Ecosystem scale evidence for the contribution of vanadium-based nitrogenase to biological nitrogen fixation.

Nitrogen is the primary limiting nutrient in high latitude ecosystems. Biological nitrogen fixation (BNF) by microorganisms associated with cryptogamic covers, such as cyanolichens and bryophytes, is an important source of new reactive nitrogen in pristine, high-latitude ecosystems. BNF is catalyzed by the enzyme nitrogenase, for which three isoforms have been described; the canonical molybdenum (Mo) nitrogenase which requires Mo in its active site and two alternative nitrogenases, the vanadium and iron-only nitrogenases. The low availability of Mo on land has been shown to limit BNF in many ecosystems from the tropical forest to the arctic tundra. Alternative nitrogenases have been suggested as viable alternatives to cope with Mo limitation of BNF, however, field data supporting this long-standing hypothesis have been lacking.

Here, we elucidated the contribution of the vanadium nitrogenase to BNF by cyanolichens across a 600 km latitudinal transect in eastern Canadian boreal forests. We report a widespread activity of the vanadium nitrogenase which contributed between 15 to 50% of total BNF rates on all sites. Vanadium nitrogenase contribution to BNF was more robust in the northern part of the transect. Vanadium nitrogenase contribution to BNF also changed during the growing season, with a three-fold increase between the early (May) and late (September) growing season. By including the contribution of the vanadium nitrogenase to BNF, estimates of new N input by cyanolichens increase by up to 30%, a significant change in these low N input ecosystems. Finally, we found that Mo availability was the primary driver for the contribution of the vanadium nitrogenase to BNF with a Mo threshold of ~ 250 ng.g_lichen^-1 for the onset of vanadium based BNF.

This study on N₂-fixing cyanolichens provides extensive field evidence, at an ecosystem scale, that vanadium-based nitrogenase greatly contributes to BNF when Mo availability is limited. The results showcase the resilience of BNF to micronutrient limitation and reveal a strong link between the biogeochemical cycle of macro- and micronutrients in terrestrial ecosystems. Given widespread findings of Mo limitation of BNF in terrestrial ecosystems, additional consideration of vanadium-based BNF is required in experimental and modeling studies of terrestrial biogeochemistry.