Micron-scale Fe²⁺/Fe³⁺, intermediate sulfur species and O₂ gradients across the biofilm-solution-sediment interface control biofilm organization

We measured micron-scale Fe²⁺/Fe³⁺ and intermediate sulfur species gradients across the biofilm-solution interface and defined the microbial community composition in natural and bioreactor-cultivated acid mine drainage biofilms to investigate how community organization correlated with geochemical conditions. Intermediate sulfur species concentrations were also measured in associated sediments. Under initial conditions of high Fe²⁺ and O₂ concentrations, the first biofilm colonists were Leptospirillum Group II, UBA genotype, and a few Archaea. Cytochrome 579 concentration in early formed biofilms was high, correlating with rapid Fe²⁺ oxidation. As biofilm thickness increased, O₂ concentrations in the middle of biofilms decreased, indicating that diffusion limitation of O₂ may control activity levels of aerobic organisms. Calculated low O₂ and high Fe³⁺ concentrations in the interior regions of biofilms may explain the previously reported suppression of the UBA genotype in mature biofilms. Instead, Leptospirillum Group II, 5-way CG genotype, dominated under these conditions. Leptospirillum Group III and eukaryotes appeared in the community as the biofilm thickened and Fe³⁺/Fe²⁺ increased. In mature biofilms, the architecture changed from planar to crenulated, perhaps to increase the surface area of biofilms and decrease O₂ diffusion limitation. In thick, mature biofilms, layering is associated with segregation of Leptospirillum Group II and Archaea and the concentration of cytochrome 579 is lower. The accumulation of Archaea close to the biofilm-air interface may facilitate their aerobic metabolism of waste carbon compounds. Sulfite, thiosulfate and polysulfides were detected in AMD sediments and thiosulfate was detected in solution. These compounds indicate the redox status of the system and represent potential energy sources. Temporal and spatial heterogeneity in community structure correlate with heterogeneity in geochemical conditions, implying active feedbacks between geochemical conditions and microbial species distribution and activity.