Measurement and modeling of Michaelis-Menten constants for PAH compounds

Sarah Sterman, Petros Dimitriou-Christidis, and Dr. Robin Autenrieth

             Many compounds within the polycyclic aromatic hydrocarbon family are toxic to ecosystems, specifically the inhabitants.  There are hundreds of PAHs of concern that are still uncharacterized.  The large number of uncharacterized PAHs of concern makes it very difficult to experimentally determine coefficients for all toxic PAHs and thus a series of sixteen was chosen from those included on the CERCLA priority list of hazardous substances.  The study set included PAHs containing up to four rings and methylated naphthalenes, phenanthrenes and fluorene.  In order to characterize some of these toxic PAHs and better understand the risks they pose, a series of biodegradation experiments was used to measure Michaelis-Menten constants.  These constants, K_m and V_max, were determined for the chosen set of sixteen PAHs in order to better understand the degradation kinetics of this organic family.  The constants were then used to develop Quantitative Structure-Activity Relationships (QSAR) that link the structure and chemistry of the compounds to their biodegradation constants.  Extant kinetic experiments were performed with Sphingomonas paucimobilis strain EPA505 pre-grown on fluoranthene.  This bacterium is known to be able to degrade a range of PAHs including several four- and five-ring members.  Duplicate 250 mL bottle reactors were used per compound.  Concentration measurements were achieved via GC/MS analysis. To monitor changes in the cell activity between experimental runs, a spectrophotometric phenanthrene uptake assay was performed.    For calculation of the kinetic constants, the Michaelis-Menten model was numerically fitted on the experimental uptake curves using a fourth order Runge-Kutta algorithm.  Results indicated that there is a relation between the structure of the compounds and their biodegradation kinetics.  With the experimentally determined coefficients two QSAR models were built using Cerius² v4.2 molecular simulation software.  The QSARs equations will allow the prediction of biodegradation kinetics for uncharacterized PAHs.

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