The Application of the Mutagen Nitrous Acid to Improve the Free Living Nitrogen Fixation Ability of Azotobacter spp.


Three previously isolated Azotobacter spp. were obtained from a previous study and their identification and sensitivity were confirmed to seven different antibiotics. They showed resistance against chloramphenicol (C15) and nalidixic acid (NA30) but all were sensitive to ampicillin (AM50), tetracycline (TE15), cefalexin (CL30), rifamycin (RA10) and streptomycin (S50). Plasmid DNA electrophoresis revealed that isolates contain a single band of DNA with a m.wt. of 14kb. The isolates were mutagenized by nitrous acid at a concentration of 0.05M for 10 min which gave mutations at a percentage of (12-21%), (8-18%) and (2-8%) towards (C15), (NA30) and (S50) respectively, (6-20%) of the mutagenized isolates showed an ability to grow at 42oC and (18%) of the isolate A3 showed inability to fix nitrogen. Three mutagenized colonies from each isolate were selected depending on its mutation frequency, all were able to fix nitrogen, had the ability to grow on 42oC and carried one or more antibiotic mutation. The amount of ammonia excreted in µg/ml at 28,37 and 42oC was recorded at intervals of three days for 15 days for the three wild type and all selected mutagenized isolates. The best amount of ammonia excreted was at 37oC at the 12th day of incubation for the mutagenized isolate A2, 15 that was 12.0, 12.5 and 7.5µg/ml at 28,37 and 42oC respectively. All mutagenized isolates had the ability to excrete ammonia at 42oC at a percentage of (4.5- 7.5)µg/ml therefore these mutagenized isolates could be used as bio-fertilizers in Iraqi soils that are affected to such temperatures for many periods of the year.