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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Elucidation of Aromatic Catabolic Pathways in White-Rot Fungi

This project aims to investigate the hypothesis that white-rot fungi can simultaneously depolymerize lignin extracellularly and catabolize depolymerization products intracellularly as carbon and energy sources. Evaluating this hypothesis will provide deeper understanding of the role of white-rot fungi in facilitating carbon sequestration in Nature. Additionally, identifying the most promising fungal strains for lignin turnover and catabolism will catalyze future efforts in genetic tool development to enable metabolic engineering in white-rot fungi for lignin bioconversion to bioproducts. Lignin is the second most abundant plant-based biopolymer on Earth and represents up to 40% of the energy density of lignocellulosic biomass. Even though lignin is a massive natural carbon and energy reservoir, only a small group of basidiomycete fungi, namely white-rot fungi (WRF), have evolved the ability to efficiently depolymerize and mineralize lignin to CO2 and H2O. Considerable research efforts have been undertaken to understand how WRF depolymerize lignin but the biochemical reactions that convert lignin into CO2 have been largely neglected. In fact, it is unclear if WRF intracellularly catabolize lignin-derived aromatic compounds to utilize them as a carbon and energy source, or rather if lignin is depolymerized and mineralized extracellularly merely to facilitate access to cellulose and hemicellulose for use as a primary carbon source. To date, we have employed 13C-isotope labeling, systems biology approaches, and in vitro enzyme assays to definitively demonstrate that two WRF, Trametes versicolor and Gelatoporia (Ceriporiopsis) subvermispora, funnel carbon from lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways [1]. Specifically, 13C-isotopic labeling approaches showed that these WRF utilize poplar-derived aromatic compounds (e.g. 4-hydroxybenzoic acid (4-HBA)) as a carbon source. In silico genome analysis led us to hypothesize a complete catabolic pathway for 4-HBA and identify multiple homologous sequences for enzymes with putative oxidative decarboxylase, hydroxylase, and ring-opening dioxygenase activities, which are among the main biochemical reactions acting on aromatic compounds. Spatial and differential proteomic and metabolomic analyses supported the proposed catabolic pathways and showed alternative catabolic steps in T. versicolor that were not present in G. subvermispora. Based on the in silico, proteomics, and transcriptomics results, we down-selected enzymes for further in vitro characterization, and we have assigned a function to six fungal enzymes (including oxidative decarboxylases, hydroxylases, and ring-opening dioxygenases). Interestingly, even though we selected homologous enzyme pairs from both WRF with similar -omics trends, in a few cases only one of the studied fungi showed activity for the proposed substrate. Based on all the observations from this study [1], we hypothesized that 4-HBA preferentially undergoes oxidative decarboxylation to hydroquinone and subsequent hydroxylation to 1,2,4-benzenetriol in G. subvermispora before ring cleavage, whereas 4-HBA would preferentially undergo hydroxylation to protocatechuate and further oxidative decarboxylation to 1,2,4-benzenetriol in T. versicolor. Examining additional decarboxylases and hydroxylases as well as enzymes from other protein families that can perform the same or similar oxidative reactions, such as cytochromes P450 with aromatic hydroxylation activity, will also be key for elucidating enzyme preferences for specific substrates. Overall, this work forms the foundation of a new research area based on lignin catabolism by WRF, which could be further exploited to convert the undervalued biopolymer lignin into value-added compounds.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗
DOE OSTI2023

Molecular insights into the evolution of woody plant decay in the gut of termites

Plant cell walls represent the most abundant pool of organic carbon in terrestrial ecosystems but are highly recalcitrant to utilization by microbes and herbivores owing to the physical and chemical barrier provided by lignin biopolymers. Termites are a paradigmatic example of an organism’s having evolved the ability to substantially degrade lignified woody plants, yet atomic-scale characterization of lignin depolymerization by termites remains elusive. We report that the phylogenetically derived termite Nasutitermes sp. efficiently degrades lignin via substantial depletion of major interunit linkages and methoxyls by combining isotope-labeled feeding experiments and solution-state and solid-state nuclear magnetic resonance spectroscopy. Exploring the evolutionary origin of lignin depolymerization in termites, we reveal that the early-diverging woodroach Cryptocercus darwini has limited capability in degrading lignocellulose, leaving most polysaccharides intact. Conversely, the phylogenetically basal lineages of “lower” termites are able to disrupt the lignin-polysaccharide inter- and intramolecular bonding while leaving lignin largely intact. These findings advance knowledge on the elusive but efficient delignification in natural systems with implications for next-generation ligninolytic agents.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Accessing monomers from lignin through carbon–carbon bond cleavage

Lignin, the heterogeneous aromatic macromolecule found in the cell walls of vascular plants, is an abundant feedstock for the production of biochemicals and biofuels. Here, many valorization schemes rely on lignin depolymerization, with decades of research focused on accessing monomers through C–O bond cleavage, given the abundance of β–O–4 bonds in lignin and the large number of available C–O bond cleavage strategies. Monomer yields are, however, invariably lower than desired, owing to the presence of recalcitrant C–C bonds whose selective cleavage remains a major challenge in catalysis. In this Review, we highlight lignin C–C cleavage reactions, including those of linkages arising from biosynthesis (β–1, β–5, β–β and 5–5) and industrial processing (5–CH 2 –5 and α–5). We examine multiple approaches to C–C cleavage, including homogeneous and heterogeneous catalysis, photocatalysis and biocatalysis, to identify promising strategies for further research and provide guidelines for definitive measurements of lignin C–C bond cleavage.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
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 OSTI2025

Integrating catalytic fractionation and microbial funneling to produce 2-pyrone-4,6-dicarboxylic acid and ethanol

Replacing biorefinery designs that use stepwise fractionation, depolymerization, and conversion processes with designed tandem process steps can greatly reduce a biorefinery's operating costs and environmental impact. Reductive Catalytic Fractionation (RCF) is a highly efficient lignin-first approach that combines biomass fractionation and lignin depolymerization to generate a hydrogenolysis oil and pulp. The oil is composed of a complex mixture of phenolic monomers, dimers, and oligomers. Intergrating this chemical deconstruction process with microbial funneling of phenolics can simplify the product mixture and make high-value products. We applied RCF to poplar biomass in a biomass-to-bioproduct processing chain in which the phenolics were funneled to 2-pyrone-4,6-dicarboxylic acid (PDC) by an engineered strain of Novosphingobium aromaticivorans DSM12444. The pulp was enzymatically digested and the glucose and xylose was funneled to ethanol by an engineered strain of Saccharomyces cerevisiae GLBRCY945. By combining biomass fractionation and lignin depolymerization we removed a costly processing step that directly translated into a 29% reduction in the minimum selling price of PDC. This work combines experimentation with process modeling of an integrated biorefinery design to show how utilizing tandem process steps can significantly reduce operating expenses and environmental impact of upgrading lignocellulosic biomass to a portfolio of high value products.

Sener, Canan [Great Lakes Bioenergy Research Cente↗
DOE OSTI2022

Cross-Kingdom Comparative Genomics of Aromatic Catabolic Pathways in Fungi and Bacteria

The overall goal of this project is to test the hypothesis that white-rot fungi can simultaneously depolymerize lignin extracellularly and catabolize depolymerization products intracellularly as carbon and energy sources. The results from this project will lead to improved understanding of lignin utilization by white-rot fungi, and enable identification of promising fungal strains for lignin catabolism and valorization. As part of this effort, we have conducted a comparative genomic study to investigate a diversity of aromatic catabolic pathways in the fungal and bacterial kingdoms. Furthermore, we have combined genomic and phylogenetic approaches to decipher the evolution of certain aromatic catabolic enzymes and discover new classes of enzymes, which represents a foundation for future biochemical and molecular genetic studies.

aromatic compounds↗
DOE OSTI2025

Co-depolymerization of lignin and plastic waste using hydrothermal liquefaction process

Sustainable valorization of underutilized lignin and plastic waste beyond incineration is critical for producing renewable fuels. Hydrothermal liquefaction (HTL) provides a promising approach to convert wet lignin and plastics into high-quality oils without the need for prior drying of wet lignin. Here, this study examines the effects of temperature (300–400 °C), residence time, and polymer molecular weight on product yield and composition during HTL of polyethylene (PE), polypropylene (PP), and lignin under subcritical and supercritical water conditions. For PE, oil yields increased with temperature, with low molecular weight (LMW) PE achieving a maximum of ∼93 % at 400 °C for 30 min, while high molecular weight (HMW) PE reached ∼86 % at 400 °C for 1 h. Lignin and PP exhibited optimal oil yields at intermediate temperatures, decreasing at higher temperatures due to secondary reactions and char formation. Lignin yielded a maximum of ∼22 % oil at 300 °C for 30 min, LMW PP ∼88 % at 375 °C for 1 h, and HMW PP ∼83 % at 400 °C for 1 h. GC–MS analysis revealed that lignin oils were rich in phenols and alkylphenols, while PE and PP oils were dominated by paraffins and olefins. Oxygenated compounds in PE decreased at higher temperatures, improving oil quality. Co-liquefaction of lignin with plastics enhanced oil yields and energy content under specific conditions, demonstrating synergistic effects dependent on plastic type, molecular weight, and HTL conditions. These findings highlight the potential of HTL to produce high-energy, value-added fuels from lignin–plastic mixtures.

09 - BIOMASS FUELS↗
DOE OSTI2023

Lignin Derived Ionic Liquids: Synthesis and Application for Biopolymer Processing (CRADA Final Report)

We have established a scalable and economically viable process to convert lignin into ionic liquids for use in biopolymer processing. We have studied the four main topics: 1) lignin depolymerization to produce vanillin through oxidation, 2) lignin reduction to produce guaiacols, 3) oxidation process scale up, and 4) ionic liquid synthesis and application for biopolymer processing. The main accomplishment is summarized below: A maximal vanillin yield of 4.9% (8.8% total product yield) was achieved under optimal oxidation conditions using design of experiments (DOE); Aminophenol with 94.5% purity was synthesized via reductive amination using vanillin isolated from ion-exchange chromatography; At a higher lignin loading (10%), the vanillin yield in the scale up reaction was lower than the bench scale reaction; The filtration-based downstream separation of the scale-up process was able to recover 76% of vanillin; A maximal lignin reduction product (guaiacols) yield of 6.5% was achieved in the presence of Ru/C, formic acid, and methanol; The lignin reduction product yield and composition were affected by the use of different solvent and catalytic reagents (methanol in combination of Ru/C and formic acid gives the highest product yield); Lignin oxidation and reduction products: vanillin, acetovanillone, guaiacols, and eugenol are successfully used for IL synthesis; XX based ILs showed cellulose solubility with up to xx%; Technoeconomic analysis results indicate the potential of producing affordable ILs from kraft lignin (MSP: $14/kg); Life-cycle assessment results show the potential to reduce GHG emissions by up to 85% relative to existing ILs ([Ch][Lys]) with GHG emission: 1.2 kg CO 2e /kg of IL produced.

09 BIOMASS FUELS↗
DOE OSTI2024

An Engineered Laccase from Fomitiporia mediterranea Accelerates Lignocellulose Degradation

Laccases from white-rot fungi catalyze lignin depolymerization, a critical first step to upgrading lignin to valuable biodiesel fuels and chemicals. In this study, a wildtype laccase from the basidiomycete Fomitiporia mediterranea (Fom_lac) and a variant engineered to have a carbohydrate-binding module (Fom_CBM) were studied for their ability to catalyze cleavage of β-O-4′ ether and C–C bonds in phenolic and non-phenolic lignin dimers using a nanostructure-initiator mass spectrometry-based assay. Fom_lac and Fom_CBM catalyze β-O-4′ ether and C–C bond breaking, with higher activity under acidic conditions (pH < 6). The potential of Fom_lac and Fom_CBM to enhance saccharification yields from untreated and ionic liquid pretreated pine was also investigated. Adding Fom_CBM to mixtures of cellulases and hemicellulases improved sugar yields by 140% on untreated pine and 32% on cholinium lysinate pretreated pine when compared to the inclusion of Fom_lac to the same mixtures. Adding either Fom_lac or Fom_CBM to mixtures of cellulases and hemicellulases effectively accelerates enzymatic hydrolysis, demonstrating its potential applications for lignocellulose valorization. We postulate that additional increases in sugar yields for the Fom_CBM enzyme mixtures were due to Fom_CBM being brought more proximal to lignin through binding to either cellulose or lignin itself.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Multi-pass flow-through reductive catalytic fractionation

Reductive catalytic fractionation (RCF) is a promising lignin-first biorefining strategy that selectively extracts and depolymerizes lignin from whole biomass. Flow-through (FT)-RCF enables physical separation of the biomass and catalyst, but this process configuration typically operates at high solvent-to-biomass ratios. Here, we demonstrate multi-pass FT-RCF, wherein the lignin-enriched solvent obtained after an initial FT-RCF step is recycled and used in subsequent FT-RCF without intermediate lignin recovery. Multi-pass FT-RCF reduces the solvent-to-biomass ratio from 48 to 1.9 L/kg, which is a lower solvent loading than is accessible in batch reactors, without negative impacts on delignification and monomer yield with up to 12 wt % lignin in the solvent. Overall, this work demonstrates that solvent demand in RCF processes, which is a key cost and energy driver to enable this process at scale, can be reduced by recycling lignin oil between RCF reactions.

09 BIOMASS FUELS↗
DOE OSTI2025

From lignin to market: a technical and economic perspective of reductive depolymerization approaches

Lignin has grown into one of the main candidates to replace fossil-based resources as it is the largest renewable source of aromatic building blocks. The complex structure of polymeric lignin, however, requires depolymerization to simpler building blocks for the chemical industry. One of the most promising depolymerization approaches is reductive depolymerization of which two process configurations are currently studied in pilot scale installations for upscaling to industrial scale: (i) reductive catalytic fractionation (RCF), and (ii) reductive catalytic depolymerization (RCD). Both technical and techno-economic aspects will be covered within this review, discussing the advantages and challenges of both approaches regarding processing, production costs, product output, and applications. In this regard, RCF benefits from its decreased energy and solvent consumption linked with being a one-step process and delivers a product with a high monomer content (∼25–45 wt%). RCD, on the other hand, has the advantage of continuous processing and reduced catalyst fouling and delivers a product that mainly consists of oligomers (<10 wt% monomers). The complete overview of both processes presented here addresses their potential, and can guide future researchers, policy makers and companies to make thoughtful decisions on lignin valorization.

09 BIOMASS FUELS↗
DOE OSTI2022

Highly stable and tunable peptoid/hemin enzymatic mimetics with natural peroxidase-like activities

Abstract Developing tunable and stable peroxidase mimetics with high catalytic efficiency provides a promising opportunity to improve and expand enzymatic catalysis in lignin depolymerization. A class of peptoid-based peroxidase mimetics with tunable catalytic activity and high stability is developed by constructing peptoids and hemins into self-assembled crystalline nanomaterials. By varying peptoid side chain chemistry to tailor the microenvironment of active sites, these self-assembled peptoid/hemin nanomaterials (Pep/hemin) exhibit highly modulable catalytic activities toward two lignin model substrates 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 3,3’,5,5’-tetramethylbenzidine. Among them, a Pep/hemin complex containing the pyridyl side chain showed the best catalytic efficiency ( V max / K m = 5.81 × 10 −3 s −1 ). These Pep/hemin catalysts are highly stable; kinetics studies suggest that they follow a peroxidase-like mechanism. Moreover, they exhibit a high efficacy on depolymerization of a biorefinery lignin. Because Pep/hemin catalysts are highly robust and tunable, we expect that they offer tremendous opportunities for lignin valorization to high value products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Molecular simulation of lignin-related aromatic compound permeation through gram-negative bacterial outer membranes

Lignin, an abundant aromatic heteropolymer in secondary plant cell walls, is the single largest source of renewable aromatics in the biosphere. Leveraging this resource for renewable bioproducts through targeted microbial action depends on lignin fragment uptake by microbial hosts and subsequent enzymatic action to obtain the desired product. Recent computational work has emphasized that bacterial inner membranes are permeable to many aromatic compounds expected from lignin depolymerization processes. In this study, we expand on these findings through simulations for 42 lignin-related compounds across a gram-negative bacterial outer membrane model. Unbiased simulation trajectories indicate that spontaneous crossing for the full outer membrane is relatively rare at molecular simulation timescales, primarily due to preferential membrane partitioning and slow diffusion within the lipopolysaccharide layer within the outer membrane. Membrane partitioning and permeability coefficients were determined through replica exchange umbrella sampling simulations to overcome sampling limitations. We find that the glycosylated lipopolysaccharides found in the outer membrane increase the permeation barrier to many lignin-related compounds, particularly the most hydrophobic compounds. However, the effect is relatively modest; at industrially relevant concentrations, uncharged lignin-related compounds will readily diffuse across the outer membrane without the need for specific porins. Together, our results provide insight into the permeability of the bacterial outer membrane for assessing lignin fragment uptake and the future production of renewable bioproducts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

2.3.2.100 - Biological Lignin Valorization (BLV)

The Biological Lignin Valorization (BLV) project develops microbial strains and associated bioprocesses to convert lignin-derived aromatic compounds into value-added bioproducts. Our main objective in the BLV project is to achieve industrially relevant bioproduction metrics that can directly contribute to the economic viability and improved sustainability of the integrated lignocellulosic biorefinery, in collaboration with complementary BETO-funded lignin valorization projects. Specifically, the BLV project works closely with the BETO-funded Lignin Utilization project, which provides bio-available aromatic compounds from chemo-catalytic lignin depolymerization. We use the robust soil bacterium, Pseudomonas putida, as our primary microbial host for the conversion of lignin-derived compounds to bioproducts. To date, we have focused on atom-efficient bioproducts that can be used as either direct replacement chemicals or performance-advantaged bioproducts, including cis,cis-muconic acid, beta-ketoadipic acid, and 2-pyrone-4,6-dicarboxylic acid. From model aromatic substrates, we have achieved titers of each of these compounds approaching 40 g/L and productivity values ranging from 0.5 to over 1 g/L/hr, all at 90% molar yield or higher. From real lignin streams, we have thus far achieved 24 g/L, 0.66 g/L/hr, and theoretical yield of beta-ketoadipic acid. A major pursuit now is to reach industrially relevant performance metrics on an expanded slate of lignin-derived streams.

BIOMASS FUELS↗