Arashida Haruka, Kugenuma Takumi, Watanabe Masanori, Maeda Isamu
Department of Applied Biological Chemistry, Faculty of Agriculture, Utsunomiya University, 350 Mine-machi, Utsunomiya, Tochigi 321-8505, Japan.
Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka, Yamagata 997-8555, Japan.
J Biosci Bioeng. 2019 May;127(5):589-593. doi: 10.1016/j.jbiosc.2018.10.010. Epub 2018 Nov 2.
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.
紫色非硫细菌（PNSB）在有氧条件下培养时，即使在无氮培养基中也不会发生固氮作用。据推测，紫色非硫细菌可能缺乏足够的能力来保护其细胞成分免受空气（含20.95体积%氧气）的影响。因此，在本研究中，将枯草芽孢杆菌与紫色非硫细菌沼泽红假单胞菌一起接种到无氮培养基中，以研究当共培养物暴露于20.95体积%氧气时，沼泽红假单胞菌是否会发生固氮作用。只有当沼泽红假单胞菌与枯草芽孢杆菌一起接种时，它才能生长并形成生物膜。当将共培养中形成的生物膜接种到无氮培养基中时，在连续传代培养中观察到了固氮生长。沼泽红假单胞菌和枯草芽孢杆菌共培养物中nifH基因的表达、固氮酶活性的去阻遏、总氮的增加以及碳氮比的降低，证明了有氧条件下固氮作用的发生。在密封培养容器中，较低的培养基与空气比例会抑制固氮生长，并且在共培养中枯草芽孢杆菌的生长先于沼泽红假单胞菌。这些结果表明，通常伴随着氧气消耗的枯草芽孢杆菌的生长，可能会导致培养基中溶解氧浓度的降低，并有助于沼泽红假单胞菌中固氮酶活性的发生。
