Linking δ¹³CDIC and microbial respiration to calcium carbonate dissolution in a complex groundwater system: evidence from a large-scale field study

The relationships between groundwater chemistry and the structure and metabolism of microbial communities inhabiting pristine aquifers remain poorly understood, as do the bidirectional interactions between groundwater pollution and microbial activity. In this study, we investigated more than 60 sites within a large groundwater system in central Italy, aiming to integrate geochemical, isotopic, and microbiological information to elucidate key biogeochemical processes.

The relationships between groundwater chemistry and the structure and metabolism of microbial communities inhabiting pristine aquifers remain poorly understood, as do the bidirectional interactions between groundwater pollution and microbial activity. In this study, we investigated more than 60 sites within a large groundwater system in central Italy, aiming to integrate geochemical, isotopic, and microbiological information to elucidate key biogeochemical processes.

The study area is the Sacco River Valley, which hosts multiple hydrogeological complexes, including Quaternary alluvial deposits, Pleistocene volcanic products and travertines, Miocene flysch sequences, and Meso–Cenozoic limestones. Aquifer potential is medium to high, with moderate vulnerability. A regional unconfined aquifer develops along the valley, mainly within volcanic deposits, alluvial sediments, and travertines, and is drained by the river along most of its course. A deeper groundwater system circulates in the Meso-Cenozoic limestones, confined beneath the Neogene-Quaternary formations.

Groundwater samples were collected from wells and springs between November 2024 and December 2025, together with in situ measurements of physical and chemical parameters. Chemical analyses included major ions, trace elements, DOC, and stable isotopes (δ¹³C_DIC, δ²H, and δ¹⁸O). Microbial communities were characterized by total cell counts (flow cytometry) and heterotrophic respiration potential (Biolog-MT2™ assay).

Most samples belong to the Ca–HCO₃ facies, and exhibited near-neutral pH. Approximately 30% of the sites showed slightly to strongly reducing conditions. δ¹³C_DIC values indicated that groundwater was predominantly influenced by biogenic CO₂ derived from soil respiration (δ¹³C_DIC < −10‰). A limited number of samples showed less negative to slightly positive δ¹³C_DIC values, associated with elevated iron and manganese concentrations, sub-neutral pH, anoxic conditions and field evidence of dissolved gases, suggesting localized interaction with deep geogenic CO₂ sources.

Preliminary statistical analyses revealed significant correlations between microbial respiration and Ca²⁺, electrical conductivity, HCO₃⁻, Mg²⁺, SO₄²⁻, δ¹³C_DIC, and iron, while a weaker negative correlation occurred with redox potential. Multivariate analyses discriminated sample groups related to redox conditions and conductivity, the latter being positively associated with heterotrophic microbial respiration. The significant correlation of microbial respiration with calcium concentration suggested a potential role of microbial activity in promoting calcium dissolution in groundwater. Overall, these results highlight the tight coupling between groundwater geochemistry and microbial metabolic activity, providing new insights into biogeochemical controls operating in complex groundwater systems.