Effect of iron and phosphate on bacterial cyanide formation determined by methemoglobin in two-dimensional gradient microcultivations

Cyanide formation by microorganisms is typically observed during early stationary growth phase and is facilitated by the presence of iron(III) and inorganic phosphate. Extracellular free cyanide in aqueous solutions might readily react with methemoglobin added and can be determined by UV/VIS spectroscopy. As alternative to existing methods, this provided the basis for an analytical method which has not been used previously for the determination of cyanide in bacterial cultivations. We successfully applied the technique to study the combined effect of both iron(III) and phosphate on the cyanide formation by Pseudomonas fluorescens, which we used as model organism known for its ability to form HCN. Information on the combined effect of iron and phosphate was obtained by using commercially available 24-well microtiter plates as two-dimensional gradient systems. After its reaction with methemoglobin, cyanide was measured reproducibly at the wavelength of 427 nm. We found a combined effect of both iron and phosphate. In the absence of inorganic phosphate, cyanide formation was stimulated considerably by increasing concentrations of iron(III), although the effect on the bacterial growth of P. fluorescens was almost insignificant. This suggests that iron is more important than inorganic phosphate for the cyanogenesis by P. fluorescens.