Analysis of the nitrogen-fixing ability of the diazotrophic community living in the rice rhizosphere using a novel test tube chemostat culture.

The rice rhizosphere is a hot spot for nitrogen fixation using carbon sources from root exudates. We herein investigated the intensity of the nitrogen-fixing ability of each diazotroph interacting with other bacteria within the rhizosphere microbiota. We established the Test Tube Chemostat replicating Rice Rhizosphere (TCRR) based on four characteristics of the rice rhizosphere: root exudates, percolation, radial oxygen loss, and soil. The TCRR flora constructed by culturing paddy soil using TCRR showed the pH and high nitrogen-fixing ability of the rice rhizosphere. Twenty-five species of diazotrophs from the TCRR flora were categorized into two types: strains producing acetate and ethanol and strains utilizing them. A co-culture of both types resulted in high nitrogen fixation, mostly through the acetate interaction. High nitrogen fixation by the TCRR flora was further increased by an inoculation of both Klebsiella pasteurii NG13 and Azospirillum lipoferum FS. These results provide novel insights to develop biofertilizers.