不同富集方法分离多环芳烃降解菌的比较研究

多环芳烃是一类普遍存在的环境污染物。本研究探讨了普通富集法,固定化富集法以及巴斯德消毒后富集法三种途径从相同红树林土壤中分离菲降解茵的差异。通过平板培养和变性梯度凝胶电泳两种方法分析分离结果。上述方法分别获得以鞘氨醇单胞茵、分枝杆菌以及红球茵为优势菌群的群落,表明分离方法对多环芳烃降解菌多样性的研究是一种重要的影响因素。     

两株四环素降解菌的分离鉴定及降解条件优化

为发掘四环素高效降解菌株,本研究从以四环素为唯一生长碳源的养鸡场粪便堆肥样品中分离筛选到2株四环素降解菌TC-04和TC-09,并通过形态特征、生理生化特征和16SrRNA基因序列分析,对其进行鉴定.采用单因素试验分别探究不同碳源、氮源、微量元素这3个培养基成分和培养时间、接种量和装液量这3个培养条件对两株菌降解四环素...     

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。     

深海多环芳烃降解菌新鞘氨醇杆菌H25的降解特性及降解基因

[目的]为了从深海环境中筛选新的多环芳烃降解菌,了解其降解基因及降解特性.[方法]以原油作为碳源从印度洋深海海水样品中富集筛选出降解能力较强的多环芳烃降解菌,并根据已报道的相关菌属的多环芳烃起始双加氧酶大亚基序列及侧翼序列设计兼并引物进行扩增.[结果]获得了1株能够高效降解原油、柴油及多种多环芳烃的菌株H25.经16S rDNA序列系统发育分析表明它属于新鞘氨醇杆菌属(Novosphingobium)(96%).并从该菌株中扩增获得2条相似度为91.0%双加氧酶基因片段.2条序列在NCBI上Blastn分析表明均与菌株N.aromaticivorans DSM12444T的降解质粒pNL1上的双加氧酶大亚基具有最高相似度,分别为99.6%和91.0%.根据pNL1上的双加氧酶序列设计引物获得了包含H25双加氧酶大亚基及上下游序列的2个基因片段H25 Ⅰ(2.9kb)和H25Ⅱ(4.5kb).另外,单碳降解实验表明H25对联苯、2-甲基萘、2,6-二甲基萘、菲、二苯并噻吩、二苯并呋喃等均有较好的降解能力.[结论]H25菌株是Novosphingobium属可能的新种.深海细菌在大洋环境多环芳烃污染的自然净化中起到一定作用,并在环境生物修复中有较大的应用前景.     

一株高浓度多环芳烃降解菌的鉴定和降解特性

采用选择性富集培养方法,从沈抚灌区土壤中分离得到多环芳烃(PAHs)高效降解菌NI2,应用此降解菌制备固定化菌剂,修复焦化厂内高浓度PAHs污染土壤,并通过生理生化和16S rDNA测序进行微生物鉴定.经过30 d的降解实验,菌N12对污染土壤中各PAH的去除率＞66％,总去除率为80％.生理生化和16S rDNA测序分析表明,分离得到的菌株N12为分支杆菌属(Mycobacterium sp.),该菌具有与其他分枝杆菌同源的双加氧酶基因nidA和pdoA2.结果表明,从土壤中筛选获得的分枝杆菌可以修复高浓度PAHs污染工业土壤.     

一株DDT降解菌的筛选、鉴定及降解特性的初步研究

从DDT污染的土壤中筛选具有DDT降解能力的细菌, 经过富集培养、分离纯化得到56株细菌, 将其接种到基础盐酵母培养基, 7 d后用紫外分光光度计法初筛得到降解率较高的一株菌, 编号为D-1。通过16S rDNA序列分析结合传统分类学方法确定该菌为寡养单胞菌属(Stenotrophomonas sp.)的一株菌。对菌体降解DDT的特性的研究表明, 在培养温度为30℃, 底物质量浓度为40 mg/L, pH 7.0, 摇床转速为200 r/min的条件下, 该菌株对DDT降解10 d的降解率为69.0%。     

一株吡虫啉杀虫剂降解菌BB-1的分离鉴定

目的:从福建安溪茶园土壤分离能降解吡虫啉杀虫剂的细菌菌株,对其进行分类鉴定.方法:采用室内培养试验方法,通过富集驯化、平板划线分离得到1株优势细菌BB-1,采用形态、生理生化特征和16S rDNA序列分析对菌株BB-1进行鉴定.结果:菌株BB-1 与多株苍白杆菌(Ochrobactrum)的亲缘关系最近,该菌株鉴定为苍白杆菌属(Ochrobactrum sp.).BB-1在吡虫啉浓度为400mg/L的LB培养基中培养0h～5h为生长延迟期,4h～20h为对数生长期,20h～42h为稳定期,42h以后为衰亡期.菌株BB-1对多种抗生素敏感,可耐受的NaCl浓度超过10%.结论:BB-1能降解吡虫啉杀虫剂,分类上应属于Ochrobactrum sp..     

LB克隆系统的构建及在鞘氨醇单胞菌基因融合表达中的应用

为摆脱限制性酶切位点不足的限制,构建可灵活改变多基因融合方向的表达载体,基于ⅡS型和ⅡT型限制性内切酶LguⅠ和BbvC Ⅰ设计开发了 LB克隆系统.该克隆系统是以广宿主质粒pBBR1MCS-3为初始载体,利用 PCR的方法,在其多克隆位点区插入LB片段(GCTCTTCCTCAGC)构建得到的.LB片段含Lgu Ⅰ和B...     

一株高分子量多环芳烃降解菌的筛选、鉴定及降解特性研究

采用富集培养方法从多环芳烃污染土壤中筛选分离得到1株能以苯并[a]芘(B[a]P)为唯一碳源和能源生长的菌株.形态特征观察和16S rDNA序列分析结果表明,该菌株为副球菌属(Paracoccus sp.),编号为HPD-2.HPD-2在3.0 mg/L的B[a]P液体培养基中生长较慢,培养5 d后B[a]P的降解率为89.7%.同时,该菌株对四环的芘和荧蒽也具有较好的降解能力,培养7 d后芘和荧蒽的降解率分别达到47.2%和84.5%.可见,该菌株对高分子量PAHs具有很好的降解潜力.     

DBP降解菌株XJ1的分离鉴定及其降解特性

目的：从湘江底泥筛选分离出能够高效降解邻苯二甲酸二丁酯（DBP）的菌株,对其进行鉴定和降解特性研究。方法：采用DBP为唯一碳源和能源的无机盐培养基,通过富集培养、平板划线分离得到一株优势菌,编号为XJ1。采用形态学、生理生化、（G＋C）mol%和16SrDNA序列分析进行鉴定。采用高效液相色谱测定菌株XJ1在摇瓶中对DBP的降解能力,并进行DBP代谢产物的分析和底物广谱性测试。结果：鉴定该菌株为Sphingomonsasp.。摇瓶实验结果表明：最佳降解条件为温度35℃,初始pH为7.0,转速150r/min;在最佳的降解条件下,40h之内DBP可完全降解。HPLC-UV检测出的中间代谢产物为邻苯二甲酸单丁酯（MBP）和邻苯二甲酸（PA）。底物广谱性实验表明菌株XJ1能够利用邻苯二甲酸二甲酯、邻苯二甲酸二乙酯等邻苯二甲酸酯类化合物。结论：菌株XJ1对DBP具有高效的降解能力,在处理含有邻苯二甲酸酯类化合物污染的生物修复方面具有独特的应用潜力。     

盐地碱蓬GST基因的克隆、序列分析及其表达特征

从盐地碱蓬 (Suaedasalsa)幼苗的cDNA文库中克隆到一个 0 .9kb的全长cDNA ,同源性分析表明该全长cDNA与已报告的大豆 (Glycinemax)GST基因相应序列的同源性达 5 5 % ,可能编码由 2 35个氨基酸组成的谷胱甘肽转移酶 (glutathioneS transferase ,GST)。Southern杂交结果证明GST基因在碱蓬基因组中可能有至少两个以上的拷贝 ;Northern杂交结果表明 ,4 0 0mmol/L的NaCl处理 4 8h ,幼叶中GSTmRNA的表达量是对照的 2～ 3倍 ,说明碱蓬中GST基因受盐诱导     

Degradation of alpha, beta, gamma and delta-hexachlorocyclohexanes by Sphingomonas paucimobilis

Sphingomonas paucimobilis degrades aerobically , , and -hexachlorocyclohexane. With -HCH, complete degradation occurred after 3 days but with and , and with -HCH, 98 and 56 % degradation occurred after 12 and 8 days of incubation, respectively. Pentachlorocyclohexene was formed as the primary metabolite during the degradation of all the HCH isomers. © Rapid Science Ltd. 1998     

降解2,4-二氯酚微生物的分离及其2,4-二氯酚羟化酶基因的克隆和表达

从土壤中分离到一株降解2,4-二氯酚能力较强的细菌菌株GT241-1,经鉴定该菌株属于假单胞菌属。菌株GT241-1在最适条件下能在48h内将90mg/L的2,4-DCP降解91%,能利用2,4-二氯酚、2,4-二氯苯氧乙酸、苯甲酸和儿茶酚为唯一碳源生长。采用Southern杂交对2,4-二氯酚羟化酶基因（dcpA）定位后构建基因组文库,再用斑点杂交筛选目的转化子,克隆了该菌株的dcpA。序列测定得知含dcpA的亚克隆片段全长2389bp,其中dcpA基因编码区1797bp。核苷酸和氨基酸序列分析表明,dcpA与已在GenBank登记的相关基因有一定的差异。dcpA基因能够在大肠杆菌转化子中成功地表达有生物活性的酶。     

Cloning, Sequencing, and Characterization of a Gene Cluster Involved in EDTA Degradation from the Bacterium BNC1

EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The emoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.     

三氯乙烯降解菌FT17的分离、鉴定及其降解特性研究

采用水-硅油双相系统, 从辽河流域浑河沈阳段底泥中筛选得到一株三氯乙烯降解菌FT17。综合形态特征、生理生化特征、16S rRNA Blast分析和系统发育分析结果, 将该菌株鉴定为Sporosarcina ginsengisoli。菌株FT17最适生长温度为34°C, 最适生长pH为7.8。苯酚作为共代谢基质可以促进该菌株对三氯乙烯的降解。该菌株的三氯乙烯降解酶在胞内和胞外均存在。采用两种质粒提取方法对该菌株进行质粒检测, 结果均没有发现质粒条带, 推测该菌株的三氯乙烯降解基因位于染色体上。     

The Role of Exopolymers Produced by Sphingomonas paucimobilis in Biofilm Formation and Composition

Exopolymers have been associated with the initial adhesion of bacteria, which is the primary step for biofilm formation. Moreover, the polymeric matrix of biofilms has a considerable influence on some of the most important physical and physiological properties of biofilms. The role of extracellular polymers in biofilm formation was studied using three mutants of Sphingomonas paucimobilis with increasing capabilities for exopolymer production. The physical, biochemical and physiological properties of three different layers of each biofilm were determined. The layers were detached by submitting the biofilm to increasing shear stress. The results revealed that the presence of exopolymers in the growth medium was essential for biofilm formation. The mutant producing the highest amount of exopolymer formed very thick biofilms, while the biofilms formed by the medium exopolymer producer were on average 8 times thinner. The lowest exopolymer producer did not form biofilm. In both types of biofilms, exopolymer density increased with depth, although this tendency was more significant in thinner biofilms. Cell distribution was also more heterogeneous in thinner biofilms, exhibiting a greater accumulation of cells in the inner layers. The thicker biofilms had very low activity in the inner layer. This was related to a high accumulation of proteins and DNA in this layer due to cell lysis and hydrolytic activity. Activity in the thin biofilm was constant throughout its depth, suggesting that there was no nutrient limitation. The production of exopolymers by each cell was constant throughout the depth of the biofilms, although it was greater in the case of the higher producer.     

Cloning, Sequencing and Characterization of a Novel Phosphatase Gene, phoI, from Soil Bacterium Enterobacter sp. 4

A gene, phoI, coding for a phosphatase from Enterobacter sp. 4 was cloned in Escherichia coli and sequenced. Analysis of the sequence revealed one open reading frame (ORF) that encodes a 269–amino acid protein with a calculated molecular mass of 29 kDa. PhoI belongs to family B acid phosphatase and exhibits 49.4% identity and 62.4% homology to the hel gene from Heamophilus influenzae, which encoded an outer membrane protein (P4). The optimum pH and temperature for phosphatase activity were pH 5.5 and 40°C, respectively. Its specific activity on ρ-nitrophenyl phosphatate was 70 U/mg at pH 5.5 and 40°C. Enzyme activity was inhibited by Al³⁺, EDTA, and DTT, but fivefold activated by Cu²⁺ ion (350 U/mg). PhoI showed a strong synergistic effect when used with a purified E. coli phytase, AppA, to estimate combination effects. Seung Ha Kang and Kwang Keun Cho contributed equally to this work.     

少动鞘氨醇单胞菌致感染性心内膜炎1例

少动鞘氨醇单胞菌(Sphingomonas paucimobilis) 是一种少见的条件致病菌,可引起手术后感染、创伤后腿部溃疡、菌血症、脑膜炎、慢性蜂窝织炎、手术后眼内炎等,未查见累及心瓣膜的报道.本文报道1例由少动鞘氨醇单胞菌所致感染性心内膜炎的病例.该患者为中年男性,因"反复发热2月余"入院.以发热伴左上腹痛为首...     

敌敌畏降解菌的分离鉴定及降解特性研究

目的:从受有机磷农药污染的土壤中分离能降解DDVP的菌株,对其进行鉴定和降解特性研究.方法:采用DDVP为惟一碳源和能源的无机盐培养基,通过富集培养、平板划线分离得到一株优势菌,编号为DDW-1,采用形态学、生理生化和16S-rDNA序列分析对其进行鉴定,采用气相色谱测定菌株DDW-1对DDVP的降解能力,并进行底物广谱性测试和降解酶定位实验.结果:该菌株鉴定为甲基杆菌属(Methylobacterium sp.).降解特性试验结果表明,其最佳生长条件为温度28℃,初始pH为7.0,在该条件下,500mg·L-> DDVP经过DDW-1菌株代谢3d后,降解率达63.7%.结论:菌株DDW-1能降解DDVP,该菌株产胞内酶.