Malachite green decolourization and detoxification by the laccase from a newly isolated strain of Trametes sp.

The decolourization and detoxification of the triarylmethane dye Malachite green (MG) by laccase from Trametes sp. were investigated. The laccase decolorized efficiently the dye down to 97% of 50 mg L^?1 initial concentration of MG when only 0.1 U mL^?1 of laccase was used in the reaction mixture. The effects of different physicochemical parameters were tested and optimal decolourization rates occurred at pH 6 and at temperatures between 50 and 60 °C. Decolourization of MG occurred in the presence of metal ions which could be found in textile industry effluent. 1-hydroxybenzotriazole (HBT) affected positively the decolourization of MG. The presence of some phenolic compounds namely ferulic, coumaric, gallic, and tannic acids was found to be inhibiting for the decolourization at a concentration of 10 mM.The effect of laccase inhibitors in the decolourization of MG was tested with l-cysteine, and ethylene diamine tetra-acetic acid (EDTA) at concentrations of 0.1, 1 and 10 mM. It was demonstrated that l-cysteine and EDTA inhibited the decolourization starting from 1 mM concentration. However, for NaCl a concentration of 100 mM was needed for the inhibition of laccase. The decolourization of MG resulted in the removal of its toxicity against Phanerochaete chrysosporium.The stability of the laccase toward temperature and HBT free radicals was also assessed during MG decolourization. It was shown that laccase was stable at 50 °C but in the presence of the laccase mediator HBT, the stability of the enzyme was severely affected resulting in a loss of 50% of the activity after 3 h incubation.     

Bio-remediation of colored industrial wastewaters by the white-rot fungi Phanerochaete chrysosporium and Pleurotus ostreatus and their enzymes

The effect of Phanerochaete chrysosporium and Pleurotus ostreatus whole cells and their ligninolytic enzymes on models of colored industrial wastewaters was evaluated. Models of acid, direct and reactive dye wastewaters from textile industry have been defined on the basis of discharged amounts, economic relevance and representativeness of chemical structures of the contained dyes. Phanerochaete chrysosporium provided an effective decolourization of direct dye wastewater model, reaching about 45% decolourization in only 1 day of treatment, and about 90% decolourization within 7 days, whilst P. ostreatus was able to decolorize and detoxify acid dye wastewater model providing 40% decolourization in only 1 day, and 60% in 7 days. P. ostreatus growth conditions that induce laccase production (up to 130,000 U/l) were identified, and extra-cellular enzyme mixtures, with known laccase isoenzyme composition, were produced and used in wastewater models decolourization. The mixtures decolorized and detoxified the acid dye wastewater model, suggesting laccases as the main agents of wastewater decolourization by P. ostreatus. A laccase mixture was immobilized by entrapment in Cu-alginate beads, and the immobilized enzymes were shown to be effective in batch decolourization, even after 15 stepwise additions of dye for a total exposure of about 1 month.     

De-colourization of textile effluent using immobilized Geotrichum candidum: an insight into mycoremediation

Textile effluent is generally complicated to manage because of its extremely noxious and recalcitrant coloured compositions. Mycoremediation is an extensively used strategy for the competent degradation of hazardous pollutants present in textile effluent. Fungus could be immobilized in synthetic or natural matrices. The current study shows the decolourization of the textile effluent by 85·5 and 98·5% within 6 h using suspended and immobilized fungus, Geotrichum candidum with optimized parameters like inoculum size (5%), pH (4·5), and temperature (30°C). To maintain a high biomass of fungal population and enhance the retention of fungal strain in the contaminated sites, the fungi need to be immobilized. Hence, the fungus was immobilized naturally onto the selected inert support that is, coconut fibres by the means of adsorption, where they grew as active films on the fibres after being grown in the culture broth. The optimized process parameters of inoculum size, fibre quantity and agitation speed for immobilized G. candidum were 5%, 2·2 g l⁻¹ of effluent and 100 rev min⁻¹ respectively. High level of laccase (22 and 25 U l⁻¹ in suspended and immobilized fungal cells treatment respectively) was observed during the process of decolourization and it was found that decolourization was directly proportional to the laccase activity. The UV–vis, FTIR, ¹H NMR and GC-MS analyses of treated textile industrial wastewater revealed the degradation of toxic pollutants in the textile effluent and formation of lower molecular weight intermediates. The study revealed a higher efficacy of immobilized G. candidum in comparison to suspended fungal culture, employing ligninolytic enzyme laccase, which catalyzes the degradation/transformation of aromatic dyes in the textile effluent thus decolourizing it.     

Reactive blue 19 decolouration by laccase immobilized on silica beads

Laccase (31.5 U of activity/g or 4.39 μg of protein/m²) from Trametes versicolor was immobilized on controlled-porosity-carrier silica beads and evaluated for the decolouration of Reactive blue 19, an anthraquinone dye. Although there was an initial, rapid adsorption of the dye to the packed bed in a recirculating reactor, about 97.5% of Reactive blue 19 removal was due to enzymatic degradation. The free enzyme lost 52% of its activity in 48 h. However, the activity of the immobilized laccase was unchanged after 4 months of storage in phosphate buffer under ambient conditions followed by three successive decolourations over 120 h. Treating the laccase immobilized beads with ethanolamine reduced dye adsorption by 40%.     

Laccase immobilization on enzymatically functionalized polyamide 6,6 fibres

Polyamide matrices, such as membranes, gels and non-wovens, have been applied as supports for enzyme immobilization, although in literature the enzyme immobilization on woven nylon matrices is rarely reported. In this work, a protocol for a Trametes hirsuta laccase immobilization using woven polyamide 6,6 (nylon) was developed. A 2⁴ full factorial design was used to study the influence of pH, spacer (1,6-hexanediamine), enzyme and crosslinker concentration on the efficiency of immobilization. The factors enzyme dosage and spacer seem to have played a critical role in the immobilization of laccase onto nylon support. Under optimized working conditions (29 U mL⁻¹ of laccase, 10% of glutaraldehyde, pH = 5.5, with the presence of the spacer), the half-life time attained was about 78 h (18% higher than that of free enzyme), the protein retention was 30% and the immobilization yield was 2%. The immobilized laccase has potential for application in the continuous decolourization of textile effluents, where it can be applied into a membrane reactor.     

Immobilization of pycnoporus coccineus laccase on Eupergit C: Stabilization and treatment of olive oil mill wastewaters

The use of olive oil mill wastewaters (OMW) as an organic fertilizer is limited by their phytotoxic effect, due to the high concentration of phenolic compounds. As an alternative to physico-chemical methods for OMW detoxification, the laccase from Pycnoporus coccineus, a white-rot fungus with the ability to decrease the chemical oxygen demand (COD) and color of the industrial effluent, is being studied. In this work, the P. coccineus laccase was immobilized on two acrylic epoxy-activated resins, Eupergit C and Eupergit C 250L. The highest activity was obtained with the macroporous Eupergit C 250L, reaching 110 U g^?1 biocatalyst. A substantial stabilization effect against pH and temperature was obtained upon immobilization. The soluble enzyme maintained ≥80% of its initial activity after 24 h at pH 7.0–10.0, whereas the immobilized laccase kept the activity in the pH range 3.0–10.0. The free enzyme was quickly inactivated at temperatures >50°C, whereas the immobilized enzyme was very stable up to 70°C. Gel filtration profiles of the OMW treated with the immobilized enzyme (for 8 h at room temperature) showed both degradation and polymerization of the phenolic compounds.     

Decolourization of recalcitrant dyes with a laccase from Streptomyces coelicolor under alkaline conditions

Colored wastewater from textile industries is a consequence of dye manufacturing processes. Two percent of dyes that are produced are discharged directly in aqueous effluent and more than 10% are subsequently lost during the textile coloration process. It is not surprising that these compounds have become a major environmental concern. In that context, we have evaluated the potential use of Streptomyces coelicolor laccase for decolourization of various dyes with and without a mediator. Results showed that in all cases the combination of laccase and the mediator acetosyringone was able to rapidly decolourize, to various degrees, all the dyes tested. In 10 min, decolourization was achieved at 94% for acid blue 74, 91% for direct sky blue 6b and 65% for reactive black 5. Furthermore, decolourization was achieved at 21% for reactive blue 19 and at 39% for the direct dye Congo red in 60 min. These results demonstrate the potential use of this laccase in combination with acetosyringone, a natural mediator, for dye decolourization.     

Decolourization of azo dyes and a dye industry effluent by a white rot fungus Thelephora sp

A white rot fungus Thelephora sp. was used for decolourization of azo dyes such as orange G (50 microM), congo red (50 microM), and amido black 10B (25 microM). Decolourization using the fungus was 33.3%, 97.1% and 98.8% for orange G, congo red and amido black 10B, respectively. An enzymatic dye decolourization study showed that a maximum of 19% orange G was removed by laccase at 15 U/ml whereas lignin peroxidase (LiP) and manganese dependent peroxidase (MnP) at the same concentration decolourized 13.5% and 10.8%, orange G, respectively. A maximum decolourization of 12.0% and 15.0% for congo red and amido black 10B, respectively, was recorded by laccase. A dye industry effluent was treated by the fungus in batch and continuous modes. A maximum decolourization of 61% was achieved on the third day in the batch mode and a maximum decolourization of 50% was obtained by the seventh day in the continuous mode. These results suggest that the batch mode of treatment using Thelephora sp. may be more effective than the continuous mode for colour removal from dye industry effluents.     

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 (IC(50)) 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 (DeltaE*) below 1.1 were measured for most dyes.     

Biodegradation of Acid Yellow Using Laccase Produced by Bacillus sp. Strain TR and its In-Silico Modeling of the Dye Degradation System

The decolourization of the azo dye (acid yellow) by laccase from Bacillus sp. strain TR under submerged fermentation (SmF) was optimized by response surface methodology (RSM). The laccase maximum yield was achieved at 96 h of SmF with pH 7.0, 1.0 g/L of maltose and 3.0 g/L of ammonium acetate at 37 °C. The enzyme yield was estimated that 570 U/mL. About 76.4% of acid yellow decolourization efficiency was observed by the laccase enzyme within 96 h. The substrate surface changes were observed before and after the laccase treatment was analyzed with the scanning electron microscope (SEM) and N?=?N transformation either nitrogen or ammonia was showed by Fourier transform infrared spectroscopy (FT-IR) analysis. The HPLC analysis explained the formation of various intermediates in the conversion of acid yellow to final products. Further, in silico studies proved the enzyme–substrate interactions and showed a better score of ??27.435 kJ/mol.

     

Green coconut fiber: a novel carrier for the immobilization of commercial laccase by covalent attachment for textile dyes decolourization

Commercial laccase formulation was immobilized on modified green coconut fiber silanized with 3-glycidoxypropyltrimethoxysilane, aiming to achieve a cheap and effective biocatalyst. Two different strategies were followed: one point (pH 7.0) and multipoint (pH 10.0) covalent attachment. The influence of immobilization time on enzymatic activity and the final reduction with sodium borohydride were evaluated. The highest activities were achieved after 2?h of contact time in all situations. Commercial laccase immobilized at pH 7.0 was found to have higher activity and higher affinity to the substrate. However, the immobilization by multipoint covalent attachment improved the biocatalyst thermal stability at 50?°C, when compared to soluble enzyme and to the immobilized enzyme at pH 7.0. The Schiff's bases reduction by sodium borohydride, in spite of causing a decrease in enzyme activity, showed to contribute to the increase of operational stability through bonds stabilization. Finally, these immobilized enzymes showed high efficiency in the continuous decolourization of reactive textile dyes. In the first cycle, the decolourization is mainly due to dyes adsorption on the support. However, when working in successive cycles, the adsorption capacity of the support decreases (saturation) and the enzymatic action increases, indicating the applicability of this biocatalyst for textile wastewater treatment.     

Laccase immobilization on enzymatically functionalized polyamide 6,6 fibres

Polyamide matrices, such as membranes, gels and non-wovens, have been applied as supports for enzyme immobilization, although in literature the enzyme immobilization on woven nylon matrices is rarely reported. In this work, a protocol for a Trametes hirsuta laccase immobilization using woven polyamide 6,6 (nylon) was developed. A 2⁴ full factorial design was used to study the influence of pH, spacer (1,6-hexanediamine), enzyme and crosslinker concentration on the efficiency of immobilization. The factors enzyme dosage and spacer seem to have played a critical role in the immobilization of laccase onto nylon support. Under optimized working conditions (29 U mL⁻¹ of laccase, 10% of glutaraldehyde, pH = 5.5, with the presence of the spacer), the half-life time attained was about 78 h (18% higher than that of free enzyme), the protein retention was 30% and the immobilization yield was 2%. The immobilized laccase has potential for application in the continuous decolourization of textile effluents, where it can be applied into a membrane reactor.     

Immobilisation of laccase on Eupergit supports and its application for the removal of endocrine disrupting chemicals in a packed-bed reactor

Laccase from Myceliophthora thermophila was covalently immobilised on Eupergit C and Eupergit C 250L yielding specific activities of up to 17 and 80 U/g, respectively. Due to its superior activity, Eupergit C 250L was chosen for further research. The somewhat lower catalytic efficiency (based on the ratio between the turnover number and the Michaelis constant, k_cat/K_M) of the immobilised enzyme in comparison with that of the free enzyme was balanced by its increased stability and broader operational window related to temperature and pH. The feasibility of the immobilised laccase was tested by using a packed bed reactor (PBR) operating in consecutive cycles for the removal of Acid Green 27 dye as model substrate. High degrees of elimination were achieved (88, 79, 69 and 57% in 4 consecutive cycles), while the levels of adsorption on the support varied from 18 to 6%, proving that dye removal took place mainly due to the action of the enzyme. Finally, a continuous PBR with the solid biocatalyst was applied for the treatment of a solution containing the following endocrine disrupting chemicals: estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2). At steady-state operation, E1 was degraded by 65% and E2 and EE2 were removed up to 80% and only limited adsorption of these compounds on the support, between 12 and 22%, was detected. In addition, a 79% decrease in estrogenic activity was detected in the effluent of the enzymatic reactor while only 14% was attained by inactivated laccase.     

Immobilization of Laccase by Alginate–Chitosan Microcapsules and its Use in Dye Decolorization

Laccase is a ligninolytic enzyme that is widespread in white-rot fungi. Alginate–chitosan microcapsules prepared by an emulsification–internal gelation technique were used to immobilize laccase. Parameters of the immobilization process were optimized. Under the optimal immobilization conditions (2% sodium alginate, 2% CaCl₂, 0.3% chitosan and 1:8 ratio by volume of enzyme to alginate), the loading efficiency and immobilized yield of immobilized laccase were 88.12% and 46.93%, respectively. Laccase stability was increased after immobilization. Both the free and immobilized laccase alone showed a very low decolorization efficiency when Alizarin Red was selected for dye decolorization test. When 0.1 mM 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was added into the decolorization system, the decolorization efficiency increased significantly. Immobilized laccase retained 35.73% activity after three reaction cycles. The result demonstrated that immobilized laccase has potential application in dyestuff treatment.     

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.     

Laccase-mediated Remazol Brilliant Blue R decolorization in a fixed-bed bioreactor

A crude laccase mixture preparation from Pleurotus ostreatus cultures supplemented with copper and ferulic acid was used to decolorize the anthraquinonic dye Remazol Brilliant Blue R (RBBR). Performance of this enzymatic system was tested, and a maximum of 70% decolorization was achievable under optimal conditions. The crude preparation was immobilized by entrapment in copper alginate beads attaining 65% yield of laccase activity. Stability of the immobilized laccases was remarkably increased in comparison with that of the free enzyme preparation. Efficiency of the immobilized system was evaluated during stepwise dye additions in batch operations. Under the best conditions, 70% RBBR decolorization was achieved even after 20 cycles, although decolorization time exponentially increased after the 10th cycle. Different fixed-bed bioreactors were prepared and analyzed in continuous decolorization processes. The best performance was obtained by decreasing the amount of enzyme loaded and by improving laccase retention using chitosan-coated alginate beads.     

Decolourization of Amaranth dye by bacterial biofilm in batch and continuous packed bed bioreactor

The aim of this study was to exploit the bacterial biofilms to remove dyes from industrial effluents. Biofilms of strains AK1, AK2, VKY1 and a consortium on sheep bone chips were examined in batch, repeated batch and continuous packed bed bioreactor. Biofilms are more efficient for decolourization of Amaranth dye at three different dye concentrations (200, 400, and 600 mg l⁻¹). 100% decolourization of Amaranth dye was observed even at higher concentrations (400 and 600 mg l⁻¹) by all the tested biofilms in 24 h than that of corresponding free cells. The biofilms were superior over those of free cells and could be reused for more than 18 repeated cycles. In a packed bed bioreactor, biofilms could be operated with much higher dilution rates and at lower hydraulic retention time. Further, the decolourization of dye was confirmed by UV–visible spectrophotometer, TLC and HPLC analysis of Amaranth dye degradation products from packed bed bioreactor effluent.     

Biodegradation of disperse textile dye Brown 3REL by newly isolated Bacillus sp. VUS

Aims:  To isolate the potential micro-organism for the degradation of textile disperse dye Brown 3 REL and to find out the reaction mechanism.
Methods and Results:  16S rDNA analysis revealed an isolate from textile effluent contaminated soil as Bacillus sp. VUS and was able to degrade (100%) dye Brown 3REL within 8 h at static anoxic condition. A significant increase in the activities of lignin peroxidase, laccase and NADH-DCIP reductase was observed up to complete decolourization of Brown 3REL. The optimum temperature required for degradation was 40°C and pH 6·5–12·0. Phyto-toxicity and chemical oxygen demand revealed nontoxic products of dye degradation. The biodegradation was monitored by UV–VIS, FTIR spectroscopy and HPLC. The final products 6,8-dichloro-quinazoline-4-ol and cyclopentanone were characterized by gas chromatography-mass spectrometry. This Bacillus sp. VUS also decolourized (80%) textile dye effluent within 12 h.
Conclusions:  This study suggests that Bacillus sp. VUS could be a useful tool for textile effluent treatment.
Significance and Impact of the Study:  The newly isolated Bacillus sp. VUS decolourized 16 textile dyes and textile dye effluent also. It achieved complete biodegradation of Brown 3REL. Phytotoxicity study demonstrated no toxicity of the biodegraded products for plants with respect to Triticum aestivum and Sorghum bicolor .     

Production and synthetic dyes decolourization capacity of a recombinant laccase from Pichia pastoris

Aims:  To produce and purify a recombinant laccase from Pichia pastoris and to test its ability in decolourization of synthetic dyes.
Methods and Results:  A cDNA encoding for a laccase was isolated from Pycnoporus sanguineus and was expressed in P. pastoris strain SMD1168H under the control of the alcohol oxidase (AOX1) promoter. The laccase native signal peptide efficiently directed the secretion of the recombinant laccase in an active form. Factors influencing laccase expression, such as cultivation temperature, pH, copper concentration and methanol concentration, were investigated. The recombinant enzyme was purified to electrophoretic homogeneity, and was estimated to have a molecular mass of about 62·8 kDa. The purified enzyme showed a similar behaviour to the native laccase produced by P. sanguineus . Four different synthetic dyes including azo, anthraquinone, triphenylmethane and indigo dyes could be efficiently decolourized by the purified recombinant laccase without the addition of redox mediators.
Conclusions:  Heterologous production of P. sanguineus laccase in P. pastoris was successfully achieved. The purified recombinant laccase could efficiently decolourize synthetic dyes in the absence of mediators.
Significance and Impact of the Study:  This study is the first report on the synthetic dye decolourization by the recombinant P. sanguineus laccase. The decolourization capacity of this recombinant enzyme suggested that it could be a useful biocatalyst for the treatment of dye-containing effluents.     

Enzymatic extract containing lignin peroxidase immobilized on carbon nanotubes: Potential biocatalyst in dye decolourization

The majority of the textile dyes are harmful to the environment and potentially carcinogenic. Among strategies for their exclusion, the treatment of dye contaminated wastewater with fungal extract, containing lignin peroxidase (LiP), may be useful. Two fungi isolates, Pleurotus ostreatus (PLO9) and Ganoderma lucidum (GRM117), produced the enzymatic extract by fermentation in the lignocellulosic residue, Jatropha curcas seed cake. The extracts from PLO9 and GRM117 were immobilized on carbon nanotubes and showed an increase of 18 and 27-fold of LiP specific activity compared to the free enzyme. Also, LiP from both fungi extracts showed higher Vmax and lower Km values. Only the immobilized extracts could be efficiently reused in the dye decolourization, contrary, the carbon nanotubes became saturated and they should be discarded over time. This device may offer a final biocatalyst with higher catalytic efficiency and capability to be reused in the dye decolourization process.