2002 Progress Report: Effectiveness of UV Irradiation for Pathogen Inactivation in Surface WatersEPA Grant Number: R829012
Title: Effectiveness of UV Irradiation for Pathogen Inactivation in Surface Waters
Investigators: Linden, Karl G. , Shin, Gwy-Am , Sobsey, Mark D.
Current Investigators: Linden, Karl G. , Sobsey, Mark D.
Institution: Duke University , University of North Carolina at Chapel Hill
EPA Project Officer: Page, Angela
Project Period: August 20, 2001 through August 19, 2004 (Extended to August 19, 2005)
Project Period Covered by this Report: August 20, 2001 through August 19, 2002
Project Amount: $524,848
RFA: Drinking Water (2000) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Ultraviolet (UV) irradiation is now recognized to be an inexpensive and relatively easy means to achieve disinfection of Cryptosporidium parvum, and does not appear to produce disinfection byproducts at practical doses. The germicidal effects of UV against emerging pathogens and challenges related to application of UV disinfection for filtered and unfiltered surface waters needs to be assessed. The objectives of this research project are to evaluate the susceptibility, repair potential, and resistance of select Contaminant Candidate List (CCL) pathogens and indicators to UV disinfection from low-pressure (LP) and medium-pressure (MP) UV sources, and to elucidate the relative germicidal effectiveness of different wavelengths of UV for these pathogens and indicators. The extent to which microbes are associated with water treatment particles typical in unfiltered systems, and the effects of this particle association and other water quality parameters on UV disinfection potential will be investigated. It is hypothesized that UV will be an effective means to inactivate CCL pathogens and that with proper system design, repair/reactivation and water quality challenges found in typical treatment scenarios will not compromise effective UV treatment.
Phase I. UV Dose-Response for Pathogens: Basic Information
In the first year of the project, we finished setting up both bench-scale LP and MP UV disinfection systems, along with proper methods for UV dosimetry. We also finished setting up the assay systems for most of the test microorganisms, and established a collaboration for obtaining and assaying the Toxoplasma gondii protozoan parasite to be used in this project. We also have performed inactivation studies on test microorganisms in phosphate-buffered water and/or filtered water. To date, inactivation studies on the indicator microorganisms Escherichia coli, coliphage MS2, bacteriophage PRD-1, and Bacillus subtilis endospores, and most of the CCL and emerging pathogens such as coxsackievirus B4, Mycobacterium terrae, adenovirus type 2, and T. gondii have been completed or are being performed.
The inactivation of E. coli by both LP and MP UV irradiation was very rapid, and more than 4 log10 inactivation was achieved within a UV dose of 10 mJ/cm2. However, the inactivation of coliphage MS2 by both LP and MP UV irradiation was relatively slow, and 4 log10 inactivation was achieved with a UV dose of 60-70 mJ/cm2. It appears that there was no significant difference between LP and MP UV in terms of their effectiveness against these two indicator microorganisms. The inactivation kinetics of these two indicator microorganisms were similar to those previously reported in the literature.
The inactivation bacteriophage PRD-1 by both LP and MP UV irradiation also was slow and 4 log10 inactivation was achieved with a UV dose of approximate 80 mJ/cm2. There was no significant difference between LP and MP UV in terms of their effectiveness against bateriophage PRD-1. However, it should be mentioned that the required UV dose to achieve a 4 log10 inactivation of PRD-1 in our study was significantly higher than previously reported in the literature. It is not certain why there is such a big difference between our data and the ones in the previous literature, but this will be explored in our future research.
Like bacteriophage PRD-1, the inactivation of B. subtilus spores by LP and MP UV irradiation was different from the ones previously reported in the literature. A 4 log10 inactivation of B. subtilus spores was achieved at a UV dose of approximately 40 mJ/cm2 by both LP and MP UV, which was significantly lower than previously reported. It should be noted, however, that the required UV dose to achieve a 4 log10 inactivation of B. subtilis spores was, in fact, widely distributed in previous reports depending on strains and the culture method used. Nonetheless, there was no significant difference between LP and MP UV in terms of their effectiveness against the strain of B. subtilis spores used in this research project.
The inactivation of coxsackievirus B4 by LP UV irradiation was rapid, and more than 4 log10 inactivation was achieved within a UV dose of approximately 20 mJ/cm2. However, the inactivation of M. terrae by LP or MP UV irradiation was relatively slow, and 3 log10 inactivation was achieved with a UV dose of 40 mJ/cm2. Compared to inactivation kinetics of other microorganisms in this research project, the inactivation kinetics of M. terrae showed a slight tailing at the high UV doses, which may be due to the aggregating nature of this microbe. Again, it appears that there was no significant difference between LP and MP UV in terms of their effectiveness against M. terrae.
The inactivation of adenovirus 2 by LP UV irradiation was very slow, and only approximately 2 log10 inactivation was achieved with a UV dose of approximately 60 mJ/cm2, which is similar to the one reported in previous report for adenoviruses 40 and 41. Unlike other microorganisms in this and other research projects, there were some remarkable differences between LP and MP UV in terms of their effectiveness against adenovirus 2. The inactivation of adenovirus 2 by MP UV generally was more rapid than that by LP UV, and it appears that the difference in terms of magnitude of inactivation increases with increasing UV doses. It is not certain at this moment why there is such a big difference between LP and MP UV inactivation for adenovirus 2, but this will be explored in our future research.
Like adenovirus 2 and M. terrae, the inactivation of T. gondii by LP and MP UV irradiation was relatively slow, and only approximately 2 log10 inactivation was achieved with a UV dose of 40 mJ/cm2. However, the data from the first two experiments are not very conclusive, so more experiments will be performed to better characterize the inactivation kinetics of this microorganism by UV irradiation.
We have developed a new assay system—long-template reverse-trascription polymerase chain reaction (LT RT-PCR)—for Norwalk virus that can amplify larger genomic regions of the virus, and it has been used to better determine the loss of infectivity of this virus with UV irradiation. Briefly, we employed an innovative approach to select RT-PCR primers (with a concept of "availability of priming sites" by analyzing the secondary structure of a genome using computer software such as MFOLD in GCG package) in developing this new RT-PCR system (LT RT-PCR) for this virus. With this approach, we were able to increase the size of PCR targets up to five-fold, while maintaining or even increasing the sensitivity of the RT-PCR detection for this virus, which would increase the possibility of detecting "viable" microorganisms. This is one of the primary concerns in using molecular biological methods in disinfection studies. The inactivation of Norwalk virus based on this new LT RT-PCR was much faster than those based on traditional RT-PCR, although it currently is impossible to determine the correlation between these data and the loss of true infectivity because of the lack of an infectivity system for this virus.
Phase II. Wavelength Effectiveness
We have established collaboration with the engineers at the Duke University Free Electron Laser (FEL) Laboratory, and developed and evaluated preliminary assessment of the UV light wavelengths available using our physical and chemical measurement instruments. In addition to using the light from the FEL, we have set up a method to perform wavelength-specific studies using a polychromatic UV light source (medium-pressure mercury vapor lamp) and a set of UV bandpass filters. These filters are capable of passing specific wavelength ranges (between 214 and 300 nm) of light from the UV source. This setup has been utilized to develop wavelength effectiveness information for coliphage MS2, bacteriophage PRD-1, and E. coli, and these data will be supplemented using the FEL system in the near future.
Phase III. Pathogen Clumping and Particle Association
Water samples were collected from raw surface waters serving various utilities across the United States. These samples were evaluated for particle-associated coliform and aerobic endospores using physical particle disruption techniques such as homogenization and blending. The counts of the indigenous microbes in the raw waters typically were low (less than 1,000 per 100 mL); thus, using these methods, it was not possible to assess whether any of the microbes were indeed particle associated. Currently, we are investigating the use of microscopic techniques (nucleic acid staining/probes along with confocal microscopy) to evaluate the issue of particle association in these raw waters. Along with the raw water particle-association testing, we have developed UV dose-response relationships for indigenous aerobic endospores in the various raw waters tested. We currently are isolating the different types of aerobic spores present in the waters, and we will develop dose-response information for them. The goal of this indigenous spore testing is to evaluate if the spores are more resistant in the native raw water as compared to their sensitivity when spiked into a water from a pure culture. Future work will utilize these and some laboratory strain spores and expose them to coagulation conditions typical to that used in water treatment plants. These flocculated microbes will be tested to evaluate the UV effectiveness for disinfection under different coagulation conditions.
Phase I. Due to the large number of microorganisms involved in this research, there is still some work to be done. First, the inactivation studies on coxsackievirus B4 and Norwalk virus by MP UV will be performed with cell culture infectivity assay and the new LT RT-PCR, respectively. Second, the inactivation study on adenovirus 40 or 41 by both LP and MP UV will most likely be performed with a newly developed molecular biology assay (RT-PCR). We have obtained several cell lines for these "fastidious" viruses, and have tried to set up an infectivity assay for them. However, as found by other researchers, we are having some difficulties in culturing these viruses and observing the cytopathic effect on these cells. In addition to continuing the effort to establish a cell culture infectivity system for these viruses, we have been considering employing a new molecular biology assay developed for these viruses, which could detect "viable" adenovirus 40 and 41. Finally, we will utilize one more filtered water matrix for some of the microorganisms in this study. We have tried one filtered water matrix, but unlike a phosphate-buffered system, there may be some differences in different (filtered) water matrices in terms of their effect on the inactivation of the microorganisms being studied.
Phase II. During the second period, we will determine wavelength effectiveness for adenovirus 2, coxsakievirus 3, and Mycobacterium terrae as proposed in the original proposal. However, due to the expensive infectivity assay of T. gondii, and the anticipated large number of samples in these (wavelength effectiveness) experiments, we must limit the number of wavelengths investigated for T. gondii to two or three wavelengths.
Phase III. We will continue the isolation of and development of UV dose-response relationships for indigenous spores identified in raw water supplies. M. terrae and B. subtilus var. niger spores will be utilized to assess the effects of microbe aggregation on disinfection efficacy. These microbes will be used because they are relatively resistant to UV radiation and because they have a tendency to grow in a clumped or aggregated state. Work will begin on evaluation of particle-associated and dispersed viruses such as Norwalk, coxsackievirus, and adenovirus using methods whereby the viruses will be prepared in a cell-associated state. Cell-associated and dispersed viruses will be compared for response to disinfection.
Phase IV. Toward the end of the second reporting period, the presence and extent of repair and recovery following UV disinfection will be evaluated for Mycobacterium spp. and the indicator E. coli as a benchmark. To assess repair following UV, microbes will be enumerated before and after the repair protocols and differences in the log10 survival will be evaluated. Both light and dark repair conditions will be evaluated to simulate conditions such as distribution system, covered clearwells, aqueducts, and uncovered supplies.