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

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

Figures

Figure 1

Figure 2

Figure 3

Figure 4

Tasks to meet objectives

1. The bench-top prototype testing involves the following tasks:
   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. Conduct a survival experiment using enterococcus to determine whether the enzyme activities change with time. Enzyme activity of the bacteria from treated and untreated effluent will also be examined and compared.
3. Finalize design specifications for fabrication of the field-portable and submersible prototypes so that SubChem Systems can fabricate the instruments and provide them for laboratory and field testing. The testing to be done includes:
   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 (MATLAB).
   4. Optimize the 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.

Progress on Tasks
Laboratory experimentation with the bench-top prototype: Most of the laboratory experimentation tasks (1-2) with the laboratory prototype, have been completed. The exceptions are the assessment of a cross-current microfiltration technique and the survival experiments using enterococcus. The results on the media clarification and antibiotic addition experiments are briefly described below (section g).

Transitioning the bench-top instrument into a submersible prototype: SubChem engineers and URI scientists exchanged information on the experimental data and operational requirements. The information was then used to complete the compilation and revision of a series of technical reports. These reports included a detailed specification document for the field-portable and submersible prototypes (Task 3). These proprietary internal reports, shared with URI investigators, included:

1. Features to consider for the SubChem’s Bioanalyzer, S. DaSilva, 04/07/06, 19p.
2. Design for SubChem’s BioAnalyzer Fluidic System, S. DaSilva, 04/20/06, 21p.
3. BioAnalyzer System Requirements, S. DaSilva, 04/20/06, 7p.

The reports indicated that two key technical improvements needed to be designed and tested for the field-portable or submersible prototype in order for the instruments to meet the requirements for the beach monitoring application. These two improvements are: 1) improved temperature control during incubation and 2) adding an automated technique for concentrating cells from a larger sample volume (i.e. cross-current micro filtration, or an alternate technique). Whereas item 1) is deemed easy to accomplish within the project time frame, the timely implementation of item 2) is more challenging. In view of this finding, it was decided to initially design and fabricate a field-portable prototype that would have the capability to measure selected microbial indicators at higher cell concentrations. Initially SubChem would build a field-portable prototype, that would be suitable for water quality monitoring near and down stream of sewer outfalls, but not sensitive enough for beach monitoring. SubChem is now in the process of completing the design and initiating fabrication of the field-portable prototype, based upon the specifications and requirements, which resulted from testing with the bench-top prototype.

Have the results/data gathered during this reporting period changed the project objectives when compared to your original proposal?
The overall project objectives remain the same, except that we intend to transition the testing of the technology with a field-portable prototype, before we conduct tests in Narragansett Bay with a submersible prototype. We expect to obtain follow-on funding to continue development of this technology beyond the end-date of this project.

Dissemination activities during this reporting period
A. Hanson, D. Smith and H. Saffert presented results and a summary of the project at a special CICEET organized meeting for water quality managers on Sept. 21, 2006 at the New Hampshire Department of Environmental Services.

Recent thesis research results, related to this project, were also presented in Heather Saffert’s student seminar requirement on September 18, 2006

Difficulties
SubChem Systems will need extra time to implement the technical enhancements required to facilitate concentrating cells to attain the beach monitoring goal and design and fabricate the submersible prototype.

Data Generated to date
Enterolert Media Clarification Experiments: An in situ instrument in development to quantify enterococci, a bacterial indicator of fecal contamination, requires a selective media that has a low background level of fluorescence. While the EPA-approved, commercially-available media, Enterolert, is useful for this application, the media emits fluorescence near the upper limit of the highly sensitive mini-fluorometer in the instrument. To decrease the background fluorescence, the media was passed through a solid-phase extraction cartridge (SepPak C-18) that is designed to remove non-polar, hydrophobic compounds, such as, modified yeast and some amino acids and antibiotics. The extraction of antibiotics from the media, however, is suspected to reduce the selectivity of the Enterolert by allowing for non-indicator bacteria to grow.
Therefore, experiments to determine if the selectivity of the media could be restored by the addition of antibiotics (amikacin sulfate, bacitracin, and monolaurin) were performed. Enterolert, clarified Enterolert and clarified Enterolert with antibiotics were tested using filtered seawater (1.2 µm) amended with Enterococcus faecalis or a raw sewage and seawater mixture (See Figure 1). Clarification of the media reduced the background level of fluorescence by 57 % (See Figure 2). The results indicate that the addition of antibiotics after clarification restores the selectivity of Enterolert, without affecting the fluorescence (See Figure 3). Figure 4 shows additional testing with cultured bacteria. It is evident that Enterolert suppresses some of the enterococci. The solution with antibiotics added after clarification indicates that this solution inhibited the enterococci too much. Additional testing to further optimize the results will be conducted. This modification will allow for the use of a sensitive fluorometer onboard the instrument resulting in a significant reduction in the time required to determine health risks associated with marine waters.

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 new field-portable prototype.

Work plan to Meet Objectives

1. A survival experiment will be conducted using with enterococcus to determine whether the enzyme activities change with time. Comparisons will be made with normal QuantiTray measurements and the mEI plating method.
2. Additional testing will be performed with the modified Enterolert solution to ensure comparable results with the normal Enterolert.
3. A field-portable prototype will become available this fall. At that time we will begin testing in the lab and field with that instrument.
   1. Test for detection of known concentrations of ATCC strains of enterococci.
   2. Test with field samples from marine waters.
   3. Develop quantification software program.
   4. Optimize the disinfection and rinsing system.
4. Prepare a standard operating procedure for using and calibrating the field-portable instrument.
5. Conduct some testing in Narragansett Bay.

Dissemination Objectives for next reporting period
Final report, paper submissions and professional meetings.

Overall Project Timeline Update
Task 1/2 ­ Laboratory Tests ­Sept/Oct 2006
Task 3 ­ Field-Portable Prototype Lab Tests ­ Nov 2006
Task 4 ­ Develop Protocol ­ Nov 2006
Task 5 ­ Field Tests with Field-Portable prototype - Nov/Dec 2006.

Expenditures
The expenditures are in the range anticipated for the work accomplished to date. We have requested, and been granted, a “no-additional cost” extension of our remaining funds that will enable us to continue working on the project until Dec. 31, 2006.