Unique genetic modifications to C. acetobutylicum altered the level of hydrogenase expression, allowing study of the resulting effects on the 2,4,6-trinitrotoluene (TNT) reduction rates to be possible. A strain designed to over-express the hydrogenase gene resulted in maintained TNT reduction during late growth phases when it is not typically observed. Strains exhibiting under-expression of hydrogenase produced slower TNT rates of reduction correlating to the expected inhibition of each strain type. Hydrogenase activity, measured by hydrogen production, in Clostridium acetobutylicum correlates strongly (R2 = 0.89) to TNT reduction rates. Indications suggested that hydrogenase potentially played an integral role in catalysis of TNT transformation by reducing its nitro substituents to the corresponding hydroxylamines. A mechanistic pathway is proposed by which this transformation takes place and may enhance the understanding of commonly found hydrogenases in other microorganisms and their ability to transform nitroaromatic compounds.