一株深海热液环境来源的PAHs降解菌TVG9-Ⅶ的系统发育与降解基因

【目的】对一株深海热液环境来源的多环芳烃(PAHs)降解菌进行系统发育分析并对其降解特性和降解机制进行研究。【方法】对16S rRNA基因进行扩增和测序,进行基于16S rRNA基因序列的系统发育分析;利用GC-MS测定其对PAHs的降解率;通过构建基因组Fosmid文库,克隆PAHs降解基因簇;并利用RT-PCR和qPCR研究关键降解酶基因在不同PAHs诱导下的表达情况。【结果】从西南太平洋劳盆地热液沉积物中分离到一株PAHs降解菌株TVG9-Ⅶ,系统发育分析结果表明,该菌株属于新鞘氨醇杆菌属(Novosphingobium),与该属的Novosphingobium indicum H25T系统发育关系最为密切,它们的16S rRNA基因序列相似性高达99.7%。该菌株在21 d内对菲、荧蒽和芘的降解率分别为95.2%,57.3%和69.6%。从Fosmid文库中筛选得到一个负责PAHs降解的上游基因簇,包含了PAHs起始降解双加氧酶大小亚基(pheA1a/b)基因和一个脱氢酶基因;RT-PCR和qPCR实验表明,双加氧酶大亚基基因pheA1a在菲的诱导下上调表达4.2倍,而在萘及高环荧蒽和芘的诱导下无上调。【结论】菌株TVG9-Ⅶ是Novosphingobium属深海热液来源的PAHs降解菌,具有良好的降解特性,特别是对高环PAHs的降解效果较好。     

一株芘降解菌的分离鉴定及其降解效果

以芘为唯一碳源,采用平板升华法,从徐州市卧牛山焦化厂周围污染土壤中分离得到一株芘降解菌SE12.经形态观察、生理生化试验和16S rDNA鉴定,该菌株属于分枝杆菌属(Mycobacterium sp.)菌株,与快速生长型非致病性南非分枝杆菌(M.austroa fricanum ATCC33464)的同源性达到98%.SE12降解芘的最适pH和温度为pH9和30℃.当土壤芘初始含量为100和200mg.kg-1,SE12接种量为107CFU.g-1时,30℃培养28d后土壤芘降解率分别达到97%和99%.利用双加氧酶基因的同源序列引物nidAF/nidAR和nidBF/nidBR进行扩增,得出了该菌株编码双加氧酶大亚基和小亚基的基因片段,它们与已知降解芘的分枝杆菌的双加氧酶基因具有高度同源性.     

大西洋洋中脊深海多环芳烃降解菌群的优势菌分析

摘要：【目的】为了分析大西洋洋中脊深海海水及表层沉积物中多环芳烃(PAHs)降解菌群中的优势菌。【方法】采用富集培养法和平板涂布法从深海样品中分离可培养细菌及PAHs降解菌。通过16S rRNA基因测序完成系统发育分析。采用变性梯度凝胶电泳（DGGE）及DNA测序分析降解菌群中的优势菌。【结果】总共分离到16株细菌,包括一株PAHs降解菌Novosphingobium sp. 4D。系统发育分析发现,可培养细菌中两个最大的类群分别与Alcanivorax dieselolei NO1A（5/16）和Tistrella mobilis TISTR 1108T（5/16）亲缘关系最近。DGGE结果表明,在菌群MC2D中菌株4L（以及4M、4N, Alcanivorax dieselolei NO1A, 99.21％）、4D（Novosphingobium pentaromativorans US6-1T,97.07％）和4B（以及4E、4H、4K,Tistrella mobilis TISTR 1108T,＞99％）是降解菌群中的优势菌。而降解菌群MC3CO中的优势菌是菌株5C（以及5H,Alcanivorax dieselolei NO1A,＞99％）、条带5-8代表的未培养菌株（Novosphingobium aromaticivorans DSM 12444T,99.41%）、5J（Tistrella mobilis TISTR 1108T,99.52％）和5F（以及5G,Thalassospira lucentensis DSM 14000T,＜97％）。【结论】本研究发现在大西洋洋中脊深海海水及表层沉积物中Alcanivorax、Novosphingobium、Thalassospira、Tistrella属的细菌是PAHs降解菌群中的优势菌,其中的主要降解菌是Novosphingobium属的细菌。     

一株海草沉积物菲降解菌的筛选、鉴定和降解特性

【背景】多环芳烃(Polycyclic Aromatic Hydrocarbons,PAHs)是一类高毒性的有机污染物,在海洋环境尤其是沿海环境中广泛分布。海草床生态系统作为沿海环境的重要组成部分,深受环境污染等人类活动的影响而处于严重衰退的状态。微生物修复是修复环境中多环芳烃污染的重要途径,具有经济简便、环境友好和无二次污染等特点。【目的】从深圳市大亚湾的海草床沉积物中筛选获得高效多环芳烃降解菌,并分析其降解特性,从而探究海草床生态系统中多环芳烃污染物的微生物修复可行性。【方法】以多环芳烃菲为唯一碳源从海草床沉积物样品中筛选菌株,再通过形态学观察、生理生化实验和16SrRNA基因序列对筛选的菌株进行鉴定,并利用特定引物扩增多环芳烃降解的功能基因——双加氧酶(nidA)基因,最后通过培养实验分析该菌株对菲的降解特性。【结果】筛选出一株高效降解菲的菌株SCSIO 43702,经鉴定为玫瑰杆菌属(Roseovarius)的潜在新菌,并成功扩增得到双加氧酶相似(nidA like)基因;培养实验结果表明,玫瑰杆菌SCSIO 43702在10 d内对100 mg/L菲的降解率最高可达96%,而且其对菲的最适降解条件为:温度30°C、pH值7.5和8.0、盐度3%。【结论】玫瑰杆菌SCSIO 43702凭借其良好的菲降解能力和较强的环境适应性,具有进一步被开发为微生物菌剂以用于多环芳烃污染修复的巨大潜力,为海草床生态系统中多环芳烃污染的微生物修复研究提供了理论依据和可利用的微生物资源。     

大连湾原油降解菌的分离和多样性分析

[目的]研究大连湾原油污染海域可培养原油降解菌的多样性,并获得新的原油降解菌.[方法]通过大连湾海水、海泥和海绵样品采集,以原油作为唯一碳源,培养、富集、分离筛选原油降解菌,根据16S rRNA基因序列确定其系统进化地位.[结果]通过形态观察和16S rRNA基因分析,共获得22个属的50株菌.其中,有6株菌的16S rRNA序列与最相近的菌株序列一致性仅为95％-97％,可能是潜在的新菌.单菌实验表明,45株菌具有石油降解能力.[结论]揭示了大连湾可培养原油降解菌的多样性,并获得了新的原油降解菌,为海洋石油污染的生物治理提供新资源.     

对硝基苯酚降解菌Pseudomonas sp. PDS-7的降解特性及 其降解相关基因的克隆

[目的]本研究的目的是分离对硝基苯酚(PNP)降解菌,研究其对PNP的降解特性;克隆其降解相关基因,并进行表达.[方法]本研究通过富集培养法和系列稀释平板涂布法分离PNP降解菌株;采用形态观察、生理生化特征测定和16S rDNA分析对菌株进行初步鉴定;通过摇瓶试验研究菌株降解特性;利用SEFA-PCR技术克隆降解相关基因,并亚克隆到表达载体pET29a中,构建重组表达质粒pETpnpC,再转入受体菌E.coli BL21(DE3)中进行诱导表达;通过分光光度法测定表达产物的酶活力.[结果]分离到一株PNP降解菌PDS-7,将该菌株鉴定为假单胞菌属(Pseudomonassp.);该菌株能够以PNP作为唯一碳源、氮源和能源生长,菌株对PNP的最高耐受浓度为80 mg/L,最适降解温度为30℃,偏碱性条件有利于菌株对PNP的降解;克隆了PNP降解过程中的偏苯三酚1,2-双加氧酶基因pnpC及马来酰醋酸还原酶基因pnpD(GenBank登陆号EU233791);将pnpC在E.coli BL21(DE3)菌株进行了诱导表达,表达产物对偏苯三酚和邻苯二酚均有邻位开环活性,比活力分别为0.45 U/mg protein和0.37 U/mg protein,表明偏苯三酚1,2-双加氧酶基因pnpC得到了活性表达.[结论]分离鉴定了一株PNP降解菌Pseudomonas sp.PDS-7,研究了该菌株的降解特性,克隆和表达了降解相关基因.     

环羟基化双加氧酶α亚基保守序列的克隆及基因定位

利用PCR法成功地克隆了不动杆菌 (Acinetobacter) L2菌株的环羟基化双加氧酶α亚基保守序列310 bp片段,并对其进行测序.序列分析结果表明该片段与3-苯基丙酸盐双加氧酶α亚基、苯1,2-双加氧酶α亚基、甲苯2,3-双加氧酶α亚基的氨基酸序列同源性分别为72%、75%和78%.Southern杂交将菌株L2的环羟基化双加氧酶α亚基基因定位在L2质粒的不同酶切片段上.     

高分子量多环芳烃降解菌筛选及在土壤电动-生物修复中应用

采用富集培养和多环芳烃双加氧酶基因检测方法,从焦化场地多环芳烃污染土壤分离筛选出9株PAHs降解菌。以高分子量多环芳烃芘为唯一碳源进行摇瓶降解实验,结果表明,J6、S5、S4、S2和B4对芘具有较好的降解能力,21 d时芘降解率均达55%以上,其中B4处理芘的降解率最高,达到70.2%。进一步研究了该5株菌及其混合菌对土壤中芘的降解效果,发现混合菌的降解效果高于单菌的降解效果,其中混合菌H4和单菌B4的降解效果较好,49 d时混合菌H4和单菌B4处理土壤中芘的降解率达29.3%和18.3%。经过16S rRNA基因序列比对,鉴定J6菌株为赤红球菌(Rhodococcus ruber),S5为芽孢杆菌属(Bacillus sp.),S4和S2是鞘脂单胞菌属(Sphingopyxis sp.),B4为假单胞菌属(Pseudomonas sp.)。在电场条件下,混合菌H4和单菌B4处理微生物数量及活性均显著提高,芘的降解率较单独H4和B4处理提高33.0%和20.1%,说明筛选出的5株高分子量多环芳烃降解菌具有较强的电场适应能力,可在高分子量多环芳烃污染土壤电动-微生物修复中应用。     

PAHs降解基因及降解酶研究进展

由于环境中的多环芳烃(PAHs)具有高遗传毒性和"三致"性(致癌、致畸和致突变),其生物降解基因和降解功能酶研究备受关注.多环芳烃双加氧酶是近年来研究较多的多环芳烃降解的关键酶系之一,主要由细菌产生,可通过氧化反应使多环芳烃开环生成小分子的中间产物并最终氧化成CO2和水.目前,有关这类酶的理化性质、结构特点、功能等的研究相继开展,本文对PAHs降解基因、降解酶的研究现状与发展趋势进行综述.     

嗜麦芽寡养单胞菌PX1的降解芘特性及定殖效能

为丰富多环芳烃降解菌菌种库、降低农作物的污染风险,本研究对一株可高效降解多环芳烃(PAHs)的植物内生菌进行筛选鉴定,并初步探究其降解途径以及定殖效能。结果表明: 菌株PX1为嗜麦芽寡养单胞菌。该菌株对多环芳烃的降解具有广谱性,7 d几乎可彻底降解PAH无机盐培养基中的萘,在分别含有50.0 mg·L^-1菲、20.0 mg·L^-1芘、20.0 mg·L^-1荧蒽和10.0 mg·L^-1苯并[a]芘的培养体系中,对菲、芘、荧蒽、苯并[a]芘的降解率分别为72.6%、50.7%、31.9%和12.9%。选取芘作为PAHs模型研究菌株PX1的降解特性。酶活性试验表明,芘可诱导菌株PX1体内邻苯二甲酸双加氧酶、邻苯二酚-1,2-双加氧酶和邻苯二酚-2,3-双加氧酶的活性。在芘降解过程中检测到4,5-环氧化芘、4,5-二羟基芘、龙胆酸/原茶儿酸、水杨酸、顺-己二烯二酸/2-羟粘糠酸半醛、顺-2′-羧基苯丙酮酸、1-羟基-2-萘甲酸、水杨醛等中间产物。浸种定殖试验表明,菌株PX1可高效定殖到空心菜和小麦体内,显著促进空心菜和小麦生长,并能够将空心菜、小麦体内及其生长基质中的芘浓度分别降低29.8%～50.7%、52.4%～67.1%和8.0%～15.3%。表明菌株PX1主要通过“水杨酸途径”和“邻苯二甲酸途径”降解芘,且可以定殖到植物体内,促进植物生长。     

厦门近海海水中多环芳烃降解菌的原位富集与降解菌多样性

摘要：【目的】为了分析厦门近海原位海水中多环芳烃降解菌的多样性。【方法】将涂有菲的聚氯乙烯（PVC）板悬挂在厦门国际邮轮码头的海水中,进行菲降解菌的原位富集。利用变性梯度凝胶电泳（Denaturing gradient gel electrophoresis,DGGE）和16S rRNA基因文库两种方法分析了在PVC板表面富集微生物的菌群结构。之后,在实验室模拟原位条件下,对PVC板表面富集的菲降解菌群进行进一步富集、分离和初步鉴定。【结果】PVC板在海水中浸没6 d后,16S rRNA基因文库分析表明,在涂菲的PVC板表面富集的菌群中解环菌属（Cycloclasticus）对应的克隆子占文库总克隆子的50%;在未涂菲的PVC板表面吸附的菌群中红杆菌科（Rhodobacteraceae）为优势菌,其对应的克隆子占文库总克隆子的47%;而解环菌属的克隆子只占文库总克隆子的2%。DGGE的分析结果也证明解环菌是菲原位富集降解菌群中的优势菌。实验室进一步富集后,从该菌群中分离鉴定出14株细菌,其中一株新鞘氨醇杆菌B14(Novosphingobium sp.B14)具有菲降解能力。但是,解环菌未能获得纯培养。【结论】菲原位富集发现,厦门近海水体中解环菌是多环芳烃的主要降解菌。     

Physiological characterization of Mycobacterium sp. strain 1B isolated from a bacterial culture able to degrade high-molecular-weight polycyclic aromatic hydrocarbons

AIM: The aim of this study was to further characterize a bacterial culture (VUN 10,010) capable of benzo[a]pyrene cometabolism. METHODS AND RESULTS: The bacterial culture, previously characterized as a pure culture of Stenotrophomonas maltophilia (VUN 10,010), was found to also contain another bacterial species (Mycobacterium sp. strain 1B), capable of degrading a similar range of PAH substrates. Analysis of its 16S rRNA gene sequence and growth characteristics revealed the strain to be a fast-growing Mycobacterium sp., closely related to other previously isolated PAH and xenobiotic-degrading mycobacterial strains. Comparison of the PAH-degrading characteristics of Mycobacterium sp. strain 1B with those of S. maltophilia indicated some similarities (ability to degrade phenanthrene and pyrene), but some differences were also noted (S. maltophilia able to degrade fluorene, but not fluoranthene, whereas Mycobacterium sp. strain 1B can degrade fluoranthene, but not fluorene). Unlike the S. maltophilia culture, there was no evidence of benzo[a]pyrene degradation by Mycobacterium sp. strain 1B, even in the presence of other PAHs (ie pyrene) as co-metabolic substrates. Growth of Mycobacterium sp. strain 1B on other organic carbon sources was also limited compared with the S. maltophilia culture. CONCLUSIONS: This study isolated a Mycobacterium strain from a bacterial culture capable of benzo[a]pyrene cometabolism. The Mycobacterium strain displays different PAH-degrading characteristics to those described previously for the PAH-degrading bacterial culture. It is unclear what role the two bacterial strains play in benzo[a]pyrene cometabolism, as the Mycobacterium strain does not appear to have endogenous benzo[a]pyrene degrading ability. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the isolation and characterization of a novel PAH-degrading Mycobacterium strain from a PAH-degrading culture. Further studies utilizing this strain alone, and in combination with other members of the consortium, will provide insight into the diverse roles different bacteria may play in PAH degradation in mixed cultures and in the environment.     

Insights into the genetic diversity of initial dioxygenases from PAH-degrading bacteria

Alpha subunit genes of initial polyaromatic hydrocarbon (PAH) dioxygenases were used as targets for the PCR detection of PAH-degrading strains of the genera Pseudomonas, Comamonas and Rhodococcus which were obtained from activated sludge or soil samples. Sequence analysis of PCR products from several Pseudomonas strains showed that alpha subunits (nahAc allele) of this genus are highly conserved. PCR primers for the specific detection of alpha subunit genes of initial PAH dioxygenases from Pseudomonas strains were not suitable for detecting the corresponding genes from the genera Comamonas and Rhodococcus. Southern analysis using a heterologous gene probe derived from the P. putida OUS82 PAH dioxygenase alpha subunit identified segments of the PAH-degradation gene cluster from C. testosteroni strain H. Parts of this gene cluster containing three subunits of the initial PAH dioxygenase were isolated. These three subunits [ferredoxin (pahAb), alpha (pahAc) and beta (pahAd) subunit] were amplified by PCR as one fragment and expressed in Escherichia coli DH5alpha, resulting in an active initial dioxygenase with the ability to transform indole and phenanthrene. The DNA sequence alignment of alpha subunits from C. testosteroni H and various PAH-degrading bacteria permitted the design of new primers and oligonucleotide probes which are useful for the detection of the initial PAH dioxygenases from strains of Pseudomonas, Comamonas and Rhodococcus.     

一株来自大西洋表层海水的烷烃降解菌Gordonia sp. S14-10的分离鉴定及其降解相关特性的分析

摘要：【目的】本研究的目的是从大西洋表层海水分离筛选新的烷烃降解菌,了解其降解基因及降解特性,为海洋石油污染的生物治理提供材料。【方法】以柴油与原油作为混合碳源从大西洋表层海水样品中富集、并分离筛选出降解能力较强的烷烃降解菌。根据16S rRNA基因和其看家基因secA1序列确定其系统进化地位。分析了烷烃降解范围、表面活性剂产生能力及其他生理生化特性;利用已报道的兼并引物进行了烷烃羟化酶基因的PCR扩增及系统进化分析。【结果】分离筛选得到1株能够降解C10?C36直链烷烃的菌株S14-10。经16S rR     

Using population dynamics analysis by DGGE to design the bacterial consortium isolated from mangrove sediments for biodegradation of PAHs

There are many PAH-degrading bacteria in mangrove sediments and in order to explore their degradation potential, surface sediment samples were collected from a mangrove area in Fugong, Longhai, Fujian Province of China. A total of 53 strains of PAH-degrading bacteria were isolated from the mangrove sediments, consisting of 14 strains of phenanthrene (Phe), 13 strains of pyrene (Pyr), 13 strains of benzo[a]pyrene (Bap) and 13 strains of mixed PAH (Phe + Pyr + Bap)-degrading bacteria. All of the individual colonies were identified by 16S rDNA sequencing. Based on the information of bacterial PCR-DGGE profiles obtained during enrichment batch culture, Phe, Pyr, Bap and mixed PAH-degrading consortia consisted of F1, F2, F3, F4 and F15 strains, B1, B3, B6, B7 and B13 strains, P1, P2, P3, P5 and P7 strains, M1, M2, M4, M12 and M13 strains, respectively. In addition, the degradation ability of these consortia was also determined. The results showed that both Phe and mixed PAH-degrading consortia had the highest ability to degrade the Phe in a liquid medium, with more than 91% being degraded in 3 days. But the biodegradation percentages of Pyr by Pyr-degrading consortium and Bap by Bap-degrading consortium were relatively lower than that of the Phe-degrading consortium. These results suggested that a higher degradation of PAHs depended on both the bacterial consortium present and the type of PAH compound. Moreover, using the bacterial community structure analysis method, where the consortia consist of different PAH-degrading bacteria, the information from the PCR-DGGE profiles could be used in the bioremediation of PAHs in the future.     

Identification and characterization of epoxide hydrolase activity of polycyclic aromatic hydrocarbon-degrading bacteria for biocatalytic resolution of racemic styrene oxide and styrene oxide derivatives

A novel epoxide hydrolase (EHase) from polycyclic aromatic hydrocarbon (PAH)-degrading bacteria was identified and characterized. EHase activity was identified in four strains of PAH-degrading bacteria isolated from commercial gasoline and oil-contaminated sediment based on their growth on styrene oxide and its derivatives, such as 2,3- and 4-chlorostyrene oxides, as a sole carbon source. Gordonia sp. H37 exhibited high enantioselective hydrolysis activity for 4-chlorostyrene oxide with an enantiomeric ratio of 27. Gordonia sp. H37 preferentially hydrolyzed the (R)-enantiomer of styrene oxide derivatives resulting in the preparation of a (S)-enantiomer with enantiomeric excess greater than 99.9 %. The enantioselective EHase activity was identified and characterized in various PAH-degrading bacteria, and whole cell Gordonia sp. H37 was employed as a biocatalyst for preparing enantiopure (S)-styrene oxide derivatives.     

Utilizing pyrene-degrading endophytic bacteria to reduce the risk of plant pyrene contamination

Aims

Endophytic bacteria are ubiquitous in plants, but little information is available on the influence of endophytic bacteria on the uptake and metabolism of PAH by plants. Thus, we seek to investigate whether the colonization of a target plant by a PAH-degrading endophytic bacterium would improve the PAH metabolism of the plant and reduce the risk of plant PAH contamination.

Methods

A pyrene-degrading endophyte was isolated from PAH-contaminated plants using enrichment culture. After root inoculation with the isolated bacterium, greenhouse container experiments were conducted. Pyrene residues in soil and plant samples were analyzed by HPLC.

Results

A pyrene-degrading endophytic bacterium, Staphylococcus sp. BJ06, was isolated from Alopecurus aequalis and could degrade 56.0 % of pyrene (50 mg?·?L^?1) within 15 days. BJ06 grew and degraded pyrene efficiently under environmental conditions. The bacterium significantly promoted ryegrass growth and pyrene removal from contaminated soil in container experiments. The pyrene concentrations in ryegrass roots and shoots in endophyte-inoculated planted soil were reduced by 31.01 % and 44.22 %, respectively, compared with endophyte-free planted soil.

Conclusions

We have provided new perspectives on the regulation and control of plant uptake of organic contaminants with endophytic bacteria. The results of this study will be valuable to risk assessments of plant PAH contamination.