Phytosynthesis of TiO 2 Nanoparticles Using E. crassipes Leaf Extracts, Their Photocatalytic Evaluation and Microbicide Effect

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Introduction
Titanium dioxide is a semiconductor material with excellent light transmission, high refractive index, and good dielectric properties [1]. Because it is not harmful to the environment and humans, it has been widely studied, but more importantly, it is used in various fields, such as medicine, cosmetics, optics [2], the packaging industry, as well as pigments in paints, inks, and coatings [3], photocatalysts, and as an antibacterial agent [4].
In general, TiO 2 NPs are an important semiconductor material with a large specific surface area and special chemical properties such as easy control, limited cost, nontoxicity, and resistance [5]. They are the best photocatalytic and microbial agents because of their environmental friendliness, low cost, high oxidative capacity, and chemical and thermal safety against photocorrosion [4].
Freshwater pollutants include bacterial microorganisms, as well as simple and complex chemicals [6]. Phenol and its derivatives are typical pollutants in wastewater and effluents from various industries, including petrochemicals, pharmaceuticals, textiles, pesticides, resins, manufacturing, steel, and herbicides [7,8].
However, the environmental policy regarding the quality of discharged effluents is increasingly tight, which leads to the need for new resolutions for the water process. Advanced oxidative processes (AOPs) are promising techniques that have the potential to be used as alternative processes or polishing steps in the effluent process [9]. Heterogeneous photocatalysis using semiconducting metal oxides, such as TiO 2 NPs, is an effective and promising approach to water purification [8,10].
The main mechanism of heterogeneous photocatalysis is based on the illumination of the photocatalyst (TiO 2 NPs) with the light of appropriate wavelength, with an energy that is equal to or greater than the bandgap, giving rise to the creation of electron/hole (e-/h+) pairs at the conduction (CB) and valence bands (VB), respectively. The photogenerated charges can react with the adsorbed H 2 O (OH-) and O 2 molecules on the semiconductor surface where it participates in redox reactions to form OH•-and O 2 •radicals, which are the active species in the photodegradation process, for removing organic contaminants by converting them into CO 2 , H 2 O, and inorganic ions [4,[8][9][10].
The antimicrobial activity of nanoparticles is strongly influenced by various intrinsic components such as their morphology, size, chemistry, source, and nanostructure, and the function of interaction with cells, their structures, and cellular metabolites [11]. Specifically, the antimicrobial activity of TiO 2 NPs strongly depends on the photocatalytic performance of TiO 2 , which is highly dependent on its morphological, structural, and textural characteristics [1].
Biosynthesis, as a means of synthesizing metal oxide nanoparticles, has proven to be one of the best synthetic processes, primarily because of its environmentally friendly principles and the potential to create new custom materials [7]. Phytosynthesis is considered a safe, cost-effective, biocompatible, nontoxic, sustainable, and environmentally friendly process [1]. The phytochemicals constituents of leaves of Eichhornia crassipes like tannins, alkaloids, flavonoids, saponins, and phenolic compounds [12,13].
In this study, the synthesis of TiNPs using Eichhornia crassipes plant extract was conducted. Their photocatalytic and antibacterial activities were investigated by measure-ment of the degradation of phenol and bacteria (E. coli and S. aureus), respectively, was investigated.

Phytosynthesis of Nanoparticles. Eichhornia crassipes
were collected from at the Valsequillo reservoir in Puebla. The leaves were washed and dehydrated in a Riossa H-33 drying oven at 50°C for 36 h. About 125 mg of the E. crassipes leaf was mixed with ethanol (80% v/v). The aqueous solution was then filtered, and its filtrate was mixed with TiO 2 (0.1 M, Sigma-Aldrich, anatase). Using solutions of NaOH (0.1 M, J.T. Baker) and HNO 3 (0.1 M, J.T. Baker), the pH = 12, 7, and 4 to stablish the pH effect [14,15] were modified, respectively, and the new solutions were homogenized in an ultrasonic bath (Bransonic Emerson; Model: M2800H). The modified nanoparticles (NPs) at pH 12, 7, and 4 values were separated from the supernatant through centrifugation (Frontier 5714 OHAUS; Model: FC571) at 5000 pm. The supernatant was decanted, and the precipitate was collected, keeping it at room temperature in a desiccator, inside amber bottles, in the dark, for later analysis.

2.2.
Characterization. The present study has described the synthesis of TiO 2 NPs by the E. crassipes leaf extract. The surface plasmon resonance (SPR) of the phytosynthesized nanoparticles was examined by using UV-Vis spectroscopy (Thermo Scientific Evolution Array) in the range of 300-700 nm. Indirect bandgap energies were determined by the Tauc equation (Equation (1)) that was used to find the energy bandgap as a function of the absorption coefficient and excitation of the transmission [16]   2 International Journal of Photoenergy where α is the extinction coefficient, hv is the photon energy, γ is a constant that can take any integer value based on the raw material and the optical transmission values β is the constant and has different values for different transitions, and E g is the energy gap [16][17][18][19].
The bioactive functional groups of TiO 2 NPs were confirmed by Fourier Transform Infrared (FT-IR) analysis (Varian 640-IR with ATR of the diamond), in the range of 4000 to 500 cm -1 . Morphology, particle size distribution, and crystallinity were conducted using Transmission Electron Microscopy (TEM-2010 FEG); Micrograph Digital software was used to determine the average particle size of tips.

Photocatalytic Degradation of Phenol.
The photocatalytic activity of the synthesized materials was evaluated by monitoring the degradation of phenol (10 mg·L -1 ). The photocatalytic degradation of phenol (determined by 4aminoantipyrine method for total phenols at λ = 510 nm) at three different concentrations (100, 50, and 10 mg·L -1 of TiNPs), at a standard dose of 1 g·L -1 . A solution with TiO 2 NPs was initially stirred for 1 h in the absence of light to obtain an adsorption-desorption equilibrium. UV irradiation was provided by one UVA lamp (UVP UVL-18 EL, λ = 254 nm, 8 w) into a lab-made photoreactor, placed at 10 cm from the samples. During the photocatalytic experiments, at precise time intervals, samples were taken out at regular time intervals, and the nanoparticles were separated from the aqueous solution by a nitrocellulose membrane of 0.45 μm (Whatman), and the supernatant solution was measured to determine the phenol decomposition rate using the UV-Vis spectrophotometer. The solution was purged with air all the time to dissolve oxygen.
The percentage of photodegradation of phenol can be estimated using the following equation: where C o is the initial concentration of phenol and C t is the concentration of phenol after photoirradiation at a given time t [4,7]. The Langmuir-Hinshelwood kinetics model was used to study the kinetics of the photocatalytic reaction on the surface of TiO 2 NPs, the equation is as follows: where C o and C t represent the concentration of phenol before degradation (C o ) and at different times during photocatalytic degradation (C t ), respectively [4,5,7].

Results and Discussion
3.1. Characterization of TiNPs. The TiO 2 nanoparticles exhibited surface plasmon resonance (SPR) at a wavelength of 327 nm (Figure 1), and the optical bandgap energy of the sample was calculated at 3.2 eV (Figure 1(a)). Changes in pH (pH 12, 7, and 4) during the synthesis of plant nanoparticles lead to changes in photocatalytic and bactericidal activity, but SPR and bandgap energy remained unchanged [5]. Ziental et al., [21] and Nobijari et al., [8], synthesized TiO 2 nanoparticles with Monsonia burkeana extract, and mentioned that the band gap value of 2.3 eV is like that of the anatase TiO 2 . This can lead to the production of highenergy electrons. Whole pairs (e-/h) in UVA light with a wavelength of 385 nm or less [1,9].  3 International Journal of Photoenergy FT-IR spectrum of TiO 2 NPs at pH 12, 7, and 4 shown in Figure 2, the peak region of 2161 cm -1 showed the presence of stretching bands of the carbonyl group [22], 1396 cm -1 corresponds to C-N stretching of the aromatic amide group [23], in general, the absorption bands that appear from 2358 to 1361 cm -1 correspond to the phytochemicals present in the extract of E. crassipes from Puebla.
For all samples, the peaks identified from 536 to 532 cm -1 confirm the formation of TiO 2 nanoparticles, through the phytosynthesis process, the intense band below 1000 cm -1 belongs to TiO 2 nanoparticles bridging stretching mode [5,8]; according to the spectra, the phytochemicals from E. crassipes extract act as coating agents in the synthesis of nanoparticles TiO 2 NPs.
According to the presented characterization results, it is not observed that the used pH value for the synthesis shows a determining influence on the size, shape, or composition of the nanoparticles, these three parameters differ for the three pH values. Similar observations were also reported by Shanavas et al., [24], Thakur et al., [25], Srinivasan et al., [23] whose synthesized nanoparticles with Phyllanthus niruri, Azadirachta indica, and Sesbania grandiflora obtained results similar to those of the present work. Figure 4 shows the phenol degradation profiles in the presence of TiO 2 NPs pH 12 (Figure 4.1), Ti O 2 NPs pH 7 (Figure 4.2), and Ti O 2 NPs pH 4 (Figure 4.3), at concentrations of 100 mg·L -1 , 50 mg·L -1 , and 10 mg·L -1 of NPs. Before turning on the light, phenol concentration was observed to drop 0.7 mg·L -1 at pH 12 and 7, and rise to 2 mg·L -1 at pH 4 due to its adsorption/desorption on the surface of TiO 2 NPs [8], observing the same phenomenon. After switching on the UV light, the phenol concentration decreases gradually with irradiation time.

Photocatalytic Degradation of Phenol.
According to the relationship of the characterization results for TiO 2 NPs and photocatalytic capacity, no determining influence on photodegradation due to the nanoparticles' size, shape, or composition. Although it cannot be generalized in any of the cases, the main influence is due to the used pH for the phytosynthesis of the nanoparticles, followed by the size diversity, then the composition, and finally the crystalline structure. Regarding the initial phenol concentration, in most photocatalytic tests, the best percentage of degradation is achieved at a lower concentration of nanoparticles.
At concentrations of 100 mg·L -1 , the degradation efficiency of phenol decreases, according to Nobijari et al., [8], when the catalyst concentration is too high, other effects such as light scattering and poor illumination of the photocatalyst particles become dominant.
Langmuir-Hinshelwood's kinetics model was used to study the kinetics of the photocatalytic degradation of phenol. Parameters of adsorption were observed in Table 1. The R 2 values of 0.9284, 0.9443, and 0.9467 that had the best fits were for TiO 2 NPs pH 12-50 mg L -1 , TiO 2 NPs pH 7-100 mg·L -1 , and TiO 2 NPs pH 4-50 mg·L -1 , respectively. The R 2 coefficient value did not indicate a good correlation between the Langmuir and Hinshelwood kinetics model. This observation could be due to the multiple processes taking place during photocatalytic degradation [5].  NPs pH 12 has shown that E. coli is the most resistant to TiO 2 NPs in comparison to S. aureus. [11] in his study synthesized titanium nanoparticles with extract of Thalictrum minus L. ssp. Elatum obtained similar results.
The inhibition of E. coli and S. aureus by TiO 2 NPs at pH 12, pH 7, and pH 4 presented the best results at concentrations of 100 mg·L -1 of nanoparticles, [26], mention that the population of bacteria declined when the concentration of nanoparticles increased. The results showed that Gramnegative are slightly less resistant to TiO 2 nanoparticles compared to Gram-positive, which may be mainly due to differences in the structures of bacterial membranes [1,27].
TiO 2 NPs interact with the surface of bacterial membranes and alter their cell permeability, penetrate bacterial cells, leading to the generation of ROS (Reactive Oxygen Species), causing damage to the cell membrane, inducing lysis, and consequently giving rise to cell death. Moreover, the antimicrobial activity of TiO 2 NPs is related to their structure, shape, and size, as well as the production of ROS [28].

Conclusions
The phytosynthesis of TiO 2 NPs using Eichhornia crassipes leaf extracts was ecological, sustainable, cheap, and free of contaminants that satisfy the requirements to be an environmentally friendly process. UV-Vis spectrophotometric analysis reveals that phytosynthesized nanoparticles have values in the appropriate range of 327 nm and 3.2 eV of bandgap energy from the Tauc plot; TiO 2 NPs show significant peaks identified at 538 and 532 cm -1 confirming the presence of TiO 2 . All the TiO 2 NPs phytosynthesized have a tetragonal form, composition of TiO 2 , and particle sizes ranging from 20 to 50 nm; TiO 2 NPs show better photocatalytic efficiencies at acidic pH and concentrations of 10 mg·L -1 . Antibacterial activity against Gram-positive and Gram-negative bacteria is efficient at concentrations of 100 mg·L -1 for all the TiO 2 NPs phytosynthesized.

Data Availability
All data generated or analyzed during this study are included in this published.

Conflicts of Interest
The authors declare that they have no conflicts of interest.  International Journal of Photoenergy