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Acidithiobacillus ferrooxidans, together with other microorganisms, has an important role on biohydrometallurgical processes. Such bacterium gets its energy from the oxidation of ferrous ion and reduced sulfur; in the first case, the accumulation of ferric ion as a product can cause its inhibition. It is known that the extracellular polymeric substances (EPS) may have an important role in the adaptation and tolerance to diverse inhibiting conditions. In the present study, it was tested how D-galactose can influence the production of extracellular polymeric substances (EPS) on At. ferrooxidans by evaluating at the same time its biooxidant activity and capacity to tolerate high concentrations of ferric ion. The visualization and quantification of EPS was done through a confocal laser scanning microscope (CLSM). The results show that at low cellular concentrations, the D-galactose inhibits the microbial growth and the biooxidation of ferrous ion; however, when the quantity of microorganisms is high enough, the inhibition is not present. By means of chemostat tests, several concentrations of D-galactose (0; 0.15; 0.25; and 0.35%) were evaluated, thus reaching the highest production of EPS when using 0.35% of this sugar. In cultures with such concentration of D-galactose, the tolerance of the bacterium was tested at high concentrations of ferric ion and it was compared with cultures in which sugar was not added. The results show that cultures with D-galactose reached a higher tolerance to ferric ion (48.15 ± 1.9 g L −1 ) compare to cultures without adding D-galactose (38.7 ± 0.47 g L −1 ferric ion). Also it was observed a higher amount of EPS on cells growing in the presence of D-galactose suggesting its influence on the greater tolerance of At. ferrooxidans to ferric ion. Therefore, according to the results, the bases of a strategy are considered to overproduce EPS by means of At. ferrooxidans in planktonic state, so that, it can be used as a pre-treatment to increase its resistance and tolerance to high concentrations of ferric ion and improve the efficiency of At. ferrooxidans when acting in biohydrometallurgical processes. Frontiers in Microbiology | www.frontiersin.org 1 April 2020 | Volume 11 | Article 759 Saavedra et al. Biooxidation of Iron by Acidithiobacillus ferrooxidans in the Presence of D-Galactose GRAPHICAL ABSTRACT | Planktonic cells of At. ferrooxidans produce EPS only when grown in the presence of D-galactose as can be seen in the CLSM images. Cells having EPS on their surface can tolerate higher iron concentrations compare to cells without a layer of EPS surrounding them.

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The presence of extracellular polymeric substances (EPS) and their relevance for biofilm formation on the mineral surface for a variety of microbial species play a fundamental role in the degradation of sulfide ores. EPS production is associated with induction or auto induction mechanisms as a response of bacteria to environmental conditions. In this study, we tested galactose as an inducer of EPS production in planktonic cells of Acidithiobacillus thiooxidans DSM 14887T and their adherence to polymetallic mineral surfaces. Cells of At. thiooxidans were first adapted to grow at different concentrations of galactose (0.15, 0.25, 0.35%) using a modified 9K liquid medium and elemental sulfur as the energy source. In order to determine EPS production, the microorganisms were grown for 24 hours at different concentrations of galactose. Our results showed a cell adherence of 84% cells within 4 hours in presence of 0.15% galactose compared to 70% without galactose. The optimal concentration of galactose for maximal EPS production was 0.25% and for the attachment of cells it was 0.15%. Higher galactose concentrations inhibited microbial growth and decreased the number of cells attached to the mineral. While with a small amount of galactose in the culture media can shift the balance between sessile cells and planktonic cells, generating an increase in adhesion and therefore a possible increase of the bioleaching rate.

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.

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