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DOE OSTI2026

Understanding Electrochemical Alcohol Hydrogenolysis Enabled by Carbonyl Reduction in Lignocellulosic Biomass-Derived Aromatic Oxygenates

Molecules derived from lignocellulosic biomass are oxygenates with multiple oxygen-containing functional groups, such as hydroxyl and carbonyl groups. Therefore, the ability to selectively reduce a specific oxygenate group is essential for the reductive upgrading of such molecules. Previous studies on electrochemical biomass conversion have shown that alcohol hydrogenolysis, which involves cleavage of the σ(C–O alcohol ) bond, is extremely challenging for furfural and 5-hydroxymethylfurfural (HMF) derivatives, including furfuryl alcohol, 5-methylfurfuryl alcohol (MFA), and 2,5-bis(hydroxymethyl)furan (BHMF). In contrast, HMF itself undergoes alcohol hydrogenolysis relatively easily in acidic aqueous media. Considering that the only structural difference between HMF and BHMF or MFA is the presence of a carbonyl group, this observation raises the question of whether the carbonyl group in HMF facilitates alcohol hydrogenolysis. In this study, we designed systematic experiments to provide a coherent explanation of when and how a carbonyl group enables hydrogenolysis of a copresent alcohol group. Specifically, we show that alcohol hydrogenolysis in HMF proceeds via reduction of the carbonyl group to a ketyl radical, followed by a spin-center shift (SCS) through extended π conjugation. We also elucidate the effect of pH on the selectivity between carbonyl hydrogenation and alcohol hydrogenolysis, both of which share the ketyl radical intermediate. This mechanistic understanding enhances our ability to predict and control alcohol hydrogenolysis in the reductive upgrading of biomass-derived oxygenates.

alcohols↗
DOE OSTI2022

Lignocellulosic Biomass-Derived Nanocellulose Crystals as Fillers in Membranes for Water and Wastewater Treatment: A Review

The improvement of membrane applications for wastewater treatment has been a focal point of research in recent times, with a wide variety of efforts being made to enhance the performance, integrity and environmental friendliness of the existing membrane materials. Cellulose nanocrystals (CNCs) are sustainable nanomaterials derived from microorganisms and plants with promising potential in wastewater treatment. Cellulose nanomaterials offer a satisfactory alternative to other environmentally harmful nanomaterials. However, only a few review articles on this important field are available in the open literature, especially in membrane applications for wastewater treatment. This review briefly highlights the circular economy of waste lignocellulosic biomass and the isolation of CNCs from waste lignocellulosic biomass for membrane applications. The surface chemical functionalization technique for the preparation of CNC-based materials with the desired functional groups and properties is outlined. Recent uses of CNC-based materials in membrane applications for wastewater treatment are presented. In addition, the assessment of the environmental impacts of CNCs, cellulose extraction, the production techniques of cellulose products, cellulose product utilization, and their end-of-life disposal are briefly discussed. Furthermore, the challenges and prospects for the development of CNC from waste biomass for application in wastewater treatment are discussed extensively. Finally, this review unraveled some important perceptions on the prospects of CNC-based materials, especially in membrane applications for the treatment of wastewater.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Molecular simulation and artificial intelligence for the circular economy of bioenergy and bioproducts

The concept of the circular bioeconomy is a carbon neutral, sustainable system with zero waste. One vision for such an economy is based upon lignocellulosic biomass. This lignocellulosic circular bioeconomy requires CO 2 absorption from biomass growth and the efficient deconstruction of recalcitrant biomass into solubilized and fractionated biopolymers, which are then used as precursors for the sustainable production of high-quality liquid fuels, chemical bioproducts, and bio-based materials. Here, in this study, we summarize the roles that molecular dynamics (MD) simulations and machine learning (ML) are playing in overcoming several fundamental challenges hindering the adoption of a circular bioeconomy. Specifically, we discuss the role of MD and ML/AI in overcoming lignocellulose recalcitrance by designing biomass pretreatment methods to efficiently produce solubilized cellulose/lignin/hemicellulose and of that in improving energy-intensive manufacturing of biomass-based materials and their structural and mechanical properties. Quantum mechanical methods and MD simulations, in addition to offering a mechanistic understanding of biomass deconstruction and biomaterials design, can provide meaningful structural, energetics, and physiochemical properties as inputs to train AI/ML models. The ML models can guide the experimental prioritization of materials/solvents and process parameters that significantly accelerate the development of biofuel and biomaterial components of the circular bioeconomy.

Smith, Jeremy C. [Oak Ridge National Laboratory (O↗
DOE OSTI2022

Catalyst Deactivation and Its Mitigation during Catalytic Conversions of Biomass

Biofuel or biochemical production from biomass, especially lignocellulosic biomass, is the most promising option to replace fossil-based products to achieve sustainability. However, biomass is currently under-utilized because biomass conversion technologies have faced significant challenges to compete with incumbent petroleum technologies. Advancement in catalysis plays a central role in increasing the readiness of biomass conversion technologies. In this respect, improving catalyst stability is one of the well-known grand challenges for biomass conversion catalysis, which impedes the scaling up and commercialization of many biomass conversion techniques. In comparison to conventional processing of fossil fuels (petroleum, coal, and natural gas), biomass conversion is largely challenged by three unique properties of biomass-derived feedstocks—high water and oxygen content, high degree and reactivity of oxygen functionalization, and high contamination by minerals and heteroatoms—which all cause greater catalyst deactivation in different ways. Therefore, research on catalyst deactivation mitigation and catalyst regeneration is extremely important for the development of biomass conversion technologies. This review aims to highlight studies on catalyst deactivation and mitigation for catalytic processes in biomass conversion, with emphasis on the deactivation caused by the three unique characteristics of biomass-derived feedstocks. Further, this work will provide information on correlating the characteristics of biomass-derived streams, their potential impact on catalyst lifetime, and a potential mitigation approach, which could guide a more rational design of a robust catalyst and processes for biomass conversion.

09 BIOMASS FUELS↗
DOE OSTI2025

Rheological Properties of Enzymatically Hydrolyzed Corn Stover Pretreated via Deacetylation and Mechanical Refining

Lignocellulosic biomass is a feedstock for fuels and chemicals that does not compete with food resources and has less contaminants than refuse-derived biomass feedstocks. To convert lignocellulosics to biofuels or value-added products, multiple processing steps are typically necessary. One method of producing biofuels from lignocellulosic biomass utilizes a deacetylation and mechanical refining pretreatment and an enzymatic hydrolysis reaction to produce fermentable sugars from cellulose and hemicellulose. The rheological properties of biomass, such as yield stress and plastic viscosity, change during enzymatic hydrolysis and alter the energy requirements for pumping and mixing, an important consideration for the design of processing equipment. The dynamic changes in rheological properties that occur in a corn stover feedstock undergoing enzymatic hydrolysis are characterized in this work, and the influence on pressure losses in piping systems is estimated. Two rheometer geometries were fabricated with stereolithography 3D printing to reduce wall slip and sample ejection. The slurries have complex rheological behaviors that include shear-thinning behavior. Shear stress ramps were performed on samples at 20 and 50 degrees C using the custom geometries, and the Herschel-Bulkley model was fit to the data. The dynamic nature of the rheological properties is correlated with changes in the average fiber length at various extents of reaction, and the influence of solids concentration on the observed rheology and piping pressure losses is discussed.

09 BIOMASS FUELS↗
DOE OSTI2021

Center for direct catalytic conversion of biomass to biofuels (Final Technical Report)

Lignocellulosic biomass has only one-third the energy density of crude oil and lacks petroleum’s versatility as a feedstock for fuels and chemicals. Since 2009, the Center for direct Catalytic Conversion of Biomass to Biofuels (C3Bio) has recognized the potential of chemical catalysis and fast-pyrolysis to overcome such limitations by transforming the main components of biomass (cellulose, xylan, and lignin) from grasses and trees directly to liquid hydrocarbons and aromatic co-products. In 2014, C3Bio proposed to develop critical systems-level understanding of how biomass structural complexity at molecular, nanoscale, and mesoscale levels impacts the yields and selectivities of desired products from catalytic and pyrolytic transformations. Our long-term goal was to gain unprecedented control of effective routing of carbon: we aimed to specify both the structures within, and the reaction products from, lignocellulosic biomass. Enabled by the EFRC high-risk, high-reward approach to grand challenge science, our interdisciplinary team of plant biologists, chemists, and chemical engineers disrupted the conventional paradigm of the cellulosic biorefinery into a new future of “no carbon left behind” - the full utilization of carbon from plant cell walls in energy-dense fuels.

09 BIOMASS FUELS↗
DOE OSTI2026

CAZyme domain architectures suggest fine-scale functional differentiation among anaerobic fungi and bacteria during lignocellulose conversion to volatile fatty acids

Anaerobic fermentation with microbial communities (microbiomes) is an emerging platform for conversion of lignocellulosic biomass to biofuels and bioproducts. The process relies on diverse anaerobic microbes that interact to deconstruct and convert lignocellulosic biomass into a range of products, such as volatile fatty acids (VFAs), which can be achieved by arresting methanogenesis during fermentation. However, defining the distinct functional roles played by various fungi and bacteria during anaerobic biodegradation remains poorly understood. Here, we performed parallel enrichment experiments from cow faeces, goat faeces, and anaerobic digester sludge, selecting for fungal or bacterial dominated communities that convert sorghum biomass into VFAs. Subsequently we reconstructed metabolic networks across these enrichments based on recovered bacterial metagenome-assembled genomes (MAGs) and fungal isolate genomes and profiled their metabolic activity using metatranscriptomics to identify potential functional niches. Our findings implicate diverse bacteria affiliated with the Bacteroidales and Lachnospiraceae in the direct conversion of lignocellulosic biomass to propionate and butyrate, respectively, whereas Neocallimastix-dominated fungal enrichments converted lignocellulose to lactate, acetate and formate. Analysis of carbohydrate-active enzymes (CAZymes) revealed fine-scale differences between microbes that expressed unique multi-functional enzymes linking two or more CAZymes together with distinct carbohydrate binding motifs, implicating lignocellulose structure as a key driver of selection and niche differentiation. Most of these multi-functional enzymes localized complementary degradation functions together, likely conferring synergistic degradation effects within and between microbiome members. We anticipate that these findings will help inform efforts to develop synthetic microbiomes with tailored functionality for low-cost conversion of lignocellulosic biomass to fuels and bio-based chemicals.

Lawson, Christopher E [University of Toronto;]↗
DOE OSTI2023

Recent advances in biomass pretreatment using biphasic solvent systems

The complexity and recalcitrance of lignocellulosic biomass seriously hinder its subsequent conversion to liquid fuels. To achieve high-value utilization of lignocellulosic biomass, the physical–chemical barrier should be overcome through appropriate pretreatment techniques to improve the accessibility of cellulose for efficient enzymatic hydrolysis. With the rapid emergence of novel pretreatment solvents, biphasic solvent pretreatments represent a nascent and green pretreatment method that has shown outstanding advantages and broad application prospects in the biorefinery of lignocellulosic substrates due to its ability to provide economically viable biomass upgrading, the separation process for products in the solvent phase, and the reutilization of solvents. Herein, different types of biphasic solvents (e.g., 2-methyltetrahydrofuran, methyl isobutyl ketone, 1-butanol, phenoxyethanol, ionic liquids, and deep eutectic solvents) were reviewed systematically, including the fundamental designs of biphasic solvents for biomass pretreatment, their effect on the fractionation of individual biomass components (e.g., carbohydrate and lignin) and the enzymatic hydrolysis performance, and the coproduction of furan and hydroxymethylfurfural. Finally, the main pros and cons of these different biphasic solvent systems are summarized, and the future development direction is also proposed. Finally, this review can provide a reference for designing and selecting effective biphasic pretreatment methods for various types of lignocellulosic biomass.

09 BIOMASS FUELS↗
DOE OSTI2020

Measurement of moisture-dependent ion diffusion constants in wood cell wall layers using time-lapse micro X-ray fluorescence microscopy

Our future bioeconomy depends on increased utilization of renewable lignocellulosic biomass. Controlling the diffusion of chemicals, such as inorganic ions, within secondary plant cell walls is central to many biomass applications. However, insufficient understanding of intra-cell-wall diffusion within secondary plant cell walls is hindering the advancement of many lignocellulosic biomass applications. In this work, X-ray fluorescence microscopy was used to measure diffusion constants of K + , Cu 2+ , and Cl - diffusing through loblolly pine (Pinus taeda) cell wall layers under 70%, 75%, or 80% relative humidity (RH). Results revealed that diffusion constants increased with RH, the larger Cu 2+ diffused more slowly than the K + , and the Cl - diffusion constant was the same as that for the counter cation, indicating cations and anions diffused together to maintain charge neutrality. Comparison with electrical conductivity measurements showed that conductivity is being controlled by ion mobility over these RH. The results further support that intra-cell-wall diffusion of inorganic ions is a Fickian diffusion process occurring through rubbery amorphous polysaccharides, which contradicts previous assertions that intra-cell-wall diffusion is an aqueous process occurring through water pathways. Researchers can now utilize polymer science approaches to engineer the molecular architecture of lignocellulosic biomass to optimize properties for specific end uses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Supply Chain Sustainability Analysis of Renewable Hydrocarbon Fuels via Indirect Liquefaction, Ex Situ Catalytic Fast Pyrolysis, Hydrothermal Liquefaction, Combined Algal Processing, and Biochemical Conversion: Update of the 2020 State-of-Technology Cases

This technical report describes the SCSAs for the production of renewable hydrocarbon transportation fuels via a range of conversion technologies in the 2020 SOTs: (1) renewable high octane gasoline (HOG) via indirect liquefaction (IDL) of woody lignocellulosic biomass (note that the IDL pathway in this SCSA represents the syngas conversion design); (2) renewable gasoline (RG) and diesel (RD) blendstocks via ex situ catalytic fast pyrolysis of woody lignocellulosic biomass; (3) RD via hydrothermal liquefaction (HTL) of wet sludge from a wastewater treatment plant; (4) renewable hydrocarbon fuels via biochemical conversion of herbaceous lignocellulosic biomass; (5) renewable diesel via HTL of a blend of algae and woody biomass; and (6) renewable diesel via combined algae processing (CAP). This technical report focuses on the environmental performance of these six biofuel production pathways in their 2020 SOT cases. The results of these renewable hydrocarbon fuel pathways in these SCSA analyses update those for the respective 2019 SOT cases. They also provide an opportunity to examine the impact of technology improvements in both biomass feedstock production and biofuel production that have been achieved in 2020 SOTs on the sustainability performance of these renewable transportation fuels. The SCSA results also reflect updates to Argonne National Laboratory’s Greenhouse gases, Regulated Emissions, and Energy use in Technologies (GREET ® ) model, which was released in October 2020. These GREET updates include the production of natural gas, electricity, and petroleum-based fuels that can influence biofuels’ supply chain greenhouse gas (GHG) (CO 2 , CH 4 , and N 2 O) emissions, water consumption, and air pollutant emissions. GHG emissions, water consumption, and nitrogen oxides (NO x ) emissions are the main sustainability metrics assessed in this analysis. In this analysis, we define water consumption as the amount of water withdrawn from a freshwater source that is not returned (or returnable) to a freshwater source at the same level of quality. Life-cycle fossil energy consumption and net energy balance, which is the life-cycle fossil energy consumption deducted from the renewable biofuel energy produced, are also assessed.

09 BIOMASS FUELS↗
DOE OSTI2020

Supply Chain Sustainability Analysis of Renewable Hydrocarbon Fuels via Indirect Liquefaction, Ex Situ Catalytic Fast Pyrolysis, Hydrothermal Liquefaction, Combined Algal Processing, and Biochemical Conversion: Update of the 2019 State-of-Technology Cases

This technical report describes the supply chain sustainability analysis (SCSAs) for the production of renewable hydrocarbon transportation fuels via a range of conversion technologies: (1) renewable high octane gasoline (HOG) via indirect liquefaction (IDL) of woody lignocellulosic biomass (note that the IDL pathway in this SCSA represents the syngas conversion design in the 2019 SOT); (2) renewable gasoline (RG) and diesel (RD) blendstocks via ex situ catalytic fast pyrolysis of woody lignocellulosic biomass; (3) RD via hydrothermal liquefaction (HTL) of wet sludge from a wastewater treatment plant; (4) renewable hydrocarbon fuels via biochemical conversion of herbaceous lignocellulosic biomass; (5) renewable diesel via HTL of a blend of algae and woody biomass; and (6) renewable diesel via combined algae processing (CAP). This technical report focuses on the environmental performance of these six biofuel production pathways in their 2019 SOT cases. The results of these renewable hydrocarbon fuel pathways in these SCSA analyses update those for the respective 2018 SOT cases. They also provide an opportunity to examine the impact of technology improvements in both biomass feedstock production and biofuel production that have been achieved in 2019 SOTs on the sustainability performance of these renewable transportation fuels, and they reflect updates to Argonne National Laboratory’s Greenhouse gases, Regulated Emissions, and Energy use in Transportation (GREET®) model, which was released in October 2019. These GREET updates include the production of natural gas, electricity, and petroleum-based fuels that can influence biofuels’ supply chain greenhouse gas (GHG) (CO₂, CH₄, and N₂O) emissions, water consumption, and air pollutant emissions. GHG emissions, water consumption, and nitrogen oxides (NO x ) emissions are the main sustainability metrics assessed in this analysis. In this analysis, we define water consumption as the amount of water withdrawn from a freshwater source that is not returned (or returnable) to a freshwater source at the same level of quality. Life-cycle fossil energy consumption and net energy balance, which is the life-cycle fossil energy consumption deducted from the renewable biofuel energy produced, are also assessed.

09 BIOMASS FUELS↗
DOE OSTI2021

A multiplexed nanostructure-initiator mass spectrometry (NIMS) assay for simultaneously detecting glycosyl hydrolase and lignin modifying enzyme activities

Lignocellulosic biomass is composed of three major biopolymers: cellulose, hemicellulose and lignin. Analytical tools capable of quickly detecting both glycan and lignin deconstruction are needed to support the development and characterization of efficient enzymes/enzyme cocktails. Previously we have described nanostructure-initiator mass spectrometry-based assays for the analysis of glycosyl hydrolase and most recently an assay for lignin modifying enzymes. Here we integrate these two assays into a single multiplexed assay against both classes of enzymes and use it to characterize crude commercial enzyme mixtures. Application of our multiplexed platform based on nanostructure-initiator mass spectrometry enabled us to characterize crude mixtures of laccase enzymes from fungi Agaricus bisporus (Ab) and Myceliopthora thermophila (Mt) revealing activity on both carbohydrate and aromatic substrates. Using time-series analysis we determined that crude laccase from Ab has the higher GH activity and that laccase from Mt has the higher activity against our lignin model compound. Inhibitor studies showed a significant reduction in Mt GH activity under low oxygen conditions and increased activities in the presence of vanillin (common GH inhibitor). Ultimately, this assay can help to discover mixtures of enzymes that could be incorporated into biomass pretreatments to deconstruct diverse components of lignocellulosic biomass.

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

CRISPR/Cas9 editing of p-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE 1 in maize alters phenolic metabolism, lignin structure, and lignin-first biomass processing

Valorization of lignocellulosic biomass for sustainable production of high-value chemicals is challenged by the complexity of lignin, a phenolic biopolymer. Beyond the classical lignin monomers derived from p-coumaryl, coniferyl, and sinapyl alcohol, grass lignins incorporate substantial amounts of monolignol p-coumarates that are produced by p-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE (PMT). Here, the CRISPR/Cas9-mediated mutation of ZmPMT1 in maize enabled the design of biomass depleted in p-coumaroylated lignin and enriched in guaiacyl lignin. Lignin-first biorefining of stem biomass from zmpmt1 mutants by reductive catalytic fractionation (RCF) generated a lignin oil depleted in carboxylates and enriched in guaiacyl-derived alcohols, which are desirable substrates for bio-based polyurethane synthesis. Furthermore, the reported lignin engineering in maize is a promising strategy for designing a dual-purpose crop, providing both food and feed, along with a renewable feedstock for the production of plant-based chemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Supply Chain Sustainability Analysis of Renewable Hydrocarbon Fuels via Indirect Liquefaction, Ex Situ Catalytic Fast Pyrolysis, Hydrothermal Liquefaction, Combined Algal Processing, and Biochemical Conversion: Update of the 2020 State-of-Technology Cases

The Department of Energy’s (DOE) Bioenergy Technologies Office (BETO) aims to develop and deploy technologies to transform renewable biomass resources into commercially viable, high-performance biofuels, bioproducts, and biopower through public and private partnerships (U.S. Department of Energy, 2016). BETO and its national laboratory teams conduct in-depth techno-economic assessments (TEA) of biomass feedstock supply and logistics and conversion technologies to produce biofuels. There are two general types of TEAs: A design case outlines a target case (future projection) for a particular biofuel pathway. It enables identification of data gaps and research and development needs and provides goals and benchmarks against which technology progress is assessed. A state of technology (SOT) analysis assesses progress within and across relevant technology areas based on actual results at current experimental scales relative to technical targets and cost goals from design cases, and includes technical, economic, and environmental criteria as available. In addition to developing a TEA for a pathway of interest, BETO also performs a supply chain sustainability analysis (SCSA). The SCSA takes the life-cycle analysis approach that BETO has been supporting for about 20 years. It enables BETO to identify energy consumption, environmental, and sustainability issues that may be associated with biofuel production. Approaches to mitigate these issues can then be developed. Additionally, the SCSA allows for comparison of energy and environmental impacts across biofuel pathways in BETO’s research and development portfolio. This technical report describes the SCSAs for the production of renewable hydrocarbon transportation fuels via a range of conversion technologies in the 2020 SOTs: (1) renewable high octane gasoline (HOG) via indirect liquefaction (IDL) of woody lignocellulosic biomass (note that the IDL pathway in this SCSA represents the syngas conversion design [Harris et al. 2021]); (2) renewable gasoline (RG) and diesel (RD) blendstocks via ex situ catalytic fast pyrolysis of woody lignocellulosic biomass [Abhijit et al. 2021]; (3) RD via hydrothermal liquefaction (HTL) of wet sludge from a wastewater treatment plant; (4) renewable hydrocarbon fuels via biochemical conversion of herbaceous lignocellulosic biomass (Davis et al. 2021; Lin et al. 2021); (5) renewable diesel via HTL of a blend of algae (Davis and Klein, 2021) and woody biomass (Hartley et al. 2020); and (6) renewable diesel via combined algae processing (CAP) (Wiatrowski and Davis, 2021). This technical report focuses on the environmental performance of these six biofuel production pathways in their 2020 SOT cases. The results of these renewable hydrocarbon fuel pathways in these SCSA analyses update those for the respective 2019 SOT cases (Cai et al. 2020). They also provide an opportunity to examine the impact of technology improvements in both biomass feedstock production and biofuel production that have been achieved in 2020 SOTs on the sustainability performance of these renewable transportation fuels. The SCSA results also reflect updates to Argonne National Laboratory’s Greenhouse gases, Regulated Emissions, and Energy use in Technologies (GREET®) model, which was released in October 2020 (Wang et al. 2020). These GREET updates include the production of natural gas, electricity, and petroleum-based fuels that can influence biofuels’ supply chain greenhouse gas (GHG) (CO 2 , CH 4 , and N 2 O) emissions, water consumption, and air pollutant emissions. GHG emissions, water consumption, and nitrogen oxides (NO x ) emissions are the main sustainability metrics assessed in this analysis. In this analysis, we define water consumption as the amount of water withdrawn from a freshwater source that is not returned (or returnable) to a freshwater source at the same level of quality. Life-cycle fossil energy consumption and net energy balance, which is the life-cycle fossil energy consumption deducted from the renewable biofuel energy produced, are also assessed.

09 BIOMASS FUELS↗
DOE OSTI2023

Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization in Pseudomonas putida M2

ABSTRACT Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds ( p- coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of crc expression via CRISPRi led to faster growth and glucose and p -coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+ p -coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose+ p -coumarate) condition compared to when repression was absent ( p -coumarate or glucose only). IMPORTANCE A newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Multi-Omics Driven Metabolic Network Reconstruction and Analysis of Lignocellulosic Carbon Utilization in Rhodosporidium toruloides

An oleaginous yeast Rhodosporidium toruloides is a promising host for converting lignocellulosic biomass to bioproducts and biofuels. In this work, we performed multi-omics analysis of lignocellulosic carbon utilization in R. toruloides and reconstructed the genome-scale metabolic network of R. toruloides . High-quality metabolic network models for model organisms and orthologous protein mapping were used to build a draft metabolic network reconstruction. The reconstruction was manually curated to build a metabolic model using functional annotation and multi-omics data including transcriptomics, proteomics, metabolomics, and RB-TDNA sequencing. The multi-omics data and metabolic model were used to investigate R. toruloides metabolism including lipid accumulation and lignocellulosic carbon utilization. The developed metabolic model was validated against high-throughput growth phenotyping and gene fitness data, and further refined to resolve the inconsistencies between prediction and data. We believe that this is the most complete and accurate metabolic network model available for R. toruloides to date.

09 BIOMASS FUELS↗
DOE OSTI2021

Towards cost-competitive middle distillate fuels from ethanol within a market-flexible C2 platform-based biorefinery concept

Ethanol to middle distillates (ETMD) is a promising pathway to produce sustainable liquid fuels to decarbonize the hard-to-electrify transportation sectors due to (1) the abundant sugar/starch and lignocellulosic biomass, (2) the existing deployment scale of fuel ethanol production (similar to 29 billion gallons per year globally), and (3) emerging opportunities in C2+ alcohol synthesis from CO2. Here we report a conceptual market-responsive biorefinery centered around a new ETMD pathway based on one-step ethanol to butene-rich olefins (ETO) over a Cu-Zn-Y/Beta catalyst. Specifically, this ethanol conversion pathway comprises one-step ETO, oligomerization, and hydrotreating. This ETO is distinct from that in the conventional ethanol-to-jet process which is based on two-step ethanol to ethylene and ethylene oligomerization to butenes. Butene-rich olefins can be shifted to butadiene-rich products by simply changing the reaction atmosphere from hydrogen to inert gas over the same ETO catalyst. Leveraging the experimental results, baseline techno-economic analysis (TEA) and sensitivity analysis indicate that the ethanol conversion cost is $0.60 per gallon gasoline equivalent (GGE), with opportunities for further cost reduction via improving the liquid hydrocarbon yield and space velocities, and process optimization on balancing dewatering of ethanol feed prior to the ETO step. The minimum fuel selling price (MFSP) of liquid hydrocarbons derived from corn starch ethanol with butadiene as coproduct is $1.64 per GGE, in the range that is cost competitive with petroleum kerosene-type jet fuel. Projected MFSP for cellulosic ethanol (corn stover) derived hydrocarbons is below $3.00 per GGE and co-production of butadiene further reduces the MFSP to $1.70 per GGE. The Well-to-Wake life-cycle analysis indicates that 85% greenhouse gas emission reduction can be achieved when using corn stover compared to petroleum reference and the associated carbon credits will provide significant economic incentives to favor the cellulosic ethanol-derived hydrocarbon fuels. This study demonstrates a low-cost pathway to middle distillate fuels leveraging existing ethanol infrastructure, where catalysis innovation drives the reduction of process complexity and flexible coproduction of a value-added chemical product.

Zhang, Junyan↗
DOE OSTI2023

Overexpression of REDUCED WALL ACETYLATION C increases xylan acetylation and biomass recalcitrance in Populus

Abstract Plant lignocellulosic biomass, i.e. secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases the level of xylan acetylation and increases the lignin content and S/G ratio, ultimately yielding poplar woody biomass with reduced saccharification efficiency. Furthermore, through gene coexpression network and expression quantitative trait loci (eQTL) analysis, we found that PtRWA-C was regulated not only by the secondary cell wall hierarchical regulatory network but also by an AP2 family transcription factor HARDY (HRD). Specifically, HRD activates PtRWA-C expression by directly binding to the PtRWA-C promoter, which is also the cis-eQTL for PtRWA-C. Taken together, our findings provide insights into the functional roles of PtRWA-C in xylan acetylation and consequently saccharification and shed light on synthetic biology approaches to manipulate this gene and alter cell wall properties. These findings have substantial implications for genetic engineering of woody species, which could be used as a sustainable source of biofuels, valuable biochemicals, and biomaterials.

59 BASIC BIOLOGICAL SCIENCES↗