Temporal Dynamics and Performance Association of the Tetrasphaera-Enriched Microbiome for Enhanced Biological Phosphorus Removal

Tetrasphaera have been recently identified based on the 16S ribosomal RNA(rRNA)gene as among the most abundant polyphosphate-accumulating organisms(PAOs)in global full-scale wastewater treatment plants(WWTPs)with enhanced biological phosphorus removal(EBPR).However,it is unclear how Tetrasphaera PAOs are selectively enriched in the context of the EBPR microbiome.In this study,an EBPR microbiome enriched with Tetrasphaera(accounting for 40%of 16S sequences on day 113)was built using a top-down design approach featuring multicarbon sources and a low dosage of allylthiourea.The microbiome showed enhanced nutrient removal(phosphorus removal~85%and nitrogen removal~80%)and increased phosphorus recovery(up to 23.2 times)compared with the seeding activated sludge from a local full-scale WWTP.The supply of 1 mg·L^(-1)allylthiourea promoted the coselection of Tetrasphaera PAOs and Microlunatus PAOs and sharply reduced the relative abundance of both ammonia oxidizer Nitrosomonas and putative competitors Brevundimonas and Paracoccus,facilitating the establishment of the EBPR microbiome.Based on 16S rRNA gene analysis,a putative novel PAO species,EBPR-ASV0001,was identified with Tetrasphaera japonica as its closest relative.This study provides new knowledge on the establishment of a Tetrasphaera-enriched microbiome facilitated by allylthiourea,which can be further exploited to guide future process upgrading and optimization to achieve and/or enhance simultaneous biological phosphorus and nitrogen removal from high-strength wastewater.

Screening of Polyphosphate Accumulating Organisms and Their Phosphorus Removal Performance

[Objectives]To study the phosphorus removal performance of phosphate accumulating organisms(PAOs).[Methods]Activated sludge from domestic sewage treatment plant was used as the strain source,and phosphate accumulating organisms were screened by plate streaking method and dilution coating plate method.Six kinds of excellent phosphate accumulating organisms were obtained by metachromatic granule staining experiment,total phosphorus experiment and simulated sewage phosphorus removal experiment to assist the observation of bac-terial morphology and experiment of phosphorus removal capacity.In addition,the influencing factors of phosphorus removal capacity(nitrogen source,trace metal ions)were analyzed.[Results]In the case of simulated sewage,the phosphorus removal rate of strain b was the highest,reaching 66.25%,while the phosphorus removal rate of strain e and f was about 10%lower than that of the phosphorus uptake experiment.[Conclusions]This study is expected to provide a theoretical reference for the gradual optimization of the screening method of phosphorus re-moval bacteria in domestic sewage treatment.

Enhanced Biological Phosphorus Removal with Pseudomonas putida GM6 from Activated Sludge

The enhanced biological phosphorus removal （EBPR） method is widely adopted for phosphorus removal from wastewater, yet little is known about its microbiological and molecular mechanisms. Therefore, it is difficult to predict and control the deterioration of the EBPR process in a large-scale municipal sewage treatment plant. This study used a novel strain isolated in the laboratory, Pseudomonas putida GM6, which had a high phosphate accumulating ability and could recover rapidly from the deteriorated system and enhance the capability of phosphorus removal in activated sludge. Strain GM6 marked with gfp gene, which was called GMTR, was delivered into a bench-scale sequencing batch reactor （SBR） of low efficiency, to investigate the colonization of GMTR and removal of phosphorus. After 21 days, the proportion of GMTR in the total bacteria of the sludge reached 9.2%, whereas the phosphorus removal rate was 96%, with an effluent concentration of about 0.2 mg L^-1. In the reactor with the addition of GMTR, phosphorus was removed quickly, in 1 h under anaerobic conditions, and in 2 h under aerobic conditions. These evidences were characteristic of EBPR processes. Field testing was conducted at a hospital sewage treatment facility with low phosphorus removal capability. Twentyone days after Pseudomonas putida GM6 was added, effluent phosphorus concentration remained around 0.3 mg L^-1, corresponding to a removal rate of 96.8%. It was therefore demonstrated that Pseudomonas putida GM6 could be used for a quick startup and enhancement of wastewater biological phosphorus removal, which provided a scientific basis for potential large-scale engineering application.

Effect of Ferric Chloride on the Properties of Biological Sludge in Co-precipitation Phosphorus Removal Process

This paper studied the effect of ferric chloride on waste sludge digestion,dewatering and sedimentation under the optimized doses in co-precipitation phosphorus removal process.The experimental results showed that the concentration of mixed liquid suspended solid(MLSS) was 2436 mg.L-1 and 2385 mg.L-1 in co-precipitation phosphorus removal process(CPR) and biological phosphorous removal process(BPR),respectively.The sludge reduction ratio for each process was 22.6% and 24.6% in aerobic digestion,and 27.6% and 29.9% in anaerobic digestion,respectively.Due to the addition of chemical to the end of aeration tank,the sludge content of CPR was slightly higher than that of BPR,but the sludge reduction rate for both processes had no distinct difference.The sludge volume index(SVI) and sludge specific resistance of BPR were 126 ml.g-1 and 11.7×1012 m.kg-1,respectively,while those of CPR were only 98 ml.g-1 and 7.1×1012 m.kg-1,indicating that CPR chemical could improve sludge settling and dewatering.

Comparing results of cultured and uncultured biological methods used in biological phosphorus removal

Increasing attention has been paid to phosphate-accumulating organisms （PAOs） for their important role in biological phosphorus removal. In this study, microbial communities of PAOs cultivated under different carbon sources （sewage, glucose, and sodium acetate） were investigated and compared through culture-dependent and culture-independent methods, respectively. The results obtained using denaturing gradient gel electrophoresis （DGGE） of polymerase chain reaction-amplified 16S rDNA fragments revealed that the diversity of bacteria in a sewage-fed reactor （1#） was much higher than in a glucose-fed one （2#） and a sodium acetate-fed one （3#）; there were common PAOs in three reactors fed by different carbon sources. Five strains were separated from three systems by using a phosphaterich medium; they were from common bacteria isolated and three isolates could not be found in DGGE profile at all. Two isolates had good phosphorus removal ability. When the microbial diversity was studied, the molecular biological method was better than the culture-dependent one. When phosphorus removal characteristics were investigated, culture-dependent approach was more effective. Thus a combination of two methods is necessary to have a comprehensive view of PAOs.

Effects of COD to Phosphorus Ratios on the Metabolism of PAOs in Enhanced Biological Phosphorus Removal with Different Carbon Sources

To elucidate the phosphorus removal and metabolism under various COD / P ratio,a sludge highly enriched in PAOs was used to investigate the impacts of COD / P in batch tests under different carbon supply conditions. Acetate,propionate and a mixture of acetate and propionate at a ratio of 3 ∶ 1( COD basis) was used as carbon sources with the COD / P of 20,15,10 and 5. 0 g COD /gP,respectively. The minimum COD / P ratios for complete P removal were found to be 8. 24 g COD /gP for acetate,11. 40 g COD /gP for propionate and9. 10 g COD /gP for the 3 ∶ 1 mixture of acetate and propionate. Converted to a mass basis,all three cases had a very similar ratio of 7. 7 g VFA /gP,which represented a useful guide for operation of EBPR plants to identify possible shortages in VFAs. The trend in PHV accumulation during the anaerobic period along with the decrease of COD / P ratios suggested that,PAOs may use the TCA pathway for anaerobic VFA uptake to maintain the required NADH production with reduced glycogen degradation. During the aerobic phase,the glycogen pool was reduced but remained enough compared to the requirement for anaerobic VFA uptake,and the synthesis and degradation of glycogen was not the inhibition factor of PAOs.

Role of extracellular exopolymers on biological phosphorus removal

Three sequencing batch reactors supplied with different carbon sources were investigated. The system supplied with glucose gained the best enhanced biological phosphorus removal although all of the three reactors were seeded from the same sludge. With the measurement of poly-β-hydroxyalkanoate （PHA） concentration, phosphorus content in sludge and extracellular exopolymers （EPS） with scanning electron microscopy （SEM） combined with energy dispersive spectrometry （EDS）, it was found that the biosorption effect of EPS played an important role in phosphorus removal and that the amount of PHA at the end of anaerobic phase was not the only key factor to determine the following phosphorus removal efficiency.

Enhanced Biological Phosphorus Removal in Anaerobic/Aerobic Sequencing Batch Reactor Supplied with Glucose as Carbon Source

Phosphorus removal performance in an aerobic/aerobic sequencing batch reactor (SBR) supplied with glucose as carbon source was investigated. It was found that there was no phosphate release concomitant with the storing of poly-β-hydroxyalkanoate (PHA) during the anaerobic phase. Whereas, glycogen was soon built up followed by rapid consumption, at the same time, glucose was depleted rapidly. Based on the analysis of different fractions of phosphorus in activated sludge, the relative ratio of organically bound phosphorus in sludge changed at the end of anaerobic and aerobic phases. The ratios were 45.3% and 51.8% respectively. This showed that the polyphosphate broke down during the anaerobic phase to supply part of energy for PHA synthesis. The reason why there was no phosphate release might be the biosorption effect of extracellular exopolymers (EPS). It was also proved by the analysis of EPS with scanning electron microscopy (SEM) combined with energy dispersive spectrometry (EDS). The phosphorus weight percentage of EPS at the end of anaerobic phase was 9.22%.

Enhanced Biological Phosphorus Removal in SBR Using Glucose as a Single Organic Substrate

Enhanced biological phosphorus removal(EBPR) was investigated in an anaerobic/aerobic sequencing batch reactor(SBR) supplied with glucose as a single organic substrate.The results illustrated that EBPR process could also occur successfully with glucose other than short chain fatty acids(SCFAs).High phosphorus release and poly-hydroxyalkanoate(PHA) accumulation in the anaerobic phase was found vital for the removal of phosphorus during the aerobic phase.The measurement of intracellular reserves revealed that glycogen had a higher chance to replace the energy role of poly-P under anaerobic conditions.Moreover,glycogen was also utilized as the carbon source for PHA synthesis,as well as a reducing power as reported earlier.The accumulated PHA in this system was mainly in the form of poly-hydroxyvalerate(PHV) instead of poly-hydroxybutyrate(PHB),and was inferred to be caused by the excess reducing power contained in glucose.Lactate as a fermentation product was also found released into the bulk solution.Applying fundamental biochemistry knowledge to the experimental results,a conceptual biochemical model was developed to explain the metabolism of the glucose-induced EBPR.

Performance of biological phosphorus removal and characteristics of microbial community in the oxic-settling-anaerobic process by FISH analysis

Performance of biological phosphorus removal in the oxic-settling-anaerobic(OSA) process was investigated. Cell staining and fluorescent in situ hybridization(FISH) were used to analyze characteristics and microbial community of sludge. Experimental results showed that phosphorus removal efficiency was near 60% and the amount of biological phosphorus accumulation in aerobic sludge of the OSA system was up to 26.9 mg/g. Biological phosphorus removal efficiency was partially inhibited by carbon sources in the continuous OSA system. Contrasted to the OSA system,biological phosphorus removal efficiency was enhanced by 14% and the average total phosphorus(TP) contents of aerobic sludge were increased by 0.36 mg/g when sufficient carbon sources were supplied in batch experiments. Staining methods indicated that about 35% of microorganisms had typical characteristics of phosphorus accumulating organisms(PAOs) . FISH analysis demonstrated that PAOMIX-binding bacteria were predominant microbial communities in the OSA system,which accounted for around 28% of total bacteria.

Enhanced Nutrient Removal with Upflow Biological Aerated Filter for Reclaimed Water

A two-stage upflow biological aerated filter was designed as an advanced treatment process to optimize the operating parameters and study the correlative factors influencing the efficiency of nitrification, denitrification and phosphorus removal. The experimental results showed that the final effluent of the two-stage upflow biofilter process operated in series could meet the stringent limits of the reclaimed water for the total nitrogen of 2 mg/L, and total phosphorus of 0.3 mg/L. The high treatment efficiency allowed the reactor operating at very high hydraulic loadings and reaching nearly complete nitrification and denitrifieation.

Nitrogen and phosphorus removal in pilot-scale anaerobic-anoxic oxidation ditch system

To achieve high efficiency of nitrogen and phosphorus removal and to investigate the rule of simultaneous nitrification and denitrification phosphorus removal （SNDPR）, a whole course of SNDPR damage and recovery was studied in a pilot-scale, anaerobicanoxic oxidation ditch （OD）, where the volumes of anaerobic zone, anoxic zone, and ditches zone of the OD system were 7, 21, and 280 L, respectively. The reactor was fed with municipal wastewater with a flow rate of 336 L/d. The concept of simultaneous nitrification and denitrification （SND） rate （rSND） was put forward to quantify SND. The results indicate that： （1） high nitrogen and phosphorus removal efficiencies were achieved during the stable SND phase, total nitrogen （TN） and total phosphate （TP） removal rates were 80% and 85%, respectively; （2） when the system was aerated excessively, the stability of SND was damaged, and rSND dropped from 80% to 20% or less; （3） the natural logarithm of the ratio of NOx to NH4^＋ in the effluent had a linear correlation to oxidation-reduction potential （ORP）; （4） when NO3^- was less than 6 mg/L, high phosphorus removal efficiency could be achieved; （5） denitrifying phosphorus removal （DNPR） could take place in the anaerobic-anoxic OD system. The major innovation was that the SND rate was devised and quantified.

Denitrifying phosphorus removal in a step-feed CAST with alternating anoxic-oxic operational strategy

A bench-scale cyclic activated sludge technology （CAST） was operated to study the biological phosphorus removal performance and a series of batch tests was carried out to demonstrate the accumulation of denitrifying polyphosphate-accumulating organisms （DNPAOs） in CAST system. Under all operating conditions, step-feed CAST with enough carbon sources in influent had the highest nitrogen and phosphorus removal efficiency as well as good sludge settling performance. The average removal rate of COD, NH4^＋-N, PO4^3--P and total nitrogen （TN） was 88.2%, 98.7%, 97.5% and 92.1%, respectively. The average sludge volume index （SVI） was 133 mL/g. The optimum anaerobic/aerobic/anoxic （AOA） conditions for the cultivation of DNPAOs could be achieved by alternating anoxic/oxic operational strategy, thus a significant denitrifying phosphorus removal occurred in step-feed CAST. The denitrification of NO^x--N completed quickly due to step-feed operation and enough carbon sources, which could enhance phosphorus release and further phosphorus uptake capability of the system. Batch tests also proved that polyphosphate-accumulating organisms （PAOs） in the step-feed process had strong denitrifying phosphorus removal capacity. Both nitrate and nitrite could be used as electron acceptors in denitrifying phosphorus removal. Low COD supply with step-feed operation strategy would favor DNPAOs accumulation.

Removal of nitrogen and phosphorus in a combined A^2/O-BAF system with a short aerobic SRT

A bench-scale anaerobic/anoxic/aerobic process-biological aerated filter （A^2/O-BAF） combined system was carded out to treat wastewater with lower C/N and C/P ratios. The A^2/O process was operated in a short aerobic sludge retention time （SRT） for organic pollutants and phosphorus removal, and denitrification. The subsequent BAF process was mainly used for nitrification. The BAF effluent was partially returned to anoxic zone of the A^2/O process to provide electron acceptors for denitrification and anoxic P uptake. This unique system formed an environment for reproducing the denitdfying phosphate-accumulating organisms （DPAOs）. The ratio of DPAOs to phosphorus accumulating organisms （PAOs） could be maintained at 28% by optimizing the organic loads in the anaerobic zone and the nitrate loads into the anoxic zone in the A^2/O process. The aerobic phosphorus over-uptake and discharge of excess activated sludge was the main mechanism of phosphorus removal in the combined system. The aerobic SRT of the A^2/O process should meet the demands for the development of aerobic PAOs and the restraint on the nitrifiers growth, and the contact time in the aerobic zone of the A^2/O process should be longer than 30 min, which ensured efficient phosphorus removal in the combined system. The adequate BAF effluent return rates should be controlled with 1--4 mg/L nitrate nitrogen in the anoxic zone effluent of A^2/O process to achieve the optimal nitrogen and phosphorus removal efficiencies.

Effect of carbon source and nitrate concentration on denitrifying phosphorus removal by DPB sludge

Effect of added carbon source and nitrate concentration on the denitrifying phosphorus removal by DPB sludge was systematically studied using batch experiments, at the same time the variation of ORP was investigated. Results showed that the denitrifying and phosphorus uptake rate in anoxic phase increased with the high initial anaerobic carbon source addition. However once the initial COD concentration reached a certain level, which was in excess to the PHB saturation of poly-P bacteria, residual COD carried over to anoxic phase inhibited the subsequent denitrifying phosphorus uptake. Simultaneously, phosphate uptake continued until all nitrate was removed, following a slow endogenous release of phosphate. High nitrate concentration in anoxic phase increased the initial denitrifying phosphorus rate. Once the nitrate was exhausted, phosphate uptake changed to release. Moreover, the time of this turning point occurred later with the higher nitrate addition. On the other hand, through on-line monitoring the variation of the ORP with different initial COD concentration, it was found ORP could be used as a control parameter for phosphorus release, but it is impossible to utilize ORP for controlling the denitrificaion and anoxic phosphorus uptake operations.

Isolation and Identification of Phosphate-accumulating Strain PAO3-1 and Its Phosphorus Removal Characteristics

A phosphate-accumulating bacteria strain PAO3-1 was isolated from biological phosphorus removal sludge supplied with sodium acetate as carbon source under stable performance. This strain has good enhanced biological phosphorus removal effect on normal activated sludge system. Phosphorus removal ratio was raised form 44% with no added strain to more than 82% with strain strengthening biological phosphorus removal. It is identified to be Alcaligenes sp. according to its morphology, biochemical characteristics and 16S rDNA sequence analysis. The cell of strain PAO3-1 is straight bacilli form, 0.4×1.1μm, no flagellum, gram negative and special aerobiotic. The optimal temperature and pH for growth are 32℃-37℃ and 5.5-9.5, respectively. The shape of slant clone is feathery. The phosphate accumulating rate of strain PAO3-1 was 8.1mgP/g cell·h, and 14.3 mgP/g cell·h when in phosphate-starving situation, which was 76.5% higher than that in non-starving situation. Its phosphate release rate of log course in anaerobic phase and in culture without phosphorus was 7.6mgP/g cell·h, while in stable course the rate was 6.1mgP/g cell·h. The rate in stable course was 19.7% lower than that in log course.

Characteristics of anoxic phosphors removal in sequence batch reactor

The characteristics of anaerobic phosphorus release and anoxic phosphorus uptake were investigated in sequencing batch reactors using denitrifying phosphorus removing bacteria （DPB） sludge. The lab-scale experiments were accomplished under conditions of various nitrite concentrations （5.5, 9.5, and 15 mg/L） and mixed liquor suspended solids （MLSS） （1844, 3231, and 6730 mg/L）. The results obtained confirmed that nitrite, MLSS, and pH were key factors, which had a significant impact on anaerobic phosphorus release and anoxic phosphorus uptake in the biological phosphorous removal process. The nitrites were able to successfully act as electron acceptors for phosphorous uptake at a limited concentration between 5.5 and 9.5 mg/L. The denitrification and dephosphorous were inhibited when the nitrite concentration reached 15 mg/L. This observation indicated that the nitrite would not inhibit phosphorus uptake before it exceeded a threshold concentration. It was assumed that an increase of MLSS concentration from 1844 mg/L to 6730 mg/L led to the increase of denitrification and anoxic P-uptake rate. On the contrary, the average P-uptake/N denitrifying reduced from 2.10 to 1.57 mg PO4^3--P/mg NO3^--N. Therefore, it could be concluded that increasing MLSS of the DEPHANOX system might shorten the reaction time of phosphorus release and anoxic phosphorus uptake. However, excessive MLSS might reduce the specific denitrifying rate. Meanwhile, a rapid pH increase occurred at the beginning of the anoxic conditions as a result of denitrification and anoxic phosphate uptake. Anaerobic P release rate increased with an increase in pH. Moreover, when pH exceeded a relatively high value of 8.0, the dissolved P concentration decreased in the liquid phase, because of chemical precipitation. This observation suggested that pH should be strictly controlled below 8.0 to avoid chemical precipitation if the biological denitrifying phosphorus removal capability is to be studied accurately.

Performance evaluation of a modified step-feed anaerobic/anoxic/oxic process for organic and nutrient removal

A pilot scale modified step-feed process was lmproved to increase nutrient/N ano P） ano organic removal operations from municipal wastewater. It combined the step-feed process and a method named ＂University of Cape Town （UCT）＂. The effect of nutrient ratios and inflow distribution ratios were studied. The highest uptake efficiency of 95% for chemical oxygen demand （COD） has been achieved at the inflow distribution ratio of 40/35/25. However, maximum removal efficiency obtained for total nitrogen （TN） and phosphorus at 93% and 78%, respectively. The average mixed liquor suspended solids （MLSS） was 5500 mg·L- 1. In addition, convenient values for dissolved oxygen （DO） concentration, and pH were obtained throughout different stages. The proposed system was identified to be an appropriate enhanced biological nutrient removal process for wastewater treatment plants owing to relatively high nutrient removal, sturdy sludge settle ability and COD removal.

Operation of three parallel AN/AO processes to enrich denitrifying phosphorus removing bacteria for low strength wastewater treatment

Three parallel anaerobic-anoxic/anaerobic-aerobic （AN/AO） processes were developed to enrich denitrifying phosphorus removal bacteria （DPB） for low strength wastewater treatment. The main body of the parallel AN/AO process consists of an AN （anaerobic-anoxic） process and an AO （anaerobic-aerobic） process. In the AO process, the common phosphorus accumulating organisms （PAOs） was dominate, while in the AN process, DPB was dominate, The volume of anaerobic zone（Vana）：anoxie zone（Vano） ： aerobic zone （Vaer） for the parallel AN/AO process is 1：1：1 in contrast with a Vana：Vaer and Vano：Vaer of 1：2 and 1：4 for a traditional biological nutrient removal process （BNR）. Process 3 excels in the 3 processes on the basis of COD, TN and TP removal. For 4 month operation, the effluent COD concentration of process 3 did not exceed 60 mg/L; the effluent TN concentration of process 3 was lower than 15 mg/L; and the effluent TP concentration of process 3 was lower than 1 mg/L.

Factors affecting biological denitrifying dephosphatation in anaerobic/anoxic/aerobic sequencing batch reactor

This study was conducted to verify and discuss the denitrifying dephosphatation under different levels of nitrate concentration and retention time of anoxic/aerobic process in a Sequencing Batch Reactor (SBR). The results of tests demonstrated that there were two kinds of phosphorus-accumulating organisms (PAOs) in the biological excess phosphorus removal (BEPR) system. One was non-DNPAOs that could only use oxygen as terminal electron acceptors, the other was denitrifying PAOs (DNPAOs) that could use both nitrate and oxygen as terminal electron acceptors. Phosphorus uptake efficiency could be attained under anoxic period ranging from 28.7%-96.7% in an anaerobic/anoxic/aerobic system. Experimental results showed that nitrate concentration and retention time of anoxic/aerobic process were the key factors affecting the course of denitrifying dephosphatation.