FIG. 1.

N₂-fixing activity (ARA) during isolation steps and fluorescence micrographs showing the structure and viability of consortia of the cells. (A) Test tube that is positive for ARA during isolation from a stem of O. officinalis. (B) Anaerobic isolate B901-1b after Gram staining. (C) Aerobic isolate B901-2 after Gram staining. (D) Growth and ARA of singly and cocultured B901-1b and B901-2 in air and N₂ gas. ARA was detected exclusively in a single culture of B901-1b in N₂ gas and in a mixed culture in air. Gas evolution and growth occurred in test tubes, except for the single culture of B901-1b in air, which sometimes caused an accumulation of agar in the uppermost layer of the medium. ARA+, test tubes that were positive for ARA (16 to 24 nmol of ethylene produced h⁻¹ tube⁻¹); −, <0.1 nmol of ethylene produced h⁻¹ tube⁻¹. (E) Fluorescence micrograph showing a reconstructed consortium. (F and G) Living (green) and dead (red) cells of Clostridium sp. strain B901-1b (large rod) cultured in RMR broth under anaerobic conditions (F) and under aerobic conditions with the accompanying bacterium Enterobacter sp. strain B901-2 (small coccus) (G). Both preparations were exposed to air for 5 min before observation. Bar, 10 μm. According to their 16S rRNA gene sequences, B901-1b and B901-2 were identified as a Clostridium sp. and an Enterobacter sp., respectively (see Fig. 3).