Endophytic bacteria that live freely in the internal tissues of plants and cause no apparent harm have a diverse range of growth promotion mechanisms including nitrogen fixation. Although 78% of the earth’s atmosphere is nitrogen, nitrogen is often a limiting factor in agriculture since it is not readily available to plants. Bacteria and Archea are the only organisms that can fix atmospheric di-nitrogen, thereby making it available for plant growth. This activity is termed biological nitrogen fixation (BNF) and is catalyzed by the oxygen sensitive nitrogenase enzyme to convert N2 to bio-available NH3. Nitrogenases are complex metalloenzymes with highly conserved structural and mechanistic features (reviewed in Alberty 1994; Burgess and Lowe 1996; Rees and Howard 2000). The enzyme is oxygen sensitive, which imposes physiological constraints on the organism. Additionally, the enzyme has a relatively slow turnover time (Thorneley and Lowe 1985), which requires the microbe to synthesize large quantities of the protein, up to twenty percent of protein in the cell (reviewed in Dixon and Khan 2004). Also, the conversion of atmospheric di-nitrogen to a form that can be used by plants requires 16 ATP to reduce one molecule of N2, making it one of the most energy demanding reactions identified in bacterial organisms (Thorneley and Lowe 1985). Together, the amount of energy, the low oxygen requirement, and the amount of protein required to create the nitrogenase enzyme, place a large burden on a nitrogen fixing endophyte. As a result, the synthesis of the nitrogenase complex is stringently regulated at the genetic level (Dixon and Khan 2004). It has been suggested that bacterial endophytes are placed in a more favorable environment compared to rhizospheric bacteria because they are less vulnerable to competition from native soil bacteria and are shielded from various biotic and abiotic stresses (Reinhold-Hurek and Hurek 1998). Perhaps the most-studied grass inoculated with free living nitrogen-fixing endophytes is sugarcane. Burkholderia MG43 inoculated sugarcane plantlets produced a 20% increase in yield over un-inoculated control (Govindarajan et al. 2006), and it was demonstrated that 60 to 80% of nitrogen accumulated in sugarcane came from atmospheric nitrogen fixation (Boddey et al. 1995). The authors also noted that farmers in Brazil have observed some varieties of sugarcane grown in fields for decades, even up to a century without showing any decline in soil N reserve or yield, despite the supply deficit of nitrogen (Boddey et al. 1995). Rice has also been studied in the context of its relationship with free-living nitrogen-fixing Burkholderia spp. In one field experiment, 31% of plant nitrogen was derived from BNF and inoculation resulted in as high as a 69% increase in biomass compared to the un-inoculated control (Baldani et al. 2000). Researchers also found Burkholderia vietnamiensis inoculated rice seedlings increased yield by 5.6 to 12.16%, and 42% of nitrogen found in the inoculated plants came from atmospheric nitrogen fixation (Govindarajan et al. 2008). In addition to rice, Burkholderia were found to be among the most common nitrogen-fixing isolates from maize plants cultivated in Mexico, and many were reported to be new species (Estrada et al. 2002). These findings support the use of free-living nitrogen-fixing endophytes in the effort to reduce the use of synthetic nitrogen fertilizer and offer hope in creating high-yielding, low — input agricultural production systems.