Marine dissolved organic matter (DOM) is the largest pool of actively cycling organic carbon and nitrogen in the ocean. Despite the importance of DOM in biogeochemical cycles, understanding of its composition and cycling is limited, in part due to difficulty in isolating representative material. Here we present a new approach to DOM isolation via a sequential combination of ultrafiltration and solid phase extraction, which allowed the selective isolation of two fractions with different compositions and reactivities. By limiting the influence of reactivity mixtures, we were able to directly investigate the composition and cycling of both labile and refractory DOM. Measurements of bulk isotopic (Δ14C, δ13C, δ15N) and compositional (C/N ratio, 13C and 15N NMR) parameters confirmed the uniqueness of the two fractions. The ultrafiltered high molecular weight (HMW) fraction was younger and dominated by carbohydrate-like and protein-like molecules. In contrast, the low molecular weight (LMW) fraction was older and dominated by alicyclic carbon and heterocyclic nitrogen containing molecules.

Spectroscopic studies have shown that the nitrogen-containing fraction of DOM (DON) is dominated by labile proteinaceous material. Despite this, other evidence suggests that a large fraction of DON is resistant to degradation. However, the paradigm of DON composition is based on measurements of HMW DON alone. Measurements of isotopic (δ15N, AA-Δ14C) and compositional (15N NMR, AA-D/L ratios) parameters of LMW DON provided a new perspective on the composition and cycling of DON in the ocean. Amino acids in LMW DON had both old radiocarbon ages and high D/L ratios, suggesting a bacterial mechanism capable of preserving proteinaceous material on millennial timescales. NMR analysis and nitrogen isotopic ratios demonstrated that LMW DON is dominated by heterocyclic nitrogen-containing molecules likely with intrinsically refractory molecular structures. These observations present a new paradigm for marine DON composition and suggest a mechanism for its long-term persistence.