Decolorization of Synthetic Dyes by Crude and Purified Laccases from Coprinus comatus Grown Under Different Cultures: The Role of Major Isoenzyme in Dyes Decolorization

Coprinus comatus laccase isoenzyme induction and its effect on decolorization were investigated. The C/N ratio, together with aromatic compounds and copper, significantly influenced laccase isoenzyme profile and enzyme activity. This fungus produced six laccase isoenzymes in high-nitrogen low-carbon cultures but much less in low-nitrogen high-carbon (LNHC) cultures. The highest laccase level (3.25 IU/ml), equivalent to a 12.6-fold increase compared with unsupplemented controls (0.257 IU/ml), was recorded after 13 days in LNHC cultures supplemented with 2.0 mM 2-toluidine. Decolorization of twelve synthetic dyes belonging to anthraquinone, azo, and triphenylmethane dyes, by crude laccases with different proportion of isoenzymes produced under selected culture conditions, illustrated that the LacA is the key isoenzyme contributed to dyes decolorization especially in the presence of 1-hydroxybenzotriazol, which was further confirmed by dyes decolorization with purified LacA in the same condition. The crude laccase only was able to decolorize over 90 % of Reactive Brilliant Blue K-3R, Reactive Dark Blue KR, and Malachite Green, and higher decolorization for broader spectrum of synthetic dyes was obtained in presence of redox mediator, suggesting that C. comatus had high potential to decolorize various synthetic dyes as well as the recalcitrant azo dyes.     

Mediator-assisted Decolorization and Detoxification of Textile Dyes/Dye Mixture by Cyathus bulleri Laccase

Laccase from basidiomycete fungus Cyathus bulleri was evaluated for its ability to decolorize a number of reactive and acidic dyes in the presence of natural and synthetic mediators. The extent of decolorization was monitored at different mediator/dye concentrations and incubation time. Among the synthetic mediators, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was effective at low mediator/dye ratios and resulted in 80-95% decolorization at rates that varied from 226 +/- 4 nmol min(-1) mg(-1) for Reactive Orange 1 to 1,333 +/- 15 nmol min(-1) mg(-1) for Reactive Red 198. Other synthetic mediators like 1-hydroxybenzotriazole and violuric acid showed both concentration- and time-dependent increases in percent decolorization. Natural mediators like vanillin, on the other hand, were found to be less effective on all the dyes except Reactive Orange 1. Computed rates of decolorization were about twofold lower than that with ABTS. The laccase-ABTS system also led to nearly 80% decolorization for the simulated dye mixture. No clear correlation between laccase activity on the mediator and its ability to decolorize dyes was found, but pH had a significant effect: Optimum pH for decolorization coincided with the optimum pH for mediator oxidation. The treated samples were also evaluated for toxicity in model microbial systems. The laccase-mediator system appears promising for treatment of textile wastewaters.     

Fabrication of metal-organic framework Universitetet i Oslo-66 (UiO-66) and brown-rot fungus Gloeophyllum trabeum biocomposite (UiO-66@GT) and its application for reactive black 5 decolorization

Many various industries use synthetic dyes as their raw materials. These dyes have triggered environmental problems because of the occurring effluents, and one of the environmentally safe solutions for this problem is biodegradation through microorganisms. Reactive Black 5 (RB5) dye degradation was performed by utilizing a metal-organic framework Universitetet i Oslo-66 (UiO-66) and Gloeophyllum trabeum (GT) fungus biocomposite. The UiO-66@GT composite was fabricated by inoculating the fungal culture in flasks with the PDB medium that contained UiO-66. This biocomposite was applied to decolorize and degrade RB5 dye, while pure GT culture can decolorize about 36.47% in five days. The percentage of RB5 decolorization was shown to be increased with the addition of UiO-66; the composite could decolorize RB5 up to 72.55% after five days incubation period. Moreover, the optimum conditions for the 100% targeted rate of RB5 decolorization found by the Response Surface Methodology (RSM) are: initial RB5 concentration (72.54 mg L^-1), pH (6.53), and temperature (38.06 °C). Two novel metabolites from RB5 decolorization by the composite were detected based on LCMS-QTOF analysis and were used to propose a degradation pathway: 6-((1-amino-7,8-dihydroxy-6-sulfonaphthalen-2-yl) diazinyl) cyclohexa-2,4-dien-1-ide (m/z = 360) and 3,4-diamino-5,6-dihydroxy-1,2,7,8-tetrahydronaphthalene-2,7-disulfonic acid (m/z = 354).     

Decolorization of Some Azo Dyes by Immobilized <Emphasis Type="Italic">Geotrichum</Emphasis> sp. Biomass in Fluidized Bed Bioreactor

Geotrichum sp. strain, which is able to decolorize azo dyes enzymatically, was used in this study for decolorization of synthetics solutions contaminated by toxic azo dyes orange G, trypan blue, azorubine, and methyl red. The biomass of Geotrichum sp. was immobilized in calcium alginate and polyacrylamide gels and used for the decolorization of tested azo dyes in fluidized bed bioreactor. The highest specific decolorization rate was obtained when the fungal biomass was entrapped in calcium alginate beads. Immobilized biomass in calcium alginate continuously decolorized azo dyes after eight repeated batch decolorization experiments without significant loss of activity whereas polyacrylamide immobilized biomass retained only 10% of its activity after 4 days of incubation. The effects of some physicochemical parameters such as temperature, pH, and dyes concentration on decolorization performance of isolated fungal strain were also investigated.     

In Silico and in Vitro Physicochemical Screening of Rigidoporus sp. Crude Laccase-assisted Decolorization of Synthetic Dyes—Approaches for a Cost-effective Enzyme-based Remediation Methodology

The objective of this paper is to compare in silico data with wet lab physicochemical properties of crude laccase enzyme isolated from Rigidoporus sp. using wheat bran as solid substrate support towards dye decolorization. Molecular docking analysis of selected nine textile and non-textile dyes were performed using laccase from Rigidoporus lignosus as reference protein. Enzyme-based remediation methodology using crude enzyme enriched from solid state fermentation was applied to screen the effect of four influencing variables such as pH, temperature, dye concentration, and incubation time toward dye decolorization. The extracellular crude enzyme decolorized 69.8 % Acid Blue 113, 45.07 % Reactive Blue 19, 36.61 % Reactive Orange 122, 30.55 % Acid Red 88, 24.59 % Direct Blue 14, 18.48 % Reactive Black B, 16.49 % Reactive Blue RGB, and 11.66 % Acid Blue 9 at 100 mg/l dye concentration at their optimal pH at room temperature under static and dark conditions after 1 h of incubation without addition of any externally added mediators. Our wet lab studies approach, barring other factors, validate in silico for screening and ranking textile dyes based on their proximity to the T1 site. We are reporting for the first time a combinatorial approach involving in silico methods and wet lab-based crude laccase-mediated dye decolorization without any external mediators.     

Enhanced Biodecolorization of Reactive Dyes by Basidiomycetes Under Static Conditions

This study presents the biodecolorization potential of basidiomycete fungi Trametes hirsuta, Pycnoporus sp., and Irpex sp. for different reactive dyes viz. Reactive Red 120, Remazol Brilliant Blue R (RBBR), Reactive Orange G, and Reactive Orange 16 under static and shaking conditions. The screening trials revealed that T. hirsuta exhibited maximum potential (83.75 %) for biodecolorization of RBBR dye under static conditions after the fifth day of incubation. However, the rate of biodecolorization of RBBR dye by Pycnoporus sp. was much slow and reached maximum (81.25 %) after 15 days of incubation under shaking conditions. By process optimization, enhanced decolorization (91.2 %) of RBBR by T. hirsuta was achieved at pH 5.5 within 24 h using a defined salt medium amended with p-coumaric acid under static conditions. pH was found to be an important parameter for the enzymatic system involved in RBBR dye decolorization by T. hirsuta and Pycnoporus sp. Biodecolorization of RBBR dye was determined by a reduction in optical density at the wavelength of maximum absorbance (λ, 578 nm) by UV–vis spectrophotometer. The shift in maximum wavelength toward shorter/longer wavelength in UV–vis scanning spectrum revealed the degradation of RBBR dye into different transformation products.     

Decolorization Potential of Some Reactive Dyes with Crude Laccase and Laccase-Mediated System

In this study, decolorization of dyestuffs, such as Reactive Red 198, Rem Blue RR, Dylon Navy 17, Rem Red RR, and Rem Yellow RR was studied using laccase and laccase-mediated system. The laccases are known to have an important potential for remediation of pollutants. Among these dyestuffs, decolorization of Rem Blue RR and Dylon Navy 17 was performed with crude laccase under optimized conditions. Vanillin was selected as laccase mediator after screening six different compounds with Rem Yellow RR, Reactive Red 198, and Rem Red RR as substrates. However, Rem Yellow RR was not decolorized by either laccase or laccase-mediated system. It is observed that the culture supernatant contained high laccase activity after treatment with catalase that was responsible for the decolorization. Besides, culture supernatant with high laccase activity as enzyme source was treated with catalase; in this way, the hypothesis that laccase was the enzyme responsible for decolorization was supported. The Rem Blue RR was decolorized with 64.84% under the optimum conditions and Dylon Navy 17 with 75.43% with crude laccase. However, using the laccase and vanillin, the decolorization of Reactive Red 198 and Rem Red RR was found to be 62% and 68%, respectively. Our study demonstrated that the decolorization abilities of laccase and/or laccase mediator systems were based on the types of mediator, the dye structure, and the standard experimental conditions. Also, the electrochemical behaviors of some samples were studied. The redox potentials of these samples were determined using cyclic voltammetry on glassy carbon electrode in phosphate buffer (pH 6) solution.     

Decolorization kinetics of the azo dye drimaren blue X3LR by laccase

Summary An extracellular Drimaren Blue X3LR decolorizing enzymatic activity was found in the crude filtrate of Funalia trogii grown by solid-state fermentation using wheat bran and soya bean waste. Decolorization of the azo dye Drimaren Blue X3LR by the crude filtrate and partially purified enzyme of Funalia trogii were investigated and compared. In the absence of additional redox mediator, maximum decolorization ratios of 81.33 % and 77.4 % were observed for Drimaren Blue X3LR using crude filtrate and partially purified enzyme respectively. Decolorization yield was found to be higher with crude enzyme preparations. Na₂S₂O₅ inhibited laccase and dye decolorizing enzyme activities but a significant peroxidase activity inhibition was not observed. Since the reaction was catalyzed in the absence of H₂O₂ as co-substrate, it could be concluded that this enzyme is not a peroxidase but may be a laccase.. The kinetic parameters of decolorization were calculated according to Michaelis constant (Km of 1.700 x 10^-5 mol dm^-3 and W_max = 8.02 x10^-7 mol dm^-3 sec^-1).     

Biodegradation of Bisphenol A and Decolorization of Synthetic Dyes by Laccase from White-Rot Fungus, Trametes polyzona

Purified laccase from Trametes polyzona WR710-1 was used as biocatalyst for bisphenol A biodegradation and decolorization of synthetic dyes. Degradation of bisphenol A by laccase with or without redox mediator, 1-hydroxybenzotriazole (HBT) was studied. The quantitative analysis by HPLC showed that bisphenol A rapidly oxidized by laccase with HBT. Bisphenol A was completely removed within 3 h and 4-isopropenylphenol was found as the oxidative degradation product from bisphenol A when identified by GC-MS. All synthetic dyes used in this experiment, Bromophenol Blue, Remazol Brilliant Blue R, Methyl Orange, Relative Black 5, Congo Red, and Acridine Orange were decolorized by Trametes laccase and the percentage of decolorization increased when 2 mM HBT was added in the reaction mixture. This is the first report showing that laccase from T. polyzona is an affective enzyme having high potential for environmental detoxification, bisphenol A degradation and synthetic dye decolorization.     

Decolorization of Reactive Black 5 and Reactive Blue 4 Dyes in Microbial Fuel Cells

Microbial fuel cells (MFCs) have potential to treat industrial wastewater containing organic compounds and simultaneously generate power. Organic compounds include textile dyes with various chromophore groups, which can be decolorized reductively by microorganisms under anaerobic conditions. In the present study, we examined the decolorization of Reactive Black 5 (RB5) azo dye and Reactive Blue 4 (RBL4) anthraquinone dye under open circuit potential in MFCs with graphite plate and graphite felt electrodes and a microbial consortium originally derived from bovine rumen fluid. RB5 dye was more than 90% decolorized in 120, 165, and 225 min at 50, 100, and 200 mg L^?1 concentrations, respectively. RBL4 dye at 50 and 100 mg L^?1 took 225 and 300 min to decolorize, while 200 mg L^?1 RBL4 dye was not decolorized at all. Under closed circuit conditions, decolorization increased with decrease in external load, whereas current generation increased with external resistance. The results demonstrate that the reductive cleavage of the chromophore was more rapid with RB5 than with RBL4.     

Degradation of textile dyes mediated by plant peroxidases

The peroxidase enzyme from the plants Ipomea palmata (1.003 IU/g of leaf) and Saccharum spontaneum (3.6 IU/g of leaf) can be used as an alternative to the commercial source of horseradish and soybean peroxidase enzyme for the decolorization of textile dyes, mainly azo dyes. Eight textiles dyes currently used by the industry and seven other dyes were selected for decolorization studies at 25–200 mg/L levels using these plant enzymes. The enzymes were purified prior to use by ammonium sulfate precipitation, and ion exchange and gel permeation chromatographic techniques. Peroxidase of S. spontaneum leaf (specific activity of 0.23 IU/mg) could completely degrade Supranol Green and Procion Green HE-4BD (100%) dyes within 1 h, whereas Direct Blue, Procion Brilliant Blue H-7G and Chrysoidine were degraded >70% in 1 h. Peroxidase of Ipomea (I. palmata leaf; specific activity of 0.827 U/mg) degraded 50 mg/L of the dyes Methyl Orange (26%), Crystal Violet (36%), and Supranol Green (68%) in 2–4 h and Brilliant Green 54%), Direct Blue (15%), and Chrysoidine (44%) at the 25 mg/L level in 1 to 2 h of treatment. The Saccharum peroxidase was immobilized on a hydrophobic matrix. Four textile dyes, Procion Navy Blue HER, Procion Brilliant Blue H-7G, Procion Green HE-4BD, and Supranol Green, at an initial concentration of 50 mg/L were completely degraded within 8 h by the enzyme immobilized on the modified polyethylene matrix. The immobilized enzyme was used in a batch reactor for the degradation of Procion Green HE-4BD and the reusability was studied for 15 cycles, and the halflife was found to be 60 h.     

Polysaccharide/Fe(III)-porphyrin hybrid film as catalyst for oxidative decolorization of toxic azo dyes: An approach for wastewater treatment

A hybrid film of chitosan/poly(vinyl alcohol)/cationic Fe(III)-porphyrin (Cht/PVA-FeTMPyP) was synthesized to act as a Fenton-like catalyst to decolorize methyl orange and methyl red azo dyes. The Cht/PVA-FeTMPyP film was characterized by different analytical and microscopy techniques, which indicated that the metalloporphyrin affects different properties of the hybrid film. Batch experiments revealed that the hybrid film exhibits enhanced catalytic activity towards dyes decolorization in the presence of H₂O₂ as compared to the “free” FeTMPyP. Fast decolorization rates as high as 90 min were observed for both azo dyes under mild conditions (pH 7 and room temperature), even at low concentrations of the catalyst in H₂O₂. After the decolorization, FTIR analysis showed that simple molecules are released as by-products. Moreover, the hybrid film performed well in cyclic runs without leaching out iron ions or losing its catalytic activity. All these features associated with its ease handling ranks the Cht/PVA-FeTMPyP hybrid film as a promising heterogeneous Fenton-like catalyst for the decomposition of azo dyes in water.     

Modeling and optimization of simultaneous photocatalysis of three dyes on ceramic-coated TiO2 nanoparticles using chemometrics methods: phytotoxicological assessment during degradation process

In this paper, ceramic plates were used as a support of TiO₂ nanoparticles for photocatalytic decolorization of a mixture of three dyes. The three textile dyes (C.I. Basic Red 46, C.I. Basic Blue 3 and Malachite Green) were quantified simultaneously during the photocatalytic degradation process. The partial least squares modeling was successfully applied for the multivariate calibration of the spectrophotometric data. Also, the central composite design has been applied to the optimization of photocatalytic decolorization of the dye solution containing three dyes using an immobilized UV/TiO₂ process. The optimum initial concentration of three dyes, reaction time, and UV light intensity were found to be 5 mg/L, 240 min, and 47.2 W/m², respectively. The chronic phytotoxicity of mixture of dyes was evaluated using aquatic species Spirodela polyrhiza (S. polyrhiza) prior to and after photocatalysis. The phytotoxicity results revealed that the photocatalysis process could effectively reduce the phytotoxicity of the dyes from their aqueous solutions.     

Aerobic Biodegradation Pathway for Remazol Orange by Pseudomonas aeruginosa

Removal of azo dyes from effluent generated by textile industries is rather difficult. Azo dyes represent a major class of synthetic colorants that are mutagenic and carcinogenic. Pseudomonas aeruginosa grew well in the presence of Remazol Orange (RO) and was able to decolorize and degrade it. In the present study, the decolorization and degradation efficiency using single culture P. aeruginosa with RO and textile wastewaters is studied. The elucidation of decolorization pathway for P. aeruginosa is of special interest. The degradation pathway and the metabolic products formed during the degradation were also predicted with the help of high performance liquid chromatography, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy analysis. The data show the cleavage of the azo dye RO to form both methyl metanilic acid and 4-aminobenzoic acid after decolorization and finally to oxidation forms benzoic acid, alkenes, aldehydes, and alkynes. The organism was able to decolorize the dye RO and wastewater effectively to the maximum of 82.4% and 62%, respectively.     

Oxidation of crocein orange G by lignin peroxidase isoenzymes

The ligninolytic enzyme system ofPhanerochaete chrysosporiun is able to decolorize several recalcitrant dyes. Three lignin peroxidase isoenzymes, LiP 3.85, LiP 4.15, and LiP 4.65, were purified by preparative isoelectric focusing from the carbon-limited culture medium ofP. chrysosporium. Based on amino terminal sequences, the purified isoenzymes correspond to the isoenzymes H8, H6, and H2, respectively, from theN-limited culture. The purified isoenzymes were used for decolorization of an azo dye, Crocein Orange G (COG). According to the kinetic data obtained, the oxidation of COG by lignin peroxidase appeared to follow Michaelis-Menten kinetics. Kinetic parameters for each isoenzyme were determined. The inactivating effect of ascending H₂O₂ concentrations on COG oxidation is shown to be exponential within the used concentration range. The best degree of decolorization of 100 μM COG was obtained when the H₂O₂ concentration was 150 μM. This was also the lowest H₂O₂ concentration for maximal decolorization of 100 μM COG, regardless of the amount of lignin peroxidase used in the reaction.     

Hydrophobic effect for anionic dye - cationic surfactant interaction in aqueous and mixed solvent

Abstract

The interaction between anionic dyes [Reactive Orange 122 (R.O 122), Reactive Blue 19 (R.B 19), Reactive Violet 5 (R.V 5) and Acid Green 20 (A.G 20)] with cationic surfactant cetyltrimethylammoniun bromide (CTAB) has been investigated by spectrophotometry and conductance technique. The used dyes are characterized by tautomeric behavior which affects the mechanism of the interaction. Various parameters such as dye structure, surfactant composition, solvent composition, temperature and pH of the medium were studied. The spectral data were applied for calculating the binding constant between dye and surfactant (K_b), fraction of micellization (?_mic), and standard free energy change of binding (ΔG°_b) in 0,10,20 and 30 v/v % acetonitile (AN). Conductance technique was constructed to estimate the ion pairing constant (K_a) at different temperatures and v/v % AN. Thermodynamic parameters (ΔG°, ΔH° and ΔS°) for ion pair formation were evaluated. The role of hydrophobic and electrostatic effect on dye-surfactant interaction was discussed.     

Photocatalytic degradation of triazine dyes over N-doped TiO2 in solar radiation

Photocatalytic degradation of the reactive triazine dyes Reactive Yellow 84 (RY 84), Reactive Red 120 (RR 120), and Reactive Blue 160 (RB 160) on anatase phase N-doped TiO₂ in the presence of natural sunlight has been carried out in this work. The effect of experimental parameters like initial pH and concentration of dye solution and dosage of the catalyst on photocatalytic degradation have also been investigated. Adsorption of dyes on N-doped TiO₂ was studied prior to photocatalytic studies. The studies show that the adsorption of dyes on N-doped TiO₂ was high at pH 3 and follows the Langmuir adsorption isotherm. The Langmuir monolayer adsorption capacity of dyes on N-doped TiO₂ was 39.5, 86.0, and 96.3 mg g^?1 for RY 84, RR 120, and RB 160, respectively. The photocatalytic degradation of the dyes follows pseudo first-order kinetics and the rate constant values are higher for N-doped TiO₂ when compared with that of undoped TiO₂. Moreover, the degradation of RY 84 on N-doped TiO₂ in sunlight was faster than the commercial Aeroxide^® P25. However, the P25 has shown higher photocatalytic activity for the other two dyes, RR 120 and RB 160. The COD of 50 mg l^?1 Reactive Yellow-84, RR 120 and RB 160 was reduced by 65.1, 73.1, and 69.6 %, respectively, upon irradiation of sunlight for 3 h in the presence of N-doped TiO₂. The photocatalyst shows low activity for the degradation of RY 84 dye, when its concentration was above 50 mg l^?1, due to the strong absorption of photons in the wavelength range 200–400 nm by the dye solution. LC–MS analysis shows the presence of some triazine compounds and formimidamide derivatives in the dye solutions after 3 h solar light irradiation in the presence of N-doped TiO₂.     

A novel WO<Subscript>3</Subscript>/MoS<Subscript>2</Subscript> photocatalyst applied to the decolorization of the textile dye Reactive Blue 198

A novel FTO/WO₃ electrode decorated with MoS₂ was constructed using two simple and low-cost techniques involving a modified single-step sol-gel method for the WO₃ film together with the electrodeposition of amorphous MoS₂. The photoelectrocatalytic performance of the material was investigated by monitoring the degradation of Reactive Blue 198 dye under visible-light irradiation. The FTO/WO₃/MoS₂ electrode exhibited excellent photocatalytic activity and afforded total decolorization of the dye after 90 min at low applied current density (5 mA cm^?2). The results described herein support the view that MoS₂ acts as a noble metal-free cocatalyst by promoting H₂ evolution and assisting in the suppression of electron/hole pair recombination in the photocatalytic material (WO₃), thereby improving the process of decolorization of the dye solution. The novel approach of combining of the WO₃ and MoS₂ materials shows particular promise and may prove to be very effective in the photocatalytic degradation of other hazardous organic compounds.     

A new fast swelling poly[DAPB-co-DMAAm-co-AASS] superabsorbent hydrogel for removal of anionic dyes from water

The objective of this research is to utilize a new poly[N,N-diallyl pyrrolidinium bromide-co-N,Ndimethyl acrylamide-co-acrylic acid sodium salt]superabsorbent hydrogel（SAH）for the removal of two anionic dyes,e.g.,Reactive Red 5B（RR5B）and Reactive Orange M2R（ROM2R）,from water.The SAH was characterized by swelling in water,FTIR,TGA and SEM.The SAH DDA6showed good swelling property and thermal stability.We have also investigated the parameters affecting dye adsorption such as pH,adsorbent dose,adsorption rate and initial dye concentration.The experimental data were also analyzed by applying the well known Langmuir and Freundlich isotherm models.     

Reductive Decolourisation of Sulphonated Mono and Diazo Dyes in One- and Two-Stage Anaerobic Systems

This work assessed the application of one- and two-stage mesophilic anaerobic systems to colour removal of sulphonated mono and diazo dyes with ethanol as electron donor. The dyes Congo Red (CR), Reactive Black 5 (RB5) and Reactive Red 2 (RR2) were selected as model compounds and tested separately in seven different periods. The one-stage system (R₁) consisted of a single up-flow anaerobic sludge blanket (UASB) reactor, whereas the two-stage system (R₂) consisted of an acidogenic UASB reactor (R_A), a settler and a methanogenic UASB reactor (R_M). For CR and RB5, no remarkable difference was observed between the colour removal performance of both anaerobic systems R₁ and R₂. The experiments with RR2 revealed that R₂ was more efficient on colour removal than R₁, showing efficiencies almost 2-fold (period VI) and 2.5-fold (period VII) higher than those found by R₁. Additionally, R₂ showed a higher stability, giving a good prospect for application to textile wastewaters. Finally, the acidogenic reactor (R_A) had an important role in the overall decolourisation achieved by R₂ during the experiments with CR and RB5 (>78 %), whereas for RR2, a more recalcitrant dye, R_A was responsible for up to 38 % of the total colour removal.