Isolation and molecular characterization of non-Azotobacter bacteria using Azotobacter isolation protocols from pastures in the south of Turkey

Ebru Çelen

doi:10.46309/biodicon.2023.1273996

Research Article

Isolation and molecular characterization of non-Azotobacter bacteria using Azotobacter isolation protocols from pastures in the south of Turkey

Year 2023, Volume: 16 Issue: 3, 252 - 257, 15.12.2023

Ebru Çelen

https://doi.org/10.46309/biodicon.2023.1273996

Abstract

Isolation and molecular characterization of non-Azotobacter bacteria using Azotobacter isolation protocols from pastures in the south of Turkey

Ebru ÇELEN
ORCID: 0000-0002-8452-5933

Department of Biology, Faculty of Arts and Science, Bolu Abant İzzet Baysal University, Gölköy, 14280 Bolu, Türkiye

Abstract
Soil is a biodiversity-rich ecosystem. Nitrogen-fixing Azotobacter bacteria, an important component of this ecosystem, are frequently isolated using various methods. The aim of the present study was to perform partial molecular characterization of non-Azotobacter isolates derived during two Azotobacter isolation protocols, as well as to determine which bacteria can be obtained using this method. A total of 800 isolates were acquired from soil samples collected from various pastures in Antalya province of Turkey, with 92 of them being molecularly characterized. These isolates were clustered through RFLP analysis of 16S rRNA gene and the DNA sequences of the isolates representing different groups were performed. According to these results, the bacteria belonging to various genera (Agrobacterium, Phyllobacterium, Variovorax, Acinetobacter, Pseudomonas, Agromyces, and Arthrobacter) were identified. The results show that similar bacteria could be obtained through two isolation protocols used in this study. However, more diverse bacteria were encountered on the Brown-medium than on soil-past.
Keywords: Azotobacter, isolate, soil, 16S rDNA

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Keywords

Azotobacter, isolate, soil, 16S rDNA

References

[1] Torsvik, V., Goksøyr, J., Daae, F. L., Sørheim, R., Michalsen, J. & Salte, K. (1994). Use of DNA analysis to determine the diversity of microbial communities. In: K. Ritz, J. Dighton & K.E. Giller (Eds.), Beyond the Biomass. Compositional and Functional Analysis of Soil Microbial Communities (pp. 39-48). Chichester: John Wiley.
[2] Falkowski, P.G., Fenchel, T. & Delong, E.F. (2008). The microbial engines that drive Earth's biogeochemical cycles. Science, 320:1034-1039. https://doi.org/10.1126/science.1153213.
[3] Schlesinger, W.H. (1991). Biogeochemistry: An Analysis of Global Change, Academic, San Diego, Calif: Academic.
[4] Young, J.P.W. (1992). Phylogenetic classification of nitrogen-fixing organisms. In: G. Stacey, R.H. Burris & H.J. Evans. (Eds.), Biological nitrogen fixation (pp. 43-86). New York: Chapman and Hall Inc.
[5] Raymond, J., Siefert, J.L., Staples, C.R. & Blankenship, R.E. (2004). The natural history of nitrogen fixation. Molecular Biology and Evolution, 21:541-554. https://doi.org/10.1093/molbev/msh047.
[6] Postgate, J.R. (1998). Nitrogen Fixation (3nd ed.), Cambridge: Cambridge University Press.
[7] Revillas, J.J., Rodelas, B., Pozo, C., Martínez‐Toledo, M.V., & González‐López, J. (2000). Production of B‐group vitamins by two Azotobacter strains with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions. Journal of Applied Microbiology, 89:486-493. https://doi.org/10.1046/j.1365-2672.2000.01139.x.
[8] Ward, D.M., Bateson, M.M., Weller, R. & Ruff-Roberts, A.L. (1992). Ribosomal RNA analysis of microorganisms as they occur in nature. Advanced Microbial Ecology, 12:220–286. https://doi.org/10.1007/978-1-4684-7609-5_5.
[9] Pham, V.H. & Kim, J. (2012). Cultivation of unculturable soil bacteria. Trends in Biotechnology, 30:475-484.
[10] Kennedy, C., Rudnick, P. Macdonald, M.L. & Melton, T. (2005). Genus III. Azotobacter Beijerinck 1901. In: N.R. Krieg, J.T. Staley & G.M. Garrity (Eds.), Bergey's Manual of Systematic Bacteriology – The Proteobacteria (2nd ed, pp 384–402). New York: Springer.
[11] Wollum, A.G. (1982). Cultural methods for soil microorganisms. In: A.L. Page, R.H. Miller & D.R. Keeney (Eds.), Methods of Soil Analysis: Part 2 Chemical and Microbiological Properties (pp. 781-802). Madison: American Society of Agronomy.
[12] Knowles, R. (1982). Free‐living dinitrogen‐fixing bacteria. In: A.L. Page, R.H. Miller & D.R. Keeney (Eds.), Methods of Soil Analysis: Part 2 Chemical and Microbiological Properties (pp. 1071-1092). Madison: American Society of Agronomy.
[13] Aquilanti, L., Favilli F. & Clementi F. (2004). Comparison of different strategies for isolation & preliminary identification of Azotobacter from soil samples. Soil Biology and Biochemistry, 36: 1475-1483. https://doi.org/10.1016/j.soilbio.2004.04.024.
[14] Ryu, E. (1938). On the Gram-differentiation of bacteria by the simplest method. Journal of the Japanese Society of Veterinary Science, 17: 205-207.
[15] Weisburg, W.G., Barns, S.M., Pelletier, D.A. & Lane D.J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173:697-703. https://doi.org/10.1128/jb.173.2.697-703.1991. [16] Sneath, P.H. & Sokal, R.R. (1973). Numerical taxonomy: The principles and practice of numerical classification. (1nd ed), San Francisco: W. H. Freeman.
[17] Felsenstein, J. (1990). PHYLIP manual version 3.3. Berkeley, CA: University of California Herbarium. [18] Nei, M. & Li, W.H. (1979). Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings of the National Academy of Sciences, 76:5269-5273. https://doi.org/10.1073/pnas.76.10.5269.
[19] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215: 403-410. https://doi.org/10.1016/S0022-2836(05)80360-2. [20] Young, J.M. & Park, D.C. (2007). Probable synonymy of the nitrogen-fixing genus Azotobacter and the genus Pseudomonas. International Journal of Systematic and Evolutionary Microbiology, 57:2894-2901. https://doi.org/10.1099/ijs.0.64969-0.
[21] Hesse, C., Schulz, F., Bull, C., Shaffer, B., Yan, Q., Shapiro, N., et al. (2018). Genome-based evolutionary history of Pseudomonas spp. Environmental Microbiology, 20: 2142– 2159. https://doi.org/10.1111/1462-2920.14130.
[22] Heo, S.M., Haase, E.M., Lesse, A.J., Gill, S.R. & Scannapieco, F.A. (2008). Genetic relationships between respiratory pathogens isolated from dental plaque and bronchoalveolar lavage fluid from patients in the intensive care unit undergoing mechanical ventilation. Clinical Infectious Diseases, 47:1562-70. https://doi.org/10.1086/593193.
[23] Chiellini, C., Chioccioli, S., Vassallo, A., Mocali, S., Miceli, E., Fagorzi, C., et al. (2019). Exploring the bacterial communities of Infernaccio waterfalls: a phenotypic and molecular characterization of Acinetobacter and Pseudomonas strains living in a red epilithic biofilm. Diversity, 11:175. https://doi.org/10.3390/d11100175.
[24] Rojas, A., Holguin, G., Glick, B. R. & Bashan, Y. (2001). Synergism between Phyllobacterium sp. (N2-fixer) and Bacillus licheniformis (P-solubilizer), both from a semiarid mangrove rhizosphere. FEMS Microbiology Ecology, 35:181-187. https://doi.org/10.1111/j.1574-6941.2001.tb00802.x.
[25] Chaudhary, H.J., Peng, G., Hu, M., He, Y., Yang, L., Luo, Y. & Tan, Z. (2012). Genetic diversity of endophytic diazotrophs of the wild rice, Oryza alta and identification of the new diazotroph, Acinetobacter oryzae sp. nov. Microbial Ecology, 63: 813-821. https://doi.org/10.1007/s00248-011-9978-5.
[26] Willems, A. Mergaert, J. & Swings, J. (2015). Variovorax. In: W.B. Whitman, P. DeVos, S. Dedysh, B. Hedlund, P. Kämpfer, F. Rainey, M.E. Trujillo, J.P. Bowman, D.R. Brown & F.O. Glöckner et al., (Eds.), Bergey’s Manual of Systematics of Archaea and Bacteria (pp. 1–9). Hoboken, NJ, USA: American Cancer Society.
[27] Finkel, O.M., Salas-González, I., Castrillo, G., Conway, J.M., Law, T.F., Teixeira, P., et al. (2020). A single bacterial genus maintains root growth in a complex microbiome. Nature, 587: 103-108. https://doi.org/10.1038/s41586-020-2778-7.
[28] Powers, E.M. (1995). Efficacy of the Ryu nonstaining KOH technique for rapidly determining gram reactions of food-borne and waterborne bacteria and yeasts. Applied and Environmental Microbiology, 61: 3756-3758. https://doi.org/10.1128/aem.61.10.3756-3758.1995.
[29] Halebian, S., Harris, B., Finegold, S.M. & Rolfe, R.D. (1981). Rapid method that aids in distinguishing Gram-positive from Gram-negative anaerobic bacteria. Journal of Clinical Microbiology, 13:444-448. https://doi.org/10.1128/jcm.13.3.444-448.1981.
[30] Çelik, P. A., Mutlu, M. B., Korkmaz, F., Yaman, B. N., Gedikli, S., & Çabuk, A. (2021). Boron mine ponds: metagenomic insight to bacterial diversity. Biyolojik Çeşitlilik ve Koruma, 14(2): 229-235. https://doi.org/10.46309/biodicon.2021.902221.
[31] Su, C., Lei, L., Duan, Y., Zhang, K. Q., & Yang, J. (2012). Culture-independent methods for studying environmental microorganisms: methods, application, and perspective. Applied microbiology and biotechnology, 93: 993-1003.
[32] Copley, J. (2000). Ecology goes underground. Nature, 406(6795): 452-455.

Türkiye'nin güneyindeki meralardan Azotobacter izolasyon protokolleri kullanılarak Azotobacter olmayan bakterilerin izolasyonu ve moleküler karakterizasyonu

Year 2023, Volume: 16 Issue: 3, 252 - 257, 15.12.2023

Ebru Çelen

https://doi.org/10.46309/biodicon.2023.1273996

Abstract

Türkiye'nin güneyindeki meralardan Azotobacter izolasyon protokolleri kullanılarak Azotobacter olmayan bakterilerin izolasyonu ve moleküler karakterizasyonu
Özet
Toprak, biyolojik çeşitlilik açısından zengin bir ekosistemdir. Bu ekosistemin önemli bir bileşeni olan azot bağlayan Azotobacter bakterileri çeşitli yöntemlerle sıklıkla izole edilir. Bu çalışmanın amacı, iki Azotobacter izolasyon protokolü sırasında elde edilen Azotobacter olmayan izolatların kısmi moleküler karakterizasyonunu gerçekleştirmek ve bu yöntemle hangi bakterilerin elde edilebileceğini belirlemektir. Türkiye'nin Antalya ilindeki çeşitli meralardan toplanan toprak örneklerinden toplam 800 izolat elde edilmiş ve bunlardan 92'si moleküler olarak karakterize edilmiştir. İzolatlar, 16S rRNA genlerinin RFLP analizi ile gruplandırılmış ve farklı grupları temsil eden izolatların DNA dizileri gerçekleştirilmiştir. Bu sonuçlara göre çeşitli cinslere (Agrobacterium, Phyllobacterium, Variovorax, Acinetobacter, Pseudomonas, Agromyces ve Arthrobacter) ait bakteriler tespit edilmiştir. Sonuçlar, bu çalışmada kullanılan iki izolasyon protokolü ile benzer bakterilerin elde edilebileceğini göstermektedir. Bununla birlikte, Brown Besi ortamında Soil-Past’a göre daha çeşitli bakteriler tespit edilmiştir.
Anahtar kelimeler: Azotobacter, izolat, toprak, 16S rDNA

Keywords

Azotobacter, izolat, toprak, 16S rDNA

Supporting Institution

Akdeniz Üniversitesi ve York Üniversitesi

Thanks

Çalışmaya konu olan bakteriler Akdeniz Üniversitesi Fen-Edebiyat Fakültesi Biyoloji Bölümü Laboratuvarlarında izole edilmiştir. İzolatların moleküler karakterizasyonu York Üniversitesi Biyoloji Bölümü’nde yapılmıştır. Çalışma boyunca Prof. Dr. J. Peter W. Young ve Dr. Cyril Bontemps bilgi ve deneyimlerini paylaşarak destek olmuşlardır. Her iki araştırmacıya ve araştırmanın deneylerini gerçekleştirmiş olduğum kurumlara teşekkür ederim.

References

[1] Torsvik, V., Goksøyr, J., Daae, F. L., Sørheim, R., Michalsen, J. & Salte, K. (1994). Use of DNA analysis to determine the diversity of microbial communities. In: K. Ritz, J. Dighton & K.E. Giller (Eds.), Beyond the Biomass. Compositional and Functional Analysis of Soil Microbial Communities (pp. 39-48). Chichester: John Wiley.
[2] Falkowski, P.G., Fenchel, T. & Delong, E.F. (2008). The microbial engines that drive Earth's biogeochemical cycles. Science, 320:1034-1039. https://doi.org/10.1126/science.1153213.
[3] Schlesinger, W.H. (1991). Biogeochemistry: An Analysis of Global Change, Academic, San Diego, Calif: Academic.
[4] Young, J.P.W. (1992). Phylogenetic classification of nitrogen-fixing organisms. In: G. Stacey, R.H. Burris & H.J. Evans. (Eds.), Biological nitrogen fixation (pp. 43-86). New York: Chapman and Hall Inc.
[5] Raymond, J., Siefert, J.L., Staples, C.R. & Blankenship, R.E. (2004). The natural history of nitrogen fixation. Molecular Biology and Evolution, 21:541-554. https://doi.org/10.1093/molbev/msh047.
[6] Postgate, J.R. (1998). Nitrogen Fixation (3nd ed.), Cambridge: Cambridge University Press.
[7] Revillas, J.J., Rodelas, B., Pozo, C., Martínez‐Toledo, M.V., & González‐López, J. (2000). Production of B‐group vitamins by two Azotobacter strains with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions. Journal of Applied Microbiology, 89:486-493. https://doi.org/10.1046/j.1365-2672.2000.01139.x.
[8] Ward, D.M., Bateson, M.M., Weller, R. & Ruff-Roberts, A.L. (1992). Ribosomal RNA analysis of microorganisms as they occur in nature. Advanced Microbial Ecology, 12:220–286. https://doi.org/10.1007/978-1-4684-7609-5_5.
[9] Pham, V.H. & Kim, J. (2012). Cultivation of unculturable soil bacteria. Trends in Biotechnology, 30:475-484.
[10] Kennedy, C., Rudnick, P. Macdonald, M.L. & Melton, T. (2005). Genus III. Azotobacter Beijerinck 1901. In: N.R. Krieg, J.T. Staley & G.M. Garrity (Eds.), Bergey's Manual of Systematic Bacteriology – The Proteobacteria (2nd ed, pp 384–402). New York: Springer.
[11] Wollum, A.G. (1982). Cultural methods for soil microorganisms. In: A.L. Page, R.H. Miller & D.R. Keeney (Eds.), Methods of Soil Analysis: Part 2 Chemical and Microbiological Properties (pp. 781-802). Madison: American Society of Agronomy.
[12] Knowles, R. (1982). Free‐living dinitrogen‐fixing bacteria. In: A.L. Page, R.H. Miller & D.R. Keeney (Eds.), Methods of Soil Analysis: Part 2 Chemical and Microbiological Properties (pp. 1071-1092). Madison: American Society of Agronomy.
[13] Aquilanti, L., Favilli F. & Clementi F. (2004). Comparison of different strategies for isolation & preliminary identification of Azotobacter from soil samples. Soil Biology and Biochemistry, 36: 1475-1483. https://doi.org/10.1016/j.soilbio.2004.04.024.
[14] Ryu, E. (1938). On the Gram-differentiation of bacteria by the simplest method. Journal of the Japanese Society of Veterinary Science, 17: 205-207.
[15] Weisburg, W.G., Barns, S.M., Pelletier, D.A. & Lane D.J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173:697-703. https://doi.org/10.1128/jb.173.2.697-703.1991. [16] Sneath, P.H. & Sokal, R.R. (1973). Numerical taxonomy: The principles and practice of numerical classification. (1nd ed), San Francisco: W. H. Freeman.
[17] Felsenstein, J. (1990). PHYLIP manual version 3.3. Berkeley, CA: University of California Herbarium. [18] Nei, M. & Li, W.H. (1979). Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings of the National Academy of Sciences, 76:5269-5273. https://doi.org/10.1073/pnas.76.10.5269.
[19] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215: 403-410. https://doi.org/10.1016/S0022-2836(05)80360-2. [20] Young, J.M. & Park, D.C. (2007). Probable synonymy of the nitrogen-fixing genus Azotobacter and the genus Pseudomonas. International Journal of Systematic and Evolutionary Microbiology, 57:2894-2901. https://doi.org/10.1099/ijs.0.64969-0.
[21] Hesse, C., Schulz, F., Bull, C., Shaffer, B., Yan, Q., Shapiro, N., et al. (2018). Genome-based evolutionary history of Pseudomonas spp. Environmental Microbiology, 20: 2142– 2159. https://doi.org/10.1111/1462-2920.14130.
[22] Heo, S.M., Haase, E.M., Lesse, A.J., Gill, S.R. & Scannapieco, F.A. (2008). Genetic relationships between respiratory pathogens isolated from dental plaque and bronchoalveolar lavage fluid from patients in the intensive care unit undergoing mechanical ventilation. Clinical Infectious Diseases, 47:1562-70. https://doi.org/10.1086/593193.
[23] Chiellini, C., Chioccioli, S., Vassallo, A., Mocali, S., Miceli, E., Fagorzi, C., et al. (2019). Exploring the bacterial communities of Infernaccio waterfalls: a phenotypic and molecular characterization of Acinetobacter and Pseudomonas strains living in a red epilithic biofilm. Diversity, 11:175. https://doi.org/10.3390/d11100175.
[24] Rojas, A., Holguin, G., Glick, B. R. & Bashan, Y. (2001). Synergism between Phyllobacterium sp. (N2-fixer) and Bacillus licheniformis (P-solubilizer), both from a semiarid mangrove rhizosphere. FEMS Microbiology Ecology, 35:181-187. https://doi.org/10.1111/j.1574-6941.2001.tb00802.x.
[25] Chaudhary, H.J., Peng, G., Hu, M., He, Y., Yang, L., Luo, Y. & Tan, Z. (2012). Genetic diversity of endophytic diazotrophs of the wild rice, Oryza alta and identification of the new diazotroph, Acinetobacter oryzae sp. nov. Microbial Ecology, 63: 813-821. https://doi.org/10.1007/s00248-011-9978-5.
[26] Willems, A. Mergaert, J. & Swings, J. (2015). Variovorax. In: W.B. Whitman, P. DeVos, S. Dedysh, B. Hedlund, P. Kämpfer, F. Rainey, M.E. Trujillo, J.P. Bowman, D.R. Brown & F.O. Glöckner et al., (Eds.), Bergey’s Manual of Systematics of Archaea and Bacteria (pp. 1–9). Hoboken, NJ, USA: American Cancer Society.
[27] Finkel, O.M., Salas-González, I., Castrillo, G., Conway, J.M., Law, T.F., Teixeira, P., et al. (2020). A single bacterial genus maintains root growth in a complex microbiome. Nature, 587: 103-108. https://doi.org/10.1038/s41586-020-2778-7.
[28] Powers, E.M. (1995). Efficacy of the Ryu nonstaining KOH technique for rapidly determining gram reactions of food-borne and waterborne bacteria and yeasts. Applied and Environmental Microbiology, 61: 3756-3758. https://doi.org/10.1128/aem.61.10.3756-3758.1995.
[29] Halebian, S., Harris, B., Finegold, S.M. & Rolfe, R.D. (1981). Rapid method that aids in distinguishing Gram-positive from Gram-negative anaerobic bacteria. Journal of Clinical Microbiology, 13:444-448. https://doi.org/10.1128/jcm.13.3.444-448.1981.
[30] Çelik, P. A., Mutlu, M. B., Korkmaz, F., Yaman, B. N., Gedikli, S., & Çabuk, A. (2021). Boron mine ponds: metagenomic insight to bacterial diversity. Biyolojik Çeşitlilik ve Koruma, 14(2): 229-235. https://doi.org/10.46309/biodicon.2021.902221.
[31] Su, C., Lei, L., Duan, Y., Zhang, K. Q., & Yang, J. (2012). Culture-independent methods for studying environmental microorganisms: methods, application, and perspective. Applied microbiology and biotechnology, 93: 993-1003.
[32] Copley, J. (2000). Ecology goes underground. Nature, 406(6795): 452-455.

There are 29 citations in total.

Details

Primary Language	English
Subjects	Structural Biology
Journal Section	Research Articles
Authors	Ebru Çelen 0000-0002-8452-5933
Early Pub Date	December 8, 2023
Publication Date	December 15, 2023
Submission Date	March 31, 2023
Acceptance Date	September 18, 2023
Published in Issue	Year 2023 Volume: 16 Issue: 3

Cite

APA	Çelen, E. (2023). Isolation and molecular characterization of non-Azotobacter bacteria using Azotobacter isolation protocols from pastures in the south of Turkey. Biological Diversity and Conservation, 16(3), 252-257. https://doi.org/10.46309/biodicon.2023.1273996

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