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- ItemSustainable ammonium recovery from agricultural waste by Donnan dialysis(2023-01-01) Fleming, Michael Anthony; Blaney, Lee; Chemical, Biochemical & Environmental Engineering; Engineering, Civil and EnvironmentalThis dissertation focused on ammonium (NH4+) recovery from agricultural waste by Donnan dialysis. The primary goals were to improve water quality and provide a sustainable source of ammonium-based fertilizers. Donnan dialysis is a separation process that exploits electrochemical potential gradients between a concentrated draw solution and a waste solution separated by a semipermeable, ion-exchange membrane. My research advanced the state of the art by employing phosphate-based draw solutions that allow for in situ precipitation of valuable fertilizers within the draw chamber of the Donnan dialysis reactor. In particular, the process was designed to recover struvite (MgNH4PO4á6H2O), a slow-release fertilizer that produces less nutrient runoff and provides better crop yields than conventional fertilizers. The broader impacts of my work will help to minimize our dependence on energy-intensive production of ammonia fertilizers and reduce eutrophication from agricultural runoff. This dissertation had three specific aims. The first aim was to experimentally measure fundamental Donnan dialysis parameters, namely the separation factors and diffusion coefficients for ammonium in three different membranes: CMI-7000; Nafion 117; and Selemion CMVN. The CMI-7000 membrane had a higher ion-exchange capacity than Nafion 117, favoring NH4+ uptake; however, Nafion 117 was thinner than CMI-7000, suggesting faster diffusion. Selemion CMVN was a thinner membrane than Nafion 117 with an ion-exchange capacity similar to CMI-7000, which suggested better potential for NH4+ recovery. For the second aim, I incorporated the Selemion CMVN membrane into Donnan dialysis reactors to recover NH4+ from synthetic wastewater as struvite. Chelating agents were used to decrease recovery of calcium phosphate products, which are not useful fertilizers. The process was further optimized for NH4+ recovery from real agricultural waste, namely poultry litter, by employing clinoptilolite in the draw chamber of the Donnan reactors. The third aim was to improve struvite collection efficiency with natural coagulants following phosphorus recovery from poultry litter. The efficacy of chitosan, bentonite, and alginate to improve coagulation and flocculation of struvite particles from poultry litter extracts was investigated and alginate was determined to be most effective.
- ItemALTERNATIVE APPROACHES TO INSULIN MANUFACTURING USING CELL-FREE SYSTEMS(2023-01-01) Borhani, Shayan; Rao, Govind; Chemical, Biochemical & Environmental Engineering; Engineering, Chemical and BiochemicalThe modular nature of cell-free protein synthesis (CFPS) has resulted in a paradigm shift in the way scientists can design, discover, and manufacture therapeutic proteins. This dissertation reports on three advances made in point-of-care (POC) manufacturing of therapeutics by using CFPS systems to address current challenges around pandemic preparedness and future therapeutic shortages. First, the utility of both prokaryotic and eukaryotic cell-free systems (CFS) was exploited to synthesize human proinsulin to assess the extent of post-translational modification and product quality achieved by each CFS. Second, an Òon-columnÓ purification approach was developed for post-translational conversion of proinsulin into mature human insulin using a specialized set of proteases. Third, a technoeconomic model was utilized to assess the cost effectiveness of cell-free manufacturing of insulin in comparison to the current state of the art. A less complex protein target, griffithsin (GRFT), was also tested using similar approaches. According to a 2022 study, approximately 1.3 million diabetic Americans are currently rationing insulin due to cost. Additionally, microsimulations have shown that by the year 2030, half of Type II diabetic patients are expected to face challenges accessing insulin. Currently, insulin manufacturing takes place in good manufacturing practice (GMP) facilities which utilize in vivo fermentations expressing proinsulin using E. coli and P. pastoris cell lines. While efficient, these fermentations take days to weeks to complete and are not achievable at the POC. For this reason, the in vitro approach enabled by CFPS systems offers a significant advantage, as it is more rapid and can be utilized for production of therapeutics at the POC and on-demand. This dissertation reports the reproducible and soluble expression of difficult-to-express proinsulin, as well as antiviral GRFT, in under 24h using both E. coli and ALiCE? (N. tabacum) CFS. Specifically, a series of cell-free reaction parameters were adjusted including, plasmid concentration, temperature, reaction time, chaperone concentration, and redox potential to achieve optimal protein titer. This dissertation also highlights a cost reduction in purification and post-translational conversion of proinsulin into recombinant human insulin by using an Òon-columnÓ immobilized metal affinity chromatography (IMAC) approach. The results suggest that by reducing the number of unit operations using this approach, a lower cost of goods sold (COGS) for insulin could be achievable with further optimization. To evaluate and optimize the manufacturing cost of the bioprocess outlined in this study, SuperPro Designer software was utilized to identify which components within the process were cost drivers. Additionally, a sensitivity analysis was performed around unit operations and parameters which were a source of increased cost and compared to the COGS of insulin using current manufacturing approaches. Keywords: human insulin; proinsulin; cell-free protein synthesis, CFPS; cell-free; transcription, translation, in vitro; protein engineering; biomanufacturing; bioprocess; point-of-care, POC; post-translational modification, PTM; biologics; pandemic preparedness; antivirals; next-generation biomanufacturing; proteases; enzymes; purification; immobilized metal affinity chromatography, IMAC
- ItemIdentifying wastewater inputs to urban streams by monitoring fluorescent dissolved organic matter and contaminants of emerging concern(2023-01-01) Batista Andrade, Jahir Antonio; Blaney, Lee; Chemical, Biochemical & Environmental Engineering; Engineering, Civil and EnvironmentalFailing sewer infrastructure introduces raw wastewater into streams, threatening public and ecological health. This dissertation presents new environmental forensics strategies to identify, locate, and quantify wastewater inputs to urban watersheds. First, analysis of fluorescent dissolved organic matter (FDOM) via excitation-emission matrix (EEM) spectroscopy and parallel factor analysis (PARAFAC) were proposed as tools to track hotspots of raw wastewater in low-order urban streams impacted by sanitary sewer leaks and overflows. Novel EEM-PARAFAC parameters, including the ratios of (i) microbial soluble product-like and humic acid-like fluorescence (R4/R5) and (ii) tryptophan-like and fulvic acid-like fluorescence (C4/C3), were developed and employed to monitor wastewater-like FDOM. The proposed EEM-PARAFAC metrics were externally validated by assessment of contaminants of emerging concern (CECs), including sucralose, antibiotics, and UV filters, and bacterial indicator organisms at select sampling sites. Second, a comprehensive study on the spatiotemporal distribution of EEM-PARAFAC components and CEC levels was conducted to assess compositional differences between sites in the main stem and tributaries of two urban watersheds. Principal component analysis (PCA) and cross-covariance analysis were applied to the EEM-PARAFAC and land cover datasets to determine relationships between FDOM sources, fate, and transport and impervious surfaces, sewer density, and septic system density. The outcomes of the PCA and cross-covariance studies suggested that geospatial data related to impervious surfaces and sewer density could be used to inform smart sampling strategies in areas most susceptible to failing sewer infrastructure. Finally, a multilinear model was developed and validated to predict wastewater content in urban streams using data extracted from fluorescence EEMs. The model was used to estimate the wastewater contents of 165 samples collected from an urban watershed, and the estimated wastewater contents ranged from 1% to 35%. The highest wastewater content was associated with an urban site known to be impacted by sanitary sewer leaks and flagged by earlier EEM and CEC analysis. The overall outcomes of this research provide alternative, rapid, and cost-effective methods to assess wastewater content in urban watersheds that do not receive wastewater effluent but are continuously affected by sanitary sewer leaks and overflows. These chemical, geospatial, and mathematical tools can also be used to estimate the potential impacts of failing sewer infrastructure on water quality in other locations.
- ItemNONINVASIVE PROCESS MONITORING FOR CELL THERAPY APPLICATIONS(2023-01-01) Rahmatnejad, Vida; Rao, Govind; Chemical, Biochemical & Environmental Engineering; Engineering, Chemical and BiochemicalCell therapies are therapies where cellular materials are injected, grafted, or implanted into the patient body to produce medicinal effects. As a growing field, cell therapy has demonstrated significant potential in the treatment of diseases ranging from diabetes and soft tissue wounds to cancer, nervous system, and genetic disorders. Despite the promising results from cell therapies, the manufacturing process of these therapies is associated with issues such as a lack of appropriate small-scale models and poorly defined manufacturing processes which contribute to the high cost of these therapies. Cell culture is the longest step in the manufacturing process of cell therapies, and the characteristics of cells could be affected by critical parameters in the cell culture environment, such as pH, dissolved carbon dioxide (DCO2), and dissolved oxygen (DO). Therefore, cell culture is one of the mostcritical steps in the manufacturing process of cell therapies because it defines the quality and efficacy of cell therapies. The integration of sensors with the manufacturing process helps in optimizing critical environmental parameters and mitigating problems at the early stages of the process. Therefore, bioreactors, as valuable platforms for cell culture processes, must be equipped with sensors to measure the critical process parameters and develop appropriate control methods. Despite the advantages that monitoring systems provide, their presence in the cell culture environment increases the risk of contamination. Avoiding contamination in the manufacturing process of cell therapies is of high importance because the final products in these processes are cells. Unlike other biologics, cells cannot be sterilized due to being fragile. Therefore, developing a noninvasive monitoring technique is beneficial because it helps eliminate the chance of contamination during the monitoring process. This dissertation is an effort to develop a technology capable of simultaneous monitoring of pH, DCO2, and DO without requiring direct contact with the cell culture environment. Sensors for monitoring pH, DCO2, and DO analytes were previously developed at CAST. The techniques were further developed to achieve noninvasive methods for monitoring pH, DCO2, and DO. The principles utilized in noninvasive techniques, the experimental studies, and the results are discussed in this report. Subsequently, the flow cell, the technology designed for simultaneous monitoring of pH, DCO2, and DO outside the bioreactor, is introduced. The flow cell was developed by combining the principles utilized in individual noninvasive techniques for monitoring pH, DCO2, and DO. The proposed flow cell prototype was investigated in multiple experiments, and the results from studies indicate the efficacy of flow cell in tracking changes inside the bioreactor.
- ItemDevelopment of animal models and molecular tools to investigate the function of prostate specific membrane antigen.(2023-01-01) Rege, Apurv; Bieberich, Charles J; Biological Sciences; Biological SciencesProstate Specific Membrane Antigen (PSMA) is a transmembrane glycoprotein expressed in human prostate luminal epithelial cells. PSMA expression increases in most prostate cancer cases. PSMA overexpression is associated with an unfavorable prognosis, biochemical recurrence, and metastatic disease in patients treated for prostate cancer. PSMA has both folate hydrolase and glutamate carboxypeptidase enzymatic activity. Given its membrane localization and increased expression in prostate cancer, PSMA has received considerable attention as a diagnostic and therapeutic target. Remarkably, the physiological roles of PSMA in the prostate gland remain unknown, and no animal models expressing human PSMA exist. Neither mice nor rats express endogenous PSMA in the prostate, precluding testing of PSMA-targeting agents in wild type Muridae species. The principal goals of this work were to develop a genetically engineered animal model expressing human PSMA in normal and malignant prostates. Multiple attempts to achieve this goal using three distinct molecular strategies in mice were unsuccessful. However, I succeeded in developing a transgenic rat model that conditionally expresses human PSMA in the prostate. By five weeks of age these rats display heterogenous PSMA expression in ventral and lateral prostate lobes. By twenty-five weeks, PSMA expression approaches homogeneity in luminal prostate epithelial cells without apparent pathological effect. Parallel efforts by others to develop a rat model of prostate cancer based on MYC oncogene expression and Pten tumor suppressor loss encountered technical roadblocks and were unsuccessful, which precluded analyses of PSMA function during malignant progression. Overcoming these technical roadblocks necessitated construction of large, complex, multifunctional transgenes that push the boundaries of extant cloning and recombineering technologies. To expedite this process, I optimized a DNA assembly technology and successfully deployed this approach to assemble up to twelve DNA fragments. I also discovered that non-specific DNA can significantly increase the likelihood of achieving success in the generation of complex assemblies. Taken together, these contributions provide a powerful framework to determine the roles of PSMA in normal and diseased prostate glands and provide new molecular tools to support development of next generation of PSMA-expressing animal models. Such models will be instrumental in advancing PSMA-directed diagnostics and therapeutics toward the clinic.