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-1DDVP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。

多环芳烃降解菌筛选及其降解特性

通过选择性富集培养,从辽河油田稠油污染土壤4号土样中,获得了能以高浓度菲(2000 mg·L-1)为唯一碳源和能源快速生长的优势菌系和优良菌株ZL5.16S rDNA核苷酸序列分析表明,ZL5菌株归类于鞘氨醇单胞菌属.分得的菌系和菌株有较强的降解菲能力,120 h混合菌系降解了投加菲的95.28％,菌株降解了69.24％,但它们对芘的降解能力均较低.外加碳源葡萄糖可提高菌系和菌株的菲、芘降解能力,加量多,提高幅度大,但超过一定量,降解速率开始下降,表现出抑制效应.所以,应用时需控制适宜的浓度.

高温放线菌属两菌株的化学分类和分子分类研究

从云南温泉和火山口分离出两株嗜热放线菌YIM60013和YIM60032。对其形态、生理生化特性、细胞壁化学组分以及16SrDNA序列进行了研究,并与高温放线菌的已知种进行了比较,初步认为这是高温放线菌属的两个新种,分别命名为白色高温放线菌(Thermoactinomycesalbussp.nov)和云南高温放线菌(Thermoactinomycesyunnanensissp.nov)。两个新种在气生菌丝和基内菌丝上均产生单孢子,纯细胞壁和全细胞糖分析表明含meso DAP和半乳糖、阿拉伯糖、木糖及少量的葡萄糖。菌丝体自溶或非自溶,气生菌丝白色或灰色,碳源利用类型有所不同。

高效石油烃降解菌的分离鉴定及降解特性

为获得更为丰富的石油降解微生物资源,从沈抚污灌区石油污染土壤和实验室高浓度柴油胁迫土壤中筛选出了4株高效石油烃降解菌SF-422、SF-428、SF-433和SYS-1.这4株菌总石油烃(Total petroleum hydrocarbon/TPH)生物降解率为67.4%~73.6%.经过16项生理生化特性实验和16S rDNA序列分析鉴定,SF-433,SF-428,SF-422和SYS-1分别为蜡状芽孢杆菌(Bacillus cereus),木糖氧化无色杆菌(Achromobacter xylosoxidans),施氏假单胞菌(Pseudomonas stutzeri)和洋葱伯克霍尔德氏菌(Burkholderia cepacia).纯烃降解定性实验表明所筛选出的4株高效降解菌均能够利用正十六烷、苯、菲和环己烷为唯一碳源生长,其中菌株SF-428和SYS-1显示了对芳烃及环烷烃较强的利用能力.

利用16SrDNA序列对十株根瘤菌系统发育的研究

为了确定本所保存的部分根瘤菌的种属地位,我们从中选出10株菌株,对其16S rDNA进行扩增并测序,利用分子生物学软件对序列进行分析,得到根瘤菌系统发育树状图,对比相应的生理生化结果,重新定位了它们在根瘤菌系统发育中的种属地位,将生物信息学应用用于根瘤菌的种属命名具有重要的科学意义。

利用DGGE指纹图谱技术考察不同营养条件下土壤微生物群落结构的变化

利用现代分子生物学技术,结合经典方法,克服传统的分离培养缺陷,探讨在不同营养条件下土壤微生物群落的基因多样性。经过直接从土壤中抽提总DNA,并对总DNA中16S rDNA及其中V6~V8可变区序列作PCR扩增、变性梯度凝胶电泳(DGGE)分析等,发现不同处理条件下的土壤微生物的基因多样性变化与土壤微生物量的波动并不一致,说明微生物群落多样性与微生物量的关系并非线形。同时发现秸秆的添加更有利于土壤微生物群落的稳定。

硫酸盐还原菌EMZY-1生理生化特性研究及16SrDNA序列分析

菌株EMZY-1是一株从中国大庆油田回注水中分离得到的硫酸盐还原菌(SRB)。通过对该菌株的形态、培养特征、生理生化特征的研究以及16S rDNA序列分析表明:该菌株为革兰氏阳性菌;细胞为棒杆状,端生孢子,大小约0.3×1.7(μm);温度低于10℃、高于60℃无明显生长;pH低于5.0、高于12.0无明显生长;NaCl质量浓度达到10%菌株不能生长;能在蔗糖、葡萄糖、甘露醇为C源的培养基上生长;NH4+、NO3-为菌株良好N源。菌株EMZY-1属于梭菌科。16S rDNA序列分析表明该菌与梭菌科中的Garciella nitratireducens菌同源性最高(99%),GenBank的注册号为EU275367,国内尚未见报道该类SRB。

深海芽孢杆菌B1394的鉴定及其产蛋白酶酶学性质

从深3 200~3 600 m的南海海底沉积物中分离到185株深海细菌,从中筛选到1株产蛋白酶活力较强的菌株B1394,酶活高达873 U/mL。采用16S rDNA分子生物学鉴定,结合细菌常规鉴定方法鉴定其为枯草芽孢杆菌(Bacillus subtilis)。对其粗酶性质进行研究发现:最适酶活温度60℃,最适pH 8.0,在低温30℃和40℃下也具有较高的酶活性,40~60℃热稳定性较好,显示出部分嗜热酶特性;Mn2+、Mg2+、Ca2+对该蛋白酶有激活作用,Hg2+、Fe3+、Cu2+、Zn2+、Fe2+对该蛋白酶有抑制作用;PMSF几乎完全抑制蛋白酶活性,推断为丝氨酸蛋白酶。

土壤中嗜酸性氧化亚铁硫杆菌的分离鉴定及其性能研究

采用改进型Leathen培养基直接从辽宁省抚顺市红透山铜矿附近的土壤中分离到了一株高度嗜酸的氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)菌株(暂命名为R2)。鉴定表明,该菌株为革兰氏阴性细菌,在扫描电镜下观察该菌株为短杆状,菌体大小为(0.4±0.2)μm×(1.6±0.4)μm。最适pH值2.0,化能自养型,能利用亚铁、单质硫和硫代硫酸钠生长,不能利用葡萄糖、蛋白胨生长。并且以16S rDNA序列同源性为基础构建了17株已报道菌种在内的系统发育树,将16S rDNA测序结果输入Genebank以Blast软件进行序列同源性比较,结果显示与氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)的多株细菌具有较高的同源性(>99%),其中与Acidithiobacillus ferrooxidans strain TGS的相似性达到100%,与标准株Acidithiobacillus ferrooxidans strain ATCC33020相似性为99.3%,结合其生理生化特性可以确定该菌为氧化亚铁硫杆菌种。序批式试验法研究表明,接种该株菌可有效溶出土壤中重金属,经过5d的生物淋滤,Cu、Cr、Zn、Cd的最高去除率分别达到30.6%、16.3%、58.4%和72%。

微生物的ARDRA检测

ARDRA（扩增性rDNA限制性酶切片段分析）是新发展起来的一项生物检测技术，可在原位下获取其有关生物性状。本文阐述了ARDRA技术的原理和方法，介绍了该技术在微生物多样性和系统发育研究中的应用，并对ARDRA技术的应用前景提出展望。

乙草胺和甲胺磷对农田黑土可培养真菌数量及种群结构的影响

应用传统及PCRDGGE方法(denaturinggradientgelelectrophoresis),分别对不同浓度乙草胺、甲胺磷胁迫下黑土中可培养真菌CFU(colonyformingunits)、种群丰富度(richness)及种群结构动态变化规律进行了研究.结果表明,在实验室微域条件下,乙草胺对黑土可培养真菌CFU的影响随处理浓度的增加而抑制作用增强,表现出由低浓度(50mg·kg-1)时的刺激生长到高浓度(250mg·kg-1)时的长期抑制效应;250mg·kg-1甲胺磷在8周处理过程中对土壤可培养真菌生长具有显著的刺激效应,使可培养真菌CFU比对照增加10倍,但50和150mg·kg-1甲胺磷处理对土壤可培养真菌CFU无显著影响.种群丰富度系数分析结果表明,高、中浓度乙草胺处理可使土壤可培养真菌种群丰富度不可逆地降低.土壤真菌rDNA特异PCRDGGE聚类分析结果表明,不同浓度乙草胺、甲胺磷处理均不同程度地对土壤可培养真菌的种群组成和结构造成影响,其中甲胺磷尤为显著.

Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China

Aims: To assess the diversity of antibiotic-resistant bacteria and their resistance genes in typical maricultural environments. Methods nand Results: Multidrug-resistant bacteria and resistance genes from a mariculture farm of China were analysed via cultivation and polymerase chain reaction (PCR) methods. Oxytetracycline (OTC)-resistant bacteria were abundant in both abalone and turbot rearing waters, accounting for 3.7% and 9.9% of the culturable microbes. Multidrug resistance was common, with simultaneous resistance to OTC, chloramphenicol and ampicillin the most common resistance phenotype. 16S rDNA sequence analyses indicate that the typical resistant isolates belonged to marine Vibrio, Pseudoalteromonas or Alteromonas species, with resistance most common in Vibrio splendidus isolates. For OTC resistance, tet(A), tet(B) and tet(M) genes were detected in some multidrug-resistant isolates, with tet(D) being the most common molecular determinant. For chloramphenicol resistance, cat II was common, and floR was also detected, especially in marine Pseudoalteromonas strains. Conclusions: There is the risk of multidrug-resistant bacteria contamination in mariculture environments and marine Vibrio and Pseudoalteromonas species serve as reservoirs of specific antibiotic resistance determinants. Significance and Impact of the Study: This paper and similar findings from Korea and Japan indicate the potential for widespread distribution of antibiotic resistance genes in mariculture environments from the East Asian region of the world.

Purification and characterization of the chitosanase from Aeromonas sp HG08

In this work, the characterization of a chitosanase-producing bacterium isolated from soil was reported and this strain was grouped under the genus Aeromonas by virtue of its morphological, physiological properties and 16S rDNA gene sequences. It is the first report that the genus Aeromonas could produce chitosanase. Aeromonas sp. HG08 could secrete the chitosanase ( named AsChi) with molecular weight of 70 kDa. The optimum pH and temperature of AsChi was 6.0 and 55 degrees C, respectively. The activity of AsChi was markedly enhanced by Mn2+ and inhibited by Fe3+, Cu2+, Ag+ and Hg2+; additionally, the activity of AsChi was increased with the degree of deacetylation ( DDA) of chitosan. Through viscosimetric assay, AsChi probably hydrolyzed chitosan in an endo-type fashion.

An efficient method for DNA isolation from red algae

A simple, inexpensive and efficient method was developed for rapid isolation of total genomic DNA from 15 red algal species. It resulted in 0.1 mug high quality DNA from 1 mg fresh algal material, with an A(260)/A(280) ratio of 1.68 - 1.90. Using this rapidly isolated DNA, the 18S ribosomal RNA genes ( rDNA) and the nuclear ribosomal DNA of the internal transcribed spacer (ITS) regions were amplified. The tested DNA was suitable for restriction endonuclease digestion, genetic marker analysis and polymerase chain reaction (PCR) amplification, and may be valid for other genetic manipulation.

Molecular phylogeny and species identification of pufferfish of the genus Takifugu (Tetraodontiformes, Tetraodontidae)

The phylogenetic relationships and species identification of pufferfishes of the genus Takifugu were examined by use of randomly amplified polymorphic DNA (RAPD) and sequencing of the amplified partial mitochondrial 16S ribosomal RNA genes. Amplifications with 200 ten-base primers under predetermined optimal reaction conditions yielded 1962 reproducible amplified fragments ranging from 200 to 3000 bp. Genetic distances between 5 species of Takifugu and Lagocephalus spadiceus as the outgroup were calculated from the presence or absence of the amplified fragments. Approximately 572 bp of the 16S ribosonial RNA gene was amplified, using universal primers, and used to determine the genetic distance values. Topological phylogenic trees for the 5 species of Takifugu and outgroup were generated from neighbor-joining analysis based on the data set of RAPD analysis and sequences of mitochondrial 16S rDNA. The genetic distance between Takifugu rubripes and Takifugu pseudommus was almost the same as that between individuals within cacti species, but much smaller than that between T. rubripes, T. pseudommus, and the other species. The molecular data gathered from both analysis of mitochondria and nuclear DNA strongly indicated that T. rubripes and T. pseudommus should be regarded as the same species. A fragment of approximately 900 bp was amplified from the genome of all 26 T. pseudommus individuals examined and 4 individuals of intermediate varieties between T. rubripes and T. pseudommus. Of the 32 T. rubripes individuals, only 3 had the amplified fragment. These results suggest that this fragment may be useful in distinguishing between T. rubripes and T. pseudommus.