Accumulation and degradation of polyphosphate in Acinetobacter sp.

Abstract

Biological phosphate removal from waste water is a biotechnological alternative to chemical phosphorus precipitation. This process is obtained by recycling the sludge through anaerobic and aerobic zones. In the anaerobic parts phosphate is released by the sludge and during anaerobiosis phosphate is taken up. Biological phosphate removal is dependent on the enrichment of activated sludge with polyphosphate accumulating Acinetobacter . Like activated sludge, pure cultures of strictly aerobic Acinetobacter sp. absorbe phosphate (up to 100 mg phosphorus per g dry biomass) during aerobic conditions and release it anaerobically. The aim of this study was to gather knowledge on the uptake and release of phosphate by Acinetobacter and the metabolic functions of polyphosphate.The accumulation of polyphosphate by pure cultures of Acinetobacter strain 210A depended on the presence of an intra- or extracellular energy source (chapter 2). The highest amount of polyphosphate was found in cells in which energy supply was not limited, namely at low growth rates under sulphur limitation, and in the stationary phase of growth when either the nitrogen or the sulphur source was depleted. Accumulation of polyphosphate was also possible during endogenous respiration. When this respiration was blocked with KCN the phosphate uptake stopped, while the inhibition of the protein synthesis with streptomycin enhanced the accumulation of phosphate, which indicated the competition between protein synthesis and polyphosphate synthesis for energy. There was a pronounced effect of the temperature on phosphorus accumulation but this effect varied from strain to strain.The role and behaviour of cations in the accumulation and release of phosphate was studied (chapter 3). PO 43-was released together with 1.8 protons. Mg 2+appeared to be the most important counterion of polyphosphate in Acinetobacter strain 210A. It was released and taken up simultaneously with phosphate. Mg 2+was not an essential polyphosphate counterion. If Mg 2+was depleted, stationary cultures of Acinetobacter strain 210A took up the same amount of phosphate with Ca 2+as the most important counterion. In the presence of Mg 2+stationary cultures did not need Ca 2+for their phosphate absorption, but the presence of K +seemed to be crucial for this process, although this cation did not play a quantitatively important role as a polyphosphate counterion. In addition, the influx and efflux of K +was independent of phosphate uptake and release. Continuous cultivation at low growth rates under K +-limitation did not result in polyphosphate accumulation, while under substrate or Mg 2+- limitation large amounts of polyphosphate were present in the cells. The same effect was found in activated sludge. 5 mg K +per litre was needed for a satisfactory biological phosphate removal in the aerobic zone of a wastewater treatment plant. Granules of Mgpolyphosphate in Acinetobacter strain 210A could serve as a Mg 2+-reserve. Cells with these granules were able to grow in a medium free of Mg 2+, whereas cells without granules were not, they only grew in the presence of extracellular Mg 2+. Polyphosphate in cell-free extracts of Acinetobacter strain 210A could be degraded by the enzymes polyphosphatase or polyphosphate:AMP phosphotransferase (chapters 4 and 6). Polyphosphate glucokinase, polyphosphate dependent NAD-kinase and polyphosphatekinase were not detectable. Polyphosphate:AMP phosphotransferase was also found in Acinetobacter strain B8, but not in Acinetobacter strain P, which contained only polyphosphatekinase. Both strains were able to accumulate large amounts of polyphosphate. In strains that cannot accumulate this biopolymer, no or very small activities of polyphosphatekinase and polyphosphate: AMP phosphotransferase were found. All strains showed activities of adenylate kinase. It was demonstrated that by the combined action of polyphosphate:AMP phosphotransferase and adenylate kinase a continuous regeneration of ATP from AMP or ADP was possible as long as polyphosphate was present. Polyphosphate:AMP phosphotransferase could use native and synthetic polyphosphate as substrate and showed a maximum activity at a pH of 8.5. Its activity was stimulated by (NH 4 ) 2 SO 4 , the K m for AMP appeared to be 0.6 mM, and V max was 60 nmol.min -1.mg -1protein. Polyphosphatase in cell-free extracts of strain 210A was able to hydrolyse native polyphosphate and synthetic Mg-polyphosphate. The K- and Na-form, however, were not degraded. The activities of polyphosphate:AMP phosphotransferase and adenylate kinase in activated sludge correlated well with the ability of the sludge to remove phosphate biologically from waste water.Degradation of polyphosphate invivo in Acinetobacter strain 210A occurred if the energy supply in the cell was stopped, for example under anaerobiosis or in the presence of KCN, α-dinitrophenol or N-N'-dicyclohexylcarbodiimid (chapter 5). The degradation and synthesis of polyphosphate was dependent on the ATP concentration in the cells. Lower ATP concentrations caused a faster phosphate release. This release was stimulated by alcohols. The transmembrane protongradient seemed to play an important role in the anaerobic energy metabolism of this strictly aerobic bacterium. Addition of α-dinitrophenol, a protonionophore, decreased the cellular ATP concentration and stimulated the polyphosphate degradation. The role of polyphosphate as an energy reserve invivo has been demonstrated by experiments in which five strains were incubated anaerobically. Cells of Acinetobacter strains 210A and B8, which were able to accumulate polyphosphate, released large amounts of ortho-phosphate anaerobically and contained high levels of ATP. Cells of two other strains of Acinetobacter and one strain of Pseudomonas which didn't accumulate polyphosphate, showed a much smaller release of phosphate and contained only low ATP concentrations. Cells of strain 210A cultivated under phosphorus limitation or at 350C did not contain detectable amounts of polyphosphate. As a result their ATP level was low and they released only small or negligible amounts of phosphate under anaerobic conditions.Mg-polyphosphate in Acinetobacter sp. is a multifunctional compound. It can serve as: (1) an energy reserve if it is degraded by a reaction with AMP, catalyzed by polyphosphate: AMP phosphotransferase, (2) a phosphorus reserve if it is hydrolyzed by polyphosphatase, and (3) a Mg 2+reserve whereby Mg 2+can be replaced by Ca 2+as a counterion. The most important role of polyphosphate in wastewater treatment plants with biological phosphate removal, and probably also in natural environments, is its use as an energy reserve to sustain temporary anaerobiosis. This property might explain the enrichment of activated sludge subjected to alternating anaerobic and aerobic conditions with polyphosphate accumulating Acinetobacter sp.