Timing of salinisation and nutrient enrichment drives freshwater microbial community metabolic responses

Biological systems face multiple stressors that impact biodiversity and ecosystem functions, with complex interactions that vary spatially and temporally, leading to unpredictable outcomes. In freshwater ecosystems, benthic microbial communities underpin vital functions like decomposition and primary productivity but are threatened by stressors such as salinisation and nutrient enrichment, which are intensifying and increasingly interacting due to climate change. We experimentally tested how stressor frequency and order affect freshwater benthic microbial community responses to these stressors over time. Communities established on tiles in 1,000 L open freshwater ponds established >10 years ago in the field were exposed to elevated salinity and nutrient enrichment, either once or repeatedly, independently or in combination, and under different orders. Controlling for frequency, we demonstrate for the first time that the order of stressor exposure is a critical driver of stressor interactions and can result in functional effects in opposing directions. When exposure to nutrient enrichment preceded elevated salinity, gross primary productivity was halved, and carbon metabolic rates increased by 50% compared to communities treated in the reverse order. These findings suggest that microbial functions, including productivity and carbon cycling, will fluctuate more dramatically as stressors increasingly interact in different sequences under global change.

Methods
This dataset contains the data in Fuggle et al (2025), Ecology, testing the hypothesis that the structure and function of freshwater benthic microbial communities are affected by salinisation and nutrient enrichment, and that combined stressor effects differ depending on frequency and order of application. Data were obtained from benthic microbial communities grown on tiles in outdoor mesocosms. It includes 1) biofilm in situ functional metrics, including oxygen production and consumption, concentration of green algae, diatoms, and cyanobacteria, total chlorophyll a concentration in biofilms, photosynthetic efficiency, 2) 16S rRNA ASV sequences from swabbed biofilms, and 3) OD590 absorbance values using Ecoplates incubated with biofilm suspensions.