Browsing by Subject "Phosphate"

Now showing 1 - 5 of 5
  • Thumbnail Image
    Item
    Capturing Stormwater Nitrate and Phosphate with Sorptive Filter Media
    (2017-07) Erickson, Andrew
    Soluble phosphate and nitrate are more bioavailable than particulate forms. These nutrients result in eutrophication in both freshwater (typically phosphate-limited) and marine (typically nitrate-limited) systems. In addition, nitrate poses a public health risk at elevated concentrations in drinking water. This research shows that sand filters mixed with 5% iron filings captured, on average, 88% of the influent phosphate in laboratory experiments. Neither incorporation of iron filings into a sand filter nor capture of phosphate had a significant effect on the hydraulic conductivity. Pond-perimeter applications of iron enhanced sand filtration (IESF) with up to 10.7% iron by weight achieved between 29% and 91% phosphate reduction for five events within the first year of operation. After five years, however, a different pond perimeter IESF retained on average 26% of the influent phosphate over three rainy seasons. Retention was best for larger filtered volume events, but negative removal was observed for events with smaller filtered volume and low influent phosphate concentration. Non-routine maintenance improved the hydraulic performance of the pond perimeter IESF and, after a rinsing event, also improved phosphate retention rates to an average of 45%. An IESF was installed to treat agricultural tile drainage and found to reduce total phosphorus loads by 42% to 95% with a flow-weighted mean reduction of 66.3% ± 6.7% (a = 0.05) for 20 events in 2016. The phosphate load reduction varied from 9% to 87% with a flow-weighted mean reduction of 63.9% ± 7.7% (a = 0.05) for 31 events in 2015 and 2016. This research also shows that nitrate is captured abiotically by granular activated carbon (GAC) in laboratory experiments designed to mimic urban and agricultural stormwater runoff. The short contact time and inorganic characteristics of the influent synthetic stormwater suggest that the nitrate was captured by ion exchange, but (bi)carbonate may have competed with nitrate for capture by GAC. Abiotic capture of nitrate requires less stormwater storage volume and less residence time to remove nitrate compared to denitrification, and thus GAC could be used to design smaller treatment practices for nitrate removal.
  • Thumbnail Image
    Item
    Fluoride and Gallein Inhibit Polyphosphate Accumulation by Oral Pathogen Rothia dentocariosa - Data Sharing Archive
    (2023-01-25) Kumar, Dhiraj; Mandal, Subhrangshu; Bailey, Jake V.; Flood, Beverly E.; Jones, Robert S.; rsjones@umn.edu; Jones, Robert, S; Collaboration between Earth and Environmental Science and School of Dentistry
    This raw data set supports publication found in Letters in Applied Microbiology: The uptake and storage of extracellular orthophosphate (Pi) by polyphosphate (polyP) accumulating bacteria may contribute to mineral dissolution in the oral cavity. To test the effect of potential inhibitors of polyP kinases on Rothia dentocariosa, gallein (0, 25, 50, 100 µM) and fluoride (0, 50, 100 ppm) were added to R. dentocariosa cultures grown in brain heart infusion broth. At late log growth phase (8h), extracellular Pi was measured using an ascorbic acid assay, and polyP was isolated from bacterial cells treated with RNA/DNAases using a neutral phenol/chloroform extraction. Extracts were hydrolyzed and quantified as above. Gallein and fluoride had minor effects on bacterial growth with NaF having a direct effect on media pH. Gallein (≥25 µM) and fluoride (≥50 ppm) attenuated the bacterial drawdown of extracellular Pi 56.7% (p <0.05) and 37.3% (p <0.01). There was a corresponding polyP synthesis decrease of 73.2% (p<0.0001) from gallein and 83.1% (p<0.0001) from fluoride. Attenuated total reflectance Fourier transform infrared spectroscopy validated the presence of polyP and its reduced concentration in R. dentocariosa bacterial cells following gallein and fluoride treatment. R. dentocariosa can directly change extracellular Pi and accumulate intracellular polyP but the mechanism is attenuated by gallein and NaF.
  • Thumbnail Image
    Item
    Monitoring an Iron-Enhanced Sand Filter Trench for the Capture of Phosphate from Stormwater Runoff
    (2015-09) Erickson, Andrew J.; Gulliver, John S.; Weiss, Peter T.
    This monitoring project was performed on an iron enhanced sand filtration (IESF) trench in the City of Prior Lake. Water from the pond and IESF trench discharges into a wetland that ultimately drains into Upper Prior Lake. In 2002, Upper Prior Lake was listed on Minnesota’s 303(d) List of Impaired Waters for nutrient/eutrophication biological indicators with aquatic recreation being impaired. Water quality has been reduced due to excessive phosphorus loading. According to the TMDL implementation plan developed for Spring Lake and Upper Prior Lake, the total phosphorus load must be reduced by 83% and 41%, respectively, to meet water quality goals. Overall, for 28 monitored natural rainfall/runoff events from 2013-2015, the IESF trench removed 26% of the phosphate mass load it received, though after non-routine maintenance in August 2014 the performance improved to 45% phosphate mass load reduction. These results indicate the importance of maintenance. A newer installation was previously monitored, and found to retain 71% of the phosphate (Erickson and Gulliver 2010). Most of the overall phosphate load reduction was achieved during larger events that had comparatively high influent phosphate concentrations (32.3 – 125.2 μg/L) and mass loads. Many small events in this investigation with low influent phosphate concentrations (3.8 – 38.4 μg/L) or mass loads exhibited negative removal (i.e., effluent mass load > influent mass load). The high effluent phosphate concentrations are suspected to be caused by the degradation of floating plants (primarily duckweed) that were deposited on the surface of the filter trench. As mentioned above, nonroutine maintenance to remove this material resulted in substantial performance improvement. After this maintenance, positive removal was observed for influent concentrations ranging from 6.3 – 44.1 μg/L. Detailed results, maintenance activities, design and operating & maintenance recommendations, and lessons learned are given within this report.
  • Thumbnail Image
    Item
    Regulation of the ESX-5 Secretion System in Mycobacterium tuberculosis
    (2017-05) Elliott, Sarah
    Mycobacterium tuberculosis (Mtb) is one of the most prolific bacterial pathogens in the history of human disease. Robert Koch discovered that Mtb was the causative agent of the disease tuberculosis in 1882, and despite intensive research and major advances, Mtb represents a major global health threat today. Worldwide in 2015, there were 10.4 million newly diagnosed active cases and 1.8 million deaths attributed to this infection. Bacterial pathogens often secrete factors to promote survival during infection, and Mtb is no exception. Mtb has evolved a unique, diderm cell membrane, which contributes to the ability of the bacterium to resist host immune responses. However, this hydrophobic barrier also presents an obstacle for the export of factors critical to success of the organism. Mycobacteria, including Mtb, have evolved the Type VII ESX secretion systems to facilitate protein export across the complex membrane. Three ESX systems have been implicated in Mtb pathogenesis, ESX-1, -3 and -5. While the regulatory mechanisms and biological functions for both ESX-1 and ESX-3 are well-defined, little was known about ESX-5 aside from a general role in Mtb virulence. The work described in chapter 2 reveals that ESX-5 secretion is directly regulated at the transcriptional level by the Pst/SenX3-RegX3 system in response to inorganic phosphate (Pi) limitation. RegX3, the response regulator, is normally activated when Pi is scarce. Disruption of a transmembrane component of the Pst Pi uptake system, through deletion of pstA1, causes constitutive activation of RegX3. We observed overexpression of esx-5 transcripts and hyper-secretion of the ESX-5 substrates EsxN and PPE41 in the Mtb ΔpstA1 mutant, and this response requires RegX3. In wild-type Erdman (WT) Mtb, transcription of esx-5 genes and secretion of ESX-5 proteins was induced by Pi limitation in a RegX3-dependent manner. Using electrophoretic mobility shift assays (EMSA), we found that RegX3 directly binds to a segment of DNA within the esx-5 locus, demonstrating that regulation of ESX-5 mediated by the Pst/SenX3-RegX3 system occurs directly. Experiments outlined in chapter 3 expand on the work reported in chapter 2. Using in vitro EMSAs, we defined the RegX3 binding sequence located within the intergenic region between ppe27 and pe19 in the esx-5 locus. RegX3 is a global response regulator, and targeted mutation of the esx-5 binding site sequence uncouples the secretion system from the myriad effects mediated by RegX3 throughout the cell. We found that mutating the esx-5 RegX3 binding site sequence reversed expression of esx-5 transcripts and secretion of EsxN and PPE41 in WT Mtb during Pi limitation. Similarly, esx-5 overexpression and ESX-5 hyper-secretion was suppressed in the ΔpstA1 mutant when the RegX3 binding site sequence was mutated or deleted. We then tested the importance of RegX3-mediated regulation of ESX-5 for Mtb virulence by infecting C57BL/6 and IrgM1-/- mice with a binding site mutant. Notably, deletion of the esx-5 RegX3 binding site partially restored virulence to the attenuated ΔpstA1 mutant. Our findings demonstrate that precise regulation of ESX-5 is critical for full Mtb virulence. Further, hyper-secretion of antigenic ESX-5 substrates sensitizes ΔpstA1 bacteria to host responses, suggesting that one or more of these aberrantly secreted proteins is responsible for attenuation. We next sought to determine whether aberrant hyper-secretion of one ESX-5 secreted factor, EsxN, sensitizes ΔpstA1 bacteria to host immune responses. Previous work has shown that the ΔpstA1 mutant is attenuated in immune competent C57BL/6 mice and immune compromised IrgM1-/- and Nos2-/- mice, while experiments described in chapter 3 demonstrated that attenuation of the ΔpstA1 mutant in C57BL/6 and IrgM1-/- mice was specifically due to constitutive activation of esx-5. Experiments in Chapter 4 evaluate the contribution of EsxN to Mtb virulence. Deletion of esxN did not reverse ΔpstA1 mutant sensitivity to reactive oxygen species, acidic pH or cell wall stress in vitro. However, WT bacteria were more sensitive to reactive nitrogen stress when esxN was deleted, suggesting a role for EsxN in resistance to reactive nitrogen species (RNS). We found that esxN does not suppress the ΔpstA1 mutant virulence defect in C57BL/6 or IrgM1-/- mice. However, deletion of esxN in the ΔpstA1 mutant did partially reverse the replication and virulence defect in Nos2-/- mice, indicating hyper-secretion of EsxN sensitizes ΔpstA1 bacteria to immune responses other than RNS production in these mice. Aberrant hyper-secretion of EsxN may influence sensitivity to other host responses in the ΔpstA1 mutant. EsxN seems to be required for survival during RNS stress in vitro in WT Mtb. Further work will be required to tease apart the potential role EsxN plays in RNS resistance. The work described in this thesis expands the knowledge of ESX-5 secretion system biology, and Mtb protein secretion in general. We have uncovered the mechanism of regulation, and also revealed a relevant environmental signal, Pi limitation, that activates this system. We have demonstrated that regulation of ESX-5 by the Pst/SenX3-RegX3 system occurs directly by identifying the RegX3 binding site sequence within the esx-5 locus. Using targeted mutation of the RegX3 binding sequence, we have shown that dysregulation of ESX-5 activity has a detrimental impact on Mtb virulence. These findings highlight the importance of proper regulation of the ESX-5 system to Mtb pathogenesis. An understanding of the regulatory mechanism and environmental signals that activate ESX-5 during infection provides an important frame-work for future studies to elucidate the functional role of this system.
  • Thumbnail Image
    Item
    Understanding the genetic requirements for Saccharomyces cerevisiae to survive at low temperatures.
    (2012-05) Haas, Kelaine C. Zimmerman
    Most organisms must possess molecular mechanisms that enable them to respond to both long-term and transient temperature fluctuations in their environment. As a first step in understanding these mechanisms, we focused on identifying molecular mechanisms that allow the budding yeast, Saccharomyces cerevisiae, to respond to low temperature. The goal of this study was to analyze the growth rate of S. cerevisiae mutant strains to identify genes required for growth at 10˚C. Within the S. cerevisiae homozygous deletion collection, we identified cold-sensitive strains representing 790 genes. The CS790 genes encode proteins that function in a wide range of biological processes, suggesting that genes required for S. cerevisiae growth at low temperatures represent complex networks of cellular and molecular functions. Two processes were statistically enriched among these 790 genes: genes required for threonine and tryptophan biosynthesis, and genes encoding components of the dynein/dynactin complex. These results provide a framework for future studies to identify the specific molecular mechanisms responsible for cold adaptation and growth of organisms. In addition to these enriched categories, previous work in our lab has also shown that three S. cerevisiae null mutants, ubc7∆, cue1∆, and doa10∆, are cold sensitive at 10˚C. UBC7, CUE1, and DOA10 encode proteins that function in the Ubc7-dependent ERAD pathway, which targets aberrant proteins in the ER lumen or membrane for ubiquitin-mediated proteasomal degradation. By introducing point mutations in the Ubc7 active site C89 residue, we created a catalytically inactive Ubc7 protein, and confirmed that the ubc7∆ strain is cold sensitive due to the loss of Ubc7 enzymatic activity. Therefore, the ability of Ubc7 to ubiquitinate appropriate target substrates is required for yeast growth at 10˚C. In an effort to identify relevant targets of this ERAD pathway, we used the Synthetic Genetic Array (SGA) protocol to create a complete set of haploid double mutants that carry both a deletion of UBC7 and a deletion of another gene. Genetic suppressors were identified by double mutant strains that no longer exhibited the cold-sensitive phenotype seen in ubc7∆ strains. This analysis identified 10 deletions that suppress the cold-sensitivity of ubc7∆, cue1∆, and doa10∆ cold-sensitivity, indicating that the suppression is not limited to the UBC7 gene, but instead affects the entire Ubc7-dependent ERAD pathway. Therefore, the proteins represented by these 10 deletion mutants are potential Ubc7-ERAD targets. Interestingly, four of these suppressors, PHO84, PHO81, SPL2 and YML122C, are members of the high-affinity phosphate transport pathway. Additional analysis of S. cerevisiae triple mutant strains with deletions in UBC7, SPL2, and the high-affinity Pi transporters, PHO84 or PHO89, shows that UBC7 may be working in a parallel pathway to SPL2. These results suggest that Ubc7 may directly or indirectly regulate the low-affinity branch of the phosphate transport pathway, via Pho87 and Pho90.