Grantee Research Project Results
Final 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 Amount: $405,811
RFA: Water and Watersheds Research (1996) RFA Text | Recipients Lists
Research Category: Watersheds , Water
Objective:
The objectives of this research project were to: (1) synthesize double-labeled fluorescent probes for rapid measurement of enzymatic hydrolysis rates and determination of enzyme structural specificities; (2) synthesize a series of spin probes, which can be used to measure enzymatic hydrolysis rates via electron paramagnetic resonance (EPR) spectroscopy (i.e., permitting direct measurement in "messy media" containing particles); and (3) develop methods using these probes with which large numbers of samples can be rapidly screened, and use these methods to determine detailed information about the hydrolysis of specific samples.
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 transformed or remineralized. The objective of this research project was to develop and test a new generation of sensitive spectroscopic probes to measure extracellular enzymatic hydrolysis rates of organic macromolecules in the water column and sediments. We have chosen 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. Our new probes represent a significant step towards the goal 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. The new analytical approaches we developed will help lead to a better understanding of major processes governing the dynamics of organic matter degradation in aquatic ecosystems.
Summary/Accomplishments (Outputs/Outcomes):
Development and Testing of Double-Labeled Fluorescent Probes. We achieved three specific objectives relating to this area:
· Synthesized and chemically characterized probes with different labeling densities and with different donor-to-acceptor fluorophore ratios;
· Photophysically characterized the probes to determine optimal probe characteristics; and
· Characterized the fluorescence response of the probes with purified enzymes in laboratory studies to correlate chromatographic and spectroscopic responses of hydrolysis.
The probes that we synthesized were derivatives of pullulan (α (1,6) linked maltotriose groups) or xylan (β (1,4) linked xylose). These polysaccharides were chosen because pullulanase and xylanase activities have been demonstrated in a range of marine bacteria and sediments (Bragger, et al., 1989; Antranikian, 1992; Arnosti and Repeta, 1994). We optimized fluorophore pair selection (ultimately selecting Texas Red (TR) and Fluoresceinamein (FLA), and developed a more effective means of attaching two labels to our probes. Probes were chemically and photophysically characterized. We determined that attachment of TR to FLA-labeled pullulan and xylan caused a decrease in FLA emission and an increase in TR emission, consistent with energy transfer from FLA to TR. However, further investigation showed that the increase in TR emission was not due to energy transfer, but to a slight increase in its emission efficiency when FLA and TR were combined in a dual-labeled probe. Our investigations showed that the quenching observed in our double-labeled high-molecular-weight polysaccharide probes was not associated with Förster energy transfer, but with another, currently unknown mechanism. To date, we are unaware of any literature that addresses this particular phenomenon.
Because fluorescein fluorescence is quenched in double-labeled probes and is directly dependent upon the presence of Texas Red. Increases in fluorescein fluorescence can be used to follow probe hydrolysis, using a different approach. In this approach, fluorescence polarization was employed the decreases in biopolymer size during the course of hydrolysis. This measurement technique uses plane-polarized light to excite selectively probe molecules whose transition moments lie in the plane of polarization. Depolarization of fluorescence emission is observed when the rotational correlation time of the labeled biopolymer is short with respect to the excited singlet state lifetime of the attached probe. Thus, for a given excited state lifetime, smaller molecules will exhibit more depolarization than larger molecules. In this manner, the degradation of high molecular weight biopolymers should be detectable.
A series of experiments showed that polarization increased in the sequence FLA → FLA-maltoheptaose → FLA-xylan → FLA-pullulan. This sequence is consistent with an increase in rotational correlation time for oligo- and polysaccharides of increasing molecular weight, as well as with a decrease in fluorescence lifetime expected from the observed decreases in relative fluorescence yield. The polarization of dual-labeled probes was higher than that of the single-labeled probes. This also is consistent with the observed decreases in relative fluorescence yield. We found that the significant differences in polarization between the higher and lower molecular weight polysaccharides and the free fluorophore indicates that fluorescence polarization measurements are a robust means of following hydrolysis, as demonstrated by a series of studies of the hydrolysis of oligo- and polysaccharides. Experiments with purified enzymes, as well as with field samples, confirmed these results.
Therefore, polarization measurements therefore can be used to monitor the hydrolysis of macromolecules in environmental systems. We envision that this approach will be particularly useful in situations where a large number of experiments are conducted simultaneously, and in remote locations where equipment transport may be limited. In these situations, using a microplate instrument capable of measuring fluorescence polarization would be especially practical.
Targeted synthesis of short-chain probes: carbohydrate biosensors. A second major part of our work arose from the discovery that quenching of fluorescence in double-labeled polysaccharides was not the result of Förster energy transfer, so ratios of donor/acceptor fluorescence could not be employed quantitatively to monitor the extent of hydrolysis. We therefore collaborated with Mike Patterson and Phil DeShong (Department of Chemistry and Biochemistry, University of Maryland) to synthesize targeted end-labeled mono- and oligosaccharides containing specific linkages. End labeling oligosaccharides should yield probes that exhibit suitable geometries for Förster energy transfer to occur. Any hydrolysis of such probes should lead to a direct loss of energy transfer. They synthesized a series of fluorescently labeled carbohydrates based on glucose and maltotriose. Procedures for the previous compounds were optimized, and the singly labeled sugars will be used to test fluorescence lifetimes. The efficacy of the biosensor in this case will be based on the degree of (fluorescence resonance energy transfer, FRET), as is seen between dansyl and fluorescein, or the amount of fluorescence quenching, such as between two fluorescein moities. Cleavage of the carbohydrate backbone should correlate with loss of FRET or fluorescence quenching. We envision that these probes can be applied as "on/off" type sensors to detect specific enzyme activities, with applications ranging from environmental testing to food-quality monitoring.
Development of Spin Probes. We developed a series of nitroxide spin-labeled (SL) polysaccharides to investigate the binding and enzymatic activity of macromolecules 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 in real time, without the need for chromatographic separation of hydrolysis products. Measurements also can be made in messy media (samples with particles, etc.), without the need for prefiltration.
The SL-probes were synthesized and chemically characterized using methods adapted from our work on synthesis of fluorescently labeled polysaccharides. In a series of experiments, we demonstrated that the higher the molecular weight of the polymer, the broader the EPR spectrum. The SL polymers were incubated with commercially available enzymes, and their hydrolysis was monitored in real-time in the EPR spectrometer. The advantage of using this method of analysis lies in its ability to allow the measurement of nontransparent samples, an impossibility for optical methods, which is of great use for the study of environmental samples.
Using EPR spectroscopy, we also investigated several questions about the dynamics and shapes of macromolecules in solution, and the extent to which such macromolecules may bind to sediment particles. Our major findings were as follows:
· The conformation of the spin probes did not differ in seawater and in phosphate buffer; therefore neither particle-binding nor the ionic composition of seawater affects probe dynamics.
· Spin probes did not associate with sandy sediment surfaces, suggesting that such relatively clean surfaces do not bind these types of organic macromolecules.
· Spin probes bound extensively to muddy sediments, and we obtained preliminary evidence of the hydrolysis of spin-probes in such sediments.
In addition, our major achievements include the following:
· We successfully synthesized and characterized a series of double-labeled fluorescent probes. These probes did not exhibit the expected Förster energy transfer, but they quenched fluorescein fluorescence upon attachment of a second fluorophore;
· Fluorescence polarization was a rapid and robust means of measuring the extent of hydrolysis of double-labeled fluorescent probes, as determined with purified enzymes as well as with porewaters from natural sediments;
· Fluorescent tags can be attached to short oligo- and monosaccharides in a directed fashion, which should yield carbohydrate biosensors, as expressed via Förster energy transfer;
· EPR-sensitive spin probes were successfully synthesized and characterized. Investigations of the behavior of these probes in buffer and in seawater demonstrate that conformation is not affected in seawater medium;
· Spin probes were rapidly hydrolyzed by purified enzymes, and the extent of hydrolysis was successfully measured in real-time in EPR tubes; and
· Investigations conducted in sediment slurries showed that spin probes do not measurably bind to sandy sediment surfaces. However, the probes bind significantly to muddy sediment surfaces. Furthermore, preliminary evidence suggests that hydrolysis of spin probes may occur in pore waters of muddy sediments.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other project views: | All 13 publications | 9 publications in selected types | All 4 journal articles |
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Type | Citation | ||
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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 |
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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 |
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Petigara BR, Blough NV. Mechanisms of hydrogen peroxide decomposition in soils. Environmental Science & Technology 2002;36:639-645. |
R825159 (Final) |
not available |
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Thomas-Smith TE, Blough NV. Photoproduction of hydrated electron from constituents of natural waters. Environmental Science & Technology 2001;35:2721-2726. |
R825159 (Final) |
not available |
Supplemental Keywords:
spectroscopic probe, spin probe, double-labeled fluorescent probe microbe, aquatic, ecosystem, 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 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.