Microwave-assisted green synthesis of silver nanoparticles using dried extracts of Chlorella vulgaris and antibacterial activity studies

Abstract Green synthesis of metallic nanoparticles (NPs) is acquiring considerable attention due to its environmental and economic superiorities over other methods. This study describes the practical synthesis of silver nanoparticles (AgNPs) through the reduction of silver nitrate solution using an algal source, Chlorella vulgaris, as the reducing as well as the stabilizing agent. The energy required for this synthesis was supplied by microwave radiation. The ultraviolet-visible spectroscopy exhibited a single peak related to the surface plasmon absorbance of AgNPs at 431 nm. The AgNPs with high stability (a zeta potential of −17 mV), hydrodynamic size distribution of 1–50 nm, and mostly spherical shape were obtained through a 10 min process. Fourier transform infrared spectroscopy analysis revealed that several functional groups, including carbonyl groups of C. vulgaris, play a significant role in the formation of functional NPs. Antibacterial features of the produced AgNPs were verified against those of Salmonella enterica subsp. enterica serovar typhimurium and Staphylococcus aureus, demonstrating a considerable growth inhibition at increasing concentrations of the NPs. As a result, the formed AgNPs can be used as a promising agent against bacterial diseases.


Introduction
Nanoscience stands out among the futuristic fields of research. Within this field, the synthesis of workable metal nanoparticles (NPs) using biological procedures has attracted significant attention worldwide [1,2]. Metal NPs present striking physicochemical features when compared to individual molecules and macro metals as a result of high surface area to volume ratios [3]. In the case of silver nanoparticles (AgNPs), based on their sizes and shapes, their fundamental properties such as electrical and thermal conductivity, catalytic activity, solubility, and sensitivity enable widespread employment of them in food packaging, water treatment tasks, and drug delivery applications [4][5][6]. A variety of methods ranging from sonoelectrochemical methods and chemical reduction to ultraviolet (UV) irradiation can be implemented for the synthesis of AgNPs [7][8][9]. The fact that applying some of these synthesis methods results in purified AgNPs cannot be neglected. In contrast, however, the synthesis cost must be considered as a fundamental factor in a formation procedure. Furthermore, using hazardous chemical materials as agents during synthesis raises serious questions about environmental toxicity and safety issues in the fabrication process. Consequently, the request has been increasing for green methods, which are more secure, practical, and environmentally friendly than current physical and chemical methods [10]. The plants along with products of plants and microorganisms (e.g., algae, yeast, fungi, and bacteria) have been used as natural alternatives to other chemicals during synthesis leading to the formation of metal NPs. Compared with other conventional methods, NPs formed by biosynthesis contain non-toxic by-products that improve NP biocompatibility in various applications [11]. Being nature friendly [2], affordable [12], maintainable [13], and free from chemical pollution [14], biological agents have become excellent candidates for the production of NPs. Furthermore, the green synthesis of NPs using microorganisms has become the center of attention over the past few years due to remarkable properties, such as lower synthesis time [15], and it is a low-priced, safe, reliable [16], and single-step [14] procedure. Due to the capping feature of components in microorganisms, addition of extra stabilizers is not required [11].
It is also known that metal NPs, as an alternative to antibiotics, exhibit outstanding antibacterial properties [15]. Besides this, their antifungal activity is considerable [17]. Among several noble metal NPs, AgNPs have been reported to have the most impressive antimicrobial features against various kinds of microorganisms, as summarized in Table 1. Being capable of stabilizing AgNPs, proteins and sugars of biological molecules used as reducing and stabilizing agents during synthesis are salient biotic components contributing to NP interactions with other organisms as well as microorganisms, which results in enhanced antimicrobial properties of the formed NPs. By making contact with the cell membrane and without penetrating it, AgNPs can apply the antibacterial effect and cause the death of microorganisms [18,19]. In the case of the energy needed for the synthesis, although numerous well-established techniques are available, the synthesis method must contain simplified and cost-effective procedures and be capable of increasing the reaction rate by orders of magnitude. Moreover, short reaction periods and higher efficiency to produce pure NPs are significant factors to be considered while choosing a method to synthesize NPs. Fulfilling the abovementioned criteria, microwave irradiation as a desirable and primary heating method surpasses conventional heating methods [20,21]. In 2,000, the required energy for the synthesis of PbTiO 3 NPs using ethylene glycol as a reducing agent was provided by microwave. This new method of supplying energy was investigated by Palchik et al. [22]. Due to striking features, including being fast, straightforward, and energy-saving, the Palchik group made use of this method for the acceleration and stabilization of the reactions. The average diameter of the NPs obtained through this method was around 15 nm, and only 1 min of microwave heating was applied in order to supply the energy required for the reaction. The microwave-assisted synthesis method produces NPs with lower dispersion, which is arranged in a more regular pattern. It is noteworthy that the shape and morphology of the formed nanostructures can be controlled in this procedure [20].
With a history of approximately 3.4 billion years, microalgae possess a distinct characteristic of high biodiversity. Among various microalgae, eukaryotic green microalgae Chlorella vulgaris from genus Chlorella has been present on earth since the Precambrian era [23]. C. vulgaris is a ball-shaped atomic type of cell with a diameter ranging from 2 to 10 μm and contains numerous constituents, including cell wall and cytoplasm with mitochondrion and chloroplast organelles. The primary components of C. vulgaris are proteins, lipids, carbohydrates, pigments, minerals, and vitamins. Proteins are incredibly significant in the algal structure and constitute half of the dry weight of the fully grown C. vulgaris cell. Besides proteins, lipids are made up of various ingredients, such as glycolipids and hydrocarbons. Carbohydrates such as starch and cellulose are examples of polysaccharides that existed in C. vulgaris [23,24]. Also, polysaccharides extracted from algae have a dual impact due to their ability to reduce silver ions and stabilize the formed AgNPs [25]. Figure 1 summarizes the main methods of synthesis along with subdivisions of green synthesis. The schematic illustration of AgNPs in this report is also presented.
In this study, the dried-biomass of C. vulgaris algae was used for the first time in the microwave-assisted green synthesis of AgNPs. As presented in Table 1, only a few studies have been conducted so far with algae for NP synthesis, and only a limited number of tests regarding antibacterial activity have been investigated. Here, we also report the antibacterial activity of the formed AgNPs against the well-known foodborne pathogens, including Salmonella enterica subsp. enterica serovar typhimurium (Gram−) and Staphylococcus aureus (Gram+).

Materials
Silver nitrate (AgNO 3 ) with a purity of 99.9% as a silver ion precursor was provided by Merck (Germany). Foodborne pathogens, Salmonella enterica subsp. enterica serovar typhimurium (ATCC® 14028™) and S. aureus (ATCC® 29213™), were acquired from American Type Culture Collection (ATCC, USA). Nutrient agar (NA) and nutrient broth (NB) as a cultivation medium were bought from Oxoid Ltd (Hampshire, England). Deionized double distilled water was used to prepare the required solutions.

Algae preparation
The cultivation of microorganisms in the BG11 medium was done by the flask-shake method [46]. C. vulgaris cells were collected through centrifugation at 10,000 rpm for 15 min. Then, the supernatant was separated, and the pellet was cleaned using sterile water. The glass plates were used for heat drying of algal biomass at 55°C for 24 h. As a final step, dried biomass was removed from the plates and powdered using a mortar. The heat-dead biomass used in this assay can be handled and stored easily without any need for nutrient supply. Finally, biomass powder (1.6 g) was mixed with 100 mL of deionized water in an Erlenmeyer flask with a magnet inside it, kept on stirring at 50°C for 3 h and at 10°C for 21 h to be well dispersed. Then the solution was filtered using a filter paper and a vacuum filtration unit. The supernatant was kept in the fridge for further reaction and analysis.

NP synthesis
For the synthesis of AgNPs, 10 mL of C. vulgaris was mixed with 90 mL of an aqueous solution of AgNO 3 (1 mM solution) and heated by microwave at the power of 180 W for various time intervals. In order to take the sample from the solution during the heating process and prevent overheating, microwave radiation was applied for 2 min by 15 s delay for a total heating time of 10 min. The bioreduction of Ag + ions in the solution was checked by regular sampling (2 mL) of the suspension, then the UV-visible spectra of the taken sample were measured. The synthesis reaction was monitored by the UV-visible spectra, as well as by monitoring the color change in the reaction solution. Gradually, the original light-green color of the solution changed to red-brown color, indicating the formation of AgNPs. In all steps, the synthesized AgNPs were sonicated and filtered.

Characterization of NPs
The change in the color of the mixture containing the silver nitrate solution and C. vulgaris was monitored by visual observation. In order 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 350-800 nm using a spectrophotometer (Cary 5000 UV-Vis-NIR, USA), which works based on surface plasmon resonance (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 AgNP concentration can be easily determined by UV-Vis spectroscopy in a 10 mm optical path quartz cuvette containing 1 mL of the target sample [18]. The Fourier transform infrared (FT-IR) analysis of AgNPs, including C. vulgaris and unmixed C. vulgaris, was done to determine the functional groups of C. vulgaris and estimate their role as reducing or stabilizing agents in AgNP synthesis using an FT-IR spectrophotometer with a deuterated triglycine sulfate detector (Thermo Scientific Nicolet iS10, USA). The range used to record the FT-IR spectra was 4,000-400 cm −1 .
The mean hydrodynamic size of the synthesized AgNPs was evaluated using a dynamic light scattering (DLS) analyzer (Zetasizer Nano ZS, Malvern, UK) with a 10 mm optical path quartz cuvette containing 1 mL of the target sample. Brownian motion, as a principle used in this device, causes the scattered light intensity fluctuations, which are measured by the DLS analyzer [13]. This measurement leads to the determination of the number of particles as well as their hydrodynamic size in a solution. In the case of the size distribution of the particles, the NP polydispersity index (PDI), as a value varying from zero to one, can be computed using this device. In order to detect and calculate the surface charge of the synthesized AgNPs, their value of zeta potential, as a demonstration of their stability, can be provided at 25°C by the DLS analyzer [13].
Further insight into the morphology of the formed AgNPs, comprising size as well as shape, was provided using a scanning electron microscope (SEM) with a focused electron beam (Zeiss Leo Supra 35 VP, Germany) along with a transmission electron microscope (TEM; CM120, Philips, Amsterdam, The Netherlands) with an acceleration voltage of 120 kV. In order to provide a sample for both SEM and TEM analyses, 5 µL of the targeted solution was dropped on a silicon wafer and left to dry at room temperature for a couple of hours.

Antibacterial activity
Antibacterial effects of the green-synthesized NPs were tested using liquid and solid nutrient media on the agar plates. For each bacterium, the suspension provided by mixing 500 µL of NB with 50 µL of target bacterium was vortexed well as a first step, and five small test tubes were taken and 5 mL of NB was added in each tube. Then, 100 µL of the suspension containing NB and bacterium was added to each test tube. Following this, 30, 60, 90, and 300 µL of the formed AgNPs was added to second, third, fourth, and fifth tubes, respectively, to monitor the effect of AgNPs on bacterium growth. The AgNP solution was not added to the first tube, which was used as a control culture. Finally, all five tubes were incubated for 24 h at 37°C. Then, 200 µL of the bacterial suspensions prepared in each of the five tubes was inoculated into plates containing culture media of NA. Finally, the plates were placed in an incubator at 37°C for 24 h.

Characterization of NP formation
As presented in Figure 2, the specific color change in the solution containing silver nitrate and C. vulgaris from pale green to reddish brown was the initial demonstration of the AgNP formation, and this color change is because of the excitation of vibration bands related to SPR [6]. The synthesis of AgNPs was verified by broad absorption peaks (λ max ) at wavelengths ranging from 380 to 450 nm. Owing to the SPR of AgNPs, the bands outlined above are ordinary absorption bands of these NPs. Noteworthy is the fact that the absorption of AgNPs was also reported between 410 and 440 nm in other research studies [2,16]. Figure 3 reveals that λ max of the synthesized AgNPs was obtained at 431 nm with a concentration of 59.22 ppm, an indication of the successfully formed AgNPs, which was in the desirable range for AgNPs. In the first 4 min, no peak was noticed, confirming that no target product was formed. After 6 min of being exposed to microwave irradiation, the SPR band for NPs 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. Thus, the synthesized NP with higher intensity was achieved at 10 min. It is worth mentioning that the irradiation time of more than 10 min led to the evaporation of all remaining solutions. Furthermore, the results in Figure S1 show the repeatability of the method applied in this report. The FT-IR spectra related to unmixed algae, as well as AgNP solutions, were analyzed to determine the various functional groups related to C. vulgaris, which are responsible for the reduction of the Ag + . Furthermore, the intensity of several regions can be noticed using this spectrum. As evidently noted in Figure 4, two sharp absorption peaks exist at 3262.98 and 1635 cm −1 . The peak with the shape of U at around 3,200-3,300 cm −1 contains both O-H hydroxyl and N-H amine groups. Moreover, a broad peak between 1,621 and 1,645 cm −1 is related to the amide I band due to the carbonyl bond. A weak peak at around 2151.54 cm −1 corresponds to the N-C compounds. Also, the -NH 2 bond of the protein or polysaccharide in C. vulgaris appears as a peak at the wavelength of 1,155 cm −1 . Following this, the absorption bands at 1,081 cm −1 and 1,036 cm −1 can be attributed to the ν(C-O-C) bond of polysaccharides that existed in C. vulgaris. Between 1,036 and 3262.98 cm −1 , the abovementioned peaks can also be noticed in the infrared band of C. vulgaris, including NPs. A considerable shift in some of these peaks indicates the possible involvement of the carbonyl group and peptides of proteins related to algae in the synthesis of target NPs. C. vulgaris is rich in proteins and carbohydrates, providing a group of polysaccharides, which are of great importance in the composition of this alga. The results obtained are consistent with those of the study by Duygu et al. [49]. The FT-IR spectrum of C. vulgaris, obtained by their group, was a confirmation for the presence of proteins, groups of carbonyls, as well as polysaccharides involved in the reduction of Ag + along with stabilization of AgNPs. Figures 5 and 6 shows the mean hydrodynamic size, PDI, and zeta potential of the formed NPs, which are 24.79 nm, 0.252, and −17 mV, respectively. With regard to the hydrodynamic size distribution pattern, particles with a size smaller than 100 nm are more noticeable after applying 2 min of microwave irradiation and increasing the   microwave exposure time up to 10 min results in NPs with smaller hydrodynamic size. Finally, particles in the interval of 10-40 nm are obtained after 10 min of exposure to microwave irradiation ( Figure 5). The AgNPs with lower PDI are more acceptable, and the PDI value of 0.252 in the current assay indicates the excellent distribution of particles in the target solution. A zeta potential of −17 mV, as an indicator of the electrical cloud around the NPs, reveals the high stability of the synthesized NPs. Due to the electric repulsion between particles, higher surface charge results in more stable suspension. It is worth mentioning that by periodic sampling of the suspension during 10 min of microwave exposure, the zeta potential values of the particles after 2, 4, 6, 8, and 10 min were measured and provided in Figure 6. The value of zeta potential related to the formed NPs was somewhere in the vicinity of −17 mV during this 10 min of reaction time. The results reported in this experiment are consistent with those of the previous experiments of Składanowski et al. and Elbeshehy et al. [37,44]. Składanowski and coworkers found the zeta potential of the formed AgNPs using Streptomyces sp. to be −19.4 mV. In the assay carried out by Elbeshehy et al., AgNPs formed by Bacillus spp. had a zeta potential of −18.5 mV. As shown in Figure S2, the reported method is repeatable, and AgNPs with a size smaller than 50 nm and a zeta potential of approximately −18 mV can be synthesized using this method.
As represented in Figures 7 and 8, TEM and SEM analyses clarify the size as well as the morphology of the  synthesized NPs. Figures 7 and 8, respectively, indicate that most of the AgNPs synthesized by C. vulgaris are spherical and quasi-spherical with flat edges. Following this, NPs with allotropic structures that have asymmetrical curves can be detected. Also, some of them are rectangular spheres, decahedral, and polygonal in shape. Being well dispersed with no aggregation, AgNPs have a size between 1 and 50 nm with an average size of 24.79 nm. Noteworthy is the fact that the difference in the size distribution of NPs can be related to the contribution of various molecules and functional groups to the reduction of silver ions and stabilization of AgNPs [12]. The formed AgNPs are surrounded by a thin capping layer of material obtained from the aqueous extract of C. vulgaris, which can be spotted by these images. The capping agent substantially prevents the Ag NP aggregation and consequently leads to the formation of NPs with higher stability [27]. The shape of particles has a significant role to play in the stability of NPs, and spherical NPs are highly stable in comparison with other shapes of NPs [18]. Also, a zeta potential value of −17 mV of the formed NPs is an excellent demonstration of high stability of particles in the nanoscale.

Antibacterial activity of the formed AgNPs
The antibacterial effect of AgNPs on S. typhimurium, as well as S. aureus bacteria, as a function of the volume of the synthesized AgNPs, is shown in Figure 9. As revealed in this figure, increasing the volume of the AgNP solution has a considerable detrimental effect on the bacterial growth rate for both bacteria. In the case of S. aureus, decrease in the number of colonies in the second, third, and fourth plates can be noticed, and the fifth tube (A5) is empty without any bacteria. In the case of S. typhimurium, the formed AgNPs have a more substantial antibacterial effect on this bacterium (Gram−) in comparison with the first one (Gram+) and the plates empty without any bacterium are shown in B3-B5 in Figure 9. The free positive ions of NPs stick to the negatively charged cell walls of both types of bacteria and apply their antibacterial effect on them. Studies in the past few years also confirmed higher resistance of Gram-positive bacteria against AgNPs in comparison with Gram-negative bacteria [50], which can be justified by the difference in the structure of the cell walls. Being covered by a layer of lipopolysaccharides and peptidoglycans, the cell of Gram-negative S. typhimurium is more susceptible to the entrance of  AgNP ions. In Gram-positive bacteria, such as S. aureus, the peptidoglycan layer as a protective coating is thicker when compared with that of Gram-negative bacteria. Furthermore, lipopolysaccharide with a negative charge can be another remarkable reason. The Gram-negative bacteria are coated with this molecule, which has a considerable affinity for NP ions. As a result, the growing uptake of ions leads to intracellular destruction. The findings in this article are in line with those of Jo et al. [40] and Priyanka Singh et al. [43], indicating that the synthesized AgNPs have a more destructive impact on Gram-negative than Gram-positive bacteria.

Concluding remarks
This study demonstrated an unsophisticated green synthesis of AgNPs, as a feasible alternative to the chemical as well as physical methods, by effectual bioreduction of Ag + using C. vulgaris, actively acting as reducing and stabilizing agents. With regard to the dried extract of C. vulgaris, various functional groups that existed in this extract are responsible for the synthesis. Instead of other time-consuming energy supplies, microwave irradiation was used as an abundant energy source. Mostly spherical NPs with size ranging from 1 to 50 nm and an average size of 24 nm could be monitored by SEM and TEM. Also, the distinctive inhibition effect of the formed NP against a variety of bacteria was represented. As future directions, another natural stabilizer beside C. vulgaris can be used to obtain AgNPs with higher stability. Due to the potential of C. vulgaris in being used as both reducing and stabilizing agents, the synthesis of other metal NPs can also be examined. There are other heating methods such as autoclave that can be assessed as an alternative energy supply to microwave irradiation to find out whether they work or not. Besides the considerable progress in nanobiotechnology, the more futuristic visions of nanoscience consist of the widespread use of NPs in biomedical and pharmaceutical applications.

Declaration
The abstract and preliminary results of this article were presented at the International Conference on Life and Engineering Sciences (ICOLES, July 2019), Istanbul Medipol University, Turkey.