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Providing access to clean drinking water will continue to be a challenge for civil engineers for generations to come. Since many communities around the world rely on groundwater as a source of drinking water, remediation technologies must be implemented at sites where groundwater contamination exists due to years of mismanagement of hazardous waste. Using nanosized zero-valent metals such as iron and zinc embedded within and on the surface of functionalized (PAA) membrane filters has shown to be an effective dechlorination technique for contaminated groundwater. Introducing a noble metal such as Pd or Ni increases reaction rates by acting a catalyst for the dechlorination reaction. This study focuses on the treatment of contaminated groundwater at a hazardous waste site in Louisville, Kentucky. Once a chlorinated organic chemical manufacturing plant, the site now operates a treatment system for the contaminated groundwater to prevent migration into the nearby Ohio River. A portable membrane treatment system, built at the University of Kentucky, incorporates this functionalized bimetallic membrane technology for treatment of the groundwater found at the former manufacturing plant. Three bench scale tests were performed with membrane treatment system using DI water spiked with the chemical trichloroethylene (TCE). Results showed that using the functionalized Fe/Pd membranes significant decreased TCE concentrations over time. While further tests should be conducted to verify the results of the preliminary bench-scale tests, the membrane treatment system shows potential for use at the hazardous waste site in Kentucky.

Functionalized polymeric membrane based techniques are becoming increasingly popular in biotechnology, food and pharmaceutical industries due to their versatility and hydrodynamic benefits over traditional materials and methods. This research work has been directed towards the development of functionalized polymeric membranes, extensive experimental and theoretical analyses of some of the fundamental aspects of accessibility, membrane fouling and enzyme catalysis, and applications in affinity based bioseparation and biocatalysis. In this research work, the impact of different types of functionalization techniques, such as functionalization of different membrane materials, covalent and electrostatic immobilization, on interaction of various biomolecules and active sites in membrane has been studied in detail. Avidin was used as model biomolecule, and covalently immobilized within acyl anhydride derivatized nylon based membrane. Quantification of the accessibility of covalently immobilized avidin sites was carried out by model biotinylated probe molecules, such as biotin 4-amidobenzoic acid and biotinylated-BSA. This study has been further extended to separate and purify a target protein, HIV-Tat, from a complex mixture of proteins (97-99 % unwanted protein) using avidin-biotin affinity interaction. It has been demonstrated that covalent immobilization of avidin in membranes reduces the accessibility of active sites for probe molecules. Accessibility decreases further for the biotinylated target protein present in the mixture of other unwanted proteins. Affinity based membrane separation of proteins is also associated with decrease in permeate flux due to fouling in membrane structure. Fouling in the membrane has been discussed by analyzing the characteristics of adsorbed protein layer in membrane. In order to improve the accessibility and fouling behavior of affinity separation of Tat protein, a pre-filtration step has been introduced prior to affinity separation. Significant enhancement in accessibility and reduction in fouling has been observed for pre-filtered cases as it removes unwanted proteins prior to affinity interaction. Contribution of the pre-filtration step in reduction of fouling has been elucidated by simple model equations. Improvement in accessibility and fouling behavior reflects in higher separation efficiency (protein recovery) and lower processing time for the pre-filtered cases. Quality of membrane purified Tat protein was examined by different analytical techniques, such as SDS-PAGE, Western Blot and biotin analysis, and then compared with that purified by traditional packed-bead column chromatography. It has been demonstrated that membrane based technique was able to isolate superior quality of pure monomeric Tat protein compare to column chromatographic technique. The other study carried out as a part of this dissertation, has involved development of high capacity, highly active, stable and reusable functionalized membrane domains for electrostatic immobilization of enzymes. Glucose oxidase (GOX) was used as a model enzyme to study the oxidation of glucose to gluconic acid and hydrogen peroxide under convective flow condition. Two different approaches of functionalization of membranes have been presented. In the first approach, alternative electrostatic attachment of cationic and anionic polyelectrolytes was carried out using Layer-By-Layer (LBL) assembly technique within a functionalized nylon based membrane. In the second one, a hydrophobic PVDF membrane was functionalized by in-situ polymerization of acrylic acid. Kinetics of glucose oxidation, effect of pH and flow rate on the activity of GOX was discussed. A comparative study was presented between the activity of free GOX, electrostatically immobilized GOX and covalently immobilized GOX, along with the advantage of convective mode of operation over soaking mode. A novel study has also been conducted on detachment and reattachment of GOX in the same membrane matrix. Further study has been directed towards implementation of the above mentioned immobilized enzymatic system for oxidative dechlorination of chloro-organics. A first time attempt was made to use a 2-stack functionalized membranes system for simultaneous enzymatic production of hydrogen peroxide in first membrane, and oxidative dechlorination of 2, 4, 6-trichlorophenol (TCP) in the Fe+2 immobilized (by ion exchange) second membrane by Fenton reaction. The technique was efficient in destruction of TCP as evident from the overall dechlorination of 70-80 %. This technique provides additional benefit of reusing the same membrane matrices by reattaching fresh GOX and Fe+2.

Red blood cells (RBCs) are the predominant cell type in blood and have a two-layered outer shell which is composed of a cytoskeleton network tethered to a cytoplasmic membrane. In this thesis, I study the structure and mechanical properties of the RBC’s cytoplasmic membrane (RBCcm) on the nanoscale and utilize this knowledge to functionalize this biological structure on a molecular level. In a first case study, I measure the membrane’s bending rigidity from thermal fluctuations observed in X-ray diffuse scattering (XDS) and Neutron Spin Echo (NSE) experiments, as well as Molecular Dynamics (MD) simulations. I provide evidence of the RBCcm's highly deformable nature with a bending rigidity that is substantially softer as compared to synthetic membranes. The methods are applied to RBCs that were stored for up to 5 weeks. I demonstrate that storage of RBCs leads to an increased fraction of liquid ordered membrane domains and an increased bending rigidity. RBCs are ideal for pharmaceutical applications as they provide access to numerous targets in the body, however lack specificity. Functionalizing the cytoplasmic membrane is thus a prerequisite to use these cells in biotechnology. I develop protocols throughout two studies to tune the membrane's lipid and protein composition. I investigate the impact of synthetic lipid molecules on the membrane's structure and demonstrate that small molecules can be encapsulated into liposomes that are formed from these hybrid membranes. Further, I provide direct evidence that the SARS-CoV 2 spike protein can be anchored into the RBCcm through a detergent mediated insertion protocol. These virus-like particles are observed to trigger seroconversion in mouse models, which demonstrates the potential of functionalized RBC in biotechnology. / Thesis / Doctor of Philosophy (PhD)