Application of stable-isotope labelling techniques for the detection of active diazotrophs

Investigating active participants in the fixation of dinitrogen gas is vital as N is often a limiting factor for primary production. Biological nitrogen fixation is performed by a diverse guild of bacteria and archaea (diazotrophs), which can be free-living or symbionts. Free-living diazotrophs are widely distributed in the environment, yet our knowledge about their identity and ecophysiology is still limited. A major challenge in investigating this guild is inferring activity from genetic data as this process is highly regulated. To address this challenge, we evaluated and improved several ¹⁵N-based methods for detecting N₂ fixation activity (with a focus on soil samples) and studying active diazotrophs. We compared the acetylene reduction assay and the ¹⁵N₂ tracer method and demonstrated that the latter is more sensitive in samples with low activity. Additionally, tracing ¹⁵N into microbial RNA provides much higher sensitivity compared to bulk soil analysis. Active soil diazotrophs were identified with a ¹⁵N-RNA-SIP approach optimized for environmental samples and benchmarked to ¹⁵N-DNA-SIP. Lastly, we investigated the feasibility of using SIP-Raman microspectroscopy for detecting ¹⁵N-labelled cells. Taken together, these tools allow identifying and investigating active free-living diazotrophs in a highly sensitive manner in diverse environments, from bulk to the single-cell level.