Silver Nanoparticles Synthesized Using Eichhornia crassipes Extract from Yuriria Lagoon, and the Perspective for Application as Antimicrobial Agent

Abstract

The antimicrobial effects of silver (Ag) ions and salts are well known. However, the antimicrobial effects, mechanism, and the cytotoxic activity in vitro of Ag nanoparticles (AgNP) has recently been validated. In this work, we report the green synthesis of AgNPs using the extract of Eichhornia crassipes as a reducing agent and evaluate its antimicrobial activity against Escherichia coli (ATCC-25922). The morphology, size, chemical composition, and inhibition properties of the nanoparticles as a function of the reduction time and temperature were analyzed. According to TEM imaging, nanoparticles with average diameters between 20–40 nm were synthesized. Antibacterial results suggest that AgNPs can be used as an effective growth inhibitor with higher antimicrobial activity against Escherichia coli after 120 min of reaction with a synthesis temperature of 95°. More extensive analysis is required for the appropriate selection of the synthesis parameters and adequate concentration for use in biomedical applications and antibacterial control systems.

1. Introduction

The use of antibiotics is required for the treatment, control, and prevention of infectious diseases. Generally, and due to genetic mutations, bacteria can develop antibiotic resistance, increasing the spreading risk [1]. Among the resistant microorganisms with greater clinical importance are [2,3]: Enterobacter cloacae, Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli, Mycobacterium tuberculosis, Neisseria gonorrheae, Salmonella, Staphylococcus aureus, and Campylobacter. To mitigate this problem, scientists are seeking to develop agents with increased antibacterial activity.

Recently, nanotechnology has revolutionized several scientific areas in search of alternatives to improve the human living conditions such as health field, social environment, and environmental [4,5,6]. Scientists have generated nanometer-scale materials with very specific properties for targeted applications [7]. In the health field, silver nanomaterials have been studied and manufactured by different synthesis methods according to their specific application [8,9,10,11,12,13]. Although pure and well-defined nanoparticles can be produced with chemical and physical methods, these methodologies are expensive and potentially dangerous to the environment because of the chemical reagents used [14,15,16,17]. On the other hand, the use of biological systems such as microorganisms, plant extracts, or plant biomass could be an alternative to produce nanoparticles in an ecological way compared with chemical and physical methods [18,19,20]. For instance, the green synthesis of silver nanomaterials has been reported using plant extracts from Aloe vera, Crocus sativus, Datura stramonium, Geranium, Ricinus communis, among others [21,22,23,24,25,26]. Characterization techniques have shown a nanoparticle size between 5 to 80 nm, with excellent antimicrobial activity.

The water lily (Eichhornia crassipes) is a free-floating, perennial plant with a root fibrous that can reach 3 m in length [27,28]. The water lily is considered among the 100 most invasive species because of its rapid growth rate and propagation. The overpopulation of this plant has negative effects, such as the hindrance of navigation through rivers and lagoons, the elevation of evaporation rates, and the impediment from the passage of light to the bottom of the ponds, which results in the elimination of microalgae that are the food of crustaceans and fish [29]. This problem has already occurred in the Yuriria Lagoon in Mexico. From a physicochemical approach, the water lily is composed mainly of cellulose, hemicellulose, and lignin, which form polysaccharides whose function is to provide structure to the plant [30]. It has been shown that the extracts from this plant can generate silver nanoparticles with antimicrobial activity. However, the information and its availability are still limited.

To fully understand this green route and take advantage of the Eichhornia crassipes from the Yuriria lagoon, silver nanomaterials have been obtained using the extract as a reducing agent. Different parameters were evaluated such as the synthesis temperature and the reaction time. These nanoparticles were subsequently characterized by UV-VIS spectroscopy and TEM, XPS, and XRD techniques. Finally, the antimicrobial activity against the gram-negative (-) microorganism Escherichia coli (ATCC-25922) was studied.

2. Materials and Methods

2.1. Extract Preparation

To obtain the extract, fresh leaves of Eichhornia crassipes from Yuriria lagoon in Mexico were collected. The leaves were washed and cut into small pieces. 20 g of processed leaf were placed in a flask with 100 mL of distilled water. The mixture was brought to boiling point and maintained at this temperature for 5 min. Finally, the solution was filtered and placed in storage at 4 °C.

2.2. Green Synthesis of Silver Nanoparticles

10 mL of leaf extract was added to 90 mL of silver nitrate (AgNO₃) solution (1 mM) as a silver ion reducing agent. The effect of the reaction was evaluated at different temperatures (75, 80, 85, 90, and 95 °C). The reduction of silver nitrate as a function of time was also evaluated at different time intervals (0, 30, 60, 120, and 180 min) for each temperature.

2.3. Nanoparticles Characterization

Measurements by UV-VIS spectrophotometry was performed with a 2000 C NanoDrop system (Thermo Fisher Scientific, Wilmington, NC, USA). All spectra were recorded from 200 to 900 nm with a resolution of 1 nm. Transmission Electron Microscopy (TEM) was carried out using a JEOL JEM-1010 microscope operated at 80 kV (JEOL JEM-1010, Peabody, MA, USA). X-ray photoelectron spectroscopy (XPS) was carried out using a K-alpha Thermo Fischer Scientific spectrometer with a monochromatic Al K radiation (1486.6 eV) as an X-ray source and were micro focused at the source to give a spot size on the sample of 400 microns in diameter (Thermo Fischer Scientific, Wilmington, USA). XPS survey and high-resolution scan spectra were collected using analyzer pass energies of 120 and 40 eV, respectively. The samples remained in the pre-chamber for 15 h and later transferred to the analytical chamber with a base pressure of 1 × 10⁻⁹ Torr. In order to calculate the proportion of metallic silver in the nanoparticles, the experimental data from detailed scan was fitted by using the same binding energies for the tree samples. XRD measurement was recorded in a diffractometer from Rigaku using an X-source of CuKα, operated with a step size 2θ = 0.01° and a step time of 1 s.

2.4. Antimicrobial Activity

Sterile filter paper sensidiscs were immersed for 2 h in the nanoparticle suspensions, placed in nutritive agar medium, and inoculated with strains of Escherichia coli (ATCC-25922) by simple streak technique. The sensidiscs were incubated for 36 h and the inhibition halos presented were analyzed.

3. Results and Discussion

Figure 1 shows the different colorations in the suspensions. It was reported that quercitin, a strong antioxidant flavonoid, is presented in Eichhornia crassipes plants at high concentration levels. This compound has keto-enol isomerism in the OH groups that release hydrogen form the molecule. The free hydrogen is believed to be responsible for the reduction of silver ions into metallic state [31]. It is noteworthy to mention that the increase in coloration is directly proportional to the reaction time, also being more intense and darker at higher temperatures. It has been proven that the increase in concentration causes the color change from transparent to yellow and later to brown. This is due to the excitation of the surface vibrations of the plasmon in the metallic nanoparticles [32]. These changes were observed in the four temperatures studied.

3.1. UV-VIS

Figure 2A shows the behavior of the absorbance and the resonant plasmon of silver for colloidal solutions obtained at a temperature of 75 °C. A plasmon shift (376 to 408 nm) and an increase in absorbance as a function of time (0, 30, 60, 120, and 180 min) were observed, reaching a maximum absorbance at 180 min of reaction. The absorbance increase has been associated with a higher concentration of metallic silver, while the displacement of the maximum peak towards longer wavelengths is mainly owing to the increase in particle size. The trends described at 75 °C were maintained at 80 °C and 85 °C, however, at these reaction temperatures the displacement towards higher wavelengths was less marked (403 to 417 nm).

At reaction temperatures of 90 and 95 °C, a gradual shift was observed in the maximum absorbance of the resonant plasmon, 404–428 nm and 390–411 nm, respectively. However, the maximum absorbance value did not continue growing for reaction times longer than 60 min. Only shifts of the maximum or changes in peak amplitude were recorded, indicating that the nanoparticle size distribution is broad or polydisperse [33,34,35]. These results are similar to those previously reported using chemical methods (398–406 nm at 65 °C using NaBH₄) and green route (434 nm with M. Wightiana) [36].

3.2. TEM Results

The micrographs acquired at 75 °C are shown in Figure 3. The main sizes obtained are 22.7 $\pm$ 9.4 nm, 28.9 $\pm$ 7.1 nm, and 33.5 $\pm$ 10.1 nm, for 10, 30, and 180 min of reaction respectively. This shows a gradual increment in the nanoparticle main size as the reaction time increased. However, the nanoparticle growth behaved differently at 95 °C (Figure 4), as the nanoparticle size remained essentially unchanged from 60 min to 120 min (26.7 $\pm$ 6.8 nm and 26.8 $\pm$ 6.8 nm, in the same order). Figure 5 shows the effect of temperature reaction after 180 min in reaction. The particle main size was similar at 75 °C and 80 °C (32.58 $\pm$ 6.4 nm and 27.2 $\pm$ 7.9 nm, respectively). This behavior was expected due to the small difference in temperature compared to the others. Besides, the size was substantially increased when the reduction temperature rinsed up to 90 °C (38.99 $\pm$ 10.1 nm). An irregular shape was observed in all temperatures and times studied. Agglomeration of nanoparticles was observed in all synthesis, and it was associated with the residues of the extract, as evidenced by the less electrodense cloud around the nanoparticles. All observations by TEM agreed with those obtained by UV-Vis.

3.3. XPS Analysis

A survey scan was acquired to identify the presence and quantification of any element in the samples obtained at low (75 °C), intermediate (90 °C), and high temperature (95 °C) (Spectrum not shown). The content of C, O, and N corresponds to $\approx$90% of the total weight of the three samples. the concentration of Ag was 1.9, 2.5, and 1.5 wt.% for the nanoparticles synthesized at 75, 90, and 95 °C, respectively. The remaining weight was conformed to the sum of K, P, Cl, Mg, Si, Zn, S, Ca, and Na elements. The high content of C, O, and N was expected because the nanoparticle purification was not carried out to maintain the nanoparticles stabilized by the biomass.

Detailed XPS analysis was performed from 364 to 370 eV (Ag 3d_5/2) with the aim of spreading the knowledge in the silver species composition (See Figure 6). The experimental data for the three temperatures were deconvoluted in two peaks centered at 368.1 eV and 367.7 eV and were assigned to the presence of Ag⁰ and Ag⁺¹, respectively. These binding energies matched with those already reported for silver nanoparticles synthesized by green methods and were the same for the tree samples [37,38]. A high Ag⁰ concentration (73%) was obtained at high temperature (95 °C), whereas at low (75 °C) and intermediate temperature (90 °C) the concentration of metallic silver was very low (28% and 18%).

3.4. XRD Analysis

In order to verify the interesting results obtained at 90 and 95 °C by XPS, where the metallic silver concentration varied significantly, XRD analysis was done, as shown in Figure 7. The corresponding diffractions centered at 2Ɵ = 38.121, 44.307, 64.45, and 77.4° correspond to the crystallographic planes [1 1 1], [2 0 0], [2 2 0], and [3 1 1] from the Ag metallic with face-centered cubic structure (JCPDS No. 040783) [38]. The peaks located at 2Ɵ = 32.8, 38.02, and 54.98° correspond to the crystallographic planes [1 1 1], [2 0 0], and [2 2 0] from silver oxide Ag₂O with cubic structure (JCPDS No. 761393) [39]. When the 100% intensity diffractions are compared between Ag⁰ y Ag₂O (indicated by arrows in the graph), it can be observed that the intensity of Ag₂O is greater than the Ag⁰ for the nanoparticles synthesized at 90 °C, while for the nanoparticles synthesized at 95 °C, the opposite result was obtained. This result indicated a higher concentration of Ag⁰ in nanoparticles synthesized at 95 °C, and confirmed the observations made by XPS.

3.5. Antibacterial Activity

It is well known that Ag ions and compounds are inorganic nanoparticles of interest because of their strong antimicrobial effects. Owing to the resistance of some microorganisms, new alternatives are required to inhibit them [40,41]. The antibacterial activity of the AgNPs was analyzed against E. Coli. The analysis considered different times (0, 30, 60, 120, and 180 min) and temperatures (75, 80, 85, and 95 °C). Positive and negative controls that can determine the validity and comparison in the tests at different times and temperatures were also included. Figure 8A,B showed a successful growth because of the absence of substances that inhibit its growth (negative control), in this case using distilled water and Eichhornia crassipes extract. On the other hand, when it is used amoxicillin as an antibiotic, the growth is null (control positive).

Figure 9 shows the antimicrobial tests for E. coli with nanoparticles synthesized at 80, 85, and 95 °C for 30, 60, 120, and 180 min of reaction. Although the first time studied in the present report was 30 min, it has been established that the inhibitory effect is initial (although not total). From the zero-exposure time, this suggests that silver nanoparticles can be used as growth inhibitors of Escherichia coli, so they can be applied in a variety of devices and antimicrobial control systems [42].

Table 1. Average diameters (mm) from inhibition halos for Escherichia coli.

		Temperature (°C)
Time (min)	75	80	85	95
0	1.33 ± 0.47	0.92 ± 0.11	1.23 ± 0.2	1.87 ± 0.12
30	1.5 ± 0.4	1.13 ± 0.18	1.16 ± 0.23	2.03 ± 0.23
60	1.66 ± 0.23	1.06 ± 0.09	1.3 ± 0.16	2.18 ± 0.23
120	1.33 ± 0.47	1.36 ± 0.44	1.1 ± 0.14	2.3 ± 0.16
180	1.16 ± 0.23	0.73 ± 0.3	0.58 ± 0.11	0.92 ± 0.11

shows the inhibition halo for all temperatures and times studied. A maximum point of inhibition is reached with the nanoparticles synthesized at 120 min for 80 °C, 85 °C, and 95 °C, while it is at 60 min for 75 °C. In general, as temperature and time increase, the diameter of the halo inhibition zone increases until it reaches a maximum point. It is noteworthy that the higher inhibitions were for temperatures of 75 °C and 95 °C. This could correlate with the size of the nanoparticle, since it was determined by TEM that the average diameters of the largest nanoparticles (superior to 32 nm) were obtained at 80 °C and 85 °C.

Interestingly, when it is compared the inhibition effect between nanoparticles synthesized at 75 and 95 °C, the degree of inhibition does not correlate with nanoparticle size. At 95 °C larger inhibition halos were obtained, but at 75 °C the particle size is smaller. This discrepancy could be related with the silver oxidation states and the inhibition mechanism: in the case of gram-negative microorganisms, such as Escherichia coli, the mechanism of action is not fully understood. However, it is believed that the positive charge of the Ag ion interacts with the negative charge of the cell membrane. In this way, the Ag nanoparticles accumulate in the membrane, causing cell death [43,44,45]. The higher proportion of metallic silver found by XPS and XRD in nanoparticles synthesized at 95 °C suggests that the nanoparticles have a high charge density. This could explain the greater inhibition effect found with these nanoparticles. Future investigations will broad the knowledge the effect of the chemical oxidation on the inhibition properties of silver-based nanoparticles. In different research works related to the synthesis of silver nanoparticles by a green methodology, they have also been evaluated in Escherichia coli bacteria as an antibacterial alternative [46,47].

Table 2. Comparison of inhibitor diameter previously reported against Escherichia coli.

References	Size (nm)	Extract	Concentration (µg/mL)	Inhibition Halo (mm)
This work	22–30	Eichhornia crassipes	7.2	2.3 ± 0.16
Chandrasekharan, et al. [46]	34–40	G. arborea	40	23.0 ± 1.73
Vinodhini. et al. [47]	50	B. alba	10	9
	55	T. divaricata	10	11
	57	A. fistulosum	10	11

shows the parameters used for antibacterial test and the average inhibitor diameter previously reported. Although the inhibition halo is smaller compared to previously reported works, the concentration used in this work is lower. This gives the suspension an opportunity to be studied and considered in future analysis against bacteria and other microorganisms, where at the same time a plant considered an invasive plant is used.

4. Conclusions

In this work, the green synthesis of silver nanoparticles (AgNPs) was performed using the extract of Eichhornia crassipes obtained from Yuriria lagoon in Mexico. The formation of the nanoparticles was confirmed by the color changes and UV-Vis spectra measurements with maximum plasmon peaks between 350–450 nm. Particle sizes between 20 to 40 nm with an irregular shape at all temperatures and reaction times were measured by TEM. The most effective antimicrobial activity against Escherichia coli was identified in the nanoparticle solution at 95 °C and 120 min of reaction. This inhibition effect was explained in terms of the size and the metallic silver proportion in the nanoparticles. Therefore, the nanoparticles analyzed in the present work could be used in biomedical applications and antibacterial control systems against gram-negative microorganisms.