ABSTRACTThe microbial carbon pump (MCP) provides a mechanistic illustration of transformation of recalcitrant dissolved organic matter (DOM) in the ocean. Here, we explored and demonstrated the key roles of algae-associated microorganisms (mainly heterotrophic bacteria) in the production and transformation of carboxyl-rich alicyclic molecule (CRAM)-like DOM through a laboratory experiment involving cultures of Skeletonema dohrnii. Without the participation of the associated bacteria, CRAM-like DOM molecules were not detected via Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in algal cultures treated with antibiotics. Similarly, CRAM-like DOM were not detected in cultures of bacteria alone. Our experimental results showed that algae-associated bacteria are important in the process of converting algal-derived organic matter into CRAM-like DOM during S. dohrnii culture. Bacteroidetes (mainly Flavobacteriia) dominated the bacterial community in the stationary and degradation phases, where the predicted metabolic pathways for bacterial assemblages were mainly involved in biosynthesis, metabolism, and degradation. Facilitated by these heterotrophic bacteria, the amount and the chemodiversity of CRAM-like DOM derived from algae varied during the growth and decomposition of algal cells, and CRAM-like DOM were enriched at the later growth stage. The properties and characteristics of these CRAM-like DOM, including molecular weight, double bond equivalent, hydrogen-carbon ratio, carbon-nitrogen ratio, carbon-sulfur ratio, and modified aromaticity index increased with the growth and decay of algal cells, indicating the transformation from active to recalcitrant DOM. In contrast, the organic matter in axenic cultures of S. dohrnii mainly existed in the form of particulate organic matters (POM), and small amounts of CRAM-like DOM were detected. This study provides the first laboratory evidence to reveal and confirm the direct involvement of algae-associated microbiomes in the production and transformation of algae-derived refractory DOM, highlighting the significance of these epiphytic bacteria in marine carbon sequestration and global carbon cycling.IMPORTANCE Dissolved organic matter (DOM) serves as a major carbon and nutrient pool in oceans, and recalcitrant DOM are the primary sources for carbon sequestration in depths. Here, we demonstrate the critical roles of algae-associated microorganisms (mainly heterotrophic bacteria) in the transformation of recalcitrant dissolved organic matter through laboratory cultures of a model diatom, Skeletonema dohrnii. Our experimental results showed that in addition to affecting the growth and the physiology of S. dohrnii, algae-associated bacteria are important in processing and converting algal DOM into CRAM-like DOM. Facilitated by the associated bacteria, the amount and the chemodiversity of DOM derived from algae varied during the growth and decomposition of algal cells, and enriched recalcitrant DOM formed in the later growth stage. The properties and diversity of DOM increased with the growth and decay of algal cells, indicating the transformation from active DOM to inert organic matter. Our results confirmed that the direct involvement of algae-associated microbes in the production of CRAM-like DOM. Detailed community structure analysis of the algae-associated bacterial community and its predicted functions confirmed the involvement of certain bacterial groups (e.g., Flavobacteriia) in biosynthesis, metabolism, and degradation.