Ectopic Production of 3,4-Dihydroxybenzoate in Planta Affects Cellulose Structure and Organization.

Lignocellulosic biomass is a highly sustainable and largely carbon dioxide neutral feedstock for the production of biofuels and advanced biomaterials. Although thermochemical pretreatment is typically used to increase the efficiency of cell wall deconstruction, genetic engineering of the major plant cell wall polymers, especially lignin, has shown promise as an alternative approach to reduce biomass recalcitrance. Poplar trees with reduced lignin content and altered composition were previously developed by overexpressing bacterial 3-dehydroshikimate dehydratase (QsuB) enzyme to divert carbon flux from the shikimate pathway. In this work, three transgenic poplar lines with increasing QsuB expression levels and different lignin contents were studied using small-angle neutron scattering (SANS) and wide-angle X-ray scattering (WAXS). SANS showed that although the cellulose microfibril cross-sectional dimension remained unchanged, the ordered organization of the microfibrils progressively decreased with increased QsuB expression. This was correlated with decreasing total lignin content in the QsuB lines. WAXS showed that the crystallite dimensions of cellulose microfibrils transverse to the growth direction were not affected by the QsuB expression, but the crystallite dimensions parallel to the growth direction were decreased by ∼20%. Cellulose crystallinity was also decreased with increased QsuB expression, which could be related to high levels of 3,4-dihydroxybenzoate, the product of QsuB expression, disrupting microfibril crystallization. In addition, the cellulose microfibril orientation angle showed a bimodal distribution at higher QsuB expression levels. Overall, this study provides new structural insights into the impact of ectopic synthesis of small-molecule metabolites on cellulose organization and structure that can be used for future efforts aimed at reducing biomass recalcitrance.

Atomic Level Interactions and Suprastructural Configuration of Plant Cell Wall Polymers in Dialkylimidazolium Ionic Liquids.

Ionic liquids (ILs) have been widely investigated for the pretreatment and deconstruction of lignocellulosic feedstocks. However, the modes of interaction between IL-anions and cations, and plant cell wall polymers, namely, cellulose, hemicellulose, and lignin, as well as the resulting ultrastructural changes are still unclear. In this study, we investigated the atomic level and suprastructural interactions of microcrystalline cellulose, birchwood xylan, and organosolv lignin with 1,3-dialkylimidazolium ILs having varying sizes of carboxylate anions. Analysis by 13C NMR spectroscopy indicated that cellulose and lignin exhibited stronger hydrogen bonding with acetate ions than with formate ions, as evidenced by greater chemical shift changes. Small-angle X-ray scattering analysis showed that while both cellulose and xylan adopted a single-stranded conformation in acetate-ILs, twice as many acetate ions were bound to one anhydroglucose unit than to an anhydroxylose unit. We also determined that a minimum of seven representative carbohydrate units must interact with an anion for that IL to effectively dissolve cellulose or xylan. Lignin is associated as groups of four polymer molecules in formate-ILs and dispersed as single molecules in acetate-ILs, which indicates that it is highly soluble in the latter. In summary, our study demonstrated that 1,3-dialkylimidazolium acetates displayed stronger binding interactions with cellulose and lignin, as compared to formates, and thus have superior potential to fractionate these polymers from lignocellulosic feedstocks.

pH-Dependent Solution Micellar Structure of Amphoteric Polypeptoid Block Copolymers with Positionally Controlled Ionizable Sites.

While solution micellization of ionic block copolymers (BCP) with randomly distributed ionization sites along the hydrophilic segments has been extensively studied, the roles of positionally controlled ionization sites along the BCP chains in their micellization and resulting micellar structure remain comparatively less understood. Herein, three amphoteric polypeptoid block copolymers carrying two oppositely charged ionizable sites, with one fixed at the hydrophobic terminus and the other varyingly positioned along the hydrophilic segment, have been synthesized by sequential ring-opening polymerization method. The presence of the ionizable site at the hydrophobic segment terminus is expected to promote polymer association toward equilibrium micellar structures in an aqueous solution. The concurrent presence of oppositely charged ionizable sites on the polymer chains allows the polymer association to be electrostatically modulated in a broad pH range (ca. 2-12). Micellization of the amphoteric polypeptoid BCP in dilute aqueous solution and the resulting micellar structure at different solution pHs was investigated by a combination of scattering and microscopic methods. Negative-stain transmission-electron microscopy (TEM), small-angle neutron scattering (SANS), and small-angle X-ray scattering (SAXS) analyses revealed the dominant presence of core-shell-type spherical micelles and occasional rod-like micelles with liquid crystalline (LC) domains in the micellar core. The micellar structures (e.g., aggregation number, radius of gyration, chain packing in the micelle) were found to be dependent on the solution pH and the position of the ionizable site along the chain. This study has highlighted the potential of controlling the position of ionizable sites along the BCP polymer to modulate the electrostatic and LC interactions, thus tailoring the micellar structure at different solution pH values in water.

Deconstruction of biomass enabled by local demixing of cosolvents at cellulose and lignin surfaces.

A particularly promising approach to deconstructing and fractionating lignocellulosic biomass to produce green renewable fuels and high-value chemicals pretreats the biomass with organic solvents in aqueous solution. Here, neutron scattering and molecular-dynamics simulations reveal the temperature-dependent morphological changes in poplar wood biomass during tetrahydrofuran (THF):water pretreatment and provide a mechanism by which the solvent components drive efficient biomass breakdown. Whereas lignin dissociates over a wide temperature range (>25 °C) cellulose disruption occurs only above 150 °C. Neutron scattering with contrast variation provides direct evidence for the formation of THF-rich nanoclusters (Rg ∼ 0.5 nm) on the nonpolar cellulose surfaces and on hydrophobic lignin, and equivalent water-rich nanoclusters on polar cellulose surfaces. The disassembly of the amphiphilic biomass is thus enabled through the local demixing of highly functional cosolvents, THF and water, which preferentially solvate specific biomass surfaces so as to match the local solute polarity. A multiscale description of the efficiency of THF:water pretreatment is provided: matching polarity at the atomic scale prevents lignin aggregation and disrupts cellulose, leading to improvements in deconstruction at the macroscopic scale.

Small Angle Neutron Scattering Shows Nanoscale PMMA Distribution in Transparent Wood Biocomposites.

Transparent wood biocomposites based on PMMA combine high optical transmittance with excellent mechanical properties. One hypothesis is that despite poor miscibility the polymer is distributed at the nanoscale inside the cell wall. Small-angle neutron scattering (SANS) experiments are performed to test this hypothesis, using biocomposites based on deuterated PMMA and "contrast-matched" PMMA. The wood cell wall nanostructure soaked in heavy water is quantified in terms of the correlation distance d between the center of elementary cellulose fibrils. For wood/deuterated PMMA, this distance d is very similar as for wood/heavy water (correlation peaks at q ≈ 0.1 Å-1). The peak disappears when contrast-matched PMMA is used, indeed proving nanoscale polymer distribution in the cell wall. The specific processing method used for transparent wood explains the nanocomposite nature of the wood cell wall and can serve as a nanotechnology for cell wall impregnation of polymers in large wood biocomposite structures.

Hemicellulose-Cellulose Composites Reveal Differences in Cellulose Organization after Dilute Acid Pretreatment.

Model hemicellulose-cellulose composites that mimic plant cell wall polymer interactions were prepared by synthesizing deuterated bacterial cellulose in the presence of glucomannan or xyloglucan. Dilute acid pretreatment (DAP) of these materials was studied using small-angle neutron scattering, X-ray diffraction, and sum frequency generation spectroscopy. The macrofibril dimensions of the pretreated cellulose alone were smaller but with similar entanglement of macrofibrillar network as native cellulose. In addition, the crystallite size dimension along the (010) plane increased. Glucomannan-cellulose underwent similar changes to cellulose, except that the macrofibrillar network was more entangled after DAP. Conversely, in xyloglucan-cellulose the macrofibril dimensions and macrofibrillar network were relatively unchanged after pretreatment, but the cellulose Iß content was increased. Our results point to a tight interaction of xyloglucan with microfibrils while glucomannan only interacts with macrofibril surfaces. This study provides insight into roles of different hemicellulose-cellulose interactions and may help in improving pretreatment processes or engineering plants with decreased recalcitrance.

Gradients in Wall Mechanics and Polysaccharides along Growing Inflorescence Stems.

At early stages of Arabidopsis (Arabidopsis thaliana) flowering, the inflorescence stem undergoes rapid growth, with elongation occurring predominantly in the apical ∼4 cm of the stem. We measured the spatial gradients for elongation rate, osmotic pressure, cell wall thickness, and wall mechanical compliances and coupled these macroscopic measurements with molecular-level characterization of the polysaccharide composition, mobility, hydration, and intermolecular interactions of the inflorescence cell wall using solid-state nuclear magnetic resonance spectroscopy and small-angle neutron scattering. Force-extension curves revealed a gradient, from high to low, in the plastic and elastic compliances of cell walls along the elongation zone, but plots of growth rate versus wall compliances were strikingly nonlinear. Neutron-scattering curves showed only subtle changes in wall structure, including a slight increase in cellulose microfibril alignment along the growing stem. In contrast, solid-state nuclear magnetic resonance spectra showed substantial decreases in pectin amount, esterification, branching, hydration, and mobility in an apical-to-basal pattern, while the cellulose content increased modestly. These results suggest that pectin structural changes are connected with increases in pectin-cellulose interaction and reductions in wall compliances along the apical-to-basal gradient in growth rate. These pectin structural changes may lessen the ability of the cell wall to undergo stress relaxation and irreversible expansion (e.g. induced by expansins), thus contributing to the growth kinematics of the growing stem.

Facile directed assembly of hollow polymer nanocapsules within spontaneously formed catanionic surfactant vesicles.

Surfactant vesicles containing monomers in the interior of the bilayer were used to template hollow polymer nanocapsules. This study investigated the formation of surfactant/monomer assemblies by two loading methods, concurrent loading and diffusion loading. The assembly process and the resulting aggregates were investigated with dynamic light scattering, small angle neutron scattering, and small-angle X-ray scattering. Acrylic monomers formed vesicles with a mixture of cationic and anionic surfactants in a broad range of surfactant ratios. Regions with predominant formation of vesicles were broader for compositions containing acrylic monomers compared with blank surfactants. This observation supports the stabilization of the vesicular structure by acrylic monomers. Diffusion loading produced monomer-loaded vesicles unless vesicles were composed from surfactants at the ratios close to the boundary of a vesicular phase region on a phase diagram. Both concurrent-loaded and diffusion-loaded surfactant/monomer vesicles produced hollow polymer nanocapsules upon the polymerization of monomers in the bilayer followed by removal of surfactant scaffolds.

Forming Micro-and Nano-Plastics from Agricultural Plastic Films for Employment in Fundamental Research Studies.

Microplastics (MPs) and nanoplastics (NPs) dispersed in agricultural ecosystems can pose a severe threat to biota in soil and nearby waterways. In addition, chemicals such as pesticides adsorbed by NPs can harm soil organisms and potentially enter the food chain. In this context, agriculturally utilized plastics such as plastic mulch films contribute significantly to plastic pollution in agricultural ecosystems. However, most fundamental studies of fate and ecotoxicity employ idealized and poorly representative MP materials, such as polystyrene microspheres. Therefore, as described herein, we developed a lab-scale multi-step procedure to mechanically form representative MPs and NPs for such studies. The plastic material was prepared from commercially available plastic mulch films of polybutyrate adipate-co-terephthalate (PBAT) that were embrittled through either cryogenic treatment (CRYO) or environmental weathering (W), and from untreated PBAT pellets. The plastic materials were then treated by mechanical milling to form MPs with a size of 46-840 µm, mimicking the abrasion of plastic fragments by wind and mechanical machinery. The MPs were then sieved into several size fractions to enable further analysis. Finally, the 106 µm sieve fraction was subjected to wet grinding to generate NPs of 20-900 nm, a process that mimics the slow size reduction process for terrestrial MPs. The dimensions and the shape for MPs were determined through image analysis of stereomicrographs, and dynamic light scattering (DLS) was employed to assess particle size for NPs. MPs and NPs formed through this process possessed irregular shapes, which is in line with the geometric properties of MPs recovered from agricultural fields. Overall, this size reduction method proved efficient for forming MPs and NPs composed of biodegradable plastics such as polybutylene adipate-co-terephthalate (PBAT), representing mulch materials used for agricultural specialty crop production.

Scattering studies of hydrophobic monomers in liposomal bilayers: an expanding shell model of monomer distribution.

Hydrophobic monomers partially phase separate from saturated lipids when loaded into lipid bilayers in amounts exceeding a 1:1 monomer/lipid molar ratio. This conclusion is based on the agreement between two independent methods of examining the structure of monomer-loaded bilayers. Complete phase separation of monomers from lipids would result in an increase in bilayer thickness and a slight increase in the diameter of liposomes. A homogeneous distribution of monomers within the bilayer would not change the bilayer thickness and would lead to an increase in the liposome diameter. The increase in bilayer thickness, measured by the combination of small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS), was approximately half of what was predicted for complete phase separation. The increase in liposome diameter, measured by dynamic light scattering (DLS), was intermediate between values predicted for a homogeneous distribution and complete phase separation. Combined SANS, SAXS, and DLS data suggest that at a 1.2 monomer/lipid ratio approximately half of the monomers are located in an interstitial layer sandwiched between lipid sheets. These results expand our understanding of using self-assembled bilayers as scaffolds for the directed covalent assembly of organic nanomaterials. In particular, the partial phase separation of monomers from lipids corroborates the successful creation of nanothin polymer materials with uniform imprinted nanopores. Pore-forming templates do not need to span the lipid bilayer to create a pore in the bilayer-templated films.

Biosynthesis and characterization of deuterated chitosan in filamentous fungus and yeast.

Deuterated chitosan was produced from the filamentous fungus Rhizopus oryzae, cultivated with deuterated glucose in H2O medium, without the need for conventional chemical deacetylation. After extraction and purification, the chemical composition and structure were determined by Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and small-angle neutron scattering (SANS). 13C NMR experiments provided additional information about the position of the deuterons in the glucoseamine backbone. The NMR spectra indicated that the deuterium incorporation at the non-exchangeable hydrogen positions of the aminoglucopyranosyl ring in the C3 - C5 positions was at least 60-80 %. However, the C2 position was deuterated at a much lower level (6%). Also, SANS showed that the structure of deuterated chitosan was very similar compared to the non-deuterated counterpart. The most abundant radii of the protiated and deuterated chitosan fibers were 54 Å and 60 Å, respectively, but there is a broader distribution of fiber radii in the protiated chitosan sample. The highly deuterated, soluble fungal chitosan described here can be used as a model material for studying chitosan-enzyme complexes for future neutron scattering studies. Because the physical behavior of non-deuterated fungal chitosan mimicked that of shrimp shell chitosan, the methods presented here represent a new approach to producing a high quality deuterated non-animal-derived aminopolysaccharide for studying the structure-function association of biocomposite materials in drug delivery, tissue engineering and other bioactive chitosan-based composites.

SANS study of cellulose extracted from switchgrass.

Lignocellulosic biomass, which is an abundant renewable natural resource, has the potential to play a major role in the generation of renewable biofuels through its conversion to bioethanol. Unfortunately, it is a complex biological composite material that shows significant recalcitrance, making it a cost-ineffective feedstock for bioethanol production. Small-angle neutron scattering (SANS) was employed to probe the multi-scale structure of cellulosic materials. Cellulose was extracted from milled native switchgrass and from switchgrass that had undergone a dilute acid pretreatment method in order to disrupt the lignocellulose structure. The high-Q structural feature (Q > 0.07 Å(-1)) can be assigned to cellulose fibrils based on a comparison of cellulose purified by solvent extraction of native and dilute acid pretreated switchgrass and a commercial preparation of microcrystalline cellulose. Dilute acid pretreatment results in an increase in the smallest structural size, a decrease in the interconnectivity of the fibrils and no change in the smooth domain boundaries at length scales larger than 1000 Å.

Breakdown of cell wall nanostructure in dilute acid pretreated biomass.

The generation of bioethanol from lignocellulosic biomass holds great promise for renewable and clean energy production. A better understanding of the complex mechanisms of lignocellulose breakdown during various pretreatment methods is needed to realize this potential in a cost and energy efficient way. Here we use small-angle neutron scattering (SANS) to characterize morphological changes in switchgrass lignocellulose across molecular to submicrometer length scales resulting from the industrially relevant dilute acid pretreatment method. Our results demonstrate that dilute acid pretreatment increases the cross-sectional radius of the crystalline cellulose fibril. This change is accompanied by removal of hemicellulose and the formation of R(g) ∼ 135 A lignin aggregates. The structural signature of smooth cell wall surfaces is observed at length scales larger than 1000 A, and it remains remarkably invariable during pretreatment. This study elucidates the interplay of the different biomolecular components in the breakdown process of switchgrass by dilute acid pretreatment. The results are important for the development of efficient strategies of biomass to biofuel conversion.

Combined Small-Angle Neutron Scattering, Diffusion NMR, and Molecular Dynamics Study of a Eutectogel: Illuminating the Dynamical Behavior of Glyceline Confined in Bacterial Cellulose Gels.

A deep eutectic solvent (DES) entrapped in a bacterial cellulose (BC) network gives rise to a gelatin-like, self-supported material termed a bacterial cellulose eutectogel (BCEG). Although this novel material holds potential for numerous industrial, environmental, energy, or medical applications, little is known about the structural features or dynamical behavior within a eutectogel. In this work, we employ X-ray diffraction (XRD), nuclear magnetic resonance (NMR), and small-angle neutron scattering (SANS) to probe the structural and diffusive behavior of the prevailing DES glyceline (1:2 molar ratio of choline chloride:glycerol) confined within bacterial cellulose. XRD investigations demonstrate that the bacterial cellulose maintains its crystallinity even as the glyceline content approaches 95 wt % in the BCEG, an outcome corroborated by molecular dynamics (MD) simulations, which suggest minimal changes in the structural features of the cellulose chains due to the presence of glyceline. SANS measurements reveal a significant reduction in the radius of gyration (Rg) for BC in a BCEG compared to its hydrogel analogue, indicating a collapse in the microfibrillar structure that we attribute to removal of waters from the interfibrillar space due to a higher affinity of DES for water than for cellulose. Furthermore, SANS experiments suggest that the vast majority of DES is hosted within large micropores in the BCEG (i.e., mesoscopic confinement). Interestingly, proton NMR experiments disclose faster diffusional rates for choline and glycerol entrapped in a BCEG compared to neat glyceline. MD simulations offer the possible explanation that this diffusional acceleration results from significant migration of chloride from the bulk to cellulose microfibrillar surfaces, thereby reducing hydrogen bonding with choline and glycerol partners. This study provides the first comprehensive investigation into the structure and diffusional dynamics of glyceline within a eutectogel, offering insights into mass transport that should be useful for tailoring these novel materials to potential applications.

Effects of soil particles and convective transport on dispersion and aggregation of nanoplastics via small-angle neutron scattering (SANS) and ultra SANS (USANS).

Terrestrial nanoplastics (NPs) pose a serious threat to agricultural food production systems due to the potential harm of soil-born micro- and macroorganisms that promote soil fertility and ability of NPs to adsorb onto and penetrate into vegetables and other crops. Very little is known about the dispersion, fate and transport of NPs in soils. This is because of the challenges of analyzing terrestrial NPs by conventional microscopic techniques due to the low concentrations of NPs and absence of optical transparency in these systems. Herein, we investigate the potential utility of small-angle neutron scattering (SANS) and Ultra SANS (USANS) to probe the agglomeration behavior of NPs prepared from polybutyrate adipate terephthalate, a prominent biodegradable plastic used in agricultural mulching, in the presence of vermiculite, an artificial soil. SANS with the contrast matching technique was used to study the aggregation of NPs co-dispersed with vermiculite in aqueous media. We determined the contrast match point for vermiculite was 66% D2O / 33% H2O. At this condition, the signal for vermiculite was ~50-100%-fold lower that obtained using neat H2O or D2O as solvent. According to SANS and USANS, smaller-sized NPs (50 nm) remained dispersed in water and did not undergo size reduction or self-agglomeration, nor formed agglomerates with vermiculite. Larger-sized NPs (300-1000 nm) formed self-agglomerates and agglomerates with vermiculite, demonstrating their significant adhesion with soil. However, employment of convective transport (simulated by ex situ stirring of the slurries prior to SANS and USANS analyses) reduced the self-agglomeration, demonstrating weak NP-NP interactions. Convective transport also led to size reduction of the larger-sized NPs. Therefore, this study demonstrates the potential utility of SANS and USANS with contrast matching technique for investigating behavior of terrestrial NPs in complex soil systems.

Configuration of PKCalpha-C2 domain bound to mixed SOPC/SOPS lipid monolayers.

X-ray reflectivity measurements are used to determine the configuration of the C2 domain of protein kinase Calpha (PKCalpha-C2) bound to a lipid monolayer of a 7:3 mixture of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine supported on a buffered aqueous solution. The reflectivity is analyzed in terms of the known crystallographic structure of PKCalpha-C2 and a slab model representation of the lipid layer. The configuration of lipid-bound PKCalpha-C2 is described by two angles that define its orientation, theta = 35 degrees +/- 10 degrees and phi =210 degrees +/- 30 degrees, and a penetration depth (=7.5 +/- 2 A) into the lipid layer. In this structure, the beta-sheets of PKCalpha-C2 are nearly perpendicular to the lipid layer and the domain penetrates into the headgroup region of the lipid layer, but not into the tailgroup region. This configuration of PKCalpha-C2 determined by our x-ray reflectivity is consistent with many previous findings, particularly mutational studies, and also provides what we believe is new molecular insight into the mechanism of PKCalpha enzyme activation. Our analysis method, which allows us to test all possible protein orientations, shows that our data cannot be explained by a protein that is orientated parallel to the membrane, as suggested by earlier work.

Production of bacterial cellulose with controlled deuterium-hydrogen substitution for neutron scattering studies.

Isotopic enrichment of biomacromolecules is a widely used technique that enables the investigation of the structural and dynamic properties to provide information not accessible with natural abundance isotopic composition. This study reports an approach for deuterium incorporation into bacterial cellulose. A media formulation for growth of Acetobacter xylinus subsp. sucrofermentans and Gluconacetobacter hansenii was formulated that supports cellulose production in deuterium (D) oxide. The level of D incorporation can be varied by altering the ratio of deuterated and protiated glycerol used during cell growth in the D2O-based growth medium. Spectroscopic analysis and mass spectrometry show that the level of deuterium incorporation is high (>90%) for the perdeuterated form of bacterial cellulose. The small-angle neutron scattering profiles of the cellulose with different amounts of D incorporation are all similar indicating that there are no structural changes in the cellulose due to substitution of deuterium for hydrogen. In addition, by varying the amount of deuterated glycerol in the media it was possible to vary the scattering length density of the deuterated cellulose. The ability to control deuterium content of cellulose extends the range of experiments using techniques such as neutron scattering to reveal information about the structure and dynamics of cellulose, and its interactions with other biomacromolecules as well as synthetic polymers used for development of composite materials.

Synergistic self-assembly of scaffolds and building blocks for directed synthesis of organic nanomaterials.

Surfactants and hydrophobic monomers spontaneously assemble into vesicles containing monomers within the bilayer. The joint action of monomers and surfactants is essential in this synergistic self-assembly. Polymerization in the bilayer formed hollow polymer nanocapsules.

Self-similar multiscale structure of lignin revealed by neutron scattering and molecular dynamics simulation.

Lignin, a major polymeric component of plant cell walls, forms aggregates in vivo and poses a barrier to cellulosic ethanol production. Here, neutron scattering experiments and molecular dynamics simulations reveal that lignin aggregates are characterized by a surface fractal dimension that is invariant under change of scale from ~1-1000 Å. The simulations also reveal extensive water penetration of the aggregates and heterogeneous chain dynamics corresponding to a rigid core with a fluid surface.