Biological Phosphorus Removal Research Articles

Effects of enhanced biological phosphorus removal on rapid control of sludge bulking and fast formation of aerobic granular sludge

This study investigated the effects of enhanced biological phosphorus removal (EBPR) on rapid sludge bulking control and fast aerobic granular sludge (AGS) formation by adding 20 % of EBPR activated sludge to the bulking activated sludge (BAS) reactor. The results indicate that activating EBPR activity swiftly improved BAS settleability within 16 days, thus resolving sludge bulking issues. Subsequently, a settling time-based selection was employed, resulting in the BAS granulation within another 16 days. The rapid achievement of EBPR activity improved the BAS settleability and facilitated the formation of sludge aggregates, thereby expediting BAS granulation. Inhibition of filamentous bacteria and enrichment of slow-growing organisms contributed to both sludge bulking control and aerobic granulation. Furthermore, the increase in proteins/polysaccharides ratio facilitated the granulation process. Additionally, total nitrogen removal increased from 59.4 % to 71.7 % because of the mature AGS formation. This study provided an approach to simultaneously control sludge bulking and promote aerobic granulation.

Effects of salinity on glycerol conversion and biological phosphorus removal by aerobic granular sludge

Industrial wastewater often has high levels of salt, either due to seawater or e.g. sodium chloride (NaCl) usage in the processing. Previous work indicated that aerobic granular sludge (AGS) is differently affected by seawater or saline water at similar osmotic strength. Here we investigate in more detail the impact of NaCl concentrations and seawater on the granulation and conversion processes for AGS wastewater treatment. Glycerol was used as the carbon source since it is regularly present in industrial wastewaters, and to allow the evaluation of microbial interactions that better reflect real conditions. Long-term experiments were performed to evaluate and compare the effect of salinity on granulation, anaerobic conversions, phosphate removal, and the microbial community. Smooth and stable granules as well as enhanced biological phosphorus removal (EBPR) were achieved up to 20 g/L NaCl or when using seawater. However, at NaCl levels comparable to seawater strength (30 g/L) incomplete anaerobic glycerol uptake and aerobic phosphate uptake were observed, the effluent turbidity increased, and filamentous granules began to appear. The latter is likely due to the direct aerobic growth on the leftover substrate after the anaerobic feeding period. In all reactor conditions, except the reactor with 30 g/L NaCl, Ca. Accumulibacter was the dominant microorganism. In the reactor with 30 g/L NaCl, the relative abundance of Ca. Accumulibacter decreased to ≤1 % and an increase in the genus Zoogloea was observed. Throughout all reactor conditions, Tessaracoccus and Micropruina, both actinobacteria, were present which were likely responsible for the anaerobic conversion of glycerol into volatile fatty acids. None of the glycerol metabolizing proteins were detected in Ca. Accumulibacter which supports previous findings that glycerol can not be directly utilized by Ca. Accumulibacter. The proteome profile of the dominant taxa was analysed and the results are further discussed. The exposure of salt-adapted biomass to hypo-osmotic conditions led to significant trehalose and PO43−-P release which can be related to the osmoregulation of the cells. Overall, this study provides insights into the effect of salt on the operation and stability of the EBPR and AGS processes. The findings suggest that maintaining a balanced cation ratio is likely to be more important for the operational stability of EBPR and AGS systems than absolute salt concentrations.

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.

Effects of microorganisms on nitrogen and phosphorus removal from wastewater

Nitrogen and phosphorus are key to plant growth. The balance of nitrogen and phosphorus is crucial in nature. Excessive nitrogen and phosphorus content in wastewater has caused great harm to the environment. Traditional nitrogen and phosphorus removal methods can no longer meet the treatment standards for nitrogen and phosphorus. This article introduces the hazards of nitrogen and phosphorus, and points out the harm to nature and human beings if they are not up to standard in wastewater treatment plants. Through the analysis and discussion of several methods, the advantages of microbial nitrogen and phosphorus removal are explained. This article further summarizes several representative new microorganisms and microbial processes through the types and mechanisms of microbial nitrogen and phosphorus removal. Anammox bacteria can accept other electron pools to increase nitrogen removal efficiency. Denitrifying phosphorus-removing bacteria can achieve energy saving and carbon reducing effects through endogenous denitrification. Besides, the enhanced biological phosphorus removal process can allow denitrifying phosphorus accumulating bacteria to absorb phosphorus in an anaerobic, anoxic, aerobic alternating environment. The moving bed biofilm reactor (MBBR) abandons the disadvantages of the traditional biofilm method, while retaining the advantages of the traditional biofilm method such as shock load resistance and low sludge production. These new microorganisms and new microbial processes have become important applications in future nitrogen and phosphorus removal processes due to their advantages of high efficiency, low energy, and no secondary pollution. This article is conducive to the promotion of microbial-based wastewater treatment and can provide suggestions for the sustainable development of wastewater treatment.

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

Abstract Candidatus Accumulibacter, a key genus of polyphosphate-accumulating organisms, plays key roles in lab- and full-scale enhanced biological phosphorus removal (EBPR) systems. Ten 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.

Effect of cement-based composite pellets on phosphorus removal and microbial community structure in eutrophic water

Phosphorus (P) removal still faces challenges in eutrophic water treatment. Combining adsorption and biological reactions can efficiently reduce the P concentrations, achieving long-term function. In this study, five kinds of pellets were produced using cement, bentonite, and organic carbon (rice husk powder) with mass ratios of 100%:0%:0% (M1), 95%:5%:0% (M2), 92.5%:5%:2.5% (M3), 90%:5%:5% (M4), and 85%:5%:10% (M5) for phosphorus removal in eutrophic water. The static adsorption experiment revealed that both Langmuir and Freundlich models were suitable for describing the adsorption characteristics. The P-bonding energy (KL) and adsorption capacity (K) followed the order of M1 < M2 < M3 < M4 < M5. The adsorption processes conformed to the Pseudo-second-order kinetic model. The adsorption capability of pellets decreased with increasing pH levels. The Cl−, NO3−, and SO42− showed negligible effects on adsorption, while CO32− reduced the adsorption amount. In the dynamic experiment, all the pellets have sustained P removal efficiency with the average removal rates of 58.54% (M1), 60.78% (M2), 63.97% (M3), 66.81% (M4), and 72.05% (M5). The presence of CaHPO4·2 H2O in pellets after P removal indicated phosphate precipitation and ion exchange. The attached microbes in pellets exhibited an increase in Proteobacteria abundance contributed to biological phosphorus removal and long-term effectiveness. Hence, this study introduces innovative cement-based pellets for phosphorus removal in eutrophic water, establishing a possibility for practical applications.

Influence of high acetate concentrations on enhanced biological phosphorus removal

Influence of high acetate concentrations on enhanced biological phosphorus removal

Converting rectangular and circular primary tanks into the AAA biologically enhanced clarification settler.

The frequent design challenge for existing water resource recovery facilities targets the accommodation of an ~50% load increase within the existing infrastructure and footprint. Off-loading this organic load at the top-end of the plant and redirection toward the digesters has proven the most efficient way of process intensification. The Triple A settler is an "activated primary treatment," stands for alternating activated adsorption, and can be retrofitted into existing rectangular or circular (mostly) primary tanks at a hydraulic retention time of 2 h and a sludge retention time of about 0.5days. Several technology implementations demonstrate flexible designs adjusting to existing tank geometries and depths of 2.5 to 5.0m. Different implementation scales from dry-weather flow rates ranging from 0.1 to 10mgd show generic applicability of the functional principles at any scale: Biosorption, bioflocculation, and assimilation provide the key added value in pretreatment efficiencies of ~60/25/33 in %COD/%N/%P removal compared with application of pure physics in primary settling with typical 33%/9%/11% removal, respectively. PRACTITIONERS POINTS: Triple A is a hybrid form of A-stage and contact stabilizer for advanced primary treatment. Besides COD and TSS, also, P and N can be removed via Triple A. Triple A can be retrofitted in existing rectangular or circular tanks. This high-rate process does not worsen the conditions for enhanced biological phosphorus removal. Energy efficiency, capacity increase, and operational benefits are the main goals of Triple A.

Enhancing wastewater treatment efficiency: A hybrid technology perspective with energy-saving strategies

The study aimed to investigate how hybrid technology, combined with various intermittent aeration (IA) strategies, contributes to reducing the energy costs of wastewater treatment while simultaneously ensuring a high treatment efficiency. Even with IA subphases lasting half as long as those without aeration, and oxygen levels reduced from 3.5 to 1.5 mg O2/L, pollutants removal efficiency remains robust, allowing for a 1.41-fold reduction in energy consumption (EO). Hybrid technology led to a 1.34–fold decrease in EO, along with improved denitrification efficiency from 74.05 ± 4.71 to 81.87 ± 2.43 % and enhanced biological phosphorus removal from 35.03 ± 4.25 to 87.32 ± 3.64 %. The high nitrification efficiency may have been attributed to the abundance of Pseudomonas, Acinetobacter, and Rhodococcus, which outcompeted the genera of autotrophic nitrifying bacteria, suggesting that the hybrid system is favorable for the growth of heterotrophic nitrifiers.

Enhanced the simultaneous removal and recovery of phosphorus in induced crystallization coupled biological phosphorus removal process

Remove and recovery of phosphorus from wastewater is expected to be a promising strategy to solve the shortage of phosphorus resources and control the water environment pollution. In this study, induced crystallization coupled biological phosphorus removal (IC-BPR) process was established to achieve simultaneous removal and recovery of phosphorus in wastewater. The effects of side stream ratio (SSR) on the removal and recovery efficiency of phosphorus and the microbial community structure were explored. The results indicated that the SSR of 40% was optimal to phosphorus removal and recovery in the IC-BPR. The total phosphorus removal efficiency was 94.23% under the SSR of 40%, of which the phosphorus removal efficiency by IC accounted for 74.87%. And the total phosphorus recovery efficiency was 67.29%. The particle size and crystal structure analysis of the crystallized products indicated that the increase of SSR was conductive to heterogeneous crystallization. Furthermore, typical polyphosphate accumulation organisms (PAOs), including Rhodocyclaceae, Acinetobacter, and Dechloromonasin, were enriched under the side stream ratio of 40%. And functional genes related to amino acid transport and metabolism, energy production and conversion were activated. The above results indicated that the IC could significantly enhance the removal and recovery of phosphorus under a suitable SSR condition. It will provide important reference for wastewater treatment coupled phosphorus resource recovery technology.

The impact of reduced sulfur compounds on aerobic granular sludge formation and biological phosphorus removal

Biological sulfur conversion combined with phosphorus removal represents an innovative biotechnology for activated sludge (AS). However, its effect on the formation of aerobic granular sludge (AGS) remains unclear. This study aimed at determining the impact of sulfide and thiosulfate on the performance of enhanced biological phosphorus removal (EBPR) and on AGS formation. Using two lab-scale sequencing batch reactors (SBR), we compared EBPR performance, the degree of granulation, and the enriched microbial community with sulfide (SBR-S, 20 mg S/L) or thiosulfate (SBR-T, 140 mg S/L) as primary sulfur source. Results indicate that both reactors achieved comparable EBPR performance, with a phosphorus removal rate of 98% in SBR-T and 99% in SBR-S. SBR-T resulted in compact granules with fast settling properties, while SBR-S resulted in dense floccular sludge, referred to as “densified sludge”. Both systems enriched a comparable microbial community with shared dominant genera, including Ca. Competibacter, Thiothrix, Ca. Accumulibacter and Dechloromonas. The higher abundance of Thiothrix unzii throughout the operating time in SBR-T, reaching 19.4%, is believed to have enhanced the granulation process. This study provides important insight into the role of reduced sulfur compounds in sludge densification and EBPR performance, relevant to sulfur-rich wastewaters such as those from industrial sources.

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.

Enhanced biological phosphorus removal sustained by aeration-free filamentous microalgal-bacterial granular sludge

The microalgal-bacterial granular sludge (MBGS) based enhanced biological phosphorus removal (EBPR) (MBGS-EBPR) was recently proposed as a sustainable wastewater treatment process. Previous work showed the possibility of obtaining an MBGS-EBPR process starting from mature MBGS and phosphate-accumulating organisms (PAOs) enriched aerobic granular sludge (AGS) and validated the effectiveness of removing carbon/nitrogen/phosphorus with mechanical aeration. The present work evaluated whether the same could be achieved starting from conventional activated sludge and operating under aeration-free conditions in an alternating dark/light photo-sequencing batch reactor (PSBR). We successfully cultivated filamentous MBGS with a high settling rate (34.5 m/h) and fast solid-liquid separation performance, which could be attributed to the proliferation of filamentous cyanobacteria and stimulation of extracellular polymeric substances (EPS) production. The process achieved near-complete steady-state removal of carbon (97.2 ± 1.9 %), nitrogen (93.9 ± 0.7 %), and phosphorus (97.7 ± 1.7 %). Moreover, improved phosphorus release/uptake driven by photosynthetic oxygenation under dark/light cycles suggests the enrichment of PAOs and the establishment of MBGS-EBPR. Batch tests showed similar phosphorus release rates in the dark but significantly lower phosphorus uptake rates in the presence of light when the filamentous granules were disrupted. This indicates that the filamentous structure of MBGS has minor limitations on substrate mass transfer while exerting protective effects on PAOs, thus playing an important role in sustaining the function of aeration-free EBPR. Microbial assays further indicated that the enrichment of filamentous cyanobacteria (Synechocystis, Leptoolybya, and Nodosilinea), putative PAOs and EPS producers (Hydrogenophaga, Thauera, Flavobacterium, and Bdellovibrio) promoted the development of filamentous MBGS and enabled the high-efficient pollutant removal. This work provides a feasible and cost-effective strategy for the startup and operation of this innovative process.

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.

Advanced nitrogen and phosphorus removal from groundwater by a composite functional particle-ceramic membrane bioreactor

Nutrients are the main environment pollution issue in groundwater, while the efficient and sustainable membrane technology is the key to solving this problem. In this work, a composite functional particle-ceramic membrane bioreactor (CFP-CMR) was developed for advanced nitrogen and phosphorus removal from groundwater. Under the condition of moderate flux (5–15 L·m−2·h−1), CFP-CMR is able to sustain the preferable water purification performance (effluent: TP < 0.1 mg/L, NH3-N < 0.5 mg/L) for long-term stable operation (>100 days) via the synergy of membrane, adsorption, and microorganism. Composite functional particle could enrich and provide the carbon source for microorganism, as well as combine with the P in the water to form the crystals. The adsorption effect of composite functional particles in the early stage and the biological phosphorus removal in the later stage could synergistically improve the TP removal. Similarly, the CFP-CMR could ensure that the NH3-N in effluent reached the Chinese drinking water standard through the synergy of adsorption and microbial. However, the excessive gel layer with high C/N (i.e. Rhodobacter) on the ceramic membrane surface brought about the dissimilation reduction of nitrate to ammonium and affected the ammonia nitrogen removal. Besides, as the main membrane fouling, the biological gel layer could be effectively removed by hydraulic backwashing, so as to reduce the TMP and restore the permeability. Whereas, the re-startup after membrane cleaning clearly reduced the microbial and organics in the system, then weakened the nitrogen and phosphorus removal efficiency. In general, the findings of this work verify the reliability of CFP-CMR for the effective and sustainable operation of groundwater purification. The CFP-CMR combines the advantages of adsorption and biological processes, and is expected to become an effective technology for nitrogen and phosphorus removal.

Comammox and unknown ammonia oxidizers contribute to nitrite accumulation in an integrated A-B stage process that incorporates side-stream EBPR (S2EBPR)

A novel integrated pilot-scale A-stage high rate activated sludge, B-stage short-cut biological nitrogen removal and side-stream enhanced biological phosphorus removal (A/B-shortcut N-S2EBPR) process for treating municipal wastewater was demonstrated with the aim to achieve simultaneous and carbon- and energy-efficient N and P removal. In this studied period, an average of 7.62 ± 2.17 mg-N/L nitrite accumulation was achieved through atypical partial nitrification without canonical known NOB out-selection. Network analysis confirms the central hub of microbial community as Nitrospira, which was one to two orders of magnitude higher than canonical aerobic oxidizing bacteria (AOB) in a B-stage nitrification tank. The contribution of comammox Nitrospira as AOB was evidenced by the increased amoB/nxr ratio and higher ammonia oxidation activity. Furthermore, oligotyping analysis of Nitrospira revealed two dominant sub-clusters (microdiveristy) within the Nitrospira. The relative abundance of oligotype II, which is phylogenetically close to Nitrospira_midas_s_31566, exhibited a positive correlation with nitrite accumulation in the same operational period, suggesting its role as comammox Nitrospira. Additionally, the phylogenetic investigation suggested that heterotrophic organisms from the family Comamonadacea and the order Rhodocyclaceae embedding ammonia monooxygenase and hydroxylamine oxidase may function as heterotrophic nitrifiers. This is the first study that elucidated the impact of integrating the S2EBPR on nitrifying populations with implications on short-cut N removal. The unique conditions in the side-stream reactor, such as low ORP, favorable VFA concentrations and composition, seemed to exert different selective forces on nitrifying populations from those in conventional biological nutrient removal processes. The results provide new insights for integrating EBPR with short-cut N removal process for mainstream wastewater treatment.

Side-Stream Enhanced Biological Phosphorus Removal (S2EBPR) enables effective phosphorus removal in a pilot-scale A-B stage shortcut nitrogen removal system for mainstream municipal wastewater treatment

While the adsorption/bio-oxidation (A/B) process has been widely studied for carbon capture and shortcut nitrogen (N) removal, its integration with enhanced biological phosphorus (P) removal (EBPR) has been considered challenging and thus unexplored. Here, full-scale pilot testing with an integrated system combining A-stage high-rate activated sludge with B-stage partial (de)nitrification/anammox and side-stream EBPR (HRAS-P(D)N/A-S2EBPR) was conducted treating real municipal wastewater. The results demonstrated that, despite the relatively low influent carbon load, the B-stage P(D)N-S2EBPR system could achieve effective P removal performance, with the carbon supplement and redirection of the A-stage sludge fermentate to the S2EBPR. The novel process configuration design enabled a system shift in carbon flux and distribution for efficient EBPR, and provided unique selective factors for ecological niche partitioning among different key functionally relevant microorganisms including polyphosphate accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs). The combined nitrite from B-stage to S2EBPR and aerobic-anoxic conditions in our HRAS-P(D)N/A-S2EBPR system promoted DPAOs for simultaneous internal carbon-driven denitrification via nitrite and P removal. 16S rRNA gene-based oligotyping analysis revealed high phylogenetic microdiversity within the Accumulibacter population and discovered coexistence of certain oligotypes of Accumulibacter and Competibacter correlated with efficient P removal. Single-cell Raman micro-spectroscopy-based phenotypic profiling showed high phenotypic microdiversity in the active PAO community and the involvement of unidentified PAOs and internal carbon-accumulating organisms that potentially played an important role in system performance. This is the first pilot study to demonstrate that the P(D)N-S2EBPR system could achieve shortcut N removal and influent carbon-independent EBPR simultaneously, and the results provided insights into the effects of incorporating S2EBPR into A/B process on metabolic activities, microbial ecology, and resulted system performance.

Revisiting the role of Acinetobacter spp. in side-stream enhanced biological phosphorus removal (S2EBPR) systems

We piloted the incorporation of side-stream enhanced biological phosphorus removal (S2EBPR) with A/B stage short-cut nitrogen removal processes to enable simultaneous carbon-energy-efficient nutrients removal. This unique configuration and system conditions exerted selective force on microbial populations distinct from those in conventional EBPR. Interestingly, effective P removal was achieved with the predominance of Acinetobacter (21.5 ± 0.1 %) with nearly negligible level of known conical PAOs (Ca. Accumulibacter and Tetrasphaera were 0.04 ± 0.10 % and 0.47 ± 0.32 %, respectively). Using a combination of techniques, such as fluorescence in situ hybridization (FISH) coupled with single cell Raman spectroscopy (SCRS), the metabolic tracing of Acinetobacter-like cells exerted PAO-like phenotypic profiling. In addition, comparative metagenomics analysis of the closely related Acinetobacter spp. revealed the EBPR relevant metabolic pathways. Further oligotyping analysis of 16s rRNA V4 region revealed sub-clusters (microdiversity) of the Acinetobacter and revealed that the sub-group (oligo type 1, identical (100 % alignment identity) hits from Acinetobacter_midas_s_49494, and Acinetobacter_midas_s_55652) correlated with EBPR activities parameters, provided strong evidence that the identified Acinetobacter most likely contributed to the overall P removal in our A/B-shortcut N-S2EBPR system. To the best of our knowledge, this is the first study to confirm the in situ EBPR activity of Acinetobacter using combined genomics and SCRS Raman techniques. Further research is needed to identify the specific taxon, and phenotype of the Acinetobacter that are responsible for the P-removal.