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Cell collectives, like other motile entities, generate and use forces to move forward. Here, we ask whether environmental configurations alter this proportional force-speed relationship, since aligned extracellular matrix fibers are known to cause directed migration. We show that aligned fibers serve as active conduits for spatial propagation of cellular mechanotransduction through matrix exoskeleton, leading to efficient directed collective cell migration. Epithelial (MCF10A) cell clusters adhered to soft substrates with aligned collagen fibers (AF) migrate faster with much lesser traction forces, compared to random fibers (RF). Fiber alignment causes higher motility waves and transmission of normal stresses deeper into cell monolayer while minimizing shear stresses and increased cell-division based fluidization. By contrast, fiber randomization induces cellular jamming due to breakage in motility waves, disrupted transmission of normal stresses, and heightened shear driven flow. Using a novel motor-clutch model, we explain that such ‘force-effective’ fast migration phenotype occurs due to rapid stabilization of contractile forces at the migrating front, enabled by higher frictional forces arising from simultaneous compressive loading of parallel fiber-substrate connections. We also model ‘ haptotaxis ’ to show that increasing ligand connectivity (but not continuity) increases migration efficiency. According to our model, increased rate of front stabilization via higher resistance to substrate deformation is sufficient to capture ‘ durotaxis’ . Thus, our findings reveal a new paradigm wherein the rate of leading-edge stabilization determines the efficiency of supracellular collective cell migration.

Switchgrass is a leading dedicated bioenergy feedstock in the United States because it is a native, high-yielding, perennial prairie grass with a broad cultivation range and low agronomic input requirements. Biomass conversion research has developed processes for production of ethanol and other biofuels, but they remain costly primarily because of the intrinsic recalcitrance of biomass. We show here that genetic modification of switchgrass can produce phenotypically normal plants that have reduced thermal-chemical ([less-than or equal to]180 °C), enzymatic, and microbial recalcitrance. Down-regulation of the switchgrass caffeic acid O-methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines require less severe pretreatment and 300-400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast. Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass. Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly. Alternatively, such modified transgenic switchgrass lines should yield significantly more fermentation chemicals per hectare under identical process conditions.

Doc number: 110 Abstract Background: Effective enzymatic hydrolysis of lignocellulosic biomass benefits from lignin removal, relocation, and/or modification during hydrothermal pretreatment. Phase transition, depolymerization/repolymerization, and solubility effects may all influence these lignin changes. To better understand how lignin is altered, Populus trichocarpa x P. deltoides wood samples and cellulolytic enzyme lignin (CEL) isolated from P. trichocarpa x P. deltoides were subjected to batch and flowthrough pretreatments. The residual solids and liquid hydrolysate were characterized by gel permeation chromatography, heteronuclear single quantum coherence NMR, compositional analysis, and gas chromatography-mass spectrometry. Results: Changes in the structure of the solids recovered after the pretreatment of CEL and the production of aromatic monomers point strongly to depolymerization and condensation being primary mechanisms for lignin extraction and redeposition. The differences in lignin removal and phenolic compound production from native P. trichocarpa x P. deltoides and CEL suggested that lignin-carbohydrate interactions increased lignin extraction and the extractability of syringyl groups relative to guaiacyl groups. Conclusions: These insights into delignification during hydrothermal pretreatment point to desirable pretreatment strategies and plant modifications. Because depolymerization followed by repolymerization appears to be the dominant mode of lignin modification, limiting the residence time of depolymerized lignin moieties in the bulk liquid phase should reduce lignin content in pretreated biomass. In addition, the increase in lignin removal in the presence of polysaccharides suggests that increasing lignin-carbohydrate cross-links in biomass would increase delignification during pretreatment.

Doc number: 150 Abstract Background: Obtaining a better understanding of the complex mechanisms occurring during lignocellulosic deconstruction is critical to the continued growth of renewable biofuel production. A key step in bioethanol production is thermochemical pretreatment to reduce plant cell wall recalcitrance for downstream processes. Previous studies of dilute acid pretreatment (DAP) have shown significant changes in cellulose ultrastructure that occur during pretreatment, but there is still a substantial knowledge gap with respect to the influence of lignin on these cellulose ultrastructural changes. This study was designed to assess how the presence of lignin influences DAP-induced changes in cellulose ultrastructure, which might ultimately have large implications with respect to enzymatic deconstruction efforts. Results: Native, untreated hybrid poplar (Populus trichocarpa x Populus deltoids ) samples and a partially delignified poplar sample (facilitated by acidic sodium chlorite pulping) were separately pretreated with dilute sulfuric acid (0.10 M) at 160°C for 15 minutes and 35 minutes, respectively . Following extensive characterization, the partially delignified biomass displayed more significant changes in cellulose ultrastructure following DAP than the native untreated biomass. With respect to the native untreated poplar, delignified poplar after DAP (in which approximately 40% lignin removal occurred) experienced: increased cellulose accessibility indicated by increased Simons' stain (orange dye) adsorption from 21.8 to 72.5 mg/g, decreased cellulose weight-average degree of polymerization (DPw ) from 3087 to 294 units, and increased cellulose crystallite size from 2.9 to 4.2 nm. These changes following DAP ultimately increased enzymatic sugar yield from 10 to 80%. Conclusions: Overall, the results indicate a strong influence of lignin content on cellulose ultrastructural changes occurring during DAP. With the reduction of lignin content during DAP, the enlargement of cellulose microfibril dimensions and crystallite size becomes more apparent. Further, this enlargement of cellulose microfibril dimensions is attributed to specific processes, including the co-crystallization of crystalline cellulose driven by irreversible inter-chain hydrogen bonding (similar to hornification) and/or cellulose annealing that converts amorphous cellulose to paracrystalline and crystalline cellulose. Essentially, lignin acts as a barrier to prevent cellulose crystallinity increase and cellulose fibril coalescence during DAP.

The hemicellulose 4- O -methyl glucuronoxylan is one of the principle components present in the secondary cell walls of eudicotyledonous plants. However, the biochemical mechanisms leading to the formation of this polysaccharide and the effects of modulating its structure on the physical properties of the cell wall are poorly understood. We have identified and functionally characterized an Arabidopsis glucuronoxylan methyltransferase (GXMT) that catalyzes 4- O -methylation of the glucuronic acid substituents of this polysaccharide. AtGXMT1, which was previously classified as a domain of unknown function (DUF) 579 protein, specifically transfers the methyl group from S-adenosyl- l -methionine to O -4 of α- d -glucopyranosyluronic acid residues that are linked to O -2 of the xylan backbone. Biochemical characterization of the recombinant enzyme indicates that GXMT1 is localized in the Golgi apparatus and requires Co ²⁺ for optimal activity in vitro. Plants lacking GXMT1 synthesize glucuronoxylan in which the degree of 4- O -methylation is reduced by 75%. This result is correlated to a change in lignin monomer composition and an increase in glucuronoxylan release during hydrothermal treatment of secondary cell walls. We propose that the DUF579 proteins constitute a previously undescribed family of cation-dependent, polysaccharide-specific O -methyl-transferases. This knowledge provides new opportunities to selectively manipulate polysaccharide O -methylation and extends the portfolio of structural targets that can be modified either alone or in combination to modulate biopolymer interactions in the plant cell wall.

In order to obtain accurate information about the ultrastructure of cellulose from native biomass by 13C cross polarization magic angle spinning (CP/MAS) NMR spectroscopy the cellulose component must be isolated due to overlapping resonances from both lignin and hemicellulose. Typically, cellulose isolation has been achieved via holocellulose pulping to remove lignin followed by an acid hydrolysis procedure to remove the hemicellulose components. Using 13C CP/MAS NMR and non-linear line-fitting of the cellulose C4 region, it was observed that the standard acid hydrolysis procedure caused an apparent increase in crystallinity of ~10% or less on the cellulose isolated from Populus holocellulose. We have examined the effect of the cellulose isolation method, particularly the acid treatment time for hemicellulose removal, on cellulose ultrastructural characteristics by studying these effects on cotton, microcrystalline cellulose (MCC) and holocellulose pulped Populus. 13C CP/MAS NMR of MCC indicated that holocellulose pulping and acid hydrolysis has little effect on the crystalline ultrastructural components of cellulose. Although any chemical method to isolate cellulose from native biomass will invariably alter substrate characteristics, especially those related to regions accessible to solvents, we found those changes to be minimal and consistent in samples of typical crystallinity and lignin/hemicellulose content. Based on the rate of the hemicellulose removal, as determined by HPLC-carbohydrate analysis and magnitude of cellulose ultrastructural alteration, the most suitable cellulose isolation methodology utilizes a treatment of 2.5 M HCl at 100 °C for a standard residence time between 1.5 and 4 h. However, for the most accurate crystallinity results this residence time should be determined empirically for a particular sample.

Background Lignin is a recalcitrant aromatic polymer that is a potential feedstock for renewable fuel and chemical production. Rhodococcus opacus PD630 is a promising strain for the biological upgrading of lignin due to its ability to tolerate and utilize lignin-derived aromatic compounds. To enhance its aromatic tolerance, we recently applied adaptive evolution using phenol as a sole carbon source and characterized a phenol-adapted R. opacus strain (evol40) and the wild-type (WT) strain by whole genome and RNA sequencing. While this effort increased our understanding of the aromatic tolerance, the tolerance mechanisms were not completely elucidated. Results We hypothesize that the composition of lipids plays an important role in phenol tolerance. To test this hypothesis, we applied high-resolution mass spectrometry analysis to lipid samples obtained from the WT and evol40 strains grown in 1 g/L glucose (glucose), 0.75 g/L phenol (low phenol), or 1.5 g/L phenol (high phenol, evol40 only) as a sole carbon source. This analysis identified > 100 lipid species of mycolic acids, phosphatidylethanolamines (PEs), phosphatidylinositols (PIs), and triacylglycerols. In both strains, mycolic acids had fewer double bond numbers in phenol conditions than the glucose condition, and evol40 had significantly shorter mycolic acid chain lengths than the WT strain in phenol conditions. These results indicate that phenol adaptation affected mycolic acid membrane composition. In addition, the percentage of unsaturated phospholipids decreased for both strains in phenol conditions compared to the glucose condition. Moreover, the PI content increased for both strains in the low phenol condition compared to the glucose condition, and the PI content increased further for evol40 in the high phenol condition relative to the low phenol condition. Conclusions This work represents the first comprehensive lipidomic study on the membrane of R. opacus grown using phenol as a sole carbon source. Our results suggest that the alteration of the mycolic acid and phospholipid membrane composition may be a strategy of R. opacus for phenol tolerance.

Main conclusion The bioenergy crop switchgrass was grown hydroponically from tiller cuttings in 50 % D₂O to obtain biomass with 34 % deuterium substitution and physicochemical properties similar to those of H₂O-grown switchgrass controls. Deuterium enrichment of biological materials can potentially enable expanded experimental use of small angle neutron scattering (SANS) to investigate molecular structural transitions of complex systems such as plant cell walls. Two key advances have been made that facilitate cultivation of switchgrass, an important forage and biofuel crop, for controlled isotopie enrichment: (1) perfusion system with individual chambers and (2) hydroponic growth from tiller cuttings. Plants were grown and maintained for several months with periodic harvest. Photosynthetic activity was monitored by measurement of CO₂ in outflow from the growth chambers. Plant morphology and composition appeared normal compared to matched controls grown with H₂O. Using this improved method, gram quantities of switchgrass leaves and stems were produced by continuous hydroponic cultivation using growth medium consisting of basal mineral salts in 50 % D₂O. Deuterium incorporation was confirmed by detection of the O-D and C-D stretching peaks with FTIR and quantified by ¹H- and ²H-NMR. This capability to produce deuterated lignocellulosic biomass under controlled conditions will enhance investigation of cell wall structure and its deconstruction by neutron scattering and NMR techniques.

Doc number: 50 Abstract Background: Agave, which is well known for tequila and other liquor production in Mexico, has recently gained attention because of its attractive potential to launch sustainable bioenergy feedstock solutions for semi-arid and arid lands. It was previously found that agave cell walls contain low lignin and relatively diverse non-cellulosic polysaccharides, suggesting unique recalcitrant features when compared to conventional C4 and C3 plants. Results: Here, we report sugar release data from fungal enzymatic hydrolysis of non-pretreated and hydrothermally pretreated biomass that shows agave to be much less recalcitrant to deconstruction than poplar or switchgrass. In fact, non-pretreated agave has a sugar release five to eight times greater than that of poplar wood and switchgrass . Meanwhile, state of the art techniques including glycome profiling, nuclear magnetic resonance (NMR), Simon's Stain, confocal laser scanning microscopy and so forth, were applied to measure interactions of non-cellulosic wall components, cell wall hydrophilicity, and enzyme accessibility to identify key structural features that make agave cell walls less resistant to biological deconstruction when compared to poplar and switchgrass. Conclusions: This study systematically evaluated the recalcitrant features of agave plants towards biofuels applications. The results show that not only does agave present great promise for feeding biorefineries on semi-arid and arid lands, but also show the value of studying agave's low recalcitrance for developments in improving cellulosic energy crops.

A comparison of the thermal decomposition and combustion characteristics of raw and scoured cottons has demonstrated a mechanistic link caused by the presence of inorganic salts in raw cotton, which enhances resistance to heat and flame. Thermogravimetry, differential thermogravimetry, and microscale combustion calorimetry were used to examine the thermal decomposition kinetics and thermal stability of cotton. During pyrolysis, both raw cotton nonwoven and woven fabrics exhibited a slower decomposition with a larger initial weight loss and produced a greater char yield, as compared to the fabrics after scouring, which removes most inorganic components from cotton. The activation energy (E ₐ ) values, calculated using the Kissinger method, the Flynn–Wall–Ozawa method, and the modified Coats–Redfern method, were consistently determined to be smaller for raw cotton than for scoured cotton. The analyses of cotton fabrics heated at elevated temperatures by ¹³C CP/MAS NMR and ATR-FTIR showed that trace quantities of inorganic components promoted the formations of oxygenated moieties at low temperatures and aliphatic intermediate char. In the combustion, raw cotton exhibited a much smaller heat release capacity and a smaller total heat release than scoured cotton, indicating enhanced thermal stability when the inorganic components are intact.