| Summary: | Postponed access: the file will be accessible after 2021-07-02 Snottites, suspended acidic biofilms forming on overhanging surfaces, represent scarcely studied microhabitats. This study aims to describe the microbial diversity and community composition of snottites and other biofilms (acid streamer – biofilm forming in acid water stream) found in subsurface acid mine drainage in the abandoned Båsmo pyrite mine in Nordland, Norway, characterised by low temperatures, absence of light and a low-pH water environment (pH ∼ 2.1). Snottites, acid streamer, stream sediment biofilm and pyrite-rich sediment samples were collected. We applied a combination of microscopy techniques (brightfield, epifluorescence, scanning electron microscopy (SEM), transmission electron microscopy (TEM)), molecular approaches with Illumina 16S rRNA sequencing and cell enumeration using fluorescence in situ hybridisation (FISH). This was combined with geochemical analyses and cultivation. Brightfield microscopy showed that the biofilm samples were composed of bacteria, filamentous fungi and protists. Scanning electron microscopy revealed a very dense bacterial community, while transmission electron microscopy revealed some very electron-dense bacteria with a lot of pili-like appendages. 16S rRNA gene community profiling revealed one, two or three bacterial taxons dominating in each sample and a low prokaryotic species richness in this extreme environment. Bacterial operational taxonomic units (OTUs) identified in biofilm samples belonged mostly to the classes Betaproteobacteria, Gammaproteobacteria and Nitrospira. 67 % to 89 % of the sequences in snottite and up to 94 % in acid streamer samples belonged to the same OTU, affiliated with the iron-oxidising betaproteobacterium Ferrovum myxofaciens, often found in acid mine drainage. Gammaproteobacteria were represented with the genus Metallibacterium. In pyrite-rich sediments and stream biofilm mixed with sediments, the majority of the sequences (63 % and 53 %, respectively) were related to the genus Leptospirillum (belonging to the class Nitrospira), another important iron-oxidising bacterium. Rare representatives were related to the families Bacteriovoraceae and Acidobacteriaceae and the genera Acidithiobacillus and Acidiphilium. No archaea were detected. The estimated number of prokaryotic cells was similar for snottites and acid streamer and ranged between 1.51x10^11 and 1.56x10^12 cells per gram (wet weight). Live/dead staining indicated that 9 % to 24 % of the prokaryotic cells in the samples were live cells. Combining Illumina sequencing and FISH allowed for a confirmation of the main players in these microhabitats. The core taxa dominating microbial communities in our samples are typically found in acid mine drainage environments. Snottites and acid streamer were mostly dominated by Ferrovum, an ironoxidising acidophilic autotroph capable of catalysing the oxidative dissolution of pyrite, CO2 and N2 fixation and producing a lot of extracellular polymeric materials. Microbes present were also acidophilic bacteria from genus Metallibacterium and Acidithiobacillus. Stream sediment biofilm and pyrite-rich sediment were more dominated by iron-oxidising Leptospirillum bacteria that have been known to have a leaching ability on pyrite. Our study shows the diversity of these acidic microhabitats, predominated by iron-oxidising bacteria using ferrous ion as e^- donor and O2 as e^- acceptor and closely connected to geochemistry. Master's Thesis in Biology MAMN-BIO BIO399
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