Purification and enzymatic characteristics of cysteine desulfurase, IscS, in Acidithiobacillus ferrooxidans ATCC 23270

A cysteine desulfurase protein, IscS, was encoded by the operon iscSUA in Acidithiobacillusferrooxidans. The gene of IscS from Acidithiobacillus ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coil The protein was purified by one-step affinity chromatography to homogeneity. The final protein yield after affinity chromatography was 12.9%. The enzyme was characterized for thermal stability, pH and kinetic parameters. The molecular mass of recombinant lscS was 46 ku by SDS-PAGE. The optimum pH was 8.0-8.5. The enzyme had a temperature optimum at 30 ℃ and was relatively stable at 40 ℃, with 67% loss of activity. 1,5-I-AEDANS significantly inhibited IscS activity. Kinetic parameters Km and Vmax were found to be 0.11 mmol/L and 2.57 μmol/（L.min）.     

生物浸铀中影响嗜酸氧化亚铁硫杆菌活性的氟毒物活性形态(英文)

为了确定浸矿菌耐氟的机制,在氟化物存在的条件下,驯化铀矿浸出菌嗜酸氧化亚铁硫杆菌ATCC23270,研究溶液中含不同氟浓度、不同pH值时铀矿浸出菌的活性变化,以及有无蛋白酶K处理时铀矿浸出菌细胞内氟浓度的变化情况。采用铂电极和Ag/AgCl参比电极测量氧化还原电位,以作为细菌不同活性的参照指标,采用氟离子选择性电极测定细胞内的氟浓度。结果表明,真正影响铀矿浸出菌活性的是HF,溶液pH值增加以及溶液中与氟有较强络合能力的离子浓度的变化,也会引起耐氟菌假象的出现。浸矿菌的耐氟能力可能与细胞壁和细胞膜上的一些蛋白密切相关。     

Bioleaching of anilite by Acidithiobacillus ferrooxidans

In order to characterize the efficiency of copper bioleaching from anilite using pure cultures of Acidithiobacillus ferrooxidans in the absence and presence of ferrous sulphate, the experiments were carried out in shake flasks with or without 4 g/L ferrous sulphate (FeSO₄·7H₂O) at pH 2.0, 150 r/min and 35°C. The tests show that Acidithiobacillus ferrooxidans is unable to attack anilite in iron-free 9K medium. Anilite is rapidly oxidized by bacterial leaching when ferrous sulphate is added. Chemical oxidation of anilite is slow compared with Acidithiobacillus ferrooxidans initiated solubilization in the presence of iron. The EDAX analysis of the surfaces of anilite confirms that sulfur coating layer is present as a reaction product on the surface of the bacterially leached mineral.     

Development and evaluation of whole-genome oligonucleotide array for Acidithiobacillus ferrooxidans ATCC 23270

To effectively monitor the characteristic of Acidithiobacillus ferrooxidans ATCC 23270 at the whole-genomic level,a whole-genome 50-mer-based oligonucleotide microarray was developed based on the 3 217 ORFs of A.ferrooxidans ATCC 23270 genome.Based on artificial oligonucleotide probes,the results showed that the optimal hybridization temperature was 45℃. Specificity tests with the purified PCR amplifications of 5 genes(Sulfide-quinone reductase,Cytochrome C,Iron oxidase,Mercuric resistance protein,Nitrog...     

Sulfur activation-related extracellular proteins of Acidithiobacillus ferrooxidans

The fractions of the extracellular proteins of Acidithiobacillus ferrooxidans grown on two different energy substrates, elemental sulfur and ferrous sulfate, were selectively prepared with hot water treatment and distinctly shown by two-dimensional gel electrophoresis. Some protein spots with apparently higher abundance in sulfur energy substrate than in ferrous sulfate energy substrate were identified by using MALDI-TOF/TOE Based on peptide mass fingerprints and bioinformatical analysis, the extracellular proteins were classified according to their functions as conjugal transfer protein, pilin, vacJ lipoprotein, polysaccharide deacetylase family protein, Ser/Thr protein phosphatase family protein and hypothetical proteins. Several extracellular proteins were found abundant in thiol groups and with CXXC functional motif, these proteins may be directly involved in the sulfur activation by use of their thiol group （Pr-SH） to bond the elemental sulfur.     

嗜酸氧化亚铁硫杆菌（ATCC 23270）浸出黄铜矿过程中的EPS、Cu2+和Fe3+的相互作用机制（英文）

采用超声-离心方法提取嗜酸氧化亚铁硫杆菌(ATCC 23270)胞外多聚物(EPS)、EPS中的Cu2+、Fe3+离子,研究生物浸出黄铜矿过程中Cu2+、Fe3+和EPS的相互作用机制。结果表明:与Fe3+离子相比,Cu2+离子可刺激细菌产生更多的EPS;当Cu2+离子浓度从0.01mol/L增加到0.04mol/L时,EPS中Fe3+/Cu2+质量比从4:1降低到2:1;从1%黄铜矿的无铁9K介质中提取的EPS中铜铁含量是从含0.04mol/LCu2+离子的9K介质中提取的量的2倍。在生物浸出黄铜矿过程中,黄铜矿表面结合黄铁钾钒的EPS层减弱了Cu2+、Fe3+离子的迁移,逐渐成为离子扩散壁垒。     

Repetitive sequence based polymerase chain reaction to differentiate close bacteria strains in acidic sites

To study the diversity of bacteria strains newly isolated from several acid mine drainage（AMD） sites in China, repetitive sequence based polymerase chain reaction （rep-PCR）, a well established technology for diversity analysis of closely related bacteria strains, was conducted on 30 strains of bacteria Leptospirillum ferriphilium, 8 strains of bacteria Acidithiobacillus ferrooxidans, as well as the Acidithiobacillusferrooxidans type strain ATCC （American Type Culture Collection） 23270. The results showed that, using ERIC and BOX primer sets, rep-PCR produced highly discriminatory banding patterns. Phylogenetic analysis based on ERIC-PCR banding types was made and the results indicated that rep-PCR could be used as a rapid and highly discriminatory screening technique in studying bacterial diversity, especially in differentiating bacteria within one species in AMD.     

Effect of Cu ions on bioleaching of marmatite

The effect of Cu^2＋ ions on bioleaching of marmatite was investigated through shake leaching experiments. The bacteria inoculated are a mixed culture ofAcidithiobacillusferrooxidans, Acidithiobacillus thiooxidans and Lepthospirillumferrooxidans. The results show that zinc is selectively leached, and the addition of appropriate content of Cu^2＋ ions has positive effect on the bioleaching of marmatite. SEM and EDX analyses of the leaching residue reveal that a product layer composed of iron sulfide, elemental sulfur and jarosite forms on the mineral surface. The biooxidation of elemental sulfur is catalyzed by the Cu^2＋ ions, which eliminate the barrier to bioleaching of marmatite and keep low pH value. With the addition of 0.5 g/L Cu^2＋ ions, the maximum zinc extraction rate reaches 73% after 23 d at the temperature of 30 ℃ with the pulp density of 10%, while that of iron is only about 10%.     

Mutagenic breeding of silver-resistant Acidithiobacillusferrooxidans and exploration of resistant mechanism

The silver-resistant Acidithiobacillus ferrooxidans were isolated from 22 acid mine drainage （AMD） samples collected from Dexing Copper Mine and Chengmen Mountain Mine, Jiangxi Province, China. Isolate DX16 is obtained from the sample taken from Dexing Copper Mine and still carries out ferrous ion oxidation when incubated in 9K medium containing silver nitrate （240 mg/L）. While isolate HI, a less resistant strain taken from Yin Mountain Mine, has a tolerate level of only 60 mg/L. Based on 16S ribosomal DNA sequencing, both bacterial 16SrDNA sequences are 100% similar to Acidithiobacillus ferrooxidans ATCC 23270. Through ultraviolet irradiation induced mutations, isolate mDX16 that is obtained from DX16 carries out ferrous ion oxidation when incubated in 9K medium containing higher concentration of silver nitrate （250 mg/L）. When silver-resistant gene （SilC） analysis is carried out on the two isolates, it is seen that this gene was absent in both.     

一种用于嗜酸性氧化亚铁硫杆菌液氮保藏的新型保护剂的保藏效果(英文)

对一种用于嗜酸性氧化亚铁硫杆菌液氮冷藏新型保护剂GP的保藏效果进行研究。依据最大细胞复苏率及最高亚铁氧化活性确定该新型保护剂的最佳使用浓度。结果表明,保护剂的最佳浓度为30%,在此浓度下细胞复苏率达到84.4%,且能在120h内完全氧化培养基中的亚铁,培养6d后菌体浓度达到5.8×107cell/mL。此外,解冻细胞在9K培养基中培养6d后,对活细胞复苏的最佳GP残留浓度为0.6%(体积分数)。在此浓度下,菌株DC完全氧化亚铁需要108h,并且最终菌体浓度为6.8×107cell/mL.因此,GP是一种简单、有效的嗜酸性氧化亚铁硫杆菌液氮保藏的冷冻保护剂。     

表面活性剂Tween-80对Acidithiobacillus ferrooxidans硫氧化和硫代谢相关蛋白质基因表达的影响(英文)

研究了表面活性剂 Tween-80 对 Acidithiobacillus ferrooxidans ATCC 23270 生长、硫氧化和硫代谢相关典型基因表达的影响。结果表明,当培养基中含有 10 2g/L Tween-80 时,A. ferrooxidans 的生长以及其对不溶性底物(S0和 CuFeS2)的代谢得到了促进。在该条件下,经过 24 d 的生物浸出,黄铜矿的铜离子浸出率比对照组(不含 Tween-80)高 16%。FT-IR 光谱分析表明,这可能是由于 Tween-80 的存在而导致胞外多聚物成分发生变化而引起的。用 RT-qPCR 来分析 17 个硫代谢相关基因在 Tween-80 存在时的表达差异。胞外蛋白质基因表达下调表明了Tween-80 对细菌硫吸附作用的影响。硫代谢相关酶基因表达水平的变化为硫代谢的研究提供了参考。     

多金属硫化精矿中铜、锌和铁的生物浸出(英文)

利用嗜温混合菌Acidithiobacillus ferrooxidans,Acidithiobacillus thiooxidans和Leptospirillum ferrooxidans对低品位复杂Cu-Zn-Pb-Fe-Ag-Au硫化精矿在曝气生物浸出反应器中进行生物浸出。该菌种为从塞尔维亚Bor地下铜矿的酸性溶液中筛选出一种嗜热嗜酸菌。营养液为p H 1.6的9K营养液。87%的矿物粒度大于10μm,矿浆密度为8%(w/v)。在测试条件下,锌、铜和铁的浸出率分别达到89%、83%和68%。动力学分析表明,浸出过程与Spencer-Topley模型相符,受局部反应扩散控制。     

Acidithiobacillus ferrooxidans enhanced heavy metals immobilization efficiency in acidic aqueous system through bio-mediated coprecipitation

This study investigated the promotion effect of A. ferrooxidans on complex heavy metals coprecipitation process. A. ferrooxidans significantly enhanced the ferrous oxidation, which also promoted the formation of iron-oxyhydroxysulphate. Cu(II) concentration reduced to 0.058 mmol/L in A. ferrooxidans inoculated system, and Cd also reduced to the lowest concentration (0.085 mmol/L). Pb was mainly immobilized as anglesite and iron-oxyhydroxysulphate promoted the removal of remanent Pb in solution. The precipitates are characterized by XRD, SEM, and FTIR analysis. The main component of the iron-oxyhydroxysulphate was well crystallized jarosite. A. ferrooxidans contributed to the formation of schwertmannite in later monovalent cation lack stage. Higher ferrous iron oxidation rate and Fe(III) supply rate in A. ferrooxidans inoculated system facilitated polyhedron crystal formation and the increase of particle diameter. Complex heavy metals could be incorporated into iron oxyhydroxysulphate crystal, and efficiently removed from acidic wastewater through A. ferrooxidans mediated coprecipitation.     

Expression, purification and molecular modeling of iron-containing superoxide dismutase from Acidithiobacillus ferrooxidans

The superoxide dismutase(SOD) from Acidithiobacillus ferrooxidans may play an important role in its tolerance to the extremely toxic and oxidative environment of bioleaching. This gene was cloned and then successfully expressed in Escherichia coli. The expressed protein was finally purified by one-step affinity chromatography to homogeneity and observed to be dimer according to SDS-PAGE and MALDI-TOF-MS. The metal content determination and optical spectra results of the recombinant protein confirmed that the protein was an iron-containing superoxide dismutase. Molecular modeling for the protein revealed that the iron atom was ligated by His26, His75, Asp158 and His162.     

Differential utilization of cyclic,orthorhombic α- and chain-like polymeric μ-sulfur by Acidithiobacillus ferrooxidans

The differential utilization of cyclic, orthorhombic α-sulfur (α-S) and chain-like polymeric μ-sulfur (μ-S) by Acidithiobacillus ferrooxidans was investigated. The growth and sulfur oxidation results indicated that utilization of μ-S by A. ferrooxidans was clearly different from α-S. Even if the planktonic cells were produced, the fall of pH and the rise of sulfate concentration were the same after 300 h on each substrate, the speeds of the planktonic cells increase, pH decrease and sulfate concentration increase in the earlier cultivation stage were faster on polymeric sulfur compared with the orthorhombic form. The adsorption capacity of the cells was higher on μ-S than on α-S. The results of SEM, DRIFTS and XRD analyses indicated that the surfaces of α-S and μ-S were modified differently by cells. Differential expression of 11 selected sulfur adsorption-activation and metabolism relevant genes was detected by RT-qPCR. The results showed that the expression of the hydrophobic substrate transport proteins and the sulfur metabolism related proteins was up-regulated, and the adsorption and activation related proteins were down-regulated when the cells were grown on μ-S, suggesting that μ-S could be more easily bio-adapted and activated than α-S.     

Bioleaching of chalcopyrite by UV-induced mutagenized Acidiphilium cryptum and Acidithiobacillus ferrooxidans

The original strains Acidithiobacillus ferrooxidans GF and Acidiphilium cryptum DX1-1 were isolated from the drainage of some caves riched in chalcopyrite in Dexing Mine in Jiangxi Province of China.The optimum temperature and pH for growth were 30℃and 3.5 for Ac.cryptum DX1-1,and 30℃and 2.0 for At.ferrooxidans GF,respectively.For Ac.cryptum DX1-1,the optimum UV radiating time was 60 s and the positive mutation rate was 22.5%.The growth curves show that Ac.cryptum after mutagenesis reached stationary phase ...     

Effects of Acidiphilium cryptum on biosolubilization of rock phosphate in the presence of Acidithiobacillus ferrooxidans

The bioleaching of pyrite and biosolubilization of rock phosphate (RP) in 9K basal salts medium were compared by the following strains of an autotrophic acidophilic bacterium, Acidithiobacillus ferrooxidans, a heterotrophic acidophilic bacterium, Acidiphilium cryptum, and mixed culture of At. ferrooxidans and A. cryptum. The results show that A. cryptum is effective in enhancing the bioleaching of pyrite and biosolubilization of RP in the presence of At. ferrooxidans, although it could not oxidize pyrite and solubilize RP by itself. This effect is demonstrated experimentally that A. cryptum enhances a decrease in pH and an increase in redox potential, concentration of total soluble iron and planktonic part bacterial number in the broth during pyrite bioleaching processes by At. ferrooxidans. The mixed culture of At. ferrooxidans and A. cryptum leads to the most extensive soluble phosphate released at 30 °C. Pulp density exceeding 3% is shown to adversely influence the release of soluble phosphate by the consortium of At. ferrooxidans and A. cryptum. It is essential to add pyrite to the 9K basal salts medium for the biosolubilization of RP by the mixed culture of At. ferrooxidans and A. cryptum, and the percentage of soluble phosphate released is the greatest when the mass ratio of RP to pyrite is 1:2 or 1:3.