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Advanced Wastewater Management Centre (AWMC), The University of Queensland, St Lucia, Brisbane 4072, Australia
Correspondence
Zhiguo Yuan
zhiguo{at}awmc.uq.edu.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-hydroxyalkanoates (PHAs) anaerobically and utilized them aerobically, demonstrating that they were putative GAOs. Some of the Alphaproteobacteria were related to Defluvicoccus vanus (16 % of Bacteria), but the specific identity of many could not be determined by FISH. Further investigation into the identity of other GAOs is necessary.
-hydroxyalkanoate; PHB, poly--hydroxybutyrate; PH2MV, poly--hydroxy-2-methylvalerate; PHV, poly--hydroxyvalerate; propionyl-CoA*, activated propionyl-CoA; SBR, sequencing batch reactor; TFO, tetrad-forming organism; TOGA, titration and off-gas analysis; VFA, volatile fatty acid
Present address: Lab. 505 - Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa (UNL), 2829-516 Caparica, Portugal.
Present address: Environment and Resources DTU, Bygningstorvet, bldg. 115, The Technical University of Denmark, DK - 2800 Lyngby, Denmark.
Present address: Department of Microbial Ecology, Aarhus University, Ny Munkegade, 8000 Aarhus C, Denmark.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Satoh et al., 1994; Thomas et al., 2003; Whang & Park, 2002). Currently, there are no pure cultures of PAOs or GAOs (Seviour et al., 2003), so their biochemical transformations have been determined through chemical analyses of enriched laboratory-scale mixed EBPR microbial communities. Metabolic models have been proposed to describe the biochemical transformations occurring in PAOs consuming acetate (Smolders et al., 1994a, b, 1995) and propionate (Oehmen et al., 2005b) as the sole carbon source. Similarly, GAO enrichments consuming acetate have also been modelled (Filipe et al., 2001a; Zeng et al., 2002, 2003b). These metabolic models provide useful tools for optimization of EBPR and for research into the competition between PAOs and GAOs.
Under anaerobic conditions, both PAOs and GAOs can take up volatile fatty acids (VFAs) and convert them into intracellular poly--hydroxyalkanoates (PHAs). To obtain the energy for anaerobic VFA uptake, the metabolic models propose that PAOs hydrolyse intracellularly stored polyphosphate and glycogen, while GAOs hydrolyse only glycogen for this purpose, as they have no stored polyphosphate. Under aerobic conditions, PAOs and GAOs oxidize the intracellular PHA to grow and to replenish their stored glycogen. PAOs also use a portion of the energy generated from PHA oxidation for orthophosphate uptake and synthesis of intracellular polyphosphate. Since GAOs consume the limited VFAs in the anaerobic period without contributing to phosphorus removal, they are highly undesirable micro-organisms in EBPR systems.
Culture-independent methods have been used to identify a PAO, named ‘Candidatus Accumulibacter phosphatis' (Accumulibacter), which is a member of the family Rhodocyclaceae within the Betaproteobacteria (Crocetti et al., 2000; Hesselmann et al., 1999). Fluorescence in situ hybridization (FISH) has shown Accumulibacter to be an abundant organism in laboratory-scale EBPR cultures with various carbon sources (Levantesi et al., 2002; Liu et al., 2001; Oehmen et al., 2004, 2005b; Onda et al., 2002; Pijuan et al., 2004; Zeng et al., 2003a). Accumulibacter has also been reported in full-scale EBPR systems (Saunders et al., 2003; Zilles et al., 2002). A similar approach has identified deeply branching members of the Gammaproteobacteria (Nielsen et al., 1999) as putative GAOs. These organisms have been called either ‘Candidatus Competibacter phosphatis' (henceforth called Competibacter; Crocetti et al., 2002) or the GB lineage (Kong et al., 2002). Competibacter can consume acetate as the sole carbon source, but a recent study has shown that they take up propionate very slowly (Oehmen et al., 2005a), and thus tend to be out-competed by Accumulibacter (Oehmen et al., 2005a, 2006; Pijuan et al., 2004).
Tetrad-forming organisms (TFOs; also called ‘G-bacteria’) have been linked to the deterioration of EBPR in laboratory-scale systems. Using culture-independent methods, two distinct groups of TFOs have recently been identified as members of the Alphaproteobacteria and shown to be putative GAOs. One group are members of the order Sphingomonadales, and the other is related to the isolate Defluvicoccus vanus within the order Rhodospirillales. Both of these organisms could consume acetate as the sole carbon source (Beer et al., 2004; Wong et al., 2004), but the D. vanus-related organism has also been recently demonstrated to consume propionate (Meyer et al., 2006), unlike Competibacter.
This study investigated the metabolism of an enriched culture of Alphaproteobacteria TFOs that were fed with propionate as the sole carbon source and demonstrated the GAO phenotype. Anaerobic and aerobic biochemical transformations as well as maintenance processes are described in detail from experimental data, and a metabolic model describing propionate uptake by GAOs is proposed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Zeng et al., 2003b) with propionate as the sole carbon source were fed during the first 6 min of the anaerobic period in each cycle, resulting in a hydraulic retention time (HRT) of 24 h. The sludge retention time (SRT) was approximately 8 days, with 250 ml mixed liquor being wasted at the end of each aerobic period. The pH was maintained at 7.0±0.1 during the anaerobic and aerobic phases using a one-way controller that dosed 0.5 M HCl when the pH was above the setpoint. The reactor was seeded with sludge from an SBR enriched for PAOs, as reported in Oehmen et al. (2005b), and maintained under identical operational conditions apart from a considerably reduced level of phosphate in the feed (2 mg PO4-P l–1 instead of 53 mg PO4-P l–1) to select for GAOs, where PO4-P is phosphate.
Cycle studies and batch experiments.
The performance of the SBR was monitored through cycle studies in which samples were collected at various points throughout a cycle and chemically analysed. Propionate, PHA, glycogen, orthophosphate and ammonia were analysed (Oehmen et al., 2005b) every 10 min for the first 40 min of the anaerobic period, followed by 20 min intervals thereafter. In the subsequent aerobic phases, PHA, glycogen, orthophosphate and ammonia analyses were carried out every 20–30 min. Total suspended solids (TSS) and volatile suspended solids (VSS) samples were determined at the end of each aerobic period (APHA, AWWA & WPCF, 1995).
Oxygen consumption and CO2 production during a cycle were determined via the titration and off-gas analysis (TOGA) sensor with 2 h anaerobic and 3 h aerobic phases (Pratt et al., 2003).
The anaerobic hydrogen ion production (or consumption) found in each test with the TOGA sensor is caused by two main factors. Propionic acid uptake by GAOs leads to the consumption of H+, and CO2 production has a pH effect through dissolving in the liquid and influencing the bicarbonate acid-base system. The total amount of hydrogen ions produced (HPtotal) can be determined as:
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A more detailed description of the TOGA and the determination of CO2 production has been reported in the literature (Oehmen et al., 2005b; Pratt et al., 2003; Zeng et al., 2003b).
Determination of the anaerobic and aerobic maintenance coefficients.
The rate of glycogen hydrolysis, PHA accumulation and CO2 production incurred by anaerobic maintenance was determined through an 8 h anaerobic batch test without propionate addition. The aerobic maintenance was found through extending the aerobic period to 9 h in one of the TOGA tests described above for the determination of oxygen uptake and CO2 production. All other operational procedures in the anaerobic and aerobic maintenance batch tests were identical to those described above.
Analytical procedures.
Orthophosphate, ammonia, nitrate and nitrite were analysed using a QuikChem8000 (Lachat, WI) flow injection analyser (FIA). Propionic acid was measured by HPLC with an HPX-87H 300x7.8 mm Bio-Rad Aminex ion exclusion HPLC column operated at 65 °C. FIA and propionic acid samples were obtained through filtering mixed liquor from the SBR using 0.22 µm Millex GP syringe-driven filters. Total suspended solids (TSS) and volatile suspended solids (VSS) concentrations were determined in accordance with standard methods (APHA, AWWA & WPCF et al., 1995). Glycogen and PHA analyses were performed as reported in Oehmen et al. (2005b). Elemental analysis of the enriched biomass was performed using 0.5 mg lyophilized sludge in a Perkin-Elmer 240 Elemental Analyser. The carbon, hydrogen, oxygen and nitrogen measurements were used to determine the active biomass composition, after subtracting the PHA and glycogen components stored in the sludge.
FISH was carried out as detailed in Amann (1995), and a list of all oligonucleotide probes used during this study is shown in Table 1. ALF1b (Manz et al., 1992) and ALF969 (R. Lemaire and others, unpublished results) probes were used for Alphaproteobacteria. ALF969 was modified from ALF968 (Neef, 1997) because Competibacter and Kouleothrix (a filamentous organism commonly found in activated sludge systems (Beer et al., 2002) are perfectly targeted by ALF968. Competitor probes targeting Competibacter and Kouleothrix were added without a fluorescent label to ensure the differentiation of these organisms from Alphaproteobacteria.
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; Crocetti et al., 2002), in which the area of cells binding the specific probe is expressed as the mean percentage of the area of the entire bacterial community (EUBMIX, Table 1
). The standard error of the mean (SEM) was calculated as the standard deviation of the area percentage divided by the square root of the number of images (n=51). Post-FISH chemical staining was carried out as detailed by Crocetti et al. (2002) using Nile Blue to stain for PHA (Ostle & Holt, 1982). Samples from the end of both the anaerobic and aerobic periods were stained to confirm intracellular PHA cycling. |
RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. The GAO phenotype is clearly displayed: VFA uptake, PHA accumulation and glycogen degradation under anaerobic conditions; and PHA oxidation and glycogen replenishment under aerobic conditions. There was no phosphorus release observed in the SBR after the first month of operation. Nitrification inhibition was achieved through addition of allyl-N-thiourea, and confirmed by the absence of NOx (below the detection limit) throughout the reactor operation. The primary fractions of PHA formed through propionate uptake were poly--hydroxyvalerate (PHV) and poly--hydroxy-2-methylvalerate (PH2MV). This differs from GAOs enriched with acetate, which produce primarily poly--hydroxybutyrate (PHB) and PHV (Filipe et al., 2001a; Zeng et al., 2003b).
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) contained PHA at the end of the anaerobic period (Fig. 2B), and the PHA was mostly absent from the cells at the end of the aerobic phase (data not shown). The anaerobic–aerobic cycling of PHA in the Alphaproteobacteria cells combined with their abundance in the biomass strongly supports the hypothesis that they were putative GAOs.
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) and Sphingomonas-related organisms (SBR9-1a) were not detected in the biomass. DF1MIX (Table 1) Defluvicoccus-related organisms accounted for 8 % (SEM=1 %) of the Bacteria. DF2MIX (Table 1) Defluvicoccus-related organisms accounted for an additional 8 % (SEM=1 %) of the Bacteria, and the cells that bound these probes were TFOs. The fact that the majority of Alphaproteobacteria in the biomass that cycled PHA did not bind the current probes for GAOs strongly suggests the presence of other GAOs, despite the similar morphology of these organisms to those described in the literature (Beer et al., 2004; Meyer et al., 2006; Wong et al., 2004). It may be that the probes from these publications do not target the diversity of the organisms within these already identified groups so that additional sequences are required in order to broaden the probe specificities; alternatively, another group of GAOs unrelated to Sphingomonas or D. vanus may exist.
A metabolic model for propionate uptake by GAOs
A metabolic model has been formulated as outlined below to describe the anaerobic metabolism of propionate by GAOs, and is presented schematically in Fig. 3. Propionate is taken up anaerobically by GAOs and converted to propionyl-CoA, using glycogen hydrolysis as the sole energy source. Glycogen glycolysis to pyruvate generates energy in the form of ATP, proceeding through what is hypothesized to be the Embden–Meyerhof (EM) pathway (Filipe et al., 2001a). A portion of the pyruvate is converted to acetyl-CoA and CO2, which produces reducing equivalents in the form of NADH2. The remainder of the pyruvate is converted to propionyl-CoA (consuming NADH2 in the process) such that the reduction-oxidation (redox) balance is maintained within the cells. Acetyl-CoA and propionyl-CoA are then reduced to form activated acetyl-CoA (acetyl-CoA*) and activated propionyl-CoA (propionyl-CoA*), the precursors of PHA. Assuming acetyl-CoA and propionyl-CoA are randomly condensed, the PHA composition is calculated based on the probability of these molecules combining together in a large pool of acetyl-CoA and propionyl-CoA (Filipe et al., 2001a). Selective condensation proposes that all of the acetyl-CoA will preferentially bind to propionyl-CoA to form PHV, while the the remainder of the propionyl-CoA molecules are condensed to PH2MV, resulting in no PHB production (Oehmen et al., 2005b). The experimental results are compared later to the model predictions for both random and selective condensation of acetyl-CoA and propionyl-CoA.
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. This model may also be applied when propionate is the carbon source fed anaerobically. The change in carbon source only affects the fractions of acetyl-CoA and propionyl-CoA metabolized aerobically. These parameters may be obtained from the anaerobic model stoichiometry.
GAO metabolic model reactions
r1, propionate is taken up into the cell and converted to propionyl-CoA, where 
GAO is defined as the energy required for transport of one carbon-mole (C-mol) of VFA across the cell membrane, which has been shown to be pH dependent (Filipe et al., 2001a; Smolders et al., 1994b):
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The aforementioned internal reactions may be expressed in terms of the measurable parameters as shown below:
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Assuming that there is no accumulation of energy, reducing power, acetyl-CoA, propionyl-CoA and pyruvate then 
, rATP=0, racetyl-CoA=0, rpropionyl-CoA=0 and rpyruvate=0. The internal and measurable reactions are then resolved with respect to propionate uptake, where the energy required for transport of 1 C-mol of propionate across the GAO cell membrane is defined as the parameter GAO:
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Therefore, the following overall relationship can be obtained (mole basis):
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Assuming that acetyl-CoA* and propionyl-CoA* are randomly condensed as PHB, PHV and PH2MV, as reported in Filipe et al., then the overall equation is shown below (C-mol basis):
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If selective condensation of acetyl-CoA* and propionyl-CoA* occurs as detailed in Oehmen et al. (2005b), then there is no PHB production, and the overall equation is adjusted to:
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Characterizing the anaerobic stoichiometry of propionate-enriched GAOs
The anaerobic biochemical transformations found experimentally in this study are summarized in Table 2, and compared to the model predictions. Glycogen, PHA and VFA were analysed through off-line samples as described above. The anaerobic CO2 production was found through equation (2) using the CO2 transfer and hydrogen ion production data obtained through the TOGA sensor (shown in Fig. 4), in conjunction with the propionate uptake. From the experimental results shown in Table 2, a carbon recovery of 97.8 % was achieved through a carbon balance, while a redox balance yielded 99.5 % recovery. Both results support the accuracy of the experimental data and strongly suggest that all relevant compounds were measured in the experiments.
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, a close fit can be observed between the experimental data and the metabolic model based on random condensation of acetyl-CoA and propionyl-CoA. This suggests that the proposed model adequately describes the stoichiometry associated with propionate uptake by GAOs. In contrast to the findings for GAOs, the stoichiometry of PAOs fed with propionate has been found to correlate closely with the model based on selective condensation of acetyl-CoA and propionyl-CoA (Oehmen et al., 2005b). The reason for this observed difference in PHA formation between PAOs (selective) and GAOs (random) is unclear at present, but seems to indicate a slight, yet distinctive, difference between the metabolic pathways of these organisms.
The energy required for transport of 1 C-mol of propionate across the GAO cell membrane is defined as the parameter GAO, which is dependent on the ambient pH of the system (Filipe et al., 2001a; Smolders et al., 1994b). The model predictions in Table 2 represent the case when GAO equals zero. Filipe et al. (2001a) estimated an GAO of 0.06 at a pH of 7.0 for an acetate-enriched GAO culture. Further investigation into the estimation of the GAO parameter for GAOs would be beneficial in order to support the hypothesis that GAO does indeed equal zero at a pH of 7.0, as suggested by this study.
Aerobic stoichiometry
The aerobic stoichiometry observed in this study is summarized in Table 3. The carbon balance closes to 99.8 %, while a redox balance yields 93.3 % recovery. Fig. 5 shows the oxygen uptake rate and CO2 transfer rate for a batch test of an extended aerobic cycle using the TOGA sensor. The total oxygen consumption shown in Table 3 was obtained through numerical integration of the oxygen uptake rate for a series of three aerobic periods. The aerobic CO2 production was calculated using equation (3) from the measured cumulative CO2 transfer and hydrogen ion production from the TOGA sensor, as well as the ammonia uptake obtained through off-line analysis and shown in Table 3.
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). Biomass growth may also be estimated using the wastage of biomass per cycle, after taking into consideration the PHA and glycogen storage products in the sludge. Using this method, the biomass growth was estimated to be 1.94 C-mmol l–1. These estimations are within 2 % of each other, supporting the hypothesis that either method may be used to calculate the biomass growth, which has been suggested by Zeng et al. (2003b).
Anaerobic and aerobic maintenance characterization
It has been hypothesized that the sole energy source for anaerobic maintenance in GAOs is obtained through glycogen hydrolysis. An equation to describe the anaerobic maintenance process by GAOs has been proposed (Filipe et al., 2001a; Zeng et al., 2003b):
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In this study, the anaerobic batch test without VFA addition allowed the estimation of the specific anaerobic maintenance coefficient (mATP) [in mol ATP (C-mol biomass)–1 h–1]. The glycogen hydrolysis rate obtained from this batch test is shown in Fig. 6, and was found to be 7.0x10–3 C-mol (C-mol biomass)–1 h–1. The corresponding mATP value is therefore determined to be 3.5x10–3 mol ATP (C-mol biomass)–1 h–1 from equation (4). The PHA accumulation observed in the same anaerobic batch test is also shown in Fig. 6. The rate of PHA formation was found to be 6.2x10–3 C-mol (C-mol biomass)–1 h–1, where the composition of PHA produced was calculated as 12 % PHB, 41 % PHV and 47 % PH2MV. The rate of CO2 formation was 2.1x10–3 C-mol (C-mol biomass)–1 h–1 from equation (2) using the TOGA data (not shown). The results obtained in this study are not well described by the proposed stoichiometry for anaerobic maintenance processes in equation (4). The reason for this is not well understood. Perhaps when the wastewater contains no VFA the maintenance process of GAOs is different to that when VFAs are present, or the maintenance of acetate-enriched GAOs (often dominated by Competibacter) may be different from that of these Alphaproteobacteria GAOs enriched on propionate. It should also be noted that equation (4) has not yet been validated experimentally, and may not describe the actual anaerobic maintenance process of GAOs.
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. After 4 h aeration, the rate of oxygen consumption (mos) [in mol O2 (C-mol biomass)–1 h–1] became relatively constant at a mean value of 0.22 mmol h–1, or 2.7x10–3 mol (C-mol biomass)–1 h–1. The oxygen uptake rate correlated well with the CO2 transfer rate during this time period (see Fig. 5), suggesting that almost all of the oxygen was converted to CO2, which is consistent with previous studies under maintenance conditions (Zeng et al., 2003b). The anaerobic and aerobic maintenance coefficients are compared with literature-reported values from previous studies with PAOs and GAOs in Table 4. It may be observed that the maintenance energy requirements are similar between PAOs and GAOs, while the mATP values are slightly higher in both PAO and GAO cultures enriched with propionate as compared to acetate.
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; Oehmen et al., 2005a, 2006; Pijuan et al., 2004; Thomas et al., 2003), likely due to a reduced level of GAO activity. The metabolic activity of PAOs and GAOs fed with different carbon sources is compared in Table 4. The maximum specific VFA uptake rate (qmax) [in C-mol (C-mol biomass)–1 h–1] and the aerobic biomass growth yield (Ygrowth) [in C-mol biomass growth (C-mol VFA uptake)–1] are factors that may influence the competition between PAOs and GAOs in wastewater systems. The qmax and Ygrowth results from this study were very similar to most of the previously reported values for PAOs and GAOs, which does not clearly suggest that PAOs possess an advantage over GAOs when propionate is the carbon source. The reason for the observed advantage of PAOs in previous studies in which propionate was the carbon source is currently unknown. Further investigation is required to assess the proliferation of these Alphaproteobacteria GAOs in EBPR plants as well as their ability to compete with PAOs in these systems.
The presence of these alphaproteobacteria GAOs in EBPR systems could have considerable ramifications for future investigations concerning the competition between PAOs and GAOs. Accumulibacter appears to be a dominant PAO in the presence of both acetate and propionate as carbon sources, and has been shown to have the capacity to switch between acetate and propionate uptake without the need for acclimation (Oehmen et al., 2005a; Pijuan et al., 2004). It is possible, however, that GAOs have a more specific preference for acetate or propionate. In one study, GAOs enriched with acetate as the sole carbon source (and dominated by Competibacter) were shown to be far less effective in propionate uptake, while GAOs enriched with propionate (dominated by Alphaproteobacteria) were shown to take up acetate at less than half the rate of propionate (Oehmen et al., 2005a). A carbon source preference exhibited by different groups of GAOs could perhaps be useful in minimizing the undesirable proliferation of GAOs in EBPR systems.
Conclusions
The anaerobic and aerobic metabolism of GAOs enriched with propionate as the sole carbon source has been described in this study. The main outcomes from this study can be summarized as follows.
The proposed metabolic model described well the anaerobic biochemical transformations by GAOs with propionate as the carbon source.
The biomass was highly enriched in Alphaproteobacteria with a tetrad morphotype. A fraction of these organisms were identified as D. vanus-related organisms. Furthermore, most Alphaproteobacteria in the biomass exhibited intracellular anaerobic–aerobic cycling of PHA.
The maximum specific VFA uptake rate and biomass growth yield of these Alphaproteobacteria GAOs were similar to values reported in the literature from PAO and GAO studies. It is unclear at present why these organisms seem to compete less effectively than PAOs when propionate is the carbon source, as has been suggested by previous studies.
The energy requirements for anaerobic and aerobic maintenance processes found in this study were similar to previously reported values for PAOs and GAOs.
Currently available oligonucleotide probes for GAO bound only 16 % of the Bacteria in this system, whereas many more appeared to be cycling PHA, suggesting the presence of other GAOs. Further investigation into the phylogenetic diversity of GAOs, and their significance in EBPR systems, is recommended.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 24 March 2005;
revised 4 April 2006;
accepted 5 May 2006.
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