Groundwater bacterial communities evolve over time in response to recharge

Time series analyses are a crucial tool for uncovering the patterns and processes shaping microbial communities and their functions, especially in aquatic ecosystems. Subsurface aquatic environments are perceived to be more stable than surface oceans and lakes, due to the lack of sunlight, the absence of photosysnthetically-driven primary production, low temperature variations, and oligotrophic conditions. However, periodic groundwater recharge should affect the structure and succession of groundwater microbiomes. To disentangle the long-term temporal changes in bacterial communities of shallow fractured bedrock groundwater, and identify the drivers of the observed patterns, we analysed bacterial 16S rRNA gene sequencing data for samples collected monthly from three groundwater wells over a six-year period (n = 230) along a hillslope recharge area. We showed that the bacterial communities in the groundwater of limestone-mudstone alternations were not stable over time and exhibited non-linear dissimilarity patterns which corresponded to periods of groundwater recharge. Further, we observed an increase in dissimilarity over time (generalized additive model P < 0.001) indicating that the successive recharge events result in communities that are increasingly more dissimilar to the initial reference time point. The sampling period was able to explain up to 29.5% of the variability in bacterial community composition and the impact of recharge events on the groundwater microbiome was linked to the strength of the recharge and local environmental selection. Many groundwater bacteria originated from the recharge-related sources (mean = 66.5%, SD = 15.1%) and specific bacterial taxa were identified as being either enriched or repressed during recharge events. Overall, similar to surface aquatic environments, the microbiomes in shallow fractured-rock groundwater vary through time, though we revealed groundwater recharges as unique driving factors for these patterns. The high temporal resolution employed here highlights the dynamics of bacterial communities in groundwater, which is an essential resource for the provision of clean drinking water; understanding the biological complexities of these systems is therefore crucial.