Nitrogen and Carbon Nitride-Doped TiO2 for Multiple Catalysis and Its Antimicrobial Activity

Nitrogen (N) and carbon nitride (C3N4)-doped TiO2 nanostructures were prepared using co-precipitation route. Fixed amount of N and various concentrations (0.1, 0.2, 0.3 wt%) of C3N4 were doped in TiO2 lattice. Through multiple techniques, structural, chemical, optical and morphological properties of samples were thoroughly investigated. XRD results verified anatase TiO2 presence along the substitutional doping of N, while higher degree of crystallinity as well as increased crystallite size were noticed after doping. HR-TEM study revealed formation of nanostructures incorporated on two dimensional (2D) C3N4 nanosheet surface. Elemental composition was checked out using EDS technique which confirmed the presence of dopant in product. Optical characteristics were evaluated with UV–vis spectroscopy which depicted representative redshift in absorption spectra resulted in a reduction in bandgap energy in N/C3N4-doped TiO2 samples. The formation of Ti–O–Ti bonds and different molecular vibrations were disclosed by FTIR. Trap sites and charge carrier’s migration in the materials were evaluated with PL spectroscopy. Multiple catalytic activities (photo, sono and photo-sono) were undertaken to evaluate the dye degradation performance of prepared specimen against methylene blue and ciprofloxacin. Further, antimicrobial activity was analyzed against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) bacteria.


Introduction
In the past few years, researchers and scientists have paid greater attention to energy crisis and environmental and aquatic pollution. In today's technology driven society, relentless consumption of fossil fuels is serving to make these issues worse [1]. Fossil fuels, the rich energy-generation source, are contracting worldwide and developed countries are switching to sustainable and environmentfriendly technologies. On the other hand, textile industry produces wastewater containing 5-15% of untreated organic dyes. Around 1 × 10 5 dyes are in use globally and 7 × 10 5 ton dyestuff is generated by the textile industry worldwide annually. Discharge of these untreated dyes not only affects the oxygen and nitrogen cycle connected to photosynthesis but also causes grave esthetic deterioration [2]. These are some of the serious environmental issues that need to be addressed to sustain human society in the long term.
Properties of bulk materials depend a lot on size and structure [3]. In this regard, nano-scaled semiconductors with diverse properties are used for photocatalytic and dye degradation applications [4]. Various transition metals (TMs) Ti, Cu, Fe, Co and non-metals (NMs) N, C, S etc., exhibit distinct physical and chemical properties [5]. In this regard, a combination of TM (titanium-dioxide) and NM (nitrogen) exhibit additive properties. Combination of semiconductors (with metals and metal oxides) that bear appropriate band arrangements possess striking applications in energy production and water treatment [6]. Rare earth metals oxides such as TiO 2 , CdO, CoO, ZnO, etc., are leading candidates for many such applications. Amongst these, TiO 2 holds limited activity in visible region due to its high band gap value (3.0 eV for rutile phase, 3.2 eV for anatase phase) [7], low surface area and high electron-hole recombination [8]. In 1972, TiO 2 was used for the decomposition of water using UV light [9]. Since then, photocatalysis with semiconductors has gained much attention due to their potential applications such as in hydrogen production and environmental cleaning [10]. Semiconductors with unique band gap (occupied valence band and unoccupied conduction band) serve as suitable catalysts for photochemical reactions. Currently, the main focus is visible light region through band gap tuning of inorganic semiconductor.
TiO 2 is a semiconductor with prominent features including tunable band gap, simple synthesis routes and ecological-friendly nature. Therefore, to improve the photocatalytic activity of TiO 2 nanostructures, modulation of its band gap was undertaken by adding nitrogen (N) for better absorption of solar spectrum. N was added through CH 4 N 2 O source which contains 46% of its concentration [7,11]. As a result, bandgap of TiO 2 varied from 3.2 to 3.06 eV (substitutional replacement of N with oxygen) or it can also be varied from 3.2 to 2.46 eV (interstitial doping) which is more preferable for visible light [12][13][14]. Dopant C 3 N 4 is a 2D material which resolves the impediment of low surface area of TiO 2 to enhance photocatalytic activity upon addition in various concentrations and exhibit broader spectral response near the visible region compared to pristine TiO 2 . Moreover, C 3 N 4 has promising applications due to a suitable band gap (2.7 eV) which has the ability to enhance photocatalytic activity of TiO 2 for use in dye degradation [15][16][17][18].
TiO 2 nanostructures can also be utilized as antibacterial agent. The antibacterial activity of TiO 2 nanostructures is assigned to reactive oxygen species (ROS) such as hydroxyl radicals and hydrogen peroxide, which induce severe oxidative stress on bacterial strain generated under illumination. Therefore, TiO 2 is a potential candidate for antibacterial agent. The generated ROS provide a contact between TiO 2 and cells which kills the cell due to damage aroused in DNA and cell membrane that ultimately results in termination of cell cycle [19]. The antimicrobial activity of prepared antibiotic also depends on surface area, morphology, crystallinity, concentration/ dosage, pH of the solution, capping agent, and also the nature of microorganisms. Combining the merits of C 3 N 4 with TiO 2 and N, prepared composite is important to solve the problems of the environmental crisis in worldwide including organic water pollutants and pathogenic microbial contaminations [20,21]. Few studies have been reported for the antibacterial activity and degradation of different dyes [5,8,11], best of our knowledge, this novel study report the efficiency of C 3 N 4 -doped N/TiO 2 nanocomposites synthesized by co-precipitation route.
In this paper, co-precipitation method was used to synthesize pristine and doped TiO 2 nanostructures. This study revealed the strong contact formation of TiO 2 with dopants (N and C 3 N 4 ) which efficiently increased the photocatalytic activities against methylene blue and ciprofloxacin as well as antibacterial property against E. coli and S. aureus bacteria.

Preparation of TiO 2 Nanostructures
Titanium-dioxide (TiO 2 ) was prepared by adopting coprecipitation method where 55 mL of ethanol, 13 mL of Ti(C 4 H 9 O) 4 and 5 mL of deionized water (DI water) were mixed and stirred vigorously for 30 min. Ethanol (~ 100 mL) and DIW (~ 100 mL) were added to the stirred solution for 2 h at 50 °C. The pH of solution was maintained up to ~ 10 using NaOH (0.5 M) solution. Then, solution was centrifuged and dried at 90 °C for 10 h. After that, sample was annealed at 450 °C for 4 h to achieve stable anatase nanostructures (Fig. 1b).

Evaluation of Photocatalytic Activity
The photocatalytic activity (PCA) of synthesized catalysts was assessed by estimating the degradation rate of a combination of two toxic dyes namely methylene blue (MB) and ciprofloxacin (CF) in aqueous solution. The stock solution of dyes was prepared in DIW (10 mg/1000 mL) and 10 mg of prepared catalyst (pristine TiO 2 , C 3 N 4 , 0:1, 0.1:1, 0.2:1, 0.3:1) was added to 50 mL stock solution. After homogeneous stirring, solution was placed in a sealed box under mercury (Hg) lamp (wavelength 400 to 700 nm and power 400 W) at ~ 15 cm distance to avoid overheating. After 20 min interval, 3 mL solution was separated to check concentration of dyes present in the solution by utilizing UV-Vis spectroscopy. The degradation efficiency was determined by the formula given as: where C o and C t initial and final concentration of dye at time t = 0 and at final time t, respectively [22].

2-Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging Assay
Free radical scavenging activity of all samples was examined using the method reported by Kibiti and Afolayan [23] with certain modifications. Various concentrations of pristine TiO 2 , C 3 N 4 , and C 3 N 4 doped TiO 2 nanoparticles (0-500 µg/mL) were prepared and mixed with equal volume of 0.1 mM DPPH solution. The reaction mixture was vortex and incubated for 30 min in dark at ambient temperature. Ascorbic acid was employed as a reference antioxidant. Absorbance of mixture was measured at 517 nm using spectrophotometer. The % scavenging ability was calculated using equation: where A 0 is absorption of control (Methanol + DPPH) and A 1 is absorbance of sample.

Bacterial Segregation and Identification
With ovine mastitic milk specimen's antibacterial evaluation was undertaken on S. aureus and E. coli isolated after initial screening at ovine blood agar (5%) and finally on mannitol salt agar (MSA) and MacConkey agar (MCA), respectively. Coagulase, catalase, and Gram' staining protocols were used to classify extracted commodities (biochemically and morphologically).

Antimicrobial Activity
Well diffusion procedure was adapted to assess antibacterial effects of N and C 3 N 4 co-doped TiO 2 by swabbing 1.5 × 10 8 CFU mL −1 of purified bacterial isolates on MCA and MSA, separately. On swabbed dishes, 6 mm deep wells were drilled with aseptic well borer. Various dilutions of doped nanomaterial's e.g., 500 and 1000 µg/50 µL were placed into wells as minimum and maximum dosage in contrast with ciprofloxacin as positive control (5 μg/50 μL) and DIW (50 μL) as negative control. The sensitivity of all prepared samples was measured with a Vernier caliper after overnight incubation (37 °C) of Petri plates. Antibacterial evaluation was contemplated by utilizing one-way analysis of variance [24].

Material Characterization
Phase transition and crystal structure of binary-doped TiO 2 was examined with XRD utilizing spectrum Bruker system with monochromatic Cu K-α (λ = 0.154 nm and 2θ = 10°-80°) with a scan rate of 0.05° per minute. The study of functional groups and chemical analysis was undertaken utilizing FTIR spectrometer (PerkinElmer 3100) with range of spectra from 4000 to 400 cm −1 in 32 scans and a resolution of 0.2 cm −1 . The optical study was carried out with Genesys 10S spectrophotometer (ranging from 200 to 800 nm). Interlayer spacing and surface morphology of prepared products were inspected through HR-TEM and EDS spectrometer, JSM-6460LV, and HRTEM Philips CM30 and JEOL JEM 2100F. Photoluminescence spectroscopy was carried out to inquire transfer and recombination of e − to h + pairs utilizing a spectrofluorometer (JASCO, FP-8300).

Results and Discussion
To analyze the structure of the crystal, phase purity and size of crystallites, XRD was employed on control and N/C 3 N 4 -doped TiO 2 in 2θ range of 10°-80° (Fig. 2a)  Various functional groups and chemical compositions present in as prepared samples were identified using FTIR analysis (Fig. 2e). In acquired spectra, it can be seen that absorption band stationed at 400-700 cm −1 corresponds to Ti-O and Ti-O-Ti stretching vibration modes, which indicated TiO 2 formation. This vibration band has been linked with physiosorbed water protons in synthesized samples [27][28][29]. Band observed at about 1635 and 3200-3500 cm −1 referred to hydroxyl (O-H) group and physically absorbed water on pristine TiO 2 surface, respectively [30,31]. In C 3 N 4 spectrum, absorption band at 1624 cm −1 was attributed to C-N heterocycle stretching vibrational modes [32], while four bands at 1232, 1304, 1411, 1556 cm −1 were referred to aromatic C-N stretching vibrational modes [33,34]. Notably, all characteristic peaks of TiO 2 and C 3 N 4 can be observed, validating the formation of C 3 N 4 :N/TiO 2 nanostructure.
The morphology and crystal structure of pristine TiO 2 (Fig. 3a), 0:1, 1:0, 0.1:1 and 0.3:1 nanostructure were studied by TEM analysis. Figure 3b represents N-TiO 2 composite with a high surface energy that leads to aggregation [35] and Fig. 3c is the illustration of C 3 N 4 , a mesoporous nanosheet (NS). Upon C 3 N 4 addition, N-TiO 2 composite was embedded and well distributed on NS which also roughly confirmed the C 3 N 4 wrapping, as illustrated in Fig. 3d. This intimate interfacial contact between N-TiO 2 composite and NS was necessary for photocatalytic activity. Upon doping, crystallinity of prepared nanostructures improved and after higher doping, NS wrapped N-TiO 2 composite and ultimately made an efficient interfacial contact (Fig. 3e). In HR-TEM image of 0.1:1 (Fig. 3c′), interlayer spacing was calculated to be 0.35 and 0.33 nm pertaining to (101) and (002) crystal planes of N-TiO 2 composite and C 3 N 4 , respectively, in consistence with XRD results. Interlayer spacing has been measured for each sample using Gatan software, given in Fig. 3á-d. In order to check further interfacial contact, EDX mapping of as-prepared C 3 N 4 (1:0) and 0.3:1 (higher doping) samples was conducted to inspect distribution pattern of its components. As revealed in Fig. 4a, five components (C, N, Ti, O, Na) were found to be uniformly dispersed in higher doped specimen. Sodium (Na) came from sodium hydroxide (NaOH) added for maintaining pH of solution up to ~ 10. Combined with HR-TEM and XRD results, it recommended that within 0.3:1 sample, N and TiO 2 nanoparticles are certainly well dispersed inside wrapped C 3 N 4 NS and indicated intimate contact as well.
Elemental composition was evaluated by EDX to confirm the purity of 0.3:1 (higher doping) and 1:0 (Fig. 4bc). As illustrated in Fig. 4b To determine optical performance of undoped and doped TiO 2 , UV-vis spectroscopy was used in the range 300-550 nm. TiO 2 has characteristic absorbance peak found around ~ 350 nm, with N-doping, slight redshift was observed in absorption spectra caused by overlapping of 2p orbitals of O 2 and N, as shown in Fig. 5a [36]. An increase in spectral absorbance was observed upon C 3 N 4 doping into N-TiO 2 composite, attributed to complete planarization of C 3 N 4 (non-overlapping of adjacent orbitals). Enhanced absorptive ability in UV-region was assigned to internal scattering and harmonious effect from N-TiO 2 and C 3 N 4 's π to π * and n to π * transitions, respectively, as depicted in Fig. 5b [37]. Peaks redshifted due to molecular engineering of C 3 N 4 in N-TiO 2 composite that potentially advanced absorption and transition ability of charge carriers [38]. Tuac transformation was applied to calculate bandgaps of prepared samples. For TiO 2 bandgap was calculated to be 3.2 eV and gradual decrease in bandgap energies were observed close to Fermi level after adding N and C 3 N 4 to ~ 2.9 eV, as given in Fig. 5c-h [39].
PL emission spectra of TiO 2 and its composites were computed from 410 to 520 nm with an excited wavelength of 350 nm at room temperature, as illustrated in Fig. 6a. Spectra unveiled migration and electron-hole (e − to h + ) pairs recombination efficiency [40]. Characteristic peak of TiO 2 at 455 nm showed the highest recombination of e − to h + pairs which significantly limited PCA of TiO 2 whereas recombination rate was decreased upon  [41]. Sub-band at the edge of conduction band facilitated PCA [42], so for higher doping sample, lower recombination rate proposed higher photo-generated charge transportation that internally enhanced PCA of 0.3:1.

Reaction Mechanism and Kinetics
The following mechanism was involved in PCA of prepared catalyst (see Fig. 7): 1. Photoexcitation: The PCA first involves photoexcitation that initiates by the photons processing equal or greater energy than bandgap energy (E g ) of material. These photons stimulate electrons of valence/lower band (VB) and migrate them to conduction/higher band (CB). Electrons leave holes behind in VB resulting in e − -h + pairs generation, as shown in equation below.
2. Ionization of water: Holes create OH • free radicals after coming into contact with water (H 2 O).
The OH • radical act as an oxidizing agent on the surface of semiconductor that targets adsorbed molecules and takes part in mineralization.
Dye (MB and CF) + OH · → CO 2 + H 2 O dye intermediates (9) Dye + h + (VB) → oxidation products (10) Dye + e − (CB) → reduction products The PCA of as-prepared samples was evaluated for degradation of MB and CF dye under replicated visible light irradiation (Fig. 6b). The blank test demonstrated that MB and CF could not be degraded under irradiation of light in the absence of catalyst thus it can be deduced that MB and CF were stable. As indicated in Fig. 6c, C3N4 degraded the targeted dye up to 45% and enhanced photocatalytic activity of nanostructures was observed with increasing C 3 N 4 concentration which effectively degraded MB and CF. In case of N-TiO 2 , relatively higher extent of degradation (58%) occurred as compared to TiO 2 (32%) while highest doped sample (0.3:1) showed maximum degradation of 85% within 80 min. The apparent reaction rate constants (k) were determined for all specimens by measuring slopes of ln (C o /C t ) against time plot. Moreover, k value of 0.3:1 was also higher than others, which was ~ 2.5 times higher than pristine TiO 2 (Fig. 6d).
The enhanced PCA of nanostructures may be attributed to these measures: firstly, the C 3 N 4 sheet has a larger surface that encouraged broad adsorption within catalyst and furnished additional active sites for surrounding reactants. Secondly, after incorporation of N and C 3 N 4 in TiO 2 , increased charge separation efficiency by inducing new energy levels within the forbidden band gap of TiO 2 . These induced levels act as trapping sites for photo induced electrons increasing the electron transfer efficiency which ultimately improved the degradation performance of nano-catalysts. Thirdly, due to intimate and well-matched band edge, N-TiO 2 collects photoinduced electrons from the CB of C 3 N 4 thus improving the charge separation efficiency. In general, C 3 N 4 produces e − to h + pairs under visible light that quickly recombine and only a small fraction of e − participate in PCA. Whereas when TiO 2 was modified by binary dopants to form a nanocomposite, photo-generated electrons in CB of C 3 N 4 can directly move to CB of N-TiO 2 composite, as shown in Fig. 7, because CB edge of C 3 N 4 was more negative than N-TiO 2 . The above-mentioned parameters eventually increased overall photocatalytic activity of as-prepared nano-composites [46][47][48].
Sonocatalytic activity (SCA) was also measured by degrading MB and CF via ultra-sonication route. Same as for PCA, samples were collected after 20 min interval in SCA. As illustrated in Fig. 8c, C3N4, TiO 2 , N-TiO 2 composites degraded the MB and CF up to 36%, 20% and 27% in 80 min while for lower doping, activity of 0.1:1 nanostructure increased up to 60% and then decreased for further and higher doping concentrations. For lower doping, catalyst formed the microbubbles and bore more active sites for growth of nucleation, further generated more reactive radicals [49] and for further doping, active sites of prepared catalysts were insufficient to be occupied by dye molecules. Second possible reason could be surplus of C 3 N 4 amount that have restricted the energy obtained from ultrasound wave [50].
The rate constants (k) have been calculated for sonodegradation kinetics by measuring slopes on ln (C o /C t ) against time as shown in Fig. 8b. PCA and SCA of pristine TiO 2 and C 3 N 4 were less efficient, thus N/C 3 N 4doped TiO 2 composite turned up as potential catalyst for dye degradation.
The combined effect of PCA and SCA has been evaluated further by adjusting sonometer under light source for all synthesized samples. The earned results unveiled that pristine C 3 N 4 , TiO 2 and N-TiO 2 composite degraded dye up to 60%, 40% and 55%, respectively. For lower doping, 0.1:1 nanostructure degraded MB and CF up to 86% caused by combined effect of PCA and SCA. But for further and higher doping (although assisted by PCA) active sites were insufficient that might be ascribed to dominant effect of SCA that subsequently decreased the degradation performance. Also, surplus amount of C 3 N 4 and inhomogeneous mixing of catalysts can cause agglomeration which might limit the energy obtained from ultrasound wave and visible light source (Fig. 9c).
For photo-sono degradation kinetics, the rate constants (k) have been estimated by computing slopes on ln (C o /C t ) against time, as manifested in Fig. 9b.
Antioxidant characteristics of compounds is tied to their electron or hydrogen atom donating capability to DPPH free radical, such that they create stable diamagnetic compounds [51]. This DPPH free radical's reduction capability can be examined by lowering the absorbance at 517 nm.
All synthesized compounds were evaluated for antioxidant activity using a DPPH radical scavenging assay. Using ascorbic acid as a reference, DPPH disappearance was evaluated spectrophotometrically at 517 nm. In this study, it was discovered that the DPPH activity of the nanoparticles increased in a dose-dependent manner (Fig. 10). It is confirmed that Pristine TiO 2 showed high scavenging activity (50.22%) at concentration of 500 µg/mL compare to C 3 N 4 . Because TiO 2 may form OH . , O 2 .− and 1 O 2 reactive oxygen species, which have the potential to bond with the DPPH free radical [51][52][53]. Some recent studies have reported 1 O 2 to be the dominant active specie in the degradation of MB dye under solar irradiation [53,54]. While N-TiO 2 showed DPPH scavenging up to 57.34% that is 7% higher than that of TiO 2 . This considerable increase is resulted from the addition of a doping agent which lowers the size of TiO 2 nanoparticles and increases their reactivity [55,56]. In case of C 3 N 4 doped TiO2 with mass ratio of 0.1:1, scavenging activity increased up to 84.45% that might be the availability of sufficient amount of nitrogen from doped C 3 N 4 . But increasing the concentration of doped C 3 N 4 on N-TiO 2 , scavenging activity was decreased. This was due to high C 3 N 4 concentration caused an increase in turbidity of test sample, which in turn caused an antagonistic interaction resulted in a decrease scavenging activity (84.45-70.75%) [50]. Antimicrobial activity of binary doped TiO 2 was conducted using well diffusion technique (Fig. 11)  Anti-bacterial effectiveness is swayed by the scale of nanoparticles so oxidative stress of invented nanocomposites is dependent on scale and concentration [57]. An electrostatic contact between bacteria and nanoscale structures results in the generation of reactive oxygen species, which are lethal to cells [24,58]. Oxygen reactive species (ROS) encircle bacteria external membrane and through extrusion and bulge of cytoplasmic components bacteria death occurs [59]. Micro pathogens ruin also proceeds when cations strongly bind with negative components of bacterial cells. Cations cause dysfunction in bacterial ribosomal activities and enzymatic degradation resulting collapse [60]. Two reactions have been identified as feasible for the bactericidal mechanism of nanomaterials, one of which involves strong interaction between the cations Ti +4 and bacterial cells, resulting in the formation of negativized sections and subsequent collapse, and the other of which involves electronic excitation of the TiO 2 valance band surface via irradiation. Additionally, the electrical O 2 reaction generates O −2 radicals, which results in the production of H 2 O 2 . The

Conclusion
Binary-doped TiO 2 was synthesized through co-precipitation method and synthesized samples were evaluated for photo, sono and photo-sono catalytic degradation of MB and CF dyes and bactericidal activities. The strong contact formation between dopants and TiO 2 efficiently increased e − to h + pairs separation efficiency induced by light. The narrow bandgap of C 3 N 4 :N/TiO 2 composite was accredited to N as well as C 3 N 4 incorporation in pristine TiO 2 . The prepared samples showed efficient degradation performance under visible light as well as under ultrasonic waves (SCA). Moreover, the combined effect of photo and sono catalysis was also evaluated for prepared catalysts for comparative study. Furthermore, prepared nanocomposites exhibited notable efficacy against S. aureus and E. coli bacteria as well. We believe that this study will open new insights into the fabrication of novel, binary doped heterojunctions for effective dye degradation and bactericidal applications in the future.