Free-living nitrogen fixation (FLNF) is the biological conversion of gaseous N2 into ammonia (NH3) by heterotrophic bacteria and archaea not in symbiosis with plants. This energy intensive process occurs predominately where carbon (C) is readily available to support these high energy demands, such as in the rhizosphere where roots exude C into the soil environment. FLNF has continually gained attention as an important and ubiquitous N source to terrestrial systems and as a potential alternative to fertilizer N addition in crop production. In particular, it has gained interest for its potential to support production of bioenergy cropping systems, like switchgrass (Panicum virgatum), particularly when grown on marginal lands. Diverse communities of N-fixing organisms (diazotrophs) have been identified in the rhizospheres of these cropping systems as well as FLNF activity. But, to harness this potential N source it is important to understand the controls on FLNF. My dissertation work characterizes biological and environmental controls on FLNF associated with the switchgrass rhizosphere.Though there are decades of research available on symbiotic N-fixation, the conditions under which FLNF occurs are quite distinct including dynamic C sources and availabilities, oxygen concentrations, and N availability. FLNF is also carried out by a diverse community of diazotrophs rather than a single population as with symbiotic N-fixation. To determine the important controls on FLNF, I first tested impacts of C source and oxygen concentration on FLNF through development of an optimized method for measuring FLNF. I found that increased diversity of C sources and lower oxygen concentrations promoted the most FLNF. I then examined the effects of legacy and short-term N additions on both FLNF rates and the composition of the diazotroph community in the switchgrass rhizosphere. Surprisingly, I found no evidence for legacy or short-term N controls on FLNF rates or diazotroph community composition. However, I found a strong rhizosphere effect on diazotroph community composition, suggesting switchgrass selects for a distinct and consistent N-fixing community. Lastly, I determined controls on FLNF under field conditions and how these controls relate to plant available N, using a variety of field and molecular data. I found that soil N availability was the dominant control on FLNF, but the direction of this control depended on the soil N pool. Together, my work highlights several important environmental and biological controls on FLNF and ultimately improves our ability to understand and predict this important N source for terrestrial systems. Lastly, my work adds to the growing body of evidence that FLNF occurs in many systems and can contribute largely to plant N demands. By extrapolating the average of my measured FLNF rates from μg N fixed g-1 dry soil day-1 to kg N ha-1 yr -1, I found FLNF has the potential to contribute upwards of 11.0 kg N ha-1 yr -1. These rates are 2x greater than the estimated contribution of N from symbiotic N-fixation in temperate grasslands and meet approximately 31% of the N deficit identified in switchgrass systems from previous work. Although these extrapolated rates are based on optimized conditions for potential FLNF rates and therefore are likely overestimates, they highlight the important role of FLNF in switchgrass cropping systems and its potential to contribute to improving the sustainability of bioenergy production.
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