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二苯砷酸高效降解菌的筛选强化及其对污染土壤的修复作用
宋芳1,2
学位类型硕士
导师骆永明研究员
2017-05-23
学位授予单位中国科学院大学
学位授予地点北京
学位专业环境科学
关键词二苯砷酸 紫金牛叶杆菌 共代谢降解 化学诱变 土壤 微生物修复 微生物功能多样性
摘要二苯砷酸(Diphenylarsinic acid,DPAA)是含砷化学武器泄漏后经水解氧化生成的新型环境污染物,因其化学性质稳定、不易降解、生物毒性强,且可通过食物链富集,进而严重威胁着土壤和地下水环境安全与人体健康。因此,亟需开展化学武器残留区DPAA污染土壤的控制修复机理与技术研究。微生物修复因具有成本低、修复面积广和不易造成二次污染等优点,已被广泛应用于有机污染土壤和地下水的修复治理中,但目前国际上有关DPAA污染土壤和地下水微生物修复的研究仍有限。本研究从实验室已保藏的3株微生物菌种资源中筛选获得了一株可好氧降解DPAA的菌株,分别通过外加碳源进行共代谢降解和采用化学诱变剂亚硝基胍(NTG)对菌株进行化学诱变,进一步提高了降解菌对DPAA的降解效率,并了解了降解菌对DPAA的降解动态特征与代谢中间产物。最后,以红壤和黑土为供试土壤,采用土壤微域实验,通过接种强化的DPAA高效降解菌,研究了降解菌对受DPAA污染土壤的修复潜力,分析了其在DPAA污染土壤修复领域的应用前景。以上研究为我国DPAA污染土壤的微生物修复技术研发提供了菌株资源和科学依据。
本研究取得的主要结论如下:
(1)对三株供试菌株的DPAA降解能力试验的结果表明:三株菌均不能利用DPAA作为唯一碳源和能源生存;添加蔗糖为共代谢基质时,只有菌株RC6b对DPAA的降解率显著提高(14%左右),确定了菌株RC6b为DPAA降解菌。
(2)在外加共代谢基质的条件下,菌株RC6b能够有效降解溶液中DPAA。分别添加蔗糖、葡萄糖和乙酸钠为外加碳源,培养28 d后,菌株RC6b对溶液中DPAA的降解率分别达到14.08%、15.21%和15.05%,不同碳源之间无显著差异。
(3)采用亚硝基胍为诱变剂对菌株RC6b进行化学诱变后,所获得诱变菌株对溶液中DPAA的降解效率,与出发菌株相比,显著提高。在以DPAA为唯一碳源培养28 d后,诱变菌株N-RC6b2对DPAA的降解率最高达36.71%;降解过程中DPAA发生了单羟基化反应,生成了代谢中间产物单羟基化DPAA。
(4)土壤微域修复试验表明,接种诱变强化后的菌株N-RC6b2对DPAA污染土壤进行微生物降解修复90 d后,红壤和黑土中DPAA的去除率分别为57.72%和52.90%,土壤的AWCD值与土壤微生物群落多样性指数较对照组均有显著提高,表明污染土壤修复的微生态环境得到了有效恢复。
 
其他摘要Diphenylarsinic acid (DPAA) is a new type of environmental pollutant produced by the hydrolysis and oxidation of arsenic containing chemical weapons after leaking. Because of its stable chemical properties, not easily degraded and strong biological toxicity, DPAA can be enriched through the food chain, thus a serious threat to the soil and groundwater environment safety and human health. Therefore, it is urgent to carry out the research on remediation mechanism and technology of DPAA contaminated soil in chemical weapons residual area. Microbial remediation has been widely used in the remediation of organically contaminated soil. However, the current international research on microbial remediation of DPAA contaminated is still limited. In this study, a strain capable of aerobic degradation of DPAA was screened from the three strains stored in the laboratory, which was subjected to co-metabolism degradation by external carbon source and by the chemical mutagens N-methyl-N’-nitro-N-nitrosoguanidine (NTG). It was shown that the degradation efficiency of DPAA was further improved through the two degradation approaches. Accordingly, the degradation characteristics and metabolic intermediates of DPAA were elucidated and the remediation potential of DPAA-contaminated soil was assessed by soil micro-domain experiment with red soil and black soil. This study provides the new microbial resource and scientific basis for further research and development in bioremediation of DPAA polluted soil.
The main conclusions of this study are as follows:
(1)The results showed that the three strains could not survive with DPAA as the sole carbon source and energy. When the sucrose was used as a co-metabolic substrate, only the strain RC6b significantly increased the DPAA degradation rate (about 14%), and the strain RC6b was identified as DPAA-degrading bacteria.
(2)The strain RC6b was able to effectively degrade DPAA in the solution under the condition of the co-metabolizing matrix. The degradation rates of DPAA in the solution were 14.08%, 15.21% and 15.05% 28 days after addition of sucrose, glucose and sodium acetate, respectively. There was no significant difference among the different carbon sources.
(3)The degradation efficiency of the obtained strain was significantly higher than that of the original strain when the strain was chemically mutated with N-methyl-N’-nitro-N-nitrosoguanidine as the mutagenic agent. The degradation rates of DPAA by using the mutant strain N-RC6b2 was up to 36.71% 28 days after culture with DPAA as the sole carbon source. It is suggested that DPAA undergo a monohydroxylation reaction during the degradation process, producing a metabolic intermediate monohydroxylated DPAA.
(4)Soil microbial remediation experiments showed that DPAA in the contaminated soils could be effectively degraded by the strain N-RC6b2. Microbial remediation of DPAA contaminated soil was carried out 90 days after addition of inoculated strain N-RC6b2. The removel rates of DPAA in red soil and in black soil increased up to 57.72% and 52.90%, respectively. The soil AWCD value and soil microbial community diversity indexs significantly increased compared with the control group, indicating that the soil microbial environment quality after bioremediation was improved effectively.
 
学科领域工学
语种中文
文献类型学位论文
条目标识符http://ir.yic.ac.cn/handle/133337/22441
专题中科院烟台海岸带研究所知识产出
作者单位1.中国科学院烟台海岸带研究所
2.中国科学院大学
第一作者单位中国科学院烟台海岸带研究所
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宋芳. 二苯砷酸高效降解菌的筛选强化及其对污染土壤的修复作用[D]. 北京. 中国科学院大学,2017.
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