This study aimed to reveal the specific mechanism of extracellular polymeric substances (EPS) in the bioleaching of copper-bearing sulfide ore by moderately thermophilic bacterium Acidithiobacillus caldus. The bioleaching performance of blank control (BC), planktonic cell deficient (PD), attached cell deficient (AD), and EPS deficient (ED) systems were compared, to investigate the specific functions of “non-contact” and “contact” (including direct contact and, EPS-mediated contact) mechanisms. The detailed mechanics of bioleaching were studied using μ_x of cell growth, scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The μ_x of cell growth demonstrated that EPS favors planktonic and attached cell growth. SEM observation revealed that intensive micro-pores on slag benefitted from the “EPS-mediated contact” mechanism. XRD identification indicated that additional chemical derivatives were produced via “EPS-mediated contact” mechanism, because of the active iron/sulfur metabolism. FTIR analysis revealed that the absorption peaks of C-O-S, sulfate, and S = O, which are closely associated with sulfur metabolism, have significant influences of EPS secretion. Taken together, the “EPS-mediated contact” mechanism contributed to almost half of the “contact” mechanism efficiency and a quarter of the total bioleaching efficiency. The proposed specific mechanism of EPS can deepen our understanding of similar bioleaching processes.

Bioleaching, an alternative environmental smelting technology, typically uses high concentrations of heavy metal ions, especially in the subsequent phase, due to metal ion accumulation from the mineral. In this study, we analyzed the overall response of the bioleaching microorganism Acidithiobacillus caldus to copper stress through physiological and transcriptomic analyses. Scanning electron microscopy results showed higher extracellular polymeric substances secretion and cell aggregation under copper stress. Intracellular levels of glutamic acid, glycine and cysteine increased, favoring the synthesis of glutathione for maintenance of the oxidation–reduction state. GSH, during copper stress conditions, the activity of GSH-PX and CAT increased, resulting in reduced oxidative damage while maintaining stable intracellular pH. Higher unsaturated and cyclopropane fatty acid levels resulted in increased membrane fluidity and compactness and decreased ATP levels to support the energy requirements for stress resistance. Initially, H⁺-ATPase activity increased to provide energy for proton output and decreased later at higher copper ion stress. From transcriptome analysis, 140 genes were differentially expressed under low copper stress (1 g/L), while 250 genes exhibited altered transcriptional levels at higher copper stress (3 g/L). These differentially expressed genes were involved primarily in metabolic pathways such as energy metabolism, two-component systems, amino acid metabolism, and signal transduction. The Sox family cluster gene cluster involved in the conversion of thiosulfate to sulfate was upregulated in the sulfur metabolism pathway. In the oxidative phosphorylation pathway, genes participating in the synthesis of NADH oxidoreductase and cytochrome c oxidase, nuoL, cyoABD (cyoA, cyoB and cyoD) and cydAB (cydA and cydB), were downregulated. The TCS element ompR, closely associated with the osmotic pressure, exhibited active response, while Cu²⁺ efflux system gene cusRS was upregulated. In the amino acid metabolism, the glnA involved in nitrogen fixation was upregulated and promoted the synthesis of glutamine synthetase for reducing excessive oxidative stress. This study provides new insights into the mechanism underlying A. caldus response to heavy-metal ion stress under harsh bioleaching conditions.