Earthern and Pot culture method for checking the stability of Azotobacter on the ocean floor
INTRODUCTION
Among the three major habitats biosphere, the marine realm that covers 70% of Eartha € ™ s surface provides the largest living space for living organisms. The study of bacterial diversity marina is important to understand the community structure and pattern of distribution (Surajit Das et al 2006). For many years, blue-green filamentous algae (Cyanobacteria) are thought to be primarily responsible for N2 fixation in the sea due to low or negligible in situ rates were observed in their absence and had a correlation of N2 fixation in situ with the intensity of light. However, evidence has accumulated that demonstrate the importance of bacterial N2 fixation the many and diverse marine habitats (Mary Lou Guerinot et al 1985). It is commonly assumed that marine bacteria, since they live in the sea, the bodies must be tolerant to salt. ZoBell Upham and identify marine bacteria and bacteria from the sea than in the initial isolation of sea water needed in an environment for growth. Therefore the analysis of seawater provide the effect of salts on the growth of marine Azotobacter. Biofertilizers are the source of microbial inoculants, which led to the hopes of many countries both economically and environmentally. Azotobacter sp is free living, knows that fix atmospheric nitrogen. There are different strains of Azotobacter each approach has chemical, biological and other characters. Azotobacter and Azospirillum are two other efficient bacteria. The response of these organisms in crop yield has been increasing commonly experienced. These are the bio-fertilizers in the cultivation of most crops. The inoculation of seeds with soil or Azotobacter is effective in increasing crop yields in soil well-paid high in organic matter. Experiments with cultures of Azotobacter 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 vegetables could be obtained in pot trials. However, under field conditions, such as same trends toward increasing performance are not always reproducible. Experiment was conducted in pot culture in order to assess the effects of Azotobacter isolated from marine source in the growth of Black gram. His shoot length, root length and chlorophyll content was measured.
MATERIAL AND METHODS:
Sample collection:
The surface water samples were collected in the area of the region Thundi (Palk Bay). Sample collection was performed in the range of about 20 days
Area â € "the water samples (to a depth of 1-2 m) were collected in sterile tube containing medium selective Azotobacter. Sediment samples were collected separately in broth medium. (Table 1 and 2)
Chemical parameters of seawater:
Water samples were analyzed for total hardness, the presence of magnesium and calcium by EDTA (0.01 M ethylene diamine tetra acetic acid) titration method. Content Total chlorine was analyzed by the method of Mohr.
EDTA method in 60 ml of water sample was inoculated to an Erlenmeyer flask. About 2 ml 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 stri. 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 titling and registration of the last instance of EDTA solution in the burette. Finally, the experimental concentration of calcium ions and magnesium sample water sample was calculated. The hardness of the water sample can be classified with a sum of all calcium and magnesium ions in solution.
In the method of Mohr 20 ml sodium chloride (0.01 M) solution was pipette into 250 ml Erlenmeyer. Approximately 2 ml dipotassium chromate indicator was added to the solution. Solution was changed bright yellow. Silver nitrate (0.01 M) solution was taken in the burette. Known chloride titrated with silver nitrate until the color changes from bright yellow to brick red color (Shake the flask constantly to see the uniform color). Finally, the experimental chloride concentration in the known solution was calculated.
To determine the unknown chloride, 5 ml water sample was taken in 250 ml Erlenmeyer. 2 ml of indicator (Disodium chromate) was added. Nitrate silver (0.01 M) solution was taken in the burette. The water sample is measured carefully 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, the reaction was stopped and recorded the final level of the solution of silver nitrate the burette. Finally, the experimental concentration of chloride in the unknown solution was calculated.
Media preparation:
Different media were used for the selective isolation of Azotobacter sp from marine source. As the isolates are of marine origin, the media were prepared by adding 3% sodium chloride (NaCl). The media used for isolation of nitrogen fixing organism (Azotobacter) of marine origin were: (Table 3)
1). Jensenâ € ™ s Agar Medium (with 3% NaCl)
2). Azotobacter Agar Medium (with 3% NaCl)
3). Burka € ™ s Medium (with 3% NaCl)
4). Marine agar.
Processing of samples (Kannan, 2002):
10 ml of water sample is mixed with 90 ml of water sterile distilled gave 10-1 dilution. Since the 10-1 dilution, the sample was diluted to 10-9 dilutions decimally. By using spread plate technique, 0.1 ml of diluted sample was planted in a sterile Petri dishes with selective media. The plates were incubated at room temperature (28a º C) for 48-72 hours.
Identification of isolates:
Grama € ™ s stain (Kannan, 2002)
The reactions were Gram stain heat fixed smears of fresh crops.
Catalase test:
catalase test was performed by adding 3% hydrogen peroxide dropwise to the slope of fresh culture Azotobacter. The presence or absence of bubbles was recorded.
Phase contrast microscopic observation:
Mobility and cell shape were determined by direct observations of wet mounts of fresh water breeding grounds, using a phase contrast microscope. (Table 4)
Acetylene Reduction Assay:
Individual colonies were picked, purified, and analyzed as pure cultures for nitrogenase activity, using half N-deficient. This technique is an indirect method of measuring nitrogen fixation at a point in time. This method provides a way simple, inexpensive procedure, highly sensitive and nondestructive measurement of nitrogen fixation rates. Cultures were randomly selected for this test. serum bottles rubber stoppers were collected, cleaned and sterilized. 30 ml of sterile broth Azotobacter was transferred to each bottle. The organisms were inoculated in the medium and incubated at Bis 28 º C for 3-4 days. incubation after 10 ml of nitrogen gas, 3 ml of acetylene gas was injected with the serum bottles using a syringe (N2 replaced air inside the bottle). Bottles incubated over a 28A Nite º C. At the end of the incubation period, 0.5 ml gas sample was removed from the bottle and injected into a FID gas chromatograph system coupled with Poro 80-100 PAK / ProPack column Q. The column temperature was maintained at 80A ° C, detector temperature of 100Â º C and injector temperature 120 ° C bis The carrier gas used was nitrogen with a flow rate of 30 ml / sec for hydrogen and air flame ionization zero rate of 30ml/sec. The area under the peak of ethylene was recorded for each culture. Randomly selected samples that showed maximum enzyme activity were selected for pot culture experiment.
Analysis of garden soil for chemical and nutrient content in pot culture experiment:
Garden Soil was collected from the region of the rhizosphere. soil was analyzed for the presence of N, P, K, copper, manganese, iron and zinc. (Table 5)
Pot experiment Culture:
The nitrogen fixing capacity of Azotobacter sp isolates was determined in a garden soil by pot experiment culture by growth assessment of black gram. After 7 days of sowing different growth characteristics, such as roots and shoot length was measured and the content chlorophyll is estimated. The experiment was conducted at the College Dr. Coimbatore.
Healthy viable seeds were selected for the experiment. Each pot contains 50 seeds viable. 10-12 kg of finely ground processed was filled in each pot. Sterilize pots with soil at a pressure of 15 pounds 4 hrs. The broth containing the active culture of Azotobacter (1 A-109 cells) was selected. Five effective strains were selected based on acetylene reduction assay for the experiment. The selected breeding grounds of Azotobacter sp were observed with phase contrast microscope before inoculation. Pots were selected for the experiment was cleaned thoroughly with disinfectant. Pots were filled with a combination Appropriate soil.
The healthy seeds were selected. Those seeds were mixed with 3 ml of Azotobacter inoculum and 3 ml of rice with fresh milk. Then the seeds were dried
Fifty seeds were sown in each pot. The pots were watered daily. Lacked control potentiometer bacterial inoculum. The effects of inoculum bacterial growth from the root of the plant, shoot length were measured at 7, 14, 21 days plant growth.
growth traits:
1. Germination percentage
2. Shoot length
3. Root length
Germination percentage:
Germination rate of all treated and control plant was calculated using the following formula: (Table 6)
The number of germinated seeds
The percentage germination = —————————————– A-100
Number of seeds sown
Shoot length:
The shoot length plant was measured in centimeters (cm) on the scale 7, 14, 21 days after planting from the ground level to the tip of the outbreak. (Table 7)
Root Length:
The plants were uprooted without disturbing the root system, then the roots were washed with tap water to remove particles soil. The root length was measured in cm scale. (Table 7)
biometric analysis, estimation of chlorophyll:
Leaf weight 1 g was finely cut into pieces, tissues were ground to a pulp with the addition of 20 ml of acetone 80%. After centrifugation at 5000 for 5 min and transfer the supernatant to the flask 100 ml. This procedure was repeated until the residue became colorless. Finally, the volume was 100 ml with 80% acetone. The absorbance was read at 645,663 nm solvent front (acetone 80%) in white. (Table 8a and 8b)
RESULTS AND DISCUSSION
Totally 100 samples were collected in the region Marine water and sediment at intervals of approximately 20 days (Table 1).
Table: 1 The total of specimens collected from the marine region.
samples water sediment
1st time 10 10
2nd time 15 5
3rd time only 15 5
4th time only 15 5
5th time only 15 5
Total 70 30
Out of 70 samples of seawater collected, all 70 samples showed the presence of Azotobacter, but were only 23 sediment marine 30 showed the presence of Azotobacter (Table 2).
Table 2: Presence of Azotobacter sp (in percent).
Source n Number of positive samples (Presence of Azotobacter) Percentage (Presence of Azotobacter)
Water 70 70 100
Sediments 30 23 76.6
Azotobacter sp is a gram-negative Soila € "housing agency with a variety of metabolic capabilities including the ability to fix atmospheric nitrogen through conversion to ammonia. These bacteria have the highest cellular respiration rate of any known organism. Its rapid oxygen consumption allows them to grow well and to fix the nitrogen under conditions of extreme ventilation. (Page et al. 1988).
initial isolation of marine bacteria prefer sea water or 3% NaCl in water in fresh medium for the growth (Robert A. MACLEOD 1965).
The total water hardness mainly represents the total concentration of calcium and magnesium ions expressed as calcium carbonate. The hardness can vary from 0-100 parts per million. Mg + + to maintain respiratory activity of Azotobacter cell, a stable body of water suspension.
Water analysis results showed that the total hardness of water was 20,200 ppm and total chloride content is 18273.98 ppm. Zobell and Upham identify bacteria and marine bacteria from the sea than in the initial isolation of sea water needed in an environment for growth.
Table: 3 colony morphology of Azotobacter
Media Details
Jenson € ™ s medium large, circular, drop mucosa, watery because as colonies.
Azotobacter half agar small, circular colonies, mucoid and in an aqueous
Burka € ™ s average surface film formation, turbidity, indicating the intensive growth of Azotobacter.
marine agar, small, circular, smooth-edged, raised colonies were observed high
Table4: Features Azotobacter sp.
Test result
Grama € ™ s negative Gram staining of cells as observed
Catalase evidence of air bubbles is
phase contrast microscopy motile cells were seen rarely non-motile cells were observed with different morphology.
The morphology of the colonies of Azotobacter strain is found to be variable based on the selective media used for isolation.
Studies on nitrogen fixation rates increased largely by the development of the acetylene reduction technique (Just et al., 1968). This test is based on the fact that the enzyme nitrogenase reduced acetylene to ethylene. The rate of formation of ethylene is a measure of nitrogenase and nitrogen â € "that fixing activity. Ethylene can be conveniently analyzed with great sensitivity using gas chromatography. Acetylene reduction in this study was performed and their maximum values noted. On the basis of this study was selected the agency for culture pot experiment.
Table 5 Chemical analysis and nutrients to garden soil
The garden soil was tested for micro and macro elements.
DATA LEVELS
pH 6.9
Electrical conductivity (DSM-1) 0446
N (kg / ha) 98
P (Kg / ha) 14.5
K (Kg / ha) 275
Copper (ppm) 0.84
Manganese (ppm) 6.32
Iron (ppm) 8.04
Zinc (ppm) 1.04
pot culture experiment
Five efficient strains were selected for pot culture experiment based on the acetylene reduction assay.
Table 6: Percentage of germination
The Results showed 85 percent germination
the pot of seed germination in%
72 control
A 80 pot
pot B 81
pot C 70
D-86 pot
82 E pot
Table: 7 length of stem and root
Stem and root length plant were measured, which ranged from 21.4 â € "
26.3 12.2 cm and 7.6 cm respectively.
the pot shoot length (cm) Root length (cm)
7 days 14 days 21 days 7 days 14th day 21st day
CONTROL 8.3 18.0 20.0 7.2 9.2 11.2
POT A 8.9 21.0 23.0 7.6 9.6 11.6
POT B 9.5 22.1 24.2 8.5 9.8 11.1
POT C 7.3 19.2 21.4 8.0 9.7 11.5
POT D 9.1 24.4 26.3 8.2 9.8 12.0
E POT 9.2 22.6 25.0 8.5 9.7 12.2
Biometric analysis:
The estimation of chlorophyll by spectrophotometric method:
Table 8: The value of absorbance at 645nm ranged from 0.102 â € "OD and 0.202 0.266 â € "0.562 OD at 663 nm
the pot DO 663 nm to 645 nm DO
0302 0103 control
A pot 0156 0423
pot B 0202 0562
pot C 0182 0522
pot D 0.154 0.455
0266 0102 E pot
Table 8: Estimation of chlorophyll content total:
Pot culture of chlorophyll a chlorophyll b chlorophyll mg / g
Control 0.3558 0.4502 0.0949
A pot 0.1592 0.4952 0.6543
B 0.8587 0.1995 0.6594 pot
0.1724 0.6139 0.7862 C pot
0.1397 0.5364 0.6759 D pot
E 0.3103 0.1091 0.4194 pot
The total chlorophyll were 0.4194-0.8587 mg total chlorophyll / g of tissue.
The pot culture experiment results showed that inoculation with Azotobacter influence the growth of black gram, increasing the stem and root length and chlorophyll content.
Experiments with cultures of Azotobacter and plants cultivated at the Agricultural Research Institute of India, New Delhi, lead us to believe that significant increases in growth and yield of wheat, rice, those and vegetable crops could be obtained in pot trials. Experiment Azotobacter in soil on growth of maize was carried out by NA Hegazi (1979) results showed significant increase in counting Azotobacter in 6 â € "Ground weeks of age.
A pot experiment was carried out by CV to evaluvating Kanchan Azotobacter inoculant effects on wheat yield. MA Kader (2002) conducted a pot experiment in the straw. He found significant increase in growth root for the treatment of Azotobacter.
SKKavimandan (1986) conducted a pot experiment with Azotobacter chroococcum along with 50 kg N / ha. He found an adverse effect of the inoculation of bacteria in the wheat yield. Choudhury. A (2005) conducted culture pot experiment in three rice cultivars N2 eight different strains of bacteria that are fixed in order to find effective nitrogen fixer. He found that Azospirillum seemed to be the best followed by Pseudomonas and Azotobacter when inoculated to rice variety. Ravikumar et al (2004) found with Azotobacter inoculation in seedlings of mangrove soil increase. root biomass, increased production of biomass, total chlorophyll of the plant. therefore it is beneficial azotobacterisation mangrove seedlings growing strong in coastal wetlands.
CONCLUSION:
A marine sample indicates that the concentration of nitrogen-fixing organisms is much lower in ocean environments in coastal environments. Without But even at low densities, the population of nitrogen-fixing microorganisms in vast areas of the seas could contribute substantially to the contribution nitrogen in the worldâ € ™ s ocean (Zehr et al 1998)
This study revealed that marine Azotobacter can be grown in conditions laboratory, which provides more information on the pattern of growth in different media. Water analysis results in a high concentration of calcium, magnesium, chloride content.
acetylene reduction assay was carried out and check the enzymatic activity of the samples randomly selected were used in the experiment pot culture. The pot experiment showed significant increase in shoot, root length of the plant. Therefore Azotobacter marine can survive in soil and fix the atmospheric nitrogen. Marina Azotobacter can be used as a suitable biofertilizer in order to reduce the use of chemical fertilizers which are powerful toxic substances, mainly petrochemicals.
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About the Author
Rajeawari.K
Department of biotechnology
Dr.G.R. Damodaran College of Science ,
Avanashi Road, Civil Aerodrome Post,
Coimbatore – 641014
Tamilnadu.
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