Seaweed Gracilaria lemaneiformis is an important species of mariculture in China. This study investigated dynamic changes in dissolved organic carbon (DOC), its molecular composition, and bacterial community structure in response to 180-day long-term degradation of G. lemaneiformis. The results showed that DOC changes in the degradation process consisted of three stages: the rising stage (0–20 days), algal lysis led to rapid DOC release, with DOC concentrations in all treatment groups reaching their peak and bacterial abundance increasing simultaneously; the decline stage (20–60 days), microbial metabolism intensified and DOC concentrations continued to fall; the stabilization stage (60–180 days): refractory dissolved organic carbon (RDOC) accumulated, and DOC concentrations tended toward stabilize. FT-ICR MS analysis indicated that during the degradation process, the three molecular categories—CHO, CHON, and CHOP—accounted for 85%–90% of the total DOC molecular composition. In all treatment groups, H/C_wa decreased, while O/C_wa and DBE_wa increased, indicating that microbial degradation accelerated DOC oxidation and generated refractory compounds, which accounted for 43% (±4%) of the total DOC. Throughout the long-term degradation process, approximately 66% of the RDOC molecular species were directly from the decomposition of G. lemaneiformis, and the other 34% were most likely newly produced by microbial metabolism from the unstable DOC derived from the seaweed, suggesting that organic matter derived from G. lemaneiformis formed aromatic structures resistant to degradation. During the degradation process, the fluorescence intensity of humic components in the treated groups remained significantly higher than that of the control (p < 0.05), indicating that microbial degradation promoted the formation and accumulation of humic-like substances derived from G. lemaneiformis. The release of algal-derived DOC directly drove changes in bacterial abundance and composition, indicating that active organic matter is the primary substrate for bacterial growth and reproduction. In the early stage, Class Gammaproteobacteria utilized proteins; in the middle stage, Class Bacteroidia and Family Flavobacteriaceae specialized in polysaccharides and sulfonic acids; and in the late stage, the bacterial community shifted to relying on microbial necromass for survival. Meanwhile, the metabolic activities of specific functional groups (such as Roseobacteraceae and Flavobacteriaceae) influenced the molecular composition of DOC, ultimately determining the accumulation efficiency of RDOC. The results indicated that DOC derived from G. lemaneiformis underwent complex changes during long-term microbial degradation, providing key evidence for the application of the microbial carbon pump theory in seaweed ecosystems.

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