Bioremediation of Textile Azo Dyes by Trichophyton rubrum LSK-27

Summary The potential of a recently isolated wood-degrading fungus, Trichophyton rubrum LSK-27, for effective decolorization of textile azo dyes was evaluated. Within two days of dye addition, the fungus was able to decolorize 83% of Remazol Tiefschwarz, 86% of Remazol Blue RR and 80% of Supranol Turquoise GGL in liquid cultures. The reactive dyes, Remazol Tiefschwarz and Remazol Blue, were removed by fungal biodegradation, while decolorization of the acid dye, Supranol Turquoise GGL, was accomplished mainly by bioadsorption. Therefore the fungus proved to be efficiently capable of both biodegradation and biosorption as the major dye removal mechanisms. The extent of biodegradation was associated with the levels of the extracellular ligninolytic enzymes such as manganese peroxidase and laccase.     

Enhanced ligninolytic enzyme production and degrading capability of Phanerochaete chrysosporium and Trametes versicolor

Ligninolytic enzyme production by the white-rot fungi Phanerochaete chrysosporium and Trametes versicolor precultivated with different insoluble lignocellulosic materials (grape seeds, barley bran and wood shavings) was investigated. Cultures of Phanerochaete chrysosporium precultivated with grape seeds and barley bran showed maximum lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) activities (1000 and 1232 U/l, respectively). Trametes versicolor precultivated with the same lignocellulosic residues showed the maximum laccase activity (around 250 U/l). For both fungi, the ligninolytic activities were about two-fold higher than those attained in the control cultures. In vitro decolorization of the polymeric dye Poly R-478 by the extracellular liquid obtained in the above-mentioned cultures was monitored in order to determine the respective capabilities of laccase, LiP and MnP. It is noteworthy that the degrading capability of LiP when P. chrysosporium was precultivated with barley bran gave a percentage of Poly R-478 decolorization of about 80% in 100 s, whereas control cultures showed a lower percentage, around 20%, after 2 min of the decolorization reaction.     

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.     

Biodegradation of diazo reactive dye Navy blue HE2R (Reactive blue 172) by an isolated <Emphasis Type="Italic">Exiguobacterium</Emphasis> sp. RD3

The diazo reactive dye Navy blue HE2R (50 mg/L) was decolorized up to 91.2% within 48 h at static condition by the Exiguobacterium sp. isolated from the dyestuff contaminated soil, collected from the textile industrial area Solapur, India. It showed ability to decolorize seven different reactive textile dyes. Maximum decolorization was observed at 30°C and pH 7. The presence and significant increase in the activity of enzymes lignin peroxidase, laccase, and azoreductase indicated prominent role of these enzymes in the decolorization of Navy blue HE2R. The degradation metabolites were analyzed by UV-Vis spectroscopy, TLC, HPLC, and FTIR spectroscopy. A possible pathway for biodegradation of this diazo reactive dye was proposed with the help of GC-MS analysis. The phytotoxicity studies confirmed the environmentally safe nature of degradation products.     

Enhanced transformation of malachite green by laccase of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> in the presence of natural phenolic compounds

In this study, we investigated the efficacy of phenolic extract of wheat bran and lignin-related phenolic compounds as natural redox mediators on laccase-mediated transformation of malachite green (MG) using purified laccase from the white-rot fungus Ganoderma lucidum. G. lucidum laccase was able to decolorize 40.7% MG dye (at 25 mg l⁻¹) after 24 h of incubation. Whereas, the addition of phenolic extract of wheat bran enhanced the decolorization significantly (p < 0.001) by two- to threefold than that of purified laccase alone. Among various natural phenolic compounds, acetovanillone, p-coumaric acid, ferulic acid, syringaldehyde, and vanillin were the most efficient mediators, as effective as the synthetic mediator 1-hydroxybenzotriazole. Characterization of MG transformation products by HPLC, UV–Vis, and liquid chromatography-mass spectrometry-electrospray ionization analysis revealed that N-demethylation was the key mechanism of decolorization of MG by laccase. Growth inhibition test based on mycelial growth inhibition of white rot fungus Phanerochaete chrysosporium revealed that treatment with laccase plus natural mediators effectively reduced the growth inhibitory levels of MG than that of untreated one. Among all the tested compounds, syringaldehyde showed the highest enhanced decolorization, as a consequence reduced growth inhibition was observed in syringaldehyde-treated samples. The results of the present study revealed that the natural phenolic compounds could alternatively be used as potential redox mediators for effective laccase-mediated decolorization of MG.     

Degradation of dye-containing textile effluent by the agaric white-rot fungus Clitocybula dusenii

Raw mixed-dye wastewater from a textile dye-producing plant was partly decolorized by the agaric white-rot fungus, Clitocybula dusenii. The fungus had higher Mn peroxidase (MnP) and laccase activities when grown with dye effluent than in control cultures. The activity of MnP increased commensurately with the proportion of the raw dye wastewater in the medium (control: 20 U l^–1; 10% v/v effluent: 67 U l^–1; 25% v/v effluent: 130 U l^–1; and 33% v/v effluent: 180 U l^–1). Maximal decolorization rates were achieved over 20 d at 28 °C using four-fold diluted dye-containing effluent on a 5 d pre-grown mycelium.     

Decolorizing Reactive Textile Dyes with White‐Rot Fungi by Temporary Immersion Cultivation

The decontamination of effluents from textile industries is problematic due to the fact that textile dyes are resistant to degradation in the environment. Enzymes from white rot fungi, especially laccase, are able to degrade various complex aromatic structures, and are therefore able to decolorize textile dyes. The white‐rot fungi Trametes versicolor and Phanerochaete chrysosporium were immobilized, separately, on both pine wood chips and palm oil fiber, and cultivated in the temporary immersion RITA® (Récipient à Immersion Temporaire Automatique) System, which was adapted to serve as a fungal bioreactor in a series of four experiments to determine optimal conditions for decolorizing the textile dyes Levafix Blue and Remazol Brilliant Red. The maximum rate of decolorization of both dyes occurred within 24 h of incubation, and laccase was detected in the system.     

Implication of <Emphasis Type="Italic">Dichomitus squalens</Emphasis> manganese-dependent peroxidase in dye decolorization and cooperation of the enzyme with laccase

Three new chromatographic forms of Dichomitus squalens manganese-dependent peroxidase (MnP) were isolated from wheat-straw cultures using Mono Q and connective interaction media (CIM) fast protein liquid chromatography. Enzymes revealed identical molar mass of 50 kDa (estimated by SDS-PAGE) and pI values of 3.5, however, they varied in Km values obtained for Mn2+ oxidation. The addition of wood and straw methanol extracts to the cultures showed that the production of MnPs in wheat-straw cultures was influenced rather by the type of cultivation than by phenolic compounds from lignocellulosic material which induced laccase production. The purified CIM1 MnP was able to decolorize selected azo and anthraquinone dyes more rapidly than laccase Lc1. In vitro dye decolorization showed a synergistic cooperation of MnP and laccase. In the case of CSB degradation MnP prevented from the production of a differently colored substance that could be produced after CSB degradation by laccase-HBT system.     

Optimization of a culture medium for ligninolytic enzyme production and synthetic dye decolorization using response surface methodology

A Box-Wilson central composite design was applied to optimize copper, veratryl alcohol and l-asparagine concentrations for Trametes trogii (BAFC 212) ligninolytic enzyme production in submerged fermentation. Decolorization of different dyes (xylidine, malachite green, and anthraquinone blue) by the ligninolytic fluids from the cultures was compared. The addition of copper stimulated laccase and glyoxal oxidase production, but this response was influenced by the medium N-concentration, with improvement higher at low N-levels. The medium that supported the highest ligninolytic production (22.75 U/ml laccase, 0.34 U/ml manganese peroxidase, and 0.20 U/ml glyoxal oxidase) also showed the greatest ability to decolorize the dyes. Only glyoxal oxidase activity limited biodecoloration efficiency, suggesting the involvement of peroxidases in the process. The addition of 1-hydroxybenzotriazole (a known laccase mediator) to the ligninolytic fluids increased both their range and rate of decolorization. The cell-free supernatant did not decolorize xylidine, poly R-478, azure B, and malachite green as efficiently as the whole broth, but results were similar in the case of indigo carmine and remazol brilliant blue R. This indicates that the mycelial biomass may supply other intracellular or mycelial-bound enzymes, or factors necessary for the catalytic cycle of the enzymes. It also implies that this fungus implements different strategies to degrade dyes with diverse chemical structures.     

Color removal ability of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in relation to lignin peroxidase and manganese peroxidase produced in molasses wastewater

Decolorization of molasses wastewater (MWW) from an ethanolic fermentation plant by Phanerochaete chrysosporium was studied. By diluting MWW properly (10%v/v) and incubating it with an appropriate concentration of the spores (2.5 × 10⁶/ml), extensive decolorization occurred (75%) on day 5 of the incubation. The colour removal ability was found to be correlated to the activity of ligninolytic enzyme system: lignin peroxidase (LiP) activity was 185 U/l while manganese peroxidase (MnP) activity equaled 25 U/l. Effects of some selected operating variables were studied: manganese(II), veratryl alcohol (VA), glucose as a carbon source and urea and ammonium nitrate, each as a source of nitrogen. Results showed that the colour reduction and LiP activity were highest (76% and 186 U/l, respectively) either when no Mn(II) was added or added at the lowest level tested (0.16 mg/l to provide 0.3 mg/l). Activity of MnP was highest (25 U/l) when Mn(II) added to the diluted MWW at the highest level (100 ppm) while activity of LiP was lowest (7.1 U/l) at this level of added Mn(II). The colour reduction in the presence of the added VA was shown to be little less than in its absence (70 vs. 75%). When urea as an organic source of nitrogen for the fungus, was added to the MWW, the decolorizing activity of P. chrysosporium decreased significantly (15 vs. 75%) and no activities were detected for LiP and MnP. Use of ammonium nitrate as an inorganic source of nitrogen did not show such a decelerating effects, although no improvements in the metabolic behavior of the fungus (i.e., LiP and MnP activities) deaccelerating was observed. Effects of addition of glucose was also discussed.     

Fungal laccase, cellobiose dehydrogenase, and chemical mediators: combined actions for the decolorization of different classes of textile dyes

Dyes belonging to the mono-, di-, tri- and poly-azo as well as anthraquinonic and mono-azo Cr-complexed classes, chosen among the most utilized in textile applications, were employed for a comparative enzymatic decolorization study using the extracellular crude culture extracts from the white rot fungus Funalia (Trametes) trogii grown on different culture media and activators able to trigger different levels of expression of oxidizing enzymes: laccase and cellobiose dehydrogenase. Laccase containing extracts were capable to decolorize some dyes from all the different classes analyzed, whereas the recalcitrant dyes were subjected to the combined action of laccase and the chemical mediator HBT, or laccase plus cellobiose dehydrogenase. Correlations among the decolorization degree of the various dyes and their electronic and structural diversities were rationalized and discussed. The utilization of cellobiose dehydrogenase in support to the activity of laccase for the decolorization of azo textile dyes resulted in substantial increases in decolorization for all the refractory dyes proving to be a valid alternative to more expensive and less environmentally friendly chemical treatments of textile dyes wastes.     

Use of bitter gourd (Momordica charantia) peroxidase together with redox mediators to decolorize disperse dyes

In this study, salt fractionated bitter gourd (Momordica charantia) peroxidase was used for the decolorization of water-insoluble disperse dyes; Disperse Red 17 and Disperse Brown 1. Effect of nine different redox mediators; bromophenol, 2,4-dichlorophenol, guaiacol, 1-hydroxybenzotriazole, m-cresol, quinol, syringaldehyde, violuric acid, and vanillin on decolorization of disperse dyes by bitter gourd peroxidase has been investigated. Among these redox mediators, 1-hydroxybenzotriazole was the most effective mediator for decolorization of both the dyes by peroxidase. Bitter gourd peroxidase (0.36 U/mL) could decolorize Disperse Red 17 maximally 90% in the presence of 0.1 mM 1-hydroxybenzotriazole while Disperse Brown 1 was decolorized 65% in the presence of 0.2 mM 1-hydroxybenzotriazole. Maximum decolorization of these dyes was obtained within 1 h of incubation at pH 3.0 and temperature 40°C. The application of such enzyme plus redox mediator systems may be extendable to other recalcitrant and water insoluble synthetic dyes using novel redox mediators and peroxidases from other new and cheaper sources.     

Decolorization of Textile Dyes and Degradation of Mono-Azo Dye Amaranth by <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis> NCIM 2890

Acinetobacter calcoaceticus NCIM 2890 (A. caloaceticus) was found to decolorize 20 different textile dyes of various classes. Decolorization of an azo dye amaranth was observed effectively (91%) at static anoxic condition, whereas agitated culture grew well but showed less decolorization (68%) within 48 h of incubation. Induction of intracellular and extracellular lignin peroxidase, intracellular laccase, dichlorophenol indophenol (DCIP) reductase and riboflavin reductase represented their involvement in the biodegradation of amaranth. The products obtained after degradation of Amaranth were characterized as naphthalene sulfamide, hydroxyl naphthalene diazonium and naphthalene diazonium. The germination and growth of Sorghum vulgare and Phaseolus mungo seeds, and the growth of E. coli and Bacillus substilis were not inhibited by the metabolic products of the dye.     

Effect of various synthetic dyes on the production of manganese-dependent peroxidase isoenzymes by immobilized <Emphasis Type="Italic">Irpex lacteus</Emphasis>

The effect of manganese and selected synthetic dyes on the production of manganese-dependent peroxidase (MnP) by Irpex lacteus immobilized on polyurethane foam was studied. In the cultures grown in a medium containing 65 μM Mn (II), up to three various isoenzymes of MnP were resolved by isolectrofocusing, with pI values within the range of 3.50–6.04. In the cultures grown in a medium containing 2.9 mM Mn (II), two new MnP isoforms (pI 3.28, 3.75) were produced. The addition of structurally different synthetic dyes, an azo dye Reactive Orange 16 (RO16), an anthraquinonic dye Remazol Brilliant Blue R (RBBR), and a triphenylmethane dye Bromophenol Blue (BPB), to the fungal cultures grown in the presence of high manganese inhibited the production of low pI MnP isoforms. However, in the presence of BPB a new MnP isoform with pI 5.67 was detected. BPB was found to induce MnP isoforms which are more effective in RBBR decolorization in vitro than the low pI isoforms present in the control cultures.     

Decolorization of textile dyes by enzymatic extract and submerged cultures of <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis>

Pleurotus sajor-caju PS2001 was screened in Petri dish plates to assess the dye-decolorizing ability of industrial textile dyes. P. sajor-caju PS2001 was also cultivated in solid-state fermentation containing sawdust of Pinus sp. and wheat bran to obtain the enzymatic extract, showing laccase and manganese-peroxidase activity, which was used to test the capacity to degrade the textile dyes. Additional tests of decolorization were performed in liquid cultures. Anthraquinone-type textile dyes proved to be substrates for the enzymatic system of P. sajor-caju PS2001. Cultures in Petri dish plates showed that the anthraquinone dye Reactive Blue 220 can act as a redox mediator for the enzymatic reactions involved in the decolorization process, and enables the azo dye degradation. Reactive Blue 220 and Acid Blue 280 were completely decolorized in 30 min and 60 min, respectively, during the tests with precipitated enzymatic extract, while the azo dyes showed resistance to degradation. Additionally, in submerged cultures with dyes, veratryl alcohol oxidases and lignin peroxidase activities were observed. These results suggest that the strain P. sajor-caju PS2001 has great potential for use in the bioremediation technology of recalcitrant pollutant such as textile effluents.     

The Effect of Fungal Morphology on Ligninolytic Enzyme Production by a Recently Isolated Wood-Degrading Fungus Trichophyton Rubrum LSK-27

The morphology and ligninolytic enzyme production of a recently isolated wood-degrading fungus Trichophyton rubrum LSK-27 was investigated. In submerged cultures, the organism appeared to be an efficient manganese peroxidase (MnP) producer. When grown in baffled and unbaffled shake flasks with three different working volume/total volume ratios (WV/TV 10, 25 and 50%), the organism displayed notable morphological differences, with variations in pellet shape and size. Cultivation in baffled flasks with 25% WV/TV resulted in higher MnP and also laccase production as well as an earlier appearance of these enzymes in culture broth. However, oxygen limitation conditions inhibited MnP and laccase production and resulted in considerable changes in the morphology of this fungus.     

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).     

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 of practical textile industry effluents by white rot fungus Coriolus versicolor IBL-04

Textile industry discharges a vast amount of unused synthetic dyes in effluents. The discharge of these effluents into rivers and lakes leads to a reduction in sunlight penetration in natural water bodies, which, in turn, decreases both photosynthetic activity and dissolved oxygen concentration rendering it toxic to living beings. This paper describes the decolorization potential of a local white rot fungus, Coriolus versicolor IBL-04 for practical industrial effluents collected from five different textile industries of Faisalabad, Pakistan. Screening of C. versicolor IBL-04 on five effluents showed best decolorization results (36.3%) for Arzoo Textile Industry (ART) effluent in 6 days followed by Crescent Textile Industry (CRT), Itmad Textile Industry (ITT), Megna Textile Industry (MGT) and Ayesha Textile Industry (AST) effluents. Optimization of different process parameters for ART effluent decolorization by C. versicolor IBL-04 showed that manganese peroxidase (MnP) (486 U/mL) was the lignolytic enzyme present in the culture filtrates with undetectable lignin peroxidase (LiP) and laccase. The MnP synthesis and effluent decolorization could be enhanced to 725 U/mL and 84.4%, respectively, with a significant time reduction to 3 days by optimizing pH and temperature and using 1% starch as a supplementary carbon source.     

Production of manganese-dependent peroxidase in a new solid-state bioreactor by Phanerochaete chrysosporium grown on wood shavings. Application to the decolorization of synthetic dyes

The production of manganese-dependent peroxidase (MnP) byPhanerochœte chrysosporium in a new solid-state bioreactor, the immersion bioreactor, operating with lignocellulosic waste, such as wood shavings, was investigated. Maximum MnP and lignin peroxidase (LiP) activity of 13.4 and 8.48 μkat/L were obtained, respectively. Thein vitro decolorization of several synthetic dyes by the extracellular liquid produced in the above-mentioned bioreactor (containing mainly MnP) was carried out and its degrading ability was assessed. The highest decolorization was reached with Indigo Carmine (98%) followed by Bromophenol Blue (56%) and Methyl Orange (36%), whereas Gentian Violet was hardly decolorized (6%).