Biomass production for the removal of heavy metals from aqueous solutions at low pH using growth-decoupled cells of a Citrobacter sp.

A Citrobacter sp. accumulates heavy metals via the activity of an acid-type phosphatase that produces inorganic phosphate, HPO42−. This ligand precipitates with heavy metals (M) as MHPO4, which is retained at the cell surface. Continuous metal deposition has been coupled to the removal of heavy metals from metal-laden solution. The pH optimum of the mediating phosphatase is 5•0–8•0, with 55% and 70–80% retention of activity at pH 4•0 and 4•5, respectively. Metal accumulation was reduced at pH 5•0, attributable to increased metal phosphate solubility and reduced metal phosphate precipitation, but this was overcome using cells of higher phosphatase activity. A 3•25-fold overproduction of enzyme compensated for a 100-fold increase in the concentration of H+. Preliminary tests enabled prediction of the increased phosphatase activity required to treat a target waste stream containing uranyl ion at pH 4•5. Enzyme over-production was achieved by growth of a phosphatase constitutive variant in a lactose-based medium, but enzyme activity was reduced at the high carbon concentration required for a high biomass yield. The latter requirement was fulfilled with enhanced enzyme production by the use of fed-batch culture, with substrate addition regulated via feedback analysis of the off-gases. The biomass removed uranyl ion efficiently from a challenge solution at pH 4•5 in a batch contactor. Lactose-grown immobilized cells also removed uranyl ion from an acidis simulated industrial wastewater.