Decadal cycling within long-lived carbon pools revealed by dual isotopic analysis of mineral-associated soil organic matter

Long-lived soil organic matter (SOM) pools are critical for the global carbon (C) cycle, but challenges in isolating such pools have inhibited understanding of their dynamics. We physically isolated particulate (>53 μm), silt-, and clay-sized organic matter from soils collected over two decades from a perennial C₃ grassland established on long-term agricultural soil with a predominantly C₄ isotopic signature. Silt- and clay-sized fractions were then subjected to a sequential chemical fractionation (acid hydrolysis followed by peroxide oxidation) to isolate long-lived C pools. We quantified ¹⁴C and the natural ¹³C isotopic label in the resulting fractions to identify and evaluate pools responsible for long-lived SOM. After removal of particulate organic matter (~14% of bulk soil C) sequential chemical treatment removed 80% of mineral-associated C. In all mineral-associated fractions, at least 55% of C₄-derived C was retained 32 years after the switch to C₃ inputs. However, C₃–C increased substantially beginning ~25 years after the switch. Radiocarbon-based turnover times ranged from roughly 1200–3000 years for chemically resistant mineral-associated pools, although some pools turned over faster under C₃ grassland than in a reference agricultural field, indicating that new material had entered some pools as early as 14 years after the vegetation switch. These findings provide further evidence that SOM chemistry does not always reflect SOM longevity and resistance to microbial decomposition. Even measureable SOM fractions that have extremely long mean turnover times (>1500 years) can have a substantial component that is dynamic over much shorter timescales.