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Sludge bulking is a common challenge in industrial biological wastewater treatment. Leading to difficulties such as bad sludge settling and washout, which is a problem also encountered in the petrochemical industry. Anaerobic feeding strategies can be used to induce the growth of storage-capable organisms, such as glycogen-accumulating organisms (GAO), leading to denser sludge flocs and better settling. In this study, the implementation of an anaerobic feeding strategy was investigated for high-salinity petrochemical wastewater (±35 g salts·L−1), using a sequencing batch reactor. Influent, effluent and sludge characteristics were analyzed throughout the operational period, which can be divided into three stages: I (normal operation), II (increased influent volume) and III (longer anaerobic mixing). Good effluent quality was observed during all stages with effluent chemical oxygen demand (COD) < 100 mgO2·L−1 and removal efficiencies of 95%. After 140 days, the sludge volume index decreased below 100 mL·g−1 reaching the threshold of good settling sludge. Sludge morphology clearly improved, with dense sludge flocs and less filaments being present. A maximum anaerobic dissolved oxygen carbon (DOC) uptake was achieved on day 80 with 74% during stage III. 16S rRNA amplicon sequencing showed the presence of GAOs, with increasing relative read abundance over time from 1 to 3.5%. HIGHLIGHTS Dense and compact sludge flocs were achieved under high-saline conditions; Filamentous organisms were suppressed in the system, favoring the growth of glycogen-accumulating organisms; Settling characteristics of the sludge were improved to the point of the sludge being characterized as well-settling;

The review deals with specific bacterial physiological groups developing under periodically varying ambient conditions and possessing a cyclic type of metabolism (CTM). CTM is characterized by unbalanced two-phase growth occurring during the period less than the generation time. During the first phase, the carbon and/or energy source are accumulated, while during the second phase biomass biosynthesis occurs using the accumulated substrate. Known CTM organisms of the groups of phosphate-accumulating and glycogen-accumulating bacteria are described, and their major biochemical models are discussed. Development of microorganisms with obligate CTM is probably impossible or significantly suppressed under stable ambient conditions, which may be the reason why the major CTM organisms can not be cultured using traditional batch and continuous cultivation, while highly enriched cultures may be obtained by cyclic cultivation. Ecological aspects of CTM are discussed, and the possible environments for CTM microorganisms in natural ecosystems are determined. [ABSTRACT FROM AUTHOR]

Recently reported kinetic and stoichiometric parameters of the Activated Sludge Model no. 2d (ASM2d) for high-temperature EBPR processes suggested that the absence of glycogen in the model contributed to underestimation of PHA accumulation at 32 °C. Here, two modified ASM2d models were used to further explore the contribution of glycogen in the process. The ASM2d-lG model incorporated glycogen metabolism by PAOs (polyphosphate-accumulating organisms), while the ASM2d-2G model further included processes by GAOs (glycogen-accumulating organisms). These models were calibrated and validated using experimental data at 32 °C. The ASM2d-lG model supported the hypothesis that the excess PHA was attributed to glycogen, but remained inadequate to capture the dynamics of glycogen without considering GAOs activities. The ASM2d-2G model performed better, but it was challenging to calibrate as it often led to wash-out of either PAOs or GAOs. Associated hurdles are highlighted and additional efforts in calibrating ASM2d-2G more effectively are proposed. [ABSTRACT FROM AUTHOR]

This article presents the results of research into the influence of one, two and three wastewater feedings in a cycle on efficiency and performance of combined biological nitrogen and phosphorus removal in an integrated fixed-film activated sludge and moving-bed sequencing batch biofilm reactor (IFAS-MBSBBR). The experiment lasted 158 days and was conducted in two laboratory models of the IFAS-MBSBBR with an active volume of 28 L. It was found that along with an increase in the number of wastewater feedings, an increase in nitrogen removal efficiency was observed (from 56.9 ± 2.30% for a single feeding to 91.4 ± 1.77% for three feedings). Moreover, the contribution of simultaneous nitrification/denitrification in nitrogen removal increased (from 2.58% for a single feeding to 69.5% for three feedings). Systems with a greater number of feedings stimulated the process of denitrifying phosphorus removal. Regardless of the way in which wastewater feeding was applied to the IFAS-MBSBBR, highly efficient chemical oxygen demand (COD) removal (94.8 ± 1.80%) and biological phosphorus removal (98.9 ± 0.87%) were achieved. [ABSTRACT FROM AUTHOR]

• Excellent NRE of 97.5% was achieved with TIN below 2.0mg/L by selective enhanced GAOs. • Intermittent phosphorus exclusion with poly-P/VSS below 0.01(w/w) enhanced GAOs. • GAOs gained organics outcompeting PAOs at low poly-P and strengthened ED. • PNA was unaffected by the enhanced ED and coupled to remove nitrogen in the anoxic stage. • EPDA was promoted by the enrichment of GAOs that reduced NO 3 − to NO 2 − exclusively. Integrating endogenous denitrification (ED) into partial nitrification-anammox (PNA) systems by adequately utilizing organics in municipal wastewater is a promising approach to improve nitrogen removal efficiency (NRE). In this study, a novel strategy to inhibit phosphorus-accumulating organisms (PAOs) by inducing phosphorus release and exclusion was adopted intermittently, optimizing organics allocation between PAOs and glycogen-accumulating organisms (GAOs). Enhanced ED-synergized anammox was established to treat real municipal wastewater, achieving an NRE of 97.5±2.2% and effluent total inorganic nitrogen (TIN) of less than 2.0 mg/L. With low poly-phosphorus (poly-P) levels (poly-P/VSS below 0.01 (w/w)), glycogen accumulating metabolism (GAM) acquired organics exceeded that of phosphorus accumulating metabolism (PAM) and dominated endogenous metabolism. Ca. Competibacter (GAO) dominated the community following phosphorus-rich supernatant exclusion, with abundance increasing from 3.4% to 5.7%, accompanied by enhanced ED capacity (0.2 to 1.4 mg N/g VSS /h). The enriched subgroups (GB4, GB5) of Ca. Competibcate r established a consistent nitrate cycle with anammox bacteria (AnAOB) through endogenous partial denitrification (EPD) at a ∆NO 2 −-N/∆NH 4 +-N of 0.91±0.11, guaranteeing the maintenance of AnAOB abundance and performance. These results provide new insights into the flexibility of PNA for the energy-efficient treatment of low-strength ammonium wastewater. [Display omitted] [ABSTRACT FROM AUTHOR]

A recently reported stable and efficient EBPR system at high temperatures around 30 °C has led to characterization of kinetic and stoichiometric parameters of the Activated Sludge Model no. 2d (ASM2d). Firstly, suitable model parameters were selected by identifiability analysis. Next, the model was calibrated and validated. ASM2d was found to represent the processes well at 28 and 32 °C except in polyhyroxyalkanoate (PHA) accumulation of the latter. The values of the kinetic parameters for PHA storage ( q), polyphosphate storage ( q) and growth ( μ) of polyphosphate-accumulating organisms (PAOs) at 28 and 32 °C were found to be much higher than those reported by previous studies. Besides, the value of the stoichiometric parameter for the requirement of polyphosphate for PHA storage ( Y) was found to decrease as temperature rose from 28 to 32 °C. Values of two other stoichiometric parameters, i.e. the growth yield of heterotrophic organisms ( Y) and PAOs ( Y), were high at both temperatures. These calibrated parameters imply that the extremely active PAOs of the study were able to store PHA, store polyphosphate and even utilize PHA for cell growth. Besides, the parameters do not follow the Arrhenius correlation due to the previously reported unique microbial clade at 28 and 32 °C, which actively performs EBPR at high temperatures. [ABSTRACT FROM AUTHOR]

Nitrite-based phosphorus (P) removal could be useful for innovative biological P removal systems where energy and carbon savings are a priority. However, using nitrite for denitrification may cause nitrous oxide (N 2 O) accumulation and emissions. A denitrifying nitrite-fed P removal system ( SBR NO 2 − ) was successfully set up in a sequencing batch reactor (SBR) and was run for 210 days. The maximum pulse addition of nitrite to SBR NO 2 − was 11 mg NO 2 − -N/L in the bulk, and a total of 34 mg NO 2 − -N/L of nitrite was added over three additions. Fluorescent in situ hybridization results indicated that the P-accumulating organisms (PAOs) abundance was 75 ± 1.1% in SBR NO 2 − , approximately 13.6% higher than that in a parallel P removal SBR using nitrate ( SBR NO 3 − ) . Type II Accumulibacter (PAOII) (unable to use nitrate as an electron acceptor) was the main PAOs species in SBR NO 2 − , contributing 72% to total PAOs. Compared with SBR NO 3 − , SBR NO 2 − biomass had enhanced nitrite/free nitrous acid (FNA) endurance, as demonstrated by its higher nitrite denitrification and P uptake rates. N 2 O accumulated temporarily in SBR NO 2 − after each pulse of nitrite. Peak N 2 O concentrations in the bulk for SBR NO 2 − were generally 6–11 times higher than that in SBR NO 3 − ; these accumulations were rapidly denitrified to nitrogen gases. N 2 O concentration increased rapidly in nitrate-cultivated biomass when 5 or 10 mg NO 2 − -N/L per pulse was added. Whereas, N 2 O accumulation did not occur in nitrite-cultivated biomass until up to 30 mg NO 2 − -N/L per pulse was added. Long-term acclimation to nitrite and pulse addition of nitrite in SBR NO 2 − reduced the risk of nitrite accumulation, and mitigated N 2 O accumulation and emissions from denitrifying P removal by nitrite. [ABSTRACT FROM AUTHOR]

Abstract: Endogenous processes such as biomass decay and intracellular polymers degradation of polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) were investigated. Cultures enriched in Accumulibacter (a well known PAO) or Competibacter (a well known GAO) were subjected to 21 and 26days of alternating anaerobic/aerobic conditions respectively. The main energy source for PAOs during starvation was their intracellular polyphosphate released into the medium during the first 14days of starvation. In contrast, GAOs used their intracellular glycogen during the 26days of starvation. Biomass decay rates were 0.029d−1 for PAOs and almost negligible for GAOs. The reduction in acetate uptake rate during the starvation period, referred to as activity decay, was 0.25 and 0.047d−1 for PAOs and GAOs, respectively. Once wastewater was reintroduced, both populations recovered their initial substrate uptake rate after 1day. The results obtained show that PAOs are more affected than GAOs by starvation conditions. [Copyright &y& Elsevier]

This study investigated the long-term adaptability of an acetate-fed anaerobic-oxic sequencing batch reactor, operated at 15 days of solid retention time (SRT), to the influent P/COD loading raised from 15/400 to 40/400 mg/mg. Experiment results showed that after four SRTs operation, the anaerobic P release, aerobic P uptake and sludge P content decreased apparently, implying the metabolism of phosphate-accumulating organisms (PAOs) was inhibited. In contrast, anaerobic glycogen consumption and polyhydroxyalkanoates (PHAs) accumulation per acetate uptake increased observably, and 3HV/PHAs ratio elevated gradually to 21.8%. These results indicated that the proliferation of glycogen-accumulating organisms (GAOs) was encouraged. When influent phosphorus was reduced back to 15 mg/L, sludge P content diminished and a low P release/acetate uptake ratio was achieved, suggesting that PAOs' metabolism was not restored. The above findings concluded that long-term operation of the high phosphorus influent inhibited the proliferation of PAOs, and more importantly provided GAOs a competitive advantage over PAOs. [ABSTRACT FROM AUTHOR]

A laboratory-scale continuous-flow system with an anaerobic/anoxic/aerobic configuration was set up to study the effect of oxygen in the internal recycle stream; of particular interest was its performance of denitrifying phosphorus-accumulating organisms (DPAOs). It was found that, by using a degas device, the dissolved oxygen in the nitrate recycle stream was effectively decreased from 0.1±0.02 to 0.01±0.01 mg/L. This provided a favorable condition for DPAOs to grow under an anoxic condition and thus be sustained successfully in the system. When the degas device was removed from the system, the dissolved oxygen concentration in the anoxic reactor increased to 0.1±0.02 mg/L. The proliferation of the denitrifying glycogen-accumulating organisms (DGAOs) population and deterioration of DPAOs performance was observed. The increased population of DGAO/GAOs, which competed for the carbon source with DPAO/PAOs, resulted in a poor performance of biological phosphorus removal. [ABSTRACT FROM AUTHOR]

Enhanced biological phosphorus removal (EBPR) was operated in a laboratory-scale sequencing batch reactor (SBR). Fluorenscent in situ hybridization (FISH) was used to study the competition between the phosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs). The influence of competition between PAOs and GAOs on phosphate removal efficiency was discussed. The results showed that 0-20 d was an adjusting stage. COD and phosphate removal efficiencies of SBR were unstable. PAOs and GAOs competed for the predominant status in reactor. During 20-60 d, the amount of PAOs increased gradually, while the amount of Alphaproteobacteria decreased and Gammaproteobacteria contents was below 8. 11% in this stage, which indicates that low temperature is beneficial to PAOs. PAOs proliferated rapidly during 50-60 d. Because the increased amount of PAOs was only 3.2% per 5 d, the phosphate removal efficiency in reactor was not delayed. [ABSTRACT FROM AUTHOR]

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Studies on the synthesis of polyhydroxyalkanoates (PHA) mainly focus on the enriched cultures of phosphorus-accumulating (PAO) or glycogen-accumulating organisms (GAO) with acetate or propionate as the sole carbon source. However, PHA production with a mixture of propionate and acetate by enriched cultures of GAO has not been reported. It is well known that the physical and mechanical properties of PHA can be improved by incorporating 3-hydroxyvalerate (3HV) into the polymer. To synthesize PHA with a high 3HV percentage, five laboratory-scale sequencing batch reactors were operated with the enriched cultures of GAO using different ratios of propionate and acetate (P/A) as the carbon sources in this study. The effects of P/A ratio on the anaerobic and aerobic transformations, composition and synthesis rate of PHA were studied. The results showed that with the increase in the P/A ratio, the anaerobic yields of total PHA declined from 4.226 to 2.469 mmol-C per gram of volatile suspended solids (VSS), and the 3-hydroxybutyrate (3HB) fraction in PHA decreased from approximately 70 to 10%, along with an increase in 3HV and 3-hydroxy-2-methylvalerate (3H2MV) percentages. When there were equal quantities (mmol-C L-1) of propionate and acetate, 3HB was produced more quickly than 3H2MV. By increasing P/A ratio from 1/10 to 10/1, the initial synthesis rate of 3H2MV rose from 0.002 to 0.029, while 3HB and the total PHA declined from 0.083 to 0.007 and 0.110 to 0.071 mmol-C g-VSS-1 min-1, respectively. Also, it was observed that more 3HV was incorporated into PHA with increasing P/A ratio. This study suggested that when the enriched cultures of GAO were applied to the synthesis of PHA, the composition, yield and synthesis rate of PHA could be manipulated by varying the feed composition. [ABSTRACT FROM AUTHOR]

Abstract: With acetate or propionate as the sole carbon source, anaerobic metabolic models describing phosphorus- and glycogen-accumulating organisms (PAO and GAO) have been developed in the literature. However, comprehensive models are in need for the description of PAO and GAO behaviors with mixed acetic and propionic acids as carbon sources since they are the two main volatile fatty acids (VFA) that coexist in real wastewater. Two metabolic models were proposed to characterize the anaerobic stoichiometry of PAO and GAO, respectively, and two groups of sequencing batch reactors (i.e. 5 PAO-SBRs and 5 GAO-SBRs) with different propionic to acetic acid ratios were used for the validation of the models. The experimental data indicated that polyhydroxyalkanoates were synthesized via random condensation in GAO cells, whereas the semi-selective/semi-random pathway was used for the integration of acetyl-CoA and propionyl-CoA in PAO cells. When the VFA was pure acetic or propionic acid, the proposed PAO (or GAO) model reverted back to the reported acetate or propionate PAO (or GAO) model. Results also showed that the energy required for the transportation of 1C-mol VFA across the membrane of both PAO and GAO cells was independent of the propionate/acetate ratio. [Copyright &y& Elsevier]

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. Water [ABSTRACT FROM AUTHOR]

In enhanced biological phosphorus removal (EBPR) systems, polyphosphate-accumulating organisms (PAOs) are primarily responsible for removing phosphate from wastewater. Propionate is an abundant carbon substrate in many EBPR plants and has been suggested to provide PAOs an advantage over their carbon competitors--the glycogen-accumulating organisms (GAOs). The aerobic metabolism of PAOs enriched with a propionate carbon source is studied in this paper. A metabolic model is proposed and experimentally validated to characterize the aerobic biochemical transformations by PAOs. The model predicts very well the experimental data obtained from the enriched PAO culture through solid-, liquid-, and gas-phase analyses. This model may be combined with previously formulated metabolic models to better describe the biochemical activity of PAOs with acetate and propionate as the primary carbon sources. Furthermore, it can also facilitate the study of the effect of different carbon sources on PAO-GAO competition. Water Environ. [ABSTRACT FROM AUTHOR]

The intracellular phosphorus content of phosphorus-accumulating organisms (PAO) was determined based on a stoichiometric equation and phosphorus balance for an enhanced biological phosphorus removal system fed with different P:COD ratios. The data indicated that a higher P:COD feeding ratio could significantly promote the growth of PAO. As the P:COD feeding ratio increased from 0. 02:1 to 0. 04:1 and 0. 16:1, the phosphorus in the sludge increased considerably from 0. 053 to 0. 084 and 0. 205mg P (mg VSSaerobic)−1, respectively, indicating a dynamic condition in the microbial population. From the calculations, the mass fractions of the PAO, glycogen-accumulating organisms, and ordinary heterotrophs changed from 0. 10–0. 15, 0. 83–0. 88, and 0. 02 at 0. 02:1 to 0. 19–0. 28, 0. 70–0. 79, and 0. 02 at 0. 04:1 and to 0. 478–0. 71, 0. 26–0. 50, 0. 03 at 0. 16:1 P:COD feeding ratios, respectively. Despite the variation in microbial diversity, the calculated phosphorus contents of the PAO at all P:COD feeding ratios were consistent between 0. 241 and 0. 378mg P (mg VSSPAO)−1. The initial specific phosphorus release and uptake rates were 84. 7–167. 9mg P (g VSSPAO)−1 h−1 and 52. 8–90. 0mgP (g VSStotal)−1 h−1, respectively. [Copyright &y& Elsevier]

Enhanced biological phosphorus removal (EBPR) has been used at many wastewater treatment plants all over the world for many years. In this study a full-scale sludge with good EBPR was tested with P-release batch tests and combined FISH/MAR (fluorescence in situ hybridisation and microautoradiography). Proposed models of PAOs and GAOs (polyphosphate- and glycogen-accumulating organisms) and microbial methods suggested from studies of laboratory reactors were found to be applicable also on sludge from full-scale plants. Dependency of pH and the uptake of both acetate and propionate were studied and used for calculations for verifying the models and results from microbial methods. All rates found from the batch tests with acetate were higher than in the batch tests with propionate, which was explained by the finding that only those parts of the bacterial community that were able to take up acetate anaerobically were able to take up propionate anaerobically. [ABSTRACT FROM AUTHOR]

Mobile gene elements associated with integrons, including as gene cassettes, have been proposed to play an important role in bacterial evolution by providing an extensive genetic resource. This study hypothesized that critical genes for enzymes involved in EBPR systems, including those involved in polyphosphate, PHA and glycogen synthesis, may be present in mobile gene cassettes. Although no such genes were identified in any of the functional and deteriorated enhanced biological phosphorus removal (EBPR) laboratory-scale SBR systems examined here, many of the open reading frames (ORFs) remained unidentified because of the incompleteness of publicly available databases. An ORF of unknown function (SBR6-2) was encountered in deteriorated EBPR system with an unexpectedly high frequency, comprising 35% of the gene cassette-associated sequences for that system. [ABSTRACT FROM AUTHOR]

Aims: To see if the compositions of the microbial communities in full scale enhanced biological phosphorus removal activated sludge systems were the same as those from laboratory scale sequencing batch reactors fed a synthetic sewage. Methods: Biomass samples taken from nine full scale enhanced biological phosphate removal (EBPR) activated sludge plants in the eastern states of Australia were analysed for their populations of polyphosphate (polyP)-accumulating organisms (PAO) using semi-quantitative fluorescence in situ hybridization (FISH) in combination with DAPI (4′-6-diamidino-2-phenylindole) staining for polyP. Results: Very few betaproteobacterial Rhodocyclus related organisms could be detected by FISH in most of the plants examined, and even where present, not all these cells even within a single cluster, stained positively for polyP with DAPI. In some plants in samples from aerobic reactors the Actinobacteria dominated populations containing polyP. Conclusions: The PAO populations in full-scale EBPR systems often differ to those seen in laboratory scale reactors fed artificial sewage, and Rhodocyclus related organisms, dominating these latter communities may not be as important in full-scale systems. Instead Actinobacteria may be the major PAO. Significance and Impact of the Study: These findings illustrate how little is still known about the microbial ecology of EBPR processes and that more emphasis should now be placed on analysis of full-scale plants if microbiological methods are to be applied to monitoring their performances. [ABSTRACT FROM AUTHOR]

Microbial population dynamics to gradual temperature change in an enhanced biological phosphorus removal system was kinetically investigated. As the temperature rose from 20.0°C to 30.0°C, and to 35.5°C, the predominant microbial group changed from the phosphorus-accumulating organisms, PAOs (47–70% of total VSS) to the glycogen-accumulating organisms (64–75% of total VSS), and to the ordinary heterotrophs (90% of total VSS), respectively. Despite the species alteration, the phosphorus contents of the PAOs appeared to be steady within 0.182–0.308 mg/mg VSSPAO regardless of the temperature level. The initial specific phosphorus release rates, which are solely due to the PAOs activities, increased with the temperature from 37.5–55.9 to 51.8–61.3, 52.0–76.9, 147.2–210.3, and 374.2–756.3 mg P/gm VSSPAO h, at 20.0°C, 25.0°C, 30.0°C, 32.5°C, and 35.0°C, respectively. On the other hand, mean initial specific phosphorus uptake rates of the biomass decreased as the temperature increased; however, the data implied that the rate of the PAOs was higher than the other two microbial groups. These results indicate that the PAOs are lower-range mesophiles or possibly psychrophiles. As the temperature rises, the portion of energy required for maintenance increases substantially which reduces the energy availability for cell reproduction; hence, the PAOs are washed out from the system. [Copyright &y& Elsevier]