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DOE OSTI2026

Island influences on plant functional traits and trait–trait associations across species‐ and community‐scales

The island rule predicts gigantism or dwarfism in body size of island species relative to their mainland counterparts. However, whether other functional traits shift and whether trait–trait associations on islands differ between species and community levels remains unclear. We measured 13 carbon- and water-related functional traits in 37 shared tree species across 35 eastern Chinese islands and 66 nearby mainland plots. We examined species-level trait value shifts and associations under the island rule and compared trait associations between species and communities. Most size-related, wood-anatomical, and hydraulic traits shifted on islands, with large values decreasing and small values increasing; yet, their associations remained stable, aligning with the global trait spectrum and trait–trait coevolution. This stability, despite trait value shifts, suggests evolutionary integration of functional strategies. By contrast, island community-scale trait associations diverged from shared species-level patterns and sometimes reversed, such as positive relationships between wood density and resource-acquisitive traits. Community-level trait associations were stronger on islands, likely reflecting constrained environmental filtering and migration limitation. These contrasting patterns suggest that dominant species can restructure trait associations at the community level, with implications for ecosystem functioning and carbon storage, thereby advancing understanding of plant trait strategies in island systems.

Archipelagos↗

DOE OSTI2024

Unlocking the distinctive enzymatic functions of the early plant biomass deconstructive genes in a brown rot fungus by cell-free protein expression

ABSTRACT Saprotrophic fungi that cause brown rot of woody biomass evolved a distinctive mechanism that relies on reactive oxygen species (ROS) to kick-start lignocellulosic polymers’ deconstruction. These ROS agents are generated at incipient decay stages through a series of redox relays that shuttle electrons from fungus’s central metabolism to extracellular Fenton chemistry. A list of genes has been suggested encoding the enzyme catalysts of the redox processes involved in ROS’s function. However, navigating the functions of the encoded enzymes has been challenging due to the lack of a rapid method for protein synthesis. Here, we employed cell-free expression system to synthesize four redox or degradative enzymes, which were identified, by transcriptomic data, as conserved players of the ROS oxidation phase across brown rot fungal species. All four enzymes were successfully expressed and showed activities that enable confident assignment of function, namely, benzoquinone reductase (BQR), ferric reductase, α-L-arabinofuranosidase (ABF), and heme-thiolate peroxidase (HTP). Detailed analysis of their catalytic features within the context of brown rot environments allowed us to interpret their roles during ROS-driven wood decomposition. Specifically, we validated the functions of BQR as the driver redox enzyme of Fenton cycles and reconstructed its interactions with the co-occurring HTP or laccase and ABF. Taken together, this research demonstrated that the cell-free expression platform is adequate for synthesizing functional fungal enzymes and provided an alternative route for the rapid characterization of fungal proteins, escalating our understanding of the distinctive biocatalyst system for plant biomass conversion. IMPORTANCE Brown rot fungi are efficient wood decomposers in nature, and their unique degradative systems harbor untapped catalysts pursued by the biorefinery and bioremediation industries. While the use of “omics” platforms has recently uncovered the key “oxidative-hydrolytic” mechanisms that allow these fungi to attack lignocellulose, individual protein characterization is lagging behind due to the lack of a robust method for rapid synthesis of crucial fungal enzymes. This work delves into the studies of biochemical functions of brown rot enzymes using a rapid, cell-free expression platform, which allowed the successful depictions of enzymes’ catalytic features, their interactions with Fenton chemistry, and their roles played during the incipient stage of brown rot when fungus sets off the reactive oxygen species for oxidative degradation. We expect this research could illuminate cell-free protein expression system’s use to fulfill the increasing need for functional studies of fungal enzymes, advancing the discoveries of novel biomass-converting catalysts.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

Biopower: Impact of Biofuels Deployment to Replace Petroleum Liquids in Stationary Power Applications

Petroleum-based liquids are used in a portion of power generation applications in the United States, predominantly in the New England, Middle Atlantic, South Atlantic, and Pacific-Noncontiguous regions. Power plants that burn petroleum liquids, such as distillate or residual fuel oils, are generally used for short periods to accommodate peak electricity demands. The Energy Information Administration (EIA) estimated the U.S. consumption of petroleum liquids for electricity generation at 27 million barrels in 2018, representing a cost of $2.4 billion annually. This study assesses the potential to displace all or part of the petroleum liquids in U.S. power generation with biofuels. The biofuels for this application are assumed to be derived from terrestrial feedstocks, with conversion routes of both fast pyrolysis (bio-oil) and hydrothermal liquefaction (bio-crude). Regional models were used to assess the availability and cost of three different base materials: clean wood, forest residues, and corn stover; each was evaluated in the laboratory at small or experimental scales for conversion to bio-oil or bio-crude. The estimated biofuel production quantities depend on equivalent heating versus the current heavy fuel. In this report, the availability of each type of biomass for each section of the U.S. Census division is estimated using a conservative broker price (in each case) of $ 80 per dry tonne. The results show that the petroleum-liquid power generation in each of the Census Divisions could be supplied by one or more of the feedstocks evaluated. For all regions, clean wood supplies (only) could provide ample supply. For all but two regions (Middle Atlantic and New England), forest residues alone are sufficient. Finally, for all regions but three (Middle Atlantic, New England, and South Atlantic), corn stover alone is adequate. The Minimum Fuel Selling Price (MFSP) of bio-oil and bio-crude were also estimated for each feedstock type and Census Division. This analysis showed that fast pyrolysis bio-oil projections to be lower (14% on average) than current wholesale petroleum-based heating oil prices in each of the regions, assuming 100 dry tonnes/day processing capacity. However, bio-crude predictions were significantly higher (2X) in all cases. The effect of biorefinery size was also quantified. Based on the preliminary results in this study, it is apparent the biofuels could be an economical alternative for current petroleum liquids in U.S. power generation. However, additional research is needed to determine the necessary biofuel characteristics to support existing generation equipment. It is recommended that both power generation and biofuel production stakeholders to be engaged to outline the research and testing needed to identify the technical hurdles to enable the opportunity.

02 PETROLEUM↗

DOE OSTI2021

The Inducible Accumulation of Cell Wall-Bound p -Hydroxybenzoates Is Involved in the Regulation of Gravitropic Response of Poplar

Lignin in Populus species is acylated with p-hydroxybenzoate. Monolignol p-hydroxybenzoyltransferase 1 (PHBMT1) mediates p-hydroxybenzoylation of sinapyl alcohol, eventually leading to the modification of syringyl lignin subunits. Angiosperm trees upon gravistimulation undergo the re-orientation of their growth along with the production of specialized secondary xylem, i.e., tension wood (TW), that generates tensile force to pull the inclined stem or leaning branch upward. Sporadic evidence suggests that angiosperm TW contains relatively a high percentage of syringyl lignin and lignin-bound p-hydroxybenzoate. However, whether such lignin modification plays a role in gravitropic response remains unclear. By imposing mechanical bending and/or gravitropic stimuli to the hybrid aspens in the wild type (WT), lignin p-hydroxybenzoate deficient, and p-hydroxybenzoate overproduction plants, we examined the responses of plants to gravitropic/mechanical stress and their cell wall composition changes. We revealed that mechanical bending or gravitropic stimulation not only induced the overproduction of crystalline cellulose fibers and increased the relative abundance of syringyl lignin, but also significantly induced the expression of PHBMT1 and the increased accumulation of p-hydroxybenzoates in TW. Furthermore, we found that although disturbing lignin-bound p-hydroxybenzoate accumulation in the PHBMT1 knockout and overexpression (OE) poplars did not affect the major chemical composition shifts of the cell walls in their TW as occurred in the WT plants, depletion of p-hydroxybenzoates intensified the gravitropic curving of the plantlets in response to gravistimulation, evident with the enhanced stem secant bending angle. By contrast, hyperaccumulation of p-hydroxybenzoates mitigated gravitropic response. These data suggest that PHBMT1-mediated lignin modification is involved in the regulation of poplar gravitropic response and, likely by compromising gravitropism and/or enhancing autotropism, negatively coordinates the action of TW cellulose fibers to control the poplar wood deformation and plant growth.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

County Electric Vehicle Home Charging Access Shares from the 2030 National Charging Network Study

This file contains modeled county-level home electric vehicle (EV) charging access shares from the study, "The 2030 National Charging Network: Estimating U.S. Light-Duty Demand for Electric Vehicle Charging Infrastructure" by Wood et al. (2023). These are based on modeling in "There's No Place Like Home: Residential Parking, Electrical Access, and Implications for the Future of Electric Vehicle Charging Infrastructure" by Ge et al. (2021). Wood, Eric, et al. 2023. The 2030 National Charging Network: Estimating U.S. Light-Duty Demand for Electric Vehicle Charging Infrastructure. Golden, CO: National Renewable Energy Laboratory. NREP/TP-5400-85654. https://www.nlr.gov/docs/fy23osti/85654.pdf . Ge, Yanbo, et al. 2021. There's No Place Like Home: Residential Parking, Electrical Access, and Implications for the Future of Electric Vehicle Charging Infrastructure. Golden, CO: National Renewable Energy Laboratory. NREL/TP-5400-81065. https://www.nlr.gov/docs/fy22osti/81065.pdf .

24 POWER TRANSMISSION AND DISTRIBUTION↗

DOE OSTI2021

Materials Engineering of Violin Soundboards by Stradivari and Guarneri

Abstract We investigated the material properties of Cremonese soundboards using a wide range of spectroscopic, microscopic, and chemical techniques. We found similar types of spruce in Cremonese soundboards as in modern instruments, but Cremonese spruces exhibit unnatural elemental compositions and oxidation patterns that suggest artificial manipulation. Combining analytical data and historical information, we may deduce the minerals being added and their potential functions—borax and metal sulfates for fungal suppression, table salt for moisture control, alum for molecular crosslinking, and potash or quicklime for alkaline treatment. The overall purpose may have been wood preservation or acoustic tuning. Hemicellulose fragmentation and altered cellulose nanostructures are observed in heavily treated Stradivari specimens, which show diminished second‐harmonic generation signals. Guarneri's practice of crosslinking wood fibers via aluminum coordination may also affect mechanical and acoustic properties. Our data suggest that old masters undertook materials engineering experiments to produce soundboards with unique properties.

Su, Cheng‐Kuan↗

DOE OSTI2021

Materials Engineering of Violin Soundboards by Stradivari and Guarneri

36 MATERIALS SCIENCE↗

DOE OSTI2025

Biotransformation of Phenolics in Spent Liquor from Aqueous Ammonia Pretreatment

Spent liquors of biomass pretreatment provide a source for renewable chemical production. These liquors require treatment before being discharged; otherwise, they negatively impact the environment. Herein, spent liquors from aqueous ammonia pretreatment of poplar wood are characterized for phenolic content via liquid chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy. The main phenolics are phenol, p-hydroxybenzamide (pHBAm), and p-hydroxybenzoic acid (pHBA), of which pHBAm and pHBA are produced from the ester-linked p-hydroxybenzoates in poplar wood. Phenol is produced from pHBA via decarboxylation. The potential biotransformation of the extracted phenolics into 2-pyrone-4,6-dicarboxylic acid (PDC) is assessed using an engineered strain of Novosphingobium aromaticivorans DSM12444 (PDC strain). Biotransformation of pHBAm to PDC is shown to be possible in the presence of pHBA, but not when pHBAm is the sole phenolic substrate, this is the first reported observation of N. aromaticivorans producing PDC from an aromatic amide. The phenol present is not transformed to PDC and does not inhibit PDC production. This study demonstrates that the phenolic amide in spent liquor from ammonia pretreatment can be valorized via biotransformation using N. aromaticivorans, which adds to the growing versatility of N. aromaticivorans as a microbial chassis for converting plant-derived compounds to useful products.

biomass↗

DOE OSTI2023

Black carbon emissions from modern automated cordwood stoves for space heating

Wood burning stove design is improving as regulation become stricter for particulate matter emissions. Here, this study describes results of black carbon (BC) emissions from three modern automated wood stoves when tested in common failure modes using a test protocol that included cold-starts, high and low fuel loading densities, and use of overly large pieces of fuel. Emission sampling was performed using a dilution sampling system in tandem with an aethalometer to measure BC with high time resolution. Results show that the time-weighted average BC emission rates were 0.91, 4.30, and 1.74 g/h for the three stoves tested (A, B, and C respectively), the values, both higher and lower than the United States 2020 certification test emission limit of 2.5 g/h. The results demonstrate good performance overall, given the rigorous nature of the testing protocol used. The aethalometer data showed that the performance was relatively poor during cold-start, when the fuel was ignited and combusted without a coalbed, and when stoves failed to optimize air delivery due to specific loading and output conditions. During the cold-start test section, the average BC emission rate was seven times higher than the overall average for Stove A, 10 times above the average for Stove B, and two times above the average emission rate for Stove C. Calculations of the Average Angstrom Absorption Exponent (AAE) of the exhaust aerosol showed little correlation with BC concentration. The results show that automation results in a quantifiable reduction in particular BC emissions.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗

DOE OSTI2025

A comprehensive review on valorization of chestnut processing wastes into bio‐based composites and bioplastics

Abstract This review examines the characterization and utilization of chestnut processing wastes (35%) in the production of bioplastics and biocomposites. In this review, a Web of Science search without any publishing year restriction on the biochemical compositions of all the components of Castanea sativa . The obtaining of bioplastics and biocomposites based on C. sativa was reviewed. First, it highlights the biochemical composition and antioxidant properties of chestnut fruit, shell, burrs, leaves, flowers, and wood focusing on the most important compounds, such as phenolic acids, flavonoids, carbohydrates, Klason lignin, cellulose, and glucan, which can enhance the properties of these materials. Then the review covers using several chestnut extracts and fillers in bioplastics production through solvent casting technique. The mechanical, structural, bioactive properties, and moisture content were optimized through the composition and production. The color, UV absorption, antioxidant, and antimicrobial activity were also discussed. Biocomposites reinforced with chestnut burs, shells, or wood flour increased the intended properties. The enhancements in tensile strength, elastic modulus, and the effects of a pre‐treatment were evaluated. Additionally, it discusses material recovery, recycling, and reuse, particularly how it affects the biodegradability of composites incorporating chestnut waste residues. Highlights Chestnut fruit, shells, and burrs are rich in starch, lignin, and cellulose. The waste of chestnut processing can be used in bioplastics and biocomposites. Chestnut‐based films and biocomposites exhibit promising mechanical properties. The antimicrobial activity, making films, and composites proper for food packaging. New techniques boost performance, offering alternatives to conventional plastics.

Silva, Simão B. [REQUIMTE/LAQV, ISEP, Polytechnic ↗

DOE OSTI2022

Effects of trunk injection with emamectin benzoate on arthropod diversity

Abstract BACKGROUND Pine wood nematode is a major plant quarantine object in the world. Trunk injection is an effective method for controlling pests that cause disease. To evaluate the ecological safety of trunk injection with emamectin benzoates in forests of Pinus massoniana , the community diversity and community composition of soil arthropods and flying insects (Hymenoptera) were studied at different stages of trunk injection. RESULTS The dominant taxonomic groups of soil arthropods were Collembola (30.80%), Insecta (26.42%), and Arachnida (23.84%). The taxonomic groups of flying insects (Hymenoptera) were Ichneumonidae (48.94%), Formicidae (14.10%), and Braconidae (8.44%). Trunk injection with emamectin benzoate has no significant effect on the community diversity indices of total soil arthropods and flying insects (Hymenoptera). However, it has a significant effect on the community diversity indices of detritivores for soil arthropods. It changed the community composition of soil arthropods but did not impact the community composition of flying insects (Hymenoptera). Redundancy analysis of arthropod community structure and environmental variables showed that total potassium, residual of green leaf, and residual of litter leaf have a significant impact on the community structure of soil arthropods, and total phosphorus, total nitrogen, water content, organic matter, and total potassium have a significant impact on the community structure of flying insects (Hymenoptera). CONCLUSION Trunk injection with emamectin benzoate is safe for the ecological environment. This study provides a new insight into the field for the prevention and control of pine wood nematode disease, which is of great significance to forest management and pest control. © 2022 Society of Chemical Industry.

Ouyang, Xianheng↗

DOE OSTI2022

Litter removal impacts on soil biodiversity and eucalypt plantation development in the seasonal tropics

The little layer of tree plantations provides primary nutrients for uptake, buffers changes in soil moisture, and provides habitat and substrate to soil epigeic fauna. However, this layer in eucalypt plantations is often removed to reduce fuel load during the fire season in the Brazilian savanna (Cerrado). Therefore, it is necessary to quantify the effects of changes in litter dynamics on the function of these plantations, on key nutrient cycling processes and on epigeic fauna diversity and abundance. In two adjacent stands (one juvenile and one mature), the consequences of two years of litter removal were quantified as monthly litterfall, leaf and fine wood litter decomposition, epigeic fauna abundance and diversity, soil biogeochemical variables, and tree diameter and basal area increments. Monthly litterfall rates in juvenile and mature stands did not change with litter removal over the study period. Annual litterfall ranged from 4.1 to 4.9 Mg ha -1 a -1 in litter removal plots and from 3.9 to 4.8 Mg ha -1 a -1 in control plots. Fine wood litter decomposition was slower in litter removal plots compared to controls, while leaf decomposition rates were similar in both. Two years of litter removal in the juvenile stand did not affect topsoil biogeochemical parameters but decreased available phosphorus at 20–40 cm depth relative to controls. In the mature stand, total cation exchange capacity (0–20 cm) was higher in controls (6.4 cmol c dm -3 ) relative to litter removal plots (6.3 cmol c dm -3 ), while soil moisture (0–40 cm depth) was lower in litter removal (25.45 m 3 m -3 ) compared to control plots (26 m 3 m -3 ) in the dry season. A non-metric multidimensional scaling ordination revealed an increased homogeneity in epigeic fauna where litter was removed. Litterfall, decomposition, diameter increment, four soil physical parameters and fourteen chemical parameters at 0–20 and 20–40 cm depth explained the differences in soil epigeic fauna composition between litter removal and control plots. Diameter increment decreased with litter removal only in the juvenile stand, which had reached its growth peak. The results indicate that removing excess litter to decrease fuel volume can alter soil biodiversity and edaphic conditions that negatively affect nutrient cycling and tree growth.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2026

Design and scale-up of 3D printed bat houses with biomass-derived polymer composites

Biomass (e.g., pine sawdust, especially high–ash content pine sawdust) is commonly disposed of as waste. Combining biomass with polymers to make composite feedstocks for 3D printing has been explored as a method to reduce or repurpose the biomass waste. Although not all biocomposite properties are known, the wood-based polylactic acid (PLA) composite has promising qualities for applications in ecological settings. In this work, pine wood–PLA composite feedstock was used to 3D print supplemental roost structures for endangered tree-roosting bats, which often face a paucity of suitable naturally occurring roosts. This material combination was selected because it is estimated to degrade faster than the synthetic material systems that are used widely in supplemental bat roosting structures to aid in the conservation of tree roosting bats. The layered, rough surface created by the 3D printing process serves as a surface that bats can grip while roosting. Computer-aided design (CAD) models were generated based on natural roost structures, and a full-size bat house was successfully additively manufactured using a pellet-fed large-scale 3D printing system. The 3D printed hexagon exhibited a tensile strength of 22–23 MPa and a Young’s modulus of 3202–3218 MPa in the x-direction. It has been demonstrated that the 3D printed bat house can be installed on a tree in a stable fashion. This successful demonstration of a bat roost manufactured using a bioderived composite should promote its use in other fish and wildlife structures and broader industrial applications such as construction and automobiles.

3D printing↗

DOE OSTI2021

Know the enemy: 2D Fermi liquids

Highlights: • Regime of validity of the Fermi liquid theory in 2D interacting Fermi systems has been investigated. • Phase diagram for hydrodynamic and ballistic regimes in the frequency–temperature space has been obtained. • Temperature correction to quasiparticle effective mass has been calculated. • 2D Wiedemann–Franz law and 2D Kadawoki–Woods relation have been discussed. We describe an analytical theory investigating the regime of validity of the Fermi liquid theory in interacting, via the long-range Coulomb coupling, two-dimensional Fermi systems comparing it with the corresponding 3D systems. We find that the 2D Fermi liquid theory and 2D quasiparticles are robust up to high energies and temperatures of the order of Fermi energy above the Fermi surface, very similar to the corresponding three-dimensional situation. We calculate the phase diagram in the frequency–temperature space separating the collisionless ballistic regime and the collision-dominated hydrodynamic regime for 2D and 3D interacting electron systems. We also provide the temperature corrections up to third order for the renormalized effective mass, and comment on the validity of 2D Wiedemann–Franz law and 2D Kadawoki–Woods relation.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

DOE OSTI2021

Comparative performance of bio-based coatings formulated with cellulose, chitin, and chitosan nanomaterials suitable for fruit preservation

Sustainable nanomaterials (SNMs) from wood, sugarcane and crab shell were prepared and used to coat selected fruits. The properties of SNMs and selected fruits were characterized and strawberry was used as an example to test antifungal activity and freshness preservation of the SNMs. The SNMs with their nano-structured morphology form strong shear-thinning dispersions for easy spraying on fruit surfaces. The fruit surface free energy was influenced by its surface morphology, predominant surface wax components, and cutin monomers. The antifungal activity of SNMs was influenced by their surface functional groups and particle size (crystals vs fibers). The coblend of wood nanocrystals (WCNCs) and chitosan nanofiber (CSNFs) exhibited the best antifungal property, which was comparable with the performance of the fungicide thiabendazole (80 mg L -1 ). The weight loss and color change of the WCNC/CSNF coated strawberries decreased by nearly half compared with the control samples, showing coating effectiveness on preserving fruit freshness.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

Overexpression of a gibberellin 20-oxidase gene in poplar xylem led to an increase in the size of nanocellulose fibrils and improved paper properties

Cellulose, the major component of secondary cell walls, is the most abundant renewable long-chain polymer on earth. Nanocellulose has become a prominent nano-reinforcement agent for polymer matrices in various industries. We report the generation of transgenic hybrid poplar overexpressing the Arabidopsis gibberellin 20-oxidase1 gene driven by a xylem-specific promoter to increase gibberellin (GA) biosynthesis in wood. X-ray diffraction (XRD) and sum frequency generation spectroscopic (SFG) analyses showed that cellulose in transgenic trees was less crystalline, but the crystal size was larger. The nanocellulose fibrils prepared from transgenic wood had an increased size compared to those from wild type. When such fibrils were used as a reinforcing agent in sheet paper preparation, the mechanical strength of the paper was significantly enhanced. Engineering the GA pathway can therefore affect nanocellulose properties, providing a new strategy for expanding nanocellulose applications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

The role of catalytic iron in enhancing volumetric sugar productivity during autothermal pyrolysis of woody biomass

Passivation of naturally occurring AAEM in biomass enhances sugar yields from the fast pyrolysis of biomass by preventing these metals from catalyzing the fragmentation of pyranose rings in cellulose and hemicellulose. However, because AAEM also catalyzes lignin depolymerization, its passivation can be accompanied by undesirable char agglomeration. Pretreatment of biomass with ferrous sulfate both passivates AAEM and substitutes ferrous ions as lignin depolymerization catalysts. This pretreatment has been particularly successful for high ash biomass like corn stover, but of limited value for low ash biomass like wood. This study explores the reasons for this discrepancy and offers a combined pretreatment of ferrous sulfate and ferrous acetate pretreatment to overcome char agglomeration in wood. Furthermore, this new pretreatment increased sugar yields from 4.4 wt% to 15.5 wt% and 5.4 wt% to 19.0 wt% for hardwood and softwood biomasses, respectively. This pretreatment produces an iron-rich biochar that catalyzes oxidation of the biochar under the oxygen-rich conditions of autothermal pyrolysis, which is preferentially consumed to provide the enthalpy for pyrolysis, preserving bio-oil as a more desirable energy product. Instead of producing carbon monoxide, which dominates oxidation of biochar from untreated biomass, the iron catalyzes oxidation to carbon dioxide, producing more energy per mole of oxygen consumed. In fact, oxygen demand to support autothermal pyrolysis of red oak and southern yellow pine was reduced 15% by the presence of iron in the biochar.

42 ENGINEERING↗

DOE OSTI2026

Catabolism of lignin-related methoxylated compounds in white-rot fungi utilizes non-canonical oxidoreductases

White-rot fungi (WRF) are the most effective lignin-degrading organisms in nature, making them essential to Earth’s carbon cycle. Lignin is a highly methoxylated, heterogeneous biopolymer derived from plants. However, the pathways WRF use to metabolize methoxylated aromatic monomericcompounds as carbon sources remain unidentified. Here, we employ a systems biology approach to elucidate the intracellular catabolism of vanillate – a monomethoxylated aromatic compound – in two white-rot fungi (WRF), Gelatoporia subvermispora and Trametes versicolor. We identified and biochemically validated a four-enzyme pathway that converts vanillate into ß-ketoadipate – a metabolite that enters central carbon metabolism. This pathway deviates from typical bacterial pathways, where vanillate is initially demethylated and ring-cleaved by intradiol dioxygenases; instead, oxidative decarboxylation occurs prior to ring cleavage by extradiol dioxygenases. Thus, we conducted an in-depth investigation of ring cleavage and further downstream catabolism by the identified fungal enzymes using biochemical and structural approaches. This revealed non-canonical enzymes, including a highly substrate-specific extradiol dioxygenase and a metal-free, promiscuous reductase, the latter capable of acting on catabolic intermediates derived from both methoxylated and non-methoxylated aromatic compounds. This work emphasizes the potential of WRF and their enzymes to advance lignin valorization and enhance our understanding of their role during wood decay.

dioxygenase↗