Evolution of biofilms during the colonization process of pyrite by Acidithiobacillus thiooxidans

We have applied epifluorescence principles, atomic force microscopy, and Raman studies to the analysis of the colonization process of pyrite (FeS₂) by sulfuroxidizing bacteria Acidithiobacillus thiooxidans after 1, 15, 24, and 72 h. For the stages examined, we present results comprising the evolution of biofilms, speciation of S_n²⁻/S⁰ species, adhesion forces of attached cells, production and secretion of extracellular polymeric substances (EPS), and its biochemical composition. After 1 h, highly dispersed attached cells in the surface of the mineral were observed. The results suggest initial non-covalent, weak interactions (e.g., van der Waal’s, hydrophobic interactions), mediating an irreversible binding mechanism to electrooxidized massive pyrite electrode (eMPE), wherein the initial production of EPS by individual cells is determinant. The mineral surface reached its maximum cell cover between 15 to 24 h. Longer biooxidation times resulted in the progressive biofilm reduction on the mineral surface. Quantification of attached cell adhesion forces indicated a strong initial mechanism (8.4 nN), whereas subsequent stages of mineral colonization indicated stability of biofilms and of the adhesion force to an average of 4.2 nN. A variable EPS (polysaccharides, lipids, and proteins) secretion at all stages was found; thus, different architectural conformation of the biofilms was observed during 120 h. The main EPS produced were lipopolysaccharides which may increase the hydrophobicity of A. thiooxidans biofilms. The highest amount of lipopolysaccharides occurred between 15–72 h. In contrast with abiotic surfaces, the progressive depletion of S_n²⁻/S⁰ was observed on biotic eMPE surfaces, indicating consumption of surface sulfur species. All observations indicated a dynamic biooxidation mechanism of pyrite by A. thiooxidans, where the biofilms stability and composition seems to occur independently from surface sulfur species depletion.     

Changes in biofilm structure during the colonization of chalcopyrite by Acidithiobacillus thiooxidans

Biofilms of Acidithiobacillus thiooxidans were grown on the surface of massive chalcopyrite electrodes (MCE) where different secondary sulfur phases were previously formed by potentiostatic oxidation of MCE at 0.780?≤?E _an?≤?0.965 V (electrooxidized MCE, eMCE). The formation of mainly S⁰ and minor amounts of CuS and S _n ^2? were detected on eMCEs. The eMCEs were incubated with A. thiooxidans cells for 1, 12, 24, 48, and 120 h in order to temporally monitor changes in eMCE's secondary phases, biofilm structure, and extracellular polymeric substance (EPS) composition (lipids, proteins, and polysaccharides) using microscopic, spectroscopic, electrochemical, and biochemical techniques. The results show significant cell attachments with stratified biofilm structure since the first hour of incubation and EPS composition changes, the most important being production after 48–120 h when the highest amount of lipids and proteins were registered. During 120 h, periodic oxidation/formation of S⁰/S _n ^2? was recorded on biooxidized eMCEs, until a stable CuS composition was formed. In contrast, no evidence of CuS formation was observed on the eMCEs of the abiotic control, confirming that CuS formation results from microbial activity. The surface transformation of eMCE induces a structural transformation of the biofilm, evolving directly to a multilayered biofilm with more hydrophobic EPS and proteins after 120 h. Our results suggest that A. thiooxidans responded to the spatial and temporal distribution and chemical reactivity of the S _n ^2?/S⁰/CuS phases throughout 120 h. These results suggested a strong correlation between surface speciation, hydrophobic domains in EPS, and biofilm organization during chalcopyrite biooxidation by A. thiooxidans.     

Influence of the surface speciation on biofilm attachment to chalcopyrite by Acidithiobacillus thiooxidans

Surfaces of massive chalcopyrite (CuFeS₂) electrodes were modified by applying variable oxidation potential pulses under growth media in order to induce the formation of different secondary phases (e.g., copper-rich polysulfides, S _n ^2?; elemental sulfur, S⁰; and covellite, CuS). The evolution of reactivity (oxidation capacity) of the resulting chalcopyrite surfaces considers a transition from passive or inactive (containing CuS and S _n ^2?) to active (containing increasing amounts of S⁰) phases. Modified surfaces were incubated with cells of sulfur-oxidizing bacteria (Acidithiobacillus thiooxidans) for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the density of cells attached to chalcopyrite surfaces, the structure of the formed biofilm, and their exopolysaccharides and nucleic acids were analyzed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy coupled to dispersive X-ray analysis (SEM-EDS). Additionally, CuS and S _n ^2?/S⁰ speciation, as well as secondary phase evolution, was carried out on biooxidized and abiotic chalcopyrite surfaces using Raman spectroscopy and SEM-EDS. Our results indicate that oxidized chalcopyrite surfaces initially containing inactive S _n ^2? and S _n ^2?/CuS phases were less colonized by A. thiooxidans as compared with surfaces containing active phases (mainly S⁰). Furthermore, it was observed that cells were partially covered by CuS and S⁰ phases during biooxidation, especially at highly oxidized chalcopyrite surfaces, suggesting the innocuous effect of CuS phases during A. thiooxidans performance. These results may contribute to understanding the effect of the concomitant formation of refractory secondary phases (as CuS and inactive S _n ^2?) during the biooxidation of chalcopyrite by sulfur-oxidizing microorganisms in bioleaching systems.     

Growth inhibition by tungsten in the sulfur-oxidizing bacterium Acidithiobacillus thiooxidans

Growth of five strains of sulfur-oxidizing bacteria Acidithiobacillus thiooxidans, including strain NB1-3, was inhibited completely by 50 microM of sodium tungstate (Na(2)WO(4)). When the cells of NB1-3 were incubated in 0.1 M beta-alanine-SO(4)(2-) buffer (pH 3.0) with 100 microM Na(2)WO(4) for 1 h, the amount of tungsten bound to the cells was 33 microg/mg protein. Approximately 10 times more tungsten was bound to the cells at pH 3.0 than at pH 7.0. The tungsten binding to NB1-3 cells was inhibited by oxyanions such as sodium molybdenum and ammonium vanadate. The activities of enzymes involved in elemental sulfur oxidation of NB1-3 cells such as sulfur oxidase, sulfur dioxygenase, and sulfite oxidase were strongly inhibited by Na(2)WO(4). These results indicate that tungsten binds to NB1-3 cells and inhibits the sulfur oxidation enzyme system of the cells, and as a result, inhibits cell growth. When portland cement bars supplemented with 0.075% metal nickel and with 0.075% metal nickel and 0.075% calcium tungstate were exposed to the atmosphere of a sewage treatment plant containing 28 ppm of H(2)S for 2 years, the weight loss of the portland cement bar with metal nickel and calcium tungstate was much lower than the cement bar containing 0.075% metal nickel.     

A direct observation of bacterial coverage and biofilm formation by Acidithiobacillus ferrooxidans on chalcopyrite and pyrite surfaces

To obtain a fundamental understanding of the population behaviour of Acidithiobacillus ferrooxidans at chalcopyrite and pyrite surfaces, the early stage attachment behaviour and biofilm formation by this bacterium on chalcopyrite (CuFeS₂) and pyrite (FeS₂) were studied by optical microscopy, Raman spectroscopy, time-of-flight secondary ion mass spectrometry (ToF-SIMS) and electron backscatter diffraction (EBSD). The results indicate there was no significant difference in selectivity of bacterial attachment between chalcopyrite and pyrite. However, the result of ToF-SIMS analysis suggests that the surface of the pyrite was covered more extensively by biofilm than that of the chalcopyrite, which may indicate more extracellular polymeric substances (EPS) formation by bacterial cells growing on pyrite. EBSD and optical image analysis indicated that selectivity of bacterial attachment to chalcopyrite was not significantly affected by crystal orientation. The results also suggest that the bacterial population in defective areas of chalcopyrite was significantly higher than on the polished surfaces.     

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. Biotechnol. Bioeng. 2013; 110: 2242–2251. © 2013 Wiley Periodicals, Inc.     

金属离子对氧化硫硫杆菌生长及产酸的影响

目的:在前期研究中选育出的氧化硫硫杆菌TT03,为了解其对各种金属离子的适应能力而进行了试验.方法:在史塔克培养基中添加规定量的各种金属离子,接种TT03后进行摇动培养,测定培养液中的产酸以代表其实际生长状况.结果:在Co<'3+>、Zn2+为100mg/L而其他微量元素为1000mg/L时没有产生抑制作用,其中有些微量元素还表现出促进作用;除pb2+、Cd2+以外,重金属在1000mg/L下普遍表现出轻微的抑制作用,对产酸没有明显影响;当pb2+、Cd2+的含量分别在10mg/L、1mg/L时,对TT03产酸没有影响.在试验的金属离子中Cd2+对TT03产酸的抑制作用最强,在浓度30mg/L时,生长产酸被完全抑制.结论:在正常土壤环境中的金属离子含量,不会对氧化硫硫杆菌TT03的生长和产酸造成致命影响.     

The effect of the introduction of exogenous strain Acidithiobacillus thiooxidans A01 on functional gene expression, structure and function of indigenous consortium during pyrite bioleaching

Acidithiobacillus thiooxidans A01 was added to a consortium of bioleaching bacteria including Acidithiobacilluscaldus, Leptospirillumferriphilum, Acidithiobacillus ferrooxidans, Sulfobacillus thermosulfidooxidans, Acidiphilium spp., and Ferroplasma thermophilum cultured in modified 9 K medium containing 0.5% (w/v) pyrite, and 10.7% increase of bioleaching rate was observed. Changes in community structure and gene expression were monitored with real-time PCR and functional gene arrays (FGAs). Real-time PCR showed that addition of At. thiooxidans caused increased numbers of all consortium members except At. caldus, and At. caldus, L. ferriphilum, and F. thermophilum remained dominant in this community. FGAs results showed that after addition of At. thiooxidans, most genes involved in iron, sulfur, carbon, and nitrogen metabolisms, metal resistance, electron transport, and extracellular polymeric substances of L. ferriphilum, F. thermophilum, and Acidiphilium spp., were up-regulated while most of these genes were down-regulated at 70-78 h in At. caldus and up-regulated in At. ferrooxidans, then down-regulated at 82-86 h.     

Inhibition of microbial concrete corrosion by Acidithiobacillus thiooxidans with functionalised zeolite-A coating

The inhibition of the corrosive action of Acidithiobacillus thiooxidans on concrete specimens coated by functionalised zeolite-A containing 14% zinc and 5% silver by weight was studied. Uncoated concrete specimens, epoxy-coated concrete specimens (EP), and functionalised zeolite-A coated concrete specimens with epoxy to zeolite weight ratios of 3:1 (Z1), 2:2 (Z2) and 1:3 (Z3) were studied. Specimens were characterised by x-ray powder diffraction and field emission scanning electron microscopy for the identification of corrosion products and morphological changes. Biomass growth at the conclusion of the 32-day experiments was 4, 179 and 193 mg volatile suspended solids g(-1) sulphur for the uncoated, EP and Z1 specimens, whereas that of Z2 and Z3 were negligible. In the uncoated, EP and Z1 specimens, sulphate production rates were 0.83, 9.1 and 8.8 mM SO(4)(2-) day(-1) and the specific growth rates, mu, were 0.14, 0.57 and 0.47 day(-1), respectively. The corresponding values for Z2 and Z3 were negligible due to their bacterial inhibition characteristics.     

Effects of inhibitors and NaCl on the oxidation of reduced inorganic sulfur compounds by a marine acidophilic, sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH

The effect of NaCl and the pathways of the oxidation of reduced inorganic sulfur compounds were studied using resting cells and cell-free extracts of Acidithiobacillus thiooxidans strain SH. This isolate specifically requires NaCl for growth. The oxidation of sulfur and sulfite by resting cells was strongly inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide. Carbonylcyanide m-chlorophenyl-hydrazone and monensin were also relatively strong inhibitors. Thiosulfate-oxidizing activity was not inhibited by these uncouplers. Valinomycin did not inhibit the oxidation of sulfur compounds. NaCl stimulated the sulfur- and sulfite-oxidizing activities in resting cells but not in cell-free extracts. The tetrathionate-oxidizing activity in resting cells was slightly stimulated by NaCl, whereas it did not influence the thiosulfate-oxidizing activity. Sulfide oxidation was biphasic, suggesting the formation of intermediate sulfur. The initial phase of sulfide oxidation was not affected by NaCl, whereas the subsequent oxidation of sulfur in the second phase was Na⁺-dependent. A model is proposed for the role of NaCl in the metabolism of reduced sulfur compounds in A. thiooxidans strain SH.     

Analysis of the elemental sulfur bio-oxidation by Acidithiobacillus ferrooxidans with sulfur K-edge XANES

Elemental sulfur bio-oxidation by the typical acidophilic sulfur-oxidizing microbe Acidithiobacillus ferrooxidans was investigated by using the technique of sulfur K-edge XANES spectroscopy. Our results showed that the majority of elemental sulfur altered by A. ferrooxidans was dissolved into the organic phase containing carbon disulfide, while a part of it floated. The fitted results of sulfur K-edge XANES spectrum of the floated sulfur showed that the floating part of the elemental sulfur powder was converted to polymeric sulfur and the relative concentration of sulfur in cyclo-octasulfur S₈ and polymeric sulfur was 37.2 and 62.8%, respectively. It seems that the cyclo-octasulfur is converted to the polymeric sulfur and this appears to be necessary for oxidation of elemental sulfur by A. ferrooxidans. The results have important implications for our understanding of the mechanisms for bio-oxidation of elemental sulfur.     

AHL signaling molecules with a large acyl chain enhance biofilm formation on sulfur and metal sulfides by the bioleaching bacterium Acidithiobacillus ferrooxidans

Biofilm formation plays a pivotal role in bioleaching activities of bacteria in both industrial and natural environments. Here, by visualizing attached bacterial cells on energetic substrates with different microscopy techniques, we obtained the first direct evidence that it is possible to positively modulate biofilm formation of the extremophilic bacterium Acidithiobacillus ferrooxidans on sulfur and pyrite surfaces by using Quorum Sensing molecules of the N-acylhomoserine lactone type (AHLs). Our results revealed that AHL-signaling molecules with a long acyl chain (12 or 14 carbons) increased the adhesion of A. ferrooxidans cells to these substrates. In addition, Card-Fish experiments demonstrated that C14-AHL improved the adhesion of indigenous A. ferrooxidans cells from a mixed bioleaching community to pyrite. Finally, we demonstrated that this improvement of cell adhesion is correlated with an increased production of extracellular polymeric substances. Our results open up a promising means to develop new strategies for the improvement of bioleaching efficiency and metal recovery, which could also be used to control environmental damage caused by acid mine/rock drainage.     

Mechanism of oxidation of inorganic sulfur compounds by thiosulfate-grown Thiobacillus thiooxidans

Thiobacillus thiooxidans was grown at pH 5 on thiosulfate as an energy source, and the mechanism of oxidation of inorganic sulfur compounds was studied by the effect of inhibitors, stoichiometries of oxygen consumption and sulfur, sulfite, or tetrathionate accumulation, and cytochrome reduction by substrates. Both intact cells and cell-free extracts were used in the study. The results are consistent with the pathway with sulfur and sulfite as the key intermediates. Thiosulfate was oxidized after cleavage to sulfur and sulfite as intermediates at pH 5, the optimal growth pH on thiosulfate, but after initial condensation to tetrathionate at pH 2.3 where the organism failed to grow. N-Ethylmaleimide (NEM) inhibited sulfur oxidation directly and the oxidation of thiosulfate or tetrathionate indirectly. It did not inhibit the sulfite oxidation by cells, but inhibited any reduction of cell cytochromes by sulfur, thiosulfate, tetrathionate, and sulfite. NEM probably binds sulfhydryl groups, which are possibly essential in supplying electrons to initiate sulfur oxidation. 2-Heptyl-4-hydroxy-quinoline N-oxide (HQNO) inhibited the oxidation of sulfite directly and that of sulfur, thiosulfate, and tetrathionate indirectly. Uncouplers, carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), inhibited sulfite oxidation by cells, but not the oxidation by extracts, while HQNO inhibited both. It is proposed that HQNO inhibits the oxidation of sulfite at the cytochrome b site both in cells and extracts, but uncouplers inhibit the oxidation in cells only by collapsing the energized state of cells, delta muH+, required either for electron transfer from cytochrome c to b or for sulfite binding.     

Draft genome sequence of the extremely acidophilic biomining bacterium Acidithiobacillus thiooxidans ATCC 19377 provides insights into the evolution of the Acidithiobacillus genus

Acidithiobacillus thiooxidans is a mesophilic, extremely acidophilic, chemolithoautotrophic gammaproteobacterium that derives energy from the oxidation of sulfur and inorganic sulfur compounds. Here we present the draft genome sequence of A. thiooxidans ATCC 19377, which has allowed the identification of genes for survival and colonization of extremely acidic environments.     

The effects of metabolites from the indigenous Acidithiobacillus thiooxidans and temperature on the bioleaching of cadmium from soil

The effect of metabolites from the indigenous Acidithiobacillus thiooxidans and temperature on the bioleaching of cadmium from soil was investigated in the present study. Bioleaching was found to be more effective than chemical leaching of cadmium. The metabolite, mainly sulfuric acid, which was shown to be growth-associated in the exponential phase, plays a major role in bioleaching. The maximum amount of cadmium leached was obtained after 8 days of precultivation when cells were directly involved in the leaching process. It indicates that cells in the exponential growth phase exhibit higher activity toward bioleaching. In contrast, the maximum amount of cadmium leached and the maximum initial rate for bioleaching were reached after 16 days of precultivation when only metabolites were involved in the bioleaching process. It implies that higher sulfuric acid concentration results in higher leaching efficiency. In addition, higher temperature leads to higher leaching efficiency. The optimal operation condition for bioleaching was determined to be a two-stage process: The first stage involves the precultivation of the indigenous A. thiooxidans at 30 degrees C for 8 days followed by 20 minutes of centrifugation to discard cells. The second stage involves the bioleaching with the subsequent supernatant at 50 degrees C.     

Kinetic model of elemental sulfur oxidation by Thiobacillus thiooxidans in batch slurry reactors

The kinetics of sulfur oxidation by T. thiooxidans has been studied in a batch well-mixed reactor and in shaker flasks. A mathematical model is proposed, which considers the attachment of the cells onto the sulfur particles' surface following Freundlich isotherm, growth of the attached bacteria, and growth inhibition by sulfates accumulation. Best-fit values of the model parameters have been calculated from the experimental data. Results show that the addition of dimethyl-dichloro-silane in the aerated reactor to prevent the formation of foam reduces the maximum specific growth rate of attached bacteria, probably because of the resulting changes in surface properties of the sulfur particles. The other model parameters are not significantly affected. The formation of clusters of sulfur particles has been observed at an initial sulfur concentration of 5% . This phenomenon reduces the rate of sulfur conversion due to the reduction of the total surface area of the particles, and the model therefore over-estimates the formation of sulfates. At lower initial sulfur concentration, the phenomenon has not been observed and the model simulations are then satisfactory.     

Characterization of tetrathionate hydrolase from the marine acidophilic sulfur-oxidizing bacterium,Acidithiobacillus thiooxidans strain SH

Tetrathionate hydrolase (4THase), a key enzyme of the S₄-intermediate (S4I) pathway, was partially purified from marine acidophilic bacterium, Acidithiobacillus thiooxidans strain SH, and the gene encoding this enzyme (SH-tth) was identified. SH-Tth is a homodimer with a molecular mass of 97 ± 3 kDa, and contains a subunit 52 kDa in size. Enzyme activity was stimulated in the presence of 1 M NaCl, and showed the maximum at pH 3.0. Although 4THases from A. thiooxidans and the closely related Acidithiobacillus caldus strain have been reported to be periplasmic enzymes, SH-Tth seems to be localized on the outer membrane of the cell, and acts as a peripheral protein. Furthermore, both 4THase activity and SH-Tth proteins were detected in sulfur-grown cells of strain SH. These results suggested that SH-Tth is involved in elemental sulfur-oxidation, which is distinct from sulfur-oxidation in other sulfur-oxidizing strains such as A. thiooxidans and A. caldus.