一株浸矿细菌的分离及其对低品位硫化镍铜矿中镍的浸出

从云南某铜矿井下酸性污水中分离到嗜酸菌株ynxd-1,对其形态、生长特性、16 S rRNA基因序列及其对低品位硫化镍铜矿的摇瓶浸出效果进行了研究.结果表明:细菌细胞呈短杆状,革兰氏染色阴性,不产芽孢,最适pH值及生长温度分别为2.0和30℃.菌株ynxd-1在10%(W/V)矿浆浓度及30℃温度的条件下摇瓶培养浸出28 d,低品位硫化镍铜矿镍的浸出率为56.4%.16 S rRNA基因序列分析表明,菌株ynxd-1与嗜酸氧化亚铁硫杆菌的同源性达到99%,可以鉴定为嗜酸氧化亚铁硫杆菌菌株.     

Isolation and characterization of Acidithiobacillus caldus from several typical environments in China

Six strains of moderately thermophilic sulfur-oxidizing bacteria were isolated from several different typical environments in China. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits. The isolates are Gram negative, rod-shaped bacteria, their optimal temperature and pH value for growth are 45-50 ℃ and 2.5-3.5 respectively. They are autotrophic and used＇elemental sulfur, sodium thiosulfate and potassium tetrathionate as electron donor, while a little glucose stimulated their growth. 16S rDNA sequences analysis reveals that the strains are phylogenetically clustered to Acidithiobacillus caldus.     

PCR-DGGE技术分析浸矿体系细菌群落结构的实验优化

为了研究低品位黄铜矿细菌搅拌浸出的浸矿菌液中的细菌群落结构,对 PCR-DGGE(变性梯度凝胶电泳)技术相关的实验方法进行了优化,主要涉及低丰度基因组 DNA提取方法优化、16SrRNA基因的 PCR扩增条件优化以及变性梯度凝胶电泳条件的优化.结果表明,采用分级离心多次浓缩的方法收集矿浆样品中的菌体,再用试剂盒提取基因组 DNA,有较好效果;使用含特定“GC夹板”的引物5′-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG-3′进行热启动降落 PCR,能显著提高目的条带的 PCR扩增效率;当 DGGE电泳设置温度为60℃左右,胶浓度为6%,尿素变性梯度为30%~50%,恒定电压为100V,时间为9h时,可以获得效果较好的 DGGE电泳图谱,该优化方案提高了对浸矿体系中细菌群落结构的检测效果     

Molecular characterization ofAcidithiobacillus ferrooxidans strains isolated from different environments by three PCR-based methods

PCR-based DNA fingerprinting, REP-PCR(repetitive element PCR), RAPD(randomly amplified polymorphic DNA) and16 S r DNA sequence analyses were used to characterize 23 Acidithiobacillus ferrooxidans strains isolated from different environments.(GTG)5 and BOXA1 R primer were selected for REP-PCR. Twenty arbitrary primers were used for RAPD to acquire DNA profiles from A. ferrooxidans. Both RAPD and REP-PCR produce complex banding patterns and show good discriminatory ability in differentiating closely related strains of A. ferrooxidans. The strains are clustered into 4 or 5 major groups and reveal genomic diversity using(GTG)5-PCR, BOX-PCR and RAPD analysis. Phylogenetic tree based on 16 S r DNA sequences of 23 strains and related strains shows that they are clustered into two distinct groups. Twelve strains are highly related to a new Acidithiobacillus named Acidithiobacillus ferrivorans. The results indicate that PCR-based methods are effective in revealing genetic diversity among A. ferrooxidans.     

Homology modeling and evolutionary trace analysis of superoxide dismutase from extremophile Acidithiobacillusferrooxidans

The gene sod in Acidithiobacillus ferrooxidans may play a crucial role in its tolerance to the extremely acidic, toxic and oxidative environment of bioleaching. For insight into the anti-toxic mechanism of the bacteria, a three-dimensional (3D) molecular structure of the protein encoded by this gene was built by homology modeling techniques, refined by molecular dynamics simulations, assessed by PROFILE-3D and PROSTAT programs and its key residues were further detected by evolutionary trace analysis. Through these procedures, some trace residues were identified and spatially clustered. Among them, the residues of Asn38, Gly103 and Glu161 are randomly scattered throughout the mapped structure; interestingly, the other residues are all distinctly clustered in a subgroup near Fe atom. From these results, this gene can be confirmed at 3D level to encode the Fe-depending superoxide dismutase and subsequently play an anti-toxic role. Furthermore, the detected key residues around Fe binding site can be conjectured to be directly responsible for Fe binding and catalytic function.     

Phenotypic and genetic characterization of a novel strain of Acidithiobacillus ferrooxidans （AF2）

A comparative study on the phenotypic and genetic characteristics among Acidithiobacillus ferrooxidans (AF2), a typic strain ATCC23270 and a previously isolated strain AF3 was performed. AF2 can use ferrous ion (Fe²⁺) or elemental sulfur (S⁰) as sole energy source, but oxidizes S⁰ more effectively than Fe²⁺, which is different from ATCC23270 and AF3. The G+C content of AF2 is 51.8% (molar fraction), however, ATCC23270 and AF3 strains have G+C content of 63.7% and 64.8% (molar fraction), respectively. The DNA-DNA hybridization results show that AF2 has 41.53% and 52.38% genome similarity to ATCC 23270 and AF3, respectively, but AF3 has a high genome similarity of 89.86% to ATCC 23270 strain. Rusticyanin (rus) and subunit III of aa₃-type cytochrome oxidase (coxC) genes are not detected in AF2, but Fe²⁺ oxidase (iro) gene can be detected. To understand the genomic organization of iro gene, a cosmid library of AF2 genome was constructed and iro gene-containing clone was screened. The sequencing result shows that although the nucleotide sequence of iro gene in AF2 is completely identical to that of ATCC 23270 strain, its genomic organization is different from that of ATCC 23270. In AF2, iro is located at downstream of purA gene, while it is located at downstream of petC-2 gene in ATCC 23270 strain. These results indicate that AF2 is a novel strain of A. ferrooxidans, and that phenotypic differences among the strains of A. ferrooxidans are closely correlated with their genetic polymorphisms.     

邻苯二甲酸酯降解菌的分离鉴定及降解特性

为了对邻苯二甲酸酯(PAEs)污染的土壤进行生物修复,从人工湿地土壤样品中分离到7株能够以PAEs为唯一碳源和能源生长的菌株D1～D7,对其综合形态特征、主要生理生化特性和16S rRNA基因序列分析结果进行鉴定,并通过3 d摇瓶间歇试验检测其对PAEs和邻苯二甲酸(PA)的降解能力．结果表明,D1、D2与假单胞菌属(Pseudomonas sp.)的同源性分别为100％和98％,D3与肠杆菌属(Enterobacter sp.)的同源性在99％以上,其余4株细菌与红球菌属(Rhodococcus sp.)的同源性都在98％以上．这些菌株对邻苯二甲酸二甲酯(DMP)、邻苯二甲酸二乙酯(DEP)和邻苯二甲酸二异辛酯(DEHP)的降解率分别在65％,60％和30％以上,且对PAEs的降解率随侧链烷基链的增长而下降．这7株PAEs降解菌均能在以PAEs的降解中间产物——PA为唯一碳源的培养液中生长,对PA的利用率在18％～39％,这说明它们可能通过PA途径实现PAEs的完全降解．     

Isolation and identification of Rhodosporidium diobovatum DS-0205 from deep-sea sediment of eastern Pacific Ocean

A facultative heterotrophic strain (DS-0205) was isolated from a deep-sea sediment sample collected at a depth of 5.2 km in the eastern Pacific Ocean, China. Strain DS-0205 is motile helmet-like single cell or pairs, forming hemisphere with the center sunken of variable size. It has a widespread carbon source and nitrogen source, including agarose, citric acid, salicin, D-glucitol nitrate, sodium nitrite and ethylamine. It can grow in the following environment: temperature 4–37 °C, pH 2.0–12.0, tolerance to NaCl⩽15%. Two phylogenetic trees, one based on the ITS and 5.8S rRNA sequences and the other based on the 18S rRNA sequences, unite strain DS-0205(=JCM 0205) to the type strain of Rhodosporidium diobovatum JCM 3787 through a considerable evolutionary distance. These results suggest that strain DS-0205 is a new strain of the Rhodosporidium diobovatum.     

Isolation and characterization of organic-sulfur degradation bacterial strain

A bacterial strain that was capable of degrading organic sulfur （dibenzothiophene） was isolated by enrichment techniques from the petroleum-contaminated soil collected from Zhongyuan Oil Field. The strain is named ZYX and is gram-positive. This strain undergoes bacilus-coccus morphological change, and forms yellow-pigment glossy circular colonies with 1.5 mm in diameter on average after 2 d incubation on Luria-Bertani（LB） plates. The full-length of 16S rDNA sequence of strain ZYX was determined and analyzed. Strain ZYX is found most relative with the genus of Arthrobacter. The similarity values between ZYX and Arthrobacter sp. P2 is 99.53%. The main morphological, biochemical and physiological features of strain ZYX accord with those of A rthrobacter. It is found that the optimal initial pH for growth is about 7.0, and the optimal concentration of dibenzothiophene（DBT） for growth is 0. 10 g/L. Additionally, the results show that the best carbon source and nitrogen source are glycerol and glutamine, respectively.     

西施舌16S rRNA基因片段PCR扩增及其核苷酸序列分析

利用酚/氯仿抽提法提取了西施舌闭壳肌总DNA,以总DNA为模板,利用两条16S rRNA基因特异引物,进行PCR扩增,扩增产物测序后进行序列分析。结果从西施舌的闭壳肌提取得到总DNA,获得一个长度约300 bp的PCR扩增产物;测序结果显示此片段为294 bp;核苷酸序列经WU-blast2分析,与Spisula subtruncata(AJ548774)和Corbicula sp(AY097259)的16S rRNA的同源性均为74%。经DNAStar分析,此核苷酸序列A,G,T,C含量分别为35.74%,25.51%,26.19%和13.59%,A T(61.90%)含量明显高于G C(38.10%)含量。     

Homology modeling and docking studies of IscS from extremophile Acidithiobacillusferrooxidans

The gene iscS-3 from Acidithiobacillus ferrooxidans may play a central role in the delivery of sulfur to a variety of metabolic pathways in this organism. For insight into the sulfur metabolic mechanism of the bacteria, an integral three-dimensional (3D) molecular structure of the protein encoded by this gene was built by homology modeling techniques, refined by molecular dynamics simulations, assessed by PROFILE-3D and PROSTAT programs and further used to search bind sites, carry out flexible docking with cofactor pyridoxal 5′-phosphate(PLP) and substrate cysteine and hereby detect its key residues. Through these procedures, the detail conformations of PLP-IscS(P-I) and cysteine-PLP-IscS(C-P-I) complexes were obtained. In P-I complex, the residues of Lys208, His106, Thr78, Ser205, His207, Asp182 and Gln185 have large interaction energies and/or hydrogen bonds fixation with PLP. In C-P-I complex, the amino group in cysteine is very near His106, Lys208 and PLP, the interaction energies for cysteine with them are very high. The above results are well consistent with those experimental facts of the homologues from other sources. Interestingly, the four residues of Glu105, Glu79, Ser203 and His180 in P-I docking and the residue of Lys213 in C-P-I docking also have great interaction energies, which are fitly conservation in IscSs from all kinds of sources but have not been identified before. From these results, this gene can be confirmed at 3D level to encode the iron-sulfur cluster assembly protein IscS and subsequently play a sulfur traffic role. Furthermore, the substrate cysteine can be presumed to be effectively recruited into the active site. Finally, the above detected key residues can be conjectured to be directly responsible for the bind and/or catalysis of PLP and cysteine.     

一株Ectoine生成菌DL031的分离及特性研究

从大连普兰店盐场盐池土壤中分离到一株产渗透压补偿溶质Ectoine的菌株DL031,通过16SrDNA序列分析,菌株DL031与Halomonas marina相似性为99%.菌株DL031可在含有0～200 g/LNaCl培养基中生长,生长最适NaCl浓度为100 g/L.DL031菌株在含有NaCl的培养基中诱导能合成Ectoine;在含3 mol/L NaCl的培养基,30℃诱导培养48 h,Ectoine的合成量为98.8 mg/L.     

一株Ectoine生成菌DL031的分离及特性研究

从大连普兰店盐场盐池土壤中分离到一株产渗透压补偿溶质Ectoine的菌株DL031,通过16SrDNA序列分析,菌株DL031与Halomonas marina相似性为99%.菌株DL031可在含有0～200 g/LNaCl培养基中生长,生长最适NaCl浓度为100 g/L.DL031菌株在含有NaCl的培养基中诱导能合成Ectoine;在含3 mol/L NaCl的培养基,30℃诱导培养48 h,Ectoine的合成量为98.8 mg/L.     

海参肠道菌HS-A38的鉴定

对一株编号为HS-A38的分离自大连海域野生海刺参样品的细菌进行鉴定和抗菌活性研究.采用传统的分类学方法观察菌株HS-A38的形态,结合16S rDNA序列分析在GenBank上进行比对确立其进化地位.利用金黄色葡萄球菌(Staphylococcus aureus)、枯草芽孢杆菌(Bacillus subtili)、溶...     

一株枯草芽孢杆菌分离鉴定及其降解稠油特性

以稠油为唯一碳源,从被稠油污染过的土壤中筛选到一株高效石油烃降解茵,经生理生化鉴定和16S rDNA鉴定确认其为枯草芽孢杆茵.在摇瓶实验中,该菌最佳降解温度为35～45℃,最佳pH值为7.5～8.5,最佳盐质量浓度为8～16 g/L.在最佳降解条件下,当油质量浓度为0.1 g/L时,稠油降解率达34.3%.利用GC-nD分析知,该茵主要降解稠油中n-C9～n-C40的烷烃组分;利用GC-MS分析得知,该茵对蔡及烷基化萘去除彻底,对二苯并噻吩、芴和稠二萘等部分芳烃类化合物有降解作用,在稠油降解过程中菲及菲的衍生物有所增加.     

Isolation and characterization of YNTC- 1, a novel Alicyclobacillus sendaiensis strain

A heterotrophic acidothermophilic bacterial strain, YNTC-1, was isolated from an acidic hot spring in Tengchong, Yunan, China. YNTC-1 grows at pH value of 1.5-8.0 and temperature of 40-70℃, with optimal pH and temperature at 3.0 and 55℃,respectively. The cells of the strain are in shape of short rod, with 1.0-1.2 μm in length and 0.7-0.8 μm in diameter, and with distinct spores at both poles of each cell. The predominant fatty acids in cellular membrane of the strain are C18:1ω7c. 16s rRNA gene analysis reveals that this strain is closely related to Alicyclobacillus sendaiensis, with over 99% sequence similarity. Based on phenotypie and genotypic analyses, YNTC-1 is identified as a member ofA. sendaiensis. Considering some important morphological and biochemical differences between strain YNTC-1 and A. sendaiensis ATCC 27009T, YNTC-1 may be proposed to be a novel subspecies of A. sendaiensis. However, this viewpoint has to be confirmed by further studies. Co-bioleaching of pyrite and chalcopyrite with strain YN22, Sulfobacillus thermosulfidooxidans, shows that strain YNTC-1 has no evident influence on bioleaching rates of these two sulphide minerals.     

Zinc bioleaching from an iron concentrate using Acidithiobacillusferrooxidans strain from Hercules Mine of Coahuila, Mexico

The iron concentrate from Hercules Mine of Coahuila,Mexico,which mainly contained pyrite and pyrrhotite,was treated by the bioleaching process using native strain Acidithiobacillus ferrooxidans(A.ferrooxidans)to determime the ability of these bacteria on the leaching of zinc.The native bacteria were isolated from the iron concentrate of the mine.The bioleaching experiments were carried out in shake flasks to analyze the effects of pH values,pulp density,and the ferrous sulfate concentration on the bioleaching process.The results obtained by microbial kinetic analyses for the evaluation of some aspects of zinc leaching show that the native bacteria A.ferrooxidans,which is enriched with a 9K Silverman medium under the optimum conditions ofpH 2.0,20 g/L pulp density,and 40 g/L FeSO4,increases the zinc extraction considerably observed by monitoring duringl 5 d,i.e.,the zinc concentration has a decrease of about 95％ in the iron concentrate.     

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.     

3DBER-S-Fe深度脱氮除磷效果

为强化三维电极生物膜(3DBER)工艺深度脱氮除磷性能,提高污水厂尾水质量,将硫磺和海绵铁作为混合填料,构建硫铁复合填料三维电极生物膜(3DBER-S-Fe)脱氮除磷工艺;在不同ρ( C)/ρ( N)、I和水力停留时间( HRT)运行条件下,探究工艺深度脱氮除磷效果.分别从反应器填料和阴极上取生物膜,通过Miseq高通量测序,构建细菌16S rRNA基因克隆文库.结果表明：在运行条件为ρ(C)/ρ(N)=2、I=150 mA和HRT=4 h时,3DBER-S-Fe对总氮和总磷的去除率分别可达85.59%和97.43%;适当增加ρ( C)/ρ( N)、I和HRT均能不同程度提高系统脱氮除磷效率.在填料和阴极上丰度最大的均为具有硫自养反硝化功能的Thiobacillus,分别占40.62%和44.75%;具有氢自养反硝化功能的Rhodocyclaceae在阴极的分布明显多于填料.因此,3DBER-S-Fe具有较高的脱氮性能主要是硫自养反硝化和氢自养反硝化共同作用的结果,且氢自养反硝化过程主要发生在阴极.     

Isolation and identification of a sulfate reducing bacteria and sequence analysis of its dissimilatory sulfite reductase gene

A sulfate reducing bacteria was isolated from mining sewage of Daqing Oilfield by Hungate anaerobic technology. Physiological-biochemical analysis showed that the strain could utilize polyacrylamide as sole carbon and nitrogen source. The sequence analysis of 16S rDNA illustrated that the similarity of F8 and Desulfovibrio desulfuricans (AF192153) was 99%, and the similarity sequence of dissimilatory sulfite reductase gene (DSR) cloned from the strain and Desulfovibrio desulfuricans (AF273034) was 98%. Their phylogenitic analysis was basically anastomosed, and thus temporarily named as Desulfovibrio desulfuricans F8. The DSR cloned from F8 strain was 2740 bp in length consisting of three ORF, DSRA, DSRB and DSRD as a single operon (DSRABD) regulated by the same operator. DSRA contained typical conservative box of sulfate—sulfite reducing enzyme (SiteⅠand SiteⅡ), which could bind siroheme and [Fe4S4]. DSRB retained a [Fe4S4] binding site, with an uncomplimentary structure for siroheme binding. There was no conservative box in DSRD. Sequence analysis of DSR will provide a theoretical basis for quantitative detection, metabolic pathway modification through gene engineering, and sulfate reducing bacteria (SRB) suppression.