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At least 343 records · Page 19
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Ethanol from Agriculture for Arkansas and America

The purpose of this project was to develop technology that would facilitate production of sugars from agricultural residues to enable biofuels and biobased product manufacturing. Our primary technology is to use genetic engineering to put bacterial and fungal cellulase genes into corn kernels, using the grain as the production system for the enzymes. At the beginning of this DoE funded program, we were producing two cellulases—E1 endocellulase from a bacterium found in a hot spring at Yellowstone National Park, and CBH I exocellulase from a wood rot fungus. Our team developed several new regulatory sequences (promoters) that increased enzyme protein accumulation in two kernel compartments (embryo and endosperm). We were also able to capitalize on the diverse genetics of corn to increase protein accumulation. High oil germplasm in particular was instrumental in this increase. A second task in the program was to produce enzymes and proteins that enhanced the activity of the E1 and CBH I enzymes. Our team produced CBH II, from the same wood rot fungus at a level that enabled highly enhanced deconstruction activity of E1 and CBH I in a synergistic manner. We analyzed an additional protein, expansin from cucumber that was expressed in the maize grain expression system. This protein had been previously shown to enhance cellulase activity (D. Cosgrove, Penn State University), and required a large-scale production platform. Our team showed that the corn production system allows industrial amounts of active expansin to be harvested from the grain. One of the challenges of any new production system is to maximize recovery of active ingredient from the raw materials at a cost compatible with its final use. Our team showed that low pH extraction of grain solubilized the enzymes without contamination of native corn protein and active product could be concentrated through ultrafiltration. The final outcomes of this project were the following: 3 cellulase enzymes and the synergistic protein expansin produced at high levels in corn grain, new promoters and combinations of promoters to enhance protein accumulation in grain, application of unique germplasm pools to enhance protein accumulation, and highly efficient processing enabling cost-effective production of cellulases that are highly active in biomass deconstruction.

09 BIOMASS FUELS↗
DOE OSTI2024

IX Cell Wall Research Conference 2022 (IXCWRC): Cell wall research for new fundamental discoveries and applications in plant biology

This application is for securing funds to offset the registration fees and lodging costs for US-based early career investigators (ERCs) to participate to the IX Cell Wall Research Conference 2022 (IXCWRC 2022): Cell wall research for new fundamental discoveries and applications in plant biology. The conference will be held in East Lansing, Michigan on June 13-17, 2022. The conference will focus on the plant cell wall. Plant cells are encapsulated by the cell wall, a dynamic structure that protects them, enables them to interface with the external environment and supports the growth of the entire organism. There is a growing interest in understanding the complex processes necessary to build and deconstruct the plant cell wall. A better understanding of how the cell wall is synthesized during plant growth and in response to the environment, and how it is deconstructed for a broad variety of applications can inform and improve biotechnological processes for increasing biomass yield and improving industrial processes for the recovery of valuable, sustainable polymers. Acquiring such knowledge requires the exchange of new ideas and results as well as the development and application of new technologies. The IXCWRC 2022 aims to reach this goal by providing an opportunity for an open forum to discuss new ideas and theories and develop collaborations for scientists across the globe in a diverse and supportive environment. A distinction of the IXCWRC 2022 from all the preceding plant cell wall conferences will be its focus on the training and inclusion of the next generation of scientists, the ERCs. The overall goal of this application is to broaden the participation of ERCs with a focus on underrepresented communities. The specific goal of this application is to secure support for registration and lodging for US-based ERCs to attend the conference. Priority will be given to ERCs from underrepresented communities in science. We expect that support from DOE will allow the development of an exciting and innovative program to discuss the latest scientific advances in cell wall biology research, advance the next generation of plant cell wall researchers and foster the foundations of an inclusive community in science.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Feedstock/Pretreatment Screening for Bioconversion of Sugars and Lignin Residues

This project will conduct biomass deconstruction (pretreatment and enzymatic saccharification) on two representative biomass feedstocks and four different pretreatment processes, including three high temperature steam/chemical pretreatments and one low temperature chemical/mechanical pretreatment. A third biomass feedstock will undergo biomass deconstruction with three different pretreatment processes, including two high temperature chemical pretreatments and one low temperature chemical/mechanical pretreatment. Several pretreatment conditions will be performed in a screening study using NLR pilot-scale pretreatment equipment to generate a range of pretreated biomass substrates. A selected number of these substrates will be chosen for enzymatic saccharification evaluation, based on standard compositional analysis of the pretreated substrates as a primary indicator of pretreatment efficacy. Resulting enzymatic hydrolysis slurries will be analyzed to determine overall biomass sugar yields. Additional compositional analysis will be performed to determine oligomeric sugar composition and structure, to analyze structural characteristics of solids fractions on native, pretreated, and enzymatically saccharified biomass residues for one of the biomass feedstocks, corn stover. The enzymatically saccharified materials will undergo 2,3-butanediol fermentation in a shaker flask as bench scale. Using relevant process performance data collected in these various conversion steps, technoeconomic analysis activities will be performed to compare the economic potential of the various biomass feedstock and pretreatment processes and to identify key economic drivers and sustainability metrics.

09 BIOMASS FUELS↗
DOE OSTI2026

Final Technical Report for DE-SC0022206

This project developed foundational genetic, genomic, and epigenetic tools for anaerobic fungi (Neocallimastigomycota), a group of microorganisms with exceptional natural abilities to deconstruct lignocellulosic biomass. Efficient biomass deconstruction remains a major barrier to economical production of renewable fuels, chemicals, and materials from agricultural and forestry residues. The project sought to enable mechanistic studies and future engineering of anaerobic fungi by improving genomic resources, establishing methods for gene expression, and investigating epigenetic regulation of biomass-degrading pathways. Major accomplishments included generation of the first chromosome-scale genome assemblies for multiple anaerobic fungal species, providing publicly available genomic resources that support both engineering and fundamental biological research. The project established the first reproducible system for heterologous gene expression in anaerobic fungi and identified genomic features and mobile genetic elements that may support future development of stable transformation technologies. In parallel, the project demonstrated direct conversion of untreated lignocellulosic biomass into fuels and specialty chemicals through a fungal-yeast bioprocess and identified anaerobic fungal enzymes with utility for metabolic engineering. The research also revealed that epigenetic regulation plays an important role in controlling fungal gene expression and enzyme production, identifying potential strategies for enhancing biomass degradation. Collectively, this work established anaerobic fungi as a tractable emerging platform for bioenergy and biomanufacturing research, generated valuable public resources, trained the next generation of researchers, and advanced DOE-BER goals related to predictive biology, sustainable bioprocessing, and the circular bioeconomy.

Solomon, Kevin [University of Delaware] (ORCID:000↗
DOE OSTI2024

Biofilm mitigation in hybrid chemical-biological upcycling of waste polymers

Accumulation of plastic waste in the environment is a serious global issue. To deal with this, there is a need for improved and more efficient methods for plastic waste recycling. One approach is to depolymerize plastic using pyrolysis or chemical deconstruction followed by microbial-upcycling of the monomers into more valuable products. Microbial consortia may be able to increase stability in response to process perturbations and adapt to diverse carbon sources, but may be more likely to form biofilms that foul process equipment, increasing the challenge of harvesting the cell biomass. To better understand the relationship between bioprocess conditions, biofilm formation, and ecology within the bioreactor, in this study a previously-enriched microbial consortium (LS1_Calumet) was grown on (1) ammonium hydroxide-depolymerized polyethylene terephthalate (PET) monomers and (2) the pyrolysis products of polyethylene (PE) and polypropylene (PP). Bioreactor temperature, pH, agitation speed, and aeration were varied to determine the conditions that led to the highest production of planktonic biomass and minimal formation of biofilm. The community makeup and diversity in the planktonic and biofilm states were evaluated using 16S rRNA gene amplicon sequencing. Results showed that there was very little microbial growth on the liquid product from pyrolysis under all fermentation conditions. When grown on the chemically-deconstructed PET the highest cell density (0.69 g/L) with minimal biofilm formation was produced at 30°C, pH 7, 100 rpm agitation, and 10 sL/hr airflow. Results from 16S rRNAsequencing showed that the planktonic phase had higher observed diversity than the biofilm, and that Rhodococcus, Paracoccus, and Chelatococcus were the most abundant genera for all process conditions. Biofilm formation by Rhodococcus sp. And Paracoccus sp. Isolates was typically lower than the full microbial community and varied based on the carbon source. Ultimately, the results indicate that biofilm formation within the bioreactor can be significantly reduced by optimizing process conditions and using pure cultures or a less diverse community, while maintaining high biomass productivity. The results of this study provide insight into methods for upcycling plastic waste and how process conditions can be used to control the formation of biofilm in bioreactors.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Factors that influence the activity of biomass-degrading enzymes in the presence of ionic liquids—a review

Ionic liquids (ILs) are seen as a more sustainable alternative to volatile organic solvents that are accelerating innovations in many industries such as energy storage, separations, and bioprocessing. The ability to effectively deconstruct lignocellulosic biomass is a significant hurdle in the biorefining/bioprocessing industry and presents limitations towards the commercial production of bioproducts (such as biofuels, biomaterials, etc. ). Certain ILs have been shown to promote effective lignin removal, cellulose recovery, and sugar yields from various biomass feedstocks such as corn stover, wheat straw, sugarcane bagasse, sorghum, switchgrass, miscanthus, poplar, pine, eucalyptus, and certain mixtures of municipal solid waste. However, these improvements are often counteracted by the limited biocompatibility of ILs, which results in an IL-induced reduction in enzyme activity and stability—an important downstream step in the conversion of biomass to biofuels/bioproducts. As a result, significant efforts have been made to discover and engineer compatible enzyme-IL systems and to improve our understanding on the effect that these ILs have on these systems. This review seeks to examine the impact of ionic liquids on enzymes involved in lignocellulosic biomass deconstruction, with a specific focus on their relevance in the context of pretreatment. Beyond presenting an overview of the ionic liquid pretreatment landscape, we outline the main factors that influence enzyme activity and stability in the presence of ILs This data is consolidated and analyzed to apply this body of knowledge towards new innovations that could lead to improvements in the processing of biomass to biofuels and bioproducts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2013

Evidence supporting dissimilatory and assimilatory lignin degradation in Enterobacter lignolyticus SCF1

Lignocellulosic biofuels are promising as sustainable alternative fuels, but lignin inhibits access of enzymes to cellulose, and by-products of lignin degradation can be toxic to cells. The fast growth, high efficiency and specificity of enzymes employed in the anaerobic litter deconstruction carried out by tropical soil bacteria make these organisms useful templates for improving biofuel production. The facultative anaerobe Enterobacter lignolyticus SCF1 was initially cultivated from Cloud Forest soils in the Luquillo Experimental Forest in Puerto Rico, based on anaerobic growth on lignin as sole carbon source. The source of the isolate was tropical forest soils that decompose litter rapidly with low and fluctuating redox potentials, where bacteria using oxygen-independent enzymes likely play an important role in decomposition. We have used transcriptomics and proteomics to examine the observed increased growth of SCF1 grown on media amended with lignin compared to unamended growth. Proteomics suggested accelerated xylose uptake and metabolism under lignin-amended growth, with up-regulation of proteins involved in lignin degradation via the 4-hydroxyphenylacetate degradation pathway, catalase/peroxidase enzymes, and the glutathione biosynthesis and glutathione S-transferase (GST) proteins. We also observed increased production of NADH-quinone oxidoreductase, other electron transport chain proteins, and ATP synthase and ATP-binding cassette (ABC) transporters. This suggested the use of lignin as terminal electron acceptor. We detected significant lignin degradation over time by absorbance, and also used metabolomics to demonstrate moderately significant decreased xylose concentrations as well as increased metabolic products acetate and formate in stationary phase in lignin-amended compared to unamended growth conditions. Our data show the advantages of a multi-omics approach toward providing insights as to how lignin may be used in nature by microorganisms coping with poor carbon availability.

4-hydroxyphenylacetate degradation pathway↗
DOE OSTI2022

Engineering Curcumin Biosynthesis in Poplar Affects Lignification and Biomass Yield

Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars mainly due to the presence of lignin. By engineering plants to partially replace traditional lignin monomers with alternative ones, lignin degradability and extractability can be enhanced. Previously, the alternative monomer curcumin has been successfully produced and incorporated into lignified cell walls of Arabidopsis by the heterologous expression of DIKETIDE-CoA SYNTHASE (DCS) and CURCUMIN SYNTHASE2 (CURS2). The resulting transgenic plants did not suffer from yield penalties and had an increased saccharification yield after alkaline pretreatment. Here, we translated this strategy into the bio-energy crop poplar. Via the heterologous expression of DCS and CURS2 under the control of the secondary cell wall CELLULOSE SYNTHASE A8-B promoter (ProCesA8-B), curcumin was also produced and incorporated into the lignified cell walls of poplar. ProCesA8-B:DCS_CURS2 transgenic poplars, however, suffered from shoot-tip necrosis and yield penalties. Compared to that of the wild-type (WT), the wood of transgenic poplars had 21% less cellulose, 28% more matrix polysaccharides, 23% more lignin and a significantly altered lignin composition. More specifically, ProCesA8-B:DCS_CURS2 lignin had a reduced syringyl/guaiacyl unit (S/G) ratio, an increased frequency of p-hydroxyphenyl (H) units, a decreased frequency of p-hydroxybenzoates and a higher fraction of phenylcoumaran units. Without, or with alkaline or hot water pretreatment, the saccharification efficiency of the transgenic lines was equal to that of the WT. These differences in (growth) phenotype illustrate that translational research in crops is essential to assess the value of an engineering strategy for applications. Further fine-tuning of this research strategy (e.g., by using more specific promoters or by translating this strategy to other crops such as maize) might lead to transgenic bio-energy crops with cell walls more amenable to deconstruction without settling in yield.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Metagenomes of tropical soil-derived anaerobic switchgrass-adapted consortia with and without iron

Tropical forest soils decompose litter rapidly with frequent episodes of anoxia, making it likely that bacteria using alternate terminal electron acceptors (TEAs) such as iron play a large role in supporting decomposition under these conditions. The prevalence of many types of metabolism in litter deconstruction makes these soils useful templates for improving biofuel production. To investigate how iron availability affects decomposition, we cultivated feedstock-adapted consortia (FACs) derived from iron-rich tropical forest soils accustomed to experiencing frequent episodes of anaerobic conditions and frequently fluctuating redox. One consortium was propagated under fermenting conditions, with switchgrass as the sole carbon source in minimal media (SG only FACs), and the other consortium was treated the same way but received poorly crystalline iron as an additional terminal electron acceptor (SG + Fe FACs). We sequenced the metagenomes of both consortia to a depth of about 150 Mb each, resulting in a coverage of 26× for the more diverse SG + Fe FACs, and 81× for the relatively less diverse SG only FACs. Both consortia were able to quickly grow on switchgrass, and the iron-amended consortium exhibited significantly higher microbial diversity than the unamended consortium. We found evidence of higher stress in the unamended FACs and increased sugar transport and utilization in the ironamended FACs. This work provides metagenomic evidence that supplementation of alternative TEAs may improve feedstock deconstruction in biofuel production.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2013

Autonomous Byte Stream Randomizer

Net-centric networking environments are often faced with limited resources and must utilize bandwidth as efficiently as possible. In networking environments that span wide areas, the data transmission has to be efficient without any redundant or exuberant metadata. The Autonomous Byte Stream Randomizer software provides an extra level of security on top of existing data encryption methods. Randomizing the data s byte stream adds an extra layer to existing data protection methods, thus making it harder for an attacker to decrypt protected data. Based on a generated crypto-graphically secure random seed, a random sequence of numbers is used to intelligently and efficiently swap the organization of bytes in data using the unbiased and memory-efficient in-place Fisher-Yates shuffle method. Swapping bytes and reorganizing the crucial structure of the byte data renders the data file unreadable and leaves the data in a deconstructed state. This deconstruction adds an extra level of security requiring the byte stream to be reconstructed with the random seed in order to be readable. Once the data byte stream has been randomized, the software enables the data to be distributed to N nodes in an environment. Each piece of the data in randomized and distributed form is a separate entity unreadable on its own right, but when combined with all N pieces, is able to be reconstructed back to one. Reconstruction requires possession of the key used for randomizing the bytes, leading to the generation of the same cryptographically secure random sequence of numbers used to randomize the data. This software is a cornerstone capability possessing the ability to generate the same cryptographically secure sequence on different machines and time intervals, thus allowing this software to be used more heavily in net-centric environments where data transfer bandwidth is limited.

Paloulian, George K.↗
DOE OSTI2013

Computational Mechanistic Studies of Acid-Catalyzed Lignin Model Dimers for Lignin Depolymerization

Lignin is a heterogeneous alkyl-aromatic polymer that constitutes up to 30% of plant cell walls, and is used for water transport, structure, and defense. The highly irregular and heterogeneous structure of lignin presents a major obstacle in the development of strategies for its deconstruction and upgrading. Here we present mechanistic studies of the acid-catalyzed cleavage of lignin aryl-ether linkages, combining both experimental studies and quantum chemical calculations. Quantum mechanical calculations provide a detailed interpretation of reaction mechanisms including possible intermediates and transition states. Solvent effects on the hydrolysis reactions were incorporated through the use of a conductor-like polarizable continuum model (CPCM) and with cluster models including explicit water molecules in the first solvation shell. Reaction pathways were computed for four lignin model dimers including 2-phenoxy-phenylethanol (PPE), 1-(para-hydroxyphenyl)-2-phenoxy-ethanol (HPPE), 2-phenoxy-phenyl-1,3-propanediol (PPPD), and 1-(para-hydroxyphenyl)-2-phenoxy-1,3-propanediol (HPPPD). Lignin model dimers with a para-hydroxyphenyl ether (HPPE and HPPPD) show substantial differences in reactivity relative to the phenyl ether compound (PPE and PPPD) which have been clarified theoretically and experimentally. The significance of these results for acid deconstruction of lignin in plant cell walls will be discussed.

09 BIOMASS FUELS↗
DOE OSTI2014

Understanding Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance (Presentation)

Fungal free enzymes and bacterial complexed cellulosomes deconstruct biomass using different physical mechanisms. Free enzymes, which typically contain a large proportion of GH7 cellobiohydrolase, diffuse throughout the substrate and hydrolyze primarily from the cellulose reducing end, resulting in 'sharpened' macrofibrils. In contrast, complexed cellulosomes contain a diverse array of carbohydrate binding modules and multiple catalytic specificities leading to delamination and physical peeling of the cellulose macrofibril structures. To investigate how cellulose structure contributes to recalcitrance, we compared the deconstruction of cellulose I, II, and III; using free and complexed enzyme systems. We also evaluated both systems on Clean Fractionation and alkaline pretreated biomass, which remove much of the lignin, to determine the impact on enzyme loading reduction. Free fungal enzymes demonstrated a swelling of the outer surface of the plant cell walls while removing localized disruptions, resulting in a smooth surface appearance. Cellulosomes produced cell wall surfaces with localized areas of disruption and little surface layer swelling. These studies contribute to the overall understanding of biomass recalcitrance and how combining different enzymatic paradigms may lead to the formulation of new enzyme cocktails to reduce the cost of producing sugars from plant cell wall carbohydrates.

09 BIOMASS FUELS↗
DOE OSTI2020

Bioelectrochemical biorefining for the conversion of hydrogenous matter to hydrogen gas and other useful products

A method for the substantially complete conversion of hydrogenous matter to higher value product, the method comprising: (i) subjecting the hydrogenous matter to a substantially complete deconstruction process in which an aqueous phase containing a multiplicity of deconstructed compounds is produced; and (ii) contacting the aqueous phase with an anode of a microbial electrolysis cell, the anode containing a community of microbes thereon which oxidatively degrade one or more of the oxygenated organic compounds in the aqueous phase to produce protons and free electrons at the anode, wherein the protons and free electrons are transported to the cathode to produce hydrogen gas or a valuable reduced organic compound at the cathode upon application of a suitable cell potential across the anode and cathode. The invention is also directed to an apparatus for practicing the method described above.

09 BIOMASS FUELS↗
DOE OSTI2022

Lignin Utilization

Valorization of lignin has massive potential economic and sustainability benefits for the lignocellulosic biorefinery. However, challenges remain to realize lignin conversion to coproducts, especially related to selective, high yield depolymerization to monomers and quantitative analytics on lignin, the latter of which is critical for accurate process modeling. Towards these goals, the Lignin Utilization project focuses on catalytic lignin deconstruction chemistries for C-O and C-C bond cleavage, development of analytical chemistry techniques to quantitatively characterize lignin, and syntheses of requisite model compounds for understanding lignin transformations. This work is done with and supports multiple BETO projects, including the Biological Lignin Valorization (BLV) project, the Separations Consortium, and others. Outcomes of Lignin Utilization for analytics and synthesis include 1) development of new mass spectrometry (MS) methods to characterize lignin dimers/oligomers in process streams, 2) deployment of a computational-experimental tool (with Biochemical Process Modeling and Simulation) to identify lignin-derived compounds with high fidelity from MS, and 3) delivery of >40 unique compounds. From a catalysis perspective, we have developed new oxidative approaches to cleave C-O and C-C bonds and produce >50% bio-available aromatic monomers for biological funneling for the BLV project and have developed recoverable bases for base-catalyzed deconstruction of lignin.

BIOMASS FUELS↗
DOE OSTI2013

Cleanup under Airlock of an Old Uranium Foundry - 13273

Since 2004, AREVA's subsidiary SICN has been conducting the cleanup and dismantling of an old uranium foundry located in the town of Annecy (France). The first operations consisted in the removal of the foundry's production equipment, producing more than 300 metric tons (MT) of waste. The second step consisted in performing the radiological characterization of the 1,600 m{sup 2} (17,200 ft{sup 2}) building, including underground trenches and galleries. The building was precisely inventoried, based on operations records and direct measurements. All sub-surfaces, which needed to be cleaned up were characterized, and a determination of the contamination migration was established, in particular with trenches and galleries. The wall thicknesses to be treated were empirically justified, knowing that the maximal migration depth inside concrete is 5 mm for a liquid transfer vector. All singularities such as cracks, anchoring points, etc. were spotted for a complete and systematic treatment. Building structures not laying directly on the soil, such as floor slabs, were not cleaned up but directly deconstructed and disposed of as waste. The facility was located within the town of Annecy. Therefore, in order to avoid the risk of dusts dispersion and public exposure during the building deconstruction and the soil treatment, a third of the building's surface was confined in a sliding airlock built from a metal structure capable of resisting to wind and snow, which are frequent in this area. This particular structure provided a static confinement over the half of the building which was covered and a dynamic confinement using a ventilation and high efficiency air filtration system, sized to provide 2.5 air changes per hour. The enclosure and its metallic structure is 33 m long (108 feet), 25 m wide (82 feet), and 13 m high (42 feet), for a volume of 10,000 m{sup 3} (353,000 ft{sup 3}). It was made up of a double skin envelope, allowing the recycling of its structure and outside envelope. After cleaning up and dismantling the first portion of the building, the enclosure was repositioned on the second and the last third of the building, by sliding it on support pads. Almost 7,000 m{sup 2} of concrete surface has been treated with no dust dispersion outside the enclosure. After treatment, all the remaining surfaces were controlled by an independent entity to verify their acceptability with regards to residual contamination (less than 0.4 Bq/cm{sup 2} (24 DPM) for alpha contamination and less than 1 Bq/g of total uranium). Approximately 1,900 MT of equipment and waste were generated in batches of 1m{sup 3}, in order to be staged on site, and then characterized and packaged in 20 foot containers for shipment to the final ANDRA repository. The package certification included the verification of the physical and chemical characteristics and the radiological characteristics (mass activity, dose rate, and residual outside surface contamination). Finally, after cleanup and dismantling of the foundry, a concrete slab was poured on the free surface as a clean base for implementation of new activities. (authors)

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗
DOE OSTI2016

Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein

Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue towards energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28 to 0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Altogether, our study provides strategies to identify highly active, low lignin-binding cellulases by either rational design or by computational screening genomic databases.

09 BIOMASS FUELS↗
DOE OSTI2025

Initiator‐Driven Tailoring of Reactivity and Inhibition in Frontally Polymerized Dicyclopentadiene‐Co‐Dihydrofuran Thermosets

Incorporating 2,3‐dihydrofuran (DHF) into dicyclopentadiene (DCPD) enables frontally polymerizable, deconstructible thermosets via acid hydrolysis. At 5–20 mol% DHF and low initiator concentration (~100 ppm), DHF inhibits front propagation, lowers the glass transition temperature ( T g ) by up to 20°C, and quenches the front, especially at 20% DHF. This paper demonstrates that increasing the initiator concentration to 250–1000 ppm overcomes inhibition, enhances front speeds by ~50%, and suppresses quenching at the expense of pot‐life. Above 5 mol% DHF, an emergence of a second exotherm is observed, attributed to increased rate dependence on the inhibition of the initiator by DHF. Void formation from volatile DHF is suppressed by applying 110 kPa nitrogen, though at the cost of reduced front speed. Dynamic mechanical analysis shows that higher initiator concentration produced more highly cross‐linked polymers, with a ~20% increase in storage modulus at 25°C and a 20°C increase in T g .

deconstructible thermosets↗
DOE OSTI2019

Heterologous co-expression of two β-glucanases and a cellobiose phosphorylase resulted in a significant increase in the cellulolytic activity of the Caldicellulosiruptor bescii exoproteome

Abstract The ability to deconstruct plant biomass without conventional pretreatment has made members of the genus Caldicellulosiruptor the target of investigation for the consolidated processing of plant lignocellulosic biomass to biofuels and bioproducts. To investigate the synergy of enzymes involved and to further improve the ability of C. bescii to degrade cellulose, we introduced CAZymes that act synergistically with the C. bescii exoproteome in vivo and in vitro. We recently demonstrated that the Acidothermus cellulolyticus E1 endo-1,4-β-D-glucanase (GH5) with a family 2 carbohydrate-binding module (CBM) increased the activity of C. bescii exoproteome on biomass, presumably acting in concert with CelA. The β-glucanase, GuxA, from A. cellulolyticus is a multi-domain enzyme with strong processive exoglucanase activity, and the cellobiose phosphorylase from Thermotoga maritima catalyzes cellulose degradation acting synergistically with cellobiohydrolases and endoglucanases. We identified new chromosomal insertion sites to co-express these enzymes and the resulting strain showed a significant increase in the enzymatic activity of the exoproteome.

09 BIOMASS FUELS↗