The role of laccase and peroxidase of <Emphasis Type="Italic">Lentinus (Panus) tigrinus</Emphasis> fungus in biodegradation of high phenol concentrations in liquid medium

The possibility of the usage of Lentinus tigrinus fungus strain VKM F-3616D for biodegradation of high (up to 5%) phenol concentrations in liquid medium and the involvement of laccase and peroxidase in this process have been studied. L. tigrinus fungus was demonstrated to effectively degrade phenol with easy biomass deletion from the liquid. Decrease in phenol concentration was accompanied by increased secretion level and laccase activity at the preliminary stages of biodegradation, while that of peroxidase was at the latest stages of biodegradation. These enzyme secretions in distinct ratios and consequences are necessary for effective phenol biodegradation. An effective approach for phenol concentration decrease in the waste water of smoking shops in meat-processing factories using L. tigrinus fungus was described.     

Isolation and properties of peroxidase produced by the fungus Panus tigrinus

Synthesis of peroxidase and laccase by the fungus Panus tigrinus was significantly stimulated by addition of the lignocellulose substrate to the culture media. Peroxidase was isolated from the culture liquid and some properties of the enzyme were investigated. P. tigrinus peroxidase belongs to a group of extracellular peroxidases similar to the plant type peroxidases.     

Effect of wood modification on lignin consumption and synthesis of lignolytic enzymes by the fungus Panus (Lentinus) tigrinus

Lignin consumption and synthesis of lignolytic enzymes by the fungus Panus (Lentinus) tigrinus cultivated on solid phase (modified and unmodified birch and pine sawdusts) were studied. The fungus grew better and consumed more readily the birch lignin than the pine wood. Peroxidase activity was higher in the case of pine sawdust; laccase and lignolytic activities, in the case of birth sawdust. Treatment with ammonia or sulfuric acid decreased lignin consumption by the fungus cultivated on either medium. Modification of sawdust by ultrasound increased lignin consumption and may be recommended for accelerating biodegradation of lignocellulose substrates.     

Laccase-mediated detoxification of phenolic compounds.

The ability of a polyphenoloxidase, the laccase of the fungus Rhizoctonia praticola, to detoxify phenolic pollutants was examined. The growth of the fungus could be inhibited by phenolic compounds, and the effective concentration was dependent on the substituents of the phenol. A toxic amount of a phenolic compound was added to a fungal growth medium in the presence or absence of a naturally occurring phenol, and half of the replicates also received laccase. The medium was then inoculated with R. praticola, and the levels of phenols in the medium were monitored by high-performance liquid chromatography analysis. The addition of the laccase reversed the inhibitory effect of 2,6-xylenol, 4-chloro-2-methylphenol, and p-cresol. Other compounds, e.g., o-cresol and 2,4-dichlorophenol, were detoxified only when laccase was used in conjunction with a natural phenol such as syringic acid. The toxicity of p-chlorophenol and 2,4,5-trichlorophenol could not be overcome by any additions. The ability of the laccase to alter the toxicity of the phenols appeared to be related to the capacity of the enzyme to decrease the levels of the parent compound by transformation or cross-coupling with another phenol.     

Laccase-mediated detoxification of phenolic compounds

The ability of a polyphenoloxidase, the laccase of the fungus Rhizoctonia praticola, to detoxify phenolic pollutants was examined. The growth of the fungus could be inhibited by phenolic compounds, and the effective concentration was dependent on the substituents of the phenol. A toxic amount of a phenolic compound was added to a fungal growth medium in the presence or absence of a naturally occurring phenol, and half of the replicates also received laccase. The medium was then inoculated with R. praticola, and the levels of phenols in the medium were monitored by high-performance liquid chromatography analysis. The addition of the laccase reversed the inhibitory effect of 2,6-xylenol, 4-chloro-2-methylphenol, and p-cresol. Other compounds, e.g., o-cresol and 2,4-dichlorophenol, were detoxified only when laccase was used in conjunction with a natural phenol such as syringic acid. The toxicity of p-chlorophenol and 2,4,5-trichlorophenol could not be overcome by any additions. The ability of the laccase to alter the toxicity of the phenols appeared to be related to the capacity of the enzyme to decrease the levels of the parent compound by transformation or cross-coupling with another phenol.     

Apparent zero-order kinetics of phenol biodegradation by substrate-inhibited microbes at low substrate concentrations

The reaction kinetics for phenol biodegradation at low substrate concentrations can be estimated based on the analysis of changes in the dissolved oxygen concentration in the bulk liquid during biodegradation. The measured oxygen concentration changes with an interesting behavior as biodegradation proceeds. The oxygen concentration in the bulk liquid decreases rapidly in the early stages of degradation and subsequently decreases linearly and then rapidly recovers to the initial saturated level. Taking into account the oxygen transfer rate between gas and liquid phases and oxygen consumption rate by microbes, the change in the dissolved oxygen concentration can be simulated with an unsteady state mass balance equation and three kinetic models for the rate of phenol metabolism: a substrate-inhibited model; a zero-order model; and a combined model. In the combined model, it is assumed that, at phenol concentrations above 10 mg/L, the degradation rate is expressed by a substrate-inhibited model; whereas at concentrations below 10 mg/L the zero-order model is applied. It was found that the characteristics of the change in the dissolved oxygen concentration, especially the rapid increase at the end of degradation, can only be described by the combined kinetic model. This result suggests that conventional Haldane-type kinetics would be unsuitable for estimating the phenol consumption rate at low phenol concentrations, in particular, at concentrations less than 10 mg/L. (c) 1996 John Wiley & Sons, Inc.     

New ways to increasing the yield of laccase from Panus tigrinus fungi

We optimized the conditions for production of laccase by lignolytic fungi Panus tigrinus 8/18. 2,4-Dimethylphenol was used as an aromatic inductor. The addition of 2,4-dimethylphenol and 2 mM CuSO4 to a rich medium was followed by a tenfold increase in the yield of this enzyme. Additional treatment of the medium with perftoran (oxygen-carrying agent) and immobilization of the fungus on polycaproamide fibers increased significantly laccase activity in the medium. The conditions for cultivation of P. tigrinus fungi were optimized. It allowed us to increase laccase activity in the medium by 25 times (compared to activity of the enzyme obtained with previously described methods).     

Bioremediation of Textile Azo Dyes by Trichophyton rubrum LSK-27

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

Submerged and solid-state production of laccase and Mn-peroxidase by Panus tigrinus on olive mill wastewater-based media

The possible use of olive-mill wastewater (OMW) as a growth medium for the production of extracellular laccase and manganese peroxidase (MnP) from the white-rot fungus Panus tigrinus (P. tigrinus) CBS 577.79 was studied using a properly formulated OMW-based medium (2-fold diluted OMW supplemented with 0.5% sucrose and 0.1% yeast extract) either in a stirred-tank or an air-lift reactor. Solid-state fermentation (SSF) was also performed in a rotary drum reactor using maize stalks moistened with the OMW-based medium. Highest levels of laccase and manganese peroxidase activity were obtained in the stirred-tank reactor (4600+/-98 U l(-1) on day 13) and in the air-lift reactor (410+/-22 on day 7), respectively. Based on total enzyme activities, SSF appears to be more suitable than LSF but the latter exhibits better volumetric productivities.     

Modeling of local dynamic behavior of phenol degradation in an internal loop airlift bioreactor by yeast Candida tropicalis

A coupled computational fluid dynamic (CFD) model, combining hydrodynamics with biochemical reactions, was developed to simulate the local transient flow patterns and the dynamic behaviors of cell growth and phenol biodegradation by yeast Candida tropicalis in an internal loop airlift reactor (ILALR). To validate this proposed model effectively, the simulated local hydrodynamic characteristics of the gas-mineral salt medium solution (gas-liquid) two-phase system, at a phenol concentration of 1,200 mg L(-1) and no presence of cells, was experimentally investigated in the ILALR using laser Doppler anemometer (LDA) measurements and conductivity probe. Furthermore, the validation of the simulated phenol biodegradation behavior by C. tropicalis at different initial concentrations of phenol and cell was also carried out in the ILALR. The time-averaged and transient results of the model simulations illustrated a satisfactory agreement with the experimental data. Finally, the local instantaneous flow and phenol biodegradation features, including gas holdup, gas velocity, liquid velocity, cell concentration, and phenol concentration inside the ILALR were successfully predicted by the proposed model.     

Biodegradation of olive oil mill wastewaterby Coriolus versicolor and Funalia trogii:effects of agitation,initial COD concentration,inoculum size and immobilization

The biodegradation of olive oil mill wastewater (OOMW) by Coriolus versicolor and Funalia trogii was investigated. Initial COD concentration, agitation and inoculum size were all found to be significant for biodegradation. Adding glucose, sulphate or nitrogen had no effect on biodegradation. During growth in optimum conditions, C.versicolor removed approximately 63% COD, 90% phenol and 65% colour within 6 days and F. trogii removed approximately 70% COD, 93% phenol and 81% colour of the OOMW used. The fungi also excreted large amounts of extracellular laccase into the medium. High biodegradation yields were also obtained by fungi immobilized in calcium alginate gels.     

Biodegradation of olive oil mill wastewaterby Coriolus versicolor and Funalia trogii:effects of agitation, initial COD concentration, inoculum size and immobilization

The biodegradation of olive oil mill wastewater (OOMW) by Coriolus versicolor and Funalia trogii was investigated. Initial COD concentration, agitation and inoculum size were all found to be significant for biodegradation. Adding glucose, sulphate or nitrogen had no effect on biodegradation. During growth in optimum conditions, C.versicolor removed approximately 63% COD, 90% phenol and 65% colour within 6 days and F. trogii removed approximately 70% COD, 93% phenol and 81% colour of the OOMW used. The fungi also excreted large amounts of extracellular laccase into the medium. High biodegradation yields were also obtained by fungi immobilized in calcium alginate gels.     

Enzymes of the xylotrophic basidiomycete Lentinus edodes F-249 in the course of morphogenesis

It has been shown that the fungus Lentinus edodes grown on a solid wort agar substrate produces intracellular enzymes, including Mn-dependent peroxidase, laccase, and tyrosinase as a family of isoforms. The composition of the complex (containing one to four forms of each enzyme) varied during the basidiomycete life cycle. The activity of oxidases was maximal at the stage of nonpigmented mycelium and at the stages of a brown mycelial mat and a fruit body. The activity of tyrosinase increased in the course of mycelium pigmentation and had two maxima: at the stage of a brown mycelial mat and at the stage of a fruit body. Laccase and tyrosinase activities were shown to increase sharply upon addition of oak sawdust extract to the culture medium as compared with the enzyme activities of mycelium grown on wort agar alone. It was established that the effect of phenol oxidase substrates on the growing mycelium consists in a twofold acceleration of the process of morphogenesis in the fungus L. edodes.     

Evaluation of toxicity and degradation of a chlorophenol mixture by the laccase produced by Trametes pubescens

In this study, the biodegradation of a mixture of 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP) using the laccase produced by the white-rot fungus Trametes pubescens CBS 696.94 was evaluated. Two laccase isoenzymes with molecular weights of about 60 and 120 kDa were identified in the enzymatic crude extract. The highest laccase activity with syringaldazine was observed with pH 6.0 and 60°C, while with 2,2-azino-bis(3-ethylbenzothiazoline-6) sulphonic acid the highest activity was observed between 50 and 60°C and 3.0-4.0 pH. A biodegradation of 100%, 99%, 82.1% and 41.1% for 2-CP, 2,4-DCP, 2,4,6-TCP and PCP, respectively, was observed after 4h of reaction. The reduction in chlorophenols concentration allowed 90% reduction in mixture toxicity. In summary, these results show the feasibility of a laccase enzymatic crude extract from T. pubescens for the reduction of concentration and toxicity of chlorophenols.     

Activity and elution profile of laccase during biological decolorization and dephenolization of olive mill wastewater

The performance and enzymatic strategy exhibited by basidiomycete Euc-1, a laccase producing strain, was investigated during the biodegradation of olive mill wastewater (OMW). This strain yielded better decolorization of solidified OMW than Phanerochaete chrysosporium and removed 90% of phenols (initial concentration=800 mg l(-1)), 73% of color (initial A465=4.4), and 45% of chemical oxygen demand in batch cultures containing OMW. Since partial phenol removal occurred before the detection of enzymatic activity, no plausible correlation could be established between them. In contrast, decolorization occurred only after the detection of laccase activity and coincided with its production over time. Two laccase fractions (Lac1 and Lac2) were separated by chromatography. OMW strongly induced Lac2 that was almost absent in defined liquid medium. Furthermore, Lac2 was the main laccase fraction in the presence of OMW. This study pointed out that basidiomycete Euc-1 and its ligninolytic system could be a useful tool for the bioremediation of wastewater generated in the process of olive oil extraction.     

苯酚高效降解菌的筛选和降解特性的研究

从天津市煤气厂的活性污泥中筛选、分离得到一株高效苯酚降解菌。经BIOLOG细菌自动鉴定系统及16SrDNA鉴定,该菌株为粪产碱杆菌(Alcaligenesfaecalis)。苯酚降解实验证实,该菌能在76h内完全降解1600mg·L-1的苯酚,并且随着苯酚浓度的增加,底物抑制作用增强,细胞得率下降。     

Increased secretion of lignolytic enzymes by the Lentinus tigrinus fungus after addition of butanol and toluene in submerged cultivation

We studied the effects of butanol and toluene on secretion of lignolytic enzymes by the Lentinus tigrinus fungus during submerged cultivation. Addition of butanol and toluene during the trophophase was followed by an increase in laccase and peroxidase activity of the culture and change in the composition of phospholipids and fatty acids. The ratio of phosphatidylcholine and phosphatidic acid decreased, while the amount of lysophosphatidylcholine, phosphatidylethanolamine, phosphoinositides, phosphatidylserine, and unsaturated fatty acids decreased. These changes resulted in an increase in the unsaturation index.     

Enzymes of the xylotrophic basidiomycete Lentinus edodes F-249 in the course of morphogenesis

It has been shown that the fungus Lentinus edodes grown on a solid wort agar substrate produces intracellular enzymes, including Mn-dependent peroxidase, laccase, and tyrosinase as a family of isoforms. The composition of the complex (containing one to four forms of each enzyme) varied during the basidiomycete life cycle. The activity of oxidases was maximal at the stage of nonpigmented mycelium and at the stages of a brown mycelial mat and a fruit body. The activity of tyrosinase increased in the course of mycelium pigmentation and had two maxima: at the stage of a brown mycelial mat and at the stage of a fruit body. Laccase and tyrosinase activities were shown to increase sharply upon addition of oak sawdust extract to the culture medium as compared with the enzyme activities of mycelium grown on wort agar alone. It was established that the effect of phenol oxidase substrates on the growing mycelium consists in a twofold acceleration of the process of morphogenesis in the fungus L. edodes.     

Use of Panus tigrinus fungi for production of pressed materials from cotton plant waste

Changes in the chemical composition of cotton plant stems used as a substrate for solid-phase cultivation of the fungus Panus tigrinus were studied as well as the effect of these changes on properties of the pressed materials made of these stems. During the first 3 days of growth, the fungus better consumed cellulose; then, the rate of cellulose consumption was comparable with that of lignin. Intensity and pattern of these changes depended on the age of inoculum. The rate of cotton plant waste biodegradation was higher when a 3-day-old incoculum was used. The pressed materials made of the raw stuff treated with a 3-day-old inoculum of P. tigrinus for 2-3 days displayed better characteristics. Annually, large amounts of lignocellulose stuff is lost while processing of agricultural waste: straw, awn, plant stems, etc. In the countries with developed cotton growing, the annual amount of only guza-paya (dry cotton plant stems) reaches several million tons. To solve this problem, bioconversion of these wastes is studied to manufacture useful products and materials.     

Increased enzymatic activities and levels of superoxide anion and phenolic compounds in cultures of basidiomycetes after temperature stress

Selected strains of basidiomycetes (Abortiporus biennis, Trametes versicolor and Cerrena unicolor) were shown to produce enhanced extracellular peroxidase (EP), superoxide dismutase (SOD) and laccase activities following the exposure of 10-day-old fungal cultures to separate high and low temperature stress. The stressful conditions also caused an increase in the concentrations of phenol compounds and superoxide anion radicals in these cultures. At first, peroxidase activity was observed at 12 hours from the moment of temperature stress application. Laccase activity appeared at 96 hours after the maximum levels of superoxide anion radicals (48 h) and SOD activity (36–72 h). The concentration of phenolic substances grew steadily during the period of cultivation. These relations between laccase, SOD and EP as well as superoxide radicals and phenol levels in the environment of ligninolytic fungi seems to be important in the course of the biosynthesis or biodegradation of lignin, as the consequence of adaptation of these basidiomycetes to environmental temperature conditions.