The majority of dissolved organic matter (DOM) in the ocean is resistant to microbial degradation, yet its formation remains poorly understood the fluorescent fraction of DOM can be used to trace the formation of recalcitrant DOM (RDOM). A long-term (> 1-year) experiment revealed 27-52% removal of dissolved organic carbon and a nonlinear increase in RDOM fluorescence associated with microbial turnover of semilabile DOM. This fluorescence was also produced using glucose as the only initial carbon source, suggesting that degradation of prokaryote remnants contributes to RDOM. Our results indicate that the formation of a fluorescent RDOM component depends on the bioavailability of the substrate: the less labile, the larger the production of fluorescent RDOM relative to organic carbon remineralized the anticipated increase in microbial carbon demand due to ocean warming can potentially force microbes to degrade less labile substrates, thereby increasing RDOM production and stimulating ocean carbon storage. Key Points Optical properties of DOM can be used to trace production of recalcitrant DOM Production of recalcitrant DOM is associated with microbial activity Less labile DOM shifts microbial activity towards production of recalcitrant DOM