Combined Application of Biological-Photocatalytic Process in Degradation of Reactive Black Dye: An Excellent Outcome

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2.2. Bacteria and Growth Medium

The bacterial strain used was isolated from dye wastewater obtained from final effluent treatment plant (FETP) of Ankleshwar, Gujarat, India. The principle of sequential selective enrichment batch culture for selection of dye decolorizing bacteria was employed in synthetic wastewater medium (SWM) with Basic Violet 3 as the carbon source. The basic composition of the synthetic wastewater medium was (g/L); (NH₄)₂SO₄ 0.28, NH₄Cl 0.23, KH₂PO₄ 0.067, MgSO₄・7H₂O 0.04, CaCl₂・2H₂O 0.022, FeCl₃・6H₂O 0.005, NaCl 0.15, NaHCO₃ 1.0, and 1 mL/L of a trace element solution containing (g/L); ZnSO₄・7H₂O 0.01, MnCl₂・4H₂O 0.1, CuSO₄・5H₂O 0.392, CoCl₂・6H₂O 0.248, NaB₄O₇・10H₂O 0.177, and NiCl₂・6H₂O 0.02. The textile wastewater used for isolation of dye-decolorizing bacteria was acclimatized for 8 weeks prior to transfer into 250mL Erlenmeyer flasks containing 100mL SWM. After incubation of the flasks, a mixed culture that showed quick and stable decolorization activity was transferred to newly prepared SWM. After five successive transfers, it was plated on SWM agar containing 20 mg/L of each dye and incubated at 30°C for 5 days. Bacterial colonies around which clear zones expanded quickly were picked for further studies and designated as ETL 1-5. To check for the dye-degrading potential of each bacterial isolate, preliminary batch experiments were carried out using sterile 250mL Erlenmeyer flasks containing 100mL of SWM spiked with dye, after which the solution was inoculated with freshly grown bacterial cells. The final pH was 7.2. The bacterial isolate that showed the highest ability to degrade the triarylmethane dyes was selected and used for subsequent investigations. The selected bacterium (ETL-1) was characterized and identified as Pseudomonas aeruginosa using Gram stain, spore test, motility test, and a battery of biochemical and physiological tests as described by Vanderzannt and Splittstoesser ^[13] and Cheesbrough ^[14] and with reference to the Bergey’s Manual of Determinative Bacteriology ^[15].

2.3. Biological Experiments and Decolorization Procedures

Examination of decolorization and biodegradation ability of Pseudomonas aeruginosa was carried out in synthetic medium containing: (glucose, 10; Yeast extract, 0.34; NH₄Cl, 0.84; KH₂PO₄, 0.134; K₂HPO₄, 0.234; MgCl₂.6H₂O, 0.084) g/L and 200 mg/L RB5. The pH of the solution was adjusted to 7±0.2 before being autoclaved. A 5% (v/v) of the culture suspension in 0.85% saline was inoculated into 100 mL of medium in 250-mL flasks and incubated in shaking incubator at 150 rpm and at 32°C for 72 h. In addition, control flasks containing the same medium without inoculums were also kept to observe the abiotic decolorization. At regular time intervals, an aliquot (4 mL) of the culture media was withdrawn from the flasks, centrifuged at 12000 rpm for 10 min and the supernatant was analyzed for the remaining dye concentration. Decolorization of dye was determined by measuring the absorbance of culture supernatant at λmax (597 nm). The percentage of decolorization was calculated according to the following formulation:

(1)

Where Ai and At are the initial absorbence of the dye solution and at sampling time (t) after inoculation, respectively ^[16]. Spectral scanning between 200 and 800 nm using a UV–vis spectrophotometer (Shimadzu, Japan) was performed in order to analyze the dye degradation process. In this study, absorbance measurement at 254 nm wavelength was used to indicate the presence of aromatic compound in the sample ^[17].

2.4. Photocatalytic Experiments

Photocatalytic reactor and light source Photocatalytic experiments were performed in a batch system (pyrex vessel) with a total capacity of 1 L (batch mode). The total volume of the dye solution in the system was 800 mL. The reactor containing reaction solution (mixture of dye solution and photocatalyst powder) was placed on a magnetic stirrer to provide appropriate mixing. A medium-pressure mercury lamp (UV-C, maximum emission at 247.3 nm) with a power of 150 W and 66 mm length (manufactured by ARDA, France) was used as the light source. The UV lamp was placed centrally and parallel to the length of the cylinder in the reactor, inside a quartz tube ^{[18, 19]}. The reactor was covered with an aluminum foil sheet to prevent UV emission around it. Due to the production of heat by the light source, the temperature of the solution was maintained constant at 21 ±3°C in all experiments by cooling water around the vessel.

2.5. Analytical Procedures

In order to perform the photocatalytic experiments, the working solution, containing 50 mg/L of dye and 0.2 g/L of TiO2 was prepared using tap water. Prior to the photocatalytic process, the suspensions were magnetically stirred in the dark for 30 min to reach adsorption-desorption equilibrium between the dye and TiO2. Samples (5 mL) were taken at specific time intervals, centrifuged at 6000 rpm for 15 min and then filtered through a 0.45 μm syringe filter in order to separate TiO2 particles ^[20]. Measurement of absorbance was carried out at maximum wavelength of RB5 (λmax =597 nm). UV–vis spectra of samples were recorded between 200 and 800 nm. Photocatalytic degradation of different concentrations of RB5, namely, 100, 200, 300 and 500 mg/L was also examined.

2.6. Combined Biological-photochemical Process

To carry out these experiments, synthetic medium was treated with Pseudomonas aeruginosa ETL-2211 cells for 24 h and after centrifugation and filtration, fed into the reactor. At different time intervals, samples were withdrawn, centrifuged and filtered through a 0.45 μm membrane filter following which UV–vis scan spectroscopy and COD measurements were carried out. COD measurements (for both biological and photocatalytic steps of the process) were performed following the closed reflux, colorimetric method using a COD reactor (Hack, USA), according to standard procedures ^[21].

3. Results

Biological experiments Pseudomonas aeruginosa ETL-2211 was able to remove about 85% of color in culture containing 200 mg/L RB5 after 12 h. Complete decolorization was observed in less than 24 h. The dye concentration in control flasks remained unchanged. UV–vis spectra (200–800 nm) were studied. Reactive Black 5 presents two main absorption peaks, one in visible region (597 nm) and another in UV region (310 nm), which can be attributed to the presence of chromophoric azo bonds and both aryl and naphthalene-like moieties, respectively ^[2]. The results of our experiments showed that the peak at 597 nm disappeared approximately after 12 h (complete removal of this peak was observed in less than 24 h). Meanwhile the main peak at UV region (310 nm) was apparently shifted to 254 nm within the first 12 h and no more changes were observed during further incubation time up to 72 h.

3.1. Photocatalytic Experiments

Photocatalytic process was employed to investigate the degradation of RB5 at 50, 100, 200, 300 and 500 mg/L concentrations. Results (Figure 2) showed that this process was effective in the removal of RB5 at low concentrations; almost complete decolorization (94.8%) was achieved within 80 min of illumination time at 50 mg/L RB5. After 90 min of exposure time, dye decolorization at 100, 200,300 and 500 mg/L RB5 concentrations was about 72.3%, 48.6%, 33.5% and 19.2%, respectively. The UV–vis absorptive spectra of RB5 (at 50 mg/L concentration) was studied at different exposure times in the photocatalytic experiments. Results (Figure 3) showed that absorbance in the visible and UV spectral regions decreased with increasing illumination time indicating the photocatalytic oxidation was effective in removal of two absorbance peaks in visible and UV regions which belong to azo bonds and aromatic rings (benzene and naphthalene), respectively.

Figure 2. Photodegradation of RB5 at different dye concentration

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Figure 3. UV-visible spectra (200-800 nm) of RB5 (50 mg/L) at different time intervals

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3.2. Decolorization Kinetics

Kinetics studies was performed for photocatalytic decolorization at 50, 100 and 200 mg/L RB5 concentrations and results (Figure 4) followed first-order kinetic model. This kinetic model was presented in equation of –Ln C/C0= kt, where C0, C, t and k are initial dye concentration, dye concentration at t (min), decolorization time (min) and decolorization rate constant (1/min), respectively ^[20]. Decolorization rate constant (k) was calculated from the slope of the curve. In Figure 4, the plot of Ln C/C0 versus t and the linear correlation between –Ln C/C0 and illumination time are shown. R2 and k values are presented in Table 1. Data showed that decolorization constant rate decreased as the initial dye concentration increased.

Table 1. Decolorization parameters in photocatalytic process at different concentrations of RBS

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Tables index

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Figure 4. The plot of In C/C₀ vs. time (at different concentrations of RB5) indicates that the kinetic studies follow first order reaction

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3.3. Combined Biological-photochemical Process

As shown in Figure 5, at 200 mg/L dye concentration, 74.9% decolorization occurred after 4 h irradiation but the peak attributed to aromatic rings in UV region did not significantly change. Results (Figure 5, curves e,f,g) showed that application of biological-photocatalytic process was more efficient than the photocalalytic and biological process solely in aromatic byproduct remediation. Absorption changes were not significant between UV–vis spectra in combined process after 2 and 3 or 4 h illumination.

Figure 5. UV-vis spectral scans, a: Control (200 mg/L RB5 without treatment), b & c: after treatment by TiO2 + UV for 2 & 4 h, respectively, d: after treatment by pseudomonas aeruginosa ETL-2211 (24 h), e: after combined treatment by pseudomonas aeruginosa ETL-2211 (24 h) & TiO2 + UV (2 h), f& g: after combined processing by pseudomonas aeruginosa ETL-2211 (24 h) & TiO2 + UV (3 & 4 h respectively)

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