Soybean peroxidase-mediated degradation of an azo dye– a detailed mechanistic study
© Ali et al.; licensee BioMed Central Ltd. 2013
Received: 27 July 2013
Accepted: 28 November 2013
Published: 5 December 2013
Peroxidases are emerging as an important class of enzymes that can be used for the efficient degradation of organic pollutants. However, detailed studies identifying the various intermediates produced and the mechanisms involved in the enzyme-mediated pollutant degradation are not widely published.
In the present study, the enzymatic degradation of an azo dye (Crystal Ponceau 6R, CP6R) was studied using commercially available soybean peroxidase (SBP) enzyme. Several operational parameters affecting the enzymatic degradation of dye were evaluated and optimized, such as initial dye concentration, H2O2 dosage, mediator amount and pH of the solution. Under optimized conditions, 40 ppm dye solution could be completely degraded in under one minute by SBP in the presence of H2O2 and a redox mediator. Dye degradation was also confirmed using HPLC and TOC analyses, which showed that most of the dye was being mineralized to CO2 in the process.
Detailed analysis of metabolites, based on LC/MS results, showed that the enzyme-based degradation of the CP6R dye proceeded in two different reaction pathways- via symmetric azo bond cleavage as well as asymmetric azo bond breakage in the dye molecule. In addition, various critical transformative and oxidative steps such as deamination, desulfonation, keto-oxidation are explained on an electronic level. Furthermore, LC/MS/MS analyses confirmed that the end products in both pathways were small chain aliphatic carboxylic acids.
KeywordsAzo dye Degradation Enzyme Mediator LC/MS Metabolites
Extensive use of synthetic dyes and their subsequent release in industrial wastewater is a growing environmental problem. It is estimated that as much as 12% of the dyestuff amounting to about 280,000 ton/ year is released to the ecosystem. These dyes are used in various industrial applications (total consumption is more than one million tons of dyes annually) and are engineered to be generally resistant to fading. They not only need to sustain alkaline or acidic environment but also need to withstand washing with soaps and bleaching agents and be resistant to light and ultraviolet irradiation.
It is well-established that some dyes are potentially carcinogenic and mutagenic, as well as genotoxic[3–5]. Furthermore, the presence of color in water bodies causes less sunlight to penetrate through water which results in reducing the photosynthetic capability of aquatic plants and microorganisms. Industrial effluents containing dyes are able to color water even in concentrations as low as 1 mg/liter, whereas in most cases, these wastewater streams typically contain a much higher amount of the dye content, about 10-200 mg/liter, which gives intense coloration. It is therefore not surprising that both international and national regulations for industrial wastewater require substantial elimination of the dyestuff content form the effluent[7, 8]. Numerous approaches have been developed to treat industrially generated wastewater, such as coagulation/flocculation, adsorption, use of activated carbon, and various forms of Advanced Oxidation Processes, e.g. ozonation, and photochemical approaches[9–16]. Most of these methods have a disadvantage of either formation of large amounts of sludge or production of possible toxic byproducts.
Removal of contaminants from the environment by biological methods using enzymes and microorganisms is considered to be closer to nature as it is an environmentally friendlier technique which leaves the ecosystem intact. The technology is scalable and can also be used to treat other organic impurities. A number of microorganisms including bacteria, fungi, and yeasts have been used to treat the dye contaminated wastewaters[19–22].
Azo dyes are electron-deficient xenobiotic components because of their azo linkage (–N=N), and in many cases, they have sulphonic (SO3−) or other electron-withdrawing groups, which generate an electron deficiency and make the dye less susceptible to degradation by microorganisms. However, under appropriate conditions, they can be degraded by azo reductase-releasing microbes.
Enzyme mediated decolorization is another newer alternative, where the enzyme can specifically react with organic pollutants by transforming them into low molecular weight products. The main advantage of using enzymes in degrading dye solutions is that they have higher reaction rates and can also work in milder reaction conditions. Some azo dyes have been decolorized by using certain peroxidases, such as soybean peroxidase (SBP), manganese peroxidase (MnP), lignin peroxidase (LiP), laccase and horseradish peroxidase (HRP)[23–26]. It has been suggested that these enzymatic treatments could oxidize the dye structures to form compounds with lower molecular weight and lower toxicity and may eventually mineralize the dyes.
Although a considerable amount of research has been published on the use of pure enzymes to degrade dyes, detailed analyses of the breakdown pathway are almost non-existent. Structures of the intermediates produced as well as the mechanisms involved in the dye degradation pathway are important to properly understand and further exploit this novel approach to pollutant-contaminated water remediation.
The objective of the present study was to study in detail the mechanism of an azo-dye degradation by soybean peroxidase enzyme (SBP). In addition to identifying the various intermediates produced and proposing possible pathways, factors affecting dye degradation such as initial dye concentration, H2O2 concentration, pH, and presence of redox mediator were also evaluated. The present study is one of the very few studies that show in detail the various intermediates produced during peroxidase-mediated degradation of an azo-dye and possible electron-level mechanisms involved.
The azo dye namely Crystal Ponceau 6R (C.I name = Acid Red 44, C.I number = 16250, Molecular Formula = C20H12N2O7S2Na2, FW = 502.446 g mol−1), herein abbreviated as CP6R was used as a model dye. The dye was procured from Sigma-Aldrich Chemicals and used as such. All the other chemicals used in this work were obtained from Sigma-Aldrich and were of high purity (< 98%).
Dye degradation studies
Stock solution (2,000 ppm) of the dye was prepared in a 250 mL flask by first dissolving an appropriate amount in deionized water. Further dilutions from this stock were done as per the requirement of the experiment. Unless otherwise indicated, the working concentration of the CP6R dye was 40 ppm. Dye degradation reactions were carried out by adding H2O2 to a buffered dye solution containing SBP enzyme. Spectrophotometric measurements were made using a CARY 50 UV/Vis spectrophotometer. The absorbance value obtained in each case was plotted against time to obtain the % degradation. The % degradation for the dye was calculated by observing the changes in λmax (510 nm) of the solution. The studies were carried out at 25°C otherwise indicated. For pH studies, the dye solution were prepared in 33.33 mM universal buffers (citrate-phosphate) adjusted to specific pH value.
Where A 0 is the initial absorbance of dye solution and A t is the absorbance of the dye solution at any given time.
Total Organic Carbon (TOC) analyses
TOC analyses were carried out using GE Sievers InnoVox TOC analyzer properly calibrated with fresh standards. The CP6R samples tested were 0% degradation sample which consisted of 400 ppm CP6R in 33 mM citrate-phosphate buffer, pH 4, 0.78 μM SBP, 0.1 mM HOBT and 100% degradation sample which was exactly the same as the 0% sample but contained 1 mM H2O2. Analyses were carried out in triplicates and the data is presented as TOC values normalized to 0% CP6R degradation sample.
HPLC and LC/MS experiments
High performance liquid chromatography (HPLC) and LC/MS analyses were carried out similar to as previously described. Briefly, samples were analyzed on an Acquity UPLC system, (Waters Corporation, Milford, MA, USA) with an Acquity UPLC BEH C18 column with 1.7 μm particle size (2.1 mm I.D. × 100 mm length, Waters Corporation, Milford, MA, USA) maintained at 35°C, coupled to Acquity tunable ultraviolet/visible detector (Waters Corporation, Milford, MA, USA) and an Acquity Tandem quadruple mass spectrometer (Waters Corporation, Milford, MA, USA). The mobile phase consisted of solution A (0.1 M ammonium formate (pH 6.7)) and solution B (1:1 acetonitrile/methanol) and a gradient of 0% B to 80% B in 13.80 minutes at the flow rate was 0.2 mL/min was used to obtain the chromatographs. The mass spectrometer was equipped with an electrospray ionization source operated in negative ion mode. The ESI conditions were as follows: capillary voltage: 3.0 kV, Cone voltage 30 V, collision energy 50 V, desolvation gas (Nitrogen at 500 L/Hr), Cone gas (Nitrogen at 2 L/Hr), desolvation temperature was set at 350°C and source temperature was 150°C. The mass range was detected from 50 to 700 m/z. Tandem MS experiment was done using Waters Masslynx V 4.1, wherein Argon gas was used as the collision gas.
Results and discussion
Optimizing the enzymatic dye degradation reaction conditions
As expected, the degradation of dye was found to be very much affected by the initial amount of dye content in solution. Studies carried out at different concentrations of the dye showed the optimum CP6R under our chosen conditions was 40 ppm (data not shown). Optimizations of other parameters are described below:
Requirement of HOBT for SBP-mediated degradation of CP6R
Initial experiments using only SBP and H2O2 showed that unlike other dyes, CP6R was unable to be degraded by SBP/H2O2 alone (data not shown). It is well known that the presence of redox mediators such as 1-hydroxybenzotriazole (HOBT), veratryl alcohol, violuric acid, 2- methoxyphenothiazone, etc. can dramatically increase the rate of dye degradation[27–30]. The mechanism involved is well known, wherein, the substrate initially undergoes an one-electron oxidation in the presence of a redox mediator and transforms into a radical cation followed by abstraction of a H-atom from the substrate by the mediator and converting it into a radical, which can then cause the substrate to co-oxidize.
Effect of hydrogen peroxide concentration on dye degradation
Since the peroxidase enzymes use H2O2 as a co-reactant, if the concentration of hydrogen peroxide used is too low, the enzyme activity becomes low; however, a very high peroxide concentration can irreversibly oxidize the enzyme and cause its inactivation. In this regard, experiments were carried out to optimize the H2O2 concentration while keeping the other parameters constant. The results obtained are shown in Additional file2: Figure S2, which showed the optimum H2O2 concentration to be 0.175 mM. Additionally, it can also be seen that at very high H2O2 concentrations, a significant reduction in dye degradation is observed (due to H2O2-mediated inactivation of SBP).
Effect of SBP enzyme concentration on dye degradation
Degradation of dye depends on the amount of catalyst added and the contact time. Thus experiments were also done to optimize the concentration of the enzyme in dye solution which was varied in the range of 0 to 2.7 μM while keeping the other parameters constant. The results are shown in Additional file3: Figure S3 and it can be noted that the enzyme dose had a significant effect on dye decoloration. At lower SBP concentrations, the dye degradation was not very significant, whereas at very high SBP concentration, the dye degraded very quickly in a very short time (almost 30% in a few seconds). Based on these data, an optimized concentration of 0.27 μM SBP was chosen for all subsequent reactions.
Effect of pH
Enzymatic driven reactions are known to be pH dependent[28, 29]. Thus experiments were done to optimize this parameter as well. SBP mediated dye degradation was studied at different pH values (from 2 to 9), while keeping the other conditions constant. The results are shown in Additional file4: Figure S4, which shows the dramatic effect of pH on SBP-mediated degradation of CP6R, with the enzyme being most active in the pH 3-5 range. A pH value of 5 was used for all the subsequent experiments. This role of pH on the peroxidase driven reactions has been reported in the literature for different dye degradation along-with its mechanism[17, 23].
Total Organic Carbon analysis
Analysis of product formation using HPLC/MS
Proposed mechanism of enzymatic degradation
The generation of CP6R radical by SBP in the presence of a mediator (HOBT) consists of four major steps. In the first step, SBP enzyme reacts with H2O2 to become an oxyl-ferric (Fe4+) cation radical, compound I (via loss of two electrons). The second step involves the abstraction of hydrogen from HOBT resulting in the formation of HOBT radical and compound II. In the third step, a second radical of HOBT is formed by the transfer of another hydrogen to compound II, leading to the regeneration of the original reduced (Fe3+) SBP enzyme and a water molecule. In the final step, HOBT radical attacks CP6R and abstracts a hydrogen, resulting in the formation of CP6R radical. Similar reactions have been previously well-documented.
Proposed mechanistic pathway of CP6R degradation by SBP-H2O2
Mass of all intermediate in asymmetrical degradation
2-carboxyvinyl -4,6disulfobenzonic acid.
3-methylhexa 2,4-diene dionic acid.
Mass of all intermediate in symmetrical degradation
2-carboxyvinyl -4,6disulfobenzonic acid.
3-methylhexa 2,4-diene dionic acid.
Asymmetrical azo bond cleavage of CP6R
Symmetrical azo bond cleavage of CP6R
Detailed electronic-level mechanism of asymmetrical cleavage of the azo bond
Oxidation of diketo
The oxidation of diketo to carboxylic acid has been observed in several photolytic degradation studies. The ring is initially opened by the attack of OH radical on alpha diketones resulting in the formation of dicarboxilic acid intermediate (A2). This is shown in the Additional file5: Figure S5.
Deamination and removal of azo group
Oxidation of phenol
Formation of amine in symmetrical cleavage
De-amination in symmetrical cleavage
In summary, we show here efficient degradation of an azo-dye, Crystal Ponceau 6R (CP6R), using the Soybean peroxidase/HOBT/H2O2 system. Under optimized conditions it was found that SBP could degrade 100% of the dye in under a minute. Dye mineralization was confirmed using TOC and HPLC experiment, and most importantly, extensive LC/MS/MS experiments were used to identify the various metabolites formed during the degradation process. Lastly, based on the LC/MS data and known radical-based reactions, we were able to develop detailed mechanisms for the various steps in the dye degradation. Our results show that the azo dye degraded via two different pathways, namely symmetric and asymmetricazo bond cleavage followed by diketo oxidation to carboxylic acids, desulfonation, deamination, and phenolic oxidation reactions.
This research was partially funded by UAEU/NRF Research Grant Program 27/11/2 (21S039 & 31S072) to SSA and MAR.
- Forgacs E, Cserhati T, Oros G: Removal of synthetic dyes from wastewaters: A review. Environ Intl. 2004, 30: 953-971. 10.1016/j.envint.2004.02.001.View ArticleGoogle Scholar
- Jin X, Liu G, Xu Z, Yao W: Decolorization of a dye industry effluent by Aspergillusfumigatus XC6. Appl Microbiol Biotechnol. 2007, 74: 239-243. 10.1007/s00253-006-0658-1.View ArticlePubMedGoogle Scholar
- Sarıkaya R, Selvi M, Erkoc F: Evaluation of potential genotoxicity of five food dyes using the somatic mutation and recombination test. Chemosphere. 2012, 88: 974-979. 10.1016/j.chemosphere.2012.03.032.View ArticlePubMedGoogle Scholar
- De Lima ROA, Bazo AP, Salvadori DMF, Rech CM, Oliveira DP, Umbuzeiro GP: Mutagenic and carcinogenic potential of a textile azo dye processing plant effluent that impacts a drinking water source. Mutat Res Genet Toxicol Environ Mutagen. 2007, 626: 53-60. 10.1016/j.mrgentox.2006.08.002.View ArticleGoogle Scholar
- Satuf ML, Pierrestegui MJ, Rossini L, Brandi RJ, Alfano OM: Kinetic modeling of azo dyesphotocatalytic degradation in aqueous TiO2 suspensions. Toxicity and biodegradability evaluation. Catal Today. 2011, 161: 121-126. 10.1016/j.cattod.2010.11.018.View ArticleGoogle Scholar
- Robinson T, McMullan G, Marchant R, Nigam P: Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. Bio Resour Technol. 2004, 77: 247-255.View ArticleGoogle Scholar
- Hessel C, Allegre C, Maisseu M, Charbit F, Moulin P: Guidelines and legislation for dye house effluents. J Environ Manag. 2007, 83: 171-180. 10.1016/j.jenvman.2006.02.012.View ArticleGoogle Scholar
- Griffiths C, Klemick H, Massey M, Moore C, Newbold S, Simpson D, Walsh P, Wheeler W: US Environmental Protection Agency valuation of surface water quality improvements. Rev Environ Econ Policy. 2012, 6: 130-146. 10.1093/reep/rer025.View ArticleGoogle Scholar
- Wu C, Wang Y, Gao B, Zhao Y, Yue Q: Coagulation performance and floccharacteristics of aluminum sulfate using sodium alginate as coagulant aid for synthetic dying wastewater treatment. Sep Purif Technol. 2012, 95: 180-187.View ArticleGoogle Scholar
- Chen T, Gao BY, Yue QY: Effect of dosing method and pH on color removal performance and floc aggregation of polyferric chloride–polyamine dualcoagulant in synthetic dyeing wastewater treatment. Colloids Surf A. 2010, 355: 121-129. 10.1016/j.colsurfa.2009.12.008.View ArticleGoogle Scholar
- Alnuaimi MM, Rauf MA, Ashraf SS: A comparative study of Neutral Red decoloration by photo-Fenton and photocatalytic processes. Dyes Pigm. 2008, 76: 332-337. 10.1016/j.dyepig.2006.08.051.View ArticleGoogle Scholar
- Mezohegyi G, Van der Zee FP, Font J, Fortuny A, Fabregat A: Towards advanced aqueous dye removal processes: A short review on the versatile role of activated carbon. J Environ Manage. 2012, 102: 148-164.View ArticlePubMedGoogle Scholar
- Gao M, Zeng Z, Sun B, Zou H, Chen J, Shao L: Ozonation of azo dye Acid Red 14 in a microporous tube-in-tube microchannel reactor: Decolorization and mechanism. Chemosphere. 2012, 89: 190-197. 10.1016/j.chemosphere.2012.05.083.View ArticlePubMedGoogle Scholar
- Meetani MA, Hisaindee SM, Abdullah F, Ashraf SS, Rauf MA: Liquid chromatography tandem mass spectrometry analysis of photodegradation of a diazo compound: A mechanistic study. Chemosphere. 2010, 80: 422-427. 10.1016/j.chemosphere.2010.04.065.View ArticlePubMedGoogle Scholar
- Rauf MA, Meetani MA, Hisaindee S: An overview on the photocatalytic degradation of azo dyes in the presence of TiO2 doped with selective transition metals. Desal. 2011, 276: 13-27. 10.1016/j.desal.2011.03.071.View ArticleGoogle Scholar
- Kalsoom U, Ashraf SS, Meetani MA, Rauf MA, Bhatti HN: Degradation and kinetics of H2O2 assisted photochemical oxidation of Remazol Turquoise Blue. Chem Eng J. 2012, 200–202: 373-379.View ArticleGoogle Scholar
- Rauf MA, Ashraf SS: Survey of recent trends in biochemically assisted degradation of dyes. Chem Eng J. 2012, 209: 520-530.View ArticleGoogle Scholar
- Saratale RG, Saratale GD, Chang JS, Govindwar SP: Bacterial decolorizationand degradation of azo dyes: A review. J Taiwan Inst Chem Eng. 2011, 42: 138-157. 10.1016/j.jtice.2010.06.006.View ArticleGoogle Scholar
- Bibi I, Bhatti HN, Asgher M: Comparative study of natural and synthetic phenolic compounds as efficient laccase mediators for transformation of cationic dye. Biochem Eng J. 2011, 56: 225-231. 10.1016/j.bej.2011.07.002.View ArticleGoogle Scholar
- Ateeq H, Rauf MA, Ashraf SS: Efficient microbial degradation of Toluidine Blue dye by Brevibacilus sp. Dyes Pigm. 2007, 75: 395-400. 10.1016/j.dyepig.2006.06.019.View ArticleGoogle Scholar
- Ali NF, El-Mohamedy RSR: Microbial decoloration of textile waste water. J Saudi Chem Soc. 2012, 16: 117-123. 10.1016/j.jscs.2010.11.005.View ArticleGoogle Scholar
- Martorell MM, Pajot HF, De Figueroa LIC: Dye-decolourizing yeasts isolated from Las Yungas rainforest. Dye assimilation and removal used as selection criteria. Inter Biodet Biodegrad. 2012, 66: 25-32. 10.1016/j.ibiod.2011.10.005.View ArticleGoogle Scholar
- Kalsoom U, Ashraf SS, Meetani MA, Rauf MA, Bhatti HN: Mechanistic study of a diazo dye degradation by soybean peroxidase. Chem Cent J. 2013, 7: 1-10. 10.1186/1752-153X-7-1.View ArticleGoogle Scholar
- Cheng XB, Jia R, Li PS, Tu SQ, Zhu Q, Tang WZ, Li XD: Studies on the properties and co-immobilization of manganese peroxidase. Enzy Microb Technol. 2007, 41: 258-264. 10.1016/j.enzmictec.2007.01.020.View ArticleGoogle Scholar
- Franciscon E, Piubeli F, Garboggini FF, De Menezes CR, Silva IS, Paulo AC, Grossman MJ, Durrant LR: Polymerization study of the aromatic amines generated by the biodegradation of azo dyes using the laccase enzyme. Enzy Microb Technol. 2010, 46: 360-365. 10.1016/j.enzmictec.2009.12.014.View ArticleGoogle Scholar
- Ryan BJ, Carolan N, Fagain CO: Horseradish and soybean peroxidases: Comparable tools for alternative niches. Trends Biotechnol. 2006, 24: 355-363. 10.1016/j.tibtech.2006.06.007.View ArticlePubMedGoogle Scholar
- Matto M, Husain Q: Decolonization of direct dyes by salt fractionated turnip proteins enhanced in the presence of hydrogen peroxide and redox mediators. Chemosphere. 2007, 69: 338-345. 10.1016/j.chemosphere.2007.03.069.View ArticlePubMedGoogle Scholar
- Jamal F, Qidwai T, Pandey PK, Singh D: Catalytic potential of cauliflower (Brassica oleracea) bud peroxidase in decolorization of synthetic recalcitrant dyes using redox mediator. Catal Commun. 2011, 15: 93-98. 10.1016/j.catcom.2011.08.020.View ArticleGoogle Scholar
- Khlifi R, Belbahri L, Woodward S, Ellouz M, Dhouib A, Sayadi S, Mechichi T: Decoloration and detoxification of textile industry wastewaterby the laccase-mediator system. J Hazard Mater. 2010, 175: 802-808. 10.1016/j.jhazmat.2009.10.079.View ArticlePubMedGoogle Scholar
- Fabbrini M, Galli C, Gentili P: Comparing the catalytic efficiency of some mediators of laccase. J Mol Catal B. 2002, 16: 231-240. 10.1016/S1381-1177(01)00067-4.View ArticleGoogle Scholar
- Pereira L, Coelho AV, Viegas CA, Dos Santos MMC, Robalo MP, Martins LO: Enzymatic biotransformation of the azo dye sudan orange G with bacterial CotA-laccase. J Biotech. 2009, 139: 68-77. 10.1016/j.jbiotec.2008.09.001.View ArticleGoogle Scholar
- Chen T, Zheng Y, Lin J, Chen G: Study on the photocatalytic degradation of methyl orange in water using Ag/ZnO as catalyst by liquid chromatography electrospray ionization ion-trap mass spectrometry. J Am Soc Mass Spectrom. 2008, 19: 997-1003. 10.1016/j.jasms.2008.03.008.View ArticlePubMedGoogle Scholar
- Dunford H: Horseradish peroxidase. II. Two-electron reactions, ferrous peroxidase, compound III, the five oxidation states, oxygen evolution and inactivation. Peroxidases and catalases: Biochemistry, biophysics, biotechnology, and physiology. Edited by: Dunford HB. 2010, New Jersey: Wiley, 41-57. 2Google Scholar
- Ozen AS, Aviyente V, Proft FD, Geerling P: Modeling the substituent effect on the oxidative degradation of azodyes. J Phys Chem A. 2004, 108: 5990-6000. 10.1021/jp037138z.View ArticleGoogle Scholar
- Rose PE, Johnson SD, Kilbourn PM: Tracer testing at Dixie Valley, Nevada, using 2-naphthalene sulfonate and 2,7-naphthalene disulfonate. Proceedings 26th Workshop on Geothermal Reservoir Engineering. 2001, Stanford, California: Stanford UniversityGoogle Scholar
- Lopez C, Valade AG, Combourieuc B, Mielgo I, Bouchon B, Lema JM: Mechanism of enzymatic degradation of the azo dyes orange II determine by ex situ 1H nuclear magnetic resonance and electrospray ionization-ion trap mass spectrometry. Anal Biochem. 2004, 335: 135-149. 10.1016/j.ab.2004.08.037.View ArticlePubMedGoogle Scholar
- Chacko JT, Subramaniam K: Enzymatic degradation of Azo dyes-A review. Int J Env Sci. 2011, 1: 1250-1260.Google Scholar
- Hisaindee S, Meetani MA, Rauf MA: Application of LC-MS to the analysis of advanced oxidation process (AOP) degradation of dye products and reaction mechanisms. Trends Anal Chem. 2013, 49: 31-44.View ArticleGoogle Scholar
- Bansal P, Singh D, Sud D: Photocatalytic degradation of azo dye in aqueous TiO2 suspension: Reaction pathway and identification of intermediates products by LC/MS. Sep Purif Techn. 2010, 72: 357-365. 10.1016/j.seppur.2010.03.005.View ArticleGoogle Scholar
- Cai M, Jin M, Weavers LK: Analysis of sonolytic degradation products of azo dye Orange G using liquid chromatography–diode array detection-mass spectrometry. Ultrasonics Sonochem. 2011, 18: 1068-1076. 10.1016/j.ultsonch.2011.03.010.View ArticleGoogle Scholar
- Brillas E, Mul E, Sauleda R, Sanchez L, Peral J, Domenech X, Casado J: Aniline mineralization by AOP’s: anodic oxidation, photocatalytic, electro-Fenton and photoelectron-fenton processes. Appl Catal B. 1998, 16: 31-42. 10.1016/S0926-3373(97)00059-3.View ArticleGoogle Scholar
- Zhang J, Feng M, Jiang Y, Hu M, Li S, Zhai Q: Efficient decolorization/degradation of aqueous azo of dyes using buffered H2O2 oxidation catalyzed by a dosage below ppm level of chloroperoxidase. Chem Eng J. 2012, 191: 236-242.View ArticleGoogle Scholar
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