Metabolism Of Polyphosphate Accumulating Organisms Research Articles

Enhanced biological phosphorus removal systems adapting to seawater intrusion: Impacts of varying carbon source on metabolic adaptation in Candidatus Accumulibacter phosphatis

In enhanced biological phosphorus removal (EBPR) systems, seawater intrusion may introduce uncertainty into the metabolism of Polyphosphate Accumulating Organisms (PAOs), especially when volatile fatty acid (VFA) profiles derived from fermentation liquids serves as the carbon source. This study investigated the impacts of varying sea salt salinity levels on the metabolism of Accumulibacter with different VFA as carbon sources. As sea salt salinity increased from 0.5 % to 3.5 %, catabolism (phosphorus release, glycogen and PHA biodegradation) was inhibited to a much lower degree compared to anabolism (phosphorus uptake, glycogen and PHA synthesis). Under seawater conditions, the intracellular free Mg2+ was partially excreted, leading to a subsequent reduction in glycogen metabolism and PHA synthesis. The decrease in PHA synthesis resulted in the diminished aerobic metabolic activity and polyphosphatase (PPX) enzyme activity, collectively causing decreased phosphorus uptake. Moreover, additional energy consumption was used to alleviate osmotic stress, which limited the energy used for esterase activity, translation activity, and phosphorus and glycogen metabolism, resulting in a decrease in growth activity. When using butyrate as a carbon source, Accumulibacter exhibited the highest metabolic activity (49.8 %), making it more adaptable to seawater conditions. The clade IIF was the dominant Accumulibacter in seawater conditions and could synthesize more PHA with butyrate, isobutyrate and valerate as a carbon source, respectively, stabilizing aerobic metabolism. Therefore, fermentation liquid containing high concentrations of butyrate and the activated sludge dominated by clade IIF have the potential to enhance the stabilization of EBPR systems in wastewater treatment plants infiltrated by seawater.

The influence of carrier material on the dominance of polyphosphate-accumulating organism metabolism in the biofilm-based biological phosphorus removal process

This study reveals that the type of carrier material affects the stoichiometry of the anaerobic conversion and the metabolism in cultures enriched with polyphosphate-accumulating organisms (PAOs) and helps increase the biological phosphorus removal performance. PAO-enriched biofilms were cultivated in two identical lab-scale sequencing batch reactors containing different carrier types: a typical moving bed bioreactor carrier (MBBR) and a novel carbon-based moving carrier type (CBMC) coated by activated carbon particles. The experiments were conducted at various influent PO43- concentrations (14, 17, 20 mg-PO43--P/L) and two aeration periods (220 and 460 min) under similar operating conditions. The results showed that due to the particular characteristics of the highly porous CBMC carriers, biomass adhesion increased by approximately 15 % compared to the MBBR carriers. It was also found that the CBMC carrier biofilm relied more on intracellularly stored polyphosphate than glycogen (which could be used by glycogen accumulating organisms (GAO)) as a source of energy for anaerobic uptake of organic substrates, which resulted in a greater dominance of PAO metabolism within the biofilm, especially at lower P concentrations. Conversely, a microbial community with mixed PAO-GAO metabolism was observed within the MBBR carrier biofilm, which changed to PAO dominated metabolism with increasing P concentration. Therefore, a higher specific PO43- uptake rate was obtained for the CBMC reactor (0.072–0.082 P-mol/C-mol.h), indicating a significant enhancement in the range of 13.1–19.6 % compared to the MBBR reactor, depending on the influent P concentration. In addition, better resistance of the CBMC biofilm to prolonged aeration corroborated that although the highly porous structure of the CBMC carriers was effective in achieving a higher P-removal efficiency by 13.5 %, the carrier material still played the major role in PAO metabolism dominance.

Accumulibacter diversity at the sub-clade level impacts enhanced biological phosphorus removal performance

Accumulibacter is a well-known group of organisms, typically considered to be polyphosphate accumulating organisms (PAOs), but potentially capable of glycogen accumulating organism (GAO) metabolism under limiting influent phosphate levels. Metabolic features of Accumulibacter are typically linked to its phylogenetic identity at the Type or clade level, though it is unclear the extent to which Accumulibacter diversity can correlate with its capacity to perform P removal. This paper investigates the fine-scale diversity of Accumulibacter and its link with enhanced biological phosphorus removal (EBPR) performance under various operating conditions, to understand the conditions and community structure leading to successful and unsuccessful EBPR operation. For this purpose, the organic carbon feeding rate and total organic carbon concentration were varied during three distinct operational periods, where influent phosphate was never limiting. Accumulibacter was always the dominant microbial group (>80% of all bacteria according to quantitative fluorescence in situ hybridisation - FISH) and low levels of Competibacter and other GAOs were consistently observed (<15% of all bacteria). Steady state was achieved in each of the three periods, with average phosphorus removal levels of 36%, 99% and >99%, respectively. Experimentally determined stoichiometric activity supported the expression of a mixed PAO/GAO metabolism in the first steady state period and the typical PAO metabolism in the other two steady state periods. FISH quantification and amplicon sequencing of the polyphosphate kinase (ppk1) functional gene indicated that Accumulibacter clade IIC was selected in the first steady state period, which shifted to clade IA after decreasing the carbon feeding rate in steady state period 2, and finally shifted back to clade IIC in the third steady state period. Fine-resolution Ppk-based phylogenetic analysis revealed three different clusters within Accumulibacter clade IIC, where clusters IICii and IICiii were linked to poor EBPR performance in period 1, and cluster IICi was linked to good EBPR performance in period 3. This study shows that the deterioration of EBPR processes through GAO activity at non-limiting P concentrations can be linked to organisms that are typically classified as PAOs, not only to known GAOs such as Competibacter. Intra-clade phylogenetic diversity within Accumulibacter showed that some clusters actually behave similarly to GAOs even without influent phosphate limitation. This study highlights the need to closely re-examine traditional interpretations regarding the link between the microbial community composition and identity with the performance and metabolism of EBPR systems.

Widespread detection of Candidatus Accumulibacter phosphatis, a polyphosphate-accumulating organism, in sediments of the Columbia River estuary.

Enhanced biological phosphorus removal (EBPR) exploits the metabolism of polyphosphate-accumulating organisms (PAOs) to remove excess phosphorus (P) from wastewater treatment. Candidatus Accumulibacter phosphatis (Accumulibacter) is the most abundant and well-studied PAO in EBPR systems. In a previous study, we detected polyphosphates throughout peripheral bay sediments, and hypothesized that an estuary is an ideal setting to evaluate PAOs in a natural system, given that estuaries are characterized by dynamic dissolved oxygen fluctuations that potentially favour PAO metabolism. We detected nucleotide sequences attributable to Accumulibacter (16S rRNA, ppk1) in sediments within three peripheral bays of the Columbia River estuary at abundances rivalling those observed in conventional wastewater treatment plants (0.01%-2.6%). Most of the sequences attributable to Accumulibacter were Type I rather than Type II, despite the fact that the estuary does not have particularly high nutrient concentrations. The highest diversity of Accumulibacter was observed in oligohaline peripheral bays, while the greatest abundances were observed at the mouth of the estuary in mesohaline sediments in the spring and summer. In addition, an approximately 70% increase in polyphosphate concentrations observed at one of the sites between dawn and dusk suggests that PAOs may play an important role in P cycling in estuary sediments.

Sulfide effects on the anaerobic metabolism of polyphosphate-accumulating organisms

Sulfate rich wastewaters can be generated from industry, use of seawater in urban environments, or by saline water infiltration into the sewerage. Under anaerobic conditions sulfate can be converted to sulfide, which may affect micro-organisms performing biological nutrient removal. The objective of this study was to evaluate the effect of sulfide on the activity of polyphosphate-accumulating organisms (PAO) in the anaerobic stage of the enhanced biological phosphorus removal process (EBPR). In this regard, a highly enriched culture of PAO was exposed in short-term activity tests to a range of sulfide concentrations at different operational pH values. The PAO activity was mainly affected by un-dissociated H2S. The specific acetate uptake rate was inhibited by 50% at around 60mg H2S.L−1. With increasing H2S concentrations, higher phosphate release rate to acetate uptake rate ratios were observed, possibly due to increased energy requirements for cell detoxification. Mathematical expressions were developed, which satisfactorily described the sulfide effects on the acetate uptake rate and phosphate release rate. The results show that, dependent on the pH, EBPR might be negatively affected by total sulfide concentrations exceeding 275mg SO4.L−1 at pH 6.5 or 1200mg SO4.L−1 at pH 7.8mg SO4.L−1 or when freshwater is partially replaced by seawater more than 45% (pH 7.8) or 10% (pH 6.5) used as secondary quality water. The findings of this study imply that sulfide, which is commonly found in different type of wastewaters, affects the anaerobic metabolism of PAO and may play an important role in the process performance of treatment plants treating wastewaters with high sulfide content.

Prevalence of 'Candidatus Accumulibacter phosphatis' type II under phosphate limiting conditions.

P-limitation in enhanced biological phosphorus removal (EBPR) systems fed with acetate, has generally been considered as a condition leading to enrichment of organisms of the genotype’ Candidatus Competibacter phosphatis’ expressing the glycogen-accumulating organisms (GAO) phenotype. Recent studies have demonstrated in short-term experiments that organisms of the genotype ‘Candidatus Accumulibacter phosphatis’ clade I and II, known to express the polyphosphate-accumulating organisms (PAO) phenotype can switch to the GAO phenotype when poly-P is absent, but are performing the HAc-uptake at lower kinetic rates, where clade I showed the lowest rates. The objective of this study was to verify whether organisms of the genotype ‘Candidatus Accumulibacter phosphatis’ can also be enriched under P-limiting conditions while expressing a GAO phenotype and more specifically to see which specific clade prevails. A sequencing batch reactor was inoculated with activated sludge to enrich an EBPR culture for a cultivation period of 128 days (16 times the solids retention time) under P-limiting conditions. A mixed culture was obtained comprising of 49 % ‘Candidatus Accumulibacter phosphatis’ clade II and 46 % ‘Candidatus Competibacter phosphatis’. The culture performed a full GAO metabolism for anaerobic HAc-uptake, but was still able to switch to a PAO metabolism, taking up excessive amounts of phosphate during the aerobic phase when it became available in the influent. These findings show that P-limitation, often used as strategy for enrichment of ‘Candidatus Competibacter phosphatis’, does not always lead to enrichment of only ‘Candidatus Competibacter phosphatis’. Furthermore, it demonstrates that ‘Candidatus Accumulibacter phosphatis’ are able to proliferate in activated sludge systems for periods of up to 128 days or longer when the influent phosphate concentrations are just enough for assimilation purposes and no poly-P is formed. The ‘Candidatus Accumulibacter phosphatis’ retain the ability to switch to the PAO phenotype, taking up phosphate from the influent as soon as it becomes available.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0214-z) contains supplementary material, which is available to authorized users.

The effect of free nitrous acid on key anaerobic processes in enhanced biological phosphorus removal systems

In this study, the effect of nitrite/FNA on the anaerobic metabolism of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) is investigated. The results clearly show that FNA has a detrimental effect on the acetate uptake rate by both PAOs and GAOs, but this adverse effect is much stronger on PAOs than on GAOs. Also, when FNA was increased, phosphate release to acetate uptake ratio by PAOs increased substantially (250–300% compared to control), which was accompanied by decreases (40–60%) in glycogen degradation and PHA production to VFA uptake. In contrast, these ratios for GAOs remained constant or increased slightly towards the highest FNA concentration applied. These results indicate that the anaerobic metabolism of PAOs is more adversely affected than that of GAOs when FNA is present. This might provide a competitive advantage to GAOs over PAOs in enhanced biological phosphorus removal systems when nitrite is present.

Response of poly-phosphate accumulating organisms to free nitrous acid inhibition under anoxic and aerobic conditions

The response of free nitrous acid (FNA)-adapted poly-phosphate accumulating organisms (PAOs) to FNA inhibition under aerobic and anoxic conditions was studied. Anoxic P-uptake was 1–6 times more sensitive to the inhibition compared to aerobic P-uptake. The aerobic nitrite reduction rate increased with FNA concentration, accompanied by an equivalent decrease in the oxygen uptake rate, suggesting under high FNA concentration conditions, electrons were channeled to nitrite reduction from oxygen reduction. In contrast, the nitrite reduction rate decreased with increased FNA concentration under anoxic conditions. Anaerobic metabolism of PAO under both anoxic and aerobic conditions was observed at high FNA concentrations. Growth of PAOs decreased sharply with FNA concentration and stopped completely at FNA concentration of 10μgHNO2–N/L. This study, for the first time, investigated the function of nitrite/FNA in an aerobic denitrifying phosphate removal process by evaluating electron as well as energy balances, and provides explanation for FNA inhibition mechanisms.

Metabolic shift of polyphosphate-accumulating organisms with different levels of polyphosphate storage

Previous studies have shown that polyphosphate-accumulating organisms (PAOs) are able to behave as glycogen-accumulating organisms (GAOs) under different conditions. In this study we investigated the behavior of a culture enriched with Accumulibacter at different levels of polyphosphate (poly-P) storage. The results of stoichiometric ratios Glydegraded/HAcuptake, PHBsynthesized/HAcuptake, PHVsynthesized/HAcuptake and Prelease/HAcuptake confirmed a metabolic shift from PAO metabolism to GAO metabolism: PAOs with high poly-P content used the poly-P to obtain adenosine tri-phosphate (ATP), and glycogen (Gly) to obtain nicotinamide adenine dinucleotide (NADH) and some ATP. In a test where poly-P depletion was imposed on the culture, all the acetate (HAc) added in each cycle was transformed into polyhydroxyalkanoate (PHA) despite the decrease of poly-P inside the cells. This led to an increase of the Glydegraded/HAcuptake ratio that resulted from a shift towards the glycolytic pathway in order to compensate for the lack of ATP formed from poly-P hydrolysis. The shift from PAO to GAO metabolism was also reflected in the change in the PHA composition as the poly-P availability decreased, suggesting that polyhydroxyvalerate (PHV) is obtained due to the consumption of excess reducing equivalents to balance the internal NADH, similarly to GAO metabolism. Fluorescence in situ hybridization analysis showed a significant PAO population change from Type I to Type II Accumulibacter as the poly-P availability decreased in short term experiments. This work suggests that poly-P storage levels and GAO-like metabolism are important factors affecting the competition between different PAO Types in enhanced biological phosphorus removal systems.

Could nitrite/free nitrous acid favour GAOs over PAOs in enhanced biological phosphorus removal systems?

Enhanced biological phosphorus removal (EBPR) normally occurs together with nitrogen removal in wastewater treatment plants (WWTPs). In recent years, efforts have been devoted to remove nitrogen via the nitrite pathway (oxidation of ammonia to nitrite and reduction of nitrite to nitrogen gas without going through nitrate), reducing the requirement for carbon and oxygen in the plant. However nitrite and free nitrous acid (FNA), the protonated species of nitrite, have been shown to cause EBPR deterioration under certain concentrations. This study provides a direct comparison between the different levels of FNA inhibition in the aerobic processes of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) by reviewing the studies published in this area. Also, new data is presented assessing the FNA effect on the anaerobic metabolism of these two groups of bacteria. Overall, FNA has shown inhibitory effects on most of the processes involved in the metabolism of PAOs and GAOs. However, the inhibition-initiation levels are different between different processes and, even more importantly between the two groups. In general, PAOs appear to be more affected than GAOs at the same level of FNA, thus giving GAOs competitive advantage over PAOs in EBPR systems when nitrite is present.

The Effect of Free Nitrous Acid on the Anaerobic Metabolism of Polyphosphate Accumulating Organisms (PAOs) and Glycogen Accumulating Organisms (GAOs)

The Effect of Free Nitrous Acid on the Anaerobic Metabolism of Polyphosphate Accumulating Organisms (PAOs) and Glycogen Accumulating Organisms (GAOs)

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-HNO 2/L (equivalent to approximately 6.3 mgN-NO 2 −/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-HNO 2/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.

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.

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.

Modeling the Aerobic Metabolism of Polyphosphate‐Accumulating Organisms Enriched with Propionate as a Carbon Source

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