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

The original strains Acidithiobacillusferrooxidans GF and Acidiphilium cryptum DXI-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 DXI-1, and 30 ℃ and 2.0 for At. ferrooxidans GF, respectively. For Ac. cryptum DXI-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 within 60 h, which was 20 h earlier than the original strain. For At. ferrooxidans GF, the optimum mutation time was 60 s and the positive mutation rate was 35%. The most active UV-mutated strain At. ferrooxidans GF oxidized all the ferrous after 48 h. The bioleaching experiments showed that bioleaching with the mixture of UV-mutated strains of At. ferrooxidans GF and A c. cryptum DX1-1 （1：1） could extract 3.01 g/L of copper after 30 d, while the extracted copper was 2.63 g/L with the mixture of the original strains before UV-mutation. At the end of the bioleaching experiments, the proportion of the cell density in the cultures ofAc. cryptum DXI-1 andAt.ferrooxidans GF was approximately 1：5.

Bioleaching of chalcopyrite by pure and mixed culture

在饮料烧瓶的黄铜矿的 bioleaching 被使用纯 Acidithiobacillus ferrooxidans 调查并且混合了在中国在 Yushui 和 Dabaoshan 铜矿从酸矿排水孤立的文化，象 YS 和 DB 显著分别地。混合文化主要由 Acidithiobacillus ferrooxidans， Acidithiobacillus thiooxidans，和 Leptospirillum spp 组成了。(Leptospirillum ferriphilum 和 Leptospirillum ferrooxians ) 。结果证明因为断然从黄铜矿增加溶解率和铜的恢复的氧化硫的文化的存在，混合文化是比纯 Acidithiobacillus ferrooxidans 更有效的。有黄铜矿沥滤的减少的 pH 价值减少评价，因为象在 bioleaching 期间的矿物质表面上的钝化层的 jarosite 的形成。在用混合文化的 bioleaching，低 pH 从氧化禁止的硫被得到， jarosite 的形成。铜抽取在混合文化到达 46.27% 并且 30.37% 在在为 75 d 沥滤以后的纯 Acidithiobacillus ferrooxidans。

A new strain Acidithiobacillus albertensis BY-05 for bioleaching of metal sulfides ores

An acidophilic,rod-shaped Gram-negative sulfur oxidizing strain BY-05 was isolated from an acid mine drainage of copper ore in Baiyin area,Gansu Province,China.Ultrastructural studies show that the isolate has a tuft of polar flagella and possesses sulfur granules with clear membrane adhering to the cell innermembrane.Physiological study shows that this isolate grows autotrophically and aerobically by oxidizing S0and reduced inorganic sulfur compounds(SO, 2 23-SO, 2 24- S2 -and ZnS)with the optimum growth at pH 3.5-4.0 and at the temperature range of 25-30℃.The 16S rRNA gene sequence(DQ 423683)of strain BY-05 has 100%sequence similarity to that of Acidithiobacillus albertensis(DSM 14366).So it is identified and named as A. albertensis BY-05.Bioleaching experiments with this new strain show that it can play an important role in recovery of metals from chalcopyrite and sphalerite.

Comparative study on bioleaching of two different types of low-grade copper tailings by mixed moderate thermophiles

The bioleaching of two different types of low-grade copper tailings,acid-leaching tailings(ALT)and copper flotation tailings(CFT)by mixed moderate thermophiles,and the variation of mineralogical and microbiological characteristics during their dissolution processes were comparatively investigated.Results showed that bioleaching behaviors of the two types of tailings were significantly different.In ALT bioleaching,lower redox potential,higher[Fe3+]/[Fe2+]ratio and higher cell density in solution were obtained.These resulted in higher total copper,primary copper sulfide and secondary copper sulfide extractions,compared with CFT bioleaching.X-ray diffraction analysis suggested that gypsum and some metal organic complexes were detected in CFT bioleaching,which could cause the sluggish oxidation of sulphide minerals.The shifts of microbial community in the leachates and leaching residues varied greatly between ALT and CFT bioleaching.The percentage of iron-oxidizing bacteria in ALT bioleaching was higher than that of CFT,but the sulfur-oxidizing bacteria percentage was the opposite.The archaeon F.thermophilum L1 was detected in ALT but not in CFT.

Thin-layer heap bioleaching of copper flotation tailings containing high levels of fine grains and microbial community succession analysis

Thin-layer heap bioleaching of copper flotation tailings containing high levels of fine grains was carried out by mixed cultures on a small scale over a period of 210 d. Lump ores as a framework were loaded at the bottom of the ore heap. The overall copper leaching rates of tailings and lump ores were 57.10wt% and 65.52wt%, respectively. The dynamic shifts of microbial community structures about attached microorganisms were determined using the Illumina MiSeq sequencing platform based on 16S rRNA amplification strategy. The results indicated that chemolithotrophic genera Acidithiobacillus and Leptospirillum were always detected and dominated the microbial community in the initial and middle stages of the heap bioleaching process; both genera might be responsible for improving the copper extraction. However, Thermogymnomonas and Ferroplasma increased gradually in the final stage. Moreover, the effects of various physicochemical parameters and microbial community shifts on the leaching efficiency were further investigated and these associations provided some important clues for facilitating the effective application of bioleaching. © 2017, University of Science and Technology Beijing and Springer-Verlag Berlin Heidelberg.

Effect of Cu^(2+)ions on bioleaching of marmatite

铁闪锌矿的 bioleaching 上的 Cu2+ 离子的效果通过饮料沥滤被调查实验。接种的细菌是 Acidithiobacillus ferrooxidans， Acidithiobacillus thiooxidans 和 Lepthospirillum ferrooxidans 的一种混合文化。结果证明那锌有选择地被沥滤，并且 Cu2+ 离子的适当内容的增加在铁闪锌矿的 bioleaching 上有积极效果。沥滤的残余的 SEM 和 EDX 分析表明产品层在矿物质表面上铁硫化物，元素的硫和 jarosite 形式创作了。元素的硫的 biooxidation 被 Cu2+ 离子催化，它消除障碍到铁闪锌矿的 bioleaching 并且使 pH 价值低。与 0.5 个 g/L Cu2+ 离子的增加，最大的锌抽取率在 30 的温度在 23 d 以后到达 73%? 吗？？

Comparative study on chalcopyrite bioleaching with assistance of different carbon materials by mixed moderate thermophiles

The catalysis of four carbon materials including artificial graphite(AG), carbon black(CB), activated carbon(AC) and carbon nanotube(CN) on chalcopyrite bioleaching by mixed moderate thermophiles was comparatively investigated. In AC and AG added bioleaching groups, low solution pH and suitable redox potential values, high total iron and ferric iron concentrations, and large number of adsorbed bacteria were obtained, resulting in high copper extractions. CB and CN inhibited the growth of bioleaching bacteria and led to the low bioleaching efficiency.X-ray diffraction analysis showed that jarosite and sulfur film were the main components of passivation layer with the addition of AG and AC,but did not hinder the dissolution of chalcopyrite. Microbial community structures of free and attached cells in AC and AG added groups changed dramatically compared with mixed moderate thermophiles. The sulfur-oxidizing bacteria of A. caldus S1 strain dominated the microbial community(93%-98%) at the end of bioleaching.The iron-oxidizing bacteria of L.ferriphilum YSK only accounted for low percentage(1%-2%).

Acidophilic bacterial community reflecting pollution level of sulphide mine impacted by acid mine drainage

To reveal the impact of mining on bacterial ecology around mining area,bacterial community and geochemical characteristics about Dabaoshan Mine(Guangdong Province,China)were studied.By amplified ribosomal DNA restriction analysis and phylogenetic analysis,it is found that mining pollution greatly impacts the bacterial ecology and makes the habitat type of polluted environments close to acid mine drainage(AMD)ecology.The polluted environment is acidified so greatly that neutrophil and alkaliphilic microbes are massively dead and decomposed.It provided organic matters that can make Acidiphilium sp.rapidly grow and become the most bacterial species in this niche.Furthermore,Acidithiobacillus ferrooxidans and Leptospirillum sp.are also present in this niche.The amount of Leptospirillum sp.is far more than that of Acidithiobacillus ferrooxidans,which indicates that the concentration of toxic ions is very high.The conclusions of biogeochemical analysis and microbiological monitor are identical. Moreover,because the growth of Acidithiobacillus ferrooxidans and Leptospirillum sp.depends on ferrous iron or inorganic redox sulfur compounds which can be supplied by continual AMD,their presence indicates that AMD still flows into the site.And the area is closer to the outfalls of AMD,their biomasses would be more.So the distinction of their biomasses among different areas can help us to find the effluent route of AMD.

Growth and surface properties of new thermoacidophilic Archaea strain Acidianus manzaensis YN-25 grown on different substrates

新 thermoacidophilic Archaea 的生长和表面性质拉紧在 Tengchong 从一个酸温泉孤立的 Acidianus manzaensis YN-25，云南省，中国被调查在包括可溶的底层的不同底层上有教养铁的硫酸盐和 nonsoluble 固体底层 S0，黄铁矿和黄铜矿。在每底层的细胞的生长特征在细胞数字与变化被描绘， pH ，啊，并且Fe2+或 SO42 的集中?，或比率[Fe2+]到[Fe3+]，并且表面性质以希腊语的第六个字母潜力被描绘并且分析， hydrophobicity ，并且出现细胞的英尺红外系列。结果证明在稳固的底层上种的房间有等电位的点的更高的价值。他们是更恐水病的并且比铁的硫酸盐表示更多的表面蛋白质成年房间。

Purification and enzymatic properties of arsenic resistance protein ArsH from heterogeneous expression in E.coli BL21

Four arsenic-resistance genes(arsB,arsC,arsH,arsR) have been discovered in Acidithiobacillus ferrooxidans.Their gene sequences have been identified and three different arsenic-resistance mechanisms have been elucidated.However,the function of the arsH gene in At.ferrooxidans remains unclear.In order to evaluate the function of the arsH gene,we cloned it and expressed it in Escherichia coli.The protein was purified and its relative molecular mass was determined by SDS-PAGE(Sodium dodecyl sulfate-polyacrylamide gel electrophoresis).The results indicated that the relative molecular mass of the purified ArsH was approximately 29 kDa.The purified protein ArsH from E.coli BL21 was a flavoprotein that oxidized in vitro NADPH with an optimal pH of 6.4.

Identification and fermentation optimization of protopectinase-overproducing strain Aspergillus niger CD-01 for pectin production

In order to solve the citrus peel resource waste problem protopectinase-overproducing strain CD-01 for pectin production and minimize the drawbacks of chemical extraction of pectin, a was isolated from a pit soil dumped with perished orange in Changde City, Hunan Province of China. The strain CD-01 had the same morphology and 28S rRNA gene sequence （FJ184995） as that of Aspergillus niger （ATCC 64028）. It was thus identified and named as Aspergillus niger CD-01. The fermentation condition was optimized based on L9（34） orthogonal experimental design and the variances analyses. The results show that the optimal condition for producing pectin is as follows： time 36 h, temperature 35 ℃, pH 5, and urea as the nitrogen source. Under this condition, the pectin yield can reach up to 24.5%. This shows a great potential of Aspergillus niger CD-01 in pectin extraction from citrus.

Bacterial leaching of chalcopyrite and bornite with native bioleaching microorganism

A native mesophilic iron-oxidizing bacterium,Acidithiobacillus ferrooxidans,has been isolated(30℃)from a typical, lead-zinc concentrate of Dachang Mine in the region of Liuzhou located in the southwest of China.Two typical copper sulfide minerals,chalcopyrite and bomite,were from Meizhou Copper Mine in the region of Guangdong Province,China.Variation of pH and cell growth on time and effects of some factors such as temperature,inoculation cell number,and pulp density on the bioleaching of chalcopyrite and bomite were investigated.The results obtained from the bioleaching experiments indicate that the efficiency of copper extraction depends on all of the mentioned variables,especially the pulp density has more effect than the other factors on the microorganism.In addition,the results show that the maximum copper recovery was achieved using a mesophilic culture.The copper dissolution reached 51.34% for the chalcopyrite while it was 72.35% for the bornite at pH 2.0,initial Fe(II)concentration 9 g/L and pulp density 5%,after 30 d.

16s rDNA based microbial diversity analysis of eleven acid mine drainages obtained from three Chinese copper mines

Eleven acid mine drainage （AMD） samples were obtained from southeast of China for the analysis of the microbial communities diversity, and the relationship with geochemical variables and spatial distance by using a culture-independent 16S rDNA gene phylogenetic analysis approach and multivariate analysis respectively. The principle component analysis （PCA） of geochemical variables shows that eleven AMDs can be clustered into two groups, relative high and low metal rich （RHMR and RLMR） AMDs. Total 1691 clone sequences are obtained and the detrended correspondence analysis （DCA） of operational taxonomic units （OTUs） shows that, ~,-Proteobacteria, Acidobacteria, Actinobacteria, Cyanobacteria, Firmicutes and Nitrospirae are dominant species in RHMR AMDs. In contrast, a-Proteobacteria, fl-Proteobacteria, Planctomycetes and Bacteriodetes are dominant species in RLMR AMD. Results also show that high-abundance putative iron-oxidizing and only putative sulfur-oxidizing microorganisms are found in RHMR AMD. Multivariate analysis shows that both geochemical variables （r=0.429 3, P=-0.037 7） and spatial distance （r=0.321 3, P=-0.018 1） are significantly positively correlated with microbial community and pH, Mg, Fe, S, Cu and Ca are key geochemistry factors in shaping microbial community. Variance partitioning analysis shows that geochemical variables and spatial distance can explain most （92%） of the variation.

Sulfur speciation transformation during bioleaching of pyrite-containing sphalerite concentrate by thermophile Sulfolobus metallicus at 65 °C

Sulfur speciation transformation during bioleaching of pyrite-containing sphalerite concentrate by thermophile Sulfolobus metallicus （S. metallicus） at 65 ℃ was investigated by X-ray diffraction （XRD）, diffuse reflectance Fourier transform infrared spectroscopy （FT-IR） and sulfur K-edge X-ray absorption near edge structure spectroscopy （XANES）. The results show that the presence of S. metallicus effectively enhances the dissolution of the mineral. The yield of zinc increases from 0.5 g/L in sterile control to 2.7 g/L in bioleaching. The pyrite in the concentrate facilitates zinc dissolution in the early stage, but has hindrance role in the late stage for the formation of jarosite. Sulfur speciation analyses show that jarosite and elemental sulfur are main products in bioleaching process, and the accumulation ofjarosite is mainly responsible for the decline of leaching efficiency.

Microwave-assisted synthesis and characterization of ZnO-nanorod arrays

High density ZnO-nanorod arrays(rod length 1.59μm)were successfully synthesized via a microwave-assisted solution-phase method using zinc chloride and ammonia solution as reactants.The influence of concentration of ammonia solution, work power,and microwave irradiation time on the morphology and size of final products was carefully investigated.The crystal structure,chemical composition and morphologies of final products were characterized using X-ray powder diffraction(XRD), scanning electron microscopy(SEM)and photoluminescence(PL).The as-synthesized ZnO is composed of single crystalline and possesses three photoluminescence emissions centered at 400,469 and 534.5 nm,respectively.

Recovery of gallium from zinc concentrate by pressure oxygen leaching

Zinc concentrate with high gallium content is one of the main resources of gallium.The gallium presents in the form of isomorphism in tetrahedron coordination with sulfur in sphalerite.The research was to investigate the amenability of zinc concentrate with high gallium to pressure oxygen leaching.The particle size,sulfuric acid concentration,oxygen partial pressure,additive amount,and time of reaction were studied.The extraction yields of gallium and zinc are 86%and 98%,respectively.The optimal condition is 100 g of zinc concentrate with particle size smaller than 38 lm,sulfuric acid concentration150 g L-1,leaching temperature 150℃,leaching time120 min,oxygen partial pressure 0.7 MPa,additive amount of 0.2 wt%.

Diversity of microbial community at acid mine drainages from Dachang metals-rich mine, China

Two acid mine drainage(AMD)samples TS and WK,which were from the Dachang metals-rich mine in Guangxi province,China,were studied using PCR-based cloning approach.A total of 44 operational taxonomic units(OTUs)were obtained from the two AMD samples.However,only three OTUs(GXDC-9,GXDC-19 and GXDC-50)detected in sample TS can also be observed in sample WK.Phylogenetic analysis revealed that the bacteria in the two samples fell into four putative divisions,which were Nitrospira,Alphaproteobacteria,Gamaproteobacteria,and Acidobacteria.Organisms of genuses Acidithiobacillus and Leptospirillum,which were in gamaproteobacteria class and Nitrospira family,were dominant in two samples,respectively.In sample TS,which was characterized by low pH,high sulfate,high iron,and high arsenide,two species(Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans)constituted 98.22% of the entire microbial community.Compared with sample TS,the microbial community in sample WK was more diversified according to the observation.Interestedly,the Legionella species,which was rarely observed in the low-pH environment,was detected in sample WK.This work helps us to further understand the diversity of microbial community living in extreme acid mine drainages with unique geochemistry and the tolerance capability of acidophiles to heavy metal.

Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide

In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.

A marked enhancement in production of amylase by Bacillus amyloliquefaciens in flask fermentation using statistical methods

A total of 126 bacterial strains were isolated from soil samples. Among them, 11 isolates were found positive for amylase production. Strain YL produced the largest zone of clearance on plate assay. The isolate YL was identified as Bacillus sp. based on morphological and physiochemical characterization. According to 16S rRNA gene sequencing data, the closest phylogenetic neighbor of strain YL was Bacillus amyloliquefaciens （99.54%）. After that, an optimization of culture conditions was carried out for the improvement of a-amylase production. Response surface methodology （RSM） was applied to evaluate the effect of medium components including wheat bran, cottonseed extract, yeast extract, starch, NaC1 and CaCl2. Three variables （wheat bran, cottonseed extract, and starch）, which were identified to significantly affect amylase production by Plackett-Burman design were further optimized using response surface methodology of Box-Behnken design （BBD）. The optimal concentrations estimated for each variable related to the maximum of amylase activity （86 kU/mL） were 10.80 g/L wheat bran, 9.90 g/L cottonseed extract, 0.5 g/L starch, 2.0 g/L yeast extract, 5.00 g/L NaCl and 2.00 g/L CaC12. The fermentation using optimized culture medium allowed a significant increase in amylase production （by 3-fold）. The improvement in the a-amylase production after optimization process can be considered adequate for large-scale applications.

Effect of gene dosage and incubation temperature on production of β-mannanase by recombinant Pichia pastoris

High-level expression ofβ-mannanase has been reported in Pichia pastoris under control of the GAP promoter.Two factors that strongly influence protein production and fermentation process development in Pichia pastoris protein expression system are gene dosage and cultivation temperature.The aim of this research was to improve the expression level ofβ-mannanase in Pichia pastoris by proper increasing the gene dosage and decreasing the culture temperature.To this end,a panel of strains harboring different copy numbers ofβ-mannanase gene were obtained by multiple zeocin concentration gradients screening,the influence of gene copy number on the expression ofβ-mannanase in Pichia pastoris X33 was investigated.With the constitutive GAP promoter,the four copies strain exhibited a 4.04-fold higherβ-mannanase yield and a 1.83-fold higher total secretion proteins than the one copy strain,but an increase of the copy number above four resulted in a decrease of expression.Furthermore,the effects of culture temperature were studied in flask.The decreased culture temperature of four copies strain resulted in a 1.8-fold(26℃)and 3.5-fold(22℃)higherβ-mannanase activity compared to that at 30℃.A fed-batch strategy was successfully used for high cell-density fermentation andβ-mannanase activity reached 2124 U/mL after cultivation for 72 h in a 5 L fermenter.