Decadal evolution of groundwater planktonic prokaryotes in a sandstone aquifer

Groundwater ecosystems host diverse and largely unexplored communities of planktonic prokaryotes that play critical roles in global biogeochemical cycling and maintaining drinking water quality. This study investigated the spatiotemporal dynamics of prokaryotic communities in a sandstone aquifer at depths of 70-338m. By integrating spatial surveys across 48 pumping boreholes in England with temporal analyses by seasonal repetition and groundwater ‘piston flow age’ analysis, we investigated how variations in age, depth, and drift thickness influence nutrient availability and microbial communities. The prokaryotic community structure was assessed by 16S rRNA gene amplicon sequencing, groundwater recharge age using CFC-12 (dichlorodifluoromethane), borehole characteristics and surface connectivity from using borehole logs, with further analysis of total dissolved nitrogen (TDN), dissolved organic carbon (DOC) and dissolved oxygen (DO) concentrations. Seasonal analyses revealed minimal shifts in dominant taxa between recharge and recession periods, with no detectable introduction or extinction of surface-derived taxa. However, there was 7% change in DOC, TDN and 35% reduction in DO between recharge and recession periods. This lack of community shift may be attributed to the high filtration capacity of the sandstone aquifer preventing surface taxa intrusion during recharge. A high Shannon diversity index (5.4) indicated a stable and highly diverse groundwater community. The microbial community structure and nutrient availability varied significantly along vertical gradients of groundwater age, screened interval depth, and drift thickness, with distinct assemblages in shallow unconfined versus deeper confined sites. Nutrient cycling patterns by these communities were inferred from nutrient profiles. Unconfined sites with thinner drift (1–10 m), shallower screen depths (23-36m) and younger water (1972–2023) exhibited abundance of ultrasmall heterotrophic families, including Omnitrophaceae, Nanoarchaeia, and classes Crenarchaea and Parcubacteria. In these primarily aerobic environments, DOC limitation (0.7 mg/L) could prevent denitrification resulting in higher legacy TDN accumulation (9.5 mg/L) from anthropogenic additions of nitrogen-based fertiliser. The dominant ultrasmall heterotrophic prokaryotes may perform cryptic (or hidden) carbon and nitrogen cycling where rapid turnover of redox species drive biogeochemical cycling or may act as parasites. Conversely, confined sites with thicker drift (10–142 m), deeper borehole perforation depths (40-124m) and older groundwater recharge ages (1953–1967) were dominated by autotrophic families such as Gallionellaceae, Rhodocyclaceae, Hydrogenophilaceae and Comamonadaceae. These autotrophs may facilitate iron and sulphur cycling in the anaerobic parts of the confined aquifer. The intermediate depth and age ranges exhibited a mix of both autotrophs and heterotrophs indicating a transition phase. These findings highlight the role of aquifer architecture and groundwater residence time in shaping the spatiotemporally heterogeneous prokaryotic communities. The spatially unique communities influence the local nutrient cycling and thus the water chemistry, which should be considered when designing sustainable groundwater management strategies.

Keywords: Groundwater, Planktonic prokaryotes, Spatio-temporal variation, Nutrient cycling, Groundwater age.