Coupled Carbonate Equilibrium and Metabolic Fractionation Drive δ¹³C-DIC Variability in Freshwater Ecosystems

Freshwater carbon cycling in oligotrophic lakes is shaped by interacting metabolic and geochemical processes, yet the way these controls vary from diel to seasonal timescales remains poorly resolved. In this study, we integrate 48-hour high-frequency measurements across four seasons with δ¹³C-DIC, water chemistry, and metabolism (GPP, ER) to evaluate the mechanisms regulating dissolved inorganic carbon (DIC) and its isotopic composition in Stephens Lake, a shallow headwater lake atop limestone in Franklin, Tennessee. Diel cycles in oxygen, pH, and temperature revealed clear metabolic patterns, with daytime photosynthetic CO₂ uptake and nighttime respiration generating characteristic shifts in DIC concentration. Corresponding δ¹³C-DIC values displayed clear diel enrichment–depletion cycles, particularly during periods of high biological activity. Seasonally, however, δ¹³C-DIC values were anchored by broader changes in carbonate equilibrium, driven by variations in Ca, Mg, DIC, and carbonate saturation state as the lake transitioned from winter dormancy to summer autotrophy. Multivariate analyses showed that winter conditions were dominated by carbonate equilibrium processes with limited metabolic influence, whereas spring and summer exhibited strong metabolic control on δ¹³C–DIC. Fall represented a transitional state in which moderate photosynthetic uptake and intermediate DIC concentrations jointly influenced δ¹³C–DIC patterns. Together, these patterns reveal two regulatory modes: (1) rapid diel metabolic forcing that drives short-term isotopic fluctuations, and (2) seasonal carbonate chemistry that sets the longer-term isotopic baseline. These findings offer a mechanistic framework for interpreting δ¹³C–DIC dynamics in carbonate-influenced, oligotrophic freshwater systems and improve understanding of how metabolism and carbonate equilibrium jointly influence carbon cycling under varying watershed conditions.