Fungal bioremediation of phenolic wastewaters in an airlift reactor

Of the various types of industry-generated effluents, those containing organic pollutants such as phenols are generally difficult to remediate. There is a need to develop new technologies that emphasize the destruction of these pollutants rather than their disposal. In this work the white rot fungus, Trametes pubescens, was demonstrated to be an effective bioremediation agent for the treatment of phenolic wastewaters. An airlift loop reactor was optimized, in terms of volumetric oxygen transfer rate (K(L)a = 0.45 s(-1)), to provide an environment suited to rapid growth of T.pubescens (mu = 0.25 day(-1)) and a particularly efficient growth yield on glucose of 0.87 g biomass.g glucose(-1). The phenolic effluent was shown to be a paramorphogen, influencing fungal pellet morphology in the reactor, as well as increasing laccase enzyme activity by a factor of 5 over the control, to a maximum of 11.8 U.mL(-1). This increased activity was aided by the feeding of nonrepressing amounts (0.5 g.L(-1)) of glucose to the reactor culture. To our knowledge the degradation results represent the highest rate of removal (0.033 g phenol.g biomass(-1).day(-1)) of phenolic compounds from water reported for white rot fungi.     

Enhancement of phenolic compounds oxidation using laccase from Trametes versicolor in a microreactor

Laccases catalyse the oxidation of a wide range of substrates by a radical-catalyzed reaction mechanism, with a corresponding reduction of oxygen to water in a four-electron transfer process. Due to that, laccases are considered environmentally friendly enzymes, and lately there has been great interest in their use for the transformation and degradation of phenolic compounds. In this work, enzymatic oxidation of catechol and L-DOPA using commercial laccase from Trametes versicolor was performed, in continuously operated microreactors. The main focus of this investigation was to develop a new process for phenolic compounds oxidation, by application of microreactors. For a residence time of 72 s and an inlet oxygen concentration of 0.271 mmol/dm³, catechol conversion of 41.3% was achieved, while approximately the same conversion of L-DOPA (45.0%) was achieved for an inlet oxygen concentration of 0.544 mmol/dm³. The efficiency of microreactor usage for phenolic compounds oxidation was confirmed by calculating the oxidation rates; in the case of catechol oxidation, oxidation rates were in the range from 76.101 to 703.935 g/dm³/d (18–167 fold higher, compared to the case in a macroreactor). To better describe the proposed process, kinetic parameters of catechol oxidation were estimated, using data collected from experiments performed in a microreactor. The maximum reaction rate estimated in microreactor experiments was two times higher than one estimated using the initial reaction rate method from experiments performed in a cuvette. A mathematical model of the process was developed, and validated, using data from independent experiments.     

Production of laccase from Trametes versicolor by solid-state fermentation using olive leaves as a phenolic substrate

The aim of the present study was to investigate whether olive leaves were feasible as a substrate for laccase production by the white-rot fungus Trametes versicolor FPRL 28A INI under solid-state fermentation conditions. Different experiments were conducted to select the variables that allow obtaining high levels of laccase activity. In particular, the effects of the initial moisture content, substrate particle size, supplementation with inorganic and organic nitrogen sources were evaluated. Highest laccase activity (276.62 ± 25.67 U/g dry substrate) was achieved with 80 % initial moisture content and 1.4–1.6 mm particle size of the substrate supplemented with yeast extract (1 % (w/w) nitrogen). Such a high activity was obtained without any addition of inducers.     

Improving laccase production by employing different lignocellulosic wastes in submerged cultures of Trametes versicolor

Laccase production by the white-rot fungus Trametes versicolor (CBS100.29) grown in submerged cultures was studied. Addition of different insoluble lignocellulosic materials into the culture medium in order to enhance laccase production was investigated. The lignocellulosic materials were grape seeds, grape stalks and barley bran, selected because of their availability and low cost, since they are agro-industrial wastes abundant in most countries. Barley bran gave the highest activities, a maximum value of 639U/l, which was 10 times the value attained in the cultures without lignocellulosics addition. The decolourisation of a model dye, Phenol Red, by the ligninolytic fluids obtained in the above-mentioned cultures was investigated. Grape stalk and barley bran cultures showed the highest ability to decolourise the dye, attaining a percentage of decolourisation of around 60% in 72 h.     

Degradation of phenanthrene by Trametes versicolor and its laccase

Phenanthrene is a three-ring polycyclic aromatic hydrocarbon and commonly found as a pollutant in various environments. Degradation of phenanthrene by white rot fungus Trametes versicolor 951022 and its laccase, isolated in Korea, was investigated. After 36 h of incubation, about 46% and 65% of 100 mg/l of phenanthrene added in shaken and static fungal cultures were removed, respectively. Phenanthrene degradation was maximal at pH 6 and the optimal temperature for phenanthrene removal was 30 degrees C. Although the removal percentage of phenanthrene was highest (76.7%) at 10 mg/l of phenanthrene concentration, the transformation rate was maximal (0.82 mg/h) at 100 mg/L of phenanthrene concentration in the fungal culture. When the purified laccase of T versicolor 951022 reacted with phenanthrene, phenanthrene was not transformed. The addition of redox mediator, 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) or 1-hydroxybenzotriazole (HBT) to the reaction mixture increased oxidation of phenanthrene by laccase about 40% and 30%, respectively.     

变色栓菌产锰过氧化物酶的条件优化

研究了多种培养基组分及培养条件对变色栓菌产锰过氧化物酶(MnP)的影响.当培养基中果糖浓度为20g/L,酒石酸铵浓度为10mmoL/L,吐温80浓度为1.0g/L,MgSO4·7H2O为0.43g/L,最终pH为4.5,500mL三角瓶装液量为100mL,接种量为10片(φ8mm)菌苔,培养温度为30℃,转速为280r/min时,MnP的活力有了很大程度的提高,最高酶活力可达2,270U/L.     

Transformation of triclosan by Trametes versicolor and Pycnoporus cinnabarinus

We investigated the ability of Trametes versicolor and Pycnoporous cinnabarinus to metabolize triclosan. T. versicolor produced three metabolites, 2-O-(2,4,4'-trichlorodiphenyl ether)-beta-D-xylopyranoside, 2-O-(2,4,4'-trichlorodiphenyl ether)-beta-D-glucopyranoside, and 2,4-dichlorophenol. P. cinnabarinus converted triclosan to 2,4, 4'-trichloro-2'-methoxydiphenyl ether and the glucoside conjugate known from T. versicolor. The conjugates showed a distinctly lower cytotoxic and microbicidal activity than triclosan did.     

Cloning of Trametes versicolor Genes Induced by Nitrogen Starvation

We have screened a genomic library of Trametes versicolor for genes whose expression is associated with nitrogen starvation, which has been shown to induce ligninolytic activity. Using two different approaches based on differential expression, we isolated 29 clones. These were shown by restriction mapping and cross-hybridization to code for 11 distinct differentially expressed genes. Northern analysis of the kinetics of expression of these genes revealed that at least four of them have kinetics of induction that parallel kinetics of induction of ligninolytic activity.     

Trametes versicolor: Potential for atrazine bioremediation in calcareous clay soil,under low water availability conditions

This study examined the feasibility of Trametes versicolor to actively degrade atrazine (0.5 μg g^?1) in non-sterile calcareous clay soil (Algarve, Portugal) microcosms for up to 24 weeks (20 °C), under low water availability (soil water potentials of ?0.7 and ?2.8 MPa). Soil respiration, laccase activity, and atrazine quantification by high-performance liquid chromatography (HPLC) were assessed. Respiration was significantly (p < 0.05) enhanced in soil containing the inoculant, particularly in the presence of atrazine, indicating that it remained metabolically active throughout the study. Furthermore, up to 98% and 85% (at ?0.7 and ?2.8 MPa, respectively) of atrazine was degraded in soil containing both the atrazine and the inoculant, compared to 96% and 50% in soil containing atrazine only. The contribution of T. versicolor to atrazine degradation was only significant (p < 0.005) under the driest soil treatment conditions. The strategies used for enhancing colonisation and biodegradation capabilities of the inoculant, as well as the selection of sawdust as carrier, were thus effective. However, there were no differences (p > 0.05) in quantified laccase activity in soil containing the inoculant and the control. Overall, this study demonstrated that T. versicolor was a strong candidate for atrazine bioremediation in soil with low moisture and organic matter contents, such as that found in semi-arid and Mediterranean-like ecosystems.     

Isolation and Identification of the Coal-Solubilizing Agent Produced by Trametes versicolor

Low-ranked coals were dissolved by using cell extracts derived from liquid cultures of Trametes versicolor. The coal-solubilizing agent (CSA) was separated from the broth components by a multistep isolation procedure including reverse-phase high-pressure liquid chromatography, size exclusion chromatography, ethanol fractionation, and recrystallization. Staircase voltammetry was used to show that two CSA moieties can coordinate to aqueous copper(II) ion. A molecular weight determination (using amperometry) gave an apparent molecular weight of 1.3₄ × 10² g/mol ± 8%. Nuclear magnetic resonance indicated that all protons on CSA are exchangeable in D₂O and that there is only one type of carbon in CSA. The infrared spectrum of recrystallized CSA is identical to that of ammonium oxalate, and X-ray studies confirmed the crystal structure and composition of CSA to be that of ammonium oxalate monohydrate. The equivalent weight of the coal in solution, when the coal was dissolved by ammonium oxalate, is 7,940 g of coal per mol of iron present in the coal.     

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.     

Optimization of laccase production from Trametes versicolor by solid fermentation

The regulation of culture conditions, especially the optimization of substrate constituents, is crucial for laccase production by solid fermentation. To develop an inexpensive optimized substrate formulation to produce high-activity laccase, a uniform design formulation experiment was devised. The solid fermentation of Trametes versicolor was performed with natural aeration, natural substrate pH (about 6.5), environmental humidity of 60% and two different temperature stages (at 37 degrees C for 3 days, and then at 30 degrees C for the next 17 days). From the experiment, a regression equation for laccase activity, in the form of a second-degree polynomial model, was constructed using multivariate regression analysis and solved with unconstrained optimization programming. The optimized substrate formulation for laccase production was then calculated. Tween 80 was found to have a negative effect on laccase production in solid fermentation; the optimized solid substrate formulation was 10.8% glucose, 27.7% wheat bran, 9.0% (NH4)2SO4, and 52.5% water. In a scaled-up verification of solid fermentation at a 10 kg scale, laccase activity from T. versicolor in the optimized substrate formulation reached 110.9 IU/g of dry mass.     

Induction of laccases in Trametes versicolor by aqueous wood extracts

The induction of laccase isoforms in Trametes versicolor HEMIM-9 by aqueous extracts (AE) from softwood and hardwood was studied. Samples of sawdust of Pinus sp., Cedrela sp., and Quercus sp. were boiled in water to obtain AE. Different volumes of each AE were added to fungal cultures to determine the amount of AE needed for the induction experiments. Laccase activity was assayed every 24 h for 15 days. The addition of each AE (50 to 150 μl) to the fungal cultures increased laccase production compared to the control (0.42 ± 0.01 U ml^?1). The highest laccase activities detected were 1.92 ± 0.15 U ml^?1 (pine), 1.87 ± 0.26 U ml^?1 (cedar), and 1.56 ± 0.34 U ml^?1 (oak); laccase productivities were also significantly increased. Larger volumes of any AE inhibited mycelial growth. Electrophoretic analysis revealed two laccase bands (lcc1 and lcc2) for all the treatments. However, when lcc2 was analyzed by isoelectric focusing, inducer-dependent isoform patterns composed of three (pine AE), four (oak AE), and six laccase bands (cedar AE) were observed. Thus, AE from softwood and hardwood had induction effects in T. versicolor HEMIM-9, as indicated by the increase in laccase activity and different isoform patterns. All of the enzymatic extracts were able to decolorize the dye Orange II. Dye decolorization was mainly influenced by pH. The optimum pH for decolorization was pH 5 (85 %), followed by pH 7 (50 %) and pH 3 (15 %). No significant differences in the dye decolorizing capacity were detected between the control and the differentially induced laccase extracts (oak, pine and cedar). This could be due to the catalytic activities of isoforms with pI 5.4 and 5.8, which were detected under all induction conditions.     

A method for the decrease of phenolic content in commercial canola meal using an enzyme preparation secreted by the white-rot fungus Trametes versicolor

An enzymatic process for upgrading the quality of canola meal (CM) by decreasing its phenolic content was investigated. The new method was based on the addition of the enzyme preparation from white-rot fungus Trametes versicolor to the meal-buffer slurry. A 98% decrease in the concentration of SAE was observed after 1 h of the treatment. The following process variables were considered for optimizing the process: pH, temperature, enzyme, meal, and oxygen concentrations. It was found that: (1) the natural buffering capacity of CM resulted in a negligible effect of the pH of the buffer, which was used as the continuous phase in the process, on the extent of decrease in sinapic acid esters (SAE); (2) the system was saturated with the enzyme when its concentration was 4 nkat/mL of the continuous phase; and (3) the optimum temperature was 50 degrees C. The process could be carried out even at higher temperatures due to the protective action of CM, which resulted in an increase in the thermal stability of the enzyme. The particle size influenced the extraction of the SAE from the meal, indicating that, at lower SAE concentrations, the process became diffusion limited. This result, together with those showing no effect of the intensity of agitation, indicated that the enzymatic process can be characterized by high Biot numbers. During the enzymatic process, the molar concentration of available oxygen can become a limiting factor when it is more than four times lower than the molar concentration of phenolics in the treated meal. The new enzymatic method was compared with other methods reported in the literature for the decrease in the phenolic content of rapeseed meals. It was found that, among the methods tested, the enzymatic treatment was the most effective, followed by the lime treatment. The enzymatic process did not reduce the quality of the protein isolates prepared from the CM. After the addition of a simple acetone-washing step, the isolate from the enzymatically treated meal had even better properties. Copyright 1999 John Wiley & Sons, Inc.     

Farnesol stimulates laccase production in Trametes versicolor

Farnesol, a quorum‐sensing molecule, was used successfully to improve laccase production in submerged cultures of Trametes versicolor. At the optimal farnesol concentration of 60 μM added at the beginning of the culture, the extracellular laccase activity reached 629.3 U L^?1 after 6 days of cultivation, which represented a 1.92‐fold increase relative to the control without farnesol treatment. The addition of farnesol resulted in an increase in the accumulation of H₂O₂ and an increased expression of the laccase (lac) gene and the RhoA gene. The RhoA gene correlated with hyperbranched mycelia, which facilitated the secretion of the intracellular laccase. This study provides a basis for understanding the induction mechanism of farnesol for enhancing laccase production.     

An oxygenase enzyme system for Trametes versicolor

Abstract An oxygenase enzyme was isolated from the basidiomycete fungus Trametes versicolor , that is capable of attacking lignin and a large number of di- and tri-substituted benzene rings containing at least one hydroxy group. This enzyme system was produced late in the growth cycle without the requirement for any inducer. This non-selective enzyme system is thermophilic and operates at pH 3–5 in the presence of air or oxygen. The action of this enzyme system caused the loss of UV absorption in ferulic acid solution, the formation of hydroxy muconic semialdehyde from catechol, and transformation with the production of CO₂ from a number of hydroxy aromatics as well as lignin.     

云芝菌株遗传多样性的ISSR分析

来自全国的34株野生菌株经形态学特征和结合ITS序列鉴定为云芝。采用ISSR标记技术对34株野生云芝菌株进行遗传多样性分析。从20条引物中筛选出7条ISSR引物,扩增得到95个扩增位点,其中多态性位点88个。多态性位点占92.6%,表明ISSR标记的多态性非常高。基于ISSR条带构建亲缘关系树状图,其中遗传变异系数范围为0.58-0.91。34个云芝菌株在相似系数0.60时分为4个类群,不同菌株的遗传差异性与地理分布有一定联系。     

Overproduction of Trametes versicolor laccase by making glucose starvation using yeast

In the present paper, overproduction of laccase by microbe interaction was studied. When Trametes versicolor was co-cultured with Candida sp. HSD07A in submerged fermentation, laccase activity could be improved significantly and reached 10500 ± 160 U/l, 11.8 times more than that of the contrast group. Fermentation tests of the yeast indicated that it could produce amylase and cellulase, but couldn’t excrete laccase and the overproductive laccase was produced by T. versicolor; the interaction mechanism between T. versicolor and Candida sp. HSD07A was investigated and the results showed that amylase and cellulose could hydrolyze cell walls of T. versicolor; however, the degree of hydrolysis was at a very low level, could not lead to overproduction of laccase; glucose starvation state made by the yeast was the real reason why T. versicolor could overproduce laccase; moreover, this study also proved that making glucose starvation using the yeast was a novel and effective method.