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Isolation Of Low Temperature Nitrogen Removal Bacteria And Preliminary Study On Nitrogen Removal Mechanism

Posted on:2023-01-11Degree:MasterType:Thesis
Country:ChinaCandidate:S Y ChenFull Text:PDF
GTID:2531307142974559Subject:Biology
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In recent years,China’s animal husbandry industry has developed rapidly.While promoting the rapid improvement of domestic economic level,it has also caused serious environmental pollution problems.Among them,water pollution and ecological imbalance caused by large-scale pig breeding have attracted much attention in China and need to be solved urgently.The commonly used denitrification process in China is often affected by environmental factors,and it is easy to cause secondary pollution in the treatment process.Compared with the traditional denitrification technology,the emerging new biological denitrification technology has become the research hotspot of scholars because of its advantages of high efficiency and environmental protection.Researchers are widely looking for different types of nitrogen removal microorganisms to meet the needs of sewage denitrification under different environmental conditions.Aiming at the problem of poor effect of sewage in winter by traditional biological nitrogen removal process,a strain D15 with high nitrogen removal performance at low temperature was isolated from water-soaked paddy soil in winter.The strain D15 was identified by morphological observation,physiological and biochemical analysis and16S r RNA gene sequence analysis.The nitrogen removal characteristics of the strain under low temperature conditions were investigated and applied to the nitrogen removal of pig manure wastewater.At the same time,the functional factors of nitrogen metabolism of strain D15 were analyzed by whole genome sequence,and the activities of nitrogen removal related enzymes were measured to preliminarily explore the nitrogen removal mechanism of strain D15.The main results are as follows:A strain of nitrogen removal bacteria D15 was isolated from paddy soil in winter.Its basic characteristics were analyzed by morphological observation,physiological and biochemical test,and the phylogenetic tree was constructed by 16S r RNA gene sequence alignment,The bacterium D15 was identified as Pseudomonas alcaliphila D15.The effects of initial nitrogen concentration,carbon source type,C/N,initial p H,culture temperature,shaking table speed and fermentation time on the nitrogen removal performance of strain D15 were investigated by single factor test method.Finally,the suitable nitrogen removal conditions of strain D15 were obtained:trisodium citrate as the only carbon source,C/N=16:1,initial p H=9.0,temperature was 15℃,shaking table speed was 190 r/min.When cultured under this condition for 36 hours,the removal rates of NH4+-N and NO3--N reached 100.0%;When cultured for 60 h,the removal rate of NO2--N can also reach 100.0%.The results of nitrogen removal from pig manure wastewater showed that after adding strain D15 and an appropriate amount of trisodium citrate,the removal rate of NO3--N could reach 98.3%when fermented at 15℃for 48 hours;At 54 hours of fermentation,the removal rates of NH4+-N and TN could reach 100.0%and 88.8%respectively.The whole genome sequencing results showed that the sequence length of strain D15 was 5173651 bp,and the number of genes was 4771.The genes were annotated through GO,COG and KEGG databases.It was analyzed that strain D15 had multiple enzyme coding genes such as Gln A,Gdh AB,Glt BD,Abr B,Nap AB,Nas A,Nir BD and hmp,and regulatory genes such as Nor R and Nnr S,which were involved in cell nitrogen metabolism.Combined with the prediction results of nitrogen removal gene,KEGG nitrogen metabolism pathway map and enzyme activity measurement results,it is speculated that the ammonia nitrogen metabolism of strain D15 includes ammonia assimilation and heterotrophic nitrification,and the metabolism of nitrate/nitrite includes nitrite/nitrate assimilation and aerobic denitrification.
Keywords/Search Tags:low temperature, HN-AD bacteria, Pseudomonas alcaliphila, Pig manure wastewater, Nitrogen metabolism pathway
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