Based on an early observation that microorganisms take up more phosphate than required for cell growth, it was found that single cells accumulate polyphosphate in granules containing a few to several thousand phosphate units (Egli and Zehnder, 1994). Phosphate constitutes up to 12% of the cell weight of polyphosphate-accumu-lating bacteria, whereas bacteria that do not accumulate polyphosphate contain only 1%-3% phosphate (van Loosdrecht et al., 1997b). The accumulated polyphosphate is assumed to be an energy source for substrate assimilation during anaerobic growth conditions and poly-p-hydroxyalkanoate (PHA) synthesis. Evidence suggests that degradation of polyphosphate may be used to regulate intracellular pH under alkaline conditions (Seviour et al., 2003). For biological phosphate removal by 'luxury phosphate uptake', the polyphosphate-accumulating bacteria in wastewater have to be subjected in sequence to an anaerobic and an aerobic environment. All previous attempts to isolate polyphosphate-accumulating bacteria (culture-dependent approach) led to recovery of Acinetobacter sp. which belongs to the y-Proteobacteria and is the model organism for explanation of the polyphosphate accumulation mechanism found in textbooks. However, pure cultures of the polyphosphate-accu-mulating Acinetobacter sp. and wastewater communities seem to behave differently (van Loosdrecht et al., 1997a). A major discrepancy is that Acinetobacter sp. do not play a dominant role in activated sludge systems, as deduced from a survey using specific gene probes (Wagner et al., 1994). The culture-independent approach led to a mixed population for enhanced biological phosphorous removal (EBPR). The phosphorous-removing members (>10% of the total population) were assigned to the p-Proteobacteria (Rhodocylus group e.g. candidatus Accumulibacter phosphatis), a-Proteobacteria and Actinobacteria (Microlunatus phosphoruvorus) and to members of the Cytophaga-Flexibacter-Bacteroides division (Seviour et al., 2003).
In Acinetobacter sp. polyphosphate accumulation is closely interconnected with poly-p-hydroxybutyrate (PHB) and glycogen metabolism (van Loosdrecht et al., 1997b). Under anaerobic conditions in the absence of nitrate, acetate is taken up by Acinetobacter cells, metabolized to p-hydroxybutyrate, polymerized to PHB, and stored intracellularly in inclusion bodies. Reducing equivalents for PHB formation from acetate are made available by glycolysis of glucose units from stored glycogen or are alternatively formed by other microorganisms for EBPR in an anaerobically operating TCA cycle. The energy for polymerization comes from hydrolysis of the anhydride bonds of ATP and polyphosphate. To supply both the maintenance metabolism and the storage metabolism of Acinetobacter with sufficient energy under anaerobic conditions, anhydride bonds in polyphosphate are hydrolyzed, and inorganic phosphate is excreted (phosphate resolubilization) (Smolders et al., 1994). Stored glycogen in microorganisms for EBPR may also provide ATP for PHA synthesis. For storage of 1 mg fatty acids, about 0.6 mg orthophosphate is released (Danesh and Oleszkiewicz, 1997). Under aerobic conditions two situations may be prevalent. In wastewater that does not contain a suitable carbon source for respira-
tion, Acinetobacter sp. hydrolyze PHB, which is stored in the granules, and respire the p-hydroxybutyrate to gain energy for growth, maintenance, formation of glycogen, and polymerization of phosphate, which is taken up from the wastewater. In the presence of oxygen much more Pi is taken up by Acinetobacter sp. from the activated sludge flora than is released under anaerobic conditions ('luxury uptake'). Danesh and Oleszkiewicz (1997) reported that approximately 6 to 9 mg volatile fatty acids are required for biological removal of 1 mg phosphorous.
In wastewater that contains suitable carbon sources for Acinetobacter sp., these carbon sources are partially oxidized by the TCA cycle and are partially used for PHB formation (Kuba et al., 1994). Since reducing equivalents are needed for PHB formation, less ATP is formed by oxidative phosphorylation. The lower ATP yield is compensated by the energy from hydrolysis of polyphosphate to Pi, which the cells use to form ADP from AMP. Two moles of ADP are disproportionated to AMP and ATP by adenylate kinase, and little Pi is released into the medium.
Except for obligately aerobic bacteria of the genus Acinetobacter, nitrate-reducing bacteria compete with microorganisms for EBPR for organic substrates, e.g., volatile fatty acids such as acetate. Nitrate-reducing bacteria also can eliminate phosphate from the aqueous environment (Kerrn-Jespersen and Henze, 1993). The simultaneous presence of obligately aerobic bacteria and nitrate-reducing bacteria in activated sludge may explain why, in the presence of nitrate in the anaerobic phosphate resolubilization phase (oxygen absent), Pi does not accumulate in the medium. The denitrifying, polyphosphate-accumulating bacteria apparently do not inhibit Pi excretion by Acinetobacter, but they take up phosphate and accumulate it as polyphosphate. This may explain why resolubilization of phosphate by degradation of stored polyphosphate is not always observed in wastewater treatment plants for biological phosphate removal. Another possibility may be the inhibitory effect of nitric oxide (Section 1.3) on adenylate kinase, which is involved in polyphosphate degradation in Acinetobacter sp. .
For biological phosphate accumulation, the ability of cells to store reserve material plays an essential role. Polymerization of substrates and storage in intracellular granules may offer the advantage that, at times of substrate shortage or in an environment with low concentrations of substrates (at low km values), such organisms can survive much better than other heterotrophic bacteria that do not have effective strategies for substrate storage.
Biological phosphate elimination is a highly effective process leading to final concentrations in the wastewater of <0.1 mg L-1 phosphate. For optimal biological phosphate removal, the COD/P ratio in the wastewater should be about 20 g COD g-1 phosphate to permit good growth of the polyphosphate-accumulating bacteria. If biological phosphate elimination is used in combination with chemical precipitation, the minimum COD/P ratio should be 2 g COD g-1 phosphate (Smolders et al., 1996) in processes with or without separation of primary sludge. If most of the particulate COD is separated in a primary sedimentation pond, ferrous ions should be added to supplement the biological phosphate removal with chemical precipitation. If no presedimentation of sludge is available or the sedimentation efficiency is low, no ferrous ions have to be added (van Loosdrecht et al., 1997b). A detailed descrip tion of the state of the art concerning biological phosphate elimination was given by Schon (1999).
Problems with the stability of the EBPR process in wastewater treatment plants were periodically reported. The reason for this may be the occurrence of microorganisms known as glycogen nonpolyphosphate-accumulating organisms (GAOs). These bacteria are selected under EBPR operation conditions (alternating anaerobic-aerobic phases during treatment) and compete with microorganisms for EBPR for the available volatile fatty acids (Blackall et al., 2002). Filipe et al. (2001) suggested an important role of pH during anaerobic and aerobic operation of reactors: growth of GAOs is favored at low pH, but at a pH of 7-7.5 the microorganisms for EBPR outcompete the GAOs. Further research is required to elucidate conditions under which GAOs are inhibited and growth of EBPR biomass is promoted.
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