Thiothrix caldifontis was the dominant microorganism (with an estimated bio-volume of 65 ± 3%) in a lab-scale enhanced biological phosphorus removal (EBPR) system containing 100 mg of sulphide per litre in the influent. After a gradual exposure to the presence of sulphide, the EBPR system initially dominated by Candidatus Accumulibacter phosphatis Clade I (98 ± 3% bio-volume) (a known polyphosphate accumulating organism, PAO) became enriched with T. caldifontis. Throughout the different operating conditions studied, practically 100% phosphate removal was always achieved. The gradual increase of the sulphide content in the medium (added to the anaerobic stage of the alternating anaerobic-aerobic sequencing batch reactor) and the adjustment of the aerobic hydraulic retention time played a major role in the enrichment of T. caldifontis. T. caldifontis exhibited a mixotrophic metabolism by storing carbon anaerobically as poly-β-hydroxy-alkanoates (PHA) and generating the required energy through the hydrolysis of polyphosphate. PHA was used in the aerobic period as carbon and energy source for growth, polyphosphate, and glycogen formation. Apparently, extra energy was obtained by the initial accumulation of sulphide as an intracellular sulphur, followed by its gradual oxidation to sulphate. The culture enriched with T. caldifontis was able to store approximately 100 mg P/g VSS. This research suggests that T. caldifontis could behave like PAO with a mixotrophic metabolism for phosphorus removal using an intracellular sulphur pool as energy source. These findings can be of major interest for the biological removal of phosphorus from wastewaters with low organic carbon concentrations containing reduced S-compounds like those (pre-)treated in anaerobic systems or from anaerobic sewers.@en

The objective of this study was to investigate the ability of a culture highly enriched with the polyphosphate-accumulating organism, “Candidatus Accumulibacter phosphatis” clade IIC, to adjust their metabolism to different phosphate availabilities. For this purpose the biomass was cultivated in a sequencing batch reactor with acetate and exposed to different phosphate/carbon influent ratios during six experimental phases. Activity tests were conducted to determine the anaerobic kinetic and stoichiometric parameters as well as the composition of the microbial community. Increasing influent phosphate concentrations led to increased poly-phosphate content and decreased glycogen content of the biomass. In response to higher biomass poly-phosphate content, the biomass showed higher specific phosphate release rates. Together with the phosphate release rates, acetate uptake rates also increased up to an optimal poly-phosphate/glycogen ratio of 0.3 P-mol/C-mol. At higher poly-phosphate/glycogen ratios (obtained at influent P/C ratios above 0.051 P-mol/C-mol), the acetate uptake rates started to decrease. The stoichiometry of the anaerobic conversions clearly demonstrated a metabolic shift from a glycogen dominated to a poly-phosphate dominated metabolism as the biomass poly-phosphate content increased. FISH and DGGE analyses confirmed that no significant changes occurred in the microbial community, suggesting that the changes in the biomass activity were due to different metabolic behavior, allowing the organisms to proliferate under conditions with fluctuating phosphate levels.@en

Candidatus Accumulibacter phosphatis is in general presented as the dominant organism responsible for the biological removal of phosphorus in activated sludge wastewater treatment plants. Lab-scale enhanced biological phosphorus removal (EBPR) studies, usually use acetate as carbon source. However, the complexity of the carbon sources present in wastewater could allow other potential poly-phosphate accumulating organism (PAOs), such as putative fermentative PAOs (e.g., Tetrasphaera), to proliferate in coexistence or competition with Ca. Accumulibacter. This research assessed the effects of lactate on microbial selection and process performance of an EBPR lab-scale study. The addition of lactate resulted in the coexistence of Ca. Accumulibacter and Tetrasphaera in a single EBPR reactor. An increase in anaerobic glycogen consumption from 1.17 to 2.96 C-mol/L and anaerobic PHV formation from 0.44 to 0.87 PHV/PHA C-mol/C-mol corresponded to the increase in the influent lactate concentration. The dominant metabolism shifted from a polyphosphate-accumulating metabolism (PAM) to a glycogen accumulating metabolism (GAM) without EBPR activity. However, despite the GAM, traditional glycogen accumulating organisms (GAOs; Candidatus Competibacter phosphatis and Defluvicoccus) were not detected. Instead, the 16s RNA amplicon analysis showed that the genera Tetrasphaera was the dominant organism, while a quantification based on FISH-biovolume indicated that Ca. Accumulibacter remained the dominant organism, indicating certain discrepancies between these microbial analytical methods. Despite the discrepancies between these microbial analytical methods, neither Ca. Accumulibacter nor Tetrasphaera performed biological phosphorus removal by utilizing lactate as carbon source.@en

Phosphorus has been successfully eliminated from wastewater by biological techniques of enhanced biological phosphorus removal (EBPR) process, which relies on a specific microbiota of polyphosphate accumulating organisms (PAOs) that accumulate phosphate as polyphosphates (poly-P). Most methods for quantification of poly-P pools suffer from low accuracy and specificity. More powerful and implementable P-analysis tools are required for poly-P quantification, which will help in improved evaluation of processes in laboratory and full-scale EBPR systems. This study developed two methods to quantify poly-P pools by releasing the poly-P from the cell. During experimental optimization, it was observed that two different methods resulted in the highest phosphate release: acetate addition at a pH of 4.8 and exposure to EDTA solution with a concentration of 1% (w/v). Treatment with EDTA resulted in a higher amount of phosphate release from all sludge samples. This was characterized by P-release of 1.5–2.5 times higher than the control tests. In contrast, treatments with acetate addition at a low pH exhibited that P-release depended upon the types of the sludge samples. The highest P-release amount and rate were found in highly-enriched PAO sludge samples, but with fewer influences on the sludge collected from WWTP, which may be attributed to the lower fraction of PAOs in the sludge. Overall, the proposed approaches to quantify the poly-P concentration can be applied in simple, user-friendly, and cost-effective ways.@en

The concentration of sulphate present in wastewater can vary from 10 to 500 mg SO4 2−/L. During anaerobic conditions, sulphate is reduced to sulphide by sulphate-reducing bacteria (SRB). Sulphide generation is undesired in wastewater treatment plants (WWTPs). Previous research indicated that SRB are inhibited by the presence of electron acceptors (such as O2, NO3 and NO2). However, the contact times and concentrations used in those studies are by far higher than occur in WWTPs. Since sulphide can influence the biological nitrogen and phosphorus removal processes, this research aimed to understand how the different electron acceptors commonly present in biological nutrient removal (BNR) systems can affect the proliferation of SRB. For this purpose, a culture of SRB was enriched in a sequencing batch reactor (approx. 88% of the total bacteria population). Once enriched, the SRB were exposed for 2 h to typical concentrations of electron acceptors like those observed in BNR systems. Their activity was assessed using three different types of electron donors (acetate, propionate and lactate). Oxygen was the most inhibiting electron acceptor regardless the carbon source used. After exposure to oxygen and when feeding acetate, an inactivation time in the sulphate reduction activity was observed for 1.75 h. Once the sulphate reduction activity resumed, only 60% of the original activity was recovered. It is suggested that the proliferation of SRB is most likely to occur in BNR plants with an anaerobic fraction higher than 15% and operating at sludge retention times higher than 20 days (at a temperature of 20 °C). These results can be used to implement strategies to control the growth of sulphate reducers that might compete for organic carbon with phosphate-accumulating organisms.@en

P-limitation in enhanced biological phosphorus removal (EBPR) systems fed with acetate, has generally been considered as a condition leading to enrichment of organisms of the genotype’ Candidatus Competibacter phosphatis’ expressing the glycogen-accumulating organisms (GAO) phenotype. Recent studies have demonstrated in short-term experiments that organisms of the genotype ‘Candidatus Accumulibacter phosphatis’ clade I and II, known to express the polyphosphate-accumulating organisms (PAO) phenotype can switch to the GAO phenotype when poly-P is absent, but are performing the HAc-uptake at lower kinetic rates, where clade I showed the lowest rates. The objective of this study was to verify whether organisms of the genotype ‘Candidatus Accumulibacter phosphatis’ can also be enriched under P-limiting conditions while expressing a GAO phenotype and more specifically to see which specific clade prevails. A sequencing batch reactor was inoculated with activated sludge to enrich an EBPR culture for a cultivation period of 128 days (16 times the solids retention time) under P-limiting conditions. A mixed culture was obtained comprising of 49 % ‘Candidatus Accumulibacter phosphatis’ clade II and 46 % ‘Candidatus Competibacter phosphatis’. The culture performed a full GAO metabolism for anaerobic HAc-uptake, but was still able to switch to a PAO metabolism, taking up excessive amounts of phosphate during the aerobic phase when it became available in the influent. These findings show that P-limitation, often used as strategy for enrichment of ‘Candidatus Competibacter phosphatis’, does not always lead to enrichment of only ‘Candidatus Competibacter phosphatis’. Furthermore, it demonstrates that ‘Candidatus Accumulibacter phosphatis’ are able to proliferate in activated sludge systems for periods of up to 128 days or longer when the influent phosphate concentrations are just enough for assimilation purposes and no poly-P is formed. The ‘Candidatus Accumulibacter phosphatis’ retain the ability to switch to the PAO phenotype, taking up phosphate from the influent as soon as it becomes available.@en

Phosphate accumulating organisms (PAO) are assumed to use nitrate as external electron acceptor, allowing an efficient integration of simultaneous nitrogen and phosphate removal with minimal organic carbon (COD) requirements. However, contradicting findings appear in literature regarding the denitrification capacities of PAO due to the lack of clade specific highly enriched PAO cultures. Whereas some studies suggest that only PAO clade I may be capable of using nitrate as external electron acceptor for anoxic P-uptake, other studies indicate that PAO clade II may be responsible for anoxic P-removal. In the present study, a highly enriched PAO clade IC culture (>99% according to FISH) was cultivated in an SBR operated under Anaerobic/Oxic conditions and subsequently exposed to Anaerobic/Anoxic/Oxic conditions using nitrate as electron acceptor. Before and after acclimatization to the presence of nitrate, the aerobic and anoxic (nitrate and nitrite) activities of the PAO I culture were assessed through the execution of batch tests using either acetate or propionate as electron donor. In the presence of nitrate, significant P-uptake by PAO I was not observed before or after acclimatization. Using nitrite as electron acceptor, limited nitrite removal rates were observed before acclimatization with lower rates in the acetate fed reactor without P-uptake and slightly higher in the propionate fed reactor with a marginal anoxic P-uptake. Only after acclimatization to nitrate, simultaneous P and nitrite removal was observed. This study suggests that PAO clade IC is not capable of using nitrate as external electron acceptor for anoxic P-removal. The elucidation of the metabolic capacities for individual PAO clades helps in better understanding and optimization of the relation between microbial ecology and process performance in enhanced biological phosphate removal processes.@en

Phosphate accumulating organisms (PAO) are assumed to use nitrate as external electron acceptor, allowing an efficient integration of simultaneous nitrogen and phosphate removal with minimal organic carbon (COD) requirements. However, contradicting findings appear in literature regarding the denitrification capacities of PAO due to the lack of clade specific highly enriched PAO cultures. Whereas some studies suggest that only PAO clade I may be capable of using nitrate as external electron acceptor for anoxic P-uptake, other studies indicate that PAO clade II may be responsible for anoxic P-removal. In the present study, a highly enriched PAO clade IC culture (>99% according to FISH) was cultivated in an SBR operated under Anaerobic/Oxic conditions and subsequently exposed to Anaerobic/Anoxic/Oxic conditions using nitrate as electron acceptor. Before and after acclimatization to the presence of nitrate, the aerobic and anoxic (nitrate and nitrite) activities of the PAO I culture were assessed through the execution of batch tests using either acetate or propionate as electron donor. In the presence of nitrate, significant P-uptake by PAO I was not observed before or after acclimatization. Using nitrite as electron acceptor, limited nitrite removal rates were observed before acclimatization with lower rates in the acetate fed reactor without P-uptake and slightly higher in the propionate fed reactor with a marginal anoxic P-uptake. Only after acclimatization to nitrate, simultaneous P and nitrite removal was observed. This study suggests that PAO clade IC is not capable of using nitrate as external electron acceptor for anoxic P-removal. The elucidation of the metabolic capacities for individual PAO clades helps in better understanding and optimization of the relation between microbial ecology and process performance in enhanced biological phosphate removal processes.@en

Sulfate rich wastewaters can be generated from industry, use of seawater in urban environments, or by saline water infiltration into the sewerage. Under anaerobic conditions sulfate can be converted to sulfide, which may affect micro-organisms performing biological nutrient removal. The objective of this study was to evaluate the effect of sulfide on the activity of polyphosphate-accumulating organisms (PAO) in the anaerobic stage of the enhanced biological phosphorus removal process (EBPR). In this regard, a highly enriched culture of PAO was exposed in short-term activity tests to a range of sulfide concentrations at different operational pH values. The PAO activity was mainly affected by un-dissociated H2S. The specific acetate uptake rate was inhibited by 50% at around 60 mg H2S.L−1. With increasing H2S concentrations, higher phosphate release rate to acetate uptake rate ratios were observed, possibly due to increased energy requirements for cell detoxification. Mathematical expressions were developed, which satisfactorily described the sulfide effects on the acetate uptake rate and phosphate release rate. The results show that, dependent on the pH, EBPR might be negatively affected by total sulfide concentrations exceeding 275 mg SO4.L−1 at pH 6.5 or 1200 mg SO4.L−1 at pH 7.8 mg SO4.L−1 or when freshwater is partially replaced by seawater more than 45% (pH 7.8) or 10% (pH 6.5) used as secondary quality water. The findings of this study imply that sulfide, which is commonly found in different type of wastewaters, affects the anaerobic metabolism of PAO and may play an important role in the process performance of treatment plants treating wastewaters with high sulfide content.@en

Enhanced Biological phosphorous removal (EBPR) processes, often operated at low temperatures, are utilised world-wide, but currently little is known regarding enrichment cultures and the characteristics of active organisms (“Candidatus Accumulibacter phosphatis” (Accumulibacter)) under psychrophilic conditions. This study assesses the long-term performance, metabolic activity, microbial community characteristics and sludge morphology in an EBPR community enriched from activated sludge at 10 °C. Long solid retention times (SRT) and low temperatures resulted in the dominance of Accumulibacter type II over type I. Despite changes in the microbial community, P-removal efficiencies did not show obvious differences and although no specific measures were implemented, the enriched Accumulibacter-PAO culture formed stable dense granules. A high level of Alginate-like exopolysaccharides (ALE) were observed, with a large number of Guluronic acid-Guluronic acid (GG) blocks derived from the biomass at 10 °C. This characteristic favors sludge granulation, increasing the mechanical strength of granules formed, which encourages solid-liquid separation and consequently, contributes to the stable operation of EBPR systems.@en

Enhanced Biological phosphorous removal (EBPR) processes, often operated at low temperatures, are utilised world-wide, but currently little is known regarding enrichment cultures and the characteristics of active organisms (“Candidatus Accumulibacter phosphatis” (Accumulibacter)) under psychrophilic conditions. This study assesses the long-term performance, metabolic activity, microbial community characteristics and sludge morphology in an EBPR community enriched from activated sludge at 10 °C. Long solid retention times (SRT) and low temperatures resulted in the dominance of Accumulibacter type II over type I. Despite changes in the microbial community, P-removal efficiencies did not show obvious differences and although no specific measures were implemented, the enriched Accumulibacter-PAO culture formed stable dense granules. A high level of Alginate-like exopolysaccharides (ALE) were observed, with a large number of Guluronic acid-Guluronic acid (GG) blocks derived from the biomass at 10 °C. This characteristic favors sludge granulation, increasing the mechanical strength of granules formed, which encourages solid-liquid separation and consequently, contributes to the stable operation of EBPR systems.@en

The objective of this study was to assess the possibility of retrofitting an existing full-scale wastewater treatment plant (WWTP) based on a sequencing batch reactor (SBR) technology with the enhanced biological phosphorus removal (EBPR) process. Wastewater characterisation showed highly variable influent composition that fluctuated throughout the year with a rather low and unstable SBOD/TP ratio (SBOD—soluble biological oxygen demand; TP—total phosphorus), which is considered unfavourable for EBPR. Characterisation of the sludge showed that the non-EBPR SBR sludge from the WWTP Koprivnica contained no detectable phosphorus accumulating organisms (PAO), but could be transformed in a laboratory into EBPR performing sludge in less than 45 days under favourable conditions for PAOs. The microbial community composition was assessed using an FISH (fluorescence in situ hybridization) analysis, which confirmed that the original sludge from the WWTP, which did not have detectable PAOs, was transformed into the sludge enriched by PAOs belonging to the genus ‘Candidatus Accumulibacter phosphatis’ after 43 days of cultivation. A plant retrofit, based on the results of laboratory experiments, was proposed with the enrichment of the wastewater with volatile fatty acids via primary anaerobic fermentation and step feeding. Results of mathematical modelling (BioWin) showed that such strategy could lead to sufficient P removal through EBPR in this WWTP.@en

The objective of this study was to assess the possibility of retrofitting an existing full-scale wastewater treatment plant (WWTP) based on a sequencing batch reactor (SBR) technology with the enhanced biological phosphorus removal (EBPR) process. Wastewater characterisation showed highly variable influent composition that fluctuated throughout the year with a rather low and unstable SBOD/TP ratio (SBOD—soluble biological oxygen demand; TP—total phosphorus), which is considered unfavourable for EBPR. Characterisation of the sludge showed that the non-EBPR SBR sludge from the WWTP Koprivnica contained no detectable phosphorus accumulating organisms (PAO), but could be transformed in a laboratory into EBPR performing sludge in less than 45 days under favourable conditions for PAOs. The microbial community composition was assessed using an FISH (fluorescence in situ hybridization) analysis, which confirmed that the original sludge from the WWTP, which did not have detectable PAOs, was transformed into the sludge enriched by PAOs belonging to the genus ‘Candidatus Accumulibacter phosphatis’ after 43 days of cultivation. A plant retrofit, based on the results of laboratory experiments, was proposed with the enrichment of the wastewater with volatile fatty acids via primary anaerobic fermentation and step feeding. Results of mathematical modelling (BioWin) showed that such strategy could lead to sufficient P removal through EBPR in this WWTP.@en