Benthic biofilms as reservoirs of recalcitrant organic matter in freshwater reservoirs: insights from FT-ICR MS

Benthic biofilms play a significant role in influencing the composition and cycling of dissolved organic matter (DOM) in freshwater reservoirs. In this study, we characterized the molecular composition of DOM using direct injection Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (DI-FT-ICR MS), with complementary insights provided by three-dimensional Excitation-Emission Matrix (3D EEM) fluorescence spectroscopy, comparing samples from bulk water and biofilms across three reservoirs. Biofilm-associated DOM exhibited higher SUVA-254 and Aromaticity Index (AI-mod) values, indicating an enrichment of recalcitrant and humic substances. 3D EEM analysis showed a 35 % increase in humic-like substances within biofilms compared to bulk water, underscoring their role in stabilizing organic compounds. FT-ICR MS analyses demonstrated that classes of aromatic compounds, including condensed aromatics, exhibited a markedly higher prevalence within biofilms (25.9 %) in comparison to bulk water (18.5 %). Biofilms also encompassed a greater quantity of nitrogenous compounds, with CHON categories constituting 17.3 % of dissolved organic matter (DOM) in biofilms in contrast to 9.2 % in bulk aquatic environments, underscoring their significance in nutrient cycling. Conversely, sulfur-containing compounds (CHOS) were less abundant in benthic biofilm samples (6.4 %) than in corresponding bulk water (12.1 %), a difference potentially attributable to various processes including differential production, consumption, or diffusion. Furthermore, biofilms displayed a higher average molecular weight (476.3 m/z) and elevated Double Bond Equivalence (DBE) (14.45) compared to bulk water (454.1 m/z; 12.99), highlighting the greater complexity of biofilm-associated DOM. Significant alterations in the composition of microbial communities observed between bulk water and biofilms provide additional evidence of the functional role that biofilms play in the alterations of dissolved organic matter (DOM). These results emphasize the significance of benthic biofilms in stabilizing organic substances, which has a substantial effect on the well-being and administration of freshwater ecosystems.