Decoloration of Amaranth by the white-rot fungus Trametes versicolor. Part II. Verification study

The involvement of lignin peroxidase (LiP) in the decoloration of the mono-azo substituted napthalenic dye Amaranth was investigated with pure enzymes and whole cultures of Trametes versicolor. The verification study confirmed that LiP has a direct influence on the initial decoloration rate and showed that another enzyme, which does not need hydrogen peroxide to function and is not a laccase, also plays a role during decoloration. These results confirm the results of a previous statistical analysis. Furthermore, the fungal mycelium affects the performance of the decoloration process.     

Contribution of manganese peroxidase and laccase to dye decoloration by Trametes versicolor

During dye decoloration by Trametes versicolor ATCC 20869 in modified Kirk’s medium, manganese peroxidase (MnP) and laccase were produced, but not lignin peroxidase, cellobiose dehydrogenase or manganese-independent peroxidase. Purified MnP decolorized azo dyes [amaranth, reactive black 5 (RB5) and Cibacron brilliant yellow] in Mn²⁺-dependent reactions but did not decolorize an anthraquinone dye [Remazol brilliant blue R (RBBR)]. However, the purified laccase decolorized RBBR five to ten times faster than the azo dyes and the addition of a redox mediator, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), did not alter decoloration rates. Amaranth and RB5 were decolorized the most rapidly by MnP since they have a hydroxyl group in an ortho position and a sulfonate group in the meta position relative to the azo bond. During a typical batch decoloration with the fungal culture, the ratio of laccase:MnP was 10:1 to 20:1 (based on enzyme activity) and increased to greater than 30:1 after decoloration was complete. Since MnP decolorized amaranth about 30 times more rapidly than laccase per unit of enzyme activity, MnP should have contributed more to decoloration than laccase in batch cultures.     

Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol

Phanerochaete chrysosporium decolorized several polyaromatic azo dyes in ligninolytic culture. The oxidation rates of individual dyes depended on their structures. Veratryl alcohol stimulated azo dye oxidation by pure lignin peroxidase (ligninase, LiP) in vitro. Accumulation of compound II of lignin peroxidase, an oxidized form of the enzyme, was observed after short incubations with these azo substrates. When veratryl alcohol was also present, only the native form of lignin peroxidase was observed. Azo dyes acted as inhibitors of veratryl alcohol oxidation. After an azo dye had been degraded, the oxidation rates of veratryl alcohol recovered, confirming that these two compounds competed for ligninase during the catalytic cycle. Veratryl alcohol acts as a third substrate (with H2O2 and the azo dye) in the lignin peroxidase cycle during oxidations of azo dyes.     

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.     

Effects of Mn2+ and NH+ 4 concentrations on laccase and manganese peroxidase production and Amaranth decoloration by Trametes versicolor

Extracellular lignin peroxidase (LiP) was not detected during decoloration of the azo dye, Amaranth, by Trametes versicolor. Approximately twice as much laccase and manganese peroxidase (MnP) was produced by decolorizing cultures compared to when no dye was added. At a low Mn²⁺ concentration (3 M), N-limited (1.2 mM NH₄ ⁺) cultures decolorized eight successive additions of Amaranth with no visible sorption to the mycelial biomass. At higher Mn²⁺ concentrations (200 M), production of MnP increased and that of laccase decreased, but the rate or number of successive Amaranth decolorations was unaffected. There was always a 6-h to 8-h lag prior to decoloration of the first aliquot of Amaranth, regardless of MnP and laccase concentrations. Although nitrogen-rich (12 mM NH₄ ⁺) cultures at an initial concentration of 200 M Mn²⁺ produced high laccase and MnP levels, only three additions of Amaranth were decolorized, and substantial mycelial sorption of the dye occurred. While the results did not preclude roles for MnP and laccase, extracellular MnP and laccase alone were insufficient for decoloration. The cell-free supernatant did not decolorize Amaranth, but the mycelial biomass separated from the whole broth and resuspended in fresh medium did. This indicates the involvement of a mycelial-bound, lignolytic enzyme or a H₂O₂-generating mechanism in the cell wall. Nitrogen limitation was required for the expression of this activity. Received: 19 May 1998 / Received revision: 22 October 1998 / Accepted: 7 November 1998     

Effect of inducers on the decolorization and biodegradation of textile azo dye Navy blue 2GL by Bacillus sp. VUS

Bacillus sp. VUS decolorized azo dye Navy blue 2GL in 48 h at static anoxic condition in yeast extract medium, whereas it took only 18 h for the decolorization in presence of CaCl₂. Different inducers played role in the decolorization of Navy blue 2GL. CaCl₂ found to be the most effective inducer among all inducers tested. The activity of enzymes like lignin peroxidase, laccase and reductases viz. NADH-DCIP, azo and riboflavin induced during decolorization represents their role in the biodegradation. Extracellular LiP and intracellular laccase activity induced with CaCl₂. Yeast extract was best medium for faster decolorization than other media. UV–vis spectrophotometer analysis and visual examinations showed decolorization of dye. High performance liquid chromatography, Fourier transforms infrared spectroscopy showed degradation of dye. Gas Chromatography-Mass Spectroscopy revealed formation of 4-Amino-3-(2-bromo-4, 6-dinitro-phenylazo)-phenol and acetic acid 2-(-acetoxy-ethylamino)-ethyl ester as final products. Bacillus sp. VUS also decolorized synthetic effluent. Phytotoxicity study showed detoxification of Navy blue 2GL.     

Cloning and expression of a lignin peroxidase gene from Streptomyces viridosporus in Streptomyces lividans.

A lignin peroxidase gene was cloned from Streptomyces viridosporus T7A into Streptomyces lividans TK64 in plasmid pIJ702. BglII-digested genomic DNA (4-10 kb) of S. viridosporus was shotgun-cloned into S. lividans after insertion into the melanin (mel+) gene of pIJ702. Transformants expressing pIJ702 with insert DNA were selected based upon the appearance of thiostrepton resistant (tsrr)/mel-colonies on regeneration medium. Lignin peroxidase-expressing clones were isolated from this population by screening of transformants on a tsr-poly B-411 dye agar medium. In the presence of H2O2 excreted by S. lividans, colonies of lignin peroxidase-expressing clones decolorized the dye. Among 1000 transformants screened, 2 dye-decolorizing clones were found. One, pIJ702/TK64.1 (TK64.1), was further characterized. TK64.1 expressed significant extracellular 2,4-dichlorophenol (2.4-DCP) peroxidase activity (= assay for S. viridosporus lignin peroxidase). Under the cultural conditions employed, plasmidless S. lividans TK64 had a low background level of 2.4-DCP oxidizing activity. TK64.1 excreted an extracellular peroxidase not observed in S. lividans TK64, but similar to S. viridosporus lignin peroxidase ALip-P3, as shown by activity stain assays on nondenaturing polyacrylamide gels. The gene was located on a 4 kb fragment of S. viridosporus genomic DNA. When peroxidase-encoding plasmid, pIJ702.LP, was purified and used to transform three different S. lividans strains (TK64, TK23, TK24), all transformants tested decolorized poly B-411. When grown on lignocellulose in solid state processes, genetically engineered S. lividans TK64.1 degraded the lignocellulose slightly better than did S. lividans TK64. This is the first report of the cloning of a bacterial gene coding for a lignin-degrading enzyme.     

Evaluation of Argentinean white rot fungi for their ability to produce lignin-modifying enzymes and decolorize industrial dyes

The decolorizing capacity of 26 white rot fungi from Argentina was investigated. Extracellular production of ligninolytic enzymes by mycelium growing on solid malt extract/glucose medium supplemented with different dyes (Malachite Green, Azure B, Poly R-478, Anthraquinone Blue, Congo Red and Xylidine), dye decolorization and the relationship between these two processes were studied. Only ten strains decolorized all the dyes, all ten strains produced laccase, lignin peroxidase and manganese peroxidase on solid medium. However, six of the strains could not decolorize any of the dyes; all six strains tested negative for lignin peroxidase, and produced less than 0.05 U/g agar of manganese peroxidase. Comparing the isolates with the well-known dye-degrader Phanerochaete chrysosporium, a new fungus was identified: Coriolus versicolor f. antarcticus, potentially a candidate for use in biodecoloration processes. Eighteen day-old cultures of this fungus were able to decolorize in an hour 28%, 30%, 43%, 88% and 98% of Xylidine (24 mg/l), Poly R-478 (75 mg/l), Remazol Brilliant Blue R (9 mg/l), Malachite Green (6 mg/l) and Indigo Carmine (23 mg/l), respectively. Laccase activity was 0.13 U/ml, but neither lignin peroxidase nor manganese peroxidase were detected in the extracellular fluids for that day of incubation.     

The role of enzymes produced by white-rot fungus Irpex lacteus in the decolorization of the textile industry effluent

The textile industry wastewater has been decolorized efficiently by the white rot fungus, Irpex lacteus, without adding any chemicals. The degree of the decolorization of the dye effluent by shaking or stationary cultures is 59 and 93%, respectively, on the 8th day. The higher level of manganese-dependent peroxidase (MnP) and non-specific peroxidase (NsP) was detected in stationary cultures than in the cultures shaken. Laccase activities were equivalent in both cultures and its level was not affected significantly by the culture duration. Neither lignin peroxidase (LiP) nor Remazol Brilliant Blue R oxidase (RBBR ox) was detected in both cultures. The absorbance of the dye effluent was significantly decreased by the stationary culture filtrate of 7 days in the absence of Mn (II) and veratryl alcohol. In the stationary culture filtrate, three or more additional peroxidase bands were detected by the zymogram analysis.     

Chemical evidence for the mechanism of the biodecoloration of Amaranth by <Emphasis Type="Italic">Trametes versicolor</Emphasis>

The white-rot fungus Trametes versicolor decolorized Amaranth. The hypothesis that the carbon structure of Amaranth was broken down in smaller mass fragments was investigated analyzing the products of decoloration. FTIR spectroscopy, ion chromatography, sulfite and ammonia analysis were used to compare the culture filtrate before dye addition, with the pure dye, the culture filtrate after dye addition, and the culture filtrate during the treatment. The hypothesis of polymerization of the decoloration products was tested by spectrophotometric analysis of dialysates of the pure dye, the culture filtrate before dye addition, and the culture filtrates after dye addition and decoloration. FTIR showed that the signals typical for the azo group disappeared after decoloration, while new peaks appeared that were characteristic of substituted naphthalenic or benzenic compounds. Ion chromatography showed that the level of sulfate in the treatment increased when compared with the level of the sulfate in control, suggesting that the sulfonic groups were being stripped from Amaranth’s structure and metabolized to sulfate. Sulfite measurements for the treatment and controls showed no significant difference, and were well below the saturation concentration for sulfite in water, confirming that the medium was aerobic. Ammonia concentration did not change with the decoloration. Absorbance scans after dialysis of decolorized samples showed no new peaks, suggesting that the decoloration products were not polymerized. These observations suggests that the decoloration mechanism starts with the azo link removal, followed by desulfonation, naphthalene ring opening, and the formation of smaller mass fragments, similar to fungal metabolites.     

Influence of aromatic substitution patterns on azo dye degradability by Streptomyces spp. and Phanerochaete chrysosporium.

Twenty-two azo dyes were used to study the influence of substituents on azo dye biodegradability and to explore the possibility of enhancing the biodegradabilities of azo dyes without affecting their properties as dyes by changing their chemical structures. Streptomyces spp. and Phanerochaete chrysosporium were used in the study. None of the actinomycetes (Streptomyces rochei A10, Streptomyces chromofuscus A11, Streptomyces diastaticus A12, S. diastaticus A13, and S. rochei A14) degraded the commercially available Acid Yellow 9. Decolorization of monosulfonated mono azo dye derivatives of azobenzene by the Streptomyces spp. was observed with five azo dyes having the common structural pattern of a hydroxy group in the para position relative to the azo linkage and at least one methoxy and/or one alkyl group in an ortho position relative to the hydroxy group. The fungus P. chrysosporium attacked Acid Yellow 9 to some extent and extensively decolorized several azo dyes. A different pattern was seen for three mono azo dye derivatives of naphthol. Streptomyces spp. decolorized Orange I but not Acid Orange 12 or Orange II. P. chrysosporium, though able to transform these three azo dyes, decolorized Acid Orange 12 and Orange II more effectively than Orange I. A correlation was observed between the rate of decolorization of dyes by Streptomyces spp. and the rate of oxidative decolorization of dyes by a commercial preparation of horseradish peroxidase type II, extracellular peroxidase preparations of S. chromofuscus A11, or Mn(II) peroxidase from P. chrysosporium. Ligninase of P. chrysosporium showed a dye specificity different from that of the other oxidative enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of aromatic substitution patterns on azo dye degradability by Streptomyces spp. and Phanerochaete chrysosporium.

Twenty-two azo dyes were used to study the influence of substituents on azo dye biodegradability and to explore the possibility of enhancing the biodegradabilities of azo dyes without affecting their properties as dyes by changing their chemical structures. Streptomyces spp. and Phanerochaete chrysosporium were used in the study. None of the actinomycetes (Streptomyces rochei A10, Streptomyces chromofuscus A11, Streptomyces diastaticus A12, S. diastaticus A13, and S. rochei A14) degraded the commercially available Acid Yellow 9. Decolorization of monosulfonated mono azo dye derivatives of azobenzene by the Streptomyces spp. was observed with five azo dyes having the common structural pattern of a hydroxy group in the para position relative to the azo linkage and at least one methoxy and/or one alkyl group in an ortho position relative to the hydroxy group. The fungus P. chrysosporium attacked Acid Yellow 9 to some extent and extensively decolorized several azo dyes. A different pattern was seen for three mono azo dye derivatives of naphthol. Streptomyces spp. decolorized Orange I but not Acid Orange 12 or Orange II. P. chrysosporium, though able to transform these three azo dyes, decolorized Acid Orange 12 and Orange II more effectively than Orange I. A correlation was observed between the rate of decolorization of dyes by Streptomyces spp. and the rate of oxidative decolorization of dyes by a commercial preparation of horseradish peroxidase type II, extracellular peroxidase preparations of S. chromofuscus A11, or Mn(II) peroxidase from P. chrysosporium. Ligninase of P. chrysosporium showed a dye specificity different from that of the other oxidative enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decolorizing an anthraquinone dye by Phlebia brevispora: Intra‐species characterization

Remazol brilliant blue R (RBBR) is an anthraquinone dye derived from anthracene that is decolorized by a white rot fungus, Phlebia brevispora. Interestingly, P. brevispora produces two phenomena of yellowish and pinkish colors during the degradation of RBBR. Here, we characterized the decolorization of RBBR by P. brevispora. The fungus was significantly different between the two colors via UV spectrophotometry, and the morphology of the hyphae observed in the respective color culture was also entirely different. Moreover, both of the two ligninolytic enzymes, laccase and manganese‐dependent peroxidase (MnP), were remarkably stimulated in the yellowish culture at the beginning of the decolorization. It is possible that the RBBR decolorizing mechanism might be primarily related to the amount of laccase and MnP produced in the yellowish culture. Thus, the decolorized color may be rapidly estimated at initial period of incubation. In addition, GeneFishing technology revealed that two genes were differentially expressed in yellowish culture.     

Phytoremediation potential of Portulaca grandiflora Hook. (Moss-Rose) in degrading a sulfonated diazo reactive dye Navy Blue HE2R (Reactive Blue 172)

Wild and tissue cultured plants of Portulaca grandiflora Hook. have shown to be able to decolorize a sulfonated diazo dye Navy Blue HE2R (NBHE2R) up to 98% in 40 h. A significant induction in the activities of lignin peroxidase, tyrosinase and DCIP reductase was observed in the roots during dye decolorization. The wild plants and tissue cultures could independently decolorize and degrade NBHE2R into metabolites viz. N-benzylacetamide and 6-diazenyl-4-hydroxynaphthalene-2-sulfonic acid. A dye mixture and a textile effluent were also decolorized efficiently by P. grandiflora. The phytotoxicity study revealed reduction in the toxicity due to metabolites formed after dye degradation.     

Characterization of a fungal strain isolated from a polyphenol polluted site

A group of fungal strains were isolated from a polyphenol polluted soil, taken from an olive oil processing plant in Attica, Greece. The fungi were tested for their ability to decolorize a polyaromatic dye Poly R-478, which was used as a model compound to test their ligninolytic activities. The strain K1.1 decolorized efficiently the dye on agar plates and was further studied. PCR amplification of the internal transcribed spacer (ITS) region of the ribosomal RNA genes from the genomic DNA isolated from mycelium grown in liquid culture resulted in amplified fragments. Via BLASTN search, the length of a 773 base pairs was identified as the basidiomycetes Coprinellus xanthothrix. The growth rates and the tolerance of the fungus were compared on solid media, containing four different concentrations of pentachlorophenol. Extracellular enzyme activities (lignin peroxidase, manganese peroxidase and laccase) were determined in defined liquid medium. The isolate expressed laccase and manganese peroxidase but not lignin peroxidase. The removal of the dye was also estimated in liquid medium. The fungus showed biosorption and biotransformation as removal mechanisms.     

Biodegradation of Grape Cluster Stems and Ligninolytic Enzyme Production by Phanerochaete chrysosporium during Semi‐Solid‐State Cultivation

The degradation undergone by grape cluster stems (woody component of vine bagasse), an agroindustrial waste, was investigated during the semi‐solid‐state cultivation of Phanerochaete chrysosporium BKM‐F‐1767 (ATCC 24725). For this, the content of lignin, cellulose and hemicellulose in grape cluster stems was determined before and after the enzymatic process. It was found that about 20% of Klason lignin, 48% of hemicellulose and 5% of cellulose were degraded during the process, being the ligninolytic enzymes (manganese‐dependent peroxidase and lignin peroxidase) produced by such cultures responsible for the degradation of grape cluster stems. In parallel, semi‐solid‐state cultures of P. chrysosporium grown on an inert support (cubes of nylon sponge), which is not susceptible to undergoing degradation during the enzymatic process, were used as reference cultures. In addition, the in vivo decolourisation of a model dye, the polymeric dye Poly R‐478, by both grape cluster stem and nylon cultures was studied in order to assess their degradative ability. A percentage of biological decolourisation higher than 90% after four days of dye addition was obtained using nylon sponge cultures, whereas grape cluster stem cultures led to a decolourisation of around 70% after eight days of dye incubation. The lower percentage of dye degradation achieved by the cultures grown on grape cluster stems was due to the enzymes produced, which were not only employed in the decolourisation of the dye but also in the degradation of the support, as indicated by the data mentioned above.     

Decoloration of a Carpet Dye Effluent Using Trametes Versicolor

Although a non-sterile, undiluted carpet dye effluent (containing two anthraquinone dyes) did not support growth of Trametes versicolor, the pre-grown fungus removed 95% of its color in shake-flasks after 10 h of incubation. After decoloration, the COD of the cell-free supernatant increased and the toxicity was unchanged as determined by the Microtox assay using Vibrio fischeri. Decoloration rates decreased when either glucose alone or Mn2+ and glucose were added. T. versicolor, immobilized on jute twine in a rotating biological contacting reactor, also decolorized four successive batches of the effluent. There was no decoloration in any of the uninoculated, non-sterile controls.     

Simultaneous Application of Glucose Oxidases and Peroxidases in Bleaching Processes

Peroxidases (POD) are used in textile decoloration and bleaching processes, but these enzymes are unfortunately inactivated rapidly at high hydrogen peroxide concentrations. A new concept has therefore been developed, which is based on a simultaneous application of glucose oxidase and peroxidase. Starting with glucose as a substrate for glucose oxidase (GOD), hydrogen peroxide was generated in situ. The freshly formed substrate H₂O₂ was immediately used by the POD oxidizing colored compounds in dyeing baths. For example, 20 mg of the dyestuff Sirius Supra Blue®FGG 200 % could be decolorized using 125 mg glucose which corresponds to 24 mg hydrogen peroxide. These experiments show that the enzyme cascade works in principle in homogeneous decoloration processes. The enzymes were not degraded by the oxidant, because under these conditions the stationary peroxide concentration is nearly zero over the whole reaction time. Moreover, experiments were carried out to check if this combined system with GOD, glucose and POD could be used even in heterogeneous systems such as the textile bleaching of natural cotton fibers. Starting from 55, a significant higher degree of whiteness (according to Berger) up to 66 could be obtained.     

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.     

Decolorization of synthetic dyes by solid state cultures of Lentinula (Lentinus) edodes producing manganese peroxidase as the main ligninolytic enzyme

The ability of the white-rot fungus Lentinula (Lentinus) edodes to decolorize several synthetic dyes was investigated using solid state cultures with corn cob as substrate. Cultures, containing amido black, congo red, trypan blue, methyl green, remazol brilliant blue R, methyl violet, ethyl violet and Poly R478 at 200 ppm, were completely decolorized after 18 days of incubation. Partial decolorization was observed in the cultures containing 200 ppm of brilliant cresyl blue and methylene blue. High manganese peroxidase activity (2600 U/g substrate), but very low lignin peroxidase (<10 U/g substrate) and laccase (<16 U/g substrate) activities were detected in the cultures. In vitro, the dye decolorization was markedly decreased by the absence of manganic ions and H2O2. These data suggest that manganese peroxidase appear to be the main responsible for the capability of L. edodes to decolorize synthetic dyes.