Multiplicity of aspartate transport in thin wastewater biofilms

This research documents the multiplicity of L-aspartate transport in thin wastewater biofilms. A Line-weaver-Burk analysis of incorporation produced a curvilinear plot (concave down) that suggested active transport by two distinct systems (1 and 2). The inactivation of system 2 with AsO4 or osmotic shock resolved system 1, which was a high-affinity, low-capacity system with an apparent Kt (Michaelis-Menten constant) of 4.3 microM (AsO4) or 4.6 microM (osmotic shock). The inactivation of system 1 with dinitrophenol resolved system 2, which was a low-affinity, high-capacity system with an apparent Kt of 116.7 microM. System 1 was more specific for aspartate than system 2 in the presence of aspartate analogs. Sodium had no discernible effect on the incorporation velocities by either system. These results indicate that system 1 is a membrane-bound proton symport coupled to the proton gradient component of the proton motive force and that system 2 is a binding protein-mediated system coupled to phosphate bond energy. Analyses of diffusional limitations on the derived transport constants indicated that internal resistances were present but that the apparent constants were close to the intrinsic values, especially for system 1. Metabolic inactivation of the biofilm with dinitrophenol and AsO4 did not completely inactivate aspartate incorporation, which indicated that some simple adsorption of the aspartate anion by the biofilm had occurred. These results show that aspartate is transported by wastewater biofilm bacteria via systems with different affinities, specificities, and mechanisms of energy coupling.