Development of cellulolytic nitrogen-fixing activity of Azotobacter spp. by transposon mutagenesis

Abstract

The chemical mutagenized microorganisms used in this study were nitrogen-fixing bacteria; Azotobacter chrococcum and Azotobacter benjerinkii which were collected from the Microbiology Laboratory, Department of Biotechnology, Kyaukse. Transposon mutagenesis was studied on these strains to get dual activities such as nitrogen-fixing and cellulolytic activities by using recombinant Escherichia coli S17 carrying cellulase gene from Rhizobium leguminosarum. The targeted strains that carry the transposon were selected on the Glucose Nitrogen Free Mineral Medium which contains chloramphenicol (0.6 mg/20ml) and kanamycin (0.4mg/20ml). Nitrogen-fixing activity of Azotobacter spp. was detected by the colour changes in Glucose Nitrogen Free Mineral Medium (G-NFM) containing trace amount of Bromothymol Blue (BTB) as indicator and ammonium test kit. Cellulolytic activity of Azotobacter spp. before and after transposon mutagenesis was detected by both plate screening method and by Dinitrosalicylic colorimetric method. The nitrogen-fixing activities of the strains were almost the same before and after transposon mutagenesis. Cellulolytic activity measured in Glucose Nitrogen Free Mineral Medium was the highest in 5 days incubation.