Browsing by Author "Alvarez, Pedro J"
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Item Co-transport of Carboxyl-functionalized Multi-walled Carbon Nanotubes and Kaolinite in Saturated Porous Media(2015-04-22) Wang, Tianxiao; Li, Qilin; Alvarez, Pedro J; Tomson, Mason BCo-transport behavior of carboxylated multiwalled carbon nanotubes (COOH-MWCNTs) and kaolinite in various solution ionic strength (IS) and pH were investigated. Results on electrophoretic mobility of CNT, kaolinite and quartz sand as well as particle size of CNT-kaolinite mixture, CNT alone and kaolinite alone were consistent with the DLVO theory. Large particle sizes of kaolinite-CNT mixture revealed potential heteroaggregations especially at 1 mM NaCl, pH=3 and 10 mM NaCl and pH=9. Deposition of CNT was enhanced during the co-transport at 1mM NaCl and pH=3 due to the low mobility of CNT-kaolinite heteroaggregate while decreased at pH=5 in 1 mM NaCl, pH=9 in 10 mM NaCl and pH=9 in 100 mM NaCl because of site blocking by kaolinite. Kaolinite mobility increased in the presence of CNT at pH=3 in 1 mM NaCl caused by site blocking and at pH=9 in 10 mM NaCl resulted from low single collector efficiency of large particles.Item Development of a rapid and portable analytical method for in-field detection of Fusobacterium necrophorum in cattle ruminal samples(2024-04-18) Delgado, Nicarao Laertes; Alvarez, Pedro JNucleic acid amplification is a crucial method to obtain microbiological information of significant relevance, ranging from biotechnology applications to the diagnosis of infectious diseases. Traditionally, DNA amplifications are performed inside the lab due to complex processes that require specialized equipment. Today, the most widely used method for DNA amplification is the polymerase chain reaction (PCR). Some of the limitations of PCR for in-field application are related to sophisticated and relatively large (non-portable) equipment requirements and the need for downstream analysis, like electrophoresis, to be useful, which hinders rapid and easily conducted in-field assessments. Loop-mediated isothermal amplification (LAMP) is an alternative to PCR, which offers some advantages as it requires non-expensive equipment due to its isothermal amplification at 65° C. Compared to PCR, it can be performed in shorter times in low-resource settings, and it has proven to have similar sensitivity and specificity. (Wong et al., 2018). The objective of this research was to develop a portable analytical method for in-field detection of DNA that can be performed in less than an hour with non-expensive and portable equipment to address the need for rapid detection of problematic bacteria in rural areas, such as Fusobacterium necrophorumm, which infects cattle and causes hepatic abscesses. Liver abscesses are a concern because infected livers are disposed of, reducing the cattle farm productivity in the overall efficiency of feed conversion, reducing the body weight gain, and producing an economic loss(Pillai et al., 2021). Reliably detecting the presence of this pathogen is important to curtail the indiscriminate overuse of antibiotics such as tylosin, which is often administered to healthy cows. This practice promotes the dissemination of antibiotic resistance in farm animals and nearby water ecosystems. The developed tool could also help identify cattle carrying tylosin-resistant strains of F. necrophorum, which is essential for monitoring infection control efficacy since some strains acquire resistance due to indiscriminate use. This thesis, therefore, addresses the need for a rapid in-field diagnosis tool to help detect the presence and identify the efficiency of F. necrophorum treatment management practices in cattle. The in-field analytical method for DNA detection developed in this thesis involves DNA extraction using magnetic beads, the design of real-time LAMP equipment for colorimetric optical analysis, and a tiered estimative quantitative method. The whole process takes 55 minutes, from sample preparation to data interpretation. An analytical method was developed for the optical analysis of the WarmStart colorimetric LAMP mastermix reaction, along with a two sensors device capable of measuring in real-time, the absorbed and scattered light variations of the light emitted through the LAMP reaction with a 520 nm wavelength LED to perform estimative qLAMP. The optical analysis of the WarmStart colorimetric LAMP reaction produces a curve with a bimodal distribution trend. A first luminance peak occurs between 3 to 6 minutes into the reaction, and a second is formed between 12 and 27 minutes. The first peak is attributed to a slight progressive clearing of the pink tone of the reaction in the colorimetric pre-transition to yellow, stimulated by the Hydrogen ions produced by DNA synthesis. Subsequently, the luminance starts decreasing to generate the first minimum as the turbidity increases due to the formation of Magnesium pyrophosphate salts due to the pyrophosphates released as a byproduct of DNA synthesis, which binds to the Magnesium in the reaction to form the precipitate. As DNA synthesis increases exponentially, the change of pH in the reaction triggers the phenol red color transition to yellow, forming the second luminance peak caused by the color transition to yellow, before a subsequent drop of luminance that may be caused by an increase in turbidity due to the magnesium pyrophosphate precipitates. This new LAMP-based method can perform tiered estimative quantitative amplifications in less than one hour with non-expensive equipment with low resource requirements, improving the accuracy compared to the visual endpoint assessment to confirm positive colorimetric LAMP reactions visible to the human eye. The detection limit for F. necrophorum in water samples was 2x104 cells/mL. Furthermore, the limit of estimative quantification was 50 cells/mL when added to ruminal samples with unknown background F. necrophorum concentrations. The detection limit is suitable for fast detection of F. necrophorum infection in cow ruminal samples in remote areas, as concentrations under 2x104 cells/mL were positively correlated with healthy cattle that do not have hepatic abscesses or need antibiotic treatment.Item Polyvalent Bacteriophages: Isolation, Modification, and Applications in Environmental Systems(2017-11-10) Yu, Ping; Alvarez, Pedro JBacteriophages are garnering significant interest for microbial control predominantly driven by the widespread and increasing onset of antibiotic-resistant bacteria (ARB). However, the scope and efficacy of phage applications in environmental systems have been hurdled due to narrow infective spectrum, development of phage resistance, inefficient biofilm penetration and infectivity loss during phage delivery. This dissertation contributes to advance our understanding of polyvalent (broad-host-range) phages and provides proof-of-concept on polyvalent phage-based antimicrobial approaches. To enhance research on polyvalent phages, we developed two sequential multi-host isolation methods and tested both culture-dependent and culture-independent phage libraries for broad infectivity. In contrast to the conventional single host enrichment method, which biased for fast-replicating narrow host-range phages, sequential multiple-host approaches enriched generalist phages in each step while left the specialist phages behind. Lytic polyvalent phages capable of interspecies or even inter-order infectivity can be readily isolated from common environmental samples without significant reduction in efficiency of infection. Due to their stable and broad host range, these phages can be enriched with a nonpathogenic host (i.e., Pseudomonas putida F1 and Escherichia coli K-12) and subsequently used to infect model problematic bacteria (Pseudomonas aeruginosa, Pseudomonas syringae). We validated the importance of phage polyvalence on phage propagation and bacterial suppression in biofilm-associated environments. The microbial community structure in biofilm had a significant impact on phage propagation and migration, and therefore the suppression of the target bacteria within the biofilm. Polyvalent phages could outcompete narrow host-range phages in the suppression of target bacteria in biofilm-associated environments, although narrow host-range phages were more effective than polyvalent phages in monoculture or simple mixed-species culture. Combining bacterial competitors with phages resulted in meaningfully greater inhibition of target bacteria than separate treatments in planktonic conditions, suggesting that phage-based biocontrol would be more effective in combination with compatible microbial control strategies. The infectivity of commonly isolated narrow-host-range phages decreases quickly because of environmental stresses. Therefore, we proposed polyvalent phage-based biocontrol approaches for safe phage production and improved ARB inhibition in environment. Polyvalent phages can proliferate and thrive in activated sludge microcosms, especially when added along with their nonpathogenic production hosts. Due to the presence of alternative hosts, polyvalent phages reached greater densities, which offset phage loss during delivery and increased the probability of ARB infection. The fraction of surviving E. coli containing the blaNDM-1 resistance gene was also significantly lower for the polyvalent phage cocktail treatment, mainly because the development of phage resistance increased the fitness costs of ARB. Biofilms may shelter pathogenic or problematic microorganisms that are difficult to eradicate due to hindered penetration of antimicrobial chemicals. Here, we demonstrated the potential for efficient bacterial suppression using polyvalent phages attached to magnetic nanoparticles that facilitate biofilm penetration under a relatively small magnetic field. The Podoviridae polyvalent phage PEL1 was immobilized onto Fe3O4-based magnetic CNCs that had been coated with chitosan (and thus functionalized with amino groups). This facilitated conjugation with phages via covalent bonding and enabled phage loading with optimal orientation. The phage-nano complex significantly improved the efficacy of phage biocontrol in biofilm compared with free phages because of high local concentration and optimized phage orientation. The conjugation also extended the application of phages for microbial control by enhancing their delivery to relatively inaccessible locations within biofilms. Overall, polyvalent phages can be preferentially isolated by sequential multiple hosts and hold the potential for ARB control and ARG mitigation. Polyvalent phages exhibit more multifaced propagation dynamics, which increase phage fitness and boost target bacterial infection in biofilm-associated systems and complex bacterial communities. Polyvalent phages are compatible with engineered functional nanomaterial and their conjugation can not only further enhance the efficacy of phage treatment, but also extend the scope of phage application in environmental systems.Item Using Environmental Biotechnology Principles and Approaches to Target Problematic Compounds in Complex Systems(2016-11-28) Gaspar, Jason T; Alvarez, Pedro JWhen put in practice, environmental engineering frequently requires the characterization and remediation of problematic compounds such as priority pollutants or other molecules that impair performance in complex systems. Here, we tackle two such complex systems: shale gas reservoirs and the human body. In shale gas wells, the presence of microbes is usually unwanted and can often have deleterious effects, including reservoir souring, plugging, equipment corrosion, and reduction in gas production volumes. Hydrocarbon souring represents the most significant financial and safety challenge to the oil and gas industry. H2S may originate from geochemical or biogenic sources, although its source is rarely discerned. Biocides are dogmatically utilized during hydraulic fracturing to prevent or inhibit H2S generation. Here we characterize whether souring in the Bakken shale play is from microbial or geological origins. We develop a regional temperature map showing that downhole temperatures in Bakken reservoir wells equal or exceed the upper known temperature limit for microbial life. Attempts to extract microbial DNA from produced water yielded little to no detectable quantities. Stable isotope analysis yielded 34Sδ values from 4.4 – 9.8‰, suggesting souring had a geochemical origin. In cases of geochemical souring, reevaluation of the need for biocide addition, based on first characterizing the H2S source as we describe here, would provide significant reductions in both operational costs and overall environmental footprint. Similar to a traditional environmental system, the human body also accumulates “garbage” (i.e., detrimental aggregates) over time. Lipofuscin (LF) is a brown-yellow, autofluorescent polymeric material that accumulates in a ceroid manner within postmitotic cells during aging. LF accumulation impairs proteosome and lysosome pathways critical to cell health and homeostasis. The ability to quickly generate LF in vitro, and identify drugs that mitigate the accumulation or clear LF would be of great benefit to aging research. Here, we developed a novel platform to quickly create LF-loaded (but otherwise healthy) cells and screen drugs for efficacy in LF bioremediation. The combination of leupeptin, iron (III) chloride and hydrogen peroxide generates significant amounts of LF within cells at a much faster rate and in a less labor-intensive manner than previous methods. We show that oxidative stress induced LF-loading is accompanied by cellular cholesterol increases, and that long-term administration of the small molecule 2-hydroxypropyl-beta-cyclodextrin (HPβCD) reduces LF accumulation (~-27%, p < 0.00001). LF-loading is associated with increases in LDLr and SREBP1 gene expression, which are mitigated by HPβCD addition. In the absence of oxidative stress, HPβCD addition induces a paradoxical response, increasing cholesterol accumulation (but not LF) via upregulation of cholesterol biosynthesis. These two distinct, but opposite effects highlight a previously overlooked therapeutic consideration: the cholesterol content of the treated cell determines which cholesterol pathways, either beneficial or harmful, are responsive to HPβCD. These results are particularly significant because they provide clarity to HPβCD’s mode of action; which until now has remained in dispute amongst the scientific community.