遗传工程菌Fhhh降解精对苯二甲酸与mnp基因表达

跨界融合构建的遗传工程菌Fhhh及其亲株黄孢显毛平革菌 (PC) ,降解精对苯二甲酸的比降解率受到Mn2 + 、酒石酸铵、H2 O2 、pH共 4因素的影响 .除pH值外 ,其余 3个因素影响 2菌株比降解率大小的排序完全一致 .pH值对PC菌的影响排序处于第 3位 ,对Fhhh则处于第 1位 .Fhhh比降解率比PC高出 11 11% ;mnp基因表达的锰过氧化物酶 (MnP)比活力水平比PC高出 15 2 2 % .降解精对苯二甲酸 4因素优化水平 ,也是mnp基因表达的优化条件 .比降解率与MnP的比活力水平之间有显著或极显著正相关性 (r>r0 .0 5( 4) ,r >r0 .0 1( 4) ) .研究结果为高效处理精对苯二甲酸废水提供了重要的分子生物学依据     

Construction of hybrid cell with Phanerochaete chrysosporium todegrade terephthalic acid wastewater (I) phenotype evidence

ＩｎｔｒｏｄｕｃｔｉｏｎＰｈａｎｅｒｏｃｈａｅｔｅｃｈｒｙｓｏｓｐｏｒｉｕｍ (ＰＣ)ｉｓｏｎｅｏｆｔｈｅｕｎｕｓｕａｌｗｈｉｔｅｒｏｔｆｕｎｇｉｔｈａｔｉｔｉｓａｂｌｅｔｏｍｉｎｅｒａｌｉｚｅｔｈｅｃｏｍｐｏｕｎｄｏｆｎａｔｉｖｅｌｉｇｎｉｎ .Ａｎｄｉｔａｌｓｏｃａｎｄｅｇｒａｄｅａｌｍｏｓｔａｌｌｈａｚａｒｄｏｕｓｏｒｇａｎｉｃｐｏｌｌｕｔａｎｔｓｉｎｃｌｕｄｉｎｇｇｅｎｅｔｉｃｍｕｔａｇｅｎｉｃａｇｅｎｔｓｓｕｃｈａｓｃｈｌｏｒｉｎａｔｅｄｏｒｇａｎｉｃｃｏｍｐｏｕｎｄｓ,ｐｏｌｙｃｙｃｌｉｃ…     

Fhhh工程菌株降解PTA废水动力学研究

研究了工程菌株Fhhh降解精对苯二甲酸(PTA)石化废水的动力学.将测得的Fhhh降解废水的6项动力学参数值、废水自然参数值、废水排放标准控制值,输入环境生物技术信息学软件(Ebis)进行计算.结果表明,来源于3种保存方法的Fhhh菌株中,所需反应器的最小体积(V_min)为1309m³,比降解率(q_A)的最高数值为0.0136h^-1,是土著菌YZ1的4倍,高于国内外同类研究的4项数值,低于同类研究的2项数值.结果表明,Fhhh工程菌株具有降解PTA废水的显著优势.     

Purified terephthalic acid wastewater biodegradation and toxicity

The biodegradation and toxicity of the purified terephthalic acid（PTA） processing wastewater was researched at NJYZ pilot with the fusant strain Fhhh in the carrier activated sludge process（ CASP）. Sludge loading rate（SLR） for Fhhh to COD of the wastewater was 1.09 d^-1 and to PTA in the wastewater was 0.29 d^-1. The results of bioassay at the pilot and calculation with software Ebis3 showed that the 48h-LC50 （median lethal concentration） to Daphnia magna for the PTA concentration in the wastewater was only 1/10 of that for the chemical PTA. There were .5 kinds of benzoate pollutants and their toxicities existing in the wastewater at least. The toxicity parameter value of the pure chemical PTA cannot be used to predicate the PTA wastewater toxicity. The toxicity of the NJYZ PTA wastewater will be discussed in detail in this paper.     

环境样品中亚硝酸细菌amoA基因的克隆与测序

对从环境样品中分离的亚硝酸细菌(Ammonia oxidizingbacteria)amoA基因进行克隆与测序,为构建基因工程菌打下基础。采用亚硝酸细菌选择性培养基,从4个不同的畜牧养殖污水处理厂采集的样品(分别编号为1,2,3,4)在室温下富集培养2个月后,采取酚氯仿抽提的方法提取DNA。根据已报道的亚硝化单胞菌(Nitrosomonassp.)amoA基因序列,设计引物AMOB AMOE,并在AMOB,AMOE的5′-端分别加上了BamHⅠ和HindⅢ的限制性酶切位点,以利于进一步酶切和克隆。用AMOB AMOE对4种样品的DNA进行PCR扩增,PCR产物进行琼脂糖凝胶电泳分析。结果表明,4种样品中1号和3号样品扩增得到预期长度的DNA片段,2号和4号样品扩增没有得到预期片段。回收纯化PCR产物与pGEM-T载体连接,构建amoA基因测序载体,并转化E.coliM15。测序结果提交GenBank进行Blast分析。结果显示,扩增得到的DNA片段均与Nitrosomonassp.GH22的amoA基因有99 7%的同源性,可从环境中分离的亚硝酸细菌中克隆出amoA基因。      

Predication of Fhhh potential in PTA wastewater treatment

Ebis is the intelligent environmental biotechnological informatics software developed for judging the effectiveness of the microorganism strain in the industrial wastewater treatment system(IWTS) at the optimal status. The parameter, as the objective function for the judgment, is the minimum reactor volume ( Vmin ) calculated by Ebis for microorganism required in wastewater treatment. The rationality and the universality of Ebis were demonstrated in the domestic sewage treatment system(DSTS) with the data published in US Aand China at first, then Fhhh strain‘s potential for treating the purified terephthalic acid(PTA) was proved. It suggests that Ebis would be useful and universal for predicating the technique effectiveness in both DSTS and IWTS.     

Pilot regulation of MnP-SA for treating PTA wastewater

In the pilot study of treating the purified terephthalic acid(PTA) wastewater with the functional Strain Fhhh in the carrier activated sludge process(CASP), the ratio of COD:TN:TP and the concentrations of Cu, Mn, Se and Zn were controlled to improve the manganese peroxidase(MnP) levels for increasing the treatment efficiency. When the ratio of COD:TN:TP was 100:0.36:0.15 and the concentrations of Cu, Mn, Se and Zn were 0.54, 5.07, 0.00 and 0.08 mg/L, the MnP specific activity(MnP-SA) reached 689 U/L, and the sludge loading rate to COD(SLRC) was 1.09 d^-1, which was 4--7 fold of that in other processes reported. The data indicated that improving MnP level could enhance the degradability of Fhhh. And the potentials of Fhhh and CASP will be also discussed in this paper.     

Ni2+对降解2-氯酚厌氧系统古细菌种群结构的影响

提取降解2-氯酚(2-chlorophenol,2-CP)的厌氧颗粒污泥在Ni2 冲击前后样品的总DNA,用古细菌特异性引物ARC21F/ARC958R进行16S rDNA的聚合酶链式反应(Polymerase Chin Reaction,PCR)、克隆、测序及序列分析等,研究了Ni2 冲击及恢复性驯化对2-CP驯化后厌氧颗粒污泥中古细菌种群多样性的影响.结果表明:Ni2 冲击对古细菌种群结构影响较大,冲击前后厌氧颗粒污泥中存在共有的古细菌菌种(Methanothrix soehngenii,Methanosaeta concilii及Uncultured archaeon等).冲击后厌氧污泥中新出现的古细菌有Toluene-degrading Methanogenic consortium archaeon M1及Candidatus Methanoregula boonei strain 6A8等.经过10 d的恢复性驯化后,厌氧污泥中又出现了新的古细菌Methanobacterium subterraneum及Methanobacterium palustre 等.2-CP厌氧降解系统经过Ni2 冲击后,古细菌种群多样性显著增加,系统能较快恢复对2-CP的降解能力.     

Isolation and characteristics of a novel biphenyl-degrading bacterial strain, Dyella ginsengisoli LA-4

A novel biphenyl-degrading bacterial strain LA-4 was isolated from activated sludge. It was identified as Dyella ginsengisoli according to phylogenetic similarity of 16S rRNA gene sequence. This isolate could utilize biphenyl as sole source of carbon and energy, which degraded over 95 mg/L biphenyl within 36 h. The major metabolites formed from biphenyl, such as 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and benzoic acid, were identified by LC-MS. The crude cell extract of strain LA-4 exhibited the activity of 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD) and the kinetic parameters were Km= 26.48 μmol/L and Vmax= 8.12 μmol/mg protein. A conserved region of the biphenyl dioxygenase gene bphA1 of strain LA-4 was amplified by PCR and confirmed by DNA sequencing.     

黄杆菌P3-2降解对硫磷的质粒

黄杆菌(Flavobacterium sp.)P3-2具有水解对硫磷农药的能力.该菌在L.B培养基或以乙酸、乙醇或甘油为碳源的合成培养基上能产生对硫磷水解酶.用碱裂解法从P3-2菌株中分离出一种质粒(PAR).P3-2菌株经丝裂霉素C处理后,消除质粒的原菌株丧失了产生对硫磷水解酶的性能.用蔗糖梯度离心纯化的质粒DNA经EcoRI和BamHI消化,分别产生16和2个DNA片段、以EcoRI消化λDNA产生的6个片段的分子量作标准,测得PAR质粒的分子量约为37.16×10~6 dalton.在电镜下观察呈环状的DNA分子.     

用于分子生态学研究的堆肥DNA提取方法

分子生态学为堆肥微生物的研究提供了新的技术手段,DNA的提取是该技术的基础,但由于腐殖酸类物质的污染,增加了堆肥微生物总DNA的提取难度.采用了3种不同的方法(溶菌酶法、超声波破碎法和蛋白酶K-CTAB法)从堆肥中提取微生物的总DNA,使用核酸和蛋白质分析仪检测后表明3种提取方法获得的DNA产量均较高;琼脂糖凝胶电泳结果表明其长度约为23 kb;使用细菌16S rRNA基因通用引物(27F和1 495R)对总DNA进行PCR扩增,都获得了几乎全长的16S rDNA序列(约1.5 kb);利用限制性内切酶(Hae Ⅲ和AluⅠ)对纯化后的PCR产物进行RFLP分析,结果表明3种方法提取的DNA反映了比较一致的微生物多样性.虽然3种方法各有优缺点,但其提取的DNA都可以用于堆肥微生物的分子生态学研究,可以根据实际需要选用某一种方法用于提取堆肥总DNA.     

生物陶粒与生物活性炭上微生物群落结构的PCR-SSCP技术解析

采用PCR-SSCP(单链构象多态性)技术,以16S rRNA基因的V4-V5区为靶对象,分析用于饮用水处理的生物陶粒和生物活性炭上的微生物群落结构.对生物陶粒和生物活性炭上的微生物分别进行超声波洗脱、R2A和LB平板培养后提取基因组DNA.结果表明,除生物活性炭超声波洗脱不能提取到DNA外,其他处理均能提取到大小在10 kb以上的基因组DNA,但所提取的量有较大差异.以提取的DNA为模板分别进行PCR,均能扩增到408 bp的基因片段.这些片段经λ核酸外切酶消化处理后进行SSCP电泳.结果显示,超声波洗脱、R2A和LB培养对试验结果影响不明显.生物陶粒的微生物基因扩增片段SSCP图谱相同,且只出现1条带.测序后与基因组数据库对比,结果显示其与uncultured Pseudomonas sp. clone FTL201 16S rDNA （GenBank登录号AF509293.1）片段同源性为92%.生物活性炭的微生物基因扩增片段SSCP图谱也相同,但有2条带.测序对比的结果表明, 这2个基因片段与Bacillus sp. JH19 16S rDNA （GenBank 登录号DQ232748.1）片段和Bacterium VA-S-11 16S rDNA （GenBank登录号AY395279.1）片段的同源性分别为100%和99%.     

中国田鼠卵巢细胞自发和亚硫酸氢钠诱发突变的gpt基因分子分析

采用标准的突变细胞克隆技术、PCR及DNA序列分析方法研究了二氧化硫在体内的衍生物—亚硫酸氢钠对CHO－AS52细胞gpt基因的致突变作用。实验结果表明,高浓度的亚硫酸氢钠能诱发该基因发生突变,且其突变频率随该化学物浓度的增加而增高。PCR分析指出,在CHO－AS52细胞自发的、5mmol/L和10mmol/L亚硫酸氢钠诱发的突变体中,gpt基因完全缺失者所占比率分别为36.00％、44.00％及65.00％。对10mmol/L亚硫酸氢钠诱发的非缺失型gpt基因突变的PCR产物直接进行DNA序列分析表明,在5个突变细胞克隆中,有1个发生基因的碱基置换型和移码型突变,其余4个突变细胞克隆的gpt基因结构未发现改变,其碱基的改变可能发生在基因启动子区。     

3-PBA降解菌BA3的降解特性及基因工程菌构建

从石油污染土壤中分离到1株能以3-苯氧基苯甲酸(3-PBA)为唯一碳源生长的细菌,命名为BA3.根据其生理生化特征和16S rDNA序列相似性分析,将该菌鉴定为鞘脂菌属(Sphingobium sp.).该菌株在60 h内对100 mg·L-1的3-苯氧基苯甲酸的降解率达到99%.降解3-苯氧基苯甲酸的最适温度为30℃...     

PCR-DGGE技术解析生物制氢反应器微生物多样性

为了揭示发酵法生物制氢反应器厌氧活性污泥的微生物种群多样性 ,从运行不同时期取厌氧活性污泥 ,通过细胞裂解直接提取活性污泥的基因组DNA .以细菌 16SrRNA基因通用引物F338GC/R5 34进行V3高变异区域PCR扩增 ,长约 200bp的PCR产物经变性梯度凝胶电泳 (DGGE)分离后 ,获得微生物群落的特征DNA指纹图谱 .研究表明 ,不同时期的厌氧活性污泥中存在共同种属和各自的特异种属 ,群落结构和优势种群数量具有时序动态性 ,微生物多样性呈现出协同变化的特征 .微生物多样性由强化到减弱 ,群落结构之间的相似性逐渐升高 ,演替速度由快速到缓慢 .优势种群经历了动态演替过程 ,最终形成特定种群构成的顶级群落 .     

菌株Cupriavidus sp. DT-1对液体和土壤中TCP的降解

采用液质联用（HPLC-MS）的方法检测菌株Cupriavidus sp.DT-1降解2-羟基吡啶（2-HP）的代谢产物.并用三亲结合、荧光定量PCR （q-PCR）方法评价降解菌对3,5,6-三氯-2-吡啶酚（TCP）污染土壤的修复效果.结果表明,菌株可以进一步降解2-HP,依次生成尼古丁蓝、马来酰胺酸和反丁烯二酸,直至转化成菌株DT-1生长的碳源.接种菌株DT-1对污染土壤中TCP的降解起到较大的促进作用,2组试验土壤中TCP （50mg/kg）降解率分别为94.4%和86.7%,未接种菌株的土壤中TCP降解率仅为20.4%和28.4%.带有绿色荧光蛋白基因gfp标记的菌株DT-1-gfp可在土壤中存活35d以上,并对TCP污染土壤的细菌群落丰度有显著的恢复作用.     

固态发酵过程中微生物总DNA提取方法比较

为了分析固态发酵过程中微生物群落的多样性及演替情况,对比研究了从固态发酵中提取细菌和真菌DNA的3种方法--溶壁酶法、超声波法、液氮研磨 CTAB法.使用紫外分光光度计测定了由不同提取方法得到的DNA的产量与纯度;使用细菌16S rDNA基因通用引物(341F和907R)和真菌18S rDNA基因通用引物(NU-SSU-0817和NU-SSU-119)对DNA进行了PCR扩增;采用DGGE(变性梯度凝胶电泳)法对固态发酵中细菌和真菌的多样性进行了分析.结果显示,3种方法得到的粗提和纯化DNA长度均约为23 kb;细菌和真菌PCR产物长度分别约为586 bp和422 bp.细菌和真菌PCR产物的DGGE分析表明,3种方法提取的DNA所反映的微生物多样性比较一致;但紫外分光光度计测定结果表明溶壁酶法提取固态发酵中微生物总DNA产量最高,超声波法次之,液氮研磨 CTAB法最低.     

通用引物PCR方法在地表水病原菌检测中的应用

为了探索通用引物PCR方法在地表水病原细菌检测中的应用价值,利用16S rRNA基因的高度保守性,设计并合成细菌的通用引物,采用合成的引物扩增参考菌株及西安市区不同地表水样,并对PCR产物分别进行序列测定及序列同源性分析,同时检测水样中的细菌总数和粪大肠杆菌浓度.结果显示,通用引物扩增4种参考菌株,320 bp处均可得到清晰的电泳条带,其特异性通过序列测定及同源性分析得到进一步验证;通用引物PCR可检出250 ng/L的埃希氏大肠杆菌标准菌株DNA,其PCR检测的灵敏度可达0.275 CFU/mL;用通用引物对西安市区不同地表水样进行PCR扩增,其中■河、兴庆湖、大唐芙蓉园北湖、北石桥污水处理厂出水样品320 bp处都得到了清晰的电泳条带,而黑河水样没有条带;与之相对应的粪大肠杆菌群检测结果显示,只有北石桥污水处理厂出水和兴庆湖水样有粪大肠杆菌检出.      

Detection of Norovirus Recombinant GII.2[P16] Strains in Oysters in Thailand

Human norovirus causes sporadic and epidemic acute gastroenteritis worldwide, and the predominant strains are genotype GII.4 variants. Recently, a novel GII.17[P17] and a recombinant GII.2[P16] strain have been reported as the causes of gastroenteritis outbreaks. Outbreaks of norovirus are frequently associated with foodborne illness. In this study, each of 75 oyster samples processed by a proteinase K extraction method and an adsorption-elution method were examined for noroviruses using RT-nested PCR with capsid primers. Thirteen (17.3%) samples processed by either method tested positive for norovirus genogroup II (GII). PCR amplicons were characterized by DNA sequencing and phylogenetic analysis as GII.2 (n?=?6), GII.4 (n?=?1), GII.17 (n?=?3), and GII.unclassified (n?=?3). Norovirus-positive samples were further amplified by semi-nested RT-PCR targeting the polymerase-capsid genes. One nucleotide sequence revealed GII.17[P17] Kawasaki strain. Five nucleotide sequences were identified as belonging to the recombinant GII.2[P16] strains by recombination analysis. The collected oyster samples were quantified for norovirus GII genome copy number by RT-quantitative PCR. Using the proteinase K method, GII was found in 13/75 (17.3%) of samples with a range of 8.83–1.85?×?10⁴ genome copies/g of oyster. One sample (1/75, 1.3%) processed by the adsorption-elution method was positive for GII at 5.00?×?10¹ genome copies/g. These findings indicate the circulation of a new variant GII.17 Kawasaki strain and the recombinant GII.2[P16] in oyster samples corresponding to the circulating strains reported at a global scale during the same period of time. The detection of the recombinant strains in oysters emphasizes the need for continuing systematic surveillance for control and prevention of norovirus gastroenteritis.

     

一种高效提取焦化废水活性污泥总DNA的方法

对焦化废水活性污泥中微生物建立高质量的总DNA提取方法是开展分子生态学研究的重要前提.通过反复冻融-蛋白酶K-SDS、溶菌酶-反复冻融-SDS及溶菌酶-反复冻融-蛋白酶K-SDS这3种综合方法对焦化废水活性污泥总DNA进行提取,以OD260/OD280、OD260/OD230、产率、片段完整性、片段大小5个指标来评价样品总DNA的提取效果.结果表明,溶菌酶-反复冻融-蛋白酶K-SDS法所提取的总DNA的OD260/OD280值约为1.8,产率为1.90～16.30 μg·g-1,片断完整性好,主带清晰,大小约为23 kb,其PCR反应抑制物少,能够直接进行16S rRNA基因的PCR扩增.溶菌酶-反复冻融-蛋白酶K-SDS法能够为焦化废水活性污泥中微生物的分子生态学研究提供高质量的总DNA.