Unique architecture of microbial snottites from a methane driven biofilm revealed by confocal microscopy

Microbial biofilms occur in many shapes and different dimensions. In natural and semi‐artificial caves they are forming pendulous structures of 10 cm and more. In this study a methane driven microbial community of a former medicinal spring was investigated. The habitat was completely covered by massive biofilms and snottites with a wobbly, gelatinous appearance. By using fluorescence techniques in combination with confocal laser scanning microscopy the architecture of these so far unknown snottites was examined. The imaging approaches applied comprised reflection of geogenic and cellular origin, possible autofluorescence, nucleic acid staining for bacterial cells, protein staining for bacteria and extracellular fine structures, calcofluor white for β 1 → 3, β 1 → 4 polysaccharide staining for possible fungi as well as lectin staining for the extracellular biofilm matrix glycoconjugates. The results showed a highly complex, intricate structure with voluminous, globular, and tube‐like glycoconjugates of different dimensions and densities. In addition, filamentous bacteria seem to provide additional strength to the snottites. After screening with all commercially available lectins, by means of fluorescence lectin barcoding and subsequent fluorescence lectin binding analysis, the AAL, PNA, LEA, and Ban lectins identified α‐Fuc, β‐Gal, β‐GlcNAc, and α‐Man with α‐Fuc as a major component. Examination of the outer boundary with fluorescent beads revealed a potential outer layer which could not be stained by any of the fluorescent probes applied. Finally, suggestions are made to further elucidate the characteristics of these unusual microbial biofilms in form of snottites. Research Highlights The gelatinous snottites revealed at the microscale a highly complex structure not seen before. The extracellular matrix of the snottite biofilm was identified as clusters of different shape and density. The matrix of snottites was examined by taking advantage of 78 fluorescently‐labeled lectins. The extracellular matrix glycoconjugates of snottites identified comprised: α‐Fuc, β‐Gal, β‐GlcNAc, and α‐Man. Probing the snottite outer surface indicated an additional unknown stratum. Confocal microscopy of gelatinous snottites (biofilms) revealed a highly complex structure not seen before with an extracellular matrix of different shape and density. The matrix was examined by employing 78 fluorescently‐labeled lectins suggesting α‐Fuc, β‐Gal, β‐GlcNAc, and α‐Man as major glycoconj