Detection of heavy metals in bacterial biofilms and microbial flocs with the fluorescent complexing agent Newport Green

The complexing agent Newport Green fluoresces upon binding of nickel, zinc or cobalt. It was used to detect nickel or zinc in MOPS buffer, in gel-like matrices, and in natural biofilms and microbial flocs cultivated in the laboratory. The response curves for increasing nickel concentrations indicated an equimolar binding capacity of Newport Green for nickel in MOPS buffer, whereas zinc fluorescence reached saturation in the presence of a 10-fold excess of zinc ions relative to Newport Green molecules. The maximum fluorescence intensity as determined by luminometry was 8-fold and 4-fold above background for nickel and zinc, respectively. The response of Newport Green to either nickel or zinc in the presence of the other metal is consistent with a different binding affinity of Newport Green for the two metals. Zinc binds more strongly to the complexing agent than nickel but it leads to a weaker fluorescent signal which was detectable by luminometry but not by confocal laser scanning microscopy (CLSM). Newport Green was able to complex nickel in the presence of 1% gelatin or agarose as determined by CLSM and image processing. Its application to fully hydrated bacterial biofilms or microbial flocs revealed the presence of nickel outside of cells. The results suggest that in addition to cellular sorption, metals are bound extracellularly by extracellular polymeric substances in intact and undisturbed microbial aggregates.