Grantee Research Project Results

2000 Progress Report: Development and Application of Spectroscopic Probes for Measurement of Microbial Activity in Aquatic Ecosystems

EPA 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 Period Covered by this Report: November 1, 1999 through October 31, 2000
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 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. We are initially using polysaccharides as our target macromolecules, because 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.

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 that 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:

Work performed during this research period focused on the use of electron paramagnetic resonance (EPR) spectroscopy and nitroxide spin-labeled (SL) polysaccharides to investigate the binding and enzymatic activity of these species in seawater and sediment media. The underlying principle is that changes in the binding, configuration, and solution dynamics of these polysaccharides will lead to changes in their EPR spectra; such changes can be measured "real time," without the need for chromatographic separation of hydrolysis products.
We synthesized a series of SL-polysaccharides and have obtained definitive evidence of the spin-labeling of polysaccharide molecules, due to the broadening of the EPR spectrum of the nitroxide-labeled polysaccharides compared to that of the free nitroxide radical. In addition, it appeared that the higher the molecular weight of the polysaccharide, the broader the EPR spectrum, with the maltoheptose exhibiting the least broadened signal, and the pullulan displaying the most broadened signal. In addition, we have demonstrated that the spin-labeled polysaccharides used in this study bind to certain natural sediments, and we have observed progressive changes in the EPR spectra of these polysaccharides as they are hydrolyzed by enzymes.

The following conclusions have been made:

• A series of nitroxide spin-labeled polysaccharides has been synthesized as evidenced by the broadening of the polysaccharide EPR spectra as compared to the free nitroxide radical.

• The degree of signal broadening appears to increase with increasing molecular weight (and thus decreased mobility) of the SL polysaccharide.

• EPR spectra of polysaccharide samples in seawater and sand sediment media are essentially identical in terms of line shape and signal intensity to those run in a phosphate buffer, indicating that no binding of the sugars to these sediments occur.

• Initial signal intensity of all polysaccharides in the Cape Lookout sediment was far less than initial signal intensities of the same molecules in phosphate buffer and seawater control, indicating that some binding of the polysaccharides to the Cape Lookout sediment was occurring.

• Signal intensities of all polysaccharides in the Cape Lookout sediment decreased as a function of time, due to the settling of the sediment particles (and thus of the bound sugars) out of the EPR detection window.

• Polysaccharide samples in seawater control left to sit at room temperature overnight produced EPR spectra similar in line shape and signal intensity to those run on the previous day, indicating the short-term stability of the SL polysaccharides.

• Mixtures of pullulan/pullulanase (or xylan/xylanase) in the Cape Lookout sediment produce EPR spectra that show a decrease in the broad region with a concomitant increase in sharper signal as a function of time. This indicates that the enzyme does indeed hydrolyze the bound polysaccharides into lower molecular weight pieces, thus sharpening the spin signal.

Future Activities:

The major objectives for the subsequent reporting period include:

• Synthesis of new spin-labeled (SL) polysaccharides1 (xylan, pullulan), with more stable2 pyrroline spin-labels:3-5

• Synthesis of SL-maltoheptaose, purification via column chromatography and dialysis to remove free SL.

• Chemical characterization (labeling density) and stability (heat, light) of these SL-polysaccharides should be determined.

• Study of binding of SL-polysaccharides on different soils, sediments and zeolites. In the case of binding events, modeling experiments for hydrolysis with enzymes will be performed. Experiments will include hydrolysis of maltoheptaose via a-amylase.

• Further experiments for hydrolysis: Competitive inhibition experiments with native substrate, nonlabeled polysaccharides and other polysaccharides. Investigation of seawater containing active xylanase/pullulanase from bacteria. Investigation of enzyme activity in whole sediments as well as in sediment porewaters.

References:

Gnewuch T, Sosnovsky G. Spin-labeled carbohydrates: past, present, and future. Chemical Reviews 1986;86:203-238.

Couet WR, Brasch RC, Sosnovsky G, Tozer TN. Factors affecting nitroxide reduction in ascorbate solution and tissue homogenates. Magnetic Resonance Imaging 1985;3(1):83-88.

Rozantsev GI. Free nitroxide radicals. Plenum Press: New York, 1970, p. 209.

Mawhinney TP, Florine KI, Feather MS, Cowan DL. Carbohydrate Research 1983;116:C1-4.

Hankovszky HO, Hideg K, Tigyi J. Acta Chimica Academiae Scieentiarium Hungaricae 1978;98:339-348.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Publications Views

Other project views:	All 13 publications	9 publications in selected types	All 4 journal articles

Publications

Type	Citation	Project	Document Sources
Journal Article	Keith SC, Arnosti C. Extracellular enzyme activity in a river-bay-shelf transect: variations in polysaccharide hydrolysis rates with substrate and size class. Aquatic Microbial Ecology 2001;24(3):243-253.	R825159 (2000) R825159 (Final)	not available

Supplemental Keywords:

environmental chemistry, new measurement methods, extracellular enzyme activity, organic carbon remineralization., 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 nutrients

Progress and Final Reports:

Original Abstract

1997

1998 Progress Report

1999 Progress Report

Final Report