PHOTOCATALYTIC DEGRADATION OF RHODAMINE B IN HETEROGE- NEOUS AND HOMOGENEOUS SYSTEMS

This study focuses on the photocatalytic degradation of Rhodamine B (RhB) in heterogeneous and homogeneous photoFenton reactions. In the heterogeneous system, iron(II) doped copper ferrite CuII (x) FeII (1−x)Fe III 2 O4 nanoparticles (NPs) prepared in our previous work were employed as potential catalysts. The photodegradation of RhB was carried out in a quartz cuvette located in a diode array spectrometer. The experimental conditions such as pH, NPs dosage and H2O2 dosage with regard to the photocatalytic degradation of RhB were optimized to be 7.5, 500 mg/L and 8.9 × 10−2 mol/L, respectively. In addition, visible light-induced photodegradation of RhB was also carried out by using H2O2 over a wide pH range in the absence of heterogeneous photocatalysts. It was observed that the reaction rate significantly increased above pH 10, resulting in a faster rate of degradation of RhB, which may be attributed to the deprotonation of hydrogen peroxide. Furthermore, the potential antibacterial property of such catalysts against the Gram-negative bacterium Vibrio fischeri in a bioluminescence assay yielded inhibition activities of more than 60% in all cases.


Introduction
Synthetic dyes have numerous applications in several industries, e.g., paper, textile, leather and paint. Besides these applications, some dyes are toxic organic compounds and their discharge into the environment causes eutrophication, aesthetic pollution and distress for marine organisms [1,2]. Some synthetic dyes are recalcitrant, that is, resistant to biological degradation and direct photolysis. In addition, many dyes contain nitrogen which produces carcinogenic as well as mutagenic aromatic amines as a result of natural anaerobic reductive degradation [3,4].
These toxic organic dyes can be mineralized into water and carbon dioxide via photocatalytic reactions using catalysts under ultraviolet or visible light irradiation [5,6]. Only a handful of research groups have developed and applied ferrite nanoparticles (NPs) as catalysts which can utilize larger bandwidths of the visible light spectrum. Manganese ferrite [7], zinc ferrite [8][9][10], aluminium doped zinc ferrite [11], manganese doped cobalt ferrite [12], barium ferrite [13], copper ferrite [14], and nickel ferrites [15,16] have been investigated with regard to the degradation of certain dyes and other toxic compounds. * Correspondence: horvath.otto@mk.uni-pannon.hu Our research group prepared and applied iron(II) doped copper ferrites Cu II (x) Fe II (1−x) Fe III 2 O 4 (where x = 0, 0.2, 0.4, 0.6, 0.8, 1) for the photo-induced degradation of Methylene Blue (MB) [17]. Here, a detailed photocatalytic study on the degradation of Rhodamine B is presented by using heterogeneous photo-Fenton systems and compared to homogeneous photocatalytic procedures. In addition, the antibacterial property of iron(II) doped copper ferrites in the Vibrio scheri bioluminescence inhibition assay was investigated.

Materials
Rhodamine B (molecular formula: C 28 H 31 ClN 2 O 3 ) was used as a model dye for visible light-induced photocatalytic degradation. Anhydrous copper(II) sulfate, ferric chloride hexahydrate, ammonium iron(II) sulfate hexahydrate and sodium hydroxide were used to prepare the catalysts. Sodium hydroxide or hydrochloric acid was added to adjust the pH during photocatalysis. Hydrogen peroxide (30%w/w) was employed as Fenton's reagent and double distilled water used as a solvent throughout the study. All the laboratory-grade chemicals were obtained from Sigma-Aldrich (Budapest, Hungary) and used without further purification.

RhB photocatalytic reactions
For photocatalysis, a stock solution of 0.5 g/L RhB was prepared. In order to perform the photocatalysis, a small cuvette used as a reactor was adjusted to a S600 UV/Vis diode array spectrophotometer. The concentration of RhB (approximately 1.8 × 10 −5 mol/L) in the cuvette was calculated by using the Beer-Lambert law [17].
Control experiments for the self-degradation of RhB were carried out without ferrite nanoparticles in the absence and presence of both light and hydrogen peroxide (for the oxidant effect). Then the NP catalyst of a given concentration was added to the RhB solution and stirred for 30 mins to ensure a good degree of dispersion and reach an adsorption equilibrium before photodegradation. The temperature of the photoreactor (25±2 • C), concentration of RhB (1.8 × 10 −5 mol/L) and duration (140 mins.) of photocatalytic experiments were kept constant. The process variables investigated were the catalyst dosage (80 to 800 mg/L), hydrogen peroxide concentration (2.2 × 10 −2 to 3.0 × 10 −1 mol/L) and pH (2 to 12). Meanwhile, the original pH of the total aqueous solution was approximately 7.5. The pH was adjusted by adding HCl or NaOH before starting the photocatalytic experiment.

Determination of reaction rate
The Beer-Lambert law was used to determine the reaction rate of each experiment. The spectral changes observed in the visible range of the absorption spectrum ( Fig. 1) indicate that the intermediates and end products formed during the photocatalytic degradation of RhB did not produce any remarkable peaks. Therefore, the reaction rate of RhB photodegradation can be determined from the reduction in absorbance at the maximum wavelength (λ max = 554 nm). The addition of heterogeneous photocatalysts caused the baseline in the recorded spectra to change as a consequence of scattering. This problem was resolved during the evaluation of the reaction rate by applying baseline corrections.

Assessment of antibacterial property
A Luminoskan Ascent microplate luminometer (Thermo Scientific) was used to measure the antibacterial property of the ferrite NPs in a Vibrio scheri bioluminescence inhibition assay. According to the manufacturer's (Hach Lange GmbH, Germany) recommendations, a test specimen of a Gram-negative Vibrio fischeri (NRRL-B-11177) suspension was prepared with a lifespan of 4 hours after being reconstituted. The same test protocol was followed as reported in the literature [19].
During the evaluation, the results obtained from 2 parallel measurements were averaged before the relative inhibition (%) was calculated using where I c (t) denotes the emission intensity of the control sample at time t and I s (t) represents the emission intensity of the test specimen at the same time.

Results and discussion
A detailed explanation regarding the control experiments concerning the photodegradation of RhB was reported in one of our previous studies [18]. The experiment used as a basis for comparisons (RhB + H 2 O 2 + Light) is shown in Fig. 2.
After the control experiments, the photocatalytic efficiency of six doped ferrite nanoparticles was investigated. Fig. 1 shows the spectral changes obtained during the photocatalytic experiment using NP-3 and the decrease in the absorbance of RhB at λ max = 554 nm (inset of Fig.  1). The degradation reaction of RhB follows apparent rstorder kinetics (Fig. 3), which is also consistent with earlier observations regarding other catalysts [20,21]. The slight deviation from the straight line is due to the complex nature of this heterogeneous system. Fig. 4 reveals that all doped ferrite NPs in the series of Cu II (x) Fe II (1−x) Fe III 2 O 4 (x = 0 − 1) delivered higher apparent rate constants for the degradation of RhB compared to the control experiment. Doped copper ferrites  NP-2 and NP-3 exhibited outstanding photocatalytic performances in the series studied. Nickel doped cobalt ferrite NPs revealed a very similar trend with regard to the photo-oxidative degradation of RhB [22]. The higher apparent rate constants for the degradation of RhB using NP-2 and NP-3 may be attributed to their special needlelike crystalline structure [17]. On the basis of the first experimental series, NP-3 was chosen to further investigate three important determinants, namely the catalyst dosage, hydrogen peroxide concentration and pH of the heterogeneous photo-Fenton system. The increase in dosage from 0−500 mg/L yielded a significant increase in the apparent rate constant. This phenomenon can be attributed to the higher number of available active sites in heterogeneous photo-Fenton processes [23]. However, increasing the dosage of NPs above 500 mg/L caused a moderate  decrease in the apparent rate constant, which may be attributed to the fact that higher concentrations of NPs can increase the turbidity of the reaction system, thereby hindering the absorption of light [4]. Therefore, for the photocatalytic experiments that followed, an optimum NP-3 dosage of 500 mg/L was used.

The effect of the hydrogen peroxide concentration
At first, the effect of H 2 O 2 on the photodegradation of RhB in the absence of NPs was investigated (Fig. 6). The concentration of H 2 O 2 was increased from 4.5 × 10 −2 to 6.7 × 10 −1 mol/L. The reaction rate was enhanced by increasing the concentration of H 2 O 2 up to 3.5 × 10 −1 mol/L. However, beyond this value, a slight decrease in the apparent rate constant was observed.
The second experimental series focused on checking the effect of increasing the concentration of H 2 O 2 from 2.2 × 10 −2 to 3 × 10 −1 mol/L in the presence of NPs  in a heterogeneous photo-Fenton system (Fig. 7). The reaction rate was remarkably improved by increasing the concentration of H 2 O 2 up to 8.9 × 10 −2 mol/L. A further increase in the concentration of H 2 O 2 did not enhance the reaction rate significantly, moreover, similar results have been published in the literature [24,25]. The excess H 2 O 2 could act as a • OH scavenger, producing the less reactive HO • 2 species instead of the highly potent • OH [4,23,25]. Hence 8.9 × 10 −2 mol/L as an optimum concentration of H 2 O 2 was used in experiments on the photocatalytic degradation of RhB that followed.

The effect of pH
The surface charge properties of the photocatalyst and the ionic species present in the photocatalytic reactor are greatly influenced by the pH. Furthermore, the photodegradation efficiency of the dye is affected by the ionic species and surface charge of the photocatalyst in the reaction mixture. Two experimental series were designed to study the effect of pH on the visible light-induced degradation of RhB. In the first series, the pH was varied from 3.8 to 12.1 while the concentrations of RhB and H 2 O 2 were kept constant in the absence of NPs. Remarkably, neutral and alkaline pHs were found to be more effective in this system concerning RhB photodegradation (Fig. 8).
In addition, the presence and absence of H 2 O 2 were also investigated at higher pH values (approximately pH 12), which can be seen from the last two data points in Fig. 8. It was observed that significantly enhancing the fraction of the more reactive deprotonated form of hydrogen peroxide (HO -2 ) at higher pH values ( pKa = 11.75 [26]) noticeably accelerated the rate of RhB degradation. On the basis of Fig. 8, it was possible to determine the individual (apparent) rate constants (under these conditions) for the differently protonated forms of peroxide, namely 1.9 × 10 −5 s −1 for H 2 O 2 and 6.2 × 10 −4 s −1 for HO -2 . Deprotonation resulted in increasing the degradation effect by 32 times.
Moreover, the effect of the pH in the presence of NPs (Fig. 9) revealed that a neutral or near alkaline pH could be optimal during this type of reaction. Although the best apparent rate constant was observed at pH ≈ 8, further increasing the pH resulted in a slight decrease in the reaction rate. By comparing Figs. 8 and 9, it can be observed that the partly hydroxylated forms of the metal ions ([Fe III (OH) 2 ] + , [Cu II (OH)] + ) could also be identified at the local maximum of approximately pH = 8 presented in Fig. 9. Therefore, the partly hydroxylated metal ions can react with H 2 O 2 , resulting in a ≈ 14times increase in the individual (apparent) rate constant (2.7 × 10 −4 s −1 compared to 1.9 × 10 −5 s −1 for H 2 O 2 in the absence of NPs).
The pH can also alter the charge state of RhB in the reaction mixture. Furthermore, at high pH values, RhB aggregates are produced as a result of the excessive concen- tration of OHions, which compete with COOto bind with N + . In addition, since the surface of the solid catalyst is negatively charged, it repels the RhB due to the presence of ionic COOgroups under basic conditions. Therefore, the degradation efficiency on the surface of the photocatalyst is decreased. The same phenomenon in the case of bismuth ferrite nanoparticles has been reported in the literature [4,27]. However, an increase in the pH above 11 significantly enhanced the reaction rate (Fig. 9) in a very similar manner to the reaction in the absence of NPs. As a result, the presence of NPs does not further increase the reactivity of HO -2 . In addition, the effect of light, hydrogen peroxide and NPs at an approximately constant pH is illustrated in Table 1. The light-induced degradation of RhB at pH 12 in the absence of both hydrogen peroxide and NP-3 yielded a very low reaction rate (Step 1). In Step 2, the addition of hydrogen peroxide in the absence of both light and NP-3 at pH 11.9 yielded a faster reaction rate. Step 3, which represents a heterogeneous Fenton system, yielded a much faster reaction rate. The heterogeneous photo-Fenton system shown in Step 4 yielded the best reaction rate as far as the degradation of RhB is concerned.
The catalyst NP-3 (Cu II (0.4) Fe II (0.6) Fe III 2 O 4 ) was able to overcome the disadvantage of the narrow pH range of conventional photo-Fenton processes. Based on this experimental series, the catalyst Cu II (0.4) Fe II (0.6) Fe III 2 O 4 is a promising candidate for the degradation of various recalcitrant dyes.

Generalized RhB degradation mechanism
A very simple schematic mechanism is proposed for the purpose of RhB degradation since the reactive species produced during irradiation, namely • OH, H + and • O − 2 , oxidize RhB molecules to intermediates of lower molecular weights. Generally speaking, the active species react with the central carbon atom in the chemical structure of RhB. Then the oxidizing agents attack the intermediates produced in the previous step, yielding smaller open-ring compounds. Subsequently, the latter compounds are mineralized to water and carbon dioxide [28]. As is displayed in Fig. 10, the UV/visible absorption spectrum of RhB degradation yields prominent peaks at 262, 358 and 554 nm. However, no significant peaks were observed following photodegradation (Fig. 10) in neither the visible nor UV region, which confirmed the complete mineralization of RhB.
The images obtained from the photoreactor (cuvette) before and after photocatalysis also confirmed the complete degradation of RhB, namely a clear, colorless solution was obtained after the removal of solid catalysts (Fig.  10) by centrifugal filtration.

Photocatalytic efficiencies under optimized conditions
Finally, the photocatalytic efficiencies of all six NPs (NP-1 to 6) were determined under optimized conditions for the degradation of RhB (Fig. 11). It was observed that all of the NPs were active photocatalysts, the application of NP-3 yielded the highest reaction rate. These results are quite comparable to those presented in Fig. 4 obtained from the first series of experiments. However, the concentration of hydrogen peroxide under the optimized conditions (8.9 × 10 −2 mol/L) is considerably lower than in the first series (1.8 × 10 −1 mol/L) and is, therefore, much more economical. Although the concentration of the photocatalyst is higher under the optimized conditions (500 vs. 400 mg/L), the NPs can be reused over several cycles. According to our results, all NPs in the series can potentially be applied for the purpose of environmental remediation.  Figure 11: Photocatalytic efficiency in terms of apparent rate constants (compared to the control experiment) for NP-1 to 6. Experimental conditions: concentration of NPs is 500 mg/L, concentration of RhB is 1.8 × 10 −5 mol/L, concentration of H 2 O 2 is 8.9 × 10 −2 mol/L, initial pH is 7.5, and irradiation time is 140 mins.

Assessment of the antibacterial activity of doped copper ferrites
The inhibition effect (%) of doped copper ferrites against Gram-negative Vibrio scheri in bioluminescence assays is illustrated in Fig. 12. The inhibition (%) of bacteria in the presence of doped nanoparticles containing varying ratios of copper (Cu II ) and iron (Fe II ) revealed that all doped copper ferrites yielded sufficient antibacterial activities. In our research, higher ratios of Cu II proved to be useful in improving antibacterial activity. The same trend in terms of bacterial inhibition against Gram-negative Escherichia coli was observed using cobalt ferrite nanoparticles synthesized by co-precipitation [29]. Generally speaking, Cu II can disrupt the functions of cells in several ways, hence the ability of microorganisms to develop resistance to Cu II is remarkably reduced. The attachment of Cu II ions to the surface of microorganisms plays a key role in their antibacterial activity [30]. The ions from the surface of doped copper ferrites, especially Cu II , are adsorbed onto bacterial cell walls, damaging the cell membrane in two possible ways, namely by altering the functions of enzymes or solidifying the structures of proteins. Therefore, the presence of copper ferrites in the bacterial growth medium immobilizes and inactivates bacteria, inhibiting their ability to replicate and ultimately leading to cell death [31]. In our study, a mechanism is proposed ( Fig. 13) in which doped copper ferrites are attached to the cell wall of the bacterium Vibrio fischeri, reducing its ability to replicate. The degree of bacterial inhibition in all cases is approximately 60%, which confirms the potential application of doped copper ferrites in terms of antibacterial developments.

Conclusion
Iron(II) doped copper ferrites Cu II (x) Fe II (1−x) Fe III 2 O 4 have been proven to be efficient catalysts for the degradation of organic pollutants under visible-light irradiation in the presence of hydrogen peroxide. The performances of NPs with copper(II) ratios of x = 0.2 and 0.4 were especially promising under optimized conditions. Contrary to conventional homogeneous photo-Fenton systems, our catalysts exhibit higher efficiencies under neutral and near alkaline conditions. Besides their advantageous photocatalytic ability, these NPs also show a sufficient degree of antibacterial activity, due to their copper(II) constituents. By taking both properties into consideration, Cu II (0.4) Fe II (0.6) Fe III 2 O 4 yields the optimum combination of these features. Therefore, from the series of NPs studied in this work, NP-3 is the most promising candidate for the combined photocatalytic purification and disinfection of water.