Earthern and Pot Culture Method to Check the Stability of Marine Azotobacter in Soil

INTRODUCTION

Among the three major habitats of the biosphere, the marine realm which covers 70% of the earth’s surface provides the largest inhabitable space for living organisms. The study of marine bacterial diversity is important in order to understand the community structure and pattern of distribution (Surajit Das et al 2006). For many years, the filamentous blue-green algae (cyanobacteria) were believed to be primarily responsible for N2 fixation in oceanic waters because low or negligible in situ rates were observed in their absence and there was a correlation of in situ N2 fixation with light intensity. However, evidence has been accumulating which documents the importance of bacterial N2 fixations in many and diverse marine habitats ( MARY LOU GUERINOT et al 1985) . It is commonly assumed that marine bacteria, since they live in the sea, must be Salt-tolerant organisms. ZoBell and Upham define marine bacteria as being bacteria from the sea which on initial isolation required seawater in the medium for growth. Therefore analysis of marine water will provide the effect of salts on the growth of marine Azotobacter. Biofertilizers are the source of microbial inoculants, which have brought hopes for many countries both economically and environmentally. Azotobacter sp is free living, known to fix atmospheric nitrogen. There are different strains of Azotobacter each has varied chemical, biological and other characters. Azotobacter and Azospirillum are two other efficient bacteria. The response of these organisms in increasing crop yield has been commonly experienced. These are the biofertilizers in the cultivation of most crops. Inoculation of soil or seed with Azotobacter is effective in increasing yields of crops in well-manured soil with high organic matter content. Experiments with Azotobacter cultures and crop plants at the Indian Agricultural Research Institute, New Delhi, lead us to believe that significant increases in growth and yield of wheat, rice and vegetable crops could be obtained in pot trials. However, under field conditions, such uniform trends towards increases in yield are not always reproducible. We carried out pot culture experiment in order to assess the effects of Azotobacter isolated from marine source on the growth of Black gram. Their shoot length, root length and their chlorophyll content were measured.

MATERIALS AND METHOD:

Sample collection:

Samples of surface water were collected in the area of Thundi region (Palk Bay) .Sample collection was accomplished at the interval of approximately 20 days

Surface –water samples (at depths of 1-2 m) were collected in sterile tube containing Azotobacter selective medium. Sediment samples were collected separately in broth medium. (Table .1 and 2)

Chemical parameter of sea water:

Collected water samples were analyzed for total hardness i.e the presence of magnesium and calcium by EDTA (0.01 M Ethylene diamine tetra acetic acid) titration method. Total Chlorine content was analyzed by Mohr method.

In EDTA method 60 ml of water sample was pipetted to an Erlenmeyer flask. About 2ml of buffer solution (mixture of ammonium chloride and ammonium hydroxide), was added to the sample. A few drops of indicator(Eriochrome black) were added and the solution was gently stried. The EDTA solution was taken in the burette and titrated with water sample until the color of the solution turns red to purple to blue. As soon as the color of the solution turned blue, stopped the titration and record the final level of EDTA solution in the burette. Finally the experimental concentration of calcium and magnesium ions in the unknown water sample was calculated. The hardness of water sample can be classified using a sum of all the calcium and magnesium ions in solution.

In Mohr method 20 ml of sodium chloride (0.01 M) solution was pipette in 250 ml Erlenmeyer flask. Approx 2ml of dipotassium chromate indicator was added to the solution. Solution was turned bright yellow color. Silver nitrate (0.01 M) solution was taken in the burette. The known chloride was titrated with silver nitrate until the color changed from bright yellow to brick red color (swirl the flask constantly to see the uniform color). Finally the experimental concentration of chloride in the known solution was calculated.

To determine unknown chloride, 5 ml of water sample was taken in 250 ml Erlenmeyer flask. 2ml of indicator (dipotassium chromate) was added. Silver nitrate (0.01 M) solution was taken in the burette. The water sample was carefully titrated with silver nitrate solution. Near the end point drop by drop was added from the burette as soon as the color of the solution turned yellow to red, stopped the reaction and recorded the final level of silver nitrate solution in the burette. Finally the experimental concentration of chloride in the unknown solution was calculated.

Media preparation:

Different selective media were used for the isolation of Azotobacter sp from marine source. As the isolates are of marine origin, the media were prepared by adding 3 % sodium chloride (NaCl). Media used for the isolation of nitrogen fixing organism (Azotobacter) from marine sources were: (Table 3)

1). Jensen’s Agar Medium (with 3% NaCl)

2). Azotobacter Agar Medium (with 3% NaCl)

3). Burk’s Medium (with 3% NaCl)

4). Marine agar medium.

Processing of samples (Kannan, 2002):

10 ml of water sample were mixed with 90 ml of sterile distilled water it gave 10-1 dilution. From the 10-1 dilution, the sample was decimally diluted up to 10-9 dilutions. By using spread plate technique, 0.1ml of diluted sample was plated in a sterile Petri plates, containing selective media. The plates were incubated at room temperature (28º C) for 48-72 hours.

Identification of isolates:

Gram’s staining (Kannan, 2002)

Gram staining reactions were recorded from heat fixed smears of fresh cultures.

Catalase test:

Catalase test was performed by adding 3% hydrogen peroxide drop by drop to the slant of fresh Azotobacter culture. Presence or absence of bubbling was recorded.

Phase contrast microscopic observation:

Motility and cell shape were determined by direct observations of wet mounts of fresh broth cultures, using phase- contrast microscopy. (Table 4)

Acetylene Reduction Assay:

Individual colonies were picked, purified, and assayed as pure cultures for nitrogenase activity, using N-deficient medium. This technique is an indirect method of measuring nitrogen fixation at a point of time. This method provides a simple, inexpensive, highly sensitive and non-destructive procedure for measuring rates of nitrogen fixation. Cultures were randomly selected for this assay. Serum bottles with rubber stoppers were collected, cleaned and sterilized. 30 ml of the sterilized Azotobacter broth was transferred to each bottle .The organisms were inoculated in the medium and incubated at 28º C for 3-4 days .after incubation 10 ml of nitrogen gas , 3 ml of acetylene gas was injected in to the serum bottles using syringe ( N2 replaces the air inside the bottle). Incubated the bottles for over nite at 28º C. at the end of the incubation period, 0.5 ml of the gas sample was withdrawn from the bottle and injected in to a gas chromatograph with FID system with 80-100 mesh Poro PAK/ propack Q column. The column temperature was maintained at 80º C, detector temperature at 100º C and injector temperature at 120º C . The carrier gas used was nitrogen with a flow rate of 30ml/ sec, for flame ionization hydrogen and zero air at the rate of 30ml/sec .the area of ethylene peak was recorded for each culture. Randomly selected samples which showed maximum enzyme activity were selected for pot culture experiment.

Analysis of garden soil for Chemical and nutrient content for pot culture experiment:

Garden soil was collected from rhizosphere region. Collected soil was analysed for the presence of N, P, K, Copper, manganese, iron, and zinc.(Table 5)

Pot culture experiment :

The nitrogen fixing ability of the isolated Azotobacter sp was determined in garden soil by pot culture experiment by assessing the growth of black gram. After 7 days of sowing various characteristics of growth such as root and shoot length was measured and chlorophyll content was estimated. Experiment was carried out in GRD College. Coimbatore.

Healthy viable seeds were selected for the experiment .Each pot contains 50 viable seeds. 10-12 kg of finely processed soil was filled in each pot .sterilized the pots with soil at 15 lb pressure for 4 hrs. The broth containing active culture of Azotobacter (1 × 109 cells) was selected. Five efficient strains were selected based on acetylene reduction assay for the experiment. The broth cultures of the selected Azotobacter sp were observed under phase contrast microscope prior to inoculation. Pots were selected for the experiment was thoroughly cleaned with disinfectant. Pots were filled with right combination of soil.

The healthy seeds were selected. Those seeds were mixed with 3ml of Azotobacter inoculums and 3ml of cool rice porridge. Then the seeds were dried

Fifty seeds were sown in each pot. The pots were watered every day. The control pot