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Open Access Scientific Reports Open Access Scientific Reports Pathak and Kalekar, 1:12 http://dx.doi.org/10.4172/scientificreports.555 Open OpenAccess Access Research Article Exploring Nitrogen Fixing, Chemo heterotrophic Oligophiles from Natural Habitats for Preparing Biofertilizers Leena Pathak* and Saylee Kalekar HPT Arts and RYK.Science College, Nashik, Maharashtra, India Introduction Protocol Chemical fertilizers have been proved to be hazardous to nature. Eutrophication is a process caused due to excess of chemical fertilizers which is an indication of gross pollution of the aquatic environment. Biofertilizers are eco friendly fertilizers [1]. They cause no pollution and are not expensive. They are suitable for all crops. They are prepared using various microorganisms such as, Nitrogen fixers-Azotobacter spp, Rhizobium spp, Blue green algae etc. and phosphate solubilizersMycorrhizal associations. Total genomic DNA was isolated using GeneElute genomic DNA isolation kit (Sigma, USA) as per the manufacturer’s instructions and used as a template for PCR. Each reaction mixture contain approximately 10 ng of DNA; 2.5 mm MgCl2, 1x PCR buffer (Genei, Banglore, India), 200 μM each dCTP, dGTP, dATP and dTTP. 2 pmol of each forward and reverse primer, and 1 U of Taq DNA polymerase (Genei, Banglore, India) in a final volume of 20 μl. FDD2 and RPP2 primers were used to amplify almost entire 16s rRNA gene as described previously (Rawlings 1995). The PCR was performed using the Eppendorf Gradient Mastercycler system with a cycle of 94°C for 5 minutes; 30 cycles of 94°C, 60°C, and 72°C for 1 minute each. Final extension was done at 72°C for 10 minute, and mixture was held at 4°C. The PCR product was precipitated using polyethylene glycol (PEG 6000, 8.5%) washed thrice-using 70% ethanol and dissolved in tris-HCl (10 mM, pH-8.0). Oligophiles are a new group of bacteria, able to grow at extremely low concentration of nutrients [2]. A substantial proportion of bacteria long thought to be unable to grow on culture media were recently shown to be oligophilic [3]. Majority of natural habitats are nutrient deficient. Numerous species of oligophiles are likely to be existing in nature. These organisms need to be explored for various beneficial purposes. Hypothesis Nitrogen fixing, chemoheterotrophic oligophiles are existing in nature. They can be isolated on culture media and have beneficial activities. They can be used for preparation of biofertilizers. These biofertilizers will be much cheaper and more effective than the existing one. Objectives Objectives of the study were to formulate suitable culture media for isolation of nitrogen fixing chemoheterotrophic oligophiles. Isolating nitrogen fixing chemoheterotrophic oligophiles from natural habitats such as soil, air, water etc. and to identify these isolates. To study the effect of various carbon sources on growth of these bacteria. Exploring the possibility of using these microbes in preparing biofertilizers. Materials Various natural sources such as soil from rhizosphere, water, air. Media used for isolating the culture were nitrogen free glucose agarNorris Medium, the most common medium used for isolation of Azotobacter spp. 1:100, 1:500, 1:1000 diluted. Nitrogen free medium with different carbon sources such as Glucose, Mannitol, and Sucrose etc. Methodology Spread suspension of soil from rhizosphere on various dilutions of nitrogen free glucose agar. Expose some plates of diluted medium to air. Incubate all plates at room temperature till some colonies appear on these media. Record colony characteristics and obtain pure cultures on slants of the respective media. Study the effect of various carbon sources on growth of these bacteria. Prepare a liquid medium with the carbon source giving maximum growth. Using the above medium prepare liquid bio-fertilizer and check their efficiency of nitrogen fixation by field trials. Compare with Azotobacter biofertilizer with respect to efficiency, cost, production, time etc. Subject the isolates to identification by latest technique. The ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, Calif.) was used for the sequencing of the PCR product. A combination of universal primers was chosen to sequence the nearly complete gene. The sequencing reaction and template preparation were performed and purified in accordance with the directions of the manufacturer (Applied Biosystems). The sequencing output was analyzed using the accompanying DNA Sequence Analyzer computer software (Applied Biosystems). The sequence was compared with National Center for Biotechnology Information GenBank entries by using the Blast algorithm. Results and Conclusions Photographs of some common bacteria unable to grow on dilute media were shown in figures 1-5. Several colonies were obtained on 1:100, 1:500 and 1:1000 dilutions of Nitrogen free glucose agar after 4-5 days of incubation at room temperature. Nitrogen free glucose agar with three different energy sources -Glucose, Sucrose and Mannitol was prepared and comparative study on their dilutions was carried out. Glucose and Mannitol were found to be suitable but not sucrose. Liquid bio fertilizer using 1:1000 dilution of the medium with Glucose was prepared but the cell density was too less after incubation for one week. Hence, lower dilution 1:500 was used. It has given promising *Corresponding author: Leena Pathak, H.P.T.Arts and R.Y.K.Science College, Nashik, Maharashtra, India, E-mail: [email protected] Received April 26, 2012; Published November 27, 2012 Citation: Pathak L, Kalekar S (2012) Exploring Nitrogen Fixing, Chemo heterotrophic Oligophiles from Natural Habitats for Preparing Biofertilizers. 1:555 doi:10.4172/scientificreports.555 Copyright: © 2012 Pathak L, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Volume 1 • Issue 12 • 2012 Citation: Pathak L, Kalekar S (2012) Exploring Nitrogen Fixing, Chemo heterotrophic Oligophiles from Natural Habitats for Preparing Biofertilizers. 1:555 doi:10.4172/scientificreports.555 Page 2 of 3 >OLIGO TCCCCCCCNCCATGGGCATTGCCAATGGGCGAAACCTGTTTGCCCGACGCCGCGTGAGTGATGACGGC CTTCGGGTTGTAACCTCTTTCATCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGATGGCCGGCT AACTACGTGCCAGCCAGCCGCGGTAAGACGTAGGGCGCAAGCGTTGTCCGGAGTTATTGGGCGTAAAT AGCTCGTAAGCGGCTTGTCGCGTCTGTTGTGCAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATA CGGGCAGGCTATATTCGGTAGGGGAGATCGNGAATTCCTGGTGTAGCGGTGAAATGCCCAGATATCAC GGAGGAACACCGGTGGCGAAGGCGGATCTCTGGTCCGCATACTGCACGCTGNATGAGGCTAAAGCGT GAGGCGGCCAAGANGATTAGATACCCTGGTAGTCCACGCCGTTAACGGTGGGCACTAGGGGTGGGCA ACATTCCACGTTGTCCGTGCCGCATCTAACCCATTAATTGCCCCGCCTGGGGAGTACGGCCGCAACGCT AAAACTCAAAGGAATTGACGGGGGCCCGCACAAGTTGCCGGAGCATGTGGCTTAATTCCGACACCAC GCGAATAACCTTNACCAAGGCTTGA Figure 1: E.coli, Bacillus spp., Klebsiella spp., Serratia marscescens replicated from normal nutrient agar on 1:1000 dilution showed no growth. Strain Total Max Description Designation score ident Streptomyces fragilis NRRL 2424 (T) 16S ribosomal RNA gene, partial sequence Oligo (AY999917) 712 93% Streptomyces viridochromogenes NBRC3113T(AB184728) 65 Streptomyces pactum NBRC13433T(AB184398) Streptomyces violascens ISP 5183T(AY999737) Streptomyces nogalater JCM 4799T(AB045886) Streptomyces fumanus NBRC13042T(AB184273) 48 Streptomyces viridiviolaceus NBRC13359T(AB184350) Streptomyces spiralis NBRC14215T(AB184575) 2 Streptomyces niveoruber NBRC 15428T(AB184675) 42 53 Streptomyces diastaticus subsp.ardesiacusNRRLB-1773T(DQ026631) 4 Streptomyces naganishii NBRC12892T(AB184224) Streptomyces coeruleofuscus NBRC12757T(AB184840) 11 Streptomyces cinereospinus NBRC15397T(AB184648) Streptomyces spinoverrucosus NBRC14228T(AB184578) 1Streptomyces fragilis NRRL2424T(AY999917) Streptomyces hyderabadensis OU-40T(FM998652) Figure 2: Colonies of Azotobacter spp. on nitrogen free glucose agar. OLIGO 0.005 Figure 5: Identification report by 16S rRNA gene sequencing approach. Reagents required Volume required ddH2O 12.0 μl 10X PCR buffer 2.0 μl dNTPs 2.0 μl Forward primer 0.4 μl Reverse primer 0.4 μl Taq DNA Polymerase 2.0 μl Template DNA 3.0 μl Total volume 20.0 μl Table 1: PCR reaction mixture set up. Figure 3: Few colonies of Azotobacter spp. transferred to 1:1000, 1:2000 dilutions of medium-no growth. results. Standardization of this biofertilizer is being done. One isolate was given to Agharkar research institute, Pune for identification by 16S r RNA method and the isolate has been identified as follows. Identification of strain by 16SrRNA gene sequencing was shown in figure 2. The cultural sample was processed for identification in the following manner – 1. Genomic DNA was isolated from the culture by using Sigma’s, “GenElute Bacterial Genomic DNA” Kit. 2. PCR was carried out using the following combination of primers – 1:1000 SOIL FDD2 – RPP2 (universal primers for 1.5 kb fragment amplification for eubacteria) 1:1000 AIR INCUBATED FOR 5 DAYS AT ROOM TEMPERATURE Template DNA – Genomic DNA Figure 4: Colonies of Oligophilic Bacteria on 1:1000 Diluted Nitrogen Free Glucose Agar. Sequences of the primer pair used for amplification RPP2 – CCAAGCTTCTAGACGGITACCTTGTTACGACTT Volume 1 • Issue 12 • 2012 Citation: Pathak L, Kalekar S (2012) Exploring Nitrogen Fixing, Chemo heterotrophic Oligophiles from Natural Habitats for Preparing Biofertilizers. 1:555 doi:10.4172/scientificreports.555 Page 3 of 3 FDD2 – CCGGATCCGTCGACAGAGTTTGATCITGGCTCAG The PCR reaction mixture was presented in table 1. 3. The PCR products obtained from above reactions were then processed for Cycle sequencing reaction (PCR performed using only one primer – SRV3-1) 4. Following the above reaction, the samples were cleaned up and loaded on the sequencer. (Avant 3100 Gene Analyser) Future Prospects • Large scale production and field trials of the product will be taken. • Development of genetic recombinant of Azotobacetr and an oligophile can be tried. References 1. Subba Rao NS (1988) Biofertilizers in agriculture 2nd edition-Oxford & IBH publishing co. pvt.ltd (pg 194) 2. Watve MG, Shejwal V, Sonawane C, Rahalkar M, Matapurkar A, et al. (2000) The ‘k’ selected oligophilic bacteria: a key to uncultured diversity? Current science 78: 1535-1542. 3. Nagarkar PP, Ravetkar SD, Watve MG (2001) Oligophilic bacteria as tools to monitor aseptic pharmaceutical production units. Appl Environ Microbiol 67: 1371-1374. Volume 1 • Issue 12 • 2012