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.

Size, shape and surface characteristics strongly affect interfacial interactions, as the presented among iron oxide nanoparticles (NPs) aqueous colloids and bacteria. In other to find the forces among this interaction, we compare three types of surface modified NPs (exposing oxalate, arginine or cysteine residues), based on a simple synthesis and derivation procedure, that allows us to obtain very similar NPs (size and shape of the magnetic core). In this way, we assure that the main difference in the synthesized NPs are the oxalate or amino acid residue exposed, an ideal situation to compare their bacterial capture performance, and so too the interactions among them. Field emission scanning electron microscopy showed homogeneous distribution of particle sizes for all systems synthesized, close to 10 nm. Magnetization, zeta potential, Fourier transformed infrared spectrometry and other studies allow us further characterization. Capture experiments of Pseudomonas putida bacterial strain showed a high level of efficiency, independently of the amino acid used to wrap the NP, when compared with oxalate. We show that bacterial capture efficiency cannot be related mostly to the bacterial and NP superficial charge relationship (as determined by z potential), but instead capture can be correlated with hydrophobic and hydrophilic forces among them.

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The presence of extracellular polymeric substances (EPS) is important in the building of biofilms on mineral surfaces, increasing the bioleaching activity, as well as protecting the cells from adverse environmental conditions. The objective of this work was to study the effect of galactose in EPS production by Acidithiobacillus ferrooxidans. The experiences were performed in shake flask of 250 mL at 30 °C, 200 rpm and at an initial pH of 1.8. In order to establish the natural tolerance of the strain, its growth behaviour was evaluated at high ferric iron concentrations by adding consecutively the equivalent of 9 g/L of ferrous iron each time it was depleted in the broth. Cell growth stopped once ferric iron concentration increased up to 38 g/L. In order to determine the optimal conditions for EPS production, experiments were run in a chemostat of 0.5 L, operated at a constant dilution rate of 0.03 h-1. Different steady states were obtained varying feeding concentrations of galactose (0.15%; 0.25% and 0.35%) and carbon dioxide (180 ppm and 360 ppm). , Cells grown in the chemostat at optimum operation conditions were used as inoculum to determine oxidative capacity of the microorganisms overproducing EPS. The EPS was quantified using confocal laser scanning microscopy (CLSM), labelling the cells with propidium iodide and EPS carbohydrates with wheat germ agglutinin (WGA). The higher volume production of EPS was observed in cells grown using 360 ppm of CO2 and 0.35% of galactose. Also it was observed a size increment of cells, compared to cells grown in culture medium having 9 g/L of ferrous iron where presence of EPS was no detected. The results revealed that EPS overproducing A. ferrooxidans showed a tolerance to ferric iron concentration almost 9.5 g/L higher than the natural tolerance of cells grown in absence of galactose. Presence of galactose in culture medium stimulated the EPS production.

Extracellular polymeric substances (EPS) play an important role in the attachment of bacteria to sulphide minerals, biofilm formation and efficiency of the bioleaching process. Previous studies have suggested a potential connection between galactose and EPS formation. In this context, the influence of exogenous galactose on EPS formation during the bioleaching of pyrite was studied. In order to fully adapt the microorganism to bioleaching conditions it was performed a total of five consecutive sub cultures, one every fifteen days, taking for each one inocula from previous culture in shake flasks with 200 ml of fermentation medium at 30°C, 200 rpm, 40 gL-1 mineral and an initial pH of 1,8. Assays were performed in a medium supplemented with exogenous galactose (0.25% w/v) and without exogenous galactose (control), both with an initial concentration of ferric sulphate in the first three sub cultures of 5 gL-1, decreasing in the last two sub cultures to 2.5 gL-1. Samples of three cultures in both conditions were analyzed using confocal laser scanning microscopy (CLSM) labelling the cells with propidium iodide and EPS carbohydrates with Wheat Germ Agglutinin (WGA). Samples obtained on the last day of the fifth culture showed that the EPS layer on the particle surface was 5.00 μm3/μm2 in the case of the control condition and 6.10 μm3/μm2 when bioleaching was carried out in the presence of exogenous galactose. Also it was observed that in the fifth sub culture the volumetric productivity of total iron in the control experiment was 0.0065 gL-1.h-1 compared with 0.0076 gL-1.h-1 obtained in presence of galactose. The results reveal that the presence of galactose in the bioleaching solution stimulates EPS's formation and apparently also favour the pyrite bioleaching process.