Abstract
Acid mine drainage (AMD) containing toxic metals exceeding effluent standard values causes a persistent environmental challenge requiring continuous and low-cost management. Iron-oxidizing bacteria (FeOB) are widely utilized to remove Fe from AMD, which metabolize ferrous ions (Fe2+) to ferric ions (Fe3+), resulting in the Fe²⁺ oxidation rate 100,000 times higher than abiotic oxidation under acidic conditions. In the Date Mine, Japan, AMD (pH: 3.0, Fe²⁺:150 mg/L, SO₄²⁻: 600 mg/L) flowing at 60 L/min is neutralized and Fe is precipitated by the addition of quicklime. Recently, a pilot-scale aeration reactor was installed to enhance the biological Fe2+ oxidation. This study quantified the bacterial abundance and characterized the microbial community in AMD and precipitates and assessed their activity in conjunction with spatial and seasonal variation in water quality. The physico-chemical parameters of AMD (temperature, dissolved oxygen (DO), pH, and Fe2+ concentration) were obtained at three sampling points (upstream of the pipeline, downstream of the pipeline, and the aeration reactor). The DO concentration in AMD was low (<1.0 mg/L) at all sampling points, whereas it increased to over 7 mg/L in the aeration reactor. Cell densities were measured using DAPI fluorescence microscopy following paraformaldehyde fixation in both original and aerated AMD samples; the density was two times higher in the aerated AMD than in the original AMD (downstream). Microbial community composition was characterized through 16S rRNA gene amplicon sequencing V4 region, which identified two distinct biological Fe2+ oxidation processes throughout the entire treatment facilities, from upstream to the aeration reactor. In the original AMD, low DO conditions are associated with a high relative abundance of Gallionellaceae, a microaerophilic FeOB family. Both Gallionellaceae abundance and a decrease in Fe2+ concentration were observed at lower temperatures (<5 ℃). In contrast, the genus Ferrovum, an aerophilic FeOB family, became dominant in the aeration reactor. This Ferrovum abundance is linked to its ability to produce extracellular polymeric substances (EPS) that facilitate precipitate formation by binding cells and iron oxides. The AMD was reacted in a beaker with biogenic precipitate obtained from the aeration reactor (5 % v/v precipitate in AMD, 120 min). These experiments confirmed rapid Fe2+ oxidation mediated by the precipitate, demonstrating the substantial contribution of high-density FeOB populations to Fe2+ removal. Furthermore, fibrous precipitates from upstream were examined using field emission scanning electron microscopy FE-SEM, JEOL 7800F, 10 kV) following freeze-drying and platinum coating. FE-SEM imaging confirmed the presence of characteristic twisted stalks produced by Gallionellaceae. These stalks prevent Fe3+ oxide encrustation of cell surfaces, indicating natural pretreatment during AMD conveyance. This study demonstrated that the AMD system at Date Mine can achieve more effective water treatment by combining natural remediation with Gallionellaceae in the water transport pipeline with adaptive Fe2+ oxidation using Ferrovum in the aeration reactor.