Evaluation of Ni-Doped Tricobalt Tetroxide with Reduced Graphene Oxide: Structural, Photocatalysis, and Antibacterial Response

Abstract

Cobalt oxide (Co₃O₄) nanoparticles were successfully prepared by sol–gel and hydrothermal methods for antibacterial and photocatalytic applications with the addition of 1%, 4% nickel (Ni), and reduced graphene oxide (rGO). The structural and morphological properties of the nanoparticles were obtained by XRD, TEM and FESEM techniques. Cobalt oxide showed typical crystallographic planes to cubic phase and particles with inferior diameter to 30 nm. The Ni-Co₃O₄ + rGO nanocrystals exhibit a band gap value of 2.0 eV. The bactericidal tests for S. aureus and E. coli revealed that the insertion rGO synthesized by the sol–gel method promoted the antimicrobial activity for both microorganisms. Afterward, the photocatalytic assay for the atrazine contaminant showed significant responses to pesticide removal attributed to the simultaneous adsorption and degradation process. In addition, the sol–gel process found a better response to Ni-Co₃O₄ in the presence of rGO, indicating a nanocomposite superior synergism.

1. Introduction

The affectation of the ecosystem and human health by pollution in the modern, technological and industrialized world is increasing worldwide. Researchers in different areas have made various efforts to apply pollution treatments in this context. However, the process still has many drawbacks that limit its practical applications [1,2,3,4,5,6,7,8,9,10]. In this way, new semiconductors of various types have been used with photocatalysts, attempting to solve the problem of environmental contamination.

Various studies of metal oxide semiconductors have attracted particular attention in different scientific areas, due to the variety of structural geometries with electronic properties that endow them with diverse physical and chemical properties [3,4,5,6]. In the literature, synthetic routes have been sought on a large economic scale, reproducibility, and environment respectfully. The sol–gel and hydrothermal techniques are evidenced in nanomaterial oxide synthesis for various physical and chemical processes [2,3].

Cobalt oxide (Co₃O₄) is an excellent candidate due to its significant electronic, optical, and magnetic properties in various applications, such as lithium-ion batteries, heterogeneous catalysis, and gas detection. In addition, Co₃O₄ has been shown as a friendly environment with low toxicity and a higher absorption in the solar spectrum. In the nanometric-scale Co₃O₄ nanoparticles has an indirect band gap of 1.5 eV. However, the Co₃O₄ photocatalytic efficiency is lower due to the rapid rate of photoelectrons and holes. An alternative is doping the oxide to elevate the photodegradation properties [1]. The material doping is favorable to increasing the number of surface effects in the samples, which may help shift the optical absorption towards the visible region. The literature [1] investigated Ag-doped Co₃O₄ for methyl orange (MO) degradation. In this study, the catalysts showed a better activity with 0.3% Ag, showing a higher photocatalyst activity of 96.4% degradation after 30 h in ultraviolet light irradiation.

Cobalt oxide (Co₃O₄) with Ni addition forms the pure spinel phase. The samples that conserved the pure phase, high surface area, and optical bactericidal properties were 4% Ni (mol mol⁻¹). Therefore, the focus of the present work consists of this percentage. These materials were obtained by sol–gel and hydrothermal methods. Additionally, for comparison, reduced graphene oxide (rGO) was used. The typical rGO applications are due to excellent electrical conductivity, high specific surface area, efficient mechanical flexibility, and ultra-thin layer characteristic.

On the other hand, rGO is commonly used as a graphene-based material with an accessible and low-cost preparation method, and it can be mass-produced [3]. As a result, the specific surface area and conductivity of the Co₃O₄/rGO nanocomposite in nickel foam are dramatically increased, resulting in a significant performance concerning specific capacitance and cyclic stability [3,10].

Photocatalysis in heterogeneous systems is a cost-effective method for decomposing toxic contaminants into non-toxic products [11]. Therefore, developing ecological and sustainable methodologies for atrazine treatment is essential for the industrialized world. In some literature studies, organic pollutants have been completely mineralized into CO₂ and H₂O during the photocatalytic process [2].

Finally, the crystallinity, morphology, elemental composition, and optical/electrical properties of the synthesized materials were characterized by XRD, FE-SEM, TEM, and UV–Vis. The experimental parameters, including the initial atrazine concentration and the catalyst dose, were determined under favorable conditions for photocatalytic degradation. Maximum degradation of 88% was achieved for continuous irradiation up to 60 min. The experiment results showed that the implementation of Ni and rGO in pure Co₃O₄ enhanced its photocatalytic activity due to a lower rate of electron–hole recombination and an increase in the specific surface area of Ni-Co₃O₄ nanocrystals and Ni-Co₃O₄ + rGO. Regarding the bactericidal tests of the S. aureus and E. coli bacteria, the best responses were obtained for the samples sintered by the sol–gel method.

2. Results

2.1. Characterization of the Samples

Two synthesis methods were carried out for this research: sol–gel and hydrothermal (two routes). The best samples were chosen for the application of catalyst and bactericide tests. The characterized samples were sintered by sol–gel (SG) and two variations in the hydrothermal synthesis route (HT). Here, the samples at 0%, 1%, and 4% nickel were characterized, plus the addition of reduced graphene oxide for the 4% samples, as shown in Figure 1.

A powder X-ray diffraction study was performed to investigate the structural and phase purity of Co₃O₄, Co₃O₄-4%Ni SG, Co₃O₄-4%Ni+rGO SG, Co₃O₄-1%Ni HT, 1-Co₃O₄-4%Ni HT, 2-Co₃O₄-4%Ni HT, and Co₃O₄-4%Ni+rGO HT nanocrystals. Strong Bragg diffraction peaks were recorded at 18.9°, 31.3°, 36°, 38°, 45°, 55°, 59°, and 65°, which can be assigned to (111) diffraction, (220), (311), (222), (400), (422), (511) and (440) planes, respectively. According to Card No. # 74-2120, these diffraction peaks reveal the pure cubic structural Co₃O₄ nanocrystals. Figure 2 shows the X-ray diffractograms for three samples as an example since they all maintain the same phase. The peaks of the diffractograms correspond to the pure spinel structure. Figure 2a shows the sample synthesized by the sol–gel method with 4% nickel addition. Figure 2b is the diffractogram for the Co₃O₄ sample with 4% nickel, and Figure 2c is the diffractogram of the Co₃O₄-4%Ni+rGO HT sample.

Figure 2 shows the X-ray diffraction patterns for Ni–Co₃O₄ prepared by hydrothermal and sol–gel routes followed by calcination at 400 °C. No secondary phases were observed in the diffraction pattern. However, with the addition of rGO, the planes are those of the pure spinel phase with a small broad baseline due to the addition of reduced graphene oxide, both for the sol–gel and hydrothermal methods, here it is presented as an example only the hydrothermal. The prominent XRD peak at 36° corresponds to the (311) plane, and the maximum intensity has decreased with the addition of rGO, decreasing the crystallinity.

We use the FESEM measurements in Figure 3 as a morphological tool to study the crystalline form, the growth, and the grain limit. In the sol–gel method, without and with the addition of rGO, no use (Figure 3a) Co₃O₄-4%Ni SG, and (Figure 3b) Co₃O₄-4%Ni+rGO SG, no significant changes are observed. However, a small agglomeration occurs in the nanoparticles. In (Figure 3c), Co₃O₄-4%Ni HT (Method 1), and (Figure 3e), Co₃O₄-4%Ni+rGO HT, we have the morphology of the nanoparticles, respectively, by the hydrothermal method with and without the addition of rGO, respectively. In (Figure 3c), without adding rGO, larger spherical-shaped particles are observed compared to the sol–gel method.

Nevertheless, for this sample with the addition of rGO, the particle size is similar to the sol–gel method. This indicates that adding rGO prevents agglomerates and agrees with the literature [3]. In (Figure 3d), Co₃O₄-4%Ni HT (Method 2), another sample is observed with the hydrothermal method 2; self-aggregating sticks with small spheres around them are observed. As can be seen, the morphological diversity changes depending on the synthesis process.

In the TEM measurements in Figure 4, it is observed that the nanoparticles are more agglomerated (Figure 4c,d) without adding rGO for the synthesis methods used in this investigation. However, in (Figure 4g,h), the addition of rGO plays an essential role in the dispersion of the agglutinated groups in many fine nanoparticles, avoiding excessive aggregation and volume expansion. This makes rGO favor a larger specific material area, providing more reactive sites. As a result, smaller nanoparticles are observed in the hydrothermal method (Figure 4c) compared to those obtained by the sol–gel method (Figure 4d). Figure 4g,h show the samples obtained by the hydrothermal and sol–gel methods with the rGO addition. However, as can be seen, the agglutination or particle size for the hydrothermal method did not change significantly (Figure 4g).

On the contrary, for the sol–gel method, the material agglutination decreased, observing smaller dispersed nanoparticles and forming a chrysanthemum-like flower. Herein, this form in the dispersion of the nanoparticles stimulates the increase in specific surface area. Due to the increased surface area, this structural arrangement and decreased agglutination of the nanoparticles is expected to be the most effective sample in bactericidal and photocatalytic applications. In addition, the TEM method is carried out to obtain a more detailed analysis of the microstructure and size of the Co₃O₄-4%Ni+rGO SG compound. The TEM compound images show that the Co₃O₄ nanoparticles were deposited on the rGO surface. Thus, it can be said that the rGO successfully combined with the Co₃O₄ system. These measures are in agreement with the FESEM results. Figure 4a,b,e,f show the lattice fringes of 0.285 nm, 0.458 nm, 0.230 nm, and 0.188 nm, corresponding to the Co₃O₄ crystal planes (220), (111), (311) and (331), respectively, which is consistent with the XRD results [10].

The FTIR analysis (Figure 5a) shows in all spectrums the stretching vibrations of the metal–oxygen bond. The spinel Co₃O₄ formation oxide is verified by two typical vibrations referred to as Co³⁺ in the octahedral hole (652 cm⁻¹) and Co²⁺ vibration localized in tetrahedral position (538 cm⁻¹) [12]. The rGO presence was more evidenced only in the sol–gel method assigned to the vibration localized between 1148 and 1068 cm⁻¹, and attributed to the Co-O bonds [13]. From the BET measurements (Figure 5b), the surface area of the samples was obtained by the 4% Ni sol–gel method, with and without rGO, thus giving values of (6.5 ± 0.9) m²/g for the Co₃O₄-4%Ni SG sample and (50.5 ± 0.9) m²/g for the Co₃O₄-4%Ni+rGO SG sample. These results indicate that the addition of rGO to the samples increases their surface area [14].

Figure 6 shows the bandgap energy of samples Figure 6a, Co₃O₄-4%Ni HT, Figure 6b, 1Co₃O₄-4%Ni HT, Figure 6c, Co₃O₄-4%Ni SG, and Figure 6d, Co₃O₄-4%Ni+rGO SG, from the linear part extrapolation. The spinel structure of Co₃O₄ is known to be a direct bandgap material. Optical absorption studies show two direct band gaps in Co₃O₄, the main and the sub-band energy gap. On the other hand, the two direct Co₃O₄ optical bands are attributed to the Co excitation in its two valence states, 2+ and 3+. The valence band has a strong O(2p) character, while the main contribution to the conduction band comes from the Co²⁺ 3d orbital. The presence of Co³⁺ cations gives rise to a sub-band inside the energy gap [15].

The first band gap corresponds to O²⁻ to Co³⁺; therefore, the second band gap is the energy gap corresponding to the transitions between bands [15]. In Figure 6, the values obtained from the first band gap energy or sub-band are 1.56 eV,1.7 eV,1.6 eV and 1.5 eV, respectively. The second or true band gap energy values obtained from the samples are 2.24 eV, 2.8 eV, 2.15 eV, and 2.0 eV. Figure 6a,b correspond to the two routes of hydrothermal synthesis, thus obtaining a band gap energy lower for the sample 2Co₃O₄-4%Ni HT. However, for the sol–gel method, the forbidden energy gap decreases even more compared to the previous samples, being much lower for the sol–gel sample with the rGO addition (Co₃O₄-4%Ni+rGO SG).

The higher band gap is assigned to the O^2– → Co²⁺ charge transfer process, basic optical band gap energy (valence-to-conduction band excitation). The lower band gap is ascribed to the O²⁻ → Co³⁺ charge transfer process (located below the conduction band). The presence of Co³⁺ centers in Co₃O₄ produces a sub-band located inside the energy gap. According to the literature [16,17,18] the band edge positions of the photocatalysts can be calculated according to the Mulliken electronegativity. The conduction band (CB) and valence band (VB) potentials of the samples can be calculated using the following equations:

E_VB = X − Ec + ½ E_g

(1)

E_CB = E_VB − E_g

(2)

Here, Ec is the energy of free electrons on the hydrogen scale (~4.5 eV), Eg is the band gap energy of the photocatalyst (2.24, 2.8, 2.15 and 2.0 eV), X is ~5.903 eV for Co₃O₄ is defined as the geometric mean of the absolute electronegativity of the constituent atom. The VB and CB values were calculated from the energy Eg (Figure 6) and electronegativity as ~5.903 eV, being 2.40 eV and 0.40 eV, respectively (

Table 1. The conduction band (CB) and valence band (VB) calculated according to the Mulliken electronegativity.

Samples	Eg (eV)	Conduction Band (CB) (eV)	Valence Band (VB) (eV)
2Co3O4-4%Ni HT	2.24	0.28	2.52
1Co3O4-4%Ni HT	2.80	0.00	2.80
Co3O4-4%Ni SG	2.15	0.33	2.48
Co3O4-4%Ni+rGO SG	2.00	0.40	2.40

).

2.2. Antibacterial Activity

Antimicrobial activity was verified from the inhibitory halo appearance, as shown in Figure 7. From the images, it is possible to confirm that only the nanocomposite Ni-doped Co₃O₄ in the rGO presence obtained by the sol–gel method (Co₃O₄-4%Ni+rGO SG) showed inhibition capacity for both microorganisms. However, Co₃O₄-4%Ni+rGO SG showed more excellent antimicrobial activity for the microorganism S. aureus (Gram-positive), with an inhibition halo value of 0.72 mm (

Table 2. Antimicrobial activity of Co₃O₄ doped with Ni and addition of rGO halo inhibition (mm).

Material	S. aureus/mm	E. coli/mm
Co3O4-4%Ni SG	-	-
1-Co3O4-4%Ni HT	-	-
2-Co3O4-4%Ni HT	-	-
Co3O4-4%Ni+rGO HT	-	-
Co3O4-4%Ni+rGO SG	0.72 ± 0.02	0.47±

), higher than that observed for E. coli (Gram-negative), 0.47 mm, see Table 2. This better performance is attributed to structural differences in the S. aureus cell membrane, which has a thicker peptidoglycan layer without an external cell wall that reduces the permeability of the antimicrobial into the [19] bacterium. In addition, recent studies have demonstrated that the rGO presence in composites with oxides allowed the gain or increase in the intensity of the antimicrobial properties [20].

Such effects can be attributed to the more excellent stability of the particles, minimizing their aggregation, and the more excellent diffusion [21]. The antimicrobial activity of rGO has been attributed to two main factors: the possibility of oxidative stress and the changes in membrane properties. In this way, it favors the loss of vital activities for cellular structures, leading to death [22].

2.3. Photocatalytic Activity

Preliminarily, all the materials were tested from the photocatalytic degradation of the atrazine emerging pollutant under UV light irradiation. Figure 8a shows the contribution of adsorption and photodegradation processes in fixed times of 30 min each. Co₃O₄-4%Ni SG, Co₃O₄-4%Ni HT, and Co₃O₄-4%Ni+rGO SG achieved a total ATZ removal of 58, 66, and 68%, respectively, which represents an improvement of up to 17% compared to the 51% removal observed for photolysis in 30 min. When separating the adsorption contribution, the photocatalytic degradation reached the highest values for Co₃O₄-4%Ni SG and Co₃O₄-4%Ni HT (~53% for both), showing that adsorption represents an essential contribution to the ATZ removal as can be verified for other emerging contaminants [23,24], due to more molecule–catalyst interactions near the surface of the material. Figure 8b shows the concentration of the byproduct HAT formed by dehalogenation followed by hydroxylation of the ATZ halogenated carbon [25]. As HAT was the only by-product formed from ATZ degradation, the highest concentrations of HAT observed in the presence of Co₃O₄-4%Ni SG and Co₃O₄-4%Ni HT corroborated the highest rate of ATZ degradation. However, the concentration of HAT found in the presence of Co₃O₄-4%Ni+rGO SG was equivalent to the concentration observed for photolysis, suggesting that the adsorbed ATZ on the catalyst was degraded to form the same amount of HAT in both processes. Furthermore, the HAT concentration obtained in Co₃O₄-4%Ni+rGO SG presence was 13% higher than that obtained in Co₃O₄ HT presence, even the latest reaching a higher ATZ photodegradation obtained for Co₃O₄-4%Ni+rGO SG. These results confirm that Co₃O₄-4%Ni+rGO SG achieves a higher total removal of ATZ due to the synergistic contribution of adsorption and photocatalysis processes to promote ATZ degradation and HAT formation efficiently.

Based on preliminary studies and the major rate of ATZ removal, the photocatalysts Co₃O₄ HT, Co₃O₄-4%Ni SG, Co₃O₄-4%Ni HT, and Co₃O₄-4%Ni+rGO SG were chosen for the kinetic studies of ATZ degradation in up to 60 min. Figure 9a shows that removal of ATZ from different materials was more efficient for photocatalysis mediated by Co₃O₄-4%Ni+rGO SG, reaching a removal of 88% compared to the 76% removal verified for photolysis. The kinetic constant values (k = min⁻¹) and their respective linear correlation coefficients (R²) were k = 2.38 × 10⁻² min⁻¹/R² = 0.992 for photolysis, k = 1.73 × 10⁻² min⁻¹/R² = 0.933 for CoNi_4_400, k = 3.22 × 10⁻² min⁻¹/R² = 0.989 for Co₃O₄-4%Ni+rGO SG, k = 2.29 × 10⁻² min⁻¹/R² = 0.959 for Co₃O₄-4%Ni SG and k = 1.53 × 10⁻² min⁻¹/R² = 0.987 for Co₃O₄ HT. As can be observed, the ATZ degradation kinetic constant for Co₃O₄-4%Ni+rGO SG was 26% higher than the constant calculated for the photolysis, reinforcing the effectiveness of the photocatalyst in the ATZ degradation process. (Figure 9b) shows the concentration of HAT formed at each ATZ degradation time, proving that all photocatalytic processes can promote the conversion of ATZ to HAT in the first 5 min of reaction. However, this formation is not observed for the photolytic process, which requires a minimum time of 10 min to convert ATZ to HAT. These results show that the conversion of ATZ into HAT is due to hydrolysis of ATZ, which, in the presence of the materials, is catalyzed from the first time of degradation (5 min). The average conversion rates of ATZ in HAT were calculated for all degradation times, where the error values represent the variation of all values in the respective time. As can be observed in Figure 9c, the average conversion values of ATZ in HAT did not show significant differences between the different processes. However, the higher error bar found for the photolytic process is due to the low conversion of ATZ in HAT, and this process presents the first degradation times (up to 10 min). On the other hand, the minor errors found for the photocatalytic processes show that all the materials follow a similar ATZ degradation mechanism, resulting in conversion values of ATZ to HAT, with little variation throughout the process.

The ATZ degradation mechanisms also were investigated in the presence of scavengers for reactive oxygen species formed in the photocatalytic degradation stage. As shown in (Figure 9d), the presence of AA and EDTA prejudice the ATZ degradation. As AA is a scavenger for ^•O₂⁻ and EDTA for h⁺, the ATZ degradation mechanism occurs by the formation of h+ in BV due to the excitation of e- for BC, which reacts with O₂ nascent to form superoxide radicals. Additionally, the ATZ degradation in the presence was 4% lower than in the absence of IPA. This result shows that although it is not the main ATZ mechanism degradation, the ^•OH formation in the system also partially contributes to the better efficiency of the photocatalytic process. In this way, the set of reactions 1 to 6 and Figure 10 present a proposal for the photocatalytic degradation mechanism of ATZ in the Co₃O₄-4%Ni+rGO SG presence.

Co₃O₄-4%Ni+rGO SG + hv → h⁺(VB) + e⁻(CB)

e⁻(CB) + O₂ → ^•O₂⁻

^•O₂⁻ + ATZ → HAT

h⁺(VB) + ATZ → HAT (main mechanism)

h⁺(VB) + H₂O → H⁺ + e⁻ + ^•OH [HAT (secondary mechanism)]

^•OH + ATZ → HAT

The stability of the materials against the photodegradation efficiency of ATZ for up to six application cycles of 60 min was shown in Figure 11. After six cycles, that is, 360 h of use, the Co₃O₄-4%Ni+rGO SG catalyst maintains its photoactivity to efficiently degrade ATZ. Therefore, in addition to being an efficient photocatalyst for ATZ degradation, recyclability highlights the sustainable use of the material in water treatment processes.

3. Materials and Methods

3.1. Synthesis

Synthesis by the sol–gel method of Co₃O₄-4%Ni SG and Co₃O₄-4%Ni+rGO SG was carried out using as precursors cobalt nitrate hexahydrate (Co (NO₃)₂·6H₂O; Acros, Geel, Belgium) with citric acid monohydrate (C₆H₈O₇; Scharlau, Barcelona, Spain) in distilled water as solvent. The mixture was stirred for 4 h, at 50 °C, and then nickel nitrate hexahydrate (Ni (NO₃)₂·6H₂O; Acros) was added at 4%. The samples obtained were dried for 10 h in an oven for dehydration. The dry product was subjected to sintering heat treatment in a muffle furnace, at 400 °C, for 3 h. Doping with reduced graphene oxide (rGO) was carried out using the same precursors with the addition of 0.050 g of rGO with a mixture stirred for 4 h, at 50 °C.

The hydrothermal method 1 of Co_3-XNi_XO₄+rGO used cobalt acetate hexahydrate CoC₄H₆O₄·4H₂O and nickel sulfate hexahydrate NiSO₄·6H₂O, urea CH₄N₂O, reduced graphene oxide (rGO) (0.050 g) and distilled water as precursors. First, stirring is started at 350 rpm and 100 °C for 3 h, grinding the reduced graphene oxide to guarantee the dispersion of the compound. Then, the solution was entered into a hermetically sealed autoclave and heated at 160 °C for 16 h. Finally, the powders were pre-calcined in the air at 300 °C for 3 h, and then at 400 °C for 3 h. Samples of this method are: 1-Co₃O₄-4%Ni HT, Co₃O₄-4%Ni+rGO HT.

Hydrothermal synthesis 2-Co₃O₄-4%Ni HT, Co₃O₄, Co₃O₄-1%Ni HT; the hydrothermal synthesis of 4% (mol mol⁻¹) Ni-doped Co₃O₄ was performed by a procedure adapted to Cardenas et al. [26] and Jin, L et al. [27]. Initially, the precursor cobalt acetate was solubilized in deionized water with nickel sulfate as the source of the doping agent. Then, 2 mL urea and the 45 mL ammonia mineralizer were added in the sequence. After homogenization, the solution was transferred to a hydrothermal reactor and heated at 180 °C for 24 h. The gray precipitate was water washed and centrifuged at 8.000 rpm, for 10 min, at 10 °C temperature. Posteriorly, the particulate was dried at 80 °C for 24 h and submitted to calcination at 300 °C for 3 h.

3.1.1. Bactericidal Test: Agar Diffusion

The antimicrobial assay was realized against bacteria S. aureus (INCQS 15 ATCC 25923) and E. coli (INCQS 33 ATCC 25922) as Gram-positive and Gram-negative, respectively. Initially, bacteria were inoculated in Müller–Hinton broth medium (brand Kasvi), incubating at 37 ± 0.5 °C for 24 h. Then, the concentration of bacteria colonies was adjusted to 10⁵ log CFU mL⁻¹ by turbidity method at 600 nm using the spectroscopy equipment in the visible ultraviolet (UV–Vis) (Shimadzu model UV-1650). In sequence, a 100 μL bacterial aliquot was used to seed on Müller–Hinton agar and incubated at 37 ± 0.5 °C for 24 h. Finally, the antimicrobial activity was verified by measuring the inhibition halos.

3.1.2. Photocatalytic Tests

For the preliminary photocatalytic tests, 10 mL of an ATZ solution (10 mg L⁻¹) was transferred to a 50 mL glass beaker to which 5 mg of catalyst was added. The mixture was kept under agitation for 30 min, in the dark, to reach the adsorption equilibrium, and then a UV (254 nm) light source was switched on and maintained for a fixed time of 30 min. After the adsorption and irradiation times, the samples were filtered in a 0.45 μm PTFE membrane, followed by the analysis of the ATZ and by-products by high-performance liquid chromatography (HPLC) according to the methodology described in the literature [18]. The materials with the best performance in the preliminary photocatalytic tests were selected for the kinetic study, varying the degradation time from 5 to 60 min.

To investigate the degradation mechanisms, Isopropanol (IPA, Sigma Aldrich, St. Louis, MI, USA, 99%), ascorbic acid (AA, Sigma Aldrich, 99%), ethylenediaminetetraacetic acid (EDTA, Sigma Aldrich, 99%), and silver nitrate (SN, Synth, 98%) were used as scavengers for hydroxyl radicals (^•OH), superoxide radicals (^•O₂⁻), holes (h⁺) and electrons (e⁻), respectively. Each 10 mL of ATZ solution (10 mg L⁻¹) was added separately, with 3 mg of SN, 3 mg of EDTA, 3 mg of AA, and 3 mL of IPA. The solutions were irradiated with UV light for 60 min, filtered, and analyzed by HPLC. The recyclability of the material was investigated for up to 6 cycles of the application under UV irradiation by applying 5mg of the material and 10 mL of ATZ (10 mg L^-1) solution at a time of 60 min for each cycle.

3.2. Characterizations

The material crystallinity was investigated by X-ray diffraction (XRD), and measurements were carried out on the PANalytical X’Pert PRO MPD, Bellaterra, Spain θ/θ Bragg–Brentano powder diffractometer with Cu Kα radiation (λ = 1.5418 Å), a working power of 45 kV–40 mA, and a secondary graphite flat crystal monochromator with X’Celerator. The morphology of the samples was characterized with the help of the field-emission scanning electron microscope (FESEM, Nijmegen, The Netherlands) technique (FESEM-JEDL J-7100). Structural details were analyzed by transmission electron microscopy (TEM), high-resolution TEM (HR-TEM), and selected area electron diffraction (SAED), using a Tecnai F20 Super-Twin TMP operating at an accelerating voltage of 200 kV. The samples were dispersed in absolute ethanol and deposited on copper grids with Lacey/Carbon 200 Mesh carbon membranes. Diffuse reflectance spectra (DRS) in the ultraviolet-visible (UV–Vis) region were recorded, at room temperature, between 200 and 1200 nm using a spectrometer (Shimadzu, Kyoto, Japan). Diffuse reflectance UV–Vis spectra were obtained. ATZ photolytic degradation in the UV region was carried out in a wooden reactor.

4. Conclusions

The samples obtain by the sol–gel route and the two hydrothermal processes resulted in the pure spinel phase in a nanometric scale. The morphological results show that the size of the nanoparticles is smaller for the sol–gel method, grouping together with the rGO presence. As consequence, the nanoparticles with rGO increased the surface area from 6.50 ± 0.9 to 50.5 ± 0.9 cm³ g⁻¹. The addition of Ni and rGo in the samples decreased the band gap energy, obtaining 2.0 eV for the Co₃O₄-4%Ni+rGO SG sample, which is the possible reason for a better bactericidal and photocatalytic response. Antimicrobial activity was verified from the appearance of the inhibitory halo, confirming that only the nanocomposite Ni-doped Co₃O₄ in the presence of rGO obtained by the sol–gel method (Co₃O₄-4%Ni+rGO SG) showed inhibition capacity for both microorganisms. The antimicrobial activity was excellent for the microorganism S. aureus (Gram-positive), with an inhibition halo value of 0.72 mm, higher than that observed for E. coli (Gram-negative), 0.47 mm. For the photocatalytic process, it was confirmed that Co₃O₄-4%Ni+rGO SG is activated by UV light. The reactive species h⁺ and ^•O₂⁻ are the main ones responsible for the ATZ photocatalytic degradation and HAT formation. The Co₃O₄-4%Ni+rGO SG catalyst maintains its photoactivity to efficiently degrade ATZ, after six cycles of reuse.