Phosphate-accumulating Organisms Research Articles

The biomass fraction of phosphate-accumulating organisms grown in anoxic environment in an enhanced biological phosphorus removal (EBPR) system

To improve the design and performance of anoxic phosphorus uptake process, the biomass fractions of PAOs grown in anoxic environment and denitrification rate, with PHAs as carbon source, was investigated in a pilot scale study of EBPR system which was based on stoichiometry and mass balance. The system, containing Modified University of Cape Town (MUCT) reactor, was maintained at steady state with COD loading of 0.25 kg COD/(kg MLVSS d), and solid retention time (SRT) of 12 d. Based on mass balance calculation, 46.74% of organic matter, 50.95% of nitrogen, and 42.03% of phosphorus in the influent were converted to carbon dioxide, nitrogen gas, and poly-phosphate, respectively. In the second anoxic stage, the biomass yield of PAOs with nitrate as electron acceptor was 4.90 gC5H7O2N/d, and its yield fraction was 20.63%. For the reaction to proceed properly, 10.48 g PHA/d of PHAs was oxidized, and 3.68 g NO3--N/d of nitrate was reduced. The denitrification rate with PHAs as carbon source was 0.058 kg NO3--N/(kg MLVSS·d). These results showed that a higher amount of PAO growth under anoxic condition was observed in the MUCT system, and 0.058 kg NO3--N/(kg MLVSS·d) of the denitrification rate with PHAs could be used as the reference values for calculation of anoxic stage volume.

Identification and quantification of microbial populations in activated sludge and anaerobic digestion processes

Eight different phenotypes were studied in an activated sludge process (AeR) and anaerobic digester (AnD) in a full-scale wastewater treatment plant by means of fluorescent in situ hybridization (FISH) and automated FISH quantification software. The phenotypes were ammonia-oxidizing bacteria, nitrite-oxidizing bacteria, denitrifying bacteria, phosphate-accumulating organisms (PAO), glycogen-accumulating organisms (GAO), sulphate-reducing bacteria (SRB), methanotrophic bacteria and methanogenic archaea. Some findings were unexpected: (a) Presence of PAO, GAO and denitrifiers in the AeR possibly due to unexpected environmental conditions caused by oxygen deficiencies or its ability to survive aerobically; (b) presence of SRB in the AeR due to high sulphate content of wastewater intake and possibly also due to digested sludge being recycled back into the primary clarifier; (c) presence of methanogenic archaea in the AeR, which can be explained by the recirculation of digested sludge and its ability to survive periods of high oxygen levels; (d) presence of denitrifying bacteria in the AnD which cannot be fully explained because the nitrate level in the AnD was not measured. However, other authors reported the existence of denitrifiers in environments where nitrate or oxygen was not present suggesting that denitrifiers can survive in nitrate-free anaerobic environments by carrying out low-level fermentation; (e) the results of this paper are relevant because of the focus on the identification of nearly all the significant bacterial and archaeal groups of microorganisms with a known phenotype involved in the biological wastewater treatment.

Polyphosphate- and glycogen-accumulating organisms in one EBPR system for liquid dairy manure.

Two enhanced biological phosphorus removal (EBPR) sequencing batch reactors (SBR1, SBR2) treating liquid dairy manure were operated with the same hydraulic retention time (HRT) and solids retention time (SRT), but with different aeration cycles. During eight months of operation, both SBRs achieved good removal of total phosphorus (P) (TP; 56.8 and 73.5% for SBR1 and SBR2 respectively) and of orthophosphate (OP; 76.2 vs. 82.7%, P < 0.05). Growth dynamics of presumptive phosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) were examined by quantitative polymerase chain reaction (qPCR). SBR1 was enriched with a greater abundance of PAOs while SBR2 was characterized by a greater abundance of GAOs. These results demonstrate the capability of EBPR of dairy manure and challenge conventional wisdom, since greater abundance of PAOs in EBPR system was not associated with improved OP removal and greater abundance of GAOs did not indicate deterioration of the EBPR system.

Metabolic characteristics of a glycogen-accumulating organism in Defluviicoccus cluster II revealed by comparative genomics.

Glycogen-accumulating organisms (GAOs) may compete with phosphate-accumulating organisms (PAOs) for short-chain fatty acids (VFAs) in anaerobic polyhydroxyalkanoates (PHA) synthesis, but no consequently aerobic polyphosphate accumulation in enhanced biological phosphorus removal (EBPR) process, thus deteriorating the EBPR process. They are detected frequently in the deteriorated EBPR process, but their metabolisms are still far from our comprehensions for there is seldom pure culture. In this study, a nearly complete draft genome of a GAOs in Defluviicoccus cluster II, GAO-HK, is recruited from the metagenome of activated sludge in a full-scale industrial anoxic/aerobic wastewater plant. Comparative genomics reveal similar metabolisms of PHA and glycogen in GAOs of GAO-HK, Defluviicoccus tetraformis TFO71 (TFO71) and Competibacter phosphatis clade IIA (CPIIA), and PAOs of Accumulibacter clade IIA UW-1 (UW-1) and Tetrasphaera elongata Lp2 (Lp2). Although there are similar gene cassettes related with polyphosphate metabolism in these GAOs and PAOs, especially for Defluviicoccus-relative bacteria and UW-1, ppk1 in GAOs are diverse from those in the identified PAOs, implying the difference of polyphosphate metabolism in GAOs and PAOs. Additionally, genes related to the dissimilatory denitrification are absent in TFO71 and GAO-HK, implying that additional nitrate or nitrite may favor PAOs over Defluviicoccus-relative GAOs. Therefore, PAOs suffering from competition of Defluviicoccus-relative GAOs might be rescued with the additional nitrate/nitrite, which is important to improve the stability of EBPR processes.

Impact of salinity on the anaerobic metabolism of phosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO).

The use of saline water as secondary quality water in urban environments for sanitation is a promising alternative towards mitigating fresh water scarcity. However, this alternative will increase the salinity in the wastewater generated that may affect the biological wastewater treatment processes, such as biological phosphorus removal. In addition to the production of saline wastewater by the direct use of saline water in urban environments, saline wastewater is also generated by some industries. Intrusion of saline water into the sewers is another source of salinity entering the wastewater treatment plant. In this study, the short-term effects of salinity on the anaerobic metabolism of phosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO) were investigated to assess the impact of salinity on enhanced biological phosphorus removal. Hereto, PAO and GAO cultures enriched at a relatively low salinity level (0.02 % W/V) were exposed to salinity concentrations of up to 6 % (as NaCl) in anaerobic batch tests. It was demonstrated that both PAO and GAO are affected by higher salinity levels, with PAO being the more sensitive organisms to the increasing salinity. The maximum acetate uptake rate of PAO decreased by 71 % when the salinity increased from 0 to 1 %, while that of GAO decreased by 41 % for the same salinity increase. Regarding the stoichiometry of PAO, a decrease in the P-release/HAc uptake ratio accompanied with an increase in the glycogen consumption/HAc uptake ratio was observed for PAO when the salinity increased from 0 to 2 % salinity, indicating a metabolic shift from a poly-P-dependent to a glycogen-dependent metabolism. The anaerobic maintenance requirements of PAO and GAO increased as the salinity concentrations risen up to 4 % salinity.

Optimal cultivation of simultaneous ammonium and phosphorus removal aerobic granular sludge in A/O/A sequencing batch reactor and the assessment of functional organisms

In this study, sequencing batch reactor (SBR) with an anaerobic/aerobic/anoxic operating mode was used to culture granular sludge. Optimal adjustment of cycle duration was achieved by the direction of pH, oxidation reduction potential and dissolved oxygen parameters. The results showed that the treating efficiency was significantly improved as the cycle was shortened from 450 to 360 min and further to 200 min. Nitrogen and phosphorus removal were nearly quantitative after 50 days operation and maintained stable to the end of the study period. The typical cycle tests revealed that simultaneous denitrification and phosphorus removal occurred when aerobic granules were gradually formed. The nitrite effect tests showed that less than 4.8 mg N/L of the nitrite could enhance superficial specific aerobic phosphate uptake rate (SAPUR) under aerobic condition, indicating that the traditional method to evaluate the capability of total phosphate-accumulating organisms (PAOs) was inaccurate. Additionally, a high level of nitrite was detrimental to PAOs. A novel method was developed to determine the activity of each kind of PAOs and other denitrifying organisms. The results showed that (1) nitrate, besides nitrite, could also enhance SAPUR and (2) aerobic granular sludge could perform denitrification even when phosphate was not supplied under anoxic condition, suggesting that other denitrifying organisms besides denitrifying phosphate-accumulating organisms also contributed to denitrification.

Microbial Ecology Pushes Frontiers in Biotechnology

Microbial Ecology Pushes Frontiers in Biotechnology

Effect of Nitrate on Inducement of Denitrifying Phosphate-Accumulating Organisms

Denitrifying phosphate accumulating (DNPA) was investigated by controlling the concentration of nitrate in anoxic stage with batch experiments. A linear relationship between the quantity of phosphorus uptaken and the consumption of nitrate was shown under anoxic condition with the same concentration of carbon source. Based on the slope of the line between phosphorus release quantity and nitrate consumption, nitrate concentration was adjusted in the anoxic stage. Excellent DNPA performance had been achieved rapidly. The removal efficiency of phosphorus was above 95%, and nitrate more than 96%. According to the maximum phosphorus uptake rate of anoxic or aerobic, it was calculated that the percent of DNPAOs to PAOs was increased from 22% to 79% after cultivation.

Start-Up of Denitrifying Dephosphatation with Nitrite as Electron Acceptor and Microbial Community Analysis

The two-stage mode was used to start up the denitrifying dephosphatation system with nitrite as electron acceptor, and the systems capability of denitrifying dephosphatation was evaluated. Phosphate-accumulating organisms were enriched under the anaerobic/aerobic condition at the first stage. Then change the condition to anaerobic/anoxic so that denitrifying phosphate-accumulating organisms (DPAO) using NO2-N as electron acceptor can be selected. The fast start-up of this system was achieved after 120 days, and the system has a good capability of denitrifying dephosphatation. The average TN and TP in the effluent were 5.39 mg/L and 1.55 mg/L with removal rates of 82.5% and 80.0%, respectively. PCR-DGGE results showed that microbial population changed in the process of domestication, while the structure of microbial community changed little in the stable stage, indicating relatively stable microbial community structure had formed after the domestication.

The Effect of PHA on Enhanced Biological Phosphorus Removal

Used A/O-SBR(anaerobic/aerobic alternating enhanced biological phosphorus removal system) to study carbon source and phosphate accumulating organisms (PAO) intracellular energy storage substance transformation, also studied PAO’s phosphorus removal capacity. PAO could uptake quick degradation organic matters and synthesis polyhydroxyalkanoates(PHA). PHA was a kind of intracellular energy storage substance, its content could affect PAO’s phosphorus removal capacity. When carbon source was plentiful, PAO could synthesis a lot of PHA, phosphorus removal capacity could reach 38.16 mgp/gvss. Sludge retention time could affect PAO synthesis PHA of quantity and structure. Along with the sludge retention time growth, intracellular PHA content was increased, and phosphorus uptake of desired aerobic time continues to decrease, so increasing the phosphorus removal efficiency. When the municipal sewage as carbon source, PHA was mainly composed of PHB(poly-ß-hydroxyvalerate) and PHV(poly-ß-hydroxyvalerate). Along with the sludge retention time prolongation, PAO could synthesis more PHV, PHB content was remain unchanged, PHA composition structure was changed.

Comparing the long‐term effect of high P/COD influent on enhancement of phosphate‐accumulating organisms between acetate‐ and propionate‐fed reactors

AbstractBackgroundHigh P/COD loading has been extensively applied to obtain a highly enriched culture of phosphate‐accumulating organisms (PAOs) in an anaerobic‐oxic (A/O) system. However, phosphorus rich influent was also observed to deteriorate the phosphorus metabolism. This study therefore operated acetate‐ and propionate‐fed A/O reactors to examine the long‐term effect of high P/COD loading on the enhancement of PAOs in both reactors.ResultsLong‐term cultivating with 40/400 mg P (mg COD)‐1 influent suppressed the proliferation of PAOs in the acetate‐fed reactor, and more importantly provided glycogen‐accumulating organisms (GAOs) with a selective advantage over PAOs. GAOs eventually dominate the A/O system even when the influent phosphorus was reduced back to 15 mg L‐1. Conversely, PAOs was encouraged, and then stably sustained in the propionate‐fed reactor with the same phosphorus influent (40/400 mg P (mg COD)‐1). Obviously, acquiring a stable PAOs‐enriched system via high phosphorus loading relied considerably on the choice of carbon source.ConclusionsPropionate was more beneficial than acetate for supporting PAOs to suffer a high phosphorus loading in an enhanced biological phosphorus removal system. © 2012 Society of Chemical Industry

Factors influencing the density of aerobic granular sludge

In the present study, the factors influencing density of granular sludge particles were evaluated. Granules consist of microbes, precipitates and of extracellular polymeric substance. The volume fractions of the bacterial layers were experimentally estimated by fluorescent in situ hybridisation staining. The volume fraction occupied by precipitates was determined by computed tomography scanning. PHREEQC was used to estimate potential formation of precipitates to determine a density of the inorganic fraction. Densities of bacteria were investigated by Percoll density centrifugation. The volume fractions were then coupled with the corresponding densities and the total density of a granule was calculated. The sensitivity of the density of the entire granule on the corresponding settling velocity was evaluated by changing the volume fractions of precipitates or bacteria in a settling model. Results from granules originating from a Nereda reactor for simultaneous phosphate COD and nitrogen removal revealed that phosphate-accumulating organisms (PAOs) had a higher density than glycogen-accumulating organisms leading to significantly higher settling velocities for PAO-dominated granules explaining earlier observations of the segregation of the granular sludge bed inside reactors. The model showed that a small increase in the volume fraction of precipitates (1-5%) strongly increased the granular density and thereby the settling velocity. For nitritation-anammox granular sludge, mainly granular diameter and not density differences are causing a segregation of the biomass in the bed.

High phosphorus shock loading induced glycogen accumulating organisms in anaerobic/oxic sequencing batch reactor

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.

Effect of oligochaete worm body fluids on biological phosphorus removal in a bench-scale EBPR system

During waste sludge reduction by oligochaetes, phosphorus (P) concentrations in the effluent have been noticed to increase. In the current study, batch experiments were carried out in order to provide explanations for this phosphorus release. The results indicated that increase in effluent phosphorus concentration might not be directly linked to the phosphorus in worm body fluids, as the phosphorus concentration in the system at the start of each operational period did not change significantly. However, the phosphorus removal efficiency rapidly dropped from 93.9%±1.9% to 62.2%±1.3% with increasing addition of oligochaete worm body fluids. Furthermore, an increase in worm body fluids induced a remarkable enhancement of anaerobic phosphorus release rate as well as anaerobic storage of poly-β-hydroxyalkanoate (PHA). At a worm density (wet weight) of 14.4 g/L, the anaerobic phosphorus release rate was elevated by 31.1%±2.8% and 57.3%±4.6% at 2.5% and 5.0% worm death rate, respectively, compared with the control. The contribution of worm body fluids to PHA production was 39.3–67.7 mg/g of dead worm (wet weight), which was mainly attributed to the extra synthesis of poly-β-hydroxyvalerate (PHV). Unfortunately, in the concomitant aerobic stage, inhibition of 3-hydroxybutyric acid (PHB) oxidation and ammonia utilization was observed along with the increasing addition of worm body fluids. Meanwhile, nitrite elevation was found at the beginning of the aerobic stage, which might be negative to the aerobic metabolic processes performed by phosphate-accumulating organisms (PAOs), namely PHB oxidation, phosphate uptake and ammonia utilization for biomass growth.

A metabolic network of a phosphate-accumulating organism provides new insights into enhanced biological phosphorous removal

Here we present a metabolic network representing the central carbon metabolism as well as the synthesis of polyhydroxyalcanohates and the polyphosphate accumulation mechanisms of the bacterium Candidatus Accumulibacter phosphatis, which was previously identified from metagenomic studies in enhanced biological phosphorous removal sludges. The reconstructed metabolic network, together with flux balance analysis can be used to provide new insights into controversial aspects of the metabolism of phosphate-accumulating organisms and is also a tool that can be used in to help enhanced biological phosphorous removal (EBPR) process design and operation.

A high throughput method and culture medium for rapid screening of phosphate accumulating microorganisms

A novel PA Medium (PAM) for efficient screening of phosphate-accumulating organisms (PAOs) was developed taking Serratia marcescens NBRI1213 as model organism. The defined National Botanical Research Institute’s growth medium (NBRI) supplemented with 0.1% maltose, designed for quantitative estimation of phosphate accumulation was designated as PAM. Our work suggested usage of PAM for efficient qualitative screening and as a microbiological medium for preferential selection of PAOs on Petri-plates. For qualitative screening of PAOs, Toluidine blue-O dye (TBO) was supplemented in PAM, designated as PAM-TBO. Qualitative analysis of phosphate accumulated by various groups correlated well with grouping based upon quantitative analysis of PAOs, effect of carbon, nitrogen, salts, and phosphate accumulation-defective transposon mutants. For significantly increasing sample throughput, efficiency of screening PAOs was further enhanced by adaptation of PAM-TBO assay to microtiter plate based method. It is envisaged that usage of this medium will be salutary for quick screening of PAOs from environment.

Selective sludge removal in a segregated aerobic granular biomass system as a strategy to control PAO–GAO competition at high temperatures

An aerobic granular sludge (AGS) reactor was run for 280 days to study the competition between Phosphate and Glycogen Accumulating Organisms (PAOs and GAOs) at high temperatures. Numerous researches have proven that in suspended sludge systems PAOs are outcompeted by GAOs at higher temperatures. In the following study a reactor was operated at 30 °C in which the P-removal efficiency declined from 79% to 32% after 69 days of operation when biomass removal for sludge retention time (SRT) control was established by effluent withdrawal. In a second attempt at 24 °C, efficiency of P-removal remained on average at 71 ± 5% for 76 days. Samples taken from different depths of the sludge bed analysed using Fluorescent in situ hybridization (FISH) microscopy techniques revealed a distinctive microbial community structure: bottom granules contained considerably more Accumulibacter (PAOs) compared to top granules that were dominated by Competibacter (GAOs). In a third phase the SRT was controlled by discharging biomass exclusively from the top of the sludge bed. The application of this method increased the P-removal efficiency up to 100% for 88 days at 30 °C. Granules selected near the bottom of the sludge bed increased in volume, density and overall ash content; resulting in significantly higher settling velocities. With the removal of exclusively bottom biomass in phase four, P-removal efficiency decreased to 36% within 3 weeks. This study shows that biomass segregation in aerobic granular sludge systems offers an extra possibility to influence microbial competition in order to obtain a desired population.

The Phosphorous Removal after Anaerobic Stages of Different Durations and under Different Organic Loadings in the Reversed A&lt;sup&gt;2&lt;/sup&gt;/O Process

The phosphorous removal behaviors under higher and moderate organic loading and after different durations of anaerobic stages were studied in the reversed A2/O process. Under both loading, the length of anaerobic stages did not significantly affect the subsequent aerobic phosphorus uptake efficiencies. Under higher loading, the available carbon sources are adequate, prolonging anaerobic stages led to more released phosphorus from phosphate accumulating organisms (PAOs). However, the phosphorus uptake rate in aerobic stage was almost the same as that of lower loading. At an anaerobic reaction time of 2.0 h, the corresponding specific aerobic phosphorus uptake rate achieved the highest value (6.13 mg/(L.h)). These results can help to determine the anaerobic hydraulic retention time in the reversed A2/O process.

Enhanced phosphorus removal by a humus soil cooperated sequencing batch reactor using acetate as carbon source

The biological phosphorus removal by humus soil sequencing batch reactor process (HS-SBR) and a conventional sequencing batch reactor (cSBR) were compared using acetate as a sole carbon source. The HS-SBR was composed of a humus soil reactor (HSR) and a conventional SBR (designated as hsSBR). The HS-SBR showed a more stable and effective phosphorus removal with the efficiency of 97.3%, while as to the cSBR, it was 80%. However, in the HS-SBR for the removal of COD and nitrogen it was not improved. Moreover, in the anaerobic phase, the hsSBR had greater soluble orthophosphate (SOP) release and poly-β-hydroxybutyrate (PHB) synthesis capacity, but lower poly-β-hydroxyvalerate (PHV) synthesis and glycogen (Gly) degradation capacity than those in the cSBR. In addition, acetate (HAc) uptake rate and SOP release rate in the hsSBR were greater than those in the cSBR. In the aerobic phase, the PHA utilization efficiency for SOP uptake in the hsSBR was higher than the cSBR. All these observations suggested that adding the HSR improved the relative dominance of the phosphate accumulating organisms (PAOs) in the hsSBR.

Comparative Performance of A2 /O and a Novel Membrane-Bioreactor-Based Process for Biological Nitrogen and Phosphorus Removal

The comparison between a novel membrane bioreactor (MBR) system and a conventional anaerobic-anoxic-aerobic (A2/O) system was conducted using synthetic wastewater (SWW) and municipal wastewater (MWW). Each system was operated at an overall hydraulic retention time of 8 hours and solids retention time of 10 days. The MBR exhibited better overall system performance than the A2/O system, in terms of phosphorus removal. Nitrogen removal efficiencies were close in the two systems at 73 to 74% in both runs, while phosphorus removal efficiencies were 96 and 74% (SWW run) and 80 and 75% (MWW run), for the MBR and A2/O, respectively. Effluent soluble chemical oxygen demand (COD) was less than 15 mg/L in the two systems during both runs. Phosphorus uptake by denitrifying phosphate-accumulating organisms accounted for 49% of the total uptake in the MBR compared with 33% in the A2/O during the SWW run. The dynamic test clearly showed that the MBR had better denitrification capacity than the A2/O system. The MWW run indicated that MBR ferments particulate COD better than A2/ O. The effect of the intermediate clarifier on MBR phosphorus removal was significant, with phosphorus uptake of 0.16 g/d in the SWW run and phosphorus release of 0.08 g/d in the MWW run, thus enhancing thetotal phosphorus removal in both cases.