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.

流经日本西南部的阿苏火山口的黑川河受到含高浓度溶解金属（Fe ²⁺和Mn ²⁺），并与上游水道中的橙色沉积物和人工水路隧道壁上厚厚的黑壳相关，中游区长度为数公里。X射线荧光分析表明，橙色沉积物中铁含量高达34.4％，黑皮中锰和铁含量分别高达25.4％和10.6％。虽然天然存在的锰氧化物和结皮在中性pH值的淡水环境中被认为是生物成因，但尚不清楚锰结皮中的整个微生物群落及其在这些环境中对锰结皮形成的贡献。对细菌和真核生物（包括真菌）进行的高通量分子测序表明，黑锰壳具有高含量的鞘氨醇单胞菌，疏水菌，芽孢杆菌，假单胞菌和Mortierella，先前报道的属，包括在其他海洋和淡水环境的Mn结皮或结核中具有Mn氧化活性的几种物种。另外，从黑锰壳中获得了一种具有锰氧化活性的细菌分离物和一种真菌分离物。这些结果表明在黑川河的贫营养环境中，包括假单胞菌和真菌在内的多种锰氧化细菌参与了暗水道隧道壁上的锰结壳的形成。