Community Diversity Of Nitrosobacteria In SBR Livestock Wastewater System With DO At 3.0mg/L

In recently,the main task of treating like-livestock weaste water is to decrease the concentration of "high ammonia nitrogen".This research was set up some simulated SBR reactors with different DOs,from which we found when DO at 3.0mg/L could reach the best removal rate of ammonia nitrogen and fulfilled the SHARON process.So,the active sludge in this condition was ours' research object,which included the repressors like hormones,pathogenic bacteria,veterinary drugs,heavy metals,etc.By using molecular biology skills,though extracted the total DNA,amplified the target gene fragment amoA and cloned the PCR product,we got the community diversity of nitrosation bacterium in the system.The aim of experiment was to connect the accumulating stage of nitrite nitrogen and the composition of nitrosation bacterium,which would offer some methods to appraise the strain and fathom its community construction.The results were summarized in the following:(1) If the SBR system kept the permanent F/M,C/N,pH,MLSS and temperature,the concentration of DO would have a good dependablity with "tri-nitrogen".The accumulating rate of nitrite nitrogen was highest,which reached 99%nearly when DO at 3.0mg/L.And the ammonia nitrogen was nearly 0 in exhalans.(2) The removal rate of COD was kept at 95%approximately excepted the low DO condition(0.5 mg/L and 1.0 mg/L).That means the removing of COD had a hapositive correlation with DO s in some degree.And in SHARON technology,the degraded time of the COD would be shorter when DO at 3.0 mg/L.(3) By combining method of "proteaseK+CTAB+SDS" with physical treatments of freeze-thawing and vibration,it could be concluded that the combination of shaker and repetitive freeze-thawing would produce the best effective and highest DNA purity with "OD₂₆₀/OD₂₈₀＞1.80".Moreover,characteristic gene fragment was amplified by establishing PCR amplification system.This indicated that the downstream molecular biological operation could be directly conducted without purification and any other special treatments. Therefore,it was a simple and efficient extraction method.(4) The orthogonal design was fit for using in PCR reaction system,which was not only elevate the efficiency but also could respond the experiment results scientifically and exactly.In this experiment,major information was got,through taking use of L₉(3⁴) orthogonal diagram and nine times 'PCR.Some factors were analyzed,including the concentration of DNA,Primers,Mg²⁺,dNTP and annealing temperature was studied.After 9 times experiments,the optimum set in 25ul reaction system was contained:1.5mmol/L Mg²⁺,200umol/L dNTP,1.0 umol/L of primers,0.32ug of genomic DNA template and 0.2U TaqDNA polymerase.Meanwhile,the optimized annealing temperature was 52℃.(5) After PCR amplification,products purification and T-A cloning,the clones were grouped by RFLP method.Restriction patterns of amoA gene fragments were obtained.The homologies were identified according to the announced sequences in Genebank by BLAST program after sequencing the representative recons.Preliminarily,it was confirmed that there were 3 genera of nitrosobacteria in this reactor:Nitrosospira sp.,Nitrosomomas sp. and Nitrosococcus sp.,and theirs distributing frequencies were 0.285,0.392 and 0.035 respectively.According to sequence analysis the Nitrosolobus multiformis and Nitrosomomas europaea had played the predominant role in accumulating the nitrite nitrogen.(6) Using the same way as above to probe the diversity of nitrosobacteria in low DO condition(0.5 mg/L),the result showed that the Nitrosomomas sp.was the only effective bacterium.And the accumulating rate of nitrite nitrogen was 16.4 times smaller than DO at 3 mg/L.Obviously,the diversity of nitrosobacteria is the important cause for keeping the high removal rate of ammonia nitrogen.(7) In the SBR active sludge at 30℃to 32℃,there were existing several genera of nitrosobacteria.Especially the Nitrosospira sp.,was not only the homology reached to 97.4 %with the standard strains,but also the frequency was the highest,which means it played the dominant role in this system.Meanwhile,the Nitrosomomas sp.,Nitrosococcus sp.and some unclutred bacteria were also contributing to the ammonia nitrogen's removal.