Carbon Dioxide Effects on Heterotrophic Dinitrogen Fixation in a Temperate Pine Forest

Increased net primary productivity (NPP) under elevated atmospheric CO₂ requires additional N inputs to sustain C sequestration. We hypothesized that heterotrophic N₂ fixation would be stimulated by enhanced litter production under elevated CO₂, thus augmenting N availability to plants. To test if N₂ fixation is limited by organic substrates alone or in combination with nutrients required for the nitrogenase enzyme, we measured nitrogenase activity (acetylene reduction) in laboratory incubations with water, nutrient, and O₂ manipulations. Response of N₂ fixation to water, glucose, P, Fe, or Mo was measured under aerobic and anaerobic conditions in forest floor and mineral soil samples from the Duke Forest, NC. Potential nitrogenase activity in forest floor and mineral soil from the Duke Forest Free Air CO₂ Enrichment (FACE) site were measured to determine if elevated CO₂ enhances N₂ fixation. In homogenized slurries with glucose additions, nitrogenase activity was 2 and 400 times greater than controls in organic and mineral soils, respectively (P < 0.01). In laboratory studies, water additions increased N₂ fixation 25-fold in intact soil cores (P < 0.01). Additions of nutrients alone or in combination with C and water did not consistently stimulate N₂ fixation in intact soil cores. We detected no CO₂ effect on potential nitrogenase activity in Duke FACE soil. Since heterotrophic N₂ fixation is not stimulated in temperate pine forests under elevated CO₂, additional N assimilation by trees will require increased acquisition of endogenous N, such as increased nutrient use efficiency or enhanced root exploration of the soil.