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Cultures of Thiobacillus ferrooxidans grown in the absence of a source of fixed nitrogen reduced acetylene to ethylene suggesting the presence of nitrogenase. This presumptive evidence has now been substantiated by the demonstration of the enrichment of 15N in the protein of the organism when grown under 15N2. The possibility that T. ferrooxidans was being grown in association with other nitrogen-fixing organisms was eliminated by the use of cultures derived from colonies whose development had been observed from single organisms.
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