Grantee Research Project Results
2008 Progress Report: Development of a Universal Microbial Collector (UMC) for Enteric Pathogens in Water and its Application for the Detection of Contaminant Candidate List Organisms in Water
EPA Grant Number: R833009Title: Development of a Universal Microbial Collector (UMC) for Enteric Pathogens in Water and its Application for the Detection of Contaminant Candidate List Organisms in Water
Investigators:
Current Investigators: Bright, Kelly R. , Gerba, Charles P.
Institution:
Current Institution: University of Arizona
EPA Project Officer: Aja, Hayley
Project Period: August 1, 2006 through July 31, 2009 (Extended to July 31, 2010)
Project Period Covered by this Report: August 1, 2007 through July 31,2008
Project Amount: $466,817
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens in Drinking Water (2005) RFA Text | Recipients Lists
Research Category: Water , Drinking Water
Objective:
While numerous methods (e.g. polymerase chain reaction - PCR, immunochemical) have been developed to detect small numbers of pathogens, their application to the detection of pathogens in water has been limited for a number of reasons including: Small assay volumes are required (usually 10 to 100 microliters), the presence of interfering substances in concentrates, the inability to determine viability, the cost for processing large volumes, low efficiency, variability of concentration methods, cost of filters, and time for concentration.
Since the development of pleated, positively charged filters for the concentration of viruses from large volumes of water in the 1980’s, there has been no significant improvement in concentration technology for viruses from water. The ultimate goal of this project is to develop a simple, efficient, low cost method for the concentration of microorganisms from water that yields a concentrate of minimal volume and with minimal substances that would interfere with the microbial detection methodology.
Progress Summary:
Our research for the first two years has focused on Tasks 1 and 2. We have also begun work on Tasks 3 and 4.
Task 1. Evaluate potential universal microbial collectors (UMC):
During the first two years, we have focused primarily on the evaluation of two innovative nanofiber filtration technologies for the retention of microorganisms from water.
The first type of filter we have worked with is a proprietary charge-modified granular carbon nanofiber available from CUNO, Inc. (Merdian, CT). The surface of this material has been modified to be highly positively charged. The enhanced retention of microorganisms on these filters is due to the combination of a large surface area (through the use of carbon nanofibers) and an enhanced cationic charge. The second filter type is comprised of nano alumina fibers on a microglass fiber matrix that is available from Argonide Corporation (Sanford, FL). The final composition has an average pore size of 2 μm and are pleated (allowing faster flow rates). This material is also highly electropositive and has a large surface area. Unlike other filters with similar pore sizes, the Argonide filters are capable of much faster flow rates.
In addition to these nanofiber filters, we also tested new sodocalcic glass wool filters supplied by Dr. Mark Borchardt of the Marshfield Clinic Research Foundation (Marshfield, WI) for the retention of microorganisms from water.
Carbon Nanofibers Results: For our evaluation of the charge-modified carbon nanofiber material, we tested various classes of microorganisms. The carbon nanofiber material was very effective at retaining multiple classes of microorganisms, with > 99.99 % retention from large volumes of water (10 to 125 liters).
Alumina Ceramic Nanofiber Results: For our initial evaluation of the alumina ceramic nanofiber pleated filters, we used the bacteriophage MS2, poliovirus 1, and Escherichia coli. Microbial retention was evaluated using dechlorinated municipal tap water. Test volumes of 20 liters were evaluated at a flow rate of 2 liters per minute.
The pleated alumina ceramic nanofiber filters were very effective at retaining MS2 bacteriophage, with retention of the virus on the filters ranging from 97.4% to > 99.99% for 40 filters (average % retention = 99.54%, standard deviation = 0.81). Likewise, these filters were very effective at retaining poliovirus 1, with an average retention of 98.39% (standard deviation = 2.41). These filters were also found to be very effective at capturing the bacterium Escherichia coli (average of 99.996% retention). These results are similar to those of more traditional filtering methods such as the use of 1MDS filters for viruses.
Glass Wool Filter Results: Retention rates for the glass wool filters were highly variable for MS2 bacteriophage, ranging from 25.0% to 86.0%. The results for poliovirus 1 were more consistent with retention ranging between 81.0 and 88.3%.
Task 2. Recovery of Organisms from the "Collector":
It is essential that the microorganisms on the carbon be recovered in a viable form. Microbial retention on the "collector" is due to a combination of electrostatic and hydrophobic forces. The relative importance of these factors depends upon the nature of the surface of the adsorbent and the specific microorganism. The organisms were eluted from the "collector" using various eluents to break down hydrophobic interactions and/or electrostatic interactions.
While the use of these filters will remove virtually all of the various types of pathogens from water, it is unrealistic to expect that all the organisms will be recovered with equal efficiency or with the same eluent. The goal is to develop a method with greater reproducibility, precision, and robustness that will be available at a lower cost than current methods. An expected recovery comparable to existing methods appears reasonable based on our results to date.
We have evaluated different elution methods for removing microorganisms from filters (Table 1). We have been able to elute virus from the alumina nanofiber filters with efficiencies similar to existing methods using eluents such as phosphate buffer, glycine, and sodium polyphosphate. It appears that the phosphate groups in the buffer may be interacting with the oxygen/hydroxyl groups on the alumina of the filter. These filters are available at a much lower cost than the filters typically used to concentrate viruses from large volumes of water [~$5 (glasss wool) to $40 (alumina nanofiber) versus ~$150 (1MDS) per filter].
* Eluent A: sodium polyphosphate (concentration 1) + phosphate buffer + glycine
Eluent B: sodium polyphosphate (concentration 2) + phosphate buffer + glycine
Eluent C: sodium polyphosphate (concentration 2) + glycine
Eluent D: phosphate buffer + glycine
Eluent E: phosphate buffer + glycine + Tween 80
Eluent F: phosphate buffer + Tween 80
Eluent G: phosphate buffer
Eluent H: glycine
Eluent I: beef extract
Eluent J: beef extract + glycine
Eluent K: NaI + phosphate buffer + glycine
Eluent L: MgCl2 + phosphate buffer + glycine
An added goal will be to minimize the volume of eluent and the amount of substances that might interact with the "detector." Our method results in an eluate of approximately 450 mL from the alumina ceramic nanofiber filters and 350 ml from the glass wool filters. We concentrated this further by centrifugation using centrifuge tube ultrafiltration (Centricon®, Millipore Corporation, Billerica, MA). Thus far, we have found that the eluate can be reduced to an average volume of 192 microliters while retaining at least 64.5% of the eluted viruses.
Future Activities:
Tasks 3 and 4. Assessment of efficiency, robustness, accuracy and precision with different microbial pathogens and water quality matrices:
For these tasks, we will test the alumina ceramic nanofiber filters and the sodocalcic glass wool filters. The carbon nanofiber filters will not be tested any further due to their poor elution efficiencies. The ability of the final protocol to recover microorganisms from the collector will be assessed using a number of waterborne pathogens, with an emphasis on organisms on the EPA’s CCL. We have already begun work with Adenovirus, Coxsackievirus, and Echovirus, and are soon to begin work with Microsporidium (Encephalitozoon intestinalis).
In addition, the 1MDS filter recommended for the concentration of viruses ("EPA Manual for Virology") will be compared with the final concentration protocol for 20 and 400 liter volumes. Poliovirus type 1 will be used for comparative purposes for 400 liter volumes. For 20 liter volumes, Coxsackievirus B5 and Adenovirus 2 will be used. All tests will be performed at least four times.
Task 5. Assessment of the occurrence of Adenoviruses and Microsporidia in drinking water from groundwater sources:
The goal of this phase of the project is to assess the occurrence of two CCL organisms in drinking water distribution systems whose source is groundwater. Adenoviruses were selected because nothing is known of their occurrence in drinking water in the United States. In addition, they are the most resistant waterborne pathogen to UV light disinfection known. Adenoviruses have been known for some time to be transmitted by recreational waters, but have recently been suspected as the cause of gastroenteritis in adults in three drinking water outbreaks. Microsporidia have been suspected in one drinking water outbreak in France among AIDS patients. Microsporidia have been previously detected in groundwater influenced by land spreading of sewage and in a source of drinking water.
The state of Arizona has the largest number of unchlorinated drinking water systems in the United States. We have identified 60 different systems supplied by groundwater that are willing to participate in the study. The supply wells are located in a wide variety of climates and geological substrata. All of these systems are not currently disinfecting water prior to its distribution. The wells are of varying depth, depth to groundwater and distances to potential contaminate sources. Our current study will provide background data on the occurrence of enteroviruses and indicator bacteria to aid us in the identification of sites that may also be experiencing contamination by adenoviruses and microsporidia.
We propose to sample each of these sites on at least one occasion over a 6 to 9 month period. Sample volumes will range from 400 to 2,000 liters. Detection of viable adenovirus 40 and 41 will be accomplished using integrated cell culture PCR (ICCPCR) so that viability may be determined. Microsopridia will be detected by PCR methods previously developed for the detection and identification of microsporidia in ground and surface waters. In addition, we will also collect the following water quality data: turbidity, pH, total dissolved solids, total coliforms, E. coli, and enteroviruses. All analyses will be with done using EPA approved methods. The following information will also be collected: well depth, age of well, distance to any possible sources of contamination, type of substrata, type of well casing (length and depth of any screens), depth to groundwater, last rainfall event prior to sampling, volume of water recovered per day and the times and duration of operation.
References:
Journal Articles:
No journal articles submitted with this report: View all 4 publications for this projectSupplemental Keywords:
water, drinking water, viruses, protozoa, bacteria, monitoring, detection, occurrence, environmental sampling, nanofiber, filtration, carbon filters, pleated filters;, RFA, Scientific Discipline, Water, POLLUTANTS/TOXICS, Environmental Chemistry, Environmental Monitoring, Drinking Water, Microorganisms, enteric viruses, aquatic organisms, bacteria, CCL, viruses, drinking water monitoring, activated carbon, parasites, contaminant removal, drinking water contaminants, drinking water treatment, contaminant candidate listProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.