【作者】 谢晴；

【导师】 邓仕槐；

【作者基本信息】 四川农业大学 ， 环境工程， 2008， 硕士

【摘要】 本研究在初设了各种不同DO条件的SBR水处理系统中,发现当DO为3.0 mg/L,（中溶解氧）时,系统的脱氮效果最好并实现了SHARON（短程硝化反硝化）工艺。因此,以该条件下的好氧活性污泥为研究对象,克服其含激素,病原菌,兽药,重金属等多种抑制物的特点,提取污泥总DNA,利用分子生物学方法,通过对亚硝化细菌目的基因片断PCR扩增,产物克隆等方法,旨在了解该畜禽废水处理系统中亚硝化细菌的群落多样性,从而将脱氮过程中亚硝态氮的积累与作用亚硝化菌组成结合起来,为废水处理系统中菌种的鉴定和结构的了解提供了一定依据。实验结果表明:（1）在保持F/M、C/N、pH、MLSS以及温度恒定的情况下,SBR系统短程工艺中,DO浓度与“三氮”浓度的变化有良好的相关性,DO越低,氨氮的去除率越低,亚硝态氮和硝态氮基本没有出现积累。当DO为3.0mg/L时,亚硝基氮积累率达到94.9%,远大于硝态氮的积累,实现了短程硝化反硝化过程,出水中氨氮几乎为0。（2）除低溶解氧（0.5mg/L,1.0mg/L）条件以外,COD的去除率基本保持在95%左右,说明有机物的去除在一定程度上与溶解氧呈正相关,在短程硝化反硝化工艺中采用曝气量（≥3.0mg/L）可以缩短COD降解的时间。（3）通过“蛋白酶K+CTAB+SDS”法提取污泥总DNA,并结合冻融处理和玻璃珠研磨震荡法进行比较,结果表明:采取摇床加反复冻融效果最好,DNA纯度最高(OD₂₆₀/OD₂₈₀大于1.80),并且通过PCR扩增,得到了目的基因,说明该DNA提取方法可以不经纯化和任何特殊处理而直接进行分子生物学下游操作,是一种简便,高效的提取方法。（4）利用正交设计,从模板浓度,Mg²⁺浓度,dNTP浓度以及引物浓度4个因素对PCR反应体系进行优化分析,并确定最适退火温度。结果表明:在PCR反应25μL体系中最佳反应组合为:模板DNA 0.32μg,dNTP200μmol/L,Mg²⁺1.5mmol/L,引物1.0μmol/L,Taq DNA聚合酶0.2U,最适退火温度则为52℃。（5）通过PCR产物回收纯化,TA克隆,在进行了RFLP分型以后,将克隆子进行测序,通过序列同源性比较表明:在DO=3.0mg/L条件下SBR畜禽废水处理系统中,主要存在欧洲亚硝化单胞菌、亚硝化螺菌和亚硝化球菌三大类亚硝化细菌,其中起主要作用的是多型亚硝化螺菌（Nitrosospira multiformis）和欧洲亚硝化单胞菌（Nitrosomomas europaea）。（6）考察低DO（0.5 mg/L）条件下的亚硝化细菌组成,将随机挑选的阳性克隆子测序后发现,该条件下亚硝化细菌仅有亚硝化单胞菌一种。同时在本试验SHARON工艺中得出DO=3.0 mg/L时亚硝态氮的积累量为DO=0.5 mg/L时的16.4倍,这说明在废水处理系统中,亚硝化细菌群落的多样性是保证高脱氮率的重要原因。（7）在中温（30℃～32℃）畜禽废水SBR好氧污泥中,多种亚硝化细菌共同发挥了作用,其中的亚硝化螺菌属,不仅与标准菌株同源性高达97.4%,而且其频度也最高,是该水处理系统中的优势菌种,同时除亚硝化螺菌外,亚硝化单胞菌,亚硝化球菌和一些未被培养的细菌也为脱氮作了贡献。更多还原

【Abstract】 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.更多还原