Enhanced Biological Phosphorus Removal Process Research Articles

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.

Integrated genomics provides insights into the evolution of the polyphosphate accumulation trait of Ca. Accumulibacter

Candidatus Accumulibacter, a prominent polyphosphate-accumulating organism (PAO) in wastewater treatment, plays a crucial role in enhanced biological phosphorus removal (EBPR). The genetic underpinnings of its polyphosphate accumulation capabilities, however, remain largely unknown. Here, we conducted a comprehensive genomic analysis of Ca. Accumulibacter-PAOs and their relatives within the Rhodocyclaceae family, identifying 124 core genes acquired via horizontal gene transfer (HGT) at its least common ancestor. Metatranscriptomic analysis of an enrichment culture of Ca. Accumulibacter revealed active transcription of 44 of these genes during an EBPR cycle, notably including the polyphosphate kinase 2 (PPK2) gene instead of the commonly recognized polyphosphate kinase 1 (PPK1) gene. Intriguingly, the phosphate regulon (Pho) genes showed minimal transcriptions, pointing to a distinctive fact of Pho dysregulation, where PhoU, the phosphate signaling complex protein, was not regulating the high-affinity phosphate transport (Pst) system, resulting in continuous phosphate uptake. To prevent phosphate toxicity, Ca. Accumulibacter utilized the laterally acquired PPK2 to condense phosphate into polyphosphate, resulting in the polyphosphate-accumulating feature. This study provides novel insights into the evolutionary emergence of the polyphosphate-accumulating trait in Ca. Accumulibacter, offering potential advancements in understanding the PAO phenotype in the EBPR process.

Discovery of a novel potential polyphosphate accumulating organism without denitrifying phosphorus uptake function in an enhanced biological phosphorus removal process

Enhanced biological phosphorus removal (EBPR) is an effective process for phosphorus removal from wastewater. In this study, two lab-scale sequencing batch reactors (SBR) were used to perform EBPR process, in which genus Propioniciclava was unexpectedly accumulated and its relative abundance was over 70 %. A series of tests were conducted to explore the role of Propioniciclava in the two EBPR systems. The two systems performed steadily throughout the study, and the phosphorus removal efficiencies were 96.6 % and 93.5 % for SBR1 and SBR2, respectively. The stoichiometric analysis related to polyphosphate accumulating organisms (PAOs) indicated that polyphosphate accumulating metabolism (PAM) was achieved in the anaerobic phase. It appeared that the Propioniciclava-dominated systems could not perform denitrifying phosphorus removal. Instead, phosphorus was released under anoxic conditions without carbon sources. According to the genomic information from Integrated Microbial Genomes (IMG) database, Propioniciclava owns ppk1, ppk2 and ppx genes that are associated with phosphorus release and uptake functions. By phylogenetic investigation of communities by reconstruction of unobserved states 2 (PICRUSt2) analysis, the abundance of genes related to phosphorus metabolism was much higher than that of genes related to denitrification. Therefore, Propioniciclava was presumed to be a potential PAO without denitrifying phosphorus uptake function. In addition to Propioniciclava, Tessaracoccus and Thiothrix were also enriched in both systems. Overall, this study proposes a novel potential PAO and broadens the understanding of EBPR microbial communities.

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

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

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

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

Interrogating the performance and microbial ecology of an enhanced biological phosphorus removal/post-anoxic denitrification process at bench and pilot scales.

Research focused on interrogating post-anoxic enhanced biological phosphorus removal (EBPR) at bench and pilot scales. Average bench-scale effluent ranged from 0.33 to 1.4 mgP/L, 0.35 to 3.7 mgNH3 -N/L, and 1.1 to 3.9 mgNOx -N/L. Comparatively, the pilot achieved effluent (50th percentile/average) of 0.13/0.2 mgP/L, 9.7/8.2 mgNH3 -N/L, and 0.38/3.3 mgNOx -N/L under dynamic influent and environmental conditions. For EBPR process monitoring, P:C ratio data indicated that 0.2-0.4 molP/molC will result in stable EBPR; relatedly, a target design influent volatile fatty acid (VFA):P ratio would exceed 15 mgCOD/mgP. Post-anoxic EBPR was enriched for Nitrobacter spp. at 1.70%-20.27%, with Parcubacteria also dominating; the former is putatively associated with nitritation and the latter is a putative fermenting heterotrophic organism. Post-anoxic specific denitrification rates (SDNRs) (20°C) ranged from 0.70 to 3.10 mgN/gVSS/h; there was a strong correlation (R2 = 0.94) between the SDNR and %Parcubacteria for systems operated at a 20-day solids residence time (SRT). These results suggest that carbon substrate potentially generated by this putative fermenter may enhance post-anoxic EBPR. PRACTITIONER POINTS: Post-anoxic EBPR can achieve effluent of <0.2 mgP/L and <12 mgN/L. The P:C and VFA:P ratios can be predictive for EBPR process monitoring. Post-anoxic EBPR was enriched for Nitrobacter spp. over Nitrospira spp. and also for Parcubacteria, which is a putative fermenting heterotrophic organism. Post-anoxic specific denitrification rates (20°C) ranged from 0.70 to 3.10 mgN/gVSS/h. BLASTn analysis of 16S rDNA PAO primer set was shown to be improved to 93.8% for Ca. Accumulibacter phosphatis and 73.2%-94.0% for all potential PAOs.

CRISPR-Cas phage defense systems and prophages in Candidatus Accumulibacter

Candidatus Accumulibacter plays a major role in enhanced biological phosphorus removal (EBPR) from wastewater. Although bacteriophages have been shown to represent fatal threats to Ca. Accumulibacter organisms and thus interfere with the stability of the EBPR process, little is known about the ability of different Ca. Accumulibacter strains to resist phage infections. We conducted a systematic analysis of the occurrence and characteristics of clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) systems and prophages in Ca. Accumulibacter lineage members (43 in total, including 10 newly recovered genomes). Results indicate that 28 Ca. Accumulibacter genomes encode CRISPR-Cas systems. They were likely acquired via horizontal gene transfer, conveying a distinct adaptivity to phage predation to different Ca. Accumulibacter members. Major differences in the number of spacers show the unique phage resistance of these members. A comparison of the spacers in closely related Ca. Accumulibacter members from distinct geographical locations indicates that habitat isolation may have resulted in the acquisition of resistance to different phages by different Ca. Accumulibacter. Long-term operation of three laboratory-scale EBPR bioreactors revealed high relative abundances of Ca. Accumulibacter with CRISPSR-Cas systems. Their specific resistance to phages in these reactors was indicated by spacer analysis. Metatranscriptomic analyses showed the activation of the CRISPR-Cas system under both anaerobic and aerobic conditions. Additionally, 133 prophage regions were identified in 43 Ca. Accumulibacter genomes. Twenty-seven of them (in 19 genomes) were potentially active. Major differences in the occurrence of CRISPR-Cas systems and prophages in Ca. Accumulibacter will lead to distinct responses to phage predation. This study represents the first systematic analysis of CRISPR-Cas systems and prophages in the Ca. Accumulibacter lineage, providing new perspectives on the potential impacts of phages on Ca. Accumulibacter and EBPR systems.

Glycerol conversion by aerobic granular sludge

Glycerol is abundantly present in wastewater from industries such as biodiesel production facilities. Glycerol is also a potential carbon source for microbes that are involved in wastewater nutrient removal processes. The conversion of glycerol in biological phosphorus removal of aerobic granular sludge processes has not been explored to date. The current study describes glycerol utilization by aerobic granular sludge and enhanced biological phosphorus removal (EBPR). Robust granules with good phosphorus removal capabilities were formed in an aerobic granular sludge sequencing batch reactor fed with glycerol. The interaction between the fermentative conversion of glycerol and product uptake by polyphosphate accumulating organisms (PAO) was studied using stoichiometric and microbial community analysis. Metagenomic, metaproteomic and microscopic analysis identified a community dominated by Actinobacteria (Tessaracoccus and Micropruina) and a typical PAO known as Ca. Accumulibacter. Glycerol uptake facilitator (glpF) and glycerol kinase (glpK), two proteins involved in the transport of glycerol into the cellular metabolism, were only observed in the genome of the Actinobacteria. The anaerobic conversion appeared to be a combination of a substrate fermentation and product uptake-type reaction. Initially, glycerol fermentation led mainly to the production of 1,3-propanediol (1,3-PDO) which was not taken up under anaerobic conditions. Despite the aerobic conversion of 1,3-PDO stable granulation was observed. Over time, 1,3-PDO production decreased and complete anaerobic COD uptake was observed. The results demonstrate that glycerol-containing wastewater can effectively be treated by the aerobic granular sludge process and that fermentative and polyphosphate accumulating organisms can form a food chain in glycerol-based EBPR processes.

Oligotyping and metagenomics reveal distinct Candidatus Accumulibacter communities in side-stream versus conventional full-scale enhanced biological phosphorus removal (EBPR) systems

Candidatus Accumulibacter phosphatis (CAP) and its clade-level micro-diversity has been associated with and implicated in functional differences in phosphorus removal performance in enhanced biological phosphorus removal (EBPR) systems. Side-stream EBPR (S2EBPR) is an emerging process that has been shown to present a suite of advantages over the conventional EBPR design, however, large knowledge gaps remain in terms of its underlying ecological mechanisms. Here, we compared and revealed the higher-resolution differences in microbial ecology of CAP between a full-scale side-stream EBPR configuration and a conventional A2O EBPR process that were operated in parallel and with the same influent feed. Even though the relative abundance of CAP, revealed by 16S rRNA gene amplicon sequencing, was similar in both treatment trains, a clade-level analysis, using combined 16S rRNA-gene based amplicon sequencing and oligotyping analysis and metagenomics analysis, revealed the distinct CAP microdiversity between the S2EBPR and A2O configurations that likely attributed to the improved performance in S2EBPR in comparison to conventional EBPR. Furthermore, genome-resolved metagenomics enabled extraction of three metagenome-assembled genomes (MAGs) belonging to CAP clades IIB (RCAB4-2), IIC (RC14) and II (RC18), from full-scale EBPR sludge for the first time, including a distinct Ca. Accumulibacter clade that is dominant and associated only with the S2EBPR configuration. The results also revealed the temporally increasing predominance of RC14, which belonged to Clade IIC, during the implementation of the S2EBPR configuration. Finally, we also show the existence of previously uncharacterized diversity of clades of CAP, namely the clades IIB and as yet unidentified clade of type II, in full-scale EBPR communities, highlighting the unknown diversity of CAP communities in full-scale EBPR systems.

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.

Evaluation of PAO adaptability to oxygen concentration change: Development of stable EBPR under stepwise low-aeration adaptation

Recent research has shown the adaptability of Biological Nutrient Removal (BNR) systems to very low level dissolved oxygen (DO) concentration, mainly focusing in the nitrification ability that maintains the nitrogen oxidation process even at very low DO levels. Although step-wise aeration decrease on Enhanced Biological Phosphorus Removal (EBPR) is not fully comprehended. This study investigated the effect of reaching micro-aeration with adaptation strategies on EBPR performance. A step-wise oxygen concentration decrease, arriving at an average aeration level of 0.4 mgO2/L was evaluated, with an outcome of approximately 90 % phosphorus removal efficiency. Compared with different aeration modes, the highest phosphorus (P)-removal efficiency, P-release and lowest effluent phosphorus was achieved in gradual DO decrease strategy. On the other hand, an instant decrease in aeration from stable EBPR process from 2 mgO2/L to 0.4 mgO2/L adversely impacted P-removal by decreasing the efficiency to average 60 % and deteriorating the phosphorus removing microbial consortium. Comparison of results between instant and gradual DO-decrease, indicated the sensitivity of microorganisms to aeration. Microbial adaptation to decreased oxygen availability is crucial to reach high process performance. This study proposes, a potential aeration mode, which contributes in reduction of energy consumption in BNR systems through wastewater treatment.

Tropical-based EBPR process: The long-term stability, microbial community and its response towards temperature stress.

Temperature is known to influence the operational efficiency of enhanced biological phosphorus removal (EBPR) systems. This study investigated the impact of thermal stress above 30°C on the properties of an EBPR community established with tropical inoculum. The results confirmed the stability of the 30°C EBPR system with high P-removal efficiency over 210 days. Accumulibacter was abundant in the community. When the EBPR sludge was subjected to a sudden temperature increase to 35°C under multiple cycles of anaerobic-aerobic phases, each lasting 4h, high P-removal was maintained over 2days, before gradually failing when the Competibacter appeared to outcompete Accumulibacter. These data suggested that the EBPR capacity is robust when subjected to occasional thermal stress. However, it could not be maintained even for a short time under temperature stress at 40°C. Thus, the threshold temperature for tropical EBPR failure is between 35°C and 40°C. PRACTITIONER POINTS: EBPR was stably maintained at 30°C with Accumulibacter being dominant. Good EBPR activities persisted for a short period at 35°C. EBPR was deteriorated at 40°C. The threshold temperature for tropical EBPR failure is between 35°C and 40°C.

Nutrient removal and phosphorus recovery performance of an anaerobic side-stream extraction based enhanced biological phosphorus removal subjected to low dissolved oxygen

An enhanced biological phosphorus removal (EBPR) system based on anaerobic side-stream phosphorus recovery was operated to investigate the nutrient removal performance of the mainstream system under low dissolved oxygen (DO = 1.0, 0.6, 0.2 mg L−1) and the corresponding phosphorus recovery efficiency with different extracting ratio (m = 0, 1/4, 1/3, 1/2) of anaerobic supernatant within 310 days. The results showed that the nutrient removal efficiency remained stable even though DO was extremely low. Nevertheless, phosphorus removal performance was found to deteriorate as side-stream ratio increased to 1/3 at DO = 0.2 mg L−1, suggesting that extracting a higher ratio of supernatant was not favorable for phosphorus removal in the mainstream process at ultra-low DO. The stoichiometric ratios of Prelease/HAcuptake during anaerobic phase which decreased from 0.125 to 0.020 P-mol/C-mol as DO decreased with increasing side-stream ratio were lower than that of typical PAO metabolism. It was very likely that the metabolic mode of PAOs changed due to long-term deprivation of phosphate. Consequently, the mainstream EBPR system failed to remove phosphorus efficiently. It was also observed that phosphorus recovery efficiency was considerable at DO ≥ 0.6 mg L−1 coupled with high side-stream ratio. Therefore, it was feasible and energy-saving to extract appropriate ratio of anaerobic supernatant for recovering phosphorus and removing nutrient efficiently in an EBPR process under low DO.

Accumulibacter diversity at the sub-clade level impacts enhanced biological phosphorus removal performance

Accumulibacter is a well-known group of organisms, typically considered to be polyphosphate accumulating organisms (PAOs), but potentially capable of glycogen accumulating organism (GAO) metabolism under limiting influent phosphate levels. Metabolic features of Accumulibacter are typically linked to its phylogenetic identity at the Type or clade level, though it is unclear the extent to which Accumulibacter diversity can correlate with its capacity to perform P removal. This paper investigates the fine-scale diversity of Accumulibacter and its link with enhanced biological phosphorus removal (EBPR) performance under various operating conditions, to understand the conditions and community structure leading to successful and unsuccessful EBPR operation. For this purpose, the organic carbon feeding rate and total organic carbon concentration were varied during three distinct operational periods, where influent phosphate was never limiting. Accumulibacter was always the dominant microbial group (>80% of all bacteria according to quantitative fluorescence in situ hybridisation - FISH) and low levels of Competibacter and other GAOs were consistently observed (<15% of all bacteria). Steady state was achieved in each of the three periods, with average phosphorus removal levels of 36%, 99% and >99%, respectively. Experimentally determined stoichiometric activity supported the expression of a mixed PAO/GAO metabolism in the first steady state period and the typical PAO metabolism in the other two steady state periods. FISH quantification and amplicon sequencing of the polyphosphate kinase (ppk1) functional gene indicated that Accumulibacter clade IIC was selected in the first steady state period, which shifted to clade IA after decreasing the carbon feeding rate in steady state period 2, and finally shifted back to clade IIC in the third steady state period. Fine-resolution Ppk-based phylogenetic analysis revealed three different clusters within Accumulibacter clade IIC, where clusters IICii and IICiii were linked to poor EBPR performance in period 1, and cluster IICi was linked to good EBPR performance in period 3. This study shows that the deterioration of EBPR processes through GAO activity at non-limiting P concentrations can be linked to organisms that are typically classified as PAOs, not only to known GAOs such as Competibacter. Intra-clade phylogenetic diversity within Accumulibacter showed that some clusters actually behave similarly to GAOs even without influent phosphate limitation. This study highlights the need to closely re-examine traditional interpretations regarding the link between the microbial community composition and identity with the performance and metabolism of EBPR systems.

Metagenomic insights into the effect of sulfate on enhanced biological phosphorus removal.

Excess phosphorus in water supplies causes eutrophication, which degrades water quality. Hence, the efficient removal of phosphorus from wastewater represents a highly desirable process. Here, we evaluated the effect of sulfate concentration on enhanced biological phosphorus removal (EBPR), in which phosphorus is typically removed under anaerobic-oxic cycles, with sulfate reduction the predominant process in the anaerobic phase. Two sequencing batch EBPR reactors operated under high- (SBR-H) vs. low-sulfate (SBR-L) concentrations for 189 days and under three periods, i.e., start-up, sufficient acetate, and limited acetate. Under acetate-rich conditions, phosphorus removal efficiency was > 90% for both reactors; however, under acetate-limited conditions, only 34% and 91.3% of the phosphorus were removed for the SBR-L and the SBR-H, respectively. Metagenomic sequencing of the reactors showed that the relative abundance of the polyphosphate-accumulating and sulfur-reducing bacteria (SRB) was higher in the SBR-H, consistent with its higher phosphorus removal activity. Ten high-quality metagenome-assembled genomes, including one closely related to the genus Thiothrix disciformis (99.81% averageamino acid identity), were recovered and predicted to simultaneously metabolize phosphorus and sulfur by the presence of phosphorus (ppk, ppx, pst, and pit) and sulfur (sul, sox, dsr, sqr, apr, cys, and sat) metabolism marker genes. The omics-based analysis provided a holistic view of the microbial ecosystem in the EBPR process and revealed that SRB and Thiothrix play key roles in the presence of high sulfate.Key points• We observed high phosphorus-removal efficiency in high-sulfate EBPR.• Metagenome-based analysis revealed sulfate-related metabolic mechanisms in EBPR.• SRB and PAOs showed interrelationships in the EBPR-sulfur systems.

Butyrate can support PAOs but not GAOs in tropical climates

Glycogen accumulating organisms (GAOs) are thought to compete with polyphosphate accumulating organisms (PAOs) for the often-limiting carbon sources available in wastewater, deteriorating enhanced biological phosphorus removal (EBPR) performance at high temperatures. Fermentation liquids are often used to provide an additional carbon source supply in EBPR processes, where butyrate is known to be an important volatile fatty acid (VFA) produced in sludge fermentation. Nevertheless, the impact of butyrate on the PAO-GAO competition is not well understood especially at high temperature. The results of this study demonstrate that butyrate, as a supplemental or sole carbon source, could be promising for EBPR in tropical climates. When the carbon source was gradually changed from acetate to butyrate, a substantial PAO population was found under both conditions, despite a substantial shift in the abundance of Candidatus Accumulibacter phosphatis (decreased from 37.4% to 13.9%) to Rhodocyclaceae (increased from 2.0% to 14.5%), where both organisms likely played an important role in P-removal. Thus, a relatively stable P removal performance was realized throughout the whole operation period. Nevertheless, butyrate had a negative impact on GAOs. The biomass concentration and microbial diversity continually decreased in the GAO reactor, and Candidatus Competibacter phosphatis reduced from 27.3% to 6.2%, where the dominant population was replaced by Zoogloea. With the addition of butyrate as carbon source, the total amount of synthesized PHAs reduced in both PAO and GAO cultures and the composition of PHA was greatly changed. The presence of a novel PHA fraction (PHH) may disturb the microbial activity in the aerobic phase, where the GAO culture was more severely affected. Glycogen cycling also seemed to be limited in both reactors. This could reduce the GAO metabolism in both cultures and favor PAOs and P removal. Furthermore, the biomass growth rate of the PAO culture was higher than that of GAO when fed with butyrate, which also provides PAO a competitive advantage. All the above results indicate that butyrate could not be well metabolized by GAOs, but could provide PAOs a competitive advantage. Thus, mixed VFAs (i.e. acetate, propionate and butyrate) are likely to favor PAOs over GAOs in EBPR processes operated in warm climates, where the impact of substantial butyrate fractions represents an advantage towards successful process operation.

Defluviicoccus vanus Glycogen-Accumulating Organisms (DvGAOs) Are Less Competitive Than Polyphosphate-Accumulating Organisms (PAOs) at High Temperature

Glycogen-accumulating organisms (GAOs) have been previously observed to be more competitive in anaerobic volatile fatty acid (VFA) uptake as compared to polyphosphate-accumulating organisms (PAOs) at high temperatures in enhanced biological phosphorus removal (EBPR) systems. However, previous evidence regarding the impact of temperature on GAOs has exclusively focused on Competibacter. Defluviicoccus vanus GAOs (DvGAOs) make up another very important GAO group and have been found in EBPR plants; however, their competitiveness at high temperature for anaerobic VFA uptake was previously unknown. The rate of uptake of VFAs by both DvGAOs reactors enriched with acetate or propionate was much lower than those of Candidatus Accumulibacter phosphatis PAOs operated under similar conditions, showing that PAOs displayed a competitive advantage at high temperature, unlike previous observations with Competibacter. This study provides new insight into our understanding of GAOs in EBPR processes, where the impact of temperature on PAO-GAO competition is highly dependent on the phylogenetic identity of the GAO present in the system. New strategies for promoting EBPR performance at high temperatures are proposed on the basis of the results of this work.

Identification and classification of the Tetrasphaera genus in enhanced biological phosphorus removal process: a review

Tetrasphaera are an attractive putative group of polyphosphate accumulating organisms (PAOs) that may play an important role in the enhanced biological phosphorus removal (EBPR) process. Unlike conventional PAOs that mostly rely on volatile fatty acids (VFAs), Tetrasphaera can assimilate a wide range of carbon substrates including glucose and various amino acids (e.g., glycine, glutamate, and aspartate). In addition, they have the ability to produce VFAs. This functional versatility confers significant performance and economic benefits for its application in EBPR processes. However, before the wastewater industry can harness these benefits, fundamental knowledge of their taxonomy, classification and identification in EBPR and activated sludge systems must be understood. This review provides a summary of the polyphasic approach: use of phenotypic, genotypic, and chemotaxonomic methods for bacterial classification and its application in the classification of Tetrasphaera. A review of molecular tools currently used to identify Tetrasphaera in activated sludge systems is also provided. In addition, this review discusses how challenges with molecular tools limit our understanding and application of Tetrasphaera in EBPR processes.

Assessment of enhanced biological phosphorus removal process inhibition

Wastewater in industrial regions frequently contains substances which are potentially inhibitory to activated sludge treatment processes. Enhanced biological phosphorus removal (EBPR) is vulnerable to process inhibition mostly because of the location at the beginning of the treatment process and complex characteristics of influent. Much knowledge has been developed on the EBPR process; nonetheless, methods used for estimation of the process inhibition vary largely. There is no easily applicable and reliable method available for rapid determination of inhibition of the EBPR process. In this study, a method for evaluation of inhibition on the EBPR process was developed and controlled. In contrast with previous research, a fresh non-acclimated inoculum was used to enhance the possibility to predict the impact for wastewater treatment plant. The efficiency of the process was evaluated based on the values of orthophosphate release and uptake, synthesis and consumption of polyhydroxyalkanoates. 3,5-dichlorophenol (3,5-DCP) was used as a model inhibitory compound. It was found that the EBPR inhibition tests using a non-acclimated sludge need wastewater as the organic carbon source. Furthermore, the length of aerobic period substantially influenced the inhibition test results—achieved maximum inhibitory concentration (IC50) values were 2.4 times higher when aerobic phase was 6 h instead of 1.5 h. Consequently, extended aeration period was suggested to diminish the inhibition effect. Finally, the impact of the 3,5-DCP on different activated sludge treatment process using respective standard methods was tested and compared. It was found that EBPR process was similarly to the nitrification a sensitive activated sludge process.

Design, operation and technology configurations for enhanced biological phosphorus removal (EBPR) process: a review

Phosphorus as a fundamental element for growth and metabolism of living organisms, yet problematic to water quality, is an irreplaceable component. Application of enhanced biological phosphorus removal (EBPR) technology in wastewater treatment plants offers phosphorus removal and recovery in addition to potential eutrophication prevention. This process is dependable on enrichment of phosphorus accumulating organisms in activated sludge to accumulate great amount of poly-phosphate inside their cell interior for enhancement of phosphorus removal. Yet, inadequate removal performance in pilot and full-scale systems, rises the need for optimization in operation and design of applicable configuration. In addition to applying advancement strategies to minimize the growth of undesirable microorganisms through cost effective phosphorus removal along with potential P-recovery and sustainability. Preceding research has certainly advanced the insight on this area of investigation. Notwithstanding, there are still numerous unresolved issues to be undertaken. This comprehensive review paper aims to revisit the current knowledge and fundamental understanding on microbiology and biochemical transformations in EBPR process. In view of application and structure, EBPR design and operation considerations along with process configurations is critically reviewed. This comprehensive review hopes to touch on the critical points of operation to help in understanding the overall EBPR process and to farther provide insights on future work onto EBPR process developments.