To Fix or Not To Fix: Controls on Free-Living Nitrogen Fixation in the Rhizosphere

Abstract

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 occurs 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.

Keywords: diazotrophs; environmental controls; free-living nitrogen fixation; rhizosphere; rhizosphere-inhabiting microbes.

FIG 1

Contrasting habitats of free-living and symbiotic nitrogen fixation. (a) FLNF is carried out by a diverse array of N fixers living in a community, while symbiotic N fixation is performed only by a few bacteria (e.g., rhizobia and Frankia) living in a population. (b) FLNF is supported by dissolved organic carbon (DOC) in the soil, a variable and complex C source, while symbiotic N fixers receive a constant supply of simple C compounds (i.e., succinate) directly from the host plant. (c) Oxygen concentration in the rhizosphere is highly variable and driven by soil structure and texture and respiration by microbes and roots. Conversely, symbiotic N fixers are supplied oxygen at low concentrations by their host plant. (d) Nutrients necessary to support FLNF (e.g., P, Fe, Mo, and V) must be acquired by the diazotroph. However, these nutrients are delivered to symbiotic N fixers by the host plant. (e) Diazotrophs in the rhizosphere can access N from soil and FLNF, while all symbiotically fixed N is delivered to the plant.

FIG 2

Environmental factors known to impact FLNF presented with triangles representing a theoretical range for each factor, low (narrow, dark-colored) to high (broad, light-colored). In contrast, symbiotic N fixation, represented by vertical hatched bar, only occurs in a narrow range of each of the environmental conditions. For example, FLNF can occur over a wide range of oxygen concentrations from low to high, while symbiotic N fixation occurs only at low oxygen concentrations.

FIG 3

Scanning electron micrograph (×20,000) showing the free-living nitrogen-fixer Azotobacter vinelandii living on a switchgrass root. Cave-in-rock variety switchgrass seedlings were grown in sterile jars and inoculated with A. vinelandii (ATCC BAA-1303).

FIG 4

Preliminary N-fixation rates from switchgrass rhizosphere soils receiving high N additions (High N; +125 kg Urea-N ha⁻¹ year⁻¹) and low N additions (Low N; +25 kg Urea-N ha⁻¹ year⁻¹). Sterile switchgrass (var. Cave-in-Rock) seeds were planted into a sterile sand and vermiculite mixture (50:50 vol/vol) containing a core of field soil as root inoculum. Field soils were collected from marginal land sites managed by the Great Lakes Bioenergy Research Center (GLBRC) in southern Michigan. Plants received one addition of N at planting and a one-half Hoagland’s nutrient solution (N free). Plants were grown in the greenhouse for 4 months prior to harvest. N-fixation rates were measured on 2-g root/rhizosphere samples via ¹⁵N₂ enrichment method (35). Samples (n = 6 per treatment) were placed in 10-ml gas vials and adjusted to 60% water holding capacity using a 4 mg C ml⁻¹ glucose solution. Vials were sealed, evacuated, and adjusted back to atmospheric pressure by adding 1 ml of ¹⁵N₂ gas, 10% equivalent volume of oxygen, and balanced with helium. Vials incubated for 7 days and were then dried and ground for ¹⁵N analysis. Final values were calculated following Warembourg (80). N additions did not significantly impact N-fixation rates (P = 0.1585).