Biofilm formation and development during the pyrite bioleaching of moderately thermophilic microorganisms

Bioleaching is a process that uses microorganisms to recover metals from intractable metal sulfide ores. Metal sulfide bioleaching is an interfacial process, and biofilm growth is crucial during the early phases. To study the interactions between microbes and minerals during the bioleaching of moderately thermophilic microorganisms, the formation and development of biofilms on pyrite coupons incubated in shake flasks for 40 days were evaluated. The biofilm biomass progressively increased as bioleaching proceeded. The most extensive biofilm was formed on day 30, when bacteria were attached to the mineral surface or submerged in extracellular polymeric substances. Confocal laser scanning microscopy indicated that the biofilm biomass of pyrite on day 40 was 6.55, 5.28, 3.17, and 1.70 times higher than that on days 5, 10, 20, and 30, respectively. Extracellular protein content exhibited an increasing trend throughout the process, whereas extracellular polysaccharides played a significant role during the early stages. Live/dead staining showed the highest mortality rate of 94% when cells were in contact with pyrite in the early stage, and the percentage of live bacteria increased in the later period. In the bioleaching environment, mature biofilms appeared to form relatively quickly after day 20. Real-time quantitative fluorescent quantitative analysis indicated that biofilm formation played an important role in defense against hostile external environments. In this study, biofilm formation and the associated RNA transcript numbers of moderately thermophilic microorganisms during pyrite bioleaching were investigated, contributing to the understanding of cell-mineral interactions.