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Item Scale-Up of a Two-Stage Cu-Catalyzed Alkaline-Oxidative Pretreatment of Hybrid Poplar(American Chemical Society, 2024-03) Dülger, Dilara N.; Yuan, Zhaoyang; Singh, Sandip K.; Omolabake, Surajudeen; Czarnecki, Celeste R.; Nikafshar, Saeid; Li, Mingfei; Bécsy-Jakab, Villő E.; Park, Seonghyun; Park, Sunkyu; Nejad, Mojgan; Stahl, Shannon S.; Hegg, Eric L.; Hodge, David B.A two-stage alkaline-oxidative pretreatment of hybrid poplar was investigated at scale (20 L reactor volume) with the goal of understanding how reaction conditions as well as interstage mechanical refining impact downstream process responses. The pretreatment comprises a first stage of alkaline delignification (alkaline pre-extraction) followed by a second delignification stage employing Cu-catalyzed alkaline hydrogen peroxide with supplemental O2 (O2-enhanced Cu-AHP). Increasing pre-extraction severity (i.e., temperature and alkali loading) and pretreatment oxidation (increasing H2O2 loading) were found to increase mass and lignin solubilization in each stage. Lignin recovered from the first stage was subjected to oxidative depolymerization and led to aromatic monomer yields as high as 23.0% by mass. Lignins recovered from the second-stage Cu-AHP pretreatment liquors were shown to exhibit aliphatic hydroxyl contents more than 6-fold higher than a typical hardwood kraft lignin, indicating that these lignins could serve as a biobased polyol for a range of polyurethane applications.Item Composition analysis of canola and intermediate wheatgrass biomass and the effects of extraction(BioResources, 2023-01) Johnsrude, Lauren M.; Scheffel, Aidan J.; Allen, Brett L.; Wettstein, Stephanie G.Knowing the composition of biomass is critical for determining accurate yields of renewable chemicals and fuels; however, nonstructural components can affect the results of standard composition procedures, leading to inaccurate reactant amounts. To remove these nonstructural components, solvent extractions can be done, but the impact on composition values has not been well-reported. For this study, compositional analysis was performed on as-received canola (Brassica napus) and intermediate wheatgrass (Thinopyrum intermedium), as well as ethanol, water, and water/ethanol extracted biomasses. Water/ethanol extraction of the intermediate wheatgrass resulted in significantly lower xylose and both acid soluble and insoluble lignin amounts when compared to the as-received analysis. Since sugar was removed during the extractions, it is recommended to use the as-received composition values for glucuronoarabinoxylans; however, the extractives may interfere with the lignin analysis and therefore, the extracted lignin values are likely more reflective of the composition.Item Influence of pretreatment, lignin extraction, and chemical modification on lignin properties and the performance of lignin-formaldehyde resins and lignin-PLA composite materials(Montana State University - Bozeman, College of Engineering, 2022) Saulnier, Brian Keith; Chairperson, Graduate Committee: David Hodge; This is a manuscript style paper that includes co-authored chapters.Bio-ethanol can be produced from lignocellulosic biomass in a biorefinery as part of a three step process, a chemical or mechanical pretreatment, enzymatic hydrolysis of the cell wall, and fermentation of these sugars to ethanol. One of the byproducts of this process is lignin, a complex biopolymer composed of a heterogeneous aromatic structure. Lignin is often burned to provide energy for the biorefinery. Incorporating lignin into higher-value products is crucial to the viability of the biorefinery process and the full utilization of the renewable carbon contained in biomass. Challenges to the inclusion of lignin in value-added products include recalcitrance of the cell wall to deconstruction and lignin extraction, heterogeneity of the lignin chemical structure, polydisperse molecular weight distributions, and low reactivity. In this thesis we address these challenges by using feedstock selection, selection of pretreatment and lignin extraction process conditions, lignin fractional precipitation, and direct chemical modification of lignin. Chapter 1 provides an overall introduction and background of previous work. Chapter 2 uses a diverse panel of corn stover genotypes subjected to dilute acid pretreatment using a variety of process conditions. The response of the biomass to pretreatment was characterized with special attention given to glucose hydrolysis yields and p-coumarate (pCA) content. Chapter 3 uses a single corn stover source pretreated using a variety of dilute acid conditions followed by two different lignin extraction methods. The influence of pretreatment and lignin extraction conditions on lignin properties was characterized with focus on lignin pCA content. This study found that lignin-formaldehyde resins using lignin from optimized process conditions achieved lap shear strengths higher than conventional phenol-formaldehyde resins. Chapter 4 addresses lignin polydispersity and heterogeneity using the fractional precipitation of lignin from formic acid liquors to obtain differing molecular weight lignin fractions while allowing for successful enzymatic hydrolysis of cellulose. Chapter 5 uses fractional precipitation of corn stover alkali liquors along with modification using propylene carbonate to obtain a panel of multi-component biopolymer fractions for manufacture of biopolymer-PLA composite materials. These materials were fully characterized finding materials made with modified biopolymers exhibited better lignin dispersion, and improved thermal and mechanical properties.Item Enzymatic strategies for controlling and harnessing the oxidative power of O 2(Montana State University - Bozeman, College of Letters & Science, 2018) Machovina, Melodie M.; Chairperson, Graduate Committee: Jennifer DuBois; Robert J. Usselman and Jennifer L. DuBois were co-authors of the article, 'Monoxygenase substrates mimic flavin to catalyze cofactorless oxygenations' in the journal 'Journal of biological chemistry' which is contained within this dissertation.; Emerald S. Ellis, Thomas J. Carney, Fikile R. Brushett and Jennifer L. DuBois were co-authors of the article, 'Understanding how a cofactor-free protein environment lowers the barrier to O 2 reactivity' in the journal 'Journal of biological chemistry' which is contained within this dissertation.; Sam J. B. Mallinson, Rodrigo L. Silveira, Marc Garcia-Borras, Nathan Gallup were authors and Christopher W. Johnson, Mark D. Allen, Munir S. Skaf, Michael F. Crowley, Ellen L. Neidle, Kendall N. Houk, Gregg T. Beckham, Jennifer L. DuBois and John E. McGeehan were co-authors of the article, 'A promiscuous cytochrome P450 aromatic O-demethylase for lignin bioconversion' in the journal 'Nature Communications' which is contained within this dissertation.; Sam J.B. Mallinson was an author and Brandon C. Knott, Marc Garcia-Borras, Alexander W. Meyers, Lintao Bu, Japheth Gado, April Oliver, Graham P. Schmidt, J. Hinchen, Michael F. Crowley, Christopher W. Johnson, Ellen L. Neidle, Christina M. Payne, Gregg T. Beckham, Kendall N. Houk, John E. McGeehan and Jennifer L. DuBois were co-authors of the article, 'Enabling microbial syringol conversion through structure-guided protein engineering' submitted to the journal 'PNAS' which is contained within this dissertation.; Dissertation contains one article of which Melodie M. Machovina is not the main author.Dioxygen, one of Nature's most powerful oxidants, is essential for countless biological reactions. To harness this oxidant's power while minimizing toxicity, enzymes evolved to interact with O 2, activate it, and poise it for catalysis with substrates. This dissertation explores how two very different enzyme families, monooxygenases and a new class of cytochrome P450s, utilize this powerful oxidant. Previously, it was thought that cofactors are essential for O 2 activation; however, a subset of O 2-utilizing enzymes that catalyze direct reactions between substrate and O 2 was recently discovered, including nogalamycin monoxygenase (NMO). To probe how the protein environment affects thermodynamic and kinetic barriers of O 2 activation, we used a suite of techniques, including: UV/vis (transient and conventional) and electron paramagnetic resonance spectroscopies, O 2 consumption, high-performance liquid chromatography (HPLC), and cyclic voltammetry. Here, we provide evidence that the NMO mechanism has similar characteristics to that in flavoenzymes; in NMO, the substrate, acting in lieu of flavin, donates an electron to O 2, activating it to superoxide with the protein environment facilitating this by lowering the reorganization energy. The last half of this dissertation describes the discovery and engineering of a new class of cytochrome P450 enzymes that employ heme-iron oxygen activation to demethylate key lignin degradation products, forming central carbon intermediates that are precursors for bioplastics. The P450 GcoAB, comprised of the oxidase GcoA and the reductase GcoB, is efficient at demethylating G-lignin, but shows poor reactivity towards S-lignin. Using a structure-guided mutagenesis approach, we generated a variant, F169A GcoA, that is more efficient than wild-type at demethylating G-lignin and the only enzyme that efficiently degrades S-lignin. We characterized this variant, and the wildtype enzyme, using biochemical (UV/vis spectroscopy, HPLC), structural (X-ray crystallography), and computational (Molecular Dynamics and Density Functional Theory). Currently, we are testing the in vitro efficiency of additional variants evolved using a directed evolution approach. The results presented in the following chapters explore the mechanisms of several enzymes. Understanding how O2 is activated and utilized across diverse enzymatic systems provides valuable knowledge that can aid in future design and engineering of systems that use this 'green' oxidant, particularly for large-scale industrial applications.Item Utilizing gene suppression technology and hay storage techniques to improve forage quality and animal performance(Montana State University - Bozeman, College of Agriculture, 2017) Staudenmeyer, Danielle Marie; Chairperson, Graduate Committee: Emily Glunk; J.G.P Bowman, R.L. Mack, A.L. Mack and E.C. Glunk were co-authors of the article, 'The effects of feeding reduced-lignin alfalfa on growing beef cattle performance' submitted to the journal 'Journal of animal science' which is contained within this thesis.; J.G.P Bowman, R.L. Mack and E.C. Glunk were co-authors of the article, 'Dry matter and forage quality losses associated with storing large round bales outside at varying geographic locations in the state of Montana' submitted to the journal 'Journal of animal science' which is contained within this thesis.Utilizing technologies such as genetic modification and forage management techniques are two ways to improve forage quality. The objective of the first study in this thesis was to determine the differences in forage quality between reduced-lignin and conventional alfalfa. To test these differences, twenty-four Crossbred Angus heifers were selected to participate in this study and their performance was evaluated based on changes in BW, ADG, DMI, and G:F. In situ digestibility was determined using four ruminally cannulated Hereford cows. Hay samples were collected and used to determine forage quality and leaf-to-stem ratio. There were no differences (P > or = 0.05) in forage quality between treatments, except for DM (P = 0.01). Means did not differ by treatment for percent leaf (P = 0.06) but did differ for leaf-to-stem ratio (P = 0.04). There were no treatment or treatment by day interactions (P > or = 0.05) for BW, ADG, DMI, or G:F. There were no treatment or treatment by time interactions (P > or = 0.05) for in situ digestibility. Overall, the results of this study suggested no difference in forage quality between reduced-lignin and conventional alfalfa. Additionally, animal performance did not differ for crossbred Angus heifers consuming reduced-lignin or conventional alfalfa. The objective of the second study in this thesis was to quantify DM and forage quality losses associated with three different methods of outdoor round bale hay storage at two different sites in Montana. Large round bales consisting of 100% grass hay wrapped in plastic net wrapping were placed into one of four storage systems at both the Bozeman Agricultural Research and Teaching farm (BART) and the Northern Agricultural Research Center (NARC). The four storage systems were: single-stack (SS), pyramid (PYR), mushroom (MSH), and inside stored bales (INSIDE). Results indicated that DM and forage quality losses differed based on geographic location in Montana. This study suggested that DM and forage quality losses differ by location and that bale placement, rather than hay storage formation, is more important for changes in DM and quality for bales stored in Montana over the winter months.Item Autohydrolysis and deligninfication of wheat straw(Montana State University - Bozeman, College of Engineering, 1985) Nakaoka, Ronald KurtItem Acid and enzymatic hydrolysis of autohydrolyzed lignocellulosic substrates(Montana State University - Bozeman, College of Engineering, 1987) Lamar, David AllenItem Seasonal changes in lignin and cellulose content of Montana range grasses(Montana State University - Bozeman, College of Letters & Science, 1941) Gieseker, L. F.Item Isolation and characterization of thermostable alkaline bacteria with ligninolytic potential(Montana State University - Bozeman, College of Engineering, 2012) Popovitch, Ari Elizabeth; Chairperson, Graduate Committee: Brent M. PeytonThermus thermophilus ST and S42 were isolated from high pH (9) and temperature (70°C) hot springs in the Alvord Desert, Oregon and the Heart Lake Geyser Basin in Yellowstone National Park, Wyoming, respectively. The two strains exhibited lignin degrading potential at pH 9 and 70°C, due to their ability to utilize the lignocellulose degradation products kraft lignin, ferulic acid, cinnamic acid, and p-coumaric acid for growth. Growth on the soluble fraction of alkaline pretreated lignocellulose sources, corn stover, corn cob and lodgepole pine was evaluated. The two isolates grew to higher cell yields due of the presence of kraft lignin, corn stover and lodge pole pine when supplemented with glucose. Dye decolorizing activity was confirmed with Remazol Brilliant Blue R (RBBR), an industrial dye and lignin analog compound. Laccase mediated 2,2' azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) activity was observed for both isolates when 0.05 mM CuSO 4 was supplemented to the growth medium. Ligninolytic bacteria capable of growth at pH 9 and 70°C are potentially useful with alkaline lignocellulose pretreatment methods to depolymerize and remove lignin prior to the production of second generation biofuel.