Bulk and spatially resolved extracellular metabolomics of free-living nitrogen fixation

Soil microorganisms drive ecosystem function, but challenges of scale between microbe and ecosystem hinder our ability to accurately quantify and predictively model the soil microbe-ecosystem function relationship. Quantifying this relationship necessitates studies that systematically characterize multi-omics of soil microorganisms and their activity across sampling scales from spatially resolved to bulk measures, and structural complexity, from liquid pure culture to in situ. To address this need, we cultured two diazotrophic bacteria in liquid and solid media, with and without nitrogen (N) to quantify differences in extracellular metabolites associated with nitrogen fixation under increasing environmental structural complexity. We also quantified extracellular metabolites across sampling scales including bulk sampling via GC-MS analysis and spatially resolved analysis via MALDI mass spectrometry imaging. We found extracellular production of inorganic and organic N during free-living nitrogen fixation activity, highlighting a key mechanism of terrestrial N contributions from this process. Additionally, our results emphasize the need to consider the structural complexity of the environment and spatial scale when quantifying microbial activity. We found differences in metabolite profiles between culture conditions, supporting previous work indicating environmental structure influences microbial function, and across scales, underscoring the need to quantify microbial scale conditions to accurately interpret microbial function. ImportanceStudying soil microorganisms, both who is present and what they are doing, is a challenge because of vast differences in scale between microorganism and ecosystem and because of inherent complexities of the soil system (e.g., opacity, chemical complexity). This makes measuring and predicting important ecosystem processes driven by soil microorganisms, like free-living nitrogen fixation, difficult. Free-living nitrogen fixing bacteria play a key role in terrestrial nitrogen contributions and may represent a significant, yet overlooked, nitrogen source in agricultural systems like bioenergy crops. However, we still know very little about how free-living nitrogen fixation contributes nitrogen to terrestrial systems. Our work provides key insight by hierarchically increasing structural complexity (liquid vs. solid culture) and scale (spatially resolved vs. bulk) to address the impact of environmental structure and sampling scale on detection of free-living nitrogen fixation and to identify the forms of nitrogen contributed to terrestrial systems by free-living nitrogen bacteria.