Grantee Research Project Results
1999 Progress Report: Development and Application of Spectroscopic Probes for Measurement of Microbial Activity in Aquatic Ecosystems
EPA Grant Number: R825159Title: Development and Application of Spectroscopic Probes for Measurement of Microbial Activity in Aquatic Ecosystems
Investigators: Arnosti, Carol , Blough, Neil V.
Institution: University of North Carolina at Chapel Hill , University of Maryland - College Park
EPA Project Officer: Packard, Benjamin H
Project Period: November 1, 1996 through October 31, 1999 (Extended to April 30, 2001)
Project Period Covered by this Report: November 1, 1998 through October 31, 1999
Project Amount: $405,811
RFA: Water and Watersheds Research (1996) RFA Text | Recipients Lists
Research Category: Watersheds , Water
Objective:
Measuring the net degradative capabilities of a complex community of microorganisms is a major challenge in understanding carbon cycling, because we lack the means to measure the rates at which bacteria hydrolyze large macromolecules to smaller pieces that can in turn be further transformed or remineralized. We are developing a new generation of sensitive spectroscopic probes to measure extracellular enzymatic hydrolysis rates of organic macromolecules in the water column and sediments. This approach is based on intramolecular energy transfer between donor and acceptor groups covalently attached to single macromolecules, so hydrolysis rates can be measured using simple fluorescence techniques. Initially, polysaccharides are being used as our target macromolecules, because they comprise a significant proportion of total organic matter, and recent studies have highlighted their importance in the global carbon cycle (Amon and Benner, 1994).Much of our current knowledge about microbial behavior and dynamics is based on laboratory studies of pure or mixed cultures of bacteria. Although these studies have yielded significant insights into microbial biochemistry and physiology, they reduce the complexity of natural aquatic systems to the point where their relevance to broader issues of biogeochemical cycling is questionable. The fluorescent-labeled probes will provide the means of making rapid, high-resolution measurements of specific enzymatic activities with minimal disturbance to the naturally complex microbial community, providing a more realistic picture of the transformations that actually take place. The new analytical approaches we are developing will lead to a better understanding of a major process governing the dynamics of organic matter degradation in aquatic ecosystems.
Progress Summary:
This year's work focused on further investigation of the photophysical characteristics of our double-labeled probes, as well as an initial application of fluorescence polarization measurements to determine the hydrolysis of biopolymers in marine systems. This work is reported in the manuscript, "Application of Fluorescence Spectroscopic Techniques and Probes to the Detection of Biopolymer Degradation in Natural Environments," submitted to Marine Chemistry.In collaboration with Dr. Phil DeShong and Mike Patterson (Department of Chemistry and Biochemistry, University of Maryland), target end-labeled synthesis of specific oligosaccharides was begun. These probes should yield double-labeled probes that can be reliably employed to observe Forster energy transfer.
Future Activities:
We plan to continue the work with Mike Patterson and Phil DeShong to obtain targeted end-labeled oligosaccharides for use as hydrolysis probes that rely on the principle of Forster energy transfer. We also are investigating the possibility of extending our probe range to include peptides. We have determined that the expertise necessary to conduct complete photophysical characterization of our existing as well as our new probes is beyond the capabilities of graduate students. Because the equipment and expertise necessary to make these measurements is available in Blough's lab, a postdoc experienced in optical sensors and photophysics is being sought to conduct this work at the University of Maryland. As reported in the Marine Chemistry manuscript, this year's investigations definitively showed that the quenching observed in the double-labeled high-molecular-weight polysaccharide probes was not associated with Forster energy transfer, but with another, unknown mechanism. This quenching was directly associated with the attachment of two different tags to polysaccharides of differing molecular weight. As described in the manuscript, these changes correlated with the molecular weight of the polymer. A qualitative association with the nature/length of the "link" between fluorophore and polysaccharide also was observed. Currently, we are unaware of any literature addressing this particular phenomena. We also will further develop our fluorescence polarization measurements to obtain a quantitative measure of polarization changes relative to cleavage sites.Journal Articles on this Report : 1 Displayed | Download in RIS Format
| Other project views: | All 13 publications | 9 publications in selected types | All 4 journal articles |
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Arnosti C, Keith SC, Blough NV. Application of fluorescence spectroscopic techniques and probes to the detection of biopolymer degradation in natural environments. Marine Chemistry 2000;71(3/4):321-330. |
R825159 (1999) R825159 (Final) |
not available |
Supplemental Keywords:
aquatic ecosystems, water, microorganisms, bacteria, spectroscopic probes, measurement methods, microbiology, microbial activity, biodegradation., RFA, Scientific Discipline, Water, Hydrology, Water & Watershed, Environmental Microbiology, Ecology and Ecosystems, Watersheds, coastal watershed, microbial pollution, aquatic ecosystems, hydrolyzing organic macromolecules, spectroscopic probes, carbon nutrientsProgress 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.