Mechanisms Used by Cycloclasticus Pugetii to Oxidize Polycyclic Aromatic Hydrocarbons
Abstract
The goal of this project is to conduct basic kinetic and structural studies on the unexpected colored intermediates that begin to appear in the culture medium immediately when Cycloclasticus pugetii is mixed with any of over 20 different polyclyclic aromatic hydrocarbons (PAHs). C. pugetii, a Gram-negative bacterium found in marine sediments around the world, oxidizes and degrades a number of different PAHs; consequently, its numbers invariably soar in salt water areas whenever large oil spills or contamination occur. The PAHs to be examined include, but are not limited to, biphenyl, fluorene, fluoranthene, dibenzofuran, dibenzo-p-dioxin, phenanthrene, and selected analogs and derivatives thereof. Two Specific Aims are proposed: Specific Aim #1 Ð to conduct sufficient kinetic studies using intact cells to quantify the rates of appearance and subsequent disappearance of each colored metabolic intermediate A novel feature of this project is that these kinetic absorbance measurements will be conducted in situ using an integrating cavity absorption meter that was custom-built using prior DoD funding. The practical goal is to determine how to maximize the production of each extracellular colored metabolite for subsequent structural studies in Specific Aim #2. These studies will also test the following 2 hypotheses: (i) steady state kinetic studies on the rate of appearance of each extracellular colored metabolite will conform to the Michaelis-Menten equation; and (ii) individual progress curves on the biphasic appearance of each colored metabolite will be mathematiccally modeled using a minimal mechanism comprised of two sequential first order reactions. Specific Aim #2 Ð to isolate or separate each spectral intermediate and determine its identity, molecular structure, and absorption coefficient These studies will test the hypothesis that the structure of each extracellular colored metabolite will be predicted by the metacleavage of one of the aromatic rings in the starting PAH. That is, that all of these structurallyrelated PAHs are initially oxidized via a meta-cleavage mechanism that is catalyzed by the same general PAH dioxygenase. It is anticipated that the results of this project will permit us to better understand the kinetic and mechanistic behavior of C. pugetii and related microorganisms during the remediation of oil spills and related environmental disasters. We anticipate that any fundamental understanding of how microorganisms degrade intractable organic aromatic hydrocarbons could be useful in designing purposeful strategies to remediate oil spills and the like no matter whether they occur on land or in the water. This project will also provide new opportunities to show how the new spectrophotometer developed with prior DOD funds can be used to examine the rates and extents of biochemical events without disrupting the complexity of the live cellular environment. The educational and research activities of STEM undergraduate students at Xavier University will benefit from this project in that numerous student support programs (including the BUILD, COE, and others) are always looking for active laboratories in which to place students for a productive research experience. It is anticipated that between 6 to 8 minority undergraduate students could enjoy productive research experiences if this 4-year project is funded by the DoD.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- May 24, 2023
- Source ID
- W911NF2310198
Entities
People
- Robert C. Blake
Organizations
- Army Contracting Command
- Office of the Secretary of Defense
- Xavier University of Louisiana