Polyphosphate-accumulating Organisms Research Articles

Effect of return activated sludge diversion ratio on phosphorus removal performance in side-stream enhanced biological phosphorus removal (S2EBPR) process

In this study, a lab-scale continuous flow side-stream enhanced biological phosphorus (P) removal (S2EBPR) reactor was operated for 247 days treating synthetic wastewater with influent carbon to phosphorus (C/P) ratio of 25.0 g COD/g P and influent PO43–-P of 7.4 ± 0.3 mg P/L. The effect of the return activated sludge (RAS) diversion ratio on S2EBPR reactor was investigated by comparing P removal performance, microbial activity, and community structure. The results showed that the RAS diversion ratio of 8.0%, by yielding a side-stream sludge retention time (SRTSS) of ∼60 h, resulted in the lowest effluent PO43–-P concentration of 0.5 ± 0.3 mg P/L. The results of in situ process profiles and ex situ P release and uptake batch tests under different RAS diversion conditions showed that the more anaerobic P release was obtained in the side-stream reactor, the higher the P removal efficiency and EBPR activity were achieved. The stoichiometric ratios observed in EBPR activity tests indicated a polyphosphate accumulating organisms (PAOs) metabolism mainly dependent on the glycolysis pathway. The results of microbial ecology analysis revealed that the optimized SRTSS would give a competitive advantage to PAOs in the S2EBPR process. By obtaining statistically reliable results, this study would provide guidance for wastewater treatment plants to achieve optimal P removal performance in S2EBPR configuration.

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

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

Towards a better understanding concerning carbon to nitrogen ratio and the carbon source in aerobic granular sludge

This study investigated the effects of the carbon-to-nitrogen (C/N) ratio on aerobic granular sludge (AGS) using two identical biological reactors (C/N = 5 and C/N = 10). After 84 days of operation, the reactors' organic substrate was switched from a simple synthetic carbon source (acetate and propionate) to a more complex carbon source (50% volatile fatty acids and 50% fermentable substrate). The C/N = 10 resulted in smaller, uniform granules (200 < Diameter(D)(μm) < 500) with improved settling characteristics (sludge volumetric index - SVI5 < 30 mL gTSS L−1). The nitrogen compounds in the influent influenced the protein/polysaccharide ratio (PN/PS), with higher PS content observed under C/N = 5 and lower PS content under C/N = 10. Both C/N ratios achieved high carbon removal rates (∼90%), but ammonia oxidation was lower under C/N = 5 (up to 45%) due to alkalinity deficiency. Insufficient alkalinity and higher nitrogen compounds in the influent negatively affected phosphorus removal by reducing the activity of phosphorus-accumulating organisms and promoting the growth of glycogen-accumulating organisms. The complexity of the organic substrate hindered microbial activities through intermediate products from glucose and amino acid metabolic pathways. EPS-producing denitrifying organisms (Thauera and Xanthomonas) were favored in C/N = 5, while at C/N = 10, organisms associated with granular biomass stability (Zoogloea and Flavobacterium) were selected. The addition of glucose in the effluent led to a significant increase in fermentative organisms (Aeromonadaceae). This study emphasizes the crucial role of the C/N ratio and organic substrate complexity in achieving optimal performance in AGS systems.

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.

Pilot-scale aerobic granular sludge reactors with granular activated carbon for effective nitrogen and phosphorus removal from domestic wastewater

Aerobic granular sludge (AGS) is a breakthrough biotechnology of 21st century and an innovative alternative to activated sludge for treating wastewater. Concerns on long-start up periods for development of AGS and stability of granules are impeding its widespread implementation for treating low-strength domestic wastewater especially in tropical climate conditions. Addition of nucleating agents have been shown to improve development of AGS while treating low-strength wastewaters. There are no previous studies on AGS development and biological nutrient removal (BNR) in the presence of nucleating agents during treatment of real domestic wastewater. This study investigated AGS formation and BNR pathways while treating real domestic wastewater in a 2 m3 pilot-scale granular sequencing batch reactor (gSBR) operated without and with granular activated carbon (GAC) particles. The gSBRs were operated under tropical climate (T ≈ 30 °C) for >4-years to evaluate the effect of GAC addition on granulation, granular stability and BNR at pilot-scale. Formation of granules was observed within 3 months. MLSS values of 4 and 8 g/L were recorded within 6 months in gSBRs without and with GAC particles, respectively. The granules had an average size of 1.2 mm and SVI5 of 22 mL/g. Ammonium was mainly removed through nitrate formation in the gSBR without GAC. But, ammonium was removed by short-cut nitrification via nitrite due to washout of nitrite oxidizing bacteria in the presence of GAC. Phosphorus removal was much higher in gSBR with GAC due to the establishment of enhanced biological phosphorus removal (EBPR) pathway. After 3 months, the phosphorus removal efficiencies were at 15 % and 75 %, respectively, without and with GAC particles. The addition of GAC led to moderation in bacterial community and enrichment of polyphosphate-accumulating organisms. This is the first ever report on pilot-scale demonstration of AGS technology in the Indian sub-continent and GAC addition on BNR pathways.

Mechanisms of persistence and impact of ordinary heterotrophic organisms in aerobic granular sludge

The stability of granules, contaminant removal and microbial structure of an aerobic granular sludge (AGS) process were investigated with a focus on ordinary heterotrophic organisms (OHOs). Long-term stable granules and high removals of COD (97 %), NH4+ (98 %), P (85 %) and total N (77 %) were achieved. Sequencing analyses identified 6.6 % of phosphorus-accumulating organisms in the sludge, concordant with the observed bio-P removal capacity. However, OHOs were the most abundant bacteria in the sludge (70–93 %) without resulting in unstable aggregates. Under current dogmas of microbial competition in activated sludge, it seemed contradictory that OHOs could persist in the long term in the AGS where COD was depleted beginning in the anaerobic phase. Microbial analyses showed that OHOs could survive in granules by micropredation, proteolysis, fermentation and EPS consumption. Heterotrophic-nitrification/ aerobic-denitrification was an active pathway in the AGS. These findings contribute to a better understanding of microbial competition in AGS and its stability.

Assessing the impact of increasing phenol loads on the performance and stability of an aerobic granular sludge system

This work evaluated the impact of phenol on the performance and stability of an aerobic granular sludge (AGS) sequencing batch reactor (SBR) operated under alternating anaerobic-aerobic conditions for simultaneous organic matter, nitrogen, and phosphorus removal. The reactor was fed with synthetic wastewater whose phenol concentration was gradually increased from 2 to 200 mg L−1, while the other carbon source (acetate) was reduced proportionally to keep the organic loading rate invariable. Up to 20 mg L−1 of phenol, the average COD removal was above 90%, while practically full ammonium, phosphorus, and phenol removal was achieved. However, at higher phenol concentrations, the activity of polyphosphate-accumulating organisms and nitrifiers was affected. Although the overall COD removal was only slightly affected by increasing phenol loads, replacing the main carbon source from acetate to phenol changed the organic matter removal profile over the cycle, with the latter being converted mainly within the aerated stage of the SBR. As a result, there was filamentous growth that deteriorated the settling properties of the granules. The establishment of an anaerobic feast-aerobic famine regime over the non-aerated and aerated phases of the AGS SBR and the presence of a readily biodegradable substrate (such as acetate) proved to be crucial for maintaining granules stability. Having phenol as the sole organic carbon source led to complete process failure regarding nutrient removal capability.

Comparison of eco-improvement on constructed wetlands with nano zero valent iron introduction under different levels of PFOA stress: Perspectives on plant, microbe, and PFOA removal

The ecological hazards of perfluoro octanoic acid (PFOA, a typical perfluoroalkyl substances) have been continually reported in constructed wetlands (CWs) for wastewater treatment. In present study, nano zero valent iron (nZVI) was adopted to alleviate PFOA stress at different levels (1 and 10 mg/L) in CWs. It was revealed that the effects of nZVI on specific ecological parameters varied at different PFOA dosages. PFOA influenced plant photosynthetic and antioxidant parameters with significant concentration-dependence. NZVI addition caused more obvious promotion of chlorophyll (25.30–31.84 %) and reduction of catalase (172.64 %) and malondialdehyde (83.01 %) with 10 mg/L PFOA exposure. For microbe, nZVI was prone to stimulate enzyme activities under 1 mg/L PFOA, in which the relative activity of dehydrogenase, urease, phosphatase, and four nitrogen cycling enzymes increased by 86.25–375.56 %, 43.10–71.16 %, 1.52–29.38 %, and 4.49–315.18 %. However, nZVI caused more abundant of functional bacteria (like nitrifying bacteria and phosphorus-accumulating organisms) and function genes (like amoA, hao, and ppx) with PFOA at 10 mg/L. On the whole, changes in bacterial community confirmed the enhancement potential of nZVI on ammonium and phosphorous removal. PFOA removal at 10 mg/L was higher compared to 1 mg/L, resulting from higher abundance of class Gammaproteobacteria, and nZVI addition further contributed to the highest removal efficiency (73.54 %). This study provided evidence on nZVI as a possible manner for optimizing eco-function in CWs with PFOA stress at different levels.

Enhanced anammox through endogenous partial denitrification/anammox (EPDA) granular sludge: Granule formation, treatment performance, and multi-perspective microbial analysis

The direct application of anammox to wastewater treatment is valuable, but the enrichment of anammox bacteria (AnAOB) is complicated due to the malignant competition of heterotrophic bacteria. Here, we propose the use of endogenous partial denitrification/anammox (EPDA) granular sludge to enhance anammox while achieving simultaneous removals of nitrogen and phosphorus. After operation for 330 days, the EPDA granules were successfully cultured with the mean size of 0.89 mm. Nitrogen and phosphorus were efficiently removed with the effluent total nitrogen (TN) and PO43--P of 6.68±0.41 mg/L and 0.06±0.03 mg/L, respectively. Anammox took a leading role in the removal of TN with a contribution of 75±3%. Moreover, high-throughput sequencing (16S rRNA) results showed that AnAOB could coexist harmoniously with glycogen accumulating organisms (GAOs) and polyphosphate accumulating organisms (PAOs) in the EPDA granules, whereas mature EPDA granules could promote in situ enrichment of AnAOB. Microbial stratification analysis of the EPDA granule found that the inner layer was resided with AnAOB, while the outer layer was employed with GAOs and PAOs. Metagenomic and metatranscriptomic analyses found that the nitrate reductase (1150.56 RPM) was 4.6 times of nitrite reductase (249.98 RPM) at the transcriptome level. Moreover, the key enzymes related to anammox (hydrazine synthase and hydrazine dehydrogenase) were much higher than the other nitrogen conversion functional genes at the transcriptome level, confirming the main role of AnAOB in the removal of nitrogen. This study presents a novel strategy for the application of anammox from EPDA granular sludge.

Growing an Enhanced Culture of Polyphosphate-Accumulating Organisms to Optimize the Recovery of Phosphate from Wastewater

Having certain bacteria called phosphorus-accumulating organisms (PAOs) is important for getting rid of phosphorus (P) in wastewater from homes. This happens in a process called enhanced biological phosphorus removal (EBPR), where PAOs are active in activated sludge. To design and make EBPR processes work better, we need to have an in-depth understanding of how PAOs work. The best way to learn about them is by studying them in a laboratory. This study undertook to culture these microorganisms in the laboratory. A University of Cape Town membrane bioreactor (UCTMBR) activated sludge (AS) system was used to grow the microorganisms and see how well it worked. This paper looked at what type of substrate PAOs like best, either acetate or propionate, and how providing them with more of their preferred substrate affects how they grow. During the process, it was observed that P was not released or taken up significantly when acetate was added to the influent. The levels were consistently low at around 5.74 ± 4.47 mgP/L infl (release) and 19.9 ± 7.17 mgP/L infl (uptake). The signs become much better when propionate was used instead of acetate. When the amount of propionate in the influent was increased from 50% to 76% (as a percentage of influent total chemical oxygen demand), the amount of P released went up to 155 ± 17.7 mgP/L infl, and the amount of P taken up went up to 213.7 ± 11.4 mgP/L infl. The proof given indicated that propionate is preferred by PAOs. This study found that when more propionate was added to the wastewater, the concentration of PAO biomass went up. This was shown by certain signs that PAOs display when they are present. Results presented in this journal article emanate from an MSc Thesis (Thela, 2022) published in open-source UCT.

Confronting Assumptions of Phosphorus-Accumulating Organisms and Glycogen-Accumulating Organisms: Peaceful Coexistence in a Carbon-Limited Sidestream EBPR Demonstration

Enhanced biological phosphorus removal (EBPR) is strongly influenced by the influent ratio of readily biodegradable carbon to soluble phosphorus due to the preferences of phosphorus-accumulating organisms (PAO). The sidestream EBPR (S2EBPR) process redirects a portion of return activated sludge (RAS) to a sidestream fermenter, increasing the availability of biodegradable carbon. In this study, we assessed the performance and microbial community structure of a full-scale S2EBPR demonstration supplemented with external carbon dosing. By the end of the 10 month study period, the demonstration achieved a median effluent orthophosphate of 0.3 mg/L. The microbial community consisted of a common core microbiome in the RAS fermenter, EBPR basin, and nitrification basin. The most abundant PAO detected were Ca. “Dechloromonas phosphorivorans”, while the canonical PAO Ca. “Accumulibacter” and Tetrasphaera were observed in lower relative abundance. In addition to non-canonical PAO enrichment, the glycogen-accumulating organism Ca. “Competibacter” proliferated throughout the study and at some points outnumbered PAO taxa by 30 to 1 with no tangible performance impacts. This study provides insights into successful S2EBPR implementation at a low-carbon facility and improves our understanding of microbial community structure and key PAO and GAO in S2EBPR systems.

Insights into simultaneous nitrogen and phosphorus removal in biofilm: The overlooked comammox Nitrospira and the positive role of glycogen-accumulating organisms

Simultaneous nitrogen and phosphorus removal (SNPR) biofilm system is an effective wastewater treatment process. However, the understanding on the mechanism of functional microorganisms driving SNPR is still limited, especially the role of complete ammonia oxidation (comammox) Nitrospira and glycogen-accumulating organisms (GAO). In this study, a sequencing batch biofilm reactor (SBBR) performing SNPR was operated for 249 d. Based on the 16S rRNA gene, comammox amoA amplicon sequencing, metagenomics and batch experiment, we found that comammox Nitrospira was the main ammonia-oxidizing microorganisms (AOM) and provided nitrite for anaerobic ammonia oxidation (anammox) bacteria (AnAOB). Besides, GAO was dominated by the bacteria of genus Defluviicoccus and played a primary role in reducing nitrate rather than nitrite. Fluorescent in situ hybridization (FISH) analysis confirmed that Nitrospira was enriched in the inner layer of the biofilm. Thus, we put forward a novel insight into the mechanism of SNPR biofilm system. Comammox Nitrospira was responsible for nitrite and nitrate production in the inner biofilm, and AnAOB consumed the produced nitrite during the anammox process. While GAO reduced nitrate to nitrite and polyphosphate-accumulating organisms (PAO) converted nitrite to dinitrogen via denitrifying phosphorus removal in the outer biofilm. These findings provide a new understanding in SNPR biofilm system.

Predicting microbial community compositions in wastewater treatment plants using artificial neural networks

BackgroundActivated sludge (AS) of wastewater treatment plants (WWTPs) is one of the world’s largest artificial microbial ecosystems and the microbial community of the AS system is closely related to WWTPs' performance. However, how to predict its community structure is still unclear.ResultsHere, we used artificial neural networks (ANN) to predict the microbial compositions of AS systems collected from WWTPs located worldwide. The predictive accuracy R21:1 of the Shannon–Wiener index reached 60.42%, and the average R21:1 of amplicon sequence variants (ASVs) appearing in at least 10% of samples and core taxa were 35.09% and 42.99%, respectively. We also found that the predictability of ASVs was significantly positively correlated with their relative abundance and occurrence frequency, but significantly negatively correlated with potential migration rate. The typical functional groups such as nitrifiers, denitrifiers, polyphosphate-accumulating organisms (PAOs), glycogen-accumulating organisms (GAOs), and filamentous organisms in AS systems could also be well recovered using ANN models, with R21:1 ranging from 32.62% to 56.81%. Furthermore, we found that whether industry wastewater source contained in inflow (IndConInf) had good predictive abilities, although its correlation with ASVs in the Mantel test analysis was weak, which suggested important factors that cannot be identified using traditional methods may be highlighted by the ANN model.ConclusionsWe demonstrated that the microbial compositions and major functional groups of AS systems are predictable using our approach, and IndConInf has a significant impact on the prediction. Our results provide a better understanding of the factors affecting AS communities through the prediction of the microbial community of AS systems, which could lead to insights for improved operating parameters and control of community structure.2jzZ-nU4sRefzvXQz6KqH6Video

Cold-resistant performance and the promoted development of functional community with flexible metabolic patterns in a Biofilm Bio-Nutrient Removal (BBNR) system amended with supplementary carbon source for phosphorus recovery

The need for recovery of phosphorus (P) from wastewater has accelerated the retrofitting of existing bio-nutrient removal (BNR) processes into bio-nutrient removal-phosphorus recovery processes (BNR-PR). A periodical carbon source supplement is needed to facilitate the P-recovery. But the impact of this amendment on the cold resistances of the reactor and the functional microorganisms (for nitrogen and phosphorus (P) removal/recovery) are still unknown. This study presents the performances of a biofilm BNR process with a carbon source regulated the P recovery (BBNR-CPR) process operating at different temperatures. When the temperature was decreased from 25 ± 1 °C to 6 ± 1 °C, the system total nitrogen and total phosphorus removals and the corresponding kinetic coefficients decreased moderately. The indicative genes of the phosphorus-accumulating organisms (e.g., Thauera spp. and Candidatus Accumulibacter spp.) increased significantly. An increase of Nitrosomonas spp. genes aligned to polyhydroxyalkanoates (PHAs), glycine, and extracellular polymeric substance synthesis were observed, which was probably related to cold resistance. The results provide a new vision for understanding the advantages of P recovery-targeted carbon source supplementation for constructing a new type of cold-resistant BBNR-CPR processes.

A novel two-stage aerobic granular sludge system for simultaneous nutrient removal from municipal wastewater with low C/N ratios

In this study, a novel two-stage aerobic granular sludge (AGS) system was developed to treat municipal wastewater. High removal efficiencies of phosphorus (91%) and nitrogen (81%) were obtained under a low influent carbon/nitrogen ratio of 5.4. The high nutrient removal was attributed to microbial segregation in the two sequencing batch reactors (SBRs). In the first reactor, the high abundance of polyphosphate-accumulating organisms (Candidatus Accumulibacter 15.3%) promoted a high rate of enhanced biological phosphorus removal (EBPR). In the second reactor, residual ammonium was autotrophically removed via partial nitritation/anammox (PN/A). Moreover, granular sludge was maintained in both SBRs with a sludge volume index of 40–80 mL/g, which reduced the settling time and improved the operational stability. Furthermore, batch tests and mass balance analysis revealed that most influent phosphorus (68%) and some organics (33%) were recovered from the waste sludge as struvite and methane, respectively. Overall, this study illustrates that the novel two-stage AGS system integrating EBPR and PN/A is promising for enhancing nutrient removal, as well as resource and bioenergy recovery from municipal wastewater.

Sulfide inhibition on polyphosphate accumulating organisms and glycogen accumulating organisms: Cumulative inhibitory effect and recoverability

Sulfate in wastewater can be reduced to sulfide and its impact on the stability of enhanced biological phosphorus removal (EBPR) is still unclear. In this study, the metabolic changes and subsequent recovery of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) were investigated at different sulfide concentrations. The results showed that the metabolic activity of PAOs and GAOs was mainly related to H2S concentration. Under anaerobic conditions, the catabolism of PAOs and GAOs was promoted at H2S concentrations below 79 mg/L S and 271 mg/L S, respectively, and inhibited above these concentrations; whereas anabolism was consistently inhibited in the presence of H2S. The phosphorus (P) release was also pH-dependent due to the intracellular free Mg2+ efflux from PAOs. H2S was more destructive to the esterase activity and membrane permeability of PAOs than those of GAOs and prompted intracellular free Mg2+ efflux of PAOs, resulting in worse aerobic metabolism and subsequent recovery of PAOs than GAOs. Additionally, sulfides facilitated the production of extracellular polymeric substances (EPS), especially tightly bound EPS. The amount of EPS in GAOs was significantly higher than that in PAOs. The above results indicated that sulfide had a stronger inhibition to PAOs than GAOs, and when sulfide was present, GAOs had a competitive advantage over PAOs in EBPR.

Insights on nitrogen and phosphorus removal mechanism in a single-stage Membrane Aeration Biofilm Reactor (MABR) dominated by denitrifying phosphorus removal coupled with anaerobic/aerobic denitrification

In the study, the denitrifying phosphorus removal process coupled with anaerobic/aerobic denitrification was started in a single-stage Membrane Aeration Biofilm Reactor (MABR). When the influent NO3−-N was 50.2 mg/L and the total phosphorus (TP) was 30.5 mg/L, the maximum Total Inorganic Nitrogen Removal Efficiency (TINRE) can reach 99.79 % and the Total Phosphorus Removal Efficiency (TPRE) can reach 82.62 % without the addition of any flocculant or precipitant. The phosphorus content in the substrate sludge was 4.10 %–6.15 %, which was higher than that in the initial biofilm or activated sludge (1.27 %–1.83 %). Based on 16S rRNA sequencing technology, it was found that Phosphorus-Accumulating Organisms (PAOs, Acinetobacter, relative abundance 0.02 % to 1.92 %) and Denitrifying Phosphorus-Accumulating Organisms (DPAOs, Flavobacterium (0.26 % to 5.39 %), Bdellovibrio (0.12 % to 1.48 %), and Pseudomonas (2.29 % to 1.28 %)) played pivotal roles in phosphorus removal. The enzyme gene reads of Complex I and Complex V processes were much more than that of Complex II- Complex IV, which indicated that Complex I and Complex V can be the main process for oxidative phosphorylation in the MABR. Moreover, four inorganic nitrogen metabolic processes, Dissimilatory Nitrate Reduction to Ammonium (DNRA), Assimilatory Nitrate Reduction Ammonium (ANRA), nitrification, and denitrification were found. Nitrifiers (Nitrosomonas (0.09 % to 0.19 %), Nitrospira (2.48 % to 0.06 %)), denitrifiers (Dokdonella (6.95 % to 2.11 %), Denitratisoma (0.42 % to1.48 %)), and aerobic denitrifiers (Pseudomonas (2.29 % to 1.28 %) and Thauera (4.75 % to 1.78 %)) contributed to nitrogen removal in the MABR. This study provided insights into the removal mechanism of nitrogen and phosphorus in a single-stage MABR.

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.

Temporal Dynamics and Performance Association of the Tetrasphaera-Enriched Microbiome for Enhanced Biological Phosphorus Removal

Tetrasphaera have been recently identified based on the 16S ribosomal RNA (rRNA) gene as among the most abundant polyphosphate-accumulating organisms (PAOs) in global full-scale wastewater treatment plants (WWTPs) with enhanced biological phosphorus removal (EBPR). However, it is unclear how Tetrasphaera PAOs are selectively enriched in the context of the EBPR microbiome. In this study, an EBPR microbiome enriched with Tetrasphaera (accounting for 40% of 16S sequences on day 113) was built using a top-down design approach featuring multicarbon sources and a low dosage of allylthiourea. The microbiome showed enhanced nutrient removal (phosphorus removal ∼85% and nitrogen removal ∼80%) and increased phosphorus recovery (up to 23.2 times) compared with the seeding activated sludge from a local full-scale WWTP. The supply of 1 mg·L−1 allylthiourea promoted the coselection of Tetrasphaera PAOs and Microlunatus PAOs and sharply reduced the relative abundance of both ammonia oxidizer Nitrosomonas and putative competitors Brevundimonas and Paracoccus, facilitating the establishment of the EBPR microbiome. Based on 16S rRNA gene analysis, a putative novel PAO species, EBPR-ASV0001, was identified with Tetrasphaera japonica as its closest relative. This study provides new knowledge on the establishment of a Tetrasphaera-enriched microbiome facilitated by allylthiourea, which can be further exploited to guide future process upgrading and optimization to achieve and/or enhance simultaneous biological phosphorus and nitrogen removal from high-strength wastewater.

Turnover of the extracellular polymeric matrix of granules performing biological phosphate removal

Polyphosphate accumulating organisms (PAOs) are responsible for enhanced biological phosphate removal (EBPR) from wastewater, where they grow embedded in a matrix of extracellular polymeric substances (EPS). EPSs comprise a mixture of biopolymers like polysaccharides or (glyco)proteins. Despite previous studies, little is known about the dynamics of EPS in mixed cultures, and their production by PAOs and potential consumption by flanking microbes. EPSs are biodegradable and have been suggested to be a substrate for other organisms in the community. Studying EPS turnover can help elucidate their biosynthesis and biodegradation cycles. We analyzed the turnover of proteins and polysaccharides in the EPS of an enrichment culture of PAOs relative to the turnover of internal proteins. An anaerobic-aerobic sequencing batch reactor (SBR) simulating EBPR conditions was operated to enrich for PAOs. After achieving a stable culture, carbon source was switched to uniformly 13C-labeled acetate. Samples were collected at the end of each aerobic phase. EPSs were extracted by alkaline treatment. 13C enrichment in proteins and sugars (after hydrolysis of polysaccharides) in the extracted EPS were measured by mass spectrometry. The average turnover rate of sugars and proteins (0.167 and 0.192 d−1 respectively) was higher than the expected value based on the solid removal rate (0.132 d−1), and no significant difference was observed between intracellular and extracellular proteins. This indicates that EPS from the PAO enriched community is not selectively degraded by flanking populations under stable EBPR process conditions. Instead, we observed general decay of biomass, which corresponds to a value of 0.048 d−1.Key Points• Proteins showed a higher turnover rate than carbohydrates.• Turnover of EPS was similar to the turnover of intracellular proteins.• EPS is not preferentially consumed by flanking populations.