Enhanced Biological Phosphorus Removal Efficiency Research Articles

Phosphorus removal performance, intracellular metabolites and clade-level community structure of Tetrasphaera-dominated polyphosphate accumulating organisms at different temperatures

Tetrasphaera are polyphosphate accumulating organisms (PAOs) that play an important role in enhanced biological phosphorus removal (EBPR) from wastewater. The effect of a wide range of temperature changes (1–30 °C) on phosphorus removal, metabolism and clade-level community structure of Tetrasphaera-dominated PAOs was investigated. At 10 °C, the bioactivities of Tetrasphaera-dominated communities were obviously inhibited and the EBPR efficiency was only 73 %. Yet at 20–30 °C, EBPR efficiency reached 99 % and the relative abundance of Tetrasphaera was up to 90 %. The temperature variation changed the community distribution of Tetrasphaera clades, which was possibly a main reason for EBPR performance. Amino acids and PHA with different contents were intracellular metabolite of Tetrasphaera-dominated communities during phosphorus release and uptake at different temperatures. Moreover, Tetrasphaera fermented protein and amino acids and released VFAs. The outcomes suggested the great potential of Tetrasphaera-PAOs in the treatment of wastewater with varying temperatures and limited carbon sources.

Genomic and in Situ Analyses Reveal the Micropruina spp. as Abundant Fermentative Glycogen Accumulating Organisms in Enhanced Biological Phosphorus Removal Systems.

Enhanced biological phosphorus removal (EBPR) involves the cycling of biomass through carbon-rich (feast) and carbon-deficient (famine) conditions, promoting the activity of polyphosphate accumulating organisms (PAOs). However, several alternate metabolic strategies, without polyphosphate storage, are possessed by other organisms, which can compete with the PAO for carbon at the potential expense of EBPR efficiency. The most studied are the glycogen accumulating organisms (GAOs), which utilize aerobically stored glycogen to energize anaerobic substrate uptake and storage. In full-scale systems the Micropruina spp. are among the most abundant of the proposed GAO, yet little is known about their ecophysiology. In the current study, genomic and metabolomic studies were performed on Micropruina glycogenica str. Lg2T and compared to the in situ physiology of members of the genus in EBPR plants using state-of-the-art single cell techniques. The Micropruina spp. were observed to take up carbon, including sugars and amino acids, under anaerobic conditions, which were partly fermented to lactic acid, acetate, propionate, and ethanol, and partly stored as glycogen for potential aerobic use. Fermentation was not directly demonstrated for the abundant members of the genus in situ, but was strongly supported by the confirmation of anaerobic uptake of carbon and glycogen storage in the absence of detectable polyhydroxyalkanoates or polyphosphate reserves. This physiology is markedly different from the classical GAO model. The amount of carbon stored by fermentative organisms has potentially important implications for phosphorus removal – as they compete for substrates with the Tetrasphaera PAO and stored carbon is not made available to the “Candidatus Accumulibacter” PAO under anaerobic conditions. This study shows that the current models of the competition between PAO and GAO are too simplistic and may need to be revised to take into account the impact of potential carbon storage by fermentative organisms.

Effect of acetate to glycerol ratio on enhanced biological phosphorus removal

Enhanced biological phosphorus removal (EBPR) is a sustainable and promising technology for phosphorus removal from wastewater. The efficiency of this technology, however, is often discounted due to the insufficient carbon sources in influent. In this work, the effect of acetate to glycerol ratio on the EBPR performance was evaluated. The experimental results showed when the ratio of acetate to glycerol decreased from 100/0% to 50/50%, the EBPR efficiency increased from 90.2% to 96.2%. Further decrease of acetate to glycerol ratio to 0/100% decreased the efficiency of EBPR to 30.5%. Fluorescence in situ hybridization analysis demonstrated appropriate increase of glycerol benefited to increase the relative abundance of phosphate accumulating organisms. Further investigation revealed the proper addition of glycerol increased the amount of polyhydroxyalkanoates synthesis, and then produced sufficient energy for oxic luxury phosphorus in the subsequent oxic phase.

"Candidatus Propionivibrio aalborgensis": A Novel Glycogen Accumulating Organism Abundant in Full-Scale Enhanced Biological Phosphorus Removal Plants.

Enhanced biological phosphorus removal (EBPR) is widely used to remove phosphorus from wastewater. The process relies on polyphosphate accumulating organisms (PAOs) that are able to take up phosphorus in excess of what is needed for growth, whereby phosphorus can be removed from the wastewater by wasting the biomass. However, glycogen accumulating organisms (GAOs) may reduce the EBPR efficiency as they compete for substrates with PAOs, but do not store excessive amounts of polyphosphate. PAOs and GAOs are thought to be phylogenetically unrelated, with the model PAO being the betaproteobacterial “Candidatus Accumulibacter phosphatis” (Accumulibacter) and the model GAO being the gammaproteobacterial “Candidatus Competibacter phosphatis”. Here, we report the discovery of a GAO from the genus Propionivibrio, which is closely related to Accumulibacter. Propionivibrio sp. are targeted by the canonical fluorescence in situ hybridization probes used to target Accumulibacter (PAOmix), but do not store excessive amounts of polyphosphate in situ. A laboratory scale reactor, operated to enrich for PAOs, surprisingly contained co-dominant populations of Propionivibrio and Accumulibacter. Metagenomic sequencing of multiple time-points enabled recovery of near complete population genomes from both genera. Annotation of the Propionivibrio genome confirmed their potential for the GAO phenotype and a basic metabolic model is proposed for their metabolism in the EBPR environment. Using newly designed fluorescence in situ hybridization (FISH) probes, analyses of full-scale EBPR plants revealed that Propionivibrio is a common member of the community, constituting up to 3% of the biovolume. To avoid overestimation of Accumulibacter abundance in situ, we recommend the use of the FISH probe PAO651 instead of the commonly applied PAOmix probe set.

Kinetic and stoichiometric characterization for efficient enhanced biological phosphorus removal (EBPR) process at high temperatures.

A recently reported stable and efficient EBPR system at high temperatures around 30°C has led to characterization of kinetic and stoichiometric parameters of the Activated Sludge Model no. 2d (ASM2d). Firstly, suitable model parameters were selected by identifiability analysis. Next, the model was calibrated and validated. ASM2d was found to represent the processes well at 28 and 32°C except in polyhyroxyalkanoate (PHA) accumulation of the latter. The values of the kinetic parameters for PHA storage (q PHA), polyphosphate storage (q PP) and growth (μ PAO) of polyphosphate-accumulating organisms (PAOs) at 28 and 32°C were found to be much higher than those reported by previous studies. Besides, the value of the stoichiometric parameter for the requirement of polyphosphate for PHA storage (Y PO4) was found to decrease as temperature rose from 28 to 32°C. Values of two other stoichiometric parameters, i.e. the growth yield of heterotrophic organisms (Y H) and PAOs (Y PAO), were high at both temperatures. These calibrated parameters imply that the extremely active PAOs of the study were able to store PHA, store polyphosphate and even utilize PHA for cell growth. Besides, the parameters do not follow the Arrhenius correlation due to the previously reported unique microbial clade at 28 and 32°C, which actively performs EBPR at high temperatures.

High-temperature EBPR process: The performance, analysis of PAOs and GAOs and the fine-scale population study of Candidatus “Accumulibacter phosphatis”

The applicability of the enhanced biological phosphorus removal (EBPR) process for the removal of phosphorus in warm climates is uncertain due to frequent reports of EBPR deterioration at temperature higher than 25 °C. Nevertheless, a recent report on a stable and efficient EBPR process at 28 °C has inspired the present study to examine the performance of EBPR at 24 °C–32 °C, as well as the PAOs and GAOs involved, in greater detail. Two sequencing batch reactors (SBRs) were operated for EBPR in parallel at different temperatures, i.e., SBR-1 at 28 °C and SBR-2 first at 24 °C and subsequently at 32 °C. Both SBRs exhibited high phosphorus removal efficiencies at all three temperatures and produced effluents with phosphorus concentrations less than 1.0 mg/L during the steady state of reactor operation. Real-time quantitative polymerase chain reaction (qPCR) revealed Accumulibacter-PAOs comprised 64% of the total bacterial population at 24 °C, 43% at 28 °C and 19% at 32 °C. Based on fluorescent in situ hybridisation (FISH), the abundance of Competibacter-GAOs at both 24 °C and 28 °C was rather low (<10%), while it accounted for 40% of the total bacterial population at 32 °C. However, the smaller Accumulibacter population and larger population of Competibacter at 32 °C did not deteriorate the phosphorus removal performance. A polyphosphate kinase 1 (ppk1)-based qPCR analysis on all studied EBPR processes detected only Accumulibacter clade IIF. The Accumulibacter population shown by 16S rRNA and ppk1 was not significantly different. This finding confirmed the existence of single clade IIF in the processes and the specificity of the clade IIF primer sets designed in this study. Habitat filtering related to temperature could have contributed to the presence of a unique clade. The clade IIF was hypothesised to be able to perform the EBPR activity at high temperatures. The clade's robustness most likely helps it to fit the high-temperature EBPR sludge best and allows it not only to outcompete other Accumulibacter clades but coexist with GAOs without compromising EBPR activity.

Metabolic Behavior and Enzymatic Aspects of Denitrifying EBPR Sludge in a Continuous-Flow Anaerobic–Anoxic System

The metabolic aspects of enhanced biological phosphorus removal (EBPR) were investigated for the first time in a continuous-flow anaerobic-anoxic plant fed with acetate, propionate, or substrates which are involved in the tricarboxylic acid and/or glyoxylate cycle, i.e., fumarate, malate, or oxaloacetate, as the sole carbon source. Although the polyphosphate-accumulating organisms (PAOs) population remained stable with any carbon source examined, no typical EBPR metabolism was observed during fumarate, malate, or oxaloacetate utilization. Specific enzymatic activities related to EBPR were determined in activated sludge homogenates and directly correlated with the nutrient metabolic rates. The experimental results indicated the direct involvement of alkaline phosphatase, pyrophosphatase, and exopolyphosphatase in the denitrifying EBPR process. Metabolic aspects of glyoxylate cycle enzymes are discussed with regard to the biomass anaerobic and anoxic activity. Process performance was highly influenced by the kind of substrate utilized, indicating that specific metabolic pathways should be followed to favor efficient EBPR.

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 2,500mg MLVSS/L resulted in the complete phosphate removal from the influent (i.e. 15mg PO(4) (3-)-P/L) for a mean hydraulic residence time (HRT) of 15h. Additionally, by increasing the influent COD/P ratio from 10 to 20g/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 2h 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.

Evidence and Long-Term Feasibility of Enhanced Biological Phosphorus Removal in Oxidation-Ditch Type of Aerated-Anoxic Activated Sludge Systems

This study investigated the potential of four full-scale oxidation ditches to accomplish enhanced biological phosphorus removal (EBPR). Despite the fact that none of the tested oxidation ditches were designed to perform EBPR, mixed liquors from all four ditches showed good specific phosphorus release and uptake rates, a typical characteristic of a typical EBPR biomass. The specific phosphorus release rates ranged from 0.042- to 0.254-mg P/mg VSS-d and the specific phosphorus uptake rates ranged from 0.023- to 0.125-mg P/mg VSS-d for the tested full-scale plants. The EBPR potential of one of the full-scale plants (Central Davis Sewer District) was further studied by changing the aeration patterns in the ditch. The mixed liquor in this full-scale plant exhibited good phosphorus release and uptake trends and dissolved phosphorus, as low as 1.26 mg/L, could be accomplished in the final effluent of this plant as a result of this optimization. The long-term feasibility of the EBPR in this full-scale was tested by running a bench-scale EBPR reactor, in which the anaerobic phase was replaced with aerated-anaerobic phase to simulate the mixed liquor environment that exists in Central Davis mixed liquor and, in general, in all oxidation-ditch-type activated sludge configurations. The bench-scale reactor showed consistent EBPR activity in the absence of an anaerobic environment and more than 85% phosphorus removal could be maintained in the reactor for more than 400 days. The intrafloc microanaerobic/anoxic zones, which may be present in the mixed liquor, did not seem to add to the EBPR efficiency under aerated-anaerobic conditions. Cloning and sequencing using Rhodocyclus specific forward primer RHC439 showed the abundance of organisms most closely falling in Rhodocyclaceae family but not related to CandidatusAccumulibacter phosphatis. Simultaneous 4′-6–Diamidino-2–phenylindole (DAPI) staining and fluorescent in situ hybridization (FISH) using RHC439 probe clearly demonstrated the participation of polyphosphate accumulating organism (PAOs) targeted by RHC439 (i.e., in Rhodocyclaceae family). Microautoradiography assisted FISH using RHC439 further confirmed the participation of PAOs in Rhodocyclaceae family.

Maintenance of phosphorus removal in an EBPR system under permanent aerobic conditions using propionate

Enhanced biological phosphorus removal (EBPR) is an efficient and sustainable technology to remove phosphorus from wastewater preventing eutrophication in natural waters. It is widely accepted that EBPR requires an optimal anaerobic hydraulic retention time to obtain stable P-removal from wastewater. Thus, it is suggested that deterioration of the EBPR efficiency regularly observed in full-scale wastewater treatment plants (WWTPs) is normally caused by an excessive aeration of activated sludge that increments the amount of oxygen recycled to the anaerobic reactor and consequently, the anaerobic conditions are not totally preserved. Furthermore, it has been reported a progressive decrease in P-removal capacity in an EBPR lab-scale system enriched with acetate as the sole carbon source under permanent aerobic conditions. Hence, to evaluate the stability of P-removal with a different carbon source, an EBPR-SBR was operated with propionate under permanent aerobic conditions. As a result, net P-removal was successfully accomplished in the SBR without any anaerobic phase during 46 days of aerobic operation. Moreover, the system was shifted after this period to the standard anaerobic–aerobic conditions and reliable P-removal was maintained. FISH (fluorescence in situ hybridisation) analysis showed a significant presence of Accumulibacter (70, 50 and 72%, in different periods) and the absence of Competibacter. The results indicate that using propionate as carbon source it is possible to maintain in a long term an enriched culture of phosphorus accumulating organisms (PAO) able to remove phosphorus under permanent aerobic conditions.

Critical factors affecting biological phosphorus removal in dairy wastewater treatment plants

Operational data from nine dairy wastewater plants (DWTPs) in Wisconsin were collected and analyzed to determine reasons for poor enhanced biological phosphorus removal (EBPR) efficiency. Several factors affecting EBPR performances in dairy were identified. Since many dairies operate five days a week, DWTPs suffer low F/M ratios during off-days, leading to sludge bulking and unstable EBPR efficiency. The most pronounced factor affecting EBPR performance was uneven organic loading caused by lack of an equalization tank or too a small volume to alleviate the fluctuation in flow and organic loading. The other factor was imbalance of nutrients, especially nitrogen. The other factors include sudden change of pH in a matter of hours and higher temperature (> 30°C) in the summer. The high temperature was thought to shift microbial population and thus lead to the loss of EBPR capability. Unexpected discharge of cleaning solution was another problem causing poor EBPR and COD removal efficiencies. Laboratory-scale tests confirmed the effects of the COD/P ratio (organic loading) and pH on EBPR efficiency. Microscopic examination showed the presence of tetrad-arranged coccoid cells, called G-bacteria in five out of nine DWTPs. Rhodocyclus-related PAOs were also detected from Fluorescent in situ hybridization (FISH) analysis.

Competition between Polyphosphate‐ and Glycogen‐Accumulating Organisms in Enhanced‐Biological‐Phosphorus‐Removal Systems: Effect of Temperature and Sludge Age

Temperature and sludge age were found to be important factors in determining the outcome of competition between polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating non-polyphosphate organisms (GAOs) and the resultant stability of enhanced-biological-phosphorus removal (EBPR). At 20 degrees C and a 10-day sludge age, PAOs were dominant in an anaerobic/aerobic (A/O) sequencing-batch reactor (SBR), as a result of their higher anaerobic-acetate-uptake rate and aerobic-biomass yield than GAOs. However, at 30 degrees C and a 10-day sludge age, GAOs were able to outcompete PAOs in the A/O SBR because of their higher anaerobic-acetate-uptake rate than PAOs. At 30 degrees C and a 5-day sludge age, GAOs coexisted with PAOs in the A/O SBR, resulting in unstable EBPR performance. At 30 degrees C, reducing the sludge age from 5 to 3 days improved the EBPR efficiency drastically, and the EBPR performance was stable. The maximum specific-anaerobic-acetate-uptake rates of GAO-enriched sludge were affected by temperature with the Arrhenius temperature coefficient theta of 0.042 (degrees C(-1) between 10 and 30 degrees C. The effect of sludge age (5 and 10 days) on the maximum specific-anaerobic-acetate-uptake rates of GAO-enriched activated sludge, however, was not significant. For the PAO-enriched activated sludge, the maximum specific-anaerobic-acetate-uptake rate did not change significantly between 20 and 30 degrees C, but significantly increased from 0.38 to 0.52 mmol-C/ mmol-C/h as the sludge age decreased from 10 to 3 days at 30 degrees C.

Evidence for Tin Inhibition of Enhanced Biological Phosphorus Removal at a Municipal Wastewater Treatment Plant

Concentrations of 34 metals were determined in the concentrated waste activated sludge from a dissolved air flotation unit at an advanced municipal wastewater treatment plant (WWTP) with biological nutrient removal. Reduction in enhanced biological phosphorus removal (EBPR) efficiency was observed at total tin concentrations greater than 4 μg L−1 in the solids fraction of the mixed liquor suspended solids. No influence on carbon or nitrogen removal efficiency was found by elevated tin concentrations. Other process control variables and metal concentrations were not correlated with reduced EBPR efficiency on dates with elevated tin levels. The known high contributions of organotin species toward total tin in activated sludges from other municipal WWTPs, and the high toxicity of these compounds, suggests elevated organotin levels may be responsible for the observed reduction in EBPR efficiency.

The competition between PAOs (phosphorus accumulating organisms) and GAOs (glycogen accumulating organisms) in EBPR (enhanced biological phosphorus removal) systems at different temperatures and the effects on system performance

It is well known and firmly established that the rate of chemical and biochemical reactions slow down as temperature decreases. Nevertheless, several studies have reported that the efficiency of enhanced biological phosphorus removal (EBPR) improves as temperature decreases. However, several recent studies have reported that EBPR reaction rates decrease with temperature decrease in accordance with the Arrhenius relationship. This study was designed to more thoroughly investigate this controversy using two UCT plants fed with a synthetic wastewater consisting primarily of acetate as the COD form, and a small amount of supplemental yeast extract. Experiments were performed over temperatures ranging from 5 to 20 degrees C. The results showed that, even though the kinetic rates decrease as temperature decreases, EBPR systems perform better at colder temperatures. The reason for better system performance is apparently related to reduced competition for substrate in the non-oxic zones, which results in an increased population of PAOs and, thus, greater EBPR efficiency. The proliferation of PAOs apparently occurs because they are psychrophilic whereas their competitors are not. The experiments showed that the EBPR sludges accumulated high concentrations of both PHA and glycogen at 20 degrees C, but accumulated more PHA and much less glycogen at 5 degrees C. Although the results could be interpreted as the result of changes in the PAO-GAO competition, Mann-Whitney non-parametric comparisons of transmission electron microscopy examinations revealed no indication of the presence of GAOs population under any temperature conditions. Regardless, mass balances of the glycogen data showed that the involvement of glycogen is less at cold temperature, even though EBPR was greater. Unlike current EBPR models (e.g. Mino model), the results suggest that glycogen metabolism is not a precursor for EBPR biochemistry. The results also indicate that temperature not only may cause selective pressure on the dominant organisms, but also may force them to use a different metabolic pathway as temperature decreases.

Glycogen-accumulating organisms in laboratory-scale and full-scale wastewater treatment processes.

Laboratory-scale sequencing batch reactors (SBRs) as models for wastewater treatment processes were used to identify glycogen-accumulating organisms (GAOs), which are thought to be responsible for the deterioration of enhanced biological phosphorus removal (EBPR). The SBRs (called Q and T), operated under alternating anaerobic-aerobic conditions typical for EBPR, generated mixed microbial communities (sludges) demonstrating the GAO phenotype. Intracellular glycogen and poly-beta-hydroxyalkanoate (PHA) transformations typical of efficient EBPR occurred but polyphosphate was not bioaccumulated and the sludges contained 1.8% P (sludge Q) and 1.5% P (sludge T). 16S rDNA clone libraries were prepared from DNA extracted from the Q and T sludges. Clone inserts were grouped into operational taxonomic units (OTUs) by restriction fragment length polymorphism banding profiles. OTU representatives were sequenced and phylogenetically analysed. The Q sludge library comprised four OTUs and all six determined sequences were 99.7% identical, forming a cluster in the gamma-Proteobacteria radiation. The T sludge library comprised eight OTUs and the majority of clones were Acidobacteria subphylum 4 (49% of the library) and candidate phylum OP10 (39% of the library). One OTU (two clones, of which one was sequenced) was in the gamma-Proteobacteria radiation with 95% sequence identity to the Q sludge clones. Oligonucleotide probes (called GAOQ431 and GAOQ989) were designed from the gamma-Proteobacteria clone sequences for use in fluorescence in situ hybridization (FISH); 92% of the Q sludge bacteria and 28% of the T sludge bacteria bound these probes in FISH. FISH and post-FISH chemical staining for PHA were used to determine that bacteria from a novel gamma-Proteobacteria cluster were phenotypically GAOs in one laboratory-scale SBR and two full-scale wastewater treatment plants. It is suggested that the GAOs from the novel cluster in the gamma-Proteobacteria radiation be named 'Candidatus Competibacter phosphatis'.

Analysis of the microbial community structure and function of a laboratory scale enhanced biological phosphorus removal reactor.

A laboratory scale sequencing batch reactor (SBR) operating for enhanced biological phosphorus removal (EBPR) and fed with a mixture of volatile fatty acids (VFAs) showed stable and efficient EBPR capacity over a four-year-period. Phosphorus (P), poly-beta-hydroxyalkanoate (PHA) and glycogen cycling consistent with classical anaerobic/aerobic EBPR were demonstrated with the order of anaerobic VFA uptake being propionate, acetate then butyrate. The SBR was operated without pH control and 63.67 +/- 13.86 mg P l-1 was released anaerobically. The P% of the sludge fluctuated between 6% and 10% over the operating period (average of 8.04 +/- 1.31%). Four main morphological types of floc-forming bacteria were observed in the sludge during one year of in-tensive microscopic observation. Two of them were mainly responsible for anaerobic/aerobic P and PHA transformations. Fluorescence in situ hybridization (FISH) and post-FISH chemical staining for intracellular polyphosphate and PHA were used to determine that 'Candidatus Accumulibacter phosphatis' was the most abundant polyphosphate accumulating organism (PAO), forming large clusters of coccobacilli (1.0-1.5 micro m) and comprising 53% of the sludge bacteria. Also by these methods, large coccobacillus-shaped gammaproteobacteria (2.5-3.5 micro m) from a recently described novel cluster were glycogen-accumulating organisms (GAOs) comprising 13% of the bacteria. Tetrad-forming organisms (TFOs) consistent with the 'G bacterium' morphotype were alphaproteobacteria, but not Amaricoccus spp., and comprised 25% of all bacteria. According to chemical staining, TFOs were occasionally able to store PHA anaerobically and utilize it aerobically.

In situ identification of polyphosphate- and polyhydroxyalkanoate-accumulating traits for microbial populations in a biological phosphorus removal process.

Polyphosphate- and polyhydroxyalkanoate (PHA)-accumulating traits of predominant microorganisms in an efficient enhanced biological phosphorus removal (EBPR) process were investigated systematically using a suite of non-culture-dependent methods. Results of 16S rDNA clone library and fluorescence in situ hybridization (FISH) with rRNA-targeted, group-specific oligonucleotide probes indicated that the microbial community consisted mostly of the alpha- (9.5% of total cells), beta- (41.3%) and gamma- (6.8%) subclasses of the class Proteobacteria, Flexibacter-Cytophaga (4.5%) and the Gram-positive high G+C (HGC) group (17.9%). With individual phylogenetic groups or subgroups, members of Candidatus Accumulibacter phosphatis in the beta-2 subclass, a novel HGC group closely related to Tetrasphaera spp., and a novel gamma-proteobacterial group were the predominant populations. Furthermore, electron microscopy with energy-dispersive X-ray analysis was used to validate the staining specificity of 4,6-diamino-2-phenylindole (DAPI) for intracellular polyphosphate and revealed the composition of polyphosphate granules accumulated in predominant bacteria as mostly P, Ca and Na. As a result, DAPI and PHA staining procedures could be combined with FISH to identify directly the polyphosphate- and PHA-accumulating traits of different phylogenetic groups. Members of Accumulibacter phosphatis and the novel gamma-proteobacterial group were observed to accumulate both polyphosphate and PHA. In addition, one novel rod-shaped group, closely related to coccus-shaped Tetrasphaera, and one filamentous group resembling Candidatus Nostocoidia limicola in the HGC group were found to accumulate polyphosphate but not PHA. No cellular inclusions were detected in most members of the alpha-Proteobacteria and the Cytophaga-Flavobacterium group. The diversified functional traits observed suggested that different substrate metabolisms were used by predominant phylogenetic groups in EBPR processes.

COMPETITION BETWEEN POLYPHOSPHATE- AND GLYCOGEN-ACCUMULATING ORGANISMS IN ENHANCED BIOLOGICAL PHOSPHORUS REMOVAL SYSTEMS—EFFECT OF TEMPERATURE AND SLUDGE AGE

Temperature and sludge age were found to be important factors in determining the outcome of competition between polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating non-polyphosphate organisms (GAOs) and the resultant stability of enhanced-biological-phosphorus removal (EBPR). At 20 degrees C and a 10-day sludge age, PAOs were dominant in an anaerobic/aerobic (A/O) sequencing-batch reactor (SBR), as a result of their higher anaerobic-acetate-uptake rate and aerobic-biomass yield than GAOs. However, at 30 degrees C and a 10-day sludge age, GAOs were able to outcompete PAOs in the A/O SBR because of their higher anaerobic-acetate-uptake rate than PAOs. At 30 degrees C and a 5-day sludge age, GAOs coexisted with PAOs in the A/O SBR, resulting in unstable EBPR performance. At 30 degrees C, reducing the sludge age from 5 to 3 days improved the EBPR efficiency drastically, and the EBPR performance was stable. The maximum specific-anaerobic-acetate-uptake rates of GAO-enriched sludge were affected by temperature with the Arrhenius temperature coefficient theta of 0.042 (degrees C(-1) between 10 and 30 degrees C. The effect of sludge age (5 and 10 days) on the maximum specific-anaerobic-acetate-uptake rates of GAO-enriched activated sludge, however, was not significant. For the PAO-enriched activated sludge, the maximum specific-anaerobic-acetate-uptake rate did not change significantly between 20 and 30 degrees C, but significantly increased from 0.38 to 0.52 mmol-C/ mmol-C/h as the sludge age decreased from 10 to 3 days at 30 degrees C.

The influence of different substrates on enhanced biological phosphorus removal in a sequencing batch reactor

All models of enhanced biological phosphorus removal (EBPR) define fermentable readily biodegradable substrate, without emphasizing the significance of its composition and the relative importance of different substrates. On the other hand, it is also known that substrates like glucose may be utilized without requiring poly-P energy, a phenomenon which deteriorates the EBPR performance. This paper reports an experimental study evaluating the effect of different organic substrates and their combinations on EBPR, in a sequencing batch reactor. Experimental data show that the EBPR efficiency is significantly affected by the increase of the glucose fraction in the feed, due to the probable dominance of G bacteria. Results of anaerobic batch tests also support this evaluation.

The influence of different substrates on enhanced biological phosphorus removal in a sequencing batch reactor

All models of enhanced biological phosphorus removal (EBPR) define fermentable readily biodegradable substrate, without emphasizing the significance of its composition and the relative importance of different substrates. On the other hand, it is also known that substrates like glucose may be utilized without requiring poly-P energy, a phenomenon which deteriorates the EBPR performance. This paper reports an experimental study evaluating the effect of different organic substrates and their combinations on EBPR, in a sequencing batch reactor. Experimental data show that the EBPR efficiency is significantly affected by the increase of the glucose fraction in the feed, due to the probable dominance of G bacteria. Results of anaerobic batch tests also support this evaluation.