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.     

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.     

Bacterial Decolorization of Azo Dyes by Rhodopseudomonas palustris

Summary The ability of Rhodopseudomonas palustris AS1.2352 possessing azoreductase activity to decolorize azo dyes was investigated. It was demonstrated that anaerobic conditions were necessary for bacterial decolorization, and the optimal pH and temperature were pH 8 and 30–35 °C, respectively. Decolorization of dyes with different molecular structures was performed to compare their degradability. The strain could decolorize azo dye up to 1250 mg l⁻¹, and the correlation between the specific decolorization rate and dye concentration could be described by Michaelis–Menten kinetics. Long-term repeated operations showed that the strain was stable and efficient during five runs. Cell extracts from the strain demonstrated oxygen-insensitive azoreductase activity in vitro.     

Decolorization of Several Polymeric Dyes by the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

The polymeric dyes Poly B-411, Poly R-481, and Poly Y-606 were examined as possible alternatives to the radiolabeled lignin previously used as a substrate in lignin biodegradation assays. Like lignin degradation, the decolorization of these dyes by the white rot basidiomycete Phanerochaete chrysosporium occurred during secondary metabolism, was suppressed in cultures grown in the presence of high levels of nitrogen, and was strongly dependent on the oxygen concentration in the cultures. A variety of inhibitors of lignin degradation, including thiourea, azide, and 4′-O-methylisoeugenol, also inhibited dye decolorization. A pleiotropic mutant of P. chrysosporium, 104-2, lacking phenol oxidase and ligninolytic activity was also not able to decolorize the polymeric dyes, whereas a phenotypic revertant strain, 424-2, regained this capacity. All of these results suggest that the ligninolytic degradation activity of the fungus was responsible for the decolorization of these dyes.     

Biodecolorization of azo,anthraquinonic and triphenylmethane dyes by white-rot fungi and a laccase-secreting engineered strain

One laccase-secreting engineered strain and four white-rot fungi were tested for their capacity to decolorize nine dyes that could be classified as azo, anthraquinonic and triphenylmethane dyes. Trametes versicolor was the most efficient of the tested strains under these experimental conditions. Anthraquinonic dyes were decolorized more easily than the other two types. Small structural differences among the dyes could significantly affect decolorization. None of the strains showed lignin peroxidase or veratryl alcohol oxidase activity. None of the dyes were decolorized completely by laccase alone. It is likely that other phenoloxidases, such as Mn-dependent and versatile peroxidase, were also involved in decolorization of the dyes.     

Decolorization of Remazol Brilliant Blue (RBBR) and Poly R-478 dyes by <Emphasis Type="Italic">Bjerkandera adusta</Emphasis> CCBAS 930

An anamorphic Bjerkandera adusta CCBAS 930 strain isolated from soil was found to decolorize two anthraquinonic dyes: Remazol Brilliant Blue R and Poly R-478. The reduction in the level of phenolic compounds in liquid B. adusta cultures containing RBBR and Poly R-478 was correlated with decolorization of studied dyes, which suggested their biodegradation. It was shown that this process was coupled with induction of secondary metabolism (idiophase) and peak peroxidase activity in culture medium, and the appearance of aerial mycelium. Decolorization of dyes depended on the presence of glucose (cometabolism).     

Overlap of laccases/cellobiose dehydrogenase activities during the decolourisation of anthraquinonic dyes with close chemical structures by Pycnoporus strains

Pycnoporus strains were used as model to understand the role of laccases in the in vivo decolourisation of three anthraquinonic dyes. The decolourisation capability of Pycnoporus sanguineus MUCL 41582 (PS7), which produces laccases as the main oxidative enzyme, was assayed and compared with the decolourisation capability of a control strain, Pycnoporus cinnabarinus MUCL 39533 (PC330) described as laccase-deficient strain. In absence of dye, laccase activity was observed during the trophophase and the idiophase with PS7, while no laccase activity was observed with PC330. Acid Blue 62 (ABu62), Acid Blue 281 (ABu281) and Reactive Blue 19 (RBu19) caused an increase in laccase activity and surprisingly laccase activity was detected with PC330. In vitro, oxidation of all three anthraquinones by a laccase preparation was obtained to a lesser extent than the whole cell process; suggesting that other factor(s) could be required for a complete decolourisation. As the time space of laccase production in the tested fungi was not perfectly coincidental with the decolourisation process, the activity of cellobiose dehydrogenase (CDH) was monitored. Present early in the broth during the growth of the fungi, CDH displayed in vitro a synergism with laccases in the decolourisation of ABu62, and an antagonism with laccases in the decolourisation of ABu281 and RBu19.     

Screening for decolorizing basidiomycetes in Mexico

A survey to isolate native white rot basidiomycetes from Northeast Mexico was conducted in the forests of the Sierra Madre Oriental in the state of Nuevo León. A total of 92 isolates from at least 20 different genera, were screened on Bran-Flakes solid plate cultures for the production of ligninolytic oxidases and/or peroxidases with guaiacol and o-anisidine as substrates; their lignin depolymerizing potential using the polymeric dye Poly R 478; their ability to decolorize anthraquinonic (Remazol Brilliant Blue Reactive), azo (Acid Red 44) and triphenylmethane (Crystal Violet) dyes. Among all fungi tested, 15 isolates showed extensive decolorization of the three dyes within a week and gave a positive reaction in guaiacol and o-anisidine tests. Nine of them were also efficient degraders of Poly R-478. Two isolates (CS5 and CU1) showed decolorization of all dyes within 5 days, comparing favorably with reference strains of P. chrysosporium, Pleurotus ostreatus, and Bjerkandera adusta. Decolorization was associated with laccase activity in both isolates and reached 90% or more for all dyes within 24 h in 8-day-old liquid cultures. The coupling of pairs 2,4-dichlorophenol + 4-aminoantipyrine and 3-dimethylaminobenzoic acid + 3-methyl-2-benzothiazolinone hydrazone, strongly suggest that the laccases of both strains correspond to those considered of high redox potential. These strains are considered good candidates for bioremediation of dye polluted effluents due to their ligninolytic potential and decolorizing performance.     

Anthraquinone dyes decolorization capacity of anamorphic Bjerkandera adusta CCBAS 930 strain and its HRP-like negative mutants

Cultures of the anamorphic fungus Bjerkandera adusta CCBAS 930 decolorizing, in stationary cultures, 0.01 % solutions of carminic acid and Poly R-478, were characterised by a strong increase in the activity of the horseradish peroxidase (HRP-like) and manganese-dependent peroxidase (MnP) at a low activity of lignin peroxidase. Genotypically modified mutants of B. adusta CCBAS 930: 930-5 and 930-14, with total or partial loss of decolorization capabilities relative to anthraquinonic dyes, showed inhibition of the activity of HRP-like peroxidase and MnP. Whereas, compared to the parental strain, in the mutant cultures there was an increase in the activity of lignin peroxidase and laccase. The paper presents a discussion of the role of the studied enzymatic activities in the process of decolorization of anthraquinonic dyes by the strain B. adusta CCBAS 930.     

Bioinformatics aided microbial approach for bioremediation of wastewater containing textile dyes

Four textile azo dyes, Joyfix Red, Remazol Red, Reactive Red and Reactive Yellow, were studied for decolorization. Of nineteen soil bacterial isolates, two novel strains were found to highly decolorize Joyfix Red and were identified as Lysinibacillus sphaericus (KF032717) and Aeromonas hydrophila (KF032718) through 16S rDNA analysis. Laccase and Azoreductase enzyme modeling and enzyme–dye interaction performed using Schrödinger Suite imitated decolorization percentage. Results based on cumulative Glide score (Dry laboratory) and decolorization percentage of the other three dyes based on ultraviolet–visible (UV–vis) spectroscopy (Wet laboratory) were reliable. Biodegradation of Joyfix Red was confirmed by high-performance liquid chromatography (HPTLC) elution profile which showed four peaks at 1.522, 1.800, 3.068 and 3.804 min with that of parent dye which showed single peak at 1.472 min. Fourier transform infrared spectroscopy (FT-IR) analysis supported the biotransformation of Joyfix Red. Gas chromatography–mass spectroscopy (GC–MS) analysis showed sodium (3E,5Z)-4-amino-6-hydroxyhexa-13,5-triene-2-sulfonate was formed as end product during biodegradation. From these findings, it can be inferred that enzyme and dye interaction studies can assist in examining decolorization efficiency of bacteria and its enzyme, thereby enhancing the bioremediation process by reducing preliminary lengthy wet laboratory screening. This is the first report of a combinatorial in silico cum in vitro approach and its validation for the bioremediation of wastewater containing these textile azo dyes.     

Decolorization of synthetic dyes by basidiomycetes isolated from woods of the Atlantic Forest (PE), Brazil

In this study, the biodegradability of synthetic dyes used in the textile industry by Basidiomycetes collected in woods of the Atlantic Forest of Pernambuco, Brazil, was evaluated. Among the fungi, the following species were identified: Caripia montagnei, Datronia caperata, Earliella scabrosa, Fomitopsis feei, Ganoderma stiptatum, Hexagonia hydnoides, Pleurotus ostreatus, Pycnoporus sanguineus and Trametes membranacea. Mycelium discs (2 cm2) of each fungal isolate were transferred to 30 ml of King medium supplemented with 0.05% m/v of each of the following dyes: methyl orange, bromophenol blue (BB), methylene blue, Congo red, phenol red (PR) and methyl green (MG). All these species showed potential to decolorize the tested dyes, with decolorization efficiency varying from 7.26 to 99.2%. H. hydnoides and T. membrancea were the only species able to degrade all dyes. BB was effectively metabolized by P. sanguineus (97.4%), H. hydnoides (95.6%), E. scabrosa (96.6%) and T. membranacea (99.2%). E. scabrosa stood out among the isolates in dyes decolorization, allowing for efficiencies >90% for MG, PR, and BB.     

Decolorization of sulfonated azo dyes with two photosynthetic bacterial strains and a genetically engineered Escherichia coli strain

Sulfonated azo dyes were decolorized by two wild type photosynthetic bacterial (PSB) strains (Rhodobacter sphaeroides AS1.1737 and Rhodopseudomonas palustris AS1.2352) and a recombinant strain (Escherichia coli YB). The effects of environmental factors (dissolved oxygen, pH and temperature) on decolorization were investigated. All the strains could decolorize azo dye up to 900 mg l⁻¹, and the correlations between the specific decolorization rate and dye concentration could be described by Michaelis–Menten kinetics. Repeated batch operations were performed to study the persistence and stability of bacterial decolorization. Mixed azo dyes were also decolorized by the two PSB strains. Azoreductase was overexpressed in E. coli YB; however, the two PSB strains were better decolorizers for sulfonated azo dyes.     

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.     

Redox-mediated decolorization of synthetic dyes by fungal laccases

Laccases from the lignin-degrading basidiomycetes Trametes versicolor, Polyporus pinisitus and the ascomycete Myceliophthora thermophila were found to decolorize synthetic dyes to different extents. Differences were attributed to the specific catalytic properties of the individual enzymes and to the structure of the dyes. Due to their higher oxidative capacities, the laccases from the two basidiomycetes decolorized dyes more efficiently than that of the ascomycete. The azo dye Direct Red 28, the indigoid Acid Blue 74 and anthraquinonic dyes were directly enzymatically decolorized within 16 h. The addition of 2 mM of the redox-mediator 1-hydroxybenzotriazole further improved and facilitated the decolorization of all nine dyes investigated. Laccases decolorized dyes both individually and in complex mixtures in the presence of bentonite or immobilized in alginate beads. Our data suggest that laccase/mediator systems are effective biocatalysts for the treatment of effluents from textile, dye or printing industries.     

Bacterial decolorization and degradation of the reactive dye Reactive Red 180 by Citrobacter sp. CK3

A bacterial strain, CK3, with remarkable ability to decolorize the reactive textile dye Reactive Red 180, was isolated from the activated sludge collected from a textile mill. Phenotypic characterization and phylogenetic analysis of the 16S rDNA sequence indicated that the bacterial strain belonged to the genus Citrobacter. Bacterial isolate CK3 showed a strong ability to decolorize various reactive textile dyes, including both azo and anthraquinone dyes. Anaerobic conditions with 4 g l^?1 glucose, pH = 7.0 and 32 °C were considered to be the optimum decolorizing conditions. Citrobacter sp. CK3 grew well in a high concentration of dye (200 mg l^?1), resulting in approximately 95% decolorization extent in 36 h, and could tolerate up to 1000 mg l^?1 of dye. UV–vis analyses and colorless bacterial cells suggested that Citrobacter sp. CK3 exhibited decolorizing activity through biodegradation, rather than inactive surface adsorption. It is the first time that a bacterial strain of Citrobacter sp. has been reported with decolorizing ability against both azo and anthraquinone dyes. High decolorization extent and facile conditions show the potential for this bacterial strain to be used in the biological treatment of dyeing mill effluents.     

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.     

Uptake of reactive textile dyes by Aspergillus foetidus

An isolated fungus, Aspergillus foetidus was found to effectively decolorize media containing azo reactive dyes namely, Drimarene dyes. The extent of color removal was greater than 95% within 48 h of growth of the fungus. The entire color was found to be strongly bioadsorbed to the rapidly settling fungal biomass pellets without undergoing significant biotransformation. Our investigations reveal that the process of decolorization is concomitant with the exponential growth phase of the fungus and has requirement for a biodegradable substrate such as glucose. The fungus was also able to decolorize media containing mixture of dyes to an extent of 85% within 72 h of growth. Kinetic analyses of fungal decolorization indicate that the process is time dependent and follows first order kinetics with respect to initial concentration of dye. The rates of color uptake (k values) decrease to a significant extent with increasing initial concentrations of dye. The fungus was able to grow and decolorize media in the presence of 5 ppm of chromium and 1% sodium chloride. An alternate and cheaper carbon source such as starch supported the growth and decolorization process. These results suggest that dye uptake process mediated by A. foetidus has a potential for large-scale treatment of textile mill discharges.     

Characterization of a salt resistant bacterial strain <Emphasis Type="Italic">Proteus</Emphasis> sp. NA6 capable of decolorizing reactive dyes in presence of multi-metal stress

Microbial biotechnologies for the decolorization of textile wastewaters have attracted worldwide attention because of their economic suitability and easiness in handling. However, the presence of high amounts of salts and metal ions in textile wastewaters adversely affects the decolorization efficiency of the microbial bioresources. In this regard, the present study was conducted to isolate salt tolerant bacterial strains which might have the potential to decolorize azo dyes even in the presence of multi-metal ion mixtures. Out of the tested 48 bacteria that were isolated from an effluent drain, the strain NA6 was found relatively more efficient in decolorizing the reactive yellow-2 (RY2) dye in the presence of 50 g L^?1 NaCl. Based on the similarity of its 16S rRNA gene sequence and its position in a phylogenetic tree, this strain was designated as Proteus sp. NA6. The strain NA6 showed efficient decolorization (>90 %) of RY2 at pH 7.5 in the presence of 50 g L^?1 NaCl under static incubation at 30 °C. This strain also had the potential to efficiently decolorize other structurally related azo dyes in the presence of 50 g L^?1 NaCl. Moreover, Proteus sp. NA6 was found to resist the presence of different metal ions (Co⁺², Cr⁺⁶, Zn⁺², Pb⁺², Cu⁺², Cd⁺²) and was capable of decolorizing reactive dyes in the presence of different levels of the mixtures of these metal ions along with 50 g L^?1 NaCl. Based on the findings of this study, it can be suggested that Proteus sp. NA6 might serve as a potential bioresource for the biotechnologies involving bioremediation of textile wastewaters containing the metal ions and salts.     

Decolorization of an industrial effluent by free and immobilized cells of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> AAP56<Emphasis Type="Italic">.</Emphasis> Implementation of efficient down flow column reactor

A bacterial strain AAP56, isolated from a polluted soil (from Kelibia city) and identified as Stentrophomonas maltophilia, was particularly interesting for its ability to decolorize recalcitrant dyes of an industrial effluent: SITEX Black. The final percentage decolorization 60% was shown by bacterial culture after incubation in LB medium at 30°C under shaking conditions. The decolorization was closely correlated with the metabolic bacterial growth. The replacement of yeast extract in LB medium composition by soya flour was clearly efficient to enhance the percentage decolorization by 20% and also to reduce the growth medium cost 60-fold. The bacteria were able to reduce 23% from the initial COD and 28% from the initial BOD₅ of the effluent. The immobilization of bacterial cells in calcium alginate beads improved by 25% the effectiveness of the biotransformation within 24 h in batch conditions. The potential of a downflow fixed column reactor (DFCR) to decolorize SITEX Black was evaluated under dilution rate. The best decolorization percentage (82%) was recorded at 0.3 h⁻¹. This bioprocess seems to be a potentially useful method to remediate the colored textile wastewater.     

Decolorization of azo dyes by marine Shewanella strains under saline conditions

Azo dye decolorization was studied with Shewanella strains under saline conditions. Growing cells of Shewanella algae and Shewanella marisflavi isolated from marine environments demonstrated better azo dye decolorization capacities than the other three strains from non-saline sources. Cell suspensions of S. algae and S. marisflavi could decolorize single or mixed azo dyes with different structures. Decolorization kinetics were described with Michaelis–Menton equation, which indicated better decolorization performance of S. algae over S. marisflavi. Lactate and formate were identified as efficient electron donors for amaranth decolorization by the two strains. S. algae and S. marisflavi could decolorize amaranth at up to 100 g?L^?1 NaCl or Na₂SO₄. However, extremely low concentration of NaNO₃ exerted strong inhibition on decolorization. Both strains could remove the color and COD of textile effluent during sequential anaerobic–aerobic incubation. Lower concentrations of NaCl (20–30 g?L^?1) stimulated the activities of azoreductase, laccase, and NADH-DCIP reductase. The decolorization intermediates were identified by high-performance liquid chromatography and Fourier transform infrared spectroscopy. Decolorization metabolites of amaranth were less toxic than original dye. These findings improved our knowledge of azo-dye-decolorizing Shewanella species and provided efficient candidates for the treatment of dye-polluted saline wastewaters.