Anaerobic Nitrogen-Fixing Consortia Consisting of Clostridia Isolated from Gramineous Plants

We report here the existence of anaerobic nitrogen-fixing consortia (ANFICOs) consisting of N2-fixing clostridia and diverse nondiazotrophic bacteria in nonleguminous plants; we found these ANFICOs while attempting to overcome a problem with culturing nitrogen-fixing microbes from various gramineous plants. A major feature of ANFICOs is that N2 fixation by the anaerobic clostridia is supported by the elimination of oxygen by the accompanying bacteria in the culture. In a few ANFICOs, nondiazotrophic bacteria specifically induced nitrogen fixation of the clostridia in culture. ANFICOs are widespread in wild rice species and pioneer plants, which are able to grow in unfavorable locations. These results indicate that clostridia are naturally occurring endophytes in gramineous plants and that clostridial N2 fixation arises in association with nondia-zotrophic endophytes. Microbes are not always culturable even though their bio-logical activities may be detectable (1, 15, 20). This is true for some N2-fixing bacteria associated with plants, such as Azoar-cus endophytes (16) and rhizobial bacteroids (22). Although the functional significance of microbial consortia in biofilms, for example (1, 3), has been emphasized, there are few con