Green synthesis of silver nanoparticles by Bacillus methylotrophicus, and their antimicrobial activity

Abstract

The synthesis of silver nanoparticles (AgNPs) by microorganisms is an area attracting growing interest in nanobiotechnology, due to the applications of these nanoparticles in various products including cosmetics and biosensors, and in the biomedical, clinical, and bioimaging fields as well. Various microorganisms have been found to be able to synthesize AgNPs when silver salts are supplied in the reaction system. The main objectives of this study were to evaluate the efficiency of synthesis of AgNPs by the strain Bacillus methylotrophicus DC3, isolated from the soil of Korean ginseng, a traditionally known oriental medicinal plant in Korea. The AgNPs showed maximum absorbance at 416 nm, when assayed by ultraviolet-visible spectroscopy (UV-vis). The field emission transmission electron micrograph (FE-TEM) results showed that the particles were spherical and 10–30 nm in size. In addition, the product was also characterized by energy dispersive X-ray spectroscopy (EDX), which displayed a 3 keV peak corresponding to the silver nanocrystal. Elemental mapping results also confirmed the presence of silver elements in the electron micrograph region. Furthermore, the AgNPs demonstrated antimicrobial activity against various pathogenic microorganisms such as Candida albicans, Salmonella enterica, Escherichia coli, and Vibrio parahaemolyticus, with enhanced antimicrobial activity being exhibited against C. albicans. Therefore, the current study describes the simple, efficient, and green method of synthesis of AgNPs by B. methylotrophicus DC3.

Keywords::

• antimicrobial activity
• Bacillus methylotrophicus
• green synthesis
• silver nanoparticles

Introduction

Metal nanoparticles have great importance due to their unique electronic, optical, and physiochemical properties, which differ significantly from those of bulk materials (Mazur 2004). These special properties could be attributed to their small size, large specific surface area, and high fraction of surface atoms. For these reasons, metal nanoparticles have broad applications in many fields, including biomedicine (Biswas et al. 2004, Govindaraju et al. 2009), and as substances with antimicrobial activity (Duran et al. 2005).

A variety of physical and chemical routes of synthesis have been reported for the preparation of metallic nanoparticles (Raut et al. 2009). However, these methods have several disadvantages, including the use of toxic chemicals and the production of hazardous waste products. There is a growing need to develop an environmentally-friendly process of nanoparticle synthesis (Whitesides 2003). Microorganisms possess the capability to reduce metals into the metal ions, and play a major role in bioremediation (Fortin and Beveridge 2000). In addition, these small nanofactories are capable of synthesizing nanoparticles in an eco-friendly way. Numerous silver, gold, and cadmium metal nanoparticles have been synthesized by various microorganisms. Bacteria have been the most commonly used microorganisms, because they are eco-friendly and relatively easy to handle (Parikh et al. 2008).

From a medical perspective, silver nanoparticles (AgNPs) present a range of anti-microbial, anti-fungal, and anti-viral applications against a broad range of microorganisms (Kim et al. 2008). In addition, AgNPs can be used as optical receptors (Schultz et al. 2000), as catalysts in chemical reactions, as spectrally selective coatings for solar energy absorption, and as intercalation material for electric batteries (Klaus-Joerger et al. 2001), cosmetics (Jin and Ye 2007), and therapeutics (Prow et al. 2006). Ginseng (Panax ginseng Meyer), is a traditionally known medicinal plant in Korea, China, and Japan. Ginseng and its crude extract have been used to cure various diseases such as cancer, diabetes, Alzheimer's, inflammation, etc. (Choi 2008, Mathiyalagan et al. 2014). The soil from the ginseng field has been used to isolate various bacteria, some of which are further utilized for biotransformation of pharmacologically active minor ginsenosides from major ginsenosides (Cheng et al. 2007), and other bacteria which promote plant growth (Deepa et al. 2010).

Due to the increasing applications and demand for AgNPs, there is a need for their green synthesis by various microorganisms. Extracellular biosynthesis of AgNPs has been conducted in Escherichia coli, Plectonema boryanum, Pseudomonas stutzeri, and Morganella sp. (Gurunathan et al. 2009). In the present study, we have described the biological synthesis and characterizations of AgNPs produced using Bacillus methylotrophicus DC3, which was isolated from ginseng field soil, and explored their antimicrobial efficacy against different pathogenic microorganisms.

Materials and methods

Media and chemicals

All media were purchased from Difco, MB cell (Seoul, Republic of Korea), including analytical grade silver nitrate (AgNO₃). Cycloheximide was purchased from Sigma-Aldrich Chemicals, USA. Vancomycin (VA30) 30 μg/disk, oleandomycin (OL15) 15 μg/disk, and penicillin G (P10) 10 μg/disk were purchased from Oxoid Ltd., England.

The pathogenic bacterial strains Vibrio parahaemolyticus [ATCC 33844], Salmonella enterica [ATCC 13076], E. coli [ATCC 10798], and Candida albicans [KACC 30062] were cultured on Tryptic soy agar (TSA) media at 30°C and preserved at − 70°C in glycerol for further study. Candida albicans was cultured on Sabouraud dextrose agar (SDA) at 28°C and preserved at − 70°C in glucose yeast peptone broth (GYP) glycerol stocks.

Isolation and molecular identification of bacteria

Soil samples were collected from the ginseng field at Kyung Hee University, Republic of Korea. The soil samples were serially diluted in sterile 0.8% NaCl and then plated onto TSA media. The colonies were further subcultured on TSA supplemented with 1 mM of filter-sterilized AgNO₃ for 48 h at room temperature, and observed for colony growth. Isolated colonies were subcultured to obtain pure cultures.

Molecular identification of the isolated strains was carried out by 16S rRNA sequencing. The genomic DNA of the isolated strain was extracted and purified with the Genomic DNA Isolation Kit (Core Bio System, Republic of Korea). The 16S rRNA gene was amplified with the universal bacterial primer pairs 27F, 518F, 800R, and 1512R (Weisburg et al. 1991). The purified PCR products were sequenced by Genotech (Daejeon, Republic of Korea). The sequences of the 16S rRNA gene were compiled with SeqMan software (version 4.1, DNASTAR, Inc., USA). The 16S rRNA gene sequences of related taxa were obtained from the GenBank database and EzTaxon-e server (Kim et al. 2012).

Extracellular biosynthesis of AgNPs

For biological synthesis of AgNPs, the selected bacterial isolate was inoculated into a 250 ml Erlenmeyer flask containing 100 ml of sterile Tryptic soy broth (TSB). The flasks were incubated in a shaking incubator at 28°C for 24 h at 120 rpm. After incubation, the culture was centrifuged at 10,000 rpm for 10 min and the bacterial pellet was removed. The supernatant was mixed with filter- sterilized AgNO₃ solution at a 1 mM final concentration, and incubated in an orbital shaker at 200 rpm and 28°C. The extracellular synthesis of AgNPs was monitored by visual observation of the color change in the culture medium. After the completion of incubation, the mixture was first centrifuged at 2000 rpm for 5 min to remove remaining medium components, and then the AgNPs were collected by high-speed centrifugation at 20,000 rpm for 10 min. The product was then washed several times by centrifugation and redispersed in water to remove the unconverted silver ions. Finally, the AgNPs were collected in a pellet and used for characterization.

Characterization of synthesized AgNPs

To verify the reduction of silver ions, the solution was scanned at 300–800 nm in a UV-vis spectrophotometer (Ultrospec 2100 pro, Amersham Biosciences Corp., USA).

The size, shape, morphology, and distribution of the nanoparticles were analyzed by field emission transmission electron microscopy (FE-TEM), energy dispersive X-ray spectroscopy (EDX), and elemental mapping with a JEM-2100F (JEOL) instrument, operated at 200 kV. The sample was prepared by placing a drop of AgNPs on a carbon-coated copper grid, and subsequently drying it in an oven at 60°C before transferring it to the microscope.

Analysis of antimicrobial activity of AgNPs

The antimicrobial activity of the biologically synthesized AgNPs against pathogenic organisms such as V. parahaemolyticus, S. enterica, E. coli, and C. albicans was measured on Mueller-Hinton agar (MHA) plates using the disk diffusion method. A 100 μL aliquot of an overnight log culture of V. parahaemolyticus, S. enterica and E. coli was spread evenly on a MHA plate with a glass spreader. The AgNPs (3 μg) were added to a plain disk on the plates. The antibiotic discs VA30, P10, and OL15 were also kept on the same plate, to act as a control, and the plate was incubated at 30°C for 24 h. Similarly, for C. albicans, an overnight log culture was spread evenly on a SDA plate and the sterile paper discs were loaded with 3 μg of cycloheximide and AgNPs, and further kept for 24-h of incubation. After incubation, the zones of inhibition were measured in all the plates.

Results and discussion

Identification of bacteria

On the basis of molecular characterization of the bacterial isolate, the isolated strain showed 99.51% similarity with B. methylotrophicus CBMB205^T, thus named as B. methylotrophicus DC3. Moreover, the 16S rRNA sequence of B. methylotrophicus DC3 has been deposited to NCBI, with the KP412492 accession number.

Synthesis and characterization of AgNPs

In the present study, AgNPs were successfully synthesized in the culture supernatant of B. methylotrophicus DC3. The formation of AgNPs by the reduction of AgNO₃ in the supernatant of B. methylotrophicus DC3 was indicated by a color change in the reaction mixture (Figure 1a and b). The color changed gradually from yellow to brown, within 48 h. The brown coloration was due to the formation of AgNPs in the reaction mixture, resulting in the excitation of surface plasmon vibrations (Anil Kumar et al. 2007).

Figure 1. Bacillus methylotrophicus culture supernatant after incubation period with AgNO₃ (1 mM) (a), control with medium and AgNO₃ (1 mM) (b).

The synthesis of AgNPs was further confirmed by UV-vis spectral analysis. The product was scanned at 300–800 nm, and the strongest peak was observed at 416 nm, which corresponded to the surface plasmon resonance band of the AgNPs (Figure 2a) (Bhainsa and D’Souza 2006). Thus, the reaction mixture indicated the formation of AgNPs. FE-TEM images of the AgNPs revealed a spherical shape, with sizes ranging from 10–30 nm (Figure 2b). The EDX analysis of silver nanocrystallites displayed an optical absorption band peak at approximately 3 keV (Figure 3), which is the typical absorption of metallic silver nanocrystallites (Magudapathy et al. 2001). Elemental mapping analysis of the AgNPs showed maximum distribution of silver (48.36%) (Figure 3). The distribution of carbon and copper were due to the TEM grid and contamination of chlorine. Overall, we found that the strain B. methylotrophicus DC3 is capable of efficient synthesis of AgNPs. Bacillus has shown activity in biosorption and biodegradation (Li et al. 2014), and B. methylotrophicus has been reported in the biosynthesis of levan by levansucrase (Zhang et al. 2014), and biocontrol of rice blast (Shan et al. 2013). To our knowledge, this is first time B. methylotrophicus DC3 has been found to be capable of synthesizing spherically shaped AgNPs. The previous studies suggest that the enzyme and proteins secreted by the microorganisms extracellularly in the culture medium play a major role in the reduction of AgNO₃ to AgNPs. The study, based on the extracellular synthesis of AgNPs by B. licheniformis, showed that the nitrate reductase secreted by the bacteria in the medium was responsible for the synthesis of AgNPs. Thus, the synthesis of AgNPs occurs extracellularly in the culture medium (Kalimuthu et al. 2008), which completely eludes the downstream processing needed for intracellular synthesis.

Figure 2. UV-vis spectra of culture supernatant of Bacillus methylotrophicus treated with AgNO₃ (1 mM) (a), transmission electron micrograph of silver nanoparticles synthesised by B. methylotrophicus showing spherical shaped silver nanoparticles, at 20 nm (b).

Figure 3. TEM micrograph of silver nanoparticles pellet solution (a), silver nanoparticles, 48.36%, green (b). EDX spectrum analysis showing major peak of silver nanoparticles at 3 keV, corresponds to silver nanoparticles (c).

Antimicrobial activity of AgNPs

In our study, the AgNPs were tested for antimicrobial activity against a range of pathogenic microorganisms, which included C. albicans (Figure 4a), S. enterica (Figure 4b), E. coli (Figure 4c), and V. parahaemolyticus (Figure 4d). After incubation, the mean diameter of the zone of inhibition was determined by measuring the diameter of the zone, and antimicrobial activity was observed against C. albicans, V. parahemolyticus, E. coli, and S. enterica. The results showed that the strains were completely resistant to these antibiotics, but sensitive to AgNPs. The AgNPs exerted more antimicrobial activity as compared to standard antibiotics. The antimicrobial activity of AgNPs has also been reported for a methicillin-resistant Staphylococcus sp. (Saravanan and Nanda 2010). In our study, we showed the synthesis of spherically shaped AgNPs by an eco-friendly method, without the use of any toxic or hazardous chemicals. Furthermore, the particles were found to exert remarkable antimicrobial activity against human pathogenic strain, and hence can be applied on a clinical platform against pathogenic and multidrug-resistant microorganisms. Moreover, the nanoparticles are well known for target specificity. Many studies highlight that apart from target specificity, AgNPs are also capable of antimicrobial activity, biofilm degradation, therapeutic action, inducing hyperthermia, enhancing radiotherapy, silencing genes, and delivering drugs; the anticoagulant effect of AgNPs has also been demonstrated, which further corresponds to their application on the biomedical platform. Thus, the AgNPs were synthesized by B. methylotrophicus DC3 in an eco-friendly, economical, and biocompatible method and showed antimicrobial activity against pathogenic microorganisms.

Figure 4. Zones of inhibition of reaction mixture containing silver nanoparticles against, Candida albicans (a), Salmonella enterica (b), Escherichia coli (c), Vibrio parahaemolyticus (d), respectively. Vancomycin 30 μg (I), penicillin G 10 μg (II), oleandomycin 15 μg (III), silver nanoparticles 3 μg (IV) and cycloheximide 3 μg (V) were loaded on each disk.

Conclusions

The study highlights the synthesis of spherically shaped AgNPs by B. methylotrophicus DC3 in an eco-friendly manner, without using any toxic or hazardous chemicals. The spherically shaped particles were well characterized by prevailing instrumentation techniques and applied for antimicrobial activity against human pathogenic microorganisms. The particles were found to exert remarkable antimicrobial activity against human pathogenic strains C. albicans, S. enterica, E. coli, and V. parahemolyticus; hence, they can be applied on a clinical platform against pathogenic and multidrug-resistant microorganisms. Additionally, the extracellular synthesis of nanoparticles could easily be adapted to large-scale operations and downstream processing. Thus, the rapid and eco-friendly synthesis of AgNPs and antimicrobial activity against human pathogenic microorganisms corresponds to their application on the biomedical platform.

Acknowledgments

This research was supported by the Korea Institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry & Fisheries (KIPET NO: 313038-03-2-SB010), and also was supported by Business for Cooperative R & D between Industry, Academy, and Research Institute funded KSMB (Grants No.C0214183).

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.