摘要
土壤中农药和重金属是经常共存且广泛分布的典型土壤环境污染物。开展农药-重金属复合污染研究,对于正确评价土壤环境中复合污染条件下污染物质的迁移转化行为,帮助人们采取合理的整治措施,解决土壤的环境污染问题具有十分重要的理论和实践意义。本文以除草剂丁草胺和镉为典型的有机物和重金属污染物,在室内模拟条件下,开展了丁草胺-镉复合污染对两种土壤(黑土和水稻土)微生物分子生态毒理效应及生物修复研究。主要研究结果如下:
1、以丁草胺、镉为目标污染物,通过室内模拟试验,研究了丁草胺-镉复合污染对土壤脲酶、磷酸酶和呼吸强度的影响。结果表明,丁草胺-镉复合污染对土壤酶活性的抑制和土壤呼吸作用的影响高于丁草胺和镉单一处理,复合污染对土壤酶的抑制作用更明显。复合污染的交互作用随着丁草胺和镉浓度比例的不同和处理时间的不同分别会产生拮抗作用和协同作用;其中,丁草胺(100 mg/L)+镉(10 mg/L)处理在整个处理过程为协同作用,而丁草胺(10 mg/L)+镉(10 mg/L)和丁草胺(50 mg/L)+镉(10 mg/L)处理初期为拮抗作用,在处理的后期则为协同作用。
2、运用RAPD分子指纹技术,对丁草胺和镉单一处理及丁草胺-镉复合污染处理对土壤微生物种群结构的影响进行了初步研究。采用酶-化学联合原位提取法,提取土壤微生物总DNA并纯化,得到产量高而且纯度也较高的土壤微生物总DNA。采用10条随机引物的RAPD实验结果表明,丁草胺-镉复合污染土壤的RAPD多态性条带明显高于单一污染土壤;复合污染土壤微生物DNA序列与对照土壤中DNA序列的差异较大。表明丁草胺-镉复合污染对土壤微生物群落结构有着明显的影响。
3、分析比较了丁草胺和镉对水稻土和黑土微生物的影响,结果表明在两种土壤中,丁草胺-镉复合污染对土壤微生物的影响有差异:水稻土脲酶、磷酸酶及呼吸强度受抑制的程度比黑土大;抑制程度出现变化的时间,水稻土中比黑土早。同时,复合污染对土壤微生物的RAPD分析结果表明,虽然黑土和水稻土中的对照处理扩增出的RAPD多态性条带数比较接近,但是丁草胺和镉处理的土样微生物DNA序列的多态性明显不同,水稻土的微生物多态性条带明显高于黑土。不同处理土样增加和缺失的DNA条带的分子量有明显差异,并且水稻土中变化的条带分子量大于黑土。微生物群落结构变化试验结果进一步验证了在研究复合污染对土壤酶和呼吸作用影响的试验中黑土比水稻土对丁草胺和镉的耐受和缓冲作用强于水稻土的结论。
4、从农药厂排污口附近采集土样,进行富集驯化和反复纯化,筛选到一株能够有效降解丁草胺的细菌,在纯培养条件下,培养3d,对添加浓度为30 mg/L丁草胺的降解率可达90%以上;根据菌体形状、菌落形态以及生理生化反应特性初步鉴定降解菌属于革兰氏阴性好氧恶臭假单胞菌属(Pseudomonsa putida),命名为RE1。确定了降解菌RE1的生长和对丁草胺降解的最适宜温度为30℃～35℃,pH值6.0～8.0。RE1对镉有一定的耐受能力,在镉浓度为1 mg/L时降解菌能够正常生长,对丁草胺的降解率达到50%以上,表明降解菌RE1在耐受一定浓度镉的条件下,仍对丁草胺产生降解作用。
5、对降解菌RE1的降解酶进行了研究,根据降解酶的降解活性结果确定降解酶既含有组成酶又含有诱导酶,组成酶具有降解丁草胺的活性,而经过丁草胺和镉诱导后产生的诱导酶增加了该菌的降解活性;酶蛋白的SDS-聚丙烯酰胺凝胶电泳实验结果表明,与对照处理相比,丁草胺单一处理和丁草胺-镉复合处理降解菌酶蛋白的含量增加了,但是两处理之间没有明显差异。该结果进一步验证了丁草胺单一处理和丁草胺-镉复合处理能够提高降解酶活性是因为降解菌产生了诱导酶,并且两种处理产生的诱导酶差异不大。
6、双向电泳技术是研究蛋白质差异表达的关键技术,本研究比较了两种双向电泳样品蛋白制备方法,确立了以液氮研磨-TCA/丙酮沉淀为主要技术的降解菌样品蛋白质的制备方法,得到的蛋白样品用pH4-7的线性IPG和12.5%的SDS-聚丙烯酰胺凝胶电泳分离,该法简便,能够有效的去除蛋白质中干扰双向电泳的杂质,得到了高分辨率的双向电泳图谱;而且第二向凝胶电泳(SDS-聚丙烯酰胺)采用普通的垂直板凝胶电泳系统替代成套的Ettan DALTsix系统,结果证明重复性良好,操作简便,降低了实验成本和劳动强度。
7、采用液氮研磨-TCA/丙酮沉淀为主的样品蛋白质制备方法进行双向电泳,研究降解菌在丁草胺和镉胁迫的条件下酶蛋白变化的特点。结果表明,丁草胺和镉处理的降解菌的蛋白发生了明显的变化,其中发生明显变化的11个蛋白点中,有10个蛋白为明显的上调蛋白,1个蛋白为新发现的蛋白。通过质谱分析和数据库搜索对差异蛋白进行鉴定,表明差异蛋白有氧化还原酶、水解酶等活性,参与细胞中物质的转移和转化作用。该研究从蛋白水平确定了丁草胺和镉能够诱导降解酶,从而提高降解能力。
Pesticide and heavy metals (HM) are frequently found together as contaminants in soils and have attracted much attention in recent decades. It is very important to investigate the compound pollution between pesticide and heavy metal for the management and diagnosis of soil pollution. In this paper, the combined effects of butachlor and cadmium (Cd) on soil microorganism were studied by using paddy and phaeozem soil. Moreover, the bioremediation of the combined pollution of butachlor and cadmium were also investigated. The main results are showed as follows:
1. The effects of butachlor and Cd on soil urease, phosphatase and soil respiration were studied under control condition. The results showed that combined inhibition of butachlor and Cd was more significant than separate exposures on soil enzyme and soil respiration. In addition, inhibition of the combined pollutants on soil enzyme was more significant than that on soil respiration. The interaction of butachlor and Cd depend largely on incubation time and the concentration ratios of the pollutants. Joint effects of pollutants may be similar (additive) or stronger (synergistic, more than additive) or weaker (antagonistic, less than additive) than expected effects from separate exposures. In this study, the interaction of butachlor (100mg/kg) and Cd (10mg/kg) was synergistic during the whole incubation time, while the interaction of butachlor (10mg/kg or 50mg/kg) and Cd (10mg/kg) was antagonistic at the beginning of the incubation and synergistic in the end of the incubation.
2. The genotoxicity of microorganism in soil contaminated with butachlor and Cd was studied by the random amplified polymorphism DNA (RAPD). The combined method with enzymatic and chemical lyses was effective for the microbial total DNA extraction. It had higher total DNA yield and purities in the products. Ten random primers were used to amplify the microbial community DNA. The results showed that combined pollution between butachlor and Cd changed the DNA sequence of soil microorganism, and caused DNA damnification. The microbial DNA damnification caused by butchlor-Cd was significantly different from their single pollution. Moreover, the number of amplified band increased with the incubation keeping on. Therefore, DNA sequence of soil microorganism was affected.
3. The effects of butachlor and Cd on soil microorganism were different between in paddy and in phaeozem. The inhibition of butachlor and Cd on soil enzyme and soil respiration was more significant in paddy than that in phaeozem. Moreover, the time of inhibition in paddy occurred earlier than that in phaeozem. In addition, the number of amplified band was more in paddy than that in phaeozem according to analysis the results of RAPD. These compared results in paddy and in phaeozem showed that phaeozem had stronger ability than paddy in enduring pollutants.
4. A butachlor-degrading bacterium (designated strain RE1) was isolated from the soil collected from a pesticide plant. The strain could degrade butachlor effectively. When the concentration of butachlor was 30mg/L, the degradation rate was about 90% within 3 days. Based on the general physiological and biochemical characteristics, strain RE1 was identified as Pseudomonsa putida. The degrading ability and the growth of strain RE1 was affected by incubation temperature and pH. The optimal temperature and pH for the strain’s growth and butachlor degradation was 30-35℃and 6.0-8.0. In addition, strain RE1 could grow normally when the concentration of Cd was below 1 mg/L. Moreover, when the concentration of Cd was 1mg/L and the concentration of butachlor was 30 mg/L, strain RE1 could degrade butchlor and the degradation rate was over 50%. The result indicated that strain RE1 could degrade butachlor at the combined condition of butachlor and Cd.
5. In this study, we tested the degradation of butachlor by the crude enzyme of ER1. In addition, we also tested butachlor and Cd as inducers of butachlor-degrading enzyme synthesis. There were significantly different in enzyme activities between the cells incubated with butachlor, butachlor + Cd and without pollutants (the controls). The activities of the enzyme protein indicated that butachlor-degrading enzyme protein was not only constitutively expressed in RE1 and was also induced by butachlor and Cd. In addition, the results of SDS-PAGE showed that the amount of some enzyme proteins increased after the strain treated with the pollutants. So it seemed that butachor and Cd induced some enzyme proteins benefit for the degrading of butachlor.
6. Two-dimensional gel electrophoresis is the key in protein analysis. The sample of strain RE1 was grinded with liquid nitrogen, 10% TCA-acetone precipitation. Proteins were analyzed by immobilize dry strip pH 4-7 (linear) and 12.5% SDS-PAGE. These methods could remove the impurity that could affect the resolving capability of 2-DE. So, with these methods high resolving capability 2-DE maps were achieved. A normal SDS-PAGE replaced the Ettan DALTsix system, which could get good protein spots effectively and also save labor costs.
7. Of the total bacteria proteins, eleven protein spots were found significantly changed after treated by butachor and Cd. Among these proteins, ten proteins were found to be up regulated, while one protein spot was only detected in butachlor and Cd treated cells but not in the control treated cells. To make the identification more confident and specific, the spots were excised from preparative 2-DE gels and analyzed by MALDI-TOF MS. In order to identify their functions, they were subjected to database searching against SWISS-PROT, TrEMBL and NCBI using profound software. The results showed that the identified proteins might have oxidoreductase or hydrolase activity. These proteins might be responsible for degradation of pollutants and played an essential role in cell defense against environmental stresses.
引文
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