Detection of Acidithiobacillus ferrooxidans in acid mine drainage environments using fluorescent in situ hybridization (FISH)

An important microorganism of acid mine drainage (AMD) and bioleaching environments is Acidithiobacillus ferrooxidans which oxidizes ferrous iron and generates ferric iron, an oxidant. Most investigations to understand microbial aspects of sulfide mineral dissolution have focused on understanding physiological, metabolic, and genetic characteristics of A. ferrooxidans. In this study, a 16S rRNA oligonucleotide probe designated S-S-T.ferr-0584-a-A-18, and labeled at the 5'-end with indocarbocyanine dye (CY3), was used in a fluorescent in situ hybridization (FISH) procedure on pure cultures of nine isolates of A. ferrooxidans. These isolates were recovered from acid mine drainage and mining environments. The probe was also used to detect cells of A. ferrooxidans, recovered from AMD samples, growing on FeTSB and FeSo solid media in a FISH procedure. In addition, the presence of cells of A. ferrooxidans in an environmental water sample from an AMD site in Copper Cliff, Ontario, Canada was analyzed using the FISH technique. Probe specificity was first confirmed with A. ferrooxidans ATCC 19859 (positive control) and Acidithiobacillus thiooxidans ATCC 19377, Acidiphilium acidophilum ATCC 27807, and Lactobacillus plantarum ATCC 8014 (negative controls). Positive and negative control cells were also used to determine optimal stringency conditions for hybridizations with the probe. Cells of the nine isolates of A. ferrooxidans stained positive, although the fluorescent signal varied in intensity from isolate to isolate. Colonies of A. ferrooxidans from the environmental water sample of the AMD site were recovered only on FeTSB solid medium after 22 days of incubation. The probe was able to detect cells of A. ferrooxidans in a FISH procedure. However, no cells of A. ferrooxidans were detected in the AMD water sample without cultivation. Thus, probe S-S-T.ferr-0584-a-A-18 hybridized effectively with cells of A. ferrooxidans recovered from pure cultures but failed to directly detect cells of A. ferrooxidans in the AMD site.     

Comparative Leaching of Chalcopyrite by Selected Acidophilic Bacteria and Archaea

A systematic study of the bioleaching of chalcopyrite (CuFeS 2 ) was conducted using axenic cultures of 11 species of acidophilic Bacteria and Archaea to obtain a direct comparison of the microbial chalcopyrite leaching capabilities of the different cultures and to determine the factors that affect Cu release. The characteristics of chalcopyrite leaching by the moderate thermophile Sulfobacillus thermosulfidooxidans , the mesophile Acidithiobacillus ferrooxidans , and the thermophile Acidianus brierleyi were used to elucidate the leaching process. Moderately thermophilic cultures of Sulfobacillus acidophilus, Acidimicrobium ferrooxidans , and Acidithiobacillus caldus were used to study the effects of different metabolic capabilities and relate those to leaching efficiency. The greatest rate of Cu solubilization from chalcopyrite was achieved at high temperatures (up to 70°C) at redox potentials below +550 mV (Ag/AgCl). The enhanced Cu solubilization observed at high temperatures resulted from accelerated chemical reaction rates, rather than from the rates at which individual acidophiles generated the mineral leaching reactants such as Fe 3+ .     

Bioleaching of realgar by Acidithiobacillus ferrooxidans using ferrous iron and elemental sulfur as the sole and mixed energy sources

The characteristics of the bioleaching of realgar by Acidithiobacillus ferrooxidans BY-3 (A. ferrooxidans) were investigated in this work. We examined the effects of using ferrous iron and elemental sulfur as the sole and mixed energy sources on the bioleaching of realgar. Under all experimental conditions, A. ferrooxidans BY-3 significantly enhanced the dissolution of realgar. Moreover, arsenic was more efficiently leached using A. ferrooxidans BY-3 in the presence of ferrous iron than in other culture conditions. A high concentration of arsenic was observed in the absence of alternative energy sources. This concentration was higher than that in cultures with sulfur only and lower than that in cultures with ferrous iron and sulfur. Linear or nonlinear models best fit the experimental data; the nonlinear model exhibited the dual effects of dissolution and removal on the bioleaching of realgar, whereas the linear model only applied to situations of slow bioleaching rather than removal.     

The role of higher polythionates in the reduction of chromium(VI) by Acidithiobacillus and Thiobacillus cultures

In this paper, we report the chromium(VI) reduction by filtrates of Acidithiobacillus and Thiobacillus cultures. Chromium(VI) reduction by filtrates of A. ferrooxidans cultures under acidic conditions was higher than that observed for A. thiooxidans. However, at pH close to 7, chromium(VI) reduction by filtrates of T. thioparus cultures was as high as that by filtrates of A. thiooxidans cultures and much higher than that observed for A. ferrooxidans cultures at the same pH. The capability of these cultures to reduce chromium(VI) was associated specifically with the fraction of cultures (cells, sulphur and associated sulphur compounds) retained by filtration through a 0.45mum filter. In the fraction that comes from A. thiooxidans culture, polythionates (S(x)O(6)(2-)) with 3-7 sulphur atoms were detected and identified (by HPLC with MS as detector). The model of vesicles containing polythionates, sulphur and water agrees with our results.     

铜蓝精矿的生物浸出

采用两种嗜酸硫杆菌(嗜酸氧化亚铁硫杆菌和喜温硫杆菌)对铜蓝进行生物浸出,实验在有或没有4 g/L硫酸亚铁pH 2．0、150转/分、35℃的三角瓶中进行。实验结果表明:用两种菌混合浸出的铜几乎等于嗜酸氧化亚铁硫杆菌单独浸出的铜;另外,亚铁的加入能提高铜的浸出。     

Recovery of scrap iron metal value using biogenerated ferric iron

The utility of employing biogenerated ferric iron as an oxidant for the recycling of scrap metal has been demonstrated using continuously growing cells of the extremophilic organism Acidithiobacillus ferrooxidans. A ferric iron rich (70 mol%) lixiviant resulting from bioreactor based growth of A. ferrooxidans readily solubilized target scrap metal with the resultant generation of a leachate containing elevated ferrous iron levels and solubilized copper previously resident in the scrap metal. Recovery of the copper value was easily accomplished via a cementation reaction and the clarified leachate containing a replenished level of ferrous iron as growth substrate was shown to support the growth of A. ferrooxidans and be fully recyclable. The described process for scrap metal recycling and copper recovery was shown to be efficient and economically attractive. Additionally, the utility of employing the E(h) of the growth medium as a means for monitoring fluctuations in cell density in cultures of A. ferrooxidans is demonstrated.     

Bacterial oxidation of ferrous iron at low temperatures

This study comprises the first report of ferrous iron oxidation by psychrotolerant, acidophilic iron-oxidizing bacteria capable of growing at 5 degrees C. Samples of mine drainage-impacted surface soils and sediments from the Norilsk mining region (Taimyr, Siberia) and Kristineberg (Skellefte district, Sweden) were inoculated into acidic ferrous sulfate media and incubated at 5 degrees C. Iron oxidation was preceded by an approximately 3-month lag period that was reduced in subsequent cultures. Three enrichment cultures were chosen for further work and one culture designated as isolate SS3 was purified by colony isolation from a Norilsk enrichment culture for determining the kinetics of iron oxidation. The 16S rRNA based phylogeny of SS3 and two other psychrotolerant cultures, SS5 from Norilsk and SK5 from Northern Sweden, was determined. Comparative analysis of amplified 16S rRNA gene sequences showed that the psychrotolerant cultures aligned within Acidithiobacillus ferrooxidans. The rate constant of iron oxidation by growing cultures of SS3 was in the range of 0.0162-0.0104 h(-1) depending on the initial pH. The oxidation kinetics followed an exponential pattern, consistent with a first order rate expression. Parallel iron oxidation by a mesophilic reference culture of Acidithiobacillus ferrooxidans was extremely slow and linear. Precipitates harvested from the 5 degrees C culture were identified by X-ray diffraction as mixtures of schwertmannite (ideal formula Fe(8)O(8)(OH)(6)SO(4)) and jarosite (KFe(3)(SO(4))(2)(OH)(6)). Jarosite was much more dominant in precipitates produced at 30 degrees C.     

氧化亚铁硫杆菌中磁小体形成相关基因在不同亚铁浓度刺激下的差异表达

氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)的生物控制矿化作用可以使其在胞内形成黑色电子致密颗粒—磁小体。本研究利用生物信息学方法对氧化亚铁硫杆菌标准菌株ATCC 23270的全基因组进行分析, 并通过Real-time PCR技术研究氧化亚铁硫杆菌中与磁小体形成相关的mpsA、magA、thy和mamB四个基因在不同亚铁浓度刺激下的差异表达, 结果发现它们在转录层面的表达量受亚铁浓度的影响, 当亚铁浓度达到150~200 mmol/L范围内达到最高表达,这对进一步深入研究氧化亚铁硫杆菌中磁小体的形成机理有积极的意义。     

生物淋滤法去除污泥中的重金属

本研究从剩余活性污泥中分离得到两株土著硫杆菌。对两株菌进行了分类鉴定。确立二者分别为嗜酸性氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans, A. f)和嗜酸性氧化硫硫杆菌(Acidithiobacillus thiooxidans, A. t)。将二者的单菌和混合菌分别接种于剩余活性污泥中, 进行了为期9 d的生物淋滤, 对淋滤过程中的pH变化、氧化还原电位(ORP)以及重金属含量进行了检测。结果表明, 生物淋滤9 d混合菌对于As、Cr、Cu、Ni和Zn的去除效果最好; 去除率分别达到了96.09%、93.47%、98.32%、97.88%和98.60%。对于Cd和Pb混合菌生物淋滤的去除率在第6天之后迅速下降, 但是A. t单菌淋滤保持较高的去除率。     

Kinetic constant variability in bacterial oxidation of elemental sulfur

Wide ranges of growth yields on sulfur (from 2.4 x 10(10) to 8.1 x 10(11) cells g(-1)) and maximum sulfur oxidation rates (from 0.068 to 1.30 mmol liter(-1) h(-1)) of an Acidithiobacillus ferrooxidans strain (CCM 4253) were observed in 73 batch cultures. No significant correlation between the constants was observed. Changes of the Michaelis constant for sulfur (from 0.46 to 15.5 mM) in resting cells were also noted.     

固体平板磁泳分离细菌新方法的研究

氧化亚铁硫杆菌（Acidithiobacillus ferrooxidans）能够在胞内形成电子致密的磁性颗粒,它的这种特性使利用氧化亚铁硫杆菌合成生物纳米磁性材料成为了可能。本课题组为了筛选出合成磁性颗粒能力强的菌株,对原有的液体磁泳进行了改进,采用了新的固体平板磁泳方法来筛选纯化目的菌株。经过磁泳分离后,细菌中含磁性颗粒的细胞比例由原始菌群的30％上升到90％,胞内含有的磁颗粒数目也由1~2颗增加至2~5颗,筛选得到的细菌在人工磁场下会进行趋磁运动。实验结果表明,氧化亚铁硫杆菌具有较弱的趋磁性,在人工磁场下会进行趋磁运动,但仅在地磁场作用下不能定向运动,利用固体平板磁泳筛选纯化含有磁性颗粒的氧化亚铁硫杆菌的方法是切实可行的,磁泳分离技术的进一步完善和改进为传统的微生物菌种分离提供了新的途径,为研究纯氧化亚铁硫杆菌菌株胞内磁性颗粒的形成条件及机理提供了前提条件,也为今后从浸矿细菌中分离筛选更多的含有磁性颗粒的菌株打下基础。     

Microbiological and geochemical dynamics in simulated-heap leaching of a polymetallic sulfide ore

The evolution of microbial populations involved in simulated-heap leaching of a polymetallic black schist sulfide ore (from the recently-commissioned Talvivaara mine, Finland) was monitored in aerated packed bed column reactors over a period of 40 weeks. The influence of ore particle size (2-6.5 mm and 6.5-12 mm) on changes in composition of the bioleaching microflora and mineral leaching dynamics in columns was investigated and compared to fine-grain (<2 microm) ore that was bioprocessed in shake flask cultures. Both column reactors and shake flasks were inoculated with 24 different species and strains of mineral-oxidizing and other acidophilic micro-organisms, and maintained at 37 degrees C. Mineral oxidation was most rapid in shake flask cultures, with about 80% of both manganese and nickel and 68% of zinc being leached within 6 weeks, though relatively little of the copper present in the ore was solubilised. The microbial consortium that emerged from the original inoculum was relatively simple in shake flasks, and was dominated by the iron-oxidizing autotroph Leptospirillum ferriphilum, with smaller numbers of Acidimicrobium ferrooxidans, Acidithiobacillus caldus and Leptospirillum ferrooxidans. Both metal recovery and (for the most part) total numbers of prokaryotes were greater in the column reactor containing the medium-grain than that containing the coarse-grain ore. The bioleaching communities in the columns displayed temporal changes in composition and differed radically from those in shake flask cultures. While iron-oxidizing chemoautotrophic bacteria were always the most numerically dominant bacteria in the medium-grain column bioreactor, there were major shifts in the most abundant species present, with the type strain of Acidithiobacillus ferrooxidans dominating in the early phase of the experiment and other bacteria (At. ferrooxidans NO37 and L. ferriphilum) dominating from week 4 to week 40. With the coarse-grain column bioreactor, similar transitions in populations of iron-oxidizing chemoautotrophs were observed, though heterotrophic acidophiles were often the most abundant bacteria found in mineral leach liquors. Four bacteria not included in the mixed culture used to inoculate the columns were detected by biomolecular techniques and three of these (all Alicyclobacillus-like Firmicutes) were isolated as pure cultures. The fourth bacterium, identified from a clone library, was related to the Gram-positive sulfate reducer Desulfotomaculum salinum. All four were considered to have been present as endospores on the dried ore, which was not sterilized in the column bioreactors. Two of the Alicyclobacillus-like isolates were found, transiently, in large numbers in mineral leachates. The data support the hypothesis that temporal and spatial heterogeneity in mineral heaps create conditions that favour different mineral-oxidizing microflora, and that it is therefore important that sufficient microbial diversity is present in heaps to optimize metal extraction.     

磁泳分离细菌新方法的研究

从酸性矿坑水中富集培养分离到的嗜酸氧化亚铁硫杆菌（Acidithiobacillus ferrooxidans,A.ferrooxidans）[1-2] 菌同趋磁细菌具有一定的相似性。通过显微镜观察发现,部分浸矿细菌在外加磁场的作用下具有微弱的趋磁性,基于菌种的这种特性,设计了磁泳分离仪,对其在磁场作用下泳动（磁泳）进行分析,经磁泳后的近磁、远磁菌的生理特性有较大的差异。从用涂布平板法获得的近磁菌纯培养A. ferrooxidans菌体中,分离得到纳米磁性颗粒,能谱分析表明,其主要成分为Fe和O元素。实验结果证明,A. ferrooxidans具有微弱趋磁性,采用磁泳分离该类菌体内含有磁性颗粒的细菌是可行的,这一分离技术的进一步完善和改进将为传统的微生物菌种分离提供一种新型分离技术,也将大大促进趋磁细菌的研究,而且它与浸矿工艺的结合将大大促进我国生物冶金的研究步伐。     

Reduction of vanadium(V) with Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans

Biotechnological leaching has been proposed as a suitable method for extraction of vanadium from spent catalysts and oil ash. In the biological leaching process, the vanadium(V) can be reduced to vanadium(IV), which is a less toxic and more soluble form of the vanadium. The present investigation showed that Acidithiobacillus ferrooxidans efficiently reduced vanadium(V) in the form of vanadium pentaoxide, to vanadyl(IV) ions, and tolerated high concentrations of vanadium(IV) and vanadium(V). A. ferrooxidans was compared with Acidithiobacillus thiooxidans, which has previously been utilized for vanadium leaching and reduction. Vanadium pentaoxide and sodium vanadate were used as model compounds. The results of this study indicate possibilities to develop an economical and technically feasible process for biotechnological vanadium recovery.     

Analysis of differential protein expression in Acidithiobacillus ferrooxidans grown under different energy resources respectively using SELDI-ProteinChip technologies

Surface-enhanced laser desorption/ionization (SELDI)-time of flight is an affinity-based mass spectrometric method in which proteins of interest are selectively absorbed to a chemically modified surface on a chip, which allows proteomic analysis with limited material requirements. This characteristic makes it a valuable technique for microbiologists handling problematic samples, such as low cell number cultures. In this study, we explored differential-expressed proteome of Acidithiobacillus ferrooxidans cultivated with Fe(2+) and elemental sulfur separately by adopting the protein biochip SELDI approach. The cell lysates of A. ferrooxidans were applied onto Ciphergen ProteinChip WCX2, SAX2 and IMAC-Cu arrays. Proteins bound to the chips were analyzed on a ProteinChip Reader Model PBS II. A summary of the molecular masses of the differentially regulated proteins found on WCX2, IMAC-Cu and SAX2 was obtained and 28 differentially expressed proteins were found on the molecular weight range of 5.0 to 25 kDa.     

正交法优化嗜酸氧化亚铁硫杆菌冷冻干燥保护剂

利用正交实验方法,以甘油、海藻糖、蔗糖和牛血清蛋白为因素,对嗜酸氧化亚铁硫杆菌（Acididfiobacillus ferrooxidans,A．ferrooxidans）冷冻干燥保护剂的最优化配比进行了研究。直观分析、因素指标分析和方差分析的结果表明：由甘油、海藻糖、蔗糖和牛血清蛋白组成的冷冻干燥保护剂中,对存活率影响的主次顺序依次为：甘油〉海藻糖〉牛血清蛋白〉蔗糖。保护剂的最优化组合为甘油5％、海藻糖15％、蔗糖18％、牛血清蛋白10％。经过验证,该组合的保护剂可使冷冻干燥嗜酸氧化亚铁硫杆菌的存活率达到94％。     

Genomic and phenotypic heterogeneity of Acidithiobacillus spp. strains isolated from diverse habitats in China

The genetic variability among 32 Chinese Acidithiobacillus spp. environmental isolates and four reference strains representing three recognized species of the genus Acidithiobacillus was characterized by using a combination of molecular methods, namely restriction fragment length polymorphisms of PCR-amplified 16S rRNA genes and 16S-23S rRNA gene intergenic spacers, repetitive element PCR, arbitrarily primed PCR and 16S rRNA gene sequence analyses. 16S rRNA gene sequences revealed that all Acidithiobacillus spp. strains could be assigned to seven groups, three of which encompassed the Acidithiobacillus ferrooxidans strains from various parts of the world. A comparative analysis of the phylogenetic Group 1 and 2 was undertaken. Restriction fragment length polymorphism results allowed us to separate the 35 Acidithiobacillus strains into 15 different genotypes. An integrated phenotypic and genotypic analysis indicated that the distribution of A. ferrooxidans strains among the physiological groups were in agreement with their distribution among the genomic groups, and that no clear correlation was found between the genetic polymorphism of the Acidithiobacillus spp. strains and either the geographic location or type of habitats from which the strains were isolated. In addition, five unidentified sulfur-oxidizing isolates may represent one or two novel species of the genus Acidithiobacillus. The results showed that the Chinese Acidithiobacillus spp. isolates exhibited a high degree of genomic and phenotypic heterogeneity.     

Purification and characterization of sulfide:quinone oxidoreductase from an acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans

Sulfide:quinone oxidoreductase (SQR) was purified from membrane of acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans NASF-1 cells grown on sulfur medium. It was composed of a single polypeptide with an apparent molecular mass of 47 kDa. The apparent K(m) values for sulfide and ubiquinone were 42 and 14 muM respectively. The apparent optimum pH for the SQR activity was about 7.0. A gene encoding a putative SQR of A. ferrooxidans NASF-1 was cloned and sequenced. The gene was expressed in Escherichia coli as a thioredoxin-fusion protein in inclusion bodies in an inactive form. A polyclonal antibody prepared against the recombinant protein reacted immunologically with the purified SQR. Western blotting analysis using the antibody revealed an increased level of SQR synthesis in sulfur-grown A. ferrooxidans NASF-1 cells, implying the involvement of SQR in elemental sulfur oxidation in sulfur-grown A. ferrooxidans NASF-1 cells.     

Global transcriptional analysis of stress-response strategies in Acidithiobacillus ferrooxidans ATCC 23270 exposed to organic extractant—Lix984n

Acidithiobacillus ferrooxidans (A. ferrooxidans) ATCC 23270 is a model bacteria for bioleaching research. Because of the use of extractant in metal extraction industry, A. ferrooxidans needs to cope with the water-organic two-phase system. To get insight into the molecular response of A. ferrooxidans to organic solvent, global gene expression pattern was examined in A. ferrooxidans ATCC 23270 cells subjected to Lix984n (an organic extractant) using the method of whole-genome DNA microarray. The data suggested that the global response of A. ferrooxidans to Lix984n stress was characterized by the up-regulation of genes involved in pentose phosphate pathway, fatty acid and glutamate biosynthesis. In further study, compared to heterotrophic bacteria in dealing with short-time stress, A. ferrooxidans has a special strategy of continuously enhancing the expression of genes encoding proteins involved in electron transport, such as petI, petII, cyo and cyd. Besides, acrAB-tolC operon encoding organic solvent efflux pump and its positive regulator gene ostR were addressed.