Synergistic effect of biological and advanced oxidation process treatment in the biodegradation of Remazol yellow RR dye

The current study investigated the efficiency of synergistic biological and Advanced Oxidation Process (AOPs) treatment (B-AOPs) using Aeromonas hydrophila SK16 and AOPs-H2O2 in the removal of Remazol Yellow RR dye. Singly, A. hydrophila and AOPs showed 90 and 63.07% decolourization of Remazol Yellow RR dye (100 mg L−1) at pH 6 and ambient temperature within 9 h respectively. However, the synergistic B-AOPs treatments showed maximum decolorization of Remazol Yellow RR dye within 4 h. Furthermore, the synergistic treatment significantly reduced BOD and COD of the textile wastewater by 84.88 and 82.76% respectively. Increased levels in laccase, tyrosinase, veratryl alcohol oxidase, lignin peroxidase and azo reductase activities further affirmed the role played by enzymes during degradation of the dye. UV–Visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), high-performance liquid chromatography (HPLC) and gas chromatography–mass spectroscopy (GC–MS) confirmed the biotransformation of dye. A metabolic pathway was proposed based on enzyme activities and metabolites obtained after GC–MS analysis. Therefore, this study affirmed the efficiency of combined biological and AOPs in the treatment of dyes and textile wastewaters in comparison with other methods.

Large volume of untreated dyestuffs and wastewaters released into the environment by textile industries pose a great danger to humans, plants and animals 1 . The threats posed by the indiscriminate disposal of dyes to environmental and health safety is attributed to their aromatic nature 2 . Textile wastewater contains dyes, disinfectants, halogen carriers, solvents, toxic heavy metals, carcinogenic amines, chlorine bleaching, biocides, pentachlorophenol, salts, softeners, surfactants, solvents and free formaldehyde 3 . Most manufacturing and allied industries (like food, leather, textile, and paper) make use of azo dyes in their day-day production of goods and products 4 . The remains of dyes are usually visible in shabbily treated textile wastewaters. This, in turn, causes shallow UV light penetration of ocean beds thus leading to poor water quality, low dissolved oxygen and decline in photosynthetic activities 5 .
Conventional physico-chemicals such as membrane filtration 6 , coagulation 7 , adsorption 8 , and chemical oxidation 9 have proven to biodegrade the dyes in textile effluent. However, the physical and chemical methods come with attendant disadvantages such as toxic residues formation, membrane fouling, bioaccumulation of sludge and formation of secondary pollutants 10 . To further enhance the reduction of toxic amines in the dyes, conventional biological treatment should be incorporated with advanced oxidation process 11 . Sarria et al. 12 reported that bioremediation of organic pollutants through AOPs in a single treatment is very arduous, relatively costly and sometimes ineffective.
To overcome the limitation, Advanced Oxidation Process (AOPs) is usually combined with biological treatment in the presence of solar radiation for the biodetoxification of textile azo dyes. This method involves the breakdown of the dye constituents followed by removal of toxic aromatic amines 13 . Bacteria like A. hydrophila have proven to be an efficient and promising tool for the removal of textile azo dyes. The use of AOPs enhance  14 . Detoxification of dyes is most pronounced of all the biological methods of treatment 15 . Advanced Oxidation Process (AOPs) is usually characterized by the production of radicals (hydroxyl) which are capable of oxidizing aromatic amines in dye wastewater 16 . AOPs are synergistically deployed alongside biological treatment to ensure rapid and efficient textile wastewater treatment 17 . Vilar et al. 18 suggested the use of sunlight energy systems as Ultraviolet source in the improvement of the oxidation process as an alternative to chemicals. There is paucity of information on the removal of Remazol Yellow RR dye through B-AOPs treatment. Although, different bacteria strains have been deployed in the combined biological and advanced oxidation process (AOPs) treatment of textile dye. However, this is the first report on the degradation of Remazol Yellow RR dye by A. hydrophila in combination with AOPs using lesser percentage of hydrogen peroxide. Hence, this present study is to investigate (1) the potency of biological treatment and advanced oxidation process in the removal of dye (2) determine the enzymes dissipated by A. hydrophila during biodegradation of Remazol Yellow RR dye (3) propose a biochemical pathway of degradation of the dye. The study revealed that the synergistic biotreatment (with bacteria) and advanced oxidation process (solar radiation) of dye has proven to be cheap, cost-effective and eco-friendly.

Results
Comparison of biological, AOPs and combined biological and AOPs. Maximum decolorization (90%) of Remazol Yellow RR was achieved by A. hydrophila after 9 h under static condition ( Fig. 1a). At present, there is a paucity of information on degradation of dye mediated by desorption and adsorption. On addition of high amount of H 2 O 2 , Advanced Oxidation Process (AOPs) showed less decolorization (63.07%) (Fig. 1b) of decolorization. Degradation becomes quite an arduous task when AOPs are deployed singly because of dye's complex aromatic structure and nature. The biologically treated sample was centrifuged at 10,000 rpm for 15 min, and subjected to Advanced Oxidation Process (AOPs) with 4% H 2 O 2 for 3-6 h. The significance values obtained for the treatment with bacteria, AOPs and B-AOPs were 0.823, 0.679 and 0.903 which further affirmed the normality of the experimental data on Remazol Yellow RR dye when subjected to Shapiro-Wilk tests. The skewness and kurtosis conducted to test the distribution of the degradation data further affirmed the normality since they were less than 1 and 2 respectively. The significant value obtained was 0.015 when the degradation data was subjected to Levene statistic. This result was able to ascertain the homogeneous nature of the experimental data. One-way Analysis of Variance (ANOVA) revealed that the percentage degradation data of the dye by bacteria, AOPs and B-AOPs were statistically significant at (P ≤ 0.05) when the means were separated with Tukey-b. The treatment showed 100% (Fig. 1c) decolorization within 4 h. After every treatment (biological, AOPs and coupled biological and AOPs) the sample was analyzed by using UV-Vis spectrophotometer in the range of 350-750 nm (Fig. 2).

Effect of combined treatment (biological, AOPs and B-AOPs) on removal of biological oxygen demand (BOD) and chemical oxygen demand (COD).
To ascertain the potency of the combined treatment, all treated samples were tested for reduction in BOD and COD. After completion of AOPs treatment, BOD and COD was reduced by 18 and 34.61% respectively. In biologically treated sample, 72.08 and 66.76% in BOD and COD reduction respectively while 84.88 and 82.76% reduction in BOD and COD was achieved when the treated sample were subjected to combined B-AOPs treatment (Fig. 3). One-way ANOVA revealed that the removal of BOD and COD under synergistic treatment was significantly different (P ≤ 0.05) in comparison with individual treatment with bacteria and advanced oxidation process.   Table 1). The skewness and kurtosis of dye degradation by the bacteria, AOPs and B-AOPs were 0.823, 0.679 and 0.903. This suggests that the data were normally distributed since they were less than 1 and 2 respectively. The significant value (0.019) was observed when the data was subjected to Levene statistic which affirmed the homogeneity in the variances in enzymes induction data. One-way ANOVA revealed substantial significant difference (P ≤ 0.05) in all the enzyme activity tests before and after treatment of the dye with A. hydrophila when the means were separated with Tukey-b. Laccase activities was found to be the highest while tyrosinase enzyme was least secreted during the decolorization experiment. Our finding revealed pivotal role played by enzymes in enhancing the decolorization of the dye.    (Fig. 4b).
HPLC analysis further confirmed appearance of various metabolites from control dye. Control sample showed a major peak at 2.029 min retention time (Fig. 5a), whereas biodegraded sample showed 5 new peaks with retentions times (1.574, 1.766, 1.867, 3.240 and 3.930 min respectively) (Fig. 5b). The HPLC analysis confirmed the single major peak in the control Remazol Yellow RR dye has been biodegraded into different peaks (five-5) evidently shown by the times (retention). This analysis further affirmed the degradation of the dye.
Remazol Yellow RR dye metabolites after biodegradation by A. hydrophila SK16 was analyzed using Gas Chromatography and Mass Spectroscopy (GCMS) elucidated with peaks in mass spectra (data not shown   (Fig. 6).

Discussion
The UV-Vis spectral results revealed that synergistic treatment with A. hydrophila and AOP accounted for the total disappearance of the major peak in Remazol Yellow RR dye (control) than individual treatment with the bacteria and AOP respectively. Kalme et al. 19 implied in an earlier work that low decolorization efficiency in aerobic state is due to the interplay of molecular forces between oxygen and azo compounds. Similar results were reported by Kalyani et al. 20 when Pseudomonas sp. SUK1 was deployed in dye degradation. The power of microbial cells to adsorb dye accounted for decolorization efficiency over a period 20 . Albeit, biodegradation via oxidation (chemical) is very expensive due to the oxidation intermediates produced during treatment. Furthermore, the intermediates are more resistant to complete chemical oxidation and furthermore consume energy relative to treatment time 21 . Harrelkas et al. 22 reported that it is highly efficient if biological treatment is incorporated to OAPs in order to enhance overall treatment efficiency. First biological treatment decreases concentration of compounds that may compete for chemical oxidation, thus increasing efficiency and lowering treatment cost 23 . In non-biodegradable textile effluent, the coupled treatment not only achieves efficient decolorization, but also significantly reduces BOD, COD and TDS 24 . The findings in this study revealed higher decolorization potency than the efficiencies recorded by Tantak and Chaudhari 25 when Reactive Blue 13 and Reactive Blue 5 was subjected to combined B-AOPs treatment. However, this study corroborated the findings of Lodha and Chaudhari 26 who reported 99% removal of color when different dye solutions were subjected to combined AOPs and biological treatment. Furthermore, this study was in agreement with the reports of Alvares et al. 27

Methods
Dyes and chemicals. Remazol Yellow RR dye was obtained from Tamil Nadu, India (Jamara textile industry). Veratryl alcohol, methyl red, L-ascorbic acid, Catechol and nutrient broth were obtained from Pvt Laboratories (Hi Media), Mumbai. Hydrogen peroxide was procured from Merck Mumbai. Other reagents used were of high purity and analytical grade.
Culture maintenance and decolorization. Aeromonas hydrophila SK16 used in the present work was isolated previously in our laboratory from textile contaminated soil 29 . The culture was maintained at 4˚C on nutrient agar slant (Nutrient Agar: NaCl-5 g, Beef extract-1.5 g, Yeast extract-1.5 g, and Agar-15 g). Decolorization study was carried out in Erlenmeyer flask (250 mL) containing nutrient broth of aforementioned composition. The dye (100 mg L −1 ) was added to a pre-grown culture of A. hydrophila and incubated at 37˚C under static condition. The pH was adjusted to 8 in pre-grown culture with the addition of NaOH. A 5 mL aliquot was withdrawn at regular intervals, centrifuged at 10,000 rpm for 15 min and decolorization was monitored with UV-Vis Spectrophotometer (Shimadzu 1800) at wavelength (λ max = 560 nm). The control experiments were made of flasks with no bacteria cells. All the experiments were performed in triplicates. Decolorization percentage was calculated using the formula: Enzyme assays. Activities of laccase and veratryl alcohol oxidase were assayed spectrophotometrically by using UV-Vis spectrophotometer (Shimadzu 1800, Japan). The total volume of 2 mL was contained 1 mM veratryl alcohol, citrate buffer (pH 3)-0.1 M and 0.2 mL enzyme source. This was done to determine veratryl alcohol oxidase. Oxidation of veratryl alcohol was monitored by increasing of absorbance at 310 nm due to formation of propanaldehyde 31 . Laccase activity was determined in a 2 mL of reaction mixture containing 10% of ABTS in 20 mM potassium phosphate buffer (pH 4) and the increased optical density was measured at 420 nm 32 . Lignin peroxidase assay was performed in a total 2.5 mL volume comprising tartaric acid (250 mM) and n-propanol (100 mM). The propanaldehyde production was estimated at 300 nm as reported by Kalyani et al. 20 . Tyrosinase activity was calculated in a reaction mixture of 2 mL, containing in 0.1 M phosphate buffer (pH 7.4) with 0.01% catechol at 495 nm 24 . Azoreductase assay was carried out using Methyl red as substrate as reported by Kurade et al. 33 .

Experiments on advanced oxidation process (AOPs
Extraction and analysis of metabolites produced during biodegradation. After complete biodegradation, the set up was centrifuged (at 10,000 rpm for 15 min). Ethyl acetate was proportionately mixed with the supernatant in order to extract the metabolites produced during biodegradation. Rotary evaporator was used to dry the extracts over anhydrous Na 2 SO 4 . The dried sample was dissolved in HPLC grade methanol and used for analytical studies. The changes of surface functional groups of Remazol Yellow RR before and after biodegradation was investigated by using FTIR Perkin Elmer (RX I) 34 . HPLC analysis was conducted using Jadhav et al. 5 method. The Shimadzu LC 40,102,010 instrument was used for HPLC study connected with C18 column. The solvent methanol was used in mobile phase with 1 mL min −1 flow rate and analysis was done at 470 nm 23 . Gas Chromatography (GC)-45XGC-44 coupled with Scion MS-40 Mass Spectroscopy (Bruker) was used in analyzing metabolites obtained after biotransformation. Voltage of (70 eV) was used while the carrier gas was made of helium with 1 mL min −1 (flow rate) and 26 min (duration). DB-WAX column (0.25 mm-30 mm) was used for the GC analysis initially set at the operating temperature mode. The column (oven) temperature was increased steadily by 10 °C per minute to 250 °C. The operating condition was kept for 26 min. from the initially programmed temperature of 80˚ C. ChemSketch software version 11.02 was deployed in sketching the metabolic products, hence the pathway (proposed) while the metabolites were identified based on comparison with mass spectra available in the NIST database.

Statistical analysis.
All experiments were performed in triplicates. Data and graphs were presented as Mean ± Standard Error of Means at 95% Confidence Interval. Normality and homogeneity of variances of the data on Remazol Yellow dye degradation and enzymes induction were conducted with Shapiro-Wilk and Levene statistic tests respectively 35 . One-way Analyses of Variance (ANOVA) was carried out to affirm the homogenous nature of the data on degradation of dye and enzyme induction. Statistical analysis (ANOVA) (P ≤ 0.05) and graphs was performed with GraphPad Prism software version 8 35 .

Conclusion
The synergistic effect of Advanced Oxidation Process (AOPs) and biological treatment developed by incorporating A. hydrophila SK16 and 4% H 2 O 2 for Remazol Yellow RR dye biodegradation was reported. This combined treatment achieved 100% decolorization, 84.88% BOD and 82.76% COD reduction. The biodegradation process elucidated substantial induction of laccase and veratryl alcohol oxidase. UV-vis spectroscopy, FTIR, HPLC and GC-MS analysis proved that degradation of Remazol Yellow RR by A. hydrophila SK16. Therefore, the combined B-AOPs provides cost effective, non-energy demanding and ecofriendly treatment for managing textile dye pollution in the environment.

Data availability
All experimental data would be made freely available and accessible upon request by the corresponding author.