阿特拉津降解菌Acinetobacter sp. DNS32对无机氮源的响应

【目的】研究Acinetobacter sp.DNS32的生长、阿特拉津降解能力和降解基因转录水平的表达对无机氮素的响应关系,为菌株的工程应用提供指导与理论基础。【方法】以Acinetobactersp.DNS32为对象,采用摇瓶法研究菌株在阿特拉津培养基中菌株生长情况及降解能力对外加硝态氮与铵态氮的响应关系,利用荧光定量PCR技术检测DNS32降解基因表达量对外加无机氮源的响应关系。【结果】外加无机氮源可以促进DNS32菌株的生长,提高阿特拉津降解能力,无机氮源对DNS32菌株的trzN、atzB和atzC 3种降解基因表达均有促进作用,加入无机氮源的试验处理中DNS32菌株trzN基因的表达量最高可达对照的11.252±2.408倍,推断DNS32菌株的这3种降解基因所编码的酶是稳定表达的组成酶。【结论】DNS32降解阿特拉津不受"氮饥饿"诱导机制调控,且无机氮源的存在对菌株的生长与降解有促进作用,因此菌株在土壤修复实践中具有广阔的应用前景。     

阿特拉津降解菌株DNS32的降解特性及分类鉴定与降解途径研究

【目的】研究阿特拉津降解菌株DNS32的菌种分类、降解特性及降解途径,丰富阿特拉津降解菌菌种资源。【方法】在长期施用阿特拉津的东北地区寒地黑土中筛选出一株以阿特拉津为唯一氮源生长的降解菌株DNS32,测定其基本降解特性,通过16S rRNA序列分析进行分类鉴定,并利用阿特拉津降解基因PCR扩增技术及降解产物生成量的测定,进一步揭示其降解途径。【结果】实验结果发现DNS32菌株具有较好的降解能力,且在相对较低温度下也具有一定的降解能力。16S rRNA序列分析结果表明DNS32与鲁氏不动杆菌(Acinetobacter lwoffii)16S rRNA序列同源性高达99%。成功地扩增降解基因trzN、atzB及atzC,实验结果表明DNS32遵循Arthrobacter aurescens TC1的降解模式,可将阿特拉津降解为氰尿酸,降解产物的生成量测定也证明了这一点。【结论】实验结果丰富了阿特拉津降解菌菌种资源,为不动杆菌属的阿特拉津降解菌研究提供了参考。     

阿特拉津降解菌株AD111的分离鉴定及其降解特性

【背景】玉豆轮作过程中,玉米田中长残留除草剂阿特拉津易对下茬大豆作物产生不良影响。【目的】从黑龙江省安达市的农田土筛选一株能适应该土壤环境生长的阿特拉津降解菌并研究其降解特性。【方法】利用富集培养法,分离、筛选一株阿特拉津高效降解菌并结合外观形态、生理生化及16SrRNA基因序列测定对其进行鉴定,通过单一变量法设置不同的碳源、pH、温度和阿特拉津浓度,研究降解菌株最佳发酵及降解条件。【结果】得到一株在BSM-G中能够以阿特拉津为唯一氮源生长的高效阿特拉津降解菌AD111,鉴定为马德普拉塔无色小杆菌(Achromobacter marplatensis)。菌株AD111降解阿特拉津的最适温度为35℃,最适pH为8.0,最佳碳源为蔗糖,24 h内对浓度为50 mg/L的阿特拉津降解率达到99.7%,对300 mg/L的阿特拉津降解率达到81.9%。【结论】降解菌AD111具有较好的环境适应及阿特拉津降解能力,为解决黑龙江偏碱土壤中阿特拉津残留提供了良好的候选菌株。     

阿特拉津降解菌株的分离、鉴定和工业废水生物处理试验

用液体无机盐培养基富集培养法和无机盐平板直接分离法, 从生产阿特拉津的农药厂的废水和污泥混合物中分离到13个能以阿特拉津为唯一氮源生长的细菌菌株。通过16S rRNA基因序列分析, 11个菌株被鉴定为Arthrobacter spp., 2个菌株被鉴定为Pseudomonas spp.。对阿特拉津降解活力最高的Arthrobacter sp. AD30和Pseudomonas sp. AD39的降解基因组成和降解特性进行了详细研究。降解基因的PCR扩增表明, AD30和AD39都含有trzN-atzBC基因, 能将有毒的阿特拉津降解成无毒的氰尿酸。降解实验表明, 向阿特拉津浓度为200 mg/L的无机盐培养基中分别接种等量的AD30、AD39和这两个菌株的混合菌液, 30°C振荡培养48 h以后, 阿特拉津去除率分别为92.5%、97.9%和99.6%, 表明混合菌的降解效果好于单菌。用AD30和AD39的混合菌液接种阿特拉津浓度为176 mg/L的工业废水, 30°C振荡培养72 h以后, 99.1%的阿特拉津被去除, 表明混合菌株在阿特拉津工业废水的生物处理中有很好的应用潜力。     

阿特拉津降解菌T_3 AB_1的分离鉴定及土壤修复

【目的】从阿特拉津污染土壤分离高效降解菌株,进行分类学鉴定、降解特性及黑土修复能力初步研究,为阿特拉津污染土壤微生物修复提供新的菌株。【方法】通过形态特征、生理生化特征和16S rDNA序列分析方法进行菌株鉴定;通过培养时间、温度、pH值等环境因素的研究得出菌株的最佳降解条件;通过降解菌株接种于不同种类除草剂为唯一碳氮源培养基获得该菌株的降解谱;通过土壤接种和敏感作物盆栽生测试验验证菌株对阿特拉津污染土壤修复能力。【结果】本试验从黑龙江省讷河市长期施用阿特拉津的玉米田地中分离出一株能以阿特拉津为唯一碳氮源生长的细菌T3AB1,初步鉴定为节杆菌属(Arthrobacter sp.),该菌株在72 h内对500 mg/L阿特拉津(pH 8.0)的降解率高达99%,其降解能力较高的条件为pH7.0-8.0、25-30℃、摇培72-108 h,该菌株能够利用甲氧咪草烟、咪唑乙烟酸、氟磺胺草醚、氟乐灵、异噁草松为唯一碳氮源进行生长,处理168 h的降解率能够达到12.66%-40.54%,该菌株处理21 d能够显著恢复敏感作物水稻的各项生物量指标,且随着处理时间的延长,其对土壤的修复作用也会逐渐增强。【结论】从黑龙江省污染土壤中筛选得到的高效降解阿特拉津的节杆菌属近缘种T3AB1,土壤接种实验表明该菌株具有很好的土壤修复作用,可为阿特拉津生物修复的研究提供适宜菌种资源。     

阿特拉津降解菌Arthrobacter sp.AG1降解基因研究

菌株Arthrobacter sp. AG1能以4000mg/L的阿特拉津(AT)为唯一碳源、氮源和能源生长。通过设计特异引物从AG1中扩增出阿特拉津氯水解酶基因trzN的全序列,该基因与已报道的trzN基因序列相似性为99%。AG1菌株中含有两个大于100kb的质粒,Southern杂交结果显示trzN和atzB基因均位于其中较大的一个质粒pAG1上。将AG1菌株在LB液体培养基中转接三代后,发现34%的细菌细胞丢失了降解活性,但却未发现丢失质粒,PCR扩增结果表明突变子丢失了trzN基因,但atzB和atzC基因未丢失,说明降解活性的缺失是trzN基因片段从质粒上丢失的结果,表明trzN基因在环境中存在水平转移现象,暗示菌株AG1中的阿特拉津降解基因是基因的水平转移重组的结果。     

阿特拉津降解菌Arthrobacter sp. AG1降解基因研究

菌株Arthrobacter sp. AG1能以4000 mg/L的阿特拉津（AT）为唯一碳源、氮源和能源生长。通过设计特异引物从AG1中扩增出阿特拉津氯水解酶基因trzN的全序列,该基因与已报道的trzN基因序列相似性为99%。AG1菌株中含有两个大于100kb的质粒,Southern杂交结果显示trzN和atzB基因均位于其中较大的一个质粒pAG1上。将AG1菌株在LB液体培养基中转接三代后,发现34%的细菌细胞丢失了降解活性,但却未发现丢失质粒,PCR扩增结果表明突变子丢失了trzN基因,但atzB和atzC基因未丢失,说明降解活性的缺失是trzN基因片段从质粒上丢失的结果,表明trzN基因在环境中存在水平转移现象,暗示菌株AG1中的阿特拉津降解基因是基因的水平转移重组的结果。     

阿特拉津降解菌Arthrobacter sp. AG1降解基因研究

菌株Arthrobacter sp. AG1能以4000 mg/L的阿特拉津（AT）为唯一碳源、氮源和能源生长。通过设计特异引物从AG1中扩增出阿特拉津氯水解酶基因trzN的全序列,该基因与已报道的trzN基因序列相似性为99%。AG1菌株中含有两个大于100kb的质粒,Southern杂交结果显示trzN和atzB基因均位于其中较大的一个质粒pAG1上。将AG1菌株在LB液体培养基中转接三代后,发现34%的细菌细胞丢失了降解活性,但却未发现丢失质粒,PCR扩增结果表明突变子丢失了trzN基因,但atzB和atzC基因未丢失,说明降解活性的缺失是trzN基因片段从质粒上丢失的结果,表明trzN基因在环境中存在水平转移现象,暗示菌株AG1中的阿特拉津降解基因是基因的水平转移重组的结果。     

反应器进水管生物膜中喹啉降解菌的分离鉴定及降解特性

【背景】喹啉是一类高毒、致癌且难降解的含氮杂环化合物,本实验室建立了一个长期高效运行的反硝化喹啉降解生物反应器。【目的】从反应器进水管富集的生物膜中筛选有氧条件下降解喹啉的菌株。【方法】通过以喹啉为唯一碳源的培养基来富集、分离、纯化菌株;利用16S rRNA基因的序列分析鉴定分离株的系统发育地位;比较不同pH及温度条件下菌株的喹啉降解特性。【结果】经鉴定,4株分离物Q1、Q3、Q7和Q8分别属于Sphingobium、Massilia、Rhodococcus和Dyadobacter属。降解实验表明,以上菌株均能在48 h内实现50 mg/L喹啉的完全去除,但各自表现出不同的降解特性,其中Q1、Q3和Q8在降解过程中都检测到了喹啉降解产物2-羟基喹啉的积累。降解喹啉的Sphingobium、Massilia和Dyadobacter属菌株尚未见报道。【结论】从喹啉降解生物反应器的进水管内分离的4株喹啉降解菌可为设计处理含喹啉工业废水的反应器提供新菌种资源,对于完善喹啉生物降解机理研究具有实际意义。     

阿特拉津降解菌株的分离和鉴定

从农药厂废水中分离到6株能以除草剂阿特拉津为唯一氮源生长的细菌,即假单胞菌（Pseudomonas spp,.)AD1,AD2和AD6,土壤杆菌（Agrobacterium sp.)AD4,黄单胞菌（Xanthomonas sp.)AD5,欧氏菌（Erwinia sp.)AD7,AD1菌株能使无机盐培养基中的0．3g/L阿特拉津在72h内降解99．9％,当以AD1,AD2,AD4,AD5,AD6和AD7菌株的总DNA为模板进行PCR扩增时,除AD2菌株以外,均得到了与献报道的假单胞菌ADP菌株的阿特拉津氯水解酶基因（atzA)同源的PCR产物。     

Isolation and Characterization of Atrazine Mineralizing Bacillus subtilis Strain HB-6

Atrazine is a widely used herbicide with great environmental concern due to its high potential to contaminate soil and waters. An atrazine-degrading bacterial strain HB-6 was isolated from industrial wastewater and the 16S rRNA gene sequencing identified HB-6 as a Bacillus subtilis. PCR assays indicated that HB-6 contained atrazine-degrading genes trzN, atzB and atzC. The strain HB-6 was capable of utilizing atrazine and cyanuric acid as a sole nitrogen source for growth and even cleaved the s-triazine ring and mineralized atrazine. The strain demonstrated a very high efficiency of atrazine biodegradation with a broad optimum pH and temperature ranges and could be enhanced by cooperating with other bacteria, suggesting its huge potential for remediation of atrazine-contaminated sites. To our knowledge, there are few Bacillus subtilis strains reported that can mineralize atrazine, therefore, the present work might provide some new insights on atrazine remediation.     

Isolation and characterization of an atrazine-degrading bacterium from industrial wastewater in China

AIMS: To isolate and characterize atrazine-degrading bacteria in order to identify suitable candidates for potential use in bioremediation of atrazine contamination. METHODS AND RESULTS: A high efficiency atrazine-degrading bacterium, strain AD1, which was capable of utilizing atrazine as a sole nitrogen source for growth, was isolated from industrial wastewater. 16S rDNA sequencing identified AD1 as an Arthrobacter sp. The atrazine chlorohydrolase gene (atzA) isolated from strain AD1 differed from that found in the Pseudomonas sp. ADP by only one nucleotide. However, it was found located on the bacterial chromosome rather than on plasmids as previously reported for other bacteria. CONCLUSIONS: Atrazine chlorohydrolase gene, atzA, either encoded by chromosome or plasmid, is highly conserved. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison analysis of atrazine degradation gene structure and arrangement in this and other bacteria provides insight into our understanding of the ecology and evolution of atrazine-degrading bacteria.     

一株阿特拉津降解菌的分离鉴定及降解特性

从农药厂废水处理池的活性污泥中分离到一株阿特拉津降解菌X-4, 根据其生理生化特性和16S rRNA基因序列相似性分析, 将其初步鉴定为节杆菌属(Arthrobacter sp.)。该菌能以阿特拉津为唯一碳氮源生长, 42 h内对100 mg/L的阿特拉津降解效果为95.7%, 降解阿特拉津的最适温度为30 °C, pH为7.0。该菌对多种重金属离子都存在抗性, 显示了其在去除阿特拉津和重金属复合污染方面的应用潜力。对其降解基因的初步研究显示, 该菌含有trzN、atzB和atzC 3个阿特拉津降解相关基因。     

Characterization of an atrazine-degrading Pseudaminobacter sp. isolated from Canadian and French agricultural soils

Atrazine, a herbicide widely used in corn production, is a frequently detected groundwater contaminant. Fourteen bacterial strains able to use this herbicide as a sole source of nitrogen were isolated from soils obtained from two farms in Canada and two farms in France. These strains were indistinguishable from each other based on repetitive extragenic palindromic PCR genomic fingerprinting performed with primers ERIC1R, ERIC2, and BOXA1R. Based on 16S rRNA sequence analysis of one representative isolate, strain C147, the isolates belong to the genus Pseudaminobacter in the family Rhizobiaceae. Strain C147 did not form nodules on the legumes alfalfa (Medicago sativa L.), birdsfoot trefoil (Lotus corniculatus L.), red clover (Trifolium pratense L.), chickpea (Cicer arietinum L.), and soybean (Glycine max L.). A number of chloro-substituted s-triazine herbicides were degraded, but methylthio-substituted s-triazine herbicides were not degraded. Based on metabolite identification data, the fact that oxygen was not required, and hybridization of genomic DNA to the atzABC genes, atrazine degradation occurred via a series of hydrolytic reactions initiated by dechlorination and followed by dealkylation. Most strains could mineralize [ring-U-(14)C]atrazine, and those that could not mineralize atrazine lacked atzB or atzBC. The atzABC genes, which were plasmid borne in every atrazine-degrading isolate examined, were unstable and were not always clustered together on the same plasmid. Loss of atzB was accompanied by loss of a copy of IS1071. Our results indicate that an atrazine-degrading Pseudaminobacter sp. with remarkably little diversity is widely distributed in agricultural soils and that genes of the atrazine degradation pathway carried by independent isolates of this organism are not clustered, can be independently lost, and may be associated with a catabolic transposon. We propose that the widespread distribution of the atrazine-degrading Pseudaminobacter sp. in agricultural soils exposed to atrazine is due to the characteristic ability of this organism to utilize alkylamines, and therefore atrazine, as sole sources of carbon when the atzABC genes are acquired.     

Isolation and characterization of atrazine-degrading Arthrobacter sp. strains from Argentine agricultural soils

Three bacterial strains capable of degrading atrazine were isolated from Manfredi soils (Argentine) using enrichment culture techniques. These soils were used to grow corn and were treated with atrazine for weed control during 3 years. The strains were nonmotile Gram-positive bacilli which formed cleared zones on atrazine solid medium, and the 16S rDNA sequences indicated that they were Arthrobacter sp. strains. The atrazine-degrading activity of the isolates was characterized by the ability to grow with atrazine as the sole nitrogen source, the concomitant herbicide disappearance, and the chloride release. The atrazine-degrader strain Pseudomonas sp. ADP was used for comparative purposes. According to the results, all of the isolates used atrazine as sole source of nitrogen, and sucrose and sodium citrate as the carbon sources for growth. HPLC analyses confirmed herbicide clearance. PCR analysis revealed the presence of the atrazine catabolic genes trzN, atzB, and atzC. The results of this work lead to a better understanding of microbial degradation activity in order to consider the potential application of the isolated strains in bioremediation of atrazine-polluted agricultural soils in Argentina.     

Genetic potential,diversity and activity of an atrazine-degrading community enriched from a herbicide factory effluent

Aims: To characterize an atrazine-degrading bacterial community enriched from the wastewater of a herbicide factory. Methods and Results: The community mineralized 81·4 ± 1·9% of [¹⁴C-ring]atrazine and 31·0 ± 1·8% of [¹⁴C-ethyl]atrazine within 6 days of batch cultivation in mineral salts medium containing atrazine as the sole nitrogen source. Degradation activity of the community towards different chloro- and methylthio-substituted s-triazine compounds was also demonstrated. Restriction analysis of amplified 16S rDNA revealed high diversity of bacterial populations forming the community, with Pseudomonas species dominating in the clone library. Atrazine-degrading genetic potential of the community determined by PCR revealed the presence of trzN, atzB, atzC and trzD genes. The trzN, atzB and atzC genes were shown to be located on a plasmid of 322 kb. Quantitative PCR showed that relative abundances of atzB, atzC and trzD genes were approx. 100-fold lower than 16S rDNA. Conclusions: The enriched community represents a complex bacterial association expressing substantial atrazine-mineralizing activity and a broad specificity towards a range of s-triazine compounds. Significance and Impact of the Study: Our study is beginning to yield insights into the richness, genetic potential and density of functional atrazine-mineralizing community that could be a potential bioaugmentation agent for improving biotransformation processes in wastewaters bearing different s-triazine compounds.     

Isolation and characterization of Arthrobacter sp. strain MCM B-436, an atrazine-degrading bacterium, from rhizospheric soil

Atrazine is one of the most environmentally prevalent s-triazine-ring herbicides. The widespread use of atrazine and its toxicity necessitates search for remediation technology. As atrazine is still used in India as a major herbicide, exploration of atrazine-degrading bacterial community is of immense importance. Considering lack of reports on well characterized atrazine-degrading bacterial cultures from India and wide diversity and density of microorganisms in rhizosphere, soil sample from rhizosphere of atrazine-resistant plant was studied. Arthrobacter sp. strain isolated in this investigation utilizes atrazine as the sole nitrogen source. In addition, the bacterium degrades other triazines such as ametryn, cyanizine, propazine and simazine. PCR analysis confirms the presence of atzBCD and triazine hydrolase (trzN) genes on chromosomal DNA. Sequencing of the trzN gene reveals high sequence similarity with trzN from Nocardioides sp. C190. An inducible and intracellular atrazine chlorohydrolase enzyme was isolated and partially purified from this isolate. This study confirms the presence of atrazine-degrading microbial population in Indian soils and could be used efficiently for remediation of contaminated soils. Presence of trzN gene indicates possible presence of bacterial community with more efficient and novel enzymatic capabilities. Comparison of enzyme and gene structure of this isolate with other geographically distinct atrazine-degrading strains will help us in the better understanding of gene transfer and evolution.