Molecular dynamics and factors governing recalcitrance of dissolved organic matter: Insights from laboratory incubation and ultra-high resolution mass spectrometry.

The oceanic dissolved organic matter (DOM) reservoir is one of Earth's largest carbon pools, yet the factors contributing to its recalcitrance and persistence remain poorly understood. Here, we employed ultra-high resolution mass spectrometry (UHRMS) to examine the molecular dynamics of DOM from terrestrial, marine and mixed sources during bio-incubation over weekly, monthly, and one year time spans. Using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), we classified DOM into three distinct categories (Consumed, Resistant and Product) based on their presence or absence at the start and end of the incubation. Our results show that molecular properties, such as hydrogen to carbon ratio (H/C), modified aromaticity index (AI), and nominal oxidation state of carbon (NOSC), strongly influence DOM lability and its biogeochemical cycling. Interestingly, Product formulas identified in the short-term incubations were often reclassified as Consumed formulas in longer-term incubations, underscoring the importance of incubation time in determining the persistence of DOM formulas. Further, we introduced a Change Ratio (CR) to identify formulas with significantly altered relative abundances. The molecular characteristics of these Increase or Decrease formulas exhibited notable differences, reinforcing their role in determining lability. In seawater samples, Decrease formulas were more abundant than Increase formulas, supporting the dilution hypothesis, which suggests low concentrations contribute to biological recalcitrance. However, the instability of relative abundance differences between Increase and Decrease formulas when CR thresholds were altered, coupled with the robustness of AI differences, highlights the dominance of molecular properties over concentration in determining DOM lability. Furthermore, the AI distribution of these Increase and Decrease formulas mirrored deep-enriched and surface-enriched formulas in the open ocean, validating our incubation results with field investigations. Overall, our study demonstrates that combining laboratory incubation with UHRMS advances our molecular-level understanding of DOM recalcitrance and thus global carbon cycling.