Involvement of Reactive Oxygen Species in the Early Stages of Biofilm Formation on Metal Sulphide Surfaces by the Acidophile Leptospirillum sp. CF-1

Bioleaching of metals from sulphide minerals is an interfacial process that is promoted by biofilm formation. Since sulphide minerals spontaneously produce H₂O₂, this study evaluated the role of this compound in the early adherence of the acidophilic bioleaching bacterium Leptospirillum sp. CF-1 to different substrates. During 8 h of incubation under acidic conditions, the minerals produced micromolar amounts of H₂O₂ (pyrite > chalcopyrite > sphalerite). As expected, suspensions containing quartz did not produce detectable amounts of H₂O₂. In agreement, adherence to pyrite, chalcopyrite, and sphalerite steadily increased during the 8 h of the assays, while no adherence to quartz was detected. After 8 h of incubation, the highest and lowest adherence were observed for pyrite (94 % ± 1.8) and sphalerite (73 % ± 4), respectively. Furthermore, incubation of the strain CF-1 in the presence of 10 μM H₂O₂ for 2 and 6 h led to an increase in early-stage biofilm formation compared to non-exposed biofilms. In agreement, 10 μM H₂O₂ also significantly stimulated the adherence of the strain to various sulphide minerals and quartz, suggesting that H₂O₂ itself has a key effect on attachment to different substrates. Exposure of strain CF-1 to 10 μM H₂O₂ also resulted in an increase in the expression of genes for flagellin (flaA), type IV pili (pilV), diffusible signal factor (dfs), and genes related to the synthesis of EPS (epsDHI). Altogether, these results suggest that H₂O₂ could represent an important environmental signal that favours the adherence of the microorganism to the mineral by regulating the expression of genes related to adherence and biofilm formation.