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At least 19 records

Effect of cellulose reducing ends and primary hydroxyl groups modifications on cellulose-cellulase interactions and cellulose hydrolysis

Cellulose reducing ends are believed to play a vital role in the cellulose recalcitrance to enzymatic conversion. However, their role in insoluble cellulose accessibility and hydrolysis is not clear. Thus, in this study, reducing ends of insoluble cellulose derived from various sources were modified by applying reducing and/or oxidizing agents. Here, the effects of cellulose reducing ends modification on cellulose reducing ends, cellulose structure, and cellulose accessibility to cellulase were evaluated along with the impact on cellulose hydrolysis with complete as well purified cellulase components. Sodium borohydride (NaBH 4 ) reduction and sodium chlorite-acetic acid (SC/AA) oxidation were able to modify more than 90% and 60% of the reducing ends, respectively, while the bicinchoninic acid (BCA) reagent applied for various cycles oxidized cellulose reducing ends to various extents. X-ray diffractograms of the treated solids showed that these treatments did not change the cellulose crystalline structure and the change in crystallinity index was insignificant. Surprisingly, it was found that the cellulose reducing ends modification, either through selective NaBH 4 reduction or BCA oxidation, had a negligible impact on cellulose accessibility as well on cellulose hydrolysis rates or final conversions with complete cellulase at loadings as low as 0.5 mg protein/g cellulose. In fact, in contrast to what is traditionally believed, modifications of cellulose reducing ends by these two methods had no apparent impact on cellulose conversion with purified cellulase components and their synergy. However, SC/AA oxidation resulted in significant drop in cellulose conversion (10%–50%) with complete as well purified cellulase components. Nonetheless, further research revealed that the cause for drop in cellulose conversion for the SC/AA oxidation case was due to primary hydroxyl groups (PHGs) oxidation and not the oxidation of reducing ends. Furthermore, it was found that the PHGs modification affects cellulose accessibility and slows the cellulase uptake as well resulting in significant drop in cellulose conversions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Data for Filling the Cellulosic Bio-economy Gap by Utilizing a Wedge Approach Combined with Stakeholder Collaboration

The price gap between the market and breakeven prices of cellulosic biomass for farmers represents a significant barrier to the development of a low-carbon cellulosic bioeconomy. Using a bottom-up, agent-based modeling tool that replicates the behaviors and interactions of key stakeholders, this study analyzes the emergence of a cellulosic bioeconomy at the local scale through a wedge approach that examines an integrated portfolio of multiple policy options, including subsidies for small-scale bioproducts and environmental credits. The role of collaboration among multiple stakeholders, such as biomass producers (farmers), bio-refinery industry, government, and society, is assessed for filling the price gap. Using the Sangamon River Basin as a case study site, we evaluate the effectiveness of the wedge approach by comparing simulation results from multiple scenarios, each incorporating different combinations of bioeconomy wedges, with and without stakeholder collaboration. Results underscore that active collaboration among stakeholders acts as a catalyst enlarging the effectiveness of bioeconomy wedges. Including the carbon credits and environmental value in the policy portfolio is found to bridge the price gap through collective contributions from diverse stakeholders, where the cellulosic biofuel and bioproduct industry plays a pivotal role. Although this study is conducted at the local watershed scale, the methodology and findings offer valuable insights for market development in other watersheds and the potential scaling of local markets to regional and national levels.

Economics↗

An Experimental and Computational Approach to Investigating CO 2 Uptake of Cellulose-producing Algae from Cellulosic Ethanol Production (Final Report)

This project combined experimental algal cultures with predictive quantum calculations to evaluate system-level CO 2 uptake and conversion efficiency of cellulose-producing Nannochloropsis sp. algae. Recently, Nannochloropsis sp. has garnered attention as a novel host organism for converting low-cost CO 2 produced from cellulosic ethanol fermentations into algal lipids for biodiesel production and microcrystalline cellulose as a high-value co-product. As depicted in the figure below, this project (1) optimized Nannochloropsis salina (N. salina) cultures on effluent gas produced directly from cellulosic ethanol fermentation, (2) characterized the fermentation products, quantify cellulose production, and calculate CO 2 uptake efficiency with predictive quantum calculations, (3) conducted a life cycle and techno economic analysis of the proposed integration, and (4) provided training opportunities to students attending UC Riverside.

09 BIOMASS FUELS↗

Five amino acid mismatches in the zinc-finger domains of Cellulose Synthase 5 and Cellulose Synthase 6 cooperatively modulate their functional properties by controlling homodimerization in Arabidopsis

Cellulose synthase 5 (CESA5) and CESA6 are known to share substantial functional overlap. In the zinc-finger domain (ZN) of CESA5, there are five amino acid (AA) mismatches when compared to CESA6. These mismatches in CESA5 were replaced with their CESA6 counterparts one by one until all were replaced, generating nine engineered CESA5s. Each N-terminal enhanced yellow fluorescent protein-tagged engineered CESA5 was introduced to prc1-1, a cesa6 null mutant, and resulting mutants were subjected to phenotypic analyses. For this work, we found that five single AA-replaced CESA5 proteins partially rescue the prc1-1 mutant phenotypes to different extents. Multi-AA replaced CESA5s further rescued the mutant phenotypes in an additive manner, culminating in full recovery by CESA5 G43R + S49T+S54P+S80A+Y88F . Investigations in cellulose content, cellulose synthase complex (CSC) motility, and cellulose microfibril organization in the same mutants support the results of the phenotypic analyses. Bimolecular fluorescence complementation assays demonstrated that the level of homodimerization in every engineered CESA5 is substantially higher than CESA5. The mean fluorescence intensity of CSCs carrying each engineered CESA5 fluctuates with the degree to which the prc1-1 mutant phenotypes are rescued by introducing a corresponding engineered CESA5. Taken together, these five AA mismatches in the ZNs of CESA5 and CESA6 cooperatively modulate the functional properties of these CESAs by controlling their homodimerization capacity, which in turn imposes proportional changes on the incorporation of these CESAs into CSCs.

59 BASIC BIOLOGICAL SCIENCES↗

Polypropylene Composites Reinforced With Recycled Waste Cellulosic Fiber/Fine Mixture: The Impact of Cellulose Sieving on Performance

This study explores how a sieving step of waste cellulosic fiber and fine (WCFF) mixture affects the performance of WCFF‐loaded polypropylene (PP) composites and whether the separation of fines from fibers offers an added benefit. The WCFF samples were downsized, and four different filler size ranges were sieved using a series of mesh sizes from 4 to 0.85 mm. The WCFF/PP composites were then compounded at 20 wt.% loading of WCFF using a twin‐screw extruder. Incorporating WCFF increased the tensile strength to 41.28 MPa and the modulus to 3207 MPa, accounting for 28% and 38% enhancements, respectively. Interestingly, the greatest improvements were associated with the nonsieved WCFF case, and the sieved WCFF fibers provided only marginal enhancements over virgin PP. The outperformance of nonsieved WCFF was attributed to the synergistic reinforcement of hybrid fibers and fines as well as the maintenance of longer fibers in the system. However, the strain at break and impact strength of PP decreased after introducing WCFF. Moreover, the complex viscosity and storage modulus increased with an increase in the filler size, due to the formation of a more effective percolative network. The PP's crystallinity exhibited a relatively strong dependency on the sieving, where WCFF samples with short‐aspect‐ratio fillers promoted the crystallinity significantly. It was also found that the WCFF degradation onset temperature increased once it was incorporated into PP. This study suggests that waste cellulosic feedstocks can be utilized as a reinforcement without additional sieving to manufacture high‐performance and cost‐effective composites.

36 MATERIALS SCIENCE↗

Tissue-specific directionality of cellulose synthase complex movement inferred from cellulose microfibril polarity in secondary cell walls of Arabidopsis

In plant cells, cellulose synthase complexes (CSCs) are nanoscale machines that synthesize and extrude crystalline cellulose microfibrils (CMFs) into the apoplast where CMFs are assembled with other matrix polymers into specific structures. We report the tissue-specific directionality of CSC movements of the xylem and interfascicular fiber walls of Arabidopsis stems, inferred from the polarity of CMFs determined using vibrational sum frequency generation spectroscopy. CMFs in xylems are deposited in an unidirectionally biased pattern with their alignment axes tilted about 25° off the stem axis, while interfascicular fibers are bidirectional and highly aligned along the longitudinal axis of the stem. These structures are compatible with the design of fiber-reinforced composites for tubular conduit and support pillar, respectively, suggesting that during cell development, CSC movement is regulated to produce wall structures optimized for cell-specific functions.

59 BASIC BIOLOGICAL SCIENCES↗

Lignin impairs Cel7A degradation of in vitro lignified cellulose by impeding enzyme movement and not by acting as a sink

Abstract Background Cellulose degradation by cellulases has been studied for decades due to the potential of using lignocellulosic biomass as a sustainable source of bioethanol. In plant cell walls, cellulose is bonded together and strengthened by the polyphenolic polymer, lignin. Because lignin is tightly linked to cellulose and is not digestible by cellulases, is thought to play a dominant role in limiting the efficient enzymatic degradation of plant biomass. Removal of lignin via pretreatments currently limits the cost-efficient production of ethanol from cellulose, motivating the need for a better understanding of how lignin inhibits cellulase-catalyzed degradation of lignocellulose. Work to date using bulk assays has suggested three possible inhibition mechanisms: lignin blocks access of the enzyme to cellulose, lignin impedes progress of the enzyme along cellulose, or lignin binds cellulases directly and acts as a sink. Results We used single-molecule fluorescence microscopy to investigate the nanoscale dynamics of Cel7A from Trichoderma reesei , as it binds to and moves along purified bacterial cellulose in vitro. Lignified cellulose was generated by polymerizing coniferyl alcohol onto purified bacterial cellulose, and the degree of lignin incorporation into the cellulose meshwork was analyzed by optical and electron microscopy. We found that Cel7A preferentially bound to regions of cellulose where lignin was absent, and that in regions of high lignin density, Cel7A binding was inhibited. With increasing degrees of lignification, there was a decrease in the fraction of Cel7A that moved along cellulose rather than statically binding. Furthermore, with increasing lignification, the velocity of processive Cel7A movement decreased, as did the distance that individual Cel7A molecules moved during processive runs. Conclusions In an in vitro system that mimics lignified cellulose in plant cell walls, lignin did not act as a sink to sequester Cel7A and prevent it from interacting with cellulose. Instead, lignin both blocked access of Cel7A to cellulose and impeded the processive movement of Cel7A along cellulose. This work implies that strategies for improving biofuel production efficiency should target weakening interactions between lignin and cellulose surface, and further suggest that nonspecific adsorption of Cel7A to lignin is likely not a dominant mechanism of inhibition.

09 BIOMASS FUELS↗

Using Solid-State NMR to Understand the Structure of Plant Cellulose

The structure of plant cellulose microfibrils remains elusive, despite the abundance of cellulose and its utility in industry. Using 2D solid-state NMR of 13 C-labeled never-dried plants, six major glucose environments are resolved, which are common to the cellulose of softwood, hardwood, and grasses. These environments are maintained in isolated holocellulose nanofibrils, allowing more detailed microfibril characterization. We show that there are only two glucose environments that reside within the microfibril core. These have the same NMR 13 C chemical shifts as tunicate cellulose Iβ center and origin chains, with no cellulose Iα being detected. The third major glucose site within spectral domain 1, previously assigned to the crystalline microfibril interior, is in close proximity to water, which could indicate that it is a surface glucose environment. The NMR peak widths of all four surface glucose environments are similar to those of the core, indicating that their glucose local order is comparable; there is no significant “amorphous” cellulose in the microfibrils. Consequently, the ratio of the carbon 4 peaks at ∼89 and ∼84 ppm, which has often provided a sample cellulose crystallinity index, is not a meaningful measure of fibril crystallinity or the interior to surface ratio. The revised ratio for poplar wood microfibrils is estimated to be 1:2, which is consistent with an 18-chain microfibril having 6 core and 12 surface chains, although other microfibril sizes are possible. These advances substantially change both the interpretation of solid-state NMR studies of cellulose and the understanding of cellulose microfibril structure and crystallinity.

carbohydrates↗

The three cellulose synthase isoforms for secondary cell wall make specific contributions to microfibril synthesis

Cellulose is synthesized at the plasma membrane by the cellulose synthase complex, a structure that contains three distinct isoforms of the catalytic subunit, cellulose synthase A (CESA). The division into three subunits appears early in land plant evolution and is highly conserved, particularly for the secondary cell wall. However, what if any unique roles each isoform plays in the complex remain unclear. Here, we assessed the contributions of specific isoforms to microfibril synthesis. First, we expressed CESA isoforms of the primary cell wall or the moss Physcomitrium patens in Arabidopsis thaliana backgrounds missing a secondary cell wall CESA. While the primary cell wall isoforms rescued the cesa knockout phenotype with partial isoform specificity, those from the moss rescued with fewer restrictions. Then, we recreated various CESA missense mutations in all three of the secondary cell wall isoforms; while results are consistent with isoform specificity, they are difficult to interpret further without molecular structures. Finally, we show that catalytically inactive CESA isoforms restore growth and cellulose content in the corresponding knockout in an isoform-specific manner; along with partial rescue of the growth and cellulose content of the inflorescence stem, the replacement lines have fiber cells with partially disorganized microfibrils and secondary cell wall cellulose with narrow crystal width. Generally, effects were more pronounced in lines where CESA8 was inactivated compared with inactivating CESA4 or 7, which tended to have similar phenotypes to each other. Here, we account for these results with a model for cellulose synthase structure with the isoforms assigned specific localization within the cellulose synthase complex.

59 BASIC BIOLOGICAL SCIENCES↗

Improving cellulose attribution by selectively removing yeast glucans from grain fermentation intermediates

Abstract This article presents an industry-relevant method for quantifying cellulose in mixed substrate samples. We built upon the cellulosic glucan measurement proposed by Sluiter et al. (2021) to investigate significant cellulose loss under cold caustic conditions, which has hindered the establishment of an industrially relevant method. To overcome this issue, we used dimethyl sulfoxide (DMSO) as an alternative solvent, which avoids mercerization reactions (Budtova and Navard 2015) but may leave some resistant starch in the sample. Treatment via an enzyme mixture removed starch and conformed to US Environmental Protection Agency (EPA) guidance on cellulose measurement by establishing quantitative de-starching via nuclear magnetic resonance (EPA 2022). The selective removal of yeast beta-glucan was accomplished using a commercially available Zymolyase. Our results demonstrated excellent reproducibility, with coefficients of variance of 7.14% or less, when measuring cellulose in low cellulose content samples. The method was tested on relevant lab and plant samples, showing an average 0.9% ethanol derived from the conversion of cellulose when cellulase was added to the process and zero response when no cellulase was added. These findings indicate that a cellulose method using DMSO and pullulanase provides a complete and accurate view of cellulose content, composition, and conversion in industrial fermentation processes.

Sluiter, Justin [National Renewable Energy Laborat↗

Deep Eutectic Solvents for Efficient Fractionation of Lignocellulose to Produce Uncondensed Lignin and High-Quality Cellulose

Simultaneously inhibiting lignin condensation and cellulose degradation remains a major challenge for achieving holistic valorization of lignocellulose. Here, we developed a deep eutectic solvent (DES), composed of l-cysteine (Cys) and lactic acid (LA), to fractionate both uncondensed lignin and high-quality cellulose from eucalyptus wood by leveraging the unique properties of Cys, i.e., highly nucleophilic groups (–SH) and hydrogen bond acceptor/donor groups (–NH 2 and –COOH). The nucleophilic –SH in Cys effectively quenches the benzylic carbocations (C α + ions, formed at the benzylic sites of lignin) that lead to lignin condensation. This enables the high yield of uncondensed lignin (81%) with high retention of β–O–4 bonds (up to 90%). The separated uncondensed lignin is further depolymerized to prepare monophenols in a satisfactory 43% yield, equivalent to 73% of the theoretical yield. Moreover, the –NH 2 and –COOH groups in Cys form extensive hydrogen bonds with the hydroxyl groups in cellulose, thus decreasing the interaction energy of DES on cellulose. As a result, the cellulose achieves an astonishing 99% retention and maintains a high degree of polymerization of 1160. The obtained high-quality cellulose is further conversed into cellulose nanofibers for strong and transparent films. Furthermore, this study provides new insights into the efficient separation of uncondensed lignin and high-quality cellulose from lignocellulose by a novel DES system.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Cellulose molecular weight of corn stover with cotreatment

Here we present a dataset of cellulose molecular weights from corn stover after a cotreatment process. The corn stover sample was subjected to a combination of mechanical disruption (ball milling) and consolidated bioprocessing (CBP) process using the bacterium C. thermocellum at 60 grams/L solids loadings. Cellulose was isolated from control, no cotreatment control, and cotreatment corn stover. Cellulose was then derivatized using anhydrous pyridine and phenyl isocyanate over 48 h at 70 ºC. The reaction was quenched by anhydrous methanol. Methanol and water mixture (7:3, v/v) was added dropwise to promote precipitation of the cellulose derivative. The solids were collected by filtration and thoroughly washed with the methanol and water mixture followed by water. The cellulose derivative was dried overnight under vacuum at 40°C. The obtained cellulose tricarbanilates was then dissolved in tetrahydrofuran (THF) at 1.0 mg/mL and the solution was filtered through a 0.45 µm PTFE filter and placed in an auto-sampler vial. The molecular weight of cellulose was measured by Agilent GPC SECurity 1200 system equipped with four Waters Styragel columns (i.e., HR0.5, HR2, HR4, and HR6) and a UV detector (270 nm). THF was used as the mobile phase. Data collection and processing was performed by Polymer Standards Service WinGPC Unity software (Build 6807). The molecular weight was calculated relative to the polystyrene calibration curve. The data on molecular weights provided information about the effects of cotreatment on corn stover cellulose molecular changes.

Cellulose, molecular weights, corn stover, cotreat↗

Investigating the Effect of Water on the Mechanical Properties of Cellulose from Multiscale Molecular Dynamics Simulations

Classical molecular dynamics (MD) simulations provide insight into the structure and physicochemical properties of materials with atomic resolution. However, the length and time scales accessible to atomistic MD are orders of magnitude smaller than many relevant processes such as the response of a bulk material to experimentally accessible strain rates, which presents challenges when comparing models to experimental measurements. Bottom-up coarse-graining provides a means for systematically mapping atomistic information to lower resolution models to increase the length and time scales achievable by simulation. Cellulose is an abundant carbohydrate biopolymer with applications to many fields of research, such as materials science and renewable energy, due to its desirable mechanical properties and viability for conversion into biofuel. The effect of moisture content on the Young's modulus of cellulose is of special interest due to its native environment often being in the hydrated secondary plant cell wall and the grinding energy requirements for biomass feedstock preprocessing. The current work investigates the effects of water solvent on the Young's modulus of cellulose calculated from coarse-grained MD mechanical stress simulations. The coarse-grained model was parametrized from atomistic MD calculations of cellulose-cellulose potentials of mean force using umbrella sampling techniques under vacuum and solvated conditions. The Young's moduli of the coarse-grained cellulose assemblies parametrized from cellulose in vacuum or solvated in water were computed via mechanical stress simulations to highlight the importance of capturing solvent interactions for modeling the mechanical behavior of cellulose.

BASIC BIOLOGICAL SCIENCES,RADIATION PROTECTION AND↗

Time-resolved tracking of cellulose biosynthesis and assembly during cell wall regeneration in live Arabidopsis protoplasts

Cellulose, the most abundant polysaccharide on earth composing plant cell walls, is synthesized by coordinated action of multiple enzymes in cellulose synthase complexes embedded within the plasma membrane. Multiple chains of cellulose fibrils form intertwined extracellular matrix networks. It remains largely unknown how newly synthesized cellulose is assembled into an intricate fibril network on cell surfaces. Here, we have established an in vivo time-resolved imaging platform to continuously visualize cellulose biosynthesis and fibril network assembly onArabidopsis thalianaprotoplast surfaces as the primary cell wall regenerates. Our observations provide the basis for a model of cellulose fibril network development in protoplasts driven by an interplay of multiscale dynamics that includes rapid diffusion and coalescence of nascent cellulose fibrils, processive elongation of single fibrils, and cellulose fibrillar network rearrangement during maturation. This study provides fresh insights into the dynamic and mechanistic aspects of cell wall synthesis at the single-cell level.

Science & Technology - Other Topics↗

Insights into the action of phylogenetically diverse microbial expansins on the structure of cellulose microfibrils

Microbial expansins (EXLXs) are non-lytic proteins homologous to plant expansins involved in plant cell wall formation. Due to their non-lytic cell wall loosening properties and potential to disaggregate cellulosic structures, there is considerable interest in exploring the ability of microbial expansins (EXLX) to assist the processing of cellulosic biomass for broader biotechnological applications. Herein, EXLXs with different modular structure and from diverse phylogenetic origin were compared in terms of ability to bind cellulosic, xylosic, and chitinous substrates, to structurally modify cellulosic fibrils, and to boost enzymatic deconstruction of hardwood pulp. Five heterogeneously produced EXLXs (Clavibacter michiganensis; CmiEXLX2, Dickeya aquatica; DaqEXLX1, Xanthomonas sacchari; XsaEXLX1, Nothophytophthora sp.; NspEXLX1 and Phytophthora cactorum; PcaEXLX1) were shown to bind xylan and hardwood pulp at pH 5.5 and CmiEXLX2 (harboring a family-2 carbohydrate-binding module) also bound well to crystalline cellulose. Small-angle X-ray scattering revealed a 20–25% increase in interfibrillar distance between neighboring cellulose microfibrils following treatment with CmiEXLX2, DaqEXLX1, or NspEXLX1. Correspondingly, combining xylanase with CmiEXLX2 and DaqEXLX1 increased product yield from hardwood pulp by ~ 25%, while supplementing the TrAA9A LPMO from Trichoderma reesei with CmiEXLX2, DaqEXLX1, and NspEXLX1 increased total product yield by over 35%. This direct comparison of diverse EXLXs revealed consistent impacts on interfibrillar spacing of cellulose microfibers and performance of carbohydrate-active enzymes predicted to act on fiber surfaces. These findings uncover new possibilities to employ EXLXs in the creation of value-added materials from cellulosic biomass.

09 BIOMASS FUELS↗

Thermobifida fusca Cel6B moves bidirectionally while processively degrading cellulose

Abstract Background Cellulose, an abundant biopolymer, has great potential to be utilized as a renewable fuel feedstock through its enzymatic degradation into soluble sugars followed by sugar fermentation into liquid biofuels. However, crystalline cellulose is highly resistant to hydrolysis, thus industrial-scale production of cellulosic biofuels has been cost-prohibitive to date. Mechanistic studies of enzymes that break down cellulose, called cellulases, are necessary to improve and adapt such biocatalysts for implementation in biofuel production processes. Thermobifida fusca Cel6B ( Tf Cel6B) is a promising candidate for industrial use due to its thermostability and insensitivity to pH changes. However, mechanistic studies probing Tf Cel6B hydrolytic activity have been limited to ensemble-scale measurements. Results We utilized optical tweezers to perform single-molecule, nanometer-scale measurements of enzyme displacement during cellulose hydrolysis by Tf Cel6B. Records featured forward motility on the order of 0.17 nm s −1 interrupted by backward motions and long pauses. Processive run lengths were on the order of 5 nm in both forward and backward directions. Motility records also showed rapid bidirectional displacements greater than 5 nm. Single-enzyme velocity and bulk ensemble activity were assayed on multiple crystalline cellulose allomorphs revealing that the degree of crystallinity and hydrogen bonding have disparate effects on the single-molecule level compared to the bulk scale. Additionally, we isolated and monitored the catalytic domain of Tf Cel6B and observed a reduction in velocity compared to the full-length enzyme that includes the carbohydrate-binding module. Applied force has little impact on enzyme velocity yet it readily facilitates dissociation from cellulose. Preliminary measurements at elevated temperatures indicated enzyme velocity strongly increases with temperature. Conclusions The unexpected motility patterns of Tf Cel6B are likely due to previously unknown mechanisms of processive cellulase motility implicating irregularities in cellulose substrate ultrastructure. While Tf Cel6B is processive, it has low motility at room temperature. Factors that most dramatically impact enzyme velocity are temperature and the presence of its native carbohydrate-binding module and linker. In contrast, substrate ultrastructure and applied force did not greatly impact velocity. These findings motivate further study of Tf Cel6B for its engineering and potential implementation in industrial processes.

Johnson, Madeline M. (ORCID:000000029518293X)↗