Green synthesis of silver nanoparticles and their antibacterial activities

2.1 Materials

Silver nitrate (AgNO₃) with an analytical specification of Ph. Eur., BP, USP, 99.8–100.5% and soluble starch from potato with a purity of 99% were purchased from Merck (Sigma-Aldrich, Germany). All chemicals were used without further purification and were at a minimum 99% purity.

All glassware was washed with distilled water and dried in oven.

2.2 Microalgae and cultivation conditions

P. kessleri, was obtained from GEPEA UMR CNRS 6144, Saint-Nazaire, France. Growth media for P. kessleri was based on modified Bold’s Basal Medium (BBM) [11]. For BBM, pH was adjusted to 6.5 before being sterilized by autoclaving at 120°C for 20 min using a pH meter (BANTE Instruments, China). All glassware used in the culture of microalgae was also autoclaved.

The cultivation was done in an Erlenmeyer where the mixing of the culture was performed by introducing ambient air continuously with a constant air flow rate of 0.5 L·min⁻¹ through a membrane into the bottom of the flask by tiny bubbles.

Fluorescent lamps were placed horizontally to the front side of the Erlenmeyer. They supply continuous illumination of 850 lx with a 16/8 h light/dark cycle.

The volume of inoculum was chosen to give an initial density of 10⁵ cells·mL⁻¹. The room temperature range during this study was from 18°C to 22°C. Samples were taken three times a week to analyze the change in pH, cell morphology and cell number using a counting chamber (Hemocytometer).

2.3 Synthesis of AgNPs using starch

Stock solutions of AgNO₃ and starch with concentrations of 5 g·L⁻¹ were prepared and stored at ambient temperature in the dark. The synthesis of AgNPs was carried out at room temperature by adding various concentrations of AgNO₃ solution (2.5 and 5 g·L⁻¹) to various starch concentrations (0.3, 0.6, and 1.2 g·L⁻¹) in test tubes. The reaction was also subjected to microwave irradiation, employing a domestic microwave oven at 400 W power. During preparation, the solution turned to the characteristic yellowish-brown color and then to grayish black, indicating the formation of AgNPs. The prepared NPs in starch were stable for 2 months without any change in the surface plasmon resonance (SPR) as indicated from the absorption spectra at room temperatures, showing that the starch was a good reducing and stabilizing agent for the AgNPs.

2.4 Synthesis of AgNPs using dry biomass

P. kessleri cells were collected through centrifugation at 4,500 rpm for 20 min. Then, the supernatant was discarded, and the pellet was washed using sterile distilled water. Glass plates were used for heat drying the algal biomass at 50°C for 48 h in an oven.

The synthesis of AgNPs was carried out using an adapted method based on a previous work [2]. Briefly, biomass powder (0.33 g) was mixed with 21 mL of distilled water in a beaker flask with a magnet inside it and kept on stirring at 50°C for 3 h and at 8°C for 21 h to be well dispersed. Then, the solution was filtered using the Whatman filter paper.

The concentration of biomass in distilled water was fixed after a series of optimization experiments, which demonstrated that a mass of dry biomass of 0.33 g in 21 mL would be optimal for maximum production of NPs.

AgNPs were prepared at room temperature by adding 4.5 mL of various concentrations of AgNO₃ solution (1 and 2.5 g·L⁻¹) to 0.5 mL of P. kessleri in a round-bottom flask. The mixture was irradiated using a domestic microwave for various time intervals at a power of 400 W.

The bioreduction of Ag⁺ ions in the solution was checked by regular sampling (3 mL) of the suspension, and then, the UV–Vis spectra of the taken sample were measured.

2.5 Characterization

The change in the color of the mixture containing the silver nitrate solution and the reductors (P. kessleri or starch) was monitored by visual observation. To confirm the formation of AgNPs, the reaction mixture was sampled at constant intervals of time. Following this, the absorption maximum (λ _max) was measured at 300–700 nm using a spectrophotometer (Rayleigh, 1800 UV–Vis), which works based on SPR. In the aforementioned range, the excitation of surface plasmon vibration bands leads to a considerable alteration in the solution color. Due to the relationship between the absorbance of the produced AgNPs and their concentration, the synthesized AgNPs’ concentration can be easily determined by UV–Vis spectroscopy in a 10 mm optical path quartz cuvette containing 1 mL of the target sample [12].

SEM images were obtained using a JOEL JSM- IT100, equipped with an embedded JEOL X-ray energy-dispersive spectroscopy (EDS) system for qualitative and quantitative elemental analysis.

The phase purity of the obtained samples was examined by (XRD, Bruker D8 Advance X-ray diffractometer) with Cu-Kα radiation (λ = 1.5406 Å) at the scan range of 0 < 2θ < 100.

The sample was prepared by drying droplets of AgNP solution at 50°C for 24 h in an oven and then placed inside carbon grids intended for observation of the surface microstructure in the SEM, while XRD samples were dried in a glass slide.

2.6 Evaluation of antibacterial activity

The green-synthesized AgNPs were tested for potential antibacterial activity against E. coli (Gram-negative), B. clausii, and S. aureus ATC29213 (Gram-positive) and compared with selected antibiotics: oxacillin (against B. clausii), amoxicillin (against S. aureus), and gentamicin (against E. coli).

B. clausii spore suspension drug (Enterogermina, Sanofi-Aventis, Morocco) consisted of a mixture of spores of four antibiotic-resistant B. clausii strains named OC, NR, SIN, and T. Each Enterogermina vial contains 2 × 10⁹ CFU of B. clausii spores.

E. coli bacterium was obtained from Professor Hamadi Fatima (Department of Biology, Faculty of Sciences, University Ibn Zohr).

Agar Disk diffusion assay was performed to assess the antibacterial activity of prepared AgNPs. Blank 6 mm disks prepared from Whatman filter paper were autoclaved prior to use. Bacterial suspensions (E. coli, B. clausii O/C, and S. aureus) were prepared from an overnight culture of each test strain and adjusted to a turbidity of 0.5 McFarland standards (EUCAST recommendations) and were then spread on the surface of a Mueller–Hinton agar plates using sterile swabs [13]. Then, five sterile filter paper disks and an antibiotic disk were placed on the dried plate. One blank disk was loaded with 20 μL of microalgae biomass solution, the second one with 20 μL of sterile distilled water as a negative control, the third one with 20 μL of AgNO₃ as positive control, and the fourth and the fifth with 20 μL of the synthesized AgNPs from starch (5 g·L⁻¹) and biomass (1 g·L⁻¹), respectively. Antibiotics used as positive controls were oxacillin (5 µg) for B. clausii, gentamicin (10 µg) for E. coli, and amoxicillin (25 µg) for S. aureus. After incubation for 20 h at 35°C, plates were examined and antibacterial activity was assessed by measuring the inhibition zone diameters around disks with a Vernier caliper. Antibacterial activity was considered if a growth inhibition halo of more than 6 mm (disk size) was produced. The experiment was carried out in triplicate for each strain.

MIC is the lowest concentration of an antimicrobial agent that inhibits visible growth of a microorganism. MIC was determined with a broth dilution method using test tubes containing sterile Mueller–Hinton broth [14,15]. Varying concentrations of AgNPs synthesized via starch (100, 50, 25, 12.5, 6.25, and 3.12 μg·mL⁻¹) and biomass (157, 78.5, 39.25, 19.6, 9.81, and 4.9 μg·mL⁻¹) were prepared and incorporated to test tubes containing the broth. Two control tubes (positive and negative) were not supplemented with the AgNPs. Each tube except negative control was inoculated with 100 μL of a 0.5 McFarland suspension of the bacterial strain. Tubes were then incubated at 37°C for 20 h. The above experiment was performed using the three bacteria: E. coli, B. clausii, and S. aureus. The next day, turbidity was evaluated by visually comparing tubes to both positive and negative controls, and thereafter, the MIC was determined.

3.1 Synthesis of AgNPs using starch

Figure 2 reveals that λ _max of the synthesized AgNPs was obtained at around 410 nm with a concentration of 0.3 g·L⁻¹, an indication of the successfully formed AgNPs, which was in the desirable range for AgNPs.

Figure 2

UV–Vis spectra of AgNPs synthesized using 2.5 g·L⁻¹ of AgNO₃ at different exposure times at microwave heating.

In the first 20 s, no peak was observed, confirming that no AgNPs were formed. After 30 s of being exposed to microwave irradiation, the SPR band for AgNPs was obtained. A consistent growth was noticed in the intensity of synthesis with an increase in irradiation time, without any considerable change in the peak position. The synthesized AgNPs with higher intensity was achieved at 40 s. It is worth mentioning that the irradiation time of more than 40 s led to the evaporation of the solutions.

To study the impact of starch concentration on the synthesis of AgNPs, different sequences were performed out by varying the amount of starch from 0.3 to 1.2 g·L⁻¹ keeping the concentration of AgNO₃ at constant. The corresponding changes in the SPR peak are shown in Figure 3.

Figure 3

UV–Vis spectra of AgNPs synthesized using 5 g·L⁻¹ of AgNO₃ at different starch concentration and with (W) or without (W/O) microwave (MW) expositions.

With the increase in starch concentration from 0.3 to 0.6 g·L⁻¹, the SPR peak intensity was increased due to the decrease in particle size. This indicates that increased starch concentration could increase the formation of AgNPs by reducing more Ag⁺ ions. As previously reported, starch controls the size of AgNPs by effectively capping the NPs [18].

The effect of concentration of AgNO₃ on the synthesis of AgNPs was investigated (Figures 2 and 3) and the results suggested that, with the increase in AgNO₃ concentration, the number of Ag⁺ ions becomes more available in the solution, which are further reduced and effectively capped by starch molecules to produce a greater number of AgNPs [19].

It should be noted that the AgNO₃ control without starch shows no AgNP formation, which confirms the importance of this reducing agent in the biosynthesis of NPs.

Starch is one of the many polysaccharides used for the green synthesis of NPs. For example, β-d-glucose has been used repeatedly as a reducing agent for the synthesis of AgNPs [20]. Starch is composed of amylose and amylopectin, and the hot water produced under microwave irradiation changes the structure of soluble amylose into smaller molecules by hydrolysis. Glucose is one of the produced smaller molecules; it is a well-known AgNO₃ reducing agent that was first investigated by Raveendran et al. [21]. The absorption diagram showed sharp peaks, Yakout and Mostafa explained this sharpness of the absorption peak, which increases in intensity with time of exposure to microwave radiation by the formation of spherical mono-dispersed AgNPs, and this mono-dispersity increases with time of exposure to microwave radiation; this was confirmed through the images taken by the SEM (Figure 6a) [22]. Our optimization to study AgNO₃ effect using 5 g·L⁻¹ is explained by Khodadi et al., who demonstrated that increasing the concentration of AgNO₃ improves the efficiency of the synthesis, because it increases the quantity of metal ions available for reduction [19].

3.2 Synthesis of AgNPs using dry biomass

Figures 4 and 5 illustrate the effect of different concentrations of AgNO₃ at different irradiation times on the formation of AgNPs and demonstrate a large absorption peak, indicating the formation of NPs. In particular, as shown in Figures 4 and 5, the tube with 1 g·L⁻¹ had more AgNPs, which increased with the time of exposure to microwaves; this concentration with an exposure of 50 s forms the best conditions for forming the AgNPs.

Figure 4

UV–Vis spectra of synthesized AgNPs with 1 g·L⁻¹ of AgNO₃ at different times of microwave exposure.

Figure 5

UV–Vis spectra of synthesized AgNPs with 2.5 g·L⁻¹ of AgNO₃ at different varying time of microwave exposure.

It is worth noting that the AgNO₃ control without microalgal extract shows no AgNPs formation; this confirms the importance of dry biomass in the biosynthesis of NPs.

Generally, microalgae are natural bioremediators; thus, they store metal and metallic pollutants. During this bioaccumulation process, NPs are generated from the trapped metal ions [23]. The biosynthesis of AgNPs using normal living microalgae is popularly studied, but this synthesis through the dry biomass of microalgae is rare; this type of synthesis is interesting, because the dry biomass used in this assay can be manipulated and stored easily without any need for nutrient intake as opposed to using microalgae culture, which needs to be fresh and alive in optimal conditions [2]. The work of Alqahtani et al. is in agreement with our results, especially on the fact that the factors which affect the synthesis of NPs are the AgNO₃/reducing agent ratio, the concentration of AgNO₃, the temperature, and the reaction time, which is demonstrated by our study at different concentrations and exposure times [24]. The work of Torabfam and Yüce using Cholerella vulgaris indicates the possible involvement of the carbonyl group and the protein peptides that make up these microalgae in the synthesis of NPs. They also confirm that C. vulgaris is rich in proteins and carbohydrates, providing a group of polysaccharides useful in synthesis, which is similar to our case and our biomass composition [2]. The exact mechanism explaining why dry biomass acts as a reducing agent could be the water-soluble nature of this biomass, which makes it one of the best solvents for microwave heating. This reduction is induced by the presence of the enzyme nitrate reductase [23]. The process is initiated with the transfer of electrons from NADH to NAD⁺ catalyzed by NADH-dependent reductase as the electron carrier, which reduces Ag⁺ ions to metallic silver [24].

3.3 Characterization of AgNPs using SEM and EDS

To characterize the structure and morphology of AgNPs, SEM was used to capture the microstructure images of these NPs. For the AgNPs synthesized by starch, small spherical and mono-dispersed NPs of size less than 100 nm are observed (Figure 6a).

Figure 6

SEM image of AgNPs synthesized by starch (a) and dry microalgae biomass (b).

For the AgNPs synthesized by microalgal biomass, the size of the NPs is about 90 nm. Their shape is a mixture of spherical and rectangular spheres (Figure 6b).

The NPs synthesized by microalgae extract have spherical shapes with sizes that vary from 40 to 150 nm. This work is in agreement with the work of Ibrahim [25].

On the other hand, the broad peaks recorded by UV–Vis spectroscopy corroborate the polydisperse form of the NPs synthesized by the biomass. Similar results were found by Rautela et al. [14]. These authors observed that most of the NPs synthesized using banana peel extract were aggregated, and this aggregation is reflected by broad peaks recorded at 400 nm [14].

For the identification of the elemental composition of AgNPs, an EDS analysis was performed, and the results are presented in Figure 7. Peaks corresponding to the silver element are observed, showing the presence of silver as an ingredient element and confirming the formation and purity of AgNPs synthesized by starch (Figure 7a).

Figure 7

EDS image of synthesized silver nanoparticles with starch (a) and with dry microalgae biomass (b).

Figure 7b illustrates the EDS results of AgNPs synthesized by microalgae biomass. Peak characteristics of silver accompanied by NA, C, and O are observed, which may be due to the emission of X-rays by the carbohydrates/proteins/enzymes present in the microalgae extracts.

3.4 Characterization of AgNPs using XRD

XRD patterns of the AgNPs were measured and are demonstrated in Figure 8. Typical XRD patterns of AgNPs, prepared from starch, are shown in Figure 8a, while those synthesized using microalgal biomass are shown in Figure 8b.

Figure 8

XRD pattern of AgNPs synthesized with starch (a) and with dry microalgae biomass (b).

Analysis of XRD results of AgNPs synthesized from starch showed diffraction peaks at 2θ = 38.1°, 44.2°, 64.5°, and 77.4°. At the same time, AgNPs synthesized from microalgae biomass showed diffraction peaks at 2θ = 31.5°, 39.7°, 41.8°, 60.5°, and 74.9°. The peaks indicating the AgNPs can be attributed to the planes (111), (200), (220), (311), and (222) facets of the silver crystal according to the standards corresponding to the ICDD file (International Center for Diffraction Data). For the previous peaks, 38.1° for starch NPs and 29.4° and 35.8° for biomass NPs are characteristic peaks of starch polysaccharides and microalgae biomass components, respectively [26,27].

3.5 Stability of AgNPs

The stability of AgNPs in solution is a necessary condition as an indication of in vitro and in vivo behavior. The stability of the synthesized AgNPs was studied by monitoring the SPR peak. As shown in Figures 9 and 10, the AgNPs are stable for more than 3 months after their synthesis and only losses of 10% and 40% were recorded for the NPs synthesized from starch and biomass, respectively. According to a previous study, AgNPs are very stable due to strong electrostatic repulsion between particles. Indeed, the AgNPs exhibited a negative zeta-potential value, indicating that the surface of the AgNPs is covered with negatively charged groups like hydroxyl and carboxyl groups [1,18].

Figure 9

UV–Vis spectra of synthesized AgNPs with 5 g·L⁻¹ of AgNO₃ and 1.2 g·L⁻¹ of starch at 40 s of microwave exposure before and after 3 months of synthesis.

Figure 10

UV–Vis spectra of microalgae biomass-mediated AgNPs’ synthesis with 1 g·L⁻¹ of AgNO₃ at 50 s of microwave exposure before and after 3 months of synthesis.

3.6 Antibacterial activity test

Antibacterial activity was evaluated by the disk diffusion method against three strains. The results were expressed as diameter of inhibition zone (Figure 11).

Figure 11

Comparative antibacterial effect of synthesized AgNPs and other substances tested against B. clausii, E. coli, and S. aureus (NIZ: no inhibition zone).

Normal growth of tested bacteria was observed around the negative control discs represented by distilled water and the biomass of microalgae, as well as around the positive control discs of AgNO₃. Antibiotics and tested AgNPs, on the other hand, show a clear zone of inhibition around the discs in all bacterial strains and with both different synthesized AgNPs.

Agar diffusion assay results indicated that synthesized AgNPs exhibited a potential antibacterial action against the three tested strains. Antibacterial activity recorded was different depending on the bacterial species. Indeed, Gram-positive bacteria B. clausii O/C and S. aureus were more sensitive to prepared AgNPs than Gram-negative bacterium E. coli. We found no noticeable difference in activity between AgNPs prepared using the two reducing agents, microalgae biomass, and starch.

AgNPs are advantageous over conventional antimicrobial agents. Their effectiveness depends on their binding to the surface of microorganisms [28]. In our agar diffusion results, a clear antibacterial effect of the green-synthesized AgNPs on the tested bacteria was highlighted, with an activity more pronounced in Gram-positive strains than in Gram-negative bacterium. These observations are consistent with results reported in other studies such as that of Yakout and Mostafa, who demonstrated a stronger antibacterial activity of AgNPs against Gram-positive bacteria (S. aureus with an inhibition zone diameter of 14 mm and Streptococcus pyogenes with an inhibition diameter of 13 mm) than against Gram-negative bacteria (Salmonella typhimurium with a diameter of 7 mm and Shigella sonnei with 8 mm) [22]. Loo et al. explained this difference in activity by the cell wall kind, and the positive charges of the AgNPs trapped by the lipopolysaccharide of Gram-negative bacteria, which made them less sensitive [28]. Whereas other studies have found higher resistance to AgNPs in Gram-positive bacteria than in Gram-negative ones [2,29]. Resistance of Gram-positive bacteria has been explained by their wall structure, which is thicker due to its peptidoglycan richness, in contrast to Gram-negative bacteria characterized by a thin wall poor in peptidoglycan. More negatively charged, peptidoglycan can bind positively charged silver ions, trapping them in the bacterial wall [30,31]. Thus, penetration of AgNPs would be easier through the wall of Gram-negative species [14]. Other studies have presented a greater effect against Gram-positive bacteria or even the absence of difference in the activity of NPs based on the cell wall kind, Gram-positive or Gram-negative [19].

MIC values of biosynthesized AgNPs showed potent antimicrobial activity against the three bacteria tested. In the case of starch-mediated AgNPs, the MIC was 6.25 μg·mL⁻¹ against both E. coli and S. aureus, while it was 12.5 μg·mL⁻¹ for B. clausii. MICs of biomass-mediated AgNPs were 19.6, 9.81, and 19.6 μg·mL⁻¹ against E. coli, B. clausii, and S. aureus, respectively. Our MIC results confirm potent antibacterial activity against the three bacteria tested [32]. Starch-mediated AgNPs showed more inhibiting activity against E. coli and S. aureus than B. clausii, whereas those synthesized using biomass were more active toward B. clausii. Biosynthesized AgNPs showed similar MICs against E. coli (Gram negative) and S. aureus (Gram positive). Thus, the bacterial wall structure would not be the only determining factor for the activity of NPs. Indeed, the inhibitory effect of AgNPs may also be attributed to their nanometric size, which allows them to easily attach to the bacterial membrane and reach the cellular content, thus disrupting its structure and making it more permeable [22]. In fact, the small size of AgNPs allows them to adhere to the wall and interact with the cell membrane and subsequently disrupt its cellular functions such as permeability and respiration and eventually leading to cell death [31]. It has been reported that AgNPs can bind to bacterial DNA and RNA and cause their denaturation, which consequently prevents the bacterial replication process [33].

More pronounced antibacterial activity shown in the dilution method could be explained by the fact that results in this advantageous technique are independent of the antimicrobial substance properties to diffuse in solid medium [34]. This AgNP activity could be due to silver ions, as AgNPs continuously produce silver ions in an aqueous environment [34]. Ag⁺ ions can adhere to the cell wall, increase its permeability, and lead to disruption of the bacterial envelope, which, after the uptake of these free silver ions, can lead to the deactivation of multiple enzymes and cause bacterium death [35].