## 3 Results
### Microbial Community Analysis of the Mn (IV) Biofilm-Colonized Pebbles  
The microbial community profile of the black biofilms on pebbles freshly collected from the stream was assessed from clone libraries which were constructed from sequence analysis of bacterial and archaeal 16S and eukaryote 18S rRNA genes. Figure 6 shows the relative abundance of microbial taxonomy (order level) of the clones obtained from the biofilm-colonized pebbles.  
Within the domain Bacteria, 16S rRNA genes sequences were dominated by _Burkholderiales_ and most of these were related to _Leptothrix (L) discophora_ (96% identity), a well-characterized Mn (II)-oxidizer. Archaeal 16S rRNA genes sequences were affiliated with ammonia-oxidizers belonging to phylum _Thaumarchea_ and dominated by the orders _Nitrososphareales_ and _Nitrosopumilles_. Three of the five taxonomic groups (order level) of eukaryotic 18S rRNA genes sequences belonged to phylum _Mollusca_. No fungi were detected using this technique. Identity of cloned archaeal, bacterial and eukaryotic genes obtained from the biofilm-colonized pebbles are shown in Supplementary Tables S2-S4, respectively. Uncultured clones found in the database were included in cases where clones were distantly related to other known species (identity <96%).  
Most of the bacterial clones (Supplementary Table S3) belonged to the phylum _Proteobacteria_, except the clone MNPBAC19, which appears to belong to the phylum _Verrucomicrobiu_, though the sequence identity match to its nearest known cultivated relative is very low (83%). The uncultured bacterial clone which closest matched clone MNPBAC19 (96% identity) was obtained from a hyporheic zone of a stream bed in California, which contained iron (hydr)oxide precipitates [17]. Another clone, MNPBAC16, showed a high level of sequence similarity to _L. discophora_ (strain SP-6), a well-documented Mn (II)-oxidizer. Most of eukaryotic clones showed high level of similarity to known organisms that belong to the phylum _Mollusca_. Clone MnE80 was suggested to belong to _Apicomplexa_ (though only 90% sequence similarity), which is a large phylum of parasitic alveolates.  
T-RFLP analysis carried out on biofilm-colonized pebbles taken directly from the stream in Snowdonia (C0) and at the end of phases C1, C2a and C3 of the tests with the MnOBs were based on bacterial 16S rRNA genes and eukaryote 18S rRNA genes. The data revealed a large number of T-RF peaks in all samples analyzed. Figure 7 shows how T-RFLP bacterial profiles on the biofilm-colonized pebbles varied amongst MnOBs. Many T-RFs of the same length were found in all samples. The 217 nt T-RF showed high relative abundance in most samples, accounting overall for ~17% of the total bacterial population  
Figure 6: Relative abundance of microbial taxonomy (order) of the clones obtained from Mn (IV) biofilm-colonized pebbles collected from a stream in Snowdonia, UK, based on archaeal and bacterial 16S and eukaryote 18S rRNA genes.  
in phases C0, C2a and C3. The exception was phase C1, where a 214 nt T-RF was more dominant, accounting for 21% of the summated T-RFs. The 223 nt T-RF matched the theoretical T-RF of the clones which were closely related to _L. discophora_. Overall, its relative abundance in the bacterial community of the colonized pebbles was \(\sim\)5%; however, more than one organism shared the same T-RF length (Supplementary Table S3) and therefore it cannot be assumed that this T-RF corresponded to only _L. discophora_.  
Supplementary Figure S6 shows how T-RFLP profiles of eukaryotes on the biofilm-colonized pebbles varied amongst MnOBs. Major changes were observed in the eukaryotic populations in the experiment. The 280 nt T-RF was dominant in all phases of the MnOB, accounting for \(\sim\)72% of the eukaryotic population in phase C3; however, this same T-RF was shared with several organisms listed in Supplementary Table S4.