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DOE OSTI2015

Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis

Here, xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis ( Arabidopsis thaliana ) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolated xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP , and receptor genes, such as HERK1 and WAK1 , were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

An alternate route for cellulose microfibril biosynthesis in plants

Similar to cellulose synthases (CESAs), cellulose synthase–like D (CSLD) proteins synthesize β-1,4-glucan in plants. CSLDs are important for tip growth and cytokinesis, but it was unknown whether they form membrane complexes in vivo or produce microfibrillar cellulose. We produced viable CESA-deficient mutants of the moss Physcomitrium patens to investigate CSLD function without interfering CESA activity. Microscopy and spectroscopy showed that CESA-deficient mutants synthesize cellulose microfibrils that are indistinguishable from those in vascular plants. Correspondingly, freeze-fracture electron microscopy revealed rosette-shaped particle assemblies in the plasma membrane that are indistinguishable from CESA-containing rosette cellulose synthesis complexes (CSCs). Our data show that proteins other than CESAs, most likely CSLDs, produce cellulose microfibrils in P. patens protonemal filaments. The data suggest that the specialized roles of CSLDs in cytokinesis and tip growth are based on differential expression and different interactions with microtubules and possibly Ca 2+ , rather than structural differences in the microfibrils they produce.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Competitive Exclusion and Metabolic Dependency among Microorganisms Structure the Cellulose Economy of an Agricultural Soil

Microorganisms that degrade cellulose utilize extracellular reactions that yield free by-products which can promote interactions with noncellulolytic organisms. We hypothesized that these interactions determine the ecological and physiological traits governing the fate of cellulosic carbon (C) in soil. We performed comparative genomics with genome bins from a shotgun metagenomic-stable isotope probing experiment to characterize the attributes of cellulolytic and noncellulolytic taxa accessing 13 C from cellulose. We hypothesized that cellulolytic taxa would exhibit competitive traits that limit access, while noncellulolytic taxa would display greater metabolic dependency, such as signatures of adaptive gene loss. We tested our hypotheses by evaluating genomic traits indicative of competitive exclusion or metabolic dependency, such as antibiotic production, growth rate, surface attachment, biomass degrading potential, and auxotrophy. The most 13 C-enriched taxa were cellulolytic Cellvibrio (Gammaproteobacteria) and Chaetomium (Ascomycota), which exhibited a strategy of self-sufficiency (prototrophy), rapid growth, and competitive exclusion via antibiotic production. Auxotrophy was more prevalent in cellulolytic Actinobacteria than in cellulolytic Proteobacteria, demonstrating differences in dependency among cellulose degraders. Noncellulolytic taxa that accessed 13 C from cellulose (Planctomycetales, Verrucomicrobia, and Vampirovibrionales) were also more dependent, as indicated by patterns of auxotrophy and 13 C labeling (i.e., partial labeling or labeling at later stages). Major 13 C-labeled cellulolytic microbes (e.g., Sorangium, Actinomycetales, Rhizobiales, and Caulobacteraceae) possessed adaptations for surface colonization (e.g., gliding motility, hyphae, attachment structures) signifying the importance of surface ecology in decomposing particulate organic matter. Our results demonstrated that access to cellulosic C was accompanied by ecological trade-offs characterized by differing degrees of metabolic dependency and competitive exclusion.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Recyclable Cellulose Fiber Reinforced Vitrimer Composite

Vitrimer is an innovative class of polymeric materials which demonstrates traditional thermoset-like mechanical and chemical resilience while still being able to flow on demand like a traditional thermoplastic through covalently adaptive dynamic linkages. Herein, high-performance cellulose fiber reinforced vitrimer composites are developed using an epoxy-based vitrimer and natural cellulose paper. The reinforced vitrimer composite was fabricated by impregnating the porous structure of cellulose paper with two curable monomers, followed by in-situ polymerization of the monomers inside the fibrous scaffold. The introduction of hydroxyl group present on the cellulosic framework assisted in a faster topological rearrangement of the crosslinked matrix through transesterification exchange reaction, thus imparting various sought-after properties like shape recovery and recyclability via simple thermal reprocessing. Moreover, the reinforced vitrimer composite exhibits superior tensile properties as high as 90 MPa with 15-25 volume% vitrimer loading due to the interfacial adhesion via ester exchange reaction between the epoxy matrix and functionalities on the cellulosic fibers. Noteworthily, the key ingredients of the resulting composite (i.e., epoxy-based vitrimer and cellulose fibers) can be comfortably recycled without using aggressive chemical treatment, enabling composite to be easily recycled or disposed of at the end of service life and assist in reducing the subsequent manufacturing cost. This study would shed light on the development of a recyclable polymer composite with exceptional mechanical properties while simultaneously demonstrating self-healing and shape memory capabilities.

Rohewal, Sargun Singh↗

DOE OSTI2022

Data for The Effects of Sequential Hydrothermal-Mechanical Refining Pretreatment on Cellulose Structure Changes and Sugar Recoveries

The recalcitrance of lignocellulosic biomass necessitates an efficient pretreatment protocol for operating a successful cellulosic biorefinery. It is critical to improve cellulose accessibility for hydrolysis and fermentation by altering the plant cell wall’s physical structure and chemical composition. Sequential hydrothermal-mechanical refining pretreatment (HMR) allows efficient recovery of cellulosic sugars without utilizing any hazardous chemicals. HMR has been successfully applied to Liberty switchgrass, a bioenergy cultivar released by the USDA, and now it is being applied to oilcane, a recently developed transgenic sugarcane variety engineered to accumulate lipids in its vegetative tissues. Sugar yields of oilcane bagasse (OCB) and switchgrass (SG) treated with HMR are 96.4% and 75.4%, respectively. This study sought to correlate cellulosic sugar yields with structural changes within the cell wall caused by HMR on two distinct bioenergy crops. Simon’s staining technique for the specific surface area analysis showed that HMR increased the specific surface area of pretreated biomass residues by 80-112%. In addition, ATR-FTIR was performed to determine the effects of HMR on physical structures based on the total crystallinity index (TCI) and hydrogen bonding intensity (HBI). Irrespective of biomass type, HMR decreased the initial crystalline cellulose contents of untreated biomass residues by 3.5% and reduced TCI and HBI by 7-13%. The study found that sugar yields were negatively correlated to reducing values of hydrogen bonding intensity, crystalline cellulose content, and total crystallinity index.

Biomass Analytics↗

DOE OSTI2010

Metallization of bacterial cellulose for electrical and electronic device manufacture

A method for the deposition of metals in bacterial cellulose and for the employment of the metallized bacterial cellulose in the construction of fuel cells and other electronic devices is disclosed. The method for impregnating bacterial cellulose with a metal comprises placing a bacterial cellulose matrix in a solution of a metal salt such that the metal salt is reduced to metallic form and the metal precipitates in or on the matrix. The method for the construction of a fuel cell comprises placing a hydrated bacterial cellulose support structure in a solution of a metal salt such that the metal precipitates in or on the support structure, inserting contact wires into two pieces of the metal impregnated support structure, placing the two pieces of metal impregnated support structure on opposite sides of a layer of hydrated bacterial cellulose, and dehydrating the three layer structure to create a fuel cell.

Evans, Barbara R↗

DOE OSTI2011

Metallization of bacterial cellulose for electrical and electronic device manufacture

25 ENERGY STORAGE↗

DOE OSTI2020

The N–terminal zinc finger of CELLULOSE SYNTHASE6 is critical in defining its functional properties by determining the level of homodimerization in Arabidopsis

Primary cell wall cellulose is synthesized by the cellulose synthase complex (CSC) containing CELLULOSE SYNTHASE1 (CESA1), CESA3 and one of four CESA6–like proteins in Arabidopsis. It has been proposed that the CESA6–like proteins occupy the same position in the CSC, but their underlying selection mechanism remains unclear. We produced a chimeric CESA5 by replacing its N–terminal zinc finger with its CESA6 counterpart to investigate the consequences for its homodimerization, a crucial step in forming higher–order structures during assembly of the CSC. We found that the mutant phenotypes of prc1–1, a cesa6 null mutant, were rescued by the chimeric CESA5, and became comparable to the wild type (WT) and prc1–1 complemented by WT CESA6 in regard to plant growth, cellulose content, cellulose microfibril organization, CSC dynamics and subcellular localization. Bimolecular fluorescence complementation assays were employed to evaluate pairwise interactions between the N–terminal regions of CESA1, CESA3, CESA5, CESA6 and the chimeric CESA5. We verified that the chimeric CESA5 explicitly interacted with all the other CESA partners, comparable to CESA6, whereas interaction between CESA5 with itself was significantly weaker than that of all other CESA pairs. Furthermore, our findings suggest that the homodimerization of CESA6 through its N–terminal zinc finger is critical in defining its functional properties, and possibly determines its intrinsic roles in facilitating higher–order structures in CSCs.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Identifying Stable Fragments of Arabidopsis thaliana Cellulose Synthase Subunit 3 by Yeast Display

Determining structures of large, complex proteins remains challenging, especially for transmembrane proteins, as the protein size increases. Additionally, Arabidopsis thaliana cellulose synthesis complex is a large, multimeric complex located in the plant cell membrane that synthesizes cellulose microfibrils in the plant cell wall. Despite the biological and economic importance of cellulose and therefore cellulose synthesis, many aspects of the cellulase synthase complex (CSC) structure and function are still unknown. Here, yeast surface display (YSD) is used to determine the full-length expression of A. thaliana cellulose synthase 3 (AtCesA3) fragments. The level of stably-folded AtCesA3 fragments displayed on the yeast surface are determined using flow cytometric analysis of differential surface expression of epitopes flanking the AtCesA3 fragment. This technique provides a fast and simple method for examining folding and expression of protein domains and fragments of complex proteins.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2014

The effect of alkaline pretreatment methods on cellulose structure and accessibility

The effects of different alkaline pretreatments on cellulose structural features and accessibility are compared and correlated with the enzymatic hydrolysis of Populus . The pretreatments are shown to modify polysaccharides and lignin content to enhance the accessibility for cellulase enzymes. The highest increase in the cellulose accessibility was observed in dilute sodium hydroxide, followed by methods using ammonia soaking and lime (Ca(OH) 2 ). The biggest increase of cellulose accessibility occurs during the first 10 min of pretreatment, with further increases at a slower rate as severity increases. Low temperature ammonia soaking at longer residence times dissolved a major portion of hemicellulose and exhibited higher cellulose accessibility than high temperature soaking. Moreover, the most significant reduction of degree of polymerization (DP) occurred for dilute sodium hydroxide (NaOH) and ammonia pretreated Populus samples. The study thus identifies important cellulose structural features and relevant parameters related to biomass recalcitrance.

09 BIOMASS FUELS↗

DOE OSTI2018

Extraction and Characterization of Nanocrystalline Cellulose from Cassava Bagasse

The objective of this study was to produce highly crystalline nanocellulose from cassava bagasse, which is a by-product from the cassava starch industry that contains 15 to 50 wt% cellulose fibers. For this purpose, the lignocellulosic fiber that was isolated from cassava bagasse was bleached with sodium chlorite and hydrolyzed with sulfuric acid. The obtained suspension was spray-dried and the resulting microparticles were characterized by X-ray diffractometry to determine the crystallinity index. Microcrystalline cellulose was subjected to the same processing conditions and considered as a reference. The crystallinity index of the nanocellulose extracted from the cassava bagasse was higher than the crystallinity index of the nanocellulose extracted from the microcrystalline cellulose; 84.1 and 78.7%, respectively. No other studies have reported cellulose nanocrystals from cassava bagasse with a high crystallinity index. The nanocrystalline cellulose produced in this study is very promising for use in innovative industrial applications.

36 MATERIALS SCIENCE↗

DOE OSTI2018

Optimal Design of Ozone Bleaching Parameters to Approach Cellulose Nanofibers Extraction from Sugarcane Bagasse Fibers

Cellulose nanofibers (CNFs) were isolated from sugarcane bagasse (SCB) through the combination of bio-refinery, sulfur-free, and totally chlorine free (TCF) chemo-mechanical pretreatments, with a focus on the optimal design of ozone bleaching parameters based on a response surface methodology (RSM). For this purpose, the most effective parameters in ozone bleaching (temperature, time, and pulp consistency) were set between 40 and 85 °C, 60 and 360 min, and 1–5 wt%, respectively. High-performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), Kappa number, and scanning electron microscopy (SEM) were used to chemically and morphologically characterize the SCB fibers. The size distribution and morphology of CNFs were also evaluated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). HPLC analysis revealed that percentage of cellulose increased from 41.5 to 91.39% after chemical pretreatments. FTIR and Kappa number analyses also confirmed the successful isolation of cellulose fibers from the SCB fibers after chemical pretreatments. Furthermore, DLS results showed that the hydrodynamic diameter of the isolated cellulose fibers reduced to 268 nm by dint of ultrasonication. Additionally, TEM images confirmed the isolation of CNFs: the average diameter of cellulose fibers decreased to about 28 nm after mechanical steps and the yield of fibrillation was found to be around 99%. According to the obtained results, the applied chemo-mechanical treatment appears to be promising for green and facile isolation of CNFs.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

DOE OSTI2011

The solvation structures of cellulose microfibrils in ionic liquids

The use of ionic liquids for non-derivatized cellulose dissolution promises an alternative method for the thermochemical pretreatment of biomass that may be more efficient and environmentally acceptable than more conventional techniques in aqueous solution. Here, we performed equilibrium MD simulations of a cellulose microfibril in the ionic liquid 1-butyl-3-methylimidazolium chloride (BmimCl) and compared the solute structure and the solute-solvent interactions at the interface with those from corresponding simulations in water. The results indicate a higher occurrence of solvent-exposed orientations of cellulose surface hydroxymethyl groups in BmimCl than in water. Moreover, spatial and radial distribution functions indicate that hydrophilic surfaces are a preferred site of interaction between cellulose and the ionic liquid. In particular, hydroxymethyl groups on the hydrophilic fiber surface adopt a different conformation from their counterparts oriented towards the fiber s core. Furthermore, the glucose units with these solvent-oriented hydroxymethyls are surrounded by the heterocyclic organic cation in a preferred parallel orientation, suggesting a direct and distinct interaction scheme between cellulose and BmimCl.

09 BIOMASS FUELS↗

DOE OSTI2022

Combining PCA and nonlinear fitting of peak models to re-evaluate C 1s XPS spectrum of cellulose

Cellulose is an example of a material that responds to XPS by the creation of new chemistry not present in the as-received sample. While improvements in instrumentation may be seen in general as beneficial to surface science, recent studies have shown that the consequences for some materials are detrimental. Here, in this work, these problems are illustrated through an analysis of cellulose spectra obtained during a degradation study. C 1s spectra are decomposed into two well-formed component curves that are open to chemical interpretation. In particular, a component-curve representative of pure cellulose is obtained as well as a second component curve that implies cellulose is degraded through the creation of carbon chemistry involving C—O, C$=$O and O—C$=$O. Since cellulose is a crystalline material, formed through the alignment of molecules under the influence of hydrogen bonds, the analysis and findings presented in this paper are relevant to any material analyzed by XPS whose properties are dependent on hydrogen bonds. The analysis techniques are based on an informed vectorial approach, which extracts directly from data spectral shapes that are used to monitor sample degradation via linear least squares optimization. Related mathematics of Principal Component Analysis and linear analysis are presented.

36 MATERIALS SCIENCE↗

DOE OSTI2018

Development of a novel cellulase biosensor that detects crystalline cellulose hydrolysis using a transcriptional regulator

Highlights: • A novel biosensor for detecting cellulolytic activity is developed using cellobiose-dependent transcriptional regulator. • CBGESS shows quantitative response to cellobiose with hyper sensitivity and specificity. • Cellulase activity including hydrolysis of crystalline cellulose is measured by CBGESS via intracellular fluorescence. Successful utilization of cellulose as renewable biomass depends on the development of economically feasible technologies, which can aid in enzymatic hydrolysis. In this study, we developed a whole-cell biosensor for detecting cellulolytic activity that relies on the recognition of cellobiose using the transcriptional factor CelR from Thermobifida fusca and transcriptional activation of its downstream gfp reporter gene. The fluorescence intensity of whole-cell biosensor, which was named as cellobiose-detectible genetic enzyme screening system (CBGESS), was directly proportional to the concentration of cellobiose. The strong fluorescence intensity of CBGESS demonstrated the ability to detect cellulolytic activity with two cellulosic substrates, carboxymethyl cellulose and p-nitrophenyl β-D-cellobioside in cellulase-expressing Escherichia coli. In addition, CBGESS easily sensed crystalline cellulolytic activity when commercial Celluclast 1.5L was dropped on an Avicel plate. Therefore, CBGESS is a powerful tool for detecting cellulolytic activity with high sensitivity in the presence of soluble or insoluble cellulosic substrates. CBGESS may be further applied to excavate novel cellulases or microbes from both genetic libraries and various environments.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2026

Cellulose-MOFs hybrid materials: Chemistry and mechanism of applications in biomedical - A review

Rising costs and performance limits of modern biomedical materials motivate the search for advanced, biocompatible alternatives. Cellulose-based metal-organic frameworks (cellulose-MOFs) emerge as distinctive hybrids combining renewable polymer chemistry with tunable porous architectures, enabling uncommon structure–function relationships. Their large surface area, controllable pore size, adaptable functional groups, and efficient host–guest interactions underpin diverse biomedical functions. Till now, no comprehensive, application-focused review has systematically summarized cellulose-MOFs synthesis for biomedical applications. This review critically analyzes cellulose-MOFs, emphasizing mechanistic links between chemistry, synthesis routes, interfacial interactions, and biomedical performance, rather than cataloging applications alone. Antibacterial action, targeted drug delivery, and sensing/biosensing are discussed through comparative insights. The article identifies unresolved challenges and proposes future research pathways to rationally design next-generation cellulose-MOFs systems, guiding researchers and clinicians alike.

Biomedical↗

DOE OSTI2010

Structure and superparamagnetic behaviour of magnetite nanoparticles in cellulose beads

Superparamagnetic magnetite nanoparticles were obtained starting from a mixture of iron(II) and iron(III) solutions in a preset total iron concentration from 0.04 to 0.8 mol l{sup -1} with ammonia at 25 and 70 {sup o}C. The regeneration of cellulose from viscose produces micrometrical spherical cellulose beads in which synthetic magnetite were embedded. The characterization of cellulose-magnetite beads by X-ray diffraction, Scanning and Transmission Electron Microscopy and magnetic measurement is reported. X-ray diffraction patterns indicate that the higher is the total iron concentration and temperature the higher is the crystal size of the magnetite obtained. Transmission Electron Microscopy studies of cellulose-magnetite beads revealed the distribution of magnetite nanoparticles inside pores of hundred nanometers. Magnetite as well as the cellulose-magnetite composites exhibit superparamagnetic characteristics. Field cooling and zero field cooling magnetic susceptibility measurements confirm the superparamagnetic behaviour and the blocking temperature for the magnetite with a mean size of 12.5 nm, which is 200 K.

36 MATERIALS SCIENCE↗

DOE OSTI2011

Thin-film Nanofibrous Composite Membranes Containing Cellulose or Chitin Barrier Layers Fabricated by Ionic Liquids

The barrier layer of high-flux ultrafiltration (UF) thin-film nanofibrous composite (TFNC) membranes for purification of wastewater (e.g., bilge water) have been prepared by using cellulose, chitin, and a cellulose-chitin blend, regenerated from an ionic liquid. The structures and properties of regenerated cellulose, chitin, and a cellulose-chitin blend were analyzed with thermogravimetric analysis (TGA) and wide-angle X-ray diffraction (WAXD). The surface morphology, pore size and pore size distribution of TFNC membranes were determined by SEM images and molecular weight cut-off (MWCO) methods. An oil/water emulsion, a model of bilge water, was used as the feed solution, and the permeation flux and rejection ratio of the membranes were investigated. TFNC membranes based on the cellulose-chitin blend exhibited 10 times higher permeation flux when compared with a commercial UF membrane (PAN10, Sepro) with a similar rejection ratio after filtration over a time period of up to 100 h, implying the practical feasibility of such membranes for UF applications.

36 MATERIALS SCIENCE↗