims: To evaluate the possibility of elephant grass acid hydrolysate converting into bacterial cellulose (BC) produced by Gluconacetobacter xylinus CH001 and to characterize the morphology and structure of the cellulose produced., Methods and Results: Acid-hydrolysed and detoxified elephant grass acid hydrolysate was inoculated with G. xylinus CH001. After 14 days of static fermentation, about 6·4 g l(-1) of BC could be generated. Meanwhile, 60·4% (w/w) of BC yield on sugar consumption was obtained. Scanning electron micrographs illustrated that the network of cellulose fibres became denser, and the diameter changed with the growth. FT-IR spectra showed almost same results for all the BC samples collected on different culture time. X-ray diffractograms demonstrated that the crystalline form of BC was cellulose I, the crystallinity increased to 53·58%, and the crystallinity index reached up to 99%., Conclusions: Elephant grass acid hydrolysate could be utilized efficiently for BC production by G. xylinus CH001. Structure analysis on the cellulose produced showed its potential of being excellent material for further application., Significance and Impact of the Study: Our studies for the first time examined the bioconversion of low-cost elephant grass into high-value BC and the changes in its morphology and structure following the culture time., (© 2013 The Society for Applied Microbiology.)

A Monte Carlo simulation was used to determine geometry correction factors that increase accuracy of quantitative X-ray microanalysis of laterally semithick biological materials. A model composed of cellulose with homogeneously distributed biological elements and lateral dimensions between 0.5-25 μm was chosen. The specimen was exposed to 5, 10, and 15 keV electrons, the net intensities of characteristic X-rays registered for the elements, and presented as a function of the lateral dimensions of the model. This showed the double decay exponential function fitted the distribution of X-ray intensities in relation to the model size. The applicability of the function as a correction method was successfully tested for 30 specimens with varying composition and dimensions. The value of relative error decreased from ±60% to ±5% when the correction was applied. Moreover, the minimal lateral size of the material was defined, below which the correction is not required. The simulation also revealed that the difference of the weighted sum of Z²/A between the unknown and the standard could reach 25% without significant influence on the quantitative results. The correction method could be helpful for accurate assessment of elemental composition in biological or organic matrices, when their lateral dimensions are smaller than the distribution range.

This research examined cellulolytic effects of fungi and other microbes present in cured sausages on the strength and stability of regenerated cellulose casings (RCC) used in the sausage industry. Occasionally during the curing process, RCC would split or fail, thereby leading to loss of product. The fungus Penicillium sp. BT-F-1, which was isolated from fermented sausages, and other fungi, which were introduced to enable the curing process, produced small amounts of cellulases on RCC in both liquid and solid cultivations. During continued incubation for 15-60 days in solid substrate cultivation (SSC) on RCC support, the fungus Penicillium sp isolate BT-F-1 degraded the casings' dry weights by 15-50% and decreased their tensile strengths by ~75%. Similarly commercial cellulase(s) resulted in 20-50% degradation of RCC in 48 h. During incubation with Penicillium sp BT-F-1, the surface structure of RCC collapsed, resulting in loss of strength and stability of casings. The matrix of industrial RCC comprised 88-93% glucose polymer residues with 0.8-4% xylan impurities. Premature casing failure appeared to result from operating conditions in the manufacturing process that allowed xylan to build up in the extrusion bath. The sausage fungus Penicillium sp BT-F-1 produced xylanases to break down soft xylan pockets prior to slow cellulosic dissolution of RCC.

This work comprises the structural characterization of Eucalyptus and Pinus radiata pulp fibers and their corresponding fibrillated materials, based on quantitative electron microscopy techniques. Compared to hardwood fibers, the softwood fibers have a relatively open structure of the fiber wall outer layers. The fibrillation of the fibers was performed mechanically and chemi-mechanically. In the chemi-mechanical process, the pulp fibers were subjected to a TEMPO-mediated oxidation to facilitate the homogenization. Films were made of the fibrillated materials to evaluate some structural properties. The thicknesses and roughnesses of the films were evaluated with standardized methods and with scanning electron microscopy (SEM), in backscattered electron imaging mode. Field-emission SEM (FE-SEM) and transmission electron microscopy (TEM) were performed to quantify the nanofibril morphology. In this study, we give additional and significant evidences about the suitability of electron microscopy techniques for quantification of nanofibril structures. In addition, we conclude that standard methods are not suitable for estimating the thickness of films having relatively rough surfaces. The results revealed significant differences with respect to the morphology of the fibrillated material. The differences are due to the starting raw material and to the procedure applied for the fibrillation.

It is generally believed that cell elongation is regulated by cortical microtubules, which guide the movement of cellulose synthase complexes as they secrete cellulose microfibrils into the periplasmic space. Transversely oriented microtubules are predicted to direct the deposition of a parallel array of microfibrils, thus generating a mechanically anisotropic cell wall that will favor elongation and prevent radial swelling. Thus far, support for this model has been most convincingly demonstrated in filamentous algae. We found that in etiolated Arabidopsis thaliana hypocotyls, microtubules and cellulose synthase trajectories are transversely oriented on the outer surface of the epidermis for only a short period during growth and that anisotropic growth continues after this transverse organization is lost. Our data support previous findings that the outer epidermal wall is polylamellate in structure, with little or no anisotropy. By contrast, we observed perfectly transverse microtubules and microfibrils at the inner face of the epidermis during all stages of cell expansion. Experimental perturbation of cortical microtubule organization preferentially at the inner face led to increased radial swelling. Our study highlights the previously underestimated complexity of cortical microtubule organization in the shoot epidermis and underscores a role for the inner tissues in the regulation of growth anisotropy.

Aims: To determine the effect of carbon sources on cellulose produced by Gluconacetobacter xylinus strain ATCC 53524, and to characterize the purity and structural features of the cellulose produced., Methods and Results: Modified Hestrin Schramm medium containing the carbon sources mannitol, glucose, glycerol, fructose, sucrose or galactose were inoculated with Ga. xylinus strain ATCC 53524. Plate counts indicated that all carbon sources supported growth of the strain. Sucrose and glycerol gave the highest cellulose yields of 3.83 and 3.75 g l(-1) respectively after 96 h fermentation, primarily due to a surge in cellulose production in the last 12 h. Mannitol, fructose or glucose resulted in consistent rates of cellulose production and yields of >2.5 g l(-1). Solid state (13)C CP/MAS NMR revealed that irrespective of the carbon source, the cellulose produced by ATCC 53524 was pure and highly crystalline. Scanning electron micrographs illustrated the densely packed network of cellulose fibres within the pellicles and that the different carbon sources did not markedly alter the micro-architecture of the resulting cellulose pellicles., Conclusions: The production rate of bacterial cellulose by Ga. xylinus (ATCC 53524) was influenced by different carbon sources, but the product formed was indistinguishable in molecular and microscopic features., Significance and Impact of the Study: Our studies for the first time examined the influence of different carbon sources on the rate of cellulose production by Ga. xylinus ATCC 53524, and the molecular and microscopic features of the cellulose produced.

Bacterial cellulose (BC) production by Acetobacter xylinum NUST4.1 was carried out in the shake flask and in a stirred-tank reactor by means of adding sodium alginate (NaAlg) into the medium. When 0.04% (w/v) NaAlg was added in the shake flask, BC production reached 6.0 g/l and the terminal yield of the cellulose was 27% of the total sugar initially added, compared with 3.7 g/l and 24% in the control, respectively. The variation between replicates in all determinations was less than 5%. During the cultivation in the stirred-tank reactor, the addition of NaAlg changed the morphology of cellulose from the irregular clumps and fibrous masses entangled in the internals to discrete masses dispersing into the broth, which indicates that NaAlg hinders formation of large clumps of BC, and enhances cellulose yield. Because the structure of cellulose is changed depending on the culture condition such as additives, structural characteristics of BC produced in the NaAlg-free and NaAlg medium are compared using scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD). SEM photographs show some differences in reticulated structures and ribbon width and FT-IR spectra indicate that there is the hydrogen bonding interaction between BC and NaAlg, then X-ray diffraction (XRD) analysis reveals that BC produced with NaAlg-added has a lower crystallinity and a smaller crystalline size. The results show that enhanced yields and modification of cellulose structure occur in the presence of NaAlg.

Enzyme engineering was performed to link the beta-glucosidase enzyme (BGL1) from Saccharomycopsis fibuligera to the cellulose-binding domain (CBD2) of Trichoderma reesei cellobiohydrolase (CBHII) to investigate the effect of a fungal CBD on the enzymatic characteristics of this non-cellulolytic yeast enzyme. Recombinant enzymes were constructed with single and double copies of CBD2 fused at the N-terminus of BGL1 to mimic the two-domain organization displayed by cellulolytic enzymes in nature. The engineered S. fibuligera beta-glucosidases were expressed in Saccharomyces cerevisiae under the control of phosphoglycerate-kinase-1 promoter (PGK1 ( P )) and terminator (PGK1 ( T )) and yeast mating pheromone alpha-factor secretion signal (MFalpha1 ( S )). The secreted enzymes were purified and characterized using a range of cellulosic and non-cellulosic substrates to illustrate the effect of the CBD on their enzymatic activity. The results indicated that the recombinant enzymes of BGL1 displayed a 2-4-fold increase in their hydrolytic activity toward cellulosic substrates like avicel, amorphous cellulose, bacterial microcrystalline cellulose, and carboxy methyl cellulose in comparison with the native enzyme. The organization of the CBD in these recombinant enzymes also resulted in enhanced substrate affinity, molecular flexibility and synergistic activity, thereby improving the ability of the enzymes to act on and hydrolyze cellulosic substrates, as characterized by adsorption, kinetics, thermal stability, and scanning electron microscopic analyses.

Using both in vivo (inhalation) and in vitro (cell culture) studies, we previously reported that p-aramid respirable fibers (RFP--defined as respirable-sized fiber-shaped particulates) are biodegraded in lungs and lung cells of rats following exposures. The current studies were undertaken to determine whether shortening mechanisms of p-aramid RFP biodegradability are also operative in human lung cells. Cultures of human A549 lung epithelial cells (A549), primary alveolar macrophages (HBAL) (collected via bronchoalveolar lavage [BAL]) from volunteers), and co-cultures (Co) of the A549 and HBAL were incubated with p-aramid RFP for either 1 h, 1 day, or 1 week to assess RFP shortening. Lengths of RFP were measured using scanning electron microscopy (SEM) following fixation, digestion of culture tissue components, and processing. Similar to findings using rat lung cells, only slight RFP shortening was measured in A549 cultures at 1-day and 1-week post-incubation. More importantly, in HBAL and Co groups, greater transverse cleavage of p-aramid RFP was measured at 1-day and 1-week postexposure compared to 1-h HBAL or Co groups, or in any A549 groups. In contrast, cellulose RFP, a biopersistent reference control fiber, were not measurably shortened under similar circumstances. Second, p-aramid RFP were incubated either with phosphate-buffered saline (PBS), or acellular BAL fluids from human volunteers or rats and processed for SEM analysis of RFP lengths. Mean lengths of p-aramid RFP incubated with human or rat BAL fluids were substantially decreased compared to PBS. Similar to our findings with rat lung cells, components of human lung fluids coat the p-aramid RFP as a prerequisite for subsequent enzymatic cleavage by human phagocytic lung cells and this finding reinforces the concept that inhaled p-aramid RFP are likely to be biodegradable in the lungs of humans.

Cortical microtubules have long been hypothesized to regulate the oriented deposition of cellulose microfibrils. However, the molecular mechanisms of how microtubules direct the orientation of cellulose microfibril deposition are not known. We have used fibers in the inflorescence stems of Arabidopsis to study secondary wall deposition and cell wall strength and found a fragile fiber (fra1) mutant with a dramatic reduction in the mechanical strength of fibers. The fra1 mutation did not cause any defects in cell wall composition, secondary wall thickening, or cortical microtubule organization in fiber cells. An apparent alteration was found in the orientation of cellulose microfibrils in fra1 fiber walls, indicating that the reduced mechanical strength of fra1 fibers probably was attributable to altered cellulose microfibril deposition. The FRA1 gene was cloned and found to encode a kinesin-like protein with an N-terminal microtubule binding motor domain. The FRA1 protein was shown to be concentrated around the periphery of the cytoplasm but absent in the nucleus. Based on these findings, we propose that the FRA1 kinesin-like protein is involved in the microtubule control of cellulose microfibril order.

The occurrence and chemical nature of the cross-links between cellulose microfibrils in outer epidermal cell walls in Pisum sativum cv. Alaska was investigated by rapid-freezing and deep-etching techniques coupled with chemical and enzymatic treatments. The cell wall in the elongating region of epidermal cells was characterized by the absence of the cross-links, while in the elongated region, the cell wall was characterized by the presence of cross-links. The cross-links remained in the cell wall of the elongated region after treatment with SDS electrophoresis sample buffer and treatment with 4% potassium hydroxide. After treatment with endo-1,4-beta-glucanase, which fragments xyloglucan, the cross-links were remarkably reduced from the cell wall of the elongated region. The endoglucanase treatment also reduced immunogold labeling of xyloglucan in the cell wall. The endoglucanase hydrolysate from the cell wall fraction of the elongated region gave spots of oligosaccharides in thin layer chromatography, which were identical to the spots of xyloglucan oligosaccharides produced by xyloglucanase from both the cell wall fraction and tamarind xyloglucan. These results indicate that the cross-links are made of xyloglucan. We discussed the possibility of cross-links involved in the control of mechanical properties of the cell wall.