ارزیابی تثبیت بیولوژیک نیتروژن برخی جدایه‌های ازتوباکتر در محیط کشت جامد و مایع LG به روش کجلدال

نویسندگان

1 دانشجوی کارشناسی ارشد بیولوژی و بیوتکنولوژی خاک، دانشکده کشاورزی، دانشگاه تبریز

2 دانشیار بیولوژی و بیوتکنولوژی خاک، دانشکده کشاورزی، دانشگاه تبریز

چکیده

روش‌های مختلفی برای اندازه‏گیری تثبیت بیولوژیک نیتروژن وجود دارد که عمدتاً شامل سنجش احیا استیلن، بکارگیری نیتروژن نشان‌دار و روش اختلاف نیتروژن می‌باشند. دقت و حساسیت این روش‌ها متفاوت است، ولی مقدور نبودن بعضی از این روش‌ها در همه آزمایشگاه‌ها ایجاب می‌نماید که از روش‌های ساده‌تر برای ارزیابی مقدار تثبیت نیتروژن استفاده نمود، هرچند برخی از این روش‌ها دقت کمتری دارند. در این تحقیق میزان تثبیت نیتروژن تعدادی جدایه‏ باکتری از جمله جنس ازتوباکتر به روش کجلدال مورد آزمایش قرار گرفت. تعداد 22 جدایه باکتریایی برای انجام آزمایش استفاده شدند. جدایه‏های مورد آزمون در محیط کشت جامد و مایع LG کشت داده شده و تثبیت نیتروژن در کشت‎های جامد و مایع اندازه‌گیری شد. نتایج حاصل از هضم، تقطیر و تیتراسیون (کجلدال) نمونه‎های رشد یافته بر محیط کشت‌ جامد نشان داد بیش‎ترین میزان تثبیت نیتروژن در جدایه‎های 14SP-Ш و 14SP2-1 به ترتیب برابر با 10/261 و 64/258 میکروگرم بر گرم محیط کشت و در ته‎نشست محیط کشت مایع در جدایه 14SP-Ι، برابر با 80 میکروگرم در میلی‎لیتر به دست آمد. نتایج حاصل از محیط کشت مایع و جامد بیانگر این است که بیش‌ترین میزان نیتروژن در جدایه‎های 14SP-Ι و 14SP2-1 متعلق به ازتوباکترکروکوکوم، سودوموناس 35SP-2، و اکروموباکتر 14SP-Ш می‎باشد. نتایج به دست آمده در هر دو محیط کشت جامد و مایع همبستگی بالایی (**69/0=r) را نشان داد. به نظر می‎رسد، استفاده از این روش برای غربالگری باکتری‏های کارآمد در تثبیت نیتروژن می‌تواند مورد استفاده قرار گیرد و از میان جدایه‌های مورد آزمون می‌توان سویه‌های 14SP-Ι، 14SP-Ш، 14SP2-1، 35SP-2 و 44SP-2 را برای انجام آزمایش‎های گلخانه‌ای و مزرعه‌ای پیشنهاد نمود.
 

کلیدواژه‌ها

عنوان مقاله [English]

Evaluation of Biological Nitrogen Fixation by Azotobacter Isolates in Solid and Liquid LG Medium by Kjeldahl method

نویسندگان [English]

  • M Leylasi Marand 1
  • MR Sarikhani 2
1 MSc Student of Soil Biology and Biotechnology, Dept. of Soil Science, Faculty of Agric., Uni. of Tabriz, Iran
2 Assoc. Prof. of Soil Biology and Biotechnology, Dept. of Soil Science, Faculty of Agric., Uni. of Tabriz, Iran
چکیده [English]

There are different methods for evaluation of the biological nitrogen fixation, which they are mainly included acetylene reduction assay, labeled nitrogen and nitrogen difference methods. Accuracy and sensitivity of these methods are different, but applying of some of these methods isn’t possible in all laboratories, so it cause the use of less accurate methods for evaluation of the nitrogen fixing content. Therefore, this article was aimed to investigate the content of nitrogen fixation (NF) of some bacteria isolates including Azotobacter using the Kjeldahl method. A total of 22 bacteria isolates were used and evaluated. The isolates were cultured in solid and liquid LG media and the content of the nitrogen fixation was measured in solid and pellet of liquid culture. According to the results of digestion, distillation and titration (Kjeldahl) of the cultured sample in the solid medium, the maximum nitrogen-fixation content 261.10 and 258.64 µg/g were found in 14SP- Ш and 14SP2-1, respectively. The highest nitrogen-fixation content in liquid culture medium was obtained in 14SP-I (80 µg/ml). The results of liquid and solid culture media indicated that the maximum nitrogen in14SP-I and 14SP2-1 isolates belonged to the Azotobacter chroococcum, Pseudomonas 35SP-2, and Achromobacter 14SP- Ш. The results of NF in both solid and liquid cultures showed a good correlation (r=0.69**). It seems that the method can be used for screening the efficient nitrogen fixing bacteria, although this is not so a precise method. 14SP-Ι, 14SP-Ш, 14SP2-1, 35SP-2 and 44SP-2 strains can be suggested among the isolates for the greenhouse and farm experiments. 

کلیدواژه‌ها [English]

  • Azotobacter
  • Difference method
  • Nitrogen fixation
  • Nitrogen free medium

مراجع

Aliasgharzad N, 2006. Methods in soil biology (translated). Tabriz university press. Tabriz. (In Persian)
Anonymous, 2000. How to measure Ammonia Nitrogen: Phenate Method HOP Technical Assistance Hydrology project, World Bank and Government of the Netherlands funded. New Delhi.
Anonymous, 2005. Fertilizer Use by Crop in the Islamic Republic of Iran. Food and Agriculture Organization of the United Nations. Rome. Italy.
Anonymous, 2006. Biofertilizer Manual, FNCA Biofertilizer Project Group. Atomic
Industrial Forum. Japan.
Aquilanti L, Favilli F and Clementi F, 2004. Comparison of different strategies for isolation and preliminary identification of Azotobacter from soil samples. Soil Biology and Biochemistry 36: 1475–1483.
Behrooz A, Olamaee M, Movahedi Naeeni SAR and Gorbani Nasrabadi T, 2016. Evaluation of plant growth promotion characteristics of native soil Azotobacter isolates from golestan province. Soil Management and Sustainable Production 6(1): 233-246. (In Persian with English abstract)
Biabani A, 2008. Formation of artificial nitrogen-fixing bacteria symbiosis with wheat. Journal of Applied Biosciences 6(2): 169-172.
Burris RH and Wilson PW. 1972. Methods for measurement of nitrogen fixation. Pp. 355-367. In: Colowick SP and Kaplan NO, (eds.) Methods in Enzymology, vol. 4.  Academic. New York.
Dalton DA and Kramer S, 2006. Plant Associated Bacteria. Springer, TheNetherlands.
Dixon ROD and Wheeler CT, 1986. Nitrogen Fixation in Plants. Chapman and Hall, New York.
Dobereiner J and Baldani VLD, 1998. Biological Nitrogen Fixation by Endophytic Diazotrophs in Non-Legumes Crops in the Topics. Academic Publishers, Dordocht, The Netherlands.
Dobereiner J, 1997. Biological nitrogen fixation in the tropics, social and economic contributions. Soil Biology and Biochemistry 29: 771-774.
Ebrahimi M, Safari Sinegani AA, Sarikhani MR, Mohammadi SA, Aliasgharzad N, 2018. Isolation, identification, and determination of plant growth promoting properties of Azotobacteria isolated from soil samples north-west of Iran under different land use. Applied Soil Researches 6 (2): 28-42.
Haahtela K, Kari K and Sundman V, 1983. Nitrogenase activity (acetylene reduction) of root-associated, cold-climate Azospirillum, Enteribacter, Klebsiella and Pseudomonas species during growth on various carbon sources and at various partial pressures of oxygen. Applied and Environmental Microbiology 45(2): 563-570.
Kanimozhi K and Panneerselvam A, 2010. Studies on isolation and nitrogen fixation ability of Azospirillum spp. isolated from Thanjavur district. Der Chemica Sinica 1(3): 138-145.
Khavazi K, 2009. Protocols in Biofertilizers Assessment. Soil and Water Research Institute. Pp 17-19.
Khosravi H, 2013. Biofertilizers containing plant growth promoting rhizobacteria: strength and weeknesses. Journal of Land Management 1(1): 33-46. (In persain)
Kizilkaya R, 2009. Nitrogen fixation capacity of Azotobacter spp. strains isolated from soils in different ecosystems and relationship between them and the microbiological properties of soils. Journal of Environmental Biology 31(1): 73- 82.
Mascarua– Esparza MA, Villa-Gonjzalez R and Caballero J, 1998. Acetylene reduction and indoleacetic acid production by Azospirillum isolates from cactaceous plants. Plant and Soil 106: 91-95.
Motsara MR and Roy RN, 2008. Guide to Laboratory Establishment for Plant Nutrient Analysis. Food and Agriculture Organization of the United Nations. The University of Michigan, USA.
Narula N, and Kupta KG, 1986. Ammonium excretion by Azotobacter chroococcum in liquid culture and soil in the presence of manganese and clayminerals. Plant and Soil 93: 205-209.
Palleroni NJ, 1984. Gram negative aerobic rods and cocci. Pp. 140-199. In: Krieg, NR, (Ed.), Bergey’s Manual of Systematic Bacteriology, vol. 1. The Archaea and the Deeply Branching and Phototrophic Bacteria, Williams and Wilkins, Baltimore.
Paul EA, 2007. Soil Microbiology and Biochemistry. Third edition. Linacre House, Jordan Hill, Oxford OX2 8DP, UK.
Rajaee S, Alikhani A and Raiesi F, 2007. Effect of plant growth promoting potentials of Azotobacter chroococcum native strains on growth, yield and uptake of nutrients in wheat. Journal of Water and Soil Science 11(41): 285-296. (In persain)
Rowell DL, 1994. Soil Science: Method and Application. Longman Scientific and Technical, Soil and Plant Analysis, Wageningen Agriculture University, The Netherland. 350 p.
Rump HH and Krist H, 1988. Laboratory Manual of the Examination of Water and Soil. VCH publishers. Escbborn, Germany.
Saribay GF, 2003. Growth and Nitrogen Fixation Dynamics of Azotobacter chroococum in Nitrogen-Free and OMW Containing Medium. Middle East Technical University, Ankara.
Sarikhani MR and Moradi S, 2015. Review on the researches of biofertilizers in Iran. The eighth national conference on agriculture research findings. University of Kurdistan, Sanandaj. Iran. (In Persian)
Shokri D, Emtiazi G and Abassi S, 2012. Investigation of free ammonium production by bacteria in nitrogen fixation process. Agronomy journal 93: 93-103. (In Persian with English abstract)
Sylvia DM, Fuhrman JJ, Hartel PG, Zuberer DA. 1999. Principles and Applications of Soil Microbiology. Prentice Hall, New Jersey.
Tejera N, Lluch C, Martinez-Toledo MV and Gonzalez-Lopez J, 2005. Isolation and characterization of Azotobacter and Azospirillum strains from the sugarcane rhizosphere. Plant and Soil 270: 223–232.
Waling I, Vark WV, Houba VJG and Van der lee JJ, 1989. Soil and Plant Analysis, a series of syllabi. Part 7. Plant Analysis Procedures. Wageningen Agriculture University, The Netherland.
 
دانش آب و خاک
دوره 28، شماره 2
تیر 1397
صفحه 207-218

فایل ها

هم رسانی

ارجاع به این مقاله

آمار