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

Abstract

We have applied epifluorescence principles, atomic force microscopy, and Raman studies to the analysis of the colonization process of pyrite (FeS(2)) 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 (n) (2-) /S(0) 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 (n) (2-) /S(0) 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.

Influence of the sulfur species reactivity on biofilm conformation during pyrite colonization by Acidithiobacillus thiooxidans

“…Results similar to these mentioned above suggest the addition of SOM to improve the overall bioleaching processes of pyrite and other MS (Sand et al 2001;Rohwerder et al 2003;Rohwerder and Sand 2007;Liu et al 2008). Despite the pivotal role of SOM in bioleaching, the mechanisms involved in the biooxidation of S n 2− and S 0 species and interfacial aspects associated with MS colonization by SOM, have been rarely studied (Sasaki et al 1998;Sand et al 2001;Liu et al 2003;Lara et al 2010;Noël et al 2010;González et al 2011). Bioleaching kinetics mainly depends on factors associated with the mineral-microorganism interfacial system set up during microbial attachment to the MS surface (Sharma et al 2003;Lizama et al 2003;Harneit et al 2006;Zeng et al 2010), and this attachment is mediated by extracellular polymeric substances (EPS) of biofilm-forming microorganisms.…”

“…Average roughness (R a , in nanometers) and root mean square roughness (R q , in nanometers) of each eMPE surface was evaluated to obtain topographic information associated with secondary phase formation. Further details concerning AFM and Raman usage are found in previous works (Lara et al 2010;González et al 2011). …”

“…After 2 h of UV irradiation, the sterile eMPE surfaces were transferred to the ATCC-125 growth culture (50 ml; pH 2) with A. thiooxidans cells (~10 8 cells/ml); these biotic assays were incubated aerobically at 28-30°C during 24 h (biooxidized surfaces). The incubation time was chosen on the basis of previous results (González et al 2011). Abiotic control experiments were also carried out to compare between the chemical and biological oxidation of S n 2− /S 0 species.…”

“…The biofilm was stained for CLSM (Leica DMI4000B with a 63× immersion objective) analysis of the exopolysaccharides (α-mannose and α-glucose) using lectin Canavalia ensiformis (Con-A, tetramethylrhodamine conjugated; Molecular Probes, Eugene, OR, USA), according to González et al (2011). Afterwards, in order to stain the nucleic acids (DNA and RNA), the fluorochrome Acridine Orange (AO, Sigma Aldrich) was applied during 30 min, in the dark at 30°C.…”

“…Since the biofilm formation on MS could be importantly affected by the application of chemical, physical or biological treatments to the MS surface (Noël et al 2010) and because the ratio of S n 2− /S 0 formed during pyrite oxidation under acidic conditions may vary (Mycroft et al 1990), the control of the resultant surface properties could either enhance or inhibit the biooxidation of pyrite and therefore influence the biofilm organization. In earlier study, we showed that the combined application of atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM) and Raman spectroscopy allows obtaining interfacial insights during colonization and biofilm formation by A. thiooxidans cells on electrochemically modified pyrite surfaces (González et al 2011). Application of a controlled potential pulse allowed the generation of S n 2− and S 0 species on modified pyrite surface, offering a colonization substrate for A. thiooxidans.…”