2，6-二硝基甲苯的微生物降解及降解途径的研究
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摘要
2,6-二硝基甲苯(2,6-DNT),英文名2,6-Dinitrotoluene,在通常条件下稳定难以降解,是一种具有较强毒性的化合物。微生物降解法,是一种有效的消除环境中污染物的方法,因其具有高效、经济、无二次污染等优点,而得以快速发展成熟。故本实验对2,6-DNT的微生物降解进行研究。分别包括活性污泥对2,6-DNT降解能力考察,单一菌种对2,6-DNT降解能力的考察和混合菌对2,6-DNT降解能力的考察,进行了详尽的研究。
本文首先对污水处理厂得到的污泥进行驯化,使通过专项驯化的活性污泥具有了较强的对2,6-DNT的降解能力,同时采用均匀设计的方法筛选微生物降解2,6-DNT的条件,通过对温度、摇床转数(需氧量)、PH值条件的考察,得出最佳降解条件为,PH值7.3、温度29℃、转速129r/min,证明在此条件下2,6-DNT浓度为90mg/L、135mg/L、180mg/L时,微生物对2,6-DNT具有很高的降解能力,其96h降解率分别达到了92.2%、90.2%、85.8%。
对活性污泥的菌液经过稀释后在扩大培养基中进行培养,对培养后的菌种进行观察挑选,将挑选后的菌重新接种在扩大培养基中进行培养,然后对培养后的菌种再次进行单一菌纯化,对于挑选出的单一菌进行2,6-DNT降解,对于降解后的菌液中的2,6-DNT进行定量检测,对检测的结果进行比较,结果得到降解率较高菌种3株编号分别为6.1、48N4、7.1,其降解率分别为64.58%、60.17%、70.14%。为了解具有较高降解2,6-DNT能力的菌是何种菌,对其进行菌种的鉴定,将单一菌用LB培养基进行扩增,基因组提取试剂盒步骤进行操作,以得到单一菌种的DNA提取物,将此提取物在2000bp Marka的指示下进行水平凝胶电泳实验,得出基因组提取物对于Marka的指示范围在1000bp到2000bp之间,在此条件下制定PCR的实验温度,将单一菌基因组提取物通过PCR扩增的产物进行基因检测得到其碱基序列,将此序列录入GeneBank中得到登录号为GU223217,在GeneBank基因库中将相似率为99%的10种菌的基因序列通过MEGA4.0生成进化树进行比对,可以得出该种菌均为铜绿假单胞菌。
对于混合菌种,采取两两分组的方法,将经过驯化筛选得到的3株单一菌进行两两分组,得到3组混合菌液,然后通过混合菌液降解2,6-DNT,对混合菌的降解能力进行考察,采用气相色谱对降解后菌液中的2,6-DNT含量进行定量检测,得出A(6.1,48N4)混合菌的降解能力大于6.1和48N4；B(6.1,7.1)混合菌的降解能力大于6.1,小于7.1；C(6.1,48N4)混合菌的降解能力大于6.1,48N4,可以得出结果A(6.1,48N4)混合菌及C(6.1,48N4)混合菌的组成单一菌之间具有相互协同的作用,降解率分别达到了67.44%,73.45%。
为了进一步解单一菌降解2,6-DNT的机制,对单一菌降解途径进行了考察,将优秀的单一菌对于2,6-DNT进行降解实验,分别在2、4、10、24、36、48、72、96、120、144、168、192h降解时进行取样,将取得的样品经过处理后通过气相色谱-质谱检测定性,可以得出其降解中间产物为2-硝基,6-氨基-甲苯,2,6-二氨基甲苯,降解途并对降解前后菌液的毒性通过MTT法进行初步考察,最终得出结果为,2,6-DNT降通过单一菌解后的毒性降低。
2,6-Dinitrotoluene (2,6-DNT for short) is a kind of compound with higher toxicity, which is hardly degradated under the normal conditions. Microbial degradation method is an effective way to eliminate environmental contaminants, and gets a rapid development because of the advantages such as the high efficiency, economy, not any secondary pollution and so on. Therefore, in this we have studied the microbial degradation of 2,6-DNT, including the degradation abilities of 2,6-DNT by the activated sludge, a single strain and the mixed bacteria respectively.
In this study, sludge from the sewage treatment plant was domesticated at the very beginning, which made a stronger degradation of 2,6-DNT. And meanwhile Uniform Design was also invited to gain the optimal degradation condition in order to make the most efficient degradation of 2,6-DNT(pH7.3,29℃,129r/min) via the observation of temperature, shaker rpm and pH value. It proved that under this condition, when the concentration of 2,6-DNT was 90mg/L,135mg/L and 180mg/L, the micro-organisms had a high degradation of 2,6-DNT, and the relevant degradation rate in 96h was 92.2%,90.2% and 85.8% respectively.
Bacteria of the activated sludge were cultivated in the expansion culture medium after the dilution and then observed and selected. And the selected bacteria were re-cultivated in the expansion culture medium, after which the bacteria were purified again. With the help of the selected single bacteria,2,6-DNT was degradated and the concentration of 2,6-DNT in the degradated bacteria solution were detected. Results showed that the three strains of bacteria with the numbers of 6.1,48N4,7.1 had great degradation rates, and the relevant rates were 64.58%,60.17% and 70.14%. In order to check out what kind of bacteria on earth they were that had the great degradation rate, evaluations were carried on. The single bacterium was enlarged cultivated by using LB medium. Following the genome extraction Kit steps, the DNA extract of the single strain bacteria would be gained. The extract was taken to carry out the horizontal gel electrophoresis experimrent under 2000bp, from which the Marka direction range of the gene extract was gained and that was between 1000bp and 2000bp. And the PCR temperature could follow this. The single bacterial gene extract was amplified by PCR, and the product was taken to carry out the gene detection to get the base sequence. And this sequence was put into the GeneBank in order to gain the logging number GU223217. Compared with the ten kinds of bacteria gene sequence of which the similarity was 99% with MEGA 4.0, it showed that the bacteria were Pseudomonas aeruginosa.
For the mixed bacteria, every two made a group. The three strains of bacteria which was domesticated and screened were grouped, following the upper grouping method. And three groups of mixed bacteria solutions. The degradation rate was observed via the degradation of 2,6-DNT by using of the gas chromatography. It showed that the degradation ability of A(6.1, 48N4) was greater than 6.1 and 48N4; B(6.1,7.1) greater than 6.1, weaker than 7.1; C(6.1, 48N4) greater than 6.1 and 48N4. It was also to say that the single bacteria in the mixed bacteria A(6.1,48N4) and C(6.1,48N4) mutual synergy effects and the degradation rates were 67.44% and 73.45%.
In order to know much about the mechanism of the degradation of 2,6-DNT, every single bacterium was observed and detected. The excellent single bacterium was taken to degradate 2,6-DNT. Samples were collected at 2，4，10，24，36，48，72，96，120，144，168，192h, when treated, they were detected and identified through GC-MS. From it, we could see that the intermediate products were 2-nitro-6-amino-toluene and 2,6-diamino toluene and the degradation pathway was The toxicity of the former and degradated bacteria solution had a initial investigation by MTT method and the result showed that the toxicity of 2,6-DNT degradated by the single bacterium decreased.
本文首先对污水处理厂得到的污泥进行驯化,使通过专项驯化的活性污泥具有了较强的对2,6-DNT的降解能力,同时采用均匀设计的方法筛选微生物降解2,6-DNT的条件,通过对温度、摇床转数(需氧量)、PH值条件的考察,得出最佳降解条件为,PH值7.3、温度29℃、转速129r/min,证明在此条件下2,6-DNT浓度为90mg/L、135mg/L、180mg/L时,微生物对2,6-DNT具有很高的降解能力,其96h降解率分别达到了92.2%、90.2%、85.8%。
对活性污泥的菌液经过稀释后在扩大培养基中进行培养,对培养后的菌种进行观察挑选,将挑选后的菌重新接种在扩大培养基中进行培养,然后对培养后的菌种再次进行单一菌纯化,对于挑选出的单一菌进行2,6-DNT降解,对于降解后的菌液中的2,6-DNT进行定量检测,对检测的结果进行比较,结果得到降解率较高菌种3株编号分别为6.1、48N4、7.1,其降解率分别为64.58%、60.17%、70.14%。为了解具有较高降解2,6-DNT能力的菌是何种菌,对其进行菌种的鉴定,将单一菌用LB培养基进行扩增,基因组提取试剂盒步骤进行操作,以得到单一菌种的DNA提取物,将此提取物在2000bp Marka的指示下进行水平凝胶电泳实验,得出基因组提取物对于Marka的指示范围在1000bp到2000bp之间,在此条件下制定PCR的实验温度,将单一菌基因组提取物通过PCR扩增的产物进行基因检测得到其碱基序列,将此序列录入GeneBank中得到登录号为GU223217,在GeneBank基因库中将相似率为99%的10种菌的基因序列通过MEGA4.0生成进化树进行比对,可以得出该种菌均为铜绿假单胞菌。
对于混合菌种,采取两两分组的方法,将经过驯化筛选得到的3株单一菌进行两两分组,得到3组混合菌液,然后通过混合菌液降解2,6-DNT,对混合菌的降解能力进行考察,采用气相色谱对降解后菌液中的2,6-DNT含量进行定量检测,得出A(6.1,48N4)混合菌的降解能力大于6.1和48N4；B(6.1,7.1)混合菌的降解能力大于6.1,小于7.1；C(6.1,48N4)混合菌的降解能力大于6.1,48N4,可以得出结果A(6.1,48N4)混合菌及C(6.1,48N4)混合菌的组成单一菌之间具有相互协同的作用,降解率分别达到了67.44%,73.45%。
为了进一步解单一菌降解2,6-DNT的机制,对单一菌降解途径进行了考察,将优秀的单一菌对于2,6-DNT进行降解实验,分别在2、4、10、24、36、48、72、96、120、144、168、192h降解时进行取样,将取得的样品经过处理后通过气相色谱-质谱检测定性,可以得出其降解中间产物为2-硝基,6-氨基-甲苯,2,6-二氨基甲苯,降解途并对降解前后菌液的毒性通过MTT法进行初步考察,最终得出结果为,2,6-DNT降通过单一菌解后的毒性降低。
2,6-Dinitrotoluene (2,6-DNT for short) is a kind of compound with higher toxicity, which is hardly degradated under the normal conditions. Microbial degradation method is an effective way to eliminate environmental contaminants, and gets a rapid development because of the advantages such as the high efficiency, economy, not any secondary pollution and so on. Therefore, in this we have studied the microbial degradation of 2,6-DNT, including the degradation abilities of 2,6-DNT by the activated sludge, a single strain and the mixed bacteria respectively.
In this study, sludge from the sewage treatment plant was domesticated at the very beginning, which made a stronger degradation of 2,6-DNT. And meanwhile Uniform Design was also invited to gain the optimal degradation condition in order to make the most efficient degradation of 2,6-DNT(pH7.3,29℃,129r/min) via the observation of temperature, shaker rpm and pH value. It proved that under this condition, when the concentration of 2,6-DNT was 90mg/L,135mg/L and 180mg/L, the micro-organisms had a high degradation of 2,6-DNT, and the relevant degradation rate in 96h was 92.2%,90.2% and 85.8% respectively.
Bacteria of the activated sludge were cultivated in the expansion culture medium after the dilution and then observed and selected. And the selected bacteria were re-cultivated in the expansion culture medium, after which the bacteria were purified again. With the help of the selected single bacteria,2,6-DNT was degradated and the concentration of 2,6-DNT in the degradated bacteria solution were detected. Results showed that the three strains of bacteria with the numbers of 6.1,48N4,7.1 had great degradation rates, and the relevant rates were 64.58%,60.17% and 70.14%. In order to check out what kind of bacteria on earth they were that had the great degradation rate, evaluations were carried on. The single bacterium was enlarged cultivated by using LB medium. Following the genome extraction Kit steps, the DNA extract of the single strain bacteria would be gained. The extract was taken to carry out the horizontal gel electrophoresis experimrent under 2000bp, from which the Marka direction range of the gene extract was gained and that was between 1000bp and 2000bp. And the PCR temperature could follow this. The single bacterial gene extract was amplified by PCR, and the product was taken to carry out the gene detection to get the base sequence. And this sequence was put into the GeneBank in order to gain the logging number GU223217. Compared with the ten kinds of bacteria gene sequence of which the similarity was 99% with MEGA 4.0, it showed that the bacteria were Pseudomonas aeruginosa.
For the mixed bacteria, every two made a group. The three strains of bacteria which was domesticated and screened were grouped, following the upper grouping method. And three groups of mixed bacteria solutions. The degradation rate was observed via the degradation of 2,6-DNT by using of the gas chromatography. It showed that the degradation ability of A(6.1, 48N4) was greater than 6.1 and 48N4; B(6.1,7.1) greater than 6.1, weaker than 7.1; C(6.1, 48N4) greater than 6.1 and 48N4. It was also to say that the single bacteria in the mixed bacteria A(6.1,48N4) and C(6.1,48N4) mutual synergy effects and the degradation rates were 67.44% and 73.45%.
In order to know much about the mechanism of the degradation of 2,6-DNT, every single bacterium was observed and detected. The excellent single bacterium was taken to degradate 2,6-DNT. Samples were collected at 2，4，10，24，36，48，72，96，120，144，168，192h, when treated, they were detected and identified through GC-MS. From it, we could see that the intermediate products were 2-nitro-6-amino-toluene and 2,6-diamino toluene and the degradation pathway was The toxicity of the former and degradated bacteria solution had a initial investigation by MTT method and the result showed that the toxicity of 2,6-DNT degradated by the single bacterium decreased.
引文
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