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. 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. 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. iii LIST OF TABLES. v LIST OF FIGURES.vi LIST OF ABBREVIATIONS. Overview of salt-contaminated soil.
Causes of salty soil formation. The actuality studies of salt-tolerance bacteria in the world and Vietnam. The actuality studies of salt-tolerance bacteria in the world. The actuality studies of salt-tolerance bacteria in Vietnam.
Soil microorganisms and roles for crops. The role of nutritional metabolism. The role of protecting crops in salty conditions. The Nitrogen Cycle.
Process of the Nitrogen Cycle. Importance of Nitrogen Cycle. Biological Nitrogen Fixation. Nitrogen-fixed microorganisms.
Types of nitrogen-fixing microorganisms. Roles of nitrogen-fixing bacteria. Overview of nitrogen-fixed bacterial isolation. MATERIALS AND METHODS.
Study time and area. Collection of samples. Preservation of the sample. Determination of soil pH.
Assess the suitability of the method of preserving freeze and dry varieties. RESULTS AND DISCUSSION. Screening and evaluation of nitrogen fixation activity. Colony morphology and cell morphology of N10.
Assessment of salt tolerance of nitrogen fixed strain N10. Nitrogen fixed strain identification N10. Assess the suitability of the method of preserving freeze-dried varieties 37 CHAPTER V. CONCLUSION AND RECOMMENDATION.
40 iv LIST OF TABLES Table 4. Salt tolerance of nitrogen fixation strain N10. Density of microorganisms of strains after storage time by freeze- drying method. 38 v LIST OF FIGURES Figure 2.
Increased salts in root zone can result in decreased water uptake by plant. The relationship between microorganisms, crops, and soil. The Nitrogen Cycle. Biological Nitrogen Fixation.
Nitrogen fixation process through nitrogenase enzyme complex. Concentration of amonium were formed by bacteria isolated. Colonies of bacteria isolated. Colony morphology of N10.
Cellular morphology of N10. 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 The Food and Agriculture Organization of the United FAO Nations ICRAF 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. 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. INTRODUCTION The situation of drought and saltwater intrusion is complicated and seriously affects the production as well as the daily life of people in the islands and archipelagos of Vietnam. Besides, the overuse of inorganic fertilizers for many years has contributed to the salinization of the soil. Saline soil has a high content of soluble salts, leading to an increase in the osmotic pressure of the soil solution, a high level of sodium exchange, resulting in very poor physical properties of the soil, and a readily digestible content of some salts.
Essential nutrients are very low. Meanwhile, the activity of soil microorganisms plays an important role in the process of soil shaping, increasing soil fertility and improving soil nutrients. In particular, the main source of nitrogen to ensure balance in the material cycle in the soil is taken up by a group of nitrogen-fixing bacteria. Salty soil is soil with a high salt content, associated with the accumulation of sea salt for a long time, leading to the influence or loss of some of the soil's properties.
Salinity is considered an environmental agent that greatly affects the ability to produce, cultivate and stabilize the land, thereby affecting food security in many regions in Vietnam and around the world. Each year it is estimated that the world's agriculture loses $27.3 billion due to this cause. Every minute that passes, about 3 hectares of land loses its ability to grow from salinity, equivalent to 1 to 2 million hectares. More than 833 million hectares of land is saline globally, or 8.7% of the planet's area.
Each year, saline soil accounts for 1.5 million hectares of arable land. Loss of agricultural productivity due to saline soil is estimated at 31 billion USD/year. This is data from the Global Map of Saline Soils, a tool released by the Food and Agriculture Organization of the United Nations (FAO) in October 2021. 2 Soil that is saline reduces both the plant's ability to take in water and the availability of micronutrients.
They also concentrate ions that are toxic to plants and can degrade soil structure. Soil can become saline for many reasons such as poor management, excessive or inappropriate fertilizer use, deforestation, sea level rise, groundwater level affecting perineum or seawater intrusion. The underground is then used for irrigation. Climate change is also compounding the problem, with models showing that by the end of the century, global drylands could increase by as much as 23% - mainly in developing countries.
The total area of Vietnam is 331,690 km2. The coastline is 3,260 km long excluding the islands. On a national scale, lowland plains, and hills (under 1,000 m) account for 85% of the area. Less than 20% of the land can be used for agriculture.
As of 2018, Vietnam's agricultural land area was 27,289,454 hectares. Vietnam has three major deltas: the Red River Delta, the Mekong River Delta, and the Central Coastal Delta. Due to the influence of the El Nino phenomenon, many coastal areas of the Mekong Delta are experiencing drought and saltwater intrusion inland. In 2019, the Mekong Delta suffered the heaviest drought and saltwater intrusion in 100 years, causing damage to more than 1,688,600 hectares.
The cultivable areas in coastal districts are affected with varying degrees of soil salinity. According to statistics: saline soil covers an area of about 1 million hectares and accounts for about 3% of the country's natural area. Concentrated mainly in the Mekong Delta. In addition, many central coastal provinces have areas of saline soil up to several tens of thousands of hectares.
Especially Truong Sa archipelago (2.08 nautical miles from the mainland) and some islands far from the mainland, saline intrusion occurs because both of these causes are very strong. The soil of Truong Sa archipelago was a terribly harsh place, arid and saline soil is not favorable for livestock farming and cultivation. The lack of green vegetables was still a 3 concern of the army and the people of Truong Sa archipelago. The island's residents had taken measures such as transporting land and food from the mainland to the island, planting trees in the area to prevent the salty water from blowing in the sea but encountered many difficulties geographic distance and natural conditions.
In recent years, there had been many units and organizations throughout the country have been ceaselessly interested, investment support for troops and people in Truong Sa archipelago to develop agricultural projects to improve production efficiency, initiative of green vegetable sources. However, new projects were being implemented to improve the conditions in the vegetable gardens on the island, using native species from mainland without any project directly exploiting the organism on the island, aiming to improve land on the island a comprehensive and long- term Although plants can evolve and survive in environments with extremely high salt concentrations in the sea and ocean, most agricultural plant varieties are incompatible with these growing conditions. High salinity affects the metabolic activities of the organism cells in a variety of ways. Key metabolic pathways can be damaged under the pressure of high salt concentrations, including damage to cell membranes, producing more free oxygen radicals, reducing the ability to regulate water intake, changing gas exchange characteristics, and creating toxic ions.
Depending on the case, such as salt levels, plant varieties, and stages of development, these metabolic processes can lead to varying degrees of damage, inhibiting growth, reducing yields, or even leading to crops that may die. Currently, to address the negative effects of salt-contaminated soil, two approaches are often adopted including modern and traditional methods. The modern method is to use genetic technology or screening to select crops that are highly salt resistant. However, using this method requires large investment in technology, effort, and economic conditions, along with high complexity that makes the likelihood of success relatively low.
In contrast, traditional 4 methods are simple methods that are easy to implement, and highly effective, such as the application of microbial interactions / soil reclamation plants or the application of interaction between plants and microflora. The study to find efficient strains of nitrogen-fixing bacteria to develop biofertilizer for crops like rice, corn, etc. is very essential. Biological nitrogen fixation is an inexpensive source of nitrogen for higher yields in non-leguminous crops, e., rice farming systems.
In this study, we will focus on isolating and selecting halophilic nitrogen-fixing bacteria. Purpose The successful isolation of nitrogen-fixing bacteria and selection of halophilic nitrogen-fixing bacteria to grow in the salty conditions. Requirements - Isolated the salt-tolerant microorganisms from soil samples taken in Truong Sa archipelago. - Evaluated the activity of nitrogen fixation of microorganisms isolated and selected microorganisms have good activity.
- Put the microorganisms has been selected to preserve. Overview of salt-contaminated soil 2. Definition Saline soil, from an agricultural point of view, is soil that contains dissolved salts at a higher-than-normal concentration, which adversely affects plants. As soils become more saline, plants become unable to draw as much water from the soil.
This is because the plant roots contain varying concentrations of ions (salts) that create a natural flow of water from the soil into the plant roots. As the level of salinity in the soil nears that of the roots, however, water becomes less and less likely to enter the root. In fact, when the soil salinity levels are high enough, the water in the roots is pulled back into the soil.