Within this research, a one-stage hybrid dual internal circulation airlift A2O (DCAL-A2O) bioreactor was designed and operated to simultaneously remove carbon, nitrogen and phosphorous (CNP) from milk processing wastewater (MPW) in different operational circumstances. The substantial operating variables monitored in this work were including hydraulic retention time (HRT), airflow rate (AFR) and aeration volume ratio (AVR) ranged from 7 to 15 h, 1–3 L/min and 0.324–0.464, respectively. From the view point of economics and process function, the optimum conditions were obtained at the HRT, AFR and AVR of 10 h, 2 L/min and 0.464, respectively. At the optimum conditions TCOD, TN, TP removal efficiencies and effluent turbidity were reported to be 97 %, 90 %, 92 % and 9 NTU, respectively. The impact of wastewater biodegradability (BOD5/COD) was evaluated on the bioreactor performance using two other wastewaters i.e. soft drink (SDW) and soybean oil plant wastewaters (SOW) in comparison with the MPW. Removal efficiencies for TCOD and TN exceeding 80 % were observed. The feeding location revealed a prominent impact on the TN and phosphorous removal efficiencies (both ≥80 %) related to the availability degree of the readily biodegradable organic substrate to denitrifiers and PAOs. The rise in HRT, AFR and AVR resulted in reducing microbial secretions as SMP present in sludge and bioreactor effluent as well as loosely bounded EPS (LB-EPS), reported to be 26, 28, 32.5 and 194.4 mg/L TOC, respectively. Different bacteria species were present at optimum conditions confirming concurrent CNP removal in a single body. Finally, the operating cost evaluation verified the effectiveness of the hybrid airlift A2O treating the MPW.@en

Methane removal is an essential step in drinking water production from methane-rich groundwaters. Conventional aeration-based stripping results in significant direct methane emissions, contributing up to one-third of a treatment plant's total carbon footprint. To address this, a full-scale trickling filter was operated for biological methane oxidation upstream of a submerged sand filter, and its performance was compared to a conventional aeration–submerged sand filtration set-up. Full-scale data were combined with ex-situ batch assays and metagenome-resolved metaproteomics to quantify the individual contribution of the main (a)biotic processes and characterize the enriched microbial communities. Both treatment setups fully removed methane, iron, ammonium, and manganese, yet the underlying mechanisms differed significantly. Methane was completely removed from the effluent after trickling filtration, with stripping and biological oxidation each accounting for half of the removal, thereby halving overall methane emissions. Methane-oxidizing bacteria not only outcompeted nitrifiers in the trickling filter, but also likely contributed directly to ammonia oxidation. In contrast to the submerged filter preceded by methane stripping, signatures of biological iron oxidation were almost completely absent in the trickling filter, suggesting that the presence of methane directly or indirectly promotes chemical iron oxidation. All systems had similar ex-situ manganese oxidation capacities, yet removal occurred only in the submerged filters but not the trickling filter. Ultimately, our results demonstrate that trickling filtration is effective in promoting biological methane oxidation at comparable produced drinking water quality, highlighting its potential for advancing sustainable drinking water production.@en

Polyhydroxyalkanoate (PHA) production is a promising technology fostering the spread of the circular bio-economy approach. However, the environmental implication of the process is usually neglected. This paper shows the results of a membrane-based PHA production pilot plant fed with no-pretreated waste activated sludge (WAS). The system was monitored for effluent water quality, nitrous oxide (N₂O), and PHA production by dynamic accumulation over a long-term period to assess the consistency of the results over several fluctuations. The experimental study was characterized by three C/N ratios of 9, 4.5, and 4 g COD/g N. The system achieved a stable and high removal efficiency for carbon and nitrogen (96.3 ± 2.6 % and 89.9 ± 6.7 %, respectively), despite the only legislation limit respected being the biological oxygen demand concentration discharge limits imposed by 2020/741/EU. Low N₂O gaseous and liquid concentrations were achieved over the 200-day experimental period, never exceeding 0.52 mg N₂O-N/L. Despite the high concentration, the N₂O emission factor accounted for only 0.21 ± 0.14 % of the influent nitrogen. Finally, the system produced an average of 36.3 ± 1.8 % g PHA/g VSS with a storage yield of up to 0.42 g COD_PHA/g COD_VFA. The system revealed a high stability over a long-term experimental period, achieving a considerable amount of PHA while maintaining a low N₂O emission. Promising effluent water quality was achieved, highlighting the potential of applying the water reuse practices.

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Water treatment facilities are bound to incorporate resource recovery in the near future, necessitating novel economic assessments that capture the full economic potential of these systems. This study evaluates three cost calculation methods—Non-allocation, Economic allocation, and Dual allocation— to improve the accuracy of the Levelized Cost for multi-product desalination and brine treatment plants. The methods were tested across three technical scenarios: Sc1) maximum water recovery, Sc2) integrated desalination with brine treatment for resource recovery and Sc3) electricity-based desalination for chemical recovery. Results reveal that the traditional Non-allocation method tends to overestimate production costs by uniformly applying fixed costs across products, leading to inflated levelized costs. The Economic allocation approach reduces the levelized costs of water and other recovered products by up to 81 %, enhancing competitiveness with conventional production methods. The Dual allocation approach is most effective for recovered salts and chemicals, ensuring fair cost distribution and fostering competitiveness with linear systems. Sc2 is the most economically feasible under both novel approaches due to its balanced mix of high-value products and moderate operational costs. These findings suggest that cost calculation methods should align with plant objectives: Economic allocation for scenarios prioritizing water recovery and Dual allocation for maximizing the value of salts and chemicals. This study provides a foundation for tailored economic assessments and guides plant design and investment decisions.

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Aerobic granular sludge (AGS) technology holds great promise of becoming the standard for biological wastewater treatment due to its lower energy consumption, small footprint, and high removal efficiency of nutrients compared to the conventional activated sludge processes. Different-sized aggregates have been shown to harbor a different microbial community composition. The central question is do full-scale AGS wastewater treatment plants (WWTPs) select for core microbial communities across different aggregate sizes and how these selected organisms differ between the different-sized aggregates. This study analyzed samples from nine geographically distributed full-scale AGS WWTPs that consistently perform well in terms of chemical oxygen demand (COD) and nutrient (N and P) removal. The main results showed that site-specific conditions highly influence microbial composition in smaller aggregates (< 1 mm), while larger granules form stable communities independent of WWTP location. Notably, all aggregates contained a small subset of 128–139 core OTUs that were both prevalent and abundant across all sizes. These core OTUs include key functional groups such as fermenters, aerobic heterotrophs, polyphosphate-accumulating organisms (PAOs), glycogen-accumulating organisms (GAOs), and nitrifiers, which play a crucial role in COD and nutrient removal. Additionally, an enrichment pattern was observed, with aerobic heterotrophs dominating in flocs, PAOs in small granules, and GAOs and nitrifiers in large granules. This study offers valuable insights into the core microbiome of different-sized aggregates in full-scale AGS WWTPs and highlights their potential role in overall system performance.

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While nowadays a lot of measurements are conducted at wastewater treatment plants, data reliability could further be improved, e.g., through data reconciliation. This study demonstrated the added value of data reconciliation to improve data quality in a full-scale wastewater treatment plant. Also, the effect of the mass balance setting (linear and bilinear mass balances) was quantitatively evaluated, considering data sets with missing measurements and with gross errors. The improvement in the precision of the key variables was higher with bilinear mass balances (40–80 %) compared to the linear setting (0–70 %). Besides, it delivered a higher number of improved key variables, especially when flow measurements were limited (minimum improved variables of 15 and 0, respectively). Bilinear mass balances were also more efficient in gross error detection and played a crucial role in cross-validation based on flow measurements, resulting in lower incorrectly-identified gross errors. Overall, it is recommended to use bilinear mass balances.

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The hydrolysis of structural extracellular polymeric substances (St-EPS) is considered a major limiting step in the anaerobic fermentation of waste activated sludge (WAS). However, the degradation of heteropolysaccharides, characterized by complex monomers of uronic acids and neutral saccharides in St-EPS, has rarely been reported. In this study, microbial-produced xanthan-like heteropolysaccharides, characterized by a blue filamentary film, were identified. The xanthan-producing bacteria comprised ∼7.2% of total genera present in WAS. An xanthan-degrading consortium (XDC) was enriched in an anaerobic batch reactor. This consortium could degrade Xanthan for over 90% and disrupt the gel structure of xanthan while promoting methane production from WAS by 29%. The xanthan degradation network consisting of extracellular enzymes and bacteria was elucidated by combining high-throughput sequencing, metagenomic, and metaproteomic analyses. Five enzymes were identified as responsible for hydrolyzing xanthan to monomers, including xanthan lyase, β-D-glucosidase, β-D-glucanase, α-D-mannosidase, and unsaturated glucuronyl hydrolase. Seven genera, including Paenibacillus (0.2%) and Clostridium (3.1%), were identified as key bacteria excreting one to five of the aforementioned enzymes. This study thus provides insights into the complex conversions in anaerobic digestion of WAS and gives a foundation for future optimization of this process.

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The objective of this study was to treat different types of industrial wastewaters (milk processing wastewater, soft drink wastewater, and soybean oil plant wastewater) using modified configurations of a bioreactor called one-stage internal dual circulation airlift A2O (DCAL-A2O) bioreactor. The modification involved the use of physical barriers (baffle and packing media) to enhance the anoxic zone of the bioreactor. The study investigated the performance of bioreactors in simultaneously removing carbon, nitrogen, and phosphorus. The bioreactor was designed in three configurations: ordinary, baffled, and hybrid DCAL-A2O bioreactors, depending on the type of manipulation. Different operating conditions, such as hydraulic retention time (HRT), air flow rate (AFR), and anaerobic volume ratio (AVR), were examined simultaneously. The hybrid DCAL-A2O bioreactor was found to be highly suitable for wastewater treatment due to its superior biological performance, ease of operation, and cost-effectiveness when compared to two other bioreactors. The chosen bioreactor demonstrated high performance in terms of TCOD, TN, phosphorus removal efficiencies, and effluent turbidity. Specifically, it achieved removal efficiencies of 97.0% for TCOD, 92% for TN (with a concentration of 179.4 mg/L), 90% for phosphorus (with a concentration of 50.33 mg/L), and an effluent turbidity of 9 NTU. These results were obtained under optimal conditions, which included a HRT of 10 h, an AFR of 2 L/min, and an AVR of 0.464. The unique setup of the bioreactor demonstrated its undeniable ability to effectively treat wastewater from different feeding points. PCR tests confirmed the co-existence of multiple functional bacterial species, including AOB, NOB, denitrifying bacteria, anammox, PAOs, DPAOs, and GAOs. This provides strong evidence for concurrent removal of organics and nutrients in all three configurations of the integrated unit. In summary, this study emphasizes the need for ongoing research in energy efficiency to safeguard our environmental resources.@en

Sensor availability and costs are nowadays no longer limiting data gathering at wastewater treatment plants (WWTPs). However, one should be aware that a higher amount of measured data gathered does not necessarily imply that also more information is obtained. In this light, this contribution assesses the general applicability and the added value of a structured experimental design approach for planning measurement campaigns at WWTPs, in view of mass-balance-based data reconciliation. To this end, the results from full-scale WWTP case studies available in the literature were compared to those obtained with the developed structured experimental design procedure. Planning measurement campaigns comprises the selection of (additional) measurements to meet a pre-set main goal. The need for a structured experimental design procedure replacing past expert judgment approaches became clear from the fact that three out of five case studies available in the literature failed to meet the main goal and/or performed unnecessary additional measurements. Translating the main goal into specific key variables was found essential in this respect. The general applicability of the procedure was proven with three outcomes. First, the procedure, involving well-defined steps, could be applied to different WWTP layouts. Second, it ensured the fulfilment of various main goals. Third, it provided useful outcomes, i.e., optimal measurement campaigns, which reduced the need for additional measurements (40-70% less) compared to expert knowledge approaches, hence more information could be obtained with less analytical data. Overall, the experimental design procedure proved a fast and useful tool ensuring the success of subsequent mass-balance-based data reconciliation.

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Extracellular Polymeric Substances (EPS) are ubiquitous in biological wastewater treatment (WWT) technologies like activated sludge systems, biofilm reactors, and granular sludge systems. EPS recovery from sludge potentially offers a high-value material for the industry. It can be utilized as a coating in slow-release fertilizers, as a bio-stimulant, as a binding agent in building materials, for the production of flame retarding materials, and more. P recovered within the extracted EPS is an intrinsic part of the recovered material that potentially influences its properties and industrial applications. P is present in EPS in different speciation (e.g., P esters, poly-P, ortho-P, etc.). Such P species are already intensively used in the chemical industry to enhance thermal stability, viscoelasticity, emulsification, water-holding capacity, and many other properties of some natural and petroleum-derived polymers. The translation of this knowledge to EPS is missing which prevents the full utilization of phosphorus in EPS. This knowledge could allow us to engineer EPS via phosphorus for specific target properties and applications. In this review, we discuss how P could affect EPS properties based on experiences from other industries and reflect on how these P species could be influenced during the EPS extraction process or in the WWTPs.

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The synthesis of chemically derived bituminous-based activated carbons (ACs) was conducted in this research. The study examined the efficacy of the KOH-2 modified AC by conducting batch experiments under different operating settings. Pollutant concentration, adsorbent dosage and contact time were considered as variables. Bovine serum albumin (BSA), dextrose, and methylene blue (MB) were used as model substances to represent protein, carbon, and textile dyes, respectively. Under optimal conditions, the activated carbon modified with a 2:1 impregnation ratio using KOH showed the best performance among two other ACs, therefore, that was introduced as chosen AC. The optimal parameters were determined as pollutant concentration, adsorbent dosage and contact time obtained to be 600 mg/L, 0.22 g/L and 220 min; 200 mg/L, 0.6 g/L and 180 min; 200 mg/L, 1 g/L and 196 min for MB, BSA and dextrose, respectively. This AC obtained the highest adsorption capacity of 2710.6, 283, and 273 mg/g adsorbing MB, BSA and dextrose, respectively. In the following, column studies were conducted using biologically treated wastewater obtained from a new one-stage hybrid internal circulation airlift A2O bioreactor to eliminate soluble microbial products (SMP). It was seen that the adsorption column, which was filled with the AC under optimal experimental conditions (bed height of 4.5 cm and solution flow rate of 5 mL/min), resulted in a decrease in residual organic substances from 35 mg-TOC/L to 6.6 mg-TOC/L. It is worth mentioning that the utilization of an integrated approach involving sophisticated biological treatment and post-treatment technology has demonstrated efficacy in the elimination of residual organic substances.

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Polyhydroxyalkanoates (PHA) can be produced using fermentation products of an excess sewage sludge fermentation process. An efficient method to enrich a PHA-producing community is an aerobic-feast/anoxic-famine enrichment strategy. The effect of different carbon to nitrogen (C/N) feed ratios of 1, 2 and 3.5 g COD/g N on the process performance was studied. The study was executed on a pilot plant scale using fermented waste activated sludge as the organic carbon source. The system's performance was monitored in terms of removing contaminants, producing PHA, and reducing N2O emissions. The results indicated that a lower C/N ratio results in lower PHA production, with PHA content in the sludge of 20, 24 and 36 % w/w for C/N ratios of 1, 2 and 3.5 g COD/g N, respectively. At the lowest C/N ratio, the highest nitrite accumulation rate (77 %), nitrification efficiency (89 %) and denitrification efficiency (89 %) were observed, but the N2O production was also the highest (0.77 mg N2O-N/L). The long-term comprehensive monitoring carried out in this study revealed high carbon and ammonia removal efficiencies (never below 80 %) despite the C/N shifts and high COD and ammonia concentrations. At the same time, the system showed relatively low PHA production and high environmental impact in terms of high gaseous N2O emission. These findings question the sustainability of the aerobic-feast/anoxic-famine enrichment strategy for PHA production in full-scale plants.@en

Division of metabolic labour is a defining trait of natural and engineered microbiomes. Denitrification-the stepwise reduction of nitrate and nitrite to nitrogenous gases-is inherently modular, catalysed either by a single microorganism (termed complete denitrifier) or by consortia of partial denitrifiers. Despite the pivotal role of denitrification in biogeochemical cycles and environmental biotechnologies, the ecological factors selecting for complete versus partial denitrifiers remain poorly understood. In this perspective, we critically review over 1500 published metagenome-assembled genomes of denitrifiers from diverse and globally relevant ecosystems. Our findings highlight the widespread occurrence of labour division and the dominance of partial denitrifiers in complex ecosystems, contrasting with the prevalence of complete denitrifiers only in simple laboratory cultures. We challenge current labour division theories centred around catabolic pathways, and discuss their limits in explaining the observed niche partitioning. Instead, we propose that labour division benefits partial denitrifiers by minimising resource allocation to denitrification, enabling broader metabolic adaptability to oligotrophic and dynamic environments. Conversely, stable, nutrient-rich laboratory cultures seem to favour complete denitrifiers, which maximise energy generation through denitrification. To resolve the ecological significance of metabolic trade-offs in denitrifying microbiomes, we advocate for mechanistic studies that integrate mixed-culture enrichments mimicking natural environments, multi-meta-omics, and targeted physiological characterisations. These undertakings will greatly advance our understanding of global nitrogen turnover and nitrogenous greenhouse gases emissions.

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Vivianite (Fe₃(PO₄)₂·8 H₂O) has emerged as a promising mineral for phosphorus (P) recovery from digested sludge, and it may also contribute to phosphate management in lake sediments and manure, given the similar anaerobic conditions across these environments. However, organic ligands in these matrices have been proposed to complex with iron (Fe), thereby reducing the efficiency of vivianite formation. This study aims to elucidate the impact of organic ligands on vivianite formation, particularly focusing on their binding strength with iron and the subsequent effects on vivianite formation in pig manure. Organic ligands not only form complexes with iron but also influence the crystal growth process. We investigated how different organic ligands affect the formation and dissolution of vivianite, assuming that ligands with higher iron-binding strength would enhance phosphate solubilization. Our findings revealed that citrate nearly completely inhibited vivianite formation (up to 100 %) and caused a 50 % dissolution of existing vivianite, while humate hindered vivianite formation by 40 % but only led to a 10 % dissolution. Interestingly, pig-derived dissolved organic matter had minimal effects on the precipitation of iron and phosphorus but significantly altered the morphology of the resulting products, which varied depending on the age of the manure filtrate. While the iron binding strength of organic ligands does influence vivianite formation, it does not solely account for the reduced vivianite formation observed in complex matrices like manure. Therefore, a more nuanced assessment of the role of organic matter in vivianite formation is warranted.

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Valuable and rare materials in seawater brine are often discarded during desalination. However, there is an increasing focus on recovering these resources, due to the economic and environmental opportunities it can bring. Despite this shift, current Sustainability Assessments (SA) in desalination overlook the brine handling and social dimensions, and brine treatment assessments remain centered on techno-economic dimensions. This work proposes a comprehensive framework for the SA of integrated desalination and resource recovery options, focusing on recovering valuable materials from brine. The framework not only evaluates pre-defined systems but supports the identification of system features of interest, such as products to assess and technologies to include, as well as the transparent selection of indicators, considering specific contexts. To develop this framework, a review of the literature on SA in desalination and brine treatment systems was conducted. Looking at the identified gaps, we synthesized the findings and key messages and proposed the integration of Multi-Criteria Analysis and Value-Sensitive Design in the decision-making process. This allows stakeholders to be involved and incorporates their values at different stages of the assessment, making it distinct from traditional SA methods. This framework offers structured guidance to stakeholders on how to carry out qualitative and quantitative assessments while ensuring transparency in the assessment process.@en

An interesting and potential property of extracellular polymeric substances (EPS) is the hydrogel formation with calcium ions. Aiming at understanding the significant difference in the hydrogel formed between EPS from flocculent and granular sludge, a targeted investigation of the lipopolysaccharides (LPS), one of the important EPS components, was performed. LPS was isolated from the EPS of flocculent and granular sludge, and both the glycan and the lipid A parts of LPS were characterized and compared. The morphology of LPS-calcium (LPS-Ca) aggregates were visualized by the polymyxin B-based fluorescent probe. The LPS constituted about 25 % and 15 % of the EPS from flocculent and granular sludge, respectively. The flocculent sludge LPS showed a lower amount of glycans, shorter glycan chain length, lower molecular weight, and higher possibility of containing unsaturated lipids than the granular sludge EPS. The flocculent sludge LPS-Ca aggregates demonstrated invert structures with the water phase in between, contributing to the fluid-like property of the respect EPS-Ca. In contrast, with the remarkably different chemical structure, LPS-Ca aggregates from granular sludge displayed bilaminar multilayered morphology, contributing to the solid, self-standing hydrogel of EPS-Ca.@en

The emphasis on phosphorus removal and recovery from wastewater treatment plants has intensified in recent years due to the urgent need to reduce dependency on nonrenewable phosphorus reserves. Enhanced biological phosphorus removal (EBPR), driven by a diverse community of polyphosphate-accumulating organisms with distinct metabolic capabilities, offers several advantages over chemical precipitation methods. These benefits include reduced chemical use, lower sludge volumes, decreased reliance on costly chemical precipitants, and improved phosphorus recovery quality. Recent advancements in recovery technologies now enable efficient phosphorus extraction from digester supernatant, dewatered digested sewage sludge, and sewage sludge ash, each yielding different recovery efficiencies. Despite these advances, a comprehensive assessment of the phosphorus recovery potential from these target streams in conjunction with EBPR remains crucial and has yet to be fully explored.@en

The post-pandemic world still faces ongoing COVID-19 infections, although international travel has returned to pre-pandemic conditions. Wastewater-based epidemiology (WBE) is considered an efficient tool for the population-wide surveillance of COVID-19 infections during the pandemic. However, the performance of WBE in post-pandemic era with travel restrictions lifted remains unknown. Utilizing weekly county-level wastewater surveillance data from June 2021-November 2022 for 222 counties in 49 states (covering 104 million people) in the United States of America, we retrospectively evaluated the correlations between SARS-CoV-2 RNA (CRNA) and reported cases, as well as the impacts of international air travel, demographics, socioeconomic aspects, test accessibility, epidemiological, and environmental factors on reported cases under the corresponding CRNA. The lifting of travel restrictions in June 2022, shifted the correlation between CRNA and COVID-19 incidence in the following 7-day and 14-day from 0.70 (IQR: 0.30–0.88) in June 2021-May 2022 (pandemic) to 0.01 (IQR: -0.31–0.36) in June-November 2022 (post-pandemic), and from 0.74 (IQR: 0.31–0.90) to -0.01 (IQR: -0.38–0.45), respectively. Besides, after lifting the travel restrictions, under the same CRNA, the reported case numbers were impacted by many factors, including the variations of international passengers, test accessibility, Omicron prevalence, ratio of population aged between 18 and 65, minority vulnerability, and healthcare system. This highlights the importance of demographics, infection testing, variants and socioeconomic status on the accuracy and implication of WBE to monitor COVID-19 infection status in post-pandemic era. Our findings facilitate the public health authorities to dynamically adjust their WBE-based tools/strategies to the local contexts to achieve optimal community surveillance.@en

In recent decades, there has been a growing focus on the management and valorization of digestate, primarily driven by its nutrient-rich composition, which positions it as a promising resource for biofertilizer production. However, several countries still restrict the direct application of digestate due to its potential environmental hazard, which includes the presence of contaminants of emerging concern (CECs) such as pharmaceuticals. This paper explores the efficacy of a novel UV/ozone-based technology, UVOX Redox®, in removing prevalent pharmaceuticals, including antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs), from the digestate of two biogas plants. In both cases, UVOX showed to be a feasible solution for pharmaceutical removal from digestate. Addition of hydrogen peroxide further increased the process efficiency, achieving > 90% removal of all compounds within an hour. The energy per order (E_EO) value for all the studied pharmaceuticals was less than the reported median E_EO for O₃ and UV treatment, showcasing notable energy efficiency in UVOX technology. Moreover, the research highlights that the presence of ions augments the removal efficiency when applying the UVOX technology. In addition, the research results revealed a significant correlation between the effectiveness of the UVOX technology and UV transmittance, with R² exceeding 90% for pharmaceuticals and 75% for Dissolved Organic Carbon (DOC). This finding suggests that UV transmittance can serve as a viable surrogate method for implementing this advanced oxidation process in practical applications.

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Currently, the most cost-effective and efficient method for phosphorus (P) removal from wastewater is enhanced biological P removal (EPBR) via polyphosphate-accumulating organisms (PAOs). This study integrates a literature review with genomic analysis to uncover the phylogenetic and metabolic diversity of the relevant PAOs for wastewater treatment. The findings highlight significant differences in the metabolic capabilities of PAOs relevant to wastewater treatment. Notably, Candidatus Dechloromonas and Candidatus Accumulibacter can synthesize polyhydroxyalkanoates, possess specific enzymes for ATP production from polyphosphate, and have electrochemical transporters for acetate and C4-dicarboxylates. In contrast, Tetrasphaera, Candidatus Phosphoribacter, Knoellia, and Phycicoccus possess PolyP-glucokinase and electrochemical transporters for sugars/amino acids. Additionally, this review explores various detection methods for polyphosphate and PAOs in activated sludge wastewater treatment plants. Notably, FISH-Raman spectroscopy emerges as one of the most advanced detection techniques. Overall, this review provides critical insights into PAO research, underscoring the need for enhanced strategies in biological phosphorus removal.

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