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Metabolic versatility in full-scale wastewater treatment plants performing enhanced biological phosphorus removal
Abstract
This study analysed the enhanced biological phosphorus removal (EBPR) microbial community and metabolic performance of five full-scale EBPR systems by using fluorescence in situ hybridisation combined with off-line batch tests fed with acetate under anaerobic-aerobic conditions. The phosphorus accumulating organisms (PAOs) in all systems were stable and showed little variability between each plant, while glycogen accumulating organisms (GAOs) were present in two of the plants. The metabolic activity of each sludge showed the frequent involvement of the anaerobic tricarboxylic acid cycle (TCA) in PAO metabolism for the anaerobic generation of reducing equivalents, in addition to the more frequently reported glycolysis pathway. Metabolic variability in the use of the two pathways was also observed, between different systems and in the same system over time. The metabolic dynamics was linked to the availability of glycogen, where a higher utilisation of the glycolysis pathway was observed in the two systems employing side-stream hydrolysis, and the TCA cycle was more active in the A(2)O systems. Full-scale plants that showed higher glycolysis activity also exhibited superior P removal performance, suggesting that promotion of the glycolysis pathway over the TCA cycle could be beneficial towards the optimisation of EBPR systems.
... The ratios of anaerobic glycogen consumption to VFA uptake (Gly/VFA) demonstrate the energy and reducing power pathways using glycolysis and/or tricarboxylic acid (TCA) cycles in anaerobic metabolism [53,54]. Gly/VFA ratios of the activated sludge from SBR-A and SBR-F were within the range of the anaerobic PAO models for the glycolysis and TCA cycles (0.0 to 0.5 mol C/mol C). ...
... Accumulibacter. The glycolysis pathway was more efficient than the TCA cycle through additional PHA production and less phosphate release with substrate uptake, potentially benefiting EBPR [53]. ...
... EBPR-F obtained a relatively higher Gly/VFA ratio than EBPR, suggesting a higher activity of the glycolytic pathway in producing reducing equivalents. The finding is consistent with studies on enriched Tetrasphaera culture [34,53,54]. EBPR-F enriched both Ca. ...
This study demonstrated the feasibility of enhanced biological phosphorus removal coupled with in-situ fermentation (EBPR-F) to improve phosphorus removal from real digested swine wastewater. We used fermentable substrates (casein hydrolysate and glucose) as the external carbon sources to promote in-situ fermentation and enhance biological phosphorus removal. Compared with conventional EBPR dominated by Candidatus Accumulibacter, EBPR-F had significantly better phosphorus removal with enriched polyphosphate-accumulating organisms (PAOs). Under supplementation with 100 mg/L glucose, total phosphorus (TP) removal was over 95% in EBPR-F, with an average TP concentration in the effluent below 1.0 mg/L, satisfying the discharge standard (8 mg P/L) in China. The PAO activity and relative abundance of Candidatus Accumulibacter (44.7% ± 3.1%) and Tetrasphaera (18.1% ± 6.6%) in EBPR-F were much higher than those in EBPR. The improvement in phosphorus removal of EBPR-F was due to the enrichment of Tetrasphaera through the enhanced in-situ fermentation, as Tetrasphaera can efficiently ferment complex organic matter and provide bioavailable organics for phosphorus removal.
... Glycogen was extracted (0.9M HCl and 3 h of digestion time) and determined using a liquid chromatography-mass spectrometer (LC-MS) (Thermo Scientific, Waltham, MA, USA). Poly-β-hydroxyalkanoates (PHAs), including poly-βhydroxybutyrate (PHB), poly-β-hydroxyvalerate (PHV), and poly-β-hydroxy-2-methylvalerate (PH2MV), were extracted (3% sulfuric acid and 3 h of digestion time) and determined by a gas chromatography-mass spectrometry (GC-MS) (Agilent, Santa Clara, CA, USA) (Lanham et al. 2013). ...
... The anaerobic P release to VFA uptake ratio (P/VFA) in fermentate and HAc feeding batch tests is 0.16 and 0.25 P-mol/Cmol, respectively (Table 2), which are considerably lower than the values predicted in the PAO models (0.37-0.50 P-mol/C-mol) (Hesselmann et al. 2000, Smolders et al. 1995 and some EBPR systems (Lanham et al. 2013, Onnis-Hayden et al. 2020). Relatively low P/VFA ratios have also been reported previously in some EBPR systems with good P removal performance, such as in an Accumulibacter clade II culture exhibiting a mixed PAO-GAO metabolism (0.22 P-mol/C-mol) (Welles et al. 2015), as well as in an S2EBPR facility run in an unmixed in-line MLSS fermentation (UMIF) configuration (0.16 P-mol/C-mol) (Onnis-Hayden et al. 2020). ...
... In addition, a high PHV fraction in generated PHAs was also observed in PAO-enriched systems fed with amino acids (Marques et al. 2017, Zengin et al. 2011. Under the subsequent aerobic phase, the P uptake to PHA utilization ratios (P/PHA) in the batch tests (0.75-0.82 P-mol/C-mol) was higher than the predicted values for the PAO model (0.41 P-mol/C-mol) yet comparable to those observed in the full-scale EBPR facilities (Lanham et al. 2013, Qiu et al. 2019. Overall, the deviations in EBPR kinetics and stoichiometric values from those typically observed in conventional EBPRs suggested that there are more diverse microbial populations and that some specific PAOs/GAOs are involved with other storage polymers rather than PHAs, which require further investigation. ...
To meet the growing interest in the development of innovative biological nutrient removal (BNR) alternatives for energy neutrality, resource recovery, and decarbonization, the adsorption/bio-oxidation (A/B) process has been widely studied for carbon capture and shortcut nitrogen (N) removal, while limited research has focused on incorporating enhanced biological phosphorus (P) removal (EBPR), mainly due to the differential carbon usage characteristics of functionally relevant microorganisms. Here, a full-scale pilot testing with an integrated system combining A-stage high-rate activated sludge (HRAS) with B-stage partial (de)nitrification/anammox and side-stream EBPR (HRAS-P(D)N/A-S2EBPR) was conducted treating real municipal wastewater. The results demonstrated that, despite the low influent carbon load, the B-stage P(D)N-S2EBPR system achieved effective and stable P removal performance, especially when the volatile fatty acid (VFA) load from A-stage was elevated. Sludge fermentation in both A-stage and B-stage promoted carbon redistribution and likely provided more competitive advantages for ammonium-oxidizing bacteria and polyphosphate accumulating organisms, leading to carbon-efficient shortcut N removal mainly through partial nitrification pathway and influent carbon-independent EBPR simultaneously. Exposure to high VFA levels was considered a potential selection factor for the suppression of nitrite-oxidizing bacteria in the system. The involvement of internal carbon-accumulating organisms would potentially play an important role in endogenous denitrification. This study provided new insights into the effects of incorporating side-stream EBPR into the A/B process on microbial ecology, metabolic activities, and system performance.
... For instance, TP removal of almost 96% was reported in a full-scale A 2 O system characterized by an average SRT of 12.7 d and a biomass concentration of 3-4 g/L (Wang et al., 2014). On the contrary, a significantly lower TP removal (43%) was reported for a A 2 O system operated at a similar SRT (12 d) but lower biomass concentration in the tanks, according to the TSS concentration in the sludge (2.9 g/L) (Lanham et al., 2013). For this application, influent characteristics were almost equal to 253 mg/L for the average COD, 81 mg/L of suspended solids, 43 mg-N/L (average N), 33 mg-N/L (average NH 3 ), 5 mg-P/L (average TP) with a resulting COD:N:P ratio of 51:9:1. ...
... An anoxic SSR was also implemented in the Biodenitro™ process, which consists of two tanks where anoxic and aerobic conditions are alternated. This has the aim of partially degrade the RAS (HRT = 20-30 h) and recirculate the previously fermented stream into the Biodenitro™ tank operating under anoxic conditions (Lanham et al., 2013;Vollertsen et al., 2006). In the two examples reported by Lanham et al. (2013), high TP removal efficiencies, i.e., 94% and 96%, were observed, despite the high differences between the operating conditions, i.e., influent TP concentration equal to 4 and 10 mg/L, influent COD concentration of 198 and 636 mg/L, average flow rate of 49ꞏ10 3 and 17ꞏ10 3 m 3 /d, and SRT maintained at 19 and 30 d, respectively. ...
... This has the aim of partially degrade the RAS (HRT = 20-30 h) and recirculate the previously fermented stream into the Biodenitro™ tank operating under anoxic conditions (Lanham et al., 2013;Vollertsen et al., 2006). In the two examples reported by Lanham et al. (2013), high TP removal efficiencies, i.e., 94% and 96%, were observed, despite the high differences between the operating conditions, i.e., influent TP concentration equal to 4 and 10 mg/L, influent COD concentration of 198 and 636 mg/L, average flow rate of 49ꞏ10 3 and 17ꞏ10 3 m 3 /d, and SRT maintained at 19 and 30 d, respectively. However, since the authors reported that FeCl 3 was added as a polishing step for P removal, it is not possible to determine the contribution of the sole Biodenitro™ configuration, combined with an anaerobic SSR, in the achievement of the overmentioned TP removal efficiencies. ...
Phosphorus (P) is an essential element to produce feed and fertilizers but also a nonrenewable resource. Both the predicted exhaustion of phosphatic rocks and the risk of eutrophication lead to an increasing necessity for P recovery methodologies to be applied in municipal wastewater treatment plants (WWTPs). One of the most promising solutions involves the precipitation of P‐based minerals reusable as slow‐release fertilizers. In this study, P recovery as struvite and hydroxyapatite from a municipal WWTP digestate liquid fraction (centrate) was investigated at varying pH (8–10), reagent typologies (MgCl2, NaOH, Ca(OH)2, and CaCl2), and concentrations under limiting magnesium doses through liquid‐ and solid‐phase analyses and thermodynamical modeling. A maximum P recovery of 87.3% was achieved at pH 9 by adding NaOH and MgCl2 at a dose of 656 mg/L (the higher tested). According to these data, it was estimated that 92.0 tons/year of struvite and 33.2 tons/year of hydroxyapatite could be recovered from the WWTP centrate with a cost for reagent consumption being almost 50% of the mean P market value. An increase in P precipitation was observed while comparing experiments with the same pH values but with a higher Mg²⁺ dose. Ca²⁺ addition led to extensive P precipitation but mainly as amorphous phases that interfere with struvite formation.
... For instance, TP removal of almost 96% was reported in a full-scale A 2 O system characterized by an average SRT of 12.7 d and a biomass concentration of 3-4 g/L (Wang et al., 2014). On the contrary, a significantly lower TP removal (43%) was reported for a A 2 O system operated at a similar SRT (12 d) but lower biomass concentration in the tanks, according to the TSS concentration in the sludge (2.9 g/L) (Lanham et al., 2013). For this application, influent characteristics were almost equal to 253 mg/L for the average COD, 81 mg/L of suspended solids, 43 mg-N/L (average N), 33 mg-N/L (average NH 3 ), 5 mg-P/L (average TP) with a resulting COD:N:P ratio of 51:9:1. ...
... An anoxic SSR was also implemented in the Biodenitro™ process, which consists of two tanks where anoxic and aerobic conditions are alternated. This has the aim of partially degrade the RAS (HRT = 20-30 h) and recirculate the previously fermented stream into the Biodenitro™ tank operating under anoxic conditions (Lanham et al., 2013;Vollertsen et al., 2006). In the two examples reported by Lanham et al. (2013), high TP removal efficiencies, i.e., 94% and 96%, were observed, despite the high differences between the operating conditions, i.e., influent TP concentration equal to 4 and 10 mg/L, influent COD concentration of 198 and 636 mg/L, average flow rate of 49ꞏ10 3 and 17ꞏ10 3 m 3 /d, and SRT maintained at 19 and 30 d, respectively. ...
... This has the aim of partially degrade the RAS (HRT = 20-30 h) and recirculate the previously fermented stream into the Biodenitro™ tank operating under anoxic conditions (Lanham et al., 2013;Vollertsen et al., 2006). In the two examples reported by Lanham et al. (2013), high TP removal efficiencies, i.e., 94% and 96%, were observed, despite the high differences between the operating conditions, i.e., influent TP concentration equal to 4 and 10 mg/L, influent COD concentration of 198 and 636 mg/L, average flow rate of 49ꞏ10 3 and 17ꞏ10 3 m 3 /d, and SRT maintained at 19 and 30 d, respectively. However, since the authors reported that FeCl 3 was added as a polishing step for P removal, it is not possible to determine the contribution of the sole Biodenitro™ configuration, combined with an anaerobic SSR, in the achievement of the overmentioned TP removal efficiencies. ...
Phosphate rocks are an irreplaceable resource to produce fertilizers, but their availability will not be enough to meet the increasing demands of agriculture for food production. At the same time, the accumulation of phosphorous discharged by municipal wastewater treatment plants (WWTPs) is one of the main causes of eutrophication. In a perspective of circular economy, WWTPs play a key role in phosphorous management. Indeed, phosphorus removal and recovery from WWTPs can both reduce the occurrence of eutrophication and contribute to meeting the demand for phosphorus-based fertilizers. Phosphorous removal and recovery are interconnected phases in WWTP with the former generally involved in the mainstream treatment, while the latter on the side streams. Indeed, by reducing phosphorus concentration in the WWTP side streams, a further improvement of the overall phosphorus removal from the WWTP influent can be obtained. Many studies and patents have been recently focused on treatments and processes aimed at the removal and recovery of phosphorous from wastewater and sewage sludge. Notably, new advances on biological and material sciences are constantly put at the service of conventional or unconventional wastewater treatments to increase the phosphorous removal efficiency and/or reduce the treatment costs. Similarly, many studies have been devoted to the development of processes aimed at the recovery of phosphorus from wastewaters and sludge to produce fertilizers, and a wide range of recovery percentages is reported as a function of the different technologies applied (from 10–25% up to 70–90% of the phosphorous in the WWTP influent). In view of forthcoming and inevitable regulations on phosphorous removal and recovery from WWTP streams, this review summarizes the main recent advances in this field to provide the scientific and technical community with an updated and useful tool for choosing the best strategy to adopt during the design or upgrading of WWTPs.
... In this approach, most of the yield coefficients are calculated theoretically through substrate, energy and reducing power balances on well-established biochemical pathways for the processes involved in polyphosphate accumulating organism (PAO) and glycogen accumulating organism (GAO) metabolism. The stoichiometric yields estimated in existing PAO-GAO metabolic models are able to describe a wide range of operational and environmental conditions as reported by several experimental studies (Carvalheira et al., 2014a(Carvalheira et al., , 2014bCarvalho et al., 2007;Lanham et al., 2013;Lu et al., 2006;Oehmen et al., 2005bOehmen et al., , 2005aPijuan et al., 2004Pijuan et al., , 2008Zeng et al., 2003b). The kinetic parameters of these models are the only parameters that require calibration because the metabolic approach mathematically correlates internal cellular reaction rates with observable conversion rates outside the cell to determine an overall stoichiometric reaction for each anaerobic, anoxic and aerobic condition (Lopez-Vazquez et al., 2009;Oehmen et al., 2005cOehmen et al., , 2010bSmolders et al., 1994aSmolders et al., , 1994bZeng et al., 2003a). ...
... Examples of these developments include the description of the activity of various types of PAOs and their competitors, e.g. GAOs, under: i) different operational conditions such as substrate, temperature, pH and dissolved oxygen (DO) (Carvalheira et al., 2014a(Carvalheira et al., , 2014bLopez-Vazquez et al., 2009); ii) different electron acceptors such as oxygen, nitrate and nitrite (Burow et al., 2007;Camejo et al., 2019Camejo et al., , 2016Carvalho et al., 2007;Flowers et al., 2009;Kim et al., 2013;Lanham et al., 2011;McIlroy et al., 2014;Rubio-Rinc on et al., 2019Skennerton et al., 2015;Wang et al., 2008); iii) metabolic shifts, e.g., glycolysis and anaerobic tricarboxylic acid (TCA) cycle, as a function of storage polymer concentrations (Cokro et al., 2017;Lanham et al., 2014Lanham et al., , 2013 as well as the role of these polymers in endogenous processes (Carvalheira et al., 2014c;Lanham et al., 2014;Liu et al., 2017;Lopez et al., 2006;Lu et al., 2007;Stokholm-Bjerregaard, 2016;Vargas et al., 2013). The incorporation of these additional factors into a model will enable the prediction of EBPR behaviour over a wide range of operational and environmental conditions. ...
... For instance, less PHA is produced and more PO 4 is released when the TCA cycle and propionyl-CoA conversion to acetyl-CoA pathways are active instead of glycolysis. In fact, the metabolic shifts between glycolysis and TCA cycle were observed in lab (Zhou et al., 2009) and full-scale plants (Cokro et al., 2017;Lanham et al., 2013;Pijuan et al., 2008), and are supported by recent metagenomics studies (Flowers et al., 2013;Martín et al., 2006;Skennerton et al., 2015) of Accumulibacter organisms. Lanham et al. (2014) demonstrated through the application of metabolic models that lower VFA concentrations in the influent as well as longer aerobic retention times favour the dominance of the TCA metabolism over glycolysis. ...
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.
... The observations under anaerobic conditions lead to question the dominant type of metabolism in full-scale EBPR system and whether one contributes more over the other. There is a scarcity of research on understanding the metabolic distribution over different plants, considering the configuration, microbial diversity and links to operational parameters (Lanham et al., 2013). ...
... An investigation on Rock Creek treatment plant, Missouri, USA, for a conventional A2O and a RAS fermentation process showed no significant difference for Tetrasphasera abundance between the two systems. Moreover, in a benchscale activity test conducted by Lanham et al. (Lanham et al., 2013), no difference was indicated for P-profile between A2O, and RAS fermentation sludge and the glycogen and polyphosphate contents were quite comparable (Dold and Conidi, 2019;Gu, 2019). These inconsistencies rise from disputes on the physiology, metabolic pathways and intracellular compounds of Tetrasphaera (Liu et al., 2019a). ...
Stringent discharge phosphorus limits and rising urge to reach very low effluent total phosphorus concentrations have challenged the available technologies to further remove phosphorus. The significance of Enhanced Biological Phosphorus Removal (EBPR) process may have been overshadowed by the design and operation limitations. These scarcities mainly root back to the lack of knowledge and understanding of fundamental mechanisms, design standards, and operational guidance. Anaerobic biomass fraction design and operation as a primary driving force for biological phosphorus removal process is commonly outweighed by aerobic and total plant sludge retention operation and design criteria. This paper tends to critically review the different perspectives of mainstream and side-stream EBPR processes and to particularly target contrasting views on hydrolysis and fermentation rates as well as anaerobic condition implementation and magnitude. Subsequently, from distinct point of views, knowledge gaps are comprehensively discussed to eventually recognize the advances and drawbacks aimed to reach a sustainable EBPR process.
... Except for days 135~200 when the water temperature experienced a baptism below 10ºC (Fig. 2), the steady COD and TP removal efficiency of 85~96% was observed and the effluent reached the Chinese National Sewage Discharge Standard Class I (grade A) level. The COD (≥86%) and TP (≥88%) removal efficiencies suggested that the activated sludge process (as a classical water treatment method) has a very stable removal effect on COD and TP at low-ratios P/C in wastewater, and its start-up time was not beyond 20 days [35,39]. ...
... Perfect phenomena of anaerobic P release and aerobic P uptake [45] both were observed in the Batch SBR tests. The 4~5 mg P/L of anaerobic P release was obviously at a low level, but its net aerobic P uptake was not low, compared with the EBPR reports [13,16,21,39]. The top 7 microorganisms, which accounted for 70.2~80.6% of the top 20 genera, showed little difference in each phase tank, and the PAOs (Candidatus Accumulibacter, Tetrasphaera and Dechloromonas) plus GAOs (Candidatus Competibacter) accounted for 7.8~12.2 ...
... Another vital PAO is Tetrasphaera, belonging to Actinobacteriota. Tetrasphaera are more abundant than Candidatus Accumulibacter in WWTPs which have lower wastewater temperatures, such as Portugal (8-25 • C) [11] and Denmark (9-18 • C) [3]. ...
... However, Tetrasphaera was detected at relative gene abundances of 0.37-0.58% in our study. Previous studies have implied that the abundance of Tetrasphaera is higher in temperate climates (3.6-28%) [11,26] than in tropical climates (0.23-1.8%) [9] in full-scale WWTPs. It is suggested that Tetrasphaera could make a comparative or even greater contribution to phosphorus removal in Denmark full-scale WWTPs compared with Candidatus Accumulibacter [10]. ...
Bacterial diversity and community composition are of great importance in wastewater treatment; however, little is known about the diversity and community structure of bacteria in tropical municipal wastewater treatment plants (WWTPs). Therefore, in this study, activated sludge samples were collected from the return sludge, anaerobic sludge, anoxic sludge, and aerobic sludge of an A2O WWTP in Haikou, China. Illumina MiSeq high-throughput sequencing was used to examine the 16S ribosomal RNA (rRNA) of bacteria in the samples. The microbial community diversity in this tropical WWTP was higher than in temperate, subtropical, and plateau WWTPs. Proteobacteria, Bacteroidota, Patescibacteria, and Chloroflexi were the dominant phyla. Nitrification bacteria Nitrosomonas, and Nitrospira were also detected. Tetrasphaera, instead of Candidatus Accumulibacter, were the dominant polyphosphate accumulating organisms (PAOs), while, glycogen accumulating organisms (GAOs), such as Candidatus Competibacter and Defluviicoccus were also detected. The bacterial community functions predicted by PICRUSt2 were related to metabolism, genetic information processing, and environmental information processing. This study provides a reference for the optimization of tropical municipal WWTPs.
... In the following aerobic phage, GAOs consume PHAs to synthesize glycogen for the growth (Zhang et al. 2008). However, some studies have reported that GAOs exit in full-scale EBPR plants and not affect the phosphorus removal efficiency (Lanham et al. 2013;Nielsen et al. 2019). Therefore, the biological characteristics of GAOs need further explore. ...
... In this study, Candidatus Contendobacter was the dominant GAO in our system rather than Candidatus Competibacter universally reported by many studies (Lanham et al. 2013;Ong et al. 2014). Candidatus Competibacter was the most common type of GAOs in both lab-scale reactors and full-scale wastewater biological treatment facilities (Nielsen et al. 2019;Yuan et al. 2020). ...
Here a stable glycogen accumulating organisms (GAOs) system was operated by anaerobic–aerobic mode in the sequencing batch reactor. We focused on the metabolic mechanisms of PHAs storage from GAOs. Our system showed the classic characteristic of glycogen accumulating metabolism (GAM). Glycogen consumption was followed by acetic acid uptake to synthesize poly-β-hydroxyalkanoates (PHAs) during the anaerobic period, and glycogen was synthesized by PHAs degradation in the aerobic stage. Microbial community structure indicated that Candidatus Contendobacter was the most prevalent GAOs. We found that the ethylmalonyl-CoA (EMC) pathway was the crucial pathway supplying the core substance propionyl-CoA for poly-β-hydroxyvalerate (PHV) synthesis in Candidatus Contendobacter. All genes in EMC pathway were mainly located in Candidatus Contendobacter by gene source analysis. The key genes expression of EMC pathway increased with Candidatus Contendobacter enrichment, further validating that propionyl-CoA was synthesized by Candidatus Contendobacter predominantly via EMC pathway. Our work revealed the novel mechanisms underlying PHV synthesis through EMC pathway and further improved the intercellular storage metabolism of GAOs.
... When PHA consumption ceased, glycogen production ceased and was then consumed, likely due to metabolic energy requirements for cell maintenance. Aerobic glycogen consumption after PHA reaches residual levels was already observed in PAOs by Lopez et al. (2006), Lanham et al. (2013a) and Carvalheira et al. (2014). ...
... The results suggest that the P uptake was mainly carried out by Accumulibacter related PAO, and accumulated as poly-P, while PHA is available. On the other hand, after PHA reaches residual levels, P removal is mainly carried out by microalgae/photosynthetic microorganisms, or other PAOs that are not PHA dependent (Lanham et al., 2013a). Microalgae and cyanobacteria both use P for growth and, in addition, cyanobacteria can accumulate it as internal poly-P granules, contributing to excess P removal beyond growth requirements (Brown and Shilton, 2014;Carvalho et al., 2018;Powell et al., 2011). ...
... When PHA consumption ceased, glycogen production ceased and was then consumed, likely due to metabolic energy requirements for cell maintenance. Aerobic glycogen consumption after PHA reaches residual levels was already observed in PAOs by Lopez et al., (2006), Lanham et al., (2013a) and Carvalheira et al., (2014.) The results suggest that the P uptake was mainly carried out by Accumulibacter related PAO, and accumulated as poly-P, while PHA is available. ...
... The results suggest that the P uptake was mainly carried out by Accumulibacter related PAO, and accumulated as poly-P, while PHA is available. On the other hand, after PHA reaches residual levels, P removal is mainly carried out by microalgae/ photosynthetic microorganisms, or other PAOs that are not PHA dependent (Lanham et al., 2013a). Microalgae and cyanobacteria both use P for growth and, in addition, cyanobacteria can accumulate it as internal poly-P granules, contributing to excess P removal beyond growth requirements (Brown and Shilton, 2014;Carvalho et al., 2018;Powell et al., 2011). ...
Conventional wastewater treatment technologies for biological nutrient removal (BNR) are highly dependent on aeration for oxygen supply, which represents a major operational cost of the process. Recently, phototrophic enhanced biological phosphorus removal (photo-EBPR) has been suggested as an alternative system for phosphorus removal, based on a consortium of photosynthetic microorganisms and chemotrophic bacteria, eliminating the need for costly aeration. However, wastewater treatment plants must couple nitrogen and phosphorus removal to achieve discharge limits. For this reason, a new microalgae-bacterial based system for phosphorus and nitrogen removal is proposed in this work. The photo-BNR system studied here consists of a sequencing batch reactor operated with dark anaerobic, light aerobic, dark anoxic and idle periods, to allow both N and P removal. Results of the study show that the photo-BNR system was able to remove 100% of the 40 mg N/L of ammonia fed to the reactor and 94 ± 3% of the total nitrogen (Influent COD:N ratio of 300:40, similar to domestic wastewater). Moreover, an average of 25 ± 9.2 mg P/L was simultaneously removed in the photo-BNR tests, representing the P removal capacity of this system, which exceeds the level of P removal required from typical domestic wastewater. Full ammonia removal was achieved during the light phase, with 67 ± 5% of this ammonia being assimilated by the microbial culture and the remaining 33 ± 5% being converted into nitrate. The assimilated P corresponded to 2.8 ± 0.23 mg P/L, which only represented, approximately, 1/9 of the P removal capacity of the system. Half of the nitrified ammonia was subsequently denitrified during the dark anoxic phase (50 ± 24%). Overall, the photo-BNR system represents the first treatment alternative for N and P from domestic wastewater with no need of mechanical aeration or supplemental carbon addition, representing an alternative low-energy technology of interest.
... When PHA consumption ceased, glycogen production ceased and was then consumed, likely due to metabolic energy requirements for cell maintenance. Aerobic glycogen consumption after PHA reaches residual levels was already observed in PAOs by Lopez et al., (2006), Lanham et al., (2013a) and Carvalheira et al., (2014.) The results suggest that the P uptake was mainly carried out by Accumulibacter related PAO, and accumulated as poly-P, while PHA is available. ...
... The results suggest that the P uptake was mainly carried out by Accumulibacter related PAO, and accumulated as poly-P, while PHA is available. On the other hand, after PHA reaches residual levels, P removal is mainly carried out by microalgae/ photosynthetic microorganisms, or other PAOs that are not PHA dependent (Lanham et al., 2013a). Microalgae and cyanobacteria both use P for growth and, in addition, cyanobacteria can accumulate it as internal poly-P granules, contributing to excess P removal beyond growth requirements (Brown and Shilton, 2014;Carvalho et al., 2018;Powell et al., 2011). ...
... Based on studies the main pathway utilized by PAOs for glycolysis is ED pathway (Maurer et al., 1997), (Smolders et al., 1995), (Wen-Tso et al., 1996), where two ATP are produced through glycogen conversion to pyruvate which is further transformed to Acetyl-CoA or Propionyl-CoA (Hesselmann et al., 2000). However several finding have indicated the dependence of PAO anaerobic metabolism on external and internal factors in utilizing the TCA cycle or glycolysis pathway as a source for reducing equivalent generation (Lanham et al., 2013). In an EBPR system, PAOs are required to be prepared for taking up either oxidized or reduced organic substances without disrupting the redox balance in the cell. ...
... Moreover, the models only indicate a single anaerobic/aerobic cycle for a single cell (Hood and Randall, 2001). Yet, investigations on full-scale EBPR systems are scarce comparing to lab-scale experiments, therefore this projects the various upcoming challenges on handling number of parameters in complex and dynamic process (Lanham et al., 2013) since single-substrate studies don't provide proper image of feeding system in real WWTPs (Ciggin et al., 2013). Even with a hint on various substrates, cycles and environmental factors, the implication of factors such as effect of additional exogenous and endogenous nutrients on polymeric substance storage and the nature and composition of the organic substrate is lacking in the emerging outlook of EBPR (Hood and Randall, 2001). ...
Enhanced biological phosphorus removal (EBPR) is one of the most promising technologies as an economical and environmentally sustainable technique for removal of phosphorus from wastewater (WW). However, with high capacity of EBPR, insufficient P-removal is a major yet common issue of many full-scale wastewater treatment plants (WWTP), due to misinterpreted environmental and microbial disturbance. By developing a rather extensive understanding on biochemical pathways and metabolic models governing the anaerobic and aerobic/anoxic processes; the optimal operational conditions, environmental changes and microbial population interaction are efficiently predicted.
Therefore, this paper critically reviews the current knowledge on biochemical pathways and metabolic models of phosphorus accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) as the most abundant microbial populations in EBPR process with an insight on the effect of available carbon source types in WW on phosphorus removal performance. Moreover, this paper critically assesses the gaps and potential future research in metabolic modeling area. With all the developments on EBPR process in the past few decades, there is still lack of knowledge in this critical sector. This paper hopes to touch on this problem by gathering the existing knowledge and to provide farther insights on the future work onto chemical transformations and metabolic strategies in different conditions to benefit the quantitative model as well as WWTP designs.
... Different predictions were obtained with the ASM-inCTRL model, where PAOs averaged 16.0% ± 5.8% and 23.0% ± 3.5% in the low and high season ( Fig. 5 -B), respectively. However, the predictions obtained with the META-ASM are in the same range as the microbial characterisation performed by Lanham et al. (2013) on this plant using quantitative fluorescence in situ hybridisation (qFISH). The authors observed that the percentages of Accumulibacter PAOs and Competibacter-lineage plus Defluviicoccus vanus GAOs were 3.2% and 0.3%, respectively. ...
... Figure D.1 (see Appendix D) shows that the preferred pathways for the origin of reducing power in the anaerobic metabolism of PAOs were the tricarboxylic acid (TCA) cycle, for the uptake of acetate and butyrate, and the conversion of propionyl-CoA to acetyl-CoA, for the uptake of propionate and valerate. The use of these pathways by PAOs is common in full-scale EBPR plants with low influent volatile fatty acid (VFA) concentrations and long aerobic retention times ( Cokro et al., 2017 ;Lanham et al., 2013 ;Zhou et al., 2009 ). In this plant, the estimated VFA concentration in the influent and the aerobic HRT averaged 20 ± 5 g COD. ...
Enhanced biological phosphorus removal (ebpr) Full-scale wastewater treatment plant (wwtp) Metabolic modelling Polyphosphate accumulating organisms (PAOs) and process optimisation a b s t r a c t 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.
... Puy2 floc was the only sample from an EBPR plant with no Accumulibacter detected. Relative Accumulibacter gene abundances of 0.2e6% had been reported for full-scale EBPR plants (Coats et al., 2017;Lanham et al., 2013;Qiu et al., 2019;Stokholm-Bjerregaard et al., 2017). Except HE1 and BM, Tetrasphaera was detected at all EBPR plants at relative gene abundances of 0.04e1.22%. ...
... Except HE1 and BM, Tetrasphaera was detected at all EBPR plants at relative gene abundances of 0.04e1.22%. Previous studies had reported Tetrasphaera abundances of 3.6e28% in full-scale plants in temperate climate (Lanham et al., 2013;Stokholm-Bjerregaard et al., 2017) and 0.23e1.8% in tropical climate (Qiu et al., 2019). The DNA extraction method used here might have caused underestimation of the Tetrasphaera abundances due to a shorter bead beating time utilized (25 s at 4 m/s) than the recommended protocols (160 s at 6 m/s) for extracting DNA from Gram-positive Actinobacteria (e.g. ...
To date, high performance of full-scale aerobic granular sludge (AGS) technology has been demonstrated on a global scale. Its further integration with existing continuous flow activated sludge (CFAS) treatment plants is the next logical step. All granular sludge reactors operated in sequencing batch reactors (SBR) mode with anaerobic feeding conditions select for growth of phosphorus and glycogen accumulating organisms (PAO and GAO, respectively), which are known to enhance sludge settling characteristics. Therefore, we hypothesized that AGS are commonly present at full-scale CFAS processes with enhanced biological phosphorus removal (EBPR) and low sludge volume index (SVI). This hypothesis was confirmed at 13 EBPR plants, where granules were found present (at plants where SVI was lower than 100 ml/g) with a strong correlation between high granule abundance and low SVI. A wide range of granule abundance was found among the plants, ranging from 0.5% to as high as 80%. Evaluations of the EBPR plant process configurations showed that high granule abundances may be related to selector design features such as high anaerobic food to mass (F/M) ratios, unmixed in-line fermentation, and high influent soluble COD fraction. Granules were also observed at a non-EBPR plant with an aerobic selector receiving high F/M feeds. Quantitative PCR and 16S rRNA gene sequencing analyses revealed higher relative gene abundance of Accumulibacter PAO and Competibacter GAO in the granules over flocs, as well as a correlation between granule abundance and some possible EPS producers such as Flavobacterium and Competibacter. Our results indicated that process configurations that select for slow-growing or EPS-producing heterotrophs play an important role for granule formation in full-scale CFAS systems as previously shown in SBR configurations.
... However, in both reactors, there was no significant trend/change in glycogen content (Table .1). It suggests that the reduction equivalent required for carbon utilization was primarily drawn from TCA-cycle instead of glycolysis (Lanham et al., 2013). The putative metabolic study testified this hypothesis. ...
... Enzymes/proteins specific to the glycolysis pathway are found limited, whereas essential enzymes required for acetate uptake, activation into acetyl-CoA, and subsequent TCA cycle were abundant. This observation suggests the TCA cycle as the primary carbon uptake mechanism of the dominant DNPAOs in SBR (Jena et al., 2016;Lanham et al., 2013). ...
... During the PHA synthesis process in the anaerobic phase, intracellularly stored glycogen could provide a significant portion of the energy for PAOs/GAOs through glycolysis [38]. Therefore, higher glycogen utilization to acetate uptake ratio (Glyc/HAc) would be related to increased relative abundance of GAOs or increased use of glycolytic pathways [39]. In this study, Glyc/HAc ratio decreased from 0.21 C-mol/C-mol in R0 to 0.14 C-mol/C-mol in R3, indicating that excessive PLA MPs may reduce glycogen utilization by PAOs, which is correlated with PHA/HAc ratio results, while the causes need further study. ...
A large number of microplastics (MPs) have been found in various stages of wastewater treatment plants, which may affect the functional microbial activity in activated sludge and lead to unstable pollutant removal performance. In this study, the effects of different concentrations of polylactic acid microplastics (PLA MPs) on system performance, nitrification and phosphorus (P) removal activities, and extracellular polymeric substances (EPS) were evaluated. The results showed that under the same influent conditions, low concentrations (50 particles/(g TS)) of PLA MPs had no significant effect on effluent quality. The average removal efficiencies of chemical oxygen demand, phosphate and ammonia were all above 80%, and the average removal efficiencies of total nitrogen remained above 70%. High concentrations (200 particles/(g TS)) of PLA MPs inhibited the activities of polyphosphate accumulating organisms (PAOs) and nitrifying bacteria. The specific anaerobic P release rate decreased from 37.7 to 23.1 mg P/(g VSS·h), and the specific aerobic P uptake rate also decreased significantly. The specific ammonia oxidation rate decreased from 0.67 to 0.34 mg N/(g VSS·h), while the change in specific nitrite oxidation rate was not significant. The dosing of PLA MPs decreased the total EPS and humic acid content. As the concentration of PLA MPs increased, microbial community diversity increased. The relative abundance of potential PAOs (i.e., Acinetobacter) increased from 0.08% to 12.57%, while the relative abundance of glycogen accumulating organisms (i.e., Competibacter and Defluviicoccus) showed no significant changes, which would lead to improved P removal performance. The relative abundance of denitrifying bacteria (i.e., Pseudomonas) decreased from 95.43% to 58.98%, potentially contributing to the decline in denitrification performance.
... Contrasting results exist in the literature regarding the effectiveness of S2EBPR vs conventional EBPR processes. Some studies have shown that the S2EBPR configuration has improved the P removal performance and stability compared to traditional EBPR configurations [22,31,49]. However, it has also been reported that S2EBPR showed high fluctuations of effluent P (0.6 ± 1.0 mgP/L) [46,49]. ...
... The differential PAO activities observed in the batch tests with different carbon sources (Table 2) suggest that the sludge may contain a large number of Competibacter GAOs that primarily utilise acetate (as shown in Figure 4), which to some extent affects the uptake of acetate by Accumulibacter PAOs and thus reduces the corresponding EBPR activity. However, compared to those in other studies [29,72,73], the P/HAc and P/HPr ratios in this study were consistently high, indicating that the presence of GAOs did not have a significant negative effect on the EBPR activity. In addition, the complex substrate (casein acid hydrolysate) in the influent was also available to other PAOs (e.g., Tetrasphaera) [74,75] and thus contributed to the overall EBPR activity. ...
Complex and high levels of various pollutants in high-strength wastewaters hinder efficient and stable biological nutrient removal. In this study, the changes in pollutant removal performance and microbial community structure in a laboratory-scale anaerobic/aerobic sequencing batch reactor (SBR) treating simulated pre-fermented high-strength wastewater were investigated under different influent loading conditions. The results showed that when the influent chemical oxygen demand (COD), total nitrogen (TN), and orthophosphate (PO43−-P) concentrations in the SBR increased to 983, 56, and 20 mg/L, respectively, the COD removal efficiency was maintained above 85%, the TN removal efficiency was 64.5%, and the PO43−-P removal efficiency increased from 78.3% to 97.5%. Partial nitrification with simultaneous accumulation of ammonia (NH4+-N) and nitrite (NO2−-N) was observed, which may be related to the effect of high influent load on ammonia- and nitrite-oxidising bacteria. The biological phosphorus removal activity was higher when propionate was used as the carbon source instead of acetate. The relative abundance of glycogen accumulating organisms (GAOs) increased significantly with the increase in organic load, while Tetrasphaera was the consistently dominant polyphosphate accumulating organism (PAO) in the reactor. Under high organic loading conditions, there was no significant PAO–GAO competition in the reactor, thus the phosphorus removal performance was not affected.
... A number of proteins that were identified throughout the entire granule structure were associated to the glycolysis pathway, including phosphoglycerate kinase, cytosolic glyceraldehyde 3-phosphate dehydrogenases and acetate kinase. In activated sludge systems, P removal performance has been observed to be better in full-scale WWTPs with higher glycolysis activity (Lanham et al., 2013). Furthermore, nitrile hydratase subunit alpha, which catalyzes the hydration of various nitrile compounds to the corresponding amides, was observed throughout the entire granule except for the outermost layers. ...
Most bacteria live in microbial assemblages like biofilms and granules, and each layer of these assemblages provides a niche for certain bacteria with specific metabolic functions. In this study, a gentle (non-destructive) extraction approach based on a cation exchange resin and defined shear was employed to gradually disintegrate biomass and collect single layers of aerobic granules from a full-scale municipal wastewater treatment plant. The microbial community composition of granule layers was characterized using next-generation sequencing (NGS) targeting the 16S rRNA gene, and protein composition was investigated using metaproteomics. The chemical composition of eroded layers was explored using Fourier Transformed Infrared Spectroscopy. On the surface of the granules, the microbial structure (flocculation-supporting Nannocystis sp.) as well as composition of extracellular polymers (extracellular DNA) and proteome (chaperonins and binding proteins) favored microbial aggregation. Extracellular polymeric substances in the granules were composed of mostly proteins and EPS-producers, such as Tetrasphaera sp. and Zoogloea sp., were evenly distributed throughout the granule structure. The interior of the granules harbored several denitrifiers (e.g., Thauera sp.), phosphate-accumulating denitrifiers (Candidatus Accumulibacter, Dechloromonas sp.) and nitrifiers (Candidatus Nitrotoga). Proteins associated with glycolytic activity were identified in the outer and middle granule layers, and proteins associated with phosphorus conversions, in the deeper layers. In conclusion, the use of an existing cation-exchange resin for gradual biomass disintegration, combined with NGS and metaproteomic analysis was demonstrated as a promising approach for simultaneously investigating the identity and functions of microbes in multilayered biofilm structures.
... In fact, Tetrasphaera is frequently identified with higher abundance than Ca. Accumulibacter in the EBPR system, reaching 30% of the total biomass [20][21][22][23]. Therefore, various researchers have now turned their interest into Tetrasphaera. ...
Free ammonia (FA) has been proposed to inhibit phosphorus (P) removal efficiency and reconfigures the microbial community in the enhanced biological phosphorus removal (EBPR) systems. However, in response to FA exposure, the microbial kinetics and the functional bacteria belonging to polyphosphate accumulating organisms (PAOs) i.e., Tetrasphaera and Candidatus Accumulibacter within the mixed communities are generally ignored. This work was conducted to characterize the effects of FA on the biological processes of P-release and its uptake, and the virtual role of PAOs in EBPR systems. Experimental data demonstrated that the FA strongly inhibited the P-release and uptake rates which was described well by Hellinga and Vadivelu models, and the determined R² in the anaerobic and aerobic period were about 0.91 and 0.96. Moreover, the abundance of key microbial communities differed significantly with various FA concentrations. Structural equation model (SEM) revealed that FA was positively associated with Ca. Accumulibacter (path coeff. = 0.30; P = 0.13) and Ca. Competibacter (path coeff. = 0.43; P < 0.05), while it was negatively associated with Tetrasphaera (path coeff. = -0.45; P < 0.001). Furthermore, Tetrasphaera was found to strongly and positively drive the rates of P-release and uptake, and was confirmed as the only virtual biomarker for indicating the response of EBPR performance towards FA inhibition. Two hypothetical mechanisms indicated that Ca. Accumulibacter under higher FA concentration could not synthesize PHA to support the EBPR process. In addition, the release of P was hindered due to the FA toxicity on the urea cycle of Tetrasphaera microbial cells. This work gives comprehensive and deep insight into the effect of FA on the EBPR system and provides a useful guide for process optimization.
... In this study, Gly/VFA ratio in both stages was over 0.95 and close to the GAO model, indicating the dominant involvement of GAO metabolism. Meanwhile, considering the high abundance of known PAOs in the system revealed by sequencing, this high Gly/VFA ratio might also be associated with the preferential use of glycolysis pathway over TCA cycle for PAOs, which was observed in side-stream hydrolysis and is considered more efficient through producing additional PHA and releasing less phosphate per substrate uptake, thus potentially beneficial for EBPR (Lanham et al., 2013a. 19 The anaerobic PHA generation to VFA uptake ratios (PHA/VFA) in stage 2 and stage 3 was 0.65 C-mol/C-mol and 0.23 C-mol/C-mol, respectively, both of which were much lower than those theoretical values predicted for either PAO or GAO models (1.33 and 1.85 respectively). ...
The enhanced biological phosphorus removal (EBPR) has been widely applied in treating domestic wastewater, while the performance on high-strength P wastewater is less investigated and the feasibility of coupling with short-cut nitrogen removal process remains unknown. This study first achieved the simultaneous high-efficient P removal and stable nitrite accumulation in one sequencing batch reactor for treating the synthetic digested manure wastewater. The average effluent P could be down to 0.8 ± 1.0 mg P/L and the P removal efficiency was 99.5 ± 0.8%. Candidatus Accumulibacter was the dominant polyphosphate accumulating organism (PAO) with the relative abundance of 14.2-33.1% in the reactor. Examination of the micro-diversity of Candidatus Accumulibacter using 16s rRNA gene-based oligotyping analysis revealed one unique Accumulibacter oligotype that different from the conventional system, which accounted for 64.2-87.9% of the total Accumulibacter abundance. The presence of high-abundant glycogen accumulating organisms (GAO) (15.6-40.3%, Defluviicoccus and Candidatus Competibacter) did not deteriorate the EBPR performance. Moreover, nitrite accumulation happened in the system with the effluent nitrite up to 20.4 ± 6.4 mg N/L and the nitrite accumulation ratio was nearly 100% maintained for 140 days (420 cycles). Nitrosomonas was the dominant ammonia-oxidizing bacteria with relative abundance of 0.3-2.4% while nitrite-oxidizing bacteria were almost undetected (<0.1%). The introduction of extended anaerobic phase and high volatile fatty acid concentrations were proposed to be the potential selector forces to promote partial nitrification. This is the first study that combined EBPR with nitrite-accumulation for digested manure wastewater treatment, and it provided new sights in strategies to combine the EBPR and short-cut nitrogen removal via nitrite to achieve simultaneous nitrogen and phosphorus removal.
... The P-removal in conventional biological wastewater treatment systems relies upon assimilation, as a direct reflection of the demand for the nutrient exerted by the synthesis of new biomass. The biological removal can also be intensified by exploiting the capacity of the heterotrophic bacteria dubbed Phosphate-Accumulating Organisms (PAOs) to accumulate the excess cellular phosphate in the form of polyphosphate grains [Lanham et al., 2013]. ...
Biological methods of removing phosphorus compounds from wastewater as applied currently at treatment plants may no longer be regarded as sufficient. They can therefore be augmented by physico-chemical methods raising the efficiency of the wastewater treatment system. Indeed, almost all large urban wastewater treatment plants practise precipitation of phosphorus using salts of iron and aluminum in the form of chemical coagulants. The search is nevertheless ongoing for new ways of assisting with the removal of the biogenic element from wastewater, e.g. by dosing bentonites, fly ash and post-technological sludge from water treatment stations, or even unconventional organic sorbents such as rice husks. A further unconventional material in P-removal from wastewater may take the form of powdered mineral materials. Against that background, the work presented here offers the results of laboratory-scale trials on P-removal using brick dust and powdered keramsite (expanded clay).
The design of biomass beds and feeding conditions is crucial for an optimal loading, microbial selection, and biological nutrient removal (BNR) in granular sludge processes. Polyphosphate- (PAOs) and glycogen-accumulating organisms (GAOs) compete for organic substrates during anaerobic phases of sequencing batch reactors (SBRs). System analysis and mathematical modeling was engaged to elucidate the hydraulic transport pattern during the up-flow feeding of wastewater in granular sludge beds and the impact of environmental conditions (pH, temperature) on PAO/GAO selection. Tracer experiments across the granular sludge column displayed a plug-flow regime with dispersion under both rapid (9 m h−1) and slow (0.9 m h−1) up-flow feedings. Fill-and-draw phases can be implemented in SBRs to feed the influent at foot and extract the treated effluent at the top. Metabolic simulations disclosed the effects of feeding duration, pH, and temperature on PAO/GAO competition for the uptake of volatile fatty acids (VFAs) under anaerobic slow feeding. Feeding time should be set in function of conversions of intracellular storage polymers that do not move with the liquid phase. The anaerobic metabolism of PAOs relies on both polyphosphate and glycogen as energy and reducing equivalents to store VFAs as poly-β-hydroxyalkanoates. PAOs withstand a feed period twice as long as GAOs in which only glycogen serves as pool of electrons and energy. Applying alkaline conditions (pH 7.25–8.0) by, e.g., dosing lime in the feed selects for PAOs independent of temperature (10–30 °C). Temperature determines the bed height necessary for optimal contact between wastewater and biomass in a full anaerobic selector for PAOs, and BNR. Almost twice higher beds are needed at 10 °C than at 20 °C for a complete anaerobic uptake of VFAs. This hydraulic-metabolic model sustains the design of bed geometries and feeding conditions to manage the microbial resource in granular sludge.
Phosphorous (P) removal in wastewater treatment is essential to prevent eutrophication in water bodies. Side‐stream enhanced biological phosphorous removal (S2EBPR) is utilized to improve biological P removal by recirculating internal streams within a side‐stream reactor to generate biodegradable carbon (C) for polyphosphate accumulating organisms (PAOs). In this study, a full‐scale S2EBPR system in a water resource recovery facility (WRRF) was evaluated for 5 months. Batch experiments revealed a strong positive correlation ( r = 0.91) between temperature and C consumption rate (3.56–8.18 mg‐COD/g‐VSS/h) in the system, with temperature ranging from 14°C to 18°C. The anaerobic P‐release to COD‐uptake ratio decreased from 0.93 to 0.25 mg‐P/mg‐COD as the temperature increased, suggesting competition between PAOs and other C‐consumers, such as heterotrophic microorganisms, to uptake bioavailable C. Microbial community analysis did not show a strong relationship between abundance and activity of PAO in the tested WRRF. An assessment of the economic feasibility was performed to compare the costs and benefits of a full scale WRRF with and without implementation of the S2EBPR technology. The results showed the higher capital costs required for S2EBPR were estimated to be compensated after 5 and 11 years of operation, respectively, compared to chemical precipitation and conventional EBPR. The results from this study can assist in the decision‐making process for upgrading a conventional EBPR or chemical P removal process to S2EBPR.
Practitioner Points
Implementation of S2EBPR presents adaptable configurations, exhibiting advantages over conventional setups in addressing prevalent challenges associated with phosphorous removal.
A full‐scale S2EBPR WRRF was monitored over 5 months, and activity tests were used to measure the kinetic parameters.
The seasonal changes impact the kinetic parameters of PAOs in the S2EBPR process, with elevated temperatures raising the carbon demand.
PAOs abundance showed no strong correlation with their activity in the full‐scale S2EBPR process in the tested WRRF.
Feasibility assessment shows that the benefits from S2EBPR operation can offset upgrading costs from conventional BPR or chemical precipitation.
A large number of microplastics (MPs) have been found in various stages of wastewater treatment plants, which may affect the functional microbial activity in activated sludge and lead to unstable pollutant removal performance. In this study, the effects of different concentrations of polylactic acid microplastics (PLA MPs) on system performance, nitrification and phosphorus (P) removal activities, and extracellular polymeric substances (EPS) were evaluated. The results showed that under the same influent conditions, low concentrations (50 particles/(g TS)) of PLA MPs had no significant effect on effluent quality. The average removal efficiencies of chemical oxygen demand, phosphate, and ammonia were all above 80%, and the average removal efficiencies of total nitrogen remained above 70%. High concentrations (200 particles/(g TS)) of PLA MPs inhibited the activities of polyphosphate-accumulating organisms (PAOs) and nitrifying bacteria. The specific anaerobic P release rate decreased from 37.7 to 23.1 mg P/(g VSS·h), and the specific aerobic P uptake rate also significantly decreased. The specific ammonia oxidation rate decreased from 0.67 to 0.34 mg N/(g VSS·h), while the change in the specific nitrite oxidation rate was not significant. The dosing of PLA MPs decreased the total EPS and humic acid content. As the concentration of PLA MPs increased, microbial community diversity increased. The relative abundance of potential PAOs (i.e., Acinetobacter) increased from 0.08 to 12.57%, while the relative abundance of glycogen-accumulating organisms (i.e., Competibacter and Defluviicoccus) showed no significant changes, which would lead to improved P removal performance. The relative abundance of denitrifying bacteria (i.e., Pseudomonas) decreased from 95.43 to 58.98%, potentially contributing to the decline in denitrification performance.
Side-stream enhanced biological phosphorus removal process (S2EBPR) has been demonstrated to improve performance stability and offers a suite of advantages compared to conventional EBPR design. Design and optimization of S2EBPR require modification of the current EBPR models that were not able to fully reflect the metabolic functions of and competition between the polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) under extended anaerobic conditions as in the S2EBPR conditions. In this study, we proposed and validated an improved model (iEBPR) for simulating PAO and GAO competition that incorporated heterogeneity and versatility in PAO sequential polymer usage, staged maintenance-decay, and glycolysis-TCA pathway shifts. The iEBPR model was first calibrated against bulk batch testing experiment data and proved to perform better than the previous EBPR model for predicting the soluble orthoP, ammonia, biomass glycogen, and PHA temporal profiles in a starvation batch testing under prolonged anaerobic conditions. We further validated the model with another independent set of anaerobic testing data that included high-resolution single-cell and specific population level intracellular polymer measurements acquired with single-cell Raman micro-spectroscopy technique. The model accurately predicted the temporal changes in the intracellular polymers at cellular and population levels within PAOs and GAOs, and further confirmed the proposed mechanism of sequential polymer utilization, and polymer availability-dependent and staged maintenance-decay in PAOs. These results indicate that under extended anaerobic phases as in S2EBPR, the PAOs may gain competitive advantages over GAOs due to the possession of multiple intracellular polymers and the adaptive switching of the anaerobic metabolic pathways that consequently lead to the later and slower decay in PAOs than GAOs. The iEBPR model can be applied to facilitate and optimize the design and operations of S2EBPR for more reliable nutrient removal and recovery from wastewater.
Enhanced biological phosphorus removal (EBPR) is an economical and sustainable process for phosphorus removal from wastewater. Despite the widespread application of EBPR for low-strength domestic wastewater treatment, limited investigations have been conducted to apply EBPR to the high-strength wastewaters, particularly, the integration of EBPR and the short-cut nitrogen removal process in the one-stage system remains challenging. Herein, we reported a novel proof-of-concept demonstration of integrating EBPR and nitritation (oxidation of ammonium to nitrite) in a one-stage sequencing batch reactor to achieve simultaneous high-strength phosphorus and short-cut nitrogen removal. Excellent EBPR performance of effluent 0.8 ± 1.0 mg P/L and >99% removal efficiency was achieved fed with synthetic high-strength phosphorus wastewater. Long-term sludge acclimation proved that the dominant polyphosphate accumulating organisms (PAOs), Candidatus Accumulibacter, could evolve to a specific subtype that can tolerate the nitrite inhibition as revealed by operational taxonomic unit (OTU)-based oligotyping analysis. The EBPR kinetic and stoichiometric evaluations combined with the amplicon sequencing proved that the Candidatus Competibacter, as the dominant glycogen accumulating organisms (GAOs), could well coexist with PAOs (15.3-24.9% and 14.2-33.1%, respectively) and did not deteriorate the EBPR performance. The nitrification activity assessment, amplicon sequencing, and functional-based gene marker quantification verified that the unexpected nitrite accumulation (10.7-21.0 mg N/L) in the high-strength EBPR system was likely caused by the nitritation process, in which the nitrite-oxidizing bacteria (NOB) were successfully out-selected (<0.1% relative abundance). We hypothesized that the introduction of the anaerobic phase with high VFA concentrations could be the potential selection force for achieving nitritation based on the literature review and our preliminary batch tests. This study sheds light on developing a new feasible technical route for integrating EBPR with short-cut nitrogen removal for efficient high-strength wastewater treatment.
The integration of biological phosphorus removal (bio‐P) and shortcut nitrogen removal (SNR) processes is challenging because of the conflicting demands on influent carbon: SNR allows for upstream carbon diversion, but this reduction of influent carbon (especially volatile fatty acids [VFAs]) prevents or limits bio‐P. The objective of this study was to achieve SNR, either via partial nitritation/anammox (PNA) or partial denitrification/anammox (PdNA), simultaneously with biological phosphorus removal in a process with upstream carbon capture. This study took place in a pilot scale A/B process with a sidestream bio‐P reactor and tertiary anammox polishing. Despite low influent rbCOD concentrations from the A‐stage effluent, bio‐P occurred in the B‐stage thanks to the addition of A‐stage WAS fermentate to the sidestream reactor. Nitrite accumulation occurred in the B‐stage via partial denitrification and partial nitritation (NOB out‐selection), depending on operational conditions, and was removed along with ammonia by the tertiary anammox MBBR, with the ability to achieve effluent TIN less than 2 mg/L.
Practitioner Points
A sidestream reactor with sufficient fermentate addition enables biological phosphorus removal in a B‐stage system with little‐to‐no influent VFA.
Enhanced biological phosphorus removal is not inhibited by intermittent aeration and is stable at a wide range of process SRTs.
Partial nitritation and partial denitrification are viable routes to produce nitrite within an A/B process with sidestream bio‐P, for downstream anammox in a polishing MBBR.
In this study, a lab-scale continuous flow side-stream enhanced biological phosphorus (P) removal (S2EBPR) reactor was operated for 247 days treating synthetic wastewater with influent carbon to phosphorus (C/P) ratio of 25.0 g COD/g P and influent PO43-P of 7.4 ± 0.3 mg P/L. The effect of the return activated sludge (RAS) diversion ratio on S2EBPR reactor was investigated by comparing P removal performance, microbial activity, and community structure. The results showed that the RAS diversion ratio of 8.0%, by yielding a side-stream sludge retention time (SRTSS) of ∼60 h, resulted in the lowest effluent PO43-P concentration of 0.5 ± 0.3 mg P/L. The results of in situ process profiles and ex situ P release and uptake batch tests under different RAS diversion conditions showed that the more anaerobic P release was obtained in the side-stream reactor, the higher the P removal efficiency and EBPR activity were achieved. The stoichiometric ratios observed in EBPR activity tests indicated a polyphosphate accumulating organisms (PAOs) metabolism mainly dependent on the glycolysis pathway. The results of microbial ecology analysis revealed that the optimized SRTSS would give a competitive advantage to PAOs in the S2EBPR process. By obtaining statistically reliable results, this study would provide guidance for wastewater treatment plants to achieve optimal P removal performance in S2EBPR configuration.
High energy consumption is impedimental for eliminating refractory organics in wastewater by current technologies. Herein, we develop an efficient self-purification process for actual non-biodegradable dyeing wastewater at pilot scale, using N-doped graphene-like (CN) complexed Cu-Al2O3 supported Al2O3 ceramics (HCLL-S8-M) fixed-bed reactor without additional input. About 36% chemical oxygen demand removal was achieved within 20 min empty bed retention time and maintained stability for almost one year. The HCLL-S8-M structure feature and its interface on microbial community structure, functions, and metabolic pathways were analyzed by density-functional theory calculation, X-ray photoelectron spectroscopy, multiomics analysis of metagenome, macrotranscriptome and macroproteome. On the surface of HCLL-S8-M, a strong microelectronic field (MEF) was formed by the electron-rich/poor area due to Cu-π interaction from the complexation between phenolic hydroxy of CN and Cu species, driving the electrons of the adsorbed dye pollutants to the microorganisms through extracellular polymeric substance and the direct transfer of extracellular electrons, causing their degradation into CO2 and intermediates, which was degraded partly via intracellular metabolism. The lower energy feeding for the microbiome produced less adenosine triphosphate, resulting in little sludge throughout reaction. The MEF from electronic polarization is greatly potential to develop low-energy wastewater treatment technology.
The integration of biological phosphorus removal (bio-P) and shortcut nitrogen removal (SNR) processes is challenging because of the conflicting demands on influent carbon: SNR allows for upstream carbon diversion but this reduction of influent carbon (especially volatile fatty acids (VFA) prevents or limits bio-P The objective of this study was to achieve SNR, either via partial nitritation/anammox (PNA) or partial denitrification/anammox (PdNA), simultaneously with biological phosphorus removal in a process with upstream carbon capture. This study took place in a pilot scale A/B process with a sidestream bio-P reactor and tertiary anammox polishing. Despite low influent rbCOD concentrations from the A-stage effluent, bio-P occurred in the B-stage thanks to the addition of A-stage WAS fermentate to the sidestream reactor. Nitrite accumulation occurred in the B-stage via partial denitrification and partial nitritation (NOB out-selection), depending on operational conditions, and was removed along with ammonia by the tertiary anammox MBBR, with the ability to achieve effluent TIN less than 2 mg/L.
Tetrasphaera-enhanced biological phosphorus removal (T-EBPR) was developed by augmenting conventional EBPR (C-EBPR) with Tetrasphaera to improve phosphorus removal from anaerobic digestate of swine wastewater. At influent total phosphorus (TP) concentrations of 45 to 55 mg/L, T-EBPR achieved effluent TP concentration of 4.17 ± 1.02 mg/L, 54% lower than that in C-EBPR (8.98 ± 0.76 mg/L). The enhanced phosphorous removal was presumably due to the synergistic effect of Candidatus Accumulibacter and Tetrasphaera occupying different ecological niches. Bioaugmentation with Tetrasphaera promoted the polyphosphate accumulation metabolism depending more on the glycolysis pathway, as evidenced by an increase in intracellular storage compounds of glycogen and polyhydroxyalkanoates by 0.87 and 0.34 mmol C/L, respectively. The enhanced intracellular storage capacity was coincidentally linked to the increase in phosphorus release and uptake rates by 1.23 and 1.01 times, respectively. These results suggest bioaugmentation with Tetrasphaera could be an efficient way for improved phosphorus removal from high-strength wastewater.
Phosphorus-accumulating organisms (PAOs), which harbor metabolic mechanisms for phosphorus removal, are widely applied in wastewater treatment. Recently, novel PAOs and phosphorus removal metabolic pathways have been identified and studied. Specifically, Dechloromonas and Tetrasphaera can remove phosphorus via the denitrifying phosphorus removal and fermentation phosphorus removal pathways, respectively. As the main PAOs in biological phosphorus removal systems, the conventional PAO Candidatus Accumulibacter and the novel PAOs Dechloromonas and Tetrasphaera are thoroughly discussed in this paper, with a specific focus on their phosphorus removal metabolic mechanisms, process applications, community abundance and influencing factors. Dechloromonas can achieve simultaneous nitrogen and phosphorus removal in an anoxic environment through the denitrifying phosphorus removal metabolic pathway, which can further reduce carbon source requirements and aeration energy consumption. The metabolic pathways of Tetrasphaera are diverse, with phosphorus removal occurring in conjunction with macromolecular organics degradation through anaerobic fermentation. A collaborative oxic phosphorus removal pathway between Tetrasphaera and Ca. Accumulibacter, or a collaborative anoxic denitrifying phosphorus removal pathway between Tetrasphaera and Dechloromonas are future development directions for biological phosphorus removal technologies, which can further reduce carbon source and energy consumption while achieving enhanced phosphorus removal.
Tetrasphaera are polyphosphate accumulating organisms (PAOs) that play an important role in enhanced biological phosphorus removal (EBPR) from wastewater. The effect of a wide range of temperature changes (10-30 °C) on phosphorus removal, metabolism and clade-level community structure of Tetrasphaera-dominated PAOs was investigated. At 10 °C, the bioactivities of Tetrasphaera-dominated communities were obviously inhibited and the EBPR efficiency was only 73 %. Yet at 20-30 °C, EBPR efficiency reached 99 % and the relative abundance of Tetrasphaera was up to 90 %. The temperature variation changed the community distribution of Tetrasphaera clades, which was possibly a main reason for EBPR performance. Amino acids and PHA with different contents were intracellular metabolite of Tetrasphaera-dominated communities during phosphorus release and uptake at different temperatures. Moreover, Tetrasphaera fermented protein and amino acids and released VFAs. The outcomes suggested the great potential of Tetrasphaera-PAOs in the treatment of wastewater with varying temperatures and limited carbon sources.
Glycogen accumulating organisms (GAOs) are closely related to the deterioration of enhanced biological phosphorus removal systems. However, the metabolic mechanisms that drive GAOs remain unclear. Here, the two-thirds supernatant of a reactor were decanted following the anaerobic period to enrich GAOs. Long-term monitoring demonstrated that the system was stable and exhibited typical characteristics of GAOs metabolism. Acetate was completely consumed after 60 min of the anaerobic phase. The level of glycogen decreased from 0.20 to 0.14 g/gSS during the anaerobic phase, whereas the level of glycogen significantly increased to 0.21g/gSS at the end of the aerobic period. Moreover, there was almost no phosphate release and absorption in the complete periods, thus confirming the successful construction of a GAOs enrichment system. Microbial community analysis demonstrated that Ca. Contendobacter was among the core functional genera and showed the highest activity among all of the communities. Furthermore, our study is the first to identify the involvement of the ethyl-malonyl-CoA pathway in the synthesis of polyhydroxyvalerate via croR, ccr, ecm, mcd, mch and mcl genes. The Embden-Meyerhof-Parnas (EMP) pathway was preferentially used via glgP. Furthermore, the glyoxylate cycle was the main source of ATP under anaerobic conditions, whereas the tricarboxylic acid cycle provided ATP under aerobic conditions. aceA and mdh appeared to be major modulators of the glyoxylate pathway for controlling energy flow. Collectively, our findings not only revealed the crucial metabolic mechanisms in a GAOs enrichment system but also provided insights into the potential application of Ca. Contendobacter for wastewater treatment.
The removal and recovery of phosphorus from wastewater are crucial for reducing eutrophication and alleviating phosphate rock depletion. In this study, nanoscale zero-valent iron (nZVI) confined in Alfa Aesar Amberlite IRA-402 (Cl) anion exchange resin composite adsorbents was developed by in situ reduction and deposition (denoted as nZVI-402-Cl) to remove phosphate from simulated and real wastewater. Surface and structure characterizations revealed that nZVI particles with a partially oxidized surface were loaded on the surface and inside the anion exchange resin. The phosphate adsorption capacity of nZVI-402-Cl was found to be high over a wide pH range (3.0–11.0), with a maximum adsorption capacity of 56.27 mg P/g at pH 7.2. Despite the presence of interfering sulfate and nitrate anions, nZVI-402-Cl maintained its high phosphate adsorption capacity owing to its excellent selectivity. The confinement of nZVI in anion exchange resins, in particular, could reduce the negative effects of humic acid on phosphate removal. After five regeneration/use cycles, the phosphate removal of nZVI-402-Cl was maintained close to 95%. In column mode tests, the nZVI-402-Cl column process generates ∼1850 bed volume (BV) clean water ([phosphorus] < 0.1 mg/L) from the wastewater treatment plant effluents. In contrast, the value of the IRA-402 column is only ∼900 BV. nZVI-402-Cl has proven to be an efficient and selective adsorbent for practical phosphate removal and recovery in different water environments.
To balance the high phosphorus concentration in recirculated solution and the stability of biofilm system, this study explored the performance and mechanism of phosphorus uptake/release for recovering phosphorus from sewage when the phosphorus content in biofilm (Pbiofilm) changed. The results showed that the maximum phosphorus concentration in the concentrated solution reached 171.2 ± 2.5 mg·L⁻¹ in harvest 1st-5th stages. Polyphosphate accumulating organisms (PAOs) performed a metabolic shift from glycogen accumulation metabolism (GAM) to polyphosphate accumulation metabolism (PAM) when Pbiofilm increased at each phosphorus enrichment stage, and more phosphorus was absorbed/released by PAOs. Nevertheless, the release of poly-phosphate from PAOs was inhibited after phosphorus concentration stabilized, and PAOs were unable to absorb phosphorus from wastewater as it reached the phosphorus saturation stage. To maintain the stability of the system, phosphorus had to be harvested so that the saturated phosphorus in PAOs was easily released in a new recirculated solution, resulting in adequate storage space for PAOs to absorb phosphorus. Meanwhile, the ³¹P NMR analysis demonstrated that phosphorus was stored in EPS and cell of PAOs, whereas EPS played a significant role than cell at the anaerobic phase. Particularly, ortho-phosphate was the major component of phosphorus release by EPS and poly-phosphate was the major part of phosphorus release by cell. Furthermore, the change of Pbiofilm had no impact on biofilm characteristics and microbial communities, whereas some PAOs would be enriched, and others that were not suitable for this process would be inhibited with repeated cycles of alternating aerobic/anaerobic operation.
This study focused on the problem that simultaneous nitrogen and phosphorus removal cannot be achieved in current urban sewage treatment systems. To address this, a pilot system was developed to first use immobilized biologically active fillers to remove nitrogen, and then use activated sludge to independently remove phosphorus. The goal was to maximize the effectiveness of biological phosphorus removal. This study mainly aimed at analyzing the post-independent phosphorus removal characteristics and mechanisms; the composition of the phosphorus removing bacteria; and the contribution rate of the system. The effluent PO4³⁻-P of the system remained stable at 0.15-0.2 mg. L⁻¹, with the phosphorus removal anaerobic tank maintaining a "micro-release" state. When the hydraulic retention time (HRT) of the aerobic phosphorus removal tank was reduced to 1 h, the contribution rate of the aerobic phosphorus removal bacteria decreased from 28.42% to 16.68%; the contribution rate of the denitrifying phosphorus removal bacteria increased from 72.58% to 83.32%; and the abundance of the dominant bacteria Dechloromonas and Thaurea significantly increased. The phosphorus removal system provides a new approach for deep phosphorus removal in main municipal wastewater systems.
Achieving enhanced biological phosphorus removal dominated by Tetrasphaera utilizing waste activated sludge (WAS) as carbon source could solve the problems of insufficient carbon source and excessive discharge of WAS in biological phosphorus removal. Up to now, the sludge reduction ability of Tetrasphaera remained largely unknown. Furthermore, the difference between traditional sludge fermentation and sludge fermentation dominated by Tetrasphaera was still unclear. In this study, two different sequencing batch reactors (SBRs) were operated. WAS from SBR-parent was utilized as sole carbon source to enrich Tetrasphaera with the relative abundance of 91.9% in SBR-Tetrasphaera. PO4³⁻-P removal and sludge reduction could simultaneously be achieved. The effluent concentration of PO4³⁻-P was 0, and the sludge reduction efficiency reached about 44.14% without pretreatment of sludge. Cell integrity detected by flow cytometry, the increase of DNA concentration in the sludge supernatant and decrease of particle size of activated sludge indicated that cell death and lysis occurred in sludge reduction dominated by Tetrasphaera. Stable structure of activated sludge was also damaged in this process, which led to the sludge reduction. By analyzing the excitation-emission matrix spectra of extracellular polymeric substances and the changes of carbohydrate and protein concentration, this study proved that slowly biodegradable organics (e.g., soluble microbial byproduct, tyrosine and tryptophan aromatic protein) could be better hydrolyzed and acidized to volatile fatty acids (VFAs) in sludge fermentation dominated by Tetrasphaera than traditional sludge fermentation, which provided carbon source for biological nutrients removal and saved operation cost in wastewater treatment.
Simultaneous partial nitrification, biological phosphorous removal and sludge fermentation in continuous–flow system faced many challenges in wastewater treatment plants (WWTPs). In this study, a novel process was developed to achieve partial nitrification, enhanced biological phosphorus removal and in–situ fermentation (PNPRF) in continuous–flow system dominated by Tetrasphaera treating low carbon/nitrogen ratio (COD/N) real domestic wastewater. After 80 days of operation, no external carbon was added into the influent. Nitrite accumulation ratio (NAR) reached 99.4% and removal efficiency of PO4³⁻–P was 100%. The effluent concentration of total inorganic nitrogen (TIN) was lower than 2 mg/L. Compared with traditional biological nitrogen and phosphorus removal processes, the daily sludge discharge in continuous–flow PNPRF decreased by 61.9%. DNA concentration of the sludge supernatant after anaerobic zone was 2.26-fold of that before anaerobic zone, and percentages of intact cells decreased by 6.9% and necrotic cells increased by 8.3%, indicating that cell lysis caused the reduction of sludge discharge. The negative effect of prolonged anaerobic phase on transcriptional response of nxrB gene was more significant than that of amoA and hao gene, which resulted in stable partial nitrification. The activities of ammonium–oxidizing bacteria (AOB) were higher than that of nitrite–oxidizing bacteria (NOB), and reverse transcriptional PCR (RT–PCR) showed that RNA expression of AOB was higher than that of NOB. Achieving partial nitrification, enhanced biological phosphorus removal and in–situ fermentation in continuous-flow system dominated by Tetrasphaera solved the problems of carbon source deficiency and large amount of sludge discharge in treating real domestic wastewater process.
An enhanced biological phosphorus removal (EBPR) system based on anaerobic side-stream phosphorus recovery was operated to investigate the nutrient removal performance of the mainstream system under low dissolved oxygen (DO = 1.0, 0.6, 0.2 mg L⁻¹) and the corresponding phosphorus recovery efficiency with different extracting ratio (m = 0, 1/4, 1/3, 1/2) of anaerobic supernatant within 310 days. The results showed that the nutrient removal efficiency remained stable even though DO was extremely low. Nevertheless, phosphorus removal performance was found to deteriorate as side-stream ratio increased to 1/3 at DO = 0.2 mg L⁻¹, suggesting that extracting a higher ratio of supernatant was not favorable for phosphorus removal in the mainstream process at ultra-low DO. The stoichiometric ratios of Prelease/HAcuptake during anaerobic phase which decreased from 0.125 to 0.020 P-mol/C-mol as DO decreased with increasing side-stream ratio were lower than that of typical PAO metabolism. It was very likely that the metabolic mode of PAOs changed due to long-term deprivation of phosphate. Consequently, the mainstream EBPR system failed to remove phosphorus efficiently. It was also observed that phosphorus recovery efficiency was considerable at DO ≥ 0.6 mg L⁻¹ coupled with high side-stream ratio. Therefore, it was feasible and energy-saving to extract appropriate ratio of anaerobic supernatant for recovering phosphorus and removing nutrient efficiently in an EBPR process under low DO.
The characteristics of enhanced biological phosphorus removal (EBPR) process under the combined actions of intracellular and extracellular polyphosphate (polyP) were investigated with the ³¹P Nuclear Magnetic Resonance (NMR) and the fractionation extracting the loosely-bound and tightly-bound extracellular polymer substances (i.e., LB-EPS and TB-EPS) and bacterial cells in EBPR sludge. The hydrolysis/synthesis of extracellular and intracellular polyP was a key step of the phosphate migration and transformation in EBPR sludge. The orthophosphate (orthoP) produced from the intracellular and extracellular polyP anaerobic-hydrolysis was partially accumulated in the bacterial cells and TB-EPS, and then the accumulated orthoP was main composition for these polyP aerobic-synthesis. Importantly, the anaerobic-hydrolysis enhancement of intracellular and extracellular ployP could promote EBPR sludge to absorb volatile fatty acids (VFAs) followed by being transformed into intracellular poly-hydroxy-alkanoates (PHAs). The mechanism for VFAs passing through the LB-EPS and TB-EPS should be an anion-exchange action between orthoP and VFAs. The orthoP accumulation in the TB-EPS kept an orthoP concentration gradient among the TB-EPS, LB-EPS and bulk solution, driving orthoP and VFAs migrations. The orthoP accumulation in the bacterial cells could keep an orthoP concentration difference between the cell-membrane two sides of phosphorus accumulating organisms (PAOs) to promote VFAs passing through the cell membrane considered as an anion exchange membrane. The intracellular PHAs continuously hydrolyzed accompanied with the average chain-length increases of the extracellular and intracellular polyP during the whole aerobic stage. Additionally, the energy of the extracellular polyP synthesized in situ should came from the intracellular PHAs hydrolysis.
Enhanced biological phosphorus removal (EBPR) can recover significant quantities of wastewater phosphorus. However, this resource recovery process realizes limited use largely due to process stability concerns. The research evaluated the effects of anaerobic HRT (τAN) and VFA concentration—critical operational parameters that can be externally controlled—on EBPR performance. Evaluated alone, τAN (1–4 h) exhibited no statistical effect on effluent phosphorus. However, PHA increased with VFA loading and biomass accumulated more phosphorus. Regarding resiliency, under increasing VFA loads PAOs hydrolyzed more phosphorus to uptake/catabolize VFAs; moreover, PHA synthesis normalized to VFA loading increased with τAN, suggesting fermentation. Kinetically, PAOs exhibited a Monod‐like relationships for qPHAAN and qVFAAN as a function of anaerobic P release; additionally, qPAE exhibited a Monod‐like relationship with end‐anaerobic PHA concentration. A culminating analysis affirmed the relationship between enhanced aerobic P uptake, and net P removal, with a parameter (phosphorus removal propensity factor) that combines influent VFA concentration with τAN.
Practitioner points
• Evaluated alone τAN exhibits no statistical effect on effluent phosphorus in an EBPR configuration.
• Increased PHA synthesis, associated with increased VFAs and/or extended τAN, enhances aerobic phosphorus removal.
• PHA synthesis normalized to VFA loading increased with τAN, suggesting fermentation in the EBPR anaerobic zone.
• Aerobic phosphorus uptake increases linearly with anaerobic phosphorus release, with the slope exceeding unity.
• Increased VFAs can be substituted for shorter anaerobic HRTs, and vice versa, to enhance EBPR performance.
Recovering energy from wastewater in addition to its treatment is a hot trend in the new concept of water resource recovery facility (WRRF). High-rate systems operating at low solid retention time (SRT) have been proposed to meet this challenge. In this paper, the integration of Enhanced Biological Phosphorus Removal (EBPR) in an anaerobic/aerobic continuous high-rate system (A-stage EBPR) was evaluated. Successful P and COD removal were obtained operating at SRT 6, 5 and 4 days treating real wastewater, while a further decrease to 3 days led to biomass washout. The best steady state operational conditions were obtained at SRT = 4d, with high removal percentage of P (94.5%) and COD (96.3%), and without detecting nitrification. COD mineralization could be reduced to 30%, while 64 % of the entering carbon could be diverted as biomass to energy recovery. Regarding nitrogen, about 69±1% of the influent N was left as ammonium in the effluent, with 30% used for biomass growth. The aerobic reactor could be operated at low dissolved oxygen (DO) (0.5 mg/L), which is beneficial to decrease energy requirements. Biochemical methane potential (BMP) tests showed better productivity for the anaerobic sludge than the aerobic sludge, with an optimal BMP of 296±2 mL CH4/gVSS. FISH analysis at SRT = 4d revealed a high abundance of Accumulibacter (33±13%) and lower proportion of GAO: Competibacter (3.0±0.3%), Defluviicoccus I (0.6±0.1%) and Defluviicoccus II (4.3±1.1%).
This study employed molecular tools and single cell Raman micro-spectroscopy techniques to reveal the single cell- and population-level phenotypic dynamics and changes in functionally relevant organisms, namely polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), in response to influent loading readily biodegradable carbon to phosphorus ratio (C/P) changes in enhanced biological phosphorus removal (EBPR) systems. The results, for the first time, provided direct and cellular evidence confirming the adaptive anaerobic metabolic pathway shifts in PAOs in response to influent loading variations. Increase in influent readily biodegradable carbon to phosphorus (C/P) ratio from 20 to 50 led to nearly 50% decline in polyphosphate content and drastic rise of intracellular polyβhydroxybutyrate (PHB) to polyphosphate (polyP) ratio by nearly 6 times in PAOs, indicating corresponding diminishing reliance on polyP hydrolysis for energy as P becomes limiting. Influent carbon availability surge also impacted the intracellular carbon polymers in GAOs, with significant increase in the mean PHB content level but no observed changes in the intracellular glycogen level. Furthermore, the Raman-based quantification of differentiated intracellular polymer content associated with PAOs and GAOs, revealed new insights into the quantitative shift in intracellular carbon storage distribution between the two populations and their variations between the two carbon polymers (PHB, Glycogen). In summary, this investigation revealed high-resolution cellular level information regarding the metabolic flexibility in PAOs, phenotypic stoichiometry changes and carbon flux and distribution among PAOs and GAOs, in response to influent loading conditions. The new information will contribute to improvement in mechanistic EBPR modeling and design.
Biological nutrient removal (BNR) plants can reduce both carbon and oxygen requirements by increasing the fraction of phosphorus (P) removed by denitrifying polyphosphate accumulating organisms (DPAOs). Contrasting findings have been reported in literature concerning whether or not PAOs and DPAOs are different microorganisms. In this study, quantitative fluorescence in situ hybridisation (FISH) measurements from different EBPR sludges support the hypothesis that PAOs and DPAOs are phyogenetically different. This experimental evidence is discussed within the context of literature findings and suggestions for future research concerning the identity of PAOs and DPAOs are proposed. Further, this paper discusses the different methodologies available for assessing the DPAO fraction through chemical analytical techniques, where the relative fraction estimated is highly dependent on the methodology employed. Thus, we recommend an alteration to previously proposed methods in order to calculate the DPAO fraction through anaerobic-anoxic and anaerobic-aerobic batch tests. This information is expected to be valuable in studies focussed on optimising the amount of phosphorus removal achieved with simultaneous denitrification.
Glycolysis has been generally accepted to be the source of reducing power used for the synthesis of polyhydroxyalkanoates (PHAs) in the anaerobic metabolism of polyphosphate accumulating organisms (PAOs). However, the tricarboxylic acid cycle (TCA) has also been suggested to contribute to the generation of reducing equivalents, creating some controversy in this research field over the last two decades. Various research approaches have been applied in order to clarify this issue, including the analysis of intracellular compounds, labelled carbon substrates and gene expression. However, the conclusions of these studies seem not to be consolidated. The extent of TCA cycle involvement could be related to the experimental methods employed, the community structure of the cultures used, and also the operational conditions employed. This mini-review analyses the historical findings related to the source of reducing power in PAOs, and highlights the different approaches used in the previous studies. Key factors influencing the generation of reducing power through different metabolic pathways are discussed, and further research directions are also proposed.
Glycogen accumulating organisms (GAO) compete for substrate with polyphosphate-accumulating organisms (PAO), which are the microorganisms responsible for the enhanced biological phosphorus removal (EBPR) in activated sludge wastewater treatment systems. This can lead to the deterioration of the EBPR process. In this paper, the long-term temperature effects on the anaerobic and aerobic stoichiometry and conversion rates on adapted enriched cultures of Competibacter (a known GAO) were evaluated from 10 to 40 degrees C. The anaerobic stoichiometry of Competibacter was constant from 15 to 35 degrees C, whereas the aerobic stoichiometry was insensitive to temperature changes from 10 to 30 degrees C. At 10 degrees C, likely due to the inhibition of the anaerobic conversions of Competibacter, a switch in the dominant bacterial population to an enriched Accumulibacter culture (a known PAO) was observed. At higher temperatures (35 and 40 degrees C), the aerobic processes limited the growth of Competibacter. Due to the inhibition or different steady-state (equilibrium) conditions reached at long-term by the metabolic conversions, the short- and long-term temperature dependencies of the anaerobic acetate uptake rate of Competibacter differed considerably between each other. Temperature coefficients for the various metabolic processes are derived, which can be used in activated sludge modeling. Like for PAO cultures: (i) the GAO metabolism appears oriented at restoring storage pools rather than fast microbial growth, and (ii) the aerobic growth rate of GAO seems to be a result of the difference between PHA consumption and PHA utilization for glycogen synthesis and maintenance. It appears that the proliferation of Competibacter in EBPR systems could be suppressed by adjusting the aerobic solids retention time while, aiming at obtaining highly enriched PAO cultures, EBPR lab-scale reactors could be operated at low temperature (e.g. 10 degrees C).
The abundance and relevance ofAccumulibacter 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(rel)/HAc(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(rel)/HAc(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.
Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphate-accumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were beta-2 Proteobacteria arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the Actinobacteria. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the beta-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing >/=98% sequence identity) related to Rhodocyclus spp. (94 to 97% identity) and Propionibacter pelophilus (95 to 96% identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the beta-2 Proteobacteria probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR sludges are bacteria closely related to Rhodocyclus and Propionibacter.
Laboratory-scale sequencing batch reactors (SBRs) as models for wastewater treatment processes were used to identify glycogen-accumulating organisms (GAOs), which are thought to be responsible for the deterioration of enhanced biological phosphorus removal (EBPR). The SBRs (called Q and T), operated under alternating anaerobic-aerobic conditions typical for EBPR, generated mixed microbial communities (sludges) demonstrating the GAO phenotype. Intracellular glycogen and poly-beta-hydroxyalkanoate (PHA) transformations typical of efficient EBPR occurred but polyphosphate was not bioaccumulated and the sludges contained 1.8% P (sludge Q) and 1.5% P (sludge T). 16S rDNA clone libraries were prepared from DNA extracted from the Q and T sludges. Clone inserts were grouped into operational taxonomic units (OTUs) by restriction fragment length polymorphism banding profiles. OTU representatives were sequenced and phylogenetically analysed. The Q sludge library comprised four OTUs and all six determined sequences were 99.7% identical, forming a cluster in the gamma-Proteobacteria radiation. The T sludge library comprised eight OTUs and the majority of clones were Acidobacteria subphylum 4 (49% of the library) and candidate phylum OP10 (39% of the library). One OTU (two clones, of which one was sequenced) was in the gamma-Proteobacteria radiation with 95% sequence identity to the Q sludge clones. Oligonucleotide probes (called GAOQ431 and GAOQ989) were designed from the gamma-Proteobacteria clone sequences for use in fluorescence in situ hybridization (FISH); 92% of the Q sludge bacteria and 28% of the T sludge bacteria bound these probes in FISH. FISH and post-FISH chemical staining for PHA were used to determine that bacteria from a novel gamma-Proteobacteria cluster were phenotypically GAOs in one laboratory-scale SBR and two full-scale wastewater treatment plants. It is suggested that the GAOs from the novel cluster in the gamma-Proteobacteria radiation be named 'Candidatus Competibacter phosphatis'.
This study investigated the role of Accumulibacter-related bacterial populations and factors influencing their distribution in enhanced biological phosphorus removal (EBPR) systems in the USA. For this purpose, five full-scale wastewater treatment facilities performing EBPR were surveyed. The facilities had different configurations but were all treating primarily domestic wastewater. Two facilities had history of poor EBPR performance. Batch-scale acetate uptake and inorganic phosphate (P(i)) release and uptake experiments were conducted to evaluate the EBPR activity of each sludge. Typical P(i) and acetate profiles were observed, and EBPR activity was found to be positively correlated to polyphosphate (polyP)-accumulating organism (PAO) abundance, as determined by staining intracellular polyP. The abundance of Accumulibacter-related organisms was investigated using fluorescent in situ hybridization. Accumulibacter-related organisms were present in all full-scale EBPR facilities, at levels ranging from 9 to 24% of total cells. More than 80% of Accumulibacter-related organisms were estimated to have high polyP content, confirming their involvement in EBPR in these five facilities. However, Accumulibacter-related PAOs were only a fraction (40-69%) of the total PAO population. The variation of Accumulibacter-related PAO abundance among these EBPR systems suggests that multiple interacting factors such as wastewater characteristics and operational conditions are structuring PAO communities.
An experimental study was carried out to investigate the metabolic pathway of acetic acid and the relationship between the phosphorus accumulation in the sludge and the metabolism of organic substrates in the anaerobic aerobic biological phosphorus removal process. Two laboratory-scale anaerobic aerobic processes were operated continuously with different phosphorus loadings and batch experiments were conducted with the sludges obtained from the continuous systems. The metabolic pathway of acetate was postulated, in which NADH2 required for PHB synthesis is supplied from the consumption of intracellular carbohydrate through the EMP pathway. The ability of the sludge to uptake acetate anaerobically was limited by the amount of polyphosphate stored in the cell, so long as the phosphorus content of sludge was below 35 mgP/gVSS. From energy balance consideration, the energy required for the accumulation of polyphosphate is found to be very little compared with the total energy produced and the sludge can maintain the yield coefficient at a required level even when a large amount of polyphosphate is accumulated.
A biochemical model is presented that explains the behaviour of Acinetobacter spp. in enhanced biological phosphorus removal activated sludge systems. The model modifies and extends the proposals of Y. Comeau et al. Two key parameters are identified in controlling poly-P and PHB synthesis and degradation, the ATP/ADP and NADH/NAD ratios. The predicted behaviour appears to be consistent with that observed.
The significance of microbial Fe(III) reduction in activated sludge was evaluated with regard to: its importance as electron acceptor; as a producer of acetate during anaerobic conditions; for phosphate release; and for its role in the floc structure. Potential Fe(III) reduction rates were measured in 6 wastewater treatment plants with and without biological P-removal and found to be in the range of 0.9–5.4 mgFe/gVSS h. Assuming an incomplete oxidation of organic matter leading to acetate formation, Fe(III) reduction was a major acetate source, providing substrate to phosphorus-accumulating organisms (PAO) during anaerobic conditions. The observed high potential Fe(III) reduction rale might also be responsible for a significant chemical phosphate release due to reduction of Fe(III) to Fe(II) in clarifiers, sludge storage tanks and anaerobic tanks in plants with biological P-removal. Investigation of the concentrations of Fe(II) in a full-scale treatment plant in anaerobic tanks, oxic/anoxic tanks and return sludge indicated that both reduction and reoxidation took place in the treatment plant. Reoxidation of Fe(II) to Fe(III) in activated sludge was shown to take place with oxygen and probably also during anoxic conditions with nitrate and nitrite as electron acceptors. The results indicate that Fe may be more involved in important processes in activated sludge than hitherto assumed, so a better understanding of Fe interactions in activated sludge is desirable.
Bulking and foaming are two frequently occurring operational problems in activated sludge wastewater treatment plants, and these problems are mainly associated with excessive growth of filamentous bacteria. In this study, a comprehensive investigation of the identity and population dynamics of filamentous bacteria in 28 Danish municipal treatment plants with nutrient removal has been carried out over three years. Fluorescence in situ hybridization was applied to quantify more than twenty probe-defined populations of filamentous bacteria that in total constituted a large fraction of the entire microbial community, on average 24%. Despite the majority being present within the flocs, they occasionally caused settling problems in most of the plants. A low diversity of probe-defined filamentous bacteria was found in the plants with Microthrix and various species belonging to phylum Chloroflexi (e.g., type 0803 and type 0092) as the most abundant. Few other filamentous probe-defined species were found revealing a large similarity between the filamentous populations in the plants investigated. The composition of filamentous populations was stable in each plant with only minor changes in relative abundances observed during the three-year study period. The relative composition of the different species was unique to each plant giving a characteristic "fingerprint". Comprehensive statistical analyses of the presence and abundance of the filamentous organisms did not reveal many correlations with a particular plant design or process parameter.
This study proposed and demonstrated the application of a new Raman microscopy-based method for metabolic state-based identification and quantification of functionally relevant populations, namely polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), in enhanced biological phosphorus removal (EBPR) system via simultaneous detection of multiple intracellular polymers including polyphosphate (polyP), glycogen, and polyhydroxybutyrate (PHB). The unique Raman spectrum of different combinations of intracellular polymers within a cell at a given stage of the EBPR cycle allowed for its identification as PAO, GAO, or neither. The abundance of total PAOs and GAOs determined by Raman method were consistent with those obtained with polyP staining and fluorescence in situ hybridization (FISH). Different combinations and quantities of intracellular polymer inclusions observed in single cells revealed the distribution of different sub-PAOs groups among the total PAO populations, which exhibit phenotypic and metabolic heterogeneity and diversity. These results also provided evidence for the hypothesis that different PAOs may employ different extents of combination of glycolysis and TCA cycle pathways for anaerobic reducing power and energy generation and it is possible that some PAOs may rely on TCA cycle solely without glycolysis. Sum of cellular level quantification of the internal polymers associated with different population groups showed differentiated and distributed trends of glycogen and PHB level between PAOs and GAOs, which could not be elucidated before with conventional bulk measurements of EBPR mixed cultures.
Enhanced biological phosphate removal (EBPR) is an established activated sludge process although many of the fundamental metabolic mechanisms are still poorly understood. Therefore, the stoichiometry and enzymatic reactions of the anaerobic phase of this process were studied in a laboratory reactor with acetate as organic substrate. Enzyme assays showed that acetate activation is performed by acetyl-CoA synthetase. Results of 13C-NMR measurements after feeding 13C-labeled acetate indicated that glycogen is degraded via the Entner–Doudoroff pathway. Energy is supplied by glycolysis, hydrolysis of polyphosphate and probably also by hydrolysis of pyrophosphate and the efflux of MgHPO4. The ratio of phosphate released to acetate taken up is variable and apparently dependent on the contents of polyphosphate and glycogen. A biochemical model is proposed explaining the experimental results in terms of carbon, redox, and energy balances. Anaerobic operation of an incomplete tricarboxylic acid cycle (TCA) is proposed to explain the generation of extra reducing equivalents.
Enhanced biological phosphorus (bio-P) removal from wastewater is a promising technology for which the fundamental mechanisms are still unclear. The purpose of this paper is to present a biochemical model that explains bio-P removal mechanisms occurring under anaerobic, aerobic and anoxic conditions of the process. A bio-P bacterium is referred to as one that can store both polyphosphate and carbon (as poly-β-hydroxybutyrate for example). In this communication, observations from the literature are first reviewed and mechanisms of bacterial bioenergetics and membrane transport are summarized. The model for bio-P metabolism under anaerobic, aerobic and anoxic conditions is then presented. The role of polyphosphate under anaerobic conditions is suggested to be as a source of energy both for the reestablishment of the proton motive force, which would be consumed by substrate transport and for substrate storage. The role of the anaerobic zone is to maximize the storage of organic substrates in bio-P bacteria. For this purpose the supply of readily available substrates should be maximized and the presence of electron acceptors (molecular oxygen or oxidized nitrogen) minimized. Under subsequent aerobic or anoxic conditions, bio-P bacteria will accumulate polyphosphates in response to the availability of electron acceptors (oxygen or oxidized nitrogen) for energy production. Carbon reserves in bio-P bacteria should provide energy for growth and for soluble phosphate accumulation as polyphosphate reserves.
In vivo13C-NMR, 31P-NMR techniques were applied to study phosphorus and carbon metabolism in activated sludge during both the anaerobic and the aerobic stages. By supplying a 13C label on the methyl group of acetate at the beginning of the anaerobic stage, the fate of the label through the subsequent aerobic/anaerobic stages was traced in vivo. It was possible to follow the flux of label from acetate to hydroxybutyrate/hydroxyvalerate co-polymer in the first anaerobic stage, then to monitor the conversion of these units into glycogen in a subsequent aerobic stage, and afterwards, by submitting the same sludge to a second anaerobic stage, to observe the flux of labelled carbon from glycogen to the hydroxyvalerate and hydroxybutyrate units. The uptake/release of inorganic phosphate and the extracellular pH were monitored by 31P-NMR in the same experiments. The data provide an unequivocal demonstration of the involvement of glycogen in the biological phosphorus removal process. On the basis of these 13C labelling data, a biochemical model for the synthesis of polyhydroxyalkanoates from acetate and glycogen was elaborated in which the tricarboxylic acid cycle is proposed as an additional source of reduction equivalents. According to this study, from 1 C-mol acetate, 1.48 C-mol are synthesized and 0.70 C-mol glycogen are degraded anaerobically, while 0.16 P-mol phosphate is released. In the aerobic stage, 1 C-mol of is converted to 0.44 C-mol glycogen.
This paper reviews microbiological and biochemical aspects of the enhanced biological phosphate removal (EBPR) process. The discussion includes: microorganisms responsible for EBPR, isolation of polyphosphate accumulating organisms (PAOs), microbial diversity of the EBPR sludge, biochemical metabolisms of PAOs, energy budget in PAOs metabolism, denitrification by PAOs, glycogen accumulating non-poly-P organisms (GAOs), etc. Since pure cultures which possess complete characteristics of PAOs have not been isolated yet, the biochemical mechanism cannot be definitively described. The criteria to obtain a pure culture isolate are proposed. Based on the review, essential characteristics of PAOs are summarized in a table and directions for future research are identified.
The microbiology of denitrifying enhanced biological phosphorus removal systems has been a subject of much debate. The question has centred on the affinities of different types of Candidatus Accumulibacter PAOs, type I and type II, towards different electron acceptors such as oxygen, nitrate and nitrite. This study used a propionate anaerobic/anoxic/aerobic lab-scale sequencing batch reactor where a microbial culture was successfully enriched in Accumulibacter type I organisms (approx. 90%). The culture was able to take up phosphorus using nitrate, nitrite and oxygen as electron acceptors, although experiments with oxygen led to the fastest P removal rate. The phosphorus uptake to nitrogen consumed ratio (P/N ratio), when using both nitrate and nitrite, was shown to be affected by pH in the range of 7-8.2, achieving higher values for lower pH values (7.0-7.5). The effect of pH on P removal seems to follow a similar trend for both nitrate and nitrite. To our knowledge, this is the first study where the impact of pH in the phosphate removal stoichiometry using the three most significant electron acceptors is shown for such a high enrichment in Accumulibacter type I.
The diversity of the putative polyphosphate-accumulating genus Tetrasphaera in wastewater treatment systems with enhanced biological phosphorus removal (EBPR) was investigated using the full-cycle rRNA approach combined with microautoradiography and histochemical staining. 16S rRNA actinobacterial gene sequences were retrieved from different full-scale EBPR plants, and the sequences belonging to the genus Tetrasphaera (family Intrasporangiaceae) were found to form three clades. Quantitative FISH analyses of the communities in five full-scale EBPR plants using 10 new oligonucleotide probes were carried out. The results showed that the probe-defined Tetrasphaera displayed different morphologies and constituted up to 30% of the total biomass. It was shown that active uptake of orthophosphate and formation of polyphosphate took place in most of the probe-defined Tetrasphaera populations. However, aerobic uptake of orthophosphate only took place after uptake of certain carbon sources under anaerobic conditions and these were more diverse than hitherto assumed: amino acids, glucose, and for some also acetate. Tetrasphaera seemed to occupy a slightly different ecological niche compared with 'Candidatus Accumulibacter' contributing to a functional redundancy and stability of the EBPR process.
The capability of "Candidatus Accumulibacter" to use nitrate as an electron acceptor for phosphorus uptake was investigated using two activated sludge communities. The two communities were enriched in Accumulibacter clade IA and clade IIA, respectively. By performing a series of batch experiments, we found that clade IA was able to couple nitrate reduction with phosphorus uptake, but clade IIA could not. These results agree with a previously proposed hypothesis that different populations of Accumulibacter have different nitrate reduction capabilities, and they will help to understand the ecological roles that these two clades provide.
The microbial populations in 25 full-scale activated sludge wastewater treatment plants with enhanced biological phosphorus removal (EBPR plants) have been intensively studied over several years. Most of the important bacterial groups involved in nitrification, denitrification, biological P removal, fermentation, and hydrolysis have been identified and quantified using quantitative culture-independent molecular methods. Surprisingly, a limited number of core species was present in all plants, constituting on average approx. 80% of the entire communities in the plants, showing that the microbial populations in EBPR plants are rather similar and not very diverse, as sometimes suggested. By focusing on these organisms it is possible to make a comprehensive ecosystem model, where many important aspects in relation to microbial ecosystems and wastewater treatment can be investigated. We have reviewed the current knowledge about these microorganisms with focus on key ecophysiological factors and combined this into a conceptual ecosystem model for EBPR plants. It includes the major pathways of carbon flow with specific organic substances, the dominant populations involved in the transformations, interspecies interactions, and the key factors controlling their presence and activity. We believe that the EBPR process is a perfect model system for studies of microbial ecology in water engineering systems and that this conceptual model can be used for proposing and testing theories based on microbial ecosystem theories, for the development of new and improved quantitative ecosystem models and is beneficial for future design and management of wastewater treatment systems.
The in situ ecophysiology of alphaproteobacterial filamentous Cluster III Defluviicoccus present in enhanced biological phosphorus removal (EBPR)-activated sludge systems was evaluated using FISH-MAR and histochemical staining methods. These organisms, sharing the Nostocoida limicola morphotype, are known to be responsible for serious episodes of activated sludge bulking. The data presented here also demonstrate an ability to assimilate short-chain fatty acids and synthesize poly-β-hydroxyalkanoates (PHA) anaerobically, and then utilize this stored PHA under aerobic conditions, but with no corresponding synthesis of polyphosphate. These features are consistent with an in situ phenotype of glycogen-accumulating organisms (GAO), populations thought to lower the efficiency of EBPR systems by outcompeting polyphosphate-accumulating organisms (PAO) for substrates in their anaerobic feed phase. Survey data indicate that these GAO are as commonly seen as the known PAO in full-scale EBPR-activated sludge systems, which suggest that they might play important roles there, and therefore should not be viewed just as laboratory curiosities.
Two alphaproteobacterial Neisser negative 'Nostocoida limicola' morphotypes differing slightly in their trichome diameter and filament regularity were dominant populations in the Bendigo, Victoria, Australia activated sludge community removing phosphorus (P). Neither responded to the FISH probes available for any of the other alphaproteobacterial 'N. limicola' morphotypes. Instead both fluoresced with the DF988 FISH probe designed originally to target alphaproteobacterial cluster II Defluviicoccus tetrad forming organisms. A 16S rRNA based clone library from this biomass revealed that the alphaproteobacterial clones grouped closely with Candidatus 'Monilibacter batavus' and Defluviicoccus clones in a cluster separate from the existing cluster I and II Defluviicoccus. When a FISH probe was designed against these, it only hybridized to the thinner and less abundant 'N. limicola' morphotype. Micromanipulation-RT-PCR was used to selectively recover the main 'N. limicola' morphotype and a FISH probe designed against the 16S rRNA clones generated from it showed only this filament fluoresced. From FISH based surveys, both 'N. limicola' variants occurred frequently in phosphorus removal activated sludge systems in Australia treating domestic waste. The data suggest that they represent two new strains of Candidatus 'Monilibacter', which on this evidence are filamentous members of the genus Defluviicoccus, a potential competitor for the polyphosphate accumulating organisms in these communities.
For decades, glycolysis has been generally accepted to supply the reducing power for the anaerobic conversion of volatile fatty acids (VFAs) to polyhydroxyalkanoates (PHAs) by polyphosphate accumulating organisms (PAOs). However, the importance of the tricarboxylic acid (TCA) cycle has also been raised since 1980s. The aim of this study is to demonstrate the involvement of the TCA cycle in the anaerobic metabolism of PAOs. To achieve this goal, the glycogen pool of an activated sludge highly enriched in Candidatus Accumulibacter Phosphatis (hereafter referred to as Accumulibacter), a putative PAO was reduced substantially through starving the sludge under intermittent anaerobic and aerobic conditions. After the starvation, acetate added was still taken up anaerobically and stored as PHA, with negligible glycogen degradation. The metabolic models proposed by Pereira, Hesselmann and Yagci, which predict the formation of reducing power through glycolysis and the full or partial TCA cycle, were used to estimate the carbon fluxes. The results demonstrate that Accumulibacter can use both glycogen and acetate to generate reducing power anaerobically. The anaerobic production of reducing power from acetate is likely through the full TCA cycle. The proportion of TCA cycle involvement depends on the availability of degradable glycogen.
Fluorescent oligonucleotide hybridization probes were used to label bacterial cells for analysis by flow cytometry. The probes, complementary to short sequence elements within the 16S rRNA common to phylogenetically coherent assemblages of microorganisms, were labeled with tetramethylrhodamine and hybridized to suspensions of fixed cells. Flow cytometry was used to resolve individual target and nontarget bacteria (1 to 5 microns) via probe-conferred fluorescence. Target cells were quantified in an excess of nontarget cells. The intensity of fluorescence was increased additively by the combined use of two or three fluorescent probes complementary to different regions of the same 16S rRNA.
In situ hybridization with rRNA-targeted oligonucleotide probes has become a widely applied tool for direct analysis of microbial population structures of complex natural and engineered systems. In such studies probe EUB338 (AMANN et al., 1990) is routinely used to quantify members of the domain Bacteria with a sufficiently high cellular ribosome content. Recent reevaluations of probe EUB338 coverage based on all publicly available 16S rRNA sequences, however, indicated that important bacterial phyla, most notably the Planctomycetales and Verrucomicrobia, are missed by this probe. We therefore designed and evaluated two supplementary versions (EUB338-II and EUB338-III) of probe EUB338 for in situ detection of most of those phyla not detected with probe EUB338. In situ dissociation curves with target and non-target organisms were recorded under increasing stringency to optimize hybridization conditions. For that purpose a digital image software routine was developed. In situ hybridization of a complex biofilm community with the three EUB338 probes demonstrated the presence of significant numbers of probe EUB338-II and EUB338-III target organisms. The application of EUB338, EUB338-II and EUB338-III should allow a more accurate quantification of members of the domain Bacteria in future molecular ecological studies.
Glycogen-accumulating organisms (GAO) have the potential to directly compete with polyphosphate-accumulating organisms (PAO) in EBPR systems as both are able to take up VFA anaerobically and grow on the intracellular storage products aerobically. Under anaerobic conditions GAO hydrolyse glycogen to gain energy and reducing equivalents to take up VFA and to synthesise polyhydroxyalkanoate (PHA). In the subsequent aerobic stage, PHA is being oxidised to gain energy for glycogen replenishment (from PHA) and for cell growth. This article describes a complete anaerobic and aerobic model for GAO based on the understanding of their metabolic pathways. The anaerobic model has been developed and reported previously, while the aerobic metabolic model was developed in this study. It is based on the assumption that acetyl-CoA and propionyl-CoA go through the catabolic and anabolic processes independently. Experimental validation shows that the integrated model can predict the anaerobic and aerobic results very well. It was found in this study that at pH 7 the maximum acetate uptake rate of GAO was slower than that reported for PAO in the anaerobic stage. On the other hand, the net biomass production per C-mol acetate added is about 9% higher for GAO than for PAO. This would indicate that PAO and GAO each have certain competitive advantages during different parts of the anaerobic/aerobic process cycle.
A novel coccobacilli group found previously in enhanced biological phosphorus removal (EBPR) systems was further revealed to have a high degree of diversity and distribution in various activated sludge systems. Phylogenetic analysis based on 14 existing and 18 newly retrieved 16S rRNA sequences revealed that these sequences formed a novel cohesive cluster with seven subgroups in the gamma-Proteobacteria. Fluorescence in situ hybridization with a set of probes designed specifically targeting the novel group at different hierarchical levels showed that the novel group with a coccoid (2-4 micro m) to occasionally long-rod (up to 20 micro m) shape widely distributed and in some cases predominated in sludge samples taken from nine lab- and full-scale EBPR systems (10-50% of total cells) and four conventional activated sludge systems (1-10%). Variation of predominance was also observed among those subgroups in systems showing deteriorated or effective EBPR activity.
In an acetate-fed anaerobic-aerobic membrane bioreactor, a deteriorated enhanced biological phosphorus removal (EBPR) community was developed (as determined based on the chemical profiles of organic substrate, soluble phosphate, and intracellular carbohydrate and polyhydroxyalkanote (PHA) concentrations). Microscopic observations revealed the dominance of tetrad-forming organisms (TFOs), of which the majority stained positively for PHA under anaerobic conditions. Fluorescence in situ hybridization (FISH) confirmed that the Alphaproteobacteria (85.0+/-7.0% of total cells) were the most dominant group. A 16S rRNA gene clone library specific for the Alphaproteobacteria indicated that most 16S rRNA gene clones (61% of total clones) were closely affiliated with 'Defluvicoccus vanus', forming a cluster within subgroup 1 of the Alphaproteobacteria. Combined PHA staining and FISH with specific probes designed for the members of the 'Defluvicoccus' cluster suggested diversity within this TFO cluster, and that these TFOs were newly identified glycogen-accumulating organisms in EBPR systems. However, these 'Defluvicoccus'-related TFOs were only seen in low abundance in 12 different EBPR and non-EBPR systems, suggesting that they were not the key populations responsible for the deterioration of full-scale EBPR processes.
Enhanced biological phosphorus removal (EBPR) performance is directly affected by the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs). This study investigates the effects of carbon source on PAO and GAO metabolism. Enriched PAO and GAO cultures were tested with the two most commonly found volatile fatty acids (VFAs) in wastewater systems, acetate and propionate. Four sequencing batch reactors (SBRs) were operated under similar conditions and influent compositions with either acetate or propionate as the sole carbon source. The stimulus for selection of the PAO and GAO phenotypes was provided only through variation of the phosphorus concentration in the feed. The abundance of PAOs and GAOs was quantified using fluorescence in situ hybridisation (FISH). In the acetate fed PAO and GAO reactors, "Candidatus Accumulibacter phosphatis" (a known PAO) and "Candidatus Competibacter phosphatis" (a known GAO) were present in abundance. A novel GAO, likely belonging to the group of Alphaproteobacteria, was found to dominate the propionate fed GAO reactor. The results clearly show that there are some very distinctive differences between PAOs and GAOs in their ability to take up acetate and propionate. PAOs enriched with acetate as the sole carbon source were immediately able to take up propionate, likely at a similar rate as acetate. However, an enrichment of GAOs with acetate as the sole carbon source took up propionate at a much slower rate (only about 5% of the rate of acetate uptake on a COD basis) during a short-term switch in carbon source. A GAO enrichment with propionate as the sole carbon source took up acetate at a rate that was less than half of the propionate uptake rate on a COD basis. These results, along with literature reports showing that PAOs fed with propionate (also dominated by Accumulibacter) can immediately switch to acetate, suggesting that PAOs are more adaptable to changes in carbon source as compared to GAOs. This study suggests that the PAO and GAO competition could be influenced in favour of PAOs through the provision of propionate in the feed or even by regularly switching the dominant VFA species in the wastewater. Further study is necessary in order to provide greater support for these hypotheses.
Fluorescent in situ hybridization (FISH) and polyphosphate (polyP) staining methods were used to characterize the microbial community structure of 13 activated sludge samples taken from nine different Japanese wastewater treatment plants with and without enhanced biological phosphorous removal (EBPR) activities. FISH with published rRNA-targeted oligonucleotide probes for important bacterial groups involving in the EBPR process revealed that Rhodocyclus-related polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms from a gammaproteobacterial lineage GB were the predominant populations detected, representing 4-18% and 10-31% of EUBmix-stained cells, respectively, in those samples. However, a considerable proportion of Rhodocyclus-related PAO cells were observed with no polyP granules accumulated based on polyP staining. This was further supported by a poor correlation between Rhodocyclus-related PAO population and sludge total phosphorous (TP) contents. In contrast, high correlations between polyP-stained cells and sludge TP contents were observed. In particular, among those polyP-stained cells in samples Ariake_A2O and Nakano_AO, more than 85% of them could not be targeted by probe PAOmix. These non-Rhodocyclus-related PAOs included populations from other bacterial divisions and members of the Betaproteobacteria other than those in Rhodocyclus-related group.
Deterioration of enhanced biological phosphorus removal (EBPR) has been linked to the proliferation of glycogen-accumulating organisms (GAOs), but few organisms possessing the GAO metabolic phenotype have been identified. An unidentified GAO was highly enriched in a laboratory-scale bioreactor and attempts to identify this organism using conventional 16S rRNA gene cloning had failed. Therefore, rRNA-based stable isotope probing followed by full-cycle rRNA analysis was used to specifically identify the putative GAOs based on their characteristic metabolic phenotype. The study obtained sequences from a group of Alphaproteobacteria not previously shown to possess the GAO phenotype, but 90 % identical by 16S rRNA gene analysis to a phylogenetic clade containing cloned sequences from putative GAOs and the isolate Defluvicoccus vanus. Fluorescence in situ hybridization (FISH) probes (DF988 and DF1020) were designed to target the new group and post-FISH chemical staining demonstrated anaerobic-aerobic cycling of polyhydroxyalkanoates, as per the GAO phenotype. The successful use of probes DF988 and DF1020 required the use of unlabelled helper probes which increased probe signal intensity up to 6.6-fold, thus highlighting the utility of helper probes in FISH. The new group constituted 33 % of all Bacteria in the lab-scale bioreactor from which they were identified and were also abundant (51 and 55 % of Bacteria) in two other similar bioreactors in which phosphorus removal had deteriorated. Unlike the previously identified Defluvicoccus-related organisms, the group identified in this study were also found in two full-scale treatment plants performing EBPR, suggesting that this group may be industrially relevant.
Temperature and sludge age were found to be important factors in determining the outcome of competition between polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating non-polyphosphate organisms (GAOs) and the resultant stability of enhanced-biological-phosphorus removal (EBPR). At 20 degrees C and a 10-day sludge age, PAOs were dominant in an anaerobic/aerobic (A/O) sequencing-batch reactor (SBR), as a result of their higher anaerobic-acetate-uptake rate and aerobic-biomass yield than GAOs. However, at 30 degrees C and a 10-day sludge age, GAOs were able to outcompete PAOs in the A/O SBR because of their higher anaerobic-acetate-uptake rate than PAOs. At 30 degrees C and a 5-day sludge age, GAOs coexisted with PAOs in the A/O SBR, resulting in unstable EBPR performance. At 30 degrees C, reducing the sludge age from 5 to 3 days improved the EBPR efficiency drastically, and the EBPR performance was stable. The maximum specific-anaerobic-acetate-uptake rates of GAO-enriched sludge were affected by temperature with the Arrhenius temperature coefficient theta of 0.042 (degrees C(-1) between 10 and 30 degrees C. The effect of sludge age (5 and 10 days) on the maximum specific-anaerobic-acetate-uptake rates of GAO-enriched activated sludge, however, was not significant. For the PAO-enriched activated sludge, the maximum specific-anaerobic-acetate-uptake rate did not change significantly between 20 and 30 degrees C, but significantly increased from 0.38 to 0.52 mmol-C/ mmol-C/h as the sludge age decreased from 10 to 3 days at 30 degrees C.
In many biological wastewater treatment systems, bacterial growth and the amount of active biomass are limited by the availability of substrate. Under these low growth conditions, endogenous processes have a significant influence on the amount of active biomass and therefore, the overall system performance. In enhanced biological phosphorus removal (EBPR) systems endogenous processes can also influence the levels of the internal storage compounds of the polyphosphate accumulating organisms (PAO), directly affecting phosphorus removal performance. The purpose of this study was to evaluate the significance of different endogenous processes that occur during the long-term starvation of EBPR sludge under aerobic and anaerobic conditions. Activated sludge obtained from a laboratory sequencing batch reactor was used to perform a series of batch starvation experiments. Under aerobic starvation conditions we observed a significant decay of PAO (first-order decay rate of 0.15/d) together with a rapid utilization of polyhydroxyalkanoates (PHA) and a slower utilization of glycogen and polyphosphate to generate maintenance energy. On the other hand, anaerobic starvation was best described by maintenance processes that rapidly reduce the levels of polyphosphate and glycogen under starvation conditions while no significant decay of PAO was observed. The endogenous utilization of glycogen for maintenance purposes is currently not included in available EBPR models. Our experimental results suggest that mathematical models for in EBPR should differentiate between aerobic and anaerobic endogenous processes, as they influence active biomass and storage products differently.
The conventional mainstream enhanced biological phosphorus removal (EBPR) process depends on the quality of the raw incoming wastewater. An alternative sidestream EBPR process is presented, where the substrates for storage by the polyphosphate accumulating organisms (PAOs) instead come from hydrolysis of the return activated sludge. This process is studied in full-scale at two treatment plants and quantified by means of phosphorus release rates and readily biodegradable COD (RBCOD) accumulation rates. It was seen that not only was a significant amount of RBCOD stored by PAOs but an approximately equal amount was accumulated in the sidestream hydrolysis tank and made available for the subsequent nitrogen removal process. The phosphorus release of the sludge with and without addition of different substrates was furthermore studied in laboratory scale. The study showed that the process is promising and in a number of cases will have significant advantages compared with the conventional mainstream EBPR
Enhanced biological phosphorus removal (EBPR) has been used at many wastewater treatment plants all over the world for many years. In this study a full-scale sludge with good EBPR was tested with P-release batch tests and combined FISH/MAR (fluorescence in situ hybridisation and microautoradiography). Proposed models of PAOs and GAOs (polyphosphate- and glycogen-accumulating organisms) and microbial methods suggested from studies of laboratory reactors were found to be applicable also on sludge from full-scale plants. Dependency of pH and the uptake of both acetate and propionate were studied and used for calculations for verifying the models and results from microbial methods. All rates found from the batch tests with acetate were higher than in the batch tests with propionate, which was explained by the finding that only those parts of the bacterial community that were able to take up acetate anaerobically were able to take up propionate anaerobically.
The anaerobic uptake of acetate and propionate as single and dual carbon sources by the putative Defluviicoccus vanus related glycogen accumulating organisms (DvGAOs) is investigated. A high enrichment of DvGAOs, representing 95+/-3% of the bacterial community bound to the EUBMIX probes, was achieved in a lab-scale reactor operated under alternating anaerobic and aerobic conditions with acetate as the sole carbon source. The culture is able to take up both acetate and propionate under anaerobic conditions, and the metabolism in both cases is well described by the metabolic models previously proposed for GAOs and verified with experimental data obtained with other types of GAO cultures. In the simultaneous presence of acetate and propionate, DvGAOs take up these two carbon sources sequentially, with propionate uptake preceding acetate uptake. Through model-based analysis, we hypothesise that DvGAOs prefer propionate in order to maximise their production of polyhydroxyalkanoates (PHAs) with the same glycogen consumption, which would enhance their growth potential in the following aerobic period. Despite a low to negligible consumption of acetate in the presence of large amounts of propionate, the presence of acetate considerably stimulated the uptake of propionate with the rate increased by over 60% in comparison to the case where only propionate was present. This property enhances the competitive capability of DvGAOs in enhanced biological phosphorus removal (EBPR) wastewater treatment systems, given the fact that wastewater typically contains both acetate and propionate.
The enhanced biological phosphorus removal (EBPR) process has been implemented in many wastewater treatment plants worldwide. While the EBPR process is indeed capable of efficient phosphorus (P) removal performance, disturbances and prolonged periods of insufficient P removal have been observed at full-scale plants on numerous occasions under conditions that are seemingly favourable for EBPR. Recent studies in this field have utilised a wide range of approaches to address this problem, from studying the microorganisms that are primarily responsible for or detrimental to this process, to determining their biochemical pathways and developing mathematical models that facilitate better prediction of process performance. The overall goal of each of these studies is to obtain a more detailed insight into how the EBPR process works, where the best way of achieving this objective is through linking together the information obtained using these different approaches. This review paper critically assesses the recent advances that have been achieved in this field, particularly relating to the areas of EBPR microbiology, biochemistry, process operation and process modelling. Potential areas for future research are also proposed. Although previous research in this field has undoubtedly improved our level of understanding, it is clear that much remains to be learned about the process, as many unanswered questions still remain. One of the challenges appears to be the integration of the existing and growing scientific knowledge base with the observations and applications in practice, which this paper hopes to partially achieve.
The metabolism of polyphosphate accumulating organisms (PAOs) has been widely studied through the use of lab-scale enrichments. Various metabolic models have been formulated, based on the results from lab-scale experiments using enriched PAO cultures. A comparison between the anaerobic stoichiometry predicted by metabolic models with that exhibited by full-scale sludge in enhanced biological phosphorus removal (EBPR) wastewater treatment plants (WWTPs) was performed in this study. Batch experiments were carried out with either acetate or propionate as the sole carbon source, using sludges from two different EBPR-WWTPs in Australia that achieved different phosphorus removal performances. The results support the hypothesis that the anaerobic degradation of glycogen is the primary source of reducing equivalents generated by PAOs, however, they also suggested a partial contribution of the tricarboxylic acid (TCA) cycle in some cases. The experimental results obtained when acetate was the carbon source suggest the involvement of the modified succinate-propionate pathway for the generation of poly-beta-hydroxyvalerate (PHV). Overall, the batch test results obtained from full-scale EBPR sludge with both substrates were generally well described by metabolic model predictions for PAOs.
This study investigated the link between the process performance of two denitrifying phosphorus (P) removal systems and their microbial community structure. Two sequencing batch reactors (SBRs) were operated with either acetate or propionate as the sole carbon source, and were gradually acclimatised from anaerobic-aerobic to anaerobic-anoxic conditions. It was found that the propionate SBR was able to sustain denitrifying P removal after acclimatisation, while the enhanced biological phosphorus removal (EBPR) activity in the acetate reactor collapsed after the aerobic phase was eliminated. The results suggested that the anoxic glycogen production rate in the acetate SBR was insufficient to support the anaerobic glycogen demand for acetate uptake. The chemical transformations in each SBR suggested that different types of polyphosphate-accumulating organisms (PAOs) were present in each system, possessing different affinities for nitrate. Microbial characterisation with fluorescence in situ hybridisation (FISH) revealed that Accumulibacter was the dominant organism in each reactor, although different cell morphotypes were observed. A coccus morphotype was predominant in the acetate SBR while the propionate SBR was enriched in a rod morphotype. It is hypothesised that the coccus morphotype corresponds to an Accumulibacter strain that is unable to use nitrate as electron acceptor but is able to use oxygen, and possibly nitrite. The rod morphotype is proposed to be a PAO able to use nitrate, nitrite and oxygen. This hypothesis is in agreement with literature studies focussed on the identity of denitrifying PAOs (DPAOs), as well as a recent metagenomic study on Accumulibacter.
The influence of operating and environmental conditions on the microbial populations of the enhanced biological phosphorus removal (EBPR) process at seven full-scale municipal activated sludge wastewater treatment plants (WWTPs) in The Netherlands was studied. Data from the selected WWTPs concerning process configuration, operating and environmental conditions were compiled. The EBPR activity from each plant was determined by execution of anaerobic-anoxic-aerobic batch tests using fresh activated sludge. Fractions of Accumulibacter as potential phosphorus accumulating organisms (PAO), and Competibacter, Defluviicoccus-related microorganisms and Sphingomonas as potential glycogen accumulating organisms (GAO) were quantified using fluorescence in situ hybridization (FISH). The relationships among plant process configurations, operating parameters, environmental conditions, EBPR activity and microbial populations fractions were evaluated using a statistical approach. A well-defined and operated denitrification stage and a higher mixed liquor pH value in the anaerobic stage were positively correlated with the occurrence of Accumulibacter. A well-defined denitrification stage also stimulated the development of denitrifying PAO (DPAO). A positive correlation was observed between Competibacter fractions and organic matter concentrations in the influent. Nevertheless, Competibacter did not cause a major effect on the EBPR performance. The observed Competibacter fractions were not in the range that would have led to EBPR deterioration. Likely, the low average sewerage temperature (12+/-2 degrees C) limited their proliferation. Defluviicoccus-related microorganisms were seen only in negligible fractions in a few plants (<0.1% as EUB), whereas Sphingomonas were not observed.
In the anaerobic phase of a biological phosphorus removal process, acetate is taken up and converted to PHB utilizing both energy generated in the degradation of polyphosphate to phosphate, which is released, and energy generated in the conversion of glycogen to poly-beta-hydroxy butyrate (PHB). The phosphate/acetate ratio cannot be considered a metabolic constant, because the energy requirement for the uptake of acetate is strongly influenced by the pH value. The observed phosphate/acetate ratio shows a variation of 0.25 to 0.75 P-mol/C-mol in a pH range of 5.5 to 8.5. It is shown that stored glycogen takes part in the metabolism to provide reduction equivalents and energy for the conversion of acetate to PHB. A structured metabolic model, based on glycogen as the source of the reduction equivalents in the anaerobic phase and the effect of the pH on the energy requirement of the uptake of acetate, is developed. The model explains the experimental results satisfactorily. (c) 1994 John Wiley & Sons, Inc.
A structured metabolic model is developed that describes the stoichiometry and kinetics of the biological P removal process. In this approach all relevant metabolic reactions underlying the metabolism, considering also components like adenosine triphosphate (ATP) and nic-otinamide-adenine dinucleotide (NADH(2)) are describedbased on biochemical pathways. As a consequence of the relations between the stoichiometry of the metabolic reactions and the reaction rates of components, the required number of kinetic relations to describe the process is reduced. The model describes the dynamics of the storage compounds which are considered separately from the active biomass. The model was validated in experiments at a constant sludge retention time of 8 days, over the anaerobic and aerobic phases in which the external oncentrations as well as the internal fractions of the relevant components involved in the P-removal process were monitored. These measurements include dissolved acetate, phosphate, and ammonium; oxygen consumption; poly-beta-hydroxybutyrate (PHB); glycogen; and active biomass. The model satisfactorily describes the dynamic behavior of all components during the anaerobicand aerobic phases.(c) 1995 John Wiley & Sons, Inc.
Full-scale Biological Phosphorus Removal: Quantification of Storage Polymers, Microbial Performance and Metabolic Modelling
- A B Lanham
Lanham, A.B., 2012. Full-scale Biological Phosphorus Removal:
Quantification of Storage Polymers, Microbial Performance
and Metabolic Modelling. PhD thesis. Universidade Nova de
Lisboa, Lisboa, pp. 51e74.