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.     

Phosphate removal under denitrifying conditions by Brachymonas sp. strain P12 and Paracoccus denitrificans PP15

In this study, we used the denitrifying phosphorus-removing bacterium Brachymonas sp. strain P12 to investigate the enhanced biologic phosphorus-removal (EBPR) mechanism involved with polyhydroxybutyrate (PHB), glycogen, and phosphorus uptake in the presence of acetate under anoxic or aerobic conditions. The results showed that excess acetate concentration and aerobic cultivation can enhance PHB formation efficiency and that PHB formation might be stimulated by glycogenolysis of the cellular glycogen. The efficiency of the uptake of anoxic phosphorus was greater when PHB production was lower. The EBPR mechanism of Brachymonas sp. strain P12 for PHB, phosphorus, and glycogen was similar to the conventional anaerobic-aerobic (or anaerobic-anoxic) EBPR models, but these models were developed under anoxic or aerobic conditions only, without an anaerobic stage. The anoxic or aerobic log phase of growth is divided into two main phases: the early log phase, in which acetate and glycogen are consumed to supply enough energy and reducing power for PHB formation and cell growth (phosphorus assimilation), and the late log phase, which ends the simultaneous degradation of PHB and remaining acetate for polyphosphate accumulation. Glycogenolysis plays a significant role in the alternate responses between PHB formation and phosphorus uptake under anoxic or aerobic conditions. After the application of the denitrifying phosphorus-removing bacterium Brachymonas sp. strain P12, aerobic cultivation increases the level of PHB production, and anoxic cultivation further increases phosphorus uptake.     

Metabolic model for glycogen-accumulating organisms in anaerobic/aerobic activated sludge systems

Glycogen-accumulating organisms (GAO) have the potential to directly compete with polyphosphate-accumulating organisms (PAO) in EBPR systems as both are able to take up VFA anaerobically and grow on the intracellular storage products aerobically. Under anaerobic conditions GAO hydrolyse glycogen to gain energy and reducing equivalents to take up VFA and to synthesise polyhydroxyalkanoate (PHA). In the subsequent aerobic stage, PHA is being oxidised to gain energy for glycogen replenishment (from PHA) and for cell growth. This article describes a complete anaerobic and aerobic model for GAO based on the understanding of their metabolic pathways. The anaerobic model has been developed and reported previously, while the aerobic metabolic model was developed in this study. It is based on the assumption that acetyl-CoA and propionyl-CoA go through the catabolic and anabolic processes independently. Experimental validation shows that the integrated model can predict the anaerobic and aerobic results very well. It was found in this study that at pH 7 the maximum acetate uptake rate of GAO was slower than that reported for PAO in the anaerobic stage. On the other hand, the net biomass production per C-mol acetate added is about 9% higher for GAO than for PAO. This would indicate that PAO and GAO each have certain competitive advantages during different parts of the anaerobic/aerobic process cycle.     

Metabolism of micro-organisms responsible for enhanced biological phosphorus removal from wastewater, Use of dynamic enrichment cultures

The removal of phosphorus from wastewater is already widely applied. In many cases use is made of micro organisms capable of accumulating phosphorus as polyphosphate inside the cell. The main characteristic providing the competitive advantage to these polyphosphate accumulating bacteria is the capability to use polyphosphate, in the absence of external electron acceptors, as energy source for the uptake and storage of acetic acid in the form of polyhydroxybutyrate (PHB). The reduction equivalents for the formation of PHB are derived from the conversion of glycogen to PHB. Despite the widespread use and study of enhanced biological phosphorus removal no pure culture, having the above mentioned characteristics, has been isolated yet. All ecophysiological studies on these type of cultures have therefore been performed by enrichment cultures. This paper reviews the research on these type of organisms, and shows that it is possible to understand a complex microbial process on a metabolic level, both stoichiometrically and kinetically, without the availability of a pure culture.     

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.     

Stoichiometry and kinetics of acetate uptake under anaerobic conditions by an enriched culture of phosphorus-accumulating organisms at different pHs

The effect of pH on the stoichiometry and kinetics of acetate uptake by phosphorus-accumulating organisms (PAOs) was studied. The stoichiometry of glycogen consumption and poly-beta-hydroxy-alkanoates (PHA) accumulation was independent of the pH over the range 6.5 to 8.0. It was again demonstrated that the amount of phosphorus released per acetate taken up (P/Hac ratio) was linearly dependent on pH, because of additional energy requirements for acetate transport at higher pH. The slope of this relationship was similar to that in previously published work, but the absolute values were different, indicating that the P/Hac ratio is the most variable stoichiometric parameter associated with the anaerobic metabolism of PAOs. A kinetic expression for acetate-uptake rate was developed and tested. It assumes a zero-order form when the polyphosphate content of the biomass is not limiting. When the polyphosphate content becomes low, the rate is significantly decreased. The expression was tested in situations in which polyphosphate was a limiting factor in the rate of acetate uptake, in which the glycogen content of the biomass became very low, and in which both glycogen and polyphosphate were present in excess. The model was able to simulate the three situations adequately. Additionally, the rate of acetate uptake was independent of the pH for the range studied (6.5 to 8.0).     

Energetics of Anaerobic Sodium Transport by the Fresh Water Turtle Bladder

Certain of the metabolic events associated with anaerobic sodium transport by the isolated bladder of the fresh water turtle have been investigated. The data suggest that energy for this transport arises from glycolysis and that endogenous glycogen was the major and perhaps the sole source of substrate. The rate of anaerobic glycolysis, as determined by lactate formation, correlates well with the rate as determined by glycogen utilization. Using lactate formation as the index of anaerobic glycolysis, a linear relationship was observed between glycolysis and net anaerobic sodium transport. In the absence of sodium transport, glycolysis decreased by approximately 45 per cent. Tissue ATP concentrations were maintained at about the same level under anaerobic as under aerobic conditions. Finally if it is assumed that in the conversion of glycogen to lactate anaerobically, 3 moles of ATP are generated per mole of glucose residue, an average of over 15 equivalents of sodium were transported for every mole of ATP generated.     

Transformation of phosphorus and relevant intracellular compounds by a phosphorus-accumulating enrichment culture in the presence of both the electron acceptor and electron donor

This study was conducted to obtain a better insight into the metabolic behavior of denitrifying phosphate-accumulating organisms relative to the transformations of relevant intracellular compounds as well as phosphorus and nitrate for enhanced biological phosphorus removal under different combinations of electron acceptor (oxygen or nitrate) and electron donor (acetate). Under anoxic conditions, the amount of polyhydroxybutyrate (PHB) produced per acetate taken up considerably increased with the increasing amount of nitrate reduced whereas the amounts of nitrate reduced and phosphorus released per acetate taken up remained almost constant. However, glycogen utilization occurred during PHB production and then was again observed in response to the initial supplementation of acetate after glycogen accumulation was transiently observed during anoxic phosphorus uptake using nitrate as an electron acceptor. On the other hand, under subsequent aerobic conditions, the additional supplementation of acetate again caused aerobic phosphorus release and PHB production, which showed that PHB production was associated with polyphosphate cleavage regardless of electron acceptor conditions. In contrast to anoxic conditions, glycogen accumulation was observed during PHB production. Based on these observations, the preliminary model for the metabolic behavior of denitrifying phosphate-accumulating organisms was proposed and could well account for the complex transformations of PHB and glycogen together with phosphorus release in the presence of acetate under different electron acceptors.     

Aerobic phosphorus release linked to acetate uptake in bio-P sludge: process modeling using oxygen uptake rate

The main processes involved in enhanced biological phosphorus removal (EBPR) under anaerobic and subsequently aerobic conditions are widely described in the literature. Polyphosphate accumulating organisms (PAO) are the organisms responsible for this process. However, the mechanisms of PAO are not fully established yet under conditions that differ from the classical anaerobic/aerobic conditions. In this work, we made a comparison between the behavior of PAO under classical EBPR conditions and its behavior when consuming substrate under only aerobic conditions. In addition, oxygen uptake rate (OUR) was measured in the set of experiments under aerobic conditions to improve the characterization of the process. A kinetic and stoichiometric model based on Activated Sludge Model No.2 (ASM2) and including glycogen economy (AnOx model), calibrated for classical anaerobic/aerobic conditions, was not able to describe the experimental data since it underestimated the acetate consumption, the PHB storage, and the OUR. Two different hypotheses for describing the experimental measurements were proposed and modeled. Both hypotheses considered that PAO, under aerobic conditions, uptake acetate coupled to PHB storage, glycogen degradation, and phosphorus release as in anaerobic conditions. Moreover, the first hypothesis (PAO-hypothesis) considered that PAO were able to store acetate as PHB linked to oxygen consumption and the second one (OHO hypothesis) considered that this storage was due to ordinary heterotrophic organisms (OHO). Both hypotheses were evaluated by simulation extending the AnOx model with additional equations. The main differences observed were the predictions for PHB degradation during the famine phase and the OUR profile during both feast and famine phases. The OHO hypothesis described the experimental profiles more accurately than the PAO hypothesis.     

The effect of dissolved oxygen on PHB accumulation in activated sludge cultures

Nitrogen removal from wastewater is often limited by the availability of reducing power to perform denitrification, especially when treating wastewaters with a low carbon:nitrogen ratio. In the increasingly popular sequencing batch reactor (SBR), bacteria have the opportunity to preserve reducing power from incoming chemical oxygen demand (COD) as poly-beta-hydroxybutyrate (PHB). The current study uses laboratory experiments and mathematical modeling in an attempt to generate a better understanding of the effect of oxygen on microbial conversion of COD into PHB. Results from a laboratory SBR with acetate as the organic carbon source showed that the aerobic acetate uptake process was oxygen-dependent, producing higher uptake rates at higher dissolved oxygen (DO) supply rates. However, at the lower DO supply rates (k(L)a 6 to 16 h(-1), 0 mg L(-1) DO), a higher proportion of the substrate was preserved as PHB than at higher DO supply rates (k(L)a 30, 51 h(-1), DO >0.9 mg L(-1)). Up to 77% of the reducing equivalents available from acetate were converted to PHB under oxygen limitation (Y(PHB/Ac) 0.68 Cmol/Cmol), as opposed to only 54% under oxygen-excess conditions (Y(PHB/Ac) 0.48 Cmol/Cmol), where a higher fraction of acetate was used for biomass growth. It was calculated that, by oxygen management during the feast phase, the amount of PHB preserved (1.4 Cmmol L(-1) PHB) accounted for an additional denitrification potential of up to 18 mg L(-1) nitrate-nitrogen. The trends of the effect of oxygen (and hence ATP availability) on PHB accumulation could be reproduced by the simulation model, which was based on biochemical stoichiometry and maximum rates obtained from experiments. Simulated data showed that, at low DO concentrations, the limited availability of adenosine triphosphate (ATP) prevented significant biomass growth and most ATP was used for acetate transport into the cell. In contrast, high DO supply rates provided surplus ATP and hence higher growth rates, resulting in decreased PHB yields. The results suggest that oxygen management is crucial to conserving reducing power during the feast phase of SBR operation, as excessive aeration rates decrease the PHB yield and allow higher biomass growth.     

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.     

Xylitol formation and reduction equivalent generation during anaerobic xylose conversion with glucose as cosubstrate in recombinant Saccharomyces cerevisiae expressing the xyl1 gene.

Glucose was used as a cosubstrate under anaerobic conditions in the conversion of xylose to xylitol by a recombinant Saccharomyces cerevisiae strain expressing the xyl1 gene. Glucose was metabolized mainly through glycolysis, with carbon dioxide, acetate, and ethanol as end products and with reduction equivalents generated in the glyceraldehyde-3-phosphate dehydrogenase and acetaldehyde dehydrogenase reactions. At a high glucose supply rate, generation of surplus reduction equivalents resulted in simultaneous ethanol formation. On the other hand, at a low glucose supply rate, additional reduction equivalents were generated by simultaneous ethanol consumption. A significantly lower xylitol formation rate was observed.     

A metabolic model of the biological phosphorus removal process: I. Effect of the sludge retention time

The biological phosphorus removal process is a process which depends basically on three internal storage compounds. Poly-beta-hydroxybutyrate (PHB) produced during the anaerobic phase is used as substrate for biomass, polyphosphate, and glycogen formation. The reaction rates of the aerobic processes are primarily determined by the PHB content of the cells. This PHB content is highly dynamic due to the conversions during the anaerobic and aerobic phase of the cycle and the ratio between substrate addition and biomass present in the reactor. The amount of biomass present in the reactor is determined by the sludge retention time and growth rate. A metabolic model of the biological phosphorus removal process was developed and verified over a wide range of growth rates. The effect of different growth rates on the internal fractions of stored components was determined and described mathematically. One set of kinetic parameters was capable of describing the measured conversions of all components observed in the reactor as a function of the sludge retention time. (c) 1995 John Wiley & Sons, Inc.     

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.     

Aerobic and anaerobic metabolism of the freshwater oligochaete Tubifex sp.

The freshwater oligochaete Tubifex shows several mechanisms of metabolic adaptations, enabling the worms to occupy saprobial habitats of extremely variable oxygen content. Under normoxic conditions the metabolism of the worms is mainly aerobic with a respiratory ratio of 0.7. Under hypoxic conditions, metabolism of energy sources via aerobic and anaerobic pathways is observed. During complete anoxia acetate and propionate are the main products of glycogen degradation and they are excreted in constant rates into the water. A retransfer of the worms to aerobic conditions enables them to regain aerobic metabolic state within about 60 min.In two Tubifex habitats, which we have characterized, concentrations of dissolved organic material (DOM) were low in the surface water, but high in the interstitial water from sediments. The short-chain fatty acids acetate and propionate reached concentrations up to 1 mmole/liter. Employing radioisotope techniques, we demonstrated that Tubifex can achieve an integumentary uptake of acetate and propionate from artificial tap water at naturally occurring concentrations of 5 to 1000 M. Levels of uptake (600 to 800 nmoles/g wwt.hr) and transport characteristics are very similar to those of marine invertebrates associated with detritus rich sediments. The uptake is susceptible to inhibition by structurally analogous compounds and to metabolic inhibition. Furthermore, DOM uptake in Tubifex is susceptible to inhibition by oxygen depletion, ouabain and Na⁺-depletion. The results may suggest that a carrier system for DOM transport exists in the integument of the worms. The uptake system is highly specific for aliphatic C2 and C3 carboxylic acids. The absorbed volatile fatty acids are rapidly metabolized. Only 15 min after absorption, a considerable amount of radioactivity is present in the glycogen storage of the animals. Depending on the substrate concentration assumed to be available for uptake, up to 40 per cent of the oxidative requirement of the worms may be attained by using dissolved organic material from the interstitial water of their habitat.Supported by the Deutsche Forschungsgemeinschaft (Ho 631/9-9).     

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.     

Anaerobic phosphate release from activated sludge with enhanced biological phosphorus removal. A possible mechanism of intracellular pH control

The biochemical mechanisms of the wastewater treatment process known as enhanced biological phosphorus removal (EBPR) are presently described in a metabolic model. We investigated details of the EBPR model to determine the nature of the anaerobic phosphate release and how this may be metabolically associated with polyhydroxyalkanoate (PHA) formation. Iodoacetate, an inhibitor of glycolysis, was found to inhibit the anaerobic formation of PHA and phosphate release, supporting the pathways proposed in the EBPR metabolic model. In the metabolic model, it is proposed that polyphosphate degradation provides energy for the microorganisms in anaerobic regions of these treatment systems. Other investigations have shown that anaerobic phosphate release depends on the extracellular pH. We observed that when the intracellular pH of EBPR sludge was raised, substantial anaerobic phosphate release was caused without volatile fatty acid (VFA) uptake. Acidification of the sludge inhibited anaerobic phosphate release even in the presence of VFA. From these observations, we postulate that an additional possible role of anaerobic polyphosphate degradation in EBPR is for intracellular pH control. Intracellular pH control may be a metabolic feature of EBPR, not previously considered, that could have some use in the control and optimisation of EBPR. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 507–515, 1999.     

Batch production of polyhydroxyalkanoate by low-polyphosphate-content activated sludge at varying pH

Polyhydroxyalkanoate (PHA), a biodegradable plastic, can be produced from excess activated sludge by utilizing intracellular glycogen and polyphosphate as energy sources under growth-limiting conditions. Activated sludge of 2%, 6%, and 8% polyphosphate with similar glycogen content of 33% was investigated for batch PHA production by varying the pH values from 6 to 8. Acetate applied at 1000 mg COD/L was almost exhausted within 80 min of anaerobic stage. The remaining glycogen in the sludge was higher at a lower pH because of less energy used for acetate uptake. Highest PHA content of 51% was obtained from sludge with an 8% polyphosphate content at pH 8. PHA production occurred rapidly within the first 20 min, with a productivity rate of 2.19 g PHA/L-h. The results in this study indicate that PHA production by using activated sludge is a promising alternative to a typical pure culture approach.     

Characterizing the biochemical activity of full-scale enhanced biological phosphorus removal systems: A comparison with metabolic models

The metabolism of polyphosphate accumulating organisms (PAOs) has been widely studied through the use of lab-scale enrichments. Various metabolic models have been formulated, based on the results from lab-scale experiments using enriched PAO cultures. A comparison between the anaerobic stoichiometry predicted by metabolic models with that exhibited by full-scale sludge in enhanced biological phosphorus removal (EBPR) wastewater treatment plants (WWTPs) was performed in this study. Batch experiments were carried out with either acetate or propionate as the sole carbon source, using sludges from two different EBPR-WWTPs in Australia that achieved different phosphorus removal performances. The results support the hypothesis that the anaerobic degradation of glycogen is the primary source of reducing equivalents generated by PAOs, however, they also suggested a partial contribution of the tricarboxylic acid (TCA) cycle in some cases. The experimental results obtained when acetate was the carbon source suggest the involvement of the modified succinate-propionate pathway for the generation of poly-beta-hydroxyvalerate (PHV). Overall, the batch test results obtained from full-scale EBPR sludge with both substrates were generally well described by metabolic model predictions for PAOs.