Formation of Tyrothricin in Submerged Cultures of Bacillus Brevis

Abstract

Since the isolation of tyrothricin from cultures of Bacillus brevis (Dubos, 1939), numerous papers have appeared dealing with the chemical and biological properties of this bactericidal agent and its components, gramicidin and tyrocidine (Robinson and Molitor, 1942). However, little is known of the physiology of the bacillus from which the bactericidal agent is obtained. The present investigation is concerned with the relation of the nitrogen nutrition of B. brevis to tyrothricin formation in stationary and submerged cultures. A method suitable for large scale production of tyrothricin in submerged cultures is described. METHODS The BG strain of Bacillus brevis was used exclusively.2 The temperature and growth period were 370 and approximately 66 hours, respectively. Qualitative tests for tyrothricin were made by adding 1 ml. of the supernatant fluid of a centrifuged cultures of B. brevis to an equal volume of a 5-hour broth culture of Micrococcus conglomeratus (Strain MY) and incubating at 370 for 2 hours. Lysis of the micrococcus, as evidenced by clearing of the tube, indicates the presence of the bactericidal agent. This test is rapid and reliable; tyrothricin has never been isolated from a culture which failed to give a positive test for lysis. Tyrothricin was determined, quantitatively, by the isolation procedure of Dubos and Hotchkiss (1941) modified to include ether extraction of the alcohol-extracted crude tyrothricin prior to precipitation with NaCl. Also, a more rapid method was developed which does not involve isolation of tyrothricin and is applicable to small amounts of culture. Cell material from an aliquot of a culture is collected by centrifugation and suspended in an amount of ethanol equivalent to the volume of the aliquot used. After 24 hours at room temperature, the alcoholic extract is diluted with water and the smallest amount necessary to cause complete lysis of the micrococcus culture is determined as described above. Usually about 4y of tyrothricin are required for lysis. Comparative experiments