Many acidophilic iron-oxidizing bacteria used in the mining industry for the bioleaching of sulfidic minerals are intolerant to high chloride concentrations, resulting in problems where chloride occurs in the deposit at high concentrations or only seawater is available. In search for strains tolerating such conditions a tetrathionate- and iron-oxidizing bacterium was isolated from a tailings-contaminated beach sample at Portman Bay, Cartagena-La Union mining district, Spain, in the presence of 20 g l-1 (0.34 M) sodium chloride. The isolate was able to form spores, did not grow in the absence of NaCl, and oxidized ferrous iron in the presence of up to 1.5 M (∼87 g l-1) NaCl. Genome sequencing based on a combination of Illumina and PacBio reads revealed two contigs, a circular bacterial chromosome of 5.2 Mbp and a plasmid of 90 kbp, respectively. The chromosome comprised seven different 16S rRNA genes. Submission of the chromosome to the Type (Strain) Genome Server (TYGS) without preselection of similar sequences revealed exclusively type strains of the genus Alicyclobacillus. In the TYGS analyses the respective most similar species were dependent on whether the final tree was derived from just 16S rRNA, from the genomes, or from the proteomes. Thus, TYGS analysis clearly showed that isolate SO9 represents a novel species of the genus Alicyclobacillus. In the presence of artificial seawater with almost 0.6 M chloride, the addition of Alicyclobacillus sp. SO9 improved copper dissolution from chalcopyrite (CuFeS2) compared to abiotic leaching without bacteria. The new isolate SO9, therefore, has potential for bioleaching at elevated chloride concentrations.

For most acidophilic iron-oxidizing bioleaching microorganisms, chloride ions are toxic even at low concentrations since they are able to induce an acidification of the cytoplasm and osmotic stress. To identify molecular determinants for osmotic tolerance of these microorganisms, a genomic analysis based on the complete genomes of twelve iron oxidizers was carried out. Genes for a complete or partial repertoire of K+ transporters Kdp and Ygg were found in most of the genomes. Representatives of Nitrospirae, Firmicutes and Actinobacteria harbor genes for the biosynthesis of (5-hydroxy)ectoine and trehalose. Members of the phylum Nitrospirae appeared to be better equipped with classical mechanisms for osmotic tolerance than representatives of other phyla. The moderately halotolerant bacteria Acidihalobacter prosperus V6 and Leptospirillum ferriphilum Sp-Cl did not show overrepresentation of these mechanisms. In agreement with the bioinformatics results, minimal inhibitory concentrations (MICs) of NaCl showed that Sulfobacillus thermosulfidooxidans, Ferrimicrobium acidiphilum and Leptospirillum ferriphilum were more tolerant than Acidithiobacillus ferrooxidans. Furthermore, external supplementation of ectoine led to an increase in NaCl MIC for L. ferriphilum and F. acidiphilum. These results may provide useful insights for designing chloride-based technologies to bioprocess minerals.

Biomining applies microorganisms to extract valuable metals from usually sulfidic ores. However, acidophilic iron (Fe)-oxidizing bacteria tend to be sensitive to chloride ions which may be present in biomining operations. This study investigates the bioleaching of pyrite (FeS2), as well as the attachment to FeS2 by Sulfobacillus thermosulfidooxidans DSM 9293T in the presence of elevated sodium chloride (NaCl) concentrations. The bacteria were still able to oxidize iron in the presence of up to 0.6M NaCl (35 g/L), and the addition of NaCl in concentrations up to 0.2M (~12 g/L) did not inhibit iron oxidation and growth of S. thermosulfidooxidans in leaching cultures within the first 7 days. However, after approximately 7 days of incubation, ferrous iron (Fe2+) concentrations were gradually increased in leaching assays with NaCl, indicating that iron oxidation activity over time was reduced in those assays. Although the inhibition by 0.1M NaCl (~6 g/L) of bacterial growth and iron oxidation activity was not evident at the beginning of the experiment, over extended leaching duration NaCl was likely to have an inhibitory effect. Thus, after 36 days of the experiment, bioleaching of FeS2 with 0.1M NaCl was reduced significantly in comparison to control assays without NaCl. Pyrite dissolution decreased with the increase of NaCl. Nevertheless, pyrite bioleaching by S. thermosulfidooxidans was still possible at NaCl concentrations as high as 0.4M (~23 g/L NaCl). Besides, cell attachment in the presence of different concentrations of NaCl was investigated. Cells of S. thermosulfidooxidans attached heterogeneously on pyrite surfaces regardless of NaCl concentration. Noticeably, bacteria were able to adhere to pyrite surfaces in the presence of NaCl as high as 0.4M. Although NaCl addition inhibited iron oxidation activity and bioleaching of FeS2, the presence of 0.2M seemed to enhance bacterial attachment of S. thermosulfidooxidans on pyrite surfaces in comparison to attachment without NaCl.

This study reports on the effect of inoculum history, growth substrates, and yeast extract on sodium chloride tolerance of Sulfobacillus thermosulfidooxidans DSM 9293T. The concentrations of NaCl for complete inhibition of Fe2+ oxidation by cells initially grown with ferrous iron sulfate, or tetrathionate, or pyrite as energy sources were 525 mM, 725 mM, and 800 mM, respectively. Noticeably, regardless of NaCl concentrations, oxygen consumption rates of S. thermosulfidooxidans with 20 mM tetrathionate were higher than with 50 mM FeSO4. NaCl concentrations of higher than 400 mM strongly inhibited the iron respiration of S. thermosulfidooxidans. In contrast, the presence of NaCl was shown to stimulate tetrathionate oxidation. This trend was especially pronounced in NaCl-adapted cells where respiration rates at 200 mM NaCl were threefold of those in the absence of NaCl. In NaCl-adapted cultures greater respiration rates for tetrathionate were observed than in non-NaCl-adapted cultures, especially at concentrations ≥ 200 mM NaCl. At concentrations of ≤ 200 mM NaCl, cell growth and iron oxidation were enhanced with the addition of increasing concentrations of yeast extract. Thus, cell numbers in cultures with 0.05% yeast extract were ∼5 times higher than without yeast extract addition. At NaCl concentration as high as 400 mM, however, iron oxidation rates improved compared to control assays without yeast extract, but there was no clear dependence on yeast extract concentrations. The initial growth of bacteria with and without yeast extract in the presence of different NaCl concentrations was shown to impact leaching of copper from chalcopyrite. Copper dissolution was enhanced in the presence of 200 mM NaCl and absence of yeast extract, while the addition of 0.02% yeast extract was shown to promote copper solubilization in the presence of 500 mM NaCl.

Reactive oxygen species (ROS) cause oxidative stress and growth inhibition by inactivation of essential enzymes, DNA and lipid damage in microbial cells. Acid mine drainage (AMD) ecosystems are characterized by low pH values, enhanced levels of metal ions and low species abundance. Furthermore, metal sulfides, such as pyrite and chalcopyrite, generate extracellular ROS upon exposure to acidic water. Consequently, oxidative stress management is especially important in acidophilic leaching microorganisms present in industrial biomining operations, especially when forming biofilms on metal sulfides. Several adaptive mechanisms have been described, but the molecular repertoire of responses upon exposure to pyrite and the presence of ROS are not thoroughly understood in acidophiles. In this study the impact of the addition of H2O2 on iron oxidation activity in Acidithiobacillus ferrooxidans DSM 14882T was investigated. Iron(II)- or sulfur-grown cells showed a higher sensitivity toward H2O2 than pyrite-grown ones. In order to elucidate which molecular responses may be involved, we used shot-gun proteomics and compared proteomes of cells grown with iron(II)-ions against biofilm cells, grown for 5 days in presence of pyrite as sole energy source. In total 1157 proteins were identified. 213 and 207 ones were found to have increased levels in iron(II) ion-grown or pyrite-biofilm cells, respectively. Proteins associated with inorganic sulfur compound (ISC) oxidation were among the latter. In total, 80 proteins involved in ROS degradation, thiol redox regulation, macromolecule repair mechanisms, biosynthesis of antioxidants, as well as metal and oxygen homeostasis were found. 42 of these proteins had no significant changes in abundance, while 30 proteins had increased levels in pyrite-biofilm cells. New insights in ROS mitigation strategies, such as importance of globins for oxygen homeostasis and prevention of unspecific reactions of free oxygen that generate ROS are presented for A. ferrooxidans biofilm cells. Furthermore, proteomic analyses provide insights in adaptations of carbon fixation and oxidative phosphorylation pathways under these two growth conditions.

In bio-hydrometallurgical operations, several studies found that besides having inhibitory effects chloride may also enhance the bacterial leaching of metal sulfides. This study compares the effect of chloride on bacterial growth, iron oxidation activity and bioleaching performance of Leptospirillum ferriphilum and Sulfobacillus thermosulfidooxidans. The exposure to elevated NaCl concentrations affected cell growth and iron oxidation rate in both strains. Sb. thermosulfidooxidans showed better tolerance to NaCl than L. ferriphilum, with the concentration that inhibited biological iron oxidation and bacterial growth completely being 325 mM and 525 mM NaCl for L ferriphilum and Sb. thermosulfidooxidans, respectively. At 100 mM NaCl, cell growth and iron oxidation of L. ferriphilum were severely slowed down while for Sb. thermosulfidooxidans, in contrast, they appeared almost unaffected. Additionally, while only 22% of L. ferriphilum cells survived after 18 h exposure to 100 mM NaCl, viability of Sb. thermosulfidooxidans was not strongly affected by 300 mM NaCl. Bioleaching of chalcopyrite and sphalerite were studied. For chalcopyrite, bioleaching by Sb. thermosulfidooxidans in the presence of 200 mM was similar to its performance in the absence of NaCl. However, bioleaching by L. ferriphilum was reduced by 50% in the presence of 200 mM NaCl. For sphalerite, bioleaching by both bacteria, L. ferriphilum and Sb. thermosulfidooxidans, was inhibited significantly in the presence of 200 mM NaCl. This study indicates that Sb. thermosulfidooxidans will be more suitable than L. ferriphilum to operate bioleaching with high chloride concentrations. Additionally, since bioleaching performance of chalcopyrite differed from bioleaching of sphalerite under similar conditions, further investigations on bioleaching with different sulfidic minerals in the presence of chloride ions are necessary.

In acidophilic microorganisms, anions like chloride have higher toxicity than their neutrophilic counterparts. In addition to the osmotic imbalance, chloride can also induce acidification of the cytoplasm. We predicted that intracellular acidification produces an increase in respiratory rate and generation of reactive oxygen species, and so oxidative stress can also be induced. In this study, the multifactorial effect as inducing osmotic imbalance, cytoplasm acidification and oxidative stress in the iron-oxidizing bacterium Leptospirillum ferriphilum DSM 14647 exposed to up to 150 mM NaCl was investigated. Results showed that chloride stress up-regulated genes for synthesis of potassium transporters (kdpC and kdpD), and biosynthesis of the compatible solutes (hydroxy)ectoine (ectC and ectD) and trehalose (otsB). As a consequence, the intracellular levels of both hydroxyectoine and trehalose increased significantly, suggesting a strong response to keep osmotic homeostasis. On the other hand, the intracellular pH significantly decreased from 6.7 to pH 5.5 and oxygen consumption increased significantly when the cells were exposed to NaCl stress. Furthermore, this stress condition led to a significant increase of the intracellular content of reactive oxygen species, and to a rise of the antioxidative cytochrome c peroxidase (CcP) and thioredoxin (Trx) activities. In agreement, ccp and trx genes were up-regulated under this condition, suggesting that this bacterium displayed a transcriptionally regulated response against oxidative stress induced by chloride. Altogether, these data reveal that chloride has a dramatic multifaceted effect on acidophile physiology that involves osmotic, acidic and oxidative stresses. Exploration of the adaptive mechanisms to anion stress in iron-oxidizing acidophilic microorganisms may result in new strategies that facilitate the bioleaching of ores for recovery of precious metals in presence of chloride.

Acidophilic leaching microorganisms have been reported to be in general intolerant to high salinity, namely high concentrations of chloride. At present this restriction hampers the use of sea water for bioleaching technology. Enrichment cultures obtained in this study from a former ore deposit near the Spanish coast oxidize ferrous iron in the presence of up to 50 gL-1 NaCl at pH 2.5 and 37°C. The presence of at least 5 gL-1 NaCl was shown to be an obligate requirement for iron oxidation. The major microbial groups comprise Alicyclobacillus and Arthrobacter . The findings may be of biotechnological relevance.

After 24h of exposure to acidic media, pyrite generates reactive oxygen species (ROS). Freshly-crushed pyrite with grain sizes between 50-100 μm at a 5 % (w/v), pulp density generated 0.17 ± 0.01 mM H2O2, while 10% pyrite generated 0.29 ± 0.01 mM and 30 % pyrite generated approximately 0.83 ± 0.06 mM. These levels of H2O2 inhibit iron oxidation in iron-grown cells of AcidithiobacillusferrooxidansT but not in pyrite-grown cells. ROS originating from pyrite, which was incubated for 24 h in acidic medium, prohibited pyrite dissolution by iron-grown cells, while pyrite-grown cells were adapted to these concentrations of ROS. Periodical addition of 100 μM H2O2 to pyrite cultures inoculated with pyrite-grown cells did not lower iron dissolution as it was observed with iron-grown cells. By high throughput proteomics analysis, an increased expression of proteins related to oxidative stress management, iron-and sulfur oxidation systems, carbon fixation and biofilm formation was observed in biofilm cells grown on pyrite compared to iron-grown cells.

Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide (O2-) and hydroxyl radicals (OH.) are known to be formed on the surface of metal sulfides in aqueous solution under oxic and anoxic conditions. Consequently bacteria which have not been adapted to their presence are metabolically inhibited [1], presumably due to the presence of these ROS. Pyrite-grown cells ofAcidithiobacillus ferrooxidansT, in contrast to iron (II)-grown cells, were able to oxidize iron (II)-ions or pyrite after 24 h starvation and contact with 1 mM externally added H2O2. In this study, similar results were obtained withAcidiferrobactersp. SPIII/3. However,Acidithiobacillus ferrivoransSS3 showed the highest tolerance towards contact with H2O2, whileLeptospirillum ferrooxidansDSM 2391 was most sensitive. Similar results were obtained after exposure to defined doses of gamma radiation, which cleaves water molecules and generates ROS. In this study members of the three aforementioned genera of mineral-oxidizing bacteria were compared regarding their ability to survive, colonize pyrite and to oxidize iron (II)-ions after exposure to different concentrations of H2O2. Pyrite colonization was studied after exposure to endogenous ROS formed on pyrite or after external addition of H2O2using confocal laser scanning microscopy (CLSM).