Combinatorial effects of non-thermal plasma oxidation processes and photocatalytic activity on the inactivation of bacteria and degradation of toxic compounds in wastewater

Abstract

The simultaneous presence of hazardous chemicals and pathogenic microorganisms in wastewater is tremendously endangering the environment and human health. Therefore, developing a mitigation strategy for adequately degrading toxic compounds and inactivating/killing microorganisms is urgently needed to protect ecosystems. In this paper, the synergetic effects of the photocatalytic activity of TiO₂ and Cu–TiO₂ nanoparticles (NPs) and the oxidation processes of non-thermal atmospheric pressure plasma (NTAPP) were comprehensively investigated for both the inactivation/killing of common water contaminating bacteria (Escherichia coli (E. coli)) and the degradation of direct textile wastewater (DTW). The photocatalytic NPs were synthesized using the hydrothermal method and further characterized employing field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), ultraviolet-visible diffuse reflection spectroscopy (UV-Vis DRS) and photoluminescence (PL). Results revealed the predominant presence of the typical anatase phase for both the flower-like TiO₂ and the multipod-like Cu–TiO₂ structures. UV-Vis DRS and PL analyses showed that the addition of Cu dopants reduced the bandgap and increased oxygen defect vacancies of TiO₂. The inactivation of E. coli in suspension and degradation of DTW were then examined upon treating the aqueous media with various plasma alone and plasma/NPs conditions (Ar plasma, Ar + O₂ plasma and Ar + N₂ plasma, Ar plasma + TiO₂ NPs and Ar plasma + Cu–TiO₂ NPs). Primary and secondary excited species such as OH˙, O, H and N₂* generated in plasma during the processes were recognized by in situ optical emission spectrometry (OES) measurements. Several other spectroscopic analyses were further employed to quantify some reactive oxygen species (ROS) such as OH, H₂O₂ and O₃ generated during the processes. Moreover, the changes in the pH and electrical conductivity (EC) of the solutions were also assessed. The inactivation of bacteria was examined by the colony-forming unit (CFU) method after plating the treated suspensions on agar, and the degradation of organic compounds in DTW was further validated by measuring the total organic carbon (TOC) removal efficiency. All results collectively revealed that the combinatorial plasma-photocatalysis strategy involving Cu–TiO₂ NPs and argon plasma jet produced higher concentrations of ROS and proved to be a promising one-step wastewater treatment effectively killing microorganisms and degrading toxic organic compounds.

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1. Introduction

The scientific community is increasingly concerned by the dangerous contamination of the aqueous environment by a wide range of microorganisms, including bacteria, protozoa, and viruses responsible for spreading various waterborne diseases to human beings. According to the World Health Organization (WHO), 80% of the reported diseases are caused by drinking contaminated water, and 50% of child deaths result from waterborne diseases.¹ Pathogenic microbes found in water can cause infectious (e.g., measles and flu) and non-infectious (e.g., heart diseases and cancer) diseases.^2,3 For instance, coliform bacteria have frequently been the cause of diarrhea, cramps, fever, nausea, fatigue, etc.⁴ Next to the high burden of microbes, the simultaneous presence of hazardous chemicals in wastewater resulting from improper industrial effluent disposal affects the environment and human health significantly.⁵ The exposure of humans to toxic chemicals can trigger carcinogenesis, genetic defects, reproductive abnormalities, central nervous system degeneration, and reduced life expectancy.^6–8 Moreover, chemicals escaping into the environment can disrupt the natural balance of indigenous microbial populations and affect the aquatic flora. Hence, a mitigation strategy for adequately degrading hazardous compounds and inactivating microorganisms is urgently needed to protect ecosystems. Water disinfection methods such as ozonation and chlorination have been conventionally adopted for the inactivation of microbes.⁹ Nonetheless, several drawbacks have emerged regarding these traditional treatments that engender the generation of potentially toxic by-products such as haloacetic acids and trihalomethanes upon interaction with halide ions and natural organic matter. Moreover, conventional technologies (adsorption, membrane filtration, coagulation, etc.) used for effluent treatment generate secondary solid waste and only partially degrade the present organic pollutants.¹⁰ In order to overcome these problems, advanced oxidation processes (AOPs) constitute an effective alternative method concurrently acting as a disinfection technology for the inactivation of microbes and a decomposition pathway of recalcitrant organic pollutants existing in wastewater.¹¹ In fact, AOPs generate reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as H₂O₂, OH, O, NO₂⁻ and NO₃⁻ that cause the degradation of organic pollutants into smaller non-toxic molecules and the killing/inactivation of microorganisms via different signaling pathways.^12–15 Those pathways trigger, amongst others, the damage of the cell membrane/wall (peptidoglycans), DNA structure and intracellular proteins. Among the numerous AOPs, non-thermal atmospheric pressure plasma (NTAPP) is distinguished, in addition to its high ability to produce ROS and RNS, by other physicochemical factors that are also actively involved in the chemical degradation and pathogen inactivation pathways such as UV radiation, high energy electrons, shock waves, and local high temperature. For instance, plasma-induced electrical fields can lead to physical destruction (electroporation or electrostatic destruction) that potentially kills pathogens present in water, and UV radiation is associated with an improved antibacterial efficiency given its capability to destroy bacterial DNA.^12–14 Nonetheless, plasma alone produces relatively low densities of reactive species (OH˙, O, H₂O₂) not effectively penetrating the gas–liquid interface to influence the target pathogens and reach a 100% degradation efficiency of organic molecules.¹⁶ To overcome this issue, few researchers have recently implemented a combinatorial strategy that couples NTAPP with various photocatalysts to generate a higher concentration of ROS and RNS in aqueous solutions.^17,18 For instance, Pandiyaraj et al. have investigated the degradation of a direct textile effluent dye using NTAPP alone or coupled with Cu-doped CeO₂ nanoparticles (NPs). Results have revealed that the efficiency of total organic carbon (TOC) removal from the direct effluent increased significantly from 7.5% for the plasma process alone to 55.3% by adding the photocatalyst.¹⁹ Zhou et al. have studied the eradication of Escherichia coli (E. coli) in aqueous solutions using an atmospheric pressure microplasma jet array sustained in different working gases, namely He, Air, N₂ and O₂. The presence of the O₂ atmosphere was revealed to inactivate 99.9% of the present E. coli within a 4 min treatment time. Nevertheless, in the presence of a TiO₂ photocatalytic film in the plasma reactor, the time required to kill 99.9% of the E. coli was reduced to 1 min.²⁰ TiO₂ is one of the most preeminent photocatalysts that has conquered a prominent position in several industries to use effluent treatment and water decontamination. The non-toxicity, easy availability, photochemical stability, economic viability and biological inertness are some of the factors that led to the supremacy of TiO₂ compared to other photocatalysts. Nonetheless, TiO₂ has associated with the two following main limitations: (1) the rapid recombination of the electron–hole pair, which hampers the generation of various reactive species such as H₂O₂, O₃, OH˙, etc. during the photodegradation and decontamination processes and (2) the relatively high bandgap restricting the optical response to the UV range. Therefore, doping TiO₂ with metal is commonly adopted to optimize the bandgap and improve the desired processes efficiency. Copper is one of the most suitable dopants for TiO₂, given its pronounced capability to enhance the light gathering capacity and prevent electron–hole recombination.^21–24

Given the above, the present study aims at enhancing the ROS and RNS production in wastewater for both an improved inactivation of common pathogens and enhanced degradation of organic pollutants. This will be achieved by the synergetic effect of the AOPs of NTAPP and the photocatalytic activity of TiO₂ and Cu-doped TiO₂ materials. Hence, the first part of the study is devoted to synthesize TiO₂ and Cu–TiO₂ NPs, making use of the hydrothermal method, and then characterize them using field emission scanning electron microscopy (FE-SEM) and X-ray diffraction (XRD). The second part of the study reports the inactivation efficiency of the most common Gram-negative (E. coli) pathogenic bacteria inoculated in water and the degradation of direct textile wastewater (DTW) by the concurrent NTAPP/photocatalyst process using various operating conditions. The reactive species generated during the inactivation process are determined by optical emission spectroscopy (OES). Moreover, the degradation efficiency of DTW is evaluated by UV-Vis spectroscopy. The formation of OH˙, and H₂O₂ in the contaminated water is investigated by several spectroscopic methods.

2. Experimental procedure

2.1. Materials

The chemicals (analytical grade) used to synthesize TiO₂, and Cu–TiO₂ NPs were purchased from MERCK, India, and were utilized as provided without any further purification. The plasma forming gases were obtained from Jayam Spl Gases, Coimbatore, India. E. coli was procured from Microbial type culture collection, Chandigarh, India (E. coli – MTCC no. 452). The direct textile wastewater was kindly provided by Junior Textile, Tirupur, Tamil Nadu, India.

2.2. Synthesis and characterization of TiO₂ and Cu–TiO₂ NPs

The synthesis of the TiO₂ and Cu/TiO₂ NPs was performed via the hydrothermal method using titanium tetraisopropoxide (TTIP) (purity ≥ 97.0%) and copper sulfate pentahydrate (purity ≥ 98.0%). Initially, 20 ml of TTIP was dissolved in 50 ml of ethanol (purity 99.9%) with a constant stirring for one hour. Subsequently, 2% of diluted hydrochloric acid (HCl, 37%) was added to the precursor solution to maintain the pH at 4.5. 4.98 g of copper sulfate was dissolved in 20 ml of ethanol, then added dropwise into the TiO₂ precursor solution with vigorous stirring to prepare Cu-doped TiO₂. Finally, the obtained solutions were transferred into the separate Teflon-lined autoclave and were heated to around 120 °C for 24 h. After that, they were allowed to cool naturally at room temperature. The precipitates were then taken out carefully and washed with ethanol several times, filtered, and dried in a hot air oven at 60 °C. Subsequently, the obtained product was calcinated at 600 °C for 3 h.^22,23 The acquired TiO₂ and Cu–TiO₂ NPs were used as a photocatalyst to deactivate microorganisms and treat DTW.

The morphology and elemental composition of TiO₂ and Cu–TiO₂ NPs were examined via FE-SEM equipped with energy-dispersive X-ray spectroscopy (EDX) (ZEISS SIGMA FESEM, Germany). The phase and structure of the NPs were investigated by a powder XRD (X'pert PRO X-ray Diffractometer, PANalytical, Netherlands) using Cu Kα (λ = 0.154 nm). The photoluminescence and optical properties of the NPs were examined and the corresponding experimental section can be found in the ESI.†

2.3. NTAPP plasma reactor

The NTAPP reactor used for the inactivation of microorganisms and degradation of organic compounds is shown in Fig. 1. A detailed description of the setup and experimental procedure is explained elsewhere.²⁴ In brief, the reactor has two major components, namely the plasma torch and the AC high voltage (40 kV) and high frequency (50 kHz) power supply. The torch consists of a ring-shaped ground electrode and a rod-shaped live electrode; both made up of copper. The rod-type live electrode is covered by quartz acting as a dielectric to prevent arc transition and is further surrounded by a quartz tube with inner and outer diameters of 11 mm and 15 mm respectively. The ring electrode is positioned underneath the live electrode at a distance of 2.5 cm. The torch is equipped with a separate provision for gas inlet and flow rate is regulated by a mass flow controller (GFC37, AALBORG, USA). The torch assembly is further covered with a Teflon enclosure to avoid electric induction during the treatment.

Fig. 1 Plasma jet during the treatment processes.

Moreover, the reactor is equipped with an optical emission spectrometer (HR4000CG UV-NIR, 1 nm, Ocean Optics, Netherlands) to identify the reactive excited species formed in plasma during the inactivation and degradation processes. The emission spectra were recorded in the wavelength region of 200–1100 nm and were analyzed using the Oceanview spectroscopy software. An optical fiber cable (QP400-2-SR-BX) was used to collect the optical signals produced during the process.

2.4. Preparation of the bacterial suspension and plasma treatment

Firstly, 1.9 g of nutrient broth and 5.2 g of nutrient agar were dissolved in 150 ml of deionized water in a separate conical flask. The broth and agar solution were sterilized for 15 min at 121 °C with 151 lbs pressure and cooled at room temperature. After the sterilization process, 20 ml of nutrient agar solution was poured evenly into a separate Petri dish and allowed to solidify. The colonies of E. coli were inoculated into a nutrient broth medium and were allowed to mix homogeneously using an orbital shaker for one hour.²⁵ Thereafter, 20 ml of bacterial suspension broth solution was poured into a Petri plate and brought beneath the plasma torch's orifice. The distance between the surface of the broth solution and the plasma torch orifice was kept constant at 3 mm. After initial arrangements, the plasma forming Ar gas was allowed into the plasma torch through the gas inlet with a constant flow rate of 9 lpm. Subsequently, a high voltage was applied between the two electrodes and adjusted until a stable plasma exited via the orifice of the torch. Finally, the Ar plasma jet plume was allowed to strike and treat the bacterial broth solution for different plasma exposure times (0–60 s) with a fixed applied potential of 24 kV.

Similarly, the bacterial inactivation process was carried out using 2 different working gas mixtures: Ar + N₂ (7 lpm + 2 lpm) and Ar + O₂ (7 lpm + 2 lpm). After that, the inactivation process was also carried out by combining Ar plasma with the photocatalytic activity of the synthesized NPs (TiO₂ and Cu–TiO₂). In this synergetic treatment process, 20 mg of TiO₂ or Cu–TiO₂ NPs was homogeneously distributed in the E. coli bacteria inoculated aqueous solutions, and the resulting solution was treated with Ar plasma using the parameters mentioned above.

After treatment, 100 μl of the treated solution was spread evenly in Petri dishes containing the solidified agar nutrient medium using an L-shaped rod. All Petri dishes were placed in an incubator for 24 h at 37 °C. The survival rate of bacteria was further assessed by the colony counting method.²⁵ Table 1 describes the typical operating parameters used in the inactivation processes. After bacterial eradication processes, the influence of plasma alone and the synergetic plasma/NPs treatment was further examined on the degradation of direct textile effluent. The same treatment procedure used for the bacterial eradication processes was followed. The treatment time, applied voltage, and distance between the orifice of the plasma jet and the DTW surface were kept constant at 20 min, 24 kV and 3 mm. After plasma treatment, the aqueous solution was filtered using a filter paper (Whatman 40 filter). Finally, the treated and untreated effluent solutions were analyzed by different analytical methods.

Table 1 Plasma operating parameters

Applied potential	24 kV
Treatment time	0–60 s
Distance between the live and the ground electrodes	2.5 cm
Distance between the plasma torch and the water surface	3 mm
Plasma-forming gas and gas mixtures	Ar, Ar + N2 and Ar + O2
Ar flow rate	9 lpm
Ar + N2 flow rate	7 lpm + 2 lpm
Ar + O2 flow rate	7 lpm + 2 lpm

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2.5. Evaluation of the untreated and plasma-treated aqueous solutions

2.5.1. Determination of ROS. A chemical dosimetry method was employed to identify the presence of OH˙ radicals in plasma-treated aqueous solutions using terephthalic acid (TA) (C₆H₄-1,4-(CO₂H)₂ – purity = 98%). The detailed description of the OH˙ measurements is described elsewhere.^25–27 TA reacts only with OH˙ radicals leading to the production of hydroxyterephthalic acid (HTA). The presence of OH˙ radicals in the plasma-treated aqueous solution was measured by UV light (wavelength: 310 nm) given the fact that the produced HTA molecules emit a fluorescence line at 425 nm as was observed by spectrophotometric measurements (Ocean Optics HR4000CG UV-NIR spectrometer). The intensity of the fluorescent emission was thus related to the increase in HTA concentration directly correlated with that of the trapped OH˙ radicals by TA. Similarly, the quantification of H₂O₂ in the plasma-treated aqueous solutions was investigated by a spectroscopic technique using potassium titanium(IV) oxalate (K₂TiO (C₂O₄)·2H₂O – purity = 99% – Sigma Aldrich, India). In order to obtain PTO solution, mixing of PTO (3.54 g), concentrated H₂SO₄ (27.2 ml) and DI water (30 ml) respectively. The volume of the solution was further increased to 100 ml by the addition of the required DI water. After that, 5 ml of the plasma-treated aqueous solution was added to 5 ml of the PTO reagent and was then treated by plasma using the above-used treatment conditions. The resulting color of the aqueous solution was changed into golden-yellow color if plasma-induced H₂O₂ species present in the plasma-treated aqueous solution. Lastly, the absorbance spectra of the aqueous solution were acquired using the spectrophotometer at a wavelength of 400 nm. The concentration of H₂O₂ was obtained based on the following expression:²⁸where A_pt and A_blank are the absorbance of the solution after the plasma treatment and the blank, respectively and l and x the pathlength (cm) of the cuvette and volume (ml) of the solution, respectively.

The presence of ozone in the plasma-treated solutions was further evaluated using the indigo method.²⁹ Before quantifying O₃ in the solution, an indigo stock solution was prepared by adding 770 mg of potassium indigo trisulfonate into a mixed solution containing 1.0 ml of conc. phosphoric acid and 500 ml of distilled water. After that, 7 ml of conc. H₂SO₄, 10 ml of the obtained indigo solution and 10 g of sodium dihydrogen phosphate were mixed thoroughly in 1000 ml volumetric flask. The mixed solution was then diluted up to the mark resulting in the formation of blue colored indigo reagent solution. Finally, the quantification of the ozone in the aqueous solution was done based on the color-changing spectroscopic method of the obtained indigo reagent solution. In this process, untreated and plasma-treated aqueous solutions were added to 10 ml of indigo reagent solution. If O₃ is present in the solution, the colored reagent will be decolorized, and the absorbance of the decolorized sample was measured at 600 nm using a spectrophotometer. The concentration of ozone was calculated as follows:²⁹

where ΔA, b and V are the difference in absorbance between the sample and the blank, pathlength (cm) and volume of the sample (ml) respectively and F = 0.42.

2.6. Assessment of the degradation, pH and electrical conductivity

The percentage of degradation of the DTW with respect to various plasma treatments was monitored via a UV-Vis spectrophotometer (Ocean Optics HR4000CG UV-NIR spectrometer) equipped with a Deuterium halogen light source using the following formula:³⁰where A₀ and A_t are the initial and final absorbances of the aqueous solution respectively.

The electrical conductivity (EC) and pH of the plasma-treated water were measured using a digital EC meter-611 (Elico Ltd, India) and a digital pH meter (pHep, HANNA Instruments, USA) respectively.

3. Result and discussion

3.1. Morphology of TiO₂ and Cu–TiO₂ NPs

SEM images revealed that TiO₂ NPs exhibited a flower-like structure, presenting groups of well-aligned nanorods having diameter of 30 nm (Fig. 2a). This flower-like TiO₂ morphology was previously reported to trigger a significant photocatalytic performance owing to its large contact areas.³¹ The morphology of the Cu–TiO₂ NPs exhibited a tripod/multipod-like structure with more or less a uniform size of each pod (approximately 300 nm in length) (Fig. 2b). This observation confirms that the Cu doping significantly affected the morphology of the TiO₂ NPs as was also observed previously.^31,32

Fig. 2 FESEM images of the synthesized (a) TiO₂ and (b) Cu–TiO₂ NPs.

3.2. Crystal structure of TiO₂ and Cu–TiO₂ NPs – XRD analysis

XRD analysis was performed to investigate the phase composition of the synthesized TiO₂ and Cu–TiO₂ NPs. Fig. 3a depicts the XRD pattern of the TiO₂ NPs that revealed various strong diffraction peaks at 2θ values of 25.70° (101), 38.05° (004), 48.11° (200), 54.81° (105), 62.67° (204), 69.76° (116) and 75.1° (215) matching well with the TiO₂ anatase phase (JCPDS 21-1272) that is considered to be the most photoactive phase.²² Fig. 3b displays the XRD pattern of the Cu–TiO₂ NPs that exhibited similar peaks of TiO₂. Nonetheless, no peaks due to Cu (CuO and Cu₂O) were observed in the XRD pattern attributed to the incorporation of Cu into the TiO₂ via replacing Ti. The absence of such peaks is presumably because the ionic radii of Cu²⁺ (0.73 pm) are very similar to the ionic radii of Ti⁴⁺ (0.68 pm).^33,34 Nevertheless, the EDX spectrum confirmed the incorporation of Cu into TiO₂ NPs (Fig. 4). Finally, the average crystallite size of TiO₂ and Cu–TiO₂ NPs were found to be 6.89 and 6.01 nm, respectively, as estimated by Debye–Scherrer's equation. Ultraviolet-visible diffuse reflection spectroscopy (UV-Vis DRS) and photoluminescence emission spectral analyses can be found in the ESI.†

Fig. 3 XRD patterns of the synthesized (a) TiO₂ and (b) Cu–TiO₂ NPs.

Fig. 4 EDX spectrum of the Cu–TiO₂ NPs.

3.3. Identification of excited species: in situ OES analysis in the presence of E. coli suspensions

The generation of excited species in plasma during the inactivation of E. coli under various treatment conditions was examined by OES as depicted in Fig. 5.

Fig. 5 OES spectra of the plasma jet in the absence and presence of the target bacterial suspension with or without photocatalytic NPs (a) Ar plasma alone, (b) Ar plasma, Ar + N₂ plasma and Ar + O₂ plasma during E. coli inactivation and (c) Ar plasma combined with TiO₂ and Cu–TiO₂ NPs during E. coli inactivation.

The OES spectrum of the Ar plasma jet alone exhibited various major peaks due to excited Ar species (690–900 nm), OH˙ (309 nm) and N₂ second positive system (NSPS) (334 and 354 nm) (Fig. 5a). The presence of hydroxyl (OH˙) radicals and NSPS may be due to the interaction of plasma species with moisture and species present in the surrounding atmosphere. The OES spectrum of the Ar plasma jet during the inactivation of E. coli also displayed similar emission lines. Furthermore, two new spectral lines were observed at 771 and 842 nm due to the formation of atomic oxygen species attributed to the interaction of high energy electrons in plasma with O₂ molecules (e⁻ + O₂ → O + O) and the fragmentation of H₂O molecules in the aqueous solution.^24,25 The OES spectra of the Ar + N₂ and Ar + O₂ plasmas presented the same emission lines attributed to OH˙, O, NSPS and Ar species. However, the intensity of the Ar emission lines was found to decrease substantially due to interaction of Ar species with N₂ and O₂ in plasma and with water molecules in the aqueous environment. These interactions triggered the increase in the intensity of the emission lines due to O, OH˙ and N₂ species (Ar* + H₂O → Ar + OH˙ + H; Ar* + N₂ → Ar + N₂*; N₂* + H₂O → N₂ + OH˙ + H) (Fig. 5b).^35–39 The observed reactive species were associated with an enhanced pathogen inactivation efficacy during the treatment. Fig. 5c portrays the OES spectra of the Ar plasma jet striking the E. coli suspension in the presence of TiO₂ and Cu–TiO₂ NPs. The spectrum corresponding to the presence of TiO₂ NPs exhibited new spectral lines at 540–570 nm due to TiO.³⁵ Finally, when Ar plasma deactivation processes were coupled with Cu–TiO₂ NPs, the OES spectrum showed various additional spectral lines due to Cu²⁺ and Cu⁺ (356, 376, 415, 518, 588, 603 and 612 nm).⁴⁰

Consequently, the intensity of the spectral lines due to Ti, O, OH˙ and NSPS were observed to increase compared to the other treatment conditions. The generation of Cu species (Cu²⁺ and Cu⁺) may be attributed to the redox reaction of Cu ions when they interact with plasma species during the inactivation processes (Cu⁺+ O ↔ Cu²⁺ + e⁻).⁴¹ The obtained Cu²⁺ and Cu⁺ presumably contributed to the increase in ROS concentration during the processes by preventing the recombination of the photo-generated electron–hole pairs that capture the energetic electrons during the interaction of UV-photons in plasma with TiO₂ (TiO₂ + hν → e⁻ + h⁺).^38,39 This prolonged lifetime of the holes in the valence bands probably led to further interaction between those photo-generated holes and water molecules, which ultimately resulted in more hydroxyl radicals (h⁺ + H₂O → OH˙ + H⁺).^39,40 Subsequently, the photo-generated electrons interacted with oxygen molecules producing superoxide ions. Besides the catalytic reactions, the energetic plasma species also contributed to the generation of various ROS and RNS during the inactivation processes via their reaction with the liquid and gas phases as presented in the following reactions.^24,42–45

Ar* + H₂O → Ar + OH˙ + H (R1)

e⁻ + H₂O → H⁺ + OH˙ + 2e⁻ (R2)

O₂ + e⁻ → O + O (R3)

Ar* + N₂ → Ar + N₂* (R4)

N₂* + H₂O_(g) → N₂ + O_(g) + H (R5)

Owing to the above-stated facts, the intensity of the emission lines corresponding to ROS significantly increased when plasma treatment was combined with Cu–TiO₂ NPs. Overall, the OES results indicated the enhanced capacity of the combinatorial strategy involving Ar plasma and Cu–TiO₂ NPs in generating significantly higher concentrations of ROS and RNS than other treatment conditions, which are expected to improve the bacteria deactivation efficiency and degradation of toxic organic pollutants.

3.4. Assessment of OH˙, H₂O₂, and O₃ generation in the treated solutions

The inactivation of the targeted pathogens in the aqueous solution is mainly due, amongst other factors, to the generation of OH˙ and H₂O₂ given their pronounced oxidation potential. Thus, it is important to investigate the presence of OH˙ and H₂O₂ in aqueous media during the different plasma inactivation processes. This in situ generation of hydroxyl radicals was initially determined by a chemical dosimetry method. Fig. 6 depicts the variation in the fluorescence emission intensity of HTA solution upon the various treatment conditions. Results showed that the fluorescence emission intensity of Ar plasma-treated HTA solution (574.2 a.u.) markedly increased when reactive gases were admixed to Ar and when NPs were added to the solution in the following ascending order: Ar + N₂ plasma < Ar + O₂ plasma < Ar plasma + TiO₂ NPs < Ar plasma + Cu–TiO₂ NPs. The results confirmed that Ar plasma treatment combined with Cu–TiO₂ NPs triggered the formation of the highest concentration of hydroxyl radicals in the aqueous solution as revealed by the obtained maximal absorbance intensity (934.4 a.u.) for this treatment condition.

Fig. 6 Variation in the concentration of H₂O₂ and OH˙ with respect to the treatment condition.

Similarly, the concentration of H₂O₂ in the different aqueous solutions was determined by a spectroscopic method using potassium titanium(IV) oxalate. The variation in the concentration of H₂O₂ followed the same trends as that of the OH˙ measurements (Fig. 6). Results also highlighted the fact that the formation of H₂O₂ in the aqueous medium depends on the generation of OH˙ that recombines to form H₂O₂ (OH˙ + OH˙ → H₂O₂).^44,45 Once again, results confirmed that the combination of plasma treatment with Cu–TiO₂ photocatalysts produced a higher concentration of reactive species which is believed to facilitate the deactivation of various pathogens present in the aqueous solution.

Fig. 7a presents the absorbance spectra of the indigo reagent mixed with various solutions subjected to the different treatment conditions. The untreated aqueous solution exhibited a strong absorbance peak at 600 nm with a maximal absorbance intensity of 1.5 a.u. The intensity of the absorbance peaks was found to decrease in the following descending order: untreated > Ar plasma > Ar + N₂ plasma > Ar plasma + TiO₂ NPs > Ar plasma + Cu–TiO₂ NPs > Ar + O₂ plasma. The formation of the highest concentration of ozone in the Ar + O₂ plasma case confirmed by the quantification of ozone using eqn (2) may be attributed to the following reaction: O₂ + O → O₃ (Fig. 7b). In addition, the obtained ozone presumably interacted with electrons producing ozonide ions (O₃⁻) that in turn generated more OH˙ radicals, as presented in the following reactions^46,47

O₃ + e⁻ → O₃^−. (R6)

O₃^−.+ 2H⁺ → H₂O₂ + O (R7)

H₂O₂ + e⁻→ 2OH˙ (R8)

Fig. 7 Estimation of O₃ concentration in solutions subjected to different treatment conditions. (a) Absorbance spectra of the indigo reaction and (b) O₃ concentration.

3.5. Inactivation/killing processes of E. coli

The influence of the different treatment conditions on the inactivation/killing of E. coli was examined by the colony-forming unit (CFU) method as portrayed in Fig. 8. It shows the photographs of E. coli plated on agar after subjecting the inoculated suspensions to the different treatment conditions and the corresponding quantitative measurements of the CFUs. A greater number of colonies (350 CFU ml⁻¹) was detected in the case of the control sample compared to all other treated samples. After 15 s of Ar plasma treatment, the number of colonies slightly decreased and continued to decrease with increasing the treatment time progressively. Finally, only 30 CFUs were observed for a plasma exposure time of 60 s, which corresponds to an inactivation efficiency of 97.1% (Fig. 8b and c). This reduction in the number of CFUs was mainly caused by increasing ROS and RNS such as OH˙, O, H₂O₂ and NO₃⁻ as the treatment time increased. At early stages, plasma treatment produced a meager amount of reactive species that could not trigger severe damage to the outer membrane of bacteria. Even if the outer membrane were affected by minor damages, E. coli bacteria would not be killed or inactivated due to their characteristic effective self-defense and self-healing mechanisms that protect them.^48–51

Fig. 8 Inactivation/killing of E. coli. (a) Growth of E. coli on agar after subjecting the suspension to different treatment conditions and exposure times; (b) CFU count as a function of the treatment condition and (c) E. coli inactivation efficiency as a function of the treatment.

In contrast, the generation of higher concentrations of reactive species upon longer plasma exposures caused severe damages to the cell membrane, which swiftly suppressed the self-protection mechanisms of the E. coli bacteria resulting in a higher inactivation/killing rate. The killing/inactivation processes were further examined by treating the bacterial suspensions with Ar + O₂ and Ar + N₂ plasmas for various exposure times. Ar + N₂ plasma treatment significantly decreased the number of colonies to 80 CFU ml⁻¹ after a relatively short treatment time of 15 s and led to a complete bacterial eradication upon a treatment time of 60 s. The inactivation/killing rate of E. coli was higher when treating the solution with Ar + O₂ compared to Ar + N₂ plasma due to the generation of higher concentrations of ROS and the emission of a more extensive extent of UV radiation during the processes (Fig. 8b and c). When the processes were carried out via the combination of the AOPs of Ar plasma with the photocatalytic reactions of TiO₂ and Cu–TiO₂ NPs, the bacterial eradication rate was significantly improved. In particular, the presence of Cu–TiO₂ NPs could lead to a complete inactivation/killing of E. coli in the aqueous solution and, as such, a 100% deactivation efficacy within only 15 s of plasma exposure (Fig. 8b and c). The observed improved results obtained when implementing the combinatorial plasma-photocatalysis strategy might be attributed to the generation of higher concentrations of ROS (e.g., H₂O₂, O, O₃, and OH˙) in the aqueous media resulting from combined plasma-electron impact phenomena and plasma-photocatalysis processes. During the inactivation/killing of bacteria by plasma-photocatalysis reactions, Ar plasma produced UV photons absorbed by TiO₂ NPs, resulting in the generation of electron–hole pairs. The photo-generated holes reacted with water molecules to form OH˙ radicals. Simultaneously, superoxide ions radical (O₂⁻˙) were formed via the interaction of photoelectrons with oxygen molecules. The formation of reactive species during the plasma catalytic process is shown in the following reactions^23,37,50,51

TiO₂ + hν → e⁻ + h⁺ (R9)

h⁺ + H₂O → H˙ + OH˙ (R10)

e⁻ + O₂ → O₂⁻˙ (R11)

2O₂⁻ + 2H⁺ → H₂O₂ + O₂ (R12)

The obtained reactive species constitute one of the main factors killing/inactivating the pathogens in aqueous media. In fact, those species oxidized the lipid peroxide to form hydroperoxide and triggered oxidative stress in the bacterial cells engendering devastating effects on the structure and activity of proteins which ultimately killed the bacteria. Moreover, Zhou et al. have reported that the positioning of the bacteria on the surface of TiO₂ photocatalysts affords an additional mechanism for their inactivation.²⁰ In fact, this contact between TiO₂ and the bacterial cell leads to a direct exchange of an electron–hole on the surface of the cell envelope that leads to formation of ROS. Once crossing the envelope, the ROS will oxidize and damage the intracellular components of the DNA and intracellular proteins, thus killing the cell. Furthermore, the direct penetration of metal ions through the cell membrane was associated with a rupture of the nucleic acid and protein, resulting in the inactivation or eradication of the bacterial cells. A schematic diagram of the microbial inactivation upon the combined plasma-NPs treatment is shown in Fig. 9. Despite the numerous stated advantages of the combined plasma treatment with TiO₂ catalysis, two major constraints exist. The first one is the fast recombination of the photo-generated electron–hole pairs during the process and the second one resides in the limited optical response within the UV range owing to the wide bandgap.²² The use of Cu–TiO₂ NPs instead of TiO₂ NPs comes to overcome the limitations in this combinatorial strategy.

Fig. 9 Schematic diagram of the effects of plasma AOPs and NP photocatalysis reactions on the inactivation/killing of a bacterial cell.

In fact, the doping of copper into the TiO₂ plays a significant role in ROS production by capturing photoelectrons that reduce Cu²⁺ oxidation states into Cu⁺ (Cu²⁺ + e⁻ → Cu⁺). In their turn, Cu⁺ ions can further re-oxidize by the presence of oxygen and hydrogen peroxide in the solution to form Cu²⁺ (Cu⁺ + O₂ → Cu²⁺ + e⁻; Cu⁺ + H₂O₂ → Cu²⁺ + 2OH˙). The obtained electrons also interact with oxygen molecules in an aqueous solution to form superoxide (O₂⁻) (e⁻ + O₂ → O₂⁻). Concurrently, the photo-generated holes react with H₂O molecules to generate OH˙ radicals (h⁺ + H₂O → OH˙ + H⁺). The ionic states of Cu (Cu²⁺ and Cu⁺) also contribute to the generation of OH˙ by reacting with H₂O and H₂O₂ (Cu²⁺ + H₂O → Cu⁺ + OH˙ + H; Cu⁺ + H₂O₂ → Cu²⁺ + 2OH˙).^23,52 Moreover, the doping of Cu can improve the light gathering capacity of TiO₂ i.e., by extending the UV range to the visible range.^22,23 Owing to the obtained evident results and the above-stated facts, one can conclude that the treatment processes carried out by the combination of plasma with Cu/TiO₂ NPs could produce significantly higher concentrations of reactive species than the plasma treatment alone enhanced the killing/inactivation of bacteria.

3.6. pH and conductivity of the untreated and treated solutions

Given the drastic effects of acidic environments on bacteria, the determination of the pH is one of the important validations indicating their successful inactivation/eradication. The pH of untreated and all treated aqueous solutions were measured, and the corresponding results are presented in Fig. 10. The pH of the untreated aqueous solution was 7.0, thus indicating a neutral medium. After performing the various treatments, the pH of the aqueous medium decreased in the following order: Ar plasma > Ar + N₂ plasma > Ar + O₂ plasma > Ar plasma + TiO₂ NPs > Ar plasma + Cu–TiO₂ NPs. Results confirmed that the natural aquatic medium was converted into acidic media due to the formation of various acidic compounds such nitric and nitrous acids (N₂ + e⁻ → N˙ + N˙; N˙ + O₂ → NO + O˙; NO + O₃ → NO₂ + O₂; NO₂ + OH˙ → HNO₃ → H⁺ + NO₃⁻; NO + OH˙ → HNO₂ → H⁺ + NO₂⁻).^42,44,53,54 In an acidic medium, the amino acids and functional groups of microorganisms are severely affected, leading to the denaturation of their proteins which compromises their activity and ultimately triggers their death.^55,56 Fig. 10 also shows the variation in the EC of the aqueous media pre-and post-treatment. Results revealed that the EC of the untreated solution was 0.11 μS cm⁻¹ and increased post-treatment in the following order of treatment conditions: Ar plasma < Ar + N₂ plasma < Ar + O₂ plasma < Ar plasma + TiO₂ NPs < Ar plasma + Cu–TiO₂ NPs. This may be ascribed to the formation of various ions in the solution, which can penetrate the cell wall of the microbes and further hamper their normal activity.⁵⁷ Given the fact that the presence of TiO₂ and Cu–TiO₂ NPs could give significantly better results compared to plasma alone, all the following investigations will be limited to the treatment conditions involving the combinatorial strategy: Ar plasma + TiO₂ NPs and Ar plasma + Cu–TiO₂ NPs. Ar plasma alone will be also tested for comparison purposes.

Fig. 10 Variation of the EC and pH as a function of the implemented treatment conditions.

3.7. Degradation of DTW

The action of the synergetic treatment was further studied by evaluating the degradation of DTW. Fig. 11a portrays the UV-Vis absorbance spectra of untreated and treated DTW under various conditions. The absorbance spectrum of untreated DTW exhibited a wide absorbance peak in the range of 551–661 nm attributed to the existence of different organic compounds in the DTW. After Ar plasma treatment alone, the intensity of the absorbance peak slightly decreased, corresponding to a degradation percentage of 16.1% (Fig. 11b).

Fig. 11 (a) UV-Vis absorption spectra and (b) degradation percentage of DTW as a function of the treatment condition (Ar plasma alone or coupled with TiO₂ or Cu–TiO₂ NPs).

The intensity of the absorbance peak further decreased when Ar plasma was combined with TiO₂ NPs, resulting in a degradation efficiency of 40.3% of the organic molecules in the DTW. Interestingly, the intensity of the absorbance peak significantly dropped when Ar plasma was coupled with Cu–TiO₂ NPs to reach a maximal degradation percentage of 55.7%. The increase in the degradation percentage of organic contaminants in the DTW is most probably attributed to the formation of various ROS (e.g., OH˙ and H₂O₂) during the different processes (Table 2). This increase was perceived to adhere to the following ascending order of treatment condition: Ar plasma < Ar plasma + TiO₂ NPs < Ar plasma + Cu–TiO₂ NPs. The degradation of DTW by the various treatment conditions was further validated by measuring the pH and EC as displayed in Table 2. Results revealed that the pH and EC of the untreated DTW were 8.9 and 0.312 μS cm⁻¹, respectively. A significant decrease in the pH values was perceived after performing the various treatments and went along with the following descending order of conditions: untreated > Ar plasma > Ar plasma + TiO₂ NPs > Ar plasma + Cu–TiO₂ NPs. A reverse ascending trend was observed for the EC of the untreated and treated DTW (Table 2). The decrease in the pH of the DTW indicated an increased acidity via the formation of various acids (HNO₂ and HNO₃) resulting from the breakdown and oxidation of the present organic molecules. The increase in the EC may be due to the generation of various ionic species in the plasma-treated DTW. Moreover, results showed that a maximum of 55.7% of TOC was removed when the DTW was synergistically subjected to the AOPs of Ar plasma and the photocatalysis processes of Cu–TiO₂ NPs. The above-observed results offered an additional strong validation that the treatment carried out by the combination of Ar plasma with Cu–TiO₂ NPs is more effective in decomposing numerous organic molecules in DTW than the Ar plasma treatment alone owing to the generation of higher concentrations of various ROS and RNS.

Table 2 Variation in the fluorescence emission intensity, pH, EC, TOC removal percentage and H₂O₂, between untreated and treated DTW for various operating conditions

Treatment conditions	Fluorescence emission intensity (a.u.)	Concentration of H2O2 (μm l^−1)	pH	EC (μS cm^−1)	% TOC removal
Untreated	—	—	8.9	0.312	—
Ar plasma	1011	0.269	8.1	0.365	16.13
Ar plasma + TiO2 NPs	2183	1.288	7.5	0.412	40.31
Ar plasma + Cu–TiO2 NPs	2556	2.137	5.9	0.564	55.76

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4. Conclusion

The research investigated the synergetic effects of the AOPs of an NTAPP treatment and the photocatalytic activity of TiO₂ and Cu–TiO₂ NPs on both the inactivation of bacteria and the degradation of organic pollutants in wastewater. Thus, the first part of the investigation was devoted to synthesize TiO₂ and Cu–TiO₂ NPs by the hydrothermal method. The morphology and phase of the NPs were then studied, making use of FE-SEM and XRD measurements. FE-SEM results revealed that TiO₂ and Cu–TiO₂ NPs exhibited flower-like and multipod-like structures, respectively. XRD analysis indicated a typical anatase structure for both TiO₂ and Cu–TiO₂ NPs. Moreover, the successful incorporation of Cu ions into the TiO₂ lattice was evidently confirmed by EDX upon Cu-doping of the NPs. The NPs bandgap, that was obtained by UV-Vis DRS, decreased when TiO₂ NPs were doped with Cu. Moreover, PL analysis revealed that the emission intensity of the Cu-doped TiO₂ NPs was higher than that of TiO₂ NPs owing to the formation of oxygen vacancies that hinder the recombination rate of electrons which improved the photocatalytic activity performance. The second part of the investigation explored the influence of various treatment conditions (Ar plasma, Ar + N₂ plasma, Ar + O₂ plasma, Ar plasma + TiO₂ NPs and Ar plasma + Cu–TiO₂ NPs) on the inactivation/killing of Gram-negative (E. coli) bacteria and degradation of DTW. OES and several other spectroscopic analyses evidently showed that the synergetic treatment implicating Cu–TiO₂ NPs and Ar plasma generated the highest density of ROS (e.g. OH˙ and H₂O₂), which led to an enhanced inactivation efficiency of the pathogens and degradation efficiency of DTW. The results were further corroborated by pH, EC, and TOC removal efficiency measurements. Overall, the present extensive analysis showed that the combinatorial strategy involving the photocatalytic activity of Cu–TiO₂ and oxidation processes of NTAPP has a great potential as a full one-step wastewater treatment concurrently killing microorganisms and degrading toxic organic compounds.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

Dr K. Navaneetha Pandiyaraj grateful to the Science and Engineering Research Board (SERB) (EMR/2016/006812 Dated: 02-Nov-2017) and Department of Science and Technology – Fund for Improvement of S&T infrastructure in universities & higher educational institutions (DST-FIST) Government of India for the financial support. Dr Rouba Ghobeira would like to thank the Research Foundation Flanders-Belgium (FWO – grant number 12ZC720N) for financing her post-doctoral position. MP would like to thank SNR Sons Charitable Trust and Sri Ramakrishna Engineering College, Coimbatore, India.

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Footnote

† Electronic supplementary information (ESI) available. See https://doi.org/10.1039/d1ra09337a

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