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.D.
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. Prof. Phi Quyet Tien, Ph.D. 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. ĐLTE00.03/21-22.
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 ..............................................................................................................i
ACKNOWLEDGEMENTS ........................................................................................... ii
TABLE OF CONTENTS .............................................................................................. iii
LIST OF TABLES ..........................................................................................................v
LIST OF FIGURES ........................................................................................................vi
LIST OF ABBREVIATIONS ...................................................................................... vii
ABSTRACT ................................................................................................................ viii
I. INTRODUCTION ......................................................................................................1
II. LITERATURE REVIEW ........................................................................................3
2.1.

Overview of silver nanoparticles ..........................................................................3

2.1.1. Nanoparticles ........................................................................................................3
2.1.2. Silver nanoparticles and their characteristics .......................................................3
2.1.3. Applications of silver nanoparticles .....................................................................5
2.2.

Synthesis of silver nanoparticles ..........................................................................7

2.2.1. Chemical and physical synthesis ..........................................................................7

2.2.2. Green synthesis of silver nanoparticles ................................................................ 7
2.3.

Modes of action against microorganisms of silver nanoparticles ........................8

2.3.1. Antimicrobial properties .......................................................................................8
2.3.2. Modes of action ...................................................................................................9
2.4.

Biosynthesis of silver nanoparticles by Streptomyces ........................................11

2.4.1. Introduction of the genus Streptomyces ............................................................. 11
2.4.2. Green synthesis of silver nanoparticles by Streptomyces ..................................12
2.5.

Overview of Crinum latifolium L. ......................................................................14

2.6.

Current status of green silver nanoparticles in Vietnam ....................................14

III. MATERIALS AND METHODS .........................................................................15
3.1.

Research subjects and materials .........................................................................15

3.1.1. Location and time of the study ...........................................................................15
3.1.2. Materials .............................................................................................................15
3.1.3. Equipment and chemicals ...................................................................................16


iii


3.1.4. Composition of media ........................................................................................16
3.2.

Methods ..............................................................................................................17

3.2.1. Cultivation of Streptomyces spp. strains associated with Crinum latifolium L. .......17
3.2.2. Intracellular silver nanoparticles biosynthesis....................................................17
3.2.3. In vitro antimicrobial activity of synthesized AgNPs ........................................18
3.2.4. Morphological, physiological, and biochemical identification of potent
strain ...................................................................................................................19
3.2.5. Molecular identification based on the 16S rRNA analysis ................................ 20
3.2.6. Data analysis .......................................................................................................22
IV. RESULTS AND DISCUSSION ...........................................................................22
4.1.

Antimicrobial screening of silver nanoparticles synthesized by endophytic
Streptomyces spp. ............................................................................................... 22

4.1.1. Observation of color change ...............................................................................22
4.1.2. Antimicrobial activity of silver nanoparticles synthesized by Streptomyces sp..........23
4.2.

Identification of potent Streptomyces spp. PCT3 ...............................................25

4.2.1. Morphological characteristics of strain PCT3 ....................................................25
4.2.2. Physiological and biochemical characterization of strain PCT3 ........................26
4.2.3. Molecular identification of strain PCT3 using 16S rRNA analysis ...................28

4.3.

Antimicrobial characterization of PCT3 silver nanoparticles ............................ 30

V. CONCLUSIONS AND SUGGESTIONS ............................................................. 33
5.1.

Conclusions ........................................................................................................33

5.2.

Suggestions .........................................................................................................33

REFERENCES ............................................................................................................34

iv


LIST OF TABLES

Table 1. Antibacterial activity of crude AgNPs synthesized by 8 Streptomyces
strains .................................................................................................. 24
Table 2. Biochemical and physiological characteristics of Streptomyces sp.
PCT3 ................................................................................................... 27
Table 3. Minimum Inhibitory Concentration of silver nanoparticles
produced from S. albus PCT3 ............................................................. 31

v



LIST OF FIGURES

Figure 1.1. The shape of AgNPs observed by transmission electron microscopy ..... 4
Figure 1.2. Routes of cytotoxicity action for AgNPs. (1) Adhesion to cell
wall; (2) Cellular internalization; (3) ROS generation; (4)
Genotoxicity. .................................................................................. 11
Figure 1.3. Mechanism of extracellular and intracellular synthesis of
AgNPs by actinomycetes ................................................................ 13
Figure 1.4. The color change observed when cell-free supernatant of the 3
representative strains were treated with 1 mM AgNO3 after 72 h. ...... 23
Figure 1.5. Antibacterial activity of crude AgNPs against Candida albicans
(A) and Enterococcus faecalis (B).................................................. 24
Figure 1.6. The colonial morphology of strain PCT3 observed after 2 days
of incubation, at 30°C. .................................................................... 26
Figure 1.7. Enzymatic activities of strain PCT3 shown on agar plates
containing skim milk (A), starch (B), and CMC (C). ..................... 28
Figure 1.8. Agarose gel electrophoresis of the 16S rRNA gene amplicons of
Streptomyces sp. PCT3. Land M: DNA marker (250 – 10,000 bp) ...... 29
Figure 1.9. Phylogenetic tree based on 16S rRNA gene sequences exhibiting
the relationship between Streptomyces spp. PCT3 and other closely
related type strains. ........................................................................... 29
Figure 1.10. Zones of inhibition of AgNPs against C. albicans ATCC 10231 (A)
and P. aeruginosa ATCC 9027 (B) ................................................ 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 freebiomass 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. albus PCT3. 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 3.9 μg/mL to 31.2
μg/mL. 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.

viii


I. 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.,
2020b). 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 anticancer 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., 2022. 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., 2021). 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.

1


Crinum latifolium L. 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 Tlymphocyte 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.-derived C. latifolium, we performed the project entitled "Biosynthesis and
antibacterial characterization of silver nanoparticles derived from
Streptomyces spp. associated with Crinum latifolium L." was investigated.
 Research purposes
Screening and evaluation of antimicrobial activity of AgNPs synthesized
by endophytic Streptomyces spp. strains from C. latifolium L.
 Research contents
- Screening of the antimicrobial activity of AgNPs made by Streptomyces
strains associated with C. latifolium L.

- Morphological and molecular identification of potent candidates.
- Synthesis and assessment of the antimicrobial activity of the AgNPs

2


II. LITERATURE REVIEW
2.1. Overview of silver nanoparticles
2.1.1. 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., 2022). 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., 2019). 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., 2019). 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.d.-b).
2.1.2. 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.C. 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 2.1). Rahman et al. 2019 proved that the surface plasmon
resonance of AgNPs was recorded at 425 and 480 nm, which is much stronger
than that of any other metal nanoparticles, such as copper, zinc, titanium, and
magnesium, which could be due to the poorer coupling to interbond transitions.
Of note, the outstanding antimicrobial properties of AgNPs have inspired their
use in a variety of products, including those designed to treat burns and ulcers,
food packaging to prevent contamination, household appliances like refrigerators
and washing machines, and a number of industrial applications (Bruna et al.,
2021). Thus, AgNPs are regarded as an important category of nanomaterials.

Figure 1.1. The shape of AgNPs observed by transmission electron microscopy
(Source: Loiseau et al., 2019)
The bioactivities of AgNPs are strongly related to particle size (surface area
and energy), particle shape (catalytic activity), particle concentration (therapeutic
index), and particle charge (oligodynamic quality). AgNPs with concentrations of
0, 25, 50, 75, and 100 μg/mL and sizes ranging from 1 - 100 nm were tested
against Escherichia coli (Bruna et al., 2021). Interestingly, 16-nm AgNPs at a
concentration of 75 μg/mL showed the strongest bacterial activity (Choi et al.,
2008). Another report showed that 5-nm AgNPs were highly active compared

with 10-nm, 15-nm, and 10-nm AgNPs. Regarding Staphylococcus mutans, 8.4nm AgNPs were preferred, indicating the relationship between NPs sizes and their
4


antimicrobial activities (Espinosa-Cristóbal et al., 2009). It could be explained
that AgNPs possess larger surface areas that promote the release of more Ag ions
(Ag+). However, the mechanisms of the antimicrobial effects of silver
nanoparticles are not clear yet.
To date, the adverse effects of AgNPs on humans and all other living beings
have been documented.As estimated, tons of silver are dumped into the
environment from industrial wastes, so the toxicity of silver in the environment is
attributed to free silver ions in the aqueous phase. Exposure to high doses of free
silver ions causes permanent bluish-gray discoloration of the skin (argyria) or the
eyes (argyrosis); liver and kidney damage; eye, skin, respiratory, and intestinal
tract irritations; and untoward changes in blood cells (Panyala et al., 2008). A
study proved that AgNPs induced toxic effects on the proliferation and cytokine
expression of peripheral blood mononuclear cells (Shin et al., 2007). The oral
toxicity of nanosilver on rats revealed that AgNPs are toxic to the liver.When
administered in considerable doses, AgNPs are not harmful to human cells (Bruna
et al., 2021) and are widely recognized as an effective antibacterial agent (Wypij
et al., 2018; Bruna et al., 2021). Due to the fact that bio-AgNPs pose a low risk of
harm to both humans and the environment, agronomists are becoming more
interested in the possibility of using them as a tool for crop protection (Jafir et al.,
2021). As a direct result of this, professionals in the fields of biology, agriculture,
and medicine have been more interested in the manufacturing of nano-scaled
particles in recent years (Khalil et al., 2021a,b; Zaki et al., 2022). Therefore, more
in vivo studies are required to assess the toxicity of nanosilver before a conclusion
on its toxicity is reached.
2.1.3. Applications of silver nanoparticles
AgNPs, which are considered to be one of the most promising types of

nanomaterials, have been widely used in agriculture. AgNPs offer antibacterial
efficacy against phytopathogen-caused plant illnesses. For example, AgNPs
reduced the severity of disease in perennial ryegrass (Lolium perenne) (Jo et al.,
2009). In addition, wheat (T. aestivum, var. UP2338), cowpea (Vigna sinensis,
5


var. Pusa Komal), and brassica (Brassica juncea, var. Pusa Jai Kisan) were treated
by AgNPs. It showed that growth and root nodulation of cowpea were
significantly improved (Pallavi et al., 2016). Several studies demonstrated
antifungal activity of AgNPs against plant fungal pathogens such as Alternaria
alternata, Penicillium digitatum, and Alternaria citri that is higher than those of
synthetic fungicides including difenoconazole and iprodione under same
conditions (Abdelmalek et al., 2016). AgNPs also decrease disease development
of fungal pathogens (Macrophomina phaseolina, Rhizoctonia solani, Curvularia
lunata, Sclerotium cepivorum, Bipolaris sorokiniana, Magnaporthe grisea)
(Nargund et al., 2021). Many efforts have been made to make AgNPs become to
be water-soluble nano-fertilizers, nano-pesticides, and nano-herbicides, which
safer than the synthetic fungicides.
AgNPs are commercially used in bioremediation. Injecting silver
nanoparticles into zeolites has hygienic applications since it prevents the
proliferation of pathogens in water (Dutta, 2011). AgNPs derived from
actinomycetes have been shown to be effective in combating biofouling.
Biosynthesized silver nanoparticles from Streptomyces rochei HMM13 suppress
bacterial biofilm formation because they decrease the number of bacterial cells in
the biofilm. AgNPs also have shown impressive promise as effective antibacterial
agents for the treatment of water (Lu et al., 2016).
On the other hand, AgNPs have shown promise for biomedical
applications. AgNPs enhance wound healing and new skin cell development
because of anti-inflammatory property, leading to applications for wound-healing

and infection-preventing dressings. AgNPs enhance the efficacy of face masks in
a remarkable way. A study involved in a face mask coated with AgNPs proved
that E. coli and S. aureus are killed after 24 hours of treatment. The synthesized
AgNPs exhibited antibacterial effectiveness against pathogenic microbes
responsible for urinary tract infections, including E. coli, Klebsiella pneumoniae,
Pseudomonas aeruginosa, and Candida albicans (Divya et al. 2019). The urgent
problem is antimicrobial-resistant microbial infections, which is on the rise at a
6


concerning rate worldwide (Shakoor et al., 2020). Accordingly, multidrugresistant bacteria have become more common due to antibiotic overuse in the
treatment of infectious disorders (Khalil et al., 2015a,b). AgNPs as a promising
option for preventing such infections, disinfecting medical equipment, and
fighting infections.
2.2. Synthesis of silver nanoparticles
2.2.1. Chemical and physical synthesis
2.2.2. Green synthesis of silver nanoparticles
The problem physical method is expensive, while of chemical method
produces hazardous chemicals that may cause pollution and biological risks.
Environmentally and economically alternative methods are required to synthesize
these nanoparticles. Thus, interest in the notion of "green synthesis pathway" has
begun to increase. The sustainable approach of green nanotechnology has a single
objective: a cleaner environment. Production of nanoparticles through an ecofriendly technique does not affect the environment or human health (Kumar et al.,
2019a). The biological method of nanoparticle synthesis employs microbes, algae,
fungus, and plants to produce high-yield, inexpensive, and environmentally
friendly nanoparticles (Narayanan et al., 2010). In addition, AgNPs have a longer
shelf life and greater stability due to natural capping. It is a cost-effective, onestep approach for the synthesis of nanoparticles with secure downstream
processing and purification. Another benefit of biological synthesis over chemical
synthesis is that it can be readily scaled up; manufactured (Kumar et al., 2019a).
By using extract from higher plants, fungi, and bacteria for producing

nanoparticles, this technique is reffered to green approach. In nature, only a small
number of bacteria can survive in a high concentration of silver and synthesize
AgNPs. The first bacteria synthesizing AgNPs is Pseudomonas stutzeri AG259
isolated from silver mine. Afterwards, various bacteria have been shown to
produce AgNPs including Enterobacteria, Bacillus licheniformis, Streptomyces
(Gnanajobitha et al., 2013). Besides, AgNPs can be synthesized by fungi
(Fusarium oxysporum, Aspergillus fumigatus) (Iravani et al., 2014; Ahmad et al.,
7


2003), and fruit extracts (Carica papaya, Vitis vinifera) (Bhainsa et al., 2006;
Karthiga et al., 2018).
It is interesting that AgNPs biosynthesis can be taken either outside or
inside of cells. The production of nanoparticles extracellularly by bacteria seems
to be dependent on nitrate reductase that converts metal ions metal nanoparticles.
Originally, nitrate reductase catalyzes nitrate to nitrite. For instances, B.
licheniformis (Sarker et al., 2007) has been used to demonstrate this technique.
The production and stability of nanoparticles in Stenotrophomonas maltophilia
through the process of charge capping has also been reported (Nayaka et al, 2020).
This process includes the electron shuttle enzymatic metal reduction process that
is generated by the nicotinamide adenine dinucleotide phosphate-dependent
reductase enzyme. It is revealved that the biosynthesis of AgNPs involves in a
nitrate reductase of Pseudomonas species reuired the presence of nicotinamide
adenine dinucleotide (NAD). The enzyme may be in charge of turning Ag + into
Ag 0 generating AgNPs. The NADH-dependent reductase is thought to act as a
carrier while the bio reduction happens with electrons from NADH (Eckhar et al.,
2013; Hulkoti et al., 2014).
2.3. Modes of action against microorganisms of silver nanoparticles
2.3.1. Antimicrobial properties
The antibacterial ability of the AgNPs against both Gram-positive and negative bacteria has been particularly described. Gram-negative and Grampositive bacteria were tested for resistance to the bactericidal and bacteriostatic

properties of nanoparticles between 5 and 100 nm (Shekhar Agnihotri et al.
2014). For three strains of E. coli, Bacillus subtilis, and S. aureus, the MIC
ranged from 20 to 110, 60 to 160, 30 to 120, and 70 to 200 μg/mL, with the
first value corresponding to the 5-nm AgNPs and the second to the 100-nm
AgNPs. These results suggeste that AgNPs always inhibit bacteria. Of note,
multidrug-resistant bacteria like Pseudomonas aeruginosa, methicillinresistant Staphylococcus aureus, also have been reported to be susceptible to
AgNPs (Subashini et al., 2014).
8


Furthemore, Derazkola et al. have developed a simple, quick, and cheap
technique of synthesizing silver nanoparticles using Crataegus microphylla fruit
extract. The results show that the antibacterial action is due, in part, to the
chemical surface of the nanoparticles. The minimum inhibitory concentrations
(MICs) of AgNPs against various bacteria were found to be 14 μg/mL against
Staphylococcus aureus, 7 μg/mL against Enterococcus, 3.5 μg/mL against
Pseudomonas aeruginosa, 3.5 μg/mL against Acinetobacter baumannii, 3.5
μg/mL against Escherichia coli, 28 μg/mL against P. mirabilis.
Apart from that, AgNPs have significant effects on fungi. AgNPs
synthesized by garlic plants (Allium sativum) suppressed Fusarium graminearum
causing head blight disease on wheat crops (Mansoor et al., 2021). Mycelium
growth, spore germination, and mycotoxin production were inhibited by AgNPs.
In support this study, Biopolar sorokiniana, a causative agent of spot blotch
disease on wheat was strongly sensitive to green AgNPs. It could be that AgNPs
are reactive and damage fungal cells at the molecular level. So far, modes of action
and toxicity of AgNPs are unknown.
2.3.2. Modes of action
The antimicrobial mechanisms of AgNPs have extensively been studied and
a heated debate, but they remain poorly understood. Many scientists supposed that
AgNPs attach firstly to the cell membrane and cell wall (Cheng et al., 2018). The

positively charged Ag+ is needed for silver's antibacterial or toxicological
properties, which must be ionized. Due to electrostatic attractions and affinity for
sulfur proteins, Ag+ ions bind to the cytoplasm and cell wall, dramatically
increasing permeability and causing bacterial casings to rupture (Khorrami et al.,
2018). Increased membrane permeability also leads to the loss of cellular
components such as proteins, carbohydrates, and adenosine triphosphates, and the
energy store of the cells (Chauhan et al., 2013; Li et al., 2013). In addition, AgNPs
damage to intracellular macromolecules such as protein, lipids, and DNA
(Morones et al., 2005; Rai et al., 2012).

9


Cellular homeostasis essential for cell survival is disturbed by reactive
oxygen species. ROS are constantly produced during oxygen-dependent aerobic
metabolism as a by-product of cellular metabolism (Jain et al., 2021). At low and
moderate concentrations, ROS serve as signaling molecules promoting cell
growth and defense mechanisms against stress conditions. In contrast, excesive
ROS cause oxidative stress leading to cell damage, especially macromolecules
such as DNA, protein and lipid. It is clear that ROS are produced as a consequence
of AgNPs toxicity, which hinder cell respiration and development (Quinteros et
al., 2016). To be more specific, AgNPs are known to mainly inhibit thiol groupcontaining enzymes, such as NADH dehydrogenase in the respiratory system,
which is concerned as a candidate for the production of reactive oxygen species.
Therefore, inhibition of this particular enzyme results in free radical production
and oxidative stress cycles, which leads to DNA damage, lipid peroxidation, an
apoptotic-like reaction, as well as the depletion of antioxidant enzymes (Lee et
al., 2014; Korshed et al., 2016).

10



Figure 1.2. Routes of cytotoxicity action for AgNPs. (1) Adhesion to cell
wall; (2) Cellular internalization; (3) ROS generation; (4) Genotoxicity.
(Source: Lee et al., 2019).
Recently, possible modes of action against microbes of AgNPs is proposed
as shown in Figure 2.2. AgNPs release Ag+ that has an affinity to attach microbial
cell wall. Since Ag+ is uptaked to the cytoplasm, antimicrobial activity is caused
by ROS generated leading to the following cascade of events: (1) inhibition of
DNA synthesis; (2) inhibition of mRNA synthesis; (3) destruction of the cell
membrane and leakage of the cell constituents; (4) inhibition of protein synthesis;
(5) inhibition of cell-wall synthesis; (6) damage to the mitochondria; and (7)
inhibition of the electron transport chain. These effects might, in the long run,
lead to the demise of cells.
2.4. Biosynthesis of silver nanoparticles by Streptomyces
2.4.1. Introduction of the genus Streptomyces
Streptomyces is the largest genus of Actinomycetes and they belong to the
family Streptomycetaceae, the order Streptomycetales (Hasani et al, 2014).
Streptomyces are Gram-positive, GC rich, spore-forming, filamentous with areal
and substrate mycelium, which are found in every environment such as soil,
marine, and plant. Streptomycetes are aerobes and chemoorganotrophic bacteria;
they need an organic carbon source, inorganic nitrogen sources, and mineral salts,
but they do not require vitamins or growth factors (Zotchev et al., 2012). The
majority of Streptomyces spp. are classified as mesophiles and may thrive at
temperatures ranging from 10 to 37 degrees Celsius. pH ranges between 6.5 and
8.0 are optimal for the growth of Streptomycetes and they are predominantly
mesophilic that growth optimally in 28 and 32°C (Hasani et al, 2014).
Streptomyces spp. are known as producer of secondary metabolites. Their
capacity to produce secondary metabolites, such as anti-infective compounds,
serves a crucial ecological function, such as the suppression of rivals during the
transition from mycelial to aerial development (Challis et al., 2003). In fact, 70–

80% of secondary metabolites in current clinical use, including antitumor agents,
11


immunosuppressive, bactericidal, fungicidal, antioxidant, cytotoxic, larvicidal,
and anti-inflammatory have been synthesized by Streptomyces (Vaishnav &
Demain, 2011).
Endophytes are microorganisms that live in plant tissues without causing
harm. Streptomyces spp. isolated from plants have been extensively investigated
owing to their potential to produce new metabolites (Zhao et al., 2011; Kadiri et
al., 2014). Streptomyces spp. was the most frequently discovered genus of
endophytic actinomycetes in medicinal plants, followed by Micromonospora,
Actinopolyspora, Nocardia, Saccharopolyspora, and Streptosporangium (Quach
et al., 2022). Compared to the terrestrial actinomyces, the endophytic
Streptomyces were reported for the higher number of active metabolites with
various applications. Endophytic Streptomyces have been shown to produce a
wide variety of metabolites with antibacterial, antimalarial, antidiabetic,
cytotoxic, and antiviral properties. In recent years, a number of studies have
shown that endophytes isolated from medicinal plants may be used to combat a
wide range of infectious illnesses by inhibiting the development of harmful
microbes (Ranjana and Jadeja, 2017).
2.4.2. Green synthesis of silver nanoparticles by Streptomyces
Streptomyces have the extraordinary capacity to reduce metal ions into
nanoparticles. AgNPs have been synthesized by Streptomyces hygroscopicus
from Pacific shore region, Streptomyces glaucus 71 MD from a soy rhizosphere,
Streptomyces sp. BDUKAS10 from mangrove sediments, Nocardiopsis sp.
MBRC-1 from marine sediments of South Korea, Streptomyces sp. LK3 from
marine sediments, Streptomyces sp. 09 PBT 005 from sugarcane rhizosphere soil
(Tsibakhashvili et al., 2011; Manivasagan et al, 2013). They indicated that
Streptomyces are still being explored from broad range of environments for

AgNPs synthesis. As for bioactivity, extracellular production of AgNPs by
Streptomyces sp LK3 was highly active against Haemaphysalis bispinosa (LC50
16.45 mg/L) and Rhipicephalus microplus (LC50 16.10 mg/L) (Karthik et al.,
2013). M. phaseolina was inhibited by the AgNPs from Streptomyces
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griseoplanus with the zone of inhibition of 13 mm at 1000 μg/mL (Vijayabharathi
et al. 2018).
To be more precise, Streptomyces possess a number of metal resistance
mechanisms that rely primarily on chemical detoxification (Jain et al., 2022).
Streptomyces may use these mechanisms to efficiently detoxify metal ions into
insoluble non-toxic metallic nanoparticles by extracellular precipitation,
biomineralization or reduction, or intracellular bioaccumulation. As shown in
Figure 2.3, the synthetic mechanism of nanoparticles requires a number of
reductive factors, including proteins, cofactors, and nitrate reductases, as well as
external enzymes and cell wall components. Since the cell wall is negatively
charged, it attracts the positively charged metal ions. Positive silver ions (Ag+) (in
the silver nitrate metal salt solution attach to the negatively charged cell wall and
are reduced to Ag0 by the aforementioned reductive species.

Figure 1.3. Mechanism of extracellular and intracellular synthesis of AgNPs by
actinomycetes
(Source: S. Kumari, 2020).

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2.5. Overview of Crinum latifolium L.
Crinum latifolium L. (Amaryllidaceace) is an exotic plant in some regions

of Vietnam and is described as a rare species of the genus Crinum. The family
Amaryllidaceae has roughly 90 genera and 1310 species distributed all throughout
the globe. With a widespread distribution over the tropics, subtropics, and warmto-hot climates, the genus Crinum is an integral component of the family
Amaryllidaceae. In 1737, Linnaeus recognized four species under the genus
Crinum: Crinum latifolium, Crinum asiaticum, Crinum americanum, and Crinum
africanum (Afroz et al., 2018).
The Crinum family has significant cultural, economic, and medical
significance. Vomiting and earaches are both alleviated by a leaf extract. Crushed
bulbs are used to treat abscesses and piles by stimulating pus production when
administered topically. As an added bonus, the roasted bulbs might be helpful in
relieving rheumatic symptoms (Afroz et al., 2018). Since C. latifolium leaf
possesses various biologically active compounds such as alkaloid and phenolic
compounds, it has been reported that extracts of C. latifolium have anti-cancer,
immune stimulating, analgesic, antiviral, antimicrobial, and antifungal effects
(Tam et al., 2019). Plants belonging to the genus C. latifolium may be found
cultivated in a number of the southern and eastern regions of Vietnam. Of note,
endophytic Streptomyces spp. recovered from C. latifolium have not been
described yet.
2.6. Current status of green silver nanoparticles in Vietnam
In Vietnam, there have been some remarkable achievements in the
fabrication of green AgNPs. Nguyen et al. (2016) study biosynthesis of AgNPs
from coconut fiber extraction. It turned out that AgNPs have a size lower than <50
nm and showed strong bactericidal effect against pathogenic microorganisms
Aspergillus niger, Penicillium, and Erwinia. In 2019, Vo Thanh Truc conducted
the project "Biosynthesis of Silver and Gold Nanoparticles Using Aqueous
Extract from Crinum latifolium Leaf and Their Applications Forward
Antibacterial Effect and Wastewater Treatment". The author explored the
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antibacterial activity and catalytic performance for degradation of pollutants of
AgNPs synthesized by leaves of C. latifolium. Surface plasmon resonance peaks
were around 402 and the spherical AgNPs had an average diameter of 20.5 nm
(Vo et al., 2019). In 2021, the extracts of medicinal herbs Piper betle and
Muntingia calabura were used to successfully fabricate AgNPs.
Recently, an outstanding result was published by Tran Do Dat et al. (2021)
revealing phytochemicals from Vietnamese Ganoderma lucidum extracts contribute
mainly to green AgNPs production. AgNPs with an average size of 11.38±5.51 nm
exhibited extraordinary antimicrobial activity against S. aureus, E. coli, P.
aeruginosa, S. enterica, and Candida albicans with IC50 values of 17.97 µg/mL,
17.06 µg/mL, 1.32 µg/mL, 54.69 µg/mL, and 27.78 µg/mL, respectively. Besides,
AgNPs possessed better anticancer activity against the human epidermic carcinoma
cancer cell line with IC50 values of 190.06 ± 3.62 µg/mL when compared to the crude
extract (Dat et al., 2021). However, AgNPs synthesized by Streptomyces spp.,
especially endophytic Streptomyces, have not been reported to date.
III. MATERIALS AND METHODS
3.1. Research subjects and materials
3.1.1. Location and time of the study
- Time: From August 2022 to January 2023
- Location: VAST - Culture Collection of Microorganisms (VCCM),
Institute of Biotechnology, Vietnam Academy of Science and Technology.
3.1.2. Materials
Streptomyces spp. strains isolated from the medicinal plant Crinum
latifolium L.: PCT3, PCT20, BCF8, PCT12, PCS32, PCF14, PCF6, BCF10 were
obtained from the VAST - Culture Collection of Microorganisms (VCCM),
Institute of Biotechnology, Vietnam Academy of Science and Technology.
Six human pathogenic bacteria (Escherichia coli ATCC 11105, Candida
albicans ATCC 10231, Enterococcus faecalis ATCC 29212, Pseudomonas
aeruginosa ATCC 9027, Staphylococcus aureus ATCC 29213 , Salmonella
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