Quantifying in situ β-glucosidase and phosphatase activity in groundwater Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/tt44pr81n

Descriptions

Attribute NameValues
Creator
Abstract or Summary
  • 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.
Resource Type
Date Available
Date Copyright
Date Issued
Degree Level
Degree Name
Degree Field
Degree Grantor
Commencement Year
Advisor
Academic Affiliation
Non-Academic Affiliation
Subject
Rights Statement
Peer Reviewed
Language
Digitization Specifications
  • File scanned at 300 ppi (Monochrome, 8-bit Grayscale) using ScandAll PRO 1.8.1 on a Fi-6670 in PDF format. CVista PdfCompressor 4.0 was used for pdf compression and textual OCR.
Replaces
Additional Information
  • description.provenance : Approved for entry into archive by Patricia Black(patricia.black@oregonstate.edu) on 2012-04-27T17:35:15Z (GMT) No. of bitstreams: 1 RadakovichKarenM2006.pdf: 1702300 bytes, checksum: 092bdc15ca531e27db110de6a84669dc (MD5)
  • description.provenance : Made available in DSpace on 2012-04-27T17:35:15Z (GMT). No. of bitstreams: 1 RadakovichKarenM2006.pdf: 1702300 bytes, checksum: 092bdc15ca531e27db110de6a84669dc (MD5) Previous issue date: 2005-05-26
  • description.provenance : Submitted by Kaylee Patterson (patterka@onid.orst.edu) on 2012-04-23T19:18:24Z No. of bitstreams: 1 RadakovichKarenM2006.pdf: 1702300 bytes, checksum: 092bdc15ca531e27db110de6a84669dc (MD5)
  • description.provenance : Approved for entry into archive by Patricia Black(patricia.black@oregonstate.edu) on 2012-04-27T17:27:25Z (GMT) No. of bitstreams: 1 RadakovichKarenM2006.pdf: 1702300 bytes, checksum: 092bdc15ca531e27db110de6a84669dc (MD5)

Relationships

In Administrative Set:
Last modified: 10/24/2017

Downloadable Content

Download PDF
Citations:

EndNote | Zotero | Mendeley

Items