VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------------- GRADUATION THESIS TOPIC: “ISOLATION AND CHARACTERIZATION OF AZOTOBACTER SPP. OBTAINED FROM YU CHOY SOIL SAMPLES IN THANH HOA CITY” Student name : Dang Hoang Son Student’s code : 637430 Class: : K63CNSHE Major: : Biotechnology Supervisor: : Pham Thi Dung, Ph.D Hanoi, 2022 GUARANTEE I hereby declare that all results in this thesis are my own work under the guidance of an Mrs. Pham Thi Dung Ph.D, Faculty of Biotechnology, Vietnam National University of Agriculture. All data, images, and results presented in this thesis are completely honest and have not been published in any other works.
The documents and information published in the works, journals, and websites referenced in this thesis have been listed in the reference list of the thesis. I take responsibility for my promises to the board and the university. Hanoi, December 2022 Student Dang Hoang Son i ACKNOWLEDGEMENT First words, in the process of completing this study, I have received great deal of helps, guidance and encouragements from teachers and friends. First of all, I would like to express my deepest thanks to my supervisor, Mrs.
Pham Thi Dung who given me suggestions on how to shape the study and always been most willing and ready to give my valuable advice, helpful comments as well as correction of my study. Next, I would like to express my gratitude to all teachers in Faculty of Biotechnology - Vietnam National University of Agriculture for their lectures 5 years that help me much in completing this study. Last but not least, I would like to thank my family and my friends who have always encouraged, supported and helped me to complete this study. Ha Noi, December 2022 Student Dang Hoang Son ii CONTENTS GUARANTEE.
iii LIST OF TABLES. v LIST OF FIGURES. The urgency of this study. Overview of nitrogen fixing microorganisms.
Nitrogen cycle in nature. Molecular nitrogen-fixing microorganisms. Molecular nitrogen fixation process. Status of research on isolation and characterization of Azotobacter spp.
Studies on Azotobacter spp. in the world. Studies on Azotobacter spp. MATERIALS, SUBJECTS AND RESEARCH METHODS 18 3.
Subjects, materials and research location. Materials, chemicals and research equipment. Place and time of the study. Isolation of microorganisms.
Method of domestication and preservation. Gram staining technique. Identification of Azotobacter spp. by using moleculer marker.
Determine the nitrogen fixation capacity of Azotobacter spp. from each sample. 28 CHAPTER IV: RESULTS AND DISCUSSION. Results of isolation and identification of strains.
Result of Identification of Azotobacter spp. by using moleculer marker. Result of DNA Total electrophoresis. Result of DNA content by OD spectroscopy of total DNA.
Results of PCR products detecting Azotobacter spp. Qualitative results with Nessler reagent. CONCLUSIONS AND RECOMMENDATIONS. 38 iv LIST OF TABLES Table 3.
Symbols, collection locations of all the soil sample. Ashby medium’s component. PCR reaction components. Colonies forms obtained in all samples.
Differential characteristics of the 3 strains of Azotobacter. Determination of DNA content by OD spectroscopy of total DNA. 34 v LIST OF FIGURES Figure 3. Soil samples collected.
The procedure of colorimetric method with Nessler reagent. pH value of soil samples utilized for the isolation of Azotobacter. Single colonies cultured in Ashby medium, at 30 oC, dark condition. Colonies were obtained when cultured in Ashby medium containing mannitol and glucose.
Result of DNA total electrophoresis. Results of PCR products detecting Azotobacter gene by 27F and 1492R primers on gel agarose 1%, 100V. The result of nitrogen fixation capacity of each sample on Ashby medium. beijerinckii: Azotobacter beijerinckii BNF: Biological Nitrogen Fixation CTAB: Cetyl Trimethyl Ammonium Bromide DNA: Deoxyribonucleic Acid PCR: Polymerase Chain Reaction SS: Soil sample YC: Yu Choy vii ABSTRACT Isolation and selection of Azotobacter spp.
from cultivated land. In this study, bacterial strains were isolated from the soil growing Yu Choy at different locations in Thanh Hoa city. These strains were biochemically identified and characterized on Ashby – specific medium based on morphological and physiological properties. Results obtained showed that all 3 isolated strains were belonging to the Azotobacter strain.
In order for molecular analysis, the 16S rDNA gene was amplified using primer pairs (including 27F and 1492R primers), and the PCR products are then subjected to electrophoresis to analyze the result. Then determine the nitrogen fixation capacity of the samples by colorimetric method with the Nessler reagent. The results showed that strain YC03 has the strongest nitrogen fixation ability, and strain YC01 has the weakest nitrogen fixation ability, and the strain YC02 was most suitable with the characteristics of Azotobacter beijerinckii. The urgency of this study Along with the continuous development of industries, our country's agriculture has also achieved many remarkable achievements thanks to the advancement of science and technology, making the productivity and quality of crops increased many times.
In agriculture, nitrogen is considered a very important source of nutrients for plants. The supply of nitrogen to plants from fertilizers is extremely important to meet the growth and development needs of plants and partly compensate for the amount of nitrogen that plants have taken from the soil through crops. When nitrogen fertilizer is applied to the soil, plants only absorb 40-50% of the fertilizer, the rest is washed away by rainwater, irrigation water, or converted and evaporated in the form of NH3, NOx, and N2. In addition, the abuse of chemical fertilizers to increase productivity has degraded the land, reduced fertility, polluted water sources, and affected the health and living environment of people and the environment of other organisms in nature (Shenoy, 2001).
Therefore, the increase in chemical nitrogen fertilizer is only a temporary solution and cannot be applied in the long term because it raises many concerns. Therefore, research and use of nutrients created from living activities of microorganisms have been interested and developed by many countries around the world. Currently, microbiological fertilizers have many advantages over chemical fertilizers. In addition to improving crop productivity and quality, reducing production costs, biofertilizers also make an important contribution to environmental protection and sustainable agricultural development.
Many research results on microbial fertilizers have confirmed that the effectiveness of microbial fertilizers depends on biological activity, ability to 1 compete with microorganisms available in the soil, and ability to adapt to soil environmental conditions of microorganisms used in feces. Microbial fertilizers are particularly useful if the microorganisms used are biologically active. One of the research directions that is of interest is the production of nitrogen-fixing microbial fertilizers derived from microorganisms, in which, Azotobacter is the most interesting strain because of many characteristics such as the ability to fix nitrogen naturally Because, in the air, many plant growth stimulants (IAA, GA3, .) are synthesized to help the root system develop firmly, absorb water and nutrients well (Okon, 1985), contribute to raising the humidity level soil fertility, stimulating growth, increasing crop yields, limiting chemical fertilizers, and developing sustainable ecological agriculture. To produce good microbial fertilizers, there must be strains of microorganisms with high biological activity, multi-activity, and high viability.
Therefore, the isolation and characterization of microbial strains is indispensable. In Vietnam, there have been a number of studies on the characteristics of Azotobacter on some soils, but currently, but currently there are not many in- depth studies on Azotobacter strains, especially on soil samples in Thanh Hoa province. Starting from the above basis, I carried out the research: “Isolation and characterization of Azotobacter spp. obtained from Yu Choy soil samples in Thanh Hoa city” 1.
Research purposes Isolation and characterization of Azotobacter spp. from 5 soil samples of Yu Choy obtained from different locations in Thanh Hoa city then determine morphological characteristics and their nitrogen fixation capacity of them. Requirements Isolation of Azotobacter spp. from soil samples.
Gram staining to determine the morphology and biological characteristics of the bacteria. By the 16S rDNA amplification to determine the Azotobacter spp. Then, determine the nitrogen fixation capacity of them by using colorimetric method with Nessler reagent. Overview of nitrogen fixing microorganisms 2.
Nitrogen cycle in nature According to Aasfar et al (2021), nitrogen is an important and indispensable nutrient source for animals, plants and even microorganisms. The amount of nitrogen stored in nature is very large; In air, nitrogen makes up 78. It is estimated that the atmosphere that covers one hectare of land contains up to 8 million tons of nitrogen. This amount of nitrogen can provide plants for tens of millions of years (if plants are able to assimilate it).
Plants do not assimilate organic nitrogen directly, but must rely on microorganisms to decompose and convert sustainable nitrogen sources into easily digestible forms (NH3 or NH4+), providing a source of nitrogen nutrients for plants. This process is called ammonification. Following the process of ammonification, the microorganisms again convert from NH3 to NO3- called nitrification. Following the nitrification process, microorganisms convert NO3- to N2 to compensate for nitrogen in the air, this is the denitrification process.
Under the action of microorganisms, nitrogen in the air is converted into nitrogen-containing organic compounds called molecular nitrogen fixation. All processes: immobilization - decomposition - metabolism and nitrification reactions always occur under the action of microorganisms and create a nitrogen balance. Thanks to that, it closes the nitrogen cycle in nature. 4 Nitrogen cycle in nature diagram Source: Sarthaks.com The nitrogen-based compounds produced from nitrogen fixation are taken up into the tissues of algae and plants.
Animals eat the algae and plants, thereby taking up the compounds in their own tissues. Animals use the compounds in their cells, or the compounds are broken down and excreted in the form of urea and other waste products. Nitrogen-based compounds released as wastes or occurring in the bodies of dead organisms are converted to ammonia and subsequently to nitrates and nitrites. These compounds are then converted again to atmospheric nitrogen by so-called denitrifying bacteria in the environment.
Molecular nitrogen-fixing microorganisms According to Patil et al (2020), nitrogen-fixing microorganisms have an important role in the N2 cycle and provide a significant amount of nitrogen to plants. According to calculations by scientists, groups of microorganisms' nitrogen fixation BNF (Biological Nitrogen Fixation) can provide up to 240 x 5 160 tons of N/year on the whole planet, 6 times more than the amount of nitrogen that the whole world produces. produced by chemical means. Nitrogen-fixing microorganisms are groups of microorganisms capable of converting the abundant N2 gas in the atmosphere (79%) into the form of NH4+ for plants.
There are two groups of nitrogen-fixing microorganisms: - Group of free-living microorganisms: Azotobacter, Clostridium bacteria,. - Group of symbiosis microorganisms: Rhizobium, Azospirillum Some other nitrogen fixing microorganisms: In addition to the above-mentioned molecular nitrogen-fixing microorganisms, there are countless other varieties that have the ability to fix molecular nitrogen, they have many meanings in agricultural, forestry and fishery production (Patil et al. - Bacteria: Azotomonas insolita, Azotomonas fluorescens, Pseudomonas azotogenis, Klabsiella pneumonia, Aerobacter aerogene, Rhodospirillum rubrum, Chromatium, Rhodomicribium, Desulfovibrio desulfuricans,… - Actinomycetes: Some species of the genera Actinomyces, Frankia, Nocardia, Actinopolyspora - Algae - Cyanobacteria: Pectonema, Anabaena azolla, Anabaena cylindrica, Calothrix elenkii, … 2. Molecular nitrogen fixation process According to Patil et al (2020), nitrogen fixation is a chemical process by which molecular nitrogen (N2), with a strong triple covalent bond, in the air is converted into ammonia (NH3) or related nitrogenous compounds, typically in soil or aquatic systems but also in industry.
Atmospheric nitrogen is molecular dinitrogen, a relatively nonreactive molecule that is metabolically useless to all but a few microorganisms. Biological nitrogen fixation or diazotrophy is an 6 important microbials mediated process that converts dinitrogen (N2) gas to ammonia (NH3) using the nitrogenase protein complex. Nitrogen fixation is essential to life because fixed inorganic nitrogen compounds are required for the biosynthesis of all nitrogen-containing organic compounds, such as amino acids and proteins, nucleoside triphosphates, and nucleic acids. As part of the nitrogen cycle, it is essential for agriculture and the manufacture of fertilizer.