| Temperature is one of the major factors influencing removal efficiency in biological system. Generally, traditional biological system may not maintain the treatment efficiency or decrease operation fee at low temperature, for it only adjusts sludge load, capacity of returns, HRT, etc. The goal of this study is to reveal and analysis the microbial community structure, function mechanism and metabolism in A/O biological treatment system using metagenomic sequencing, and propose an agent construction strategy in terms of microbial ecology in order to provide justification and rationale for the further development of biotechnology applicationA/O, SBR and oxidation ditch processes are selected of Northeast China in this study and quality of effluent is monitored all year. The effluent COD and NH4+-N concentration of biological system can meet the first level B criteria in winter and spring. Especially when temperature falls below15℃in A/O process of winter, removal efficiency of NH4+-N can be more than80%; PCR-DGGE cluster analysis shows that A/O process can provide highest diversity and largest amount of dominant bacteria, with the highest stability of microbial community. According to the16S rDNA sequencing result, dominant microbe communities belong to Proteobacteria. Combination of traditional culture dependent and independent technologies in detection for carbohydrate, lipid and protein usage of, A/O process shows more advantages in biological system efficiency and microbial structure stability.The result of three-dimensional fluorescence spectrum for optimized extracting indicate that combining grinding with liquid nitrogen and extracting with DNA extraction kit can acquire metagenome DNA for high-throughput sequencing efficiently. Illumina sequencing of active sludge metagenome DNA from three seasons (S1, autumn, S2, winter and S3, summer) offers190million,280million and200million available bases (bp), with the utilization of96.8%,98.1%and93.7%respectively. A large amount of microbes are identified through comparison to Blast-nr data base, and the dominant species in A/O system are determined. The abundance of β-Proteobacteria in S1(autumn) is about44.9%, α-Proteobacteria in S2(winter) is about54.37%and β-Proteobacteria in S3(summer) is about43.88%. The α-diversities of the three seasons are463.98,443.08and473.13respectively, among which S3(summer) has the highest α-diversity and highest evenness of species.KEGG results of MG-RAST data base show that polysaccharide is an important energy source during growth and reproduction of eucarya. The metabolism path of polysaccharide for some of the microorganism communities shows that bacteria can synthesize extracellular polymeric substances using polysaccharide in the winter under low temperature. The abundance of key enzyme of polysaccharide synthese, EC1.1.1.22(UDP-glucose6-dehydrogease, UGD), follows the order of S1(autumn)<S3(summer)<S2(winter), which suggests syntheses increase of extracellular polymeric substances during the winter. Furthermore, genes of flocculant secretion initializing enzyme EC2.7.7.9(UDP glucose pyrophosphorylase) won’t be lost as temperature drops. The genes of degradation enzymes for steroid, anthracene, naphthalene and other special organic pollutant are rare in microbial communities, which may be because the lack of HRT and SRT of biological system, which limit the time for bacteria growth.According to microbial communities’ metabolism analyses of elements such as N, P and S in oxidation of A/O process, microbial communities contain a full range of genes of nitrification enzymes. The key enzyme, EC1.7.3.4(hydroxylamine oxidase), enables oxidization from nitrite to nitrate, while the existence of EC1.7.99.4(nitrate reductas), EC1.7.2.1(nitrite reductase) and EC1.7.99.7(Oxidoreductases) enhances the possibility of denitrification. Besides, metabolism path of phosphorus is very complete in the microbial communities. The abundance of EC2.3.1(PHB systhetase) key enzymes, follows the order of S3(summer)<S1(autumn)<S2(winter), which indicates a higher vitality of PAOs in lower temperature. Microbial communities mainly participate in the oxidizing of sulfide in sulfur metabolism, illustrating that oxidization from sulfate to sulfur could not happen during oxidizing process of A/O.Comparison on SEED result shows that microbial communities of S2winter contain the largest amount of cold shock protein (CSPs), which is3times of that of S1autumn and1.2times of that of S3summer. The abundant existence of CSPs in biological system in the winter can assist microbes with defensing against physical damages caused by temperature variation, ensuring high activity of microbes at low temperature.Microbial ecosystem technologies are used in analyzing abundance, structure and function of microbial communities in A/O biological system, and strategies of inoculants establishing are founded. Full-scale metabolic inoculants establishment strategy can be used for metabolism with clear enzyme genes and abundance. Using physical process before biochemical process and substrate-inducing is suitable for special organic pollutant with fewer enzyme genes. For polysaccharide anabolism, which is easier to be influenced by temperature, adding flocculants to the system is an appropriate strategy to maintain settle ability of biological system in winter. |
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