Polyphosphate-accumulating Organisms Research Articles

Microbiome structure and function in parallel full-scale aerobic granular sludge and activated sludge processes

Aerobic granular sludge (AGS) and conventional activated sludge (CAS) are two different biological wastewater treatment processes. AGS consists of self-immobilised microorganisms that are transformed into spherical biofilms, whereas CAS has floccular sludge of lower density. In this study, we investigated the treatment performance and microbiome dynamics of two full-scale AGS reactors and a parallel CAS system at a municipal WWTP in Sweden. Both systems produced low effluent concentrations, with some fluctuations in phosphate and nitrate mainly due to variations in organic substrate availability. The microbial diversity was slightly higher in the AGS, with different dynamics in the microbiome over time. Seasonal periodicity was observed in both sludge types, with a larger shift in the CAS microbiome compared to the AGS. Groups important for reactor function, such as ammonia-oxidising bacteria (AOB), nitrite-oxidising bacteria (NOB), polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs), followed similar trends in both systems, with higher relative abundances of PAOs and GAOs in the AGS. However, microbial composition and dynamics differed between the two systems at the genus level. For instance, among PAOs, Tetrasphaera was more prevalent in the AGS, while Dechloromonas was more common in the CAS. Among NOB, Ca. Nitrotoga had a higher relative abundance in the AGS, while Nitrospira was the main nitrifier in the CAS. Furthermore, network analysis revealed the clustering of the various genera within the guilds to modules with different temporal patterns, suggesting functional redundancy in both AGS and CAS.Key points• Microbial community succession in parallel full-scale aerobic granular sludge (AGS) and conventional activated sludge (CAS) processes.• Higher periodicity in microbial community structure in CAS compared to in AGS.• Similar functional groups between AGS and CAS but different composition and dynamics at genus level.Graphical abstract

Towards in-site carbon utilization and highly efficient nutrients removal: Effects of ammonium on the phosphorus recovery performance in a biofilm-based system

An alternatively anaerobic–aerobic biofilm system was operated for treating synthetic municipal wastewater for 150 days, and the effect of ammonium concentration in range of 15 mg∙L−1 - 50 mg∙L−1 on the phosphorus recovery performance was discussed. The ammonium concentration was confirmed as an important factor for balancing phosphorus accumulating organisms (PAOs) and denitrifying phosphorus-accumulating organisms (DPAOs) to affect nutrients removal and phosphorus recovery. The PAOs dominated in phosphorus removal with the ammonium less than 30 mg∙L−1, the ortho-P concentration in recovery steam was plateaued at 52 mg∙L−1. The increase in ammonium to 40 mg∙L−1 caused the seriously deterioration in PAOs activity; however, the phosphorus removal and recovery performance was decreased slightly which attributed to the vital contribution of DPAOs in phosphorus removal. The adverse effect of ammonium can be eliminated by decreasing the volume exchange ratio (VER) from 3.0 to 1.5, leading to the phosphorus concentration in recovery steam of 72.21 mg∙L−1. Meanwhile, the phosphorus removal efficiency (PRE) and total nitrogen removal efficiency (NRE) were stable at 97.8 % and 99.2 %, respectively, indicating highly efficient phosphorus recovery and nutrients removal in the biofilm system. The outcome of the present study could facilitate the establishment of an integrative technology to realize in-site carbon utilization, highly efficient nutrients removal and phosphorus recovery simultaneously in the mainstream treatment of sewage wastewater, and the study reveals the transformation pathways of P and N in the BPRR system, as well as the complex interactions between functional microorganisms.

Turning alkalinity deficiency-induced metabolic disorder into an opportunity for promoting granulation and enhancing heat generation

Shortage of alkalinity is a common problem in treating low carbon/nitrogen (C/N) wastewater and industrial influents. It negatively affects system stability and efficiency, but its positive effects remain unclear. To address this issue, insufficient (R1: 400 mg CaCO3/L) and sufficient alkalinity (R2: 750 mg CaCO3/L) levels were established in our study, where R1 served as the experimental group and R2 as the control group. In the experimental group, alkalinity deficiency deteriorated the nitrification and phosphorus removal process by reducing the abundance of functional microbes (Nitrosomonas, genus ammonia-oxidizing bacteria, AOB, and Dechloromonas, genus polyphosphate-accumulating organisms, PAOs) and genes (hao). At the same time, the alkalinity deficiency enriched slow-growing functional microbes (Candidatus_Competibacter, genus glycogen-accumulating organisms, GAOs) and key genes (nirK, norB, nosZ, acnB, fumA, fumB) increased the content of extracellular polymeric substances (EPS) and the ratio of protein to polysaccharide (PN/PS). By day 113, over 87 % of the particles were larger than 200 μm, and complete granulation was achieved. Microbial energy metabolism further indicated that alkalinity deficiency diverted some energy to non-growth metabolism, thereby enhancing microbial heat generation. Our results highlight an innovative approach, using the setting of insufficient alkalinity conditions to improve heat recovery and address the challenges of WWTPs. These research findings are in favor of wastewater treatment plants seeking to meet multiple sustainable objectives, including improving water quality, saving footprint, and maximizing energy recovery.

Insights into the mechanism of phosphorus enrichment and stripping in side-stream enhanced biological phosphorus removal and enrichment process

In-depth research on the side-stream enhanced biological phosphorus removal (S2EBPR) process holds promise for addressing the challenge of low phosphorus (P) enrichment concentration. This study developed a side-stream enhanced biological phosphorus enrichment/removal (S2EBPE/R) reactor by improving a S2EBPR process. The P removal, enrichment, and mass balance were used to evaluate polyphosphate-accumulating organisms (PAOs) activity. Microbial community structures were investigated to reveal the mechanism of P enrichment and stripping. The results showed that the P removal efficiency reached up to 93% in the S2EBPE/R process. Approximately 13% of P-rich liquid could be stably obtained from the side-stream system, with a concentration of 108.85 ± 4.53 mg P/L. Apart from responses to EBPR activity (P/HAc) of up to 0.41 P-mol/C-mol, enrichment of typical PAOs of up to 12.0% enhanced the P enrichment and removal performance. The abundance of functional microorganisms (PAOs and glycogen accumulating organisms (GAOs)) related to P was as high as 13.4%. These findings highlighted the outstanding advantages of the S2EBPE/R process in P enrichment and removal, thereby making significant contributions to P recovery from wastewater.

Influence of operation sequences on phosphorus recovery by polyphosphate-accumulating organisms biofilm: Performance, kinetics and metabolic response

Two biofilm sequencing batch reactors (BSBRs) were conducted under alternating aerobic/anaerobic (Ae/An) sequence and alternating anaerobic/aerobic (An/Ae) sequence to investigate the effects of operation sequences on phosphorus removal and recovery performance. The Ae/An BSBR had advantages in phosphate enrichment, phosphorus concentration of enrichment solution can reach 99.08 mg/L. While An/Ae BSBR exhibited a higher kinetic rate for phosphate uptake and release up to 0.48 mmol/gVSS. In An/Ae BSBR, the remarkable increase of phosphate concentration in aerobic stage put a great strain on the phosphate uptake of biofilms that led to an increase of phosphorus accumulation in the biofilm (Pbiofilm). However, the poly-P proportion in TPbiofilm decreased which triggered the metabolisim of polyphosphate accumulating organisms (PAOs) shifting from polyphosphate accumulating metabolism (PAM) to glycogen accumulating metabolism (GAM) and suppressed the phosphorus enrichment performance of the An/Ae BSBR. The phosphate uptake rate (PUR) of biofilms was in alignment with the bulk phosphate concentration. PAOs were dominated by Rhodoplanes and Saprospiraceae in the Ae/An BSBR, whereas Thiothrix was predominant in the An/Ae BSBR. Notably, the Candidatus Competibacter, considered as a denitrifying glycogen accumulating organisms (DGAOs), showed a dominant proportion in the microbial community of both BSBRs. The higher expression of actP in the An/Ae system indicated a greater capacity for the uptake of acetate, while the Ae/An system was is more adept for degrading acetate represented by the higher relative abundance of ackA, pta and acsA genes The high phosphorus loading in the An/Ae system suppressed the ppk expression but enhanced the ppx expression.

Synergistic degradation efficiency of nitrogen, phosphorus and microbial dynamic succession driven by a novel strain Gordonia sp. D4 during synthetic wastewater treatment

Nitrogen (N) and phosphorus (P) coexist in the industrial and domestic wastewater posed a pressing challenge for synergistic removal of N and P by bioenhancement. In this study, a novel denitrifying phosphorus-accumulating organism (DPAO) of Gordonia sp. D4, exhibited peak NO3--N, NO2--N, NH4+-N and TP removal efficiencies of 98.11%, 65.02%, 85.34% and 100%, corresponding to 9.30 mg/(L·h), 6.08 mg/(L·h), 3.63 mg/(L·h) and 0.47 mg/(L·h) removal rates, respectively. Strain D4 contained the nitrogen cycling genes narK, narI, narJ, narH, narG, nirB and nirD, as well as the phosphorus cycling genes ppk1, ppk2 and ppx-gppA, which contributes to its excellent simultaneous removal of N and P. More importantly, the addition of D4 into the sequencing batch reactor (SBR) significantly improved the P, TN and NO3--N removal efficiencies by 18.91%, 7.35%, and 18.55%, respectively. This was closely related to the clear enrichment of Gordonia and its contribution to the increasing abundances of Pararhodobacter, unclassified_f_Cyclobacteriaceae, unclassified_o_Rhodospirillales, Thauera and Candidatus Promineofilum and their associated functional genes that drive P or N removal, thereby enhancing the N and P synergistic degradation efficiency. This study provides a valuable strain resource and process reference for the simultaneously removing N and P from the wastewater.

Cultivation of phosphate-accumulating biofilm: Study of the effects of acyl-homoserine lactones (AHLs) and cyclic dimeric guanosine monophosphate (c-di-GMP) on the formation of biofilm and the enhancement of phosphate metabolism capacity

This study investigated the mechanisms of microbial growth and metabolism during biofilm cultivation in the biofilm sequencing batch reactor (BSBR) process for phosphate (P) enrichment. The results showed that the sludge discharge was key to biofilm growth, as it terminated the competition for carbon (C) source between the nascent biofilm and the activated sludge. For the tested reactor, after the sludge discharge on 18 d, P metabolism and C source utilization improved significantly, and the biofilm grew rapidly. The P concentration of the recovery liquid reached up to 157.08 mg/L, which was sufficient for further P recovery via mineralization. Meta-omics methods were used to analyze metabolic pathways and functional genes in microbial growth during biofilm cultivation. It appeared that the sludge discharge activated the key genes of P metabolism and inhibited the key genes of C metabolism, which strengthened the polyphosphate-accumulating metabolism (PAM) as a result. The sludge discharge not only changed the types of polyphosphate-accumulating organisms (PAOs) but also promoted the growth of dominant PAOs. Before the sludge discharge, the necessary metabolic abilities that were spread among different microorganisms gradually concentrated into a small number of PAOs, and after the sludge discharge, they further concentrated into Candidatus_Contendobacter (P3) and Candidatus_Accumulibacter (P17). The messenger molecule C-di-GMP, produced mostly by P3 and P17, facilitated P enrichment by regulating cellular P and C metabolism. The glycogen-accumulating organism (GAO) Candidatus_Competibacter secreted N-Acyl homoserine lactones (AHLs), which stimulated the secretion of protein in extracellular polymeric substances (EPS), thus promoting the adhesion of microorganisms to biofilm and improving P metabolism via EPS-based P adsorption. Under the combined action of the dominant GAOs and PAOs, AHLs and C-di-GMP mediated QS to promote biofilm development and P enrichment. The research provides theoretical support for the cultivation of biofilm and its wider application.

A comprehensive floc model for simulating simultaneous nitrification, denitrification, and phosphorus removal

A comprehensive floc model for simultaneous nitrification, denitrification, and phosphorus removal (SNDPR) was designed, incorporating polyphosphate-accumulating organisms (PAOs), glycogen-accumulating organisms (GAOs), intrinsic half-saturation coefficients, and explicit external mass transfer terms. The calibrated model was able to effectively describe experimental data over a range of operating conditions. The estimated intrinsic half-saturation coefficients of oxygen values for ammonia-oxidizing bacteria, nitrite-oxidizing bacteria, ordinary heterotrophic organisms (OHOs), PAOs, and GAOs were set at 0.08, 0.18, 0.03, 0.07, and 0.1 mg/L, respectively. Simulation suggested that low dissolved oxygen (DO) environments favor K-strategist nitrifying bacteria and PAOs. In SNDPR, virtually all influent and fermentation-generated volatile fatty acids were assimilated as polyhydroxyalkanoates by PAOs in the anaerobic phase. In the aerobic phase, PAOs absorbed 997 % and 171 % of the benchmark influent total phosphorus mass loading through aerobic growth and denitrification via nitrite. These high percentages were because they were calculated relative to the influent total phosphorus, rather than total phosphorus at the end of the anaerobic period. When considering simultaneous nitrification and denitrification, about 23.1 % of influent total Kjeldahl nitrogen was eliminated through denitrification by PAOs and OHOs via nitrite, which reduced the need for both oxygen and carbon in nitrogen removal. Moreover, the microbial and DO profiles within the floc indicated a distinct stratification, with decreasing DO and OHOs, and increasing PAOs towards the inner layer. This study demonstrates a successful floc model that can be used to investigate and design SNDPR for scientific and practical purposes.

Evaluation of various carbon sources on ammonium assimilation and denitrifying phosphorus removal in a modified anaerobic-anoxic-oxic process from low-strength wastewater

A pilot-scale continuous-flow modified anaerobic-anoxic-oxic (MAAO) process examined the impact of external carbon sources (acetate, glucose, acetate/propionate) on ammonium assimilation, denitrifying phosphorus removal (DPR), and microbial community. Acetate exhibited superior efficacy in promoting the combined process of ammonia assimilation and DPR, enhancing both to 50.0 % and 60.0 %, respectively. Proteobacteria and Bacteroidota facilitated ammonium assimilation, while denitrifying phosphorus-accumulating organisms (DPAOs) played a key role in nitrogen (N) and phosphorus (P) removal. Denitrifying glycogen-accumulating organisms (DGAOs) aided N removal in the anoxic zone, ensuring stable N and P removal and recovery. Acetate/propionate significantly enhanced DPR (77.7 %) and endogenous denitrification (37.9 %). Glucose favored heterotrophic denitrification (29.6 %) but had minimal impact on ammonium assimilation. These findings provide valuable insights for wastewater treatment plants (WWTPs) seeking efficient N and P removal and recovery from low-strength wastewater.

Global abundance patterns, diversity, and ecology of Patescibacteria in wastewater treatment plants

BackgroundMicroorganisms are responsible for nutrient removal and resource recovery in wastewater treatment plants (WWTPs), and their diversity is often studied by 16S rRNA gene amplicon sequencing. However, this approach underestimates the abundance and diversity of Patescibacteria due to the low coverage of commonly used PCR primers for this highly divergent bacterial phylum. Therefore, our current understanding of the global diversity, distribution, and ecological role of Patescibacteria in WWTPs is very incomplete. This is particularly relevant as Patescibacteria are considered to be associated with microbial host cells and can therefore influence the abundance and temporal variability of other microbial groups that are important for WWTP functioning.ResultsHere, we evaluated the in silico coverage of widely used 16S rRNA gene-targeted primer pairs and redesigned a primer pair targeting the V4 region of bacterial and archaeal 16S rRNA genes to expand its coverage for Patescibacteria. We then experimentally evaluated and compared the performance of the original and modified V4-targeted primers on 565 WWTP samples from the MiDAS global sample collection. Using the modified primer pair, the percentage of ASVs classified as Patescibacteria increased from 5.9 to 23.8%, and the number of detected patescibacterial genera increased from 560 to 1576, while the detected diversity of the remaining microbial community remained similar. Due to this significantly improved coverage of Patescibacteria, we identified 23 core genera of Patescibacteria in WWTPs and described the global distribution pattern of these unusual microbes in these systems. Finally, correlation network analysis revealed potential host organisms that might be associated with Patescibacteria in WWTPs. Interestingly, strong indications were found for an association between Patescibacteria of the Saccharimonadia and globally abundant polyphosphate-accumulating organisms of the genus Ca. Phosphoribacter.ConclusionsOur study (i) provides an improved 16S rRNA gene V4 region-targeted amplicon primer pair inclusive of Patescibacteria with little impact on the detection of other taxa, (ii) reveals the diversity and distribution patterns of Patescibacteria in WWTPs on a global scale, and (iii) provides new insights into the ecological role and potential hosts of Patescibacteria in WWTPs.9DpTXjuT--dysnV-FwKdBeVideo

Novel three-sludge municipal wastewater treatment process coupling denitrifying phosphorus removal with anaerobic ammonium oxidation

The two-sludge anoxic dephosphation (DEPHANOX) process frequently encounters the challenge of elevated effluent ammonia levels in practical applications. In this study, the anaerobic ammonium oxidation (anammox) biofilm was introduced into the DEPHANOX system, transforming it into a three-sludge system, enabling synchronous nitrogen and phosphorus elimination, particularly targeting ammonia. Despite a chemical oxygen demand/total nitrogen ratio of 4.3 ± 0.8 in the actual municipal wastewater and 4.5 h of aeration, the effluent total nitrogen was 13.7 mg/L, lower than the parallel wastewater treatment plant. Additionally, the effluent ammonia reduced to 5.1 ± 2.5 mg/L. Notably, denitrifying phosphorus removal and anammox were coupled in the anoxic zone, yielding 74.5 % nitrogen and 87.8 % phosphorus removal. 16S rRNA gene sequencing identified denitrifying phosphorus-accumulating organisms primarily in floc sludge (Saprospiraceae 7.07 %, Anaerolineaceae 1.95 %, Tetrasphaera 1.57 %), while anammox bacteria inhabited the biofilm (Candidatus Brocadia 4.00 %). This study presents a novel process for efficiently treating municipal wastewater.

Refinement of the "Candidatus Accumulibacter" genus based on metagenomic analysis of biological nutrient removal (BNR) pilot-scale plants operated with reduced aeration.

Members of the "Candidatus Accumulibacter" genus are widely studied as key polyphosphate-accumulating organisms (PAOs) in biological nutrient removal (BNR) facilities performing enhanced biological phosphorus removal (EBPR). This diverse lineage includes 18 "Ca. Accumulibacter" species, which have been proposed based on the phylogenetic divergence of the polyphosphate kinase 1 (ppk1) gene and genome-scale comparisons of metagenome-assembled genomes (MAGs). Phylogenetic classification based on the 16S rRNA genetic marker has been difficult to attain because most "Ca. Accumulibacter" MAGs are incomplete and often do not include the rRNA operon. Here, we investigate the "Ca. Accumulibacter" diversity in pilot-scale treatment trains performing BNR under low dissolved oxygen (DO) conditions using genome-resolved metagenomics. Using long-read sequencing, we recovered medium- and high-quality MAGs for 5 of the 18 "Ca. Accumulibacter" species, all with rRNA operons assembled, which allowed a reassessment of the 16S rRNA-based phylogeny of this genus and an analysis of phylogeny based on the 23S rRNA gene. In addition, we recovered a cluster of MAGs that based on 16S rRNA, 23S rRNA, ppk1, and genome-scale phylogenetic analyses do not belong to any of the currently recognized "Ca. Accumulibacter" species for which we propose the new species designation "Ca. Accumulibacter jenkinsii" sp. nov. Relative abundance evaluations of the genus across all pilot plant operations revealed that regardless of the operational mode, "Ca. A. necessarius" and "Ca. A. propinquus" accounted for more than 40% of the "Ca. Accumulibacter" community, whereas the newly proposed "Ca. A. jenkinsii" accounted for about 5% of the "Ca. Accumulibacter" community.IMPORTANCEOne of the main drivers of energy use and operational costs in activated sludge processes is the amount of oxygen provided to enable biological phosphorus and nitrogen removal. Wastewater treatment facilities are increasingly considering reduced aeration to decrease energy consumption, and whereas successful BNR has been demonstrated in systems with minimal aeration, an adequate understanding of the microbial communities that facilitate nutrient removal under these conditions is still lacking. In this study, we used genome-resolved metagenomics to evaluate the diversity of the "Candidatus Accumulibacter" genus in pilot-scale plants operating with minimal aeration. We identified the "Ca. Accumulibacter" species enriched under these conditions, including one novel species for which we propose "Ca. Accumulibacter jenkinsii" sp. nov. as its designation. Furthermore, the MAGs obtained for five additional "Ca. Accumulibacter" species further refine the phylogeny of the "Ca. Accumulibacter" genus and provide new insight into its diversity within unconventional biological nutrient removal systems.

Aerobic and anoxic utilization of organic matter for flexible nitrite supply in nutrient conversion pathways based on anaerobic ammonium oxidation: Microbial interactive mechanism

This study investigated nutrient conversion pathways and corresponding interactive mechanisms in a mainstream partial-nitritation (PN)/anaerobic ammonium oxidation (anammox)/partial-denitrification-(PD)-enhanced biological phosphorus-removal (EBPR) (PN/A/PD-EBPR) process. A laboratory-scale sequencing batch reactor was operated for 301 days under different operational strategies. Mainstream PN/A/PD-EBPR was successfully operated with aerobic and anoxic utilization of organic matter. Aerobic utilization of organic matter was an effective strategy for conversion to denitrifying polyphosphate-accumulating organism-based phosphorus removal, referring to a biological reaction that outperformed nitrite-oxidizing bacteria. Aerobically adsorbed organic matter could be used as a carbon source for PD, which further enhanced nitrogen removal by PN/A. Ultimately, the interaction between complex nutrient conversion pathways served to achieve stable performance. High-throughput sequencing results elucidated the core microbe functioning in the mainstream PN/A/PD-EBPR process with respect to various nutrients. The outcomes of this study will be beneficial to those attempting to implement mainstream PN/A/PD-EBPR.

Partial denitrifying phosphorus removal coupling with anammox (PDPRA) enables synergistic removal of C, N, and P nutrients from municipal wastewater: A year-round pilot-scale evaluation

Applying anaerobic ammonium oxidation (anammox) in municipal wastewater treatment plants (MWWTPs) can unlock significant energy and resource savings. However, its practical implementation encounters significant challenges, particularly due to its limited compatibility with carbon and phosphorus removal processes. This study established a pilot-scale plant featuring a modified anaerobic-anoxic-oxic (A2O) process and operated continuously for 385 days, treating municipal wastewater of 50 m3/d. For the first time, we propose a novel concept of partial denitrifying phosphorus removal coupling with anammox (PDPRA), leveraging denitrifying phosphorus-accumulating organisms (DPAOs) as NO2− suppliers for anammox. 15N stable isotope tracing revealed that the PDPRA enabled an anammox reaction rate of 6.14 ± 0.18 μmol-N/(L·h), contributing 57.4 % to total inorganic nitrogen (TIN) removal. Metagenomic sequencing and 16S rRNA amplicon sequencing unveiled the co-existence and co-prosperity of anammox bacteria and DPAOs, with Candidatus Brocadia being highly enriched in the anoxic biofilms at a relative abundance of 2.46 ± 0.52 %. Finally, the PDPRA facilitated the synergistic conversion and removal of carbon, nitrogen, and phosphorus nutrients, achieving remarkable removal efficiencies of chemical oxygen demand (COD, 83.5 ± 5.3 %), NH4+ (99.8 ± 0.7 %), TIN (77.1 ± 3.6 %), and PO43− (99.3 ± 1.6 %), even under challenging operational conditions such as low temperature of 11.7 °C. The PDPRA offers a promising solution for reconciling the mainstream anammox and the carbon and phosphorus removal, shedding fresh light on the paradigm shift of MWWTPs in the near future.

Improving BioWin Modeling of Phosphorus Solubilization in Acid-Phase Digesters

BioWin 6.0 does not accurately predict phosphorus (P) speciation in acidogenic anaerobic digesters under default kinetics characterization and parameterization. The accurate modeling of acid-phase digestion is needed to predict the performance of novel nutrient recovery technologies that act on these digester effluents. The main thrust of this work was to identify and correct the causes of inaccurate P partitioning and precipitation within BioWin models of acid-phase digestion reactors. A BioWin configuration including an organic acid digester was parameterized and recalibrated based on the known traits of acid-phase digestion and then validated against a full-scale digester in a municipal wastewater treatment plant. This digester, with pH 5.14 and 61–74% solubilized P, was predicted by BioWin default parameters to have only 27% soluble P and a net formation of P precipitates. Corrections to the polyphosphate-accumulating organism decay, endogenous product decay, hydrolysis rate, and brushite behavior resulted in 67% solubilization with no precipitate formation. Cabinet configurations showed similar behavior when modified to include an acid-phase digester under default parameters, but predictions were similarly amended by our parameter changes. This improved modeling technique should allow operators to effectively characterize acid digesters for their own treatment trains and allow engineers to predict the performance of novel nutrient recovery technologies acting on acidogenic digest.

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.

Simulation assisted process simplification and energy recovery from a cryptic biological nutrient removal plant

In this study, simulation-based analysis is conducted for nitrogen removal mechanism in a biological nutrient removal plant designed in an encrypted manner. In the first phase, the simulation study elucidates how nitrogen removal occurs in a complex design using a series of 4 carousel bioreactors with additional nitrate recycle. It is demonstrated that pre-anoxic volume fed with limited internal recirculation only would not be sufficient to meet the discharge Total Nitrogen (TN) limit of 10 mg N/L. However, the addition of simultaneous nitrification-denitrification (SND) processes in aerobic reactors enables compliance with TN discharge limits. In the second phase, the configuration is simplified by means of process simulation, where the complex design is streamlined by eliminating internal recycle while achieving nitrogen removal through the SND process. Plant data confirmed that the simplified configuration can achieve nitrogen removal providing the same discharge quality. The simulations resulted that TN concentration achieved at the plant effluent is comparable, while effluent total phosphorus concentration is decreased by approximately 50 % due to increased activity of Phosphorus Accumulating Organisms (PAOs) under reduced sludge retention time (SRT). In the third phase, the application of the SND process in the field using a simplified and new activated sludge configuration yields similar results to the simulation. According to plant records, 28 % energy savings and an equivalent chemical cost reduction of €945,000 per year were achieved with the new process configuration.

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.

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.

Phosphorus mining from marine sediments adopting different carbon/nitrogen strategies driven by anaerobic reactors: The exploration of potential mechanism and microbial activities

To investigate the possibility of phosphorus (P) recovery from marine sediment and explore the role of the carbon: nitrogen ratio in affecting the internal P release under anaerobic conditions, we experimented with the external addition of carbon (acetic acid and glucose) and ammonia nitrogen (NH4-N) to expose P release mechanisms. The 24-day anaerobic incubations were conducted with four different carbon: nitrogen dosing groups including no NH4-N addition and COD/N ratios of 100, 50, and 10. The P release showed that extra NH4-N loading significantly suppressed the decomposition of P (p < 0.05) from the marine sediment, the maximum P release was 4.07 mg/L and 7.14 mg/L in acetic acid- and glucose-fed systems, respectively, without extra NH4-N addition. Additionally, the results exhibited that the imbalance of carbon: nitrogen not only failed to induce the production of organic P mineralization enzyme (alkaline phosphatase) in the sediment but also suppressed its activity under anaerobic conditions. The highest enzyme activity was observed in the group without additional NH4-N dosage, with rates of 1046.4 mg/(kg∙h) in the acetic acid- and 967.8 mg/(kg∙h) in the glucose-fed system, respectively. Microbial data analysis indicated that a decrease in the abundance of P release-regulating bacteria, including polyphosphate-accumulating organisms (Rhodobacteraceae) and sulfate-reducing bacteria (Desulfosarcinaceae), was observed in the high NH4-N addition groups. The observed reduction in enzyme activity and suppression of microbial activity mentioned above could potentially account for the inhibited P decomposition in the presence of high NH4-N addition under anaerobic conditions. The produced P-enriched solution from the bioreactors may offer a promising source for future recovery endeavors.