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 was devoid of the bacterial inoculums. The effects of bacterial inoculums on the growth of plant root, shoot length were measured on the 7th, 14th, 21st day of plant growth.
Growth characters:
1. Percentage of germination
2. Shoot length
3. Root length
Percentage of germination:
The germination rate of all treated and control plant was calculated by using the following formula: (Table 6)
Number of seeds germinated
Percentage of germination = —————————————– × 100
Number of seeds sown
Shoot length:
The shoot length of the plant was measured in centimeter (cm) scale on 7th, 14th, 21st day of sowing from ground level to the shoot tip. (Table 7)
Root length:
The plants were uprooted without disturbing the root system, and then the roots were washed with tap water to remove the soil particles. The length of the root was measured in cm scale. (Table 7)
Biometric analysis; Estimation of chlorophyll :
Weighed 1g of leaf was finely cut in to pieces; tissues were ground to a pulp with the addition of 20ml of 80% acetone. Then centrifuged at 5000 for 5 min and transferred the supernatant to the 100ml volumetric flask. This procedure was repeated until the residue turned colorless. Finally the volume was made to 100ml with 80% acetone. The absorbance was read at 645,663nm against the solvent (80% acetone) blank.( Table 8a and 8b)
RESULT AND DISCUSSION:
Totally 100 samples were collected in marine region of both water and sediments at the intervals of approximately 20 days (Table: 1).
Table :1 The total samples collected from marine region.
samples water sediment
1st time 10 10
2nd time 15 5
3rd time 15 5
4th time 15 5
5th time 15 5
Total 70 30
Out of 70 marine water samples collected, all the 70 samples were showed the presence of Azotobacter, but only 23 marine sediments out of 30 were showed the presence of Azotobacter (Table no: 2).
Table no 2: presence of Azotobacter sp (in percentage).
Source No of samples POSITIVE (Presence of Azotobacter) Percentage (Presence of Azotobacter)
Water 70 70 100
Sediment 30 23 76.6
Azotobacter sp is a gram-negative soil–dwelling organism with a wide variety of metabolic capabilities which includes the ability to fix atmospheric nitrogen by converting it to ammonia. These bacteria possess the highest cellular respiratory rate of any known organism. Their rapid consumption of oxygen allows them to grow well and to fix nitrogen under extreme aeration condition. (Page et al. 1988).
Initial isolation of marine bacteria prefers sea water or 3 % NaCl to fresh water in the medium for growth (ROBERT A. MACLEOD 1965).
The total hardness of water represents primarily the total concentration of calcium and magnesium ions expressed as calcium carbonate. Hardness may range from 0-100 of parts per million. Mg++ to maintain the respiratory activity of cell Azotobacter, an organism stable in water suspension.
Water analysis result showed that the total hardness of water was 20200 ppm and total chloride content was 18273.98 ppm. Zobell and upham define marine bacteria as being bacteria from the sea which on initial isolation required seawater in the medium for growth.
Table: 3 colony morphology of Azotobacter
Media Details
Jenson’s medium Large, circular, mucoid, watery due drop like colonies.
Azotobacter agar medium Small, circular, mucoid and watery colonies in a medium
Burk’s medium Surface Pellicle formation, turbidity indicating the Heavy growth of Azotobacter.
Marine agar medium Small, circular, smooth edged, raised elevated colonies were observed
Table4: characteristics of Azotobacter sp.
Test Result
Gram’s staining Gram negative rod shaped cells were seen
Catalase test Air bubbles were seen
Phase contrast microscopy Motile cells were seen/rarely non-motile cells were seen with different morphology.
The colony morphology of Azotobacter strain is found to be varying based on the selective media used for isolation.
Studies on the rates of nitrogen fixation were greatly enhanced by development of the acetylene reduction assay (Hardly et al., 1968). This assay is based on the fact that nitrogenase enzyme will reduce acetylene to ethylene. The rate of formation of ethylene is a measure of nitrogenase or nitrogen –fixing activity. Ethylene can be conveniently assayed with great sensitivity using a gas chromatography. In this study acetylene reduction was performed and their peak values were noted. Based on this assay the organism was selected for pot culture experiment.
Table: 5 Chemical and nutrient analysis of garden soil
The garden soil was tested for micro and macro elements.
PARTICULARS LEVELS
pH 6.9
Electrical conductivity(dSm-1) 0.446
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 acetylene reduction assay.
Table 6: Percentage of germination
Result showed 85 percent of germination
pot culture seed germination in %
control 72
pot A 80
pot B 81
pot C 70
pot D 86
pot E 82
Table: 7 shoot and root length
Shoot and root length of the plant were measured, which ranged from 21.4 –
26.3 cm and 7.6-12.2cm respectively.
pot culture Shoot length(cm) Root length(cm)
7th day 14th day 21st day 7th day 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
POT E 9.2 22.6 25.0 8.5 9.7 12.2
Biometric analysis:
Estimation of chlorophyll by spectrophotometric method:
Table 8a : The optical density value at 645nm ranged from 0.102 – 0.202 OD and 0.266 – 0.562 OD at 663 nm
pot culture OD AT 645 nm OD AT 663 nm
control 0.103 0.302
pot A 0.156 0.423
pot B 0.202 0.562
pot C 0.182 0.522
pot D 0.154 0.455
pot E 0.102 0.266
Table 8a: Estimation of total chlorophyll content:
Pot culture Chlorophyll a Chlorophyll b Total chlorophyll mg/g
control 0.3558 0.0949 0.4502
pot A 0.4952 0.1592 0.6543
pot B 0.6594 0.1995 0.8587
pot C 0.6139 0.1724 0.7862
pot D 0.5364 0.1397 0.6759
pot E 0.3103 0.1091 0.4194
The total chlorophyll ranged from 0.4194-0.8587 mg total chlorophyll/g tissue.
The pot culture experiment results showed that, inoculation with Azotobacter influence the growth of black gram by increasing their shoot and root length and chlorophyll 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. Experiment on soil Azotobacter on the growth of maize was carried out by N.A Hegazi(1979) result showed significant increase in the count of Azotobacter in 6 –week- old plant.
A pot culture experiment was conducted by C.V Kanchan to evaluvating the effects of Azotobacter inoculants on the yield of wheat. M.A kader(2002) was conducted a pot culture experiment on straw. He found significant increase in root growth by the treatment of Azotobacter .
S.K.Kavimandan (1986) was carried out a pot experiment with an Azotobacter chroococcum along with 50 Kg N /Ha. He found an adverse effect of bacterial inoculation on the yield of wheat. Choudhury .A(2005) carried out pot culture experiment on three rice cultivars with eight different N2 fixing bacteria strains with the objective to find out effective nitrogen fixer. He found that Azospirillum appeared to be the best followed by Pseudomonas and Azotobacter when inoculated to rice variety. Ravikumar et al (2004) found inoculation with Azotobacter in mangrove soil increase seedlings,. Root biomass, shoot biomass ,total chlorophyll of plant . thus azotobacterisation is beneficial in raising vigorous seedlings of mangrove in coastal wetlands.
CONCLUTION :
A marine sample indicates that the concentration of nitrogen-fixing organisms is much lower in oceanic environments than in coastal environments. However, even at low densities, active population of nitrogen- fixing microorganism over vast areas of the open ocean could contribute substantially to the nitrogen inputs in the world’s ocean (Zehr et al 1998)
This study revealed that marine Azotobacter can be cultivated in laboratory condition, which provides more information on growth pattern on different media. Water analysis result showed high concentration of calcium, magnesium, chloride content.
Acetylene reduction assay was performed and checked the enzyme activity of randomly selected samples and were used for pot culture experiment. The pot culture experiment showed significant increase in shoot, root length of the plant. Hence marine Azotobacter can survive in soil and fix atmospheric nitrogen. Marine Azotobacter can be used as a suitable biofertilizer in order to reduce the usage of chemical fertilizer which is potent harmful substances mainly petrochemicals.
LITERATURE CITED:
Bedford, R.H, 1933. Marine bacteria of the northern pacific ocean. The temperature range of growth. Contrib. can. Bio. Fisheries 8: 433-438.
Burk, D., and Horner, C.K. 1940. Molybdenum and calcium in Azotobacter nutrition. Proe. Third Intern. Congr. Microbiol. (New York), p 489-490.
CHOUDHURY . A 2005. Screening of rice cultivars and diazotrops combination for better N(2) fixing system. Indian journal of plant physiology Vol 10 p 82-85
Eisenstarh A., K.J. McMahon and Roma Eisenstarh, 1949. Department of Bacteriology, Oklahoma. A cytological study of pleomorphic strain of Azotobacter with the electron and phase Microscope and the Robinson Nuclear – Staining Technique.
Guerinot, M.L., and Patriquin D.G. 1981. The association of N2-fixing bacteria with sea urchins. Mar. Biol. Vol 62: 197-207.
Hans W.Parel. Microbially Mediated Nitrogen Cycling. Techniques in Microbial Ecology, P 4-27 – ND.
Hardy R.W.F. 1968. Acetylene ethylene assay for nitrogen fixation: Laboratory and field evaluation. Plant physiology vol.43, 1185-1207.
Hegazi, M. Monib and Vlassak K, 1978. Effect of inoculation with N2-Fixing Spirilla and Azotobacter on Nitrogenase Activity on Roots of Maize Grown Under subtropical conditions, vol.38 No.4. P 621-625.
James. A. Coyer, Alejandrocabello – Pasini, Hewson Swift and Randall. S. Alberte, 1996. N2 fixation in marine hetrotrophic bacteria dynamic of environmental and molecular regulation. Vol 93: P 3575 – 3580.
Jensen. H.L. 1954, The Azotobacteriaceae. Bacteriological Rev. 18: 195-214.
S.K Kavimandan 1986 Influence of rhizobia,azotobacter and blue green algae on n content and yield of rice .Vol 96 133-135
M.A Kader 2002. Effects of Azotobacter inoculant on the yield and nitrogen uptake by wheat ,Journal of biological sciences , vol 2(4) p 259-261
Lewis I.M. 1937. Cell inclusions and life cycle of Azobacter J. Bacteriol 34: 191-205.
MacLeod. R.A. and Onofrey, 1957. Nutrition and metabolism of marine bacteria III. The relation of sodium and potassium to growth J. Cell. Comp. Physiol. Vol 50: 398-409.
Maria GT – Rubio, Sandra AVP., Jaime Bernal – Castilo, Patrica Martinel- Nieto, 2000. Association Latinoamericana de microbiologia. Vol 42: 171-176.
Mary LG and Rita R Colwell 1985, Enumeration, isolation and characterization of N2 fixing bacteria from sea water. Department of Microbiology, University of Maryland. Vol 50 No.2.
Murray C.M.J.P. Riley: and T.R.S. Wilson 1969. The solubility of gases in distilled water and sea water I Nitrogen. Deep sea. 16: 297-310.
Page W.J. and H.L. Sadoff, 1975. Relationship Between Calcium and Uronic Acids in the Encysment of Azobacter vinelandii. Journal of Bacteriology, Vol.122, No.1 p 145-151.
Postgate, J.R (1982). Fundamentals of nitrogen fixation (Cambridge Univ. Press. Cambridge, U.K).
Rai. M.K, Handbook of Microbial Biofertilizers, an imprint of the Haworth Press, Inc. New York, London, Oxford.
Ramos J.L and R.L. Robinson, 1985. Isolation and properties of mutant of Azobacter chroococum defective in aerobic nitrogen fixation J. Gen. Microbial. 131: 1449-1458.
Ravikumar et al 2004. Nitrogen fixing Azotobacter from mangrove habitate and their utility as marine biofertilizera, journal of experimental marine biology and ecology Vol312 p5-17
Richter. O. 1928. Natrium: Ein notwendiges N’ahrelement fur eine marine mikroarophile Leuchbakterie. Anz. Oesterr. Akad. Wiss. Math. Naturw. KI. 101: 261-292.
Robert A Macleod, 1965. The question of the existence of specific Marine bacteria, department of bacteriology, American society for microbiology, McGill University, Canada. Vol.29 No.1.
Robinson G.G.C.L.L. Hendzea and D.C. Giillespie 1973. A relation between heterotrophic utilization of organic acids and bacterial population in West Blue iake, Manitoba, Limnon, Oceanogr 18: 264-269.
Tyler. M.E., M.C.Bielling and D.B. Pratt, 1960. Mineral requirements and other characters of selected marine bacteria Jou. Gen. Microbiol. Vol.23: 153-161.
Vela G.R. and Rosenthal R.S., 1972. Effect of Peplon on Azotobacter Morphology, America Society for Microbiology. Vol.111 No.1.
Zobell. C.E and Upham H.C., 1994. A list of marine bacteria including description of sixty new species. Bull. Scripps Inst. Oceanography. Vol. 5: 239-292.
Standard method for the examination of water and waste water, 16th edition, APHA, AWWA, WPCF.
* Cristian G.D. Analytical Chemistry 4th Edition, J. Wiley and Sons.
* Harris D.C. Quantitative Chemical Analysis 5th Edition, W.H. Freeman.
* History of nitrogen fixation. From: Biology 446, Uni. Of Watterloo (Biology 446).
* http://www.indiaagronet.com/
* http://www.thekrib.com/plant/co2/hardness-larryfr.,html.
* The microbial world: The nitrogen cycle and nitrogen fixation produced by Jim Deacon, Institute of cell and molecular biology, the University of Edinburgh.
* Determination of hardness of water method WHO/M/26.RI, revised 10 Dec. 1999.
* htt://www.nalms.org/
Carlson, R.E. and J. Simson, 1996. A coordinators Guide to volunteer lake Monitoring Methods, North American lake Management Society, P 96.
* Determination of water hardness by EDTA Titration from Gannon University SIM.
* http:/www.bact.wisc.edu/The world of Microbes.htm
*web.centre.edu/shiba/che117L/exp8_hardness.htm:
Pages from web.centre.edu.
* http://www/tau/ac/
The nitrogenous complex
* http://www.bookrags.com
Azotobacter compete article
*Shri Dorji Tenzing Bhutia, 2004. Joint Director Skims, Biofertilizers for Nutrient Management in Organic Production of Agri / Horticultural crops.
* http://cos.colstate.edu/stokes/chlorophyll.htm.
*http://www.usoe.k12.ut.us/curr/science/scriber/00/8th/energy/scriber/chlorophyll.htm.
Chlorophyll : why?
** Atlas R. and Bartha R, 1998. Microbial Ecology Fundamentals and Applications 4th Edition Benjamin Cummings. Menlopark. Ca.694pp.
** Camphell. N. 1993. Biology 3rd Edition. Benjamin Cummings, Redwood City, Ca 1190.
** N.S.Subba Rao, Soil Microbiology and plant growth 4th edition.
** Robert L. Tate, 1995. Soil Microbiology. Jhon Willey and Sons, Inc.
** Jan Dirk Van Elsas, Jack T. Trevors, Elizabeth, M.H.Willington 1997, Modern Microbiology.
**S.Sadasivam and A.Manikam, 2004. Biochemical methods, 2nd Edi., Centre for plant molecular biology, TNAU/
(* Net reference)
(** Book Reference)
