## 3 Results
### Characterization of a Mn (II)-Oxidizing Fungal Isolate  
A brown/black-colored fungal isolate, coded MnI1, was obtained on solid medium that had been inoculated with a biofilm from a pebble removed from the MnOB after 6 days of phase C2b. Colonies were evident on solid medium within 5 days and showed the characteristic brown/black color of the mycelia (Supplementary Figure S5a). The colonies had rhizoidal morphologies and filiform margins, with vegetative and aerial/reproductive mycelia, characteristic of filamentous fungus. Colonies adhered firmly to the solid medium and presented low emulsification in water. Colonies of isolate MnI1 grown on Mn-free solid medium had nonpigmented hyphae (Supplementary Figure S5b). Even though the fungal mycelia on Mn-free plates were also dark-colored in the center of the colonies, the difference between pigmentation of the colonies on Mn-containing and Mn-free plates was clearly noticeable, suggesting that brown/black-colored insoluble Mn (IV) oxides were deposited onto fungal mycelium on plates containing soluble manganese. Micrographic images of the isolate growing on solid medium in presence and absence of Mn (II) showed that the isolate formed nonseptate hyphae and putative manganese oxides were found as particulate materials that were associated with the fungal hyphae (Supplementary Figure S7a). Microscopic examination of the pigmented centers of these colonies did not reveal the presence of particulate materials, as found on Mn (II)-containing plates (Supplementary Figure S7b). Phylogenetic analysis revealed that isolate MnI1 belongs to the _Ascomycota_ phylum and clusters with members of the order _Pleosporales_, class _Dothidemocytes_. The 18S rRNA genes sequence from MnI1 was closely related (98% gene similarity) to those from _Ullospora bilgramii_ and several other species. The phylogenetic tree (Figure 8) showed  
Figure 7: Terminal restriction enzyme fragment length polymorphism (T-RFLP) analysis (HaeIII digests) of amplified 165 rRNA bacterial genes from the biofilm-colonized pebbles collected from the stream (C0) and at different phases (C1, C2a and C3) of operating MnOBs. The T-RFs are stacked upwards in each column in increasing fragment lengths. MnI1 as a single member of a clade forming a branch with low bootstrap values of 47%, supporting the monophily of the clade. Based on these findings, it appears that this fungal species has not been previously described. Oxidation of Mn (II) by isolate MnI1 was tested in liquid media at initial pH 6.5 and 25 \({}^{\circ}\)C, and results are shown in Figure 9. Concentrations of soluble manganese in noninoculated control remained constant throughout the experiment, whereas in the inoculated flasks, concentrations of soluble Mn decreased gradually, from 450 to 97 \(\upmu\)M after 13 days. Parallel trends were observed with pH values. The final pH of noninoculated control was 6.5, whereas the final pH of the inoculated flasks was \(\sim\)4.0. Figure 8: Phylogenetic tree showing the relationship of the fungal isolate MnII to other microorganisms based on 185 rRNA sequence homology (values in brackets are NCBI accession numbers). Scale bar is number of nucleotides substituted per base pair. Bootstrap values are given at the respective nodes. The tree was rooted with the 185 rRNA gene sequence of Saccharomyces cerevisiae (J01353.1). Figure 9: Changes in soluble manganese concentrations (\(\blacktriangle\), \(\blacktriangle\)) and pH (\(\bullet\), \(\bullet\)) in shake flasks cultures inoculated with isolate MnII (4, \(\bullet\)) and noninoculated controls (\(\blacktriangle\), \(\bullet\)) with isolate MnI1. The experiment was carried out in duplicate and different values between replicates are occluded by the symbols. Isolate MnI1 was able to grow between 15 and 30 \({}^{\circ}\)C and appeared (from visual inspection of cultures) to grow optimally at 25 \({}^{\circ}\)C. No growth was observed at 45 \({}^{\circ}\)C. The isolate formed dark brown/black-colored mycelia in liquid media at all temperatures where growth was observed.