Peptides, not vitamins: Yeast extract components sustaining glucose respiration of the halotolerant acidophile Sulfobacillus thermosulfidooxidans

The use of Sulfobacillus thermosulfidooxidans DSM 9293 T in bioleaching of sulfidic ores under saline conditions is limited by chloride-induced stress, despite some inherent halotolerance of the species. While yeast extract supplementation can improve chloride tolerance, its undefined composition hinders process standardization. To address this, we systematically investigated which components of yeast extract are essential for supporting respiration of Sulfobacillus thermosulfidooxidans , under assay conditions without additional NaCl, aiming to replace it with compositionally simpler medium supplementation. Our comparative assessments confirmed Sulfobacillus thermosulfidooxidans as the most halotolerant among the tested Sulfobacillus species, tolerating up to 500 mM NaCl (approximately 17.7 g Cl⁻ l⁻¹) while maintaining Fe(II)-oxidizing activity. Respiration assays revealed that casamino acids (CA), glucose, and potassium supported substantial respiratory activity and reached an endpoint comparable to that obtained with YE by day 7, although with a delayed onset, whereas vitamins alone had minimal effect. Further omission experiments identified the glutamate, aspartate, histidine, and alanine/branched-chain amino acid groups as important contributors to respiration. Molecular weight fractionation indicated that yeast extract fractions between 0.5 and 1 kDa primarily stimulated respiration. Separately, FT-ICR-MS analysis of CA-derived material identified 118 unique elemental formulas compatible with di- or tripeptide compositions, of which 26 showed culture-associated disappearance relative to uninoculated controls. Although high-resolution FT-ICR-MS provides accurate elemental compositions, without fragmentation data it cannot distinguish between isomeric peptide sequences or isobaric residues sharing the same molecular formula. Therefore, these assignments were interpreted as candidate peptide compositions rather than sequence-resolved peptides. Together, these findings demonstrate that CA, glucose, and potassium can sustain glucose-dependent respiration to an endpoint comparable to YE, although with slower respiratory activation. CA nevertheless remains chemically complex and therefore represents a compositionally simpler rather than fully defined supplementation strategy. Whether this supplementation provides comparable support under chloride stress remains to be tested directly.