Three kinds of samples (acid mine drainage, coal mine wastewater, and thermal spring) derived from different sites were collected in China. Thereafter, these samples were combined and then inoculated into a basal salts solution in which different substrates (ferrous sulfate, elemental sulfur, and chalcopyrite) served as energy sources. After that, the mixed cultures growing on different substrates were pooled equally, resulting in a final mixed culture. After being adapted to gradually increasing pulp densities of chalcopyrite concentrate by serial subculturing for more than 2 years, the final culture was able to efficiently leach the chalcopyrite at a pulp density of 20% (wt/vol). At that pulp density, the culture extracted 60.4% of copper from the chalcopyrite in 25 days. The bacterial and archaeal diversities during adaptation were analyzed by denaturing gradient gel electrophoresis and constructing clone libraries of the 16S rRNA gene. The results show that the culture consisted mainly of four species, including Leptospirillum ferriphilum , Acidithiobacillus caldus , Sulfobacillus acidophilus , and Ferroplasma thermophilum , before adapting to a pulp density of 4%. However, L. ferriphilum could not be detected when the pulp density was greater than 4%. Real-time quantitative PCR was employed to monitor the microbial dynamics during bioleaching at a pulp density of 20%. The results show that A. caldus was the predominant species in the initial stage, while S. acidophilus rather than A. caldus became the predominant species in the middle stage. F. thermophilum accounted for the greatest proportion in the final stage.

Human milk is the main source of nutrition for infants and the transmission of various microorganisms. The lactic acid bacteria (LAB) in breast milk allow for the establishment of the gut microflora of infants. In this study, we aimed to assess the probiotic potential of LAB strains isolated from breast milk of healthy Chinese women. Two strains, Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus) LHL6 and LHL7, were selected and identified through morphology observation, Gram staining, and 16S rDNA phylogenetic analysis. Using Limosilactobacillus fermentum (formerly Lactobacillus fermentum) CECT5716 as the standard reference strain, the screened strains were characterized for aspects of growth, production of lactic acid and H₂O₂, antibiotic susceptibility, survival under simulated gastrointestinal conditions, and tolerance to cadmium (Cd). In de Man, Rogosa, and Sharpe (MRS) broth, LHL6 and LHL7 showed longer lag phases than CECT5716 but higher specific growth rates. For the production of lactic acid and H₂O₂, LHL7 performed better than LHL6 and CECT5716, indicating better antimicrobial ability. Strain LHL7 generated 9.99 mg/L H₂O₂, considerably higher than 1.25 mg/L for LHL6 and 2.33 mg/L for CECT5716. According to European Food Safety Authority minimum inhibitory concentrations, all of the investigated strains were resistant to chloramphenicol, streptomycin, and kanamycin. However, unlike LHL6 and CECT5716, LHL7 was susceptible to ampicillin and resistant to tetracycline. Resistance to azithromycin, cephalexin, and penicillin G were similar for all 3 strains, whereas CECT5716 was resistant to a higher concentration of roxithromycin. All 3 strains were able to survive in a simulated gastric-like solution, but a low percentage survived in the presence of 0.4% bile salt and 7% pancreatin. Encapsulation with protectants may enhance the survival rate. All 3 strains were tolerant to 500 mg/L Cd in MRS broth and to 1,000 mg/L Cd on MRS agar medium. In summary, 2 novel strains of LAB were obtained that have similar characteristics to the reference strain CECT5716. This work identified potential probiotic candidates for application in the food and pharmaceutical industries and facilitated identification of further probiotics.