Phosphorus Removal System Research Articles

Quaternary ammonium compounds inhibited phosphorus removal performance and aggravated the spread of resistance genes in enhanced biological phosphorus removal systems

As a common surfactant, quaternary ammonium compounds (QACs) are often used as the main ingredient in disinfection products. Hence, the three most commonly used QACs were selected for this study, including alkyltrimethylammonium compounds (ATMACs), alkylbenzyldimethylammonium compounds (BACs) and dialkyldimethylammonium compounds (DADMACs). Typical QACs at low (0.2 mg/L), medium (1 mg/L) and high (5 mg/L) concentrations to the enhanced biological phosphorus removal (EBPR) systems were studied. The QACs exposure study was conducted for a total of 93 days using four sequential batch reactors. DADMAC-C12 had the greatest inhibitory effect on EBPR performance, followed by BAC-C12 and ATMAC-C12. On day 51, R1 (stimulated by ATMAC-C12) and R2 (stimulated by BAC-C12) recovered their EBPR performance, whereas the P removal efficiency of R3 (stimulated by DADMAC-C12) was still only 65.9 %. Furthermore, the dosage of both ATMAC-C12 and BAC-C12 promoted sludge granulation, whereas DADMAC-C12 caused slight breakdown of formed aerobic granular sludge. The median volumetric diameter in R3 initially increased to a maximum of 409.9 µm and finally decreased to 352.6 µm at the end of the operation. The stress of QACs resulted in an enrichment of pathogenic bacteria (e.g., Flavobacterium) and antibiotic resistant bacteria (e.g., Pseudomonas) in the systems. The abundance of Pseudomonas in R1 and R3 finally reached up to 9.40 % and 9.44 %, respectively. Additionally, QACs disrupted bacterial cell membranes and resulted in the release of intracellular resistent genes in sludge (si-RGs). Then, si-RGs converted to extracellular RGs in sludge and RGs in water. Zoogloea, Rhizobium, Turneriella and Aquimonas were the dominant potential hosts of RGs in EBPR systems dosed with QACs. This study can serve as an important reference for the control of RGs in EBPR processes.

Substrate preference of Tetrasphaera in dual PAOs synergistic EBPR system and its phosphorus removal performance

In this study, an alternated anaerobic/aerobic sequencing batch reactor (SBR) was employed to investigate the effect of the substrate conversion from casein hydrolysate (Cas aa) to a mixture of amino acids (glutamate, aspartate, glycine and proline) on the efficacy of nitrogen and phosphorus removal in an enhanced biological phosphorus removal (EBPR) system characterized by dual polyphosphate-accumulating organisms (PAOs), mainly focusing on the substrate preference of fermentative PAOs. The results indicated that a stable removal efficiency was found to be above 90 % as the substrate was converted to the mixture of amino acids. Meanwhile, the removal efficiency of ammonium eventually increased to over 95 % in stage III, despite an increase of ammonia nitrogen by hydrolysis with the rising proportion of mixed amino acids in influent. Moreover, shortening the anaerobic duration did not significantly affect the phosphorus removal performance, even enhancing anaerobic phosphorus release capacity. Nevertheless, the ammonia nitrogen removal was influenced. 16 s rRNA high-throughput sequencing results demonstrated that the detected fermentative PAO like Tetrasphaera in the EBPR system exhibited a clear preference for utilizing mixed amino acids and could cooperate with traditional PAO like Accumulibacter for phosphorus removal. In addition, the fermentation of amino acids mixture by Tetrasphaera was more pronounced and its abundance increased under the condition of a shorter anaerobic duration, leading to a relatively stable and high abundance of Accumulibacter in the presence of sufficient substrates from Tetrasphaera. However, considering the nitrogen removal performance and steady operation of the EBPR system, a longer anaerobic duration was more reasonable.

Candidatus Thiothrix phosphatis SCUT-1: A novel polyphosphate-accumulating organism abundant in the enhanced biological phosphorus removal system

A novel coccus Thiothrix-related polyphosphate-accumulating organism (PAO) was enriched in an acetate-fed enhanced biological phosphorus removal (EBPR) system. High EBPR performance was achieved for an extended period (>100 days). A high-quality draft genome (completeness 97.2 %, contamination 3.26 %) was retrieved, representing a novel Thiothrix species (with similarity<93.2 % to known Thiothrix species), and was denoted as 'Candidatus Thiothrix phosphatis SCUT-1'. Its acetate uptake rate (6.20 mmol C/g VSS/h) surpassed most Ca. Accumulibacter and known glycogen-accumulating organisms (GAOs), conferring their predominance in the acetate-fed system. Metatranscriptomic analysis suggested that Ca. Thiothrix phosphatis SCUT-1 employed both low- and high-affinity pathways for acetate activation, and both the conventional (PhaABC) pathway and the fatty acid β-oxidation pathway for PHA synthesis; additionally, a much more efficient FAD-dependent malate: quinone oxidoreductase (MQO) were encoded and employed than the traditional malate dehydrogenase (MDH) to oxidize malate to oxaloacetate in the TCA and glyoxylate cycle, collectively contributing to a higher acetate utilization and processing rate of this microorganism. Batch tests further demonstrated the versatile ability of this PAO in using VFA (acetate, propionate, and butyrate), lactate, amino acids (aspartate and glutamate), and glucose as carbon sources for EBPR, showing a partially overlapped but unique ecological niche of this microorganism comparing to Ca. Accumulibacter and known GAOs. A metabolic model was built for Ca. Thiothrix phosphatis SCUT-1 using the above-mentioned carbon sources for EBPR. Overall, this study represents the first comprehensive characterization of the physiology and metabolic characteristics of representative coccus Thiothrix-related PAOs, which are expected to provide new insights into PAO microbiology in EBPR systems.

Distinct microdiversity of phosphate accumulating organisms (PAOs) between side-stream and conventional enhanced biological phosphorus removal (EBPR) systems with performance implications

Polyphosphate Accumulating Organisms (PAOs) microdiversity is a key factor to elucidate the mechanisms involved in the side-stream enhanced biological phosphorus removal (S2EBPR) systems, which has been shown to improve the process stability over conventional EBPR. However, fast, effective and cost-efficient methods to resolve PAO microdiversity in real-world activate sludge samples is still in absence. In this study, we applied oligotyping analysis following the regular 16S rRNA gene amplicon sequencing standard operation pipeline (SOP) to resolve subgenus-level PAO oligotypes, which cannot be achieved using traditional 16S rRNA sequencing SOP. The identified oligotype profiles of PAO-containing genera Ca. Accumulibacter, Tetrasphaera and Comamonas showed distinguished community-level differences across 12 water resource recovery facilities (WRRFs), which would not be revealed at the genus level. The WRRF-level differences were observed larger than the temporal differences in the same WRRF, indicating intrinsic sub-genus level microdiversity fingerprint between EBPR/S2EBPR systems. The identified oligotypes can be associated with known PAO clades phylogenetically, suggesting that oligotyping can suffice as a fast and cost-efficient approach for PAO microdiversity profiling. In addition, network analysis can be used to identify coexistence patterns between oligotypes with respect to EBPR/S2EBPR configurations and performance, enabling more detailed analysis between EBPR system performance and PAOs microdiversity. Correlation analyses between oligotype profiles and key EBPR performance parameters revealed potential different biological functional traits among these PAO species with P-removal performance implications.

Lag in pollutant removal efficiency relative to microbial community dynamics in a denitrifying phosphorus removal bioreactor

Determining the system status in advance is critical for implementing timely control strategies, preventing wastewater treatment instability due to overload stress, and enhancing system resilience to impact loads. Unlike traditional instability monitoring methods, identifying temporal relationships between pollutant removal efficiency and microbial community dynamics can offer novel insights into identifying optimal intervention windows. Here, we chose nitrite, a vital intermediate of nitrogen reaction in wastewater treatment, as a stress factor. Then, we investigated the effects of nitrite stress on denitrifying phosphorus removal system performance and microbiome. By monitoring carbon, nitrogen, and phosphorus removal efficiency and analyzing microbiome information, we found that variations in bacterial ecology preceded pollutant removal efficiency. The lag in pollutant removal efficiency relative to microbial community dynamics reflects the complex dynamics of species traits, metabolic functions, and functional redundancy. Species in clusters may drove specific pollutant removal; e.g., Betaproteobacteria are more closely associated with nitrogen removal, while Gammaproteobacteria are more linked to phosphorus removal. The cellular secretion function displayed greater susceptibility to overload stress compared to the substrate conversion function, resulting in decreased stability observed after 30 cycles, preceding the decline in substrate conversion efficiency. The lag in performance relative to community stability can be attributed to functional redundancy. Under overload stress conditions, functional redundancy within the microbiome decreased by 30.77% from 60 cycles to 120 cycles. Our results support a novel strategy for system collapse prediction via changes in the microbiome,enabling proactive regulation and ensuring system stability before irreversible deterioration occurs.

Effect of perfluorooctanoic acid on denitrifying phosphorus removal system under short-term stress

Effect of perfluorooctanoic acid on denitrifying phosphorus removal system under short-term stress

Molecular evidence of internal carbon-driven partial denitrification in a mainstream pilot A-B system coupled with side-stream EBPR treating municipal wastewater

Achieving mainstream short-cut nitrogen removal via nitrite has become a carbon and energy efficient way, but still remains challenging for low-strength municipal wastewaters. This study integrated sidestream enhanced biological phosphorus removal system in a pilot-scale adsorption/bio-oxidation (A-B) process (named A-B-S2EBPR system) and nitrite accumulation was successfully achieved for treating the municipal wastewater. Nitrite could accumulate to 5.5 ± 0.3 mg N/L in the intermittently aerated tanks of B-stage with the nitrite accumulation ratio (NAR) of 79.1 ± 6.5 %. The final effluent concentration and removal efficiency of total inorganic nitrogen (TIN) were 4.6 ± 1.8 mg N/L and 84.9 ± 5.6 %, respectively. In-situ process performance of nitrogen conversions, routine batch nitrification/denitrification activity tests and functional gene abundance of nitrifiers collectively suggested that the nitrite accumulation was mainly caused by partial denitrification rather than out-selection of nitrite oxidizing bacteria (NOB). Moreover, the single-cell Raman spectroscopy analysis first demonstrated that there was a specific microbial population that could utilize polyhydroxyalkanoates (PHA) as the potential internal carbon source during the partial denitrification process. The integration of S2EBPR brings unique features to the conventional A-B process, such as extended anaerobic retention time, lower oxidation–reduction potential (ORP), much higher and complex volatile fatty acids (VFAs) etc., which can largely reshape the microbial communities. The dominant genera were Acinetobacter and Comamonadaceae, which accounted for (17.8 ± 15.5)% and (6.7 ± 3.4)%, respectively, while the relative abundance of conventional nitrifiers was less than 0.2%. This study provides insights into phylogenetic and phenotypic shifts of microbial communities when incorporating S2EBPR into the sustainable A-B process to achieve mainstream short-cut nitrogen removal.

Inhibition of phosphorus removal performance in activated sludge by Fe(III) exposure: transitions in dominant metabolic pathways.

Simultaneous chemical phosphorus removal process using iron salts (Fe(III)) has been widely utilized in wastewater treatment to meet increasingly stringent discharge standards. However, the inhibitory effect of Fe(III) on the biological phosphorus removal system remains a topic of debate, with its precise mechanism yet to be fully understood. Batch and long-term exposure experiments were conducted in six sequencing batch reactors (SBRs) operating for 155 days. Synthetic wastewater containing various Fe/P ratios (i.e., Fe/P = 1, 1.2, 1.5, 1.8, and 2) was slowly poured into the SBRs during the experimental period to assess the effects of acute and chronic Fe(III) exposure on polyphosphate-accumulating organism (PAO) growth and phosphorus metabolism. Experimental results revealed that prolonged Fe(III) exposure induced a transition in the dominant phosphorus removal mechanism within activated sludge, resulting in a diminished availability of phosphorus for bio-metabolism. In Fe(III)-treated groups, intracellular phosphorus storage ranged from 3.11 to 7.67 mg/g VSS, representing only 26.01 to 64.13% of the control. Although the abundance of widely reported PAOs (Candidatus Accumulibacter) was 30.15% in the experimental group, phosphorus release and uptake were strongly inhibited by high dosage of Fe(III). Furthermore, the abundance of functional genes associated with key enzymes in the glycogen metabolism pathway increased while those related to the polyphosphate metabolism pathway decreased under chronic Fe(III) stress. These findings collectively suggest that the energy generated from polyhydroxyalkanoates oxidation in PAOs primarily facilitated glycogen metabolism rather than promoting phosphorus uptake. Consequently, the dominant metabolic pathway of communities shifted from polyphosphate-accumulating metabolism to glycogen-accumulating metabolism as the major contributor to the decreased biological phosphorus removal performance.

Metabolic response and clade variation in Accumulibacter under phosphorus deprivation stress

Two enhanced biological phosphorus removal systems (R1 and R2) fed with different carbon sources (acetic acid and propionic acid) were used to investigate the metabolic response of nitrite-type denitrifying phosphorus removal functional bacteria Accumulibacter to different phosphorus concentrations and the changes of its clade community structure. The results indicated that the systems almost lost the ability to release phosphorus after 15 d phosphorus deprivation, the contents of poly-P in the sludge of R1 and R2 decreased from 0.63 to 0.02 mgP/mgVSS and from 1.23 to 0.05 mgP/mgVSS, respectively. According to the analysis of stoichiometric parameters, the functional bacteria in both systems performed glycogen accumulating organism metabolism to survive under phosphorus-poor condition. Besides, the two systems exhibited a stronger EBPR ability even after 42 d phosphorus deprivation, and the maximum anoxic phosphorus uptake rate was 1.92 times and 2.29 times of those under phosphorus-rich conditions. Real-time quantitative Polymerase Chain Reaction detection indicated that the proportion of Accumulibacter in the two systems decreased from 16.45 % to 8.78 % and from 31.00 % to 8.43 %, respectively, as phosphorus was deprived. Clade IIC maintained its dominance in both systems, while clade IID was gradually washed out from propionic acid system, suggesting that clade IIC had a more flexible metabolic response to phosphorus deprivation stress.

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.

Forming densified activated sludge in a modified simultaneous nitrification–denitrification and phosphorus removal (SNDPR) system by introducing shift work mode

Forming granular-like sludge in the Simultaneous Nitrification-Denitrification and Phosphorus Removal (SNDPR) process would promote nutrient removal and improve the sludge settleability. This study introduced the “shift work mode” into an SNDPR system, which featured that 50 % of sludge was temporarily transferred into and “rested” in a side-stream reactor a cyclic time rather than let it keep “working” in the mainstream SNDPR reactor. Results showed that 77.6 % of inorganic nitrogen and 80.6 % of orthophosphate were removed well even after the combination, which attributed to the co-work of Candidatus_Competibacter (10.1 %–29.0 %), nitrifiers, and phosphate accumulating organisms. The 30-min settled sludge volume and sludge volume index were 8 % and 45.8 mL/g, respectively, the sludge density was 1.045 g/mL, and cumulative 90 % of the particle diameter was less than 80 µm; these indicated the densified activated sludge was formed. After analyses, the increase in the food-to-microorganisms ratio and temporary enrichment of filamentous bacteria Thiothrix might be the causes. Such shift work mode promisingly serves as a simple and practical way to improve settleability and promote granulation.

Assessment of three different approaches for integrating phosphorus recovery from sewage sludge and derived products in existing wastewater treatment plants

Three different technological solutions, namely acidogenic fermentation and chemical extraction (alkaline or acidic), followed by precipitation with 1% Ca(OH)2, were investigated in the view of integrating phosphorus recovery into existing wastewater treatment plants. Experiments were conducted at the lab-scale using (i) sludge taken from biologically and chemically promoted phosphorus removal activated sludge processes and (ii) ashes obtained from sludge muffle incineration. Results highlighted the benefits of enhanced biological phosphorus removal (EBPR) systems rather than chemically promoted phosphorus removal in not only phosphorus extraction (up to 40% with EBPR) and recovery directly from secondary sludge (P precipitation between 66 and 92%), but after sludge incineration as well (P extraction up to 96% and precipitation above 96%). Acidogenic fermentation ensured the highest phosphorus release from EBPR sludge (equal to a concentration in solution of 122 mg/L P-PO43−), while the derived ashes had a lower level of metal contamination (particularly Fe and Al content < 2%). The phosphorus-rich product obtained by means of the recovery process showed relevant metal contamination (Cu, Zn, and Ni) under some operating conditions, suggesting the need for further treatments.

Phosphorus Release Dynamics from Ashes during a Soil Incubation Study: Effect of Feedstock Characteristics and Combustion Conditions

Recovering phosphorus (P) through combustion from waste streams, like wastewater sludge and animal manure, offers a promising solution. This research explores the P release patterns in different ashes derived from secondary raw materials, using a long-term soil incubation lasting 160 days. The study evaluated the P release dynamics in five types of ashes from enhanced biological phosphorus removal (EBPR) systems and pig slurry burned at different temperatures. According to the results, a primary effect was observed on P bioavailability during the initial incubation period. All tested ashes release more than 50% of the total P applied between days 5 and 10. Ashes from EBPR exhibited higher P release than those from pig manure, indicating ash origin as a key factor in P release. Additionally, combustion temperature was crucial, with higher temperatures resulting in increased P release rates. Furthermore, the Pearson correlation revealed a strong relationship between the characteristics of the ashes and the amount of P release. Overall, these findings suggest that ashes could be a valuable P-source for agriculture avoiding the process of wet chemical P extraction, thus reducing both economic and environmental costs.

Microplastics shaped performance, microbial ecology and community assembly in simultaneous nitrification, denitrification and phosphorus removal process

The widespread use of microplastics (MPs) has led to an increase in their discharge to wastewater treatment plants. However, the knowledge of impact of MPs on macro-performance and micro-ecology in simultaneous nitrification, denitrification, and phosphorus removal (SNDPR) systems is limited, hampering the understanding of potential risks posed by MPs. This study firstly comprehensively investigated the performance, species interactions, and community assembly under polystyrene (PS) and polyvinyl chloride (PVC) exposure in SNDPR systems. The results showed under PS (1, 10 mg/L) and PVC (1, 10 mg/L) exposure, total nitrogen removal was reduced by 3.38–10.15 %. PS and PVC restrained the specific rates of nitrite and nitrate reduction (SNIRR, SNRR), as well as the activities of nitrite and nitrate reductase enzymes (NIR, NR). The specific ammonia oxidation rate (SAOR) and activity of ammonia oxidase enzyme (AMO) were reduced only at 10 mg/L PVC. PS and PVC enhanced the size of co-occurrence networks, niche breadth, and number of key species while decreasing microbial cooperation by 5.85–13.48 %. Heterogeneous selection dominated microbial community assembly, and PS and PVC strengthened the contribution of stochastic processes. PICRUSt prediction further revealed some important pathways were blocked by PS and PVC. Together, the reduced TN removal under PS and PVC exposure can be attributed to the inhibition of SAOR, SNRR, and SNIRR, the restrained activities of NIR, NR, and AMO, the changes in species interactions and community assembly mechanisms, and the suppression of some essential metabolic pathways. This paper offers a new perspective on comprehending the effects of MPs on SNDPR systems.

Metabolomic reveals the responses of sludge properties and microbial communities to high nitrite stress in denitrifying phosphorus removal systems

Nitrite, as an electron acceptor, plays a good role in denitrifying phosphorus removal (DPR); however, high nitrite concentration has adverse affects on sludge performance. We investigated the precise mechanisms of responses of sludge to high nitrite stress, including surface characteristics, intracellular and extracellular components, microbial and metabolic responses. When the nitrite stress reached 90 mg/L, the sludge settling performance was improved, but the activated sludge was aging. FTIR and XPS analysis revealed a significant increase in the hydrophobicity of the sludge, resulting in improve settling performance. However, the intracellular carbon sources synthesis was inhibited. In addition, the components in the tightly bound extracellular polymeric substances (TB-EPS) of sludge were significantly reduced and indicated the disturb of metabolism. Notably, Exiguobacterium emerged as a new genus when face high nitrite stress that could maintaining survival in hostile environments. Moreover, metabolomic analysis demonstrated strong biological response to nitrite stress further supported above results that include the inhibited of carbohydrate and amino acid metabolism. More importantly, some lipids (PS, PA, LysoPA, LysoPC and LysoPE) were significantly upregulated that related enhanced membrane lipid remodeling. The comprehensive analyses provide novel insights into the high nitrite stress responses mechanisms in activated sludge systems.

Aerobic granulation in a continuous-flow simultaneous nitrification, endogenous denitrification, and phosphorus removal system fed with low-strength wastewater: Granulation mechanism and microbial succession

The culture of aerobic granular sludge (AGS) in continuous-flow system for treating low-strength municipal wastewater is still a challenge for its widespread application. This study developed a continuous-flow reactor based on metabolic and hydraulic selection pressure to explore the feasibility of culturing AGS in conventional continuous-flow mode. The results showed that aerobic granulation was successfully achieved with a mean particle size of 373 μm and almost all granules (95.6 %) were larger than 200 μm. An obvious three-stages granulation process comprised of initial adhesion, the formation of early aggregates that acted as nuclei of granules, and the final AGS maturation, was observed in this work. The selective enrichment of slow-growing bacteria in alternating feast/famine condition played a critical role in the aerobic granulation. The analysis results of extracellular polymeric substances revealed that β-polysaccharides and proteins distributed uniformly within the mature granules, while α-polysaccharides were found mainly concentrated around the granules, which was believed to enhance the mechanical strength of granules. The continuous-flow AGS system exhibited an excellent simultaneous carbon, nitrogen, and phosphorus removal performance with high removal efficiencies of NH4+-N (97.6 %), TIN (91.0 %), COD (93.7 %), and TP (94.8 %). Moreover, a low sludge yield of 0.1 gMLSS/gCOD was obtained for dramatic in situ sludge reduction. Illumina MiSeq sequencing results confirmed the outstanding accumulation of GAOs (Candidatus_Competibacter) and PAOs (Rhodocyclaceae) in the system, which were responsible for simultaneous C, N, P removal and sludge reduction. Overall, this study demonstrated the feasibility of aerobic granulation in conventional continuous-flow mode and offered insights into the granulation mechanism, providing implications and references for the application of AGS technology in existing wastewater treatment plants.

Combined Phototrophic Simultaneous Nitrification-Endogenous Denitrification with Phosphorus Removal (P-SNDPR) System Treating Low Carbon to Nitrogen Ratio Wastewater for Potential Carbon Neutrality.

Conventional biological nutrient removal processes rely on external aeration and produce significant carbon dioxide (CO2) emissions. This study constructed a phototrophic simultaneous nitrification-denitrification phosphorus removal (P-SNDPR) system to treat low carbon to nitrogen (C/N) ratios wastewater and investigated the impact of sludge retention time (SRT) on nutrient removal performance, nitrogen conversion pathway, and microbial structure. Results showed that the P-SNDPR system at SRT of 15 days had the highest nutrient removal capacity, achieving over 85% and 98% removal of nitrogen and phosphorus, respectively, meanwhile maintaining minimal CO2 emissions. Nitrogen removal was mainly through assimilation at SRTs of 5 and 10 days, and nitrification-denitrification at SRTs of 15 and 20 days. Stable partial nitrification was facilitated by photoinhibition and low DO levels. Flow cytometry sorting technique results revealed SRT drove community structural changes in translational activity (BONCAT+) microbes, where BONCAT+ microbes were mainly simultaneous nitrogen and phosphorus removal bacteria (Candidatus Accumulibacter), denitrifying bacteria (Candidatus Competibacter and Plasticicumulans), ammonia-oxidizing bacteria (Nitrosomonas), and microalgae (Chlorella and Dictyosphaerium). The P-SNDPR system represents a novel, carbon-neutral process for efficient nutrient removal from low C/N ratio wastewater without aeration and external carbon source additions.

Metabolic shifts in glycogen-accumulating organisms: Exploring carbon and phosphorus dynamics under two distinct phosphorus limitation modes

Glycogen-accumulating organisms (GAOs) are recognized as competitors of phosphorus-accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) systems. However, different types of GAOs exhibit unique metabolic traits in different environments. To characterize the metabolic activity of GAOs, two distinct modes were constructed. Long-term operation with the mode of anaerobic post-drainage demonstrated the successful establishment of a rapid and enduring GAOs system dominated by Candidatus Contendobacter community. Acetate was completely consumed within 60 min of the anaerobic phase. Concurrently, glycogen levels decreased by 0.062 g Glycogen/g SS, while intracellular carbon storage of poly-hydroxybutyrate (PHB) and poly-hydroxyvalerate (PHV) increased by 0.027 g PHB/g SS and 0.007 g PHV/g SS, respectively. Various concentrations of phosphorus loads illustrated the resistance of the system to high phosphorus loads, while maintaining glycogen accumulating metabolism (GAM). Under complete phosphorus limitation, no correlation between glycogen and acetate was observed during the initial 140 d. The addition of 1 mg/L phosphate to the influent preserved the typical GAM characteristics. Moreover, the pathway involved in glycogen conversion to trehalose may be pivotal in enhancing anaerobic glycogen degradation. The complete tricarboxylic acid cycle ensured efficient energy production, thereby sustaining the foundational metabolism. Notably, sucC and mdh were critical regulators of energy flux. This study provides a distinctive metabolic profile of a robust and resilient GAOs model with Ca. Contendobacter.

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.

Blind spots of universal primers and specific FISH probes for functional microbe and community characterization in EBPR systems.

Fluorescence in situ hybridization (FISH) and 16S rRNA gene amplicon sequencing are commonly used for microbial ecological analyses in biological enhanced phosphorus removal (EBPR) systems, the successful application of which was governed by the oligonucleotides used. We performed a systemic evaluation of commonly used probes/primers for known polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs). Most FISH probes showed blind spots and covered nontarget bacterial groups. Ca. Competibacter probes showed promising coverage and specificity. Those for Ca. Accumulibacter are desirable in coverage but targeted out-group bacteria, including Ca. Competibacter, Thauera, Dechlorosoma, and some polyphosphate-accumulating Cyanobacteria. Defluviicoccus probes are good in specificity but poor in coverage. Probes targeting Tetrasphaera or Dechloromonas showed low coverage and specificity. Specifically, DEMEF455, Bet135, and Dech453 for Dechloromonas covered Ca. Accumulibacter. Special attentions are needed when using these probes to resolve the PAO/GAO phenotype of Dechloromonas. Most species-specific probes for Ca. Accumulibacter, Ca. Lutibacillus, Ca. Phosphoribacter, and Tetrasphaera are highly specific. Overall, 1.4% Ca. Accumulibacter, 9.6% Ca. Competibacter, 43.3% Defluviicoccus, and 54.0% Dechloromonas in the MiDAS database were not covered by existing FISH probes. Different 16S rRNA amplicon primer sets showed distinct coverage of known PAOs and GAOs. None of them covered all members. Overall, 520F-802R and 515F-926R showed the most balanced coverage. All primers showed extremely low coverage of Microlunatus (<36.0%), implying their probably overlooked roles in EBPR systems. A clear understanding of the strength and weaknesses of each probe and primer set is a premise for rational evaluation and interpretation of obtained community results.