CICEET Progress Report for the period 9/16/05 Through 3/15/06

Project Title: A New Autonomous Technology for Monitoring Microbial Indicators of Fecal Con-tamination in Coastal Waters
Principal Investigator(s): Alfred K. Hanson, David C. Smith
Additional Investigator(s): Heather L. Saffert
Project Start Date: September 1, 2004

Figures

Figure 1

Figure 2

Figure 3a

Figure 3b

Figure 3c

Figure 3d

Figure 3e

Figure 4a

Figure 4b

Figure 5a

Figure 5b

Tasks to meet objectives

1. The testing of the bench-top prototype involved the following tasks:
   1. Test in the lab for detection of known concentrations of ATCC strains of E. coli and enterococci.
   2. Evaluate a disinfection step between samples with solutions containing bromine.
   3. Test with field samples from marine waters.
   4. Assess whether a filtration step is necessary.
   5. Develop quantification software program and test further.
2. Prepare a specification document for the instrument manufacturer (SubChem Systems, Inc.) to use as a guide for designing and fabricating the submersible prototype.
3. Laboratory testing of the submersible prototype will be conducted, once it becomes available (revised delivery date May, 2006).
   1. Test for the detection of known concentrations of ATCC strains of E. coli and enterococci.
   2. Evaluate the bromine solution disinfection technique.
   3. Test with field samples from marine waters.
   4. Refine quantification software program.
4. Prepare a standard operating procedure for using and calibrating the field instrument.
5. Conduct some preliminary tests in situ in Narragansett Bay.

Progress on Tasks

1. Testing with the bench-top prototype;
   1. To evaluate the influence of improved temperature control on instrument performance, the prototype was reconfigured to fit within a small incubator (Figure 1). The temperature variability during incubation was reduced to (+ 1.0° C) by this experiment, which is more conducive for both the target bacteria growth and enzyme activity. Calibrations with MU and Enterolert were made during testing. Different abundances of laboratory cultured Enterococci faecalis were tested simultaneously with control samples. The time to detection had an exponential relationship with the number of bacteria present (Figure 2). Concentrations of 10-30,000 enterococci per 100 ml were detected in 2.5- 6 hours (Figure 2 and Figures 3a - 3e).
   2. Disinfection with a bromine solution was tested again and found not be completely effective within a 1 hour timeframe. A 10% bleach solution was used instead to disinfect the instrument. Test results are shown in Figure 4a and Figure 4b.
   3. We are continuing progress on subtasks c-e.
2. To examine whether enzyme activity was affected by the aging or starvation of bacteria, a survival experiment was conducted. E. coli were placed into 2 large flasks containing 500 ml of sterilized artificial seawater. Over 60 days, subsamples were taken and analyzed for enzyme activities. Figure 5a and Figure 5b shows the relationship between the time to detection based on enzyme acitivities and the abundance of E. coli.
   1. B-glucuronidse activity positively correlated to the number of bacteria present, which decreased over time.
   2. In contrast, B-galactosidase activity increased over this same period and was inversely correlated with the bacterial abundance.
3. Transitioning the bench-top into a submersible prototype;
   1. SubChem engineers have compiled and revised a detailed specification document for the submersible prototype. Two key improvements that are being designed and tested for the submersible prototype are: 1) improved temperature control during incubation and 2) cell concentration by cross-current microfiltration. SubChem is in the process of fabricating a submersible prototype, based upon these specifications and requirements, which resulted from testing with the bench-top prototype.
   2. The first generation submersible prototype for the SubBioAnalyzer is expected to be completed by May/June 2006. At that time it will become available for testing, evaluation, and optimization over the summer.
4. In other related activities, Heather Saffert teamed with other URI CICEET project investigators, David Smith and Marek Kirs, who are working on Microbial Source Tracking using F-specific coliphage and Real-time QPCR.
   1. The water quality in the Narrow River and Narragansett Beach and to identify potential pollution sources in the watershed was completed. The Mumford Brook a tributary to the estuary with very high levels was investigated further.
   2. Results were presented at a local public meeting at URI in January.
   3. A paper on the study is in preparation and almost ready for submission for publication.

Difficulties
1. Calibrations of the instrument over time varied slightly more than expected. This appears to be due more to differences in temperature during calibrations more than instrument drift.

2. Changes in enzyme activities due to aging or starvation of bacteria is a concern that needs to be addressed further by testing effluent and field samples.

3. Disinfecting with the bromine solution was surprisingly difficult this time. A bleach solution was used instead. However, for field operation an improved disinfection technique will need to be developed.

Project Objectives for Next Reporting Period

Objectives
For the next reporting period, we plan to continue working to accomplish the previously stated objectives. We expect to finish laboratory testing with the bench-top prototype and then work with the submersible prototype this spring and summer.

Tasks to Meet Objectives

1. To complete the bench-top prototype testing, the following tasks will be performed:
   1. Further optimize the disinfectant solution
   2. Test with field samples from marine waters
   3. Assess the efficiency of the cross-current microfiltration technique
   4. Further develop quantification software and test
2. A survival experiment will be conducted using with enterococcus to determine whether the enzyme activities change with time. Enzyme activity of the bacteria from treated and and untreated effluent will also be examined and compared.
3. The submersible prototype will be built by May/June 2006. At that time we will commence laboratory testing of the submersible prototype.
   1. Test for detection of known concentrations of ATCC strains of E. coli and enterococci.
   2. Test with field samples from marine waters.
   3. Develop quantification software program.
   4. Optimize a disinfection and rinsing system.
4. Prepare a standard operating procedure for using and calibrating the field instrument.
5. Conduct some field tests, in situ in Narragansett Bay.

Work Plan for Next Reporting Period
We have revised out project timeline.

Anticipated Success in Meeting Project Objectives
We expect that we will meet all the project objectives during the following year.

Overall Project Timeline Update
Task 1 ­ Bench-top Tests - October-April 06
Task 2 ­ Submersible Tests ­ May 06 ­ August 06
Task 3 ­ Develop Protocol ­ March - May 06
Task 4 ­ Field Tests ­ July-August 06

Preliminary Data
See figures.

Dissemination
Conferences: Heather Saffert presented a poster “An Submersible Analyzer for Indicator Bacteria” at the Ocean Sciences Conference in Honolulu, HI in February, 2006.

Outreach Activities: Ms. Saffert organized a Water Quality and New Technologies Meeting that was open to the public January 17th, 2006. About 50 people attended including many end-users, local volunteers, and others were working on water quality issues in the region. The following non-profits hosted the event: URI Watershed Watch Program, Narrow River Preservation Association, and RI SurfRider. Heather presented results from the Narrow River and Narragansett Beach study conducted with Marek Kirs. She also explained the novel Microbial Source Tracking method developed by Kirs with CICEET funding. Details about the meeting and some of the presentations can be found at http://www.narrowriver.org/water_quality_problems_and_solutions.htm

Marek Kirs and Heather identified some pollution problems (extensive spray cans and painting supplies, and camping debris) while surveying the Mumford Brook watershed. Heather organized a Saturday morning Coastal Clean-up effort in the watershed in October 2005 in coordination with the Audubon Society and the Narrow River Preservation Association. Advocacy letters to protect the watershed were sent out to local community leaders about the cleanup and the problems in the region.

Contact with End Users: End users at both RIDOH and RI DEM were consulted while arranging the January meeting and were in attendance.

We have also been working with Annette DeSilva who is involved in the Narrow River Preservation Association and leads the URI Watershed Watch sampling of the river. With DeSilva’s help and contacts, we gained additional funding to further investigate the Mumford Brook area, which has a history of very high indicator levels. Charlie Vandemoer, the refuge manager of the US Fish and Wildlife Service, which owns part of the land surrounding the Mumford Brook, provided us with $1,000 worth of supplies for this study.

Expenditures
The expenditures are in the range anticipated for the work accomplished to date

End User Advisor Feedback
Name: Bonnie Blair
Organization: Rhode Island Dept. of Health
Location:
3 Capitol Hill, Room 203
Providence, RI 02908
Phone number: 401-222-1406
E-mail: ribeaches@doh.state.ri.us

1) At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.

2) What, if anything, has changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?

3) Do you see any key challenges that the researchers may want to address or keep in mind?

4) Does this report offer you enough information to adequately address the above questions?

5) Other feedback?