All activated sludge systems for removing phosphate microbiologically are configured so the biomass is cycled continuously through alternating anaerobic and aerobic zones. This paper describes a novel aerobic process capable of decreasing the amount of phosphate from 10 to 12 mg P liter ؊1 to less than 0.1 mg P liter ؊1(when expressed as phosphorus) over an extended period from two wastewaters with low chemical oxygen demand. One wastewater was synthetic, and the other was a clarified effluent from a conventional activated sludge system. Unlike anaerobic/aerobic enhanced biological phosphate removal (EBPR) processes where the organic substrates and the phosphate are supplied simultaneously to the biomass under anaerobic conditions, in this aerobic process, the addition of acetate, which begins the feed stage, is temporally separated from the addition of phosphate, which begins the famine stage. Conditions for establishing this process in a sequencing batch reactor are detailed, together with a description of the changes in poly--hydroxyalkanoate (PHA) and poly(P) levels in the biomass occurring under the feed and famine regimes, which closely resemble those reported in anaerobic/aerobic EBPR processes. Profiles obtained with denaturing gradient gel electrophoresis were very similar for communities fed both wastewaters, and once established, these communities remained stable over prolonged periods of time. 16S rRNA-based clone libraries generated from the two communities were also very similar. Fluorescence in situ hybridization (FISH)/microautoradiography and histochemical staining revealed that "Candidatus Accumulibacter phosphatis" bacteria were the dominant poly(P)-accumulating organisms (PAO) in both communities, with the phenotype expected for PAO. FISH also identified large numbers of betaproteobacterial Dechloromonas and alphaproteobacterial tetrad-forming organisms related to Defluviicoccus in both communities, but while these organisms assimilated acetate and contained intracellular PHA during the feed stages, they never accumulated poly(P) during the cycles, consistent with the phenotype of glycogen-accumulating organisms.High levels of phosphate in effluents from activated sludge systems not designed to remove it can lead to toxic cyanobacterial blooms in receiving bodies of water. Consequently, efforts have been directed towards removing phosphate during treatment by microbiological means with a process called enhanced biological phosphorus removal (EBPR), where phosphate is removed from the wasted biomass as intracellular poly(P) (5, 37, 45). Such treatment processes are based on the underlying principle that the biomass needs to be recycled repeatedly through alternating anaerobic and aerobic stages (37), a requirement regarded as crucial for successful EBPR operation. Only after repeated recycling are poly(P)-accumulating organisms (PAO) thought to have a selective advantage over other populations, eventually allowing them to become dominant (5,37,45). In the anaerobic (feed) stage, PAO are belie...
This study was conducted to obtain a better insight into the metabolic behavior of denitrifying phosphate-accumulating organisms relative to the transformations of relevant intracellular compounds as well as phosphorus and nitrate for enhanced biological phosphorus removal under different combinations of electron acceptor (oxygen or nitrate) and electron donor (acetate). Under anoxic conditions, the amount of polyhydroxybutyrate (PHB) produced per acetate taken up considerably increased with the increasing amount of nitrate reduced whereas the amounts of nitrate reduced and phosphorus released per acetate taken up remained almost constant. However, glycogen utilization occurred during PHB production and then was again observed in response to the initial supplementation of acetate after glycogen accumulation was transiently observed during anoxic phosphorus uptake using nitrate as an electron acceptor. On the other hand, under subsequent aerobic conditions, the additional supplementation of acetate again caused aerobic phosphorus release and PHB production, which showed that PHB production was associated with polyphosphate cleavage regardless of electron acceptor conditions. In contrast to anoxic conditions, glycogen accumulation was observed during PHB production. Based on these observations, the preliminary model for the metabolic behavior of denitrifying phosphate-accumulating organisms was proposed and could well account for the complex transformations of PHB and glycogen together with phosphorus release in the presence of acetate under different electron acceptors.
Long-term influences of different steady-state pH conditions on microbial community composition were determined by fluorescence in situ hybridization (FISH) in a laboratory scale reactor configured for enhanced biological phosphorus removal (EBPR). Chemical profiles were consistent with shifts in populations from polyphosphate-accumulating organisms (PAO) to glycogen-accumulating organisms (GAO) when pH fell from pH 7.5 to 7.0 and then to 6.5. While biomass was both dispersed and flocculated at pH 7.5, almost complete granulation occurred gradually after pH was dropped to 7.0, and these granules increased in size as the pH was reduced further to 6.5. Reverting back to pH 7.5 led to granule breakdown and corresponding increases in anaerobic phosphate release. Granules consisted almost entirely of Accumulibacter PAO cells, while putative GAO populations were always present in small numbers. Results suggest that low pH may contribute to granulation under these operational conditions. While chemical profiles suggested the PAO:GAO balance was changing as pH fell, FISH failed to reveal any marked corresponding increase in GAO abundances. Instead, TEM evidence suggested the Accumulibacter PAO phenotype was becoming more like that of a GAO. These data show how metabolically adaptable the Accumulibacter PAO can be under anaerobic:aerobic conditions in being able to cope with marked changes in plant conditions. They suggest that decreases in EBPR capacity may not necessarily reflect shifts in community composition, but in the existing population metabolism.
We investigated the effects of free ammonia (FA) and free nitrous acid (FNA) concentrations on the predominant ammonia-oxidizing bacteria (AOB) and the emission of nitrous oxide (NO) in a lab-scale sequencing batch reactor for partial nitrification. The reactor was operated with stepwise increases in the NH loading rate, which resulted in a maximum FA concentration of 29.3 mg-N/L at pH 8.3. Afterwards, FNA was increased by a gradual decrease of pH, reaching its maximum concentration of 4.1 mg-N/L at pH 6.3. Fluorescence in situ hybridization indicated that AOB remained predominant during the operation, achieving specific nitrification rates of 1.04 and 0.99 g-N/g-VSS/day at the highest accumulations of FA and FNA, respectively. These rates were in conjunction with partial nitrification efficiencies of >84%. The NO emission factor of oxidized NH was 0.90% at pH 7.0, which was higher than those at pH 8.3 (0.11%) and 6.3 (0.12%), the pHs with the maximum FA and FNA concentrations, respectively. High-throughput sequencing of 16S ribosomal RNA genes showed that increases in FNA drastically changed the predominant AOB species, although increased FA produced no significant changes. This study demonstrates that the FNA concentration and pH are the main drivers that determine the predominant AOB species and NO-emission in a partial nitrifying bioreactor.
Aims: To investigate the ecophysiology of populations of polyphosphate‐accumulating organisms (PAO) and glycogen‐accumulating organisms (GAO) in communities of a novel acetate fed process removing phosphate from wastewater. Attempts were made to see if acetate could be replaced by an alternative carbon source which did not support the growth of the GAO. Methods and Results: A continuously aerated sequencing batch reactor was operated with different acetate feed levels. Fluorescence in situ hybridization (FISH) showed that Defluviicoccus GAO numbers increased at lower acetate feed levels. With FISH/microautoradiography (MAR) both detected morphotypes of Defluviicoccus assimilated a wider range of substrates aerobically than Accumulibacter PAO. Their uptake profile differed from that reported for the same phylotype in full scale anaerobic : aerobic EBPR plants. Conclusions: This suggests that replacing acetate with another substrate is unlikely to provide Accumulibacter with a selective advantage in this process. Why Defluviicoccus appeared to out‐compete Accumulibacter at lower acetate concentrations was not clear. Data suggest physiological and morphological diversity may exist within a single Defluviicoccus phylotype. Significance and Impact of the Study: This study implies that the current FISH probes for Defluviicoccus GAO may not reveal the full extent of their biodiversity, and that more information is required before strategies for their control can be devised.
The effects of various types of electron acceptors on anoxic phosphorus uptake were investigated in detail to obtain a better insight into the metabolic behavior of denitrifying phosphate-accumulating organisms. Batch experimental tests under three different electron acceptor conditions, i.e., nitrate, nitrite and mixtures of nitrate and nitrite, were carried out using activated sludge cultivated in a sequencing batch reactor. The experimental results confirmed no inhibition of the utilization of nitrate or nitrite as an electron acceptor for anoxic phosphorus uptake. Anoxic phosphorus uptake occurred provided there was an electron acceptor present regardless of whether it was nitrate or nitrite. However, for nitrite a relatively small amount of anoxic phosphorus was taken up per nitrogen denitrified compared to nitrate. On the other hand, the amount of anoxic phosphorus taken up per nitrogen denitrified increased with an increase in the initial loading amount of electron acceptor in the case of nitrate, whereas it slightly decreased nitrite. Moreover, the amount of phosphorus taken up per nitrogen denitrified decreased with increasing mixed liquor suspended solid (MLSS) concentration in the case of nitrate, while it slightly increased for nitrite. From these results, it was confirmed that the activity of anoxic phosphorus uptake is strongly associated with the type and the initial loading amount of electron acceptor and the MLSS concentration under anoxic conditions.
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