Ecological aspects of the biological phosphate removal from waste waters

Phosphate emission into surface waters can be reduced by treating sewage in wastewater treatment plants which are run alternately anaerobic and aerobic. Under these conditions, sludge in wastewater treatment plants becomes enriched with polyphosphate accumulating bacteria. Phosphate is released by the sludge under anaerobic conditions. During aerobiosis, this amount of phosphate is taken up by the sludge together with phosphate from the influent, resulting in very low phosphorus concentrations in the effluent of wastewater treatment plants. The aim of the research presented in this thesis was to obtain more knowledge about the complex bacterial interactions within the activated sludge process removing biologically phosphate. A simple system was developed, in which biological phosphate removal could be studied under well defined conditions. The so- called fill and draw system consists of a one reactor vessel in which sludge is fed intermittently with a chemically defined medium containing acetate and glucose as carbon sources. The sludge was subjected to cycles with an anaerobic period, an aerobic period and a period allowing the sludge to settle. The resulting sludges were extremely enriched with polyphosphate accumulating bacteria. The phosphorus content reached up to 110 mg P/g dry weight. The amount of polyphosphate in the cells during steady state depended on the acetate:glucose ratio, the nitrate, and phosphate concentration in the medium. Highest phosphate accumulation was obtained with an acetate:glucose ratio of 9:1. Intracellular polyphosphate was formed during the aerobic period and was anaerobically hydrolysed and released as phosphate into the medium. In the absence of oxygen and in the presence of 2 g acetate-COD/l, 80-90% of phosphate was released by sludge containing 100 mg P/g dry weight. In the absence of acetate only 2-19% of the accumulated phosphate was excreted. The amount of precipitated metal phosphates and the acid soluble polyphosphate concentration were measured in several sludges and pure cultures of Acinetobacter strains. The percentage metal phosphates of the total phosphorus content of the sludges varied between negligible and about 80%. Sludges from fill and draw systems with over 100 mg P/g dry weight, were almost completely devoid of these precipitates. The same was true for polyphosphate- accumulating cultures of Acinetobacter. Besides high molecular weight polyphosphate, the sludges contained also low polymeric polyphosphates (LPP), ranging from 0 to 50% of the total phosphate content. The LPP fraction in polyphosphate accumulating cultures of Acinetobacter strains was between 3 and 23% of their total phosphorus content. Sludges developed in the fill and draw systems could release in the presence of acetate more than 50% of the accumulated phosphate within 60 min. About 66% of this phosphate originated from LPP. Pure cultures of Acinetobacter strains released in the same time less than 1 mg P/g dry weight. The initial rate of phosphate release by sludge of a wastewater treatment plant designed to remove biologically phosphate (Renpho sludge) in the presence of acetate was reduced by addition of nitrate. In the presence of 16 mM acetate, maximum inhibition of 40 to 50% occurred at a nitrate concentration of 3.6 mM. Increasing the amount of acetate 10 times above the theoretical amount needed for complete denitrification, did not overcome the effect of nitrate. After depletion of nitrate, the phosphate release rate recovered slightly. In the presence of KCN and azide, inhibitors of denitrification, nitrate did not inhibit phosphate release. After completion of phosphate release and with sufficient acetate present, phosphate was taken up after addition of 3.6 mM nitrate. This phosphate uptake was too small to explain the reduction of the initial phosphate release rate, when nitrate was added at the start of the experiments. The formation of precipitated phosphates by an increase of the pH in the medium as a result of denitrification could only partly explain the reduction of the initial phosphate release rate. Nitrous oxide added at a concentration of 3.6 mm in the liquid phase had no negative effect on the phosphate release. Nitrite showed about the same effect as nitrate. Nitric oxide at a constant partial pressure inhibited phosphate release by 50% at a concentration of 30 μM. The inhibitory effect of nitric oxide on the phosphate release was slightly reduced by addition of 5 mM KCN. Nitric oxide production by sludges removing biologically phosphate was dependent on the sludge type, the nitric oxide precursor, the carbon source and the pH. At pH 7.5, rates of nitric oxide production by Renpho sludge were 390 μmol/g sludge dry weight . h upon incubation with acetate plus nitrite, 16 μmol/g dry weight . h with acetate plus nitrate and 25 μmol/g dry weight . h with glucose plus nitrate. The highest rate of nitric oxide production upon incubation with acetate plus nitrite was 550 μmol/g dry weight . h at pH 8. At pH 7.5, rates of nitric oxide production by sludge from the fill and draw systems were 100-160 μmol/g dry weight . h upon incubation with acetate plus nitrite, 6-7 μmol/g dry weight . h, with acetate plus nitrate and 13-16 μmol/g dry weight . h with glucose plus nitrate. Pasteurized sludge produced nitric oxide only upon incubation with nitrite (12 μmol/g dry weight . h). The results show that nitric oxide is formed by biological processes in sludges from wastewater treatment plants removing biologically phosphate and that in the systems tested nitric oxide is responsible for the reduced phosphate release under denitrifying conditions.