Chemical and Biological Engineering, 1931-2025

H2 is a clean fuel that can circumvent the dispersed and intermittent nature of renewable energy sources, yet challenges in storing and transporting H2 restrict its current utilization. H2 can be converted to the more easily liquefied NH3 for distribution to its point of use where it is decomposed back to H2 sustainably via electrified processes, e.g., dielectric barrier discharge (DBD)-assisted reactors. Catalyst packed beds within the DBD can improve the low energy yields associated with these reactors by selectively facilitating surface reactions and interacting synergistically with the DBD. This thesis studies DBD-assisted NH3 decomposition with earth-abundant, inexpensive zeolites, as their ordered aluminosilicate nature and high dielectric constants are favored in DBD systems. Specifically, we probe zeolite elemental composition and pore structure effects on the H2 energy yield by systematically quantifying dilute NH3 decomposition rates and efficiencies within a single-stage, coaxial AC-powered reactor under similar experimental conditions in the presence of different zeolites. We systematically evaluate a suite of MFI-framework samples (X-MFI-Y, where X represents the cation (H+ or NH4+), and Y represents the Si/Al ratio (40 or 25)) as well as previously reported LTA (5A) and FAU (13X) frameworks. H-MFI-25 has higher steady-state decomposition rates and H2 energy yield than H-MFI-40, as well as the zeolite 13X benchmark. H-MFI-25 further has a higher H2 energy yield than the zeolite 5A benchmark, with a slightly lower but comparable steady-state decomposition rate. Steady-state decomposition rates on H-MFI-40 and H-MFI-25 trend logarithmically with NH3 feed concentration, similar to DBD-only reactions, yet the rates are higher in the presence of MFI zeolites at similar residence times. On average, steady-state DBD power is highest for the empty tube and lowest for H-MFI-25, while the inverse is true for H2 energy yield. H-MFI-25 further exhibits higher NH3 decomposition rates (0.41 μmol/s) and H2 energy yield (22 g/kWh) as well as a lower steady-state DBD power across feed concentrations compared to H-MFI-40. These results suggest that a lower Si/Al ratio (higher Al content) influences the bulk DBD properties, which could affect both the DBD-phase reaction rate as well as the DBD-zeolite interactions mediating the surface-facilitated reactions. Future deconvolution of zeolite and bulk DBD contributions to the decomposition rate is key for quantifying the overall surface-facilitated reaction kinetics and the underlying DBD-assisted mechanism of NH3 decomposition; this better understanding of the complex interactions in plasma-assisted chemistries is needed to overcome limiting efficiencies in sustainable green H2 production.

In addition to the onslaught of environmental challenges posed by the warming climate, eutrophication is encouraging the growth of the cyanobacteria that cause harmful algal blooms (HABs) and release toxins that threaten human, wildlife, and environmental health. To track the emergence of and protect against cyanobacterial toxins, as well as to inform remediation processes, a robust and real-time detection system is first required. Here, I design, construct, and evaluate a sensor system that integrates Shewanella oneidensis’ naturally occurring extracellular electron transport (EET) pathways with nanobodies’ capacity for antigen-specific binding to digitize information on contamination events via a modulated electrical signal. This report outlines the expression, purification, and binding-capability assessment of the biological components for such a real-time bioelectronic detection device. In addition to creating a biohybrid device to detect microcystin-LR – a well-characterized cyanobacterial toxin within the microcystin family – I also designed a model system to detect green fluorescent protein (GFP) and validate the approach to cell-surface binding. The final design relied on the direct display of nanobodies on the surface of S. oneidensis for target detection. After confirming target binding to the cells via microscopy, I performed an electrochemical analysis of the sensor in response to target exposure. This preliminary study indicated that the engineered strain of Shewanella underpinning the sensor design demonstrates a change in current distinct from that of the control strain in both chronoamperometry and cyclic voltammetry experiments upon addition of the target.

The rhizosphere is a dynamic chemical environment shaped by secondary metabolites that influence microbial interactions, nutrient availability, and plant health. Among these, ribosomally synthesized, post-translationally modified peptides (RiPPs) represent a largely unexplored class of natural products. DUF692 multinuclear non-heme iron-dependent oxidative enzymes (MNIOs) are an emerging family of tailoring enzymes involved in the biosynthesis of RiPPs. These MNIO-modified RiPPs have shown growing evidence for roles in copper chelation, antivirulence functions, and microbial community dynamics, though their full potential remains underexplored. To address this gap, this study presents a scalable bioinformatic pipeline for identifying MNIO-associated biosynthetic gene clusters (BGCs) across bacterial genomes. Application of this framework uncovered over 1,200 putative MNIO-RiPP BGCs. To probe their functional relevance, we selected a candidate cluster from [Actinomadura] parvosata subsp. kistnae for experimental study. Mass spectrometry analysis revealed a unique mass shift consistent with MNIO-mediated modification, and a putative structure has been proposed. This work expands the known chemical space of MNIO-RiPPs and underscores their potential as bioactive mediators of rhizosphere interactions with translational relevance in environmental and agricultural contexts.

This thesis establishes the fundamentals for designing a radiation shield and heat exchanger device for the Princeton Field-Reversed Configuration (PFRC) reactor. The device must capture neutron radiation, X-rays, and microwaves while effectively carrying heat out of the system using a coolant. Two design concepts were proposed: layered shell and packed bed. The thermal performance of a layered shell device was evaluated under varying flow rates and heat loads with a parametric study on design parameters, such as the number of cooling channels and the total coolant volume. Using numerical simulations, key performance metrics including maximum temperature and pressure drop were evaluated. For the layered shell, a nondimensional parameter Π is defined to represent the ratio of cooling capacity to thermal load. A logarithmic relationship between maximum temperature and Π is devised such that, given a maximum temperature limit, a critical value Π* can be calculated under which the system overheats. To start development on a packed bed design, a method was developed to randomly generate a slice of the packed bed which uses periodic boundaries and symmetry to represent a full bed. This method provides a starting point for CFD simulations to prepare for large-scale experiments. These findings provide insight into the design and optimization of a joint shielding and heat exchanger device, providing a basis for future improvements in swiftly designing and manufacturing an outer shell for the PFRC.

This thesis investigates electrolytically driven calcium carbonate precipitation as a pathway for carbon dioxide mineralization through induced alkalization. Using a three-electrode setup with nickel foil as the working electrode, electrolysis of potassium bicarbonate solutions was conducted at controlled current densities to generate hydroxide ions via the hydrogen evolution reaction (HER). In-situ Raman spectroscopy was used to monitor interfacial speciation and quantify local pH near the electrode surface. Starting from an open-circuit pH of 8.35, the pH rose to 9.42 at two minutes and reached 10.83 after six minutes of electrolysis at −2 mA/cm2. Calcium carbonate precipitation experiments were conducted using solutions of sodium chloride and calcium chloride mimicking seawater concentrations. A key finding was that precipitation occurred primarily in the bulk of the solution, rather than directly at the electrode surface. This was confirmed by visual observations during electrolysis and supported by electrochemical impedance spectroscopy (EIS) measurements. While charge transfer resistance (Rct) remained high throughout, the relatively stable solution resistance (Rs) and increasing Warburg impedance over time indicated minimal surface blockage and a dominant role of diffusion-driven precipitation in solution. To further understand the precipitation process, Raman spectroscopy, scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDXS) were used to characterize the solid calcium carbonate products. The predominant polymorphs identified were calcite and vaterite. SEM images showed that the crystals had well-defined morphologies, with spherical vaterite and rhombohedral calcite observed. Taken together, these results demonstrate the effectiveness of electrolytically driven alkalization for inducing carbonate formation and provide mechanistic insight into spatial precipitation behavior. The findings offer valuable direction for optimizing electrode and cell design in future electrochemical carbon capture systems.

This thesis proposes a model for the effects of YAP signaling on Sox2 and Sox9 expression and lung morphology during the E12 stage of branching morphogenesis in mouse lungs. Results of culturing experiments demonstrate that inhibiting YAP causes growth of the epithelium into cystic structures rather than distinct branches. Results of immunostaining experiments suggest that YAP constrains Sox2 and Sox9 to the airway epithelium, and that inhibiting YAP allows expansion of Sox2 and Sox9 expression into the mesenchyme, which leads to abnormal branching morphogenesis. Results of qPCR experiments provide quantitative confirmation that YAP inhibition causes additional expression of Sox2 and Sox9. Together, these results suggest that normal branching morphogenesis requires the absence of Sox2 and Sox9 expression in the mesenchyme to promote smooth muscle wrapping. Future research should examine YAP signaling at stages of development before and after E12, study different downstream proteins of the YAP signaling pathway, and use other experimental techniques to separately quantify protein expression in the epithelium and in the mesenchyme. This research has important implications for engineering of complex branched lung tissues to treat lung diseases.

Biomolecular condensates present a compelling frontier in biological engineering due to their dynamic material properties and ability to spatially and temporally organize biochemical reactions without membrane boundaries. These structures form through liquid-liquid phase separation, a thermodynamically driven process in which proteins and RNAs spontaneously form reversible, concentrated droplets. Stress granules (SGs) are a class of condensates that self-assemble in response to cellular stress and play a critical role in the regulation of mRNA triage during translational arrest. However, aberrant SGs have been implicated in the progression of neurodegenerative diseases such as Alzheimer’s, frontotemporal lobar degeneration, and amyotrophic lateral sclerosis, where persistent SG assemblies are associated with the pathological aggregation of TDP-43. Despite the growing recognition of this correlation, the biophysical mechanisms that drive SGs toward disease-relevant states remain poorly understood. To address this knowledge gap, this thesis presents a coarse-grained, reaction-diffusion model that integrates phase separation theory and reaction kinetics to investigate SG formation and aging during different stress regimes. The model tracks the interplay between SG components, including mRNA and RNA-binding proteins, and aggregation-prone proteins such as TDP-43. By using numerical computing to systematically vary key biophysical parameters, including interaction strength, cytoplasmic mRNA levels, and mRNP dissolution rates, we simulate how SG morphology, domain size, and composition evolve over time. Our model provides a quantitative and mechanistic framework for exploring how SGs shift from functional condensates to sites for fibril formation, offering insights into the physical principles linking phase separation to protein aggregation in neurodegenerative diseases.

Chondroitinase ABC is an enzyme that has shown therapeutic potential in treating spinal cord injuries through the breakdown of glial scarring, which inhibits axonal regrowth. However, it is unstable at human body temperature, and has been difficult to stabilize, which makes it challenging to implement as a therapeutic. Thus, a collaboration between the Webb and Gormley labs identified and tested the ability of copolymers to serve as stabilizing agents. Many of these copolymers successfully boosted the retained enzyme activity (REA) of chABC over 24 hours at 310K, or human body temperature. However, the experiments involved in this study did not reveal the mechanism by which these copolymers endowed thermodynamic stability. If this mechanism of stabilization were better understood, it would contribute to the understanding of chABC’s thermal instabilities and forward the goal of implementing chABC as a therapeutic. Molecular dynamics simulations were utilized to computationally interpret the mechanism of copolymer stabilization of chABC. These simulations revealed that there are facets of chABC’s wet lab behavior that can be understood and interpreted computationally, as well as contribute to the understanding of copolymer chABC stabilization. Investigating the interactions between chABC and high REA copolymers revealed that high levels of intermolecular contact can lead to a maintained protein structure, while low REA copolymers can exhibit distinctly destabilizing behavior not exhibited in a laboratory setting. These impacts may be related to the ability of copolymers to block the collapse of chABC from a crescent into a torus. Successful blocking of destabilizing intra-protein interactions, along with high contact bracing, seems to result in the most successful stabilization of chABC. Conversely, blocking the destabilizing intra-protein interactions without any additional support results in the most significant destabilization of chABC.

Understanding how antibiotics affect bacterial cells beyond their primary target is essential for developing next-generation therapeutics. Traditional profiling methods primarily focus on identifying a drug’s direct target, often overlooking the downstream cellular effects of antibiotic treatment. This study evaluates a modified Bacterial Cytological Profiling (BCP) approach that uses a panel of Escherichia coli mutants with deletions in transcription-associated genes to characterize antibiotic mechanisms of action beyond their primary target. By integrating GFP-based translational readouts with measurements of DNA content (DAPI), membrane structure (FM4-64FX), and cell size (FSC), analyzed via flow cytometry, we examined cellular responses to two transcription-blocking rifamycins: rifampicin (RIF) and rifabutin (RFB).

We found that many mutants exhibited consistent and gene-specific cytological responses to RIF, highlighting the method’s ability to reveal downstream effects of RNAP inhibition. Additionally, drug-specific phenotypes emerged between RIF and RFB, demonstrating that the modified BCP approach can resolve subtle mechanistic differences between closely related antibiotics.

Together, these findings validate the modified BCP approach as a tool for uncov- ering antibiotic action with greater depth and resolution, offering valuable insights for future drug development.

Bioelectrochemical systems (BESs) interface bacterial metabolic processes with electronic systems for purposes including biosensing, bioremediation, and modulating biological activity. A key bottleneck that currently hinders development of these systems is the difficulty of interfacing bacteria with electronics, resulting in the limited mutual transfer of charge associated with the contact between the bacteria's natural biofilms and an electrode surface. I developed a formulation using biocompatible conductive polymers and designed a device in which to electropolymerize an engineered living hydrogel that incorporates electrically active microbes into its polymer matrix, increasing the surface area contact between the bacteria and the electrode. With a connected potentiostat, the device allows for repeatable, consistent electrochemical systems for hydrogel synthesis and measurement via cyclic voltammetry and chronoamperometry. Engineering a new bridge between biology and electronics requires material that can syncretize the two worlds and an engineering design-build-test-learn cycle that can fine-tune this bridge to create a path toward more environmentally-friendly energy production in the future.

Biofuel development can serve as a possible approach to reduce GHG emissions and help mitigate the effects of climate change on the environment and human health. In the development of biofuels involving microorganisms, there have been various trade-offs between growth and ethanol production. While previous studies have proposed different approaches to assess these trade-offs, temperature-sensitive (ts) mutants have not been widely explored in this context. These mutants lack the function of a certain essential gene at nonpermissive temperatures and display full to partial function at semi-permissive temperatures. In this project, we performed screenings on a library collection consisting of 600 temperature-sensitive mutants of Saccharomyces cerevisiae essential genes at permissive (21°C) and nonpermissive temperatures (38°C). The aim is to identify strains that exhibit impaired growth at elevated temperatures, but can still maintain active metabolism to efficiently produce ethanol. This is based on the assumption that high temperatures can disrupt cellular processes and functions linked to growth, but not ethanol fermentation because of the changes in metabolic pathways. Initial screenings were conducted using alcohol dehydrogenase assays, and high-performance liquid chromatography was used to verify the high specific production ratios of 15 ts mutants that were selected. The gene ontology analysis confirmed that many of these selected genes are involved in growth-related processes, such as cell cycle regulation and ribosomal biogenesis. Further work should be conducted to dynamically control these strains using optogenetic techniques to improve the fermentation efficiency. Additional experiments should also investigate whether these ts alleles can produce more ethanol than the wild-type (BY4741) through fed-batch fermentations.

Intrinsically disordered proteins are key components of many liquid-like biomolecular condensates that are thought to contribute to a broad range of cellular functions, including mRNA processing, stress responses, and signal transduction. These same proteins also form pathological aggregates which are hallmarks of neurodegenerative diseases. However, how the same components exist in both material states in vivo remain poorly understood. To investigate how a liquid-like and arrested assembly might coexist within the cell, this work utilized Forever Corelets, a constitutive 24-mer oligomer, as a model arrested assembly. Comparing to its liquid-like, light-induced counterpart the Corelet system, we investigated how tuning homotypic interaction strength, phosphorylation state, and valence influenced the phase coexistence behavior of these engineered oligomeric constructs through mutations that perturb self interaction strength and mimic endogenous phosphorylation, as well as by modulating the fixed valence of the Forever Corelets oligomeric core. Particle tracking of individual puncta throughout the course of hypotonic dissolution treatments performed on the various populations revealed that the Forever Corelets display two kinetically distinct populations: a rapidly dissolving reversible phase and a persistent arrested phase. Decreasing homotypic interaction strength and increasing phosphomimic content rendered the Corelets populations more susceptible to hypotonic dissolution, in line with previous work that has shown that those specific biophysical manipulations decrease the propensity of the system to phase separate. Applied to the Forever Corelets, these manipulations decreased the clustering propensity of the assemblies and increased the fraction of assemblies resistant to hypotonic dissolution. However, increasing the valence of the Forever Corelets did increase the irreversibility of the system, pointing to valence as a universal driver of assembly formation regardless of the formation paradigm. Together, these data establish the Forever Corelets system as a model system for studying liquid-arrested phase coexistence within the cell. Additionally, this work establishes how valence, transient interaction energy, and electrostatic repulsion can modulate the phase coexistence behavior of the Forever Corelets system. This framework may offer insight into the phase behavior of native condensates, inform design principles for multiphase synthetic organelles, and work to clarify the role of liquid-arrested phase coexistence in driving pathological aggregation in neurodegenerative diseases.

The ability to spatially control the presentation of proteins on substrates is essential for investigating cellular responses to microenvironmental cues and for engineering functional biomaterials. This paper reviews and compares several protein patterning techniques, including photopatterning using quartz photomasks, digital micromirror devices (DMDs), microcontact stamping, and patterning with stencils. Each method is evaluated based on its throughput, accessibility, and suitability for generating both defined patterns. Each method was tested experimentally, and it was found that the cells were able to adhere onto substrates regardless of where the protein patterns were created for techniques that used Poly-L-lysine polyethylene glycol (PLL-g-PEG). In order to more reliably control the protein pattern and cell adhesion, physical barriers such as silicone stencils were also used to dictate the protein patterning. This allowed for easy and reliable control over the protein patterns, which enabled the electrotaxis experiment that was conducted. The protein patterned substrates are used to control the shape and size of cells to study the effect of tissue size on the response of the tissue when placed in an electric field. We found that increasing tissue size leads to an increase in the velocity of the cells along the direction of the stimulation direction, and we provide several explanations for why this phenomenon is observed.

Mismanagement of plastic waste presents significant environmental challenges, with only a small fraction of plastic being recycled and a majority ending in landfills or natural environments. To address these concerns, this study explores catalytic upcycling of polyolefin plastics using hierarchical FAU-type zeolite catalysts. Specifically, it investigates how mesoporous structures within FAU catalysts influence catalytic cracking performance, which is essential for converting bulky plastic polymers into economically valuable hydrocarbons. Four distinct hierarchical FAU materials were synthesized via varied post-synthetic desilication methods, each generating unique mesoporous networks characterized through X-ray diffraction, N2-physisorption, and acidity measurements via temperature-programmed desorption. Catalytic cracking reactions of polyethylene (PE), polypropylene (PP), and hexatriacontane (C36) were conducted to evaluate performance differences attributable to mesopore size, volume, and connectivity. Results revealed significant improvements in cracking rates for catalysts synthesized with surfactants (H-FAU-CTA, H-FAU-TPA), while hydrothermally treated catalysts showed enhanced reactivity specifically for larger, branched PP molecules, suggesting that mesopore accessibility and geometry significantly modulate catalytic efficacy. Importantly, the presence of mesopores alone did not universally enhance reaction rates; instead, performance depended critically on mesopore characteristics and reactant molecular size. These findings demonstrate the nuanced relationship between hierarchical porosity and catalytic activity, emphasizing the importance of targeted mesopore design for optimizing plastic waste upcycling processes. The insights gained lay foundational knowledge for developing advanced catalysts that could significantly improve environmental sustainability and facilitate a circular economy for plastics.

Global agricultural production must increase by up to 70% by 2050 to meet the demands of a growing population (1). Sustainable agriculture, harnessing the power of naturally occurring plant-associated microbes, offers a promising way to improve agricultural productivity. Optimizing the performance of beneficial microbes will require a deeper understanding of how these bacteria colonize roots and interact with the plant immune system. Traditional tools for microbial tracking such as 16S rRNA sequencing, fluorescence markers, or antibiotic resistance are often limited by resolution, bias, and poor compatibility with soil environments (17-19). This study applies Wild-Type Isogenic Standardized Hybrid (WISH) tagging, a novel microbial barcoding technique developed by Daniel et al. (2024), to track and quantify root colonization dynamics with (20). In this study, WISH tags were inserted into the following bacterial strains: Pseudomonas simiae WCS417, Dyella japonica MF79 wildtype, and its mutant, a knockout of the immunosuppressive subtilase A (IssA) gene. Sequencing of WISH-tagged populations revealed a competitive advantage of the ΔIssA mutant over the wild-type, indicating that immune suppression traits may incur fitness trade-offs. Overall, this work demonstrates the power of WISH-tagging for high-resolution analysis of plant–microbe interactions and underscores its potential as a tool for future microbiome engineering and sustainable agricultural innovation.

Agricultural systems around the world are using increasingly large amounts of nitrogen, phosphate, and potassium synthetic fertilizers to support modern-day demands. Unfortunately, these fertilizers contribute to greenhouse gas emissions and wash off into natural waterways, posing environmental harm. Climate change further exacerbates the stresses on agricultural systems, leaving them susceptible to crop disease and reduced productivity. A consequence of excessive synthetic fertilizer use and climate change stress is plant nutrient imbalance. Nutrients are essential for a variety of necessary functions; without them, the plant will face stunted growth, low yield production, and other negative side effects. While the Earth’s soil is abundant in nutrients, most of them are bound in soil minerals. Geochemical release processes like mineral weathering are very slow, but minerals can also be processed for plant uptake via microbial mineralization mechanisms. Rhizospheric microbes play a crucial role in making nutrients available for plant uptake. Nitrogen-fixing, phosphate-solubilizing, and potassium-solubilizing bacteria are essential in making these macronutrients bioavailable to support plant growth. This project sought to uncover the potassium-solubilizing bacteria (KSB) within the Arabidopsis and Brachypodium root-microbiome and elucidate the key genes and mechanisms that these KSB employ to make nutrients available for plant uptake. Furthermore, using transposon mutagenesis, this project constructed a mutant library of a select strain, Rhizobacterium sp. PA090, to further investigate the KSB genome. This work will be used to inform further research and guide the development of biofertilizers as a sustainable alternative to the environmentally harmful chemical fertilizers.

Genome mining methodologies were used to advance three high impact projects. In project 1, homology-based genome mining was used to discover a new thermostable poly(ethylene terephthalate) (PET)-degrading enzyme. Rational engineering aided by structural modeling was then used to improve enzyme activity. In project 2, a custom genome mining script was used to discover 1094 examples of lasso pep- tides with putative anti-cyanobacterial activity. These lasso peptides are examples of RiPPs—ribosomally synthesized and post-translationally modified peptides—and contain a unique lariat-like knot. In the 1094 anti-cyanobacterial examples, the lasso peptides likely bind a critical metal needed by cyanobacterial metabolism, after which the metal-lasso complex is imported by the lasso-producing organism and linearized via an isopeptidase. The presence of this isopeptidase gene was used to find the anti-cyanobacterial lasso peptides. Project 3 also involved RiPPs; typically, RiPPs use one class defining enzyme to install a modification onto a ribosomally synthesized peptide precursor. To expand the range of chemistries observed in nature, large-scale genome mining and bioinformatic analysis was used to identify the first examples of hybrid RiPPs, which putatively employ more than one class-defining enzyme to create unique structures. The first examples of hybrid RiPPs were heterologously expressed and purified in E. coli. Nomenclature for these new molecules was created after further bioinformatic exploration of several dozen other putative hybrid RiPPs. The successful computational and experimental workflows used to identify and syn- thesize the first hybrid RiPPs can be applied to find more hybrid RiPPs with unique structures and applications in natural product-based pharmaceuticals.

Sustainable biofuels can be produced using agricultural feedstocks rich in lignocellulose, such as corn stalks and barley straw—low-cost, renewable feedstocks that are typically discarded. However, lignocellulose is both physically and chemically challenging to degrade. Anaerocellum bescii, a thermophilic bacterium that thrives at ~75°C, is a promising candidate for biorefining processes due to its powerful arsenal of carbohydrate-active enzymes (CAZymes), which can efficiently break down lignocellulose and convert it to ethanol and other biofuels. However, efforts to metabolically engineer A. bescii are hindered by a limited understanding of its ATP-binding cassette (ABC) sugar transport systems, which play a vital role in importing extracellular oligosaccharides into the cell. The Xylan Degradation Locus (XDL) and Conserved Xylan Utilization Locus (CXUL) are involved in xylose utilization and together encode three putative ABC sugar transporters. The XDL includes AxoFGE (Athe_0174–0176) and XloEFG (Athe_0179–0181), with Athe_0174 and Athe_0181 as the associated substrate binding proteins, while the CXUL gene cluster xynUVW (Athe_0614–0616) encodes Athe_0614. By heterologously expressing and purifying these proteins, followed by ligand screening using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC), we identified their hemicellulosic sugar preferences and roles in transport. Additionally, ROSIE docking simulations, contact residue mapping, and sequence alignments with homologous proteins revealed conserved amino acids involved in xylo-oligosaccharide recognition. This work advances our understanding of how A. bescii utilizes diverse hemicellulosic substrates, accelerating its development as a platform for sustainable bioproduction of biofuels and other valuable chemicals.