Putative metabolism of Ca. Accumulibacter via the utilization of glucose.

Ca. Accumulibacter was the predominant microorganism (relative FISH bio-abundance of 67 ± 5%) in a lab-scale sequential batch reactor that accomplished enhanced biological phosphorus removal (EBPR) while using glucose and acetate as the carbon sources (1:1 COD-based ratio). Both organic compounds were completely anaerobically consumed. The reactor's performance in terms of P/C ratio, phosphorous release and uptake, and overall kinetic and stoichiometric parameters were on the high end of the reported spectrum for EBPR systems (100:9.3 net mg phosphate removal per mg COD consumed when using glucose and acetate in a 1:1 ratio). The batch tests showed that, to the best of our knowledge, this is the first time a reactor enriched with Ca. Accumulibacter can putatively utilize glucose as the sole carbon source to biologically remove phosphate (COD:P (mg/mg) removal ratio of 100:6.3 when using only glucose). Thus, this research proposes that Ca. Accumulibacter directly anaerobically stored the fed glucose primarily as glycogen by utilizing the ATP provided via the hydrolysis of poly-P and secondarily as PHA by balancing its ATP utilization (glycogen generation) and formation (PHA storage). Alternative hypotheses are also discussed. The reported findings could challenge the conventional theories of glucose assimilation by Ca. Accumulibacter, and can be of significance for the biological removal of phosphorus from wastewaters with high contents of fermentable compounds or low VFAs.

Recent advances in bio-P modelling - a new approach verified by full-scale observations.

This paper summarizes recent developments in biological phosphorus removal modelling, with special attention to side-stream enhanced biological phosphorus removal (S2EBPR) systems on which previous models proved to be ineffective without case-by-case parameter adjustments. Through the research and experience of experts and practitioners, a new bio-kinetic model was developed including an additional group of biomass (glycogen accumulating organisms - GAOs) and new processes (such as aerobic and anoxic maintenance for PAO and GAO; enhanced denitrification processes; fermentation by PAOs which - along with PAO selection - is driven by oxidation-reduction potential (ORP)). This model successfully described various conditions in laboratory measurements and full plant data. The calibration data set is provided by Clean Water Services from Rock Creek Facility (Hillsboro, OR) including two parallel trains: conventional A2O and Westbank configurations, allowing the model to be verified on conventional and side-stream EBPR systems as well.

Denitrification of nitrate and nitrite by 'Candidatus Accumulibacter phosphatis' clade IC.

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.

Temperature effects on glycogen accumulating organisms.

Glycogen accumulating organisms (GAO) compete for substrate with polyphosphate-accumulating organisms (PAO), which are the microorganisms responsible for the enhanced biological phosphorus removal (EBPR) in activated sludge wastewater treatment systems. This can lead to the deterioration of the EBPR process. In this paper, the long-term temperature effects on the anaerobic and aerobic stoichiometry and conversion rates on adapted enriched cultures of Competibacter (a known GAO) were evaluated from 10 to 40 degrees C. The anaerobic stoichiometry of Competibacter was constant from 15 to 35 degrees C, whereas the aerobic stoichiometry was insensitive to temperature changes from 10 to 30 degrees C. At 10 degrees C, likely due to the inhibition of the anaerobic conversions of Competibacter, a switch in the dominant bacterial population to an enriched Accumulibacter culture (a known PAO) was observed. At higher temperatures (35 and 40 degrees C), the aerobic processes limited the growth of Competibacter. Due to the inhibition or different steady-state (equilibrium) conditions reached at long-term by the metabolic conversions, the short- and long-term temperature dependencies of the anaerobic acetate uptake rate of Competibacter differed considerably between each other. Temperature coefficients for the various metabolic processes are derived, which can be used in activated sludge modeling. Like for PAO cultures: (i) the GAO metabolism appears oriented at restoring storage pools rather than fast microbial growth, and (ii) the aerobic growth rate of GAO seems to be a result of the difference between PHA consumption and PHA utilization for glycogen synthesis and maintenance. It appears that the proliferation of Competibacter in EBPR systems could be suppressed by adjusting the aerobic solids retention time while, aiming at obtaining highly enriched PAO cultures, EBPR lab-scale reactors could be operated at low temperature (e.g. 10 degrees C).

Temperature effects on the aerobic metabolism of glycogen-accumulating organisms.

Short-term temperature effects on the aerobic metabolism of glycogen-accumulating organisms (GAO) were investigated within a temperature range from 10 to 40 degrees C. Candidatus Competibacter Phosphatis, known GAO, were the dominant microorganisms in the enriched culture comprising 93 +/- 1% of total bacterial population as indicated by fluorescence in situ hybridization (FISH) analysis. Between 10 and 30 degrees C, the aerobic stoichiometry of GAO was insensitive to temperature changes. Around 30 degrees C, the optimal temperature for most of the aerobic kinetic rates was found. At temperatures higher than 30 degrees C, a decrease on the aerobic stoichiometric yields combined with an increase on the aerobic maintenance requirements were observed. An optimal overall temperature for both anaerobic and aerobic metabolisms of GAO appears to be found around 30 degrees C. Furthermore, within a temperature range (10-30 degrees C) that covers the operating temperature range of most of domestic wastewater treatment systems, GAOs aerobic kinetic rates exhibited a medium degree of dependency on temperature (theta = 1.046-1.090) comparable to that of phosphorus accumulating organisms (PAO). We conclude that GAO do not have metabolic advantages over PAO concerning the effects of temperature on their aerobic metabolism, and competitive advantages are due to anaerobic processes.

Factors affecting the microbial populations at full-scale enhanced biological phosphorus removal (EBPR) wastewater treatment plants in The Netherlands.

The influence of operating and environmental conditions on the microbial populations of the enhanced biological phosphorus removal (EBPR) process at seven full-scale municipal activated sludge wastewater treatment plants (WWTPs) in The Netherlands was studied. Data from the selected WWTPs concerning process configuration, operating and environmental conditions were compiled. The EBPR activity from each plant was determined by execution of anaerobic-anoxic-aerobic batch tests using fresh activated sludge. Fractions of Accumulibacter as potential phosphorus accumulating organisms (PAO), and Competibacter, Defluviicoccus-related microorganisms and Sphingomonas as potential glycogen accumulating organisms (GAO) were quantified using fluorescence in situ hybridization (FISH). The relationships among plant process configurations, operating parameters, environmental conditions, EBPR activity and microbial populations fractions were evaluated using a statistical approach. A well-defined and operated denitrification stage and a higher mixed liquor pH value in the anaerobic stage were positively correlated with the occurrence of Accumulibacter. A well-defined denitrification stage also stimulated the development of denitrifying PAO (DPAO). A positive correlation was observed between Competibacter fractions and organic matter concentrations in the influent. Nevertheless, Competibacter did not cause a major effect on the EBPR performance. The observed Competibacter fractions were not in the range that would have led to EBPR deterioration. Likely, the low average sewerage temperature (12+/-2 degrees C) limited their proliferation. Defluviicoccus-related microorganisms were seen only in negligible fractions in a few plants (<0.1% as EUB), whereas Sphingomonas were not observed.

A practical method for quantification of phosphorus- and glycogen-accumulating organism populations in activated sludge systems.

Enhanced biological phosphorus removal (EBPR) from wastewater relies on the enrichment of activated sludge with phosphorus-accumulating organisms (PAOs). The presence and proliferation of glycogen-accumulating organisms (GAOs), which compete for substrate with PAOs, may be detrimental for EBPR systems, leading to deterioration and, in extreme cases, failure of the process. Therefore, from both process evaluation and modeling perspectives, the estimation of PAO and GAO populations in activated sludge systems is a relevant issue. A simple method for the quantification of PAO and GAO population fractions in activated sludge systems is presented in this paper. To develop such a method, the activity observed in anaerobic batch tests executed with different PAO/GAO ratios, by mixing highly enriched PAO and GAO cultures, was studied. Strong correlations between PAO/GAO population ratios and biomass activity were observed (R2 > 0.97). This served as a basis for the proposal of a simple and practical method to quantify the PAO and GAO populations in activated sludge systems, based on commonly measured and reliable analytical parameters (i.e., mixed liquor suspended solids, acetate, and orthophosphate) without requiring molecular techniques. This method relies on the estimation of the total active biomass population under anaerobic conditions (PAO plus GAO populations), by measuring the maximum acetate uptake rate in the presence of excess acetate. Later, the PAO and GAO populations present in the activated sludge system can be estimated, by taking into account the PAO/GAO ratio calculated on the basis of the anaerobic phosphorus release-to-acetate consumed ratio. The proposed method was evaluated using activated sludge from municipal wastewater treatment plants. The results from the quantification performed following the proposed method were compared with direct population estimations carried out with fluorescence in situ hybridization analysis (determining Candidatus Accumulibacter Phosphatis as PAO and Candidatus Competibacter Phosphatis as GAO). The method showed to be potentially suitable to estimate the PAO and GAO populations regarding the total PAO-GAO biomass. It could be used, not only to evaluate the performance of EBPR systems, but also in the calibration of potential activated sludge mathematical models, regarding the PAO-GAO coexistence.

Short-term temperature effects on the anaerobic metabolism of glycogen accumulating organisms.

Proliferation of glycogen accumulating organisms (GAO) has been identified as a potential cause of enhanced biological phosphorus removal (EBPR) failure in wastewater treatment plants (WWTP). GAO compete for substrate with polyphosphate accumulating organisms (PAO) that are the microorganisms responsible for the phosphorus removal process. In the present article, the effects of temperature on the anaerobic metabolism of GAO were studied in a broad temperature range (from 10 to 40 degrees C). Additionally, maximum acetate uptake rate of PAO, between 20 and 40 degrees C, was also evaluated. It was found that GAO had clear advantages over PAO for substrate uptake at temperatures higher than 20 degrees C. Below 20 degrees C, maximum acetate uptake rates of both microorganisms were similar. However, lower maintenance requirements at temperature lower than 30 degrees C give PAO metabolic advantages in the PAO-GAO competition. Consequently, PAO could be considered to be psychrophilic microorganisms while GAO appear to be mesophilic. These findings contribute to understand the observed stability of the EBPR process in WWTP operated under cold weather conditions. They may also explain the proliferation of GAO in WWTP and thus, EBPR instability, observed in hot climate regions or when treating warm industrial effluents. It is suggested to take into account the observed temperature dependencies of PAO and GAO in order to extend the applicability of current activated sludge models to a wider temperature range.