Nitrogen fixation in purple non-sulfur bacteria (PNSB) does not take place even in N-free medium when they are cultured under aerobic conditions. It is assumed that PNSB might possess inadequate capability to protect their cellular components from exposure to air (20.95 vol.% oxygen). In this study, therefore, Bacillus subtilis was inoculated together with a purple non-sulfur bacterium Rhodopseudomonas palustris in N-free medium in order to examine whether nitrogen fixation in Rps. palustris takes place when the co-culture is exposed to 20.95 vol.% oxygen. Rps. palustris grew and formed biofilm only when it was inoculated together with B. subtilis. When the biofilm formed in the co-culture was inoculated in N-free medium, diazotrophic growth was observed in the sequential subcultures. Expression of nifH gene, derepression of nitrogenase activity, an increase of total nitrogen, and a decrease of C/N in the co-culture of Rps. palustris and B. subtilis demonstrated the occurrence of nitrogen fixation under aerobic conditions. The diazotrophic growth was suppressed at a lower medium-to-air ratio in a sealed culture vessel, and growth of B. subtilis preceded growth of Rps. palustris in the co-culture. These results suggest that growth of B. subtilis, which is usually accompanied with oxygen consumption, might cause a decrease of dissolve oxygen concentration in medium and contribute to the occurrence of nitrogenase activity in Rps. palustris. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.