Irpex lacteus, a white-rot fungus with biotechnological potential--review.

White-rot fungi that are efficient lignin degraders responsible for its turnover in nature have appeared twice in the center of biotechnological research - first, when the lignin degradation process started being systematically investigated and major enzyme activities and mechanisms involved were described, and second, when the huge remediation potential of these organisms was established. Originally, Phanerochaete chrysosporium became a model organism, characterized by a secondary metabolism regulatory pattern triggered by nutrient (mostly nitrogen) limitation. Last decade brought evidence of more varied regulatory patterns in white-rot fungi when ligninolytic enzymes were also abundantly synthesized under conditions of nitrogen sufficiency. Gradually, research was focused on other species, among them Irpex lacteus showing a remarkable pollutant toxicity resistance and biodegradation efficiency. Systematic research has built up knowledge of biochemistry and biotechnological applicability of this fungus, stressing the need to critically summarize and estimate these scattered data. The review attempts to evaluate the information on I. lacteus focusing on various enzyme activities and bioremediation of organopollutants in water and soil environments, with the aim of mediating this knowledge to a broader microbiological audience.

Degradation of polyesteramides during composting under standard and isothermal conditions.

Polyesteramides based on epsilon-caprolactam and epsilon-caprolactone differing in the content of ester-amide structural units were subjected to biodegradation - composting in a big compost pile under controlled conditions (controlled composting) and in small composters at a steady temperature of 60 degrees C (isothermal composting). Both types of composting resulted in degradation of the polyesteramides depending on copolymer composition, isothermal composting being more robust. The contribution of abiotic hydrolysis to the degradation of polyesteramides was studied at 60 degrees C in buffer solutions with pH 5.4, 7.4 and 8.4. The prevailing effect of abiotic hydrolysis over biological one was shown at 60 degrees C. Ester bonds in polyestramide chains were preferentially cleaved; therefore, the scope of degradation increased with the content of ester units in the copolymer.

Degradation of PAHs by ligninolytic enzymes of Irpex lacteus.

The ligninolytic fungus Irpex lacteus was shown as an efficient degrader of oligocyclic aromatic hydrocarbons (PAHs; 'polycyclic aromatic hydrocarbons') possessing 3-6 aromatic rings in complex liquid media. The strain produced mainly Mn-dependent peroxidase in media without pollutants. Activity of ligninolytic enzymes was higher in a N-limited medium. However, after contamination with PAHs (especially pyrene) the values increased and significant activity of Mn-independent peroxidase appeared in the complex medium. Other factors (such as the increase in nitrogen concentration or the presence of solvent(s) for dissolution of PAHs) had no effect. Cytochrome P-450 was detected in the microsomal fraction of biomass grown in the complex medium. The rate of PAH degradation was also affected by the presence of various combinations of PAHs. However, independently of the enzyme activities, anthracene was shown to have a positive influence on degradation of pyrene and fluoranthene.

Mobilizing agents enhance fungal degradation of polycyclic aromatic hydrocarbons and affect diversity of indigenous bacteria in soil.

The impact of several mobilizing agents (MAs) (i.e., soybean oil, Tween-20, Tween-80, olive-oil mill wastewaters, and randomly methylated beta-cyclodextrins) on the degradation performances of the white-rot fungi Irpex lacteus and Pleurotus ostreatus was comparatively assessed in a soil spiked with a mixture of seven polycyclic aromatic hydrocarbons (PAHs). Among the different MAs, soybean oil best supported the growth of both fungi that was twice that observed in soil in the absence of MAs. In addition, soybean oil positively affected PAH degradation by both fungi. In this case, the total weight of organic contaminants (TWOC) was lower than that in the absence of MAs (57.7 vs. 201.3 and 26.3 vs. 160.4 mg kg(-1) with I. lacteus and P. ostreatus, respectively). On the other hand, the number of cultivable heterotrophic bacteria was significantly lower in the soil with soybean oil augmented with either one of the two fungi (5.21 vs. 8.71 and 0.22 vs. 0.51 x 10(7) CFU g(-1) soil with I. lacteus and P. ostreatus, respectively). The effect of soybean oil was confirmed by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes that showed a general decrease in biodiversity. The impact of the other MAs on bacterial diversity was either slightly negative or positive in incubation controls. Both richness and Shannon-Weaver index decreased upon treatment with P. ostreatus. Moreover, with this fungus the composition of the indigenous bacteria was not significantly affected by the type of MA used. By contrast, both indices increased in soil with I. lacteus in the presence of randomly methylated beta-cyclodextrins (39 vs. 33 and 1.43 vs. 1.26, respectively) and soybean oil (19 vs. 5 and 1.01 vs. 0.65, respectively).

Biodegradation of synthetic polymers by composting and fungal treatment.

Two types of copolymers--poly(ester-amide)s--by the anionic copolymerization of epsilon-caprolactam and epsilon-caprolactone, and aromatic-aliphatic copolyesters based on glycolyzed polyethylene terephthalate from used beverage bottles and epsilon-caprolactone were prepared. Biodegradation tests of these copolymers were performed by two methods, viz. composting under controlled conditions and treatment with ligninolytic fungi. Both methods resulted in degradation of the copolymers, composting being more robust. Out of fungal strains tested Inonotus hispidus degraded aromatic-aliphatic copolyesters most intensively.

Compost-mediated removal of polycyclic aromatic hydrocarbons from contaminated soil.

Compost-assisted remediation of a manufactured-gas plant soil contaminated with polycyclic aromatic hydrocarbons (PAHs) was performed in thermally insulated composting chamber using mushroom compost consisting wheat straw, chicken manure, and gypsum. The degradation of individual PAHs was in range of 20-60% at the end of 54 days of composting followed by further increase of PAH removal (37-80%) after another 100 days of maturation. Both chemical analysis of the contaminated soil for PAHs and ecotoxicity tests on bioluminescent bacteria, earthworms, and plant seeds were performed before and after the composting. After the composting, inhibition of bioluminescence decreased, whereas no significant change in toxicity was observed for earthworm survival and seed germination. Using bacterial culture of Escherichia coli K12 genotoxicity tests were performed on samples taken from different parts of the composting pile; after the composting the decrease in genotoxicity was observed only in the sample taken from upper part of the composted pile.

Mycoremediation of PAH-contaminated soil.

Out of a number of white-rot fungal cultures, strains of Irpex lacteus and Pleurotus ostreatus were selected for degradation of 7 three- and four-ring unsubstituted aromatic hydrocarbons (PAH) in two contaminated industrial soils. Respective data for removal of PAH in the two industrial soils by I. lacteus were: fluorene (41 and 67%), phenanthrene (20 and 56%), anthracene (29 and 49%), fluoranthene (29 and 57%), pyrene (24 and 42%), chrysene (16 and 32%) and benzo[a]anthracene (13 and 20%). In the same two industrial soils P. ostreatus degraded the PAH with respective removal figures of fluorene (26 and 35%), phenanthrene (0 and 20%), anthracene (19 and 53%), fluoranthene (29 and 31%), pyrene (22 and 42%), chrysene (0 and 42%) and benzo[a]anthracene (0 and 13%). The degradation of PAH was determined against concentration of PAH in non-treated contaminated soils after 14 weeks of incubation. The fungal degradation of PAH in soil was studied simultaneously with ecotoxicity evaluation of fungal treated and non-treated contaminated soils. Compared to non-treated contaminated soil, fungus-treated soil samples indicated decrease in inhibition of bioluminescence in luminescent bacteria (Vibrio fischerii) and increase in germinated mustard (Brassica alba) seeds.

Bioremediation of PAH-contaminated soil by composting: a case study.

Composting technique was used for bioremediation of industrial soil originating from a former tar-contaminated site. The composting process was regulated by aeration to keep optimal temperature gradient and concentrations of O2 and CO2 inside the composting pile. The efficiency of bioremediation was evaluated by performing analysis of 11 individual three- to six-ring unsubstituted aromatic hydrocarbons (PAH) and estimating of changes in ecotoxicity of the contaminated soil. After 42 d of composting, PAH with 3-4 rings were removed from 42 to 68%, other higher-molar mass PAH from 35 to 57%. Additional 100 d of compost maturation in open-air field did not result in a further decrease of PAH. Ecotoxicity tests performed with bioluminescent bacteria Vibrio fischerii showed a decrease in toxicity both after composting and maturation phases. However, toxicity tests on mustard-seed germination did not reveal any significant changes during composting and maturation phases.

Capacity of Irpex lacteus and Pleurotus ostreatus for decolorization of chemically different dyes.

The rate and efficiency of decolorization of poly R-478- or Remazol Brilliant Blue R (RBBR)-containing agar plates (200 microg x g(-1)) were tested to evaluate the dye degradation activity in a total of 103 wood-rotting fungal strains. Best strains were able to completely decolorize plates within 10 days at 28 degrees C. Irpex lacteus and Pleurotus ostreatus were selected and used for degradation of six different groups of dyes (azo, diazo, anthraquinone-based, heterocyclic, triphenylmethane, phthalocyanine) on agar plates. Both fungi efficiently degraded dyes from all groups. Removal of RBBR, Bromophenol blue, Cu-phthalocyanine, Methyl red and Congo red was studied with I. lacteus also in liquid medium. Within 14 days, the following color reductions were attained: RBBR 93%, Bromophenol blue 100%, Cu-phthalocyanine 98%, Methyl red 56%, Congo red 58%. The ability of I. lacteus to degrade RBBR spiked into sterile soil was checked, the removal being 77% of the dye added within 6 weeks. The capacity of selected white rot fungal species to remove efficiently diverse synthetic dyes from water and soil environments is documented.

Hydrocarbon deposition and soil microflora as affected by highway traffic.

The proximity of a busy highway (90,000 vehicles/day) increased the amount of polycyclic aromatic hydrocarbons (PAHs) in soil at the depth of 5-15 cm from 106 ng/g as a grassland background to 3095 ng/g dry soil at the highway verge (a sum of 10 PAH species). The PAH concentration was related to the distance from the source and exhibited a biphasic character, which is interpreted in terms of bimodal distribution of the exhaust microparticles with different rates of deposition. Similarly, the tendency of benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, and indeno(1,2,3-cd)pyrene to decrease their proportion with distance from the highway, in contrast to phenanthrene, fluoranthene, pyrene, benzo(a)pyrene, and benzo(g,h,i)perylene, was attributed to their prevalent localisation on the heavier particle fraction. The abundance of bacteria (8.33 x background) and fungi (3.17 x background) close to the highway is thought to be a consequence of hydrocarbon deposition from the traffic that serves as a significant energetic input into the soil. The elevated concentrations of hydrocarbon substrates, as indicated by PAHs, increased both the absolute and relative numbers of the microbial degraders of diesel fuel, biphenyl, naphthalene, and pyrene. Their maximum numbers at 0.5-1.5 m from the pavement reached 1.3 x 10(4), 1.2 x 10(5), 1.1 x 10(4), and 6.6 x 10(3) colony-forming units (CFU) or infection units per gramme dry soil, respectively. On the other hand, the number of anthracene degraders (1.1 x 10(3) CFU per g dry soil) remained close to the detection limit of the enumeration technique used (0.1-0.2 x 10(3) per g dry soil), consistently with the absence of anthracene and higher linear PAHs in the investigated soil samples. The amounts of persisting PAHs justify artificial inoculation with effective degrader strains in the vicinity of motorways.

PCB congener selective biodegradation by the white rot fungus Pleurotus ostreatus in contaminated soil.

Six strains of white rot fungi were tested for their biodegradation ability of low chlorinated polychlorinated biphenyl (PCB) commercial mixture (Delor 103) in real soil system. Phanerochaete chrysosporium and Trametes versicolor did not show any ability to degrade PCBs in soil. On the contrary, four strains of Pleurotus ostreatus were able to remove about 40% of Delor 103 in two months. All P. ostreatus strains decomposed PCBs selectively with the preference for congeners with chlorine atoms in ortho > meta > para position. Degradation efficiency decreased with increasing number of chlorination.

Irpex lacteus, a white rot fungus applicable to water and soil bioremediation.

Growth parameters, ligninolytic enzyme activities and ability to degrade polycyclic aromatic hydrocarbons by the fungus Irpex lacteus were characterized and compared with those of other white rot fungi capable of rapid decolorization of poly R-478 and Remazol Brilliant Blue R dyes. I. lacteus was able to grow on mineral and complex media and efficiently colonized sterile and non-sterile soil by exploratory mycelium growing from a wheat straw inoculum. In shallow stationary cultures growing on high nitrogen mineral medium containing 45 mM ammonium as nitrogen source, the fungus produced lignin peroxidase (LIP), Mn-dependent peroxidase (MnP) and laccase simultaneously, the respective maximal activities of 70, 970 and 36 U/l being attained around day 18. Growing in nitrogen-limited medium (2.4 mM ammonium), no LIP was formed and levels of MnP and laccase decreased significantly. During growth in sterile soil, the fungus synthesized LIP and laccase but not MnP. I. lacteus efficiently removed three- and four-ringed PAHs from liquid media and artificially spiked soil. The variety of ligninolytic enzymes, robust growth, capability of soil colonization and resistance to inhibitory action of soil bacteria make I. lacteus a suitable fungal organism for use in bioremediation.

Extracellular oxidative enzyme production and PAH removal in soil by exploratory mycelium of white rot fungi.

Selected strains of three species of white rot fungi, Pleurotus ostreatus, Phanerochaete chrysosporium and Trametes versicolor, were grown in sterilized soil from straw inocula. The respective colonization rates and mycelium density values decreased in the above mentioned order. Three- and four-ringed PAHs at 50 ppm inhibited growth of fungi in soil to some extent. The activities of fungal MnP and laccase (units per g dry weight of straw or soil), extracted with 50 mM succinate-lactate buffer (pH 4.5), were 5 to 20-fold higher in straw compared to soil. The enzyme activities per g dry soil in P. ostreatus and T. versicolor were similar, in contrast to P. chrysosporium, where they were extremely low. Compared to the aerated controls, P. ostreatus strains reduced the levels of anthracene, pyrene and phenanthrene by 81-87%, 84-93% and 41-64% within 2 months, respectively. During degradation of anthracene, all P. ostreatus strains accumulated anthraquinone. PAH removal rates in P. chrysosporium and T. versicolor soil cultures were much lower.

Investigation into PCB biodegradation using uniformly 14C-labelled dichlorobiphenyl.

Biodegradation of polychlorinated biphenyls (PCBs) in soil is considered to be very complex due to various physico-chemical factors involved. Isotope labelling technique is the best to trace fate of the xenobiotic in the environment. In this work, the uniformly 14C-labelled PCB congener 11 (3,3'-chlorobiphenyl) was chosen as a low chlorinated coplanar biphenyl which was assumed to be readily degraded by microorganisms. Pleurotus ostreatus and two Pseudomonas species, representing white rot fungi and soil bacteria were used separately or in a consortium. The amount of liberated 14CO2 and radio-HPLC, HPLC, GC-MS, and radio-TLC analyses of extracts at the end of a two-month experiment showed that the mineralization of PCB 11 was < 0.4%, volatilization < 3.1%, and 30% of radioactivity was irreversibly bound to the soil matrix. The respective contents of all intermediate metabolites were 4.7 to 10.5 and 2.5 to 2.7% where Pseudomonas alcaligenes alone or in combination with P. putida was applied. 3-Chlorobenzoic acid was the major biodegradation product.

Removal of PCBs by various white rot fungi in liquid cultures.

The ability of Phanerochaete chrysosporium, Trametes versicolor, Coriolopsis polyzona, and Pleurotus ostreatus growing in a mitogen-limited mineral medium (NMM) to degrade PCBs in a commercial, Delor 106 mixture at a concentration of 0.9 ppm was compared. The respective amount of PCBs removed from the fungal cultures within 3 weeks were 25, 50, 41 and 0%. The capacities of the individual fungal species to remove PCBs correlated to some extent with their capabilities of decolorization of NMM agar containing both Poly R-478 or Remazol Brilliant Blue R dyes. Enzyme estimations indicated that both high and relatively stable activities of Mn-dependent peroxidase, Mn-independent peroxidase, lignin peroxidase, and laccase characterized efficient PCB degraders.

Physical factors negatively affecting evaluation of long-term biodegradation experiments of polychlorinated biphenyls.

Behaviour of PCB added in the form of a technical mixture (Delor 106) and [14C]PCB 77 into different aqueous model cultivation media in long-term experiments was studied. The work was focused on monitoring PCB recovery from liquid media in the absence of micro-organisms. GC-ECD and radioanalysis were utilised for determination of PCB in the examined systems. Strong relations between duration of experiments, composition of media, degree of chlorination of biphenyls, and recovery of PCB were found. The lowest yields of PCB were observed for the extracts from model solutions based on N-limited cultivation media without veratryl alcohol and Tween 80. The addition of these components, mainly of Tween 80, caused a significant increase of PCB recoveries.

Production of thermozymocidin (myriocin) by the pyrenomycete Melanconis flavovirens.

Submerged culture of the pyrenomycete Melanconis flavovirens produces a strongly active antifungal antibiotic. The antibiotic was isolated from the culture mash. In purified compound the physico-chemical characteristics, including 1H NMR spectrum, were estimated. The antibiotic was found to be identical with thermozymocidin (myriocin) as confirmed by comparison with synthetically prepared thermozymocidin.

Flavovirin--a new antifungal antibiotic produced by the pyrenomycete Melanconis flavovirens.

The pyrenomycete Melanconis flavovirens was found to produce a mixture of two antifungal antibiotics. The first has been already described; the antibiotic is identical with thermozymocidin (myriocin). The second antibiotic is a new compound, whose isolation and identification is presented in this paper. The chemical structure of this substance (C21H37NO5) was determined to be 2-amino-2-hydroxymethyl-3,4-trans-epoxy-14-oxo-eicos-trans-6-enoic acid. After the producer it was named flavovirin and it possesses strong activity against yeasts and a moderate effectivity against filamentous fungi.

Antibiotic production by submerged culture of Melanconis flavovirens (Otth) Wehm.

The pyrenomycete Melanconis flavovirens under submerged cultivation produced antibiotics active against both bacteria and fungi. Glucose and corn-steep liquor were the best C and N sources for the antibiotic production, respectively. Supplementation with Tween-80 and enthylene glycol stimulated both antifungal and antibacterial antibiotic production, whereas oleic acid only the antifungal one. Addition of K2HPO4 also showed a positive effect. The optimal conditions for fermentation of the antifungal component are given.

Antibiotic activity of pyrenomycetes under submerged conditions.

Twenty-three pyrenomycete species were tested for antibiotic activity in submerged cultures. When they were screened against bacteria and fungi, 15 showed positive results. Among these, Eutypa acharii, Diaporthe pustulata, Melanconis flavovirens and Camarops microspora were quite promising against bacteria and/or fungi. An antibacterial antibiotic from Camarops microspora was partially purified and characterized.