Screening Of Aerobic Denitrifying Bacteria Based On LAMP And Its Denitrification Performance

The aerobic denitrification process is a process in which denitrifying bacteria sequentially reduce nitrate nitrogen or nitrite nitrogen to nitrogen under aerobic conditions.The aerobic denitrifying bacteria are catalyzed by four kinds of metal reductases in the reduction process of different nitrogen forms,and the nitrates are reduced to nitrite by the action of periplasmic nitrate reductase Nap or membranous nitrate reductase Nar.Nitrite is reduced to NO by the action of nitrite reductase Nir,and NO is reduced to N2 O by the action of nitrite oxidoreductase Nor.N2 O is reduced to N2 by the action of nitrous oxide reductase Nos.The denitrification process is controlled by nitrite reductase is the key rate-limiting step of denitrification,and the nitrous oxide reductase is the last step of denitrification to nitrogen.Therefore,the nir S,nir K and nosZ genes can be detected from the molecule by LAMP system.From the biological point of view,the characteristics of aerobic denitrifying bacteria and the transformation of nitrogen in denitrification process are studied in depth,which can optimize the denitrification capacity of aerobic denitrifying bacteria and provide convenient strain screening methods and strain resources for environmental treatment of sewage wastewater.Provide data support for scientific research and production and life of biological nitrogen removal.Loop-mediated Isothermal Amplification(LAMP)is a novel in vitro amplified DNA technique that uses two pairs(or three pairs)of special primers and Bst DNA polymerase with strand displacement activity at constant temperature.Under the(60 ? 65 ° C),a biochemical detection method for nucleic acid amplification can be completed in 60 min.Compared with other conventional gene detection technologies,the LAMP reaction system is fast,simple,highly specific and does not depend on expensive detection equipment.In order to quickly and accurately screen seawater aerobic denitrification strains,this paper designed LAMP primers for nir S gene,nir K gene and nosZ gene using LAMP special software,and used the specific LAMP reaction system to make a preliminary aerobic denitrification strain.The sieve was filtered by the PCR method and the gas detection method,and the denitrification performance of the selected denitrification strain was tested and the environmental factors of denitrification capacity were optimized.The main findings are as follows:(1)By enriching under aerobic denitrification conditions,13 candidate strains were isolated from the biofilter of P.vannamei shrimp breeding system,numbered KFDX1,KFDX2,KFDX3,KFDX4,KFDX5,KFDX6,KFDX7,KWDX1,KWDX2,KWDX3,FJ3-1,FJ3-2 and FJ6-1.(2)According to the nitrite reductase encoding gene nir S gene and nir K gene,the oxidized nitro-reductase-encoding gene nosZ gene sequence designed LAMP primer,LAMP system was reacted at 60 ° C for 60 min,by Gene Finder TM nucleic acid dye direct staining and agar Two methods of sugar gel electrophoresis were used to specifically detect the amplified products.Seven strains of denitrifying strains carrying three genes,such as KFDX1,KFDX2,KFDX3,KFDX4,KWDX1,FJ3-1 and FJ3-2,were initially screened.In order to avoid false positives of the strains screened by LAMP,further verification by PCR showed that the results were consistent with the results of the LAMP screening.Three tests were performed on the gas detection by a digital differential pressure meter to screen for strains that did produce gas.Based on the results of three steps,it was confirmed that KFDX1,KFDX2,KFDX3,KWDX1,FJ3-2 5 N2 aerobic denitrification strains were screened from the whitewater culture system of P.vannamei.These strains were sequenced by 16 S r RNA gene to identify KFDX1.Vibrio alginolyticus,KFDX2 is Paracoccus homiensis,KFDX3 is Paracoccus zeaxanthinifaciens,KWDX1 is Bacillus subtilis,and FJ3-2 is Psudoaltermonus lipolytica.(3)The results of growth and denitrification of strains under different nitrogen sources showed that the strains containing the nosZ gene and producing gas,KFDX1,KFDX2,KFDX3,KWDX1 and FJ3-2,were nitrite,nitrate and ammonia.The removal efficiency of total nitrogen and total nitrogen was good,the removal rates were 99%,90%,97% and 90% respectively,and the maximum removal rates were up to 4.9 mg/(L·h),4.03 mg/(L·h),4.15 mg /(L·h)and 6.03 mg/(L·h).The removal rate was significantly higher than the strains KFDX4 and FJ3-1 without the gene.When the nitrogen source was the only variable,the removal of nitrite by 5 strains was better than that of ammonia nitrogen,which was better than nitrate nitrogen.(4)Effects of environmental conditions on the growth and denitrification of 5 strains The experimental results show that the growth and denitrification of different strains have different environmental requirements and different tolerance to environmental factors.5 strains at a temperature of 22 ° C ? 40 ° C,p H value of 6.0 ? 9.0,salinity of 20 ? 50,C / N of 4 ? 24,with sodium citrate,glucose,sucrose,ethanol,sodium acetate as the sole carbon Both the source conditions maintain good growth and maintain high denitrification characteristics.The optimal growth conditions of 1 strain KFDX1 were temperature 34 °C,p H 7.0,salinity 30 g·L-1,C/N 20,carbon source ethanol;optimal nitrogen removal conditions were temperature 22 ° C,p H 7.0,salinity 30 g·L-1,C/N 20,and the carbon source is sucrose or sodium citrate.The nitrite removal rate of 24 h can reach 94%,and the nitrate removal rate can reach 95%.The optimal growth conditions of 2 strain KFDX2 were temperature 28 °C,p H 7.5,salinity 50 g·L-1,C/N 20,carbon source ethanol;optimal nitrogen removal conditions were temperature 40 ° C,p H 8.5 and 6.0,The salinity is 50 g·L-1,the C/N is 20,and the carbon source is glucose.The nitrite removal rate was 91% at 24 h and the nitrate removal rate was 93%.The optimal growth conditions for the 3 strain KFDX3 were temperature 28 ° C,p H 7.5,salinity 50 g·L-1,C/N 16.carbon source;optimal nitrogen removal conditions were temperature 28 ° C,p H 7.5,salinity 50 g·L-1,C/N is 16,and the carbon source is glucose or citrate.The nitrite removal rate was 83% at 24 h and the nitrate removal rate was 88%.The optimal growth conditions of 4 strains KWDX1 were temperature 28 °C,p H 8.0,salinity 20 g·L-1,C/N 30,carbon source ethanol;optimal nitrogen removal conditions were temperature 22 ° C,p H 7.5,salinity 20 g·L-1,C/N is 30,and the carbon source is sucrose or sodium citrate.The nitrite removal rate was 87% at 24 h and the nitrate removal rate was 88%.The optimum growth conditions for the 5 strain FJ3-2 were temperature 34 ° C,p H 7.5,salinity 25 g·L-1,C/N 24,carbon source ethanol;optimal nitrogen removal conditions were temperature 22 ° C,p H 6.5,The salinity is 0 g·L-1,the C/N is 12,and the carbon source is glucose.The nitrite removal rate was 92% at 24 h and the nitrate removal rate was 89%.The rapid and accurate aerobic denitrification strain screening method and the high-efficiency strains established in this thesis are of great significance for improving the screening technology of denitrifying strains and nitrogen removal and purification of high nitrogen wastewater.