Isolation and Use of Lytic Bacteria against Nitrogen-Fixing Cyanobacteria for Protoplast Preparation

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The ability to reduce the dinitrogen molecule to ammonia is restricted to prokaryotes where it is relatively scarce but spread widely through the systematic groups. Among these organisms, a prominent role is occupied by the cyanobacteria. They fix N_2 both in the free-living state and in symbiosis with a wide range of partners, and they are important contributors to the global supply of fixed nitrogen, including use in agriculture as a source of "green fertilizer."The screening and isolation of microorganisms capable of lysing cyanobacteria were performed on Dermocapra sp. and Anabaena cylindrica with the conventional double-agar overlay technique. Lysed zones were caused by dissolution of the cyanobacteria after digestion of cell walls by the lytic nicroorganisms.All of the single-colony isolations were finally retested on medium containing cyanobacteria whole cells, after the microorganisms had been examined for the presence of contamination on several different media. By this procedure, six strains of bacteria were iso;ated from paddy and upland soil samples, of which bacterium CL-101, CL-105 and CL-106 showed superior lytic activity.Morphological and physiological properties of the three strains were examined and identified as Flexithrix sp., Arthrobacter sp. and Bacillus sp., respectively.Protoplasts were obtained from Anabaena cyrindrica and Anabaena variabilis by using lytic enzyme from Bacillus CL-106. Several buffer sustems and osmotic agents were tried for effectiveness.Sucrose-Tris HCl buffer was finally chosen for the incubation period for the enzyme digestion. The MDM was added as stabilizers.