Isolation and selection of halophilic nitrogen fixing bacteria

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY � � � UNDERGRADUATE THESIS TOPIC: “ISOLATION AND SELECTION OF HALOPHILIC NITROGEN-FIXING BACTERIA” HANOI – 2022 i VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY � � � UNDERGRADUATE THESIS TOPIC: “ISOLATION AND SELECTION OF HALOPHILIC NITROGEN-FIXING BACTERIA” Full name : DO VAN THAO Class : K62CNSHE Student code : 620416 Supervisors : Bui Thi Thu Huong, Dr Dong Huy Gioi, Assoc Prof Dr HANOI - 2022 ii COMMITMENT I commit this is the work of myself The data and results mentioned in the thesis are honest and have never been published in any other study I commit that the information cited in the thesis has been specified origin Hanoi, June 7th 2022 Student Do Van Thao i ACKNOWLEDGEMENTS First and foremost, I would like to express my deepest gratitude to my supervisors, Dr Bui Thi Thu Huong, and Assoc Prof Dr Dong Huy Gioi Faculty of Biology, Vietnam National University of Agriculture for their insight and constructive direction, passionate support, and useful critiques of this thesis, as well as their advice Secondly, I gratefully acknowledge the Faculty of Biotechnology from Vietnam National University of Agriculture and Apart from that, I am grateful to my wonderful classmates in class K62CNSHE for the time we spent together over the previous four years Finally, I express my gratitude to my loved ones for their unwavering and unconditional love, as well as their emotional support through times of adversity in our lives They are the reason I was able to finish my graduation thesis Student Do Van Thao ii CONTENTS COMMITMENT i ACKNOWLEDGEMENTS .ii CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS vii ABSTRACT CHAPTER I INTRODUCTION CHAPTER II LITERATURE REVIEW 2.1 Overview of salt-contaminated soil 2.1.1 Definition 2.1.2 Causes of salty soil formation 2.2 The actuality studies of salt-tolerance bacteria in the world and Vietnam 2.2.1 The actuality studies of salt-tolerance bacteria in the world 2.2.2 The actuality studies of salt-tolerance bacteria in Vietnam 2.3 Soil microorganisms and roles for crops 10 2.3.1 The role of nutritional metabolism 10 2.3.2 The role of protecting crops in salty conditions 11 2.4 The Nitrogen Cycle 14 2.4.1 Definition 15 2.4.2 Process of the Nitrogen Cycle 15 2.4.3 Importance of Nitrogen Cycle 17 2.5 Biological Nitrogen Fixation 18 2.5.1 Introduction 18 2.5.2 The Process 19 2.5.3 Nitrogen-fixed microorganisms 20 iii 2.5.4 Types of nitrogen-fixing microorganisms 23 2.5.5 Roles of nitrogen-fixing bacteria 26 2.6 Overview of nitrogen-fixed bacterial isolation 27 2.6.1 Solid fermentation 27 2.6.2 Liquid fermentation 27 CHAPTER III MATERIALS AND METHODS 29 3.1 Study time and area 29 3.2 Materials 29 3.3 Methods 29 3.3.1 Collection of samples 29 3.3.2 Sampling Procedure 30 3.3.3 Preservation of the sample 30 3.3.4 Determination of soil pH 30 3.3.5 Isolation 30 3.3.6 Assess the suitability of the method of preserving freeze and dry varieties 31 CHAPTER IV RESULTS AND DISCUSSION 32 4.1 Screening and evaluation of nitrogen fixation activity 32 4.2 Colony morphology and cell morphology of N10 34 4.3 Assessment of salt tolerance of nitrogen fixed strain N10 34 4.4 Nitrogen fixed strain identification N10 35 4.5 Assess the suitability of the method of preserving freeze-dried varieties 37 CHAPTER V CONCLUSION AND RECOMMENDATION 39 5.1 Conclusion 39 5.2 Recommendation 39 REFERENCES 40 iv LIST OF TABLES Table 4.1 Salt tolerance of nitrogen fixation strain N10 35 Table 4.2 Density of microorganisms of strains after storage time by freezedrying method 38 v LIST OF FIGURES Figure 2.1 Increased salts in root zone can result in decreased water uptake by plant Figure 2.2 The relationship between microorganisms, crops, and soil 11 Figure 2.3 The Nitrogen Cycle 14 Figure 2.4 Biological Nitrogen Fixation 18 Figure 2.5 Nitrogen fixation process through nitrogenase enzyme complex 21 Figure 4.1 Concentration of amonium were formed by bacteria isolated 32 Figure 4.2 Colonies of bacteria isolated 33 Figure 4.3 Colony morphology of N10 34 Figure 4.4 Cellular morphology of N10 34 Figure 4.5 Phylogenetic plants of the N10 strain with species with close kinship based on RNA16S sequencing analysis 37 vi LIST OF ABBREVIATIONS BNF Biological Nitrogen Fixation ATP Adenosine Triphosphate FAO ICRAF The Food and Agriculture Organization of the United Nations International Council for Research in Agroforestry vii ABSTRACT Adaptation characteristic of microorganisms in different extreme conditions seeks discoveries in field of pharma, food, bioenergy sectors and fresh agriculture In the present study were successfully isolated 28 different saline tolerant microorganism strains approach was proposed to prove adaptation ability in extreme conditions with growth and development of agricultural soil bacterial species in stepwise adaptation in 6% salt (NaCl) conditions from soil surrounding the Truong Sa archipelago Screening Plant growth-promoting selected a bacterium fixation nitrogen N10 ranged 21,61 mg/l Biochemical and molecular (16S rDNA sequencing) characterization revealed the strains to be N10 was located on a small branch with Bacillus megaterium NA10.2 (MZ404904) This experiment provides the base to link the adaptation capabilities of soil microorganisms in salty environment and production of salt-tolerant microbial organic fertilizer At a salt concentration of 6%, the growth capacity of microorganisms is good, adapted to the salty conditions of the environment The freeze-drying method can be used to preserve isolated strains of microorganisms 3.3.2 Sampling Procedure To soil sample collection, a sampling kit consisting of several sterile polythene bags, a marking pen, spatula, alcohol, knives, etc were taken Enough soils were collected from each site; kept in a polythene bag and tagged Soil samples were collected from the top cm of the soil surface, as this is where most of microbial activities take place At each time of collection, hands were sterilized with alcohol (95%) and the bags were partially filled with soil; then the bags were properly tied and labeled Special care was always taken to avoid contamination as far as practicable 3.3.3 Preservation of the sample After collection, the samples were brought to the laboratory carefully and preserved in the refrigerators for immediate use; however, for long-term usage, samples were stored at 4oC 3.3.4 Determination of soil pH 25 g (field moist) was taken in a clean dry 150 ml beaker and 50ml distilled water was added The contents were thoroughly stirred with a vortex machine pH of the suspension was measured with a digital pH meter 3.3.5 Isolation Nitrogen-fixed microorganisms isolated on Ashby’s Mannitol Agar medium supplemented with NaCl 3% 10 g of soil is diluted with 90 ml of sterile distilled water in a 250 ml triangle jar After that, ml of the fluid at a concentration of 10-3-10-5 is implanted on the agar plate and brewed at a temperature of 28 ± 2ºC for - days Colonies grow on transplanted agar plates that are transferred, cleaned, and stored on Ashby’s Mannitol Agar medium 30 To select a strain of bacteria with high protein fixation, ml of fluid (108 CFU/ml) of each strain is cultured shake (120 rpm) on the Ashby fluid medium at a temperature of 30ºC for days The culture fluid is then recovered and N-NH4+ content is determined by coloring the Nessler reagent The residue is dried to determine bacterial biomass 3.3.6 Assess the suitability of the method of preserving freeze and dry varieties Protected strains of microorganisms include fixed nitrogen strains preserved by long-term storage methods for microorganisms in agriculture, made in accordance with TCVN 9299:2014 To assess the suitability of the storage method, live cell density is assessed at the time before dry freezing, after dry freezing, after a storage period of week, weeks, weeks, and 12 weeks in the appropriate nutritional environment National standard TCVN 9299:2014 on Microbiology - Long-term preservation of microorganisms for agricultural use - Liquid nitrogen method - Sample are cultured on prepared slant agar at suitable temperature and time (from 28°C to 30°C for not less than days for bacteria) for uniform growth of microorganisms on inoculation, collection of microbial biomasses for preservation; carried out in a sterile incubator - The microbiological suspension is prepared by adding ml of the prepared protective medium into an inclined agar tube in which the microorganisms have been cultured, then gently stirring with a sterile culture rod to create a homogenous suspension ensure that the microbial cell density is not less than 108 cells/ml Use a sterile pipette to take ml of the small suspension into the bottom of the prepared storage tube, then close the cap of the storage tube, to prevent microorganisms from sticking to the edge or wall of the storage tube Conducted in a sterile incubator - Place the polypropylene storage tube containing the seed sample in the refrigerator at °C for 30 Then immediately transfer the storage tube containing the seed sample to the storage position in the liquid nitrogen flask 31 CHAPTER IV RESULTS AND DISCUSSION 4.1 Screening and evaluation of nitrogen fixation activity From soil samples on the Truong Sa archipelago, the project has successfully isolated 28 strains capable of developing on the Ashby’s medium To assess protein retention, these strains of bacteria are fed on the Ashby’s medium to determine the levels of ammonium produced The results of the assessment of nitrogen fixability by measuring ammonium content are as follows: Figure 4.1 Concentration of amonium were formed by bacteria isolated Excreted ammonium concentration released by isolated strains was estimated using spectrophotometer at OD625nm Single colony of isolated 32 strains was inoculated in Ashby medium and incubated at 37ºC for days After eight days incubation, culture broth was centrifuged at 10000 rpm for mins and 1ml of supernatant was transferred into a test tube The results showed that the measured ammonium concentrations of 28 strains of microorganisms were isolated between 6,66 and 21,61 mg/l In which the strain N10 with nitrogen-fixed activity achieved the highest results of 21,61 ± 1.14 mg/l after 96 hours of culture on a specific environment to assess the ability to fix ammonium proteins In some salt-contaminated lands, about 5% of microbial strains are likely to survive in a non-nitrogen environment The isolation of these microorganisms offers great potential for the development of microbial compost used in island waters Figure 4.2 Colonies of bacteria isolated According to research by Liu J et al (2012), when conducting the isolation of N-fixed bacteria from mangrove root soil samples at Dongzhaigang Mangrove National Nature Reserve (China) obtained 135 strains of species Dhara P Sachdev et al (2009), isolated only nine strains of N-fixed bacteria from wheat root soils According to Pham Thi Ngoc Lan et al (1999) when studying Azotobacter bacteria on the hilly land of Thua Thien Hue province, 37 strains were isolated from 03 acres of land of ear glue roots, yellow flower, and controlled soil 33 4.2 Colony morphology and cell morphology of N10 Figure 4.3 Colony morphology of N10 - Source: near the kitchen landfill in island - Colony morphology: circle, glossy, smooth margin, white - Cell morphology: rod-shaped, chain - Gram: (-) Figure 4.4 Cellular morphology of N10 4.3 Assessment of salt tolerance of nitrogen fixed strain N10 To assess salinity tolerance, the N10 strain is cultured on Ashby fluid environments with different salt concentrations (0%, 2%, 4%, 6%, 8%, 10%) The results showed that when the salt concentration increased from 0% to 4%, the growth performance of strain N10 was not significantly affected When 34 salt levels increase by 4-6%, the growth capacity of this strain decreases from OD600 = 0.832 to OD600 = 0.562 At salt concentrations of 8% and 10%, the N10 strain is virtually impossible to grow and develop In general, microbial strains isolated from salt-contaminated lands are often more salt resistant than conventional strains A study that isolated bacteria from the salt-contaminated region found that when comparing fermentation environments that not contain NaCl and contain 5% NaCl, the protein fixation and growth capacity of these microorganisms varies negligibly Table 4.1 Salt tolerance of nitrogen fixation strain N10 Ability to grow (OD600) Microorganism 0% 2% 4% 6% 8% strain NaCl NaCl NaCl NaCl NaCl N10 0.903 0.881 0.832 0.562 0.125 10% NaCl 0.025 4.4 Nitrogen fixed strain identification N10 The results of the RNA 16S sequencing analysis of the N10 strain are as follows: AATACATGCAAGTCGAGCGAACTGATTAGAAGCTTGCTTCTA TGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCT GTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAG GATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATC ACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAAC GGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCG GCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAG CAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACG CCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAG GGAAGAACAAGTACGAGAGTAACTGCTCGTACCTTGACGGTACCT AACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATA 35 CGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGC GCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGT GGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAA GCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGA ACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAG GCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTC CACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTT TAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGG TCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGC GGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACC AGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTT CGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTG TCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATC TTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGA CAACCGGAGGAAGGTGGGGATGACGTCAATCATCTGCC The N10 strain were resistant to 3% NaCl and had nitrogen-fixing activity on Ashby's medium To identify the species name, primer pair 27F/1525R was used in PCR reaction to multiply the 16S ribosomal RNA gene segment of bacteria Based on the obtained nucleotide sequence, conduct a search for nucleotide sequences that are close to the sequence of the analyzed samples using an online tool (BLAST search) on the Gene Bank This sequence is 100% similar to Bacillus megaterium The pedigree tree was built using the Neighbor-Joining method and the reliability of the pedigree relationships was calculated using the "bootstrap" technique with 1,000 iterations The results are shown in figure 4.5 36 Figure 4.5 Phylogenetic plants of the N10 strain with species with close kinship based on RNA16S sequencing analysis 4.5 Assess the suitability of the method of preserving freeze-dried varieties The results of the study showed that the microbial density of bacillus megaterium strains of bacteria did not change significantly when stored by the freeze-drying method This suggests that the method of freezing and drying the breed is suitable for preserving isolated strains of microorganisms 37 Table 4.2 Density of microorganisms of strains after storage time by freezedrying method Microbial density (CFU/ml) No Strains Bacillus megaterium N10 Pre- After Storage Storage Storage Storage freeze freeze- for for for for 12 dry dried week weeks weeks weeks 4.6 × 4.3 × 3.9 × 4.0 × 3.6 × 3.8 × 107 107 107 107 107 107 Freeze-dried microorganisms are assessed for survival density before and immediately after freezing dry for approximately the same density results (4.3-4.6 x 107 CFU/ml) After freezing and drying, the sample was evaluated once a month for samples within months All results obtained by the N10 strain have a bacterial density at post-freeze dry times >106 CFU/ml, meeting the criteria set out by TCVN 9299:2014 (from 3.6-4.0 x 107 CFU/ml) 38 CHAPTER V CONCLUSION AND RECOMMENDATION 5.1 Conclusion From the results achieved, we draw some of the following conclusions: From 10 acres of soil in the Truong Sa archipelago has isolated 28 strains of bacteria capable of fixing nitrogen In particular, the selection of a strong nitrogen fixed strain of bacteria is N10: the highest peak nitrogen fixation activity reached 21,61 ± 1.14 mg/l after 96 hours of culture on Ashby's medium Strains of microorganisms isolated from salt-contaminated lands are often more salt resistant than conventional strains At a salt concentration of 6%, the growth capacity of microorganisms is good, adapted to the salty conditions of the environment The freeze-drying method can be used to preserve isolated strains of microorganisms 5.2 Recommendation Conduct research on the production of preparations from the N10 strain for application to microbial compost Put into production probiotics from selected microbial sources to achieve economic efficiency and develop and 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