Abstract

Agave tequilana Weber var. azul fibers (ATF) are widely used as a reinforcement material despite their polarity makes them incompatible with hydrophobic matrices. Consequently, ATF are commonly modified employing different chemical processes (e.g., mercerization and coupling agents) to change their surface characteristics to improve the interface between the fibers and the polymeric matrix. Nevertheless, these treatments could damage the fibers during the process, negatively affecting their natural properties. The use of nanotechnology to repair this natural material could help to restore its intrinsic properties and give it new ones as antibacterial activity. In this work, chemically treated ATF were used as templates for the biosynthesis of silver nanoparticles (AgNPs) using a natural extract obtained from Agave tequilana Weber var. azul leaves (ATL) as reducing agent. Scanning and transmission electron microscopy images as well as dynamic light scattering results indicate that stable nanometric particles were successfully synthesized on all fibers. X-ray photoelectron spectroscopy and X-ray diffraction results confirm the composition of the nanoparticles. Tensile tests indicate that AgNPs improved the mechanical properties of fibers previously mercerized and treated with maleic anhydride grafted polyethylene as coupling agent. Additionally, an antibacterial effect against S. enterica was conferred to ATF due to the presence of AgNPs.

1. Introduction

Natural fibers are widely used as a reinforcement material in different kinds of composite materials (e.g., wood plastic composites (WPC)) despite lignocellulosic fibers are incompatible with hydrophobic matrices due to their polarity [1, 2]. To enhance the poor interaction between natural fibers and hydrophobic matrices, different chemical treatments are employed (e.g., mercerization and coupling agents); as a result, the mechanical properties of the final product are improved due to a better interface between the fibers and the polymeric matrix. Mercerization consists in immersing fibers in an alkaline solution removing pectin, waxy substances, and natural oils present on the surface layer of the fibers. Subsequently, a rougher surface with a more porous structure is produced. However, it could decrease the mechanical properties of natural fibers due to fibrillation. Mercerization is frequently used as a pretreatment to increase the efficiency of any further chemical treatment (e.g., coupling agents). Coupling agents are easier to handle than alternative methods used to improve the interfacial bonding between wood and thermoplastics. Coupling agents chemically interact with natural fibers and mechanically with the hydrophobic matrices enhancing the compatibility and consequently achieving a better interfacial bonding [26].

On the other hand, during the last two decades, metallic nanoparticle (MNP) research has increased due to the wide range of properties that make them suitable for different applications [7]. Among the available synthesis methods, green routes constitute cost-effective and environmentally friendly alternatives to traditional processes (i.e., chemical and physical) used to obtain MNPs. Particularly, plant-mediated methods offer potential benefits due to the absence of toxic chemicals, nonbiological risk, low cost of production, and the use of natural capping and reducing agents [811]. Currently, a wide variety of plant extracts have been successfully used in the synthesis of metallic nanoparticles due to the presence of different phytochemicals and biopolymers that act as reducing agents (e.g., polysaccharides, terpenoids, flavonoids, phenolic compounds, saponins, and lignocellulosic materials). Although the mechanistic aspects of the green synthesis process are still unknown, it has been reported that phytochemicals reduce metallic ions through functional groups such as –CO, -OH, and -NH present at their constituent molecules [10]. A possible chemical reaction on how silver nanoparticles (AgNPs) could be produced using Agave tequilana Weber var. azul extract (ATE) is proposed and described in Figure 1. As a part of the synthesis process to obtain AgNPs, Ag+ must be reduced to Ag° in the presence of reducing molecules. To do so, sucrose present in Agave tequilana Weber var. azul leaves (ATL) needs to be hydrolyzed to obtain glucose and fructose. Glucose belongs to the family of aldoses, which are characterized by the presence of an aldehyde group, which in the presence of Ag+, water and heating promotes the oxidation of carbon up to the formation of a carboxylic acid group, releasing two hydrogen protons (H+) and the reduction of silver (Ag°) to obtain AgNPs. An alternative process would be focused on the reducing molecule fructose, which belongs to the ketose family. The ketone’s carbon in contrast to the aldehyde’s carbon is at its maximum degree of oxidation, which nulls out the possibility that Ag+ could oxidize this carbon, as well as the possibility that this substrate could be the first oxidized molecule during the synthesis of nanoparticles. However, in contrast to glucose, fructose shows two terminal –OH groups, which can be more easily oxidized. From this analysis, it is proposed that during Ag+ reduction process, for each glucose oxidized, two Ag° nanoparticles can be reduced, and once glucose is no longer available in the media, fructose will be now oxidized.

Among different MNPs, AgNPs have caught the world’s attention due to their novel chemical, physical, and biological properties. So far, green synthesized AgNPs have been used in pharmacological applications and wastewater treatment, as well as in the cosmetic, textile, and food industry [1214]. However, potential applications of AgNPS in the development of new lignocellulosic composite materials have been scarcely studied despite the incorporation of AgNPs could improve the mechanical properties of natural fibers commonly used as reinforcement material in lignocellulosic composites and confer them new properties, expanding their possible applications [1521]. Thus, in this work, the effect of biosynthesized AgNPs on the tensile properties and antibacterial activity of chemically surface-modified Agave tequilana Weber var. Azul fibers (ATF) is reported.

2. Materials and Methods

The materials are as follows: AgNO3 (Sigma-Aldrich), NaOH (Lavoisier-Chem), maleic anhydride grafted polyethylene, (MAPE; DuPont), and xylene (Golden-Bell). Agave tequilana Weber var. azul plant was purchased from a nursery in Tequila-Mexico. ATF were donated by a tequila company of the Mexican state of Jalisco, Mexico. Experimental research and collection of plant material were performed in accordance with relevant institutional, national, and international guidelines and regulations.

2.1. Fiber Preparation
2.1.1. Agave Fiber Pretreatment

Fibers were soaked in water and washed repeatedly until all impurities were eliminated; subsequently, fibers were dried outdoors and manually separated. In a second stage, fibers were milled and sieved; untreated fibers (UF) retained on mesh 50 were collected for subsequent treatments.

2.1.2. Mercerization

Mercerized fibers (MF) were obtained soaking UF in a 2 wt% NaOH aqueous solution (fibers/solution wt. ratio: 1/10) at room temperature for 30 min. After this process, fibers were repeatedly washed and dried outdoors.

2.1.3. Coupling Agent Treatment

Mercerized fibers treated with MAPE (MMF) were obtained treating MF with a MAPE solution in xylene (3 wt%; fibers/xylene wt. ratio: 1/20) at 90°C during 30 min. After MAPE treatment, fibers were removed from the solution and dried at room temperature in an extraction hood.

3. Synthesis of AgNPs Using ATF as Template

3.1. Preparation of ATE

ATL were washed, cut and boiled (30 g/100 mL of distilled water) for 1 h. The solution was filtered to remove solid residues.

3.2. AgNP Synthesis Using ATF as Templates

ATF (UF, MF, or MMF) were poured into 100 mL of AgNO3 solution 20 mM (AgNPs20) or 30 mM (AgNPs30) during 1 h. Later, fibers were filtered to eliminate AgNO3 solution excess. Fibers impregnated with Ag+ ions were soaked in 80 mL of ATE and autoclaved during 5 min at 110°C. Finally, fibers were removed and dried at room temperature.

Figure 2 illustrates the process followed to obtain the testing samples analyzed in this work, which are listed in Table 1.

4. Characterization

Fourier-transform infrared spectroscopy: attenuated total reflectance spectroscopy was performed using a Nicolet iS50 FTIR. For each sample, 30 scans were recorded (4000-400 cm-1). Electron microscopy: SEM and TEM micrographs as well as energy dispersive X-ray spectroscopy (EDS) analysis were obtained using a scanning electron microscope FE-SEM TESCAN MIRA-3 LMU equipped with EDS operating at 20 kV and a JEOL JEM1010 microscope operating at 80 kV, respectively. Contact angle: to characterize the wetting behavior of fibers, contact angle was analyzed with a Tantec Contact Angle Meter model CAM-Micro taking TAPPI T458 test method as reference. Tests were performed at room temperature with at least five specimens per fiber. X-ray diffraction: XRD tests were performed using a Panalytical Empyream device with Cu, operating at 40 kV and 30 mA, and the scanning range (2θ) from 30° to 70°. X-ray photoelectron spectroscopy: XPS tests were performed using an X-ray photoelectron spectrometer (PHOIBOS 150, SPECS Germany) with a monochromatic Al Kα ( eV) XR50M source operated at 250 W. Wide spectra were obtained using 1.0 eV of step size and 70 eV of pass energy. High-resolution spectra data of core levels Ag3d, C1s, and O1s were collected using 0.1 eV step size and 15 eV pass energy. Higher resolution spectra of peaks were deconvoluted using software AAnalyzer1 v2.21. The C1s spectra were fitted and adjusted with a corresponding shift using reference position at 284.8 eV due to adventitious carbon presented in sample. Tensile testes: tensile properties of ATF were evaluated using a universal machine Instron model 3345 according to ASTM C1557 test method using a crosshead speed of 5 mm min-1 for five specimens of each sample.

4.1. Antibacterial Activity

Agar overlay test was used to determine the antibacterial activity of UF, MF, and MMF against gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Salmonella enterica). A culture grown overnight at 37°C of each bacterial strain was reinoculated in fresh trypticase soy broth in ratio 1 : 1000. This new culture was grown at 200 rpm and 37°C to reach an O.D. at 600 nm of 0.4. Subsequently, 100 μL of overnight culture was inoculated into 25 mL of non-gelified trypticase soy agar at 45°C, poured into the Petri dish, and allowed to cool until the first layer was formed. Subsequently, ATF with the different treatments were placed on the gelled agar. A new layer of agar not inoculated with the strain was placed to fix the fibers and allowed to cool until the overlayed gelled. The Petri dishes thus formed were incubated at 37°C for 16 h, and the inhibition halo was determined. The experiments were run in triplicate.

Additionally, minimum inhibitory concentrations (MICs) and percentage growth inhibition assays were performed. First, overnight cultures of S. aureus and S. enterica grown in LB broth media under agitation (250 rpm) at 37°C were done. Bacterial growth was monitored by measuring the optical density at 595 nm (OD595). Typical parameters derived from growth curves are as follows: length of lag phase (λ), growth rate represented by the maximum slope (μ), and the maximum cell growth (A) appears in equation (1), in which indicates growing period and indicates the growing parameter. The integral (area under the curve) is also used as growth parameter [22].

In a second stage, to determine the MICs at different concentrations (1, 0.5, 0.25, and 0.125 mg fiber per milliliter of LB medium) of modified fibers (MF20, MF30, MMF20, and MMF30), a modified standard MIC method was used [23]. To evaluate the lowest concentration of fiber (mg/mL) required to inhibit the visible growth of those bacterial cultures after overnight incubation (24 h), 2 μL of each overnight culture was added to each well of a 96-well plate and allowed to incubate under agitation (250 rpm) at 37°C for 24 h. After incubation, the 96-well plate was analyzed at OD595.

Percentage growth inhibition was calculated by subtracting mean OD values of respective blank from the mean OD value of an experimental set. Percentage growth in presence of compound was calculated considering the growth in the absence of any test material as 100% according to equation (2). The growth in the presence of compound was calculated by the following equation [24].

4.2. Statistics

Mechanical test data were analyzed with statistical software (GraphPad Software Prism 7). Data are shown as the mean and the corresponding standard error. Kruskal-Wallis test of one-way analysis of variance was performed considering tensile modulus and strength as variables. Dunn’s multiple comparison test was used for the determination of statistical significance, which was defined as a value of .

5. Results and Discussion

5.1. Agave Fibers

The qualitative surface chemical composition of UF, MF, and MMF is presented in Figure 3. The intensity of bands around 1030 cm-1 commonly attributed to C-O deformation in cellulose, symmetric C-O-C stretching, and aromatic C-H deformation of lignin, and the hydroxyl region (3080-3500 cm-1) corresponding to wood components decreased in the following order MF > UF > MMF. As it was expected, MMF showed the lowest concentration of wood-associated chemical groups due to the use of MAPE, which completely covered wood surface. Otherwise, MF showed the highest concentration of wood-associated bands, since mercerization produced a rougher surface with a more porous structure, exposing a higher surface area to the environment [25]. On the other hand, CH2 asymmetric and symmetric bands (2919 and 2851 cm-1, respectively) corresponding to main absorptions of polyethylene are clearly observed only in MMF samples due to the presence of MAPE [21, 26].

Figure 4 shows SEM micrographs of treated ATF. It can be observed that MMF (Figure 4(a)) show a smooth surface, since the coupling agent (i.e., MAPE) filled the empty space between the broken fibers, producing a hydrogen-rich surface due to the presence of polyethylene. On the other hand, MF (Figure 4(b)) show a rough surface due to the removal of lignin, pectin, and hemicelluloses, which leads to fibrillation and breaking of fiber bundles exposing a heterogeneous/oxygen-rich surface structure.

Droplet formation during contact angle tests is illustrated in Figure 5; when MF were tested, an instant absorption of water upon contact area was observed (Figure 5(a)) due to the high hydrophilic nature of the fibers as a result of the mercerization process. According to reported literature [27], contact angle could not be obtained for substrates that show such behavior. Regarding MMF (Figure 5(b)), contact angle (θ) was calculated according to equation (3), where corresponds to the height of a droplet and to the radius of the droplet’s base. θ was found to be equal to 73.72°, indicating that the fiber treated with NaOH and MAPE (MMF) shows a less hydrophilic surface than the mercerized fiber (MF). The presence of polyethylene modifies the surface of the fiber due to its intrinsic hydrophobic behavior [28]. Finally, Figure 5(c) shows an image of a droplet deposited on a fiber during contact angle tests.

Figures 6(a)6(f) show the SEM micrographs of UF, MF, and MMF reinforced with biosynthesized AgNPs, indicating that all samples were successfully used as templates no matter the previous surface treatment applied, since AgNPs are clearly observed on all samples. According to SEM images, there is not a defined distribution pattern of AgNPs in any type of fiber. In the case of UF and MF samples, the oxygen-rich surface structure of natural fibers contributed to the synthesis of nanoparticles, while in the case of MMF samples, the excellent metal-binding characteristics of polyethylene promoted the superficial presence of AgNPs [21, 29]. To analyze the morphology of the biosynthesized AgNPs, particles were detached from the fibers. SEM and TEM results indicate that a wide size distribution and different shaped nanosized particles (i.e., spheres and rods) were obtained as observed in Figures 6(g)6(i) and Figure 7, respectively, using ATE as reducing agent. So far, a wide variety of different shaped AgNPs have been synthesized as indicated in Table 2. Differences in the morphologies of biosynthesized AgNPs are due to variables such as the vast combination of biomolecules such as proteins, enzymes, polysaccharides, alkaloids, terpenoids, and vitamins obtained from different parts of the plant resources that reduce and stabilize silver ions; the reaction parameters of each particular synthesis process (e.g., temperature, stirring, and reactant ratio); and the different synthesis methods employed (e.g., autoclave, incubator, and microwave) [8].

Regarding the surface treatment previously applied to the fiber, it seems that the heterogeneous-porous surface structure of wood promoted the formation of a wider range of morphologies as well as larger particles than the polyethylene surface of MMF produced due to the presence of MAPE. EDS analysis of detached AgNPs is shown in Figure 8. AgNPs show a strong optical absorption peak at approximately 3 keV due to their surface plasmon resonance and the following composition: silver (54.44%), oxygen (30.47%), and sodium (15.10%). The presence of sodium in silver nanoparticles may be due to the mercerization treatment of the fibers used as templates and from which the AgNPs were detached. Regarding the presence of oxygen, it could represent surface biomolecules originated from ATE [30, 31].

XRD was carried out to confirm the composition and the crystalline nature of biosynthesized AgNPs. Characteristic peaks of face cubic centered (fcc) silver corresponding to the diffraction of (111) and (200) at 38.42° and 44.23°, respectively, are shown in Figure 9. Results indicate that AgNPs showed no preferential growing direction [32].

XPS tests were performed to corroborate the nature of the biosynthesized AgNPs; results are shown in Figure 10. Main peaks observed during survey scan correspond to Ag3d, Ag3p, and O1s. Due to a high concentration of AgNPs on the film layer, the Auger zone was observed and appeared at higher binding energy; also, the peak Ag3s is observed around 720 eV. Good intensity of three core levels, 3d, 3p, and 3s, of silver is presented, corresponding to the main cross sections of the element. The insert in Figure 8 of Ag3d shows the doublet of 3/2 and 5/2 spin orbitals. Once deconvoluted, the signal presents two components: the right component, which is positioned exactly at 367.9 eV with a splitting of 6.00 eV that corresponds to metallic silver, and the main component, positioned at 368.4 eV corresponding to Ag+1 due to Ag2O that matches with the oxygen content [33].

Table 3 shows the DLS measurements results, confirming the nanometric dimensions of the synthesized AgNPs. Moreover, the -potential results evidence a negative charged surface and the stability of AgNPs in water suspensions. A positive or negative charge on the surface of AgNPs gives the particle stability and prevents aggregation. According to the literature, -potentials higher than +30 mV or lower than -30 mV indicate very stable nanoparticles, which can preserve their structure over a long time [34]. According to DLS results, stable nanometric particles were successfully synthesized.

Tensile properties of untreated and treated ATF are presented in Table 1. Regarding UF/20 and UF/30 compared to UF, statistical analysis indicated that neither tensile strength nor tensile modulus show a significant change due to the presence of AgNPs. With respect to MF, it is clear that mercerization process produced a drop in the tensile properties compared to UF due to the fibrillation and breaking of fiber bundles. The presence of AgNPs in samples MF/20 and MF/30 did not improve significantly the tensile strength compared to MF. However, a significant improvement was observed in tensile modulus for MF/30 samples. Finally, it can also be observed that MMF show higher tensile properties compared to MF, indicating that although the presence of a coupling agent does not fully compensate the negative effect of mercerization, it certainly reinforces the fiber and helps to restore its original properties. Furthermore, the presence of AgNPs significantly improved both, tensile strength and modulus of MMF. According to Dunn’s tests, in the case of tensile modulus, a significant improvement was observed for MMF/20 and MMF/30 samples. On the other hand, tensile strength showed a significant change in the case of MMF/30 samples. In light of the registered micrographs and the obtained tensile results, the hydrophobic surface of MMF compared to the hydrophilic surface of both UF and MF promoted the synthesis of comparatively smaller nanoparticles, which were more homogeneously distributed in the MMF system, resulting in a better attachment of AgNPs due to hydrophobic interactions and, consequently, in a more notorious effect on the mechanical behavior.

The antibacterial activity was evaluated against different gram-positive and gram-negative bacteria (Figure 11). Biosynthesized AgNPs were found to inhibit the growth of S. enterica as shown in Figure 11(a). It can be clearly observed the inhibition halos corresponding to MF/20, MF/30, MMF/20, and MMF/30 due to the presence of AgNPs. It is known that Ag breakdown bacteria cell wall, inhibiting intracellular enzyme activity, since AgNPs are attached to ATF, the inhibition halo is produced due to the releasing of Ag ions to the agar medium [21]. In our experiments, we could not find antimicrobial activity against the gram-positive bacteria evaluated, S. aureus (Figure 11(b)); however, it is known that among the mechanisms of action of AgNPs for the inhibition of bacterial growth, gram-negative bacteria are usually more susceptible due to their own morphological characteristics and cell wall composition. In the case of gram-positive bacteria, a rigid cell wall composed of a thick layer of peptidoglycan consisting of polysaccharide chains cross linked by short peptides is observed, whereas in the case of gram-negative bacteria, the presence of a thinner layer (i.e., less rigid cell wall) of peptidoglycan facilitates the penetration of AgNPs [3537]. A comparison of our results and those of some other works regarding the antibacterial activity of biosynthesized AgNPs using plant extracts against different bacterial strains is provided in Table 4. It can be observed that gram-negative bacteria are preferentially inhibited by AgNPS. On the other hand, the variation observed in the inhibition halos is a natural process of fiber treatments; other authors have reported that the change in the distribution of nanoparticles on the fiber can affect the release of these to achieve bacterial inhibition [38, 39].

Regarding minimum inhibitory concentration (MIC) and percentage growth inhibition, the modified fibers showed inhibitory specificity against S. enterica. In the case of MF20, MF30, and MMF20 samples, a MIC of 0.5 mg of the fiber per milliliter of LB medium can inhibit up to 50% of the bacterial growth. In the case of MMF30, an MIC of 0.25 mg of fiber/mL of medium was able to inhibit the growth bacteria up to 60%.

However, despite observing that these modified fibers could have great inhibitory activity in the growth rate of gram-negative bacteria, for the gram-positive bacteria such S. aureus did not show a significant change in the rate of percentage growth. This suggests that the MIC could be above the concentration of 1 mg/mL or definitively does not have inhibitory action on the bacterial growth of S. aureus.

6. Conclusions

In the present work, the effect of biosynthesized AgNPs on the properties of surface-modified ATF was evaluated. Results indicate that the presence of AgNPs significantly increased the tensile modulus and strength of mercerized fibers treated with MAPE, especially in the case of MMF/30 samples. A less hydrophilic surface caused by the presence of polyethylene could have promoted a better attachment of AgNPs to AT fibers. AgNPs also conferred antibacterial properties to MF and MMF due to the presence of Ag ions. XRD and XPS results confirm the composition of the synthesized particles. SEM, TEM, and DLS results indicate that stable nanometric particles were successfully obtained.

Data Availability

Data will be available on request (contact information: Edgar López, [email protected]).

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

The authors want to thank Sergio Oliva-León and Ricardo Martínez-Magallanes for their technical support. This work was supported by the Centro de Ciencias Exactas e Ingenierías Universidad de Guadalajara.