Isolation of Nitrogen Fixing Free Living Bacteria

Nitrogen fixation by bacteria Nitrogen is one of the major nutrient required by all living organisms. Nitrogen in the elemental form is abundant in atmospheric air. However, most of the living organisms cannot uptake this free nitrogen. Plants play as the major nitrogen source for higher animals and for the plants nitrogen is available either from atmospheric inputs or from biological fixation, mostly they relay on Biological nitrogen fixation (BNF). Only prokaryotes can fix nitrogen biologically and a large number of microbes are reported to be involved in BNF. Nitrogen fixation is a part of nitrogen cycle, which involves three steps (1) Nitrification, (2) Denitrification, and (3) Nitrogen fixation. Mostly nitrogen fixation is performed by three group of organisms (a) Free living bacteria (Eg. Azotobacter, Klebsiella, Clostridium, and Methanococcus), (b) Symbiotic bacteria (Living in association with plants Eg. Rhizobium) (c) Cyanobacteria under anaerobic condition (Eg. Nostoc and Anabaena) During nitrogen fixation atmospheric gaseous nitrogen is reduced to ammonia. Nitrogen is highly inert molecule owing to its strong triple bond and reduction is highly energy requiring process. Reduction of nitrogen to ammonia is performed by an enzyme complex called as nitrogenase. It consists of two proteins a, Dinitrogenase and b, Dinitrogenase reductase. Isolation of Nitrogen Fixing free living bacteria Medium used for the isolation should be nitrogen free, and different media are available in the literature for the purpose. Ashby N-Free Sucrose Medium (1L) Sucrose 20.0 g K2HPO4 0.5 g MgSO4.7H2O 0. 2 g NaCl 0.2 g CaCO3 5.0g 5% Na2MoO4.2H2O 0.002 g MnSO4.4H2O 0.002 g CaSO4.2H2O 0.2 g Benzoate Medium Benzoformyl K2HPO4 MgSO4.7H2O NaCl CaSO4.2H2O 5 % Na2MoO4 1% FeSO4 1 % MnSO4 10 g 0.2 g 0.2 g 0.2 g 0.2 g 1 ml 1 ml 1 ml Dobereiner N-Free Malate Semi-Solid Medium Mannitol 10.0 g Sucrose 10.0 g K2HPO4 0.5 g MgSO4.7H2O 0.2 g NaCl 0.2 g CaCO3 5.0 g FeSO4 0.001g MnSO4.4H2O 0.002 g Malate 0.002 g
Glucose Nitrogen Free Mineral Medium Glucose 20g K2HPO4 1g CaCl2 0.1 g NaCl 0.5 g MgSO4.7H2O 0.25 g FeSO4.7H2O 0.01 g Na2MoO4.2H2O 0.01 g MnSO4.5H2O 0.01g

We also did the isolation of Azotobacter spp from soil. We used Jensen's Nitrogen free medium to isolate. But, for screening on nitrogen fixing activity of isolates, we used and BTB as color indicator. First, we adjusted medium as green color with BTB and we inoculated strains on this medium. After 3-7 days, the medium color will change to blue color, if your isolates have Nitrogen fixing activity. Here is the composition of GNFMM; Composition (gram per liter)

Agar 20 g I hope this information is useful for you. Good luck! lynn