Ecophysiology of polyphosphate-accumulating organisms and glycogen-accumulating organisms in a continuously aerated enhanced biological phosphorus removal process

Aims: To investigate the ecophysiology of populations of polyphosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO) in communities of a novel acetate fed process removing phosphate from wastewater. Attempts were made to see if acetate could be replaced by an alternative carbon source which did not support the growth of the GAO. Methods and Results: A continuously aerated sequencing batch reactor was operated with different acetate feed levels. Fluorescence in situ hybridization (FISH) showed that Defluviicoccus GAO numbers increased at lower acetate feed levels. With FISH/microautoradiography (MAR) both detected morphotypes of Defluviicoccus assimilated a wider range of substrates aerobically than Accumulibacter PAO. Their uptake profile differed from that reported for the same phylotype in full scale anaerobic : aerobic EBPR plants. Conclusions: This suggests that replacing acetate with another substrate is unlikely to provide Accumulibacter with a selective advantage in this process. Why Defluviicoccus appeared to out-compete Accumulibacter at lower acetate concentrations was not clear. Data suggest physiological and morphological diversity may exist within a single Defluviicoccus phylotype. Significance and Impact of the Study: This study implies that the current FISH probes for Defluviicoccus GAO may not reveal the full extent of their biodiversity, and that more information is required before strategies for their control can be devised.     

Identity and ecophysiology of uncultured actinobacterial polyphosphate-accumulating organisms in full-scale enhanced biological phosphorus removal plants

Microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was used to screen for potential polyphosphate-accumulating organisms (PAO) in a full-scale enhanced biological phosphorus removal (EBPR) plant. The results showed that, in addition to uncultured Rhodocyclus-related PAO, two morphotypes hybridizing with gene probes for the gram-positive Actinobacteria were also actively involved in uptake of orthophosphate (Pi). Clone library analysis and further investigations by MAR-FISH using two new oligonucleotide probes revealed that both morphotypes, cocci in clusters of tetrads and short rods in clumps, were relatively closely related to the genus Tetrasphaera within the family Intrasporangiaceae of the Actinobacteria (93 to 98% similarity in their 16S rRNA genes). FISH analysis of the community biomass in the treatment plant investigated showed that the short rods (targeted by probe Actino-658) were the most abundant (12% of all Bacteria hybridizing with general bacterial probes), while the cocci in tetrads (targeted by probe Actino-221) made up 7%. Both morphotypes took up P(i) aerobically only if, in a previous anaerobic phase, they had taken up organic matter from wastewater or a mixture of amino acids. They could not take up short-chain fatty acids (e.g., acetate), glucose, or ethanol under anaerobic or aerobic conditions. The storage compound produced during the anaerobic period was not polyhydroxyalkanoates, as for Rhodocyclus-related PAO, and its identity is still unknown. Growth and uptake of Pi took place in the presence of oxygen and nitrate but not nitrite, indicating a lack of denitrifying ability. A survey of the occurrence of these actinobacterial PAO in 10 full-scale EBPR plants revealed that both morphotypes were widely present, and in several plants more abundant than the Rhodocyclus-related PAO, thus playing a very important role in the EBPR process.     

Involvement of Rhodocyclus-related organisms in phosphorus removal in full-scale wastewater treatment plants

The participation of organisms related to Rhodocyclus in full-scale enhanced biological phosphorus removal (EBPR) was investigated. By using fluorescent in situ hybridization techniques, the communities of Rhodocyclus-related organisms in two full-scale wastewater treatment plants were estimated to represent between 13 and 18% of the total bacterial population. However, the fractions of these communities that participated in polyphosphate accumulation depended on the type of treatment process evaluated. In a University of Cape Town EBPR process, the percentage of Rhodocyclus-related cells that contained polyphosphate was about 20% of the total bacterial population, but these cells represented as much as 73% of the polyphosphate-accumulating organisms (PAOs). In an aerated-anoxic EBPR process, Rhodocyclus-related PAOs were less numerous, accounting for 6% of the total bacterial population and 26% of the total PAO population. In addition, 16S ribosomal DNA sequences 99.9% similar to the sequences of Rhodocyclus-related organisms enriched in acetate-fed bench-scale EBPR reactors were recovered from both full-scale plants. These results confirmed the involvement of Rhodocyclus-related organisms in EBPR and demonstrated their importance in full-scale processes. In addition, the results revealed a significant correlation between the type of EBPR process and the PAO community.     

Microautoradiographic study of Rhodocyclus-related polyphosphate-accumulating bacteria in full-scale enhanced biological phosphorus removal plants

The ecophysiology of uncultured Rhodocyclus-related polyphosphate-accumulating organisms (PAO) present in three full-scale enhanced biological phosphorus removal (EBPR) activated sludge plants was studied by using microautoradiography combined with fluorescence in situ hybridization. The investigations showed that these organisms were present in all plants examined and constituted 5 to 10, 10 to 15, and 17 to 22% of the community biomass. The behavior of these bacteria generally was consistent with the biochemical models proposed for PAO, based on studies of lab-scale investigations of enriched and often unknown PAO cultures. Rhodocyclus-related PAO were able to accumulate short-chain substrates, including acetate, propionate, and pyruvate, under anaerobic conditions, but they could not assimilate many other low-molecular-weight compounds, such as ethanol and butyrate. They were able to assimilate two substrates (e.g., acetate and propionate) simultaneously. Leucine and thymidine could not be assimilated as sole substrates and could only be assimilated as cosubstrates with acetate, perhaps serving as N sources. Glucose could not be assimilated by the Rhodocyclus-related PAO, but it was easily fermented in the sludge to products that were subsequently consumed. Glycolysis, and not the tricarboxylic acid cycle, was the source that provided the reducing power needed by the Rhodocyclus-related PAO to form the intracellular polyhydroxyalkanoate storage compounds during anaerobic substrate assimilation. The Rhodocyclus-related PAO were able to take up orthophosphate and accumulate polyphosphate when oxygen, nitrate, or nitrite was present as an electron acceptor. Furthermore, in the presence of acetate growth was sustained by using oxygen, as well as nitrate or nitrite, as an electron acceptor. This strongly indicates that Rhodocyclus-related PAO were able to denitrify and thus played a role in the denitrification occurring in full-scale EBPR plants.     

A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants

Enhanced biological phosphorus removal (EBPR) from wastewater can be more-or-less practically achieved but the microbiological and biochemical components are not completely understood. EBPR involves cycling microbial biomass and influent wastewater through anaerobic and aerobic zones to achieve a selection of microorganisms with high capacity to accumulate polyphosphate intracellularly in the aerobic period. Biochemical or metabolic modelling of the process has been used to explain the types of carbon and phosphorus transformations in sludge biomass. There are essentially two broad-groupings of microorganisms involved in EBPR. They are polyphosphate accumulating organisms (PAOs) and their supposed carbon-competitors called glycogen accumulating organisms (GAOs). The morphological appearance of microorganisms in EBPR sludges has attracted attention. For example, GAOs as tetrad-arranged cocci and clusters of coccobacillus-shaped PAOs have been much commented upon and the use of simple cellular staining methods has contributed to EBPR knowledge. Acinetobacter and other bacteria were regularly isolated in pure culture from EBPR sludges and were initially thought to be PAOs. However, when contemporary molecular microbial ecology methods in concert with detailed process performance data and simple intracellular polymer staining methods were used, a betaproteobacteria called ‘Candidatus Accumulibacter phosphatis’ was confirmed as a PAO and organisms from a novel gammaproteobacteria lineage were GAOs. To preclude making the mistakes of previous researchers, it is recommended that the sludge ‘biography’ be well understood – i.e. details of phenotype (process performance and biochemistry) and microbial community structure should be linked. This revised version was published online in August 2006 with corrections to the Cover Date.     

Improvement strategy on enhanced biological phosphorus removal for municipal wastewater treatment plants: full-scale operating parameters, sludge activities, and microbial features

The poor quality of effluent discharged by municipal wastewater treatment plants (WWTPs) is threatening the safety of water ecology. This study, which integrated a field survey, batch tests, and microbial community identification, was designed to improve the effectiveness of the enhanced biological phosphorus removal (EBPR) process for WWTPs. Over two-thirds of the investigated WWTPs could not achieve total P in effluent lower than 0.5 mg/L, mainly due to the high ratio of chemical oxygen demand to P (28.6-196.2) in the influent. The rates of anaerobic P release and aerobic P uptake for the activated sludge varied from 0.22 to 7.9 mg/g VSS/h and 0.43 to 8.11 mg/g VSS/h, respectively. The fraction of Accumulibacter (PAOs: polyphosphate accumulating organisms) was 4.8 ± 2.0% of the total biomass, while Competibacter (GAOs: glycogen-accumulating organisms) accounted for 4.8 ± 6.4%. The anaerobic P-release rate was found to be an effective indicator of EBPR. Four classifications of the principal components were identified to improve the EBPR effluent quality and sludge activity.     

A metagenome of a full-scale microbial community carrying out enhanced biological phosphorus removal

Enhanced biological phosphorus removal (EBPR) is widely used for removal of phosphorus from wastewater. In this study, a metagenome (18.2 Gb) was generated using Illumina sequencing from a full-scale EBPR plant to study the community structure and genetic potential. Quantitative fluorescence in situ hybridization (qFISH) was applied as an independent method to evaluate the community structure. The results were in qualitative agreement, but a DNA extraction bias against gram positive bacteria using standard extraction protocols was identified, which would not have been identified without the use of qFISH. The genetic potential for community function showed enrichment of genes involved in phosphate metabolism and biofilm formation, reflecting the selective pressure of the EBPR process. Most contigs in the assembled metagenome had low similarity to genes from currently sequenced genomes, underlining the need for more reference genomes of key EBPR species. Only the genome of ‘Candidatus Accumulibacter'', a genus of phosphorus-removing organisms, was closely enough related to the species present in the metagenome to allow for detailed investigations. Accumulibacter accounted for only 4.8% of all bacteria by qFISH, but the depth of sequencing enabled detailed insight into their microdiversity in the full-scale plant. Only 15% of the reads matching Accumulibacter had a high similarity (>95%) to the sequenced Accumulibacter clade IIA strain UW-1 genome, indicating the presence of some microdiversity. The differences in gene complement between the Accumulibacter clades were limited to genes for extracellular polymeric substances and phage-related genes, suggesting a selective pressure from phages on the Accumulibacter diversity.     

Anaerobic metabolism of propionate by polyphosphate-accumulating organisms in enhanced biological phosphorus removal systems

Propionate, a carbon substrate abundant in many prefermenters, has been shown in several previous studies to be a more favorable substrate than acetate for enhanced biological phosphorus removal (EBPR). The anaerobic metabolism of propionate by polyphosphate accumulating organisms (PAOs) is studied in this paper. A metabolic model is proposed to characterize the anaerobic biochemical transformations of propionate uptake by PAOs. The model is demonstrated to predict very well the experimental data from a PAO culture enriched in a laboratory-scale reactor with propionate as the sole carbon source. Quantitative fluorescence in-situ hybridization (FISH) analysis shows that Candidatus Accumulibacter phosphatis, the only identified PAO to date, constitute 63% of the bacterial population in this culture. Unlike the anaerobic metabolism of acetate by PAOs, which induces mainly poly-beta-hydroxybutyrate (PHB) production, the major fractions of poly-beta-hydroxyalkanoate (PHA) produced with propionate as the carbon source are poly-beta-hydroxyvalerate (PHV) and poly-beta-hydroxy-2-methylvalerate (PH2MV). PHA formation correlates very well with a selective (or nonrandom) condensation of acetyl-CoA and propionyl-CoA molecules. The maximum specific propionate uptake rate by PAOs found in this study is 0.18 C-mol/C-mol-biomass . h, which is very similar to the maximum specific acetate uptake rate reported in literature. The energy required for transporting 1 carbon-mole of propionate across the PAO cell membrane is also determined to be similar to the transportation of 1 carbon-mole of acetate. Furthermore, the experimental results suggest that PAOs possess a similar preference toward acetate and propionate uptake on a carbon-mole basis.     

Impact of nitrite on aerobic phosphorus uptake by poly-phosphate accumulating organisms in enhanced biological phosphorus removal sludges

Impact of nitrite on aerobic phosphorus (P) uptake of poly-phosphate accumulating organisms (PAOs) in three different enhanced biological phosphorus removal (EBPR) systems was investigated, i.e., the enriched PAOs culture fed with synthetic wastewater, the two lab-scale sequencing batch reactors (SBRs) treating domestic wastewater for nutrient removal through nitrite-pathway nitritation and nitrate-pathway nitrification, respectively. Fluorescence in situ hybridization results showed that PAOs in the three sludges accounted for 72, 7.6 and 6.5 % of bacteria, respectively. In the enriched PAOs culture, at free nitrous acid (FNA) concentration of 0.47 × 10^?3 mg HNO₂-N/L, aerobic P-uptake and oxidation of intercellular poly-β-hydroxyalkanoates were both inhibited. Denitrifying phosphorus removal under the aerobic conditions was observed, indicating the existence of PAOs using nitrite as electron acceptor in this culture. When the FNA concentration reached 2.25 × 10^?3 mg HNO₂-N/L, denitrifying phosphorus removal was also inhibited. And the inhibition ceased once nitrite was exhausted. Corresponding to both SBRs treating domestic wastewater with nitritation and nitrification pathway, nitrite inhibition on aerobic P-uptake by PAOs did not occur even though FNA concentration reached 3 × 10^?3 and 2.13 × 10^?3 mg HNO₂-N/L, respectively. Therefore, PAOs taken from different EBPR activated sludges had different tolerance to nitrite.     

High and stable substrate specificities of microorganisms in enhanced biological phosphorus removal plants

Microbial communities are typically characterized by conditions of nutrient limitation so the availability of the resources is likely a key factor in the niche differentiation across all species and in the regulation of the community structure. In this study we have investigated whether four species exhibit any in situ short‐term changes in substrate uptake pattern when exposed to variations in substrate and growth conditions. Microautoradiography was combined with fluorescence in situ hybridization to investigate in situ cell‐specific substrate uptake profiles of four probe‐defined coexisting species in a wastewater treatment plant with enhanced biological phosphorus removal. These were the filamentous ‘Candidatus Microthrix’ and Caldilinea (type 0803), the polyphosphate‐accumulating organism ‘Candidatus Accumulibacter’, and the denitrifying Azoarcus. The experimental conditions mimicked the conditions potentially encountered in the respective environment (starvation, high/low substrate concentration, induction with specific substrates, and single/multiple substrates). The results showed that each probe‐defined species exhibited very distinct and constant substrate uptake profile in time and space, which hardly changed under any of the conditions tested. Such niche partitioning implies that a significant change in substrate composition will be reflected in a changed community structure rather than the substrate uptake response from the different species.     

Activity and identity of fermenting microorganisms in full-scale biological nutrient removing wastewater treatment plants

Hydrolysis and fermentation are of key importance in biological nutrient removal (BNR) wastewater treatment plants as they provide polyphosphate-accumulating organisms and denitrifying bacteria with carbon and energy sources (e.g. short chain fatty acids). Little information, however, exists about the microbiology of the microorganisms involved in hydrolysis and fermentation. In this study, fermentation of monosaccharides was found to be a universal process taking place in all full-scale BNR plants investigated, where glucose and other monosaccharides were consumed and fermented during anaerobic conditions. The removal rates of glucose were in the range of 0.05–0.32 mmol gVSS⁻¹ h⁻¹ and only slightly lower than glucose removal under aerobic conditions. The main fermentation products detected were (in descending order) propionic acid, lactic acid, acetic acid and formic acid. The fermentation was diverse, consisting of at least three fermentation metabolisms, including lactic acid (homolactic), mixed acid and propionic acid fermentations. Possible existence of alcohol and/or butyric acid fermentations could not be excluded. Fermentation organisms in Aalborg East treatment plant were identified by using microautoradiography combined with fluorescence in situ hybridization. All microorganisms involved in monosaccharide fermentation belonged to either Gram-positive Firmicutes or Actinobacteria . Most of them were related either to Streptococcus , hybridizing to the oligonucleotide probe Str, or to uncultured Actinobacteria with a phenotype of polyphosphate-accumulating organisms. The fermenting bacteria were widespread in the nine full-scale BNR plants investigated and constituted 3–21% of the total bacterial biovolume.     

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.     

Ozonolysate of excess sludge as a carbon source in an enhanced biological phosphorus removal for low strength wastewater

Potential use of the municipal sludge ozonolysate as a carbon source was examined for phosphorus removal from low strength wastewater in a modified intermittently decanted extended aeration (IDEA) process. At ozone dosage of 0.2 g O₃/g solids, readily biodegradable COD accounted for about 36% of COD from sludge ozonolysate. The denitrification potential of ozonolysate as a carbon source was comparable to that of acetate. Although, the first order constant for phosphorus release with the ozonolysate was half that of acetate, it was much higher than that of wastewater. Continuous operation of the modified IDEA process showed that the removals of nitrogen and phosphorus were simultaneously enhanced by addition of the ozonolysate. Phosphorus release was significantly induced after complete denitrification indicating that phosphorus release was strongly depended on nitrate concentration. Effectiveness of the ozonolysate as a carbon source for EBPR was also confirmed in a track study of the modified IDEA.     

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.     

Methanol‐driven enhanced biological phosphorus removal with a syntrophic consortium

The presence of suitable carbon sources for enhanced biological phosphorus removal (EBPR) plays a key role in phosphorus removal from wastewater in urban WWTP. For wastewaters with low volatile fatty acids (VFAs) content, an external carbon addition is necessary. As methanol is the most commonly external carbon source used for denitrification it could be a priori a promising alternative, but previous attempts to use it for EBPR have failed. This study is the first successful report of methanol utilization as external carbon source for EBPR. Since a direct replacement strategy (i.e., supply of methanol as a sole carbon source to a propionic‐fed PAO‐enriched sludge) failed, a novel process was designed and implemented successfully: development of a consortium with anaerobic biomass and polyphosphate accumulating organisms (PAOs). Methanol‐degrading acetogens were (i) selected against other anaerobic methanol degraders from an anaerobic sludge; (ii) subjected to conventional EBPR conditions (anaerobic + aerobic); and (iii) bioaugmented with PAOs. EBPR with methanol as a sole carbon source was sustained in a mid‐term basis with this procedure. Biotechnol. Bioeng. 2013; 110: 391–400. © 2012 Wiley Periodicals, Inc.     

Recent advances in understanding the ecophysiology of enhanced biological phosphorus removal

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增强型生物除磷过程中聚磷酸盐积累微生物的研究进展

从磷污染控制、污水脱磷和磷资源角度论述了生物除磷的作用,并着重论述了增强型生物除磷过程中聚磷酸盐微生物(PAO)的研究历史、代谢特征及研究方法．聚磷酸盐广泛存在于自然界,但只有少数PAO微生物被分离、培养、鉴定出来．培养基能否分离出PAO和PAO能否在实验室条件下表现出polyP积累特征,均至关重要．糖原积累微生物(GAO)与PAO对碳源存在竞争关系,影响EBPR的效率．原位荧光分子杂交、激光共聚焦扫描电镜、微量放射自显影术、活体核磁共振光谱等现代科学技术的发展。使我们能够观察原位微生物群落组成、空间结构和功能变化．对PAO的深入研究,可改进污水脱磷的效率,提高对磷在环境中迁移转化的认识     

Thermophilic biological nitrogen removal in industrial wastewater treatment

Nitrification is an integral part of biological nitrogen removal processes and usually the limiting step in wastewater treatment systems. Since nitrification is often considered not feasible at temperatures higher than 40 °C, warm industrial effluents (with operating temperatures higher than 40 °C) need to be cooled down prior to biological treatment, which increases the energy and operating costs of the plants for cooling purposes. This study describes the occurrence of thermophilic biological nitrogen removal activity (nitritation, nitratation, and denitrification) at a temperature as high as 50 °C in an activated sludge wastewater treatment plant treating wastewater from an oil refinery. Using a modified two-step nitrification–two-step denitrification mathematical model extended with the incorporation of double Arrhenius equations, the nitrification (nitrititation and nitratation) and denitrification activities were described including the cease in biomass activity at 55 °C. Fluorescence in situ hybridization (FISH) analyses revealed that Nitrosomonas halotolerant and obligatehalophilic and Nitrosomonas oligotropha (known ammonia-oxidizing organisms) and Nitrospira sublineage II (nitrite-oxidizing organism (NOB)) were observed using the FISH probes applied in this study. In particular, this is the first time that Nitrospira sublineage II, a moderatedly thermophilic NOB, is observed in an engineered full-scale (industrial) wastewater treatment system at temperatures as high as 50 °C. These observations suggest that thermophilic biological nitrogen removal can be attained in wastewater treatment systems, which may further contribute to the optimization of the biological nitrogen removal processes in wastewater treatment systems that treat warm wastewater streams.