September 21, 2008

Functionally Relevant Microorganisms to Enhanced Biological Phosphorus Removal Performance at Full-Scale Wastewater Treatment Plants in the United States

By Gu, April Z Saunders, A; Neethling, J B; Stensel, H D; Blackall, L L

ABSTRACT: The abundance and relevance of Accumulibacter phosphatis (presumed to be polyphosphate-accumulating organisms [PAOs]), Competibacter phosphatis (presumed to be glycogen- accumulating organisms [GAOs]), and tetrad-forming organisms (TFOs) to phosphorus removal performance at six full-scale enhanced biological phosphorus removal (EBPR) wastewater treatment plants were investigated. Coexistence of various levels of candidate PAOs and GAOs were found at these facilities. Accumulibacter were found to be 5 to 20% of the total bacterial population, and Competibacter were 0 to 20% of the total bacteria population. The TFO abundance varied from nondetectable to dominant. Anaerobic phosphorus (P) release to acetate uptake ratios (P^sub rel^/HAc^sub up^) obtained from bench tests were correlated positively with the abundance ratio of Accumulibacter/(Competibacter +TFOs) and negatively with the abundance of (Competibacter +TFOs) for all plants except one, suggesting the relevance of these candidate organisms to EBPR processes. However, effluent phosphorus concentration, amount of phosphorus removed, and process stability in an EBPR system were not directly related to high PAO abundance or mutually exclusive with a high GAO fraction. The plant that had the lowest average effluent phosphorus and highest stability rating had the lowest P^sub rel^/ HAc^sub up^ and the most TFOs. Evaluation of full-scale EBPR performance data indicated that low effluent phosphorus concentration and high process stability are positively correlated with the influent readily biodegradable chemical oxygen demand-to- phosphorus ratio. A system-level carbon-distribution-based conceptual model is proposed for capturing the dynamic competition between PAOs and GAOs and their effect on an EBPR process, and the results from this study seem to support the model hypothesis.

Water Environ. Res., 80, 688 (2008).

KEYWORDS: enhanced biological phosphorus removal, glycogen- accumulating organisms, polyphosphate-accumulating organisms, tetradforming organisms, Accumulibacter, Competibacter.



Enhanced biological phosphorus removal (EBPR) has been applied worldwide in full-scale activated sludge facilities to achieve effluent phosphorus (P) concentrations. However, process reliability varies among wastewater treatment plants (WWTPs), and many facilities suffer from periodic upsets (Gu et al., 2004, 2005; Neethling et al., 2005; Stephens et al., 2004).

Successful EBPR processes rely on the selection of polyphosphate- accumulating organisms (PAOs) via uptake of volatile fatty acids (VFAs) and phosphorus release in an anaerobic zone followed by phosphorus uptake into polyphosphate storage granules in a subsequent aerobic zone. Lack or deterioration of phosphorus removal performance was attributed to the proliferation of glycogen- accumulating organisms (GAOs) in laboratory-scale EBPR reactors (Cech and Hartman, 1990,1993; Satoh et al., 1994) and also at full- scale facilities (Gu et al., 2005; Saunders et al., 2003). The GAOs are able to compete with PAOs for VFAs in the anaerobic zone without phosphorus release and with phosphorus uptake only for cell synthesis. Because VFAs are often the limiting substrate for EBPR processes, an abundance of GAOs could potentially affect phosphorus removal efficiency.

The candidate PAOs identified include Accumulibacter phosphatis in the Rhodocyclaceae group of the Betaproteobacteria (Crocetti et al., 2000; Hesselmann et al., 1999; Oehmen et al., 2007), Actinobacteria (Kong et al., 2005), and Malikia spp. (Spring et al., 2005). The candidate GAOs identified include Amaricoccus spp. and Defluvicoccus vanus-related organisms in the Alphapmteobacteria, Betaproteobacteria (Quadracoccus sp.), Competibacter in the Gammapmteobacteria, and possibly some Actinobacteria (Micropruina glycogenica, Tetrasphaera spp., and Kineosphaera limosa) (Crocetti et al., 2002; Kong, Beer, Seviour, Lindrea, and Rees, 2002; Kong et al., 2001, Kong, Ong, Ng, and Liu, 2002; Liu et al., 2001; Meyer et al., 2006; Seviour et al., 2000, Tsai and Liu, 2002; Wong et al., 2004). Candidate GAOs consist of several different morphologic types. Among them, characteristic cocci-arranged tetrads or sheets are commonly observed, and they are referred to as tetrad-forming organisms (TFOs) (Cech and Hartman, 1990, 1993). However, not all TFOs have the GAO phenotype. Investigation of TFOs in two laboratory- scale sequence batch reactors revealed diverse phylogenetic affiliation and physiological traits, in terms of intracellular polyphosphate and polyhydroxyalkanoates (PHA) accumulation (Tsai and Liu, 2002; Wong et al., 2004).

Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes allows the detection and quantification of candidate PAOs and GAOs in EBPR activated sludges. Rhodocyclus- group organisms were found to account for 26 and 73% of the total PAO populations in two plants surveyed by Zilles et al. (2002). Saunders et al. (2003) also found various levels of Accumulibacter in the plants they studied, suggesting the important role they may play in EBPR processes. However, a study of PAOs in full-scale EBPR systems in Australia by Beer et al. (2006) indicated that Actinobacteria comprised dominant populations containing polyphosphate in many plants instead of Accumulibacter.

This study investigated the abundance of candidate PAOs and GAOs, including Accumulibacter, Competibacler, and TFOs at six full-scale EBPR plants in the United States, for which, operation and performance data were collected over a 2-year period. The VFA uptake and phosphorus release and uptake characteristics were evaluated with mixed liquor from each plant in batch tests. Both light microscope and molecular techniques were used to examine the presence of candidate PAOs and GAOs in the plant mixed liquor samples. From these results, possible relationships between the population distribution of PAOs and GAOs and phosphorus removal efficiency and stability were evaluated.


This study evaluated the phosphorus removal performance, relative PAO/GAO populations, and mixed liquor characteristics of VFA uptake and phosphorus release under anaerobic conditions and phosphorus uptake under aerobic conditions for six full-scale EBPR WWTPs in the United States. Operation and performance data were collected and summarized for the 2001 through 2003 time period. Mixed liquor samples were obtained from each facility in August through September 2003, to evaluate the composition and characteristics of the VFA- utilizing populations. The performance data collected from the WWTPs include influent flow, temperature, solids retention time (SRT), and influent and effluent total and soluble phosphorus across the EBPR systems. In addition, intensive sampling was conducted for 2 to 3 months at plants to determine other wastewater characteristic and system performance data, such as metals, flocculated and filtered chemical oxygen demand (ffCOD), and VFA. The flocculation- filtration method by Mamais et al. (1993) was applied to both secondary influent and effluent samples to obtain their ffCOD concentrations, with their difference being the readily biodegradable COD (rbCOD) concentration. The secondary influent rbCOD to soluble orthophosphorus (rbCOD/P) ratios were then determined.

Batch tests were conducted to evaluate anaerobic VFA uptake and phosphorus release rates and aerobic phosphorus uptake characteristics using mixed liquor samples from the aerobic basin of each facility that were shipped overnight on ice. Thee samples were purged with nitrogen for 15 to 60 minutes to reach the initial anaerobic condition before carbon (sodium acetate) addition. Sodium acetate (60 to 70 mg/L as acetate) was added to 3.5 to 4 L mixed liquor in a 5-L reactor mixed with a magnetic stir plate and stirring bar. During the initial anaerobic period (45 minutes), the reactor was sparged with nitrogen gas, and, in the following aerobic period (200 to 250 minutes), the reactor was sparged with air. Samples taken at the start of the experiment and then at 10- to 15- minute intervals were analyzed for soluble phosphorus and VFAs. Oxidation-reduction potential and pH were continuously monitored, but not controlled.

The same mixed liquor that was used for bench batch phosphorus release and uptake and release tests was subject to microscopic observation of chemical staining of cells with Neisser stain and 4', 6-diamidino-2-phenylindole (DAPI) to monitor the presence of PAOs and TFOs. The TFOs were semiquantified by counting the number of fields out of 10 fields in which they were present. For DAPI stain, the activated sludge from the aerobic zone was fixed, stained, and observed using confocal laser-scanning microscopy (Zeiss, LSM 5 PASCAL, AxioSkop 2, Oberkochen, Germany) equipped with epifluorescence. An estimate of PAO abundance level number was given based on visual observation, with 1 = some found, 2 = easily found, and 3 = abundant. The same mixed liquor samples were also fixed and prepared on microslides (Paul Marienfeld, Lauda-Konigshofen, Germany) for FISH analyses. For FISH, bacteria were probed with a EUBMIX probe (Daims et al., 1999); Accumulibacter were probed with PAO421, PAO651, and PAO846 together as so-called "PAOMIX"(Crocetti et al., 2000); Competibacter were probed with GAOQ989 and GB-G2 (Crocetti et al., 2002, Kong, Beer, Seviour, Lindrea, and Rees, 2002); and Alphapmteobacteria were probed with ALF969. Details of the FISH and quantification protocol used are described in Saunders et al. (2003). Briefly, microscopic slides that were covered with sludge samples were subject to FISH hybridization and then visualized with a BioRad Radiance 2000 confocal laser scanning microscope (Hercules, California) using an Olympus 60 x oil- immersion objective collecting 8-bit, 512 x 512 pixel images. The area containing Cy3-labeled specific probe (PAOMIX or GAOQ989) cells was quantified as a percentage of the area of Cy5-labeled bacterial probe (EUBMIX) within each image using Image Pro Plus 4 Software (Media Cybernetics, Bethesda, Maryland). Dissolved phosphorus was analyzed by inductively coupled plasma via an atomic emission spectrophotometer (model 955, Jarrell Ash, Franklin, Massachusetts) following filtration through a 0.22-[mu]m glass-fiber filter. The VFAs (acetic, propionic, isobutyric, butyric, and valeric acids) were analyzed by gas-liquid chromatography using a flame ionization detector and a 0.32-mm internal diameter x 30 m Hewlett Packard (Palo Alto, California) capillary-free-fatty-acid-phase column (Agilent Technologies; Wilmington, Delaware) with helium carrier gas at 2.7 mL/min on a Perkin Elmer auto-system gas Chromatograph (Perkin Elmer, Waltham, Massachusetts). Samples were filtered through 0.22-[mu]m glass-fiber filters and acidified to pH 2 with 5.25 N sulfuric acid, then stored at 4[degrees]C until analysis. The following oven temperature program was used: 80[degrees]C for 1.5 minutes, 20[degrees]C/min to 160[degrees]C for 1 minute, then 10[degrees]C/ min to 220[degrees]C for 3 minutes. The detection limit was 3 to 5 mg/L for a 1-[mu]L sample injection.

Results and Discussion

Full-Scale Enhanced Biological Phosphorus Removal Performance. Phosphorus removal performance and key operating parameters are summarized for the EBPR WWTPs in Table 1. The facilities represent a range of EBPR process configurations, including anaerobic-anoxic- aerobic (A2O), Virginia initiative plant (VIP), University of Cape Town (UCT), and modified UCT (MUCT). Their treatment capacities ranged from 18 900 to 113 600 m^sup 3^/d (5 to 30 mgd). The EBPR process influent readily biodegradable COD to soluble phosphorus ratio (rbCOD/P) ranged from 5 to 38 mg/mg. Secondary effluent total phosphorus (TP) concentration was less than 1.0 mg/L for all the WWTPs, except one that averaged 1.2 mg-P/L, indicating effective EBPR processes for all the facilities. The reliability of phosphorus removal in the EBPR facilities was assessed from a cumulative frequency of percent occurrence versus secondary clarifier effluent total phosphorus concentration. For an effluent total phosphorus concentration of less than or equal to 0.50 mg/L, the cumulative frequency of occurrence ranged from 24 to 95%.

Acetate Uptake and Phosphorus Release and Phosphorus Uptake in Batch Tests. The results of anaerobic acetate uptake and phosphorus release rates, aerobic phosphorus uptake rates and specific net phosphorus uptake, and release per unit of biomass in the batch tests are summarized in Table 2. Also shown in Table 2 are the anaerobic-phosphorus-release-to-acetate-uptake-rate ratio (P^sub up^/ HAc^sub rel^), aerobic-phosphorus-uptake-to-anaerobic-phosphorus- release-rate ratio (P^sub up^/P^sub rel^), and the range of pH recorded during the tests. Phosphorus release and uptake rates varied greatly among the WWTPs, ranging from 5.6 to 31.9 mg-P/gVSS * h and from 2.4 to 9.7 mgP/gVSS * h, respectively. The aerobic phosphorus uptake rates were found to be lower than anaerobic phosphorus release rates for all plants studied, and the ratio of phosphorus uptake rate to phosphorus release rate ranged from 0.24 to 0.69. The specific net phosphorus release and net phosphorus uptake ranged from 0.005 to 0.024 g-P/gVSS and from 0.004 to 0.015 g- P/gVSS, respectively. Three plant samples showed higher net phosphorus release than phosphorus uptake. Sludge transportation and storage could result in secondary phosphorus release (as evidenced by the relatively high initial soluble phosphorus concentrations in the tests) that was not associated with intracellular carbon energy synthesis. The disturbance of the balance between the intracellular energy and carbon storage could lead to reduced phosphorus uptake in the following cycle (Stephens et al., 1998; Wang and Park, 2001), as observedin this study. Initial acetate uptake rates varied from 16.1 to 42.5 mgHAc/gVSS*h. The P^sub rel^/HAc^sub up^ obtained in this study ranged from 0.27 to 0.75 mole/C-mole, which is in the range of values reported in the literature (0.01 to 0.93 moleP/C-mole acetate) (Bond et al., 1998; Saunders et al., 2003; Schuler and Jenkins, 2003; Wentzel et al., 1988). Based on current understanding of the biochemical pathways of the phosphorus-removal process, the theoretical P^sub rel^/HAc^sub up^ ratio is proposed to be 0.5 to 0.7 mol/C-mol for PAOs (see summary in Schuler and Jenkins, 2003). Values that are lower than theoretical predictions indicate that there are GAOs present in the sludge because they compete for VFA utilization without contributing to phosphorus release.

In previous reports, the P^sub rel^/HAc^sub up^ ratio was correlated with both the initial phosphorus release and the initial acetate uptake rates (Saunders et al., 2003; Schuler and Jenkins, 2003). Although we found a linear correlation between the P^sub rel^/ HAc^sub up^ ratios and initial phosphorus release rates (Figure 1), there was no clear correlation between P^sub rel^/HAc^sub up^ ratios and initial acetate uptake rates (data not shown). The latter correlation is likely expected for systems where the increase in P^sub rel^/HAc^sub up^ and in initial acetate uptake rates was caused by an increase in PAO abundance, such as the laboratoryscale reactors (Liu et al., 1997; Schuler and Jenkins, 2003). In those systems, the influent rbCOD/P ratios were varied by keeping the influent rbCOD concentration constant and changing the influent phosphorus concentration. An increase in influent phosphorus led to higher PAO abundance, which then led to both higher acetate uptake rate and a higher P^sub rel^/HAc^sub up^ ratio. However, for full- scale systems that have varying influent rbCOD and phosphorus levels, increases in P^sub rel^/HAc^sub up^ and initial acetate uptake rates do not always co-occur, because the P^sub rel^/HAc^sub up^ ratio is dictated by the relative PAO-to-GAO abundance level. For example, elevation of GAO abundance would lead to higher acetate uptake rates and a lower P^sub rel^/HAc^sub up^ ratio, if PAO abundance remains the same. The unclear correlation between P^sub rel^/HAc^sub up^ ratios and initial acetate uptake rates implies that the plants surveyed in this study had various combinations of PAOs and GAOs, as confirmed by the microscopic observation of candidate PAOs and GAOs (Table 2). Further evaluation also revealed positive correlation between the anaerobic P^sub rel^/HAc^sub up^ ratio and the specific anaerobic net phosphorus release and between the P^sub rel^/HAc^sub up^ ratio and the specific aerobic net phosphorus uptake, as shown in Figures 2 and 3. These results imply that the systems that had a higher relative PAO-to-GAO abundance level had a higher fraction of PAOs in the biomass also (Table 3).

Factors that may affect phosphorus and acetate uptake rates include temperature, SRT, and pH. The temperature was kept constant for all experiments at 22 to 25[degrees]C and therefore was not expected to affect the acetate uptake rates significantly (Whang and Park, 2001). Even though SRT was considered as a factor influencing the selective enrichment of PAOs/GAOs and affecting the acetate uptake rate (Whang and Park, 2001; Zilles et al., 2002), association of SRT with acetate uptake rates and with the P^sub rel^/HAc^sub up^ ratio was not evident in this study (data not shown). This was probably because the SRT values at the WWTPs studied (6.7 to 11 days) were beyond the range that could affect the competition of PAOs and GAOs (3 to 5 days; Whang and Park, 2001). The pH was found to be a key factor that affected the metabolic rates and competition between PAOs and GAOs (Filipe et al., 2001). The acetate uptake rate by GAOs was shown to increase with pH up to 6.5, being the greatest at pH 7, and starting to decrease at pH 7.5, whereas, for PAOs, the acetate uptake rate was shown to increase with pH up to 6.5, remain nearly constant from pH 7.0 to 8.5, and start to decrease at pH 8.5 (Filipe et al., 2001; Schuler and Jenkins, 2002). Therefore, the correlation patterns of anaerobic phosphorus release rate and acetate uptake rate with pH varies depending on relative PAO and GAO levels in the system. In our experiments, the pH was between 7.2 and 8.3 (Table 2). The extent of differences in pH values among plant samples could contribute to, but could not account for, the great variation of acetate uptake and phosphorus release and uptake rates observed (Table 2).

Observation of Candidate Polyphosphate- and Glycogen- Accumulating Organisms. Both candidate PAOs and GAOs were found to coexist at various levels in all the WWTPs (Table 3). Accumulibacter were found to be 5 to 20% of the total bacterial population, and Competibacter were 0 to 20% of the total bacteria population. The abundance of TFOs varied from nondetectable to dominant.

Accumulibacter were found at all plants with various process configurations and influent wastewater characteristics, suggesting that they could be important to EBPR. However, no correlation was observed between the number of cells containing polyphosphorus (according to Neisser and DAPI staining) and Accumulibacter abundance. This could indicate that Accumulibacter are not the only PAOs present in full-scale EBPR systems, and the diversity of PAO populations is larger than our current understanding (Beer et al., 2006; Oehmen et al., 2007; Saunders et al., 2003; Wong et al., 2005; Zilles et al., 2002). However, because we did not confirm that all cells containing polyphosphorus actually participated in the well- known EBPR transformations of anaerobic phosphorus release (in a stoichiometric relationship with acetate uptake) and aerobic phosphorus uptake, it is difficult to be emphatic about the latter conclusion. It is well-known that polyphosphorus is a common component in many cells, where it has myriad functions (McInerney et al., 2006). Higher enrichments of Rhodocyclus-related organisms were proposed to be associated with the UCT process compared with the A/ O process (Zilles et al., 2002). This association was not really supported in our study. Plants E and F, which were operated in modified UCT and UCT mode, did not contain a higher abundance of Accumilibacter than those of plants A and B, which were run in A2O mode. This suggests that there are factors other than process configuration that affect Accumulibacter abundance. It is also possible that the difference, if any, is finer than any of the investigative methods could detect in this study.

Competibacter, a candidate GAO in the Gammaproteobacteria lineage, was found in 3 of the 6 plants investigated. The TFOs were found at various levels in all plants except one. The TFOs were frequently found in laboratory systems that exhibited no or deteriorated EBPR activities (Crocetti et al., 2002; Kong et al., 2001; Liu et al., 1997; Tsai and Liu, 2002). Although TFOs apparently often possess the GAO phenotype, a recent study revealed that the physiological traits of TFOs are quite diverse, in terms of their ability to accumulate carbon and/or polyphosphate (Tsai and Liu, 2002). The presence of both Competibacter and TFOs were observed in full-scale EBPR plants by Wong et al. (2005). Interestingly, only Competibacter (but no TFOs) were found at plant D. The reason the Competibacter is selected at this plant is not completely clear. One noticeable feature of plant D, however, is that it had a much lower rbCOD/P ratio (5 mg/mg) compared with the other plants, as a result of its high influent phosphorus (14 mg/ L). The rbCOD/P ratio seemed to affect the selection of different GAO populations, as shown by Crocetti et al. (2002). In their study, two sequence batch reactors that were fed with different influent rbCOD/P ratios had different microbial community and potential GAOs identified. Whether the rbCOD/P ratio had an affect on the selection of different GAO populations needs further investigation.

The presence of Alphaproteobacteria was investigated because a number of TFOs reported belong to Alphaproteobacteria (Beer et al., 2002; Meyer et al., 2006; Seviour et al., 2000; Wong et al., 2004). The abundance of Alphaproteobacteria was not able to be correlated with the level of total TFOs. However, this finding is expected, considering the diversity of bacteria in this subdivision and the likely diversity of TFOs.

Correlation of P^sub rel^/HAc^sub up^ Ratio with Accumulibacter, Competibacter, and Tetrad-Forming Organism Abundance. The P^sub rel^/ HAc^sub up^ ratio has previously been used as an indicator of PAO abundance in relation to GAO abundance, in both laboratory-scale sequencing batch reactors (SBR) and full-scale processes (Liu et al., 1997; Saunders et al., 2003; Schuler and Jenkins, 2003). It was proposed that P^sub rel^/HAc^sub up^ ratios higher than 0.5 indicate PAO dominance, values less than 0.25 indicate GAO dominance, and values in between indicate mixed populations. In reality, the P^sub rel^/HAc^sub up^ is an indication of organism activity, which may not necessarily be proportional to organism abundance. In any case, results in the present study are consistent with the previous findings. Five of the six plants surveyed had P^sub rel^/HAc^sub up^ ratios in the range 0.25 to 0.5 P-mole/C-mole and were found to have mixed Accumulibacter (presumed to the abundant PAOs), Competibacter (presumed to be the abundant GAOs), and TFO populations (Table 3). Excluding plant B, there seem to be a positive correlation between P^sub rel^/HAc^sub up^ ratio and the Accumulibacter/(Competibacter + TFO) ratio (Figure 4). Plant F, which had the highest P^sub rel^/ HAc^sub up^ ratio of 0.75, had the highest Accumulibacter/ (Competibacter + TFO) ratio. Plants D and E, which showed the lowest P^sub rel^/HAc^sub up^ ratios, had the lowest Accumulibacter/ (Competibacter + TFOs) ratios in their systems.

No consistent correlation between P^sub rel^/HAc^sub up^ ratio and Accumulibacter abundance was observed for all the plants studied (Table 3). Plants B and F had higher P^sub rel^/HAc^sub up^ ratios and did not contain a higher abundance of Accumulibacter, indicating the existence of other PAOs.

Effect of Glycogen-Accumulating Organisms on Carbon-Source- Utilization Efficiency in Enhanced Biological Phosphorus Removal. The P^sub rel^/HAc^sub up^ is an indicator of carbon-utilization efficiency, because it shows the amount of phosphorus that can be removed per unit amount of carbon utilization. The proliferation of GAOs reduce the P^sub rel^/HAc^sub up^ ratio as a result of their contribution to acetate uptake, but not to the phosphorus release. A general reverse correlation between the P^sub rel^/HAc^sub up^ ratio and the overall abundance of Competibacter and TFOs (presuming they were the important GAOs in the systems) was observed (Figure 5). This implies that Competibacter and TFOs together might represent the dominant GAO population in these full-scale plants. The negative correlation between the P^sub rel^/HAc^sub up^ ratio and TFO abundance was observed at all except one plant (plant D) (Figure 5b). Plant D had a low P^sub rel^/ HAc^sub up^ ratio, did not contain TFOs, and had the highest abundance of Competibacter. Despite the diverse phylogenetic affiliations and physiological traits of the TFOs (Tsai and Liu, 2002), the correlation of P^sub rel^/HAc^sub up^ ratio with TFO abundance might suggest that TFOs represent the dominant GAOs in most full-scale plants in the United States.

Because molecular-technique-based analyses are typically not accessible for most WWTPs, simple microscopic observations of TFOs could potentially help operators monitor possible GAO proliferation. Of course, extreme caution must be taken for exceptions, such as plant D, which did not have any TFOs, but had a high abundance of Competibacter. Phosphorus release and uptake tests should be conducted with microbial assessments to monitor the status of the EBPR process.

Competition Between Polyphosphate- and Glycogen-Accumulating Organisms and Its Effect on Enhanced Biological Phosphorus Removal Performance and Stability. Good phosphorus removal with effluent

(1) The relative anaerobic VFA uptake rates (kinetics) of PAOs versus the GAOs will determine what percentage of the influent carbon the PAOs and GAOs will obtain. As carbon (volatile fatty acids or rbCOD) flows (Q^sub c^) into the anaerobic phase of an EBPR system, PAOs (biomass as X^sub PAO^)and GAOs (biomass as X^sub GAO^) will compete for carbon uptake and storage. The VFA uptake rates (q^sub c^^sub PAO^ and g^sub c^^sub GAO^) differ for PAOs and GAOs, and they are affected by many parameters, including pH, temperature, substrate type, and SRT (Filipe et al., 2001; Schuler and Jenkins, 2002; Whang and Park, 2001; Whang et al., 2002; Zilles et al., 2002). As a result, before entering the following aerobic phase, the carbon is already distributed between PAOs and GAOs in the form of intracellular carbon polymers (PHA).

(2) Then, in the aerobic phase, the intracellular carbon polymers (such as PHA or glycogen, noted as C^sub PAO^^sub PHA^, C^sup GAO^^sub PHA^) will be used for cell synthesis, for both PAOs and GAOs. The amount of phosphorus that can be removed depends on the net biomass production of PAOs, which depends on the amount of carbon that is stored and available for growth and yield values (Y^sub PAO^ and Y^sub GAO^). The net amount of carbon available for growth is the total amount, C (i.e., in PHAs), minus the need for maintenance and for restoring intracellular polymers (glycogen or polyphosphorus) used in the anaerobic phase, as follows:

(C^sup PAO^^sub PHA^ - C^sub main^ - C^sub gly^ - C^sup pp^) for PAOs (1)

(C^sup GAO^^sub PHA^ - C^sub main^ - C^sub gly^) for GAOs (2)

The environmental and operational factors (pH, temperature, and SRT) in the aerobic phase can affect the PHA utilization rate and the energy need for maintenance, and, therefore, they will affect the biomass production of PAOs and GAOs. (3) The maximum PAO abundance in a system depends on the influent phosphorus concentration and the carbon source available. There is a stoichiometric requirement of carbon for a given amount phosphorus to be removed. The minimum rbCOD/P ratio for complete phosphorus removal was found to be 8 to 10 g/g in an acetate-fed SBR EBPR by Schuler and Jenkins (2003). Under the condition that gives competitive advantage to PAOs, the PAOs will take up sufficient VFA in the anaerobic phase to drive subsequent aerobic phosphorus uptake for complete phosphorus removal. If the influent carbon is more than what is required for complete phosphorus removal, the spare carbon can be sequestered by GAOs and allow GAOs to coexist with PAOs, without negatively affecting the EBPR performance, as shown in all the plants studied. Based on this understanding, the PAO fraction in the sludge biomass in a system should be proportional to the influent phosphorus concentration, provided that there is sufficient influent carbon. Examination of the relationship between the influent phosphorus concentrations and net phosphorus release measured in the phosphorus uptake and release batch test (which is an indicator of PAO abundance or activity level) showed positive correlation for all plants except D, as shown in Figure 9. Similar correlation was also demonstrated by Schuler and Jenkins (2003). These results support the proposed hypothesis. The exception of plant D was likely the result of the insufficient influent rbCOD/P ratio (5 g/g) at plant D, which limited the PAO proliferation.

(4) On the other hand, if the process conditions change, such that they become favorable for GAOs for VFA uptake, GAOs will continue to proliferate and eventually outcompete PAOs and cause a system failure. This is because the growth of GAOs will only depend on the carbon available, unlike PAOs, whose proliferation depends not only on carbon available, but also the influent phosphorus level.

Correlation of rbCOD/P Ratio with Enhanced Biological Phosphorus Removal Performance. For EBPR WWTPs, the effluent phosphorus concentration and phosphorus removal stability are of the greatest concern. A positive correlation between phosphorus removal performance stability and influent rbCOD/P ratio was observed in this study (Figure 10), which was also shown in other investigations (Liu et al., 1997; Schuler and Jenkins, 2003). An influent rbCOD/P ratio greater than 15 to 20 was recommended to achieve effluent phosphorus

A higher influent rbCOD/P ratio correlates with stable phosphorus removal performance, but, at the same time, leads to higher GAO populations and therefore lower carbon utilization efficiency. A rbCOD/P ratio of 50 led to GAO dominance; and a rbCOD/P ratio between these values led to coexistence of both PAOs and OAOs (Ahn, Lee, and Kwon, 2006; Liu et al., 1997; Schuler and Jenkins, 2003). The plants surveyed in the current study had carbon-to-phosphorus ratios ranging between 5 and 38, therefore predicting that our evaluated plants contained a combination of PAO and GAO populations, which is consistent with our observation.

For most municipal WWTPs, the influent carbon-to-phosphorus ratios range from 5 to 40, and this is where coexistence of both PAOs and GAOs is expected. As previously discussed, an EBPR process in which both PAOs and GAOs coexist can maintain good and stable performance, as long as the conditions are favorable for PAOs to preferably uptake sufficient carbon to removal all the influent phosphorus, only leaving the spare carbon for GAOs to sequester. The positive correlation of process stability with the increasing influent rbCOD/P ratio, and by extension with the elevating GAOs abundance in the system, suggests the possibility that GAOs may actually serve as a buffer to attenuate the effect of fluctuation of influent carbon loading on the PAOs, because PAOs seem to be sensitive to process disturbances (Ann, Park, and Kim, 2006). However, changes in conditions of a stable EBPR system that becomes more favorable for the GAOs could potentially result in proliferation of GAOs and outcompete PAOs, eventually leading to deterioration of phosphorus removal. Therefore, understanding and controlling the operation conditions favorable for anaerobic VFA uptake and aerobic growth of PAOs is necessary to maintain the system stability. Competition between PAOs and GAOs can be influenced by many factors, including rbCOD/P ratio, substrate type, SRT, anaerobic hydraulic retention time, temperature, pH, dissolved oxygen, and feeding strategy (and probably more). It is worth mentioning that facilities that have higher rbCOD/P ratios, such as plants B and E, may achieve higher stability, but might also be more prone to upsets, as a result of GAO proliferation, because a higher carbon-to-phosphorus ratio means more GAOs in the system. Periodic summer upsets experienced at plant C are an example of phosphorus removal deterioration that could be related to GAO proliferation, possibly induced by higher temperatures in the summer, which favor development of GAOs. This was evidenced by three consecutive analyses of Accumulibacter and TFOs at plant C. An increase in GAOs (TFOs) was observed when samples taken in July and September 2003 were compared (Tables 2 and 3). The increase of TFOs correlated with a decrease in P^sub rel^/HAc^sub up^ ratio and an increase in effluent total phosphorus. The average effluent total phosphorus at plant C increased from


The following conclusions can be drawn from this study:

* Quantitative observation of candidate PAOs and GAOs, in combination with batch phosphorus release and uptake tests, and full- scale plant performance evaluation are necessary to reveal the effects of these two populations on EBPR performance and stability. The candidate PAOs and GAOs studied, including Accumulibacter phosphatis, Competibacter phosphatis, and TFOs, were relevant to EBPR processes in the United States, although the results also suggested that the diversity of PAOs and GAOs are larger than our current understanding.

* The effluent phosphorus concentration, amount of phosphorus removed, and process stability in an EBPR system are not directly related to high PAO abundance or mutually exclusive with a high GAO fraction. Effective phosphorus removal is achieved with coexistence and a great variation of combined PAO and GAO populations in the six full-scale EBPR plants in the United States. The presence of GAOs does not necessarily negatively affect EBPR performance, provided that the process condition favors the PAOs to uptake sufficient carbon for complete removal of influent phosphorus, leaving only the spare carbon to be sequestered by GAOs. Understanding and controlling the factors that affect VFA uptake rate of PAOs versus GAOs, including pH, temperature, SRT, and carbon substrate type, is critical.

* Stability of EBPR processes was shown to be correlated with higher influent rbCOD/P ratios, even though a higher rbCOD/P ratio often selected for a higher GAO level also. The EBPR process that has higher influent carbon (rbCOD), either by nature or from additional supplement, is expected to have more stable phosphorus removal. It should be noted, however, that facilities that have higher rbCOD/P ratios, although they may achieve higher stability, might also be more prone to upsets if conditions change to be favorable for GAOs, as a result of the relatively higher GAO abundance in the system.

* More research should be directed toward the study of maintaining EBPR stability with a combined PAO and GAO microbial community at rbCOD/P ratios common in WWTPs. Experimental design of laboratory studies on EBPR processes and mechanism should consider the system feed rbCOD/P ratios and effect on the PAO and potential PAO/GAO populations that may be encouraged. Such studies should include population characterization of GAOs and PAOs in interpreting results on phosphorus removal mechanisms, kinetics, and simulation model developments.


This study was funded by the Water Environment Research Foundation (Alexandria, Virginia) project 01-CTS-3. Special thanks to Ann Conklin at the University of Washington (Seattle) for her assistance in VFA analysis and to Mario Benisch and Heather Stephens at HDR Engineering Inc. (Folsom, California) for their contribution of the plant performance survey.

Submitted for publication June 12, 2006; revised manuscript submitted manuscript submitted October 24, 2007; accepted for publication February 14, 2008.

The deadline to submit Discussions of this paper is November 15, 2008.


Ahn, C. H.; Park, J. K.; Kim, K. S. (2006) Microbial Adaptability to Organic Loading Changes in an Enhanced Biological Phosphorus Removal Process. ASCE J. Environ. Eng., 132 (8), 909-917.

Ahn, J.; Lee, M.; Kwon, H. (2006) Changes in Respiratory Quinone Profiles of Enhanced Biological Phosphorus Removal Activated Sludge Under Different Influent Phosphorus/Carbon Ratio Conditions. Bioprocess Biosystems Eng., 29 (3), 143-148.

Beer, M.; Seviour, E. M.; Kong, Y.; Cunningham, M.; Blackall, L. L.; Seviour, R. J. (2002) Phytogeny of the Filamentous Bacterium Eikelboom Type 1851, and Design and Application of a 16S rRNA Targeted Oligonuceotide Probe for Its Fluorescence In Situ Identification in Activated Sludge. FEMS Microbiol. Lett., 207, 179- 183. Beer, M.; Stratton, H. M.; Griffiths, P. C.; Seviour, R. J. (2006) Which are the Polyphosphate Accumulating Organisms in Full- Scale Activated Sludge Enhanced Biological Phosphate Removal Systems in Australia? J. Appl. Microbiol, 100 (2), 233-243.

Bond, P. L.; Keller, J.; Blackall, L. L. (1998) Characterization of Enhanced Biological Phosphorus Removal Activated Sludges with Dissimilar Phosphorus Removal Performances. Water Sci. Technol., 37, 567-571.

Cech, J. S.; Hartman, P. (1993) Competition Between Polyphosphate and Polysaccharide Accumulating Bacteria in Enhanced Biological Phosphate Removal Systems. Water Res., 27, 1219-1225.

Cech, J. S.; Hartman, P. (1990) Glucose Induced Break Down of Enhanced Biological Phosphate Removal. Environ. Technol., 11, 651- 656.

Crocetti, G. R.; Banfield, J. F.; Keller, J.; Bond, P. L.; Blackall, L. L. (2002) Glycogen-Accumulating Organisms in Laboratory- Scale and Full-Scale Wastewater Treatment Processes. Microbiol, 148, 3353-3364.

Crocetti, G. R.; Hugenholtz, P.; Bond, P. L.; Schuler, A.; Keller, J.; Jenkins, D.; Blackall, L. L. (2000) Identification of Polyphosphate-Accumulating Organisms and Design of 16s rDNA Directed Probes for Their Detection and Quantification. Appl. Environ. Microbiol, 66 (3), 1175-1182.

Daims, H.; Bruhl, A.; Amann, R.; Schleifer, K. H.; Wagner, M. (1999) The Domain-Specific Probe EUB338 is Insufficient for the Detection of All Bacteria: Development and Evaluation of a More Comprehensive Probe Set. Systematic Appl. Microbiol. 22 (3), 431- 444.

Filipe, C. D. M.; Daigger, G. T.; Grady, C. P. L., Jr. (2001) pH as a Key Factor in the Competition Between Glycogen-Accumulating Organisms and Phosphorus-Accumulating Organisms. Water Environ. Res., 73, 223-232.

Gu, A. Z.; Saunders, A.; Neethling, J. B.; Stensel, H. D.; Blackall, L. L. (2005) Investigation of PAOs and GAOs and Their Effects on EBPR Performance at Full-Scale Wastewater Treatment Plants. Proceedings of the 78th Annual Water Environment Federation Technical Exposition and Conference, Washington, D.C., Oct. 29-Nov. 2; Water Environment Federation: Alexandria, Virginia.

Gu, A. Z.; Stensel, H. D.; Neethling, J. B.; Benisch, M.; Stephens, H.; Jenkins, D. (2004) Evaluation of Metal Cation Stoichiometry with Biological Phosphorus Removal in Full-Scale EBPR Processes. Proceedings of the 77th Annual Water Environment Federation Technical Exposition and Conference, New Orleans, Louisiana, Oct. 2-6; Water Environment Federation: Alexandria, Virginia.

Hesselmann, R. P. X.; Werten, C.; Hahn, D.; van der Meer, J. R.; Zehnder, A. J. B. (1999) Enrichment, Phylogenetic Analysis and Detection of a Bacterium that Performs Enhanced Biological Phosphorus Removal in Activated Sludge. Syst. Appl. Microbiol., 22, 454-465.

Kong, Y. H.; Beer, M.; Seviour, R. J.; Lindrea, K. C.; Rees, G. A. (2002) Role of "G-Bacteria" in Anaerobic Substrate Uptake in a SBR with No Phosphorus Removal. Water Sci. Technol., 46 (1-2), 171- 178.

Kong, Y. H.; Beer, M.; Seviour, R. J.; Lindrea, K. C.; Rees, G. A. (2001) Structure and Functional Analysis of the Microbial Community in an Aerobic-Anaerobic Sequencing Batch Reactor (SBR) with No Phosphorus Removal. Syst. Appl. Microbiol., 24, 597-609.

Kong, Y. H.; Nielsen, J. L.; Nielsen, P. H. (2005) Identity and Ecophysiology of Uncultured Actinobacterial PolyphosphateAccumulating Organisms in Full-Scale Enhanced Biological Phosphorus Removal Plants. Appl. Environ. Microbiol., 71, 4076-4085.

Kong, Y. H.; Ong, S. L.; Ng, W. J.; Liu, W. T. (2002) Diversity and Distribution of a Deeply Branched Novel Proteobacterial Group Found in Anaerobic-Aerobic Activated Sludge Processes. Environ. Microbiol., 4 (11), 753-757.

Lindrea, K. C.; Pigdon, S. P.; Boyd, B.; Lockwood, G. A. (1994) Biomass Characterization in a Nitrification-Denitrification Biological Enhanced Phosphorus Removal (NDBEPR) Plant during Start- Up and Subsequent Periods of Good and Poor Phosphorus Removal. Water Sci. Technol, 29, 91-100.

Liu, W. T.; Nakamura, K.; Matsuo, T.; Mino, T. (1997) Internal Energy-Based Competition Between Polyphosphate and Glycogen Accumulating Bacteria in Biological Phosphorus Removal Reactors- Effect of P/C Feeding Ratio. Water Res., 31 (6), 1430-1438.

Liu, W. T.; Nielsen, A. T.; Wu, J. H.; Tsai, C. S.; Matsuo, Y.; Molin, S. (2001) In Situ Identification of Polyphosphate- and PolyhydroxyalkanoateAccumulating Traits for Microbial Populations in a Biological Phosphorus Removal Process. Environ. Microbiol., 3, 110- 122.

Mamais, D.; Jenkins, D.; Pitt, P. (1993) A Rapid Physical- Chemical Method for the Determination of Readily Biodegradable Soluble COD in Municipal Wastewater. Water Res., 27 (1), 195-197.

McInerney, P.; Mizutani, T.; Shiba, T. (2006) Inorganic Polyphosphate Interacts with Ribosomes and Promotes Translation Fidelity In Vitro and In Vivo. Mol. Microbiol., 60, 438-447.

Meyer, R. L.; Saunders, A. M.; Blackall, L. L. (2006) Putative Glycogen-Accumulating Organisms Belonging to the Alphaproteobacteria Identified Through rRNA-Based Stable Isotope Probing. Microbiol. UK, 152, 419-429.

Neethling, J. B.; Bakke, B.; Benisch, M.; Gu, A. Z.; Stephens, H.; Stensel, H. D.; Moore, R. (2005) Factors Influencing the Reliability of Enhanced Biological Phosphorus Removal, report Ol- CTS-3; Water Environment Research Foundation: Alexandria, Virginia.

Oehmen, A.; Lemos, P. C.; Carvalho, G.; Yuan, Z.; Keller, J.; Blackall, L. L.; Reisa, M. A. M. (2007) Advances in Enhanced Biological Phosphorus Removal: From Micro to Macro Scale. Water Res., 41 (11), 2271-2300.

Randall, C. W.; Barnard, J. L.; Stensel, H. D. (Eds.) (1992) Design and Retrofit of Wastewater Treatment Plants for Biological Nutrient Removal; Technomic Publishing: Lancaster, Pennsylvania.

Satoh, H.; Mino, T.; Matsuo, T. (1994) Deterioration of Enhanced Biological Phosphorus Removal by the Domination of Microorganisms without Polyphosphate Accumulation. Water Sci. Technol, 30 (6), 203- 211.

Saunders, A. M.; Oehmen, A.; Blackall, L. L.; Yuan, Z.; Keller, J. (2003) The Effect of GAOs (Glycogen Accumulating Organisms) on Anaerobic Carbon Requirements in Full-Scale Australian EBPR (Enhanced Biological Phosphorus Removal) Plants. Water Sci. Technol., 47 (11), 37-43.

Schuler, A.; Jenkins, D. (2002) Effects of pH on Enhanced Biological Phosphorus Removal Metabolisms. Water Sci. Technol., 46 (4-5), 171-178.

Schuler, A.; Jenkins, D. (2003) Enhanced Biological Phosphorus Removal from Wastewater with Different Phosphorus Contents, Part I and II. Water Environ. Res., 75, 485-498.

Seviour, R. J.; Maszenan, A. M.; Soddell, J. A.; Tandoi, V.; Patel, B. K. C.; Kong, Y.; Schumann, P. (2000) Microbiology of the 'G-Bacteria' in Activated Sludge. Environ. Microbiol, 2 (6), 581- 593.

Spring, S.; Wagner, M.; Schumann, P.; Kampfer, P. (2005) Malikia granosa gen. Nov., sp.nov., a Novel Polyhydroxyalkanoates and Polyphosphate Accumulating Bacterium Isolated from Activated Sludge, and Reclassification of Pseudomonas apinosa as Malikia spinosa Comb. Nov. Int. J. Syst. Ecol. Microbiol, 55, 621-629.

Stephens, H. L.; Stensel, H. D. (1998) Effect of Operating Conditions on Biological Phosphorus Removal. Water Environ. Res., 70, 362-369.

Stephens, H. M.; Neethling, J. B.; Benisch, M.; Gu, A. Z.; Stensel, H. D. (2004) Comprehensive Analysis of Full-Scale Enhanced Biological Phosphorus Removal Facilities. Proceedings of the 77th Annual Water Environment Federation Technical Exposition and Conference, New Orleans, Louisiana, Oct. 2-6; Water Environment Federation: Alexandria, Virginia.

Tetreault, M. J.; Benedict A. H.; Kaempfer, C.; Borth, E. F. (1986) Biological Phosphorus Removal: A Technology Evaluation. J. Water Pollut. Control Fed., 58, 823-837.

Tsai, C. S.; Liu, W. T. (2002) Phylogenetic and Physiological Diversity of Tetrad-Forming Organisms in Deteriorated Biological Phosphorus Removal Systems. Water Sci. Technol., 46 (1-2), 179-184.

Wang, J. C.; Park, J. K. (2001) Effect of Anaerobic-Aerobic Contact Time on the Change of Internal Storage Energy in Two Different Phosphorus-Accumulating Organisms. Water Environ. Res., 73, 436-443.

Wentzel, M. C.; Loewenthal, R. E.; Ekama, G. A.; Marais, G. v. R. (1988) Enhanced Polyphosphate Organism Cultures in Activated-Sludge Systems. 1. Enhanced Culture Development. Water SA, 14, 81-92.

Whang, L. M.; Filipe, C. D. M.; Park, J. K. (2002) Model-Based Evaluation of Competition Between Polyphosphate and Glycogen Accumulating Organisms. Proceedings of the 75th Annual Water Environment Federation Technical Exposition and Conference, Chicago, Illinois, Sept. 28-Oct. 2; Water Environment Federation: Alexandria, Virginia.

Whang, L-M.; Park, J. K. (2001) Competition Between Polyphosphateand Glycogen-Accumulating Organisms in Enhanced Biological Phosphorus Removal Systems-Effect of Temperature and Sludge Age. Proceedings of the 74th Annual Water Environment Federation Technical Exposition and Conference, Atlanta, Georgia, Oct. 13-17; Water Environment Federation: Alexandria, Virginia.

Wong, M-T.; Mino, T.; Seviour, R. J.; Onuki, M.; Liu, W-T. (2005) In Situ Identification and Characterization of the Microbial Community Structure of Full-Scale Enhanced Biological Phosphorus Removal Plants in Japan. Water Res., 39, 2901-2914.

Wong, M-T.; Tan, F. M.; Liu, W-T. (2004) Identification and Occurrence of Tetrad-Froming Alphaproteobacteria in Anaerobic: Aerobic Activated Sludge Processes. Microbiol., 150, 3741-3748.

Zilles, J. L.; Peccia, J.; Kim, M. W.; Hung, C. H.; Noguera, D. R. (2002) Involvement of Rhodocyclus-Related Organisms in Phosphorus Removal in Full-Scale Wastewater Treatment Plants. Appl. Environ, Microbiol, 68 (6), 2763-2769.

April Z. Gu1,2*, A. Saunders3, J. B. Neethling2, H. D. Stensel4, L. L. Blackall3

1 Department of Civil and Environmental Engineering, Northeastern University, Boston, Massachusetts. 2 HDR Engineering Inc., Folsom, California.

3 Advanced Wastewater Management Centre, University of Queensland, St. Lucia, Australia.

4 Department of Civil and Environmental Engineering, University of Washington, Seattle, Washington.

* Department of Civil and Environmental Engineering, Northeastern University, Boston, Massachusetts; e-mail: [email protected]

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