Biodegradation of Cyanide by a White Rot Fungus, Trametes versicolor

The cyanide degradation abilities of three white rot fungi, Trametes versicolor ATCC 200801, Phanerochaete chrysosporium ME 496 and Pleurotus sajor-caju, were examined. T. versicolor was the most effective with 0.35 g dry cell/100 ml degrading 2 mm KCN (130 mg/l) over 42 h, at 30°C, pH 10.5 with stirring at 150 rpm.     

Solid-phase treatment with the fungus Trametes versicolor substantially reduces pharmaceutical concentrations and toxicity from sewage sludge

For safe biosolid-land-applying, sludge should be contaminant-free. However, it may contain important amounts of micropollutants, not removed in the wastewater-treatment-processes. An alternative treatment with the fungus Trametes versicolor was applied in sterile solid-phase systems consisting of sludge and a lignocellulosic substrate. Fungal colonization and activity were demonstrated during the process, according to monitoring of ergosterol, laccase activity and the naproxen-degradation test (ND24). Fourteen out of 43 analyzed pharmaceuticals were found in the raw sludge. After treatment, phenazone, bezafibrate, fenofibrate, cimetidine, clarithromycin, sulfamethazine and atenolol were completely removed, while removals between 42% and 80% were obtained for the remaining pharmaceuticals. Toxicological analyses (Daphnia magna, Vibrio fischeri and seed germination) showed an important reduction in sludge toxicity after treatment. Results suggest that a solid-phase treatment with T. versicolor may reduce the ecotoxicological impact of micropollutants present in sewage sludge. This is the first report of a fungal-approach for elimination of emerging pollutants from biosolids.     

Enhanced production of laccase in the fungus Trametes versicolor by the addition of xenobiotics

Agrochemicals, industrial compounds and their transformation products have been assayed for their ability to enhance laccase production in liquid cultures of Trametes versicolor, when added at 0.5 mM. After 3 days of treatment, enzymatic activity in the culture medium was increased 14-fold by 4-n-nonylphenol and 24-fold by aniline. Laccase activity was enhanced 10-fold by oxidised derivatives of the herbicide diquat, 17-fold by N,N-dimethyl-N-(5-chloro,4-hydroxyphenyl)urea and 22-fold by 9-fluorenone.     

Decoloration of textile dyes by Trametes versicolor and its effect on dye toxicity

Amaranth, Tropaeolin O, Reactive Blue 15, Congo Red, and Reactive Black 5 were completely decolorized with no dye sorption by Trametes versicolor. Cibacron Brilliant Red 3G-P, Cibacron Brilliant Yellow 3B-A, and Remazol Brilliant Blue R were partially decolorized with some dye sorbed to the biomass. The Microtox assay before decoloration showed that Amaranth and Tropaeolin O were not toxic [the percent concentration to decrease 20% of the luminescence of Vibrio fischeri (EC₂₀) was greater than 100%]; Cibacron Brilliant Yellow 3B-A, Reactive Blue 15 and Cibacron Brilliant Red 3G-P were moderately non-toxic (100% > EC₂₀ > 75%); Remazol Brilliant Blue R was toxic (75% > EC₂₀ > 50%); and Congo Red and Reactive Black 5 were moderately toxic (50% > EC₂₀ > 25%). After decoloration the toxicity of the solutions containing Amaranth, Tropaeolin O and Reactive Black 5 was unchanged; Reactive Blue 15, Remazol Brilliant Blue R and Cibacron Brilliant Red 3G-P decreased to non-toxic levels; and Cibacron Brilliant Yellow 3B-A and Congo Red became very toxic (EC₂₀ < 25%).     

Different proportions of laccase isoenzymes produced by submerged cultures of Trametes versicolor grown on lignocellulosic wastes

The white-rot fungus Trametes versicolor grown in submerged culture produced two laccase isoenzymes, LacI and LacII. Addition of insoluble lignocellulosic materials into the culture medium increased the total laccase activity. The proportion of laccase isoenzymes also changed depending on the lignocellulosic material employed, with ratios of activity LacII/LacI from 0.9 (barley straw) to 4.4 (grape stalks). Besides, this proportion played an important role in the dye decolourisation.     

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.     

Production of sterigmatocystin by Aspergillus versicolor isolated from roughage

A total of 69 samples of hay and straw collected during the winter period of 1984/85 were surveyed for their contamination by Aspergillus versicolor. The percentage of A. versicolor-positive samples was 14.5%. Nineteen A. versicolor strains mainly isolated from roughage were tested for the production of sterigmatocystin. All of the isolates examined were capable of producing different levels of sterigmatocystin on a cracked corn substrate. The majority of these strains were highly toxigenic; 53% of the isolates produced more than 500 mg/kg of sterigmatocystin. These findings suggest that corn is a very suitable substrate for sterigmatocystin production and that particularly in the surface layers of feed stocks and corn silos such toxigenic strains of A. versicolor can produce considerable growth and possibly sterigmatocystin, too.     

Biological treatment of a pulp and paper industry effluent by Fomes lividus and Trametes versicolor

White rot fungi Fomes lividus and Trametes versicolor, isolated from the Western Ghats region of Tamil Nadu, India, were used to treat pulp and paper industry effluents on a laboratory scale and in a pilot scale. On the laboratory scale a maximum decolourization of 63.9% was achieved by T. versicolor on the fourth day. Inorganic chloride at a concentration of 765 mg/l, which corresponded to 227% of that in the untreated effluent, was liberated by F. lividus on the 10th day. The chemical oxygen demand (COD) was also reduced to 1984 mg/l (59.3%) by each of the two fungi. On the pilot scale, a maximum decolourization of 68% was obtained with the 6-day incubation by T. versicolor, inorganic chloride 475 mg/l (103%) was liberated on the seventh day by T. versicolor, and the COD was reduced to 1984 mg/l corresponding to 59.32% by F. lividus. These results suggested that F. lividus seems to be another candidate efficient for dechlorination of wastewater.     

Solid-state fermentation of sugarcane bagasse with Flammulina velutipes and Trametes versicolor

The mushroom Flammulina velutipes and the white-rot fungus Trametes versicolor were cultivated separately on sugarcane bagasse for 40 days. Trametes versicolor produced laccase and manganese-peroxidase activities, showing a simultaneous degradation of lignin and holocellulose. However, only phenoloxidase activity was found with Flammulina velutipes. A preferential degradation of lignin was detected in F. velutipes, which exhibited a greater reduction in the ratio of weight loss to lignin loss than T. versicolor. A decrease in the syringyl/guaiacyl ratio observed with both fungi indicated the preferential degradation of non-condensed (syringyl-type) lignin units. An increase in the relative abundance of aromatic carboxylic acids suggested that the oxidative transformation of lignin unit side-chains was occurring. This was more noticeable with Flammulina velutipes than with T. versicolor.     

Amaranth decoloration by Trametes versicolor in a rotating biological contacting reactor

Sequential batch and continuous operation of a rotating biological contacting (RBC) reactor and the effects of dissolved oxygen on the decoloration of amaranth by Trametes versicolor were evaluated. Amaranth belongs to the group of azo dyes which are potential carcinogens and/or mutagens that can be transformed into toxic aryl amines under anaerobic conditions. Cultivation of T. versicolor in a stirred tank reactor was found to be unsuitable for amaranth decoloration due to significant biomass fouling and increase in medium viscosity. Assuming that decoloration follows first-order kinetics, amaranth was decolorized more rapidly when T. versicolor was immobilized on jute twine in a RBC reactor operated either in a sequential batch (k=0.25 h^–1) or in a continuous (0.051 h⁻¹) mode compared to a stirred tank reactor (0.015 h⁻¹). Oxygen was found to be essential for decoloration with the highest decoloration rates occurring at oxygen saturation. Although longer retention times resulted in more decoloration when the RBC was operated in the continuous mode (about 33% amaranth decoloration), sequential batch operation gave better results (>95%) under similar nutrient conditions. Our data indicate that the fastest decoloration should occur in the RBC using nitrogen-free Kirk’s medium with 1 g/l glucose in sequential batch operation at rotational speeds and/or aeration rates which maintain oxygen saturation in the liquid phase.     

Biological pretreatment with a cellobiose dehydrogenase-deficient strain of Trametes versicolor enhances the biofuel potential of canola straw

The use of Trametes versicolor as a biological pretreatment for canola straw was explored in the context of biofuel production. Specifically, the effects on the straw of a wild-type strain (52 J) and a cellobiose dehydrogenase (CDH)-deficient strain (m4D) were investigated. The xylose and glucose contents of the straw treated with 52 J were significantly reduced, while only the xylose content was reduced with m4D treatment. Lignin extractability was greatly improved with fungal treatments compared to untreated straw. Saccharification of the residue of the m4D-treated straw led to a significant increase in proportional glucose yield, which was partially attributed to the lack of cellulose catabolism by m4D. Overall, the results of this study indicate that CDH facilitates cellulose access by T. versicolor. Furthermore, treatment of lignocellulosic material with m4D offers improvements in lignin extractability and saccharification efficacy compared to untreated biomass without loss of substrate due to fungal catabolism.     

Enhanced expression of laccase during the degradation of endocrine disrupting chemicals in Trametes versicolor

A putative laccase cDNA from a white-rot basidiomycete, Trametes versicolor, that consisted of 1,769 nucleotides was cloned using the rapid amplification of cDNA ends (RACE)-PCR method. The deduced amino acid sequence had 4 putative copper binding regions, which are common to fungal laccases. In addition, the sequence was 57 approximately 97 % homologous to sequences of other T. versicolor laccases. Additionally, the expression of laccase and manganese peroxidase in this fungus were both greatly increased under degrading conditions for bisphenol A, nonylphenol and two phthalic esters (benzylbutylphthalate and diethylphthalate), all of which are reportedly endocrine disrupting chemicals (EDCs). Furthermore, the estrogenic activities of the EDCs also decreased rapidly during incubation when examined in a two-hybrid yeast system. Finally, kojic acid inhibited the removal of estrogenic activities generated by bisphenol A and nonylphenol, which confirmed that laccase was involved in the degradation of EDCs in T. versicolor.     

The effect of pH on the transformation of syringic and vanillic acids by the laccases of Rhizoctonia praticola and Trametes versicolor

Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) from Rhizoctonia praticola and Trametes versicolor formed different products from syringic and vanillic acids at different pH values, but both enzymes generated the same chemicals at a particular pH. The products were separated by thin-layer and high-performance liquid chromatography. Four compounds were determined from syringic acid (m/z 168, 334, 350 and 486) at pH 6.9, but only two of these (m/z 168 and 334) were found at pH 3.5. In the case of vanillic acid two isomeric dimers (m/z 304) and 2-methoxy-1,4-benzoquinone (m/z 138) were identified. The dimers were formed at both pH values (pH 3.5 and 6.9), but 2-methoxy-1,4-benzoquinone appeared only at pH 3.5.     

Enhanced production of laccase activity by <Emphasis Type="Italic">Trametes versicolor </Emphasis>immobilized into alginate beads by the addition of different inducers

In the present paper the effect of adding veratryl alcohol and copper sulphate on laccase activity production by Trametes versicolor immobilized into alginate beads has been investigated. Employing copper sulphate as laccase inducer or supplementing the culture medium with veratryl alcohol, led to maximum values of laccase activity. However, the highest laccase activity (around 4,000 U l⁻¹) was obtained in cultures simultaneously supplemented with copper sulphate (3 mM) and veratryl alcohol (20 mM). These values implied a considerable enhancement in relation to␣control cultures without any inducer (around 200 U l⁻¹). The production of laccase by immobilized T. versicolor in a 2-l airlift bioreactor with the optimized inducer has been evaluated. Laccase activities around 1,500 U l⁻¹ were attained. The bioreactor operated for 44 days without operational problems and the bioparticles (fungus grows in alginate beads) maintained their shape throughout the fermentation. Moreover, the extracellular liquid obtained was studied in terms of pH and temperature activity and stability. On the other hand, anthracene was added in two-repeated batches in order to determine the efficiency of this process to degrade pollutants. Near complete degradation was reached in both batches. Moreover, in vitro degradation of several polycyclic aromatic hydrocarbons by crude laccase was also performed.     

Comparative characterization of four laccases from Trametes versicolor concerning phenolic C-C coupling and oxidation of PAHs

The laccase genes lccα, lccβ, lccγ and lccδ encoding four isoenzymes from Trametes versicolor have been cloned and expressed in Pichia pastoris. Biochemical characterization allowed classification of these laccases into two distinct groups: Lccα and Lccβ possessed higher thermal stability, but lower catalytic activity towards 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) compared to Lccγ and Lccδ. Activities of the laccases were quite different as well. Laccase Lccδ showed highest phenolic C-C coupling activity with sinapic acid, but lowest oxidizing activity towards polycyclic aromatic hydrocarbons (PAHs). Highest activity towards PAHs was observed with Lccβ. After 72 h, more than 80% of fluorene, anthracene, acenaphthene and acenaphthylene were oxidized by Lccβ in the presence of ABTS. Investigation of the structural basis of the different activities of the laccases demonstrated the impact of positions 164 and 265 in the substrate binding site on oxidation of PAHs.     

Biosolubilization of low-rank coal by aTrametes versicolor siderophore-like product and other complexing agents

Summary A heat stable, low molecular weight (<1000) extracellular product inTrametes versicolor (=Coriolus versicolor=Polyporous versicolor) cultures was demonstrated to be a principal factor in the solubilization of leonardite and other low-rank coals. The solubilization of leonardite byT. versicolor cell-free cultures and active fractions was inhibited by Fe³⁺ and was mimicked by the siderophore desferal mesylate and the iron chelating agents EDTA and 8-hydroxyquinoline. Leonardite solubilization by these later compounds was also inhibited by Fe³⁺. The ferrated and unferrated form of the partially purified active component fromT. versicolor cultures demonstrated absorption spectra that were similar to the ferrated and unferrated form of desferal mesylate.     

Degradation of the antibiotics norfloxacin and ciprofloxacin by a white-rot fungus and identification of degradation products

More than 90% of the antibiotics ciprofloxacin (CIPRO) and norfloxacin (NOR) at 2 mg L⁻¹ were degraded by Trametes versicolor after 7 days of incubation in malt extract liquid medium. In in vitro assays with purified laccase (16.7 nkat mL⁻¹), an extracellular enzyme excreted constitutively by this fungus, 16% of CIPRO was removed after 20 h. The addition of the laccase mediator 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt led to 97.7% and 33.7% degradation of CIPRO and NOR, respectively. Inhibition of CIPRO and NOR degradation by the cytochrome P450 inhibitor 1-aminobenzotriazole suggests that the P450 system also plays a role in the degradation of the two antibiotics. Transformation products of CIPRO and NOR were monitored at different incubation times by triple-quadrupole and quadrupole time-of-flight mass spectrometry, and can be assigned to three different reaction pathways: (i) oxidation of the piperazinyl substituent, (ii) monohydroxylation, and (iii) formation of dimeric products.     

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.     

Microarray analysis of differential gene expression elicited in Trametes versicolor during interspecific mycelial interactions

Trametes versicolor is an important white rot fungus of both industrial and ecological interest. Saprotrophic basidiomycetes are the major decomposition agents in woodland ecosystems, and rarely form monospecific populations, therefore interspecific mycelial interactions continually occur. Interactions have different outcomes including replacement of one species by the other or deadlock. We have made subtractive cDNA libraries to enrich for genes that are expressed when T. versicolor interacts with another saprotrophic basidiomycete, Stereum gausapatum, an interaction that results in the replacement of the latter. Expressed sequence tags (ESTs) (1920) were used for microarray analysis, and their expression compared during interaction with three different fungi: S. gausapatum (replaced by T. versicolor), Bjerkandera adusta (deadlock) and Hypholoma fasciculare (replaced T. versicolor). Expression of significantly more probes changed in the interaction between T. versicolor and S. gausapatum or B. adusta compared to H. fasciculare, suggesting a relationship between interaction outcome and changes in gene expression.