Biofilm Formation on Excavation Damaged Zone Fractures in Deep Neogene Sedimentary Rock

Deep underground galleries are used to access the deep biosphere in addition to mining and other engineering applications, such as geological disposal of radioactive waste. Fracture networks developed in the excavation damaged zone (EDZ) are concerned with accelerating mass transport, where microbial colonization might be possible due to the availability of space and nutrients. In this study, microbial biofilms at EDZ fractures were investigated by drilling from a 350-m-deep gallery and subsequent borehole logging at the Horonobe Underground Research Laboratory (URL). By using microscopic and spectroscopic techniques, the dense colonization of microbial cells was demonstrated at the surfaces of the EDZ fractures with high hydraulic conductivity. 16S rRNA gene sequence analysis revealed the dominance of gammaproteobacterial lineages, the cultivated members of which are aerobic methanotrophs. The near-complete genomes from Horonobe groundwater, affiliated with the methanotrophic lineages, were fully equipped with genes involved in aerobic methanotrophy. Although the mediation of aerobic methanotrophy remains to be demonstrated, microbial O₂ production was supported by the presence of genes in the near-complete genomes, such as catalase and superoxide dismutase that produce O₂ from reactive oxygen species and a nitric oxide reductase gene with the substitutions of amino acids in motifs. It is concluded that the EDZ fractures provide energetically favorable subsurface habitats for microorganisms.