UMD Theses and Dissertations
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Item PREVENTION AND TREATMENT OF PERSISTENT ORGANIC POLLUTANTS IN STORMWATER AND SEDIMENT(2023) Yuan, Chen; Kjellerup, Birthe V; Davis, Allen P; Civil Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Polycyclic aromatic hydrocarbons (PAHs) and Polychlorinated biphenyls (PCBs) are two groups of persistent organic pollutants (POPs) with toxicity, carcinogenicity, and teratogenicity. Those compounds are harmful to human health and wildlife. Stormwater is one of the important sources of PAHs and PCBs to aquatic environments. Stormwater control measures (SCMs) have already been used to remove PAHs and PCBs from stormwater, however traditional SCMs can remove PAHs and PCBs in the particle phase, but there still are dissolved PAHs and PCBs in the outflow of SCMs. This study focused on reducing the influence of PAHs and PCBs in stormwater on the environment by 1) improve the treatment performance by adding a polishing treatment procedure after traditional SCMs, and remove the PAHs and PCBs accumulated in the polishing treatment media by bioaugmentation of Pseudomonas putida ATCC 17484 and Paraburkholderia xenovorans LB400 and 2) dechlorination of PCBs in the sediment of aquatic environments by biofilm Dehalobium chlorocoercia DF1 inoculum. The results of polishing treatment showed that all black carbon materials, namely biochar, granular activated carbon (GAC), and regenerated GAC (RAC), were effective to remove dissolved PAHs with removal > 95%. However, all materials had lower removal efficiency on PCBs with removal > 84%, By the comparation of cost and lifetime under the condition that 50% polishing media are used in the polishing treatment facility. RAC which has a lifetime>147 years based on the precipitation of Maryland and Washington and cost <3.79 $-m3-yr-1, was the best material for polishing treatment. Results of treatment train with a traditional SCM media column and polishing treatment column indicated that average removal of PAHs can be improved from 94.56% of BSM columns to 99.61% of polishing treatment columns, and removal of PCBs can be improved from 84.61% to 95.16%. Results of bioaugmentation of polishing treatment media showed no biodegradation took place in the mesocosms with polishing media. However, the liquid mesocosms showed P.putida degraded 97.9% of pyrene. The bacteria colony on plates after the biodegradation experiment showed that there were less P.putida and P.xenovorans colony of polishing media mesocosms than liquid mesocosms. Therefore, the limitation of biodegradation of polishing media mesocosms may cause by the limited bioavailability and less active inoculated bacteria. The results of dechlorination by Dehalobium chlorocoercia DF1 biofilm shows that there were native bacteria, such as Gemmatimonadetes, Actinobacteria, Proteobacteria and Firmicutes in the sediment that can dechlorinate PCBs. The three treated mesocosm groups (addition of biochar, bioaugmentation with DF1 biofilm and liquid DF1 culture) all can improve dechlorination, of 28.09%, 21.30%, and 17.10%, respectively. Those three groups had dechlorination extent higher than negative control (4.60%), and abiotic control (-1.02%). The microbial community analysis indicated that biofilm inoculation improved abundance of DF1 and had a more stable influence on the community than liquid inoculation. Overall, biofilm inoculation and addition of biochar dechlorinate PCBs in sediment efficiently, and polishing treatment is an efficient approach to improve traditional SCMs, while treating the polishing media with bioaugmentation need further study.Item Investigations of Substrate Recognition of the Biofilm Glycosidase Enzyme Dispersin B(2022) Peterson, Alexandra Breslawec; Poulin, Myles B; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacterial biofilms, which are comprised of bacterial cells embedded in a thick extracellular polymeric substance (EPS), is a type of survival mechanism used by a variety of medically relevant bacteria to endure harsh conditions, the immune system of the host organism, and medical intervention such as antibiotics. Biofilms confer additional protection to these bacteria, protecting them from a number of stressors, and contribute to the growing problem of antibiotic-resistant infections. Biofilm EPS is comprised of extracellular polysaccharides, proteins, DNA, and other small molecules such as enzymes and nutrients, and the strength and structure of biofilms are often attributed to extracellular polysaccharides such as poly-β-D-(1→6)-N-acetyl-glucosamine (PNAG). Glycoside hydrolase enzymes that are produced as part of the biofilm’s life cycle are being explored as possible anti-biofilm compounds, due to their ability to destabilize biofilms through degradation of the polysaccharide components. The enzyme Dispersin B (DspB), a family 20 glycoside hydrolase produced by Aggregatibacter actinomycetemcomitans, hydrolyzes partially de-N-acetylated PNAG (dPNAG), and shows promise as a potential anti-biofilm agent. Here, we use a variety of techniques to investigate the interactions between DspB and PNAG, leading to a greater understanding of the binding interactions and mechanisms used by DspB to hydrolyze PNAG (Chapter 2). First, the activity of DspB on a monosaccharide probe, 4-methylumbelliferone-GlcNAc (4muGlcNAc) was observed over a pH range to determine the ideal conditions for DspB activity (2.2). Specifically acetylated PNAG trisaccharide analogs were then used to determine the substrate specificity of DspB, which supported the existing hypothesis that DspB uses a substrate-assisted mechanism to hydrolyze PNAG (2.4-2.6). These studies also indicated the possibility of electrostatic interactions between anionic amino acids on the binding surface of DspB and cationic deacetylated residues on PNAG that stabilize the substrate-binding interactions and allow for additional cleavage activities of DspB, namely improved cleavage of partially deacetylated PNAG and the ability to perform endo- or exoglycosidic cleavage activity, dependent on the substrate acetylation patterns present (2.5). Mutagenesis of amino acid residues on the binding surface of DspB was performed to investigate these interactions (Chapters 3-4), resulting in the discovery of an improved DspB mutant. This E248Q mutant of DspB also has an improved ability to clear Staphylococcus epidermidis biofilms, indicating that it may have improved anti-biofilm activity (3.3). Finally, a high-throughput assay for anti-PNAG activity has been developed for use with a degenerate DspB mutant library in order to identify additional DspB mutants with improved anti-biofilm activity.Item SYNTHESIS OF PNAG ANALOGS TO PROFILE BIOFILM GLYCOSIDASES(2021) Wang, Shaochi; Poulin, Myles; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacteria biofilms consisting of surface-attached bacterial communities embedded in an extracellular matrix serve as a defense mechanism for many medically important bacterial species. Exopolysaccharides of partially de-N-acetylated poly-β-D-(1->6)-N-acetyl-glucosamine (dPNAG) are key structural components in the biofilm of many human pathogens. Dispersin B (DspB), a family 20 glycoside hydrolase produced by the Aggregatibacter actinomycetemcomitans, catalyzes the hydrolysis of dPNAG to disrupt biofilm formation leading to its use as an aniti-biofilm agent. Yet little is known of substrate recognition by DspB.Here, we describe the synthesis of two series of PNAG trisaccharide analogs with defined N-acetylation pattern (2.1 – 2.5) or containing glucose moiety (2.32 and 2.33) prepared through an iterative one-pot glycosylation approach and used to profile the activity and substrate preference of DspB (Chapter 2). These studies suggest that DspB hydrolyzes dPNAG polysaccharides via both exo- or endoglycosidase mechanisms and has a substrate preference for cationic substrates at the +2 position of the binding site. Understanding the activity and specificity of DspB provides a valuable guide to develop biocatalyst with improved biofilm dispersal activity. Next, colorimetric (Chapter 3) and fluorogenic (Chapters 4 & 5) PNAG analogs were developed as substrates for high-throughput PNAG glycosidase assay development. PNAG disaccharide probes (3.1 and 5.1) demonstrate exclusive specificity for enzymes capable of hydrolyzing PNAG and monosaccharide analog AMC-GlcNAc (4.1) acts as a general hexosaminidases enzyme substrate. We showed that all the analogs can detect DspB activity in crude E. coli cell lysates, and thus could be applied for functional metagenomic screening to discover novel PNAG glycosidase enzyms. Finally, a series of PNAG triazinyl glycosides (6.1, 6.2 and 6.3) were designed, synthesized and evaluated as affinity labeling reagents for PNAG binding proteins, using a catalytically inactive DspB E184Q mutant as a model PNAG binding protein (Chapter 6). However, only non-specific background signal was observed. In the future, recombinant enzymes or lectins that have higher binding affinity to the PNAG might be used to revisit these labeling results.Item INTEGRATED MICROSYSTEM-BASED APPROACH FOR DETECTION AND TREATMENT OF BACTERIAL BIOFILMS ON URINARY CATHETERS(2020) Huiszoon, Ryan Cornelis; Ghodssi, Reza; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Biofilms are a ubiquitous mode of growth for bacteria and present a significant challenge in healthcare due to their resistant nature towards traditional antibiotic therapy. Particularly, biofilms can form on indwelling urinary catheters, leading to catheter-associated urinary tract infections, which are one of the most prevalent healthcare-acquired infections. In recent years, microsystems-based approaches have been developed to measure and study bacterial biofilms. In this dissertation, microsystems are adapted for the catheterized urinary tract environment to address biofilm infections in situ. Specifically, a proof-of-concept device comprised of gold interdigitated electrodes on a flexible polyimide substrate is fabricated and characterized in vitro. This substrate allows the device to conform seamlessly with the cylindrical surface of a catheter. Real-time impedance sensing is demonstrated, showing an average decrease in impedance of 30.3% following 24 hours of biofilm growth. The device also applies the bioelectric effect, which yields an increase in impedance of 12% and the lowest biomass relative to control treatments. Furthermore, 3D-printed molds and commercial modeling software show that the cylindrical conformation does not have an appreciable impact on performance. This device is integrated with a commercially available Foley catheter using two disparate approaches: (1) integration of the flexible proof-of-concept device using a 3D-printed catheter insert and (2) electroless plating directly onto the catheter lumen. In addition to electrode integration, miniaturized electronic systems are developed to control sensing and treatment wirelessly with a minimal form factor. A smartphone mobile application is developed in conjunction with this effort, to provide a user-friendly interface for the system. Several functions are verified with the integrated system, including biofilm sensing, wireless signal transmission, bladder drainage, and balloon inflation. To decrease the risk associated with this system for future research in vivo and in a clinical setting, sensing and treatment are evaluated under realistic conditions. The biochemical aspect of the catheterized environment is recreated using artificial urine, and the physical aspect is recreated using a silicone model of a human bladder and a programmable pump. A 13.0% decrease in impedance is associated with bacterial growth; this decreased magnitude relative to the proof-of-concept device is due to the reduced degree of growth in artificial urine. The bioelectric effect is demonstrated as well, showing a reduction in planktonic bacteria of 1.50×107 CFU/ml and adhered biomass equivalent to OD590nm = 0.072 relative to untreated samples. This work provides a framework for developing microsystem-based tools for biofilm infection management and research from proof-of-concept to integrated system, particularly for CAUTI. The results demonstrate that the cylindrical conformation does not interfere with device sensing or treatment performance and that the system maintains functionality under realistic conditions, laying the groundwork for future in vivo and clinical testing. The system will provide in situ and real-time data regarding catheter biofilm colonization in a way that is not possible with existing techniques. Finally, the system can serve to reduce reliance on antibiotics and reduce the spread of antibiotic resistance in CAUTI and other vulnerable areas.Item Evaluation of treatment and resource recovery potential of bioelectrochemical systems to DC Water process streams by bench and pilot system(2018) Leininger, Aaron Matthew; Kjellerup, Birthe V; Civil Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Microbial fuel cell and microbial electrolysis cell systems were developed and tested with different wastewater process streams from DC Water Blue Plains Advanced Wastewater Treatment Plant. These biofilm-based systems provide an alternative to the conventional activated sludge system by oxidizing wastewater organics without the need for mechanical aeration. In bench-scale systems, the application of high-strength solids-dewatering wastewater as a feedstock was shown to increase both treatment energy savings and energy recovery. Current densities in meso-scale microbial electrolysis cells were 3.3 and 3.6 times higher when fed dewatering-filtrate or a blend of filtrate and primary effluent as compared to reactors operating with primary effluent. An integrated 800L pilot biocathode microbial fuel cell system was designed and constructed, and initial results are reported. Over the first 43 days of operation, the system averaged 15% removal of chemical oxygen demand and a load removal of 110 g_tCOD/(m^3*day).Item Role of gonococcal surface glycoconjugates, their diversity and their role in bacteria-bacteria interaction and bacteria-host interaction(2015) Bhoopalan, Senthil Velan; Stein, Daniel C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacterial interactions with each other and with host cells play a critical role in the pathogenesis of gonorrhea. Bacterial aggregation was observed to be mediated by the interaction between lipooligosaccharide (LOS) and the opacity-associated protein. In this study, I identified a gene encoding a beta-hexosaminidase (NagZ) in Neisseria gonorrhoeae that modulates gonococcal aggregation and biofilm formation. In comparison to the parental strain, a strain with the nagZ gene deleted produced a biofilm with increased mass. Scanning electron microscopy and confocal laser microscopy were able to visualize differences in the biofilms formed by the two strains. Biofilms formed by a strain deficient in nagZ were disrupted by addition of exogenously added purified NagZ. This is the first study to demonstrate that an enzyme thought to be restricted to peptidoglycan recycling is able to moonlight as biofilm modulator. NagZ could play an important role in promoting bacterial escape from a biofilm, along with previously characterized agents such as Nuc thermonuclease. Using strains defective in surface protein glycosylation, I demonstrate that the increase in biofilm formation seen in nagZ mutant is dependent on PglC-mediated surface protein glycosylation. This is the first study demonstrating the role played by surface glycoconjugates in gonococcal biofilm formation. I used bioinformatic analysis to study the diversity of the other major glycoconjugate on gonococcal surface, LOS. I identified significant differences in the LOS core structure between commensals and pathogens within the Neisseriaceae. I generated preliminary data suggesting that N. gonorrhoeae activate inflammasomes in epithelial cells, resulting in production of IL-18. While activation of inflammasomes does not affect production of other cytokines such as IL-8, IL-8 levels were reduced by using MyD88-inhibitors. Gonococcal-induced inflammasome activation in epithelial cells and production of inflammasome-dependent cytokines were further confirmed in human cervix explants.Item Design and Implementation of Microfluidic Systems for Bacterial Biofilm Monitoring and Manipulation(2014) Meyer, Mariana; Ghodssi, Reza; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacterial biofilms - pathogenic matrices formed through bacterial communication and subsequent extracellular matrix secretion - characterize the majority of clinical bacterial infections. Biofilms exhibit increased resistance to conventional antibiotics, necessitating development of alternative treatments. Standard microbiological methods for studying biofilms often rely on in vitro systems with involved instrumentation for biofilm quantification, or destroy the biofilm in the process of characterization. Additionally, biofilm formation is sensitive to many growth parameters, and can exhibit a large degree of variability between repeated experiments. This dissertation presents the development of systems designed to address these challenges through integration of continuous biofilm monitoring in a microfluidic platform, and through creation of a microfluidic platform for multiple assays performed on one biofilm formed in a single channel. The microsystems developed in this work provide building blocks for developing controlled, high throughput testbeds for development and evaluation of drugs targeting bacterial biofilms. The first platform developed relied on optical density monitoring as a means for evaluating biofilm formation. This method was noninvasive, as it used an external light source and array of photodiodes to evaluate biofilms by the amount of light transmitted through the microfluidic channel where they were grown. The optical density biofilm measurement method and microfluidic platform were used to evaluate the dependence of biofilm formation on quorum sensing, an autoinducer-mediated intercellular communication process. This system was also used in the first demonstration of biofilm inhibition and reduction by two different autoinducer-2 analogs. The second microfluidic system developed addressed the challenge of variability in biofilm formation. Biofilms formed in a single microfluidic channel were partitioned by hydraulically actuated valves into three separate segments, which were then treated as representatives of the original biofilm in further experiments. A novel photoresist passivation process was developed in order to create the multi-depth channels needed to accommodate both valve actuation and biofilm formation. Biofilms grown in the device were uniform throughout, providing reliable experimental controls within the system. Biofilm partitioning was demonstrated by exposing three segments of one biofilm to varying detergent concentrations.Item Biofilm formation by Escherichia coli O157:H7(2007-12-14) Silagyi, Karen Suzanne; Lo, Y. Martin; Kim, Shin-Hee; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Escherichia coli O157:H7 from cattle was evaluated for its ability to produce biofilm on food contact surfaces and quorum sensing signals in various raw meat, raw poultry, and produce broths. Generally, the strain was able to attach and form the most biofilm on stainless steel. Transfer of cells attached to stainless steel was observed onto various raw meat, raw poultry, ready-to-eat deli meats, and produce products as high as 104 CFU/cm2. E. coli O157:H7 isolated from 14 animal, food, and human sources were characterized on antimicrobial susceptibility, ability to form biofilm, and production of curli fimbriae and cellulose. Strains isolated from cattle, retail chicken, and retail beef were able to form strong biofilms in addition to curli and cellulose production. Additionally, E. coli O157:H7 from retail chicken showed considerable antimicrobial resistance. This study suggests E. coli O157:H7 biofilms pose significant risk to continuous contamination of a variety of food products.