Possible contribution of alternative nitrogenases to nitrogen fixation by asymbiotic N₂-fixing bacteria in soils

The relative contributions of the alternative (V- and Fe-only) and canonical (Mo-) nitrogenases to N₂ fixation in natural environments are still largely unknown. Here we combine chemical and molecular approaches to examine their role in terrestrial ecosystems. Pure-culture studies show that while the R ratio (defined as R=acetylene reduction rate/N₂ fixation rate by nitrogenase) is highly variable, low R ratios (0.5<R<2) are usually an indication of alternative nitrogenase activity. Microcosm experiments with temperate soils showed low R ratios and a large increase in N₂ fixation rates in response to vanadium amendments, indicating V-nitrogenase activity. Using RT-PCR, we were able to demonstrate expression of the V-nitrogenase, together with the Mo-nitrogenase, in these soils. An analysis of published R ratios showed that the contribution of alternative nitrogenases to asymbiotic N₂ fixation in soils may be more prevalent than previously recognized.