Biofilm development on fractured rock in oligotrophic nitrate-rich groundwater: An in-situ bioreactor study

Biofilms drive all biogeochemical processes and represent the main mode of existence for active microbial life. Many past studies examined biofilm formation under static and eutrophic conditions, but those conditions are not representative of typical groundwater environments. In this study, we developed in situ bioreactors and methodologies to examine the influence of subsurface properties such as redox condition and lithology on the properties of naturally formed biofilms in two adjacent wells, a 30-m deep well completed in alluvium and a 120-m deep well in gneiss bedrock. The bulk chemistry of groundwater from the wells was similar, with neutral pH and abundant nitrate (21.9-24.6 mg/L), but redox conditions differed with depth (alluvial: oxic, gneiss bedrock: anoxic). Microbial community analysis revealed distinct clustering of biofilm community composition with the groundwater environment. Biofilm communities were consistently assembled by deterministic processes whereas planktonic communities had a higher influence of stochastic processes. Alluvial biofilms exhibited more diverse communities mainly composed of organotrophic aerobes capable of nitrate utilization. Bedrock biofilms indicated similar community compositions with groundwater where anaerobic denitrifiers coupled with sulfur oxidizers were dominant. Visualization and biomass quantification revealed distinct morphologies and development of biofilm along rock types and groundwater environments. Biofilm on gneiss surface had more biomass and formed a thin layered structure, compared to sandstone biofilm which had a randomly distributed pattern, implying that the morphology of biofilm was governed by the properties of the rock. Attached to unattached (planktonic) microbe ratios ranged from 3.9 × 10³ to 1.2 × 10⁴: 1 in the gneiss surface and 3.4 × 10² to 4.2 × 10²: 1 in the sandstone surface in bedrock groundwater environment. Taken together, this study advances our understanding of subsurface biomass abundance and demonstrates that the in-situ bioreactors are effective for cultivating and analyzing of subsurface biofilms. Based on the specific field conditions tested, we found that biofilm can form stably on fractured rock surfaces within a year, with groundwater redox conditions shaping community composition and rock types determining biofilm volume and morphology. The methodologies presented here can be extended to other subsurface environments with varying groundwater geochemistry and lithology, which will help further refine estimates of microbial life and its role in subsurface ecosystems.