VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ---------- ---------- GRADUATION THESIS TITLE: “BIOSYNTHESIS AND ANTIBACTERIAL CHARACTERIZATION OF SILVER NANOPARTICLES DERIVED FROM STREPTOMYCES ASSOCIATED WITH CRINUM LATIFOLIUM L.” Hanoi - 2023 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ---------- ---------- GRADUATION THESIS TITLE: “BIOSYNTHESIS AND ANTIBACTERIAL CHARACTERIZATION OF SILVER NANOPARTICLES DERIVED FROM STREPTOMYCES ASSOCIATED WITH CRINUM LATIFOLIUM L.” Student name : DINH THI LINH CHI Class : K63CNSHE Student’s code : 637406 Supervisor : QUACH NGOC TUNG, Ph. NGUYEN THANH HUYEN, Msc. Department : MICROBIAL BIOTECHNOLOGY Hanoi - 2023 COMMITMENT I hereby declare that the data and results stated in the thesis are honest and have never been published by anyone in other studies. In the references section, the graduations with references to papers and action information are mentioned.
I am completely responsible for the data of this thesis. Hanoi, January 9th, 2023 Sincerely Dinh Thi Linh Chi i ACKNOWLEDGEMENTS First and foremost, I would like to express my heartfelt gratitude to my main supervisor Dr. Quach Ngoc Tung from VAST - Culture Collection of Microorganisms (VCCM), Institute of Biotechnology, Vietnam Academy of Science and Technology. The inspiration, valuable guidance, and support he provided was vital driving force behind the theiss, which helped me to achieve the best and strive towards my goals.
I would also like to thank Msc. Nguyen Thanh Huyen, a Vice head of the Department of Microbial biotechnology at the Vietnam National University of Agriculture, for co-supervising the thesis, for giving me the golden opportunity to work at VCCM and keeping me on track. A special thank you to Assoc. Phi Quyet Tien, Ph.
Vu Thi Hanh Nguyen, and researchers at the VCCM-Institute of Biotechnology, who have provided technical support and imparted valuable knowledge as well as research experience. The knowledge and skills gained would be beneficial in the future it had been a warm and fruitful experience. Without the immense support of the Board of Directors at Vietnam National University of Agriculture and the lecturers of Biotechnology Department, Vietnam National University of Agriculture, I would not have been able to acquire the professional foundation necessary to complete this report, and the wealth of experience that has helped me take my first confident steps along my chosen career path. Thank you too to all my friends in the laboratory who have made coming to VCCM so enjoyable.
The research leading to these results has received funding from Vietnam Academy of Science and Technology under grant agreement no. To wrap up, I'd want to express my gratitude to my family and all who have journeyed with me, encouraged me, and shared in my experiences. I sincerely thank you! Hanoi, Janury 9th, 2023 Sincerely Dinh Thi Linh Chi ii TABLE OF CONTENTS COMMITMENT. ii TABLE OF CONTENTS.
iii LIST OF TABLES .v LIST OF FIGURES .vi LIST OF ABBREVIATIONS. Overview of silver nanoparticles. Silver nanoparticles and their characteristics. Applications of silver nanoparticles.
Synthesis of silver nanoparticles. Chemical and physical synthesis. Green synthesis of silver nanoparticles. Modes of action against microorganisms of silver nanoparticles.
Modes of action. Biosynthesis of silver nanoparticles by Streptomyces. Introduction of the genus Streptomyces. Green synthesis of silver nanoparticles by Streptomyces.
Overview of Crinum latifolium L. Current status of green silver nanoparticles in Vietnam. MATERIALS AND METHODS. Research subjects and materials.
Location and time of the study. Equipment and chemicals. Composition of media. Cultivation of Streptomyces spp.
strains associated with Crinum latifolium L. Intracellular silver nanoparticles biosynthesis. In vitro antimicrobial activity of synthesized AgNPs. Morphological, physiological, and biochemical identification of potent strain.
Molecular identification based on the 16S rRNA analysis. RESULTS AND DISCUSSION. Antimicrobial screening of silver nanoparticles synthesized by endophytic Streptomyces spp. Observation of color change.
Antimicrobial activity of silver nanoparticles synthesized by Streptomyces sp. Identification of potent Streptomyces spp. Morphological characteristics of strain PCT3. Physiological and biochemical characterization of strain PCT3.
Molecular identification of strain PCT3 using 16S rRNA analysis. Antimicrobial characterization of PCT3 silver nanoparticles. CONCLUSIONS AND SUGGESTIONS .34 iv LIST OF TABLES Table 1. Antibacterial activity of crude AgNPs synthesized by 8 Streptomyces strains.
Biochemical and physiological characteristics of Streptomyces sp. Minimum Inhibitory Concentration of silver nanoparticles produced from S. 31 v LIST OF FIGURES Figure 1. The shape of AgNPs observed by transmission electron microscopy.
Routes of cytotoxicity action for AgNPs. Mechanism of extracellular and intracellular synthesis of AgNPs by actinomycetes. The color change observed when cell-free supernatant of the 3 representative strains were treated with 1 mM AgNO3 after 72 h. Antibacterial activity of crude AgNPs against Candida albicans (A) and Enterococcus faecalis (B).
The colonial morphology of strain PCT3 observed after 2 days of incubation, at 30°C. Enzymatic activities of strain PCT3 shown on agar plates containing skim milk (A), starch (B), and CMC (C). Agarose gel electrophoresis of the 16S rRNA gene amplicons of Streptomyces sp. Land M: DNA marker (250 – 10,000 bp).
Phylogenetic tree based on 16S rRNA gene sequences exhibiting the relationship between Streptomyces spp. PCT3 and other closely related type strains. Zones of inhibition of AgNPs against C. albicans ATCC 10231 (A) and P.
32 vi LIST OF ABBREVIATIONS Abbreviation Full word AgNPs Silver nanoparticles Bio-AgNPs Bio silver nanoparticles CFS Cell-free supernatant CMC Carboxymethyl cellulose MIC Minimum Inhibitory Concentration NADH Nicotinamide Adenine Dinucleotide rDNA Ribosomal DNA ROS Reactive oxygen species SPR Surface plasmon resonance vii ABSTRACT Nanotechnology holds an emerging domain of medical science as it can be utilized virtually in all areas. Among metallic nanoparticles, silver nanoparticles (AgNPs) are widely used in biomedical sciences, healthcare, drug–gene delivery, space industries, cosmetics, chemical industries, optoelectronics. The applications of AgNPs attribute to their own physical, chemical and biological activities. Recently, green approach of AgNPs synthesis is gained attentions due to growing need for developing cost-effective, bio-compatible, and eco-friendly approaches to synthesis AgNPs.
In this study, 8 endophytic strains from medicinal plant Crinum latifolium L. were screened for their ability to synthesize AgNPs with antibacterial property. Among them, the AgNPs synthesized by mixing free- biomass filtrate of strain PCT3 with 1 mM AgNO3 were the most promising as indicated by the color intensity and microbial activity. Strain PCT3 grew well on ISP2 agar with irregular margin, filamentous, white, umbonate, and rough colonies, which exhibited the typical morphology of Streptomyces.
Besides, it utilized glucose, fructose, sucrose as carbon sources for growth and produced protease and amylase. Phylogenetic analysis of PCT3 based on 16S rRNA gene analysis revealed that PCT3 was closely related to Streptomyces albus. Combining morphological, biochemical, and molecular analysis, strain PCT3 was identified as S. Antibacterial characterization of AgNPs from S.
albus PCT3 revealed that obtained AgNPs were effective against 2 Gram-positive, 3 Gram-negative bacteria with MIC values ranging from 3. In addition, AgNPs strongly inhibited yeast Candida albicans ATCC 10231. In conclusion, the current study is a demonstration of an efficient synthesis of PCT3 by endophytic Streptomyces from C. latifolium, which is an interesting subject for development of antimicrobial agents to combat infection.
INTRODUCTION Antimicrobial resistance is the ability of microbes such as bacteria and fungi to grow in the presence of antibiotics that might normally kill them or at least limit their growth. Antimicrobial resistance threatens to destabilize progress in human health by reducing the ability to treat common infectious diseases (Mitchell et al. There is an urgent need to find new antibiotics and non-antibiotic compounds to prevent global threats. Silver has long been known for its various applications in many fields such as food technology, medicine, cosmetics, and pharmaceuticals.
Recent advancements in nanotechnology have led to the widespread use of silver nanoparticles (AgNPs) as antimicrobial, antioxidant, anti-inflammatory, and anti- cancer agents. In addition, AgNPs have been employed as catalysts, biopharmaceuticals, waste treatment reagents, fertilizer additives, and biomedical materials. The synthesis of metal nanoparticles involves the employment of a wide variety of methods, the majority of which are prohibitively costly and generate hazardous chemical wastes (Madani et al. Synthesis of AgNPs by using bacteria has gained huge attention over chemical and physical methods since the biological method is eco-friendly.
The genus Streptomyces, belonging to the order Actinomycetales, is known as an important source of antibiotics. Exploiting Streptomyces spp. from untapped environments can be an effective way to meet the everlasting demand for novel antibiotics and non-antibiotic agents (Ma et al. Endophytic Streptomyces from medicinal plants have been extensively explored and gained attention from microbiologists all over the world due to the limitations of finding novel compounds.
Of note, green AgNPs synthesis has been reported in some members of the Streptomyces genus, such as Streptomyces rochei, Streptomyces coelicolor, Streptomyces griseorubens, and Streptomyces viridodiastaticus. is a valuable medicinal herb found in certain regions of Vietnam. In traditional medicine, it is used for the treatment of allergic disorders and tumor diseases. The alkaloid compounds extracted from C.
latifolium are reported to exhibit remarkable antitumor and immune-stimulating activities. In addition, leaf extracts of C. latifolium in Vietnam showed in vitro and in vivo T- lymphocyte activation and retarded the growth of chemically induced tumors in rats. However, endophytes associated with C.
latifolium and their capability to synthesize AgNPs have not been reported yet. With the aim of shedding light on the ability to synthesize AgNPs with antimicrobial activity from Streptomyces spp. latifolium, we performed the project entitled "Biosynthesis and antibacterial characterization of silver nanoparticles derived from Streptomyces spp. associated with Crinum latifolium L.
Research purposes Screening and evaluation of antimicrobial activity of AgNPs synthesized by endophytic Streptomyces spp. Research contents - Screening of the antimicrobial activity of AgNPs made by Streptomyces strains associated with C. - Morphological and molecular identification of potent candidates. - Synthesis and assessment of the antimicrobial activity of the AgNPs 2 II.
Overview of silver nanoparticles 2. Nanoparticles Nanotechnology is known as an interdisciplinary field that focuses on the development, manufacture, characterization, and use of nanoscale materials. It is believed that the term "nanoparticles," approved by the International Organization for Standardization (ISO), is used to describe a particle having a size ranging from 1 to 100 nm with at least one of the three possible dimensions (ISO 2015a). Organic and inorganic nanomaterials are the two primary classifications that may be found (Harish et al.
The organic nanomaterials include carbon-bearing particles like liposomes, while the inorganic nanomaterials consist of magnetic nanoparticles, noble metal nanoparticles (gold and silver), and semi-conductor nanoparticles (titanium oxide and zinc oxide). Recently, noble metal nanoparticles such as silver have gained the attention of researchers around the world because of their remarkable optical, electrical, magnetic, catalytic, biological, and mechanical properties (Khan et al. Due to their size, improved properties, and advantages, nanoparticles are particularly promising for application in diagnostics, sensing, energy storage, medicine, drug delivery, and many other fields (Sim et al., 2021; Niu et al. As a result, more than 1,800 nanoparticle products have been commercialized, and many more are being developed.
The global value of nanotechnology products reached 2 trillion euros in 2015, demonstrating nanotechnology's significant impact on global economics (Nanomaterials, n. Silver nanoparticles and their characteristics Silver nanoparticles (AgNPs) have received countless accolades due to their various applications in different sectors such as cancer therapeutics, biosensors, antibiotics, anti-inflammatory therapy, and drug delivery. For example, silver has been used for antimicrobial treatment since 1000 B. Silver salts and their derivaties are commercially utilized as antimicrobial agents since silver is active against a wide range of 116 microorganisms.
It has been shown 3 that the silver at the nanoscale has improved properties as compared to silver salts due to its small size, large surface area, and strong toxicity to a wide range of microorganisms (Figure 2. Rahman et al.