EGU22-10896
https://doi.org/10.5194/egusphere-egu22-10896
EGU General Assembly 2022
© Author(s) 2022. This work is distributed under
the Creative Commons Attribution 4.0 License.

Methodological and analytical improvement of the ISotopic Acetylene Reduction Assay for the assessment of complementary biological nitrogen fixation in low activity samples

Shannon Haynes, Romain Darnajoux, Eunah Han, and Xinning Zhang
Shannon Haynes et al.
  • Princeton University, Department of Geosciences, Princeton, United States of America (sjhaynes@princeton.edu)

Understanding the enzymes responsible for biological nitrogen fixation in the natural environment is crucial for understanding the global nitrogen cycle. The isotopic acetylene reduction assay (ISARA) is currently one of the only ways to distinguish between nitrogenase enzymes and it involves measuring the δ13C of ethylene generated via the reduction of acetylene. However, the classical method can only be applied to samples with ethylene concentrations >1,000 ppm which is limiting for environmental samples, where N2 fixation activity is generally low resulting in a low headspace ethylene concentration (<300 ppm).

Here we describe an improved analytical method for analyzing δ13C of ethylene using a homemade gas pre-concentration system and reproducible in-house standards developed from commercially available ethylene tanks. We also present a simple methodology using mutants of Azotobacter vinelandii (Mo-only and V-only nitrogenase) and the removal of headspace acetylene by chemical precipitation to easily scale the ISARA experiment from δ13C to complementary nitrogenase contribution without the uncertainty and tediousness surrounding measurement of the source acetylene.

The new Low activity - ISARA (LISARA) method can now estimate contribution of complementary nitrogenase from environmental samples with as little as 10 ppm of ethylene. Updated limit of quantification for δ13C of ethylene is < 2 ppm. Finally, we demonstrate the applicability of the method using samples with characteristically low N2 fixation activity (termites, wood, leaf litter, soil, moss), with substantial contribution of complementary nitrogenase across multiple sites in the northeastern United States.

Our results expand our knowledge of the contribution of complementary nitrogenase to temperate ecosystems. The new methodology will allow broader access to the classical ISARA method for pure culture experiments and high activity samples through the outsourcing of δ13C ethylene measurements, facilitating the study of complementary nitrogenases.

How to cite: Haynes, S., Darnajoux, R., Han, E., and Zhang, X.: Methodological and analytical improvement of the ISotopic Acetylene Reduction Assay for the assessment of complementary biological nitrogen fixation in low activity samples, EGU General Assembly 2022, Vienna, Austria, 23–27 May 2022, EGU22-10896, https://doi.org/10.5194/egusphere-egu22-10896, 2022.