Development and Application of Spectroscopic Probes for Measurement of Microbial Activity in Aquatic EcosystemsEPA Grant Number: R825159
Title: 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 Amount: $405,811
RFA: Water and Watersheds Research (1996) RFA Text | Recipients Lists
Research Category: Watersheds , Water
Description:Microbial degradation of organic matter is a key part of carbon cycling in aquatic systems. Bacteria are responsible for a significant portion of organic carbon turnover in water and sediments, hydrolyzing organic macromolecules to smaller pieces that can be remineralized, and interconverting organic structures through a variety of reactions. Measuring the net degradative capabilities of a complex community of microorganisms is one of the major challenges in understanding the dynamics of carbon cycling, because we lack the means to measure accurately the rates at which bacteria hydrolyze large macromolecules to smaller pieces that can then in turn be further transformed or remineralized.
The objective of the proposed research is to develop a new generation of sensitive and versatile spectroscopic probes that can be used to measure extracellular enzymatic hydrolysis rates of organic macromolecules in the water column and sediments. This approach is based on efficient intramolecular energy transfer between donor and acceptor groups covalently attached to single macromolecules, so hydrolysis rates can be measured using simple fluorescence techniques. The sensitivity of these probes should permit measurement of hydrolysis rates at true tracer (picomolar) levels; the ease of analysis means that a large number of samples can be surveyed rapidly. Spin-labeled probes will be synthesized and used to follow enzymatic hydrolysis in intact sediment samples. The fluorescent- and spin-labels will initially be covalently linked to polysaccharides, since they comprise a significant proportion of total organic matter, and recent studies (Amon and Benner, 1994) have highlighted their importance in the global carbon cycle. The fluorescent- and spin-labeled probes will be synthesized, spectroscopically characterized, and calibrated in the lab. The effects of physical factors (salinity, pH, interaction with humic materials and particles) on probe dynamics will also be examined. The probes will then be used to investigate extracellular enzymatic hydrolysis rates at specific sites in Pamlico Sound, one of the most productive estuarine systems in the U.S., where measurements of organic matter turnover rates are central to understanding human impact on the natural ecosystem.
The proposed research incorporates several new analytical approaches that will lead to better understanding of a major process governing the dynamics of organic matter degradation in aquatic ecosystems. 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- and spin-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 which actually take place.