Polyphosphate-accumulating Organisms Research Articles

Machine learning application reveal dynamic interaction of polyphosphate-accumulating organism in full-scale wastewater treatment plant

Enhanced biological phosphorous removal (EBPR) represents one of the most widely used wastewater systems for treating phosphorous-bearing waste streams. This study aimed to advance the current understanding of the diversity and interactions of polyphosphate-accumulating organisms (PAOs), playing a key role for the EBPR performance. Frequently occurring and predominant organisms consisted of PAOs such as Tetrasphaera, Candidatus Accumulibacter, Acinetobacter, Pseudomonas, and glycogen-accumulating organisms (GAOs) such as Defluvicoccus, Microlunatus, Nakamurella, and Kineosphaera. The degree of immigrating PAOs/GAOs carried with the influent was associated with their succession within the EBPR processes. Ca Accumulibacter and Nakamurella were transported to a greater degree with the influents of combined and separate sewers, respectively. Although no significant shifts in the PAO-GAO community structure were observed across seasons using conventional ordination-based analyses, machine learning (ML) modeling using the PAO-GAO microbiome data could successfully predict the EBPR system's environmental condition (water temperature) with linear and non-linear models (R2 of 0.4–0.7). ML-based feature importance analysis identified two specific species populations, Ca Accumulibacter (PAO) and Defluvicoccus (GAO), strongly correlated with high water temperatures, in contrast to the existing understanding based on laboratory experiments. The positive association among Ca Accumulibacter, Defluvicoccus, and the water temperature in full scale EBPR processes was validated with microbial association network analysis as well as the ML-based results. Overall, our ML-enabled analysis could provide new findings about the interplay between PAOs and environmental factors, which have practical implications for sustainable design and operation of full scale EBPR processes.

Role of carrier characteristics affecting microbial density and population in enhanced nitrogen and phosphorus removal from wastewater

This research aims to improve simultaneous nitrification-denitrification and phosphorus removal (SNDPR) using novel carriers and to demonstrate the effect of carrier characteristics on nutrient removal in a biofilm reactor. For this purpose, biofilms enriched with both polyphosphate-accumulating organisms (PAOs) and nitrifiers were cultivated in two parallel sequencing batch reactors containing conventional moving bed bioreactor carriers (MBBR) and a novel type of carriers (carbon-based moving carriers (CBMC)). The new carriers were produced based on recycled waste materials via a chemical-thermal process and their specific surface area were 10.4 times higher than typical MBBR carriers of similar dimensions. The results showed that the use of CBMC carriers increased bacterial adhesion by about 18.5% and also affected the microbial population inside the biofilms, leading to an increase in PAOs abundancy and thus an increase in biological phosphorus removal up to 12.5%. Additionally, it was corroborated that the volume of the anoxic zones with dynamic behavior is strictly influenced by the carrier structure and biofilm thickness due to a limitation in oxygen penetration. Accordingly, the formation of broader anoxic zones and shrinkage of these zones to a lesser extent resulted in the continuation of anoxic reactions for longer periods using the novel carriers. Thereby, an increase in nitrogen removal by about 15% was obtained mainly by denitrifying PAOs. The results also exhibited that a higher simultaneous nitrification-denitrification (SND) efficiency can be achieved by selecting an appropriate aeration program influencing the dynamic changes of anoxic zones. Overall, a biofilm system using the new carriers, with phosphorus and nitrogen removal efficiencies of 97.5% and 92.3%, was presented as an efficient, compact, and simple operation SNDPR process.

Diclofenac biotransformation in the enhanced biological phosphorus removal process

Diclofenac is a pharmaceutical active compound frequently detected in wastewater and water bodies, and often reported to be persistent and difficult to biodegrade. While many previous studies have focussed on assessing diclofenac biodegradation in nitrification and denitrification processes, this study focusses on diclofenac biodegradation in the enhanced biological phosphorus removal (EBPR) process, where the efficiency of this process for diclofenac biodegradation as well as the metabolites generated are not well understood. An enrichment of Accumulibacter polyphosphate accumulating organisms (PAOs) was operated in an SBR for over 300 d, and acclimatized to 20 μg/L of diclofenac, which is in a similar range to that observed in domestic wastewater influents. The diclofenac biotransformation was monitored in four periods of stable operation and linked to the microbial community and metabolic behaviour in each period. Nitrification was observed in two of the four periods despite the addition of a nitrification inhibitor, and these periods were positively correlated with increased diclofenac biodegradation. Interestingly, in two periods with excellent phosphorus removal (>99%) and no nitrification, different levels of diclofenac biotransformation were observed. Period 2, enriched in Accumulibacter Type II achieved more significant diclofenac biotransformation (3.4 μg/gX), while period 4, enriched in Accumulibacter Type I achieved lower diclofenac biotransformation (0.4 μg/gX). In total, 23 transformation products were identified, with lower toxicity than the parent compound, enabling the elucidation of multiple metabolic pathways for diclofenac biotransformation. This study showed that PAOs can contribute to diclofenac biotransformation, yielding less toxic transformation products, and can complement the biodegradation carried out by other organisms in activated sludge, particularly nitrifiers.

Adsorption-Release Characteristics of Phosphorus and the Community of Phosphorus Accumulating Organisms of Sediments in a Shallow Lake

One of the most challenging issues for developing countries in modern times is the care and management of clean, potable drinking water sources. Accordingly, this study singled out potential contributing factors to harmful algae blooms with a particular focus on phosphorus (P) release. The potential risks of P release for the drinking water from a lake in Fujian were assessed by investigating the spatial-temporal distribution of P, its exchange capacity, and its discharge capacity in sediment, including the community composition of phosphorus accumulating organisms (PAOs) and the phosphate’s initial migration effects on sediments. Different mixed materials, including soil aggregate distributions, sorbent, fractions, adsorption-desorption of P, and the community composition of PAOs were assessed. Total phosphorus (TP) content was measured at 24.4 ± 1.2 to 563.9 ± 38.2 mg/kg, but contents displayed some spatial differences. The dominant Ps found in the sample sediments were organic phosphorus and inorganic phosphorus associated with hydroxide Fe/Al-P, which accounted for 48.6% and 43.6%, respectively, of the TP content in the lake’s central waters. The TP concentration in vertical sediment ranged from 436.2 ± 21.3 to 602.9 ± 31.4 mg/kg. The TP spatio-temporal distribution inputs varied with rainfall (p < 0.05). P deposition occurred throughout most water bodies (p < 0.05), covering extensive areas and also decreasing at lower depths. Forty-four operational taxonomic unit (OTU) phosphorus-accumulating organism types from 11 phyla were detected in the sediment samples obtained from the Sanshiliujiao Lakes region. Proteobacteria also dominated compared to the organisms with the strongest PAOs. The diversity of PAOs in summer samples was significantly higher than that of the autumn samples. These findings provide a scientific foundation for determining the future discovery of the microbial mechanisms involved in the phosphorus metabolic cycle found in reservoir sediments. Various forms of phosphorus influenced the PAO diversity, especially Fe/Al-P. Thus, the abundance of PAOs in the sediment proved to be an essential component of the P cycle and may even play a key role in regional material circulation and in causing other environmental issues.

Integrating electrochemical and biological phosphorus removal processes via electrokinetic-based technology

Excessive phosphorus levels in aquatic environments create harmful algal bloom that produces cultural eutrophication and poor water quality. The phosphorus removal from municipal wastewater treatment plants (WWTPs) is required to control this undesirable phenomenon. Removing nutrients from influent wastewater necessitates the construction of various operating units to provide different conditions (i.e., anoxic and aerobic) to promote the removal efficiency of each single-nutrient compound. Unfortunately, such designs have a high energy consumption rate and leave a big carbon footprint. The present work investigates the possibility of simultaneous electrochemical and biological phosphorus removal in a single reactor. A bench-scale electro-bioreactor was fed synthetic wastewater containing phosphorus ranging from 14 to 18 mg PO4−P/L. At steady-state conditions, the phosphorus removal efficiency (PO4−P) reached over 99%. Furthermore, a single membrane electro-bioreactor (MEBR) created anoxic and aerobic conditions suitable for polyphosphate-accumulating organisms (PAOs) growth. The operation of electrocoagulation inside MEBR also generated Al3+ due to the electrolytic dissolution of aluminum anode, increasing the system’s electrochemical phosphorus removal. Therefore, findings suggest that such a high percentage of phosphorus removal was due to both the electrocoagulation process and proliferation of PAOs in the reactor. The developed MEBR is particularly suitable for small-scale plants for various applications, including mining, remote and rural communities, settlements, and temporary (e.g., exploration or military).

Molecular Coordination, Structure, and Stability of Metal-Polyphosphate Complexes Resolved by Molecular Modeling and X-ray Scattering: Structural Insights on the Biological Fate of Polyphosphate.

Polyphosphate-accumulating organisms (PAOs), which can store high levels of phosphate (Pi) in the form of polyphosphate (polyP), are employed to engineer enhanced biological P removal (EBPR) from wastewaters. Co-localization of Mg and K in polyP granules of PAOs has been reported, and higher abundance of Mg-polyP granules relative to other metal complexes was correlated positively with EBPR performance stability. However, the underlying mechanism remains unknown. Here, we obtained molecular structural information of hydrated polyP complexes with four physiologically relevant metal cations (Na+, K+, Ca2+, and Mg2+) using computational and experimental techniques. Molecular dynamics simulations revealed that Mg-polyP and K-polyP complexes were the most and least stable of the complexes, respectively, suggesting that the co-occurrence of these complexes facilitates variable polyP bioavailability. The relative thermodynamic stability reflected the strength of metal chelation whereby the coordination distance between the polyP ligand O and the metal was 1.71-2.01 Å for Mg2+ but this distance was 2.64-2.70 Å for K+. Pair distribution function analysis of X-ray scattering data obtained with a Mg-polyP solution corroborated the theoretical Mg-polyP coordination geometry. These findings implied a possible mechanistic role of metal complexation in the P cycling traits of PAOs in engineered and natural systems.

N-acyl-homoserine-lactones signaling as a critical control point for phosphorus entrapment by multi-species microbial aggregates

Quorum sensing (QS) has been extensively studied in pure stains of microorganisms, but the ecological roles of QS in multi-species microbial aggregates are poorly understood due to the aggregates’ heterogeneity and complexity, in particular the phosphorus (P) entrapment, a key aspect of element cycling. Using periphytic biofilm as a microbial-aggregate model, we addressed how QS signaling via N-acyl-homoserine-lactones (AHLs) regulated P entrapment. The most-abundant AHLs detected were C8-HSL, 3OC8-HSL, and C12-HSL, are the primary regulator of P entrapment in the periphytic biofilm. QS signaling-AHL is a beneficial molecule for bacterial growth in periphytic biofilm and the addition of these three AHLs optimized polyphosphate accumulating organisms (PAOs) community. Growth promotion was accompanied by up-regulation of pyrimidine, purine and energy metabolism. Both intra- and extra-cellular P entrapment were enhanced in the addition of AHLs. AHLs increased extracellular polymeric substances (EPS) production to drive extracellular P entrapment, via up-regulating amino acids biosynthesis and amino sugar/nucleotide sugar metabolism. Also, AHLs improved intracellular P entrapment potential by regulating genes involved in inorganic-P accumulation (ppk, ppx) and P uptake and transport (pit, pstSCAB). This proof-of-concept evidence about how QS signaling regulates P entrapment by microbial aggregates paves the way for managing QS to enhance P removal by microbial aggregates in aquatic environments.

The storage compounds associated with Tetrasphaera PAO metabolism and the relationship between diversity and P removal

In enhanced biological phosphorus removal (EBPR), Tetrasphaera can potentially be an abundant and important polyphosphate accumulating organism (PAO), however ongoing questions remain concerning its storage compounds, phosphorus (P) removal capabilities and metabolic behaviour. This study investigated each of these points in an enriched Tetrasphaera culture (95% biovolume). The enriched Tetrasphaera culture fermented amino acids, while also converting and storing diverse amino acids as aspartic and glutamic acid within cells. Subsequent intracellular consumption of these two amino acids during the aerobic phase supports their importance in the metabolism of Tetrasphaera. Polyhydroxyalkanoate (PHA) cycling was also observed in this study, in contrast to some previous studies on Tetrasphaera. While exhibiting anaerobic phosphorus release and aerobic uptake, the highly enriched Tetrasphaera culture was unable to completely remove phosphorus in sequencing batch reactors (SBR) cycles, with an average removal efficiency of 72.3 ± 7.8%. This is unlike a previous study containing both Tetrasphaera (70%) and Accumulibacter (22%), which regularly performed complete phosphorus removal under otherwise similar operational conditions, at efficiencies of > 99%. Notably, the phylodiversity of organisms belonging to Tetrasphaera was substantially different in the present work, consisting mainly of organisms within Clade 2, likely impacting PHA cycling. These results suggest that the contribution of Tetrasphaera towards P removal is highly dependent on the composition of its Clades within this microbial group and an observed higher abundance of Tetrasphaera in WWTPs does not necessarily imply overall higher P removal. This study improves our understanding of the role of Tetrasphaera within EBPR systems and key factors impacting its metabolism.

Effects of seasonal temperature variations on phosphorus removal, recovery, and key metabolic pathways in the suspended biofilm

The effects of seasonal temperature variations on phosphorus removal performance and community structure of dominant polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) were explored. Such impacts on the dominance of PAOs and GAOs involved in carbon and phosphorus metabolic pathways and gene abundance of key enzymes in biofilm sequencing batch reactor (BSBR) were studied. Enzyme assay and Miseq high-throughput sequencing revealed that typical GAOs hold a competitive advantage, the activities of phosphate kinase (PPK) and phosphate hydrolase (PPX) were decreased, and typical GAOs deteriorated phosphorus removal performance in summer (27–30 °C). In spring, autumn, and winter (15–22 °C), PAOs dominated the competitive advantage, the activities of PPK and PPX were increased, and atypical PAOs dominated the improvement of phosphorus removal performance. Metagenomic sequencing showed that the reducing power of PAOs for polyhydroxyalkanoate ( PHA) synthesis mainly came from the glycolysis pathway, followed by hexose monophosphate pathway (HMP) and tricarboxylic acid (TCA) cycles. The change of phosphorus removal performance was not only related to the carbon and phosphorus metabolic pathways activated by PAOs, but also closely associated with the change of the competitive pathways between GAOs and PAOs. Collectively, our findings provided a temperature reference for the improvement of BSBR process performance and added the first in-depth information for the understanding of the phosphorus removal mechanism and the competition mechanism with GAOs in BSBR.

Tetrasphaera, rather than Candidatus Accumulibacter as performance indicator of free ammonia inhibition during the enhanced biological phosphorus removal processes

Free ammonia (FA) has been proposed to inhibit phosphorus (P) removal efficiency and reconfigures the microbial community in the enhanced biological phosphorus removal (EBPR) systems. However, in response to FA exposure, the microbial kinetics and the functional bacteria belonging to polyphosphate accumulating organisms (PAOs) i.e., Tetrasphaera and Candidatus Accumulibacter within the mixed communities are generally ignored. This work was conducted to characterize the effects of FA on the biological processes of P-release and its uptake, and the virtual role of PAOs in EBPR systems. Experimental data demonstrated that the FA strongly inhibited the P-release and uptake rates which was described well by Hellinga and Vadivelu models, and the determined R2 in the anaerobic and aerobic period were about 0.91 and 0.96. Moreover, the abundance of key microbial communities differed significantly with various FA concentrations. Structural equation model (SEM) revealed that FA was positively associated with Ca. Accumulibacter (path coeff. = 0.30; P = 0.13) and Ca. Competibacter (path coeff. = 0.43; P < 0.05), while it was negatively associated with Tetrasphaera (path coeff. = − 0.45; P < 0.001). Furthermore, Tetrasphaera was found to strongly and positively drive the rates of P-release and uptake, and was confirmed as the only virtual biomarker for indicating the response of EBPR performance towards FA inhibition. Two hypothetical mechanisms indicated that Ca. Accumulibacter under higher FA concentration could not synthesize PHA to support the EBPR process. In addition, the release of P was hindered due to the FA toxicity on the urea cycle of Tetrasphaera microbial cells. This work gives comprehensive and deep insight into the effect of FA on the EBPR system and provides a useful guide for process optimization.

Achieving combined biological short-cut nitrogen and phosphorus removal in a one sludge system with side-stream sludge treatment

Biological nitrogen (N) removal via the short-cut pathway (NH4+-N→NO2−-N→N2) is economically attractive in wastewater treatment plants (WWTPs). However, biological phosphorus (P) removal processes remain a bottleneck in these systems due to the strong inhibitory effect of nitrite or its protonated form (HNO2, free nitrous acid - FNA) on polyphosphate accumulating organisms (PAOs). In this study, a novel combined nitrogen and phosphorus removal strategy was verified and achieved in a biological short-cut nitrogen removal system via side-stream sludge treatment with FNA, and the mechanisms impacting this process were investigated. The side-stream FNA treatment process applied here led to a significant reduction in the real sludge retention time (SRT) in the mainstream (approximately 2.7 days) based on the biocidal effect of FNA to the majority of the organisms. This work also found that around 40% of the P uptake activity was still maintained at a much higher FNA level of 38 μg N/L with potential PAOs, which highly broadened the current knowledge of PAOs community. An economic analysis revealed advantages of the proposed as compared to conventional biological nitrogen and phosphorus removal (13% savings in total cost), biological short-cut nitrogen removal (via FNA treatment) with chemical phosphorus precipitation (21% savings) and conventional biological nitrogen removal with chemical precipitation (27% savings). Overall, this study presents a novel and viable retrofit strategy in integrating biological short-cut nitrogen removal with EBPR for next generation WWTPs.

Partitioning of nutrient removal contribution between granules and flocs in a hybrid granular activated sludge system

Sludge granulation in continuous-flow systems is an emerging technology to intensify existing activated sludge infrastructure for nutrient removal. In these systems, the nutrient removal contributions and partitioning of microbial functions between granules and flocs can offer insights into process implementations. To this end, a reactor system that simulates the continuous-flow environment using an equal amount of initial granule and floc biomass was investigated. The two operational strategies for maintaining granule growth in the continuous-flow system were (a) the higher solids retention time (SRT) for the granules versus flocs, as well as (b) selective feeding of carbon to the granules. The SRT of the large granule fractions (>425 µm, LG) and floc/small granule fractions (<425 µm, FSG) were controlled at 20 and 2.7–6.0 days, respectively. Long term operation of the hybrid granule/floc system achieved high PO43- and NH4+ removal efficiencies. Higher polyphosphate-accumulating organisms (PAO) activity was observed in the FSG than LG, while ammonia-oxidizing bacteria (AOB) activities were similar in the two biomass fractions. Nitrite shunt was observed in the FSG, possibly due to out-competition by the high NOB activity in LG. More importantly, washing out the FSG caused a reduction in LG's AOB and PAO activity, indicating a possible dependency of LG on FSG for maintaining its nutrient removal capacity. Our findings highlighted the partitioning and potential competition/cooperation of key microbial functional groups between LG and FSG, facilitating nutrient removal in a hybrid granular activated sludge system, as well as implications for practical application of the treatment platform.

Rapidly achieving partial nitrification of municipal wastewater in enhanced biological phosphorus removal (EBPR) reactor: Effect of heterotrophs proliferation and microbial interactions

Stable nitritation is the major challenge for short-cut nitrogen removal from municipal wastewater. This paper demonstrated a rapid achievement of partial nitrification (PN) in an enhanced biological phosphorus removal (EBPR) reactor treating domestic wastewater. Polyphosphate accumulating organisms (PAOs) were enriched operated at a short aerobic HRT (2.0 h) and SRT (10 d), with satisfactory phosphorus removal efficiency (95.9%). Both of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) were elutriated simultaneously. Interestingly, AOB recovered much faster than NOB by a subsequent extension of aerobic HRT and SRT, resulting in a rapid development of PN within 15 days. Ammonia oxidation rates of AOB significantly increased by 44.2%, facilitating a high nitrite accumulation rate (NAR) of 95.8%. Genus Tetrasphaera, Halomonas, Paracoccus and Candidatus_Accumulibacter belonging to PAOs accounted for 4.6%. The proliferation of heterotrophs, typically as PAOs, maximized the microbial competition against NOB by favoring AOB activity and synergy with functional bacteria.

Assessing biomass activities, sludge characteristics and membrane fouling in the University of Cape Town membrane bioreactor under ferric chloride addition

Membrane bioreactor (MBR) technology has been commonly employed in the treatment of domestic wastewater. However, total phosphorus (TP) removal performance and membrane fouling are two major challenges for MBR application. In this study, FeCl 3 ⋅ 6H2O was added to the membrane tank (MT) to improve the overall performance of University of Cape Town (UCT)-MBR. When the Fe(III) dosing concentration was increased from 0 to 1.8 mmol/L, the concentration of TP in the effluents was declined from 3.0 to 0.1 mg/L, and the high transmembrane pressure was reduced by 21.9%. The total nitrogen (TN) removal efficiency remained similar. However, when the Fe(III) dosing concentration was increased to 2.2 and 2.6 mmol/L, the phosphorus accumulating organisms (PAOs) and ammonia oxidizing bacteria (AOB) activities were greatly inhibited. Based on the overall nutrient removal and membrane fouling mitigation performances, the optimal Fe(III) dosing concentration was determined to be 1.8 mmol/L

Start-up of an aerobic granular sludge system from stored granules: Evaluating the impact of storage period on biomass activity and stability and the effect of temperature on nitrification and phosphorus removal rates

Aerobic granular sludge (AGS) has been considered a breakthrough within the wastewater treatment sector. However, the long reactor start-up for the formation of granules is challenging and may hinder the spread of this technology. To circumvent this obstacle, inoculation of the reactors with pre-formed granules from existing plants is an interesting approach. In this context, issues related to biomass storage becomes very relevant. In this study, reactivation of aerobic granular biomass after storage was evaluated in a sequencing batch reactor (SBR) designed for achieving simultaneous organic matter, nitrogen and phosphorus removal. Two different scenarios, short (40 days) and long (180 days) storage periods, were assessed, and their influence on the granules physical properties and bioactivity was addressed. The results revealed that the granules stored for a shorter period showed higher resistance to breakage and underwent smooth color changes. On the other hand, the biomass stored for a longer period acquired a dark color and was more susceptible to disruption during reactivation. The granules stored for 6 months become swollen and exhibited an irregular morphology and fluffy structure within the first days of reactivation. Consequently, their settling properties were adversely affected, and some parameters such as the food-to-microorganism ratio had to be adjusted to prevent granules disintegration. Regarding the bioactivity of important microbial functional groups, COD removal was rapidly restored within a few days of SBR operation with the biomass stored for a shorter period. However, it took longer for the biomass stored for 180 days to reach the same performance observed for the granules stored for 40 days. A similar trend is valid for nitrification. In the experiments with sludge stored for a longer time, it took almost twice as long to reach effluent ammonium concentrations lower than 1 mg NH4+-N L−1 compared to the test using biomass stored for 40 days. Phosphate removal was strongly affected by biomass storage, especially after 180 days of inactivity, a condition found to be detrimental for polyphosphate-accumulating organisms. Finally, cycle tests were also conducted to assess substrate conversion rates for comparison between different trials and evaluate the influence of temperature (10–35 °C) on nitrification and phosphate removal rates.

Metabolic Differentiation of Co-occurring Accumulibacter Clades Revealed through Genome-Resolved Metatranscriptomics.

ABSTRACTNatural microbial communities consist of closely related taxa that may exhibit phenotypic differences and inhabit distinct niches. However, connecting genetic diversity to ecological properties remains a challenge in microbial ecology due to the lack of pure cultures across the microbial tree of life. “Candidatus Accumulibacter phosphatis” (Accumulibacter) is a polyphosphate-accumulating organism that contributes to the enhanced biological phosphorus removal (EBPR) biotechnological process for removing excess phosphorus from wastewater and preventing eutrophication from downstream receiving waters. Distinct Accumulibacter clades often coexist in full-scale wastewater treatment plants and laboratory-scale enrichment bioreactors and have been hypothesized to inhabit distinct ecological niches. However, since individual strains of the Accumulibacter lineage have not been isolated in pure culture to date, these predictions have been made solely on genome-based comparisons and enrichments with varying strain compositions. Here, we used genome-resolved metagenomics and metatranscriptomics to explore the activity of coexisting Accumulibacter strains in an engineered bioreactor environment. We obtained four high-quality genomes of Accumulibacter strains that were present in the bioreactor ecosystem, one of which is a completely contiguous draft genome scaffolded with long Nanopore reads. We identified core and accessory genes to investigate how gene expression patterns differed among the dominating strains. Using this approach, we were able to identify putative pathways and functions that may confer distinct functions to Accumulibacter strains and provide key functional insights into this biotechnologically significant microbial lineage.IMPORTANCE “Candidatus Accumulibacter phosphatis” is a model polyphosphate-accumulating organism that has been studied using genome-resolved metagenomics, metatranscriptomics, and metaproteomics to understand the EBPR process. Within the Accumulibacter lineage, several similar but diverging clades are defined by the shared sequence identity of the polyphosphate kinase (ppk1) locus. These clades are predicted to have key functional differences in acetate uptake rates, phage defense mechanisms, and nitrogen-cycling capabilities. However, such hypotheses have largely been made based on gene content comparisons of sequenced Accumulibacter genomes, some of which were obtained from different systems. Here, we performed time series genome-resolved metatranscriptomics to explore gene expression patterns of coexisting Accumulibacter clades in the same bioreactor ecosystem. Our work provides an approach for elucidating ecologically relevant functions based on gene expression patterns between closely related microbial populations.

Response of simultaneous nitrification-denitrification to DO increments in continuously aerated biofilm reactors for aquaculture wastewater treatment

Abstract Intensive aquaculture usually produces large volumes of nutrient-rich wastewater, which is essential to treat to avoid eutrophication. This study aimed to evaluate the performance of five continuously aerated biofilm reactors treating simulated high-strength aquaculture wastewater under different dissolved oxygen (DO) levels, and the effects of DO increments on simultaneous nitrification-denitrification (SND). Continuous aeration was beneficial to complete nitrification. Total inorganic nitrogen (TIN), principally ammonium, was mainly removed by SND. The SND rate response to different DO levels was fitted well by the power function of y = 54.81 + 371.58/(1 − 0.16*x)^(−1/0.24) (R2 = 0.897, P = 0.000). When the TIN was removed completely, the optimal SND rate was defined and corresponded to a value of 121.8%. Accordingly, the optimal DO concentration was calculated as 2.10 mg/L, close to the actual level of 1.83 mg/L, at which the highest proportional removals of total nitrogen (58.0%) and TIN (57.3%) were obtained. Phosphorus was also removed by denitrifying polyphosphate-accumulating organisms.

Nitrite and nitrate inhibition thresholds for a glutamate-fed bio-P sludge

Enhanced biological phosphorus removal (EBPR) is an efficient and sustainable technology to remove phosphorus from wastewater. A widely known cause of EBPR deterioration in wastewater treatment plants (WWTPs) is the presence of nitrate/nitrite or oxygen in the anaerobic reactor. Moreover, most existing studies on the effect of either permanent aerobic conditions or inhibition of EBPR by nitrate or free nitrous acid (FNA) have been conducted with a “Candidatus Accumulibacter” or Tetrasphaera-enriched sludge, which are the two major reported groups of polyphosphate accumulating organisms (PAO) with key roles in full-scale EBPR WWTPs. This work reports the denitrification capabilities of a bio-P microbial community developed using glutamate as the sole source of carbon and nitrogen. This bio-P sludge exhibited a high denitrifying PAO (DPAO) activity, in fact, 56% of the phosphorus was uptaken under anoxic conditions. Furthermore, this mixed culture was able to use nitrite and nitrate as electron acceptor for P-uptake, being 1.8 μg HNO2–N·L−1 the maximum FNA concentration at which P-uptake can occur. Net P-removal was observed under permanent aerobic conditions. However, this microbial culture was more sensitive to FNA and permanent aerobic conditions compared to “Ca. Accumulibacter”-enriched sludge.

Advanced nitrogen and phosphorus removal by combining endogenous denitrification and denitrifying dephosphatation in constructed wetlands

To achieve high-efficiency nutrient removal in constructed wetlands (CWs), a novel simultaneous nitrogen and phosphorus removal (SNPR) process was developed by combining nitrification, endogenous denitrification, and denitrifying phosphorus removal. In SNPR process, denitrifying glycogen-accumulating organisms (DGAOs) and denitrifying polyphosphate-accumulating organisms (DPAOs) utilized NOx--N(NO3--N or NO2--N) as electron acceptor and poly-beta-hydroxy-alkanoates (PHAs) as carbon sources for endogenous denitrification and denitrifying phosphorus removal processes. Results from 217 days of operation showed that a high-level of nitrogen removal efficiency of 83.73% was achieved with influent COD/N of 4. The success was attributed to the fact that most of influent carbon sources could be transformed into PHAs before nitrification via enriching DGAOs and DPAOs in CW, which simultaneously improved nitrification and denitrification due to reducing oxygen and carbon sources consumption by aerobic heterotrophs. Phosphorus was mainly removed via denitrifying phosphorus removal, and PO43--P removal efficiency reached up to 87.84% with even common gravel used as substrate. Stoichiometry analysis revealed that DGAOs were the main organisms providing nitrite to DPAOs, suggesting that the effective PO43--P removal under high DGAO abundance condition might be attributed to the coordination of DGAOs and DPAOs in SNRP processes.

Integrated omics analyses reveal differential gene expression and potential for cooperation between denitrifying polyphosphate and glycogen accumulating organisms

Unusually high accumulation of the potent greenhouse gas nitrous oxide (N2 O) has previously been documented in denitrifying biological phosphorus (P) removal bioprocesses, but the roles of differential denitrification gene expression patterns and ecological interactions between key functional groups in driving these emissions are not well understood. To address these knowledge gaps, we applied genome-resolved metagenomics and metatranscriptomics to a denitrifying bioprocess enriched in as-yet-uncultivated denitrifying polyphosphate accumulating organisms (PAOs) affiliated with Candidatus Accumulibacter. The six transcriptionally most active populations in the community included three co-occurring Accumulibacter strains affiliated with clades IF (a novel clade identified in this study), IA and IC, a competing glycogen accumulating organism (GAO) affiliated with Competibacteraceae (GAO1), a Gammaproteobacteria PR6 and an Anaerolineae CH7. Strongly elevated expression of nitrite reductase genes compared to nitrous oxide reductase genes was observed in the overall community and in Accumulibacter populations, suggesting a strong role for differential gene expression in driving N2 O accumulation. Surprisingly, while ~90% of the nirS gene transcripts were expressed by the three co-occurring PAO populations, ~93% of the norB gene transcripts were expressed by GAO1 and ~75% of the norZ gene transcripts were mapped to PR6 and several other non-PAO flanking populations. This suggests the potential for cooperation between flanking populations and PAOs in reducing denitrification intermediates. Such cooperation may benefit the community by reducing the accumulation of toxic nitric oxide.