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At least 469 records · Page 26

Final Technical Report

The Department of Energy is interested in technologies that support the sustainable production of fuels, chemicals, and other bioproducts from plant biomass, to offset the nation’s reliance on fossil resources. The plant cell wall of energy crops provides the largest reservoir of raw materials for bioproducts. However, the widespread use of plant cell walls is hampered by their complexity and resistance to breakdown. To improve the productivity and cost-effectiveness of using energy crops to generate bioproducts, the fundamental problem of deconstructing plant cell walls must be addressed. This project developed and evaluated an innovative genetic modification technology to produce strategically designed enzymes that specifically accumulate in the plant cell wall. The resulting enzyme-engineered energy crops are expected to grow normally under natural conditions but break down more quickly and easily under high temperature during the production of biobased products. As such, this plant cell wall targeting enzyme engineering effort will reduce the cost of plant cell wall deconstruction and ultimately improve the economics of bioproducts. The overall objective of this project is to develop and evaluate the in-planta enzyme engineering technology to reduce lignocellulose deconstruction cost. The concept was first validated using tobacco plant, a model plant system that is typically used in lab testing for initial concept validation. Then the enzyme optimization was validated using switchgrass, the energy crop to be used to produce bioproducts. There are three specific objectives in this Phase I project: (1) validate the enzyme optimization concept using tobacco plant, a model plant system. (2) validate the enzyme optimization concept using switchgrass. (3) techno-economic analysis (TEA) for further scale-up application. By the end of this project, in-planta enzyme engineering was validated in both tobacco and switchgrass plants, with improved enzyme activity and saccharification efficiency. The in-planta enzyme engineering in Tabacco didn’t have a significant impact on plant growth and development. Transgenic tobacco plants with in-planta cellulose degrading enzymes showed higher biomass digestibility than wild type. Gene construction and transformation in switchgrass was much longer than expected, which delayed the research progress. Besides, in-planta engineering of lignin degrading enzyme is more challenging than cellulose degrading enzyme, in terms of expression detection. Expression of lignin degrading enzyme and cellulose degrading enzyme improved biomass yield and saccharification efficiency of switchgrass, respectively. It is promising to express both genes in switchgrass for optimized overall performance. According to the results of TEA, switchgrass biomass production cost is mainly attributed to by fertility and harvesting. Biomass production profit can increase up to 10-fold depending on biomass price. The PHA production profit is also sensitive to the biomass price. The proposed technology could potentially reduce the biomass deconstruction cost from 33% to 9% of PHA revenue, making the biomass-based PHA competitive to petroleum-based polymers even in case of relatively high biomass price of biomass. Therefore, cultivation of the genetically engineered self-deconstruction switchgrass for Polyhydroxyalkanoate (PHA) production could benefit switchgrass grower and PHA producer with attractive profits for both sectors. This new enzyme optimization approach will be beneficial for bioindustries that use energy crops as feedstocks. It will improve the economic viability of converting energy crops to renewable products that support a sustainable society and helps address the Nation’s long-term strategic needs for renewable products and reduction of reliance on fossil resources.

42 ENGINEERING↗

Biohydrogen Generation from Un-Pretreated Spent Coffee Grounds with Clostridium Thermocellum

Coffee is among the world's most widely consumed beverages, generating 18 million wet tonnes of waste spent coffee grounds annually. Disposal of SCGs is complicated by their high moisture content, their recalcitrant chemical composition, and the presence of caffeine and other bioactive compounds. Dark fermentation is a promising technology for conversion of waste cellulosic biomass into renewable hydrogen, carbon dioxide, and volatile fatty acids. Typical SCGs have high moisture content, are pre-milled to fine particle sizes, and contain 8%-19% cellulose and up to 40% hemicellulose, making them an attractive feedstock for dark fermentation. In this study, we investigate biohydrogen production from unpretreated SCGs using Clostridium thermocellum strain KJC19-9, a cellulolytic bacterium engineered to co-utilize xylose, specifically examining the inhibitory effects of residual caffeine and high solids loadings on microbial growth and hydrogen production efficiency. While SCGs were initially resistant to both cell growth and biohydrogen production, a strategy to co-ferment with low concentrations of cellobiose dramatically enhanced process performance, reducing lag phase duration and enabling cellulosome production for efficient cellulose hydrolysis. This co-substrate approach generated up to 944 mL hydrogen per L reaction from 50 g/L of spent coffee grounds over 120 h (1.34 mol H2/mol carbohydrate), validating SCGs as a promising dark fermentation feedstock and underscoring the value of mixed substrate approaches in consolidated bioprocessing.

08 HYDROGEN↗

Nanostructural Analysis of Enzymatic and Non-enzymatic Brown Rot Fungal Deconstruction of the Lignocellulose Cell Wall†

Brown rot (BR) decay mechanisms employ carbohydrate-active enzymes (CAZymes) as well as a unique non-enzymatic chelator-mediated Fenton (CMF) chemistry to deconstruct lignocellulosic materials. Unlike white rot fungi, BR fungi lack peroxidases for lignin deconstruction, and also lack some endoglucanase/cellobiohydrolase activities. The role that the CMF mechanism plays in “opening up” the wood cell wall structure in advance of enzymatic action, and any interaction between CMF constituents and the selective CAZyme suite that BRs possess, is still unclear. Expression patterns for CMF redox metabolites and lytic polysaccharide monooxygenase (LPMO–AA9 family) genes showed that some LPMO isozymes were upregulated with genes associated with CMF at early stages of brown rot by Gloeophyllum trabeum. In the structural studies, wood decayed by the G. trabeum was compared to CMF-treated wood, or CMF-treated wood followed by treatment with either the early-upregulated LPMO or a commercial CAZyme cocktail. Structural modification of decayed/treated wood was characterized using small angle neutron scattering. CMF treatment produced neutron scattering patterns similar to that of the BR decay indicating that both systems enlarged the nanopore structure of wood cell walls to permit enzyme access. Enzymatic deconstruction of cellulose or lignin in raw wood samples was not achieved via CAZyme cocktail or LPMO enzyme action alone. CMF treatment resulted in depolymerization of crystalline cellulose as attack progressed from the outer regions of individual crystallites. Multiple pulses of CMF treatment on raw wood showed a progressive increase in the spacing between the cellulose elementary fibrils (EFs), indicating the CMF eroded the matrix outside the EF bundles, leading to less tightly packed EFs. Peracetic acid delignification treatment enhanced subsequent CMF treatment effects, and allowed both enzyme systems to further increase spacing of the EFs. Moreover, even after a single pulse of CMF treatment, both enzymes were apparently able to penetrate the cell wall to further increase EF spacing. The data suggest the potential for the early-upregulated LPMO enzyme to work in association with CMF chemistry, suggesting that G. trabeum may have adopted mechanisms to integrate non-enzymatic and enzymatic chemistries together during early stages of brown rot decay.

59 BASIC BIOLOGICAL SCIENCES↗

A Swollenin From Talaromyces leycettanus JCM12802 Enhances Cellulase Hydrolysis Toward Various Substrates

Swollenins exist within some fungal species and are candidate accessory proteins for the biodegradation of cellulosic substrates. Here, we describe the identification of a swollenin gene, Tlswo , in Talaromyces leycettanus JCM12802. Tlswo was successfully expressed in both Trichoderma reesei and Pichia pastoris . Assay results indicate that Tl SWO is capable of releasing reducing sugars from lichenan, barley β-glucan, carboxymethyl cellulose sodium (CMC-Na) and laminarin. The specific activity of Tl SWO toward lichenan, barley β-glucan, carboxymethyl cellulose sodium (CMC-Na) and laminarin is 9.0 ± 0.100, 8.9 ± 0.100, 2.3 ± 0.002 and 0.79 ± 0.002 U/mg, respectively. Additionally, Tl SWO had disruptive activity on Avicel and a synergistic effect with cellobiohydrolases, increasing the activity on pretreated corn stover by up to 72.2%. The functional diversity of Tl SWO broadens its applicability in experimental settings, and indicating that it may be a promising candidate for future industrial applications.

59 BASIC BIOLOGICAL SCIENCES↗

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

The present invention relates to isolated polypeptides having cellobiohydrolase activity, catalytic domains, and cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains, and cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains, or cellulose binding domains.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Polypeptides having endoglucanase activity and polynucleotides encoding same

The present invention relates to isolated polypeptides having endoglucanase activity, catalytic domains, cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

Spodsberg, Nikolaj↗

Chemical structure–property relationships in nanocelluloses

Abstract Nanocelluloses, nanoscale cellulosic particles or fibrils, are an appealing class of nanomaterials with enormous potential for sustainable development. They have great promise to target some of the 17 Sustainable Development Goals outlined by the United Nations. Nanocelluloses are derived from the most abundant biopolymer in the world, cellulose, which have multiple hydroxyl groups on their surface, enabling a vast range of chemical modifications. The interplay between the chemical structure and physicochemical properties of nanocelluloses is crucial to engineering the next generation of green, functional nanomaterials. In this review paper, we provide a comprehensive account of the structure–property relationships in nanocelluloses, which may establish the basis for the design of precision bio‐based materials. Chemical modifications of nanocelluloses, including hydrolysis, oxidation, esterification, amidation, and polymer grafting, provide an array of chemical and physical properties that open opportunities in a diverse spectrum of applications. We review how chemical modifications affect the physicochemical properties of nanocelluloses, such as thermal stability, hydrophobicity, and dispersibility in nonpolar media. Understanding nanocellulose chemical structure–property relationships is integral to the development of sustainable material platforms based on the most renewable biopolymer on earth.

59 BASIC BIOLOGICAL SCIENCES↗

Carbon source-induced changes in the transcriptional landscape of Novacetimonas hansenii

Novacetimonas hansenii is a model organism for bacterial cellulose (BC) production, a biopolymer with broad industrial applications. To better understand its carbon source-dependent metabolism, we conducted the first time-resolved transcriptomic analysis of N. hansenii ATCC 23769 grown on five carbon sources (glucose, fructose, mannitol, galactose, and sucrose) across five time points (24–120h). Glucose, fructose, and mannitol supported growth and efficient BC production, whereas galactose and sucrose resulted in poor yields, likely due to limited transport and enzymatic capacity. Solid-state NMR revealed that cellulose crystallinity and polymorphism were carbon source-dependent, with reduced bcsD expression correlating with lower crystallinity. Transcriptomic profiling showed distinct metabolic responses. Cultures containing glucose and mannitol activated core metabolic pathways, fructose induced a delayed adaptive shift, and galactose and sucrose triggered stress-related gene expression. These results highlight the metabolic plasticity of N. hansenii and offer insights for enhancing BC production through carbon source optomization.

Bacterial cellulose↗

Rapid High-Resolution Analysis of Polysaccharide-Lignin Interactions in Secondary Plant Cell Walls Using Proton-Detected Solid-State NMR

The plant secondary cell wall, a complex matrix composed of cellulose, hemicellulose, and lignin, is crucial for the mechanical strength and water-proofing properties of plant tissues, and serves as a primary source of biomass for biorenewable energy and biomaterials. Structural analysis of these polymers and their interactions within the secondary cell wall has been heavily relying on 13 C-based solid-state NMR techniques. In this study, we explore the application of 1 H-detected solid-state NMR techniques for rapid, high-resolution structural characterization of polysaccharides and lignin, demonstrated on the stems of hardwood eucalyptus. We explored the use of synthesized 2D spectra to resolve central 1 H resonances and the combined application of 3D hCCH and hCHH experiments for complete resonance assignment and unambiguous identification of lignin-carbohydrate interactions. Our findings emphasize the central role of acetylated three-fold xylan conformers, rather than two-fold, in stabilizing the carbohydrate-lignin interface, with glucuronic acid sidechains in eucalyptus glucuronoxylan colocalizing with lignin, revised cellulose-lignin interactions involving uncoated microfibril surfaces, and pectin-lignin interactions indicative of early-stage lignification. These results present a novel approach for rapid structural analysis of lignocellulosic biomaterials without the need for solubilization or extraction.

09 BIOMASS FUELS↗

High-Biomass Sorghums as a Feedstock for Renewable Fuels and Chemicals

High-biomass sorghums are intended for use in biorefineries that convert vegetative biomass into renewable fuels and chemicals. The majority of plant biomass consists of cell walls, a complex matrix of cellulose, hemicellulosic polysaccharides and lignin. In the biorefinery, the biomass is ground and then subjected to a thermo-chemical pretreatment (elevated temperature, high pressure, chemical catalysts) that disrupts the cell wall matrix and solubilizes some of the cell wall polymers, followed by enzymatic saccharification of the cellulose, which produces D-glucose. The glucose and sometimes also the xylose derived from the hydrolysis of the hemicellulosic polysaccharides, are subsequently converted to fuels or other useful chemicals by microbial biocatalysts. The genetic improvement of high-biomass sorghums has as its ultimate goals to maximize the yield of fermentable sugars on a per-hectare basis, to minimize the inputs of fertilizer, irrigation, fungicides and pesticides and to reduce the environmental footprint. Breeding strategies thus need to target biomass yield, biomass composition, maturity, pest and disease resistance and nutrient use efficiency. Furthermore, this chapter reviews the genetic basis of these traits and their potential application in breeding programs.

60 APPLIED LIFE SCIENCES↗

Multi-catalytic active site biochar-based catalysts for glucose isomerized to fructose: Experiments and density functional theory study

In this study, this work provides an innovative method for preparing different isomerization catalysts by impregnating different proportions of MgCl 2 and AlCl 3 and combining different K compounds on cellulose-derived biochar, followed by pyrolysis. Results show MgO and Al(OH) 3 existing in 4 Mg- 1 Al-C catalyst can obtain better catalytic effect on glucose isomerization than the singe of Al presenting in 0 Mg- 1 Al-C catalyst. Moreover, the synergism effects of the multi-catalytic active sites such as β-, γ -Al(OH) 3 , KCl, MgO, and K 4 H 2 (CO 3 ) 3 in Mg-Al-KHCO 3 -C catalyst can further lead to an increase in glucose isomerization, compared to the 4 Mg- 1 Al-C catalyst. The X-ray diffraction results present that the value of O/Al in Mg-Al-KHCO 3 -C catalyst is as high as 13.38, which provides many unsaturated acidic catalysis sites and benefits the glucose isomerization. Simultaneously, the TPD results reveal that the main active sites (MgO, Al(OH) 3 , and K 4 H 2 (CO 3 ) 3 ) in Mg-Al-KHCO 3 -C catalyst can provide weakly acidic and basic sites and avoid strongly acidic and basic sites to excessively attack the glucose. Based on the DFT analysis, the results indicate that the MgO has a great effect on the ring-opening reaction to form acyclic glucose, while Al(OH) 3+ has a great effect on promoting acyclic glucose hydrogen transfer isomerized to form fructose. Compared to other carbon-based metal catalysts, the prepared Mg-Al-KHCO 3 -C has excellent catalytic performance, which gives a higher fructose yield (38.7%) and selectivity (87.72%) and glucose conversion (44.12%) at 100 °C in 30 min. In this study, we develop a highly efficient Mg-Al-K-biochar catalyst for glucose isomerization and provide an efficient method for cellulose valorization.

36 MATERIALS SCIENCE↗

Hot-melt extrudability of amorphous solid dispersions of flubendazole-copovidone: An exploratory study of the effect of drug loading and the balance of adjuvants on extrudability and dissolution

The FDA-approved anthelmintic flubendazole has shown potential to be repositioned to treat cancer and dry macular degeneration; however, its poor water solubility limits its use. Amorphous solid dispersions may overcome this challenge, but the balance of excipients may impact the preparation method and drug release. Here, we evaluate the influence of adjuvants and drug loading on the development of an amorphous solid dispersion of flubendazole-copovidone by hot-melt extrusion. The drug, copovidone, and adjuvants (magnesium stearate and hydroxypropyl cellulose) mixtures were statistically designed, and the process was performed in a twin-screw extruder. The study showed that flubendazole and copovidone mixtures were highly extrudable, except when drug loading was high (>40%). Furthermore, magnesium stearate positively impacted the extrusion and was more effective than hydroxypropyl cellulose. The extruded materials were evaluated by modulated differential scanning calorimetry and X-ray powder diffraction, obtaining positive amorphization and physical stability results. Pair distribution function analysis indicated the presence of drug rich domains with medium-range order structure and no evidence of polymer-drug interaction. All extrudates presented faster dissolution (HCl, pH 1.2) than pure flubendazole, and both adjuvants had a notable influence on the dissolution rate. In conclusion, hot-melt extrusion may be a viable option to obtain stable flubendazole: copovidone amorphous dispersions.

60 APPLIED LIFE SCIENCES↗

The physiochemical alteration of flax fibers structuring components after different scouring and bleaching treatments

Scouring and bleaching are essential steps in flax yarn production; however, in many cases, these treatments have relatively low selectivity on non-cellulosic polysaccharides (NCPs) and lignin. Herein, the specific chemical properties of flax fibers through a variety of scouring and/or bleaching procedures are reviewed to understand their correlations with the mechanical and morphological characteristics. The hydrolysis behaviors of hemicellulose and lignin are different from those of cellulose during chlorine-alkali-oxygen (Cl-AO), enzyme-alkali-oxygen (EAO), and alkali-oxygen (AO) treatments, while the structural hemicellulose polysaccharides fractions were not affected by acid pretreatment followed by alkali scouring (AA) treatment. It was found that the unbleached AA treatment improved scouring efficiency to the greatest extent by decreasing 73.3 % of hemicellulose content and 59.8 % of lignin content compared to the untreated flax fibers and obtained the highest tensile strength (6.58 cN/dtex) and elongation (3.73 %). Delignification was more crucial on fiber tenacity and fineness than hemicellulose removal (R 2 = 0.71, 0.79 vs R 2 = 0.46, 0.61). Within the hemicellulose fractions, a corresponding beneficial relationship between the xylan (XYL) composition and flax fiber properties is presented. The correlation of GM and GGM contents in hemicellulose with flax fiber length was significant with R 2 value 0.97 and 0.99, respectively. This work provides insight into the hydrolysis behavior of hemicellulose and a basis for developing scouring and/or bleaching models that can predict fiber properties.

60 APPLIED LIFE SCIENCES↗

Carbon-Negative Biofuel Production

Achievement of the 1.5 °C limit for global temperature increase relies on the large-scale deployment of carbon dioxide removal (CDR) technologies. In this article, we explore two CDR technologies: soil carbon sequestration (SCS), and carbon capture and storage (CCS) integrated with cellulosic biofuel production. These CDR technologies are applied as part of decentralized biorefinery systems processing corn stover and unfertilized switchgrass grown in riparian zones in the Midwestern United States. Cover crops grown on corn-producing lands are chosen from the SCS approach, and biogenic CO 2 in biorefineries is captured, transported by pipeline, and injected into saline aquifers. The decentralized biorefinery system using SCS, CCS, or both can produce carbon-negative cellulosic biofuels (≤-22.2 gCO 2 MJ –1 ). Meanwhile, biofuel selling prices increase by 15–45% due to CDR costs. Economic incentives (e.g., cover crop incentives and/or a CO 2 tax credit) can mitigate price increases caused by CDR technologies. Lastly, a combination of different CDR technologies in decentralized biorefinery systems is the most efficient method for greenhouse gas (GHG) mitigation, and its total GHG mitigation potential in the Midwest is 0.16 GtCO 2 year –1 .

09 BIOMASS FUELS↗

Water Availability for Biorefineries in the Contiguous United States and the Implications for Bioenergy Production Distribution

Renewable biofuel production depends on many factors, including feedstock availability, refinery and shipment infrastructure, and in particular, water availability. This study assesses water requirement and availability for mainstream biorefinery technologies in the contiguous United States (CONUS). The assessment is conducted in newly defined spatial units, namely, biorefinery planning boundaries, considering feedstock availability, transportation cost, and refinery capacity requirement for cost-effectiveness. The results suggest that the total biorefinery water use in the CONUS by 2030 will be low compared to the total water availability. However, biorefinery water requirements can aggravate the water stress situation in many regions, including the Great Plains, California Central Valley, and the upper Columbia-Snake River basin in Washington. Bioenergy productions in these regions can be largely constrained by water. Here, it is projected that biofuel production will concentrate in Northern Plains, Lake States, and Corn Belt regions, which contribute 94.4% of the conventional, 86.1% of biodiesel, and 54.8% of cellulosic biofuel production mandated by the renewable fuel standard. If biorefineries are constrained to use less than 10% of the locally available water, up to 7% of planned cellulosic biofuel production will be affected. Findings from this study can aid the sustainable planning of national bioenergy production.

54 ENVIRONMENTAL SCIENCES↗

Cost and Carbon Intensity Implications of Coprocessing Sustainable Aviation Fuel at Petroleum Refineries

Sustainable aviation fuel (SAF) will play a critical role in decarbonizing the aviation industry. Among SAF production pathways, alcohol-to-jet (ATJ) stands out for its scalability, supported by abundant feedstock availability and a well-established bioethanol industry. However, significant reductions in SAF carbon intensity (CI) require the use of future feedstocks (e.g., cellulosic) whose adoption is hindered by high capital costs for feedstock processing and ethanol upgrading. Here, we evaluate the financial viability and environmental implications of integrating an ATJ SAF biorefinery within a petroleum refinery, utilizing miscanthus and switchgrass as example feedstocks. Three scenarios are evaluated: standalone (benchmark), colocated, and repurposing (coprocessing SAF within the petroleum refinery). Results show repurposing reduces baseline capital costs by 36% and SAF minimum selling price (MSP) by 12% to 8.14 USD·gal −1 ; the superior performance of repurposing is consistent across both feedstocks. Integration has a limited effect on SAF CI, which remains stable across scenarios, whereas using cellulosic feedstocks reduces CI by over 70% relative to corn, with baseline values of 17.01 g CO 2 e· MJ −1 for miscanthus and 12.23 g CO 2 e·MJ −1 for switchgrass. Global sensitivity analysis reveals MSP declines with greater coprocessing levels.

09 BIOMASS FUELS↗

Cross-Sections of Nanocellulose from Wood Analyzed by Quantized Polydispersity of Elementary Microfibrils

Bio-based nanocellulose has been shown to possess impressive mechanical properties and simplicity for chemical modifications. The chemical properties are largely influenced by the surface area and functionality of the nanoscale materials. However, finding the typical cross-sections of nanocellulose, such as cellulose nanofibers (CNFs), has been a long-standing puzzle, where subtle changes in extraction methods seem to yield different shapes and dimensions. Here, we extracted CNFs from wood with two different oxidation methods and variations in degree of oxidation and high-pressure homogenization. Additionally, the cross-sections of CNFs were characterized by small-angle X-ray scattering and wide-angle X-ray diffraction in dispersed and freeze-dried states, respectively, where the results were analyzed by assuming that the cross-sectional distribution was quantized with an 18-chain elementary microfibril, the building block of the cell wall. We find that the results agree well with a pseudosquare unit having a size of about 2.4 nm regardless of sample, while the aggregate level strongly depends on the extraction conditions. Furthermore, we find that aggregates have a preferred cohesion of phase boundaries parallel to the (110)-plane of the cellulose fibril, leading to a ribbon shape on average.

36 MATERIALS SCIENCE↗

Residual Strain and Nanostructural Effects during Drying of Nanocellulose/Clay Nanosheet Hybrids: Synchrotron X-ray Scattering Results

Cellulose nanofibrils (CNF) with 2D silicate nanoplatelet reinforcement readily form multifunctional composites by vacuum-assisted self-assembly from hydrocolloidal mixtures. The final nanostructure is formed during drying. The crystalline nature of CNF and montmorillonite (MTM) made it possible to use synchrotron X-ray scattering (WAXS, SAXS) to monitor structural development during drying from water and from ethanol. Nanostructural changes in the CNF and MTM crystals were investigated. Changes in the out-of-plane orientation of CNF and MTM were determined. Residual drying strains previously predicted from theory were confirmed in both cellulose and MTM platelets due to capillary forces. The formation of tactoid platelet stacks could be followed. We propose that after filtration, the constituent nanoparticles in the swollen, solid gel already have a “fixed” location, although self-assembly and ordering processes take place during drying.

36 MATERIALS SCIENCE↗