Fe^2＋对嗜铁钩端螺旋菌L.ferriphilum生长活性的影响

以分离于江西某铜矿的嗜铁钩端螺旋菌(L.ferriphilum YSK)菌株为研究对象,报道不同Fe2+浓度对L.ferriphilum生长活性的影响。结果表明,YSK菌株生长最适宜的Fe2+浓度约为0.1 mol/L。当初始Fe2+浓度为0.4 mol/L时,进入对数生长期前很明显需要一个较长的延迟期,表明该Fe2+浓度对细胞的生长产生较强的抑制作用;当初始Fe2+浓度为0.6 mol/L时,YSK菌株的生长完全受到抑制。尽管高浓度的Fe2+抑制细胞的生长,但受到抑制而不生长繁殖的YSK细胞仍然具有氧化Fe2+的能力。

Surface characterization of chalcopyrite interacting with Leptospirillum ferriphilum

The alteration of surface properties of chalcopyrite after biological conditioning with Leptospirillum ferriphilum was studied by adsorption,zeta-potential,contact angle and bioleaching tests.The strains of L.ferriphilum cultured using different energy sources（either soluble ferrous ion or chalcopyrite） were used.The adhesion of bacteria to the chalcopyrite surface was a fast process.Additionally,the adsorption of substrate-grown bacteria was greater and faster than that of liquid-grown ones.The isoelectric point（IEP） of chalcopyrite moved toward that of pure L.ferriphilum after conditioning with bacteria.The chalcopyrite contact angle curves motioned diversely in the culture with or without energy source.The results of X-ray diffraction patterns（XRD）,scanning electron microscopy（SEM） and energy-dispersive X-ray spectroscopy（EDS） analysis indicate that the surface of chalcopyrite is covered with sulfur and jarosite during the bioleaching process by L.ferriphilum.Furthermore,EDS results imply that iron phase dissolves preferentially from chalcopyrite surface during bioleaching.The copper extraction is low,resulting from the formation of a passivation layer on the surface of chalcopyrite.The major component of the passivation layer that blocked continuous copper extraction is sulfur instead of jarosite.

Effects of mutation on a new strain Leptospirillum ferriphilum YXW and bioleaching of gold ore

Leptospirillum ferriphilum YXW was isolated through serial dilution from mixed microorganisms enriched in AMD from Dexing copper mine in Jiangxi Province, China. It was mutated by ultrasonic, UV and microwave to collect more efficient strain for bioleaching of gold ore. Physiological and biochemical characteristics indicate that strain YXW is a strict chemoautotrophic microorganism, and the optimal condition for its growth is temperature of 40 °C and pH 1.5. After mutation by ultrasonic, UV and microwave, the density of bacterial cells reached 9×109, 8.4×109 and 4.3×108 mL-1, increased by 291%, 265%and 87%, respectively, compared with the original culture. The bacterial total protein activity was improved by microwave and UV mutations, but was reduced by ultrasonic. Mutations had effects on bioleaching of gold ore in sequence of microwave〉UV〉ultrasonic. During gold ore bioleaching, the bacterial mutant after mutation by microwave had the best effect on the extraction rates of arsenic and iron, which were 19.6%and 17.7%higher than that of the original strain after bioleaching for 10 d, respectively. The results suggested that the effects of mutation on bioleaching of gold ore may not be mainly due to increase of bacterial cells density, but may be mainly attributed to the improvement of bacterial total protein activity.

嗜铁钩端螺旋菌(Leptospirillum ferriphilum)gyrB基因的PCR扩增、克隆与序列分析

根据GenBank和ICB数据库中gyrB蛋白氨基酸序列的两个保守区域TPGMYIG和QRY(F)KGLGEM设计简并引物,以L.ferriphilum菌株YSK基因组DNA为模板,PCR扩增出获得大小约为2.2kb的DNA片段。序列分析表明,菌株YSK的扩增片断的开放阅读框(ORF)能够推导出一个编码分子量约为82.24kD、氨基酸数目为731个的蛋白质片断。这个氨基酸序列与所研究的gyrB蛋白氨基酸序列显示出高度的同源性,尤其是与菌株AMC_Cont91的gyrB蛋白氨基酸序列的相似性高达92%,而与M.xanthus的gyrB的相似性最低,仅为37%。基于氨基酸序列的同源性及其所预测的蛋白质大小,可以推断出该扩增片段属于gyrB基因。

Bio-leaching effects of Leptospirillum ferriphilum on the surface chemical properties of pyrite

Leptospirillum ferriphilum cultured using different energy sources(either soluble ferrous ion or pyrite) changed the surface properties of pyrite.Cell adsorption,zeta-potential,hydrophobicty,FT-IR spectra and surface morphology were investigated.Adhesion of bacterial cells to the pyrite surface is a fast process.Furthermore,the adsorption of cells grown in pyrite is greater than of cells grown in soluble ferrous ion.The Iso-Electric Point(IEP) of pyrite treated with L.ferriphilum approaches that of the cells themselves.The contact angle of the pyrite surface was observed to decrease due to the surface formation of some hydrophilic substances during bio-leaching.Changes in pyrite surface properties after bacterial treatment support the idea that ferric ion plays an important role and that indirect bio-oxidation is the principal mechanism of pyrite bio-leaching.

Isolation of Leptospirillum ferriphilum by single-layered solid medium

According to physiological and biochemical characteristics of Leptospirillum ferriphilum, a strain of object bacteria was isolated successfully. Bacteria were enriched by selective liquid medium and plated on designed single-layered agar solid medium. Colony was cultured and bacteria were collected. The morphologies of the object bacteria were observed using crystal violet staining, scanning electron microscope（SEM） and transmission electron microscope （TEM）. The result of 16S rDNA identification shows that this bacterium belongs to Leptospirillum ferriphilum and it is named as Leptospirillum ferriphilum strain D1. These results indicate that this new single-layered agar solid medium is efficient and physiological-biochemical characteristics show that the optimum simple for isolation of Leptospirillum ferriphilum. Additionally, initial pH value and its growth temperature are 1.68 and 40℃.

Comparative genome analysis on intraspecific evolution and nitrogen fixation of Leptospirillum ferriphilum isolates

To reveal the intraspecific evolution of Leptospirillum ferriphilum isolates which thrived in industrial bioleaching ecosystems and acid mine drainages,genome sequences of L.ferriphilum YSK,L.ferriphilum DX and L.ferriphilum ZJ were determined to compare with complete genome of L.ferriphilum ML-04.The genome comparisons reveal that extensive intraspecific variation occurs in their genomes,and that the loss and insertion of novel gene blocks of probable phage origin may mostly contribute to heterogeneity of gene content among L.ferriphilum genomes.Surprisingly,a nif gene cluster is identified in L.ferriphilum YSK and L.ferriphilum ZJ genomes.Intensive analysis and further experiments indicate that the nif gene cluster in L.ferriphilum YSK inherits from ancestor rather than lateral gene transfer.Overall,results suggest that the population of L.ferriphilum undergoes frequent genetic recombination,resulting in many closely related genome types in recent evolution.The combinatorial processes profoundly shape their physiologies and provide the basis for adaptation to different niches.

Bioleaching of pyrite by A.ferrooxidans and L.ferriphilum

黄铁矿氧化率面对 A 在各种各样的条件下面被检验。ferrooxidans 和 L。ferriphilum，不同的肉集中，接种数量， Fe3+ 的外部增加和起始的 pH 在珍视被执行。它被发现那 A。ferrooxidans 和 L。在 bioleaching 的类似的行为处理的 ferriphilum 表演。增加的肉集中减少铁的沥滤的率，和高集中 Fe3+ 的外部增加对沥滤也不利黄铁矿。增加的接种数量和高起始的 pH 价值对沥滤有益黄铁矿，和这些改变的条件在沥滤上带更明显的效果由 L 的黄铁矿。ferriphilum 比由 A。ferrooxidans。结果也证明调整在沥滤的值处理的 pH 由于形成的 jarosite 困惑沥滤的黄铁矿。在沥滤形成的 Jarosite 过程与水晶的形态学用 XRD， SEM 和精力光谱分析，和碎片的可观的数量被观察在黄铁矿的表面上是在场的。结果暗示间接行动为 bioleaching 黄铁矿是更重要的。

Stepwise dissolution of chalcopyrite bioleaching by thermophile A.manzaensis and mesophile L. ferriphilum

Chalcopyrite dissolution was evaluated by bioleaching and electrochemical experiments with thermophile A. manzaensis（Acidianus manzaensis） and mesophile L. ferriphilum（Leptospirillum ferriphium） cultures at 65 ℃ and 40 ℃, respectively. It was investigated that the bioleaching of chalcopyrite was stepwise. It was reduced to Cu2 S at a lower redox potential locating in the whole bioleaching process by A. manzaensis at high temperature while only at initial days of bioleaching by L. ferriphilum at a relative low temperature. No reduced product was detected when the redox potential was beyond a high level（e.g., 550 m V（vs SCE）） bioleached by L. ferriphilum. Chalcopyrite bioleaching efficiency was substantially improved bioleached by A. manaensis compared to that by L. ferriphilum, which was mainly attributed to the reduction reaction occurring during bioleaching. The reductive intermediate Cu2 S was more amenable to oxidation than chalcopyrite, causing enhanced copper extraction.

Extraction and purification of magnetic nanoparticles from strain of Leptospirillum ferriphilum

The magnetic nanoparticles were extracted from Leptospirillum ferriphilum,strain YSK,isolated from acid mine drainages by treatment with sodium dodecyl sulfate(SDS)and centrifugation through a sucrose density gradient.Transmission electron microscopy(TEM)indicates that the nanoparticles are approximately spherical with a mean diameter of 44 nm,and magnetite crystals in this size range are single magnetic domains.Energy-dispersive X-ray analysis shows that the nanoparticles primarily contain two kinds of elements,iron and oxygen.Thus it can be concluded that the magnetic particles are magnetosomes.Generally,it is thought that cellular magnetotaxis is a direct consequence of the cell possessing magnetosomes.The discovery of magnetosomes in strain YSK can provide the theoretical basis for screening efficient bioleaching bacteria which are specific to different magnetic minerals under an outer magnetic field.

Effect of introduction of exogenous strain Leptospirillum ferriphilum YSK on functional gene expression,structure,and function of indigenous consortium during pyrite bioleaching

Leptospirillum ferriphilum YSK was added to a native consortium of bioleaching bacteria including Acidithiobacillus caldus,A.thiooxidans,A.ferrooxidans,Sulfobacillus thermosulfidooxidans,Acidiphilium spp.,and Ferroplasma thermophilum cultured in modified 9K medium containing 0.5%(W/V)pyrite.The bioleaching efficiency markedly increased.Changes in community structure and gene expression were monitored with real-time PCR and functional gene arrays.Dynamic changes that varied in different populations in the consortium occurred after the addition of L.ferriphilum YSK,with growth of A.caldus S1,A.thiooxidans A01,Acidiphillum spp.DX1-1 promoted the growth of Ferroplasma L1,inhibited that of S.thermosulfidooxidans ST,and exerted little effect on that of A.ferrooxidans CMS.Genes encoding ADP heptose,phosphoheptose isomerase,glycosyltransferase,biotin carboxylase,and protoheme ferrolyase from L.ferriphilum,acetyl-CoA carboxylase from Acidiphillum spp.,and doxD from A.caldus were up-regulated in 0-20 h.Genes encoding lipid A disaccharide synthase LpxB,glycosyl transferase,and ADP heptose synthase from A.ferrooxidans were up-regulated in 0-8 h and then down-regulated in 8-20 h.Genes encoding ferredoxin oxidoreductase from Ferroplasma sp.were up-regulated in 0-4 h,down-regulated in 4-16 h,and again up-regulated in 16-20 h.CbbS from A.ferrooxidans was down-regulated in 0-20 h.

某铀矿石微生物浸出性能及影响因素研究

为评估微生物浸出某铀矿石的应用前景,设计正交实验,在不同初始pH值、接种量、浸出时间和固液比条件下,分别开展了嗜铁钩端螺旋菌、嗜酸氧化亚铁硫杆菌和嗜酸喜温硫杆菌浸出某铀矿石的研究。三株微生物对某铀矿石的最高浸出率均高于97%。浸出过程中,微生物浸出体系的pH值均呈下降趋势,Eh值均呈上升趋势。初始pH值、接种量、浸出时间和固液比四个因素对三株微生物的浸出均有影响,但对不同微生物浸铀的影响存在区别。影响嗜铁钩端螺旋菌浸出的主要因素是接种量,而嗜酸氧化亚铁硫杆菌和嗜酸喜温硫杆菌的浸出主要受初始pH值的影响。

不同能源条件下中度嗜热嗜酸细菌多样性分析

采用黄铁矿、黄铜矿、硫酸亚铁和硫粉混合物作为主要能源物质在50℃条件下分别培养中度嗜热细菌混合物,研究其细菌多样性.提取细菌基因组总DNA,采用PCR结合限制性酶切片段多态性分析（RFLP）方法进行细菌16S rRNA基因的系统发育分析,比较不同能源条件下富集培养的混合细菌群落构成的差异.从3个培养物中共获得阳性克隆303个并进行RFLP分析,对29种不同酶切谱型的克隆插入序列进行测定和系统发育分析.大部分序列与已报道的浸矿微生物16S rRNA序列相似性较高（89.1%～99.7%）,归属于硫化叶菌属的耐温氧化硫化杆菌（Sulfobacillus thermotolerans）和热氧化硫化杆菌（Sulfobacillus thermosulfidooxidans）,嗜酸硫杆菌属的喜温硫杆菌（Acidithiobacillus caldus）,钩端螺旋菌属的嗜铁钩端螺旋菌（Leptospirillum ferriphilum）以及uncultured forest soil bacterium、uncultured proteobacterium.其中Acidithiobacillus caldus,Sulfobacillus thermotolerans,Leptospirillum ferriphilum 3种细菌为三类能源物质培养物中的优势细菌类群.L.ferriphilum在黄铁矿培养体系（53.8%）和硫酸亚铁和硫粉为能源的培养体系中（45.9%）中丰度最高;在以黄铜矿为能源物质的培养体系中,S.thermotolerans的比例大幅上升（70.1%）.

氯离子对氧化亚铁硫杆菌和氧化亚铁钩端微螺菌活性的影响

采用连续稀释法分别从江西和广东某铀矿生物冶金现场筛选得到两株亚铁氧化菌。16srDNA鉴定结果表明,细菌分别为氧化亚铁硫杆菌（Acidimicrobium ferrooxidans）和氧化亚铁钩端微螺菌（Leptospirillum ferriphilum）。在氯离子浓度在1g/L以内,对细菌亚铁氧化活性基本没有影响;1-2g/L铁氧化活性有所降低,但是细菌通过调节依然能够适应并维持比较高的活性;氯离子浓度大于2g/L后亚铁氧化活性急速下降,当氯离子浓度大于3g/L后细菌生长受到抑制,亚铁氧化活性很低。

螺旋状专性化能自养铁氧化细菌ML-04菌株的分子鉴定

对 1株螺旋状专性化能自养铁氧化细菌ML 0 4的 16SrDNA基因PCR扩增产物进行了序列分析 ,并以 16SrD NA序列同源性为基础构建系统发育树 ,结果表明 ,ML 0 4菌株与氧化亚铁钩端螺旋菌 (Leptospirillumferrooxidans)具有较大的差异性 ,而与嗜铁钩端螺旋菌 (Leptospirillumferriphilum)的相似度达 99.7%以上 。

金属离子对中等嗜热混合菌活性的影响

通过金属耐受试验,测定了金属离子Mo^(6+)、Cu^(2+)、Zn^(2+)对中等嗜热混合菌(喜温嗜酸硫杆菌和氧化亚铁微螺菌共培养体系)活性的影响。结果表明,随着金属离子浓度的提高,细菌的活性降低,受试菌对上述3种金属离子的耐受浓度分别为0.6g/L、10 g/L和30 g/L,3种金属离子的毒性顺序为:Mo^(6+)>Cu^(2+)>Zn^(2+)。喜温嗜酸硫杆菌对金属离子的耐受能力高于氧化亚铁微螺菌。结果说明喜温嗜酸硫杆菌和氧化亚铁微螺菌共培养体系具有较强的金属离子耐受能力,特别是耐受钼的能力很强,适于含钼硫化矿、含钼废料以及含钼污泥等的生物浸出。

嗜铁钩端螺旋菌氧化难处理金矿的研究

采用改良双层板方法从酸性矿坑水（AMD）中筛选、鉴定并命名为氧化效果最佳的分离株—嗜铁钩端螺旋菌（Leptospirillum ferriphilum）LJ-02。当温度在36~46℃范围内,矿石氧化率呈现出先升后降的趋势;降低pH对提高细菌氧化能力有着显著影响;在接种量不超过10%（v/v）的条件下,接种量的提高有利于矿石的生物氧化。该菌株对难处理金矿的吸附属于Langmuir等温吸附,其吸附平衡常数为8.72×10-10 mL/cells,单位质量矿物颗粒的最大吸收能力为1.99×109 cells/g。

培养条件对三种细菌在黄铁矿表面吸附的影响

实验研究了中等嗜热浸矿菌(A.c菌、S.t菌和L.f菌)在黄铁矿表面的吸附影响,通过测定在不同溶液pH值(1.6、2.0和2.5)和温度(40℃和45℃)条件下细菌在黄铁矿表面的吸附,分析培养条件对细菌吸附的影响。研究结果表明,L.f菌在矿表面具有较强的吸附能力,在温度为40℃,pH=2.0时,吸附量达到89.47%;pH=1.6时,混合菌(A.c菌∶S.t菌∶L.f菌为1∶1∶1)的吸附量达到75.54%。溶液初始温度的变化,对矿驯化菌的接触角和Zeta电位影响不明显,但是随着溶液pH值的升高,菌的接触角和Zeta电位均有所下降。静力显微镜和扫描电子显微镜检测结果显示,细菌在黄铁矿表面呈单细胞或簇状排列,且大量的细菌吸附在黄铁矿的裂缝或凹陷处,这对矿物的浸出具有重要意义。

铜离子胁迫对嗜铁钩端螺旋菌生理特性的影响

研究铜离子胁迫对嗜铁钩端螺旋菌ML-04生理特性的影响。以野生菌嗜铁钩端螺旋菌ML-04和驯化菌嗜铁钩端螺旋菌ML-04-ADP为材料,比较吸附及胞内的总铜含量,驯化菌对闪锌矿的吸附率、zeta电位、傅立叶变换红外光谱、疏水性和氨基酸组成的影响。结果表明:初始时,驯化菌吸附及胞内总铜含量明显高于野生菌,在0.5 mol·L-1 CuSO4培养基中培养1h后,驯化菌增加的铜也远比野生菌多,当将驯化菌放入到无铜培养基中培养1h后,总铜含量又显著减少;驯化菌对闪锌矿的吸附率提高了20.76%,驯化菌的等电点从野生菌的4.0提高到5.0。FT-IR结果表明,驯化菌表面产生了更多的蛋白物质,疏水性增强;正十六烷-9K培养基两相分层试验表明,驯化菌和野生菌水相中菌体的数量分别减少了14%和2%;氨基酸组成分析表明,两株菌的蛋白在种类上是有差别的。在高浓度铜离子环境下,驯化菌表面产生更多的蛋白物质,使细菌的疏水性增强,细胞表面的电荷数和等电点发生变化。在pH值2.0时,驯化菌表面带的正电荷大于野生菌,此时闪锌矿带负电荷。静电引力和疏水性的共同作用使驯化菌对闪锌矿的吸附能力大于野生菌对闪锌矿的吸附能力。

嗜铁钩端螺旋菌中铁硫簇相关蛋白的生物信息学分析

利用在线分析软件对嗜铁钩端螺旋菌中9种与铁硫簇结构、功能及生物合成相关蛋白质基本性质、活性位点、结构和蛋白相互作用网络等方面进行了预测。9种蛋白质包含7种酶和2种非酶蛋白,酶类蛋白与机体蛋白质裂解,翻译、信号转导和能量代谢相关,非酶类蛋白为生长因子和转运结合蛋白。所有蛋白的稳定性和等电点均有差异。WP_014960240.1、WP_014960241.1、WP_014960239.1和WP_014960235.1在ISC和NIF系统中发挥着重要的作用,参与铁硫簇的组装;WP_014959988.1可能主要参与细胞生长;WP_014960813.1和WP_014961639.1可能在能量代谢中发挥重要的作用;WP_014961659.1与铜金属解毒机制有关;WP_014962089.1参与亚硝酸盐的还原。该研究为进一步用分子生物学方法验证其相互作用机制提供给了理论依据,同时为菌株改良和生物浸矿奠定理论基础。