2,4,6-Trinitrotoluene Transformation Using Spinacia Oleracea: Saturation Kinetics of the Nitrate Reductase Enzyme

A series of aqueous phase and soil-slurry phase microcosm studies were conducted on 2,4,6-trinitrotoluene (TNT) to obtain kinetic data for optimizing a treatment protocol using an enzyme extract from spinach (Spinacia oleracea). Crude extract was obtained by homogenization of fresh leaves with a buffered protease inhibitor, and employed as phytoremediation agent. Aqueous phase microcosms containing $20\mathrm{mg}∕\mathrm{L}$ TNT and soil–slurry microcosms containing 1 g of a characterized sandy loam soil contaminated with $\mathrm{2,500}\mathrm{mg}∕\mathrm{kg}$ TNT and $\mathrm{1,000}\mathrm{mg}∕\mathrm{kg}$ hexahydro-1,3,5-trinitro-1,3,5-triazine were dosed with fixed aliquots of extract and analyzed for TNT transformation over time. The TNT concentration was monitored using a colorimetric method for nitroaromatic compounds based on EPA Method 8515. Nitrate reductase activity of the applied crude extract was simultaneously quantified. The transformation of TNT was described by a pseudofirst-order reaction. Coupling kinetic rate information with enzyme activity allowed for estimation of a second-order rate constant with respect to activity. A rectangular hyperbola function normalized for enzyme activity described observed kinetic data based on enzyme saturation, similar to a Michaelis–Menten relationship. Pseudofirst-order rate constants for the aqueous phase and soil–slurry phase experiments were fit to this function. The maximum rate of reaction $\left(k_{\max }\right)$ for TNT transformation was 0.50 and $0.04{\mathrm{h}}^{-1}$ for aqueous phase and soil–slurry phase experiments, respectively, while respective half-saturation constants $\left(K_{\mathrm{sat}}^{*}\right)$ were comparable in value at 0.63 and $0.28\mathrm{U}∕\mu \mathrm{mol}\text{–}{\mathrm{NO}}_2$ , respectively. A Hanes–Woolf plot of reaction velocity versus TNT concentration with and without soil suggests an uncompetitive inhibition mechanism may be affecting overall nitrate reductase efficacy. Temperature effects for both aqueous phase and soil–slurry phase microcosms followed the Arrhenius relationship with estimated activation energies of 54.7 and $26.1\mathrm{kJ}∕\mathrm{mol}$ , respectively.