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The Kurokawa river flowing through the Aso Caldera in Southwest Japan is influenced by groundwater containing high concentration of dissolved metals (Fe2+ and Mn2+) and is associated with orange deposits in the upstream and thick black crusts on the wall of artificial waterway tunnel with several km lengths in the midstream regions. X-ray fluorescence analysis revealed that the orange deposits contained up to 34.4% Fe and black crusts comprised up to 25.4% and 10.6% Mn and Fe, respectively. Although naturally occurring Mn-oxides and crusts are considered biogenic in pH-neutral freshwater environments, the whole microbial community in Mn crust and their contribution to Mn crust formation in these environments remain unclear. High-throughput molecular sequencing for bacteria and eukaryotes including fungi revealed black Mn crusts had a high abundance of Sphingomonas , Hyphomicrobium, Bacillus , Pseudomonas , and Mortierella , previously reported genera including several species with Mn-oxidizing activity in Mn crusts or nodules of other marine and freshwater environments. In addition, one bacterial isolate and one fungal isolate with Mn-oxidizing activity were obtained from black Mn crust. These results suggest that multiple Mn-oxidizing bacteria including Pseudomonas and fungi are involved in Mn crust formation on the wall of dark waterway tunnel in the oligotrophic environment of the Kurokawa river. [ABSTRACT FROM AUTHOR]