Introduction
Background and Motivation
As a core unit, the Caltech Solar Toilet electrochemical reactor uses semiconductor anodes for the oxidation of chloride to chlorine, leading to the disinfection of microorganisms (Huang et al., 2016). The main requirements for a reliable assessment are specificity, sensitivity, reproducibility, speed, automation and low cost (Kostić et al., 2011).
Thesis Organization
Linear-regression fit to re-injury data is shown in Figure 2.5; the fluence-based first-order kinetic rate constants, k, were determined from the slopes, which are summarized in Table 2.5 of Section 2.5.5. This is due to the oxidation of Ti3+-Ovac pairs back to Ti4+ (ie the conversion from conducting TiO2-x to insulating TiO2).
Removal of Antibiotic Resistant Bacteria and Genes by UV Assisted
Introduction
ARB and ARGs originate from different sources, e.g. municipal waste, hospital and livestock waste, which are ultimately affected by treatment plants (Karkman et al., 2018). Additional treatment steps after ozonation may be necessary to avoid regrowth of ARB (Iakovides et al., 2019).
Experimental
- Chemicals and Materials
- Construction and Preparation of Plasmids
- Treatment of intracellular ARGs (i-ARGs)
- Treatment of extracellular ARGs (e-ARGs)
- Treatment of ARGs in Wastewater
- Quantitative PCR
- Radical Generation Probed by Benzoic Acid Degradation
The plasmid stock for pEB1-tetA or pEB1-sul1 was spiked in 30 mM NaClO4 or 30 mM NaCl to reach a concentration of ~10 ng/µL. The filtered wastewater with a volume of 30 mL was directly added into the reactor for UV-enhanced electrolysis under optimized electrical conditions, i.e., the current density of 10 mA/cm2, with a duration of 0-30 min.
Results And Discussion
- Inactivation of ARB and Degradation of ARGs
- Mechanisms on ARG damage
- Simulation on Radical Generation
- UV Assisted Electrochemical Oxidation of ARGs in Wastewater
For EO treatment in the presence of Cl-, Cl2·- is the dominant radical, followed by ·OH and Cl· (Figure 2.10a). Second, all photo-based techniques, like UV disinfection, can be significantly affected by water quality parameters, for example, transmission, color, and presence of particulate matter (US Environmental Protection Agency, 2006).
Conclusion
This result suggests that UV-EO treatment and other photo-based techniques should be applied as the last step after other water treatments for the effectiveness of ARB and ARGs removal. Combinations of different techniques will certainly become more common in future water treatment processes aimed at the elimination of ARB and ARGs. Further attempts should be made to understand the effects of different water quality parameters on the removal of ARB and ARGs treated with UV-EO and to monitor the ARB and ARGs with other traditional water treatment followed by UV-EO treatment.
Furthermore, to better understand the efficacy of UV-EO treatment in controlling ARG proliferation and the accuracy of using qPCR to assess ARG shedding, the change in transformation activity for UV-EO-treated ARGs should be investigated.
Supporting Information
- Detailed Method on Plasmid Construction
- Preparation and Characterization of Blue Nanotube Array (BNTA)
- Effect of electrolytes on the DNA analysis by gel electrophoresis
- List of Primers
- Summary of Kinetic Parameters from Literature and This Study
A control aliquot was extracted from the same commercial DNA extraction kit as a comparison. This may be mainly due to DNA damage during the ECL process (e.g., high local pH which will be discussed further with the simulation in this study), as we rule out leakage-induced PCR inhibition electrolyzed cathodic. The most common water quality parameters that affect the effectiveness of UV disinfection (eg, water light transmission, color, and particle material) may also play an important role in the application of other UV methods or photolysis.
After modification, the oxygen electrode functions as a selective transducer for the detection of an enzymatic reaction, that is, the oxidation of glucose by glucose oxidase.
Electrochemical Cell Lysis of Gram-Positive and Gram-Negative
Introduction
In recent years, the use of biomolecular techniques such as polymerase chain reaction (PCR) has led to rapid, accurate and sensitive methods for quantifying aquatic bacteria (Xie et al., 2016; Heid et al., 1996; Kim et al., 2013). In addition, removal of reagents after cell lysis is necessary to avoid interference with further detection (Di Carlo et al., 2005; Lee et al., 2010). The downside of electroporation is the use of strong electric fields to achieve irreversible electroporation (e.g. 10 kV/cm as reported by Poudineh et al. in 2014).
Electrochemical cell lysis (ECL) relies on the cathodically generated hydroxide (i.e. localized high pH) to disrupt microbial cell membranes by breaking fatty acid-glycerol ester bonds in phospholipids (Di Carlo et al., 2005; Nevill et al., 2007).
Experimental
- Reagents
- Bacterial Sample Preparation
- Electrochemical Cell Lysis Experiment
- Analysis of Cell Lysis by Fluorescent Microscope
- DNA Quantification by qPCR
- pH Effect Tests
- Electrochemical Cell Lysis of Bacteria in Environmental Water
As a negative control, an aliquot of the initial sample without ECL was treated in the same way to remove all the cells. Another aliquot of the initial sample was extracted for each bacterial strain using a commercial DNA extraction kit (PureLink® Genomic DNA Mini Kit, Invitrogen by Thermo Fisher Scientific, USA) as a positive control. To evaluate the DNA extraction efficiency, !CT values of the ECL-treated samples were calculated by subtracting CT values of the suspended DNA in the ECL-treated samples from those in the untreated ones.
Three different environmental water samples were tested to evaluate the performance of the ECL technique on DNA extraction of bacteria from ambient environmental water.
Theory and Simulation
Pond water was added directly to the cathodic chamber for the ECL reaction without any pretreatment while 50 mM Na2SO4 was added to the anodic chamber. Then, filtered wastewater was added to the cathodic chamber for the ECL reaction while 50 mM Na2SO4 was added to the anodic chamber. Suspended total bacterial DNA from all environmental water samples was then collected by centrifugation at 10,000 g for 10 min.
All of the above environmental water samples were also extracted using the same commercial DNA extraction kit (PureLink®. Genomic DNA Mini Kit) as the positive control.
Results and Discussion
- Electrochemical Cell Lysis of Different Bacteria
- pH Effects on Cell Lysis and DNA Extraction
- Simulations of pH Profiles at the Cathode
- Electrochemical Cell Lysis in Environmental Water
For the ECL-treated samples, !CT values were calculated by subtracting CT values of the suspended DNA in ECL-treated samples from those in the untreated samples. For the samples extracted by the commercial kit, !CT values were calculated by subtracting CT values of the total DNA extracted by the commercial kit from those of the suspended DNA in the untreated samples. There is no significant difference between the !CT values of the samples treated with 1 min ECL and of those extracted by the commercial kit (P = 0.72 for E. coli and P = 0.48 for S. Typhi).
However, !CT values were still significantly increased to 7.9 and 6.5 for B after 1 min of ECL.
Conclusion
Supporting information
- Detailed Methods and Information of qPCR Measurements
- Detailed Simulation Methods
- The ECL Effects on DNA Damage and PCR Inhibition
- Summary of Experimental Setups for Previous Studies on ECL
Five calibration solutions were detected by qPCR in triplicate based on the same method described in Section 3.2.5. Extracted DNA samples were prepared in the same manner as for the DNA damage assay above. It suggests that hardly any PCR inhibitors were generated in the ECS, which could contribute to the decrease in DNA extraction efficiency.
Thus, even increased pH in ECS with different ECL durations should not have an inhibitory effect on qPCR detection.
Introduction
86 With these limitations, polymerase chain reaction (PCR)-based methods have been developed and increasingly used for rapid, sensitive and specific detection of pathogens (Girones et al., 2010; He and Jiang, 2005; Heid et al., 1996). . . Pretreatment with propidium monoazide (PMA) prior to PCR can help distinguish DNA from dead cells whose cell membranes are irreparably damaged (Nocker et al., 2006a). The mechanism is that PMA penetrates the compromised cell membrane but is excluded from living cells with intact cell membranes (Nocker et al., 2006a).
Cross-linked DNA strands become insoluble and can be easily removed during the DNA extraction process (Nocker et al., 2006a).
Materials and Methods
- Bacterial Sample Preparation
- PMA Treatment
- DNA Extraction and qPCR Measurement
- Data Analysis
Species (Gram stain) Media T (oC) ATCC No. A typical PMA pretreatment includes the following three steps: 1) add PMA stock solution (20 mM in water, Biotium Inc.) to samples to a final concentration of 80 µM in an ice bath; 2) incubate samples in the dark for 10 min; and 3) expose samples to light for 10 min to photoactivate PMA. 90 extracted using the PureLink® Genomic DNA Mini Kit (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. Real-time PCR assays were performed with MasterCycler RealPlex 4 (Eppendorf Inc.) to quantify the presence of universal bacterial 16S rRNA gene.
The reaction mix contains 10 µL PerfeCTa® qPCR ToughMix® (Quanta BioSciences Inc.), 0.25 µM forward primer (1369F, 5'CGGTGAATACGTTCYCGG3′, where Y is either C or T, Integrated DNA Technologies Inc.), µM reverse primer (µM reverse primer). 1492R, 5'GGWTACCTTGTTACGACTT3' where W is either A or T, Integrated DNA Technologies Inc.), 0.25 µM TaqMan probe (FAM-5'CTTGTACACACCCGCCGTC3', Integrated DNA Technologies Inc.), 1 µL template, DNA -free, and µL template water (Promega Corporation) to a final volume of 20 µL (Suzuki et al., 2000).
Results and Discussion
- Validation of Sunlight-Activated PMA Pretreatment
- Optimization of Pretreatment Conditions
- Effect of Light Intensity
- Effect of Multiple Sequential Treatments
- PMA-qPCR Application on Wastewater Samples
The PMA-qPCR to distinguish between viable and dead cells was performed by the Ct. The black dotted line indicates the expected Ct values for 100% efficiency of amplification of viable cells without any interference from dead cells. The goal was to find a condition that resulted in the highest DCt value for dead cells, but the lowest DCt values for viable cells.
In general, the DCt value for dead cells increased with PMA concentration and exposure time (Figure 4.3A).
Conclusion
100 cathodic effluents as indicators of the optimal operational duration for the practical use of ECL. Effect of catalyst type on the kinetics of the photoelectrochemical disinfection of water inoculated with E. Their formation by means of radiation pulses and their role in the mechanism of the Fricke dosimeter”.
Kinetic study of the reactions of chlorine atoms and Cl2•- radical anions in aqueous solutions.
Conclusion and Future Direction
Conclusion
Therefore, we suggest that, as in the case of UV-C disinfection, UV-assisted electrochemical methods should also be applied in the last step after operations and other processes of the water treatment unit for the efficient removal of ARGs in water environmental or designed. The development of new dyes that provide more sensitive differentiation or other methods based on the difference in motility between living and dead cells should be explored in the future. Third, relatively lower DNA extraction efficiency of gram-positive bacteria was found in buffered samples, which may limit the application of ECL to gram-negative bacteria.
It will be challenging but interesting to make more efforts in the future study to improve the DNA extraction efficiency of gram-positive bacteria by electrochemical methods based on different mechanisms (e.g., oxidation).
Future Direction
- Development of Electrochemical DNA Sensor for Waterborne
- Introduction
- Development of Electrochemical DNA Sensor
- Integration of Electrochemical DNA Sensing Platform
Electrochemical cleavage of the thiol-gold linkage facilitated the elution of DNA hybrids for further analysis. In order to take advantage of the large specific surface area of the modified nanoporous electrode, a single-stranded DNA probe sequence will be immobilized on the surface of the nanochannel wall. The ability of the modified electrochemical DNA sensor to detect ARGs will also be investigated, as they remain serious and growing challenges for human health and have attracted attention in recent years (as described in Chapter 2 of this thesis).
Water samples first flow into the cathodic chamber of the cell lysis unit, after which the effluent is distributed across multiple electrochemical DNA sensors for separate detection of specific pathogens. The challenges related to the integrated platform include the pH adjustment of the cell lysis unit effluents, miniaturization of power supplies, affordability and scalability, device control and the development of the appropriate workstation. Proton transfer and the mobilities of the H+ and OH− ions from studies of a dissociative model for water”.
