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 Notably, ATU withdrawn created opportunistic conditions for the unexpected explosive growth and predominance of Thiothrix filaments, leading to a serious bulking event.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.

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.

Layered Extraction and Adsorption Performance of Extracellular Polymeric Substances from Activated Sludge in the Enhanced Biological Phosphorus Removal Process.

A large amount of phosphorus was found in the extracellular polymeric substances (EPS) of activated sludge used in enhanced biological phosphorus removal (EBPR), so the role of EPS and extracellular phosphorus in EBPR should not be neglected. The composition and properties of tightly bound EPS (TB-EPS) and loosely bound EPS (LB-EPS) were significantly different, and it was necessary to study the adsorption performance of EPS through the fractionating of activated sludge into LB-EPS, TB-EPS and microbial cells. In this study, the adsorption performance of LB-EPS and TB-EPS for phosphate was explored by extracting LB-EPS and TB-EPS via sonication and cation exchange resin (CER), respectively. The results indicated that the sonication-CER method was an efficient and reliable extraction method for EPS with a synergistic effect. The performance of EPS in the adsorption/complexing of phosphate was excellent because of its abundant functional groups. Specifically, the type and content of metal elements and functional groups in TB-EPS were much greater than those in LB-EPS, which led to the key role of TB-EPS in the adsorption/complexing of phosphate. Finally, a metabolic model for EBPR with consideration of the adsorption performance of LB-EPS and TB-EPS was proposed.

The link between the microbial ecology, gene expression, and biokinetics of denitrifying polyphosphate-accumulating systems under different electron acceptor combinations.

The emission of the greenhouse gas nitrous oxide (N2O) can occur during biological nutrient removal. Denitrifying enhanced biological phosphorus removal (d-EBPR) systems are an efficient means of removing phosphate and nitrogen, performed by denitrifying polyphosphate-accumulating organisms (d-PAOs). The aim of this work was to study the effect of various combinations of electron acceptors, nitrate (NO3-), nitrite (NO2-), and N2O, on the denitrification pathway of a d-EBPR system. Batch tests were performed with different electron acceptor combinations, to explore the denitrification pathway. Reverse transcriptase-qPCR (RT-qPCR) and high-throughput sequencing, combined with chemical analysis, were used to study gene expression, microbial diversity, and denitrification kinetics. The potential for N2O production was greater than the potential for its reduction in most tests. A strong correlation was observed between the N2O reduction rate and the relative gene expression of nitrous oxide reductase per nitrite reductase (nosZ/(nirS + nirK)), suggesting that the expression of denitrifying marker genes is a strong predictor of the N2O reduction rate. The d-EBPR community maintained a core population with low variations throughout the study. Furthermore, phylogenetic analyses of the studied marker genes revealed that the organisms actively involved in denitrification were closely related to Thauera sp., Candidatus Accumulibacter phosphatis, and Candidatus Competibacter denitrificans. Moreover, Competibacter-related OTUs seem to be important contributors to the N2O reduction capacity of the system, likely scavenging the N2O produced by other organisms. Overall, this study contributes to a better understanding of the microbial biochemistry and the genetics involving biological denitrification removal, important to minimize N2O emissions in wastewater treatment plants.

Community structures and population dynamics of "Candidatus Accumulibacter" in activated sludges of wastewater treatment plants using ppk1 as phylogenetic marker.

Candidatus Accumulibacter has been identified as dominant polyphosphate-accumulating organisms (PAOs) in enhanced biological phosphorus (P) removal (EBPR) from wastewater. This study revealed the relevance of community structure, abundance and seasonal population dynamics of Candidatus Accumulibacter to process operation of wastewater treatment plants (WWTPs) in China using ppk1 gene as phylogenetic marker. All sludge samples had properties of denitrifying P removal using nitrate as an electron acceptor. Accumulibacter abundance in the anaerobic-anoxic-oxic (A2O) process was the highest (26% of total bacteria), and higher in winter than in summer with a better EBPR performance. Type-II was the dominant Accumulibacter in all processes, and type-I accounted for a small proportion of total Accumulibacter. The abundance of Clade-IIC as the most dominant clade reached 2.59×109 cells/g MLSS and accounted for 87.3% of total Accumulibacter. Clade IIC mainly contributed to denitrifying P removal. Clades IIA, IIC and IID were found in all processes, while clade-IIF was only found in oxidation ditch process through phylogenetic analysis. High proportion of clade IID to total Accumulibacter led to poor performance of aerobic P-uptake in inverted A2O process. Therefore, Accumulibacter clades in WWTPs were diverse, and EBPR performance was closely related to the clade-level community structures and abundances of Accumulibacter.

Sulphide effects on the physiology of Candidatus Accumulibacter phosphatis type I.

Sulphate-rich wastewaters can be generated due to (i) use of saline water as secondary-quality water for sanitation in urban environments (e.g. toilet flushing), (ii) discharge of industrial effluents, (iii) sea and brackish water infiltration into the sewage and (iv) use of chemicals, which contain sulphate, in drinking water production. In the presence of an electron donor and absence of oxygen or nitrate, sulphate can be reduced to sulphide. Sulphide can inhibit microbial processes in biological wastewater treatment systems. The objective of the present study was to assess the effects of sulphide concentration on the anaerobic and aerobic physiology of polyphosphate-accumulating organisms (PAOs). For this purpose, a PAO culture, dominated by Candidatus Accumulibacter phosphatis clade I (PAO I), was enriched in a sequencing batch reactor (SBR) fed with acetate and propionate. To assess the direct inhibition effects and their reversibility, a series of batch activity tests were conducted during and after the exposure of a PAO I culture to different sulphide concentrations. Sulphide affected each physiological process of PAO I in a different manner. At 189 mg TS-S/L, volatile fatty acid uptake was 55% slower and the phosphate release due to anaerobic maintenance increased from 8 to 18 mg PO4-P/g VSS/h. Up to 8 mg H2S-S/L, the decrease in aerobic phosphorus uptake rate was reversible (Ic60). At higher concentrations of sulphide, potassium (>16 mg H2S-S/L) and phosphate (>36 mg H2S-S/L) were released under aerobic conditions. Ammonia uptake, an indicator of microbial growth, was not observed at any sulphide concentration. This study provides new insights into the potential failure of enhanced biological phosphorus removal sewage plants receiving sulphate- or sulphide-rich wastewaters when sulphide concentrations exceed 8 mg H2S-S/L, as PAO I could be potentially inhibited.

Effects of different carbon sources on enhanced biological phosphorus removal and "Candidatus Accumulibacter" community composition under continuous aerobic condition.

Previous studies have shown that enhanced biological phosphorus removal (EBPR) performance under continuous aerobic conditions always eventually deteriorates; however, the speed at which this happens depends on the carbon source supplied. The published data suggest that propionate is a better carbon source than acetate is for maintaining operational stability, although it is not clear why. A lab-scale sequencing batch reactor was run initially under conventional anaerobic/aerobic conditions with either acetate or propionate as the carbon source. Chemical and microbiological analyses revealed that both sources performed as expected for such systems. When continuous aerobic conditions were imposed on both these established communities, marked shifts of the "Candidatus Accumulibacter" clades were recorded for both carbon sources. Here, we discuss whether this shift could explain the prolonged EBPR stability observed with propionate.

Enhanced biological phosphorus removal with different carbon sources.

Enhanced biological phosphorus removal (EBPR) process is one of the most economical and sustainable methods for phosphorus removal from wastewater. However, the performance of EBPR can be affected by available carbon sources types in the wastewater that may induce different functional microbial communities in the process. Glycogen accumulating organisms (GAOs) and polyphosphate accumulating organisms (PAOs) are commonly found by coexisting in the EBPR process. Predominance of GAO population may lead to EBPR failure due to the competition on carbon source with PAO without contributing phosphorus removal. Carbon sources indeed play an important role in alteration of PAOs and GAOs in EBPR processes. Various types of carbon sources have been investigated for EBPR performance. Certain carbon sources tend to enrich specific groups of GAOs and/or PAOs. This review summarizes the types of carbon sources applied in EBPR systems and highlights the roles of these carbon sources in PAO and GAO competition. Both single (e.g., acetate, propionate, glucose, ethanol, and amino acid) and complex carbon sources (e.g., yeast extract, peptone, and mixed carbon sources) are discussed in this review. Meanwhile, the environmental friendly and economical carbon sources that are derived from waste materials, such as crude glycerol and wasted sludge, are also discussed and compared.

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.

Two new clades recovered at high temperatures provide novel phylogenetic and genomic insights into Candidatus Accumulibacter.

Candidatus Accumulibacter, a key genus of polyphosphate-accumulating organisms, plays key roles in lab- and full-scale enhanced biological phosphorus removal (EBPR) systems. A total of 10 high-quality Ca. Accumulibacter genomes were recovered from EBPR systems operated at high temperatures, providing significantly updated phylogenetic and genomic insights into the Ca. Accumulibacter lineage. Among these genomes, clade IIF members SCELSE-3, SCELSE-4, and SCELSE-6 represent the to-date known genomes encoding a complete denitrification pathway, suggesting that Ca. Accumulibacter alone could achieve complete denitrification. Clade IIC members SSA1, SCUT-1, SCELCE-2, and SCELSE-8 lack the entire set of denitrifying genes, representing to-date known non-denitrifying Ca. Accumulibacter. A pan-genomic analysis with other Ca. Accumulibacter members suggested that all Ca. Accumulibacter likely has the potential to use dicarboxylic amino acids. Ca. Accumulibacter aalborgensis AALB and Ca. Accumulibacter affinis BAT3C720 seemed to be the only two members capable of using glucose for EBPR. A heat shock protein Hsp20 encoding gene was found exclusively in genomes recovered at high temperatures, which was absent in clades IA, IC, IG, IIA, IIB, IID, IIG, and II-I members. High transcription of this gene in clade IIC members SCUT-2 and SCUT-3 suggested its role in surviving high temperatures for Ca. Accumulibacter. Ambiguous clade identity was observed for newly recovered genomes (SCELSE-9 and SCELSE-10). Five machine learning models were developed using orthogroups as input features. Prediction results suggested that they belong to a new clade (IIK). The phylogeny of Ca. Accumulibacter was re-evaluated based on the laterally derived polyphosphokinase 2 gene, showing improved resolution in differentiating different clades.

The phototrophic metabolic behaviour of Candidatus accumulibacter.

The phototrophic capability of Candidatus Accumulibacter (Accumulibacter), a common polyphosphate accumulating organism (PAO) in enhanced biological phosphorus removal (EBPR) systems, was investigated in this study. Accumulibacter is phylogenetically related to the purple bacteria Rhodocyclus from the family Rhodocyclaceae, which belongs to the class Betaproteobacteria. Rhodocyclus typically exhibits both chemoheterotrophic and phototrophic growth, however, limited studies have evaluated the phototrophic potential of Accumulibacter. To address this gap, short and extended light cycle tests were conducted using a highly enriched Accumulibacter culture (95%) to evaluate its responses to illumination. Results showed that, after an initial period of adaptation to light conditions (approximately 4-5 h), Accumulibacter exhibited complete phosphorus (P) uptake by utilising polyhydroxyalkanoates (PHA), and additionally by consuming glycogen, which contrasted with its typical aerobic metabolism. Mass, energy, and redox balance analyses demonstrated that Accumulibacter needed to employ phototrophic metabolism to meet its energy requirements. Calculations revealed that the light reactions contributed to the generation of, at least more than 67% of the ATP necessary for P uptake and growth. Extended light tests, spanning 21 days with dark/light cycles, suggested that Accumulibacter generated ATP through light during initial operation, however, it likely reverted to conventional anaerobic/aerobic metabolism under dark/light conditions due to microalgal growth in the mixed culture, contributing to oxygen production. In contrast, extended light tests with an enriched Tetrasphaera culture, lacking phototrophic genes in its genome, clearly demonstrated that phototrophic P uptake did not occur. These findings highlight the adaptive metabolic capabilities of Accumulibacter, enabling it to utilise phototrophic pathways for energy generation during oxygen deprivation, which holds the potential to advance phototrophic-EBPR technology development.

Demystifying polyphosphate-accumulating organisms relevant to wastewater treatment: A review of their phylogeny, metabolism, and detection.

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.

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.

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.

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.

CRISPR-Cas phage defense systems and prophages in Candidatus Accumulibacter.

Candidatus Accumulibacter plays a major role in enhanced biological phosphorus removal (EBPR) from wastewater. Although bacteriophages have been shown to represent fatal threats to Ca. Accumulibacter organisms and thus interfere with the stability of the EBPR process, little is known about the ability of different Ca. Accumulibacter strains to resist phage infections. We conducted a systematic analysis of the occurrence and characteristics of clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) systems and prophages in Ca. Accumulibacter lineage members (43 in total, including 10 newly recovered genomes). Results indicate that 28 Ca. Accumulibacter genomes encode CRISPR-Cas systems. They were likely acquired via horizontal gene transfer, conveying a distinct adaptivity to phage predation to different Ca. Accumulibacter members. Major differences in the number of spacers show the unique phage resistance of these members. A comparison of the spacers in closely related Ca. Accumulibacter members from distinct geographical locations indicates that habitat isolation may have resulted in the acquisition of resistance to different phages by different Ca. Accumulibacter. Long-term operation of three laboratory-scale EBPR bioreactors revealed high relative abundances of Ca. Accumulibacter with CRISPSR-Cas systems. Their specific resistance to phages in these reactors was indicated by spacer analysis. Metatranscriptomic analyses showed the activation of the CRISPR-Cas system under both anaerobic and aerobic conditions. Additionally, 133 prophage regions were identified in 43 Ca. Accumulibacter genomes. Twenty-seven of them (in 19 genomes) were potentially active. Major differences in the occurrence of CRISPR-Cas systems and prophages in Ca. Accumulibacter will lead to distinct responses to phage predation. This study represents the first systematic analysis of CRISPR-Cas systems and prophages in the Ca. Accumulibacter lineage, providing new perspectives on the potential impacts of phages on Ca. Accumulibacter and EBPR systems.

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.

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.

The impact of pH on the anaerobic and aerobic metabolism of Tetrasphaera-enriched polyphosphate accumulating organisms.

Members of the genus Tetrasphaera are putative polyphosphate accumulating organisms (PAOs) that have been found in greater abundance than Accumulibacter in many full-scale enhanced biological phosphorus removal (EBPR) wastewater treatment plants worldwide. Nevertheless, previous studies on the effect of environmental conditions, such as pH, on the performance of EBPR have focused mainly on the response of Accumulibacter to pH changes. This study examines the impact of pH on a Tetrasphaera PAO enriched culture, over a pH range from 6.0 to 8.0 under both anaerobic and aerobic conditions, to assess its impact on the stoichiometry and kinetics of Tetrasphaera metabolism. It was discovered that the rates of phosphorus (P) uptake and P release increased with an increase of pH within the tested range, while PHA production, glycogen consumption and substrate uptake rate were less sensitive to pH changes. The results suggest that Tetrasphaera PAOs display kinetic advantages at high pH levels, which is consistent with what has been observed previously for Accumulibacter PAOs. The results of this study show that pH has a substantial impact on the P release and uptake kinetics of PAOs, where the P release rate was >3 times higher and the P uptake rate was >2 times higher at pH 8.0 vs pH 6.0, respectively. Process operational strategies promoting both Tetrasphaera and Accumulibacter activity at high pH do not conflict with each other, but lead to a potentially synergistic impact that can benefit EBPR performance.

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.