Introduction
Nitrogen fixation is the reduction of N2 (atmospheric nitrogen) to NH3 (ammonia). Free living prokaryotes with the ability to fix atmospheric dinitrogen (diazotrophs) are ubiquitous in soil. But our knowledge of their ecological importance and their diversity remains incomplete. In natural ecosystems, biological N2 fixation is most important source of N. The capacity for nitrogen fixation is widespread among bacteria and archaea. The estimated contribution of free-living N-fixing prokaryotes to the N input of soil ranges from 0-60 kg/ha /year (Burgmann et al., 2003). Dinitrogen (N2)-fixing microorganisms (diazotrophs) play important roles in ocean biogeochemistry and plankton productivity (Church et al., 2005).

Nitrogen fixation can be an important source of nitrogen for biological productivity in the marine environment. Biological nitrogen fixation is catalyzed by the enzyme nitrogenase, which is possessed by diverse microorganisms representing virtually all phylogenetic groups. Interest in nitrogen fixation in the sea has usually been focused on rates of nitrogen fixation, but information on the types of species present with the capability for nitrogen fixation can be important for predicting nitrogen fixation rates in situ (Zehr et al., 1998).

Nitrogenase catalyzes the reduction of nitrogen gas to ammonium in an ATP-and reductant dependent reaction. It is one of the best characterized metalloenzyme and is an excellent model for elucidating metalloprotein assembly. Nitrogenase is composed of two oxygen-labile metallo protein; dinitrogenase and dinitrogenase reductase. Dinitrogenase is a 240-KDa alpha2-beta2 tetramer of the nifD and nifK gene products. Dinitrogenase reductase is a 60-KDa alpha2 dimer of the nifH gene products that contains a single 4Fe-4S center coordinated between the two subunits (Rubio et al., 2005). Understanding how fixed N regulates nitrogenase availability is necessary for devising strategies to increase the amount of ammonium synthesized by nitrogen fixing bacteria with the potential to be used in agriculture (Kennedy et al., 2004).

Molecular tools for detection and characterization of the nitrogenase (Nif) genes and immunoassays for nitrogenase protein can provide new information on the factors regulating the distribution and activity of diverse nitrogen fixing organisms in the marine environment. Amplification and characterization of NifH sequences has made it possible to identify the type(s) of organism responsible for nitrogen fixation, such as in aggregates of the cyanobacterium and Trichodesmium. Differences in nitrogen fixation patterns have been linked to genetic differences between Trichodesmium strains. Further development of these approaches will provide new and powerful ways to link the genetic potential for nitrogen fixation to nitrogen fixation rates in the ocean (Zehr et al., 1998)

Nitrogenase gene (NifH) sequences amplified directly from oceanic waters showed that the open ocean contains more diverse diazotrophic microbial populations and more diverse habitats for nitrogen fixers than previously observed by classical microbiological techniques (Zehr et al., 1998). Understanding how fixed N regulates nitrogenase availability is necessary for devising strategies to increase the amount of ammonium synthesized by nitrogen fixing bacteria with the potential to be used in agriculture (Kennedy et al., 2004).

The commercial history of biofertilizer began with the launch of "Nitrogin" by Nobbe and Hiltner; a laboratory culture of rhizobia in 1895, followed by the discovery of Azotobacter and then the blue green algae and a host of other microorganisms. Azotobacter is used as a biofertilizer in the cultivation of most crops. Azotobacter is an obligate aerobic diazotrophic soil-dwelling organism with a wide variety of metabolic capabilities, which include the ability to fix atmospheric nitrogen by converting it to ammonia. Azotobacter naturally, fixes atmospheric nitrogen in the rhizosphere. There are different strains of Azotobacter each has varied chemical, biological and other characters. However, some strains have higher nitrogen fixing ability than others (Burgmann et al., 2003). Besides, nitrogen fixation, Azotobacter also produces, Thiamin, Riboflavin, indol acetic acid and gibberellins. When Azotobacter is applied to seeds, seed germination is improved to a considerable extent, so also it controls plant diseases due to above substances produced by Azotobacter (Kader et al., 2002.)

This NifH gene has been largely studied by culture-independent approaches. These approaches provide a more complete picture of the diazotrophic community than culture-based approaches. Various techniques, such as PCR cloning, denaturing gradient gel electrophoresis, PCR-restriction fragment length polymorphism (RFLP), and fluorescently labeled terminal (FLT)-RFLP, have been used to analyze the composition of NifH gene pools in various environments. These studies found that the NifH gene is present in diverse environments: forest soil, the rhizosphere of native wetland species, such as Spartina, or of crop species, such as rice, aquatic or polar cyanobacteria, and the bacteria found in termite guts. All these studies described a large number of unknown sequences which correspond to diverse unidentified diazotrophs. Some NifH genes are characteristic of an ecological niche (Shaffer et al., 2000) evoked the possible relationship between the habitats of soil nitrogen-fixing bacteria and the structure of NifH gene pools (Poly et al., 2001).

Nitrogen fixation in A. vinelandii is complicated by the presence of three biochemically and genetically distant nitrogenase enzymes, each of which is synthesized under different conditions of metal supply. The regulation of conventional molybdenum nitrogenase, whose subunits are encoded by the Nif-HDK genes and which is similar to the enzyme purified from number of other nitrogen-fixing organisms. (Sabra et al., 2000). The Nif-HDK genes are located in a large cluster of nif genes, which includes, in order, NifHDKTYENXUSVWZMF (Bali et al., 1992). Molecular methods based on universal PCR detection of nifH marker genes have been successfully applied to describe diazotroph population in the environment (Burgmann et al., 2003).

Materials and Methods

Sample collection
Samples were collected in different locations of Rameshwaram marine region (Gulf of Mannar) at the depth of 1–5 m. The randomly collected samples in the sterile plastic bags (soil sample) and water sampling bottles (water sample) bottles were kept in an ice-cold box and transported safely to the lab for further analysis with in 12 hrs. The sample with media tubes were packed and transported safely to the laboratory.

Isolation of Azotobacter from water and sediment samples (Mary et al., 1985)
Different selective media were used for the isolation of Azotobacter sp from marine source as described previously. Azotobacter strains used for this study were maintained and cultured in Burk medium as previously described (Joerger et al., 1988). As the isolates are of marine origin, the media were prepared by the 3.5% sodium chloride (NaCl). Media used for the isolation of nitrogen fixing organism (Azotobacter) from marine sources were Jensen’s agar medium, Azotobacter agar medium, Burk’s Medium and marine agar medium.

Culture characteristics (Bagwell et al., 1988)
Gram-staining characteristics and cell morphologies were determined by standard methods(Gerhardt et al., 1981). Motility was observed in wet mount using phase contrast microscope. Preliminary physiological characterization such as catalase test, starch hydrolysis test were also carried out.

Extraction and purification of DNA (Kelly et al ., 1990)
Azotobacter genomic DNA was isolated as previously described (Robson et al., 1980). Linear DNA fragments were analyzed by electrophoresis in agarose gel in TEB buffer (Maniatis et al., 1982). The purity of the DNA was checked spectrophotometric method by using the formula OD at 260 nm/ OD at 280 nm (Wilfinger et al., 1997).

PCR amplification of the NifH gene fragment

Nitrogenase Fe protein genes (NifH) were amplified from Azotobacter sp derived genomic DNA, using the primer from OPERON diagnostic Ltd, USA. The samples were amplified by PCR in a mixture containing reaction buffer 5.0 µl, 10mM dNTP 1.0 µl, primer 1 (25 mer) 1.0 µl, primer 2 (24 mer) 1.0 µl, template DNA 1.0 µl, enzyme Taq polymerase 1.0 µl for 35 cycles ( 1 min at 94° C,1 min at 54° C and 1 min at 72° C) (Zehr et al., 1988).

Results and Discussion
Totally 100 samples were collected in marine region of both water and sediments in the intervals of approximately 20 days .Out of 70 marine water samples collected, all the 70 samples were showing the presence of Azotobacter, but only 23 marine sediments out of 30 were showing the presence of Azotobacter. These samples were processed through the commonly used procedures such as selective media. Gram’s staining, Phase contrast observation for motility, starch hydrolysis test and Catalase test for identification of free- living diazotrophic organism i.e Azotobacter from the above samples, and that can be processed, result shows that Azotobacter sp are motile, gram negative, catalase and starch hydrolysis positve.

The colony morphology of Azotobacter strains were varying during the isolation in the selective media. The colonies were very clear, large, mucoid, watery due drops like initially i.e. from the marine source. The mother culture was sub cultured in the same media; the colony morphology differs slightly i.e. small, and circular, convex in nature. All the isolated Azotobacter strains were numbered for the easy identification and convenience. From these isolates, well defined pure culture of Azotobacter strains (1, 16, 27, 82, 101, 103, 108, 115, 125, and 132) were selected for the nucleic acid analysis.

The reference (standard) cultures such as Azotobacter beijerinckii (123), A. chroococcum (446), and A. vinelandii (124) procured from MTCC, (Chandigarh, India) were also used along with marine isolates for the nucleic acid extraction and purification. The DNA of the selected strains was isolated and estimated OD at 260 nm, the value ranges from 0.141 to 0.177. The estimated value of the extracted DNA was ranging from 0.70 to 0.88.The purity of the DNA was analyzed by spectrophotometric method using OD at 260 and OD at 280 nm. The presence and purity of DNA was checked by OD at 260nm/ OD at 280 nm, the value ranges from 1.13 to 2.21. If the estimated value is 1.8 conforms the presence of pure DNA. If the estimated value is lesser /greater than 1.8 conforms the presence of DNA to protein / RNA contamination, according to respective values DNA was purified using the enzymes proteases and RNase. The purified form of the DNA was separated by the agarose gel electrophoresis for the comparison the banding pattern between the randomly selected marine samples and the standard strains. There is no substantial difference between the banding patterns of the chromosomal DNA on the gel as shown in the plates. This result confirms that molecular weight of chromosomal DNA in all strains is similar.

One µl of DNA was used as template in PCR. selected primers, primer1:5-GGAATTCCTGYGAYCCNAARGCCNA-3,
Primer2:5-CGGATCCGDNGCCATCATYTCNCC-3 procured from OPERON diagnostics LTD, USA, respectively was used to amplify a 324-bp region between sequence positions 336 and 660. All N2 fixers carry a NifH gene, which encodes the Fe protein of the nitrogenase (Poly et al., 2001). The results of the PCR products were compared on 2% agarose gel electrophoresis. Selective NifH primer from Anabaena sp strain PCC7120 was used for the amplification of the Azotobacter sp, the primers used in my study was exactly matching the Azotobacter genome. Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the NifH marker gene have been successfully applied to describe diazotroph populations in the environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all NifH genes (Bürgmann et al., 2003).

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