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At least 325 records · Page 18
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

An integrated biorefinery approach to obtain xylo-oligosaccharides from corncob using lactic acid-rich fermentation broth

Although xylo-oligosaccharides production from lignocelluloses via lactic acid hydrolysis is advantageous in terms of high yields with less by-products, the use of high purity lactic acid inevitably increases the cost of xylo-oligosaccharides production. The utilization of lactic acid-rich fermentation broth for xylo-oligosaccharides production can avoid the separation and purification of lactic acid. However, the feasibility of lactic acid-rich fermentation broth for xylo-oligosaccharides production from lignocelluloses was unclear. In this work, the results indicated that the highest xylo-oligosaccharides yield of 72% was obtained from corncob by lactic acid-rich fermentation broth pretreatment and xylanase hydrolysis. Subsequently, monosaccharides-rich hydrolysate (90.9 g/L) was obtained from solid residues of corncob by cellulase hydrolysis. Lactic acid-rich fermentation broth was produced by Weizmannia coagulans fermentation of the monosaccharide-rich hydrolysate. Besides, the obtained xylo-oligosaccharides and lactic acid mixture might be used as feed additives, avoiding the purification and separation of lactic acid or xylo-oligosaccharides. Meanwhile, the production cost of feed grade xylo-oligosaccharides from this work was much lower than the current market price. Overall, this work proposed a new strategy for efficient and environmental-friendly production of xylo-oligosaccharides from corncob.

09 BIOMASS FUELS↗
DOE OSTI2024

Improving dairy manure hydrolysis and acidification through microbial community restructuring by adaptation to hyperthermophilic conditions

Dairy manure (DM) contributes significantly to greenhouse gas emissions and ecosystem degradation, yet its resistance to biodegradation hinders widespread bioprocessing applications. Lignocellulosic materials in DM pose a particular challenge because of their recalcitrance. Bioprocessing under hyperthermophilic (≥70 °C) conditions potentially offers an advantage over traditional fermentation temperatures due to enhanced activity of enzymes and the kinetics of enzymatic reactions. This can lead to a higher conversion rate and a greater extent of biomass hydrolysis and acidification. To test the validity of this hypothesis, the current study evaluated the efficacy of anaerobic hydrolysis and acidogenic fermentation of DM under mesophilic, thermophilic, and hyperthermophilic conditions. All inocula were adapted to corresponding temperatures but were derived from the same mesophilic source. Hyperthermophilic conditions resulted in superior DM hydrolysis efficiency (53%) compared to mesophilic (34%) and thermophilic (42%) conditions. The hyperthermophilic environment was particularly favorable to the decomposition of crude proteins and hemicellulose, which were reduced by 64% and 54%, respectively. Furthermore, hyperthermophilic fermentation also yielded the highest volatile fatty acid (VFA) production rate of 460 mg/L/day during the first four days, representing improvements of 50% and 90% over mesophilic and thermophilic conditions. In part, this was attributed to the enhanced production of branched-chain VFAs, including an increase of 6–10% in isobutyric acid and 12–13% in isovaleric acid. At hyperthermophilic conditions, however, there was no accumulation of VFAs during the days 5–8 of fermentation, which could be due to acetate conversion by the syntrophic acetate-oxidizing bacteria. A considerable gain in hydrolysis efficiency and VFA production rate were accompanied by a reduction in microbial diversity, which suggests that hyperthermophilic temperature is a favorable environment for the selection of organisms with enhanced DM hydrolysis and fermentation capabilities. A significantly increased relative abundance of xylanolytic Caldicoprobacter (23% of population) and proteolytic Thermovirga (9% of population) could be the major contributors to improved decomposition of hemicellulose and protein. As revealed by the techno-economic analysis, acidogenic fermentation of DM at 70 °C and a retention period of 4 days provides the greatest positive net present value, highest internal rate of return of 9.2%, and shortest investment payback period of 9 years. Furthermore, this study demonstrates that hyperthermophilic conditions enable superior deconstruction and bioconversion of lignocellulose-containing biomass into VFAs under reduced retention times, offering a promising approach for improving DM management and generating bioproducts.

09 BIOMASS FUELS↗
DOE OSTI2024

Highly silanized cellulose biocomposites for sustainable insulation materials

Microfibrillated lignocellulose networks, derived from agricultural byproducts, represent an environmentally friendly biogenic material production due to their abundant availability to circular bioeconomy and inherent carbon sink in life cycle analysis. Yet, its vulnerability to moisture and flammability, coupled with challenges in creating highly reinforced insulation materials, poses challenges for the carbon-zero green building sector. Here we address these challenges with a new concept of in-situ grafting polymerization of nanoporous silica in pre-formed lignocellulosic fiber networks. The seamlessly integrating nanoporous silica with cellulose through hydrogen bonding networks enabled us to prepare highly reinforced biogenic composites for green building insulations. A high reinforcement biocomposite with hierarchal arrangements of nanoporous silica within the cellulose network exhibits remarkable attributes. It boasts a thermal conductivity of 24.2 mW·m –1 ·K –1 , a flexural modulus of 942 MPa, and soundproofing with a 20.8 % noise reduction, as well as the fire resistance characterized by an extended time to ignition and a reduced peak heat release rate of 144 kW·m –2 at 35 kW·m –2 of incident radiant heat flux. Furthermore, it demonstrates a reduced water absorption capacity, dropping from 5.12 g·g –1 to 0.75 g·g –1 . Altogether, this study opens the new pathways towards sustainable carbon-zero building materials in the context of circular bioeconomy.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Process Intensification for the Biological Production of the Fuel Precursor Butyric Acid from Biomass

The production of fuels from lignocellulosic biomass is key to reduce our reliance on petroleum and to promote a sustainable bioeconomy. Butyric acid (BA) is a promising chemical precursor for the production of renewable diesel and jet fuels. BA can be biologically produced from lignocellulosic sugars. However, challenges associated with product selectivity and recovery must be overcome to achieve industrially relevant metrics. Here, we evaluate various fermentation configurations and demonstrate near-homo-butyrate production by using the biocatalyst Clostridium tyrobutyricum. We also develop an advanced in situ product recovery process based on hybrid extraction-distillation (HED-ISPR) and conduct techno-economic analyses and life cycle assessments. We demonstrate that the HED-ISPR process lowers the overall capital and operating expenses and environmental impact compared to other traditional fermentation processes. Overall, BA minimum product selling price from biomass is 55% of the current BA selling price from petroleum, a significant decrease toward viable renewable fuel production.

09 BIOMASS FUELS↗
DOE OSTI2025

Advancing sustainable aviation fuel with high-energy-density bicycloalkanes production from corn stover mixed sugars

Bicycloalkanes stand out as a replacement for aromatics from conventional jet fuel (CJF) because of their high energy density and lower freezing point. Most reports to date have focused on synthesizing bicycloalkanes using model compounds or single sugar components of lignocellulose. Here, we utilize all lignocellulosic sugars of corn stover (CS) to produce bicycloalkanes. First, furfural (FFR) and 5-hydroxymethyl furfural (5-HMF) are produced via acid-catalyzed dehydration, achieving combined FFRs molar yields ranging from 65% to 73%, respectively. The resulting FFRs are converted to cyclopentanones with molar yields within the range of 57%–63% via hydrogenation and Piancatelli rearrangement. Further aldol condensation of cyclopentanones yields C 10 -C 12 oxygenates with 80 mol %. Lastly, these oxygenates are directly hydrodeoxygenated to bicycloalkanes in a yield of 83.8 mol %. The resulting bicycloalkanes exhibit tier α fuel properties consistent with Jet A fuel (CJF) specifications and blend compatibility with CJF at a 37% volumetric ratio.

09 BIOMASS FUELS↗
DOE OSTI2023

Atomic Level Interactions and Suprastructural Configuration of Plant Cell Wall Polymers in Dialkylimidazolium Ionic Liquids

Ionic liquids (ILs) have been widely investigated for the pretreatment and deconstruction of lignocellulosic feedstocks. However, the modes of interaction between IL-anions and cations, and plant cell wall polymers, namely, cellulose, hemicellulose, and lignin, as well as the resulting ultrastructural changes are still unclear. In this study, we investigated the atomic level and suprastructural interactions of microcrystalline cellulose, birch wood xylan, and organosolv lignin with 1,3-dialkylimidazolium ILs having varying sizes of carboxylate anions. Analysis by 13 C NMR spectroscopy indicated that cellulose and lignin exhibited stronger hydrogen bonding with acetate ions than with formate ions, as evidenced by greater chemical shift changes. Small-angle X-ray scattering analysis showed that while both cellulose and xylan adopted a single-stranded conformation in acetate-ILs, twice as many acetate ions were bound to one anhydroglucose unit than to an anhydroxylose unit. Further, we also determined that a minimum of seven representative carbohydrate units must interact with an anion for that IL to effectively dissolve cellulose or xylan. Lignin is associated as groups of four polymer molecules in formate-ILs and dispersed as single molecules in acetate-ILs, which indicates that it is highly soluble in the latter. In summary, our study demonstrated that 1,3-dialkylimidazolium acetates displayed stronger binding interactions with cellulose and lignin, as compared to formates, and thus have superior potential to fractionate these polymers from lignocellulosic feedstocks.

13C NMR↗
DOE OSTI2026

Understanding and Mitigating Stickiness in Biochar Produced through Acid Hydrolysis and Dehydration

Levulinic acid is a platform chemical with significant potential for conversion into a wide range of biobased chemicals and fuels. A common process for producing levulinic acid from lignocellulosic feedstocks involves acid hydrolysis and dehydration (AHDH), where hexose polymers are hydrolyzed into monomeric sugars and subsequently dehydrated to levulinic acid and formic acid in the presence of dilute sulfuric acid. However, scaling the AHDH process is challenging because of the formation of byproducts such as sticky biochar, which accumulates in continuous-flow reactors, reducing effective reaction volume and increasing process downtime. This study investigates the effect of a chemical preconditioning step on mitigating sticky biochar formation. Woody biomass was preconditioned at 170 °C with 0.26 wt % sulfuric acid for 30 min, resulting in substantial removal of hemicellulose and acid-soluble lignin. AHDH of these preconditioned solids produced biochar that did not adhere to reactor surfaces. TGA analysis confirmed that the chemical preconditioning step minimized interactions between hemicellulose-derived degradation products and lignin side chains, reducing sticky char formation. Additionally, the study observed a 6% higher yield of organic acids from softwood species compared to hardwoods, with bark content shown to negatively impact yield. These findings suggest that targeted preconditioning of lignocellulosic biomass can enhance reactor operability and improve organic acid production efficiency in AHDH processes.

biopolymers↗
DOE OSTI2026

Quantifying Membrane Structure and Dynamics during Bioproduct Production in Zymomonas mobilis by Molecular Simulation

The conversion of lignocellulosic biomass into biofuels and bioproducts by microbial biorefineries is central to a sustainable chemical industry. Zymomonas mobilis is one such biorefinery chassis and is resistant to ethanol stress, leading to its use in biomass conversion to biofuels and bioproducts. However, Z. mobilis growth is often inhibited by organic acids, aldehydes, alcohols, ketones, and amides found in biomass hydrolysate. The resulting slow growth inhibits production and as a result drives up the price for the resulting products. One hypothesis is that these molecules interact with or disrupt the bacterial membrane, triggering stress responses and hindering growth. To test this hypothesis at the molecular level, we employ all-atom molecular dynamics (MD) simulations to investigate lignocellulose-derived small molecules and their impact on a biologically relevant Z. mobilis membrane model. Simulations were conducted across a range of inhibitor concentrations from 0 to 2.5 mol %, analyzing key membrane properties such as area per lipid (APL), membrane thickness, lipid-order parameter (−S CH ), lateral diffusion coefficient (D xy ), and permeability coefficient (Pm). From simulation, we observed altered membrane structure and dynamics at these modest small molecule concentrations commonly found in hydrolysates. Generally, the membranes become thinner, with a higher area per lipid and lower-order parameter as the small molecule concentration increases. These trends are stronger for more hydrophobic molecules with greater hydrophobic bulk, as isobutanol, propanol, and propanoic acid showed greater membrane perturbations as the concentration increased compared to other small molecules. Tracking small molecule distributions directly in our equilibrium simulations allows us to determine concentration-dependent free energy profiles for these molecules. While the trends are noisy, generally the barriers to crossing the membrane decrease as the concentration increases, indicating that the membranes become leakier as small molecule concentrations rise. Comparing between native Z. mobilis membranes with hopanoids and membranes sharing the same phospholipid composition but without hopanoids, hopanoids stabilize and order the membrane for smaller molecules to maintain membrane structure but appear insufficient for larger hydrophobic molecules like isobutanol. These findings provide a mechanistic understanding of how small molecules found in biomass degradation streams interact with the Z. mobilis membrane, offering valuable insights for future strain engineering efforts to optimize biofuel and bioproduct synthesis from biomass feedstocks by highlighting limits to small molecule tolerance. This knowledge can guide the modification of membrane composition to develop more robust microbes, thereby improving microbial survival and yields in industrial contexts.

Singh, Nitin Kumar [Michigan State Univ., East Lan↗
DOE OSTI2026

Postanaerobic Digestion Manure Fibers as a Renewable Source of Lignin-Derived Aromatic Compounds for Microbial Upgrading

Abstract Postanaerobic digestion manure fibers are an abundant solid residue that remains underutilized. The substantial lignocellulosic content of digested manure opens the possibility of using it as a renewable resource for producing valuable products. In this study, we performed compositional and nuclear magnetic resonance analyses to determine lignin content and aromatic composition of these fibers. Additionally, alkaline-based treatment and reductive catalytic fractionation were evaluated as two different processes to produce aromatic-rich streams. The liquor from the alkaline treatment was subsequently utilized for microbial upgrading to produce 2-pyrone-4,6-dicarboxylic acid (PDC). The results showed that lignin in manure fibers represents 30% of the biomass (dry weight basis), with a ∼26% abundance of β-ether-linked aromatics and ∼13% p-coumarate moieties, indicating that the amount of lignin in the postanaerobic biomass is comparable to a moderately pretreated plant biomass. The alkaline treatment released 5.6 g aromatics/kg fibers, while reductive catalytic fractionation produced 14.0 g aromatics/kg fibers. A bioreactor operated in fed-batch mode produced a yield of ∼1 mol of PDC/mol measured aromatics at a rate of 0.2 g PDC/L·h and a titer of 2.6 g PDC/L. This study demonstrates how postanaerobic digestion manure fibers could be used in a circular bioeconomy by harnessing their lignocellulosic content to extract aromatic compounds and use them for obtaining valuable chemicals via microbial upgrading.

Riascos, Brayan D. [University of Wisconsin-Madiso↗
DOE OSTI2020

Synthesis, Characterization, and Utilization of a Lignin-Based Adsorbent for Effective Removal of Azo Dye from Aqueous Solution

How to effectively remove toxic dyes from the industrial wastewater using a green low-cost lignocellulose-based adsorbent, such as lignin, has become a topic of great interest but remains quite challenging. In this study, cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment and Mannich reaction were combined to generate an aminated CELF lignin which is subsequently applied for removal of methylene blue and direct blue (DB) 1 dye from aqueous solution. 31P NMR was used to track the degree of amination, and an orthogonal design was applied to determine the relationship between the extent of amination and reaction parameters. The physicochemical, morphological, and thermal properties of the aminated CELF lignin were characterized to confirm the successful grafting of diethylenetriamine onto the lignin. The aminated CELF lignin proved to be an effective azo dye-adsorbent, demonstrating considerably enhanced dye decolorization, especially toward DB 1 dye (>90%). It had a maximum adsorption capacity of DB 1 dye of 502.7 mg/g, and the kinetic study suggested the adsorption process conformed to a pseudo-second-order kinetic model. The isotherm results also showed that the modified lignin-based adsorbent exhibited monolayer adsorption. The adsorbent properties were mainly attributed to the incorporated amine functionalities as well as the increased specific surface area of the aminated CELF lignin.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Deconstruction of Woody Biomass via Protic and Aprotic Ionic Liquid Pretreatment for Ethanol Production

Ionic liquids (ILs) have emerged as important solvents for conversion of lignocellulosic feedstocks to fuels and chemicals due to their ability to enable efficient biomass deconstruction and fractionation. Woody biomass derived from forest and agricultural residues has the potential to be used for production of biofuels and its removal from forests can help mitigate disastrous wildfires in fire-prone states like California. This study evaluated woody biomass types (pine, almond, walnut, and fir) from California as potential biofuel feedstocks. The feedstocks were pretreated with the ILs cholinium lysinate ([Ch][Lys]) and ethanolamine acetate ([EOA][OAc]), followed by enzymatic hydrolysis and fermentation of lignocellulosic sugars to produce ethanol. Under optimal conditions, [EOA][OAc] pretreatment and enzymatic hydrolysis generated glucose and xylose yields in the range of 24-82 and 14-80%, respectively, while glucose and xylose yields for the [Ch][Lys] ranged between 28-83 and 23-80%, respectively. Maximum fermentable sugar was released from almond wood, and the lowest amount was from pine and fir. Further, blends of feedstocks were also explored, and a blend with a mass ratio of 2/2/1 (almond/walnut/pine) resulted in maximum glucose and xylose (>90%) yields using [Ch][Lys]. Fermentation of this hydrolysate using a C5-utilizing strain of Saccharomyces cerevisiae resulted in a maximum ethanol concentration of 17.9 g/L for mixture biomass hydrolysate, corresponding to 60.8% fermentation efficiency. This study represents the first demonstration of the use of these ILs for pretreatment of woody biomass blends that resulted in a high overall conversion efficiency for ethanol production.

09 BIOMASS FUELS↗
DOE OSTI2022

Chemical and Morphological Structure of Transgenic Switchgrass Organosolv Lignin Extracted by Ethanol, Tetrahydrofuran, and $\mathcal{γ}$-Valerolactone Pretreatments

We report the recalcitrance of lignocellulosic biomass is a challenge in biological-based biorefinery systems due to the complex physicochemical structure of plant cell walls. Pretreatment and genetic modification are two approaches in biomass conversion that have succeeded in modifying the structure of lignocellulose to enable better enzymatic deconstruction. However, the structural differences among pretreatment-solubilized lignin isolated from switchgrass genotypes have not been extensively investigated. Here, three organosolv pretreatment systems-ethanol (EtOH), tetrahydrofuran (THF), and γ-valerolactone (GVL)-were used on wild-type (WT) and two transgenic switchgrasses. All organosolv pretreatments caused a significant reduction in the molecular mass of lignins; particularly, up to ~90% decrease was observed in EtOH-pretreated lignin compared to untreated lignin. The WT EtOH lignin also presented the smallest particle size among all WT lignins. THF pretreated transgenic lignins showed a higher molecular mass, β-O-4 linkages, and aliphatic hydroxyl content compared to EtOH and GVL pretreated lignin. The number of hydrogen bonds between lignin and the organic solvents calculated from the molecular dynamics simulations followed the same trend as the experimentally determined reduction in lignin molecular mass. The results revealed the structural changes of solubilized lignin isolated from wild-type and transgenic switchgrass after different organosolv pretreatments.

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

Greenhouse Gas Emissions Associated with Pretreatment Techniques Utilized for Biosugar Production

Less carbon-intensive production of biosugars from lignocellulosic materials can lead to improved manufacturing of biofuels and bioproducts. In this study, the production of biosugars is analyzed to understand the potential of producing bioproducts from biosugars with a low carbon intensity. Life-cycle assessment is conducted on five lignocellulosic feedstocks along with seven pretreatment techniques to produce biosugars as an important platform intermediate for producing bioproducts. The production of electricity using lignin with and without heat integration was also incorporated. In addition, seven bioproducts are analyzed for the estimation of the GHG emissions budget, which represents the emissions available to convert, separate, and upgrade biosugars into bioproducts within the 70% emissions reduction target. Corn stover-deacetylation and dilute acid pretreatment (CS-DDA) provide the lowest GHG emissions for biosugar (0.03 kg CO 2 eq/kg biosugar) and thereby the highest GHG emissions budget for the case of lactic acid production (3.76 kg CO 2 eq/kg lactic acid). Natural gas and chemicals are the major contributors to all of the pretreatment techniques under study. The outcomes from this study can benefit the advancement of upcoming biobased processes that meet decarbonization targets.

Biopolymers↗
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↗