Location, speciation, and quantification of carbon in silica phytoliths using synchrotron scanning transmission X-ray microspectroscopy.

Phytoliths of biogenic silica play a vital role in the silicon biogeochemical cycle and occlude a fraction of organic carbon. The location, chemical speciation, and quantification of this carbon within phytoliths have remained elusive due to limited direct experimental evidence. In this work, phytoliths (bilobate morphotype) from the sugarcane stalk epidermis are sectioned with a focused ion beam to produce lamellas (≈10 × 10 µm2 size, <500 nm thickness) and probed by synchrotron scanning transmission X-ray microspectroscopy (≈100-200 nm pixel size; energies near the silicon and carbon K-absorption edges). Analysis of the spectral image stacks reveals the complementarity of the silica and carbon spatial distributions, with carbon found at the borders of the lamellas, in islands within the silica, and dispersed in extended regions that can be described as a mixed silica-carbonaceous matrix. Carbon spectra are assigned mainly to lignin-like compounds as well as to proteins. Carbon contents of 3-14 wt.% are estimated from the spectral maps of four distinct phytolith lamellas. The results provide unprecedented spatial and chemical information on the carbon in phytoliths obtained without interference from wet-chemical digestion.

Exploring the compatibility between hydrothermal depolymerization of alkaline lignin from sugarcane bagasse and metabolization of the aromatics by bacteria.

Although hydrothermal treatments for biomass fractionation have been vastly studied, their effect on the depolymerization of isolated lignins in terms of yield, composition, and compatibility of the produced lignin bio-oils with bioconversion is still poorly investigated. In this study, we evaluated the hydrothermal depolymerization of an ß-O-4'-rich lignin extracted from sugarcane bagasse by alkaline fractionation, investigating the influence of temperature (200-350 °C), time (30-90 min), and solid-liquid ratio (1:10-1:50 m.v-1) on yield of bio-oils (up to 31 wt%) rich in monomers (light bio-oils). Principal Components Analysis showed that the defunctionalization of the aromatic monomers was more pronounced in the most severe reaction conditions and that the abundance of more hydrophobic monomers increased in more diluted reactions. While the high-molecular-weight (heavy) bio-oil generated at 350 °C, 90 min, and 1:50 m.v-1 failed to support bacterial growth, the corresponding light bio-oil rich in aromatic monomers promoted the growth of bacteria from 9 distinct species. The isolates Pseudomonas sp. LIM05 and Burkholderia sp. LIM09 showed the best growth performance and tolerance to lignin-derived aromatics, being the most promising for the future development of biological upgrading strategies tailored for this lignin stream.

How lignin sticks to cellulose-insights from atomic force microscopy enhanced by machine-learning analysis and molecular dynamics simulations.

Elucidating cellulose-lignin interactions at the molecular and nanometric scales is an important research topic with impacts on several pathways of biomass valorization. Here, the interaction forces between a cellulosic substrate and lignin are investigated. Atomic force microscopy with lignin-coated tips is employed to probe the site-specific adhesion to a cellulose film in liquid water. Over seven thousand force-curves are analyzed by a machine-learning approach to cluster the experimental data into types of cellulose-tip interactions. The molecular mechanisms for distinct types of cellulose-lignin interactions are revealed by molecular dynamics simulations of lignin globules interacting with different cellulose Iß crystal facets. This unique combination of experimental force-curves, data-driven analysis, and molecular simulations opens a new approach of investigation and updates the understanding of cellulose-lignin interactions at the nanoscale.

The Effect of Sugarcane Straw Aging in the Field on Cell Wall Composition.

Cellulosic ethanol is an alternative for increasing the amount of bioethanol production in the world. In Brazil, sugarcane leads the bioethanol production, and to improve its yield, besides bagasse, sugarcane straw is a possible feedstock. However, the process that leads to cell wall disassembly under field conditions is unknown, and understanding how this happens can improve sugarcane biorefinery and soil quality. In the present work, we aimed at studying how sugarcane straw is degraded in the field after 3, 6, 9, and 12 months. Non-structural and structural carbohydrates, lignin content, ash, and cellulose crystallinity were analyzed. The cell wall composition was determined by cell wall fractionation and determination of monosaccharide composition. Non-structural carbohydrates degraded quickly during the first 3 months in the field. Pectins and lignin remained in the plant waste for up to 12 months, while the hemicelluloses and cellulose decreased 7.4 and 12.4%, respectively. Changes in monosaccharide compositions indicated solubilization of arabinoxylan (xylose and arabinose) and ß-glucans (ß-1,3 1,4 glucan; after 3 months) followed by degradation of cellulose (after 6 months). Despite cellulose reduction, the xylose:glucose ratio increased, suggesting that glucose is consumed faster than xylose. The degradation and solubilization of the cell wall polysaccharides concomitantly increased the level of compounds related to recalcitrance, which led to a reduction in saccharification and an increase in minerals and ash contents. Cellulose crystallinity changed little, with evidence of silica at the latter stages, indicating mineralization of the material. Our data suggest that for better soil mineralization, sugarcane straw must stay in the field for over 1 year. Alternatively, for bioenergy purposes, straw should be used in less than 3 months.

Directed Assembly of Cellulose Nanocrystals in Their Native Solid-State Template of a Processed Fiber Cell Wall.

Nanoparticle assembly is intensely surveyed because of the numerous applications within fields such as catalysis, batteries, and biomedicine. Here, directed assembly of rod-like, biologically derived cellulose nanocrystals (CNCs) within the template of a processed cotton fiber cell wall, that is, the native origin of CNCs, is reported. It is a system where the assembly takes place in solid state simultaneously with the top-down formation of the CNCs via hydrolysis with HCl vapor. Upon hydrolysis, cellulose microfibrils in the fiber break down to CNCs that then pack together, resulting in reduced pore size distribution of the original fiber. The denser packing is demonstrated by N2 adsorption, water uptake, thermoporometry, and small-angle X-ray scattering, and hypothetically assigned to attractive van der Waals interactions between the CNCs.

Developing fibrillated cellulose as a sustainable technological material.

Cellulose is the most abundant biopolymer on Earth, found in trees, waste from agricultural crops and other biomass. The fibres that comprise cellulose can be broken down into building blocks, known as fibrillated cellulose, of varying, controllable dimensions that extend to the nanoscale. Fibrillated cellulose is harvested from renewable resources, so its sustainability potential combined with its other functional properties (mechanical, optical, thermal and fluidic, for example) gives this nanomaterial unique technological appeal. Here we explore the use of fibrillated cellulose in the fabrication of materials ranging from composites and macrofibres, to thin films, porous membranes and gels. We discuss research directions for the practical exploitation of these structures and the remaining challenges to overcome before fibrillated cellulose materials can reach their full potential. Finally, we highlight some key issues towards successful manufacturing scale-up of this family of materials.

Location of water in fresh sugarcane bagasse observed by synchrotron X-ray microtomography.

Sugarcane bagasse is a vast lignocellulosic byproduct generated in the industry with ~50% humidity (1 kg dry matter associated with 1 kg water). Although the presence of water brings deleterious consequences for combustion, storage and sugar extraction, the location of water in fresh bagasse remains unknown. In this work, we use synchrotron X-ray microtomography for non-invasive 3D imaging of fresh bagasse particles, which allows the visualization of intraparticle water. The sclerified fiber cells in the sheaths surrounding xylem vessels are often found full of water. We suggest this can be juice preserved from the native stalks as many sclerified fibers seem to keep their structural integrity despite the mechanical action during sugarcane crushing. The microtomograms of fresh bagasse also shows mineral particles adhered to biomass surfaces, with adhesion presumably favored by the presence of water. In summary, this work unveils the location of water in fresh bagasse, solving an old mystery of sugarcane technology.

Toward an understanding of the increase in enzymatic hydrolysis by mechanical refining.

BACKGROUND: Mechanical refining is a low-capital and well-established technology used in pulp and paper industry to improve fiber bonding for product strength. Refining can also be applied in a biorefinery context to overcome the recalcitrance of pretreated biomass by opening up the biomass structure and modifying substrate properties (e.g., morphology, particle size, porosity, crystallinity), which increases enzyme accessibility to substrate and improves carbohydrate conversion. Although several characterization methods have been used to identify the changes in substrate properties, there is no systematic approach to evaluate the extent of fiber cell wall disruption and what physical properties can explain the improvement in enzymatic digestibility when pretreated lignocellulosic biomass is mechanically refined. This is because the fiber cell wall is complex across multiple scales, including the molecular scale, nano- and meso-scale (microfibril), and microscale (tissue level). A combination of advanced characterization tools is used in this study to better understand the effect of mechanical refining on the meso-scale microfibril assembly and the relationship between those meso-scale modifications and enzymatic hydrolysis. RESULTS: Enzymatic conversion of autohydrolysis sugarcane bagasse was improved from 69.6 to 77.2% (11% relative increase) after applying mechanical refining and an increase in enzymatic digestibility is observed with an increase in refining intensity. Based on a combination of advanced characterizations employed in this study, it was found that the refining action caused fiber size reduction, internal delamination, and increase in pores and swellability. CONCLUSIONS: A higher level of delamination and higher increase in porosity, analyzed by TEM and DSC, were clearly demonstrated, which explain the faster digestibility rate during the first 72 h of enzymatic hydrolysis for disc-refined samples when compared to the PFI-refined samples. In addition, an increased inter-fibrillar distance between cellulose microfibrils at the nano-meso-scale was also revealed by SFG analysis, while no evidence was found for a change in crystalline structure by XRD and solid-state NMR analysis.

Tunable Structural and Mechanical Properties of Cellulose Nanofiber Substrates in Aqueous Conditions for Stem Cell Culture.

Thin cellulose nanofiber (CNF) nanostructured substrates with varying roughness, stiffness (Young's modulus), porosity, and swelling properties were produced by varying the conditions used during fabrication. It was shown that with increased heat exposure, CNF substrate porosity in an aqueous state decreased while Young's modulus in a water submerged state increased. In this study, the adhesion and viability of mesenchymal stem cells (MSCs) cultured on this CNF substrate will be presented. Viability of D1/BALBc MSCs were assessed for 24 and 48 h, and it was shown that depending on the CNF substrate the viability varied significantly. The adhesion of MSCs after 6 and 24 h was conditional on material mechanical properties and porosity of the CNF in cell culture conditions. These results suggest that material properties of CNF nanostructured substrate within the aqueous state can be easily tuned with curing step without any chemical modification to the CNF and that these changes can affect MSC viability in cell culture.

Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis.

As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus.

Hydrated fractions of cellulosics probed by infrared spectroscopy coupled with dynamics of deuterium exchange.

This article presents a novel method to selectively probe the non-crystalline, hydrated fractions of cellulosic biomass. The method is based on time-resolved infrared spectra analyzed to provide information on spectral and dynamical features of deuterium exchange (OH → OD) in D2O atmosphere. We assign deuterium exchange spectral regions (700-3800 cm(-1)) and explore changes due to relative humidity, different cellulosic samples, and infrared polarization. Here, two results are highlighted. First, a wide range of celluloses isolated from plants show remarkable spectral similarities whatever the relative amounts of cellulose and xylan. This result supports an inherent type of hydrated disorder which is mostly insensitive to the molecular identities of the associated polysaccharides. Second, polarized infrared analysis of cotton reveals hydrated cellulose having chains preferentially aligned with those of crystals, while the hydroxyls of hydrated cellulose present much more randomized orientation. Our results provide new insights on molecular and group orientation and on hydrogen bonding in hydrated fractions of cellulosic biomass.

Tissue-specific cell wall hydration in sugarcane stalks.

Plant cell walls contain water, especially under biological and wet processing conditions. The present work characterizes this water in tissues of sugarcane stalks. Environmental scanning electron microscopy shows tissue deformation upon drying. Dynamic vapor sorption determines the equilibrium and kinetics of moisture uptake. Thermoporometry by differential scanning calorimetry quantifies water in nanoscale pores. Results show that cell walls from top internodes of stalks are more deformable, slightly more sorptive to moisture, and substantially more porous. These differences of top internode are attributed to less lignified walls, which is confirmed by lower infrared spectral signal from aromatics. Furthermore, cell wall nanoscale porosity, an architectural and not directly compositional characteristic, is shown to be tissue-specific. Nanoscale porosities are ranked as follows: pith parenchyma > pith vascular bundles > rind. This ranking coincides with wall reactivity and digestibility in grasses, suggesting that nanoscale porosity is a major determinant of wall recalcitrance.

Crystallite width determines monolayer hydration across a wide spectrum of celluloses isolated from plants.

Relating cellulose structure to its water uptake is a classical problem with many investigations done through measurements of cellulose "crystallinity". However, there is presently a growing consensus that crystallinity measurements are appreciably uncertain, leading to ambiguous interpretations of underlying cellulose organization. In this scenario, this article revisits the relations between cellulose structure and water uptake, moving the emphasis away from degree of crystallinity and directing it toward crystallite width, which is inferred with less ambiguity from the broadening of 200 X-ray diffraction peaks. With this approach, analysis of a wide spectrum of celluloses isolated from plants (preserving cellulose I phase and having variable contents of residual hemicelluloses) reveals a simple linear relation (R(2) = 0.98) between reciprocal crystallite width and monolayer hydration (determined from vapor sorption). The primary role of crystallite width supports that most water-accessible polysaccharides are laterally associated with the crystallites, with a minor fraction in disordered domains along the fibrils. Furthermore, the secondary role left to hemicellulosic contents indicates cellulose being partly decrystallized to complement the disordered amount required to interface the crystallites. Finally, a substantial part of hydration is attributed to polysaccharides in voids left by the imperfect packing of aggregated crystallites.

Insights on how the activity of an endoglucanase is affected by physical properties of insoluble celluloses.

Cellulose physical properties like crystallinity, porosity, and particle size are known to influence cellulase activity, but knowledge is still insufficient for activity prediction from such measurable substrate characteristics. With the aim of illuminating enzyme-substrate relationships, this work evaluates a purified hyperthermophilic endo-1,4-beta-glucanase (from Pyrococcus furiosus) acting on 13 celluloses characterized for crystallinity and crystal width (by X-ray diffraction), wet porosity (by thermoporometry), and particle size (by light scattering). Activities are analyzed by the Michaelis-Menten kinetic equation, which is justified by low enzyme-substrate affinity. Michaelis-Menten coefficients K(m) and k(cat) are reinterpreted in the context of heterogeneous cellulose hydrolysis. For a set of as-received and milled microcrystalline celluloses, activity is successfully described as a function of accessible substrate concentration, with accessibility proportional to K(m)(-1). Accessibility contribution from external particle areas, pore areas, and crystalline packing are discriminated to have comparable magnitudes, implying that activity prediction demands all these substrate properties to be considered. Results additionally suggest that looser crystalline packing increases the lengths of released cello-oligomers as well as the maximum endoglucanase specific activity (k(cat)).