New Strategies for the Isolation of Microorganisms Responsible for Phosphate Accumulation

Abstract

Several strategies were used to isolate organisms involved in the uptake and subsequent release of inorganic phosphate from waste water sludge. These included direct staining for polyphosphates (polyP), growing in 32P inorganic phosphate followed by autoradiography, resistance to dicyclohexyl carbodiimide (DCCD), an ATPase inhibitor, and isolation on the basis of the buoyant density of the cell. Among those microorganisms isolated, three were identified as Acinetobacter lwoffii, A. calcoaceticus and Pseudomqnas vesicularis. The Ps. vesicularis culture had 31% of phosphate as polyP. 31P NMR analysis of the whole cells revealed the presence of polyP when the cultures were grown aerobic-ally to the late stationary phase and its subsequent loss during anaerobic incubation. Loss of polyP was also associated with a decrease in buoyant density of the cell. In the presence of DCCD, there was a decrease in the polyP peak, but a substantial increase in the sugar phosphates which is consistent with a hypothesis that polyP is used as a reserve energy source, Ps. vesicularis cells showed a two-fold increase in the level of polyphosphatase during early stationary phase, but a thirty fold increase in polyphosphate kinase activity during late stationary phase. This increased enzyme activity is consistent with the increased polyP synthesis during late stationary phase.