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。     

工业废水中阿特拉津降解细菌的遗传和生理多样性

【目的】通过遗传学和生理学实验,揭示分离自工业废水的阿特拉津降解细菌具有遗传和生理多样性,为阐明阿特拉津生物降解的分子机理和阿特拉津降解细菌在污染环境生物修复中的应用提供新见解。【方法】用普通PCR方法检测菌株的阿特拉津降解基因,分析其降解基因组成;用基因组重复序列PCR技术(rep-PCR)分析降解菌株的基因组类型;用Western blot方法检测菌株阿特拉津降解途径的第一个酶三嗪水解酶(TrzN);用不同氮源(阿特拉津、莠灭净、扑草净、西玛津、氰草净、阿特拉通和氰尿酸)和碳源(蔗糖、葡萄糖、麦芽糖、乳糖、柠檬酸钠、乙酸钠和琥珀酸钠)培养降解菌株,通过检测培养液的OD600值,证明菌株能够利用的氮源和碳源种类。【结果】对分离自工业废水的27个阿特拉津降解菌株所进行的阿特拉津降解基因PCR检测表明,其降解基因组成分别为trzN-atzBC、trzN-atzABC和atzADEF;通过rep-PCR实验将27个阿特拉津降解菌株分为7个群;Western blot结果表明,27个菌株中有24个含有三嗪水解酶TrzN;氮源利用实验表明,2个菌株能够利用所有7种氮源生长,其余25个菌株只能利用其中的2-6种;碳源利用实验表明,10个菌株能够利用所有7种碳源生长,其余17个菌株只能利用其中的3-6种。【结论】分离自某工业废水的27株阿特拉津降解功能菌存在相当广泛的遗传和生理学上的多样性,trzN-atzABC降解基因组成为首次发现。     

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。     

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。     

阿特拉津降解菌SYSA的分离筛选和鉴定

从长期施用阿特拉津的土壤中筛选到1株能够以阿特拉津为惟一碳源生长的菌株SYSA,经生理生化特性鉴定和16S rDNA序列分析,该菌为阴沟肠杆菌(Enterobacter cloacae).对SYSA菌的生物学特性研究表明,pH 7-8,30℃时,在以阿特拉津(20 mg/L)为惟一碳源的培养基上经146 h培养,降解率为87%.     

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ATR3的分离鉴定与土壤修复

阿特拉津因效率高、价格低廉,是我国玉米田施用最广泛的除草剂之一,但其结构稳定,残留时间长,因此对生态环境和人类健康造成了一定的危害。从长期受阿特拉津污染的玉米田土壤中筛选并鉴定阿特拉津降解菌,明确其在不同类型土壤中的去除能力。对分离出的阿特拉津降解菌ATR3进行生理生化分析和16S rRNA序列鉴定,确定菌株ATR3为节杆菌属（Arthrobacter sp.）。该菌株以阿特拉津为唯一氮源,培养48 h后对1 000 mg/L阿特拉津的去除率达到97%以上。敏感作物盆栽试验结果表明,阿特拉津在棕壤上去除最快,褐土次之,黑土最慢,说明阿特拉津在土壤中的去除过程与土壤本身的理化性质呈相关关系。同时,该菌株处理14 d后,能明显恢复玉米的各项生物学指标,说明该菌株对阿特拉津污染土壤具有良好的修复能力。为阿特拉津降解菌剂的推广利用提供参考。     

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。     

Metabolic ability and gene characteristics of Arthrobacter sp. strain DNS10, the sole atrazine-degrading strain in a consortium isolated from black soil

Strain DNS10 was the only member that could utilize atrazine as the sole nitrogen source for growth in an atrazine-degrading consortium which was isolated from black soil previously in our laboratory. It belongs to the genus Arthrobacter according to the sequence of 16S rRNA gene and is designated as Arthrobacter sp. DNS10. 16S rRNA gene phylogenetic analysis showed that strain DNS10 was located in a different evolutionary branch comparing with other Arthrobacter sp. atrazine-degrading strains. The degrading genes such as trzN, atzB and atzC harbored in strain DNS10 revealed high sequence similarity with those in Arthrobacter aurescens TC1 and Pseudomonas sp. ADP. These genes enabled the strain DNS10 to decompose atrazine to cyanuric acid. This was further proved by the results that the strain DNS10 (10⁸ CFU mL⁻¹) could degrade the whole atrazine (100 mg L⁻¹) in the medium within 24 h at 30 °C and there was 66.13 ± 2.11 mg L⁻¹ cyanuric acid accumulated at 24 h. These results imply that the strain DNS10 seems to be an excellent atrazine-degrading strain. Furthermore, this paper helps us in the better understanding of the strain evolution by comparing the metabolic ability and gene characteristics of strain DNS10 with other geographically distinct atrazine-degrading strains.     

Isolation and characterization of an atrazine-degrading Rhodococcus sp. strain MB-P1 from contaminated soil

Aims:  The aim of this study is to isolate and characterize organisms capable of utilizing high concentration atrazine from the contaminated sites.
Methods and Results:  A selective enrichment was used for isolating atrazine-degrading organisms from the contaminated sites resulting in isolation of an efficient atrazine-degrading organism designated as strain MB-P1. On the basis of 16S rRNA gene sequencing, total cellular fatty acid analysis and physiological and biochemical tests, strain MB-P1 was identified as a member of genus Rhodococcus . High performance liquid chromatography was performed to identify the atrazine degradation intermediates demonstrating that the degradation proceeds via formation of 'de-ethylatrazine' and 'de-isopropylatrazine'. Further, plasmid curing by SDS method showed atrazine-degrading gene(s) to be plasmid-encoded.
Conclusions:  We have successfully isolated a Rhodococcus sp. strain MB-P1 which is capable of utilizing atrazine as sole source of carbon and energy at very high concentrations of 1000 ppm. The pathway for degradation of atrazine has also been determined. The metabolic gene(s) responsible for atrazine degradation was found to be plasmid-encoded.
Significance and Impact of the Study:  Rhodococcus sp. strain MB-P1 could be used as an ideal model system for in-situ degradation and restoration of ecological niches which are heavily contaminated with atrazine.     

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 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.     

小龙虾肠道产木聚糖酶细菌的分离与鉴定

【背景】小龙虾肠道微生物是小龙虾降解纤维素和半纤维素的主要驱动力。【目的】研究肠道内细菌的相对丰度,为揭示肠道微生物在小龙虾纤维素降解过程中的作用提供理论支撑。【方法】采用纯培养法从小龙虾肠道筛选产木聚糖酶细菌,并且对小龙虾肠道细菌进行16S高通量测序。【结果】形态学和16SrRNA基因分子鉴定表明,筛选到的4株产木聚糖酶细菌均属于芽孢杆菌科芽孢杆菌属;结合进一步的生理生化特征鉴定,结果为:菌株Z-3为枯草芽孢杆菌(Bacillus subtilis),菌株Z-4为贝莱斯芽孢杆菌(Bacillus velezensis),菌株Z-29为蜡状芽孢杆菌(Bacillus cereus),菌株Z-30为高地芽孢杆菌(Bacillus altitudinis);16S rRNA基因高通量测序结果表明:在属水平上,小龙虾肠道细菌主要是Candidatus Bacilloplasma、拟杆菌属、弧菌属、不动杆菌属、Dysgonomonas、Tyzzerella3、气单胞菌属和希瓦氏菌属细菌。【结论】小龙虾肠道内细菌资源丰富,且芽孢杆菌属细菌在木质纤维素降解过程中发挥一定功能。     

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.     

Evidence of atrazine mineralization in a soil from the Nile Delta: Isolation of Arthrobacter sp. TES6, an atrazine-degrading strain

The s-triazine herbicide atrazine was rapidly mineralized (i.e., about 60% of ¹⁴C-ring-labelled atrazine released as ¹⁴CO₂ within 21 days) by an agricultural soil from the Nile Delta (Egypt) that had been cropped with corn and periodically treated with this herbicide. Seven strains able to degrade atrazine were isolated by enrichment cultures of this soil. DNA fingerprint and phylogenetic studies based on 16S rRNA analysis showed that the seven strains were identical and belonged to the phylogeny of the genus Arthrobacter (99% similarity with Arthrobacter sp. AD38, EU710554). One strain, designated Arthrobacter sp. strain TES6, degraded atrazine and mineralized the ¹⁴C-chain-labelled atrazine. However, it was unable to mineralize the ¹⁴C-ring-labelled atrazine. Atrazine biodegradation ended in a metabolite that co-eluted with cyanuric acid in HPLC. This was consistent with its atrazine-degrading genetic potential, shown to be dependent on the trzN, atzB, and atzC gene combination. Southern blot analysis revealed that the three genes were located on a large plasmid of about 175 kb and clustered on a 22-kb SmaI fragment. These results reveal for the first time the adaptation of a North African agricultural soil to atrazine mineralization and raise interesting questions about the pandemic dispersion of the trzN, atzBC genes among atrazine-degrading bacteria worldwide.