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一株丁草胺降解菌及其在废水处理和土壤污染修复中的应用基础

A Butachlor-degrading Bacterium and Its Application in Wastewater Treatment and Soil Pollution Remediation

【作者】 楚小强

【导师】 虞云龙;

【作者基本信息】 浙江大学 , 农药学, 2009, 博士

【摘要】 丁草胺是我国使用量最大的几种除草剂之一,由于大量生产和广泛使用,造成了水体和土壤的污染,对生态系统和人体健康构成了威胁。因此对水体和土壤中丁草胺污染净化的研究引起了人们的关注。本文分离到了一株丁草胺降解细菌,明确了该菌对丁草胺和丙草胺的降解特性及机理,对其在丁草胺废水处理和污染土壤修复中的应用进行了研究。主要研究结果如下:分离到一株能以丁草胺为唯一碳源和能源生长的细菌BD-1,通过形态特征、生理生化特性、Biolog鉴定结果及16S rDNA序列分析将其鉴定为施氏假单胞菌(Pseudomonas stutzeri)。在纯培养的条件下测定了细菌BD-1对丁草胺的降解特性,在接种量0.2(OD415)、pH 7.0、30℃条件下,BD-1对丁草胺的降解符合一级动力学特征,1.0、10.0和100.0mg L-1丁草胺的降解半衰期分别0.11 d、0.60 d、0.96 d。细菌BD-1在不同pH和温度下对10 mg L-1丁草胺的降解作用为:pH 7.0>pH 6.0>pH 8.0、30℃>20℃>40℃。BD-1菌降解丁草胺的主要产物为2-氯-2′,6′-二乙基乙酰苯胺和2,6-二乙基乙酰苯胺,主要降解机制有脱氯、脱烷基、水解、羟基化等。鉴于BD-1能以丁草胺为唯一碳源和能源生长,因此丁草胺的降解产物可能被进一步分解生成二氧化碳,为一矿化过程。细菌BD-1能以丙草胺为唯一碳源和能源生长,在纯培养条件下BD-1对丙草胺的降解受初始浓度、pH和温度的影响。不同条件下BD-1对丙草胺的降解作用顺序为:100 mg L-1>10 mg L-1>1 mg L-1、pH 7.0>pH 6.0>pH 8.0、30℃>20℃>40℃。丙草胺微生物降的主要解产物为2-氯2′,6′-二乙基乙酰苯胺,BD-1对丙草胺的作用方式主要有脱氯、羟基化、脱烷基、水解等。利用细菌BD-1构建的膜生物反应器对污染物的去除受进水pH及进水CODCr影响较大,营养液添加对污染物的去除影响不大。在进水CODCr为1500 mg L-1、HRT为24 h、pH值8.0,膜生物反应器对废水中CODCr的去除率在81%左右;对废水中丁草胺等四种农药的总去除率在95%以上,其中丁草胺和对丙草胺的去除率分别高达99%和97%。膜生物反应器经过一年多的运行,对丁草胺等酰胺类除草剂的混合废水及丁草胺的生产废水中的CODCr、农药、浊度等污染物有稳定的去除效果。采用海藻酸钙凝胶将Pseudomonas stutzeri BD-1进行固定化,研究了固定化BD-1对丁草胺的降解效果。在pH 7.0、30℃条件下,固定化和游离态的BD-1对丁草胺的降解符合一级动力学特征,固定化和游离BD-1对100.0 mg L-1的丁草胺的降解半衰期分别为1.55 h和1.93 h。固定化BD-1在试验pH和温度范围内均对丁草胺均有理想去除效果,与游离的BD-1相比有较宽的pH和温度适应性。在经过五次重复使用之后,固定化BD-1对丁草胺降解明显加快,丁草胺的降解半衰期由第一次的1.56 h缩短至第五次的0.91 h。用固定化细菌BD-1构建了固定化微生物反应器,研究了该反应器对水中丁草胺的去除效果。结果表明固定化微生物反应器对丁草胺的去除受pH和流速的影响较大,在pH 7.0、流速小于50 mL min-1时,反应器对5 mg L-1丁草胺的去除率在99%以上。建立了一种膜生物反应器-固定化微生物复合系统,在最佳操作条件下该系统对丁草胺生产废水中的CODCr、BOD5、丁草胺、浊度和臭的去除率分别为97.68%、98.90%、100%、100%和100%,同时废水中的其他化合物也得到了有效的去除。室内模拟条件下2.0 mg kg-1、4.0 mg kg-1和10.0 mg kg-1的丁草胺在土壤中的降解符合一级动力学特征,其降解半衰期分别为6.53 d、8.81 d和11.27 d,随着丁草胺浓度的提高,其在土壤中的降解半衰期明显延长。丁草胺处理初期均会对土壤微生物多样性产生明显的抑制作用。接种Pseudomonas stutzeri BD-1能明显加快土壤中丁草胺的降解2.0 mg kg-1、4.0mg kg-1和10.0 mg kg-1的丁草胺在BD-1接种土壤中的降解半衰期分别为0.37 d、0.46 d和0.99 d。与未接种土壤相比,丁草胺的降解半衰期分别缩短了94.33%,94.78%和91.22%。BD-1的加入消除了丁草胺对土壤微生物的抑制,提高了土壤微生物多样性。田间条件下接种细菌BD-1能有效促进土壤中丁草胺的降解。丁草胺在各处理中的降解均符合一级动力学特征,其在喷药浓度为1.26 kg a.i.ha-1、2.52 kg a.i.ha-1和6.30kg a.i.ha-1土壤中的降解半衰期分别为11.17 d、11.67 d和15.71 d,接种BD-1后3个处理(1.26 kg a.i.ha-1+BD-1、2.52 kg a.i.ha-1+BD-1和6.30 kg a.i.ha-1+BD-1)丁草胺的降解半衰期为4.95 d、4.41 d和5.99 d,分别是丁草胺单独处理时的44.32%,37.79%和38.37%。丁草胺能降低土壤微生物的多样性,而BD-1的加入则能恢复并提高土壤微生物多样性。结果表明接种细菌BD-1进行生物强化是丁草胺污染土壤修复的一种有效方法。

【Abstract】 Butachlor is one of the most widely used herbicides in China. As a result of massproduction and extensive use, it has caused considerable pollution to the water and soil andposes a threat to ecosystem and human health. Therefore, there is an increasing concern onthe cleanup of water and soil contaminated by butachlor. One bacterial strain capable ofutilizing butachlor as sole carbon and energy source was isolated. The biodegradationcharacteristics and mechanisms of butachlor and pretilachlor were investigated, and theapplication of the bacterium strain in butachlor wastewater treatment and soil pollutionremediation were also investigated. The results are summarized as follows:A bacterial strain BD-1 capable of utilizing butachlor as sole carbon and energysources was isolated from the sludge of a membrane bioreactor after successive enrichmentcultures. BD-1 was identified as Pseudomonas stutzeri based on morphology, physiologicaland biochemical characteristics, Biolog GN2, and the results of the 16S rDNA homologuesequence analysis.The degradation characteristics of butachlor by bacterial strain BD-1 was investigatedin pure cultures. The degradation of butachlor by BD-1 was fitted to the fisr-order function.Degradation half-lives of butachlor at concentration of 1.0、10.0 and 100.0 mg L-1 by BD-1were measured to be 0.11 d, 0.60 d and 0.96 d, under conditions of pH 7.0, 25℃andbiomass of 0.2 (OD415). The degradation rates of butachlor by bacterial strain BD-1 wereaffected by pH and temperature following an order of pH 7.0>pH 6.0>pH8.0, and of 30℃>20℃>40℃, respectively. Seven metabolites were identified by GC/MS based onmass spectra data and fragmentation patterns. The main biodegradation products ofbutachlor were 2-chloro-2’,6’-diethylacetanilide and 2,6-diethylacetanilide. The metabolismof butachlor by strain BD-1 involved dechlorination, dealkylation, hydrolyzation andhydroxylation. According to the capability of the bacterial strain BD-1 to utilize butachloras sole carbon and energy sources, it is reasonable to propose that butachlor is mineralizedby the BD-1.Bacterial strain BD-1 was able to use pretilachlor as sole carbon and energy source.The ability of BD-1 to degrade pretilachlor in pure cultures was affected by initialconcentration, pH and temperature. The degradation half-lives of pretilachlor weremeasured to be 0.10 d, 0.23 d and 0.46 d at concentrations of 1, 10 and 100 mg L-1,respectively. The optimal conditions for biodegradation of pretilachlor were pH 7.0 and 30 ℃. Five metabolites were detected and identified by GC/MS. 2-chloro-2’,6’-diethylacetanilidewas the major biodegradation products of pretilachlor by BD-1. The mainmetabolisms of pretilachlor by BD-1 included dechlorination, dealkylation, hydrolyzationand hydroxylation.One membrane bioreactor (MBR) was constructed with the bacterial strain BD-1. TheMBR was used for the treatment of pesticide wastewater containing butachlor and otherchloroacetanilide herbicides. The results showed that MBR had high removal efficiency forCODCr, pesticide, turbidity and odor in the pesticide wastewater. The pollutant removalefficiency of MBR was significantly affected by influent pH and CODCr. The optimaloperating conditions were pH 8.0 and 1500 mg L-1 of influent CODCr. The addition ofnutrient salts had little effects on the removal efficiency of MBR. About 81%, 95 %, 99 %and 100 % of CODCr, total pesticide, butachlor and turbidity in pesticide wastewater wereremoved under the optimal operating conditions. Continuous operation of MBR for morethan 12 months showed stable performance for butachlor wastewater treatment.Pseudomonas stutzeri BD-1 was immobilized with calcium-alginate. Degradation ofbutachlor by immobilized BD-1 proved to be more efficient than free BD-1. Thedegradation half lives of butachlor by the immobilized and free BD-1 at concentrations100.0 mg L-1 were measured to be 1.55 h and 1.93 h, respectively, under conditions of pH7.0 and 30℃. Immobilized BD-1 showed ideal performance for butachlor removing in testpH and temperature. Compared to the free BD-1, immobilized BD-1 could withstand a widerange of pH and temperature change. Butachlor degradation using immobilized BD-1 wasreused for five times, and enhancing degradation rate was observed. The degradationhalf-life of butachlor was shorten from 1.91 h to 0.91 h after 5th reuse.Immobilized microorganism reactor was set up to determine the feasibility of usingimmobilized strain BD-1 to remove of butachlor from simulated wastewater. The butachlorremoval efficiency of the immobilized microorganism reactor was significantly affected bypH and flow rate. A butachlor removal efficiency of greater than 99 % was achieved underthe condition of pH 7.0 and flow rate of below 50 mL min-1.A membrane bioreactor-immobilized microorganism combined system was constructedby connecting immobilized column to the effluent port of MBR and its use in butachlorwastewater treatment was investigated. At the optimal operating conditions, the CODCr,BOD5, butachlor, turbidity and odor removal efficiency of this system were 97.68 %, 98.90% 100 %, 100 %, 100 %, respectively, Other chemicals in the butachlor wastewater were also effectively removed.Degradation of butachlor at levels of 2.0, 4.0, and 10.0 mg kg-1 in laboratory soil wereall fitted to the first-order kinetics, and the corresponding degradation half-lives weredetermined to be 6.53 d, 8.81 d, and 11.27 d, respectively. The degradation half-lives ofbutachlor in soil were extended significantly with the increasing concentration of butachlor.The soil microbial diversity was inhibited significantly 1d after butachlor treatment. Theinhibitory effects were enhanced with the increasing concentration of butachlor.The degradation of butachlor in laboratory soil was significantly enhanced by theinoculation of bacterial strain BD-1. Degradation of butachlor in strain BD-1 inoculatedtreatments (2.0 mg kg-1+BD-1, 4.0 mg kg-1+BD-1, and 10.0 mg kg-1 mg kg-1+BD-1) werefitted to fist-order kinetics and the corresponding degradation half-lives were measured tobe 6.53 d, 8.81 d, and 11.27 d, respectively. Compared to the un-inoculated controls, thedegradation half-lives of butachlor were shorted by 94.33 %, 94.78 % and 91.22 %,respectively. The addition of bacterial strain BD-1 could eliminate the inhibitive effects ofbutachlor on soil microbes and enhance soil microbial diversity.Under the field conditions, bacterial strain BD-1 could effectively enhance thedegradation of butachlor in soil. Degradation of butachlor in all treatments was fitted to thefirst-order kinetics. The degradation half-lives of butachlor in control soil and strain BD-1inoculated soil were 11.17 d, 4.95 d at 1.26 kg a.i. ha-1, 11.67 d, 4.95 d at 2.52 kg a.i. ha-1and 15.71 d, 5.99 d at 6.30 kg a.i. ha-1, respectively. Biolog analysis showed that butachlorcould reduce the microbial diversity in the soil during initial time of experiment, while theaddition of BD-1 could restore and enhance the soil microbial diversity. The resultsindicated that bacterial strain BD-1 inoculation is a promising method for the remediation ofbutachlor contaminated soil.

  • 【网络出版投稿人】 浙江大学
  • 【网络出版年期】2009年 11期
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