VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY --- --- GRADUATION THESIS TITLE: “BIOSYNTHESIS AND ANTIBACTERIAL CHARACTERIZATION OF SILVER NANOPARTICLES DER
INTRODUCTION
Antimicrobial resistance refers to the capability of bacteria and fungi to thrive despite the presence of antibiotics that would typically eliminate them or inhibit their growth This growing resistance poses a significant risk to advancements in human health, as it diminishes our ability to effectively treat common infectious diseases (Mitchell et al., 2020b) Consequently, there is an urgent necessity to discover new antibiotics and alternative compounds to combat this global threat.
Silver is widely recognized for its diverse applications in food technology, medicine, cosmetics, and pharmaceuticals Recent advancements in nanotechnology have popularized silver nanoparticles (AgNPs) for their antimicrobial, antioxidant, anti-inflammatory, and anti-cancer properties Additionally, AgNPs serve as catalysts, biopharmaceuticals, waste treatment agents, fertilizer additives, and biomedical materials While various methods exist for synthesizing metal nanoparticles, many are costly and produce hazardous waste In contrast, the biological synthesis of AgNPs using bacteria has gained significant attention due to its eco-friendly nature.
The genus Streptomyces, part of the Actinomycetales order, is a vital source of antibiotics Exploring Streptomyces species from unexplored environments offers a promising approach to satisfy the ongoing need for new antibiotics and non-antibiotic compounds (Ma et al., 2021) Endophytic Streptomyces from medicinal plants have attracted significant interest from microbiologists due to the challenges in discovering novel compounds Notably, the synthesis of green silver nanoparticles (AgNPs) has been documented in several Streptomyces species, including Streptomyces rochei and Streptomyces coelicolor.
Streptomyces griseorubens, and Streptomyces viridodiastaticus
Crinum latifolium L is a valuable medicinal herb found in certain regions of
In Vietnam, C latifolium is traditionally utilized for treating allergic disorders and tumor diseases, with its alkaloid compounds demonstrating significant antitumor and immune-stimulating properties Research indicates that leaf extracts of C latifolium can activate T-lymphocytes and inhibit the growth of chemically induced tumors in rats However, the potential of endophytes associated with C latifolium to synthesize silver nanoparticles (AgNPs) has not been explored This study aims to investigate the biosynthesis and antibacterial properties of AgNPs derived from Streptomyces spp.-associated C latifolium.
Streptomyces spp associated with Crinum latifolium L." was investigated
Screening and evaluation of antimicrobial activity of AgNPs synthesized by endophytic Streptomyces spp strains from C latifolium L
- 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
LITERATURE REVIEW
Overview of silver nanoparticles
Nanotechnology is an interdisciplinary field dedicated to the development, manufacture, characterization, and application of nanoscale materials The term "nanoparticles," recognized by the International Organization for Standardization (ISO), refers to particles with sizes in the nanoscale range.
Nanomaterials, classified into organic and inorganic types, range from 1 to 100 nm in at least one dimension (ISO 2015a) Organic nanomaterials include carbon-based particles like liposomes, while inorganic types encompass magnetic nanoparticles, noble metal nanoparticles (such as gold and silver), and semiconductor nanoparticles (like titanium oxide and zinc oxide) Recently, noble metal nanoparticles, particularly silver, have attracted global research interest due to their exceptional optical, electrical, magnetic, catalytic, biological, and mechanical properties (Khan et al., 2019) Their unique size and enhanced properties make nanoparticles highly promising for applications in diagnostics, sensing, energy storage, medicine, and drug delivery (Sim et al., 2021; Niu et al., 2019) Consequently, over 1,800 nanoparticle products have been commercialized, with many more in development, contributing to a global nanotechnology market valued at 2 trillion euros in 2015, highlighting its significant economic impact (Nanomaterials, n.d.-b).
2.1.2 Silver nanoparticles and their characteristics
Silver nanoparticles (AgNPs) are highly praised for their diverse applications in fields like cancer treatment, biosensing, antibiotics, anti-inflammatory therapy, and drug delivery Historically, silver has been employed for antimicrobial purposes since 1000 B.C., with silver salts and their derivatives being widely used as effective antimicrobial agents against over 116 microorganisms At the nanoscale, silver exhibits enhanced properties compared to traditional silver salts, attributed to its small size, large surface area, and potent toxicity to various microorganisms Research by Rahman et al (2019) demonstrated that AgNPs exhibit strong surface plasmon resonance at 425 and 480 nm, surpassing that of other metal nanoparticles such as copper, zinc, titanium, and magnesium, likely due to their superior coupling to interbond transitions.
The remarkable antimicrobial properties of silver nanoparticles (AgNPs) have led to their incorporation in various products, such as burn and ulcer treatments, food packaging to inhibit contamination, household appliances like refrigerators and washing machines, and numerous industrial applications.
2021) Thus, AgNPs are regarded as an important category of nanomaterials
Figure 1.1 The shape of AgNPs observed by transmission electron microscopy
The bioactivities of silver nanoparticles (AgNPs) are significantly influenced by various factors, including particle size, shape, concentration, and charge Specifically, the surface area and energy associated with particle size, the catalytic activity linked to particle shape, the therapeutic index determined by particle concentration, and the oligodynamic quality related to particle charge all play crucial roles in their effectiveness.
In a study by Bruna et al (2021), various concentrations of silver nanoparticles (AgNPs) ranging from 0 to 100 μg/mL and sizes from 1 to 100 nm were evaluated for their antibacterial effects against Escherichia coli Notably, 16-nm AgNPs at a concentration of 75 μg/mL exhibited the most significant antibacterial activity, as reported by Choi et al.
Research indicates that 5-nm silver nanoparticles (AgNPs) exhibit higher antimicrobial activity compared to larger sizes such as 10-nm and 15-nm AgNPs Specifically, for Staphylococcus mutans, 8.4-nm AgNPs are preferred, highlighting the correlation between nanoparticle size and antimicrobial effectiveness This enhanced activity may be attributed to the larger surface areas of smaller AgNPs, which facilitate the release of more silver ions (Ag⁺) However, the exact mechanisms underlying the antimicrobial effects of silver nanoparticles remain unclear.
The adverse effects of silver nanoparticles (AgNPs) on humans and other living beings have been well-documented, with significant amounts of silver released into the environment from industrial waste The toxicity of silver is primarily due to free silver ions in water, which can lead to serious health issues such as argyria, liver and kidney damage, and irritations in various body systems Research has shown that AgNPs can negatively affect the proliferation and cytokine expression of peripheral blood mononuclear cells While AgNPs exhibit toxicity to the liver in animal studies, they are generally considered safe for human cells at certain doses and are recognized for their antibacterial properties Consequently, bio-AgNPs are gaining attention among agronomists for their potential use in crop protection due to their low risk to human health and the environment.
In recent years, there has been a growing interest among professionals in biology, agriculture, and medicine regarding the production of nano-scaled particles This increased focus highlights the need for more in vivo studies to evaluate the toxicity of nanosilver before definitive conclusions can be drawn about its safety.
Silver nanoparticles (AgNPs) are highly regarded nanomaterials with significant applications in agriculture They demonstrate effective antibacterial properties against plant diseases caused by phytopathogens For instance, AgNPs have been shown to decrease disease severity in perennial ryegrass (Lolium perenne) (Jo et al.).
In recent studies, the application of silver nanoparticles (AgNPs) has shown significant benefits for various crops, including wheat (T aestivum, var UP2338), cowpea (Vigna sinensis, var Pusa Komal), and brassica (Brassica juncea, var Pusa Jai Kisan) Notably, AgNPs significantly enhanced the growth and root nodulation of cowpea (Pallavi et al., 2016) Furthermore, AgNPs exhibited superior antifungal activity against plant pathogens such as Alternaria alternata, Penicillium digitatum, and Alternaria citri, outperforming synthetic fungicides like difenoconazole and iprodione under similar conditions (Abdelmalek et al., 2016) Additionally, AgNPs were effective in reducing the development of various fungal diseases caused by pathogens including Macrophomina phaseolina, Rhizoctonia solani, Curvularia lunata, Sclerotium cepivorum, Bipolaris sorokiniana, and 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
Silver nanoparticles (AgNPs) are widely utilized in bioremediation due to their ability to inhibit pathogen growth in water when injected into zeolites Research indicates that AgNPs produced from actinomycetes, particularly Streptomyces rochei HMM13, effectively reduce bacterial biofilm formation by decreasing the bacterial cell count within the biofilm Additionally, AgNPs have demonstrated significant potential as antibacterial agents for water treatment.
Silver nanoparticles (AgNPs) demonstrate significant potential in biomedical applications, particularly in wound healing and skin cell regeneration due to their anti-inflammatory properties This makes them ideal for use in wound-healing and infection-preventing dressings Additionally, AgNPs significantly enhance the effectiveness of face masks; a study showed that a face mask coated with AgNPs effectively killed E coli and S aureus within 24 hours Furthermore, synthesized AgNPs exhibit strong antibacterial activity against pathogenic microbes linked to urinary tract infections, such as E coli and Klebsiella pneumoniae.
Pseudomonas aeruginosa and Candida albicans are significant pathogens contributing to the growing issue of antimicrobial-resistant infections, which are increasing at an alarming rate globally The rise of multidrug-resistant bacteria is largely attributed to the overuse of antibiotics in treating infectious diseases In this context, silver nanoparticles (AgNPs) emerge as a promising solution for preventing infections, disinfecting medical equipment, and combating these resistant pathogens.
Synthesis of silver nanoparticles
2.2.2 Green synthesis of silver nanoparticles
The traditional physical methods for nanoparticle synthesis are costly, while chemical methods generate hazardous byproducts that pose environmental and health risks Therefore, there is a growing need for environmentally friendly and economically viable alternatives, leading to increased interest in "green synthesis pathways." The primary goal of green nanotechnology is to promote a cleaner environment by producing nanoparticles through eco-friendly techniques that do not harm human health or the ecosystem (Kumar et al., 2019a) Biological methods utilize microbes, algae, fungi, and plants to create high-yield, cost-effective, and sustainable nanoparticles (Narayanan et al., 2010) Additionally, silver nanoparticles (AgNPs) benefit from enhanced stability and longer shelf life due to natural capping, making this one-step synthesis approach not only economical but also suitable for easy scaling and efficient downstream processing (Kumar et al., 2019a).
The green approach to nanoparticle production involves using extracts from higher plants, fungi, and bacteria Notably, only a few bacteria can thrive in high concentrations of silver and synthesize silver nanoparticles (AgNPs) The first identified bacterium capable of producing AgNPs is Pseudomonas stutzeri AG259, which was isolated from a silver mine Since then, several other bacteria, including Enterobacteria, Bacillus licheniformis, and Streptomyces, have also been found to synthesize AgNPs.
(Gnanajobitha et al., 2013) Besides, AgNPs can be synthesized by fungi (Fusarium oxysporum, Aspergillus fumigatus) (Iravani et al., 2014; Ahmad et al.,
2003), and fruit extracts (Carica papaya, Vitis vinifera) (Bhainsa et al., 2006; Karthiga et al., 2018)
AgNPs can be synthesized both extracellularly and intracellularly by bacteria, with the extracellular production relying on nitrate reductase, which converts metal ions into nanoparticles This enzyme typically catalyzes the reduction of nitrate to nitrite, as demonstrated by the use of B licheniformis in research (Sarker et al., 2007).
The production and stability of nanoparticles in Stenotrophomonas maltophilia through charge capping has been documented (Nayaka et al., 2020) This process involves an electron shuttle enzymatic metal reduction facilitated by the nicotinamide adenine dinucleotide phosphate-dependent reductase enzyme Additionally, the biosynthesis of silver nanoparticles (AgNPs) in Pseudomonas species requires nitrate reductase and the presence of nicotinamide adenine dinucleotide (NAD), which is essential for converting Ag\(^+\) into nanoparticles.
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).
Modes of action against microorganisms of silver nanoparticles
The antibacterial properties of silver nanoparticles (AgNPs) have been extensively studied, demonstrating effectiveness against both Gram-positive and Gram-negative bacteria Research conducted by Shekhar Agnihotri et al evaluated the resistance of these bacteria to the bactericidal and bacteriostatic effects of nanoparticles ranging in size from 5 to 100 nm.
In a study examining the antimicrobial effects of silver nanoparticles (AgNPs), the minimum inhibitory concentration (MIC) for three strains of E coli, Bacillus subtilis, and S aureus ranged from 20 to 110, 60 to 160, 30 to 120, and 70 to 200 μg/mL, respectively, with the lower values corresponding to 5-nm AgNPs and the higher values to 100-nm AgNPs These findings indicate that AgNPs consistently inhibit bacterial growth Notably, multidrug-resistant bacteria, including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus, have also shown susceptibility to AgNPs.
Derazkola et al have introduced an efficient and cost-effective method for synthesizing silver nanoparticles using Crataegus microphylla fruit extract Their findings indicate that the antibacterial properties of these nanoparticles are partially attributed to their chemical surface Notably, the minimum inhibitory concentrations (MICs) of the synthesized AgNPs against different bacterial strains were determined to be 14 μg/mL.
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
Silver nanoparticles (AgNPs) exhibit notable antifungal properties, particularly against Fusarium graminearum, the pathogen responsible for head blight disease in wheat crops Research by Mansoor et al (2021) indicates that AgNPs synthesized from garlic plants (Allium sativum) effectively inhibit mycelium growth, spore germination, and mycotoxin production.
Bipolaris sorokiniana, the pathogen responsible for spot blotch disease in wheat, exhibits significant sensitivity to green silver nanoparticles (AgNPs) This suggests that AgNPs may interact with and damage fungal cells at the molecular level However, the specific mechanisms of action and toxicity of AgNPs remain largely unexplored.
The antimicrobial mechanisms of silver nanoparticles (AgNPs) have been widely researched, yet they remain inadequately understood It is believed that AgNPs initially attach to the cell membrane and wall, with the positively charged Ag\(^+\) ions being crucial for their antibacterial properties These ions, through electrostatic attractions and a strong affinity for sulfur proteins, bind to the cytoplasm and cell wall, significantly increasing permeability and leading to the rupture of bacterial casings.
Increased membrane permeability results in the loss of essential cellular components, including proteins, carbohydrates, and adenosine triphosphates, which are crucial for energy storage (Chauhan et al., 2013; Li et al., 2013) Furthermore, silver nanoparticles (AgNPs) cause significant damage to intracellular macromolecules, affecting proteins, lipids, and DNA (Morones et al., 2005; Rai et al., 2012).
Cellular homeostasis, crucial for cell survival, is disrupted by reactive oxygen species (ROS), which are continuously generated during aerobic metabolism (Jain et al., 2021) While low to moderate levels of ROS act as signaling molecules that promote cell growth and stress defense, excessive ROS lead to oxidative stress and damage to vital macromolecules like DNA, proteins, and lipids Notably, silver nanoparticles (AgNPs) contribute to ROS production, impairing cell respiration and development (Quinteros et al., 2016) AgNPs primarily inhibit thiol group-containing enzymes, such as NADH dehydrogenase, which is linked to ROS generation This inhibition triggers free radical production and oxidative stress cycles, resulting in DNA damage, lipid peroxidation, apoptotic-like reactions, and depletion of antioxidant enzymes (Lee et al., 2014; Korshed et al., 2016).
Figure 1.2 Routes of cytotoxicity action for AgNPs (1) Adhesion to cell wall; (2) Cellular internalization; (3) ROS generation; (4) Genotoxicity
Recent studies suggest that silver nanoparticles (AgNPs) exert their antimicrobial effects through several mechanisms As illustrated in Figure 2.2, AgNPs release silver ions (Ag⁺), which have a strong affinity for microbial cell walls Once inside the cytoplasm, Ag⁺ induces the generation of reactive oxygen species (ROS), triggering a series of detrimental events: (1) inhibition of DNA synthesis, (2) inhibition of mRNA synthesis, (3) destruction of the cell membrane leading to leakage of cellular contents, and (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.
Biosynthesis of silver nanoparticles by Streptomyces
2.4.1 Introduction of the genus Streptomyces
Streptomyces, the largest genus of Actinomycetes within the family Streptomycetaceae and order Streptomycetales, are Gram-positive, GC-rich, spore-forming, filamentous bacteria found in diverse environments such as soil, marine, and plant habitats These aerobic, chemoorganotrophic bacteria require an organic carbon source, inorganic nitrogen, and mineral salts for growth, but do not need vitamins or growth factors Most Streptomyces species are mesophiles, thriving at temperatures between 10 to 37 degrees Celsius, with optimal growth occurring at pH levels between 6.5 and 8.0, particularly at 28 to 32°C.
Streptomyces spp are renowned for their ability to produce secondary metabolites, which play a vital ecological role by suppressing competitors during the transition from mycelial to aerial development Notably, 70–80% of secondary metabolites currently used in clinical applications, including antitumor agents, immunosuppressants, and various antimicrobial compounds, are synthesized by these bacteria.
Endophytes are beneficial microorganisms residing in plant tissues without causing harm Among them, Streptomyces spp have been widely studied for their ability to produce novel metabolites (Zhao et al., 2011; Kadiri et al., 2014) This genus is the most commonly found among endophytic actinomycetes in medicinal plants, followed by Micromonospora, Actinopolyspora, Nocardia, Saccharopolyspora, and Streptosporangium (Quach et al., 2022) In comparison to terrestrial actinomycetes, endophytic varieties offer unique advantages and potential applications.
Endophytic Streptomyces are known for their production of diverse active metabolites with significant applications, including antibacterial, antimalarial, antidiabetic, cytotoxic, and antiviral properties Recent studies indicate that these endophytes, isolated from medicinal plants, can effectively combat various infectious diseases by inhibiting the growth 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 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 can detoxify metal ions into non-toxic metallic nanoparticles through mechanisms such as extracellular precipitation, biomineralization, reduction, or intracellular bioaccumulation The synthesis of these nanoparticles involves various reductive factors, including proteins, cofactors, nitrate reductases, external enzymes, and cell wall components The negatively charged cell wall of Streptomyces attracts positively charged metal ions, such as silver ions (Ag\(^+\)), which then attach to the cell wall and are reduced to metallic silver (Ag\(^0\)) by the reductive species present.
Figure 1.3 Mechanism of extracellular and intracellular synthesis of AgNPs by actinomycetes (Source: S Kumari, 2020).
Overview of Crinum latifolium L
Crinum latifolium L., belonging to the Amaryllidaceae family, is an exotic and rare species found in certain regions of Vietnam The Amaryllidaceae family comprises approximately 90 genera and 1,310 species globally, thriving in tropical, subtropical, and warm climates The genus Crinum plays a vital role within this diverse 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 holds considerable cultural, economic, and medical importance, with leaf extracts providing relief from vomiting and earaches Crushed bulbs are applied topically to treat abscesses and piles by promoting pus production, while roasted bulbs may alleviate rheumatic symptoms (Afroz et al., 2018) The leaves of C latifolium contain various biologically active compounds, including alkaloids and phenolic compounds, which have been shown to possess anti-cancer, immune-stimulating, analgesic, antiviral, antimicrobial, and antifungal properties (Tam et al., 2019) C latifolium is cultivated in several southern and eastern regions of Vietnam, yet the endophytic Streptomyces spp associated with this plant remain uncharacterized.
Current status of green silver nanoparticles in Vietnam
Vietnam has made significant strides in the production of green silver nanoparticles (AgNPs) A study by Nguyen et al (2016) demonstrated the successful biosynthesis of AgNPs using coconut fiber extraction, resulting in nanoparticles smaller than 50 nm These AgNPs exhibited a potent 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
The article discusses the antibacterial effects and wastewater treatment capabilities of silver nanoparticles (AgNPs) synthesized from the leaves of C latifolium, highlighting their catalytic performance in pollutant degradation The study identified surface plasmon resonance peaks at approximately 402 nm, with spherical AgNPs averaging 20.5 nm in diameter (Vo et al., 2019) Additionally, in 2021, extracts from the medicinal herbs Piper betle and Muntingia calabura were utilized to effectively produce AgNPs.
A recent study by Tran Do Dat et al (2021) highlighted that phytochemicals from Vietnamese Ganoderma lucidum extracts play a significant role in the production of green silver nanoparticles (AgNPs) These AgNPs, averaging 11.38±5.51 nm in size, demonstrated remarkable antimicrobial properties against various pathogens, including 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 Additionally, AgNPs exhibited superior anticancer activity against the human epidermic carcinoma cell line, showing an IC50 value of 190.06 ± 3.62 µg/mL, outperforming the crude extract Notably, the synthesis of AgNPs using Streptomyces spp., particularly endophytic Streptomyces, remains unexplored.
MATERIALS AND METHODS
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
Streptomyces spp strains, including PCT3, PCT20, BCF8, PCT12, PCS32, PCF14, PCF6, and BCF10, were isolated from the medicinal plant Crinum latifolium L These strains are part of the VAST - Culture Collection of Microorganisms (VCCM) at the Institute of Biotechnology, Vietnam Academy of Science and Technology.
The VCCM Institute of Biotechnology provided six human pathogenic bacteria: Escherichia coli ATCC 11105, Candida albicans ATCC 10231, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 29213, and Salmonella enterica ATCC 14028.
Vietnam Academy of Science and Technology
The primary equipment utilized for experiments included a microcentrifuge from Eppendorf (Germany), an incubator shaker from Parmer (Germany), an autoclave from Clea (Japan), a dryer from Steelco (Spain), a spectrophotometer from Hitachi (Japan), a microscope from Carson (USA), microscope slides from Vietnam, an incubator from Esco (Singapore), a laminar airflow cabinet from Esco (Australia), a PCR thermocycler from BIO-RAD (USA), a -86°C freezer from Thermo (USA), an analytical balance from Presisa (Switzerland), and a cork borer from Eisco (USA).
The primary chemicals utilized in the experiments include analytical grade silver nitrate (AgNO₃) sourced from SBC, China, along with agarose and agar from Merck, Germany, and Vietnam, respectively Additional reagents include NaOH, HCl, ethanol, phenol, and isoamyl alcohol, all from Xilong, China Other essential components are glycerol from Xilong, yeast extract and peptone from Himedia, India, as well as fructose and sorbitol from India Carboxymethyl cellulose (CMC), skim milk, and ethylene diamine tetra-acetic acid (EDTA) are also obtained from Himedia, India, while the GeneJET Gel Extraction Kit and Taq DNA Polymerase are provided by Thermo, Singapore.
The following are a list of media used during the experimental courses:
- Meat-and-peptone agar (MPA) medium (g/L): 5 g Meat extract; 5 g NaCl;
10 g peptone; 20 g agar; 1000 mL of sterile water; pH 7
The YIM 38 medium formulation consists of 10 g of malt extract, 4 g of yeast extract, and 4 g of glucose, supplemented with a vitamin mixture that includes 0.5 mg each of thiamine-HCl, riboflavin, niacin, pyridoxine-HCl, inositol, calcium pantothenate, and p-aminobenzoic acid, along with 0.25 mg of biotin Additionally, the medium contains 20 g of agar and is prepared with 1000 mL of sterile water, adjusted to a pH of 7.2 (Hayakawa, 1987).
- ISP9 medium (g/L): 2.64g (NH4)2SO4; 2.38g KH2PO4; 1.0ml of solution B; 5.65g K2HPO4.3H2O; 1g MgSO4.7H2O ; 20 g agar; pH 7.0
- Luria Bertani medium (g/L): 10 g tripton, 10 g NaCl and 5 g yeast extract, pH 7.0.
Methods
3.2.1 Cultivation of Streptomyces spp strains associated with Crinum latifolium L
8 Streptomyces strains that associated with C.latifolium L.: PCT3, PCT20,
BCF8, PCT12, PCS32, PCF14, PCF6, and BCF10 were preserved in a 20% glycerol solution and cultivated on YIM 38 agar at 28°C for 5 days Purification involved repeated transfers of actively growing bacterial material until uniform colonies were obtained The purity of each strain was confirmed using a light microscope, and these pure cultures were subsequently utilized for silver nanoparticle synthesis and taxonomic studies.
Preparation of cell-free supernatant
All pure strains were incubated onto an Erlenmeyer flask 250 mL containing
Under aseptic conditions, 100 mL of YIM38 broth was incubated for four days at 28°C with shaking at 130 rpm The cultures were then centrifuged at 10,000 g for 15 minutes to collect the mycelia, which were washed with distilled water to eliminate residual culture medium The cells were resuspended in 100 mL of sterile water and incubated for an additional 48 hours at 28°C, followed by centrifugation at 5000 rpm for 10 minutes The resulting cell-free supernatant was collected for the biosynthesis of nanosilver.
Green synthesis of silver nanoparticles
The cell-free supernatant was combined with a 1 mM AgNO3 solution and incubated at 30 °C for 120 hours, covered with aluminum foil to block light A control sample of distilled water with 1 mM AgNO3 was also prepared The synthesis of silver nanoparticles was indicated by a color change in the solution from light yellow to dark brown by the end of the incubation period, with all experiments conducted in triplicates.
Following the green synthesis of silver nanoparticles (AgNPs), the samples were centrifuged at 15,000 rpm for 20 minutes at 4°C, and the supernatant was discarded to isolate the nanoparticles The AgNPs powder was then washed twice with 90% ethanol to remove any potential contaminants The precipitated AgNPs were air-dried and subsequently evaluated for their antimicrobial properties.
3.2.3 In vitro antimicrobial activity of synthesized AgNPs
The agar-well diffusion method was carried out to evaluate the antibacterial activity of the AgNPs (Heatley,1944) Six bacterial pathogen including E coli
In this study, bacterial strains including C albicans ATCC 10231, E faecalis ATCC 29212, P aeruginosa ATCC 9027, S aureus ATCC 29213, and S enterica ATCC 14028 were cultured overnight in MPA medium at 4°C using a rotary shaker MPA agar plates were prepared, and the freshly cultured pathogens were evenly swabbed onto the plates with a sterile cell spreader Six-millimeter wells were created using a sterile cork borer and filled with 100 µL of bioAgNPs, while AgNO3 solution and cell-free supernatant served as controls The plates were incubated at 4°C for 2 hours to allow nanoparticle diffusion, followed by a 24-hour incubation at 37°C The antimicrobial activity of the nanoparticles was assessed by measuring the zone of inhibition, with the experiment being repeated three times for accuracy.
The broth micro-dilution technique, as recommended by the Clinical Laboratory Standards Institute (CLSI), was utilized to determine the minimum inhibitory concentration (MIC) of silver nanoparticles (AgNPs), defined as the lowest concentration preventing observable bacterial or fungal growth The assay was conducted in triplicate using 96-well microtiter plates with LB broth for microbial growth, maintaining a final concentration of 5 × 10² CFU/mL for bacteria or yeast in each well Various concentrations of AgNPs were prepared, including 250 µg/mL, 125 µg/mL, 62.5 µg/mL, 31.25 µg/mL, 15.63 µg/mL, 7.8 µg/mL, 3.9 µg/mL, and 1.9 µg/mL, with control samples lacking AgNPs The microbial inoculum density was assessed at an OD600 of 0.05, and the inoculated plates were incubated at 37 °C for 24 hours, after which MIC values were visually estimated.
3.2.4 Morphological, physiological, and biochemical identification of potent strain 3.2.4.1 Morphological characteristics
Strain PCT3 was cultured on ISP2 agar at 28°C to examine its aerial mycelium, substrate mycelium, and pigment After 5 days of incubation, the colony morphology, including size, shape, margin, texture, and form, was carefully observed and photographed.
The strain PCT3 demonstrated the ability to utilize various carbon sources, as evaluated by Benedict et al (1955) Cultured on ISP-9 medium with 1% carbon sources such as glucose, fructose, inositol, sorbitol, mannitol, and sucrose, glucose acted as the positive control, while ISP-9 without carbon served as the negative control The assessment of carbon source assimilation was based on the viability and growth of strain PCT3 after 5-7 days of culture.
The enzymatic activity of strain PCT3 was assessed using a Petri plate assay, as described by Tang-um et al (2012) ISP-9 agar with 1% (w/v) skimmed milk was prepared and punctured with a sterile cork borer A 100 µL aliquot of cell-free supernatant from PCT3 was added and incubated at 4 °C for 2 hours to allow diffusion, followed by incubation at 28 °C for 20 hours Proteolytic activity was indicated by clear zones around the wells after adding 10% (w/v) TCA Similarly, to evaluate amylase activity, 100 µL of cell-free supernatant was added to ISP-9 agar with 1% (w/v) starch, and 1% Lugol solution was applied, resulting in clear zones against a dark blue background For cellulase activity, ISP-9 agar containing 1% (w/v) CMC was used, and substrate hydrolysis was detected with 1% Lugol solution, which also produced clear zones surrounded by a dark blue backdrop.
The growth and development of the strain PCT3 were influenced by varying temperature, pH, and NaCl concentration Culturing the strain on ISP-2 medium at temperatures of 22°C, 30°C, 37°C, and 45°C revealed significant effects on its growth Additionally, the pH levels tested included 3 and 4, further impacting the strain's development.
5, 6, 7, 8, 9, and 10), and NaCl (0, 2, 4, 6, 8, and 10%) Three independent experiments were performed
3.2.5 Molecular identification based on the 16S rRNA analysis
Total DNA extraction of bacteria was performed using the Khosravi Babadi et al (2020) method as follows:
- Take a single colony into 5 mL of YIM38 broth and incubated for 48 h at 37°C with sharking at 200 rpm
- The culture was centrifuged at 5000 rpm for 2 min Cells were dissolved in
600 àL TE buffer (pH 8.0); vortex 1 min
- Supplement with 28 àL lysozyme (50 mg/mL); vortex and followed by incubation at 37°C for 1 h
- Add 40 àL of 10% sodium dodecyl sulfate (SDS) and mix well Add 5–7 àL protease K, mix well, and incubate at 56°C for 1 h
- Subsequently, address 90 àL of 5M NaCl into the mixture, blend up, and add 90 àL of CTAB (cetyltrimethylammonium bromide), followed by incubation at 65ºC for 15 min
- Add 700 - 800 àL of Phenol: Chloroform: Isoamyl alcohol (25:24:1) mixture, blend it Centrifuge at 12,000 rpm for 10 min
- The upper aqueous phase was transferred to a clean Eppendorf tube After that, a mixture of chloroform: isoamyl alcohol (24:1) in an equal volume (ratio of 1:1) was added to it
Transfer V1 (mL) of the top aqueous phase into a new Eppendorf tube, then add V2 (mL) of isopropyl alcohol to the mixture, where V2 equals 0.6 times V1 Mix thoroughly and incubate the solution at -20°C for 1 hour Finally, centrifuge the mixture at 12,000 rpm for 15 minutes.
- The DNA was washed with 700 àL of 70% cold ethanol and air-dried Centrifuge at 12000 rpm for 15 min
- The pellet of crude DNA extracts was re-suspended in 30 – 35 μL distilled water
- The resulting DNA extract was electrophorized in 1X TAE buffer (0.04 M Tris-acetate, 0.002 M EDTA, pH 8.0) with 1.0% agarose for integrity examination
- The DNA sample was stored at -20 °C for further experiments
3.2.5.2 PCR amplification and analysis of 16S rRNA sequence
The bacterial 16S rRNA gene was amplified by using forward primer 27F (5’ AGAGTTTGATCCTGGCTCAG 3’) and reverse primer 1492R (5’ GGTTACCTTGTTACGACTT 3’) (Valentin et al., 2021) The reaction mixture
The PCR amplification of the 16S rRNA gene was performed using a total volume of 50 μl, which included 5 μl of buffer (composed of 0.2 M Tris-HCl at pH 8.3, 0.25 M KCl, and 20 mM MgCl2), 4 μl of a dNTP mix (at 100 μM per type), 1 μl of each primer, 0.5 μl of 1U Taq DNA polymerase, 1 μl of DNA template, and distilled water to complete the volume The amplification conditions involved an initial denaturation at 95 °C for 10 minutes, followed by 30 cycles of denaturation at 95 °C for 120 seconds, annealing at 58 °C for 60 seconds, and extension at 72 °C for 60 seconds, concluding with a final extension at 72 °C for 10 minutes.
The amplified product was stored at 4 °C indefinitely after being heated to 72 °C Its purity was assessed using 1% agarose gel electrophoresis, and the resulting PCR product was purified before being sent to First Base Laboratories Sdn Bhd in Seri Kembangan, Selangor, Malaysia for sequencing.
The strain PCT3 sequences were imported into BioEdit software (Version 7.2) for error checking and correction The 16S rRNA sequence was then submitted to GenBank (NCBI) for comparison with published 16S rRNA gene sequences of closely related species Phylogenetic neighbor identification and pairwise 16S rRNA gene sequence similarity calculations were performed using BLAST analysis.
The sequences were aligned using CLUSTALW (Thompson et al., 1997) and a phylogenetic tree was constructed from the aligned sequences utilizing the neighbor joining method with Kimura-2-parameter distances in MEGA 7 software (Labeda et al., 2016).
RESULTS AND DISCUSSION
Antimicrobial screening of silver nanoparticles synthesized by endophytic
To synthesize silver nanoparticles (AgNPs), free-biomass filtrates from eight Streptomyces species associated with Crinum latifolium were added to a 1 mM AgNO3 solution The formation of AgNPs was indicated by a color change in the solution from clear to reddish brown, as illustrated in Figure 4.1.
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
After a 20-minute incubation of AgNO3 with free-biomass filtrates, a significant color change was observed in 3 out of 8 samples, while controls with AgNO3 or cell-free supernatant remained colorless The reddish-brown color formed during incubation persisted for up to 72 hours, indicating the successful conversion of ionic silver (Ag\(^+\)) to metallic silver (Ag\(^0\)) This finding aligns with previous research demonstrating that endophytic Streptomyces laurentii from Achillea fragrantissima can act as a biocatalyst for the green synthesis of AgNPs, evidenced by a color change from colorless to yellowish-brown Notably, the intensity of the color was directly related to the concentration of reduced silver ions (Eid et al 2020) Similarly, exposure of AgNO3 to an extracellular extract of Streptomyces sp SSUT88A resulted in a color shift from light yellow to brown, confirming the reduction of Ag\(^+\) to Ag\(^0\) (Rosyidah et al 2022).
4.1.2 Antimicrobial activity of silver nanoparticles synthesized by Streptomyces sp
Antimicrobial effects of crude AgNPs solution were investigated against 6 pathogens through agar-well diffusion assay Given that free-biomass filtrates of
Streptomyces spp and silver nitrate (AgNO3) exhibit natural antimicrobial properties that can effectively inhibit microbial pathogens To assess their efficacy, free-biomass filtrates of Streptomyces spp and AgNO3 were utilized as controls The antimicrobial activity of crude silver nanoparticles (AgNPs) was demonstrated through the observed zones of inhibition against various microbial strains, as illustrated in Figure 4.2.
Figure 1.5 Antibacterial activity of crude AgNPs against Candida albicans (A) and Enterococcus faecalis (B)
The biosynthesized silver nanoparticles (AgNPs) from eight Streptomyces spp strains exhibited antimicrobial activity against various pathogens Among these, the extracellular solutions of seven strains—PCT20, BCF8, PCT3, PCF14, PCT12, BCF10, and PCS32—showed no antimicrobial effects, while only the PCF6 strain demonstrated inhibition against E faecalis.
ATCC 29212, C albicans ATCC 10231, S epidermidis ATCC 12228, and Pseudomonas aeruginosa ATCC 9027 In addition, 1 mM AgNO3 inhibited all tested pathogens (Figure 4.2)
Table 1 Antibacterial activity of crude AgNPs synthesized by 8 Streptomyces strains
Diameter of inhibition zone (mm)
The PCT3 mixture demonstrated the most significant inhibitory effects against six pathogens, with inhibition zones ranging from 4.3 ± 2.0 mm to 7.3 ± 1 mm, particularly showing strong inhibition of yeast C albicans ATCC 10231 Additionally, the BCF8 mixture exhibited activity against E faecalis ATCC 29212 (5.0 ± 1.1 mm), C albicans ATCC 10231 (3.7 ± 2.9 mm), E coli ATCC 11105 (3.0 ± 0.2 mm), S epidermidis ATCC 12228 (3.3 ± 0.6 mm), S enterica ATCC 14028 (3.3 ± 1.2 mm), and P aeruginosa at lower levels.
ATCC 9027 (1.3 ± 0.6 mm) The lowest activity against tested pathogens was recorded in PCT20 mixture
The study revealed that 3 out of 8 starved Streptomyces cells could synthesize antimicrobial silver nanoparticles (AgNPs), evidenced by a color change from clear to reddish brown Additionally, prior research demonstrated that AgNPs produced from marine Streptomyces rocheii MHM13 displayed significant antibacterial activity.
B subtilis, E coli, and K pneumoniae (Abd-Elnaby et al., 2016) Similar findings were found in Streptomyces sp isolated from acid forest soil that produced AgNPs with antibacterial activity against K pneumoniae, P mirabilis, S infantis, P aeruginosa, and B subtilis (Składanowski et al., 2017) As predicted, the antimicrobial activity of AgNPs was around 3-fold higher than that of 1 mM AgNO3 used in this study, highlighting the bioactivity of AgNPs The inhibitory action of AgNPs against pathogens may be due to their smaller size and larger surface area, which improve their binding ability to microbial membranes.
Identification of potent Streptomyces spp PCT3
4.2.1 Morphological characteristics of strain PCT3
Strain PCT3 was cultivated on ISP2 agar at 30°C for 3 days to examine its morphological characteristics After 2 days, PCT3 demonstrated good growth on ISP2 agar, forming medium-sized colonies with irregular margins, filamentous structures, and a rough texture The aerial mycelium appeared white to light gray, while the substrate mycelium was pale yellow No diffusible pigment was observed on ISP2 agar, and the isolate displayed typical morphological features.
Streptomyces sp Streptomyces genera is known for its ability to produce the aerial spore mass, vegetative, and aerial mycelia They are distinct characteristics to identify Streptomyces through visual observation
Figure 1.6 The colonial morphology of strain PCT3 observed after 2 days of incubation, at 30°C
4.2.2 Physiological and biochemical characterization of strain PCT3
The growth characteristics of strain PCT3 were assessed under varying incubation temperatures, pH levels, and salt concentrations Strain PCT3 demonstrated robust growth at pH levels between 3 and 10, with optimal growth at pH 8 The isolate thrived within a temperature range of 20 to 45 °C, with 37 °C identified as the ideal temperature Additionally, PCT3 was capable of growing in NaCl concentrations up to 10%, with the best growth observed at 4% NaCl These physiological traits of PCT3 align with the native environment of C latifolium, corroborating findings by Ameerah et al., which reported that actinomycetes from mangrove environments also thrived in up to 10% NaCl (optimal at 5%), a pH range of 5 to 9 (optimal at pH 8.0), and a temperature range of 25–40 °C (optimal at 30 °C).
Table 2.Biochemical and physiological characteristics of Streptomyces sp PCT3
*Note: -: Negative reaction, +: Positive reaction
Figure 1.7 Enzymatic activities of strain PCT3 shown on agar plates containing skim milk (A), starch (B), and CMC (C)
Streptomyces sp PCT3 is capable of producing protease and amylase, but it does not produce CMCase This strain utilizes glucose, fructose, and sucrose as carbon sources for optimal growth, while mannitol and inositol are not utilized It is estimated that 78% of Streptomyces species can produce more than two extracellular enzymes, which may serve as mechanisms for survival in competitive environments.
4.2.3 Molecular identification of strain PCT3 using 16S rRNA analysis
Genomic DNA of PCT3 was extracted to identify the species level, and agarose gel electrophoresis confirmed a high yield suitable for PCR amplification The 16S rRNA gene was successfully amplified using the 27F and 1492R primers, resulting in a clear band of approximately 1500 bp, indicating specificity and compliance with DNA purification standards The amplified 16S rRNA gene was then gel purified and sent to First BASE Laboratories Sdn Bhd in Malaysia for sequencing via Sanger technology Sequence analysis, conducted with both forward and reverse primers, produced a consensus sequence with 300–500 overlapping base pairs.
The 16S rRNA sequence analysis revealed that strain PCT3 exhibits a high similarity of 99% to both Streptomyces albus NRRL B-1811T and Streptomyces albus NRRC 13015T, suggesting that PCT3 may be classified within the Streptomyces genus Phylogenetic analysis further demonstrated that the 16S rRNA gene sequences of PCT3 align differentially with various species, forming a distinct clade alongside five type strains, including S albus NRRL B-1811T, S albus NRRC 13015T, and S albus NBRC.
13014 T , S albus NBRC 15415 T , S albus NBRC 13078 T Therefore, strain PCT3 was identified as Streptomyces albus PCT3
Figure 1.9 Phylogenetic tree based on 16S rRNA gene sequences exhibiting the relationship between Streptomyces spp PCT3 and other closely related type strains.
Figure 1.8 Agarose gel electrophoresis of the 16S rRNA gene amplicons of
Streptomyces sp PCT3 Land M: DNA marker (250 – 10,000 bp)
S albus was discovered for the first time by Nagatsu and co-worker in 1962
The S albus strain is readily isolated from environmental sources like self-heated compost and soil It effectively degrades oil cake and straw waste by producing xylanase, which plays a crucial role in biogas production.
In 2012, S albus was identified in grass and shown to be effective against the cotton aphid Aphis gossypii This strain produces various insecticidal secondary metabolites, including flavensomycin, antimycin A, piericidins, macrotetralides, and prasinons Additionally, S albus serves as a preferred host for the heterologous production of diverse secondary metabolites from other bacteria and for the expression of metagenomic DNA clones It is also capable of producing commercial antibiotics such as steffimycin, fredericamycin, isomigrastatin, napyradiomycin, cyclooctatin, thiocoraline, and moenomycin through recombinant methods Notably, the green synthesis of silver nanoparticles (AgNPs) by S albus has not been previously reported, underscoring the significance of this research.
Antimicrobial characterization of PCT3 silver nanoparticles
Silver nanoparticles (AgNPs) synthesized from the free-biomass filtrate of S albus PCT3 were purified and evaluated for their antimicrobial properties The minimum inhibitory concentration (MIC) results, detailed in Table 4.3, indicate that these AgNPs effectively inhibited 2 Gram-positive bacteria, 3 Gram-negative bacteria, and yeast Notably, the Gram-positive bacterium S aureus ATCC 29213 exhibited an MIC value of 31.2 μg/mL, while E faecalis ATCC 29212 demonstrated a lower MIC of 15.6 μg/mL.
S enterica ATCC 14028 exhibited a minimum inhibitory concentration (MIC) of 15.6 μg/mL, while E coli ATCC 11105 and P aeruginosa ATCC 9027 showed lower MIC values of 3.9 μg/mL, indicating that Gram-negative bacteria are more susceptible to silver nanoparticles (AgNPs) compared to Gram-positive bacteria Additionally, the MIC value of AgNPs for C albicans ATCC was also assessed.
10231was determined to be 1.9 àg/mL, which was highest concentration against microbe recorded (Table 4.3)
Table 3.Minimum Inhibitory Concentration of silver nanoparticles produced from S albus PCT3
Antimicrobial activity of AgNPs from S albus PCT3 was quite promising
Gmelina arborea-mediated silver nanoparticles (AgNPs) demonstrated a minimum inhibitory concentration (MIC) of 90 µg/mL against Pseudomonas aeruginosa and 20 µg/mL against Escherichia coli Additionally, another study reported that AgNPs derived from S chiangmaiensis SSUT88A exhibited MIC values against Acinetobacter baumannii, Klebsiella pneumoniae 1617, Pseudomonas aeruginosa N90PS, and Escherichia coli 8564.
The antibacterial properties of silver nanoparticles (AgNPs) derived from PCT3 were found to be superior to those reported in previous studies, with values of 6.8, 27, 13, and 27 àg/mL (Rosyidah et al., 2022b) In contrast, AgNPs synthesized from Vitis vinifera leaf extract demonstrated a minimum inhibitory concentration (MIC) of 0.331 àg/mL against Candida albicans, indicating a significantly higher efficacy compared to the green AgNPs examined in this study (ACAY et al., 2019b) Additionally, the MIC for Staphylococcus aureus was lower than that reported for extracts from Artemisia tournefortiana Rchb (12.5 àg/mL), Pleurotus ostreatus (16.1 àg/mL), and Desmodium triflorum.
Figure 1.10 Zones of inhibition of AgNPs against C albicans ATCC 10231
The agar-well diffusion method confirmed the minimum inhibitory concentration (MIC) results, showing that all pathogens were sensitive to silver nanoparticles (AgNPs) at concentrations ranging from 7.8 to 62.5 µg/mL Notably, Gram-positive bacteria exhibited greater resistance compared to Gram-negative bacteria Contrary to several reports, AgNPs demonstrated more effectiveness against Gram-positive bacteria due to differences in cell wall structure The positive charges of AgNPs compromised the integrity and permeability of the bacterial cell walls, allowing them to attach and penetrate the cells.
Gram-positive bacteria possess a negative charge on their cell walls due to teichoic acids associated with peptidoglycan or the plasma membrane In contrast, Gram-negative bacteria feature an outer layer of phospholipids and lipopolysaccharides, which diminishes the effect of the negative charge compared to Gram-positive bacteria The AgNPs from S albus PCT3 exhibited a distinct pattern, suggesting unknown mechanisms of action against pathogenic bacteria that warrant further investigation.
CONCLUSIONS AND SUGGESTIONS
Conclusions
In this study, 3 out of 8 Streptomyces species isolated from C latifolium demonstrated the ability to synthesize silver nanoparticles (AgNPs), evidenced by a color change from clear to reddish-brown Notably, the crude AgNPs produced by strain PCT3 exhibited significant inhibitory effects against six pathogens, including E faecalis ATCC.
29212, C albicans ATCC 10231, E coli ATCC 11105, S epidermidis ATCC
12228, S enterica ATCC 14028, and P aeruginosa ATCC 9027
- Based on morphological and biochemical characteristics, and 16S rRNA gene analysis, the most potent strain was identified as S albus PCT3
The purified silver nanoparticles (AgNPs) synthesized from S albus PCT3 exhibited significant antibacterial activity against both Gram-positive and Gram-negative bacteria, with minimum inhibitory concentration (MIC) values between 3.9 μg/mL and 31.2 μg/mL Notably, C albicans ATCC 10231 demonstrated the highest sensitivity, with an MIC value of 1.9 μg/mL.
Suggestions
Based on the findings, I offer the following suggestions for further research:
- Optimization of cultural and reaction conditions will be required further to improve green synthesis of AgNPs from S albus PCT3
- Spectroscopic and microscopic characterization studies are needed to discover functional groups and the morphological properties of synthesized AgNPs
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