Lignin-Derived Compounds as Efficient Laccase Mediators for Decolorization of Different Types of Recalcitrant Dyes

Ten phenols were selected as natural laccase mediators after screening 44 different compounds with a recalcitrant dye (Reactive Black 5) as a substrate. Their performances were evaluated at different mediator/dye ratios and incubation times (up to 6 h) by the use of Pycnoporus cinnabarinus and Trametes villosa laccases and were compared with those of eight known synthetic mediators (including -NOH- compounds). Among the six types of dyes assayed, only Reactive Blue 38 (phthalocyanine) was resistant to laccase-mediator treatment under the conditions used. Acid Blue 74 (indigoid dye), Reactive Blue 19 (anthraquinoid dye), and Aniline Blue (triarylmethane-type dye) were partially decolorized by the laccases alone, although decolorization was much more efficient and rapid with mediators, whereas Reactive Black 5 (diazo dye) and Azure B (heterocyclic dye) could be decolorized only in the presence of mediators. The efficiency of each natural mediator depended on the type of dye to be treated but, with the only exception being Azure B (<50% decolorization), nearly complete decolorization (80 to 100%) was attained in all cases. Similar rates were attained with the best synthetic mediators, but the reactions were significantly slower. Phenolic aldehydes, ketones, acids, and esters related to the three lignin units were among the best mediators, including p-coumaric acid, vanillin, acetovanillone, methyl vanillate, and above all, syringaldehyde and acetosyringone. The last two compounds are especially promising as ecofriendly (and potentially cheap) mediators for industrial applications since they provided the highest decolorization rates in only 5 to 30 min, depending on the type of dye to be treated.     

Biodegradation of diazo dye Direct brown MR by Acinetobacter calcoaceticus NCIM 2890

Acinetobacter calcoaceticus was employed for the degradation of Direct brown MR (DBMR), commercially used azo dye in the textile industry in order to analyze mechanism of the degradation and role of inhibitors, redox mediators and stabilizers of lignin peroxidase during decolorization. Induction of intracellular and extracellular lignin peroxidase, intracellular laccase and DCIP reductase represented their involvement in the biodegradation of DBMR. Decolorization and biodegradation of azo dye DBMR in broth were monitored by UV–visible spectrophotometer and TLC. The products obtained from A. calcoaceticus degradation were characterized by FTIR and identified by GC/MS as biphenyl amine, biphenyl, 3-amino 6-hydroxybenzoic acid and naphthalene diazonium. Germination (%) and growth efficiency of Sorghum vulgare and Phaseolus mungo seeds revealed the degradation of DBMR into less toxic products than original dye. A. calcoaceticus also has a potential to degrade diverse dyes present in the textile effluent, into nontoxic metabolites, hence A. calcoaceticus can be applied for the commercial application.     

Potential of Trametes trogii culture fluids and its purified laccase for the decolorization of different types of recalcitrant dyes without the addition of redox mediators

Trametes trogii BAFC 463 culture fluids (containing 110 U ml⁻¹ laccase; 0.94 U ml⁻¹ manganese peroxidase), as well as its purified laccase were capable of decolorizing azoic, indigoid, triphenylmethane, anthraquinonic and heterocyclic dyes, in the absence of redox mediators. Six dyes: RBBR, Indigo Carmine, Xylidine, Malachite Green, Gentian Violet and Bromophenol Blue were almost completely degraded (more than 85% decolorization after 1 d) by either laccase or T. trogii itself in culture, proving the role of the enzyme in dye decolorization. The purified laccase also decolorized 65% of Fast Blue RR and 30% of Azure B and Methylene Blue after 24 h. The use of redox mediators significantly increased the decolorization rates (90% decolorization of Azure B after 1 h). 1-hydroxybenzotriazole resulted the best redox mediator, but the natural mediator p-hydroxybenzoic acid also demonstrated its efficiency for dye decolorization. Due to their ability to decolorize recalcitrant dyes without addition of redox mediators, high laccase activities, high thermostability and efficient decolorization at 70 °C and pH 7.0, even in the presence of high concentrations of heavy metals (100 mM Cu⁺², Pb⁺² or Cd⁺²) or in a synthetic dyebath, T. trogii culture fluids could be effectively used to decolorize synthetic dyes from effluents.     

Azo dye decolorization by a laccase/mediator system in a membrane reactor: enzyme and mediator reusability

This paper presents the use of a membrane-integrated reactor system with recycling of laccase and mediator for azo dye decolorization. From initial screening of different laccases and mediators, Trametes versicolor laccase and syringaldehyde provided the best system for decolorization. Decolorization yields of 98, 88, 80 and 78% were obtained for Red FN-2BL, Red BWS, Remazol Blue RR and Blue 4BL, respectively. The reaction parameters were optimized and a membrane reactor was set up for dye decolorization in batch mode with reuse of the enzyme. Between 10 and 20 batches could be run with decolorization yields from 95 to 52% depending on the dye type. To study the possibility of reusing both enzyme and mediator, the reactor was run using 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) coupled to polyethylene glycol (PEG). Nine batches were run for the treatment of Remazol Blue RR, providing decolorization yields of 96-78%. Cost analysis of the processes showed that the costs of laccase/syringaldehyde or laccase/TEMPO were almost equal when running 20 batches, but the cost for the PEG-TEMPO was higher. However, the advantages associated with reuse of the mediator should motivate further development of the concept.     

Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes

Ten phenols were selected as natural laccase mediators after screening 44 different compounds with a recalcitrant dye (Reactive Black 5) as a substrate. Their performances were evaluated at different mediator/dye ratios and incubation times (up to 6 h) by the use of Pycnoporus cinnabarinus and Trametes villosa laccases and were compared with those of eight known synthetic mediators (including -NOH- compounds). Among the six types of dyes assayed, only Reactive Blue 38 (phthalocyanine) was resistant to laccase-mediator treatment under the conditions used. Acid Blue 74 (indigoid dye), Reactive Blue 19 (anthraquinoid dye), and Aniline Blue (triarylmethane-type dye) were partially decolorized by the laccases alone, although decolorization was much more efficient and rapid with mediators, whereas Reactive Black 5 (diazo dye) and Azure B (heterocyclic dye) could be decolorized only in the presence of mediators. The efficiency of each natural mediator depended on the type of dye to be treated but, with the only exception being Azure B (< 50% decolorization), nearly complete decolorization (80 to 100%) was attained in all cases. Similar rates were attained with the best synthetic mediators, but the reactions were significantly slower. Phenolic aldehydes, ketones, acids, and esters related to the three lignin units were among the best mediators, including p-coumaric acid, vanillin, acetovanillone, methyl vanillate, and above all, syringaldehyde and acetosyringone. The last two compounds are especially promising as ecofriendly (and potentially cheap) mediators for industrial applications since they provided the highest decolorization rates in only 5 to 30 min, depending on the type of dye to be treated.     

Decolorization of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the sole phenol-oxidizing enzyme

The ability of a Brazilian strain ofPleurotus pulmonarius to decolorize structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes) was investigated in solid and submerged cultures. Both were able to decolorize completely or partially 8 of 10 dyes (Amido Black, Congo Red, Trypan Blue, Methyl Green, Remazol Brilliant Blue R, Methyl Violet, Ethyl Violet, Brilliant Cresyl Blue). No decolorization of Methylene Blue and Poly R 478 was observed. Of the four phenol-oxidizing enzymes tested in culture filtrates (lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, laccase),P. pulmonarius produced only laccase. Both laccase activity and dye decolorization were related to glucose and ammonium starvation or to induction by ferulic acid. The decolorizationin vivo was tested using three dyes — Remazol Brilliant Blue R, Trypan Blue and Methyl Green. All of them were completely decolorized by crude extracellular extracts. Decolorization and laccase activity were equally affected by pH and temperature. Laccase can thus be considered to be the major enzyme involved in the ability ofP. pulmonarius to decolorize industrial dyes.     

Predicting dye biodegradation from redox potentials

Two biological approaches for decolorization of azo sulfonated dyes have been compared: reductive decolorization with the ascomycete yeast Issatchenkia occidentalis and enzymatic oxidative decolorization with Trametes villosa laccase alone or in the presence of the mediator 1-hydroxybenzotriazole. The redox potential difference between the biological cofactor involved in the reductive activity of growing cells and the azo dye is a reliable indication for the decolorization ability of the biocatalyst. A linear relationship exists between the redox potential of the azo dyes and the decolorization efficiency of enzyme, enzyme/mediator, and yeast. The less positive the anodic peak of the dye, the more easily it is degraded oxidatively with laccase. The more positive the cathodic peak of the dye, the more rapidly the dye molecule is reduced with yeast.     

Biodecolorization of textile azo dyes by isolated yeast from activated sludge: Issatchenkia orientalis JKS6

An ascomycetous yeast strain isolated from activated sludge could decolorize Reactive Black 5 azo dye at 200 mg l^?1 up to 90 % within 12–18 h under agitated condition. Yeast decolorization ability was investigated at different RB5 concentrations and, at higher dye concentration, 500 mg l^?1, the decolorization was found to be 98 % after 36 h incubation time. Extensive decolorization (95–99 %) was obtained in presence of five other azo dyes, Reactive Orange 16, Reactive Red 198, Direct Blue 71, Direct Yellow 12, and Direct Black 22, by isolated yeast. HPLC analysis, UV–vis spectra and colorless biomass obtained after complete decolorization showed that the decolorization occured through a biodegradation mechanism. Decolorization was occurred during the exponential growth phase which is associated to primary metabolism. Laccase production by the yeast cells was not detected. The isolated yeast was characterized according to phenotypical and molecular procedures and was closely related (99 % identity) to Issatchenkia orientalis.     

Rapid decolorization of azo dyes by crude manganese peroxidase from Schizophyllum sp. F17 in solid-state fermentation

The production of ligninolytic enzymes by the fungus Schizophyllum sp. F17 using a cost-effective medium comprised of agro-industrial residues in solid-state fermentation (SSF) was optimized. The maximum activities of the enzymes manganese peroxidase (MnP), laccase (Lac), and lignin peroxidases (LiP) were 1,200, 586, and 109 U/L, respectively, on day 5 of SSF. In vitro decolorization of three structurally different azo dyes by the extracellular enzymes was monitored to determine its decolorization capability. The results indicated that crude MnP, but not LiP and Lac, played a crucial role in the decolorization of azo dyes. After optimization of the dye decolorization system with crude MnP, the decolorization rates of Orange IV and Orange G, at an initial dye concentration of 50 mg/L, were enhanced to 76 and 57%, respectively, after 20 min of reaction at pH 4 and 35°C. However, only 8% decolorization of Congo red was observed. This enzymatic reaction system revealed a rapid decolorization of azo dyes with a low MnP activity of 24 U/L. Thus, this study could be the basis for the production and application of MnP on a larger scale using a low-cost substrate.     

Autocatalysis in Reactive Black 5 biodecolorization by <Emphasis Type="Italic">Rhodopseudomonas palustris</Emphasis> W1

Autocatalysis in biological decolorization of Reactive Black 5 (RB5) by Rhodopseudomonas palustris W1 was investigated in batch assays. An improvement of 1.5-fold in decolorization rate of RB5 was obtained by supplementing decolorization metabolites from 200 mg l(-1) RB5. Liquid chromatography-mass spectrometry and cyclic voltammetric analysis revealed that the constituent of dye precursors, from azo bonds breakage, with quinone-like structure and reversible oxidation-reduction activity can be used as redox mediators and was responsible for the catalytic reduction of RB5. The required amount of metabolites for catalytic decolorization was quite small, indicating its possible application in real textile wastewater treatment. Furthermore, decolorization metabolites of RB5 were shown as effective in catalyzing anaerobic decolorization of Direct Yellow 11, an azo dye without autocatalyic activity.     

Anthraquinone dye assisted the decolorization of azo dyes by a novel Trametes trogii laccase

A new Trametes trogii laccase was purified and its biochemical properties were subsequently characterized. After a survey of other T. trogii laccases, this laccase showed a lower isoelectric point, different N-terminal sequence and kinetic parameters. Recently most laccase-catalyzed decolorizations of synthetic dyes are single-solute studies with commercially available dyes as model pollutants and need the employment of redox mediators. In this study, to simulate the real industry wastewaters, experiments of laccase-catalyzed decolorization of mixed dyes constituted by azo and anthraquinone dyes were carried out. The results showed that anthraquinone dyes, playing the role of mediators, dramatically promoted the degradation of azo dyes when there was no exogenous mediator in the reaction mixture. This study represents the first attempt to decolorize the mixtures of azo and anthraquinone dyes by purified T. trogii laccase, suggesting great potential for laccase to decolorize textile industry wastewaters.     

Decolorization and Detoxification of Textile Dyes with a Laccase from Trametes hirsuta

Trametes hirsuta and a purified laccase from this organism were able to degrade triarylmethane, indigoid, azo, and anthraquinonic dyes. Initial decolorization velocities depended on the substituents on the phenolic rings of the dyes. Immobilization of the T. hirsuta laccase on alumina enhanced the thermal stabilities of the enzyme and its tolerance against some enzyme inhibitors, such as halides, copper chelators, and dyeing additives. The laccase lost 50% of its activity at 50 mM NaCl while the 50% inhibitory concentration (IC₅₀) of the immobilized enzyme was 85 mM. Treatment of dyes with the immobilized laccase reduced their toxicities (based on the oxygen consumption rate of Pseudomonas putida) by up to 80% (anthraquinonic dyes). Textile effluents decolorized with T. hirsuta or the laccase were used for dyeing. Metabolites and/or enzyme protein strongly interacted with the dyeing process indicated by lower staining levels (K/S) values than obtained with a blank using water. However, when the effluents were decolorized with immobilized laccase, they could be used for dyeing and acceptable color differences (ΔE*) below 1.1 were measured for most dyes.     

Purification and characterization of laccase produced by a white rot fungus Pleurotus sajor-caju under submerged culture condition and its potential in decolorization of azo dyes

An extracellular laccase was isolated and purified from Pleurotus sajor-caju grown in submerged culture in a bioreactor, and used to investigate its ability to decolorize three azo dyes. The extracellular laccase production was enhanced up to 2.5-fold in the medium amended with xylidine (1 mM). Purification was carried out using ammonium sulfate (70% w/v), DEAE-cellulose, and Sephadex G-100 column chromatography. The enzyme was purified up to 10.3-fold from the initial protein preparation with an overall yield of 53%. The purified laccase was monomeric with an apparent molecular mass of 61.0 kDa. The purified enzyme exerted its optimal activity with 2,2-azino–bis(3-ethylbenzo-thiazoline-6-sulfonate (ABTS) and oxidized various lignin-related phenols. The catalytic efficiencies k _cat/K _m determined for ABTS and syringaldazine were 9.2×10⁵ and 8.7×10⁵, respectively. The optimum pH and temperature for the purified enzyme was 5.0 and 40 °C, respectively. Sodium azide completely inhibited the laccase activity. The absorption spectrum revealed type 1 and type 3 copper signals. The purified enzyme decolorized azo dyes such as acid red 18, acid Black 1, and direct blue 71 up to 90, 87, and 72%, respectively. Decolorization ability of P. sajor-caju laccase suggests that this enzyme could be used for decolorization of industrial effluents.     

Differential decolorization of textile dyes in mixtures and the joint effect of laccase and cellobiose dehydrogenase activities present in extracellular extracts from Funalia trogii

The largest part of the bio-decolorization investigations have been performed to date on a single dye without exploring the behavior in complex mixtures as the real dyeing baths. Therefore, mixtures of dyes belonging to azo and anthraquinonic classes, chosen among the most utilized in textile wool dyeing, were employed for comparative enzymatic decolorization studies using the extracellular extracts from the white rot fungus Funalia trogii, to understand how the concomitant presence of more than one dye could influence their degradation course and yield.Fungal extracts containing laccase activity only were capable to partially decolorize dyes mixtures from the different classes analyzed. The deconvolution of the decolorization with time allowed to monitor the degradation of the single dyes in the mixtures evidencing a time dependent differential decolorization not observed for the singles alone. Some dyes in the blend were in fact decolorized only when the most easily converted dyes were largely transformed. These experiments would allow to help the dyeing factories in the selection of the most readily degraded dyes.Since F. trogii grown on different media and activators shows diverse levels of expression of the redox enzymes laccase and cellobiose dehydrogenase (CDH), the dyes mixtures recalcitrant to decolorization by laccase activity alone, were subjected to the combined action of extracts containing laccase and CDH. The use of CDH, in support to the activity of laccase, resulted in substantial decolorization increases (>84%) for all the refractory dyes mixtures.     

Decolorization and degradation mechanism of Amaranth by Polyporus sp. S133

Polyporus sp. S133 decolorized the Amaranth in 72 h (30 mg L^?1) under static and shaking conditions. Liquid medium containing glucose has shown the highest decolorization of Amaranth by Polyporus sp. S133. When the effect of increasing inoculum concentration on decolorization of Amaranth was studied, maximum decolorization was observed with 15 % inoculum concentration. Significant increase in the enzyme production of laccase (102.2 U L^?1) was observed over the period of Amaranth decolorization compared to lignin peroxidase and manganese peroxidase. Germination rate of Sorghum vulgare and Triticum aestivum was less with Amaranth treatment as compared to metabolites obtained after its decolorization. Based on the metabolites detected by GC–MS, it was proposed that Amaranth was bio-transformed into two intermediates, 1-hydroxy-2-naphthoic acid and 1,4-naphthaquinone. Overall findings suggested the ability of Polyporus sp. S133 for the decolorization of azo dye and ensured the ecofriendly degradation of Amaranth.     

Decolorization of azo, triphenylmethane and anthraquinone dyes by a newly isolated Trametes sp. SQ01 and its laccase

A white-rot fungus, strain SQ01, was isolated from decayed wood in a temperate forest. The strain was identified as a member of genus Trametes, based on the morphological characteristics and a complete sequence analysis of its 18S rRNA gene and ITS region. Strain SQ01 was capable of decolorizing a variety of synthetic dyes, including azo, triphenylmethane, and anthraquinone dyes, with an optimal efficiency of decolorization obtained when dyes added after 5 days of culture, with the exception of Cresol Red, showing that the point of dye addition was an important influencing factor for decolorization by this fungus. All of the tested dyes were decolorized by the purified laccase in the absence of any redox mediators, but only a few were completely removed, while others were not completely degraded even with increased decolorization time.     

Laccase induction in fungi and laccase/N-OH mediator systems applied in paper mill effluent

There has been increasing interest in extracellular enzymes from white rot fungi, such as lignin and manganese peroxidases, and laccases, due to their potential to degrade both highly toxic phenolic compounds and lignin. The optimum cultivation conditions for laccase production in semi-solid and liquid medium by Trametes versicolor, Trametes villosa, Lentinula edodes and Botrytis cinerea and the effects of laccase mediator system in E1 effluent were studied. The higher laccase activity (12756 U) was obtained in a liquid culture of T. versicolor in the presence of 1 mM of 2,5-xylidine and 0.4 mM copper salt as inducers. The effluent biotreatments were not efficient in decolorization with any fungal laccases studied. Maximum phenol reduction was approximately 23% in the absence of mediators from T. versicolor. The presence of 1-hydroxybenzotriazole did not increase phenol reduction. However, acetohydroxamic acid, which was not degraded by laccase, acted very efficiently on E1 effluent, reducing 70% and 73% of the total phenol and total organic carbon, respectively. Therefore, acetohydroxamic acid could be applied as a mediator for laccase bioremediation in E1 effluent.     

Overexpression of a Novel Thermostable and Chloride-Tolerant Laccase from Thermus thermophilus SG0.5JP17-16 in Pichia pastoris and Its Application in Synthetic Dye Decolorization

Laccases have been used for the decolorization and detoxification of synthetic dyes due to their ability to oxidize a wide variety of dyes with water as the sole byproduct. A putative laccase gene (LacTT) from Thermus thermophilus SG0.5JP17-16 was screened using the genome mining approach, and it was highly expressed in Pichia pastoris, yielding a high laccase activity of 6130 U/L in a 10-L fermentor. The LacTT open reading frame encoded a protein of 466 amino acid residues with four putative Cu-binding regions. The optimal pH of the recombinant LacTT was 4.5, 6.0, 7.5 and 8.0 with 2,2''-azino-bis(3-ethylbenzothazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ), guaiacol, and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The optimal temperature of LacTT was 90°C with guaiacol as the substrate. LacTT was highly stable at pH 4.0–11.0 and thermostable at 40°C–90°C, confirming that it is a pH-stable and thermostable laccase. Furthermore, LacTT also exhibited high tolerance to halides such as NaCl, NaBr and NaF, and decolorized 100%, 94%, 94% and 73% of Congo Red, Reactive Black B and Reactive Black WNN, and Remazol Brilliant Blue R, respectively. Interestingly, addition of high concentration of NaCl increased the RBBR decolorization efficiency of LacTT. These results suggest that LacTT is a good candidate for industrial applications such as dyestuff processing and degradation of dyes in textile wastewaters.     

Decolorization of textile dyes by whole cultures of Ischnoderma resinosum and by purified laccase and Mn-peroxidase

Ischnoderma resinosum produced extracellular ligninolytic enzymes laccase and MnP. The activity of laccase achieved the maximum on day 10 (29.4 U L⁻¹), the MnP on day 14 (34.5 U L⁻¹). Laccase and Mn-peroxidase were purified from the culture liquid using gel permeation and ion-exchange chromatographies. Purified Mn-peroxidase performed decolorization of all textile dyes tested (Reactive Black 5, Reactive Blue 19, Reactive Red 22 and Reactive Yellow 15). Laccase was inactive with Reactive Black 5 and Reactive Red 22, while all dyes were decolorized after addition of the redox mediators violuric acid (VA) and hydroxybenzotriazole (HBT). The culture liquid from I. resinosum cultures was also able to decolorize all dyes as well as the synthetic dyebaths in the presence of VA and HBT. The highest decolorization rates were detected in acidic pH (3–4).     

Decolorization of Azo, Triphenyl Methane, Heterocyclic, and Polymeric Dyes by Lignin Peroxidase Isoenzymes from Phanerochaete chrysosporium

The ligninolytic enzyme system of Phanerochaete chrysosporium decolorizes several recalcitrant dyes. Three isolated lignin peroxidase isoenzymes (LiP 4.65, LiP 4.15, and LiP 3.85) were compared as decolorizers with the crude enzyme system from the culture medium. LiP 4.65 (H2), LiP 4.15 (H7), and LiP 3.85 (H8) were purified by chromatofocusing, and their kinetic parameters were found to be similar. Ten different types of dyes, including azo, triphenyl methane, heterocyclic, and polymeric dyes, were treated by the crude enzyme preparation. Most of the dyes lost over 75% of their color; only Congo red, Poly R-478, and Poly T-128 were decolorized less than the others, 54, 46, and 48%, respectively. Five different dyes were tested for decolorization by the three purified isoenzymes. The ability of the isoenzymes to decolorize the dyes in the presence of veratryl alcohol was generally comparable to that of the crude enzyme preparation, suggesting that lignin peroxidase plays a major role in the decolorization and that manganese peroxidase is not required to start the degradation of these dyes. In the absence of veratryl alcohol, the decolorization activity of the isoenzymes was in most cases dramatically reduced. However, LiP 3.85 was still able to decolorize 20% of methylene blue and methyl orange and as much as 60% of toluidine blue O, suggesting that at least some dyes can function as substrates for isoenzyme LiP 3.85 but not to the same extent for LiP 4.15 or LiP 4.65. Thus, the isoenzymes have different specificities towards dyes as substrates.