Competition between polyphosphate and glycogen accumulating organisms in enhanced biological phosphorus removal systems with acetate and propionate as carbon sources

Enhanced biological phosphorus removal (EBPR) is a widely used process for achieving phosphorus removal from wastewater. A potential reason for EBPR failure is the undesirable growth of glycogen accumulating organisms (GAOs), which can compete for carbon sources with the bacterial group responsible for phosphorus removal from wastewater: the polyphosphate accumulating organisms (PAOs). This study investigates the impact of carbon source on EBPR performance and the competition between PAOs and GAOs. Two sequencing batch reactors (SBRs) were operated during a 4-6 month period and fed with a media containing acetate or propionate, respectively, as the sole carbon source. It was found that the acetate fed SBR rarely achieved a high level of phosphorus removal, and that a large portion of the microbial community was comprised of "Candidatus Competibacter phosphatis", a known GAO. The propionate fed SBR, however, achieved stable phosphorus removal throughout the study, apart from one brief disturbance. The bacterial community of the propionate fed SBR was dominated by "Candidatus Accumulibacter phosphatis", a known PAO, and did not contain Competibacter. In a separate experiment, another SBR was seeded with a mixture of PAOs and a group of alphaproteobacterial GAOs, both enriched with propionate as the sole carbon source. Stable EBPR was achieved and the PAO population increased while the GAOs appeared to be out-competed. The results of this paper suggest that propionate may provide PAOs with a selective advantage over GAOs in the PAO-GAO competition, particularly through the minimisation of Competibacter. Propionate may be a more suitable substrate than acetate for enhancing phosphorus removal in EBPR systems.     

Comparison of acetate and propionate uptake by polyphosphate accumulating organisms and glycogen accumulating organisms

Enhanced biological phosphorus removal (EBPR) performance is directly affected by the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs). This study investigates the effects of carbon source on PAO and GAO metabolism. Enriched PAO and GAO cultures were tested with the two most commonly found volatile fatty acids (VFAs) in wastewater systems, acetate and propionate. Four sequencing batch reactors (SBRs) were operated under similar conditions and influent compositions with either acetate or propionate as the sole carbon source. The stimulus for selection of the PAO and GAO phenotypes was provided only through variation of the phosphorus concentration in the feed. The abundance of PAOs and GAOs was quantified using fluorescence in situ hybridisation (FISH). In the acetate fed PAO and GAO reactors, "Candidatus Accumulibacter phosphatis" (a known PAO) and "Candidatus Competibacter phosphatis" (a known GAO) were present in abundance. A novel GAO, likely belonging to the group of Alphaproteobacteria, was found to dominate the propionate fed GAO reactor. The results clearly show that there are some very distinctive differences between PAOs and GAOs in their ability to take up acetate and propionate. PAOs enriched with acetate as the sole carbon source were immediately able to take up propionate, likely at a similar rate as acetate. However, an enrichment of GAOs with acetate as the sole carbon source took up propionate at a much slower rate (only about 5% of the rate of acetate uptake on a COD basis) during a short-term switch in carbon source. A GAO enrichment with propionate as the sole carbon source took up acetate at a rate that was less than half of the propionate uptake rate on a COD basis. These results, along with literature reports showing that PAOs fed with propionate (also dominated by Accumulibacter) can immediately switch to acetate, suggesting that PAOs are more adaptable to changes in carbon source as compared to GAOs. This study suggests that the PAO and GAO competition could be influenced in favour of PAOs through the provision of propionate in the feed or even by regularly switching the dominant VFA species in the wastewater. Further study is necessary in order to provide greater support for these hypotheses.     

Effect of different carbon sources on the enhanced biological phosphorus removal in a sequencing batch reactor

The effect of the different carbon sources acetate, acetate/glucose or glucose on the enhanced biological phosphorus removal (EBPR) process was studied by experiments under alternating anaerobic–aerobic conditions in one sequencing batch reactor for each carbon source. The glucose was consumed completely within the first 30 min of the anaerobic phase whereas acetate degradation was slow and incomplete. Phosphate was released independently of the carbon source during the whole anaerobic phase. The highest phosphate release (27 mg P l⁻¹) and polyhydroxyalkanoate (PHA) storage (20 mg C g⁻¹ dry matter (DM)) during the anaerobic phase as well as the highest polyphosphate (poly-P) (8 mg P g⁻¹ DM) and glycogen storage (17 mg C g⁻¹ DM) during the aerobic phase were observed with acetate. In contrast to other investigations, glycogen storage did not increase with glucose as substrate but was significantly smaller than with acetate. The PHA composition was also influenced strongly by the carbon source. The polyhydroxyvalerate (PHV) portion of the PHA was maximal 17% for acetate and 82% for glucose. Due to the strong influence of the carbon source on the PHA concentration and composition, PHA storage seems to regulate mainly the phosphate release and uptake. This revised version was published online in July 2006 with corrections to the Cover Date.     

Accumulation of polyhydroxyalkanoates by Microlunatus phosphovorus under various growth conditions

Microlunatus phosphovorus is an activated-sludge bacterium with high levels of phosphorus-accumulating activity and phosphate uptake and release activities. Thus, it is an interesting model organism to study biological phosphorus removal. However, there are no studies demonstrating the polyhydroxyalkanoate (PHA) storage capability of M. phosphovorus, which is surprising for a polyphosphate-accumulating organism. This study investigates in detail the PHA storage behavior of M. phosphovorus under different growth conditions and using different carbon sources. Pure culture studies in batch-growth systems were conducted in shake-flasks and in a bioreactor, using chemically defined growth media with glucose as the sole carbon source. A batch-growth system with anaerobic–aerobic cycles and varying concentrations of glucose or acetate as the sole carbon source, similar to enhanced biological phosphorus removal processes, was also employed. The results of this study demonstrate for the first time that M. phosphovorus produces significant amounts of PHAs under various growth conditions and with different carbon sources. When the PHA productions of all cultivations were compared, poly(3-hydroxybutyrate) (PHB), the major PHA polymer, was produced at about 20–30% of the cellular dry weight. The highest PHB production was observed as 1,421 mg/l in batch-growth systems with anaerobic–aerobic cycles and at 4 g/l initial glucose concentration. In light of these key results regarding the growth physiology and PHA-production capability of M. phosphovorus, it can be concluded that this organism could be a good candidate for microbial PHA production because of its advantages of easy growth, high biomass and PHB yield on substrate and no significant production of fermentative byproducts.     

Role of glycogen in acetate uptake and polyhydroxyalkanoate synthesis in anaerobic-aerobic activated sludge with a minimized polyphosphate content

The role of glycogen in the uptake of acetate in anaerobic-aerobic activated sludge without enhanced biological phosphorus removal were investigated. Although the polyphosphate content of the sludge was minimized by lowering the phosphorus feeding concentration, significant acetate uptake and accumulation of polyhydroxyalkanoates (PHAs) were observed in proportion to glycogen consumption under anaerobic conditions. The results of anaerobic inhibition studies, which showed suppressive effects on acetate uptake by a glycolysis inhibitor (iodoacetate) but not by a membrane ATPase inhibitor (N,N′-dicyclohexyl carbodiimide), supported an assumption that glycogen degradation through glycolysis supplies the required ATP and reducing power for PHA synthesis from acetate and consumed glycogen. Under subsequent aerobic conditions, the accumulated PHAs were depleted and the consumed glycogen recovered to the same level as that at the start of the anaerobic phase. Iodoacetate also inhibited the recovery of glycogen under aerobic conditions, suggesting that nearly 50% of the PHAs depleted was used for glycogen synthesis through reversed glycolysis.     

Recent developments in the biochemistry and ecology of enhanced biological phosphorus removal

Most of the genes encoding the enzymes involved in polyP synthesis and degradation and in phosphate transport have been studied in various Gram-negative bacteria. Progress has also been made in studying the biochemical mechanisms underlying the process of enhanced biological phosphorus removal (EBPR), in particular in lab-scale systems fed with acetate or acetate plus glucose as the sole carbon and energy sources. By applying 13C-NMR, previous models concerning anaerobic carbon metabolism have been advanced and the role of glycogen in providing reducing equivalents in EBPR is definitely demonstrated. The role of the citric acid cycle in supplying reducing equivalents for the conversion of acetyl-CoA into poly-beta-hydroxybutyrate and poly-beta-hydroxyvalerate has been discussed. An incomplete citric acid cycle has been proposed to provide a small part of the reducing equivalents. Polyphosphate:AMP phosphotransferase and polyphosphatase were readily detectable in EBPR sludge fed with acetate plus glucose, but polyphosphate kinase remained undetected. In a lab-scale EBPR system, fed for several months with only acetate as carbon source, a Rhodocyclus-like bacterium (R6) was highly enriched and is therefore probably responsible for EBPR in systems fed with acetate only. This R6-type bacterium was however also present in other EBPR sludges (but to a lesser extent), and may therefore play an important role in EBPR in general. This organism accumulates polyhydroxyalkanoates anaerobically and polyP under aerobic conditions. Unlike members of the genus Rhodocyclus, bacterium R6 cannot grow phototrophically. Therefore a provisional new genus Candidatus and species Accumulibacter phosphatis was proposed.     

A metabolic model for acetate uptake under anaerobic conditions by glycogen accumulating organisms: Stoichiometry, kinetics, and the effect of pH

A metabolic model for the stoichiometry of acetate uptake under anaerobic conditions by an enriched culture of glycogen accumulating organisms (GAOs) was developed and tested by experimental studies. Glycogen served as the source of both reducing power and energy to drive the process of acetate uptake. The amount of glycogen consumed and poly-beta-hydroxyvalerate (PHV) accumulated in the cells increased with increasing pH, indicating that the energy requirements for acetate uptake increased with pH. The composition of the accumulated poly-beta-hydroxyalkanoates (PHAs) was adequately predicted using the assumption that acetyl-CoA and propionyl-CoA condense randomly to produce PHA. In addition, the rate of acetate uptake was strongly affected by the pH. The rate decreased with increasing pH and this dependence could be described with a saturation type of expression. A comparison of the rate of acetate uptake at low pH with the rates observed in enriched cultures of phosphorus accumulating organisms (PAOs) indicated that GAOs are able to compete effectively with PAOs in nutrient removal systems under certain conditions.     

Metabolic model for acetate uptake by a mixed culture of phosphate- and glycogen-accumulating organisms under anaerobic conditions

This paper proposes a new metabolic model for acetate uptake by a mixed culture of phosphate- and glycogen-accumulating organisms (PAOs and GAOs) under anaerobic conditions. The model uses variable overall stoichiometry based on the assumption that PAOs may have the ability of using the glyoxylate pathway to produce the required reducing power for polyhydroxyalkonate (PHA) synthesis. The proposed model was tested and verified by experimental results. A sequencing batch reactor system was operated for enhanced biological phosphorus removal (EBPR) with acetate as the sole carbon source at different influent acetate/phosphate ratios. The resulting experimental data supported the validity of the proposed model, indicating the presence of GAOs for all tested HAc/P ratios, especially under P-limiting conditions. Strong agreement is observed between experimental values and model predictions for all model components, namely, PHB production, PHA composition, glycogen utilization, and P release.     

Model of the anaerobic metabolism of the biological phosphorus removal process: Stoichiometry and pH influence

In the anaerobic phase of a biological phosphorus removal process, acetate is taken up and converted to PHB utilizing both energy generated in the degradation of polyphosphate to phosphate, which is released, and energy generated in the conversion of glycogen to poly-beta-hydroxy butyrate (PHB). The phosphate/acetate ratio cannot be considered a metabolic constant, because the energy requirement for the uptake of acetate is strongly influenced by the pH value. The observed phosphate/acetate ratio shows a variation of 0.25 to 0.75 P-mol/C-mol in a pH range of 5.5 to 8.5. It is shown that stored glycogen takes part in the metabolism to provide reduction equivalents and energy for the conversion of acetate to PHB. A structured metabolic model, based on glycogen as the source of the reduction equivalents in the anaerobic phase and the effect of the pH on the energy requirement of the uptake of acetate, is developed. The model explains the experimental results satisfactorily. (c) 1994 John Wiley & Sons, Inc.     

Metabolic influence of lead on polyhydroxyalkanoates (PHA) production and phosphate uptake in activated sludge fed with glucose or acetic acid as carbon source

Sludge in a sequential batch reactor (SBR) system was used to investigate the effect of lead toxicity on metabolisms of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) communities fed with acetic acid or glucose as their sole carbon source, respectively. Results showed that the effect of lead on substrate utilization of both PAOs and GAOs was insignificant. However, lead substantially inhibited both of phosphate release and uptake of PAOs. In high concentration of acetic acid trials, an abnormal aerobic phosphate release was observed instead of phosphate uptake and the release rate increased with increasing lead concentration. Results also showed that PAOs could normally synthesize polyhydroxybutyrate (PHB) in the anaerobic phase even though lead concentration was 40 mg L⁻¹. However, they could not aerobically utilize PHB normally in the presence of lead. On the other hand, GAOs could not normally metabolize polyhydroxyvalerate (PHV) in both the anaerobic and aerobic phases.     

Evaluation of the feasibility of alcohols serving as external carbon sources for biological phosphorus removal induced by the oxic/extended‐idle regime

Recently, a novel operational regime (i.e., the oxic/extended‐idle [OEI] regime) has been reported to successfully achieve enhanced biological phosphorus removal (EBPR) when employing glucose and volatile fatty acids as the sole substrate. In the OEI regime, polyphosphate accumulating organisms (PAOs) could get a selective advantage over other populations during the extended‐idle period where polyphosphate released but polyhydroxyalkanoates and glycogen transformations were negligible/low, thus energy requirements for maintenance purposes in the period could be covered by polyphosphate release. This study further evaluated the feasibility of alcohols as external carbon sources for EBPR induced by the OEI regime, as the available substrate in the raw wastewater is often deficient. First, phosphorus removal in the OEI process was compared, respectively, with methanol and ethanol as the sole substrate. The results showed that the ethanol‐reactor achieved 90.8 ± 2.3% of phosphorus removal, which was approximate twofold than the methanol‐reactor. Further studies displayed that the cells in the ethanol‐reactor contained more PAOs, and had higher activities of exopolyphosphatase and polyphosphate kinase than those in the methanol‐reactor. Also, the aerobic transformations of polyhydroxyalkanoates and glycogen in the ethanol‐reactor were, respectively, higher and lower than those in the methanol‐reactor, which were consistent with the reactors performances. Then, the feasibility of using ethanol as external substrate to enhance EBPR in the OEI process was confirmed for a municipal wastewater. Finally, EBPR performance and metabolic transformation values between the OEI and the anaerobic/oxic (A/O) regimes with ethanol as the sole substrate were compared. The results showed that EBPR in the ethanol‐OEI reactor was higher than that in the ethanol‐A/O reactor. All the above results proved that ethanol was a favorable external substrate to the OEI regime for EBPR enhancement. Biotechnol. Bioeng. 2013; 110: 827–837. © 2012 Wiley Periodicals, Inc.     

Effects of different carbon supplements on phosphorus removal in low C/P ratio industrial wastewater

This study focuses on the effects of different carbon supplements on biological phosphorus removal in the optonics and semiconductor industrial wastewater treatment. Experimental results demonstrate that the addition of a carbon source (glucose, acetate, and digester supernatant) improved phosphorus removal effectively. When the COD/P ratios were controlled in the range of 18-20 (using glucose and supernatant as supplement), the acclimated sludge showed more than 98% removal of phosphorus. In addition, different organic carbons induce dissimilar behavior in anaerobic release and aerobic uptake of phosphorus. The glucose supplement induces significant phosphorus release in anaerobic phase and then an increased phosphorus uptake in aerobic phase. The released phosphorus descended in anaerobic phase when acetate and supernatant were added. There was a good linear relationship of first order reaction between initial COD concentration and specific substrate utilization rate in anaerobic phase.     

Stoichiometric model of the aerobic metabolism of the biological phosphorus removal process

In the aerobic phase of the biological phosphorus removal process, poly-beta-hydroxybutyrate, produced during anaerobic conditions, is used for cell growth, phosphate uptake, and glycogen formation. A metabolic model of this process has been developed. The yields for growth, polyphosphate and glycogen formation are quantified using the coupling of all these conversions to the oxygen consumption. The uptake of phosphate and storage as polyphosphate is shown to have a direct effect on the observed oxygen consumption in the aerobic phase. The overall energy requirements for the P-metabolism are substantial: 25% of the acetate consumed during anaerobic conditions and 60% of the oxygen consumptions is used for the synthesis of polyphosphate and glycogen. (c) 1994 John Wiley & Sons, Inc.     

Effects of acetate and nitrite addition on fraction of denitrifying phosphate-accumulating organisms and nutrient removal efficiency in anaerobic/aerobic/anoxic process

The effects of acetate and nitrite on the performance of sequencing batch reactors (SBRs) employing an anaerobic/aerobic/anoxic (AOA) process were investigated. Three types of SBR operations were used: sodium acetate addition at the start of anoxic condition for heterotrophic denitrification (Type 1); sodium acetate addition at the start of aerobic condition for anoxic phosphate removal by denitrifying phosphate-accumulating organisms (DNPAOs) (Type 2: conventional AOA process); and nitrite addition at the start of aerobic condition for inhibition of phosphate-accumulating organisms (PAOs) (Type 3). A track experiment shows that Type 2 led to the best performance of SBRs among the three types. An analysis by fluorescence in situ hybridization (FISH) revealed that nitrite addition decreased the ratio of PAOs with a decrease in phosphorus removal efficiency. The fraction of DNPAOs in Type 2 was the highest at 13%, indicating that Type 2 is suitable for the simultaneous nitrogen and phosphorus removal in the AOA process.     

Polyhydroxyalkanoate synthesis using different carbon sources by two enhanced biological phosphorus removal microbial communities

Polyhydroxyalkanoate (PHA) is a biodegradable plastic synthesised by bacteria as energy and carbon storage material. PHA production is mostly based on pure cultures operated under sterile conditions, which increase the costs of this biopolymer. The use of inexpensive mixed culture biomass, such as activated sludge, to produce biodegradable plastics from renewable waste streams has been proposed as an alternative.The effect of carbon sources (acetate, propionate, butyrate and glucose) on the type and quantity of PHA synthesis obtained with different enhanced biological phosphorus removal (EBPR) microbial communities enriched with acetate and propionate are reported in this work. Two sequencing batch reactors (SBRs) were seeded with biomass withdrawn from a non-EBPR wastewater treatment plant (WWTP). The same operational conditions were kept, but using acetate or propionate as the sole carbon source of each reactor. These conditions produced two microbial communities with different P-removal capacity. The results presented in this study show the effect of the carbon source on the PHA composition (amount of polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV) and polyhydroxy-2-methylvalerate (PH2MV)), which differed not just between substrates but also between the two EBPR communities used. In addition, some monomers not always analysed contribute significantly to the total amount of PHA, especially when using butyrate, showing that if they are not considered this can lead to erroneous calculated yields.     

The fate of phosphate under anoxic conditions in biological nutrient removal activated sludge systems

The nitrogen removal potential of phosphate accumulating organisms under anoxic conditions has been evaluated using a laboratory scale sequencing batch reactor fed with synthetic wastewater and operated in a sequence of anaerobic, anoxic and aerobic periods. The phosphate uptake rate under anoxic conditions was lower than that under aerobic conditions. However, in the presence of an external substrate such as glucose and acetate, the fate of phosphate was dependent on the substrate type; phosphate release occurred in the presence of nitrate as long as acetate was present and glucose did not cause any phosphate release. The nitrate uptake rate was also much lower with glucose than acetate. The results implied that poly-hydroxyalkanoates could be oxidized by nitrate and phosphate uptake during the anoxic phase should be introduced into process modeling. © Rapid Science Ltd. 1998     

Effect of basic operating parameters on biological phosphorus removal in a continuous-flow anaerobic–anoxic activated sludge system

A continuous-flow anaerobic–anoxic (A2) activated sludge system was operated for efficient enhanced biological phosphorus removal (EBPR). Because of the system configuration with no aeration zones, phosphorus (P) uptake takes place solely under anoxic conditions with simultaneous denitrification. Basic operating conditions, namely biomass concentration, influent carbon to phosphorus ratio and anaerobic retention time were chosen as variables in order to assess their impact on the system performance. The experimental results indicated that maintenance of biomass concentration above 2,500 mg MLVSS/L resulted in the complete phosphate removal from the influent (i.e. 15 mg PO₄ ³⁻-P/L) for a mean hydraulic residence time (HRT) of 15 h. Additionally, by increasing the influent COD/P ratio from 10 to 20 g/g, the system P removal efficiency was improved although the experimental results indicated a possible enhancement of the competition between phosphorus accumulating organisms (PAOs) and other microbial populations without phosphorus uptake ability. Moreover, because of the use of acetate (i.e. easily biodegradable substrate) as the sole carbon source in the system feed, application of anaerobic retention times greater than 2 h resulted in no significant release of additional P in the anaerobic zone and no further amelioration of the system P removal efficiency. The application of anoxic P removal resulted in more than 50% reduction of the organic carbon necessitated for nitrogen and phosphorus removal when compared to a conventional EBPR system incorporating aerobic phosphorus removal.     

Comparison between acetate and propionate as carbon sources for phosphorus removal in the aerobic/extended-idle regime

Lately, we have proved that biological phosphorus (P) removal can be achieved in the aerobic/extended-idle (AEI) regime using glucose as the sole carbon source, which might develop a potential simple strategy for simultaneous removal of P and organic substrates from wastewater. Since acetate and propionate are the two most common substrates present in real domestic wastewater, this paper further assesses the P removal performances in the AEI process using acetate and propionate as the sole carbon source. The results showed that 3.91 and 3.64 mg of P/g of total suspended solids were, respectively, removed in the acetate-reactor and propionate-reactor after 50 days, respectively. After 90 days P removal in the propionate-reactor increased to 4.91 mg P/g of total suspended solids whereas that in the acetate-reactor kept in the same level (3.98 mg/g). Though both acetate and propionate could be used as carbon sources for P removal in such a novel system, the latter was more effective after long-term operation. Further investigations showed that, after 90 days’ acclimatization, sludge poly-P content in the propionate-system was more than that in the acetate-system, which was the primarily reason for the propionate-system showing higher P removal.     

Comparison of polyhydroxyalkanoates production by activated sludges from anaerobic and oxic zones of an enhanced biological phosphorus removal system: effect of sludge retention time

This study compared the PHAs production behavior of sludges from the anaerobic and oxic phases of an enhanced biological phosphorus removal (EBPR) system. This was accomplished by using the kinetics and stoichiometric coefficients obtained from aerobic batch tests to evaluate the performance of these two sludges. Experimental results indicated that the metabolic behavior of the sludges for PHAs production depend significantly on the operating sludge retention time (SRT) of the EBPR system. The oxic sludge with 5 days of SRT exhibited better PHAs production performance than anaerobic sludge. Conversely, the anaerobic sludge with 15 days of SRT had superior PHAs production capability compared to oxic sludge. These comparisons suggest that whether anaerobic or oxic sludge should be employed for PHAs production depends mainly on the operating SRT of the EBPR system.     

A new interpretation of ASM2d for modeling of SBR performance for enhanced biological phosphorus removal under different P/HAc ratios

This study evaluated the prediction capability of Activated Sludge Model No. 2d (ASM2d), for the enhanced biological phosphorus removal (EBPR) performance of a sequencing batch reactor (SBR) receiving variable influent phosphate load. For this purpose, a laboratory-scale SBR was operated with a synthetic feed containing acetate as the sole carbon source. The experiments were conducted in four different Runs to ensure a range of different phosphate/acetate ratios in the influent. Model evaluations were carried out using concentration profiles measured throughout a representative cycle at steady state. An iterative calibration methodology was developed based on sensitivity analysis and applied to four different sets of experimental data on relevant model parameters reflecting SBR performance. ASM2d was able to predict the steady state behavior of the SBR system receiving variable influent phosphate loads only with the recalibrated parameter set. The regular changing pattern of the coefficients could be interpreted with the ability of the SBR system to sustain glycogen accumulating microorganisms, GAOs, which can store substrate under anaerobic conditions without polyphosphate energy, but deriving energy from the degradation of glycogen. Thus they are capable of prevailing at lower P/Ac ratios. The results indicate the need to include glycogen and GAOs as model components for processes involving both phosphate accumulating organisms, (PAOs) and GAOs, in order to obtain a better prediction of X(PHA) and oxygen uptake rate (OUR) profiles in the system.