Study On Functionand Community Structure Of Candidatus Accumulibacter In Denitrifying Phosphorus Removal System

Denitrifying phosphorus removal process is using nitrate （NO₃^--N） or nitrite（NO₂^--N） as an electron acceptor in phosphorus uptake stage， rather than oxygen（O2）.Meanwhile， NO₃^--N or NO₂^--N is reduced to nitrogen， in order to removenitrogen and phosphorus from wastewater at the same time. Carbon played a"one-carbon dual" role in denitrifying phosphorus removal process， not only the beused to synthesizing of PHAs， but also for the denitrification of electron donor，making fμll use of the raw water source of carbon to improve nitrogen andphosphorus remove efficiency， is a cost-effective water treatment technologies."Candidatus Accumulibacter" is a wild-known bactria in EBPR systems.Accumulibacter evolutionary branch numerous and various evolutionary branch onNO₃^--N and NO₂^--N utilization capabilities were significantly different. ThereforeAccumulibacter flora and its evolutionary branch proportional change in thedistribution and wastewater treatment systems can have a major impact on theperformance of phosphorus removal. In order to further investigate the mechanism ofdenitrifying phosphorus removing bacteria and denitrifying phosphorus flora functionand community distribution， using Real-time PCR methods for dealing withdomestic wastewater by a MUCT process and SBRs using NO₃^--N or NO₂^--N aselectron acceptor to detective the dominant branch of "Candidatus Accumulibacter"and total bacterial communities in different EBPR systems the advantage ofdenitrifying phosphorus removal systems， establishing of phosphorus flora and thecorrelation between the operating parameters for EBPR stable operation of the systemto lay the theoretical foundation.Poly-P kinase1（PPK1） is the key enzyme in PAOs intracellμlar catalytically andsynthesising of poly-P， and aerobic phosphorus uptake rate and PPK1activity arelinear. The ppk1is the functional gene of synthesis of this enzyme， ppk1processdirectly affects the expression of the metabolic activity of PAOs and phosphorusremoves efficiency. Accumulibacter intracellμlar ppk1is a single copy gene， as it’sevolution speed is4times faster than16S rRNA. It becomes the best marker gene inthe study of evolutionary Accumulibacter branch. Therefore， this study ppk1functional genes as genetic markers， not only can further clarify Accumulibactereach clade， but it can create ppk1gene expression on EBPR systems dynamic response to changing environmental conditions mechanism. Studies were usedprimers （Acc-ppk1-763f， Acc-ppk1-1170r），（Acc-ppk1-893f， Acc-ppk1-997r），（Acc-ppk1-870f， Acc-ppk1-1002r），（Acc-ppk1-254f， Acc-ppk1-460r） and（Acc-ppk1-375f， Acc-ppk1-522r） to establish QPCR standard curve ofAccumulibacter branch I， IIA， IIB， IIC and IID. During the establishment of thestandard curves it found that: The temperature gradient PCR experiment can bedetermined the corresponding optimal annealing temperature only by a particμlarsystem.Which can improve QPCR experiments efficiency in a short time， especiallyfor Accumulibacter which has so many branchs. Ingenious solution to the four-stepprimer-dimers and other non-specific amplification of the target product wasquantified impact.5standard curves correlation coefficients were all greater than0.998. The amplification efficiency was98.2%，105.6%，93.9%，96.7%and99.7%.The standard curve shows the correlation coefficient and the amplification efficiencyof the method in accordance with the requirements of precise quantification.In order to study the dynamic response of the DPAOs using different electronacceptors and carbon sources， according to the physiological characteristics of thedenitrifying phosphorus removal bacteria （DPAOs）， the SBR reactors were usedunder anaerobic/anoxic environment for the enriched of DPAOs. Also， qualitativeanalysis and quantitative analysis of "Candidatus Accumulibacter" have been used.SBR1and SBR2were used acid and propionic acid as carbon source， respectively，and nitrate as electron acceptor enriching DPAOs.The study resμlts showed that:Using acid as the sole carbon source and NO₃^-N as the only electron acceptor for theenrichment of DPAOs.Acid as sole carbon source， NO₃^-N as electron acceptorenrichment of denitrifying phosphorus removal bacteria adapts to run after about80cycles，the system showed good phosphorus uptake and release performance. SystemsTab.ility of the Gram stain showed that the system flora mainly spherical bacteria."Candidatus Accumulibacter" quantitatived by QPCR shows that the ratio of branchAcc-I was gradually increased. The proportion of Acc-1up from the original0.6%increased to3.6%after120cycles， became the dominant branch. The SBR1PAOsproportion of total bacteria decreased from the initial2.8%to0.6%; but phosphorusremoval performance of the system has been enhanced. It may be due to the acc-1using NO3-N as electron acceptor has been enriched in the SBR system. When acidas the sole carbon source， the dosing of NO₃^--N portion converted to NO₂^-N， itwoμld be the DPAOs in SBR reactors using NO₂^--N as the electron acceptor （e.x. the branch IIA of Accumulibacter） always existed and the proportion increased.Using Propionic acid as the sole carbon source and NO₃^-N as the only electronacceptor for the enrichment of DPAOs.Although phosphorus release reaction can beobserved in the anaerobic， phosphorus uptake reaction cannot be found in anoxicstage. Sometimes， the effluent phosphorus concentration is even greater than theinfluent phosphorus concentration. Gram stains test resμlts show that there are a lot offilamentous bacteria in the system. Most of the remaining bacteria were Bacillus.QPCR resμlts show that: SBR2the Accumulibacter branch IID dominate theAccumulibacter in SBR2and the ratio was always larger than96%. TheAccumulibacter IID quantity is gradually increased with the reaction run， which isthe main reason for the increase of Accumulibacter in total bacteria. Fμll-cycle testshowed that: excess carbon source into the anoxic zone led to the large popμlation ofheterotrophic bacteria. After a period of time to adapt to increased consumption ofNO₃^--N， the absence of the electron acceptor and did not add timely stopped thefurther absorption of phosphorus， and it is the main reason for phosphorus removaldeterioration in SBR2system.SBR3and SBR4were used acid and propionic acid as carbon source，respectively， and nitrite as electron acceptor enriching DPAOs.The study resμltsshowed that: Using acid as the sole carbon source and NO₂^--N as the only electronacceptor for the enrichment of DPAOs.After eight cycles of short-term the P removaleffection of the system can be enhanced， but with the increase of the operatingcycle， Phosphorus removal of the system gradually decreased. QPCR resμlts showedthat: after eight cycles of operation， the proportion of DPAOs in the systemsincreased from initial0.66%to2%; and after phosphorus removal performance of thesystem disappeared， DPAOs in total bacteria dropped to0.7%.Using propionic acidas a solo carbon source and NO₂^--N as only electron acceptor for the enrichmentDPAOs， Phosphorus removal of the system disappeared after15cycles， thencontinue acclimation to60cycles， and phosphorus removal performance of thesystem is still not recovered. QPCR quantitative resμlts show that the proportionDPAOs in the reactor was reduced from0.66%to0.15%of the final. The conclusionis DPAOs Can not survive using propionic acid as an only carbon source and NO₂^-Nelectron acceptor， it can cause the collapse of phosphorus removal in EPBR systems.Nitritation and denitrifying phosphorus （P） removal was achieved in MUCTprocess treating domestic wastewater with a low C/N ratio．The effect of nitrite Accumulation on performance of phosphorus removal and popμlation structureof＂Candidatus Accumulibacter＂was investigated during nitritation establishmentand destruction. Res μ lts indicated that P removal was mainly completed bydenitrifying P removal of about88%．The P removal efficiency had a clear correlationwith the nitrite Accumulation rate. Under nitritation，the P removal was30%higherthan that under complete nitrification，suggesting that nitrite was appropriate to beused as electron acceptor for denitrifying P removal when treating low C/Nwastewater. Real time quantitative PCR assays were carried out using poly-P kinase1（ppk1） as phylogenetic marker to characterize the abundance of total Accumulibacterand the relative distributions and abundances of the Accumulibacter clades．Undercomplete nitrification，a very few Acc-I clade of below5%in total Accumulibacterwas present using nitrate as electron acceptor．When the reactor transformed intonitritation，Acc-I clade gradually disappeared．The Acc-IID clade using nitrite aselectron acceptor for denitrifying P removal was always the dominant Accumulibacterthroughout the operational period， with above92%on average in totalAccumulibacter，even up to nearly100%，which led to sTab. performance ofdenitrifying P removal using nitrite as electron acceptor．The nitrite concentrationssignificantly affected the abundances of Acc-IID clade．Shortcut nitrification was achieved in MUCT process treating domesticwastewater with a low C/N ratio by controlling DO concentration and hydraμlicretention time. The total nitrogen removal rate of the system finally achieved up to90%of stability. Analysts believe that the nitrite pathway SND causes a stable highTN removal，which can be up to35.5%. The low DO provided process conditions forthe realization of the SND. It not only prompted AOB become the dominant nitrifyingbacteria，thus ensuring a stable nitrification implementation，as well as provided thehypoxic microenvironment for the realization of the SND. Anammox bacteria werethe function microbial to achieve nitrite SND， and they played a catalytic role for thestability TN removal. The abundance of anammox rose to from2.0×105copies/g VSS to1.32×106copies/g VSS maximum in the shortcut nitrification stage， theTN removal efficiency through SND increased by15%compared to. the species mud.17mg/L of NO₂^--N concentration woμld produce inhibition to anammox. Therealization of partial SND saved carbon source， which ensures a stable phosphorusremoval in low C/N wastewater.