Study On The Start-up Of Anammox Reactor And Microbial Community Analysis

The anaerobic ammonium oxidation （ANAMMOX） process is considered to beefficient and cost-effective alternative to the conventional nitrification-denitrificationprocess, due to lower oxygen demand, less sludge production and no requirement ofexternal carbon sources. However, one of main drawbacks common to application of theanammox process is requirement of a long start-up period due to mainly strict survivalconditions and slow growth rates of anammox bacteria （the doubling time was reportedto be approximately11d）. To date, the start-up time of anammox reactors usingconventional activated sludge as inoculum was reported to be several years.The primarily goal of this study is therefore to solve the bottleneck problem of thelong start-up period of cultivating anammox bacteria in reactor, and provide animportant guidance for the extensive application of anammox process in future. Toachieve this objective, the seeding sludge, reactor configuration and control optionswere discussed using some modern molecular biological techniques, such as TerminalRestriction Fragment Length Polymorphisms （T-RFLP）, Real-time Quantitative PCR（Q-PCR）. The main results of this thesis were as follows:（1） In order to rapid identification of anammox bacteria in environmental samples,a cloning-independent method based on T-RFLP was proposed. Based on SILVA （R10⁸）SSU Ref database containing40016S rRNA gene sequences of anammox bacteria,defined lengths of terminal restriction fragments （T-RFs） were predicted by virtual PCRand restriction enzyme digestion using suitable PCR primers （amx368f-amx820r） andrestriction enzyme （MspI&RsaI）. Then the genus of anammox bacteria was identifiedby comparative analysis of T-RFs. Through repeatability and sensibility analysis, ourdata suggest that anammox bacteria specific T-RFLP analysis is a reliable tool to rapidlyassess the complexity of anammox bacteria in environmental samples.（2） Four sludge samples were selected as study object from different wastewatertreatment plants （WWTPs）: Harbin high-tech soybean WWTP （EGSB+SBR process）and Harbin Taiping municipal WWTP （A/O process）. The results of anammox bacteriaspecific T-RFLP analysis showed that there are three main types in the granule sludgefrom EGSB process:“Anammoxoglobus”,“Brocadia” and “Jettenia”. In contrast, onlythe genus of “Brocadia” was detected in activated sludge from SBR process. Thecompositions of anammox bacteria community in the sludge from anoxic basin and oxicbasin of A/O process were found to be similar, and contained “Anammoxoglobus”,“Brocadia” and “Scalindua”. Furthermore, the16S rRNA gene copy numbers ofanammox bacteria in the sludge samples were quantified by Q-PCR. There were1.50×10⁷,1.05×10⁸,6.80×10⁷and8.20×10⁷copies/mg-dry sludge in the different sludge samples from EGSB process, SBR process, anoxic and oxic basin ofA/O process, respectively. In addition, the anammox activity tests revealed that thesludge from SBR process was the fastest to observe clear anammox activity. Thus, wedecided to use the sludge from SBR process as an inoculum for the following anammoxreactor.（3） The completely mixed reactor （CSTR） with sludge totally recycles, inoculatedwith the sludge of SBR process in Harbin high-tech soybean WWTP, was operated for127days under mesophilic temperature and low nitrogen load （35℃and0.06kg （m³·d）, respectively）. The total nitrogen removal rate was reached65%,lower than theoretically removal rate of80%, probably due to nitrite was oxidated tonitrate in the reactor. Moreover, based on the variation of NH4+, NO2-and NO3-duringthe start-up of anammox reactor, the experimental period could be divided into threestages: denitrifying active period, transition period and anammox activity improveperiod. In addition, five parameters reflecting the performance of anammox reactorwere proposed to provide a reference for the rapid start-up of anammox reactor in future.They are the variation of16S rRNA gene copy numbers of anammox （C₀）, theproduction of nitrate （δN）, and the variation of pH, ORP and sludge characteristics.（4） In view of molecular ecology, the target bacteria （anammox bacteria） and totalbacteria communities were analyzed by T-RFLP during the start-up of anammox reactor.The results of anammox bacteria specific T-RFLP analysis showed that the genus of“Brocadia” became dominated in the anammox reactor. In addition, the diagram of totalbacteria T-RFLP revealed that the microbial community structure had changedsignificantly during the start-up of anammox reactor. The appearance andever-increasing abundance of320bp, predicted the accumulation of anammox bacteria.Furthermore, the microbial diversity in the anammox reactor was discussed bystatistical method. The variation of Shannon-Wiener Index indicated the decrease ofmicrobial diversity during the start-up of anammox reactor. And the results of PCAanalysis showed that the microbial community structure changed dramatically in theinitial stage, then with the operation of the reactor, the microbial community structure istend to stabilize.