Free-living nitrogen-fixation (FLNF) in the rhizosphere, or N-fixation by heterotrophic bacteria living on/near root surfaces, is ubiquitous and a significant source of N in some terrestrial systems. FLNF is also of interest in crop production as an alternative to chemical fertilizer, potentially reducing production costs and ameliorating negative environmental impacts of fertilizer N additions. Despite this interest, a mechanistic understanding of controls (e.g. carbon, oxygen, nitrogen, and nutrient availability) on FLNF in the rhizosphere is lacking, but necessary. FLNF is distinct from and occurring under more diverse and dynamic conditions than symbiotic N-fixation, therefore predicting FLNF rates and understanding controls on FLNF has proven difficult. This has led to large gaps in our understanding of FLNF and studies aimed at identifying controls on FLNF are needed. Here we provide a mechanistic overview of FLNF, including how various controls may influence FLNF in the rhizosphere in comparison with symbiotic N-fixation occurring in plant nodules where environmental conditions are moderated by the plant. We apply this knowledge to a real-world example, the bioenergy crop switchgrass (Panicum virgatum), to provide context of how FLNF may function in a managed system. We also highlight future challenges to assessing FLNF and understanding how FLNF functions in the environment and significantly contributes to plant N availability and productivity.

Soil microorganisms play a key role in driving major biogeochemical cycles and in global responses to climate change. However, understanding and predicting the behavior and function of these microorganisms remains a grand challenge for soil ecology due in part to the microscale complexity of soils. It is becoming increasingly clear that understanding the microbial perspective is vital to accurately predicting global processes. Here, we discuss the microbial perspective including the microbial habitat as it relates to measurement and modeling of ecosystem processes. We argue that clearly defining and quantifying the size, distribution and sphere of influence of microhabitats is crucial to managing microbial activity at the ecosystem scale. This can be achieved using controlled and hierarchical sampling designs. Model microbial systems can provide key data needed to integrate microhabitats into ecosystem models, while adapting soil sampling schemes and statistical methods can allow us to collect microbially-focused data. Quantifying soil processes, like biogeochemical cycles, from a microbial perspective will allow us to more accurately predict soil functions and address long-standing unknowns in soil ecology.

Cellulosic bioenergy crops, like switchgrass (Panicum virgatum), have potential for growth on lands unsuitable for food production coupled with potential for climate mitigation. Sustainability of these systems lies in identifying conditions that promote high biomass yields on marginal lands under low-input agricultural practices. Associative nitrogen fixation (ANF) is a potentially important nitrogen (N) source for these crops, yet ANF contributions to plant N, especially under fertilizer N addition are unclear. In this study, we assess structure (nifH) and function (ANF) of switchgrass root-associated diazotrophic communities to long-term and short-term N additions using soil from three marginal land sites. ANF rates were variable and often unexpectedly high, sometimes 10× greater than reported in the literature, and did not respond in repeatable ways to long-term or short-term N. We found few impacts of N addition on root-associated diazotrophic community structure or membership. Instead, we found a very consistent root-associated diazotrophic community even though switchgrass seeds were germinated in soil from field sites with distinct diazotrophic communities. Ultimately, this work demonstrates that root-associated diazotrophic communities have the potential to contribute to switchgrass N demands, independent of N addition, and this may be driven by selection of the diazotrophic community by switchgrass roots.

Plants and soil microorganisms interact closely in the rhizosphere where plants may exchange carbon (C) for functional benefits from the microbial community. For example, the bioenergy crop, switchgrass (Panicum virgatum) is thought to exchange root-exuded C for nitrogen (N) fixed by diazotrophs (free-living N-fixers). However, this interaction is not well characterized and it is not known how or if switchgrass responds to diazotrophs or their activity. To explore this question, we assessed rhizosphere metabolite chemistry of switchgrass grown in a hydroponic system under two N levels and under inoculated or uninoculated conditions. Plants were grown with the inoculum Azotobacter vinelandii DJ for 3 days before harvest. We found switchgrass root exudate chemistry to be driven by N availability. Total metabolite concentrations were generally greater under high N versus low N and unaffected by inoculation. Examination of rhizosphere chemical fingerprints indicates metabolite chemistry was also driven strongly by N availability with a greater relative abundance of carbohydrates under high N and greater relative abundance of organic acids under low N. We also found evidence of changes in rhizosphere chemical fingerprints by inoculation treatment. However, we found little evidence of N treatment and inoculation interaction effects that suggests this response is not directly mediated by N availability. [ ] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .