Reactive oxygen species (ROS) play a crucial role in the biogeochemistry of aquatic environments, yet their occurrence and accumulation in the biofilm of submerged macrophytes have been poorly documented. Herein, we first investigated the light-dark cycling fluctuations of biofilm microenvironment and the temporal variations of a representative ROS (O2•-) during biofilm succession on the macrophyte leaves and subsequently quantified the photochemical processes in biofilms. The sustained production of O2•- exhibited a distinct rhythmic fluctuation from 32.49±0.56 μmol/kg to 72.56±0.92 μmol/kg FW, which simultaneously fluctuated with the dissolved oxygen, redox potential, and pH, all driven by the alternating oxic-anoxic conditions of biofilms. The intensities of O2•- and ROS firstly increased and then decreased throughout biofilm succession. The O2•- concentrations in biofilms from different waters followed the order of rural river water >landscape lake water >aquaculture pond water, and the leaf photosynthesis and microbial community played a key role. ROS production was significantly associated with Actinobacteria, Proteobacteria and Bacteroidetes, with contributions of 44.6%, 32.8%, and 15.2%, respectively. Partial least squares path modeling structural equation analysis showed that ROS production in leaf biofilms was mainly related to the microenvironment and microbial metabolism. These findings will facilitate the development of ecological restoration strategies in aquatic environments.

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