Siderite and vivianite as energy sources for the extreme acidophilic bacterium Acidithiobacillus ferrooxidans in the context of mars habitability.

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, Fe2+ 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 Fe2+ that came from the solubilization of these minerals under low pH. Additionally, in sealed flasks without CO2, 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.

Establishing a green biodesulfurization process for iron ore concentrates in stirred tank and leaching column bioreactors using Acidithiobacillus thiooxidans.

The presence of sulfur impurities in complex iron ores represents a significant challenge for the iron mining and steel-making industries as their removal often necessitates the use of hazardous chemicals and energy-intensive processes. Here, we examined the microbial and mineralogical composition of both primary and secondary iron concentrates, identifying the presence of Sulfobacillus spp. and Leptospirillum spp., while sulfur-oxidizing bacteria were absent. We also observed that these concentrates displayed up to 85% exposed pyrrhotite. These observations led us to explore the capacity of Acidithiobacillus thiooxidans to remove pyrrhotite-sulfur impurities from iron concentrates. Employing stirred tank bioreactors operating at 30°C and inoculated with 5·106 (At. thiooxidans cells mL-1), we achieved 45.6% sulfur removal over 16 days. Then, we evaluated packed leaching columns operated at 30°C, where the At. thiooxidans enriched system reached 43.5% desulfurization over 60 days. Remarkably, sulfur removal increased to 80% within 21 days under potassium limitation. We then compared the At. thiooxidans-mediated desulfurization process, with and without air supply, under potassium limitation, varying the initial biomass concentration in 1-m columns. Aerated systems facilitated approximately 70% sulfur removal across the entire column with minimal iron loss. In contrast, non-aerated leaching columns achieved desulfurization levels of only 6% and 26% in the lower and middle sections of the column, respectively. Collectively, we have developed an efficient, scalable biological sulfur-removal technology for processing complex iron ores, aligning with the burgeoning demand for sustainable practices in the mining industry.

Pangenome-level analysis of nucleoid-associated proteins in the Acidithiobacillia class: insights into their functional roles in mobile genetic elements biology.

Mobile genetic elements (MGEs) are relevant agents in bacterial adaptation and evolutionary diversification. Stable appropriation of these DNA elements depends on host factors, among which are the nucleoid-associated proteins (NAPs). NAPs are highly abundant proteins that bind and bend DNA, altering its topology and folding, thus affecting all known cellular DNA processes from replication to expression. Even though NAP coding genes are found in most prokaryotic genomes, their functions in host chromosome biology and xenogeneic silencing are only known for a few NAP families. Less is known about the occurrence, abundance, and roles of MGE-encoded NAPs in foreign elements establishment and mobility. In this study, we used a combination of comparative genomics and phylogenetic strategies to gain insights into the diversity, distribution, and functional roles of NAPs within the class Acidithiobacillia with a special focus on their role in MGE biology. Acidithiobacillia class members are aerobic, chemolithoautotrophic, acidophilic sulfur-oxidizers, encompassing substantial genotypic diversity attributable to MGEs. Our search for NAP protein families (PFs) in more than 90 genomes of the different species that conform the class, revealed the presence of 1,197 proteins pertaining to 12 different NAP families, with differential occurrence and conservation across species. Pangenome-level analysis revealed 6 core NAP PFs that were highly conserved across the class, some of which also existed as variant forms of scattered occurrence, in addition to NAPs of taxa-restricted distribution. Core NAPs identified are reckoned as essential based on the conservation of genomic context and phylogenetic signals. In turn, various highly diversified NAPs pertaining to the flexible gene complement of the class, were found to be encoded in known plasmids or, larger integrated MGEs or, present in genomic loci associated with MGE-hallmark genes, pointing to their role in the stabilization/maintenance of these elements in strains and species with larger genomes. Both core and flexible NAPs identified proved valuable as markers, the former accurately recapitulating the phylogeny of the class, and the later, as seed in the bioinformatic identification of novel episomal and integrated mobile elements.

Electrotrophic perchlorate reduction by a psychrotolerant acidophile isolated from an acid rock drainage in Antarctica.

A new extremophilic isolate (USS-CCA7) was obtained from an acidic environment (pH âˆ¼ 3.2) in Antarctica phylogenetically related to Acidithiobacillus ferrivorans; its electrotrophic capacities were evaluated in a three-electrode electrochemical cell. Cyclic voltammetry showed cathodic peaks of -428 mV, -536 mV, and -634 mV (vs. Ag/AgCl; pH = 1.7; 3 M KCl) for nitrate, oxygen, and perchlorate, respectively. The catalytic role of this microorganism was also observed by a decrease in the charge transfer resistance registered via electrochemical impedance spectroscopy. Five-day chronoamperometry of culture at pH = 1.7, USS-CCA7 showed a perchlorate removal rate of 19.106 ± 1.689 mgL-1 day-1 and a cathodic efficiency of 112 ± 5.2  %. Growth on electrodes was observed by epifluorescence and scanning electron microscopy. Interestingly, the results showed that toward higher pH, the cathodic peak of perchlorate is reduced in the voltammetric profiles. This study highlights the use of this psychrotolerant acidophile for the bioremediation of harsh perchlorate-pressured terrestrial under acidic conditions.

Absence of trehalose synthesis in Acidithiobacillus ferrooxidans ATCC 23270 and Acidithiobacillus ferrivorans CF27 at low temperature

Trehalose is a type of carbohydrate that protects against different types of stress and is also used as a source of carbon storage in prokaryotes. There are four different ways of synthesizing trehalose in Acidithiobacillus ferrivorans and two in Acidithiobacillus ferrooxidans, but its purpose remains unknown. This study aimed to measure the production of trehalose under different conditions by quantifying it in three culture media at two different temperatures. The growth kinetics of both species were also assessed, and the trehalose concentration was analysed during the early stationary phase using an enzymatic method. The results showed that the modified 9K medium with ferrous iron at 28°C had the highest production of trehalose, with A. ferrivorans CF27 having a higher production of 0.34 µmol/mg protein compared to A. ferrooxidans ATCC 23270 at 0.31 µmol/mg protein. When using CuS, the production of trehalose was lower, with 0.02 and 0.03 µmol/mg protein for A. ferrivorans CF27 and A. ferrooxidans ATCC 23270, respectively, while no trehalose was detected in the presence of zinc. At 15°C, the enzymatic method did not detect any trehalose in all three culture media, this would indicate that this carbohydrate does not protect against low temperatures in either species.

La trehalosa es un tipo de carbohidrato, que en procariotas protege contra diferentes tipos de estrés y también se utiliza como fuente de almacenamiento de carbono. Hay cuatro formas diferentes de sintetizar trehalosa en Acidithiobacillus ferrivorans y dos en Acidithiobacillus ferrooxidans, pero su propósito sigue siendo desconocido. Este estudio tuvo como objetivo medir la producción de trehalosa en diferentes condiciones mediante su cuantificación en tres medios de cultivo a dos temperaturas diferentes. También se evaluó la cinética de crecimiento de ambas especies y se analizó la concentración de trehalosa durante la fase estacionaria temprana mediante un método enzimático. Los resultados mostraron que el medio 9K modificado con hierro ferroso a 28 °C tuvo la mayor producción de trehalosa, con A. ferrivorans CF27 con una mayor producción de 0.34 µmol/mg de proteína en comparación con A. ferrooxidans ATCC 23270 a 0.31 µmol/mg de proteína. Al utilizar CuS, la producción de trehalosa fue menor, con 0.02 y 0.03 µmol/mg de proteína para A. ferrivorans CF27 y A. ferrooxidans ATCC 23270, respectivamente, mientras que en presencia de zinc no se detectó trehalosa. A 15°C, el método enzimático no detectó trehalosa en los tres medios de cultivo, lo que indicaria que este carbohidrato no protege contra las bajas temperaturas en ninguna de las especies.

Membrane vesicles in Acidithiobacillia class extreme acidophiles: influence on collective behaviors of 'Fervidacidithiobacillus caldus'.

Membrane vesicles (MVs) are envelope-derived extracellular sacs that perform a broad diversity of physiological functions in bacteria. While considerably studied in pathogenic microorganisms, the roles, relevance, and biotechnological potential of MVs from environmental bacteria are less well established. Acidithiobacillaceae family bacteria are active players in the sulfur and iron biogeochemical cycles in extremely acidic environments and drivers of the leaching of mineral ores contributing to acid rock/mine drainage (ARD/AMD) and industrial bioleaching. One key aspect of such a role is the ability of these bacteria to tightly interact with the mineral surfaces and extract electrons and nutrients to support their chemolithotrophic metabolism. Despite recent advances in the characterization of acidithiobacilli biofilms and extracellular matrix (ECM) components, our understanding of its architectural and mechanistic aspects remains scant. Using different microscopy techniques and nano-tracking analysis we show that vesiculation is a common phenomenon in distant members of the Acidithiobacillaceae family, and further explore the role of MVs in multicellular colonization behaviors using 'Fervidacidithiobacillus caldus' as a bacterial model. Production of MVs in 'F. caldus' occurred in both planktonic cultures and biofilms formed on sulfur surfaces, where MVs appeared individually or in chains resembling tube-shaped membranous structures (TSMSs) important for microbial communication. Liquid chromatography-mass spectrometry data and bioinformatic analysis of the MV-associated proteome revealed that 'F. caldus' MVs were enriched in proteins involved in cell-cell and cell-surface processes and largely typified the MVs as outer MVs (OMVs). Finally, microbiological assays showed that amendment of 'F. caldus' MVs to cells and/or biofilms affects collective colonizing behaviors relevant to the ecophysiology and applications of these acidophiles, providing grounds for their exploitation in biomining.

Progress in bioleaching: fundamentals and mechanisms of microbial metal sulfide oxidation - part A.

Bioleaching of metal sulfides is performed by diverse microorganisms. The dissolution of metal sulfides occurs via two chemical pathways, either the thiosulfate or the polysulfide pathway. These are determined by the metal sulfides' mineralogy and their acid solubility. The microbial cell enables metal sulfide dissolution via oxidation of iron(II) ions and inorganic sulfur compounds. Thereby, the metal sulfide attacking agents iron(III) ions and protons are generated. Cells are active either in a planktonic state or attached to the mineral surface, forming biofilms. This review, as an update of the previous one (Vera et al., 2013a), summarizes some recent discoveries relevant to bioleaching microorganisms, contributing to a better understanding of their lifestyle. These comprise phylogeny, chemical pathways, surface science, biochemistry of iron and sulfur metabolism, anaerobic metabolism, cell-cell communication, molecular biology, and biofilm lifestyle. Recent advances from genetic engineering applied to bioleaching microorganisms will allow in the future to better understand important aspects of their physiology, as well as to open new possibilities for synthetic biology applications of leaching microbial consortia. KEY POINTS: • Leaching of metal sulfides is strongly enhanced by microorganisms • Biofilm formation and extracellular polymer production influences bioleaching • Cell interactions in mixed bioleaching cultures are key for process optimization.

Characterization and genomic analysis of two novel psychrotolerant Acidithiobacillus ferrooxidans strains from polar and subpolar environments.

The bioleaching process is carried out by aerobic acidophilic iron-oxidizing bacteria that are mainly mesophilic or moderately thermophilic. However, many mining sites are located in areas where the mean temperature is lower than the optimal growth temperature of these microorganisms. In this work, we report the obtaining and characterization of two psychrotolerant bioleaching bacterial strains from low-temperature sites that included an abandoned mine site in Chilean Patagonia (PG05) and an acid rock drainage in Marian Cove, King George Island in Antarctic (MC2.2). The PG05 and MC2.2 strains showed significant iron-oxidation activity and grew optimally at 20°C. Genome sequence analyses showed chromosomes of 2.76 and 2.84 Mbp for PG05 and MC2.2, respectively, and an average nucleotide identity estimation indicated that both strains clustered with the acidophilic iron-oxidizing bacterium Acidithiobacillus ferrooxidans. The Patagonian PG05 strain had a high content of genes coding for tolerance to metals such as lead, zinc, and copper. Concordantly, electron microscopy revealed the intracellular presence of polyphosphate-like granules, likely involved in tolerance to metals and other stress conditions. The Antarctic MC2.2 strain showed a high dosage of genes for mercury resistance and low temperature adaptation. This report of cold-adapted cultures of the At. ferrooxidans species opens novel perspectives to satisfy the current challenges of the metal bioleaching industry.

Sticky bacteria: Combined effect of galactose and high ferric iron concentration on extracellular polymeric substances production and the attachment of Acidithiobacillus ferrooxidans on a polymetallic sulfide ore surface.

Adaptation and microbial attachment mechanisms for the degradation of sulfide ores are mediated by the production of extracellular polymeric substances (EPS) and their role in biofilm formation. EPS production responds to induction mechanisms associated with environmental conditions. In this study, the double induction of EPS with galactose and high ferric iron concentrations in planktonic cells of Acidithiobacillus ferrooxidans, and their attachment on the surface of a polymetallic sulfide ore from Bella Rica-Azuay in Ecuador were evaluated. A. ferrooxidans cells were previously adapted to different concentrations of galactose [0, 0.15, and 0.25% (w/v)], using two ferrous iron concentrations as an energy source (9 and 18 g L-1) in a 9K culture medium. EPS production and its effect on mineral attachment were determined at the time point of maximal growth. The results obtained show a maximum cell attachment of 94.1% within 2 h at 0.15% of galactose and 18 g⋅L-1 of ferric iron concentration, compared to 71.4% without galactose and 9 g⋅L-1 of ferric iron. The maximum concentration of EPS was obtained with a 0.25% galactose concentration; however, it did not result in greater attachment compared to 0.15% galactose concentration. Through the combined induction of low galactose concentration and high ferric iron concentration, the percentage of bacterial attachment can be increased and, therefore, a possible increase in the rate of biooxidation and bioleaching could be obtained.

Biofilm Formation Is Crucial for Efficient Copper Bioleaching From Bornite Under Mesophilic Conditions: Unveiling the Lifestyle and Catalytic Role of Sulfur-Oxidizing Bacteria.

Biofilm formation within the process of bioleaching of copper sulfides is a relevant aspect of iron- and sulfur-oxidizing acidophilic microorganisms as it represents their lifestyle in the actual heap/dump mining industry. Here, we used biofilm flow cell chambers to establish laminar regimes and compare them with turbulent conditions to evaluate biofilm formation and mineralogic dynamics through QEMSCAN and SEM-EDS during bioleaching of primary copper sulfide minerals at 30°C. We found that laminar regimes triggered the buildup of biofilm using Leptospirillum spp. and Acidithiobacillus thiooxidans (inoculation ratio 3:1) at a cell concentration of 106 cells/g mineral on bornite (Cu5FeS4) but not for chalcopyrite (CuFeS2). Conversely, biofilm did not occur on any of the tested minerals under turbulent conditions. Inoculating the bacterial community with ferric iron (Fe3+) under shaking conditions resulted in rapid copper recovery from bornite, leaching 40% of the Cu content after 10 days of cultivation. The addition of ferrous iron (Fe2+) instead promoted Cu recovery of 30% at day 48, clearly delaying the leaching process. More efficiently, the biofilm-forming laminar regime almost doubled the leached copper amount (54%) after 32 days. In-depth inspection of the microbiologic dynamics showed that bacteria developing biofilm on the surface of bornite corresponded mainly to At. Thiooxidans, while Leptospirillum spp. were detected in planktonic form, highlighting the role of biofilm buildup as a means for the bioleaching of primary sulfides. We finally propose a mechanism for bornite bioleaching during biofilm formation where sulfur regeneration to sulfuric acid by the sulfur-oxidizing microorganisms is crucial to prevent iron precipitation for efficient copper recovery.

Potential application of a knowledgebase of iron metabolism of Acidithiobacillus ferrooxidans as an alternative platform

BACKGROUND: Acidithiobacillus ferrooxidans is a facultative anaerobe that depends on ferrous ion oxidation as well as reduced sulfur oxidation to obtain energy and is widely applied in metallurgy, environmental protection, and soil remediation. With the accumulation of experimental data, metabolic mechanisms, kinetic models, and several databases have been established. However, scattered data are not conducive to understanding A. ferrooxidans that necessitates updated information informed by systems biology. RESULTS: Here, we constructed a knowledgebase of iron metabolism of A. ferrooxidans (KIMAf) system by integrating public databases and reviewing the literature, including the database of bioleaching substrates (DBS), the database of bioleaching metallic ion-related proteins (MIRP), the A. ferrooxidans bioinformation database (Af-info), and the database for dynamics model of bioleaching (DDMB). The DBS and MIRP incorporate common bioleaching substrates and metal ion-related proteins. Af-info and DDMB integrate nucleotide, gene, protein, and kinetic model information. Statistical analysis was performed to elucidate the distribution of isolated A. ferrooxidans strains, evolutionary and metabolic advances, and the development of bioleaching models. CONCLUSIONS: This comprehensive system provides researchers with a platform of available iron metabolism-related resources of A. ferrooxidans and facilitates its application.

Redox stress response and UV tolerance in the acidophilic iron-oxidizing bacteria Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans.

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 Fe3+, H2O2, K2CrO4, 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.

RNA transcript response by an Acidithiobacillus spp. mixed culture reveals adaptations to growth on arsenopyrite.

Biooxidation of gold-bearing refractory mineral ores such as arsenopyrite (FeAsS) in stirred tanks produces solutions containing highly toxic arsenic concentrations. In this study, ferrous iron and inorganic sulfur-oxidizing Acidithiobacillus strain IBUN Ppt12 most similar to Acidithiobacillus ferrianus and inorganic sulfur compound oxidizing Acidithiobacillus sp. IBUNS3 were grown in co-culture during biooxidation of refractory FeAsS. Total RNA was extracted and sequenced from the planktonic cells to reveal genes with different transcript counts involved in the response to FeAsS containing medium. The co-culture's response to arsenic release during biooxidation included the ars operon genes that were independently regulated according to the arsenopyrite concentration. Additionally, increased mRNA transcript counts were identified for transmembrane ion transport proteins, stress response mechanisms, accumulation of inorganic polyphosphates, urea catabolic processes, and tryptophan biosynthesis. Acidithiobacillus spp. RNA transcripts also included those encoding the Rus and PetI proteins involved in ferrous iron oxidation and gene clusters annotated as encoding inorganic sulfur compound metabolism enzymes. Finally, mRNA counts of genes related to DNA methylation, management of oxidative stress, chemotaxis, and motility during biooxidation were decreased compared to cells growing without mineral. The results provide insights into the adaptation of Acidithiobacillus spp. to growth during biooxidation of arsenic-bearing sulfides.

Comparative genomic analysis of two novel plasmids from Acidithiobacillus ferrivorans strain PQ33 / Análisis genómico comparativo de dos nuevos plásmidos de la cepa PQ33 de Acidithiobacillus ferrivorans

Abstract Acidithiobacillus ferrivorans is a psychrotolerant acidophile capable of growing and oxidizing ferrous and sulphide substrates at low temperatures. To date, six genomes of this organism have been characterized; however, evidence of a plasmid in this species has been reported only once, whereby there is no conclusive role of the plasmids in the species. Herein, two novel plasmids of A. ferrivorans PQ33 were molecularly characterized and compared at a genomic scale. The genomes of two plasmids (12 kbp and 10 kbp) from A. ferrivorans PQ33 (NZ_LVZL01000000) were sequenced and annotated. The plasmids, named pAfPQ33-1 (NZ_CP021414.1) and pAfPQ33-2 (NZ_CP021415.1), presented 9 CDS and 13 CDS, respectively. In silico analysis showed proteins involved in conjugation (TraD, MobA, Eep and XerD), toxin-antitoxin systems (HicA and HicB), replication (RepA and DNA binding protein), transcription regulation (CopG), chaperone DnaJ, and a virulence gene (vapD). Furthermore, the plasmids contain sequences similar to phosphate-selective porins O and P and a diguanylate cyclase-phosphodiesterase protein. The presence of these genes suggests the possibility of horizontal transfer, a regulatory system of plasmid maintenance, and adhesion to substrates for A. ferrivorans species and PQ33. This is the first report of plasmids in this strain.

Resumen Acidithiobacillus ferrivorans es un acidófilo psicrotolerante capaz de hacer crecer y oxidar sustratos ferrosos y sulfurosos a bajas temperaturas. Hasta la fecha se han caracterizado seis genomas de este organismo; sin embargo, la evidencia de un plásmido en esta especie ha sido informado solo una vez, por lo que no hay un rol concluyente de los plásmidos en la especie. Aquí, dos plásmidos novedosos de A. ferrivorans PQ33 se caracterizaron molecularmente y se compararon a escala genómica. Se secuenciaron y anotaron los genomas de dos plásmidos (12 kpb y 10 kpb) de A. ferrivorans PQ33 (NZ_LVZL01000000). Los plásmidos, denominados pAfPQ33-1 (NZ_CP021414.1) y pAfPQ33-2 (NZ_CP021415.1), presentaron 9 CDS y 13 CDS, respectivamente. El análisis in silico mostró proteínas involucradas en la conjugación (TraD, MobA, Eep y XerD), sistemas de toxina-antitoxina (HicA y HicB), replicación (RepA y proteína de unión al ADN), regulación de la transcripción (CopG), chaperona DnaJ y un gen de virulencia (vapD). Además, los plásmidos contienen secuencias similares a las porinas selectivas de fosfato O y P y una proteína diguanilato ciclasa-fosfodiesterasa. La presencia de estos genes sugiere la posibilidad de transferencia horizontal, un sistema regulador de mantenimiento de plásmidos y adhesión a sustratos para especies de A. ferrivorans y PQ33. Este es el primer informe de plásmidos en esta cepa.

High copper concentration reduces biofilm formation in Acidithiobacillus ferrooxidans by decreasing production of extracellular polymeric substances and its adherence to elemental sulfur.

Acidithiobacillus ferrooxidans is an acidophilic bacterium able to grow in environments with high concentrations of metals. It is a chemolithoautotroph able to form biofilms on the surface of solid minerals to obtain its energy. The response of both planktonic and sessile cells of A. ferrooxidans ATCC 23270 grown in elemental sulfur and adapted to high copper concentration was analyzed by quantitative proteomics. It was found that 137 proteins varied their abundance when comparing both lifestyles. Copper effllux proteins, some subunits of the ATP synthase complex, porins, and proteins involved in cell wall modification increased their abundance in copper-adapted sessile lifestyle cells. On the other hand, planktonic copper-adapted cells showed increased levels of proteins such as: cupreredoxins involved in copper cell sequestration, some proteins related to sulfur metabolism, those involved in biosynthesis and transport of lipopolysaccharides, and in assembly of type IV pili. During copper adaptation a decreased formation of biofilms was measured as determined by epifluorescence microscopy. This was apparently due not only to a diminished number of sessile cells but also to their exopolysaccharides production. This is the first study showing that copper, a prevalent metal in biomining environments causes dispersion of A. ferrooxidans biofilms. SIGNIFICANCE: Copper is a metal frequently found in high concentrations at mining environments inhabitated by acidophilic microorganisms. Copper resistance determinants of A. ferrooxidans have been previously studied in planktonic cells. Although biofilms are recurrent in these types of environments, the effect of copper on their formation has not been studied so far. The results obtained indicate that high concentrations of copper reduce the capacity of A. ferrooxidans ATCC 23270 to form biofilms on sulfur. These findings may be relevant to consider for a bacterium widely used in copper bioleaching processes.

Early reprecipitation of sulfate salts in coal biodesulfurization processes using acidophilic chemolithotrophic bacteria.

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).

Attachment of Acidithiobacillus ferrooxidans to pyrite in fresh and saline water and fitting to Langmuir and Freundlich isotherms.

OBJECTIVE: This study aims to investigate the attachment of Acidithiobacillus ferrooxidans to pyrite in two different environments: fresh and saline water (water with 35 g/L of NaCl or 0.6 M). Adsorption isotherms were analyzed using the Langmuir and Freundlich models. Saline water is water with 35 g/L of NaCl (0.6 M), which is the concentration of NaCl in seawater. The use of raw seawater in mining is becoming relevant in leaching and flotation process. At the same time the use of microorganisms in both processes is gaining attention. For this reason, it is important to study the behavior of adherence of microorganisms to minerals in saline aqueous environments, similar to seawater. RESULTS: The bacteria showed a higher level of attachment to pyrite in fresh water than in saline water. The Langmuir model fitted better the experimental data obtained in fresh water than in saline water with a coefficient of determination (R2) of 0.85 and 0.61 for fresh and saline water, respectively. CONCLUSIONS: This suggests that the bacteria tend to adhere more as a monolayer in fresh than in saline water in the early stage of adhesion.

Bacterial influence on storage and mobilisation of metals in iron-rich mine tailings from the Salobo mine, Brazil.

In this study we investigated the potential effects of promoting bacterial activity on tailings from the Salobo iron-oxide copper­gold (IOCG) mine, Brazil. In particular we focussed on (1) the potential for mobilising additional Cu and (2) the effects of long-term storage on other metals. Unlike typical sulphide-ore tailings, the pH of the Salobo tailings is circumneutral and these tailings are dominated by Fe-bearing silicates and magnetite, with minor micrometre-scale encapsulated Cu-bearing sulphides. While these tailings do not produce acid mine drainage, an endemic strain of Acidithiobacillus ferrooxidans was isolated from the mine site. These bacteria were used in laboratory column leaching experiments of tailings material, which ran for up to 395 days, without the addition of ferrous iron. Bacteria-tailings interactions were typically maintained at a pH > 5, due to silicate-mediated pH buffering. This was eventually overcome after ~200 days by regular addition of acidic (pH 2.2) nutrient solution, as well as growth and acid generation by bacteria. Copper dissolution was not significantly enhanced by bacterial activity compared to abiotic control experiments while pH was >5. However, as the experiments were progressively acidified, additional Cu was mobilised in the biotic systems. The mineral alteration reactions produced abundant ferrihydrite precipitates within the tailings, which was enhanced by bacterial activity as the pH decreased. Adsorption of metal cations to these precipitates ensured that effluent solutions had only low levels (<0.5 mg/l) of dissolved trace metals such as As, Co, Pb, Zn, Se, Ni and Cr. These adsorption processes will strongly inhibit metal leaching from the tailings during long-term storage, as long as the iron oxidising bacteria are producing the requisite excess of ferrihydrite and the pH is >5. This case study shows that bacterially-mediated silicate weathering, in Fe(II)-bearing silicate rich tailings with only minor sulphides and Acidithiobacillus ferrooxidans can enhance the environmental stability of the tailings.

Effect of CO2 Concentration on Uptake and Assimilation of Inorganic Carbon in the Extreme Acidophile Acidithiobacillus ferrooxidans.

This study was motivated by surprising gaps in the current knowledge of microbial inorganic carbon (Ci) uptake and assimilation at acidic pH values (pH < 3). Particularly striking is the limited understanding of the differences between Ci uptake mechanisms in acidic versus circumneutral environments where the Ci predominantly occurs either as a dissolved gas (CO2) or as bicarbonate (HCO3 -), respectively. In order to gain initial traction on the problem, the relative abundance of transcripts encoding proteins involved in Ci uptake and assimilation was studied in the autotrophic, polyextreme acidophile Acidithiobacillus ferrooxidans whose optimum pH for growth is 2.5 using ferrous iron as an energy source, although they are able to grow at pH 5 when using sulfur as an energy source. The relative abundance of transcripts of five operons (cbb1-5) and one gene cluster (can-sulP) was monitored by RT-qPCR and, in selected cases, at the protein level by Western blotting, when cells were grown under different regimens of CO2 concentration in elemental sulfur. Of particular note was the absence of a classical bicarbonate uptake system in A. ferrooxidans. However, bioinformatic approaches predict that sulP, previously annotated as a sulfate transporter, is a novel type of bicarbonate transporter. A conceptual model of CO2 fixation was constructed from combined bioinformatic and experimental approaches that suggests strategies for providing ecological flexibility under changing concentrations of CO2 and provides a portal to elucidating Ci uptake and regulation in acidic conditions. The results could advance the understanding of industrial bioleaching processes to recover metals such as copper at acidic pH. In addition, they may also shed light on how chemolithoautotrophic acidophiles influence the nutrient and energy balance in naturally occurring low pH environments.

Electrochemical monitoring of Acidithiobacillus thiooxidans biofilm formation on graphite surface with elemental sulfur.

Inorganic wastewaters and sediments from the mining industry and mineral bioleaching processes have not been fully explored in bioelectrochemical systems (BES). Knowledge of interfacial changes due to biofilm evolution under acidic conditions may improve applications in electrochemical processes, specifically those related to sulfur compounds. Biofilm evolution of Acidithiobacillus thiooxidans on a graphite plate was monitored by electrochemical techniques, using the graphite plate as biofilm support and elemental sulfur as the only energy source. Even though the elemental sulfur was in suspension, S0 particles adhered to the graphite surface favoring biofilm development. The biofilms grown at different incubation times (without electric perturbation) were characterized in a classical three electrode electrochemical cell (sulfur and bacteria free culture medium) by non-invasive electrochemical impedance spectroscopy (EIS) and cyclic voltammetry. The biofilm structure was confirmed by Environmental Scanning Electrode Microscopy, while the relative fractions of exopolysaccharides and extracellular hydrophobic compounds at different incubation times were evaluated by Confocal Laser Scanning Microscopy. The experimental conditions chosen in this work allowed the EIS monitoring of the biofilm growth as well as the modification of Extracellular Polymeric Substances (EPS) composition (hydrophobic/ exopolysaccharides EPS ratio). This strategy could be useful to control biofilms for BES operation under acidic conditions.