Enhanced biological phosphorus removal sustained by aeration-free filamentous microalgal-bacterial granular sludge.

The microalgal-bacterial granular sludge (MBGS) based enhanced biological phosphorus removal (EBPR) (MBGS-EBPR) was recently proposed as a sustainable wastewater treatment process. Previous work showed the possibility of obtaining an MBGS-EBPR process starting from mature MBGS and phosphate-accumulating organisms (PAOs) enriched aerobic granular sludge (AGS) and validated the effectiveness of removing carbon/nitrogen/phosphorus with mechanical aeration. The present work evaluated whether the same could be achieved starting from conventional activated sludge and operating under aeration-free conditions in an alternating dark/light photo-sequencing batch reactor (PSBR). We successfully cultivated filamentous MBGS with a high settling rate (34.5 m/h) and fast solid-liquid separation performance, which could be attributed to the proliferation of filamentous cyanobacteria and stimulation of extracellular polymeric substances (EPS) production. The process achieved near-complete steady-state removal of carbon (97.2 ± 1.9 %), nitrogen (93.9 ± 0.7 %), and phosphorus (97.7 ± 1.7 %). Moreover, improved phosphorus release/uptake driven by photosynthetic oxygenation under dark/light cycles suggests the enrichment of PAOs and the establishment of MBGS-EBPR. Batch tests showed similar phosphorus release rates in the dark but significantly lower phosphorus uptake rates in the presence of light when the filamentous granules were disrupted. This indicates that the filamentous structure of MBGS has minor limitations on substrate mass transfer while exerting protective effects on PAOs, thus playing an important role in sustaining the function of aeration-free EBPR. Microbial assays further indicated that the enrichment of filamentous cyanobacteria (Synechocystis, Leptoolybya, and Nodosilinea), putative PAOs and EPS producers (Hydrogenophaga, Thauera, Flavobacterium, and Bdellovibrio) promoted the development of filamentous MBGS and enabled the high-efficient pollutant removal. This work provides a feasible and cost-effective strategy for the startup and operation of this innovative process.

An integrated system combining Tetrasphaera-dominated enhanced biological phosphorus removal with sulfur autotrophic denitrification to enhance biological nutrients removal.

This study developed a two-stage process, including Tetrasphaera-dominated enhanced biological phosphorus-removal (EBPR(T)) sequencing batch reactor (SBR), followed by sulfur autotrophic denitrification (SADN) SBR, to achieve advanced nutrients removal from low VFAs wastewater. The removal efficiencies of nitrogen and phosphorus (PO43--P) reached 99 % with effluent PO43--P and total inorganic nitrogen (TIN) below 0.5 mg/L and 1 mg/L in EBPR(T) and SADN SBR, respectively. Mechanism analysis indicated that as increasing drainage ratio and complex carbon sources, free amino acids, glycogen, and PHA served as the endogenous carbon sources of Tetrasphaera to store energy. SADN contributed to approximately 80 % of nitrogen removal. DNA and cDNA results indicated Tetrasphaera was shifted from clade 2 to clade 1 after increasing the drainage ratio and the complexity of the carbon source, and Tetrasphaera (50.95 %) and Ca. Accumulibacter (9.12 %) were the most important functional microorganisms synergized to remove phosphorus at the transcriptional level in EBPR(T). Thiobacillus (45.97 %) and Sulfuritalea (9.24 %) were the dominant sulfur autotrophic denitrifiers at gene and transcriptional level in SADN. The results suggested that the EBPR(T) - SADN SBRs have great nutrient removal performance in treating low VFAs wastewater without additional carbon sources.

Evaluating the opportunities for mainstream P-recovery in anaerobic/anoxic/aerobic systems.

Mainstream P-recovery can help wastewater treatment plants (WWTPs) to effectively maintain good enhanced biological phosphorus removal (EBPR) while helping to recover P. In this study, a pilot-scale anaerobic-anoxic-aerobic (A2O) process was operated for simultaneous COD/N/P removal and P-recovery under different operational conditions. The operation with conventional extraction of waste activated sludge (WAS) from the aerobic reactor was compared to the mainstream P-recovery strategy of WAS extraction from the anaerobic reactor. Successful nutrient removal was obtained for both scenarios, but the anaerobic WAS extraction results improved polyphosphate accumulating organisms (PAOs) activity by increasing almost 27 % P concentration in the anaerobic reactor. WAS fermentation was also evaluated, showing that anaerobic WAS required only 3 days to reach a high P concentration, while the aerobic WAS fermentation required up to 7 days. The fermentation process increased the amount of soluble P available for precipitation from 24.4 % up to 51.6 % in the fermented anaerobic WAS scenario. Results obtained by precipitation modelling of these streams showed the limitations for struvite precipitation due to Ca2+ interference and Mg2+ and NH4+ as limiting species. The optimum precipitation scenario showed that P-recovery could reach up to 51 % of the input P, being 90 % struvite.

Candidatus Accumulibacter use fermentation products for enhanced biological phosphorus removal.

Previous research suggested that two major groups of polyphosphate-accumulating organisms (PAOs), i.e., Ca. Accumulibacter and Tetrasphaera, play cooperative roles in enhanced biological phosphorus removal (EBPR). The fermentation of complex organic compounds by Tetrasphaera provides carbon sources for Ca. Accumulibacter. However, the viability of the fermentation products (e.g., lactate, succinate, alanine) as carbon sources for Ca. Accumulibacter and their potential effects on the metabolism of Ca. Accumulibacter were largely unknown. This work for the first time investigated the capability and metabolic details of Ca. Accumulibacter cognatus clade IIC strain SCUT-2 (enriched in a lab-scale reactor with a relative abundance of 42.8%) in using these fermentation products for EBPR. The enrichment culture was able to assimilate lactate and succinate with the anaerobic P release to carbon uptake ratios of 0.28 and 0.36 P mol/C mol, respectively. In the co-presence of acetate, the uptake of lactate was strongly inhibited, since two substrates shared the same transporter as suggested by the carbon uptake bioenergetic analysis. When acetate and succinate were fed at the same time, Ca. Accumulibacter assimilated two carbon sources simultaneously. Proton motive force (PMF) was the key driving force (up to 90%) for the uptake of lactate and succinate by Ca. Accumulibacter. Apart from the efflux of proton in symport with phosphate via the inorganic phosphate transport system, translocation of proton via the activity of fumarate reductase contributed to the generation of PMF, which agreed with the fact that PHV was a major component of PHA when lactate and succinate were used as carbon sources, involving the succinate-propionate pathway. Metabolic models for the usage of lactate and succinate by Ca. Accumulibacter for EBPR were built based on the combined physiological, biochemical, metagenomic, and metatranscriptomic analyses. Alanine was shown as an invalid carbon source for Ca. Accumulibacter. Instead, it significantly and adversely affected Ca. Accumulibacter-mediated EBPR. Phosphate release was observed without alanine uptake. Significant inhibitions on the aerobic phosphate uptake was also evident. Overall, this study suggested that there might not be a simply synergic relationship between Ca. Accumulibacter and Tetrasphaera. Their interactions would largely be determined by the kind of fermentation products released by the latter.

Microbiome assembly mechanism and functional potential in enhanced biological phosphorus removal system enriched with Tetrasphaera-related polyphosphate accumulating organisms.

Tetrasphaera-related polyphosphate accumulating organisms (PAOs) are the key functional guilds for enhanced biological phosphorus removal (EBPR) systems. Their biomass enrichment can be enhanced by the nitrification inhibitor allylthiourea (ATU). However, the underlying assembly mechanism and the functional potential of the EBPR microbiome regulated by ATU are unclear. This study investigates the effect of ATU on microbiome assembly and functional potential by closely following the microbiota dynamics in an EBPR system enriched with Tetrasphaera-related PAOs for 288-days before, during and after ATU addition. The results showed that ATU addition increased microbiota structural similarity and compositional convergence, and enhanced determinism in the assembly of EBPR microbiome. During exposure to ATU, Tetrasphaera-related PAOs were governed by homogeneous selection and the dominant species revealed by 16S rRNA gene-based phylogenetic analysis shifted from clade III to clade I. Meanwhile, ATU supply promoted significant enrichment of functional genes involved in phosphate transport (pit) and polyphosphate synthesis and degradation (ppk1 and ppk2), whereas both Nitrosomonas and ammonia monooxygenase-encoding genes (amoA/B/C) assignable to this group of nitrifying bacteria decreased. Moreover, ATU addition relieved the significant abundance correlation between filamentous bacteria Ca. Promineofilum and denitrifying Brevundimonas (FDR-adjusted P < 0.01), damaging their potential synergic or cooperative interactions, thus weakening their competitiveness against Tetrasphaera-related PAOs. Notably, ATU withdrawn created opportunistic conditions for the unexpected explosive growth and predominance of Thiothrix filaments, leading to a serious bulking event. Our study provides new insights into the microbial ecology of Tetrasphaera-related PAOs in EBPR system, which could guide the establishment of an efficient microbiota for EBPR.

Reduction of SARS-CoV-2 by biological nutrient removal and disinfection processes in full-scale wastewater treatment plants.

Detection of SARS-CoV-2 RNA in wastewater poses people's concerns regarding the potential risk in water bodies receiving wastewater treatment effluent, despite the infectious risk of SARS-CoV-2 in wastewater being speculated to be low. Unlike well-studied nonenveloped viruses, SARS-CoV-2 in wastewater is present abundantly in both solid and liquid fractions of wastewater. Reduction of SARS-CoV-2 in past studies were likely underestimated, as SARS-CoV-2 in influent wastewater were quantified in either solid or liquid fraction only. The objectives of this study were (i) to clarify the reduction in SARS-CoV-2 RNA during biological nutrient removal and disinfection processes in full-scale WWTPs, considering the SARS-CoV-2 present in both solid and liquid fractions of wastewater, and (ii) to evaluate applicability of pepper mild mottle virus (PMMoV) as a performance indicator for reduction of SARS-CoV-2 in WWTPs. Accordingly, large amount of SARS-CoV-2 RNA were partitioned in the solid fraction of influent wastewater for composite sampling than grab sampling. When SARS-CoV-2 RNA in the both solid and liquid fractions were considered, log reduction values (LRVs) of SARS-CoV-2 during step-feed multistage biological nitrogen removal (SM-BNR) and enhanced biological phosphorus removal (EBPR) processes ranged between>2.1-4.4 log and did not differ significantly from those in conventional activated sludge (CAS). The LRVs of SARS-CoV-2 RNA in disinfection processes by ozonation and chlorination did not differ significantly. PMMoV is a promising performance indicator to secure reduction of SARS-CoV-2 in WWTPs, because of its higher persistence in wastewater treatment processes and abundance at a detectable concentration even in the final effluent after disinfection.

Enhancing phosphorus removal of photogranules by incorporating polyphosphate accumulating organisms.

Photogranules are a novel wastewater treatment technology that can utilize the sun's energy to treat water with lower energy input and have great potential for nutrient recovery applications. They have been proven to efficiently remove nitrogen and carbon but show lower conversion rates for phosphorus compared to established treatment systems, such as aerobic granular sludge. In this study, we successfully introduced polyphosphate accumulating organisms (PAOs) to an established photogranular culture. We operated photobioreactors in sequencing batch mode with six cycles per day and alternating anaerobic (dark) and aerobic (light) phases. We were able to increase phosphorus removal/recovery by 6 times from 5.4 to 30 mg/L/d while maintaining similar nitrogen and carbon removal compared to photogranules without PAOs. To maintain PAOs activity, alternating anaerobic feast and aerobic famine conditions were required. In future applications, where aerobic conditions are dependent on in-situ oxygenation via photosynthesis, the process will rely on sunlight availability. Therefore, we investigated the feasibility of the process under diurnal cycles with a 12-h anaerobic phase during nighttime and six short cycles during the 12 h daytime. The 12-h anaerobic phase had no adverse effect on the PAOs and phototrophs. Due to the extension of one anaerobic phase to 12 h the six aerobic phases were shortened by 47% and consequently decreased the light hours per day. This resulted in a decrease of phototrophs, which reduced nitrogen removal and biomass productivity up to 30%. Finally, we discuss and suggest strategies to apply PAO-enriched photogranules at large-scale.

Exploring the stability of an A-stage-EBPR system for simultaneous biological removal of organic matter and phosphorus.

This work evaluates the performance and stability of a continuous anaerobic/aerobic A-stage system with integrated enhanced biological phosphorus removal (A-stage-EBPR) under different operational conditions. Dissolved oxygen (DO) in the aerobic reactor was tested in the 0.2-2 mgDO/L range using real wastewater amended with propionic acid, obtaining almost full simultaneous COD and P removal without nitrification in the range 0.5-1 mgDO/L, but failing at 0.2 mgDO/L. Anaerobic purge was tested to evaluate a possible mainstream P-recovery strategy, generating a P-enriched stream containing 22% of influent P. COD and N mass balances indicated that about 43% of the influent COD could be redirected to the anaerobic digestion for methane production and 66% of influent NH4+-N was discharged in the effluent for the following N-removal B-stage. Finally, when the system was switched to glutamate as sole carbon source, successful EBPR activity and COD removal were maintained for two months, but after this period settleability problems appeared with biomass loss. Microbial community analysis indicated that Propionivibrio, Thiothrix and Lewinella were the most abundant species when propionic acid was the carbon source and Propionivibrio was the most favoured with glutamate. Thiothrix, Hydrogenophaga, Dechloromonas and Desulfobacter appeared as the dominant polyphosphate-accumulating organisms (PAOs) under different operation stages.

Phosphorus removal performance, intracellular metabolites and clade-level community structure of Tetrasphaera-dominated polyphosphate accumulating organisms at different temperatures.

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 (1-30 °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.

Formation and stability of aerobic granular sludge in a sequential batch reactor for the simultaneous removal of organic matter and nutrients from low-strength domestic wastewater.

Simultaneous removal of organic matter, nitrogen, and phosphorus, via simultaneous nitrification and denitrification (SND) and enhanced biological phosphorus removal processes, was evaluated in a pilot-scale sequential batch reactor. The focus was on granule's morphology, stability, microbiological composition, and reactor performance while treating diluted domestic wastewater with total chemical oxygen demand (CODt) of ≈ 200 mg.L-1. The applied organic loading rate was 0.9 ± 0.3 kg CODt.m-3.d-1 in the experiment. Aerobic granular sludge developed gradually. After 87-day operation, granules (diameter ≥ 0.2 mm) were ≥ 50 % of the biomass, and after 168 days, complete granulation was obtained (≥ 80 % of biomass). In the third period (days 168-247, complete granulation), mixed liquor biomass reached a volatile suspended solids (VSS) concentration of 1.2 ± 0.3 g VSS.L-1, with the granules remaining stable until the experimental end. In this period, low effluent concentrations of COD, nitrogen (NH4+-N, NO2--N and NO3--N) and phosphate (PO43-P) were obtained (mg.L-1): 36 ± 11; 4 ± 5; 3 ± 3, 4 ± 5; and 0.9 ± 0.4, respectively. COD, NH4+-N, and PO43--P removal efficiencies (%) were 80 ± 11; 83 ± 20; and 55 ± 24, respectively. Heterotrophic nitrification and SND were observed, resulting in a process efficiency of 31 % even with dissolved oxygen applied to saturation. The phosphate removal was mainly attributed to denitrifying phosphorus accumulating organisms. Pseudomonas, the dominant genus found, acted in nitrogen and phosphorus removal. Pseudoxanthomonas also assisted in phosphorus removal. Bacterial communities in the flocs (≈ 20 % of biomass) during the last period were similar to those in the granules; therefore, they constituted the basis for granule formation, directly contributed to the simultaneous good removal of organic matter and nutrients.

Biological phosphorus removal and its microbial community in a modified full-scale activated sludge system under dry and wet weather dynamics.

Enhanced biological phosphorus removal (EBPR) performance and microbial community dynamics during dry and wet-weather conditions of a full-scale treatment plant was evaluated by converting a section of activated sludge basins using low-cost operational modifications into an anoxic/anaerobic zone to promote EBPR. Two trains of the activated sludge system at the Des Moines, Iowa Metropolitan Wastewater Reclamation Facility were used for the study with one train modified for EBPR, and the other remained as nitrification-only for comparison. In addition to measuring the modification effectiveness for phosphorus removal, performance was compared during dry and wet weather conditions over the course of two summer seasons to improve understanding of wet and dry weather dynamics for EBPR. DNA sequencing and qPCR tests were conducted to develop an understanding of microbial population changes between control and modified basins and wet and dry weather conditions. Basin hydraulic retention times varied from 2.6 to 12.7 hours with an average of 8.9 hours. EBPR activity was successfully established in the modified basins with average phosphorus content of the return activated sludge 0.032 ± 0.002 compared to 0.016 ± 0.001 mg TP/mg TSS (95% confidence) in the control basins. Phosphorus removal was significantly decreased by prolonged wet weather conditions, particularly in year two of the study, however the modified basin maximum removal of 96% and average of 43.7 ± 5.3% remained significantly higher than the maximum of 46% and average 12.6 ± 2.4% removal in the control basins. DNA sequencing showed a significant increase in relative abundance of phyla Chloroflexi, Nitrospirae, and Verrucomicrobia in the modified basins, but no correlation to EBPR performance. qPCR indicated significant increase in relative quantity of Accumulibacter, but not for Actinetobacter-like phosphorus accumulating organisms (PAOs), which includes the PAO Tetrasphaera. Significant abundance of some Accumulibacter clades found within the modified basins was contrary to previous literature which focused on small-scale and batch studies. A higher than expected dominance of clade I and increased relative quantities of clades IIB and IIC during extended wet weather was observed which may have contributed to rapid recovery of phosphorus removal when dry weather resumed. The abundance of PAOs did not significantly correlate with changes in phosphorous removal performance, contrary to reports from previous small-scale and batch studies.

Enrichment of phosphate-accumulating organisms (PAOs) in a microfluidic model biofilm system by mimicking a typical aerobic granular sludge feast/famine regime.

Wastewater treatment using aerobic granular sludge has gained increasing interest due to its advantages compared to conventional activated sludge. The technology allows simultaneous removal of organic carbon, nitrogen, and phosphorus in a single reactor system and is independent of space-intensive settling tanks. However, due to the microscale, an analysis of processes and microbial population along the radius of granules is challenging. Here, we introduce a model system for aerobic granular sludge on a small scale by using a machine-assisted microfluidic cultivation platform. With an implemented logic module that controls solenoid valves, we realized alternating oxic hunger and anoxic feeding phases for the biofilms growing within. Sampling during ongoing anoxic cultivation directly from the cultivation channel was achieved with a robotic sampling device. Analysis of the biofilms was conducted using optical coherence tomography, fluorescence in situ hybridization, and amplicon sequencing. Using this setup, it was possible to significantly enrich the percentage of polyphosphate-accumulating organisms (PAO) belonging to the family Rhodocyclaceae in the community compared to the starting inoculum. With the aid of this miniature model system, it is now possible to investigate the influence of a multitude of process parameters in a highly parallel way to understand and efficiently optimize aerobic granular sludge-based wastewater treatment systems.Key points• Development of a microfluidic model to study EBPR.• Feast-famine regime enriches polyphosphate-accumulating organisms (PAOs).• Microfluidics replace sequencing batch reactors for aerobic granular sludge research.

A review on the integration of mainstream P-recovery strategies with enhanced biological phosphorus removal.

Phosphorus (P), an essential nutrient for all organisms, urgently needs to be recovered due to the increasing demand and scarcity of this natural resource. Recovering P from wastewater is a feasible and promising way widely studied nowadays due to the need to remove P in wastewater treatment plants (WWTPs). When enhanced biological P removal (EBPR) is implemented, an innovative option is to recover P from the supernatant streams obtained in the mainstream water line, and then combine it with liquor-crystallisation recovery processes, being the final recovered product struvite, vivianite or hydroxyapatite. The basic idea of these mainstream P-recovery strategies is to take advantage of the ability of polyphosphate accumulating organisms (PAO) to increase P concentration under anaerobic conditions when some carbon source is available. This work shows the mainstream P-recovery technologies reported so far, both in continuous and sequenced batch reactors (SBR) based configurations. The amount of extraction, as a key parameter to balance the recovery efficiency and the maintenance of the EBPR of the system, should be the first design criterion. The maximum value of P-recovery efficiency for long-term operation with an adequate extraction ratio would be around 60%. Other relevant factors (e.g. COD/P ratio of the influent, need for an additional carbon source) and operational parameters (e.g. aeration, SRT, HRT) are also reported and discussed.

Unexpected phosphorous removal in a Candidatus_Competibacter and Defluviicoccus dominated reactor.

Competition between polyphosphate- and glycogen-accumulating organisms (PAOs and GAOs) is problematic in the enhanced biological phosphorus removal (EBPR) process. Aiming at a high phosphorus removal efficiency (PRE), the phosphorus release amount (PRA) is considered an essential evaluating indicator. However, the correlations between PRE and PRA and the abundance of PAOs are not clear. In this study, the EBPR was established and optimized via adjusting influent carbon to phosphorus ratio (C/P). After 110-day operation, 17.67 mg/L of PRA and 75.86% of PRE simultaneously achieved with influent C/P of 40 mgCOD/mgP. As for PAOs, Candidatus_Accumulibacter and Tetrasphaera were absent, while Hypomicrobium (3.69%), Pseudofulvimonas (1.02%), and unclassified_f_Rhodobacteraceae (2.41%) were found at a low level. On the contrary, Candidatus_Competibacter and Defluviicoccus were unexpectedly enriched with high abundance (24.94% and 16.04%, respectively). These results also suggested that it was difficult to distinguish whether PAOs were enriched merely based on the variations of PRA and PRE.

Rapidly achieving partial nitrification of municipal wastewater in enhanced biological phosphorus removal (EBPR) reactor: Effect of heterotrophs proliferation and microbial interactions.

Stable nitritation is the major challenge for short-cut nitrogen removal from municipal wastewater. This paper demonstrated a rapid achievement of partial nitrification (PN) in an enhanced biological phosphorus removal (EBPR) reactor treating domestic wastewater. Polyphosphate accumulating organisms (PAOs) were enriched operated at a short aerobic HRT (2.0 h) and SRT (10 d), with satisfactory phosphorus removal efficiency (95.9%). Both of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) were elutriated simultaneously. Interestingly, AOB recovered much faster than NOB by a subsequent extension of aerobic HRT and SRT, resulting in a rapid development of PN within 15 days. Ammonia oxidation rates of AOB significantly increased by 44.2%, facilitating a high nitrite accumulation rate (NAR) of 95.8%. Genus Tetrasphaera, Halomonas, Paracoccus and Candidatus_Accumulibacter belonging to PAOs accounted for 4.6%. The proliferation of heterotrophs, typically as PAOs, maximized the microbial competition against NOB by favoring AOB activity and synergy with functional bacteria.

Characteristics of enhanced biological phosphorus removal (EBPR) process under the combined actions of intracellular and extracellular polyphosphate.

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.

Nitrite and nitrate inhibition thresholds for a glutamate-fed bio-P sludge.

Enhanced biological phosphorus removal (EBPR) is an efficient and sustainable technology to remove phosphorus from wastewater. A widely known cause of EBPR deterioration in wastewater treatment plants (WWTPs) is the presence of nitrate/nitrite or oxygen in the anaerobic reactor. Moreover, most existing studies on the effect of either permanent aerobic conditions or inhibition of EBPR by nitrate or free nitrous acid (FNA) have been conducted with a "Candidatus Accumulibacter" or Tetrasphaera-enriched sludge, which are the two major reported groups of polyphosphate accumulating organisms (PAO) with key roles in full-scale EBPR WWTPs. This work reports the denitrification capabilities of a bio-P microbial community developed using glutamate as the sole source of carbon and nitrogen. This bio-P sludge exhibited a high denitrifying PAO (DPAO) activity, in fact, 56% of the phosphorus was uptaken under anoxic conditions. Furthermore, this mixed culture was able to use nitrite and nitrate as electron acceptor for P-uptake, being 1.8 µg HNO2-N·L-1 the maximum FNA concentration at which P-uptake can occur. Net P-removal was observed under permanent aerobic conditions. However, this microbial culture was more sensitive to FNA and permanent aerobic conditions compared to "Ca. Accumulibacter"-enriched sludge.

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

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

Achieving simultaneous biological COD and phosphorus removal in a continuous anaerobic/aerobic A-stage system.

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%).

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

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