【作者】 郑金伟；

【导师】 李顺鹏；

【作者基本信息】 南京农业大学 ， 生物技术， 2011， 博士

【摘要】 氯代乙酰胺类除草剂是一类高效、高选择性的触杀性除草剂,对禾本科杂草具有非常显著的杀除效果,其主要代表品种乙草胺和丁草胺是我国使用最多的三种除草剂中的两种,年使用量分别超过1万吨和5千吨。氯代乙酰胺类除草剂对鱼类有较强的毒性,乙草胺和丁草胺被美国环保局定为B-2类致癌物,它们还有不易挥发、不易光解、土壤残留期长的特点,对生态环境和人体健康有着巨大威胁。另外,氯代乙酰胺类除草剂对作物存在隐性药害,对农业造成严重的损失。因此,该类除草剂在环境中的吸附、迁移、转化和降解等生态行为越来越受到关注。氯代乙酰胺类除草剂在土壤中主要是通过微生物的降解作用而消失,国内外报道了多株该类除草剂降解菌株,但能降解多种氯代乙胺类除草剂并以其为碳源生长的菌株的分离筛选、降解特性和代谢途径方面的研究还不多,其分子结构对其微生物可降解性的影响的研究还未见报道。本文以氯代乙酰胺类除草剂的常用品种丁草胺为降解对象,从长期施用丁草胺的稻田表层土壤及生产丁草胺的农药厂污水处理车间的生化污泥中分离到丁草胺降解菌株14株,其中10株只能以共代谢的方式降解丁草胺,另外4株能以丁草胺作为唯一碳源生长,菌株DCA-1和FLY-8对丁草胺的降解率最高,在无机盐培养基中,DCA-1和FLY-8在5d内对100mg·L-1的丁草胺的降解率为80.3%和68.5%。14株丁草胺降解菌分属于9个不同的属,这一结果显示自然环境中能降解该类除草剂的微生物种类具有丰富的多样性。本文对菌株DCA-1和FLY-8的生物学特性和分类地位进行了详细研究。菌株DCA-1不产芽孢、不运动、严格好氧,革兰氏染色阴性,短杆状不产水溶性色素,未发现内膜结构及光能生长。LB平板上菌落显淡黄色,呈圆形,中间凸起,边缘整齐,不透明。接触酶和氧化酶阳性硝酸盐还原、脲酶、吲哚、产H2S反应显阴性。主要呼吸醌为泛醌10,主要细胞脂肪酸为C18:1ω7c and 11-methyl C18:1ω7c,DNA G+C含量为62.5 mol%。16S rRNA基因同源性及系统发育分析结果表明菌株DCA-1为红球菌科成员,且和Catellibacterium aquatile同源性最近(96.5%),在系统发育树上能很好地聚类成一个分支。根据表型特征、生理生化特性和16S　rRNA基因序列系统发育分析,将其鉴定为Catellibacterium属的一个新种,命名为Catellibacterium caeni sp. nov..菌株FLY-8为不产芽孢、革兰氏染色阴性及不运动的杆菌。最适生长温度为25-30℃,最适pH值为7.0-7.5,最适盐浓度(NaCl)为0.5%。氧化酶、接触酶和硝酸盐还原阳性,脲酶阴性。对庆大霉素和壮观霉素有抗性。DNA G+C含量为62.5 mol%。16S rRNA基因同源性及系统发育分析结果表明菌株FLY-8为副球菌属成员,且和Paracoccus kocurii JCM 7684T (similarity 99.4%)同源性最近,在系统发育树上能很好地聚类成一个分支,将其鉴定为Paracoccus sp.。降解菌株DCA-1能快速降解甲草胺、乙草胺和丁草胺,5d降解率分别达到89.6%、83.2%、75.9%,对异丙草胺也有一定的降解能力,5d降解率为36.4%,并且降解菌株DCA-1能以这四种氯代乙酰胺类除草剂为碳源生长。降解菌株DCA-1不能降解异丙甲草胺和丙草胺。降解菌株FLY-8降解谱要比降解菌株DCA-1的降解谱广,对甲草胺、乙草胺、异丙草胺、丁草胺、丙草胺和异丙甲草胺,5d降解率分别达到98.7%、88.2%、78.3%、65.2%、35.9%和21.4%,并且菌株FLY-8能以这六种氯代乙酰胺类除草剂为碳源生长。菌株DCA-1和FLY-8在温度为20-35℃,pH值为6-9时对丁草胺的降解效果最好。菌株DCA-1和FLY-8对低于100mg·L-1的丁草胺有较好的降解效果；丁草胺浓度超过200mmg·L-1时会对菌株DCA-1和FLY-8产生毒害作用。接种量越大降解效果越好。通气量试验结果表明,在一定转速范围内菌株DCA-1和FLY-8对丁草胺的降解效果随着摇床转速的增加而增加。探讨了氯代乙酰胺类除草剂分子结构对其生物可降解性的影响。结果表明不同烷氧烷基取代对氯代乙酰胺类除草剂的微生物可降解性有非常显著的影响,烷氧甲基取代要比烷氧乙基取代更容易降解；氯代乙酰胺类除草剂分子中的烷氧甲基中烷基的长度对氯代乙酰胺类除草剂的微生物可降解性有明显的影响,烷基链长度越长,降解速率越慢；烷氧甲基中烷基分支结构也影响到其生物可降解性；而氯代乙酰胺类除草剂中苯环上的烷基取代对除草剂的微生物可降解性似乎没有显著影响。通过HPLC和GC-MS技术鉴定了降解菌株DCA-1和FLY-8对丁草胺降解过程中产生的中间代谢产物并对推测了可能的降解代谢途径。推测菌株DCA-1降解丁草胺的代谢途径为：首先丁草胺通过脱去丁氧甲基支链中的丁基,转化为N-hydroxymethyl-2-chloro-N(2,6-diethyl-phenyl)-acctamide (A),然后物质A可以通过脱掉N上的-hydroxymethyl(羟甲基)转化为2,6-二乙基氯代乙酰替苯胺(B),或者A通过脱去氯原子转化为N-(2,6-Diethyl-pheny)-N-hydroxymethyl-acetamide (D);物质D还可以脱掉一个甲基生成N-(2,6-Diethyl-pheny)-N-hydroxymethyl-formamide (E)。丁草胺还可能通过脱氯和脱乙基被直接转化成(2,6-Diethyl-phenyl)-ethoxymethyl-carbamic acid (C).推测菌株FLY-8降解丁草胺的代谢途径为：首先丁草胺脱丙烷基转化为2-chloro-N-(2,6-dimethylphenyl)-N-(methoxymethyl) acetamide (甲草胺),甲草胺进而N原子上脱烷基转化为2-chloro-N-(2,6-dimethylphenyl) acetamide,接着2-chloro-N-(2,6-dimethylphenyl) acetamid又被转化成2,6-diethylaniline,2,6-diethylaniline经过一些列未知方式的降解进一步被转化成了苯胺,苯胺在苯胺双加氧酶的作用下生成邻苯二酚,再在邻苯二酚双加氧酶的作用下开环,最终被完全矿化为二氧化碳和水。通过土壤试验证实微生物降解作用是土壤环境中丁草胺残留降解的主要动力。外源添加丁草胺降解菌株DCA-1能显著促进各类土壤中丁草胺残留的降解,但降解效果受到土壤性质的影响,其中pH值、有机质含量和粘粒含量是相对重要的影响因素,偏中性的pH值,较高的有机质和粘粒含量有利于菌株DCA-1对丁草胺的降解,而淹水状态不利于菌株DCA-1对土壤中丁草胺残留的降解。更多还原

【Abstract】 Chloroacetamide herbicides are among the most important class of pre-emergence herbicides used for the control of annual grass and broadleaf weeds. The most commonly used chloroacetamide herbicides in the world were acetochlor and butachlor. Chloroacetamide herbicides persist for a long time in soil, and the residues consistently injure subsequent rotation crops, especially in sandy soils with low organic matter. Several studies have demonstrated that these herbicides were highly toxic to some aquatic organisms and were carcinogenic in mammal:acetochlor and alachlor caused tumors in the nasal turbinates, butachlor caused stomach tumors, and metolachlor caused liver tumors. Thus, great concerns have been raised about the behavior and fate of chloroacetamide herbicides and their degradation metabolites in the environment.Studies have demonstrated that biodegradation was the most important factor in the dissipation of chloroacetamide herbicides in environment. Many microorganisms capable of degrading chloroacetamide herbicides have been isolated and the metabolic metabolites were also identified. However, up to now, most reported pure microbial strains co-metabolized chloroacetamide herbicides and only a partial biodegradation was achieved, resulting in the accumulation of their metabolites, which contaminated the soil, surface and ground water. Moreover, up to now, the influence of the molecular structure of chloroacetanilide herbicides on their biodegradability has not been studied extensively.In this study,14 butachlor-degrading strains were isolated form rice field soil and activated sludge. Among these strains,10 of the 14 strains could only grow and degrade butachlor in LB medium, indicating that these strains co-metabolized butachlor. Four strains were able to utilize butachlor as the sole carbon source for growth. All of these strains distributed in nine of bacteria genus, which imply the diversity of the butachlor-degradating bacteria. Two strains, designated DCA-1 and FLY-8, were selected for further study due to their high degradation efficiencies. Strain DCA-1 and FLY-8 were able to degrade about 80.3% and 68.5% of the initially added 100 mg L-1 butachlor in MSM medium within 5 d at 30℃.The taxonomic position of strain DCA-1 was determined using a polyphasic taxonomic approach. Cells of strain DCA-1 are non-sporulating, non-motile, strictly aerobic and Gram-negative. No diffusible pigments are produced. Vesicular internal membrane structures and photoheterotrophic growth were not observed. The major respiratory quinone was ubiquinone-10 and the major cellular fatty acids were C18:1ω7c and 11-methyl C18:1ω7c. The genomic DNA G + C content of strain DCA-1 was 62.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences comparison revealed that strain DCA-1 was a member of the family Rhodobacteraceae and was related most closely to the type strain of Catellibacterium aquatile (sequence similarity 96.5%). The combination of phylogenetic analysis, phenotypic characteristics and chemotaxonomic data supports the suggestion that strain DCA-1 represents a novel species of the genus Catellibacterium, for which the name Catellibacterium caeni sp. nov. is proposed.Strain FLY-8 is a non-spore-forming, gram-negative, nonmotile and rod-shaped bacterium. The DNA G+C content is 69.5 mol%. Phylogenetic analysis of the 16S rRNA gene sequences revealed that strain FLY-8 groupes among Paracoccus species and forms a subclade with Paracoccus kocurii JCM 7684T (similarity 99.4%) with a high bootstrap value of 100%. Thus, based on the results of phenotypic, genotypic and phylogenetic properties, strain FLY-8 was identified as Paracoccus sp.Strain DCA-1 was able to degrade alachlor, acetochlor, propisochlor and butachlor and utilized these herbicides as carbon source for growth. When the initial concentration of different chloroacetamide herbicides were 100 mg L-1,89.6% of alachlor,83.2% of acetochlor,36.4% of propisochlor and 75.9% of butachlor were degraded by strain FLY-8, respectively, after 5 d incubation at 30℃. Strain FLY-8 was able to degrade the six chloroacetamide herbicides used in study and utilized these herbicides as carbon source for growth; and the order of degradation rates was:alachlor> acetochlor> propisochlor> butachlor> pretilachlor> metolachlor. When the initial concentration of different chloroacetamide herbicides were 100 mg L-1,98.7% of alachlor,88.2% of acetochlor,78.3 % of propisochlor,65.2% of butachlor,35.9% of pretilachlor and 24.1% of metolachlor were degraded by strain FLY-8, respectively, after 5 d incubation at 30℃.The influence of the molecular structure of chloroacetanilide herbicides on their biodegradability was studied. The results indicated that the substitutions of alkoxymethyl side chains with alkoxyethyl side chain greatly reduced the degradation efficiencies; the length of amide nitrogen’s alkoxymethyl significantly affected the biodegradability of these herbicides, the longer the alkyl was, the slower the degradation efficiencies occurred; the phenyl alkyl substituents have no obviously influence on the degradation efficiency.The optimal pH and temperature for the butachlor degradation by the two strains were 6-9 and 20-35℃, respectively. The degradation efficiency was related positively to initial inoculum size and ventilation. Low concentrations of butachlor did not inhibit the butachlor degradation. However, high concentrations of butachlor (above 200 mg L-1) reduced the degradation rate.The pathway of butachlor degradation by strain DCA-1 and strain FLY-8 were studied by metabolite identification and enzymatic studies. In strain DCA-1, butachlor was degraded to N-hydroxymethyl-2-chloro-N(2,6-diethyl-phenyl)-acctamide, which then converted to 2-chloro-N-(2,6-diethyl-phenyl)-acetamide,2,6-diethyl-phenyl)-ethoxymethyl-carbamic acid or to N-(2,6-diethyl-pheny)-N-hydroxymethyl-acetamide. N-(2,6-diethyl-pheny)-N-hydroxymethyl-acetamide was transformed to N-(2,6-diethyl-pheny)-N-hydroxymethyl-formamide. In strain FLY-8, butachlor was degraded to alachlor by the partial C-dealkylation and then converted to 2-chloro-N-(2,6-dimethylphenyl) acetamide by N-dealkylation, which subsequently transformed to 2,6-diethylaniline,2,6-diethylaniline was further degraded via the metabolite aniline and catechol, and catechol was oxidized through an ortho-cleavage pathway.Inoculation of strain DCA-1 into soils was found to significantly promote the removal of butachlor residue in soil. The moderate pH, high concentration of organic matter and clay could promote the degradation efficiencies of DCA-1. The degradation of butachlor in the soils need an appropriate soil moisture, high content of soil water would inhibit the degradation efficiencies.更多还原