Reduced Inorganic Sulfur Compounds Research Articles

Characterize the Growth and Metabolism of Acidithiobacillus ferrooxidans under Electroautotrophic and Chemoautotrophic Conditions.

Acidophiles are capable of surviving in extreme environments with low pH. Acidithiobacillus ferrooxidans is a typical acidophilic bacterium that has been extensively studied when grown chemoautotrophically, i.e., when it derives energy from oxidation of Fe2+ or reduced inorganic sulfur compounds (RISCs). Although it is also known to grow with electrons supplied by solid electrodes serving as the sole source of energy, the understanding of its electroautotrophic growth is still limited. This study aimed to compare the growth characteristics of A. ferrooxidans under electroautotrophic (ea) and chemoautotrophic (ca) conditions, with an attempt to elucidate the possible mechanism(s) of extracellular electron flow into the cells. Jarosite was identified by Raman spectroscopy, and it accumulated when A. ferrooxidans used Fe2+ as the electron donor, but negligible mineral deposition occurred during electroautotrophic growth. Scanning electron microscopy (SEM) showed that A. ferrooxidans possesses more pili and extracellular polymeric substances (EPSs) under electroautotrophic conditions. A total of 493 differentially expressed genes (DEGs) were identified, with 297 genes being down-regulated and 196 genes being up-regulated in ea versus ca conditions. The genes known to be essential for chemoautotrophic growth showed a decreased expression in the electroautotrophic condition; meanwhile, there was an increased expression of genes related to direct electron transfer across the cell's outer/inner membranes and transmembrane proteins such as pilin and porin. Joint analysis of DEGs and differentially expressed metabolites (DEMs) showed that galactose metabolism is enhanced during electroautotrophic growth, inducing A. ferrooxidans to produce more EPSs, which aids the cells in adhering to the solid electrode during their growth. These results suggested that electroautotrophy and chemoautotrophy of A. ferrooxidans have different extracellular electron uptake (EEU) pathways, and a model of EEU during electroautotrophic growth is proposed. The use of extracellular electrons as the sole energy source triggers A. ferrooxidans to adopt metabolic and subsequently phenotypic modifications.

Genome sequencing and metabolic network reconstruction of a novel sulfur-oxidizing bacterium Acidithiobacillus Ameehan.

Sulfur-oxidizing bacteria play a crucial role in various processes, including mine bioleaching, biodesulfurization, and treatment of sulfur-containing wastewater. Nevertheless, the pathway involved in sulfur oxidation is highly intricate, making it complete comprehension a formidable and protracted undertaking. The mechanisms of sulfur oxidation within the Acidithiobacillus genus, along with the process of energy production, remain areas that necessitate further research and elucidation. In this study, a novel strain of sulfur-oxidizing bacterium, Acidithiobacillus Ameehan, was isolated. Several physiological characteristics of the strain Ameehan were verified and its complete genome sequence was presented in the study. Besides, the first genome-scale metabolic network model (AMEE_WP1377) was reconstructed for Acidithiobacillus Ameehan to gain a comprehensive understanding of the metabolic capacity of the strain.The characteristics of Acidithiobacillus Ameehan included morphological size and an optimal growth temperature range of 37-45°C, as well as an optimal growth pH range of pH 2.0-8.0. The microbe was found to be capable of growth when sulfur and K2O6S4 were supplied as the energy source and electron donor for CO2 fixation. Conversely, it could not utilize Na2S2O3, FeS2, and FeSO4·7H2O as the energy source or electron donor for CO2 fixation, nor could it grow using glucose or yeast extract as a carbon source. Genome annotation revealed that the strain Ameehan possessed a series of sulfur oxidizing genes that enabled it to oxidize elemental sulfur or various reduced inorganic sulfur compounds (RISCs). In addition, the bacterium also possessed carbon fixing genes involved in the incomplete Calvin-Benson-Bassham (CBB) cycle. However, the bacterium lacked the ability to oxidize iron and fix nitrogen. By implementing a constraint-based flux analysis to predict cellular growth in the presence of 71 carbon sources, 88.7% agreement with experimental Biolog data was observed. Five sulfur oxidation pathways were discovered through model simulations. The optimal sulfur oxidation pathway had the highest ATP production rate of 14.81 mmol/gDW/h, NADH/NADPH production rate of 5.76 mmol/gDW/h, consumed 1.575 mmol/gDW/h of CO2, and 1.5 mmol/gDW/h of sulfur. Our findings provide a comprehensive outlook on the most effective cellular metabolic pathways implicated in sulfur oxidation within Acidithiobacillus Ameehan. It suggests that the OMP (outer-membrane proteins) and SQR enzymes (sulfide: quinone oxidoreductase) have a significant impact on the energy production efficiency of sulfur oxidation, which could have potential biotechnological applications.

From Genes to Bioleaching: Unraveling Sulfur Metabolism in Acidithiobacillus Genus.

Sulfur oxidation stands as a pivotal process within the Earth's sulfur cycle, in which Acidithiobacillus species emerge as skillful sulfur-oxidizing bacteria. They are able to efficiently oxidize several reduced inorganic sulfur compounds (RISCs) under extreme conditions for their autotrophic growth. This unique characteristic has made these bacteria a useful tool in bioleaching and biological desulfurization applications. Extensive research has unraveled diverse sulfur metabolism pathways and their corresponding regulatory systems. The metabolic arsenal of the Acidithiobacillus genus includes oxidative enzymes such as: (i) elemental sulfur oxidation enzymes, like sulfur dioxygenase (SDO), sulfur oxygenase reductase (SOR), and heterodisulfide reductase (HDR-like system); (ii) enzymes involved in thiosulfate oxidation pathways, including the sulfur oxidation (Sox) system, tetrathionate hydrolase (TetH), and thiosulfate quinone oxidoreductase (TQO); (iii) sulfide oxidation enzymes, like sulfide:quinone oxidoreductase (SQR); and (iv) sulfite oxidation pathways, such as sulfite oxidase (SOX). This review summarizes the current state of the art of sulfur metabolic processes in Acidithiobacillus species, which are key players of industrial biomining processes. Furthermore, this manuscript highlights the existing challenges and barriers to further exploring the sulfur metabolism of this peculiar extremophilic genus.

Identification of Sulfate Sources and Biogeochemical Processes in an Aquifer Affected by Peatland: Insights from Monitoring the Isotopic Composition of Groundwater Sulfate in Kampinos National Park, Poland

Temporal and spatial variations of the concentration and the isotopic composition of groundwater sulfate in an unconfined sandy aquifer covered by peatland have been studied to better understand the sources and biogeochemical processes that affect sulfate distribution in shallow groundwater systems influenced by organic rich sediments. The groundwater monitoring was carried out for one year at hydrogeological station Pożary located within the protected zone of the Kampinos National Park. Sulfur (δ34SSO4) and oxygen (δ18OSO4) isotopic composition of dissolved sulfates were analyzed together with oxygen (δ18OH2O) and hydrogen (δ2HH2O) isotopic composition of water and major ions concentration at monthly intervals. The research revealed three main sources of sulfates dissolved in groundwater, namely, (a) atmospheric sulfates—supplied to the aquifer by atmospheric deposition (rain and snow melt), (b) sulfates formed by dissolution of evaporite sulfate minerals, mainly gypsum—considerably enriched in 34S and 18O, and (c) sulfate formed during oxidation of reduced inorganic sulfur compounds (RIS), mainly pyrite—depleted in 34S and 18O. The final isotopic composition and concentration of dissolved SO42− in groundwater are the result of overlapping processes of dissimilatory sulfate reduction, oxidation of sulfide minerals, and mixing of water in aquifer profile.

Insight Into Interactions of Thermoacidophilic Archaea With Elemental Sulfur: Biofilm Dynamics and EPS Analysis.

Biooxidation of reduced inorganic sulfur compounds (RISCs) by thermoacidophiles is of particular interest for the biomining industry and for environmental issues, e.g., formation of acid mine drainage (AMD). Up to now, interfacial interactions of acidophiles with elemental sulfur as well as the mechanisms of sulfur oxidation by acidophiles, especially thermoacidophiles, are not yet fully clear. This work focused on how a crenarchaeal isolate Acidianus sp. DSM 29099 interacts with elemental sulfur. Analysis by Confocal laser scanning microscopy (CLSM) and Atomic force microscopy (AFM) in combination with Epifluorescence microscopy (EFM) shows that biofilms on elemental sulfur are characterized by single colonies and a monolayer in first stage and later on 3-D structures with a diameter of up to 100 μm. The analysis of extracellular polymeric substances (EPS) by a non-destructive lectin approach (fluorescence lectin-barcoding analysis) using several fluorochromes shows that intial attachment was featured by footprints rich in biofilm cells that were embedded in an EPS matrix consisting of various glycoconjugates. Wet chemistry data indicate that carbohydrates, proteins, lipids and uronic acids are the main components. Attenuated reflectance (ATR)-Fourier transformation infrared spectroscopy (FTIR) and high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) indicate glucose and mannose as the main monosaccharides in EPS polysaccharides. EPS composition as well as sugar types in EPS vary according to substrate (sulfur or tetrathionate) and lifestyle (biofilms and planktonic cells). This study provides information on the building blocks/make up as well as dynamics of biofilms of thermoacidophilic archaea in extremely acidic environments.

Sulfur Oxidation in the Acidophilic Autotrophic Acidithiobacillus spp.

Sulfur oxidation is an essential component of the earth’s sulfur cycle. Acidithiobacillus spp. can oxidize various reduced inorganic sulfur compounds (RISCs) with high efficiency to obtain electrons for their autotrophic growth. Strains in this genus have been widely applied in bioleaching and biological desulfurization. Diverse sulfur-metabolic pathways and corresponding regulatory systems have been discovered in these acidophilic sulfur-oxidizing bacteria. The sulfur-metabolic enzymes in Acidithiobacillus spp. can be categorized as elemental sulfur oxidation enzymes (sulfur dioxygenase, sulfur oxygenase reductase, and Hdr-like complex), enzymes in thiosulfate oxidation pathways (tetrathionate intermediate thiosulfate oxidation (S4I) pathway, the sulfur oxidizing enzyme (Sox) system and thiosulfate dehydrogenase), sulfide oxidation enzymes (sulfide:quinone oxidoreductase) and sulfite oxidation pathways/enzymes. The two-component systems (TCSs) are the typical regulation elements for periplasmic thiosulfate metabolism in these autotrophic sulfur-oxidizing bacteria. Examples are RsrS/RsrR responsible for S4I pathway regulation and TspS/TspR for Sox system regulation. The proposal of sulfur metabolic and regulatory models provide new insights and overall understanding of the sulfur-metabolic processes in Acidithiobacillus spp. The future research directions and existing barriers in the bacterial sulfur metabolism are also emphasized here and the breakthroughs in these areas will accelerate the research on the sulfur oxidation in Acidithiobacillus spp. and other sulfur oxidizers.

Effect of elevated pressure on ferric iron reduction coupled to sulfur oxidation by biomining microorganisms

Acidophiles, including a mixed mesophilic, iron-oxidizing culture (FIGB) and a thermophilic enrichment culture (TK65) from a black shale and sandstone copper ore (Kupferschiefer), were used to evaluate microbial ferric iron reduction coupled to oxidation of reduced inorganic sulfur compounds (RISCs) at elevated pressure to simulate potential biogeochemical processes in deep in situ leaching operations. Experiments were performed from atmospheric pressure (0.1 MPa) up to 36 MPa in different incubation chambers and high-pressure reactors. Microbial abundance and activity were determined by quantifying iron and sulfur species concentrations, by direct cell counting, terminal restriction fragment length polymorphism (T-RFLP) analysis and quantitative PCR (qPCR). The data indicate that the tested acidophilic cultures were able to reduce soluble ferric iron coupled to oxidation of sulfur compounds under anaerobic conditions. Even at high pressure (up to 36 MPa) these acidophiles were capable of growing, and microbial ferric iron reduction was only partially inhibited. These results show that the tested acidophiles are barotolerant and their activities may contribute to sulfur and iron cycling in anaerobic environments including deep ore deposits, which is relevant for in situ leaching operations.

Reduction of Iron(III) Ions at Elevated Pressure by Acidophilic Microorganisms

A composed mixed acidophilic, iron-oxidizing culture (FIGB) and a thermo-acidophilic enrichment culture (TK65) were used to evaluate microbial iron(III) reduction coupled to oxidation of reduced inorganic sulfur compounds (RISCs) under high pressure. Experiments were done in batch culture in high pressure vessels at 1 and 100 bar. Microbial abundance and activity were determined by measuring iron(II) concentration, direct cell counting, T-RFLP and quantitative real-time PCR. The data indicate that both cultures are able to reduce soluble iron(III) by oxidation of sulfur compounds under anaerobic conditions. At high pressure (100 bar) these acidophiles were capable of growing and microbial ferric iron reduction was only partially inhibited. These results indicate that acidophiles can be barotolerant and their activities are contributing to sulfur and iron cycling in anaerobic environments including deep ore deposits which is highly relevant for in situ leaching operations.

Potential use of sulfite as a supplemental electron donor for wastewater denitrification

Biological denitrification typically requires the addition of a supplemental electron donor, which can add a significant operating expense to wastewater treatment facilities. Most common electron donors are organic, but reduced inorganic sulfur compounds (RISCs), such as sulfide (HS−) and elemental sulfur (S0), may be more cost-effective. S0 is an inexpensive and well characterized electron donor, but it provides slow denitrification rates due to its low solubility. A lesser-known RISC is sulfite ( $${\text{SO}}_{3}^{2 - }$$ ), which can be easily produced from S0 by a simple combustion process. Unlike S0, $${\text{SO}}_{3}^{2 - }$$ is highly soluble, and therefore may provide higher denitrification rates. However, very little is known about microbial denitrification with $${\text{SO}}_{3}^{2 - }$$ . Also, $${\text{SO}}_{3}^{2 - }$$ is a strong reductant that reacts abiotically with oxygen and has toxic effects on microorganisms. This paper reviews $${\text{SO}}_{3}^{2 - }$$ in the environment, $${\text{SO}}_{3}^{2 - }$$ chemistry, microbiology, toxicity, and its potential use for denitrification. Since $${\text{SO}}_{3}^{2 - }$$ is an intermediate in the sulfur oxidation pathway of most sulfur-oxidizing microorganisms, it is an energetic electron donor and it should select for a $${\text{SO}}_{3}^{2 - }$$ -oxidizing community. Our review of the literature, as well as our own lab experience, suggests that $${\text{SO}}_{3}^{2 - }$$ can effectively serve as an electron donor for denitrification. Further research is needed to determine the kinetics of $${\text{SO}}_{3}^{2 - }$$ -based denitrification, its toxic threshold for sulfur-oxidizing microorganisms, and its potential inhibition of sensitive species such as nitrifying microorganisms and potential formation of nitrous oxide. Its effect on sludge settling efficiency also should be explored.

Long-term stability of bioelectricity generation coupled with tetrathionate disproportionation

To prevent uncontrolled acidification of the environment, reduced inorganic sulfur compounds (RISCs) can be bioelectrochemically removed from water streams. The long-term stability of bioelectricity production from tetrathionate (S4O62−) was studied in highly acidic conditions (pH<2.5) in two-chamber fed-batch microbial fuel cells (MFCs). The maximum current density was improved from previously reported 80mAm−2 to 225mAm−2 by optimizing the external resistance. The observed reaction products of tetrathionate disproportionation were sulfate and elemental sulfur. In long-term run, stable electricity production was obtained for over 700days with the average current density of 150mAm−2. The internal resistance of the MFC decreased over time and no biofouling was observed. This study shows that tetrathionate is an efficient substrate also for long-term bioelectricity production.

Life in heaps: a review of microbial responses to variable acidity in sulfide mineral bioleaching heaps for metal extraction

Industrial heap leaching of low grade mineral sulfide ores is catalysed by the use of acidophilic microorganisms. These microorganisms obtain energy for growth from the oxidation of reduced inorganic or organic compounds, including soluble ferrous ion, reduced inorganic sulfur compounds (RISC) and acid-stable organic compounds. By-products of these oxidative processes, such as soluble ferric ion and sulfuric acid create favourable chemical conditions for leaching. This review is focused on the behaviour of common bioleaching microorganisms, their responses to changing pH in an industrial setting, and how both changes and microbial responses can impact the micro and macro environment.

Organic Carbon Utilisation by Iron(II)-Oxidising Bacteria &lt;i&gt;Sulfobacillus&lt;/i&gt; &lt;i&gt;thermosulfidooxidans&lt;/i&gt; and &lt;i&gt;Alicyclobacillus&lt;/i&gt; Strain FP1 that Inhabit Copper Sulfide Leaching Heaps

Strains ofSulfobacillus (S.)andAlicyclobacillusspecies have been identified/detected in managed bioleaching heaps and agitated tanks using culture-dependent and culture-independent methods.S thermosulfidooxidansoxidises both iron(II) and reduced inorganic sulfur compounds (RISC). Several strains were isolated from a copper sulfide heap in North Western Australia.Alicyclobacillusstrain FP1 (FP1) was also isolated from the heap and oxidises iron(II) but not RISC. However, the species exhibit differentiating characteristics during growth on D-glucose, which has been explored using a suite of monitoring and measurement techniques. As examples, a growth factor (yeast extract) is essential for FP1 but not forS. thermosulfidooxidans, although yeast extract is beneficial to the latter. FP1 grows well on glucose, compared with the poor-to-no growth ofS. thermosulfidooxidanson the substrate. Solution pH strongly influences the activity of both species when grown on organic substrates, suggesting a pivotal role for solution acidity in the growth and activity of heterotrophs or mixotrophs in heap leach systems. This research forms part of an ongoing development of a data base with which to interpret the impacts of leaching conditions in heaps on microbial activity without having to disrupt metal production by invasive sampling campaigns. The insights gained will assist in understanding the effects that changing conditions in heaps due to acid consumption and/or increased element concentrations in process water may have on microbial activity.

Study and assessment of microbial communities in natural and commercial bioleaching systems

The exceptional diversity of ecogeographical conditions of Armenia and the richness of non-ferrous metals represent a great and valuable potential for the investigation of the biodiversity of acidophilic chemolithotrophic bacteria as well as for the isolation of new highly efficient strains and their communities. The studies of physiological and biotechnological properties of these relevant strains from the bioleaching communities will allow us to achieve comprehensive insight of interspecies relationships as a substantial prerequisite for the enhancement of the efficiency of biohydrometallurgical processes.In this study, novel strains of acidophilic chemolithotrophic bacteria belonging to genera Acidithiobacillus as well as Leptospirillum and Sulfobacillus have been isolated from sulfide ores of different geoformations in Armenia. The results showed that: (1) high efficiency of the isolated bacteria in bioleaching of pyrite was determined by their biochemical properties, especially enzyme activities involved in oxidation of ferrous iron and reduced inorganic sulfur compounds (RISCs). (2) Bioleaching of pyrite was directly connected with the ability of isolated strains to adhere on pyrite surfaces. Moreover, the efficiency of bioleaching of pyrite was correlated with the amount of adhered/attached bacterial cells of the isolated strains.

Construction and characterization of tetH overexpression and knockout strains of Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is a major participant in consortia of microorganisms used for bioleaching. It can obtain energy from the oxidation of Fe(2+), H2, S(0), and various reduced inorganic sulfur compounds (RISCs). Tetrathionate is a key intermediate during RISC oxidation, hydrolyzed by tetrathionate hydrolase (TetH), and used as sole energy source. In this study, a tetH knockout (ΔtetH) mutant and a tetH overexpression strain were constructed and characterized. The tetH overexpression strain grew better on sulfur and tetrathionate and possessed a higher rate of tetrathionate utilization and TetH activity than the wild type. However, its cell yields on tetrathionate were much lower than those on sulfur. The ΔtetH mutant could not grow on tetrathionate but could proliferate on sulfur with a lower cell yield than the wild type's, which indicated that tetrathionate hydrolysis is mediated only by TetH, encoded by tetH. The ΔtetH mutant could survive in ferrous medium with an Fe(2+) oxidation rate similar to that of the wild type. For the tetH overexpression strain, the rate was relatively higher than that of the wild type. The reverse transcription-quantitative PCR (qRT-PCR) results showed that tetH and doxD2 acted synergistically, and doxD2 was considered important in thiosulfate metabolism. Of the two sqr genes, AFE_0267 seemed to play as important a role in sulfide oxidation as AFE_1792. This study not only provides a substantial basis for studying the function of the tetH gene but also may serve as a model to clarify other candidate genes involved in sulfur oxidation in this organism.

Draft Genome Sequence of the Extremophile Acidithiobacillus thiooxidans A01, Isolated from the Wastewater of a Coal Dump

The draft genome of Acidithiobacillus thiooxidans A01 contains 3,820,158 bp, with a G+C content of 53.08% and 3,660 predicted coding sequences (CDSs). The bacterium contains a series of specific genes involved in the oxidation of elemental sulfur and reduced inorganic sulfur compounds (RISCs).

Whole-genome sequencing reveals novel insights into sulfur oxidation in the extremophile Acidithiobacillus thiooxidans.

BackgroundAcidithiobacillus thiooxidans (A. thiooxidans), a chemolithoautotrophic extremophile, is widely used in the industrial recovery of copper (bioleaching or biomining). The organism grows and survives by autotrophically utilizing energy derived from the oxidation of elemental sulfur and reduced inorganic sulfur compounds (RISCs). However, the lack of genetic manipulation systems has restricted our exploration of its physiology. With the development of high-throughput sequencing technology, the whole genome sequence analysis of A. thiooxidans has allowed preliminary models to be built for genes/enzymes involved in key energy pathways like sulfur oxidation.ResultsThe genome of A. thiooxidans A01 was sequenced and annotated. It contains key sulfur oxidation enzymes involved in the oxidation of elemental sulfur and RISCs, such as sulfur dioxygenase (SDO), sulfide quinone reductase (SQR), thiosulfate:quinone oxidoreductase (TQO), tetrathionate hydrolase (TetH), sulfur oxidizing protein (Sox) system and their associated electron transport components. Also, the sulfur oxygenase reductase (SOR) gene was detected in the draft genome sequence of A. thiooxidans A01, and multiple sequence alignment was performed to explore the function of groups of related protein sequences. In addition, another putative pathway was found in the cytoplasm of A. thiooxidans, which catalyzes sulfite to sulfate as the final product by phosphoadenosine phosphosulfate (PAPS) reductase and adenylylsulfate (APS) kinase. This differs from its closest relative Acidithiobacillus caldus, which is performed by sulfate adenylyltransferase (SAT). Furthermore, real-time quantitative PCR analysis showed that most of sulfur oxidation genes were more strongly expressed in the S0 medium than that in the Na2S2O3 medium at the mid-log phase.ConclusionSulfur oxidation model of A. thiooxidans A01 has been constructed based on previous studies from other sulfur oxidizing strains and its genome sequence analyses, providing insights into our understanding of its physiology and further analysis of potential functions of key sulfur oxidation genes.

Anaerobic Growth of &lt;i&gt;Acidithiobacillus ferrooxidans&lt;/i&gt; on Pyrite

In the bioleaching of mineral sulphides under the catalytic action ofAt. ferrooxidans,ferrous ion oxidation and sulfides/sulfur solubilization uses oxygen as the final electron acceptor. Also, under anaerobic conditions,At. ferrooxidanscan alternatively catalize the oxidation of sulfur or reduced inorganic sulfur compounds (RISC) using ferric iron as electron acceptor [1]. The formation of Fe (II) from pyrite and covellite in the ferric anaerobic bioleaching withA. ferrooxidans,has been studied and well documented [2,3]. The requirements of ferric iron as electron acceptor for the anaerobic growth ofAt. ferrooxidanson elemental sulfur has been demonstrated and a linear relationship was obtained between the concentration of ferrous iron accumulated in the cultures and the increase in cell density [4]. It has been suggested a possible role in the solubilization of metals from sulfide ores involving the participation of the enzyme sulfur (sulfide): Fe (III) oxidoreductase [5]. Bacterial growth ofAt. ferrooxidanshas also been reported in the oxidative anaerobic respiration using hydrogen as electron donor and ferric iron as electron acceptor [6]. Anaerobic reduction of ferric iron and ferrous iron production from pyrite byAt. ferrooxidanshas been demonstrated [2], however there are no reports about bacterial growth using this mineral. In this work, we studied the anaerobic bioleaching of pyrite with the aim to determine ifAt. ferrooxidansis capable to anaerobic growth on pyrite using ferric iron as electron acceptor.

Stoichiometric modeling of oxidation of reduced inorganic sulfur compounds (Riscs) in Acidithiobacillus thiooxidans

The prokaryotic oxidation of reduced inorganic sulfur compounds (RISCs) is a topic of utmost importance from a biogeochemical and industrial perspective. Despite sulfur oxidizing bacterial activity is largely known, no quantitative approaches to biological RISCs oxidation have been made, gathering all the complex abiotic and enzymatic stoichiometry involved. Even though in the case of neutrophilic bacteria such as Paracoccus and Beggiatoa species the RISCs oxidation systems are well described, there is a lack of knowledge for acidophilic microorganisms. Here, we present the first experimentally validated stoichiometric model able to assess RISCs oxidation quantitatively in Acidithiobacillus thiooxidans (strain DSM 17318), the archetype of the sulfur oxidizing acidophilic chemolithoautotrophs. This model was built based on literature and genomic analysis, considering a widespread mix of formerly proposed RISCs oxidation models combined and evaluated experimentally. Thiosulfate partial oxidation by the Sox system (SoxABXYZ) was placed as central step of sulfur oxidation model, along with abiotic reactions. This model was coupled with a detailed stoichiometry of biomass production, providing accurate bacterial growth predictions. In silico deletion/inactivation highlights the role of sulfur dioxygenase as the main catalyzer and a moderate function of tetrathionate hydrolase in elemental sulfur catabolism, demonstrating that this model constitutes an advanced instrument for the optimization of At. thiooxidans biomass production with potential use in biohydrometallurgical and environmental applications.

Acidithiobacillus caldus sulfur oxidation model based on transcriptome analysis between the wild type and sulfur oxygenase reductase defective mutant.

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.ResultsAn A. caldus mutant lacking the sulfur oxygenase reductase gene (sor) was created and its growth abilities were measured in media using elemental sulfur (S0) and tetrathionate (K2S4O6) as the substrates, respectively. Then, comparative transcriptome analysis (microarrays and real-time quantitative PCR) of the wild type and the Δsor mutant in S0 and K2S4O6 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 S4I 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.ConclusionAn integrated sulfur oxidation model with various sulfur oxidation pathways of A. caldus is proposed and the features of this model are summarized.

Acidithiobacillus ferrooxidans oxidizes ferrous iron before sulfur likely through transcriptional regulation by the global redox responding RegBA signal transducing system

The mechanisms of ferrous iron (Fe(II)) and reduced inorganic sulfur compounds (RISC) oxidation under oxic conditions of the bioleaching bacterium Acidithiobacillus ferrooxidans have been deciphered. The genes involved in these pathways are more transcribed in the presence than in the absence of the corresponding electron donor, i.e. the genes involved in Fe(II) oxidation are expressed preferentially in the presence of Fe(II) than sulfur (S0) and those involved in RISC oxidation are more transcribed in S0- than in Fe(II)-grown cells. In this study, oxidation of Fe(II) and S0 and expression of the main genes involved in these pathways have been followed when both electron donors are present in the medium. The results obtained clearly indicate that (i) S0 oxidation takes place only when iron is totally oxidized and (ii) the genes involved in Fe(II) are expressed before those involved in RISC oxidation, even in the presence of S0. Therefore, a transcriptional regulator is likely inducing the genes involved in Fe(II) oxidation and repressing those involved in RISC oxidation when Fe(II) is present. Data presented in this paper suggest that the redox sensing sensor/regulator two-component signal transducing system RegBA could be responsible for this regulation since (i) the redox potential increases during Fe(II) oxidation but remains stable during S0 oxidation and (ii) the regulator RegA is able to bind to the regulatory region of a number of genes/operons of which the expression is regulated by Fe(II). The understanding of how RegBA controls the energetic pathways depending on the overall redox state of the cell is of primordial importance since it could help in controlling the dynamics of Fe(II) and RISC oxidation during bioleaching processes.