Screening Of Denitrogenation Microorganism And Its Application In The Biofilm Technique

With the rapid acceleration of urbanization,the ammonia-nitrogen pollution in rivers has become a common phenomenon in China,which will threaten the water environmental safety.Microorganism as the main driver of the bioremediation which decided the remediation performance directly.Therefore,the screening of microbes with excellent nitrogen removal ability and eco-friendly characteristics has become a focus of interest for researchers recently.The biofilm technology utilize the microorganisms colonized on the biofilms attached to the carrier materials to remove pollutants through adsorption and degradation.Base on the advantages of great efficiency,low operation cost and good safety performance,biofilm technology shows broad prospects for bioremediation of polluted water.While the application of biofilm technology to the in situ restoration engineering in China is still in the exploring stage.Therefore,the specific aim of the present work is to isolate novel indigenous microorganism with both excellent ammonium removal capability and biofilm-forming ability and enhanced the ammonium removal performance of biofilm technique by the immobilization of the functional microbes.The major contents and findings of the dissertation are as follows:1.116 strains of aerobic denitrifying bacteria were isolated from the overlying water,sediment and biofilm samples in Liangshui River of Beijing through specific media.These bacteria were classified into 14 genera based on the 16S rRNA sequences.The Pseudomonas sp.composed the major culturable aerobic denitrifiers of the river,and the Venn graph and cluster analysis indicated that three species including OTU1?Pm.mendocina?,OTU2?Pm.stutzeri?and OUT3?Pm.putida?were the universal culturable aerobic denitrifying bacteria in all the phases of river.The PCA plot showed that these aerobic denitrifiers had different habitat-preferable characteristics among the three phases of river system.Totally eight OTUs representative strains contained OTU2?Pseudomonas stutzeri?,OTU4?unclassified Ochrobactrum sp.?,OTU11?Gordonia malaquae?,OTU13?Stenotrophomonas acidaminiphila?and so on exhibited heterotrophic nitrification and aerobic denitrification characteristics by preliminary identification.2.Based on the above results,the further research were conducted on a strain identified as Pseudomonas stutzeri W12 which was ubiquitous in different habitats and was characterized with the ability of heterotrophic nitrification and aerobic denitrification.The sequence amplification results suggested the presence of the denitrification genes napA,nirS,norB and nosZ in strain W12,provided additional evidence that strain W12 was capable of aerobic denitrification.The nitrogen removal experiments showed that 90%of the NH₄⁺-N and 97.4%of NO₃^--N were removed in30 h and 24 h,respectively,and only a slight NO₂^--N accumulation was observed.The biofilm formation ability of Pm.stutzeri W12 and P.denitrificans DSM413 were assessed by the crystal violet assay,the quantified results depicted that the ability to form biofilm by strain Pm.stutzeri W12 was better than P.denitrificans DSM413.3.The effect of nutritional conditions and start-up method on the biofilm augmented purification performance of ammonia-polluted water was investigated in simulated Setup 1?contained reactor Blank,reactor Raw and reactor PCC?.The microbial community structures were assessed by terminal restriction fragment length polymorphism?T-RFLP?and the succession process of community was analyzed using the plot of nonmetric multidimensional scaling?NMDS?.The ammonia removal efficiencies of both the biofilm reactors Raw and PCC were higher than 95%when the C/N ratio was adjusted to 2:1.The NMDS results showed that the microbial community structures of Raw and PCC had high similarity when the C/N was 2:1,and the dominant bacteria were composed of?-Proteobacteria,Actinobacteria and Nitrospira.And the C/N ratio was not only a key factor impacting on the ammonia removal efficiencies of biofilm reactors but also a driving force for the shifts of microbial community structures in the biofilm reactors.The dominant position of the initial-immobilized Paracoccus denitrificans PD1222 had been replaced because of the competition by autochthonous microbes,and the biofilm system improved the degradation potential of the inhabiting microbial population.4.Based on the above studies,we combined the strain Pm.stutzeri W12 with the biofilm technique by the cell immobilization technique and developed another simulated Setup 2?contained reactor Blank,reactor Raw,reactor DSM413 and reactor W12?to assess the practical feasibility of the augmented biofilm technique with immobilized indigenous microbe Pm.stutzeri W12 and investigate the effects of C/N ratio,DO and HRT on the system performance and the microbial community structures.The main conclusions are as follows:?1?Setup 2 were started up with the operational parameters as follows:C/N=2:1,DO 3-5 mg/L and HRT=20 h.During the stable operation period,ammonium removal efficiency of biofilm reactors Raw,DSM413 and W12 were 96.0%,97.07%and96.47%,respectively.Moreover,the total removal efficiency of biofilm reactor W12was greater than the biofilm reactors Raw and DSM413.Meanwhile,the T-RFLP analysis results showed that the immobilized Pm.stutzeri W12 and Paracoccus denitrificans PDDSM413 both could persist stably in the biofilm system with the relative abundance of 20.36%and 6.0%,which reflected the advantages of application of indigenous microbes in biofilm augmentation.The augmented biofilm technique successfully overcome the problems of bacterial consortium washing off from the target site and the problem of incompatibility with the environment.?2?Compared with the operation performance at C/N=4:1,the biofilm reactors of Raw,DSM413 and W12 exhibited better nitrification activities both at C/N ratio 1:1and 2:1.While the C/N ratio 4:1 was the optimal value for the three biofilm reactors to removal the total nitrogen,and the TN removal efficiency of biofilm reactor W12 was higher than reactor Raw and reactor DSM413.?3?The dissolved oxygen tests suggested that the biofilm reactors of Raw,DSM413 and W12 presented a satisfactory performance with DO 3-5 mg/L.With the decrease of DO concentration from 3-5 mg/L to 1-2 mg/L,the nitrifying and denitrifying activities of biofilm reactors were inhibited simultaneously.?4?The Setup 2 was operated at four different HRTs?20 h,15 h,10 h and 5h?,the performance of the biofilm reactors showed that the removal of ammonia nitrogen reached the highest efficiency exceeded 96%at HRT 20 h,with the decrease of HRT from 15 h to 10 h,the NH₄⁺-N removal efficiency almost kept unchanged.The biofilm reactors presented a satisfactory TN removal at HRT of 10 h.And an obvious decline of ammonia-nitrogen removal efficiency and total removal efficiency were found with the further decreasing HRT of 5 h.Above knowable,changes in C/N ratio,DO concentration and HRT significantly influenced the performance of the biofilm reactors.It is found that the biofilm reactors of Raw,DSM413 and W12 exhibited good nitrification activities with the NH₄⁺-N removal efficiency of 96.0%,97.07%and 96.47%at C/N ratio 2:1,DO 3-5 mg/L and HRT 20 h.?5?The population dynamics of the reactors were revealed by the Illumina HiSeq high-throughput sequencing technology.Results demonstrated that the microbial community structures of biofilm reactors were correlated with C/N ratio,DO and HRT.In biofilm reactors the microbial diversity in water niches were lower than that in biofilm niches,and Proteobacteria was the prominent phylum in biofilm reactors.The analysis of bacterial compositions at the class level showed that?-Proteobacteria,?-Proteobacteria,?-Proteobacteria and Actinobacteria were widespread with high percentages in the biofilms.The changes of microbial community succession with different operation conditions implied that the?-Proteobacteria and?-Proteobacteria bacteria were the main contributors to the nitrification process and?-Proteobacteria bacteria ware the main contributors to the denitrification process.It could be speculated that the nitrification were carried out by autotrophic bacteria and heterotrophic bacteria synergistically,and aerobic denitrification maybe a primary model of denitrification process.