@article{2a64759c216143c991781a7adfb2da92,
title = "Quantification of biological nitrogen fixation by Mo-independent complementary nitrogenases in environmental samples with low nitrogen fixation activity",
abstract = "Biological nitrogen fixation (BNF) by canonical molybdenum and complementary vanadium and iron-only nitrogenase isoforms is the primary natural source of newly fixed nitrogen. Understanding controls on global nitrogen cycling requires knowledge of the isoform responsible for environmental BNF. The isotopic acetylene reduction assay (ISARA), which measures carbon stable isotope (13C/12C) fractionation between ethylene and acetylene in acetylene reduction assays, is one of the few methods that can quantify isoform-specific BNF fluxes. Application of classical ISARA has been challenging because environmental BNF activity is often too low to generate sufficient ethylene for isotopic analyses. Here we describe a high sensitivity method to measure ethylene δ13C by in-line coupling of ethylene preconcentration to gas chromatography-combustion-isotope ratio mass spectrometry (EPCon-GC-C-IRMS). Ethylene requirements in samples with 10% v/v acetylene are reduced from > 500 to ~ 20 ppmv (~ 2 ppmv with prior offline acetylene removal). To increase robustness by reducing calibration error, single nitrogenase-isoform Azotobacter vinelandii mutants and environmental sample assays rely on a common acetylene source for ethylene production. Application of the Low BNF activity ISARA (LISARA) method to low nitrogen-fixing activity soils, leaf litter, decayed wood, cryptogams, and termites indicates complementary BNF in most sample types, calling for additional studies of isoform-specific BNF.",
author = "Haynes, {Shannon J.} and Romain Darnajoux and Eunah Han and Sergey Oleynik and Ezra Zimble and Xinning Zhang",
note = "Funding Information: We gratefully acknowledge funding support from the Watershed Institute (https://thewatershed.org), the Carbon Mitigation Initiative (Princeton High Meadows Environmental Institute to XZ), the Tuttle Fund (Princeton Geoscience Department to XZ), NSF EAR grant 1631814 (to XZ), and a Simons Foundation Life Science Research Postdoctoral Fellowship (to RD). We thank collaborators at the Watershed institute, the Rutgers Pineland Field Station, the Stony Ford Center for Ecological Studies at Princeton University, the Institute of Advanced Studies, and the Dartmouth College-Mt. Moosilauke Advisory Committee for permission and access to field samples; Katja Luxem and Linta Reji for help with field work, Anne Kraepiel, F. Morel, J.P. Bellenger, and all members of the Zhang laboratory for discussions. Funding Information: We gratefully acknowledge funding support from the Watershed Institute ( https://thewatershed.org ), the Carbon Mitigation Initiative (Princeton High Meadows Environmental Institute to XZ), the Tuttle Fund (Princeton Geoscience Department to XZ), NSF EAR grant 1631814 (to XZ), and a Simons Foundation Life Science Research Postdoctoral Fellowship (to RD). We thank collaborators at the Watershed institute, the Rutgers Pineland Field Station, the Stony Ford Center for Ecological Studies at Princeton University, the Institute of Advanced Studies, and the Dartmouth College-Mt. Moosilauke Advisory Committee for permission and access to field samples; Katja Luxem and Linta Reji for help with field work, Anne Kraepiel, F. Morel, J.P. Bellenger, and all members of the Zhang laboratory for discussions. Publisher Copyright: {\textcopyright} 2022, The Author(s).",
year = "2022",
month = dec,
doi = "10.1038/s41598-022-24860-9",
language = "English (US)",
volume = "12",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",
number = "1",
}