The influence of aqueous copper speciation on bioluminosity

Studies were carried out on Luria Bertani (LB) nutrient broth, typical of growth media used to grow strains of luminescent bacteria such as E. coli pCulux, in order to ascertain the chemical speciation of added copper and infer the effect of speciation on luminescent response. Both acid-base and complexometric (with Cu) titrations were conducted. Data were analyzed using the binding spectrum approach in which the concentration and pK value of the reactions of interest are estimated. Results of acid-base titration indicated that LB contains both inorganic and a suite of organic functional groups, the latter probably associated with proteins and amino acids contained in the materials used in making the medium. Titration with cupric ion suggested the presence of two copper-binding groups for freshly prepared LB, but three groups for media in which the bacteria had been allowed to grow for several hours, indicating either the introduction of a Cu-binding substance associated with microbial metabolism, or the ability to detect a weaker binding group as the concentrations of the other groups declined during degradation. Organic-Cu complexes dominate the computed speciation of Cu in these systems. Interpretation of luminescence in such systems is complicated by temporal changes in ligand type and concentration, and differential complexation of Cu by differing ligands. If free cupric ion is assumed to be the activating form for luminescence, these results indicate that the bacteria are approximately six orders more sensitive in detecting Cu than results based on total Cu alone.