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At least 1,387 records · Page 77
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Surface Characterization of Populus during Caldicellulosiruptor bescii Growth by TOF-SIMS Analysis

Caldicellulosiruptor bescii is a thermophilic, anaerobic bacterium that is capable of utilizing unpretreated biomass in addition to breaking down cellulose and hemicellulose into simple sugars. Despite the fact that C. bescii must first bind to the surface of the biomass, there has been no analysis of the morphological or chemical changes to the biomass surface as a result of incubation with the micro-organism. To understand more about C. bescii growth, juvenile poplar stems were sectioned (80 μm thick) and incubated with C. bescii beyond the typical 24 h experiment length. Monitoring the cell counts during incubation revealed a biphasic growth pattern. The impact the micro-organism had on the surface was determined by scanning electron microscopy (SEM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), which showed physical crevices in the cell wall caused by the C. bescii along with a decrease of polysaccharide ions and an increase in lignin ions on the poplar surface. Employing infrared microspectroscopy, the decreasing trend was corroborated.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Evaluating the Transport Mechanism for Lignocellulosic Inhibitors in Zymomonas mobilis

The conversion of lignocellulosic biomass to biofuels and bioproducts is limited by small inhibitory molecules generated during the pretreatment process. The diverse compounds within the cell wall hydrolysate including aldehydes, carboxylic acids, phenolics, and alcohols inhibit microbial growth by disrupting microbial membranes. Although Zymomonas mobilis combines high ethanol productivity with hopanoid-rich membranes that confer solvent tolerance, hydrolysate compounds still inhibit microbial growth. One proposed mitigation strategy is to pump these molecules out through active transport processes, reducing direct toxicity. However, if passive permeation rates are high, active transport would create a futile cycle where the exported molecules diffuse back in, creating a net drag on fitness. To investigate this hypothesis, we measure the passive permeation for thirty-three lignocellulose derived compounds using atomistic molecular dynamics simulations with enhanced sampling. Permeability coefficients determined spanned more than eight orders of magnitude driven largely by the balance between hydrophobicity and polarity. Hydrophobic inhibitors such as aromatic acids, aldehydes, and short chain alcohols readily partitioned into the membrane and showed high permeability consistent with rapid passive influx and strong toxicity. Most polar molecules have lower but still appreciable permeation rates, with only the most polar sugars having low permeabilities. By integrating these predictions together with hydrolysate composition information, we find that most of these molecules are so permeable that futile cycles where passive permeation overwhelms efflux is highly likely. These findings provide a predictive framework for strain engineering, highlighting that, for highly permeable inhibitors, reducing extracellular inhibitor concentrations through hydrolysate detoxification, pretreatment optimization, or dilution may be necessary to mitigate futile cycling and improve microbial productivity in industrial biorefineries.

Zymomonas mobilis↗
DOE OSTI2018

Multiscale Alterations in Sugar Cane Bagasse and Straw Submitted to Alkaline Deacetylation

Alkaline deacetylation has emerged as a promising chemistry for pretreatments performed prior to enzymatic saccharification of lignocellulosic biomass. This process avoids complex pressurized reactors and opens new opportunities for lignin covalorization. In this work, we evaluate the chemical and morphological response of sugar cane bagasse and straw submitted to alkaline treatments. Alkaline solutions for deacetylation (0.4% w/w NaOH, 70 degrees C, 3 h) as well as proximal conditions (0.1-0.7% NaOH, 55-85 degrees C, 1-5 h) chosen by 23 experimental design were evaluated. The deacetylation treatment removes ~90% of the acetyl groups and 20-30% of the lignin from both bagasse and straw, while removal of ~20% of the xylan and glucan is observed in straw, but not in bagasse. Considering nanoscale structural alterations, neither cellulose cocrystallization (evaluated by X-ray diffraction) nor formation of lignin aggregates (evaluated by thermoporometric signature) are observed after the alkaline conditions, in contrast to observations after hydrothermal treatments. Furthermore, calorimetric thermoporometry as well as scanning and transmission electron microscopies show substantial introduction of nanoscale porosity and loosening of the tissue and cell wall structures, indicating desirable mechanical weakening and gains in enzyme accessibility. These results provide fundamental and practical knowledge for biorefineries based on alkaline deacetylation of sugar cane bagasse and straw.

09 BIOMASS FUELS↗
DOE OSTI2018

Understanding Lignin Fractionation and Characterization from Engineered Switchgrass Treated by an Aqueous Ionic Liquid

Aqueous ionic liquids (ILs) have received increasing interest because of their high efficacy in fractionating and pretreating lignocellulosic biomass while at the same time mitigating several challenges associated with IL pretreatment such as IL viscosity, gel formation during pretreatment, and the energy consumption and costs associated with IL recycling. This paper investigated the fate of lignin, its structural and compositional changes, and the impact of lignin modification on the deconstruction of cell wall compounds during aqueous IL (10% w/w cholinium lysinate) pretreatment of wild-type and engineered switchgrass. The 4CL genotype resulting from silencing of 4-coumarate:coenzyme A ligase gene (Pv4CL1) had a lower lignin content, relatively higher amount of hydroxycinnamates, and higher S/G ratio and appeared to be less recalcitrant to IL pretreatment likely due to the lower degree of lignin branching and more readily lignin solubilization. The results further demonstrated over 80% of lignin dissolution from switchgrass into the liquid fraction under mild conditions while the remaining solids were highly digestible by cellulases. The soluble lignin underwent partial depolymerization to a molecular weight around 500–1000 Da. 1 H– 13 C HSQC NMR results demonstrated that the variations in lignin compositions led to different modes of lignin dissolution and depolymerization during pretreatment of engineered switchgrass. Finally, these results provide insights into the impact of lignin manipulation on biomass fractionation and lignin depolymerization and lead to possible ways toward developing a more selective and efficient lignin valorization process based on aqueous IL pretreatment technology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Characteristics of Hot Water Extracts from the Bark of Cultivated Willow ( Salix sp.)

Willow bark is a rich source of heterogeneous polyphenolic compounds and a potential feedstock for biorefinery processes aiming at chemicals and fiber production. Here, mild hot water treatment of willow hybrid Karin was studied to find a practical means of isolating its non-cell-wall components for their utilization in a willow biorefinery proposed to aid valorization of the willow biomass. A short aqueous treatment of the bark at 80 °C liberated the extract in >20% yield under unpressurized conditions. The extract was characterized using mainly gas chromatography-mass spectrometry and one- and two-dimensional NMR techniques. Authentic analytes were applied to confirm the identification and quantification of the main components that were picein, (+)-catechin, triandrin, glucose, and fructose. Fructose was converted into 5-hydroxymethylfurfural in an acidic treatment which led to its condensation with the phenolic components and formation of a recalcitrant precipitate that should be avoided.

(+)-Catechin↗
DOE OSTI2019

Chemical and Structural Effects on the Rate of Xylan Hydrolysis during Dilute Acid Pretreatment of Poplar Wood

Economic biofuel production requires high sugar yields during biomass pretreatment, however, the chemical and structural features of biomass can be obstructive toward efficient xylose hydrolysis. Here, we tested the hindrance imposed by the multiscale structure of biomass on the hydrolysis of xylan during dilute acid pretreatment by studying the effects of both the chemical nature of xylan and physical structure of biomass. Dilute acid pretreatment of poplar wood at particle sizes ranging from 10 um to 10 mm was conducted, however, no significant differences in the rates of xylan hydrolysis were observed over more than 2 orders of magnitude in particle size. A significant reduction in the rate of xylan hydrolysis was observed when compared to the intrinsic rate of hydrolysis for isolated xylan. Thus, it appears likely that the chemical structure of xylan and/or the interaction of xylan with other polymers in the cell wall matrix have greater effects on xylan hydrolysis rates than mass transfer limitations.

09 BIOMASS FUELS↗
DOE OSTI2018

Automated Transformation of Lignin Topologies into Atomic Structures with LigninBuilder

Lignin is an abundant aromatic heteropolymer found in secondary plant cell walls and is a potential feedstock for conversion into bioderived fuels and chemicals. Lignin chemical diversity complicates traditional structural studies, and so, relatively little experimental evidence exists for how lignin structure exists in aqueous solution or how lignin polymers respond to changes in their chemical environment. Molecular modeling can address these concerns; however, prior computational structural lignin models typically did not capture lignin heterogeneity, as only a few polymers were considered. LigninBuilder creates a framework for building structural libraries for lignin from existing topological libraries, permitting significantly greater diversity of lignin structures to be sampled at atomic detail. As a demonstration of its capabilities, LigninBuilder was applied to three libraries of lignin from hardwood, softwood, and grass, and the resulting polymer structures were simulated in an aqueous environment. The lignins adopted compact globular structures, as would be expected for polymers in poor solvents. The differences between the libraries were largest when quantifying the packing of nonadjacent aromatic residues, with greater branching within the polymer resulting in poorer aromatic packing. Individual lignin polymers were also found to undergo rapid conformational changes, with the dwell time within a state growing as the square of the molecular weight. This first application of LigninBuilder demonstrates the potential for atomic-level insight into lignin interactions. LigninBuilder is distributed as a plugin to the visualization software VMD, lowering the barrier for modeling lignin structure in diverse environments.

09 BIOMASS FUELS↗
DOE OSTI2019

A Quantitative Molecular Atlas for Interactions Between Lignin and Cellulose

Lignin and cellulose are two of the most abundant polymers on Earth, and are found in close proximity within plant cell walls. Despite their ubiquity, relatively little is known quantitatively about their interactions within plants, and by extension how their interaction may affect industrial biomass utilization. Given the inherent heterogeneity of the lignin polymer and the structural complexity of cellulose, quantitative relationships between given cellulose faces and specific lignin chemistries have been challenging to ascertain experimentally. In this study, we use molecular simulation to examine individual combinations of cellulose binding face and lignin chemistry to build a quantitative lignin-cellulose interaction atlas, including contributions from both specific hydrogen bonds observed in simulation and nonspecific interactions between lignin and cellulose driven by solvent considerations. Over all monomeric and dimeric lignin chemistries tested, the hydrophobic 100 face is the preferred cellulose interaction site. Among the hydrophilic crystalline faces, binding of these molecules is strongest to the 110 cellulose face. The chemical composition of lignin monomers and dimers is also found to modulate the binding affinity, with additional methoxy groups increasing the contact area to the cellulose surface, which we hypothesize may protect native cellulose from degradation. Additional methoxylation and monomer linkages that facilitate planar aromatic ring orientations increase the binding affinity between small lignin-related molecules and cellulose, with the planar linkages between guaiacyl-type monomers leading to higher binding affinities. These trends can be extended to larger lignin polymers, where we quantify the relationships between lignin polymer size and binding affinity through replica exchange umbrella sampling simulations. Rather than molecular weight, the binding surface area between the lignin polymer and the cellulose surface dictates the interaction strength. As a result, based on the findings here, we expect there to be abundant and durable interaction between cellulose and lignin, particularly on the hydrophobic face of cellulose where cellulase enzymes typically bind.

09 BIOMASS FUELS↗
DOE OSTI2019

Vitamin A Production by Engineered Saccharomyces cerevisiae from Xylose via Two-Phase in Situ Extraction

Vitamin A is an essential human micronutrient and plays critical roles in vision, reproduction, immune system, and skin health. Current industrial methods for the production of vitamin A rely on chemical synthesis from petroleum-derived substrates, such as acetone and acetylene. In this study, we developed a biotechnological method for production of vitamin A from an abundant and non-edible sugar. Specifically, we engineered Saccharomyces cerevisiae to produce vitamin A from xylose—the second most abundant sugar in plant cell wall hydrolysates—by introducing a β-carotene biosynthetic pathway, and a gene coding for β-carotene 15, 15’-dioxygenase (BCMO) into a xylose-fermenting S. cerevisiae . The resulting yeast strain produced vitamin A from xylose at a titer four-fold higher than from glucose. When a two-phase in situ extraction strategy with dodecane, or olive oil as an extractive agent was employed, vitamin A production improved additional two-fold. Furthermore, a xylose fed-batch fermentation with dodecane in situ extraction achieved a final titer of 3,350 mg/L vitamin A, which consisted of retinal (2,094 mg/L) and retinol (1,256 mg/L). These results suggest that potential limiting factors of vitamin A production in yeast, such as insufficient supply of isoprenoid precursors, and limited intracellular storage capacity, can be effectively addressed by using xylose as a carbon source, and two-phase in situ extraction. The engineered S. cerevisiae and fermentation strategies described in this study might contribute to sustainable and economic production of vitamin A, and vitamin A-enriched bioproducts from renewable biomass.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Structural Studies of Deuterium-Labeled Switchgrass Biomass

Small-angle neutron scattering (SANS) is being used to study the molecular structure of tiller biomass from switchgrass, a perennial North American prairie grass that is being developed as a bioenergy crop. To enable the expanded application of SANS methods, switchgrass plants were cultivated in hydroponic media containing 50% D2O to produce tiller biomass with 34% deuterium incorporation. Characterization of the deuterated biomass found chemical and physical properties similar to the same cultivar grown hydroponically in H2O but with slightly increased lignin content. Surprisingly, the deuterated switchgrass was found to be less recalcitrant to enzymatic hydrolysis. Investigation with transmission electron microscopy found changes in lignin deposition patterns in deuterated cell walls. The partially deuterated switchgrass and its control were studied by contrast variation SANS to evaluate deuterium incorporation into the different plant polymers.

Evans, Barbara↗
DOE OSTI2019

Integrating Density Functional Theory Calculations with Vibrational and Nuclear Magnetic Resonance Spectroscopy

This review focuses on the complementary nature of density functional theory calculations and spectroscopic studies related to cellulose and its interactions within plant cell walls (PCWs). The complexity, structural disorder, and nanoscale nature of PCWs make them difficult to analyze and understand, but advances in sum-frequency generation and nuclear magnetic resonance spectroscopies as well as in density functional theory have made it possible to elucidate structure not observable previously. Combined with other techniques such as wide-angle X-ray scattering and neutron diffraction, details of PCW components (e.g., cellulose, lignin, pectin) and their interactions with one another can be elucidated. Furthermore, these techniques provide new insight into biological and biochemical studies and provide a basis for creating improved models of the PCWs.

36 MATERIALS SCIENCE↗
DOE OSTI2012

Suite of Activity-Based Probes for Cellulose-Degrading Enzymes

Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degradation of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome producing bacterium that couples rapid reproduction rates to highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chemistry, and to increase enzyme active site inclusion for LC-MS analysis. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes in addition to a large collection of integral cellulosome-containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic cellulose degrading systems, and facilitates a greater understanding of the organismal role associated within biofuel development.

Chauvigne-Hines, Lacie M↗
DOE OSTI2015

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism

Yeasts, which have been a component of the human diet for at least 7000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for Bacteroides thetaiotaomicron (Bt), a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by Bt presents a ‘selfish’ model for the catabolism of this recalcitrant polysaccharide. This report shows how a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Structure and mechanism of the essential two-component signal-transduction system WalKR in Staphylococcus aureus

Most low GC Gram-positive bacteria possess an essential walKR two-component system (TCS) for signal transduction involved in regulating cell wall homoeostasis. Despite the well-established intracellular regulatory mechanism, the role of this TCS in extracellular signal recognition and factors that modulate the activity of this TCS remain largely unknown. Here we identify the extracellular receptor of the kinase ‘WalK’ (erWalK) as a key hub for bridging extracellular signal input and intracellular kinase activity modulation in Staphylococcus aureus . Characterization of the crystal structure of erWalK revealed a canonical Per-Arnt-Sim (PAS) domain for signal sensing. Single amino-acid mutation of potential signal-transduction residues resulted in severely impaired function of WalKR. A small molecule derived from structure-based virtual screening against erWalK is capable of selectively activating the walKR TCS. Lastly, the molecular level characterization of erWalK will not only facilitate exploration of natural signal(s) but also provide a template for rational design of erWalK inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Functional metagenomics reveals abundant polysaccharide-degrading gene clusters and cellobiose utilization pathways within gut microbiota of a wood-feeding higher termite

Plant cell-wall polysaccharides constitute the most abundant but recalcitrant organic carbon source in nature. Microbes residing in the digestive tract of herbivorous bilaterians are particularly efficient at depolymerizing polysaccharides into fermentable sugars and play a significant support role towards their host’s lifestyle. Here, we combine large-scale functional screening of fosmid libraries, shotgun sequencing, and biochemical assays to interrogate the gut microbiota of the wood-feeding “higher” termite Globitermes brachycerastes. A number of putative polysaccharide utilization gene clusters were identified with multiple fibrolytic genes. Our large-scale functional screening of 50,000 fosmid clones resulted in 464 clones demonstrating plant polysaccharide-degrading activities, including 267 endoglucanase-, 24 exoglucanase-, 72 β-glucosidase-, and 101 endoxylanase-positive clones. We sequenced 173 functionally active clones and identified ~219 genes encoding putative carbohydrate-active enzymes (CAZymes) targeting cellulose, hemicellulose and pectin. Further analyses revealed that 68 of 154 contigs encode one or more CAZyme, which includes 35 examples of putative saccharolytic operons, suggesting that clustering of CAZymes is common in termite gut microbial inhabitants. Biochemical characterization of a representative xylanase cluster demonstrated that constituent enzymes exhibited complementary physicochemical properties and saccharolytic capabilities. Furthermore, diverse cellobiose-metabolizing enzymes include β-glucosidases, cellobiose phosphorylases, and phopho-6-β-glucosidases were identified and functionally verified, indicating that the termite gut micro-ecosystem utilizes diverse metabolic pathways to interconnect hydrolysis and central metabolism. Collectively, these results provide an in-depth view of the adaptation and digestive strategies employed by gut microbiota within this tiny-yet-efficient host-associated ecosystem.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Molecular basis of an agarose metabolic pathway acquired by a human intestinal symbiont

In red algae, the most abundant principal cell wall polysaccharides are mixed galactan agars, of which agarose is a common component. While bioconversion of agarose is predominantly catalyzed by bacteria that live in the oceans, agarases have been discovered in microorganisms that inhabit diverse terrestrial ecosystems, including human intestines. Here we comprehensively define the structure–function relationship of the agarolytic pathway from the human intestinal bacterium Bacteroides uniformis (Bu) NP1. Using recombinant agarases from Bu NP1 to completely depolymerize agarose, we demonstrate that a non-agarolytic Bu strain can grow on GAL released from agarose. This relationship underscores that rare nutrient utilization by intestinal bacteria is facilitated by the acquisition of highly specific enzymes that unlock inaccessible carbohydrate resources contained within unusual polysaccharides. Intriguingly, the agarolytic pathway is differentially distributed throughout geographically distinct human microbiomes, reflecting a complex historical context for agarose consumption by human beings.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Visualizing conformation transitions of the Lipid II flippase MurJ

The biosynthesis of many polysaccharides, including bacterial peptidoglycan and eukaryotic N-linked glycans, requires transport of lipid-linked oligosaccharide (LLO) precursors across the membrane by specialized flippases. MurJ is the flippase for the lipid-linked peptidoglycan precursor Lipid II, a key player in bacterial cell wall synthesis, and a target of recently discovered antibacterials. However, the flipping mechanism of LLOs including Lipid II remains poorly understood due to a dearth of structural information. Here we report crystal structures of MurJ captured in inward-closed, inward-open, inward-occluded and outward-facing conformations. Together with mutagenesis studies, we elucidate the conformational transitions in MurJ that mediate lipid flipping, identify the key ion for function, and provide a framework for the development of inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗