VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY THESIS TITLE: ISOLATION AND IDENTIFICATION OF AGAR- DEGRADING MICROORGANISMS FROM DIVERSE ENVIRONMENTS IN VIETNAM Hanoi, 1/2021 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY THESIS TITLE: ISOLATION AND IDENTIFICATION OF AGAR- DEGRADING MICROORGANISMS FROM DIVERSE ENVIRONMENTS IN VIETNAM Student : Vu Thi My Duyen ID : 610710 Department : Biotechnology Supervisor : Vu Nguyen Thanh, Assoc. Nguyen Van Giang, Assoc. Hanoi, 1/2021 COMMITMENT I hereby declare that: This is my study, which was conducted under the guidance of the supervisors; All data provided are true and accurate; All published data and information have been duly cited. Hanoi, January 2021 Student Vu Thi My Duyen i ACKNOWLEDGEMENTS Firstly, I am grateful to express my gratitude to the Food Industries Research Institute, especially to the Center for Industrial Microbiology for granting me support to pursue my thesis as well as to the Department of Biotechnology, Vietnam National University of Agriculture, who provided me opportunities to pursue higher education and to prepare myself to better.
I also would like to express my gratitude to my supervisor Assoc. Nguyen Van Giang for providing me with an opportunity to do the final work in the Vietnam National University of Agriculture and giving me all support, which made me complete the project. I owe my deep gratitude to Assoc. Vu Nguyen Thanh, who has given me all support, guidance, all the necessary information that made me complete the project.
He allowed me to do the necessary research work and use the lab equipment needed at the Center for Industrial Microbiology, Food Industries Research Institute. Besides, I would like to extend our sincere esteems to all members in the laboratory for their timely support. It also gives my thankfulness to my family, to all of my friends, for sharing my difficulties, and giving me various useful pieces of advice during the process of learning and studying. Thank you very much! Hanoi, 2nd February 2021 Student Vu Thi My Duyen ii TABLE OF CONTENTS COMMITMENT.
ii TABLE OF CONTENTS. iii LIST OF FIGURES .v LIST OF TABLES. Introduction of agar. Properties of agar.
Agar-degrading enzymes. Applications of agarases. Agar production and agar-degrading enzymes research in Vietnam. MATERIALS AND METHODS.
Study laboratory and time. Samples for isolation. Method of isolation. Purification and maintenance of isolates.
Observation of colonies and cells. Protein fingerprinting by SDS-PAGE method. DNA extraction and purification. Agarase assay by Congo red staining.
RESULTS AND DISCUSSION. Selection of agar degrading microorganisms. Observation of colonies, cells. Grouping by protein fingerprinting.
Identification of microorganisms by rDNA sequencing. CONCLUSION AND SUGGESTION. 39 iv LIST OF FIGURES Figure 2. Structure of agarose.
Diagram of the cleavage site of agarase (Kazłowski, Pan and Ko, 2008). 3 Strategies for agarose hydrolysis and the various physiological activities of agarose-derived sugars. Some kinds of samples. Isolation origins of 109 agar-degrading strains.
Enrichment samples were transferred on agar medium at 28 °C after 10 days. The morphology of agar-degradable microorganisms. Electrophoresis images of Protein fingerprinting products of 79 strains. Neighbour-joining phylogram depicting the relationships between isolated strains and neighbouring taxa.
Pie chart comparing frequency appears of microorganisms degrading agar in different samples in Vietnam. Congo red staining results of strains cultured on MSYA agar at 28 ° C after 7 days.25 v LIST OF TABLES Table 2. Some agarolytic microorganisms: origins and characteristic of the enzyme. Characterization of some recombinant β-agarases (Veerakumar and Manian, 2018).
Group of isolated strains by morphology and protein fingerprinting results. Qualitative agarase activity of representative strains.26 vi ABBREVIATIONS Abbreviations Full name DNA Deoxyribose nucleic acid PCR Polymerase chain reaction rDNA Ribosomal DNA TAE Tris-acetate-EDTA MSYA Minimal salt yeast extract agar CBB Coomassie Brilliant Blue AOS Agarooligosaccharides NAOS Neoagarooligosaccharides vii ABSTRACT In this study, samples were collected from regions in Vietnam to isolate agar- degrading microorganisms. As we know agar production process still has many shortcomings such as causing environmental pollution, high production costs, etc. The research direction of using biotechnology to agar production is currently being interested, in particular the use of enzymes from microorganisms.
The thesis research for agar-degrading microorganisms, target to produce secondary products of higher value agar, and searched for microorganisms capable of producing enzymes that cut agar into other products such as AOS- agar oligosaccharides and NAOS- neoagarooligosaccharides. These products are used in pharmaceuticals and cosmetics. From 90 samples collected, 109 agar-degrading microorganisms were isolated using morphology and protein fingerprinting method, 44 representative strains randomly of each group one or two strains selected from 61 groups for 16S sequence analysis. The results of the sequence analysis identified 19 species including Microbulbifer elongatus, Enterobacter huaxiensi, Alteromonas abrolhosensis, Bacillus flexus, Bacillus tenquilensis, Catenococcus thiocycli, Luteibacter jiangsuensis, Lysinibacillus fusiformis, Microbacterium aquimaris, Nitratireductor indicus, Paracoccus homiensis, Pseudomonas pachastrellae, Salipiger pacificus, Pseudomonas plecoglossicida, Shinella curvata, Sinomicrobium oceani, Shingopyxis granuli, Stappia indica, and Advenellas kashmirensis.
Four undescribed species of the genera Bacillus, Microbacterium, Microbulbifer, and Shingopyxis have been identified. The agarolytic activity of the strains was also investigated. INTRODUCTION Agar, mainly extracted from the species of marine red algae including Gelidium and Gracilaria, is a polysaccharide well known as an important gelifying agent for the food, cosmetic, and medical industries, and is composed of agarose and agaropectin. However, the current agar production process still has many shortcomings such as causing environmental pollution, high production costs, etc.
The research direction of using biotechnology to agar production is currently being interested, in particular the use of enzymes from microorganisms. A number of agar-hydrolyzing bacteria have been isolated from marine and other environments and several agarases have been purified and characterized in the past decade from isolates of Cytophaga, Pseudomonas, Alteromonas, Pseudoalteromonas, Streptomyces, Vibrio, Agarovirans, Saccharophagus, Microscilla, etc. Agarases catalyze agar hydrolysis and include two types, α-agarase and β-agarase depending on the pattern of its cleavage. Agarooligosaccharides are produced when α- agarases cleave α-1,3 linkages of agar polymer and neoagarooligosaccharides are produced when β-agarases cleave β-1,4 linkages of agar polymer.
Agar-derived sugars, including agar oligosaccharides and neoagarooligosaccharides, have industrial potentials as pharmaceuticals, prebiotics, and cosmetic ingredients owing to their various physiological activities. Agarases have been applied in a wide range of biotechnological and industrial applications. Agarases have been used for agar hydrolysis to produce oligosaccharides which have essential physiological and biological activities that are helpful for human health. Agarase was also applied in DNA purification from agarose gel.
Protoplasts from seaweeds have been obtained by using agarase, and for understanding the composition and structure of the cell wall of seaweeds. Thus, isolating microorganisms producing agarase would be of great importance in providing a valuable understanding of this enzyme, also widening the use of this enzyme in medical, cosmetic, life sciences, and industrial fields. In this study, the research topic "Isolation and identification of agar- degrading microorganisms from diverse environments in Vietnam" was conducted to 1 understand the diversity of the group of microorganisms, and exploring novel species for various applications. Research objective The objective of this research is to understand the diversity of agar-degrading microorganisms in Vietnam.
More specifically: - To isolate agar-degrading microorganisms from diverse environments like soil, plants, seaweeds … - To identify the isolates by rDNA sequencing - To determine the agar-degrading activity of the isolates. Introduction of agar 2. Agar structure Agar is a type of heterogeneous polysaccharide whose main component is agarose, accounting for about 70%, the rest is agaropectin. Agarose is a linear polymer consisting of alternating two molecules β-D-galactose and 3,6-anhydro-α-L- galactopyranose, linked together by 1,3-linked β-d-galactose (neoagarobiose) and 1,4- linked 3,6-anhydro-α-l-galactose (agarobiose) (Figure 2.
Whereas agaropectin is a heterogeneous mixture with a short-chain structure, the composition is similar to agarose and has additional bonds with -OSO3-, -OCH3, glucuronate, or pyruvate… at the C2 or C6 position of the 3,6- anhydro-α-L-galactose molecule. The physical properties of agar are closely related to its chemical properties like agar gel strength depends greatly on the ratio of agarose and agaropectin as well as the composition of the bonding groups. Structure of agarose. In mass terms, agarose has a high molecular weight of above 100 kDa.
While an agaropectin molecule weighs less than 20 kDa, the composition contains from 5 to 8% sulfate, so it is often not used in food processing (Armisen and Galatas, 1987). However, agaropectin could be modified into agarose by removing the sulfate groups of agaropectin (Phillips and Williams, 2020). A high concentration of agaropectin is found in red algae Gracilaria, Porphyra. (Lu et al.
Agar source Agar is mainly extracted from the cell walls of the red algae Gelidium and Gracilaria. Gelidium is a good source of agar but these species are small, slow- growing plants, and only well-cultivated in a few countries and regions. Whereas Gracilaria species are capable of growing rapidly and cultivated widely in the world. Thus, Gracilaria has become the most important source of agar production (Armisen, 1995) 2.
Properties of agar Agar is a mixture of agarose and agaropectin in different proportions depending on the alga species of raw materials and manufacturing process. Therefore, different types of agar have not similar properties, but all types of agar have some physical and chemical properties (Hải, 2016) Solubility: Agar is insoluble in cold water, but it swells considerably. It is slightly soluble in ethanolamine and well soluble in formamide. Agar is soluble in water and other solvents at temperatures between 95ºC to 100ºC.
Gel-forming ability: Agar forms a gel after being heated and cooled without the addition of gel-forming agents, while carrageenan needs potassium or protein to form gels, alginate needs calcium or divalent cations. The agarose molecules change from a coil structure to the helices. Gel-forming ability and gel strength depend on the agar concentration and the average molecular weight (the larger the average molecule, the more stable the gel is formed).5% agar begins to gel at 32 - 43°C and will not melt below 85ºC. This gel-forming ability has led to a large number of practical applications where agar is used as a food additive or in other applications in microbiology, biochemistry, or molecular biology, as well as in industrial applications.
Viscosity: Agar viscosity depends on the agaropectin content of the raw material. In general, the viscosity of agar is relatively stable at pH 4. Agar can be used well in the pH range of 5-8. 4 Stability: Agar is capable of reversing gelation and melting at high temperature without changing the initial properties.
Agar in the dry state is not subject to contamination by microorganisms. Thanks to the above properties, agar is often used in food processing technology such as a substitute for processed cheese or as an additive to baked goods, confectionery, products from meat and dairy products. Agar is a polysaccharide that is indigestible to most microorganisms and should be used as a culture medium. Besides, agar has been experimented on producing agar-soybean protein biofilm for enhanced chemical and physical properties.
Agar is also used in the textile industry, functional foods, and other applications. With its useful properties in smart materials development for composites and adsorption applications, agar is believed to become a promising material in the future (Chew et al. Agarolytic microorganisms The first agarolytic bacterium was isolated from seawater at the beginning of the 20th century by Gran, 1902. Since then, a number of microorganisms have been reported to degrade agars, mainly in the marine environment, either in the water column, in coastal marine sediments, or associated with red algae (Humm, 1946), (Stanier, 1941).
Agarolytic bacteria have also been identified in brackish water and salt marshes (Ekborg et al., 2005) and, more surprisingly, in freshwater (Van der Meulen, Harder, and Veldkamp, 1974) and soils (Buttner et al. 1987), (Suzuki et al. This unexpected presence of agarases in non-marine environments was also confirmed by the metagenomic approach on soil samples (Voget et al. Despite the number of isolated species, the agarolytic bacteria represent only a few phyla and classes.