Enzymes play an important role in the environment, they breakdown natural-occurring
and anthropogenic molecules so that they can be transported into cells and
utilized. Enzyme assays are routinely used in soil science and oceanography to measure
the activities of specific processes and to serve as general indicators of microbial activity.
Conventional enzyme assays are conducted as batch incubation of sediment and water
samples. During these assays the concentration of product is measured and enzyme
activity is then determined as the rate of product formation. Few studies have measured
enzyme activities of groundwater. This work investigates the use of β-glucosidase and
phosphatase assays for quantifying in situ enzyme activities in groundwater.
Improvements to conventional enzyme assays using p-nitrophenyl substituted compounds
were made by developing a high performance liquid chromatography method to improve
quantitation limits of the product and to quantify concentrations of both the substrate and
the product. An in situ single-well push pull test was then conducted to measure
β-glucosidase activity in situ and to estimate the Michaelis constant (K[subscript m]) and the maximum
reaction velocity (V[subscript max]) in petroleum-contaminated groundwater at a field site near
Newberg, Oregon. An important feature of the single-well push pull test is the nonlinear
drop in pore water velocity that the test solution experiences as it moves out from the
injection point. The nonlinear drop in pore water velocity is of particular interest because
enzyme-mediated reactions are very fast and changes in the hydraulic properties during
the test may give rise to mass-transport limitations. Fast reactions lead to the
simultaneous depletion of substrate and accumulation of product at the site of the reaction
so substrate and product concentrations near the enzyme can be different then the
concentrations in bulk solution. And the rates obtained from a single-well push pull tests
may be a combination of the rates at which substrate diffuses to the microorganism and at
which the reaction occurs. Laboratory experiments with sediment-packed columns were
conducted with a range of pore water velocities typically achieved in the subsurface
during as push-pull test as a means for examining the potential effects of inhibition and
diffusion on phosphatase enzyme kinetics. In this set of column experiments rates of
phosphatase-mediated reactions were investigated instead of β-glucosidase, which is an
inducible enzyme. Numerical investigations were then conducted to examine the
importance of diffusion limitations for describing the influence of transport processes on
the observed rates of reaction. The theoretical investigation was conducted by formally
upscaling the proposed sub-pore-scale processes to develop a macroscale (or Darcy scale)
description of the transport of the substrate. These results indicate that mass-transfer
limitations due to the diffusion of the substrate to the enzyme cause an increase in the
apparent K[subscript m] but have no effect on V[subscript max]. In this study an analytical method was
developed to measure rates of enzyme-mediated reaction in situ so that the measured rates
reflected actual rates of microorganism in their natural environment. More carefully
controlled laboratory experiments demonstrated that rates of enzyme-mediated reactions
measured at low substrate concentrations depended on the flow properties of the test
solution. / Graduation date: 2006
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/28917 |
| Date | 26 May 2005 |
| Creators | Radakovich, Karen M |
| Contributors | Field, Jennifer A. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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