Comparative Study on Enhanced Photocatalytic Activity of Visible Light-Active Nanostructures for Degradation of Oxytetracycline and COD Removal of Licorice Extraction Plant Wastewater

Abstract

This study evaluates the effects of carbon, nitrogen, and sulfur dopants on the photocatalytic activity of TiO₂ for degradation of oxytetracycline (OTC) and chemical oxygen demand (COD) removal from licorice extraction plant wastewater (LEPW). Three novel visible-light-responsive nanostructures, including L-Histidine-TiO₂, L-Methionine-TiO₂ and L-Asparagine-TiO₂, were successfully synthesized. The results showed that the modification of TiO₂ with these three amino acids made the catalyst active in the visible light region and reduced the recombination rate of e⁻/h⁺ pairs according to PL analysis. The photodegradation efficiency of L-Histidine (2 wt.%)-TiO₂ was 100% and 94% for OTC and COD, respectively. It showed the highest photocatalytic activity under illumination, compared to L-Methionine (1.5 wt.%)-TiO₂ and L-Asparagine (2 wt.%)-TiO₂. Synthesized composites were characterized with SEM, XRD, FTIR, DRS, and PL analyses. The biological oxygen demand to COD (BOD₅/COD) ratio for treated LEPW was determined to be 0.5–0.6, confirming the enhanced biodegradability of the treated effluent. The effect of the independent variables, namely, initial concentration of OTC and COD, catalyst dosage, irradiation time, pH of solution, and light intensity, on the photocatalytic process was evaluated by Response Surface Methodology (RSM), and the optimum value of each independent parameter for maximum degradation of OTC and COD by L-Histidine (2 wt.%)-TiO₂ was determined. The radical trapping experiment was performed with various scavengers in order to propose a photocatalytic mechanism, showing that hydroxyl radicals were the main active species. L-Histidine (2 wt.%)-TiO₂ showed a stable and reusable structure even after four cycles of COD removal under the following optimal conditions of [COD]: 300 mg/L, [catalyst]: 1 g/L, light intensity: 25 W/cm² at pH = 4 after 180 min irradiation.

1. Introduction

Contaminants discharged from industries and through anthropogenic activities into aqueous media are responsible for water pollution [1], water shortage crises [2], and contamination of the environment [3]. In wastewater, most contaminants are non-biodegradable and resistant to natural degradation, such as OTC [4] and licorice [5]. Tetracycline-based OTC antibiotics are used worldwide for human and veterinary medicine [6]; however, OTC is poorly absorbed in the digestive tract (30%) and most of it enters the environment through body excretions [7]. Ultimately, these residues can cause serious health problems and threaten the ecosystem. They can contribute significantly to the increase in antibiotic resistance genes [8,9]. As a result of their hydrophilic properties and stable naphthalene rings, OTC residues cannot be conventionally treated by biodegradation or chlorination [10].

The compound licorice is widely used in tobacco, dietary supplements, sweets, and medicines [11]. Due to this, large quantities end up in water, wastewater, and soil as residues. Environmental pollution with licorice causes serious problems for the ecosystem [11,12,13].

Photocatalysis is a useful and practical method for degrading non-biodegradable contaminants, such as OTC and licorice, among other treatment methods [14]. The ability of the photocatalysis method to convert contaminants to harmless compounds, its affordability, reusability, and sustainability make it an environmentally friendly and effective water treatment method [15]. As a result of its strong oxidizing power [15] and chemical and biological inertness [16], its wide band gap energy leads to poor performance under visible light irradiation, and its high recombination rate of electron/hole pairs negatively affects its photocatalytic activity [17,18]. To address these problems, non-metal doping is an appropriate method because it reduces band gap energy and makes TiO₂ a visible light-driven photocatalyst [19,20]. In contrast to metal doping, non-metal doping enhances the photocatalytic properties of TiO₂ because it creates new levels near the valence band [21]. Some studies have investigated the photocatalytic activity of C-N and S-doped TiO₂, Sushma et al., investigated the effect of tri-doping C, N and S on the photocatalytic activity of TiO₂ [22]. Wan et al. assessed the influence of tri-doping on the performance of TiO₂ nanorods [23]. Their findings showed that non-metal modifications of TiO₂ resulted in increased active sites, longer lifetimes of holes and electron pairs, and a red shift to the visible region, as well as a narrowing of the band gap [21,22]. L-amino acids are rich sources of N, C and S that can be used for co-doping and tri-doping TiO₂. Zanganeh et al., studied the role of L-proline [24], L-Lysine [25], L-Histidine [26] in improvement of the photocatalytic performance of TiO₂.

The purpose of this study was to assess the effect of co-doping and tri-doping TiO₂ on photocatalytic degradation of OTC and COD in LEPW. L-Histidine, L-Methionine and L-Asparagine were used as sources of nonmetals to modify TiO₂, and their positive effects were compared. Based on the photocatalytic performance of L-amino acid-TiO₂ networks for removal of OTC with different contents of dopants, the best weight percent was selected. L-histidine-TiO₂ which showed the highest OTC and COD degradations were selected for investigating the optimal conditions based on response surface methodology (RSM) as a statistical technique. The prepared photocatalysts were characterized with X-ray diffraction (XRD), Fourier transform infrared (FT-IR), diffuse reflectance spectra (DRS), field emission scanning electron microscopy (FESEM), energy dispersive analysis of X-ray (EDX) and photoluminescence analysis (PL). The novelty of this study was its fabrication of three reusable visible-light responsive photocatalysts, including L-Histidine (C, N) codoped-TiO₂, L-Asparagine (C, N) codoped-TiO₂, and L-Methionine (C, N, S) tri-doped-TiO₂. The impact of variables, including OTC and COD concentration, irradiation time, light intensity, catalyst concentration, and pH, for the photodegradation process were evaluated, based on central composite design (CCD). The variables were optimized to obtain the maximum COD removal and OTC removal efficiency. The reusability of the prepared catalyst, the biodegradability of the treated LEPW and total organic carbon (TOC) for OTC were evaluated.

2. Experimental

2.1. Materials

Tetra-n-butylorthotitanate (TBOT (C₁₆H₃₆O₄Ti, 99%)), ethanol (C₂H₅OH, 99.0%) and acetic acid (CH₃COOH, 99.8%) were provided by Merck (Germany). OTC (C₂₂H₂₄N₂O₉, 99%), L-Methionine (C₅H₁₁NO₂S, 99%), L-Asparagine (C₄H₈N₂O₃, 99%), L-Histidine (C₆H₉N₃O₂, 99%) were purchased from Sigma-Alderich (UK). Licorice (liquorice) extraction plant wastewater was taken from a Zagros liquorice company, Kermanshah, Iran. The characteristics of the licorice extraction plant wastewater samples are shown in

Table 1. Characteristics of licorice extraction plant wastewater.

Wastewater Properties	Initial Conditions before Treatment
Color	Brown
COD (mg/L)	700–800
BOD5/COD ratio (mg/L)	0.17–0.19
pH	5.7–6.4
TSS (mg/L)	120–250

. Since the BOD₅/COD ratio of raw LEPW was 0.19 and the minimum BOD₅/COD ratio, as an acceptable factor for the wastewater to be biodegradable, was 0.4 [12,13] raw LEPW was considered non-biodegradable.

2.2. Synthesis of L-Asparagine-TiO₂, L-Histidine-TiO₂ and L-Methionine-TiO₂

All photocatalysts were prepared by the sol-gel method [27]. Initially, different amounts (1.0, 1.5, 2.0, 2.5 wt.%) of L-Asparagine, L-Methionine, L-Histidine were dissolved in mixtures of 15 mL ethanol, 0.2 mL deionized water, and 3.4 mL acetic acid and mixed for 1 h with a magnetic stirrer (solutions A₁–A₃). Then, 5 mL TBOT was added to 10 mL ethanol and dispersed for 20 min by an ultrasonic bath (solution B). The solutions A₁–A₃ were added dropwise to solution B and were sonicated for 1.5 h. The solutions were kept in darkness at room temperature for 24 h. Finally, the solutions were dried in an electric oven at 120 °C for 12 h and were calcined at 500 °C for 2 h inside a muffle furnace to obtain L-Asparagine-TiO₂ (C,N co-doped TiO₂), L-Histidine-TiO₂ (C,N co-doped TiO₂) and L-Methionine-TiO₂ (C,N,S tri-doped TiO₂) nanostructures.

2.3. Catalysts Characterization

The X-ray diffraction (XRD) patterns were obtained by using Rigaku D-max C III (Tokyo, Japan) with Ni-filtered Kα radiation (λ = 1.542 °A). The functional group of catalysts were determined by Fourier transform infrared (FT-IR) spectra (Shimadzu Varian 4300, Kyoto, Japan) with KBr pellets containing the powder samples. The UV–Vis diffuse reflectance spectra (DRS) were obtained using a UV–Vis spectrophotometer (Shimadzu 1800, Kyoto, Japan). The morphologies of synthesized composites were studied by field emission scanning electron microscopy energy dispersive analysis of X-rays (FESEM-EDX) (Philips XL 30 and S-4160, Eindhoven, Netherlands) and to make the samples conductive, they were covered with gold. The photoluminescence (PL) analysis was performed in a (fluorescence spectrophotometer Perkin Elmer/LS55, Waltham, Massachusetts, USA) at the excited wavelength of 410 nm.

2.4. Photocatalytic Activity Experiments

The photocatalytic experiments were conducted in a cylindrical Pyrex equipped with a cooling jacket to adjust the temperature of the solution to 25 °C. Irradiation was performed using LED lamps (50 W, emission: 405 nm and light intensity of 13 lm per m²). The specific amount of catalyst was suspended to the solution of OTC or LEPW, followed by adjusting the pH with NaOH and H₂SO₄ (1 M). The solution was stirred in dark conditions for 30 min to obtain the adsorption–desorption equilibrium. After which, the light sources were switched on and the photocatalytic experiments were conducted under visible light irradiation and the solution continuously stirred. At different time intervals 2 mL of the OTC solution or LEPW was sampled and centrifuged to remove the remaining nanoparticles. The COD concentration of LEPW samples was determined by a colorimetric method and COD calibration curve. The COD removal was measured by means of Equation (1):

$$\mathrm{Removal} \left(\%\right)=\left(1-\frac{{\mathrm{C}}_{\mathrm{t}}}{{\mathrm{C}}_0}\right)\times 100$$

(1)

where C₀ and C_t are the initial and the final COD concentrations of LEPW in the solution, respectively. OTC concentration was determined using the spectrophotometry method in a spectrophotometer device V-570 (Jasco, Japan) and the degradation efficiency was calculated according to Equation (1) (C0 and Ct are the initial and the final OTC concentration). Total organic carbon (TOC) of the OTC solutions was measured with a TOC analyzer (2200, Shimadzu, Kyoto, Japan).

2.5. Experimental Design Methodology

The CCD with five independent factors at three levels was applied to design the experiments in the present study. The number of experiments was determined according to Equation (2) [15]:

$$\mathrm{N}=2^{\mathrm{k}-\mathrm{q}}+2\times \mathrm{k}+{\mathrm{n}}_{\mathrm{c}}$$

(2)

where k stands for the number of independent factors and q and n_c represent a fraction of the number of factor and the repetition number of the central point, respectively.

The independent variables for this study were OTC concentration, COD in LEPW, catalyst dosage, irradiation time, pH of solution, and light intensity, and OTC photodegradation and COD removal were chosen as the responses (

Table 2. Experimental ranges and levels of the processing factors for OTC and COD removal.

Factors	Levels
	−1	0	1
OTC
A: [OTC] (mg/L)	50	75	100
B: [Catalyst] (g/L)	0.5	1	1.5
C: pH	4	7	10
D: Irradiation time (min)	15	30	45
E: Light Intensity (W/cm2)	15	20	25
LEPW
A: [COD] (mg/L)	300	500	700
B: [Catalyst] (g/L)	1	1.5	2
C: pH	4	7	10
D: Irradiation time (min)	60	120	180
E: Light Intensity (W/cm2)	15	20	25

). The factors and their levels were chosen based on the screening experiments and the literature. A total number of 50 experiments were designed for photodegradation of OTC (Table S1) and also COD removal from LEPW (Table S2), according to Equation (2) (q is zero for a full factorial design). The effect of independent variables, their interaction, the mathematical model and optimization of variables were determined by CCD.

3. Results and Discussion

3.1. Optimization of L-Asparagine, L-Histidine and L-Methionine Loading in TiO₂ Network

In order to determine the optimum content of L-Asparagine, L-Histidine, and L-Methionine in composites, their ability to photodegrade OTC at an initial concentration of 50 mg/L, pH = 4.5, and 1 g/L of catalyst dosage was assessed (Figure 1). After 60 min illumination, the removal efficiency of L-Methionine-TiO₂ increased from 69% to 90% after increasing the weight percentage of L-Methionine from 0.5 wt.% to 1.5 wt.% (Figure 1b). A further increase in L-Methionine loading (2.0 wt.%), however, resulted in a drop in OTC removal efficiency to 86.6% (Figure 1b). In the case of L-Asparagine and L-Histidine, increasing L-amino acids content improved the photocatalytic performance of TiO₂, and the degradation efficiency of L-Histidine (2.0 wt.%)-TiO₂ and L-Asparagine (2.0 wt.%) in removal of OTC reached 100% and 74% (Figure 1a,c). Nonetheless, more increment of L-Asparagine, as well as L-Histidine loading, to 2.5 wt.% had negative effects on the OTC removal efficiency and it declined to about 2–4%. The improvement could be attributed to reduction of the band gap because of formation of new levels between VB and CB due to the introduction of C, N and S to the oxygen sites of TiO₂ [15,23]. However, the downward trends of removal efficiency with excessive doping could be explained by the reduction of the distance between trapping sites in photocatalysts, and increase in the recombination centers and the recombination rates of electron/hole pairs [28,29].The results showed that L-Histidine at 2 wt.%, L-Methionine at 1.5 wt.% and L-Asparagine at 2 wt.% were the optimal amounts of loading in the TiO₂ network. Meanwhile, the role of the adsorption process on OTC removal was assessed and OTC removal efficiency after 30 min stirring in darkness was about 15–22% for L-Histidine (2 wt.%)-TiO₂, L-Methionine (1.5 wt.%)-TiO₂ and L-Asparagine (2 wt.%)-TiO₂.

To further investigate the photocatalytic activities of L-Histidine (2 wt.%)-TiO₂, L-Methionine (1.5 wt.%)-TiO₂ and L-Asparagine (2 wt.%)-TiO₂, the reaction kinetics of photodegradation of OTC were studied and are illustrated in Figure S1. The photocatalytic removal rate constant (K₁ and k₂ for Pseudo first and second order models, respectively, and determination of the coefficient (R²) are also represented in

Table 3. Kinetic constant and determination of coefficient of pseudo first and second order models for photocatalytic removal of OTC.

Pseudo First Order Model	K1	R2
L-Histidine-TiO2	0.0251	0.977
L-Methionine-TiO2	0.0149	0.983
L-Asparagine-TiO2	0.0111	0.977
Pseudo Second Order Model	k2	R2
L-Histidine-TiO2	0.0108	0.824
L-Methionine-TiO2	0.0082	0.902
L-Asparagine-TiO2	0.0080	0.947

. The results exhibited that the degradation reaction for all the prepared nanocomposites followed pseudo first-order kinetics based on Equation (3) [30]:

$$\mathrm{Ln}\left(\frac{{\mathrm{C}}_0}{{\mathrm{C}}_{\mathrm{t}}}\right)={\mathrm{K}}_1\mathrm{t}$$

(3)

$$\frac{\mathrm{t}}{{\mathrm{C}}_{\mathrm{t}}}=\frac{1}{{\mathrm{k}}_2{\mathrm{C}}_0}+\frac{\mathrm{t}}{{\mathrm{C}}_0}$$

(4)

where C₀, C_t, K₁, k₂ and t are concentrations at time zero and t, rate constant and irradiation time, respectively. The k₁ values of pseudo first order kinetic models were achieved at about 0.0251, 0.0149 and 0.0111 min⁻¹ for L-Histidine-TiO₂, L-Methionine-TiO₂ and L-Asparagine-TiO₂, respectively. It was confirmed that there was more photocatalytic activity of L-Hisitindine-TiO₂, as explained earlier. Therefore, L-Histidine (2 wt.%)-TiO₂, L-Methionine (1.5 wt.%)-TiO₂ and L-Asparagine (2 wt.%)-TiO₂ showed the highest photocatalytic performances for OTC and COD photodegradation. Consequently, the characterization tests were carried out for these optimum loading catalysts

The photocatalytic ability of the three synthesized catalysts at optimum weight percentages for COD removal was also studied at an initial COD concentration of 500 mg/L, 2 g/L of catalysts and original pH (Figure 2a,c). The biodegradability of the treated LEPW was studied by classification of various possible intermediates, including COD mineralized (the complete conversion of a compounds to CO₂ and H₂O and other inorganic compound), bCOD (Biodegradable COD concentration), nbCOD (Non-biodegradable COD concentration), and CODe (COD contents remained as parent form) as defined in the following Equations:

Total COD = COD mineralized + bCOD + nbCOD + CODe

(5)

CODe = bCOD + nbCOD

(6)

bCOD = UBOD + 1.42f_dY_xCODe

(7)

BOD₅ = 0.68UBOD

(8)

where UBOD, f_d and Y_x are, ultimate biochemical oxygen demand, fraction of cell mass remaining as cell debris (0.15 g/g) and biomass yield coefficient (0.2 gVSS/gCOD).

The results showed that L-Histidine (2.0 wt.%)-TiO₂ was the most effective catalyst for the photocatalytic removal of COD (Figure 2a). As can seen in Figure 2 the mineralized COD was achieved at 470 mg/L by using L-Histidine (2 wt.%)-TiO₂ nanoparticles after 120 min visible irradiation. It was obtained at 400 and 330 mg/L for L-Methionine (1.5 wt.%)-TiO₂ and L-Asparagine (2 wt.%)-TiO₂, respectively. The results showed that the nbCOD content of raw LEPW was much higher than the bCOD amount, and after photocatalytic removal of COD by all synthesized photocatalysts the nbCOD values reduced more than bCOD, which showed increasing biodegradability of the treated LEPW.

Moreover, the biodegradability of the produced unknown products was studied by determination of the BOD₅/COD ratio for the treated wastewater. The COD removal and BOD₅/COD ratio versus irradiation time are represented in Figure 3a,b, respectively. The maximum ratio of BOD₅/COD was determined to be 0.6, 0.54 and 0.5 after 120 min visible irradiation for L-Histidine (2 wt.%)-TiO₂, L-Methionine (1.5 wt.%)-TiO₂ and L-Asparagine (2 wt.%)-TiO₂ respectively (Figure 3b). This confirmed the effectiveness of the photocatalytic process in enhancing the biodegradability of the treated LEPW. The COD removal efficiencies were about 94, 80 and 74% for L-Histidine (2 wt.%)-TiO₂, L-Methionine (1.5 wt.%)-TiO₂ and L-Asparagine (2 wt.%)-TiO₂ catalysts under 120 min visible light irradiation time, respectively. The contribution of the adsorption process for LEPW using synthesized nanoparticles was insignificant (5–8%).

The higher photocatalytic activity of L-Histidine (2 wt.%)-TiO₂ in elimination of OTC and COD, in comparison with L-Methionine (1.5 wt.%)-TiO₂ and L-Asparagine (2 wt.%)-TiO₂ nanostructures, can be explained by PL analysis, which was used for evaluation of the recombination rate of the photo-induced electron/hole pairs. The weaker emission peak intensity of PL spectra signified lower recombination rate and higher lifetime of charge carriers [31]. The PL spectra for the three prepared nanoparticles is illustrated in Figure 4. The intensity of emission peak for L-Histidine (2 wt.%)-TiO₂ was far lower in comparison with the two other modified catalysts (L-Methionine (1.5 wt.%)-TiO₂ and L-Asparagine (2 wt.%)-TiO₂). Consequently, the modification of TiO₂ with L-Histidine (2 wt.%) had a more significant role in reduction of the e⁻/h⁺ recombination rate than those of the two other nanoparticles.

3.2. Characterization of Catalysts

The XRD patterns of three catalysts, including L-Histidine-TiO₂, L-Methionine-TiO₂ and L-Asparagine-TiO₂ are illustrated in Figure 5a. The peaks at 2θ = 25°, 38°, 48°, 54.5°, 55.3° and 63° that correspond with (101), (103), (200), (105), (204) and (213) diffraction planes, respectively, could be attributed to TiO₂ [32]. There were no peaks related to L-Methionine, L-Asparagine and L-Histidine, because the amounts of loading on TiO₂ were low.

FTIR analysis (Figure 5b) showed that the bands at 522 and 713 cm⁻¹ (range 400 and 800 cm⁻¹) were characteristic bands of TiO₂ and related to the vibration band of Ti-O and Ti-O-Ti bonds [33].The absorption peaks around at 3420 cm⁻¹ could be attributed to the hydroxyl groups (O-H) of water molecules absorbed on the surface of the catalysts [34]. The bands observed around 2355 cm⁻¹ were related to the presence of dissolved, or atmospheric, CO₂ in the samples [35].

The UV–Vis diffuse reflectance spectra (DRS) and Tauc plots of the as-synthesized TiO₂ samples are illustrated in Figure 5c,d, respectively. The noticeable red shift with C–N-codoped-TiO₂ and C–N–S-tridoped-TiO₂ catalysts to the visible light region were observed (Figure 5c). The extension of the light absorption edge of catalysts into the UV-visible region could be a decisive factor in increasing the number of photogenerated electrons and holes to generate more reactive species which improved the photocatalytic activity of TiO₂. The optical band gap energy of each of the three L-Histidine (2 wt.%)-TiO₂, L-Methionine (1.5 wt.%)-TiO₂ and L-Asparagine (2 wt.%)-TiO₂ catalysts were obtained at 2.1, 2.3 and 2.17 eV (Figure 5d), which was lower than the band gap of pure TiO₂ (3.1 eV) [30]. The reduction of the band gap was due to the introduction of doping species (C, N and S) into the TiO₂ lattice. Similar results were obtained for co-doped TiO₂, and the band gap energy of L-Arginine (1 wt.%)-TiO₂, L-Lysine (C, N co-doped)-TiO₂/WO₃ and L-Proline (2 wt.%)-TiO₂ were obtained as 1.7, 2.1, and 1.7, respectively [25,36].

The SEM images of the synthesized catalysts are illustrated in Figure 6. The SEM images of catalysts showed the formation of spherical-like particles and uniform distribution without agglomeration structures. The composition of elements in L-Histidine (2 wt.%)-TiO₂ nano photocatalysts were estimated as shown in the EDX spectroscopy results (Figure 6d). Based on an EDX spectrum, L-Histidine (2 wt.%)-TiO₂ mainly consisted of Ti and O, along with a small amount of C and N, indicating that nonmetals had been successfully incorporated into TiO₂. The uniformity and size of nanoparticles for all the photocatalysts were also investigated by image processing software (ImageJ 1.44p software) and the result is given in Table S3.

3.3. Statistical Analysis and Modeling for Photodegradation of OTC and COD by L-Histidine (2 wt.%)-TiO₂

The effects of the independent variables on the OTC and COD removal were statistically assessed by applying CCD. The analysis of variance (ANOVA) verified that the quadratic models of the photocatalytic process with p-value < 0.0001 and F-value of 129.72 for OTC removal and 49.06 for COD removal were significant (

Table 4. ANOVA results for models of COD and OTC photocatalytic degradation.

Source	Sum of Squares		df		F-Value		p-Value
	LEPW	OTC	LEPW	OTC	LEPW	OTC	LEPW	OTC
Model	12,391.4	27,990.10	8	8	49.06	129.72	<0.0001	<0.0001
A: [dye]	3790.6	2647.06	1	1	120.05	98.14	<0.0001	<0.0001
B: [catalyst]	1882.6	995.76	1	1	59.62	36.92	<0.0001	<0.0001
C: pH	1794.4	6250.62	1	1	56.83	231.75	<0.0001	<0.0001
D: Irradiation time	1284.7	984.97	1	1	40.96	36.52	<0.0001	<0.0001
E: light Intensity	2542.2	3790.62	1	1	80.52	140.54	<0.0001	<0.0001
CE	-	399.03	-	1	-	14.79	-	0.0004
A2	181.8	-	1	-	5.76	-	0.0210	-
B2	-	178.29	-	1	-	6.61	-	0.0139
C2	189.2	2951.64	1	-	5.99	109.44	0.0187	<0.0001
D2	230.7	-	1	1	7.31	9.99	0.0100	-
Residual	1294.5	1105.82	41	42
Lack of Fit	1127	876.94	34	35	1.39	0.79	0.3457	0.7047
Pure Error	167.5	228.88	7	7
LEPW: R2 = 0.91, adjusted R2 = 0.9, Std. Dev = 5.62, Adeq precision = 33.06, C.V.% = 8.92, Lack of fit = 0.34
OTC: R2 = 0.96, adjusted R2 = 0.95, Std. Dev = 5.19, Adeq precision = 39.67, C.V.% = 10.38, Lack of fit = 0.7

). The reduced quadratic models, based on the significant parameters, are depicted with the following Equations:

$${\mathrm{Y}}_{\mathrm{OTC}}\left(\%\right)=+74.19-8.82\ast \mathrm{A}+5.41\ast \mathrm{B}-13.56\ast \mathrm{C}+5.38\ast \mathrm{D}+10.56\ast \mathrm{E}-3.53\ast \mathrm{CE}-7.01\ast {\mathrm{B}}^2-28.51\ast {\mathrm{C}}^2$$

(9)

$${\mathrm{Y}}_{\mathrm{COD}}\left(\%\right)=+68.92-10.56\ast \mathrm{A}+7.44\ast \mathrm{B}-7.26\ast \mathrm{C}+6.15\ast \mathrm{D}+8.65\ast \mathrm{E}-7.92\ast {\mathrm{A}}^2+8.08\ast {\mathrm{C}}^2-8.92\ast {\mathrm{D}}^2$$

(10)

The p-values of independent variables indicated that all five factors, including COD or OTC concentration (A), catalyst concentration (B), pH (C), irradiation time (D), and light intensity (E), were significant.

According to their F-values (Table 4) and the regression coefficients of Equations (9) and (10) the degree of effect and significance of independent variables on OTC removal had the following orders: C > E > A > B > D. It should be noted, however, that the importance of independent parameters for the removal of COD was A > E > B > C > D.

The ANOVA results also confirmed that there were interactions between A and C for OTC removal, whereas no interaction between variables was found for COD removal. The quadratic terms, including B² and C² for OTC removal and A², C², and D² for COD removal, were statistically significant. The coefficient of A and C were negative, implying that the increase of the OTC or COD concentration and pH could lead to the decline of OTC and COD removal efficiency (Table 4).

The coefficient of determination (R²) was 0.97 for OTC removal and 0.91 for COD removal, close to 1, confirming that the predicted data were matched with experimental values [37,38]. The lack of fit for both models was non-significant, indicating the suitability of the models. Adeq precision was obtained as 39.90 for OTC and 33.61 for COD removal, which represented a sufficient signal [39]. The adjusted R² of 0.96 for OTC removal and 0.90 for COD removal were in good agreement with their R², implying the adequacy of the proposed models. The normal probability plot of the residuals (Figure S2) further confirmed that the models fitted the experimental runs well, as the distribution of the experimental data was normal and the points were near the straight lines [38]. The value of coefficient of variation (CV) and standard deviation for both of the proposed models indicated the high reliability and precision of the models, respectively [39].

3.4. Photodegradation Modeling and Optimization of Independent Variables

As shown in Figure 7, Figure 8 and Figure 9, the counter plots illustrate the relationship between OTC and COD removal efficiency and the five independent variables. The effects of A (OTC or COD concentration) and B (L-Histidine (2 wt.%)-TiO₂ catalyst concentration) on responses are illustrated in Figure 7a,b, respectively. By increasing the OTC concentration from 50 to 100 mg/L, the removal efficiency decreased from 83% to 55%. With an increase in COD from 300 to 700 mg/L, around 35% of COD removal efficiency was lost. This reduction could be explained by occupation of the active sites with pollutants at higher concentration levels. Furthermore, the presence of more pollutants inhibited photocatalytic activity by blocking visible light photons, preventing them from reaching the surface of the photocatalysts [40].

The effect of L-Histidine (2 wt.%)-TiO₂ catalyst dosage on response was also assessed and higher removal efficiency for both OTC and COD removal were obtained at higher levels of catalyst dosage. The enhancement of activity could be attributed to the availability of more active sites for the adsorption of pollutants. In addition, these sites could also absorb more photons to produce active species [21,40].

The photodegradation of OTC and COD, as a function of pH and irradiation time, is shown in Figure 8a,b. The photodegradation efficiency of OTC increased significantly to around 80% by bringing the pH from acidic to neutral, however it decreased significantly to almost 35% when the pH was increased to alkaline (Figure 8a). The reason why the maximum removal efficiency of OTC was observed at pH = 7 could be attributed to the properties of both the catalyst and the OTC (pHpzc of the catalyst and surface charge of the OTC). The pHpzc of L-Histidine (2 wt.%)-TiO₂ catalyst was determined as 6.9 (Figure S3), which meant that it was positively charged at a pH less than 6.9 and it had negative charge at a pH more than 6.9. Further, the OTC had pKa values of 3.22, 7.46, and 8.94 [41] and, therefore, under acidic conditions, it existed as both neutral (H₂OTC) and positively charged (H₃OTC+) forms. At pH levels greater than 9, the OTC had a negatively charged form [42]. Therefore, the repulsive force caused by equal charges of catalyst and OTC dissociations led to a decreased degradation yield, both in acidic and basic media. The COD removal of efficiency was also highly influenced by pH and Figure 8b shows that the acidic condition (pH = 4) was the most favorable to the COD degradation compared to neutral or alkaline pH, owing to the attraction forces between licorice (pKa = 2.6) [43] and L-Histidine (2 wt.%)-TiO₂ at this pH. Moreover, Figure 8a,b also depicts the effect of irradiation time on the responses. The positive effects of longer irradiation time on the responses were observed for both OTC and COD removal. The photodegradation removal efficiency improved by almost 20–30% when the irradiation time went up from 15 to 60 min for OTC and 60 to 180 min for COD removal. The reason for this was that there was more time for radical hydroxyls to form and for reactions to occur between the generated species and the pollutant molecules, which could lead to enhanced photodegradation.

The effect of light intensity on the photodegradation efficiency are depicted in Figure 9. The upward trend of the OTC removal efficiency was observed by increasing the light intensity from 15 to 25 W/cm² (Figure 9a). This upward trend was also observed for the COD removal from LEPW at higher light intensity and it reached around 90% removal at the light intensity of 25 W/cm² (Figure 9b). This improvement in removal efficiency was because of increased photon absorption by L-histidine (2 wt.%)-TiO₂ in higher light intensities [44]. The optimum conditions for OTC photodegradation and COD removal from LEPW were determined using CCD while considering the desirability of 1. The COD in LEPW was completely removed under the optimal conditions of initial COD of 300 mg/L, catalyst concentration of 2 g/L at pH = 4 and light intensity of 25 W/cm² after 180 min visible light irradiation, which was close to the predicted removal efficiency. Further, the optimization results determined that, at an initial concentration of 50 mg/L of OTC, and at a light intensity of 25 W/cm² and a L-Histidine (2 wt.%)-TiO₂ concentration of 1 g/L, OTC was completely removed after 60 min of irradiation, which was quite close to the predicted removal efficiency (99.5%), confirming the suitability of the proposed models.

3.5. Photocatalytic Mechanism of L-Histidine (2 wt.%)-TiO₂

The first active species that are produced in photocatalysis are e⁻ and h⁺, and then these photoinduced electrons and holes react with H₂O and O₂ on the conduction band (CB) and valance band (VB) of photocatalysts [45]. To suggest a photocatalytic mechanism, finding the main reactive species is necessary. Therefore, a radical trapping experiment, by introducing different scavengers to the reaction media, was performed. In this study 1,4-benzoquinone (BQ), sodium azide (SA), isopropanol alcohol (IPA), and ammonium oxalate (AO) were introduced as scavengers of superoxide radical (^•O₂⁻), singlet oxygen (¹O₂), hydroxyl radical (^•OH), and hole (h⁺), respectively [46,47,48]. The effect of scavengers on photocatalytic removal of OTC and COD were evaluated at conditions of initial concentrations of 50 and 300 mg/L, photocatalyst loadings of 1 and 2 g/L, pH of 7 and 4, and at 25 W/cm² light intensity 25 W/cm² for OTC and COD, respectively. Without any scavengers, OTC and COD were completely degraded after 60 and 120 min, respectively. Among different radical trappings, the introduction of SA had no effect on photodegradation efficiency, which showed that ¹O₂ did not have any role in removal of pollutants. However, the photocatalytic performance of L-Histidine (2 wt. %)-TiO₂ was significantly reduced by adding IPA. Therefore, hydroxyl radicals (^•OH) were the main species in degradation of OTC and COD, as seen in Figure 10a,b. Similarly, introducing AO and BQ reduced removal efficiency of OTC and COD, which indicated that h⁺ and ^•O₂⁻ played roles in the photodegradation mechanism. The results of the radical trapping experiments provided information which suggested a photocatalytic mechanism for removal of OTC and COD. The possible mechanism could explain the degradation pathway in two ways. At first, the reaction between dissolved oxygen (O₂) and photogenerated electrons produced superoxide radical (^•O₂⁻), which later transformed to ^•OH and H₂O₂, resulting in degradation of OTC molecules [49]. Then, the photoinduced holes and adsorbed OTC molecules on the photocatalyst surface could directly oxidize OTC radicals to ^•OTC⁺. In addition, N-Ti-O bonds were formed when N was doped into the TiO₂ lattice. In the meantime, the combination of the N2p and O2p orbitals created a new level above VB, resulting in a reduction in the band gap of TiO₂ [50]. Additionally, doped C could serve as a photosensitizer for TiO₂, by injecting electrons into its CB [51], as seen in Figure 11.

3.6. Reusability Performance

The reusability and photostability of L-Histidine (2 wt.%)-TiO₂ for COD removal from LEPW under optimum conditions for four following cycles was studied to evaluate its potential for industrial usage. Figure 12a shows that the COD photodegradation efficiency reduced slightly (only 7–8%) after four cycles. This indicated that L-Histidine (2 wt.%)-TiO₂ nanostructure could be used as an effective and potentially reusable catalyst for photocatalytic treatment of water and wastewater.

Further, the structural decomposition and mineralization effectiveness of L-Histidine (2 wt.%)-TiO₂ catalyst was assessed for OTC removal under the optimum conditions through a TOC analyzer. The initial OTC solution (50 mg/L) had 22.4 mg/L of TOC and the complete removal of TOC was observed after 300 min irradiation (Figure 12b). The mineralization results verified that OTC could be effectively degraded to byproducts and then it could be completely converted to harmless compounds, which indicated the effectiveness of the L-Histidine (2 wt.%)-TiO₂/UV–Vis degradation process. In order to distinguish the contribution of adsorption, photolysis and photocatalysis in the removal of the pollutants, experiments were done in darkness, without catalysts, under visible light illumination and in visible light/L-Histidine (2 wt.%)-TiO₂ system, respectively. In the photolysis process, the COD and OTC removals from LEPW and OTC were achieved 3 and 8% after 180 and 60 min, respectively. The role of adsorption was more significant than photolysis, which could eliminate 47% OTC and 39% OTC after 60 and 180 min, respectively.

4. Conclusions

L-Histidine-TiO₂, L-Methionine-TiO₂ and L-Asparagine-TiO₂ novel photocatalysts were successfully synthesized through the sol-gel method. The optimum weight ratio of amino acid to TiO₂ was determined by assessing their photocatalytic performances in degradation of OTC and COD, which obtained L-Histidine (2 wt.%), L-Methionine (1.5 wt.%) and L-Asparagine (2 wt.%). The performance evaluation of the synthesized photocatalysts showed L-Histidine (2 wt.%)-TiO₂ had the best photocatalytic activity and the lowest recombination rate. Characterization of as-prepared photocatalysts proved enhancement of TiO₂ photocatalytic performance through reducing the band gap energy of TiO₂ from 3.1 to 2.1, 2.3 and 2.17 eV for L-Histidine (2 wt.%)-TiO₂, L-Methionine (1.5 wt.%)-TiO₂ and L-Asparagine (2 wt.%)-TiO₂. PL analysis showed reduction of the recombination rate of the co-doped TiO₂ photocatalyst.

Analysis of variance (ANOVA) showed the accuracy of the proposed models for photodegradation of OTC and COD by L-Histidine (2 wt.%)-TiO₂. The models showed that decreasing initial concentration and increasing catalyst dosage, irradiation time and light intensity positively influenced photocatalysis. The complete OTC removal was obtained at optimal conditions of 50mg/L OTC, 1g/L of L-Histidine (2 wt.%)-TiO₂, at neutral pH and light intensity of 25 W/cm⁻¹ after 60 min visible light irradiation and 100% reduction of TOC after 300 min irradiation time. The increase of BOD₅/COD of treated LEPW from 0.19 to about 0.6 confirmed the improvement of the biodegradability. The scavenger study showed the role of radicals in the degradation process followed the order of (^•OH) > (^•O₂⁻) > (h⁺). The slight decline of COD removal efficiency of LEPW (about 7–8%) after four cycles verified the reusability and stability of the catalyst. Based on the results, L-Histidine (2 wt.%)-TiO₂ could be considered a promising photocatalyst for industrial and practical wastewater and water treatments.