Grantee Research Project Results
Final Report: Use of a Genetically Modified Organism To Assess Sewer Line Integrity
EPA Contract Number: EPD04030Title: Use of a Genetically Modified Organism To Assess Sewer Line Integrity
Investigators: Steck, Todd R.
Small Business: BioTrackers
EPA Contact: Richards, April
Phase: I
Project Period: March 1, 2004 through August 31, 2004
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2004) RFA Text | Recipients Lists
Research Category: Pollution Prevention/Sustainable Development , SBIR - Water and Wastewater , Small Business Innovation Research (SBIR)
Description:
The overall goal of this research project was to test the feasibility of a novel method for leak detection and for tracking bacteria in the environment. The specific goals of this research project were to: (1) estimate the bacterial dosage for use in the pilot study; (2) determine if genetically modified bacteria added to a leaking sewer line could be detected outside the sewer line; (3) determine if the number of bacteria found outside the sewer line could be enumerated; and (4) evaluate the ability of this method for determining whether any living bacteria found outside the sewer line were in a dormant or culturable physiological form.
A novel method for leak detection and fecal source tracking was tested. This method is based on using a living bacterial tracer that can be dosed to a sewer reach and subsequently sought in surrounding soil, groundwater, and surface water. The organism is a natural isolate of Escherichia coli that was to be modified to carry a green fluorescent protein ( GFP) gene in its chromosome. GFP expression causes a cell to fluoresce green, allowing the bacterium to be detected microscopically and, given that this gene is not normally found in bacteria, by polymerase chain reaction (PCR). The U.S. Environmental Protection Agency granted permission for a pilot study in which the bacterial tracer was to be added to two sewer lines suspected of having leaks and to one leak-free line. Soil and water samples from sites adjacent to the sewer lines were to be examined for the presence and enumeration of the genetically modified organism. The study was a joint collaborative effort with the Mecklenburg County Department of Environmental Protection; the North Carolina Department of Natural and Environmental Resources; BioTrackers, Inc.; and the University of North Carolina, Charlotte.
Summary/Accomplishments (Outputs/Outcomes):
Dye tracer studies indicated that the estimated time it would take for the genetically modified bacterium to migrate from a leaking sewer line to an adjacent polluted stream was much longer than originally estimated. The original estimate was that it would take 2-7 days for a bacterium to travel from a sewer line to a stream located approximately 20 feet away. It is now estimated that it will take 1-2 weeks. The travel time, in turn, affects the method of bacteria detection that would be used in field studies. Although the GFP gene chosen for addition to the bacterium was the variant with the longest half-life (published as being more than 24 hours, but empirically determined to be up to 1 week), the gene variant may not fluoresce for 30 days. This parameter is one that will be examined when the strain is constructed and the pilot study is completed.
The majority of the proposed experiments could not be attempted because of problems constructing the genetically modified organisms. These difficulties have been detailed in monthly reports. Briefly, the bacterium was to contain both a GFP gene (to be used for bacteria detection in environmental samples) and the mer TPAB genes (encodes resistance to mercury, for use as a selectable marker in strain construction, and potentially for selection of the bacterium in environmental samples). Two different plasmid systems were used to deliver the GFP/mer genes. The first system was based on a suicide vector. The second system was based on using a commercially available transposon delivery system. Selection of E. coli cells on medium containing mercuric acetate led to a very high background of false positives. None of the plasmid/chromosome DNA isolated from mercury-resistant cells that were examined using the molecular methods of restriction endonuclease digestion and PCR analysis have resulted in the isolation of the desired E. coli strain, although strains containing just GFP have been obtained.
Conclusions:
The use of a genetically modified bacterium as an environmental tracer has the potential for use in multiple applications, such as monitoring sewer line integrity, or any application in which the goal is to follow the movement and physiological status of bacteria in the environment over time. Although the goals stated in the application were not accomplished, BioTrackers, Inc., can draw the following conclusions that relate to the stated goals:
- Dye tracer studies indicated that the potential movement of bacteria from a leaking sewer line to an adjacent stream could take weeks, instead of days. This long time frame suggests that the bacterium detection method most likely to be used in the field is PCR and not microscopic examination of green fluorescing bacteria.
- PCR can be used to detect small numbers of the bacterium, on the order of approximately 100 genetically modified cells extracted from environmental samples.
- Based on results with PCR, a genetically modified E. coli strain carrying just GFP as the marker gene would be sufficient for use in any proposed applications.
- Before conclusions on specific commercial applications can be made, the experiments described in the proposal need to be completed.
Supplemental Keywords:
genetically modified organism, sewer line integrity, wastewater, monitoring, bacteria, Escherichia coli, groundwater, surface water, soil, green fluorescent protein, GFP, polymerase chain reaction, PCR, tracer, SBIR,, Scientific Discipline, Water, POLLUTANTS/TOXICS, Wastewater, Ecology and Ecosystems, Microorganisms, bacteria, bacterial tracer, sewer leak detection, municipal sewers, genetically modified organism, municipal wastewater, wastewater systems, groundwater contamination, wastewater discharges, fecal contamination detectionThe 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.