Doctor of Philosophy, The Ohio State University, 2022, Chemistry

Peptide self-assembly offers a powerful tool in the development of nanomedicines. We present a CPT based tetrapeptide scaffold, containing cysteines, which offers high stability and controlled release at low concentrations due to the crosslinking disulfides. The inclusion of cysteines allows for crosslinking under oxidative conditions (10% DMSO in PBS). The formation of covalent bonds allows for the structure to remain intact even at low concentrations and provides an opportunity to control the release of CPT. Many cancers are known to overexpress reducing agents, GSH, which would selectively break the disulfide bonds to release the CPT in the presence of cancer cells. Dual drug delivery systems have the potential to be a powerful tool in the treatment of cancers. Currently, combinations of anticancer agents are prescribed together, in many instances, 5-Fu and irinotecan are used together. We present a dual drug scaffold using both 5-Fu and CPT that can quickly release 5-Fu, while a secondary structure forms to afford the slow release of CPT. The initial system is assembled into a nanotube with a diameter of 84 nm, and as the 5-Fu is released, the nanotubes morph into a second assembly with a diameter of 72 nm that allows the CPT to be released more slowly. Previous studies have demonstrated peptide based self-assembled system can have a stabilizing effect on enzymes. We report the use of electro-spun CNCs, graciously given to us by the National Research Council of Canada, to test the long-term stability of Rubisco over the period of 49 days. Further studies are warranted to determine the potential use of PDA as a polymer coat of the CNC scaffolds.

Committee: Jonathan Parquette (Advisor); Christopher Hadad (Committee Member); Jovica Badjic (Committee Member) Subjects: Chemistry

Master of Science in Chemistry, Youngstown State University, 2020, Department of Chemistry

Immobilized multienzyme systems catalyzing cascade reactions have shown to be an effective industrial strategy. These systems reduce production cost by increasing both reusability and stability of enzymes under extreme conditions. Development of a stable support for enzyme immobilization further improves the process. The objective of this study was to improve lactose hydrolysis by designing a two-component system comprised of lactase and glucose oxidase immobilized independently on a modified chitosan support. The structural stability of chitosan was enhanced by addition of fortifying agents such as activated charcoal, silica, and magnetic particles followed by crosslinking beads with glutaraldehyde. Lactase was immobilized on chitosan-magnetic beads, chitosan-charcoal beads, and chitosan-silica beads. Glucose oxidase was immobilized on chitosan-charcoal beads and chitosan-silica beads. The physical properties, immobilization efficiency, and enzymatic activity were determined for each immobilized enzyme. Both enzymes, lactase and glucose oxidase, retained their activity after immobilization. Assays performed with the combinations of the chitosan beads indicated that the immobilized enzymes are capable of catalyzing two sequential reactions - lactose hydrolysis and glucose oxidation.

Committee: Nina Stourman PhD (Advisor); Michael Serra PhD (Committee Member); Douglas Genna PhD (Committee Member) Subjects: Biochemistry; Chemistry

Doctor of Philosophy, University of Akron, 2020, Chemistry

The receptors embedded in cell membranes are essential for signaling in response to various stimuli or communicating with neighboring cells. In order to target these membrane proteins for drug discovery, it is essential to understand their interactions with each other in the live cell. Techniques such as structural studies, interactions in solution, and live cell fluorescence have all aided in determining the details of protein-protein complex formation. Here fluorescence cross-correlation spectroscopy (FCCS) is used to elucidate these events in live cells. This method allows analysis of fluorescent protein intensity fluctuations to gain insight on protein diffusion and oligomerization in the 2D cell membrane. The research presented here aimed to determine homotypic and heterotypic interactions for the plexin/neuropilin/semaphorin family of proteins. These membrane proteins are involved with tissue patterning in the developing embryo and play various roles in disease. While truncated, soluble domains have been studied in some capacity, the full-length receptor complexes have not been completely explored and FCCS allows insight to their network of interactions. Chapter I and II give an introduction to these receptors and an overview of the FCCS methods used. Chapters III and IV focus on the degree of interaction between Neuropilin-1 (Nrp1), Plexin A2, Plexin A4, and Plexin D1 when stimulated by Semaphorin 3A or Semaphorin 3C. These combinations have been implicated in various cancers and this work resolves interactions which may aid in the design of new therapeutic strategies. Chapter V looks at the oligomerization of the transmembrane domains of Nrp1, Plexin B1, Plexin B2, and Plexin D1 using FCCS and computational predictions. Most experiments agreed with the simulations, showing individual motifs which enhanced or disrupted helix interactions, while also revealing that competition in the live cell may prevent some of the interactions predicted in isolation. Chapte (open full item for complete abstract)

Committee: Adam Smith (Advisor); Leah Shriver (Committee Member); Michael Konopka (Committee Member); Sailaja Paruchuri (Committee Member); Nic Leipzig (Committee Member) Subjects: Biochemistry; Biophysics; Chemistry

Master of Science in Chemistry, Youngstown State University, 2018, Department of Chemistry

Enzyme immobilization refers to any technique by which an enzyme is restrained or localized to a support system. This can provide retention of catalytic activity and reusability of the enzyme, both of which are important in industrial processes. Enzyme supports can be made of organic materials, typically naturally occurring polysaccharides, such as cellulose, agarose, or chitosan. The objective of this study was to immobilize beta glycosidase BglX and its mutant E293Q on chitosan gel beads while retaining catalytic activity. The beads were fortified with activated charcoal or silica and cross-linked with glutaraldehyde to increase their mechanical stability and immobilization efficiency. Lactase was used as a model enzyme to determine which type of chitosan gel bead was the most suitable for immobilization. The chromogenic substrate ortho-nitrophenyl ß-D-galactopyranoside (oNPGal) and a lactose solution were used to test the catalytic activity of immobilized lactase. Upon successfully immobilizing lactase, BglX and E293Q were tested similarly. Since BglX showed higher percent conversion of substrate, it was used in a packed bed column and the ability of the immobilized enzyme to hydrolyze the lactose present in milk whey was tested in a small-scale continuous production system.

Committee: Nina Stourman PhD (Advisor); Michael Serra PhD (Committee Member); Clovis Linkous PhD (Committee Member) Subjects: Biochemistry; Chemistry; Food Science

Master of Science (M.S.), University of Dayton, 2016, Chemical Engineering

Through biotic-abiotic interactions, it has been shown that peptides can recognize and selectively bind to a wide variety of materials dependent on both their surface properties and the environment. Better understanding of these peptides and the materials to which they bind can be beneficial in the development of biofunctionalization approaches for creating hybrid materials and sensors. Several research groups have identified material binding peptides using biopanning with phage or cell peptide display libraries. However, limitations with sequence diversity of traditional bacteriophage (phage) display libraries and loss of unique phage clones during the amplification cycles results in a smaller pool of peptide sequences identified. In order to overcome some of the limitations of traditional biopanning methodology, a modified method using phage display along with high-throughput next generation sequencing to select for unique peptides specific for different classes of single wall carbon nanotubes has been devised. The process, analysis and characterization of peptide sequences identified using the modified method is described and compared to peptides identified using the traditional methods. Selected sequences from this study were immobilized on surfaces and used in site-specific capture of metallic and/or semiconducting carbon nanotubes. A dispersion experiment was carried out to identify chiral specific peptides. From this research, successful methods have been identified to select and confirm binding peptides specific to various materials. Knowledge of chiral specific recognizing peptides can allow for the potential purification and separation of specific chirality carbon nanotubes, thus opening the door for a number of carbon nanotube applications which had been previously hindered by mixed carbon nanotube samples.

Committee: Kristen Comfort Dr. (Advisor); Rajesh Naik Dr. (Advisor); Kevin Myers Dr. (Committee Member); Christina Harsch Dr. (Committee Member) Subjects: Biochemistry; Bioinformatics; Chemical Engineering; Materials Science

Master of Science, University of Akron, 2015, Polymer Science

Biomimetic catalysis has great impact on the development of the research of organic catalysis1. Immobilized enzyme used as catalysts has attracted a lot of attention because of its high stability and the excellent ability of recovering from the reaction environment. Furthermore, the activity of immobilized enzyme can be retained after long-term storage or exposure to high temperatures2,3. In this study, Immobilization of GOx was achieved by adsorption and encapsulation of GOx onto porous polymer support, and catalytic activity of GOx immobilized porous support was evaluated by testing the formation of H2O2. On the other hand, biological sequestration of carbon dioxide (CO2) is one of the proposed approaches for CO2 unitization. Carbonic anhydrase was chose as catalyst to study the catalytic reaction of CO2. The reaction was in situ monitored by Attenuated total reflectance (ATR), the preliminary results indicated the formation of bicarbonate (HCO3-), which was evidenced by the IR characteristic band at 1400 cm-1 4.

Committee: Steven S.C. Chuang Dr. (Advisor); Toshikazu Miyoshi Dr. (Committee Member) Subjects: Polymers

Doctor of Philosophy, University of Akron, 2014, Chemical Engineering

Millions of individuals are affected by nerve damage to either their central nervous system (brain, spinal cord and eyes) or their peripheral nervous system. Endogenous positive and negative biochemical cues are expressed during trauma, working in conjunction with one another, regenerating tissue and protecting the body from further damage. However, because of the inherent nature of the nervous system, these mechanisms are typically skewed toward deleterious results and functional recovery is limited or unlikely to occur. Additionally, topographical features and electrical signals aid in nervous system patterning, and they could therefore be beneficial in axonal regeneration as well. The goal of this dissertation project is to develop initial axon guidance models to serve as a platform for neural regenerative strategies. The major component of this research is the development of an immobilization scheme where guidance proteins, notably nerve growth factor (NGF) and semaphorin3A (Sema3A), are synthesized and immobilized to a chitosan biomaterial film in order to evaluate axon and growth cone responses. Characteristic axon outgrowth responses were seen for single protein cues, attraction toward immobilized NGF sources and inhibition and axon turning away from immobilized Sema3A sources. These responses were not seen when proteins were adsorbed to the chitosan substrate. These axonal results showed the validity of the immobilization platform, which was further examined by tethering NGF and Sema3A in the same region in order to fine-tune axon guidance. Axons were more sensitive to lower concentrations of Sema3A signals compared to NGF, when these proteins were co-immobilized, as noted by axon turning and breakdown of axonal cytoskeletal structure. Another axon guidance model was formed through topographical contact guidance of micropatterned channels on coumarin polyester films. These features were able to align processes of central nervous system neurons and glia p (open full item for complete abstract)

Committee: Nic Leipzig Dr. (Advisor); Gang Cheng Dr. (Committee Member); William Landis Dr. (Committee Member); Bi-min Newby Dr. (Committee Member); Rebecca Willits Dr. (Committee Member) Subjects: Biomedical Engineering; Chemical Engineering

PhD, University of Cincinnati, 2014, Engineering and Applied Science: Chemical Engineering

Polymers containing perfluorosulfonic acid (PSA) groups have been used as solid acid catalysts for many years. These materials have unique and extensively studied hetero-phase morphology of hydrophilic PSA domains dispersed within a hydrophobic hydrocarbon matrix. However, the correlation between the intrinsic activity of the PSA domains and changes in solid-state morphology remains uncertain. This dissertation investigates the immobilization of suitable organic molecules within the PSA hydrophilic domains from the gas and liquid phases and by means of acid catalyzed reaction that forms a colored product. This approach permits direct comparisons of immobilization site to active site to PSA group concentration and how changes in membrane morphology influence the kinetics and thermodynamics of the solid-state reaction process. The approach is also shown to produce sensing elements for the portable, real-time detection and measurement of highly toxic trimellitic anhydride (TMA) vapors.

Committee: Anastasios Angelopoulos Ph.D. (Committee Chair); Jonathan Bernstein M.D. (Committee Member); Junhang Dong Ph.D. (Committee Member); Stephen Thiel Ph.D. (Committee Member) Subjects: Chemical Engineering

Doctor of Philosophy, University of Akron, 2014, Polymer Science

Porous materials comprising polymeric and inorganic segments have attracted interest from the scientific community due to their unique properties and functionalities. The physical and chemical characteristics of these materials can be effectively exploited for adsorption applications. This dissertation covers the experimental techniques for fabrication of poly(vinyl alcohol) (PVA) and silica (SiO2) porous supports, and their functionalization with polyamines for developing adsorbents with potential applications in separation of CO2 and catalysis of organic reactions. The supports were synthesized by processes involving (i) covalent cross-linking of PVA, (ii) hydrolysis and poly-condensation of silica precursors (i,e,. sol-gel synthesis), and formation of porous structures via (iii) direct templating and (iv) phase inversion techniques. Their physical structure was controlled by the proper combination of the preparation procedures, which resulted in micro-structured porous materials in the form of micro-particles, membranes, and pellets. Their adsorption characteristics were tailored by functionalization with polyethyleneimine (PEI), and their physicochemical properties were characterized by vibrational spectroscopy (FTIR, UV-vis), microscopy (SEM), calorimetry (TGA, DSC), and adsorption techniques (BET, step-switch adsorption). Spectroscopic investigations of the interfacial cross-linking reactions of PEI and PVA with glutaraldehyde (GA) revealed that PEI catalyzes the cross-linking reactions of PVA in absence of external acid catalysts. In-situ IR spectroscopy coupled with a focal plane array (FPA) image detector allowed the characterization of a gradient interface on a PEI/PVA composite membrane and the investigation of the cross-linking reactions as a function of time and position. The results served as a basis to postulate possible intermediates, and propose the reaction mechanisms. The formulation of amine-functionalized CO2 capture sorbents was ba (open full item for complete abstract)

Committee: Steven Chuang Dr. (Advisor); Matthew Becker Dr. (Committee Member); Mesfin Tsige Dr. (Committee Member); Darrell Reneker Dr. (Committee Member); Jie Zheng Dr. (Committee Member) Subjects: Chemical Engineering; Chemistry; Climate Change; Energy; Engineering; Environmental Engineering; Experiments; Fluid Dynamics; Materials Science; Molecules; Nanotechnology; Organic Chemistry; Polymer Chemistry; Polymers; Scientific Imaging; Technology

PhD, University of Cincinnati, 2013, Arts and Sciences: Chemistry

The goal of this project is to investigate the interfacial effect in different systems including polymer nanocomposites and surface immobilized proteins. In order to find out how the interaction between filler and polymer affects the polymer's properties in polymer nanocomposites, different polymers including polydimethylsiloxane and polybutadiene were filled with modified graphene, and mechanical and thermal properties were studied in section one. In addition, interfacial interactions between immobilized protein and modified surface affecting the protein's orientation and protein's activity were studied in section two. Since polymer nanocomposite was discovered by Toyota research group at 1990, it opened a new dimension in the field of polymer and materials science. Polymer nanocomposites consist of a polymer having nanofillers dispersed in the polymer matrix. The use of nanofillers including carbon nanotube and graphene has attracted increasing interest due to their unique properties and potential applications in aerospace, electronic and automotive industries. Polymer nanocomposites show substantial property enhancement at lower filler loading than polymer composites with conventional micro-scale fillers due to strong polymer-filler interaction and better filler dispersion. In protein surface immobilization, the interfacial effect will affect the immobilized protein orientation and morphology. There is an unmet need to develop biocompatible, non-adherent surface coatings for medical devices to combat the complications seen after cardiovascular stenting, a procedure used for hundreds of thousands of Americans each year. Among current post-stenting therapies, including anti-inflammatory drugs and drug eluting stents, long term administration of anti-platelet drugs is required to prevent thrombosis. In order to solve these unmet needs, we synthesized different coatings to covalently couple an enzyme, specifically human soluble calcium-activated nucle (open full item for complete abstract)

Committee: Neil Ayres Ph.D. (Committee Chair); Ruxandra Dima Ph.D. (Committee Member); James Mark Ph.D. (Committee Member); Dale Schaefer Ph.D. (Committee Member) Subjects: Chemistry

PhD, University of Cincinnati, 2011, Engineering and Applied Science: Chemical Engineering

The interactions between biomolecules and solid surfaces are complex phenomena. Understanding the nature of these interactions can allow engineering highly efficient systems for bioseparation and biocatalysis. However, there is still a lack of understanding of these fundamental interactions due to the complexity and fragility of biomolecules, especially proteins. The overall goal in this research is to improve the current understanding of these interactions as functions of the properties of mobile phases and stationary phases by investigating adsorption isotherms, adsorption thermodynamics, and biocatalytic activity of immobilized proteins. Mesoporous silica and alumina were used as stationary phases (adsorbents). In particular, mesostructured cellular foam (MCF) silica, which has an open 3-dimensional pore structure with superior physical properties, was used to immobilize biomolecules. The surface chemistry of the synthesized MCF silica was engineered to control the immobilization of biomolecules by grafting functional groups, including charge-terminated (amine-terminated, mecapto-terminated) and hydrophobic-terminated groups (methyl-terminated) groups, to the surface. The interactions between biomolecules and prepared adsorbents were also investigated using flow microcalorimetry (FMC) to reveal the adsorption mechanisms during the immobilization of biomolecules at different levels of pH and ionic strength and several functionalized solid surfaces. Adsorption thermodynamics and mechanism can be modulated by changing ionic strength by adding a neutral salt (sodium sulfate) and by changing the pH. Biomolecule adsorption is a complex phenomenon, exhibiting multiple heat events. However, similar thermograms were observed for the interactions between protein and MCF silicas in most cases. Also, the driving force for protein adsorption was investigated by using semi-empirical analysis. Adsorption energetics were affected significantly by surface modification. Also, t (open full item for complete abstract)

Committee: Stephen Thiel PhD (Committee Chair); Neville Degouvea-Pinto PhD (Committee Member); Vadim Guliants PhD (Committee Member); Peixuan Guo PhD (Committee Member); Chia Chi Ho PhD (Committee Member) Subjects: Chemical Engineering

MS, University of Cincinnati, 2010, Engineering and Applied Science: Electrical Engineering

Carbon nanotubes have excellent electrical and mechanical properties, which are ideally suited for field emission and sensor/actuator applications. The catalyst layer needed for CNT growth (Fe, Ni or Co) once coated on the substrate is subject to an annealing step, which results in the formation of tiny globules of randomly aligned particles. CNTs finally grow on these randomly placed catalyst particles after the substrate annealing. The disadvantage of the bottom-up approach is that the catalyst globules are susceptible to migration on the substrate during thermal annealing and the CNT growth process. The scope of this thesis includes: (1) Patterning arrays of nano-/micro- features by e-beam lithography, (2) shallow etches of the holes by plasma etching in these features (3) deposition of the catalyst material into the shallow holes, (4) CNT growth, and (5) characterization of the patterned nano/micro-scale CNT catalysts and CNT growth. The main objective in this thesis is to immobilize the catalysts on the substrate at a specific location with an array of shallow holes. We believe this will localize and anchor the catalyst producing patterned arrays of CNT's. This method process will also be compared with the current methods of catalyst immobilization developed by Dr. Shanov's group where an alumina layers acts as the catalyst anchor layer. Also CNT growth is compared and with a substrate with no immobilization of the catalyst. The differences in catalyst morphology between annealing the substrate in air and nitrogen will also be compared. All the comparisons are done across different diameters of patterned features on the substrate. This new process will allow for the controlled patterning of CNT growth and enable CNT's to be integrated into manufacture able devices.

Committee: Marc Cahay PhD (Committee Chair); Vesselin Shanov PhD (Committee Member); Robert Jones PhD (Committee Member) Subjects: Materials Science

PhD, University of Cincinnati, 2010, Engineering : Materials Science

Lipases have been proven to be versatile and efficient biocatalysts which can be used in a broad variety of esterification, transesterification, and ester hydrolysis reactions. Due to the high chemo-, regio-, and stereo-selectivity and the mild conditions of lipase-catalyzed reactions, the vast potential of these biocatalysts for use in industrial applications has been increasingly recognized. Polysiloxanes (silicones) are well known for their unique physico-chemical properties and can be prepared in the form of fluids, elastomers, gels and resins for a wide variety of applications. However, the enzymatic synthesis of silicone polyesters and copolymers is largely unexplored.In the present investigations, an immobilized Candida antarctica lipase B (CALB) on macroporous acrylic resin beads (Novozym-435®) has been successfully employed as a catalyst to synthesize silicone polyesters and copolymers under mild reaction conditions. The silicone aliphatic polyesters and the poly(dimethylsiloxane)–poly(ethylene glycol) (PDMS-PEG) copolymers were synthesized in the bulk (without using a solvent), while the silicone aromatic polyesters, the silicone aromatic polyamides and the poly(ε-caprolactone)–poly(dimethylsiloxane)–poly(ε-caprolactone) (PCL-PDMS-PCL) triblock copolymers were synthesized in toluene. The synthesized silicone polyesters and copolymers were characterized by Gel Permeation Chromatography (GPC), Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD). This dissertation also describes a methodology for physical immobilization of the enzyme pepsin from Porcine stomach mucosa in silicone elastomers utilizing condensation-cure room temperature vulcanization (RTV) of silanol-terminated poly(dimethylsiloxane) (PDMS). The activity and the stability of free pepsin and pepsin immobilized in silicone elastomers were studied with respect to pH, temperature, cross-lin (open full item for complete abstract)

Committee: Stephen Clarson PhD (Committee Chair); Jude Iroh PhD (Committee Member); Gregory Beaucage PhD (Committee Member); James Mark PhD (Committee Member) Subjects: Materials Science

PhD, University of Cincinnati, 2006, Arts and Sciences : Chemistry

Microfabricated devices offer a number of important benefits for chemical analysis, and they have the potential for integrating several chemical processes, including sample preparation, chemical reactions, separation and detection, directly onto a single device. Polymer–based microfabricated devices offer a number of advantages as compared to silica-based devices. These advantages include lower cost, easier fabrication, and their potential to be mass-produced. We have been exploring the use of polymer-based microfabricated devices for the analysis of biological samples. Of particular interest to our laboratory is the coupling of these devices to mass spectrometry for protein identification. Here, I describe suitable approaches for covalent immobilization of biological analytes, such as enzymes, via amide linkage to poly(methyl methacrylate) (PMMA) surfaces. The results reveal that enzymes retain their activities after immobilization and can be used for the digestion of proteins and peptides. However, the kinetic parameters (Km and Vmax) of these enzymes are affected by the immobilization to PMMA. After the surface of PMMA is derivatized, the immobilization of enzymes is found to be extremely efficient with immobilized enzyme showing long-term stability. Enzymes can be immobilized to channel walls and PMMA microspheres, which are inserted in a reaction chamber, in PMMA microfabricated devices. These microfabricated devices are used for on-line enzymatic digestion of biological compounds and are interfaced with electrospray ionization-mass spectrometry (ESI-MS). Integration of on-chip enzymatic digestion and electrophoresis to separate components of a sample can be accomplished. It is envisioned that an integration of on-chip enzymatic digestion, separation by microchip electrophoresis and ESI-MS can be used to create a microchip-based proteomics sample preparation tool that will be available for the characterization of proteins.

Committee: Dr. Patrick Limbach (Advisor) Subjects: Chemistry, Analytical

PhD, University of Cincinnati, 2003, Arts and Sciences : Chemistry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful analytical tool for the structural characterization of biomolecules. However, nearly all proteomic or genomic methodologies that employ MALDI-MS require external sample manipulation which limits the overall throughput of analysis. In this work functional MALDI sample plates were fabricated to permit in-situ characterization of nucleic acids. Poly(methylmethacrylate) (PMMA) sample plates were fabricated by micromilling technique. The key structural feature of the microfabricated sample plate is the presence of individual cylindrical posts (360 µm x 360 µm), which serve as individual sample targets within the overall PMMA-based sample plate. Functionality is added to these microposts via the covalent attachment of enzymes. Enzymatic digestion of ribonucleic acids (RNAs) was performed in-situ with subsequent analysis by MALDI-MS. Advantages to such an approach include reduction in sample handling and reduction in the amount of sample required for analysis due to the small surface area of the microposts.The gas phase dissociation of biomolecules can be used for sequence analysis, structural characterization and modification analysis. MALDI-post-source decay (PSD) was used for the characterization and understanding of the fragmentation patterns that are observed in synthetic oligoribonucleotides. The results show that the PSD of synthetic oligoribonucleotides is dependent on the base composition. It was also observed that partial sequence can be obtained from the PSD fragment products.Coupling of PSD with on-probe digestion of intact RNAs was also investigated. The results show that this approach can be used for sequencing oligonucleotides and for screening the presence of known posttranscriptional modifications. To simplify PSD data analysis, stable isotope labeling was investigated by incorporating an 18 O-label at the 3'-phosphate of oligoribonucleotides during the enzymatic process (open full item for complete abstract)

Committee: Dr. Patrick A. Limbach (Advisor) Subjects: Chemistry, General

Doctor of Philosophy, The Ohio State University, 2007, Chemical Engineering

Galacto-oligosaccharides (GOS) contain two to five molecules of galactose and one glucose connected through glycosidic bonds. They are classified as non-digestible dietary fibers and widely used in the nutritional market because of their beneficial health effects on stimulating Bifidobacteriain the lower part of the human intestine. GOS can be produced by enzymatic hydrolysis of lactose using β-galactosidase in either free or immobilized form. The goal of this research was to evaluate the feasibility of immobilizing Aspergillus oryzaeand Bacillus circulansβ-galactosidases on commercial nanofiltration (NF) membranes for the production and purification of GOS from lactose. These enzymes were immobilized on NF membranes by adsorption with or without the binding support of polyethyleneimine (PEI).The immobilized enzyme showed the same GOS formation kinetics as that of the free enzyme, indicating that there was no diffusion limitation of the immobilized enzyme reaction with the catalytic membrane. The nanofiltration membranes with immobilized lactases were then studied in the dead-end filtration system for the simultaneous production and separation of GOS. The results showed that the GOS formation kinetics in the simultaneous reaction/separation at the early stage of lactose conversion was the same as that in the conventional method without separation. However, as lactose was further converted beyond 50% conversion, the GOS content in the simultaneous system increased as a result of the removal of inhibiting glucose from the reaction mixture.The GOS content increased approximately 15% in the simultaneous reaction/separation, with approximately 28-32% of monosaccharides removed. Nanofiltration of GOS products from the enzymatic process was evaluated for further removing monosaccharides (glucose and galactose) from the GOS product mixture under diafiltration conditions. A high degree of removal of monosaccharides (~94%) from the GOS mixture could be achieved with three to (open full item for complete abstract)

Committee: Shang-Tian Yang (Advisor) Subjects: Engineering, Chemical

Doctor of Philosophy, The Ohio State University, 2006, Chemical Engineering

The goal of this study is to investigate the application of immobilization technology in various systems: immobilized cell/enzyme bioreactors, affinity chromatography, and BioMEM surface modification. These systems were investigated to solve a particular problem. A novel method for the co-immobilization of whole cells and LDH enzyme on cotton cloth was developed using poly (ethyleneimine) (PEI), which induced the formation of PEI-enzyme-cell aggregates and their adsorption onto cotton cloth, leading to multilayer co-immobilization of cells and enzyme with a high loading amount (0.5 g cell and 8 mg LDH per gram of cotton cloth) and activity yield (>95%). A fibrous bed bioreactor with cells and enzyme co-immobilized on the cotton cloth was then evaluated for R-HPBA production in fed-batch and repeated batch modes, which gave relatively stable reactor productivity. A novel surface treatment method using poly(ethyleneimine) (PEI), an amine-bearing polymer, was developed to enhance antibody binding on the poly(methyl methacrylate) (PMMA) microfluidic immunoassay device. By treating the PMMA surface of the microchannel on the microfluidic device with PEI, 10 times more active antibodies can be bound to the microchannel surface as compared to those without treatment or treated with the small amine-bearing molecule, hexamethylene diamine (HMD). Consequently, PEI surface modification greatly improved the immunoassay performance of the microfluidic device, making it more sensitive and reliable in the detection of IgG. The surface modification method was further simplified and optimized to enhance polymer-based microchannel ELISA for E. coli O157:H7 detection. By applying an amine-bearing polymer, poly (ethyleneimine) (PEI), onto a poly (methyl methacrylate) (PMMA) surface at pH higher than 11, PEI molecules were covalently attached and their amine groups were introduced to the PMMA surface. Zeta potential analysis and X-ray photoelectron spectroscopy (XPS) demonstrated that t (open full item for complete abstract)

Committee: Shang-Tian Yang (Advisor) Subjects: Engineering, Chemical

Doctor of Philosophy, The Ohio State University, 2005, Chemical Engineering

Propionic acid is widely used in food and dairy industries. The demand of propionic acid for use as a natural preservative in foods and grains is high. Fermentation by propionibacteria produces propionic acid from sugars; however, the fermentation suffers from low propionic acid production due to by-product formation and strong propionate inhibition on cell growth and the fermentation. In this work, fed-batch fermentation of glucose by Propionibacterium acidipropionici immobilized in a fibrous-bed bioreactor (FBB) with a high cell density (>45 g/L) produced a high propionate concentration of 72 g/L and a high propionate yield of up to 0.65 g/g. A mutant with improved propionate tolerance was obtained by adaptation in the FBB, resulting in significant physiological and morphological changes. The mutant was less sensitive to propionate inhibition and had a higher saturated fatty acid content in the cell membrane and a slimmer shape with an increased specific surface area. Metabolic stoichiometric analysis was applied to quantitatively describe the global cellular mechanism in propionic acid fermentation. By feeding carbon sources with different oxidation states, different fermentation end-product compositions were obtained, indicating different controlling mechanisms involving various acid-forming enzymes with significant changes in their activities and overall protein expression pattern. The metabolic pathway generally shifted toward more propionate formation with a more-reduced substrate. Gene inactivation via gene disruption and integrational mutagenesis was used to knock out the acetate kinase (ack) gene with the goal of eliminating acetate formation and further enhancing propionate production. Mutants were obtained by transforming the cells with a partial ack gene introduced either as a linear fragment with a tetracycline resistance cassette within the partial ack gene or in a non-replicative integrational plasmid containing the tetracycline resistance cassette. (open full item for complete abstract)

Committee: Shang-Tian Yang (Advisor) Subjects: Engineering, Chemical

Doctor of Philosophy, The Ohio State University, 2005, Chemical Engineering

Lactic acid has been widely used in food and pharmaceutical industries. Recently, the worldwide demand has been increasing due to many new industrial applications. Lactic acid bacteria have been used in lactic acid production because of their high growth rate and product yield. However, the limitations including costly substrates and complicated product recovery make bacterial fermentation economically unattractive. In contrast, filamentous fungi such as Rhizopus oryzae can directly produce optically pure L(+)-lactic acid from carbohydrates present in agricultural residues and plant biomass; therefore, can overcome the problems in bacterial fermentation. However, change and diversity of fungal morphology during fermentation cause many problems in reactor control and operation, and affect lactic acid production. In this research, fungal morphology was controlled by immobilization in a Rotating Fibrous Bed Bioreactor (RFBB). It was found that RFBB provided good morphological control and improved oxygen transfer resulting in increased lactic acid production, limited undesirable ethanol production, and stable long-term production in the RFBB. Lactic acid production cost can be minimized by using low-value substrates derived from agricultural residues and plant biomass. The results showed that R. oryzae was capable of utilizing both starchy materials present in agricultural residues and pentose sugars derived from hemicellulose to produce lactic acid. Process engineering techniques were used to improve lactic acid production. It was found that overgrown immobilized cells in the RFBB caused oxygen limitation and lowered lactic acid production. Oxygen limitation was prevented by increasing oxygen transfer rate using high aeration rate or supplying oxygen-enriched air. Controlling cell growth and biofilm thickness by shaving-off the fungal mycelia under high shear rates and limiting the nitrogen source in the medium was also studied. To achieve controlled growth and immobil (open full item for complete abstract)

Committee: Shang-Tian Yang (Advisor) Subjects: Engineering, Chemical

Doctor of Philosophy, The Ohio State University, 2003, Environmental Science

Cr(VI) reduction and immobilization by Hanford sediment minerals (biotite, magnetite etc.) were investigated under high pH and high ionic strengths conditions similar to the tank waste fluids at the Hanford site. In the homogeneous system, Cr(VI) reduction by aqueous Fe(II) at high pH proceed very quickly, Cr(VI) removal and reduction increase with Fe(II):Cr(VI) ratio. Reduced Cr(III) precipitate out while all the remaining Cr in the solution phase is Cr(VI). Cr(VI) reduction was non-stoichiometric, either due to the trace amount of O2 oxidizing Fe(II) to Fe(III) or due to the passivation effect of Fe-Cr precipitates on the Fe(OH)2 formed in the alkaline pH conditions. The Fe-Cr precipitates may have spinel structure similar as chromite, with Cr-O distance 1.98 A, Cr-Cr distance 3.01 A. Under alkaline conditions, biotite dissolution increases with NaOH concentration. Cr(VI) reduction by Fe(II) containing silicate minerals such as biotite is closely related to mineral dissolution in different NaOH concentrations. Ionic strength will increase both the biotite dissolution and Cr(VI) reduction. Secondary precipitates formation will have important effect on both biotite dissolution and Cr(VI) reduction. Coprecipitation is an important mechanism of Cr(VI) immobilization at the Hanford site. Under alkaline pH conditions, in addition to maghemite, goethite formation from magnetite was more prominent, and goethite formation increases with NaOH concentration. Compared to acid and neutral pH conditions, Cr(VI) reduction was much less significant under alkaline pH conditions, and it seems that Cr(VI) reduction decreases with amount of NaOH added. Maghemite and goethite formation might passivate the magnetite surface, inhibit and stop the Cr(VI) reduction by magnetite. Cr(VI) reduction by magnetic fraction, clay fraction and the Hanford sediment is closely related to dissolution of silicate minerals releasing Fe2+ into solution. Clay fraction dissolution under alkaline pH condit (open full item for complete abstract)

Committee: Samuel Traina (Advisor) Subjects: Environmental Sciences