Which are the polyphosphate accumulating organisms in full-scale activated sludge enhanced biological phosphate removal systems in Australia?

AIMS: To see if the compositions of the microbial communities in full scale enhanced biological phosphorus removal activated sludge systems were the same as those from laboratory scale sequencing batch reactors fed a synthetic sewage. METHODS: Biomass samples taken from nine full scale enhanced biological phosphate removal (EBPR) activated sludge plants in the eastern states of Australia were analysed for their populations of polyphosphate (polyP)-accumulating organisms (PAO) using semi-quantitative fluorescence in situ hybridization (FISH) in combination with DAPI (4'-6-diamidino-2-phenylindole) staining for polyP. RESULTS: Very few betaproteobacterial Rhodocyclus related organisms could be detected by FISH in most of the plants examined, and even where present, not all these cells even within a single cluster, stained positively for polyP with DAPI. In some plants in samples from aerobic reactors the Actinobacteria dominated populations containing polyP. CONCLUSIONS: The PAO populations in full-scale EBPR systems often differ to those seen in laboratory scale reactors fed artificial sewage, and Rhodocyclus related organisms, dominating these latter communities may not be as important in full-scale systems. Instead Actinobacteria may be the major PAO. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings illustrate how little is still known about the microbial ecology of EBPR processes and that more emphasis should now be placed on analysis of full-scale plants if microbiological methods are to be applied to monitoring their performances.     

A metabolic model of the biological phosphorus removal process: II. Validation during start-up conditions

A metabolic model of the biological phosphorus removal process has been developed and validated previously for complex conversions during the process under anaerobic and aerobic conditions at different growth rates in sequencing batch reactors in steady state. For additional validation of the metabolic model, the model was applied to the dynamic conditions which occur during the start-up phase of the biological P removal in the presence and absence of non-polyP heterotrophic microorganisms. In a laboratory scale sequencing batch reactor, experiments were performed to examine the enrichment of the population with polyphosphate organisms during the start-up and the subsequent shift from non-polyP, heterotrophic organisms to polyP organisms in the sludge. The effect of different influent loading patterns for acetate and phosphate was studied. In these experiments, the maximal growth rate of the polyP organisms and the behavior of the internal storage compounds could be derived. The metabolic model was capable of describing the experimental results, without the need to adjust the kinetic or stoichiometric parameters obtained under steady state conditions. (c) 1995 John Wiley & Sons, Inc.     

Phosphorus removal characteristics of granular and flocculent sludge in SBR

Aerobic granulation technology has become a novel biotechnology for wastewater treatment. However, the distinct properties and characteristics of phosphorus removal between granules and flocculent sludge are still sparse in enhanced biological phosphorus removal process. Two identical sequencing batch reactors (SBRs) were operated to compare phosphorus removal performance with granular sludge (R1) and flocculate activated sludge (R2). Results indicated that the start-up period was shorter in R2 than R1 for phosphorus removal, which made R2 reach the steady-state condition on day 21, while R1 was on day 25, and R2 released and took up more phosphorus than R1. As a result, the phosphorus removal was around 90% in R2 while 80% in R1 at the steady-state system. The special phosphorus release rate and special phosphorus uptake rate were 8.818 mg P/g volatile suspended solids (VSS)/h and 9.921 mg P/g VSS/h in R2, which were consistently greater than those (0.999 and 3.016 mg P/g VSS/h) in R1. The chemical oxygen demand removal in two reactors was similar. The granular SBR had better solid-separation performance and higher removal efficiency of NH₄⁺–N than flocculent SBR. Denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA fragment analysis revealed that the diversity and the level of phosphorus-accumulating bacteria in flocculent sludge were much more than those in the granular sludge.     

Analysis of the polyphosphate-accumulating microflora in phosphorus-eliminating, anaerobic-aerobic activated sludge systems by using diaminopropane as a biomarker for rapid estimation of Acinetobacter spp.

Polyphosphate-accumulating gram-negative bacteria were isolated from different anaerobic-aerobic activated sludge systems with diverse processes for enhanced biological phosphorus (P) elimination. Of 22 isolates, 10 were allocated to the genus Acinetobacter by using multiple-test systems and soluble protein and polyamine patterns. As diaminopropane (DAP) appears to be the characteristic main polyamine compound produced by Acinetobacter spp., it was used as a biomarker for the genus. The high DAP contents of representative samples from municipal wastes with enhanced biological P elimination indicated that Acinetobacter spp. can be dominant organisms in sewage treatment plants with low organic loading and nitrification and denitrification steps. Contrary to accepted opinion, sludge from treatment plants with efficient P removal and high organic loading had a low DAP content, indicating that bacteria other than Acinetobacter spp. are responsible for enhanced biological P elimination in these plants.     

Analysis of the polyphosphate-accumulating microflora in phosphorus-eliminating, anaerobic-aerobic activated sludge systems by using diaminopropane as a biomarker for rapid estimation of Acinetobacter spp.

Polyphosphate-accumulating gram-negative bacteria were isolated from different anaerobic-aerobic activated sludge systems with diverse processes for enhanced biological phosphorus (P) elimination. Of 22 isolates, 10 were allocated to the genus Acinetobacter by using multiple-test systems and soluble protein and polyamine patterns. As diaminopropane (DAP) appears to be the characteristic main polyamine compound produced by Acinetobacter spp., it was used as a biomarker for the genus. The high DAP contents of representative samples from municipal wastes with enhanced biological P elimination indicated that Acinetobacter spp. can be dominant organisms in sewage treatment plants with low organic loading and nitrification and denitrification steps. Contrary to accepted opinion, sludge from treatment plants with efficient P removal and high organic loading had a low DAP content, indicating that bacteria other than Acinetobacter spp. are responsible for enhanced biological P elimination in these plants.     

Long-term population dynamics and in situ physiology in activated sludge systems with enhanced biological phosphorus removal operated with and without nitrogen removal

Quantitative fluorescence in situ hybridization (FISH) and the combination of FISH with microautoradiography (MAR) were used in order to study the long-term population dynamics (2.5 years) and the in situ physiology in two parallel activated sludge pilot systems with enhanced biological phosphorus removal (EBPR). The two systems received the same influent wastewater, but were differently operated (with and without nitrogen removal, respectively). Both systems showed a significant P removal that increased when different substrates (phosphorus (P), acetate and glucose, respectively) were added to the influent wastewater. Rhodocyclus-related bacteria were present in both systems in significant numbers (ranging from 4 to 28%) throughout the whole period. This supports the hypothesis that these bacteria occur in significant numbers in different types of well-operating EBPR activated sludge processes. However, we observed a lower correlation (< 0.5) for the amount of Rhodocyclus-related bacteria to the P content in activated sludge than previous studies (> 0.9). The Actinobacteria were the only additional group of bacteria which showed a similar degree of correlation to the P content in activated sludge as the Rhodocyclus-related bacteria--but only for the system without nitrogen removal. Significant amounts (< or = 12%) of glycogen-accumulating bacteria (GAOs) were detected in the system with nitrogen removal (but not in the other system), but had no, in contrast to previous observations, apparent negative effect on the overall EBPR performance. FISH-MAR indicated that a significant part of the Betaproteobacteria (part of them identified as Rhodocyclus-related bacteria) as well as the Actinobacteria were able to take up 33Pi, [3H]-acetate and [3H]-glucose under anaerobic-aerobic conditions. The contribution of anoxic 33Pi uptake under alternating anaerobic-anoxic conditions was significantly lower. Interestingly, not all Rhodocyclus-related bacteria showed uptake of these three radioactive substrates. This may be due to differences in metabolic state, physiological potential or genotype, not detectable by the present probe set for Rhodocyclus-related bacteria. Comparison of the 33Pi, [3H]-acetate and [3H]-glucose uptake by activated sludge after different fixation and incubation procedures showed that a part of the observed 33Pi uptake may have been caused by a combination of a biological and chemical or biologically induced chemical P adsorption.     

Phosphate Removal from Wastewaters: A Novel Approach

The conditions necessary for the establishment and maintenance of Enhanced Biological Phosphate Removal (EBPR) from wastewaters are discussed in the light of our inability to achieve levels of EBPR from artificial sewage in a laboratory‐scale system. Adequate levels of P removal and polyP accumulation by sludge biomass could only be restored by the imposition of stringent anaerobiosis (Eh < –120 mV) and by increasing the short chain fatty acid composition of the influent. Subsequent laboratory‐scale investigations into several possible alternative strategies to achieve enhanced levels of P removal and polyP accumulation from artificial sewage medium indicated that a reduction in the operational pH of the system to approximately 5.5 could achieve comparable levels of P removal under fully‐aerobic conditions. Acid stimulated P uptake and polyP formation might serve as the basis of novel alternative technologies for eutrophication control at wastewater treatment facilities.     

Biotechnology for phosphorus removal during wastewater treatment

Advanced biological wastewater treatment for the removal of phosphorus in excess of the normal metabolic requirements of activated sludge type processes has been developed as an alternative to chemical addition. Current laboratory and pilot plant investigations have confirmed that a preliminary anaerobic zone and plug-flow type configuration are necessary for good enhanced biological phosphorus removal. Nitrate in the anaerobic stage inhibits the process whereas acetate enhances phosphorus uptake. The bacteria probably responsible are of the Acinetobacter genus and the presence of stored polyphosphate within these bacteria has been demonstrated. It has also been shown that pure cultures of Acinetobacter do not necessarily take up soluble substrate as phosphate is released during the anaerobic phase, in contrast to the current proposed mechanism, and that in certain cases natural chemical precipitation could make a significant contribution towards overall phosphorus removal. Several studies of pilot and full-scale plants have been reported.     

Possible complication regarding phosphorus removal with a continuous flow biofilm system: diffusion limitation

Diffusion limitation of phosphate possibly constitutes a serious problem regarding the use of a biofilm reactor for enhanced biological phosphorus removal. A lab-scale reactor for simultaneous removal of phosphorus and nitrate was operated in a continuous alternating mode of operation. For a steady-state operation with excess amounts of carbon source (acetate) during the anaerobic phase, the same amount of phosphate was released during the anaerobic phase as was taken up during the anoxic phase. The measured phosphorus content of the biomass that detached during backwash after an anoxic phase was low, 2.4 +/- 0.4% (equal to 24 +/- 4 mg P/g TS). A simplified computer model indicated the reason to be phosphate diffusion limitation and the model revealed a delicate balance between the obtainable phosphorus contents of the biomass and operating parameters, such as backwash interval, biofilm thickness after backwash, and phase lengths. The aspect of diffusion is considered of crucial importance when evaluating the performance of a biofilter for phosphate removal.     

Polyphosphate-accumulating bacteria from sewage plants with different proceses for biological phosphorus removal

Abstract Activated sludge samples from pilot plants using different processes for enhanced biological phosphorus removal were investigated for the occurrence of polyphosphate-accumulating bacteria. All samples showed a direct correlation between the relative number of phosphate-accumulating bacteria and phosphate uptake. Various species of bacteria with polyphosphate granules were found in sludge flocs, but in every case Acinetobacter -like cells formed part or the main part of the polyphosphate-accumulating bacterial population. The spectrum of the bacteria with stored polyphosphates varied, depending on the sewage composition on the one hand and on the processes used for phosphorus removal on the other hand.     

Application of polyphosphate metabolism to environmental and biotechnological problems

The synthesis and degradation of polyphosphate (polyP) are influenced by the energy state of the cell and extracellular phosphate levels. The import of excess phosphate and its incorporation into polyP under phosphate- and energy-rich growth conditions allows organisms to survive when phosphate or energy are depleted. Under phosphate-starvation conditions, phosphate can be recovered from polyP by hydrolysis. When the organism is energy starved, energy can be recovered either by regenerating the high-energy phosphoanhydride bond donor (ATP in most cases) or by hydrolysis of polyP and subsequent secretion of orthophosphate to recharge the transmembrane proton gradient. Understanding how the energy state of the cell and environmental phosphate levels affect polyP metabolism is essential to improving such environmental processes as enhanced biological phosphorus removal, a treatment process that is widely used to remove excess phosphate from wastewater. Manipulation of the genes responsible for polyP metabolism can also be used to improve gene expression from phosphate-starvation promoters and to remove heavy metals from contaminated environments.     

The effects of hydraulic retention time and sludge retention time on the fate of di-(2-ethylhexyl) phthalate in a laboratory-scale anaerobic-anoxic-aerobic activated sludge system

A laboratory-scale anaerobic-anoxic-aerobic (AAA) activated sludge wastewater treatment system was employed to investigate the effects of hydraulic retention time (HRT) and sludge retention time (SRT) on the removal and fate of di-(2-ethylhexyl) phthalate (DEHP). In the range from 5 to 14h, HRT had no significant effect on DEHP removal. However, longer HRT increased DEHP accumulation in the system and DEHP retention in the waste sludge. When SRT was increased from 15 to 25d, DEHP removal efficiency stayed above 96%. Compared to the removal of only 88% at SRT of 10d, longer SRT enhanced DEHP degradation efficiency. The optimal HRT and SRT for both nutrients (nitrogen and phosphorus) and DEHP removal were 8h and 15d. At these retention times, about 71% of DEHP was degraded by the activated sludge process, 26% was accumulated in the system, 2% was released in the effluent, and 1% remained in the waste sludge. The anaerobic, anoxic and aerobic reactors were responsible for 15%, 19% and 62% of the overall DEHP removal, respectively.     

Comparative study of biological nutrient removal (BNR) processes with sedimentation and membrane-based separation

A membrane-enhanced biological phosphorus removal (MEBPR) process was operated in parallel with a conventional EBPR (CEBPR) process under challenging operating conditions to uncover fundamental differences in their ability to remove chemical oxygen demand (COD), nitrogen (N), and phosphorus (P) from municipal wastewater. Both systems exhibited the same potential to achieve excellent soluble-P removal when a favorable COD to P ratio was maintained in the influent. The MEBPR train generated a superior effluent quality when measured as total P. The CEBPR effluent contained significantly lower levels of nitrates due to the extra denitrification occurring in the sludge blanket of the secondary clarifier. The observed sludge yield in the MEBPR system was estimated to be between 0.23 and 0.28 g VSS/g COD, and this was 15% lower than the CEBPR sludge yield. When the influent volatile fatty acids (VFAs) became limiting, the CEBPR train exhibited better performance in the removal of soluble-P, due to the higher observed sludge yield and an overall greater denitrification activity that led to a more efficient use of VFAs in the anaerobic zone. After experiencing a severe deterioration of the biological P activity in both processes, the MEBPR train exhibited faster recovery than the CEBPR side. In this experimental work, it was demonstrated that an MEBPR process can sustain long-term satisfactory bio-P performance at HRTs as low as 7 h. However, the lower sludge yield and the reduced denitrification capacity are two important factors that impact the design of high rate membrane-assisted biological nutrient removal (BNR) processes.     

Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed probes for their detection and quantitation

Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphate-accumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were beta-2 Proteobacteria arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the Actinobacteria. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the beta-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing >/=98% sequence identity) related to Rhodocyclus spp. (94 to 97% identity) and Propionibacter pelophilus (95 to 96% identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the beta-2 Proteobacteria probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR sludges are bacteria closely related to Rhodocyclus and Propionibacter.     

Inducing mechanism of biological phosphorus removal driven by the aerobic/extended-idle regime

Recently, it was found that excess phosphorus (Pi) removal could be achieved in activated sludge with an aerobic/extended‐idle (AEI) process. In this study, batch tests were performed to further reveal the inducing mechanism of Pi removal involved in the AEI process. Unlike the classical anaerobic/aerobic process where an anaerobic Pi release along with a significant polyhydroxyalkanoate (PHA) accumulation drives polyphosphate (poly‐P) accumulating organisms (PAOs) to over‐store Pi as poly‐P, an idle Pi release accompanied by a low‐idle PHA production, which is usually considered to be detrimental for biological Pi removal, was observed to induce some cells to effectively uptake Pi in excess of metabolic requirement in the AEI process. With the increase of idle Pi release, Pi removal efficiency linearly increased. The results also showed that a long idle period with a low level of intracellular glycogen could significantly increase Pi release contents, thus remarkably enhancing Pi removal performances. Fluorescence in situ hybridization analysis further revealed that activated sludge in the AEI process contained 37.6% of Accumulibacter (PAOs) and 28.2% of Competibacter and Defluviicoccus‐related organisms (glycogen accumulating organisms). This study revealed an actually existent, yet previously unrecognized, inducing mechanism of poly‐P accumulation, and this mechanism behind the AEI regime may provide a scientific basis for the development of an alternative strategy for Pi removal from wastewaters. Biotechnol. Bioeng. 2012; 109: 2798–2807. © 2012 Wiley Periodicals, Inc.     

Influence of the Addition of Different Metals to an Activated Sludge System on the Enhanced Biological Phosphorus Removal

Activated sludge systems can be operated to select bacteria which accumulate phosphorus as polyphosphate. By these means, phosphate can be removed without the addition of chemical precipitants. This contribution presents results of experiments with a bench-scale purification plant for domestic sewage. The goal was to find the concentrations of relevant metal cations at which the biological P removal may be affected by competing chemical (precipitation) or physical (adsorption) processes. For this goal, increasing amounts of iron and calcium, respectively, were added into the pilot plant. During the addition of iron, the proportion of chemically (iron-) bound phosphorus increased from 10 to more than 50%. The P-release rate substantially decreased with increasing amount of added iron. An addition of small amounts of iron enhanced the long-term stability of the P elimination as a whole. During the experimental period with addition of calcium, the proportion of Ca-bound phosphorus increased from 1 to 2% to almost 15%. In batch experiments a measurable Ca-phosphate precipitation took place at a pH value of at least 8.0 and a Ca-concentration of at least 100 mg/l. The increase in hardness of the influent waste water didn't produce any positive effect on the stability of the enhanced biological phosphorus removal. The metal ions Ca²⁺, Mg²⁺ and K⁺ serve as counter-ions in the polyhosphate chains. They were identified and quantified by X-ray spectrometry in combination with scanning transmission electron microscopy. A release of Mg²⁺ and K⁺ occured simultaneously with the degration of polyphosphates (PP). The PP bound to Ca was not redox sensitive.     

Inhibition of chemical dose in biological phosphorus and nitrogen removal in simultaneous chemical precipitation for phosphorus removal

A study on the influence of chemical dosing on biological phosphorus and nitrogen removal was carried out through batch experimental tests by lab-scale and a full-scale wastewater treatment plant (employing a typical anaerobic-anoxic-oxic treatment). Results indicated that the inhibition of aluminum salt on biological phosphorus release and uptake processes is significant, as well as the inhibition of aluminum salt on Ammonia-Oxidizing Bacteria (AOB) is dominantly observed in the nitrification process and is recoverability. The inhibition of iron salt in biological phosphorus and nitrogen removal is weak, and only the inhibition of iron salt on phosphorus release at anaerobic periods emerge under large dosing. Evidence shows persistent inhibition from the accumulation of chemical doses in sludge mass. Intermittent chemical dosing proves recommendable for simultaneous chemical phosphorus removal.     

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

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

亚硝酸盐对污水生物除磷影响的研究进展

亚硝酸盐作为生物硝化和反硝化的中间产物, 存在于污水生物脱氮除磷系统中。对于生物强化除磷工艺亚硝酸盐既是电子受体用于反硝化除磷, 同时又是抑制剂影响生物除磷过程。本文综述了聚磷菌在厌氧、好氧和缺氧环境中的代谢机理, 在此基础上分别从好氧除磷和反硝化除磷两方面介绍了亚硝酸盐对污水生物除磷影响的研究, 同时概述了亚硝酸盐对生物除磷的抑制机理, 并对该领域的研究提出了个人见解。     

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

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