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At least 343 records · Page 19
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Engineered Accumulation of Protocatechuate in Corn Biomass to Enhance Biomanufacturing

The in-planta accumulation of coproducts in crops can enhance the value of lignocellulosic biomass and facilitate a sustainable bioeconomy. Corn stover represents a major renewable source of lignocellulose for the production of advanced biofuels and bioproducts. In this study, we engineered corn with a bacterial gene encoding a dehydroshikimate dehydratase (QsuB) to overproduce protocatechuate (DHBA). Transgenic corn lines accumulate up to 2.9% DHBA on a dry weight basis in leaf and stem biomass. DHBA occurs in the form of glucosides that are extractable from biomass using aqueous methanol as the solvent. The analysis of lignin did not show any evidence for the incorporation of DHBA; however, an increase in the lignin syringyl to guaiacyl ratio and a higher relative abundance of p-coumarate groups compared with total lignin units were observed in QsuB-modified corn. Alkaline hydrolysates prepared from QsuB corn were enriched in DHBA compared to the hydrolysates obtained from wild-type biomass, which contained mostly p-coumarate and ferulate. Using engineered Novosphingobium aromaticivorans as a production host, a 375% improvement in 2-pyrone-4,6-dicarboxylate titers was achieved through biological upgrading of alkaline hydrolysates derived from QsuB corn compared to unmodified biomass. Our data demonstrate an engineering strategy to overproduce DHBA in corn that can facilitate sustainable manufacturing of other valuable bioproducts using stover as a feedstock.

2-pyrone-4,6-dicarboxylate↗
DOE OSTI2022

Muconic acid production from glucose and xylose in Pseudomonas putida via evolution and metabolic engineering

Muconic acid is a bioprivileged molecule that can be converted into direct replacement chemicals for incumbent petrochemicals and performance-advantaged bioproducts. In this study, Pseudomonas putida KT2440 is engineered to convert glucose and xylose, the primary carbohydrates in lignocellulosic hydrolysates, to muconic acid using a model-guided strategy to maximize the theoretical yield. Using adaptive laboratory evolution (ALE) and metabolic engineering in a strain engineered to express the D-xylose isomerase pathway, we demonstrate that mutations in the heterologous D-xylose:H + symporter (XylE), increased expression of a major facilitator superfamily transporter (PP_2569), and overexpression of aroB encoding the native 3-dehydroquinate synthase, enable efficient muconic acid production from glucose and xylose simultaneously. Using the rationally engineered strain, we produce 33.7 g L -1 muconate at 0.18 g L -1 h -1 and a 46% molar yield (92% of the maximum theoretical yield). This engineering strategy is promising for the production of other shikimate pathway-derived compounds from lignocellulosic sugars.

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 OSTI2022

Understanding the structure and composition of recalcitrant oligosaccharides in hydrolysate using high-throughput biotin-based glycome profiling and mass spectrometry

Novel Immunological and Mass Spectrometry Methods for Comprehensive Analysis of Recalcitrant Oligosaccharides in AFEX Pretreated Corn Stover. Lignocellulosic biomass is a sustainable alternative to fossil fuel and is extensively used for developing bio-based technologies to produce products such as food, feed, fuel, and chemicals. The key to these technologies is to develop cost competitive processes to convert complex carbohydrates present in plant cell wall to simple sugars such as glucose, xylose, and arabinose. Since lignocellulosic biomass is highly recalcitrant, it must undergo a combination of thermochemical treatment such as Ammonia Fiber Expansion (AFEX), dilute acid (DA), Ionic Liquid (IL) and biological treatment such as enzyme hydrolysis and microbial fermentation to produce desired products. However, when using commercial fungal enzymes during hydrolysis, only 75–85% of the soluble sugars generated are monomeric sugars, while the remaining 15–25% are soluble recalcitrant oligosaccharides that cannot be easily utilized by microorganisms. Previously, we successfully separated and purified the soluble recalcitrant oligosaccharides using a combination of charcoal and celite-based separation followed by size exclusion chromatography and studies their inhibitory properties on enzymes. We discovered that the oligosaccharides with higher degree of polymerization (DP) containing methylated uronic acid substitutions were more recalcitrant towards commercial enzyme mixtures than lower DP and neutral oligosaccharides. Here, we report the use of several complementary techniques that include glycome profiling using plant biomass glycan specific monoclonal antibodies (mAbs) to characterize sugar linkages in plant cell walls and enzymatic hydrolysate, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using structurally-informative diagnostic peaks offered by negative ion post-secondary decay spectra, gas chromatography followed by mass spectrometry (GC–MS) to characterize oligosaccharide sugar linkages with and without derivatization. Since oligosaccharides (DP 4–20) are small, it is challenging to mobilize these molecules for mAbs binding and characterization. To overcome this problem, we have applied a new biotin-coupling based oligosaccharide immobilization method that successfully tagged most of the low DP soluble oligosaccharides on to a micro-plate surface followed by specific linkage analysis using mAbs in a high-throughput system. This new approach will help develop more advanced versions of future high throughput glycome profiling methods that can be used to separate and characterize oligosaccharides present in biomarkers for diagnostic applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Electron tomography unravels new insights into fiber cell wall nanostructure; exploring 3D macromolecular biopolymeric nano-architecture of spruce fiber secondary walls

Lignocellulose biomass has a tremendous potential as renewable biomaterials for fostering the “bio-based society” and circular bioeconomy paradigm. It requires efficient use and breakdown of fiber cell walls containing mainly cellulose, hemicellulose and lignin biopolymers. Despite their great importance, there is an extensive debate on the true structure of fiber walls and knowledge on the macromolecular nano-organization is limited and remains elusive in 3D. We employed dual-axis electron tomography that allows visualization of previously unseen 3D macromolecular organization/biopolymeric nano-architecture of the secondary S2 layer of Norway spruce fiber wall. Unprecedented 3D nano-structural details with novel insights into cellulose microfibrils (~2 nm diameter), macrofibrils, nano-pore network and cell wall chemistry (volume %) across the S2 were explored and quantified including simulation of structure related permeability. Matrix polymer association with cellulose varied between microfibrils and macrofibrils with lignin directly associated with MFs. Simulated bio-nano-mechanical properties revealed stress distribution within the S2 and showed similar properties between the idealized 3D model and the native S2 (actual tomogram). Present work has great potential for significant advancements in lignocellulose research on nano-scale understanding of cell wall assembly/disassembly processes leading to more efficient industrial processes of functionalization, valorization and target modification technologies.

3-D reconstruction↗
DOE OSTI2020

Ammonia-salt solvent promotes cellulosic biomass deconstruction under ambient pretreatment conditions to enable rapid soluble sugar production at ultra-low enzyme loadings

In this paper, we report a novel ammonia : ammonium salt solvent based pretreatment process that can rapidly dissolve crystalline cellulose into solution and eventually produce highly amorphous cellulose under near-ambient conditions. Pre-activating the cellulose I allomorph to its ammonia–cellulose swollen complex (or cellulose III allomorph) at ambient temperatures facilitated rapid dissolution of the pre-activated cellulose in the ammonia-salt solvent ( i.e. , ammonium thiocyanate salt dissolved in liquid ammonia) at ambient pressures. For the first time in reported literature, we used time-resolved in situ neutron scattering methods to characterize the cellulose polymorphs structural modification and understand the mechanism of crystalline cellulose dissolution into a ‘molecular’ solution in real-time using ammonia-salt solvents. We also used molecular dynamics simulations to provide insight into solvent interactions that non-covalently disrupted the cellulose hydrogen-bonding network and understand how such solvents are able to rapidly and fully dissolve pre-activated cellulose III. Importantly, the regenerated amorphous cellulose recovered after pretreatment was shown to require nearly ~50-fold lesser cellulolytic enzyme usage compared to native crystalline cellulose I allomorph for achieving near-complete hydrolytic conversion into soluble sugars. Lastly, we provide proof-of-concept results to further showcase how such ammonia-salt solvents can pretreat and fractionate lignocellulosic biomass like corn stover under ambient processing conditions, while selectively co-extracting ~80–85% of total lignin, to produce a highly digestible polysaccharide-enriched feedstock for biorefinery applications. Unlike conventional ammonia-based pretreatment processes ( e.g. , Ammonia Fiber Expansion or Extractive Ammonia pretreatments), the proposed ammonia-salt process can operate at near-ambient conditions to greatly reduce the pressure/temperature severity necessary for conducting effective ammonia-based pretreatments on lignocellulose.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Ethanol to diesel: a sustainable alternative for the heavy-duty transportation sector

The combustion of middle distillates (diesel and jet fuel) is responsible for the emission of more than 2 GTon of CO 2 per year worldwide. While sustainable alternatives exist for gasoline and jet fuel, we still lack sustainable alternatives for diesel. This fact is especially relevant if we consider that electrification of sectors where diesel is used is challenging. One sustainable approach for diesel production is the catalytic upgrading of ethanol. While most work in this field has focused on the dehydration/oligomerization of ethanol, this approach is limited to producing fuels that have a high degree of branching and low cetane number. Another approach is the sequential use of ethanol Guerbet coupling, leading to higher alcohols, followed by etherification, leading to large ethers which, importantly, result in a product with high cetane number. In this work, we explore the catalytic upgrading of ethanol into diesel following an approach based on the initial Guerbet coupling of ethanol followed by etherification. The results presented are a collaborative and synergic effort among process and systems engineers, experimentalist in the area of catalysis, and fuel property modelers. Here, we demonstrate experimentally the feasibility of upgrading ethanol into a diesel fuel with properties that surpass its fossil counterpart. The diesel produced has a predicted cetane number of ~70 and outstanding cold flow properties, while maintaining other properties (viscosity, density, and flash point) within expected ranges. A technoeconomic analysis performed based on a detailed biorefinery model shows that the MFSP is ~5.89$ per Gal in 2021 dollars when lignocellulosic ethanol is used, with the most relevant economic driver the cost of the ethanol feedstock. The upgrading process can be performed with a net energy gain (EROI = 1.49 > 1). An LCA analysis of greenhouse gas (GHG) emissions reveals that the use of lignocellulosic ethanol may lead to more than 50% reduction in GHG emissions compared to fossil diesel. Depending on the CO 2 emissions associated with the production of ethanol we show that in some instances the production of carbon neutral diesel fuel is possible.

09 BIOMASS FUELS↗
DOE OSTI2025

Evaluating the industrial potential of emerging biomass pretreatment technologies in bioethanol production and lipid recovery from transgenic sugarcane

The selection of pretreatment methods is critical to achieving high product yields during bioconversion of lignocellulosic biomass. Hydrothermal, soaking-in-aqueous ammonia, and ionic liquid pretreatment methods are viable candidates for minimizing sugar decomposition, permitting the effective hydrolysis of structural carbohydrates, and producing a fermentable substrate suitable for achieving industrial ethanol titers and yields. In this study, the effect of these three pretreatment methods on non-modified sugarcane cultivar CP88-1762 and two transgenic lipid-accumulating sugarcane lines, oilcane 1565 and oilcane 1566, were investigated and compared in terms of lipid recovery, sugar yield, and ethanol yields within the lignocellulosic biomass conversion pipeline. Fed-batch enzymatic hydrolysis at high solid loading yielded hydrolysates capable of supporting industrial bioethanol titers across all conditions. The highest sugar yields were obtained on ammonia-pretreated biomass hydrolysate (253.73 g L −1 ), followed by hydrothermally pretreated hydrolysate (213.10 g L −1 ) and ionic liquid-pretreated hydrolysate (154.20 g L −1 ). Commercially viable ethanol titers of 100.62 g L −1 , 64.47 g L −1 , and 52.95 g L −1 were achieved from ammonia, hydrothermal, and ionic liquid pretreated hydrolysate with the corresponding ethanol productivities of 2.08 g L −1 h −1 , 0.53 g L −1 h −1 , and 0.36 g L −1 h −1 . The lower acetic acid concentration in ammonia-pretreated hydrolysate may have enhanced its fermentability relative to the hydrothermal pretreatment condition, as indicated by the differences in ethanol titer and productivity. Lower sugar yields and ethanol productivities under the ionic liquid conditions likely resulted from the inhibitory effect of cholinium lysinate. Oilcane 1565 and oilcane 1566 bagasse accumulated over 16- and 3 times higher lipids than the non-modified sugarcane CP88-1762. The total fatty acid content in the oilcane samples was reduced in ammonia and ionic liquid-pretreated bagasse relative to the hydrothermal pretreatment condition. While all pretreatment techniques tested are industrially viable, the observed differences in titer, productivity, and lipid content indicate that careful selection and validation of upstream processing methods can contribute to improved economic and environmental outcomes.

09 BIOMASS FUELS↗
DOE OSTI2021

The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein–encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain ?cre-1, while glucose repression was still mostly functional in ?exo-1. Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in ?exo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in ?exo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

Gabriel, Raphael↗
DOE OSTI2021

Evolutionary innovations through gain and loss of genes in the ectomycorrhizal Boletales

Summary We aimed to identify genomic traits of transitions to ectomycorrhizal ecology within the Boletales by comparing the genomes of 21 symbiotrophic species with their saprotrophic brown‐rot relatives. Gene duplication rate is constant along the backbone of Boletales phylogeny with large loss events in several lineages, while gene family expansion sharply increased in the late Miocene, mostly in the Boletaceae. Ectomycorrhizal Boletales have a reduced set of plant cell‐wall‐degrading enzymes (PCWDEs) compared with their brown‐rot relatives. However, the various lineages retain distinct sets of PCWDEs, suggesting that, over their evolutionary history, symbiotic Boletales have become functionally diverse. A smaller PCWDE repertoire was found in Sclerodermatineae. The gene repertoire of several lignocellulose oxidoreductases (e.g. laccases) is similar in brown‐rot and ectomycorrhizal species, suggesting that symbiotic Boletales are capable of mild lignocellulose decomposition. Transposable element (TE) proliferation contributed to the higher evolutionary rate of genes encoding effector‐like small secreted proteins, proteases, and lipases. On the other hand, we showed that the loss of secreted CAZymes was not related to TE activity but to DNA decay. This study provides novel insights on our understanding of the mechanisms influencing the evolutionary diversification of symbiotic boletes.

Wu, Gang↗
DOE OSTI2020

Rewired phenolic metabolism and improved saccharification efficiency of a Zea mays cinnamyl alcohol dehydrogenase 2 (zmcad2) mutant

Lignocellulosic biomass is an abundant byproduct from cereal crops that can potentially be valorized as a feedstock to produce biomaterials. Zea mays CINNAMYL ALCOHOL DEHYDROGENASE 2 (ZmCAD2) is involved in lignification, and is a promising target to improve the cellulose–to–glucose conversion of maize stover. Here, we analyzed a field–grown zmcad2 Mutator transposon insertional mutant. Zmcad2 mutant plants had an 18% lower Klason lignin content, whereas their cellulose content was similar to that of control lines. The lignin in zmcad2 mutants contained increased levels of hydroxycinnamaldehydes, i.e. the substrates of ZmCAD2, ferulic acid and tricin. Ferulates decorating hemicelluloses were not altered. Phenolic profiling further revealed that hydroxycinnamaldehydes are partly converted into (dihydro)ferulic acid and sinapic acid and their derivatives in zmcad2 mutants. Syringyl lactic acid hexoside, a metabolic sink in CAD–deficient dicot trees, appeared not to be a sink in zmcad2 maize. The enzymatic cellulose–to–glucose conversion efficiency was determined after 10 different thermochemical pre–treatments. Zmcad2 yielded significantly higher conversions compared with controls for almost every pre–treatment. However, the relative increase in glucose yields after alkaline pre–treatment was not higher than the relative increase when no pre–treatment was applied, suggesting that the positive effect of the incorporation of hydroxycinnamaldehydes was leveled off by the negative effect of reduced p–coumarate levels in the cell wall. Here, taken together, our results reveal how phenolic metabolism is affected in CAD–deficient maize, and further support mutating CAD genes in cereal crops as a promising strategy to improve lignocellulosic biomass for sugar–platform biorefineries.

59 BASIC BIOLOGICAL SCIENCES↗
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 OSTI2024

yuyao-jia/Bio-based_HMF_coproduction

5-Hydroxymethyl furfurals (HMF) is one of the versatile platform chemicals. However, green routes to produce it directly from lignocellulosic biomass are lacking. A significant amount of HMF produced during the hydrothermal valorization of lignocellulosic biomass is considered undesired and ends up in a waste stream. The study transformed the undesired byproduct into a valuable coproduct by advancing the existing biofuel production process. A detailed economic and environmental sustainability analysis of the integrated biorefinery design was performed. The evaluation showed that the biorefinery could afford a maximum feedstock purchasing price of $115.17 per MT and produce HMF with a minimum selling price of $4.54 per kg which is ∼75% lower than the commercial price of HMF. The median global warming potential of HMF was estimated to be 3.92 kg CO2-eq. per kg HMF which was ∼32% less than its counterpart bio-based p-xylene. Diverse coproducts produced in the biorefinery using transgenic feedstock positively impacted sustainability.

Jia, Yuyao↗
DOE OSTI2023

Partial wet oxidation of dairy manure as a pretreatment process to produce acetic acid ‘a Source Growth of Methanogens’

Wet oxidation can be an effective process for the pretreatment of complex biomass such as lignocellulose. However, studies on the use of wet oxidation for treating solid waste such as dairy manure are limited. The use of partial wet oxidation to convert dairy manure into low molecular weight carboxylic acids as final products were investigated. This work focuses on the performance of the sub-critical wet oxidation treatment of dairy cattle manure as a conversion/pretreatment process to release matter from the lignocellulosic fraction rather than a destructive process. The operating conditions were controlled at the short residence time and optimal temperature in the presence of oxygen under a pressure of 120 psi. The thermal hydrolysis under wet oxidation significantly affected conversion manure slurry into organic acids. The concentration of acetic acid reached 1778 mg L –1 , achieved at 190°C (60 minutes reaction time) as the reaction temperature increased within the range of 150°C–200°C, total organic carbon was reduced and monomers in the process liquids decreased. On the other hand, soluble COD in process liquids increased with an increment in reaction temperature. The results provide insights into technical options to pretreat dairy manure to improve biochemical conversion yield.

09 BIOMASS FUELS↗
DOE OSTI2020

Conversion of poplar biomass into high-energy density tricyclic sesquiterpene jet fuel blendstocks

In an effort to ensure future energy security, reduce greenhouse gas emissions and create domestic jobs, the US has invested in technologies to develop sustainable biofuels and bioproducts from renewable carbon sources such as lignocellulosic biomass. Bio-derived jet fuel is of particular interest as aviation is less amenable to electrification compared to other modes of transportation and synthetic biology provides the ability to tailor fuel properties to enhance performance. Specific energy and energy density are important properties in determining the attractiveness of potential bio-derived jet fuels. For example, increased energy content can give the industry options such as longer range, higher load or reduced takeoff weight. Energy-dense sesquiterpenes have been identified as potential next-generation jet fuels that can be renewably produced from lignocellulosic biomass. We developed a biomass deconstruction and conversion process that enabled the production of two tricyclic sesquiterpenes, epi-isozizaene and prespatane, from the woody biomass poplar using the versatile basidiomycete Rhodosporidium toruloides. We demonstrated terpene production at both bench and bioreactor scales, with prespatane titers reaching 1173.6 mg/L when grown in poplar hydrolysate in a 2 L bioreactor. Additionally, we examined the theoretical fuel properties of prespatane and epi-isozizaene in their hydrogenated states as blending options for jet fuel, and compared them to aviation fuel, Jet A. Our findings indicate that prespatane and epi-isozizaene in their hydrogenated states would be attractive blending options in Jet A or other lower density renewable jet fuels as they would improve viscosity and increase their energy density. Saturated epi-isozizaene and saturated prespatane have energy densities that are 16.6 and 18.8% higher than Jet A, respectively. These results highlight the potential of R. toruloides as a production host for the sustainable and scalable production of bio-derived jet fuel blends, and this is the first report of prespatane as an alternative jet fuel.

09 BIOMASS FUELS↗
DOE OSTI2021

Solid-state NMR of unlabeled plant cell walls: high-resolution structural analysis without isotopic enrichment

Abstract Background Multidimensional solid-state nuclear magnetic resonance (ssNMR) spectroscopy has emerged as an indispensable technique for resolving polymer structure and intermolecular packing in primary and secondary plant cell walls. Isotope ( 13 C) enrichment provides feasible sensitivity for measuring 2D/3D correlation spectra, but this time-consuming procedure and its associated expenses have restricted the application of ssNMR in lignocellulose analysis. Results Here, we present a method that relies on the sensitivity-enhancing technique Dynamic Nuclear Polarization (DNP) to eliminate the need for 13 C-labeling. With a 26-fold sensitivity enhancement, a series of 2D 13 C– 13 C correlation spectra were successfully collected using the unlabeled stems of wild-type Oryza sativa (rice). The atomic resolution allows us to observe a large number of intramolecular cross peaks for fully revealing the polymorphic structure of cellulose and xylan. NMR relaxation and dipolar order parameters further suggest a sophisticated change of molecular motions in a ctl1 ctl2 double mutant: both cellulose and xylan have become more dynamic on the nanosecond and microsecond timescale, but the motional amplitudes are uniformly small for both polysaccharides. Conclusions By skipping isotopic labeling, the DNP strategy demonstrated here is universally extendable to all lignocellulose materials. This time-efficient method has landed the technical foundation for understanding polysaccharide structure and cell wall assembly in a large variety of plant tissues and species.

09 BIOMASS FUELS↗
DOE OSTI2021

Coculture with hemicellulose-fermenting microbes reverses inhibition of corn fiber solubilization by Clostridium thermocellum at elevated solids loadings

Abstract Background The cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber > 95% in 5 days, but solubilization declines markedly at substrate concentrations higher than 20 g/L. This differs for model cellulose like Avicel, on which the maximum solubilization rate increases in proportion to substrate concentration. The goal of this study was to examine fermentation at increasing corn fiber concentrations and investigate possible reasons for declining performance. Results The rate of growth of C. thermocellum on corn fiber, inferred from CipA scaffoldin levels measured by LC–MS/MS, showed very little increase with increasing solids loading. To test for inhibition, we evaluated the effects of spent broth on growth and cellulase activity. The liquids remaining after corn fiber fermentation were found to be strongly inhibitory to growth on cellobiose, a substrate that does not require cellulose hydrolysis. Additionally, the hydrolytic activity of C. thermocellum cellulase was also reduced to less-than half by adding spent broth. Noting that > 15 g/L hemicellulose oligosaccharides accumulated in the spent broth of a 40 g/L corn fiber fermentation, we tested the effect of various model carbohydrates on growth on cellobiose and Avicel. Some compounds like xylooligosaccharides caused a decline in cellulolytic activity and a reduction in the maximum solubilization rate on Avicel. However, there were no relevant model compounds that could replicate the strong inhibition by spent broth on C. thermocellum growth on cellobiose. Cocultures of C. thermocellum with hemicellulose-consuming partners— Herbinix spp. strain LL1355 and Thermoanaerobacterium thermosaccharolyticum —exhibited lower levels of unfermented hemicellulose hydrolysis products, a doubling of the maximum solubilization rate, and final solubilization increased from 67 to 93%. Conclusions This study documents inhibition of C. thermocellum with increasing corn fiber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn fiber fermentation broth. Our results support the importance of hemicellulose-utilizing coculture partners to augment C. thermocellum in the fermentation of lignocellulosic feedstocks at high solids loading.

09 BIOMASS FUELS↗
DOE OSTI2021

THF co-solvent pretreatment prevents lignin redeposition from interfering with enzymes yielding prolonged cellulase activity

Background: Conventional aqueous dilute sulfuric acid (DSA) pretreatment of lignocellulosic biomass facilitates hemicellulose solubilization and can improve subsequent enzymatic digestibility of cellulose to fermentable glucose. However, much of the lignin after DSA pretreatment either remains intact within the cell wall or readily redeposits back onto the biomass surface. This redeposited lignin has been shown to reduce enzyme activity and contribute to rapid enzyme deactivation, thus, necessitating significantly higher enzyme loadings than deemed economical for biofuel production from biomass. Results: In this study, we demonstrate how detrimental lignin redeposition on biomass surface after pretreatment can be prevented by employing Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment that uses THF–water co-solvents with dilute sulfuric acid to solubilize lignin and overcome limitations of DSA pretreatment. We first find that enzymatic hydrolysis of CELF-pretreated switchgrass can sustain a high enzyme activity over incubation periods as long as 5 weeks with enzyme doses as low as 2 mg protein/g glucan to achieve 90% yield to glucose. A modified Ninhydrin-based protein assay revealed that the free-enzyme concentration in the hydrolysate liquor, related to enzyme activity, remained unchanged over long hydrolysis times. DSA-pretreated switchgrass, by contrast, had a 40% drop in free enzymes in solution during incubation, providing evidence of enzyme deactivation. Furthermore, measurements of enzyme adsorption per gram of lignin suggested that CELF prevented lignin redeposition onto the biomass surface, and the little lignin left in the solids was mostly integral to the original lignin–carbohydrate complex (LCC). Scanning electron micrographs and NMR characterization of lignin supported this observation. Conclusions: Enzymatic hydrolysis of solids from CELF pretreatment of switchgrass at low enzyme loadings was sustained for considerably longer times and reached higher conversions than for DSA solids. Analysis of solids following pretreatment and enzymatic hydrolysis showed that prolonged cellulase activity could be attributed to the limited lignin redeposition on the biomass surface making more enzymes available for hydrolysis of more accessible glucan.

09 BIOMASS FUELS↗