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Past and present habitability of Mars have been intensely studied in the context of the search for signals of life. Despite the harsh conditions observed today on the planet, some ancient Mars environments could have harbored specific characteristics able to mitigate several challenges for the development of microbial life. In such environments, Fe 2+ minerals like siderite (already identified on Mars), and vivianite (proposed, but not confirmed) could sustain a chemolithoautotrophic community. In this study, we investigate the ability of the acidophilic iron-oxidizing chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans to use these minerals as its sole energy source. A. ferrooxidans was grown in media containing siderite or vivianite under different conditions and compared to abiotic controls. Our experiments demonstrated that this microorganism was able to grow, obtaining its energy from the oxidation of Fe 2+ that came from the solubilization of these minerals under low pH. Additionally, in sealed flasks without CO 2 , A. ferrooxidans was able to fix carbon directly from the carbonate ion released from siderite for biomass production, indicating that it could be able to colonize subsurface environments with little or no contact with an atmosphere. These previously unexplored abilities broaden our knowledge on the variety of minerals able to sustain life. In the context of astrobiology, this expands the list of geomicrobiological processes that should be taken into account when considering the habitability of environments beyond Earth, and opens for investigation the possible biological traces left on these substrates as biosignatures., (© 2024. The Author(s).)

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that is commonly reported to exhibit diauxic population growth behavior where ferrous iron is oxidized before elemental sulfur when both are available, despite the higher energy content of sulfur. We have discovered sulfur dispersion formulations that enables sulfur oxidation before ferrous iron oxidation. The oxidation of dispersed sulfur can lower the culture pH within days below the range where aerobic ferrous iron oxidation can occur. Thus, ferric iron reduction can be observed quickly which had previously been reported over extended incubation periods with untreated sulfur. Therefore, we demonstrate that this substrate utilization pattern is strongly dependent on the cell loading in relation to sulfur concentration, sulfur surface hydrophobicity, and the pH of the culture. Our dispersed sulfur formulation, lig-sulfur, can be used to support the rapid antibiotic selection of plasmid-transformed cells, which is not possible in liquid cultures where ferrous iron is the main source of energy for these acidophiles. Furthermore, we find that media containing lig-sulfur supports higher production of green fluorescent protein compared to media containing ferrous iron. The use of dispersed sulfur is a valuable new tool for the development of engineered A. ferrooxidans strains and it provides a new method to control iron and sulfur oxidation behaviors., (© 2021 Wiley Periodicals LLC.)

The oxidative stress response represents a sum of antioxidative mechanisms that are essential for determining the adaptation and abundance of microorganisms in the environment. Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans are chemolithotrophic bacteria that obtain their energy from the oxidation of ferrous ion. Both microorganisms are important for bioleaching of sulfidic ores and both are tolerant to high levels of heavy metals and other factors that can induce oxidative stress. In this work, we compared the tolerance and response of L. ferriphilum and At. ferrooxidans to Fe 3+ , H 2 O 2 , K 2 CrO 4 , and UV-C radiation. We evaluated growth, generation of reactive oxygen species (ROS), oxidative damage to lipid membranes and DNA, and the activity of antioxidative proteins in cells exposed to these stressors. L. ferriphilum had higher cell density, lower ROS content and less lipid and DNA damage than At. ferrooxidans. Consistent with this, the activity levels of thioredoxin and superoxide dismutase in L. ferriphilum were upregulated and higher than in At. ferrooxidans. This indicated that L. ferriphilum has a higher capacity to respond to oxidative stress and to manage redox homeostasis. This capacity could largely contribute to the high abundance of this species in natural and anthropogenic sites., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

In the actual mining scenario, copper bioleaching, mainly raw mined material known as run-of-mine (ROM) copper bioleaching, is the best alternative for the treatment of marginal resources that are not currently considered part of the profitable reserves because of the cost associated with leading technologies in copper extraction. It is foreseen that bioleaching will play a complementary role in either concentration-as it does in Minera Escondida Ltd. (MEL)-or chloride main leaching plants. In that way, it will be possible to maximize mines with installed solvent-extraction and electrowinning capacities that have not been operative since the depletion of their oxide ores. One of the main obstacles for widening bioleaching technology applications is the lack of knowledge about the key events and the attributes of the technology's critical events at the industrial level and mainly in ROM copper bioleaching industrial operations. It is relevant to assess the bed environment where the bacteria-mineral interaction occurs to learn about the limiting factors determining the leaching rate. Thus, due to inability to accurately determine in-situ key variables, their indirect assessment was evaluated by quantifying microbial metabolic-associated responses. Several candidate marker genes were selected to represent the predominant components of the microbial community inhabiting the industrial heap and the metabolisms involved in microbial responses to changes in the heap environment that affect the process performance. The microbial community's predominant components were Acidithiobacillus ferrooxidans , At. thiooxidans , Leptospirillum ferriphilum , and Sulfobacillus sp. Oxygen reduction, CO 2 and N 2 fixation/uptake, iron and sulfur oxidation, and response to osmotic stress were the metabolisms selected regarding research results previously reported in the system. After that, qPCR primers for each candidate gene were designed and validated. The expression profile of the selected genes vs. environmental key variables in pure cultures, column-leaching tests, and the industrial bioleaching heap was defined. We presented the results obtained from the industrial validation of the marker genes selected for assessing CO 2 and N 2 availability, osmotic stress response, as well as ferrous iron and sulfur oxidation activity in the bioleaching heap process of MEL. We demonstrated that molecular markers are useful for assessing limiting factors like nutrients and air supply, and the impact of the quality of recycled solutions. We also learned about the attributes of variables like CO 2 , ammonium, and sulfate levels that affect the industrial ROM-scale operation.

Biosorption is a potential tool for the extraction of metals from contaminated water and recovery of precious metals, which is a convenient alternative to conventional processes. In the present study, molybdenum recovery by Acidithiobacillus ferrooxidans strain ZT-94 was evaluated. Additionally, the effects of pH initial concentration of molybdenum, contact time, adsorbent concentration, and temperature on the biosorption were investigated. As revealed by the results, the greatest amount of molybdenum sorption was achieved at pH 5. By increasing the concentration of molybdenum from 2 to 45 mg/l, the molybdenum removal increases from 71.13 to 150 mg/g dry weight of biomass, but biosorption efficiency decreased. Also, increasing the dry weight of biomass from 0.008 to 0.06 g/l degreased the biosorption efficiency from 20.68 to 85.69%. The results of molybdenum biosorption were evaluated by Langmuir and Freundlich adsorption isotherm. The maximum biosorption capacity for molybdenum extraction was 150.497 mg/g and amount which is very suitable for a biosorbent. The biosorption was examined by scanning electron microscopy-energy-dispersive X-ray spectroscopy. Because of the elevated biosorption properties of molybdenum by this biosorbent, it can be concluded that Acidithiobacillus ferrooxidans strain ZT-94 is a promising candidate for the removal and recovery of molybdenum from aqueous systems.

A LuxI/R-like quorum sensing (QS) system (AfeI/R) has been reported in the acidophilic and chemoautotrophic Acidithiobacillus spp. However, the function of AfeI/R remains unclear because of the difficulties in the genetic manipulation of these bacteria. Here, we constructed different afeI mutants of the sulfur- and iron-oxidizer A. ferrooxidans, identified the N-acyl homoserine lactones (acyl-HSLs) synthesized by AfeI, and determined the regulatory effects of AfeI/R on genes expression, extracellular polymeric substance synthesis, energy metabolism, cell growth and population density of A. ferrooxidans in different energy substrates. Acyl-HSLs-mediated distinct regulation strategies were employed to influence bacterial metabolism and cell growth of A. ferrooxidans cultivated in either sulfur or ferrous iron. Based on these findings, an energy-substrate-dependent regulation mode of AfeI/R in A. ferrooxidans was illuminated that AfeI/R could produce different types of acyl-HSLs and employ specific acyl-HSLs to regulate specific genes in response to different energy substrates. The discovery of the AfeI/R-mediated substrate-dependent regulatory mode expands our knowledge on the function of QS system in the chemoautotrophic sulfur- and ferrous iron-oxidizing bacteria, and provides new insights in understanding energy metabolism modulation, population control, bacteria-driven bioleaching process, and the coevolution between the acidophiles and their acidic habitats., (© 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The use of microorganisms in mining processes is a technology widely employed around the world. Leaching bacteria are characterized by having resistance mechanisms for several metals found in their acidic environments, some of which have been partially described in the Acidithiobacillus genus (mainly on ferrooxidans species). However, the response to copper has not been studied in the psychrotolerant Acidithiobacillus ferrivorans strains. Therefore, we propose to elucidate the response mechanisms of A. ferrivorans ACH to high copper concentrations (0-800 mM), describing its genetic repertoire and transcriptional regulation. Our results show that A. ferrivorans ACH can grow in up to 400 mM of copper. Moreover, we found the presence of several copper-related makers, belonging to cop and cus systems, as well as rusticyanins and periplasmatic acop protein in the genome. Interestingly, the ACH strain is the only one in which we find three copies of copB and copZ genes. Moreover, transcriptional expression showed an up-regulation response ( acop, copZ, cusA, rusA, and rusB ) to high copper concentrations. Finally, our results support the important role of these genes in A. ferrivorans copper stress resistance, promoting the use of the ACH strain in industrial leaching under low temperatures, which could decrease the activation times of oxidation processes and the energy costs.

This study evaluated the effect of three sulfate salt-based culture media on the reprecipitation of sulfur under the action of two types of bacterial inoculum, a pure strain of Acidithiobacillus ferrooxidans (ATCC 23270) and a consortium of this strain and Acidithiobacillus thiooxidans (ATCC 15494), in a biodesulfurization process for coal (particle size < 0.25 mm) from the 'La Guacamaya' mine (Puerto Libertador, Córdoba, Colombia). All of the experiments were periodically monitored, with measurements taken of pH, cell concentration, iron concentration, and pyrite oxidation. Additionally, mineralogical analyses were conducted on the initial and final coal samples, through scanning electron microscopy with an energy-dispersive X-ray spectrometer. The results showed that sulfate reprecipitation occurred primarily, and nearly entirely, during the first 3 days of the process. While all the treatments obtained high levels of mineral oxidation, the reprecipitation processes decreased in media with low concentrations of sulfate, leading to the higher final removal of inorganic sulfur. The bioassays revealed that after 15 days, the maximum pyrite oxidation (86%) and inorganic sulfur removal (53%) was obtained with the treatments using the Kos and McCready culture media. The bacteria evaluated were found to have a great ability to adapt to very simple culture media with minimal nutrient concentrations, and even with some nutrients absent (as in the case of magnesium).

In the present work, the effects of different energy substrates and nickel ions (Ni 2+ ) and cadmium ions (Cd 2+ ) on the growth of Acidithiobacillus ferrooxidans (A. ferrooxidans) were investigated. Ferrous sulphate (FeSO 4 ) was the optimum energy substrate for A. ferrooxidans growth, among the selected substrates. When cultured together with FeSO 4 and sulphur (S), A. ferrooxidans first oxidised the ferrous ions (Fe 2+ ), and the S was utilised as the concentration of Fe 2+ decreased. After adapting to culture with Ni 2+ and Cd 2+ , A. ferrooxidans presented good tolerance to both ions, with the maximum concentration reaching 4.11 g/L Ni 2+ and 1.69 g/L Cd 2+ . A preliminary simulation of industrial application was also performed on used Ni-Cd batteries. With bioleaching, the highest concentrations of Cd 2+ and Ni 2+ were 3003 mg/L at day 8 and 1863 mg/L at day 14, respectively.

The formation of acid mine drainage (AMD), which results from the oxidation of sulfur minerals by air and water, can be accelerated by acidophilic and chemolithotrophic bacteria such as Acidithiobacillus ferrooxidans. Our previous study revealed that walnut shell powder and its phenolic component inhibit the growth of A. ferrooxidans. However, their inhibitory effect on AMD formation in the environment needs verification. We established a bioleaching system to test whether walnut shell powder and its phenolic component can limit AMD formation. Our results showed that lignin and cellulose isolated from walnut shell decreased metal ion concentrations through absorption, whereas the phenolic component increased pH by downregulating the expression of Fe 2+ -oxidizing genes and rus operon genes of A. ferrooxidans. Only walnut shell powder showed an excellent ability to curb AMD by binding metal ions and increasing the pH value. On probing deeper into the alteration of the bacterial community structure in the bioleaching system, we found that the bacterial community became more diverse-the amount of A. ferrooxidans decreased and that of some non-acidophilic bacteria increased. The bacterial community in samples treated with walnut shell powder or its phenolic component had low abundance in the pathways of metabolism and energy production, as determined by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt). In other words, preponderant microbes, mainly A. ferrooxidans, lacked energy to grow well in the treated samples. Our findings provide a practical applicability of walnut shell powder to reduce leaching from a complex environmental community.

To explore engineering platforms towards 'active bacterial baths', we grow and characterize native and commercial strains of Acidithiobacillus ferrooxidans to promote swimming locomotion. Three different energy sources were used, namely elemental sulfur, ferrous sulfate, and pyrite. The characteristics of the culture, such as pH, Eh, and the concentration of cells and ions, are monitored to seek correlations between the oxidation route and the transport mechanism. We found that only elemental sulfur induces swimming mobility in the commercial DSMZ - 24,419 strain, while ferrous sulfate and the sulfide mineral, pyrite, did not activate swimming on any strain. The bacterial mean squared displacement and the mean velocity are measured to provide a quantitative description of the bacterial mobility. We found that, even if the A. ferrooxidans strain is grown in a sulfur-rich environment, it preferentially oxidizes iron when an iron-based material is included in the media. Similar to other species, once the culture pH decreases below 1.2, the active locomotion is inhibited. The engineering control and activation of swimming in bacterial cultures offer fertile grounds towards applications of active suspensions such as energy-efficient bioleaching, mixing, drug delivery, and bio-sensing.

Acidithiobacillus ferrooxidans is a gram-negative, autotrophic and rod-shaped bacterium. It can gain energy through the oxidation of Fe(II) and reduced inorganic sulfur compounds for bacterial growth when oxygen is sufficient. It can be used for bio-leaching and bio-oxidation and contributes to the geobiochemical circulation of metal elements and nutrients in acid mine drainage environments. The iron and sulfur oxidation pathways of A. ferrooxidans play key roles in bacterial growth and survival under extreme circumstances. Here, the electrons transported through the thermodynamically favourable pathway for the reduction to H 2 O (downhill pathway) and against the redox potential gradient reduce to NAD(P)(H) (uphill pathway) during the oxidation of Fe(II) were reviewed, mainly including the electron transport carrier, relevant operon and regulation of its expression. Similar to the electron transfer pathway, the sulfur oxidation pathway of A. ferrooxidans, related genes and operons, sulfur oxidation mechanism and sulfur oxidase system are systematically discussed.

The ability of acidophilic bacteria to grow in the presence of elevated concentrations of cationic transition metals, though varying between species, has long been recognized to be far greater than that of most neutrophiles. Conversely, their sensitivity to both inorganic and organic anions, with the notable exception of sulfate, has generally been considered to be far more pronounced. We have compared the tolerance of different species of mineral-oxidizing Acidithiobacillus and Sulfobacillus, and the heterotrophic iron-reducer Acidiphilium cryptum, to copper and chloride when grown on ferrous iron, hydrogen or glucose as electron donors at pH values between 2.0 and 3.0. While tolerance of copper varied greatly between species, these were invariably far greater at pH 2.0 than at pH 3.0, while their tolerance of chloride showed the opposite pattern. The combination of copper and chloride in liquid media appeared to be far more toxic than when these elements were present alone, which was thought to be due to the formation of copper-chloride complexes. The results of this study bring new insights into the understanding of the physiological behaviour of metal-mobilising acidophilic bacteria, and have generic significance for the prospects of bioleaching copper ores and concentrates in saline and brackish waters., (Copyright © 2018 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The present study was aimed to test an electrochemical sensing approach for the detection of an active chemolithotrophic metabolism (and therefore the presence of chemolithotrophic microorganisms) by using the corrosion of pyrite by Acidithiobacillus ferrooxidans as a model. Different electrochemical techniques were combined with adhesion studies and scanning electron microscopy (SEM). The experiments were performed in presence or absence of A. ferrooxidans and without or with ferrous iron in the culture medium (0 and 0.5 g L -1 , respectively). Electrochemical parameters were in agreement with voltammetric studies and SEM showing that it is possible to distinguish between an abiotically-induced corrosion process (AIC) and a microbiologically-induced corrosion process (MIC). The results show that our approach not only allows the detection of chemolithotrophic activity of A. ferrooxidans but also can characterize the corrosion process. This may have different kind of applications, from those related to biomining to life searching missions in other planetary bodies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The increasing production of graphene raised concerns about their releasing into sewage sludge, however, there is little information about graphene impacting on the growth of bacteria and hence their bioleaching of metal ions from sewages sludge. In this study, we reported that Acidithiobacillus sp., isolated from sewages, were used to bioleach Cu 2+ and Zn 2+ from sewages sludge in the presence of graphene. The negative effect on the growth of Acidithiobacillus sp. and dose-dependent were observed in presence of graphene, where the optical density (OD 420 ) of the culture decreased from 0.163 to 0.045, while the bioleaching efficiency of Cu 2+ (70%-16%) and Zn 2+ (80%-48%) were also reduced when the graphene dose decreased from 50 mg L -1 to 1 mg L -1 . Furthermore, scanning electron microscopy (SEM) and atomic force microscopy (AFM) confirmed that the direct contacts between graphene and cell at 1 mg L -1 graphene caused cell membrane disruption, while Acidithiobacillus sp. grew better by forming dense biofilms around the suspended graphene at a 50 mg L -1 . LIVE/DEAD staining further demonstrated that almost no live cells were detected at 1 mg L -1 graphene. The toxicity of graphene could generally be explained by depending on the concentration of graphene. The new findings provide an insight into dose dependence, which impacted on the growth of Acidithiobacillus sp. and their bioleaching of metal ion from sludge., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Acidithiobacillus ferrooxidans is a strict acidophilic chemolithoautotrophic bacterium that obtains its energy from reduced inorganic sulfur species or ferrous iron oxidation under aerobic conditions. Carbon felt electrodes were pre-colonized by A. ferrooxidansATCC 23270 T using ferrous iron or sulfur as electron donors, via the addition (or not) of a mixture of C14 acyl-homoserine lactones (C14-AHLs). Electrode coverage during pre-colonization was sparse regardless of the electron donor source, whereas activation of quorum sensing significantly enhanced it. Microbial fuel cells (MFCs) inoculated with pre-colonized electrodes (which behaved as biocathodes) were more efficient in terms of current production when iron was used as an electron donor. Biocathode coverage and current output were remarkably increased to -0.56 A m -2 by concomitantly using iron-based metabolism and C14-AHLs. Cyclic voltammetry displayed different electrochemical reactions in relation to the nature of the electron donor, underlying the implication of different electron transfer mechanisms., (© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Acidithiobacillus ferrooxidans is an important iron- and sulfur-oxidizing acidophilic chemolithoautotroph that is used extensively in metal extraction and refining, and more recently in the bioproduction of chemicals. However, a lack of genetic tools has limited the further development of this organism for industrial bioprocesses. Using prior microarray studies that identified genes, which may express differentially in response to the availability of iron and sulfur, the cycA1 and tusA promoter sequences have been characterized for their ability to drive green fluorescent protein expression. The promoters exhibited opposite control behavior, where the cycA1 sequence was repressed and the tusA promoter was induced by the presence of sulfur in the growth medium. Sulfur was found to be the dominant signal. The sulfur IC 50 for cycA1 was 0.56 mM (18 mg/L), whereas the sulfur EC 50 of tusA was 2.5 mM (80 mg/L). Together these sequences provide two new tools to selectively induce or repress gene expression in A. ferrooxidans. Acidithiobacillus ferrooxidans is an important industrial organism; however, genetic tools for control of gene expression do not exist. Here, we report the identification of promoter sequences that allow for the development of control of gene expression for engineering this organism., (© 2016 International Union of Biochemistry and Molecular Biology, Inc.)

Acidithiobacillus caldus is a chemolithoautotrophic sulfur-oxidizing bacterium that is widely used for bioleaching processes. Acidithiobacillus spp. are suggested to contain sulfur dioxygenases (SDOs) that facilitate sulfur oxidation. In this study, two putative sdo genes (A5904&#95;0421 and A5904&#95;1112) were detected in the genome of A. caldus MTH-04 by BLASTP searching with the previously identified SDO (A5904&#95;0790). We cloned and expressed these genes, and detected the SDO activity of recombinant protein A5904&#95;0421 by a GSH-dependent in vitro assay. Phylogenetic analysis indicated that A5904&#95;0421and its homologous SDOs, mainly found in autotrophic bacteria, were distantly related to known SDOs and were categorized as a new subgroup of SDOs. The potential functions of genes A5904&#95;0421 (termed sdo1) and A5904&#95;0790 (termed sdo2) were investigated by generating three knockout mutants (Δsdo1, Δsdo2 and Δsdo1&2), two sdo overexpression strains (OE-sdo1 and OE-sdo2) and two sdo complemented strains (Δsdo1/sdo1' and Δsdo2/sdo2') of A. caldus MTH-04. Deletion or overexpression of the sdo genes did not obviously affect growth of the bacteria on S0, indicating that the SDOs did not play an essential role in the oxidation of extracellular elemental sulfur in A. caldus. The deletion of sdo1 resulted in complete inhibition of growth on tetrathionate, slight inhibition of growth on thiosulfate and increased GSH-dependent sulfur oxidation activity on S0. Transcriptional analysis revealed a strong correlation between sdo1 and the tetrathionate intermediate pathway. The deletion of sdo2 promoted bacterial growth on tetrathionate and thiosulfate, and overexpression of sdo2 altered gene expression patterns of sulfide:quinone oxidoreductase and rhodanese. Taken together, the results suggest that sdo1 is essential for the survival of A. caldus when tetrathionate is used as the sole energy resource, and sdo2 may also play a role in sulfur metabolism.

Acidithiobacillus ferrivorans is an acidophile that often occurs in low temperature acid mine drainage, e.g., that located at high altitude. Being able to inhabit the extreme environment, the bacterium must possess strategies to copy with the survival stress. Nonetheless, information on the strategies is in demand. Here, genomic and transcriptomic assays were performed to illuminate the adaptation mechanisms of an A. ferrivorans strain YL15, to the alpine acid mine drainage environment in Yulong copper mine in southwest China. Genomic analysis revealed that strain has a gene repertoire for metal-resistance, e.g., genes coding for the mer operon and a variety of transporters/efflux proteins, and for low pH adaptation, such as genes for hopanoid-synthesis and the sodium:proton antiporter. Genes for various DNA repair enzymes and synthesis of UV-absorbing mycosporine-like amino acids precursor indicated hypothetical UV radiation-resistance mechanisms in strain YL15. In addition, it has two types of the acquired immune system-type III-B and type I-F CRISPR/Cas modules against invasion of foreign genetic elements. RNA-seq based analysis uncovered that strain YL15 uses a set of mechanisms to adapt to low temperature. Genes involved in protein synthesis, transmembrane transport, energy metabolism and chemotaxis showed increased levels of RNA transcripts. Furthermore, a bacterioferritin Dps gene had higher RNA transcript counts at 6°C, possibly implicated in protecting DNA against oxidative stress at low temperature. The study represents the first to comprehensively unveil the adaptation mechanisms of an acidophilic bacterium to the acid mine drainage in alpine regions.

In Acidithiobacillus ferrooxidans, one of the most important bioleaching bacterial species, the proteins encoded by the rus operon are involved in the electron transfer from Fe 2+ to O 2 . To obtain further knowledge about the mechanism(s) involved in the adaptive responses of the bacteria to growth on the different uranium ore pulp densities, we analyzed the expression of the four genes from the rus operon by real-time PCR, when Acidithiobacillus sp. FJ2 was grown in the presence of different uranium concentrations. The uranium bioleaching results showed the inhibitory effects of the metal pulp densities on the oxidation activity of the bacteria which can affect Eh, pH, Fe oxidation and uranium extractions. Gene expression analysis indicated that Acidithiobacillus sp. FJ2 tries to survive in the stress with increasing in the expression levels of cyc2, cyc1, rus and coxB, but the metal toxicity has a negative effect on the gene expression in different pulp densities. These results indicated that Acidithiobacillus sp. FJ2 could leach the uranium even in high pulp density (50%) by modulation in rus operon gene responses.

The toxicities of Cr (III) and Cr (VI) in water were evaluated using sulfur-oxidizing bacterial (SOB) bioassays both in batch and fed-batch conditions. Two days were enough for a quick buildup of SOB consortium in the master culture reactor (MCR). At concentrations up to 100 mg L(-1), Cr (III) was found to be nontoxic in both conditions, while Cr (VI) at very low concentrations (0.1-2 mg L(-1)) was very toxic to the SOB. Literature review suggested that the nontoxic nature of Cr (III) might be due to the absence of the iron uptake pathway in Acidithiobacillus caldus (the predominant bacteria in our reactors), which is required for Cr (III) uptake. The 2-h median effective concentration (EC50) values obtained for Cr (VI) in the batch and fed-batch tests were 2.7 mg L(-1) and 1.5 mg L(-1), respectively., (Copyright © 2016. Published by Elsevier Ltd.)

The genus Acidithiobacillus includes iron-oxidizing lithoautotrophs that thrive in acidic mine environments. Acidithiobacillus ferrooxidans is a representative species and has been extensively studied for its application to the bioleaching of precious metals. In our attempts to cultivate the type strain of A. ferrooxidans (ATCC 23270(T)), repeated transfers to fresh inorganic media resulted in the emergence of cultures with improved growth traits. Strains were isolated from the resultant culture by forming colonies on inorganic silica-gel plates. A representative isolate (strain NU-1) was unable to form colonies on agarose plates and was more sensitive to organics, such as glucose, than the type strain of A. ferrooxidans. Strain NU-1 exhibited superior growth traits in inorganic iron media to those of other iron-oxidizing acidithiobacilli, suggesting its potential for industrial applications. A draft genome of NU-1 uncovered unique features in catabolic enzymes, indicating that this strain is not a mutant of the A. ferrooxidans type strain. Our results indicate that the use of inorganic silica-gel plates facilitates the isolation of as-yet-unexamined iron-oxidizing acidithiobacilli from environmental samples and enrichment cultures.

We adapted a mixed culture of acidophiles to high arsenic concentrations to confirm the possibility of achieving more than 70% biooxidation of refractory gold concentrates containing high arsenic (As) concentration. The biooxidation process was applied to refractory gold concentrates containing approximately 139.67 g/kg of total As in a stirred tank reactor using an adapted mixed culture of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. The percentage of the biooxidation process was analyzed based on the total As removal efficiency. The As removal was monitored by inductively coupled plasma (ICP) analysis, conducted every 24 h. The results obtained with the adapted culture were compared with the percentage of biooxidation obtained with a non-adapted mixed culture of A. ferrooxidans and A. thiooxidans, and with their respective pure cultures. The percentages of biooxidation obtained during 358 h of reaction were 72.20%, 38.20%, 27.70%, and 11.45% for adapted culture, non-adapted culture, and pure cultures of A. thiooxidans and A. ferrooxidans, respectively. The adapted culture showed a peak maximum percentage of biooxidation of 77% at 120 h of reaction, confirming that it is possible to obtain biooxidation percentages over 70% in gold concentrates containing high As concentrations., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Acidithiobacillus ferrooxidans is an acidophile that thrives in metal-contaminated environments and tolerates high levels of uranium. To gain a better understanding of the processes involved in U(VI) resistance, comparative proteomics was used. The proteome of A. ferrooxidans was grown in the presence and absence of 0.5 mM U(VI); expression of 17 proteins was upregulated and one was downregulated. Most proteins with increased expression are part of the general stress response or are involved in reactive oxygen species detoxification. Four novel proteins showed increased expression in the presence of U(VI) and may contribute to U(VI) resistance via thiol homoeostasis and U(VI) binding., (Copyright © 2016 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

This study investigates the synergy of Rhizobium phaseoli and Acidithiobacillus ferrooxidans in the bioleaching process of copper. The results showed that additional R. phaseoli could increase leaching rate and cell number of A. ferrooxidans. When the initial cell number ratio between A. ferrooxidans and R. phaseoli was 2 : 1, A. ferrooxidans attained the highest final cell number of approximately 2 × 10(8) cells/mL and the highest copper leaching rate of 29%, which is 7% higher than that in the group with A. ferrooxidans only. R. phaseoli may use metabolized polysaccharides from A. ferrooxidans, and organic acids could chelate or precipitate harmful heavy metals to reduce their damage on A. ferrooxidans and promote its growth. Organic acids could also damage the mineral lattice to increase the leaching effect.

Application of bioleaching process for metal recovery from electronic waste has received an increasing attention in recent years. In this work, a column bioleaching of copper from waste printed circuit boards (WPCBs) by Acidithiobacillus ferrooxidans has been investigated. After column bioleaching for 28d, the copper recovery reached at 94.8% from the starting materials contained 24.8% copper. Additionally, the concentration of Fe(3+) concentration varied significantly during bioleaching, which inevitably will influence the Cu oxidation, thus bioleaching process. Thus the variation in Fe(3+) concentration should be taken into consideration in the conventional kinetic models of bioleaching process. Experimental results show that the rate of copper dissolution is controlled by external diffusion rather than internal one because of the iron hydrolysis and formation of jarosite precipitates at the surface of the material. The kinetics of column bioleaching WPCBs remains unchanged because the size and morphology of precipitates are unaffected by maintaining the pH of solution at 2.25 level. In bioleaching process, the formation of jarosite precipitate can be prevented by adding dilute sulfuric acid and maintaining an acidic condition of the leaching medium. In such way, the Fe(2)(+)-Fe(3+) cycle process can kept going and create a favorable condition for Cu bioleaching. Our experimental results show that column Cu bioleaching from WPCBs by A. ferrooxidans is promising., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In this study, a Candida digboiensis strain was isolated from a heap leaching plant in Zambia and used in double-layer agar plate to efficiently isolate and purify leaching bacteria. Unlike Acidiphilium sp., the yeast strain was tetrathionate tolerant and could metabolize a great range of organic compounds including organic acids. These properties allowed the yeast strain to enable and fasten the growth of iron and sulfur oxidizers on double-layer agar plate. The isolates were identified as Acidithiobacillus ferrooxidans FOX1, Leptospirillun ferriphilum BN, and Acidithiobacillus thiooxidans ZMB. These three leaching bacteria were inhibited by organic acids such as acetic and propionic acids; however, their activities were enhanced by Candida digboiensis NB under dissolved organic matter stress.

This paper presents a study on the relation between bacterial adhesion force and bioleaching rate of chalcopyrite, which sheds light on the influence of interfacial interaction on bioleaching behavior. In our research, Acidithiobacillus ferrooxidans (A. ferrooxidans) were adapted to grow with FeSO4 · 7H2O, element sulfur or chalcopyrite. Then, surface properties of Acidithiobacillus ferrooxidans and chalcopyrite were analyzed by contact angle, zeta potential and Fourier transform infrared spectroscopy (FTIR). Adhesion force between bacteria and chalcopyrite was measured by atomic force microscopy (AFM). Attachment and bioleaching behaviors were also monitored. The results showed that A. ferrooxidans adapted with chalcopyrite exhibited the strongest adhesion force to chalcopyrite and the highest bioleaching rate. Culture adapted with sulfur bacteria took second place and FeSO4 · 7H2O-adapted bacteria were the lowest. Bioleaching rate and bacterial attachment capacity were positively related to bacterial adhesion force, which is affected by the nature of energy source. According to this work, the attachment of bacteria to chalcopyrite surface is one of the most important aspects that influence the bioleaching process of chalcopyrite., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this study, the process of pyrite colonization and leaching by three iron-oxidizing Acidithiobacillus species was investigated by fluorescence microscopy, bacterial attachment, and leaching assays. Within the first 4-5 days, only the biofilm subpopulation was responsible for pyrite dissolution. Pyrite-grown cells, in contrast to iron-grown cells, were able to oxidize iron(II) ions or pyrite after 24 h iron starvation and incubation with 1 mM H₂O₂, indicating that these cells were adapted to the presence of enhanced levels of reactive oxygen species (ROS), which are generated on metal sulfide surfaces. Acidithiobacillus ferrivorans SS3 and Acidithiobacillus ferrooxidans R1 showed enhanced pyrite colonization and biofilm formation compared to A. ferrooxidans (T). A broad range of factors influencing the biofilm formation on pyrite were also identified, some of them were strain-specific. Cultivation at non-optimum growth temperatures or increased ionic strength led to a decreased colonization of pyrite. The presence of iron(III) ions increased pyrite colonization, especially when pyrite-grown cells were used, while the addition of 20 mM copper(II) ions resulted in reduced biofilm formation on pyrite. This observation correlated with a different extracellular polymeric substance (EPS) composition of copper-exposed cells. Interestingly, the addition of 1 mM sodium glucuronate in combination with iron(III) ions led to a 5-fold and 7-fold increased cell attachment after 1 and 8 days of incubation, respectively, in A. ferrooxidans (T). In addition, sodium glucuronate addition enhanced pyrite dissolution by 25%.

Computer printed circuit boards (CPCBs) have a rich metal content and are produced in high volume, making them an important component of electronic waste. The present study used a pure culture of Acidithiobacillus ferrooxidans to leach Cu and Ni from CPCBs waste. The adaptation phase began at 1g/l CPCBs powder with 10% inoculation and final pulp density was reached at 20g/l after about 80d. Four effective factors including initial pH, particle size, pulp density, and initial Fe(3+) concentration were optimized to achieve maximum simultaneous recovery of Cu and Ni. Their interactions were also identified using central composite design in response surface methodology. The suggested optimal conditions were initial pH 3, initial Fe(3+) 8.4g/l, pulp density 20g/l and particle size 95μm. Nearly 100% of Cu and Ni were simultaneously recovered under optimum conditions. Finally, bacterial growth characteristics versus time at optimum conditions were plotted., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The response of Acidithiobacillus ferrooxidans ATCC 23270 to copper was analyzed in sulfur-grown cells by using quantitative proteomics. Forty-seven proteins showed altered levels in cells grown in the presence of 50 mM copper sulfate. Of these proteins, 24 were up-regulated and 23 down-regulated. As seen before in ferrous iron-grown cells, there was a notorious up-regulation of RND-type Cus systems and different RND-type efflux pumps, indicating that these proteins are very important in copper resistance. Copper also triggered the down-regulation of the major outer membrane porin of A. ferrooxidans in sulfur-grown bacteria, suggesting they respond to the metal by decreasing the influx of cations into the cell. On the contrary, copper in sulfur-grown cells caused an overexpression of putative TadA and TadB proteins known to be essential for biofilm formation in bacteria. Surprisingly, sulfur-grown microorganisms showed increased levels of proteins related with energy generation (rus and petII operons) in the presence of copper. Although rus operon is overexpressed mainly in cells grown in ferrous iron, the up-regulation of rusticyanin in sulfur indicates a possible role for this protein in copper resistance as well. Finally, copper response in A. ferrooxidans appears to be influenced by the substrate being oxidized by the microorganism., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that is important in biomining and other biotechnological operations. The cells are able to oxidize inorganic iron, but the insolubility and product inhibition by Fe(3+) complicates characterization of these cultures. Here we explore the growth kinetics of A. ferrooxidans in iron-based medium in a pH range from 1.6 to 2.2. It was found that as the pH was increased from 1.6 to 2.0, the maintenance coefficient decreased while both the growth kinetics and maximum cell yield increased in the precipitate-free, low Fe(2+) concentration medium. In higher iron media a similar trend was observed at low pH, but the formation of precipitates at higher pH (2.0) hampered cell growth and lowered the specific growth rate and maximum cell yield. In order to eliminate ferric precipitates, chelating agents were introduced into the medium. Citric acid was found to be relatively non-toxic and did not appear to interfere with iron oxidation at a maximum concentration of 70 mM. Inclusion of citric acid prevented precipitation and A. ferrooxidans growth parameters resumed their trends as a function of pH. The addition of citrate also decreased the apparent substrate saturation constant (KS ) indicating a reduction in the competitive inhibition of growth by ferric ions. These results indicate that continuous cultures of A. ferrooxidans in the presence of citrate at elevated pH will enable enhanced cell yields and productivities. This will be critical as these cells are used in the development of new biotechnological applications such as electrofuel production., (© 2014 Wiley Periodicals, Inc.)

Contribution rates of factors controlling sludge dewaterability during bioleaching, such as sludge pH, microbial quantity, extracellular polymeric substances (EPS), etc., were investigated in this study. Results showed that the dewaterability of bioleached sludge was jointly enhanced by the growth of Acidithiobacillus sp., the increase of Fe(3+) concentration, the decreases of sludge pH, heterotrophic microorganism quantity change, and the decreases of EPS and bound water contents. Ridge regression analysis further revealed that the contribution rates of microbial quantity change, bound water content and slime EPS content on sludge dewaterability enhancement were 32.50%, 24.24%, and 22.37%, respectively, all of which are dominant factors. Therefore, the enhancement of sludge dewaterability was mainly controlled by microbial quantity change and the decrease of bound water and slime EPS contents during bioleaching., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Acidithiobacillus ferrooxidans showed the compensate growth and oxidation after re-feeding with sufficient ferrous materials after starvation. Compensatory phenomena were first detected in chemoautotrophic organisms. Starvation stress of Acidithiobacillus ferrooxidans was achieved via culturing in low concentrations of iron. During compensation, growth and ferrous oxidation took place faster than in controls. In addition, some genes related to ferrous oxidation (such as rus) and carbon assimilation (cbbR, csoS3) were expressed in different patterns in the low energy environments. Their expression patterns can account for this increased growth and oxidation. Other groups of genes (cspAB, feoAB, fur) were suppressed in response to starvation stress. The presence of pyrite and joint cold stress can render compensation nearly undetectable. This may be why the compensation phenomena observed under these conditions was not the same as that observed under single starvation stress conditions. Gene expression reflected a possible mechanism of tolerance to starvation in Acidithiobacillus ferrooxidans, which would allow the organism to adapt and survive in ferrous-limited environments., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A large non-coding RNA, termed α-Fur, of ~1000 nt has been detected in the extreme acidophile Acidithiobacillus ferrooxidans encoded on the antisense strand to the iron-responsive master regulator fur (ferric uptake regulator) gene. A promoter for α-fur was predicted bioinformatically and validated using gene fusion experiments. The promoter is situated within the coding region and in the same sense as proB, potentially encoding a glutamate 5-kinase. The 3' termination site of the α-fur transcript was determined by 3' rapid amplification of cDNA ends to lie 7 nt downstream of the start of transcription of fur. Thus, α-fur is antisense to the complete coding region of fur, including its predicted ribosome-binding site. The genetic context of α-fur is conserved in several members of the genus Acidithiobacillus but not in all acidophiles, indicating that it is monophyletic but not niche specific. It is hypothesized that α-Fur regulates the cellular level of Fur. This is the fourth example of an antisense RNA to fur, although it is the first in an extreme acidophile, and underscores the growing importance of cis-encoded non-coding RNAs as potential regulators involved in the microbial iron-responsive stimulon.

An oxidized lateritic ore which contained 0.8 % (by weight) copper was bioleached in pH- and temperature-controlled stirred reactors under acidic reducing conditions using pure and mixed cultures of the acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans. Sulfur was provided as the electron donor for the bacteria, and ferric iron present in goethite (the major ferric iron mineral present in the ore) acted as electron acceptor. Significantly more copper was leached by bacterially catalysed reductive dissolution of the laterite than in aerobic cultures or in sterile anoxic reactors, with up to 78 % of the copper present in the ore being extracted. This included copper that was leached from acid-labile minerals (chiefly copper silicates) and that which was associated with ferric iron minerals in the lateritic ore. In the anaerobic bioreactors, soluble iron in the leach liquors was present as iron (II) and copper as copper (I), but both metals were rapidly oxidized (to iron (III) and copper (II)) when the reactors were aerated. The number of bacteria added to the reactors had a critical role in dictating the rate and yield of copper solubilised from the ore. This work has provided further evidence that reductive bioprocessing, a recently described approach for extracting base metals from oxidized deposits, has the potential to greatly extend the range of metal ores that can be biomined.

Metal-related genes (afe&#95;0654, afe&#95;0671, afe&#95;0674, afe&#95;1143, afe&#95;1144, and afe&#95;2126) were cloned to identify whether those genes existed in Acidithiobacillus ferrooxidans strain DC (A. ferrooxidans DC). The deduced amino acid sequences of those genes were analyzed by bioinformatics. The tolerance levels of A. ferrooxidans DC to Mn(2+), Zn(2+), and Cd(2+) were determined, which were 0.52, 0.42, and 0.16 mol/L for ferrous iron-grown cells and 0.38, 0.18, and 0.08 mol/L for sulfur-grown cells, respectively. Real-time quantitative PCR was employed to analyze the transcriptional levels of the metal-related genes when ferrous iron- and sulfur-grown cells of A. ferrooxidans DC, respectively, exposed to Mn(2+), Zn(2+), and Cd(2+). The metal-related genes were up-regulated when A. ferrooxidans DC exposed to Mn(2+). When A. ferrooxidans DC exposed to Zn(2+), the metal-related genes were up-regulated in sulfur-grown cells; afe&#95;0654 and afe&#95;0674 were down-regulated, and the others were up-regulated in ferrous iron-grown cells. Afe&#95;2126 was down-regulated, and the others were up-regulated when A. ferrooxidans DC exposed to Cd(2+). According to experimental results and bioinformatics analysis, the proteins encoded by afe&#95;0654 and afe&#95;0674 may relate with Mn(2+) and Cd(2+) efflux. It needed further study whether they relate with Zn(2+) transport. Proteins encoded by afe&#95;0671, afe&#95;1143, and afe&#95;1144 may relate with the efflux of Mn(2+), Zn(2+), and Cd(2+). The protein encoded by afe&#95;2126 may relate with Mn(2+) and Zn(2+) efflux and Cd(2+) uptake.

Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile that oxidizes ferrous iron or sulfur compounds to obtain energy in the presence of various ions. To investigate the potassium ion response of A. ferrooxidans, we conducted a proteomics analysis. We identified eight proteins that were differentially expressed in the presence of high potassium concentration, including four up-regulated and four down-regulated proteins. Transcription levels of the genes encoding differential expressed proteins were subsequently analyzed by Northern blot in the presence of high potassium concentration. Among the up-regulated proteins, GDP-mannose 4,6-dehydratase, ribose 5-phosphate isomerase A and ribose-phosphate pyrophosphokinase were known to be implicated in the synthesis of glycocalyx, suggesting that the formation of glycocalyx might be involved in the A. ferrooxidans response to high potassium concentration. Thickening of the glycocalyx layer was also observed in cells cultivated under high potassium concentration via transmission electronic microscopy (TEM) analysis. Among the down-regulated proteins, ATP synthase F1 delta subunit and ATP synthase F1 beta subunit were two important components of ATP synthase. ATP synthase (P-ATPase) is directly linked to the transport of potassium into the cell, thus Acidithiobacillus ferrooxidans might just reduce the quantity of ATP synthase to offset the high potassium level in the culture medium. Therefore, the results obtained here provide some new clues to improve our understanding of the response of A. ferrooxidans to high potassium concentration., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

In contrast to iron-oxidizing Acidithiobacillus ferrooxidans, A. ferrooxidans from a stationary phase elemental sulfur-oxidizing culture exhibited a lag phase in pyrite oxidation, which is similar to its behaviour during ferrous iron oxidation. The ability of elemental sulfur-oxidizing A. ferrooxidans to immediately oxidize ferrous iron or pyrite without a lag phase was only observed in bacteria obtained from growing cultures with elemental sulfur. However, these cultures that shifted to ferrous iron oxidation showed a low rate of ferrous iron oxidation while no growth was observed. Two-dimensional gel electrophoresis was used for a quantitative proteomic analysis of the adaptation process when bacteria were switched from elemental sulfur to ferrous iron. A comparison of total cell lysates revealed 39 proteins whose increase or decrease in abundance was related to this phenotypic switching. However, only a few proteins were closely related to iron and sulfur metabolism. Reverse-transcription quantitative PCR was used to further characterize the bacterial adaptation process. The expression profiles of selected genes primarily involved in the ferrous iron oxidation indicated that phenotypic switching is a complex process that includes the activation of genes encoding a membrane protein, maturation proteins, electron transport proteins and their regulators.

The psychrotolerant acidophile Acidithiobacillus ferrivorans has been identified from cold environments and has been shown to use ferrous iron and inorganic sulfur compounds as its energy sources. A bioinformatic evaluation presented in this study suggested that Acidithiobacillus ferrivorans utilized a ferrous iron oxidation pathway similar to that of the related species Acidithiobacillus ferrooxidans. However, the inorganic sulfur oxidation pathway was less clear, since the Acidithiobacillus ferrivorans genome contained genes from both Acidithiobacillus ferrooxidans and Acidithiobacillus caldus encoding enzymes whose assigned functions are redundant. Transcriptional analysis revealed that the petA1 and petB1 genes (implicated in ferrous iron oxidation) were downregulated upon growth on the inorganic sulfur compound tetrathionate but were on average 10.5-fold upregulated in the presence of ferrous iron. In contrast, expression of cyoB1 (involved in inorganic sulfur compound oxidation) was decreased 6.6-fold upon growth on ferrous iron alone. Competition assays between ferrous iron and tetrathionate with Acidithiobacillus ferrivorans SS3 precultured on chalcopyrite mineral showed a preference for ferrous iron oxidation over tetrathionate oxidation. Also, pure and mixed cultures of psychrotolerant acidophiles were utilized for the bioleaching of metal sulfide minerals in stirred tank reactors at 5 and 25°C in order to investigate the fate of ferrous iron and inorganic sulfur compounds. Solid sulfur accumulated in bioleaching cultures growing on a chalcopyrite concentrate. Sulfur accumulation halted mineral solubilization, but sulfur was oxidized after metal release had ceased. The data indicated that ferrous iron was preferentially oxidized during growth on chalcopyrite, a finding with important implications for biomining in cold environments.

Zinc can occur in extremely high concentrations in acidic, heavy metal polluted environments inhabited by acidophilic prokaryotes. Although these organisms are able to thrive in such severely contaminated ecosystems their resistance mechanisms have not been well studied. Bioinformatic analysis of a range of acidophilic bacterial and archaeal genomes identified homologues of several known zinc homeostasis systems. These included primary and secondary transporters, such as the primary heavy metal exporter ZntA and Nramp super-family secondary importer MntH. Three acidophilic model microorganisms, the archaeon 'Ferroplasma acidarmanus', the Gram negative bacterium Acidithiobacillus caldus, and the Gram positive bacterium Acidimicrobium ferrooxidans, were selected for detailed analyses. Zinc speciation modeling of the growth media demonstrated that a large fraction of the free metal ion is complexed, potentially affecting its toxicity. Indeed, many of the putative zinc homeostasis genes were constitutively expressed and with the exception of 'F. acidarmanus' ZntA, they were not up-regulated in the presence of excess zinc. Proteomic analysis revealed that zinc played a role in oxidative stress in At. caldus and Am. ferrooxidans. Furthermore, 'F. acidarmanus' kept a constant level of intracellular zinc over all conditions tested whereas the intracellular levels increased with increasing zinc exposure in the remaining organisms.

At an initial pH of 2, while abiotic oxidation of aqueous Fe(2+) was enhanced by a flux of H2O2 at micromolar concentrations, bio-oxidation of aqueous Fe(2+) could be impeded due to oxidative stress/damage in Acidithiobacillus ferrooxidans caused by Fenton reaction-derived hydroxyl radical, particularly when the molar ratio of Fe(2+) to H2O2 was low. When pyrite cubes were intermittently exposed to fluxes of micromolar H2O2, the reduced Fe(2+)-Fe(3+) conversion rate in the solution (due to reduced microbial activity) weakened the Fe(3+)-catalyzed oxidation of cubic pyrite and added to relative importance of H2O2-driven oxidation in the corrosion of mineral surfaces for the treatments with high H2O2 doses. This had effects on reducing the build-up of a passivating coating layer on the mineral surfaces. Cell attachment to the mineral surfaces was only observed at the later stage of the experiment after the solutions became less favorable for the growth of planktonic bacteria.

While multiple theories exist regarding the effect of dissolved oxygen (DO) on the biooxidation of minerals, few studies have been performed the cellular or molecular scale (e.g., genetics) and the mechanism remains unclear. In this paper, the effects of DO concentration on the biooxidation process of refractory sulfide gold ores by Acidithiobacillus ferrooxidans were investigated in the experimental stirred tank bioreactors (STRs). The results indicated that higher biooxidation and cell growth rates were correlated with higher DO concentration. The biooxidation process was restricted at 1.2 ppm DO due to oxygen limitation. Furthermore, the effects of DO on cellular and molecular scale were studied for the first time. The results demonstrated that the oxygen uptake rate (OUR), the Fe(2+) oxidation activity and the rus gene expression of A. ferrooxidans all increased with the DO concentration, which might be responsible for the increase of the biooxidation rates with the DO concentration. This study provides insight into the potential impact of molecular-level mechanisms of DO in the biooxidation process of minerals., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Understanding how bioleaching systems respond to the availability of CO(2) is essential to developing operating conditions that select for optimum microbial performance. Therefore, the effect of inlet gas and associated dissolved CO(2) concentration on the growth, iron oxidation and CO(2) -fixation rates of pure cultures of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum was investigated in a batch stirred tank system. The minimum inlet CO(2) concentrations required to promote the growth of At. ferrooxidans and L. ferriphilum were 25 and 70 ppm, respectively, and corresponded to dissolved CO(2) concentrations of 0.71 and 1.57 µM (at 30°C and 37°C, respectively). An actively growing culture of L. ferriphilum was able to maintain growth at inlet CO(2) concentrations less than 30 ppm (0.31-0.45 µM in solution). The highest total new cell production and maximum specific growth rates from the stationary phase inocula were observed with CO(2) inlet concentrations less than that of air. In contrast, the amount of CO(2) fixed per new cell produced increased with increasing inlet CO(2) concentrations above 100 ppm. Where inlet gas CO(2) concentrations were increased above that of air the additional CO(2) was consumed by the organisms but did not lead to increased cell production or significantly increase performance in terms of iron oxidation. It is proposed that At. ferrooxidans has two CO(2) uptake mechanisms, a high affinity system operating at low available CO(2) concentrations, which is subject to substrate inhibition and a low affinity system operating at higher available CO(2) concentrations. L. ferriphilum has a single uptake system characterised by a moderate CO(2) affinity. At. ferrooxidans performed better than L. ferriphilum at lower CO(2) availabilities, and was less affected by CO(2) starvation. Finally, the results demonstrate the limitations of using CO(2) uptake or ferrous iron oxidation data as indirect measures of cell growth and performance across varying physiological conditions., (Copyright © 2012 Wiley Periodicals, Inc.)

Phototrophy and chemotrophy are two dominant modes of microbial metabolism. To date, non-phototrophic microorganisms have been excluded from the solar light-centered phototrophic metabolism. Here we report a pathway that demonstrates a role of light in non-phototrophic microbial activity. In lab simulations, visible light-excited photoelectrons from metal oxide, metal sulfide, and iron oxide stimulated the growth of chemoautotrophic and heterotrophic bacteria. The measured bacterial growth was dependent on light wavelength and intensity, and the growth pattern matched the light absorption spectra of the minerals. The photon-to-biomass conversion efficiency was in the range of 0.13-1.90‰. Similar observations were obtained in a natural soil sample containing both bacteria and semiconducting minerals. Results from this study provide evidence for a newly identified, but possibly long-existing pathway, in which the metabolisms and growth of non-phototrophic bacteria can be stimulated by solar light through photocatalysis of semiconducting minerals.

During bioleaching, Acidithiobacillus ferrooxidans is subjected to different types of stress, including heat stress, which affect bacterial growth. In this work, real time quantitative PCR was used to analyze the expression of heat shock genes, as well as genes that encode proteins related to several functional categories in A. ferrooxidans. Cells were submitted to long-term growth and heat shock, both at 40°C. The results showed that heat shock affected the expression levels of most genes investigated, whilst long-term growth at 40°C resulted in minor changes in gene expression, except for certain genes related to iron transport, which were strongly down-regulated, suggesting that the iron processing capability of A. ferrooxidans was affected by long-term growth at 40°C. A bioinformatic analysis of the genes' promoter regions indicated a putative transcriptional regulation by the σ(32) factor in 12 of the 31 genes investigated, suggesting the involvement of other regulatory mechanisms in the response of A. ferrooxidans to heat stress.

The extremely acidophilic, chemolithoautotrophic Acidithiobacillus ferrooxidans is an important bioleaching bacterium of great value in the metallurgical industry and environmental protection. In this report, a mutagenesis system based on the homing endonuclease I-SceI was developed to produce targeted, unmarked gene deletions in the strain A. ferrooxidans ATCC 23270. A targeted phosphofructokinase (PFK) gene (pfkB) mutant of A. ferrooxidans ATCC 23270 was constructed by homologous recombination and identified by PCR with specific primers as well as Southern blot analysis. This potential pfkB gene (AFE&#95;1807) was also characterized by expression in PFK-deficient Escherichia coli cells, and heteroexpression of the PFKB protein demonstrated that it had functional PFK activity, though it was significantly lower (about 800-fold) than that of phosphofructokinase-2 (PFK-B) expressed by the pfkB gene from E. coli K-12. The function of the potential PFKB protein in A. ferrooxidans was demonstrated by comparing the properties of the pfkB mutant with those of the wild type. The pfkB mutant strain displayed a relatively reduced growth capacity in S(0) medium (0.5% [wt/vol] elemental sulfur in 9K basal salts solution adjusted to pH 3.0 with H(2)SO(4)), but the mutation did not completely prevent A. ferrooxidans from assimilating exogenous glucose. The transcriptional analysis of some related genes in central carbohydrate metabolism in the wild-type and mutant strains with or without supplementation of glucose was carried out by quantitative reverse transcription-PCR. This report suggests that the markerless mutagenesis strategy could serve as a model for functional studies of other genes of interest from A. ferrooxidans and multiple mutations could be made in a single A. ferrooxidans strain.

Background: Acidithiobacillus caldus (A. caldus) is widely used in bio-leaching. It gains energy and electrons from oxidation of elemental sulfur and reduced inorganic sulfur compounds (RISCs) for carbon dioxide fixation and growth. Genomic analyses suggest that its sulfur oxidation system involves a truncated sulfur oxidation (Sox) system (omitting SoxCD), non-Sox sulfur oxidation system similar to the sulfur oxidation in A. ferrooxidans, and sulfur oxygenase reductase (SOR). The complexity of the sulfur oxidation system of A. caldus generates a big obstacle on the research of its sulfur oxidation mechanism. However, the development of genetic manipulation method for A. caldus in recent years provides powerful tools for constructing genetic mutants to study the sulfur oxidation system., Results: An A. caldus mutant lacking the sulfur oxygenase reductase gene (sor) was created and its growth abilities were measured in media using elemental sulfur (S(0)) and tetrathionate (K(2)S(4)O(6)) as the substrates, respectively. Then, comparative transcriptome analysis (microarrays and real-time quantitative PCR) of the wild type and the Δsor mutant in S(0) and K(2)S(4)O(6) media were employed to detect the differentially expressed genes involved in sulfur oxidation. SOR was concluded to oxidize the cytoplasmic elemental sulfur, but could not couple the sulfur oxidation with the electron transfer chain or substrate-level phosphorylation. Other elemental sulfur oxidation pathways including sulfur diooxygenase (SDO) and heterodisulfide reductase (HDR), the truncated Sox pathway, and the S(4)I pathway for hydrolysis of tetrathionate and oxidation of thiosulfate in A. caldus are proposed according to expression patterns of sulfur oxidation genes and growth abilities of the wild type and the mutant in different substrates media., Conclusion: An integrated sulfur oxidation model with various sulfur oxidation pathways of A. caldus is proposed and the features of this model are summarized.

Although the synergetic interactions between chemolithoautotroph Acidithiobacillus ferrooxidans and heterotroph Acidiphilium acidophilum have drawn a share of attention, the influence of Aph. acidophilum on growth and metabolic functions of At. ferrooxidans is still unknown on transcriptional level. To assess this influence, a co-culture composed by At. ferrooxidans and Aph. acidophilum was successfully acclimated in this study. Depending on the growth dynamics, At. ferrooxidans in co-culture had 2 days longer exponential phase and 5 times more cell number than that in pure culture. The ferrous iron concentration in culture medium and the expression of iron oxidation-related genes revealed that the energy acquisition of At. ferrooxidans in co-culture was more efficient than that in pure culture. Besides, the analysis of CO2 fixation-related genes in At. ferrooxidans indicated that the second copy of RuBisCO-encoding genes cbbLS-2 and the positive regulator-encoding gene cbbR were up-regulated in co-culture system. All of these results verified that Aph. acidophilum could heterotrophically grow with At. ferrooxidans and promote the growth of it. By means of activating iron oxidation-related genes and the second set of cbbLS genes in At. ferrooxidans, the Aph. acidophilum facilitated the iron oxidation and CO2 fixation by At. ferrooxidans.

There is great interest in understanding how extremophilic biomining bacteria adapt to exceptionally high copper concentrations in their environment. Acidithiobacillus ferrooxidans ATCC 53993 genome possesses the same copper resistance determinants as strain ATCC 23270. However, the former strain contains in its genome a 160-kb genomic island (GI), which is absent in ATCC 23270. This GI contains, amongst other genes, several genes coding for an additional putative copper ATPase and a Cus system. A. ferrooxidans ATCC 53993 showed a much higher resistance to CuSO(4) (>100 mM) than that of strain ATCC 23270 (<25 mM). When a similar number of bacteria from each strain were mixed and allowed to grow in the absence of copper, their respective final numbers remained approximately equal. However, in the presence of copper, there was a clear overgrowth of strain ATCC 53993 compared to ATCC 23270. This behavior is most likely explained by the presence of the additional copper-resistance genes in the GI of strain ATCC 53993. As determined by qRT-PCR, it was demonstrated that these genes are upregulated when A. ferrooxidans ATCC 53993 is grown in the presence of copper and were shown to be functional when expressed in copper-sensitive Escherichia coli mutants. Thus, the reason for resistance to copper of two strains of the same acidophilic microorganism could be determined by slight differences in their genomes, which may not only lead to changes in their capacities to adapt to their environment, but may also help to select the more fit microorganisms for industrial biomining operations., (© Springer-Verlag 2011)