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 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 i ACKNOWLEDGEMENT ii CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi ABBREVIATIONS vii ABSTRACT viii CHAPTER I INTRODUCTION 1.1 The urgency of this study 1.2 Research purposes 1.3 Requirements CHAPTER II LITERATURE REVIEW 2.1 Overview of nitrogen fixing microorganisms 2.1.1 Nitrogen cycle in nature 2.1.2 Molecular nitrogen-fixing microorganisms 2.1.3 Molecular nitrogen fixation process 2.2 Azotobacter species 2.2.1 Scientific classification 2.2.2 Biological characteristics 10 2.3 Status of research on isolation and characterization of Azotobacter spp from soil 16 2.3.1 Studies on Azotobacter spp in the world 16 2.3.1 Studies on Azotobacter spp in Vietnam 16 CHAPTER III MATERIALS, SUBJECTS AND RESEARCH METHODS 18 3.1 Subjects, materials and research location 18 3.1.1 Research subjects 18 3.1.2 Materials, chemicals and research equipment 19 iii 3.1.3 Place and time of the study 20 3.2 Research Methods 21 3.2.1 Isolation of microorganisms 21 3.2.2 Method of domestication and preservation 22 3.2.3 Gram staining technique 23 3.2.4 Identification of Azotobacter spp by using moleculer marker 25 3.2.5 Determine the nitrogen fixation capacity of Azotobacter spp from each sample 28 CHAPTER IV: RESULTS AND DISCUSSION 30 4.1 Soil sampling 30 4.2 Results of isolation and identification of strains 30 4.3 Result of Identification of Azotobacter spp by using moleculer marker 33 4.3.1 Result of DNA Total electrophoresis 33 4.3.2 Result of DNA content by OD spectroscopy of total DNA 34 4.3.3 Results of PCR products detecting Azotobacter spp 35 4.4 Qualitative results with Nessler reagent 35 CHAPTER V CONCLUSIONS AND RECOMMENDATIONS 37 5.1 Conclusions 37 5.2 Recommendations 37 REFERENCES 38 iv LIST OF TABLES Table 3.1 Symbols, collection locations of all the soil sample 18 Table 3.2 Ashby medium’s component 20 Table 3.3 PCR reaction components 28 Table 4.1 Colonies forms obtained in all samples 31 Table 4.2 Differential characteristics of the strains of Azotobacter 31 Table 4.3 Determination of DNA content by OD spectroscopy of total DNA 34 v LIST OF FIGURES Figure 3.1 Soil samples collected 18 Figure 3.2 The procedure of colorimetric method with Nessler reagent 29 Figure 4.1 pH value of soil samples utilized for the isolation of Azotobacter 30 Figure 4.2 Single colonies cultured in Ashby medium, at 30 oC, dark condition 31 Figure 4.3 Colonies were obtained when cultured in Ashby medium containing mannitol and glucose 32 Figure 4.4 Result of DNA total electrophoresis 34 Figure 4.5 Results of PCR products detecting Azotobacter gene by 27F and 1492R primers on gel agarose 1%, 100V 35 Figure 4.6 The result of nitrogen fixation capacity of each sample on Ashby medium 36 vi ABBREVIATIONS A 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 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 viii CHAPTER I INTRODUCTION 1.1 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 The amplification condition was: - at 94 °C for initial denaturation - 30 cycles of 45s at 94 °C - 45s at 52 °C, and at 72 °C - The final extension at 72 °C for 3.2.4.4 Run electrophoresis for PCR products DNA quality and concentration were checked by electrophoresis with 1% agarose gel at 100V for 30 (Khosravi & Dolatabad, 2020) Table 3.3 PCR reaction components Chemical V (μl) PCR 2X Master Mix Free water Primer – F 0.5 Primer – R 0.5 DNA sample 3.2.5 Determine the nitrogen fixation capacity of Azotobacter spp from each sample Determination of NH4+ content in culture solution by colorimetric method with Nessler reagent (Yousuf & Mahmud, 2022) Prepare Nessler reagent: - Weigh 15g HgI2 and 10g KI into 15ml H2O, put in a fume hood, stir to dissolve, add 80ml of 50% NaOH solution, then add H2O to 500ml, stir and filter the residue Store in a dark bottle Protocol: - Prepare test tubes containing autoclaved Ashby medium (121 oC, atm), each containing 10 ml of medium - Inoculate isolated samples of Azotobacter spp into test tubes, control sample without bacteria, containing only Ashby medium 28 - Incubate at room temperature for days - Filter the culture with filter paper and add Nessler reagent to the filtrate - The experiment was repeated times with each sample Observe the color change of the filtrate and compare the color with the control sample Figure 3.2 The procedure of colorimetric method with Nessler reagent 29 CHAPTER IV: RESULTS AND DISCUSSION 4.1 Soil sampling Since the presence of Azotobacter in the soil is highly influenced by the pH value, and their populations increase in soils with pH above 6.5, pH values of soil samples collected measured that showed pH values within the range of 7.2–7.5 and were therefore used for Azotobacter isolation (Figure 4.1.) Figure 4.1 pH value of soil samples utilized for the isolation of Azotobacter 4.2 Results of isolation and identification of strains Results of isolation from soil samples, all samples showed Azotobacter colonies All samples showed 2-3 forms of colonies After the subculture of single colonies, is purified, the isolates have diverse colors, morphology, and size colonies Bacterial strains after being purified are named after the sample part and numbered A total of strains of bacteria were obtained Bacterial strains after cleaning are named after Yu Choy's abbreviation YC and numbered A total of bacterial strains were obtained 30 Table 4.1 Colonies forms obtained in all samples Figure 4.2 Single colonies cultured in Ashby medium, at 30oC, dark condition Table 4.2 Differential characteristics of the strains of Azotobacter Characteristics Colony morphology Cell shape Gram reaction Motility Carbon sources utilization Glucose Mannitol YC01 Large, transparent, spherical, slimy, convex Rod Negative + + + YC02 YC03 Small, pale-white in Small, dullthe outside, dullwhite, circular, white in the middle, slimy circular, flat, slimy Rod Rod Negative Negative + + + 31 + + Figure 4.3 Colonies were obtained when cultured in Ashby medium containing mannitol and glucose From Table 4.2, it can be seen that the isolated strains have characteristics consistent with previous studies on Azotobacter bacteria It was observed that isolated strains of Azotobacter had the following characteristics: Young colonies in all strains were milky white, milky white or clear white, this result is consistent with the study of Do Thu Ha (2008) and Nguyen Thi Luyen (2011) After the colony matures, it has the following characteristics: - YC01: Colonies are spherical, transparent, convex and slightly mucilaginous The largest colony diameter measured was less than or equal to 3mm - YC02: Colonies are round, milky white, slightly flat, slightly mucilaginous Colony with the largest diameter measured is less than or equal to 1.5mm - YC03: Colonies are round, off-white, with opalescent dots in the middle, slightly flat, mucilaginous Colony with the largest diameter measured is less than or equal to 2.5mm 32 4.3 Result of Identification of Azotobacter spp by using moleculer marker 4.3.1 Result of DNA Total electrophoresis In this study, we chose the CTAB method of Minas et al (2011) to extract DNA from the Azotobacter spp Not only Cetyltrimethylammonium bromide (CTAB) buffer is used for DNA extraction from plants with various other extraction buffers but although CTAB for DNA extraction not only from plants but various other samples like fungi, algae, bacteria, and human blood The basic principle of this method is to use cetyl-trimethylamoniumbromide (CTAB), which is able to dissolve substances that escape the cell membrane after their membrane is disrupted, DNA is much more soluble than other substances Therefore, CTAB plays an important role in nucleic acid extraction After obtaining the DNA, check the DNA quality by electrophoresis on 1% agarose gel at 100V The electrophoresis results (Figure 4.1) show that the ribbons are neat and sharp, the DNA is not broken, and the purity is quite high, ensuring the implementation of PCR reactions and subsequent experiments From isolates of Azotobacter (YC01/02/03), conduct total electrophoresis (using DNA samples with each strain) 33 Figure 4.4 Result of DNA total electrophoresis 4.3.2 Result of DNA content by OD spectroscopy of total DNA Table 4.3 Determination of DNA content by OD spectroscopy of total DNA Soil No Colonies samples SS01 SS02 SS03 SS04 SS05 YC01 YC02 YC03 YC01 YC02 YC03 YC01 YC02 YC03 YC01 YC02 YC03 YC01 YC02 YC03 dsDNA concentration (ng/µl) 135 104 102 133 107 99 129 111 103 126 101 105 140 104 107 34 Ratio A260/A280 Ratio A260/A230 2.05 1.84 1.96 2.03 1.85 1.93 2.02 1.88 1.95 2.00 1.83 1.96 2.09 1.84 1.86 2.01 1.81 1.87 1.99 1.83 1.91 1.98 1.85 1.92 1.96 1.82 1.92 2.02 1.82 1.86  The results of the DNA extraction procedure show that the DNA samples have been removed and purified, which is a good product in the PCR process 4.3.3 Results of PCR products detecting Azotobacter spp Agarose gel visualization of gene fragments amplified by 27F and 1492R primers M stands for the marker 1: YC01, 2: YC02, 3: YC03 Figure 4.5 Results of PCR products detecting Azotobacter gene by 27F and 1492R primers on gel agarose 1%, 100V PCR products obtained with primers 27F and 1492R carrying the Azotobacter gene range in the size from 1400 - 1500 bp Survey results using primers showed that all samples carried genes from 1300-1500bp (Khosravi & Dolatabad, 2020) 4.4 Qualitative results with Nessler reagent The colorimetric results with Nessler reagent showed that the isolates all reacted with the reagent and had a yellow color, the characteristic color of Nessler reagent The yellow color has different intensity depending on the NH4+ content that Azotobacter strains produce in the culture solution Of the four isolates, strain YC03 had the darkest yellow color, and strain YC01 had the lightest color Strains YC02 has a darker coloration level than strains YC01 The 35 degree of nitrogen fixation of the strains gradually decreased with the degree of color decrease is presented as follows: YC03, YC02, YC01 Figure 4.6 The result of nitrogen fixation capacity of each sample on Ashby medium 36 CHAPTER V CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions In this study, soil samples were isolated on Ashby agar, and bacterial strains were obtained The isolated bacteria were diverse in color, morphology, and size The strains after being domesticated are named after the target vegetable species in the study, numbered from 01 to 03 They are YC01, YC02, YC03 All strains were gram-negative and had morphological features consistent with several previously studied Azotobacter strains All strains showed bright, clear bands, the size matching the band size of Azotobacter that were studied (1300 - 1500bp) Strain YC03 has the strongest nitrogen fixation ability, strain YC01 has the weakest nitrogen fixation ability  From the above results, it can be determined that strain YC02 is likely to be Azotobacter beijerinckii 5.2 Recommendations From the above results, conduct accurate identification of bacteria based on the 16S rRNA gene sequencing method, to finalize the conclusion 37 REFERENCES Aasfar, A., Bargaz, A., Yaakoubi, K., & Hilali, A (2021) Nitrogen Fixing Azotobacter Species as Potential Soil Biological Enhancers for Crop Nutrition and Yield Stability 12(February), 1–19 https://doi.org/10.3389/fmicb.2021.628379 Adams, E (1975) Studies Histochemistry, in gram staining Biotechnic 50(4), and 227–231 https://doi.org/10.3109/10520297509117063 Amaresan, N., Patel, P., & Amin, D (n.d.) Practical Handbook on Agricultural Microbiology Aquilanti, L., Favilli, F., & Clementi, F (2004) Comparison of different strategies for isolation and preliminary identification of Azotobacter from soil samples 36, 1475–1483 https://doi.org/10.1016/j.soilbio.2004.04.024 Chen, S L., Tsai, M K., Huang, Y M., & Huang, C H (2018) Diversity and characterization of Azotobacter isolates obtained from rice rhizosphere soils in Taiwan Annals of Microbiology, 68(1), 17–26 https://doi.org/10.1007/s13213-017-1312-0 Chosewood, L C (2020) Biosafety in Microbiological and Biomedical Laboratories December 2009 Evstatieva, Y (2019) CHARACTERIZATION AND MORPHOLOGICAL STUDY OF AZOTOBACTER SP STRAIN 104, 383–393 Fallik, E., Chan, Y., & Robert, L (1991) Detection of Alternative Nitrogenases in Aerobic Gram-Negative Nitrogen-Fixing Bacteria E ~~ ThiiEJ Kl 173(1), 365–371 Ghoniem, A A., El-naggar, N E., Saber, W I A., El-hersh, M S., & Elkhateeb, A Y (2020) Statistical modeling-approach for optimization of Cu + biosorption by Azotobacter nigricans NEWG-1 ; characterization 38 and application of immobilized cells for metal removal 1–16 https://doi.org/10.1038/s41598-020-66101-x 10 Gupta, R P., & Pandher, M S (1998) 32 Poster Effect of inoculation of 1954, 271–273 11 Hindersah, R., Setiawati, M R., Asmiran, P., & Natalie, B (2020) Formulation of Bacillus and Azotobacter Consortia in Liquid Cultures : Preliminary Research on Microbes-Coated Urea 8(1), 1–10 https://doi.org/10.20956/ijas.v8i1.2283 12 Hu, H (1979) Effect of Oxygen Concentration and Growth Rate on Glucose Metabolism, Poly-p-hydroxybutyrate Biosynthesis and Respiration of Azotobacter beijerinckii 393–400 13 Hu, H (1976) Regulation of the Tricarboxylic Acid Cycle and Poly-phydroxybutyrate Metabolism in Azotobacter beijerinckii Grown under Nitrogen or Oxygen Limitation 14 Itis, P M (1904) Integrated Taxonomic Information System - Report Species Azotobacter beijerinckii Lipman , 1904 contains : 1–2 15 Krieg N.R., Holt J.G., Sneath P.H.A., Staley J.T., and Williams S.T (1994) Bergey’s Manual of Determinative Bacteriology (9th ed.), Williams & Wilkins, Baltimore, Md, USA 16 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, 30(1), 73–82 17 Mazinani, Z., & Asgharzadeh, A (2014) Genetic diversity of Azotobacter strains isolated from soils by amplified ribosomal DNA restriction analysis Cytology and Genetics, 48(5), 293–301 https://doi.org/10.3103/S0095452714050041 39 18 Mehta, A., Mehta, Y R., & Rosato, Y B (2002) ERIC- and REP-PCR amplify non-repetitive fragments from the genome of Drechslera avenae and Stemphylium solani 211 19 Minas, K., Mcewan, N R., Newbold, C J., & Scott, K P (2011) Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures FEMS Microbiology Letters, 325(2), 162–169 https://doi.org/10.1111/j.1574-6968.2011.02424.x 20 Nosrati, R., Owlia, P., Saderi, H., Olamaee, M., Rasooli, I., & A, A T (2012) Correlation between nitrogen fixation rate and alginate productivity of an indigenous Azotobacter vinelandii from Iran 4(3), 153–159 21 Palkova, L., Tomova, A., Repiska, G., Babinska, K., Bokor, B., Mikula, I., & Minarik, G (2021) Evaluation of 16S rRNA primer sets for characterisation of microbiota in paediatric patients with autism spectrum disorder Scientific Reports, 1–13 https://doi.org/10.1038/s41598-02186378-w 22 Patil, S V., Mohite, B V., Patil, C D., Koli, S H., Borase, H P., & Patil, V S (2020) Azotobacter Beneficial Microbes in Agro-Ecology: Bacteria and Fungi, 397–426 https://doi.org/10.1016/B978-0-12-823414-3.00019-8 23 Pham, T H., Nguyen, T H., Truong, M P., Thuy, T., Le, H., & Quan, Q D (2019) Constructing a molecular genotyping assay for rs11077 based on real-time polymerase chain reaction high resolution melting ( PCR HRM ) technique for the prognosis of hepatocellular carcinoma ( HCC ) patients June 2018 https://doi.org/10.31276/VJSTE.60(2).44 24 Poly, F., Monrozier, L J., & Bally, R (2001) Improvement in the RFLP procedure for studying the diversity of nifH genes in communities of nitrogen fixers in soil 152, 95–103 40 25 Ross, D J (1960) A note on the occurrence of Azotobacter beijerinckii in a market-garden soil New Zealand Journal of Agricultural Research, 3(1), 137–140 https://doi.org/10.1080/00288233.1960.10419867 26 Rubio, E J., Montecchia, M S., Tosi, M., Cassán, F D., Perticari, A., & Correa, O S (2013) Genotypic Characterization of Azotobacteria Isolated from Argentinean Soils and Plant-Growth-Promoting Traits of Selected Strains with Prospects for Biofertilizer Production The Scientific World Journal, 2013 https://doi.org/10.1155/2013/519603 27 Science, E (2021) Bacillus and Azotobacter counts in solid biofertilizer with different concentration of zeolite and liquid inoculant Bacillus and Azotobacter counts in solid biofertilizer with different concentration of zeolite and liquid inoculant 0–6 https://doi.org/10.1088/1755- 1315/667/1/012010 28 Senior, B P J., & Dawes, E A (1973) The Regulation of Poly-Phydroxybutyrate Metabolism in Azotobacter beijerinckii 134, 225–238 29 Trần Thị Linh (2012) Tuyển chọn chủng vi khuẩn Azotobacter cho sản xuất phân bón hữu vi sinh vật 30 Upadhyay, S., Kumar, N., Singh, V K., & Singh, A (2015) Isolation, characterization and morphological study of Azotobacter isolates Journal of Applied and Natural Science, 7(2), 984–990 https://doi.org/10.31018/jans.v7i2.718 31 Vitkute, J., Maneliene, Z., & Janulaitis, A (1998) AbeI , a restriction endonuclease from Azotobacter beijerinckii , which recognizes the asymmetric heptanucleotide sequence ′ -CCTCAGC-3 ′ (– /– ) 26(21), 4917–4918 32 Wakarera, P W., Ojola, P., & Njeru, E M (2022) Characterization and diversity of native Azotobacter 41 spp isolated from semi-arid agroecosystems of Eastern Kenya Biology Letters, 18(3) https://doi.org/10.1098/rsbl.2021.0612 33 Ward, A (2018) Transfer of transposable drug-resistance elements Tn5 , Tn7 , and Tn76 to Azotobacter beijerinckii : Use of plasmid RP4 :: Tn76 as a suicide vector Transfer of Transposable October 1985 https://doi.org/10.1016/0147-619X(85)90076-9 34 Yamagata, U (1923) Physiological study of Azotobacter chroo- coccum, VIII(6), 521–531 35 Yousuf, M F., & Mahmud, M S (2022) Review on Detection Methods of Nitrogen Species in Air, Soil and Water Nitrogen, 3(1), 101–117 https://doi.org/10.3390/nitrogen3010008 36 Yu, S S., Kyaw, E P., Lynn, T., Latt, Z K., Aung, A., Sev, T M., & Thet, M (2017) The correlation of carbon source and ammonium accumulation in culture broth by nitrogen-fixing bacterial isolates The correlation of carbon source and ammonium accumulation in culture broth by nitrogen-fixing bacterial isolates June https://doi.org/10.31254/jsir.2017.6205 42