Dissolved organic matter (DOM) in the ocean is composed of a small portion of labile compounds, but the main part is refractory or recalcitrant and thus not remineralized to CO2 for thousands of years. To understand the cycling of marine DOM, a mechanistic understanding of its transformation processes is crucial but so far largely unexplored. Ultrahigh resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) enables to determine the molecular elemental composition of thousands of DOM molecules. Based on this technique, we tested the hypothesis that the microbial degradation of different substrates leads to non-labile DOM with similar molecular fingerprints. In our experiments we studied the changes in DOM concentrations and shifts in its molecular composition during the microbial utilization of glucose and an algal exudate over a period of 2 years. Although glucose was quickly degraded, a non-labile DOM background was generated which was dependent on the substrate concentrations: higher substrate levels resulted in a higher amount of background DOM. In contrast, only 20% of the organic carbon in the algal exudate was degraded within the 2 years of incubation. The molecular signatures of DOM derived from algal exudates or glucose after 70 days of incubation differed considerably from refractory DOM. After 2 years, however, the molecular patterns of DOM in the glucose incubations were very similar to refractory DOM whereas the degraded exudate was still strongly different. We conclude that the amount and composition of non-labile DOM is dependent on concentration and composition of the initial substrate which is an important result with regard to carbon sequestration in the ocean. It must be considered, however, that 2 years of incubation are far away from the average DOC age of several thousand years in the deep ocean. Therefore, the material potentially could be further degraded and transformed by prolonged incubation, other microbial communities, or photo-degradation.