Insights on biological phosphorus removal with partial nitrification in single sludge system via sidestream free ammonia and free nitrous acid dosing.

The sidestream sludge treatment by free ammonium (FA)/free nitrous acid (FNA) dosing was frequently demonstrated to maintain the nitrite pathway for the partial nitrification (PN) process. Nevertheless, the inhibitory effect of FA and FNA would severely influence polyphosphate accumulating organisms (PAOs), destroying the microbe-based phosphorus (P) removal. Therefore, a strategic evaluation was proposed to successfully achieve biological P removal with a partial nitrification process in a single sludge system by sidestream FA and FNA dosing. Through the long-term operation of 500 days, excellent phosphorus, ammonium and total nitrogen removal performance were achieved at 97.5 ± 2.6 %, 99.1 ± 1.0 % and 75.5 ± 0.4 %, respectively. Stable partial nitrification with a nitrite accumulation ratio (NAR) of 94.1 ± 3.4 was attained. The batch tests also reported the robust aerobic phosphorus uptake based on FA and FNA adapted sludge after exposure of FA and FNA, respectively, suggesting the FA and FNA treatment strategy could potentially offer the opportunity for the selection of PAOs, which synchronously have the tolerance to FA and FNA. Microbial community analysis suggested that Accumulibacter, Tetrasphaera, and Comamonadaceae collectively contributed to the phosphorus removal in this system. Summarily, the proposed work presents a novel and feasible strategy to integrate enhanced biological phosphorus removal (EBPR) and short-cut nitrogen cycling and bring the combined mainstream phosphorus removal and partial nitrification process closer to practical application.

Diclofenac biotransformation in the enhanced biological phosphorus removal process.

Diclofenac is a pharmaceutical active compound frequently detected in wastewater and water bodies, and often reported to be persistent and difficult to biodegrade. While many previous studies have focussed on assessing diclofenac biodegradation in nitrification and denitrification processes, this study focusses on diclofenac biodegradation in the enhanced biological phosphorus removal (EBPR) process, where the efficiency of this process for diclofenac biodegradation as well as the metabolites generated are not well understood. An enrichment of Accumulibacter polyphosphate accumulating organisms (PAOs) was operated in an SBR for over 300 d, and acclimatized to 20 µg/L of diclofenac, which is in a similar range to that observed in domestic wastewater influents. The diclofenac biotransformation was monitored in four periods of stable operation and linked to the microbial community and metabolic behaviour in each period. Nitrification was observed in two of the four periods despite the addition of a nitrification inhibitor, and these periods were positively correlated with increased diclofenac biodegradation. Interestingly, in two periods with excellent phosphorus removal (>99%) and no nitrification, different levels of diclofenac biotransformation were observed. Period 2, enriched in Accumulibacter Type II achieved more significant diclofenac biotransformation (3.4 µg/gX), while period 4, enriched in Accumulibacter Type I achieved lower diclofenac biotransformation (0.4 µg/gX). In total, 23 transformation products were identified, with lower toxicity than the parent compound, enabling the elucidation of multiple metabolic pathways for diclofenac biotransformation. This study showed that PAOs can contribute to diclofenac biotransformation, yielding less toxic transformation products, and can complement the biodegradation carried out by other organisms in activated sludge, particularly nitrifiers.

Intracellular polyphosphate length characterization in polyphosphate accumulating microorganisms (PAOs): Implications in PAO phenotypic diversity and enhanced biological phosphorus removal performance.

Polyphosphate (polyP) accumulating organisms (PAOs) are the key agent to perform enhanced biological phosphorus removal (EBPR) activity, and intracellular polyP plays a key role in this process. Potential associations between EBPR performance and the polyP structure have been suggested, but are yet to be extensively investigated, mainly due to the lack of established methods for polyP characterization in the EBPR system. In this study, we explored and demonstrated that single-cell Raman spectroscopy (SCRS) can be employed for characterizing intracellular polyPs of PAOs in complex environmental samples such as EBPR systems. The results, for the first time, revealed distinct distribution patterns of polyP length (as Raman peak position) in PAOs in lab-scale EBPR reactors that were dominated with different PAO types, as well as among different full-scale EBPR systems with varying configurations. Furthermore, SCRS revealed distinctive polyP composition/features among PAO phenotypic sub-groups, which are likely associated with phylogenetic and/or phenotypic diversity in EBPR communities, highlighting the possible resolving power of SCRS at the microdiversity level. To validate the observed polyP length variations via SCRS, we also performed and compared bulk polyP length characteristics in EBPR biomass using conventional polyacrylamide gel electrophoresis (PAGE) and solution 31P nuclear magnetic resonance (31P-NMR) methods. The results are consistent with the SCRS findings and confirmed the variations in the polyP lengths among different EBPR systems. Compared to conventional methods, SCRS exhibited advantages as compared to conventional methods, including the ability to characterize in situ the intracellular polyPs at subcellular resolution in a label-free and non-destructive way, and the capability to capture subtle and detailed biochemical fingerprints of cells for phenotypic classification. SCRS also has recognized limitations in comparison with 31P-NMR and PAGE, such as the inability to quantitatively detect the average polyP chain length and its distribution. The results provided initial evidence for the potential of SCRS-enabled polyP characterization as an alternative and complementary microbial community phenotyping method to facilitate the phenotype-function (performance) relationship deduction in EBPR systems.

The storage compounds associated with Tetrasphaera PAO metabolism and the relationship between diversity and P removal.

In enhanced biological phosphorus removal (EBPR), Tetrasphaera can potentially be an abundant and important polyphosphate accumulating organism (PAO), however ongoing questions remain concerning its storage compounds, phosphorus (P) removal capabilities and metabolic behaviour. This study investigated each of these points in an enriched Tetrasphaera culture (95% biovolume). The enriched Tetrasphaera culture fermented amino acids, while also converting and storing diverse amino acids as aspartic and glutamic acid within cells. Subsequent intracellular consumption of these two amino acids during the aerobic phase supports their importance in the metabolism of Tetrasphaera. Polyhydroxyalkanoate (PHA) cycling was also observed in this study, in contrast to some previous studies on Tetrasphaera. While exhibiting anaerobic phosphorus release and aerobic uptake, the highly enriched Tetrasphaera culture was unable to completely remove phosphorus in sequencing batch reactors (SBR) cycles, with an average removal efficiency of 72.3 ± 7.8%. This is unlike a previous study containing both Tetrasphaera (70%) and Accumulibacter (22%), which regularly performed complete phosphorus removal under otherwise similar operational conditions, at efficiencies of > 99%. Notably, the phylodiversity of organisms belonging to Tetrasphaera was substantially different in the present work, consisting mainly of organisms within Clade 2, likely impacting PHA cycling. These results suggest that the contribution of Tetrasphaera towards P removal is highly dependent on the composition of its Clades within this microbial group and an observed higher abundance of Tetrasphaera in WWTPs does not necessarily imply overall higher P removal. This study improves our understanding of the role of Tetrasphaera within EBPR systems and key factors impacting its metabolism.

Achieving combined biological short-cut nitrogen and phosphorus removal in a one sludge system with side-stream sludge treatment.

Biological nitrogen (N) removal via the short-cut pathway (NH4+-N→NO2--N→N2) is economically attractive in wastewater treatment plants (WWTPs). However, biological phosphorus (P) removal processes remain a bottleneck in these systems due to the strong inhibitory effect of nitrite or its protonated form (HNO2, free nitrous acid - FNA) on polyphosphate accumulating organisms (PAOs). In this study, a novel combined nitrogen and phosphorus removal strategy was verified and achieved in a biological short-cut nitrogen removal system via side-stream sludge treatment with FNA, and the mechanisms impacting this process were investigated. The side-stream FNA treatment process applied here led to a significant reduction in the real sludge retention time (SRT) in the mainstream (approximately 2.7 days) based on the biocidal effect of FNA to the majority of the organisms. This work also found that around 40% of the P uptake activity was still maintained at a much higher FNA level of 38 µg N/L with potential PAOs, which highly broadened the current knowledge of PAOs community. An economic analysis revealed advantages of the proposed as compared to conventional biological nitrogen and phosphorus removal (13% savings in total cost), biological short-cut nitrogen removal (via FNA treatment) with chemical phosphorus precipitation (21% savings) and conventional biological nitrogen removal with chemical precipitation (27% savings). Overall, this study presents a novel and viable retrofit strategy in integrating biological short-cut nitrogen removal with EBPR for next generation WWTPs.

Accumulibacter diversity at the sub-clade level impacts enhanced biological phosphorus removal performance.

Accumulibacter is a well-known group of organisms, typically considered to be polyphosphate accumulating organisms (PAOs), but potentially capable of glycogen accumulating organism (GAO) metabolism under limiting influent phosphate levels. Metabolic features of Accumulibacter are typically linked to its phylogenetic identity at the Type or clade level, though it is unclear the extent to which Accumulibacter diversity can correlate with its capacity to perform P removal. This paper investigates the fine-scale diversity of Accumulibacter and its link with enhanced biological phosphorus removal (EBPR) performance under various operating conditions, to understand the conditions and community structure leading to successful and unsuccessful EBPR operation. For this purpose, the organic carbon feeding rate and total organic carbon concentration were varied during three distinct operational periods, where influent phosphate was never limiting. Accumulibacter was always the dominant microbial group (>80% of all bacteria according to quantitative fluorescence in situ hybridisation - FISH) and low levels of Competibacter and other GAOs were consistently observed (<15% of all bacteria). Steady state was achieved in each of the three periods, with average phosphorus removal levels of 36%, 99% and >99%, respectively. Experimentally determined stoichiometric activity supported the expression of a mixed PAO/GAO metabolism in the first steady state period and the typical PAO metabolism in the other two steady state periods. FISH quantification and amplicon sequencing of the polyphosphate kinase (ppk1) functional gene indicated that Accumulibacter clade IIC was selected in the first steady state period, which shifted to clade IA after decreasing the carbon feeding rate in steady state period 2, and finally shifted back to clade IIC in the third steady state period. Fine-resolution Ppk-based phylogenetic analysis revealed three different clusters within Accumulibacter clade IIC, where clusters IICii and IICiii were linked to poor EBPR performance in period 1, and cluster IICi was linked to good EBPR performance in period 3. This study shows that the deterioration of EBPR processes through GAO activity at non-limiting P concentrations can be linked to organisms that are typically classified as PAOs, not only to known GAOs such as Competibacter. Intra-clade phylogenetic diversity within Accumulibacter showed that some clusters actually behave similarly to GAOs even without influent phosphate limitation. This study highlights the need to closely re-examine traditional interpretations regarding the link between the microbial community composition and identity with the performance and metabolism of EBPR systems.

Butyrate can support PAOs but not GAOs in tropical climates.

Glycogen accumulating organisms (GAOs) are thought to compete with polyphosphate accumulating organisms (PAOs) for the often-limiting carbon sources available in wastewater, deteriorating enhanced biological phosphorus removal (EBPR) performance at high temperatures. Fermentation liquids are often used to provide an additional carbon source supply in EBPR processes, where butyrate is known to be an important volatile fatty acid (VFA) produced in sludge fermentation. Nevertheless, the impact of butyrate on the PAO-GAO competition is not well understood especially at high temperature. The results of this study demonstrate that butyrate, as a supplemental or sole carbon source, could be promising for EBPR in tropical climates. When the carbon source was gradually changed from acetate to butyrate, a substantial PAO population was found under both conditions, despite a substantial shift in the abundance of Candidatus Accumulibacter phosphatis (decreased from 37.4% to 13.9%) to Rhodocyclaceae (increased from 2.0% to 14.5%), where both organisms likely played an important role in P-removal. Thus, a relatively stable P removal performance was realized throughout the whole operation period. Nevertheless, butyrate had a negative impact on GAOs. The biomass concentration and microbial diversity continually decreased in the GAO reactor, and Candidatus Competibacter phosphatis reduced from 27.3% to 6.2%, where the dominant population was replaced by Zoogloea. With the addition of butyrate as carbon source, the total amount of synthesized PHAs reduced in both PAO and GAO cultures and the composition of PHA was greatly changed. The presence of a novel PHA fraction (PHH) may disturb the microbial activity in the aerobic phase, where the GAO culture was more severely affected. Glycogen cycling also seemed to be limited in both reactors. This could reduce the GAO metabolism in both cultures and favor PAOs and P removal. Furthermore, the biomass growth rate of the PAO culture was higher than that of GAO when fed with butyrate, which also provides PAO a competitive advantage. All the above results indicate that butyrate could not be well metabolized by GAOs, but could provide PAOs a competitive advantage. Thus, mixed VFAs (i.e. acetate, propionate and butyrate) are likely to favor PAOs over GAOs in EBPR processes operated in warm climates, where the impact of substantial butyrate fractions represents an advantage towards successful process operation.

Long-term simulation of a full-scale EBPR plant with a novel metabolic-ASM model and its use as a diagnostic tool.

This study evaluates the predictive capacity of the META-ASM model, a new integrated metabolic activated sludge model, in describing the long-term performance of a full-scale enhanced biological phosphorus removal (EBPR) system that suffers from inconsistent performance. In order to elucidate the causes of EBPR upsets and troubleshoot the process accordingly, the META-ASM model was tested as an operational diagnostic tool in a 1336-day long-term dynamic simulation, while its performance was compared with the ASM-inCTRL model, a version based on the Barker & Dold model. Overall, the predictions obtained with the META-ASM without changing default parameters were more reliable and effective at describing the active biomass of polyphosphate accumulating organisms (PAOs) and the dynamics of their storage polymers. The primary causes of the EBPR upsets were the high aerobic hydraulic retention times (HRTs) and low organic loading rates (OLRs) of the plant, which led to periods of starvation. The impact of these factors on EBPR performance were only identified with the META-ASM model. Furthermore, the first signs of process upsets were predicted by variations in the aerobic PAO maintenance rates, suggesting that the META-ASM model has potential to provide an early warning of process upset. The simulation of a new viable operational strategy indicated that troubleshooting the process could be achieved by reducing the aerated volume by switching off air in the first half of the aeration tank. In this new strategy, the META-ASM model predicted a simultaneous improvement in the biological phosphorus (P) and nitrogen (N) removal due to the enhancement of the hydrolysis and fermentation of the mixed liquor sludge in the new unaerated zone, which increased the availability of volatile fatty acids (VFAs) for PAOs. This study demonstrates that the META-ASM model is a powerful operational diagnostic tool for EBPR systems, capable of predicting and mitigating upsets, optimising performance and evaluating new process designs.

The impact of temperature on the metabolism of volatile fatty acids by polyphosphate accumulating organisms (PAOs).

This study investigated the effects of different carbon sources on enriched Accumulibacter PAO cultures at high temperature (30 °C) and compared the carbon transformation with low temperature (20 °C) cases reported in literature, revealing several key metabolic differences. While PAOs seemed to prefer propionate anaerobically as compared to other VFAs at high temperature, high aerobic glycogen replenishment was realized with propionate as the anaerobic carbon source, a trait not previously observed at low temperatures. Therefore, it was found that propionate is not correlated with high P removal by Accumulibacter PAO at high temperatures. A combined substrate of acetate, propionate and perhaps butyrate seemed to be a better carbon source combination, since the total VFA uptake rate increased by up to 46%, and this increased the aerobic P-removal efficiency by up to 38.4% and reduced the glycogen recovery by more than 63% compared to the use of only propionate as substrate. This study improves our understanding of how to stimulate successful EBPR operation in warm climates by augmenting the P removal performance of PAOs.

Purple phototrophic bacteria for resource recovery: Challenges and opportunities.

Sustainable development is driving a rapid focus shift in the wastewater and organic waste treatment sectors, from a "removal and disposal" approach towards the recovery and reuse of water, energy and materials (e.g. carbon or nutrients). Purple phototrophic bacteria (PPB) are receiving increasing attention due to their capability of growing photoheterotrophically under anaerobic conditions. Using light as energy source, PPB can simultaneously assimilate carbon and nutrients at high efficiencies (with biomass yields close to unity (1 g CODbiomass·g CODremoved-1)), facilitating the maximum recovery of these resources as different value-added products. The effective use of infrared light enables selective PPB enrichment in non-sterile conditions, without competition with other phototrophs such as microalgae if ultraviolet-visible wavelengths are filtered. This review reunites results systematically gathered from over 177 scientific articles, aiming at producing generalized conclusions. The most critical aspects of PPB-based production and valorisation processes are addressed, including: (i) the identification of the main challenges and potentials of different growth strategies, (ii) a critical analysis of the production of value-added compounds, (iii) a comparison of the different value-added products, (iv) insights into the general challenges and opportunities and (v) recommendations for future research and development towards practical implementation. To date, most of the work has not been executed under real-life conditions, relevant for full-scale application. With the savings in wastewater discharge due to removal of organics, nitrogen and phosphorus as an important economic driver, priorities must go to using PPB-enriched cultures and real waste matrices. The costs associated with artificial illumination, followed by centrifugal harvesting/dewatering and drying, are estimated to be 1.9, 0.3-2.2 and 0.1-0.3 $·kgdry biomass-1. At present, these costs are likely to exceed revenues. Future research efforts must be carried out outdoors, using sunlight as energy source. The growth of bulk biomass on relatively clean wastewater streams (e.g. from food processing) and its utilization as a protein-rich feed (e.g. to replace fishmeal, 1.5-2.0 $·kg-1) appears as a promising valorisation route.

A novel metabolic-ASM model for full-scale biological nutrient removal systems.

This study demonstrates that META-ASM, a new integrated metabolic activated sludge model, provides an overall platform to describe the activity of the key organisms and processes relevant to biological nutrient removal (BNR) systems with a robust single-set of default parameters. This model overcomes various shortcomings of existing enhanced biological phosphorous removal (EBPR) models studied over the last twenty years. The model has been tested against 34 data sets from enriched lab polyphosphate accumulating organism (PAO)-glycogen accumulating organism (GAO) cultures and experiments with full-scale sludge from five water resource recovery facilities (WRRFs) with two different process configurations: three stage Phoredox (A2/O) and adapted Biodenitro™ combined with a return sludge sidestream hydrolysis tank (RSS). Special attention is given to the operational conditions affecting the competition between PAOs and GAOs, capability of PAOs and GAOs to denitrify, metabolic shifts as a function of storage polymer concentrations, as well as the role of these polymers in endogenous processes and fermentation. The overall good correlations obtained between the predicted versus measured EBPR profiles from different data sets support that this new model, which is based on in-depth understanding of EBPR, reduces calibration efforts. On the other hand, the performance comparison between META-ASM and literature models demonstrates that existing literature models require extensive parameter changes and have limited predictive power, especially in the prediction of long-term EBPR performance. The development of such a model able to describe in detail the microbial and chemical transformations of BNR systems with minimal adjustment to parameters suggests that the META-ASM model is a powerful tool to predict and mitigate EBPR upsets, optimise EBPR performance and to evaluate new process designs.

Phosphorus and ammonium removal characteristics from aqueous solutions by a newly isolated plant growth-promoting bacterium.

An indigenous plant growth-promoting bacterium isolated from Peganum Harmala rhizosphere in the arid ecosystem was found to solubilize and accumulate phosphates. This isolate was identified as Pseudomonas sp. (PHR6) by partial 16S rRNA gene sequence analysis. Controlled batch experiments on nutrients removal by this isolate in mineral medium showed relatively high efficiencies after 24 h of aerobic incubation with average values of 117.59 and 335.38 mg gVSS-1 for phosphorus (P-PO4) and nitrogen (N-NH4), respectively. Furthermore, the strain performed heterotrophic nitrification ranging from 48.81% to 84.24% of the total removed nitrogen. On the other hand, the experimental results showed that a short idle period (24 h) significantly enhanced P accumulation (up to 95%) and N assimilation (up to 50%) of the total removed amounts. However, long idle period (20 days) revealed firstly aerobic phosphorous release phase succeeded by another removal one within 24 h of incubation. Overall, the idle treatment enhances P removal efficiency from the mineral liquid medium without significant effects on N-NH4 removal performance. The isolated strain showed also significant nutrient removal ability from synthetic wastewater providing an accumulated fraction of 98% from the total removed phosphorus amount. This study highlights the potential contribution of the selected rhizobacterium PHR6 to both environmental nutrient recycling and pollution control especially regarding phosphorus.

Elucidating functional microorganisms and metabolic mechanisms in a novel engineered ecosystem integrating C, N, P and S biotransformation by metagenomics.

Denitrifying sulfur conversion-associated enhanced biological phosphorous removal (DS-EBPR) system is not only a novel wastewater treatment process, but also an ideal model for microbial ecology in a community context. However, it exists the knowledge gap on the roles and interactions of functional microorganisms in the DS-EBPR system for carbon (C), nitrogen (N), phosphorus (P) and sulfur (S) bioconversions. We use genome-resolved metagenomics to build up an ecological model of microbial communities in a lab-scale DS-EBPR system with stable operation for more than 400 days. Our results yield 11 near-complete draft genomes that represent a substantial portion of the microbial community (39.4%). Sulfate-reducing bacteria (SRB) and sulfide-oxidizing bacteria (SOB) promote complex metabolic processes and interactions for C, N, P and S conversions. Bins 1-4 and 10 are considered as new potential polyphosphate-accumulating organisms (PAOs), in which Bins 1-4 can be considered as S-related PAOs (S-PAOs) with no previously cultivated or reported members. Our findings give an insight into a new ecological system with C, N, P and S simultaneous bioconversions and improve the understanding of interactions among SRB, SOB, denitrifiers and PAOs within a community context.

Denitrifying capabilities of Tetrasphaera and their contribution towards nitrous oxide production in enhanced biological phosphorus removal processes.

Denitrifying enhanced biological phosphorus removal (EBPR) systems can be an efficient means of removing phosphate (P) and nitrate (NO3-) with low carbon source and oxygen requirements. Tetrasphaera is one of the most abundant polyphosphate accumulating organisms present in EBPR systems, but their capacity to achieve denitrifying EBPR has not previously been determined. An enriched Tetrasphaera culture, comprising over 80% of the bacterial biovolume was obtained in this work. Despite the denitrification capacity of Tetrasphaera, this culture achieved only low levels of anoxic P-uptake. Batch tests with different combinations of NO3-, nitrite (NO2-) and nitrous oxide (N2O) revealed lower N2O accumulation by Tetrasphaera as compared to Accumulibacter and Competibacter when multiple electron acceptors were added. Electron competition was observed during the addition of multiple nitrogen electron acceptors species, where P uptake appeared to be slightly favoured over glycogen production in these situations. This study increases our understanding of the role of Tetrasphaera-related organisms in denitrifying EBPR systems.

Denitrification activity of polyphosphate accumulating organisms (PAOs) in full-scale wastewater treatment plants.

A comprehensive assessment of full-scale enhanced biological phosphorus removal (EBPR) plants (five plants, 19 independent tests) was undertaken to determine their effectiveness in terms of aerobic and anoxic P removal. By comparing parallel P uptake tests under only aerobic or under anoxic-aerobic conditions, results revealed that introducing an anoxic stage led to an overall P removal of on average 90% of the P removed under only aerobic conditions. This was achieved with negligible higher PHA and glycogen requirements, 30% lower overall oxygen consumption and with the simultaneous removal of nitrate, reducing up to an estimate of 70% of carbon requirements for simultaneous N and P removal. Varying fractions of denitrifying polyphosphate accumulating organisms (DPAOs), from an average of 25% to 84%, were found in different plants. No correlation was found between the DPAO fractions and EBPR configuration, season, or the concentration of any of the microbial groups measured via quantitative fluorescence in situ hybridisation. These included Type I and Type II Ca. Accumulibacter and glycogen accumulating organisms, suggesting that chemical batch tests are the best methodology for quantifying the potential of anoxic P removal in full-scale wastewater treatment plants.

Metabolism and ecological niche of Tetrasphaera and Ca. Accumulibacter in enhanced biological phosphorus removal.

Tetrasphaera and Candidatus Accumulibacter are two abundant polyphosphate accumulating organisms in full-scale enhanced biological phosphorus removal (EBPR) systems. However, little is known about the metabolic behaviour and ecological niche that each organism exhibits in mixed communities. In this study, an enriched culture of Tetrasphaera and Ca. Accumulibacter was obtained using casein hydrolysate as sole carbon source. This culture was able to achieve a high phosphorus removal efficiency (>99%), storing polyphosphate while consuming amino acids anaerobically. Microautoradiography and fluorescence in situ hybridisation confirmed that more than 90% Tetrasphaera cells were responsible for amino acid consumption while Ca. Accumulibacter likely survived on fermentation products. Tetrasphaera performed the majority of the P removal (approximately 80%) in this culture, and batch tests showed that the metabolism of some carbon sources could actually lead to anaerobic orthophosphate (Pi) uptake (9.0 ± 2.1 mg-P/L) through energy generated by fermentation of glucose and amino acids. This anaerobic Pi uptake may lead to lower net Pi release to C uptake ratios and reduce the Pi needed to be removed aerobically in WWTPs. Intracellular metabolites such as amino acids, sugars, volatile fatty acids and small amines were observed as potential storage products, which may serve as energy sources in the aerobic phase. Evidence of the urea cycle was found, which could be involved in reducing the intracellular nitrogen content. This study improves our understanding of how phosphorus is removed in EBPR systems and can enable novel process optimisation strategies.

Survival strategies of polyphosphate accumulating organisms and glycogen accumulating organisms under conditions of low organic loading.

Enhanced biological phosphorus removal (EBPR) is usually limited by organic carbon availability in wastewater treatment plants (WWTPs). Polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) were operated under extended periods with low organic carbon loading in order to examine its impact on their activity and survival. The decrease in organic carbon load affected PAOs and GAOs in different ways, where the biomass decay rate of GAOs was approximately 4times higher than PAOs. PAOs tended to conserve a relatively high residual concentration of polyhydroxyalkanoates (PHAs) under aerobic conditions, while GAOs tended to deplete their available PHA more rapidly. This slower oxidation rate of PHA by PAOs at residual concentration levels enabled them to maintain an energy source for aerobic maintenance processes for longer than GAOs. This may provide PAOs with an advantage over GAOs in surviving the low organic loading conditions commonly found in full-scale wastewater treatment plants.

Metabolic modelling of full-scale enhanced biological phosphorus removal sludge.

This study investigates, for the first time, the application of metabolic models incorporating polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) towards describing the biochemical transformations of full-scale enhanced biological phosphorus removal (EBPR) activated sludge from wastewater treatment plants (WWTPs). For this purpose, it was required to modify previous metabolic models applied to lab-scale systems by incorporating the anaerobic utilisation of the TCA cycle and the aerobic maintenance processes based on sequential utilisation of polyhydroxyalkanoates, followed by glycogen and polyphosphate. The abundance of the PAO and GAO populations quantified by fluorescence in situ hybridisation served as the initial conditions of each biomass fraction, whereby the models were able to describe accurately the experimental data. The kinetic rates were found to change among the four different WWTPs studied or even in the same plant during different seasons, either suggesting the presence of additional PAO or GAO organisms, or varying microbial activities for the same organisms. Nevertheless, these variations in kinetic rates were largely found to be proportional to the difference in acetate uptake rate, suggesting a viable means of calibrating the metabolic model. The application of the metabolic model to full-scale sludge also revealed that different Accumulibacter clades likely possess different acetate uptake mechanisms, as a correlation was observed between the energetic requirement for acetate transport across the cell membrane with the diversity of Accumulibacter present. Using the model as a predictive tool, it was shown that lower acetate concentrations in the feed as well as longer aerobic retention times favour the dominance of the TCA metabolism over glycolysis, which could explain why the anaerobic TCA pathway seems to be more relevant in full-scale WWTPs than in lab-scale systems.

The impact of aeration on the competition between polyphosphate accumulating organisms and glycogen accumulating organisms.

In wastewater treatment plants (WWTPs), aeration is the major energetic cost, thus its minimisation will improve the cost-effectiveness of the process. This study shows that both the dissolved oxygen (DO) concentration and aerobic hydraulic retention time (HRT) affect the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs). At low DO levels, Accumulibacter PAOs were shown to have an advantage over Competibacter GAOs, as PAOs had a higher oxygen affinity and thus largely maintained their aerobic activity at low DO levels, while GAO activity decreased. Bioreactor operation at low DO levels was found to increase the PAO fraction of the sludge. Furthermore, an increase in aerobic HRT (at a DO level of 2 mg O2/L), promoted the proliferation of GAOs over PAOs, decreasing the EBPR efficiency. Overall, this study shows that low aeration can be beneficial for EBPR performance through selecting for PAOs over GAOs, which should be incorporated into WWTP models in order to minimise energetic costs and improve WWTP sustainability.

Monitoring intracellular polyphosphate accumulation in enhanced biological phosphorus removal systems by quantitative image analysis.

A rapid methodology for intracellular storage polyphosphate (poly-P) identification and monitoring in enhanced biological phosphorus removal (EBPR) systems is proposed based on quantitative image analysis (QIA). In EBPR systems, 4',6-diamidino-2-phenylindole (DAPI) is usually combined with fluorescence in situ hybridization to evaluate the microbial community. The proposed monitoring technique is based on a QIA procedure specifically developed for determining poly-P inclusions within a biomass suspension using solely DAPI by epifluorescence microscopy. Due to contradictory literature regarding DAPI concentrations used for poly-P detection, the present work assessed the optimal DAPI concentration for samples acquired at the end of the EBPR aerobic stage when the accumulation occurred. Digital images were then acquired and processed by means of image processing and analysis. A correlation was found between average poly-P intensity values and the analytical determination. The proposed methodology can be seen as a promising alternative procedure for quantifying intracellular poly-P accumulation in a faster and less labour-intensive way.