Isolation of the key intermediate in biological nitrogen fixation by Clostridium.

Investigations with the isotopic tracer N16 have furnished definite evidence that ammonia is the key intermediate in nitrogen fixation by Azotobacter vinelundii (1). Direct isolation of the intermediate, however, has not been possible, apparently because ammonia is used so rapidly by this bacterium that it does not accumulate. In a study of the comparative biochemistry of nitrogen fixation, we have extended these experiments to other agents; in the course of these trials it was established that Clostridium pasteurianum excreted into the medium a considerable portion of the nitrogen fixed (2). This excretion differed materially both quantitatively and qualitatively from that previously noted with Azotobacter. Up to 50 per cent of the total nitrogen fixed may be excreted by the anaerobe, and, even more significant, the excretion begins in the early stages of growth, long before the exogenous supply of energy is exhausted. These observations suggested that the excreted nitrogen might be “juvenile;” i.e., recently fixed rather than arising from breakdown of cell protein. Experiments with labeled Nz not only allowed a decision to be made between these two possibilities but also resulted in the actual isolation of the key intermediate, ammonia.