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

将二氮分子还原为氨的能力仅限于原核生物，原核生物相对较少，但在系统群中广泛传播。在这些生物中，蓝藻占据着突出的作用。它们在自由生活状态和与广泛的伙伴共生的情况下固定n2，它们是全球固定氮供应的重要贡献者，包括在农业中作为“绿色肥料”的来源。采用常规的双琼脂覆盖技术，对Dermocapra sp.和Anabaena ica进行了能够裂解蓝藻的微生物的筛选和分离。溶解区是由溶解微生物消化细胞壁后蓝藻溶解造成的。在几种不同的培养基上检查了微生物是否存在污染后，所有的单菌落分离最终在含有蓝藻全细胞的培养基上重新测试。通过这种方法，6株细菌被分离；其中CL-101、CL-105和CL-106细菌具有较强的裂解活性。对这3株菌株的形态和生理特性进行了检测，鉴定为Flexithrix sp.、Arthrobacter sp.和Bacillus sp.。利用芽孢杆菌CL-106的裂解酶，对水蛭和变水蛭进行原生质体分离。试验了几种缓冲系统和渗透剂的效果。最后选择蔗糖- tris HCl缓冲液作为酶消化的孵育期。添加MDM作为稳定剂。

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.