Increased chalcopyrite bioleaching capabilities of extremely thermoacidophilic Metallosphaera sedula inocula by mixotrophic propagation

Extremely thermoacidophilic Crenarchaeota belonging to the order Sulfolobales, such as Metallosphaera sedula, are metabolically versatile and of great relevance in bioleaching. However, the impacts of extreme thermoacidophiles propagated with different energy substrates on subsequent bioleaching of refractory chalcopyrite remain unknown. Transcriptional responses underlying their different bioleaching potentials are still elusive. Here, it was first showed that M. sedula inocula propagated with typical energy substrates have different chalcopyrite bioleaching capabilities. Inoculum propagated heterotrophically with yeast extract was deficient in bioleaching; however, inoculum propagated mixotrophically with chalcopyrite, pyrite or sulfur recovered 79%, 78% and 62% copper, respectively, in 12 days. Compared with heterotrophically propagated inoculum, 937, 859 and 683 differentially expressed genes (DEGs) were identified in inoculum cultured with chalcopyrite, pyrite or sulfur, respectively, including upregulation of genes involved in bioleaching-associated metabolism, e.g., Fe2+ and sulfur oxidation, CO2 fixation. Inoculum propagated with pyrite or sulfur, respectively, shared 480 and 411 DEGs with chalcopyrite-cultured inoculum. Discrepancies on repertories of DEGs that involved in Fe2+ and sulfur oxidation in inocula greatly affected subsequent chalcopyrite bioleaching rates. Novel genes (e.g., Msed_1156, Msed_0549) probably involved in sulfur oxidation were first identified. This study highlights that mixotrophically propagated extreme thermoacidophiles especially with chalcopyrite should be inoculated into chalcopyrite heaps at industrial scale.