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.

Hypothesis for metabolites of denitrifying phosphate accumulating organisms by the restriction of denitrifying bacteria in anoxic phase

The objective of this work is to verify a hypothesis that nitrite accumulation comes from the metabolites of denitrification phosphate accumulating organisms (DPAOs),not denitrifying bacteria.On the precondition of the restriction of denitrifying bacteria in anoxic phase,static experimental test was designed using NO3-as electron acceptor,effluent was removed after sedimentation in anaerobic phase,and the same concentration solution of PO43--P was returned,so that TOC was excluded and denitrification was inhibited in the next phases.A parallel experiment was carried out simultaneously with the normal anaerobic-anoxic progress.The results showed that,in static test,by keeping the normal growth of DPAO and inhibiting denitrification of denitrifying bacteria,P-release in anaerobic and P-uptake in anoxic phase proceeded normally.DPAO had obvious effect on P-removal and the P-removal efficiency was 69%.The effluent concentration of NO3--N and NO2--N was 7.62 mg/L and 6.05 mg/L respectively,compared with parallel experiments,and nitrogen removal rate was lower.No nitrite residue was found in parallel test.Therefore,it can confirm the hypothesis that the metabolites of DPAO are both nitrogen and nitrite when nitrate is taken as electron acceptor,and nitrite is subsequently converted to nitrogen by denitrifying bacteria.

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.

Effects of Substitution of Organic Manure for Chemical Fertilizer on Phosphorus Accumulation and Grain Yield in Maize

In order to explore the response of maize phosphorus absorption to the appropriate proportion of organic fertilizer substitution for reduced chemical fertilizer,a field experiment with eight treatments was conducted in 2018.The eight treatments in the trial were:(1)CK;(2)M0+F;(3)M1+F1;(4)M1+F2;(5)M2+F1;(6)M2+F2;(7)M3+F1 and(8)M3+F2.In these treatments,M0,M1,M2 and M3 were organic fertilization of 0,15(low),30(medium)and 45(high)m3•hm-2,respectively.F1 and F2 indicated 20%and 40%reduction of conventional chemical fertilization.The soil nutrient content,phosphorus accumulation,dry weight,yield and yield components of maize were analyzed.The results showed that the phosphorus accumulation of maize stalks and leaves were 3.30%-43.17%and 10.98%-84.95%higher in M3+F1 treatment(20%reduction of chemical fertilizer with organic fertilizer of 45 m3•hm-2)than those in the conventional treatment(M0+F)during the maize reproductive period.At R2 stage,stalk and shoot P accumulation of M3+F1 treatment were 24.67%-43.18%and 20.34%-25.19%higher than other treatments,respectively.At V12 stage,leaf P accumulation of M3+F1 increased by 84.95%compared with other treatments.The maize yield of M3+F1 was significantly higher than that of other treatments,even though the partial productivity of the total phosphorus fertilizer of M3+F2 was the highest in the treatments.It could be found that the substitution of organic fertilizer for chemical fertilizer improved crop yield,phosphorus fertilizer efficiency and accumulation.M3+F1 treatment had the most positive effects on improving maize phosphorus accumulation and yield.

Demystifying polyphosphate-accumulating organisms relevant to wastewater treatment: A review of their phylogeny, metabolism, and detection

Currently,the most cost-effective and efficient method for phosphorus(P)removal from wastewater is enhanced biological P removal(EPBR)via polyphosphate-accumulating organisms(PAOs).This study integrates a literature review with genomic analysis to uncover the phylogenetic and metabolic diversity of the relevant PAOs for wastewater treatment.The findings highlight significant differences in the metabolic capabilities of PAOs relevant to wastewater treatment.Notably,Candidatus Dechloromonas and Candidatus Accumulibacter can synthesize polyhydroxyalkanoates,possess specific enzymes for ATP production from polyphosphate,and have electrochemical transporters for acetate and C4-dicarboxylates.In contrast,Tetrasphaera,Candidatus Phosphoribacter,Knoellia,and Phycicoccus possess PolyP-glucokinase and electrochemical transporters for sugars/amino acids.Additionally,this review explores various detection methods for polyphosphate and PAOs in activated sludge wastewater treatment plants.Notably,FISH-Raman spectroscopy emerges as one of the most advanced detection techniques.Overall,this review provides critical insights into PAO research,underscoring the need for enhanced strategies in biological phosphorus removal.

Strain Pseudomonas putida PAO-1 Isolate with Polyphosphate Accumulating and Elongation Ability

Filamentous bacteria(FB)overgrowth is an important cause of sludge bulking in wastewater treatment plants(WWTPs).However,to date,methods for the cultivation and preservation of isolated FB in the laboratory have not been completely described.Furthermore,research on whether FB can function as phosphorus accumulating organisms(PAOs)is limited.In this study,a pure strain,a Pseudomonas putida PAO-1(P.putida PAO-1)isolate with phosphorus removal functions was isolated from the biofilm of an alternating anaerobic/aerobic biofilter(AABF),and its physiological characteristics were studied.Nitrate or nitrite could be used by the strain P.putida PAO-1 as electron acceptors for denitrification during phosphorus anoxic uptake,and 0.63 mg NO-3-N was consumed to reduce 1 mg soluble orthophosphate(SOP)by P.putida PAO-1.The strain P.putida PAO-1 consumed phosphorus within the optimal pH range of 6 to 8 and the temperature range of 25℃to 35℃.Cell deformity was a main morphological trait of the strain P.putida PAO-1,and it could elongate(with an elongation rate of 300%-500%)when it was subjected to oligotrophic or high-salt stress(15 g·L-1 NaCl).The findings in this study provide a microbiological reference for understanding the special characteristics of a denitrifying PAO.

SPNED-PR系统内PAOs-GAOs的竞争关系及其氮磷去除特性

为研究同步短程硝化内源反硝化除磷(SPNED-PR)系统的脱氮除磷特性及系统内聚磷菌(PAOs)和聚糖菌(GAOs)在氮磷去除的贡献和竞争关系,本研究以实际低C/N比(4左右)生活污水为处理对象,考察了不同浓度的溶解氧(DO)(0.5~2.0mg/L)、NO2--N(4.7~39.9mg/L)和NO3--N(5.0~40.0mg/L)对延时厌氧(150min)/低氧(180min,溶解氧0.5~0.7mg/L)运行的SPNED-PR系统氮磷去除特性和底物转化特性的影响.结果表明,DO浓度均不影响PAOs和GAOs的好氧代谢活性,且两者之间几乎不存在DO竞争.不同NO2--N浓度条件下,GAOs较PAOs更具竞争优势,NO2--N主要是通过GAOs去除的(约占58%);且GAOs所具有的高内源反硝化活性和亚硝耐受力,减弱了高NO2--N浓度(26.2~39.9mg/L)对PAOs反硝化吸磷的抑制,保证了系统的脱氮除磷性能.不同NO3--N浓度条件下,PAOs较GAOs处于竞争优势,其在NO3--N去除中的贡献比例达61.2%.此外,SPNED-PR系统的PURDO>PURnitrate>PURnitrite,PAOs对DO的优先利用保证了低氧条件下系统的高效除磷,且GAOs的内源短程反硝化特性保证了系统的高效脱氮.

EBPR中两类细菌PAOs和GAOs竞争的研究进展

强化生物除磷(EBPR)工艺可以获取高效的除磷效果,已在很多污水处理厂得到广泛应用。但是大型污水处理厂在相当多的条件下,EBPR工艺也会出现周期性除磷效果的波动和不充分。针对这一难题,研究者试图采用许多手段来研究工艺中的主要微生物。文章针对典型的EBPR工艺和碳源、pH值、温度等因素对EBPR工艺中两类细菌聚磷菌(PAOs)和聚糖菌(GAOs)竞争的研究进展进行了论述,并展望了未来的研究方向。

Phosphorus accumulation by bacteria isolated from a continuous-flow two-sludge system

In this article, polyphosphate-accumulating organisms （PAOs） from a lab-scale continuous-flow two-sludge system was isolated and identified, the different phosphorus accumulation characteristics of the isolates under anoxic and aerobic conditions were investigated. Two kinds of PAOs were both found in the anoxic zones of the two-sludge system, one of them utilized only oxygen as electron aeceptor, and the other one utilized either nitrate or oxygen as electron aeceptor. Of the total eight isolates, five isolates were capable of utilizing both nitrate and oxygen as electron acceptors to uptake phosphorus to some extent. And three of the five isolates showed good phosphorus accumulative capacities both under anoxic or aerobic conditions, two identified as Alcaligenes and one identified as Pseudomonas. Streptococcus was observed weak anoxic phosphorus accumulation because of its weak denitrification capacity, but it showed good phosphorus accumulation capacity under aerobic conditions. One isolates identified as Enterobacteriaceae was proved to be a special species of PAOs, which could only uptake small amounts of phosphorus under anoxic conditions, although its denitrification capacity and aerobic phosphorus accumulation capacity were excellent.

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.

聚磷菌PAO1-1的筛选及除磷特性

从运行稳定的以生活污水为碳源的生物除磷污泥中筛选出一株聚磷菌PAO1-1,该菌株对普通活性污泥系统具有很好的强化作用,驯化10 d后可使除磷率由投菌前的44%提高到90%以上。对该菌株的形态、生理生化特征及16S rDNA序列进行分析后,鉴定该菌株为产碱杆菌属。该菌株对磷的平均吸收速率为13.8 mg/(g.h),处于“磷酸盐饥饿期”时对磷的吸收速率为19.2 mg/(g.h),比“非饥饿期”提高了39.1%。处于对数期的PAO1-1在厌氧条件下的无磷培养基中的释磷速率为11.8 mg/(g.h),稳定期释磷速率为7.0 mg/(g.h),释磷速率下降了40.7%。

Effect of Land Use Conversion from Rice Paddies to Vegetable Fields on Soil Phosphorus Fractions

Excess phosphorus （P） from agricultural soils contributes to eutrophication in water bodies. Samples （n = 60） were taken from sites where rice paddies have been converted to vegetable fields for 0, 〈 10, 10-20, and 〉 20 years and analyzed for five inorganic P （Pi） fractions, three organic P （Po） fractions, and several soil parameters to investigate how land use conversion affects Pi and Po fractions in a peri-urban area of China with soils characteristic of many agricultural areas of Asia. Significant increases of 33, 281, 293, and 438 mg kg-i were found for soluble and loosely bound Pi （SL-Pi）, aluminum-bound Pi （Al-Pi）, calcium-bound Pi （Ca-Pi）, and iron-bound Pi （Fe-Pi）, respectively, after conversion from rice paddies to vegetable fields. Most of the increase in Pi was in the form of Fe-Pi, which increased from 8% of total P （TP） on paddy soil to 31% on the soil with 〉 20-year vegetable cultivation, followed by Al-Pi, which increased from 2% to 19% of TP. For Po fractions, there was no significant change in P concentrations. The conversion of land use from paddy fields to high intensity vegetable fields was causing significant changes in soil P fractious. Management practices were causing a buildup of soil P, primarily in the Fe-Pi fraction, followed by Ca-Pi and Al-Pi fractions. If current trends continue, a 30%-70% increase in TP could be expected in the next 20 years. Farmers in the area should reduce P application and use to maximize P uptake.

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.

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.

Effect of temperature on anoxic metabolism of nitrites to nitrous oxide by polyphosphate accumulating organisms

Temperature is an important physical factor, which strongly influences biomass and metabolic activity. In this study, the effects of temperature on the anoxic metabolism of nitrite （NO2） to nitrous oxide （N2O） by polyphosphate accumulating organisms, and the process of the accumulation of N2O （during nitrite reduction）, which acts as an electron acceptor, were investigated using 91% ：e 4% Candidatus Accumulibacterphosphatis sludge. The results showed that N2O is accumulated when Accumulibacter first utilize nitrite instead of oxygen as the sole electron acceptor during the denitrifying phosphorus removal process. Properties such as nitrite reduction rate, phosphorus uptake rate, N2O reduction rate, and polyhydroxyalkanoate degradation rate were all influenced by temperature variation （over the range from 10 to 30℃ reaching maximum values at 25℃）. The reduction rate of N2O by N2O reductase was more sensitive to temperature when N2O was utilized as the sole electron acceptor instead of NO2, and the N2O reduction rates, ranging from 0.48 to 3.53 N2O-N/（hr.g VSS）, increased to 1.45 to 8.60 mg N2O-N/（hr·g VSS）. The kinetics processes for temperature variation of 10 to 30℃ were （01 = 1.140-1.216 and θ2 = 1.139-1.167）. In the range of 10℃ to 30℃, almost all of the anoxic stoichiometry was sensitive to temperature changes. In addition, a rise in N2O reduction activity leading to a decrease in N2O accumulation in long term operations at the optimal temperature （27℃ calculated by the Arrhenius model）.

Acclimation and Characterization of a Pseudomonas Strain for Improved Phosphorus Removal

A Pseudomonas strain(named as P.PAO-1) with phosphorous removal function was isolated and characterized.A new method of two-stage cultivation was applied to the strain to induce the synthesis of intracellular polyhydroxylbutyrate(PHB) in the cells.In the first stage,bacterial cells were enriched under a high carbon source condition;in the second stage,the bacteria cells were cultivated under the nutrient-imbalanced conditions with the carbon source-regulated medium.As a result,the PHB content of strain P.PAO-1 reached 22.8% compared with the normal 7.41% in the non-acclimated strain.This change had led to the rising of P uptake from 1 to 25 mg·L^(-1).When added the strain P.PAO-1into the activated sludge(with the addition proportion of 1:1),the ratio of biological phosphorus removal increased by 14.9%under the normal alternating anaerobic/aerobic conditions with low-carbon consumption.The results demonstrated that the isolated pure culture strain P.PAO-1 belonged to the functional group of poly-P accumulating microorganism.When cultured by the two-stage cultivation method,the initial accumulation of PHB in the strain cells could be achieved and the phosphorous removal capacity of strain P.PAO-1 could be induced subsequently.When applied to the wastewater,strain P.PAO-1 performed phosphorus removal from the wastewater with or without the addition of activated sludge.

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.

MUCT工艺反硝化除磷效率与Candidatus Accumulibacter的群落演替

以处理实际生活污水的连续流MUCT工艺为基础,探究了全程硝化和短程硝化状态下优势聚磷菌Candidatus Accumulibacter的群落动态与系统反硝化除磷效果之间的相互联系.流式细胞仪定量检测结果表明,系统中聚磷菌占全菌的比例为22.1%,Accumulibacter是处于全程硝化状态下的优势聚磷菌,对系统中氮磷的去除起到重要作用.q PCR定量结果表明,短程硝化状态下的优势种IIC和IIC(ppk1 excluding OTU NS D3)以及IIF的增长率较快,占总Accumulibacter比例分别为11.53%、34.31%和17.97%,其利用亚硝进行反硝化除磷的能力比其他分支强,对系统化中亚硝的去除起到重要作用.IID分支在反应器整个运行阶段一直处于优势地位,是系统内氮磷去除最重要的分支.其在全程硝化状态下占Accumulibacter分支总和的80%,但在短程状态下的竞争能力不如其他的TypeⅡ型分支.

SRT对酪蛋白水解物为碳源的EPBR系统的影响

为了探究污泥龄(SRT)对以酪蛋白水解物(Cas aa)为唯一碳源的强化生物除磷(EPBR)系统性能的影响,采用序批式反应器(SBR),在低温条件下考察了SRT对Cas aa为碳源的EPBR中微生物菌群的变化及其污染物去除性能的影响。结果表明,SRT为15 d时,系统除磷性能最佳,除磷率可达95%以上,显著高于其他SRT条件。系统运行典型周期分析表明:当SRT为15 d时,系统的厌氧释磷质量浓度最大,为34.03 mg/L,好氧吸磷质量浓度最大,为48.45 mg/L,除磷效果最优。高通量测序分析结果表明,系统内聚磷菌(PAOs)的组成和结构会随着SRT的变化而变化,SRT为15 d时,系统内全体PAOs相对丰度最高。

短程硝化-强化生物除磷序批式反应器处理畜禽养殖废水

畜禽养殖废水有机物水质水量变化大,有机物、氨氮与磷的浓度较高,直接排放会严重危害环境。通过构建厌氧-好氧序批式反应器(SBR)处理预酸化畜禽养殖废水,分析了不同进水负荷条件下反应器对污染物的去除性能和微生物群落结构的变化规律。结果表明:SBR反应器对高负荷进水中TN、PO_(4)^(3-)—P和COD的平均去除率可分别达到64.5%、97.5%和94.5%。反应器出现NH_(4)^(+)—N和NO_(2)^(—)-N亚硝酸同时积累的短程硝化现象,这可能与高进水负荷对氨氧化菌和亚硝酸盐氧化菌的活性和种群的影响有关。与乙酸盐相比,以丙酸盐作碳源时污泥的强化生物除磷活性更高。随着进水负荷的增大,聚糖菌(GAOs)的相对丰度明显升高。四联球状菌(Tetrasphaera)为反应器中始终占优势的聚磷菌(PAOs),对反应器除磷性能有重要贡献。在高有机负荷条件下,SBR内PAOs与GAOs之间不存在明显的底物竞争关系,系统脱氮除磷性能未受影响。