Browsing by Subject "Disinfection"

The growth and proliferation of invasive bacteria in engineered systems is an ongoing problem. While there are a variety of physical and chemical processes to remove and inactivate bacterial pathogens, there are many situations in which these tools are no longer effective or appropriate for the treatment of a microbial target. For example, certain strains of bacteria are becoming resistant to commonly used disinfectants, such as chlorine and UV. Additionally, the overuse of antibiotics has contributed to the spread of antibiotic resistance, and there is concern that wastewater treatment processes are contributing to the spread of antibiotic resistant bacteria.

Due to the continually evolving nature of bacteria, it is difficult to develop methods for universal bacterial control in a wide range of engineered systems, as many of our treatment processes are static in nature. Still, invasive bacteria are present in many natural and engineered systems, where the application of broad acting disinfectants is impractical, because their use may inhibit the original desired bioprocesses. Therefore, to better control the growth of treatment resistant bacteria and to address limitations with the current disinfection processes, novel tools that are both specific and adaptable need to be developed and characterized.

In this dissertation, two possible biological disinfection processes were investigated for use in controlling invasive bacteria in engineered systems. First, antisense gene silencing, which is the specific use of oligonucleotides to silence gene expression, was investigated. This work was followed by the investigation of bacteriophages (phages), which are viruses that are specific to bacteria, in engineered systems.


For the antisense gene silencing work, a computational approach was used to quantify the number of off-targets and to determine the effects of off-targets in prokaryotic organisms. For the organisms of Escherichia coli K-12 MG1655 and Mycobacterium tuberculosis H37Rv the mean number of off-targets was found to be 15.0 + 13.2 and 38.2 + 61.4, respectively, which results in a reduction of greater than 90% of the effective oligonucleotide concentration. It was also demonstrated that there was a high variability in the number of off-targets over the length of a gene, but that on average, there was no general gene location that could be targeted to reduce off-targets. Therefore, this analysis needs to be performed for each gene in question. It was also demonstrated that the thermodynamic binding energy between the oligonucleotide and the mRNA accounted for 83% of the variation in the silencing efficiency, compared to the number of off-targets, which explained 43% of the variance of the silencing efficiency. This suggests that optimizing thermodynamic parameters must be prioritized over minimizing the number of off-targets. In conclusion for the antisense work, these results suggest that off-target hybrids can account for a greater than 90% reduction in the concentration of the silencing oligonucleotides, and that the effective concentration can be increased through the rational design of silencing targets by minimizing off-target hybrids.

Regarding the work with phages, the disinfection rates of bacteria in the presence of phages was determined. The disinfection rates of E. coli K12 MG1655 in the presence of coliphage Ec2 ranged up to 2 h-1, and were dependent on both the initial phage and bacterial concentrations. Increasing initial phage concentrations resulted in increasing disinfection rates, and generally, increasing initial bacterial concentrations resulted in increasing disinfection rates. However, disinfection rates were found to plateau at higher bacterial and phage concentrations. A multiple linear regression model was used to predict the disinfection rates as a function of the initial phage and bacterial concentrations, and this model was able to explain 93% of the variance in the disinfection rates. The disinfection rates were also modeled with a particle aggregation model. The results from these model simulations suggested that at lower phage and bacterial concentrations there are not enough collisions to support active disinfection rates, which therefore, limits the conditions and systems where phage based bacterial disinfection is possible. Additionally, the particle aggregation model over predicted the disinfection rates at higher phage and bacterial concentrations of 108 PFU/mL and 108 CFU/mL, suggesting other interactions were occurring at these higher concentrations. Overall, this work highlights the need for the development of alternative models to more accurately describe the dynamics of this system at a variety of phage and bacterial concentrations. Finally, the minimum required hydraulic residence time was calculated for a continuous stirred-tank reactor and a plug flow reactor (PFR) as a function of both the initial phage and bacterial concentrations, which suggested that phage treatment in a PFR is theoretically possible.

In addition to determining disinfection rates, the long-term bacterial growth inhibition potential was determined for a variety of phages with both Gram-negative and Gram-positive bacteria. It was determined, that on average, phages can be used to inhibit bacterial growth for up to 24 h, and that this effect was concentration dependent for various phages at specific time points. Additionally, it was found that a phage cocktail was no more effective at inhibiting bacterial growth over the long-term than the best performing phage in isolation.

Finally, for an industrial application, the use of phages to inhibit invasive Lactobacilli in ethanol fermentations was investigated. It was demonstrated that phage 8014-B2 can achieve a greater than 3-log inactivation of Lactobacillus plantarum during a 48 h fermentation. Additionally, it was shown that phages can be used to protect final product yields and maintain yeast viability. Through modeling the fermentation system with differential equations it was determined that there was a 10 h window in the beginning of the fermentation run, where the addition of phages can be used to protect final product yields, and after 20 h no additional benefit of the phage addition was observed.

In conclusion, this dissertation improved the current methods for designing antisense gene silencing targets for prokaryotic organisms, and characterized phages from an engineering perspective. First, the current design strategy for antisense targets in prokaryotic organisms was improved through the development of an algorithm that minimized the number of off-targets. For the phage work, a framework was developed to predict the disinfection rates in terms of the initial phage and bacterial concentrations. In addition, the long-term bacterial growth inhibition potential of multiple phages was determined for several bacteria. In regard to the phage application, phages were shown to protect both final product yields and yeast concentrations during fermentation. Taken together, this work suggests that the rational design of phage treatment is possible and further work is needed to expand on this foundation.

The primary goals of this research were to investigate the electrochemical behavior of carbon nanostructures of varying morphology, identify morphological characteristics that improve electrochemical capacitance for applications in energy storage and neural stimulation, and engineer and characterize a boron-­doped diamond (BDD) electrode based electrochemical system for disinfection of human liquid waste.

Carbon nanostructures; ranging from vertically aligned multiwalled carbon nanotubes (MWCNTs), graphenated carbon nanotubes (g-­CNTs) to carbon nanosheets (CNS); were synthesized using a MPECVD system. The nanostructures were characterized by using scanning electron microscopy (SEM) and Raman spectroscopy. In addition to employing commonly used electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), a new technique was developed to evaluate the energy and power density of individual electrodes. This facilitated comparison of a variety of electrode materials without having to first develop complex device packaging schemes. It was found that smaller pore size and higher density of carbon foliates on a three-dimensional scaffold of carbon nanotubes increased specific capacitance. A design of experiments (DOE) study was conducted to explore the parametric space of the MWCNT system. A range of carbon nanostructures of varying morphology were obtained. It was observed that the capacitance was dependent on defect density. Capacitance increased with defect density.

A BDD electrode was characterized for use in a module designed to disinfect human liquid waste as a part of a new advanced energy neutral, water and additive-free toilet designed for treating waste at the point of source. The electrode was utilized in a batch process system that generated mixed oxidants from ions present in simulated urine and inactivated E. Coli bacteria. Among the mixed oxidants, the concentration of chlorine species was measured and was found to correlate to the reduction in E. Coli concentration. Finally, a new operating mode was developed that involved pulsing the voltage applied to the BDD anode led to 66% saving in energy required for disinfection and yet successfully reduced E. Coli concentration to less than the disinfection threshold.

In order to meet disinfection guidelines, wastewater utilities must achieve a high level of treatment before discharging treated water for irrigation or industrial use. However, public pressure to reduce disinfection by-products and pharmaceutically-active compounds, recently-promulgated regulations on chlorine-resistant microorganisms such as Cryptosporidium parvum, and growth in population and water demand have driven an interest in alternatives to chlorination. The WateReuse Foundation has funded WRF 02-009 (Innovative Treatment Technologies for Reclaimed Water), which is a survey of current and emerging reuse water treatment technologies. The goal of the project is to evaluate treatment technologies can provide adequate recycled water effluent without the cost of reverse osmosis (RO) or the disinfection by-products (DBPs) formed during chlorination.

The inactivation of indigenous microorganisms (total and fecal coliform bacteria, and total aerobic spores) and spiked surrogate, respiratory, and enteric viruses (MS-2 bacteriophage, adenovirus type 4, reovirus type 3, and coxsackievirus type B5) and chemical degradation by wastewater treatment technologies was evaluated on the bench-scale. These include: low- and medium-pressure UV, LPUV/H₂O₂, ozonation, O₃/H₂O₂, peracetic acid (PAA), LPUV/PAA, chlorination, chloramination, and ultrafiltration. The applicability of the candidate disinfection methods, especially emerging and comparatively untested methods such as PAA and advanced oxidation processes (AOPs), was studied through comparison of their performance and the important water matrix parameters (e.g., alkalinity, BOD, TSS, etc.).

Of the chemical disinfectants, molecular ozone and free chlorine were the most effective, with substantial coliform and virus kill at low doses. Combined chlorine in the form of monochloramine had a reduced disinfectant capacity than free chlorine, and peracetic acid (PAA) performed equally as well as free chlorine with respect to coliform bacteria in some instances but had little to no impact on spiked MS2 bacteriophage. None of the aforementioned disinfectants had an appreciable impact on indigenous aerobic spore-forming bacteria due to their physiology. UV and O₃ rapidly killed human enteric and respiratory viruses, but a consistent benefit by AOPs over their base technologies was not observed for any of their base technologies.

Low and medium-pressure UV inactivated free-floating indigenous coliform bacteria almost immediately, while slower inactivation rates at higher UV fluences illustrated the "tailing" behavior observed when bacteria are embedded in or shielded by particulate matter. Log-linear inactivation of spiked viruses and indigenous aerobic spores by UV was consistent across the utility waters. The UV-based advanced oxidation processes (UV/H₂O₂ and UV/PAA) destroyed spiked organic compounds at much higher rates than direct UV photolysis, while O₃, with or without H₂O₂ , oxidized spiked compounds and reduced estrogenicity (EEQ) at low doses. Recalcitrant chlorinated hydrocarbons such as TCEP were only moderately removed by the tested AOPs, but low doses of O₃ (3 ppm residual O₃) reduced estrogenic activity by 99%. Like other disinfection processes, AOP performance is dependant on pretreatment, especially concerning particulates.