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We have previously conducted a microflora analysis and examined the biofilm-forming activity of bacteria isolated from toilet bowl biofilms. In the present investigation, to reveal the strain involved in the formation of black dirt in toilet bowls, we performed a microflora analysis of the bacteria and fungi isolated from the black dirt of toilet bowls at ten homes. Among samples from different isolation sites and sampling seasons, although a similar tendency was not seen in bacterial microflora, Exophiala sp. was detected in the fungal microflora from all samples of black dirt except for one, and constituted the major presence. By scanning electron microscope (SEM) analysis of the formed black dirt, SEM image at × 1,000 and × 5,000 magnification showed objects like hyphae and many bacteria adhering to them, respectively. Micro fourier transform infrared spectroscopy (micro FT-IR) and SEM with X-ray microanalysis (SEM-XMA) were used to investigate the components of black dirt. IR spectra of micro-FT-IR showed typical absorptions associated with amide compounds and protein, and the elements such as C, N, O, Na, Mg, Al, Si, P, S, K, and Ba were detected with SEM-XMA. These results showed that black dirt had living body ingredients. Furthermore, Exophiala sp. and Cladosporium sp. strains, which were observed at a high frequency, accumulated 2-hydroxyjuglone (2-HJ) and flaviolin as one of the intermediates in the melanin biosynthetic pathway by the addition of a melanin synthesis inhibitor (tricyclazole) at the time of cultivation. These results suggested strongly that the pigment of black dirt in toilet bowls was melanin produced by Exophiala sp. and Cladosporium sp. strains.

Characterization of Microorganisms Isolated from the Black Dirt of Toilet Bowls and Componential Analysis of the Black Dirt