Incorporating microbial ecology into the metabolic modelling of polyphosphate accumulating organisms and glycogen accumulating organisms

In the enhanced biological phosphorus removal (EBPR) process, the competition between polyphosphate accumulating organisms (PAO) and glycogen accumulating organisms (GAO) has been studied intensively in recent years by both microbiologists and engineers, due to its important effects on phosphorus removal performance and efficiency. This study addresses the impact of microbial ecology on assessing the PAO-GAO competition through metabolic modelling, focussing on reviewing recent developments, discussion of how the results from molecular studies can impact the way we model the process, and offering perspectives for future research opportunities based on unanswered questions concerning PAO and GAO metabolism. Indeed, numerous findings that are seemingly contradictory could in fact be explained by the metabolic behaviour of different sub-groups of PAOs and/or GAOs exposed to different environmental and operational conditions. Some examples include the glycolysis pathway (i.e. Embden-Meyerhof-Parnas (EMP) vs. Entner-Doudoroff (ED)), denitrification capacity, anaerobic tricarboxylic acid (TCA) cycle activity and PAOs’ ability to adjust their metabolism to e.g. a GAO-like metabolism. Metabolic modelling may further yield far-reaching influences on practical applications as well, and serves as a bridge between molecular/biochemical research studies and the optimisation of wastewater treatment plant operation.     

Involvement of the TCA cycle in the anaerobic metabolism of polyphosphate accumulating organisms (PAOs)

For decades, glycolysis has been generally accepted to supply the reducing power for the anaerobic conversion of volatile fatty acids (VFAs) to polyhydroxyalkanoates (PHAs) by polyphosphate accumulating organisms (PAOs). However, the importance of the tricarboxylic acid (TCA) cycle has also been raised since 1980s. The aim of this study is to demonstrate the involvement of the TCA cycle in the anaerobic metabolism of PAOs. To achieve this goal, the glycogen pool of an activated sludge highly enriched in Candidatus Accumulibacter Phosphatis (hereafter referred to as Accumulibacter), a putative PAO was reduced substantially through starving the sludge under intermittent anaerobic and aerobic conditions. After the starvation, acetate added was still taken up anaerobically and stored as PHA, with negligible glycogen degradation. The metabolic models proposed by Pereira, Hesselmann and Yagci, which predict the formation of reducing power through glycolysis and the full or partial TCA cycle, were used to estimate the carbon fluxes. The results demonstrate that Accumulibacter can use both glycogen and acetate to generate reducing power anaerobically. The anaerobic production of reducing power from acetate is likely through the full TCA cycle. The proportion of TCA cycle involvement depends on the availability of degradable glycogen.     

Temperature effects on glycogen accumulating organisms

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

Obtaining highly enriched cultures of Candidatus Accumulibacter phosphates through alternating carbon sources

Candidatus Accumulibacter Phosphatis is widely considered to be a polyphosphate accumulating organism (PAO) of prime importance in enhanced biological phosphorus removal (EBPR) systems. This organism has yet to be isolated, despite many attempts. Previous studies on the biochemical and physiological aspects of this organism, as well as its response to different EBPR operational conditions, have generally relied on the use of mixed culture enrichments. One frequent problem in obtaining highly enriched cultures of this organism is the proliferation of glycogen accumulating organisms (GAO) that can compete with PAOs for limited carbon sources under similar operational conditions. In this study, Candidatus Accumulibacter Phosphatis has been enriched in a lab-scale bioreactor to a level greater than 90% as quantified by fluorescence in situ hyrbridisation (FISH). This is the highest enrichment of this organism that has been reported thus far, and was obtained by alternating the sole carbon source in the feed between acetate and propionate every one to two sludge ages, and operating the bioreactor within a pH range of 7.0–8.0. Simultaneously, the presence of two known groups of GAOs was eliminated under these operational conditions. Excellent phosphorus removal performance and stability were maintained in this system, where the phosphorous concentration in the effluent was below 0.2 mg/L for more than 7 months. When a disturbance was introduced to this system by adding sludge from an enriched GAO culture, Candidatus Accumulibacter Phosphatis once again became highly enriched, while the GAOs were out-competed. This feeding strategy is recommended for future studies focused on describing the physiology and biochemistry of Accumulibacter, where a highly-enriched culture of this organism is of high importance.     

In situ identification and characterization of the microbial community structure of full-scale enhanced biological phosphorous removal plants in Japan

Fluorescent in situ hybridization (FISH) and polyphosphate (polyP) staining methods were used to characterize the microbial community structure of 13 activated sludge samples taken from nine different Japanese wastewater treatment plants with and without enhanced biological phosphorous removal (EBPR) activities. FISH with published rRNA-targeted oligonucleotide probes for important bacterial groups involving in the EBPR process revealed that Rhodocyclus-related polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms from a gammaproteobacterial lineage GB were the predominant populations detected, representing 4-18% and 10-31% of EUBmix-stained cells, respectively, in those samples. However, a considerable proportion of Rhodocyclus-related PAO cells were observed with no polyP granules accumulated based on polyP staining. This was further supported by a poor correlation between Rhodocyclus-related PAO population and sludge total phosphorous (TP) contents. In contrast, high correlations between polyP-stained cells and sludge TP contents were observed. In particular, among those polyP-stained cells in samples Ariake_A2O and Nakano_AO, more than 85% of them could not be targeted by probe PAOmix. These non-Rhodocyclus-related PAOs included populations from other bacterial divisions and members of the Betaproteobacteria other than those in Rhodocyclus-related group.     

亚硝酸盐对聚磷菌厌氧代谢的影响

以2种强化生物除磷(EBPR)系统中的活性污泥为研究对象,考察亚硝酸盐对聚磷菌厌氧代谢的影响,结果表明：不同EBPR系统中的聚磷菌对于亚硝酸盐的耐受能力不同。人工配水富集聚磷菌的活性污泥,当亚硝态氮浓度超过10 mg/L时,聚磷菌吸收VFA受到抑制, PHA的合成减少,磷酸盐的释放增加;处理生活污水的SBR短程脱氮除磷活性污泥,亚硝酸盐的浓度高达30 mg/L时,未对聚磷菌的厌氧代谢造成抑制,但引起异养反硝化菌与聚磷菌竞争VFA,导致PHA合成量和释磷量的减少。富集聚磷菌的活性污泥投加亚硝酸盐后P/VFA     

污水强化生物除磷的生化模型研究进展

阐述了在污水强化生物除磷过程中生化模型的建立与发展,重点介绍了与聚磷菌有关的Mino模型,强调了在理解生化模型时应该注意的问题：所有聚磷菌生化模型都只是在试图描述发生在EBPR（Enhanced Biological Phosphorus Removal）生物群中的一些生物化学变化现象和规律（多聚磷酸盐的吸收和水解、PHA的合成与再利用）.另外,还都假设所有具有EBPR能力的细菌都有着共同的代谢特征.所以,尽管这些生化模型有助于对整个EBPR过程的理解,但不应该拘泥于这些生化模型,只有通过对聚磷菌纯培养获得最终成功才能够彻底解决整个问题.     

Advancing post-anoxic denitrification for biological nutrient removal

The objective of this research was to advance a fundamental understanding of a unique post-anoxic denitrification process for achieving biological nutrient removal (BNR), with an emphasis on elucidating the impacts of surface oxygen transfer (SOT), variable process loadings, and bioreactor operational conditions on nitrogen and phosphorus removal. Two sequencing batch reactors (SBRs) were operated in an anaerobic/aerobic/anoxic mode for over 250 days and fed real municipal wastewater. One SBR was operated with a headspace open to the atmosphere, while the other had a covered liquid surface to prevent surface oxygen transfer. Process performance was assessed for mixed volatile fatty acid (VFA) and acetate-dominated substrate, as well as daily/seasonal variance in influent phosphorus and ammonia loadings. Results demonstrated that post-anoxic BNR can achieve near-complete (>99%) inorganic nitrogen and phosphorus removal, with soluble effluent concentrations less than 1.0 mgN L⁻¹ and 0.14 mgP L⁻¹. Observed specific denitrification rates were in excess of typical endogenous values and exhibited a linear dependence on the glycogen concentration in the biomass. Preventing SOT improved nitrogen removal but had little impact on phosphorus removal under normal loading conditions. However, during periods of low influent ammonia, the covered reactor maintained phosphorus removal performance and showed a greater relative abundance of polyphosphate accumulating organisms (PAOs) as evidenced by quantitative real-time PCR (qPCR). While GAOs were detected in both reactors under all operational conditions, BNR performance was not adversely impacted. Finally, secondary phosphorus release during the post-anoxic period was minimal and only occurred if nitrate/nitrite were depleted post-anoxically.     

Effect of nitrite on phosphate uptake by phosphate accumulating organisms

In biological nitrogen removal processes, nitrite can be formed and accumulated through both nitrification and denitrification. Despite the fact that, in practice, biological phosphate removal (BPR) is often combined with biological nitrogen removal, there are only a few publications reporting the effect of nitrite on BPR. In this study, phosphate-accumulating organisms (PAO) were cultivated in an anaerobic-anoxic-aerobic sequencing batch reactor (SBR). The effect of nitrite on the enrichment of the sludge with PAO, the phosphate uptake rates and the sludge respiration was investigated. The results indicate that (1) presence of nitrite inhibits both aerobic and anoxic (denitrifying) phosphate uptake, (2) aerobic phosphate uptake was more affected than anoxic phosphate uptake, (3) presence of nitrite could be one of the factors enhancing the presence of glycogen accumulating organisms (GAO)--competitors to PAO for substrate in the anaerobic phase, and (4) it is required to monitor and control nitrite accumulation in a full-scale wastewater treatment plants.     

A novel post denitrification configuration for phosphorus recovery using polyphosphate accumulating organisms

Enhanced biological phosphorus removal (EBPR) has been widely used to remove phosphorus (P) from wastewater. In this study we report a novel modification to the EBPR approach, namely enhanced biological phosphorus removal and recovery (EBPR-r) that facilitates biological recovery of P from wastewater using a post denitrification configuration. The novel approach consists of two major steps. In the first step, a biofilm of phosphorus accumulating organisms (PAOs) is exposed to a wastewater stream in the absence of active aeration, during which P is taken up by the biofilm using nitrate and residual dissolved oxygen as electron acceptors. Thus, P and nitrogen (N) removal from wastewater is achieved. During the second step, the P enriched biofilm is exposed to a smaller recovery stream supplemented with an external carbon source to facilitate P release under anaerobic conditions. This allows P to be recovered as a concentrated liquid. The EBPR-r process was able to generate a P recovery stream four time more concentrated (28 mg-P/L) than the wastewater stream (7 mg-P/L), while removing nitrate (denitrification) from the wastewater stream. Repeated exposure of the biofilm (10 P-uptake and release cycles) to a recovery stream yielded up to 100 mg-P/L. Overall, EBPR-r is the first post denitrification strategy that can also facilitate P recovery during secondary wastewater treatment.     

Could polyphosphate-accumulating organisms (PAOs) be glycogen-accumulating organisms (GAOs)?

Polyphosphate (poly-P) is known to be a key compound in the metabolism of polyphosphate-accumulating organisms (PAOs). In this study, a sludge highly enriched (80%) in Candidatus Accumulibacter phosphatis (hereafter referred to as Accumulibacter), a widely known PAO, was used to study the ability of these microorganisms to utilize acetate anaerobically under poly-P-limiting conditions. The biomass was subject to several anaerobic and aerobic cycles, during which the poly-P pool of PAOs was gradually emptied by supplying feed deficient in phosphate and washing the biomass at the end of each anaerobic period using media containing no phosphorus. After three cycles, phosphorus was hardly released but PAOs were still able to take up acetate and stored it as polyhydroxyalkanoates (PHA), as demonstrated by post-FISH chemical staining. Glycogen degradation increased substantially, suggesting PAOs were using glycogen as the main energy source. This is a key feature of glycogen-accumulating organisms (GAOs), which are known to compete with PAOs in enhanced biological phosphorus removal (EBPR) systems. The ratios between acetate uptake, polyhydroxybutyrate (PHB) and polyhydroxyvalerate (PHV) production, and glycogen consumption agree well with the anaerobic models previously proposed for GAOs.     

Endogenous processes during long-term starvation in activated sludge performing enhanced biological phosphorus removal

In many biological wastewater treatment systems, bacterial growth and the amount of active biomass are limited by the availability of substrate. Under these low growth conditions, endogenous processes have a significant influence on the amount of active biomass and therefore, the overall system performance. In enhanced biological phosphorus removal (EBPR) systems endogenous processes can also influence the levels of the internal storage compounds of the polyphosphate accumulating organisms (PAO), directly affecting phosphorus removal performance. The purpose of this study was to evaluate the significance of different endogenous processes that occur during the long-term starvation of EBPR sludge under aerobic and anaerobic conditions. Activated sludge obtained from a laboratory sequencing batch reactor was used to perform a series of batch starvation experiments. Under aerobic starvation conditions we observed a significant decay of PAO (first-order decay rate of 0.15/d) together with a rapid utilization of polyhydroxyalkanoates (PHA) and a slower utilization of glycogen and polyphosphate to generate maintenance energy. On the other hand, anaerobic starvation was best described by maintenance processes that rapidly reduce the levels of polyphosphate and glycogen under starvation conditions while no significant decay of PAO was observed. The endogenous utilization of glycogen for maintenance purposes is currently not included in available EBPR models. Our experimental results suggest that mathematical models for in EBPR should differentiate between aerobic and anaerobic endogenous processes, as they influence active biomass and storage products differently.     

Model-based evaluation of competition between polyphosphate- and glycogen-accumulating organisms

Many studies show that glycogen-accumulating non-polyphosphate organisms (GAOs) can compete with polyphosphate-accumulating organisms (PAOs) for organic substrate under anaerobic conditions and may indeed cause the deterioration of enhanced biological phosphorus removal (EBPR) systems. Understanding their behaviors in an anaerobic/aerobic (A/O) system at different operational conditions is essential in developing control strategies that ensure EBPR. A model-based evaluation of competition between PAOs and GAOs under different operational conditions was presented in this study. At 30 degrees C and a 10-day sludge age, the dominance of GAOs in the A/O sequencing batch reactor (SBR) was strongly dependent upon their considerable kinetic advantage in anaerobic acetate uptake. At 20 degrees C and a 10-day sludge age, the kinetic advantage of GAOs in anaerobic acetate uptake could be less, compared to that at 30 degrees C and a 10-day sludge age, leading to the relative dominance of PAOs and a stable phosphorus removal in the A/O system. At 30 degrees C and a 3-day sludge age, the parameters responsible for determining the aerobic distribution of anaerobically stored X(PHA) for both PAOs and GAOs, other than kinetic parameters of anaerobic acetate uptake, are important for them being dominant in the A/O SBR. In a situation when the q(PHA,P) value is lower than q(PHA,G) but comparable, PAOs may still be dominant in the A/O SBR, presumably because their aerobic conversion fraction of biomass production from PHA was higher than that of the GAOs.     

The effect of pH on the competition between polyphosphate-accumulating organisms and glycogen-accumulating organisms

In enhanced biological phosphorus removal (EBPR) processes, glycogen-accumulating organisms (GAOs) may compete with polyphosphate-accumulating organisms (PAOs) for the often-limited carbon substrates, potentially resulting in disturbances to phosphorus removal. A detailed investigation of the effect of pH on the competition between PAOs and GAOs is reported in this study. The results show that a high external pH ( approximately 8) provided PAOs with an advantage over GAOs in EBPR systems. The phosphorus removal performance improved due to a population shift favouring PAOs over GAOs, which was shown through both chemical and microbiological methods. Two lab-scale reactors fed with propionate as the carbon source were subjected to an increase in pH from 7 to 8. The phosphorus removal and PAO population (as measured by quantitative fluorescence in situ hybridisation analysis of "Candidatus Accumulibacter phosphatis") increased in each system, where the PAOs appeared to out-compete a group of Alphaproteobacteria GAOs. A considerable improvement in the P removal was also observed in an acetate fed reactor, where the GAO population (primarily "Candidatus Competibacter phosphatis") decreased substantially after a similar increase in the pH. The results from this study suggest that pH could be used as a control parameter to reduce the undesirable proliferation of GAOs and improve phosphorus removal in EBPR systems.     

新型双泥生物反硝化除磷脱氮工艺

在对生物脱氮与除磷机理进行深入研究后发现，生物脱氮与除磷是两个相对独立而又相互交叉的生理过程，其交叉点是部分聚磷菌在缺氧状态下的反硝化吸磷脱氮。在此基础上提出的新型双泥生物反硝化除磷脱氮工艺（由两个不同功能的SBR反应器组成）成功地解决了硝化菌与聚磷菌的泥龄之争。反硝化与聚磷菌厌氧释磷的矛盾等难题，该工艺运行稳定且处理效果良好，特别适合于处理BOD5/TP值低的污水。     

Changes in phosphorus removing performance and bacterial community structure in an enhanced biological phosphorus removal reactor

A lab-scale-enhanced biological phosphorus removal (EBPR) reactor was operated for 204 days to investigate the correlation between phosphorus removing performance and bacterial community structure. The phosphorus removing performance was good from day 1 to 92 and from day 172 to 204. However, the removal activity was in a deteriorated state from day 93 to 171. From day 69 (2 weeks before the beginning of the deterioration) to 118 (2 weeks after the beginning of the deterioration), sludge P content decreased. The amounts of ubiquinone-8 and menaquinone-8 (H(4)) decreased during this period while the amount of ubiquinone-10 increased. The comparison of these changes and the general attribution of each quinone to the bacterial phylogenetic groups suggested that beta proteobacteria and Actinobacteria contributed to EBPR positively, and that alpha proteobacteria were related to this EBPR deterioration. Glycogen accumulating organisms (GAOs) are considered to detrimentally affect EBPR ability by outcompeting the phosphorus accumulating organisms by using aerobically synthesized glycogen as the energy source to assimilate organic substrates anaerobically to form polyhydroxyalkanoates. However, in this research, there was nearly no substrate uptake during the anaerobic period at the middle of the deteriorated performance period. This suggests that the deterioration observed in this research does not agree with the GAOs inhibition model. In this research, the excess P release at the anaerobic period was concluded to cause the deterioration.     

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

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

The effect of free nitrous acid on the anabolic and catabolic processes of glycogen accumulating organisms

Nitrite/Free Nitrous Acid (FNA) has previously been shown to inhibit aerobic and anoxic phosphate uptake by polyphosphate accumulating organisms (PAOs). The inhibitory effect of FNA on the aerobic metabolism of Glycogen Accumulating Organisms (GAOs) is investigated. A culture highly enriched (92 ± 3%) in Candidatus Competibacter phosphatis (hereafter called Competibacter) was used. The experimental data strongly suggest that FNA likely directly inhibits the growth of Competibacter, with 50% inhibition occurring at 1.5 × 10⁻³ mgN-HNO₂/L (equivalent to approximately 6.3 mgN-NO₂⁻/L at pH 7.0). The inhibition is well described by an exponential function. The organisms ceased to grow at an FNA concentration of 7.1 × 10⁻³ mgN-HNO₂/L. At this FNA level, glycogen production, another anabolic process performed by GAOs in parallel to growth, decreased by 40%, while the consumption of polyhydroxyalkanoates (PHAs), the intracellular carbon and energy sources for GAOs, decreased by approximately 50%. FNA likely inhibited either or both of the PHA oxidation and glycogen production processes, but to a much less extent in comparison to the inhibition on growth. The comparison of these results with those previously reported on PAOs suggest that FNA has much stronger inhibitory effects on the aerobic metabolism of PAOs than on GAOs, and may thus provide a competitive advantage to GAOs over PAOs in enhanced biological phosphorus removal (EBPR) systems.     

Long-term impact of anaerobic reaction time on the performance and granular characteristics of granular denitrifying biological phosphorus removal systems

Removal of nitrogen and phosphorus (P) from wastewater is successfully and widely practiced in systems employing both granular sludge technology and enhanced biological P removal (EBPR) processes; however, the key parameter, anaerobic reaction time (AnRT), has not been thoroughly investigated. Successful EBPR is highly dependent on an appropriate AnRT, which induces carbon and polyphosphate metabolism by phosphorus accumulating organisms (PAOs). Therefore, the long-term impact of AnRT on denitrifying P removal performance and granular characteristics was investigated in three identical granular sludge sequencing batch reactors with AnRTs of 90 (R1), 120 (R2) and 150 min (R3). The microbial community structures and anaerobic stoichiometric parameters related to various AnRTs were monitored over time. Free nitrite acid (FNA) accumulation (e.g., 0.0008–0.0016 mg HNO₂–N/L) occurred frequently owing to incomplete denitrification in the adaptation period, especially in R3, which influenced the anaerobic/anoxic intracellular intermediate metabolites and activities of intracellular enzymes negatively, resulting in lower levels of poly-P and reduced activity of polyphosphate kinase. As a result, the Accumulibacter-PAOs population decreased from 51 ± 2.5% to 43 ± 2.1% when AnRT was extended from 90 to 150 min, leading to decreased denitrifying P removal performance. Additionally, frequent exposure of microorganisms to the FNA accumulation and anaerobic endogenous conditions in excess AnRT cases (e.g., 150 min) stimulated increased extracellular polymeric substances (EPS) production by microorganisms, resulting in enhanced granular formation and larger granules (size of 0.6–1.2 mm), but decreasing anaerobic PHA synthesis and glycogen hydrolysis. Phosphorus removal capacity was mediated to some extent by EPS adsorption in granular sludge systems that possessed more EPS, longer AnRT and relatively higher GAOs.